Authentically phosphorylated α-synuclein at Ser129 accelerates neurodegeneration in a rat model of familial Parkinson's disease

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra (SN) and the appearance of fibrillar aggregates of insoluble α-synuclein (α-syn) called Lewy bodies (LBs). Approximately 90% of α-syn deposited in LBs is phosphorylated at serine 129 (Ser129). In contrast, only 4% of total α-syn is phosphorylated in normal brain, suggesting that accumulation of Ser129-phosphorylated α-syn is involved in the pathogenesis of PD. However, the role of Ser129 phosphorylation in α-syn neurotoxicity remains unclear. In this study, we coexpressed familial PD-linked A53T α-syn and G-protein-coupled receptor kinase 6 (GRK6) in the rat SN pars compacta using recombinant adeno-associated virus 2. Coexpression of these proteins yielded abundant Ser129-phosphorylated α-syn and significantly exacerbated degeneration of dopaminergic neurons when compared with coexpression of A53T α-syn and GFP. Immunohistochemical analysis revealed that Ser129-phosphorylated α-syn was preferentially distributed to swollen neurites. However, biochemical analysis showed that the increased expression of Ser129-phosphorylated α-syn did not promote accumulation of detergent-insoluble α-syn. Coexpression of catalytically inactive K215R mutant GRK6 failed to accelerate A53T α-syn-induced degeneration. Furthermore, introducing a phosphorylation-incompetent mutation, S129A, into A53T α-syn did not alter the pace of degeneration, even when GRK6 was coexpressed. Our study demonstrates that authentically Ser129-phosphorylated α-syn accelerates A53T α-syn neurotoxicity without the formation of detergent-insoluble α-syn, and suggests that the degenerative process could be constrained by inhibiting the kinase that phosphorylates α-syn at Ser129.     

β淀粉样蛋白（1-42）促进α-突触核蛋白在Ser129位点磷酸化和聚集

目的：通过转染人α-突触核蛋白（α-syn）A53T突变基因的PC12细胞（A53T-PC12细胞）模型,探讨β-淀粉样蛋白1-42（Aβ1-42）对A53T-PC12细胞在Ser29位点α-syn磷酸化（Pα-syn）水平、聚集程度的影响。方法：分别予H2O2、PBS、Aβ1-42干预A53T-PC12细胞（将A53T-PC12细胞分为：PBS干预组;5μmol.L-1Aβ1-42干预组;5μmol.L-1Aβ1-42＋300μmol.L-1维生素C干预组;200μmol.L-1H2O2干预组）,观察细胞内活性氧自由基（ROS）水平变化。并通过双重免疫荧光染色观察A53T-PC12细胞内α-syn聚集情况,并通过Western blot半定量分析A53T-PC12细胞内Ser129位点磷酸化Pα-syn水平。结果：Aβ1-42增加细胞内ROS。免疫荧光染色提示Aβ1-42干预后促进A53T-PC12细胞内α-syn聚集;Western blot半定量分析显示Pα-syn较PBS干预组升高（P〈0.001,P〈0.05）。结论：Aβ1-42促进A53T-PC12细胞内α-syn在Ser129位点磷酸化和聚集。     

Forebrain overexpression of α-synuclein leads to early postnatal hippocampal neuron loss and synaptic disruption

Transgenic (Tg) mouse models of Parkinson's disease (PD) generated to date have primarily been designed to overexpress human alpha-synuclein (α-syn) to recapitulate PD-like motor impairments as well as PD-like nigrostriatal degeneration and α-syn pathology. However, cognitive impairments and cortical α-syn pathology are also common in PD patients. To model these features of PD, we created forebrain-specific conditional Tg mice that overexpress human wild type (WT) or A53T mutant α-syn. Here we show that both WT and A53T mutant α-syn lead to massive degeneration of postmitotic neurons in the hippocampal dentate gyrus (DG) during postnatal development, with hippocampal synapse loss as evidenced by reduced levels of pre- and postsynaptic markers. However, when mutant and WT α-syn expression was repressed until the Tg mice were mature postnatally and then induced for several months, no hippocampal neuron loss was observed. These data imply that developing neurons are more vulnerable to degenerate than mature neurons as a consequence of forebrain WT and mutant α-syn overexpression.     

