PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Synuclein”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed2005

Inhibition of alpha-synuclein fibrillization by dopamine analogs via reaction with the amino groups of alpha-synuclein. Implication for dopaminergic neurodegeneration.

Fibrillization of alpha-synuclein (alpha-Syn) is closely associated with the formation of Lewy bodies in neurons and dopamine (DA) is a potent inhibitor for the process, which is implicated in the causative pathogenesis of Parkinson's disease (PD). To elucidate any molecular mechanism that may have biological relevance, we tested the inhibitory abilities of DA and several analogs including chemically synthetic and natural polyphenols in vitro. The MS and NMR characterizations strongly demonstrate that DA and its analogs inhibit alpha-Syn fibrillization by a mechanism where the oxidation products (quinones) of DA analogs react with the amino groups of alpha-Syn chain, generating alpha-Syn-quinone adducts. It is likely that the amino groups of alpha-Syn undergo nucleophilic attack on the quinone moiety of DA analogs to form imino bonds. The covalently cross-linked alpha-Syn adducts by DA are primarily large molecular mass oligomers, while those by catechol and p-benzoquinone (or hydroquinone) are largely monomers or dimers. The DA quinoprotein retains the same cytotoxicity as the intact alpha-Syn, suggesting that the oligomeric intermediates are the major elements that are toxic to the neuronal cells. This finding implies that the reaction of alpha-Syn with DA is relevant to the selective dopaminergic loss in PD.

Cross-Linking Reagents↗
PubMed2002

Differential localization of alpha-, beta- and gamma-synucleins in the rat CNS.

Alpha-synuclein is a presynaptic protein that normally participates in the homeostasis of synaptic vesicles. Missense mutations in its gene cause the protein to participate actively in the development of heritable forms of Parkinson's disease. Moreover, its metabolism is perturbed in all cases of Parkinson's disease where alpha-synuclein accumulates in a filamentous form in the Lewy body nerve cell lesion. Lewy bodies also develop in other common neurodegenerative disorders, like dementia with Lewy bodies and Lewy body variant of Alzheimer's disease. In the present study, we have studied the detailed distribution of alpha-, beta- and gamma-synuclein in the rat CNS. Alpha-synuclein was not observed in perikarya, but was distributed with high intensity in nerve terminals in the caudate and putamen and ventral pallidum, where beta-synuclein was much weaker and less densely distributed in the caudate and putamen. Gamma-synuclein was not found in the caudate and putamen. Alpha-synuclein was robustly distributed in the substantia nigra pars reticulata, but was very weak or virtually absent from the perikarya of the neurons in the pars compacta. In contrast, beta-synuclein was very weak or absent from the substantia nigra. gamma-Synuclein was absent from the terminals of substantia nigra pars reticulata, but sparsely distributed gamma-synuclein-containing neurons were detected in the substantia nigra pars compacta. In the brainstem, alpha-synuclein as well as gamma-synuclein were present in the locus coeruleus with high intensity, while beta-synuclein was very weak. In addition, alpha-synuclein was intense in the vagus nucleus, but weak in the oculomotor, facial, hypoglossal, accessory and ambiguous nuclei, where beta-synuclein was very intensely present. Furthermore, gamma-synuclein was localized in the terminals and in cell bodies of the Edinger-Westphal nucleus, the red nucleus, locus coeruleus, and most cranial nerve-related nuclei. In the spinal cord, alpha- and gamma-synucleins were intensely present in laminae I and II and in the preganglionic sympathetic nuclei, whereas beta-synuclein was very weak. These results indicate that alpha-synuclein is abundant in central catecholaminergic regions. Beta-synuclein is more localized in the somatic cholinergic components, while it is particularly weak or absent from catecholaminergic neurons. Gamma-synuclein appears to be present in both cholinergic and catecholaminergic regions, but very weak in the forebrain.

Animals↗
PubMed2001

Induction of alpha-synuclein aggregation by intracellular nitrative insult.

Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.

Ammonium Chloride↗
PubMed2000

Synucleins are expressed in the majority of breast and ovarian carcinomas and in preneoplastic lesions of the ovary.

