SGK1 inhibition restores excitability of cortical neurons derived from Alzheimer's disease patients

Figures

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  Fig. 1.. Diminished voltage-gated Na+ currents in AD cortical neurons. (A-B) Human iPSC-derived cortical neurons from healthy control (top) and sAD patients (bottom) at day 110 were costained as indicated. Scale bar, 10 μm. (C) A representative phase contrast image of iPSC-derived human cortical neurons on day 100. Scale bar, 30 μm. (D) Cell capacitance of control and AD neurons. CON: n = 4 (8, 10, 19, 11 cells / line); AD: n = 4 (10, 11, 15, 9 cells / line); p = 0.8432. (E) Resting membrane potential of control and AD neurons. CON: n = 4 (8, 9, 19, 11 cells / line); AD: n = 4 (10, 11, 14, 9 cells / line); p = 0.6094. (F) Representative voltage-gated Na+ and K+ currents (black) induced by depolarizing voltage steps (grey) in control (top) and AD neurons (bottom). Insets, enlarged traces for control (blue) and AD (red) neurons. (G) Averaged I-V curves (colored) for Na+ currents in individual neurons (grey) derived from control subjects (top) and AD patients (bottom). (H) Peak Na+ currents evoked at −20 mV in control and AD neurons. CON: n = 4 (10, 6, 14, 6 cells / line); AD: n = 4 (10, 6, 10, 12 cells / line); p = 0.0030. (I) Averaged I-V curves (colored) for fast K+ currents in individual…

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  Fig. 2.. Reduced action potential amplitudes in AD cortical neurons. (A) Representative traces of evoked action potentials (eAPs) elicited by 20 pA current injection (grey) from control (blue) and AD neurons (red), with RMP held at −60 mV. (B) AP frequencies in response to this stimulation in control and AD neurons (p = 0.2564). (C-F) The first AP in the burst was used for spike shape analysis. AP shape analysis for peak amplitudes (p = 0.0166) (C), overshoots (p = 0.0274) (D), threshold (p = 0.3522) (E), and half-width (p = 0.7210) (F). CON: n = 4 (8, 6, 9, 10 cells / line); AD: n = 4 (7, 5, 10, 9 cells / line). *p < 0.05,unpaired two-tailed student’s t-test; error bars, standard error of measurement; ns, non-significant (p > 0.05), n lists the number of human iPSC lines per group, with the number of cortical neurons from each line of CON (CW70305, CW70256, CW70344, CW50040) or AD group (CW50018, CW50024, CW50158, CW50170) listed in order inside the brackets. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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  Fig. 3.. Decreased frequencies of sEPSC and sAP in AD neurons. (A) Representative traces of spontaneous excitatory postsynaptic currents (sEPSC) recorded at -60 mV in control (blue) and AD neurons (red). (B, C) sEPSC frequency (p = 0.0040) (B) and amplitude (p = 0.3728) (C) in control and AD neurons. CON: n = 4 (15, 14, 33, 11 cells / line); AD: n = 4 (16, 19, 13, 18 cells / line) for (B), CON: n = 4 (15, 14, 33, 11 cells / line); AD: n = 4 (16, 14, 10, 17 cells / line) for (C). (D) Representative traces of spontaneous Action Potentials (sAP) recorded from control (blue) and AD neurons (red). (E, F) sAP frequency (p = 0.0045) (E) and amplitude (p = 0.9543) (F) in control and AD neurons. CON: n = 4 (8, 11, 6, 5 cells / line); AD: n = 4 (8, 9, 6, 7 cells / line) for (E), CON: n = 4 (8, 11, 5, 4 cells / line); AD: n = 4 (1, 2, 1, 4 cells / line) for (F). **p < 0.01, unpaired two-tailed student’s t-test; error bars, standard error of measurement; ns, non-significant (p > 0.05), n lists the number of human iPSC lines per group, with the number of cortical neurons from each line of CON (CW70305, CW70256, CW70344, CW50040) or AD group (CW50018, CW50024, CW50158, CW50170) listed in order inside…

