Aopwiki

SNAPSHOT

Created at: 2017-10-12 14:34

AOP ID and Title:


AOP 3: Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits
Short Title: Mitochondrial dysfunction and Neurotoxicity

Authors


Anna Bal-Price, Chemical Safety and Alternative Methods Unit/EURL ECVAM of the European Commission, Ispra, Italy

Marcel Leist, University of Konstanz, Konstanz, Germany

Stefan Schildknecht, University of Konstanz, Konstanz, Germany

Florianne Tschudi-Monnet, University of Lausanne and SCAHT, Lausanne, Switzerland

Alicia Paini, Chemical Safety and Alternative Methods Unit/EURL ECVAM of the European Commission, Ispra, Italy

Andrea Terron, European Food Safety Authority, Parma Italy (corresponding author: Andrea.TERRON@efsa.europa.eu)


Status

Author status OECD status OECD project SAAOP status
Open for citation & comment EAGMST Under Review 1.33 Included in OECD Work Plan

Abstract


This Adverse outcome Pathway (AOP) describes the linkage between inhibition of complex I (CI) of the mitochondrial respiratory chain and motor deficit as in parkinsonian disorders. Binding of an inhibitor to complex I has been defined as the molecular initiating event (MIE) that triggers mitochondrial dysfunction, impaired proteostasis, which then cause degeneration of dopaminergic (DA) neurons of the nigro-striatal pathway. Neuroinflammation is triggered early in the neurodegenerative process and exacerbates it significantly. These causatively linked cellular key events result in motor deficit symptoms, typical for parkinsonian disorders, including Parkinson's disease (PD), described in this AOP as an Adverse Outcome (AO). Since the release of dopamine in the striatum by DA neurons of the Substantia Nigra pars compacta (SNpc) is essential for motor control, the key events refer to these two brain structures. The weight-of-evidence supporting the relationship between the described key events is based mainly on effects observed after an exposure to the chemicals rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), i.e. two well-known inhibitors of complex I. Data from experiments with these two chemicals reveal a significant concordance in the dose-response relationships between the MIE and AO and within key events (KEs). Also essentiality of the described KEs for this AOP is strong since there is evidence from knock out animal models, engineered cells or replacement therapies that blocking, preventing or attenuating an upstream KE is mitigating the AO. Similarly, there is proved experimental support for the key event relationships (KERs) as multiple studies performed with modulating factors that attenuate (particularly with antioxidants) or augment (e.g. overexpression of viral-mutated α-synuclein) a KE up show that such interference leads to an increase of KE down or the AO. Information from in vitro and in vivo experiments is complemented by human studies in brain tissues from individuals with sporadic Parkinson's disease (Keeney et al., 2006) to support the pathways of toxicity proposed in this AOP.



Summary of the AOP


Stressors


Name Evidence
MPP+ Strong
Rotenone Strong

MPP+

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a meperidine analog intended for illecit use as a ricreative drug resulted in life-long parkinsonism in a number of abusers. MPTP is able to cross the blood-brain-barrier and is actually a pro-toxin which is converted by monoamine oxidase B in astrocytes to the neurotoxic active species 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP+). MPP+ has high affinityfor the DA transporter (DAT), thus preferentially enters dopaminergic neurons where is accumulating in mitochondria to directely inhibit CI and indirectly CII in the respiratory transport chain. (Aguilar et al.2015; pasquali et al.2014).

Rotenone

The insecticide rotenone is a potent Inhibitor of complex I (CI) in the respiratory transport chain of mitochondroa with a relative selectivity for dopaminergig neurons in low dosage.Additional non-specific treatment related effects include: vascular damage with secondary ischemic neurodegeneration, tissue damage in heart and testicles and interstitial haemorrhages in lungs and kidneys. Mitochondrial dysfunction is considered to be the initial step in rotenone toxicity; though, multiple pathways are involved in the process associated with rotenone-induced neurotoxicity. however, the impact of rotenone on glial cells also impact on neuronal outcomes. (Aguilar et al.2015). Rotenone provided the first proof-of-concept that a systemic defect in mitochondrial function could lead to selective nigrostriatal neurodegeneration.  The rotenone animal model accurately recapitulates many other features of Parkinson's disease , including: accumulation and aggregation of endogenous, wildtype alpha-synuclein; α-synuclein- and polyubiquitin-positive Lewy bodies and Lewy neurites; apomorphine responsive behavioral deficits; early and sustained activation of microglia; oxidative modification and translocation of DJ-1 into mitochondria in vivo; impairment of the nigral ubiquitin-proteasome system; accumulation of iron in the substantia nigra through a mechanism involving transferrin and transferrin receptor 2; α-synuclein pathology in enteric neurons and functional deficits in GI function, including gastroparesis (Greenamyre et al. 2010). 

Molecular Initiating Event

Title Short name
Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I) Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)

888: Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)

Short Name: Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I)

Key Event Component

Process Object Action
mitochondrial electron transport, NADH to ubiquinone NADH-ubiquinone oxidoreductase chain 1 decreased

AOPs Including This Key Event

Stressors

Name
1',2'-dihydrorotenone

Biological Organization

Level of Biological Organization
Molecular

Cell term

Cell term
eukaryotic cell

Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

Numerous hydrophobic, amphipathic compounds are known to inhibit the proton pumping NADH:ubiquinone oxidoreductase, also known as the ubiquinone reductase reaction of respiratory chain complex I (Fendel et al., 2008). However, the most studied examples of chemicals that inhibit CI are: rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Desplats et al., 2012; Lin et al., 2012; Sava et al., 2007). Both, rotenone (pesticide) and MPP+ (the active metabolite of MPTP) are well known to reproduce the anatomical, neurochemical, behavioural and neuropathological features of PD-like syndrome (Betarbet et al., 200; Greenamyre et al., 2001). Indeed, an overwhelming evidence has accumulated in the existing literature suggesting such a link and therefore these two inhibitors of CI will be discussed in the context of all KE identified in this AOP.

1. Rotenone affinity to complex I binding sites.

Rotenone (a flavonoid, extracted from the several plants e.g. Derris scandens)is one of the most powerful, an irreversible inhibitor of CI, binding with high affinity to CI and is typically used to define the specific activity of this complex. Rotenone is extremely lipophilic, it crosses biological membrane easily and it gets into brain very rapidly. Rotenone inhibits 20 kDa subunit of complex I (PSST) labeling without effect on 36 kDa subunit of complex I (ND1) (Schuler and Casida, 2001). The interaction of rotenone with active ('pulsed') and thermally de-activated ('resting') membrane-bound Complex I as revealed by inhibition of NADH-ubiquinone- and ubiquinol-NAD+ reductase activities was studied. Ki = 1 x 10(-9) M, k(on) = 5 x 10(7) M-1 min-1 and k(off) = 0.02 min-1 (inhibitory effect of rotenone on NADH oxidation) and Ki = 2 x 10(-8) M (inhibition of reverse electron transfer) were determined for pulsed enzyme. The equilibrium between de-activated and active enzyme is reached (K approximately 100) after the slow strongly temperature-dependent de-activation process has completed. Rotenone partially prevents and reverses the enzyme de-activation. About two order of magnitude difference in affinity of rotenone to the active and de-activated forms of the enzyme was demonstrated (Grivennikova et al., 1997). Dose-dependent relative affinities of rotenone to the inhibitory site of CI is shown in Fig. 3B (for more detail Grivennikova et al., 1997).

Most of the studies suggest that hydrophobic inhibitors like rotenone or Piericidin A most likely disrupt the electron transfer between the terminal Fe-S cluster N2 and ubiquinone (Fig. 3A).

 

MIE Fig. 3A.jpg

 

Fig. 3A. Rotenone structure and a schematic representation of its binding site (and other Rotenone-like compounds) to CI. IMS: inter-membrane space (based on Lummen, 1998)

 

MIE Fig. 3B.jpg

 

Fig. 3B. Fig. 2. Relative affinities of rotenone to the inhibitory site(s) of Complex I. Panel (A): activated submitochondrial particles (SMP) (2.8 mg/ml, approx. 0.4 microM Complex I) were incubated in the standard reaction mixture for 20 min at 25oC and residual initial rate of NADH oxidation was measured. 100% correspond to the specific activity of 1.0 micromol/min per mg of protein. Panel (B): curve 1 (o), SMP (48 microg/ml, approx. 8 nM Complex I) were activated in the assay cuvette and pre-incubated with rotenone in the presence of gramicidin and 10 mM malonate for 20 min at 25oC and the residual NADH oxidase activity was then measured; black circle: the same as (o), except that pre-incubation with rotenone was made in the presence of 10 mM succinate (no gramicidin and malonate), 10 mM malonate and gramicidin were added simultaneously with 100 microM NADH to measure the residual activity. Curve 2, presents the reverse electron transfer activity and curve 3, de-activated SMP were preincubated with rotenone as described for curve 1(o) (for further details see Grivennikova et al., 1997). Panel (C): The same as Panel B, curve 3, except for enzyme concentration was 0.5 mg/ml and rotenone concentration range which was increased to show interaction of the inhibitor with de-activated enzyme. The activity was measured after 200-fold dilution into the assay mixture. All the continues lines corresponds to the theoretical titration curves for the reversible single site inhibition with Ki values of 1 nM, 20 nM and 80 nM for the curves 1, 2 and 3, respectively (for further details see Grivennikova et al., 1997).


2. MPTP affinity to complex I binding sites. MPTP is not directly binding to CI and it is therefore non-toxic to DA neurons. MPTP exerts its toxicity after it is metabolized by mono-amino-oxidase, type B (MAO B), in astrocytes to 1-methyl-4-phenylpyridinium (MPP+). This metabolite binds to CI, and is toxic. MPP+ is a good substrate for dopamine transporters (DAT), expressed selectively by DA neurons (Greenamyre et al (2001). Due to both a positive charge and an amphoteric character, MPP+ specifically accumulates in mitochondria, where despite a lower affinity to the binding site of complex I than rotenone, it reaches high enough intra-mitochondrial concentrations to inhibit CI activity (Ramsay et al., 1991). The binding affinity of MPP+ is low (mM range), and it can be totally reversed by washing out.  Competitive binding experiments with rotenone and MPP+ suggest that the two compounds bind to the same site of the CI (Ramasay et al., 1991). Schuler and Casida (2001) reported that MPP+ inhibits PSST and elevates ND1 labelling subunits of the mitochondrial complex I.

3. General characteristics of other complex I inhibitors There is a variety of CI inhibitors, both naturally occurring besides rotenone such as Piericidin A (from Streptomyces mobaraensis), acetogenins (from various Annonaceae species) as well as their derivatives, and synthetically manufactured compounds like pyridaben and various piperazin derivatives (Ichimaru et al. 2008). They have been used to probe the catalytic activity of complex I especially in order to clarify its ubiquinone binding site and indeed, most of these compounds inhibit the electron transfer step from the Fe-S clusters to ubiquinone (Friedrich et al. 1994). Therefore, classification of CI inhibitors is based on their types of action. Type A inhibitors, like piericidin A, 2-decyl-4-quinazolinyl amine (DQA), annonin VI and rolliniastatin-1 and -2, are considered to be antagonists of the ubiquinone substrate. For piericidin A, it has been shown that it inhibits NADH:Q2 activity in a partially competitive manner. Contrary to type A, type B inhibitors, like the commonly used rotenone, have hydrogen-bonding acceptors only in the cyclic head of the molecule and are non-competitive towards UQ (ubiquinone), but are believed to displace the semiquinone intermediate during the catalysis (Fig. 2). Finally, inhibitors classified as type C, like stigmatellin and capsaicin, form a third group of hydrophobic CI inhibitors that are believed to act as antagonists of reduced ubiquinone (Degli Esposti 1998, Friedrich et al. 1994, Haefeli 2012) (Fig. 2). Competition studies with representatives of all three different types of inhibitors revealed that type A and B and type B and C, but not type A and C, compete with each other for binding. This led to a suggestion that all CI inhibitors acting at the ubiquinone binding pocket share a common binding domain with partially overlapping sites (Okun et al. 1999).

Some inhibitors bind to the outside of the ubiquinone reduction site and do not fit the preceding classification. Examples of such compounds are ADP-ribose, which competes for substrate binding at the NADH site (Zharova and Vinogradov, 1997), and diphenyleneiodonium (DPI) that covalently binds to reduced flavin mononucleotide (FMN) in the hydrophilic part of the enzyme blocking the electron transfer to the Fe-S clusters (Majander et al., 1994). There are also new, commercially available insecticides/acaricides with potential to inhibit mitochondrial respiration such as benzimidazole, bullatacin, 6-chlorobenzothiadiazole, cyhalothrin, Fenazaquin Fenpyroximate, Hoe 110779, Pyridaben, Pyrimidifen, Sandoz 547A, Tebufenpyrad and Thiangazole (Greenamyre et al., 2001). It is clear that they are capable of inhibiting the mammalian CI of mitochondrial respiratory chain, by binding to and blocking ubiquinone-dependent NADH oxidation with high efficacy (Lummen, 1998).



Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Strong NCBI

CI has a highly conserved subunit composition across species, from lower organisms to mammals (Cardol, 2011). Fourteen subunits are considered to be the minimal structural requirement for physiological functionality of the enzyme. These units are well conserved among bacterial (E. coli), human (H. sapiens), and Bovine (B. taurus) (Vogel et al., 2007b; Ferguson, 1994). However, the complete structure of CI is reported to contain between 40 to 46 subunits and the number of subunits differs, depending on the species (Gabaldon 2005; Choi et al., 2008). In vertebrates CI consists of at least 46 subunits (Hassinen, 2007), particularly, in humans 45 subunits have been described (Vogel et al, 2007b). Moreover, enzymatic and immunochemical evidence indicate a high degree of similarity between mammalian and fungal counterparts (Lummen, 1998). Mammalian CI structure and activity have been characterized in detail (Vogel et al., 2007a; Vogel et al., 2007b), referring to different human organs including the brain. There is also a substantial amount of studies describing CI in human muscles, brain, liver, as well as bovine heart (Janssen et al., 2006; Mimaki et al. 2012) (Okun et al., 1999).


How this Key Event Works

Electron transport through the mitochondrial respiratory chain (oxidative phosphorylation) is mediated by five multimeric complexes (I–V) that are embedded in the mitochondrial inner membrane (Fig. 1). NADH-ubiquinone oxidoreductase is the Complex I (CI) of electron transport chain (ETC). It is a large assembly of proteins that spans the inner mitochondrial membrane. In mammals, it is composed of about 45-47 protein subunits (human 45) of which 7 are encoded by the mitochondrial genome (ND1, ND2, ND3, ND4, ND4L, ND5, and ND6) and the remainder by the nuclear genome (Greenamyre, 2001). CI oxidizes NADH elevating the NAD+/NADH ratio by transferring electrons via a flavin mononucleotide (FMN) cofactor and several iron-sulfur centers to ubiquinone (Friedrich et al., 1994) (Fig. 1). Binding of an inhibitor to CI inhibits the NADH–ubiquinone oxido-reductase activity, i.e. blocks the electron transfer. Recent studies suggest that a wide variety of CI inhibitors share a common binding domain at or close to the ubiquinone reduction site (Ino et al., 2003). Furthermore, the structural factors required for inhibitory actions have been characterized on the basis of structure-activity relationships (Miyoshi, 1998, Hideto, 1998). Based on molecular docking simulations, in silico models mimicking the binding of chemicals to the pocket of NADH ubiquinone oxidoreductase have been created according to the crystal structure of mitochondrial CI. To investigate the ability of chemicals to bind to the active pocket, around 100 individual docking simulations have been performed. These confirmed the possible site of interaction between the chemical and the pocket of CI. In particular, Miao YJ and coworkers recently investigated the IC50 values of 24 chemicals (annonaceous acetogenins) for inhibition of mitochondrial CI (Miao et al., 2014).

Based on their binding sites, CI inhibitors are classified as follows (Degli Esposti, 1998) (Fig. 2): (i) type A inhibitors are antagonists of fully oxidized ubiquinone binding; (ii) type B inhibitors displace the partially reduced ubisemiquinone intermediate; (iii) type C inhibitors are antagonists of the fully reduced ubiquinol product. The affinity of the different types of CI inhibitors to their diverse CI binding sites is described in the paragraph Evidence for Chemical Initiation of this Molecular Initiating Event (see below) in the context of a specific type of inhibitor.


AOP-003-Figure1-smaller.JPG

Fig. 1. The electron transport chain in the mitochondrion. CI (NADH-coenzyme Q reductase or NADH dehydrogenase) accepts electrons from NADH and serves as the link between glycolysis, the citric acid cycle, fatty acid oxidation and the electron transport chain. Complex II also known as succinate-coenzyme Q reductase or succinate dehydrogenase, includes succinate dehydrogenase and serves as a direct link between the citric acid cycle and the electron transport chain. The coenzyme Q reductase or Complex III transfers the electrons from CoQH2 to reduce cytochrome c which is the substrate for Complex IV (cytochrome c reductase). Complex IV transfers the electrons from cytochrome c to reduce molecular oxygen into water. Finally, this gradient is used by the ATP synthase complex (Complex V) to make ATP via oxidative phosphorylation. mtDNA: mitochondrial DNA; nDNA: nuclear DNA.

MIE Fig. 2.jpg

Fig. 2. Schematic representation of CI and proposed inhibition binding sites by inhibitors of class A, B and C. Nicotinamide adenine dinucleotide (NADH, reduced and NAD, oxidized), flavin mononucleotide (FMN) and Ubiquinone (Q) (taken from Haefeli, 2012).


How it is Measured or Detected

Two different types of approaches have been used. The first is to measure binding as such, and the corresponding assays are described below; the second is to infer binding indirectly from assays that quantify e.g. CI activity and to assume that the activity can only be altered upon binding. The second type of approach is dealt with in the chapter entitled KE1: Inhibition of NADH ubiquinone oxidoreductase (complex I). However, it has to be noted here that indirect assays can lead to wrong conclusions. For instance, some compounds may trigger oxidative stress without actually binding to CI. Such compounds, by triggering the generation of reactive oxygen species (ROS), may damage CI protein components, thus causing a reduction of CI activity. Measurement of binding by quantitative autoradiography To assess binding of an inhibitor at the rotenone binding site of CI in tissues (e.g. in the substantia nigra or in the striatum), the standard approach is to quantify the displacement of a radioactively labelled ligand of this binding site by the toxicant under evaluation. Most commonly, binding of [3H]-labeled dihydrorotenone (DHR) is measured and compared in control tissue and treated tissue. Binding of this rotenone-derivative is detected by autoradiography. Unselective binding is determined by measurement of [3H]-DHR binding in the presence of an excess of unlabeled rotenone. Since a rotenone-derivative is used for the assay, only CI inhibitors that bind to the rotenone-binding site in CI are detected. This was observed for e.g., meperdine, amobarbital, or MPP+. This method allows a spatial resolution of CI expression and the mapping of the binding of a competitive inhibitor on CI.

The method can be used for (a) in vitro measurements and for (b) ex vivo measurements:

a) In vitro measurements. Tissues are embedded in a matrix for cutting by a cryostat. The tissue slices are then mounted onto slides. For the binding experiment, they are incubated with the test compound in the presence of labeled [3H]-DHR. Then the tissue slices are washed and prepared for autoradiographic detection (Greenamyre et al. 1992; Higgins and Greenamyre, 1996). b) Ex vivo measurements. As rotenone can pass the blood brain barrier, the in vitro method was further extended for in vivo labeling of CI in the brains of living animals, and detection of binding after preparation of the tissue from such animals. Animals are exposed to test compounds and [3H]-DHR is applied intraventricularly for 2-6 h before the brain is dissected and arranged for the preparation of tissue slices (Talpade et al. 2000). In untreated animals, this method allows a precise spatial resolution of the expression pattern of CI. In animals with impaired CI activity, either as a result of CI deficiencies, or upon treatment with CI inhibitors, the assay allows an assessment of the degree of CI inhibition.

Complex I Enzyme Activity (Colorimetric)

The analysis of mitochondrial OXPHOS CI enzyme activity can be performed using human, rat, mouse and bovine cell and tissue extracts (abcam: http://www.abcam.com/complex-i-enzyme-activity-microplate-assay-kit-colorimetric-ab109721). Capture antibodies specific for CI subunits are pre-coated in the microplate wells. Samples are added to the microplate wells which have been pre-coated with a specific capture antibody. After the target has been immobilized in the well, CI activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be evaluated.


References

 

Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. (2000). Chronic systemic pesticide exposure reproduces features of Parkinson's disease. Nat Neurosci 3:1301-1306.

Cardol, P., (2011) Mitochondrial NADH:ubiquinone oxidoreductase (complex I) in eukaryotes: A highly conserved subunit composition highlighted by mining of protein databases Biochimica et Biophysica Acta 1807, 1390–1397.

Choi WS., Kruse S.E., Palmiter R, Xia Z., (2008) Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP, or paraquat. PNAS, 105, 39, 15136-15141.

Degli Esposti (1998) Inhibitors of NADH-ubiquinone reductase: an overview Biochimica et Biophysica Acta 1364-222-235.

Desplats P, Patel P, Kosberg K, Mante M, Patrick C, Rockenstein E, Fujita M, Hashimoto M, Masliah E. (2012). Combined exposure to Maneb and Paraquat alters transcriptional regulation of neurogenesis-related genes in mice models of Parkinson’s disease. Mol Neurodegener 7:49.

Fendel UTocilescu MAKerscher SBrandt U. (2008) Exploring the inhibitor binding pocket of respiratory complex I.Biochim Biophys Acta. 2008, 1777(7-8):660-5.

Ferguson SJ. Similarities between mitochondrial and bacterial electron transport with particular reference to the action of inhibitors. Biochem Soc Trans. 1994 Feb;22(1):181-3.

Friedrich T, van Heek P, Leif H, Ohnishi T, Forche E, Kunze B, Jansen R, TrowitzschKienast W, Hofle G & Reichenbach H (1994) Two binding sites of inhibitors in NADH: ubiquinone oxidoreductase (complex I). Relationship of one site with the ubiquinone-binding site of bacterial glucose:ubiquinone oxidoreductase. Eur J Biochem 219(1–2): 691–698.

Gabaldon, T., Rainey, D., Huynen, M.A. (2005) Tracing the evolution of a large protein complex in the eukaryotes, NADH:ubiquinone oxidoreductase (Complex I), J. Mol. Biol. 348; 857–870.

Greenamyre, J T., Sherer, T.B., Betarbet, R., and Panov A.V. (2001) Critical Review Complex I and Parkinson’s Disease Life, 52: 135–141.

Greenamyre JT, Higgins DS, Eller RV (1992) Quantitative autoradiography of dihydrorotenone binding to complex I of the electron transport chain. J Neurochem. 59(2):746-9.

Grivennikova, V.G., Maklashina, E.O., E.V. Gavrikova, A.D. Vinogradov (1997) Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition Biochim. Biophys. Acta, 1319 (1997), pp. 223–232.

Haefeli, RH (2012) Molecular Effects of Idebenone. Doctoral thesis http://edoc.unibas.ch/19016/1/Molecular_Effects_of_Idebenone_Roman_Haefeli.pdf.

Hassinen I (2007) Regulation of Mitochondrial Respiration in Heart Muscle. In Mitochondria – The Dynamic Organelle Edited by Schaffer & Suleiman. Springer ISBN-13: 978-0-387-69944-8.

Hideto M. Structure–activity relationships of some complex I inhibitors. Biochimica et Biophysica Acta 1364 _1998. 236–244.

Higgins DS Jr1, Greenamyre JT. (1996). [3H]dihydrorotenone binding to NADH: ubiquinone reductase (complex I) of the electron transport chain: an autoradiographic study. J Neurosci. 1996 Jun 15;16(12):3807-16.

Ichimaru, N., Murai, M., Kakutani, N., Kako, J., Ishihara, A., Nakagawa, Y., … Miyoshi, H. (2008). Synthesis and Characterization of New Piperazine-Type Inhibitors for Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I). Biochemistry, 47(40), 10816–10826.

Ino T, Takaaki N, Hideto M. Characterization of inhibitor binding sites of mitochondrial complex I using fluorescent inhibitor. Biochimica et Biophysica Acta 1605 (2003) 15– 20.

Janssen RJ, Nijtmans LG, van den Heuvel LP, Smeitink JA. Mitochondrial complex I: structure, function and pathology. J Inherit Metab Dis. 2006 Aug;29(4):499-515.

Keeney PM, Xie J,Capaldi RA,Bennett JP Jr. (2006) Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci. 10;26(19):5256-64.

Lin CJ, Lee CC, Shih YL, Lin CH, Wang SH, Chen TH, Shih CM. (2012). Inhibition of Mitochondria- and Endoplasmic Reticulum Stress-Mediated Autophagy Augments Temozolomide-Induced Apoptosis in Glioma Cells. PLoS ONE 7:e38706.

Lümmen, P., (1998) Complex I inhibitors as insecticides and acaricides1, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1364, Issue 2, Pages 287-296.

Majander A, Finel M, Wikstrom M. (1994) Diphenyleneiodonium inhibits reduction of iron–sulfur clusters in the mitochondrial NADH–ubiquinone oxidoreductase (complex I) J Biol Chem. 269:21037–21042.

Miao YJ, Xu XF, Xu F, Chen Y, Chen JW, Li X. (2014) The structure-activity relationships of mono-THF ACGs on mitochondrial complex I with a molecular modelling study. Nat Prod Res.28(21):1929-35.

Mimaki M, Wang X, McKenzie M, Thorburn DR, Ryan MT. Understanding mitochondrial complex I assembly in health and disease. Biochim Biophys Acta. 2012 Jun;1817(6):851-62. doi: 10.1016/j.bbabio.2011.08.010.

Miyoshi H. Structure-activity relationships of some complex I inhibitors. Biochim Biophys Acta. 1998, 6:236-244.

Okun, J.G, Lümmen, P and Brandt U., (1999) Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) J. Biol. Chem. 274: 2625-2630. doi:10.1074/jbc.274.5.2625.

Ramsay R., Krueger MJ., Youngster SK., Gluck MR., Casida J.E. and Singer T.P. Interaction of 1-Methyl-4-Phenylpyridinium Ion (MPP+) and Its Analogs with the Rotenone/Piericidin Binding Site of NADH Dehydrogenase. Journal of Neurochemistry, 1991, 56: 4, 1184–1190.

Sava V, Velasquez A, Song S, Sanchez-Ramos J. (2007). Dieldrin elicits a widespread DNA repair and antioxidative response in mouse brain. J Biochem Mol Toxicol 21:125-135.

Schuler, F. and Casida, JE. (2001) Functional coupling of PSST and ND1 subunits in NADH:ubiquinone oxidoreductase established by photoaffinity labeling, Biochimica et Biophysica Acta (BBA) - Bioenergetics, Volume 1506, Issue 1, 2 July 2001, Pages 79-87, ISSN 0005-2728, https://doi.org/10.1016/S0005-2728(01)00183-9.

Talpade DJ, Greene JG, Higgins DS Jr, Greenamyre JT (2000) In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone. J Neurochem. 75(6):2611-21.

Vogel R.O., van den Brand M.A., Rodenburg R.J., van den Heuvel L.P., Tsuneoka M., Smeitink J.A., Nijtmans L.G. (2007a). Investigation of the complex I assembly chaperones B17.2L and NDUFAF1 in a cohort of CI deficient patients. Mol. Genet. Metab. 91:176–182.

Vogel, R.O. Smeitink, J.A. Nijtmans L.G. (2007b) Human mitochondrial complex I assembly: a dynamic and versatile process Biochim. Biophys. Acta, 1767-. 1215–1227.

Zharova, TV, and Vinogradov, A.(1997) A competitive inhibition of the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) by ADP-ribose. Biochimica et Biophysica Acta, 1320:256-64.


Key Events

Title Short name
Inhibition, NADH-ubiquinone oxidoreductase (complex I) Inhibition, NADH-ubiquinone oxidoreductase (complex I)
N/A, Mitochondrial dysfunction 1 N/A, Mitochondrial dysfunction 1
Impaired, Proteostasis Impaired, Proteostasis
N/A, Neuroinflammation N/A, Neuroinflammation
Degeneration of dopaminergic neurons of the nigrostriatal pathway Degeneration of dopaminergic neurons of the nigrostriatal pathway

887: Inhibition, NADH-ubiquinone oxidoreductase (complex I)

Short Name: Inhibition, NADH-ubiquinone oxidoreductase (complex I)

Key Event Component

Process Object Action
NADH dehydrogenase (ubiquinone) activity NADH-ubiquinone oxidoreductase chain 1 decreased

Stressors

Name
1',2'-dihydrorotenone

Biological Organization

Level of Biological Organization
Cellular

Cell term

Cell term
eukaryotic cell

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
Rattus sp. Rattus sp. Strong NCBI
mouse Mus musculus Strong NCBI

The CI is well-conserved across species from lower organisms to mammals. The central subunits of CI harboring the bioenergetic core functions are conserved from bacteria to humans. CI from bacteria and from mitochondria of Yarrowia lipolytica, a yeast genetic model for the study of eukaryotic CI (Kerscher et al., 2002) was analyzed by x-ray crystallography (Zickermann et al., 2015, Hofhaus et al., 1991; Baradaran et al., 2013). The CI of the mitochondria of eukaryotes and in the plasma membranes of purple photosynthetic bacteria are closely related to respiratory bacteria and the close homology of sequences, function, and prosthetic groups shows a common ancestry (Friedrich et al., 1995).


