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Extracellular vesicles (EVs) are important mediators in intercellular communication. However, understanding the biological origin and functional effects of EVs subtypes has been challenging due to the moderate differences in their physical properties and absence of reliable markers. Here, we characterize the proteomes of ectosomes and exosomes using an improved differential ultracentrifugation protocol and quantitative proteomics. Our analyses revealed singular proteomic profiles for ectosomes and exosomes that enabled us to establish specific protein markers that can be used for their biochemical distinction. Cytoskeleton and glycolytic proteins are distinctively present in ectosomes, while endosomal sorting complexes proteins and tetraspanins are enriched in exosomes. Furthermore, annexin-A2 was identified as a specific marker for ectosomes derived from cell media and human cerebrospinal fluid. Expression of EGFP as a cytosolic reporter leads to its incorporation in EVs and enables their imaging with higher resolution. Assessment of neuronal network activity using multi-electrode array recordings demonstrated that spontaneous neuronal activity can be modulated by EVs. Ectosomes and exosomes internalization in neuronal cells disrupted their regular synchronized bursting activity, resulting in overall lower and more disorganized spiking activity. Our findings suggest that EVs cargoes reflect core intracellular processes, and their functional properties might regulate basic biological and pathological processes. SIGNIFICANCE: This article presents novel approaches for studying the origin, composition, and biological effects in neuronal activity of ectosomes and exosomes. Our findings suggest that EVs cargoes reflect core intracellular processes, and their functional properties might regulate basic biological and pathological processes. Ultimately, our study also forms the foundation for future biomarker studies and for the understanding of the molecular basis of different diseases., Competing Interests: Declaration of Competing Interest The authors report no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) and Alzheimer's disease (AD) are characterized by pathological accumulation and aggregation of different amyloidogenic proteins, α-synuclein (aSyn) in PD, and amyloid-β (Aβ) and Tau in AD. Strikingly, few PD and AD patients' brains exhibit pure pathology with most cases presenting mixed types of protein deposits in the brain. Bimolecular fluorescence complementation (BiFC) is a technique based on the complementation of two halves of a fluorescent protein, which allows direct visualization of protein-protein interactions. In the present study, we assessed the ability of aSyn and Tau to interact with each other. For in vitro evaluation, HEK293 and human neuroblastoma cells were used, while in vivo studies were performed by AAV6 injection in the substantia nigra pars compacta (SNpc) of mice and rats. We observed that the co-expression of aSyn and Tau led to the emergence of fluorescence, reflecting the interaction of the proteins in cell lines, as well as in mouse and rat SNpc. Thus, our data indicates that aSyn and Tau are able to interact with each other in a biologically relevant context, and that the BiFC assay is an effective tool for studying aSyn-Tau interactions in vitro and in different rodent models in vivo., (© 2022. The Author(s).)

Background: Various cellular pathways have been implicated in the transfer of disease-related proteins between cells, contributing to disease progression and neurodegeneration. However, the overall effects of protein transfer are still unclear., Objective: Here, we performed a systematic comparison of basic molecular mechanisms involved in the release of alpha-synuclein, Tau, and huntingtin, and evaluated functional effects upon internalization by receiving cells., Methods: Evaluation of protein release to the extracellular space in a free form and in extracellular vesicles using an optimized ultracentrifugation protocol. The extracellular effects of the proteins and extracellular vesicles in primary neuronal cultures were assessed using multi-channel electrophysiological recordings combined with a customized spike sorting framework., Results: We demonstrate cells differentially release free-forms of each protein to the extracellular space. Importantly, neuronal activity is distinctly modulated upon protein internalization in primary cortical cultures. In addition, these disease-related proteins also occur in extracellular vesicles, and are enriched in ectosomes. Internalization of ectosomes and exosomes by primary microglial or astrocytic cells elicits the production of pro-inflammatory cytokines, and modifies spontaneous electrical activity in neurons., Objective: Overall, our study demonstrates that released proteins can have detrimental effects for surrounding cells, and suggests protein release pathways may be exploited as therapeutic targets in different neurodegenerative diseases.

The accumulation of misfolded alpha-synuclein (aSyn) throughout the brain, as Lewy pathology, is a phenomenon central to Parkinson's disease (PD) pathogenesis. The stereotypical distribution and evolution of the pathology during disease is often attributed to the cell-to-cell transmission of aSyn between interconnected brain regions. The spreading of conformationally distinct aSyn protein assemblies, commonly referred as strains, is thought to result in a variety of clinically and pathologically heterogenous diseases known as synucleinopathies. Although tremendous progress has been made in the field, the mechanisms involved in the transfer of these assemblies between interconnected neural networks and their role in driving PD progression are still unclear. Here, we present an update of the relevant discoveries supporting or challenging the prion-like spreading hypothesis. We also discuss the importance of aSyn strains in pathology progression and the various putative molecular mechanisms involved in cell-to-cell protein release. Understanding the pathways underlying aSyn propagation will contribute to determining the etiology of PD and related synucleinopathies but also assist in the development of new therapeutic strategies.

