Impact of neural cell type and differentiation state on alpha-synuclein aggregation

Background and Aims Alpha synucleinopathies, including Parkinson’s disease (PD) and multisystem atrophy (MSA), are a class of neurodegenerative movement disorders characterized by pathological aggregation and deposition of the protein alpha-synuclein (aSyn). In PD, aggregated aSyn forms the major component of Lewy bodies (LB) and Lewy neurites (LN) in neurons of PD. Similarly, in MSA, it constitutes the primary component of glial cytoplasmatic inclusions (GCI) in oligodendrocytes (OL). These aSyn aggregates leads to neuronal or oligodendroglial dysfunction and subsequent neuronal loss in specific brain regions, resulting in disease-typical motor and non-motor symptoms. However, the precise mechanisms and causes of aSyn aggregation remain incompletely understood. It is particularly noteworthy that specific cell types are vulnerable to aSyn aggregation. In PD, midbrain dopaminergic neurons (mDAN) are significantly affected by aSyn accumulation. Interestingly, aSyn also accumulates in OLs in MSA, even though OLs scarcely synthesize aSyn. This cell type-selective vulnerability raises the question of whether distinct cellular milieus influence aSyn aggregation. This work investigates the propensity for aSyn aggregation in various neural cell types, including cortical projection neurons (CPN) and mDANs, as well as in OLs. Given the crucial role of the autophagy-lysosomal pathway (ALP) in clearing aSyn, differences in the ALP were also examined to understand its impact on cell type-dependent aSyn aggregation. Methods To explore the effect of cell type, CPNs and mDANs differentiated from human-induced pluripotent stem cells (hiPSC) were used. These hiPSCs were generated from two PD patients with a heterozygous duplication of the aSyn coding gene (SNCA) and from a healthy control, allowing comparison of aSyn aggregation in autologous CPNs and mDANs. Further, to compare aSyn aggregation patterns in OLs and neurons, rat central glia 4 (CG4) cells overexpressing the wild-type aSyn (CG4 WTS) and Lund human mesencephalic (LUHMES) cells overexpressing the wild-type aSyn (LUHMES WTS), as well as their respective control cells without aSyn overexpression were used. Differentiated CG4 and LUHMES cells of various variants served as OL and neuronal models, respectively. aSyn overexpression in CG4- and LUHMES WTS cells was achieved using an identical lentiviral construct, facilitating a comparison of aSyn aggregation in OLs and neurons in vitro. Additionally, transgenic mouse models overexpressing aSyn in OLs or neurons, driven by MBP (MBP-SNCA) or Thy1 (Thy1-SNCA) promoters, respectively, were employed to compare the impact of cell type on aSyn aggregation in vivo. aSyn aggregation in cells and the striatum of mouse brains were evaluated via a solubility analysis. This involved separating aSyn based on its solubility through a series of ultracentrifugation steps, followed by Western blot analysis using antibodies against aSyn. To elucidate mechanisms underlying cell type-dependent aSyn aggregation, the ALP was characterized using Western blot analysis of key ALP markers, p62 and LC3-II. Results In hiPSC-derived neurons, overexpression of aSyn due to SNCA-gene duplication in mDANs led to increased aSyn aggregation and an elevated oligomer/monomer ratio compared to the CPNs. In vivo study demonstrated that MBP-driven aSyn overexpression led to more strongly elevated aSyn aggregation levels as compared to Thy1-driven aSyn overexpression or non-transgenic control animals. This finding suggests an increased aSyn aggregation propensity, when aSyn overexpression was induced in OLs compared to neurons. In vitro study of CG4 WTS and LUHMES WTS cells demonstrated a remarkably increase in total aSyn levels in CG4 WTS cells compared to LUHMES WTS cells. However, no significant differences in aSyn aggregation, as assessed by the solubility assay, were observed between CG4- and LUHMES WTS cells. Interestingly, a differentiation-dependent effect was detected in either CG4 or LUHMES models regardless of aSyn overexpression, demonstrating differentiated cells being more prone to aSyn aggregation. The differences in aSyn aggregation patterns linked to different cell types and differentiation states appeared to be associated to distinct ALP patterns. Specifically, MBP-SCNA mice, where aSyn is overexpressed in OLs, exhibited significantly higher levels of p62 and lower levels of LC3-II compared to neuronal aSyn overexpressing Thy1-SNCA mice. Additionally, distinct ALP patterns were observed between proliferating and differentiated LUHMES and CG4, with differentiated cells showing markedly lower levels of LC3-II under basal conditions. Interesting, upon ALP inhibition with Bafilomycin A1, a stronger aSyn-dependent effect was noted in differentiated CG4 WTS cells compared to their proliferating counterparts. In differentiated CG4 WTS cells, aSyn overexpression led to increased LC3-II levels, while p62 levels remained unchanged. Conclusions and discussion The results of this study highlight the significant impact of neural cell type and differentiation state on aSyn aggregation. Specifically, aSyn overexpressing mDANs are more susceptible to aSyn aggregation than autologous CPNs and neurons with normal aSyn levels. Additionally, both in vivo and in vitro data support the notion that aSyn in OLs tends to be more prone to aggregation (in vivo) or exhibit a higher level (in vitro) compared to neurons, likely due to reduced clearance capacity or declined turnover rate in OLs. Furthermore, aSyn aggregation in differentiated cells is more prone to aggregation than in proliferating cells. Moreover, this study underscores the association of aSyn aggregation propensity and the ALP. The elevated levels of aggregated aSyn in the striatum of MBP-SNCA mice compared to Thy1-SNCA mice are accompanied by reduced LC3-II levels however elevated p62 levels, suggesting a diminished capacity for autophagosome formation and a blockage in autophagy flux, thereby indicating reduced ALP function in MBP-SNCA mice. Similarly, the ALP in differentiated cells appears to be limited, as evidenced by lower LC3-II levels, pointing to a reduced capacity for autopahgosome formation. Additionally, differentiated OLs with aSyn overexpression seem more susceptible to Bafilomycin A1-induced ALP impairments compared to proliferating cells. This is indicated by increased autophagosome formation (elevated LC3-II levels) but decreased fusion with lysosomes for protein degradation (accumulation of p62), which coincides with their susceptibility to aSyn aggregation compared to proliferating cells. These findings provide mechanistic insight that aligns with the observed increase in aSyn pathology with ageing and reduced efficiency of protein clearance in neural cells. This work emphasizes the crucial role of ALP as a decisive pathway in cell type-dependent aSyn aggregation, reinforcing its importance as a target for developing future therapeutic approaches for distinct synucleinopathies. Partial results presented in this thesis were previously published in: Brazdis RM, Alecu JE, Marsch D, Dahms A, Simmnacher K, Lorentz S, et al. Demonstration of brain region-specific neuronal vulnerability in human iPSC-based model of familial Parkinson's disease. Hum Mol Genet. 2020;29(7):1180-91.