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Background: Sarcopenia is common in patients with Parkinson's disease (PD), showing mitochondrial oxidative stress in skeletal muscle. The aggregation of α-synuclein (α-Syn) to induce oxidative stress is a key pathogenic process of PD; nevertheless, we know little about its potential role in regulating peripheral nerves and the function of the muscles they innervate., Methods: To investigate the role of α-Syn aggregation on neuromuscular system, we used the Thy1 promoter to overexpress human α-Syn transgenic mice (mThy1-hSNCA). hα-Syn expression was evaluated by western blot, and its localization was determined by confocal microscopy. The impact of α-Syn aggregation on the structure and function of skeletal muscle mitochondria and neuromuscular junctions (NMJs), as well as muscle mass and function were characterized by flow cytometry, transmission electron microscopy, Seahorse XF24 metabolic assay, and AAV9 in vivo injection. We assessed the regenerative effect of mitochondrial-targeted superoxide dismutase (Mito-TEMPO) after skeletal muscle injury in mThy1-hSNCA mice., Results: Overexpressed hα-Syn protein localized in motor neuron axons and NMJs in muscle and formed aggregates. α-Syn aggregation increased the number of abnormal mitochondrial in the intramuscular axons and NMJs by over 60% (P < 0.01), which inhibited the release of acetylcholine (ACh) from presynaptic vesicles in NMJs (P < 0.05). The expression of genes associated with NMJ activity, neurotransmission and regulation of reactive oxygen species (ROS) metabolic process were significantly decreased in mThy1-hSNCA mice, resulting in ROS production elevated by ~220% (P < 0.05), thereby exacerbating oxidative stress. Such process altered mitochondrial spatial relationships to sarcomeric structures, decreased Z-line spacing by 36% (P < 0.05) and increased myofibre apoptosis by ~10% (P < 0.05). Overexpression of α-Syn altered the metabolic profile of muscle satellite cells (MuSCs), including basal respiratory capacity (~170% reduction) and glycolytic capacity (~150% reduction) (P < 0.05) and decreased cell migration and fusion during muscle regeneration (~60% and ~40%, respectively) (P < 0.05). We demonstrated that Mito-TEMPO treatment could restore the oxidative stress status (the complex I/V protein and enzyme activities increased ~200% and ~150%, respectively), which caused by α-Syn aggregation, and improve the ability of muscle regeneration after injury. In addition, the NMJ receptor fragmentation and ACh secretion were also improved., Conclusions: These results reveal that the α-synuclein aggregation plays an important role in regulating acetylcholine release from neuromuscular junctions and induces intramuscular mitochondrial oxidative stress, which can provide new insights into the aetiology of muscle atrophy in patients with Parkinson's disease., (© 2022 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of Society on Sarcopenia, Cachexia and Wasting Disorders.)

Iron deposition is present in main lesion areas in the brains of patients with Parkinson's disease (PD) and an abnormal iron content may be associated with dopaminergic neuronal cytotoxicity and degeneration in the substantia nigra of the midbrain. However, the cause of iron deposition and its role in the pathological process of PD are unclear. In the present study, we investigated the effects of the nasal mucosal delivery of synthetic human α-synuclein (α-syn) preformed fibrils (PFFs) on the pathogenesis of PD in Macaca fascicularis. We detected that iron deposition was clearly increased in a time-dependent manner from 1 to 17 months in the substantia nigra and globus pallidus, highly contrasting to other brain regions after treatments with α-syn PFFs. At the cellular level, the iron deposits were specifically localized in microglia but not in dopaminergic neurons, nor in other types of glial cells in the substantia nigra, whereas the expression of transferrin (TF), TF receptor 1 (TFR1), TF receptor 2 (TFR2), and ferroportin (FPn) was increased in dopaminergic neurons. Furthermore, no clear dopaminergic neuron loss was observed in the substantia nigra, but with decreased immunoreactivity of tyrosine hydroxylase (TH) and appearance of axonal swelling in the putamen. The brain region-enriched and cell-type-dependent iron localizations indicate that the intranasal α-syn PFFs treatment-induced iron depositions in microglia in the substantia nigra may appear as an early cellular response that may initiate neuroinflammation in the dopaminergic system before cell death occurs. Our data suggest that the inhibition of iron deposition may be a potential approach for the early prevention and treatment of PD.

