Dopaminergic (DA) cell death in Parkinson’s disease (PD) is associated with the gradual appearance of neuronal protein aggregates termed Lewy bodies (LBs) that are comprised of vesicular membrane structures and dysmorphic organelles in conjunction with the protein alpha-Synuclein (α-Syn). Although the exact mechanism of neuronal aggregate formation and death remains elusive, recent research suggests α-Syn-mediated alterations in the lysosomal degradation of aggregated proteins and organelles – a process termed autophagy. Here, we used a combination of molecular biology and immunochemistry to investigate the effect of α-Syn on autophagy turnover in cultured human DA neurons and in human post-mortem brain tissue. We found α-Syn overexpression to reduce autophagy turnover by compromising the fusion of autophagosomes with lysosomes, thus leading to a decrease in the formation of autolysosomes. In accord with a compensatory increase in the plasma membrane fusion of autophagosomes, α-Syn enhanced the number of extracellular vesicles (EV) and the abundance of autophagy-associated proteins in these EVs. Mechanistically, α-Syn decreased the abundance of the v-SNARE protein SNAP29, a member of the SNARE complex mediating autophagolysosome fusion. In line, SNAP29 knockdown mimicked the effect of α-Syn on autophagy whereas SNAP29 co-expression reversed the α-Syn-induced changes on autophagy turnover and EV release and ameliorated DA neuronal cell death. In accord with our results from cultured neurons, we found a stage-dependent reduction of SNAP29 in SNc DA neurons from human post-mortem brain tissue of Lewy body pathology (LBP) cases. In summary, our results thus demonstrate a previously unknown effect of α-Syn on intracellular autophagy-associated SNARE proteins and, as a consequence, a reduced autolysosome fusion. As such, our findings will therefore support the investigation of autophagy-associated pathological changes in PD
Extracellular vesicles (EVs) are endogenous membrane‐derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch‐clamp electrophysiology and pH‐sensitive dye imaging is used to examine stimulus‐evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high‐frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus‐evoked MVB‐PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v‐SNARE vesicle‐associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking‐down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB‐PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release.
Genetic approaches to complex diseases are subject to the currently available technological innovations, and successes or failures using these approaches influence our hypotheses for the genetic contributions to complex diseases. Common complex diseases with a genetic contribution result in the bulk of healthcare expenses through chronic care. It is thought that an understanding of the genetic contributions to common complex diseases will allow for advances in disease prevention, mitigation of disease pathogenesis and curative treatments. The authors discuss genetic approaches to complex diseases from the perspective of idiopathic scoliosis, a prevalent vertebral deformity syndrome that involves the integration of clinical, psychological, mechanical, and basic science disciplines. The authors focus specifically on the different hypotheses and approaches for genetic study, drawing on past studies and discussing possible future studies, with consideration of technological innovations. Key Concepts: The genetic basis of idiopathic scoliosis is not well understood. Idiopathic scoliosis is a complex deformity syndrome with a complex genetic component. Idiopathic scoliosis is the most common form of human spinal deformity. IS imposes a substantial healthcare cost through bracing, hospitalisations, surgery and chronic back pain. Identification of IS genes might lead to innovations in screening and treatment.
Spreading of alpha-synuclein (αSyn) may play an important role in Parkinson’s disease and related synucleinopathies. Passive immunization with anti-αSyn antibodies is a promising method to slow down the spreading process and thereby the progression of synucleinopathies. Currently, it remains elusive which specific characteristics are essential to render therapeutic antibodies efficacious. Here, we established a neuronal co-culture model, in which αSyn species are being released from αSyn-overexpressing cells and induce toxicity in a priori healthy GFP-expressing cells. In this model, we investigated the protective efficacy of three anti-αSyn antibodies. Only two of these antibodies, one C-terminal and one N-terminal, protected from αSyn-induced toxicity by inhibiting the uptake of spreading-competent αSyn from the cell culture medium. Neither the binding epitope nor the affinity of the antibodies towards recombinant αSyn could explain differences in biological efficacy. However, both protective antibodies formed more stable antibody-αSyn complexes than the non-protective antibody. These findings indicate that the stability of antibody-αSyn complexes may be more important to confer protection than the binding epitope or affinity to recombinant αSyn.
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