A53T Α-syn Research Articles

The role of LRRK2 inhibition in α-synuclein-induced neurotoxicity and neuroinflammation in vivo.

Event Abstract Back to Event The role of LRRK2 inhibition in α-synuclein-induced neurotoxicity and neuroinflammation in vivo. Diego Cabezudo1*, Anke Van Der Perren1, Géraldine Gelders1, Chris Van Den Haute1, Veerle Baekelandt1 and Evy Lobbestael1 1 KU Leuven, Belgium Parkinson’s disease (PD) is the most common neurodegenerative motor disorder, affecting 10 million people worldwide. A major challenge is the development of disease-modifying therapies, for which a better understanding of PD pathogenesis is indispensable. Leucine-rich repeat kinase 2 (LRRK2) and α-synuclein (α-SYN) are key players in PD and previous studies indicate that targeting LRRK2 can modify α-SYN-induced neurotoxicity and neuroinflammation in preclinical PD models. To further explore the functional interaction between both proteins and evaluate the therapeutic potential of LRRK2, we studied the effects of LRRK2 kinase inhibition and total LRRK2 protein loss in different viral vector-mediated α-SYN-based PD models. We used recombinant adeno-associated viral vectors (rAAV2/7) to overexpress human wild-type or pathogenic A53T α-SYN in the substantia nigra of wild-type and LRRK2 knock out rats. By applying different vector doses, we modelled early and late stage neurodegeneration. In addition, by using an in-diet treatment protocol with MLi-2, we assessed the therapeutic potential of LRRK2 kinase inhibition. Effects on motor behaviour deficits were evaluated by the cylinder test, nigrostriatal neurodegeneration and α-SYN pathology were assessed by immunohistochemistry. Given the role of both LRRK2 and α-SYN in neuroinflammation, we also examined whether loss of LRRK2 (kinase activity) can affect α-SYN-induced neuroinflammatory changes in the different models. Keywords: LRRK2, A-synuclein, Neurodegenaration, Parkinson ' s disease, Neuroinflammantion Conference: 13th National Congress of the Belgian Society for Neuroscience , Brussels, Belgium, 24 May - 24 May, 2019. Presentation Type: Poster presentation Topic: Cellular/Molecular Neuroscience Citation: Cabezudo D, Van Der Perren A, Gelders G, Van Den Haute C, Baekelandt V and Lobbestael E (2019). The role of LRRK2 inhibition in α-synuclein-induced neurotoxicity and neuroinflammation in vivo.. Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience . doi: 10.3389/conf.fnins.2019.96.00020 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Mr. Diego Cabezudo, KU Leuven, Leuven, Belgium, diego.cabezudo@kuleuven.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael Google Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael Google Scholar Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael PubMed Diego Cabezudo Anke Van Der Perren Géraldine Gelders Chris Van Den Haute Veerle Baekelandt Evy Lobbestael Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Secretion and Uptake of \u03b1-Synuclein Via Extracellular Vesicles in Cultured Cells

In Parkinson’s disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.

Low-Dose Aspirin Upregulates Tyrosine Hydroxylase and Increases Dopamine Production in Dopaminergic Neurons: Implications for Parkinson's Disease.

Increasing the function of residual dopaminergic neurons in the nigra of PD patients is an important area of research as it may eventually compensate the loss. Although tyrosine hydroxylase (TH) is the rate-limiting enzyme in the dopamine (DA) biosynthesis pathway, there are no effective drugs/molecules to upregulate TH and increase the production of DA in nigral dopaminergic neurons. This study underlines the importance of aspirin in stimulating the expression of TH and increasing the level of DA in dopaminergic neurons. At low doses, aspirin increased the expression of TH and the production of DA in mouse MN9D dopaminergic neuronal cells. Accordingly, oral administration of aspirin increased the expression of TH in the nigra and upregulated the level of DA in striatum of normal C57/BL6 mice and aged A53T α-syn transgenic mice. Oral aspirin also improved locomotor activities of normal mice and A53T transgenic mice. While investigating mechanisms, we found the presence of cAMP response element (CRE) in the promoter of TH gene and the rapid induction of cAMP response element binding (CREB) activation by aspirin in dopaminergic neuronal cells. Aspirin treatment also increased the level of phospho-CREB in the nigra of C57/BL6 mice. The abrogation of aspirin-induced expression of TH by siRNA knockdown of CREB and the recruitment of CREB to the TH gene promoter by aspirin suggest that aspirin stimulates the transcription of TH in dopaminergic neurons via CREB. These results highlight a new property of aspirin in stimulating the TH-DA pathway, which may be beneficial in PD patients. Graphical Abstract ᅟ.

