Deregulation of calcium homeostasis mediates secreted aesynuclein - induced neurotoxicity

α-Synuclein (AS) plays a crucial role in Parkinson's disease pathogenesis. AS is normally secreted from neuronal cells and can thus exert paracrine effects. We have previously demonstrated that naturally secreted AS species, derived from SH-SY5Y cells inducibly overexpressing human wild type AS, can be toxic to recipient neuronal cells. In the current study, we show that application of secreted AS alters membrane fluidity and increases calcium (Ca2+) entry. This influx is reduced on pharmacological inhibition of voltage-operated Ca2+ channels. Although no change in free cytosolic Ca2+ levels is observed, a significantly increased mitochondrial Ca2+ sequestration is found in recipient cells. Application of voltage-operated Ca2+ channel blockers or Ca2+ chelators abolishes AS-mediated toxicity. AS-treated cells exhibit increased calpain activation, and calpain inhibition greatly alleviates the observed toxicity. Collectively, our data suggest that secreted AS exerts toxicity through engagement, at least in part, of the Ca2+ homeostatic machinery. Therefore, manipulating Ca2+ signaling pathways might represent a potential therapeutic strategy for Parkinson's disease

Assessment of α-Synuclein Secretion in Mouse and Human Brain Parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease

Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world‐wide survey to ISEV members to assess methods considered ‘best practices’ for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies

Targeting intracellular and extracellular alpha-synuclein as a therapeutic strategy in Parkinson's disease and other synucleinopathies

Introduction: alpha-Synuclein is a neuronal presynaptic protein that regulates neurotransmitter release. Genetic, neuropathological, biochemical and animal model data indicate that it plays a major role in Parkinson’s disease and other neurodegenerative disorders, acting through a toxic gain of function. Although the mechanism of the toxic function of alpha-Synuclein is not yet certain, it may involve multiple intracellular targets of the aberrantly misfolded, aggregated protein. It is generally thought that specific soluble oligomeric alpha-Synuclein species are the offending toxic agents. The total amount of alpha-Synuclein is a significant factor that determines its toxicity. alpha-Synuclein can also be secreted and can thus affect neuronal and glial function. Propagation of alpha-Synuclein pathology via neuron-to-neuron transmission and seeding may also contribute to Parkinson’s disease pathogenesis. Areas covered: Key mechanisms of deregulation of alpha-Synuclein that could be relevant to neurodegeneration, and could offer opportunities for therapeutic intervention. Expert opinion: Counteracting intracellular and extracellular effects of alpha-Synuclein represents a valid therapeutic target in neurodegeneration. In particular, strategies that target alpha-Synuclein through limitation of its burden at the transcriptional and post-transcriptional level, inhibition of its aggregation or of aberrant phosphorylation states, immunization or attenuation of its secretion and propagation may be therapeutic options

Cell-produced alpha-synuclein oligomers are targeted to, and impair, the 26S proteasome

Proteasomal dysfunction may play a role in neurodegenerative conditions and protein aggregation. Overexpression in neuronal cells of alpha-synuclein, a molecule linked to Parkinson’s Disease, may lead to proteasomal dysfunction. Using PCl2 cells stably expressing wild-type or mutant alpha-synuclein and gel filtration, we demonstrate that soluble, intermediate size oligomers of alpha-synuclein co-elute with the 26S proteasome. These soluble oligomers associate with the 26S proteasome and are significantly increased following treatment with proteasomal, but not lysosomal, inhibitors, indicating specific degradation of these particular species by the 26S proteasome. Importantly, expression of alpha-synuclein resulted in a significant inhibition of all proteasomal activities without affecting the levels or assembly of the 26S proteasome. Pharmacological dissociation of alpha-synuclein oligomers restored proteasomal function and reduced polyubiquitinated protein load in intact cells. Our findings suggest a model where only a subset of specific soluble cell-derived alpha-synuclein oligomers is targeted to the 26S proteasome for degradation, and simultaneously inhibit its function, likely by impeding access of other proteasomal substrates. (C) 2008 Elsevier Inc. All rights reserved

The promise of the TGF-β superfamily as a therapeutic target for Parkinson's disease

A large body of evidence underscore the regulatory role of TGF-β superfamily in the central nervous system. Components of the TGF-β superfamily modulate key events during embryonic brain development and adult brain tissue injury repair. With respect to Parkinson's disease (PD), TGF-ß signaling pathways are implicated in the differentiation, maintenance and synaptic function of the dopaminergic neurons, as well as in processes related to the activation state of astrocytes and microglia. In vitro and in vivo studies using toxin models, have interrogated on the dopaminotrophic and protective role of the TGF-β superfamily members. The evolution of genetic and animal models of PD that more closely recapitulate the disease condition has made possible the dissection of intracellular pathways in response to TGF-β treatment. Although the first clinical trials using GDNF did not meet their primary endpoints, substantial work has been carried out to reappraise the TGF-β superfamily's clinical benefit

alpha-synuclein degradation by autophagic pathways - A potential key to Parkinson's disease pathogenesis

