神经元活性调控TrkB受体细胞膜表面插入机制的研究
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摘要
「研究背景和目的」
神经元突触可塑性对神经环路的建立和控制脑的认知功能及复杂行为具有重要作用。脑源性神经营养因子(BDNF)属于神经营养因子家族的重要成员,能够促进神经元的存活、分化、树突和轴突的生长,尤其在调节突触的可塑性中起关键作用。BDNF参与包括从神经肌肉接头到中枢神经系统在内的突触发育和功能执行;尤其重要的是中枢神经系统的长时程增强(LTP);BDNF对于维持突触间的LTP是必需的。此外,它还参与了早老性痴呆、抑郁症和药物成瘾等神经精神疾病的病理生理过程,有望成为治疗这些疾病的一种手段。
更多证据表明神经元活性能够显著影响BDNF生物学效应的发挥。比如通过NMDA型谷氨酸受体的作用提高神经元活性及增加Ca~(2+)内流对于BDNF调节发育视觉皮层的树突复杂度是必需的。另外,如果没有高钾离子导致去极化、谷氨酸促效剂刺激或提高胞内cAMP浓度这些过程,BDNF就不能发挥促进视网膜神经节神经细胞发育的作用。所以神经元的活性能调节神经元对BDNF的反应,即BDNF能选择性地作用于高活性的神经元。
然而BDNF作为一种易于扩散的分子,它如何能做到优先作用于高活性的神经元或神经突触的?一种可能性是BDNF可以在高活性突触部位局部合成和分泌;另一种是高活性神经元及其突触对于BDNF比非活性状态的神经元有更好的反应性,后者成为是我们研究的聚焦点。
BDNF的功能主要由两类不同的受体介导:p75神经营养素受体及TrkB酪氨酸激酶受体。迄今所有文献报道的BDNF对突触的生物学作用都是由TrkB受体调节的。TrkB是一种高亲和性的酪氨酸激酶受体。BDNF与TrkB受体结合后,TrkB受体胞内域的酪氨酸残基被激活,继而激活MAPK、PI3K和PLC-γ等一系列胞内信号转导通路,介导其生物学效应。中枢神经系统的神经元,其膜表面TrkB受体含量的增加不仅可以增强BDNF的作用,还可参与并提高LTP效应。膜表面TrkB受体的含量只占到细胞TrkB受体总量的一小部分,大量的TrkB受体存在于胞内。当视网膜神经节细胞发生去极化或高频电刺激均可导致神经元的活化及突触传递,增加神经元膜表面TrkB的分布。以上说明神经元活性升高能增加TrkB受体在细胞膜表面的分布。因而BDNF能选择性地对活性较高的神经元尤其突触发挥作用。是哪些分子参与调控神经元活性促进TrkB受体胞内运输以至膜表面聚集的?也就是其分子细胞学调控机制如何尚不清楚!
受体胞内运输与其胞内域和相关分子的结合及调控有关,因此我们通过酵母双杂交及及蛋白质免疫沉淀的方法,发现并证实TrkB受体的胞内域能和carboxypeptidase E(CPE)结合。CPE是羧肽酶的一种,在高尔基体TGN中CPE具有分选受体的作用,可分选目的蛋白:如BDNF、POMC等分泌性蛋白进入可调性分泌途径,这一过程受胞内蛋白激酶的调控。实验表明缺失C末端4个或15个氨基酸参基的CPE不能参与到RSP中、CPE的敲基因小鼠有肥胖、神经突触传递障碍等表型,这些表型与TrkB基因敲除小鼠的表型有相似性。我们推测CPE介导神经元活性促进TrkB受体插入到细胞膜表面的过程。
总之神经元活性能促进TrkB受体在胞膜表面的分布,但其复杂的分子调控机制还不清楚。本研究旨在深入认识神经元活性对TrkB受体胞内运输调控的分子细胞学机制及其相应的生物学意义,这不仅有助于我们深入认识BDNF作用的机制,也能为今后临床应用BDNF治疗神经精神疾病提供指导。
「方法与结果」
1、TrkB受体细胞膜表面表达水平免疫荧光定量分析方法的建立
本课题首先通过构建FLAG-TrkB-GFP表达质粒建立了“TrkB受体细胞膜表面分布比例的荧光定量分析方法”,该方法能够直接从形态学角度进行不同时空条件下TrkB受体膜表面分布水平的检测,且通过膜表面生物素化法确定了其有效性,从而为获得TrkB受体活性依赖性膜表面插入机制的依据奠定了一定的基础。通过该方法结合膜表面生物素化的方法我们发现LTP的化学性诱导剂—glycin能有效地提高TrkB受体在神经元膜表面的含量(1.82±0.08倍),从而将glycine作为后续研究TrkB受体活性依赖性膜表面插入机制的神经元活性诱导剂—被称为cLTP。
2、神经元细胞膜表面TrkB受体分布比例的测定
为了更加准确地了解不所构建的神经元表达系统中红绿两种荧光强度的比值,利用此参数换算得到在基础状态下神经元膜表面TrkB受体占总体TrkB的比例为34%同条件下TrkB受体在细胞膜上变化的量化指标,我们通过免疫荧光定量分析方法分别测定,证明TrkB在神经元非活性状态下大都存在于胞质内的TrkB受体存储池中,只有在神经元被激活后才会快速聚集并插入到膜表面。通过此定量方法我们发现TrkB受体活性依赖性膜表面插入过程在神经元突起明显多于胞体(1.98倍vs.1.35倍),可以用于解释BDNF突触可塑性等功能的发挥。因此后续实验中采用突起中的荧光强度值定量分析TrkB的膜表面分布水平。
3、TrkB的近膜区是其活性依赖性膜表面插入的充分必要条件
更有意义的是,我们通过构建TrkB的缺失突变体pEGFP-FLAGTrkBΔJM,pEGFP-FLAGTrkBΔTK,pEGFP-FLAGTrkBΔCT即缺失相应胞内区域:近膜区、激酶区和C末端区的突变体质粒并转染入海马神经元,结合前述免疫荧光定量分析方法发现只有表达pEGFP-FLAGTrkBΔJM的海马神经元丧失了活性依赖性TrkB受体胞膜表面插入现象,说明近膜区是TrkB受体活性依赖性向膜表面插入的必要条件;用同样的方法我们又证实神经细胞中天然存在的TrkB的截短突变体T1不具有活性依赖性的膜表面插入行为;在T1受体胞内区末端分别嫁接近膜区、激酶区和C末端区构建pEGFP-FLAGT1JM,pEGFP-FLAGT1TK,pEGFP-FLAGT1CT质粒并转染神经元后发现:只有嫁接了JM区的T1在cLTP发生后出现了膜表面TrkB的明显增加,这一结果说明近膜区是TrkB受体活性依赖性膜表面聚集的充分条件。因此,TrkB的JM区既是其活性依赖性膜表面插入过程的充分条件又是必要条件,在此过程中发挥了重要的结构性作用。
