CN115109138B - 一种sTREM2多肽类似物及其在治疗阿尔兹海默病中的应用 - Google Patents
一种sTREM2多肽类似物及其在治疗阿尔兹海默病中的应用 Download PDFInfo
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Abstract
本发明公开了一种sTREM2多肽类似物及其在治疗阿尔兹海默病中的应用，属于生物技术领域。本发明的sTREM2多肽类似物的氨基酸序列如SEQ ID NO.1所示，其能明显抑制tau的过度磷酸化，改善突触功能障碍和认知功能障碍，可用于制备治疗阿尔兹海默病及tau相关疾病的药物。
Description
技术领域
本发明属于生物技术领域，涉及一种sTREM2多肽类似物及其在治疗阿尔兹海默病中的应用。
背景技术
阿尔兹海默病(Alzheimer disease,AD)是临床上最常见的神经变性疾病，也是导致老年人痴呆最常见的原因。60岁以上老年人患病率高达5％，给社会和家庭带来沉重负担[1,2]。但迄今为止，AD尚无缺乏针对发病机制、延缓病程的治疗措施。
遗传和环境因素均在AD的发生中发挥重要作用。全基因组测序研究发现髓样细胞激发受体-2(triggering receptor expressed on myeloid cells 2,TREM2)基因突变会使AD的发病率增加2到3倍，因此TREM2在AD发病中的作用受到越来越多的关注[3]。小胶质细胞表面的TREM2还能被α-分泌酶水解，形成可溶性的TREM2(soluble TREM2,sTREM2)，释放到细胞外间隙[4]。并且AD患者脑脊液(cerebrospinal fluid,CSF)中sTREM2的含量随着病程的进展发生规律性的改变[5-8]。研究发现，sTREM2可以通过结合TG2降低神经元内tau磷酸化水平。而tau病变的严重程度与AD中神经损伤和认知障碍的关系非常密切[9,10]。
目前AD尚无有效的治疗措施，找到一种有效的治疗方法来减轻tau的病理，改善认知至关重要。鉴于sTREM2对于tau病变的抑制作用，开发sTREM2多肽类似物对AD的治疗具有极大的应用前景。
参考文献：
1.Chan KY,Wang W,Wu JJ,Liu L,Theodoratou E,Car J,Middleton L,Russ TC,Deary IJ,Campbell H,Wang W,Rudan I.Epidemiology of Alzheimer's disease andother forms of dementia in China,1990-2010:a systematic review andanalysis.Lancet.2013,381:2016-2023.
2.Jia J,Wang F,Wei C,Zhou A,Jia X,Li F,Tang M,Chu L,Zhou Y,Zhou C,CuiY,Wang Q,Wang W,Yin P,Hu N,Zuo X,Song H,Qin W,Wu L,Li D,Jia L,Song J,Han Y,Xing Y,Yang P,Li Y,Qiao Y,Tang Y,Lv J,Dong X.The prevalence of dementia inurban and rural areas of China.Alzheimers Dement.2014,10:1-9.
3.Jonsson T,Stefansson H,Steinberg S,Jonsdottir I,Jonsson PV,SnaedalJ,Bjornsson S,Huttenlocher J,Levey AI,Lah JJ,Rujescu D,Hampel H,Giegling I,Andreassen OA,Engedal K,Ulstein I,Djurovic S,Ibrahim-Verbaas C,Hofman A,IkramMA,van Duijn CM,Thorsteinsdottir U,Kong A,Stefansson K.Variant of TREM2associated with the risk of Alzheimer’s disease[J].The New England Journal ofMedicine,2013,368(2):107–116.
4.Feuerbach D,Schindler P,Barske C,Joller S,Beng-Louka E,Worringer KA,Kommineni S,Kaykas A,Ho DJ,Ye C,Welzenbach K,Elain G,Klein L,Brzak I,MirAK,Farady C J,Aichholz R,Popp S,George N,Neumann U.ADAM17 is the mainsheddase for the generation of human triggering receptor expressed in myeloidcells(hTREM2)ectodomain and cleaves TREM2 after Histidine 157[J].NeuroscienceLetters,2017,660:109–114.
