Postdoc University of Antwerp Wilrijk, Antwerpen, Belgium
Abstract: Synucleinopathies are a group of neurodegenerative disorders characterized by the abnormal accumulation of misfolded alpha-synuclein (αSyn) protein in the brain. While significant insights have been gained so far into the aggregation, prion-like transmission, and toxicity of αSyn fibrils, most of these findings stem from animal models and traditional 2D in vitro cultures. To enable the study of αSyn pathology in a more human brain-like environment, we evaluated different strategies for inducing αSyn pathology in human iPSC-derived neurospheroids. Hereto, five month-old human iPSC-derived neurospheroids, composed of both neurons and astrocytes, were exposed to three conditions: (i) AAV-mediated overexpression of wild-type αSyn, (ii) addition of pre-formed αSyn fibrils (PFFs), or (iii) a combination of both, and were analysed by immunocytochemistry after 1 week, 1, 2 and 3 months. Immunostainings for αSyn, phosphorylated αSyn (p-αSyn) and thioflavin S were performed to evaluate αSyn overexpression, fibril uptake and the formation of pathological inclusions. Immunostaining for αSyn antigen confirmed successful αSyn overexpression in the AAV-transduced group and demonstrated fibril uptake by the outer cell layers of the neurospheroids in the PFF-treated groups. Staining for p-αSyn, a marker of pathological αSyn accumulation, revealed induction of αSyn pathology in the PFF-treated conditions, with accelerated spread and accumulation observed when PFFs were combined with αSyn overexpression. Thioflavin S staining to detect β-sheet-rich structures characteristic of pathological inclusions further confirmed these findings. In conclusion, the addition of PFFs to five month-old human iPSC-derived neurospheroids, with or without AAV-mediated αSyn overexpression, proved to be the most effective in inducing widespread pathological changes, resembling those seen in human synucleinopathies. Further transcriptomic and proteomic analyses will now have to confirm that neurospheroids treated with PFFs represent a promising and physiologically relevant platform for studying αSyn pathology and potential therapies in a human brain-like context.
