CHRM3基因與孤獨(dú)癥譜系障礙*

巨興達(dá) 宋 偉 徐 婧

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基因與孤獨(dú)癥譜系障礙*

巨興達(dá)1宋 偉1徐 婧2

(1東北師范大學(xué)心理學(xué)院, 長(zhǎng)春 130024) (2長(zhǎng)春中醫(yī)藥大學(xué)臨床醫(yī)學(xué)院, 長(zhǎng)春 130117)

孤獨(dú)癥譜系障礙是一類具有遺傳基礎(chǔ)的兒童發(fā)展障礙疾病。近些年, 研究者們從分子病理學(xué)層面發(fā)現(xiàn)中樞膽堿能神經(jīng)系統(tǒng)異常與孤獨(dú)癥患者認(rèn)知和行為異常存在相關(guān)性。尸檢研究、臨床案例、動(dòng)物模型研究均發(fā)現(xiàn)毒蕈堿型(M型)乙酰膽堿受體異常和孤獨(dú)癥的發(fā)生有著密切的關(guān)系。在以小鼠為模型的行為學(xué)研究中, 編碼毒蕈堿型乙酰膽堿受體Ⅲ亞型的基因突變會(huì)導(dǎo)致小鼠出現(xiàn)認(rèn)知障礙、刻板行為等孤獨(dú)癥樣表現(xiàn)。深入了解基因的功能將能夠幫助研究者進(jìn)一步解釋孤獨(dú)癥的相關(guān)行為特征, 為孤獨(dú)癥兒童教育方案的制定提供新的思路和方法。

孤獨(dú)癥譜系障礙;基因; 臨床特征; 動(dòng)物模型

1 引言

孤獨(dú)癥譜系障礙(Autism Spectrum Disorders, ASD), 簡(jiǎn)稱孤獨(dú)癥, 是一種發(fā)病于嬰幼兒時(shí)期的、常見(jiàn)的社會(huì)性發(fā)展障礙, 與大腦的神經(jīng)化學(xué)機(jī)制異常有著密切的關(guān)系。美國(guó)精神疾病手冊(cè)第五版(Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, DSM-V)指出孤獨(dú)癥患者的核心癥狀表現(xiàn)為：持續(xù)性的社會(huì)交流和社會(huì)互動(dòng)能力缺失, 以及興趣狹窄和重復(fù)刻板的行為方式。美國(guó)疾病控制與預(yù)防中心(Christensen et al., 2012)最新調(diào)查結(jié)果顯示, 兒童孤獨(dú)癥患病率已達(dá)14.4‰, 即每68名8歲以下兒童中就有一名孤獨(dú)癥患兒, 與2000年相比, 患病比率增長(zhǎng)了2.18倍。因此, 探究孤獨(dú)癥的發(fā)病原因已經(jīng)成為醫(yī)學(xué)、生物學(xué)界的重要議題之一。

生物遺傳學(xué)研究表明, 大約10%~30%孤獨(dú)癥發(fā)病是由基因異常導(dǎo)致的(Huguet, Ey, & Bourgeron, 2013; Gaugler et al., 2014; Sanders et al., 2015), 即基因異常影響了其編碼的蛋白質(zhì)的結(jié)構(gòu)和功能, 進(jìn)而改變了腦的特定功能, 最終表現(xiàn)為患者的認(rèn)知和行為異常。雙生子研究也證明遺傳因素在孤獨(dú)癥發(fā)病中起著非常重要的作用, 同卵雙生的孤獨(dú)癥共患率大約為77%~95%, 顯著高于異卵雙生子31% (Ronald, Happe, & Plomin, 2005; Taniai, Nishiyama, Miyachi, Imaeda, & Sumi, 2008; Rosenberg et al., 2009)。家族聚集性研究顯示, 同胞患孤獨(dú)癥的幾率為10%~20%, 大約是家庭中出現(xiàn)新生孤獨(dú)癥概率的20倍(Ozonoff et al., 2011; Wood et al., 2015), 據(jù)此推測(cè)父母某一方患孤獨(dú)癥其子代患病風(fēng)險(xiǎn)大概為10%~15%, 且男嬰患病率高于女?huà)?Vorstman et al., 2017)。根據(jù)同卵雙生、異卵雙生共患的差異以及患者同胞再患的危險(xiǎn)度推斷, 孤獨(dú)癥的遺傳幾率可達(dá)91%~93% (Bailey et al., 1995)。借助基因二代測(cè)序技術(shù), 已發(fā)現(xiàn)多個(gè)染色體區(qū)域上的拷貝數(shù)變異(Copy Number Variants, CNV)會(huì)增加孤獨(dú)癥患病風(fēng)險(xiǎn)。到目前為止, 有4%~20% 的孤獨(dú)癥患者攜帶疾病相關(guān)的CNV (Schaaf & Zoghbi, 2011; Pinto et al., 2014), 已發(fā)現(xiàn)的包含CNV的染色體片段達(dá)2223個(gè), 遍及所有染色體。除此之外, 基因新生突變(de novo mutations)也被認(rèn)為是孤獨(dú)癥發(fā)生的一個(gè)重要原因。SFARI (Simons Foundation Autism Research Initiative)目前已經(jīng)收錄了990個(gè)孤獨(dú)癥相關(guān)基因, 包括和等(Michaelson et al., 2012; Pinto et al., 2011; Roohi et al., 2008; Neale et al., 2012; Bernier et al., 2014)。其中部分已經(jīng)實(shí)驗(yàn)驗(yàn)證為孤獨(dú)癥易感基因, 如基因突變影響神經(jīng)元突觸發(fā)育過(guò)程, 導(dǎo)致該基因缺失小鼠表現(xiàn)出多項(xiàng)典型的孤獨(dú)癥行為特征(Durand et al., 2007)。無(wú)義突變使轉(zhuǎn)錄過(guò)程提前終止, 導(dǎo)致編碼產(chǎn)物縮短, 破壞了蛋白質(zhì)原有功能, 影響神經(jīng)元增殖、樹(shù)突發(fā)育和突觸形成過(guò)程, 被認(rèn)為是導(dǎo)致孤獨(dú)癥發(fā)病的重要風(fēng)險(xiǎn)因素(Bernier et al., 2014; O’Roak, Vives, Fu, et al., 2012; Neale et al., 2012)?；蛲蛔兏淖兞藢?duì)組蛋白H2A的化學(xué)修飾, 使得小鼠出現(xiàn)孤獨(dú)癥類似行為(Gao et al., 2014)。此類研究均證實(shí)了基因功能異常是孤獨(dú)癥發(fā)生的重要原因。

