No treatment for frontotemporal dementia (FTD), the second most common early-onset dementia, is available but therapeutics are being investigated to target the two main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hamstrung by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human iPSC-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease sub-types marked by TDP-43 inclusions. Lastly, we validated that truncated STMN2 RNA is elevated in the frontal cortex of a cohort of FTLD-TDP cases but not in controls or cases with progressive supranuclear palsy (PSP), a type of FTLD-tau. Further, in FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

TDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9ORF72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9ORF72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9ORF72 iPSC-MNs have higher Ca 2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca 2+ release. We identify here, by RNA sequencing, that both C9ORF72 and TARDBP iPSC-MNs have upregulation of Ca 2+ -permeable AMPA and NMDA subunits and impairment of mitochondrial Ca 2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca 2+ uniporter, indicating that impaired mitochondrial Ca 2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9ORF72 or TARDBP mutations.

The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and non-homologous end-joining by deleting the repeat region, with the risk of creating indels and genomic instability. In this study, we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/frontotemporal dementia patient using CRISPR/Cas9 genome editing and homology-directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal gene expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC-derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared with its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the Kyoto Encyclopedia of Genes and Genomes ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9-mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.