5.Suárez-Calvet M,Kleinberger G,Araque CaballeroBrendel M,Rominger A,Alcolea D,Fortea J,/>A,Blesa R,Gispert JD,Sánchez-Valle R,Antonell A,Rami L,Molinuevo JL,Brosseron F,Traschütz A,Heneka MT,Struyfs H,Engelborghs S,Sleegers K,Van Broeckhoven C,Zetterberg H,/>B,Blennow K,Crispin A,Ewers M,Haass C.sTREM2cerebrospinal fluid levels are a potentialbiomarker for microglia activity in early-stage Alzheimer’s disease andassociate with neuronal injury markers[J].EMBO molecular medicine,2016,8(5):466–476.
6.Heslegrave A,Heywood W,Paterson R,Magdalinou N,Svensson J,JohanssonP,A,Blennow K,Hardy J,Schott J,Mills K,Zetterberg H.Increasedcerebrospinal fluid soluble TREM2 concentration in Alzheimer’s disease[J].Molecular Neurodegeneration,2016,11:3.
7.Ma LZ,Tan L,Bi YL,Shen XN,Xu W,Ma YH,Li HQ,Dong Q,Yu JT.Dynamicchanges of CSF sTREM2 in preclinical Alzheimer's disease:the CABLE study[J].Molecular Neurodegeneration,2020,15(1):25.
8.Suárez-Calvet M,Araque Caballero MKleinberger G,Bateman R J,Fagan A M,Morris J C,Levin J,Danek A,Ewers M,Haass C,Dominantly InheritedAlzheimer Network.Early changes in CSF sTREM2 in dominantly inheritedAlzheimer’s disease occur after amyloid deposition and neuronal injury[J].Science Translational Medicine,2016,8(369):369ra178.
9.Ossenkoppele R,Smith R,Ohlsson T,Strandberg O,Mattsson N,Insel PS,Palmqvist S,Hansson O.Associations between tau,Abeta,and cortical thicknesswith cognition in Alzheimer disease[J].Neurology,2019,92(6):e601-e612.
10.Smirnov DS,Salmon DP,Galasko D,Goodwill VS,Hansen LA,Zhao Y,EdlandSD,Léger GC,Peavy GM,Jacobs DM,Rissman R,Pizzo DP,Hiniker A.Association ofNeurofibrillary Tangle Distribution With Age at Onset-Related ClinicalHeterogeneity in Alzheimer Disease:An Autopsy Study[J].Neurology,2022,98(5):e506–e517.
发明内容
本发明的目的是开发一种sTREM2多肽类似物，用于AD和其他tau相关疾病的治疗。
本发明的目的通过以下技术方案实现：
一种可穿过血脑屏障的sTREM2多肽类似物，其氨基酸序列如SEQ ID NO.1所示，该多肽由位于N端的Tat和位于C端的sTREM2(77-89aa)组成，其中Tat为11个氨基酸的穿膜结构序列，具体如SEQ ID NO.2所示；sTREM2(77-89aa)是与TG2亲和力最高的sTREM2蛋白序列上的13个氨基酸片段，具体如SEQ ID NO.3所示。
一种sTREM2多肽，其氨基酸序列如SEQ ID NO.3所示。
上述sTREM2多肽或sTREM2多肽类似物在制备治疗阿尔兹海默病的药物中的应用。
