Wild-type Α-synuclein Research Articles

LMK235, a small molecule inhibitor of HDAC4/5, protects dopaminergic neurons against neurotoxin- and α-synuclein-induced degeneration in cellular models of Parkinson's disease

Epigenetic modifications in neurodegenerative disease are under investigation for their roles in disease progression. Alterations in acetylation rates of certain Parkinson's disease (PD)-linked genes have been associated with the pathological progression of this disorder. In light of this, and given the lack of disease-modifying therapies for PD, HDAC inhibitors (HDIs) are under consideration as potential pharmacological agents. The neuroprotective effects of pan-HDACs and some class-specific inhibitors have been tested in in vivo and in vitro models of PD, with varying outcomes. Here we used gene co-expression analysis to identify HDACs that are associated with human dopaminergic (DA) neuron development. We identified HDAC3, HDAC5, HDAC6 and HDAC9 as being highly correlated with the DA markers, SLC6A3 and NR4A2. RT-qPCR revealed that mRNA expression of these HDACs exhibited similar temporal profiles during embryonic mouse midbrain DA (mDA) neuron development. We tested the neuroprotective potential of a number of class-specific small molecule HDIs on human SH-SY5Y cells, using neurite growth as a phenotypic readout of neurotrophic action. Neither the class I-specific HDIs, RGFP109 and RGFP966, nor the HDAC6 inhibitor ACY1215, had significant effects on neurite outgrowth. However, the class IIa HDI, LMK235 (a HDAC4/5 inhibitor), significantly increased histone acetylation and neurite outgrowth. We found that LMK235 increased BMP-Smad-dependent transcription in SH-SY5Y cells and that this was required for its neurite growth-promoting effects on SH-SY5Y cells and on DA neurons in primary cultures of embryonic day (E) 14 rat ventral mesencephalon (VM). These effects were also seen in SH-SY5Y cells transfected with HDAC5 siRNA. Furthermore, LMK235 treatment exerted neuroprotective effects against degeneration induced by the DA neurotoxin 1-methyl-4-phenylpyridinium (MPP+), in both SH-SY5Y cells and cultured DA neurons. Treatment with LMK235 was also neuroprotective against axonal degeneration induced by overexpression of wild-type (WT) or A53T mutant α-synuclein in both SH-SY5Y cells and primary cultures of DA neurons. In summary, these data show the neuroprotective potential of the class IIa HDI, LMK235, in cell models of relevance to PD.

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson's diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

No Detectable Effect on Visual Responses Using Functional MRI in a Rodent Model of α-Synuclein Expression.

Parkinson’s disease (PD) is a progressive neurodegenerative disease that is typically diagnosed late in its progression. There is a need for biomarkers suitable for monitoring the disease progression at earlier stages to guide the development of novel neuroprotective therapies. One potential biomarker, α-synuclein, has been found in both the familial cases of PD, as well as the sporadic cases and is considered a key feature of PD. α-synuclein is naturally present in the retina, and it has been suggested that early symptoms of the visual system may be used as a biomarker for PD. Here, we use a viral vector to induce a unilateral expression of human wild-type α-synuclein in rats as a mechanistic model of protein aggregation in PD. We employed functional magnetic resonance imaging (fMRI) to investigate whether adeno-associated virus (AAV) mediated expression of human wild-type α-synuclein alter functional activity in the visual system. A total of 16 rats were injected with either AAV-α-synuclein (n = 7) or AAV-null (n = 9) in the substantia nigra pars compacta (SNc) of the left hemisphere. The expression of α-synuclein was validated by a motor assay and postmortem immunohistochemistry. Five months after the introduction of the AAV-vector, fMRI showed robust blood oxygen level-dependent (BOLD) responses to light stimulation in the visual systems of both control and AAV-α-synuclein animals. However, our results demonstrate that the expression of AAV-α-synuclein does not affect functional activation of the visual system. This negative finding suggests that fMRI-based read-outs of visual responses may not be a sensitive biomarker for PD.

MMP13 Expression Is Increased Following Mutant α-Synuclein Exposure and Promotes Inflammatory Responses in Microglia.

