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Wirdefeldt, K., N. Bogdanovic, et al. (2001). "Expression of alpha-synuclein in the human brain: relation to Lewy body disease." Brain Res Mol Brain Res 92(1-2): 58-65.
alpha-Synuclein is mutated in some hereditary cases of Parkinson's disease and the protein precipitates in Lewy bodies, the pathological hallmark of both Parkinson's disease and Lewy body disease. Transgenic mice overexpressing human wild-type alpha-synuclein develop alpha-synuclein-immunoreactive inclusions in brain regions typically affected with Lewy body disease. We used in situ hybridization to characterize alpha-synuclein expression and examine mRNA levels in patients affected with Lewy body disease and controls. Substantia nigra was avoided because of the extensive neuronal loss and cingulate gyrus was chosen as it is one of the diagnostic regions in Lewy body disease where Lewy bodies most frequently are demonstrated. beta-tubulin was used to control for neuronal degeneration. The alpha-synuclein probe showed intense labeling of pyramidal cells in lamina III and V in both patients and controls. We found no difference in alpha-synuclein mRNA levels and beta-tubulin mRNA was not significantly altered (P=0.06) in patient brains. There was no difference in the ratio of alpha-synuclein and beta-tubulin mRNA levels between patients and controls. Further, we found no relationship between alpha-synuclein mRNA levels and Lewy bodies. Great variability in alpha-synuclein mRNA levels among patients indicates that Lewy body disease may be a heterogeneous disorder with regard to alpha-synuclein involvement.

Wenning, G. K. and K. Seppi (2001). "[In Process Citation]." Schweiz Rundsch Med Prax 90(23): 1035-40.
Numerous studies of the past years have established the clinical features, course and neuropathology characterizing multiple system atrophy (MSA). Clinically, two motor subtypes can be classified based on the predominance of a parkinsonian syndrome refractory to L-dopa and cerebellar ataxia. 80% of the cases involve MSA-P (the parkinsonian variant of MSA) and 20% MSA-C (cerebellar variant of MSA). Virtually all of these patients show disturbances of autonomic and urogenital function, half of the patients also exhibit pyramidal signs. Neuropathologically, MSA-C is based on an olivopontocerebellar atrophy (OPCA) and MSA-P on striatonigral degeneration (SND). However, a combination of OPCA and SND pathologies is observed in most cases. Recent evidence suggests that a key pathogenetic role may be played by glial alpha synuclein-containing inclusion bodies, which might lead to neuronal dysfunction and ultimately to cell loss. There is no therapy known to be effective in treating the motor disorders of MSA-C. By contrast, L-dopa replacement is at least transiently effective in about 30% of patients with MSA-P. Currently, initial efforts are being undertaken throughout Europe to develop neuroprotective solutions. Experiments are underway to test whether neurotransplantation by striatal grafting is a suitable method for inducing a clinically relevant response to L-dopa. Neurologically, the options for treating orthostatic hypertension and urogenital disorders are often overlooked.

Walker, R. H., M. F. Brin, et al. (2001). "Distribution and immunohistochemical characterization of torsinA immunoreactivity in rat brain." Brain Res 900(2): 348-54.
A mutation of the DYT1 gene on chromosome 9q34 has recently been identified as the cause of one form of autosomal-dominantly inherited dystonia. TorsinA, the protein product of this gene, has homology with the family of heat shock proteins, and is found in many peripheral tissues and brain regions. We used a polyclonal antibody to torsinA, developed in our laboratory, to systematically examine the regional distribution of torsinA in rat brain. We find that neurons in all examined structures are immunoreactive for this protein. There is intense immunoreactivity in most neuronal nuclei, with slightly less labeling of cytoplasm and proximal processes. Terminals also are labeled, especially in striatum, neocortex and hippocampus. Double-labeling fluorescence immunohistochemistry using antibodies to neurotransmitters and other neurochemical markers demonstrated that the majority of neurons of all studied neurochemical types are immunoreactive for torsinA. Our findings indicate that torsinA is widely distributed in the central nervous system implicating additional, localized factors, perhaps within the basal ganglia, in the development of dystonia. Many other proteins have a similar widespread distribution, including some which have been implicated in other movement disorders and neurodegenerative processes, such as parkin, alpha-synuclein, ubiquitin and huntingtin. The distribution of torsinA in rat brain as demonstrated by immunohistochemistry contrasts with the results of in situ hybridization studies of torsinA mRNA in human postmortem brain in which a more limited distribution was found.

Waelter, S., A. Boeddrich, et al. (2001). "Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation." Mol Biol Cell 12(5): 1393-407.
The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

Volles, M. J., S. J. Lee, et al. (2001). "Vesicle Permeabilization by Protofibrillar alpha-Synuclein: Implications for the Pathogenesis and Treatment of Parkinson's Disease." Biochemistry 40(26): 7812-9.
Fibrillar alpha-synuclein is a component of the Lewy body, the characteristic neuronal inclusion of the Parkinson's disease (PD) brain. Both alpha-synuclein mutations linked to autosomal dominant early-onset forms of PD promote the in vitro conversion of the natively unfolded protein into ordered prefibrillar oligomers, suggesting that these protofibrils, rather than the fibril itself, may induce cell death. We report here that protofibrils differ markedly from fibrils with respect to their interactions with synthetic membranes. Protofibrillar alpha-synuclein, in contrast to the monomeric and the fibrillar forms, binds synthetic vesicles very tightly via a beta-sheet-rich structure and transiently permeabilizes these vesicles. The destruction of vesicular membranes by protofibrillar alpha-synuclein was directly observed by atomic force microscopy. The possibility that the toxicity of alpha-synuclein fibrillization may derive from an oligomeric intermediate, rather than the fibril, has implications regarding the design of therapeutics for PD.

Uversky, V. N., J. Li, et al. (2001). "Pesticides directly accelerate the rate of alpha-synuclein fibril formation: a possible factor in Parkinson's disease." FEBS Lett 500(3): 105-8.
Parkinson's disease involves intracellular deposits of alpha-synuclein in the form of Lewy bodies and Lewy neurites. The etiology of the disease is unknown, however, several epidemiological studies have implicated environmental factors, especially pesticides. Here we show that several pesticides, including rotenone, dieldrin and paraquat, induce a conformational change in alpha-synuclein and significantly accelerate the rate of formation of alpha-synuclein fibrils in vitro. We propose that the relatively hydrophobic pesticides preferentially bind to a partially folded intermediate conformation of alpha-synuclein, accounting for the observed conformational changes, and leading to association and subsequent fibrillation. These observations suggest one possible underlying molecular basis for Parkinson's disease.

Uversky, V. N., J. Li, et al. (2001). "Evidence for a partially folded intermediate in alpha-synuclein fibril formation." J Biol Chem 276(14): 10737-44.
Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.

Turnbull, S., B. J. Tabner, et al. (2001). "alpha-synuclein implicated in Parkinson's disease catalyses the formation of hydrogen peroxide in vitro." Free Radic Biol Med 30(10): 1163-70.
Some rare inherited forms of Parkinson's disease (PD) are due to mutations in the gene encoding a 140-amino acid presynaptic protein called alpha-synuclein. In PD, and some other related disorders such as dementia with Lewy bodies, alpha-synuclein accumulates in the brain in the form of fibrillar aggregates, which are found inside the neuronal cytoplasmic inclusions known as Lewy bodies. By means of an electron spin resonance (ESR) spin trapping method, we show here that solutions of full-length alpha-synuclein, and a synthetic peptide fragment of alpha-synuclein corresponding to residues 61-95 (the so-called non-Abeta component or NAC), both liberate hydroxyl radicals upon incubation in vitro followed by the addition of Fe(II). We did not observe this property for the related beta- and gamma-synucleins, which are not found in Lewy bodies, and are not linked genetically to any neurodegenerative disorder. There is abundant evidence for the involvement of free radicals and oxidative stress in the pathogenesis of nigral damage in PD. Our new data suggest that the fundamental molecular mechanism underlying this pathological process could be the production of hydrogen peroxide by alpha-synuclein.

Trojanowski, J. Q. and V. M. Lee (2001). "Parkinson's disease and related neurodegenerative synucleinopathies linked to progressive accumulations of synuclein aggregates in brain." 7(3): 247-251.
Parkinson's disease (PD) is the most common neurodegenerative movement disorder and the classic clinical-neuropathological features of PD have been well established, including many aspects of the morphology and distribution the filamentous hallmark intraneuronal inclusions of PD known as Lewy bodies (LBs). Nonetheless, the mechanisms underlying brain degeneration in PD are unknown, while only partially effective symptomatic treatments for PD are available, and there are no known therepeutic interventions that are able to prevent PD or block or retard the progression of this relentless disorder. However, dramatic new insights into pathobiology of PD have emerged recently with recognition that alpha-synuclein abnormalities play a role in the onset and/or progression of PD. Moreover, continuing advances in this new research arena provide fresh research opportunities to advance understanding of PD, and these novel breakthroughs will accelerate discovery of more effective therapies for PD.

Touchman, J. W., A. Dehejia, et al. (2001). "Human and mouse alpha-synuclein genes: comparative genomic sequence analysis and identification of a novel gene regulatory element." Genome Res 11(1): 78-86.
The human alpha-synuclein gene (SNCA) encodes a presynaptic nerve terminal protein that was originally identified as a precursor of the non-beta-amyloid component of Alzheimer's disease plaques. More recently, mutations in SNCA have been identified in some cases of familial Parkinson's disease, presenting numerous new areas of investigation for this important disease. Molecular studies would benefit from detailed information about the long-range sequence context of SNCA. To that end, we have established the complete genomic sequence of the chromosomal regions containing the human and mouse alpha-synuclein genes, with the objective of using the resulting sequence information to identify conserved regions of biological importance through comparative sequence analysis. These efforts have yielded approximately 146 and approximately 119 kb of high-accuracy human and mouse genomic sequence, respectively, revealing the precise genetic architecture of the alpha-synuclein gene in both species. A simple repeat element upstream of SNCA/Snca has been identified and shown to be necessary for normal expression in transient transfection assays using a luciferase reporter construct. Together, these studies provide valuable data that should facilitate more detailed analysis of this medically important gene.

Togo, T., E. Iseki, et al. (2001). "Glial involvement in the degeneration process of Lewy body-bearing neurons and the degradation process of Lewy bodies in brains of dementia with Lewy bodies." J Neurol Sci 184(1): 71-5.
Glial involvement in the degeneration process of Lewy body (LB)-bearing neurons and the degradation process of LBs in the cerebral cortex and amygdala in brains of dementia with Lewy bodies was investigated immunohistochemically. HLA-DR-positive microglia frequently extended their processes to degenerated neurons with alpha-synuclein-positive LBs, while some GFAP-positive astroglial processes attached to weakly alpha-synuclein-positive extracellular LBs. Some intracellular LBs were immunoreactive to anti-C4d antibody, and these LB-bearing neurons were involved by activated microglia. About half of the intracellular LBs were immunoreactive to anti-chromogranin-A (CGA) antibody, and most of CGA-positive LB-bearing neurons were surrounded by microglia. Although we could find no evident participation of TNF-alpha, a candidate cytokine that is up-regulated by microglia following CGA stimulation, in the degeneration process of LB-bearing neurons, some intracellular LBs were immunoreactive to the antibody to NF-kappaB, a transcriptional factor activated by cytokines. These findings suggest that microglia participate in the degeneration process of LB-bearing neurons via varying immunogenic elements including complement proteins, CGA and probably some cytokines, and that astroglia participate in the degradation process of LBs.

Terai, K., A. Iwai, et al. (2001). "beta-amyloid deposits in transgenic mice expressing human beta-amyloid precursor protein have the same characteristics as those in Alzheimer's disease." Neuroscience 104(2): 299-310.
A transgenic mouse expressing the human beta-amyloid precursor protein with the "Swedish" mutation, Tg2576, was used to investigate the mechanism of amyloid-beta peptide (Abeta) deposition. We characterized Abeta deposits in the cerebral cortex biochemically and pathologically. A surface-enhanced laser desorption/ionization affinity mass spectrometric study using the 6E10 monoclonal antibody demonstrated that the major species of Abeta in a formic acid-extracted fraction of the cortex were Abeta(1-38), Abeta(1-40) and Abeta(1-42). Immunohistochemistry using antibodies to the carboxy-terminal epitopes of Abeta(1-40) and Abeta(1-42), as well as 6E10, showed that plaques containing Abeta(1-42) were more numerous than those containing Abeta(1-40) throughout the cortex. Laser confocal analysis of the immunoreactivities in the plaques demonstrated that Abeta(1-40) was preferentially located in the central part of the Abeta(1-42) positive plaques. Enzyme-linked immunosorbent assay measurements of Abeta(1-40) and Abeta(1-42) showed that Abeta(1-40) was several-fold more abundant than Abeta(1-42).From these data we suggest that Abeta(1-42) deposition may precede Abeta(1-40) deposition, while Abeta(1-40) begins to deposit in the central part of the plaques and accumulates there. Furthermore, localization of Abeta(1-40) corresponded almost exactly to congophilic structures, which were associated with aberrant swollen synapses detected with antibodies to synaptophysin and alpha-synuclein. Thus, Abeta deposits in Tg2576 mice have similar characteristics to those in Alzheimer's disease.

