CN102076866A - 光学控制第二信使的细胞系、***和方法 - Google Patents
光学控制第二信使的细胞系、***和方法 Download PDFInfo
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Abstract
许多方法、装置和组合物用于光活化分子。一种此类方法是在细胞内产生第二信使。用一个或多个异源受体亚单位{例如,肾上腺素能受体(α1,β2)}修饰表达光反应性嵌合膜蛋白(例如,视紫质)的核苷酸序列。在响应光产生第二信使的细胞内表达光反应性膜蛋白。
Description
相关专利文件
依据35U.S.C.§119(e),本专利文件要求2008/5/29提交的题为“光学控制第二信使的细胞系、***和方法”的美国临时专利申请序列号61/057,108的优先权,所述临时申请通过引用完整纳入本文。
通过引用纳入电子提交材料
通过引用完整纳入随本文一起提交的计算机可读形式核苷酸/氨基酸序列表,具体是:一份在2009/4/29生成的名为“STFD195PCT_ST25.txt”的12,342字节的ASCII(文本)文件。
发明领域
本发明一般涉及响应光学刺激产生第二信使的***和方法,更具体涉及各与响应光产生第二信使有关的细胞系、核苷酸序列、嵌合蛋白以及它们的应用。
背景
鸟嘌呤核苷酸-结合蛋白(G蛋白)被认为能在无活性的鸟苷二磷酸(GDP)状态和有活性的鸟苷三磷酸(GTP)结合状态之间发生改变。这两种状态与细胞内第二信使的释放相关联。释放的第二信使能够调节下游细胞过程。
第二信使包括迅速产生/释放的信号传导分子。这些分子通过激活细胞内的效应蛋白产生细胞反应。细胞信号传导***的例子包括磷酸肌醇体系、环腺苷酸(cAMP)体系和花生四烯酸体系。
称为G蛋白-偶联受体(GPCR)、G蛋白-连接受体(GPLR)、七跨膜结构域受体(7TM受体)或七螺旋受体的蛋白质可触发G蛋白不同状态之间的变化。该蛋白质家族包括各种跨膜受体。这些受体通过激活细胞内的信号转导途径而响应外部刺激(例如,光、神经递质、气味或激素)。具体地说,配体结合并激活转导途径从而造成G蛋白改变状态。GPCR-相关活性与许多疾病有关,因此GPCR是许多药物和治疗的靶点。
据信,市场上超过30%的药物寻靶G-蛋白偶联受体(GPCR),因此其中的大多数涉及第二信使cAMP的产生或抑制。有许多病理过程直接涉及cAMP,包括神经生理学疾病、内分泌学疾病、心脏疾病、代谢疾病和免疫疾病。在研究哺乳动物复杂行为时,技术局限性导致不能在时空上精确控制胞内信号传导过程。调节第二信使水平,如cAMP水平的现有化学方法起效较慢,导致不能在第一时间研究与某些组织如如神经或心脏组织有关的机体的活性。这些化学方法通常缺乏探测这些快速时间量程(例如,在筛选新疗法时)的速度。
概述
本发明涉及克服上述挑战,还涉及第二信使的产生以及有关成像装置和它们的应用。本发明有许多实例和应用,其中一些概述在下文中。
与本发明的一个实施方式一致,一种方法用于在细胞内产生第二信使。用一个或多个异源受体亚单位{例如,肾上腺素能受体(α1,β2)}修饰表达光反应性嵌合膜蛋白(例如,视紫质)的核苷酸序列。光反应性膜蛋白在细胞内表达以便响应光产生第二信使。
与本发明的一个实施方式一致,一种方法用于评价涉及胞内信使相关的假定治疗方案(例如,药物或电刺激或经由这些第二信使发挥作用的任何其他物质)的效果。用一个或多个异源受体亚单位{例如,肾上腺素能受体(α1,β2)}修饰表达光反应性嵌合膜蛋白(例如,视紫质)的核苷酸序列。光反应性膜蛋白在细胞内表达以便响应光产生第二信使。将所述蛋白暴露于光。评价治疗效果。
本发明的实施方式涉及表达具有一个或多个异源受体亚单位{例如,肾上腺素能受体(α1,β2)}的光反应性嵌合膜蛋白(视紫质)的细胞。
本发明的实施方式涉及表达具有一个或多个异源受体亚单位{例如,肾上腺素能受体(α1,β2)}的光反应性嵌合膜蛋白(视紫质)的核苷酸序列。
上述本发明的概述不是要描述本发明的每个示例性实施方式或每个应用。下面的附图和详细描述更具体阐述了这些实施方式。
附图简述
结合附图,通过本发明各个实施方式的详细描述可以更全面地理解本发明,图中:
图1A是与本发明的示例性实施方式一致的optoGs和optoGq的示意图;
图1B是与本发明的示例性实施方式一致的未被转染或者用optoGs或optoGq转染的细胞的cAMP、cGMP和IP1的酶联免疫吸附试验(ELISA)示意图;
图1C是与本发明的示例性实施方式一致的被optoGs和optoGq的mCherry融合蛋白转染的细胞的Ca-成像示意图;
图2是与本发明的示例性实施方式一致的被optoGs和optoGq的mCherry融合蛋白转染的细胞的Ca-成像示意图;
图3A是与本发明的示例性实施方式一致的表达各种构建物的HEK细胞的cAMP、IP1和IP3水平的示意图;