The neuronal protein alpha-synuclein is thought to be central in the pathogenesis of Parkinson’s disease (PD). Excessive wild type alpha-synuclein levels can lead to PD in select familial cases and alpha-synuclein protein accumulation occurs in sporadic PD. Therefore, elucidation of the mechanisms that control alpha-synuclein levels is critical for PD pathogenesis and potential therapeutics. The subject of alpha-synuclein degradation has been controversial. Previous work shows that, in an assay with isolated liver lysosomes, purified wild type alpha-synuclein is degraded by the process of chaperone-mediated autophagy (CMA). Whether this actually occurs in a cellular context has been unclear. In our most recent work, we find that wild type alpha-synuclein, but not the closely related protein alpha-synuclein, is indeed degraded by CMA in neuronal cells, including primary postnatal ventral midbrain neurons. Macroautophagy, but not the proteasome, also contributes to alpha-synuclein degradation. Therefore, two separate lysosomal pathways, CMA and macroautophagy, degrade wild type alpha-synuclein in neuronal cells. It is hypothesized that impairment of either of these two pathways, or of more general lysosomad function, may be an initiating factor in alpha-synuclein accumulation and sporadic PD pathogenesis

Assessment of cerebrospinal fluid α-synuclein as a potential biomarker in Parkinson’s disease and synucleinopathies

The discovery of diagnostic and prognostic biomarkers for neurodegenerative diseases represents an unmet clinical challenge. For example, the diagnosis of Parkinson’s disease (PD) relies mainly on the presence of clinical symptoms. Therefore, the identification and use of novel PD biomarkers would allow the application of disease-modifying treatments at the very early stages of neurodegeneration. The presynaptic protein, α-synuclein, has been genetically and biochemically linked with PD pathogenesis and has been considered as a potential biomarker for the diagnosis of PD and the related synucleinopathies. The vast majority of studies have assessed the measurement of α-synuclein, alone or in combination with other biomarkers in the cerebrospinal fluid (CSF), since it is the biofluid that most closely reflects the pathophysiology of the brain. The diagnostic value of the monomeric α-synuclein but also the oligomeric, the phosphorylated and the aggregated forms of the protein has been evaluated using a variety of immunoassays. The results have so far been reproducible but the assays used are still lacking the required diagnostic accuracy. Recent reports have shown that Protein misfolding cyclic amplification is a technique that has the potential to detect α-synuclein seeds in samples of CSF with high sensitivity and across different synucleinopathies. In an effort to increase the source of biomarker for PD and related synucleinopathies, α-synuclein has also been measured in neuronal exosomes, small vesicles of endosomal origin that are secreted from neurons into the CSF or the periphery. The potential diagnostic value of exosomes stems from the notion that exosomes carry a disease-specific repertoire of marker proteins. Therefore, the assessment of exosome-associated α-synuclein species may also open up new avenues for disease diagnosis in different synucleinopathies

Wild type alpha-synuclein is degraded by chaperone-mediated autophagy and macroautophagy in neuronal cells

alpha-Synuclein ( ASYN) is crucial in Parkinson disease ( PD) pathogenesis. Increased levels of wild type ( WT) ASYN expression are sufficient to cause PD in humans. The manner of posttranscriptional regulation of ASYN levels is controversial. Previously, we had shown that WT ASYN can be degraded by chaperone- mediated autophagy ( CMA) in isolated liver lysosomes. Whether this occurs in a cellular and, in particular, in a neuronal cell context is unclear. Using a mutantASYNform that lacks the CMA recognition motif and RNA interference against the rate- limiting step in the CMA pathway, Lamp2a, we show here that CMA is indeed involved in WT ASYN degradation in PC12 and SH- SY5Y cells, and in primary cortical and midbrain neurons. However, the extent of involvement varies between cell types, potentially because of differences in compensatory mechanisms. CMA inhibition leads to an accumulation of soluble high molecular weight and detergent- insoluble species of ASYN, suggesting that CMA dysfunction may play a role in the generation of such aberrant species in PD. ASYN and Lamp2a are developmentally regulated in parallel in cortical neuron cultures and in vivo in the central nervous system, and they physically interact as indicated by co- immunoprecipitation. In contrast to previous reports, inhibition of macroautophagy, but not the proteasome, also leads toWTASYN accumulation, suggesting that this lysosomal pathway is also involved in normal ASYN turnover. These results indicate thatCMAand macroautophagy are important pathways for WT ASYN degradation in neurons and underline the importance of CMA as degradation machinery in the nervous system