VPS41, a protein involved in lysosomal trafficking,is protective in Caenorhabditis elegans and mammalian cellular models of Parkinson's disease

VPS41 is a protein identified as a potential therapeutic target for Parkinson's disease (PD) as a result of a high-throughput RNAi screen in Caenorhabditis elegans. VPS41 has a plausible mechanistic link to the pathogenesis of PD, as in yeast it is known to participate in trafficking of proteins to the lysosomal system and several recent lines of evidence have pointed to the importance of lysosomal system dysfunction in the neurotoxicity of alpha-synuclein (α-syn). We found that expression of the human form of VPS41 (hVPS41) prevents dopamine (DA) neuron loss induced by α-syn overexpression and 6-hydroxydopamine (6-OHDA) neurotoxicity in C. elegans. In SH-SY5Y neuroblastoma cell lines stably transfected with hVPS41, we determined that presence of this protein conferred protection against the neurotoxins 6-OHDA and rotenone. Overexpression of hVPS41 did not alter the mitochondrial membrane depolarization induced by these neurotoxins. hVPS41 did, however, block downstream events in the apoptotic cascade including activation of caspase-9 and caspase-3, and PARP cleavage. We also observed that hVPS41 reduced the accumulation of insoluble high-molecular weight forms of α-syn in SH-SY5Y cells after treatment with rotenone. These data show that hVPS41 is protective against both α-syn and neurotoxic-mediated injury in invertebrate and cellular models of PD. These protective functions may be related to enhanced clearance of misfolded or aggregated protein, including α-syn. Our studies indicate that hVPS41 may be a useful target for developing therapeutic strategies for human PD.     

Membrane Lipid Modification by Docosahexaenoic Acid (DHA) Promotes the Formation of α-Synuclein Inclusion Bodies Immunopositive for SUMO-1 in Oligodendroglial Cells After Oxidative Stress

α-Synuclein (α-syn) is the major constituent of Lewy bodies and glial cytoplasmic inclusions which are pathological hallmarks of neurodegenerative disorders like Parkinson’s disease or multiple system atrophy (MSA), respectively. It accumulates and aggregates during the pathogenic process, and missense mutations, such as A53T, are increasing its probability of aggregate formation. Furthermore, α-syn interacts with polyunsaturated fatty acids, and this interaction may promote the oligomerization process. To investigate whether membrane lipid modification by docosahexaenoic acid (DHA) modifies the aggregation process of α-syn in oligodendroglial cells, we have used OLN-93 cells stably expressing the human α-syn A53T mutation. Cells were supplemented with DHA (25 μM) for 3 days and then subjected to oxidative stress (OS) exerted by hydrogen peroxide. The data show that modification of the oligodendroglial cell membranes by DHA followed by OS caused the formation of fibrillary α-syn inclusions, a decrease in α-syn solubility, and an increase in phosphorylation at serine 129, which has been suggested to play a proaggregatory role. The aggregates contain αB-crystallin and ubiquitinated proteins and SUMO-1 immunoreactivity. SUMO-1 has been implicated in protein aggregation and identified as a constituent in inclusion bodies in MSA. Hence, membrane lipid modification in oligodendroglial cells promotes the formation of α-syn inclusion bodies resembling protein aggregates in neurodegenerative disease. This effect is not only attributable to the A53T mutation but also is observable in OLN cells expressing wild-type α-syn.     