How this Key Event Works

Under physiological conditions complex I (CI) couples the oxidation of NADH to NAD+ by reducing flavin mononucleotide (FMN) to FMNH2. FMNH2 is then oxidized through a semiquinone intermediate. Each electron moves from the FMNH2 to Fe-S clusters, and from the Fe-S clusters to ubiquinone (Q). Transfer of the first electron results in the formation of the free-radical (semiquinone) form of Q, and transfer of the second electron reduces the semiquinone form to the ubiquinol form (CoQH2). Altogether, four protons are translocated from the mitochondrial matrix to the inter-membrane space for each molecule of NADH oxidized at CI. This leads to the establishment of the electrochemical potential difference (proton-motive force) that may be used to produce ATP (Garrett and Grisham, 2010). Binding of an inhibitor attenuates or completely blocks the activity of CI, i.e. the oxidation of NADH is impaired and protons are not moved. This causes two major consequences: first, electrons are channelled toward oxygen instead Q. This impairs normal oxygen reduction into water at complex IV and leads to the formation of the ROS superoxide at other sites of the respiratory chain. Superoxide may cause damage of proteins, lipid and DNA of the cell, or damage components of the mitochondria after transformation into e.g. hydrogen peroxide. These processes result in mitochondrial dysfunction (Voet and Voet., 2008). The second consequence is the increase of the NADH/NAD+ ratio in mitochondria. This affects the function of key dehydrogenase enzymes in the citric acid cycle and can lead to its block, resulting in an inhibition of mitochondrial ATP production and mitochondrial respiration. Prolonged treatment with an inhibitor results in a severe, progressive and irreversible inhibition of complex I, most likely by indirect mechanisms involving oxidative damage (Cleeter et al., 1992). The functional consequences of CI inhibition have been titrated in a time- and dose-dependent manner (Barrientos and Moraes, 1999), with mitochondrial dysfunction measured by a range of different assays (Barrientos and Moraes, 1999; Greenamyre et al., 2001). These included quantification of ROS derived from mitochondria, and of cellular respiration (see KE2: Mitochondrial dysfunction).


How it is Measured or Detected

As CI has an enzymatic function as such, but also contributes to the overall function of oxidative phosphorylation, there are two fundamental approaches to assess CI inhibition. The first approach measures the enzymatic activity of the complex itself; the second one assesses the overall activity of oxidative phosphorylation of entire mitochondria, and indirectly infers from this a potential dysfunction of CI.

I. Direct detection of complex I activity. This type of assay is always performed in homogenates of cells or tissues, and requires at least a partial purification of mitochondria or respiratory chain components. In order to focus on CI activity, the activities of Complexes III (e.g. antimycin A) and complex IV (e.g. cyanide) need to be blocked by pharmacological inhibitors in these setups.

1. Forward Electron Transfer. Submitochondrial particles or intact isolated mitochondria are incubated with NADH as electron donor and with an electron acceptor to measure the flow of electrons from NADH, through CI to the acceptor. As readout, either the consumption of NADH, or the reduction of the electron acceptor is followed photometrically or fluorometrically (Lenaz et al. 2004; Spinazzi et al. 2012; Long et al. 2009; Kirby et al. 2007). The physiological electron acceptor of CI is Coenzyme Q10 (CoQ10). Due to its hydrophobicity, it is not suitable for use in an experimental in vitro setup. Short-chain analogs of CoQ10, such as CoQ1 or decylubiquinone (DB) with a 10 carbon-atom linear saturated side chain are hence applied as alternatives. With these non-physiological electron acceptors, it is important to consider that the activity of CI can easily be underestimated. As water-soluble electron acceptors, either ferricyanide or 2,6-dichlorophenolidophenol (DCIP) are used. However the reduction of such compounds is not strictly coupled to the transduction of energy. To identify the portion of rotenone-inhibitable CI activity, all samples investigated are assayed in parallel following treatment with rotenone. In contrast to the autoradiography assays, direct CI activity detection allows the identification also of CI inhibitors that bind to sites of CI different from the rotenone binding site.

2. Reverse Electron Transfer. An alternative setup for the direct measurement of CI activity with minimal interference by the activities of complex III and complex IV make use of the observation of a general reversibility of oxidative phosphorylation and electron flow across the mitochondrial respiratory chain (Ernster et al. 1967). With this method, electrons enter the respiratory chain via complex II. Based on the reverse flux, this method allows the complete circumvention of complexes III and IV. As electron donor, succinate is applied, together with NAD+ as electron acceptor. Formation of NADH from NAD+ can be determined photometrically. The succinate-linked NAD+ reduction can be performed either with intact isolated mitochondria or with submitochondrial particles. For the direct assessment of CI activity, submitochondrial particles are used. For assays with intact mitochondria, the succinate-linked reduction of NAD+ is performed in the presence of ATP as energy source. Potassium cyanide (KCN) is added for inhibition of forward electron transport towards complex IV.

3. Complex I activity dipstick assay. To assess CI activity and its inhibition in cell or tissue homogenates without interference by other components of the respiratory chain, CI-selective antibodies attached to a matrix (e.g. multiwell plates) are used (Willis et al., 2009). Homogenized tissue can directly be added for capturing of CI, the unbound supernatant is washed away and leaves a complex of the antibody and mitochondrial CI. For activity determination, NADH as electron donor and nitroblue tetrazolium (NBT) as acceptor are added. Reduced NBT forms a colored precipitate, its signal intensity is proportional to the amount of CI bound to the antibody. CI inhibitors can directly be added for an assessment of their inhibitory potential. This method, when applied in e.g. 96-well or 384-well plates, allows screening of large sets of potential CI inhibitors without any interference by other elements of the mitochondrial respiratory chain.

II. Indirect measurements of complex I activity. Such assays mostly require / allow the use of live cells.

1. Oxygen consumption. Electrons, fed into the mitochondrial respiratory chain either by CI or complex II, ultimately reduce molecular oxygen to water at complex IV. In a closed system, this consumption of oxygen leads to a drop of the overall O2 concentration, and this can serve as parameter for mitochondrial respiratory activity. Measurements are traditionally done with a Clark electrode, or with more sophisticated optical methods. At the cathode of a Clark electrode, oxygen is electrolytically reduced, which initiates a current in the electrode, causing a potential difference that is ultimately recorded. Clark electrodes however have the disadvantage that oxygen is consumed. Furthermore, interferences with nitrogen oxides, ozone, or chlorine are observed (Stetter et al., 2008). To circumvent these limitations, optical sensors have been developed that have the advantage that no oxygen is consumed, combined with a high accuracy and reversibility. Optical oxygen sensors work according to the principle of dynamic fluorescence quenching. The response of the respective fluorescence dye is proportional to the amount of oxygen in the sample investigated (Wang and Wolfbeis, 2014). In a model of isolated mitochondria in the absence of complex II substrates, oxygen consumption can serve as surrogate readout for the assessment of the degree of CI inhibition. It is however essential to realize that also complex III and complex IV activities are involved and their inhibition also results in a decline in O2 consumption. In addition to that, CI inhibitors can lead to a one-electron reduction of molecular oxygen at the site of CI to yield superoxide. The amount of superoxide formed hence contributes to the consumption of oxygen, but this must not be interpreted as oxygen consumption as a result of controlled and coupled electron flux through the complexes of the mitochondrial respiratory chain. A modern convenient method to measure oxygen consumption is provided by the Seahorse technology of extracellular flux (XF) analysis, in which cells are kept in a very small volume, so that changes of oxygen levels can be detected very sensitively by an oxygen sensor. To allow manipulation of the mitochondria in cells, the cell membrane can be permeabilized with saponin (SAP), digitonin (DIG) or recombinant perfringolysin O (rPFO) (XF-plasma membrane permeabilizer (PMP) reagent), to allow addition of specific substrates to measure activity of different respiratory chain complexes, including CI. (Salabei et al., 2014).

2. Intracellular ATP levels. Intracellular ATP levels originate both from mitochondria and from glycolysis. If glycolytic ATP production is impaired or inhibited, the cellular production of ATP is a measure of mitochondrial function. If it is assumed that the ATP consumption remains constant, then the steady state ATP levels can serve as indirect readout for mitochondrial activity, and the latter depends on the functioning of CI. Inhibitors of CI reduce cellular ATP levels, but it has to be remembered that intracellular ATP levels are also affected by inhibitors of other parts of the respiratory chain, of the citric acid cycle or of the transport of energy substrates. For a proper interpretation of assay results, it has to be ascertained in each particular test system, that ATP production from other sources is excluded and that the cellular ATP consumption remains constant. ATP levels can be easily measured from lysates of in vitro cell cultures or from tissues by a luminometric luciferase/luciferin assay. The amount of light emitted is proportional to the amount of ATP in the sample (Nguyen et al. 1988, Leist et al., 1997).

3. Other approaches As mitochondrial activity is coupled to many cellular functions, there is a multitude of other indirect assays that are sensitive to inhibitors of CI. Some of these tests may indeed be very sensitive, while they have a low specificity. Thus, their application requires usually a good control of the experimental system and care with the interpretation of the data. One exemplary approach is the measurement of NADH/NAD+ ratios in mitochondria by imaging methods. This provides resolution on the level of individual mitochondria within a living cell (van Vliet et al., 2014).


References


Baradaran R., John M. Berrisford, Gurdeep S. Minhas , Leonid A. Sazanov. Crystal structure of the entire respiratory complex I. Nature , 2013,| 494,443–448.

Barrientos A., and Moraes C.T. (1999) Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. Vol. 274, No. 23, pp. 16188–16197.

Cleeter MW, Cooper JM, Schapira AH. Irreversible inhibition of mitochondrial complex I by 1-methyl-4-phenylpyridinium: evidence for free radical involvement. J Neurochem. 1992 Feb;58(2):786-9.

Degli Esposti (1998) Inhibitors of NADH-ubiquinone reductase: an overview Biochimica et Biophysica Acta 1364-222-235.

Ernster L, Lee C (1967) Energy-linked reduction of NAD+ by succinate. Methods Enzym. 10:729-738.

Friedrich, T., Steinmüller, K. & Weiss, H. (1995) The proton-pumping respiratory complex I of bacteria and mitochondria and is homologue of chloroplasts. FEBS Lett. (Minireview), 367, 107-111.

Garrett and Grisham, Biochemistry, Brooks/Cole, 2010, pp 598-611.

Greenamyre, J T., Sherer, T.B., Betarbet, R., and Panov A.V. (2001) Critical Review Complex I and Parkinson’s Disease Life, 52: 135–141.

Hofhaus, G., Weiss, H. and Leonard, K. (1991): Electron microscopic analysis of the peripheral and the membrane parts of mitochondrial NADH dehydrogenase (Complex I). J. Mol. Biol. 221, 1027-1043.

Kerscher, S. Dröse, K. Zwicker, V. Zickermann, U. Brandt Yarrowia lipolytica, a yeast genetic system to study mitochondrial complex I. Biochim. Biophys. Acta 1555, 83–91 (2002).

Kirby DM, Thorburn DR, Turnbull DM, Taylor RW (2007) Biochemical assays of respiratory chain complex activity. Methods Cell Biol. 80:93-119.

Leist M, Single B, Castoldi AF, Kühnle S, Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. J Exp Med. 185:1481-6.

Leist M. Current approaches and future role of high content imaging in safety sciences and drug discovery. ALTEX. 2014;31(4):479-93.

Lenaz G, Fato R, Baracca A, Genova ML (2004) Mitochondrial quinone reductases: complex I. Methods Enzymol. 382:3-20.

Long J, Ma J, Luo C, Mo X, Sun L, Zang W, Liu J (2009) Comparison of two methods for assaying complex I activity in mitochondria isolated from rat liver, brain and heart. Life Sci. 85(7-8):276-80.

Nguyen VT, Morange M, Bensaude O. (1988) Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells. Anal Biochem. 171(2):404-8.

van Vliet E, Daneshian M, Beilmann M, Davies A, Fava E, Fleck R, Julé Y, Kansy M, Kustermann S, Macko P, Mundy WR, Roth A, Shah I, Uteng M, van de Water B, Hartung T, Spinazzi M, Casarin A, Pertegato V, Salviati L, Angelini C (2012) Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc. 7(6):1235-46.

Salabei J.K., Gibb A.A. and Hill BG. (2014) Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nature Protocols, 9, 421–438.

Stetter JR, Li J (2008) Amperometric gas sensors--a review. Chem Rev. 108(2):352-66.

Wang XD, Wolfbeis OS (2014) Optical methods for sensing and imaging oxygen: materials, spectroscopies and applications. Chem Soc Rev. 43(10):3666-761.

Voet DJ and Voet JG; Pratt CW (2008). Chapter 18, Mitochondrial ATP synthesis. Principles of Biochemistry, 3rd Edition. Wiley. p. 608. ISBN 978-0-470-23396-2.

Willis JH, Capaldi RA, Huigsloot M, Rodenburg RJ, Smeitink J, Marusich MF (2009) Isolated deficiencies of OXPHOS complexes I and IV are identified accurately and quickly by simple enzyme activity immunocapture assays. Biochim Biophys Acta. 1787(5):533-8.

Zickermann V., Christophe Wirth, Hamid Nasiri, Karin Siegmund, Harald Schwalbe, Carola Hunte, Ulrich Brandt. Mechanistic insight from the crystal structure of mitochondrial complex I. Science 2 January 2015: Vol. 347 no. 6217 pp. 44-49.


177: N/A, Mitochondrial dysfunction 1

Short Name: N/A, Mitochondrial dysfunction 1

Key Event Component

Process Object Action
mitochondrion functional change

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Cellular

Cell term

Cell term
eukaryotic cell

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
mouse Mus musculus Strong NCBI
rat Rattus norvegicus Strong NCBI

Mitochondrial dysfunction is a universal event occurring in cells of any species (Farooqui and Farooqui, 2012). Many invertebrate species (drosophila, C, elegans) are considered as potential models to study mitochondrial function. New data on marine invertebrates, such as molluscs and crustaceans and non-Drosophila species, are emerging (Martinez-Cruz et al., 2012). Mitochondrial dysfunction can be measured in animal models used for toxicity testing (Winklhofer and Haass, 2010; Waerzeggers et al 2010) as well as in humans (Winklhofer and Haass, 2010).


How this Key Event Works

Mitochondrial dysfunction is a consequence of inhibition of the respiratory chain leading to oxidative stress.

Mitochondria can be found in all cells and are considered the most important cellular consumers of oxygen. Furthermore, mitochondria possess numerous redox enzymes capable of transferring single electrons to oxygen, generating the superoxide (O2-). Some mitochondrial enzymes that are involved in reactive oxygen species (ROS) generation include the electron-transport chain (ETC) complexes I, II and III; pyruvate dehydrogenase (PDH) and glycerol-3-phosphate dehydrogenase (GPDH). The transfer of electrons to oxygen, generating superoxide, happens mainly when these redox carriers are charged enough with electrons and the potential energy for transfer is elevated, like in the case of high mitochondrial membrane potential. In contrast, ROS generation is decreased if there are not enough electrons and the potential energy for the transfer is not sufficient (reviewed in Lin and Beal, 2006).

Cells are also able to detoxify the generated ROS due to an extensive antioxidant defence system that includes superoxide dismutases, glutathione peroxidases, catalase, thioredoxins, and peroxiredoxins in various cell organelles (reviewed in Lin and Beal, 2006). It is worth mentioning that, as in the case of ROS generation, antioxidant defences are also closely related to the redox and energetic status of mitochondria. If mitochondria are structurally and functionally healthy, an antioxidant defence mechanism balances ROS generation, and there is not much available ROS production. However, in case of mitochondrial damage, the antioxidant defence capacity drops and ROS generation takes over. Once this happens, a vicious cycle starts and ROS can further damage mitochondria, leading to more free-radical generation and further loss of antioxidant capacity. During mitochondrial dysfunction the availability of ATP also decreases, which is considered necessary for repair mechanisms after ROS generation.

A number of proteins bound to the mitochondria or endoplasmic reticulum (ER), especially in the mitochondria-associated ER membrane (MAM)are playing an important role of communicators between these two organelles (reviewed Mei et al., 2013). ER stress induces mitochondrial dysfunction through regulation of Ca2+ signaling and ROS production (reviewed Mei et al., 2013). Prolonged ER stress leads to release of Ca2+ at the MAM and increased Ca2+ uptake into the mitochondrial matrix, which induces Ca2+-dependent mitochondrial outer membrane permeabilization and apoptosis. At the same, ROS are produced by proteins in the ER oxidoreductin 1 (ERO1) family. ER stress activates ERO1 and leads to excessive production of ROS, which, in turn, inactivates SERCA and activates inositol-1,4,5- trisphosphate receptors (IP3R) via oxidation, resulting in elevated levels of cytosolic Ca2+, increased mitochondrial uptake of Ca2+, and ultimately mitochondrial dysfunction. Just as ER stress can lead to mitochondrial dysfunction, mitochondrial dysfunction also induces ER Stress (reviewed Mei et al., 2013). For example, nitric oxide disrupts the mitochondrial respiratory chain and causes changes in mitochondrial Ca2+ flux which induce ER stress. Increased Ca2+ flux triggers loss of mitochondrial membrane potential (MMP), opening of mitochondrial permeability transition pore (MPTP), release of cytochrome c and apoptosis inducing factor (AIF), decreasing ATP synthesis and rendering the cells more vulnerable to both apoptosis and necrosis (Wang and Qin, 2010).

Summing up: Mitochondria play a pivotal role in cell survival and cell death because they are regulators of both energy metabolism and apoptotic/necrotic pathways (Fiskum, 2000; Wieloch, 2001; Friberg and Wieloch, 2002). The production of ATP via oxidative phosphorylation is a vital mitochondrial function (Kann and Kovács, 2007; Nunnari and Suomalainen, 2012). The ATP is continuously required for signalling processes (e.g. Ca2+ signalling), maintenance of ionic gradients across membranes, and biosynthetic processes (e.g. protein synthesis, heme synthesis or lipid and phospholipid metabolism) (Kang and Pervaiz, 2012), and (Green, 1998; McBride et al., 2006). Inhibition of mitochondrial respiration contributes to various cellular stress responses, such as deregulation of cellular Ca2+ homeostasis (Graier et al., 2007) and ROS production (Nunnari and Suomalainen, 2012; reviewed Mei et al., 2013).). It is well established in the existing literature that mitochondrial dysfunction may result in: (a) an increased ROS production and a decreased ATP level, (b) the loss of mitochondrial protein import and protein biosynthesis, (c) the reduced activities of enzymes of the mitochondrial respiratory chain and the Krebs cycle, (d) the loss of the mitochondrial membrane potential, (e) the loss of mitochondrial motility, causing a failure to re-localize to the sites with increased energy demands (f) the destruction of the mitochondrial network, and (g) increased mitochondrial Ca2+ uptake, causing Ca2+ overload (reviewed in Lin and Beal, 2006; Graier et al., 2007), (h) the rupture of the mitochondrial inner and outer membranes, leading to (i) the release of mitochondrial pro-death factors, including cytochrome c (Cyt. c), apoptosis-inducing factor, or endonuclease G (Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013), which eventually leads to apoptotic, necrotic or autophagic cell death (Wang and Qin, 2010). Due to their structural and functional complexity, mitochondria present multiple targets for various compounds.


How it is Measured or Detected

Mitochondrial dysfunction can be detected using isolated mitochondria, intact cells or cells in culture as well as in vivo studies. Such assessment can be performed with a large range of methods (revised by Brand and Nicholls, 2011) for which some important examples are given. All approaches to assess mitochondrial dysfunction fall into two main categories: the first assesses the consequences of a loss-of-function, i.e. impaired functioning of the respiratory chain and processes linked to it. Some assay to assess this have been described for KE1, with the limitation that they are not specific for complex I. In the context of overall mitochondrial dysfunction, the same assays provide useful information, when performed under slightly different assay conditions (e.g. without addition of complex III and IV inhibitors). The second approach assesses a ‘non-desirable gain-of-function’, i.e. processes that are usually only present to a very small degree in healthy cells, and that are triggered in a cell, in which mitochondria fail.

I. Mitochondrial dysfunction assays assessing a loss-of function.

1. Cellular oxygen consumption.

See KE1 for details of oxygen consumption assays. The oxygen consumption parameter can be combined with other endpoints to derive more specific information on the efficacy of mitochondrial function. One approach measures the ADP-to-O ratio (the number of ADP molecules phosphorylated per oxygen atom reduced (Hinkle, 1995 and Hafner et al., 1990). The related P/O ratio is calculated from the amount of ADP added, divided by the amount of O2 consumed while phosphorylating the added ADP (Ciapaite et al., 2005; Diepart et al., 2010; Hynes et al., 2006; James et al., 1995; von Heimburg et al., 2005).

2. Mitochondrial membrane potential (Δψm ).

The mitochondrial membrane potential (Δψm) is the electric potential difference across the inner mitochondrial membrane. It requires a functioning respiratory chain in the absence of mechanisms that dissipate the proton gradient without coupling it to ATP production. The classical, and still most quantitative method uses a tetraphenylphosphonium ion (TPP+)-sensitive electrode on suspensions of isolated mitochondria. The Δψm can also be measured in live cells by fluorimetric methods. These are based on dyes which accumulate in mitochochondria because of Δψm. Frequently used are tetramethylrhodamineethylester (TMRE), tetramethylrhodaminemethyl ester (TMRM) (Petronilli et al., 1999) or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide (JC-1). Mitochondria with intact membrane potential concentrate JC-1, so that it forms red fluorescent aggregates, whereas de-energized mitochondria cannot concentrate JC-1 and the dilute dye fluoresces green (Barrientos et al., 1999). Assays using TMRE or TMRM measure only at one wavelength (red fluorescence), and depending on the assay setup, de-energized mitochondria become either less fluorescent (loss of the dye) or more fluorescent (attenuated dye quenching).

3. Enzymatic activity of the electron transport system (ETS).

Determination of ETS activity can be dene following Owens and King's assay (1975). The technique is based on a cell-free homogenate that is incubated with NADH to saturate the mitochondrial ETS and an artificial electron acceptor [l - (4 -iodophenyl) -3 - (4 -nitrophenyl) -5-phenylte trazolium chloride (INT)] to register the electron transmission rate. The oxygen consumption rate is calculated from the molar production rate of INT-formazan which is determined spectrophotometrically (Cammen et al., 1990).

4. ATP content.

For the evaluation of ATP levels, various commercially-available ATP assay kits are offered  based on luciferin and luciferase activity. For isolated mitochondria various methods are available to continuously measure ATP with electrodes (Laudet 2005), with luminometric methods, or for obtaining more information on different nucleotide phosphate pools (e.g. Ciapaite et al., (2005).


II. Mitochondrial dysfunction assays assessing a gain-of function.


1. Mitochondrial permeability transition pore opening (PTP).

The opening of the PTP is associated with a permeabilization of mitochondrial membranes, so that different compounds and cellular constituents can change intracellular localization. This can be measured by assessment of the translocation of cytochrome c, adenylate kinase or AIF from mitochondria to the cytosol or nucleus. The translocation can be assessed biochemically in cell fractions, by imaging approaches in fixed cells or tissues or by life-cell imaging of GFP fusion proteins (Single 1998; Modjtahedi 2006). An alternative approach is to measure the accessibility of cobalt to the mitochondrial matrix in a calcein fluorescence quenching assay in live permeabilized cells (Petronilli et al., 1999).

2. mtDNA damage as a biomarker of mitochondrial dysfunction.

Various quantitative polymerase chain reaction (QPCR)-based assays have been developed to detect changes of DNA structure and sequence in the mitochondrial genome. mtDNA damage can be detected in blood after low-level rotenone exposure, and the damage persists even after CI activity has returned to normal. With a more sustained rotenone exposure, mtDNA damage is also detected in skeletal muscle. These data support the idea that mtDNA damage in peripheral tissues in the rotenone model may provide a biomarker of past or ongoing mitochondrial toxin exposure (Sanders et al., 2014a and 2014b).

3. Generation of ROS and resultant oxidative stress.

a. General approach. Electrons from the mitochondrial ETS may be transferred ‘erroneously’ to molecular oxygen to form superoxide anions. This type of side reaction can be strongly enhanced upon mitochondrial damage. As superoxide may form hydrogen peroxide, hydroxyl radicals or other reactive oxygen species, a large number of direct ROS assays and assays assessing the effects of ROS (indirect ROS assays) are available (Adam-Vizi, 2005; Fan and Li 2014). Direct assays are based on the chemical modification of fluorescent or luminescent reporters by ROS species. Indirect assays assess cellular metabolites, the concentration of which is changed in the presence of ROS (e.g. glutathione, malonaldehyde, isoprostanes,etc.) At the animal level the effects of oxidative stress are measured from biomarkers in the blood or urine.

b. Measurement of the cellular glutathione (GSH) status. GSH is regenerated from its oxidized form (GSSH) by the action of an NADPH dependent reductase (GSSH + NADPH + H+ à 2 GSH + NADP+). The ratio of GSH/GSSG is therefore a good indicator for the cellular NADH+/NADPH ratio (i.e. the redox potential).. GSH and GSSH levels can be determined by HPLC, capillary electrophoresis, or biochemically with DTNB (Ellman’s reagent). As excess GSSG is rapidly exported from most cells to maintain a constant GSH/GSSG ratio, a reduction of total glutathione (GSH/GSSG) is often a good surrogate measure for oxidative stress.

c. Quantification of lipid peroxidation. Measurement of lipid peroxidation has historically relied on the detection of thiobarbituric acid (TBA)-reactive compounds such as malondialdehyde generated from the decomposition of cellular membrane lipid under oxidative stress (Pryor et al., 1976). This method is quite sensitive, but not highly specific.. A number of commercial assay kits are available for this assay using absorbance or fluorescence detection technologies. The formation of F2-like prostanoid derivatives of arachidonic acid, termed F2-isoprostanes (IsoP) has been shown to be more specific for lipid peroxidation. A number of commercial ELISA kits have been developed for IsoPs, but interfering agents in samples requires partial purification before analysis. Alternatively, GC/MS may be used, as robust (specific) and sensitive method.


d. Detection of superoxide production. Generation of superoxide by inhibition of complex I and the methods for its detection are described by Grivennikova and Vinogradov (2014). A range of different methods is also described by BioTek (http://www.biotek.com/resources/articles/reactive-oxygen-species.html). The reduction of ferricytochrome c to ferrocytochrome c may be used to assess the rate of superoxide formation (McCord, 1968). Like in other superoxide assays, specificity can only be obtained by measurements in teh absence and presence of superoxide dismutase. Chemiluminescent reactions have been used for their increased sensitivity. The most widely used chemiluminescent substrate is lucigenin. Coelenterazine has also been used as a chemiluminescent substrate. Hydrocyanine dyes are fluorogenic sensors for superoxide and hydroxyl radical, and they become membrane impermeable after oxidation (trapping at sit of formation). The best characterized of these probes are Hydro-Cy3 and Hydro-Cy5. generation of superoxide in mitochondria can be visualized using fluorescence microscopy with MitoSOX™ Red reagent (Life Technologies). MitoSOX™ Red reagent is a cationic derivative of dihydroethidium that permeates live cells and accumulates in mitochondria.

e. Detection of hydrogen peroxide (H2O2) production. There are a number of fluorogenic substrates, which serve as hydrogen donors that have been used in conjunction with horseradish peroxidase (HRP) enzyme to produce intensely fluorescent products in the presence of hydrogen peroxide (Zhou et al., 1997: Ruch et al., 1983). The more commonly used substrates include diacetyldichloro-fluorescein, homovanillic acid, and Amplex® Red. In these examples, increasing amounts of H2O2 form increasing amounts of fluorescent product (Tarpley et al., 2004).

Summing up, mitochondrial dysfunction can be measured by: • ROS production: superoxide (O2-), and hydroxyl radicals (OH−) • Nitrosative radical formation such as ONOO− or directly by: • Loss of mitochondrial membrane potential (MMP) • Opening of mitochondrial permeability transition pores (MPTP) • ATP synthesis • Increase in mitochondrial Ca2+ • Cytochrome c release • AIF (apoptosis inducing factor) release from mitochondria • Mitochondrial Complexes enzyme activity • Measurements of mitochondrial oxygen consumption • Ultrastructure of mitochondria using electron microscope and mitochondrial fragmentation measured by labelling with DsRed-Mito expression (Knott et al, 2008) Mitochondrial dysfunction-induced oxidative stress can be measured by: • Reactive carbonyls formations (proteins oxidation) • Increased 8-oxo-dG immunoreactivity (DNA oxidation) • Lipid peroxidation (formation of malondialdehyde (MDA) and 4- hydroxynonenal (HNE) • 3-nitrotyrosine (3-NT) formation, marker of protein nitration • Translocation of Bid and Bax to mitochondria • Measurement of intracellular free calcium concentration ([Ca2+]i): Cells are loaded with 4 μM fura-2/AM). • Ratio between reduced and oxidized form of glutathione (GSH depletion)(Promega assay, TB369; Radkowsky et al., 1986 • Neuronal nitric oxide synthase (nNOS) activation that is Ca2+-dependent All above measurements can be performed as the assays for each readout are well established in the existing literature (e.g. Bal-Price and Brown, 2000; Bal-Price et al., 2002; Fujikawa, 2015; Walker et al., 1995). See also KE Oxidative Stress, Increase


References

 

Adam-Vizi V. Production of reactive oxygen species in brain mitochondria: contribution by electron transport chain and non-electron transport chain sources. Antioxid Redox Signal. 2005, 7(9-10):1140-1149.

Bal-Price A. and Guy C. Brown. Nitric-oxide-induced necrosis and apoptosis in PC12 cells mediated by mitochondria. J. Neurochemistry, 2000, 75: 1455-1464.

Bal-Price A, Matthias A, Brown GC., Stimulation of the NADPH oxidase in activated rat microglia removes nitric oxide but induces peroxynitrite production. J. Neurochem. 2002, 80: 73-80.

Brand MD, Nicholls DG. Assessing mitochondrial dysfunction in cells. Biochem J. 2011 Apr 15;435(2):297-312.

Braun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182.

Barrientos A., and Moraes C.T. (1999) Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. Vol. 274, No. 23, pp. 16188–16197.

Cammen M. Corwin, Susannah Christensen. John P. (1990) Electron transport system (ETS) activity as a measure of benthic macrofaunal metabolism MARINE ECOLOGY PROGRESS SERIES- (65) : 171-182.

Ciapaite, Lolita Van Eikenhorst, Gerco Bakker, Stephan J.L. Diamant, Michaela. Heine, Robert J Wagner, Marijke J. V. Westerhoff, Hans and Klaas Krab (2005) Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver Mitochondria Diabetes 54:4 944-951.