目前已發(fā)現(xiàn)的孤獨(dú)癥易感基因多與神經(jīng)系統(tǒng)發(fā)育有關(guān), 涉及神經(jīng)細(xì)胞的運(yùn)動(dòng)與增殖、神經(jīng)元的軸突投射、樹(shù)突棘可塑性、突觸形成和維持等, 與核染色質(zhì)重組、基因轉(zhuǎn)錄調(diào)控、酶的活性調(diào)控、細(xì)胞骨架調(diào)控、蛋白化學(xué)修飾等過(guò)程密切相關(guān)(Pinto et al., 2010; Sanders et al., 2012; Sakai et al., 2011; O’Roak, Vives, Fu, et al., 2012; King et al., 2013; Donato, Chowdhury, Lahr, & Caroni, 2015), 所涉及的分子信號(hào)通路包括Wnt信號(hào)通路(O’Roak, Vives, Girirajan, et al., 2012; Mine, Yuskaitis, King, Beurel, & Jope, 2010; Okerlund & Cheyette, 2011)、鈣離子信號(hào)通路(Yun & Trommer, 2011; Moretti et al., 2006)、神經(jīng)生長(zhǎng)因子(nerve growth factor, NGF)信號(hào)通路(Riikonen & Vanhala, 1999; Nelson et al., 2001)、以及G蛋白偶聯(lián)受體(G Protein-Coupled Receptor, GPCR)信號(hào)通路等(Zhang & Alger, 2010; Maccarrone et al., 2010; Chen et al., 2011; Silverman et al., 2012)。由此可見(jiàn), 基因異常影響了關(guān)鍵的神經(jīng)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo), 因此被視作孤獨(dú)癥發(fā)生的高風(fēng)險(xiǎn)因素之一。近年來(lái)以基因?yàn)榘悬c(diǎn)開(kāi)展孤獨(dú)癥研究已成為了相關(guān)領(lǐng)域研究者關(guān)注的重點(diǎn)。

長(zhǎng)期以來(lái), 人們對(duì)孤獨(dú)癥的認(rèn)識(shí)多是從異常行為入手。有學(xué)者指出, 孤獨(dú)癥患者個(gè)體之間存在巨大差異, 且不同基因突變可導(dǎo)致不同孤獨(dú)癥行為特征(Happe, Ronald, & Plomin, 2006), 一些針對(duì)刻板行為和交流障礙的研究已證實(shí)了該現(xiàn)象(Cuccaro et al., 2003; Buxbaum et al., 2001)。所以將基因功能和行為研究聯(lián)系起來(lái), 不但能揭示孤獨(dú)癥發(fā)病機(jī)制, 更能促進(jìn)孤獨(dú)癥治療和康復(fù)(State & Sestan, 2012)。

2 毒蕈堿型乙酰膽堿受體Ⅲ亞型(cholinergic receptor, muscarinic 3, CHRM3)

作為一種神經(jīng)遞質(zhì), 乙酰膽堿(acetylcholine, ACh)在信號(hào)傳遞中扮演著重要角色, 可調(diào)節(jié)神經(jīng)系統(tǒng)發(fā)育和神經(jīng)元興奮性變化。膽堿能神經(jīng)元廣泛分布于全腦, 涉及學(xué)習(xí)記憶、認(rèn)知調(diào)節(jié)、情緒控制以及社會(huì)交往等過(guò)程(Bentley, Vuilleumier, Thiel, Driver, & Dolan, 2003; Dani & Bertrand, 2007; Karva & Kimchi, 2014), 膽堿能信號(hào)通路異常與多種精神類疾病的發(fā)生有關(guān)(Bowen, Smith, White, & Davison, 1976; Whitehouse et al., 1982; Deng, & Reiner., 2016)。動(dòng)物模型研究發(fā)現(xiàn)膽堿能相關(guān)基因突變會(huì)導(dǎo)致小鼠出現(xiàn)孤獨(dú)癥癥狀(Zhang et al., 2016), 基因功能異常影響腦內(nèi)膽堿能信號(hào)通路的信號(hào)傳遞以及膽堿能相關(guān)因子的表達(dá)水平, 進(jìn)而引發(fā)孤獨(dú)癥。同時(shí), 還有研究發(fā)現(xiàn)孤獨(dú)癥患者腦內(nèi)灰質(zhì)和顳葉腦區(qū)膽堿能信號(hào)通路異常(Perry et al., 2001; Lee et al., 2002; Martin-Ruiz et al., 2004; Ray et al., 2005; Friedman et al., 2006; Deutsch, Urbano, Neumann, Burket, & Katz, 2010; Petersen et al., 2013), 藥物學(xué)研究中利用VPA (valproic acid)大鼠模型發(fā)現(xiàn), 給孕期大鼠注射VPA能夠?qū)е麓笫蠹捌渥哟哪憠A能神經(jīng)系統(tǒng)紊亂, 增加患孤獨(dú)癥的風(fēng)險(xiǎn), 而使用ACh酯酶抑制劑藥物對(duì)緩解其出現(xiàn)的社交障礙、認(rèn)知障礙和重復(fù)刻板行為問(wèn)題十分有效(Kim er al., 2014)。目前美國(guó)食品藥物管理局(Food and Drug Administration, FDA)已批準(zhǔn)使用ACh酯酶抑制劑緩解孤獨(dú)癥癥狀(Dineley, Pandya, & Yakel, 2015), 因此, 膽堿能相關(guān)通路應(yīng)在孤獨(dú)癥研究和治療中受到更多關(guān)注, 檢測(cè)其正常與否在未來(lái)也許可以成為研究、診斷和治療孤獨(dú)癥或是區(qū)分孤獨(dú)癥不同亞型的一個(gè)重要參考指標(biāo)。