Characterization of kinases involved in the phosphorylation of aggregated α-synuclein

α-Synuclein (α-syn) is the major component of pathological inclusions characteristic of several neurodegenerative disorders, such as Parkinson's disease. The major posttranslational modification of α-syn is phosphorylation at S129, and previous studies estimate that approximately 90% of α-syn in proteinaceous, pathological inclusions is phosphorylated at this site. α-Syn can be phosphorylated by polo-like kinases (PLKs) 1-3 and casein kinases (CK) 1 and 2; however, the kinases associated with the hyperphosphorylation of aggregated α-syn are still under debate. Using a high-efficiency cellular model of α-syn aggregate formation, we found that selective inhibitors for CK2 and PLKs each partially inhibited S129 phosphorylation of soluble (nonaggregated) α-syn, but only PLK inhibitors modestly attenuated the phosphorylation of aggregated α-syn. In addition, none of the kinase inhibitors used had a substantial effect on the propensity of α-syn to aggregate. Overexpression of all PLKs promoted robust phosphorylation of soluble α-syn, but none altered the propensity of α-syn to aggregate. Overexpression of only PLK2 increased phosphorylation of aggregated α-syn at S129, which likely is due to increased phosphorylation of soluble α-syn, which then was incorporated into aggregates. Overexpression of PLK1 and treatment with BI2536 resulted in a significant reduction of phosphorylated, aggregated α-syn protein, beyond that of BI2536 treatment alone. These studies suggest that phosphorylation of α-syn is independent of α-syn aggregate formation, that PLK1 is involved in the phosphorylation of aggregated α-syn at S129 in this system, and that mechanisms resulting in hyperphosphorylation of aggregated α-syn appear to be independent of those responsible for the phosphorylation of soluble α-syn.     

Role of Synucleins in Alzheimer’s Disease

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common causes of dementia and movement disorders in the elderly. While progressive accumulation of oligomeric amyloid-β protein (Aβ) has been identified as one of the central toxic events in AD leading to synaptic dysfunction, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers has been linked to PD. Most of the studies in AD have been focused on investigating the role of Aβ and Tau; however, recent studies suggest that α-syn might also play a role in the pathogenesis of AD. For example, fragments of α-syn can associate with amyloid plaques and Aβ promotes the aggregation of α-syn in vivo and worsens the deficits in α-syn tg mice. Moreover, α-syn has also been shown to accumulate in limbic regions in AD, Down’s syndrome, and familial AD cases. Aβ and α-syn might directly interact under pathological conditions leading to the formation of toxic oligomers and nanopores that increase intracellular calcium. The interactions between Aβ and α-syn might also result in oxidative stress, lysosomal leakage, and mitochondrial dysfunction. Thus, better understanding the steps involved in the process of Aβ and α-syn aggregation is important in order to develop intervention strategies that might prevent or reverse the accumulation of toxic proteins in AD.     

Monoamine Oxidase-B Inhibition Facilitates α-Synuclein Secretion In Vitro and Delays Its Aggregation in rAAV-Based Rat Models of Parkinson's Disease

Cell-to-cell transmission of α-synuclein (α-syn) pathology is considered to underlie the spread of neurodegeneration in Parkinson''s disease (PD). Previous studies have demonstrated that α-syn is secreted under physiological conditions in neuronal cell lines and primary neurons. However, the molecular mechanisms that regulate extracellular α-syn secretion remain unclear. In this study, we found that inhibition of monoamine oxidase-B (MAO-B) enzymatic activity facilitated α-syn secretion in human neuroblastoma SH-SY5Y cells. Both inhibition of MAO-B by selegiline or rasagiline and siRNA-mediated knock-down of MAO-B facilitated α-syn secretion. However, TVP-1022, the S-isomer of rasagiline that is 1000 times less active, failed to facilitate α-syn secretion. Additionally, the MAO-B inhibition-induced increase in α-syn secretion was unaffected by brefeldin A, which inhibits endoplasmic reticulum (ER)/Golgi transport, but was blocked by probenecid and glyburide, which inhibit ATP-binding cassette (ABC) transporter function. MAO-B inhibition preferentially facilitated the secretion of detergent-insoluble α-syn protein and decreased its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Moreover, in a rat model (male Sprague Dawley rats) generated by injecting recombinant adeno-associated virus (rAAV)-A53T α-syn, subcutaneous administration of selegiline delayed the striatal formation of Ser129-phosphorylated α-syn aggregates, and mitigated loss of nigrostriatal dopaminergic neurons. Selegiline also delayed α-syn aggregation and dopaminergic neuronal loss in a cell-to-cell transmission rat model (male Sprague Dawley rats) generated by injecting rAAV-wild-type α-syn and externally inoculating α-syn fibrils into the striatum. These findings suggest that MAO-B inhibition modulates the intracellular clearance of detergent-insoluble α-syn via the ABC transporter-mediated non-classical secretion pathway, and temporarily suppresses the formation and transmission of α-syn aggregates.SIGNIFICANCE STATEMENT The identification of a neuroprotective agent that slows or stops the progression of motor impairments is required to treat Parkinson''s disease (PD). The process of α-synuclein (α-syn) aggregation is thought to underlie neurodegeneration in PD. Here, we demonstrated that pharmacological inhibition or knock-down of monoamine oxidase-B (MAO-B) in SH-SY5Y cells facilitated α-syn secretion via a non-classical pathway involving an ATP-binding cassette (ABC) transporter. MAO-B inhibition preferentially facilitated secretion of detergent-insoluble α-syn protein and reduced its intracellular accumulation under chloroquine-induced lysosomal dysfunction. Additionally, MAO-B inhibition by selegiline protected A53T α-syn-induced nigrostriatal dopaminergic neuronal loss and suppressed the formation and cell-to-cell transmission of α-syn aggregates in rat models. We therefore propose a new function of MAO-B inhibition that modulates α-syn secretion and aggregation.     