Correia SC, Santos RX, Perry G, Zhu X, Moreira PI, Smith MA. (2012). Mitochondrial importance in Alzheimer’s, Huntington’s and Parkinson’s diseases. Adv Exp Med Biol 724:205 – 221.

Cozzolino M, Ferri A, Valle C, Carri MT. (2013). Mitochondria and ALS: implications from novel genes and pathways. Mol Cell Neurosci 55:44 – 49.

Diepart, C, Verrax, J Calderon, PU, Feron, O., Jordan, BF, Gallez, B (2010) Comparison of methods for measuring oxygen consumption in tumor cells in vitroAnalytical Biochemistry 396 (2010) 250–256.

Farooqui T. and . Farooqui, A. A (2012) Oxidative stress in Vertebrates and Invertebrate: molecular aspects of cell signalling. Wiley-Blackwell,Chapter 27, pp:377- 385.

Fan LM, Li JM. Evaluation of methods of detecting cell reactive oxygen species production for drug screening and cell cycle studies. J Pharmacol Toxicol Methods. 2014 Jul-Aug;70(1):40-7.

Fiskum G. Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma. 2000 Oct;17(10):843-55.

Friberg H, Wieloch T. (2002). Mitochondrial permeability transition in acute neurodegeneration. Biochimie 84:241–250.

Fujikawa DG, The Role of Excitotoxic Programmed Necrosis in Acute Brain Injury. Computational and Structural Biotechnology Journal, 2015, 13: 212–221.

Graier WF, Frieden M, Malli R. (2007). Mitochondria and Ca2+ signaling: old guests, new functions. Pflugers Arch 455:375–396.

Green DR. (1998). Apoptotic pathways: the roads to ruin. Cell 94:695-698.

Grivennikova VG, Vinogradov AD. Generation of superoxide by the mitochondrial Complex I. Biochim Biophys Acta. 2006, 1757(5-6):553-61.

Hafner RP, Brown GC, Brand MD: Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the ‘top-down’ approach of metabolic control theory. Eur J Biochem188 :313 –319,1990.

Hinkle PC (1995) Measurement of ADP/O ratios. In Bioenergetics: A Practical Approach. Brown GC, Cooper CE, Eds. Oxford, U.K., IRL Press, p.5 –6.

Hynes, J.. Marroquin, L.D Ogurtsov, V.I. Christiansen, K.N. Stevens, G.J. Papkovsky, D.B. Will, Y. (2006)) Investigation of drug-induced mitochondrial toxicity using fluorescence-based oxygen-sensitive probes, Toxicol. Sci. 92 186–200.

James, P.E. Jackson, S.K.. Grinberg, O.Y Swartz, H.M. (1995) The effects of endotoxin on oxygen consumption of various cell types in vitro: an EPR oximetry study, Free Radic. Biol. Med. 18 (1995) 641–647.

Kang J, Pervaiz S. (2012). Mitochondria: Redox Metabolism and Dysfunction. Biochem Res Int 2012:896751.

Kann O, Kovács R. (2007). Mitochondria and neuronal activity. Am J Physiol Cell Physiol 292:C641-576.

Knott Andrew B., Guy Perkins, Robert Schwarzenbacher & Ella Bossy-Wetzel. Mitochondrial fragmentation in neurodegeneration. Nature Reviews Neuroscience, 2008, 229: 505-518.

Llaudet E, Hatz S, Droniou M, Dale N. Microelectrode biosensor for real-time measurement of ATP in biological tissue. Anal Chem. 2005, 77(10):3267-73.

Lee HC, Wei YH. (2012). Mitochondria and aging. Adv Exp Med Biol 942:311-327.

Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, et al. Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J Biol Chem.2003;278:8516–8525.

Lin MT, Beal MF. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 2006. 443:787-795.

Martin LJ. (2011). Mitochondrial pathobiology in ALS. J Bioenerg Biomembr 43:569 – 579.

Martinez-Cruz, Oliviert Sanchez-Paz, Arturo Garcia-Carreño, Fernando Jimenez-Gutierrez, Laura Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, ISBN 978-953-51-0090-4, Publisher InTech, 2012, 181-218.

McBride HM, Neuspiel M, Wasiak S. (2006). Mitochondria: more than just a powerhouse. Curr Biol 16:R551–560.

McCord, J.M. and I. Fidovich (1968) The Reduction of Cytochrome C by Milk Xanthine Oxidase. J. Biol. Chem. 243:5733-5760.

Mei Y, Thompson MD, Cohen RA, Tong X. (2013) Endoplasmic Reticulum Stress and Related Pathological Processes. J Pharmacol Biomed Anal.. 1:100-107.

Modjtahedi N, Giordanetto F, Madeo F, Kroemer G. Apoptosis-inducing factor: vital and lethal. Trends Cell Biol. 2006 May;16(5):264-72.

Nunnari J, Suomalainen A. (2012). Mitochondria: in sickness and in health. Cell 148:1145–1159. Hajnóczky G, Csordás G, Das S, Garcia-Perez C, Saotome M, Sinha Roy S, Yi M. (2006). Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis. Cell Calcium 40:553-560.

Oliviert Martinez-Cruz, Arturo Sanchez-Paz, Fernando Garcia-Carreño, Laura Jimenez-Gutierrez, Ma. de los Angeles Navarrete del Toro and Adriana Muhlia-Almazan. Invertebrates Mitochondrial Function and Energetic Challenges (www.intechopen.com), Bioenergetics, Edited by Dr Kevin Clark, ISBN 978-953-51-0090-4, Publisher InTech, 2012, 181-218.

Owens R.G. and King F.D. The measurement of respiratory lectron-transport system activity in marine zooplankton. Mar. Biol. 1975, 30:27-36.

Petronilli V, Miotto G, Canton M, Brini M, Colonna R, Bernardi P, Di Lisa F: Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 1999, 76:725-734.

Promega GSH-Glo Glutathione Assay Technical Bulletin, TB369, Promega Corporation, Madison, WI.

Pryor, W.A., J.P. Stanley, and E. Blair. (1976) Autoxidation of polyunsaturated fatty acids: II. A Suggested mechanism for the Formation of TBA-reactive materials from prostaglandin-like Endoperoxides. Lipids, 11:370-379.

Radkowsky, A.E. and E.M. Kosower (1986) Bimanes 17. (Haloalkyl)-1,5-diazabicyclo[3.3.O]octadienediones (halo-9,10- dioxabimanes): reactivity toward the tripeptide thiol, glutathione, J. Am. Chem. Soc 108:4527-4531.

Ruch, W., P.H. Cooper, and M. Baggiollini (1983) Assay of H2O2 production by macrophages and neutrophils with Homovanillic acid and horseradish peroxidase. J. Immunol Methods 63:347-357.

Sanders LH, McCoy J, Hu X, Mastroberardino PG, Dickinson BC, Chang CJ, Chu CT, Van Houten B, Greenamyre JT. (2014a). Mitochondrial DNA damage: molecular marker of vulnerable nigral neurons in Parkinson's disease. Neurobiol Dis. 70:214-23.

Sanders LH, Howlett EH2, McCoy J, Greenamyre JT. (2014b) Mitochondrial DNA damage as a peripheral biomarker for mitochondrial toxin exposure in rats. Toxicol Sci. Dec;142(2):395-402.

Single B, Leist M, Nicotera P. Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ. 1998 Dec;5(12):1001-3.

Tahira Farooqui and Akhlaq A. Farooqui. (2012) Oxidative stress in Vertebrates and Invertebrate: molecular aspects of cell signalling. Wiley-Blackwell,Chapter 27, pp:377- 385.

Tarpley, M.M., D.A. Wink, and M.B. Grisham (2004) Methods for detection of reactive Metabolites of Oxygen and Nitrogen: in vitro and in vivo considerations. Am . J. Physiol Regul Integr Comp Physiol. 286:R431-R444.

von Heimburg, D. Hemmrich, K. Zachariah S.,. Staiger, H Pallua, N.(2005) Oxygen consumption in undifferentiated versus differentiated adipogenic mesenchymal precursor cells, Respir. Physiol. Neurobiol. 146 (2005) 107–116.

Waerzeggers, Yannic Monfared, Parisa Viel, Thomas Winkeler, Alexandra Jacobs, Andreas H. (2010) Mouse models in neurological disorders: Applications of non-invasive imaging, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Volume 1802, Issue 10, Pages 819-839.

Walker JE, Skehel JM, Buchanan SK. (1995) Structural analysis of NADH: ubiquinone oxidoreductase from bovine heart mitochondria. Methods Enzymol.;260:14–34.

Wang A, Costello S, Cockburn M, Zhang X, Bronstein J, Ritz B. (2011). Parkinson’s disease risk from ambient exposure to pesticides. Eur J Epidemiol 26:547-555.

Wang Y., and Qin ZH., Molecular and cellular mechanisms of excitotoxic neuronal death, Apoptosis, 2010, 15:1382-1402.

Wieloch T. (2001). Mitochondrial Involvement in Acute Neurodegeneration 52:247–254.

Winklhofer, K. Haass,C (2010) Mitochondrial dysfunction in Parkinson's disease, Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1802: 29-44.

Zhou, M., Z.Diwu, Panchuk-Voloshina, N. and R.P. Haughland (1997), A Stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: application in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem 253:162-168.


889: Impaired, Proteostasis

Short Name: Impaired, Proteostasis

Key Event Component

Process Object Action
Proteostasis deficiencies protein abnormal

Stressors

Name
1',2'-dihydrorotenone

Biological Organization

Level of Biological Organization
Cellular

Cell term

Cell term
eukaryotic cell

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Moderate NCBI

The ubiquitin proteasome system is highly conserved in eukaryotes, from yeast to human. Ubiquitin is a small (8.5 kDa) regulatory protein that has been found in almost all tissues of eukaryotic organisms. For instance, drosophila has been used as PD model to study the role of ubiquitin in α-synuclein induced-toxicity (Lee et al., 2009). Human and yeast ubiquitin share 96% sequence identity. Neither ubiquitin nor the ubiquitination machinery are known to exist in prokaryotes. Autophagy is ubiquitous in eukaryotic cells and is the major mechanism involved in the clearance of oxidatively or otherwise damaged/worn-out macromolecules and organelles (Esteves et al., 2011). Due to the high degree of conservation, most of the knowledge on autophagy proteins in vertebrates is derived from studies in yeast (Klionsky et al., 2007). Autophagy is seen in all eukaryotic systems, including fungi, plants, slime mold, nematodes, fruit flies and insects, rodents (i.e., laboratory mice and rats), and humans. It is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation.


How this Key Event Works

The concept of proteostasis refers to the homeostasis of proteins in space and time, i.e. the correct balance between protein synthesis, modification, transport and degradation. Disturbance of proteostasis results in pathological changes either by loss of function events (lack of a pivotal protein/protein function) or by a gain of undesired functions (aggregation of a protein leading to the formation of inclusions and new structures in cells and disturbing turnover of many unrelated proteins).

Proteostasis regulation is the main defence mechanism against toxic proteins, whose accumulation could greatly compromise normal cellular function and viability. Therefore, the chaperone and degradation systems assuring the removal of misfolded and aggregated proteins, as well as damaged, dysfunctional cellular organelles (e.g., defective mitochondria) play a key role in cellular homeostasis (Lee et al., 2012). The two major degradation systems are the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway (ALP) (Korolchuk et al., 2010; Kroemer et al., 2010; Ravikumar et al., 2010). The UPS works through the attachment of multiple ubiquitin molecules to a protein substrate, followed by the subsequent degradation of the tagged polyubiquitinated protein by the proteasome (Ciechanover, 1998; Ciechanover and Brundin, 2003). A compromised function of the UPS leads to the accumulation of ubiquitylated proteins, such as α-synuclein, (Ii et al. 1997; Spillantini et al. 1997; Sulzer and Zecca 2000). The accumulation of polyubiquitinated proteins, as a consequence of a dysfunctional proteasome activity, is observed in some pathologies, and experimental inhibition of the proteasome has been shown to trigger parkinsonian neurodegeneration (McNaught and Jenner 2001; Hardy et al., 2001).

ALP involves the engulfment of cytoplasmic materials into autophagosomes, which are degraded by lysosomal enzymes after fusion of autophagosomes with lysosomes (Kuma et al., 2004) or direct import of proteins into lysosomes (Cuervo, 2004; Mizushima et al., 2008). Autophagy also plays an essential role for the removal of damaged organelles, such as mitochondria. Both, excessive autophagy or reduced autophagic flux can compromise cell survival (Rothermel and Hill, 2007), and several genetic forms of PD are linked to the autophagy-related genes Pink1, Parkin or Uchl1. Autophagy enables cell survival during mitochondrial stress by clearing the damaged organelles (Lee et al., 2012).

One of the main aggregated proteins found to accumulate in nigrostriatal cells during Parkinson's disease is α-synuclein. Aggregation of α-synuclein can obstruct normal cellular transport, leading to impaired intracellular trafficking and/or trapping of cellular organelles in inappropriate locations, this resulting in synaptic and cell dysfunctions (Bartels et al., 2011) (Bellucci A., et al., 2012; Cookson MR., 2005; Games D., et al., 2013; Hunn BH., et al., 2015). Importantly, accumulation of α-synuclein affects mitochondrial trafficking. The polarity and correct function of different types of cells depend on an efficient transport of mitochondria to areas of high energy consumption (Sheng, 2014). Therefore, the correct distribution of mitochondria to various parts of a cell is essential to preserve cell function (Schwarz, 2013; Zhu et al., 2012).


How it is Measured or Detected

1. Evaluation of UPS function. General turnover assays Quantitative evaluation can be based on the detection of increased ubiquitin or ubiquinated proteins, as well as proteasomal subunits, either by immunocyto/histochemistry or by western blotting (Rideout et al., 2001; Ortega and Lucas, 2014). UPS activity can be continuously monitored by quantitating (by mean of flow cytometry or microscopy) the level of e.g. EGFP-degron fusion proteins (green fluorescent protein) that are selectively degraded by the proteasome (Bence et al., 2001).

Proteasome activity assay. Various fluorogenic substrates (e.g., Suc-Leu-Leu-Val-Tyr-AMC for the chymotrypsin-like activity) can be used for the determination of proteasomal activity in in vivo or in vitro applications. These substrates may be applied to tissue or cell homogenates, but specific measurements require partial purification of the proteasome (Kisselev and Goldberg, 2005).

Detection of α-synuclein (AS) aggregates. The most common methods to detect AS aggregates use immunostaining for AS (in cells or in tissues). In cell culture, AS may also be epitope-tagged or coupled to GFP to allow an indirect detection. The detection of small, not microscopically-visible AS aggregates is indicative of protease-resistance. Tissue slices may be exposed to proteases before immunostaining for AS. Alternatively, small or large aggregates may be biochemically enriched by differential centrifugation and proteolytic treatment, and then analyzed, e.g., by western blot, mass spectrometry or ELISA-like immunoquantification.

2. Evaluation of ALP function. Quantification of lysosomes or autophagosomes Disturbances of ALP often result in counter-regulations that can be visualized by staining of lysosomes or parts of the autophagy system. Several weakly basic dyes can be used to stain acidic organelles (lysosomes) in live cells. For example, the dye LysoTracker Red stains lysosomes and can be used to monitor autophagy (Klionsky et al., 2007; Klionsky et al., 2008). The autofluorescent drug monodansylcadaverine (MDC) has also been used as autophago-lysosome marker (Munafó and Colombo, 2002). A convenient way to stain lysosomes in tissue or fixed cells is the use of antibodies against the Lysosomal-Associated Membrane Protein 1 (LAMP-1) (Rajapakshe et al., 2015) or against cathepsins (Foghsgaard et al., 2001). For qualitative or semiquantitative estimates of lysosomes and related organelles, transmission electron microscopy has been frequently used (Barth et al., 2010).

Monitoring of autophagy-related molecules. The amount and the localization of autophagy-related proteins can change during disturbance of the ALP. Especially in cell culture, but also in transgenic mice, various techniques have been used to monitor autophagy by mean of fluorescence-tags or other substrates, e.g., ATG, autophagy-related protein or autophagy substrates, to monitor their fate in cells and thus provide information on disturbed ALP, or the over-expression of GFP–LC3, in which GFP (green fluorescent protein) is expressed as a fusion protein at the amino terminus of LC3 (microtubule-associated protein 1A/1B-light chain 3), which is the a mammalian homologue of S. cerevisiae ATG8 (Kadowaki and Karim, 2009).

Monitoring autophagic flux. The lysosomal degradation of the autophagic cargo constitutes the autophagic flux, which can be measured by assessing the rate of turnover of long-lived proteins that are normally turned over by autophagy (Bauvy et al., 2009). This is performed by labelling intracellular proteins with either [14C]-leucine or [14C]-valine, followed by a long culture period in standard medium. The release of radioactive leucin or valin into the culture medium corresponds to the protein degradation rate in cells, and it may be measured by liquid scintillation counting.

Monitoring the conversion of LC3-I to LC3-II. The progression of autophagy (autophagic flux) can be studied by the conversion of LC3-I into LC3-II (i.e. a post-translational modification specific for autophagy) by mean of Western blot analysis. The amount of LC3-II correlates with the number of autophagosomes. Conversion of LC3 can be used to examine autophagic activity in the presence or absence of lysosomal activity (Klionsky et al., 2007; Klionsky et al., 2008). The technology can also be used in vivo, e.g. by the use of transgenic mice that overexpress GFP–LC3 (Kuma et al., 2004).

3. Evaluation of intracellular transport of mitochondria and other organelles.

A range of technologies has been used to visualize mitochondrial dynamics in live cells (Jakobs, 2006; Grafstein and Forman, 1980). They usually employ a combination of mitochondrial labelling with fluorescent dyes (e.g. DiOC6 (3, 3′-Dihexyloxacarbocyanine iodide), JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′ tetraethylbenzimida-zolylcarbo-cyanine iodide), MitoTracker, MitoFluor probes, etc.), followed by video- or confocal microscopy for live cell imaging (Schwarz, 2013; Pool et al., 2006). Most frequently, mitochondrial mobility is observed along neurites, and measurable endpoints may be mitochondrial speed and direction with regard to the cell soma (Schildknecht et al. 2013). Additionally, also mitochondrial fusion and fission have been monitored by such methods (Exner et al., 2012). The transport of other organelles along neurites may be monitored using similar methods, and the microtubule structures that serve as transport scaffold may be co-stained.


References

Barth S., Danielle Glick, and Kay F Macleod, Autophagy: assays and artifacts. J Pathol. 2010 Jun; 221(2): 117–124.

Bartels T, Choi JG, Selkoe DJ (Sep 2011). "α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation". Nature 477 (7362): 107–10.

Bauvy C, Meijer AJ, Codogno P. Assaying of autophagic protein degradation. Methods Enzymol. 2009;452:47–61.

Bellucci A., M. Zaltieri, L. Navarria, J. Grigoletto, C. Missale, and P. Spano, “From α-synuclein to synaptic dysfunctions: new insights into the pathophysiology of Parkinson’s disease,” Brain Research, vol. 1476, pp. 183–202, 2012.

Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin–proteasome system by protein aggregation. Science 2001;292:1552–5.

Ciechanover A. (1998) The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 17, 7151±7160.

Ciechanover A., and Brundin P., 2003, The Ubiquitin Proteasome System in Neurodegenerative Diseases: Sometimes the Chicken, Sometimes the Egg. Neuron, 427–446

Cookson MR., “The biochemistry of Parkinson’s disease,” Annual Review of Biochemistry, vol. 74, pp. 29–52, 2005.

Cuervo A.M., “Autophagy: many paths to the same end,” Molecular and Cellular Biochemistry, vol. 263, no. 1, pp. 55–72, 2004.

Exner N, Lutz AK, Haass C, Winklhofer KF. Mitochondrial dysfunction in Parkinson's disease: molecular mechanisms and pathophysiological consequences. EMBO J. 2012 Jun 26;31(14):3038-62.

Esteves AR, Arduíno DM, Silva DF, Oliveira CR, Cardoso SM. 2011. Mitochondrial Dysfunction: The Road to Alpha-Synuclein Oligomerization in PD. Parkinsons Dis. 2011:693761.

Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, Elling F, Leist M, Jäättelä M. Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol. 2001 May 28;153(5):999-1010.

Games D., P. Seubert, E. Rockenstein et al., “Axonopathy in an α-synuclein transgenic model of Lewy body disease is associated with extensive accumulation of c-terminal-truncated α-synuclein,” American Journal of Pathology, vol. 182, no. 3, pp. 940–953, 2013.

Grafstein B., and Forman DS. Intracellular transport in neurons. Physiological Reviews Published 1 October 1980 Vol. 60 no. 4.

Hardy J. Rideout, Kristin E. Larsen, David Sulzer and Leonidas Stefanis, Proteasomal inhibition leads to formation of ubiquitin/a-synuclein-immunoreactive inclusions in PC12 cells. Journal of Neurochemistry, 2001, 78, 899±908

Hunn BH., S. J. Cragg, J. P. Bolam, M. G. Spillantini, and R. Wade-Martins, “Impaired intracellular trafficking defines early Parkinson’s disease,” Trends in Neurosciences, vol. 38, no. 3, pp.178–188, 2015.

Ii K., Ito H., Tanaka K. and Hirano A. (1997) Immunocytochemical co-localization of the proteasome in ubiquitinated structures in neurodegenerative diseases and the elderly. J. Neuropathol. Exp. Neurol. 56, 125-131.

Jakobs S., High resolution imaging of live mitochondria, 2006, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1763, Issues 5–6 Pages 561–575

Kadowaki M, Karim MR. Cytosolic LC3 ratio as a quantitative index of macroautophagy. Methods Enzymol. 2009;452:199–213. [PubMed]

Kisselev AF, Goldberg AL. Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates. Methods Enzymol. 2005;398:364–378.

Klionsky DJ., Ana Maria Cuervo & Per O. Seglen. Methods for Monitoring Autophagy from Yeast to Human. Autophagy 2007, 3:3, 181-206; Klionsky D.J., Abeliovich H., Agostinis P., Agrawal D.K., Aliev G., Askew D.S., Baba M., Baehrecke E.H., Bahr B.A., Ballabio A., et al Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 2008;4:151-175.

Korolchuk VI, Menzies FM, Rubinsztein DC (2010) Mechanisms of cross-talk between the ubiquitin–proteasome and autophagy–lysosome systems. FEBS Lett 584:1393–1398

Kroemer G, Mariño G, Levine B (2010) Autophagy and the integrated stress response. J. Molecular cell 40:280–293.

Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, et al. The role of autophagy during the early neonatal starvation period. Nature. 2004;432:1032–1036.

Lee J, Giordano S, Zhang J; Giordano; Zhang (January 2012). "Autophagy, mitochondria and oxidative stress: cross-talk and redox signalling". Biochem. J. 441 (2): 523–40.

Lee FK, Wong AK, Lee YW, Wan OW, Chan HY, Chung KK. The role of ubiquitin linkages on alpha-synuclein induced-toxicity in a Drosophila model of Parkinson's disease. J Neurochem. 2009 Jul;110(1):208-19

McNaught K. S. and Jenner P. (2001) Proteasomal function is impaired in substantia nigra in Parkinson's disease. Neurosci. Lett. 297, 191-194.

Mizushima N. et al., 2008. Autophagy fights disease through cellular self-digestion. Nature. 451(7182):1069-75. Review.

Munafó DB, Colombo MI. Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24. Traffic. 2002 Jul;3(7):472-82.

Ortega Z. and Lucas J.J. (2014) Ubiquitin–proteasome system involvement in Huntington’s disease Front Mol Neurosci. 2014; 7: 77.

Pool M., Rippstein P., Mcbride H. Kothary R., 2006 Trafficking of Macromolecules and Organelles in Cultured Dystonia musculorum Sensory Neurons Is Normal. J. Comparative Neurology 494:549–558 (2006)

Rajapakshe AR, Podyma-Inoue KA, Terasawa K, Hasegawa K, Namba T, Kumei Y, Yanagishita M, Hara-Yokoyama M. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells. Exp Cell Res. 2015 Feb 1;331(1):211-22. doi: 10.1016/j.yexcr.2014.09.014.

Ravikumar B, Sarkar S, Davies JE et al (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90:1383–1435. doi:10.1152/physrev.00030.2009

Rothermel BA, Hill JA (2007) Myocyte autophagy in heart disease: friend or foe? Autophagy 3:632–634.

Rideout HJ, Larsen KE, Sulzer D, Stefanis L. 2001.Proteasomal inhibition leads to formation of ubiquitin/a-synuclein-immunoreactive inclusions in PC12 cells. Journal of Neurochemistry. 78, 899-908.

Schildknecht S, Karreman C, Pöltl D, Efrémova L, Kullmann C, Gutbier S, Krug A, Scholz D, Gerding HR, Leist M. Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases. ALTEX. 2013;30(4):427-44.

Schwarz TL.Mitochondrial trafficking in neurons. Cold Spring Harb Perspect Biol. 2013 Jun 1;5(6). pii: a011304.

Sheng ZH., Mitochondrial trafficking and anchoring in neurons: new insight and implications. J of Cell Biology, vol. 204. No.7 pp. 1087-1098, 2014.

Spillantini M. G., Schmidt M. L., Lee V. M., Trojanowski J. Q., Jakes R. and Goedert M. (1997) Alpha-synuclein in Lewy bodies.Nature 388, 839-840.

Sulzer D. and Zecca L. (2000) Intraneuronal dopamine-quinonem synthesis: a review. Neurotoxicity Res. 1, 181-195.

Zhu XH, Qiao H, Du F, Xiong Q, Liu X, Zhang X, Ugurbil K, Chen W. Quantitative imaging of energy expenditure in human brain. Neuroimage. 2012;60(4):2107-17)


188: N/A, Neuroinflammation

Short Name: N/A, Neuroinflammation

Key Event Component

Process Object Action
brain inflammation microglial cell pathological
brain inflammation astrocyte pathological

Biological Organization

Level of Biological Organization
Tissue

Organ term

Organ term
brain

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Strong NCBI
human Homo sapiens Moderate NCBI

Neuroinflammation is observed in human, monkey, rat, mouse, and zebrafish, in association with neurodegeneration or following toxicant exposure. Some references (non-exhaustive list) are given below for illustration:

In human: Vennetti et al., 2006

In monkey (Macaca fascicularis): Charleston et al., 1994, 1996

Iin rat: Little et al., 2012; Zurich et al., 2002; Eskes et al., 2002

In mouse: Liu et al., 2012

In zebrafish: Xu et al., 2014.


How this Key Event Works

Neuroinflammation or brain inflammation differs from peripheral inflammation in that the vascular response and the role of peripheral bone marrow-derived cells are less conspicuous. The most easily detectable feature of neuroinflammation is activation of microglial cells and astrocytes. It is evidenced by changes in shape, increased expression of certain antigens, and accumulation and proliferation of the glial cells in affected regions (Aschner, 1998; Graeber & Streit, 1990; Monnet-Tschudi et al, 2007; Streit et al, 1999; Kraft and Harry, 2011; Claycomb et al., 2013). Upon stimulation by cytokines or inflammogens (e.g. from pathogens or from damaged neurons), both glial cell types activate inflammatory signalling pathways, which result in increased expression and/or release of inflammatory mediators such as cytokines, eicosanoids, and metalloproteinases (Dong & Benveniste, 2001), as well as in the production of reactive oxygen (ROS) and nitrogen species (RNS) (Brown & Bal-Price, 2003). Different types of activation states are possible for microglia and astrocytes, resulting in pro-inflammatory or anti-inflammatory signalling and other cellular functions (such as phagocytosis) (Streit et al., 1999; Nakajima and Kohsaka, 2004).

Therefore, neuroinflammation can have both neuroprotective/neuroreparative and neurodegenerative consequences (Carson et al., 2006 ; Monnet-Tschudi et al, 2007; Aguzzi et al., 2013 ; Glass et al., 2010). Under normal physiological conditions, microglial cells scan the nervous system for neuronal integrity (Nimmerjahn et al, 2005) and for invading pathogens (Aloisi, 2001; Kreutzberg, 1995; Kreutzberg, 1996; Rivest, 2009). They are the first type of cell activated (first line of defence), and can subsequently induce astrocyte activation (Falsig, 2008). Two distinct states of microglial activation have been described (Gordon, 2003; Kigerl et al, 2009; Maresz et al, 2008; Mosser & Edwards, 2008; Perego et al; Ponomarev et al, 2005): The M1 state is classically triggered by interferon-gamma and/or other pro-inflammatory cytokines, and this state is characterized by increased expression of integrin alpha M (Itgam) and CD86, as well as the release of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6), and it is mostly associated with neurodegeneration. The M2 state is triggered by IL-4 and IL-13 (Maresz et al, 2008; Perego et al, 2011; Ponomarev et al, 2007) and induces the expression of mannose receptor 1 (MRC1), arginase1 (Arg 1) and Ym1/2; it is involved in repair processes. The activation of astrocytes by microglia-derived cytokines or TLR agonists resembles the microglial M1 state (Falsig 2006). Although classification of the M1/M2 polarization of microglial cells may be considered as a simplification of authentic microglial reaction states (Ransohoff, 2016), a similar polarization of reactive astrocytes has been descibed recently Liddlelow et al., 2017): Interleukin-1 alpha (IL-1Þ), TNF and subcomponent q (C1q) released by activated microglial cells induce A1-reactive astrocytes, which lose the ability to promote neuronal survival, outgrowth, synaptogenesis and phagocytosis and induce the death of neurons and oligodendrocytes.


