以建立CRMP-1基因剔除小鼠動物模型研究其在腦部生理功能的角色

在神經發育的過程中，調控神經細胞軸突和樹突延展及潰縮的機制引導神經纖維精準達到標的組織或臟器關係到正常生理功能的運作。多年來已有許多的蛋白家族被發現與此機制有高度相關性，最具代表性即Semaphorin家族，其中又以Semaphorin 3A研究最為明確；經由細胞膜上Semaphorin 3A所傳導的訊息，可被膜內一群名為CRMP（Collapsin Response Mediator Protein）家族所承續。CRMP可以藉由和細胞骨架相關蛋白質或是和細胞骨架本體的交互聯結，促使骨架聚合化或是去聚合化達到神經纖維延展或潰縮。CRMP-1為CRMP蛋白家族的成員之一，過去研究顯示CRMP-1高度專一表現於神經系統中，尤其以大腦的海馬迴表現量最多，然而體外細胞培養的結果對於CRMP-1參與神經纖維延展或是潰縮的訊息傳遞仍有待釐。2001年發現CRMP-1的表現量和肺癌轉移和臨床癒後有負相關性，且定義為腫瘤轉移抑制基因。為研究CRMP-1在生物體內所扮演的角色及參與之功能，本論文以條件式基因剔除策略建立了CRMP-1基因缺乏小鼠；CRMP-1基因剔除小鼠可以正常生長及繁衍子代，外觀上與基礎血液生化檢驗亦和野生型小鼠無異，且不因老化而易產生自發性腫瘤。根據CRMP-1高表現的區域進一步分析，發現基因剔除小鼠的海馬迴及胼胝體雖然其結構和神經叢數量與野生型小鼠無異，但是經由MAP2（Microtubule-Associate Protein）螢光染色，發現在基因剔除小鼠CA1區域的樹突發育有潰縮及發育不整的表型，且和樹突相關的PSD95（Post-Synaptic Density）表現量也有明顯下降；此外，雖然CRMP-1基因剔除小鼠在軸突的發育以及構造上和野生型小鼠無明顯差異，然而和軸突以及神經電位可塑性有關的GAP-43（Growth-Associated Protein）在CA1區域表現量有顯著減少。這些表型也使得CRMP-1基因缺乏的小鼠在海馬迴CA1區域的長效性神經電位受到了抑制，相對影響到小鼠在水迷宮實驗中的空間學習和記憶能力。除此之外，在強制性游泳的實驗中，CRMP-1基因缺乏小鼠也表現出相對沮喪、停滯不動的情緒和行為反應，推測CRMP-1可能也參與了情緒調控的機制。綜言之，本論文成功以條件式基因剔除策略建立了CRMP-1基因缺乏小鼠，發現其樹突發育受到影響，使得與記憶相關的長效性神經電位受到抑制，造成空間學習與記憶能力缺損。另一方面，CRMP-1基因缺乏小鼠小腦發育也受到影響，除了重量較野生型小鼠輕外，小腦十個由顆粒層形成的摺葉中，編號第VIb的摺葉在發育上也有缺損，不明顯，推測CRMP-1亦調控小腦的生長與發育。

關鍵字

CRMP-1 ；基因剔除；海馬迴；軸突形成；長效性電位；空間學習記憶

並列摘要

Brain function needs the precise anatomical and histological connections generated by exquisitely specific axonal guidance system during development. Neuronal outgrowth is directed by attracting by attracting and repelling signaling molecules. Well known guidance cues are semaphorins/collapsins. Semaphorin 3A, also known as Collapsin-1 is thought to be an initiator of signaling involved in neural growth cone outgrowth. Collapsin response mediator proteins (CRMPs) are a family of cytosolic phosphoproteins that mediate the signal from Semaphorin 3A. Collapsing response mediator protein-1 (CRMP-1) was initially identified in brain and has been implicated in plexin-dependent neuronal function. The high amino acid sequence identity among the five CRMPs has hindered determination of the functions of each individual CRMP. In 2001, CRMP-1 had also been characterized as a tumor metastasis suppressor gene dependent on its role in lung cancer metastasis and clinical outcome. In order to study the physiological function of CRMP-1 in vivo, we generated viable and fertile CRMP-1 knockout (CRMP-1-/-) mice with no evidence of gross abnormality in the major organs and difference in hematological and biochemical analysis between wild-type mice. According to high CRMP-1 expression in the hippocampus, we analyzed the formation of axon and dendrite there by staining with neural markers. CRMP-1-/- mice exhibited intense MAP2 staining in the proximal portion of the dendrites, but reduced and disorganized MAP2 staining in the distal dendrites of hippocampal CA1 pyramidal cells. Immunoreactivity to GAP-43 and PSD95 (a postsynaptic membrane adherent cytoskeletal protein) was also decreased in the CA1 region of the knockout mice. These changes were consistent with the mutant mice showing a reduction in long-term potentiation (LTP) in the CA1 region and impaired performance in hippocampal-dependent spatial learning and memory tests. CRMP-1-/- mice showed a normal synapsin I labeling pattern in CA1 and normal paired pulse facilitation. In addition, CRMP-1-/- also showed more depressed in forced swimming test suggested that CRMP-1 may involved in emotion control. These findings provide the first evidence suggesting that CRMP-1 may be involved in proper neurite outgrowth in the adult hippocampus and that loss of CRMP-1 may affect LTP maintenance and spatial learning and memory. Furthermore, CRMP-1-/- mice also exhibit more depressed in forced swimming test compared with wild-type mice suggested that CRMP-1 may regulate emotion response. On the other hand, CRMP-1-/- mice also show defect in cerebellum development. Mice deficient in CRMP-1 not only showed reduced weight in cerebellum but also lack the normal organic structure in VIb lobe suggested that CRMP-1 also involved in cerebellum development.

並列關鍵字

CRMP-1 ； gene targeting ； cre-loxP ； hippocampus ； axon guidance ； apical dendrite formation ； long-term potentiation ； Morris Water Maze ； learning and memory

參考文獻

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以建立CRMP-1基因剔除小鼠動物模型研究其在腦部生理功能的角色

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