4、Cdk5激酶活性影响TrkB的活性依赖性膜表面插入
为了进一步探讨何种激酶参与了TrkB受体的调节并促进其活性依赖性向膜表面运输的机制,我们采用Cdk5激酶的抑制剂和Cdk5的RNA干扰质粒这两种降低Cdk5作用的方法,同时结合以上免疫荧光分析方法发现Cdk5的激酶活性参与了TrkB的活性依赖性膜表面插入;有报道Cdk5磷酸化TrkB在BDNF促进神经元树突生长中发挥重要作用,将此关键位点:JM区478位的Ser突变为Ala构建点突变体FLAG-TrkBS478A-GFP,或将T1的胞内域嫁接JMS478A构建T1JMS478A,结果是它们均丧失了cLTP活性促进膜表面受体插入的现象;同时Cdk5在cLTP刺激下,其15位Tyr和159位Ser有显著的磷酸化现象。以上均提示Cdk5激酶磷酸化TrkB478位点的酪氨酸对于TrkB活性依赖性膜表面插入过程具有重要的调节作用。另一方面,利用免疫荧光分析方法结合Trk激酶的抑制剂及TrkB酪氨酸激酶活性缺失突变体FLAG-TrkBKD-GFP我们发现TrkB活性依赖性膜表面聚集并不依赖于Trk激酶的活性。
5、cLTP促进TrkB受体明显趋向于聚集到突触后膜
为了明确cLTP导致的TrkB膜表面插入是否有空间分布的差别,通过突触后标志蛋白PSD95标记突触后膜区,结合免疫荧光检测共定位的方法发现:比较非突触后区域,在cLTP刺激后TrkB以大约2倍的差别更多地聚集于突触后区域,而对于近膜区缺失突变体TrkBΔJM并无差异。进而TrkB膜表面含量的免疫荧光定量检测实验证明TrkB以2.13比1.75倍的差别显著地聚集在突触后膜上,同时提示JM区对于此过程也是必需的结构基础。
6、羧肽酶E的C末端能够与TrkB受体胞内区结合
在通过酵母双杂交方法首次发现CPE能够与TrkB受体结合后,构建MYC-CPE的质粒后我们又通过蛋白质免疫共沉淀的方法证明了不论外源转染过表达的蛋白还是内源性表达蛋白,这两种蛋白间都能够结合。除此之外,通过激光共聚焦显微镜技术进一步证实了二者在神经元中具有共定位的特性,即二者存在于共同的亚细胞结构中。因TrkB胞内区是调控其胞内运输的关键区域,分别构建CPE与TrkB的缺失突变体以得到只保留胞内区的FLAG-TrkB IC和只保留胞质侧25个氨基酸的MYC-CPE25AA-RFP,通过蛋白质免疫共沉淀(CO-IP)的方法发现CPE胞内区的25个氨基酸可以与TrkB受体胞内区结合。
7、羧肽酶E参与TrkB受体活性依赖性细胞膜表面插入过程的调节
通过设计和构建CPE的小干扰RNA质粒(siRNA)并通过免疫荧光和Western blot分别检测其干扰效率。通过共转染CPE siRNA与FLAG-TrkB-GFP质粒,结合TrkB膜表面含量的免疫荧光定量分析方法我们发现:CPE的表达被干扰后,cLTP激活的细胞膜表面TrkB受体聚集明显被抑制。这一结果使我们进一步推测CPE很可能参与了TrkB受体的活性依赖性膜表面插入的过程。本课题后续的实验还在进行中。
「结论」
1、所建立的细胞膜表面TrkB受体定量检测的免疫荧光分析方法,可以从空间即细胞的不同区域和时间上定量分析不同条件下TrkB膜表面的变化情况,为后续研究TrkB活性依赖性膜表面插入的机制奠定了基础。
2、定量分析了在非活化的基础状态下海马神经元膜表面TrkB受体占胞内总体TrkB的比例为34%,说明TrkB的在被激活后是从胞内存储池中插入到膜表面的,同时此定量分析更能准确掌握细胞在不同条件下膜表面TrkB受体的变化情况。
3、明确了TrkB近膜区(JM)是其活性依赖性膜表面插入过程的关键调控区域,是此过程的必要和充分条件,并且对于TrkB快速聚集到突触后膜并插入细胞膜也是关键性调控区域。
4、发现Cdk5激酶活性对于cLTP诱导的TrkB膜表面聚集的增加是重要影响因素,Cdk5对于TrkB JM区478位点Tyr的磷酸化参与了TrkB活性依赖性膜表面插入过程。
5、发现cLTP诱导TrkB膜表面插入的增强在突起尤其突触后膜更加明显,这种活性刺激在突触快速调集TrkB受体的现象可以用于解释活性神经元突触对于BDNF作用的高度反应性。
6、首次发现羧肽酶E胞内C-末端的25个氨基酸能够结合TrkB的胞内区;并且初步证明CPE在cLTP诱导的活性依赖性膜表面插入中具有重要的调控作用。
「Background and objective」
Neurotrophins are best known for their ability to promote neuronal survival and differentiation,Brain-derived neurotrophic factor(BDNF) is an important member in neurotrophins family which has a novel function in synapse development and plasticity,particularly in the central nervous system.It suggests that BDNF facilitates both early-phase and late-phase LTP is necessary for LTP.Remarkably,neuronal activity could influence the effectiveness of BDNF,for example,BDNF cannot enhance the survival of retinal ganglion neurons unless depolarized by high K~+ or glutamate agonists,or increased intracellular cAMP.
