DYNAMICS OF REST TURNOVER AND ASTROCYTE-MEDIATED REGULATION IN IPSC-DERIVED MODELS

This study uses iPSC-derived human neuronal models to investigate the mechanisms of RE-1 silencing transcription factor (REST) turnover in neurodegenerative disease, and how glial neuroinflammatory secretions influence REST expression and neuroprotective capabilities. Mature neurons were established using patient-derived iPSC and iNeuron systems to assess REST stability and expression through cellular stress assays. In parallel, iPSC-derived reactive astrocytes were generated to produce conditioned media (ACM). Reactive ACM and IL1B was applied to neurons, and REST transcriptional expression was assessed at both early (30 minutes) and later (24 hours) time points following treatment. REST was identified in two distinct molecular weight populations: a ~130 kDa form and a ~250 kDa form. The lower molecular weight pool is stable across cell types, including neurons, as confirmed through cycloheximide chase assays (2-24 hours). By contrast, the higher molecular weight population showed measurable turnover within 2 hours cycloheximide treatment in HeLa cells. BAFA1 and MG132 treatment confirmed both autophagic and proteasomal pathways exhibit control over REST degradation. Rotenone induced expression of the higher molecular weight REST species. ACM and IL1B induced a transient increase in REST transcription at 30 minutes, followed by a dramatic reduction by 24 hours in neurons. These findings highlight the complexity of REST biology, including the presence of a stable neuronal pool. The higher molecular weigh may represent the functional relevant species mediating neuronal restriction and neuroprotection. Reactive astrocytic secretion significantly alters REST expression dynamics. Future work will employ transcriptomic approaches in knockout and overexpression systems to further define REST-regulated pathways.