王玉杰，周小坤，徐丹

綜述

常染色體隱性遺傳小頭畸形相關(guān)蛋白研究進展

王玉杰，周小坤，徐丹

福州大學(xué)生物科學(xué)與工程學(xué)院，福州 350108

腦發(fā)育相關(guān)疾病是一類影響大腦或中樞神經(jīng)系統(tǒng)生長和發(fā)育的疾病。常染色體隱性遺傳小頭畸形(autosomal recessive primary microcephaly, MCPH)是一種神經(jīng)系統(tǒng)發(fā)育障礙疾病，病人主要表現(xiàn)為頭圍減小，并伴隨一定程度的智力衰退。迄今為止已發(fā)現(xiàn)至少有25個基因突變都會導(dǎo)致MCPH，根據(jù)它們發(fā)現(xiàn)的順序分別命名為~。MCPH蛋白作為重要的成份參與調(diào)控大腦發(fā)育相關(guān)信號通路。本文對目前發(fā)現(xiàn)的25個MCPH相關(guān)蛋白的表達模式、細胞定位、分子生物學(xué)功能、表型及動物模型進行了綜述，旨在提升人們對腦發(fā)育相關(guān)疾病的致病機制的認知，促進對神經(jīng)元生成、腦尺寸大小及腦功能調(diào)控等分子機制的研究。

小頭畸形；表達模式；細胞組分；表型概述；分子生物學(xué)功能；動物模型

大腦是人體最重要和最復(fù)雜的器官，擁有上千億個神經(jīng)元和彼此間相互作用形成的百萬億個連接點。大腦通過形成龐大而復(fù)雜的神經(jīng)網(wǎng)絡(luò)控制著其他器官或系統(tǒng)的正常功能。理解大腦的運轉(zhuǎn)機制，是人類與科學(xué)屆面臨的最偉大的挑戰(zhàn)之一。腦科學(xué)是21世紀各種前沿科學(xué)中最為引人關(guān)注的領(lǐng)域之一，同時也是多學(xué)科交叉的重要前沿科學(xué)領(lǐng)域。2013年，美國啟動“腦計劃”旨在探索人類大腦工作機制，繪制腦活動全圖，并最終開發(fā)出針對大腦疾病的療法。2018年以來，“北京腦科學(xué)中心”和“上海腦科學(xué)中心”相繼成立，標志著“中國腦計劃”也正式拉開了帷幕。

腦發(fā)育異常會導(dǎo)致其功能異常，并最終導(dǎo)致嚴重的神經(jīng)疾病如自閉癥、精神分裂癥及小頭癥等[1~7]。小頭癥即小頭畸形(microcephaly)是一種比較罕見的大腦疾病，是由于大腦神經(jīng)系統(tǒng)發(fā)育障礙導(dǎo)致的，發(fā)病率在2~12/萬[8]。胎兒出生后的頭圍測量是診斷小頭癥最常用的方式之一[8]。診斷小頭癥的臨床標準是病人的頭圍相對于其同年齡與性別的平均值明顯小2個標準差以上[9]。通常在孕28周左右應(yīng)用超聲波技術(shù)和核磁共振掃描檢測即可發(fā)現(xiàn)患兒的頭圍測量值及腦容量低于正常同齡胎兒。小頭癥的主要表現(xiàn)是腦的重量明顯輕于正常、腦回過小或根本無腦回、大腦的發(fā)育明顯遲緩，甚至在嬰兒第3~5個月時就停止發(fā)育，最后導(dǎo)致患兒的頭頂變得小而尖、鼻梁凹陷、耳大、下額后縮、前額狹小并且頭圍明顯小于正常嬰兒，最大的頭圍不足43 cm。小頭癥患兒大腦發(fā)育障礙常常伴有不同程度的智力低下，有的患兒還會出現(xiàn)癲癇、運動障礙、語言障礙及其他行為異常[10]。造成小頭癥的原因有很多，大致可分成兩類：一類是由遺傳因素引起，即染色體或基因突變所導(dǎo)致；另一類是由環(huán)境因素引起，即胎兒在妊娠早期受到各種有害因素影響包括營養(yǎng)不良、中毒、物理或化學(xué)影響以及子宮感染(弓形蟲病，風(fēng)疹，皰疹，梅毒，巨細胞病毒及艾滋病毒)所導(dǎo)致。2015年，一種蟲媒病毒(寨卡病毒)在美洲和熱帶地區(qū)爆發(fā)導(dǎo)致小頭癥新生兒的大量產(chǎn)生引發(fā)了全世界的關(guān)注[11]。通過動物模型驗證，科學(xué)家已經(jīng)證實寨卡病毒感染確實會導(dǎo)致小頭癥的發(fā)生[12~14]。

