Sex-dimorphic neuroprotective effect of CD163 in an α-synuclein mouse model of Parkinson's disease.

Alpha-synuclein (α-syn) aggregation and immune activation represent hallmark pathological events in Parkinson's disease (PD). The PD-associated immune response encompasses both brain and peripheral immune cells, although little is known about the immune proteins relevant for such a response. We propose that the upregulation of CD163 observed in blood monocytes and in the responsive microglia in PD patients is a protective mechanism in the disease. To investigate this, we used the PD model based on intrastriatal injections of murine α-syn pre-formed fibrils in CD163 knockout (KO) mice and wild-type littermates. CD163KO females revealed an impaired and differential early immune response to α-syn pathology as revealed by immunohistochemical and transcriptomic analysis. After 6 months, CD163KO females showed an exacerbated immune response and α-syn pathology, which ultimately led to dopaminergic neurodegeneration of greater magnitude. These findings support a sex-dimorphic neuroprotective role for CD163 during α-syn-induced neurodegeneration.

Monomeric α-synuclein activates the plasma membrane calcium pump.

Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.

Protein kinase R dependent phosphorylation of α-synuclein regulates its membrane binding and aggregation.

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-syn's membrane binding region. This allows enhancement of the "double-anchor" vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation, and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from α-syn aggregate-related diseases.

Alpha-synuclein activates the classical complement pathway and mediates complement-dependent cell toxicity.

BACKGROUND: Synucleinopathies are characterized by neurodegeneration and deposition of the presynaptic protein α-synuclein in pathological protein inclusions. Growing evidence suggests the complement system not only has physiological functions in the central nervous system, but also is involved in mediating the pathological loss of synapses in Alzheimer's disease. However, it is not established whether the complement system has a similar role in the diseases Parkinson's disease, Dementia with Lewy bodies, and multiple system atrophy (MSA) that are associated with α-synuclein aggregate pathology. METHODS: To investigate if the complement system has a pathological role in synucleinopathies, we assessed the effect of the complement system on the viability of an α-synuclein expressing cell model and examined direct activation of the complement system by α-synuclein in a plate-based activation assay. Finally, we investigated the levels of the initiator of the classical pathway, C1q, in postmortem brain samples from MSA patients. RESULTS: We demonstrate that α-synuclein activates the classical complement pathway and mediates complement-dependent toxicity in α-synuclein expressing SH-SY5Y cells. The α-synuclein-dependent cellular toxicity was rescued by the complement inhibitors RaCI (inhibiting C5) and Cp20 (inhibiting C3). Furthermore, we observed a trend for higher levels of C1q in the putamen of MSA subjects than that of controls. CONCLUSION: α-Synuclein can activate the classical complement pathway, and the complement system is involved in α-synuclein-dependent cellular cytotoxicity suggesting the system could play a prodegenerative role in synucleinopathies.

Multiple system atrophy-associated oligodendroglial protein p25α stimulates formation of novel α-synuclein strain with enhanced neurodegenerative potential.

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

PKR kinase directly regulates tau expression and Alzheimer's disease-related tau phosphorylation.

Deposition of extensively hyperphosphorylated tau in specific brain cells is a clear pathological hallmark in Alzheimer's disease and a number of other neurodegenerative disorders, collectively termed the tauopathies. Furthermore, hyperphosphorylation of tau prevents it from fulfilling its physiological role as a microtubule-stabilizing protein and leaves it increasingly vulnerable to self-assembly, suggestive of a central underlying role of hyperphosphorylation as a contributing factor in the etiology of these diseases. Via in vitro phosphorylation and regulation of kinase activity within cells and acute brain tissue, we reveal that the inflammation associated kinase, protein kinase R (PKR), directly phosphorylates numerous abnormal and disease-modifying residues within tau including Thr181, Ser199/202, Thr231, Ser262, Ser396, Ser404 and Ser409. Similar to disease processes, these PKR-mediated phosphorylations actively displace tau from microtubules in cells. In addition, PKR overexpression and knockdown, respectively, increase and decrease tau protein and mRNA levels in cells. This regulation occurs independent of noncoding transcriptional elements, suggesting an underlying mechanism involving intra-exonic regulation of the tau-encoding microtubule-associated protein tau (MAPT) gene. Finally, acute encephalopathy in wild type mice, induced by intracranial Langat virus infection, results in robust inflammation and PKR upregulation accompanied by abnormally phosphorylated full-length- and truncated tau. These findings indicate that PKR, independent of other kinases and upon acute brain inflammation, is capable of triggering pathological modulation of tau, which, in turn, might form the initial pathologic seed in several tauopathies such as Alzheimer's disease and Chronic traumatic encephalopathy where inflammation is severe.