Aberrant α-synuclein (α-syn) accumulation has been shown to impair mitochondrial function by reducing mitochondrial membrane potential (MMP). However, the underlying mechanisms remain elusive. Transient receptor potential canonical (TRPC) channels are a diverse group of non-selective Ca 2+ channels, among which TRPC3 is the only one that is localized in mitochondria and plays a role in maintaining the normal MMP. This raises a possibility that altered TRPC3 expression may play a role in the mitochondrial dysfunction induced by α-syn accumulation. To demonstrate this possibility, we first examined the expressions of mitochondrial TRPC3 in the brains of aging monkeys and α-syn transgenic and wild-type mice. We showed that α-syn levels increased in mitochondria in an age-dependent manner that was positively correlated to an elevation of mitochondrial TRPC3. This correlation was more prominent in the striatum than in the cerebellum, possibly due to the greater age-dependent α-syn accumulation in the striatum than in the cerebellum. We then used primary neurons overexpressing α-syn to investigate the effect of the α-syn-induced elevation of mitochondrial TRPC3 on the MMP and apoptotic cell death. We found that neurons with overexpressed α-syn had increased mitochondrial TRPC3 and decreased MMP, which were accompanied by increased number of apoptotic neurons. Suppressing TRPC3 expression partially reversed the reduction of MMP and alleviated the apoptotic cell death, indicating that the mitochondrial TRPC3 may play a role in the mitochondrial dysfunction in neurons with α-syn accumulation that may occur in not only the aged brain but also the brain with PD.

Objectives: To investigate the demographic features, clinical features, and potential mechanism in patients with Parkinson disease (PD) with pure apathy., Method: A total of 145 patients with PD without depression and dementia and 30 age-matched controls were consecutively recruited. Patients with PD were evaluated by Apathy Scale (AS), scales for motor symptoms and quality of life. The levels of iron, oxidative and neuroinflammatory factors, α-synuclein oligomer, and dopamine in cerebrospinal fluid (CSF) from patients with PD and controls were detected by enzyme-linked immunosorbent assay, chemical colorimetric method, and high-performance liquid chromatography. Comparisons between PD with pure apathy and with no pure apathy groups and correlation between AS score and the levels of above factors were analyzed., Results: There were 64 (44.14%) cases in PD-apathy group. The PD-apathy group had older age, (97.81 ± 10.82) years versus (61.86 ± 10.80) years, and severer quality of life (P < .05). The PD-apathy and PD without apathy groups presented no remarkable differences in motor symptoms (P > .05). The levels of iron, hydroxyl radical (·OH), hydrogen peroxide (H2O2), and α-synuclein oligomer in CSF in PD-apathy group were significantly higher than that in PD without the apathy group (P < .05). In patients with PD, the AS score was positively correlated with the levels of iron, ·OH, H2O2 and α-synuclein oligomer in CSF (r = 19.838, .063, 1.046, and 0.498, respectively, P < .05). In PD-apathy group, iron level was positively correlated with ·OH level (r = .011, P < .05), and H2O2 level was positively correlated with α-synuclein oligomer level in CSF (r = .045, P < .05)., Conclusion: Patients with PD had high prevalence of pure apathy. Patients with PD having pure apathy had older age. Pure apathy reduced quality of life for patients without worsening motor function. Excessive iron and α-synuclein oligomer in brain commonly contributed to pure apathy of PD through potential mechanism of oxidative stress., (© The Author(s) 2016.)

Synucleinopathies and abnormalities in the nerves of the enteric nervous system are hypothesized to be involved in age-associated motility disorders. The aim of the present study was to investigate the expression of various antigens, including α‑synuclein (Syn) and its post‑translational modified forms, in the human colon at various ages. In addition, the study aimed to correlate the expression of Syn with neurodegeneration. Immunohistochemistry was used to detect the expression of neurofilament (NF), Syn, as well as its nitrated (N) form in the healthy colonic tissue of 12 young (34.08±5.12 years), 10 middle‑aged (51.80±3.52 years), and 11 elderly (75.82±7.70 years) individuals. To the best of our knowledge, the current study is the first to demonstrate the presence of N‑Syn in the colonic tissue. N‑Syn was identified in the upper layer of the mucosa and submucosa layer. Furthermore, Syn (wild‑type) was present in the mucosa and submucosa. The number of NF‑positive neurons in the submucosal layer declined significantly with age (P<0.01). In addition, Syn and N‑Syn significantly increased during aging (P<0.01). Furthermore, a negative correlation was identified between neuron number and synucleinopathies, indicating the abnormal accumulation of both wild-type Syn and N‑Syn in the mucosa, submucosa, muscle layer and myenteric plexus. The present study demonstrates that the Syn pathology may be linked to colic neuronal degeneration during normal aging, and this link may cause functional deficits.