图3B是与本发明的示例性实施方式一致的慢病毒表达载体,opto-α1AR-表达细胞的GAD免疫染色,以及光刺激10分钟后在optoXR-表达细胞(mCherry+)中观察到的pCREB活化;
图4A是与本发明的示例性实施方式一致的转导的伏核(accumbens)的光电极(optrode)寻靶,波峰波形以及所示构建物的基线代谢率;
图4B是与本发明的示例性实施方式一致的用光刺激记录的体内光电极示意图;
图4C是与本发明的示例性实施方式一致的波峰频率与光和基线的变化情况的示意图;
图4D是与本发明的示例性实施方式一致的代谢率变化(firing rate change)动力学的示意图;
图5A是与本发明的示例性实施方式一致的转导区的立体定向寻靶(stereotactic targeting),植入了光学纤维的自由移动小鼠,位置偏好装置和试验的示意图以及自由移动小鼠的轨迹;
图5B是与本发明的示例性实施方式一致的对照和opto-α1AR的偏好的示意图;和
图5C是与本发明的示例性实施方式一致的各种旷场试验的总距离结果的示意图。
尽管本发明可包括各种修饰和改变形式,但其具体形式已在附图中通过举例形式列出,并将在下文中详细描述。然而,应理解,这并不是要将本发明限于所述具体实施方式。相反,本发明包括落入本发明精神和范围内的所有修饰、等价形式和改变。
发明详述
本发明被认为可用于各种光学***和方法的实际应用,并且发现本发明特别适用于对细胞内第二信使的水平进行光学控制的***和方法。由于本发明不必局限于这些应用,因此通过对本文中各种实施例的讨论可以了解本发明的各个方面。
本发明的实施方式涉及通过在细胞内释放第二信使而响应光学刺激的嵌合膜蛋白。在具体例子中,所述嵌合蛋白是异源受体亚单位与通过光异构化与光起反应而经历构象(变化)并因此被光激活的蛋白的组合。视紫质或亚视黄基(retinylidene)蛋白是可被修饰以包含异源受体亚单位的光反应性蛋白的例子。
根据本发明的实施方式,对据信含有七跨膜α-螺旋结构域的蛋白质进行修饰以包含与第二信使相关的异源受体亚单位。当在细胞膜内表达时,所述蛋白质通过共形(conformal)改变与光发生反应。共形改变引发第二信使的释放/产生。
本发明的实施方式涉及通过在细胞内释放第二信使而响应光学刺激的嵌合膜蛋白的编码核苷酸序列。
本发明的实施方式涉及表达异源嵌合膜蛋白的细胞。所述嵌合膜蛋白通过引发第二信使在细胞内的释放而响应光学刺激。在某些实施方式中,嵌合膜蛋白的表达在体内发生。在其他实施方式中,嵌合膜蛋白的表达在体外发生。
本发明的实施方式可用于通过修饰鸟嘌呤核苷酸-结合蛋白偶联的受体蛋白(GPCR)以包含合适的受体亚单位而产生任何合适的第二信使。
本发明的实施方式可使用响应各种波长和强度的光的蛋白质。
本发明的实施方式涉及使用本文所述的嵌合GPCR蛋白来确定感兴趣的第二信使活性的任何下游效应。
本发明的实施方式涉及在各种细胞类型中表达嵌合GPCR蛋白,所述细胞类型包括但不限于哺乳动物细胞、干细胞、植物细胞以及酵母和大肠杆菌等单细胞生物。
本发明的一个具体实施方式涉及优化表达连接有荧光蛋白以利于目测观察的嵌合蛋白,以及形态的优化应用以研究光诱导的第二信使活性的下游效应。
发明的实施方式涉及本文所述的遗传靶向嵌合GPCR蛋白,涉及在其中表达蛋白质的具体细胞群。存在的细胞类型特异性启动子在靶细胞类型中选择性表达(例如,突触蛋白-1靶向神经元;肌钙蛋白变体靶向心脏组织)。将这些启动子置于表达载体中嵌合GPCR蛋白的上游可使蛋白质在感兴趣的细胞类型内靶向表达。其中包括可诱导、可逆、或其他可控制的启动子***,如Tet-响应、ER-响应和Cre/Lox***。
根据本发明的示例性实施方式,开发了遗传可编码蛋白质,从而,当这些蛋白质在感兴趣细胞类型中表达时就会响应光而产生环腺苷酸(cAMP)。例如,这可用于目测观察细胞生理学的下游效应,包括但不限于药物筛选。其他实施方式采用导致响应光而释放第二信使的嵌合和异源GPCR。示例性的第二信使包括cAMP、环鸟苷酸(cGMP)、肌醇三磷酸/肌醇1,4,5-三磷酸/三磷酸肌醇(IP3)和花生四烯酸。
与本发明的实施方式一致,一种方法被用于评价与胞内形式相关的假定治疗方案(例如,药物或电刺激或通过这些第二信使发挥作用的任何其他物质)的功效。用一个或多个异源受体亚单位{例如,肾上腺素能受体(α1,β2)}修饰核苷酸序列以表达光反应性嵌合膜蛋白(例如,视紫质)。所述光反应性膜蛋白在细胞内表达以便响应光产生第二信使。所述蛋白质暴露于光。评价治疗效果。
可按照所需刺激曲线施加光。在一种实施方式中,表达的膜蛋白在数十毫秒内响应光。因此,这种刺激曲线可包括一系列快速交替的光脉冲,并可利用例如Ca2+敏感染料监测所得效果。
在一个例子中,首先可刺激细胞而不作处理。一旦给予处理后再次刺激细胞。可比较各次测试的结果以评价治疗效果。
