張佳惠 劉恒 王玉田 姜宏
[摘要] 目的 研究帕金森病轉(zhuǎn)基因模型小鼠中腦黑質(zhì)(SN)區(qū)磷酸化GluN2B(p-GluN2B)亞基的蛋白含量變化。
方法 實驗所用動物為12~15月齡α-突觸核蛋白(α-syn)A53T轉(zhuǎn)基因小鼠和同窩野生型(WT)小鼠。利用開放曠場實驗檢測小鼠的運動行為能力,采用蛋白免疫印跡法檢測小鼠SN區(qū)p-GluN2B及磷酸化α-syn(pS129 α-syn)蛋白表達。
結(jié)果 與WT小鼠相比,α-syn A53T轉(zhuǎn)基因小鼠曠場總運動距離減少(t=2.920,P<0.05),平均運動速度減慢(t=2.518,P<0.05),中腦SN區(qū)p-GluN2B和pS129 α-syn蛋白表達量明顯升高(t=2.470、3.533,P<0.05)。
結(jié)論 α-syn A53T 轉(zhuǎn)基因小鼠SN區(qū)GluN2B亞基活性增強且可能參與多巴胺能神經(jīng)元的變性死亡過程。
[關(guān)鍵詞] 帕金森??;小鼠,轉(zhuǎn)基因;黑質(zhì);α突觸核蛋白;GluN2B
[中圖分類號] R338.2
[文獻標志碼] A
[文章編號] 2096-5532(2022)05-0643-03
doi:10.11712/jms.2096-5532.2022.58.166
[開放科學(xué)(資源服務(wù))標識碼(OSID)]
[網(wǎng)絡(luò)出版] https://kns.cnki.net/kcms/detail/37.1517.R.20221021.1358.001.html;2022-10-24 09:36:11
CHANGE IN THE EXPRESSION OF PHOSPHORYLATED GLUN2B SUBUNIT IN THE SUBSTANTIA NIGRA OF α-SYNUCLEIN A53T TRANSGENIC MICE
ZHANG Jiahui, LIU Heng, WANG Yutian, JIANG Hong
(State Key Disciplines: Physiology (in Incubation), Department of Physiology, Qingdao University, Qingdao 266071, China);
[ABSTRACT] Objective To investigate the change in the expression of phosphorylated GluN2B (p-GluN2B) subunit in the substantia nigra (SN) of transgenic mice with Parkinsons disease (PD).
Methods The α-synuclein (α-syn) transgenic mice and wild-type (WT) littermates, aged 12-15 months, were used in this experiment. The open field test was used to observe motor behavior abilities, and Western blotting was used to measure the protein expression of p-GluN2B and phosphorylated α-syn (pS129 α-syn) in the SN of mice.
Results The open field test showed that compared with the WT mice, the α-syn A53T transgenic mice had significant reductions in total movement distance (t=2.920,P<0.05) and mean movement speed (t=2.518,P<0.05), and Western blotting showed that compared with the WT mice, the α-syn A53T transgenic mice had significant increases in the protein expression levels of p-GluN2B and pS129 α-syn in the SN of the midbrain (t=2.470,3.533;P<0.05).
Conclusion The activity of GluN2B subunit in the SN of α-syn A53T transgenic mice is enhanced, which may be involved in the degeneration and death of dopaminergic neurons.