毒蕈堿型乙酰膽堿受體Ⅲ亞型(cholinergic receptor, muscarinic 3, CHRM3)是介導(dǎo)ACh信號(hào)傳遞的受體之一, 是毒蕈堿型乙酰膽堿受體(muscarinic acetylcholine receptor, mAChR)家族一員, 廣泛分布于前腦、海馬以及下丘腦等區(qū)域, 在腦內(nèi)神經(jīng)信號(hào)傳導(dǎo)和行為調(diào)節(jié)中具有重要作用(Levey, Edmunds, Heilman, Desmond, & Frey, 1994)。CHRM3屬于G蛋白偶聯(lián)受體, 是一種大量分布在神經(jīng)系統(tǒng)中的突觸后膜促代謝型受體。在正常生理狀況下, CHRM3接收到乙酰膽堿信號(hào)刺激后通過(guò)Gq蛋白激活磷脂酶C (PLC, phospholipase C), 進(jìn)而作用于第二信使二酰甘油(DAG, diacylglycerol)和三磷酸肌醇(IP3, inositol 1, 4, 5-triphosphate), 調(diào)控細(xì)胞的增殖、代謝、細(xì)胞骨架和突觸可塑性(Matsui et al., 1999)。由于CHRM3分布廣泛, 對(duì)個(gè)體高級(jí)神經(jīng)活動(dòng)的發(fā)生有著關(guān)鍵性的作用, 因此基因突變會(huì)對(duì)神經(jīng)系統(tǒng)生長(zhǎng)發(fā)育產(chǎn)生重要的影響, 可能導(dǎo)致癲癇(Koeleman, 2018)、精神分裂癥(Devor et al., 2017)、阿爾茨海默癥(Tsang et al., 2008)等多種神經(jīng)系統(tǒng)疾病。近年來(lái), 越來(lái)越多的研究者開(kāi)始關(guān)注GPCRs以及Gq-PLC信號(hào)通路異常與孤獨(dú)癥的關(guān)系(Chen et al., 2011; Silverman et al., 2012; O'Connor, Bariselli, & Bellone, 2014)。遺傳學(xué)研究證實(shí), 位于Gq-PLC信號(hào)通路下游的基因是孤獨(dú)癥易感基因(Spinelli, Black, Berg, Eickholt, & Leslie, 2015; Cupolillo et al., 2015)。藥物研究發(fā)現(xiàn)給孤獨(dú)癥模型小鼠BTBR T~(+)tf/J注射mGlu5R拮抗劑對(duì)于改善小鼠的刻板行為和社交行為有明顯的效果(Silverman et al., 2012)。值得注意的是, mGlu5R與CHRM3同為G蛋白偶聯(lián)受體, 均通過(guò)與Gq蛋白偶聯(lián)激活PLC。這一系列研究暗示CHRM3及Gq-PLC信號(hào)通路可能對(duì)孤獨(dú)癥發(fā)生發(fā)展有重要影響。

臨床報(bào)道與基因檢測(cè)結(jié)果均表明基因所在的1q43染色體區(qū)域缺陷與孤獨(dú)癥相關(guān)(見(jiàn)表1, Perrone et al., 2012; Petersen et al., 2013; Soueid et al., 2016)。該基因突變患者會(huì)表現(xiàn)出不同程度的行為異常、認(rèn)知障礙、言語(yǔ)障礙以及運(yùn)動(dòng)發(fā)育遲緩等問(wèn)題(Silipigni et al., 2017; Luukkonen et al., 2017)。Gai等人在(2012)年通過(guò)單核苷酸多態(tài)性微陣列(SNP microarray)技術(shù)對(duì)1224名孤獨(dú)癥患者的染色體進(jìn)行分析, 結(jié)果顯示有患者的編碼區(qū)內(nèi)存在CNV (Gai et al., 2012)。此外, 利用全基因組關(guān)聯(lián)分析等方法, 多項(xiàng)研究都提出基因可能是孤獨(dú)癥易感基因(Hussman et al., 2011; De Rubeis et al., 2014; Butler, Rafi, & Manzardo, 2015; Ch'ng, Kwok, Rogic, & Pavlidis, 2015; Li et al., 2017), 從統(tǒng)計(jì)學(xué)角度證實(shí)了基因突變會(huì)提高孤獨(dú)癥患病風(fēng)險(xiǎn)。同時(shí)研究者在動(dòng)物模型研究中也發(fā)現(xiàn), 抑制或過(guò)度激活CHRM3都將會(huì)導(dǎo)致小鼠出現(xiàn)不同程度的孤獨(dú)癥樣異常行為(Alexander et al., 2009; Wang & McGinty, 1997; Amodeo, Sweeney, & Ragozzino, 2014)。上述結(jié)果說(shuō)明基因與孤獨(dú)癥發(fā)生之間存在密切聯(lián)系。