Neuroprotection of α-synuclein under acute and chronic rotenone and maneb treatment is abolished by its familial Parkinson's disease mutations A30P, A53T and E46K

α-Synuclein (α-Syn) plays a crucial role in the pathophysiology of Parkinson's disease (PD). α-Syn has been extensively studied in many neuronal cell-based PD models but has yielded mixed results. The objective of this study was to re-evaluate the dual cytotoxic/protective roles of α-Syn in dopaminergic SH-SY5Y cells. Stable SH-SY5Y cells overexpressing wild type or familial α-Syn mutants (A30P, E46K and A53T) were subjected to acute and chronic rotenone and maneb treatment. Compared with untransfected SH-SY5Y cells, wild type α-Syn attenuated rotenone and maneb-induced cell death along with an attenuation of toxin-induced mitochondrial membrane potential changes and Reactive Oxygen Species level, whereas the mutant α-Syn constructs exacerbated environmental toxins-induced cytotoxicity. After chronic treatment, wild type α-Syn but not the mutant variants was found to rescue cells from subsequent acute hydrogen peroxide insult. These results suggest that the fundamental property of wild type α-Syn may be protective, and such property may be lost by its familial PD mutations.     

Age-Dependent Alpha-Synuclein Accumulation and Phosphorylation in the Enteric Nervous System in a Transgenic Mouse Model of Parkinson’s Disease

The enteric nervous system (ENS) controls the function of the gastrointestinal tract and has been implicated in various diseases, including Parkinson’s disease (PD). PD is a neurodegenerative disease with Lewy bodies (LBs) and Lewy neurites (LNs) as the main pathological features. In addition to the typical motor symptoms in PD, attention has been drawn to non-motor symptoms, such as constipation, implying dysfunction of the ENS. In the present study, we characterized the age-dependent morphological alterations and aggregation of α-synuclein (α-syn), the primary protein component in LBs and LNs, in the ENS in an α-syn transgenic mouse model. We found that the expression and accumulation of α-syn increased gradually in neurons of Meissner’s and Auerbach’s plexuses of the gastrointestinal tract with age (from 1 week to 2 years). In addition, α-syn was increasingly phosphorylated at the serine 129 residue, reflecting pathological alterations of the protein over time. Furthermore, α-syn was present in different subtypes of neurons expressing vasoactive intestinal polypeptide, neuronal nitric oxide synthase, or calretinin. The results indicated that BAC-α-Syn-GFP transgenic mice provide a unique model in which to study the relationship between ENS and PD pathogenesis.     

