巨興達 宋 偉 徐 婧

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基因與孤獨癥譜系障礙*

巨興達1宋 偉1徐 婧2

(1東北師范大學心理學院, 長春 130024) (2長春中醫(yī)藥大學臨床醫(yī)學院, 長春 130117)

孤獨癥譜系障礙是一類具有遺傳基礎的兒童發(fā)展障礙疾病。近些年, 研究者們從分子病理學層面發(fā)現(xiàn)中樞膽堿能神經系統(tǒng)異常與孤獨癥患者認知和行為異常存在相關性。尸檢研究、臨床案例、動物模型研究均發(fā)現(xiàn)毒蕈堿型(M型)乙酰膽堿受體異常和孤獨癥的發(fā)生有著密切的關系。在以小鼠為模型的行為學研究中, 編碼毒蕈堿型乙酰膽堿受體Ⅲ亞型的基因突變會導致小鼠出現(xiàn)認知障礙、刻板行為等孤獨癥樣表現(xiàn)。深入了解基因的功能將能夠幫助研究者進一步解釋孤獨癥的相關行為特征, 為孤獨癥兒童教育方案的制定提供新的思路和方法。

孤獨癥譜系障礙;基因; 臨床特征; 動物模型

1 引言

孤獨癥譜系障礙(Autism Spectrum Disorders, ASD), 簡稱孤獨癥, 是一種發(fā)病于嬰幼兒時期的、常見的社會性發(fā)展障礙, 與大腦的神經化學機制異常有著密切的關系。美國精神疾病手冊第五版(Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, DSM-V)指出孤獨癥患者的核心癥狀表現(xiàn)為：持續(xù)性的社會交流和社會互動能力缺失, 以及興趣狹窄和重復刻板的行為方式。美國疾病控制與預防中心(Christensen et al., 2012)最新調查結果顯示, 兒童孤獨癥患病率已達14.4‰, 即每68名8歲以下兒童中就有一名孤獨癥患兒, 與2000年相比, 患病比率增長了2.18倍。因此, 探究孤獨癥的發(fā)病原因已經成為醫(yī)學、生物學界的重要議題之一。

生物遺傳學研究表明, 大約10%~30%孤獨癥發(fā)病是由基因異常導致的(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的結構和功能, 進而改變了腦的特定功能, 最終表現(xiàn)為患者的認知和行為異常。雙生子研究也證明遺傳因素在孤獨癥發(fā)病中起著非常重要的作用, 同卵雙生的孤獨癥共患率大約為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, 同胞患孤獨癥的幾率為10%~20%, 大約是家庭中出現(xiàn)新生孤獨癥概率的20倍(Ozonoff et al., 2011; Wood et al., 2015), 據(jù)此推測父母某一方患孤獨癥其子代患病風險大概為10%~15%, 且男嬰患病率高于女嬰(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度推斷, 孤獨癥的遺傳幾率可達91%~93% (Bailey et al., 1995)。借助基因二代測序技術, 已發(fā)現(xiàn)多個染色體區(qū)域上的拷貝數(shù)變異(Copy Number Variants, CNV)會增加孤獨癥患病風險。到目前為止, 有4%~20% 的孤獨癥患者攜帶疾病相關的CNV (Schaaf & Zoghbi, 2011; Pinto et al., 2014), 已發(fā)現(xiàn)的包含CNV的染色體片段達2223個, 遍及所有染色體。除此之外, 基因新生突變(de novo mutations)也被認為是孤獨癥發(fā)生的一個重要原因。SFARI (Simons Foundation Autism Research Initiative)目前已經收錄了990個孤獨癥相關基因, 包括和等(Michaelson et al., 2012; Pinto et al., 2011; Roohi et al., 2008; Neale et al., 2012; Bernier et al., 2014)。其中部分已經實驗驗證為孤獨癥易感基因, 如基因突變影響神經元突觸發(fā)育過程, 導致該基因缺失小鼠表現(xiàn)出多項典型的孤獨癥行為特征(Durand et al., 2007)。無義突變使轉錄過程提前終止, 導致編碼產物縮短, 破壞了蛋白質原有功能, 影響神經元增殖、樹突發(fā)育和突觸形成過程, 被認為是導致孤獨癥發(fā)病的重要風險因素(Bernier et al., 2014; O’Roak, Vives, Fu, et al., 2012; Neale et al., 2012)?；蛲蛔兏淖兞藢M蛋白H2A的化學修飾, 使得小鼠出現(xiàn)孤獨癥類似行為(Gao et al., 2014)。此類研究均證實了基因功能異常是孤獨癥發(fā)生的重要原因。

