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Phospholipase Cdelta3 regulates RhoA/Rho kinase signaling and neurite outgrowth.

ABSTRACT: Phospholipase C?3 (PLC?3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLC?3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLC?3 in neuronal migration and outgrowth. PLC?3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLC?3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in the cerebral cortex. In addition, PLC?3KD inhibits axon and dendrite outgrowth in primary cortical neurons. PLC?3KD also suppresses neurite formation of Neuro2a neuroblastoma cells induced by serum withdrawal or treatment with retinoic acid. This inhibition is released by the reintroduction of wild-type PLC?3. Interestingly, the H393A mutant lacking phosphatidylinositol 4,5-bisphosphate hydrolyzing activity generates supernumerary protrusions, and a constitutively active mutant promotes extensive neurite outgrowth, indicating that PLC activity is important for normal neurite outgrowth. The introduction of dominant negative RhoA (RhoA-DN) or treatment with Y-27632, a Rho kinase-specific inhibitor, rescues the neurite extension in PLC?3KD Neuro2a cells. Similar effects were also detected in primary cortical neurons. Furthermore, the RhoA expression level was significantly decreased by serum withdrawal or retinoic acid in control cells, although this decrease was not observed in PLC?3KD cells. We also found that exogenous expression of PLC?3 down-regulated RhoA protein, and constitutively active PLC?3 promotes the RhoA down-regulation more significantly than PLC?3 upon differentiation. These results indicate that PLC?3 negatively regulates RhoA expression, inhibits RhoA/Rho kinase signaling, and thereby promotes neurite extension.

SUBMITTER: Kouchi Z

PROVIDER: S-EPMC3048730 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Phospholipase Cdelta3 regulates RhoA/Rho kinase signaling and neurite outgrowth.

Kouchi Zen Z Igarashi Takahiro T Shibayama Nami N Inanobe Shunichi S Sakurai Kazuyuki K Yamaguchi Hideki H Fukuda Toshifumi T Yanagi Shigeru S Nakamura Yoshikazu Y Fukami Kiyoko K

The Journal of biological chemistry 20101227 10

Phospholipase Cδ3 (PLCδ3) is a key enzyme regulating phosphoinositide metabolism; however, its physiological function remains unknown. Because PLCδ3 is highly enriched in the cerebellum and cerebral cortex, we examined the role of PLCδ3 in neuronal migration and outgrowth. PLCδ3 knockdown (KD) inhibits neurite formation of cerebellar granule cells, and application of PLCδ3KD using in utero electroporation in the developing brain results in the retardation of the radial migration of neurons in th ...[more]

PMID: 21187285

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Project description:BACKGROUND:DNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIβ have not been clarified yet. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases. METHODS AND RESULTS:For this purpose, topo IIβ was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIβ deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIβ gene to elucidate the possible neuroprotective effect of topo IIβ overexpression on neural-induced hMSCs. Topo IIβ overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIβ in both topo IIβ-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIβ. CONCLUSION:Our results suggest that topo IIβ deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIβ overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

Dataset Information

Phospholipase Cdelta3 regulates RhoA/Rho kinase signaling and neurite outgrowth.

Publications

Phospholipase Cdelta3 regulates RhoA/Rho kinase signaling and neurite outgrowth.

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