Tanji, K., T. Imaizumi, et al. (2001). "Expression of alpha-synuclein in a human glioma cell line and its up-regulation by interleukin-1beta." Neuroreport 12(9): 1909-12.
Although alpha-synuclein is expressed primarily in neurons, it is a major component of oligodendroglial and astrocytic inclusions in several neurodegenerative diseases. Recent study has further demonstrated that alpha-synuclein is expressed in cultured rat oligodendrocytes. We determined whether alpha-synuclein might be expressed in astrocytic cells. alpha-Synuclein mRNA and protein were detected in U251 human astrocytic glioma cells and normal human astrocytes, and the levels were increased in the former, but not in the latter, by stimulation with interleukin-1beta in a time- and concentration-dependent manner. Serum deprivation also led to an increase of alpha-synuclein mRNA and protein in U251 cells. Immunofluorescent staining confirmed the cell-associated alpha-synuclein. These findings suggest that human astrocytes can produce alpha-synuclein in culture and that certain inflammatory cytokines and cell stress increase alpha-synuclein expression.

Tanaka, Y., S. Engelender, et al. (2001). "Inducible expression of mutant alpha-synuclein decreases proteasome activity and increases sensitivity to mitochondria-dependent apoptosis." Hum Mol Genet 10(9): 919-26.
Parkinson's disease (PD) is a common progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra. Although mutations in alpha-synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic neural cell death occurs remains unknown. Proteins encoded by two other genes in which mutations cause familial PD, parkin and UCH-L1, are involved in regulation of the ubiquitin-proteasome pathway, suggesting that dysregulation of the ubiquitin-proteasome pathway is involved in the mechanism by which these mutations cause PD. We established inducible PC12 cell lines in which wild-type or mutant alpha-synuclein can be de-repressed by removing doxycycline. Differentiated PC12 cell lines expressing mutant alpha-synuclein showed decreased activity of proteasomes without direct toxicity. Cells expressing mutant alpha-synuclein showed increased sensitivity to apoptotic cell death when treated with sub-toxic concentrations of an exogenous proteasome inhibitor. Apoptosis was accompanied by mitochondrial depolarization and elevation of caspase-3 and -9, and was blocked by cyclosporin A. These data suggest that expression of mutant alpha-synuclein results in sensitivity to impairment of proteasome activity, leading to mitochondrial abnormalities and neuronal cell death.

Takenouchi, T., M. Hashimoto, et al. (2001). "Reduced neuritic outgrowth and cell adhesion in neuronal cells transfected with human alpha-synuclein." Mol Cell Neurosci 17(1): 141-50.
Since recent reports have suggested that alpha-synuclein might play a role in neuronal plasticity, the main objective of this study was to determine the effects of alpha-synuclein on neuritic outgrowth. We stably transfected either human (h) alpha- or beta-synuclein cDNA in B103 rat neuronal cells. Expression of h(alpha)-synuclein resulted in reduced neurite extension and weak adhesion compared to vector-transfected and h(beta)-synuclein expressing cells. To investigate the potential pathways involved, we studied the effects of reagents known to modulate B103 proliferation and differentiation. Neither phorbol 12-myristate 13-acetate nor forskolin or antioxidants (catalase, superoxide dismutase, or vitamin E) were able to restore the reduced length of neurites in h(alpha)-synuclein-expressing cells. These results suggest that reduced neuritic activity in the h(alpha)-synuclein-expressing cells might be due, in part, to alterations in cell adhesion capacity. This might be attributed to alpha-synuclein affecting a signal transduction pathway distinct from protein kinase C and protein kinase A.

Swerdlow, R. H., J. K. Parks, et al. (2001). "Biochemical analysis of cybrids expressing mitochondrial DNA from Contursi kindred Parkinson's subjects." Exp Neurol 169(2): 479-85.
Complex I activity is reduced in cytoplasmic hybrid (cybrid) cell lines that contain mitochondrial DNA (mtDNA) from sporadic Parkinson's disease (PD) patients. This implies that mtDNA aberration occurs in sporadic PD. To assess the integrity of mtDNA in autosomal dominant PD arising from mutation of the alpha-synuclein gene, we transferred mitochondrial genes from PD-affected members of the Italian-American Contursi kindred to cells previously depleted of their endogenous mtDNA. Unlike cybrid cell lines expressing mtDNA from persons with sporadic or maternally inherited PD, the resultant Contursi cybrid lines did not manifest complex I deficiency, indicating that in Contursi PD mtDNA integrity is relatively preserved. Compared to control cybrids, however, Contursi cybrid lines did show some evidence of oxidative stress. For reasons that are unclear, at least a limited amount of mtDNA damage may nevertheless develop in PD patients with alpha-synuclein mutation. Copyright 2001 Academic Press.

Surguchov, A., B. McMahan, et al. (2001). "Synucleins in ocular tissues." J Neurosci Res 65(1): 68-77.
Synucleins are small proteins associated with neurodegenerative diseases and some forms of cancer. Most studies of this group of proteins have been directed to the elucidation of their role in the brain and their connection to the formation of depositions in brain tissues. Here we describe the localization of different types of synucleins in ocular tissues. By Western blot analysis, all members of the synuclein family are found in the retina and optic nerve, where their relative ratio varies. The data on immunohistochemical staining show that different members of the synuclein family have different localizations in ocular tissues. Alpha-synucleins and beta-synucleins are present predominantly in the inner plexiform layer, whereas gamma-synuclein is in the nerve fiber layer. In transgenic mice overexpressing alpha-synuclein, a different pattern of localization depending on the promoter used for the expression was observed. In Alzheimer's disease patients, immunohistochemical staining for gamma-synuclein revealed the loss of immunoreactivity in the nerve fiber layer and the nerve fiber layer and the appearance of immunopositive cells in or near the outer nuclear layer. We conclude that, in mature eyes, synucleins are present predominantly in the retina and optic nerve, and the immunoreactivity of gamma-synuclein changes specifically in the retina of Alzheimer's disease patients. In transgenic mice overexpressing alpha-synuclein, immunopositive deposits in the optic nerve and accumulation of immunoreactivity in specific retinal cells were found. Copyright 2001 Wiley-Liss, Inc.

Sung, J. Y., J. Kim, et al. (2001). "Induction of neuronal cell death by Rab5A-dependent endocytosis of alpha-synuclein." J Biol Chem 276(29): 27441-8.
The presynaptic alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. Here we examined the pathogenic mechanism of neuronal cell death induced by alpha-synuclein. The exogenous addition of alpha-synuclein caused a marked decrease of cell viability in primary and immortalized neuronal cells. The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of alpha-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of alpha-synuclein.

Stefanis, L., N. Kholodilov, et al. (2001). "Synuclein-1 is selectively up-regulated in response to nerve growth factor treatment in PC12 cells." J Neurochem 76(4): 1165-76.
Mutations in the alpha-synuclein gene have recently been identified in families with inherited Parkinson's disease and the protein product of this gene is a component of Lewy bodies, indicating that alpha-synuclein is involved in Parkinson's disease pathogenesis. A role for normal alpha-synuclein in synaptic function, apoptosis or plasticity responses has been suggested. We show here that in rat pheochromocytoma PC12 cells synuclein-1, the rat homolog of human alpha-synuclein, is highly and selectively up-regulated at the mRNA and protein levels after 7 days of nerve growth factor treatment. Synuclein-1 expression appears neither sufficient nor necessary for the neuritic sprouting that occurs within 1-2 days of nerve growth factor treatment. Rather, it likely represents a component of a late neuronal maturational response. Synuclein-1 redistributes diffusely within the cell soma and the neuritic processes in nerve growth factor-treated PC12 cells. Cultured neonatal rat sympathetic neurones express high levels of synuclein-1, with a diffuse intracellular distribution, similar to neuronal PC12 cells. These results suggest that levels of synuclein-1 may be regulated by neurotrophic factors in the nervous system and reinforce a role for alpha-synuclein in plasticity-maturational responses. In contrast, there is no correlation between synuclein expression and apoptotic death following trophic deprivation.

Spira, P. J., D. M. Sharpe, et al. (2001). "Clinical and pathological features of a Parkinsonian syndrome in a family with an Ala53Thr alpha-synuclein mutation." Ann Neurol 49(3): 313-9.
We describe an Australian family of Greek origin with a parkinsonian syndrome and an Ala53Thr alpha-synuclein gene mutation. Five of 9 siblings were affected, the average age of onset was 45 years, and the initial symptoms were variable, including resting tremor, bradykinesia, and gait disturbance, as previously described in families with the same point mutation. Affected family members responded well to levodopa, developed progressive cognitive impairment, and had a disease duration of 5 to 16 years. Pathologic features typical of idiopathic Parkinson's disease were found at autopsy. However, there were several additional features not previously reported in families with this gene mutation. These features included severe central hypoventilation, orthostatic hypotension, prominent myoclonus, and urinary incontinence. An abundance of alpha-synuclein-immunoreactive Lewy neurites were found in the brainstem pigmented nuclei, hippocampus, and temporal neocortex. The Lewy neurites were associated with temporal lobe vacuolation. Subcortical basal ganglia cell loss and gliosis were seen. These additional clinical and pathological features suggest that the Ala53Thr alpha-synuclein mutation can produce a more widespread disorder than found in typical idiopathic Parkinson's disease.

Sopher, B. L., K. L. Koszdin, et al. (2001). "Genomic organization, chromosome location, and expression analysis of mouse beta-synuclein, acandidate for involvement in neurodegeneration." Cytogenet Cell Genet 93(1-2): 117-23.
The synuclein family of proteins is a group of primarily brain-expressed polypeptides that show a high degree of amino acid conservation. alpha-Synuclein is the best known of the synuclein family, as it is a major component of the Lewy body, a cytoplasmic inclusion characteristic of Parkinson's disease as well as a variety of related neurodegenerative disorders. With the discovery that mutations in alpha-synuclein can cause Parkinson's disease, a potential role for the other synuclein family members in neurodegenerative disease is being considered. beta-Synuclein in particular may deserve special attention, as it is co-expressed with alpha-synuclein at presynaptic nerve terminals, is subject to phosphorylation by Ca(2+) calmodulin protein kinase II, appears important for neural plasticity, and forms aggregates in the brains of patients with Parkinson's disease and a related disorder. To facilitate study of beta-synuclein, we have cloned the mouse beta-synuclein gene (Sncb) and determined its genomic organization, size, and intron-exon structure. Using an interspecific backcross mapping panel from The Jackson Laboratory, we were then able to localize Sncb to chromosome 13 at the MGD 35.0 cM position. Like the human beta-synuclein gene, Sncb appears to consist of six exons separated by five introns. Unlike the human beta-synuclein gene, the mouse ortholog possesses a variant GC 5' splice donor sequence at the exon 4 - intron 4 boundary in a highly conserved splice junction consensus. Northern blot analysis and Western blot analysis both indicate that Sncb is highly expressed in the brain. Knowledge of the genomic organization and expression pattern of Sncb will allow functional studies of its potential role in neurodegeneration to commence in the mouse. Copyright 2001 S. Karger AG, Basel

Simon, H. H., H. Saueressig, et al. (2001). "Fate of midbrain dopaminergic neurons controlled by the engrailed genes." J Neurosci 21(9): 3126-34.
Deficiencies in neurotransmitter-specific cell groups in the midbrain result in prominent neural disorders, including Parkinson's disease, which is caused by the loss of dopaminergic neurons of the substantia nigra. We have investigated in mice the role of the engrailed homeodomain transcription factors, En-1 and En-2, in controlling the developmental fate of midbrain dopaminergic neurons. En-1 is highly expressed by essentially all dopaminergic neurons in the substantia nigra and ventral tegmentum, whereas En-2 is highly expressed by a subset of them. These neurons are generated and differentiate their dopaminergic phenotype in En-1/En-2 double null mutants, but disappear soon thereafter. Use of an En-1/tau-LacZ knock-in mouse as an autonomous marker for these neurons indicates that they are lost, rather than that they change their neurotransmitter phenotype. A single allele of En-1 on an En-2 null background is sufficient to produce a wild type-like substantia nigra and ventral tegmentum, whereas in contrast a single allele of En-2 on an En-1 null background results in the survival of only a small proportion of these dopaminergic neurons, a finding that relates to the differential expression of En-1 and En-2. Additional findings indicate that En-1 and En-2 regulate expression of alpha-synuclein, a gene that is genetically linked to Parkinson's disease. These findings show that the engrailed genes are expressed by midbrain dopaminergic neurons from their generation to adulthood but are not required for their specification. However, the engrailed genes control the survival of midbrain dopaminergic neurons in a gene dose-dependent manner. Our findings also suggest a link between engrailed and Parkinson's disease.

Shimura, H., M. G. Schlossmacher, et al. (2001). "Ubiquitination of a new form of alpha-synuclein by parkin from human brain: implications for Parkinson's disease." Science 293(5528): 263-9.
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.

