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Dopamine and synuclein

(70 References)

Alves Da Costa, C., E. Masliah, et al. (2003). "beta -synuclein displays an antiapoptotic p53-dependent phenotype and protects neurons from 6-hydroxydopamine-induced caspase 3 activation: Cross-talk with alpha -synuclein and implication for parkinson's diseases l." J Biol Chem.

            We have established stable transfectants expressing beta-synuclein in TSM1 neurons. We show that in basal andstaurosporine -induced conditions, the number of Tunel- positive beta-synuclein -expressing neurons was drastically lower than in mock-transfected TSM1 cells. This was accompanied by a lower DNA fragmentation as evidenced by the reduction of propidium iodide incorporation measured by FACS analysis. beta-synuclein strongly reduces staurosporine-induced caspase 3 activity and immuno-reactivity. We establish that beta-synuclein triggers drastic reduction of p53 expression and transcriptional activity. This was accompanied by increased Mdm2-immunoreactivity while p38 expression appears enhanced, indicating that beta-synuclein-induced p53 down-regulation likely occurs at a post-transcriptional level. We previously showed that alpha-synuclein displays an antiapoptotic function that was abolished by the dopaminergic-derived toxin, 6Hydroxy-dopamine (6OHDA). Interestingly, beta-synuclein reta ins its ability to protect TSM1 neurons even after 6OHDA- treatment. Furthermore, beta-synuclein restores the antiapoptotic function of alpha-synuclein in 6OHDA-treated neurons. Altogether our data document for the first time, that beta-synuclein protects neurons from staurosporine and 6OHDA-stimulated caspase activation in a p53-dependent manner. Our observation that beta-synuclein contributes to restore the alpha-synuclein antiapoptotic function abolished by 6OHDA may have direct implications for Parkinsons disease pathology. In this context, the cross-talk between these two parent proteins is discussed.

Alves Da Costa, C., E. Paitel, et al. (2002). "Alpha-synuclein lowers p53-dependent apoptotic response of neuronal cells. Abolishment by 6-hydroxydopamine and implication for Parkinson's disease." J Biol Chem 277(52): 50980-4.

            We have examined the influence of alpha-synuclein on the responsiveness of TSM1 neuronal cells to apoptotic stimulus. We show that alpha-synuclein drastically lowers basal and staurosporine-stimulated caspase 3 immunoreactivity and activity. This is accompanied by lower DNA fragmentation and reduced number of terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)-positive neurons. Interestingly, alpha-synuclein also diminishes both p53 expression and transcriptional activity. We demonstrate that the antiapoptotic phenotype displayed by alpha-synuclein can be fully reversed by the Parkinson's disease-associated dopamine derivative 6-hydroxydopamine. Thus, 6-hydroxydopamine fully abolishes the alpha-synuclein-mediated reduction of caspase 3 activity and reverses the associated decrease of p53 expression. 6-Hydroxydopamine triggers thioflavin T-positive deposits in alpha-synuclein, but not mock-transfected TSM1 neurons, and drastically increases alpha-synuclein immunoreactivity. Altogether, we suggest that alpha-synuclein lowers the p53-dependent caspase 3 activation of TSM1 in response to apoptotic stimuli and we propose that the natural toxin 6-hydroxydopamine abolishes this antiapoptotic phenotype by triggering alpha-synuclein aggregation, thereby likely contributing to Parkinson's disease neuropathology.

Asanuma, M., I. Miyazaki, et al. (2003). "Dopamine- or L-DOPA-induced neurotoxicity: the role of dopamine quinone formation and tyrosinase in a model of Parkinson's disease." Neurotox Res 5(3): 165-76.

            Dopamine (DA)- or L-dihydroxyphenylalanine-(L-DOPA-) induced neurotoxicity is thought to be involved not only in adverse reactions induced by long-term L-DOPA therapy but also in the pathogenesis of Parkinson's disease. Numerous in vitro and in vivo studies concerning DA- or L-DOPA-induced neurotoxicity have been reported in recent decades. The reactive oxygen or nitrogen species generated in the enzymatical oxidation or auto-oxidation of an excess amount of DA induce neuronal damage and/or apoptotic or non-apoptotic cell death; the DA-induced damage is prevented by various intrinsic and extrinsic antioxidants. DA and its metabolites containing two hydroxyl residues exert cytotoxicity in dopaminergic neuronal cells mainly due to the generation of highly reactive DA and DOPA quinones which are dopaminergic neuron-specific cytotoxic molecules. DA and DOPA quinones may irreversibly alter protein function through the formation of 5-cysteinyl-catechols on the proteins. For example, the formation of DA quinone-alpha-synuclein consequently increases cytotoxic protofibrils and the covalent modification of tyrosine hydroxylase by DA quinones. The melanin-synthetic enzyme tyrosinase in the brain may rapidly oxidize excess amounts of cytosolic DA and L-DOPA, thereby preventing slowly progressive cell damage by auto-oxidation of DA, thus maintainng DA levels. Since tyrosinase also possesses catecholamine-synthesizing activity in the absence of tyrosine hydroxylase (TH), the double-edged synthesizing and oxidizing functions of tyrosinase in the dopaminergic system suggest its potential for application in the synthesis of DA, instead of TH in the degeneration of dopaminergic neurons, and in the normalization of abnormal DA turnover in the long-term L-DOPA-treated Parkinson's disease patients.

Auluck, P. K. and N. M. Bonini (2002). "Pharmacological prevention of Parkinson disease in Drosophila." Nat Med 8(11): 1185-6.

Auluck, P. K., H. Y. Chan, et al. (2002). "Chaperone suppression of alpha-synuclein toxicity in a Drosophila model for Parkinson's disease." Science 295(5556): 865-8.

            Parkinson's disease is a movement disorder characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta. Dopaminergic neuronal loss also occurs in Drosophila melanogaster upon directed expression of alpha-synuclein, a protein implicated in the pathogenesis of Parkinson's disease and a major component of proteinaceous Lewy bodies. We report that directed expression of the molecular chaperone Hsp70 prevented dopaminergic neuronal loss associated with alpha-synuclein in Drosophila and that interference with endogenous chaperone activity accelerated alpha-synuclein toxicity. Furthermore, Lewy bodies in human postmortem tissue immunostained for molecular chaperones, also suggesting that chaperones may play a role in Parkinson's disease progression.

Baptista, M. J., C. O'Farrell, et al. (2003). "Co-ordinate transcriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines." J Neurochem 85(4): 957-68.

            Abnormal accumulation of alpha-synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) alpha-synuclein using microarrays, with confirmation of selected genes by quantitative RT-PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis-inducing molecules, transcription factors and membrane-bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co-ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild-type but not mutant alpha-synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co-ordinate regulation of dopamine synthesis is regulated through this transcription factor.

Betarbet, R., T. B. Sherer, et al. (2002). "Animal models of Parkinson's disease." Bioessays 24(4): 308-18.

            Animal models are important tools in experimental medical science to better understand pathogenesis of human diseases. Once developed, these models can be exploited to test therapeutic approaches for treating functional disturbances observed in the disease of interest. On the basis of experimental and clinical findings, Parkinson's disease (PD) was the first neurological disease to be modeled and, subsequently, to be treated by neurotransmitter replacement therapy. Agents that selectively disrupt or destroy catecholaminergic systems, such as reserpine, methamphetamine, 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine have been used to develop PD models. Recently, it has been found that agricultural chemicals, such as rotenone and paraquat, when administered systemically, can reproduce specific features of PD in rodents, apparently via oxidative damage. Transgenic animals that over-express alpha-synuclein are used to study the role of this protein in dopaminergic degeneration. This review critically discusses animal models of PD and compares them with characteristics of the human disease.

Burke, W. J. (2003). "3,4-dihydroxyphenylacetaldehyde: a potential target for neuroprotective therapy in Parkinson's disease." Curr Drug Target CNS Neurol Disord 2(2): 143-8.

            The simplest explanation for the selective loss of substantia nigra (SN) dopamine (DA) neurons in Parkinson's disease (PD) is that DA or a metabolite is neurotoxic. Recently, a series of investigations implicate the MAO metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), as the critical endogenous toxin which triggers DA neuron loss in PD: 1. Hereditary PD contains mutations in the gene for alpha-synuclein (alpha-syn). Investigations implicate a DA metabolite as mediator of alpha-syn neurotoxicity, and DOPAL is 1000-fold more toxic than DA in vivo. 2. A deficit in mitochondrial complex I is found in PD SN. Inhibition of complex I causes increases in DOPAL levels and death of DA neurons in vitro and in vivo. 3. L-DOPA, the precursor of DA, which is used to treat PD, is toxic and contributes to the progression of PD. L-DOPA-treated rats have an 18-fold increase in striatal DOPAL. 4. Free hydroxyl radicals (.OH) trigger aggregation of alpha-syn to its toxic form. DOPAL with H(2)O(2) generates.OH radicals. These investigations provide several therapeutic strategies to limit DOPAL toxicity and progression of PD: 1. Delaying the start of L-DOPA therapy by early use of DA receptor agonists, which may also be free radical scavengers, limits the amount of DOPAL formed from L-DOPA. 2. Nonspecific MAO inhibitors may more effectively decrease production of DOPAL from DA than MAO-B inhibitors. 3. Newer more potent and targeted free radical scavengers could block DOPAL toxicity. 4. Coenzyme Q(10) increases complex I activity and nicotine adenine dinucleotide (NAD) synthesis, and thereby could enhance DOPAL catabolism by aldehyde dehydrogenase, which uses NAD as a cofactor. 5. DA uptake blockers could be used to limit intraneuronal DOPAL production. 6. Tauroursodeoxycholic acid, an inhibitor of apoptosis shown to be effective in models of Huntington's disease, may also prove effective in blocking DOPAL toxicity in PD. 7. Agents which block aggregation of alpha-syn should limit DOPAL toxicity.

Cabin, D. E., K. Shimazu, et al. (2002). "Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein." J Neurosci 22(20): 8797-807.

            Although the mutation of alpha-synuclein, a protein associated with presynaptic vesicles, is implicated in the etiology and pathogenesis of Parkinson's disease, the biological function of the normal protein is unknown. Mice that lack alpha-synuclein have been generated by homologous recombination in embryonic stem cells. Electron microscopic examination of hippocampal synapses revealed a striking selective deficiency of undocked vesicles without affecting docked vesicles. Field recording of CA1 synapses in hippocampal slices from the mutant mice demonstrated normal basal synaptic transmission, paired-pulse facilitation, and response to a brief train of high-frequency stimulation (100 Hz, 40 pulses) that exhausts only docked vesicles. In contrast, the alpha-synuclein knock-out mice exhibited significant impairments in synaptic response to a prolonged train of repetitive stimulation (12.5 Hz, 300 pulses) capable of depleting docked as well as reserve pool vesicles. Moreover, the replenishment of the docked vesicles by reserve pool vesicles after depletion was slower in the mutant synapses. Thus, alpha-synuclein may be required for the genesis and/or maintenance of a subset of presynaptic vesicles, those in the "reserve" or "resting" pools. These results reveal, for the first time, the normal function of endogenous alpha-synuclein in regulating synaptic vesicle mobilization at nerve terminals.

