CN107779437B - 自噬诱导剂作为微管稳定药物促进神经再生的用途 - Google Patents
自噬诱导剂作为微管稳定药物促进神经再生的用途 Download PDFInfo
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Abstract
本发明提供自噬诱导剂作为微管稳定药物促进神经再生的用途。具体而言,本发明提供促进体外培养中枢神经元的突起生长并减弱髓磷脂对突起生长的抑制作用,或稳定中枢神经受损神经元的微管,或减少中枢神经轴突损伤后的回撤或回撤小泡形成,或促进中枢神经受损后轴突再生的方法,所述方法包括使所述离体神经元与自噬诱导剂接触以诱导自噬的步骤。本发明还提供自噬诱导剂在制备药物中的用途。采用本发明的方法,能显著减少脊髓受损后轴突的回撤,促进轴突再生和运动功能恢复。
Description
技术领域
本发明涉及自噬诱导剂作为微管稳定药物促进神经再生的用途。
背景技术
中枢神经系统由脑和脊髓组成,是人体神经系统最主体的部分。脊髓是周围神经和脑之间连接的通路。脊髓损伤指由于创伤(如跌落、车祸),疾病或退化(如癌症、炎症)等造成的脊髓损坏。据世界卫生组织估计,每年的全球发生率为每百万人40至80例,且呈现逐年增高的趋势。脊髓中的主轴突干受损后难以再生,导致损伤节段以下肢体严重的功能障碍和一系列并发症及较高的死亡率,给患者本人带来身心的严重伤害,也对整个社会造成巨大的经济负担。脊髓损伤后的神经再生修复和功能重建仍缺乏有效的治疗手段。
目前普遍认为成年哺乳动物中枢神经系统的神经元在受损后缺乏再生能力,其原因既包括外环境中有多种再生抑制因子,又包括神经元内部再生能力的匮乏。目前研究已经找到了许多外界生长抑制因子,例如来源于少突胶质细胞的髓磷脂相关糖蛋白(MAG)、Nogo、OMg或者来源于星形胶质细胞的硫酸软骨素蛋白多糖(CSPG)等等,它们通过与各自受体结合抑制轴突生长与再生。然而,中和这些抑制因子或者基因敲除它们的受体仍然无法促进轴突再生或者仅有有限的促进作用。
许多抑制因子通过级联放大的信号通路调节细胞骨架。中枢神经元轴突受损后,末端产生很多回撤小泡(retraction bulb),包含着无序排列的微管。相比之下,外周神经系统(PNS)神经元轴突在受损后可快速形成生长锥(growth cone),并维持稳定和有序的微管束。与之相对应,在脊髓损伤后,通过药物手段稳定微管能够促进其轴突的再生。此外,通过分析基因敲除小鼠已经发现一些在成年中枢神经中抑制轴突再生的内在因子,这些因子包括PTEN(Phosphate and tensin homolog)和SOCS3(Suppressor of cytokinesignaling 3)等。然而,不论是操控单个因子或是同时改变多个因子的表达都只能获得有限的轴突再生或出芽能力,使得动物获得短暂的功能恢复。同时,通过操控某些特定基因表达从而获得轴突再生的方法在目前仍然无法应用于临床。基于此,目前仍然亟待开发出新的促进轴突再生的有效方法。
轴突损伤会在胞体和轴突上引起剧烈的应激反应,这类应激反应包括染色质溶解(chromatolysis),轴突细胞膜的封闭(axonal membrane sealing),生长锥或回撤小泡的生成以及沃勒变性(Wallerian degeneration)。生长锥的形成以及轴突的再生都依赖于胞质成分和轴突结构的广泛重构,牵涉到一系列蛋白的合成和降解。自噬是细胞在压力环境下,大量快速降解和有效周转细胞质成分的主要途径。自噬通过溶酶体途径降解受损的细胞器或致病蛋白,在维持细胞内稳态中起着重要的作用。通过敲除自噬相关基因抑制自噬,能导致小鼠小脑浦肯野细胞(Purkinje cells)或者其他脑区中神经元出现退化(degeneration)。与之相反的是,通过药物抑制自噬却能够缓解视网膜神经节细胞(retinal ganglion cell,RGC)轴突的急性退化。这些现象看似相悖,却提示了自噬在维持轴突稳态中发挥着关键作用。而目前,调节自噬水平是否能够促进成年哺乳动物中枢神经的轴突再生仍然缺少定论。
发明内容
本发明第一方面提供一种促进体外培养中枢神经元的突起生长并减弱髓磷脂对突起生长的抑制作用,或稳定中枢神经受损神经元的微管,或减少中枢神经轴突损伤后的回撤或回撤小泡形成,或促进中枢神经受损后轴突再生的方法,其特征在于,所述方法包括使所述离体神经元与自噬诱导剂接触以诱导自噬的步骤。
在一个或多个实施方案中,所述自噬诱导剂选自:SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列,或SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列与促进穿膜的肽组成的氨基酸序列,组蛋白去乙酰化酶(HDAC)抑制剂,他莫昔芬,EB1089,抗血管生成剂,酪氨酸激酶抑制剂,白藜芦醇及其类似物RSVA,烷化剂,三氧化砷,Akt抑制剂〔如10-(4’-N-二乙基氨基)丁基-2-氯吩噁嗪和Akti-1/2〕,HSP90-CDC37伴娘蛋白复合物抑制剂(如17-AAG),HIV蛋白酶抑制剂,哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂(如雷帕霉素、KU-0063794和Torin 1),MTORC1抑制剂(如依维莫斯),NAE抑制剂(如MLN4924),NAADP-AM,PMI,ATP2A/SERCA抑制剂(如柴胡皂甙d),顺式-3,5,3-三甲氧基芪,海藻糖,依霉素,氟司必林,三氟拉嗪,哌迷清,尼卡地平,尼古地平,洛哌丁胺,胺碘酮,维拉帕米,米诺地尔,可乐定,PP242,MG-132,ESC8和亚精胺中的一种或多种。
在一个或多个实施方案中,所述促进穿膜的肽选自:RQIKIWFQNRRMKWKK(SEQ IDNO:5);YGRKKRRQRRR(SEQ ID NO:6);KQAIPVAK(SEQ ID NO:7);RRRRNRTRRNRRRVR(SEQ IDNO:8);寡精氨酸(R9-R12);KLTRAQRRAAARKNKRNTRGC(SEQ ID NO:9);ALWKTLLKKVLKAPKKKRKVC(SEQ ID NO:10);RKKRRQRRR(SEQ ID NO:11);DAATATRGRSAASRPTERPRAPARSASRPRRPVE(SEQ ID NO:12);GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO:13);AGYLLGKINLKALAALAKKIL(SEQ ID NO:14);和YTAIAWVKAFIRKLRK(SEQ ID NO:15)。
