(F1110) EVALUATING ASCL1, LMX1A, AND NURR1 IN COMPARISON TO PTBP1 KNOCKDOWN FOR HUMAN GLIA-TO-DOPAMINERGIC NEURON REPROGRAMMING ACROSS CELLULAR CONTEXTS
Abstract: Parkinson's Disease (PD) is characterized by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra, resulting in debilitating motor symptoms. While stem cell-based therapies have reached clinical trials, direct reprogramming of resident glial cells into functional DA neurons presents a promising alternative. However, critical questions remain regarding the efficacy and specificity of reprogramming strategies in human cells. In this study, we compared two reprogramming approaches: Ascl1, Lmx1a, and Nurr1 with REST inhibition (ALNRi) versus polypyrimidine tract-binding protein 1 (PTBP1) knockdown, for their ability to generate DA neurons from human stem cell-derived glial progenitor cells (GPCs) in vitro. Reprogrammed cells were evaluated using immunocytochemistry to assess neuronal and DA-specific markers, RNA sequencing for transcriptional profiles, and electrophysiology for functional maturation. ALNRi-induced neurons exhibited distinct electrophysiological and transcriptional signatures of induced DA neurons, whereas PTBP1 knockdown failed to generate neurons with the desired specificity or functionality. To investigate whether extrinsic microenvironmental cues provided by different brain regions could influence or improve DA neuron reprogramming, human GPCs were transplanted into 3D organoid models mimicking the forebrain and midbrain. Single-nuclei transcriptomic analyses showed that ALNRi activation post-transplantation consistently generated DA neurons within these organoid environments, while PTBP1 knockdown remained ineffective and did not yield DA neurons. These findings suggest that intrinsic mechanisms engaged by ALNRi are critical for generating DA neurons and that extrinsic microenvironmental cues alone cannot overcome the limitations of PTBP1 knockdown as a reprogramming strategy. Our results establish ALNRi as a robust strategy for glia-to-neuron reprogramming, capable of reliably generating DA neurons. By demonstrating efficacy in vitro and after transplantation into regionalized organoid models, this approach provides a strong foundation for advancing to in vivo studies, with the goal of developing innovative therapies for PD.
