Sarkosyl differentially solubilizes patient-derived alpha-synuclein fibril strains.

Insoluble α-synuclein (αSyn) filaments in brain tissue are a hallmark of Parkinson's disease (PD) and Multiple system atrophy (MSA), and for structural studies, they have for decades been extracted using the detergent sarkosyl. We asked if PD and MSA patient-derived αSyn filament strains display different stability to sarkosyl extraction as this may confound our interpretation of the landscape of structural strains present in patients' tissue. We compared the stability of cerebrospinal fluid-derived strains from four PD and four MSA patients using sedimentation and immunoassays and tested the seeding competence and strain-specific characteristics of the sarkosyl-soluble fractions using a seed amplification assay (SAA) and Thioflavin T (ThT) fluorescence. We demonstrate that filaments from PD are less resistant to sarkosyl than from MSA after they have been subjected to freezing and sonication. An enhanced release of monomers from PD filaments was the major difference between PD and MSA, but the sarkosyl-soluble fraction released from both PD and MSA filaments contained aggregates that displayed aggregate-specific epitopes and seeding activity with preserved disease-specific strain characteristics. Our results demonstrate that sarkosyl differentially destabilizes patient derived αSyn filament strains, which may compromise our ability to fully appreciate the landscape of αSyn filament currently being uncovered by high resolution cryoEM analyses. This should motivate an effort to develop more gentle extraction protocols.

Alpha-Synuclein Strain Variability in Body-First and Brain-First Synucleinopathies.

Pathogenic alpha-synuclein (asyn) aggregates are a defining feature of neurodegenerative synucleinopathies, which include Parkinson's disease, Lewy body dementia, pure autonomic failure and multiple system atrophy. Early accurate differentiation between these synucleinopathies is challenging due to the highly heterogeneous clinical profile at early prodromal disease stages. Therefore, diagnosis is often made in late disease stages when a patient presents with a broad range of motor and non-motor symptoms easing the differentiation. Increasing data suggest the clinical heterogeneity seen in patients is explained by the presence of distinct asyn strains, which exhibit variable morphologies and pathological functions. Recently, asyn seed amplification assays (PMCA and RT-QuIC) and conformation-specific ligand assays have made promising progress in differentiating between synucleinopathies in prodromal and advanced disease stages. Importantly, the cellular environment is known to impact strain morphology. And, asyn aggregate pathology can propagate trans-synaptically along the brain-body axis, affecting multiple organs and propagating through multiple cell types. Here, we present our hypothesis that the changing cellular environments, an asyn seed may encounter during its brain-to-body or body-to-brain propagation, may influence the structure and thereby the function of the aggregate strains developing within the different cells. Additionally, we aim to review strain characteristics of the different synucleinopathies in clinical and preclinical studies. Future preclinical animal models of synucleinopathies should investigate if asyn strain morphology is altered during brain-to-body and body-to-brain spreading using these seeding amplification and conformation-specific assays. Such findings would greatly deepen our understanding of synucleinopathies and the potential link between strain and phenotypic variability, which may enable specific diagnosis of different synucleinopathies in the prodromal phase, creating a large therapeutic window with potential future applications in clinical trials and personalized therapeutics.

Low dose DMSO treatment induces oligomerization and accelerates aggregation of α-synuclein.

Dimethyl sulfoxide (DMSO) is a highly utilized small molecule that serves many purposes in scientific research. DMSO offers unique polar, aprotic and amphiphilic features, which makes it an ideal solvent for a wide variety of both polar and nonpolar molecules. Furthermore, DMSO is often used as a cryoprotectant in cell-based research. However, recent reports suggest that DMSO, even at low concentration, might interfere with important cellular processes, and cause macromolecular changes to proteins where a shift from α-helical to ß-sheet structure can be observed. To investigate how DMSO might influence current research, we assessed biochemical and cellular impacts of DMSO treatment on the structure of the aggregation-prone protein α-synuclein, which plays a central role in the etiology of Parkinson's disease, and other brain-related disorders, collectively termed the synucleinopathies. Here, we found that addition of DMSO increased the particle-size of α-synuclein, and accelerated the formation of seeding-potent fibrils in a dose-dependent manner. These fibrils made in the presence of DMSO were indistinguishable from fibrils made in pure PBS, when assessed by proteolytic digestion, cytotoxic profile and their ability to seed cellular aggregation of α-synuclein. Moreover, as evident through binding to the MJFR-14-6-4-2 antibody, which preferentially recognizes aggregated forms of α-synuclein, and a bimolecular fluorescence complementation assay, cells exposed to DMSO experienced increased aggregation of α-synuclein. However, no observable α-synuclein abnormalities nor differences in neuronal survival were detected after oral DMSO-treatment in either C57BL/6- or α-synuclein transgenic F28 mice. In summary, we demonstrate that low concentrations of DMSO makes α-synuclein susceptible to undergo aggregation both in vitro and in cells. This may affect experimental outcomes when studying α-synuclein in the presence of DMSO, and should call for careful consideration when such experiments are planned.

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.

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.

Evidence for bidirectional and trans-synaptic parasympathetic and sympathetic propagation of alpha-synuclein in rats.

The conversion of endogenous alpha-synuclein (asyn) to pathological asyn-enriched aggregates is a hallmark of Parkinson's disease (PD). These inclusions can be detected in the central and enteric nervous system (ENS). Moreover, gastrointestinal symptoms can appear up to 20 years before the diagnosis of PD. The dual-hit hypothesis posits that pathological asyn aggregation starts in the ENS, and retrogradely spreads to the brain. In this study, we tested this hypothesis by directly injecting preformed asyn fibrils into the duodenum wall of wild-type rats and transgenic rats with excess levels of human asyn. We provide a meticulous characterization of the bacterial artificial chromosome (BAC) transgenic rat model with respect to initial propagation of pathological asyn along the parasympathetic and sympathetic pathways to the brainstem, by performing immunohistochemistry at early time points post-injection. Induced pathology was observed in all key structures along the sympathetic and parasympathetic pathways (ENS, autonomic ganglia, intermediolateral nucleus of the spinal cord (IML), heart, dorsal motor nucleus of the vagus, and locus coeruleus (LC)) and persisted for at least 4 months post-injection. In contrast, asyn propagation was not detected in wild-type rats, nor in vehicle-injected BAC rats. The presence of pathology in the IML, LC, and heart indicate trans-synaptic spread of the pathology. Additionally, the observed asyn inclusions in the stomach and heart may indicate secondary anterograde propagation after initial retrograde spreading. In summary, trans-synaptic propagation of asyn in the BAC rat model is fully compatible with the "body-first hypothesis" of PD etiopathogenesis. To our knowledge, this is the first animal model evidence of asyn propagation to the heart, and the first indication of bidirectional asyn propagation via the vagus nerve, i.e., duodenum-to-brainstem-to-stomach. The BAC rat model could be very valuable for detailed mechanistic studies of the dual-hit hypothesis, and for studies of disease modifying therapies targeting early pathology in the gastrointestinal tract.