3 CHRM3基因異常的孤獨(dú)癥患者臨床研究

近期已有兩例與基因異常密切相關(guān)的典型孤獨(dú)癥病例被相繼報(bào)道。

患者一：Perrone等人(2012)報(bào)道了一名7歲的意大利男性孤獨(dú)癥患者。該患者為非近親生獨(dú)子, 足月分娩出生。出生體重3.4 kg, 身高34 cm, 哺乳時(shí)吸入困難, 同時(shí)伴有運(yùn)動(dòng)功能發(fā)育遲緩(12月齡獨(dú)坐, 4歲獨(dú)走)、智力低下、隱睪、身體矮小, 生長(zhǎng)發(fā)育遲緩以及孤獨(dú)癥行為等特征。查體顯示枕骨周圍有脫發(fā)斑點(diǎn), 出現(xiàn)脫發(fā)跡象; 腳趾拇指和第五指先天性趾側(cè)彎; 有內(nèi)斜視和咬手的問(wèn)題特征; 在喂養(yǎng)方面由于患者有咀嚼困難的問(wèn)題, 因此只能吃混合食物?；驒z測(cè)結(jié)果顯示患者1號(hào)染色體丟失91172 bp, 為新生突變, 該缺失區(qū)域包含基因、基因、基因。其中,為假基因, 即在基因組上的非功能性基因組DNA拷貝, 一般情況下不被轉(zhuǎn)錄, 沒(méi)有明確的生理意義?；蚝突蚓c中樞神經(jīng)系統(tǒng)發(fā)育有關(guān), 是潛在的致病基因?；颊進(jìn)RI (Magnetic Resonance Imaging)、心電圖和腹部超聲檢查正常。

表1 孤獨(dú)癥家系研究中的CHRM3突變

圖1 CHRM3信號(hào)傳導(dǎo)模式圖。CHRM3可能通過(guò)“Gq-PLC-第二信使”信號(hào)通路調(diào)控神經(jīng)細(xì)胞的增殖、運(yùn)動(dòng)、分化、突起生長(zhǎng)和興奮性

患者二：Petersen等人(2013)報(bào)道的是一名3歲7個(gè)月的男性患者, 患者系G3P1A1 (懷孕3次; 分娩1次; 流產(chǎn)1次)母親足月生胎兒, 出生體重3.3 kg。4個(gè)月時(shí)常規(guī)查體和MRI檢查發(fā)現(xiàn)患者表現(xiàn)出斜視和顱神經(jīng)麻痹的癥狀, 12個(gè)月左右被發(fā)現(xiàn)語(yǔ)言發(fā)育遲緩, 3歲7個(gè)月時(shí)經(jīng)ADOS (Autism Diagnostic Observation Schedule)診斷為孤獨(dú)癥?；颊弑憩F(xiàn)出多動(dòng)、易怒、注意力差、自傷行為傾向、對(duì)觸覺(jué)/視覺(jué)刺激異常敏感、行為刻板、社交能力嚴(yán)重受損等行為缺陷?；驒z測(cè)結(jié)果顯示患者1號(hào)染色體丟失473 kb, 為新生突變, 丟失區(qū)域內(nèi)只含有基因。此外, 患者母親報(bào)告在產(chǎn)前曾出現(xiàn)子癇前期的癥狀。

將兩名基因缺失的孤獨(dú)癥患者的臨床表現(xiàn)進(jìn)行對(duì)比, 發(fā)現(xiàn)患者均表現(xiàn)出認(rèn)知功能受損、發(fā)育遲緩、進(jìn)食困難的特征(表2)。此外, 在目前報(bào)道的其他基因缺失的臨床案例中, 患者還出現(xiàn)了癲癇、中風(fēng)、發(fā)育遲緩以及注意力缺陷等與神經(jīng)系統(tǒng)功能異常有關(guān)的特征(Shimojima et al., 2012; Luukkonen et al., 2017)。

表2 兩名CHRM3基因缺失的孤獨(dú)癥患者的臨床表現(xiàn)對(duì)比

注：NA, date not available

4 CHRM3相關(guān)動(dòng)物模型研究

4.1 CHRM3異常與孤獨(dú)癥刻板行為

重復(fù)刻板行為是孤獨(dú)癥診斷中的一項(xiàng)重要標(biāo)準(zhǔn)。在《精神疾病診斷與統(tǒng)計(jì)手冊(cè)第五版》(DSM-V)中, 刻板行為被定義為：一種重復(fù)性、限制性的行為、興趣或活動(dòng)。其主要表現(xiàn)為自我刺激行為, 如尖叫、轉(zhuǎn)圈等和自傷行為, 還包括一些儀式性、規(guī)則性的行為, 具體表現(xiàn)為每天在固定的時(shí)間完成某項(xiàng)任務(wù), 或者固定地以某種方式進(jìn)行某項(xiàng)活動(dòng)等?？贪逍袨闀?huì)嚴(yán)重影響患者的正常生活, 對(duì)患者的社交和學(xué)習(xí)造成阻礙。