Dauer, W., N. Kholodilov, et al. (2002). "Resistance of alpha -synuclein null mice to the parkinsonian neurotoxin MPTP." Proc Natl Acad Sci U S A 99(22): 14524-9.

            Parkinson's disease (PD) is most commonly a sporadic illness, and is characterized by degeneration of substantia nigra dopamine (DA) neurons and abnormal cytoplasmic aggregates of alpha-synuclein. Rarely, PD may be caused by missense mutations in alpha-synuclein. MPTP, a neurotoxin that inhibits mitochondrial complex I, is a prototype for an environmental cause of PD because it produces a pattern of DA neurodegeneration that closely resembles the neuropathology of PD. Here we show that alpha-synuclein null mice display striking resistance to MPTP-induced degeneration of DA neurons and DA release, and this resistance appears to result from an inability of the toxin to inhibit complex I. Contrary to predictions from in vitro data, this resistance is not due to abnormalities of the DA transporter, which appears to function normally in alpha-synuclein null mice. Our results suggest that some genetic and environmental factors that increase susceptibility to PD may interact with a common molecular pathway, and represent the first demonstration that normal alpha-synuclein function may be important to DA neuron viability.

Dawson, T., A. Mandir, et al. (2002). "Animal models of PD: pieces of the same puzzle?" Neuron 35(2): 219-22.

            Parkinson's disease (PD) is a common neurodegenerative disorder with no known cure. The etiology of PD is likely due, in part, to combinations of genetic susceptibilities and environmental factors. In rare familial cases, PD is due to genetic mutations. A number of new genetic and toxin models of PD and advances in older models are yielding important new information about the pathogenesis of PD. This has prompted us to critically review the current animal models for PD and discuss how these models may yield fresh insights into the pathogenesis of PD, as well as new therapeutic opportunities.

Di Monte, D. A., M. Lavasani, et al. (2002). "Environmental factors in Parkinson's disease." Neurotoxicology 23(4-5): 487-502.

            Evidence discussed in this review article lends strong support in favor of an etiologic role of environmentalfactors in Parkinson's disease. First, thanks to the discovery of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), it is now clear that, by targeting the nigrostriatal system, neurotoxicants can reproduce the neurochemical and pathological features of idiopathic parkinsonism. The sequence of toxic events triggered by MPTP has also provided us with intriguing clues concerning mechanisms of toxicant selectivity and nigrostriatal vulnerability. Relevant examples are (i) the role of the plasma membrane dopamine transporter in facilitating the access of potentially toxic species into dopaminergic neurons; (ii) the vulnerability of the nigrostriatal system to failure of mitochondrial energy metabolism; and (iii) the contribution of inflammatory processes to tissue lesioning. Epidemiological and experimental data suggest the potential involvement of specific agents as neurotoxicants (e.g. pesticides) or neuroprotective compounds (e.g. tobacco products) in the pathogenesis of nigrostriatal degeneration, further supporting a relationship between the environment and Parkinson's disease. A likely scenario that emerges from our current knowledge is that neurodegeneration results from multiple events and interactive mechanisms. These may include (i) the synergistic action of endogenous and exogenous toxins (e.g. the ability of the pesticide diethyldithiocarbamate to promote the toxicity of other compounds); (ii) the interactions of toxic agents with endogenous elements (e.g. the protein alpha-synuclein); (iii) the tissue response to an initial toxic insult; and, last but not least, (iv) the effects of environmental factors on the background of genetic predisposition and aging.

Dong, Z., B. Ferger, et al. (2002). "Overexpression of Parkinson's disease-associated alpha-synucleinA53T by recombinant adeno-associated virus in mice does not increase the vulnerability of dopaminergic neurons to MPTP." J Neurobiol 53(1): 1-10.

            Mutations in the alpha-synuclein gene are linked to a rare dominant form of familial Parkinson's disease, and alpha-synuclein is aggregated in Lewy bodies of both sporadic and dominant Parkinson's disease. It has been proposed that mutated alpha-synuclein causes dopaminergic neuron loss by enhancing the vulnerability of these neurons to a variety of insults, including oxidative stress, apoptotic stimuli, and selective dopaminergic neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test this hypothesis in vivo, we overexpressed human alpha-synuclein(A53T) in the substantia nigra of normal and MPTP-treated mice by rAAV-mediated gene transfer. Determination of dopaminergic neuron survival, striatal tyrosine hydroxylase fiber density, and striatal content of dopamine and its metabolites in rAAV-injected and uninjected hemispheres demonstrated that alpha-synuclein(A53T) does not increase the susceptibility of dopaminergic neurons to MPTP. Our findings argue against a direct detrimental role for (mutant) alpha-synuclein in oxidative stress and/or apoptotic pathways triggered by MPTP, but do not rule out the possibility that alpha-synuclein aggregation in neurons exposed to oxidative stress for long periods of time may be neurotoxic.

Eells, J. B. (2003). "The control of dopamine neuron development, function and survival: insights from transgenic mice and the relevance to human disease." Curr Med Chem 10(10): 857-70.

            Transgenic technology, especially the use of homologous recombination to disrupt specific genes to produce knockout mice, has added considerably to the understanding of dopamine (DA) neuron develop, survival and function. The current review summarizes results from knockout mice with the target disruption of genes involved in the development of DA neurons (engrailed 1 and 2, lmx1b, and Nurr1), in maintaining DA neurotransmission (tyrosine hydroxylase, vesicular monoamine transporter, DA transporter, DA D2 and D3 receptors) and important for DA neuron survival (alpha-synuclein, glia cell line-derived neurotrophic factor and superoxide dismutase). As alterations in DA neurotransmission have been implicated in a number of human neuropathologies including Parkinson's disease, schizophrenia and attention deficit/hyperactivity disorder, understanding how specific genes are involved in the function of DA neurons and the compensatory changes that result from loss or reduction in gene expression could provide important insight for the treatment of these diseases.

Elkon, H., J. Don, et al. (2002). "Mutant and wild-type alpha-synuclein interact with mitochondrial cytochrome C oxidase." J Mol Neurosci 18(3): 229-38.

            Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in both inherited and sporadic Parkinson's disease (PD). Furthermore, rare mutations of alpha-synuclein cause autosomal-dominant PD. However, it is unknown how alpha-synuclein is involved in the pathogenesis of nigral degeneration in PD. In this study, we examine the protein-protein interactions of wild-type and mutant (A53T) a-synuclein with adult human brain cDNA expression library using the yeast two-hybrid technique. We found that both normal and mutant alpha-synuclein specifically interact with the mitochondrial complex IV enzyme, cytochrome C oxidase (COX). Wild-type and mutant alpha-synuclein genes were further fused with c-Myc tag and translated in rabbit reticulocyte lysate. Using anti-c-Myc antibody, we demonstrated that both wild-type and mutant alpha-synuclein, coimmunoprecipitated with COX. We also showed that potassium cyanide, a selective COX inhibitor, synergistically enhanced the sensitivity of SH-SY5Y neuroblastoma cells to dopamine-induced cell death. In conclusion, we found specific protein-protein interactions of alpha-synuclein, a major LB protein, to COX, a key enzyme of the mithochondrial respiratory system. This interaction suggests that alpha-synuclein aggregation may contribute to enhance the mitochondrial dysfunction, which might be a key factor in the pathogenesis of PD.

Fornai, F., M. Gesi, et al. (2002). "Striatal postsynaptic ultrastructural alterations following methylenedioxymethamphetamine administration." Ann N Y Acad Sci 965: 381-98.

            Amphetamine derivatives, such as methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), act as monoaminergic neurotoxins in the central nervous system. Although there are slight differences in their mechanism of action, these compounds share a final common pathway, which involves dopamine release and oxidative stress. Apart from striatal toxicity involving monoamine axons, no previous report evidenced any alteration at the striatal level concerning postsynaptic sites. Given the potential toxicity for extracellular dopamine at the striatal level, and the hypothesis for neurotoxic effects of dopamine on striatal medium-sized neurons in Huntington's disease, we evaluated at an ultrastructural level the effects of MDMA on intrinsic striatal neurons of the mouse. In this study, administering MDMA, we noted ultrastructural alterations of striatal postsynaptic GABAergic cells consisting of neuronal inclusions shaped as whorls of concentric membranes. These whorls stained for ubiquitin but not for synuclein and represent the first morphologic correlate of striatal postsynaptic effects induced by MDMA.

Gomez-Isla, T., M. C. Irizarry, et al. (2003). "Motor dysfunction and gliosis with preserved dopaminergic markers in human alpha-synuclein A30P transgenic mice." Neurobiol Aging 24(2): 245-58.

            Alpha-synuclein is a major component of Lewy bodies (LBs) in the substantia nigra and cortex in Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and in glial inclusions in multiple systems atrophy (MSA). Mutations in alpha-synuclein have been associated with autosomal dominant forms of PD. We investigated the clinical and neuropathological effects of overexpression of human alpha-synuclein, alpha-synuclein A30P, and alpha-synuclein A53T under the control of the hamster prion protein (PrP) promoter; 5-15x endogenous levels of protein expression were achieved with widespread neuronal, including nigral, transgene expression. High expression of alpha-synuclein A30P in the Tg5093 line was associated with a progressive motor disorder with rigidity, dystonia, gait impairment, and tremor. Histological analysis of this line showed aberrant expression of the protein in cell soma and progressive CNS gliosis, but no discrete Lewy body-like alpha-synuclein inclusions could be identified. Biochemical analysis demonstrated alpha-synuclein fragmentation. Despite strong expression of the transgene in the nigra, there was no specific deterioration of the nigrostriatal dopaminergic system as assessed by quantitation of nigral tyrosine hydroxylase (TH) containing neurons, striatal TH immunoreactivity, dopamine levels, or dopamine receptor number and function. Lower expressing lines had no specific behavioral or histopathological phenotype. Thus, high expression of mutant human alpha-synuclein resulted in a progressive motor and widespread CNS gliotic phenotype independent of dopaminergic dysfunction in the Tg5093 line.

Gomez-Santos, C., I. Ferrer, et al. (2003). "Dopamine induces autophagic cell death and alpha-synuclein increase in human neuroblastoma SH-SY5Y cells." J Neurosci Res 73(3): 341-350.

            Free cytoplasmic dopamine may be involved in the genesis of neuronal degeneration in Parkinson's disease and other such diseases. We used SH-SY5Y human neuroblastoma cells to study the effect of dopamine on cell death, activation of stress-induced pathways, and expression of alpha-synuclein, the characteristic protein accumulated in Lewy bodies. We show that 100 and 500 microM dopamine causes a 40% and 60% decrease of viability, respectively, and triggers autophagy after 24 hr of exposure, characterized by the presence of numerous cytoplasmic vacuoles with inclusions. Dopamine causes mitochondrial aggregation in adherent cells prior to the loss of functionality. Plasma membrane and nucleus also maintain their integrity. Cell viability is protected by the dopamine transporter blocker nomifensine and the antioxidants N-acetylcysteine and ascorbic acid. Dopamine activates the stress-response kinases, SAPK/JNK and p38, but not ERK/MAPK or MEK, and increases alpha-synuclein expression. Both cell viability and the increase in alpha-synuclein expression are prevented by antioxidants; by the specific inhibitors of p38 and SAPK/JNK, SB203580 and SP600125, respectively; and by the inhibitor of autophagy 3-methyladenine. This indicates that oxidative stress, stress-activated kinases, and factors involved in autophagy up-regulate alpha-synuclein content. The results show that nonapoptotic death pathways are triggered by dopamine, leading to autophagy. These findings should be taken into account in the search for strategies to protect dopaminergic neurons from degeneration. Copyright 2003 Wiley-Liss, Inc.

