Holbling, Benedikt Vinzenz; (2023) Identifying modulators of C9orf72 DPRs and STMN2 levels in ALS/FTD using high throughput screening. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases on the same continuum. In this thesis, I will focus on two key genetic and pathological hallmarks of ALS/FTD which have strong therapeutic potential. A hexanucleotide repeat expansion in the gene C9orf72 is the most common genetic cause of familial and sporadic FTD and ALS. Repeat-associated non-AUG (RAN) translation of the repeat containing sequence gives rise to five dipeptide repeat (DPR) proteins, which cause neural toxicity and are hypothesized to contribute to neurodegeneration. RAN translation is an unconventional translation process that may serve as a therapeutic target for C9 ALS/FTD, however, it is not well understood yet. In this study, I generated the first endogenous DPR reporter cell line by genome editing patient-derived iPSCs to tag RAN translation products. Thereafter, I utilized this reporter system to establish a high-throughput screening platform to identify modulators of C9orf72 RAN translation in proof-of-concept screens. By genetic CRISPR nuclease screening, I identified the helicases ERCC8, RTEL1 and UPF1 as selective genetic enhancers of DPR expression. Further, small molecule screening revealed four compounds either increasing or decreasing endogenous DPR levels. These pilot screens validate the capability of our platform that will now be utilized for larger screening campaigns and further be made publicly available. Cytoplasmic mislocalization and aggregation of TDP-43 is a pathological hallmark present in approximately 97% of ALS patients and 50% of FTD patients. Besides a potential gain of toxicity from cytoplasmic aggregates, nuclear depletion significantly disrupts RNA processing. The RNA transcript of the microtubule binding factor STMN2 is amongst the genes most affected by TDP-43 depletion. Enhancing STMN2 levels in the context of TDP43 pathology rescues multiple cellular phenotypes and is therefore an interesting target for therapeutic approaches. Here, I developed a high-throughput screening platform to detect small molecule enhancers of STMN2 protein levels in inducible TDP-43 knockdown cells. After two pilot screens, I performed a high-throughput screening campaign by primary, secondary, and counter screening to characterize compound effects on STMN2. The most potent and selective compounds will now be deeply characterized, chemically optimized, and tested in a phenotypic assay, established during the work of this PhD project. Overall, the work in this thesis generated new tools for the FTD/ALS research community that will hopefully contribute to therapy development. Further, it has enhanced our understanding of the regulation of DPR expression by identifying novel endogenous and small molecule modulators.

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