Wild-type Α-synuclein Research Articles

Synergistic Amyloid Switch Triggered by Early Heterotypic Oligomerization of Intrinsically Disordered α-Synuclein and Tau

Amyloidogenic intrinsically disordered proteins, α-synuclein and tau are linked to Parkinson's disease and Alzheimer's disease, respectively. A body of evidence suggests that α-synuclein and tau, both present in the presynaptic nerve terminals, co-aggregate in many neurological ailments. The molecular mechanism of α-synuclein-tau hetero-assembly is poorly understood. Here we show that amyloid formation is synergistically facilitated by heterotypic association mediated by binding-induced misfolding of both α-synuclein and tau K18. We demonstrate that the intermolecular association is largely driven by the electrostatic interaction between the negatively charged C-terminal segment of α-synuclein and the positively charged tau K18 fragment. This heterotypic association results in rapid formation of oligomers that readily mature into hetero-fibrils with a much shorter lag phase compared to the individual proteins. These findings suggested that the critical intermolecular interaction between α-synuclein and tau can promote facile amyloid formation that can potentially lead to efficient sequestration of otherwise long-lived lethal oligomeric intermediates into innocuous fibrils. We next show that a well-known familial Parkinson's disease mutant (A30P) that is known to aggregate slowly via accumulation of highly toxic oligomeric species during the long lag phase converts into amyloid fibrils significantly faster in the presence of tau K18. The early intermolecular interaction profoundly accelerates the fibrillation rate of A30P α-synuclein and impels the disease mutant to behave similar to wild-type α-synuclein in the presence of tau. Our findings suggest a mechanistic underpinning of bypassing toxicity and suggest a general strategy by which detrimental amyloidogenic precursors are efficiently sequestered into more benign amyloid fibrils.

HMGB1 promotes the starvation-induced autophagic degradation of α-synuclein in SH-SY5Y cells Atg 5-dependently

Impaired autophagic clearance of aggregated α-synuclein is considered as one of key mechanisms underlining Parkinson disease (PD). High-mobility group protein B1 (HMGB1) has recently been demonstrated to mediate persistent neuroinflammation and consequent progressive neurodegeneration by promoting multiple inflammatory and neurotoxic factors. In this study, we examined the influence of the overexpression of wild-type (WT) and mutant-type (MT, A53T and A30P) α-synuclein on the autophagy in neuroblastoma SH-SY5Y cells under starvation, and then investigated the regulation of endogenous HMGB1 on the α-synuclein degradation and on the starvation-induced autophagy in the α-synuclein-overexpressed SH-SY5Y cells. It was demonstrated that the overexpression of WT or MT α-synuclein significantly downregulated the starvation-induced conversion of LC3I to LC3II and autophagy protein (Atg) 5 expression, whereas markedly inhibited the starvation-downregulated mTOR in SH-SY5Y cells. On the other side, the lentivirus-mediated upregulation of endogenous HMGB1 promoted the degradation of WT or MT α-synuclein in SH-SY5Y cells autophagy-dependently via promoting Atg 5, but not mTOR, the Atg 5 knockdown downregulated the HMGB1-mediated promotion to α-synuclein degeneration. Thus, we concluded that α-synuclein inhibited the starvation-induced autophagy in neuroblastoma SH-SY5Y cells via inhibiting the mTOR/Atg 5 signaling. However, the endogenous HMGB1 promoted the autophagic degradation of α-synuclein via the Atg 5-dependent autophagy-initiation pathway, implying the protective role of endogenous HMGB1 in the neuroblastoma cells against the α-synuclein accumulation.

