Astrocyte-to-neuron transdifferentiation and the PTB/REST axis

: developing tools for Parkinson’s research

Abstract

Parkinson’s is the second most common neurodegenerative disease, characterised by the appearance of Lewy bodies and the loss of midbrain dopaminergic neurons (mDANs) in the substantia nigra pars compacta. There is no known cure for Parkinson’s, emphasising the need for novel in vitro human models for its study and to create novel therapeutics. Qian et al. (2020) showed a reversal of Parkinson’s phenotypes in a mouse model by triggering an astrocyte-to-neuron transdifferentiation by depletion of PTB, a splicing regulator with roles in neuronal differentiation. Using inspiration from Qian et al. (2020), this project sought to create an inducible system to convert hiPSC-derived ventral midbrain astrocytes (vmAstros) into mDANs in vitro. This could create a more efficient and scalable method of creating mDANs for Parkinson’s research and contributions towards novel therapeutics.

The initial aim of this project was to integrate a controllable system(s) for knockdown of PTB in hiPSC-derived vmAstros, in order to characterize changes in cell morphology and the proteomic landscape during transdifferentiation. I trialled several systems, including inducible CRISPRi, inducible shRNA, AID-mediated degradation, and stable shRNA expression, applying immunoblotting, immunofluorescence, TMT-proteomics, and live-cell imaging for characterisation of PTB-depleted vmAstros. In my hands, PTB suppression did not constitute an effective mode of neuronal transdifferentiation in hiPSC-derived vmAstros but instead revealed PTB to be a key regulator of the expression of neuronal genes (among others). My data support the findings of several reports published since the work of Qian et al. (2020) that also contradict the statement that PTB-depletion is sufficient to convert astrocytes to neurons. Instead, my data suggest that the findings reported by Qian et al. (2020) could instead be attributed to components included in the neuronal media. The remainder of this project shifted focus to the regulation of REST, a repressor of neuronal genes and core component of the PTB-regulated neuronal induction loop. I follow up on previous work on aged human and mouse neurons, to investigate whether REST is regulated by autophagy in vmAstros, and if REST is astro-protective in this setting. Here, I report that REST is in part regulated by autophagy, but that it does not provide protection in vmAstros. However, my findings do suggest that REST is downregulated following ⍺-synuclein PFF treatment, highlighting Parkinson’s stresses may influence REST levels and/or activity in human vmAstros.

Date of Award	13 May 2025
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jon D Lane (Supervisor) & Jan Frayne (Supervisor)