Coactivation of GSK3β and IGF-1 Attenuates Amyotrophic Lateral Sclerosis Nerve Fiber Cytopathies in SOD1 Mutant Patient-Derived Motor Neurons

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive nervous system disease that causes motor neuron (MN) degeneration and results in patient death within a few years. To recapitulate the cytopathies of ALS patients' MNs, SOD1^G85R mutant and corrected SOD1^G85G isogenic-induced pluripotent stem cell (iPSC) lines were established. Two SOD1 mutant ALS (SOD1^G85R and SOD1^D90A), two SOD1 mutant corrected (SOD1^G85G and SOD1^D90D), and one sporadic ALS iPSC lines were directed toward MNs. After receiving ~90% purity for MNs, we first demonstrated that SOD1^G85R mutant ALS MNs recapitulated ALS-specific nerve fiber aggregates, similar to SOD1^D90A ALS MNs in a previous study. Moreover, we found that both SOD1 mutant MNs showed ALS-specific neurite degenerations and neurotransmitter-induced calcium hyperresponsiveness. In a small compound test using these MNs, we demonstrated that gastrodin, a major ingredient of Gastrodia elata, showed therapeutic effects that decreased nerve fiber cytopathies and reverse neurotransmitter-induced hyperresponsiveness. The therapeutic effects of gastrodin applied not only to SOD1 ALS MNs but also to sporadic ALS MNs and SOD1^G93A ALS mice. Moreover, we found that coactivation of the GSK3β and IGF-1 pathways was a mechanism involved in the therapeutic effects of gastrodin. Thus, the coordination of compounds that activate these two mechanisms could reduce nerve fiber cytopathies in SOD1 ALS MNs. Interestingly, the therapeutic role of GSK3β activation on SOD1 ALS MNs in the present study was in contrast to the role previously reported in research using cell line- or transgenic animal-based models. In conclusion, we identified in vitro ALS-specific nerve fiber and neurofunctional markers in MNs, which will be useful for drug screening, and we used an iPSC-based model to reveal novel therapeutic mechanisms (including GSK3β and IGF-1 activation) that may serve as potential targets for ALS therapy.

Keywords: GSK3β; IGF-1; SOD1; amyotrophic lateral sclerosis (ALS); drug screening; gastrodin; induced pluripotent stem cell (iPSC); motor neuron (MN).

Figure 1

iPSC generation and SOD1 mutant site correction. (a) Morphology of SOD1^G85R ALS patient’s PBMC-derived iPSC clone. (b) Karyotype of SOD1^G85R iPSCs. (c) DNA sequencing of SOD1^G85R iPSCs (upper panel) and corrected SOD1^G85G iPSCs (lower panel). Mutant and corrected site are emphasized with arrows. (d–g) SOD1^G85R iPSC-expressed pluripotent stem cell-specific markers including Oct-4 (d), Nanog (e), Sox-2 (f), and SSEA4 (g). (h–j) In vitro differentiation of SOD1^G85R iPSCs into cell types with three germ layer cell markers including the ectoderm markers N-cadherin and Sox-1 (h), mesoderm marker Brachyury (i), and endoderm marker Sox-17 (j). Scale bar, 100 μm.

Figure 2

Robust generation of MNs from iPSCs. (a) Procedure for CHSF-based MN differentiation. (b–m) Identification of SOD1^G85R (b–d), SOD1^G85G (e–g), SOD1^D90A (h–j), and SOD1^D90D (k–m) iPSC line-derived MN progenitors and MNs. Every line was identified with neural stem cell markers (N-cadherin and Sox-1) and MN progenitor markers (Oligo-2 and Islet-1) at day 15 of differentiation, and with an MN marker (HB9) and a neuronal marker (NF) at days 23–25 of differentiation. (n) Calculation of the HB9 expression ratio of DAPI⁺ cells. Scale bar, 100 μm.

Figure 3

Nerve fiber beads and neurite degeneration in SOD1 ALS MNs. (a–d) Nerve fiber beads (a) and neurite density (b) of SOD1^G85R and SOD1^G85G (nerve fiber beads: c; neurite density: d) MNs identified with NF immunocytochemistry staining. (e) Number of nerve fiber beads of SOD1^G85R and SOD1^G85G MNs at day 35 of differentiation. (f) Neurite density of SOD1^G85R and SOD1^G85G MNs at day 28 of differentiation. (g–j) Nerve fiber beads (g) and neurite density (h) of SOD1^D90A and SOD1^D90D (nerve fiber beads: (i); neurite density: (j)) MNs. (k) Number of nerve fiber beads of SOD1^D90A and SOD1^D90D MNs at day 35 of differentiation. (l) Neurite density of SOD1^D90A and SOD1^D90D MNs at day 28 of differentiation. The nerve fiber beads are indicated by arrow heads. * p < 0.05 and ** p < 0.01. Scale bar, 100 μm.