Drp-1 dependent mitochondrial fragmentation and protective autophagy in dopaminergic SH-SY5Y cells overexpressing alpha-synuclein

Parkinson's disease is a neurodegenerative movement disorder caused by the loss of dopaminergic neurons from substantia nigra. It is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. Additional common features of this disease are the mitochondrial dysfunction and the activation/inhibition of autophagy both events associated to the intracellular accumulation of α-synuclein. The mechanism by which these events contribute to neural degeneration remains unknown. In the present work we investigated the effect of α-synuclein on mitochondrial dynamics and autophagy/mitophagy in SH-SY5Y cells, an in vitro model of Parkinson disease. We demonstrated that overexpression of wild type α-synuclein causes moderated toxicity, ROS generation and mitochondrial dysfunction. In addition, α-synuclein induces the mitochondrial fragmentation on a Drp-1-dependent fashion. Overexpression of the fusion protein Opa-1 prevented both mitochondrial fragmentation and cytotoxicity. On the other hand, cells expressing α-synuclein showed activated autophagy and particularly mitophagy. Employing a genetic strategy we demonstrated that autophagy is triggered in order to protect cells from α-synuclein-induced cell death. Our results clarify the role of Opa-1 and Drp-1 in mitochondrial dynamics and cell survival, a controversial α-synuclein research issue. The findings presented point to the relevance of mitochondrial homeostasis and autophagy in the pathogenesis of PD. Better understanding of the molecular interaction between these processes could give rise to novel therapeutic methods for PD prevention and amelioration.

Membrane-induced initial structure of α-synuclein control its amyloidogenesis on model membranes

Amyloid fibrillation causes serious neurodegenerative diseases and amyloidosis; however, the detailed mechanisms by which the structural states of precursor proteins in a lipid membrane-associated environment contribute to amyloidogenesis still remains to be elucidated. We examined the relationship between structural states of intrinsically-disordered wild-type and mutant α-synuclein (αSN) and amyloidogenesis on two-types of model membranes. Highly-unstructured wild-type αSN (αSNWT) and a C-terminally-truncated mutant lacking negative charges (αSN103) formed amyloid fibrils on both types of membranes, the model membrane mimicking presynaptic vesicles (Mimic membrane) and the model membrane of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC membrane). Unstructured αSNWT and αSN103 both bound to Mimic membranes in a helical conformation with similar binding affinity. Promotion and then inhibition of amyloidogenesis of αSNWT were observed as the concentration of Mimic lipids increased. We explain this by the two-state binding model: at lower lipid concentrations, binding of αSNWT to membranes enhances amyloidogenicity by increasing the local concentration of membrane-bound αSN and so promoting amyloid nucleation; at higher lipid concentrations, membrane-bound αSNWT is actually in a sense diluted by increasing the number of model membranes, which blocks amyloid fibrillation due to an insufficient bound population for productive nucleation. Meanwhile, αSN103 formed amyloid fibrils over the whole concentration of Mimic lipids used here without inhibition, revealing the importance of helical structures for binding affinity and negatively charged unstructured C-terminal region for modulating amyloidogenesis. We propose that membrane binding-induced initial conformations of αSN, its overall charge states, and the population of membrane-bound αSN are key determinants of amyloidogenesis on membranes.

α-synuclein Induces Mitochondrial Dysfunction through Spectrin and the Actin Cytoskeleton

Genetics and neuropathology strongly link α-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related α-synucleinopathies. Here we describe a new Drosophila model of α-synucleinopathy based on widespread expression of wild-type human α-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant α-synuclein aggregation. We use results of forward genetic screening and genetic analysis in our new model to demonstrate that α-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse α-synucleinopathy model and in postmortem brain tissue from patients with α-synucleinopathy. Importantly, we provide evidence that the interaction of α-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstreamneurotoxicity. These findings suggest new therapeutic approaches for α-synuclein induced neurodegeneration.

Subthalamic Nucleus Deep Brain Stimulation Does Not Modify the Functional Deficits or Axonopathy Induced by Nigrostriatal \u03b1-Synuclein Overexpression