PET imaging reveals early and progressive dopaminergic deficits after intra-striatal injection of preformed alpha-synuclein fibrils in rats.

Alpha-synuclein (a-syn) can aggregate and form toxic oligomers and insoluble fibrils which are the main component of Lewy bodies. Intra-neuronal Lewy bodies are a major pathological characteristic of Parkinson's disease (PD). These fibrillar structures can act as seeds and accelerate the aggregation of monomeric a-syn. Indeed, recent studies show that injection of preformed a-syn fibrils (PFF) into the rodent brain can induce aggregation of the endogenous monomeric a-syn resulting in neuronal dysfunction and eventual cell death. We injected 8 µg of murine a-syn PFF, or soluble monomeric a-syn into the right striatum of rats. The animals were monitored behaviourally using the cylinder test, which measures paw asymmetry, and the corridor task that measures lateralized sensorimotor response to sugar treats. In vivo PET imaging was performed after 6, 13 and 22 weeks using [11C]DTBZ, a marker of the vesicular monoamine 2 transporter (VMAT2), and after 15 and 22 weeks using [11C]UCB-J, a marker of synaptic SV2A protein in nerve terminals. Histology was performed at the three time points using antibodies against dopaminergic markers, aggregated a-syn, and MHCII to evaluate the immune response. While the a-syn PFF injection caused only mild behavioural changes, [11C]DTBZ PET showed a significant and progressive decrease of VMAT2 binding in the ipsilateral striatum. This was accompanied by a small progressive decrease in [11C]UCB-J binding in the same area. In addition, our histological analysis revealed a gradual spread of misfolded a-syn pathology in areas anatomically connected to striatum that became bilateral with time. The striatal a-syn PFF injection resulted in a progressive unilateral degeneration of dopamine terminals, and an early and sustained presence of MHCII positive ramified microglia in the ipsilateral striatum and substantia nigra. Our study shows that striatal injections of a-syn fibrils induce progressive pathological synaptic dysfunction prior to cell death that can be detected in vivo with PET. We confirm that intrastriatal injection of a-syn PFFs provides a model of progressive a-syn pathology with loss of dopaminergic and synaptic function accompanied by neuroinflammation, as found in human PD.

Glycogen synthase kinase 3 ß activity is essential for Polo-like kinase 2- and Leucine-rich repeat kinase 2-mediated regulation of α-synuclein.

Parkinson's disease (PD) is a currently incurable disease and the number of patients is expected to increase due to the extended human lifespan. α-Synuclein is a pathological hallmark of PD and variations and triplications of the gene encoding α-synuclein are strongly correlated with the risk of developing PD. Decreasing α-synuclein is therefore a promising therapeutic strategy for the treatment of PD. We have previously demonstrated that Polo-like kinase 2 (PLK-2) regulates α-synuclein protein levels by modulating the expression of α-synuclein mRNA. In this study, we further expand the knowledge on this pathway and show that it depends on down-stream modulation of Glycogen-synthase kinase 3 ß (GSK-3ß). We show that PLK-2 inhibition only increases α-synuclein levels in the presence of active GSK-3ß in both cell lines and primary neuronal cultures. Furthermore, direct inhibition of GSK-3ß decreases α-synuclein protein and mRNA levels in our cell model and overexpression of Leucine-rich repeat kinase 2, known to activate GSK-3ß, increases α-synuclein levels. Finally, we show an increase in endogenous α-synuclein in primary neurons when increasing GSK-3ß activity. Our findings demonstrate a not previously described role of endogenous GSK-3ß activity in the PLK-2 mediated regulation of α-synuclein levels. This finding opens up the possibility of GSK-3ß as a novel target for decreasing α-synuclein levels by the use of small molecule compounds, hereby serving as a disease modulating strategy.

Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates.

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed α-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous α-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using an adeno-associated viral (AAV) vector in α-synuclein knockout slices. The processes of aggregation and spreading of α-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of α-synuclein aggregates and their prion-like spreading.

Stabilization of α-synuclein oligomers using formaldehyde.

The group of neurodegenerative diseases, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) all exhibit inclusions containing amyloid-type α-synuclein (α-syn) aggregates within degenerating brain cells. α-syn also exists as soluble oligomeric species that are hypothesized to represent intermediates between its native and aggregated states. These oligomers are present in brain extracts from patients suffering from synucleinopathies and hold great potential as biomarkers. Although easily prepared in vitro, oligomers are metastable and dissociate over time, thereby complicating α-syn oligomer research. Using the small amine-reactive cross-linker, formaldehyde (FA), we successfully stabilized α-syn oligomers without affecting their size, overall structure or antigenicity towards aggregate-conformation specific α-syn antibodies FILA and MJFR-14-6-4-2. Further, cross-linked α-syn oligomers show resistance towards denaturant like urea and SDS treatment and remain fully functional as internal standard in an aggregation-specific enzyme-linked immunosorbent assay (ELISA) despite prior incubation with urea. We propose that FA cross-linked α-syn oligomers could serve as important calibrators to facilitate comparative and standardized α-syn biomarker studies going forward.

Reduced Cytosolic Calcium as an Early Decisive Cellular State in Parkinson's Disease and Synucleinopathies.

The more than 30-year-old Calcium hypothesis postulates that dysregulation in calcium dependent processes in the aging brain contributes to its increased vulnerability and this concept has been extended to Alzheimer's disease and Parkinson's disease. Central to the hypothesis is that increased levels of intracellular calcium develop and contributes to neuronal demise. We have studied the impact on cells encountering a gradual build-up of aggregated α-synuclein, which is a central process to Parkinson's disease and other synucleinopathies. Surprisingly, we observed a yet unrecognized phase characterized by a reduced cytosolic calcium in cellular and neuronal models of Parkinson's disease, caused by α-synuclein aggregates activating the endoplasmic calcium ATPase, SERCA. Counteracting the initial phase with low calcium rescues the subsequent degenerative phase with increased calcium and cell death - and demonstrates this early phase initiates decisive degenerative signals. In this review, we discuss our findings in relation to literature on calcium dysregulation in Parkinson's disease and dementia.

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation.

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

ELISA method to detect α-synuclein oligomers in cell and animal models.

Soluble aggregates of α-synuclein, so-called oligomers, are hypothesized to act as neurotoxic species in Parkinson's disease, Lewy body dementia and multiple systems atrophy, but specific tools to detect these aggregated species are only slowly appearing. We have developed an α-synuclein oligomer ELISA that allows us to detect and compare α-synuclein oligomer levels in different in vivo and in vitro experiments. The ELISA is based on commercially available antibodies and the epitope of the capture antibody MJF14-6-4-2 is folding- and aggregate-dependent and not present on monomers.

Alpha-synuclein aggregates activate calcium pump SERCA leading to calcium dysregulation.

Aggregation of α-synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α-synuclein aggregation. Analyses of cell lines and primary culture models of α-synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α-synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+ CPA protects the cells against α-synuclein-aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo Proximity ligation assays also reveal an increased interaction between α-synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α-synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down-stream processes may be therapeutic targets for treating α-synucleinopathies.

Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death.

Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy comprise a group of neurodegenerative diseases termed synucleinopathies. Synucleinopathie are, characterized by presence of inclusion bodies in degenerating brain cells which contain aggregated α-synuclein phosphorylated on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease cellular degeneration. In vitro phosphorylation demonstrates that PKR can directly bind and phosphorylate monomeric and filamenteous α-synuclein on Ser129. Inhibition and knockdown of PKR reduce Ser129 phosphorylation in different models (SH-SY5Y ASYN cells, OLN-AS7 cells, primary mouse hippocampal neurons, and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-ß-1b, lead to increased levels of phosphorylated Ser129 α-synuclein that is completely blocked by simultaneous PKR inhibition. These results reveal a direct link between PKR and the phosphorylation and toxicity of α-synuclein, and they support that neuroinflammatory processes play a role in modulating the pathogenicity of α-synuclein.

Polo-like kinase 2 modulates α-synuclein protein levels by regulating its mRNA production.

Variations in the α-synuclein-encoding SNCA gene represent the greatest genetic risk factor for Parkinson's disease (PD), and duplications/triplications of SNCA cause autosomal dominant familial PD. These facts closely link brain levels of α-synuclein with the risk of PD, and make lowering α-synuclein levels a therapeutic strategy for the treatment of PD and related synucleinopathies. In this paper, we corroborate previous findings on the ability of overexpressed Polo-like kinase 2 (PLK-2) to decrease cellular α-synuclein, but demonstrate that the process is independent of PLK-2 phosphorylating S129 in α-synuclein because a similar reduction is achieved with the non-phosphorable S129A mutant α-synuclein. Using a specific PLK-2 inhibitor (compound 37), we demonstrate that endogenous PLK-2 phosphorylates S129 only in some cells, but increases α-synuclein protein levels in all tested cell cultures and brain slices. PLK-2 is found to regulate the transcription of α-synuclein mRNA from both the endogenous mouse SNCA gene and transgenic vectors that only contain the open reading frame. Moreover, we are the first to show that regulation of α-synuclein by PLK-2 is of physiological importance since 10days' inhibition of endogenous PLK-2 in wt C57BL/6 mice increases endogenous α-synuclein protein levels. Our findings collectively demonstrate that PLK-2 regulates α-synuclein levels by a previously undescribed transcription-based mechanism. This mechanism is active in cells and brain tissue, opening up for alternative strategies for modulating α-synuclein levels and thereby for the possibility of modifying disease progression in synucleinopaties.

