Emerging Mechanisms Underpinning Neurophysiological Impairments in C9ORF72 Repeat Expansion-Mediated Amyotrophic Lateral Sclerosis/Frontotemporal Dementia

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are characterized by degeneration of upper and lower motor neurons and neurons of the prefrontal cortex. The emergence of the C9ORF72 hexanucleotide repeat expansion mutation as the leading genetic cause of ALS and FTD has led to a progressive understanding of the multiple cellular pathways leading to neuronal degeneration. Disturbances in neuronal function represent a major subset of these mechanisms and because such functional perturbations precede degeneration, it is likely that impaired neuronal function in ALS/FTD plays an active role in pathogenesis. This is supported by the fact that ALS/FTD patients consistently present with neurophysiological impairments prior to any apparent degeneration. In this review we summarize how the discovery of the C9ORF72 repeat expansion mutation has contributed to the current understanding of neuronal dysfunction in ALS/FTD. Here, we discuss the impact of the repeat expansion on neuronal function in relation to intrinsic excitability, synaptic, network and ion channel properties, highlighting evidence of conserved and divergent pathophysiological impacts between cortical and motor neurons and the influence of non-neuronal cells. We further highlight the emerging association between these dysfunctional properties with molecular mechanisms of the C9ORF72 mutation that appear to include roles for both, haploinsufficiency of the C9ORF72 protein and aberrantly generated dipeptide repeat protein species. Finally, we suggest that relating key pathological observations in C9ORF72 repeat expansion ALS/FTD patients to the mechanistic impact of the C9ORF72 repeat expansion on neuronal function will lead to an improved understanding of how neurophysiological dysfunction impacts upon pathogenesis.

Disrupting the Molecular Pathway in Myotonic Dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults. It consists of two forms: type 1 (DM1) and type 2 (DM2). DM1 is associated with a trinucleotide repeat expansion mutation, which is transcribed but not translated into protein. The mutant RNA remains in the nucleus, which leads to a series of downstream abnormalities. DM1 is widely considered to be an RNA-based disorder. Thus, we consider three areas of the RNA pathway that may offer targeting opportunities to disrupt the production, stability, and degradation of the mutant RNA.

CRISPR-Cas9 targeted deletion of the C9orf72 repeat expansion mutation corrects cellular phenotypes in patient-derived iPS cells

The large hexanucleotide (GGGGCC) repeat expansion in the non-coding promoter region of C9orf72 is the leading cause of Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Mechanisms underlying neurodegeneration are not clear, and both a C9orf72 loss of function and a gain of toxicity, in the form of RNA foci or dipeptide repeat deposition, are implicated. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9-mediated genome editing is an attractive strategy for disease modeling and therapeutic intervention. Here we show that this system can be utilized to completely remove the large repeat expansion mutation within C9orf72 in patient-derived induced pluripotent stem (iPS) cells. Removal of the mutation prevented RNA foci formation and promoter hypermethylation, two phenotypes of the C9orf72 mutation. Interestingly, these changes did not significantly alter C9orf72 expression at the mRNA or protein level. This work provides a proof-of-principle for the use of CRISPR-Cas9-mediated excision of the pathogenic C9orf72 repeat expansion as a therapeutic strategy in FTD/ALS.One Sentence SummaryCRISPR-Cas9-mediated excision of the large C9orf72 repeat expansion mutation rescues RNA foci formation and promoter hypermethylation without altering C9orf72 transcript and protein expression.