Previous studies have shown that active neurons/synapses might respond better to BDNF than less active one,a plausible mechanism would be local synthesis and/or secretion of BDNF at the active synapse or active neurons/synapses might respond better to BDNF than less active ones, and this could be achieved by activity dependent control of BDNF signaling. Neuronal activity dependent signaling of BDNF is virtually all attributed to TrkB receptor tyrosine kinase.Neuronal activity could increase the number of TrkB receptors on cell surface or enhance the signaling transduction for BDNF.Depolarization or Electric stimulation has been shown to increase the levels of the TrkB receptor on the cell surface of neurons. Activity-dependent enhancement of the quantity of surface TrkB receptor may define an important mechanism by which the specificity of BDNF activity-dependant modulation is achieved.However the detailed cellular and molecular mechanism underlying this effect is unclear.
For further provide insights into the mechanistic link between activity-dependent and neurotrophic modulation of CNS neurons and synapses,acquire of the intracellular trafficking mechanisms of TrkB is the key research point.For finding out the molecules which could not only interact with TrkB but also assistant with TrkB's intracellular trafficking,we performed Yeast Two-hybrid assay and found TrkB could interact with the carboxypeptidase E,a proneuropeptide/prohormone processing enzyme which is also a sorting receptor associated with BDNF vesicles and other neuropeptide or prohormone targeting into the regulated secretory pathway (RSP).It is proved the C-terminal cytoplasmic tail of CPE functioned as the key region of the membrane binding and sorting.There is also work suggested the 4 or 15-residue sequence in the C-terminal region of CPE is necessary to sorting of CPE to the RSP.Considering for there are some similar characters between TrkB knock out mice and CPE knock out mice, we hypothesize CPE participates in the regulated progress of TrkB activity dependent membrane surface insertion.
Here we employed a dual tag TrkB fluorescence assay to investigate the molecular and cellular mechanisms which regulate the trafficking progress of TrkB activity dependent membrane surface insertion to provided new insights regarding neuronal activity dependent surface delivery of TrkB receptor,which will advance our understanding the role of TrkB in synaptic plasticity modulation.
「Methods and results」
1.Ratiometric fluorescence assay to measure surface TrkB receptor level
To employed the FLAG-TrkB-GFP construct and the "surface FLAG-TrkB-GFP fluorescence ratiometric assay" to monitor the TrkB quick recruitment to the plasma membrane.With this assay to provided temporal, structure and spatial information regarding neuronal activity dependent surface delivery of TrkB receptor,Through this assay we found chemical LTP(glycine) induced the most robust increase(1.82±0.08 fold) in surface TrkB levels and 200μM glycine was then chosen as the stimulation condition in our subsequent studies.
2.Plasma surface TrkB fraction measurement
To measure the percentage of surface TrkB receptor in total TrkB at basal state,we stained FLAG tag of FLAG-TrkB-GFP under permeabilized and non-permeabilized conditions.From plotting the anti-FLAG fluorescence signal in the whole cell region inpermeabilized neurons against the GFP fluorescence signal we got the relationship between the anti-FLAG and the GFP signals by immunofluorescence(IF) is 1.01.With this constant we calculate the fraction of TrkB on hippocampal neurons surface is about 34%.By this assay we found activity dependent cell surface TrkB increase was much more obvious in neuronal processes than in cell body(1.35 vs.1.98 fold) which could be used to understand the mechanisms of BDNF's function in synapse plasticity.
3.Juxtamembrane domain of TrkB is necessary and sufficient for cLTP induced TrkB plasma membrane insertion
To define the potential region in TrkB receptor that is required for its activity-dependent plasma membrane insertion,we generated a series of constructs in which various TrkB domains(juxtamembrane,kinase,and C terminal) were deleted from TrkBFL or different TrkB domains (juxtamembrane,kinase,and C terminal) were grafted to the C-terminal of T1 receptor(a truncated TrkBFL isoform).All the mutant constructs (TrkBΔJM,TrkBΔTK,TrkBΔCT,T1JM,T1TK and T1CT) contain the same FLAG and GFP epitope tag.With the ratiometric fluorescence assay we found only the TrkBΔJM abolished glycine-induced TrkB receptor surface insertion in the deletion constructs and only T1JM shown activity-dependent surface recruitment upon cLTP stimuli in the grafting constructs.It is suggested that TrkB cytoplasmic juxtamembrane region is not only necessary for efficient TrkB surface recruitment but also sufficient to promote rapid insertion of TrkB to plasma membrane upon cLTP stimulation.
