宋彬彬，王清，宋秋萍，段立疆，武香梅，楊璇，于佳

運(yùn)動(dòng)神經(jīng)元病（motor neuron disease，MND）是一種由上、下運(yùn)動(dòng)神經(jīng)元損傷變性而導(dǎo)致的進(jìn)行性神經(jīng)變性疾病，其選擇性侵犯脊髓前角細(xì)胞、腦干運(yùn)動(dòng)神經(jīng)元及錐體束，使運(yùn)動(dòng)系統(tǒng)受累。脊髓中的運(yùn)動(dòng)調(diào)控神經(jīng)環(huán)路由運(yùn)動(dòng)神經(jīng)元和中間神經(jīng)元組成，通過(guò)精細(xì)復(fù)雜的突觸連接支配骨骼肌收縮和隨意運(yùn)動(dòng)。1969年Conradi從形態(tài)上定義了一種位于脊髓前角的可支配運(yùn)動(dòng)神經(jīng)元的特殊膽堿能C型突觸終扣[1]，也稱C-bouton。C-bouton結(jié)構(gòu)復(fù)雜，突觸前后分布有多種蛋白受體、離子通道及特殊結(jié)構(gòu)，可精確調(diào)控α-運(yùn)動(dòng)神經(jīng)元的興奮性，影響肌肉活動(dòng)而與運(yùn)動(dòng)神經(jīng)元病緊密相關(guān)。本文就C-bouton的結(jié)構(gòu)（圖1）、功能及其與運(yùn)動(dòng)神經(jīng)元病的關(guān)系進(jìn)行綜述。

1 C-bouton結(jié)構(gòu)和組成

研究者根據(jù)解剖結(jié)構(gòu)將支配運(yùn)動(dòng)神經(jīng)元的突觸分為5種類型，分別為C、F、M、S和T型[2]。其中C型突觸終扣即C-bouton，主要分布在α-運(yùn)動(dòng)神經(jīng)元（尤其是支配快肌纖維的運(yùn)動(dòng)神經(jīng)元）的細(xì)胞體和樹突近端，可被乙酰膽堿轉(zhuǎn)移酶（choline acetyl transferase，ChAT）、乙酰膽堿酯酶（acetyl cholinesterase，AChE）和囊泡乙酰膽堿轉(zhuǎn)運(yùn)體（vesicular acetylcholine transporter，VAChT）抗體特異性標(biāo)記[3]，屬于膽堿能突觸。研究發(fā)現(xiàn)C-bouton來(lái)源于脊髓中央管附近的特異性表達(dá)轉(zhuǎn)錄因子Pitx2的V0（Dbx1+）中間神經(jīng)元，單個(gè)V0中間神經(jīng)元可產(chǎn)生大量C-bouton，支配一個(gè)或多個(gè)α-運(yùn)動(dòng)神經(jīng)元——這種多對(duì)一或多對(duì)多的突觸連接模式使突觸前軸突對(duì)于α-運(yùn)動(dòng)神經(jīng)元有更多的遞質(zhì)釋放位點(diǎn)，保證膽堿能神經(jīng)調(diào)節(jié)的安全性和可靠性[4]。C-bouton體積較大，人和嚙齒動(dòng)物中C-bouton的直徑分別為3～6 μm和1～8 μm。突觸前區(qū)域具有密集明亮的球形突觸囊泡和大量線粒體；突觸后高度特化，突觸后膜下方5～8 nm處具有標(biāo)志性的內(nèi)質(zhì)網(wǎng)衍生結(jié)構(gòu)（subsynaptic cisterns，SSC）。其是與整個(gè)突觸前結(jié)構(gòu)相對(duì)的10～15 nm厚的盤狀結(jié)構(gòu)，呈“C”型，故稱為C-bouton。SSC下方存在粗面內(nèi)質(zhì)網(wǎng)，游離的核糖體也分布于突觸后區(qū)域[2]。

