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Zebrafish diras1 Promoted Neurite Outgrowth in Neuro-2a Cells and Maintained Trigeminal Ganglion Neurons In Vivo via Rac1-Dependent Pathway

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Abstract

The small GTPase Ras superfamily regulates several neuronal functions including neurite outgrowth and neuron proliferation. In this study, zebrafish diras1a and diras1b were identified and were found to be mainly expressed in the central nervous system and dorsal neuron ganglion. Overexpression of green fluorescent protein (GFP)-diras1a or GFP-diras1b triggered neurite outgrowth of Neuro-2a cells. The wild types, but not the C terminus truncated forms, of diras1a and diras1b elevated the protein level of Ras-related C3 botulinum toxin substrate 1 (Rac1) and downregulated Ras homologous member A (RhoA) expression. Glutathione S-transferase (GST) pull-down assay also revealed that diras1a and diras1b enhanced Rac1 activity. Interfering with Rac1, Pak1, or cyclin-dependent kinase 5 (CDK5) activity or with the Arp2/3 inhibitor prevented diras1a and diras1b from mediating the neurite outgrowth effects. In the zebrafish model, knockdown of diras1a and/or diras1b by morpholino antisense oligonucleotides not only reduced axon guidance but also caused the loss of trigeminal ganglion without affecting the precursor markers, such as ngn1 and neuroD. Co-injection with messenger RNA (mRNA) derived from mouse diras1 or constitutively active human Rac1 restored the population of trigeminal ganglion. In conclusion, we provided preliminary evidence that diras1 is involved in neurite outgrowth and maintains the number of trigeminal ganglions through the Rac1-dependent pathway.

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Acknowledgments

These authors thank Dr. Yi-Shuian Huang (Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan) for providing pCMV-mCherry plasmid and Addgene for providing plasmids as mentioned in “Materials and method” section. This study was supported by grants obtained from the Ministry of Science and Technology of Taiwan (MOST-103-2311-B-040-001). The authors thank the Zebrafish Core in Academia Sinica (ZCAS), Institute of Cellular and Organismic Biology (ICOB), which is supported by grant NSC-103-2321-B-001-050 from the National Science Council (NSC), the Taiwan Zebrafish Core Facility (TZCF) for providing the zebrafish AB strain and Tg(huc:gfp), and the National Health Research Institute (NHRI), which is supported by grant 100-2321-B-400-003 from National Science Council (NSC). Upright fluorescent microscopy and confocal microscopy were performed in the Instrument Center of Chung Shan Medical University, which is supported by the National Science Council, Ministry of Education and Chung Shan Medical University.

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  1. Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, No. 110, Sec.1, Jianguo N. Rd., Taichung, 40201, Taiwan

    Chi-Wei Yeh & Li-Sung Hsu

  2. Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, 40201, Taiwan

    Li-Sung Hsu

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  1. Chi-Wei Yeh
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Yeh, CW., Hsu, LS. Zebrafish diras1 Promoted Neurite Outgrowth in Neuro-2a Cells and Maintained Trigeminal Ganglion Neurons In Vivo via Rac1-Dependent Pathway. Mol Neurobiol 53, 6594–6607 (2016). https://doi.org/10.1007/s12035-015-9550-2

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