所述处理可包括多种不同方法,其中包括但不限于药物、对细胞的修饰(遗传修饰或其他修饰)、细胞的物理参数(例如,温度变化或电刺激)或施加于生物的治疗方案。
在一种实施方式中,所述处理是光刺激表达的膜蛋白。在该例子中,例如,可通过监测与被治疗疾病相关的症状来监测效果。
在另一实施方式中,所述治疗方案被用作疾病或病症建模的一部分。例如,可使用疾病模型(细胞或动物)并评估背景/基线状态,然后表达蛋白质并评价治疗方案。
实验结果显示,可通过用cAMP-诱导物和cAMP-靶阳离子通道转染细胞并用Ca2+-敏感染料目测观察所得结果来目测观察光引发的cAMP对靶离子通道的调节。第二信使活性的这种遗传可编码的、光活化调节物的组合可用于筛选新的疗法,且其本身就可作为一种新的治疗形式,暗示了cAMP涉及ADHD和心脏通道病变等众多疾病状态。可工程构建该蛋白质用于各种其他第二信使(例如,IP3),通过工程构建视黄醛结合位点或选择具有不同吸收/作用光谱的视紫质或锥形视蛋白的嵌合体用于光活化的其它颜色,以及第二信使的其他下游效应,如钙信号传导和/或激酶活性。
图1A、1B和1C显示了从optoGs和optoGq获得的实验数据,它们是已经开发出来的第二信使信号传导的光活化诱导物的两个例子(‘optoXR’)。这些光活化诱导物是视紫质/GPCR嵌合体。OptoGq为Gq信号传导提供光反应性控制,而OptoGs为Gs信号传导提供光反应性控制。
在optoGs和optoGq中都显示出在黑暗中基线cAMP和IP3水平的差异可忽略,且与其他第二信使途径如cGMP途径没有交叉。光刺激optoGq时看到的cAMP水平的升高是IP3产生的预期下游效应。
图1A是与本发明的示例性实施方式一致的optoGs和optoGq的示意图。对于每种蛋白,视紫质的胞内环被那些通常偶联于Gs(β2)或Gq(α1)的肾上腺素能蛋白代替。遗传编码序列为在人和鼠细胞中表达最优化。所得序列的例子包括optoGs:Seq.Id.No.1和Seq.Id.No.2;optoGq:Seq.Id No.3和Seq.Id.No 4。
如本领域技术人员已知的,提供的蛋白质的氨基酸序列是支持实施方式的非限制性例子,其范围涵盖提供一致、可互换或等价结果的遗传序列的变化形式(例如,点突变)。
图1B是与本发明的示例性实施方式一致的未被转染或者用optoGs或optoGq转染的细胞的cAMP(上图)、cGMP(中图)和IP1(下图;IP3的降解产物)的酶联免疫吸附试验(ELISA)示意图。如图所示,图1B的结果在从在每个点上用504nm光(波长20nm)刺激1分钟或保持在黑暗中的细胞获得的。
用环境控制倒式培养显微镜(莱卡(Leica)DMI6000B)进行刺激。在cAMP试验中,一些细胞用10uM福司柯林处理30分钟作为试验的饱和、阳性对照。OptoGs响应光而显著提高cAMP水平。在OptoGs作用下未发现cAMP基线水平显著升高,也未发现cGMP或IP3水平有差异。OptoGq响应光而显著提高IP3水平,但未显著改变cGMP水平。cAMP水平和IP3产量的提高被认为是胞内Ca2+释放的共有序列。
图1C是与本发明的示例性实施方式一致的被optoGs和optoGq的mCherry融合蛋白转染的细胞的Ca-成像示意图。为检测cAMP,转染超过optoGs的环核苷酸门控Ca2+通道CNGA2的cAMP-选择性突变体。IP3激活胞内Ca2+储存的释放,从而提供可靠的Gq活化信号。对照群也仅用mCherry转染,存在过量突变体CNGA2。细胞用fura-2(培育20-25分钟)加载,并且每2秒钟在340nm和380nm下暴露2毫秒。在optoGs和optoGq每种情况下,单独照射都足以产生Ca信号,而在对照群中未检测到显著信号。
图1显示了从具体实验装置获得的数据,但本发明不限于此。例如,可采用除转染外的各种递送技术,其中包括但不限于病毒转导、弹道基因递送(基因枪)以及自发核酸摄入。
可修饰基础-视紫质以用于任何合适的异源受体亚单位,如Gi-偶联受体如α2-肾上腺素能受体或多巴胺D2受体或血清素5HT2A受体;或其他Gs-或Gq-偶联受体如多巴胺D1A受体或亲代谢性谷氨酸盐受体。
根据本发明的一个实施方式,所述基础-视紫质是衍生自欧洲牛(Bostaurus)的蛋白质。
根据一个实施方式,也可使用除上述基础-视紫质之外的基础-蛋白质,包括各种7-跨膜蛋白,如锥形视蛋白(红色、绿色或蓝色)、其他物种的视紫质、以及配体-门控受体如多巴胺或血清素受体。
各种实现方式涉及在哺乳动物体内应用。这些实现方式包括,但不限于,测试以及证实神经回路和疾病模式。
图3A和3B显示了在体内应用第二信使信号传导光活化诱导物的两个例子-optoGs(opto-β2AR)和optoGq(opto-α1AR)的实验数据。本发明的一些方面涉及遗传编码光学工具(‘optoXR’)万能家族的应用和开发,所述家族能平衡G-蛋白-偶联受体(GPCR)的一般构效关系,以便以高时空精确度募集和控制受体引发的生物化学信号传导途径。
