This Key Event Relationship is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

Relationship: 905

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Impaired, Proteostasis leads to Degeneration of dopaminergic neurons of the nigrostriatal pathway

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Impaired, Proteostasis

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Degeneration of dopaminergic neurons of the nigrostriatal pathway

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes.Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

AOPs Referencing Relationship

AOP Name	Adjacency	Weight of Evidence	Quantitative Understanding	Point of Contact	Author Status	OECD Status
Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits	adjacent	High	Moderate	Andrea Terron (send email)	Open for citation & comment	WPHA/WNT Endorsed

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Sex Applicability

An indication of the the relevant sex for this KER. More help

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

One of the critical functions in the long-lived cells such as neurons is the clearing system for the removal of the unfolded proteins. This function is provided by two major systems, the Ubiquitin Proteosome System (UPS) and the Autophagy-Lysosome Pathway (ALP) (Tai HC et al. 2008; Korolchuck VI et al. 2010 and Ravikumar B et al. 2010). Impaired proteostasis with formation of misfolded α-synuclein aggregates deregulates microtubule assembly and stability with reduction in axonal transport and impairment of mithocondrial trafficking and energy supply (Esposito et al. 2007; Chen et al. 2007; Borland et al. 2008; O’Malley 2010; Fujita et al. 2014; Weihofen et al. 2009).

Pathological consequences of these deregulated process include interference with the function of synapses, formation of toxic aggregates of proteins, impaired energy metabolism and turnover of mitochondria and chronic endoplasmic reticulum stress; all eventually leading to degeneration of DA neurons in the nigrostriatal pathway (Fujita et al. 2010, Shulman et al. 2011, Dauer et al. 2003, Orimo et al.2008, Raff et al. 2005; Schwarz 2015).

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

The weight of evidence for the relationship between impaired proteostasis and degeneration of dopaminergic neurons of the nigrostriatal pathway is strong. The biological plausibility is based on the knowledge of the physiological cellular process governing the cleaning processes of degradated proteins and organells and on the observations done in genetic and idiopathic forms of Parkinson's disease. Dose and time concordance support a strong response-respose relationships which is also supported by the very well known chronic and progressive behviour of the Parkinson's disease. Although essentiality has been demonstrated in multiple models and lines of evidence, including knockout animals, a single molecular chain of events cannot be established; therefore essentiality for this KEs relationship was considered moderate.

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

The fact that impaired proteostasis can induce degeneration of DA neurons of the nigrostriatal pathway is well known and based on the understanding of the physiological cellular processes involved in removing degraded/misfolded proteins as they are critical for normal mitochondria and axonal transport. Accumulation of misfolded and/or aggregated α-synuclein and the presence of abnormal mitochondria is a consequence of deregulation of this clearing process, and the Lewy bodies, a pathological hallmark of sporadic PD, stain specifically for proteins associated with UPS (Fornai et al., 2003; Gai et al., 2000; McNaught et al., 2002).

Impaired proteostasis has been described in humans affected by sporadic PD (McNaught et al.; 2001, 2003), and changes induced by excess cellular levels of degraded proteins in nigral dopaminergic neurons cause a progressive decline in lysosome function, i.e. ALP system, contributing to neurodegeneration (Decressac et al. 2013). In this context, the ALP system is likely working in a complementary way, with the UPS being the major cleaning system in the soma and the ALP playing a role at pre-synaptic sites (Friedman et al., 2012). Pathological observations from patients affected by PD and from animal models show an increased number of autophagic vacuoles or autophagic markers (Alvarez-Erviti et al., 2010; Crews et al. 2010). Additional observations support the role of impaired proteostasis in nigrostriatal toxicity such as : several genetic variants of sporadic PD are due to susceptible genes able to participate in or modify proteostasis (Shulman et al. 201, Fornai et al. 2003, Shimura et al. 2000, Leroy et al.1998) and striatal microinfusion of proteasome inhibitors induce selective nigrostriatal toxicity with loss of DA and DA metabolites (DA, DOPAC and HVA) in the striatum, retrograde loss of nigral DA cell and intracytoplasmatic inclusions positive for protein of the UPS (Fornai et al. 2003).

Transgenic overexpression of mutant or wild-type forms of α-synuclein in mice causes neuropathological changes including dystrophic neurites and α-synuclein positive LB-inclusion (Dauer et al. 2003; Masiliah et al. 2000). However, they fail to reproduce specific cell death in the nigrostriatal pathway. In contrast, injection of human α-synuclein expressing viral vectors into the SN of adult rats causes a selective death of dopaminergic neurons and formation of LB inclusions (Dauer et al. 2003; Kirik et al. 2002; Lo Bianco et al. 2002). These effects were observed with adeno-associated virus –mediated expression of A30P α-synuclein and with lentiviral-mediated expression of α-synuclein in rats, mice and non-human primates (Shulman 2010; Kirk et al.2003; Klein et al. 2002; Lo Bianco et al. 2002 and 2004; Lauwerset al. 2003).