4.Cdk5 but not TrkB kinase activity is involved in cLTP induced TrkB plasma membrane insertion
Protein kinases play important roles in neuronal activity and mediating BDNF signaling and biological activity.By the ratiometric fluorescence assay together with selective inhibitor of Cyclin-dependent kinase 5(Cdk5) Roscovitin and Cdk5 siRNA,the data shown the cLTP-induced TrkB surface recruitment was significantly abrogated.Furthermore TrkBS478A (ser478 mutant to alanine) and T1JMS478A neuronal surface level could not increase in response to cLTP stimuli.And cLTPstimuli could lead to an acute Cdk5 phosphorylation at Tyr15 and Ser159 site.It suggested Cdk5 is crucial in activity dependent TrkB plasma membrane insertion.From the results of both pre-treatment of Trk inhibitor K252a and kinase dead(KD) TrkB construct retained the ability to insert into surface in response to glycine stimuli,it suggested tyrosine kinase activity is not required for this behavior.
5.cLTP induced TrkB surface recruitment occurs more efficiently in postsynaptic region
With the ratiometric fluorescence assay combined with transfection with the constructs of PSD-95-mCherry and FLAG-TrkBFL-GFP or FLAG-TrkBΔJM-GFP it present that the neuronal activity could facilitate more TrkBFL receptors trafficking to the postsynaptic region which shown it dramatically insertion into the PSD-95(postsynaptic marker) positive region than in PSD-95 negative region(2.13 vs.1.75 fold),but the TrkBΔJM receptor abolished this process which suggested the JM domain is essential for TrkB's activity-dependent intracellular trafficking.
6.CPE cytoplasmic tail interact with TrkB receptor's intracellular domain
For investigating the molecule regulating TrkB activity-dependent insertion into membrane surface,Yeast Two-hybrid assay and Co-immunoprecipitation(CO-IP) were performed and found TrkB could interact with carboxypeptidase E.This result was confirmed by the images of confocal fluorescent microscope.Established constructs of MYC-CPE25AA-RFP(intracellular domain) and FLAG-TrkB IC,which only has the intracellular domain of CPE or TrkB and further performed CO-IP,it is suggested that c-terminal 25aa of CPE could associate with TrkB intracellular domain.
7.CPE is involved into the regulating of cLTP induced TrkB membrane surface insertion
Design and generate siRNA of CPE together with the ratiometric fluorescence assay to test the activity dependent cell surface TrkB increase was significantly restrained.This result confirmed us cLTP induced TrkB surface insertion is regulated by the biologic function of CPE.
「Conclusion」
1.Established the "Surface TrkB receptor fluorescence ratiometric assay" to investigate the mechanism underlying TrkB plasma membrane recruitment upon chemical LTP(cLTP) stimuli.
2.Calculated and defined the percentage of surface TrkB receptor in total TrkB at basal state is 34%.
3.Revealed the juxtamembrane(JM) domain of TrkB is necessary and sufficient for its activity-dependent plasma membrane insertion.
4.The kinase activity of Cyclin-dependent kinase 5(Cdk5) is crucial to cLTP induced TrkB surface recruitment;and the Tyr478 of TrkB JM domain which could be phosphorylated by Cdk5 is the most key site.
5.cLTP induced TrkB surface recruitment occurs more efficiently on neuronal processes especially at the postsynaptic membrane,which may present a mechanism for rapid enhancement of postsynaptic sensitivity to incoming BDNF signaling.
6.The C-terminal 25aa of Carboxypeptidase E was confirmed associate with the intracellular domain of TrkB and this interaction is crucial in regulating LTP induced TrkB surface recruitment.
神经元突触可塑性对神经环路的建立和控制脑的认知功能及复杂行为具有重要作用。脑源性神经营养因子(BDNF)属于神经营养因子家族的重要成员,能够促进神经元的存活、分化、树突和轴突的生长,尤其在调节突触的可塑性中起关键作用。BDNF参与包括从神经肌肉接头到中枢神经系统在内的突触发育和功能执行;尤其重要的是中枢神经系统的长时程增强(LTP);BDNF对于维持突触间的LTP是必需的。此外,它还参与了早老性痴呆、抑郁症和药物成瘾等神经精神疾病的病理生理过程,有望成为治疗这些疾病的一种手段。
更多证据表明神经元活性能够显著影响BDNF生物学效应的发挥。比如通过NMDA型谷氨酸受体的作用提高神经元活性及增加Ca~(2+)内流对于BDNF调节发育视觉皮层的树突复杂度是必需的。另外,如果没有高钾离子导致去极化、谷氨酸促效剂刺激或提高胞内cAMP浓度这些过程,BDNF就不能发挥促进视网膜神经节神经细胞发育的作用。所以神经元的活性能调节神经元对BDNF的反应,即BDNF能选择性地作用于高活性的神经元。
然而BDNF作为一种易于扩散的分子,它如何能做到优先作用于高活性的神经元或神经突触的?一种可能性是BDNF可以在高活性突触部位局部合成和分泌;另一种是高活性神经元及其突触对于BDNF比非活性状态的神经元有更好的反应性,后者成为是我们研究的聚焦点。
BDNF的功能主要由两类不同的受体介导:p75神经营养素受体及TrkB酪氨酸激酶受体。迄今所有文献报道的BDNF对突触的生物学作用都是由TrkB受体调节的。TrkB是一种高亲和性的酪氨酸激酶受体。BDNF与TrkB受体结合后,TrkB受体胞内域的酪氨酸残基被激活,继而激活MAPK、PI3K和PLC-γ等一系列胞内信号转导通路,介导其生物学效应。中枢神经系统的神经元,其膜表面TrkB受体含量的增加不仅可以增强BDNF的作用,还可参与并提高LTP效应。膜表面TrkB受体的含量只占到细胞TrkB受体总量的一小部分,大量的TrkB受体存在于胞内。当视网膜神经节细胞发生去极化或高频电刺激均可导致神经元的活化及突触传递,增加神经元膜表面TrkB的分布。以上说明神经元活性升高能增加TrkB受体在细胞膜表面的分布。因而BDNF能选择性地对活性较高的神经元尤其突触发挥作用。是哪些分子参与调控神经元活性促进TrkB受体胞内运输以至膜表面聚集的?也就是其分子细胞学调控机制如何尚不清楚!