本发明第二方面提供自噬诱导剂在制备药物中的用途,所述药物用于:促进中枢神经受损神经元的突起生长并减弱髓磷脂对突起生长的抑制作用,和/或减少中枢神经轴突损伤后的回撤或回撤小泡形成,和/或稳定中枢神经受损神经元的微管,和/或恢复对象中枢神经受损后的运动能力,和/或促进中枢神经受损后轴突再生,和/或治疗中枢神经轴突损伤。
在一个或多个实施方案中,所述自噬诱导剂选自:SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列,或SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列与促进穿膜的肽组成的氨基酸序列,组蛋白去乙酰化酶(HDAC)抑制剂,他莫昔芬,EB1089,抗血管生成剂,酪氨酸激酶抑制剂,白藜芦醇及其类似物RSVA,烷化剂,三氧化砷,Akt抑制剂〔如10-(4’-N-二乙基氨基)丁基-2-氯吩噁嗪和Akti-1/2〕,HSP90-CDC37伴娘蛋白复合物抑制剂(如17-AAG),HIV蛋白酶抑制剂,哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂(如雷帕霉素、KU-0063794和Torin 1),MTORC1抑制剂(如依维莫斯),NAE抑制剂(如MLN4924),NAADP-AM,PMI,ATP2A/SERCA抑制剂(如柴胡皂甙d),顺式-3,5,3-三甲氧基芪,海藻糖,依霉素,氟司必林,三氟拉嗪,哌迷清,尼卡地平,尼古地平,洛哌丁胺,胺碘酮,维拉帕米,米诺地尔,可乐定,PP242,MG-132,ESC8和亚精胺中的一种或多种。
在一个或多个实施方案中,所述促进穿膜的肽选自:RQIKIWFQNRRMKWKK(SEQ IDNO:5);YGRKKRRQRRR(SEQ ID NO:6);KQAIPVAK(SEQ ID NO:7);RRRRNRTRRNRRRVR(SEQ IDNO:8);寡精氨酸(R9-R12);KLTRAQRRAAARKNKRNTRGC(SEQ ID NO:9);ALWKTLLKKVLKAPKKKRKVC(SEQ ID NO:10);RKKRRQRRR(SEQ ID NO:11);DAATATRGRSAASRPTERPRAPARSASRPRRPVE(SEQ ID NO:12);GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO:13);AGYLLGKINLKALAALAKKIL(SEQ ID NO:14);和YTAIAWVKAFIRKLRK(SEQ ID NO:15)。
在一个或多个实施方案中,所述回撤为脊髓背侧轴突和/或皮质脊髓束的回撤;所述轴突再生为脊髓下行轴突的再生。
在一个或多个实施方案中,所述自噬诱导剂选自:SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列,或SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列与促进穿膜的肽组成的氨基酸序列,雷帕霉素和亚精胺。
本发明第三方面提供一种筛选作为治疗中枢神经轴突损伤的候选药物的方法,所述方法包括:
(1)使待筛选物质与受损的中枢神经神经元接触;
(2)检测该待筛选物质是否在该受损中枢神经神经元中诱导了自噬,以及该神经元胞质中的SCG10是否减少;
如果诱导了自噬且胞质中的SCG10减少,则将该待筛选物质作为治疗中枢神经轴突损伤的候选药物。
在一个或多个实施方案中,所述待筛选物质是核酸、多肽或小分子化合物;优选地,所述待筛选物质是SCG10的抑制剂。
附图说明
图1:自噬诱导对培养的皮层神经元的轴突生长的影响。(A)用所示的肽(5μM,3h)处理体外培养1天(DIV1)皮层神经元的代表性电子显微镜图。N,核;M,线粒体;A,自噬体;A1,自溶体。标尺,体细胞为0.5μm,轴突为0.2μm。(B)Tat-Bec或Tat-Scr(5μM,3h)处理的DIV1皮层神经元中LC3和p62的免疫印迹。使用β-肌动蛋白作为加样对照。(C)Tat-Bec或Tat-Scr(5μM,3h)处理的DIV1皮层神经元中β-微管蛋白III(Tuj1)的免疫染色。标尺为50μm。(D)BSA或髓磷脂底物上培养的DIV2皮层神经元经所示处理(10nM Rapa,1μM Spd,或2.5μM Tat-Scr或Tat-Bec)后β-微管蛋白III(Tuj1)的免疫染色。标尺为100μm。(E和F)轴突长度的定量。数据来自3次实验,每组70-80个细胞,以平均值±SEM表示。*P<0.05,***P<0.001,E图采用T检验,F图采用ANOVA和图基事后检验法。
图2:增强的自噬稳定微管。(A)诺考达唑(1μg/ml)及所示试剂(40nM Rapa、2μMSpd或5μM Tat-Scr或Tat-Bec)处理DIV1皮层神经元3小时后乙酰化α-微管蛋白(A-Tub)和酪氨酸化α-微管蛋白(T-Tub)的免疫染色。显示的是代表性的图片。标尺为10μm。(B)乙酰化α-微管蛋白和酪氨酸化α-微管蛋白之比(A/T比)。数据来自3次实验,每组70-80个细胞,以平均值±SEM表示。*P<0.05,***P<0.001,采用ANOVA和图基事后检验法。(C)5μM Tat-Bec处理之前及之后DIV2皮层神经元中表达的EB3-GFP的动态图像叠加。底图显示轴突(c1和c3)和体细胞(c2)中EB3-GFP信号。标尺为10μm。(D)Tat-Bec处理之前及处理后2小时EB3-GFP纤维长度。数据来自3次实验,每组18-20个细胞,以平均值±SEM表示。(E)所示条件下轴突中EB3-GFP的荧光强度。
