Hyperexcitability in young iPSC-derived C9ORF72 mutant motor neurons is associated with increased intracellular calcium release

Abstract

A large hexanucleotide repeat expansion in the C9ORF72 gene is the most prevalent cause of amyotrophic lateral sclerosis (ALS). To better understand neuronal dysfunction during ALS progression, we studied motor neuron (MN) cultures derived from iPSC lines generated from C9ORF72 (C9) expansion carriers and unaffected controls. C9 and control MN cultures showed comparable mRNA levels for MN markers SMI-32, HB9 and ISL1 and similar MN yields (> 50% TUJ1/SMI-32 double-positive MNs). Using whole-cell patch clamp we showed that C9-MNs have normal membrane capacitance, resistance and resting potential. However, immature (day 40) C9-MNs exhibited a hyperexcitable phenotype concurrent with increased release of calcium (Ca²⁺) from internal stores, but with no changes to Na_V and K_V currents. Interestingly, this was a transient phenotype. By day 47, maturing C9-MNs demonstrated normal electrophysiological activity, displaying only subtle alterations on mitochondrial Ca²⁺ release. Together, these findings suggest the potential importance of a developmental component to C9ORF72-related ALS.

Figure 1

Characterization of C9ORF72 MNs and CTRL MNs. (a) Diagram of the MN protocol adapted from Du et al.. (b) iPSC-derived MNs immunostained for SMI-32 and TUJ1. Scale bars = 100 µm. (c) Flow cytometry of cells stained with TUJ1 and SMI-32 to verify MN counts (mean ± SEM), analysed using FlowJo v10.4 Software (BD Life Sciences). Two-way RM ANOVA: Genotype p = 0.0233, Time p = ns, Interaction p = ns with Sidak’s multiple comparisons, **p < 0.01, N = 3 lines, 3 differentiations (d) RT-qPCRs for HB9, ISL1 and SMI-32 (mean ± SEM). Two-way RM ANOVA: Genotype p = ns, Time p = < 0.0001, Interaction p = ns, N = 3 lines, 2 differentiations.

Figure 2

Hyperexcitability is present in young C9ORF72 MNs vs CTRL MNs. Whole-cell patch clamp of iPSC-derived MNs showed that (a) membrane capacitance (C_m), (b) membrane resistance (R_m), and (c) resting membrane voltage were not significantly different between C9 and CTRL MNs at day 40 nor at day 47. Two-Way RM ANOVA: p = ns, N = 3 lines, 1–2 differentiations. (di,ei) Representative traces of evoked action potential firing. (dii,eii) Average action potential firing per 500 ms for each current step (mean ± SEM). Non-linear regression ****p = < 0.0001.

Figure 3

Transient calcium dyshomeostasis is evident in C9ORF72 MNs vs CTRL MNs. Example traces for Na_V at − 20 mV (a) and K_V at + 50 mV. (b) Current densities show no significant difference between the CTRL or C9 groups (Na_V IV graph, ci; K_V IV graph, di). The same measures also show no difference at day 47, (Na_V IV graph, cii; K_V IV graph, dii). However, a temporal comparison shows a significant increase of average peak current density of Na_V current and K_V current over time. Two-way RM ANOVA: Interaction Na⁺/K⁺ p = ns, Time *Na⁺ p = 0.0282, *K⁺ p = 0.0145, Genotype Na⁺/K⁺ p = ns with Sidak’s multiple comparisons Na⁺/K⁺ p = ns, N = 3 lines, 1–2 differentiations. Investigation of calcium store release (ei, eii; mean ± SEM) evoked by ionomycin injection showed a significant increase in the delta Fura-2 AM fluorescence of C9 versus CTRL at Day 40 (eiii; Two-way RM ANOVA: Interaction **** p = < 0.0001, Time p = < 0.0001, Genotype p = ns) but not Day 47 (eiv; Interaction p = ns, Time p = < 0.0001, Genotype p = ns.) Unpaired T-Test p * < 0.05, N = 3 lines, 2–3 differentiations.