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.

Parkinson disease (PD) is the second most common neurodegenerative disorder and the leading neurodegenerative cause of motor disability. Pathologic accumulation of aggregated alpha synuclein (AS) protein in brain, and imbalance in the nigrostriatal system due to the loss of dopaminergic neurons in the substantia nigra- pars compacta, are hallmark features in PD. AS aggregation and propagation are considered to trigger neurotoxic mechanisms in PD, including mitochondrial deficits and oxidative stress. The eukaryotic elongation factor-2 kinase (eEF2K) mediates critical regulation of dendritic mRNA translation and is a crucial molecule in diverse forms of synaptic plasticity. Here we show that eEF2K activity, assessed by immuonohistochemical detection of eEF2 phosphorylation on serine residue 56, is increased in postmortem PD midbrain and hippocampus. Induction of aggressive, AS-related motor phenotypes in a transgenic PD M83 mouse model also increased brain eEF2K expression and activity. In cultures of dopaminergic N2A cells, overexpression of wild-type human AS or the A53T mutant increased eEF2K activity. eEF2K inhibition prevented the cytotoxicity associated with AS overexpression in N2A cells by improving mitochondrial function and reduced oxidative stress. Furthermore, genetic deletion of the eEF2K ortholog efk-1 in C. elegans attenuated human A53T AS induced defects in behavioural assays reliant on dopaminergic neuron function. These data suggest a role for eEF2K activity in AS toxicity, and support eEF2K inhibition as a potential target in reducing AS-induced oxidative stress in PD.

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.

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.

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus-mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

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 (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.

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.

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.

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 β (GSK-3β). We show that PLK-2 inhibition only increases α-synuclein levels in the presence of active GSK-3β in both cell lines and primary neuronal cultures. Furthermore, direct inhibition of GSK-3β decreases α-synuclein protein and mRNA levels in our cell model and overexpression of Leucine-rich repeat kinase 2, known to activate GSK-3β, increases α-synuclein levels. Finally, we show an increase in endogenous α-synuclein in primary neurons when increasing GSK-3β activity. Our findings demonstrate a not previously described role of endogenous GSK-3β activity in the PLK-2 mediated regulation of α-synuclein levels. This finding opens up the possibility of GSK-3β as a novel target for decreasing α-synuclein levels by the use of small molecule compounds, hereby serving as a disease modulating strategy.

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as 'the master regulator of cellular anti-oxidant response', both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

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.

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.

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.

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.

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.

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.

Multiple system atrophy (MSA) is a progressive neurodegenerative disease clinically characterized by parkinsonism and cerebellar ataxia, and pathologically by oligodendrocyte α-synuclein inclusions. Genetic variants of COQ2 are associated with an increased risk for MSA in certain populations. Also, deficits in the level of coenzyme Q10 and its biosynthetic enzymes are associated with MSA. Here, we measured ATP levels and expression of biosynthetic enzymes for coenzyme Q10, including COQ2, in multiple regions of MSA and control brains. We found a reduction in ATP levels in disease-affected regions of MSA brain that associated with reduced expression of COQ2 and COQ7, supporting the concept that abnormalities in the biosynthesis of coenzyme Q10 play an important role in the pathogenesis of MSA.

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.

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.