目前已發(fā)現(xiàn)的孤獨癥易感基因多與神經系統(tǒng)發(fā)育有關, 涉及神經細胞的運動與增殖、神經元的軸突投射、樹突棘可塑性、突觸形成和維持等, 與核染色質重組、基因轉錄調控、酶的活性調控、細胞骨架調控、蛋白化學修飾等過程密切相關(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), 所涉及的分子信號通路包括Wnt信號通路(O’Roak, Vives, Girirajan, et al., 2012; Mine, Yuskaitis, King, Beurel, & Jope, 2010; Okerlund & Cheyette, 2011)、鈣離子信號通路(Yun & Trommer, 2011; Moretti et al., 2006)、神經生長因子(nerve growth factor, NGF)信號通路(Riikonen & Vanhala, 1999; Nelson et al., 2001)、以及G蛋白偶聯(lián)受體(G Protein-Coupled Receptor, GPCR)信號通路等(Zhang & Alger, 2010; Maccarrone et al., 2010; Chen et al., 2011; Silverman et al., 2012)。由此可見, 基因異常影響了關鍵的神經細胞信號轉導, 因此被視作孤獨癥發(fā)生的高風險因素之一。近年來以基因為靶點開展孤獨癥研究已成為了相關領域研究者關注的重點。

長期以來, 人們對孤獨癥的認識多是從異常行為入手。有學者指出, 孤獨癥患者個體之間存在巨大差異, 且不同基因突變可導致不同孤獨癥行為特征(Happe, Ronald, & Plomin, 2006), 一些針對刻板行為和交流障礙的研究已證實了該現(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, 不但能揭示孤獨癥發(fā)病機制, 更能促進孤獨癥治療和康復(State & Sestan, 2012)。

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

作為一種神經遞質, 乙酰膽堿(acetylcholine, ACh)在信號傳遞中扮演著重要角色, 可調節(jié)神經系統(tǒng)發(fā)育和神經元興奮性變化。膽堿能神經元廣泛分布于全腦, 涉及學習記憶、認知調節(jié)、情緒控制以及社會交往等過程(Bentley, Vuilleumier, Thiel, Driver, & Dolan, 2003; Dani & Bertrand, 2007; Karva & Kimchi, 2014), 膽堿能信號通路異常與多種精神類疾病的發(fā)生有關(Bowen, Smith, White, & Davison, 1976; Whitehouse et al., 1982; Deng, & Reiner., 2016)。動物模型研究發(fā)現(xiàn)膽堿能相關基因突變會導致小鼠出現(xiàn)孤獨癥癥狀(Zhang et al., 2016), 基因功能異常影響腦內膽堿能信號通路的信號傳遞以及膽堿能相關因子的表達水平, 進而引發(fā)孤獨癥。同時, 還有研究發(fā)現(xiàn)孤獨癥患者腦內灰質和顳葉腦區(qū)膽堿能信號通路異常(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能夠導致大鼠及其子代的膽堿能神經系統(tǒng)紊亂, 增加患孤獨癥的風險, 而使用ACh酯酶抑制劑藥物對緩解其出現(xiàn)的社交障礙、認知障礙和重復刻板行為問題十分有效(Kim er al., 2014)。目前美國食品藥物管理局(Food and Drug Administration, FDA)已批準使用ACh酯酶抑制劑緩解孤獨癥癥狀(Dineley, Pandya, & Yakel, 2015), 因此, 膽堿能相關通路應在孤獨癥研究和治療中受到更多關注, 檢測其正常與否在未來也許可以成為研究、診斷和治療孤獨癥或是區(qū)分孤獨癥不同亞型的一個重要參考指標。

