單羧基運輸蛋白在斑馬魚中樞神經系統發育過程中所扮演之角色

Abstract

星狀細胞(Astrocyte)為一類神經膠質細胞，近年來被證明可以運送來自微血管的乳酸至神經細胞，以提供神經細胞的能量需求。而運送乳酸的運輸蛋白為單羧基運輸蛋白 (monocarboxylate transporter)，目前在哺乳類已發現有十四型異構物，其中第一至四型單羧基運輸蛋白可運輸乳酸，而僅第一、二及四型表現在哺乳類腦部，且已知第一及四型表現於星狀細胞，而第二型則表現在神經細胞；根據這三型異構物的表現及功能，提出了“astrocyte-neuron lactate shuttle”假說，然而，目前尚無任何in vivo的證據支持此假說。 在本篇論文中，利用斑馬魚作為模式動物，以分子生物技術提供in vivo的證據證明單羧基運輸蛋白對於中樞神經系統發育的重要性；運用PCR技術，斑馬魚的第一至四型單羧基運輸蛋白第一次完全被選殖出來，經由RT-PCR及in situ hybridization，發現第一至四型單羧基運輸蛋白均表現於斑馬魚早期的中樞神經，且均可表現於神經細胞及星狀細胞。為了確認單羧基運輸蛋白的功能，利用基因專一morpholino將單羧基運輸蛋白的表現抑制後，發現腦部神經細胞的分化會被抑制，藉此證明單羧基運輸蛋白對於中樞神經發育的重要性；其次更進一步利用mRNA補償被抑制的單羧基運輸蛋白，證明第二型單羧基運輸蛋白在斑馬魚中樞神經系統發育過程中扮演著不可或缺的角色。

Astrocytes provide energy to neuron mainly with lactate, which is transported through monocarboxylate transporters (MCTs). Among 14 isoforms of MCTs reported in mammals, only MCT1, 2 and 4 are expressed in brain and the three isoforms are found to differentially expressed in neuron and astrocyte, respectively. Based on these, “astrocyte-neuron lactate shuttle” has been proposed. However, no in vivo evidence was available so far to support this hypothesis. In the present study, zebrafish was used as a model to provide in vivo molecular physiological evidence for the involvements of MCTs in the development and functioning of central nervous system (CNS). Full-length cDNAs of the zebrafish MCT1-4 were cloned from zebrafish. In RT-PCR and double in situ hybridization/immunocytochemistry experiments, the zMCT1-4 were all expressed in brain of embryos, and were further found to localize in both neuron and astrocyte. Morpholino knockdown experiments provided for the 1st time the in vivo evidence to indicate that the zMCT1-4 may be involved in energy translocation and functioning of the developing brain. Subsequent rescue experiments with mRNAs of specific isoforms further indicated zMCT2 to be an indispensable transporter for CNS development and function.