In vivo quantification of glial activation in minipigs overexpressing human α-synuclein.

Parkinson's disease is characterized by a progressive loss of substantia nigra (SN) dopaminergic neurons and the formation of Lewy bodies containing accumulated alpha-synuclein (α-syn). The pathology of Parkinson's disease is associated with neuroinflammatory microglial activation, which may contribute to the ongoing neurodegeneration. This study investigates the in vivo microglial and dopaminergic response to overexpression of α-syn. We used positron emission tomography (PET) and the 18 kDa translocator protein radioligand, [11 C](R)PK11195, to image brain microglial activation and (+)-α-[11 C]dihydrotetrabenazine ([11 C]DTBZ), to measure vesicular monoamine transporter 2 (VMAT2) availability in Göttingen minipigs following injection with recombinant adeno-associated virus (rAAV) vectors expressing either mutant A53T α-syn or green fluorescent protein (GFP) into the SN (4 rAAV-α-syn, 4 rAAV-GFP, 5 non-injected control minipigs). We performed motor symptom assessment and immunohistochemical examination of tyrosine hydroxylase (TH) and transgene expression. Expression of GFP and α-syn was observed at the SN injection site and in the striatum. We observed no motor symptoms or changes in striatal [11 C]DTBZ binding potential in vivo or striatal or SN TH staining in vitro between the groups. The mean [11 C](R)PK11195 total volume of distribution was significantly higher in the basal ganglia and cortical areas of the α-syn group than the control animals. We conclude that mutant α-syn expression in the SN resulted in microglial activation in multiple sub- and cortical regions, while it did not affect TH stains or VMAT2 availability. Our data suggest that microglial activation constitutes an early response to accumulation of α-syn in the absence of dopamine neuron degeneration.

Lipid metabolism alterations in the neuronal response to A53T α-synuclein and Fe-induced injury

Pathological α-synuclein (α-syn) overexpression and iron (Fe)-induced oxidative stress (OS) are involved in the death of dopaminergic neurons in Parkinson's disease (PD). We have previously characterized the role of triacylglycerol (TAG) formation in the neuronal response to Fe-induced OS. In this work we characterize the role of the α-syn variant A53T during Fe-induced injury and investigate whether lipid metabolism has implications for neuronal fate. To this end, we used the N27 dopaminergic neuronal cell line either untransfected (UT) or stably transfected with pcDNA3 vector (as a transfection control) or pcDNA-A53T-α-syn (A53T α-syn). The overexpression of A53T α-syn triggered an increase in TAG content mainly due to the activation of Acyl-CoA synthetase. Since fatty acid (FA) β-oxidation and phospholipid content did not change in A53T α-syn cells, the unique consequence of the increase in FA-CoA derivatives was their acylation in TAG moieties.Control cells exposed to Fe-induced injury displayed increased OS markers and TAG content. Intriguingly, Fe exposure in A53T α-syn cells promoted a decrease in OS markers accompanied by α-syn aggregation and elevated TAG content.We report here new evidence of a differential role played by A53T α-syn in neuronal lipid metabolism as related to the neuronal response to OS.

Chronic Caffeine Treatment Protects Against α-Synucleinopathy by Reestablishing Autophagy Activity in the Mouse Striatum.

Despite converging epidemiological evidence for the inverse relationship of regular caffeine consumption and risk of developing Parkinson's disease (PD) with animal studies demonstrating protective effect of caffeine in various neurotoxin models of PD, whether caffeine can protect against mutant α-synuclein (α-Syn) A53T-induced neurotoxicity in intact animals has not been examined. Here, we determined the effect of chronic caffeine treatment using the α-Syn fibril model of PD by intra-striatal injection of preformed A53T α-Syn fibrils. We demonstrated that chronic caffeine treatment blunted a cascade of pathological events leading to α-synucleinopathy, including pSer129α-Syn-rich aggregates, apoptotic neuronal cell death, microglia, and astroglia reactivation. Importantly, chronic caffeine treatment did not affect autophagy processes in the normal striatum, but selectively reversed α-Syn-induced defects in macroautophagy (by enhancing microtubule-associated protein 1 light chain 3, and reducing the receptor protein sequestosome 1, SQSTM1/p62) and chaperone-mediated autophagy (CMA, by enhancing LAMP2A). These findings support that caffeine—a strongly protective environment factor as suggested by epidemiological evidence—may represent a novel pharmacological therapy for PD by targeting autophagy pathway.