Shimo, Y., M. Takanashi, et al. (2001). "[A-56-year-old woman with parkinsonism, whose mother had Parkinson's disease]." No To Shinkei 53(5): 495-505.
We report a 56-year-old woman with progressive gait disturbance. Her mother had Parkinson's disease with onset at age 70. She died at age 74 and the post-mortem examination confirmed the diagnosis of Lewy body positive Parkinson's disease. The patient was well until the age of 50(1995) when she noted an onset of resting tremor and difficulty of gait. She also developed delusional ideation and was admitted to a psychiatric service of another hospital, where a major tranquilizer was given. The delusion disappeared but she developed marked rigidity. The major tranquilizer was discontinued and an anticholinergic and amantadine HCl were given. She showed marked improvement to Hoehn and Yahr stage II and was discharged. In 1995, when she was 52 years of the age, she developed delusion again and a major tranquilizer was given. She developed marked parkinsonism again and became Hoehn and Yahr stage V. The major tranquilizer was discontinued and she was treated with levodopa/carbidopa, trihexyphenidyl, bromocriptine, and dops. She improved remarkably to stage II. She was admitted to our service on October 8, 1996 for drug adjustment. She was alert and not demented. She was anxious but delusion or hallucination was noted. Higher cerebral functions were intact. Cranial nerve functions were also intact except for masked face and small voice. Her posture was stooped and steps were small. She showed retropulsion and moderate bradykinesia. Resting tremor was noted in her left hand. Rigidity was noted in both legs. No cerebellar ataxia or weakness was noted. Deep tendon reflexes were within normal range and sensation was intact. Her cranial MRI revealed some atrophic changes in the putamen, in which a T 2-high signal linear lesion was seen along the lateral border of the putamen bilaterally. In addition, posterior part of the putamen showed T 2-low signal intensity change. She was treated with 1.6 mg of talipexole, 6 mg of trihexyphenidyl, and 100 mg of L-dops. She was in stage III of Hoehn and Yahr. She developed neurogenic bladder with a large amount of residual urine for which she required catheterization. She was transferred to another hospital. Despite drug adjustment, she lost response to levodopa and her parkinsonism deteriorated gradually. She also developed syncope orthostatic hypotension. In April of 1998, she developed intracerebral hemorrhage and was admitted again on April 19, 1998. She was unable to stand and showed marked akinesia and rigidity. She was in stage V of Hoehn and Yahr. Her cranial CT scan revealed bilateral high-density lesions in the posterior parietal lobes. She developed dysphagia for which she required gastrostomy. She was transferred to another hospital but her clinical condition deteriorated further. On December 22, 1999, she developed fever and dyspnea and was admitted to our service again. She developed cardial arrest at the emergency room from hypoxia. She was resuscitated; however, she was comatose with loss of brain stem reflexes. Later on she developed generalized myoclonus. She developed cardiac arrest and pronounced dead on December 28, 1999. The patient was discussed in a neurological CPC. The chief discussant arrived at the conclusion that the patient had striatonigral degeneration because of poor response to levodopa in the later course, autonomic failures, and MRI changes. Some other participants thought that the patient had a form of familial Parkinson's disease. Opinions were divided into these two possibilities. Post-mortem examination revealed that the substantia nigra showed intense neuronal loss and gliosis, however, no Lewy bodies were seen. In addition, intracytoplasmic inclusions were seen in oligodendrocytes. The putamen was markedly atrophic in its posterior part with marked gliosis and neuronal loss. The ventromedial part of the pontine nucleus also showed neuronal loss and intracytoplasmic glial inclusions. Pathologic diagnosis was multiple system atrophy. In the parietal lobe, an arteriovenous malformation with bleeding was noted. This is very unique case. Although her mother had Lewy body-positive Parkinson's disease, the patient had Lewy body-negative multiple system atrophy with a-synuclein-positive glial inclusions. Whether this is just a coincidental occurrence or the presence of a genetic load for Parkinson's disease might triggered her multiple system atrophy is an interesting question to be answered in future.

Sharon, R., M. S. Goldberg, et al. (2001). "alpha -Synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins." Proc Natl Acad Sci U S A 98(16): 9110-5.
alpha-Synuclein (alphaS) is a 140-residue neuronal protein that forms insoluble cytoplasmic aggregates in Parkinson's disease (PD) and several other neurodegenerative disorders. Two missense mutations (A53T and A30P) are linked to rare forms of familial PD. The normal function of alphaS is unknown, and cultured cell systems that model its modification from soluble monomers to aggregated forms have not been reported. Through a systematic centrifugal fractionation of mesencephalic neuronal cell lines and transgenic mouse brains expressing wild-type or A53T human alphaS, we observed unusual, previously unrecognized species of alphaS that migrate well above the 17-kDa monomeric form in denaturing gels. Incubation at 65 degrees C of high-speed cytosols from cells or brains revealed a modified alphaS species migrating at approximately 36 kDa and an extensive higher molecular mass alphaS-reactive smear. Extraction of the cytosols with chloroform/methanol or with a resin (Lipidex 1000) that binds fatty acids resulted in a similar pattern of higher molecular mass alphaS forms. On the basis of this effect of delipidation, we reexamined the primary structure of alphaS and detected a motif at the N and C termini that is homologous to a fatty acid-binding protein signature. In accord, we found that purified human alphaS binds oleic acid, with an apparent K(d) of 12.5 &mgr;M. We also observed an enhanced association of A53T alphaS with microsomal membranes in both mesencephalic cells and transgenic mouse brains. We conclude that alphaS has biochemical properties and a structural motif that suggest it is a novel member of the fatty acid-binding protein family and may thus transport fatty acids between the aqueous and membrane phospholipid compartments of the neuronal cytoplasm.

Sharma, N., J. Hewett, et al. (2001). "A close association of torsinA and alpha-synuclein in Lewy bodies: a fluorescence resonance energy transfer study." Am J Pathol 159(1): 339-44.
TorsinA, a novel protein in which a mutation causes dominant, early onset torsion dystonia, may serve as a chaperone for misfolded proteins that require refolding or degradation. It has been hypothesized that misfolded alpha-synuclein, a protein in which two mutations cause autosomal dominantly inherited Parkinson's disease, serves as a nidus for the development of a Lewy body. We hypothesized that torsinA plays a role in the cellular processing of alpha-synuclein. We demonstrate that anti-torsin antibodies stain Lewy bodies and Lewy neurites in the substantia nigra and cortex. Using sensitive fluorescent resonance energy transfer (FRET) techniques, we find evidence of a close association between torsinA and alpha-synuclein in Lewy bodies.

Seppi, K., Z. Puschban, et al. (2001). "Overstimulation of the alpha1B-adrenergic receptor causes a "seizure plus" syndrome." Nat Med 7(2): 132.

Satoh, J. and Y. Kuroda (2001). "Alpha-synuclein expression is up-regulated in NTera2 cells during neuronal differentiation but unaffected by exposure to cytokines and neurotrophic factors." 8(1): 7-17.
Increasing evidence has indicated that proinflammatory cytokines such as TNF-alpha and IL-1beta, produced by activated microglia and astrocytes, play a key role in progressive degeneration of the nigrostriatal dopaminergic neurons in Parkinson's disease (PD). Since alpha-synuclein is a major component of Lewy bodies in PD brains, we studied the constitutive and cytokine/neurotrophic factor-regulated expression of alpha-synuclein in cultured human neurons by Northern blot and Western blot analyses. The constitutive expression of alpha-synuclein mRNA was identified in a variety of human neural and non-neural cell lines. The levels of alpha-synuclein expression were elevated markedly in NTera2 teratocarcinoma cells following retinoic acid-induced neuronal differentiation, accompanied with an increased expression of synphilin-1, while they were unaltered in NTera2-derived differentiated neurons by exposure to TNF-alpha, IL-1beta, BDNF or GDNF. These results indicate that alpha-synuclein expression in human neurons is up-regulated during differentiation, but is unaffected by a panel of cytokines and neurotrophic factors which are supposed to be involved in the nigral neuronal death and survival.

Satoh, J. I. and Y. Kuroda (2001). "Ubiquitin C-terminal hydrolase-L1 (PGP9.5) expression in human neural cell lines following induction of neuronal differentiation and exposure to cytokines, neurotrophic factors or heat stress." Neuropathol Appl Neurobiol 27(2): 95-104.
Dysfunction of the ubiquitin-dependent proteolytic pathway contributes to progressive accumulation of ubiquitinated protein inclusions in neurodegenerative disorders, such as Parkinson's disease (PD). Ubiquitin C-terminal hydrolase-L1 (UCH-L1), alternatively designated protein gene product 9.5 (PGP9.5), is a neural deubiquitinating enzyme which is identified as a principal constituent of Lewy bodies. To clarify the regulatory mechanism of UCH-L1 expression in human neural cells, we studied the constitutive, cytokine/neurotrophic factor-regulated, and heat stress-induced expression of UCH-L1 in cultured human neural cell lines by Western blot analysis. The constitutive expression of UCH-L1 was identified in SK-N-SH neuroblastoma cells, IMR-32 neuroblastoma cells, U-373MG astrocytoma cells, and NTera2 teratocarcinoma-derived differentiated neurones (NTera2-N). The levels of UCH-L1 expression were unaltered in these cell lines following treatment with TNF-alpha, IL-1beta, BDNF, GDNF, dibutyryl cyclic AMP, or phorbol 12-myristate 13-acetate, and remained unchanged by exposure to heat stress. In contrast, its levels were elevated substantially in NTera2 teratocarcinoma cells following retinoic acid-induced neuronal differentiation, accompanied with an increased expression of alpha-synuclein and synaptophysin. These results indicate that UCH-L1 is expressed constitutively in human neual cell lines, where it is upregulated following induction of neuronal differentiation, but unaffected by exposure to heat stress, cytokines, or growth/differentiation factors which are supposed to be invloved in the nigral neuronal death and survival in PD.

Rathke-Hartlieb, S., P. J. Kahle, et al. (2001). "Sensitivity to MPTP is not increased in Parkinson's disease-associated mutant alpha-synuclein transgenic mice." J Neurochem 77(4): 1181-4.
Environmental and genetic factors that contribute to the pathogenesis of Parkinson's disease are discussed. Mutations in the alpha-synuclein (alphaSYN ) gene are associated with rare cases of autosomal-dominant Parkinson's disease. We have analysed the dopaminergic system in transgenic mouse lines that expressed mutant [A30P]alphaSYN under the control of a neurone-specific Thy-1 or a tyrosine hydroxylase (TH) promoter. The latter mice showed somal and neuritic accumulation of transgenic [A30P]alphaSYN in TH-positive neurones in the substantia nigra. However, there was no difference in the number of TH-positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum between these transgenic mice and non-transgenic littermates. To investigate whether forced expression of [A30P]alphaSYN increased the sensitivity to putative environmental factors we subjected transgenic mice to a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) regimen. The MPTP-induced decrease in the number of TH-positive neurones in the substantia nigra and the concentrations of catecholamines in the striatum did not differ in any of the [A30P]alphaSYN transgenic mouse lines compared with wild-type controls. These results suggest that mutations and forced expression of alphaSYN are not likely to increase the susceptibility to environmental toxins in vivo.

Przedborski, S., Q. Chen, et al. (2001). "Oxidative post-translational modifications of alpha-synuclein in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease." J Neurochem 76(2): 637-640.
Structural and functional alterations of alpha-synuclein is a presumed culprit in the demise of dopaminergic neurons in Parkinson's disease (PD). alpha-Synuclein mutations are found in familial but not in sporadic PD, raising the hypothesis that effects similar to those of familial PD-linked alpha-synuclein mutations may be achieved by oxidative post-translational modifications. Here, we show that wild-type alpha-synuclein is a selective target for nitration following peroxynitrite exposure of stably transfected HEK293 cells. Nitration of alpha-synuclein also occurs in the mouse striatum and ventral midbrain following administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Conversely, ss-synuclein and synaptophysin were not nitrated in MPTP-intoxicated mice. Our data demonstrate that alpha-synuclein is a target for tyrosine nitration, which, by disrupting its biophysical properties, may be relevant to the putative role of alpha-synuclein in the neurodegeneration associated with MPTP toxicity and with PD.