Impaired proteostasis and formation of proteins aggregates also affect the axonal transport and mitochondrial trafficking. α-synuclein mutants accumulate in the neuronal soma when overexpressed, reducing the axonal transport (Kim-Han et al. 2011; Saha et al.2004); in addition, overexpressed vesicle-associated α-synuclein binds to the microtubules with a detrimental role on axonal transport (Kim-Han et al. 2011; Yang et al. 2010). Postmortem studies on PD patients are indicative of axonal damage. It appears that axonal changes precede neuronal loss, supporting the idea that axonal impairments are early events in neurodegenerative disorders (Orimo et al. 2005 and 2008, Raff 2002, Braak et al. 2004). These changes, and observation from animals models using the chemical stressor MPTP (Meissner et al. 2003, Serra et al. 2002, Hasbani et al. 2006) are supporting the notion that DA neurons of the nigrostriatal pathway degenerate through a “dying back” axonopathy (Raff et al. 2002). It was demonstrated that axonal degeneration follows an active process distinct from cell body loss in a Wallerian degeneration slow (WldS) mutant mouse transgenic model. In this model, axonal degeneration in a variety of disorders is inhibited. In WldS mice, acute treatment with MPTP (20 mg/kg ip for 7 days) resulted in attenuated nigrostriatal axon degeneration, and attenuated DA loss, but cell bodies were not rescued (Hasbani et al. 2006). Indeed, multiple evidences from genetic and experimental models (particularly using MPTP as a chemical stressor) support an early and critical role of axonal impairment with early occurrence of Lewy neurites preceding Lewy bodies formation and cell death (O’Malley 2010).

In addition, a strong link between mitochondrial dysfunction and PD came from the discovery that mutations in PINK1, α-synuclein, LRRK2, parkin and DJ-1, all linked with genetic causes of PD, can affect mitochondrial function (Rappold et al.2014, O’Malley 2010). Deregulation of mitochondrial dynamics (fission, fusion and movement of mitochondria) can affect neuronal activity and viability and imbalance of mitochondrial dynamics have been reported in experimental models of PD with mutated α-synuclein (Tieu, 2014) or chronic model of primary neuronal cells treated with low concentrations (0.1-1 nM) of rotenone (Arnold et al. 2011). Progression of neuronal changes with formation of Lewy neurites and reduction of mitochondrial movement leading to cell death has been also observed in-vitro in a chronic cell-based model (SH-SY5Y neuroblastoma cell line) treated with Rotenone (50nM for 21 days). In this assay, reduction in mitochondrial movement was associated with a progressive damage, first including formation of Lewy neurites, followed by cell death (Borland et al.2008).

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

Degeneration of DA neurons of the nigrostriatal pathway, similar to the one observed in PD, have been reproduced in human and experimental animal models following exposure to MPTP (Dauer 2003; Kitamura et al. 2000; Meissner et al. 2003; Serra et al. 2002; Langston et al. 1983; Rose et al. 1993; Irwin et al. 1993; Forno et al. 1993; Ovadia et al. 1995; Porras et al. 2012 ) and in animals following administration of rotenone through multiple routes of exposure (Betarbet et al. 2000 and 2006, Fleming et al.2004, Schmidt et al. 2002, Inden et al.2007, Saravanan et al. 2005, Sherer et al. 2003; Pan-Montojo et al. 2010 and Johnson et al. 2015). This indicates that both chemicals can be used as a tool compound for experimental investigations on PD and exploring the key event relationship between impaired proteostasis and degeneration of DA neurons of nigrostriatal pathway. Also, similar to PD, susceptibility to MPTP increases with age in both non-human primates and mice (Rose et al.1993, Irwin et al. 1993, Ovadia et al. 1995). It should be noted that the upstream key event includes multiple pathological events, eventually leading to the downstream key event. As it is difficult to assess real time changes for a series of complex and dynamic events in a single experiment, most of the empirical supporting evidences are performed by exploring single factors (e.g. impairment of ALP or UPS or axonal transports) and their role in the degeneration of DA neurons. A selection of studies supporting the empirical evidence is reported below.