How it is Measured or Detected

Neuroinflammation, i.e. the activation of glial cells can be measured by quantification of cellular markers (most commonly), or of released mediators (less common). As multiple activation states exist for the two main cell types involved, it is necessary to measure several markers of neuroinflammation:

  1. Microglial activation can be detected based on the increased numbers of labeled microglia per volume element of brain tissue (due to increase of binding sites, proliferation, and immigration of cells) or on morphological changes. A specific microglial marker, used across different species, is CD11b. Alternatively various specific carbohydrate structures can be stained by lectins (e.g. IB4). Beyond that, various well-established antibodies are available to detect microglia in mouse tissue (F4/80), phagocytic microglia in rat tissue (ED1) or more generally microglia across species (Iba1). Transgenic mice are available with fluorescent proteins under the control of the CD11b promoter to easily quantify microglia without the need for specific stains.
  2. The most frequently used astrocyte marker is GFAP (99% of all studies) (Eng et al., 2000). This protein is highly specific for astrocytes in the brain, and antibodies are available for immunocytochemical detection. In neuroinflamatory brain regions, the stain becomes more prominent, due to an upregulation of the protein, a shape change/proliferation of the cells, and/or better accessibility of the antibody. Various histological quantification approaches can be used. Occasionally, alternative astrocytic markers, such as vimentin of the S100beta protein, have been used for staining of astrocytes (Struzynska et al., 2007). Antibodies for complement component 3 (C3), the most characteristic and highly upregulated marker of A1 neurotoxic reactive astrocytes are commercially available.
  3. All immunocytochemical methods can also be applied to cell culture models.
  4. In patients, microglial accumulation can be monitored by PET imaging, using [11C]-PK 11195 as a microglial marker (Banati et al., 2002).
  5. Activation of glial cells can be assessed in tissue or cell culture models also by quantification of sets of activation markers. This can for instance be done by PCR quantification of inflammatory factors, by measurement of the respective mediators, e.g. by ELISA-related immuno-quantification. Such markers include:
  • Pro- and anti-inflammatory cytokine expression (IL-1β; TNF-α, Il-6, IL-4); or expression of immunostimmulatory proteins (e.g. MHC-II)
  • Itgam, CD86 expression as markers of M1 microglial phenotype
  • Arg1, MRC1, as markers of M2 microglial phenotype

(for descriptions of techniques, see also  Falsig 2004; Lund 2006 ; Kuegler 2010; Monnet-Tschudi et al., 2011; Sandström et al., 2014; von Tobel et al.,  2014)

Regulatory example using the KE:Measurement of glial fibrillary acidic protein (GFAP) in brain tissue, whose increase is a marker of astrocyte reactivity, is required by the US EPA in rodent toxicity studies for fuel additives (40 CFR 79.67), but is optional for other toxicant evaluations..


References

Aschner M (1998) Immune and inflammatory responses in the CNS: modulation by astrocytes. ToxicolLett 103: 283-287

Banati, R. B. (2002). "Visualising microglial activation in vivo." Glia 40: 206-217.     

Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355

Charleston JS, Body RL, Bolender RP, Mottet NK, Vahter ME, Burbacher TM. 1996. Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure. NeuroToxicology 17: 127-138.

Charleston JS, Bolender RP, Mottet NK, Body RL, Vahter ME, Burbacher TM. 1994. Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure. ToxicolApplPharmacol 129: 196-206.

Dong Y, Benveniste EN (2001) Immune Function of Astrocytes. Glia 36: 180-190

Eng LF, Ghirnikar RS, Lee YL (2000) Glial Fibrillary Acidic Protein: GFAP-Thirty-One Years (1969-2000). NeurochemRes 25: 1439-1451

Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. 2002. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 37(1): 43-52.

Falsig J, Latta M, Leist M. Defined inflammatory states in astrocyte cultures correlation with susceptibility towards CD95-driven apoptosis. J Neurochem. 2004  Jan;88(1):181-93.

Falsig J, Pörzgen P, Lund S, Schrattenholz A, Leist M. The inflammatory transcriptome of reactive murine astrocytes and implications for their innate immune function. J Neurochem. 2006 Feb;96(3):893-907.

Falsig J, van Beek J, Hermann C, Leist M. Molecular basis for detection of invading pathogens in the brain. J Neurosci Res. 2008 May 15;86(7):1434-47.

Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH (2010). Mechanisms underlying inflammation in neurodegeneration. Cell. 2010 Mar 19;140(6):918-34.

Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35

Graeber MB, Streit WJ (1990) Microglia: immune network in the CNS. Brain Pathol 1: 2-5

Kigerl KA, Gensel JC, Ankeny DP, Alexander JK, Donnelly DJ, Popovich PG (2009) Identification of two distinct macrophage subsets with divergent effects causing either neurotoxicity or regeneration in the injured mouse spinal cord. J Neurosci 29: 13435-13444

Kuegler PB, Zimmer B, Waldmann T, Baudis B, Ilmjärv S, Hescheler J, Gaughwin P, Brundin P, Mundy W, Bal-Price AK, Schrattenholz A, Krause KH, van Thriel C, Rao MS, Kadereit S, Leist M. Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing. ALTEX. 2010;27(1):17-42

Kreutzberg GW (1995) Microglia, the first line of defence in brain pathologies. Arzneimttelforsch 45: 357-360

Kreutzberg GW (1996) Microglia : a sensor for pathological events in the CNS. Trends Neurosci 19: 312-318

Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. 2017. Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638): 481-487.

Little AR, Miller DB, Li S, Kashon ML, O'Callaghan JP. 2012. Trimethyltin-induced neurotoxicity: gene expression pathway analysis, q-RT-PCR and immunoblotting reveal early effects associated with hippocampal damage and gliosis. Neurotoxicol Teratol 34(1): 72-82.

Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.

Lund S, Christensen KV, Hedtjärn M, Mortensen AL, Hagberg H, Falsig J, Hasseldam H, Schrattenholz A, Pörzgen P, Leist M. The dynamics of the LPS triggered inflammatory response of murine microglia under different culture and in vivo conditions. J Neuroimmunol. 2006 Nov;180(1-2):71-87.

Maresz K, Ponomarev ED, Barteneva N, Tan Y, Mann MK, Dittel BN (2008) IL-13 induces the expression of the alternative activation marker Ym1 in a subset of testicular macrophages. J Reprod Immunol 78: 140-148

Monnet-Tschudi F, Zurich MG, Honegger P (2007) Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum Exp Toxicol 26: 339-346

Monnet-Tschudi, F., A. Defaux, et al. (2011). "Methods to assess neuroinflammation." Curr Protoc Toxicol Chapter 12: Unit12 19.         

Mosser DM, Edwards JP (2008) Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8: 958-969

Nakajima K, Kohsaka S. 2004. Microglia: Neuroprotective and neurotrophic cells in the central nervous system. Current Drug Targets-Cardiovasc & Haematol Disorders 4: 65-84.

Perego C, Fumagalli S, De Simoni MG (2011) Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice. J Neuroinflammation 8: 174

Ponomarev ED, Maresz K, Tan Y, Dittel BN (2007) CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J Neurosci 27: 10714-10721

Ponomarev ED, Shriver LP, Maresz K, Dittel BN (2005) Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J Neurosci Res 81: 374-389

Ransohoff RM. 2016. A polarizing question: do M1 and M2 microglia exist? Nat Neurosci 19(8): 987-991.

Sandstrom von Tobel, J., D. Zoia, et al. (2014). "Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures." Toxicol Lett. DOI : 10.1016/j.toxlet.2014.02.001

Struzynska L, Dabrowska-Bouta B, Koza K, Sulkowski G (2007) Inflammation-Like Glial Response in Lead-Exposed Immature Rat Brain. Toxicol Sc 95:156-162

von Tobel, J. S., P. Antinori, et al. (2014). "Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype." Neurotoxicology 44C: 61-70.

Venneti S, Lopresti BJ, Wiley CA. 2006. The peripheral benzodiazepine receptor (Translocator protein 18kDa) in microglia: from pathology to imaging. Prog Neurobiol 80(6): 308-322.

Xu DP, Zhang K, Zhang ZJ, Sun YW, Guo BJ, Wang YQ, et al. 2014. A novel tetramethylpyrazine bis-nitrone (TN-2) protects against 6-hydroxyldopamine-induced neurotoxicity via modulation of the NF-kappaB and the PKCalpha/PI3-K/Akt pathways. Neurochem Int 78: 76-85.

Zurich M-G, Eskes C, Honegger P, Bérode M, Monnet-Tschudi F. 2002. Maturation-dependent neurotoxicity of lead aceate in vitro: Implication of glial reactions. J Neurosc Res 70: 108-116.

 


890: Degeneration of dopaminergic neurons of the nigrostriatal pathway

Short Name: Degeneration of dopaminergic neurons of the nigrostriatal pathway

Key Event Component

Process Object Action
loss of dopaminergic neurons increased

Biological Organization

Level of Biological Organization
Organ

Organ term

Organ term
brain

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Strong NCBI

Parkinson’s disease (PD), one of the best characterized parkinsonian disorder, is a progressive age-related human neurodegenerative disease with a multi-factorial pathogenesis implicating various genetic and environmental factors and is more prevalent in males (Fujita et al. 2014). However, the anatomy and function of the nigrostriatal pathway is conserved across mammalian species (Barron et al. 2010).

Pathological changes, similar to the one described in human PD, have been reproduced with chemicals such as rotenone and MPTP. These chemicals have been tested successfully in multiple mammalian species, including primates, rats and mice. The mouse C57BL/6 strain is the most frequently used strain in the reported experiments. A difference in vulnerability was observed, particularly for rats, depending on the strain and route of administration, possibly indicating the relevance of genetic factors in the development of this pathology. The Lewis strain gives more consistency in terms of sensitivity when compared to the Sprague Dawley. In addition to rodents, the pesticide rotenone has been also studied in Caenorhabditis elegans (C.elegans), Drosophila, Zebrafish and Lymnaea Stagnalis (L.stagnalis) (Johnson et al., 2015).


How this Key Event Works

Degeneration of dopaminergic neurons (DA neurons) within the Substantia Nigra pars compacta (SNpc) i.e. the nigrostriatal pathway, paralleled by the formation of cytoplasmic fibrillar inclusions called Lewy bodies (LB), is regarded as a key event in Parkinson’s disease (PD) and is, in a quantitative manner, directly linked to the occurrence of clinical signs indicative of PD, i..e impaired motor behavior (Shulman et al. 2011; Jellinger et al. 2009, Dickinson 2012, Dauer et al. 2003). The severity of the clinical signs correlates with the degree of nigral cell loss, and the reduced level of dopamine in the striatum. It is estimated that at the onset of clinical signs, 60% of SNpc neurons are lost, corresponding to an 80% depletion of striatal dopamine (Jellinger et al. 2009). PD is clinically and pathologically defined as a progressive disorder: There is a temporally progress, according to a specific pattern, from the brain stem to the nigrostriatal areas and to cortical locations (Braak et al. 2004 and 2009) and there is a temporal increase in the occurrence of LB, of dopamine depletion in the striatum and of loss of DA neurons in the SNpc (Shulman et al. 2012). Indeed, in patients with PD there is a more evident loss of dopamine in striatum compared to SNpc, indicating that striatal dopaminergic nerve terminals are the primary target of the degenerative process in the nigrostriatal pathway and that neuronal loss in SNpc would result as a final outcome (Hornykiewicz et al.1966; Dauer et al. 2003; Bernhaimer et al. 1973; Pavese. et al. 2009). Studies in PD patients and experimental models are also suggesting that progression from striatal terminal to loss of DA neurons occurs through a “dying back” axonopathy pathology and that axonal dysfuction may be an important hallmark in PD (Orimo et al. 2005; Raff et. al. 2002; Kim-Han et al. 2011; O’Malley 2010).

In human brain, the classical Lewy body (LB) is characterized at light microscopy by eosinophilic, spherical, intra-cytoplasmatic inclusion and it stains for α-synuclein and ubiqutin proteins which form the ultrastructural fibrillar core of LB visible at transmission electron microscopy. On autopsy, from individuals affected by PD, accumulation of aggregates positive for α-synuclein protein are also observed within neuronal processes, called Lewy neurites (LN), as well as by neurons showing a more diffuse or granular peri-nuclear pattern (Dickson 2012). Because dopaminergic cells are rich in melanin, their loss is detectable by depigmentation of the midbrain at gross pathology examination (Dickson 2012; Shulman et al. 2010). However, it should be noted that, although LB are recognized as characteristic of PD, they are not found in a minority of clinically defined PD cases (Dauer 2003) and they can also be observed in other diseases (Dickson 2012).

The biological function of the nigrostriatal pathway depends on the intactness of its anatomical structure. Preservation of the striatum terminals and of neuronal cell bodies of DA neurons in the SNpc is a prerequisite for the maintenance of the physiological function (Fujita et al. 2014). The nigrostriatal system is anatomically located in the basal ganglia loop which comprises the motor system structures caudate nucleus, putamen, globus pallidum and subsatantia nigra. The caudate nucleus and the putamen are collectively called striatum (David Robinson in: Neurobiology, Springer edition, 1997). The system plays a unique integrative role in the control of movement as part of a system called the “basal ganglia motor loop”. This anatomical loop includes structures in the thalamus, motor and somatosensory cortex and wide regions of surrounding cortex. Neurons of the SN produce dopamine ( DA) and project to the striatum. They give dopaminergic excitatory (D1 receptors) and inhibitory (D2 receptors) inputs to striatal interneurons (GABAergic). These control thalamic output to the motor cortex. Degeneration within the SNpc leads to a decreased thalamic activation of the motor cortex. (Shulman et al, 2011).

The dopaminergic cells localized in the SNpc synthesize the transmitter substance dopamine (DA) and make extensive contacts within the caudate and putamen (the striatum). These DA neurons have a complex morphology and high energy demand. They are provided with very long and dense arborisations projecting into the striatum where DA is released. This unique morphological characteristics demand a high level of energy to maintain the activity at the synaptic level, to compensate for the risk of depolarization of the poorly myelinated fibres and to support a long distance axonal transport. This puts a tremendous burden on mitochondrial functions (Pissadaki et al. 2013). SNpc neurons are provided with specific calcium channels, the L-type Cav 1.3 which are intended to regulate the autonomous firing as “pacemaker”. The high demand of calcium buffering arising from this is handled by the endoplasmic reticulum (ER) and by the mitochondria. This is a function specific for SNpc DA neurons, as the dopaminergic neurons belonging to the ventral tegmental area (VTA) are using Na+ channels as a pacemaker. Additional peculiarities of the neurons of the nigrostriatal pathway are the high number of synapses and the higher probability of these neurons to accumulate misfolded proteins, including α-synuclein. Furthermore, the nigrostriatal pathway metabolism of DA is known to induce oxidative and nitrative stress (Fujita et al.2014; Asanuma et al. 2003; Cantuti-Castelvetri et al. 2003; Pissadaki et al. 2013) making DA neurons particularly sensitive to oxidative stress (Lotharius and Brundin, 2002). DA neurons in SNpc also have a relatively low mitochondria mass which may contribute to the vulnerability of these neurons (Liang et al. 2007). In addition, increased levels of iron have been observed in SN of PD patients (Gotz et al. 2004) and the high content of iron in dopamine neurons has been reported to trigger oxidative/nitrosative stress and subsequent neurodegeneration (Ayton and Lei 2014; Benshachar et al. 1991). As a consequence, these neurons are particularly sensitive to various stressors that can contribute to their vulnerability and preferential loss (Fujita et al. 2014).


How it is Measured or Detected

The presence of DA cells in the SNpc and DA terminals in the striatum can be visualized using different phenotypic histological markers. Changes can be captured by measurement of markers specific for dopaminergic neurons such as tyrosine hydroxylase (TH),dopamine transporter (DAT) and vesicular monoamine transporter type 2 (VMAT2). Degenerating and/or degenerated neurons can be detected by the silver stains and the Fluoro Jade stains. Depending on the chemical stressor, study design and animal species/strain, the methodology required for the analysis of neuropathology and function may vary as it is recognized that different endpoints can be quantitatively and/or qualitatively affected differently. However, when testing potential neurotoxic chemicals, it is important to assess the integrity and function of the nigra-striatal pathway. Some well-developed and accepted techniques that are commonly used for these purposes are described (Tieu, 2011):  Quantification of dopaminergic neurons in the substantia nigra par compacta, quantification of dopaminergic terminals in the striatum, quantification of dopamine content in the striatum and detection of Lewy Body-like aggregation.

• The silver degeneration stain is a method to trace degeneration of axons. By this matter, products from disintegrated cells are visualized (Switzer R., 2000; Betarbet et al. 2000). The mechanism by which the siver degeneration stain labels degenerating neurons is unknown.

• Fluoro Jade stain is a fluorochrome derived from fluorescein used in neuroscience disciplines to label degenerating neurons. It is an alternative technique to traditional methods for labeling degenerating neurons such as silver degeneration staining. Fluoro-Jade may be preferred to other degeneration stains due to the simplicity of staining procedures, which are a common drawbacks of conventional stains. However, the mechanism by which fluoro-jade labels degenerating neurons is unknown (Betarbet et al. 2000, Schmued et al. 1997).

• Detection of TH, the enzyme responsible for catalyzing the conversion of the amino acid L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), a precursor for dopamine. Detection of TH can be done either by immunocytochemistry followed by cell counting (quantitative evaluation) or by western blot followed by densitometry analysis (Betarbet et al. 2000, Lee 1987, Fetissov 1999).

• Counting of cells, immunostained for TH, or counting of nuclei by e.g. with Nissel’s , DAPI (Kapuscinski, 1995) or Hoechst stain (Latt et al. 1976) should be done following standard morphometric protocols. However, inclusion of stereological cell counts to assess neurodegeneration is representing the most sensitive method to confirm quantitatively specific morphological changes (Dauer 2008, Brooks 1992, Thiruchelvam 2000a and 2000b).

• Quantification of dopaminergic neurons in SNpc: the average number of DA neurons in adult mouse SN is approximately 8.000 to 14.000, depending on strain (Zaborszky and Vadasz 2001). Their distribution is not homogeneous with difference in density between the caudal and rostral part of the SN. The gold standard for counting neurons is then to use an unbiased stereological protocol for cell counting with an optical dissector system (Tieu et al. 2011). This requires a computerized stereology software. The count should include TH+ neurons as well the total count of neurons using a non-specific cell stain (e.g. Nisell’s, Fox3).

• Quantification of dopaminergic terminals in the striatum: the density of dopaminergic terminals is not homogeneous in the striatum, increasing from the rostral to the caudal part and representative regions of the striatum should be assessed. This can be done by digitalization of the fibres and quantification by optical density or quantification of the fiber density identifyied by by TH+ immunoreactivity (Tieu et al. 2011; Fernagut et al. 2007). Alternatively, striatal tissue can be isolated for immunobloting of TH or DAT.

• DA transporters (DAT) and vesicular monoamine transporter type 2 (VMAT2) can be visualized and quantified using immunocytochemistry (single cell levels) or western blot followed by densitometry analysis, to quantify the changes in their expression. (Hirata et al. 2007; Fornai et al. 2003; Tong et al. 2011; Ciliax et al. 1995). • DA, DOPAC (DA metabolite) and homovanillic acid (HVA) content in the striatum can be quantified through several methodologies such as capillary electrophoresis, spectrofluorimetry and high performance liquid chromatography (HPLC). The commonly used detectors for chromatography include MS, UV, optical fiber detector, electrochemical detector and fluorescence detector (Zhao et al. 2011, Fornai et al. 2005, Magnusson et al. 1980). • Indentification of LB in standard histological sections stained with haematoxylin and eosin, they are characterized by the presence of pale eosinophilic vacuoles (Betarbet et al. 2000 and 2006; Pappolla et al. 1988; Dale et al. 1992).

• Immuno staining for α-synuclein and ubiquitin to identify and quantify Lewy bodies presence. In vivo, α-synuclein and ubiqutin can be evaluated in the fixed tissue and quantified for fluorescence intensity (Betarbet et al. 2000 and 2006; Forno et al. 1996, Tiller-Borcich 1988; Galloway et al. 199;, Kuzuhara et al. 1988; Kuusisto eyt al. 2003).

• Imaging techniques: 18-fluoro-dopa positron emission tomography (PET) quantification of various dopamine presynaptic markers (e.g. dopamine transporter DAT, vescicular monoamine transporter type 2 VAT2) identified by single photon emission tomography (SPECT). They permit to visualize the loss of nigrostriatal DA neurons in patients(Shapira et al. 2013) .


References

Ayton S and Lei P. 2014. Nigral Iron Elevation Is an Invariable Feature of Parkinson’s Disease and Is a Sufficient Cause of Neurodegeneration. Biomed Research Inernational. (2014) 1-9.

Asanuma M, Miyazaki I, Ogawa N. (3003). Dopamine or L-DOPA-induced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson’s disease. Neurotox Res (5) 165-76.

Barron AB, Søvik E, Cornish JL (2010). The roles of dopamine and related compounds in reward-seeking behavior across animal phyla". Frontiers in Behavioral Neuroscience 4: 163.

Ben-Shachar D, Youdim MBH. 1991.Intranigral Iron Injection Induces Behavioral and Biochemical “Parkinsonism” in Rat. Journal of Neurochemistry . 57(6), 2133–5.

Bernhaimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. 1973. Brain dopamine and the syndrome of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol.Sci.(20) 415-5.

Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. 2000. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nature neuroscience. 3 (12) 1301-6.

Betarbet R, Canet-Aviles RM, Sherer TB, Mastroberardino PG,Mc Lendon C, Kim JH, Lund S, Na HM, taylor G, Bence NF, kopito R, seo BB, Yagi T, Yagi A, Klinfelter G, Cookson MR, Greenmyre JT. 2006. Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the pesticide rotenone on DJ-1, α-synuclein, and the ubiquitin-proteasome system. Neurobiology disease. (22) 404-20.

Braak H, Ghebremedhin E, Rub U, Bratzke H, Del Tredici K. 2004.Stages in the development of Parkinson’s disease-related pathology. CellTissue Res. (318) 121-4.

Braak H, Del Tredici K. 2009. Neuroanatomy and pathology of sporadic Parkinson’s disease. Adv Anat Embryol Cell Biol (201) 1-119. Brooks AI, Chadwick CA, Gelbard HA, Cory-Slechta DA, Federoff HJ. 1999. Paraquat elicited neurobehavioral syndrome caused by dopaminergic neuron loss. Brain Research (823) 1-10.

Canuti-Silvestri I, Shukitt-Hale B, Joseph JA. (2003). Dopamine neurotoxicity: age dependent behavioural and histological effects. Neurobiol aging (24) 697-6.

Dale GE, Probst A, Luthert P, Martin J, Anderton BH, Leigh PN. 1992.Relationships between Lewy bodies and pale bodies in Parkinson's disease. Acta Neuropathol. 83(5):525-9.

Dauer W, Przerdborski S. 2003. Parkinson’sdisease: Mechanisms and Models.Neuron. 39, 889-9.

Dickinson D. 2012. Parkinson’s disease and parkinsonism: Neuropathology.Cold Spring Harb Perspect Med. 2:a009258.

Efremova L, Scildknecht S, Adam M, pape R, Gutbier S, Hanf B, Burkle A, Leist M. 2015. Prevention of the degeneration of dopaminergic neurons in an astrocyte co-culture system allowing endogenous drug metabolism. British Journal of Pharmacology; 172; 4119-32.

Fetissov SO, Marsais F. 1998. Combination of immunohistochemical and in situ hybridization methods to reveal tyrosine hydroxylase and oxytocin and vasopressin mRNA in magnocellular neurons of obese Zucker rats. Braian research Protocols. 4, 36-3.

Fornai F, Lenzi P, Gesi M, Ferrucci M, Lazzeri G, Busceti C, Ruffoli R, Soldani P, Ruggieri S, Alessandri’ MG, Paparelli A. 2003. Fine structure and mechanisms underlying nigrostriatal inclusions and cell death after proteasome inhibition. The journal of neuroscience. 23 (26) 8955-6.

Forno LS, DeLanney LE, Irwin I, Langston JW. (1992). Electron microscopy of lewy bodies in the amigdala-parahippocampal region. Comparison eith inclusion bodies in the MPTP-treated squirrel monkey. Adv.neurol.(69) 217-8.

Forno LS. (1969). Concentric hyaline intraneuronal inclusion of Lewy type in brains of elderly person (50 incident cases): Relationship to parkinsonism. J Am Geriatr Soc. (6) 557-5.

Fujita KA, Ostaszewski M, Matsuoka Y, Ghosh S, Glaab E, Trefois C, Crespo I, Perumal TM, Jurkowski W, Antony PM, Diederich N, Buttini M, Kodama A, Satagopam VP, Eifes S, Del Sol A, Schneider R, Kitano H, Balling R. Integrating pathways of Parkinson's disease in a molecular interaction map. Mol Neurobiol. 2014 Feb;49(1):88-102.

Galloway PG, Mulvihill P, perry G. 1992. Filaments of Lewy bodies contain insoluble cytoskeletal elements. AmJ Pathol. (140) 809-2. Gotz ME, Double K, Gerlach M, Youdim MBH, Riederer P. 2004. The relevance of iron in the pathogenesis of Parkinson’s disease. Ann.N.Y. Acad. Sci. (1012) 193-8.

Hirata Y, suzuno S, tsuruta T, OH-hashi K, Kiuchi K. 2008. The role of dopamine transporter in selective toxicity of manganese and rotenone. Toxicology. (244). 249-6.

Hornykiewicz O, Kish SJ. 1987. Biochemical pathophysiology of parkinson’s disease. In Parkinson’s Disease. M Yahr and K.J. Bergmann, eds (New.York: Raven Press) 19-34.

Kapuscinski J. 1995. Biotechnic and Histochemistry, 70(5), 220-3.

Kim-Han JS, Dorsey JA, O’Malley KL. 2011. The parkinsonian mimetic MPP+, specifically impairs mitochondrial transport in dopamine axons. The Journal of Neuroscience. 31(19) 7212-1.

Kuusisto E, Parkkinen L, Alafuzoff I. 2003. Morphogenesis of Lewy bodies: dissimilar incorporation of α-synuclein, ubiquitin and 62. J Neuropathol Exp Neurol. (62)1241-3.

Kuzuhara S, mori H, Izumiyama N, Yoshimura M, Ihara Y. 1988. Lewy bodues are ubiquinated. A light and electron microscopic immunocytochemical study. Acta Neuropathol. (75) 345-3.

Jellinger KA. 2009. A critical evaluation of current staging of α-synuclein pathology in Lewy body disorders. Biochemica and Biophysica Acta. 730-0.

Johnson ME, Bobrovskaya L. 2015. An update on the rotenone models of parkinson’s disease: Their ability to reproduce features of clinical disease and model gene-environment interactions. 946). 101-16.

Latt, SA; Stetten, G; Juergens, LA; Willard, HF; Scher, CD .1975. Recent developments in the detection of deoxyribonucleic acid synthesis by 33258 Hoechst fluorescence. The journal of histochemistry and cytochemistry : Official Journal of the Histochemistry Society 23 (7): 493–5.

Liang CL; wang TT; Luby-Phelps K, German DC. 2007. Mitochondria mass is low in mouse substantia nigra dopamine neurons: Implication for Parkinson’s disease. Experimental Neurology (203) 370-80.

Lotharius, J., Brundin, P., 2002. Pathogenesis of Parkinson’s disease: dopamine, vesicles and α-synuclein. Nat. Rev., Neurosci. 3, 932– 2.

Magnusson O., Nilsson LB, Westerlund D. 1980). Simultaneous determination of dopamine, DOPAC, and homovanilic acid. Direct injection of supernatants from the brain tissue homogenates in a liquid chromatography-electrochemical detection system. J.Chromatogr, 221, 237-47.

Minnema DJ, Travis KZ, Breckenridge CB, Sturgess NC, Butt M, Wolf JC, Zadory D, Beck MJ, Mathews JM, tisdel MO, Cook AR, Botham PA, Smith LL. 2014. Dietary administration of paraquat for 13 weeks does not result in a loss of dopaminergic neurons in the substantia nigra of C57BL/6J mice. (68). 250-8.

O’Malley KL. 2010. The role of axonopathy in Parkinson’s disease. 2010. Experimental Neurobiology. (19). 115-19.

Orimo S, Amino T, Itoh Y, Takahashi A, Kojo T, Uchihara T, Tsuchiya K, Mori F, Wakabayashi K, Takahashi h. 2005. Cardiac sympathetic denervation precedes neuronal loss in the sympathetic ganglia in Lewy body disease. Acta Neuropathol (109) 583-8.

Ossowska K., Wardas J, Smialowska M, Kuter K, Lenda T, Wieronska JM, Zieba B, Nowak P, Dabrowska J, Bortel A, Kwiecinski A, Wolfarth S. 2005. A slowly developing dysfunction of dopaminergiv nigrostriatal neurons induced by long-term paraquat administration in rats: an animal model of preclinical stages of Parkinso’s disease ? European Journal of Neurosciences. (22) 1294-04.

Pappolla MA, Shank DL, Alzofon J, Dudley AW.1988. Colloid (hyaline) inclusion bodies in the central nervous system: their presence in the substantia nigra is diagnostic of Parkinson's disease. Hum. Pathol.19(1):27-1.

Pavese N, Brooks DJ. 2009. Imaging neurodegeneration in parkinson’s disease.. Biochimica and Biophysica Acta (1972) 722-9. Pissadaki EK, Bolam JP. 2013. The energy cost of action potential propagation in dopamine neurons: clues to susceptibility in Parkinson’s disease. (7) 1-17.

Raff MC, Whitemore AV, Finn JT. 2002. Axonal self-destruction and neurodegeneration. Science (296) 868-1.

Robinson D.1997. Neurobiology. Published by Springer-Verlag. 245-247.

Schapira AHV. 2013. Recent developments in biomarkers in Parkinson disease. Neurology. 26 (4) 395-0.

Shmued LC, Albertson C, Sikker W. 1997. Fluoro-jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain research. 751, 37-6.

Shulman JM, DeJager PL, Feany MB. 2011. Parkinson’s disease: Genetics and Pathogenesis. Annu.Rev.Pathol.Mech.Dis. 6:193-2.

Switzer RC. 2000. Application of silver degeneration stains for neurotoxicity testing. Toxicologic pathology. 28(1) 70-83.