We previously reported that the levels of α-syn oligomers, which play pivotal pathogenic roles in age-related Parkinson's disease (PD) and dementia with Lewy bodies, increase heterogeneously in the aging brain. Here, we show that exogenous α-syn incubated with brain extracts from older cynomolgus monkeys and in Lewy body pathology (LBP)-susceptible brain regions (striatum and hippocampus) forms higher amounts of phosphorylated and oligomeric α-syn than that in extracts from younger monkeys and LBP-insusceptible brain regions (cerebellum and occipital cortex). The increased α-syn phosphorylation and oligomerization in the brain extracts from older monkeys and in LBP-susceptible brain regions were associated with higher levels of polo-like kinase 2 (PLK2), an enzyme promoting α-syn phosphorylation, and lower activity of protein phosphatase 2A (PP2A), an enzyme inhibiting α-syn phosphorylation, in these brain extracts. Further, the extent of the age- and brain-dependent increase in α-syn phosphorylation and oligomerization was reduced by inhibition of PLK2 and activation of PP2A. Inversely, phosphorylated α-syn oligomers reduced the activity of PP2A and showed potent cytotoxicity. In addition, the activity of GCase and the levels of ceramide, a product of GCase shown to activate PP2A, were lower in brain extracts from older monkeys and in LBP-susceptible brain regions. Our results suggest a role for altered intrinsic metabolic enzymes in age- and brain region-dependent α-syn oligomerization in aging brains.

Aging is associated with an increased risk for Parkinson's disease and dementia with Lewy bodies, in which α-synuclein (α-syn) oligomerization plays key pathogenic roles. Here, we show that oligomeric α-syn levels increase with age in the brain of cynomolgus monkeys and are accompanied by a decrease in the expression and activity of glucocerebrosidase (GCase), a lysosomal enzyme whose dysfunction is linked to accumulation of oligomeric α-syn. Besides, levels of α-syn phosphorylated at serine 129 (pS129 α-syn), a modification that promotes α-syn oligomerization also increase with age in the brain and is associated with a reduction in the activity of protein phosphatase 2A (PP2A), an enzyme that facilitates α-syn dephosphorylation. The inverse relationship between levels of oligomeric α-syn and pS129 α-syn and activity of GCase and PP2A was more evident in brain regions susceptible to neurodegeneration (i.e., the striatum and hippocampus) than those that are less vulnerable (i.e., cerebellum and occipital cortex). In vitro experiments showed that GCase activity was more potently inhibited by oligomeric than by monomeric α-syn in the lysosome-enriched fractions isolated from brain tissues and cultured neuronal cells. Inhibition of GCase activity induced an elevation of oligomeric α-syn levels, which was shown to increase pS129 α-syn levels and reduce PP2A activity in cultured neuronal cells. The alterations in oligomeric and pS129 α-syns and their association with GCase and PP2A in aging brains may explain the vulnerability of certain brain regions to neurodegeneration in Parkinson's disease and dementia with Lewy bodies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is a very common neurodegenerative disorder characterized by the accumulation of α-synuclein (α-syn) into Lewy body (LB) inclusions and the loss of neuronmelanin (NM) containing dopamine (DA) neurons in the substantia nigra (SN). Pathological α-syn and NM are two prominent hallmarks in this selective and progressive neurodegenerative disease. Pathological α-syn can induce dopaminergic neuron death by various mechanisms, such as inducing oxidative stress and inhibiting protein degradation systems. Therefore, to explore the factors that trigger α-syn to convert from a non-toxic protein to toxic one is a pivotal question to clarify the mechanisms of PD pathogenesis. Many triggers for pathological α-syn aggregation have been identified, including missense mutations in the α-syn gene, higher concentration, and posttranslational modifications of α-Syn. Recently, the role of NM in inducing α-syn expression and aggregation has been suggested as a mechanism for this pigment to modulate neuronal vulnerability in PD. NM may be responsible for PD and age-associated increase and aggregation in α-syn. Here, we reviewed our previous study and other recent findings in the area of interaction between NM and α-syn.

