Category: Assay Development and Screening
TAR DNA-binding protein 43 (TDP-43) is a splicing factor of the hnRNP family (heterogeneous nuclear ribonucleoprotein family) involved in many aspects of RNA metabolism, including splicing of POLDIP3 (polymerase delta interacting protein 3). Aggregates of ubiquitinated, misfolded and hyperphosphorylated TDP-43 are commonly found in the brains of ALS (amyotrophic lateral sclerosis), FTLD (frontotemporal lobar degeneration) and some Alzheimer's disease patients.
Although it is not fully understood whether TDP-43 aggregate formation is a protective mechanism or has a detrimental effect, the identification of compounds which induce aggregate clearance at the phenotypic level may help with the understanding of TDP-43 proteinopathies if followed up by bioinformatic, cheminformatic and mechanism of action studies.
In this study, a phenotypic high content imaging assay was used to screen for compounds which promote TDP-43 aggregate clearance regardless of their putative mode of action. The development of cellular models for TDP-43 expression has been difficult due to the cellular toxicity caused by overexpression systems therefore models are limited to those which are sufficiently robust for screening.
The model used in this study is a novel system expressing an inducible fluorescently tagged TDP-43 construct containing 12Q/N repeats in HEK293 cells. This construct is sufficient to cause TDP-43 aggregation, sequestration of endogenous TDP-43, TDP-43 splicing loss of function and locomotive defects in a Drosophila model, thus mimicking ALS disease phenotype.
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 in a live cell, multiparameter high content imaging assay with readouts measuring 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).
Several chemical clusters have been identified in the primary screen assay which induce aggregate clearance in a dose dependent manner, without affecting the other cell physiology parameters. The active compounds also restore TDP-43 function as an intronic splicing enhancer of POLDIP3 in a secondary functional assay indicative of on target effects.
Since an age-related decline of both the UPS (ubiquitin proteasome system) and autophagy pathways have been suggested to play a role in TDP-43 aggregation, mechanism of action and target deconvolution studies focusing on these pathways will help characterizing the compound’s mode of action.
Further follow up studies in more physiologically relevant cellular systems on a neuronal background and in the Drosophila model will strengthen this proof of concept study and prove beneficial to advance research on a variety of neurodegenerative diseases where aggregates formation occurs.
Emilie Bureau– Scientist, Cellular Pharmacology, LifeArc, Stevenage, England, United Kingdom
Scientist, Cellular Pharmacology
Stevenage, England, United Kingdom
Dr Emilie Bureau worked on stem cells during her PhD at Cancer Research UK in London and graduated from UCL (University College London) in 2008. Since then she has been working as a scientist at LifeArc (the new name for MRC Technology) in the Compound Profiling and Cellular Pharmacology department, where she has been involved in a number of small molecule and therapeutic antibody projects, focusing mainly on the areas of oncology and neuroscience.