受体胞内运输与其胞内域和相关分子的结合及调控有关,因此我们通过酵母双杂交及及蛋白质免疫沉淀的方法,发现并证实TrkB受体的胞内域能和carboxypeptidase E(CPE)结合。CPE是羧肽酶的一种,在高尔基体TGN中CPE具有分选受体的作用,可分选目的蛋白:如BDNF、POMC等分泌性蛋白进入可调性分泌途径,这一过程受胞内蛋白激酶的调控。实验表明缺失C末端4个或15个氨基酸参基的CPE不能参与到RSP中、CPE的敲基因小鼠有肥胖、神经突触传递障碍等表型,这些表型与TrkB基因敲除小鼠的表型有相似性。我们推测CPE介导神经元活性促进TrkB受体插入到细胞膜表面的过程。
总之神经元活性能促进TrkB受体在胞膜表面的分布,但其复杂的分子调控机制还不清楚。本研究旨在深入认识神经元活性对TrkB受体胞内运输调控的分子细胞学机制及其相应的生物学意义,这不仅有助于我们深入认识BDNF作用的机制,也能为今后临床应用BDNF治疗神经精神疾病提供指导。
「方法与结果」
1、TrkB受体细胞膜表面表达水平免疫荧光定量分析方法的建立
本课题首先通过构建FLAG-TrkB-GFP表达质粒建立了“TrkB受体细胞膜表面分布比例的荧光定量分析方法”,该方法能够直接从形态学角度进行不同时空条件下TrkB受体膜表面分布水平的检测,且通过膜表面生物素化法确定了其有效性,从而为获得TrkB受体活性依赖性膜表面插入机制的依据奠定了一定的基础。通过该方法结合膜表面生物素化的方法我们发现LTP的化学性诱导剂—glycin能有效地提高TrkB受体在神经元膜表面的含量(1.82±0.08倍),从而将glycine作为后续研究TrkB受体活性依赖性膜表面插入机制的神经元活性诱导剂—被称为cLTP。
2、神经元细胞膜表面TrkB受体分布比例的测定
为了更加准确地了解不所构建的神经元表达系统中红绿两种荧光强度的比值,利用此参数换算得到在基础状态下神经元膜表面TrkB受体占总体TrkB的比例为34%同条件下TrkB受体在细胞膜上变化的量化指标,我们通过免疫荧光定量分析方法分别测定,证明TrkB在神经元非活性状态下大都存在于胞质内的TrkB受体存储池中,只有在神经元被激活后才会快速聚集并插入到膜表面。通过此定量方法我们发现TrkB受体活性依赖性膜表面插入过程在神经元突起明显多于胞体(1.98倍vs.1.35倍),可以用于解释BDNF突触可塑性等功能的发挥。因此后续实验中采用突起中的荧光强度值定量分析TrkB的膜表面分布水平。
3、TrkB的近膜区是其活性依赖性膜表面插入的充分必要条件
更有意义的是,我们通过构建TrkB的缺失突变体pEGFP-FLAGTrkBΔJM,pEGFP-FLAGTrkBΔTK,pEGFP-FLAGTrkBΔCT即缺失相应胞内区域:近膜区、激酶区和C末端区的突变体质粒并转染入海马神经元,结合前述免疫荧光定量分析方法发现只有表达pEGFP-FLAGTrkBΔJM的海马神经元丧失了活性依赖性TrkB受体胞膜表面插入现象,说明近膜区是TrkB受体活性依赖性向膜表面插入的必要条件;用同样的方法我们又证实神经细胞中天然存在的TrkB的截短突变体T1不具有活性依赖性的膜表面插入行为;在T1受体胞内区末端分别嫁接近膜区、激酶区和C末端区构建pEGFP-FLAGT1JM,pEGFP-FLAGT1TK,pEGFP-FLAGT1CT质粒并转染神经元后发现:只有嫁接了JM区的T1在cLTP发生后出现了膜表面TrkB的明显增加,这一结果说明近膜区是TrkB受体活性依赖性膜表面聚集的充分条件。因此,TrkB的JM区既是其活性依赖性膜表面插入过程的充分条件又是必要条件,在此过程中发挥了重要的结构性作用。
4、Cdk5激酶活性影响TrkB的活性依赖性膜表面插入
为了进一步探讨何种激酶参与了TrkB受体的调节并促进其活性依赖性向膜表面运输的机制,我们采用Cdk5激酶的抑制剂和Cdk5的RNA干扰质粒这两种降低Cdk5作用的方法,同时结合以上免疫荧光分析方法发现Cdk5的激酶活性参与了TrkB的活性依赖性膜表面插入;有报道Cdk5磷酸化TrkB在BDNF促进神经元树突生长中发挥重要作用,将此关键位点:JM区478位的Ser突变为Ala构建点突变体FLAG-TrkBS478A-GFP,或将T1的胞内域嫁接JMS478A构建T1JMS478A,结果是它们均丧失了cLTP活性促进膜表面受体插入的现象;同时Cdk5在cLTP刺激下,其15位Tyr和159位Ser有显著的磷酸化现象。以上均提示Cdk5激酶磷酸化TrkB478位点的酪氨酸对于TrkB活性依赖性膜表面插入过程具有重要的调节作用。另一方面,利用免疫荧光分析方法结合Trk激酶的抑制剂及TrkB酪氨酸激酶活性缺失突变体FLAG-TrkBKD-GFP我们发现TrkB活性依赖性膜表面聚集并不依赖于Trk激酶的活性。