Objectives: To investigate clinical features and potential mechanisms involving α-synuclein oligomer and inflammation in patients with Parkinson disease (PD) and probable REM sleep behavior disorder (PRBD)., Methods: We used the REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) to evaluate patients with PD and classified each as PRBD or not probable (NPRBD). Data collection included demographic information and evaluation of clinical symptoms using a series of rating scales. We tested for α-synuclein oligomer and inflammatory factors in CSF and serum. Data analyses included comparisons between PRBD and NPRBD groups and correlation analyses among RBDSQ score and levels of the above factors., Results: The frequency of PRBD in patients with PD was 30.67%. The PRBD group had longer disease duration, more advanced disease stage, more severe motor symptoms, and other more severe nonmotor symptoms, including depression, anxiety, and fatigue. Levels of α-synuclein oligomer in CSF and serum in the PRBD group were elevated compared with NPRBD and control groups. RBDSQ score was increased with the elevated α-synuclein oligomer level in CSF, interleukin 1β and nitric oxide levels in CSF, and prostaglandin E2 level in serum in the PD group. The level of α-synuclein oligomer in CSF was enhanced with the deterioration of motor symptoms, and the elevated levels of interleukin 1β, nitric oxide, and tumor necrosis factor α in CSF in the PRBD group., Conclusions: PRBD is common in patients with PD, especially those with longer disease duration and more severe motor and nonmotor symptoms. Elevated α-synuclein levels in CSF and serum may be correlated with PRBD through inflammation in central and peripheral nervous systems., (© 2015 American Academy of Neurology.)

DNA methylation of a CpG island located in intron 1 of the α-synuclein gene (SNCA) has been reported to play an essential role in the regulation of α-synuclein transcription, and probably has a close association with Parkinson's disease (PD). However, there is no simple and cost effective method to quantify DNA methylation in this region. Additionally, whether this CpG island is hypomethylated in peripheral blood in PD and can be used as PD biomarker is still under debate. In the present study, we developed a set of bisulfite pyrosequencing assays which can be used to examine the DNA methylation level of 13 CpG sites in intron 1 of SNCA. We compared DNA methylation levels at these sites in leukocytes from 50 PD patients and 50 healthy controls. Our results indicated that there were no significant differences in DNA methylation between PD patients and controls., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

α-Syn (α-synuclein) is a small soluble acidic protein that is extensively expressed in the nervous system. Genetic, clinical and experimental studies demonstrate that α-syn is strongly implicated in the pathogenesis of PD (Parkinson's disease). However, the pathogenic mechanism remains elusive. In the present chapter, we first describe the normal expression and potential physiological functions of α-syn. Then, we introduce recent research progress related to the pathogenic role of α-syn in PD, with special emphasis on how α-syn oligomers cause the preferential degeneration of dopaminergic neurons in the substantia nigra and the spreading of α-syn pathology in the brain of PD patients.

We previously showed that alpha-synuclein (α-Syn), a protein implicated in the pathogenesis of several neurodegenerative diseases, is a microtubule-associated protein (MAP), facilitating the polymerization of tubulin into microtubules. Therefore, we hypothesized that α-Syn might promote neurite outgrowth, a process that requires microtubule assembly. To test this hypothesis, recombinant human wild type (WT) and mutant (A30P and A53T) α-Syn proteins were added to cultured primary rat cortical neurons, and their effects on early neurite outgrowth were observed. The WT and mutant α-Syn proteins entered the neurons after 1-4 h of incubation. However, a significant increase in neurite outgrowth was observed only in neurons treated with WT α-Syn. MES23.5 dopaminergic neuronal cells overexpressing WT α-Syn also exhibited enhanced neurite outgrowth, indicating that the ability of α-Syn to promote neurite outgrowth was not due to a direct action on the cell membrane or by the membrane translocation process. Co-immunoprecipitation demonstrated that the recombinant human α-Syn was bound to tubulin. In addition, the α-Syn-treated neurons displayed increased levels of polymerized tubulin. Because α-Syn's MAP functionality is mediated by specific domains, we generated N-terminal (a.a. 1-65), non-amyloid-β (non-Aβ) component (NAC) (a.a. 61-95) and C-terminal (a.a. 96-140) fragments and added them to the primary neurons. After 1-4 h of incubation, the various α-Syn fragments had entered the neurons. However, only the NAC and C-terminal fragments, which have been previously shown to mediate MAP functionality, promoted neurite outgrowth. These results suggest that α-Syn promotes neurite outgrowth by facilitating the polymerization of tubulin into microtubules.

