Human iPSC-derived motoneurons harbouring TARDBP or C9ORF72 ALS mutations are dysfunctional despite maintaining viability

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease for which a greater understanding of early disease mechanisms is needed to reveal novel therapeutic targets. We report the use of human induced pluripotent stem cell (iPSC)-derived motoneurons (MNs) to study the pathophysiology of ALS. We demonstrate that MNs derived from iPSCs obtained from healthy individuals or patients harbouring TARDBP or C9ORF72 ALS-causing mutations are able to develop appropriate physiological properties. However, patient iPSC-derived MNs, independent of genotype, display an initial hyperexcitability followed by progressive loss of action potential output and synaptic activity. This loss of functional output reflects a progressive decrease in voltage-activated Na(+) and K(+) currents, which occurs in the absence of overt changes in cell viability. These data implicate early dysfunction or loss of ion channels as a convergent point that may contribute to the initiation of downstream degenerative pathways that ultimately lead to MN loss in ALS.

Figure 1. Differentiation of MNs from control and patient iPSC lines.

(a) Immunohistochemical staining of differentiated iPSCs (control, TARDBP and C9ORF72 lines) using antibodies raised against β3-tubulin, Hb9, SMI-32 and glial fibrillary acidic protein (GFAP). (b) Proportion of differentiated iPSCs expressing β3-tubulin, Hb9 and GFAP (total cells counted: control, 1,126 cells; TARDBP, 1,224 cells; C9ORF72, 1,534 cells; scale bar, 50 μm).

Figure 2. Equivalent viability of control and patient iPSC-derived MNs.

(a) IR-DIC images of iPSC-derived MNs from control, TARDBP and C9ORF72 lines at weeks 9–10 post plating (scale bar, 20 μm). (b) LDH activity plotted for control and patient iPSC-derived cultures from weeks 3–10 post plating (Control lines (D6: two experiments, M2 and R6), TARDBP lines (D1 and D3), C9ORF72 (S6: two experiments and R2); data are plotted as ; *P<0.05; factorial ANOVA).

Figure 3. Hyperexcitability followed by loss of action potential output in patient iPSC-derived MNs.

(a) Fluorescent image of a cell filled with Alexa Fluor 488 dye during whole-cell patch-clamp recordings and immunolabelling with an antibody raised against SMI-32 (arrow heads point to cell soma; scale bar, 10 μm). (b) Repetitive firing in response to square current injection in iPSC-derived MNs from control, TARDBP and C9ORF72 lines. (c) Frequency-current (f–I) relationships generated for repetitively firing iPSC-derived MNs from control (n=62), TARDBP (n=19) and C9ORF72 (n=19) lines recorded from weeks 2–6 post plating (data are plotted as with lines of best fit; *significantly different to control, P<0.05; ***significantly different to control, P<0.0001; linear model with multiple contrast for the gradient values, and adjusted with Bonferroni correction). (d) Examples of the four categories of firing observed in iPSC-derived MNs (repetitive, adaptive, single or no firing). (e) Proportion of cells exhibiting each firing category in iPSC-derived MNs from control (n=702), TARDBP (n=380) and C9ORF72 (n=239) lines across weeks 3–10 post plating (***significantly different to control, P<0.0001; logistic regression with multiple Wald’s test and Bonferroni correction).

Figure 4. Loss of synaptic input to patient iPSC-derived MNs.

(a) Current responses in iPSC-derived MNs during bath application of glutamate (100 μM), GABA (100 μM) and glycine (100 μM). (b) Voltage-clamp recordings of spontaneous synaptic activity in iPSC-derived MNs at different holding potentials. (c) Proportion of cells displaying synaptic activity from weeks 3–10 post plating in iPSC-derived MNs from control (n=845), TARDBP (n=417) and C9ORF72 (n=265) lines. (*significantly different to control, P<0.05; ***significantly different to control, P<0.0001; logistic regression with multiple Wald’s tests and Bonferroni correction). (d) Graphs of inter-event interval and amplitude of synaptic events recorded from control and patient iPSC-derived MNs.

Figure 5. Loss of fast-inactivating Na⁺ currents in patient iPSC-derived MNS.

(a) Raw data showing fast, inactivating Na⁺ currents in control, TARDBP and C9ORF72 iPSC-derived MNs at week 3–4 and weeks 9–10. (b) Current–voltage relationships of peak Na⁺ currents recorded from control and patient iPSC-derived MNs at weeks 3–4. (c) Current–voltage relationships of peak Na⁺ currents recorded from control and patient iPSC-derived MNs at weeks 9–10. (d) Peak fast, inactivating Na⁺ currents plotted from weeks 3–10 for control (n=847), TARDBP (n=452) and C9ORF72 (n=264) iPSC-derived MNs. (data are plotted as ; *significantly different to controls, P<0.05; ***significantly different to controls, P<0.0001; #significant difference between patient lines, P<0.05; ##significant difference between patient lines, P<0.001; linear model with multiple Wald’s tests and Bonferroni correction).

Figure 6. Loss of persistent voltage-activated K⁺ currents in patient iPSC-derived MNs.

(a) Raw data showing persistent K⁺ currents in control, TARDBP and C9ORF72 iPSC-derived MNs at week 3–4 and weeks 9–10. (b) Current–voltage relationships of peak K⁺ currents recorded from control and patient iPSC-derived MNs at weeks 3–4. (c) Current–voltage relationships of peak K⁺ currents recorded from control and patient iPSC-derived MNs at weeks 9–10. (d) Peak K⁺ currents plotted from weeks 3–10 for control (n=847), TARDBP (n=452) and C9ORF72 (n=264) iPSC-derived MNs (data are plotted as ; *significantly different to control, P<0.05; ***significantly different to control, P<0.0001; #significant difference between patient lines, P<0.05; ###significant difference between patient lines, P<0.0001; linear model with multiple Wald’s tests and Bonferroni correction).

Figure 7. Firing categories of iPSC-derived MNs are predicted by peak Na⁺ and K⁺ currents.

(a) Relationship between peak Na⁺ and K⁺ currents and action potential firing category were determined using multinomial logistic regressions in control (n=448), TARDBP (n=230) and C9ORF72 (n=176) iPSC-derived MNs at 3–6 weeks post plating. Values in parentheses denote the proportion, as a percentage, of iPSC-derived MNs exhibiting each firing category. (b) Predicted probability of each firing category calculated over a range of peak Na⁺ currents. (c) Predicted probability of each firing category calculated over a range of peak K⁺ currents.