Thiruchelvam M, Brockel BJ, Richfield EK, Bags RB, Cory-Slechta DA. (2000a). potential and preferential effects of combined paraquat and maneb on nigrostriatal dopamine system: environmental risk factor for Parkinson’s disease? Brian research (873) 225-4.

Thiruchelvam M, Richfield EK, Bags RB, tank AW, Cory-Slechta DA. (2000b). The nigrostriatal dopaminergic system as a potential target of repeated exposuresto combined paraquat and maneb: implication for Parkinson’s disease. J.neurosci. (20). 9207-4.

Tieu K. 2011. A guide to neurotoxic animal models of Parkinson's disease.Cold Spring Harb Perspect Med. 2011 Sep;1(1):a009316. doi: 10.1101/cshperspect.a009316.

Tong J, Boileau I, Furukawa Y, Chang LJ, Wilson AA, Houle S, Kish S. 2011. Distribution of vescicular monoamine transporter 2 protein in human brain: implications for brain imaging studies. Journal of cerebral blood flow & metabolism. (31) 20165-5.

Tiller-Borcich JK, Forno LS.(1988) Parkinson’s disease and dementia with neuronal inclusions in the cerebral cortex: lewy bodies or Pick bodies. J Neuropathol Exp. Neurol. (5) 526-5.

Zhao Hong-Xia, Mu Hui, Bai Yan-Hong, Hu Yu, Hu Ying-Mei.2011. A rapid method for the determination in porcine muscle by pre-column derivatization and HPLC with fluorescin detection. Journal of pharmaceutical analysis. 1(3); 208-12.


Adverse Outcomes

Title Short name
Parkinsonian motor deficits Parkinsonian motor deficits

896: Parkinsonian motor deficits

Short Name: Parkinsonian motor deficits

Key Event Component

Process Object Action
Parkinsonian disorders occurrence

AOPs Including This Key Event


Biological Organization

Level of Biological Organization
Individual

Evidence Supporting Applicability of this Event


Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
mouse Mus musculus Moderate NCBI

Parkinson’s disease (PD) is a progressive age-related human neurodegenerative disease with a multi-factorial pathogenesis implicating various genetic and environmental factors and is more prevalent in males (Fujita et al. 2014). There are no sex and species restriction for the application of this AO; however aged animals showed to be more suscieptible to parkinsonian motor deficits induced by chemical stressors (Rose et al.1993, Irwin et al. 1993, Ovadia et al. 1995)


How this Key Event Works

A large number of neurological disorders are characterized by a clinical syndrome with motor symptoms of bradykinesia, tremor, rigidity and postural instability. As these clinical features are common to multiple disorders, the clinical syndrome is referred as “parkinsonism” and when parkinsonism is representing the prevalent part of the syndrome, these are referred as “parkinsonian disorders”. Parkinson’s Disease (PD) is one of parkinsonian disorders and can have an idiopathic, genetic or toxic (i.e. MPTP induced parkinsonism) cause (Dickson 2012). All these disorders include a deregulation of the extrapyramidal system.

The pyramidal motor system comprises bundles of neurons originating in the motor centers of the cerebral cortex to terminate in the brainstem or in the spinal cord where they are responsible for voluntary control of motor functions (Brooks 1971). The extrapyramidal system, which is the anatomical organization of the AO, is the part of the motor system primarily involved in the control and regulation of involuntary motor control, and in fine tuning (Barnes et al. 1983). Especially the initiation and maintenance of complex movement patterns or of neuronal regulatory pathways involved in postural control of the body are regulated by the nigrostriatal system that is affected in parkinsonian states. The CNS input is modulated by extrapyramidal circuits before the execution of complex motor movements. The modulated information from the basal ganglia is looped back through the thalamus to the cortex, from where final motor signals are sent via the pyramidal system; i.e. the basal ganglia system is not involved in the control of motor neurons and striatal muscles, but it modulates the signals from the cortex to these systems. Thus, an impaired input of dopamine into the striatum leads to an impairment of this modulation loop, and a disturbance of basal ganglia feedback to the thalamus and cortex. This ultimately manifests in key parkinsonian symptoms such as tremor, rigidity, or bradykinesia (Bernheimer et al. 1973). These conditions can be generated experimentally by dopamine depletion with reserpine, by inhibition of dopamine receptors, by mechanical or chemical ablation of nigrostriatal dopamine neurons (cut of the median forebrain bundle or injection of the toxicant 6-OH-dopamine) or the application of toxicants that leading to a relatively selective death of dopaminergic neurons in the substantia nigra (e.g. MPTP) and therefore a reduction of dopamine in the striatum (Kolata et al. 1983).

The basal ganglia include the ventral striatum, the neostriatum composed of the putamen and the caudate nucleus, the globus pallidus pars externa (GPe), the globus pallidus pars interna (GPi), the subthalamic nucleus (STN), the substantia nigra pars reticulata (SNpr) and the substantia nigra pars compacta (SNpc) (Obeso et al.2008). The main input sites into basal ganglia are the striatum and the STN where cortical (glutamatergic) innervations terminate in a topographically organized manner that largely reflects the organization in the cortex (Fallon et al. 1978; Takada et al. 1998). Both the GPi and the SNpr represent the main output nuclei projecting into the thalamus (Parent et al.1999; Alexander et al. 1990). The connection between input and output nuclei is functionally organized into a “direct” and an “indirect” pathway (Silverdale et al. 2003). These two pathways in parallel regulate the activity of the basal ganglia output neurons of the GPi and STN and are modulated by dopamine in the striatum. The dopaminergic terminals in teh striatum originate from dopaminergic projections from the SNpc. Striatal dopamine modulates the activity of inhibitory GABAergic medium spiny neurons that make up 90% of all neurons in the striatum (Smith et al. 1994). Medium spiny neurons that preferentially express the D1 dopamine receptor are involved in the direct pathway and directly project into the two main output nuclei (GPi and SNpr). Activation of the D1 medium spiny neuronal direct pathway results in a reduction of the inhibitory basal ganglia output (GPi and SNpr) leading to a dis-inhibition of thalamic target neurons (Bolam et al.2000). These events ultimately lead to an elevated activity in the respective cortical neurons, i.e. D1 signalling in the striatum leads to an increase in motor activity.

Medium spiny neurons predominantly expressing the D2 dopamine receptor mostly project to the GPe (Gerfen et al. 1990). Activation of D2 expressing neurons leads to an inhibition of their activity. D2 neurons of the indirect pathway connect the striatum with GPi/SNpr via synaptic connections in the GPe and the STN. Activating neurons originating in the STN project into the GPi/SNpr are glutamatergic. From the STN, activating glutamatergic neuronal projections into the GPi/SNpr lead to a basal, low activation. Activation of the indirect pathway by striatal dopamine from the substantia nigra hence leads to a low basal inhibitory GABAergic output into thalamic structures, and thus allows a strong motor cortex activation of the thalamus.

AOpicture.jpg

Functional anatomy of basal ganglia. A) Normal conditions. Striatal (STR) dopamine mainly originates from projections originating in the substantia nigra pars compacta (SNc). The STR is mainly composed of inhibitory GABAergic medium spiny neurons (MSN). MSN involved in the direct pathway directly project to the globus pallidus pars interna (GPi) and the sunstantia nigra pars reticulata (SNpr) leading to a basal inhibition of these output nuclei. MSN involved in the indirect pathway send inhibitory projections to the globus pallidus pars externa (GPe). Their activity is dampened by dopamine binding to D2 receptor expressing MSN in the striatum. B) Lack of striatal dopamine. Under conditions of a lack of striatal dopamine, inhibitory GABAergic neurons, originating in the striatum, receive less activation, resulting in a declined inhibition of GPi and SNpr inhibitory output. In the indirect pathway, the lack of dopamine causes a lack of its inhibitory influence on inhibitory GABAergic projections into the GPe. This accelerated inhibition of the GPe results in a decline in its inhibitory output into the STN. The decline in STN inhibition allows its overactivation, resulting in an excessive activation of stimulatory glutamatergic projections into the GPi and SNpr. (according to Silverdale 2003).

Parkinson’s Disease is characterized by a decline in striatal dopamine input from the substantia nigra pars compacta (Smith t al. 1994). Under normal conditions, ganglial output via GPi/SNpr nuclei causes a moderate inhibitory influence on cortical and brainstem motor neurons. A reduction in striatal dopamine leads to an underactivation of D1 receptor-expressing medium spiny neurons of the direct pathway. This insufficient activation of the inhibitory GABAergic medium spiny neurons results in a reduction of its normal inhibitory influence on GPi and SNpr output nuclei. As a consequence, dopamine depletion leads to the overactivation of the inhibitory GABAergic GPi/SNpr output via the direct pathway (Mitchell et al.1989).

In the indirect pathway, the reduced activation of D2 receptors expressing neurons leads to an overactivation of inhibitory output nuclei projecting into the GPe. The resulting inhibitory output of the GPe is hence reduced, thus leading to a declined inhibition of the STN. Overactivation of the stimulatory glutamatergic projections originating in the STN leads to the hyperactivation of the output GPi/SNpr nuclei. As a consequence of striatal dopamine depletion, the direct pathway becomes underactivated and the indirect pathway becomes overactivated. This leads to an overactivation of the basal ganglia output nuclei. Due to their inhibitory influence on thalamocortical motor centers, the resulting reduced cortical activation leads to the prominent impairment of motor functions in parkinsonian states (Silverdale et al. 2003).

The model of direct and indirect pathways linking striatal dopamine content with the basal ganglia output nuclei has been criticized in recent years as it ignores the influence of extrastriatal dopamine (Smith et al. 2000), or the fact that some medium spiny neurons express dopamine receptors of both the D1 and of the D2 type (Surmeier et al. 1996). Principal validity of the model and the central role of striatal dopamine was e.g. demonstrated by L-DOPA-mediated supplementation of striatal dopamine content in unprimed PD patients that causes a partial reduction in the overactivation of GPi/SNpr output (Yuan et al. 2010; Heimer et al. 2006). As an alternative way for symptomatic treatment of parkinsonian conditions, deep brain stimulation of either the STN or the GPi was shown to relieve from parkinsonian motor features (Mazzone 2003, Odekerken 2013).


How it is Measured or Detected

For the analysis of striatal dopamine content and its correlation with motor control, both biochemical analysis methods on the cellular and tissue level as well as behavioral tests are required. Available test models are mice and rats on the one hand and non-human primates and humans on the other. Motor impairment features associated with parkinsonian states in man serve as reference standard. Monkey models have the advantage to largely reflect complex motor impairment patterns observed in humans which are rather difficult to assess in rodents. Rodent models in contrast are cost-efficient and allow both biochemical analysis that require major invasive methods as well as basic behavioral tests. Due to the limitations in the assessment of moderate motor impairment in rodents and the well-established correlation between striatal dopamine content and impaired motor output, analysis of striatal dopamine is often applied as surrogate readout for the assessment of motor deficits.

Detection of striatal dopamine (total or extracellular).

The standard method used in the majority of experimental work is the determination of total contents of dopamine and its two degradation metabolites HVA and DOPAC. For this purpose, the striatum is quickly removed from experimental animals, homogenized in a suitable acidic buffer, and the dopamine (metabolites) determined by HPLC with electrochemical detector or by HPLC-MS. For live in vivo detection of extracellular dopamine levels, a microdialysis probe is inserted into the striatum. Microdialysis can be performed in anesthetized animals or freely moving animals; basal dopamine levels or stimulated levels (amphetamine, KCl) can be recorded. Dopamine and its metabolites are detected in the dialysate either by HPLC or by HPLC-mass spectrometry analysis (Saraswat 1981, Cui 2009, Gonzalez 2011).

Detection of dopamine neuron terminals in the striatum.

As alternative to the detection of striatal dopamine that is to a large extent limited to live detection setups due to its instability in tissues, the number of remaining dopamine neurons in the substantia nigra pars compacta was suggested as alternative readout (Burns 1983). It allows the analysis of ex vivo samples without the limitations associated with the instability and reactivity of extravesicular dopamine. Although the number of surviving dopamine neurons in the SNpc in PD or in complex-I inhibitor challenged test animals is a valuable parameter on its own, it was discovered that the number of DA neurons in the SNpc not necessarily correlates with the amount of dopamine released in the striatum. Tyrosine hydroxylase (TH) was regularly stained as marker for DA neurons, however it was observed that TH expression was very variable following MPTP intoxication in the absence of cell death and therefore has only limited suitability for the assessment of DA neuronal numbers (Aznavour 2012). Second, many DA neurites and terminals displayed damage or degradation in the absence of death of the corresponding neuronal cell (Ling 2015). Hence, even in the presence of viable DA neurons in the SNpc, their corresponding terminals could no longer be able to release dopamine into the striatum. Staining of DA neuronal terminals in the striatum is therefore used as a more reliable indirect marker for striatal dopamine content. For the analysis of nigrostriatal terminals, the dopamine transporter (DAT) is visualized either by antibody-mediated staining in tissue slices or by the application of radioactively labeled DAT ligands that allow their application both in vivo and in ex vivo samples (Morris 1996).

Behavioral tests: Rodent models.

Rotation: the rotation model of Ungerstedt et al (Ungerstedt 1970) is based on the unilateral lesion of the nigrostriatal dopamine neuron system either in rodents or in non-human primates. The lesion can be produced either surgivally, or by stereotaxic infusion of e.g. 6-OHDA into the nigrostriatal system of one hemisphere, or by infusion of MPTP through one carotid (single sided). After the lesion, animals are left to recover, then the dopamine system is stimulated by injection of amphetamine. The asymmetry of remaining dopamine neurons (only on one side) triggers spontaneous asymmetric motor behaviour, i.e. rotations of the animals. Each full turn of an animal is recorded, the respective numbers of left- and right turns are plotted versus time, respectively. In the standard rotation model, monkeys become hypokinetic in the limbs on the contralateral side of the brain hemisphere treated. Rats preferentially rotate towards the side of the lesion upon treatment with drugs that trigger activation of the remaining dopamine neurons.

Rotarod: assessment of motor coordination. The animals are placed on a rotating rod that is subjected to linear acceleration. The latency to fall from the rod is detected (Jones 1968). Hang test: Detection of neuromuscular strength. Mice are placed on a horizontal grid. When the animals grabbed the grid with their fore- and hindpaws, the grid is inverted with the animal hanging upside down. In a typical setup, mice are required to remain on the grid for at least 30 s (Tillerson 2002).

Forepaw Stride length during walking. Ink is applied to the forepaws and the mice walk across a blank sheet of paper. Training of the animals to walk across the white paper in a straight line without stopping is performed before the respective treatment. The distance between single steps on each side are measured (Klapdor 1997).

Grid test: Mice hang upside down for 30 s on the grid that is also used for the Hang test and are recorded on video for closer analysis. With this method, the average forepaw distance is measured by assessing the distance covered, divided by the number of successful forepaw steps. In the course of the analysis, the number of unsuccessful forepaw steps are detected and displayed as percentage of the total number of steps performed (Crawley 1999).

Akinesia: the animal is placed on a flat surface and the latency until it has moved all of its four limbs is assessed. Open field test: Infrared beams detect the animals activity for the determination of parameters such as the time spent locomoting, the distance travelled, or the number of rearings.

Pole test: the animal is placed on a gauze-taped pole with the head upwards below the top of the pole. Two parameters are detected: 1) time until animals turn by 180°; 2) time until the animals reach the floor.

Non-invasive imaging of DA neuron terminals

Positron emission tomography (PET): Based on its appropriate half life time of ca. 2 h for clinical investigations, fluorine-18 labeled L-[18F]-fluorodopa is routinely used in trace amounts for intravenous administration. Striatal uptake of L-[18F]-fluorodopa is followed by applying positron emission tomography (PET) (Leenders 1986).

Single photon emission computed tomography (SPECT): monitoring of dopamine transporter (DAT). Iodine-123-β-CIT is used as a sensitive ligand for dopamine and serotonin transporters and was applied in monkeys and humans (Winogrodzka 2003).

Human neurological tests

A recent systematic review and evaluation of currently used rating scales for the assessment of motor impairment and disability in PD patients identified the 1) Columbia University rating scale, 2) the Northwestern University Disability Scale, and 3) the Unified Parkinsons Disease rating scale as the most evaluated and reliable scales available (Ramaker 2002). All scales evaluate several parameters, some of which are not motor related. Thus, only subscales are useful for readout of motor symptoms (e.g. 13 of the 42 UPDRS parameters). Of these, not all are equally dependent on nigrostriatal dopamine. Examination needs to be done by a trained neurologist.


Regulatory Examples Using This Adverse Outcome

Neurotoxic effects shall be carefully addressed and reported in routine required regulatory toxicological studies (acute toxicity studies,short-term toxicity studies, long term toxicity and carcinogenicity studies and reproductive toxicity studies). Regarding neurotoxicity in rodents, inclusion of neurotoxicity investigations in routine toxicology studies shall also be considered. For pesticide active substances the circumstances in which neurotoxicity studies should be performed are listed in Regulation (EU) No 283/2013:

Specific neurotoxicity studies in rodents shall be performed in case of one those following conditions:

  • there is indication of neurotoxicity in routine toxicity studies carried out with the active substance;
  • the active substance is a structurally related to known neurotoxic compound;
  • the active substance has a neurotoxic mode of pesticidal action.

As a result, specific neurotoxicity studies are not routinely required for all pesticide active substances. Specific neurotoxicity testing becomes obligatory only if neurotoxicity has been observed during histopathological evaluation or in case of structural analogy with a known neurotoxic compound. Motor activity should be measured once in short-term repeated dose toxicity studies (OECD 407, 408 and 422) and several times in specific neurotoxicity studies (OECD 424, OECD 426 and cohort 2 of OECD 443). However, this is not a requirement in chronic toxicity studies unless neurotoxic effects have been reported in the shorter studies. The same test (measures horizontal and/or vertical movements in a test chamber) is implemented in both routine studies and neurotoxicity studies. Coordination and balance are evaluated by rotation or rotarod or pole tests, and gait abnormalities by forepaw stride length test. Those tests are not required by any repeated dose toxicity OECD guidelines and they can be optionally incorporated in the design of neurotoxicity studies OECD 424 and OECD 426.

Although motor deficits is the AO in this AOP, degeneration of DA neurons, is also considered an adverse effect in the regulatory framework, even in the absence of clear clinical symptoms or motor deficits. Morphological assessment of brain structures is a standard requirement in the regulatory toxicological studies supporting the risk assessment of chemical substances and it is a regulatory expectation that the anatomical structures belonging to the nigrostriatal pathway would be included and evaluated as part of the standard evaluation of the brain. Treatment related neuronal degeneration, when occurring as a consequence of the treatment, is generally dose-dependent in incidence and severity. However, if not accompanied by clinical signs or behavioral changes indicative of central nervous system pathology, minimal loss of DA neurons would likely remain undetected in the standard histological evaluation, due to the presence of non DA neurons or as a consequence of the subjectivity of non-quantifiable analysis, unless specific markers are used. As multiple forms of perturbation can affect the neurons, some changes are potentially still reversible (e.g. loss of TH or DA) and irreversibility should be confirmed as part of the assessment. It is then important to apply a sensitive and appropriate method (Switzer 2000) and evaluation of the phenotypic markers in the striatum and in the SNpc should be always performed as a minimum standard (Minnema et al 2014) when investigating perturbation of the nigrostriatal pathway. It should additionally consider that rat is likely to be a poor model to capture this kind of hazard, as demonstrated by the poor sensitivity of rat to MPTP or related compounds and this should be taken into account for the design and interpretation of the studies.

Dissimilarities of chemical induced animal models to human disease are also important and should be carefully weighted when considering the duration and schedule of the study/treatment. Differently from the human disease, with the MPTP animal model, the damage occurs rapidly, is hardly progressive, is little age-dependent and formation of Lewy bodies is sometime not occurring (Efremova et al. 2015). Therefore, for different animals models, the standard 90 days toxicity study could not match with the chronic and progressive characteristics of the human disease and compensatory changes influencing DA metabolism and turnover and protein catabolism can occur during the treatment period with an impact on the time of onset of the lesion (Ossowska et al. 2005).


References

Alexander GE, Crutcher MD. Functional architecture of basal ganglia circuits: neural substrates of parallel processing. Trends Neurosci. 1990 Jul;13(7):266-71.

Aznavour N, Cendres-Bozzi C, Lemoine L, Buda C, Sastre JP, Mincheva Z, Zimmer L, Lin JS. MPTP animal model of Parkinsonism: dopamine cell death or only tyrosine hydroxylase impairment? A study using PET imaging, autoradiography, and immunohistochemistry in the cat. CNS Neurosci Ther. 2012 Nov;18(11):934-41.

Barnes CD. The basal ganglia in extrapyramidal dysfunction. Brain Res Bull. 1983 Aug;11(2):271-5.

Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci. 1973 Dec;20(4):415-55.

Bolam JP, Hanley JJ, Booth PA, Bevan MD. Synaptic organisation of the basal ganglia. J Anat. 2000 May;196 ( Pt 4):527-42. Brain Res. 1998 Apr 27;791(1-2):335-40.

Brooks VB, Stoney SD Jr. Motor mechanisms: the role of the pyramidal system in motor control. Annu Rev Physiol. 1971;33:337-92.

Burns RS, Chiueh CC, Markey SP, Ebert MH, Jacobowitz DM, Kopin IJ. A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4546-50.

Crawley JN. Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res. 1999 Jul 17;835(1):18-26.

Cui M, Aras R, Christian WV, Rappold PM, Hatwar M, Panza J, Jackson-Lewis V, Javitch JA, Ballatori N, Przedborski S, Tieu K. The organic cation transporter-3 is a pivotal modulator of neurodegeneration in the nigrostriatal dopaminergic pathway. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8043-8.

Dickinson D. 2012. Parkinson’s disease and parkinsonism: Neuropathology.Cold Spring Harb Perspect Med. 2:a009258. Fallon JH, Moore RY. Catecholamine innervation of the basal forebrain. IV. Topography of the dopamine projection to the basal forebrain and neostriatum. J Comp Neurol. 1978 Aug 1;180(3):545-80.

Efremova L, Scildknecht S, Adam M, pape R, Gutbier S, Hanf B, Burkle A, Leist M. 2015. Prevention of the degeneration of dopaminergic neurons in an astrocyte co-culture system allowing endogenous drug metabolism. British Journal of Pharmacology; 172; 4119-32

Fujita KA, Ostaszewski M, Matsuoka Y, Ghosh S, Glaab E, Trefois C, Crespo I, Perumal TM, Jurkowski W, Antony PM, Diederich N, Buttini M, Kodama A, Satagopam VP, Eifes S, Del Sol A, Schneider R, Kitano H, Balling R. Integrating pathways of Parkinson's disease in a molecular interaction map. Mol Neurobiol. 2014 Feb;49(1):88-102

Gerfen CR, Engber TM, Mahan LC, Susel Z, Chase TN, Monsma FJ Jr, Sibley DR. D1 and D2 dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons. Science. 1990 Dec 7;250(4986):1429-32.

González RR, Fernández RF, Vidal JL, Frenich AG, Pérez ML. Development and validation of an ultra-high performance liquid chromatography-tandem mass-spectrometry (UHPLC-MS/MS) method for the simultaneous determination of neurotransmitters in rat brain samples. J Neurosci Methods. 2011 Jun 15;198(2):187-94.

Heimer G, Rivlin-Etzion M, Bar-Gad I, Goldberg JA, Haber SN, Bergman H. Dopamine replacement therapy does not restore the full spectrum of normal pallidal activity in the 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine primate model of Parkinsonism. J Neurosci. 2006 Aug 2;26(31):8101-14.

Irwin JK, 1993, Parkinson’s disease: Past, Present and Future. Neuropsycopharmacology. 9(1). 1-1.

Jones BJ, Roberts DJ. The quantiative measurement of motor inco-ordination in naive mice using an acelerating rotarod. J Pharm Pharmacol. 1968 Apr;20(4):302-4.

Klapdor K, Dulfer BG, Hammann A, Van der Staay FJ. A low-cost method to analyse footprint patterns. J Neurosci Methods. 1997 Jul 18;75(1):49-54.

Kolata G. Monkey model of Parkinson's disease. Science. 1983 May 13;220(4598):705.

Leenders KL, Palmer AJ, Quinn N, Clark JC, Firnau G, Garnett ES, Nahmias C, Jones T, Marsden CD. Brain dopamine metabolism in patients with Parkinson's disease measured with positron emission tomography. J Neurol Neurosurg Psychiatry. 1986 Aug;49(8):853-60.

Ling H, Kearney S, Yip HL, Silveira-Moriyama L, Revesz T, Holton JL, Strand C, Davey K, Mok KY, Polke JM, Lees AJ. Parkinson's disease without nigral degeneration: a pathological correlate of scans without evidence of dopaminergic deficit (SWEDD)? J Neurol Neurosurg Psychiatry. 2015 Jul 24. pii: jnnp-2015-310756. doi: 10.1136/jnnp-2015-310756.

Mazzone P. Deep brain stimulation in Parkinson's disease: bilateral implantation of globus pallidus and subthalamic nucleus. J Neurosurg Sci. 2003 Mar;47(1):47-51.

Minnema DJ, Travis KZ, Breckenridge CB, Sturgess NC, Butt M, Wolf JC, Zadory D, Beck MJ, Mathews JM, tisdel MO, Cook AR, Botham PA, Smith LL. 2014. Dietary administration of paraquat for 13 weeks does not result in a loss of dopaminergic neurons in the substantia nigra of C57BL/6J mice. (68). 250-8.

Mitchell IJ, Clarke CE, Boyce S, Robertson RG, Peggs D, Sambrook MA, Crossman AR. Neural mechanisms underlying parkinsonian symptoms based upon regional uptake of 2-deoxyglucose in monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Neuroscience. 1989;32(1):213-26.

Morris ED, Babich JW, Alpert NM, Bonab AA, Livni E, Weise S, Hsu H, Christian BT, Madras BK, Fischman AJ. Quantification of dopamine transporter density in monkeys by dynamic PET imaging of multiple injections of 11C-CFT. Synapse. 1996 Nov;24(3):262-72.

Neural mechanisms underlying parkinsonian symptoms based upon regional uptake of 2-deoxyglucose in monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Obeso JA, Rodríguez-Oroz MC, Benitez-Temino B, Blesa FJ, Guridi J, Marin C, Rodriguez M. Functional organization of the basal ganglia: therapeutic implications for Parkinson's disease. Mov Disord. 2008;23 Suppl 3:S548-59.

Odekerken VJ, van Laar T, Staal MJ, Mosch A, Hoffmann CF, Nijssen PC, Beute GN, van Vugt JP, Lenders MW, Contarino MF, Mink MS, Bour LJ, van den Munckhof P, Schmand BA, de Haan RJ, Schuurman PR, de Bie RM. Subthalamic nucleus versus globus pallidus bilateral deep brain stimulation for advanced Parkinson's disease (NSTAPS study): a randomised controlled trial. Lancet Neurol. 2013 Jan;12(1):37-44.

Ossowska K., Wardas J, Smialowska M, Kuter K, Lenda T, Wieronska JM, Zieba B, Nowak P, Dabrowska J, Bortel A, Kwiecinski A, Wolfarth S. 2005. A slowly developing dysfunction of dopaminergiv nigrostriatal neurons induced by long-term paraquat administration in rats: an animal model of preclinical stagesof Parkinso’s disease.

Ovadia A, Zhang Z, Gash DM. 1995. Increased susceptibility to MPTP in middle-aged Rhesus Monkeys. Neurobiology of aging. 16 (6) 931-7

Parent A, Hazrati LN. Functional anatomy of the basal ganglia. I. The cortico-basal ganglia-thalamo-cortical loop. Brain Res Brain Res Rev. 1995 Jan;20(1):91-127.

Ramaker C, Marinus J, Stiggelbout AM, Van Hilten BJ. Systematic evaluation of rating scales for impairment and disability in Parkinson's disease. Mov Disord. 2002 Sep;17(5):867-76.

Rose S N M, Jackson EA, Gibb WR, Jaehnig P, Jenner P, Marsden CD. 1993. Age-related effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment of common marmosets. Eur J Pharmacol. 230(2).177-85.

Saraswat LD, Holdiness MR, Justice JB, Salamone JD, Neill DB. Determination of dopamine, homovanillic acid and 3,4-dihydroxyphenylacetic acid in rat brain striatum by high-performance liquid chromatography with electrochemical detection. J Chromatogr. 1981 Mar 13;222(3):353-62.

Silverdale MA, Fox SH, Crossman AR, Brotchie JM. Potential nondopaminergic drugs for Parkinson's disease. Adv Neurol. 2003;91:273-91.

Smith Y, Bennett BD, Bolam JP, Parent A, Sadikot AF. Synaptic relationships between dopaminergic afferents and cortical or thalamic input in the sensorimotor territory of the striatum in monkey. J Comp Neurol. 1994 Jun 1;344(1):1-19.

Smith Y, Kieval JZ. Anatomy of the dopamine system in the basal ganglia. Trends Neurosci. 2000 Oct;23(10 Suppl):S28-33.

Surmeier DJ, Song WJ, Yan Z. Coordinated expression of dopamine receptors in neostriatal medium spiny neurons. J Neurosci. 1996 Oct 15;16(20):6579-91.

Switzer RC. 2000. Application of silver degeneration stains for neurotoxicity testing. Toxicologic pathology. 28(1) 70-83.

Takada M, Tokuno H, Nambu A, Inase M. Corticostriatal input zones from the supplementary motor area overlap those from the contra- rather than ipsilateral primary motor cortex.

Tillerson JL, Caudle WM, Reverón ME, Miller GW. Detection of behavioral impairments correlated to neurochemical deficits in mice treated with moderate doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Exp Neurol. 2002 Nov;178(1):80-90.

Ungerstedt U, Arbuthnott GW. Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res. 1970 Dec 18;24(3):485-93.

Winogrodzka A, Bergmans P, Booij J, van Royen EA, Stoof JC, Wolters EC. [(123)I]beta-CIT SPECT is a useful method for monitoring dopaminergic degeneration in early stage Parkinson's disease. J Neurol Neurosurg Psychiatry. 2003 Mar;74(3):294-8.