常染色體隱性遺傳小頭畸形(autosomal recessive primary microcephaly, MCPH)是一種比較少見的神經(jīng)分裂異常引起的腦發(fā)育疾病。病人主要表現(xiàn)為頭圍減小并伴有不同程度的智力衰退[8]。目前發(fā)現(xiàn)的與MCPH有關(guān)的25個基因包括：()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()、()和()[15]。全世界范圍內(nèi)，超過50%的MCPH患者是由于()和)基因突變造成的[8]。本文通過查詢小鼠基因組信息(Mouse Genome Informatics, MGI)和美國國立生物技術(shù)信息中心(National Center for Biotechnology Information, NCBI)數(shù)據(jù)庫對已命名的25個MCPH相關(guān)蛋白的表達模式、細胞定位、分子生物學(xué)功能、表型及動物模型等進行了綜述，旨在提升人們對MCPH發(fā)病機制的認知并加深人們對神經(jīng)元生成及腦尺寸大小調(diào)控機理的理解。

1 MCPH基因的時空特異性表達

結(jié)合MGI和NCBI網(wǎng)站數(shù)據(jù)分析發(fā)現(xiàn)，相關(guān)基因存在非常明顯的時空特異性表達(圖1，圖2)。MCPH作為一類神經(jīng)發(fā)育疾病相關(guān)蛋白，大多數(shù)與細胞分裂相關(guān)，因此在細胞分裂活躍的組織表達較高。MGI數(shù)據(jù)庫(基于免疫組化和RNA原位雜交結(jié)果)結(jié)果顯示，有21個基因都在神經(jīng)系統(tǒng)表達，僅和未在神經(jīng)系統(tǒng)中檢測到表達。除神經(jīng)系統(tǒng)外，大部分在視覺系統(tǒng)、生殖系統(tǒng)和消化系統(tǒng)中也有表達。NCBI數(shù)據(jù)(基于正常組織RNAseq)顯示在人體組織中，已發(fā)現(xiàn)的25個中有11個基因在睪丸(相對其他組織)中表達量最高，分別是()、()、()、()、(STIL)、(Cep152)、()、(MFSD2A)、(ANKLE2)、()和()。另外，還有4個基因在睪丸(相對其他組織)中表達量次高，其中包括()、(SAS-6)、(CIT)和()。主要原因可能是基因在分裂旺盛的組織表達較高，而睪丸在產(chǎn)生精子過程中首先會產(chǎn)生很多精原干細胞。

圖1 Mcph在小鼠不同組織的表達模式

根據(jù)小鼠基因組信息(Mouse Genome Informatics, MGI)數(shù)據(jù)庫總結(jié)得到22個基因在不同組織的表達模式。

此外，通過分析NCBI數(shù)據(jù)庫RNA-seq結(jié)果，將25個基因在小鼠腦發(fā)育不同階段的表達情況進行匯總，結(jié)果發(fā)現(xiàn)大部分在腦發(fā)育早期(胚胎11.5天)表達量較高，隨著發(fā)育的進行(胚胎18天)表達量逐漸下降，在成年腦皮質(zhì)中表達量更低甚至幾乎檢測不到(圖2)。基因的時空特異性表達決定其在調(diào)控腦發(fā)育和育性方面起著非常重要的作用。