Subthalamic nucleus deep brain stimulation (STN DBS) protects dopaminergic neurons of the substantia nigra pars compacta (SNpc) against 6-OHDA and MPTP. We evaluated STN DBS in a parkinsonian model that displays α-synuclein pathology using unilateral, intranigral injections of recombinant adeno-associated virus pseudotype 2/5 to overexpress wildtype human α-synuclein (rAAV2/5 α-syn). A low titer of rAAV2/5 α-syn results in progressive forelimb asymmetry, loss of striatal dopaminergic terminal density and modest loss of SNpc dopamine neurons after eight weeks, corresponding to robust human-Snca expression and no effect on rat-Snca, Th, Bdnf or Trk2. α-syn overexpression increased phosphorylation of ribosomal protein S6 (p-rpS6) in SNpc neurons, a readout of trkB activation. Rats received intranigral injections of rAAV2/5 α-syn and three weeks later received four weeks of STN DBS or electrode implantation that remained inactive. STN DBS did not protect against α-syn-mediated deficits in forelimb akinesia, striatal denervation or loss of SNpc neuron, nor did STN DBS elevate p-rpS6 levels further. ON stimulation, forelimb asymmetry was exacerbated, indicating α-syn overexpression-mediated neurotransmission deficits. These results demonstrate that STN DBS does not protect the nigrostriatal system against α-syn overexpression-mediated toxicity. Whether STN DBS can be protective in other models of synucleinopathy is unknown.

DJ-1 Inhibits α-Synuclein Aggregation by Regulating Chaperone-Mediated Autophagy.

α-Synuclein misfolding and aggregation play an important role in the pathogenesis of Parkinson’s disease (PD). Loss of function and mutation of the PARK7/DJ-1 gene cause early-onset familial PD. DJ-1 can inhibit α-synuclein aggregation, and may function at an early step in the aggregation process. Soluble wild-type (WT) α-synuclein is mainly degraded by chaperone-mediated autophagy (CMA), and impairment of CMA is closely related to the pathogenesis of PD. Here, we investigated whether DJ-1 could reduce α-synuclein accumulation and aggregation by CMA. DJ-1 knockout mice and DJ-1 siRNA knockdown SH-SY5Y cells were used to investigate the potential mechanisms underlying the relationship between DJ-1 deficiency and α-synuclein aggregation. First, we confirmed that DJ-1 deficiency increased the accumulation and aggregation of α-synuclein in both SH-SY5Y cells and PD animal models, and overexpression of DJ-1 in vitro effectively decreased α-synuclein levels. α-Synuclein overexpression activated CMA by elevating the levels of lysosome-associated membrane protein type-2A (LAMP2A), but DJ-1 deficiency suppressed upregulation of LAMP2A. DJ-1 deficiency downregulated the level of lysosomal 70 kDa heat-shock cognate protein (HSC70) but not the levels of that in homogenates. Further studies showed that DJ-1 deficiency accelerated the degradation of LAMP2A in lysosomes, leading to the aggregation of α-synuclein. Our study suggests that DJ-1 deficiency aggravates α-synuclein aggregation by inhibiting the activation of CMA and provides further evidence of the molecular interaction between PD-related proteins via the CMA pathway.

Proteasome impairment by α-synuclein.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder worldwide and characterized by the loss of dopaminergic neurons in the patients’ midbrains. Both the presence of the protein α-synuclein in intracellular protein aggregates in surviving neurons and the genetic linking of the α-synuclein encoding gene point towards a major role of α-synuclein in PD etiology. The exact pathogenic mechanisms of PD development are not entirely described to date, neither is the specific role of α-synuclein in this context. Previous studies indicate that one aspect of α-synuclein-related cellular toxicity might be direct proteasome impairment. The 20/26S proteasomal machinery is an important instrument of intracellular protein degradation. Thus, direct proteasome impairment by α-synuclein might explain or at least contribute to the formation of intracellular protein aggregates. Therefore this study investigates direct proteasomal impairment by α-synuclein both in vitro using recombinant α-synuclein and isolated proteasomes as well as in living cells. Our experiments demonstrate that the impairment of proteasome activity by α-synuclein is highly dependent upon the cellular background and origin. We show that recombinant α-synuclein oligomers and fibrils scarcely affect 20S proteasome function in vitro, neither does transient α-synuclein expression in U2OS ps 2042 (Ubi(G76V)-GFP) cells. However, stable expression of both wild-type and mutant α-synuclein in dopaminergic SH-SY5Y and PC12 cells results in a prominent impairment of the chymotrypsin-like 20S/26S proteasomal protein cleavage. Thus, our results support the idea that α-synuclein in a specific cellular environment, potentially present in dopaminergic cells, cannot be processed by the proteasome and thus contributes to a selective vulnerability of dopaminergic cells to α-synuclein pathology.