Yuan H, Zhang ZW, Liang LW, Shen Q, Wang XD, Ren SM, Ma HJ, Jiao SJ, Liu P. Treatment strategies for Parkinson's disease. Neurosci Bull. 2010 Feb;26(1):66-76.


Scientific evidence supporting the linkages in the AOP

Upstream Event Relationship Type Downstream Event Evidence Quantitative Understanding
N/A, Mitochondrial dysfunction 1 directly leads to Impaired, Proteostasis Moderate Weak
Impaired, Proteostasis directly leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway Strong Moderate
N/A, Neuroinflammation directly leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway Moderate Moderate
Degeneration of dopaminergic neurons of the nigrostriatal pathway directly leads to N/A, Neuroinflammation Moderate Moderate
N/A, Mitochondrial dysfunction 1 indirectly leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway Moderate Weak
Degeneration of dopaminergic neurons of the nigrostriatal pathway directly leads to Parkinsonian motor deficits Strong Strong
Binding of inhibitor, NADH-ubiquinone oxidoreductase (complex I) directly leads to Inhibition, NADH-ubiquinone oxidoreductase (complex I) Strong Weak
Inhibition, NADH-ubiquinone oxidoreductase (complex I) directly leads to N/A, Mitochondrial dysfunction 1 Strong Moderate

Graphical Representation

Overall Assessment of the AOP


Domain of Applicability

Life Stage Applicability
Life Stage Evidence
Adult Strong
Taxonomic Applicability
Term Scientific Term Evidence Links
human Homo sapiens Strong NCBI
rat Rattus norvegicus Strong NCBI
Sex Applicability
Sex Evidence
Mixed Strong

This proposed AOP is neither sex-dependent nor associated with certain life stage; however, aged animals may be more sensitive. The relevance of this AOP during the developmental period has not been investigated. In vivo testing has no species restriction. The mouse was the species most commonly used in the experimental models conducted with the chemical stressors; though experimental studies using alternative species have been also performed. (Johnson et al. 2015). However, animal models (rodents in particular) would have limitations as they are poorly representative of the long human life-time as well as of the human long-time exposure to the potential toxicants. Human cell-based models would likely have better predictivity for humans than animal cell models. In this case, toxicokinetics information from in-vivo studies would be essential to test the respective concentrations in-vitro on human cells.

Essentiality of the Key Events

Essentiality of KEs for this AOP is strong. There is ample evidence from knock out animal models, engineered cells or replacement therapies that blocking, preventing or attenuating an upstream KE is mitigating the AO. In addition, there is experimental support for the KERs as multiple studies performed with modulating factors that attenuate (particularly with antioxidants) or augment (e.g. overexpression of viral-mutated α-synuclein) a KE show that such interference leads to an increase of KE down or the AO.

 

2 Support for Essentiality of KEs

Defining Question

Are downstream KEs and/or the AO prevented if an upstream KE is blocked ?

High (Strong)

Moderate

Low(Weak)

 

 

Direct evidence from specifically designed experimental studies illustrating essentiality for at least one of the important KEs (e.g. stop/reversibility studies, antagonism, knock out models, etc.)

Indirect evidence that sufficient modification of an expected modulating factor attenuates or augments a KE leading to increase in KE down or AO

No or contradictory experimental evidence of the essentiality of any of the KEs

KE1

Inhibition of complex I

STRONG

Rationale: Inactivation of the NADH:Ubiquinone Oxidoreductase Core Subunit S7 (Ndufs 4 gene knockout mice) that produces CI deficiency causes encephalomyopathy, including ataxia and loss of motor skills (Kruse et al., 2008). NDI1-transducted SK-N-MC cells expressing the rotenone-insensitive single subunit NADH dehydrogenase of yeast (NDI1) that acts as a replacement for the entire CI in mammalian cells were completely resistant to 100 nM rotenone-mediated cell death (at 48 hrs of exposure) indicating that rotenone – induced toxicity requires rotenone biding of CI (Sherer et al., 2003). In all rotenone models, mitochondria CI is inhibited at the dose that cause neurodegeneration (Betarbet et al 2000 and 2006).

 

KE2

Mitochondrial dysfunction

STRONG

Rationale: Many studies showing that antioxidants protect the cells against rotenone or MPTP induced oxidative stress are published (Chen et al. 2015; Lu et al., 2015; Saravanan et al., 2006; Chiu et al., 2015, Sherer et al.2003, Nataraj et al.2015, Wu et al. 1994; Tseng et al. 2014; Li et al. 2010; Kim-Han et al. 2011). This provides (indirect) evidence for essentiality of KE2, if production of reactive oxygen species (ROS) is assumed as direct consequence/sign of mitochondrial dysfunction. Additional evidence comes from experiments with overexpression or activation of antioxidative enzymes (e.g.SOD or ALDH2) , which also prevent rotenone and MPTP/MPP+ induced neurotoxicity (Mudo et al. 2012; Ciu CC et al. 2015). Furthermore, promotion of mitochondrial fusion or blocking of mitochondrial fission prevents or attenuates rotenone and MPTP/MPP+ induced neurotoxicity (Tieu K. et al. 2014).

KE3

Impaired proteostasis

MODERATE

Rationale: Indirect evidence for the role of disturbed alpha-synuclein proteostasis: Lacking of alpha-synuclein expression in mice prevented induction of behavioural symptoms, neuronal degeneration in the nigrostriatal pathway and loss of DA neurons after chronic treatment with MPTP/MPP+ (Fornai et al. 2004; Dauer et al. 2002) . Injection of adeno/lenti-associated virus that expresses wild-type or mutant α-synuclyn into rat, mice or non-human primate SN produced loss of dopaminergic neurons, but the effect is not easily reproduced in transgenic mice overexpressing alpha-synuclein (Kirk, 2002; Klein, 2002; Lo Bianco, 2002; Lauwers, 2003; Kirk, 2003).

Rationale for the role of autophagy: Early dendritic and axonal dystrophy, reduction of striatal dopamine content, and the formation of somatic and dendritic ubiquitinated inclusions in DA neurons were prevented by ablation of Atg7 (an essential autophagy related gene (Friedman et al. 2012)).

Rationale for the role of Ubiquitin Proteosomal System/Autophagic Lysosomal Pathway (UPS/ALP): Protection from DA neuronal death was also observed in multiple experiments through the pharmacological modulation of the UPS, ALP system; however, there are also contradicting data in the literature. (Inden et al. 2007; Fornai et al. 2003; Dehay et al. 2010; Zhu et al. 2007, Fornai et al. 2005).

However, although many lines of evidence exist to support essentiality of impaired proteostasis, a single molecular chain of events cannot be established.

KE4 

Degeneration of DA neurons of nigrostriatal pathway

MODERATE

Receptors for advanced glycated end product (AGEs) can activate NF-kB (a transcription factor involved in the inflammatory response) and they are found on microglia cells and astrocytes. Ablation of receptor for advanced glycated end product (RAGE) proved to be protective against MPTP-induced decreases of TH+ neurons and mitigation of microglia and astrocytes reactivity was observed (Teismann et al. 2012). Inhibition of RAGE, which is upregulated in the striatum following rotenone exposure and in response to neuroinflammation, decreases rotenone-induced apoptosis by suppressing NF(Nuclear Factor)-kB activation, as well as the downstream inflammatory markers TNF-alpha, iNOS and myeloperoxidase (Abdelsalam and Safar, 2015). This showed intermingled links between neuronal injury/death and neuroinflammation. Rotenone-induced neurotoxicity was less pronounced in neuron-enriched cultures than in neuron-glia co-cultures (Gao et al., 2002), suggesting that neuron-glia interactions are critical for rotenone-induced neurodegeneration. In addition, in in vitro systems, a decrease in thyroxine hydrosilase (TH) mRNA expression has been observed to be a sufficient signal to trigger microglial reactivity (Sandström et al., 2017).

KE5

Neuroinflammation

MODERATE

Rationale: Following treatment with Rotenone or MPTP/ MPP+, protection of DA neurons and terminals was observed in vivo and in vitro by inhibiting different feature of neuroinflammation (microglia/astrocyte); however, inhibition was different in different models and considered as an indirect evidence of essentiality (Zhou et al., 2007; Gao et al., 2002 and 2003 and 2015; ; Emmrich et al., 2013; Salama et al., 2012; Chang et al., 2013; Wang et al., 2014; Liu et al., 2012, 2015; Borrajo et al., 2013; Brzozowski et al., 2015; Wang et al., 2006; Chung et al., 2011; Sriram et al., 2014; Feng et al., 2002; Sathe et al., 2012; Khan et al., 2014; Ros-Bernal et al., 2011; Ferger et al., 2004; Chao et al., 2009; Rojo et al., 2010; Qian et al., 2011; Dehmer et al., 2000; Bodea et al., 2014). Mice lacking the type-1 Interferons receptor showed an attenuated pro-inflammatory response and reduced loss of dopaminergic neurons induced by MPTP/MPP+. The neuro-protective potential was also confirmed by treatment with a blocking monoclonal antibody against type-1A IFN receptor (interferon receptor) that increased survival of dopaminergic neurons of TH+ (Main et al., 2016).

KE4 

Degeneration of DA neurons of nigrostriatal pathway

STRONG

Rationale: Clinical and experimental evidences show that the pharmacological replacement of the dopamine (DA) neurofunction by allografting fetal ventral mesencephalic tissues is successfully replacing degenerated DA neurons resulting in the total reversibility of motor deficit in animal model and partial effect is observed in human patient for PD (Widner et al., 1992; Henderson et al., 1991; Lopez-Lozano et al., 1991; Freed et al., 1990; Peschanski et al., 1994; Spencer et al., 1992).

Also, administration of L-DOPA or DA agonists results in an improvement of motor deficits (Calne et al 1970; Fornai et al. 2005). The success of these therapies in man as well as in experimental animal models clearly confirms the causal role of dopamine depletion for PD motor symptoms ( Connolly et al., 2014; Lang et al., 1998Silva et al., 1997; Cotzias et al., 1969; Uitti et al., 1996; Ferrari-Tonielli et al., 2008; Kelly et al., 1987; Walter et al., 2004; Narabayashi et al., 1984; Matsumoto et al., 1976; De Bie et al., 1999; Uitti et al., 1997; Scott et al., 1998; Moldovan et al., 2015; Deuschl et al., 2006; Fasano et al., 2010; Castrito et al., 2011; Liu et al., 2014; Widner et al., 1992; Henderson et al., 1991; Lopez-Lozano et al., 1991; Freed et al., 1990; Peschanski et al., 1994;Spencer et al., 1992).

Furthermore, experimental evidence from animal models of PD and from in-vitro systems indicate that prevention of apoptosis through ablation of BCL-2 family genes prevents or attenuates neurodegeneration of DA neurons (Offen D et al., 1998; Dietz GPH et al. 2002).

 

 

Weight of Evidence Summary

Concordance of dose-response relationship.

Data from experiments with the stressor compounds rotenone and MPTP (known inhibitors of the mitochondrial Complex I (CI)) reveal a good concordance of the dose-response relationships between the MIE and AO and within KEs. Although the different KEs have been measured using different methodologies, comparison of data from multiple in-vitro/in-vivo studies shows a general agreement in dose-relationship (see table 1 and 2). There is a good consistency when comparing data on KE4 and the AO after exposure to rotenone and MPTP. However, in vivo rodent studies proved that only exposure to low concentrations of rotenone (rat brain concentration between 20-30 nM of rotenone; Betrabet et al., 2000) or MPTP (mice striatum concentration of approximately 12-47 µM MPP+; Fornai et al., 2005; Thomas et al. 2012) after chronic exposure (approximately 5 weeks) reproduced the anatomical, neurochemical behavioural and neuropathological features similar to the ones observed in Parkinson’s disease (PD). Because of the variability of experimental protocols used, a clear no-effect threshold could not be established; nevertheless, these brain concentrations of rotenone (20-30 nM) and MPP+ (approximately 12-47µM) could serve as probabilistic thresholds for chronic exposure that could reproduce features of PD as both concentrations trigger approximately a 50% inhibition of Complex I (see table 3). Generally, a strong response-response relationship is observed within studies. Some exceptions for this rule are observed between KE3/KE5 and KE4, likely because of the all biological complexity associated with these KEs. In this AOP, neuroinflammation was considered to have a direct effect on degeneration of DA neurons. However, it was not clear at which conditions it would become a modulatory factor and for practical reasons was not included in table 1, 2 and 3 but considered in the weight of evidence analysis.

Table1 Dose-response and temporality table for rotenone.

 

Rotenone Concentration 

KE1aaa

Inhibition of C I

KE2aaa

Mitochondrial dysfunction

KE3aaa

Impaired proteostasis

KE4

Degeneration of DA neurons of nigrostriatal pathway

AO

Parkinsonian motor symptoms

5-10 nM in-vitro

[1]

+

4-72 hours [1]

+

4-72 hours [4]

+

24 hours [3]

-

-

20-30 nM ex-vivo, rat brain concentration

[4-5-2-6]

++

4-72 hours (4-5)

++

4-72 hours [4-5]

++

24 hours [3-2-6]

++a

5 weeks [2-6]

+++aa

5 weeks [2-6]

100 nM in-vitro

[4]

+++

4-72 hours [4]

+++

4-72 hours [4]

+++

24 hours [3]

Corresponding to a concentration above the maximum tolerated dose in-vivo [2-6]

Corresponding to a concentration above the maximum tolerated dose in vivo [2-6]

References: Choi et al. 2008 [1]; Betarbet et al. 2006 [2]; Chou et al. 2010 [3]; Barrientos and Moraes 1999 [4]; Okun et al. 1999 [5]; Betarbet et al. 2000 [6]

-no data available

+: low severity score, ++ intermediate severity score, +++ high severity score

a: 50% of treated animals showed loss of DA neurons in SNpc

aa: All animals affected in KE4 showed impaired motor symptoms

aaa: KE 1, 2 and 3 showed a dose-related severity in the effect and the score ++ was normalized vs. the KE4

Table 2. Dose-Response and Temporality table for MPTP/MPP+

MPTP Administered Dose

MPPBrain Concentration

KE1bb

Inhibition of C I

KE2bb

Mitochondrial dysfunction

KE3b

Impaired proteostasis

KE4

Degeneration of DA neurons of nigrostriatal pathway

AO

Parkinsonian motor symptoms

1 mg/kg sc infusion [1]

-

-

-

+

4 weeks[1]

+aaa

4 weeks [1]

No effect

5 mg/kg sc infusion [1]

-

-

-

++

4 weeks[1]

++aa

4 weeks [1]

+++

4 weeks [1]

20-30 mg/kg sporadic ip. injections (4 times every 2 hours)

[2, 1]

 

47µM [2]^

12µM [1]

+++

4 hrs [2]

+++

4hrs [2]

+++

4 weeks [1]

+++a

1-4 weeks[2,1]

 

+++

4 weeks [1]


References. Fornai et al. 2005 [1]; Thomas et al. 2012 [2]

-no data available

a: approx 50% loss of DA neurons in SNpc

aa: approx 30% loss of DA neurons SN pc

aaa: no loss of DA neurons in SN pc. Reduced level of striata DA

b: for KE3, a dose response effect was observed.

bb: for KE 1 and 2 the severity of the effect was normalized vs. the KE4

^ After single dose MPTP administration, brain concentration was approx. 5.15 µM

Temporal concordance among the MIE, KEs and AO.

There is a strong agreement that loss of DA neurons of the SNpc that project into the putamen is preceded by reduction in DA and degeneration of DA neuronal terminals in the striatum (Bernheimer et al. 1973). The clinical symptoms of a motor deficit are observed when 80% of striatal DA is depleted (Koller et al. 1992) and the sequence of pathological events leading to the adverse outcome has been well-documented (Fujita, et al.2014; O’Malley 2010, Dexter et al. 2013). Temporal concordance (see table 1 and 2) among the KEs can be observed in the experimental models of PD using the chemical stressors rotenone and MPTP (Betarbet 2000 and 2006; Sherer et al. 2003, Fornai et al. 2005). The acute administration of the chemical stressors can trigger a dose-related change from the MIE to impaired proteostasis; however, to trigger KE4 (i.e. degeneration of DA neurons in SNpc with presence of intracytoplasmatic Lewy-like bodies) and motor deficits (AO), proteostasis needs to be disturbed for a minimum period of time (Fornai et al. 2005).

Strength, consistency, and specificity of association of AO and MIE.

Strength and consistency of the association of the AO with the MIE is strong. There is a large body of evidence from in-vitro and in-vivo studies with chemical stressors, showing association between the MIE that triggers an inhibition of CI and the AO (Sherer et al. 2003; Betarbet et al. 2000 and 2006, Fornai et al. 2005). Human data also suggest a link between inhibition of CI and AO (Greenamyre et al. 2001; Schapira et al. 1989; Shults, 2004). Using the two different chemical stressors, rotenone and MPTP, data are consistent and the pattern of activation of the MIE leading of the AO is similar. For rotenone and MPTP, specificity is high; however, there are many inhibitors of the mitochondrial CI without evidence of triggering the AO. When considering  the limited amount of chemical stressors for which empirical data are available for supporting the full sequence of KEs, kinetic and metabolic considerations should be taken into account to demonstrate specificity for these compounds; therefore, kinetic and metabolic considerations should be taken into account to fully demonstrate specificity for these compounds. The issue of specificity was also debated during the external review of this AOP and the following information was added:

The vast majority of empirical support available in the literature is based on complex I inhibitors, such as rotenone and MPTP/MPP+, as well as on studies involving genetic impairment of complex I activity. A relatively wide spectrum of structurally different complex I inhibitors have been described over the course of recent decades. Prominent examples are acetogenins (Nat Prod Rep 2005, 22, 269-303); alkaloids (J Neurochem 1996, 66, 1174-1181); antibiotics (BBA 1998, 1364, 222-235; Eur J Biochem 1994, 219, 691-698; JBC 1970, 245, 1992-1997; Bioorg Med Chem 2003, 11, 4569-4575); pesticides (Biochem Soc Trans 1994, 22, 230-233); quinones (JBC 1971, 246, 2346-2353); or vanilloids (ABB 1989, 270, 573-577). Additional information can be also retreived from Fato et al 2009, Espositi et al. 1993, Lagoa et al. 2011 and Park et al. 2003.

All of these structurally different complex I inhibitors were characterized with isolated mitochondria or with submitochondrial particles. Application of bovine heart mitochondria revealed IC50 values in the range of 20-70 nM for piericidin A, fenpyroximate, rotenone, and phenoxan (Eur. J. Biochem 1994, 219, 691-698). IC50 values in the range of 1-10 nM were detected by application of submitochondrial particles with rotenone, molvizarin, rollinstatin-1 and -2, and piericidin A (Biochem J. 1994, 301, 161-167).

Studies involving neuronal cell cultures or in vivo models are in fact rather rare. A systematic comparison of the IC50 values for complex I inhibition and EC50 values for the reduction of ATP levels; cell death was performed with rat fetal striatal neurons (Exp Neurol 2009, 220, 133-142). Due to the lipophilicity of most of the complex I inhibitors tested, the detected EC50 values were in most cases lower than the IC50 values detected for complex I inhibition. EC50 values detected were: annonacin (60 nM), fenazaquin (45 nM), piericidin A (1.6 nM), rollinstatin- 2 (1 nM), rotenone (8 nM), and squamocin (1 nM).

A systematic investigation involving mesencephalic cultures as well as rats was performed for the complex I inhibitor annonacin, a major acetogenin of soursop, a plant suspected to cause an atypical form of PD in Guadeloupe. Mesencephalic cultures treated for 24 h with annonacin revealed EC50 values of 20 nM (annonacin), 34 nM (rotenone), and 1900 nM (MPP+) (Neurosci 2003, 121(2), 287-296). Intravenous application by minipumps over the course of 28 days indicated a passage of annonacin across the blood-brain barrier, and an energy-dependent loss of ca. 30 % of DA neurons in the substantia nigra (Champi et al.2004)).

 

Weight of Evidence (WoE).

Biological plausibility, coherence, and consistency of the experimental evidence.

The biological plausibility of this AOP is overall considered strong. When using multiple stressors in different studies and assays, the coherence and consistency of the experimental data is well established. Furthermore, in-vivo and in-vitro studies are also in line with the human evidence from PD patients. In addition, although the mechanistic understanding of parkinsonian disorders (and PD in particular) are not fully clear, the KEs and KERs described in this AOP are considered critical for the development of the disease (Fujita et al. 2015, Shulman et al. 2011, Dexter et al. 2013, Dauer et al. 2003).

 

1 Support for Biological Plausibility of KERs

Defining Question

High (Strong)

Moderate

Low(Weak)

 

Is there a mechanistic (i.e. structural or functional) relationship between KEup and KE down consistent with established biological knowledge?

Extensive understanding of the KER based on extensive previous documentation and broad acceptance

The KER is plausible based on analogy to accepted biological relationships, but scientific understanding is not completely established

There is empirical support for a statistical association between KEs but the structural or functional relationship between them is not understood

MIE => KE1

Binding of inhibitor to NADH-ubiquinone oxidoreductase leads of complex I

STRONG

Rationale: As describe in this KER there is an extensive understanding of the functional relationship between binding of an inhibitor to NADH-ubiquinone oxidoreductase (CI) and its inhibition. Different complex I ligands, both naturally occurring, like rotenone (from Derris scandens), piericidin A (from Streptomyces mobaraensis), acetogenins (from various Annonaceae species) and their derivatives, and synthetically manufactured like pyridaben and various piperazin derivatives inhibit the catalytic activity of complex I (Degli Esposti, 1994: Ichimaru et al. 2008; Barrientos and Moraes, 1999; Betarbet et al., 2000).

KE1 => KE2

Inhibition of complex I leads to mitochondrial dysfunction

STRONG

Rationale: There is extensive understanding of the mechanisms explaining how the inhibition of complex I lead to mitochondrial dysfunction (i.e. failure to produce ATP, increase in production of ROS etc). It is well documented that CI inhibition is one of the main sites at which electron leakage to oxygen occurs resulting in oxidative stress (Efremov and Sazanow, 2011; lauren et al. 2010; Greenamyre et al. 2001). These pathological mechanisms are well studied as they are used as readouts for evaluation of mitochondrial dysfunction (Graier et al., 2007; Braun, 2012; Martin, 2011; Correia et al., 2012; Cozzolino et al., 2013

KE2 => KE3

Mitochondrial dysfunction results in impaired proteostasis

MODERATE

Rationale: The weight of evidence supporting the biological plausibility behind the relationship between mitochondrial dysfunction and impaired proteostasis, including the impaired function of UPS and ALP that results in decreased protein degradation and increase protein aggregation is well documented but not fully understood. It is well established that the two main mechanisms that normally remove abnormal proteins (UPS and ALP) rely on physiological mitochondrial function. The role of oxidative stress, due to mitochondrial dysfunction, burdens the proteostasis with oxidized proteins and impairs the chaperone and the degradation systems. This leads to a vicious circle of oxidative stress inducing further mitochondrial impairment (Powers et al., 2009; Zaltieri et al., 2015; McNaught and Jenner, 2001). Therefore, the interaction of mitochondrial dysfunction and UPS /ALP deregulation plays a pivotal role in the pathogenesis of PD (Dagda et al., 2013; Pan et al., 2008; Fornai et al., 2005; Sherer et al., 2002).

KE2 => KE4

Mitochondrial dysfunction leads to the degeneration of dopaminergic neurons of the nigrostriatal pathway

 

STRONG

Rationale: Mitochondrial are essential for ATP production, ROS management, calcium homeostasis and control of apoptosis. Mitochondrial homeostasis by mitophagy is also an essential process for cellular maintenance (Fujita et al. 2014). Because of their anatomical and physiological characteristics, SNpc DA neurons are considered more vulnerable than other neuronal populations (Sulzer et al. 2013; Surmeier et al.2010). Mechanistic evidence of mutated proteins relate the mitochondrial dysfunction in familial PD with reduced calcium capacity, increased ROS production, increase in mitochondrial membrane permeabilization and increase in cell vulnerability (Koopman et al. 2012; Gandhi et al. 2009). Human studies indicate mitochondrial dysfunction in human idiopathic PD cases in the substantia nigra (Keeney et al., 2006; Parker et al., 1989, 2008; Swerdlow et al., 1996). In addition, systemic application of mitochondrial neurotoxicants such as rotenone or MPTP leads to a preferential loss of nigrostriatal DA neurons (Langston et al., 1983).

KE3 => KE4

Impaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway

MODERATE

Rationale: It is well known that impaired proteostasis refers to misfolded and aggregated proteins including alfa-synuclein, deregulated axonal transport of mitochondria and impaired trafficking of cellular organelles. Evidences are linked to PD and experimental PD models as well as from genetic studies (McNaught et al. 2001, 2003; Tieu et al. 2014; Arnold 2011; Rappold et al. 2014). Strong evidence for degeneration of the nigrostriatal pathway comes from the experimental manipulations that directly induce the same disturbances of proteostasis as observed in PD patients (e.g. viral mutated alpha-synuclein expression) or in chronic rotenone/MPTP models trigger degeneration of the nigrostriatal pathway (Kirk et al. 2003; Betarbet et al. 2000 and 2006; Fornai et al. 2005). However, a clear mechanistic proof for the understanding of the exact event triggering cell death is lacking. There is only moderate evidence showing that interventions that correct disturbances of proteostasis after exposure to rotenone would prevent neuronal degeneration and that the disturbances of proteostasis correlate quantitatively under many conditions with the extent of nigrostriatal neuronal degeneration.

KE4 => KE5

Degeneration of DA neurons of the nigrostriatal pathway leads to neuroinflammation

MODERATE

Rationale: The fact that neuronal injury/death can trigger neuroinflammation is supported by evidence in human and experimental models. NeThe evidence that neuroinflammation triggered by neuronal damage can cause neuronal death (vicious circle), is mostly indirect (blockade of any feature of neuroinflammation) or by analogy (Hirsch and Hunot, 2009; Tansey and Goldberg, 2009; Griffin et al., 1998; McGeer and Mc Geer, 1998; Blasko et al., 2004; Cacquevel et al., 2004; Rubio-Perez and Morillas-Ruiz, 2012; Thundyil and Lim, 2014; Barbeito et al., 2010). Neuroinflammation is observed in idiopathic and in genetic human PD as well as in complex I inhibitor exposed humans, non-human primates, and rodent.Components of damaged neurons lead to glial cells activation via Toll-like receptors.Several chemokines and chemokine receptors (fraktalkine, CD200) control the neuron-microglia interactions. Neuroinflammation in response to damaged neurons is not confined to PD, but is common to several neurodegenerative diseases 

KE5 => KE4

Neuroinflammation leads to degeneration of DA neurons of the nigrostriatal pathway

MODERATE

Rationale: The fact that reactive glial cells (microglia and astrocytes) may kill neurons is well accepted. The mechanisms underlying this effect may include the release of cytotoxic signals (e.g. cytokines) or production of ROS and RNS (Chao et al., 1995 ; Brown and Bal-Price, 2003 ; Kraft and Harry, 2011 ; Taetzsch and Block, 2013). However, the responsible mediators differ from model to model. In humans or non-human primates, an inflammatory activation of glial cells is observed years after exposure to complex I inhibitors. Activated microglia and astrocytes form pro-inflammatory cytokines and free radical species, mostly responsible for neuronal damage. Glial reactivity promotes an impairment of blood brain barrier integrity, allowing an infiltration of peripheral leukocytes that exacerbate the neuroinflammatory process and contribute to neurodegeneration.The debris of degenerating neurons causes neuroinflammation, which in turn can trigger neurodegeneration, thus leading to a self-perpetuating vicious cycle.

KE4 => AO

Degeneration of DA neurons of the nigrostriatal pathway leads to parkinsonian motor symptoms

STRONG

Rationale: The mechanistic understanding of the regulatory role of striatal DA in the extrapyramidal motor control system is well established. The loss of DA in the striatum is characteristic of all aetiologies of PD and is not observed in other neurodegenerative diseases (Bernheimer et al. 1973; Reynolds et al. 1986). Characteristic motor symptoms such as bradykinesia, tremor, or rigidity are manifested when more than 80 % of striatal DA is depleted as a consequence of SNpc DA neuronal degeneration (Koller et al. 1992).

Empirical support.

Empirical support is strong. Many studies show evidence for the KERs by showing temporal concordance and dose concordance when using different stressors.

 

3 Empirical support for KERs

Defining Question

Does the empirical evidence support that a change in the KEup leads to an appropriate change in the KE down? Does KEup occur at lower doses and earlier time points than KE down and is the incidence of KEup higher than that for KE down?

Are inconsistencies in empirical support cross taxa, species and stressors that don’t align with expected pattern of hypothesized AOP?

High (Strong)

Moderate

Low(Weak)

 

 

Multiple studies showing dependent change in both exposure to a wide range of specific stressors (extensive evidence for temporal, dose-response and incidence concordance) and no or few critical data gaps or conflicting data.

Demonstrated dependent change in both events following exposure to a small number of specific stressors and some evidence inconsistent with expected pattern that can be explained by factors such as experimental design, technical considerations, differences among laboratories, etc.

Limited or no studies reporting dependent change in both events following exposure to a specific stressor (ie endpoints never measured in the same study or not at all); and/or significant inconsistencies in empirical support across taxa and species that don’t align with expected pattern for hypothesized AOP

MIE => KE1

Binding of inhibitor to NADH-ubiquinone oxidoreductase leads to partial or total inhibition of complex I

STRONG

Rationale: The inhibition of complex I is well documented in a variety of studies using isolated mitochondria or cells as well as in in vivo experiments and in human post mortem PD brains. In many experiments using different inhibitors ie rotenone and MPTP, the observed relationship between the two events was temporal, response and dose concordant (Betarbet et al., 2000 and 2006, Okun et al., 1999, Koopman et al., 2007, Choi et al., 2008, Grivennikova et al., 1997, Barrientos and Moraes 1999).