α-Synuclein (α-syn) and oxidative stress play pivotal roles in the pathogenesis of Parkinson's disease (PD). However, the mechanisms underlying the interaction between α-syn and oxidative stress remain poorly understood. The present study provides evidence to suggest that the nuclear translocation of α-syn increases death of dopaminergic neurons in response to oxidative stress. We found that administration of H2O2 induced a rapid cleavage and nuclear translocation of α-syn in cultured MES23.5 cells. Inhibition of calpain proteolysis, using a calpain inhibitor (MDL-28170), significantly blocked cleavage and nuclear translocation of α-syn and attenuated H2O2-induced cell death in MES23.5 cells. Expression of a truncated fragment of α-syn (58-140) significantly increased the cell death induced by H2O2 treatment. These results suggest that calpain proteolysis is involved in the process of nuclear translocation of α-syn in MES23.5 dopaminergic cells induced by oxidative stress, and that nuclear translocation of α-syn increases susceptibility of these cells to oxidative stress. Taken together, our findings provide new insight into the interaction between α-syn and oxidative stress through activation of calpain proteolytic activity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although the increased prevalence of Parkinson's disease (PD) with aging suggests that aging processes predispose dopamine neurons to degeneration, the mechanism involved remains unknown. Dopamine neurons contain significant amounts of neuromelanin, and the amount of neuromelanin increases with aging. In the present study, age-related changes in the number of nigral neurons expressing neuromelanin (NM), α-synuclein, and tyrosine hydroxylase (TH) were stereologically analyzed in the postmortem brains of 28 healthy humans with an age range of 17-84 years. Stereological counting of NM content, α-synuclein content, and TH immunoreactivity revealed significant accumulation of NM and α-synuclein in neurons during the aging process. In cells containing a large amount of NM, α-synuclein-immunoreactive cells in aged individuals outnumbered those of younger individuals. In non-NM cells, the α-synuclein expression profile was similar across age groups. Furthermore, TH-immunoreactive neurons decreased significantly with aging, which was associated with accumulation of NM and α-synuclein. Our results suggest that age related accumulation of NM might induce α-synuclein over-expression and thereby make dopamine neurons more vulnerable to injuries.

Although abnormal aggregation of α-synuclein (α-syn) is involved in several neurodegenerative diseases, its biological functions remain poorly understood, which limits our understanding of its pathogenic mechanisms. α-Syn exhibits MAP-like activity and promotes the assembly of microtubules. Since microtubules play a pivotal role in proliferative cell division, it is possible that α-syn affects cell proliferation by facilitating microtubule assembly. The role of α-syn in promoting cell proliferation was reported previously in PC12 dopaminergic cells overexpressing α-syn. Here, we extended this study aiming at finding the association between the cell proliferation effect of α-syn and its microtubule assembly activity, and identifying the potential active domain for the effect of α-syn on cell proliferation. By exploiting the property that the 11-mer repeats of synuclein molecules are able to mediate a rapid intracellular translocation of these proteins across the plasma membrane without being degraded by the cellular proteolytic system, we added recombinant full-length α-syn (wild type and A53T and A30P mutants) and β-syn to the culture medium of MES23.5 dopaminergic cells, and observed their intracellular translocation, subcellular distribution and effects on cell proliferation. We found that all the synuclein molecules could enter the cells where they were localized in both the cytoplasm and nucleus. However, only the wild-type α-syn, which had been shown to have microtubule assembly activity, was able to promote proliferation of the MES23.5 cells. The A53T and A30P mutant α-syn as well as β-syn, which had been proved not to possess microtubule assembly activity, did not exhibit any effect on cell proliferation. Since the α-syn activity in microtubule assembly was shown to be related to its specific functional domain, we then generated different functional fragments (N-terminal aa1-65, NAC aa61-95 and C-terminal aa96-140) and tested their activities in cell proliferation. We showed that all the α-syn fragments could enter the cells, but with different subcellular localizations. The N-terminal and NAC fragments were localized in the cytoplasm and the C-terminal fragment mainly in the nucleus. In accordance with the activity for the C-terminal part of α-syn in microtubule assembly, only the NAC and C-terminal fragments exhibited the activity in cell proliferation. The N-terminal fragment without microtubule assembly activity did not promote cell proliferation. The above results suggest that the α-syn function in promoting cell proliferation is associated with its microtubule assembly activity with the functional domain localized in its C-terminal part.