C-bouton的突觸前后區(qū)域具有不同的標(biāo)志性蛋白[3]（圖1）：①ATP門控離子通道P2X7受體和煙堿乙酰膽堿受體（nicotinic a cetylcholine receptor，nAChR）分布于突觸前膜；②蕈堿樣乙酰膽堿受體2（muscarinic acetylcholine receptor 2，m2 receptor）、小電導(dǎo)鈣激活鉀離子通道（small-conductance calcium-activated potassium channel，SK channel）、電壓門控鉀離子通道Kv2.1和電壓門控鈣離子通道位于突觸后膜，SK通道蛋白和m2受體分別聚集成簇，并與突觸前囊泡釋放位點(diǎn)緊密對(duì)應(yīng)，Kv2.1填充在突觸后膜未被m2受體和SK通道蛋白占據(jù)的位置；③SSC上具有大量受體型分子伴侶Sigma-1受體蛋白（Sig-1R）和神經(jīng)調(diào)節(jié)素1（neuregulin 1，NRG1）等。

2 C-bouton功能和調(diào)控

C-bouton的主要功能是調(diào)節(jié)運(yùn)動(dòng)神經(jīng)元的電活動(dòng)，從而影響肌肉收縮力的大小和持續(xù)時(shí)間。研究發(fā)現(xiàn)，使用m2受體拮抗劑抑制小鼠脊髓切片中C-bouton的乙酰膽堿能神經(jīng)傳導(dǎo)，會(huì)降低運(yùn)動(dòng)神經(jīng)元輸出；使用膽堿酯酶抑制劑增強(qiáng)C-bouton的乙酰膽堿能神經(jīng)傳導(dǎo)，會(huì)升高運(yùn)動(dòng)神經(jīng)元的輸出[5]。另外，C-bouton以高度任務(wù)依賴方式調(diào)節(jié)運(yùn)動(dòng)神經(jīng)元放電來(lái)適應(yīng)不同運(yùn)動(dòng)強(qiáng)度的需要，如低強(qiáng)度運(yùn)動(dòng)時(shí)（走路）運(yùn)動(dòng)神經(jīng)元低輸出、高強(qiáng)度運(yùn)動(dòng)（游泳）時(shí)運(yùn)動(dòng)神經(jīng)元高輸出。Zagoraiou等[6]發(fā)現(xiàn)，敲除乙酰膽堿合成酶抑制V0中間神經(jīng)元的C-bouton輸出，導(dǎo)致實(shí)驗(yàn)組小鼠游泳時(shí)相對(duì)于走路時(shí)的肌電活動(dòng)增加程度顯著低于對(duì)照組小鼠。C-bouton突觸前后的多種蛋白參與調(diào)控α-運(yùn)動(dòng)神經(jīng)元產(chǎn)生的后超極化電位（after hyperpolarizing potential，AHP）和放電率，并且形成內(nèi)部安全機(jī)制以避免運(yùn)動(dòng)神經(jīng)元興奮性毒性損傷的發(fā)生。

2.1 突觸前的nAChR和P2X7受體

nAChR和P2X7受體分別與釋放到突觸間隙的乙酰膽堿（acetylcholine，ACh）和三磷酸腺苷（ade-nosine triphosphate，ATP）結(jié)合后介導(dǎo)大量鈣離子內(nèi)流，引起囊泡和突觸前膜活性區(qū)域的融合和胞吐，從而進(jìn)一步增強(qiáng)C-bouton的ACh釋放[7]。

2.2 突觸后膜的m2受體、SK通道和Kv通道

m2受體是G蛋白偶聯(lián)受體，特異性與ACh結(jié)合發(fā)生激活后通過(guò)調(diào)節(jié)SK和Kv2.1等離子通道蛋白的活性，降低動(dòng)作電位的后超極化而增加運(yùn)動(dòng)神經(jīng)元的興奮性[5]。

SK通道對(duì)鈣離子高度敏感，可快速將細(xì)胞內(nèi)鈣離子濃度的改變轉(zhuǎn)化為細(xì)胞膜電位的變化，SK通道被激活后，對(duì)鉀離子通透性增加，使鉀離子外流，可在產(chǎn)生動(dòng)作電位之后引起AHP，調(diào)節(jié)運(yùn)動(dòng)神經(jīng)元的放電率。C-bouton中SK通道蛋白主要有SK2和SK3兩種亞型，所有嚙齒動(dòng)物的α-運(yùn)動(dòng)神經(jīng)元都表達(dá)SK2，而SK3僅表達(dá)在體積較小的α-運(yùn)動(dòng)神經(jīng)元中，這類α-運(yùn)動(dòng)神經(jīng)元產(chǎn)生的AHP半衰期較長(zhǎng)，幅度較大，軸突傳導(dǎo)速度明顯減慢，主要支配慢肌纖維。SK3陰性、SK2陽(yáng)性的α-運(yùn)動(dòng)神經(jīng)元往往體積較大，且主要支配快肌纖維[8]。m2受體激活，抑制N型鈣離子通道介導(dǎo)的鈣內(nèi)流，下調(diào)SK通道的活性，從而降低AHP，增強(qiáng)神經(jīng)元興奮性[2]。另外，m2受體激活可能直接引起蛋白激酶C（protein kinase C，PKC）和CK2（casein kinase 2）對(duì)SK的磷酸化，減弱其對(duì)鈣離子的敏感性而抑制其活性，調(diào)節(jié)運(yùn)動(dòng)神經(jīng)元的興奮性[2,9,10]。

