Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II.

Julianna Lilienberg,András Málnási-Csizmadia,Zoltán Hegyi,Eszter Szabó,Ágota Apáti,László Homolya,Edit Hathy,János M Réthelyi

doi:10.3389/fcell.2021.719636

Abstract

Studies on neural development and neuronal regeneration after injury are mainly based on animal models. The establishment of pluripotent stem cell (PSC) technology, however, opened new perspectives for better understanding these processes in human models by providing unlimited cell source for hard-to-obtain human tissues. Here, we aimed at identifying the molecular factors that confine and modulate an early step of neural regeneration, the formation of neurites in human neural progenitor cells (NPCs). Enhanced green fluorescent protein (eGFP) was stably expressed in NPCs differentiated from human embryonic and induced PSC lines, and the neurite outgrowth was investigated under normal and injury-related conditions using a high-content screening system. We found that inhibitors of the non-muscle myosin II (NMII), blebbistatin and its novel, non-toxic derivatives, initiated extensive neurite outgrowth in human NPCs. The extracellular matrix components strongly influenced the rate of neurite formation but NMII inhibitors were able to override the inhibitory effect of a restrictive environment. Non-additive stimulatory effect on neurite generation was also detected by the inhibition of Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), the upstream regulator of NMII. In contrast, inhibition of c-Jun N-terminal kinases (JNKs) had only a negligible effect, suggesting that the ROCK1 signal is dominantly manifested by actomyosin activity. In addition to providing a reliable cell-based in vitro model for identifying intrinsic mechanisms and environmental factors responsible for impeded axonal regeneration in humans, our results demonstrate that NMII and ROCK1 are important pharmacological targets for the augmentation of neural regeneration at the progenitor level. These studies may open novel perspectives for development of more effective pharmacological treatments and cell therapies for various neurodegenerative disorders.

Highlights

Neurodegenerative disease conditions, such as Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease are characterized by a progressive loss of various types of neurons
We investigated the dynamics of neurite outgrowth in human stem cell-derived neural progenitor cell (NPC) and examined the effect of BS and its derivatives on this process in permissive and inhibitory environments
Insertion of enhanced green fluorescent protein (eGFP) cDNA into the NPC genome caused no alteration in the cell morphology or the proliferative capacity (Figures 1A,B)

Summary

Introduction

Neurodegenerative disease conditions, such as Huntington’s disease, Alzheimer’s disease, and Parkinson’s disease are characterized by a progressive loss of various types of neurons. Regenerative mechanisms, limited in adult neural tissues, may delay or even halt disease progression. Extensive studies have been performed to elucidate the process of axonal growth impairment (Allingham et al, 2005; Medeiros et al, 2006; Tornieri et al, 2006; Rosner et al, 2007; Kubo et al, 2008; Kollins et al, 2009; Hur et al, 2011; Pool et al, 2011; Yu et al, 2012; Roland et al, 2014; Spedden et al, 2014; Xie et al, 2014; Evans et al, 2017; Wang et al, 2017, 2020; Tanaka et al, 2018; Basso et al, 2019; Dupraz et al, 2019; Costa et al, 2020), and, in addition to the intrinsic factors, revealed the importance of environmental inhibitory mechanisms in retarded axonal growth (Ichikawa et al, 2009; Hur et al, 2011; Tan et al, 2011; Beller and Snow, 2014; Baldwin and Giger, 2015; Nakamura et al, 2019). Because of the limited availability of human neural tissues, most of these studies used rodent or avian models

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