Fluorescence Lifetime-Based FRET Biosensors for Monitoring N Terminal Domain-Dependent Interactions of TDP-43 in Living Cells: A Novel Approach for ALS and FTD Drug Discovery
Noah Nathan Kochen, Marguerite Murray, Sophia Zafari, Nagamani Vunnam, Elly E. Liao, Lihsia Chen, Anthony R. Braun, Jonathan N. Sachs
Abstract
High Resolution Image Download MS PowerPoint Slide Pathological aggregates of TDP-43 are implicated in Alzheimer’s disease, frontotemporal dementia, and amyotrophic lateral sclerosis. While therapeutic efforts have traditionally focused on mitigating end-stage TDP-43 aggregation, recent evidence highlights an upstream and potentially targetable event: the loss of functional nuclear TDP-43 multimers due to disrupted N-terminal domain (NTD) interactions. To address this, we developed fluorescence lifetime (FLT)-based FRET biosensors to monitor TDP-43 multimerization in living cells that couple a full-length TDP-43 FLT-FRET biosensor screen with an NTD-deletion counter screen, forming the foundation of a novel high-throughput screening (HTS) platform. Screening the 2682 compound FDA-approved Selleck library, we identified the small molecule ketoconazole, which stabilizes functional nuclear TDP-43 multimers in an NTD-dependent manner with low micromolar potency. Ketoconazole rescues TDP-43 mislocalization and aggregation, restores SREBP2 mRNA levels under TDP-43 overexpression, improves neuronal health, and partially restores motor function in a TDP-43 C. elegans model. These findings establish both the biosensors and the HTS platform as innovative tools for TDP-43 drug discovery and support an exciting translational approach for targeting TDP-43 proteinopathies.