图3A和3B所示结果涉及两种响应光而选择性募集不同的靶信号传导途径的特定optoXR。在体内,这两种optoXR对伏核的波峰形成具有相反作用,其本身对伏核的精确定时optoXR光刺激足以驱动自由移动小鼠的条件位置偏好。optoXR法能够以可寻靶和时间上精确的方式测试与行为哺乳动物中生化信号传导因果影响有关的假说。
对胞内信号传导的光学控制被用于哺乳动物,采用GPCR的共有构效关系在体内开发和表达具有将信号偶联于效应物的新转导逻辑(transduction logic)的多种不同视蛋白/GPCR2嵌合体。与各种应用一致,工程构建一种或多种嵌合视蛋白-受体蛋白在哺乳动物体内发挥功能,可被特定细胞寻靶,以及响应精确定时的光脉冲。这种方法能够在精确确定且行为相关的时刻使用高速光学刺激(和蛋白质响应)来测试和表征胞内生化事件。一些非限制性的例子包括,脉动与补充调节(tonic modulation),不同调节***之间的同步,以及指定细胞类型在一段时间量程内的其他基础生理与病理过程。
哺乳动物应用已被成功实现。在一个示例性应用中,首先通过比对Gq-偶联人αIa肾上腺素能受体(α1AR)和Gs-偶联仓鼠β2-肾上腺素能受体(β2AR)与Gt-偶联牛视紫质的保守残基,用特定肾上腺素能受体的那些环取代视紫质的胞内环(图1A)。基于结构模型工程改造交换每个受体的胞内区(包括羧基末端结构域)以便从Gt转移G-蛋白偶联,并为在哺乳动物体内表达最优化各受体。用各种配体活化后,天然受体可具有多种总体状态以募集配体-偏好信号传导现象的经典和非经典途径。optoXR在读出灯(sensing light)下可能以生物依赖性方式选择单一活性总体状态。
编码嵌合体(opto-α1AR和optoβ2AR)的基因融合于荧光蛋白。用仅被opto-α1AR转染的HEK细胞(预计通过Gq募集[Ca2+]i)或同时被opto-β2AR(预计通过Gs募集环状AMP)和cAMP-门控Ca2+通道CNGA2-C460W/E583M转染的HEK细胞中的成像[Ca2+]i(胞内钙浓度)确认功能性optoXR表达。参比[Ca2+]i成像证实60秒绿光刺激(504+/-6nm,7mW mm-2)足以驱动显著的optoXR下游[Ca2+]i信号,但对照条件下除外(图2),显示功能性表达。为测试每种optoXR控制的信号传导的特异性,转导的HEK细胞用3mW mm-2504+/-6nm光照射60秒,然后裂解并通过免疫测定分析cGMP、cAMP和IP1(IP3降解产物)的水平。opto-β2AR的预计经典模式对应于其分子设计,即光学刺激在opto-β2AR-表达细胞中导致显著产生cAMP(图3A,上图),类似于野生型β2AR药理学刺激且不募集IP3(图3A,中图)、[Ca2+]i所实现的(图2)或实质上的暗活性。相反,产生的光刺激显著上调opto-α1AR-表达细胞中的IP3信号传导(图3A,中图),类似于野生型α1AR药理学刺激诱导的水平。结合[Ca2+]i评估(图2),这些数据揭示了Gq募集的预期模式,一种在opto-β2AR-表达细胞中未发现的模式(图3A,上图)。光学刺激表达任一构建物的细胞不能调节cGMP水平(图3A,下图),进一步说明了嵌合蛋白的信号传导特异性。类似试验显示,optoXR的作用光谱非常接近天然视紫质,能够整合生物适用的光通量范围内的信号,并且能够激活非-经典途径到与野生型受体类似的程度,如p42/p44-MAPK信号传导。
已经测试了完整神经组织内OptoXR的性能,包括是否需要补充视黄醛辅因子。在该测试中,携带受突触蛋白-I启动子控制的optoXR融合基因的慢病毒载体(为了将生化调节靶向局部神经元而非其他潜在的Gs/Gq-反应性细胞组织元件,如神经胶质和内皮细胞;图3B,上左)被立体定位注入成年小鼠的伏核。这种方法的目标是用伏核内的躯体树突区室对神经元进行生化调节(~95%GABA能中型棘神经元,无进一步的亚型特异性;图3B,左图),并且,由于这些慢病毒属不能通过轴突转导细胞,从而排除通道纤维或传入突触前末稍。转导2周后,在人造脑脊液内制备伏核的急性冠状切片(acute coronal slice),光学刺激10分钟,立即固定并用Ser 133-磷酸化CREB(pCREB,cAMP和Ca2+-偶联信号传导级联的生化整合物)染色。在没有补充外源类视色素时,在表达optoXR的细胞群内观察到pCREB显著升高(图3B,右图),但在未光照组织内无此现象。