2 MCPH蛋白在細胞中的定位

通過蛋白序列分析或結(jié)構(gòu)預(yù)測發(fā)現(xiàn)，大部分MCPH蛋白定位在細胞骨架和細胞核中(圖3)。中心體是微管組織中心，大部分MCPH蛋白都定位在有絲分裂裝置如中心體或紡錘體上[16]。在腦發(fā)育過程中，中心體和紡錘體的正確組裝對于產(chǎn)生和維持正常的神經(jīng)細胞的數(shù)量起著非常關(guān)鍵的作用。MCPH蛋白缺失或突變會干擾中心體或紡錘體的正常形成，影響細胞周期及DNA復(fù)制等過程，進而影響神經(jīng)前體細胞的增殖、分化和凋亡等過程，最終導(dǎo)致神經(jīng)元數(shù)量減少并形成偏小的大腦[17~20]。MCPH2/WDR62表現(xiàn)出非常明顯的細胞周期依賴性表達。在有絲分裂中期或前中期，WDR62主要集中在紡錘體極點，而在有絲分裂間期WDR62則彌散地分布在細胞質(zhì)中[21]。在細胞有絲分裂間期，高爾基體是除中心體外的另一個微管組織中心。有些MCPH蛋白定位在高爾基體上，如MCPH3 (CDK5RAP2)通過ATP及中心體依賴的形式定位在高爾基體上[22]。MCPH19 (COPB2，β-輔酶亞基)以GTP依賴形式結(jié)合在高爾基體膜上并對高爾基體的形成及囊泡運輸過程起重要作用[23]。還有些MCPH蛋白(MCPH15/MFSD2A和MCPH16/ANKLE2)則定位在內(nèi)質(zhì)網(wǎng)上[24,25](圖3)。

圖2 Mcph基因在小鼠腦發(fā)育不同階段的表達情況

根據(jù)美國國立生物技術(shù)信息中心(National Center for Biotechnology Information, NCBI)數(shù)據(jù)庫總結(jié)得到不同基因在小鼠不同發(fā)育階段中樞神經(jīng)系統(tǒng)的表達情況。藍色不同強度代表表達強弱，越接近藍色表達越強，越接近白色表達越弱。

3 MCPH蛋白參與的生物學(xué)過程

動物個體發(fā)育是一個復(fù)雜而精細的過程。大部分MCPH蛋白在發(fā)育過程參與細胞成分組裝進而調(diào)控細胞的增殖分化及系統(tǒng)發(fā)育等過程(圖4)。大腦的大小是通過調(diào)節(jié)神經(jīng)干細胞增殖、分化和凋亡的平衡來控制的[26]。目前研究得比較多的一些MCPH蛋白(如MCPH5/ASPM、MCPH2/WDR62和MCPH6/ CENPJ等)在神經(jīng)干細胞的增殖或分化過程中起著重要作用[20,27~30]。神經(jīng)遷移與神經(jīng)發(fā)生和大腦尺寸調(diào)控密切相關(guān)。在大腦皮層形成過程中，正常的神經(jīng)遷移是至關(guān)重要的一個環(huán)節(jié)，是構(gòu)成大腦皮層復(fù)雜的組織結(jié)構(gòu)及特殊回路的前提和基礎(chǔ)。一些MCPH蛋白(如MCPH5和MCPH6)參與調(diào)控大腦皮層神經(jīng)細胞的遷移[27,28]。一些MCPH蛋白(如MCPH1)則參與調(diào)控細胞凋亡或DNA損傷過程[31]。一些MCPH蛋白(如MCPH2/WDR62、MCPH3/ CDK5RAP2和MCPH5/ASPM)參與調(diào)控微管的組裝、聚合或解聚[32~34]。MCPH7/STIL參與小鼠胚胎體軸的特化和神經(jīng)管的發(fā)育[35]。MCPH18/WDFY3通過調(diào)控自噬過程來影響大腦發(fā)育[36]。