Piao, Y. S., S. Hayashi, et al. (2001). "Co-localization of alpha-synuclein and phosphorylated tau in neuronal and glial cytoplasmic inclusions in a patient with multiple system atrophy of long duration." Acta Neuropathol (Berl) 101(3): 285-93.
Neuronal and glial cytoplasmic inclusions (NCIs and GCIs), which contain alpha-synuclein as a major component, are characteristic cytopathological features of multiple system atrophy (MSA). We report MSA of 19 years' duration in a 73-year-old woman. Her initial symptom was parkinsonism, with dementia appearing about 8 years later. Postmortem examination showed marked atrophy of the frontal and temporal white matter and limbic system, in addition to the pathology typical of MSA. In the limbic system, severe neuronal loss and astrocytosis were observed, and the remaining neurons often had lightly eosinophilic, spherical cytoplasmic inclusions. Interestingly, a double-labeling immunofluorescence study revealed that the NCIs in the dentate gyrus and amygdaloid nucleus, and the GCIs in the frontal and temporal white matter often expressed both alpha-synuclein NACP-5 and phosphorylated tau AT8 epitopes. Double-immunolabeling electron microscopy of the NCIs in the dentate gyrus and the GCIs in the temporal white matter clearly revealed labeling of their constituent granule-associated filaments with NACP-5, and some of them were also labeled with AT8. These findings strongly suggested that some alpha-synuclein filaments were decorated with phosphorylated tau without formation of fibrils such as paired helical filaments. Immunoblotting of sarkosyl-insoluble tau indicated that the accumulated tau consisted mainly of four-repeat tau isoforms of 383 amino acids and 412 amino acids. We consider that the limbic system can be a major site of neurodegeneration in MSA of long duration. The mechanisms of such abnormal tau accumulation in the NCIs and GCIs are unknown.

Perry, G., J. Avila, et al. (2001). "Biochemistry of neurodegeneration." Science 291(5504): 595-7.

Papapetropoulos, S., C. Paschalis, et al. (2001). "Clinical phenotype in patients with alpha-synuclein Parkinson's disease living in Greece in comparison with patients with sporadic Parkinson's disease." J Neurol Neurosurg Psychiatry 70(5): 662-5.
OBJECTIVE: An Ala53Thr mutation of the alpha-synuclein gene has been recently identified as a rare cause of autosomal Parkinson's disease (PD). The clinical characteristics of 15 patients with PD living in Greece with the Ala53Thr alpha-synuclein mutation (alpha-synPD) were compared with patients with sporadic Parkinson's disease (sPD). METHODS: An investigator, blind to the results of the genetic analysis, examined 15 patients with alpha-synPD and 52 consecutive patients with sPD. Demographic data, age at onset of the illness, modality of presentation, and duration of PD were collected. The unified Parkinson's disease rating scale, the Hoehn and Yahr scale, and the Schwab-England scale were completed. The patients with alpha-synPD were matched for duration of disease with 32 of the 52 patients with sporadic PD (MsPD group). RESULTS: Patients with the alpha-synuclein mutation were significantly younger (mean 7.6 years), showed the first sign of the disease significantly earlier in life (mean 10.8 years), and had significantly longer duration of the disease compared with patients with sPD. Tremor at onset of the disease was present in only one (6.7%) of the patients with alpha-synPD, whereas it was present in 32 (61.5%) of the patients with sPD (p=0.0006). During the course of the disease one patient in the alpha-synPD group went on to develop tremor compared with six patients in the sPD group. Rigidity, bradykinesia, postural instability, orthostatic hypotension, intellectual impairment, depression, complications of therapy, and clinical severity of the disease at the time of examination did not differ significantly between patients with alpha-synPD and those with sPD, or between patients with alpha-synPD and the MsPD group. CONCLUSION: The younger age at onset of the illness, the much lower prevalence of tremor, and the longer duration of the disease characterise the clinical phenotype in this sample of patients with alpha-synPD. The other symptoms and signs of the illness did not seem to differentiate the patients with alpha-synPD from those with sPD.

Nielsen, M. S., H. Vorum, et al. (2001). "Ca2+ binding to alpha-synuclein regulates ligand binding and oligomerization." J Biol Chem 276(25): 22680-4.
alpha-Synuclein is a protein normally involved in presynaptic vesicle homeostasis. It participates in the development of Parkinson's disease, in which the nerve cell lesions, Lewy bodies, accumulate alpha-synuclein filaments. The synaptic neurotransmitter release is primarily dependent on Ca(2+)-regulated processes. A microdialysis technique was applied showing that alpha-synuclein binds Ca(2+) with an IC(50) of about 2-300 microm and in a reaction uninhibited by a 50-fold excess of Mg(2+). The Ca(2+)-binding site consists of a novel C-terminally localized acidic 32-amino acid domain also present in the homologue beta-synuclein, as shown by Ca(2+) binding to truncated recombinant and synthetic alpha-synuclein peptides. Ca(2+) binding affects the functional properties of alpha-synuclein. First, the ligand binding of (125)I-labeled bovine microtubule-associated protein 1A is stimulated by Ca(2+) ions in the 1-500 microm range and is dependent on an intact Ca(2+) binding site in alpha-synuclein. Second, the Ca(2+) binding stimulates the proportion of (125)I-alpha-synuclein-containing oligomers. This suggests that Ca(2+) ions may both participate in normal alpha-synuclein functions in the nerve terminal and exercise pathological effects involved in the formation of Lewy bodies.

Narayanan, V. and S. Scarlata (2001). "Membrane Binding and Self-Association of alpha-Synucleins." Biochemistry 40(33): 9927-34.
Although its function is unknown, alpha-synuclein is widely distributed in neural tissue and is the major component in the pathological aggregates found in patients with Parkinson's disease, Alzheimer's disease, Down's syndrome, and multiple system atrophy. In this report, we have quantified the binding alpha-synucleins to lipid membranes. In contrast to previous studies, we find, using real time equilibrium fluorescence methods, that alpha-synuclein binds strongly to large, unilamellar vesicles with either anionic or zwitterionic headgroups. Membrane binding is also strong for beta-synuclein, phosphorylated alpha-synuclein, and a synuclein mutant that is associated with familial Parkinson's disease. In solution at less than 400 nM, synuclein has a tendency to undergo concentration-dependent oligomerization as determined by changes in intrinsic fluorescence and fluorescence resonance energy transfer. Above this concentration, the protein begins to aggregate into structures visible by light scattering. Although membrane binding does not affect the secondary structure of alpha-synuclein, it greatly inhibits the ability of this protein to self-associate. Taken together, our results indicate that pathological conditions may be associated with a disruption in synuclein-membrane interactions.

Nakamura, T., H. Yamashita, et al. (2001). "Activated Fyn phosphorylates alpha-synuclein at tyrosine residue 125." Biochem Biophys Res Commun 280(4): 1085-92.
alpha-Synuclein is a presynaptic protein of unknown function that has been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. To gain insight into the functions of alpha-synuclein, we sought protein kinases that phosphorylate alpha-synuclein in the central nervous system. In contrast to Lyn, PYK2, FAK, MAPK/ERK1, SAPK/JNK, and Cdk5, only Fyn could phosphorylate alpha-synuclein. In addition, A30P and A53T mutations did not affect the phosphorylation of alpha-synuclein by Fyn. Mutation analysis revealed that activated Fyn phosphorylates specifically tyrosine residue 125 of alpha-synuclein. The distribution of alpha-synuclein and Fyn expression was similar in various parts of the brain and was colocalized in subcellular structures. Since Fyn regulates various signal transduction pathways in the central nervous system and plays an essential role in the neuronal cell differentiation, survival, and plasticity, results of this paper indicate that phosphorylation of alpha-synuclein might be involved in one of the Fyn-mediated signaling pathways in neuronal cells. Copyright 2001 Academic Press.

Nagar, S., R. C. Juyal, et al. (2001). "Mutations in the alpha-synuclein gene in Parkinson's disease among Indians." Acta Neurol Scand 103(2): 120-2.
OBJECTIVE: To investigate the prevalence of G88C, G209A and any other mutation(s) in exons 3 and 4 of the alpha-synuclein gene in Indian patients with Parkinson's disease (PD). METHODS: A total of 169 PD patients comprising 18 familial, 3 juvenile, 48 early onset and 100 sporadic cases were included in this study. Genomic DNA was amplified by PCR using primers specific for Exons 3 and 4. Mutations at G88C and G209A were screened following restriction enzyme digestion of the PCR product. Direct PCR product sequencing of entire exons 3 and 4 was carried out for at least one proband each from the 10 familial cases. RESULTS: Neither G88C and G209A mutations nor any other mutation in exons 3 and 4 was found in the PD patients analysed. CONCLUSION: The G88C and G209A mutations do not seem to be the predominant genetic determinant of PD among Indians.

McNaught, K. S. and P. Jenner (2001). "Proteasomal function is impaired in substantia nigra in Parkinson's disease." Neurosci Lett 297(3): 191-4.
The accumulation of alpha-synuclein, ubiquitin and other proteins in Lewy bodies in degenerating dopaminergic neurones in substantia nigra in idiopathic Parkinson's disease (PD) suggest that inhibition of normal/abnormal protein degradation may contribute to neuronal death. We now show for the first time that the chymotrypsin- (39%), trypsin- (42%) and postacidic-like (33%) hydrolysing activities of 20/26S proteasome are impaired in substantia nigra in PD. Proteasome inhibition does not appear to result from drug treatment since high concentrations of L-3,4-dihydroxyphenylalanine had no effect on enzymatic activity in vitro. These observations provide the first direct evidence that inhibition of the ubiquitin-proteasome pathway leading to altered protein handling and Lewy body formation may be responsible for degeneration of the nigrostriatal pathway in idiopathic PD.

McLean, P. J., H. Kawamata, et al. (2001). "alpha-Synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons." Neuroscience 104(3): 901-12.
alpha-Synuclein accumulates in the brains of sporadic Parkinson's disease patients as a major component of Lewy bodies, and mutations in alpha-synuclein are associated with familial forms of Parkinson's disease. The pathogenic mechanisms that precede and promote the aggregation of alpha-synuclein into Lewy bodies in neurons remain to be determined. Here, we constructed a series of alpha-synuclein-enhanced green fluorescent protein (alpha-synucleinEGFP, SynEGFP) fusion proteins to address whether the Parkinson's disease-associated mutations alter the subcellular distribution of alpha-synuclein, and to use as a tool for experimental manipulations to induce aggregate formation. When transfected into mouse cultured primary neurons, the 49-kDa alpha-synucleinEGFP fusion proteins are partially truncated to a approximately 27-kDa form. This non-fluorescent carboxy-terminally modified fusion protein spontaneously forms inclusions in the neuronal cytoplasm. A marked increase in the accumulation of inclusions is detected following treatment with each of three proteasome inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly, Ala30Pro alpha-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and Ala53Thr alpha-synucleinEGFP, supporting the idea that the Ala30Pro alpha-synuclein protein conformation differs from wild-type alpha-synuclein. Similar inclusions are formed if alpha-synuclein carboxy-terminus is modified by the addition of a V5/6xHistidine epitope tag. By contrast, overexpression of unmodified alpha-synuclein does not lead to aggregate formation. Furthermore, synphilin-1, an alpha-synuclein interacting protein also found in Lewy bodies, colocalizes with the carboxy-terminally truncated alpha-synuclein fusion protein in discrete cytoplasmic inclusions.Our finding that manipulations of the carboxy-terminus of alpha-synuclein lead to inclusion formation may provide a model for studies of the pathogenic mechanisms of alpha-synuclein aggregation in Lewy bodies.

Matsuoka, Y., M. Vila, et al. (2001). "Lack of nigral pathology in transgenic mice expressing human alpha-synuclein driven by the tyrosine hydroxylase promoter." Neurobiol Dis 8(3): 535-9.
alpha-Synuclein has been identified as a major component of Lewy body inclusions, which are one of the pathologic hallmarks of idiopathic Parkinson's disease. Mutations in alpha-synuclein have been found to be responsible for rare familial cases of Parkinsonism. To test whether overexpression of human alpha-synuclein leads to inclusion formation and neuronal loss of dopaminergic cells in the substantia nigra, we made transgenic mice in which the expression of wild-type or mutant (A30P and A53T) human alpha-synuclein protein was driven by the promoter from the tyrosine hydroxylase gene. Even though high levels of human alpha-synuclein accumulated in dopaminergic cell bodies, Lewy-type-positive inclusions did not develop in the nigrostriatal system. In addition, the number of nigral neurons and the levels of striatal dopamine were unchanged relative to non-transgenic littermates, in mice up to one year of age. These findings suggest that overexpression of alpha-synuclein within nigrostriatal dopaminergic neurons is not in itself sufficient to cause aggregation into Lewy body-like inclusions, nor does it trigger overt neurodegenerative changes. Copyright 2001 Academic Press.