图3:SCG10为自噬的底物。(A)5μM Tat-Scr或Tat-Bec处理DIV1皮层神经元3小时后所示蛋白的免疫印迹。显示的是3次独立实验获得的代表性印迹,具有相似的结果。使用β-肌动蛋白作为加样对照。(B)所示蛋白相对于β-肌动蛋白的水平定量。数据以平均值±SEM(n=3)。***P<0.001(Tat-Bec vs.Tat-Scr),T检验。(C)Tat-Scr或Tat-Bec处理的DIV1皮层神经元中SCG10分布的免疫电镜分析。Tat-Scr组胞质中和Tat-Bec组自噬体中SCG10信号如箭头所示。标尺为200nm。(D)胞质和自噬体内标记SCG10的金颗粒的数量。数据来自3次实验,每组30个细胞,以平均值±SEM表示。(E)SCG10-GFP转染的DIV2皮层神经元经Tat-Scr或Tat-Bec(5μM,3h)处理后A-Tub和T-Tub的免疫染色。过表达SCG10-EGFP的神经元(GFP阳性)如箭头所示。标尺为10μm。(F)A/T比值。数据来自3次实验,每组70-80个细胞,以平均值±SEM表示。***P<0.001(SCG10-EGFP vs.对照组),T检验。
图4:小鼠神经损伤后,局部给予Tat-Bec能保护轴突,并稳定微管。(A)成年Thy1-YFP小鼠损伤后(0h)以及给予Tat肽治疗后1-5小时背柱轴突的实时成像观察。底图,顶图中损伤后0h到5h靠近损伤位点的框图区的放大图。大的红色箭头指示损伤部位,小的白色箭头指示回撤小泡(RB);虚线指示完整轴突的末端。标尺为100μm。(B)损伤后1-5小时后具有回撤小泡(RB)的轴突的百分比。数据来自每组10只小鼠的实验,以平均值±SEM表示。(C)损伤1-5小时后轴突回撤长度。(D)成年Emx1-Cre;Ai9小鼠在脊髓颈部C4-C5节段双侧脊髓半横切后立即给予Tat-Bec或Tat-Scr肽。4周后,以GFAP标记的胶质瘢痕标记损伤部位,分析损伤区域的纵切面中tdTomato标记的CST。虚线为切口边缘;箭头指示完整轴突的末端;星号指示回撤小泡。标尺为200μm。(E)脊髓切口边缘起的CST回撤距离。数据来自每组12只小鼠,以平均值±SEM表示。(F和G)SCI 3小时后脊髓纵切中Glu-tub和Tuj1的免疫染色结果。方框区域为接近损伤部位的区域;箭头指向Tuj1染色的轴索。标尺为10μm。(H)SCI及施与多肽3小时后CST中Glu-tub标记的微管相对于Tuj1标记的微管的长度之比值。数据来自每组6-8只小鼠,以平均值±SEM表示。(I)损伤后轴突藕断的电镜图片,线条部分为通过软件手动描绘的微管示意图。样品获自SCI后经多肽处理3小时的Emx1-Cre;Ai9小鼠。箭头指示自噬体,标尺为0.5μm。(J)微管相对轴突轴线的偏移角的定量。显示的是来自各组3只小鼠至少15个视野内的分布及微管偏移角度的平均值±SEM。
图5:Tat-Beclin1促进脊髓损伤后的单胺能轴突再生,及运动功能恢复。(A)脊髓双侧半横切模型的示意图。红色方框区域表示C4-C5节段的纵向切面,蓝色方框区域(上图右侧的小方框)表示C6节段的腹角的横向切面。(B)损伤8周后脊髓C4-5节段纵向切面中5-羟色胺(5-HT)和GFAP的免疫染色。方框区域表示损伤部位的尾侧区域(虚线)。标尺为100μm。(C)损伤部位头侧和尾侧区域中5-HT+纤维的比例。显示为每组7只小鼠的平均值±SEM。(D)脊髓C6节段横切面5-HT、胆碱乙酰转移酶(ChAT)和突触素(Syn)的免疫染色。方框区域内的箭头指示5-HT+轴突支配的ChAT标记的运动神经元。左图标尺为100μm,方框区域的标尺为50μm。(E)腹角内5-HT阳性纤维的面积。数据以每组4只小鼠的平均值±SEM表示。(F)损伤及给予多肽后在所示时间点进行Rotarod测试获得的恢复指数。数据以每组14只小鼠的平均值±SEM表示。(G)损伤及给予多肽之后在所示时间点统计5分钟内小鼠在网格上走动时四肢跌落的次数。数据以每组12只小鼠的平均值±SEM表示。(H和I)步态分析中的协调指数(H)以及步伐长度(I)。数据以每组12-15只小鼠的平均值±SEM表示。(J)本发明提出的Tat-Bec诱导自噬后促进损伤后轴突再生的模型。
具体实施方式
轴突受损后,通过诱导自噬来研究自噬在轴突再生中的作用是一种直接的手段,但大多数诱导自噬的化合物存在不可控的副作用。在本发明中,我们主要利用一种可以在体内和体外特异增强自噬的多肽Tat-beclin1来诱导自噬。我们在体外培养的大鼠皮层神经元中发现,提高自噬水平能够通过降解一个神经元特有的微管去稳定蛋白(SCG10)而稳定微管,并促进神经元突起的生长。此外,在脊髓损伤的小鼠模型中,局部给予Tat-beclin1处理能显著减少脊髓受损后轴突的回撤,促进轴突再生和小鼠运动功能恢复。本发明发现了自噬可以稳定神经元微管的关键功能,并提出以Tat-beclin1为代表的自噬诱导剂作为一种有潜力的中枢神经轴突损伤后的治疗药物。
因此,本发明涉及促进中枢神经元的突起生长并减弱髓磷脂对突起生长的抑制作用的方法,或稳定中枢神经受损神经元的微管的方法,或减少中枢神经轴突损伤后的回撤或回撤小泡形成的方法,或促进中枢神经受损后轴突再生的方法。本发明的各种方法可以是体外或离体方法,也可以是体内方法。本发明中,中枢神经包括大脑和脊髓。
本发明的方法包括给予所述中枢神经神经元自噬诱导剂的步骤。
适用于本文的自噬诱导剂可以是本领域周知的能诱导细胞形成自噬体的任何制剂,可以是核酸分子,多肽,也可以是小分子化合物。例如,可用于本发明的自噬诱导剂可以是SEQ ID NO:1第12-31位氨基酸所示的氨基酸序列或其功能变体。本文中,所谓“功能变体”,是指在SEQ ID NO:1第12-31位所示的氨基酸序列中具有5个以内、优选3个以内的氨基酸突变(如缺失、插入或取代),但仍保留SEQ ID NO:1第12-31位所示序列诱导自噬功能的突变体。优选的突变是在SEQ ID NO:1第12-31位残基的非关键性位点上发生的突变;更优选地,突变是本领域周知的保守性取代。在某些实施方案中,可直接给予SEQ ID NO:1第12-31位所示的多肽。