4 MCPH突變表型

4.1 MCPH突變病人的表型

MCPH病人一般比較矮小，身高和體重亦低于正常值下限。MCPH是一種神經(jīng)發(fā)育障礙疾病，并且大部分基因在神經(jīng)系統(tǒng)高表達，因此基因突變病人的表型主要集中在神經(jīng)系統(tǒng)(圖5)。大腦體積減小和皮質(zhì)發(fā)育不良是MCPH病人的主要特征。同時一些MCPH病人還表現(xiàn)出小腦、腦干及胼胝體發(fā)育不良[15]。MCPH病人在行為上主要表現(xiàn)為不同程度的智力障礙，部分表現(xiàn)出運動及語言障礙，也有部分病人表現(xiàn)出共濟失調(diào)、癲癇及先天性耳聾等癥狀[37~39]。

圖3 MCPH蛋白的細胞定位

根據(jù)小鼠基因組信息(Mouse Genome Informatics, MGI)數(shù)據(jù)庫總結(jié)得到25個MCPH蛋白在不同細胞組分中的定位。

有絲分裂過程和分離缺陷、微管和紡錘體異常以及DNA損傷和細胞周期異常等過程在病人來源或基因突變的細胞中被發(fā)現(xiàn)[21,40~44]。也有研究表明由于神經(jīng)前體細胞中RNA加工模式的不同是導(dǎo)致MCPH突變影響神經(jīng)系統(tǒng)的原因[45]。根據(jù)MGI數(shù)據(jù)庫顯示大部分突變會影響有絲分裂引起不同組織的多種表型，同時也會影響減數(shù)分裂引起生殖系統(tǒng)出現(xiàn)問題(圖5)。

4.2 MCPH相關(guān)動物模型的構(gòu)建

研究MCPH蛋白功能的動物模型主要有斑馬魚()、果蠅()和小鼠()。MCPH除在神經(jīng)系統(tǒng)發(fā)育上表現(xiàn)出小頭癥的表型外，在個體生長發(fā)育過程中也起著重要作用(表1)。另外，MCPH蛋白在生殖系統(tǒng)中高表達，因此，MCPH蛋白缺失會導(dǎo)致小鼠生殖細胞發(fā)育缺陷進而引起育性降低，甚至不育[46~49]。近年來，有研究表明缺失也會影響小鼠聽力并引發(fā)中耳炎[50]。由于MCPH蛋白對動物生長發(fā)育的關(guān)鍵作用，一些MCPH蛋白缺失甚至?xí)?dǎo)致動物胚胎致死或細胞凋亡。基因敲除小鼠的構(gòu)建為表型分析和MCPH致病機制的研究提供了有效的動物模型。但由于小鼠大腦與人腦相比沒有腦回結(jié)構(gòu)及更多類型的神經(jīng)前體細胞，基因敲除小鼠有時不能很好地模擬小頭癥的表型。2016年，科學(xué)家利用TALEN技術(shù)制備了突變體食蟹猴()，等位基因突變食蟹猴表現(xiàn)出頭圍減小、胼胝體發(fā)育不良以及上肢痙攣等特征模擬了大部分小頭癥病人的臨床表型[51]。近年來有研究在雪貂()中敲除，敲除雪貂表現(xiàn)出更為明顯的小頭癥表型[52]。

圖4 MCPH蛋白參與的生物學(xué)過程

根據(jù)小鼠基因組信息(Mouse Genome Informatics, MGI)數(shù)據(jù)庫總結(jié)得到25個MCPH蛋白參與的生物學(xué)過程。

