anp32a促進斑馬魚胚胎脊髓受損後神經的修復

Abstract

脊髓損傷( spinal cord injury，SCI )是指會對脊髓造成功能缺失的傷害，如感覺神經或運動神經功能的喪失。在哺乳動物受到SCI後，神經再生能力相當有限；但在低等脊椎動物如斑馬魚受到SCI後，神經功能則可恢復。Acidic nuclear phosphoprotein 32 family member A ( ANP32A )參與神經發育的功能，但作用卻不一致，例如在人類和小鼠大腦發育過程中促進神經元的分化；但在Alzheimer's disease中，ANP32A卻是損害microtubule network及neurite outgrowth。還有，ANP32A在SCI時對神經再生的影響也尚不清楚，所以，本研究想要探討斑馬魚胚胎受到SCI後，ANP32A是否參與脊髓神經的再生和及其角色。首先，我將anp32a mRNA顯微注射在運動神經會發螢光的Tg(mnx 1:GFP)斑馬魚轉殖品系胚胎後進行SCI，再觀測胚胎傷口處的神經再生和泳動能力的恢復，發現過量表現ANP32A具有幫助促進SCI後motor neurons的再生和泳動能力的恢復。相反地，若將anp32a knockdown 後進行SCI，則會顯著降低motor neuron的再生及泳動距離。進一步地，我用免疫螢光染色標記PH3 (proliferation marker)和fluorescence activated cell sorting，發現ANP32A會促進SCI後radial glial cells這類cell type的proliferation。最後，我將anp32a過量表現在radial glial cells會發螢光的Tg(gfap:GFP)斑馬魚轉殖品系後進行SCI，並進行標記PH3，觀察到radial glial cells和PH3-positive cells 有colocalization的現象，這結果支持ANP32A是增進radial glial cells的proliferation來幫助motor neurons的再生。綜合以上，本研究證實了ANP32A參與斑馬魚胚胎脊髓損傷後神經再生和功能恢復的過程乃是透過促進radial glial cells的proliferation來幫助motor neurons再生，因為radial glial cells這類neural precursors可以分化為neurons。

Spinal cord injury (SCI) is injuries that causes functional loss of the spinal cord, such as loss of sensory or motor function. After SCI in mammals, the ability of neuronal regeneration is quite limited; but in lower vertebrates such as zebrafish, neuronal function can be recovered after SCI. Acidic nuclear phosphoprotein 32 family member A (ANP32A) is involved in neuroal development, but its function is not consistent. For example, ANP32A plays an important role in neuronal differentiation during mammalian brain development, while ANP32A impairs microtubule network and neurite outgrowth in Alzheimer's disease. Furthermore, the involvement of ANP32A in zebrafish SCI remains unknown. Therefore, this study aimed to investigate whether ANP32A is involved in the neuronal regeneration of injured zebrafish larvae. First, I microinjected anp32a mRNA into embryos of the transgenic zebrafish strain, Tg(mnx 1:GFP), which expresses the fluoresces in the motor neurons, followed by SCI, and then observed the regeneration of neurons around the SCI site and the recovery of swimming ability, and found that overexpression of anp32a enhanced the regeneration of motor neurons and swimming capability in the SCI-treated zebrafish larvae. Conversely, knockdown of anp32a followed by SCI significantly reduced regeneration of motor neurons and swimming distance. Furthermore, I used immunofluorescence staining to label PH3 (proliferation marker) and fluorescence activated cell sorting (FACS), found that ANP32A can promote the proliferation of cell types such as radial glial cells after SCI. Finally, I overexpressed anp32a in the transgenic zebrafish strain, Tg(gfap:GFP),which expresses the fluoresces in the radial glial cells, followed by SCI and labeled PH3, and observed colocalization between radial glial cells and PH3-positive cells , which supports that ANP32A promotes the proliferation of radial glial cells to help the regeneration of motor neurons.