上述sTREM2多肽或sTREM2多肽类似物在制备治疗tau相关疾病的药物中的应用，所述的tau相关疾病包括额颞叶痴呆、进行性核上性麻痹、皮质基底节变性、慢性创伤性脑病等疾病。
一种治疗阿尔兹海默病或tau相关疾病的药物，包含上述sTREM2多肽或sTREM2多肽类似物。
本发明相对于现有技术具有如下优点及效果：
(1)本发明的多肽在体外HEK293 tau细胞中明显抑制tau的过度磷酸化。
(2)本发明的多肽在tau病模型上明显抑制了tau的过度磷酸化。
(3)本发明的多肽在tau病模型上改善了突触功能障碍。
(4)本发明的多肽在tau病模型上改善了认知功能障碍。
附图说明
图1为sTREM2多肽(SEQ ID NO.3)在体外HEK293 tau细胞中明显抑制tau的过度磷酸化。HEK293-tau细胞加入不同浓度sTREM2多肽(0μM，1μM，10μM，20μM)处理24h，Westernblot检测tau磷酸化及GSK3β磷酸化水平(A)，并定量分析p-tau S202/total tau、p-tauS396/total tau、p-GSK3βS9/GSK3β和p-GSK3βY216/GSK3β水平(B)。n＝4次独立重复实验。方差分析，*P<0.05，**P<0.01，***P<0.001。
图2为sTREM2多肽类似物(SEQ ID NO.1)穿过血脑屏障的能力。tau P301S小鼠腹腔注射Tat-sTREM2多肽-FITC或Tat-反序对照多肽-FITC，一周后脑组织内FITC荧光表达。比例尺，20μm。
图3为sTREM2多肽类似物在AD动物模型上改善了认知功能障碍。(A，B)在训练阶段1-4天逃往平台所耗时间(A)以及在第4天训练时逃往平台所耗时间(B)。(C)探测阶段小鼠在岛所在目标象限耗时。(D)小鼠在新异臂中探索的时间。数据表示为mean±s.e.m.，*P<0.05；**P<0.01；***P<0.001，n＝8，t-test。
图4为sTREM2多肽类似物在AD动物模型上明显抑制了tau的过度磷酸化。Westernblot检测tau P301S小鼠注射对照多肽和sTREM2多肽后脑组织中tau磷酸化和GSK3β磷酸化水平(A)，并定量分析p-tau S202/total tau、p-tau S396/total tau、p-GSK3βS9/GSK3β和p-GSK3βY216/GSK3β水平(B)。数据表示为mean±s.e.m.，*P<0.05；**P<0.01；***P<0.001，n＝4，t-test。
图5为sTREM2多肽类似物增加AD动物模型中突触与树突棘密度。电镜结果显示tauP301S小鼠注射对照多肽和sTREM2(77-89aa)多肽后海马区域的突触密度。星号指示突触后致密区。数据表示为mean±s.e.m.，**P<0.01，n＝6，t-test。
具体实施方式
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
【实施例1】sTREM2多肽类似物和sTREM2多肽的序列
一种sTREM2多肽类似物由位于N端的Tat和位于C端的sTREM2(77-89aa)组成，其氨基酸序列为：YGRKKRRQRRRRWNGSTAITDDTL(SEQ ID NO.1)。其中，Tat为11个氨基酸的穿膜结构序列，其为转录反式激活蛋白Tat中的第47-57位氨基酸片段，序列为YGRKKRRQRRR(SEQID NO.2)。sTREM2(77-89aa)多肽为与TG2亲和力最高的sTREM2蛋白序列上的13个氨基酸片段aa.77-89，序列为RWNGSTAITDDTL(SEQ ID NO.3)。
【实施例2】sTREM2(77-89aa)多肽对HEK293 tau细胞中tau磷酸化的影响
(1)利用LV-EF1a-EGFP-Tau-1-441-CMV-puro-WPRE病毒(购买于武汉枢密脑科学技术有限公司)感染HEK293细胞构建稳定表达EGFP-tau(1-441aa)的细胞系(HEK293-tau)。
(2)PBS溶解sTREM2(77-89aa)多肽(SEQ ID NO.3)，配置成1mg/mL母液。将HEK293-tau细胞在无血清培养基中饥饿12h后，在培养基中加入不同浓度的sTREM2(77-89aa)多肽(终浓度为0μM、1μM、10μM、20μM)。
(3)制样：24h后收集细胞，PBS洗一遍，加裂解液冰上裂解30min，取上清至新的EP管中，加入5×SDS loading buffer，混匀，100℃沸水煮10min。
(4)跑胶：将制好的样品按照图1的顺序进行点样，80V恒压跑胶20min后，120V恒压跑胶到Marker分开至合适的位置。
(5)转膜：提前配好转膜液，4℃预冷，按照负极-海绵-滤纸-胶-NC膜-滤纸-海绵-正极的顺序将胶和膜用转膜夹固定好，220mA恒流转膜75min。