Mimicking phosphorylation at serine 87 inhibits the aggregation of human α-synuclein and protects against its toxicity in a rat model of Parkinson's disease

Several lines of evidence suggest that phosphorylation of α-synuclein (α-syn) at S87 or S129 may play an important role in regulating its aggregation, fibrillogenesis, Lewy body formation, and neurotoxicity in vivo. However, whether phosphorylation at these residues enhances or protects against α-syn toxicity in vivo remains unknown. In this study, we investigated the cellular and behavioral effect of overexpression of wild-type (WT), S87A, and S87E α-syn to block or to mimic S87 phosphorylation, respectively, in the substantia nigra of Wistar rats using recombinant adeno-associated vectors. Our results revealed that WT and S87A overexpression induced α-syn aggregation, loss of dopaminergic neurons, and fiber pathology. These neuropathological effects correlated well with the induction of hemi-parkinsonian motor symptoms. Strikingly, overexpression of the phosphomimic mutant S87E did not show any toxic effect on dopaminergic neurons and resulted in significantly less α-syn aggregates, dystrophic fibers, and motor impairment. Together, our data demonstrate, for the first time, that mimicking phosphorylation at S87 inhibits α-syn aggregation and protects against α-syn-induced toxicity in vivo, suggesting that phosphorylation at this residue would play an important role in controlling α-syn neuropathology. In addition, our results provide strong evidence for a direct correlation between α-syn-induced neurotoxicity, fiber pathology, and motor impairment and the extent of α-syn aggregation in vivo, suggesting that lowering α-syn levels and/or blocking its aggregation are viable therapeutic strategies for the treatment of Parkinson's disease and related synucleinopathies.     

α-Synuclein phosphorylation as a therapeutic target in Parkinson's disease

Phosphorylation is a key post-translational modification necessary for normal cellular signaling and, therefore, lies at the heart of cellular function. In neurodegenerative disorders, abnormal hyperphosphorylation of pathogenic proteins is a common phenomenon that contributes in important ways to the disease process. A prototypical protein that is hyperphosphorylated in the brain is α-synuclein (α-syn) - found in Lewy bodies and Lewy neurites - the pathological hallmarks of Parkinson's disease (PD) and other α-synucleinopathies. The genetic linkage of α-syn to PD as well as its pathological association in both genetic and sporadic cases have made it the primary protein of interest. In understanding how α-syn dysfunction occurs, increasing focus is being placed on its abnormal aggregation and the contribution of phosphorylation to this process. Studies of both the kinases and phosphatases that regulate α-syn phosphorylation are beginning to reveal the roles of this post-translational modification in disease pathogenesis. Modulation of α-syn phosphorylation may ultimately prove to be a viable strategy for disease-modifying therapeutic interventions. In this review, we explore mechanisms related to α-syn phosphorylation, its biophysical and functional consequences, and its role in neurodegeneration.     

The Role of Transient Receptor Potential Vanilloid 1 (Trpv1) Receptors in Dextran Sulfate-Induced Colitis in Mice

Mutations in the α-synuclein gene have been linked to rare cases of familial Parkinson's disease (PD). α-Synuclein, a 140 amino acid polypeptide, is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice, Drosophila and marmosets (Challitrix jacchus) expressing either wild type (WT) or mutant human α-synuclein develop motor deficits, LB-like inclusions in some neurons and neuronal degeneration. The effects of human α-synuclein were investigated in a neuronal rat cell line (B103). Plasmids expressing WT and mutant human α-synuclein regulated by the cytomegalovirus (CMV) promoter were prepared and used for creating stably transfected neuronal rat cell lines. For localizing α-synuclein expression, stably transfected neuronal rat cell lines, expressing α-synuclein enhanced green fluorescent protein fusion proteins, regulated by either the CMV or the human platelet-derived growth factor ß promoter were generated. Over-expression of WT and A53T α-synuclein regulated by CMV promoter in stable transfectants resulted in formation of α-synuclein-immunopositive inclusion-like structures and mitochondrial alterations. Taken together, these results suggest that abnormal accumulation of α-synuclein could lead to mitochondrial alterations that might result in oxidative stress and eventually, cell death.     