Petersen等人(2013)報(bào)道的基因異常的患者表現(xiàn)出刻板行為：經(jīng)常抓自己的頭發(fā)、用頭撞墻, 只吃固定的食物; 同時(shí)患者也出現(xiàn)咬手的自傷行為。因此孤獨(dú)癥患者的刻板行為可能與基因異常有關(guān)。在孤獨(dú)癥的動(dòng)物模型研究中, 改變基因的功能不僅會(huì)影響孤獨(dú)癥小鼠的刻板行為, 還會(huì)影響正常小鼠是否會(huì)出現(xiàn)孤獨(dú)癥樣行為特征。

BTBR T~(+)tf/J (簡(jiǎn)稱BTBR)小鼠是一種近交系小鼠, 即不同個(gè)體間98%以上的基因座為純和狀態(tài)的小鼠品系, 因此具有穩(wěn)定的基因型。該品系小鼠能在不同代子代中穩(wěn)定地表現(xiàn)出社會(huì)交往交流障礙和重復(fù)刻板的行為、興趣等孤獨(dú)癥樣行為, 以及與孤獨(dú)癥患者類似的腦發(fā)育異常、免疫生化指標(biāo)異常的問(wèn)題特征(Yang et al., 2007; Bolivar, Walters, & Phoenix, 2007), 是一種良好的孤獨(dú)癥研究動(dòng)物模型。研究發(fā)現(xiàn)BTBR小鼠腦內(nèi)乙酰膽堿水平顯著低于野生型小鼠(McTighe, Neal, Lin, Hughes, & Smith, 2013), 給小鼠注射M型受體激動(dòng)劑氧化震顫素(Oxotremorine)可以顯著減少小鼠的自我理毛和埋珠子等刻板行為(Amodeo et al., 2014)。另外在臨床藥理學(xué)研究中也曾發(fā)現(xiàn), 當(dāng)給孤獨(dú)癥患者使用拮抗M型乙酰膽堿受體的精神類藥物后, 患者重復(fù)刻板問(wèn)題行為顯著增加(Martin, Koenig, Scahill, & Bregman, 1999; Hardan, Jou, & Handen, 2005)。但是以上有關(guān)研究只是發(fā)現(xiàn)改變M型受體的信號(hào)轉(zhuǎn)導(dǎo)功能會(huì)影響孤獨(dú)癥的重復(fù)刻板行為出現(xiàn), 并沒(méi)有詳細(xì)探究這種異常是否是由于CHRM3功能異常所致。

Alexander等人(2009)的研究證明, 改變CHRM3功能將會(huì)影響小鼠出現(xiàn)重復(fù)刻板的孤獨(dú)癥樣行為。研究者使改造后的人(human M3 muscarinic DREADD receptor coupled to Gq, hM3Dq)基因在小鼠前腦中正常表達(dá), 由于hM3Dq無(wú)法接受內(nèi)源性乙酰膽堿的信號(hào)刺激, 因此注射疊氮平-N-氧化物(clozapine-N-oxide, CNO)可以誘導(dǎo)激活CHRM3下游信號(hào)通路, 起到過(guò)度激活CHRM3的效果。研究者發(fā)現(xiàn)當(dāng)不給hM3Dq小鼠注射外源性配體CNO時(shí), hM3Dq小鼠與野生型小鼠的各項(xiàng)行為指標(biāo)均無(wú)顯著差異。當(dāng)給小鼠注射較高濃度CNO后, CHRM3被過(guò)度激活, hM3Dq小鼠的刻板行為顯著增加, 多動(dòng)行為增多且出現(xiàn)癲癇癥狀。上述研究不僅揭示了CHRM3功能與孤獨(dú)癥刻板行為間的關(guān)系, 也為孤獨(dú)癥患者的行為干預(yù)提出了新的思路和方法。

4.2 CHRM3異常與認(rèn)知功能受損

認(rèn)知功能受損并非孤獨(dú)癥診斷標(biāo)準(zhǔn)中的核心癥狀, 但是絕大多數(shù)孤獨(dú)癥患者都伴有不同程度的認(rèn)知功能受損問(wèn)題(Wing, 1981; Crane, Pring, Jukes, & Goddard, 2012)。美國(guó)疾病控制與預(yù)防中心(CDCP 2012)的調(diào)查結(jié)果顯示42%~60%的孤獨(dú)癥患者表現(xiàn)出認(rèn)知功能受損的特征, 具體體現(xiàn)為患者在基本概念認(rèn)知、記憶力、注意力等方面的表現(xiàn)低于正常兒童。缺乏正常的認(rèn)知能力導(dǎo)致孤獨(dú)癥兒童無(wú)法對(duì)圖形符號(hào)或語(yǔ)言指令做出正確的識(shí)別、理解和應(yīng)答, 且由于孤獨(dú)癥患者均存在不同程度的語(yǔ)言溝通困難, 進(jìn)而也無(wú)法與老師或家長(zhǎng)進(jìn)行溝通, 患者的學(xué)習(xí)過(guò)程受到了極大的阻礙。因此提高孤獨(dú)癥患者的認(rèn)知能力有利于提高患者的生活技能、適應(yīng)人際交往活動(dòng)。腦發(fā)育過(guò)程中在大腦皮層和海馬等區(qū)域大量表達(dá)(Levey, Edmunds, Koliatsos, Wiley, & Heilman, 1995),意味著基因可能與認(rèn)知功能有關(guān)。Perrone和Petersen等人報(bào)道的兩例基因變異的孤獨(dú)癥患者也都出現(xiàn)了智力發(fā)育落后、注意力缺陷等認(rèn)知功能受損的問(wèn)題。