Gosavi, N., H. J. Lee, et al. (2002). "Golgi fragmentation occurs in the cells with prefibrillar alpha-synuclein aggregates and precedes the formation of fibrillar inclusion." J Biol Chem 277(50): 48984-92.

            Amyloid-like fibrillar aggregates of intracellular proteins are common pathological features of human neurodegenerative diseases. However, the nature of pathogenic aggregates and the biological consequences of their formation remain elusive. Here, we describe (i) a model cellular system in which prefibrillar alpha-synuclein aggregates and fibrillar inclusions are naturally formed in the cytoplasm with distinctive kinetics and (ii) a tight correlation between the presence of prefibrillar aggregates and the Golgi fragmentation. Consistent with the structural abnormality of Golgi apparatus, trafficking and maturation of dopamine transporter through the biosynthetic pathway were impaired in the presence of alpha-synuclein aggregates. Reduction in cell viability was also observed in the prefibrillar aggregate-forming condition and before the inclusion formation. The fibrillar inclusions, on the other hand, showed no correlation with Golgi fragmentation and were preceded by these events. Furthermore, at the early stage of inclusion formation, active lysosomes and mitochondria were enriched in the juxtanuclear area and co-aggregate into a compact inclusion body, suggesting that the fibrillar inclusions might be the consequence of an attempt of the cell to remove abnormal protein aggregates and damaged organelles. These results support the hypothesis that prefibrillar alpha-synuclein aggregates are the pathogenic species and suggest that Golgi fragmentation and subsequent trafficking impairment are the specific consequence of alpha-synuclein aggregation.

Helfand, S. L. (2002). "Neurobiology. Chaperones take flight." Science 295(5556): 809-10.

Jakobsen, L. D. and P. H. Jensen (2003). "Parkinson's Disease: alpha -Synuclein and Parkin in Protein Aggregation and the Reversal of Unfolded Protein Stress." Methods Mol Biol 232: 57-66.

            Parkinson's disease (PD) is the second most common neurodegenerative disorder. The prevalence of PD increases dramatically with age, affecting 1-2% of the population above 65 years (1,2). PD is characterized by a progressive loss of dopaminergic neurons in the substantia nigra, pars compacta in the brain stem. The classical symptoms are tremor, rigidity, and slowness of movements. The symptoms appear when about 70% of the dopaminergic neurons are lost, demonstrating that the degenerative process is active long before the patients become aware of the disease. In the degenerating neurons, proteinaceous inclusions named Lewy bodies are found in the cell body and Lewy neurites in the neuronal processes (3). The inclusions exhibit filamentous protein aggregates with alpha- synuclein being a prominent component (4-7). The definitive diagnosis of PD requires both the clinical symptoms and post mortem examination. No preventive or neuroprotective therapies exist for PD, of which treatment primarily relies on substitution therapy with dopamine precursors or agonists. Genetic predispositions as well as environmental factors increase the risk of PD, but the etiology of PD is largely unknown. In rare familial cases, PD is linked to mutations in single genes encoding alpha-synuclein and parkin proteins. These cases have shed light on mechanisms leading to the selective dopaminergic cell loss.

Jellinger, K. A. (2002). "Recent developments in the pathology of Parkinson's disease." J Neural Transm Suppl(62): 347-76.

            Parkinson's disease (PD) is morphologically characterized by progressive loss of neurons in the substantia nigra pars compacta (SNpc) and other subcortical nuclei associated with intracytoplasmic Lewy bodies and dystrophic (Lewy) neurites mainly in subcortical nuclei and hippocampus und, less frequently in cerebral cortex. SN cell loss is significantly related to striatal dopamine (DA) deficiency as well as to both the duration and clinical severity of disease, The two major clinical subtypes of PD show different morphologic lesion patterns: the akinetic-rigid form has more severe cell loss in the ventrolateral part of SN with negative correlation to DA loss in the posterior putamen, and motor symptoms related to overacitivty of the GABAergic "indirect" motor loop, which causes inhibition of the glutamatergic thalamocortical pathway and reduced cortical activation. The tremor-dominant type shows more severe cell loss in the medial SNpc and retrorubal field A 8, which project to the matrix of the dorsolateral striatum and ventromedial thalamus, thus causing hyperactivity of thalamomotor and cerebellar projections. These and experimental data suggesting different pathophysiological mechanisms for the major clinical subtypes of PD may have important therapeutic implications. Lewy bodies, the morphologic markers of PD, are composed of hyperphosphorylated neurofilament proteins, lipids, redox-active iron, ubiquitin, and alpha-synuclein, showing a continuous accumulation in the periphery and of ubiquitin in the central core. Alpha-synuclein, is usually unfolded in alpha-helical form. By gene mutation, environmental stress or other factors it can be transformed to beta-folding which is sensible to self-aggregation in filamentous fibrils and formation of insoluble intracellular inclusions that may lead to functional disturbances and, finally, to death of involved neurons. While experimental and tissue culture studies suggest that apoptosis, a genetically determined form of programmed cell death, represents the most common pathway in neurodegeneration, DNA fragmentation, overexpression of proapoptotic proteins and activated caspase-3, the effector enzyme of the terminal apopoptic cascade, have only extremely rarely been detected in SN of PD brains. This is in accordance with the rapid course of apoptotis and the extremely slow progression of the neurodegenerative process in PD. The biological role of Lewy bodies and other intracellular inclusions, the mechanisms of the intracellular aggregation of insoluble protein deposits, and their implication for cellular dysfunction resulting in neurodegeneration and cell demise are still unresolved. Further elucidation of the basic molecular mechanisms of cytoskeletal lesions will provide better insight into the pathogenesis of neurodegeneration in PD and related disorders.

Junn, E. and M. M. Mouradian (2002). "Human alpha-synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine." Neurosci Lett 320(3): 146-50.

            alpha-Synuclein is a major component of Lewy bodies found in the brains of patients with Parkinson's disease (PD). Two point mutations in alpha-synuclein (A53T and A30P) are identified in few families with dominantly inherited PD. Yet the mechanism by which this protein is involved in nigral cell death remains poorly understood. Mounting evidence suggests the importance of oxidative stress in the pathogenesis of PD. Here we investigated the effects of wild-type and two mutant forms of alpha-synuclein on intracellular reactive oxygen species (ROS) levels using clonal SH-SY5Y cells engineered to over-express these proteins. All three cell lines, and particularly mutant alpha-synuclein-expressing cells, had increased ROS levels relative to control LacZ-engineered cells. In addition, cell viability was significantly curtailed following the exposure of all three alpha-synuclein-engineered cells to dopamine, but more so with mutant alpha-synuclein. These results suggest that over-expression of alpha-synuclein, and especially its mutant forms, exaggerates the vulnerability of neurons to dopamine-induced cell death through excess intracellular ROS generation. Thus, these findings provide a link between mutations or over-expression of alpha-synuclein and apoptosis of dopaminergic neurons by lowering the threshold of these cells to oxidative damage.

Kaufmann, H. (2003). "[The most common dysautonomias]." Rev Neurol 36: 93-6.

            Aims. To review the classification and the clinical and pathological characteristics of the most common dysautonomias. Method. Primary dysautonomia includes neurodegenerative diseases of unknown causes that are characterised by the intracytoplasmic accumulations of alpha synuclein that manifest with four different phenotypes: pure autonomic failure, Parkinson s disease, dementia with Lewy bodies and multiple system atrophy. Of the secondary dysautonomias, diabetes mellitus is the most common cause of autonomic neuropathy in developed countries. Familiar dysautonomia is a recessive autosomic disease; the gene responsible for it has been located in the long arm of chromosome 9 (9q31). Paraneoplasic dysautonomia is associated with cancer of the lungs, the pancreas, Hodgkin s disease and testicular cancer. In Lambert Eaton myasthenic syndrome and in botulism the release of acetylcholine is deficient in both the somatic and the autonomic neurons. There are other diseases that affect autonomic cholinergic neurotransmission without bringing about any disorders in neuromuscular transmission. Chagas disease affects the neurons of the parasympathetic ganglion and produces megaesophagus, megacolon and myocardiopathy. Dopamine beta hydroxylase enzyme deficiency is a congenital disease characterised by the failure to convert dopamine into noradrenaline which results in orthostatic hypotension. Conclusions. Dysautonomias can be classified, according to their aetiology, as primary or secondary; according to the deficient neurotransmitter, as cholinergic, adrenergic and mixed (pan dysautonomia) or, according to the anatomical distribution of the neurons that are affected, as central and peripheral.

Kirik, D., L. E. Annett, et al. (2003). "Nigrostriatal alpha-synucleinopathy induced by viral vector-mediated overexpression of human alpha-synuclein: a new primate model of Parkinson's disease." Proc Natl Acad Sci U S A 100(5): 2884-9.

            We used a high-titer recombinant adeno-associated virus (rAAV) vector to express WT or mutant human alpha-synuclein in the substantia nigra of adult marmosets. The alpha-synuclein protein was expressed in 90-95% of all nigral dopamine neurons and distributed by anterograde transport throughout their axonal and dendritic projections. The transduced neurons developed severe neuronal pathology, including alpha-synuclein-positive cytoplasmic inclusions and granular deposits; swollen, dystrophic, and fragmented neuritis; and shrunken and pyknotic, densely alpha-synuclein-positive perikarya. By 16 wk posttransduction, 30-60% of the tyrosine hydroxylase-positive neurons were lost, and the tyrosine hydroxylase-positive innervation of the caudate nucleus and putamen was reduced to a similar extent. The rAAV-alpha-synuclein-treated monkeys developed a type of motor impairment, i.e., head position bias, compatible with this magnitude of nigrostriatal damage. rAAV vector-mediated alpha-synuclein gene transfer provides a transgenic primate model of nigrostriatal alpha-synucleinopathy that is of particular interest because it develops slowly over time, like human Parkinson's disease (PD), and expresses neuropathological features (alpha-synuclein-positive inclusions and dystrophic neurites, in particular) that are similar to those seen in idiopathic PD. This model offers new opportunities for the study of pathogenetic mechanisms and exploration of new therapeutic targets of particular relevance to human PD.

Kirik, D., C. Rosenblad, et al. (2002). "Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system." J Neurosci 22(7): 2780-91.

            Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human alpha-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including alpha-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinson's disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30-80% of the nigral dopamine neurons, a 40-50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50-60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that alpha-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant alpha-synuclein, pointing to a key role for alpha-synuclein in the pathogenesis of Parkinson's disease. Targeted overexpression of alpha-synuclein in the nigrostriatal system may provide a new animal model of Parkinson's disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.

Kitamura, Y., J. Kakimura, et al. (2002). "Antiparkinsonian drugs and their neuroprotective effects." Biol Pharm Bull 25(3): 284-90.

            In Parkinson's disease, while dopamine (DA) replacement therapy, such as with L-DOPA (levodopa), improves the symptoms, it does not inhibit the degeneration of DA neurons in the substantia nigra. Numerous studies have suggested that both endogenous and environmental neurotoxins and oxidative stress may participate in this disease, but the detailed mechanisms are still unclear. Recent genetic studies in familial Parkinson's disease and parkinsonism have shown several gene mutations. This new information regarding its pathogenesis offers novel prospects for effective strategies involving the neuroprotection of vulnerable DA neurons. This review summarizes current findings regarding the pathogenesis and antiparkinsonian drugs, and discusses their possibilities of targets to develop novel neuroprotective drugs.

Klein, R. L., M. A. King, et al. (2002). "Dopaminergic cell loss induced by human A30P alpha-synuclein gene transfer to the rat substantia nigra." Hum Gene Ther 13(5): 605-12.

            Somatic cell gene transfer was used to express a mutant form of alpha-synuclein (alpha-syn) that is associated with Parkinson's disease (PD) in the rat substantia nigra (SN), a brain region that, in humans, degenerates during PD. DNA encoding the A30P mutant of human alpha-syn linked to familial PD was incorporated into an adeno-associated virus vector, which was injected into the adult rat midbrain. The cytomegalovirus/chicken beta-actin promoter was used to drive transgene expression. Over a 1-year time course, this treatment produced three significant features relevant to PD: (1) accumulation of alpha-syn in SN neuron perikarya, (2) Lewy-like dystrophic neurites in the SN and the striatum, and (3) a 53% loss of SN dopamine neurons. However, motor dysfunction was not found in either rotational or rotating rod testing. The lack of behavioral deficits, despite the significant cell loss, may reflect pathogenesis similar to that of PD, where greater than 50% losses occur before motor behavior is affected.

Kuhn, K., J. Wellen, et al. (2003). "The mouse MPTP model: gene expression changes in dopaminergic neurons." Eur J Neurosci 17(1): 1-12.

            Parkinson's disease (PD) is a common neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although valuable animal models have been developed, our knowledge of the aetiology and pathogenic factors implicated in PD is still insufficient to develop causal therapeutic strategies aimed at halting its progression. The neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is one of the most valuable models for analysing pathological aspects of PD. In this paper we studied the gene expression patterns underlying the pathogenesis of MPTP-induced neurodegeneration. We treated young and old C57BL/6 mice with different schedules of MPTP to induce degenerative processes that vary in intensity and time-course. During the first week after intoxication we used nonradioactive in situ-hybridization to investigate the expression patterns of genes associated with (i) dopamine metabolism and signalling; (ii) familial forms of PD; (iii) protein folding and (iv) energy metabolism. MPTP injections induced different severities of neuronal injury depending on the age of the animals and the schedule of administration as well as a significant degeneration in the striatum. In situ hybridization showed that MPTP intoxication initiated a number of gene expression changes that (i) were restricted to the neurons of the substantia nigra pars compacta; (ii) were correlated in intensity and number of changes with the age of the animals and the severity of histopathological disturbances; (iii) displayed in each a significant down-regulation by the end of one week after the last MPTP injection, but (iv) varied within one MPTP regimen in expression levels during the observation period. The subacute injection of MPTP into one-year-old mice induced the most severe changes in gene expression. All genes investigated were affected. However, alpha-synuclein was the only gene that was exclusively up-regulated in MPTP-treated animals displaying cell death.

Lee, E. N., S. Y. Lee, et al. (2003). "Lipid interaction of alpha-synuclein during the metal-catalyzed oxidation in the presence of Cu2+ and H2O2." J Neurochem 84(5): 1128-42.

            Alpha-synuclein co-exists with lipids in the Lewy bodies, a pathological hallmark of Parkinson's disease. Molecular interaction between alpha-synuclein and lipids has been examined by observing lipid-induced protein self-oligomerization in the presence of a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Lipids such as phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and even arachidonic acid induced the self-oligomerization whereas phosphatidylcholine did not affect the protein. Because the oligomerizations occurred from critical micelle concentrations of the lipids, the self interaction of alpha-synuclein was shown to be a lipid-surface dependent phenomenon with head group specificity. By employing beta-synuclein and a C-terminally truncated alpha-synuclein (alpha-syn97), the head-group dependent self-oligomerization was demonstrated to occur preferentially at the N-terminal region while the fatty acid interaction leading to the protein self-association required the presence of the acidic C-terminus of alpha-synuclein. In the presence of Cu2+ and H2O2, phosphatidylinositol (PI), along with other acidic lipids, actually enhanced the metal-catalyzed oxidative self-oligomerization of alpha-synuclein. The dityrosine crosslink formation responsible for the PI-enhanced covalent self-oligomerization was more sensitive to variation of copper concentrations than that of H2O2 during the metal-catalyzed oxidation. The enhancement by PI was shown to be due to facilitation of copper localization to the protein because actual binding affinity between copper and alpha-synuclein increased from Kd of 44.7 microm to 5.9 microm in the presence of the lipid. Taken together, PI not only affects alpha-synuclein to be more self-interactive by providing the lipid surface, but also enhances the metal-catalyzed oxidative protein self-oligomerization by facilitating copper localization to the protein when the metal and H2O2 are provided. This observation therefore could be implicated in the formation of Lewy bodies as lipids and metal-catalyzed oxidative stress have been considered to be a part of pathological causes leading to the neurodegeneration.

Lehmensiek, V., E. M. Tan, et al. (2002). "Expression of mutant alpha-synucleins enhances dopamine transporter-mediated MPP+ toxicity in vitro." Neuroreport 13(10): 1279-83.

            Mutations in the alpha-synuclein gene (A30P and A53T) are reported to cause familial Parkinson's disease (PD), but it is not known how they result in selective dopaminergic cell death. Here we report on effects of mutant alpha-synucleins on dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) in vitro. We established human embryonic kidney HEK-293 cell lines stably co-expressing each alpha-synuclein isoform and the human DAT. We demonstrate that expression of all alpha-synuclein isoforms enhances toxicity of general complex I inhibition (rotenone), but only the expression of mutant alpha-synucleins induces significant increased DAT-dependent toxicity of very low concentrations of MPP+ compared to wild-type protein. Proteasomal inhibition by lactacystin does not alter MPP+-toxicity in all cell lines. Our data suggest a new mechanism of MPP+-induced dopaminergic toxicity by an interaction between mutant alpha-synucleins and the DAT, which is independent of the function of the proteasome.

Lim, K. L., V. L. Dawson, et al. (2003). "The cast of molecular characters in Parkinson's disease: felons, conspirators, and suspects." Ann N Y Acad Sci 991: 80-92.

            Parkinson's Disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of dopamine neurons and the accumulation of Lewy bodies and neurites. Recent advances indicate that PD is due in some individuals to genetic mutations in alpha-synuclein, parkin, and ubiquitin C-terminal hydrolase L1 (UCHL1). All three PD-linked gene products are related directly or indirectly to the functioning of the cellular ubiquitin proteasomal system (UPS), suggesting that UPS dysfunction may be important in PD pathogenesis. Indeed, emerging evidence indicates that derangements of the UPS may be one of the underlying mechanisms of PD pathogenesis. The function of parkin as an ubiquitin protein ligase positions it as an important player in both familial and idiopathic PD. We recently demonstrated that parkin mediates a nondegradative form of ubiquitination on synphilin-1 that could contribute to synphilin-1's aggregation in PD. Our results implicate parkin involvement in the formation of Lewy bodies associated with sporadic PD. This review discusses the role of the UPS, as well as the modus operandi of the three PD candidate felons (alpha-synuclein, parkin, and UCHL1) along with their conspirators in bringing about dopaminergic cell death in PD.

Lo Bianco, C., J. L. Ridet, et al. (2002). "alpha -Synucleinopathy and selective dopaminergic neuron loss in a rat lentiviral-based model of Parkinson's disease." Proc Natl Acad Sci U S A 99(16): 10813-8.

            Parkinson's disease (PD) is characterized by the progressive loss of substantia nigra dopaminergic neurons and the presence of cytoplasmic inclusions named Lewy bodies. Two missense mutations of the alpha-synuclein (alpha-syn; A30P and A53T) have been described in several families with an autosomal dominant form of PD. alpha-Syn also constitutes one of the main components of Lewy bodies in sporadic cases of PD. To develop an animal model of PD, lentiviral vectors expressing different human or rat forms of alpha-syn were injected into the substantia nigra of rats. In contrast to transgenic mice models, a selective loss of nigral dopaminergic neurons associated with a dopaminergic denervation of the striatum was observed in animals expressing either wild-type or mutant forms of human alpha-syn. This neuronal degeneration correlates with the appearance of abundant alpha-syn-positive inclusions and extensive neuritic pathology detected with both alpha-syn and silver staining. Lentiviral-mediated expression of wild-type or mutated forms of human alpha-syn recapitulates the essential neuropathological features of PD. Rat alpha-syn similarly leads to protein aggregation but without cell loss, suggesting that inclusions are not the primary cause of cell degeneration in PD. Viral-mediated genetic models may contribute to elucidate the mechanism of alpha-syn-induced cell death and allow the screening of candidate therapeutic molecules.

Lotharius, J., S. Barg, et al. (2002). "Effect of mutant alpha-synuclein on dopamine homeostasis in a new human mesencephalic cell line." J Biol Chem 277(41): 38884-94.

            Mutations in alpha-synuclein have been linked to rare, autosomal dominant forms of Parkinson's disease. Despite its ubiquitous expression, mutant alpha-synuclein primarily leads to the loss of dopamine-producing neurons in the substantia nigra. alpha-Synuclein is a presynaptic nerve terminal protein of unknown function, although several studies suggest it is important for synaptic plasticity and maintenance. The present study utilized a new human mesencephalic cell line, MESC2.10, to study the effect of A53T mutant alpha-synuclein on dopamine homeostasis. In addition to expressing markers of mature dopamine neurons, differentiated MESC2.10 cells are electrically active, produce dopamine, and express wild-type human alpha-synuclein. Lentivirus-induced overexpression of A53T mutant alpha-synuclein in differentiated MESC2.10 cells resulted in down-regulation of the vesicular dopamine transporter (VMAT2), decreased potassium-induced and increased amphetamine-induced dopamine release, enhanced cytoplasmic dopamine immunofluorescence, and increased intracellular levels of superoxide. These results suggest that mutant alpha-synuclein leads to an impairment in vesicular dopamine storage and consequent accumulation of dopamine in the cytosol, a pathogenic mechanism that underlies the toxicity of the psychostimulant amphetamine and the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium. Interestingly, cells expressing A53T mutant alpha-synuclein were resistant to amphetamine-induced toxicity. Because extravesicular, cytoplasmic dopamine can be easily oxidized into reactive oxygen species and other toxic metabolites, mutations in alpha-synuclein might lead to Parkinson's disease by triggering protracted, low grade dopamine toxicity resulting in terminal degeneration and ultimately cell death.