Modification of C Terminus Provides New Insights into the Mechanism of α-Synuclein Aggregation

Aggregation of neuronal protein α-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson’s disease. The mechanism of α-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to α-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such α-synuclein sequence extension on α-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of α-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of α-synuclein. To address these questions, we prepared mutants of α-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type α-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of α-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of α-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).

Protective efficacy of phosphodiesterase-1 inhibition against alpha-synuclein toxicity revealed by compound screening in LUHMES cells

α-synuclein-induced neurotoxicity is a core pathogenic event in neurodegenerative synucleinopathies such as Parkinson’s disease, dementia with Lewy bodies, or multiple system atrophy. There is currently no disease-modifying therapy available for these diseases. We screened 1,600 FDA-approved drugs for their efficacy to protect LUHMES cells from degeneration induced by wild-type α-synuclein and identified dipyridamole, a non-selective phosphodiesterase inhibitor, as top hit. Systematic analysis of other phosphodiesterase inhibitors identified a specific phosphodiesterase 1 inhibitor as most potent to rescue from α-synuclein toxicity. Protection was mediated by an increase of cGMP and associated with the reduction of a specific α-synuclein oligomeric species. RNA interference experiments confirmed PDE1A and to a smaller extent PDE1C as molecular targets accounting for the protective efficacy. PDE1 inhibition also rescued dopaminergic neurons from wild-type α-synuclein induced degeneration in the substantia nigra of mice. In conclusion, this work identifies inhibition of PDE1A in particular as promising target for neuroprotective treatment of synucleinopathies.

Role of Sporadic Parkinson Disease Associated Mutations A18T and A29S in Enhanced α-Synuclein Fibrillation and Cytotoxicity.

Deposition of presynaptic protein α-synuclein in Lewy bodies and Lewy neurites in the substantia nigra region of brain has been linked with the clinical symptoms of the Parkinson's disease (PD). Proteotoxic stress conditions and mutations that cause abnormal aggregation of α-synuclein have close association with onset of PD and its progression. Therefore, studies pertaining to α-synuclein mutations play important roles in mechanistic understanding of aggregation behavior of the protein and subsequent pathology. Herein, guided by this fact, we have studied the aggregation kinetics, morphology, and neurotoxic effects of the two newly discovered sporadic PD associated mutants A18T and A29S of α-synuclein. Our studies demonstrate that both of the mutants are aggregation prone and undergo rapid aggregation compared to wild-type α-synuclein. Further, it was found that A18T mutant followed faster aggregation kinetics compared to A29S substitution. Additionally, we have designed three point mutations of α-synuclein for better understanding of the effects of substitutions on protein aggregation and demonstrated that substitution of alanine at the 18th position is highly sensitive compared to adjacent positions. Our results provide better understanding of the effects of α-synuclein mutations on its aggregation behavior that may be important in development of PD pathology.

Computational investigation on the effects of H50Q and G51D mutations on the α-Synuclein aggregation propensity

The aggregation of α-synuclein is linked directly to the histopathology of Parkinson’s disease (PD). However, several missense mutations present in the α-synuclein gene (SNCA) have been known to be associated with PD. Several studies have highlighted the effect of SNCA mutations on the α-synuclein aggregation, but their pathological roles are not completely established. In this study, we have focused on the effects of the recently discovered α-synuclein missense mutants (H50Q and G51D) on the aggregation using computational approaches. We performed all atom molecular dynamics (MD) simulation on these mutants and compared their conformational dynamics with Wild-Type (WT) α-synuclein. We noticed the solvent accessible surface area (SASA), radius of gyration, atomic fluctuations, and beta strand content to be higher in H50Q than G51D and WT. Using PDBSum online server; we analyzed the inter-molecular interactions that drive the association of monomeric units of H50Q, WT, and G51D in forming the respective homo-dimer. We noticed the interface area, number of interacting residues and binding free energy to be higher for H50Q homo-dimer than the WT and G51D homo-dimers. Our findings in this study suggest that in comparison to WT and G51D, H50Q mutation to have a positive effect on increasing the α-synuclein aggregation propensity. Hence, we see that H50Q and G51D mutation show conflicting effect on the aggregation propensity of α-synuclein.