α-Synuclein is a 140-amino acid protein that readily misfolds and is associated with the Lewy body pathology found in sporadic and genetic forms of Parkinson's disease. We and others have shown that wild-type α-synuclein is a damage-associated molecular pattern that directly elicits a proinflammatory response in microglia through toll-like receptor activation. Here we investigated the direct effect of oligomeric mutant α-synuclein (A53T) on microglia morphology and activation. We found that misfolded A53T increased quantitative measures of amoeboid cell morphology, NFκB nuclear translocation and the expression of prototypical proinflammatory molecules. We also demonstrated that A53T increased expression of MMP13, a matrix metalloproteinase that remodels the extracellular matrix. To better understand the role of MMP13 in synucleinopathies, we further characterized the role of MMP13 in microglial signaling. We showed exposure of microglia to MMP13 induced a change in morphology and promoted the release of TNFα and MMP9. Notably, IL1β was not released indicating that the pathway involved in MMP13 activation of microglia may be different than the A53T pathway. Lastly, MMP13 increased the expression of CD68 suggesting that the lysosomal pathway might be altered by this MMP. Taken together this study shows that mutant α-synuclein directly induces a proinflammatory phenotype in microglia, which includes the expression of MMP13. In turn, MMP13 directly alters microglia supporting the need for multi-target therapies to treat Parkinson's disease patients.

Polyphenol Honokiol and Flavone 2′,3′,4′-Trihydroxyflavone Differentially Interact with α-Synuclein at Distinct Phases of Aggregation

The association between protein aggregation and neurodegenerative diseases such as Parkinson's disease continues to be well interrogated but poorly elucidated at a mechanistic level. Nevertheless, the formation of amyloid fibrils from the destabilization and misfolding of native proteins is a molecular hallmark of disease. Consequently, there is ongoing demand for the identification and development of small molecules which prevent fibril formation. This study comprehensively assesses the inhibitory properties of two small molecules, the lignan polyphenol honokiol and the flavonoid 2',3',4'-trihydroxyflavone, in preventing α-synuclein fibrilization. The data shows that honokiol does not prevent α-synuclein fibril elongation, while 2',3',4'-trihydroxyflavone is effective at inhibiting fibril elongation and induces oligomer formation (for both wild-type α-synuclein and the disease-associated A53T mutation). Moreover, the exposed hydrophobicity of α-synuclein fibrils is reduced in the presence of 2',3',4'-trihydroxyflavone, whereas the addition of honokiol did not reduce the hydrophobicity of fibrils. In addition, ion mobility-mass spectrometry revealed that the conformation of α-synuclein wild-type and A53T monomers after disassembly is restored to a nonaggregation-prone state upon 2',3',4'-trihydroxyflavone treatment. Collectively, this study shows that the mechanisms by which these polyphenols and flavonoids prevent fibril formation are distinct by their interactions at various phases of the fibril-forming pathway. Furthermore, this study highlights how thorough biophysical interrogation of the interaction is required for understanding the ability of inhibitors to prevent protein aggregation associated with disease.

Increased telomerase improves motor function and alpha-synuclein pathology in a transgenic mouse model of Parkinson's disease associated with enhanced autophagy.

Protective effects of the telomerase protein TERT have been shown in neurons and brain. We previously demonstrated that TERT protein can accumulate in mitochondria of Alzheimer's disease (AD) brains and protect from pathological tau in primary mouse neurons. This prompted us to employ telomerase activators in order to boost telomerase expression in a mouse model of Parkinson's disease (PD) overexpressing human wild type α-synuclein. Our aim was to test whether increased Tert expression levels were able to ameliorate PD symptoms and to activate protein degradation. We found increased Tert expression in brain for both activators which correlated with a substantial improvement of motor functions such as gait and motor coordination while telomere length in the analysed region was not changed. Interestingly, only one activator (TA-65) resulted in a decrease of reactive oxygen species from brain mitochondria. Importantly, we demonstrate that total, phosphorylated and aggregated α-synuclein were significantly decreased in the hippocampus and neocortex of activator-treated mice corresponding to enhanced markers of autophagy suggesting an improved degradation of toxic alpha-synuclein. We conclude that increased Tert expression caused by telomerase activators is associated with decreased α-synuclein protein levels either by activating autophagy or by preventing or delaying impairment of degradation mechanisms during disease progression. This encouraging preclinical data could be translated into novel therapeutic options for neurodegenerative disorders such as PD.