Lotharius, J. and P. Brundin (2002). "Pathogenesis of Parkinson's disease: dopamine, vesicles and alpha-synuclein." Nat Rev Neurosci 3(12): 932-42.

Lotharius, J. and P. Brundin (2002). "Impaired dopamine storage resulting from alpha-synuclein mutations may contribute to the pathogenesis of Parkinson's disease." Hum Mol Genet 11(20): 2395-407.

            Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the inability to initiate, execute and control movement. Neuropathologically, there is a striking loss of dopamine-producing neurons in the substantia nigra pars compacta, accompanied by depletion of dopamine in the striatum. Most forms of PD are sporadic, though in some cases familial inheritance is observed. In the late 1990s, two mutations in the alpha-synuclein gene were linked to rare, autosomal dominant forms of PD. Previously cloned from cholinergic vesicles of the Torpedo electric ray, alpha-synuclein is highly enriched in presynaptic nerve terminals and appears to be involved in synapse maintenance and plasticity. It is expressed ubiquitously in the brain, raising the important question of why dopaminergic neurons are primarily targeted in persons carrying mutations in alpha-synuclein. In this article, we review the current literature on alpha-synuclein and suggest a possible role for this protein in vesicle recycling via its regulation of phospholipase D2, its fatty acid-binding properties, or both. Exogenous application of dopamine, as well as redistribution of vesicular dopamine to the cytoplasm, can be toxic to dopaminergic neurons. Thus, impaired neurotransmitter storage arising from mutations in alpha-synuclein could lead to cytoplasmic accumulation of dopamine. The breakdown of this labile neurotransmitter in the cytoplasm could, in turn, promote oxidative stress and metabolic dysfunction, both of which have been observed in nigral tissue from PD patients.

Lykkebo, S. and P. H. Jensen (2002). "Alpha-synuclein and presynaptic function: implications for Parkinson's disease." Neuromolecular Med 2(2): 115-29.

            This article focuses on alpha-synuclein's role in normal and pathological axonal and presynaptic functions and its relationship to Parkinson's disease. It is not possible to mention all the contributions to aspects of this area. Readers interested in alpha-synuclein's relation to aggregation, Lewy lesions, and pathological modifications are referred to the many reviews (see Goldberg and Lansbury 2000; Galvin 2001a; Goedert 2001).

Mash, D. C., Q. Ouyang, et al. (2003). "Cocaine abusers have an overexpression of alpha-synuclein in dopamine neurons." J Neurosci 23(7): 2564-71.

            Alpha-synuclein is a presynaptic protein that has been implicated as a possible causative agent in the pathogenesis of Parkinson's disease. The native protein is a major component of nigral Lewy bodies in Parkinson's disease, and full-length alpha-synuclein accumulates in Lewy neurites. Here we present evidence that alpha-synuclein levels are elevated in midbrain dopamine (DA) neurons of chronic cocaine abusers. Western blot and immunoautoradiographic studies were conducted on postmortem neuropathological specimens from cocaine users and age-matched drug-free control subjects. The results demonstrated that alpha-synuclein levels in the DA cell groups of the substantia nigra/ventral tegmental complex were elevated threefold in chronic cocaine users compared with normal age-matched subjects. The increased protein levels in chronic cocaine users were accompanied by changes in the expression of alpha-synuclein mRNA in the substantia nigra and ventral tegmental area. Although alpha-synuclein expression is prominent in the hippocampus, there was no increase in protein expression in this brain region. The levels of beta-synuclein, a possible negative regulator of alpha-synuclein, also were not affected by cocaine exposure. Alpha-synuclein protein levels were increased in the ventral tegmental area, but not the substantia nigra, in victims of excited cocaine delirium who experienced paranoia, marked agitation, and hyperthermia before death. The overexpression of alpha-synuclein may occur as a protective response to changes in DA turnover and increased oxidative stress resulting from cocaine abuse. However, the accumulation of alpha-synuclein protein with long-term cocaine abuse may put addicts at increased risk for developing the motor abnormalities of Parkinson's disease.

McCormack, A. L. and D. A. Di Monte (2003). "Effects of L-dopa and other amino acids against paraquat-induced nigrostriatal degeneration." J Neurochem 85(1): 82-6.

            Exposure to the herbicide paraquat causes selective nigrostriatal degeneration and aggregation of alpha-synuclein in the mouse brain. The purpose of this study was to assess mechanisms of paraquat entry into the CNS and, in particular, the effects of substrates of the blood-brain barrier (BBB) neutral amino acid transporter (System L carrier) on paraquat accumulation and neurotoxicity. Using a paraquat antibody, robust immunoreactivity was observed in the midbrain of mice injected with the herbicide. This immunoreactivity was abolished by administration of l-valine or l-phenylalanine, two System L substrates, immediately before paraquat exposure. Pre-treatment with these amino acids completely protected against paraquat-induced loss of nigrostriatal dopaminergic cells and formation of thioflavine S-positive intracellular deposits. Interestingly, the anti-parkinsonian drug l-dopa, which is transported across the BBB through the same neutral amino acid carrier, was also neuroprotective when administered 30 min prior to paraquat. In contrast, paraquat-induced toxicity was unaffected if animals (i) were pre-treated with d-valine, the biologically inactive d-isomer of l-valine, or with l-lysine, a substrate of the basic rather than the neutral amino acid carrier, or (ii) were injected with l-dopa 24 h after paraquat exposure. Data are consistent with a critical role of uptake across the BBB in paraquat neurotoxicity, and suggest that dietary elements (e.g. amino acids) or therapeutic agents (e.g. l-dopa) may modify the effects of toxicants targeting the nigrostriatal system.

McKeith, I. G., D. J. Burn, et al. (2003). "Dementia with Lewy bodies." Semin Clin Neuropsychiatry 8(1): 46-57.

            The objective was to summarize recent findings about the clinical features, diagnosis and investigation of dementia with Lewy (DLB) bodies, together with its neuropathology, neurochemistry and genetics. Dementia with Lewy bodies (DLB) is a primary, neurodegenerative dementia sharing clinical and pathological characteristics with both Parkinson's disease (PD) and Alzheimer's disease (AD). Antiubiquitin immunocytochemical staining, developed in the early 1990s, allowed the frequency and distribution of cortical LBs to be defined. More recently, alpha-synuclein antibodies have revealed extensive neuritic pathology in DLB demonstrating a neurobiological link with other "synucleinopathies" including PD and multiple system atrophy (MSA). The most significant correlates of cognitive failure in DLB appear to be with cortical LB and Lewy neurites (LNs) rather than Alzheimer type pathology. Clinical diagnostic criteria for DLB, published in 1996, have been subjected to several validation studies against autopsy findings. These conclude that although diagnostic specificity is high (range 79- 100%, mean 92%), sensitivity is lower (range 0- 83 %, mean, 49%). Improved methods of case detection are therefore required. Fluctuating impairments in attention, visual recognition and construction are more indicative of DLB than AD. Relative preservation of medial temporal lobe volume on structural MRI and the use of SPECT tracers for regional blood flow and the dopamine transporter are the most reliable current biomarkers for DLB. There are no genetic or CSF tests recommended for the diagnosis of DLB at present. Between 15 and 20% of all elderly demented cases reaching autopsy have DLB, making it the most common cause of degenerative dementia after AD. Exquisite, not infrequently fatal, sensitivity to neuroleptic drugs and encouraging reports of the effects of cholinesterase inhibitors on cognitive, psychiatric and neurological features, mean that an accurate diagnosis of DLB is more than merely of academic interest. Dementia developing late in the course of PD shares many of the same clinical and pathological characteristics.

McNaught, K. S., R. Belizaire, et al. (2003). "Altered proteasomal function in sporadic Parkinson's disease." Exp Neurol 179(1): 38-46.

            Parkinson's disease (PD) is characterized pathologically by preferential degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNc). Nigral cell death is accompanied by the accumulation of a wide range of poorly degraded proteins and the formation of proteinaceous inclusions (Lewy bodies) in dopaminergic neurons. Mutations in the genes encoding alpha-synuclein and two enzymes of the ubiquitin-proteasome system, parkin and ubiquitin C-terminal hydrolase L1, are associated with neurodegeneration in some familial forms of PD. We now show that, in comparison to age-matched controls, alpha-subunits (but not beta-subunits) of 26/20S proteasomes are lost within dopaminergic neurons and 20S proteasomal enzymatic activities are impaired in the SNc in sporadic PD. In addition, while the levels of the PA700 proteasome activator are reduced in the SNc in PD, PA700 expression is increased in other brain regions such as the frontal cortex and striatum. We also found that levels of the PA28 proteasome activator are very low to almost undetectable in the SNc compared to other brain areas in both normal and PD subjects. These findings suggest that failure of the ubiquitin-proteasome system to adequately clear unwanted proteins may underlie vulnerability and degeneration of the SNc in both sporadic and familial PD.

McNaught, K. S., L. M. Bjorklund, et al. (2002). "Proteasome inhibition causes nigral degeneration with inclusion bodies in rats." Neuroreport 13(11): 1437-41.

            Structural and functional defects in 26/20S proteasomes occur in the substantia nigra pars compacta and may underlie protein accumulation, Lewy body formation and dopaminergic neuronal death in Parkinson's disease. We therefore determined the pathogenicity of proteasomal impairment following stereotaxic unilateral infusion of lactacystin, a selective proteasome inhibitor, into the substantia nigra pars compacta of rats. These animals became progressively bradykinetic, adopted a stooped posture and displayed contralateral head tilting. Administration of apomorphine to lactacystin-treated rats reversed behavioral abnormalities and induced contralateral rotations. Lactacystin caused dose-dependent degeneration of dopaminergic cell bodies and processes with the cytoplasmic accumulation and aggregation of alpha-synuclein to form inclusion bodies. These findings support the notion that failure of the ubiquitin-proteasome system to degrade and clear unwanted proteins is an important etiopathogenic factor in Parkinson's disease.

McNaught, K. S., C. Mytilineou, et al. (2002). "Impairment of the ubiquitin-proteasome system causes dopaminergic cell death and inclusion body formation in ventral mesencephalic cultures." J Neurochem 81(2): 301-6.