毒蕈堿型乙酰膽堿受體Ⅲ亞型(cholinergic receptor, muscarinic 3, CHRM3)是介導ACh信號傳遞的受體之一, 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, 在腦內神經信號傳導和行為調節(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刺激后通過Gq蛋白激活磷脂酶C (PLC, phospholipase C), 進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), 調控細胞的增殖、代謝、細胞骨架和突觸可塑性(Matsui et al., 1999)。由于CHRM3分布廣泛, 對個體高級神經活動的發(fā)生有著關鍵性的作用, 因此基因突變會對神經系統(tǒng)生長發(fā)育產生重要的影響, 可能導致癲癇(Koeleman, 2018)、精神分裂癥(Devor et al., 2017)、阿爾茨海默癥(Tsang et al., 2008)等多種神經系統(tǒng)疾病。近年來, 越來越多的研究者開始關注GPCRs以及Gq-PLC信號通路異常與孤獨癥的關系(Chen et al., 2011; Silverman et al., 2012; O'Connor, Bariselli, & Bellone, 2014)。遺傳學研究證實, 位于Gq-PLC信號通路下游的基因是孤獨癥易感基因(Spinelli, Black, Berg, Eickholt, & Leslie, 2015; Cupolillo et al., 2015)。藥物研究發(fā)現(xiàn)給孤獨癥模型小鼠BTBR T~(+)tf/J注射mGlu5R拮抗劑對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, 均通過與Gq蛋白偶聯(lián)激活PLC。這一系列研究暗示CHRM3及Gq-PLC信號通路可能對孤獨癥發(fā)生發(fā)展有重要影響。

臨床報道與基因檢測結果均表明基因所在的1q43染色體區(qū)域缺陷與孤獨癥相關(見表1, Perrone et al., 2012; Petersen et al., 2013; Soueid et al., 2016)。該基因突變患者會表現(xiàn)出不同程度的行為異常、認知障礙、言語障礙以及運動發(fā)育遲緩等問題(Silipigni et al., 2017; Luukkonen et al., 2017)。Gai等人在(2012)年通過單核苷酸多態(tài)性微陣列(SNP microarray)技術對1224名孤獨癥患者的染色體進行分析, 結果顯示有患者的編碼區(qū)內存在CNV (Gai et al., 2012)。此外, 利用全基因組關聯(lián)分析等方法, 多項研究都提出基因可能是孤獨癥易感基因(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)計學角度證實了基因突變會提高孤獨癥患病風險。同時研究者在動物模型研究中也發(fā)現(xiàn), 抑制或過度激活CHRM3都將會導致小鼠出現(xiàn)不同程度的孤獨癥樣異常行為(Alexander et al., 2009; Wang & McGinty, 1997; Amodeo, Sweeney, & Ragozzino, 2014)。上述結果說明基因與孤獨癥發(fā)生之間存在密切聯(lián)系。

3 CHRM3基因異常的孤獨癥患者臨床研究

近期已有兩例與基因異常密切相關的典型孤獨癥病例被相繼報道。

患者一：Perrone等人(2012)報道了一名7歲的意大利男性孤獨癥患者。該患者為非近親生獨子, 足月分娩出生。出生體重3.4 kg, 身高34 cm, 哺乳時吸入困難, 同時伴有運動功能發(fā)育遲緩(12月齡獨坐, 4歲獨走)、智力低下、隱睪、身體矮小, 生長發(fā)育遲緩以及孤獨癥行為等特征。查體顯示枕骨周圍有脫發(fā)斑點, 出現(xiàn)脫發(fā)跡象; 腳趾拇指和第五指先天性趾側彎; 有內斜視和咬手的問題特征; 在喂養(yǎng)方面由于患者有咀嚼困難的問題, 因此只能吃混合食物?；驒z測結果顯示患者1號染色體丟失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, 一般情況下不被轉錄, 沒有明確的生理意義?；蚝突蚓c中樞神經系統(tǒng)發(fā)育有關, 是潛在的致病基因?；颊進RI (Magnetic Resonance Imaging)、心電圖和腹部超聲檢查正常。

