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, 36 (1), 142-52

Human iPS Cell-Derived Neurons Uncover the Impact of Increased Ras Signaling in Costello Syndrome

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Human iPS Cell-Derived Neurons Uncover the Impact of Increased Ras Signaling in Costello Syndrome

Gemma E Rooney et al. J Neurosci.

Abstract

Increasing evidence implicates abnormal Ras signaling as a major contributor in neurodevelopmental disorders, yet how such signaling causes cortical pathogenesis is unknown. We examined the consequences of aberrant Ras signaling in the developing mouse brain and uncovered several critical phenotypes, including increased production of cortical neurons and morphological deficits. To determine whether these phenotypes are recapitulated in humans, we generated induced pluripotent stem (iPS) cell lines from patients with Costello syndrome (CS), a developmental disorder caused by abnormal Ras signaling and characterized by neurodevelopmental abnormalities, such as cognitive impairment and autism. Directed differentiation toward a neuroectodermal fate revealed an extended progenitor phase and subsequent increased production of cortical neurons. Morphological analysis of mature neurons revealed significantly altered neurite length and soma size in CS patients. This study demonstrates the synergy between mouse and human models and validates the use of iPS cells as a platform to study the underlying cellular pathologies resulting from signaling deficits.

Significance statement: Increasing evidence implicates Ras signaling dysfunction as a major contributor in psychiatric and neurodevelopmental disorders, such as cognitive impairment and autism, but the underlying cortical cellular pathogenesis remains unclear. This study is the first to reveal human neuronal pathogenesis resulting from abnormal Ras signaling and provides insights into how these phenotypic abnormalities likely contribute to neurodevelopmental disorders. We also demonstrate the synergy between mouse and human models, thereby validating the use of iPS cells as a platform to study underlying cellular pathologies resulting from signaling deficits. Recapitulating human cellular pathologies in vitro facilitates the future high throughput screening of potential therapeutic agents that may reverse phenotypic and behavioral deficits.

Keywords: Costello syndrome; Ras; cortical development; iPS cells; stem cells.

