Abstract
Background Spinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy.
Methods We induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy.
Results We generate highly pure and functional mRNA-induced MNs (miMNs) from normal and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment.
Conclusions This study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Disclosure of Potential Conflicts of Interest: The authors declare no potential conflicts of interest.
(liqianpumch{at}163.com), (orchidfy{at}hotmail.com), (yxue8{at}jhmi.edu), (xiping.zhan{at}howard.edu), (fuy{at}kennedykrieger.org), (guigege01{at}gmail.com), (JeanPhilippe.Richard{at}reprocell.com), (ataga1{at}jhmi.edu), (xmao4{at}jhmi.edu), (nmaragak{at}jhmi.edu), (ying{at}kennedykrieger.org)
List of abbreviations
- iPSC
- induced pluripotent stem cell
- ESC
- embryonic stem cell
- PSC
- pluripotent stem cell
- MN
- motor neuron
- ALS
- amyotrophic lateral sclerosis
- miMN
- mRNA-induced motor neuron
- FDA
- the Food and Drug Administration
- TF
- transcription factor