BACKGROUND: The synucleins (alpha, beta, and gamma) are a family of small cytoplasmic proteins that are expressed predominantly in neurons. alpha synuclein has attracted considerable attention due to its involvement in neurodegenerative diseases. Abnormal expression of gamma synuclein has recently been reported in some breast tumors. In this study, the authors examined a panel of breast and ovarian carcinomas for expression of alpha, beta, and gamma synucleins. METHODS: Normal breast and ovary tissue samples, tissue from ovaries of women at high risk of ovarian carcinoma, and tissue from breast and ovarian carcinomas were screened by immunohistochemistry and Western blot analysis for synuclein expression. RESULTS: Synucleins were not detectable by Western blot analysis in normal breast tissue. Eighty-two percent (14 of 17) of Stage III/IV breast ductal carcinomas expressed beta synuclein, gamma synuclein, or both simultaneously. Expression of alpha synuclein was not detected in breast carcinomas by Western blot analysis. Synuclein (alpha, beta, and gamma) expression was not detectable by immunohistochemistry in normal ovarian epithelium. Eighty-seven percent (39 of 45) of ovarian carcinomas were found to express at least 1 type of synuclein, and 42% (19 of 45) expressed all 3 synucleins (alpha, beta, and gamma) simultaneously. Highly punctate gamma synuclein expression was also observed in 20% of preneoplastic lesions of the ovary, including epithelial inclusion cysts, hyperplastic epithelium, and papillary structures, suggesting that gamma synuclein up-regulation may occur early in the development of some ovarian tumors. CONCLUSIONS: alpha, beta, and gamma synuclein are expressed in a high percentage of ovarian and breast carcinomas, and abnormal gamma synuclein expression may occur early in the development of ovarian carcinoma.

Adenocarcinoma↗
PubMed2004

An antiaggregation gene therapy strategy for Lewy body disease utilizing beta-synuclein lentivirus in a transgenic model.

Current experimental gene therapy approaches for Parkinson's disease (PD) and dementia with Lewy bodies (DLB) include the use of viral vectors expressing antiapoptosis genes, neurotrophic factors and dopaminergic system enzymes. However, since increasing evidence favors a role for alpha-synuclein accumulation in the pathogenesis of these disorders, an alternative therapy might require the transfer of genes that might block alpha-synuclein accumulation. beta-Synuclein, the nonamyloidogenic homologue of alpha-synuclein, has recently been identified as a potential candidate. Thus, in vivo transfer of genes encoding beta-synuclein might provide a novel approach to the development of experimental treatments for PD and DLB. To assess this possibility and to better understand the mechanisms involved, a lentiviral vector expressing human (h) beta-synuclein (lenti-beta-synuclein) was tested in a transgenic (tg) mouse model of halpha-synuclein aggregation. This study showed that unilateral intracerebral injection of lenti-beta-synuclein reduced the formation of halpha-synuclein inclusions and the accumulation of halpha-synuclein in synapses and ameliorated the neurodegenerative alterations in the tg mice. Both in vivo and in vitro coimmunoprecipitation and immunoblot experiments show that the mechanisms of beta-synuclein neuroprotection involve binding of this molecule to halpha-synuclein and Akt, resulting in the decreased aggregation and accumulation of halpha-synuclein in the synaptic membrane. Together, these data further support a role for beta-synuclein in regulating the conformational state of alpha-synuclein and suggest that this gene transfer approach might have potential for the development of alternative therapies for PD and DLB.

Animals↗
PubMed2005

Alpha-synuclein phosphorylation enhances eosinophilic cytoplasmic inclusion formation in SH-SY5Y cells.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that alpha-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of alpha-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of alpha-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type alpha-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A alpha-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with alpha-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A alpha-synuclein instead of wild-type alpha-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A alpha-synuclein with synphilin-1 compared with wild-type alpha-synuclein. Expression of S129A alpha-synuclein instead of wild-type alpha-synuclein also decreased the association of synphilin-1 and parkin and subsequently reduced the parkin-mediated ubiquitination of synphilin-1 and the formation of ubiquitinated inclusions. Treatment of SH-SY5Y cells with H(2)O(2) increased alpha-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of alpha-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of alpha-synuclein at S129 may be important for the formation of inclusions in PD and related alpha synucleinopathies.

Carrier Proteins↗
PubMed2003

Distribution of alpha- and gamma-synucleins in the adult rat brain and their modification by high-dose cocaine treatment.