Transient transfection of WT-αS and A53T-αS brought about a mild apoptosis due to degradation of released cytochrome c through PARC

α-Synuclein (αS) aggregates plays a pivotal role in the pathogenesis of synucleinopathies including Parkinson's Disease. The toxicity of αS aggregates has been broadly studied and variant defects have been reported through which these aggregates lead in cell death. Although cell death through apoptosis pathway has been proposed in many studies, the molecular details underlying in this pathway have not been uncovered. To shed a light on the relationships between αS aggregates and apoptotic cell death, changes in levels and behavior of molecular indicators of the intrinsic apoptotic pathway was investigated in HEK-293T cells overexpressing wild-type α-synuclein and A53T-α-synuclein. Overexpression of both WT-αS and A53T-αS resulted in the increase of caspase-9 activity, and rise in Cytochrome c (Cyt c) and PARC content, concurrently. We assume that rising in PARC level may result in Cyt c degradation, and consequently suppressing/attenuating intrinsic apoptosis pathway. Besides, increasing of Casp-9 activity can be related to αS aggregates and subsequent degradation of Cyt c. To understand the mechanisms behind this using theoretical model, molecular dynamic simulation was also applied to investigate the possible interaction of Casp-9 with α-synuclein aggregates. The results showed that the interaction between Casp-9 with αS aggregates could activate Casp-9 by changing the conformation of some crucial residues.

Rapid Structural, Kinetic, and Immunochemical Analysis of Alpha-Synuclein Oligomers in Solution.

Oligomers comprised of misfolded proteins are implicated as neurotoxins in the pathogenesis of protein misfolding conditions such as Parkinson's and Alzheimer's diseases. Structural, biophysical, and biochemical characterization of these nanoscale protein assemblies is key to understanding their pathology and the design of therapeutic interventions, yet it is challenging due to their heterogeneous, transient nature and low relative abundance in complex mixtures. Here, we demonstrate separation of heterogeneous populations of oligomeric α-synuclein, a protein central to the pathology of Parkinson's disease, in solution using microfluidic free-flow electrophoresis. We characterize nanoscale structural heterogeneity of transient oligomers on a time scale of seconds, at least 2 orders of magnitude faster than conventional techniques. Furthermore, we utilize our platform to analyze oligomer ζ-potential and probe the immunochemistry of wild-type α-synuclein oligomers. Our findings contribute to an improved characterization of α-synuclein oligomers and demonstrate the application of microchip electrophoresis for the free-solution analysis of biological nanoparticle analytes.

Inhibition of α-Synuclein Aggregation and Mature Fibril Disassembling With a Minimalistic Compound, ZPDm.

Synucleinopathies are a group of disorders characterized by the accumulation of α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α-Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson’s Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities.

Changes in the cellular fatty acid profile drive the proteasomal degradation of α-synuclein and enhance neuronal survival.

Parkinson's disease is biochemically characterized by the deposition of aberrant aggregated α-synuclein in the affected neurons. The aggregation properties of α-synuclein greatly depend on its affinity to bind cellular membranes via a dynamic interaction with specific lipid moieties. In particular, α-synuclein can interact with arachidonic acid (AA), a polyunsaturated fatty acid, in a manner that promotes the formation of α-helix enriched assemblies. In a cellular context, AA is released from membrane phospholipids by phospholipase A2 (PLA2 ). To investigate the impact of PLA2 activity on α-synuclein aggregation, we have applied selective PLA2 inhibitors to a SH-SY5Y cellular model where the expression of human wild-type α-synuclein is correlated with a gradual accumulation of soluble oligomers and subsequent cell death. We have found that pharmacological and genetic inhibition of GIVA cPLA2 resulted in a dramatic decrease of intracellular oligomeric and monomeric α-synuclein significantly promoting cell survival. Our data suggest that alterations in the levels of free fatty acids, and especially AA and adrenic acid, promote the formation of α-synuclein conformers which are more susceptible to proteasomal degradation. This mechanism is active only in living cells and is generic since it does not depend on the absolute quantity of α-synuclein, the presence of disease-linked point mutations, the expression system or the type of cells. Our findings indicate that the α-synuclein-fatty acid interaction can be a critical determinant of the conformation and fate of α-synuclein in the cell interior and, as such, cPLA2 inhibitors could serve to alleviate the intracellular, potentially pathological, α-synuclein burden.

The effect of copy number on α-synuclein's toxicity and its protective role in Bax-induced apoptosis, in yeast.