Mesenchymal Stem Cells Stabilize Axonal Transports for Autophagic Clearance of α-Synuclein in Parkinsonian Models.

Genome-wide association studies have identified two loci, SNCA and the microtubule (MT)-associated protein tau, as common risk factors for Parkinson's disease (PD). Specifically, α-synuclein directly destabilizes MT via tau phosphorylation and induces axonal transport deficits that are the primary events leading to an abnormal accumulation of α-synuclein that causes nigral dopaminergic cell loss. In this study, we demonstrated that mesenchymal stem cells (MSCs) could modulate cytoskeletal networks and trafficking to exert neuroprotective properties in wild-type or A53T α-synuclein overexpressing cells and mice. Moreover, we found that eukaryotic elongation factor 1A-2, a soluble factor derived from MSCs, stabilized MT assembly by decreasing calcium/calmodulin-dependent tau phosphorylation and induced autophagolysosome fusion, which was accompanied by an increase in the axonal motor proteins and increased neuronal survival. Our data suggest that MSCs have beneficial effects on axonal transports via MT stability by controlling α-synuclein-induced tau phosphorylation, indicating that MSCs may exert a protective role in the early stages of axonal transport defects in α-synucleinopathies. Stem Cells 2017;35:1934-1947.

Axonal dystrophy in the brain of mice with Sanfilippo syndrome

Axonal dystrophy has been described as an early pathological feature of neurodegenerative disorders including Alzheimer's disease and amyotrophic lateral sclerosis. Axonal inclusions have also been reported to occur in several neurodegenerative lysosomal storage disorders including Mucopolysaccharidosis type IIIA (MPS IIIA; Sanfilippo syndrome). This disorder results from a mutation in the gene encoding the lysosomal sulphatase sulphamidase, and as a consequence heparan sulphate accumulates, accompanied by secondarily-stored gangliosides. The precise basis of symptom generation in MPS IIIA has not been elucidated, however axonal dystrophy may conceivably lead to impaired vesicular trafficking, neuronal dysfunction and/or death. We have utilised a faithful murine model of MPS IIIA to determine the spatio-temporal profile of neuronal inclusion formation and determine the effect of restoring normal lysosomal function. Dopaminergic (tyrosine hydroxylase-positive), cholinergic (choline acetyltransferase-positive) and GABAergic (glutamic acid decarboxylase65/67-positive) neurons were found to exhibit axonal dystrophy in MPS IIIA mouse brain. Axonal lesions present by ~seven weeks of age were Rab5-positive but lysosomal integral membrane protein-2 negative, suggesting early endosomal involvement. By 9–12-weeks of age, immunoreactivity for the autophagosome-related proteins LC3 and p62 and the proteasomal subunit 19S was noted in the spheroidal structures, together with wildtype α-synuclein, phosphorylated Thr-181 Tau and amyloid precursor protein, indicative of impaired axonal trafficking. Sulphamidase replacement reduced but did not abrogate the axonal lesions. Therefore, if axonal dystrophy impairs neuronal activity and ultimately, neuronal function, its incomplete resolution warrants further investigation.

A Molecular Tweezer Ameliorates Motor Deficits in Mice Overexpressing α-Synuclein.

Aberrant accumulation and self-assembly of α-synuclein are tightly linked to several neurodegenerative diseases called synucleinopathies, including idiopathic Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Deposition of fibrillar α-synuclein as insoluble inclusions in affected brain cells is a pathological hallmark of synucleinopathies. However, water-soluble α-synuclein oligomers may be the actual culprits causing neuronal dysfunction and degeneration in synucleinopathies. Accordingly, therapeutic approaches targeting the toxic α-synuclein assemblies are attractive for these incurable disorders. The "molecular tweezer" CLR01 selectively remodels abnormal protein self-assembly through reversible binding to Lys residues. Here, we treated young male mice overexpressing human wild-type α-synuclein under control of the Thy-1 promoter (Thy1-aSyn mice) with CLR01 and examined motor behavior and α-synuclein in the brain. Intracerebroventricular administration of CLR01 for 28days to the mice improved motor dysfunction in the challenging beam test and caused a significant decrease of buffer-soluble α-synuclein in the striatum. Proteinase-K-resistant, insoluble α-synuclein deposits remained unchanged in the substantia nigra, whereas levels of diffuse cytoplasmic α-synuclein in dopaminergic neurons increased in mice receiving CLR01 compared with vehicle. More moderate improvement of motor deficits was also achieved by subcutaneous administration of CLR01, in 2/5 trials of the challenging beam test and in the pole test, which requires balance and coordination. The data support further development of molecular tweezers as therapeutic agents for synucleinopathies.