Figures

Figure 1.
Figure 1.
E18.5 mouse model of Ras hyperactivation reveals aberrant cortical lamination. a–e, Immunohistochemical analysis of coronal brain sections of WT and HRASG12V E18.5 mice. Immunohistochemical analysis of layer-specific markers used to quantify neuronal cells per cortical area: Cux1, layers II-III (green) (a, b); Ctip2, layer V motor neurons (red) (a, c); SatB2, layers II-V callosal projection neurons (green) (a, d); and Tbr1, layers V/VI (red) (a, e). f, g, Immunohistochemical analysis of PAX6 expression around the lateral ventricles in WT and HRASG12V E18.5 mouse. a–g, WT, n = 5; HRASG12V, n = 4. Data are mean ± SEM. *p < 0.05 (nonparametric Kruskal–Wallis test).
Figure 2.
Figure 2.
P7 mouse model of Ras hyperactivation reveals aberrant cortical lamination and cortical neuronal morphology. a–d, Immunohistochemical analysis of coronal brain sections of WT and HRASG12V P7 mice. Immunohistochemical analysis of layer-specific markers used to quantify neuronal cells per cortical area: SatB2, layers II-V callosal projection neurons (green) (a, b); Ctip2, layer V motor neurons (red) (a, c); and Tbr1, layers V/VI (red) (a, d). a–d, WT, n = 5; HRASG12V, n = 5. Data are mean ± SEM. *p < 0.05 (nonparametric Kruskal–Wallis test). e–g, Morphological analysis of WT and HRASG12V P7 cortical mouse neurons. Golgi staining of cortical pyramidal neurons in coronal brain sections of WT and HRASG12V P7 mice (e). ImageJ analysis of total neurite length per neuron in WT and HRASG12V P7 neurons (f). ImageJ analysis of soma size in WT and HRASG12V P7 neurons (g). f, g, WT, n = 36 neurons; HRASG12V, n = 20 neurons. Data are mean ± SEM. *p < 0.05 (nonparametric Kruskal–Wallis test).
Figure 3.
Figure 3.
CS-derived fibroblasts and iPS cells exhibit aberrant Ras signaling. a–f, Proliferation rates in control and CS fibroblasts. Control (a, c) and CS (b, d) fibroblasts were assayed for BrdU inclusion (c–e) and doubling time (f). Data are mean ± SEM. *p < 0.05 (t test). g, Fold changes in mki67 and RB mRNA levels in CS fibroblasts relative to control fibroblasts (red line). Data are mean ± SEM. *p < 0.05 (t test). h, Total baseline HRAS-GTP levels in serum-starved fibroblasts reveals elevated levels in CS cells. Levels are normalized to HRAS in control lines. i, j Immunostaining of pluripotent markers OCT4 and TRA-1–81 in control (i) and CS (j) iPS cells. k–n, Proliferation rates in control and CS iPS cells. Control (k) and CS (l) iPS cells were assayed for BrdU inclusion (k, l, m) and doubling time (n). Data are mean ± SEM. o, Fold changes in mki67 and RB mRNA levels in CS iPS cells relative to control iPS cells (red line). Data are mean ± SEM. *p < 0.05 (t test). n = 4 patients per group for all analyses. p, Total baseline HRAS-GTP levels in iPS cells reveals elevated levels in CS cells. Levels are normalized to HRAS in control lines.
Figure 4.
Figure 4.
CS-derived neurons exhibit morphological alterations. a–d, Morphological analysis of embryoid body and rosette formation. Embryoid body (a, b) and rosette (c, d) formation in control and CS populations. e–i, Morphological analysis of control- and CS-derived neurons. Fluorescent microscopy analysis of GFP-labeled control-derived (e) and CS-derived (f) neurons. ImageJ analysis of total neurite length per neuron in control- and CS-derived neurons (g). Sholl analysis of the number of intersecting neurites relative to the cell soma in control- and CS-derived neurons (h). ImageJ analysis of soma size in control- and CS-derived neurons (i). Data are mean ± SEM. n = 40 neurons per patient group (at 4 patients per group). *p < 0.05 (t test). j, Cell density as calculated from DAPI immunostaining in control- and CS-derived neurons. k, Fold changes in HRAS mRNA levels in CS cells relative to control cells at each developmental stage. Data are mean ± SEM. *p < 0.05 (t test). n = 4 patients per group for all analyses.
Figure 5.
Figure 5.
CS-derived neuronal populations express elevated levels of laminar specific markers and persistent expression of PAX6 progenitors. a–h, Immunostaining of TBR1, CTIP2, and PAX6 expression in TUJ1+ and MAP2+ neurons at day 32 of differentiation. g, Fraction of neurons (TUJ1+) expressing lower layer markers TBR1 (layer V) and CTIP2 (layer V). h, Fraction of cells expressing PAX6. i, Fold changes in TBR1, CTIP2, CUX1, PAX6, TBR2, mki67, and RB mRNA levels in CS-derived relative to control-derived (red line) neuronal cultures. Data are mean ± SEM. *p < 0.05 (t test). j, k, Immunostaining of Synapsin1+ puncta on TUJ1+ control- and CS-derived neurons at day 67 of differentiation. l, Quantification of Synapsin1+ puncta per 50 μm neurite length in control- and CS-derived neurons. Data are mean ± SEM. *p < 0.05 (t test). n = 4 patients per group for all analyses. m, Fold changes in NXN1, SHANK2, SHANK3, NLGN3, and NLGN4 mRNA levels in CS-derived relative to control-derived (red line) neuronal cultures. Data are mean ± SEM. *p < 0.05 (t test).
Figure 6.
Figure 6.
Electrophysiological properties of Costello iPS cell-derived neurons. A, Representative 3 s recording trace of spontaneous EPSCs recorded from iPS cell-derived human neuron. Calibration: 20 pA, 200 ms. Currents were completely blocked by the AMPA receptor antagonist DNQX mean EPSC event, averaged from >50 isolated. BE, Summary graphs of EPSC parameters from control (N = 6) and HRAS (N = 9) cells. Each circle represents the mean response from individual neurons. The event amplitude (pA) (B), activation kinetics (time from 10% to 90% of peak, ms) (C), decay (event half-width, ms) (D), and event frequency (Hz) (E) were not statistically different between control and mutant neurons.

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