表1 孤獨癥家系研究中的CHRM3突變

圖1 CHRM3信號傳導模式圖。CHRM3可能通過“Gq-PLC-第二信使”信號通路調控神經細胞的增殖、運動、分化、突起生長和興奮性

患者二：Petersen等人(2013)報道的是一名3歲7個月的男性患者, 患者系G3P1A1 (懷孕3次; 分娩1次; 流產1次)母親足月生胎兒, 出生體重3.3 kg。4個月時常規(guī)查體和MRI檢查發(fā)現(xiàn)患者表現(xiàn)出斜視和顱神經麻痹的癥狀, 12個月左右被發(fā)現(xiàn)語言發(fā)育遲緩, 3歲7個月時經ADOS (Autism Diagnostic Observation Schedule)診斷為孤獨癥。患者表現(xiàn)出多動、易怒、注意力差、自傷行為傾向、對觸覺/視覺刺激異常敏感、行為刻板、社交能力嚴重受損等行為缺陷?；驒z測結果顯示患者1號染色體丟失473 kb, 為新生突變, 丟失區(qū)域內只含有基因。此外, 患者母親報告在產前曾出現(xiàn)子癇前期的癥狀。

將兩名基因缺失的孤獨癥患者的臨床表現(xiàn)進行對比, 發(fā)現(xiàn)患者均表現(xiàn)出認知功能受損、發(fā)育遲緩、進食困難的特征(表2)。此外, 在目前報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、發(fā)育遲緩以及注意力缺陷等與神經系統(tǒng)功能異常有關的特征(Shimojima et al., 2012; Luukkonen et al., 2017)。

表2 兩名CHRM3基因缺失的孤獨癥患者的臨床表現(xiàn)對比

注：NA, date not available

4 CHRM3相關動物模型研究

4.1 CHRM3異常與孤獨癥刻板行為

重復刻板行為是孤獨癥診斷中的一項重要標準。在《精神疾病診斷與統(tǒng)計手冊第五版》(DSM-V)中, 刻板行為被定義為：一種重復性、限制性的行為、興趣或活動。其主要表現(xiàn)為自我刺激行為, 如尖叫、轉圈等和自傷行為, 還包括一些儀式性、規(guī)則性的行為, 具體表現(xiàn)為每天在固定的時間完成某項任務, 或者固定地以某種方式進行某項活動等。刻板行為會嚴重影響患者的正常生活, 對患者的社交和學習造成阻礙。

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

BTBR T~(+)tf/J (簡稱BTBR)小鼠是一種近交系小鼠, 即不同個體間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)出社會交往交流障礙和重復刻板的行為、興趣等孤獨癥樣行為, 以及與孤獨癥患者類似的腦發(fā)育異常、免疫生化指標異常的問題特征(Yang et al., 2007; Bolivar, Walters, & Phoenix, 2007), 是一種良好的孤獨癥研究動物模型。研究發(fā)現(xiàn)BTBR小鼠腦內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)。另外在臨床藥理學研究中也曾發(fā)現(xiàn), 當給孤獨癥患者使用拮抗M型乙酰膽堿受體的精神類藥物后, 患者重復刻板問題行為顯著增加(Martin, Koenig, Scahill, & Bregman, 1999; Hardan, Jou, & Handen, 2005)。但是以上有關研究只是發(fā)現(xiàn)改變M型受體的信號轉導功能會影響孤獨癥的重復刻板行為出現(xiàn), 并沒有詳細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功能將會影響小鼠出現(xiàn)重復刻板的孤獨癥樣行為。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, 由于hM3Dq無法接受內源性乙酰膽堿的信號刺激, 因此注射疊氮平-N-氧化物(clozapine-N-oxide, CNO)可以誘導激活CHRM3下游信號通路, 起到過度激活CHRM3的效果。研究者發(fā)現(xiàn)當不給hM3Dq小鼠注射外源性配體CNO時, hM3Dq小鼠與野生型小鼠的各項行為指標均無顯著差異。當給小鼠注射較高濃度CNO后, CHRM3被過度激活, hM3Dq小鼠的刻板行為顯著增加, 多動行為增多且出現(xiàn)癲癇癥狀。上述研究不僅揭示了CHRM3功能與孤獨癥刻板行為間的關系, 也為孤獨癥患者的行為干預提出了新的思路和方法。