Apoptosis is a form of programmed cell death which is essential for the growth of dividing human cells whereas, in contrast, it is deleterious for post-mitotic cells such as neurons. Bax and α-synuclein are two human proteins which play a role in the induction of neuronal apoptosis in neurodegenerative diseases like Alzheimer’s and Parkinson’s. Human Bax and α-synuclein also induce cell death when expressed in baker’s yeast, Saccharomyces cerevisiae. Quite unexpectedly, the human α-synuclein gene had been identified as an inhibitor of pro-apoptotic Bax using a yeast-based screen of a human hippocampal cDNA library. Plasmids were constructed with different promoters, which allow expression of wildtype and Parkinson’s disease (PD)-related mutant α-synuclein genes, from (i) multi-copy 2µ (episomal) plasmids and (ii) integrative plasmids that compel expression of genes from chromosomal sites in varying copy numbers (1–3). All α-synuclein-containing plasmids were introduced, through transformation, into a yeast strain which already contained a chromosomally integrated copy of Bax. It is for the first time that it was observed that, depending on gene dosage, only wildtype α-synuclein is anti-apoptotic while mutant α-synuclein is not. The results also indicate that wildtype α-synuclein has a remarkable ability to manifest two contrasting effects depending on its level of expression: (i) normally, it would negate apoptosis but (ii) when overexpressed, it tends to induce apoptosis which is probably what happens in PD.

Comprehensive Structural and Thermodynamic Analysis of Prefibrillar WT α-Synuclein and Its G51D, E46K, and A53T Mutants by a Combination of Small-Angle X-ray Scattering and Variational Bayesian Weighting.

The in solution synchrotron small-angle X-ray scattering SAXS technique has been used to investigate an intrinsically disordered protein (IDP) related to Parkinson’s disease, the α-synuclein (α-syn), in prefibrillar diluted conditions. SAXS experiments have been performed as a function of temperature and concentration on the wild type (WT) and on the three pathogenic mutants G51D, E46K, and A53T. To identify the conformers that populate WT α-syn and the pathogenic mutants in prefibrillar conditions, scattering data have been analyzed by a new variational bayesian weighting method (VBWSAS) based on an ensemble of conformers, which includes unfolded monomers, trimers, and tetramers, both in helical-rich and strand-rich forms. The developed VBWSAS method uses a thermodynamic scheme to account for temperature and concentration effects and considers long-range protein–protein interactions in the framework of the random phase approximation. The global analysis of the whole set of data indicates that WT α-syn is mostly present as unfolded monomers and trimers (helical-rich trimers at low T and strand-rich trimers at high T), but not tetramers, as previously derived by several studies. On the contrary, different conformer combinations characterize mutants. In the α-syn G51D mutant, the most abundant aggregates at all the temperatures are strand-rich tetramers. Strand-rich tetramers are also the predominant forms in the A53T mutant, but their weight decreases with temperature. Only monomeric conformers, with a preference for the ones with the smallest sizes, are present in the E46K mutant. The derived conformational behavior then suggests a different availability of species prone to aggregate, depending on mutation, temperature, and concentration and accounting for the different neurotoxicity of α-syn variants. Indeed, this approach may be of pivotal importance to describe conformational and aggregational properties of other IDPs.

Anti-α-synuclein ASO delivered to monoamine neurons prevents α-synuclein accumulation in a Parkinson's disease-like mouse model and in monkeys.

BackgroundProgressive neuronal death in monoaminergic nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Given that α-synuclein may be an early mediator of the pathological cascade that ultimately leads to neurodegeneration, decreased α-synuclein synthesis will abate neurotoxicity if delivered to the key affected neurons.MethodsWe used a non-viral gene therapy based on a new indatraline-conjugated antisense oligonucleotide (IND-ASO) to disrupt the α-synuclein mRNA transcription selectively in monoamine neurons of a PD-like mouse model and elderly nonhuman primates. Molecular, cell biology, histological, neurochemical and behavioral assays were performed.FindingsIntracerebroventricular and intranasal IND-ASO administration for four weeks in a mouse model with AAV-mediated wild-type human α-synuclein overexpression in dopamine neurons prevented the synthesis and accumulation of α-synuclein in the connected brain regions, improving dopamine neurotransmission. Likewise, the four-week IND-ASO treatment led to decreased levels of endogenous α-synuclein protein in the midbrain monoamine nuclei of nonhuman primates, which are affected early in PD.Conclusions: The inhibition of α-synuclein production in dopamine neurons and its accumulation in cortical/striatal projection areas may alleviate the early deficits of dopamine function, showing the high translational value of antisense oligonucleotides as a disease modifying therapy for PD and related synucleinopathies.FundingGrants SAF2016-75797-R, RTC-2014-2812-1 and RTC-2015-3309-1, Ministry of Economy and Competitiveness (MINECO) and European Regional Development Fund (ERDF), UE; Grant ID 9238, Michael J. Fox Foundation; and Centres for Networked Biomedical Research on Mental Health (CIBERSAM), and on Neurodegenerative Diseases (CIBERNED).