Environmental neurotoxic challenge of conditional alpha-synuclein transgenic mice predicts a dopaminergic olfactory-striatal interplay in early PD

The olfactory bulb (OB) is one of the first brain regions in Parkinson’s disease (PD) to contain alpha-synuclein (α-syn) inclusions, possibly associated with nonmotor symptoms. Mechanisms underlying olfactory synucleinopathy, its contribution to progressive aggregation pathology and nigrostriatal dopaminergic loss observed at later stages, remain unclear. A second hit, such as environmental toxins, is suggestive for α-syn aggregation in olfactory neurons, potentially triggering disease progression. To address the possible pathogenic role of olfactory α-syn accumulation in early PD, we exposed mice with site-specific and inducible overexpression of familial PD-linked mutant α-syn in OB neurons to a low dose of the herbicide paraquat. Here, we found that olfactory α-syn per se elicited structural and behavioral abnormalities, characteristic of an early time point in models with widespread α-syn expression, including hyperactivity and increased striatal dopaminergic marker. Suppression of α-syn reversed the dopaminergic phenotype. In contrast, paraquat treatment synergistically induced degeneration of olfactory dopaminergic cells and opposed the higher reactive phenotype. Neither neurodegeneration nor behavioral abnormalities were detected in paraquat-treated mice with suppressed α-syn expression. By increasing calpain activity, paraquat induced a pathological cascade leading to inhibition of autophagy clearance and accumulation of calpain-cleaved truncated and insoluble α-syn, recapitulating biochemical and structural changes in human PD. Thus our results underscore the primary role of proteolytic failure in aggregation pathology. In addition, we provide novel evidence that olfactory dopaminergic neurons display an increased vulnerability toward neurotoxins in dependence to presence of human α-syn, possibly mediating an olfactory-striatal dopaminergic network dysfunction in mouse models and early PD.     

Conditional expression of Parkinson's disease-related mutant α-synuclein in the midbrain dopaminergic neurons causes progressive neurodegeneration and degradation of transcription factor nuclear receptor related 1

α-Synuclein (α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson's disease (PD). However, only a few studies on α-syn have been performed in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We also systematically examined the subcellular abnormalities that appeared in the mDA neurons of mutant mice and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and the impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that overexpression of α-syn promoted a proteasome-dependent degradation of nuclear receptor-related 1 protein (Nurr1), whereas inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.     

Alpha-Synuclein Transmission and Mitochondrial Toxicity in Primary Human Foetal Enteric Neurons In Vitro

Parkinson’s disease (PD) is a multicentred neurodegenerative disorder characterised by the accumulation and aggregation of alpha-synuclein (α-syn) in several parts of the central nervous system. However, it is well established that PD can generate symptoms of constipation and other gastrointestinal problems and α-syn containing lesions have been identified in intestinal nerve cells. In this study, we show that α-syn can be taken up and accumulate in primary human foetal enteric neurons from the gastrointestinal tract and can be transferred between foetal enteric neurons. Impaired proteosomal/lysosomal degradation can promote the uptake and accumulation of α-syn in enteric neurons. Enteric neurons exposed to α-syn can also lead to impaired mitochondrial complex I activity, reduced mitochondrial function, and NAD⁺ depletion culminating in cell death via energy restriction. These findings demonstrate neuron-to-neuron transmission of α-syn in enteric neurons, providing renewed evidence for Braak’s hypothesis and the aetiology of PD.     

Phosphorylation of α-Synuclein at Y125 and S129 Alters Its Metal Binding Properties: Implications for Understanding the Role of α-Synuclein in the Pathogenesis of Parkinson's Disease and Related Disorders