Synucleins have attracted much attention because of their involvement in several neurodegenerative disorders. In a screening for genes differentially expressed after high-dose cocaine exposure, we found gamma-synuclein as a major upregulated candidate in the tegmentum. Overexpression of both alpha- and gamma-synuclein after drug treatment was confirmed by means of microarrays, yielding an increase in the hippocampus, the striatum and the tegmentum (2.65 x, 1.96 x and 3.5 x, respectively, for alpha-synuclein vs. 2.7 x, 1.96 x and 7.16 x for gamma-synuclein), but no change in the nucleus accumbens. Investigation of the distribution of mRNA (by in situ hybridization) and of the proteins (by immunocytochemistry) shows in both cases a clearly distinct pattern of expression for alpha- and gamma-synuclein. alpha-synuclein displays a very characteristic distribution, confined to specific nuclei, whereas gamma-synuclein is more widely expressed throughout the brain. mRNA of both alpha- and gamma-synucleins display a complementary pattern of expression all over the cortex. In contrast to gamma-synuclein, alpha-synuclein is neuronal, being only found in NeuN-expressing cells, and is expressed in the basal ganglia (faintly) and in the substantia nigra compacta where it is highly correlated with tyrosine hydroxylase. Immunocytochemistry shows that gamma-synuclein generally colocalizes with glial fibrillary acidic protein-expressing cells and is abundant in the red nucleus, the substantia nigra reticulata and the anterior commissure, while gamma-synuclein mRNA labels the matrix compartments of the caudate-putamen. The role of synucleins in relation to cocaine-induced plasticity or neurotoxicity is discussed.

Anesthetics, Local↗
PubMed2002

alpha-Synuclein exhibits competitive interaction between calmodulin and synthetic membranes.

alpha-Synuclein, a pathological component of Parkinson's disease by constituting the Lewy bodies, has been suggested to be involved in membrane biogenesis via induction of amphipathic alpha-helices. Since the amphipathic alpha-helix is also known as a recognition signal of calmodulin for its target proteins, molecular interaction between alpha-synuclein and calmodulin has been investigated. By employing a chemical coupling reagent of N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline, alpha-synuclein has been shown to yield a heterodimeric 1 : 1 complex with calmodulin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and even absence of calcium, whereas beta-synuclein was more dependent upon calcium for its calmodulin interaction. The selective calmodulin interaction of alpha-synuclein in the absence of calcium was also demonstrated with the aggregation kinetics of the synucleins in which only the alpha-synuclein aggregation was affected by calmodulin. A reversible binding assay confirmed that alpha-synuclein interacted with the Ca2+-free as well as the Ca2+-bound calmodulins with almost identical Kds of 0.35 micro m and 0.31 micro m, respectively, while beta-synuclein preferentially recognized the Ca2+-bound form with a Kd of 0.68 micro m. By using a C-terminally truncated alpha-synuclein of alpha-syn97, the calmodulin binding site(s) on alpha-synuclein was(were) shown to be located on the N-terminal region where the amphipathic alpha-helices have been suggested to be induced upon membrane interaction. By employing liposome and calmodulin in a state of being either soluble or immobilized on agarose, actual competition of alpha-synuclein between membranes and calmodulin was demonstrated with the observation that alpha-synuclein previously bound to the liposome was released upon specific interaction with the calmodulins. Taken together, these data may suggest that alpha-synuclein could act not only as a negative regulator for calmodulin in the presence and even absence of calcium, but it could also exert its activity at the interface between calmodulin and membranes.

Animals↗
PubMed2006

Levels of mRNA coding for alpha-, beta-, and gamma-synuclein in the brains of newborn, juvenile, and adult rats.

Synucleins are proteins known for their malfunction in a group of illnesses called synucleopathies, which includes Alzheimer's and Parkinson's disease. To learn more about the role of synucleins in the CNS, we have studied levels of message coding for alpha-, beta-, and gamma-synuclein using quantitative RT-PCR. Levels of synuclein mRNAs were studied in the cerebral cortex (left and right, anterior and posterior), hippocampus, striatum, and cerebellum, obtained from 5-d-old (newborn), 1-mo (juvenile)-, and 6-, and 9-mo (adult)-old rats. The mRNA levels for all synucleins varied significantly among structures. The rank order of mRNA levels in different structures was cortex = hippocampus > striatum > cerebellum for alpha-synuclein; cortex > hippocampus = cerebellum > striatum for beta-synuclein; and hippocampus = striatum > cortex = cerebellum for gamma-synuclein. There was significant effect of age for mRNA levels for all synucleins. The dynamics of these changes were different depending on type of synuclein and brain structure. Levels of mRNA for alpha-synuclein were significantly reduced with age in all structures except hippocampus. For beta- and gamma-synuclein, levels increased significantly only in the cerebral cortex and only from 5 d to 1 mo of age. In contrast, gamma-synuclein levels in the cerebellum were very high at 5 d and significantly reduced at 1 mo of age. The revealed pattern and dynamics of changes in the levels of mRNA coding for synucleins would support the conclusion for an important role of these molecules during development and the aging process.

Aging↗
PubMed2004

Inhibiting aggregation of alpha-synuclein with human single chain antibody fragments.

The alpha-synuclein protein has been strongly correlated with Parkinson's disease (PD) and is a major component of the hallmark Lewy body aggregates associated with PD. Two different mutations in the alpha-synuclein gene as well as increased gene dosage of wild-type alpha-synuclein all associate with early onset cases of PD; and transgenic animal models overexpressing alpha-synuclein develop PD symptoms. Alpha-synuclein, a natively unfolded protein, can adopt a number of different folded conformations including a beta-sheet form that facilitates formation of numerous aggregated morphologies, including long fibrils, spherical and linear protofibrils, and smaller aggregates or oligomers. The roles of the various morphologies of alpha-synuclein in the progression of PD are not known, and different species have been shown to be toxic. Here we show that single chain antibody fragments (scFv's) isolated from naïve phage display antibody libraries can be used to control the aggregation of alpha-synuclein. We isolated an scFv with nanomolar affinity for monomeric alpha-synuclein (K(D) = 2.5 x 10(-8) M). When co-incubated with monomeric alpha-synuclein, the scFv decreased not only the rate of aggregation of alpha-synuclein, but also inhibited the formation of oligomeric and protofibrillar structures. The scFv binds the carboxyl terminal region of alpha-synuclein, suggesting that perturbation of this region can influence folding and aggregation of alpha-synuclein in vitro along with the previously identified hydrophobic core region of alpha-synuclein (residues 61-95, particularly residues 71-82). Since the scFv has been isolated from an antibody library based on human gene sequences, such scFv's can have potential therapeutic value in controlling aggregation of alpha-synuclein in vivo when expressed intracellularly as intrabodies in dopaminergic neurons.

Amino Acid Sequence↗
PubMed2001

Self-oligomerization and protein aggregation of alpha-synuclein in the presence of Coomassie Brilliant Blue.

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.

Binding Sites↗
PubMed2000

Alpha-synuclein up-regulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP.

Mutations in alpha-synuclein cause a form of familial Parkinson's disease (PD), and wild-type alpha-synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for alpha-synuclein in PD. Thus far, however, little is known about the importance of alpha-synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein-1, the rodent homologue of human alpha-synuclein, in both normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein-positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein-positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP-induced dopaminergic neurodegeneration. In these MPTP-injected mice, there was also a dramatic increase in the number of synuclein-immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein-positive neurons were tyrosine hydroxylase-positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up-regulated following MPTP-induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2002

alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells.

alpha-Synuclein has been implicated in the pathogenesis of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Although the function of alpha-synuclein remains largely unknown, recent studies have demonstrated that this protein can interact with phospholipids. To address the role of alpha-synuclein in neurodegenerative disease, we have investigated whether it binds phospholipase D (PLD) and affects PLD activity in human embryonic kidney (HEK)-293 cells overexpressing wild type alpha-synuclein or the mutant forms of alpha-synuclein (A53T, A30P) associated with Parkinson's disease. Tyrosine phosphorylation of alpha-synuclein appears to play a modulatory role in the inhibition of PLD, because mutation of Tyr(125) to Phe slightly increases inhibitory effect of alpha-synuclein on PLD activity. Treatment with pervanadate or phorbol myristate acetate inhibits PLD more in HEK 293 cells overexpressing alpha-synuclein than in control cells. Binding of alpha-synuclein to PLD requires phox and pleckstrin homology domain of PLD and the amphipathic repeat region and non-Abeta component of alpha-synuclein. Although biologically important, co-transfection studies indicate that the interaction of alpha-synuclein with PLD does not influence the tendency of alpha-synuclein to form pathological inclusions. These results suggest that the association of alpha-synuclein with PLD, and modulation of PLD activity, is biologically important, but PLD does not appear to play an essential role in the pathophysiology of alpha-synuclein.

