Origin and cell type specificity of mitochondrial DNA mutations in <i>C9ORF72</i> ALS-FTLD human brain organoids

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are primarily genetic in ~20% of patients. Mutations in <i>C9ORF72</i> are the most frequent cause, but it is not understood why there is notable regional pathology. An increased burden of mitochondrial DNA (mtDNA) mutations in ALS-FTLD brains implicates mitochondrial mechanisms; however, it remains unclear how and when these mutations arise. To address this, we generated cerebral organoids derived from human-induced pluripotent stem cells (hiPSCs) of patients with ALS-FTLD harboring the <i>C9ORF72</i> hexanucleotide repeat expansion alongside CRISPR-corrected isogenic and healthy controls. Here, we show a higher mtDNA single-nucleotide variant (mtSNV) burden in astroglia derived from <i>C9ORF72</i>-mutant organoids, with some de novo mtSNVs likely due to the <i>C9ORF72</i> repeat and others evading selection to reach higher heteroplasmy levels. Thus, the functional consequences of the regional accumulation of mtSNVs in <i>C9ORF72</i> ALS-FTLD brains are likely to manifest through astroglial mitochondrial dysfunction.