在其它实施方案中,可给予SEQ ID NO:1所示的多肽,其由促进穿膜的肽(即SEQID NO:1第1-11位氨基酸残基,本文中编号为SEQ ID NO:6)与诱导自噬的肽(即SEQ IDNO:1第12-31位氨基酸残基)组成。应理解,可使用本领域周知的其它促进穿膜的肽替换SEQ ID NO:1的第1-11位氨基酸残基。例如,可参见Siegmund Reissmann,Cellpenetration:scope and limitations by the application of cell-penetratingpeptides,J.Pept.Sci.2014;20:760–784。本文将其全部内容以引用的方式纳入本文。尤其是,适用于本发明的促进穿膜的肽段包括上述文献表1所列的编号为第1-92的序列,包括但不限于RQIKIWFQNRRMKWKK(SEQ ID NO:5)、KQAIPVAK(SEQ ID NO:7)、RRRRNRTRRNRRRVR(SEQ ID NO:8)、寡精氨酸(R9-R12)、KLTRAQRRAAARKNKRNTRGC(SEQ ID NO:9)、ALWKTLLKKVLKAPKKKRKVC(SEQ ID NO:10)、RKKRRQRRR(SEQ ID NO:11)、DAATATRGRSAASRPTERPRAPARSASRPRRPVE(SEQ ID NO:12)、GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO:13)、AGYLLGKINLKALAALAKKIL(SEQ ID NO:14)、YTAIAWVKAFIRKLRK(SEQ ID NO:15)。
在合适的情况下,也可给予该多肽的表达载体。合适的表达载体及其构建为本领域所周知。
合适的自噬诱导剂的例子还可参见例如Guidelines for the use andinterpretation of assays for monitoring autophagy,Autophagy,2016(第三版);CN201180053316.1;CN201380022280.X;CN200980129095.4;http://www.selleck.cn/pathways_autophagy.html等,本文将其全部内容,尤其是涉及自噬诱导剂的内容以引用的方式纳入本文。例如,合适的自噬诱导剂包括但不限于组蛋白去乙酰化酶(HDAC)抑制剂,他莫昔芬,EB1089,抗血管生成剂,酪氨酸激酶抑制剂,白藜芦醇及其类似物RSVA,烷化剂,三氧化砷,Akt抑制剂〔如10-NCP(10-(4’-N-二乙基氨基)丁基-2-氯吩噁嗪)和Akti-1/2〕,HSP90-CDC37伴娘蛋白复合物抑制剂(如17-AAG),HIV蛋白酶抑制剂,哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂(如雷帕霉素、KU-0063794、Torin 1),MTORC1抑制剂(如依维莫斯),NAE抑制剂(如MLN4924(Pevonedistat)),NAADP-AM,PMI,ATP2A/SERCA抑制剂(如柴胡皂甙d),TMS(顺式-3,5,3-三甲氧基芪),海藻糖,依霉素,氟司必林,三氟拉嗪,哌迷清,尼卡地平,尼古地平,洛哌丁胺,胺碘酮,维拉帕米,米诺地尔,可乐定,PP242,MG-132,ESC8,和亚精胺中的一种或任意多种。可单独给予一种自噬诱导剂,也可给予两种以上自噬诱导剂的组合。在某些实施方案中,适用于本发明的自噬诱导剂是雷帕霉素和/或亚精胺。
所给予的自噬诱导剂的量应足以在受损的中枢神经神经元中诱导自噬体的形成且不会导致细胞死亡。更优选的,诱导形成的自噬体能优先适当降解微管解聚蛋白SCG10,使微管稳定。可采用本领域周知的方法来测定不同自噬诱导剂的合适的给予量。例如,可参照本申请实验和材料部分所提供的相关实验进行。
因此,本发明也包括自噬诱导剂的用途,包括但不限于用于促进中枢神经受损神经元的突起生长并减弱髓磷脂对突起生长的抑制作用,和/或减少中枢神经轴突损伤后的回撤或回撤小泡形成,和/或稳定中枢神经受损神经元的微管,和/或恢复对象中枢神经受损后的运动能力,和/或促进中枢神经受损后轴突再生,和/或治疗中枢神经轴突损伤;以及用于制备药物的用途,这些药物可用于促进中枢神经受损神经元的突起生长并减弱髓磷脂对突起生长的抑制作用,和/或减少中枢神经轴突损伤后的回撤或回撤小泡形成,和/或稳定中枢神经受损神经元的微管,和/或恢复对象中枢神经受损后的运动能力,和/或促进中枢神经受损后轴突再生,和/或治疗中枢神经轴突损伤。
本发明的方法和用途可适用于哺乳动物,包括但不限于脊椎动物和啮齿动物,如人、小鼠、大鼠、兔和家畜等。因此,所述中枢神经神经元损伤或中枢神经轴突损伤可以是任意哺乳动物的中枢神经神经元损伤或轴突损伤。在某些实施方案中,轴突回撤是脊髓背侧轴突和/或皮质脊髓束的回撤。在某些实施方案中,轴突再生为脊髓下行轴突的再生。神经元或轴突的损伤可以本领域已知的各种原因导致的损伤,如机械性损伤或化学性损伤,包括外科手术导致的损伤,或者意外如车祸导致的损伤等。
本发明的药物除含有自噬诱导剂外,还可含有本领域周知的赋形剂或运载体,尤其是那些适用于神经给药的赋形剂或运载体。本发明药物中自噬诱导剂的含量可由本领域技术人员根据不同自噬诱导剂、不同使用目的等容易地确定。
基于本发明的发现,本发明还提供一种筛选作为治疗中枢神经轴突损伤的候选药物的方法,所述方法包括:
(1)使待筛选物质与受损的中枢神经神经元接触;
(2)检测该待筛选物质是否在该受损中枢神经神经元中诱导了自噬,以及该神经元胞质中的SCG10是否减少;
如果诱导了自噬且胞质中的SCG10减少,则将该待筛选物质作为治疗中枢神经轴突损伤的候选药物。
可采用本发明的方法筛选各种物质,包括但不限于各种核酸、多肽或小分子化合物。本文中,小分子化合物通常指多肽以外的其它有机化合物。在某些实施方案中,待筛选物质可以是例如SCG10的抑制剂。
以下将以具体实施例的方式阐述本发明。应理解,这些实施例仅仅是阐述性的,并非限制本发明。实施例中所用到的方法和试剂,除非另有说明,否则为本领域常规的方法和试剂。
一、实验材料和方法
(一)小鼠神经损伤及药物应用
1、脊髓背侧神经损伤后活体连续观察
脊髓背侧神经受损后的活体连续观察运用了成年的Thy1-YFP转基因小鼠(8周左右),该小鼠在Thy1启动子的驱动下,在包括DRG的感觉神经元在内的部分类型神经元的胞浆中高效表达黄色荧光蛋白(YFP)。小鼠麻醉后咬开C4-C5节段的椎骨,暴露出脊髓,用维纳斯眼科剪在单侧脊髓的背侧表面的神经(上行的感觉神经)上剪一道小口。在损伤后立即给药,用人工脑脊液(ACSF)配制终浓度为3μM的Tat-Bec或对照组Tat-Scr,加入到小鼠背部暴露形成的凹槽中,使受损的脊髓浸泡在液体中。1小时后,吸去药物,用ACSF清洗。神经受损前,以及受损后0、1、2、3、4、5小时用Prairie双光子显微镜进行拍摄。小鼠在整个过程中每两个小时通过腹腔注射麻药(三溴乙醇和叔戊醇的混合物)处于麻醉状态,并用加热板来保持体温。