[KEY WORDS] Parkinson disease; mice, transgenic; substantia nigra; alpha-synuclein; GluN2B
在帕金森?。≒D)中,α-突觸核蛋白(α-syn)在Ser-129 位點的磷酸化促進了α-syn的積累及路易小體的形成,對PD的發(fā)生發(fā)展起到重要作用[1-4]。N-甲基-D-天冬氨酸(NMDA)受體為離子型谷氨酸受體,具有高鈣離子通透性,在學(xué)習(xí)與記憶中發(fā)揮重要作用[5-6]。該受體過度激活會導(dǎo)致細胞功能障礙和死亡,被認為是與許多神經(jīng)系統(tǒng)疾病相關(guān)的神經(jīng)元損傷的介質(zhì)[7-8]。NMDA受體GluN2B亞基的C末端可以被酪氨酸激酶Fyn激活而發(fā)生磷酸化聚集在突觸后膜上,而細胞內(nèi)Fyn的表達可能受到α-syn的調(diào)控[9-10]。然而,NMDA 受體參與PD的發(fā)病機制仍然不明確。本實驗旨在通過檢測α-syn A53T 轉(zhuǎn)基因小鼠和野生型(WT)小鼠黑質(zhì)區(qū)(SN)磷酸化GluN2B(p-GluN2B)蛋白表達水平,探究隨PD進展GluN2B亞基活性變化,以期為PD提供潛在的治療靶點。
1 材料與方法
1.1 實驗材料
1.1.1 實驗動物 α-syn A53T轉(zhuǎn)基因小鼠購自南京大學(xué)模式動物研究所,進行基因型鑒別,獲得α-syn A53T+/+小鼠和α-syn A53T+/-小鼠。本實驗選擇12~15月齡的α-syn A53T+/+小鼠和同窩WT小鼠各5只作為研究對象。小鼠飼養(yǎng)于SPF級環(huán)境中,設(shè)定室溫為(22±2)℃、白晝與黑夜的光照時間為1∶1。
1.1.2 儀器及試劑 p-GluN2B抗體(ABcam);磷酸化α-syn(pS129 α-syn)抗體、GAPDH抗體(Cell Signaling Technology);山羊抗兔二抗(Absin);BCA試劑盒、SDS-PAGE loading buffer、RIPA裂解液、分離膠緩沖液和濃縮膠緩沖液(康為世紀);ECL顯色發(fā)光液、PVDF膜(Millipore);電泳儀、電轉(zhuǎn)儀(BioRad);凝膠成像系統(tǒng)(General Electric Company)。
1.2 實驗方法
1.2.1 曠場實驗 應(yīng)用50 cm×50 cm的方盒曠場,方盒周壁及底部的顏色均不透明,將方盒底部平均分為4×4的16個小方格。攝像頭置于方盒上方,可觀察整個曠場,將小鼠輕輕放置在方盒中,黑暗中進行10 min的視頻錄制。每只小鼠測試結(jié)束后,清理方盒并應(yīng)用乙醇消除氣味后晾干,以防對下一只小鼠測試產(chǎn)生影響。采用SMART軟件分析小鼠總運動距離及平均運動速度等數(shù)據(jù)。
1.2.2 蛋白免疫印跡法檢測SN區(qū)蛋白表達 小鼠麻醉后斷頭取腦,在冰上按照腦圖譜完整取出中腦SN區(qū)。向腦組織中加入100 μL RIPA蛋白裂解液靜置,置于研磨機中研磨。在4 ℃下以12 000 r/min離心20 min,離心結(jié)束后取上清液80 μL,在酶標儀上用BCA 法測定蛋白濃度。加入5×蛋白loading buffer混勻,100 ℃金屬浴煮沸,根據(jù)BCA法檢測數(shù)據(jù)確定上樣量。按照所需蛋白分子量配膠,電泳完成以后轉(zhuǎn)膜(300 mA、100 min)。按照Marker切下需要的目標條帶,用100 g/L脫脂奶粉封閉,室溫孵育2 h;分別加入用一抗稀釋液配制的p-GluN2B抗體(1∶1 000)、pS129 α-syn 抗體(1∶1 000)、GAPDH抗體(1∶10 000),置低速搖床上4 ℃孵育過夜;以TBST緩沖液洗脫3次,半小時后加入山羊抗兔的二抗(1∶10 000,TBST緩沖液配制),置搖床上室溫孵育1 h;以TBST緩沖液洗脫3次,擦干TBST后,加入適量ECL化學(xué)發(fā)光液進行顯影。采用Image J軟件分析條帶灰度值,結(jié)果以p-GluN2B、pS129 α-syn與內(nèi)參GAPDH 灰度值的比值表示。