Overexpression of α-synuclein in an astrocyte cell line promotes autophagy inhibition and apoptosis.

α-Synuclein is the major component of neuronal cytoplasmic aggregates called Lewy bodies, the main pathological hallmark of Parkinson disease. Although neurons are the predominant cells expressing α-synuclein in the brain, recent studies have demonstrated that primary astrocytes in culture also express α-synuclein and regulate α-synuclein trafficking. Astrocytes have a neuroprotective role in several detrimental brain conditions; we therefore analyzed the effects of the overexpression of wild-type α-synuclein and its A30P and A53T mutants on autophagy and apoptosis. We observed that in immortalized astrocyte cell lines, overexpression of α-synuclein proteins promotes the decrease of LC3-II and the increase of p62 protein levels, suggesting the inhibition of autophagy. When these cells were treated with rotenone, there was a loss of mitochondrial membrane potential, especially in cells expressing mutant α-synuclein. The level of this decrease was related to the toxicity of the mutants because they show a more intense and sustained effect. The decrease in autophagy and the mitochondrial changes in conjunction with parkin expression levels may sensitize astrocytes to apoptosis.

Viral-mediated oligodendroglial alpha-synuclein expression models multiple system atrophy.

MSA is a fatal neurodegenerative disorder characterized by a combination of autonomic dysfunction, cerebellar ataxia, and l-dopa unresponsive parkinsonism. The hallmark of MSA is the accumulation of α-synuclein, forming cytoplasmic inclusions in oligodendrocytes. Adeno-associated viruses allow efficient targeting of disease-associated genes in selected cellular ensembles and have proven efficient for the neuronal overexpression of α-synuclein in the substantia nigra in the context of PD. We aimed to develop viral-based models of MSA. Chimeric viral vectors expressing either human wild-type α-synuclein or green fluorescent protein under the control of mouse myelin basic protein were injected in the striatum of rats and monkeys. Rats underwent a longitudinal motor assessment before histopathological analysis at 3 and 6 months. Injection of vectors expressing α-synuclein in the striatum resulted in >80% oligodendroglial selectivity in rats and >60% in monkeys. Rats developed progressive motor deficits that were l-dopa unresponsive when assessed at 6 months. Significant loss of dopaminergic neurons occurred at 3 months, further progressing at 6 months, together with a loss of striatal neurons. Prominent α-synuclein accumulation, including phosphorylated and proteinase-K-resistant α-synuclein, was detected in the striatum and substantia nigra. Viral-mediated oligodendroglial expression of α-synuclein allows replicating some of the key features of MSA. This flexible strategy can be used to investigate, in several species, how α-synuclein accumulation in selected oligodendroglial populations contributes to the pathophysiology of MSA and offers a new framework for preclinical validation of therapeutic strategies. © 2017 International Parkinson and Movement Disorder Society.

Environmental neurotoxicant manganese regulates exosome-mediated extracellular miRNAs in cell culture model of Parkinson's disease: Relevance to α-synuclein misfolding in metal neurotoxicity