4.2 CHRM3異常與認知功能受損

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

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)實驗中依賴海馬的環(huán)境聯(lián)系性記憶能力均顯著低于野生型小鼠。由于小鼠的痛覺和焦慮反應與野生型小鼠沒有顯著差異, 因此研究者推測小鼠表現(xiàn)出來的這種認知功能受損可能源于海馬CHRM3功能異常。通過對基因突變小鼠的研究, Poulin等人認為突變小鼠的認知功能受損是由CHRM3不能正常磷酸化導致的。CHRM3受體磷酸化發(fā)生在第384號絲氨酸位點上, 當編碼該位點氨基酸的基因突變后, CHRM3無法正常磷酸化, 影響了β-arrestin與CHRM3的結合過程, 導致受體內在化過程受阻, 最終阻斷了神經信號通路的信號傳遞過程, 小鼠表現(xiàn)出認知能力受損的特征。為了進一步了解CHRM3如何影響小鼠的學習記憶能力, 研究者測定了小鼠海馬神經元中基因的表達水平。在環(huán)境聯(lián)系性學習過程中, 突觸后神經元興奮產生長時程增強(long term potentiation, LTP)激活基因?；蚓幋a的磷酸蛋白可作為轉錄因子與DNA結合, 促進或抑制相關基因的表達, 從而把由外界刺激所誘發(fā)的短暫的細胞內信息與由基因改變所產生的突觸可塑性過程偶聯(lián)起來, 一旦再次接受該環(huán)境刺激時,基因的表達水平會迅速增加, 因此誘導mRNA的表達可能是形成長時記憶的必要條件(Beck & Fibiger, 1995; Tischmeyer, Kaczmarek, Strauss, Jork, & Matthies, 1990)。Poulin等人的結果顯示突變小鼠海馬和齒狀回內基因表達水平顯著低于野生型小鼠。Rosethorne、Nahorski和Challiss (2008)也曾發(fā)現(xiàn)CHRM3對表達起著調節(jié)作用：CHRM3可以促進CREB (cAMP response-element binding protein)磷酸化, 而CREB磷酸化能夠誘導基因表達, 因此激活CHRM3可以提高的表達水平。值得注意的是, CREB在神經元發(fā)育、突觸可塑性建立、學習記憶過程中起著重要的調節(jié)功能(Silva, Kogan, Frankland, & Kida, 1998; Lonze & Ginty, 2002; Carlezon, Duman, & Nestler, 2005)。綜合以上研究推測,突變小鼠學習記憶能力較低的原因可能是由于學習記憶相關神經元內依賴Gq-PLC的鈣離子信號通路信號傳遞受阻抑制了CREB磷酸化, 進而抑制了基因啟動應對環(huán)境刺激反應的下游基因的表達, 因此無法激活與學習記憶相關神經元, 特定腦區(qū)功能受損, 最終表現(xiàn)為個體學習記憶能力較低, 無法在短時間內習得應對環(huán)境刺激的反應。除此之外, 在Karvat和Kimchi (2014)的研究中還發(fā)現(xiàn), 向BTBR小鼠背內側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習能力缺陷的問題(Karvat & Kimchi, 2014)。由此可見, 在后續(xù)研究中可以通過向BTBR小鼠的海馬或背內側紋狀體注射CHRM3特異性激動劑, 觀察小鼠是否表現(xiàn)出學習記憶能力變化, 并測定表達量來進一步探究基因在孤獨癥患者認知活動中的作用。

當前關于認知功能機制的研究大多集中于邊緣系統(tǒng),突變的孤獨癥患者的認知功能受損主要被認為與海馬功能異常有關, 但對此也有不同的觀點, 有研究者認為CHRM3介導的信號傳遞過程可能是小腦浦肯野細胞突觸形成的主要機制(Rinaldo & Hansel, 2013), 因此突變的孤獨癥患者的認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一步探討。

4.3 CHRM3異常與孤獨癥生長發(fā)育遲緩

研究發(fā)現(xiàn), 孤獨癥患者中出現(xiàn)生長發(fā)育遲緩問題的比例較高(Haglund & Kallen, 2010), 因此有學者提出生長發(fā)育遲緩可能是導致孤獨癥發(fā)生的中介因素之一(Haglund & Kallen, 2010)。在已報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。

