Mechanisms of gain-of-repeat-toxicity and loss-of-C9ORF72-function in FTD and ALS - PROJECT SUMMARY/ABSTRACT Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are incurable, adult-onset neurodegenerative diseases. Our understanding of their etiology is incomplete, and no treatment can halt their progression. This project focuses on the most common genetic cause of FTD and ALS–the GGGGCC repeat expansion in the C9orf72 gene (known as C9FTD/ALS). This expansion can mediate both gain-of-toxicity (via repeat RNAs and accumulation of dipeptide-repeat proteins) and loss of the normal function of the C9ORF72 protein. Whether and how gain and loss mechanisms interact with each other and whether there are convergent pathways to provoke the accelerated decline that characterizes these two distinct diseases remain unresolved questions. The objective of this project is therefore to define the contributions of gain-of-repeat-toxicity and loss- of-C9ORF72-function to the cognitive and motor phenotypes using mouse models (our established model of FTD and our new mouse model of C9ALS, which for the first time successfully recapitulates C9ALS hallmarks). Our preliminary data show that (i) repeat expression by itself leads to ALS phenotypes, which sheds light into a long-standing question in the field; (ii) suppressing microglial activation delays the onset of paralysis in our C9ALS model; and (iii) reduction of C9ORF72 accelerates motor function decline and paralysis onset, increases microgliosis, and associates with profound and regionalized transcriptional changes in microglia in the C9ALS model. Because microglia have regional specificity in the central nervous system, it is possible that C9ORF72 reduction would affect C9FTD and C9ALS microglia differently. Altogether, we hypothesize that microglial overactivation mediated by C9ORF72-reduction accelerates the development of cognitive and motor deficits in FTD and ALS triggered by expanded repeat expression. To test this hypothesis, we will first identify the neuropathological changes linked to neuronal gain-of-toxicity in our C9ALS model. Next, we will define the effects of suppressing microglial activation to dissect their role (non- cell autonomous). We will also determine whether reduced C9ORF72 function worsens the motor deficits in C9ALS, as we previously showed in C9FTD. We will then confirm that microglial reduction of C9ORF72 is necessary for the accelerated phenotype, by defining the effects of knocking out C9orf72 exclusively in microglia on both cognitive/motor phenotypes and neuropathology. Last, we will identify microglial transcriptional changes and subpopulations in both models as neurodegeneration progresses, using spatial transcriptomics at single- cell level and validation in C9FTD/ALS postmortem tissue. Once completed, the proposed research is expected to uncover molecular mechanisms underlying these disorders with the potential to be developed into novel therapeutic interventions. Since the C9orf72 repeat expansion is also implicated in sporadic cases of ALS and FTD, our findings can have broad relevance.