1.3 統(tǒng)計學(xué)分析
應(yīng)用GraphPad Prism 8軟件進行統(tǒng)計學(xué)分析。計量資料結(jié)果以±s表示,兩組比較采用t檢驗,以P<0.05為差異有統(tǒng)計學(xué)意義。
2 結(jié) 果
2.1 α-syn A53T+/+小鼠運動行為能力改變
α-syn A53T+/+小鼠和WT小鼠在開放曠場的總運動距離分別為(2 575.0±293.7)、(3 655.0±225.1)cm(n=5),α-syn A53T+/+小鼠曠場總運動距離較WT小鼠減少,差異具有統(tǒng)計學(xué)意義(t=2.920,P<0.05)。α-syn A53T+/+小鼠和WT小鼠的平均運動速度分別為(4.302±0.480)、(5.898±0.415)cm/s(n=5),α-syn A53T+/+小鼠平均運動速度較WT小鼠減慢,差異具有統(tǒng)計學(xué)意義(t=2.518,P<0.05)。
2.2 α-syn A53T+/+小鼠中腦SN區(qū)p-GluN2B 和pS129 α-syn蛋白表達變化
α-syn A53T+/+小鼠和WT 小鼠中腦SN區(qū)p-GluN2B蛋白的表達水平分別為1.115±0.141和0.706±0.087(n=5),α-syn A53T+/+小鼠SN區(qū)p-GluN2B蛋白的表達水平較WT小鼠明顯升高,差異具有統(tǒng)計學(xué)意義(t=2.470,P<0.05)。α-syn A53T+/+小鼠和WT 小鼠SN區(qū)pS129 α-syn 蛋白的表達水平分別為1.277±0.219和0.433±0.094(n=5),差異具有統(tǒng)計學(xué)意義(t=3.533,P<0.05)。
3 討 論
NMDA受體為谷氨酸門控離子通道,在中樞神經(jīng)系統(tǒng)中廣泛表達,并在興奮性突觸傳遞中起著關(guān)鍵作用[11]。NMDA受體共包含3種類型的亞基(GluN1~3),亞基結(jié)構(gòu)間具有高度同源性,均由氨基端、配體結(jié)合區(qū)、跨膜區(qū)及羧基末端4個結(jié)構(gòu)域組成[12-13]。越來越多的研究表明,在PD細胞模型和PD病人腦組織中,紋狀體和伏隔核中谷氨酸與NMDA受體結(jié)合顯著增加,而這可能會加速神經(jīng)元退化過程[14-15]。有文獻報道,在亞急性1-甲基-4-苯基-1,2,3,6-四氫吡啶(MPTP)小鼠模型中,可以通過抑制mGlu2/3受體配體影響突觸后Fyn/NMDA受體的功能[16]。最新的研究結(jié)果表明,在魚藤酮小鼠模型中,GluN2B在mGluR5介導(dǎo)的內(nèi)質(zhì)網(wǎng)應(yīng)激和DNA損傷中發(fā)揮著重要作用[17]。
上述研究揭示,在PD的進展過程中,NMDA受體介導(dǎo)的谷氨酸興奮性毒性發(fā)揮作用。本文研究結(jié)果顯示,12~15月齡α-syn A53T+/+小鼠運動能力受損,與之前有關(guān)研究報道相吻合[18]。本文結(jié)果還顯示,α-syn A53T+/+小鼠SN區(qū)pS129 α-syn和p-GluN2B蛋白表達水平明顯升高,說明在SN區(qū)出現(xiàn)了α-syn聚集,從而可能過度激活Fyn使GluN2B亞基表達過磷酸化。有研究報道,F(xiàn)yn在Tyr1472處磷酸化GluN2B會抑制與網(wǎng)格蛋白接頭AP-2的結(jié)合,最終會抑制細胞表面含有GluN2B的NMDA受體的內(nèi)化[19-20]。因此我們推測,當p-GluN2B增多時,由于Fyn活性增強使GluN2B內(nèi)化減少,導(dǎo)致大量Ca2+內(nèi)流,這可能成為神經(jīng)元損傷的重要原因。本研究為闡明NMDA受體參與PD發(fā)生發(fā)展的機制提供了思路,其具體機制還有待進一步探討。
[參考文獻]
[1]PRZEDBORSKI S. The two-century journey of Parkinson di-
sease research[J]. Nature Reviews Neuroscience, 2017,18(4):251-259.