動物模型研究發(fā)現(xiàn)敲除小鼠會出現(xiàn)體重減輕, 攝食量減少、血清內瘦素和胰島素水平顯著降低等一系列生長發(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, CHRM3主要分布在下丘腦, 而敲除小鼠下丘腦內CHRM3數(shù)量與野生型小鼠相比下降了近50%, 同時免疫組化研究顯示小鼠下丘腦內黑色素聚集激素(melanin- concentrating hormone, MCH)的表達水平也顯著低于野生型小鼠(Yamada et al., 2001)。已有研究證實MCH對于調控攝食和體重變化具有重要作用(Qu et al., 1996), 且CHRM3與MCH被證實在外側下丘腦細胞內共表達, 因此Ymada等人推測在有關飲食調節(jié)的信號通路中, 瘦素和胰島素作為上游的信號因子刺激下丘腦弓形核, 激活MCH細胞, 從而激活了下丘腦信號通路, 開啟信號轉導過程。在該信號通路下游的外側下丘腦內, CHRM3通過控制MCH細胞分泌MCH從而調控個體的攝食行為, 即當外側下丘腦內的MCH細胞接收到乙酰膽堿信號刺激后, CHRM3被激活, MCH釋放量迅速提高, 個體出現(xiàn)攝食行為。因此在敲除小鼠體內, CHRM3缺失導致MCH細胞無法被激活釋放MCH, 小鼠攝食量下降, 進而表現(xiàn)出體重減輕等發(fā)育遲緩的問題癥狀。

由于瘦素是激活下丘腦飲食調節(jié)信號通路的主要因子, 因此瘦素含量降低也會導致個體出現(xiàn)生長發(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致發(fā)育遲緩的小鼠在成年期出現(xiàn)運動能力降低、社交興趣喪失、認知能力受損、以及杏仁核體積增大等孤獨癥樣的異常特征(Meyer et al., 2014)。因此嬰兒期個體瘦素水平降低可能與孤獨癥的發(fā)生有關。結合在Ymada等人的研究中敲除小鼠血清內瘦素含量顯著降低這一結果, 推測瘦素含量下降與基因表達水平降低有關, 早期營養(yǎng)不足可能是后期行為問題出現(xiàn)的原因之一, 即缺失會降低個體的攝食行為, 在一定程度上影響身體生長和腦的發(fā)育過程, 最終導致問題行為出現(xiàn)。

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

圖2 CHRM3異常在腦與個體不同水平上的影響

5 總結與展望

作為G蛋白偶聯(lián)受體家族一員, CHRM3介導Gq-PLC信號通路參與突觸信號傳遞, 對于調控細胞增殖、代謝、細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元信號傳導是學習、記憶等高級心理活動的生理基礎, 因此CHRM3可能與人的認知能力發(fā)展以及社會化等發(fā)育過程密切相關。

臨床案例和動物模型研究均發(fā)現(xiàn)改變CHRM3功能會引發(fā)動物出現(xiàn)認知缺陷以及刻板行為等孤獨癥特征(見圖2)。抑制基因的表達將會影響受體磷酸化過程, 降低海馬、杏仁核、嗅球等組織中神經元的活躍水平, 進而導致一系列異常行為特征出現(xiàn)。而過表達會導致海馬內興奮性神經元被過度激活, 也會影響孤獨癥樣行為出現(xiàn), 因此無論CHRM3所介導的神經信號通路被抑制或是增強, 一旦神經系統(tǒng)內環(huán)境穩(wěn)態(tài)被破壞都有可能引發(fā)孤獨癥的發(fā)生。鑒于此, 控制Gq-PLC信號通路活動水平適中對于特定行為的發(fā)展有重要作用。但選擇哪一項指標作為衡定信號通路適中的標準, 尚有待今后的深入研究。除此之外, 當孤獨癥高風險基因發(fā)生突變時,的表達也會受到影響(Forrest, Waite, Martin-Rendon, & Blake, 2013; Chan et al., 2015)。另外在對孤獨癥患者家系全基因組檢測中, 發(fā)現(xiàn)了一個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及其所調控信號通路對孤獨癥發(fā)生發(fā)展有重要影響。但是目前有關基因突變在孤獨癥發(fā)生發(fā)展中的作用以及在腦發(fā)育過程中的機制還有待進一步探討。

在接下來的研究中, 可以在建立小鼠動物模型的基礎上, 通過檢測基因分子水平變化、細胞組織器官發(fā)育分化、形態(tài)差異以及分析行為特征來研究基因在神經系統(tǒng)發(fā)育中的作用, 及其對孤獨癥發(fā)生的影響。另外, 關注CHRM3所介導的Gq-PLC信號通路在孤獨癥發(fā)生中的作用, 可為孤獨癥的基因靶向干預提供新的思路和方法, 為教育方案的制定提供科學的幫助和指導。

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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

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

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

B845

10.3724/SP.J.1042.2018.02141