2、小鼠脊髓颈段双侧半横切模型的建立
为检测脊髓损伤后皮质脊髓束(CST)的回撤,应用了在CST中表达红色荧光蛋白的Emx1-Cre;Ai9小鼠(8-10周)。其余实验(下行的单胺能轴突的再生,行为实验,CST轴突的再生)均用8周的C57BL/6J小鼠建立模型。小鼠通过腹腔注射麻药(三溴乙醇和叔戊醇的混合物)处于麻醉状态,刮掉头颈部的毛后,剪开皮肤,剥开肌肉,剔除椎骨上的肌肉和粘连,暴露出C4-C8节段。用咬骨钳移去背侧椎骨,撕去硬脊膜。用竖直眼科角膜剪,从脊髓背侧进入,在脊髓双侧的C4-C5节段剪下2mm深度,造成双侧半横切。利用明胶海绵止血后,在损伤伤口处,运用微量注射器注射Tat肽链(500μM,5μl)。重新整理肌肉和皮肤的结构,进行缝合。小鼠在加热板上保持体温直至苏醒。取材、形态、行为学的观察在手术后相应的时间点进行。
3、小鼠坐骨神经挤压模型的建立
8周的C57BL/6J小鼠通过腹腔注射麻药(三溴乙醇和叔戊醇的混合物)处于麻醉状态,刮去腿部的毛。剪开坐骨隆起附近的皮肤,剥开肌肉,暴露出坐骨神经。用解剖镊在神经上挤压15秒,连续挤压三次,直至神经挤压处变透明,神经断开,仅剩下髓鞘包裹。重新整理肌肉和皮肤的结构,进行缝合。小鼠在加热板上保持体温直至苏醒。取材在手术后相应的时间点进行。
(二)质粒、试剂及抗体
Tat多肽序列如下:
Tat-Beclin 1:YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT(SEQ ID NO:1);
Tat-Scrambled:YGRKKRRQRRRGGVGNDFFINHETTGFATEW(SEQ ID NO:2)。
在吉尔生化(上海)有限公司合成Tat多肽。本研究所使用的寡核苷酸序列如下:
SCG10siRNA序列:5’-GCAGATCAACAAGCGTGCTT-3’(SEQ ID NO:3),
用作对照的scrambled序列:5’-AGCTAGCGAGACACTCGATT-3’(SEQ ID NO:4)。
siRNA序列经过退火处理,克隆到pSUPER载体。pEGFP-SCG10质粒载体由北京大学陈建国研究员组提供。GFP-EB3质粒是将微管末端结合蛋白EB3基因的cDNA克隆到腺病毒载体pAdeno-CMV-EGFP-3×FLAG上。
抑制性中枢髓磷脂提取物(CME)是采用之前文献报道的方法(Larocca,J.N.和W.T.Norton,Isolation of myelin,Curr Protoc Cell Biol,2007,第3章:p.Unit325)从SD大鼠(200-300g)大脑中提取。
诺考达唑和亚精胺来自Sigma Aldrich,雷帕霉素来自Selleck。
人工脑脊液(ACSF)成分如下:125mM NaCl、5mM KCl、1.25mM NaH2PO4、1mM MgSO4、2mM CaCl2、25mM NaHCO3和20mM D-(+)-葡萄糖(试剂均来自Sigma公司),pH 7.4,310mOsm1-1。
抗体来自:Sigma(乙酰化的微管蛋白,LC3b,β-肌动蛋白,5-羟色胺),Abcam(突触素,纤维粘连蛋白,Glu-微管蛋白,酪氨酸化微管蛋白),Millipore(酪氨酸羟化酶,GFAP),Covance(Tuj1),BD(p62),Protech(SCG10),Chemicon(ChAT)以及Invitrogen(Alexa fluor488,555or 647荧光标记的二抗)。
0.1M磷酸缓冲液(PB)的配制:磷酸二氢钠(NaH2PO4·H2O)1.3g,磷酸氢二钠(Na2HPO4·12H2O)14.5g,重蒸馏水加至500ml,调pH至7.4。
(三)细胞培养,转染和处理
从E16.5的SD大鼠大脑皮层中取下的神经元铺在多聚赖氨酸(PDL)预包被的玻璃片上,用全效培养基(80%DMEM,10%FBS和10%F12)进行培养。种板后4小时后,培养基换成含有2%B27和1%L-谷氨酰胺的神经元基础培养基。对于需要电转的神经元,在种板之前,用Amaxa核转染装置对分离的单个神经元混合相应质粒进行电穿孔处理。神经元在培养箱中培养1天或2天(DIV1或DIV2)后,分别用文章中提及的试剂进行处理。在髓磷脂对神经元突起生长影响的实验中,玻璃片在用PDL处理后,再用1.67μg/ml中枢髓磷脂提取物(CME)进行包被。诺考达唑以1μg/ml的终浓度加入到培养基中。
(四)在培养的皮层神经元中观察荧光标记的EB3的运动
DIV1的大鼠皮层神经元利用表达pAdeno-CMV-EGFP-EB3-3×FLAG的腺病毒转染。24小时后,在Tat-Bec(5μM)给药前,以及给药后1、2小时分别观察EB3荧光亮点的运动情况,每5秒拍摄一次,每次拍摄持续30秒。细胞放置在37℃饱和5%CO2的活细胞培育装置中,利用倒置NIKON A1R激光扫描共焦显微镜,在60x油镜下观察。
(五)活细胞观察皮层神经元激光损伤后的轴突回撤实验
E16.5的大鼠皮层神经元通过电转pCAG-YFP质粒来标记神经元形态,在打孔培养皿中培养。3天后,用Tat肽链(5μM)预处理1小时。之后,将细胞放置在37℃饱和5%CO2的活细胞培育装置中,应用405nm波长的显微镜激光距离胞体约100μm的轴突上进行损伤照射20秒。立即进行活细胞成像观察受损后轴突的形态变化。在损伤后每5秒观察一次,共观察125秒。利用倒置NIKON A1R激光扫描共焦显微镜进行激光损伤,并在60x油镜下观察。
(六)免疫印迹、免疫染色和轴突示踪
为确定蛋白的相对水平,采用了免疫印迹(Western blotting)的方法:组织和培养的神经元在含有蛋白酶抑制剂(cocktail)的RIPA缓冲液中进行匀浆裂解以提取总蛋白,之后通过标准SDS-PAGE,以及Western blotting结合相应一抗、辣根过氧化酶标记的二抗进行蛋白定量分析。Western blotting结果采用化学发光法(试剂来自ThermoFisherScientific)显色,并通过Multi Gauge软件分析。
