探討 ARHGEF10 於血小板功能及自閉症相關行為所扮演之角色

Abstract

Rho GTPase 在人體內調控許多生理反應，外部刺激會透過G蛋白偶聯受體(G protein-coupled receptor)來活化Rho GTPase，進而調控各種細胞的功能，而鳥嘌呤核苷酸交換因子(guanine nucleotide exchange factors, GEFs) 是調控 Rho GTPase的重要因子之一。而鳥嘌呤核苷酸交換因子透過結合到G蛋白來啟動Rho GTPase 呈現活化的狀態，藉由將Rho GTPase的GDP轉換為GTP而使Rho蛋白活化。進而能夠繼續相關的訊號傳遞。ARHGEF10是GEFs其中的一員，在臨床上發現該基因的單核苷酸多型性(single-nucleotide polymorphism)與缺血性中風有很大的關聯性。但Arhgef10是如何調節血栓形成仍未知。我們於是建立了Arhgef10基因剔除小鼠，藉由此動物模式來研究Arhgef10與血小板功能的相關性。在本研究中發現，剔除Arhgef10並不會影響小鼠的血小板數目，和其他血液相關的細胞 。但 Arhgef10基因剔除的血小板在利用膠原酶(collagen), 腺苷二磷酸(ADP), 血小板凝血酶(thrombin), U46619 和 PAR4 等刺激下的凝集反應顯著的減少。另外，在塗有纖維蛋白原的(fibrinogen-coated)玻片的貼附反應的實驗中Arhgef10基因剔除的血小板表現較少板狀偽足狀(lamellipodia)的形態。正常的血小板在受到刺激後活化、貼附後會進一步凝集成凝塊，而在利用凝血酶刺激劑來形成凝塊的實驗中Arhgef10缺失的血小板則出現較為鬆散凝塊。從凝塊形成的實驗結果和凝集反應的實驗所觀察到的現象相類似，顯示ARHGEF10對於血小板從活化到凝集的過程中扮演了重要的角色。因此我們也檢驗了ARHGEF10主要調控的目標蛋白RhoA和其下游相關的蛋白，發現RhoA直接作用的蛋白ROCK有顯著減少，此外磷酸化的肌球蛋白輕鏈激酶(myosin light chain)也有顯著的減少。在動物體內的實驗中 利用氯化鐵引起的頸動脈栓塞動物模型及腦中主動脈的缺血性中風模式中，Arhgef10基因剔除對氯化鐵所引起頸動脈栓塞有顯著地抑制作用而對缺血性中風有顯著的保護作用。基於以上的實驗結果我們發現，Arhgef10在血小板凝集的過程中扮演了重要的角色。 此外，Arhgef10基因剔除小鼠也顯示出自閉症相關的行為。從動物行 為實驗的觀察中我們發現， 在社交行為測試中Arhgef10基因剔除小鼠表現出較不喜歡與其他小鼠互動，在社交再認的行為測試中也顯現出對社交行為的不敏感。此外，與野生型的小鼠相比，Arhgef10基因剔除小鼠也表現較為過動的行為特徵。這些行為特徵亦與臨床上自閉症病人的行為相似。生理上我們針對與自閉症相關的腦區進行神經傳導物質的檢驗。在高效液相層析儀的分析中發現，Arhgef10基因剔除小鼠在前額葉和杏仁核兩個腦區中的去甲腎上腺素和血清張素有顯著的增加。進一步分析與神經傳導物質合成與降解相關的蛋白也發現，單胺氧化酶在前額葉及杏仁核的表現量顯著的降低，但與神經傳導物質合成相關的蛋白並無改變。因此可以推論Arhgef10基因剔除小鼠的自閉症相關行為的表現型可能與前額葉和杏仁核兩個腦區的單胺氧化酶表現量降低進而導致神經傳導物質改變有關。 透過Arhgef10基因剔除小鼠的研究，由血小板的實驗可以發現，Arhgef10不僅參與了血小板凝集的過程，更可能在缺血性中風的病理現象中扮演了重要角色; 在中樞神經系統部分，Arhgef10亦影響了神經傳導物質代謝相關的蛋白表現，而神經傳導物質與單胺氧化酶表現量之改變則可能是造成Arhgef10基因剔除小鼠表現出自閉症相關行為的原因。

A Rho guanine nucleotide exchange factors (ARHGEF10), a member of Rho guanine nucleotide exchange factors (GEF) family, stimulates the Rho GTPases. Rho GTPases have been reported to regulate a variety of cellular behaviors such as cell polarity, cytoskeletal organization and gene transcription. Our previous copy number variation (CNV) study on CNV discovered that ARHGEF10 was deleted in a patient with an autism spectrum disorder (ASD). ARHGEF10 Single-neucleotide-polymorphisms (SNPs) are also linked with the risk of ischemic stroke. A missense mutation of ARHGEF10 has been reported to be one of the contributory factors in several diseases of the central nervous system. However, the relationship between the loss of ARHGEF10 and platelet function as well as the clinical symptoms of ASD is unclear. To examine the role of ARHGEF10 in platelet function, we generated Arhgef10 knockout mice to study the in vitro and in vivo effect of Arhgef10 knockout on the platelet function and arterial thrombosis formation. Arhgef10 knockout mice had normal platelet counts, but displayed altered aggregation in response to thrombin, collagen, adenosine diphosphate (ADP), proteinase-activated receptor 4 (PAR-4 ) peptide and U46619 stimulation. Knockout of Arhgef10 influenced platelets spreading on fibrinogen - coated surface and formed less lamellipodia-like extension than wild-type platelets from wild-type mice. Arhgef10 deficiency also inhibited platelet clot retraction induced by thrombin stimulation. Knockout of Arhgef10 resulted in prolonged tail bleeding time and inhibited the stable thrombus formation induced by FeCl3 in carotid artery in vivo. In the second part, Arhgef10 knockout mice were used as a model of ASD and characterized the social behaviors and the biochemical changes in the brains of the knockout mice. Compared with their wild-type littermates, the Arhgef10-KO mice showed impaired social interaction, hyperactivity and decreased depression-like and anxiety-like behaviors. Behavioral measurements of learning in the Morris water maze were not affected by the Arhgef10 deficiency. Moreover, neurotransmitters including serotonin, norepinephrine, and dopamine were significantly increased in different brain regions of the Arhgef10 knockout mice. In addition, monoamine oxidase A (MAO-A) decreased in several brain regions. In summary, ARHGEF10 serves as an important regulator in platelet shape change, spreading and aggregation. Moreover, the results in autistic-like symptoms suggest that ARHGEF10 is a candidate risk gene for ASD. Taken together, Arhgef10 knockout mice model could be a tool for studying the mechanisms of arterial thrombosis formation and neurotransmission in ASD.