KE1 => KE2

Inhibition of complex I leads to mitochondrial dysfunction

STRONG

Rationale: There is a large amount of studies showing that the inhibition of CI inhibition results in mitochondrial dysfunctions in a response and dose dependent manner (Barriento and Moraes, 1999).

KE2 => KE3

Mitochondrial dysfunction results in impaired proteostasis

STRONG

Rationale: Based on the existing in vitro and in vivo data it is suggested that mitochondrial dysfunction impairs protein homeostasis (impairment of the UPS and ALP system) through oxidative and nitrosative stress resulting in accumulation of misfolded proteins (including α-synuclein), disruption of microtubule assembly and damaged intracellular transport of proteins and cell organelles. A number of studies performed with chemical stressors showed evidence of temporal, response and dose concordance (Chou et al. 2010; Betarbet et al. 2000 and 2006; Fornai et al. 2005).

KE2 => KE4

 

Mitochondrial dysfunction directly leads to degeneration of DA neurons of nigrostriatal pathway

 

STRONG

Rationale: Multiple in vitro studies indicate dose and response-response concordance. As most of the studies were conducted in vitro, the temporal concordance is difficult to establish; however, can be expected based on the well know temporal sequence of the two KEs. (Park et al., 2014; Choi et al., 2014; Marella et al., 2008; Du et al. 2010; Hajieva et al., 2009; Sherer et al., 2003; Sherer et al., 2007; Wen et al. 2011; Swedlow et al., 1996; Jana et al., 2011; Jha et al., 2000; Chinta et al., 2006)

KE3 => KE4a

Impaired proteostasis leads to degeneration of DA neurons of the nigrostriatal pathway

STRONG

Rationale: The empirical support linking impaired proteostasis with degeneration of DA neurons of the nigrostriatal pathway is strong and comes from in-vivo and in-vitro studies performed with different stressor (i.e. Rotenone, MPTP or proteasome inhibitors) and post-mortem human evidences in PD patients supporting a causative link between the two key events. Temporal, effect and dose concordance was established in a number of experiments (Fornai et al. 2005; Fornai et al. 2003; Betabret et al. 2000 and 2006).

KE4a => KE5

Degeneration of DA neurons of nigrostriatal pathway leads to neuroinflammation

MODERATE

Rationale: multiple in vivo and in vitro experiments support the link between degeneration of DA neurons in the nigrostriatal pathway and neuroinflammation. The observation of concomitant presence of reactive microglial and astrocytic cells and degenerated/degenerating DA neurons is also reported in many studies with a good temporal and response concordance. ATP and other damage associated molecular patterns (DAMPs), released from degenerating cells, stimulate P2Y receptors on microglia, leading to their activation. Experimental injection of DAMPs, fraktalkine, or neuromelanin, released by degenerating DA neurons evokes neuroinflammation. Neutralization of DAMPs (e.g. antibodies against HMGB1 or CX3CR1) decreases MPTP-induced neuroinflammation .Toll-like receptor 4 deficient mice display a reduced neuroinflammatory response upon MPTP treatment. Inhibition of RAGE, which is upregulated in striatum upon rotenone exposure, suppresses NF-kB activation and downstream inflammatory markers.

KE5 => KE4b

Neuroinflammation leads to degeneration of DA neurons of nigrostriatal pathway.

MODERATE

Rationale: multiple in vivo and in vitro experiments support the link between neuroinflammation and degeneration of DA neurons in the nigrostriatal pathway. The observation of concomitant presence of reactive microglial and astrocytic cells and degenerated/degenerating DA neurons is also reported in many studies with a good temporal and response concordance. Neuroinflammation has been implicated in dopaminergic neuronal cell death in PD patients (Vivekanantham et al., 2014). LPS injection into the CNS, or applied systemically, evokes glial inflammation and a preferential degeneration of DA neurons. In mouse models with a knockout of either IL-1b, IFN-g, or TNF-a receptors 1 and 2, LPS no longer evokes neuroinflammation and DA neurodegeneration. Experimental interference with CD4+ T cell activation protects from DA neurodegeneration.Transfer of immunosuppressive regulatory T cells protect from DA neurodegeneration. Anti-inflammatory TGF-b1 signaling protects from DA neurodegeneration . Clinical trials indicate a protective influence on DA neuron survival by the antibiotic minocycline blocking microglial reactivity, in association with rasagiline (prevents DA degeneration), and coenzyme Q10/creatine (restoration of cellular ATP).

KE4b => AO

Degeneration of DA neurons of nigrostriatal pathway leads to parkinsonian motor symptoms

STRONG

Rationale: The experimental support linking the degeneration of DA neurons of nigrostriatal pathways with the manifestation of motor symptoms of PD comes from human in vivo observations as well as from monkey, mice and rat in vivo models exposed to an experimental toxin ie rotenone and MPTP. Observations in human allow defining correlation between the levels of striatal DA with the onset of motor dysfunction (Lloyd et al. 1975; Hornykiewicz et al. 1986; Bernheimer et al. 1973). Temporal, effect and dose concordance comes from studies performed in multiple animal species following administration of rotenone and MPTP ( Bezard et al. 2001; Cannon et al. 2009; Petroske et al. 2001; Alvarez-Fischer et al. 2008; Blesa et al. 2012; Lloyd et a. 1975).

Uncertainties and Inconsistencies.

  • The strength of this AOP is mainly based on MPP+ and rotenone and only limited information on whether other mitochondrial complex I inhibitors also perturb the KEs (specifically degeneration of DA neurons in the SNpc)  or produce a similar AO.
  • Conflicting data exists (Choi et al.2008) showing that mitochondrial complex I inhibition is not required for DA neuron death induced by rotenone, MPTP/MPP+, or paraquat, challenging the current AOP. The cited research article shows that abolishment of complex I’s activity by inactivation of a gene that codes for a subunit of complex I does not impact the survival of DA neurons in culture. The actions of rotenone, MPTP/MMP+ are independent of complex I. Since some complex I inhibitors also target other complexes, it is possible that impairment of other respiratory complexes may be involved. It was noted that this paper used the approach of genetically deleting an essential chaperone in complex I assembly, and the authors found that absence of complex I activity in this model did not affect the toxicity of rotenone and MPP+. However,  the findings have never been confirmed/ continued, neither in the originating laboratory, nor by others. Second, the work did not consider the possibility that some functions of complex I were not affected by the absence of the chaperone (e.g. reverse electron transfer from complex II and III), and that rotenone and MPTP/MPP+ may well cause toxicity by interfering with such residual function (e.g. by favoring channeling of electrons to molecular oxygen). In light of this situation, the publication of Choi et al (2008) should be considered weak in the overall weight of evidence and therefore considered a minor inconsistency. 
  • There is no strict linear relationship between inhibitor binding and reduced mitochondrial function. Low doses of rotenone that inhibit CI activity partially do not alter mitochondrial oxygen consumption. Therefore, bioenergetics defect cannot account alone for rotenone-induced neurodegeneration. Instead, under such conditions, rotenone neurotoxicity may result from oxidative stress (Betarbet et al., 2000). Few studies used human brain cells/human brain mitochondria. Therefore, full quantitative data for humans are not available.
  • It is molecularly unclear how rotenone binding alter CI function, switching it to ROS production. It is still unclear whether the site of superoxide production in CI inhibited mitochondria is complex I itself or not (Singer and Ramsay, 1994).
  • Some studies suggest that rotenone and MPTP/MPP+ may have effects other than CI inhibition, e.g. MPTP and rotenone can induce microtubule disruption (Feng, 2006; Ren et al., 2005Cappelletti et al., 1999; Cappelletti et al., 2001, Brinkley et al., 1974; Aguilar et al. 2015).
  • There are additional feedback possible between KEs, e.g. ROS production from KE2 may damage CI, this leads to enhancement of KE1.
  • Some KEs e.g. KE 2, 3, 5 pool molecular processes that may need to be evaluated individually at a later stage.
  • The exact molecular link from mitochondrial dysfunction to disturbed proteostasis is still unclear (Malkus et al 2009; Zaltieri et al. 2015).
  • The role of ATP depletion vs. other features of mitochondrial dysfunction is not clear.
  • The role of a α-synuclein in neuronal degeneration is still unclear as well as the mechanisms leading to its aggregation.
  • It is not clear under which conditions KE3 and KE5 become modulatory factors, and when they are essential. MPTP/MPP+ can induce damage to nigrostriatal neurons without formation of Lewy bodies (Dauer 2003; Forno 1986, 1993). Similarly, discontinuous administration of rotenone, even at high doses, damages the basal ganglia but produce no inclusions (Heikkila et al. 1985; Ferrante et al. 1997, Lapontine 2004). To reproduce the formation of neuronal inclusions, continuous sc infusion of MPTP/MPP+ or rotenone is necessary. Acute intoxication with rotenone seems to spare dopaminergic neurons (Dauer et al 2003, Ferrante 1997). In addition, in rats chronically infused with rotenone showed a reduction in striatal DARPP-32-positive (dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32,000), cholinergic and NADPH diaphorase-positive neurons (Hoglinger 2003) or in other brain regions. These results would suggest that Rotenone can induce a more widespread neurotoxicity (Aguilar 2015) or the model is not reproducible in all laboratories.
  • Inconsistent effects of MPTP/MPP+ on autophagy (up or down regulation) are reported (Drolet et al., 2004: Dauer et al., 2002). There is conflicting literature on whether increased autophagy would be protective or enhances damage. Similarly, a conflicting literature exists on extent of inhibition or activation of different protein degradation system in PD and a clear threshold of onset is unknown (Malkus et al. 2009; Fornai et al. 2005).
  • The selective vulnerability of the SN pc dopaminergic pathway does not have a molecular explanation.
  • in some instances, the differential vulnerability of various brain regions towards a generalized complex Iinhibition found non-dopaminergic lesions, particularlyin the striatum, in all animals with nigral lesion, as seen inatypical parkinsonism but not in idiopathic parkinson's disease (Hoglinger et al.2003)

  • Priority of the pattern leading to cell death could depend on concentration, time of exposure and species sensitivity; these factors have to be taken into consideration for the interpretation of the study’s result and extrapolation of potential low-dose chronic effect as this AOP refers to long-time exposure.
  • The model of striatal DA loss and its influence on motor output ganglia does not allow to explain specific motor abnormalities observed in PD (e.g. resting tremor vs bradykinesia) (Obeso et al. 2000). Other neurotransmitters (Ach) may play additional roles. Transfer to animal models o PD symptoms is also representing an uncertainties.
  • There are some reports indicating that in subacute rotenone or MPTP models (non-human primates), a significant, sometimes complete, recovery of motor deficits can be observed after termination of toxicant treatment. The role of neuronal plasticity in intoxication recovery and resilience is unclear.
  • This AOP is a linear sequence of KEs. However, mitochondrial dysfunction (and oxidative stress) and impaired proteostasis are influencing each other and this is considered an uncertainties (Malkus et al. 2009).

 

Quantitative Consideration

The quantitative understanding of this AOP includes a clear response-response relationship and the identification of a threshold effect. The WoE analysis clearly supports the qualitative AOP as a means to identify and characterize the potential of a chemical to induce DA neuronal loss and the AO. Importantly, both the AO and the KE4 are considered relevant regulatory endpoints for this AOP. The empirical evidence supports existence of a response-response relationship. This response-response is likely triggered by a the brain concentrations of approximately 20-30 nM and 17-47 µM of rotenone and MPTP/MPP+ respectively and both concentrations trigger approx. a 50% inhibition of mitochondrial complex I and this could be considered as a “threshold”. However, a more detailed dose-response analysis for each KE is lacking as well as it is not clear which temporal relationship exists for lower CI inhibitory effects. It is clear from the analysis of the AOP that for the identification of these AOs, the design of the in-vivo studies should be tailored as to a MIE which leads to a long-lasting perturbation of the KEs. This provides the most specific and definite context to trigger neuronal death. To observe KEs relevant for this AOP, new endpoints need to be introduced. Although a dose, response and temporal relationship is evident for most KEs, the quantitative relationship between impaired proteostasis and degeneration of DA neurons has yet to be elucidated. Moving from a qualitative AOP to quantitative AOP would need a clear understanding of effect thresholds and this is still representing a major hurdle for several KEs of this AOP.


Table 3 Summary of quantitative effects at the concentration of rotenone and MPTP triggering the AO.

Concentration

KE1

Inhibition of C I

KE2

Mitochondrial dysfunction

KE3

Impaired proteostasis

KE4

Degeneration of DA neurons of nigrostriatal pathway

AO

Parkinsonian motor symptoms

Rotenone 20-30 nM rat brain concentration

[1-2]

Approx. 53%[4-5]

Approx. 20-53% (decrease in respiration rate)[1-2]

Approx. 20-60% (decrease in UPS (26S) activity) [3]

Neuronal loss (50% of animal affected) [2]

Motor impairment (100% of animals with neuronal loss) [2]

MPP+ 12-47 µM rat brain concentration [4-5]

Approx. 50-75% [5]

Approx. 38% (reduction in phosphorylating respiration) [5]

Approx. 60% (decrease in UPS activity) [4]

Approx. 50% of neuronal loss [4-5]

Motor impairment [4]


[1]; Okun et al. 1999 [2]; Barrientos and Moraes 1999; [3] Borland et al.2008 [4] Thomas et al 2012; [5] Betarbet et al 2000 and 2006.


Summary of the proposed Key Events in this AOP:

Final graph.jpg

Chronic, low level of exposure to environmental chemicals that inhibit complex I could result in mitochondrial dysfunction and oxidative stress, triggering proteasomal dysfunction strongly implicated in parkinsonian disorders, including aggregation/modifications in α-synuclein protein and organelles trafficking. These cellular key events cause DA terminals degeneration in striatum and progressive cell death of DA neurons in SNpc, accompanied by neuroinflammation that potentiates neuronal cell death, finally leading to parkinsonian's motor symptoms. Important to notice that at each step, the effects become regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway.

Considerations for Potential Applications of the AOP (optional)


  1. This AOP has been developed in order to evaluate the biological plausibility that the adverse outcome i.e. parkinsonian motor deficits, is linked to a MIE that can be triggered by chemical substances i.e. pesticides and chemicals in general. The relevance of the AOP has been documented by tools compounds known to trigger the described AOP. By means of using a human health outcome that has been shown in epidemiological studiesto be association with pesticide exposure, the authors intend to draw attention on this AO in the process of hazard identification. This AOP can be used to support the biological plausibility of this association during the process of evaluation and integration of the epidemiological studies into the risk assessment. It is biologically plausible that a substance triggering the pathway, can be associated with the AO and ultimately with the human health outcome, pending the MoA analysis.
  2. In addition, this AOP can be used to support identification of data gaps that should be explored when a chemical substance is affecting the pathway. Moreover, the AOP provides a scaffold for recommendations on the most adequate study design to investigate the apical endpoints. It is important to note that, although the AO is defined in this AOP as parkinsonian motor deficits, degeneration of DA neurons is already per se an adverse outcome even in situations where it is not leading to parkinsonian motor deficits, and this should be taken into consideration for the regulatory applications of this AOP.
  3. The MIE and KEs identified in this AOP could serve as a basis for assays development that could contribute to an AOP informed-IATA construction which can be applied for different purposes such as: screening and prioritization of chemicals for further testing, hazard characterization or even risk assessment when combined with exposure and ADME information.
  4. This AOP can be used for neurotoxicity assessment, since it is plausible that a compound that binds to the mitochondrial complex I may eventually lead to Parkinsonian motor deficits.

  5. The regulatory applicability of this AOP would be to use experimental findings in model systems representing the MIE and KEs as indicators/alerts for the AO. Risk assessment may be possible if bioavailability at the target cells can be estimated, the toxic concentrations in vitro can be extrapolated to in vivo and exposure scenarios can be simulated.

  6. This AOP can be applied for chemicals that have structural similarities to rotenone or MPTP. However, this AOP may not at the moment be used for chemicals that do not resemble rotenone or MPTP. It is however expected that compounds acting on the same MIE, but belonging to different chemical classes and those that are structurally different, will be tested in the near future in order to substantiate a broader specificity for this AOP. However, it remains evident that chemicals affecting the MIE are potential risk factors for this AO.

 

References


Abdelsalam RMSafar MM J Neurochem. Neuroprotective effects of vildagliptin in rat rotenone Parkinson's disease model: role of RAGE-NFκB and Nrf2-antioxidant signaling pathways. 2015 Jun;133(5):700-7. doi: 10.1111/jnc.13087. Epub 2015 Mar 26.

Aguilar JS, Kostrzewa RM. Neurotoxin mechanisms and processes relevant to parkinson’s disease: un update. Neurotox Res. DOI 10.1007/s12640-015-9519-y.

Alvarez-Fischer D, Guerreiro S, Hunot S, Saurini F, Marien M, Sokoloff P, Hirsch EC, Hartmann A, Michel PP. Modelling Parkinson-like neurodegeneration via osmotic minipump delivery of MPTP and probenecid. J Neurochem. 2008 Nov;107(3):701-11. doi: 10.1111/j.1471-4159.2008.05651.x. Epub 2008 Sep 16.

Arnold, B., et al. (2011). "Integrating Multiple Aspects of Mitochondrial Dynamics in Neurons: Age-Related Differences and Dynamic Changes in a Chronic Rotenone Model." Neurobiology of Disease 41(1): 189-200.

Barbeito AG, Mesci P, Boillee S. 2010. Motor neuron-immune interactions: the vicious circle of ALS. J Neural Transm 117(8): 981-1000.

Barrientos A., and Moraes C.T. (1999) Titrating the Effects of Mitochondrial Complex I Impairment in the Cell Physiology. Vol. 274, No. 23, pp. 16188–16197.

Bernheimer H, Birkmayer W, Hornykiewicz O, Jellinger K, Seitelberger F. Brain dopamine and the syndromes of Parkinson and Huntington. Clinical, morphological and neurochemical correlations. J Neurol Sci. 1973 Dec;20(4):415-55

Betarbet R, Sherer TB, MacKenzie G, Garcia-Osuna M, Panov AV, Greenamyre JT. 2000. Chronic systemic pesticide exposure reproduces features of Parkinson’s disease. Nat Neurosci. 3:1301–6

Betarbet R, Canet-Aviles RM, Sherer TB, Mastroberardino PG,Mc Lendon C, Kim JH, Lund S, Na HM, taylor G, Bence NF, kopito R, seo BB, Yagi T, Yagi A, Klinfelter G, Cookson MR, Greenmyre JT. 2006. Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the pesticide rotenone on DJ-1, α-synuclein, and the ubiquitin-proteasome system. Neurobiology disease. (22) 404-20.

Bezard E, Dovero S, Prunier C, Ravenscroft P, Chalon S, Guilloteau D, Crossman AR, Bioulac B, Brotchie JM, Gross CE (2001) Relationship between the appearance of symptoms and the level of nigrostriatal degeneration in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease. J Neurosci. 21(17):6853-61.

Blasko I, Stampfer-Kountchev M, Robatscher P, Veerhuis R, Eikelenboom P, Grubeck-Loebenstein B. 2004. How chronic inflammation can affect the brain and support the development of Alzheimer's disease in old age: the role of microglia and astrocytes. Aging cell 3(4): 169-176.

Blesa J, Pifl C, Sánchez-González MA, Juri C, García-Cabezas MA, Adánez R, Iglesias E, Collantes M, Peñuelas I, Sánchez-Hernández JJ, Rodríguez-Oroz MC, Avendaño C, Hornykiewicz O, Cavada C, Obeso JA (2012) The nigrostriatal system in the presymptomatic and symptomatic stages in the MPTP monkey model: a PET, histological and biochemical study. Neurobiol Dis. 48(1):79-91.

Bodea LG, Wang Y, Linnartz-Gerlach B, Kopatz J, Sinkkonen L, Musgrove R, et al. 2014. Neurodegeneration by activation of the microglial complement-phagosome pathway. J Neurosci 34(25): 8546-8556.

Borrajo A, Rodriguez-Perez AI, Villar-Cheda B, Guerra MJ, Labandeira-Garcia JL. 2014. Inhibition of the microglial response is essential for the neuroprotective effects of Rho-kinase inhibitors on MPTP-induced dopaminergic cell death. Neuropharmacology 85: 1-8

Brinkley BR, Barham SS, Barranco SC, and Fuller GM. 1974. Rotenone inhibition of spindle microtubule assembly in mammalian cells,” Experimental Cell Research. 85(1)41–46.

Brown GC, Bal-Price A (2003) Inflammatory neurodegeneration mediated by nitric oxide, glutamate, and mitochondria. Mol Neurobiol 27: 325-355

Braun RJ. (2012). Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182.

Brzozowski MJ, Jenner P, Rose S. 2015. Inhibition of i-NOS but not n-NOS protects rat primary cell cultures against MPP(+)-induced neuronal toxicity. J Neural Transm 122(6): 779-788.

Cacquevel M, Lebeurrier N, Cheenne S, Vivien D. 2004. Cytokines in neuroinflammation and Alzheimer's disease. Curr Drug Targets 5(6): 529-534.

Calne DB, Sandler M (1970) L-Dopa and Parkinsonism. Nature. 226(5240):21-4.

Cannon JR, Tapias V, Na HM, Honick AS, Drolet RE, Greenamyre JT (2009) A highly reproducible rotenone model of Parkinson's disease. Neurobiol Dis. 34(2):279-90.

Cappelletti G, Maggioni MG, Maci R. 1999. Influence of MPP+ on the state of tubulin polymerisation in NGF-differentiated PC12 cells. J Neurosci Res. 56(1):28-35.

Cao S, Theodore S, Standaert DG.2010. Fc gamma receprors are required for NF-kB signaling, microglial activation and dopaminergic neurodegeneration in an AAV-synuclein mouse model of Parkinson's disease. molecular neurodegeneration.5-42.

Cappelletti G, Pedrotti B, Maggioni MG, Maci R. 2001. Microtubule assembly is directly affected by MPP(+)in vitro. Cell Biol Int.25(10):981-4.

Castrioto A, Lozano AM, Poon YY, Lang AE, Fallis M, Moro E. 2011. Ten-year outcome of subthalamic stimulation in Parkinson disease: a blinded evaluation. Arch Neurol. 68(12):1550-6.

Chang CY, Choi DK, Lee DK, Hong YJ, Park EJ. 2013. Resveratrol confers protection against rotenone-induced neurotoxicity by modulating myeloperoxidase levels in glial cells. PLoS One 8(4): e60654.

Champy et al. (2004). Annonacin, a lipophilic inhibitor of mitochondrial complex I, induces nigral and striatal neurodegeneration in rats: possible relevance for atypical Parkinsonism in Guadeloupe. J Neurochem 88: 63-69. 

Chao YX, He BP, Tay SS. 2009. Mesenchymal stem cell transplantation attenuates blood brain barrier damage and neuroinflammation and protects dopaminergic neurons against MPTP toxicity in the substantia nigra in a model of Parkinson's disease. J Neuroimmunol 216(1-2): 39-50.

Chen Y, Zhang DQ, Liao Z, Wang B, Gong S, Wang C, Zhang MZ, Wang GH, Cai H, Liao FF, Xu JP 2015. Anti-oxidant polydatin (piceid) protects against substantia nigral motor degeneration in multiple rodent models of Parkinson's disease. Mol Neurodegener. 2;10(1):4.

Chinta SJ, Andersen JK (2006) Reversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: implications for Parkinson's disease. Free Radic Biol Med. 41(9):1442-8.

Choi WS., Kruse S.E., Palmiter R, Xia Z., (2008) Mitochondrial complex I inhibition is not required for dopaminergic neuron death induced by rotenone, MPP, or paraquat. PNAS, 105, 39, 15136-15141

Choi BS, Kim H, Lee HJ, Sapkota K, Park SE, Kim S, Kim SJ (2014) Celastrol from 'Thunder God Vine' protects SH-SY5Y cells through the preservation of mitochondrial function and inhibition of p38 MAPK in a rotenone model of Parkinson's disease. Neurochem Res. 39(1):84-96.

Chiu CC, Yeh TH, Lai SC, Wu-Chou YH, Chen CH, Mochly-Rosen D, Huang YC, Chen YJ, Chen CL, Chang YM, Wang HL, Lu CS. 2015. Neuroprotective effects of aldehyde dehydrogenase 2 activation in rotenone-induced cellular and animal models of parkinsonism. Exp Neurol. 263:244-53.

Chou AP, Li S, Fitzmaurice AG, Bronstein JM. 2010. Mechanisms of rotenone-induced proteasome inhibition. NeuroToxicology. 31:367–372. Chung YC, Kim SR, Park JY, Chung ES, Park KW, Won SY, et al. 2011. Fluoxetine prevents MPTP-induced loss of dopaminergic neurons by inhibiting microglial activation. Neuropharmacology 60(6): 963-974.

Correia SC, Santos RX, Perry G, Zhu X, Moreira PI, Smith MA. (2012). Mitochondrial importance in Alzheimer’s, Huntington’s and Parkinson’s diseases. Adv Exp Med Biol 724:205 – 221.

Cotzias GC, Papavasiliou PS, Gellene R. 1969. L-dopa in parkinson's syndrome. N Engl J Med. 281(5):272.

Cozzolino M, Ferri A, Valle C, Carri MT. (2013). Mitochondria and ALS: implications from novel genes and pathways. Mol Cell Neurosci 55:44 – 49.

Dagda RK, Banerjee TD and Janda E. 2013. How Parkinsonian Toxins Dysregulate the Autophagy Machinery. Int. J. Mol. Sci. 14:22163-22189.

Dauer W, Kholodilov N, Vila M, Trillat AC, Goodchild R, Larsen KE, Staal R, Tieu K, Schmitz Y, Yuan CA, Rocha M, Jackson-Lewis V, Hersch S, Sulzer D, Przedborski S, Burke R, Hen R. 2002. Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. Proc Natl Acad Sci U S A. 99(22):14524-9.

Dauer W, Kholodilov N, Vila M, Trillat AC, Goodchild R, Larsen KE, Staal R, Tieu K, Schmitz Y, Yuan CA, Rocha M, Jackson-Lewis V, Hersch S, Sulzer D, Przedborski S, Burke R, Hen R. 2002. Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP. Proc Natl Acad Sci U S A. 99(22):14524-9.

Dauer W, Przerdborski S. 2003. Parkinson’sdisease: Mechanisms and Models.Neuron. 39, 889-9.

De Bie RM, de Haan RJ, Nijssen PC, Rutgers AW, Beute GN, Bosch DA, Haaxma R, Schmand B, Schuurman PR, Staal MJ, Speelman JD. 1999. Unilateral pallidotomy in Parkinson's disease: a randomised, single-blind, multicentre trial. Lancet. 354(9191):1665-9.

Degli Esposti M, Ghelli A. 1994. The mechanism of proton and electron transport in mitochondrial complex I. Biochim Biophys Acta.1187(2):116–120.

Dehay B, Bove J, Rodriguez-Muela N, Perier C, Recasens A, Boya P, Vila M. 2010. Pathogenic lysosomal depletion in Parkinson’s disease. J. Neurosci. 30:12535–12544.

Dehmer T, Lindenau J, Haid S, Dichgans J, Schulz JB. 2000. Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo. J Neurochem 74(5): 2213-2216.

Deuschl G, Schade-Brittinger C, Krack P, Volkmann J, Schäfer H, Bötzel K, Daniels C, Deutschländer A, Dillmann U, Eisner W, Gruber D, Hamel W, Herzog J, Hilker R, Klebe S, Kloss M, Koy J, Krause M, Kupsch A, Lorenz D, Lorenzl S, Mehdorn HM, Moringlane JR, Oertel W, Pinsker MO, Reichmann H, Reuss A, Schneider GH, Schnitzler A, Steude U, Sturm V, Timmermann L, Tronnier V, Trottenberg T, Wojtecki L, Wolf E, Poewe W, Voges J; German Parkinson Study Group, Neurostimulation Section. 2006. A randomized trial of deep-brain stimulation for Parkinson's disease. N Engl J Med. 355(9):896-908.

Dexter D. T., Jenner P.. Parkinson disease: from pathology to molecular disease mechanisms. Free Radical Biology and Medicine 62 (2013) 132-144

Dietz GPH, Stockhausen KV, Dietz B et al. (2008) Membrane-permeable Bcl-xL prevents MPTP-induced dopaminergic neuronal loss in the substantia nigra. J Neurochem 104:757-765. Doi:10.1111/j.1471-4159.2007.05028.

Drolet RE, Behrouz B, Lookingland KJ, Goudreau JL, 2004. Mice lacking α-synuclein have an attenuated loss of striatal dopamine following prolonged chronic MPTP administration. Neurotoxicology. 25(5):761-9.

Drouin-Ouellet J, St-Amour I, Saint-Pierre M, Lamontagne-Prolux J, Kriz J, Barker R, Cicchetti F.2015. Toll-like receptor expression in the blood and brain of patients and a mouse of Parkinson's disease. International Journal of Neuropsychopharmacology. 1-11.

Du T, Li L, Song N, Xie J, Jiang H (2010) Rosmarinic acid antagonized 1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity in MES23.5 dopaminergic cells. Int J Toxicol. 29(6):625-33.

Efremov RG, Sazanov LA. Respiratory complex I: 'steam engine' of the cell? Curr Opin Struct Biol. 2011 Aug;21(4):532-40. doi: 10.1016/j.sbi.2011.07.002. Epub 2011 Aug 8. Review.

Efremov RG, Sazanov LA. Structure of the membrane domain of respiratory complex I. Nature. 2011 Aug 7;476(7361):414-20. doi: 10.1038/nature10330.

Emmrich JV, Hornik TC, Neher JJ, Brown GC. 2013. Rotenone induces neuronal death by microglial phagocytosis of neurons. The FEBS journal 280(20): 5030-5038.

Esposti et al. (1993) Complex I and Complex III of mitochondria have common inhibitors acting as ubiquinone antagonists. Biochem Biophys Res Commun 190(3): 1090-6. 