Self- and Cross-Seeding on α-Synuclein Fibril Growth Kinetics and Structure Observed by High-Speed Atomic Force Microscopy.

Fibril formation is an obligatory process in amyloid diseases and is characterized by nucleation and elongation phases that result in the formation of long filaments with cross-β sheet structure. The kinetics of this process, as well as that of secondary nucleation, is controlled by a variety of factors, including nucleus (seed) structure, monomer conformation, and biochemical milieu. Some fibrillar amyloid assemblies act as prions, replicating themselves from protein monomers templated by existing prion seeds. Prion strains, which are characterized by distinct physicochemical and pathologic properties, may also form due to perturbation of the templating process within the susceptible organism. Understanding the types and effects of perturbations occurring during the development and progression of Parkinson's disease is an area requiring more study. Here, we used high-speed atomic force microscopy to determine the kinetics and structural dynamics of α-synuclein fibril elongation initiated by self-seeding or cross-seeding of wild-type (WT) or mutant α-synuclein with WT or mutant α-synuclein seeds. We found that cross-seeding modulated not only elongation rates but also the structures of the growing fibrils. Some fibrils produced in this manner had structures distinct from their "parent" seeds. In other cases, cross-seeding was not observed at all. These findings suggest that α-synuclein sequence variants can produce different types of strains by self- or cross-seeding. Perpetuation of specific strains then would depend on the relative rates of fibril growth and the relative stabilities of the fibrils formed by each strain.

α-Synuclein-112 Impairs Synaptic Vesicle Recycling Consistent With Its Enhanced Membrane Binding Properties.

Synucleinopathies are neurological disorders associated with α-synuclein overexpression and aggregation. While it is well-established that overexpression of wild type α-synuclein (α-syn-140) leads to cellular toxicity and neurodegeneration, much less is known about other naturally occurring α-synuclein splice isoforms. In this study we provide the first detailed examination of the synaptic effects caused by one of these splice isoforms, α-synuclein-112 (α-syn-112). α-Syn-112 is produced by an in-frame excision of exon 5, resulting in deletion of amino acids 103–130 in the C-terminal region. α-Syn-112 is upregulated in the substantia nigra, frontal cortex, and cerebellum of parkinsonian brains and higher expression levels are correlated with susceptibility to Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). We report here that α-syn-112 binds strongly to anionic phospholipids when presented in highly curved liposomes, similar to α-syn-140. However, α-syn-112 bound significantly stronger to all phospholipids tested, including the phosphoinositides. α-Syn-112 also dimerized and trimerized on isolated synaptic membranes, while α-syn-140 remained largely monomeric. When introduced acutely to lamprey synapses, α-syn-112 robustly inhibited synaptic vesicle recycling. Interestingly, α-syn-112 produced effects on the plasma membrane and clathrin-mediated synaptic vesicle endocytosis that were phenotypically intermediate between those caused by monomeric and dimeric α-syn-140. These findings indicate that α-syn-112 exhibits enhanced phospholipid binding and oligomerization in vitro and consequently interferes with synaptic vesicle recycling in vivo in ways that are consistent with its biochemical properties. This study provides additional evidence suggesting that impaired vesicle endocytosis is a cellular target of excess α-synuclein and advances our understanding of potential mechanisms underlying disease pathogenesis in the synucleinopathies.

Ferulic Acid Ameliorates MPP+/MPTP-Induced Oxidative Stress via ERK1/2-Dependent Nrf2 Activation: Translational Implications for Parkinson Disease Treatment.