Previous studies have shown that over-expression of alpha-Synuclein (alpha-Syn), a protein whose abnormality is implicated in the pathogenesis of Parkinson's disease (PD), reduces tyrosine hydroxylase (TH) expression and dopamine synthesis. To explore the possible mechanism for the regulation of TH expression by alpha-Syn, luciferase reporter gene carrying a -493/+27bp fragment of human TH gene (pGL3-TH520) and pcDNA carrying halpha-Syn gene (pcDNA-halpha-Syn) were co-transfected into T293 cells. The results showed that alpha-Syn was only detected in pcDNA-halpha-Syn-transfected cells but not in pcDNA vector control cells. In alpha-Syn-transfected cells, the luciferase activity was dramatically reduced compared with the vector control cells. These results suggest that alpha-Syn may function as a negative regulator for TH expression by affecting the activity of TH promoter.

α-Synuclein (α-syn), a protein involved in the pathogenesis of Parkinson's disease (PD), is known to accumulate in mitochondria, disrupt mitochondrial function. However, the molecular mechanisms that link these pathological responses have not been investigated. In rats overexpressing α-syn in the substantia nigra (SN) through adeno-associated virus (AAV) transduction, about 50% of tyrosine hydroxylase positive neurons were lost after 24 weeks. Overexpression of α-syn was also associated with morphological deformation of mitochondria and depolarization of the mitochondrial membrane potential (ΔΨm). Both co-immunoprecipitation and confocal microscopy demonstrated that mitochondrial α-syn associated with adenylate translocator (ANT), a component of the mitochondrial permeability transition pore (mPTP). The depolarization of ΔΨm was partially reversed in vitro by bongkrekic acid (BKA), an inhibitor of ANT, suggesting that the molecular association between α-syn and ANT facilitated ΔΨm depolarization. Concomitant with α-syn accumulation in mitochondria, abnormal mitochondrial morphology, ΔΨm depolarization, and loss of TH-positive neurons, there was a decrease in apoptosis-inducing factor (AIF) within the mitochondrial matrix, suggesting possible translocation to the cytosol. Our findings suggest that overexpression of α-syn may cause mitochondrial defects in dopaminergic neurons of the substantia nigra through an association with adenylate translocator and activation of mitochondria-dependent cell death pathways. Disruption of normal mitochondrial function may contribute to the loss of dopaminergic neurons in Parkinson's disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hyperphosphorylated α-synuclein is considered an important event in the pathogenesis of Parkinson's disease but its function remains elusive. In this study we provide evidence that tyrosine hydroxylase (TH) expression was unaffected by overexpression of wild-type and phospho-mimic mutant α-synuclein (S129D) in dopaminergic MN9D cells. However, α-synuclein overexpression evidently inhibited TH phosphorylation at Ser40 and dopamine synthesis, while α-synuclein (S129D) mutant enhanced TH phosphorylation and dopamine synthesis. This phospho-mimic mutant prevented wild-type α-synuclein cytotoxicity to MN9D cells, which might be due to aggregation of mutant α-synuclein in the cytoplasm and nuclei. These results demonstrated that phosphorylation at Ser129 was involved in the regulation of TH activity, as well as in eliminating the neurotoxicity of wild-type α-synuclein overexpression in MN9D cells., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