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  Fig. 4.. SGK1 inhibition restores voltage-gated Na+ and K+ currents in AD neurons without affecting control neurons. (A) Representative traces of voltage-gated Na+ and K+ currents (black traces and scale bar) induced by depolarizing voltage steps (grey inset) in AD or control neurons treated with DMSO or the SGK1 inhibitor (SGK1i) GSK650394 (100 nM, 72 h). Colour insets, enlarged traces for each condition coded with colour. (B, C) Averaged I-V curves for voltage-gated Na+ currents (B) and peak Na+ currents at −20 mV in AD (p = 0.0201) or control neurons (p > 0.9999) treated with DMSO or SGK1i (C). (D, E) Averaged I-V curves for voltage-gated K+ currents (D) and peak K+ currents at +40 mV (E) in AD (p = 0.0428) or control neurons (p = 0.6564) treated with DMSO or SGK1i. * p < 0.05, two-way ANOVA followed by Bonferroni’s multiple comparisons. AD+DMSO: n = 4 (6, 6, 12, 9 cells / line); AD+SGK1i: n = 4 (6, 8, 11, 14 cells / line); CON+DMSO: n = 4 (9, 6, 13, 7 cells / line); CON+SGK1i: n = 4 (7, 3, 12, 9 cells / line). n lists the number of human iPSC lines per group, with the number of cortical neurons from each line of CON (CW70305, CW70256, CW70344, CW50040) or AD group (CW50018, CW50024,…

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  Fig. 5.. SGK1 inhibition restores action potential amplitudes in AD neurons without affecting control neurons. (A) Representative traces of evoked action potentials (eAPs) elicited by 20 pA current injection (grey inset) in AD or control neurons treated with DMSO or the SGK1 inhibitor (SGK1i) GSK650394 (100 nM, 72 h). (B) Frequency of eAP for each condition (AD: p = 0.8517, control: p > 0.9999). (C-F) The first eAP in the burst were analyzed for amplitude (AD: p = 0.0124, control: p > 0.9999) (C), overshoot (AD: p = 0.0173, control: p > 0.9999) (D), threshold (AD: p = 0.7484, control: p > 0.9999) (E), and half-width (AD: p = 0.3898, control: p = 0.6082) (F). * p < 0.05, two-way ANOVA followed by Bonferroni’s multiple comparisons. AD+DMSO: n = 4 (20, 8, 11, 8 cells / line); AD+SGK1i: n = 4 (20, 8, 9, 13 cells / line); CON+DMSO: n = 4 (6, 7, 13, 12 cells / line); CON+SGK1i: n = 4 (7, 4, 16, 18 cells / line). n lists the number of human iPSC lines per group, with the number of cortical neurons from each line of CON (CW70305, CW70256, CW70344, CW50040) or AD group (CW50018, CW50024, CW50158, CW50170) listed in order inside the brackets.

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  Fig. 6.. SGK1 inhibition rescues sEPSC and sAP frequencies in AD neurons without affecting control neurons. (A) Representative traces of sEPSCs recorded at −60 mV in AD or control neurons treated with DMSO or the SGK1 inhibitor (SGK1i) GSK650394 (100 nM, 72 h). (B, C) Quantification of sEPSC frequency (sAD: p = 0.0324, control: p > 0.9999) (B) and amplitude (sAD: p = 0.9724, control: p = 0.9996) (C). AD+DMSO: n = 4 (8, 11, 14, 13 cells / line); AD+SGK1i: n = 4 (10, 18, 19, 15 cells / line); CON+DMSO: n = 4 (8, 10, 14, 8 cells / line); CON+SGK1i: n = 4 (8, 11, 20, 10 cells / line) for (B). AD+DMSO: n = 4 (8, 9, 12, 10 cells / line); AD+SGK1i: n = 4 (10, 18, 14, 13 cells / line); CON+DMSO: n = 4 (8, 10, 14, 8 cells / line); CON+SGK1i: n = 4 (8, 11, 20, 10 cells / line) for (C). (D, E) Representative traces (D) and frequency (sAD: p = 0.2975, control: p = 0.4396) (E) of sAPs recorded in AD or control neurons treated with DMSO or SGK1i. AD+DMSO: n = 4 (6, 10, 8, 6 cells / line); AD+SGK1i: n = 4 (2, 8, 13, 8 cells / line); CON+DMSO: n = 4 (2, 2, 10, 6 cells / line); CON+SGK1i: n = 4 (3, 1, 14, 8 cells / line). * p < 0.05, two-way ANOVA followed by Bonferroni’s multiple comparisons. #p < 0.05,…