Matsubara, M., H. Yamagata, et al. (2001). "Genetic association between Alzheimer disease and the alpha-synuclein gene." Dement Geriatr Cogn Disord 12(2): 106-9.
alpha-Synuclein has been isolated as a component of amyloid in addition to the major A beta peptide in Alzheimer disease (AD). However, there are conflicting reports regarding the association of alpha-synuclein gene polymorphism with AD. Using a novel and common polymorphism in intron 3, we examined the relationship between AD and alpha-synuclein and apolipoprotein E (ApoE) genes in 183 Japanese AD patients and 210 controls. Carriers of the alpha-synuclein deletion (D) allele had a 2.2-fold increased risk of developing AD than noncarriers in women. The odds ratio for the ApoE epsilon 4 and the alpha-synuclein D allele was 11.4 in women. The results showed that the alpha-synuclein gene is associated with sporadic AD in women, independent of ApoE epsilon 4 status. Copyright 2001 S. Karger AG, Basel

Martin, J. L. and R. S. Sloviter (2001). "Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P." J Comp Neurol 436(2): 127-52.
Episodes of prolonged seizures or head trauma produce chronic hippocampal network hyperexcitability hypothesized to result primarily from inhibitory interneuron loss or dysfunction. The possibly causal role of inhibitory neuron failure in the development of epileptiform pathophysiology remains unclear because global neurologic injuries produce such a multitude of effects. The recent finding that Substance P receptors (SPRs) are expressed exclusively in the rat hippocampus by inhibitory interneurons provided the rationale for attempting to ablate interneurons selectively by using neurotoxic conjugates of SPR ligands and the ribosome inactivating protein saporin that specifically target Substance P receptor-expressing cells. Whereas intrahippocampal microinjection of a conjugate of native SP and saporin produced significant nonspecific damage at concentrations needed to produce even limited selective loss of SPR-positive cells, a conjugate of saporin and the more potent and peptidase-resistant SP analog [Sar(9), Met(O(2))(11)] Substance P (SSP-saporin) caused negligible nonspecific damage at the injection site, and a virtually complete loss of SPR-like immunoreactivity (LI) up to 1 mm from the injection site. Within the SPR depletion zone, immunoreactivities for most GABA-, parvalbumin-, somatostatin-, and cholecystokinin-immunoreactive cells and fibers were eliminated. The few interneurons detectable within the affected zone were devoid of SPR-LI. The apparent loss of interneurons was selective in that calbindin- and glutamate receptor subunit 2 (GluR2) -positive principal cells survived within the affected zone, as did myelinated fibers and the extrinsic calretinin- and tyrosine hydroxylase-immunoreactive terminals of subcortical afferents. An apparent lack of reactive synaptic reorganization in response to interneuron loss was indicated by zinc transporter-3 (ZnT3)- and beta-synuclein-LI, as well as by Timm staining, all of which revealed relatively normal patterns of excitatory terminal distribution. Control injections produced minor damage at the injection site, but no apparent specific loss of SPR-LI. One to 12 weeks after injection of SSP-saporin, extracellular electrophysiological field responses recorded in the CA1 pyramidal and dentate granule cell layers in response to afferent stimulation were blindly evaluated simultaneously in two sites 1-2 mm apart along the longitudinal hippocampal axis. SSP-saporin-treated rats exhibited relatively normal responses in some sites, whereas disinhibition and hyperexcitability indistinguishable from the pathophysiology produced by experimental status epilepticus were simultaneously recorded at adjacent sites. Anatomic analysis of the recording sites in each animal revealed that epileptiform pathophysiology was consistently observed only within areas of SPR ablation, whereas relatively normal evoked responses were recorded from immediately adjacent and relatively unaffected regions. These data establish the efficacy of [Sar(9), Met(O(2))(11)] Substance P-saporin for producing a selective and spatially extensive ablation of hippocampal inhibitory interneurons in vivo and a highly focal disinhibition that was restricted to the site of interneuron loss. These results also demonstrate that the "epileptic" pathophysiology produced by experimental status epilepticus or head trauma can be replicated by focal interneuron loss per se, without involving principal cell loss and other interpretive confounds inherent in the use of global neurologic injury models. Copyright 2001 Wiley-Liss, Inc.

Londos, E., U. Passant, et al. (2001). "Neuropathological correlates to clinically defined dementia with Lewy bodies." Int J Geriatr Psychiatry 16(7): 667-79.
OBJECTIVES: To analyse the neuropathological changes behind clinically defined dementia with Lewy bodies (clinDLB) compared with clinically diagnosed Alzheimer's disease (clinAD). METHODS: The prevalence of neuropathological findings in 48 clinDLB and 45 clinAD cases was compared. Sixteen clinDLB and 10 clinAD cases were reassessed with alpha-synuclein staining for Lewy bodies (LB). RESULTS: Alzheimer pathology was found in 81% of the clinDLB and 93% of the clinAD cases. The clinDLB group had a higher prevalence of frontal white matter pathology, mostly of ischemic type, and a more severe degeneration of the substantia nigra compared with the clinAD group. In hematoxylin-eosin staining, LBs were identified in seven (15%) of the clinDLB and in four (9%) of the clinAD group. In alpha-synuclein staining, 38% of the clinDLB and 40% of the clinAD cases exhibited LBs. The cases without LBs, in the clinDLB group, had AD pathology in combination with frontal white matter disease. Vascular pathology of significant degree was prevalent in more than 40% of all the cases with verified LBs regardless of clinical diagnosis. CONCLUSION: Consecutive dementia cases, fulfilling the clinical consensus criteria for DLB, may exhibit combinations of neuropathological changes which in themselves can explain the clinical picture of DLB even when LBs are absent. Copyright 2001 John Wiley & Sons, Ltd.

Lev, N. and E. Melamed (2001). "Heredity in Parkinson's disease: new findings." Isr Med Assoc J 3(6): 435-8.
Multiple factors have been hypothesized over the last century to be causative or contributory for Parkinson's disease. Hereditary factors have recently emerged as a major focus of Parkinson's disease research. Until recently most of the research on the etiology of Parkinson's disease concentrated on environmental factors, and the possibility that genetic factors contribute significantly to the pathogenesis of Parkinson's disease has been neglected. However, it has become increasingly apparent that even in sporadic cases, the disease most likely reflects a combination of genetic susceptibility and an unknown environmental insult. Moreover, the identification of genes and proteins that may cause hereditary parkinsonism substantially contributes to our ability to understand the pathogenesis of Parkinson's disease and may help in the early identification of the disease and its treatment. The discovery of alpha-synuclein mutations in families with autosomal dominant Parkinson's disease sheds light on its role in sporadic Parkinson's disease. It seems that this protein tends to aggregate when the cellular milieu is altered [14-16]. The question as to the exact changes that cause its deposition remains open. One of the major possibilities is oxidative stress [16]. The role of these aggregates in neuronal cell death is also still unclear. Transgenic mice expressing wild-type human alpha-synuclein developed progressive accumulation of alpha-synuclein and ubiquitin-immunoreactive inclusions in neurons in the neocortex, hippocampus and the substantia nigra. These alterations were associated with loss of dopaminergic terminals and motor impairments [24]. This finding suggests that accumulation of alpha-synuclein may play a causal role in sporadic Parkinson's disease as well. The parkin protein seems to be a crucial survival factor for nigral neurons [15]. The parkin protein is related to the ubiquitin pathway, which is important in the elimination of damaged proteins. Ubiquitin-mediated degradation of proteins plays a central role in the control of numerous processes, including signal transduction, receptor and transcriptional regulations, programmed cell death, and breakdown of abnormal proteins that may interfere with normal cell functions. Further studies on the function of Parkin protein and its relation to the ubiquitin pathway could elucidate at least one of the molecular mechanisms of nigral neuronal death. A mutation in the ubiquitin carboxy-teminal hydrolase L1 gene also implies the importance of the ubiquitin pathway in Parkinson's disease. Abnormal tau protein was found to be the cause of familial frontotemporal dementia and parkinsonism. It tends to form filamentous structures, which may lead to neuronal death. Elucidation of the molecular mechanism of neuronal death in this disease may contribute to our understanding of sporadic diseases with tau accumulation, such as corticobasal degeneration, progressive supranuclear palsy, Pick's disease, Alzheimer's disease and possibly also the pathogenesis of Parkinson's disease. Other genetic loci have been identified by linkage analysis of patients with familial parkinsonism. These loci conceal other genes and proteins that may be pivotal factors in the pathogenesis of Parkinson's disease. The discovery of genetic mutations in patients with parkinsonism may offer us new insights into the understanding of the pathways leading to neuronal death and development of Parkinson's disease. It may also help in the early identification of susceptible people to this disease and possibly in developing new treatment strategies.

Leng, Y., T. N. Chase, et al. (2001). "Muscarinic Receptor Stimulation Induces Translocation of an alpha -Synuclein Oligomer from Plasma Membrane to a Light Vesicle Fraction in Cytoplasm." J Biol Chem 276(30): 28212-8.
The close correspondence between the distribution of brain alpha-synuclein and that of muscarinic M(1) and M(3) receptors suggests a role for this protein in cholinergic transmission. We thus examined the effect of muscarinic stimulation on alpha-synuclein in SH-SY5Y, a human dopaminergic cell line that expresses this protein. Under basal conditions, alpha-synuclein was detected in all subcellular compartments isolated as follows: plasma membrane, cytoplasm, nucleus, and two vesicle fractions. The lipid fractions contained only a 45-kDa alpha-synuclein oligomer, whereas the cytoplasmic and nuclear fractions contained both the oligomer and the monomer. This finding suggests alpha-synuclein exists physiologically as a lipid-bound oligomer and a soluble monomer. Muscarinic stimulation by carbachol reduced the alpha-synuclein oligomer in plasma membrane over a 30-min period, with a concomitant increase of both the oligomer and the monomer in the cytoplasmic fraction. The oligomer was associated with a light vesicle fraction in cytoplasm that contains uncoated endocytotic vesicles. The carbachol-induced alteration of alpha-synuclein was blocked by atropine. Translocation of the alpha-synuclein oligomer in response to carbachol stimulation corresponds closely with the time course of ligand-stimulated muscarinic receptor endocytosis. The data suggest that the muscarine receptor stimulated release of the alpha-synuclein oligomer from plasma membrane, and its subsequent association with the endocytotic vesicle fraction may have a role in muscarine receptor endocytosis. We propose that its function may be a transient release of membrane-bound phospholipase D(2) from alpha-synuclein inhibition, thus allowing this lipase to participate in muscarinic receptor endocytosis.

Lee, F. J., F. Liu, et al. (2001). "Direct binding and functional coupling of alpha-synuclein to the dopamine transporters accelerate dopamine-induced apoptosis." Faseb J 15(6): 916-26.
Mutations in alpha-synuclein, a protein highly enriched in presynaptic terminals, have been implicated in the expression of familial forms of Parkinson's disease (PD) whereas native alpha-synuclein is a major component of intraneuronal inclusion bodies characteristic of PD and other neurodegenerative disorders. Although overexpression of human alpha-synuclein induces dopaminergic nerve terminal degeneration, the molecular mechanism by which alpha-synuclein contributes to the degeneration of these pathways remains enigmatic. We report here that alpha-synuclein complexes with the presynaptic human dopamine transporter (hDAT) in both neurons and cotransfected cells through the direct binding of the non-A beta amyloid component of alpha-synuclein to the carboxyl-terminal tail of the hDAT. alpha-Synuclein--hDAT complex formation facilitates the membrane clustering of the DAT, thereby accelerating cellular dopamine uptake and dopamine-induced cellular apoptosis. Since the selective vulnerability of dopaminergic neurons in PD has been ascribed in part to oxidative stress as a result of the cellular overaccumulation of dopamine or dopamine-like molecules by the presynaptic DAT, these data provide mechanistic insight into the mode by which the activity of these two proteins may give rise to this process.

Lee, M., D. Hyun, et al. (2001). "Effect of the overexpression of wild-type or mutant alpha-synuclein on cell susceptibility to insult." J Neurochem 76(4): 998-1009.
Mutations in alpha-synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of alpha-synuclein overexpression on the response of cells to various insults. Wild-type alpha-synuclein and alpha-synuclein mutations associated with FPD were overexpressed in NT-2/D1 and SK-N-MC cells. Overexpression of wild-type alpha-synuclein delayed cell death induced by serum withdrawal or H(2)O(2), but did not delay cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)). By contrast, wild-type alpha-synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4-hydroxy-2-trans-nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild-type alpha-synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant alpha-synucleins increased levels of 8-hydroxyguanine, protein carbonyls, lipid peroxidation and 3-nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant alpha-synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal alpha-synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.

Lee, D., E. K. Lee, et al. (2001). "Self-oligomerization and protein aggregation of alpha-synuclein in the presence of Coomassie Brilliant Blue." Eur J Biochem 268(2): 295-301.
alpha-Synuclein has been implicated in various neurodegenerative disorders, including Parkinson's and Alzheimer's diseases, by its participation in abnormal protein depositions. As the protein has been suggested to play a significant role in the formation of the deposits which might be responsible for neurodegeneration, there is a strong demand to screen for alpha-synuclein-interactive small molecules. In this report, Coomassie Brilliant Blue (CBB) interaction of alpha-synuclein has been investigated with respect to induction of protein self-oligomerization in the presence of the chemical coupling reagent N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. Both CBB-G and CBB-R, which differ by only two methyl groups, induced the self-oligomerization of alpha-synuclein in a biphasic manner with optimal dye concentrations of 250 microM and 150 microM, respectively. The protein aggregates of alpha-synuclein induced by the dyes in the absence of the coupling reagent were analysed by electron microscopy. Whereas CBB-G induced formation of protein aggregates with a worm-like structure, CBB-R induced clear fibrilization of alpha-synuclein on a background of granular structures. CBB-R interacted with alpha-synuclein approximately twice as effectively as CBB-G (dissociation constants 0.63 microM and 1.37 microM, respectively). These dye interactions were independent from the acidic C-terminus of alpha-synuclein, which was reminiscent of the Alphabeta25-35 interaction of alpha-synuclein. However, the metal-catalysed oxidative self-oligomerization of alpha-synuclein in the presence of Cu2+/H2O2, which was augmented synergistically by Alphabeta25-35, was not affected by the dyes. This indicates that the dye binding site is also distinctive from the Alphabeta25-35 interaction site on alpha-synuclein. These biochemically specific interactions between alpha-synuclein and the dyes indicate that alpha-synuclein-interactive small molecules could provide a tool with which to approach development of diagnostic, preventive, or therapeutic strategies for various alpha-synuclein-related neurodegenerative disorders.