在微管状态分析实验中,体外培养的皮层神经元转染特定质粒或经特定试剂处理后,细胞样品用乙酰化、酪氨酸化、或总的微管蛋白抗体进行免疫荧光实验。简要步骤如下:培养的皮层神经元用4%多聚甲醛固定(4%PFA),同时瞬时在含有0.25%戊二醛,3.7%多聚甲醛,3.7%蔗糖,以及0.1%Triton X-100的PHEM缓冲液(60mM Pipes,25mM Hepes,5mMEGTA和1mM MgCl2)中透化处理7-10分钟,并且在50mM氯化铵中处理30分钟,以淬灭未聚合的微管单体的信号。PBS清洗后,用10%山羊血清封闭,在4℃过夜孵育一抗,室温下孵育荧光标记的二抗(1:1000)2小时。DAPI或荧光鬼笔环肽(fluorescent phalloidin)在室温下处理20-30分钟标记神经元的细胞核和β-肌动蛋白。制好的样品在NIKON A1R激光扫描共聚焦显微镜下观察。
对于体内受损轴突的组织学分析实验,将老鼠用4%PFA灌注后,取出受损节段的脊髓,用4%PFA后固定1天后,用30%蔗糖脱水过夜至标本下沉。随后进行连续冰冻切片,厚度为20μm,贴在载玻片上,40℃烤片1-2小时。标本在含有0.1%Triton的PBS(0.1%PBST)中洗涤,用配制在0.3%PBST中的10%山羊血清封闭2小时,在4℃过夜孵育一抗,室温下孵育荧光标记的二抗(1:1000)2小时。处理好的样品在Nikon TiE-A1激光扫描共聚焦显微镜下观察。
在受损后CST示踪实验中,脊髓半横切(SCH)后6周的小鼠通过腹腔麻醉后在小鼠立体定位仪下固定。通过电钻钻孔,在小鼠右侧头盖骨上钻取6个直径为1.5mm的小孔,其坐标为:前卤后0.1、0.6、1.1mm;横向1.0和1.4mm;深度0.7mm。在右侧的感觉运动皮层的4-5层注射BDA(10%,10000MW,Invitrogen)。注射2周以后,左侧脊髓做矢状切片,以分析CST的再生情况。
(七)质谱实验步骤及数据处理
准备以下四组样品进行质谱实验,分别是:1、空白组DIV1的皮层神经元;2、经15μMTat-Scr处理3小时的DIV1的皮层神经元;3、经5μM Tat-Bec处理3小时的DIV1的皮层神经元;4、经15μM Tat-Bec处理3小时的DIV1的皮层神经元。神经元在SDT缓冲液(2%SDS,0.1MDTT,0.1M Tris/HCl,pH 7.6)中进行总蛋白的抽提。抽提得到的蛋白通过过滤器辅助制样方法(FASP)进行消化〔Wisniewski,J.R.等,Universal sample preparation method forproteome analysis,Nat Methods,2009,6(5):p.359-62.〕。消化产物通过nano-emitter分离柱(15cm长,75μM内径,包含3μM C18ReproSil颗粒)进行分离,并用于之后的质谱分析(采用电喷雾离子源,电压为1.7-2.2kV)。肽段通过4%到26%的线性梯度缓冲液以250nL/min的速率进行分离,离子强度前20位的离子被依次分离进入MS/MS测序。测序获得的原始数据利用MaxQuant software(Version 1.5.2.8)进行分析处理,得到的峰数据在Uniprot数据库中搜索相应的蛋白名称。蛋白鉴定的阈值参数设定为20ppm或0.5Da;FDR设定为1%;最小可接受的肽段长度设定为7个氨基酸。鉴定得到的所有蛋白表达数据通过β-actin进行标准化处理。对比经标准化处理后的蛋白表达值,我们通过如下标准选定目标蛋白:1、蛋白在Tat-Scr组和Blank组之间的表达变化<1.2倍;2、蛋白在Tat-Bec组的表达值小于Tat-Scr组;3、蛋白在15μM Tat-Bec组的表达值小于5μM Tat-Bec组。
(八)电镜研究
体外培养1天的大鼠皮层神经元、成年小鼠脊髓或坐骨神经在0.1M PBS溶液(pH7.4)中配制的2.5%戊二醛固定过夜。固定后的样品用1%四氧化锇后固定30分钟,用0.1MPB洗三遍,在梯度乙醇中脱水,最后在环氧树脂中包埋2天。样品经过甲醇醋酸双氧铀和柠檬酸铅染色后在Joel JEM-1230透射电镜下进行观察。
对SCG10的亚细胞定位的观察运用了免疫电镜。用固定液(4%多聚甲醛、0.1%苦味酸和0.05%戊二醛配制在0.1M PB中)在4℃下固定培养的神经元2小时。固定后的样品切片到200μm薄片,并与1%氧化锇共孵育30分钟。孵育后样品进行脱水处理并在Epon 812中包埋,在37℃下聚合12小时,45℃下聚合12小时,60℃下聚合24小时。之后对样品进行超薄切片,利用200目镍网收集样品,用SCG抗体(1:100)在4℃下孵育48小时,用PB洗样品(10分钟/次×10次),再与12-nm金颗粒(1:100,Jackson Immuno Research)偶联的二抗在室温下共孵育2小时。孵育后用PB洗样品(10分钟/次×10次),利用甲醇醋酸双氧铀和柠檬酸铅对样品进行染色,之后通过Joel JEM-1230透射电镜进行观察。
(九)动物行为分析
通过Rotarod和Gridwalk检测SCI后1、3、7、14、21、28天小鼠的运动协调性。Rotarod测试以8分钟内从5rpm加速到80rpm的速度进行。小鼠在手术前一周在Rotarod上适应训练3天,每天练习3-5次,取小鼠在转轮上停留直到落下的平均时间作为损伤前的对照。以损伤后的停留时间/损伤前的停留时间的比值作为Rotarod的恢复指标。
Gridwalk测试中,小鼠在全程摄像情况下,在长宽为52cm×32cm的网格(间隙为2.5cm)上自由行走2分钟,通过录像回放,以单盲方式,手动记录小鼠四肢跌落网格的次数。统计时,换算成单位行走距离中跌落次数。
步态分析实验中,SCI后8周的小鼠在CatWalk(Noldus)系统上分析步态。记录的脚印由系统自带的分析软件进行分析,计算双后肢的步距以及协调性指数。
(十)统计分析
所有结果都以平均值±SEM表示。两组之间的显著性分析采用two-tailedunpaired Student's t-test,多组之间的显著性分析采用One-way ANOVA和post hocTurkey's test。所有的统计分析都采用Windows下的GraphPad Prism5软件进行。
二、实验结果
1、诱导自噬能够促进神经元突起生长
前期实验中,我们通过western检测中枢和外周神经受损后自噬水平的变化。我们发现再生能力较强的外周神经元受损后自噬水平出现明显升高。而与之相反,再生能力较弱的中枢神经受损后自噬水平没有明显变化。自噬水平与再生能力的这种正相关性促使我们进一步研究在中枢神经受损情况下,诱导自噬是否能够提高其轴突的再生能力。