Poulin等人(2010)在研究中發(fā)現(xiàn),基因敲除小鼠在恐懼性條件反射(fear conditioning)實(shí)驗(yàn)中依賴海馬的環(huán)境聯(lián)系性記憶能力均顯著低于野生型小鼠。由于小鼠的痛覺(jué)和焦慮反應(yīng)與野生型小鼠沒(méi)有顯著差異, 因此研究者推測(cè)小鼠表現(xiàn)出來(lái)的這種認(rèn)知功能受損可能源于海馬CHRM3功能異常。通過(guò)對(duì)基因突變小鼠的研究, Poulin等人認(rèn)為突變小鼠的認(rèn)知功能受損是由CHRM3不能正常磷酸化導(dǎo)致的。CHRM3受體磷酸化發(fā)生在第384號(hào)絲氨酸位點(diǎn)上, 當(dāng)編碼該位點(diǎn)氨基酸的基因突變后, CHRM3無(wú)法正常磷酸化, 影響了β-arrestin與CHRM3的結(jié)合過(guò)程, 導(dǎo)致受體內(nèi)在化過(guò)程受阻, 最終阻斷了神經(jīng)信號(hào)通路的信號(hào)傳遞過(guò)程, 小鼠表現(xiàn)出認(rèn)知能力受損的特征。為了進(jìn)一步了解CHRM3如何影響小鼠的學(xué)習(xí)記憶能力, 研究者測(cè)定了小鼠海馬神經(jīng)元中基因的表達(dá)水平。在環(huán)境聯(lián)系性學(xué)習(xí)過(guò)程中, 突觸后神經(jīng)元興奮產(chǎn)生長(zhǎng)時(shí)程增強(qiáng)(long term potentiation, LTP)激活基因?；蚓幋a的磷酸蛋白可作為轉(zhuǎn)錄因子與DNA結(jié)合, 促進(jìn)或抑制相關(guān)基因的表達(dá), 從而把由外界刺激所誘發(fā)的短暫的細(xì)胞內(nèi)信息與由基因改變所產(chǎn)生的突觸可塑性過(guò)程偶聯(lián)起來(lái), 一旦再次接受該環(huán)境刺激時(shí),基因的表達(dá)水平會(huì)迅速增加, 因此誘導(dǎo)mRNA的表達(dá)可能是形成長(zhǎng)時(shí)記憶的必要條件(Beck & Fibiger, 1995; Tischmeyer, Kaczmarek, Strauss, Jork, & Matthies, 1990)。Poulin等人的結(jié)果顯示突變小鼠海馬和齒狀回內(nèi)基因表達(dá)水平顯著低于野生型小鼠。Rosethorne、Nahorski和Challiss (2008)也曾發(fā)現(xiàn)CHRM3對(duì)表達(dá)起著調(diào)節(jié)作用：CHRM3可以促進(jìn)CREB (cAMP response-element binding protein)磷酸化, 而CREB磷酸化能夠誘導(dǎo)基因表達(dá), 因此激活CHRM3可以提高的表達(dá)水平。值得注意的是, CREB在神經(jīng)元發(fā)育、突觸可塑性建立、學(xué)習(xí)記憶過(guò)程中起著重要的調(diào)節(jié)功能(Silva, Kogan, Frankland, & Kida, 1998; Lonze & Ginty, 2002; Carlezon, Duman, & Nestler, 2005)。綜合以上研究推測(cè),突變小鼠學(xué)習(xí)記憶能力較低的原因可能是由于學(xué)習(xí)記憶相關(guān)神經(jīng)元內(nèi)依賴Gq-PLC的鈣離子信號(hào)通路信號(hào)傳遞受阻抑制了CREB磷酸化, 進(jìn)而抑制了基因啟動(dòng)應(yīng)對(duì)環(huán)境刺激反應(yīng)的下游基因的表達(dá), 因此無(wú)法激活與學(xué)習(xí)記憶相關(guān)神經(jīng)元, 特定腦區(qū)功能受損, 最終表現(xiàn)為個(gè)體學(xué)習(xí)記憶能力較低, 無(wú)法在短時(shí)間內(nèi)習(xí)得應(yīng)對(duì)環(huán)境刺激的反應(yīng)。除此之外, 在Karvat和Kimchi (2014)的研究中還發(fā)現(xiàn), 向BTBR小鼠背內(nèi)側(cè)紋狀體注射乙酰膽堿酯酶抑制劑后可以有效改善小鼠的學(xué)習(xí)能力缺陷的問(wèn)題(Karvat & Kimchi, 2014)。由此可見(jiàn), 在后續(xù)研究中可以通過(guò)向BTBR小鼠的海馬或背內(nèi)側(cè)紋狀體注射CHRM3特異性激動(dòng)劑, 觀察小鼠是否表現(xiàn)出學(xué)習(xí)記憶能力變化, 并測(cè)定表達(dá)量來(lái)進(jìn)一步探究基因在孤獨(dú)癥患者認(rèn)知活動(dòng)中的作用。