圖5 Mcph基因突變相關(guān)表型

根據(jù)小鼠基因組信息(Mouse Genome Informatics, MGI)數(shù)據(jù)庫總結(jié)得到25個基因突變的表型。

表1 MCPH相關(guān)動物模型

續(xù)表

5 MCPH分子機制研究

5.1 MCPH蛋白功能預(yù)測

MCPH蛋白分子的功能是多樣化的。根據(jù)MGI網(wǎng)站生物信息學(xué)分析預(yù)測一些MCPH蛋白如MCPH3、MCPH6、MCPH13、MCPH20和MCPH25主要參與結(jié)合細胞骨架蛋白。一些MCPH蛋白結(jié)合DNA (MCPH3、MCPH10和MCPH11)和糖類及其衍生物(MCPH12、MCPH13、MCPH17和MCPH20)；還有一些蛋白則作為各種酶如水解酶(MCPH13和MCPH20)、轉(zhuǎn)移酶(MCPH6、MCPH12、MCPH17和MCPH18)催化各種生物化學(xué)反應(yīng)來調(diào)控生物體內(nèi)的新陳代謝和能量轉(zhuǎn)換(圖6)。

5.2 MCPH參與的信號通路

MCPH蛋白通常調(diào)控細胞周期，因此很多MCPH蛋白都參與細胞周期相關(guān)信號通路的調(diào)控。MCPH1通過Chk1–Cdc25–Cdk1調(diào)控中心體及紡錘體的形成進而調(diào)控神經(jīng)前體細胞的分裂[53]。MCPH1/ BRIT1還可以與E2F1結(jié)合調(diào)控CHK1和BRCA1參與DNA修復(fù)和細胞凋亡調(diào)控[71]。MCPH2/WDR62通過JNK信號通路調(diào)控神經(jīng)前體細胞的增殖和分化[20]。同時，MEKK3和FBW7雙向調(diào)控WDR62蛋白的穩(wěn)定性進而調(diào)控大腦皮層神經(jīng)干細胞穩(wěn)態(tài)平衡[72]。MCPH3/CDK5RAP2與EB1結(jié)合調(diào)控微管的動態(tài)組裝[32]。MCPH5/ASPM通過Wnt信號通路調(diào)控神經(jīng)前體細胞的增殖和分化[27]。MCPH6/CPAP在大腦發(fā)育過程中作用于Ascl1下游來調(diào)控神經(jīng)前體細胞的分裂及神經(jīng)遷移[28]。MCPH7通過調(diào)控Sonic hedgehog (Shh)信號通路來調(diào)控胚胎體軸的發(fā)育[36]。MCPH9/CEP152與PLK4結(jié)合調(diào)控中心粒的復(fù)制[52]。MCPH9/CEP152和MCPH17/CITK被報道與p53信號通路相關(guān)[47,67]。MCPH18/ALFY通過調(diào)控自噬來調(diào)控經(jīng)典Wnt信號通路[35]。MCPH19/COPB2通過上調(diào)YAP表達來調(diào)控細胞增殖[73]。

圖6 MCPH蛋白的分子生物學(xué)功能

根據(jù)小鼠基因組信息(Mouse Genome Informatics, MGI)數(shù)據(jù)庫總結(jié)得到25個MCPH蛋白的分子生物學(xué)功能。