Synucleinopathies are a group of disorders characterized by the accumulation of inclusions rich in the a-synuclein (aSyn) protein. This group of disorders includes Parkinson's disease, dementia with Lewy bodies (DLB), multiple systems atrophy, and pure autonomic failure (PAF). In addition, genetic alterations (point mutations and multiplications) in the gene encoding for aSyn (SNCA) are associated with familial forms of Parkinson's disease, the most common synucleinopathy. The Synuclein Meetings are a series that has been taking place every 2 years for about 12 years. The Synuclein Meetings bring together leading experts in the field of Synuclein and related human conditions with the goal of discussing and advancing the research. In 2019, the Synuclein meeting took place in Ofir, a city in the outskirts of Porto, Portugal. The meeting, entitled "Synuclein Meeting 2019: Where we are and where we need to go", brought together >300 scientists studying both clinical and molecular aspects of synucleinopathies. The meeting covered a many of the open questions in the field, in a format that prompted open discussions between the participants, and underscored the need for additional research that, hopefully, will lead to future therapies for a group of as of yet incurable disorders. Here, we provide a summary of the topics discussed in each session and highlight what we know, what we do not know, and what progress needs to be made in order to enable the field to continue to advance. We are confident this systematic assessment of where we stand will be useful to steer the field and contribute to filling knowledge gaps that may form the foundations for future therapeutic strategies, which is where we need to go., (© 2020 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Alpha-synuclein (aSyn) was identified as the main component of inclusions that define synucleinopathies more than 20 years ago. Since then, aSyn has been extensively studied in an attempt to unravel its roles in both physiology and pathology. Today, studying the mechanisms of aSyn toxicity remains in the limelight, leading to the identification of novel pathways involved in pathogenesis. In this chapter, we address the molecular mechanisms involved in synucleinopathies, from aSyn misfolding and aggregation to the various cellular effects and pathologies associated. In particular, we review our current understanding of the mechanisms involved in the spreading of aSyn between different cells, from the periphery to the brain, and back. Finally, we also review recent studies on the contribution of inflammation and the gut microbiota to pathology in synucleinopathies. Despite significant advances in our understanding of the molecular mechanisms involved, we still lack an integrated understanding of the pathways leading to neurodegeneration in PD and other synucleinopathies, compromising our ability to develop novel therapeutic strategies., (© 2020 Elsevier B.V. All rights reserved.)

The aggregation of α-synuclein (α-syn) into amyloid fibrils is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. The mechanisms underlying the structural transition of soluble and innocuous α-syn to aggregated neurotoxic forms remains largely unknown. The disordered nature of α-syn has hampered the use of structure-based protein engineering approaches to elucidate the molecular determinants of this transition. The recent 3D structure of a pathogenic α-syn fibril provides a template for this kind of studies. The structure supports the NAC domain being a critical element in fibril formation, since it constitutes the core of the fibril, delineating a Greek-key motif. Here, we stapled the ends of this motif with a designed disulfide bond and evaluated its impact on the conformation, aggregation and toxicity of α-syn in different environments. The new covalent link biases the native structural ensemble of α-syn toward compact conformations, reducing the population of fully unfolded species. This conformational bias results in a strongly reduced fibril formation propensity both in the absence and in the presence of lipids and impedes the formation of neurotoxic oligomers. Our study does not support the Greek-key motif being already imprinted in early α-syn assemblies, discarding it as a druggable interface to prevent the initiation of fibrillation. In contrast, it suggests the stabilization of native, compact ensembles as a potential therapeutic strategy to avoid the formation of toxic species and to target the early stages of PD., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

A functional protein quality control machinery is crucial to maintain cellular and organismal physiology. Perturbation in the protein homeostasis network can lead to the formation of misfolded and aggregated proteins that are a hallmark of protein conformational disorders and aging. Protein aggregation is counteracted by the action of chaperones that can resolubilize aggregated proteins. An alternative protein aggregation clearance strategy is the elimination by proteolysis employing the ubiquitin proteasome system (UPS) or autophagy. Little is known how these three protein aggregate clearance strategies are regulated and coordinated in an organism with the progression of aging or upon expression of disease-associated proteins. To unravel the crosstalk between the protein aggregate clearance options, we investigated how autophagy and the UPS respond to perturbations in protein disaggregation capacity. We found that autophagy is induced as a potential compensatory mechanism, whereas the UPS exhibits reduced capacity upon depletion of disaggregating chaperones in C. elegans and HEK293 cells. The expression of amyloid proteins Aβ 3-42 and Q 40 result in an impairment of autophagy as well as the UPS within the same and even across tissues. Our data indicate a tight coordination between the different nodes of the proteostasis network (PN) with the progression of aging and upon imbalances of the capacity of each clearance mechanism.

