A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.

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.

In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT 3 Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonistevoked responses of 5-HT 3 Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT 3 Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT 3A Rs (64 6 7% and 80 6 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT 3A Rs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT 3A Rs or are able to confer this property on mouse 5-HT 3A Rs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT 3A Rs by 5-HT. We conclude that 5-HT 3A Rs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies.