Molecular effects of metformin, PKR and TBI on C9orf72 ALS/FTD

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Project Summary The G4C2 repeat expansion mutation in the C9orf72 gene is the most common, known cause of familial and sporadic amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD)1,2. There are no effective treatments for C9-ALS/FTD or >50 other expansion disorders3,4. Our work identified two mechanisms now thought to play major roles in C9-ALS/FTD and other expansion diseases: 1) bidirectional transcription of expansion mutations5; and 2) repeat associated non-AUG (RAN) translation6. More than 90% of ALS presents as sporadic disease with no family history or genetic diagnosis. Surprisingly, our data show RAN proteins accumulate in ~44% of C9orf72-negative [C9(-)] sALS autopsy cases. The accumulation of toxic RAN proteins in C9(-) sALS and a growing number of other diseases highlights the need to develop drugs that block their production7-9. Activation of the integrated stress response (ISR) increases RAN translation10,11 and expansion RNAs activate the ISR kinase protein kinase R (PKR)12,13. We showed targeting RAN proteins in transgenic C9orf72 BAC mice with antibodies14 or PKR inhibition13 mitigates disease. We also discovered that metformin, a well-tolerated diabetes drug inhibits PKR, decreased RAN protein levels, improved behavior, histopathology, and motor neuron survival in C9-ALS/FTD mice13 but the mechanisms for these effects are not yet understood. These data combined with increased rates of ALS in patients with traumatic brain injury (TBI) suggest the possibility that stress and injury trigger ALS in vulnerable populations through increased RAN translation. This proposal aims to understand the mechanisms of PKR and metformin on RAN translation and to test TBI as a trigger of RAN translation and ALS that can be mitigated by PKR inhibition and metformin. In this proposal we will test the following hypothesis: (1) that metformin improves disease by reducing RAN proteins in C9orf72 ALS/FTD in a PKR dependent manner. Understanding the mechanism of action of metformin will provide important insights to advance its clinical use for C9-ALS/FTD and to improve drug efficacy; (2) that AAV-PKR-K296R is a highly effective therapeutic strategy to reduce RAN proteins in C9-ALS/FTD through p- eIF2α-dependent and independent pathways. Understanding how PKR regulates RAN translation and if AAV- PKR-K296R improves disease in adult C9-BAC mice will facilitate the development therapeutic strategies for C9orf72 ALS/FTD; and (3) that injury will increase RAN translation and exacerbate disease in C9orf72 mice and brain organoids from C9+ and genetically unknown, sporadic RAN+ sALS (gsr+ALS) patients and that reducing RAN protein levels will be protective. Taken together these innovative series of experiments could fundamentally change the diagnostics and treatment options for patients with C9-ALS, genetically unknown RAN positive sALS as well as the larger family of RAN protein associated expansions diseases. 1