Glycation is the non-enzymatic reaction between reactive dicarbonyls and amino groups, and gives rise to a variety of different reaction products known as advanced glycation end products (AGEs). Accumulation of AGEs on proteins is inevitable, and is associated with the aging process. Importantly, glycation is highly relevant in diabetic patients that experience periods of hyperglycemia. AGEs also play an important role in neurodegenerative diseases including Alzheimer's (AD) and Parkinson's disease (PD). Huntington's disease (HD) is a hereditary neurodegenerative disease caused by an expansion of a CAG repeat in the huntingtin gene. The resulting expanded polyglutamine stretch in the huntingtin (HTT) protein induces its misfolding and aggregation, leading to neuronal dysfunction and death. HD patients exhibit chorea and psychiatric disturbances, along with abnormalities in glucose and energy homeostasis. Interestingly, an increased prevalence of diabetes mellitus has been reported in HD and in other CAG triplet repeat disorders. However, the mechanisms underlying the connection between glycation and HD progression remain unclear. In this review, we explore the possible connection between glycation and proteostasis imbalances in HD, and posit that it may contribute to disease progression, possibly by accelerating protein aggregation and deposition. Finally, we review therapeutic interventions that might be able to alleviate the negative impact of glycation in HD.

The budding yeast Saccharomyces cerevisiae (S. cerevisiae) has been a remarkable experimental model for the discovery of fundamental biological processes. The high degree of conservation of cellular and molecular processes between the budding yeast and higher eukaryotes has made it a valuable system for the investigation of the molecular mechanisms behind various types of devastating human pathologies. Genetic screens in yeast provided important insight into the toxic mechanisms associated with the accumulation of misfolded proteins. Thus, using yeast genetics and high-throughput screens, novel molecular targets with therapeutic potential have been identified. Here, we describe a yeast screen protocol for the identification of genetic modifiers of alpha-synuclein (aSyn) toxicity, thereby accelerating the identification of novel potential targets for intervention in Parkinson's disease (PD) and other synucleinopathies.

Alpha-synuclein (aSyn) is a key player in a group of neurodegenerative diseases commonly known as synucleinopathies. Recent findings indicate phosphorylation in several aSyn residues can modulate its aggregation and subcellular localization, thereby affecting pathological processes. However, the precise molecular mechanisms governing aSyn phosphorylation are still unclear. Recent studies investigated the role of various families of protein kinases, such as the polo-like kinases, G protein-coupled receptor kinases or casein kinases. In contrast, our understanding of the phosphatases involved in the dephosphorylation of aSyn is rather limited. Here, we exploited the unique toolbox of the yeast Saccharomyces cerevisiae in order to identify novel phosphatases capable of modulating aSyn phosphorylation, inclusion formation and toxicity of human aSyn. In summary, given the association between aSyn phosphorylation and pathology in Parkinson's disease and other synucleinopathies, modulation of this post-translational modification may constitute an attractive target for therapeutic intervention.

Parkinson's disease and other synucleinopathies are characterized by the accumulation of aggregated α-synuclein in intracellular proteinaceous inclusions. The progressive nature of synucleinopathies seems to be related to the cell-to-cell spreading of α-synuclein pathology, and several possible mechanisms have been put forward to explain this phenomenon. In our recent study, we found that α-synuclein oligomers interact with cellular prion protein in glutamatergic synapses. This interaction triggered a signaling cascade involving phosphorylation of Fyn kinase and activation of the N-methyl-d-aspartate receptor, thereby leading to synaptic dysfunction. Here, we present relevant plasma membrane proteins that have been described to interact with α-synuclein and discuss the possible pathological implications. We focus primarily on the prion protein and propose a pathological mechanism in which the interaction between α-synuclein and prion protein leads to the formation of cofilin/actin rods, culminating in long-term potentiation impairment and cognitive dysfunction. We posit that deciphering the mechanisms involved in sensing specific forms of extracellular α-synuclein and transducing this information may prove invaluable in our quest to devise novel diagnostic and therapeutic approaches in PD and other synucleinopathies. © 2018 International Parkinson and Movement Disorder Society., (© 2018 International Parkinson and Movement Disorder Society.)