Alpha-Synuclein and ligodendroglia in MSA pathogenesis

a-Synuclein (a-Syn) is a protein of 140 amino acids enriched in the pre-synaptic terminals. The normal function of a-Syn is incompletely understood. -Synucleinopathies are neurodegenerative diseases characterized by -Syn- positive cytoplasmic inclusions in the CNS. a-Synucleinopathies include Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). a-Syn is considered a neuronal pre-synaptic protein that is absent in adult healthy oligodendroglia. Therefore, the origin and formation of -Syn inclusions (glial cytoplasmic inclusions, GCIs) in MSA oligodendroglia is presently unknown and poses a specific challenge in MSA pathogenesis research. Cell-to-cell transmission of a-Syn has been recently reported, suggesting a novel propagation mechanism in -synucleinopathies. Exogenous -Syn uptake by oligodendroglia seems to be the likely mechanism of GCI formation as suggested also by negative a-Syn mRNA expression studies in MSA oligodendroglia. Based on the evidence in the current literature and our own preliminary results, we hypothesise that abnormal uptake and accumulation of a-Syn by oligodendroglia is promoted by primary oligodendroglial dysfunction. The main body of experiments will be performed in vitro in purified primary murine oligodendroglial cell culture. In the first step of this proposal we will characterize a-Syn uptake by healthy oligodendroglia in cell cultures exposed to recombinant (soluble or fibrillized) and cell-derived a-Syn. In the second step we will systematically assess candidate mechanisms leading to primary oligodendroglial dysfunction which may promote exogenous a- Syn uptake and GCI formation. Oligodendroglial cell cultures will be manipulated: 1) to abnormally enhance endocytosis by Rab5 overexpression, 2) to induce cytoskeletal disruption, to trigger 3) proteasome and 4) autophagy malfunction. The generated dysfunctional oligodendroglia will be exposed to recombinant (soluble or fibrillized) and cell-derived a-Syn and the protein uptake and GCI formation will be monitored. In the final stage of this proposal we will implant dysfunctional oligodendroglia derived from the previous experiments into the brains of transgenic mice overexpressing human a-Syn and study the in vivo dynamics of GCI formation. The proposed experiments will provide novel insights on GCI formation based on propagation of a-Syn to oligodendroglia. The role of oligodendroglial dysfunction related to Rab5 ectopic expression, cytoskeletal disruption and/or malfunctional protein degradation on GCI formation will be systematically analyzed for the first time. The in vitro results will be expanded by a novel in vivo analysis of GCI formation in "dysfunctional oligodendroglia" associated with cell-derived a-Syn propagation.

?-Synuclein (?-Syn) is a protein of 140 amino acids enriched in the pre-synaptic terminals. ?- Synucleinopathies are neurodegenerative diseases characterized by ?-Syn-positive cytoplasmic inclusions in the CNS. ?-Synucleinopathies include Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). ?-Syn is considered a neuronal pre-synaptic protein that is thought to be absent in adult healthy oligodendroglia. Therefore, the origin and formation of ?-Syn inclusions (glial cytoplasmic inclusions, GCIs) in MSA oligodendroglia is presently unknown and poses a specific challenge in MSA pathogenesis research. Cell-to-cell transmission of ?-Syn has been recently reported, suggesting a novel propagation mechanism in ?-synucleinopathies. Exogenous ?-Syn uptake by oligodendroglia seems to be the likely mechanism of GCI formation. We hypothesise that abnormal uptake and accumulation of ?-Syn by oligodendroglia is promoted by primary oligodendroglial dysfunction. Furthermore, we test the hypothesis that increased clearance of ?-Syn may ameliorate the pathology in MSA transgenic mice. Part of the experiments were performed in vitro in oligodendroglial cell culture. In the first step we characterized ?-Syn uptake by healthy oligodendroglia in cell cultures exposed to recombinant (soluble or fibrillized) and cell-derived ?-Syn. In the second step we assessed macroautophagy dysfunction as candidate mechanism leading to primary oligodendroglial dysfunction which may promote exogenous ?-Syn uptake and GCI formation. The generated dysfunctional oligodendroglia were exposed to recombinant (soluble or fibrillized) and cell- derived ?-Syn and the protein uptake and GCI formation were monitored. Our results suggest that macroautophagy dysfunction in oligodendrocytes is not sufficient to trigger and promote GCI formation from exogenously uptaken ?-Syn. In the final stage of the project running we studied the role of TLR4 agonists and passive immunization with anti-?-Syn antibodies to reduce ?-Syn load and exert beneficial effects on the pathological phenotype of transgenic mice overexpressing human ?-Syn in oligodendrocytes.The experiments provide novel insights on GCI formation based on propagation of ?-Syn to oligodendroglia. The role of improved clearance of ?-Syn was shown to be a promising strategy for disease modification in MSA.