The expression of KATP channel subunits in alpha-synuclein-transfected MES23.5 cells.

SUR1, one of the subunits of ATP-sensitive potassium (KATP) channels, was found to be highly expressed in mRNA levels in the substantia nigra (SN) of Parkinson's disease (PD) brains. Though the mechanism of the selective dopamine (DA) neurons death is still unknown, some studies have demonstrated that selective activation of the KATP channels in the SN might be associated with the degeneration of DA neurons. The objective of our study is to examine the expressions of KATP channel subunits in dopaminergic cells with alpha-synuclein (α-Syn) transfection. In this study, we detected the KATP channel subunits mRNA levels in MES23.5 cells by real-time quantitative PCR after the cells transfected with α-Syn. Our results showed that the mRNA levels of SUR1 subunit were markedly increased by 35% in WT α-Syn overexpression cells and by 31% in A53T α-Syn overexpression cells, respectively. However, the mRNA levels of SUR2B and Kir6.2 subunit have no obviously differences from the controls. We showed that the mRNA levels of SUR1 but not SUR2B or Kir6.2 were selectively upregulated in MES23.5 cells over-expressed with α-Syn. The findings demonstrated that the SUR1 overexpressed might be involved in the process of PD.

Cardiolipin exposure on the outer mitochondrial membrane modulates α‐synuclein proteostasis in hPSC‐derived Parkinson's Disease neurons

IntroductionThe decline of voluntary motor function in Parkinson's disease (PD) is caused by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta. The death of these neurons is preceded by the formation of intracellular proteinaceous aggregates known as Lewy Bodies, which contain a variety of misfolded protein components, including α‐synuclein (α‐syn). Although SNCA mutations (α‐syn gene) are causal in several rare familial forms of PD, mutations in the SNCA locus are also consistently identified in genome‐wide association studies (GWAS) of sporadic PD, and α‐syn protein is commonly found in Lewy‐neurites of idiopathic disease cases. These findings emphasize the need to understand how altered α‐syn structure and function relate to PD pathogenesis in general terms.ApproachThe generation of human isogenic induced pluripotent stem cell (hiPSC) and embryonic stem cell (hESC) models of familial PD has facilitated the analysis of PD pathology at the cellular level, allowing us to contrast A9‐type DA neurons (hNs) harboring the SNCA‐A53T or SNCA‐E46K mutations with isogenic, mutation‐corrected controls. Using these systems in combination with SNCA‐A53T transgenic animals, we describe a novel mechanism in which the mitochondrial membrane lipid cardiolipin plays a vital role in preventing accumulation of α‐syn protein aggregates. This protective process is disrupted by both SNCA‐A53T and SNCA‐E46K mutations.ResultsHere, we demonstrate that cardiolipin translocates to the outer mitochondrial membrane (OMM) in both A53T and E46K SNCA‐mutant hNs at the onset of α‐syn deposition, where it binds α‐syn, facilitating the folding of intrinsically disordered α‐syn to an a‐helical conformation. This finding was confirmed in SNCA‐A53T transgenic mouse brains. Furthermore, we show that OMM‐localized cardiolipin can pull α‐syn monomers away from multimeric‐fibrils and facilitate their re‐folding, from multimeric b‐sheet forms back to monomers comprising a‐helices, effectively buffering α‐syn pathology. Surprisingly, both the A53T and E46K α‐syn mutations reduced the kinetic rate of cardiolipin‐mediated α‐syn re‐folding relative to wild‐type (WT), increasing the level of α‐syn present at the OMM in A53T and E46K hNs. Binding of A53T and E46K α‐syn to cardiolipin led to significantly increased LC3 recruitment to the OMM relative to WT α‐syn, which in turn increased mitochondrial stress, triggering excessive mitophagy.SignificanceThis work represents the first demonstration of α‐syn as a modulator of LC3 function, and identifies cardiolipin exposure on mitochondrial membranes as a key regulator of PD pathogenesis.Support or Funding InformationThis work was supported in part by the Parkinson Society of Canada (2014‐685 to SDR), the Natural Sciences and Engineering Research Council of Canada (RG060805 to SDR, RG121541 to GH), the CRC Program (GH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity.

Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD.Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system.Results: Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression.Conclusion: In A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD.

Induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient CNS pathology

Alpha-Synuclein (α-syn) is by far the most highly vetted pathogenic and therapeutic target in Parkinson's disease. Aggregated α-syn is present in sporadic Parkinson's disease, both in the central nervous system (CNS) and peripheral nervous system (PNS). The enteric division of the PNS is of particular interest because 1) gastric dysfunction is a key clinical manifestation of Parkinson's disease, and 2) Lewy pathology in myenteric and submucosal neurons of the enteric nervous system (ENS) has been referred to as stage zero in the Braak pathological staging of Parkinson's disease. The presence of Lewy pathology in the ENS and the fact that patients often experience enteric dysfunction before the onset of motor symptoms has led to the hypothesis that α-syn pathology starts in the periphery, after which it spreads to the CNS via interconnected neural pathways. Here we sought to directly test this hypothesis in rodents and non-human primates (NHP) using two distinct models of α-syn pathology: the α-syn viral overexpression model and the preformed fibril (PFF) model. Subjects (rat and NHP) received targeted enteric injections of PFFs or adeno-associated virus overexpressing the Parkinson's disease associated A53T α-syn mutant. Rats were evaluated for colonic motility monthly and sacrificed at 1, 6, or 12 months, whereas NHPs were sacrificed 12 months following inoculation, after which the time course and spread of pathology was examined in all animals. Rats exhibited a transient GI phenotype that resolved after four months. Minor α-syn pathology was observed in the brainstem (dorsal motor nucleus of the vagus and locus coeruleus) 1 month after PFF injections; however, no pathology was observed at later time points (nor in saline or monomer treated animals). Similarly, a histopathological analysis of the NHP brains revealed no pathology despite the presence of robust α-syn pathology throughout the ENS which persisted for the entirety of the study (12 months). Our study shows that induction of α-syn pathology in the ENS is sufficient to induce GI dysfunction. Moreover, our data suggest that sustained spread of α-syn pathology from the periphery to the CNS and subsequent propagation is a rare event, and that the presence of enteric α-syn pathology and dysfunction may represent an epiphenomenon.

Ginkgolide K promotes the clearance of A53T mutation alpha-synuclein in SH-SY5Y cells.

Alpha-synuclein (α-syn) is associated to Parkinson's disease (PD). The aggregated form of α-syn has potential neurotoxicity. Thus, the clearance of α-syn aggregation is a plausible strategy to delay disease progression of PD. In our study, we found that the treatment of Ginkgolide B (GB) and Ginkgolide K (GK) reduced cell death, and enhanced cell proliferation in SH-SY5Y cells, which overexpressed A53T mutant α-syn. Surprisingly, GK, but not GB, promoted the clearance of A53T α-syn, which can be abolished by autophagy inhibitor 3-methyladenine, indicating that GK-induced autophagy intervened in the clearance of A53T α-syn. However, GK did not affect the NEDD4 that belongs to the ubiquitin ligase in the endosomal-lysosomal pathway. Furthermore, GK treatment inhibited the p-NF-kB/p65 and induced the PI3K, BDNF, and PSD-95. Taken together, GK increased the clearance of α-syn, reduced cell death, and triggered complex crosstalk between different signaling pathways. Although our results show a potentially new therapeutic candidate for PD, the details of this mechanism need to be further identified.

