Novel repeat associated non-AUG (RAN) proteins in sALS sFTD and SBMA: shared pathological features and unifying therapeutic opportunities - ABSTRACT Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are complex neurodegenerative diseases that affect motor neurons in various regions of brain and spinal cord with devastating impacts on a patient’s health and lifespan. While research has identified ALS and FTD mutations in a number of genes (i.e. C9orf72, MAPT, SOD, and GRN), approximately 90% of ALS and 60% of FTD patients present as sporadic cases (sALS & sFTD) with unknown genetic etiology. Complex disease mechanisms coupled with a large genetically and phenotypically heterogeneous patient population have severely limited research and therapeutic success for these diseases. The discovery of the intronic C9orf72 G4C2 repeat expansion mutation as the most common genetic cause of ALS and FTD links these diseases to the larger family of microsatellite expansion disorders. Amongst these diseases is spinal-bulbar muscular atrophy (SBMA), a CAG•CTG disease that, like ALS and FTD, also affects motor neurons. A growing number of expansion disorders are reported to express proteins in multiple reading frames by repeat associated non-AUG (RAN) translation. Our recent unpublished findings show that novel polySer and polyLeu RAN proteins accumulate in at least six of the ten CAG•CTG polyGln diseases. These observations raise the possibility that novel polySer and polyLeu RAN proteins accumulate in the spinal-bulbar muscular atrophy (SBMA) and that other unidentified RAN proteins may contribute to sALS and sFTD. Our central hypothesis is that repeat expansion mutations that express novel RAN proteins substantially contribute to sALS, sFTD and spinobulbar muscular atrophy (SBMA) and that therapeutic approaches that reduce RAN protein levels will improve disease in preclinical models. To address this hypothesis, we have developed an innovative pathology-to-genetics strategy that enables rapid and direct identification of novel RAN protein producing expansion mutations from patient DNA. We are excited to report that in an initial screen, ~30% of sALS autopsy cases of unknown genetic etiology (i.e. C9 and SCA36 negative) were positive for GR or PR RAN protein aggregates suggesting the presence of novel expansion mutations. In this proposal, we will test the hypothesis that novel types of RAN proteins contribute to sALS, sFTD and SBMA (Aim 1) using immunoassays and patient blood and autopsy tissue samples. In Aim 2, we will utilize an innovative dCas9READ method to identify novel repeat expansion mutations in RAN(+) sALS and sFTD cases and study the toxic effects of putative disease-causing expansion mutations. Lastly, we will test the hypothesis that decreasing RAN translation using AAV-PKR(K296R) or metformin will improve disease phenotypes in patient derived induced models and mouse models of sALS, sFTD and SBMA (Aim 3). Taken together, these studies will provide critical insights into the molecular mechanisms of sALS, sFTD and SBMA and facilitate the development of unifying therapeutic approaches to fight these devastating diseases.
