- 學位論文
自閉症相關基因Dlgap2突變小鼠在海馬迴突觸蛋白表達異常及結構改變並影響空間記憶能力
Dlgap2 mutant mice exhibit altered synaptic protein expression, aberrant spine morphology and impaired spatial memory
摘要
泛自閉症障礙 (ASDs)的特徵是溝通與社會互動障礙，以及出現侷限性的重複行為。在近期的研究中發現，一位台灣自閉症男孩的染色體8p23末端區域發現了長度大約2.4 Mb的缺失。在鑑定該區域缺失的基因後，發現一種對應在腦中高表達的突觸後支架蛋白DLGAP2基因，可能與泛自閉症障礙的發病機制有關。為了闡明DLGAP2的功能，我們鑑定了Dlgap2異型合子(Dlgap2 Het)和同型合子(Dlgap2 Homo)的基因敲除小鼠的表現型。在開放場地試驗(open field test)中，Dlgap2異型合子與同型合子突變小鼠的活動能力均沒有明顯差異，惟Dlgap2同型合子小鼠表現出較焦慮的行為。在新物體辨別測試 (novel object recognition test)和Y型迷宮測試 (Y-maze test)中，Dlgap2突變小鼠表現了正常的短期辨別記憶和工作記憶。然而，在本實驗室張荷清小姐先前所做之水迷宮 (Morris water maze)實驗中發現，Dlgap2同型合子突變小鼠空間記憶的能力不良，表示缺乏DLGAP2會造成海馬迴的功能異常。因此，我們檢測了海馬迴中，包括突處後支架蛋白 (postsynaptic scaffolding proteins)、谷氨酸受體 (glutamate receptors)、信號分子 (signaling molecules)和突觸前蛋白 (presynaptic protein)在內的蛋白質表現。我們發現在Dlgap2 同型合子突變小鼠中，包括 SHANK3、PSD95、Homer1、GluR2、GluN2A、βCaMKII、Akt、Erk1/2、BDNF和Arc在內的許多突觸後蛋白表達量都降低了；然而，在Dlgap2同型合子突變小鼠中，mGluR5的表達量卻都增加了。接下來，我們檢測了齒狀迴(dentate gyrus)中顆粒細胞(granular cell)的形態特徵。我們發現在Dlgap2突變小鼠中，齒狀迴顆粒細胞的複雜性和分支模式，與野生型小鼠(Dlgap2 WT)之間，只有些微的變化。在樹突棘 (dendritic spine)的結構分析中，Dlgap2同型合子突變小鼠的樹突棘，在齒狀迴顆粒細胞的近端樹突處密度提高。同時，在Dlgap2同型合子突變小鼠中，成熟的蕈菇型樹突棘 (mushroom-type spine)百分比降低。隨後，我們檢測了海馬迴中的神經新生 (neurogenesis)，並沒有發現齒狀迴顆粒層底部 (subgranular zone)表現Ki67的細胞密度，在Dlgap2突變小鼠中有任何變化。我們的結果，說明了DLGAP2在突觸後蛋白質的表達，和齒狀迴顆粒細胞的樹突棘成熟過程中，扮演重要的角色。此外，DLGAP2也影響了許多維持細胞功能相關的幾個下游信號分子。在本研究中，我們在Dlgap2同型合子突變小鼠發現，海馬迴突觸後蛋白的表達異常，以及齒狀迴中樹突棘結構的改變，證明DLGAP2在海馬迴的重要性，這些改變可能與智能障礙(intellectual disability)相關的空間記憶缺損有關。Dlgap2基因突變小鼠，表現出自閉症及智能障礙的共病性，可做為一個合適的泛自閉症障礙動物模型。在未來的研究中，這些小鼠將更利於我們對泛自閉症障礙的瞭解。
並列摘要
Autism spectrum disorders (ASDs) are characterized by impaired communication, reciprocal social interaction, restricted repetitive behavior or interests and cognitive dysfunctions. A deletion of approximately 2.4 Mb at 8p23 terminal region in a Taiwanese autistic boy has been identified. Among genes mapped in this region, DLGAP2, which is a postsynaptic scaffold protein, highly expressed in the hippocampus, might be involved in the pathogenesis of ASDs. In order to elucidate the function of DLGAP2, Dlgap2 gene knockout mice were generated. In the open field test, no obvious difference in the locomotor activity was noted in heterozygous and homozygous Dlgap2 knockout (Dlgap2 Het and Dlgap2 Homo) mice, while Dlgap2 Homo mice exhibited anxiety-like behavior. In the novel object recognition test and Y-maze test, Dlgap2 mutant mice exhibited intact short-term recognition memory and working memory. However, in the previous experiments conducted by Ho-Chin Chang, Dlgap2 Homo mice exhibited impaired spatial memory in the Morris water maze test, indicating the impairment of hippocampal function in the absence of DLGAP2. We then examined the expression of proteins including scaffolding proteins, glutamate receptors, signaling molecules and presynaptic protein in the hippocampus. Notably, numerous proteins including SHANK3, PSD95, Homer1, GluR2, GluN2A, βCaMKII, Akt, Erk1/2, BDNF, and Arc were reduced in Dlgap2 Homo mice, in contrast, the level of mGluR5 was increased in Dlgap2 Homo mice. We then examined the morphological features of the granule cells in the dentate gyrus (DG). The complexity and branching pattern were slightly altered in the DG granular cells of Dlgap2 mutant mice. Surprisingly, the spine density in proximal dendritic segments was increased in Dlgap2 Homo mice. Meanwhile, the percentage of mature mushroom-type spines was decreased in Dlgap2 Homo mice. We also examined the neurogenesis in the hippocampus. There was no change in the density of Ki67-positive cells in the subgranular zone in Dlgap2 mutant mice. Our results suggested DLGAP2 plays important roles in the expression of postsynaptic proteins and the spinogenesis in the DG granular cells. In addition, DLGAP2 affect several downstream signaling molecules linked to numerous cellular processes. Here we demonstrated the importance of DLGAP2 by showing aberrant synaptic protein expressions and structures of dendritic spines in the hippocampus of Dlgap2 Homo mice, which might be linked to impaired hippocampus-related spatial memory associated with intellectual disability. Mice with Dlgap2 deficiency, showing signs of ASDs and ID, could be an appropriate ASDs animal model, which may advance our understanding of ASDs.