5、cLTP促进TrkB受体明显趋向于聚集到突触后膜
为了明确cLTP导致的TrkB膜表面插入是否有空间分布的差别,通过突触后标志蛋白PSD95标记突触后膜区,结合免疫荧光检测共定位的方法发现:比较非突触后区域,在cLTP刺激后TrkB以大约2倍的差别更多地聚集于突触后区域,而对于近膜区缺失突变体TrkBΔJM并无差异。进而TrkB膜表面含量的免疫荧光定量检测实验证明TrkB以2.13比1.75倍的差别显著地聚集在突触后膜上,同时提示JM区对于此过程也是必需的结构基础。
6、羧肽酶E的C末端能够与TrkB受体胞内区结合
在通过酵母双杂交方法首次发现CPE能够与TrkB受体结合后,构建MYC-CPE的质粒后我们又通过蛋白质免疫共沉淀的方法证明了不论外源转染过表达的蛋白还是内源性表达蛋白,这两种蛋白间都能够结合。除此之外,通过激光共聚焦显微镜技术进一步证实了二者在神经元中具有共定位的特性,即二者存在于共同的亚细胞结构中。因TrkB胞内区是调控其胞内运输的关键区域,分别构建CPE与TrkB的缺失突变体以得到只保留胞内区的FLAG-TrkB IC和只保留胞质侧25个氨基酸的MYC-CPE25AA-RFP,通过蛋白质免疫共沉淀(CO-IP)的方法发现CPE胞内区的25个氨基酸可以与TrkB受体胞内区结合。
7、羧肽酶E参与TrkB受体活性依赖性细胞膜表面插入过程的调节
通过设计和构建CPE的小干扰RNA质粒(siRNA)并通过免疫荧光和Western blot分别检测其干扰效率。通过共转染CPE siRNA与FLAG-TrkB-GFP质粒,结合TrkB膜表面含量的免疫荧光定量分析方法我们发现:CPE的表达被干扰后,cLTP激活的细胞膜表面TrkB受体聚集明显被抑制。这一结果使我们进一步推测CPE很可能参与了TrkB受体的活性依赖性膜表面插入的过程。本课题后续的实验还在进行中。
「结论」
1、所建立的细胞膜表面TrkB受体定量检测的免疫荧光分析方法,可以从空间即细胞的不同区域和时间上定量分析不同条件下TrkB膜表面的变化情况,为后续研究TrkB活性依赖性膜表面插入的机制奠定了基础。
2、定量分析了在非活化的基础状态下海马神经元膜表面TrkB受体占胞内总体TrkB的比例为34%,说明TrkB的在被激活后是从胞内存储池中插入到膜表面的,同时此定量分析更能准确掌握细胞在不同条件下膜表面TrkB受体的变化情况。
3、明确了TrkB近膜区(JM)是其活性依赖性膜表面插入过程的关键调控区域,是此过程的必要和充分条件,并且对于TrkB快速聚集到突触后膜并插入细胞膜也是关键性调控区域。
4、发现Cdk5激酶活性对于cLTP诱导的TrkB膜表面聚集的增加是重要影响因素,Cdk5对于TrkB JM区478位点Tyr的磷酸化参与了TrkB活性依赖性膜表面插入过程。
5、发现cLTP诱导TrkB膜表面插入的增强在突起尤其突触后膜更加明显,这种活性刺激在突触快速调集TrkB受体的现象可以用于解释活性神经元突触对于BDNF作用的高度反应性。
6、首次发现羧肽酶E胞内C-末端的25个氨基酸能够结合TrkB的胞内区;并且初步证明CPE在cLTP诱导的活性依赖性膜表面插入中具有重要的调控作用。
「Background and objective」
Neurotrophins are best known for their ability to promote neuronal survival and differentiation,Brain-derived neurotrophic factor(BDNF) is an important member in neurotrophins family which has a novel function in synapse development and plasticity,particularly in the central nervous system.It suggests that BDNF facilitates both early-phase and late-phase LTP is necessary for LTP.Remarkably,neuronal activity could influence the effectiveness of BDNF,for example,BDNF cannot enhance the survival of retinal ganglion neurons unless depolarized by high K~+ or glutamate agonists,or increased intracellular cAMP.
