Characterization of TDP-43-related de novo proteins in ALS/FTD

Thesis

Mislocalization of the DNA/RNA-binding protein TDP-43, characterized by its nuclear loss and cytosolic accumulation, is the predominate pathology in amyotrophic lateral sclerosis (ALS) and occurs in up to 50% of frontotemporal dementia (FTD) cases. TDP-43 plays a major role in splicing regulation, and its loss from the nucleus leads to the erroneous expression of intronic sequences, called cryptic exons. Cryptic exons are non-conserved sequences that often result in degradation of affected transcripts, with potential deleterious consequences for cellular function. The aim of my dissertation work was to address whether certain cryptic exon-harboring transcripts could generate de novo proteins in settings of TDP-43 deficiency. I first discuss the development of a proteogenomic informatic pipeline, leveraging parallel transcriptomic and proteomic data from TDP-43-depleted human iPSC-derived neurons to identify 65 putative cryptic peptides from 12 transcripts. The cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were highly predictive of dysregulated splicing in postmortem brain tissue from patients with TDP-43 proteinopathy, thus validating the physiological relevance of our in vitro model. I then demonstrate the orthogonal validation of cryptic peptides identified by proteogenomics using antibody- and targeted proteomics-based assays in TDP-43-depleted iPSC-derived neurons. I next show that expression of cryptic exon sequences can alter the interactome of affected proteins, thereby likely affecting their function. Finally, using an ultra-sensitive target proteomics approach, I demonstrate that at least 18 de novo peptides across 13 genes are unequivocally present in the CSF of individuals with ALS/FTD spectrum disorders. The discovery of cryptic exon translation suggests potential new mechanisms of disease pathogenesis downstream of TDP-43 dysfunction in ALS/FTD and provides a novel strategy for the development of assays to monitor TDP-43 function in living individuals.

Authors: Seddighi, S

• DOI: 10.5287/ora-wv98kaoo2
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English