α-Synuclein (α-syn) is a 140-amino acid protein that plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. However, the molecular determinants that are responsible for triggering and/or propagating α-syn aggregation and toxicity remain poorly understood. Several studies have suggested that there are direct interactions between different metals and α-syn, but the role of metal ions and α-syn in the pathogenesis of PD is not firmly established. Interestingly, the majority of disease-associated post-translational modifications (PTMs) (e.g., truncation, phosphorylation, and nitration) of α-syn occur at residues within the C-terminal region (Y125, S129, Y133, and Y136) and in very close proximity to the putative metal binding sites. Therefore, we hypothesized that phosphorylation within this domain could influence the α-syn-metal interactions. In this paper, we sought to map the interactions between the di- and trivalent cations, Cu(II), Pb(II), Fe(II), and Fe(III), and the C-terminal region of α-syn encompassing residues 107-140 and to determine how phosphorylation at S129 or Y125 alters the specificity and binding affinity of metals using electrospray ionization-mass spectrometry (ESI-MS) and fluorescence spectroscopy. We demonstrate that D115-M116 and P128-S129 act as additional Cu(II) binding sites and show for the first time that the residues P128-S129 and D119 are also involved in Pb(II) and Fe(II) coordination, although D119 is not essential for binding to Fe(II) and Pb(II). Furthermore, we demonstrate that phosphorylation at either Y125 or S129 increases the binding affinity of Cu(II), Pb(II), and Fe(II), but not Fe(III). Additionally, we also show that phosphorylations at these residues lead to a shift in the binding sites of metal ions from the N-terminus to the C-teminus. Together, our findings provide critical insight into and expand our understanding of the molecular and structural bases underlying the interactions between α-syn and metal ions, including the identification of novel metal binding sites, and highlight the potential importance of cross-talk between post-translational modifications and metal ion binding in modulating α-syn functional and aggregation properties that are regulated by its C-terminal domain.     

Neuromelanin enhances the toxicity of α-synuclein in SK-N-SH cells

The key pathological feature of Parkinson's disease (PD) is selective degeneration of the neuromelanin (NM)-pigmented dopaminergic neurons in the substantia nigra (SN). NM, like other risk factors, such as oxidative stress (OS) and α-synuclein (α-syn), is involved in the pathogenesis of PD. But whether or not NM synergizes with α-syn or OS in the pathogenesis of PD remains unexplored. In the present study, we examined the effects of NM on cellular viability, apoptosis and free radical production in α-syn over-expressing human neuroblastoma cell line (SK-N-SH) in the presence or absence of the oxidizer Fenton's Reagent (FR). We showed that NM synergized with FR in suppressing cell viability, and in inducing apoptosis and hydroxyl radical production in all SK-N-SH cell lines. α-Syn over-expressing cells exhibited more pronounced effect, especially the A53T mutation. Our findings suggest that NM synergizes with both OS and α-syn in conferring dopaminergic vulnerability, adding to our understanding of the pathogenesis of PD.     

pVAX1-IL4/SYN-B核酸疫苗对鱼藤酮慢性帕金森病小鼠的治疗作用

目的探讨优化人α-突触核蛋白(humanα-synuclein,hα-syn)核酸疫苗pVAX1-IL4/SYN-B对鱼藤酮慢性帕金森病小鼠的治疗效果。方法采用鱼藤酮1 mg/kg给予背部皮下注射,1次/d,连续注射40 d,建立慢性帕金森病小鼠模型。建模后小鼠随机分为3组:核酸疫苗组、空质粒组、PBS组(n=8)。于小鼠双侧后肢胫前肌部位,分别注射pVAX1-IL4/SYN-B核酸疫苗,空质粒pVAX1,PBS各100μL,共免疫3次,每次间隔3周。末次免疫后2周,观察小鼠行为学变化;免疫组化方法检测小鼠脑黑质致密部α-syn表达和酪氨酸羟化酶阳性细胞数目变化;RT-PCR检测α-syn mRNA表达情况。结果行为学观察各组小鼠自由活动实验显示,核酸疫苗组与空质粒组及PBS组小鼠移动格子数和下肢站立次数均有明显改善(P<0.05)。免疫组化检测发现核酸疫苗组小鼠与空质粒组、PBS组相比,TH阳性细胞数增加51.43%、59.00%(P<0.05),α-syn表达减少45.64%、43.28%,(P<0.05)。RT-PCR检测发现核酸疫苗组α-syn mRNA表达水平(0.37±0.17)较空质粒组(0.80±0.01)及PBS组(0.74±0.05)明显减少(P<0.05)。结论 hα-syn核酸疫苗有较强改善帕金森病小鼠行为学的作用;能有效改善鱼藤酮神经毒素对黑质多巴胺能神经元的损害,能对帕金森病小鼠产生较好的免疫治疗作用。