當(dāng)前關(guān)于認(rèn)知功能機(jī)制的研究大多集中于邊緣系統(tǒng),突變的孤獨(dú)癥患者的認(rèn)知功能受損主要被認(rèn)為與海馬功能異常有關(guān), 但對(duì)此也有不同的觀點(diǎn), 有研究者認(rèn)為CHRM3介導(dǎo)的信號(hào)傳遞過(guò)程可能是小腦浦肯野細(xì)胞突觸形成的主要機(jī)制(Rinaldo & Hansel, 2013), 因此突變的孤獨(dú)癥患者的認(rèn)知障礙或許是由小腦功能異常所致, 這還需要在今后的研究中進(jìn)一步探討。

4.3 CHRM3異常與孤獨(dú)癥生長(zhǎng)發(fā)育遲緩

研究發(fā)現(xiàn), 孤獨(dú)癥患者中出現(xiàn)生長(zhǎng)發(fā)育遲緩問(wèn)題的比例較高(Haglund & Kallen, 2010), 因此有學(xué)者提出生長(zhǎng)發(fā)育遲緩可能是導(dǎo)致孤獨(dú)癥發(fā)生的中介因素之一(Haglund & Kallen, 2010)。在已報(bào)道的基因異常的臨床案例中, 患者均出現(xiàn)了發(fā)育遲緩的癥狀。

動(dòng)物模型研究發(fā)現(xiàn)敲除小鼠會(huì)出現(xiàn)體重減輕, 攝食量減少、血清內(nèi)瘦素和胰島素水平顯著降低等一系列生長(zhǎng)發(fā)育遲緩的特征(Yamada et al., 2001; Matsui et al., 2000; Meyer, Zhu, Miller, & Roghair, 2014), 這與Perrone等人(2012)和Shimojima等人(2012)報(bào)道的患者的臨床表現(xiàn)相似。研究人員發(fā)現(xiàn)在野生型小鼠腦內(nèi), CHRM3主要分布在下丘腦, 而敲除小鼠下丘腦內(nèi)CHRM3數(shù)量與野生型小鼠相比下降了近50%, 同時(shí)免疫組化研究顯示小鼠下丘腦內(nèi)黑色素聚集激素(melanin- concentrating hormone, MCH)的表達(dá)水平也顯著低于野生型小鼠(Yamada et al., 2001)。已有研究證實(shí)MCH對(duì)于調(diào)控?cái)z食和體重變化具有重要作用(Qu et al., 1996), 且CHRM3與MCH被證實(shí)在外側(cè)下丘腦細(xì)胞內(nèi)共表達(dá), 因此Ymada等人推測(cè)在有關(guān)飲食調(diào)節(jié)的信號(hào)通路中, 瘦素和胰島素作為上游的信號(hào)因子刺激下丘腦弓形核, 激活MCH細(xì)胞, 從而激活了下丘腦信號(hào)通路, 開(kāi)啟信號(hào)轉(zhuǎn)導(dǎo)過(guò)程。在該信號(hào)通路下游的外側(cè)下丘腦內(nèi), CHRM3通過(guò)控制MCH細(xì)胞分泌MCH從而調(diào)控個(gè)體的攝食行為, 即當(dāng)外側(cè)下丘腦內(nèi)的MCH細(xì)胞接收到乙酰膽堿信號(hào)刺激后, CHRM3被激活, MCH釋放量迅速提高, 個(gè)體出現(xiàn)攝食行為。因此在敲除小鼠體內(nèi), CHRM3缺失導(dǎo)致MCH細(xì)胞無(wú)法被激活釋放MCH, 小鼠攝食量下降, 進(jìn)而表現(xiàn)出體重減輕等發(fā)育遲緩的問(wèn)題癥狀。

由于瘦素是激活下丘腦飲食調(diào)節(jié)信號(hào)通路的主要因子, 因此瘦素含量降低也會(huì)導(dǎo)致個(gè)體出現(xiàn)生長(zhǎng)發(fā)育遲緩的癥狀(Meyer et al.,2014)。研究發(fā)現(xiàn), 嬰兒期瘦素缺失將導(dǎo)致發(fā)育遲緩的小鼠在成年期出現(xiàn)運(yùn)動(dòng)能力降低、社交興趣喪失、認(rèn)知能力受損、以及杏仁核體積增大等孤獨(dú)癥樣的異常特征(Meyer et al., 2014)。因此嬰兒期個(gè)體瘦素水平降低可能與孤獨(dú)癥的發(fā)生有關(guān)。結(jié)合在Ymada等人的研究中敲除小鼠血清內(nèi)瘦素含量顯著降低這一結(jié)果, 推測(cè)瘦素含量下降與基因表達(dá)水平降低有關(guān), 早期營(yíng)養(yǎng)不足可能是后期行為問(wèn)題出現(xiàn)的原因之一, 即缺失會(huì)降低個(gè)體的攝食行為, 在一定程度上影響身體生長(zhǎng)和腦的發(fā)育過(guò)程, 最終導(dǎo)致問(wèn)題行為出現(xiàn)。

另外, 免疫組化研究證實(shí)小鼠唾液腺上2/3的M型受體為CHRM3受體, 說(shuō)明CHRM3對(duì)于調(diào)控唾液分泌也具有重要作用(Matsui et al., 2000; Bymaster et al., 2003), 因此突變的生長(zhǎng)發(fā)育遲緩小鼠出現(xiàn)進(jìn)食障礙有可能是由于唾液分泌過(guò)程異常引起的食物消化功能受損所致。以上研究表明CHRM3與生長(zhǎng)發(fā)育之間有著緊密的聯(lián)系, 一方面CHRM3可以通過(guò)調(diào)節(jié)攝食行為來(lái)影響生長(zhǎng)發(fā)育, 另一方面可以通過(guò)調(diào)節(jié)消化能力影響生長(zhǎng)發(fā)育。