(6)封闭：转膜完成后，将膜用TTBS溶液洗涤一次后，置于5％奶粉中，放置于摇床上室温封闭1h。
(7)孵育一抗：封闭完成后，孵育一抗，一抗采用3％的奶粉进行稀释(anti-p-tauS202抗体(abcam,ab108387)：1:1000；anti-p-tau S396抗体(abcam,ab109390)：1:1000；anti-p-GSK3βS9抗体(abcam,EPR2286Y)：1:1000；anti-p-GSK3βY216抗体(abcam,ab75745)：1:1000；anti-GSK3β抗体(abcam,ab32391)：1:1000)，4℃摇床孵育过夜。
(8)洗膜：室温采用TTBS溶液洗膜40min，每10min换一次洗膜液。
(9)孵育二抗：采用3％的奶粉将二抗按照1:10000比例进行稀释，室温孵育1h。
(10)洗膜：室温采用TTBS溶液洗膜1h，每10min换一次洗膜液。
(11)显影：使用ECL显影液对膜进行曝光显影。通过比较p-tau S202/total tau、p-tau S396/total tau、p-GSK3βS9/GSK3β和p-GSK3βY216/GSK3β水平检测sTREM2(77-89aa)多肽对tau磷酸化和GSK3β磷酸化的影响。结果(图1)表明sTREM2(77-89aa)多肽处理细胞后，tau的磷酸化水平降低，并且介导tau磷酸化的激酶GSK3β活性降低。
【实施例3】sTREM2多肽类似物在AD模型上对tau磷酸化、突触功能和认知功能的影响
该实施例中使用的sTREM2多肽类似物和反序多肽对照采用了异硫氰酸荧光素酯(FITC)标记，序列如下：
Tat-sTREM2多肽-FITC：YGRKKRRQRRRRWNGSTAITDDTL(FITC)，
Tat-反序对照多肽-FITC：YGRKKRRQRRRLTDDTIATSGNWR(FITC)。
(1)将3月龄tau P301S小鼠分为两大组，Tat-sTREM2多肽-FITC组和Tat-反序对照多肽-FITC组(以下简称为多肽组与对照组)，每组各20只。注射Tat-sTREM2多肽-FITC与Tat-反序对照多肽-FITC，给药浓度为10mg/kg，给药方式为腹腔注射，每周两次，持续4个月。
(2)注射多肽1周后，每组各取一只小鼠，用4％多聚甲醛灌注固定后，将脑组织进行冰冻切片，荧光显微镜下观察脑组织FITC荧光，神经元内可观察到绿色荧光，说明多肽穿过了血脑屏障(图2)。
(3)注射多肽4个月后，分别对每组小鼠进行水迷宫和Y迷宫实验，观察不同处理小鼠学习记忆能力。水迷宫结果表明多肽组小鼠相比于对照组在训练的第四天上岛时间更短(图3A、B)，并且在岛所在象限停留时间更长(图3C)。Y迷宫结果表明多肽组小鼠在新异臂中停留时间更长(图3D)。表明多肽组小鼠学习记忆能力更好。
(4)行为学完成后每组取5只小鼠海马分离取材，进行Western blot检测tau和GSK3β的磷酸化水平变化。结果(图4)表明多肽组tau磷酸化程度明显低于对照组，GSK3β活性被抑制。
(5)每组取5只小鼠用2％戊二醛灌注固定，取小鼠海马组织切片，在1％四氧化锇溶液中固定1h后，切片染色，电镜观察突触密度与突触结构。结果(图5)表明多肽组突触密度高于对照组。
序列表
<110> 武汉大学
<120> 一种sTREM2多肽类似物及其在治疗阿尔兹海默病中的应用
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<170> SIPOSequenceListing 1.0
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Thr Ala Ile Thr Asp Asp Thr Leu
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Claims (5)
1. 一种sTREM2多肽类似物，其特征在于：其氨基酸序列如SEQ ID NO.1所示。
2. 一种sTREM2多肽，其特征在于：其氨基酸序列如SEQ ID NO.3所示。
3.权利要求1所述的sTREM2多肽类似物或权利要求2所述的sTREM2多肽在制备治疗阿尔兹海默病的药物中的应用。
4.权利要求1所述的sTREM2多肽类似物或权利要求2所述的sTREM2多肽在制备治疗tau相关疾病的药物中的应用，其特征在于：所述的tau相关疾病选自额颞叶痴呆、进行性核上性麻痹、皮质基底节变性、慢性创伤性脑病。
5.一种治疗阿尔兹海默病或tau相关疾病的药物，其特征在于：包含权利要求1所述的sTREM2多肽类似物或权利要求2所述的sTREM2多肽；所述的tau相关疾病选自额颞叶痴呆、进行性核上性麻痹、皮质基底节变性、慢性创伤性脑病。
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