Modeling Multiple System Atrophy Prion Propagation in Astrocytes From Transgenic Mice

Abstract

Background/AimsThere is a growing body of evidence that astrocytes contain α‐synuclein deposits in Parkinson's disease (PD), multiple system atrophy (MSA), and other α‐synucleinopathies at advanced stages of disease. The native protein isoform of α‐synuclein misfolds into a prion conformation that aggregates and spreads throughout the brain, resulting in neurodegeneration in each of these conditions. We aim to use a new in vitro model of α‐synuclein prion propagation to clarify the role of astrocytes in disease progression of MSA. This system may also prove useful for elucidating the molecular mechanisms of neurotoxicity.MethodsWe isolated primary cell culture astrocytes from the brains of transgenic mice expressing either the wild‐type (wt) form of human α‐synuclein or with the A53T mutation, which is associated with familial PD. Astrocytes were exposed to brain homogenates from MSA patients, mouse‐passaged MSA, recombinant wt α‐synuclein fibrils, and cell lysate from MSA‐infected astrocyte cultures. Following exposure, cells were thoroughly washed and cultured in fresh medium. We used confocal microscopy and high content analysis to examine α‐synuclein intracellular morphology and phosphorylation at multiple time points.ResultsWhile exogenously added α‐synuclein fibrils persisted unphosphorylated, they efficiently induced aggregation and phosphorylation of endogenously expressed α‐synuclein in exposed astrocytes. We observed rapid, progressive, and dose‐dependent α‐synuclein aggregate formation in astrocytes expressing either wt or A53T human α‐synuclein exposed to fibrils or MSA homogenates. Moreover, confocal microscopy revealed two morphologically distinct types of α‐synuclein assemblies – filamentous and granular. Both types progressively formed glial cytoplasmic inclusions, were hyperphosphorylated, thioflavin S positive, ubiquitinated, and co‐localized with the p62 marker, as is also seen in the brains of human MSA patients.ConclusionsOur studies investigate the formation of α‐synuclein aggregates in astrocytes and report these cells as potential candidates in which to examine various pathological forms of α‐synuclein prions. This cell culture model may allow studies on mechanisms of neurotoxicity and might provide a novel approach for drug discovery against MSA and other α‐synucleinopathies.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.