            Mutations in alpha-synuclein, parkin and ubiquitin C-terminal hydrolase L1, and defects in 26/20S proteasomes, cause or are associated with the development of familial and sporadic Parkinson's disease (PD). This suggests that failure of the ubiquitin-proteasome system (UPS) to degrade abnormal proteins may underlie nigral degeneration and Lewy body formation that occur in PD. To explore this concept, we studied the effects of lactacystin-mediated inhibition of 26/20S proteasomal function and ubiquitin aldehyde (UbA)-induced impairment of ubiquitin C-terminal hydrolase (UCH) activity in fetal rat ventral mesencephalic cultures. We demonstrate that both lactacystin and UbA caused concentration-dependent and preferential degeneration of dopaminergic neurons. Inhibition of 26/20S proteasomal function was accompanied by the accumulation of alpha-synuclein and ubiquitin, and the formation of inclusions that were immunoreactive for these proteins, in the cytoplasm of VM neurons. Inhibition of UCH was associated with a loss of ubiquitin immunoreactivity in the cytoplasm of VM neurons, but there was a marked and localized increase in alpha-synuclein staining which may represent the formation of inclusions bodies in VM neurons. These findings provide direct evidence that impaired protein clearance can induce dopaminergic cell death and the formation of proteinaceous inclusion bodies in VM neurons. This study supports the concept that defects in the UPS may underlie nigral pathology in familial and sporadic forms of PD.

Meredith, G. E., S. Totterdell, et al. (2002). "Lysosomal malfunction accompanies alpha-synuclein aggregation in a progressive mouse model of Parkinson's disease." Brain Res 956(1): 156-65.

            We have detected granular and filamentous inclusions that are alpha-synuclein- and ubiquitin-immunoreactive in the cytoplasm of dopaminergic and cortical neurons of C57/black mice treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and probenecid. The immunoreactive aggregates only become evident several weeks after large-scale dopaminergic cell death and a downregulation of alpha-synuclein gene expression. Numerous lipofuscin granules accumulate alpha-synuclein in the nigral and limbic cortical neurons of treated mice. These data provide evidence that insoluble proteins, such as alpha-synuclein, build up as granular and filamentous inclusions in dopaminergic neurons that survive the initial toxic MPTP insult. They further suggest that defective protein degradation rather than altered gene expression underlies deposition of alpha-synuclein and that abundant lysosomal compartments are present to seal off the potentially toxic material.

Mishizen-Eberz, A. J., R. P. Guttmann, et al. (2003). "Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro." J Neurochem 86(4): 836-847.

            Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.

Mizuno, Y. (2002). "[Progress in diagnosis and therapy for Parkinson's disease]." Nippon Naika Gakkai Zasshi 91(8): 2344-8.

Mouradian, M. M. (2002). "Recent advances in the genetics and pathogenesis of Parkinson disease." Neurology 58(2): 179-85.

            The identification of three genes and several additional loci associated with inherited forms of levodopa-responsive PD has confirmed that this is not a single disorder. Yet, analyses of the structure and function of these gene products point to the critical role of protein aggregation in dopaminergic neurons of the substantia nigra as the common mechanism leading to neurodegeneration in all known forms of this disease. The three specific genes identified to date--alpha-synuclein, Parkin, and ubiquitin C terminal hydrolase L1--are either closely involved in the proper functioning of the ubiquitin-proteasome pathway or are degraded by this protein-clearing machinery of cells. Knowledge gained from genetically transmitted PD also has clear implications for nonfamilial forms of the disease. Lewy bodies, even in sporadic PD, contain these three gene products, particularly abundant amounts of fibrillar alpha-synuclein. Increased aggregation of alpha-synuclein by oxidative stress, as well as oxidant-induced proteasomal dysfunction, link genetic and potential environmental factors in the onset and progression of the disease. The biochemical and molecular cascades elucidated from genetic studies in PD can provide novel targets for curative therapies.

Perez, R. G., J. C. Waymire, et al. (2002). "A role for alpha-synuclein in the regulation of dopamine biosynthesis." J Neurosci 22(8): 3090-9.

            The alpha-synuclein gene is implicated in the pathogenesis of Parkinson's disease. Although alpha-synuclein function is uncertain, the protein has homology to the chaperone molecule 14-3-3. In addition, alpha-synuclein can bind to 14-3-3, and both alpha-synuclein and 14-3-3 bind to many of the same proteins. Because 14-3-3 binds to and activates tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) biosynthesis, we explored whether alpha-synuclein also bound to tyrosine hydroxylase and influenced its activity. Immunoprecipitation revealed an interaction between alpha-synuclein and tyrosine hydroxylase in brain homogenates and MN9D dopaminergic cells. Colocalization of alpha-synuclein with tyrosine hydroxylase was confirmed by immunoelectron microscopy. To explore the consequences of the interaction, we measured the effect of recombinant alpha-synuclein on tyrosine hydroxylase activity in a cell-free system and observed a dose-dependent inhibition of tyrosine hydroxylase by alpha-synuclein. To measure the impact of alpha-synuclein on tyrosine hydroxylase in dopaminergic cells, we stably transfected MN9D cells with wild-type or A53T mutant alpha-synuclein. Overexpression of wild-type or A53T mutant alpha-synuclein did not significantly alter tyrosine hydroxylase protein levels in our stably transfected cells. However, overexpressing cell lines had significantly reduced tyrosine hydroxylase activity and a corresponding reduction in dopamine synthesis. The reduction in cellular dopamine levels was not caused by increased dopamine catabolism or dopamine efflux. These data suggest that alpha-synuclein plays a role in the regulation of dopamine biosynthesis, acting to reduce the activity of tyrosine hydroxylase. If so, a loss of soluble alpha-synuclein, by reduced expression or aggregation, could increase dopamine synthesis with an accompanying increase in reactive dopamine metabolites.

Richfield, E. K., M. J. Thiruchelvam, et al. (2002). "Behavioral and neurochemical effects of wild-type and mutated human alpha-synuclein in transgenic mice." Exp Neurol 175(1): 35-48.

            Human alpha-synuclein (halpha-SYN) is implicated in the Parkinson's disease phenotype (PDP) based on a variety of studies in man, animal models, and in vitro studies. The normal function of halpha-SYN and the mechanism by which it contributes to the PDP remains unclear. We created transgenic mice expressing either wild-type (hwalpha-SYN) or a doubly mutated (hm2alpha-SYN) form of halpha-SYN under control of the 9-kb rat tyrosine hydroxylase promoter. These mice expressed halpha-SYN in cell bodies, axons, and terminals of the nigrostriatal system. The expression of halpha-SYN in nigrostriatal terminals produced effects in both constructs resulting in increased density of the dopamine transporter and enhanced toxicity to the neurotoxin MPTP. Expression of hm2alpha-SYN reduced locomotor responses to repeated doses of amphetamine and blocked the development of sensitization. Adult hwalpha-SYN-5 transgenic mice had unremarkable dopaminergic axons and terminals, normal age-related measures on two motor coordination screens, and normal age-related measures of dopamine (DA) and its metabolites. Adult hm2alpha-SYN-39 transgenic mice had abnormal axons and terminals, age-related impairments in motor coordination, and age-related reductions in DA and its metabolites. Expression of hm2alpha-SYN adversely affects the integrity of dopaminergic terminals and leads to age-related declines in motor coordination and dopaminergic markers.

Rideout, H. J., P. Dietrich, et al. (2003). "Regulation of alpha-synuclein by bFGF in cultured ventral midbrain dopaminergic neurons." J Neurochem 84(4): 803-13.

            Alpha-synuclein is a neuronal protein that is implicated in the control of synaptic vesicle function and in Parkinson's disease (PD). Consequently, alterations of alpha-synuclein levels may play a role in neurotransmission and in PD pathogenesis. However, the factors that regulate alpha-synuclein levels are unknown. Growth factors mediate neurotrophic and plasticity effects in CNS neurons, and may play a role in disease states. Here we examine the regulation of alpha-synuclein levels in primary CNS neurons, with particular emphasis on dopaminergic neurons. E18 rat cortical neurons and dopaminergic neurons of E14 rat ventral midbrain showed an induction of alpha-synuclein protein levels with maturation in culture. Application of basic Fibroblast growth factor (bFGF) promoted alpha-synuclein expression selectively within dopaminergic, and not GABAergic or cortical neurons. This induction was blocked by actinomycin D, but not by inhibition of bFGF-induced glial proliferation. alpha-Synuclein levels were not altered by glial-derived neurotrophic factor (GDNF), or by apoptotic stimuli. We conclude that bFGF promotes alpha-synuclein expression in cultured ventral midbrain dopaminergic neurons through a direct transcriptional effect. These results suggest that distinct growth factors may thus mediate plasticity responses or influence disease states in ventral midbrain dopaminergic neurons.

Riobo, N. A., F. J. Schopfer, et al. (2002). "The reaction of nitric oxide with 6-hydroxydopamine: implications for Parkinson's disease." Free Radic Biol Med 32(2): 115-21.

            Oxidation of catecholamines is suggested to contribute to oxidative stress in Parkinson's disease. Nitric oxide (*NO) is able to oxidize cyclic compounds like ubiquinol; moreover, recent lines of evidence proposed a direct role of *NO and its by-product peroxynitrite in the pathophysiology of Parkinson's disease. The aim of this study was to analyze the potential reaction between 6-hydroxydopamine, a classic inducer of Parkinson's disease, and *NO. The results showed that *NO reacts with the deprotonated form of 6-hydroxydopamine at pH 7 and 37 degrees C with a second-order rate constant of 1.5 x 10(3) M(-1) x s(-1) as calculated by the rate of *NO decay measured with an amperometric sensor. Accordingly, the rates of formation of 6-hydroxy-dopamine quinone were dependent on *NO concentration. The coincubation of *NO and 6-hydroxydopamine with either bovine serum albumin or alpha-synuclein led to tyrosine nitration of the protein, in a concentration dependent-manner and sensitive to superoxide dismutase. These findings suggest the formation of peroxynitrite during the redox reactions following the interaction of 6-hydroxydopamine with *NO. The implications of this reaction for in vivo models are discussed in terms of the generation of reactive nitrogen and oxygen species within a propagation process that may play a significant role in neurodegenerative diseases.

Schluter, O. M., F. Fornai, et al. (2003). "Role of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in mice." Neuroscience 118(4): 985-1002.

            In humans, mutations in the alpha-synuclein gene or exposure to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produce Parkinson's disease with loss of dopaminergic neurons and depletion of nigrostriatal dopamine. alpha-Synuclein is a vertebrate-specific component of presynaptic nerve terminals that may function in modulating synaptic transmission. To test whether MPTP toxicity involves alpha-synuclein, we generated alpha-synuclein-deficient mice by homologous recombination, and analyzed the effect of deleting alpha-synuclein on MPTP toxicity using these knockout mice. In addition, we examined commercially available mice that contain a spontaneous loss of the alpha-synuclein gene. As described previously, deletion of alpha-synuclein had no significant effects on brain structure or composition. In particular, the levels of synaptic proteins were not altered, and the concentrations of dopamine, dopamine metabolites, and dopaminergic proteins were unchanged. Upon acute MPTP challenge, alpha-synuclein knockout mice were partly protected from chronic depletion of nigrostriatal dopamine when compared with littermates of the same genetic background, whereas mice carrying the spontaneous deletion of the alpha-synuclein gene exhibited no protection. Furthermore, alpha-synuclein knockout mice but not the mice with the alpha-synuclein gene deletion were slightly more sensitive to methamphetamine than littermate control mice. These results demonstrate that alpha-synuclein is not obligatorily coupled to MPTP sensitivity, but can influence MPTP toxicity on some genetic backgrounds, and illustrate the need for extensive controls in studies aimed at describing the effects of mouse knockouts on MPTP sensitivity.