用靶向转导的伏核的光电极通过记录多单位体内神经元开通(multi-unit in vivo neuronal firing)来确定optoXR激活对伏核局部电活性的功能后果(图4A)。在黑暗中用任一构建物均未观察到基线代谢率有显著差异(图4A,右下)。光学刺激导致表达opto-β2AR的伏核中网络开通降低(图4B左图显示了效应动力学;总结数据分别显示在图4C和4D中),与之前靶向Gs的药理学研究一致。光学刺激增加了表达opto-α1AR的伏核中的开通(图4B右图;图4C,4D)。波峰频率直方图显示,optoXR对代谢率的影响的动力学与生物化学一致,而非信号的电引发(图4D)。这些电生理学数据以及早期生化验证结果都支持optoXR能在体内功能性表达,允许对胞内级联进行差异性光可激活控制,以及调节网络生理学。
在一种应用中,光遗传学(optogenetic)被用来评价精确定时的optoXR刺激调节自由移动小鼠行为的能力。将便携式固态光传送与转基因表达optoXR组合以便以用于操作性行为的时间精确方式对伏核神经元内的胞内信号传导进行光学控制(图5A)。共焦分析显示表达局限于局部伏核;尤其在传入纤维中、在伏核的远端突出区域、在神经胶质内或在周围区域内未观察到标记。作为三天操作性条件化位置偏好试验的一部分,光学刺激靶向转导的伏核(图5A)。在该测试的每一天让动物自由探索位置偏好装置(图5A,下图)。在第1天,动物自由探索该装置而不作光学刺激。在第2天,一旦动物自由进入指定的条件室,安装在转导区的激光二极管-偶联光学纤维传递10Hz的光脉冲以接近强奖励期间单胺能输入类似的强度。路径追踪显示,柔性光学纤维法能够完全地不受障碍地探索整个室(图5A,下图)。在第3天再让动物自由探索没有光刺激的装置,通过两个独立的不知情记录员量化动物花费在条件室内的时间。显然,表达opto-α1AR的动物在光学刺激后对装置的条件化侧有强烈偏好(图5B)。这种时间上精确的生化调节效果在两次独立opto-α1AR动物测试中是可重现的(每次测试斯氏t检验为:n=5-6,P<0.05,对条件室中的时间;n=11,P<0.01,对所有群体),而其他视蛋白基因,opto-β2AR和ChR2,显示出较少驱使偏好效应。opto-α1AR对伏核的刺激效应特异于奖励相关行为,并且不能延伸至直接调节焦虑-相关行为或运动行为,因为在旷场测试中输送到相同动物组的相同光学刺激显示对朝墙壁的移动距离或偏好没有显著影响(图5C)。
现在将描述与上述实验一致的一个具体的非限制性实施方式。体内记录和分析采用光电极,其由与记录电极(1MV钨,A-M***(A-M Systems))偶联的直径200mm的多模光学纤维(托尔实验室(Thor labs))构成,降低200-400mm的电极/纤维端点到端点的距离从而进入小鼠转导的伏核(电极端点低于前卤4.8-5.2mm),小鼠被放置在立体定向框架内(DK仪器公司(David Kopf Instruments))并用异氟醚麻醉。473nm二极管激光器(CL公司(CrystaLaser))的光经纤维传送。使电信号通过带通滤波器并放大(0.3-1kHz,1800微电极交流放大器,A-M***),并用pClamp 10.0(分子装置公司(Molecular Devices))分析。通过阈值检测波峰,并由检测人员独立验证。
采用光刺激进行行为分析,通过偶联于473nm蓝色二极管激光器(CL公司)的光学纤维(直径200mm,托尔实验室)施加光刺激,并用套管寻靶伏核(距离端点0-100mm)登记。通过函数发生器(Agilent 33220A)给optoXR输送脉冲宽度为50ms的光。在标准装置(SD仪器公司(SD Instruments))内进行位置偏好(测试),移开两室之间的墙壁以允许自由探索。采用MATLAB(Mathworks)运行定制的计数轨迹(tallying script),由两名独立的不知情的观察者根据录像分析数据,以了解动物花费在每个室上的时间量。对于旷场测试,将动物放在40340cm的方形旷场内;用与位置偏好实验相同的参数输送光刺激。采用自动软件(Viewpoint)分析录像,以了解在中间15315cm方形区域以及外部环状区域(场地的其余部分)内的总时间和总距离。
用双尾斯氏t检验(用Microsoft Excel计算)或单向ANOVA加Tukey后验测试(GraphPad Prism)进行上述统计分析。所有归纳性柱状图用平均值+/-s.e.m.表示,显著性如下:*P<0.05,**P<0.01,***P<0.001。
支持本发明各种实施方式的令人惊讶的结果和功效的进一步细节见《时间上精确体内控制胞内信号传导》(Temporally precise in vivo control of in vivo signalling),Raag D.Airan等,Nature 458,1025-1029(2009/4/23),将其通过引用完整纳入本文。
下面的内容详细描述了与本发明实施方式一致的具体非限制性方法。该方法的诸多变化也包含在本发明范围之内。
载体构建