alpha-Synuclein (alpha-Syn) abnormality and mitochondrial deficiency are two major changes in the brain of patients with Parkinson's disease (PD). A link between alpha-Syn and mitochondria in PD has been demonstrated by a recent study showing that accumulation of alpha-Syn in the mitochondria from the PD-vulnerable brain regions was associated with decreased complex I activity of these mitochondria. In this study, we examined the normal expressions of alpha-Syn in mitochondria from different regions of the rat brain. We showed that alpha-Syn was highly expressed in the mitochondria in olfactory bulb, hippocampus, striatum, and thalamus, where the cytosolic alpha-Syn was also rich. However, the cerebral cortex and cerebellum were two exceptions, which contained rich cytosolic alpha-Syn but very low or even undetectable levels of mitochondrial alpha-Syn. The close quantitative association between mitochondrial and cytosolic alpha-Syn in most brain regions, suggests that the concentration of cytosolic alpha-Syn may determine the amount of alpha-Syn in mitochondria. This is partially supported by the in vitro experiment showing that incubation of alpha-Syn with endogenous alpha-Syn-undetectable cerebellar mitochondria caused a dose-dependent transport of alpha-Syn to the mitochondria. Moreover, we found that the inhibitory effect of alpha-Syn on complex I activity of mitochondrial respiratory chain was also dose-dependent. These results suggest that alpha-Syn in mitochondria is differentially expressed in different brain regions and the background levels of mitochondrial alpha-Syn may be a potential factor affecting mitochondrial function and predisposing some neurons to degeneration.

Alpha-synuclein (alpha-Syn) is a brain-enriched protein of 140 amino acids. Despite of strong evidence showing the implication of the protein in the pathogenesis of several neurodegenerative diseases, its physiological function remains poorly understood. To study the physiological function of alpha-Syn, a depiction of its precise subcellular localization is necessary. Although alpha-Syn expression in the brain has been extensively investigated using several different antibodies, its precise subcellular localization in neurons remains elusive. In this study, immunogold electron microscopy with a newly produced 3D5 monoclonal antibody recognizing the C-terminal 115-121 amino acids of alpha-Syn was used to examine its subcellular localization in rat brain neurons. In addition, the relative amount of the protein in different subcellular pools of the neurons in several brain regions was evaluated and compared. The results showed that alpha-Syn-positive gold particles were unevenly distributed in axons, presynaptic terminals, cytoplasm and nucleus in the neuron, with the density of gold particles being greater in presynaptic terminals and nucleus than in other subcellular pools. In the cytoplasmic region, relatively dense gold particles were seen in some mitochondria. In the same subcellular pools, the density of gold particles was varied among the neurons from different brain regions. Although the cortical neurons showed much higher density of gold particles in the presynaptic terminals and nuclei than in striatal, hippocampal and substantia nigral neurons, the density of gold particles in their mitochondria was much lower compared with the mitochondria of striatal, hippocampal and substantia nigral neurons. The relative high level of mitochondrial alpha-Syn in hippocampus, striatum and substantia nigral neurons may have special pathophysiological significance, which deserves further investigation.

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease (PD). Previous studies have shown that alpha-synuclein is involved in the regulation of dopamine (DA) metabolism, possibly by down-regulating the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in DA biosynthesis. In this study, we constructed alpha-synuclein stably silenced MN9D/alpha-SYN(-) cells by vector mediated RNA interference and examined its effects on DA metabolism. We found that there were no significant differences in TH protein and mRNA levels between MN9D, MN9D/alpha-SYN(-) and MN9D/CON cells, suggesting that silencing alpha-synuclein expression does not affect TH gene expression. However, significant increases in phosphorylated TH, cytosolic 3, 4-dihydroxyphenylalanine (L-DOPA) and DA levels were observed in MN9D/alpha-SYN(-) cells. Our data show that TH activity and DA biosynthesis were enhanced by down-regulation of alpha-synuclein, suggesting that alpha-synuclein may act as a negative regulator of cytosolic DA. With respect to PD pathology, a loss of functional alpha-synuclein may result in increased DA levels in neurons that may lead to cell injury or even death.

Growing evidence suggests that oxidative stress is involved in the neuronal degeneration and can promote the aggregation of alpha-synuclein. However, the role of alpha-synuclein under physiological and pathological conditions remains poorly understood. In the present study, we examined the possible interaction between the alpha-synuclein and oxidative stress. In a dopaminergic cell line MES23.5, we have found that the 200microM H(2)O(2) treatment induced the translocation of alpha-synuclein from cytoplasm to nuclei at 30min post-treatment. The immunoactivity of alpha-synuclein became highly intensive in the nuclei after 2h treatment. The protein translocated to nucleus was a 10kDa fragment of C-terminus region of alpha-synuclein, while full-length alpha-synuclein remained in cytoplasm. Thioflavine-S staining suggested that the C-terminal fragment in the nuclei has no beta-sheet structures. Our present results indicated that 200microM H(2)O(2) treatment induces the intranuclear accumulation of the C-terminal fragment of alpha-synuclein in dopaminergic neurons, whose role remains to be investigated.