Inhibiting Glycogen Synthase Kinase 3 Suppresses TDP-43-Mediated Neurotoxicity in a Caspase-Dependent Manner

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative diseases characterised by TAR DNA-binding protein 43 kDa (TDP-43) pathology. We previously showed that deletion of glycogen synthase kinase-3 (GSK3) suppresses TDP-43-mediated motor neuron degeneration in Drosophila . Here, we investigated the potential of GSK3 inhibition to ameliorate TDP-43-mediated toxicity in mammalian neurons. We show that TDP-43 activates GSK3 and promotes caspase-dependent cleavage of TDP-43, generating C-terminal fragments. We determine the functional importance of the N-terminal Asp89 caspase cleavage site in regulating TDP-43 proteostasis in both wild-type and ALS-linked TDP-43 variants and show that GSK3 inhibition selectively reduces truncated TDP-43 species, lowers nuclear TDP-43 levels, and improves neuronal survival. Neuroprotective effects were conserved in primary rodent cortical neurons, primary mouse motor neurons, and human iPSC-derived cortical neurons, highlighting the potentially broad therapeutic potential of GSK3 inhibition. We also find that the GSK3 inhibitor CHIR99021 reduces GSK3 RNA and protein expression and increases GSK3 phosphorylation, indicating novel mechanisms by which it acts to inhibit GSK3 activity. Unexpectedly, an N-terminally truncated variant (TDP-43 ^N-Del ), originally designed as a negative transfection control, exerted modest toxicity, potentially through retained susceptibility to caspase cleavage. Together, our findings uncover a caspase-mediated mechanism linking GSK3 activity to TDP-43 turnover, localisation, and neurotoxicity, and position GSK3 inhibition as a promising strategy to mitigate TDP-43-driven neurodegeneration in ALS-FTD.