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, SOD1G85R mutant and corrected SOD1G85G isogenic-induced pluripotent stem cell (iPSC) lines were established. Two SOD1 mutant ALS (SOD1G85R and SOD1D90A), two SOD1 mutant corrected (SOD1G85G and SOD1D90D), and one sporadic ALS iPSC lines were directed toward MNs. After receiving ~90% purity for MNs, we first demonstrated that SOD1G85R mutant ALS MNs recapitulated ALS-specific nerve fiber aggregates, similar to SOD1D90A 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 SOD1G93A 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.

Gastrodin ameliorates synaptic impairment, reestablishes mitochondrial membrane potential and reduces oxidative stress in N2a/APP cells through ERK1/2 and GSK-3β pathways

“…We found that gastrodin treatment increased the levels of p-ERK and p-JNK and reduced the level of p-GSK-3β, while t-ERK, t-GSK-3β and t-JNK remained stable compared to the control group. Our results are in line with earlier findings of the gastrodin effect mediated via downregulation of GSK-3β phosphorylation [26,35,48,78,79] and upregulation of ERK1/2 phosphorylation [34,46,47,78,80,81] in AD cellular and animal models. Intraneuronal…”

Robust Generation of Ready-to-Use Cryopreserved Motor Neurons from Human Pluripotent Stem Cells for Disease Modeling

“…For MND modeling, we first generated an ALS iPSC line with the SOD1 G256C point mutation (SOD1 G85R ). In our previous study, we employed CRISPR/Cas9 gene editing to correct the SOD1 G256C mutant site to the normal nucleic acid, producing healthy SOD1 protein (SOD1 G85G iPSCs) [ 24 ]. In another previous study, we established an SFEB-based robust neuronal differentiation protocol known as the BiSF method to induce neuronal differentiation by adding FGFb, activin inhibitor SB, and Wnt ligand BIO ( Figure 1 a) [ 22 , 23 ].…”

“…To evaluate the applicability of the CHSF-MN protocol on other PSC lines, two hESC lines, H9 (from WiCell; Figure 2 a–c) [ 30 ] and TW1 (established in Taiwan; Figure 2 d–f) [ 31 ]; and 5 ALS-related iPSC lines, SOD1 G85R ( Figure 2 g–i) [ 24 ], SOD1 G85G ( Figure 1 ) [ 24 ], SOD1 D90D (WiCell; Figure 2 j–l) [ 6 ], SOD1 D90A (WiCell; Figure 2 m–o) [ 6 ], and sporadic ALS ( Figure 2 p–r) [ 24 ] iPSC lines were directed toward MNs using this protocol. The differentiated cells expressed 95.43% ± 3.01% to 98.19% ± 1.05% Sox1 and 92.93% ± 1.36 to 98.13% ± 0.96% Oligo2 on day 15 with rosette-like structures in all hESC and iPSC lines ( Figure 2 s).…”

“…TW1 hESCs were kindly provided by Lee Women’s Hospital (Taichung, Taiwan) [ 31 ]. SOD1 G85R and sporadic ALS iPSC lines were generated from patients’ peripheral blood mononuclear cells (PBMCs) with the approval of the Ethical Institutional Review Board at Hualien Tzu Chi Hospital (IRB105-131-A), and SOD1 G85G iPSCs were generated from SOD1 G85R iPSCs [ 24 ]. Informed consent was obtained from all the patients.…”

Coating-Free Culture Medium for Establishing and Maintaining Human Induced Pluripotent Stem Cells

“…We steered the dissociated hPSCs differentiation to neural lineages by embryoid body (EB) formation 25 – 27 . In brief, the 1-2 × 10 5 /ml cells were cultured in Essential 6 medium (E6 medium, A1516401, Gibco) to form EB within the first 2 days.…”