合成opto-α1AR和opto-β2AR的哺乳动物密码子最佳序列(图1A中的氨基酸序列),将其克隆入pcDNA3.1,并用NotI位点与mCherry或YFP(删除其起始密码子)的N-末端融合。optoXR和mCherry/YFP之间的接头是5’GCGGCCGCC 3’。通过将每种optoXR mCherry的转基因克隆入pLenti突触蛋白I hChR2 mCherry WPRE载体的AgeI和EcoRI位点来构建含有突触蛋白IoptoXR mCherry的慢病毒载体。
慢病毒制造
制造了高效价慢病毒。简言之,HEK 293FT细胞在4-层细胞工厂(Nunc)内用含10%FBS的DMEM平铺培养至90%汇合。细胞用690μg上述慢病毒载体和两种辅助质粒(690μg pΔCMVR8.74和460μg pMD2.G)共转染。转染15小时后更换培养基。转染24小时后将培养基更换成200-220mL含5mM丁酸钠的无血清UltraCULTURE(坎布莱克斯公司(Cambrex))。在转染后40小时将现在含有病毒的培养上清液以1000rpm离心5分钟以除去细胞碎片,然后用0.45μm低蛋白结合抽滤瓶过滤。澄清的上清液然后用SW 28转子(贝克曼公司(Beckman))以55,000g超滤2小时以沉淀病毒。离心之后弃去上清液并将所得病毒沉淀溶于总共100μL冷的(4℃)PBS。将重悬后的病毒以7000rpm离心5分钟以除去剩余细胞和病毒碎片。将等分样品于-80℃冷冻直至使用。
动物外科学和行为学
按照斯坦福大学的实验脊椎动物手册的规定关养和处理10-12周龄的雌性C57BL/6小鼠。如下将病毒溶液输送到右伏核。用异氟醚麻醉动物并剪除头顶的毛。在异氟醚麻醉下将动物的头放入立体定向框架(DK仪器公司)。在头皮正中线上切一切口并在前卤前1.10mm、侧1.45mm处在颅骨上钻一个直径约1mm的孔。然后将预先加入病毒的斜角33号针(NanoFil,WP仪器公司(World Precision Instruments))***伏核下部(针尖距离前卤腹侧4.70-4.80mm)并用自动注射泵(NanoFil,WP仪器公司)以100nL/分的速度注射1.0μL病毒。注射后让组织放松以及让液体扩散3-5分钟,然后取出针头。对进行急性切片或体内记录实验的动物,用牙粘固粉(朗氏牙科(Lang Dental))填充开颅部分并用VetBond(3M公司)封闭切口。对进行行为分析的动物,插管(C316G,在基座下切开4.5mm;PlasticsOne)和基座一起放入头盖骨。插管用Metabond(帕卡公司(Parkell))和牙粘固粉(朗氏牙科)固定。将VetBond或牙粘固粉干燥,然后将动物从框架中取出,使其恢复至少1周,再进行操作。对行为实验的对照动物进行与试验动物相同的操作(手术、植入插管、光刺激),但仅注射载体(PBS)代替病毒。在位置偏好实验中,研究采用了对任一侧室(>70%或<10%)或对中间室(>40%)均未显示出基线偏好的动物;超过90%的动物满足关于非偏好、平衡位置偏好设计的标准。
急性切片制备
动物用异氟醚麻醉并用外科剪刀(精细科学工具公司(Fine Science Tools))断头。切下275μm厚的包含伏核的冠状切片并储存于含有64mM NaCl、2.5mM KCl、1.25mM NaH2PO4、25mM NaHCO3、10mM葡萄糖、120mM蔗糖、0.5mM CaCl2和7mM MgCl2的切割溶液(用95%O2/5%CO2平衡)。切片之后将切片在切片溶液内32-35℃培育30分钟,然后室温培育直至实验。离体optoXR刺激时将切片放在直立式显微镜(BX51W,奥林巴斯(Olympus))的载物台上并用含有124mM NaCl、3mM KCl、1.25mM NaH2PO4、26mM NaHCO3、10mM葡萄糖、2.4mM CaCl2和1.3mM MgCl2的人工脑脊液(用95%O2/5%CO2平衡)灌注。使300W Lambda DG-4(苏特公司(Sutter))产生的光通过473nm±20nm带通滤波器(萨姆洛克公司(Semrock))并施加于切片,采用4X目镜(0.28NA),照射10分钟,然后立即固定进行随后的分析。
信号传导确认试验
HEK293FT细胞(英杰公司(Invitrogen))在24孔板内用脂转染胺2000(英杰公司)转染,转染4-6小时后更换成无血清培养基。为进行Ca2+成像,将细胞铺放在基质胶涂覆的盖玻片上,并加入配制在Tyrode(含1μM ATR)中的F-127普流尼克/DMSO(探针公司(Probes))中的5μg/ml fura-2AM,在37℃和5%CO2气氛中培育20-25分钟。加载后将盖玻片在奥林巴斯BX51W上340nm/380nm成像,采用Metafluor(艾克森仪器公司(Axon Instruments))控制300W Lambda DG-4(苏特公司)。为进行免疫测定,转染18-24小时后加入1μM ATR和50mM LiCl(以防止IP1降解)并将平板转移至环境控制显微镜(莱卡DMI6000;37℃,5%CO2)。每次光学刺激每个孔中的5个区域(苏特300W Lambda DG-4;萨姆洛克504/12nm带通滤波器;10X 0.30NA目镜);3孔/条件。培育(cAMP/cGMP:20分钟;IP1:1小时),之后裂解细胞并通过HTRF(CB公司(CisBio))分析,用Biotek Synergy4读数。
