![]() ![]() ![]() To validate the use of this iPSC model, we confirmed these expanded C9ORF72-related phenotypes in postmortem human ALS CNS tissue. In addition, we discovered aberrantly expressed genes in C9ORF72 cells and determined that C9ORF72 ALS iPSNs are highly susceptible to glutamate-mediated excitotoxicity. Furthermore, we identified several protein binding partners for the expanded GGGGCC RNA (GGGGCC exp) and confirmed that the RNA binding protein ADARB2 interacts with nuclear GGGGCC RNA foci. Using this model system, we discovered intranuclear C9ORF72 repeat-containing RNA fociin all tested human C9ORF72 iPSN cell lines. To understand the pathogenesis of the C9ORF72 expansion and to develop possible therapeutics, we generated a collection of C9ORF72 ALS induced pluripotent stem cells (iPSCs) and differentiated them into neurons (iPSNs). Notably, intranuclear GGGGCC RNA foci have also been found in the motor cortex and spinal cord of C9ORF72 ALS/FTD patients ( DeJesus-Hernandez et al., 2011), suggesting that, like myotonic dystrophy, RNA toxicity plays a role in C9ORF72 neurodegeneration. MBLN1 is sequestered in the nucleus by the repeat-containing RNA resulting in the formation of a pathogenic protein:RNA complex that, when visualized by RNA fluorescent in situ hybridization, form an intranuclear RNA foci, which leads to a loss of protein activity and reduces alternative splicing of other genes ( Kanadia et al., 2003, 2006). The repeat expansion in DM1 alters activities of RNA binding proteins (RBPs), including muscleblind-like 1 (MBLN1) ( Fardaei et al., 2002 Grammatikakis et al., 2011 Miller et al., 2000). Repeat expansions are classified as coding or noncoding according to their gene location, and the disease-causing mechanisms include protein gain-of-function (Huntington’s disease, HD), protein loss-of-function (FRAXA, FRDA), toxic RNA gain-of-function (DM1&2) (for reviews, see Brouwer et al., 2009 Gatchel and Zoghbi, 2005 Todd and Paulson, 2010), and non-ATG-initiated translation (RAN) peptides ( Mori et al., 2013b) ( Ash et al., 2013). Since the discovery of pathogenic repeat expansions as a mechanism of disease in the 1990s, the list of neurodegenerative and neuromuscular disorders characterized by unstable repeat expansions has grown to over 20 ( Brouwer et al., 2009 Pearson et al., 2005 Todd and Paulson, 2010). These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.Ī recently discovered hexanucleotide ‘‘GGGGCC’’ repeat’’ expansion in the noncoding region of the C9ORF72 gene has been found in at least 8% of sporadic ALS (sALS) and FTD cases and more than 40% of familial ALS (FALS) and FTD cases ( DeJesus-Hernandez et al., 2011 Majounie et al., 2012 Renton et al., 2011). These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCC exp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCC exp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ![]()
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