\u03b1Synuclein control of mitochondrial homeostasis in human-derived neurons is disrupted by mutations associated with Parkinson\u2019s disease

The etiology of Parkinson’s disease (PD) converges on a common pathogenic pathway of mitochondrial defects in which α-Synuclein (αSyn) is thought to play a role. However, the mechanisms by which αSyn and its disease-associated allelic variants cause mitochondrial dysfunction remain unknown. Here, we analyzed mitochondrial axonal transport and morphology in human-derived neurons overexpressing wild-type (WT) αSyn or the mutated variants A30P or A53T, which are known to have differential lipid affinities. A53T αSyn was enriched in mitochondrial fractions, inducing significant mitochondrial transport defects and fragmentation, while milder defects were elicited by WT and A30P. We found that αSyn-mediated mitochondrial fragmentation was linked to expression levels in WT and A53T variants. Targeted delivery of WT and A53T αSyn to the outer mitochondrial membrane further increased fragmentation, whereas A30P did not. Genomic editing to disrupt the N-terminal domain of αSyn, which is important for membrane association, resulted in mitochondrial elongation without changes in fusion-fission protein levels, suggesting that αSyn plays a direct physiological role in mitochondrial size maintenance. Thus, we demonstrate that the association of αSyn with the mitochondria, which is modulated by protein mutation and dosage, influences mitochondrial transport and morphology, highlighting its relevance in a common pathway impaired in PD.

Alpha-Synuclein Suppresses Retinoic Acid-Induced Neuronal Differentiation by Targeting the Glycogen Synthase Kinase-3β/β-Catenin Signaling Pathway

Alpha-synuclein (α-SYN) is expressed during neuronal development and is mainly involved in the modulation of synaptic transmission. Missense mutations and amplifications of this gene have been associated with the pathogenesis of Parkinson's disease. Here, we evaluate whether α-SYN plays a detrimental role in the phenotypic and morphological regulation of neurons. We also identify the underlying mechanisms of this process in all-trans-retinoic acid (RA)-induced differentiated SH-SY5Y cells, which represents dopaminergic (DAergic) phenotype. Our results indicate that overexpression of wild-type or mutant A53T α-SYN attenuated the RA-induced upregulation of tyrosine hydroxylase and dopamine transporter as well as neurite outgrowth in SH-SY5Y cells. In addition, GSK-3β inactivation and downstream β-catenin stabilization were associated with RA-induced differentiation, which was attenuated by α-SYN. Moreover, protein phosphatase 2A was positively regulated by α-SYN and was implicated in the α-SYN-mediated interference with RA signaling. The results obtained from SH-SY5Y cells were verified in primary cultures of mesencephalic DAergic neurons from A53T α-SYN transgenic mice, which represent high levels of α-SYN and protein phosphatase 2A in the midbrain. The number and length of neurites in tyrosine hydroxylase-positive as well as Tau-positive cells from A53T α-SYN transgenic mice were significantly lower than those in littermate controls. The current results provide novel insight into the role of α-SYN in the regulation of neuronal differentiation, including DAergic neurons. Identifying the signaling pathway involved in the α-SYN-mediated dysregulation of neuronal differentiation could lead to a better understanding of the developmental processes underlying α-SYN-related pathologies and facilitate the discovery of specifically targeted therapeutics.

MPTP-induced vulnerability of dopamine neurons in A53T α-synuclein overexpressed mice with the potential involvement of DJ-1 downregulation.

Familial Parkinson's disease (PD) has been linked to point mutations and duplication of the α-synuclein (α-syn) gene. Mutant α-syn expression increases the vulnerability of neurons to exogenous insults. In this study, we developed a new PD model in the transgenic mice expressing mutant hemizygous (hemi) or homozygous (homo) A53T α-synuclein (α-syn Tg) and their wildtype (WT) littermates by treatment with sub-toxic (10 mg/kg, i.p., daily for 5 days) or toxic (30 mg/kg, i.p., daily for 5 days) dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Tyrosine hydroxylase and Bcl-2 levels were reduced in the α-syn Tg but not WT mice by sub-toxic MPTP injection. In the adhesive removal test, time to remove paper was significantly increased only in the homo α-syn Tg mice. In the challenging beam test, the hemi and homo α-syn Tg mice spent significantly longer time to traverse as compared to that of WT group. In order to find out responsible proteins related with vulnerability of mutant α-syn expressed neurons, DJ-1 and ubiquitin enzyme expressions were examined. In the SN, DJ-1 and ubiquitin conjugating enzyme, UBE2N, levels were significantly decreased in the α-syn Tg mice. Moreover, A53T α-syn overexpression decreased DJ-1 expression in SH-SY5Y cells. These findings suggest that the vulnerability to oxidative injury such as MPTP of A53T α-syn mice can be explained by downregulation of DJ-1.