免疫组织化学和共焦分析
体内刺激结束之后小鼠用冰冷却的4%低聚甲醛(PFA)的PBS(pH 7.4)溶液经心脏灌注90分钟。取出大脑并用4%PFA固定过夜,然后用30%蔗糖的PBS溶液平衡。在冷冻切片机上切下40μm厚的冠状切片,储存于4℃的冷冻保护剂直至进行免疫组织化学处理。自由流动切片用PBS洗涤,然后在0.3%Tx100和3%标准驴血清(NDS)中培育30分钟。为进行急性切片实验,刺激后立即将275μm厚切片在冰冷却的4%PFA中固定1小时并用0.5%Tx100和3%NDS培育。为进行MAPK实验,HEK293细胞刺激后立即将盖玻片固定15分钟,用0.6%H2O2培育,然后用3%NDS配制的0.1%Tx100渗透化处理。在0.01%Tx100和3%NDS中与第一抗体培育过夜:小鼠抗-GAD67 1∶500(马萨诸塞州比尔里卡的密里博公司(Millipore,Billerica,MA));大鼠抗-cfos 1∶500(加利福尼亚州圣迭戈的卡尔生化公司(Calbiochem,San Diego,CA));大鼠抗-磷酸-CREB Ser133 1∶500(密里博公司)。洗涤切片并与偶联于FITC或Cy5(宾夕法尼亚州西格罗夫的杰克逊实验室(Jackson Laboratories,West Grove,PA))的第二抗体(1∶1000)室温培育3小时。与DAPI(1∶50,000)培育20分钟后洗涤切片并用PVD-DABCO固定在显微镜载玻片上。其余与第一抗体(兔抗-磷酸Erk1/2;抗-磷酸-MAPK p38 1∶500(威斯康星州麦迪逊的普利马公司(Promega,Madison,WI));小鼠单克隆抗-多巴胺D1受体1∶50(开米肯公司(Chemicon));兔多克隆抗-多巴胺D2受体1∶50(密里博公司);山羊多克隆抗-胆碱乙酰基转移酶1∶200(密里博公司))培育过夜,然后与生物素化第二抗体(1∶500,杰克逊实验室)培育,用链霉抗生物素蛋白-生物素-辣根过氧化物酶(ABC试剂盒(加利福尼亚州伯林格姆的载体实验室(Vector Labs,Burlingame,CA)))处理,并按照制造商的说明进行TSA检测(康涅狄格州舍尔顿的帕金埃尔默公司(Perkin Elmer,Shelton,CT))。
用20X/0.70NA或40X/1.25NA油镜在莱卡TCS SP5扫描激光显微镜上获得共焦荧光图像。在插管通道下500μm区域内获得每种条件的4张连续叠加图像。采用Volocity(Improvision)软件,用DAPI染色来描绘伏核以确定cfos或pCREB免疫反应性的平均像素强度。阳性或pCREB-活性细胞通过强度阈确定,获取图像并在不知晓实验条件的情况下进行分析。
表S1
数据的pCREB强度原始数值(au)列在图3B中。每个亚组的平均值和SEM用黑体表示;各亚组相比对照的双尾和t检验p-值用斜体表示。
表S2
数据的基线代谢率(Hz)的原始数值列在图4A中。每个亚组的平均值和SEM用黑体表示;各亚组相比对照的t检验p-值用斜体表示。
表S3
数据的代谢率变化(Hz)的原始数值列在图4C中,计算基线代谢率本身的(‘基线’)以及基线和光刺激阶段(‘光照’)之间的。
据此,本发明的实施方式涉及光遗传控制胞内信号传导,以及用于动物行为体内操作时的时间精确性,同时显示非常低的暗活性,而且能募集天然受体下游的多种信号传导分子,藉此在一种技术中集合了其他方法的许多有利方面。类似的实施方式直接探测其他调节物(包括众多神经递质和内分泌激素)引发的七跨膜依赖性信号传导途径的因果显著性。其他实施方式采用optoXR法,这些方式超越可兴奋细胞以利用光学纤维深度寻靶与光遗传靶向光敏感性的万能组合。一种此类实施方式涉及探测不同可兴奋组织内时间上精确生化信号传导的因果显著性。
本发明的实施方式利用了配体偏好信号传导现象,其中各种配体可稳定总体受体构象状态从而使受体的胞内作用偏向于偶联备用转导级联。用optoXR来诱导这些备用级联至药理学操作类似水平(例如,opto-β2AR可诱导与天然配体在野生型β2AR中作用类似的MAPK活性变化);然而,可能不是总能发现个体optoXR能控制导致配体偏好信号传导的所有构象状态。由于哺乳动物组织中存在内源生色团,且在黑暗中的活性尤其低,因此基于视黄醛的工具尤其有用。光遗传可采取连接于快速单组分视黄醛结合模块的不同效应物的形式,利用光学的时间精确性。