首先,我们在体外培养的皮层神经元中验证自噬诱导多肽Tat-beclin 1(Tat-Bec)的作用。我们采用beclin1的随机扰乱序列的多肽Tat-scrambled(Tat-Scr)作为对照。为了避免过度诱导自噬引起对细胞的毒害作用,我们优化了Tat-Bec的使用浓度。如图1(A)所示,在体外培养1天(DIV1)的皮层神经元中,5μM的Tat-Bec处理3h后,细胞胞体和突起中都有较多的自噬体(autophagosome)和自噬溶酶体(autolysosome),而对照组中则很少。同时,我们观察到Tat-Bec处理组相比对照组,LC3-II(自噬体的marker)的水平显著增加,而p62(自噬的底物)的水平显著降低(图1,B)。有趣的是,Tat-Bec处理3h可以促进神经元突起的生长(图1,C和E),同时也能对抗髓磷脂对突起生长的抑制作用(图1,D和F)。我们又分别用雷帕霉素(Rapa)和亚精胺(Spd)这两种常用的自噬诱导剂代替Tat-Bec进行实验,同样也能够显著缓解髓磷脂对突起生长的抑制作用(图1,D和F)。据此,我们认为,提高自噬水平可以增强神经元突起的生长能力。
2、诱导自噬能够稳定神经元的微管
细胞骨架的重构是轴突生长和修复的基础。已有研究发现受损的CNS轴突含有许多回撤小泡,这与微管的紊乱以及自噬相关蛋白有关。我们试图弄清神经元中自噬与微管稳定性之间的关系。根据文献,我们用乙酰化/酪氨酸化α-微管蛋白的比值(A/T比)来表示稳定态和不稳定态微管的比例。在微管去稳定药物诺考达唑(NCDZ)的作用下,神经元稳定态的微管解聚。然而经过Tat-Bec处理后,我们发现相比对照组,稳定态的微管保存得更好,A/T比显著增加(图2,A和B)。用雷帕霉素或亚精胺处理神经元时,我们同样观察到了A/T比增加的现象(图2,A和B)。这些结果说明诱导自噬稳定了神经元细胞的微管。我们使神经元过表达GPF-EB3(偶联GFP的微管正向末端结合蛋白EB3),做Time-lapse分析,发现Tat-Bec的处理能够促进微管聚合,并向细胞外周分布,延长到突触的远端(图2,C-E)。这些结果证明神经元中诱导自噬能够增加微管的稳定性。
3、自噬通过降解微管解聚蛋白SCG10稳定微管
我们进一步希望探寻自噬稳定微管的分子机制。微管的稳定性与动态性是由多种微管辅助蛋白共同调节的,其中有与微管蛋白结合从而稳定微管的微管相关蛋白(MAPs),也有使微管去稳定化的蛋白,包括能与游离的微管蛋白结合阻碍微管组装的stathmin蛋白家族,以及能促进微管解聚的kinesin-13家族蛋白等。为了寻找自噬的底物,我们将DIV1的皮层神经元用Tat-Bec处理后,进行蛋白质质谱分析。我们从2232个有效蛋白中发现,有219个蛋白在Tat-Bec处理后显著下降。进一步通过Gene Ontology数据库对这些蛋白进行功能分析,我们发现19个蛋白具有与微管调节相关的功能。这19个蛋白中包括kinesin-13家族蛋白KIF2A、神经元特异的stathmin家族蛋白Stathmin-2(也称为上颈神经节蛋白10,即SCG10(Superior Cervical Ganglia protein 10))。接下来,我们用Western blot的方法验证质谱的结果,发现SCG10在Tat-Bec处理的细胞中发生明显下降(图3,A和B)。然而,MAP2和Tau这两种主要的神经元表达的MAPs以及Stathmin-1和KIF2A的表达水平没有显著变化(图3,A和B),提示我们SCG10受到自噬的降解。我们利用免疫电镜分析SCG10的细胞定位,发现经过Tat-Bec处理后,神经元上的SCG10被募集到自噬体和自噬溶酶体上,同时胞质中的SCG10减少(图3,C和D)。这些结果显示SCG10是神经元中特异的自噬底物。我们接着试图通过调控SCG10水平来研究其在神经元微管动态调节中的功能。在体外培养的神经元中过表达SCG10(箭头所指GFP阳性的为过表达的细胞),A/T比显著降低(图3,E和F)。而用Tat-Bec处理后,A/T比升高(图3,E和F),表明Tat-Bec可以挽救由于过表达SCG10造成的微管不稳定。这些结果提示,诱导自噬可以有效清除神经元内源和外源表达的SCG10。
4、Tat-Bec处理减少轴突损伤后的退化回撤
之前的研究表明,受损的CNS轴突会形成回撤小泡(RBs),其中含有分散紊乱的微管,同时发现自噬相关蛋白与RBs相关联。在上述研究中发现诱导自噬可调控微管稳定性的结果,使我们进一步去研究自噬在轴突受损后的作用。
我们利用脊髓损伤(SCI)的小鼠为模型研究Tat-Bec的作用。首先,我们采用成年的Thy1-YFP-M转基因小鼠〔Feng,G.等,Imaging neuronal subsets in transgenic miceexpressing multiple spectral variants of GFP,Neuron,2000,28(1):p.41-51〕来观察背根神经节(DRG)在脊髓中上行的轴突。在脊髓颈段C4-C5单侧半横切后,利用双光子显微镜观察受损后轴突的形态变化。在轴突损伤后,立即用Tat-Bec(3μM,配于人工脑脊液中)处理1h,之后用人工脑脊液清洗,并持续观察5h(图4,A)。与前人的研究结果一致〔Erturk,A.等,Disorganized microtubules underlie the formation of retraction bulbs andthe failure of axonal regeneration,J Neurosci,2007,27(34):p.9169-80〕,我们在受损的轴突上观察到明显的RBs的存在,而受损的轴突也逐渐回撤(图4,A,Tat-Scr)。有趣的是,相比对照组,Tat-Bec处理组中出现RBs的轴突比例明显减少(图4,B),同时也减少了轴突回撤距离(图4,C)。
进一步地,我们利用Emx1-Cre;Ai9转基因小鼠的皮质脊髓束(CST)表达tdTomato〔Bareyre,F.M.等,Transgenic labeling of the corticospinal tract for monitoringaxonal responses to spinal cord injury,Nat Med,2005,11(12):p.1355-60〕,即CST有自发红色荧光标记来研究CST的回撤。我们同样发现Tat-Bec处理能够在SCI后28天明显减弱轴突的回撤(图4,D和E)。因此,这些结果显示Tat-Bec处理能够有效防止损伤引起的轴突退化。