Kv2.1通道介導(dǎo)的鉀離子流具有延遲整流特性，被激活后會(huì)降低神經(jīng)元興奮性。Kv2.1通道可在C-bouton突觸后膜上聚集成簇，高密度的Kv2.1通道蛋白是低或者非導(dǎo)電的。Kv2.1通道蛋白N端和C端存在多個(gè)磷酸化位點(diǎn)，磷酸化水平調(diào)控Kv2.1的激活和聚集狀態(tài)[11]。不同強(qiáng)度運(yùn)動(dòng)過(guò)程中，Kv2.1磷酸化水平和聚集程度發(fā)生變化，從而影響運(yùn)動(dòng)神經(jīng)元的興奮性和放電率。m2受體活化可抑制高電壓激活鈣離子電流而阻止鈣離子/鈣調(diào)磷酸酶（calcineurin）依賴的去磷酸化通路，上調(diào)Kv2.1磷酸化水平和聚集程度，降低其介導(dǎo)的電流幅度從而增加運(yùn)動(dòng)神經(jīng)元興奮性[2,12]。

2.3 SSC上的Sig-1R和NRG1

Sig-1R在神經(jīng)系統(tǒng)中高表達(dá)，其N端的內(nèi)質(zhì)網(wǎng)停泊信號(hào)介導(dǎo)其在內(nèi)質(zhì)網(wǎng)和SSC上的定位，調(diào)節(jié)鈣離子通道、鉀等離子通道、肌醇三磷酸（IP3）受體和NMDA受體等，參與調(diào)控內(nèi)質(zhì)網(wǎng)、線粒體功能及神經(jīng)遞質(zhì)釋放等。SSC衍生于內(nèi)質(zhì)網(wǎng)，作為運(yùn)動(dòng)神經(jīng)元內(nèi)的鈣離子儲(chǔ)備池，在鈣離子的穩(wěn)態(tài)和動(dòng)員的調(diào)控中發(fā)揮重要作用：SSC可從空間和功能上制約游離鈣離子的擴(kuò)散；神經(jīng)元活性增高時(shí)，SSC從細(xì)胞質(zhì)中吸收和清除過(guò)多的游離鈣離子；SSC上的Sig-1R和鈣釋放通道Ryanodine受體（RyR受體）調(diào)控鈣離子向細(xì)胞質(zhì)的釋放，上調(diào)細(xì)胞質(zhì)局部游離鈣離子濃度[2,13]。Sig-1R不僅對(duì)鈣離子的可用性有潛在調(diào)節(jié)作用，還可調(diào)節(jié)m2受體對(duì)Ach的敏感性及Kv通道在細(xì)胞內(nèi)和細(xì)胞膜上的分布，進(jìn)而影響運(yùn)動(dòng)神經(jīng)元的興奮性[14,15]。

NRG1是神經(jīng)營(yíng)養(yǎng)因子，調(diào)節(jié)細(xì)胞間信號(hào)傳導(dǎo)、軸突運(yùn)輸、突觸功能、神經(jīng)遞質(zhì)受體表達(dá)、神經(jīng)膠質(zhì)細(xì)胞的增殖和活化等，對(duì)于神經(jīng)系統(tǒng)的發(fā)育和成熟以及神經(jīng)炎癥具有重要作用[16]。NRG1位于突觸后區(qū)域，而C-bouton突觸前膜上存在NRG1受體ErbB2/4，提示NRG1可能是C-bouton中的逆行信號(hào)調(diào)節(jié)分子[17]。