Empirical support using MPTP/MPP⁺

Inhibition of the UPS was observed following continuous infusion of MPTP at 1, mg/kg/day for 28 days in mice. A dose related decrease in the enzyme activity of the UPS was observed and this effect was associated with a dose-related decrease of TH positive terminals (densitometry analysis) in the dorsal and ventral striatum. This effect was accompanied by a dose-related cell loss in the SN (counting of TH positive cells) at 5 and 30 mg/kg/day. At 30 mg/kg/day the authors reported cytoplasmic inclusions positively staining for ubiquitin and α-synuclein in neurons of the SN (and locus coeruleus). In the same experiment, acute administration of MPTP (single injection of 30 mg/kg/ or 4 separate injections of 20 mg/kg) induced a transient inhibition of the UPS activity, neuronal loss but no intracytoplasmatic inclusions, indicating that a continuous infusion is necessary to induce permanent inhibition and pathological changes similar to the one observed in PD (Fornai et al. 2005).
In mice lacking α-synuclein, continuous infusion of up to 30 mg/kg/day for 28 days of MPTP neuronal cell death and behavioral symptoms were almost alleviated (Fornai et al. 2005, Dauer et al. 2002).
Administration of MPTP to mice (30 mg/kg/day ip for 5 days) produced autophagosome (AP) accumulation (increase in LC3II) and dopaminergic cell death which was preceded by a decrease in the amount of lysosomes in DA neurons. MPTP also induced mitochondrial- derived ROS and permeabilization of the lysosomal membrane. This resulted in a decrease in Lamp 1 lysosome structural protein and accumulation of undegraded AP and release of lysosomal enzymes into the cytosol. The effect observed in-vivo was quantitatively confirmed in-vitro (human neuroblastoma cell line BEM17(M17EV)). MPP+ was tested in-vitro at the concentrations of 0.25 to 2.5 µM and induced a concentration- related decrease in Lamp1, increase in LC3II, increase in cell death and decrease in lysotracker. In the same in-vitro system, MPP+ also induced lysosome membrane permeabilization. In the same experiment, induction of lysosome biogenesis by the autophagy-enhancer compound rapamycin attenuated the dopaminergic neurodegeneration, both in vitro and in vivo, by restoring lysosomal levels (Dehay et al. 2010).
In an in-vitro microchamber that allowed specific exposure of neuritis of murine mesencephalic neurons, treatment with 1 to 5 µM of MPP+ induced impairment of mitochondrial transport, neurite degeneration (degeneration of proximal dendrites) and autophagy, before cell death (Kim-Han et al. 2011). The number of TH positive cell bodies and neurites was reduced at 1 µM, and axonal fragmentation and LC3 dots increased while tubulin density decreased (Kim-Han et al. 2011).
Mice treated with MPTP at 20mg/kg/day ip for 5 days showed loss of DA neurons in SN which was attenuated by the pharmacological block of mitochondrial fission protein Drp1. Drp 1 blockade also promoted mitochondrial fusion and enhanced the release of DA from the striatal terminals in a PINK1 knockout model showing a defective DA release (Rappold et al. 2014; Tieu et al. 2014).
In differentiated (d6) LUHMENS cell system stably expressing eGFP/mito-tRFP, treatement with MPP+ (5µM) for 24 hours revealed a reuction in the total number of mitochondria in neuritis and a significant reduction in velocity. Partial protection from MPP+ dependent mitochondrial immobilization in neuritis as well as from drop in mitochondria numbers in neuritis was detects following co-treatment with the anti-oxidant Vitamin C (Schildknecht et al. 2013)

Proteasome inhibitors

Intracerebral microinfusion of proteasome inhibitors (lactacystein or epoxomycin at , 100 and 1000 µM) induced loss of TH and DAT immunostaining and decrease in DA and DOPAC in DA terminals in the striatum and loss of nigral cells in SN (counting of TH positive cells). Formation of cell inclusions (positively immunostained for α-synuclein and ubiquitin) and apoptosis were observed after treatment with proteasome inhibitors (0.1 to 50 µM) in an in-vitro system (PC 12 cells). The concentration response curve for apoptosis was shifted to the right compared to the concentration response curve for cellular inclusions indicating that inclusions occurred earlier and independently of cell death. A maximum effect was reached between 1 and 10 µM (Fornai et al.2003).