Parkinson's disease (PD) is a neurodegenerative disorder closely associated with oxidative stress. The biochemical and cellular alterations that occur after cell and mouse treatment with the parkinsonism-inducing neurotoxin MPP+/MPTP are remarkably similar to those observed in idiopathic PD. Previously, we showed that ferulic acid (FA) has antioxidant properties and the ability to activate nuclear factor E2-related factor 2 (Nrf2). The present study tested the hypothesis that FA attenuates MPP+/MPTP-induced oxidative stress by regulating crosstalk between sirtuin 2 (SIRT2) and Nrf2 pathways. To test this hypothesis, we performed in vitro and in vivo studies using MPP+/MPTP-challenged SH-SY5Y cells or mice treated with or not with FA. FA marginally inhibited SIRT2 in parallel with α-synuclein at levels of transcription and translation in SH-SY5Y cells challenged with MPP+. Moreover, FA attenuated MPP+-induced oxidative stress, as indicated by reactive oxygen species, lipid hydroperoxides, GSH/GSSG ratio, and NAD+/NADH ratio. Mechanistically, FA strongly upregulated the glutamate cysteine ligase catalytic subunit and heme oxygenase-1 expression at the levels of transcription and translation. Interestingly, FA-mediated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation contributed to nuclear accumulation of Nrf2 via de novo synthesis, which was validated by the use of dominant negative ERK2. Surprisingly, activation of the ERK1/2 and inhibition of SIRT2 by FA are mediated by independent mechanisms. Furthermore, FA ameliorated motor deficits and oxidative stress in the ventral midbrain in MPTP-treated (25mg/kg, i.p., daily for 5days) wild-type mice and α-synuclein knockout mice, but not in Nrf2 knockout mice. Collectively, FA exerts antioxidant effects through ERK1/2-mediated activation of the Nrf2 pathway, and these results may have important translational value for the treatment of PD.

α-Synuclein Overexpression in SH-SY5Y Human Neuroblastoma Cells Leads to the Accumulation of Thioflavin S-positive Aggregates and Impairment of Glycolysis.

Deterioration of energy metabolism in affected cells is an important feature of synucleinopathies, including Parkinson's disease. Here, we studied the association between α-synuclein accumulation and glycolysis using SH-SY5Y neuroblastoma cell lines stably expressing wild-type α-synuclein or its A53T mutant linked to the autosomal dominant form of the disease. Overexpression of both proteins led to the accumulation of thioflavin S-positive aggregates, more pronounced for α-synuclein A53T. It also caused changes in the cell energy metabolism manifested as a decrease in the lactate accumulation and glucose uptake. Impairments in glycolysis were also accompanied by a decrease in the activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In vitro experiments with purified proteins indicated that GAPDH inactivation might be caused by its binding to the monomeric and oligomeric forms of α-synuclein. Therefore, a decrease in the GAPDH activity induced by its interaction with α-synuclein, might be one of the causes of glucose metabolism deterioration in synucleinopathies.

Ultrasensitive RT-QuIC assay with high sensitivity and specificity for Lewy body-associated synucleinopathies

The clinical diagnosis of synucleinopathies, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA), is challenging, especially at an early disease stage, due to the heterogeneous and often non-specific clinical manifestations. The discovery of reliable specific markers for synucleinopathies would consequently be of great aid to the diagnosis and management of these disorders. Real-Time Quaking-Induced Conversion (RT-QuIC) is an ultrasensitive technique that has been previously used to detect self-templating amyloidogenic proteins in the cerebrospinal fluid (CSF) and other biospecimens in prion disease and synucleinopathies. Using a wild-type recombinant α-synuclein as a substrate, we applied RT-QuIC to a large cohort of 439 CSF samples from clinically well-characterized, or post-mortem verified patients with parkinsonism or dementia. Of significance, we also studied patients with isolated REM sleep behavior disorder (iRBD) (n = 18) and pure autonomic failure (PAF) (n = 28), representing clinical syndromes that are often caused by a synucleinopathy, and may precede the appearance of parkinsonism or cognitive decline. The results show that our RT-QuIC assay can accurately detect α-synuclein seeding activity across the spectrum of Lewy Body (LB)-related disorders (LBD), including DLB, PD, iRBD, and PAF, with an overall sensitivity of 95.3%. In contrast, all but two patients with MSA showed no α-synuclein seeding activity in the applied experimental setting. The analysis of the fluorescence response reflecting the amount of α-synuclein seeds revealed no significant differences between the clinical syndromes associated with LB pathology. Finally, the assay demonstrated 98% specificity in a neuropathological cohort of 101 cases lacking LB pathology. In conclusion, α-synuclein RT-QuIC provides an accurate marker of synucleinopathies linked to LB pathology and may have a pivotal role in the early discrimination and management of affected patients. The finding of no α-synuclein seeding activity in MSA seems to support the current view that MSA and LBD are associated with different conformational strains of α-synuclein.