3 C-bouton與運(yùn)動(dòng)神經(jīng)元病

3.1 運(yùn)動(dòng)神經(jīng)元病中C-bouton的病理改變和功能障礙

肌萎縮側(cè)索硬化（amyotrophic lateral sclerosis，ALS）是運(yùn)動(dòng)神經(jīng)元病中最常見的類型，患者皮質(zhì)、腦干和脊髓的運(yùn)動(dòng)神經(jīng)元發(fā)生變性死亡，支配運(yùn)動(dòng)神經(jīng)元的中間神經(jīng)元也會(huì)發(fā)生病理改變。目前，對(duì)于ALS中C-bouton的數(shù)量和體積變化的確切結(jié)論仍存在爭(zhēng)議，可能與研究對(duì)象實(shí)驗(yàn)設(shè)計(jì)、分析方法和疾病病程不一致等有關(guān)[18]。早期研究發(fā)現(xiàn)散發(fā)性ALS患者脊髓運(yùn)動(dòng)神經(jīng)元的膽堿能輸入突觸C-bouton數(shù)量減少[19]。而SOD1G93A ALS小鼠模型中發(fā)現(xiàn)C-bouton數(shù)量增加[20]、不變[21]和減少[22]的現(xiàn)象。最近Milan等[23]發(fā)現(xiàn)C-bouton數(shù)量的改變與病程緊密相關(guān)：疾病早期C-bouton數(shù)量不變，隨后增加，晚期減少，這種變化可能與機(jī)體維持細(xì)胞內(nèi)穩(wěn)態(tài)的自我補(bǔ)償機(jī)制有關(guān)。多數(shù)研究認(rèn)為SOD1G93A ALS小鼠模型中C-bouton體積變大[2,3,23]。Herron等[21]指出C-bouton體積變大出現(xiàn)在SOD1G93AALS小鼠模型的發(fā)育早期（8～30 d），早于運(yùn)動(dòng)癥狀出現(xiàn)（90～100 d），雄性中變化更加明顯。由于ALS中去神經(jīng)支配增加，C-bouton體積變大可能是補(bǔ)償乙酰膽堿能神經(jīng)輸出的一種神經(jīng)保護(hù)機(jī)制[24]，這種改變也帶來(lái)潛在的風(fēng)險(xiǎn)——會(huì)使運(yùn)動(dòng)神經(jīng)元興奮性增強(qiáng)，隨著時(shí)間推移又可能會(huì)產(chǎn)生興奮毒性而導(dǎo)致運(yùn)動(dòng)神經(jīng)元損傷和變性。筆者在前期工作中發(fā)現(xiàn)，過(guò)表達(dá)VAPBP56S的ALS轉(zhuǎn)基因小鼠模型與過(guò)表達(dá)VAPBWT的轉(zhuǎn)基因小鼠及非轉(zhuǎn)基因小鼠模型相比，C-bouton的體積不變但最大直徑變長(zhǎng)，C-bouton的長(zhǎng)效放電顯著減弱[25]。另外，脊髓性肌萎縮（spinal muscular atrophy，SMA）是一類因脊髓前角運(yùn)動(dòng)神經(jīng)元變性導(dǎo)致的肌無(wú)力、肌萎縮的運(yùn)動(dòng)神經(jīng)元病。Ling等[26]發(fā)現(xiàn)SMA（SMNΔ7）小鼠出生后12～14 d脊髓L3～L5節(jié)段中運(yùn)動(dòng)神經(jīng)元對(duì)應(yīng)的C-bouton數(shù)量和密度減少及活化的小膠質(zhì)細(xì)胞增生，活化的小膠質(zhì)細(xì)胞可促進(jìn)突觸剝離（synaptic stripping）使C-bouton損傷，突觸輸出減少。