我们也分析了SCI后,CST中微管的变化。免疫组化结果显示,相比对照组,Tat-Bec处理后的CST中,标记聚合状态微管的去酪氨酸化α-微管蛋白(Glu-tub)得到较好的保留(图4,G和H)。透射电镜结果显示,相比起对照组中微管的无序排列,在Tat-Bec处理组中,微管大多数呈现平行排列(图4,I),且这些微管与轴突轴的偏差角较对照组微管明显减小(图4,J)。同时,我们在Tat-Bec处理的小鼠轴突CST上观察到许多自噬体的聚集(图4,I,箭头所示)。这些结果进一步支持了Tat-Bec诱导的自噬具有在受损的轴突上稳定微管作用的结论。
5、Tat-Bec处理促进脊髓损伤后小鼠单胺能神经的轴突再生和运动能力的恢复
在CST以外,缝合脊髓束和红核脊髓束等其他的下行通路同样对SCI之后的运动能力恢复有帮助。因此,在脊髓C4-C5双侧半横切的小鼠模型中(图5,A),我们通过对5羟色胺(5-HT)的染色对5羟色胺能轴突纤维的再生进行研究(图5,A)。我们发现在SCI之后,立即局部Tat-Bec处理能够显著提高受损位点尾侧5-HT阳性纤维的量(图5,B,C,D,E)。同时,再生的5-HT纤维可以与脊髓腹侧乙酰胆碱转移酶(ChAT)标记的运动神经元形成突触连接(图5,D)。因此,Tat-Bec处理能够在SCI后下行的脊髓神经轴突的再生。
最后,我们测定了Tat-Bec对SCI后小鼠运动功能恢复的作用。我们发现Tat-Bec的处理的小鼠从SCI 1周之后在Rotarod上停留的时间开始明显增加(图5,F);从SCI 3天之后在Grid walk测试上四肢跌落的数量开始明显减少(图5,G)。对SCI 8周之后的小鼠进行CatWalk测试,检测小鼠的运动协调性。发现Tat-Bec处理明显提升了协调指数(RI)以及步伐长度(前脚和后脚)(图5,H和I)。综上所述,Tat-Bec给药促进了SCI小鼠运动功能的恢复。
目前为止自噬在轴突再生中的作用仍然了解得不够清楚。本研究中,我们利用一个特异诱导自噬多肽Tat-Bec,发现诱导自噬可以稳定神经元的微管,且具有显著促进SCI后轴突再生和小鼠运动功能恢复的作用。
逐渐有证据表明微管在轴突自噬体的形成以及运输中发挥重要作用。然而,自噬是否对微管有调节作用仍然不得而知。在我们的研究中,我们发现诱导自噬能够通过降解SCG10这一微管去稳定蛋白从而增加微管的稳定性,并进而促进损伤后的轴突再生。因此,基于自噬对SCG10的降解从而调控微管稳定性,可以进行药物设计。开发特异和靶向作用的自噬诱导剂,具有对轴突受损后修复潜在的应用价值。
Claims (6)
1.一种稳定中枢神经受损神经元的微管,并促进体外培养中枢神经元的突起生长并减弱髓磷脂对突起生长的抑制作用、减少中枢神经轴突损伤后的回撤或回撤小泡形成的方法,其特征在于,所述方法包括使离体神经元与自噬诱导剂接触以诱导自噬的步骤,其中,所述自噬诱导剂选自:SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列,SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列与促进穿膜的肽组成的氨基酸序列,哺乳动物雷帕霉素靶蛋白抑制剂和亚精胺中的一种或多种。
2.如权利要求1所述的方法,其特征在于,所述促进穿膜的肽选自:
RQIKIWFQNRRMKWKK(SEQ ID NO:5);
YGRKKRRQRRR(SEQ ID NO:6);
KQAIPVAK(SEQ ID NO:7);
RRRRNRTRRNRRRVR(SEQ ID NO:8);
寡精氨酸(R9-R12);
KLTRAQRRAAARKNKRNTRGC(SEQ ID NO:9);
ALWKTLLKKVLKAPKKKRKVC(SEQ ID NO:10);
RKKRRQRRR(SEQ ID NO:11);
DAATATRGRSAASRPTERPRAPARSASRPRRPVE(SEQ ID NO:12);
GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO:13);
AGYLLGKINLKALAALAKKIL(SEQ ID NO:14);和
YTAIAWVKAFIRKLRK(SEQ ID NO:15)。
3.如权利要求1所述的方法,其特征在于,所述哺乳动物雷帕霉素靶蛋白抑制剂选自雷帕霉素、KU-0063794和Torin 1。
4.自噬诱导剂在制备药物中的用途,其中,所述药物用于以下用途中的一种或多种:
恢复对象中枢神经受损后的运动能力,和
促进中枢神经受损后轴突再生;
其中,所述自噬诱导剂选自:SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列和SEQ ID NO:1第12-31位氨基酸残基所示的氨基酸序列与促进穿膜的肽组成的氨基酸序列中的一种或多种。
5.如权利要求4所述的用途,其特征在于,所述促进穿膜的肽选自:
RQIKIWFQNRRMKWKK(SEQ ID NO:5);
YGRKKRRQRRR(SEQ ID NO:6);
KQAIPVAK(SEQ ID NO:7);
RRRRNRTRRNRRRVR(SEQ ID NO:8);
寡精氨酸(R9-R12);
KLTRAQRRAAARKNKRNTRGC(SEQ ID NO:9);
ALWKTLLKKVLKAPKKKRKVC(SEQ ID NO:10);
RKKRRQRRR (SEQ ID NO:11);
DAATATRGRSAASRPTERPRAPARSASRPRRPVE(SEQ ID NO:12);
GWTLNSAGYLLGKINLKALAALAKKIL(SEQ ID NO:13);
AGYLLGKINLKALAALAKKIL(SEQ ID NO:14);和
YTAIAWVKAFIRKLRK(SEQ ID NO:15)。
6.如权利要求4-5中任一项所述的用途,其特征在于,所述轴突再生为脊髓下行轴突的再生。
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