Many chronic neurodegenerative disorders share a common pathogenic mechanism involving the aggregation and deposition of misfolded proteins. Recently, it was shown that these aggregated proteins could be transferred from one cell to another via extracellular nanovesicles called exosomes. Initially thought to be a means of cellular waste removal, exosomes have since been discovered to actively participate in cell-to-cell communication. Importantly, various inflammatory and signaling molecules, as well as small RNAs are selectively packaged in these vesicles. Considering the important role of environmental manganese (Mn) in Parkinson's disease (PD)-like neurological disorders, we characterized the effect of Mn on exosome content and release using an MN9D dopaminergic cell model of PD, which was generated to stably express wild-type human α-synuclein (αSyn). Mn exposure (300μM MnCl2) for 24h induced the release of exosomes into the extracellular media prior to cytotoxicity, as determined by NanoSight particle analysis and electron microscopy. Strikingly, Western blot analysis revealed that Mn treatment in αSyn-expressing cells increases the protein Rab27a, which regulates the release of exosomes from cells. Moreover, next-generation sequencing showed more small RNAs in exosomes isolated from Mn-exposed cells than from control exosomes. Our miRNA profiling analysis led to the discovery of increased expression of certain miRNAs previously shown to regulate key biological pathways, including protein aggregation, autophagy, inflammation and hypoxia. Collectively, our results provide a glimpse of Mn's role in modulating extracellular miRNA content through exosomal release from dopaminergic neuronal cells and thus potentially contributing to progressive neurodegeneration. Further characterization of extracellular miRNAs and their targets will have major impacts on biomarker discovery and translational strategies for environmentally linked neurodegenerative diseases including PD.

α-Synuclein Enhances Cadmium Uptake and Neurotoxicity via Oxidative Stress and Caspase Activated Cell Death Mechanisms in a Dopaminergic Cell Model of Parkinson's Disease.

This study examined the role of alpha-synuclein in regulating cadmium (Cd)-induced neurotoxicity using the N27 dopaminergic neuronal model of Parkinson's disease (PD) that stably expresses wild-type human α-synuclein (α-Syn) or empty vector (Vec) control. We report that α-Syn significantly increased Cd-induced cytotoxicity as compared to Vec control cells upon 24h exposure. To explore the cellular mechanisms, we examined oxidative stress, caspase activation, and Cd uptake and intracellular accumulation. Expression of α-Syn coupled with Cd-induced cytotoxicity increased oxidative stress. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed an increase in Cd uptake and intracellular accumulation in α-Syn-expressing cells upon Cd exposure. Analysis of the mitochondrial mediated apoptotic pathway showed greater activation of caspase-9 and caspase-3 in α-Syn cells. To functionally evaluate the role of metal transporters in the altered Cd phenotype, we examined Cd toxicity in the presence of nontoxic levels of divalent manganese Mn(II) and iron Fe(II). Co-treatment with Fe(II) or Mn(II) did not significantly attenuate Cd-induced cytotoxicity. We report that Cd exposure decreased the divalent metal transporter 1 and Akt protein levels in the α-Syn-expressing cells without altering native PKCδ protein levels in both Vec control and α-Syn lines. In addition, we show decreased basal metallothionein-3 protein expression in α-Syn-expressing cells. Co-treatment with N-acetyl-L-cysteine was sufficient to attenuate and abolish the α-Syn×Cd-induced cytotoxicity. Collectively, these results demonstrate that α-Syn exhibits neurotoxic properties upon acute Cd exposure to cause cell death by causing oxidative stress, increasing Cd uptake, altering caspase-9 and caspase-3 activation, and diminishing the neuroprotective effect of Akt in a dopaminergic neuronal model of PD.

α-Synuclein increases β-amyloid secretion by promoting β-/γ-secretase processing of APP.

α-Synuclein misfolding and aggregation is often accompanied by β-amyloid deposition in some neurodegenerative diseases. We hypothesised that α-synuclein promotes β-amyloid production from APP. β-Amyloid levels and APP amyloidogenic processing were investigated in neuronal cell lines stably overexpressing wildtype and mutant α-synuclein. γ-Secretase activity and β-secretase expression were also measured. We show that α-synuclein expression induces β-amyloid secretion and amyloidogenic processing of APP in neuronal cell lines. Certain mutations of α-synuclein potentiate APP amyloidogenic processing. γ-Secretase activity was not enhanced by wildtype α-synuclein expression, however β-secretase protein levels were induced. Furthermore, a correlation between α-synuclein and β-secretase protein was seen in rat brain striata. Iron chelation abolishes the effect of α-synuclein on neuronal cell β-amyloid secretion, whereas overexpression of the ferrireductase enzyme Steap3 is robustly pro-amyloidogenic. We propose that α-synuclein promotes β-amyloid formation by modulating β-cleavage of APP, and that this is potentially mediated by the levels of reduced iron and oxidative stress.