Dipeptide Repeat Proteins Associated with C9orf72 ALS/FTD: Understanding How They Are Made and How They Potentiate Disease

Thomas Raymond Stefeson Westergard, Thomas Jefferson University


Nucleotide repeat expansions (NREs) are common mutations found in a multitude of neurodegenerative diseases. The RNA transcripts of NREs have been discovered to undergo repeat-associated non-ATG (RAN) translation, producing repeat poly-peptides that have been shown to drive disease pathology. Another common element in neurodegenerative diseases is the propensity for disease relevant proteins to transmit between cells, propagating disease pathology. Recently, an hexanucleotide repeat expansions (GGGGCC) in the non-coding region of the C9orf72 gene has been discovered to be the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The RAN translation of the sense and antisense transcripts of this NRE form five dipeptide repeat proteins (DPRs): glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), and proline-arginine (PR). Severe toxicity has been demonstrated by DPR models, particularly the arginine-rich species, in cellular cultures, drosophila, and mouse models. The studies presented in this thesis sought to explore two aspects of DPR pathophysiology: 1) To study whether DPRs are transmitted and propagate between cells as most of the pathogenic proteins do in neurodegenerative diseases; and 2) To understand the mechanisms and triggers of RAN translation, which leads to their production. In the studies related to DPR transmission, we utilized different experimental platforms in which we found evidence for cell-to-cell spreading for each DPR with varying modality and efficiency. The primary modality in which DPRs were transmitted between cells is through microvesicles known as exosomes. Exosomes isolated from DPR-expressing cells showed varying amounts of DPRs and evidence of transmission to neurons. We demonstrated anterograde and retrograde transmission between connecting neurons and found evidence of DPR transmission in iPSCs from C9orf72-ALS patients. Expanding on these findings, we found that poly-GR transmits in vivo when spatially expressed in a novel GR mouse model that we generated. In the studies involving mechanisms and triggers of DPRs productionour RAN translation studies, we developed a reporter construct consisting of 188 G4C2 repeats with a Dendra2 fluorescent tag and determined that a variety of inducers of cellular stress trigger RAN translation and production of DPRs in in vitro cell models. We found that increased neuronal activity and excito-stress specifically induced DPR production in primary cortical neurons and patient derived sMNs. Induction of RAN translation was concomitant with increases in the ISR, increasing levels of phosphorylated-eif2α. Finally, targeting these pathways with FDA approved small molecules reduced DPR production. Together, these studies deepen our understanding on the pathogenic properties of DPRs and the mechanisms involved in their production, both revealing potential therapeutic targets to prevent disease progression.

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Recommended Citation

Westergard, Thomas Raymond Stefeson, "Dipeptide Repeat Proteins Associated with C9orf72 ALS/FTD: Understanding How They Are Made and How They Potentiate Disease" (2018). ETD Collection for Thomas Jefferson University. AAI10936369.