Fasano A, Romito LM, Daniele A, Piano C, Zinno M, Bentivoglio AR, Albanese A. 2010. Motor and cognitive outcome in patients with Parkinson's disease 8 years after subthalamic implants. Brain. 133(9):2664-76.

Fato et al. (2009) Differential effects of mitochondrial complex I inhibitors on production of reactive oxygen species. Biochim Biophys Acta 1787(5): 384-392. 

Feng ZH, Wang TG, Li DD, Fung P, Wilson BC, Liu B, et al. 2002. Cyclooxygenase-2-deficient mice are resistant to 1-methyl-4-phenyl1, 2, 3, 6-tetrahydropyridine-induced damage of dopaminergic neurons in the substantia nigra. Neurosci Lett 329(3): 354-358.

Feng J. Mictrotubule. A common target for parkin and Parkinson's disease toxins. Neuroscientist 2006, 12.469-76.

Ferger B, Leng A, Mura A, Hengerer B, Feldon J. 2004. Genetic ablation of tumor necrosis factor-alpha (TNF-alpha) and pharmacological inhibition of TNF-synthesis attenuates MPTP toxicity in mouse striatum. J Neurochem 89(4): 822-833.

Ferrante RJ, Schulz JB, Kowall NW, Beal MF. 1997. Systematic administration of rotenone produces selective damage in the striatum and globus pallidus, but not in the substantia nigra. Brain Research. (753). 157-2.

Ferrari-Toninelli G, Bonini SA, Cenini G, Maccarinelli G, Grilli M, Uberti D, Memo M. 2008. Dopamine receptor agonists for protection and repair in Parkinson's disease. Curr Top Med Chem. 8(12):1089-99.

Friedman LG, lachenmayer ML, Wang J, He L, Poulose SM, Komatsu M, Holstein GR, Yue Z. 2012. Disrupted autophagy leads to dopaminergic axon and dendrite degeneration and promotes presynaptic accumulation of α-synuclein and LRRK2 in the brain. The Journal of Neuroscience. 32 (22) 7585-93.

Fornai F, Lenzi P, Gesi M, Ferrucci M, Lazzeri G, Busceti C, Ruffoli R, Soldani P, Ruggieri S, Alessandri’ MG, Paparelli A. 2003. Fine structure and mechanisms underlying nigrostriatal inclusions and cell death after proteasome inhibition. The journal of neuroscience. 23 (26) 8955-6.

Fornai F., P. Lenzi, M. Gesi et al., “Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells,” Journal of Neurochemistry, vol. 88, no. 1, pp. 114–123, 2004.

Fornai F, Schlüter OM, Lenzi P, Gesi M, Ruffoli R, Ferrucci M, Lazzeri G, Busceti CL, Pontarelli F, Battaglia G, Pellegrini A, Nicoletti F, Ruggieri S, Paparelli A, Südhof TC. 2005. Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitinproteasome system and _α-synuclein. PNAS. 102: 3413–3418.

Freed CR, Breeze RE, Rosenberg NL, Schneck SA, Wells TH, Barrett JN, Grafton ST, Huang SC, Eidelberg D, Rottenberg DA. 1990.

Transplantation of human fetal dopamine cells for Parkinson's disease. Results at 1 year. Arch Neurol. 47(5):505-12.

Fujita KA, Ostaszewski M, Matsuoka Y, Ghosh S, Glaab E, Trefois C, Crespo I, Perumal TM, Jurkowski W, Antony PM, Diederich N, Buttini M, Kodama A, Satagopam VP, Eifes S, Del Sol A, Schneider R, Kitano H, Balling R. 2014. Integrating pathways of Parkinson's disease in a molecular interaction map. Mol Neurobiol.49(1):88-102.

Gandhi S, Wood-Kaczmar A, Yao Z, et al. PINK1-associated Parkinson’s disease is caused by neuronal vulnerability to calcium-induced cell death. Molecular Cell. 2009;33:627–638.

Gao HM, Hong JS, Zhang W, Liu B. 2002. Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 22(3): 782-790.

Gao HM, Liu B, Hong JS. 2003. Critical role for microglial NADPH oxidase in rotenone-induced degeneration of dopaminergic neurons. J Neurosci 23(15): 6181-6187.

Gao L, Brenner D, Llorens-Bobadilla E, Saiz-Castro G, Frank T, Wieghofer P, et al. 2015. Infiltration of circulating myeloid cells through CD95L contributes to neurodegeneration in mice. J Exp Med 212(4): 469-480.

Graier WF, Frieden M, Malli R. (2007). Mitochondria and Ca2+ signaling: old guests, new functions. Pflugers Arch 455:375–396.

Greenamyre, J T., Sherer, T.B., Betarbet, R., and Panov A.V. (2001) Critical Review Complex I and Parkinson’s Disease Life, 52: 135–141.

Greenamayre et al. 2010. Lessons from the rotenone model of Parkinson's disease. Trends pharmacol. Sci. 31(4):141-2

Griffin WS, Sheng JG, Royston MC, Gentleman SM, McKenzie JE, Graham DI, et al. 1998. Glial-neuronal interactions in Alzheimer's disease: the potential role of a 'cytokine cycle' in disease progression. Brain Pathol 8(1): 65-72.


Grivennikova, V.G., Maklashina, E.O., E.V. Gavrikova, A.D. Vinogradov (1997) Interaction of the mitochondrial NADH-ubiquinone reductase with rotenone as related to the enzyme active/inactive transition Biochim. Biophys. Acta, 1319 (1997), pp. 223–232

Hajieva P, Mocko JB, Moosmann B, Behl C (2009) Novel imine antioxidants at low nanomolar concentrations protect dopaminergic cells from oxidative neurotoxicity. J Neurochem. 110(1):118-32.

Hoglinger G.U. et al.2003.Chronic systemic complex I inhibition induces a hypokynetic multisystem degeneration in rats. J.neurochem 84:491-502.

Hornykiewicz O, Kish SJ. 1987. Biochemical pathophysiology of parkinson’s disease. In Parkinson’s Disease. M Yahr and K.J. Bergmann, eds (New.York: Raven Press) 19-34.

Jana S, Sinha M, Chanda D, Roy T, Banerjee K, Munshi S, Patro BS, Chakrabarti S (2011) Mitochondrial dysfunction mediated by quinone oxidation products of dopamine: Implications in dopamine cytotoxicity and pathogenesis of Parkinson's disease. Biochim Biophys Acta. 1812(6):663-73.

Jha N, Jurma O, Lalli G, Liu Y, Pettus EH, Greenamyre JT, Liu RM, Forman HJ, Andersen JK (2000) Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease. J Biol Chem. 275(34):26096-101.

Johnson ME, Bobrovskaya L. 2015. An update on the rotenone models of parkinson’s disease: Their ability to reproduce features of clinical disease and model gene-environment interactions. 946). 101-16.

Heikkila RE, Nicklas WJ, Vyas I, Duvoisin RC. 1985. Dopaminergic toxicity of rotenone and the 1-methyl-4-phenylpyridinium ion after their stereotaxic administration to rats: implication for the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity. Neurosci Lett. 62(3):389-94.

Henderson BT, Clough CG, Hughes RC, Hitchcock ER, Kenny BG. 1991. Implantation of human fetal ventral mesencephalon to the right caudate nucleus in advanced Parkinson's disease. Arch Neurol. 48(8):822-7.

Hirsch EC, Hunot S. 2009. Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol 8(4): 382-397. Hoglinger GU, Feger J, Annick P, Michel PP, Karine P, Champy P, Ruberg M, Wolfgang WO, Hirsch E. 2003. Chronic systemic complexI inhibition induces a hypokinetic multisystem degeneration in rats. J. Neurochem.. (84) 1-12.

Ichimaru N, Murai M, Kakutani N, Kako J, Ishihara A, Nakagawa Y, Miyoshi H. 2008.. Synthesis and Characterization of New Piperazine-Type Inhibitors for Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I). Biochemistry. 47(40)10816–10826.

Inden M, Yoshihisa Kitamura, Hiroki Takeuchi, Takashi Yanagida, Kazuyuki Takata, Yuka Kobayashi, Takashi Taniguchi, Kanji Yoshimoto, Masahiko Kaneko, Yasunobu Okuma, Takahiro Taira, Hiroyoshi Ariga and Shun Shimohama. 2007. Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone. Journal of Neurochemistry. 101.(6).1491–4.

Keeney PM,Xie J,Capaldi RA,Bennett JP Jr. (2006) Parkinson's disease brain mitochondrial complex I has oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci. 10;26(19):5256-64.

Kelly PJ, Ahlskog JE, Goerss SJ, Daube JR, Duffy JR, Kall BA. 1987. Computer-assisted stereotactic ventralis lateralis thalamotomy with microelectrode recording control in patients with Parkinson's disease. Mayo Clin Proc. 62(8):655-64.

Khan MM, Kempuraj D, Zaheer S, Zaheer A. 2014. Glia maturation factor deficiency suppresses 1-methyl-4-phenylpyridinium-induced oxidative stress in astrocytes. J Mol Neurosci 53(4): 590-599.

Kim-Han JS, Dorsey JA, O’Malley KL. 2011. The parkinsonian mimetic MPP+, specifically impairs mitochondrial transport in dopamine axons. The Journal of Neuroscience. 31(19) 7212-1.

Kirk D, Rosenblad C, Burger C, Lundberg C, Johansen TE, Muzyczka N, Mandel R, Bijorklund A. 2002. Parkinson-like neurodegeneration induced by targeted overexpression of α-synuclein in the nigrostriatal system. 22(7) 2780-91.

Kirk D, Annett L, Burger C, Muzyczka N, Mandel R, Bijorklund A. 2003. Nigrostriatal α-synucleinopathy induced by viral vector-mediated overexpression of human α-synuclein: A new primate model of parkinson’s disease. PNAS (100) 2884-9.

Klein RL, King MA, Hamby ME, Meyer EM. 2002. Dopaminergic cell loss induced by human A30P α-synuclein gene transfer to the rat substantia nigra. Hum.Gene.Ther. (13) 605-2.

Koller WC (1992) When does Parkinson's disease begin? Neurology. 42(4 Suppl 4):27-31 Koopman W, Hink M, Verkaart S, Visch H, Smeitink J, Willems P. 2007. Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy. Biochimica et Biophysica Acta 1767:940-947.

Koopman W, Willems P (2012) Monogenic mitochondrial disorders. New Engl J Med. 22;366(12):1132-41. doi: 10.1056/NEJMra1012478.

Kraft AD, Harry GJ. 2011. Features of microglia and neuroinflammation relevant to environmental exposure and neurotoxicity. International journal of environmental research and public health 8(7): 2980-3018.

Lagoa et al. (2011) Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria. Biochimica et Biophys Acta 1807: 1562-1572.

Lang AE, Lozano AM. 1998. Parkinson's disease. Second of two parts. N Engl J Med. 339(16):1130-43.

Langston JW, ballard P, Irwin I. 1983. Chronic parkinsonism in human due to a product of meperidine-analog synthesis. Science. (219) 979-0.

Lapointe N, StHilaire M, martinoli MG, Blanchet J, gould P, Rouillard C, Cicchetti F. 2004. Rotenone induces non-specific central nervous system and systemic toxicity. The FASEB Journal express article 10.1096/fj.03-0677fje

Lauwers E, Debyser Z, Van Drope J, DeStrooper B, Nuttin B. 2003. Neuropathology and neurodegeneration in rodent brain induced by lentiviral vector-mediated overexpression of α-synuclein. Brain pathol. (13) 364-72.

Liu Y, Hu J, Wu J, Zhu C, Hui Y, Han Y, et al. 2012. alpha7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation. J Neuroinflammation 9: 98.

Liu Y, Li W, Tan C, Liu X, Wang X, Gui Y, Qin L, Deng F, Hu C, Chen L. 2014. Meta-analysis comparing deep brain stimulation of the globus pallidus and subthalamic nucleus to treat advanced Parkinson disease. J Neurosurg. 121(3):709-18.

Liu Y, Zeng X, Hui Y, Zhu C, Wu J, Taylor DH, et al. 2015. Activation of alpha7 nicotinic acetylcholine receptors protects astrocytes against oxidative stress-induced apoptosis: implications for Parkinson's disease. Neuropharmacology 91: 87-96.

Liu W, Kong S, Xie Q, Su J, Li W, Guo H, Li S, Feng X, Su Z, Xu Y, Lai X. Protective effects of apigenin against 1-methyl-4-phenylpyridinium ion induced neurotoxicity in PC12 cells. Int J Mol Med. 2015, 35(3):739-46.

Lloyd KG, Davidson L, Hornykiewicz O (1975) The neurochemistry of Parkinson's disease: effect of L-dopa therapy. J Pharmacol Exp Ther. 195(3):453-64.

Lo Bianco C, Ridet JL, Deglon N, Aebischer P. 2002. Alpha-synucleopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson’s disease. Proc.natl.Sci.USA (99)10813-8.

López-Lozano JJ, Bravo G, Abascal J. 1991. Grafting of perfused adrenal medullary tissue into the caudate nucleus of patients with Parkinson's disease. Clinica Puerta de Hierro Neural Transplantation Group. J Neurosurg. 75(2):234-43.

Mangano EN, Litteljohn D, So R, Nelson E, Peters S, Bethune C, et al. 2012. Interferon-gamma plays a role in paraquat-induced neurodegeneration involving oxidative and proinflammatory pathways. Neurobiol Aging 33(7): 1411-1426.

Marella M, Seo BB, Nakamaru-Ogiso E, Greenamyre JT, Matsuno-Yagi A, Yagi T (2008) Protection by the NDI1 gene against neurodegeneration in a rotenone rat model of Parkinson's disease. PLoS One. 3(1):e1433.

Martin LJ. (2011). Mitochondrial pathobiology in ALS. J Bioenerg Biomembr 43:569 – 579.

Matsumoto K, Asano T, Baba T, Miyamoto T, Ohmoto T. 1976. Long-term follow-up results of bilateral thalamotomy for parkinsonism. Appl Neurophysiol. 39(3-4):257-60.

McGeer PL, McGeer EG. 1998. Glial cell reactions in neurodegenerative diseases: Pathophysiology and therapeutic interventions. Alzheimer DisAssocDisord 12 Suppl. 2: S1-S6.

McNaught KS, Jenner P. 2001. Proteasomal function is impaired in substantia nigra in Parkinson’s disease. Neurosci. Lett. 297, 191– 194. McNaught KSC, Olanow W, Halliwell B. 2001. Failure of the ubiquitine-proteasome system in parkinson’s disease. Nature Rev. Neurosci. (2) 589-4.

McNaught KS, Belizaire R, Isacson O, Jenner P, Olanow CW. 2003. Altered proteasomal function in sporadic Parkinson’s disease. Exp. Neurol. 179, 38– 46.

Moldovan AS, Groiss SJ, Elben S, Südmeyer M, Schnitzler A, Wojtecki L. 2015. The treatment of Parkinson's disease with deep brain stimulation: current issues. Neural Regen Res. 10(7):1018-22.

Mudò G, Mäkelä J, Di Liberto V, Tselykh TV, Olivieri M, Piepponen P, Eriksson O, Mälkiä A, Bonomo A, Kairisalo M, Aguirre JA, Korhonen L, Belluardo N, Lindholm D. (2012) Transgenic expression and activation of PGC-1α protect dopaminergic neurons in the MPTP mouse model of Parkinson's disease. Cell Mol Life Sci. 69(7):1153-65.

Narabayashi H, Yokochi F, Nakajima Y. 1984. Levodopa-induced dyskinesia and thalamotomy. J Neurol Neurosurg Psychiatry. 47(8):831-9. Nataraj J, Manivasagam T, Justin Thenmozhi A, Essa MM 2015. Lutein protects dopaminergic neurons against MPTP-induced apoptotic death and motor dysfunction by ameliorating mitochondrial disruption and oxidative stress. Nutr Neurosci. 2015 Mar 2. [Epub ahead of print].

Obeso JA, Rodríguez-Oroz MC, Rodríguez M, Lanciego JL, Artieda J, Gonzalo N, Olanow CW (2000) Pathophysiology of the basal ganglia in Parkinson's disease. Trends Neurosci. 23(10 Suppl):S8-19.

Offen D, Beart PM, Cheung NS et al. (1998) Transegnic mice expressing human Bcl-2 in their neurons are resistant to 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine neurotoxicity. PNAS 95:5789-5794

O’Malley KL. 2010. The role of axonopathy in Parkinson’s disease. 2010. Experimental Neurobiology. (19). 115-19.

Okun, J.G, Lümmen, P and Brandt U., (1999) Three Classes of Inhibitors Share a Common Binding Domain in Mitochondrial Complex I (NADH:Ubiquinone Oxidoreductase) J. Biol. Chem. 274: 2625-2630. doi:10.1074/jbc.274.5.2625

Pan T, Kondo S, Le W, Jankovic J. 2008. The role of autophagy-lysosome pathway in neurodegeneration associated with Parkinson’s disease. Brain. 131, 1969-1978.

Park C.et al. (2003) Quercetin protects the hydrogen peroxide-induced apoptosis via inhibition of mitochondrial dysfunction in H9c2 cardiomyoblast cells. Biochem Pharmacol 66(7): 1287-1295.

Park SE, Sapkota K, Choi JH, Kim MK, Kim YH, Kim KM, Kim KJ, Oh HN, Kim SJ, Kim S (2014) Rutin from Dendropanax morbifera Leveille protects human dopaminergic cells against rotenone induced cell injury through inhibiting JNK and p38 MAPK signaling. Neurochem Res. 39(4):707-18.

Parker WD Jr, Boyson SJ, Parks JK. 1989. Abnormalities of the electron transport chain in idiopathic Parkinson's disease. Ann Neurol.26(6):719-23.

pasqualiL, Caldarazzo-Ienco Fornai . (2014). MPTP neurotoxicity:actions, mechanisms, and animal modeling of Parkinson's disease. In: Kostrzewa RM (ed) Handbook of neurotoxicity. Springer, Heidelberg, pp237-275.

Peschanski M, Defer G, N'Guyen JP, Ricolfi F, Monfort JC, Remy P, Geny C, Samson Y, Hantraye P, Jeny R. 1994. Bilateral motor improvement and alteration of L-dopa effect in two patients with Parkinson's disease following intrastriatal transplantation of foetal ventral mesencephalon. Brain. 117 ( Pt 3):487-99.

Petroske E, Meredith GE, Callen S, Totterdell S, Lau YS (2001) Mouse model of Parkinsonism: a comparison between subacute MPTP and chronic MPTP/probenecid treatment. Neuroscience. 106(3):589-601.

Powers ET1, Morimoto RI, Dillin A, Kelly JW, Balch WE. 2009.. Biological and Chemical Approaches to Diseases of Proteostasis Deficiency. Ann. Rev. Biochem 78: 959–91.

Purisai MG, McCormack AL, Cumine S, Li J, Isla MZ, Di Monte DA. 2007. Microglial activation as a priming event leading to paraquat-induced dopaminergic cell degeneration. Neurobiol Dis 25(2): 392-400. Parker WD Jr, Parks JK, Swerdlow RH (2008) Complex I deficiency in Parkinson's disease frontal cortex. Brain Res. 1189:215-8.

Qian L, Wu HM, Chen SH, Zhang D, Ali SF, Peterson L, et al. 2011. beta2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway. J Immunol 186(7): 4443-4454.

Rappold PM et al.2014. Drp1 inhibition attenuates neurotoxicity and dopamine release deficits in vivo. Nature Communications. 5:5244 doi: 10.1038/ncomms6244.

Ren Y. et al., 2005. Selectivwe vulnerabity of dopaminergic neurons to microtubule depolymerisation. J. Bio. Chem. 280:434105-12. Reynolds GP, Garrett NJ (1986) Striatal dopamine and homovanillic acid in Huntington's disease. J Neural Transm. 65(2):151-5.

Rojo AI, Innamorato NG, Martin-Moreno AM, De Ceballos ML, Yamamoto M, Cuadrado A. 2010. Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson's disease. Glia 58(5): 588-598.

Ros-Bernal F, Hunot S, Herrero MT, Parnadeau S, Corvol JC, Lu L, et al. 2011. Microglial glucocorticoid receptors play a pivotal role in regulating dopaminergic neurodegeneration in parkinsonism. Proc Natl Acad Sci U S A 108(16): 6632-6637.

Rubio-Perez JM, Morillas-Ruiz JM. 2012. A review: inflammatory process in Alzheimer's disease, role of cytokines. ScientificWorldJournal 2012: 756357.

Salama M, Helmy B, El-Gamal M, Reda A, Ellaithy A, Tantawy D, et al. 2013. Role of L-thyroxin in counteracting rotenone induced neurotoxicity in rats. Environmental toxicology and pharmacology 35(2): 270-277.

Sandström JBroyer AZoia DSchilt CGreggio C1, Fournier MDo KQMonnet-Tschudi FPotential mechanisms of development-dependent adverse effects of the herbicide paraquat in 3D rat brain cell cultures.Neurotoxicology. 2017 May;60:116-124. doi: 10.1016/j.neuro.2017.04.010. Epub 2017 Apr 30.

Saravanan KS, Sindhu KM, Senthilkumar KS, Mohanakumar KP. 2006. L-deprenyl protects against rotenone-induced, oxidative stress-mediated dopaminergic neurodegeneration in rats. Neurochem Int.49(1):28-40.

Sathe K, Maetzler W, Lang JD, Mounsey RB, Fleckenstein C, Martin HL, et al. 2012. S100B is increased in Parkinson's disease and ablation protects against MPTP-induced toxicity through the RAGE and TNF-alpha pathway. Brain 135(Pt 11): 3336-3347.

Schapira AH, Cooper JM, Dexter D, Jenner P, Clark JB, and Marsden CD. 1989. Mitochondrial complex I de. ciency in Parkinson’s disease. Lancet. 1,1269.

Scott R, Gregory R, Hines N, Carroll C, Hyman N, Papanasstasiou V, Leather C, Rowe J, Silburn P, Aziz T. 1998. Neuropsychological, neurological and functional outcome following pallidotomy for Parkinson's disease. A consecutive series of eight simultaneous bilateral and twelve unilateral procedures. Brain. 121 ( Pt 4):659-75.

Sherer TB, Betarbet R, Stout AK, Lund S, Baptista M, Panov AV, Cookson MR, Greenamyre JT. 2002. An in vitro model of Parkinson's disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J Neurosci. 22(16):7006-15.

Sherer TB, Betarbet R, Testa CM, Seo BB, Richardson JR, Kim JH, et al. 2003. Mechanism of toxicity in rotenone models of Parkinson’s disease. J Neurosci. 23:10756–64.

Sherer TB, Richardson JR, Testa CM, Seo BB, Panov AV, Yagi T, Matsuno-Yagi A, Miller GW, Greenamyre JT (2007) Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson's disease. J Neurochem. 100(6):1469-79.

Shulman JM, DeJager PL, Feany MB. 2011. Parkinson’s disease: Genetics and Pathogenesis. Annu.Rev.Pathol.Mech.Dis. 6:193-2

Shults CW. 2004. Mitochondrial dysfunction and possible treatments in Parkinson’s disease–a review. Mitochondrion 4:641– 648.

Singer TP, Ramsay RR.The reaction sites of rotenone and ubiquinone with mitochondrial NADH dehydrogenase. Biochim Biophys Acta. 1994 Aug 30;1187(2):198-202.

Spencer DD, Robbins RJ, Naftolin F, Marek KL, Vollmer T, Leranth C, Roth RH, Price LH, Gjedde A, Bunney BS. 1992. Unilateral transplantation of human fetal mesencephalic tissue into the caudate nucleus of patients with Parkinson's disease. N Engl J Med. 1992 Nov 26;327(22):1541-8

Sriram K, Matheson JM, Benkovic SA, Miller DB, Luster MI, O'Callaghan JP. 2002. Mice deficient in TNF receptors are protected against dopaminergic neurotoxicity: implications for Parkinson's disease. Faseb J 16(11): 1474-1476.

Sulzer D, Surmeier DJ. 2013. Neuronal vulnerability, pathogenesis, and Parkinson’s disease. Movement Disorders. 28 (6) 715-24.

Surmeier DJ1, Guzman JN, Sanchez-Padilla J, Goldberg JA. 2010. What causes the death of dopaminergic neurons in Parkinson's disease? Prog Brain Res. 2010;183:59-77. doi: 10.1016/S0079-6123(10)83004

Silva MA, Mattern C, Häcker R, Tomaz C, Huston JP, Schwarting RK. 1997. Increased neostriatal dopamine activity after intraperitoneal or intranasal administration of L-DOPA: on the role of benserazide pretreatment. Synapse. 27(4):294-302.

Swerdlow RH, Parks JK, Miller SW, Tuttle JB, Trimmer PA, Sheehan JP, Bennett JP Jr, Davis RE, Parker WD Jr (1996) Origin and functional consequences of the complex I defect in Parkinson's disease. Ann Neurol. 40(4):663-71.

Taetzsch T, Block ML. 2013. Pesticides, microglial NOX2, and Parkinson's disease. J Biochem Mol Toxicol 27(2): 137-149.

Tansey MG, Goldberg MS. 2009. Neuroinflammation in Parkinson's disease: Its role in neuronal death and implications for therapeutic intervention. Neurobiol Dis.

Thomas B., Banerjee R.,Starkova NN., Zhang S., Calingasan NY, Yang L., Wille E., Lorenzo B., Ho D., Beal M., Starkov A. 2012. Mitochondrial permeability transition pore component cyclophilin D distinguishes nigrostriatal dopaminergic death paradigms in the MPTP mouse model of Parkinson's disease. Antioxidants & redox signaling 16 (9) 855-68

Thundyil J, Lim KL. 2014. DAMPs and Neurodegeneration. Ageing research reviews.

Tieu Kim, Imm Jennifer. 2014. Mitochondrial dynamics as potential therapeutic target for Parkinson’s disease? ACNR 14 (1) 6-8.

Tseng YT, Chang FR, Lo YC2. 2014. The Chinese herbal formula Liuwei dihuang protects dopaminergic neurons against Parkinson's toxin through enhancing antioxidative defense and preventing apoptotic death. Phytomedicine. 21(5):724-33.

Uitti RJ, Ahlskog JE. 1996. Comparative Review of Dopamine Receptor Agonists in Parkinson's Disease. C NS Drugs. 5(5):369-88.

Uitti RJ, Wharen RE Jr, Turk MF, Lucas JA, Finton MJ, Graff-Radford NR, Boylan KB, Goerss SJ, Kall BA, Adler CH, Caviness JN, Atkinson EJ. 1997. Unilateral pallidotomy for Parkinson's disease: comparison of outcome in younger versus elderly patients. Neurology. 49(4):1072-7.

Vivekanantham S, Shah S, Dewji R,Dewji A, Khatri C & Ologunde R.2014. Neuroinflammation in Parkinson's disease: role in neurodegeneration and tissue repair. International Journal Of NeuroscienceAccepted. Author Version Posted Online.

Walter BL, Vitek JL. 2004. Surgical treatment for Parkinson's disease. Lancet Neurol. 3(12):719-28.

Widner H, Tetrud J, Rehncrona S, Snow B, Brundin P, Gustavii B, Björklund A, Lindvall O, Langston JW. 1992. Bilateral fetal mesencephalic grafting in two patients with parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). N Engl J Med. 26;327(22):1556-63.

walker DG, Lue LF, Serrano G, Adler CH, Caviness JN, Sue LI, Beach T.2016. altered expression patterns of inflammation-associated and trophic molecules in substantia nigra and striatum brain samples from Parkinson's disease, incidental Lewy Body disease and normal control cases. Frontiers in Neuroscience. 9:507.

Wang Q, Chu CH, Oyarzabal E, Jiang L, Chen SH, Wilson B, et al. 2014. Subpicomolar diphenyleneiodonium inhibits microglial NADPH oxidase with high specificity and shows great potential as a therapeutic agent for neurodegenerative diseases. Glia 62(12): 2034-2043.

Wang T, Zhang W, Pei Z, Block M, Wilson B, Reece JM, et al. 2006. Reactive microgliosis participates in MPP+-induced dopaminergic neurodegeneration: role of 67 kDa laminin receptor. Faseb J 20(7): 906-915.

Wen Y, Li W, Poteet EC, Xie L, Tan C, Yan LJ, Ju X, Liu R, Qian H, Marvin MA, Goldberg MS, She H, Mao Z, Simpkins JW, Yang SH (2011) Alternative mitochondrial electron transfer as a novel strategy for neuroprotection. J Biol Chem. 286(18):16504-15.

Wu XF, Block ML, Zhang W, Qin L, Wilson B, Zhang WQ, et al. 2005. The role of microglia in paraquat-induced dopaminergic neurotoxicity. Antioxidants & redox signaling 7(5-6): 654-661.

Wu RM, Mohanakumar KP, Murphy DL, Chiueh CC. 1994. Antioxidant mechanism and protection of nigral neurons against MPP+ toxicity by deprenyl (selegiline). Ann N Y Acad Sci. 17;738:214-21.

Yadav S, Gupta SP, Srivastava G, Srivastava PK, Singh MP. 2012. Role of secondary mediators in caffeine-mediated neuroprotection in maneb- and paraquat-induced Parkinson's disease phenotype in the mouse. Neurochem Res 37(4): 875-884.

Zaltieri M, Longhena F, Pizzi M, Missale C, Spano P, Bellucci A. 2015. Mitochondrial Dysfunction and α-Synuclein Synaptic Pathology in Parkinson's Disease: Who's on First? Parkinsons Dis. 2015:108029.

Zhou F, Wu JY, Sun XL, Yao HH, Ding JH, Hu G. 2007. Iptakalim alleviates rotenone-induced degeneration of dopaminergic neurons through inhibiting microglia-mediated neuroinflammation. Neuropsychopharmacology 32(12): 2570-2580.

Zhu JH, Horbinski C, Guo F, Watkins S, Uchiyama Y, Chu CT. 2007.Regulation of autophagy by extracellular signal-regulated protein kinases during 1-methyl-4-phenylpyridinium-induced cell death. Am. J. Pathol. 170:75–86.