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

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.

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

10.21203/rs.3.rs-443732/v1 ◽

2021 ◽

Author(s):

Julianna Lilienberg ◽

Zoltán Hegyi ◽

Eszter Szabó ◽

Edit Hathy ◽

András Málnási-Csizmadia ◽

...

Keyword(s):

Stem Cell ◽

Neurite Outgrowth ◽

Progenitor Cells ◽

Pluripotent Stem Cell ◽

Neural Progenitor Cells ◽

Myosin Ii ◽

Neural Progenitor ◽

Neural Regeneration ◽

Human Neural Progenitor Cells ◽

Muscle Myosin

Abstract Studies on neural development and neuronal regeneration are mainly based on animal models. The establishment of pluripotent stem cell technology, however, opened new perspectives for better understanding these processes in humans 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). eGFP was stably expressed in NPCs differentiated from human embryonic and induced pluripotent stem cell lines, and the neurite outgrowth was investigated under permissive and restrictive conditions using a high-content screening system. We found that the non-muscle myosin II (NMII) inhibitor blebbistatin and its novel, non-toxic derivatives initiate extensive neurite outgrowth in human NPCs. We observed that the extracellular matrix components greatly influence the rate of neurite formation, but NMII inhibitors are able to override the inhibitory effect of the restrictive environment. Similar, non-additive stimulatory effect on neurite generation was detected by the inhibition of ROCK1 kinase, the upstream regulator of NMII, whereas inhibition of JNKs had negligible effect, suggesting that ROCK1 signal is dominantly manifested by the 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, and may open novel perspectives in to development of more effective pharmacological and cell therapies for various neurodegenerative disorders.

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Corrections to “Light Supports Neurite Outgrowth of Human Neural Progenitor Cells In Vitro: The Role of P2Y Receptors” [Jan/Feb 08 118-125]

IEEE Journal of Selected Topics in Quantum Electronics ◽

10.1109/jstqe.2008.921188 ◽

2008 ◽

Vol 14 (2) ◽

pp. 521-521 ◽

Cited By ~ 1

Author(s):

J. J. Anders ◽

T. B. Romanczyk ◽

I. K. Ilev ◽

H. Moges ◽

L. Longo ◽

...

Keyword(s):

Neurite Outgrowth ◽

Progenitor Cells ◽

Neural Progenitor Cells ◽

P2y Receptors ◽

Neural Progenitor ◽

Human Neural Progenitor Cells

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HDAC Inhibitors Induce BDNF Expression and Promote Neurite Outgrowth in Human Neural Progenitor Cells-Derived Neurons

International Journal of Molecular Sciences ◽

10.3390/ijms20051109 ◽

2019 ◽

Vol 20 (5) ◽

pp. 1109 ◽

Cited By ~ 4

Author(s):

Amir Bagheri ◽

Parham Habibzadeh ◽

Seyedeh Razavipour ◽

Claude-Henry Volmar ◽

Nancy Chee ◽

...

Keyword(s):

Neurite Outgrowth ◽

Progenitor Cells ◽

Neural Progenitor Cells ◽

Hdac Inhibitors ◽

Long Term Potentiation ◽

Neural Progenitor ◽

Synaptic Function ◽

Human Skin Fibroblast ◽

Bdnf Expression ◽

Human Neural Progenitor Cells

Besides its key role in neural development, brain-derived neurotrophic factor (BDNF) is important for long-term potentiation and neurogenesis, which makes it a critical factor in learning and memory. Due to the important role of BDNF in synaptic function and plasticity, an in-house epigenetic library was screened against human neural progenitor cells (HNPCs) and WS1 human skin fibroblast cells using Cell-to-Ct assay kit to identify the small compounds capable of modulating the BDNF expression. In addition to two well-known hydroxamic acid-based histone deacetylase inhibitors (hb-HDACis), SAHA and TSA, several structurally similar HDAC inhibitors including SB-939, PCI-24781 and JNJ-26481585 with even higher impact on BDNF expression, were discovered in this study. Furthermore, by using well-developed immunohistochemistry assays, the selected compounds were also proved to have neurogenic potential improving the neurite outgrowth in HNPCs-derived neurons. In conclusion, we proved the neurogenic potential of several hb-HDACis, alongside their ability to enhance BDNF expression, which by modulating the neurogenesis and/or compensating for neuronal loss, could be propitious for treatment of neurological disorders.

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Myelin basic protein enhances axonal regeneration from neural progenitor cells

Cell & Bioscience ◽

10.1186/s13578-021-00584-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Zhengjian Yan ◽

Lei Chu ◽

Xiaojiong Jia ◽

Lu Lin ◽

Si Cheng

Keyword(s):

Myelin Basic Protein ◽

Neurite Outgrowth ◽

Progenitor Cells ◽

Axonal Regeneration ◽

Neural Progenitor Cells ◽

Basic Protein ◽

Neural Progenitor ◽

Dependent Manner ◽

Cord Injury

Abstract Introduction Stem cell therapy using neural progenitor cells (NPCs) shows promise in mitigating the debilitating effects of spinal cord injury (SCI). Notably, myelin stimulates axonal regeneration from mammalian NPCs. This led us to hypothesize that myelin-associated proteins may contribute to axonal regeneration from NPCs. Methods We conducted an R-based bioinformatics analysis to identify key gene(s) that may participate in myelin-associated axonal regeneration from murine NPCs, which identified the serine protease myelin basic protein (Mbp). We employed E12 murine NPCs, E14 rat NPCs, and human iPSC-derived Day 1 NPCs (D1 hNPCs) with or without CRISPR/Cas9-mediated Mbp knockout in combination with rescue L1-70 overexpression, constitutively-active VP16-PPARγ2, or the PPARγ agonist ciglitazone. A murine dorsal column crush model of SCI utilizing porous collagen-based scaffolding (PCS)-seeded murine NPCs with or without stable Mbp overexpression was used to assess locomotive recovery and axonal regeneration in vivo. Results Myelin promotes axonal outgrowth from NPCs in an Mbp-dependent manner and that Mbp’s stimulatory effects on NPC neurite outgrowth are mediated by Mbp’s production of L1-70. Furthermore, we determined that Mbp/L1-70’s stimulatory effects on NPC neurite outgrowth are mediated by PPARγ-based repression of neuron differentiation-associated gene expression and PPARγ-based Erk1/2 activation. In vivo, PCS-seeded murine NPCs stably overexpressing Mbp significantly enhanced locomotive recovery and axonal regeneration in post-SCI mice. Conclusions We discovered that Mbp supports axonal regeneration from mammalian NPCs through the novel Mbp/L1cam/Pparγ signaling pathway. This study suggests that bioengineered, NPC-based interventions can promote axonal regeneration and functional recovery post-SCI.

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