本发明的实施方式采用optoXR法补充微生物视蛋白策略,提供了另一种对行为哺乳动物进行快速、可寻靶细胞控制的方法。
与本发明的其他实施方式一致,可使用基于已知视蛋白基因和不同作用光谱的optoXR的波长变化形式。这种optoXR对于提供生化和电学控制的可分离通道尤其有用。
本文所述具体蛋白质序列的变体与本发明的实施方式一致。一些变体与这些蛋白质序列的同源性大于约75%,而其他变体的同源性大于约80%、85%或90%。在一些实施方式中,同源性可高达约93%至约95%或约98%。本发明的组合物包括本文提供的蛋白质和核酸序列,包括与所提供序列的同源性大于约50%的变体,并最高包括100%同源的变体。
本文所述的各种实施方式可与快速回路读出技术组合,以便在正常操作和疾病状态中进行神经回路的高级询问和反向构建。
上述各种实施方式仅作为例子提供,不构成对本发明的限制。基于上述描述和列举,本领域技术人员将了解,可对本发明进行各种修饰和变化而不必严格遵照文中列举和描述的示例性实施方式和应用。例如,这种变化可包括改变制造的第二信使。这种修饰和变化不背离由以下权利要求列出的本发明的真实精神和范围。
Claims (12)
1.一种在细胞内产生第二信使的方法,所述方法包括:
用一个或多个异源受体亚单位修饰表达基于视紫质的光反应性嵌合膜蛋白的核苷酸序列;和
在响应光而产生第二信使的细胞内表达该光反应性膜蛋白。
2.如权利要求1所述的方法,其特征在于,所述一个或多个异源受体亚单位包括肾上腺素能受体。
3.如权利要求1所述的方法,其特征在于,所述基于视紫质的光反应性嵌合膜蛋白是七跨膜蛋白。
4.如权利要求1所述的方法,其特征在于,所述第二信使是cAMP、环鸟苷酸(cGMP)、肌醇三磷酸/肌醇1,4,5-三磷酸/三磷酸肌醇(IP3)和花生四烯酸中的一个。
5.如权利要求1所述的方法,其特征在于,所述表达步骤在体内完成。
6.如权利要求1所述的方法,其特征在于,所述表达步骤在体外完成。
7.如权利要求1所述的方法,还包括光学刺激表达的光反应性膜蛋白的步骤。
8.一种评价涉及胞内信使相关的假定治疗方案的效果的方法,所述方法包括:
用一个或多个异源受体亚单位修饰表达基于视紫质的光反应性嵌合膜蛋白的核苷酸序列;
在响应光而产生第二信使的细胞内表达该光反应性膜蛋白;
将该蛋白暴露于光;和
评价治疗效果。
9.一种表达具有一个或多个异源受体亚单位的基于视紫质的光反应性嵌合膜蛋白的细胞。
10.如权利要求9所述的细胞,其特征在于,所述一个或多个异源受体亚单位是肾上腺素能受体α1和β2中的至少一个。
10.一种表达具有一个或多个异源受体亚单位的基于视紫质的光反应性嵌合膜蛋白的核苷酸序列。
11.一种如Seq.Id.No.1或Seq.Id.No.3及其点突变体所述的核苷酸序列。
12.一种表达如Seq.Id.No.1或Seq.Id.No.3及其点突变体所述的氨基酸的细胞。
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2009
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- 2009-05-29 MY MYPI20105597 patent/MY152003A/en unknown
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- 2009-05-29 EP EP13160448.0A patent/EP2610350B1/en not_active Not-in-force
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- 2009-05-29 WO PCT/US2009/045611 patent/WO2009148946A2/en active Application Filing
- 2009-05-29 KR KR1020107029438A patent/KR20110018924A/ko not_active Application Discontinuation
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- 2009-05-29 CN CN2009801259133A patent/CN102076866A/zh active Pending
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- 2016-09-07 US US15/258,876 patent/US20170081388A1/en not_active Abandoned
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