Extracts from two ubiquitous Mediterranean plants ameliorate cellular and animal models of neurodegenerative proteinopathies

A signature feature of age-related neurodegenerative proteinopathies is the misfolding and aggregation of proteins, typically amyloid-β (Aβ) in Alzheimer’s disease (AD) and α-synuclein (α-syn) in Parkinson’s disease (PD), into soluble oligomeric structures that are highly neurotoxic. Cellular and animal models that faithfully replicate the hallmark features of these disorders are being increasing exploited to identify disease-modifying compounds. Natural compounds have been identified as a useful source of bioactive molecules with promising neuroprotective capabilities. In the present report, we investigated whether extracts derived from two ubiquitous Mediterranean plants namely, the prickly pear Opuntia ficus-indica (EOFI) and the brown alga Padina pavonica (EPP) alleviate neurodegenerative phenotypes in yeast (Saccharomyces cerevisiae) and fly (Drosophila melanogaster) models of AD and PD. Pre-treatment with EPP or EOFI in the culture medium significantly improved the viability of yeast expressing the Arctic Aβ42 (E22G) mutant. Supplementing food with EOFI or EPP dramatically ameliorated lifespan and behavioural signs of flies with brain-specific expression of wild-type Aβ42 (model of late-onset AD) or the Arctic Aβ42 variant (model of early-onset AD). Additionally, we show that either extract prolonged the survival of a PD fly model based on transgenic expression of the human α-syn A53T mutant. Taken together, our findings suggest that the plant-derived extracts interfere with shared mechanisms of neurodegeneration in AD and PD. This notion is strengthened by evidence demonstrating that EOFI and to a greater extent EPP, while strongly inhibiting the fibrillogenesis of both Aβ42 and α-syn, accumulate remodelled oligomeric aggregates that are less effective at disrupting lipid membrane integrity. Our work therefore opens new avenues for developing therapeutic applications of these natural plant extracts in the treatment of amyloidogenic neurodegenerative disorders.

Bee venom phospholipase A2 ameliorates motor dysfunction and modulates microglia activation in Parkinson's disease alpha-synuclein transgenic mice.

α-Synuclein (α-Syn) has a critical role in microglia-mediated neuroinflammation, which leads to the development of Parkinson's disease (PD). Recent studies have shown that bee venom (BV) has beneficial effects on PD symptoms in human patients or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxin-induced PD mice. This study investigated whether treatment with BV-derived phospholipase A2 (bvPLA2) would improve the motor dysfunction and pathological features of PD in human A53T α-Syn mutant transgenic (A53T Tg) mice. The motor dysfunction of A53T Tg mice was assessed using the pole test. The levels of α-Syn, microglia and the M1/M2 phenotype in the spinal cord were evaluated by immunofluorescence. bvPLA2 treatment significantly ameliorated motor dysfunction in A53T Tg mice. In addition, bvPLA2 significantly reduced the expression of α-Syn, the activation and numbers of microglia, and the ratio of M1/M2 in A53T Tg mice. These results suggest that bvPLA2 could be a promising treatment option for PD.

Aberrant adenosine A2A receptor signaling contributes to neurodegeneration and cognitive impairments in a mouse model of synucleinopathy

Synucleinopathy is characterized by abnormal accumulation of misfolded α-synuclein (α-Syn)-positive cytoplasmic inclusions and by neurodegeneration and cognitive impairments, but the pathogenesis mechanism of synucleinopathy remains to be defined. Using a transmission model of synucleinopathy by intracerebral injection of preformed A53T α-Syn fibrils, we investigated whether aberrant adenosine A2A receptor (A2AR) signaling contributed to pathogenesis of synucleinopathy. We demonstrated that intra-hippocampal injection of preformed mutant α-Syn fibrils triggered a striking and selective induction of A2AR expression which was closely co-localized with pSer129 α-Syn-rich inclusions in neurons and glial cells of hippocampus. Importantly, by abolishing aberrant A2AR signaling triggered by mutant α-Syn, genetic deletion of A2ARs blunted a cascade of pathological events leading to synucleinopathy, including pSer129 α-Syn-rich and p62-positive aggregates, NF-κB activation and astrogliosis, apoptotic neuronal cell death and working memory deficits without affecting motor activity. These findings define α-Syn-triggered aberrant A2AR signaling as a critical pathogenesis mechanism of synucleinopathy with dual controls of cognition and neurodegeneration by modulating α-Syn aggregates. Thus, aberrant A2AR signaling represents a useful biomarker as well as a therapeutic target of synucleinopathy.