Previous studies have shown that active neurons/synapses might respond better to BDNF than less active one,a plausible mechanism would be local synthesis and/or secretion of BDNF at the active synapse or active neurons/synapses might respond better to BDNF than less active ones, and this could be achieved by activity dependent control of BDNF signaling. Neuronal activity dependent signaling of BDNF is virtually all attributed to TrkB receptor tyrosine kinase.Neuronal activity could increase the number of TrkB receptors on cell surface or enhance the signaling transduction for BDNF.Depolarization or Electric stimulation has been shown to increase the levels of the TrkB receptor on the cell surface of neurons. Activity-dependent enhancement of the quantity of surface TrkB receptor may define an important mechanism by which the specificity of BDNF activity-dependant modulation is achieved.However the detailed cellular and molecular mechanism underlying this effect is unclear.
For further provide insights into the mechanistic link between activity-dependent and neurotrophic modulation of CNS neurons and synapses,acquire of the intracellular trafficking mechanisms of TrkB is the key research point.For finding out the molecules which could not only interact with TrkB but also assistant with TrkB's intracellular trafficking,we performed Yeast Two-hybrid assay and found TrkB could interact with the carboxypeptidase E,a proneuropeptide/prohormone processing enzyme which is also a sorting receptor associated with BDNF vesicles and other neuropeptide or prohormone targeting into the regulated secretory pathway (RSP).It is proved the C-terminal cytoplasmic tail of CPE functioned as the key region of the membrane binding and sorting.There is also work suggested the 4 or 15-residue sequence in the C-terminal region of CPE is necessary to sorting of CPE to the RSP.Considering for there are some similar characters between TrkB knock out mice and CPE knock out mice, we hypothesize CPE participates in the regulated progress of TrkB activity dependent membrane surface insertion.
Here we employed a dual tag TrkB fluorescence assay to investigate the molecular and cellular mechanisms which regulate the trafficking progress of TrkB activity dependent membrane surface insertion to provided new insights regarding neuronal activity dependent surface delivery of TrkB receptor,which will advance our understanding the role of TrkB in synaptic plasticity modulation.
「Methods and results」
1.Ratiometric fluorescence assay to measure surface TrkB receptor level
To employed the FLAG-TrkB-GFP construct and the "surface FLAG-TrkB-GFP fluorescence ratiometric assay" to monitor the TrkB quick recruitment to the plasma membrane.With this assay to provided temporal, structure and spatial information regarding neuronal activity dependent surface delivery of TrkB receptor,Through this assay we found chemical LTP(glycine) induced the most robust increase(1.82±0.08 fold) in surface TrkB levels and 200μM glycine was then chosen as the stimulation condition in our subsequent studies.
2.Plasma surface TrkB fraction measurement
To measure the percentage of surface TrkB receptor in total TrkB at basal state,we stained FLAG tag of FLAG-TrkB-GFP under permeabilized and non-permeabilized conditions.From plotting the anti-FLAG fluorescence signal in the whole cell region inpermeabilized neurons against the GFP fluorescence signal we got the relationship between the anti-FLAG and the GFP signals by immunofluorescence(IF) is 1.01.With this constant we calculate the fraction of TrkB on hippocampal neurons surface is about 34%.By this assay we found activity dependent cell surface TrkB increase was much more obvious in neuronal processes than in cell body(1.35 vs.1.98 fold) which could be used to understand the mechanisms of BDNF's function in synapse plasticity.
3.Juxtamembrane domain of TrkB is necessary and sufficient for cLTP induced TrkB plasma membrane insertion
To define the potential region in TrkB receptor that is required for its activity-dependent plasma membrane insertion,we generated a series of constructs in which various TrkB domains(juxtamembrane,kinase,and C terminal) were deleted from TrkBFL or different TrkB domains (juxtamembrane,kinase,and C terminal) were grafted to the C-terminal of T1 receptor(a truncated TrkBFL isoform).All the mutant constructs (TrkBΔJM,TrkBΔTK,TrkBΔCT,T1JM,T1TK and T1CT) contain the same FLAG and GFP epitope tag.With the ratiometric fluorescence assay we found only the TrkBΔJM abolished glycine-induced TrkB receptor surface insertion in the deletion constructs and only T1JM shown activity-dependent surface recruitment upon cLTP stimuli in the grafting constructs.It is suggested that TrkB cytoplasmic juxtamembrane region is not only necessary for efficient TrkB surface recruitment but also sufficient to promote rapid insertion of TrkB to plasma membrane upon cLTP stimulation.
4.Cdk5 but not TrkB kinase activity is involved in cLTP induced TrkB plasma membrane insertion
Protein kinases play important roles in neuronal activity and mediating BDNF signaling and biological activity.By the ratiometric fluorescence assay together with selective inhibitor of Cyclin-dependent kinase 5(Cdk5) Roscovitin and Cdk5 siRNA,the data shown the cLTP-induced TrkB surface recruitment was significantly abrogated.Furthermore TrkBS478A (ser478 mutant to alanine) and T1JMS478A neuronal surface level could not increase in response to cLTP stimuli.And cLTPstimuli could lead to an acute Cdk5 phosphorylation at Tyr15 and Ser159 site.It suggested Cdk5 is crucial in activity dependent TrkB plasma membrane insertion.From the results of both pre-treatment of Trk inhibitor K252a and kinase dead(KD) TrkB construct retained the ability to insert into surface in response to glycine stimuli,it suggested tyrosine kinase activity is not required for this behavior.
5.cLTP induced TrkB surface recruitment occurs more efficiently in postsynaptic region
With the ratiometric fluorescence assay combined with transfection with the constructs of PSD-95-mCherry and FLAG-TrkBFL-GFP or FLAG-TrkBΔJM-GFP it present that the neuronal activity could facilitate more TrkBFL receptors trafficking to the postsynaptic region which shown it dramatically insertion into the PSD-95(postsynaptic marker) positive region than in PSD-95 negative region(2.13 vs.1.75 fold),but the TrkBΔJM receptor abolished this process which suggested the JM domain is essential for TrkB's activity-dependent intracellular trafficking.
