【摘要】：包含SH2結(jié)構(gòu)域的酪氨酸磷酸酶2(Src homology 2 domain containing phosphotyrosine phosphatase 2,Shp2)是非受體酪氨酸磷酸酶的一種,在進(jìn)化中高度保守。在患病人群中大約50%的Noonan綜合征(Noonan syndrom,NS)是由Shp2的激活突變引起的,而大約80%的LEOPARD綜合征(LEOPARD syndrom,LS)則是由它的失活突變引起。這兩種疾病都是全身性的發(fā)育障礙病,并且均有一定比例的病人表現(xiàn)出智力障礙。雖然已有報(bào)道稱Shp2能夠通過(guò)調(diào)控下游Ras/ERKMAP激酶(MAPK)來(lái)參與記憶過(guò)程,但是內(nèi)在機(jī)制依然不是很清楚。有報(bào)道稱Shp2存在于N-甲基-D-門(mén)冬氨酸(N-methyl-D-aspartate,NMDA)受體復(fù)合物中,并且有它和NMDA受體的GluN2B亞基直接相互作用的證據(jù)。NMDA受體是一類興奮性離子型谷氨酸受體,主要通透Ca2+。能夠發(fā)揮功能的NMDA受體由2個(gè)組構(gòu)性的GluN1亞基和兩個(gè)調(diào)節(jié)性的GluN2或者GluN3亞基構(gòu)成。它是配體和電壓雙門(mén)控的通道,能夠響應(yīng)突觸前的谷氨酸釋放和突觸后的去極化,在突觸可塑性中發(fā)揮了重要的作用�；诖�,我們采用Shp2 FloxP/FloxP小鼠與CaMKⅡα-Cre小鼠雜交得到在前腦興奮性神經(jīng)元中特異敲除Shp2的小鼠(Shp2 Floxp/Floxp:CaMKⅡα-Cre +/-,CaSKO)來(lái)研究 Shp2 是否通過(guò)調(diào)節(jié) NMDA 受體亞基磷酸化來(lái)調(diào)控突觸可塑性乃至學(xué)習(xí)記憶功能。我們進(jìn)行的蛋白檢測(cè)表明CaSKO小鼠海馬組織中Src激酶416位酪氨酸(Src Y416)磷酸化水平升高,這一位點(diǎn)代表了其酶活性,相應(yīng)的NMDA受體的GluN2B亞基的1472位酪氨酸(GluN2B Y1472)和GluN2A亞基的1325位酪氨酸(GluN2A Y1325)的磷酸化水平也特異性的升高。這使得突觸部位NMDA受體的電流增加并且CaSKO小鼠海馬CA1的錐體神經(jīng)元上興奮性突觸傳遞也增加。我們進(jìn)一步的分析表明,這些在CaSKO小鼠突觸上增加的NMDA受體是三異聚體GluN1/GluN2A/GluN2B,并且這些受體對(duì)于鋅離子抑制的敏感性增強(qiáng),導(dǎo)致在高頻刺激時(shí)通過(guò)該受體的電量減少。在場(chǎng)電位長(zhǎng)時(shí)程突觸增強(qiáng)(long-term potentiation,LTP)的測(cè)試中,我們發(fā)現(xiàn)敲除Shp2會(huì)削弱海馬CA1由100 Hz/s-高頻刺激誘導(dǎo)的LTP,而這種LTP的削弱正是與突觸部位的NMDA受體對(duì)于鋅離子抑制的敏感性增加相關(guān)。此外,我們進(jìn)一步發(fā)現(xiàn)CaSKO小鼠的長(zhǎng)時(shí)程場(chǎng)景恐懼記憶被削弱,而場(chǎng)景恐懼記憶的形成和短時(shí)記憶則不受影響。對(duì)于場(chǎng)景恐懼記憶前后小鼠海馬組織中這些蛋白的磷酸化檢測(cè)發(fā)現(xiàn)WT小鼠在場(chǎng)景恐懼記憶檢測(cè)后SrcY416的磷酸化升高,并且GluN2B Y1472的磷酸化也相應(yīng)上升,而在CaSKO小鼠中這些位點(diǎn)已經(jīng)升高的磷酸化水平并未出現(xiàn)活性依賴的升高。通過(guò)對(duì)CaSKO小鼠進(jìn)行Src激酶抑制劑PP2的處理,我們發(fā)現(xiàn)能夠反轉(zhuǎn)Shp2缺失所引起的一系列后果。據(jù)此,我們推斷是Shp2敲除引起的Src激酶活性增高導(dǎo)致了 NMDA受體亞基磷酸化水平的升高,從而使得更多的NMDA受體在突觸部位表達(dá),且功能性突觸數(shù)目和其強(qiáng)度也增多。這種飽和作用導(dǎo)致了 LTP的下降,并進(jìn)一步影響長(zhǎng)時(shí)程場(chǎng)景恐懼記憶。這為我們理解酪氨酸磷酸酶在突觸可塑性調(diào)控中的作用以及NS和LS中智力障礙的表型提供了新的思路。
[Abstract]:The tyrosine phosphatase 2 (SH2), which contains the SH2 domain, is one of the non-receptor tyrosine phosphatases and is highly conserved in the evolution. Noonan syndrome (NS), which is about 50% of the affected population, is caused by the activation mutation of Shp2, while about 80% of the LEOPARD syndrom (LS) is caused by its inactivation mutation. Both of these diseases are systemic developmental disorders and there are a proportion of patients exhibiting mental disorders. Although it has been reported that Shp2 is able to participate in the memory process by regulating downstream Ras/ ERKMAP kinase (MAPK), the intrinsic mechanism remains unclear. It is reported that Shp2 is present in the N-methyl-D-aspartate (NMDA) receptor complex and has evidence of its direct interaction with the GluN2B subunit of the NMDA receptor. The NMDA receptor is an excitable ionic glutamate receptor, which is mainly permeable to Ca2 +. The NMDA receptor capable of functioning is composed of 2 fabric GluN1 subunits and two regulatory GluN2 or GluN3 subunits. It is a double-gated channel of ligand and voltage, which can respond to the glutamate release and postsynaptic depolarization before the synapse, and play an important role in the synaptic plasticity. Based on this, we used the SH2 FloxP/ FloP mice to cross with CaMK 鈪，

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