α-Synuclein Mutation Inhibits Endocytosis at Mammalian Central Nerve Terminals.

α-Synuclein (α-syn) missense or multiplication mutations may cause neurodegenerative diseases, such as Parkinson's disease and dementia with Lewy bodies. The initial impact of α-syn mutations before neuronal loss is impairment of exocytosis, which may contribute to eventual neurodegeneration. The mechanism underlying impairment of exocytosis is poorly understood. Here we report that an α-syn mutant, the human α-syn A53T, inhibited two of the most commonly observed forms of endocytosis, slow and rapid endocytosis, at a mammalian central synapse. We also found that α-syn A53T inhibited vesicle replenishment to the readily releasable pool. These results may contribute to accounting for the widely observed early synaptic impairment caused by α-syn mutations in the progression toward neurodegeneration.

Effects of Serine 129 Phosphorylation on α-Synuclein Aggregation, Membrane Association, and Internalization

Although trace levels of phosphorylated α-synuclein (α-syn) are detectable in normal brains, nearly all α-syn accumulated within Lewy bodies in Parkinson disease brains is phosphorylated on serine 129 (Ser-129). The role of the phosphoserine residue and its effects on α-syn structure, function, and intracellular accumulation are poorly understood. Here, co-expression of α-syn and polo-like kinase 2 (PLK2), a kinase that targets Ser-129, was used to generate phosphorylated α-syn for biophysical and biological characterization. Misfolding and fibril formation of phosphorylated α-syn isoforms were detected earlier, although the fibrils remained phosphatase- and protease-sensitive. Membrane binding of α-syn monomers was differentially affected by phosphorylation depending on the Parkinson disease-linked mutation. WT α-syn binding to presynaptic membranes was not affected by phosphorylation, whereas A30P α-syn binding was greatly increased, and A53T α-syn was slightly lower, implicating distal effects of the carboxyl- on amino-terminal membrane binding. Endocytic vesicle-mediated internalization of pre-formed fibrils into non-neuronal cells and dopaminergic neurons matched the efficacy of α-syn membrane binding. Finally, the disruption of internalized vesicle membranes was enhanced by the phosphorylated α-syn isoforms, a potential means for misfolded extracellular or lumenal α-syn to access cytosolic α-syn. Our results suggest that the threshold for vesicle permeabilization is evident even at low levels of α-syn internalization and are relevant to therapeutic strategies to reduce intercellular propagation of α-syn misfolding.

Fasudil, a Rho kinase inhibitor, promotes the autophagic degradation of A53T α-synuclein by activating the JNK 1/Bcl-2/beclin 1 pathway

Accumulation of α-synuclein (α-syn) is pivotally implicated in the pathogenesis of Parkinson׳s disease (PD), and enhancing its clearance might be a promising strategy in PD treatment. It has recently been shown that Rho kinase (ROCK) activation is involved in many neurodegenerative diseases, and some ROCK inhibitors might promote the degradation of abnormal protein aggregates. However, it is not known if fasudil, the only ROCK inhibitor available in clinical setting, could promote the degradation of α-syn, and ameliorate the α-syn induced neurotoxicity. In this regard, we investigated the effect of fasudil on neurite injury caused by A53T α-syn overexpression and the implicated pathway it might mediate. In the current study, we found that under the condition of A53T α-syn overexpression, the neurite outgrowth decreased significantly with the increasing expression of ROCK2. Fasudil, the ROCK inhibitor, ameliorated such neurotoxicity and promoted the clearance of A53T α-syn. Its underlying mechanism was supported by that fasudil could increase the macroautophagy activation via JNK 1 and Bcl-2 phosphorylation and beclin 1/Vps34 complex formation. Taken together, fasudil might be able to provide a novel and promising strategy for PD treatment by enhancing α-syn clearance and activating the JNK 1/Bcl-2/beclin 1 pathway.