Kuusisto, E., A. Salminen, et al. (2001). "Ubiquitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies." Neuroreport 12(10): 2085-90.
We examined the immunoreactivity of ubiquitin-binding protein p62 and its association with ubiquitin (Ub), alpha-synuclein, and paired helical filament (PHF)-tau in the affected brain areas of human tauopathies and synucleinopathies. Ubiquitin-binding protein p62 is a widely expressed protein that can bind to Ub noncovalently and is involved in several signalling pathways, making p62 a candidate regulator of Ub-mediated proteolysis. We show that p62 immunoreactivity co-localizes with neuronal and glial Ub-containing inclusions in Alzheimer's disease, Pick's disease, dementia with Lewy bodies, Parkinson's disease, and multiple system atrophy. This is the first demonstration of a common protein component, apart from Ub, that is present in both PHF-tau and alpha-synuclein inclusions. In both tauo- and synucleinopathies, the staining patterns for p62 and Ub were markedly similar, suggesting that a common mechanism which requires interaction of p62 and Ub contributes to the formation of PHF-tau and alpha-synuclein inclusions.

Kruger, R., W. Kuhn, et al. (2001). "Familial parkinsonism with synuclein pathology: clinical and PET studies of A30P mutation carriers." Neurology 56(10): 1355-62.
BACKGROUND: The authors identified the second known mutation in the alpha-synuclein(SNCA) gene, an alanine-to-proline exchange in amino acid position 30 (A30P), that cosegregates with the disease in one German family with autosomal dominantly inherited parkinsonism (ADP). The authors studied carriers of the A30P mutation to compare the phenotype of this mutation with idiopathic PD (IPD) and to assess nigrostriatal dopaminergic function in symptomatic and preclinical mutation carriers. METHODS: The pedigree of the A30P family spans five generations with five affected individuals. The authors performed detailed neurologic examinations followed by mutation analysis in 11 living individuals. In three mutation carriers, two individuals with definite PD and one person at risk for PD, they used L-[18]F-fluoro-3,4-dihydroxyphenylalanine (F-DOPA), [11]C-raclopride (RAC), and [18]F-fluorodeoxyglucose (FDG) PET to investigate presynaptic dopaminergic function, dopamine D2 receptors, and cerebral energy metabolism. The authors studied the cognitive functions of carriers of the A30P mutation using neuropsychological screening. RESULTS: PET studies revealed striatal presynaptic dopaminergic alterations consistent with sporadic IPD in two affected family members and no evidence for nigrostriatal dopaminergic dysfunction in one presymptomatic mutation carrier. Neuropsychological testing in four mutation carriers provided evidence for cognitive impairment as a frequent and early symptom of the A30P mutation; this is also supported by regional cerebral energy metabolism alterations in the clinically presymptomatic subject. CONCLUSIONS: The phenotype of the A30P mutation in the SNCA gene is similar to that of sporadic IPD, including a high variability of the age at disease onset, ranging from 54 to 76 years. The follow-up of presymptomatic carriers of the A30P mutation may give insight into preclinical disease stages and early manifestations of PD.

Klein, C. (2001). "[In Process Citation]." Schweiz Rundsch Med Prax 90(23): 1015-23.
A genetic contribution to the etiology of Parkinson's disease was first suspected by Charcot and later confirmed by case control, family, and twin studies, as well as by the description of large parkinsonian families with Mendelian inheritance of the disease. Recent progress in the field of molecular neurogenetics has led to the identification of several Parkinson disease genes and gene loci. Mutations in the alpha-Synuclein gene (PARK1) and in the gene for the ubiquitin C-terminal hydrolase I (PARK5), along with two gene loci harboring currently unknown genes (PARK3 and PARK4), have been linked to very rare autosomal dominantly inherited parkinsonian syndromes. Mutations in the parkins gene (PARK2), causing autosomal recessive early-onset parkinsonism, are much more common and therefore of clinical relevance. A second gene locus for an autosomal dominantly inherited Parkinsonian syndrome was recently localized on chromosome 1 (PARK6). All three parkinson genes identified thus far imply the involvement of the ubiquitin pathway of protein degradation in the pathogenesis of Parkinson's disease.

Kitamura, Y., Y. Ishida, et al. (2001). "Alpha-synuclein protein is not scavenged in neuronal loss induced by kainic acid or focal ischemia." Brain Res 898(1): 181-5.
Alpha-synuclein, a presynaptic protein, is markedly included in Lewy bodies (LB) in Parkinson's and LB diseases. In this study, neuronal loss and the activation of glial cells such as microglia and astrocytes were induced by neurodegenerative insults such as the injection of kainic acid and occlusion of the middle cerebral artery. In contrast, immunoreactivity for alpha-synuclein did not change even at 7 days after these insults. These results suggest that alpha-synuclein protein may be so scarcely scavenged by glial cells that it readily condenses in neurodegenerative regions.

Kim, Y., D. Lee, et al. (2001). "Multiple ligand interaction of alpha-synuclein produced various forms of protein aggregates in the presence of Abeta25-35, copper, and eosin." Brain Res 908(1): 93-8.
Various protein aggregates of alpha-synuclein developed by way of the common protein self-oligomerization in the presence of Abeta25-35, copper, and eosin were examined. All the aggregates exhibited congo red birefringence although the actual amounts of the aggregates were varied as determined by thioflavin T binding fluorescence. When their morphologies were analyzed in relation to in vitro cytotoxicity, the smallest granular aggregates obtained with copper exhibited the highest cytotoxicity, while the fibrous structures by eosin did not affect the cell.

Khan, N., E. Graham, et al. (2001). "Parkinson's disease is not associated with the combined alpha-synuclein/apolipoprotein E susceptibility genotype." Ann Neurol 49(5): 665-8.
A recent study showed significant association of sporadic Parkinson's disease with a polymorphism within the alpha-synuclein gene and closely linked DNA markers on chromosome 4q and the APOE epsilon4 allele. A combined alpha-synuclein/APOE-epsilon4 genotype increased the relative risk of developing Parkinson's disease 12-fold. We failed to confirm this association in a much larger sample of histopathologically proven cases of Parkinson's disease and controls.

Kawamata, H., P. J. McLean, et al. (2001). "Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations." J Neurochem 77(3): 929-34.
alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.

Kaufmann, H., K. Hague, et al. (2001). "Accumulation of alpha-synuclein in autonomic nerves in pure autonomic failure." Neurology 56(7): 980-1.

Kakimura, J., Y. Kitamura, et al. (2001). "Release and aggregation of cytochrome c and alpha-synuclein are inhibited by the antiparkinsonian drugs, talipexole and pramipexole." Eur J Pharmacol 417(1-2): 59-67.
Recently, it has been shown that release of cytochrome c from the mitochondria to the cytosol is required for activation of the caspase-3-dependent cascade in apoptosis, and also for alpha-synuclein aggregation. In the present study, we examined the effects of talipexole and pramipexole on the release of cytochrome c and alpha-synuclein, their aggregations, and activation of caspases. Treatment of human neuroblastoma SH-SY5Y cells with 1-methyl-4-phenylpyridinium (MPP(+), 1 mM) induced the first event, which was the release of cytochrome c from the organellar fraction to the cytosolic fraction, then came the DNA fragmentation, and caused the last event, which was the accumulation of alpha-synuclein protein in the cytosolic fraction. Talipexole and pramipexole at low concentration (0.1-1 mM) significantly inhibited the accumulation of cytochrome c or alpha-synuclein in the cytosolic fraction. These drugs at high concentration (3-10 mM) inhibited in vitro aggregation of cytochrome c by hydrogen peroxide or that of alpha-synuclein by cytochrome c and hydrogen peroxide. In addition, in vitro activation of caspase-3 induced by cytochrome c and/or dATP was also inhibited by drugs at high concentration (5-10 mM). These results suggest that talipexole and pramipexole may have protective effects against the neurodegeneration, which is induced by intracellular accumulation of cytochrome c and alpha-synuclein.

Jimenez Del Rio, M. and C. Velez Pardo (2001). "[Apoptosis in neurodegenerative diseases: facts and controversies]." Rev Neurol 32(9): 851-860.
OBJECTIVES. In this article, the authors analyzed critically the morphological and biochemical evidences of cell death by apoptosis from post?mortem studies in Alzheimer s, Parkinson s, Huntington s and Wilson s diseases. DEVELOPMENT.During the last few years, apoptosis has been postulated as a type of neuronal death responsible for the neurodegenerative process in those heterogeneous, chronic and progressive neurological disorders, which are characterized by a selective and a symmetric loss of neurons in motor, sensory or cognitive systems. With regard to neuronal death mechanism and the contribution of the mutated or metabolic altered proteins such as bA, P?tau, a?synuclein, Parkin, Huntingtin, ATP?7B, proteins in the pathogenesis of those disorders are still unknown. CONCLUSIONS. We consider that the morphological (e.g. DNA fragmentation without showing classical apoptotic morphology) and biochemical evidences are still insufficient and contradictory to formally indict apoptosis as the mechanism of neuronal cell death in those neurological disorders. The establishment of the molecular mechanisms leading neurons to cell death (?by apoptosis?) could provide significant information for the design of therapeutic strategies to retard or prevent the development of such neurodegenerative diseases in affected individuals.

Jensen, P. H. and W. P. Gai (2001). "Alpha-synuclein. Axonal transport, ligand interaction and neurodegeneration." Adv Exp Med Biol 487: 129-34.

Izumi, Y., H. Morino, et al. (2001). "Genetic studies in Parkinson's disease with an alpha-synuclein/NACP gene polymorphism in Japan." Neurosci Lett 300(2): 125-7.
Dinucleotide repeat polymorphism has been observed in the promoter of the alpha-synuclein (alpha-SYN)/NAC precursor protein (NACP) gene. Alpha-SYN/NACP allele 3 (described by Xia et al. (Ann. Neurol., 40 (1996) 207), equivalent to allele 1 described by Kruger et al. (Ann. Neurol. 45 (1999) 611) is reported to be significantly more frequent among patients with sporadic Parkinson's disease (sPD) than controls. In this study, we genotyped the same alpha-SYN/NACP polymorphism in Japanese sPD patients and healthy controls, but found that any aliele showed no significant difference between the two groups.

Iwata, A., S. Miura, et al. (2001). "alpha-Synuclein forms a complex with transcription factor Elk-1." J Neurochem 77(1): 239-52.
alpha-Synuclein has been identified as a component of Lewy bodies in Parkinson's disease and diffuse Lewy body disease, and glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA). To explore the role of alpha-synuclein in the pathogenesis, we searched for molecules interacting with alpha-synuclein and discovered that GCIs are stained by anti-Elk-1 antibody. To seek the role of Elk-1 in synucleinopathies, we cotransfected alpha-synuclein and Elk-1 to cultured cells, and found small granular structure complexes where the two molecules colocalized. Moreover, alpha-synuclein and Elk-1 were co-immunoprecipitated from the cell lysates. For formation of the complex, the presence of both ETS and B-box domains of Elk-1 was required. Although there was no evidence of direct binding between alpha-synuclein and Elk-1, we discovered that alpha-synuclein and Elk-1 both bind to ERK-2, a MAP kinase. The effect of alpha-synuclein on the MAP kinase pathway was assessed using the Pathdetect system, which showed prominent attenuation of Elk-1 phosphorylation with alpha-synuclein, and especially A53T mutant. Our results suggest that alpha-synuclein reacts with the MAP kinase pathway, which might cause dysfunction of neurons and oligodendrocytes and lead to neurodegeneration in Parkinson's disease and MSA.

Iseki, E., M. Kato, et al. (2001). "A neuropathological study of the disturbance of the nigro-amygdaloid connections in brains from patients with dementia with Lewy bodies." J Neurol Sci 185(2): 129-34.
We neuropathologically and immunohistochemically investigated characteristics of the central amygdaloid nucleus lesion and its relationship with the substantia nigra lesion in dementia with Lewy bodies (DLB) brains. Nine DLB, four Parkinson's disease (PD) and four Alzheimer-type dementia (ATD) cases were examined. The degree of neuronal loss in the substantia nigra was (+)-(+++) in DLB cases, (+++) in PD cases and (+) in ATD cases. All DLB cases showed spongy change and ubiquitin-positive spheroids in the central nucleus. The degree of spongy change was (+)-(+++) in DLB cases, (+) in PD cases and (-)-(+) in ATD cases, which was correlated with the degree of neuronal loss in the substantia nigra in DLB cases. The number of ubiquitin-positive spheroids was parallel to the degree of spongy change. The central nucleus receives dense dopaminergic fibers from the substantia nigra. Many ubiquitin-positive spheroids were also positive to alpha-synuclein and tyrosine-hydroxylase, suggesting that they derive from the degeneration of terminal or distal axons of Lewy body-bearing dopaminergic neurons in the substantia nigra. The disturbance of the dopaminergic connections from the substantia nigra to the central nucleus may be responsible for psychotic symptoms in DLB patients.