Empirical support using Rotenone

Administration of rotenone, via osmotic mini pumps implanted to rats (3 mg/kg/day for 7 days) induced decrease of TH in substantia nigra and striatum and decrease in α-synuclein, in its native form, in substantia nigra and striatum, while monoubiquitinated alpha-synuclein increased in the same regions. Valproic acid (VPA) treatment (effective inhibitor of histone deacetylases) significantly counteracted the death of nigral neurons and the 50% drop of striatal dopamine levels caused by rotenone administration.VPA treatment also counteracted both type of α-synuclein alterations. Furthermore, monoubiquitinated alpha-synuclein increased its localization in nuclei isolated from substantia nigra of rotenone-treated rats, an effect also prevented by VPA treatment. Nuclear localization of alpha-synuclein has been recently described in some models of PD and its neurodegenerative effect has been ascribed to histone acetylation inhibition (Monti et al. 2010).
Chronic oral administration of rotenone at 30mg/kg/day in mice produced neuronal loss and degeneration of TH positive terminals in the striatum accompanied by an increase in α-synuclein, ubiquinated proteins and decrease in proteaosomal activity. Concomitant treatment with 4-PBA (a chemical chaperone able to reverse the mislocalization and/or aggregation of proteins) inhibited rotenone-induced neuronal death and decreased protein level of α-synuclein (Inden et al. 2007).
Treatment of Lewis rat with 2 mg/kg/day of rotenone, administered sc for 8 weeks impaired autophagic flux, induced lysosomal dysfunction and degeneration of DA neurons (decrease in number of TH positive cells and decrease in density of TH positive fibers ) in SNpc . The effect of rotenone was paralleled by an increase in LC3 immunopositive dots and upregulation of the LC3II in DA neurons. A concomitant effect was observed and characterized by a decrease in LAMP2 and catepsin immunodots with a diffuse morphological pattern, possibly indicative of decreased lysosomal membrane integrity and leaking to cytosol. In-vitro (PC12 cells) at 500 nM, rotenone also induced increases in α-synuclein, microtubule associated protein 1, light chain 3-II, Beclin 1, p62, increased lysosome permeability and induced cell death. In PC12 cell, the concomitant treatment with trehalose (autophagic inducer) attenuated the rotenone-induced cell death while in-vivo trehalose treatment decreased the rotenone-induced dopaminergic neurons loss (Wu et al. 2015).
Rotenone LD50 of 10 nM in differentiated SH-SY5Y cells decreased autophagic flux at both 2 and 24h. Up-regulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3-methyladenine exacerbated cell death (Giordano et al. 2014)
Treatment of embryonic midbrain neuronal cells with 0.1 to 10 µM rotenone for 30 minutes induces a decrease in polymerized tubulin and increased the number of apoptotic TH+ cells. Similar effects were observed with colchicine treatment, a well-known microtubule-depolyrizing agent and prevented by taxol, a well-known microtubule –stabilizing agent. The effect was considered specific to DA neurons as the effect on apoptosis and cell death was much less evident in GABAergic and glutamatergic neurons (Ren et al. 2005).

Human studies

Inclusion bodies in DA neurons (ie Lewy bodies), a pathological hallmark for sporadic PD, stains specifically for proteins associated with the UPS (Fornai et al. 2003, Gai et al. 2000, Mcnaught et al. 2002), including α-synuclein, parkin and ubiquitin; possibly indicating that failure of the UP system represents a common step in the pathogenesis of PD and impairment of the proteasome system was found in humans affected by sporadic PD (McNaught et al. 2001, 2003).
Lysosomal breakdown and autophagosome (AP) accumulation with co-localization of lysosomal markers in Lewy Bodies is reported to occur in PD brain samples where Lewy bodies were strongly immunoreactive for the autophagosome markers (LC3II), (Dehay et al. 2010).
Postmortem studies on PD patients show axonal pathology that is likely to precede the loss of neuronal bodies In this investigation, TH immunoreactive fibers had almost entirely disappeared with preservation of neuronal bodies (Orimo et al. 2005 and 2008).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

MPTP can induce damage to nigrostriatal neurons without formation of Lewy bodies (hall mark of PD). Acutely intoxicated humans and primates with MPTP lack LB-like formation (Dauer et al. 2003; Forno et al. 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 infusion of MPTP 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, cholinergic and NADPH diaphorase-positive neurons (Hoglinger et al. 2003) or in other brain regions. These results would suggest that Rotenone can induce a more widespread neurotoxicity (Aguilar et al. 2015).

The vulnerability of the dopaminergic pathway still remains circumstantial. The selectivity of MPP+ for dopaminergic neurons is due to its selective uptake via dopamine transporter (DAT), which terminates the synaptic actions of dopamine (Javitch et al. 1985, Pifl et al. 1993, Gainetdinov et al.1997, Hirata et al. 2008). Selectivity of rotenone for dopaminergic neurons is not fully understood (Hirata 2008).

Transgenic overexpression of α-synuclein induces neurotoxicity (ie neuronal atrophy, distrophic neuritis, astrocytosis and LB-like formation). However they fail to cause death of dopaminergic neurons. Nevertheless, injection of the human protein or mutated form expressing viral vectors into the SN, are able to induce all the pathological changes characteristic of PD. This discrepancy could be due to the higher expression of α-synuclein in the viral vector model or because in these models, α-synuclein overexpression would occur suddenly in adult animals (Dauer et al. 2003). In addition, transgenic expression of C-terminal truncated α-synuclein also leads to motor symptoms but neuronal degeneration is not reported (Halls et al. 2015).

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 (Fornai et al. 2005).

Several mechanisms may affect the axonal transport in neurons showing swelling of neurites positive for α-synuclein. These include e.g. ROS production, lysosome and mitochondria membranes depolarization, increased permeability and microtubule depolymerization (Kim-Ham et al.2011, Borland et al.2008, Choi et al.2008). As both MPTP and rotenone could directly trigger these effects, a clear mechanistic understanding leading to cell death is difficult to identify (Aguilar et al. 2015).