6 結(jié)語與展望

現(xiàn)代人大腦的尺寸大小約是3百萬年前人類祖先南方古猿人大腦的3倍，特別是大腦皮層及腦回數(shù)量增加達100倍[74]。大腦的尺寸大小及結(jié)構(gòu)復(fù)雜度的增加必然伴隨認知功能的增加[75]。由于大腦的尺寸大小及腦回數(shù)量與神經(jīng)元的數(shù)量密切相關(guān)，因此研究那些影響神經(jīng)細胞增殖、分化及凋亡的基因有利于了解腦發(fā)育過程及人類的進化過程。MCPH是一種常染色體隱性遺傳小頭畸形病癥。本文對已報道的25個MCPH蛋白的表達、定位和功能進行了總結(jié)和概述，為研究大腦發(fā)育相關(guān)蛋白特別是MCPH蛋白的致病機制提供了理論依據(jù)和線索。在已報道的25個MCPH中，和突變導(dǎo)致的MCPH病人較多，因此相關(guān)研究也較多。同時根據(jù)被發(fā)現(xiàn)的順序，一些較早發(fā)現(xiàn)的MCPH蛋白(MCPH1~MCPH10)的動物模型建立較多，而較晚發(fā)現(xiàn)的MCPH11~MCPH25只有少部分蛋白的動物模型已經(jīng)建立。在已建立的動物模型中，大部分MCPH缺失或突變模型都會導(dǎo)致小頭癥的典型特征即大腦明顯變小，還有一部分動物模型影響了生殖系統(tǒng)。導(dǎo)致小頭癥的分子機制研究主要集中在調(diào)控中心體或紡錘體的形成進而影響神經(jīng)前體細胞的增殖分化和細胞凋亡。也有一些MCPH突變小鼠表現(xiàn)出小腦發(fā)育不良或神經(jīng)元軸突發(fā)育異常。近年來，大腦類器官的建立為研究大腦疾病包括小頭癥的發(fā)病機制提供了很好的模型[76,77]。Gabriel等[77,78]利用人iPSC誘導(dǎo)的類腦模型研究MCPH6/CPAP的致病機制。鑒于大腦類器官模型在體外培養(yǎng)的局限性，構(gòu)建小頭癥相關(guān)蛋白缺失或突變的動物模型仍是未來研究小頭癥發(fā)病機制的有效手段。

大腦的尺寸在一定程度上反應(yīng)腦重量和腦容量的大小[79]。但是擁有一個更大的大腦并不意味著人類或動物在認知方面就表現(xiàn)得更好。病人的頭圍相對于其同年齡與性別的平均值明顯大2個標準差以上會導(dǎo)致另外一種腦發(fā)育疾病稱為巨腦癥(macro-cephaly)[80]。巨腦癥主要是由于神經(jīng)增殖和遷移異常引起，病人表現(xiàn)出智力低下并有部分伴有自閉癥樣行為[80~83]。因此，維持大腦尺寸的正常發(fā)育對于腦功能的正常發(fā)揮起著非常重要的作用。

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Update on autosomal recessive primary microcephaly (MCPH)-associated proteins

Yujie Wang, Xiaokun Zhou, Dan Xu

Brain development diseases refer to a group of diseases that affect the development of the brain or the central nervous system. Autosomal recessive primary microcephaly (MCPH) is a typical neurodevelopmental disorder characterized by a decreased brain size, mental retardation and abnormal behaviors. To date, at least 25 genes have been discovered to cause MCPH when mutated. These genes were namedaccording to the discovery order. MCPH proteins play important roles in regulating brain developmental signaling pathways. Here, we provide a timely review of the expression patterns, cellular localization, molecular functions, phenotypes, as well as animal models of these 25 MCPH proteins that will expedite our understanding of the pathogenesis of brain disorders at both molecular and cellular levels.

microcephaly; expression pattern; cellular component; phenotype overview; molecular function; animal models

2019-04-18；

2019-05-15

福建省自然科學(xué)基金項目（編號：2018J01730）資助[Supported by the Natural Science Foundation of the Fujian Province (No. 2018J01730)]

王玉杰，碩士研究生，專業(yè)方向：細胞生物學(xué)。E-mail: yjwang9412@163.com

徐丹，博士，助理研究員，研究方向：腦發(fā)育相關(guān)疾病。E-mail: xu200828@163.com

10.16288/j.yczz.19-070

2019/6/11 17:00:20

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20190611.1700.001.html

(責(zé)任編委: 許執(zhí)恒)