Animals↗
PubMed2002

A role for alpha-synuclein in the regulation of dopamine biosynthesis.

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

Amino Acid Substitution↗
PubMed2006

alpha-Synuclein is colocalized with 14-3-3 and synphilin-1 in A53T transgenic mice.

alpha-Synuclein is a major constituent of Lewy bodies, the neuropathological hallmark of Parkinson's disease (PD). Three types of alpha-synuclein mutations, A53T, A30P, and E46K, have been reported in familial PD. Wild-type alpha-synuclein accumulates at high concentrations in Lewy bodies, and this process is accelerated with mutated A53T alpha-synuclein. The accumulation of alpha-synuclein is thought to be toxic, and causes neuronal death when alpha-synuclein aggregates into protofibrils and fibrils. Lewy bodies contain not only alpha-synuclein, but also other proteins including 14-3-3 proteins and synphilin-1. 14-3-3 Proteins exist mainly as dimers and are related to intracellular signal transduction pathways. Synphilin-1 is known to interact with alpha-synuclein, promoting the formation of cytoplasmic inclusions like Lewy bodies in vitro. To investigate the colocalization of alpha-synuclein, synphilin-1, and 14-3-3 proteins, we performed immunohistochemical studies on alpha-synuclein, 14-3-3 proteins, and synphilin-1 in the brain and spinal cord of A53T transgenic mice. In homozygous mouse brains, alpha-synuclein immunoreactivity was observed in the neuronal somata and processes in the medial part of the brainstem, deep cerebellar nuclei, and spinal cord. The distribution of 14-3-3 proteins and synphilin-1 immunoreactivity was similar to that of alpha-synuclein in the homozygous mice. Double immunofluorescent staining showed that alpha-synuclein and synphilin-1 or 14-3-3 proteins were colocalized in the pons and spinal cord. These results indicate that the accumulation of mutant alpha-synuclein occurs in association with 14-3-3 proteins and synphilin-1, and may cause the sequestration of important proteins including 14-3-3 proteins and synphilin-1. The sequestration and subsequent decrease in 14-3-3 proteins and synphilin-1 levels may account for neuronal cell death.

14-3-3 Proteins↗
PubMed2006

Synuclein proteins of the pufferfish Fugu rubripes: sequences and functional characterization.

In humans, three genes encode the related alpha-, beta-, and gamma-synucleins, which function as lipid-binding proteins in vitro. They are being widely studied, mainly because of the central involvement of alpha-synuclein in a number of neurodegenerative diseases, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In these diseases, the normally soluble alpha-synuclein assembles into abnormal filaments. Here, we have identified and characterized the synuclein gene family from the pufferfish Fugu rubripes. It consists of four genes, which encode alpha-, beta-, gamma1-, and gamma2-synucleins. They range from 113 to 127 amino acids in length and share many of the characteristics of human synucleins, including the presence of imperfect amino-terminal repeats of 11 amino acids, a hydrophobic middle region, and a negatively charged carboxy-terminus. All four synucleins are expressed in the Fugu brain. Recombinant Fugu synucleins exhibited differential liposome binding, which was strongest for alpha-synuclein, followed by beta-, gamma2-, and gamma1-synucleins. In assembly experiments, Fugu alpha-, gamma1-, and gamma2-synucleins formed filaments more readily than human alpha-synuclein. Fugu beta-synuclein, by contrast, failed to assemble in bulk. Filament assembly of synucleins was directly proportional to their degree of hydrophobicity and their tendency to form beta-sheet structure, and correlated inversely with their net charge.

Amino Acid Sequence↗
PubMed2006

alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or toxicity.

Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.

Animals↗