Different features of imbalanced proteostasis can trigger one another (e.g. disturbed protein degradation, pathological protein aggregation, microtubule dysfunction); and each of them can lead to cell death. Therefore, the “single” triggering event triggering axonal degeneration or neuronal death is not known. For instance, for α-synuclein aggregation, it is not clear whether this causes death because some vital function of neurons is lost, or whether some protein increases e.g. because of inhibited chaperone-mediate autophagy (Kaushik et al. 2008, Cuervo et al. 2014).

Real-time changes in DA axons are difficult to assess, accounting for the limitation of testing models of structural or trafficking impairment in-vivo.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

As described in the empirical support, a quantitative relationship has been established between chemical stressors inducing impaired proteostasis and loss of DA neurons of nigrostriatal pathway. The response-response relationship was evident in most of the studies and, where possible a relationship in dose-response could be also observed. A chronic dose regimen for the chemical stressor was necessary in most of the studies and this is confirming that a long lasting perturbation of the key event up is necessary to affect neuronal loss consistent with the presence of intracytoplasmatic inclusions. However, some inconsistency in the measurement of the endpoints relevant for impaired proteostasis were observed, probably because they also act as compensatory factors (Betarbet et al. 2006). The acute administration of MPTP (single injection of 30 mg/kg/ or 4 separate injections of 20 mg/kg) induced a transient inhibition of the UPS activity and neuronal loss but no intracytoplasmatic inclusions ie Lewy body were observed, supporting the temporal relationship among the two events (Fornai et al. 2005).

Measured endpoint relevant for the KEup (KE3)	Measured endpoint relevant for the KEdown (KE4)	Model	Reference
Approx. 40% inhibition of UPS	Approx. 38% decrease in TH density in dorsal striatum	MPTP 1mg/kg/day IV infusion for 28 days in mice	Fornai et al. 2005
Approx.50% inhibition of UPS	Approx. 40% decrease in number of TH positive cells/mm2 in SN and approx. 25% decrease in TH in dorsal striatum	MPTP 5mg/kg/day IV infusion for 28 days in mice
Approx.60% inhibition of UPS	Approx. 86% decrease in number of TH positive cells/mm2 in SN and approx. 50% decrease in TH in dorsal striatum and approx. 50% in ventral striatum	MPTP 30mg/kg/day IV infusion for 28 days in mice
Approx. 40% proteasome inhibition	Approx. 70% decrease in DA and 50% decrease in DOPAC in striatum and 30% cell loss in SN	ic infusion of lactacystin (proteasome inhibitors) in rats 100 µM	Fornai et al. 2003
Approx. 50% increase in mRNA expression for α-synuclein	Decrease in TH immunoreactivity (approx. 50%), in TH-positive nerve terminals in the striatum	Transgenic model overexpressing α-synuclein	Kirk et al. 2002
Approx.16-13% reduction in proteosomal activity	Degeneration of nigrostriatal dopaminergic neurons in 50% of animals	Chronic iv treatment (up to 5 weeks) of Lewis rat with rotenone at 2-3 mg/kg day (free brain Rotenone 20-30 nM)	Betabret et al. 2000 and 2006
Approx. 50% increase in α-synuclein	Approx. 57% reduction in TH immunoreactivity in SNpc neurons at 30 mg/kg/day Decrease in TH and DATin the striatum (approx. 30% and 70% respectively) and ventral midbrain area (approx. 60%) at 30 mg/kg/day	Oral chronic administration (28 days) of rotenone (0.25, 1, 2.5, 5, 10 or 30 mg/kg/day) to mice	Inden et al. 2007
Increase in LC3 positive dots in nigral DA neurons (approx. 380%), upregulation of LC3II ( approx. 40%), Beclin 1 (approx. 33%) and P62 (approx. 50%) autophagic substrate	Approx.40% decrease in the number of TH neurons (SNpc) and density of TH positive fibers (approx.50%) (striatum).	2mg/kg/day for 8 wks sc of Rotenone in Levis rats	Wu F. et al., 2015
Approx. 8 fold increase in the number of TH+ neurons with granular LC3	Approx. 40 % decrease in the number of TH immunoreactive neurons.	Primary dopaminergic neurons following treatment with MPP+ (LD50 of 5µM/L)	Zhu et al. 2007
Decrease in mitochondrial speed (approx. 100% decrease in anterograde speed and approx. 28% increase in retrograde speed)	Approx. 70% decrease in positive TH neuronal bodies at 48hours	Treatment with up to 5 µM (1 to 5 µM) of MPP+ in TH positive murine mesencephalic neurons in an in-vitro microchamber segregating system	Kim-Ham et al. 2011
Reduction in mitochondrial movement was statistically significant from day 8 and was greatest on day 16 at 50 nM (approx. day 3 19%, day 6 7%, day 8 62%, day 14 37%, day 16 200%)	Approx 60% of cell loss by day 21	In vitro SH-SY5Y neural cells treated with 50 nM rotenone for 21 days	Borland K. et al., 2008
30% increase over control in static mitochondria and 50 decrease over control in number of mitochondria	Significant decline of intracellular ATP at 24 hours	differentiated (d6) LUHMENS stably expressing eGFP/mito-tRFP, treated with MPP+ (5µM) for 24 hours	Schildknecht S. et al. 2013