[2]JANKOVIC J, TAN E K. Parkinsons disease: etiopathoge-
nesis and treatment[J]. Journal of Neurology, Neurosurgery, and Psychiatry, 2020,91(8):795-808.
[3]HENDERSON M X, TROJANOWSKI J Q, LEE V M Y. α-Synuclein pathology in Parkinsons disease and related α-synucleinopathies[J]. Neuroscience Letters, 2019,709:134316.
[4]ROCHA E M, DE MIRANDA B, SANDERS L H. Alpha-synuclein: pathology, mitochondrial dysfunction and neuroinflammation in Parkinsons disease[J]. Neurobiology of Di-
sease, 2018,109:249-257.
[5]ZHANG Z, ZHANG S Q, FU P F, et al. Roles of glutamate receptors in Parkinsons disease[J]. International Journal of Molecular Sciences, 2019,20(18):4391.
[6]IOVINO L, TREMBLAY M E, CIVIERO L. Glutamate-induced excitotoxicity in Parkinsons disease: the role of glial cells[J]. Journal of Pharmacological Sciences, 2020,144(3):151-164.
[7]HARDINGHAM G. NMDA receptor C-terminal signaling in development, plasticity, and disease[J]. F1000Research, 2019,8:F1000 Faculty Rev-1547.
[8]BINVIGNAT O, OLLOQUEQUI J. Excitotoxicity as a target against neurodegenerative processes[J]. Current Pharmaceutical Design, 2020,26(12):1251-1262.
[9]ROY B, JACKSON G R. Interactions between Tau and α-synuclein augment neurotoxicity in a Drosophila model of Parkinsons disease[J]. Human Molecular Genetics, 2014,23(11):3008-3023.
[10]YAMASAKI T, FUJINAGA M, KAWAMURA K, et al. Dynamic changes in striatal mGluR1 but not mGluR5 during pathological progression of Parkinsons disease in human alpha-synuclein A53T transgenic rats: a multi-PET imaging study[J]. The Journal of Neuroscience: the Official Journal of the Society for Neuroscience, 2016,36(2):375-384.
[11]PAOLETTI P, NEYTON J. NMDA receptor subunits: function and pharmacology[J]. Current Opinion in Pharmacology, 2007,7(1):39-47.
[12]HANSEN K B, YI F, PERSZYK R E, et al. Structure, function, and allosteric modulation of NMDA receptors[J]. The Journal of General Physiology, 2018,150(8):1081-1105.
[13]SHERWANI Z A, KHALIL R, NUR-E-ALAM M, et al. Structure-based virtual screening to identify negative allosteric modulators of NMDA[J]. Medicinal Chemistry (Shariqah (United Arab Emirates)), 2022,18(9):990-1000.
[14]GUO H Q, CAMARGO L M, YEBOAH F, et al. A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine[J]. Scientific Reports, 2017,7(1):11608.
[15]WANG J, WANG F S, MAI D M, et al. Molecular mechanisms of glutamate toxicity in Parkinsons disease[J]. Frontiers in Neuroscience, 2020,14:585584.
[16]TAN Y, XU Y, CHENG C, et al. LY354740 reduces extracellular glutamate concentration, inhibits phosphorylation of Fyn/NMDARs, and expression of PLK2/pS129 α-synuclein in mice treated with acute or sub-acute MPTP[J]. Frontiers in Pharmacology, 2020,11:183.
[17]GU L, LUO W Y, XIA N, et al. Upregulated mGluR5 induces ER stress and DNA damage by regulating the NMDA receptor subunit NR2B[J]. Journal of Biochemistry, 2022,171(3):349-359.
[18]LU J, DOU F F, YU Z H. The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinsons disease[J]. Journal of Neuroinflammation, 2019,16(1):273.
[19]PRYBYLOWSKI K, CHANG K, SANS N, et al. The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2[J]. Neuron, 2005,47(6):845-857.
[20]BLAND T, ZHU M Y, DILLON C, et al. Leptin controls glutamatergic synaptogenesis and NMDA-receptor trafficking via Fyn kinase regulation of NR2B[J]. Endocrinology, 2020,161(2):bqz030.
(本文編輯 馬偉平)
青島大學(xué)學(xué)報(醫(yī)學(xué)版)2022年5期