C9orf72 Hexanucleotide Expansions Are Associated with Altered Endoplasmic Reticulum Calcium Homeostasis and Stress Granule Formation in Induced Pluripotent Stem Cell-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia

Abstract

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC‐derived motor neurons, decreased cell survival is correlated with dysfunction in Ca2+ homeostasis, reduced levels of the antiapoptotic protein Bcl‐2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC‐derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063–2078

A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism

“…First, as opposed to findings in C9orf72 iPSC-derived neurons [1,13], we observed no increased levels of p62. However, p62 was upregulated by overexpression of Pur-alpha but not by the Pur-alpha deletion constructs lacking the G-rich or PUR2 domain.…”

“…p62 is upregulated by Pur-alpha and prevents repeat RNA toxicity As p62 protein levels have previously been shown to be increased in C9orf72 iPSC-derived neurons [1,13], we investigated the possible involvement of p62 in our zebrafish model. Injection of sense repeat RNA did not modify the protein (Fig.…”

Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease

“…All other iPSC lines (Parkinson's disease and additional matched healthy control patients) were derived from skin biopsies or monocytes and reprogrammed in the same laboratory as described previously (33,35). iPSC lines were grown in mTeSR1 on Matrigel (Corning)-coated tissue culture dishes, passaged using 0.5 mM EDTA or using TrypLE, and plated with the Rho-kinase inhibitor Y-27632 (10 μmol/l).…”

“…Previously uncharacterized iPSC lines (JR036-2 and OX1-40) were characterized as described previously (35,40). Briefly, genome integrity was assessed by Illumina Human CytoSNP-12v2.1 beadchip array (∼300 000 markers) and analyzed using Karyostudio to generate karyograms and Genomestudio software (Illumina) to track samples to confirm parentage.…”

Pathogenic determinants and mechanisms of ALS/FTD linked to hexanucleotide repeat expansions in the C9orf72 gene

“…Most studies reported that individual DPRs shared similar localization patterns but displayed different abundances: from highest to lowest are poly-GA, poly-GP, poly-GR, and poly-PA/poly-PR [89, 90]. Similar findings were also reported in C9 patient iPSCs derived neurons and C9 patient CSF (poly-GP was detected) [33, 49, 91–94]. Fundamental questions regarding the role of these C9-DPRs remain: Is the presence of DPRs a mere pathological hallmark?…”