Neurotoxicity induced by continuous MPTP administration. (a) Representative tyrosine hydroxylase (TH)-stained sections of the substantia nigra from mice that were continuously treated for 28 days with control pump infusions or with infusions of 1, 5, or 30 mg MPTP/kg daily. (Scale bar, 600 μm.) (b and c) TH-positive cell counts in the substantia nigra (b) and semiquantitative densitometric measurements of the TH signal in striatum (c)(n = 10 mice per group). (d) Striatal monoamine levels in MPTP-treated mice (n = 10 mice per group). Asterisks indicate statistically significant differences (P < 0.05) of a sample compared to control (single asterisks) or to both the control and the lower MPTP dose (double asterisks).(From Fornai et al.2005).

Effect of an α-synuclein deletion on MPTP toxicity. (b) Uptake of [14C]2-DG in littermate wild-type and α-synuclein KO mice that were continuously infused for 7 days with control or MPTP (30 mg/kg daily) solution. Pictures display false-color autoradiograms. (c) Proteasome activity in control and α-synuclein KO mice continuously infused with MPTP (30 mg per kg of body weight daily). Proteasome activities in the substantia nigra are depicted as percent of control (means ± SEMs) as a function of time after beginning of the infusions (five mice per group). In a and c, asterisks indicate statistically significantly different values (P < 0.05) from controls. (From Fornai et al. 2005).

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Multiple animal modeles have been used to mimic PD (Johnson et al. 2015). There are no sex restriction; however, susceptibility to MPTP increases with age in both non-human primates and mice (Rose et al.1993, Irwin et al. 1993, Ovadia et al. 1995).

References

List of the literature that was cited for this KER description. More help

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

Alvarez-Erviti L, Rodriguez-Oroz MC, Cooper JM, Caballero JD, Ferrer I, Obeso JI, Schapira AHV. 2010. Chaperone-Mediated Autophagy Markers in Parkinson Disease Brains. Arch Neurol. 67(12). 1464-2.

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.

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.2006. Neurobiology disease. (22) 404-20.

Borland MK, Trimmer PA, Rubinstein JD, et al (2008). Chronic, low dose rotenone reproduces Lewy neuritis found in early stages of Parkinson’s disease, reduces nitochondrial movement and slowly kill differentiated SH-SY5Y neural cells. Mol Neurodegener 3-21.

Chen L, Jin J, Davis J (2007). Oligomeric α-synuclein inhibits tubulin polymerization. Biochem Biophys Res Commun (356) 548-3.

Choi WS, Kruse SE, Palmiter RD, Xia Z. 2008. Mitochondrial complexI inhibition is not required for dopaminergic neuron death induced by rotenone, MPP+, or paraquat. PNAS. 105 (39) 15136-41.

Crews L, Spencer B, Desplats P, Patrick C, Paulino A, Rockenstein E, Hansen L, Adame A, Galasko D, Malsiah E. 2010. Selective molecular alterations in the autophagy pathway in patients with lewy body disease and in models of α-synucleinopathy. 5(2)1-16.

Cuervo AM, Wong E. 2014. Chaperone-mediated autophagy: roles in disease and aging. Cell Research. (24); 92-104.

Decressac M, Björklund A. 2013. Pathogenic role and therapeutic target in Parkinson disease. Autophagy. 9, (8). 1–3.

Dehay B, bove J, Rodriguez-manuela N, perier C, Recasens A, boya P, vila M. 2010. Pathogenic lysosomal depletion in parkinson’s disease. The journal of neuroscience. 30(37) 12535-12544.

Dauer W, Kholodilov N, Vila M, Trillat AC, Goodchild R, Larsen KE, Staal R, Tieu K, Schmitz Y, Yuan CA, Rocha M, Lewis VJ, Hersch S, Sulzer D, Przedborski S, burke R, Hen R. 2002. Resistance of α-synuclein null mice to the parkinsonian neurotoxicity MPTP. PNAS. (99) 14524-9.

Dauer W, Przerdborski S. 2003. Parkinson’sdisease: Mechanisms and Models.Neuron. 39, 889-9. Esposito A, Dohm CP, Kermer P. (2007). α-synuclein and its disease-related mutants interact differentially with the microtubule protein tau and associate with actin cytoskeleton. Neurobiol Dis. (26) 521-1.

Fleming SM, Zhu C, Fernagut PO, Mehta A, DiCarlo CD, Seaman R, Chesselet MF. 2004. Behavioral and immunohistochemical effects of chronic intravenous and subcutaneous infusion of varying doses of rotenone. Experimental neurology. (187). 418-9.