圖2 CHRM3異常在腦與個(gè)體不同水平上的影響

5 總結(jié)與展望

作為G蛋白偶聯(lián)受體家族一員, CHRM3介導(dǎo)Gq-PLC信號(hào)通路參與突觸信號(hào)傳遞, 對(duì)于調(diào)控細(xì)胞增殖、代謝、細(xì)胞骨架建立和突觸可塑性形成具有重要作用。由于突觸依賴性的神經(jīng)元信號(hào)傳導(dǎo)是學(xué)習(xí)、記憶等高級(jí)心理活動(dòng)的生理基礎(chǔ), 因此CHRM3可能與人的認(rèn)知能力發(fā)展以及社會(huì)化等發(fā)育過(guò)程密切相關(guān)。

臨床案例和動(dòng)物模型研究均發(fā)現(xiàn)改變CHRM3功能會(huì)引發(fā)動(dòng)物出現(xiàn)認(rèn)知缺陷以及刻板行為等孤獨(dú)癥特征(見(jiàn)圖2)。抑制基因的表達(dá)將會(huì)影響受體磷酸化過(guò)程, 降低海馬、杏仁核、嗅球等組織中神經(jīng)元的活躍水平, 進(jìn)而導(dǎo)致一系列異常行為特征出現(xiàn)。而過(guò)表達(dá)會(huì)導(dǎo)致海馬內(nèi)興奮性神經(jīng)元被過(guò)度激活, 也會(huì)影響孤獨(dú)癥樣行為出現(xiàn), 因此無(wú)論CHRM3所介導(dǎo)的神經(jīng)信號(hào)通路被抑制或是增強(qiáng), 一旦神經(jīng)系統(tǒng)內(nèi)環(huán)境穩(wěn)態(tài)被破壞都有可能引發(fā)孤獨(dú)癥的發(fā)生。鑒于此, 控制Gq-PLC信號(hào)通路活動(dòng)水平適中對(duì)于特定行為的發(fā)展有重要作用。但選擇哪一項(xiàng)指標(biāo)作為衡定信號(hào)通路適中的標(biāo)準(zhǔn), 尚有待今后的深入研究。除此之外, 當(dāng)孤獨(dú)癥高風(fēng)險(xiǎn)基因發(fā)生突變時(shí),的表達(dá)也會(huì)受到影響(Forrest, Waite, Martin-Rendon, & Blake, 2013; Chan et al., 2015)。另外在對(duì)孤獨(dú)癥患者家系全基因組檢測(cè)中, 發(fā)現(xiàn)了一個(gè)CHRM3下游分子PLC家族成員(磷酯酶)的編碼基因存在新生突變, 這暗示CHRM3及其所調(diào)控信號(hào)通路對(duì)孤獨(dú)癥發(fā)生發(fā)展有重要影響。但是目前有關(guān)基因突變?cè)诠陋?dú)癥發(fā)生發(fā)展中的作用以及在腦發(fā)育過(guò)程中的機(jī)制還有待進(jìn)一步探討。

在接下來(lái)的研究中, 可以在建立小鼠動(dòng)物模型的基礎(chǔ)上, 通過(guò)檢測(cè)基因分子水平變化、細(xì)胞組織器官發(fā)育分化、形態(tài)差異以及分析行為特征來(lái)研究基因在神經(jīng)系統(tǒng)發(fā)育中的作用, 及其對(duì)孤獨(dú)癥發(fā)生的影響。另外, 關(guān)注CHRM3所介導(dǎo)的Gq-PLC信號(hào)通路在孤獨(dú)癥發(fā)生中的作用, 可為孤獨(dú)癥的基因靶向干預(yù)提供新的思路和方法, 為教育方案的制定提供科學(xué)的幫助和指導(dǎo)。

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gene and autism spectrum disorder

JU Xingda1; SONG Wei1; XU Jing2

(1School of Psychology, Northeast Normal University, Changchun 130024, China) (2School of Clinical Medicine, Changchun University of Chinese Medicine, Changchun 130117, China)

Autism Spectrum Disorder is one of the most complex developmental disorders with a strong genetic impact. In recent years, researchers have increasingly linked effects of central cholinergic system dysfunction to autism-related cognitive and behavioral abnormalities at the molecular pathological level. Results from autopsy studies, clinical cases and animal experiments revealed that aberrant muscarinic acetylcholine receptors have a strong relationship with autism. In behavioral studies using mouse models, the variations ofgene, which encodes the muscarinic acetylcholine receptor subtype III receptor, can cause autistic phenotypes such as cognitive impairment and stereotypic behavior. Accordingly, in-depth functional understanding ofgene may have important implications to further explain the characteristics and mechanisms of autistic behavior and may potentially provide new ideas and methods for the development of educational programs for autistic children.

autism spectrum disorder;gene; clinical features; animal models

2018-05-19

* 全國(guó)教育科學(xué)“十二五”規(guī)劃教育部青年專項(xiàng)課題“兒童孤獨(dú)癥的基因靶向教育策略研究”(EBA140364)資助。巨興達(dá)、宋偉為共同第一作者

巨興達(dá), E-mail: juxd513@nenu.edu.cn 徐婧, E-mail: xuj391@nenu.edu.cn

B845

10.3724/SP.J.1042.2018.02141