Key Event Title
|Level of Biological Organization|
Key Event Components
|loss of dopaminergic neurons||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Mitochondrial dysfunction and Neurotoxicity||KeyEvent|
Key Event Description
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 such as 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, which are commonly used for these purposes, are described below (Tieu, 2011):
• 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) .
Domain of Applicability
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).
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.