Hu, H. Y., Q. Li, et al. (2001). "beta-sheet structure formation of proteins in solid state as revealed by circular dichroism spectroscopy." Biopolymers 62(1): 15-21.
Cross beta-sheet structure formation and abnormal aggregation of proteins are thought to be pathological characteristics of some neurodegenerative disorders. To investigate the novel structural transformation and aggregation, the solid-state secondary structures of some proteins and peptides associated in thin films were determined by circular dichroism spectroscopy. Insulin, lysozyme, DsbA protein, luciferase, and ovalbumin peptide fall into one group; they show no or slight structural rearrangement from solution to the solid state. Another group, including bovine serum albumin, ovalbumin, alpha-synuclein, and plasminogen activator inhibitor-1 (PAIRC) peptide, undergo structural transformation with an increase of beta-sheet structure in the solid state. The beta-sheet formation of PAIRC peptide may reflect the structural transformation of the serpin reactive center that is relevant to the inhibitor activity. The beta-sheet structure of alpha-synuclein in the solid state may correspond to the amyloid-like aggregates, which are implicated in the pathogenesis of some neurodegenerative diseases.

Hasegawa, K., M. Funayama, et al. (2001). "Analysis of alpha-synuclein, parkin, tau, and UCH-L1 in a Japanese Family with Autosomal Dominant Parkinsonism." Eur Neurol 46(1): 20-4.
We examined whether autosomal dominant parkinsonism of a Japanese family, Sagamihara family, was due to the mutations of alpha-synuclein, parkin, tau, and UCH-L1, which have been reported as the causal genes for parkinsonism in other families. Restriction-enzyme digestion of polymerase-chain reaction (PCR) amplified genomic DNA fragments of alpha-synuclein exons 3 and 4 detected no point mutation. PCR-amplification of parkin exons 3, 4, 5, 6 and 7 detected no exon deletion. Direct sequencing of PCR-amplified DNA fragments of tau exons 9, 10, 12, and 13 and intron 10, and of UCH-L1 exon 4 revealed that all these exons and intron were normal including a polymorphic nucleotide substitution. These results indicated that the parkinsonism of the Sagamihara family seems not to be due to previously identified point mutations of alpha-synuclein, tau, or UCH-L1, or to exon deletion of parkin. Copyright 2001 S. Karger AG, Basel

Hartman, V. N., M. A. Miller, et al. (2001). "Testosterone regulates alpha-synuclein mRNA in the avian song system." Neuroreport 12(5): 943-6.
Alpha-synuclein is a small, highly conserved protein in vertebrates that has been linked to several neurodegenerative diseases. The avian song control system is one of the model systems in which the protein was independently discovered. Alpha-synuclein is dynamically regulated in the song system during song learning, a process in which sex steroids play a central role. We compared alpha-synuclein mRNA expression in the brains of 12 adult male chipping sparrows (Spizella passerina) treated with either testosterone or blank s.c. implants. We saw pronounced upregulation of alpha-synuclein mRNA in, as well as an increase in the volume of, the song control nucleus area X in response to exogenous testosterone. To our knowledge this is the first report of steroid regulation of synuclein gene expression in any model system.

Haass, C. and P. J. Kahle (2001). "Neuroscience. Parkin and its substrates." Science 293(5528): 224-5.

Gomez-Tortosa, E., J. L. Sanders, et al. (2001). "Cortical neurons expressing calcium binding proteins are spared in dementia with Lewy bodies." Acta Neuropathol (Berl) 101(1): 36-42.
The consistent regional and laminar distribution of cortical Lewy bodies (LB) in brains of patients with dementia with Lewy bodies (DLB) suggests that only a certain subpopulation of neurons develops these alpha-synuclein-immunoreactive cytoplasmic inclusions. This study examined whether four non-overlapping neuronal subpopulations, defined by the expression of non-phosphorylated neurofilaments (SMI-32) and several calcium binding proteins (parvalbumin, calretinin and calbindin D28k), are vulnerable to LB formation. We performed peroxidase immunostaining to examine the distribution and to quantitate each neuronal subpopulation within the superior temporal sulcus (STS) area, and double-label immunohistochemistry to test for colocalization of alpha-synuclein and each neuronal marker in the STS and the entorhinal cortex. There were no significant differences between DLB brains and controls in the proportional quantity or laminar distribution of each neuronal subpopulation. Parvalbumin-immunoreactive neurons represented around 7%, calbindin D28k 8%, calretinin 10%, and SMI-32 about 20% of the total neuronal population in the STS cortex. Neurons expressing parvalbumin and SMI-32 showed a widespread distribution across layers II to VI. Neurons expressing calretinin were present in superficial layers (II to IV), and calbindin D28k-immunoreactive neurons were mostly distributed within granular layers II and IV. None of the LB observed in the STS or the entorhinal cortex were located in neurons expressing calcium binding proteins; 25% of the LB were contained in SMI-32 immunoreactive neurons. In conclusion, cortical neurons expressing calcium binding proteins are spared in DLB, while SMI-32-positive neurons are affected in proportion to their density in the cortex. However, the majority of cortical LB develop in neurons not identified by any of these markers.

Golbe, L. I., A. M. Lazzarini, et al. (2001). "The tau A0 allele in Parkinson's disease." Mov Disord 16(3): 442-7.
Parkinson's disease (PD) is primarily an alpha-synucleinopathy, rather than a tauopathy, but there is evidence for an indirect association of tau with the pathogenetic process in PD. We therefore assessed the frequency in PD of the tau A0 allele, a dinucleotide repeat marker that has been associated with a sporadic tauopathy, progressive supranuclear palsy (PSP). We found the A0 allele to comprise 79.2% of 758 alleles from PD patients and 71.2% of 264 control alleles (P = 0.008). We also performed a meta-analysis of three previous reports, two of which failed to produce statistically significant results. Taken together, they also support a PD/A0 allelic association, even after correction for misdiagnosis of PSP as PD (P< 0.001). The A0/A0 genotype frequency in our patients (62.3%) did not differ significantly from that in controls (53.0%, P = 0.062), but the meta-analysis, even after correction for misdiagnosis, showed a significant result, with P = 0.002. The frequency of A0 allele and the A0/A0 genotype were compatible with Hardy-Weinberg equilibrium. The frequency of the A0 allele and the A0/A0 genotype in our patients with familial PD was not significantly greater than in those with sporadic PD. We conclude that the tau protein may play a small role in the pathogenesis of PD and that biochemical characterization of this role may suggest opportunities for PD prophylaxis. Copyright 2001 Movement Disorder Society.

Goedert, M. (2001). "Alpha-synuclein and neurodegenerative diseases." Nat Rev Neurosci 2(7): 492-501.

Goedert, M. (2001). "Parkinson's disease and other alpha-synucleinopathies." Clin Chem Lab Med 39(4): 308-12.
Parkinson's disease is the most common movement disorder and the second most common neurodegenerative disease. Neuropathologically, it is characterized by the degeneration of nerve cells that develop filamentous inclusions in the form of Lewy bodies and Lewy neurites. Recent work has shown that rare, familial forms of Parkinson's disease are caused by missense mutations in the alpha-synuclein gene and that the filamentous lesions of Parkinson's disease are made of alpha-synuclein. The same is true of the Lewy body pathology that is associated with other neurodegenerative diseases, such as dementia with Lewy bodies. The filamentous inclusions of multiple system atrophy have also been found to be made of alpha-synuclein, thus providing an unexpected molecular link with Lewy body diseases. Recombinant alpha-synuclein assembles into filaments with similar morphologies to those found in the human diseases and with a cross-beta diffraction pattern characteristic of amyloid. The related proteins beta-synuclein and gamma-synuclein are poor at assembling into filaments. They are not present in the pathological filamentous lesions and have not been found to be linked to genetic disease. The new work has established the alpha-synucleinopathies as a major class of neurodegenerative disease.

Goedert, M. (2001). "The significance of tau and alpha-synuclein inclusions in neurodegenerative diseases." Curr Opin Genet Dev 11(3): 343-51.
Intracellular filamentous inclusions made of either the microtubule-associated protein tau or the protein alpha-synuclein define the majority of cases of neurodegenerative disease. Mutations in the tau gene in familial forms of frontotemporal dementia and in the alpha-synuclein gene in familial cases of Parkinson's disease have provided causal links between the dysfunction of these proteins and neurodegeneration. Over the past year, several novel tau gene mutations have been identified and more has been learned about possible mechanisms by which tau gene mutations lead to frontotemporal dementia. Experimental animal models have provided a link between tau filament formation and nerve cell degeneration. Along similar lines, animal models have been produced that result in the formation of alpha-synuclein filaments and the degeneration of dopaminergic nerve cells. Building on previous work, synthetic alpha-synuclein filaments have been shown to exhibit the characteristics of amyloid.

Goedert, M., M. G. Spillantini, et al. (2001). "From genetics to pathology: tau and alpha-synuclein assemblies in neurodegenerative diseases." Philos Trans R Soc Lond B Biol Sci 356(1406): 213-27.
The most common degenerative diseases of the human brain are characterized by the presence of abnormal filamentous inclusions in affected nerve cells and glial cells. These diseases can be grouped into two classes, based on the identity of the major proteinaceous components of the filamentous assemblies. The filaments are made of either the microtubule-associated protein tau or the protein alpha-synuclein. Importantly, the discovery of mutations in the tau gene in familial forms of frontotemporal dementia and of mutations in the alpha-synuclein gene in familial forms of Parkinson's disease has established that dysfunction of tau protein and alpha-synuclein can cause neurodegeneration.

Giasson, B. I., I. V. Murray, et al. (2001). "A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly." J Biol Chem 276(4): 2380-6.
Neuronal and oligodendrocytic aggregates of fibrillar alpha-synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise viability of affected cells. Here, we report that a 12-amino acid stretch ((71)VTGVTAVAQKTV(82)) in the middle of the hydrophobic domain of human alpha-synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human beta-synuclein is highly homologous to alpha-synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of alpha-synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of alpha-synuclein filaments, because it is resistant to proteolytic digestion in alpha-synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human alpha-synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human alpha-synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit alpha-synuclein fibrillization and might arrest disease progression.

Garcia de Yebenes, J. (2001). "[Genetics of Parkinson's disease]." Neurologia 16(1): 1-6.

Galvin, J. E., T. M. Schuck, et al. (2001). "Differential expression and distribution of alpha-, beta-, and gamma-synuclein in the developing human substantia nigra." Exp Neurol 168(2): 347-55.
Although the functions of alpha-, beta-, and gamma-synuclein (alphaS, betaS, gammaS, respectively) are unknown, these synaptic proteins are implicated in the pathogenesis of Parkinson's disease (PD) and related disorders. For example, alphaS forms Lewy bodies (LBs) in substantia nigra (SN) neurons of PD. However, since it is not known how these hallmark PD lesions contribute to the degeneration of SN neurons or what the normal function of alphaS is in SN neurons, we studied the developing human SN from 11 weeks gestational age (GA) to 16 years of age using immunohistochemistry and antibodies to alphaS, betaS, gammaS, other synaptic proteins, and tyrosine hydoxylase (TH). SN neurons expressed TH at 11 weeks GA and alphaS, betaS, and gammaS appeared initially at 15, 17, and 18 weeks GA, respectively. These synucleins first appeared in perikarya of SN neurons after synaptophysin, but about the same time as synaptotagmin and synaptobrevin. Redistribution of alphaS from perikarya to processes of SN neurons occurred by 18 weeks GA in parallel with synaptophysin, while betaS and synaptotagmin were redistributed similarly between 20 and 28 weeks GA and this also occurred with gammaS and synaptobrevin between 33 weeks GA and 9 months postnatal. These data suggest that alphaS, betaS, and gammaS may play a functional role in the development and maturation of SN neurons, but it remains to be determined how sequestration of alphaS as LBs in PD contributes to the degeneration of SN neurons.

Galvin, J. E., V. M. Lee, et al. (2001). "Synucleinopathies: clinical and pathological implications." Arch Neurol 58(2): 186-90.
The synucleinopathies are a diverse group of neurodegenerative disorders that share a common pathologic lesion composed of aggregates of insoluble alpha-synuclein protein in selectively vulnerable populations of neurons and glia. Growing evidence links the formation of abnormal filamentous aggregates to the onset and progression of clinical symptoms and the degeneration of affected brain regions in neurodegenerative disorders. These disorders may share an enigmatic symmetry, i.e., missense mutations in the gene encoding for the disease protein (alpha-synuclein) cause familial variants of Parkinson disease as well as its hallmark brain lesions, but the same brain lesions also form from the corresponding wild-type brain protein in the more common sporadic varieties of Parkinson disease. It is likely that clarification of this enigmatic symmetry in 1 form of synucleinopathy will have a profound impact on understanding the mechanisms underlying all these disorders. Furthermore, these efforts will likely lead to novel diagnostic and therapeutic strategies in regard to the synucleinopathies.