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.

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, Schluter OM, lenzi P, Gesi M, Ruffoli R, Ferrucci M, Lazzeri G, Busceti CL, pontarelli F, battaglia G, pellegrini A, Nicoletti F, Ruggeri S, paparelli A, Sudhof TC. 2005. Parkinson-like syndrome induced by continuous MPTP infusion: Convergent roles of the ubiquitin-proteasome system and α-synuclein. PNAS.102(9) 3413-18.

Forno LS, DeLanney LE, Irwin I, Langston JW.1993. Similarities and differences between MPTP-induced parkinsonsim and Parkinson's disease. Neuropathologic considerations. Adv Neurol. (60). 600-8.

Forno LS, Langston JW, DeLanney LE, Irwin I, Ricaurte GA. 1986. Locus ceruleus lesions and eosinophilic inclusions in MPTP-treated monkeys. 20,( 4) 449–5.

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.

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.

Gai WP, Yuan HX, Li XQ, Power JT, Blumbergs PC, Jensen PH. 2000. In situ and in vitro study of colocalization and segregation of alpha-synuclein, ubiquitin, and lipids in Lewy bodies. Exp Neurol. 166(2):324-33.

Gainetdinov RR, Fumagalli F, Jones SR, Caron MG.1997. Dopamine transporter is required for in vivo MPTP neurotoxicity: evidence from mice lacking the transporter. J.Neurochem.(69). 1322-5.

Giordano, S., et al. (2014). "Bioenergetic Adaptation in Response to Autophagy Regulators During Rotenone Exposure." Journal of Neurochemistry 131: 625-633.

Hall K, Yang S, Sauchanka O, Spillantini MG, Anichtchik O. 2015. Behavioural deficits in transgenic mice expressing human truncated (1-120 amino acid) alpha-synuclein. Exp Neurol. 264:8-13.

Hasbani DM, O’malley KL. 2006. Wlds mice are protected against Parkinsonisn mimetic MPTP. Experimental Neurology. (202) 93-9. 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.

Hirata Y, Suzuno H, Tsuruta T, Oh-hashi K, Kiuchi K. 2008. The role of dopaminergic transporter in selective toxicity of manganese and rotenone.Toxicology.(244). 249-6.

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.

Javitch JA, D’Amato RJ, Strittmatter SM, Snyder SH. 1985. Parkinson inducing neurotoxin, MPTP,: uptake of the metabolite MPP+ by dopamine neurons explains selective toxicity. Proc. Natl.Acad.Sci.(82). 2173-77

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.

Kaushik S. and A. M. Cuervo. 2008. Chaperone Mediated Autophagy. Methods Mol Biol. 2008 ; 445: 227–244.

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.

Kitamura Y, Shimohama S, Akaike A, Taniguchi T. The Parkinsonian Models: Invertebrates to Mammals. 2000 The Japanese Journal of Pharmacology. 84 (3) 237-3.

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.

Korolochuk VI, Menzies FM, Rubinsztein DC. 2010. Mechanism of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems. FEBS Lett. (584) 1393-8.

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.

Irwin JK, 1993, Parkinson’s disease: Past, Present and Future. Neuropsycopharmacology. 9(1). 1-1. 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. 2013. Neuropathology and neurodegeneration in rodent brain induced by lentiviral vector-mediated overexpression of α-synuclein. Brain pathol. (13) 364-72.

Leroy E, Boyer R, Auburger G, Leube B, Ulm G, Mezey E, Harta G, Brownstein MJ, Jonnalagada S, Chernova T, Dehejia A, Lavedan C, gasser T, Steinbach PI, Wilkinson KD, Polymeopoulos MH. 1998. The ubiquitin pathway in parkinson’s disease. Nature. (395) 451-2.

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.

Lo Bianco C, Schneider BL, bauer M, Sajadi A, Brice A. 2004. Lentiviral vector delivery of parkin prevents dopaminergic degeneration in a α-synuclein rat model of parkinson’s disease. Proc.Natl.Acad.Sci. (101). 17510-15.

Masiliah E, Rockenstein E, Veibergs I, malloty M, Hashimoto M, takeda A, Sagara Y, Sisk A, Mucke L. 2000. Dopaminergic loss and inclusion body formation in α-synuclein mice: inmplications for neurodegenerative disorders. Science (287) 1265-9.

Meissner W, prunier C, Guilloteau D, Chalon S, Gross CE, bezard E. 2003. Time-course of nigrostriatal degeneration in a progressive MPTP-lesioned macaque model of parkinson’s disease. Molecular Neurobiology. (3) 209-8.

McNaught KSC, Olanow W, Halliwell B. 2001. Failure of the ubiquitine-proteasome system in parkinson’s disease. Nature Rev. Neurosci. (2) 589-4.