3.2 運(yùn)動(dòng)神經(jīng)元病中C-bouton標(biāo)志性蛋白的改變

在ALS患者中發(fā)現(xiàn)Sig-1RE102Q突變，突變位點(diǎn)位于蛋白跨膜保守區(qū)域，過(guò)表達(dá)Sig-1RE102Q的運(yùn)動(dòng)神經(jīng)元樣細(xì)胞系NSC34會(huì)上調(diào)內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)細(xì)胞凋亡[27]。ALS患者α-運(yùn)動(dòng)神經(jīng)元中Sig-1R重新分布且表達(dá)水平降低[28]。Sig-1R敲除小鼠表現(xiàn)出運(yùn)動(dòng)功能障礙、肌力下降和軸突變性[29]。且Sig-1R表達(dá)下降使m2受體激活，抑制SK和Kv2.1通道，運(yùn)動(dòng)神經(jīng)元興奮性增強(qiáng)[30]，可能產(chǎn)生興奮毒性。敲減原代運(yùn)動(dòng)神經(jīng)元和NSC34細(xì)胞中的Sig-1R導(dǎo)致細(xì)胞內(nèi)鈣信號(hào)紊亂、內(nèi)質(zhì)網(wǎng)應(yīng)激增強(qiáng)、線粒體活性和軸突運(yùn)輸受損[28,31]。在攜帶VAPB P56S突變的ALS患者的成纖維細(xì)胞中Sig-1R形成積聚，且與VAPB、20S蛋白酶體和泛素共定位；Sig-1R激動(dòng)劑具有一定的神經(jīng)保護(hù)作用，并可顯著降低VAPBP56S的蛋白沉積[28]。

在ALS患者中還發(fā)現(xiàn)了ErbB4的p.R927Q和p.R1275W突變，提出NRG1-ErbB4信號(hào)通路異常參與疾病發(fā)生[32]。Song等[33]發(fā)現(xiàn)SOD1G93AALS小鼠模型在發(fā)病早期即表現(xiàn)出NRG1-I水平增加和小膠質(zhì)細(xì)胞活化，提出NRG1是神經(jīng)損傷后脊髓中小膠質(zhì)細(xì)胞活化的重要信號(hào)。Casanovas等[34]認(rèn)為NRG1是小膠質(zhì)細(xì)胞的趨化因子，神經(jīng)損傷后NRG1-ErbB特異性激活小膠質(zhì)細(xì)胞，引起神經(jīng)損傷早期的炎癥反應(yīng)。而且Lobsiger 等[35]研究發(fā)現(xiàn)，參與免疫應(yīng)答和炎癥的補(bǔ)體免疫系統(tǒng)中關(guān)鍵組分C1q與ALS中C-bouton損傷有關(guān)。在SOD1G37R/C1q-/-小鼠模型中膠質(zhì)細(xì)胞活性未增加，小鼠的疾病起始期、發(fā)生期和生存期沒有明顯差異，C1q介導(dǎo)的補(bǔ)體系統(tǒng)異常不能增加SOD1G37R小鼠神經(jīng)毒性。但在疾病末期SOD1G37R/C1q-/-比SOD1G37R小鼠C-bouton損傷更明顯，這提示膠質(zhì)細(xì)胞的活化引起的神經(jīng)炎癥對(duì)于ALS發(fā)生發(fā)展有重要調(diào)控作用[36]，但C-bouton損傷程度與ALS進(jìn)程關(guān)系復(fù)雜，需要深入研究。

4 總結(jié)和展望

來(lái)源于脊髓V0中間神經(jīng)元的軸突投射到α-運(yùn)動(dòng)神經(jīng)元的細(xì)胞體和樹突近端形成乙酰膽堿能C-bouton，控制運(yùn)動(dòng)神經(jīng)元的興奮性。C-bouton的結(jié)構(gòu)及功能受到nAChR、P2X7受體、m2受體、SK和Kv2.1離子通道、Sig-1R和NRG1等多種突觸前后蛋白的調(diào)控，并在運(yùn)動(dòng)神經(jīng)元病中發(fā)生顯著的病理改變和功能異常，導(dǎo)致運(yùn)動(dòng)神經(jīng)元電活動(dòng)受損，影響肌肉收縮特性。C-bouton中Sig-1R和NRG1等蛋白是參與ALS發(fā)生發(fā)展的重要因子。隨著形態(tài)學(xué)、電生理和生化學(xué)等技術(shù)的發(fā)展，C-bouton運(yùn)動(dòng)依賴性調(diào)節(jié)運(yùn)動(dòng)神經(jīng)元興奮性的具體機(jī)制、C-bouton中標(biāo)志性結(jié)構(gòu)SSC的具體功能以及C-bouton在運(yùn)動(dòng)神經(jīng)元病發(fā)病過(guò)程中的具體作用將得到更加深入的了解。

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