The Structure of the RAGE:S100A6 Complex Reveals a Unique Mode of Homodimerization for S100 Proteins.

S100 proteins are calcium-dependent regulators of homeostatic processes. Upon cellular response to stress, and notably during tumorigenesis, they relocalize to the extracellular environment where they induce pro-inflammatory signals by activating the receptor for advanced glycation end products (RAGE), thereby facilitating tumor growth and metastasis. Despite its importance in sustaining inflammation, the structural basis for RAGE-S100 crosstalk is still unknown. Here we report two crystal structures of the RAGE:S100A6 complex encompassing a full-length RAGE ectodomain. The structures, in combination with a comprehensive interaction analysis, suggest that the primary S100A6 binding site is formed by the RAGE C1 domain. Complex formation with S100A6 induces a unique dimeric conformation of RAGE that appears suited for signal transduction and intracellular effector recruitment. Intriguingly, S100A6 adopts a dimeric conformation radically different from all known S100 dimers. We discuss the physiological relevance of this non-canonical homodimeric form in vivo.

Protein Partners of α-Synuclein in Health and Disease.

α-synuclein is normally situated in the nerve terminal but it accumulates and aggregates in axons and cell bodies in synucleinopathies such as Parkinson's disease. The conformational changes occurring during α-synucleins aggregation process affects its interactions with other proteins and its subcellular localization. This review focuses on interaction partners of α-synuclein within different compartments of the cell with a focus on those preferentially binding aggregated α-synuclein. The aggregation state of α-synuclein also affects its catabolism and we hypothesize impaired macroautophagy is involved neuronal excretion of α-synuclein species responsible for the prion-like spreading of α-synuclein pathology.

Identification of synaptosomal proteins binding to monomeric and oligomeric α-synuclein.

Monomeric α-synuclein (αSN) species are abundant in nerve terminals where they are hypothesized to play a physiological role related to synaptic vesicle turn-over. In Parkinson's disease (PD) and dementia with Lewy body (DLB), αSN accumulates as aggregated soluble oligomers in terminals, axons and the somatodendritic compartment and insoluble filaments in Lewy inclusions and Lewy neurites. The autosomal dominant heritability associated to mutations in the αSN gene suggest a gain of function associated to aggregated αSN. We have conducted a proteomic screen to identify the αSN interactome in brain synaptosomes. Porcine brain synaptosomes were fractionated, solubilized in non-denaturing detergent and subjected to co-immunoprecipitation using purified recombinant human αSN monomers or oligomers as bait. The isolated αSN binding proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using Windows client application, Skyline Targeted Proteomic Environment. Data are available via ProteomeXchange with identifier PXD001462. To quantify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. We identified 10 proteins preferentially binding monomer, and 76 binding preferentially to oligomer and a group of 92 proteins not displaying any preferred conformation of αSN. The proteomic data were validated by immunoprecipitation in both human and porcine brain extracts using antibodies against monomer αSN interactors: Abl interactor 1, and myelin proteolipid protein, and oligomer interactors: glutamate decarboxylase 2, synapsin 1, glial fibrillary acidic protein, and VAMP-2. We demonstrate the existence of αSN conformation selective ligands and present lists of proteins, whose identity and functions will be useful for modeling normal and pathological αSN dependent processes.

How epigallocatechin gallate can inhibit α-synuclein oligomer toxicity in vitro.

Oligomeric species of various proteins are linked to the pathogenesis of different neurodegenerative disorders. Consequently, there is intense focus on the discovery of novel inhibitors, e.g. small molecules and antibodies, to inhibit the formation and block the toxicity of oligomers. In Parkinson disease, the protein α-synuclein (αSN) forms cytotoxic oligomers. The flavonoid epigallocatechin gallate (EGCG) has previously been shown to redirect the aggregation of αSN monomers and remodel αSN amyloid fibrils into disordered oligomers. Here, we dissect EGCG's mechanism of action. EGCG inhibits the ability of preformed oligomers to permeabilize vesicles and induce cytotoxicity in a rat brain cell line. However, EGCG does not affect oligomer size distribution or secondary structure. Rather, EGCG immobilizes the C-terminal region and moderately reduces the degree of binding of oligomers to membranes. We interpret our data to mean that the oligomer acts by destabilizing the membrane rather than by direct pore formation. This suggests that reduction (but not complete abolition) of the membrane affinity of the oligomer is sufficient to prevent cytotoxicity.