McNaught KSP, Belizaire R, Isacson O, Jenner P, Olanow CW. 2002. Altered proteasomal function in sporadic Parkinson’s disease. Experimental Neurology (179) 38-46.

McNaught KSP, Olanow CW. 2003). Proteolytic Stress: A Unifying Concept for the Etiopathogenesis of Parkinson’s Disease. Ann Neurol ;53 (3):73–6.

Monti, B. Gatta V, Piretti F, Raffaelli S,Virgili M, Contestabile A. (2010). "Valproic Acid Is Neuroprotective in the Rotenone Rat Model of Parkinson's Disease: Involvement of Alpha-Synuclein." Neurotoxicity Research 17: 130-41.

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.

Ovadia A, Zhang Z, Gash DM. 1995. Increased susceptibility to MPTP in middle-aged Rhesus Monkeys. Neurobiology of aging. 16 (6) 931-7 Pan-Montojo F, Anichtchik O, Dening Y, knels L, pursche S, Jung R, Jackson S, gille G, Spillantini MG, Reichmann H, Funk RHW. 2010. Progression of Parkinson’s disease pathology is reproduced by intragastric administration of rotenone in mice. PloS ONE. 5(1) 1-10.

Pifl C, Giros B, Caron MG. 2004. Dopamine transporter expression confers cytotoxicity to low doses of the parkinsonism-inducing neurotoxin MPTP. J.Neurosci. (13) 4246-3.

Porras G, Bezard E, 2012. Modelling Parkinson’s disease in primates: The MPTP model. Cold Spring Harb Perspect Med. 2. 1-0. Raff MC, Whitemore AV, Finn JT. 2002. Axonal self-destruction and neurodegeneration. Science (296) 868-1.

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

Ravikumar Bb, Sarkar S, Davies JE et al . 2010. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol rev. (90) 1383-435.

Ren Y, liu W, Jiang H, jiang Q, Feng J. 2005. Selective vulnerability of dopaminergic neurons to microtubule depolymerisation. 280(40). 34105-12. 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.

Saha AR, Utton MA, Asuni AA, Ackerley S, Grierson AJ, Miller CC, Davies AM, Bucham VI, Anderton BH, Hanger DP. 2004. Parkinson’s disease alpha-synuclein mutation exhibit defective axonal transport in cultured neurons J.Cell Sci (117) 1017-24.

Saravan KS, Sindhu K, Mohanakumar P. 2005. Acute intranigral infusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to parkinson’s disease. Brian research. (1049). 147-5.

Serra PA, Sciola L, Delogu MR, Spano A, Monaco G, Miele E, Rocchitta G, Miele M, Migheli R, Desole MS. 2002. The neurotoxin MPTP induces apoptosis in mouse nigrostriatal glia.. The Journal of Biological Chemestry. 277(37) 34451-61

Sherer TB, kim JH, betarbet R, Greenmayre JT. 2002. Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and α synuclein aggregation. Experimental neurology. (179). 9-16.

Schildknecht S, Karreman C, Pöltl D, Efrémova L, Kullmann C, Gutbier S, KrugA, Scholz D, Gerding HR, Leist M. 2013.Generation of genetically-modified human differentiated cells for toxicological tests and the study of neurodegenerative diseases. ALTEX. ;30(4):427-44. Shimura H, hattori N, Kubo S, Mizuno Y, Asakawa S, Minoshima S, Shimizu N, Chiba IK, Tanaka K, Suzuki T. 2000. Familial Parkinson’s disease gene product, parkin, is an ubiquitin-protein ligase. Nat Genett.(25). 302-5.

Schmidt MA. 2002. Rotenone destroys dopaminergic neurons and induces parkinsonian symptoms in rats. Behavioural brain research. (136) 317-4.

Schwarz TL. 2015. Mitochondrial trafficking in neurons. Cold Spring Harb Perspect Biol. 2013-5.

Shulman JM, DeJager PL, Feany MB. 2011. Parkinson’s disease: Genetics and Pathogenesis. Annu.Rev.Pathol.Mech.Dis. 6:193-2. Tai HC, Schuman EM. 2008. Ubiquitin, the proteasome and the protein degradation in neuronl function and dysfunction. Nat.Rev. Neurosci (9) 826-38.

Tieu Kim, Imm Jennifer. 2014. Mitochondrial dynamics as potential therapeutic target for Parkinson’s disease? ACNR 14 (1) 6-8. Yang MI, Hasasdri L, Woods WS, George JM. 2010. Dynamic transport and localization of alpha-synuclein in primary hippocampal neurons. Molneurodegener 5-9.

Weihofen A, Thomas KJ, Ostaszewski BL. (2009). Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking. Biochemistry (48). 2045-2. Wu F, Xu HD, Guan JJ, Hou YS, Gu JH, Zhen XC, Qin ZH. 2015. Rotenone impairs autophagic flux and lysosomal functions in parkinson’s disease. (284) 900-11.'