PHENOTYPIC HIGH-CONTENT SCREEN MEASURING CLEARANCE OF TDP - 43 AGGREGATES

The presence of aggregates of ubiquitinated, misfolded and hyperphosphorylated transactive response DNA-binding protein of 43 kDa (TDP-43) is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In addition, TDP-43 aggregates have now been detected in a number of other neurodegenerative diseases, many associated with tau pathology, including Guam Parkinson dementia complex and Alzheimer's disease (AD). Although it is not fully understood whether TDP-43 aggregate formation is a protective mechanism or has a detrimental effect, looking for chemical probes that clear aggregates at the phenotypic level regardless of the mechanism of action should help advance understanding of TDP-43 proteinopathies. In this study, we used HEK293 cells expressing an inducible fluorescently tagged TDP-43 construct containing 12Q/N repeats that are sufficient to cause TDP-43 aggregation, sequestration of endogenous TDP-43, TDP-43 splicing loss of function and locomotive defect in Drosophila model. After induction of aggregate formation for 24h, the cells were exposed to compounds from our in-house library or from our annotated compound set for 48h. Effects of these compounds were assessed by high content imaging on live cells looking at total number of aggregates per well (green fluorescent tag; measure of aggregate clearance), total number of cells per well (Hoechst staining; measure of cell viability) and TMRM intensity (measure of mitochondrial toxicity). We identified 161 compounds from our in-house library and 94 compounds from our annotated compound set that induce aggregate clearance in a dose dependent manner without affecting the other cell physiology parameters. As part of our effort to understand the mechanism of action of the identified compounds, effect of exemplar compounds on POLDIP3 splicing and LC3 cellular localisation were investigated. The use of a cell line producing TDP-43 aggregates without relying on TDP-43 overexpression is a good tool to identify chemical probes leading to aggregate clearance without cell toxicity. Follow up of compounds and understanding of their mechanisms of action will prove beneficiary to advance research on a variety of neurodegenerative diseases where aggregates formation occurs.