Sharma, S. K. and M. Ebadi (2003). "Metallothionein attenuates 3-morpholinosydnonimine (SIN-1)-induced oxidative stress in dopaminergic neurons." Antioxid Redox Signal 5(3): 251-64.

            Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other subcortical nuclei associated with a widespread occurrence of Lewy bodies. The causes of cell death in Parkinson's disease are still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative stress have been proposed. We have examined 3-morpholinosydnonimine (SIN-1)-induced apoptosis in control and metallothionein-overexpressing dopaminergic neurons, with a primary objective to determine the neuroprotective potential of metallothionein against peroxynitrite-induced neurodegeneration in Parkinson's disease. SIN-1 induced lipid peroxidation and triggered plasma membrane blebbing. In addition, it caused DNA fragmentation, alpha-synuclein induction, and intramitochondrial accumulation of metal ions (copper, iron, zinc, and calcium), and enhanced the synthesis of 8-hydroxy-2-deoxyguanosine. Furthermore, it down-regulated the expression of Bcl-2 and poly(ADP-ribose) polymerase, but up-regulated the expression of caspase-3 and Bax in dopaminergic (SK-N-SH) neurons. SIN-1 induced apoptosis in aging mitochondrial genome knockout cells, alpha-synuclein-transfected cells, metallothionein double-knockout cells, and caspase-3-overexpressed dopaminergic neurons. SIN-1-induced changes were attenuated with selegiline or in metallothionein-transgenic striatal fetal stem cells. SIN-1-induced oxidation of dopamine to dihydroxyphenylacetaldehyde was attenuated in metallothionein-transgenic fetal stem cells and in cells transfected with a mitochondrial genome, and enhanced in aging mitochondrial genome knockout cells, in metallothionein double-knockout cells and caspase-3 gene-overexpressing dopaminergic neurons. Selegiline, melatonin, ubiquinone, and metallothionein suppressed SIN-1-induced down-regulation of a mitochondrial genome and up-regulation of caspase-3 as determined by reverse transcription-polymerase chain reaction. The synthesis of mitochondrial 8-hydroxy-2-deoxyguanosine and apoptosis-inducing factors were increased following exposure to 1-methyl-4-phenylpyridinium ion or rotenone. Pretreatment with selegiline or metallothionein suppressed 1-methyl-4-phenylpyridinium ion-, 6-hydroxydopamine-, and rotenone-induced increases in mitochondrial 8-hydroxy-2-deoxyguanosine accumulation. Transfection of aging mitochondrial genome knockout neurons with mitochondrial genome encoding complex-1 or melanin attenuated the SIN-1-induced increase in lipid peroxidation. SIN-1 induced the expression of alpha-synuclein, caspase-3, and 8-hydroxy-2-deoxyguanosine, and augmented protein nitration. These effects were attenuated by metallothionein gene overexpression. These studies provide evidence that nitric oxide synthase activation and peroxynitrite ion overproduction may be involved in the etiopathogenesis of Parkinson's disease, and that metallothionein gene induction may provide neuroprotection.

Sherer, T. B., J. H. Kim, et al. (2003). "Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha-synuclein aggregation." Exp Neurol 179(1): 9-16.

            Previous studies demonstrated that chronic systemic exposure to the pesticide and mitochondrial toxin rotenone through jugular vein cannulation reproduced many features of Parkinson's disease (PD) in rats, including nigrostriatal dopaminergic degeneration and formation of alpha-synuclein-positive cytoplasmic inclusions in nigral neurons (R. Betarbet et al., 2000, Nat. Neurosci. 3, 1301-1306). Although novel and conceptually important, the rotenone model of PD suffered from being extremely labor-intensive. The current paper demonstrates that these same features of PD can be reproduced by chronic, systemic exposure to rotenone following implantation of subcutaneous osmotic pumps. Chronic subcutaneous exposure to low doses of rotenone (2.0-3.0 mg/kg/day) caused highly selective nigrostriatal dopaminergic lesions. Striatal neurons containing DARPP-32 (dopamine and cAMP-regulated phosphoprotein) remained intact with normal morphology, and NeuN staining revealed normal neuronal nuclear morphology. Neurons of the globus pallidus and subthalamic nucleus were spared. Subcutaneous rotenone exposure caused alpha-synuclein-positive cytoplasmic aggregates in nigral neurons. This new protocol for chronic rotenone administration is a substantial improvement in terms of simplicity and throughput.

Siderowf, A. and M. Stern (2003). "Update on Parkinson disease." Ann Intern Med 138(8): 651-8.

            This Update reviews developments in the pathophysiology and treatment of Parkinson disease during the past several years. In the area of pathophysiology, studies have addressed the contribution of environmental factors such as caffeine and pesticides. Large-scale epidemiologic studies have also expanded the role genetic factors are thought to play. Detailed studies of kindreds with familial Parkinson disease due to alpha-synuclein and parkin have catalyzed basic science investigations into the pathologic mechanisms of the disease. These studies have led to the development of a pathophysiologic model of Parkinson disease that emphasizes abnormal protein aggregation. Studies of treatment have clarified the relative roles of l-dopa and dopamine agonists in early Parkinson disease and shown the potential for surgical interventions, particularly deep-brain stimulation, to relieve the symptoms of advanced, medically refractory disease.

Simon, H. H., L. Bhatt, et al. (2003). "Midbrain dopaminergic neurons: determination of their developmental fate by transcription factors." Ann N Y Acad Sci 991: 36-47.

            Midbrain dopaminergic neurons are the main source of dopamine in the mammalian central nervous system and are associated with one of the most prominent human neurological disorders, Parkinson's disease. During development, they are induced in the ventral midbrain by an interaction between two diffusible factors, SHH and FGF8. The local identity of this part of the midbrain is probably determined by the combinatorial expression of three transcription factors, Otx2, Pax2, and Pax5. After the last cell division, the neurons start to express transcription factors that control further differentiation and the manifestation of cellular properties characteristic for adult dopaminergic neurons of the substantia nigra compacta and the ventral tegmentum. The first to appear is the LIM-homeodomain transcription factor, Lmx1b. It is essential for the survival of these neurons, and it regulates the expression of another transcription factor, Pitx3, an activator of tyrosine hydroxylase. Lmx1b is followed by the orphan steroid receptor Nurr1. It is essential for the expression of the dopaminergic phenotype. Several genes involved in dopamine synthesis, transport, release, and reuptake are regulated by Nurr1. This requirement is specific to the midbrain dopaminergic neurons, since other populations of the same neurotransmitter phenotype develop normally in absence of the gene. A day after Nurr1, two homeodomain transcription factors, engrailed-1 and -2, are expressed. In animals deficient in the two genes, the midbrain dopaminergic neurons are generated, but then fail to differentiate and disappear very rapidly. Interestingly, alpha-synuclein, a gene recently linked to familial forms of Parkinson's disease, is regulated by engrailed-1 and -2.

Smith, R., J. Lotharius, et al. (2003). "[Free radicals and ailing proteins--the culprits behind Parkinson disease?]." Lakartidningen 100(15): 1324-6, 1329-30.

            Parkinson's disease is one of the most common neurodegenerative diseases, and affects approximately 1% of the population over 65 years of age. Many different insults appear to be involved in the etiology of the disease, among them environmental toxins and mitochondrial dysfunction. During the past five years, mutations in five different genes have been linked to rare, familial forms of Parkinson's disease. One of the mutated proteins, alpha-synuclein is normally implicated in synaptic plasticity and vesicle function. Dysfunction of this protein might lead to increased cytoplasmic dopamine levels. Since cytoplasmic dopamine is readily prone to autooxidation and enzymatic degradation--processes which generate reactive oxygen species--failure to properly store dopamine into vesicles might lead to oxidative stress. Indeed, nigral tissue from idiopathic Parkinson's disease patients shows signs of oxidative damage. In this article we propose that dopamine-induced oxidative stress might be a common final pathway in the pathogenesis of the disease.

Speciale, S. G. (2002). "MPTP: insights into parkinsonian neurodegeneration." Neurotoxicol Teratol 24(5): 607-20.

            MPTP burst upon the medical landscape two decades ago, first as a mysterious parkinsonian epidemic, triggering an unparalleled quest for the toxin's identity, and closely followed by an intense pursuit of its cellular mechanisms of action. MPTP treatment created an animal model of many features of Parkinson's disease (PD), used primarily in primates and later in mice. The critical role of oxidative stress damage to vulnerable dopamine neurons, as well as for neurodegenerative diseases in general, emerged from MPTP neurotoxicity. A remarkable cross-fertilization of basic and clinical findings, including genetic and epidemiologic studies, has greatly advanced our understanding of PD and revealed multiple factors contributing to the parkinsonian phenotypes. Brain imaging localizes sites of action and provides potential presymptomatic diagnostic testing. Epidemiologic reports linking PD with pesticide exposure were complimented by supportive evidence from biochemical studies of MPTP and structurally related compounds, especially after low-level, long-term exposure. Genetic studies on the role of risk genes, such as alpha-synuclein or parkin, have been validated by biochemical, anatomical and neurochemical investigations showing factors interacting to produce pathophysiology in the animal model. Focusing on the pivotal role of mitochondria, subcellular pathways participating in cell death have been clarified by unraveling similar sites of action of MPTP. Along the way, compounds antagonizing or potentiating MPTP effects indicated new PD therapies, some of the former achieving clinical trials. The future is encouraging for combating PD and will continue to benefit from the MPTP neurotoxicity model.

Steece-Collier, K., E. Maries, et al. (2002). "Etiology of Parkinson's disease: Genetics and environment revisited." Proc Natl Acad Sci U S A 99(22): 13972-4.

Welch, K. and J. Yuan (2002). "Releasing the nerve cell killers." Nat Med 8(6): 564-5.

Wenning, G. K., K. Seppi, et al. (2002). "A novel grading scale for striatonigral degeneration (multiple system atrophy)." J Neural Transm 109(3): 307-20.

            Striatonigral degeneration (SND) is commonly thought to represent the neuropathological substrate of L-Dopa unresponsive parkinsonism in patients with multiple system atrophy (MSA). Other neuropathological hallmarks of MSA include olivopontocerebellar atrophy (OPCA) and preganglionic sympathetic spinal cord lesions. Clinicopathological evaluation of MSA patients recruited into ongoing natural history studies or neuroprotective intervention trials will require standardized grading of MSA pathology. Based on 25 autopsy cases of MSA, we propose a novel SND grading scale which allows semiquantitative assessment of lesion severity based on neuronal loss, astrogliosis and presence of alpha-synuclein positive glial cytoplasmic inclusions (GCIs) in substantia nigra, putamen, caudate nucleus, and globus pallidus. SND grade I is defined as degeneration of the substantia nigra pars compacta (SNC) with relative preservation of the striatum except for minimal gliosis and GCIs in the posterior putamen ("minimal change MSA"). SND grade II is characterized by neuronal loss, astrogliosis and presence of GCIs in SNC and posterior/dorsolateral putamen. Caudate nucleus and external globus pallidus may exhibit slight gliosis. Striatal pathology is severe and extends to anterior ventromedial subregions in SND grade III. There is neuronal loss in caudate nucleus and globus pallidus. GCIs are more abundant in grade II than grade III SNC and putamen. Preliminary clinicopathologic correlation studies suggest milder parkinsonian disability and better initial L-Dopa responsiveness in SND grade I and II cases compared to grade III cases. Prospective clinicopathologic studies are required to validate the proposed SND grading scale and may result in further subdivisions, particularly of SND grade III.