Figure 4

Gastrodin reduces nerve fiber abnormalities of SOD1 ALS MNs. (a–d) Nerve fiber beads (a) and neurite density (b) of untreated and gastrodin-treated SOD1^G85R (nerve fiber beads: c; neurite density: d) MNs identified with NF immunocytochemistry staining. (e) Number of nerve fiber beads of untreated (ctrl) and gastrodin-treated SOD1^G85R MNs at day 35 of differentiation. (f) Neurite density of untreated (ctrl) and gastrodin-treated SOD1^G85R MNs at day 28 of differentiation. (g–j) Nerve fiber beads (g) and neurite density (h) of untreated and gastrodin-treated SOD1^D90A (nerve fiber beads: (i); neurite density: (j)) MNs. (k) Number of nerve fiber beads of untreated (ctrl) and gastrodin-treated SOD1^D90A MNs at day 35 of differentiation. (l) Neurite density of untreated (ctrl) and gastrodin-treated SOD1^D90A MNs at day 28 of differentiation. * p < 0.05, and *** p < 0.001. Scale bar, 100 μm.

Figure 5

Gastrodin reverses SOD1^D90A MN hyper-calcium flux after glutamate stimulation. (a) SOD1^G85G, SOD1^G85R, and gastrodin-treated SOD1^G85R MNs were subjected to Fluo-4 calcium imaging at day 38 of differentiation. The calcium flux stimulated by potassium chloride (presented as orange bars) and glutamate (yellow bars) was revealed with ΔF/F0. (b,c) Average calcium flux stimulated by potassium chloride (b) and glutamate (c) of SOD1^G85G and SOD1^G85R MNs. (d,e) Average calcium flux stimulated by potassium chloride (d) and glutamate (e) of untreated and gastrodin-treated SOD1^G85R MNs. (f) SOD1^D90D, SOD1^D90A, and gastrodin-treated SOD1^D90A MNs were subjected to calcium imaging. (g,h) Average calcium flux stimulated by potassium chloride (g) and glutamate (h) of SOD1^D90D and SOD1^D90A MNs. (i,j) Average calcium flux stimulated by potassium chloride (i) and glutamate (j) of untreated and gastrodin-treated SOD1^D90A MNs. * p < 0.05, *** p < 0.001.

Figure 6

Gastrodin reverses nerve fiber degeneration in sporadic ALS MNs. (a) Morphology of sporadic ALS (sALS) iPSC clones. (b) Karyotype of sALS iPSCs. (c–f) sALS iPSC-expressed pluripotent stem cell markers including Oct-4 (c), Nanog (d), Sox-2 (e), and SSEA4 (f). (g–i) In vitro differentiation of sALS iPSCs into three germ layer cell types with markers including the ectoderm markers N-cadherin and Sox-1 (g), mesoderm marker Brachyury (h), and endoderm marker Sox-17 (i). (j–l) sALS iPSC line-derived MNs were identified with neural stem cell markers (N-cadherin and Sox-1) and MN progenitor markers (Oligo-2 and Islet-1) at day 15 of differentiation, and with an MN marker (HB9) and a neuronal marker (NF) at day 25 of differentiation. (m,n) Nerve fiber beads (m) and cell body inclusions (n) of NF protein on sALS MNs. (o,p) Number of nerve fiber beads (o) and neurite intensity (p) of untreated (ctrl) and gastrodin-treated sALS MNs at day 62 of differentiation. * p < 0.05. Scale bar, 100 μm.

Figure 7

Gastrodin improves the motor function and prolongs the life span of SOD1^G93A ALS mice. ALS mice were treated with gastrodin from 60 days of age. Data on the rotarod test (a–c), grip strength (d), survival rate (e), and BBB score (f) were recorded from 90 days-of-age. Rotarod data are presented as the average latency to fall time (a), latency to fall time of acceleration (b), and latency to fall time of maximum (c). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 8

Gastrodin promotes GSK3β and MAPK pathway activation in SOD1^G93A-NSC34 MN-like cells. (a) Dendrogram showing clustering of gastrodin-treated and untreated SOD1^G93A-NSC34 cells based on transcriptional changes detected by RNA-microarray. (b) Gene sets enrichment in gastrodin-treated SOD1^G93A-NSC34 cells. (c) Graph of expression fold change for Wnt and MAPK signaling-related genes in gastrodin-treated and untreated control SOD1^G93A-NSC34. (d–h) Western blotting of GAPDH, GSK3β, phospho-GSK3β, MAPK, phospho-MAPK, MEK, and phospho-MEK proteins from gastrodin-treated and untreated (ctrl) SOD1^G93A-NSC34 cells. * p < 0.05.

Figure 9

Activation of the GSK3β and IGF-1 pathway reduces axonal cytopathies in SOD1 mutant ALS MNs. (a) Neurite densities of day-28 SOD1^G85R MNs treated with Wnt3a, CHIR99021, IWP-2, and IGF-1, and their combinations were analyzed with NF immunocytochemistry staining and calculated as (a8). (b) Nerve fiber beads of day-35 SOD1^G85R MNs treated with the aforementioned compounds and their combinations were analyzed and calculated as (b8). (c) Neurite density of day-28 SOD1^D90A MNs treated with gastrodin and its combinations with CHIR99021 or Wnt3a, were analyzed and calculated as (c5). (d) Nerve fiber beads of day-35 SOD1^D90A MNs treated with gastrodin and combinations were analyzed and calculated as (d5). * p < 0.05. Scale bar, 100 μm.