Saturday, May 4, 2024
5/4/2024
PROJECT SUMMARY The C9orf72-mediated ALS (amyotrophic lateral sclerosis) and FTD (frontotemporal dementia) (C9- ALS/FTD) are two fatal neurodegenerative diseases with no curative treatment. We aim to address a new mechanism at the core of C9-ALS/FTD to improve the therapeutic potential. While ALS is an adult-onset disease with progressive degeneration of motor neurons, and while FTD is characterized with progressive decline of the frontal and temporal lobes of the brain, the two share a common genetic cause – repeat expansion of the nucleotide sequence G4C2 in the first intron of the C9orf72 gene. Of the proposed causes of C9-ALS/FTD, the most significant is the synthesis of dipeptide repeat (DPR) proteins from ribosome translation of the expanded repeats. These DPRs are consistently observed in patient tissues and in various model systems. While the initiation of DPR synthesis is non-canonical, the elongation of protein synthesis is by the classical ribosome machinery that translates each codon using a set of tRNA species with the matching anticodon, which is provided by the cellular tRNA pool. Notably, translation of repeated sequences is highly challenging, requiring repeated use of the tRNAs for the same codon, where each species must be efficiently charged and post-transcriptionally modified and matured. This challenge is further intensified in patient cells, where the repeat length can run up to thousands, raising the question of how the cellular tRNA pool responds to such an unusual demand. This is an unexplored, but critical, question in C9-ALS/FTD, because the deficiency of quality tRNAs can shift the ribosome translational reading-frame, leading to frameshifting and synthesis of chimeric DPRs that would alter the disease pathology. Here we will address this question, based on our extensive expertise in tRNA and in tRNA-associated ribosome frameshifting. In Aim 1, we will determine changes of the tRNA pool in C9 patient-derived cells relative to an isogenic control, using our improved tRNA-seq. We will also decipher changes of the ribosome-mediated global protein synthesis that underlie the disease. We will determine changes both in the iPSC (induced pluripotent stem cell) state and in the differentiated neuron (iPSN) state, due to their fundamental differences in regulation of protein synthesis, and due to the potential to produce new insight into the disease during differentiation. In Aim 2, we will test the hypothesis that the course of DPR synthesis can be induced to undergo a desired frameshifting to produce less toxic proteins that can reduce the disease pathology. We will test how changes of the abundance and charging levels of the tRNAs required for DPR synthesis can induce the desired frameshifting. This work will serve as the foundation for understanding the therapeutic implications of tRNA and ribosome protein synthesis in the development of C9-ALS/FTD, providing a template that is generalizable to other aging and neurodegenerative diseases associated with nucleotide repeat expansions.
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