Nilotinib Fails to Prevent Synucleinopathy and Cell Loss in a Mouse Model of Multiple System Atrophy.

Multiple system atrophy (MSA) is a rare, untreatable neurodegenerative disorder characterized by accumulation of α-synuclein in oligodendroglial inclusions. As such, MSA is a synucleinopathy along with Parkinson's disease (PD) and dementia with Lewy bodies. Activation of the abelson tyrosine kinase c-Abl leads to phosphorylation of α-synuclein at tyrosine 39, thereby promoting its aggregation and subsequent neurodegeneration. The c-Abl inhibitor nilotinib used for the treatment of chronic myeloid leukemia based on data collected in preclinical models of PD might interfere with pathogenic mechanisms that are relevant to PD and dementia with Lewy bodies, which motivated its assessment in an open-label clinical trial in PD and dementia with Lewy bodies patients. The objective of this study was to assess the preclinical efficacy of nilotinib in the specific context of MSA. Mice expressing human wild-type α-synuclein in oligodendrocytes received daily injection of nilotinib (1 or 10 mg/kg) over 12 weeks. Postmortem analysis included the assessment of c-Abl activation, α-synuclein burden, and dopaminergic neurodegeneration. α-Synuclein phosphorylated at tyrosine 39 was detected in glial cytoplasmic inclusions in MSA patients. Increased activation of c-Abl and α-synuclein phosphorylation at tyrosine 39 were found in transgenic mice. Despite significant inhibition of c-Abl and associated reduction of α-synuclein phosphorylation at tyrosine 39 by 40%, nilotinib failed to reduce α-synuclein aggregate burden (including phosphorylation at serine 129) in the striatum and cortex or to lessen neurodegeneration in the substantia nigra. This preclinical study suggests that partial inhibition of c-Abl and reduction of α-synuclein phosphorylation at tyrosine 39 may not be a relevant target for MSA. © 2020 International Parkinson and Movement Disorder Society.

Insight into Parkinson’s Disease from Yeasts: Combined Impact of Covalent Modifications and Familial Mutations on α‐Synuclein

Parkinson’s Disease (PD) is a neurodegenerative disorder linked to the loss of dopaminergic neurons in the midbrain. A key pathological marker of PD is the presence of Lewy bodies, which are composed of misfolded α‐synuclein protein. α‐Synuclein is highly post‐translationally modified in healthy and diseased states. While phosphorylation and nitration of α‐synuclein are well‐studied as contributors to PD pathology, less is known about SUMOylation, acetylation, and glycation. Also, the combined effects of these modifications remain largely unclear, on both wildtype α‐synuclein (linked with sporadic PD) and the six mutant forms (A30P, E46K, H50Q, G51D, A53T, A53E) linked with early‐onset familial PD. We first evaluated the effects of blocking SUMOylation on α‐synuclein in the well‐established budding yeast model for PD and found that α‐synuclein becomes more toxic and aggregated, losing its membrane localization. Second, we found that SUMOylation and phosphorylation counteract each other in toxicity and localization. Third, we expanded our investigation to two newly reported modifications – acetylation and glycation. We found that acetylation is protective and glycation is harmful. When we combined acetylation or glycation manipulations with SUMOylation and phosphorylation alterations on the α‐synuclein level, we found that the effects of these modifications are not additive – the impacts of acetylation and glycation depends on phosphorylation status. Furthermore, we found that decreased nitration protects against toxicity and reduces α‐syn aggregation in the mutants modifying SUMOylation and phosphorylation. Finally, we investigated the effects of the familial mutants in tandem with altered acetylation, glycation, or nitration. We report two familial mutant‐specific effects: H50Q is surprisingly sensitive and non‐toxic in a hyper‐acetylation environment, while A53E is highly toxic with hypo‐glycation. We also found that too much nitration exacerbates toxicity of the familial mutants relative to low nitration. Specifically, mutant A53T is highly sensitive to excess nitration while H50Q is non‐toxic in such environment. These studies show the relevance of covalent modifications in sporadic and familial PD, and their in‐tandem effects that underlie toxicity mechanisms.Support or Funding InformationYG= supported by Parkinson's foundation‐American Parkinson's disease association, A.B,C.M, A.R and P.J: Research grants from Neurosci