Furler, S., J. C. Paterna, et al. (2001). "Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons." Gene Ther 8(11): 864-73.
Recombinant adeno-associated viruses (rAAVs) are promising vectors for gene therapy since they efficiently and stably transduce a variety of tissues of immunocompetent animals. The major disadvantage of rAAVs is their limited capacity to package foreign DNA (< or =5 kb). Often, co-expression of two or more genes from a single viral vector is desirable to achieve maximal therapeutic efficacy or to track transduced cells in vivo by suitable reporter genes. The internal ribosome entry site (IRES) sequence of encephalomyocarditis virus has been widely used to construct bicistronic viral vectors. However, the IRES is rather long and IRES-mediated translation can be relatively inefficient when compared with cap-dependent translation. As an alternative to the IRES for in vivo gene expression, we studied the 16 amino-acid long 2A peptide of foot and mouth disease virus (FMDV). The 2A peptide mediates the primary cis-'cleavage' of the FMDV polyprotein in a cascade of processing events that ultimately generate the mature FMDV proteins. We have generated several different rAAV genomes in which two coding regions are fused in-frame via the FMDV 2A sequence. We show that FMDV 2A efficiently mediates the generation of the expected cleavage products from the artificial fusion proteins in cells. Furthermore, we find that both EGFP and alpha- synuclein are expressed at substantially higher levels from 2A vectors than from the corresponding IRES-based vectors, while SOD-1 is expressed at comparable or slightly higher levels. Finally, we demonstrate for the first time, that the 2A sequence results in effective bicistronic gene expression in vivo after injection of 2A-dependent rAAVs into the rat substantia nigra. We conclude that 2A-containing rAAVs may represent an attractive alternative to IRES-dependent vectors for ex vivo and in vivo gene expression and gene therapy.

Ferrer, I. (2001). "[Alpha-synucleinopathies]." Neurologia 16(4): 163-170.
The term a-synucleinopathy is used to name a group of disorders having in common the abnormal deposition of a-synuclein in the cytoplasm of neurons or glial cells, as well as in extracellular deposits of amyloid. In Parkinson's disease and Lewy body dementia, a-synuclein is the main component of Lewy bodies and dystrophic neurites; a-synuclein also accumulates in the cytoplasm of glial cells. In multiple system atrophy, a-synuclein conforms the cytoplasmic oligodendroglial inclusions and the neuronal inclusions which are the hallmark of this disease. Finally, the amyloidogenic fragment 61-95 amino acids of a-synuclein is the non-AB component of senile plaque amyloid in Alzheimer disease. Accumulations of a-synuclein in all these disorders have in common a fibrilar configuration, but they differ in the binding of a-synuclein to distinct proteins with the exception of ubiquitin whose binding to a-synuclein is common to all a-synuclein inclusions. The mechanisms leading to a-synuclein fragmentation and aggegation into extracellular amyloid are not known, although a-synuclein fragment and BA4 aggregates are the result of abnormal cleavage of large precursors. On the other hand, several studies have shown that a-synuclein may adopt a fibrilar conformation and give rise to insoluble forms and high molecular weight aggregates in vitro. Similar complexes have also been observed in a-synucleinopathies. Although studies in vitro and in vivo have shown toxic effects of a-synuclein, the consequence of a-synuclein deposition on cell survival in a-synucleinopathies is not known.

Ferrarese, C., L. Tremolizzo, et al. (2001). "Decreased platelet glutamate uptake and genetic risk factors in patients with Parkinson's disease." Neurol Sci 22(1): 65-6.
Genetic risk factors seem to play a role in sporadic Parkinson's disease (PD), maybe triggering oxidative stress and excitotoxicity within substantia nigra. However, genetic factors act at systemic level: reduced activity of mitochondrial enzymes and decreased glutamate uptake have been shown in platelets from PD patients. In this study we investigated glutamate uptake in platelets from 38 sporadic PD patients, 13 patients with parkinsonian syndromes and 28 controls and assessed polymorphisms of alpha-synuclein and ApoE genes. A 48% reduction of glutamate uptake p)<0.0001) was observed in PD patients which, with respect to control groups, correlated with the disease severity (r = -0.44, p < 0.05). Genetic studies of this population did not show differences between PD and controls, nor correlations with platelet glutamate uptake.

Farrer, M., A. Destee, et al. (2001). "Genetic analysis of synphilin-1 in familial Parkinson's disease." Neurobiol Dis 8(2): 317-23.
alpha-Synuclein is present in Lewy bodies of patients with both sporadic and familial Parkinson's disease. However, pathogenic mutations Ala30Pro and Ala53Thr in alpha-synuclein are rare causes of disease. Synphilin-1 has been demonstrated to associate with alpha-synuclein and promote the formation of cytosolic inclusions in vitro. Two-point genetic linkage analysis of a dinucleotide repeat within the synphilin-1 gene initially implicated this locus as a cause of Parkinson's disease in three of nine families. However, subsequent haplotype, sequencing, and association analyses in these three families and an independent case-control series suggest that variability within the locus does not confer susceptibility to Parkinson's disease. Copyright 2001 Academic Press.

Ellis, C. E., P. L. Schwartzberg, et al. (2001). "alpha-synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases." J Biol Chem 276(6): 3879-84.
alpha-Synuclein (alpha-Syn) is implicated in the pathogenesis of Parkinson's Disease, genetically through missense mutations linked to early onset disease and pathologically through its presence in Lewy bodies. alpha-Syn is phosphorylated on serine residues; however, tyrosine phosphorylation of alpha-Syn has not been established (, ). A comparison of the protein sequence between Synuclein family members revealed that all four tyrosine residues of alpha-Syn are conserved in all orthologs and beta-Syn paralogs described to date, suggesting that these residues may be of functional importance (). For this reason, experiments were performed to determine whether alpha-Syn could be phosphorylated on tyrosine residue(s) in human cells. Indeed, alpha-Syn is phosphorylated within 2 min of pervanadate treatment in alpha-Syn-transfected cells. Tyrosine phosphorylation occurs primarily on tyrosine 125 and was inhibited by PP2, a selective inhibitor of Src protein-tyrosine kinase (PTK) family members at concentrations consistent with inhibition of Src function (). Finally, we demonstrate that alpha-Syn can be phosphorylated directly both in cotransfection experiments using c-Src and Fyn expression vectors and in in vitro kinase assays with purified kinases. These data suggest that alpha-Syn can be a target for phosphorylation by the Src family of PTKs.

Eliezer, D., E. Kutluay, et al. (2001). "Conformational properties of alpha-synuclein in its free and lipid-associated states." J Mol Biol 307(4): 1061-73.
alpha-Synuclein (alphaS) is a presynaptic terminal protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). We have used NMR spectroscopy to characterize the conformational properties of alphaS in solution as a free monomer and when bound to lipid vesicles and lipid-mimetic detergent micelles. Free wild-type alphaS is largely unfolded in solution, but exhibits a region with a preference for helical conformations that may be important in the aggregation of alphaS into fibrils. The N-terminal region of alphaS binds to synthetic lipid vesicles and detergent micelles in vitro and adopts a highly helical conformation, consistent with predictions based on sequence analysis. The C-terminal part of the protein does not associate with either vesicles or micelles, remaining free and unfolded. These results suggest that one function of alphaS may be to tether as of yet unidentified partners to lipid surfaces via interactions with its C-terminal tail. Copyright 2001 Academic Press.

Ebadi, M., P. Govitrapong, et al. (2001). "Ubiquinone (coenzyme q10) and mitochondria in oxidative stress of parkinson's disease." Biol Signals Recept 10(3-4): 224-53.
Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease affecting approximately1% of the population older than 50 years. There is a worldwide increase in disease prevalence due to the increasing age of human populations. A definitive neuropathological diagnosis of Parkinson's disease requires loss of dopaminergic neurons in the substantia nigra and related brain stem nuclei, and the presence of Lewy bodies in remaining nerve cells. The contribution of genetic factors to the pathogenesis of Parkinson's disease is increasingly being recognized. A point mutation which is sufficient to cause a rare autosomal dominant form of the disorder has been recently identified in the alpha-synuclein gene on chromosome 4 in the much more common sporadic, or 'idiopathic' form of Parkinson's disease, and a defect of complex I of the mitochondrial respiratory chain was confirmed at the biochemical level. Disease specificity of this defect has been demonstrated for the parkinsonian substantia nigra. These findings and the observation that the neurotoxin 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP), which causes a Parkinson-like syndrome in humans, acts via inhibition of complex I have triggered research interest in the mitochondrial genetics of Parkinson's disease. Oxidative phosphorylation consists of five protein-lipid enzyme complexes located in the mitochondrial inner membrane that contain flavins (FMN, FAD), quinoid compounds (coenzyme Q10, CoQ10) and transition metal compounds (iron-sulfur clusters, hemes, protein-bound copper). These enzymes are designated complex I (NADH:ubiquinone oxidoreductase, EC 1.6. 5.3), complex II (succinate:ubiquinone oxidoreductase, EC 1.3.5.1), complex III (ubiquinol:ferrocytochrome c oxidoreductase, EC 1.10.2.2), complex IV (ferrocytochrome c:oxygen oxidoreductase or cytochrome c oxidase, EC 1.9.3.1), and complex V (ATP synthase, EC 3.6.1.34). A defect in mitochondrial oxidative phosphorylation, in terms of a reduction in the activity of NADH CoQ reductase (complex I) has been reported in the striatum of patients with Parkinson's disease. The reduction in the activity of complex I is found in the substantia nigra, but not in other areas of the brain, such as globus pallidus or cerebral cortex. Therefore, the specificity of mitochondrial impairment may play a role in the degeneration of nigrostriatal dopaminergic neurons. This view is supported by the fact that MPTP generating 1-methyl-4-phenylpyridine (MPP(+)) destroys dopaminergic neurons in the substantia nigra. Although the serum levels of CoQ10 is normal in patients with Parkinson's disease, CoQ10 is able to attenuate the MPTP-induced loss of striatal dopaminergic neurons.

Dickson, D. W. (2001). "alpha-Synuclein and the Lewy body disorders." Curr Opin Neurol 14(4): 423-32.
alpha-Synuclein has risen to prominence during the past 5 years because of its association with several neurodegenerative diseases that have come to be known as the synucleinopathies. The clinical phenotype of the synucleinopathies is variable, with the most common being parkinsonism, autonomic dysfunction, and dementia. Progress has been made in clinical, neuropathologic and biochemical characterization of the synucleinopathies and their differentiation from other neurodegenerative disorders. At the molecular level, the synucleinopathies have conformational and post-translational modifications of synuclein that favor its fibrillization and aggregation in inclusions in neurons and glia. Whether inclusion body formation is an adaptive response or is directly related to degeneration of neuronal and glial cells is a topic of current research.

D'Andrea, M. R., S. Ilyin, et al. (2001). "Abnormal patterns of microtubule-associated protein-2 (MAP-2) immunolabeling in neuronal nuclei and Lewy bodies in Parkinson's disease substantia nigra brain tissues." Neurosci Lett 306(3): 137-40.
Parkinson's disease (PD) is a neurodegenerative disorder associated with the appearance of cytoplasmic Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra and the progressive loss of these neurons. Cytoskeleton alterations and associated impairments of neuronal transport may contribute to neuronal death. Microtubule-associated protein-2 (MAP-2), a cytoskeleton protein is localized primarily in neuronal dendrites and is known to stabilize microtubule assembly and mediate their interactions with other neuronal cell components. To determine if alterations in MAP-2 morphology are present in PD neurons, we used single and double immunohistochemical and immunofluorescent techniques to characterize MAP-2 in PD neuronal tissues. We report abnormal MAP-2 immunolabeling in some neurons of the substantia nigra of PD brain tissues, which were not observed in the normal, age-matched, control brain tissues. Furthermore, MAP-2 was co-localized with alpha-synuclein and ubiquitin in cytoplasmic LBs of neurons. Surprisingly, MAP-2 was also found to form fibrous aggregates and crystal-like structures within neuronal nuclei. These PD-associated alterations in MAP-2 morphology and distribution suggest that impaired neuronal transport may contribute to the progression of neuronal loss in the brains of PD patients.

Campbell, B. C., C. A. McLean, et al. (2001). "The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease." J Neurochem 76(1): 87-96.
Intracellular inclusions containing alpha-synuclein (alpha SN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease-associated changes of alpha SN, this study compared the levels, solubility and molecular weight species of alpha SN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent-soluble and detergent-insoluble alpha SN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of alpha SN in the detergent-soluble fraction of brain samples from pons and white matter but detergent-insoluble alpha SN was not detected. There was an inverse correlation between buffered saline-soluble and detergent-soluble levels of alpha SN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of alpha SN between grey and white matter in disease may result from different processing of alpha SN in neurons compared with oligodendrocytes. Highly inso