Wersinger, C. and A. Sidhu (2003). "Differential cytotoxicity of dopamine and H2O2 in a human neuroblastoma divided cell line transfected with alpha-synuclein and its familial Parkinson's disease-linked mutants." Neurosci Lett 342(1-2): 124-8.

            alpha-Synuclein accumulates in Lewy bodies and two missense mutations, A30P and A53T, have been linked to familial Parkinson's disease. Neither the normal function of alpha-synuclein nor the pathomechanism of alpha-synuclein-induced neuropathy are known. SK-N-MC neuroblastoma cells were transiently transfected with either wt alpha-synuclein, or its mutants, and their abilities to protect against oxidative stress were assessed. At low expression levels (1 microg cDNA/10(5) cells), all three synuclein variants were devoid of any effect on dopamine-induced cytotoxicity and nitrite production, whereas at higher expression (5 microg cDNA/10(5) cells), the variants enhanced dopamine-mediated effects. Low levels of wt alpha-synuclein blocked H(2)O(2)-induced cytotoxicity and nitrite production, a protective effect that was partly decreased upon higher expression. Both A30P and A53T increased in a dose-dependent manner H(2)O(2)-induced nitrite production and cell death. These results show an absence of protective effects for the A30P/A53T mutants, and a differential cytoprotective role of alpha-synuclein against oxidants, which varies according to expression levels.

Wersinger, C. and A. Sidhu (2003). "Attenuation of dopamine transporter activity by alpha-synuclein." Neurosci Lett 340(3): 189-92.

            Alpha-synuclein accumulates in Lewy bodies in idiopathic Parkinson's disease. Neither the normal function nor contribution of alpha-synuclein to the pathophysiology of neurodegeneration is known. Here we show that a normal function of alpha-synuclein is the negative modulation of human dopamine transporter (hDAT) activity. In cotransfected Ltk(-) cells, alpha-synuclein attenuated the reuptake of dopamine by hDAT, in a manner dependent on expression levels of alpha-synuclein. Alpha-synuclein-mediated inhibition of hDAT activity was independent of expression vectors, cell types and methods of transfection. The alpha-synuclein-mediated decrease in DAT activity occurred through diminished uptake velocity of dopamine, without changes in the affinity of hDAT for dopamine. Co-immunoprecipitation studies confirmed the formation of a stable complex between alpha-synuclein and DAT, through direct protein:protein interactions. Thus, under normal (non-toxic) expression conditions, alpha-synuclein negatively modulates dopamine uptake by DAT.

Wirdefeldt, K., C. E. Burgess, et al. (2003). "A Linkage Study of Candidate Loci in Familial Parkinson's Disease." BMC Neurol 3(1): 6.

            BackgroundParkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. Most cases are sporadic, however familial cases do exist. We examined 12 families with familial Parkinson's disease ascertained at the Movement Disorder clinic at the Oregon Health Sciences University for genetic linkage to a number of candidate loci. These loci have been implicated in familial Parkinson's disease or in syndromes with a clinical presentation that overlaps with parkinsonism, as well as potentially in the pathogenesis of the disease.MethodsThe examined loci were PARK3, Parkin, DRD (dopa-responsive dystonia), FET1 (familial essential tremor), BDNF (brain-derived neurotrophic factor), GDNF (glial cell line-derived neurotrophic factor), Ret, DAT1 (the dopamine transporter), Nurr1 and Synphilin-1. Linkage to the alpha-synuclein gene and the Frontotemporal dementia with parkinsonism locus on chromosome 17 had previously been excluded in the families included in this study. Using Fastlink, Genehunter and Simwalk both parametric and model-free non-parametric linkage analyses were performed. ResultsIn the multipoint parametric linkage analysis lod scores were below -2 for all loci except FET1 and Synphilin-1 under an autosomal dominant model with incomplete penetrance. Using non-parametric linkage analysis there was no evidence for linkage, although linkage could not be excluded. A few families showed positive parametric and non-parametric lod scores indicating possible genetic heterogeneity between families, although these scores did not reach any degree of statistical significance. ConclusionsWe conclude that in these families there was no evidence for linkage to any of the loci tested, although we were unable to exclude linkage with both parametric and non-parametric methods.

Xu, J., S. Y. Kao, et al. (2002). "Dopamine-dependent neurotoxicity of alpha-synuclein: a mechanism for selective neurodegeneration in Parkinson disease." Nat Med 8(6): 600-6.

            The mechanism by which dopaminergic neurons are selectively lost in Parkinson disease (PD) is unknown. Here we show that accumulation of alpha-synuclein in cultured human dopaminergic neurons results in apoptosis that requires endogenous dopamine production and is mediated by reactive oxygen species. In contrast, alpha-synuclein is not toxic in non-dopaminergic human cortical neurons, but rather exhibits neuroprotective activity. Dopamine-dependent neurotoxicity is mediated by 54 83-kD soluble protein complexes that contain alpha-synuclein and 14-3-3 protein, which are elevated selectively in the substantia nigra in PD. Thus, accumulation of soluble alpha-synuclein protein complexes can render endogenous dopamine toxic, suggesting a potential mechanism for the selectivity of neuronal loss in PD.

Yang, Y., I. Nishimura, et al. (2003). "Parkin suppresses dopaminergic neuron-selective neurotoxicity induced by Pael-R in Drosophila." Neuron 37(6): 911-24.

            Parkin, an E3 ubiquitin ligase that degrades proteins with aberrant conformations, is associated with autosomal recessive juvenile Parkinsonism (AR-JP). The molecular basis of selective neuronal death in AR-JP is unknown. Here we show in an organismal system that panneuronal expression of Parkin substrate Pael-R causes age-dependent selective degeneration of Drosophila dopaminergic (DA) neurons. Coexpression of Parkin degrades Pael-R and suppresses its toxicity, whereas interfering with endogenous Drosophila Parkin function promotes Pael-R accumulation and augments its toxicity. Furthermore, overexpression of Parkin can mitigate alpha-Synuclein-induced neuritic pathology and suppress its toxicity. Our study implicates Parkin as a central player in the molecular pathway of Parkinson's disease (PD) and suggests that manipulating Parkin expression may provide a novel avenue of PD therapy.

Zeng, B. Y., B. Dass, et al. (2002). "6-Hydroxydopamine lesioning differentially affects alpha-synuclein mRNA expression in the nucleus accumbens, striatum and substantia nigra of adult rats." Neurosci Lett 322(1): 33-6.

            The effect of a unilateral 6-hydroxydopamine (6-OHDA) lesion and/or repeated administration of levodopa (L-DOPA) to normal and 6-OHDA-lesioned rats on alpha-synuclein mRNA expression was investigated by in situ hybridization histochemistry. A 6-OHDA lesion of the nigro-striatal pathway alone, confirmed by the loss of nigral tyrosine hydroxylase mRNA expression, markedly decreased alpha-synuclein mRNA in the lesioned substantia nigra (SN). In contrast, the levels of alpha-synuclein mRNA in the denervated striatum and nucleus accumbens were not altered. Chronic administration of L-DOPA to normal or 6-OHDA-lesioned rats had no effect on alpha-synuclein mRNA expression in the SN, striatum or nucleus accumbens. These data confirm that alpha-synuclein is localized in the nigro-striatal tract but that its gene expression is not regulated by dopamine.

Zhou, W., J. Schaack, et al. (2002). "Overexpression of human alpha-synuclein causes dopamine neuron death in primary human mesencephalic culture." Brain Res 926(1-2): 42-50.

            Mutations in the alpha-synuclein gene have been linked to rare cases of familial Parkinson's disease (PD). Alpha-synuclein is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice and Drosophila expressing either wild-type or mutant human alpha-synuclein develop motor deficits, LB-like inclusions in some neurons, and neuronal degeneration. However, the relationship between abnormal aggregates of alpha-synuclein and human dopamine (DA) neuron degeneration remains unclear. In this report, we have investigated the influence of alpha-synuclein expression on DA neurons in primary culture of embryonic human mesencephalon. Two days after culture, human DA cells were transduced with wild-type or mutant human (Ala(53)Thr) alpha-synuclein adenoviruses and maintained for 5 days. Overexpression of mutant and wild-type human alpha-synuclein resulted in 49% (P<0.01) and 27% (P<0.05) loss of DA neurons, respectively, while not affecting viability of other cells in the culture. Overexpression of rat alpha-synuclein or GFP (green fluorescent protein) had no effect on DA neuron survival. Cytoplasmic inclusions of alpha-synuclein were detected immunohistochemically in DA cells transduced with mutant human alpha-synuclein, but not wild-type alpha-synuclein. These results show that overexpression of human alpha-synuclein, particularly the mutant form, can cause human DA neuron death, suggesting that alpha-synuclein may have a primary role in the pathogenesis of PD.

Zigmond, M. J., T. G. Hastings, et al. (2002). "Increased dopamine turnover after partial loss of dopaminergic neurons: compensation or toxicity?" Parkinsonism Relat Disord 8(6): 389-93.

            6-Hydroxydopamine (6-OHDA) has proven a valuable tool in the study of Parkinson's disease (PD); it has also served to emphasize the possibility that this disorder may result in part from the sort of oxidative stress that 6-OHDA exerts on dopamine neurons. In this review we comment on several lines of our research related to the role of oxidative stress in PD, research that has benefited greatly from the work of Gerald Cohen whose memory we honor at this symposium. First, we discuss our use of 6-OHDA to produce an animal model of PD; second, we comment on our studies on dopamine's neurotoxic effects; and finally, we discuss our finding that tyrosine hydroxylase is regulated in part by an interaction with alpha-synuclein. We suggest that PD is associated with an increase in dopamine turnover, which may not only reduce the immediate symptoms of the disease but also contribute to its progression.

Zourlidou, A., M. D. Payne Smith, et al. (2003). "Modulation of cell death by alpha-synuclein is stimulus-dependent in mammalian cells." Neurosci Lett 340(3): 234-8.

            alpha-synuclein is a neuronally-expressed protein which is mutated in familial Parkinson's disease. Previous studies have suggested that over-expression of alpha-synuclein can either enhance, reduce or have no effect on the degree of cell death in response to death-inducing stimuli. We resolve this discrepancy by using a well-characterised cell system to demonstrate that wild type alpha-synuclein can enhance cell death in response to ischaemia/reoxygenation or staurosporine treatment whilst protecting against serum removal and dopamine-induced cell death. In contrast, the two mutant forms of alpha-synuclein uniformly enhance cell death. Hence, the disease-associated mutations appear to convert alpha-synuclein from a protein which modulates cell death differently in different circumstances to forms which have a universal damaging effect.

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