6.CPE cytoplasmic tail interact with TrkB receptor's intracellular domain
For investigating the molecule regulating TrkB activity-dependent insertion into membrane surface,Yeast Two-hybrid assay and Co-immunoprecipitation(CO-IP) were performed and found TrkB could interact with carboxypeptidase E.This result was confirmed by the images of confocal fluorescent microscope.Established constructs of MYC-CPE25AA-RFP(intracellular domain) and FLAG-TrkB IC,which only has the intracellular domain of CPE or TrkB and further performed CO-IP,it is suggested that c-terminal 25aa of CPE could associate with TrkB intracellular domain.
7.CPE is involved into the regulating of cLTP induced TrkB membrane surface insertion
Design and generate siRNA of CPE together with the ratiometric fluorescence assay to test the activity dependent cell surface TrkB increase was significantly restrained.This result confirmed us cLTP induced TrkB surface insertion is regulated by the biologic function of CPE.
「Conclusion」
1.Established the "Surface TrkB receptor fluorescence ratiometric assay" to investigate the mechanism underlying TrkB plasma membrane recruitment upon chemical LTP(cLTP) stimuli.
2.Calculated and defined the percentage of surface TrkB receptor in total TrkB at basal state is 34%.
3.Revealed the juxtamembrane(JM) domain of TrkB is necessary and sufficient for its activity-dependent plasma membrane insertion.
4.The kinase activity of Cyclin-dependent kinase 5(Cdk5) is crucial to cLTP induced TrkB surface recruitment;and the Tyr478 of TrkB JM domain which could be phosphorylated by Cdk5 is the most key site.
5.cLTP induced TrkB surface recruitment occurs more efficiently on neuronal processes especially at the postsynaptic membrane,which may present a mechanism for rapid enhancement of postsynaptic sensitivity to incoming BDNF signaling.
6.The C-terminal 25aa of Carboxypeptidase E was confirmed associate with the intracellular domain of TrkB and this interaction is crucial in regulating LTP induced TrkB surface recruitment.
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37. Kovalchuk, Y, et al., Postsynaptic Induction of BDNF-Mediated Long-Term Potentiation. Science, 2002. 295(5560): p. 1729-34.
38. Poo, M.M., Neurotrophins as synaptic modulators. Nat Rev Neurosci, 2001. 2(1): p. 24-32.
39. Lu, B., BDNF and activity-dependent synaptic modulation. Learn Mem, 2003. 10(2): p. 86-98.
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41. Du, J., et al., Activity- and Ca(2+)-dependent modulation of surface expression of brain-derived neurotrophic factor receptors in hippocampal neurons. J Cell Biol, 2000. 150(6): p. 1423-34.
42. Huang, E.J. and L.F. Reichardt, Trk receptors: roles in neuronal signal transduction. Annu Rev Biochem, 2003. 72: p. 609-42.
43. Chao, M.V., Neurotrophins and their receptors: a convergence point for many signalling pathways. Nat Rev Neurosci, 2003. 4(4): p. 299-309.
44. Casaccia-Bonnefil, P., H. Kong, and M.V. Chao, Neurotrophins: the biological paradox of survival factors eliciting apoptosis. Cell Death Differ, 1998. 5(5): p. 357-64.
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48. Meyer-Franke, A., et al., Depolarization and cAMP elevation rapidly recruit TrkB to the plasma membrane of CNS neurons. Neuron, 1998. 21 (4): p. 681-93.
49. Ehlers, M.D., et al., NGF-stimulated retrograde transport of trkA in the mammalian nervous system. J Cell Biol, 1995. 130(1): p. 149-56.
50. Grimes, M.L., et al., Endocytosis of activated TrkA: evidence that nerve growth factor induces formation of signaling endosomes. J Neurosci, 1996. 16(24): p. 7950-64.
51. Dhanvantari, S., et al., Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion. Biochemistry, 2002. 41(1): p. 52-60.
52. Zhang, C.F., et al., Sorting of carboxypeptidase E to the regulated secretory pathway requires interaction of its transmembrane domain with lipid rafts. Biochem J, 2003. 369(Pt 3): p. 453-60.
53. Arnaoutova, I., et al., Recycling of Raft-associated prohormone sorting receptor carboxypeptidase E requires interaction with ARF6. Mol Biol Cell, 2003. 14(11): p. 4448-57.
54. Dhanvantari, S., et al., Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia. Mol Endocrinol, 2003.17(9): p. 1856-67.
55. Park, J.J., N.X. Cawley, and Y.P. Loh, Carboxypeptidase E cytoplasmic tail-driven vesicle transport is key for activity-dependent secretion of peptide hormones. Mol Endocrinol, 2008. 22(4): p. 989-1005.
56. Lou, H., et al., Sorting and activity-dependent secretion of BDNF require interaction of a specific motif with the sorting receptor carboxypeptidase e. Neuron, 2005. 45(2): p. 245-55.
57. Dhanvantari, S. and Y.P. Loh, Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor. J Biol Chem, 2000. 275(38): p. 29887-93.
58. Assadi, M., et al., The C-terminus of prohormone convertase 2 is sufficient and necessary for Raft association and sorting to the regulated secretory pathway. Biochemistry, 2004. 43(24): p. 7798-807.
59. Cawley, N.X., et al., The carboxypeptidase E knockout mouse exhibits endocrinological and behavioral deficits. Endocrinology, 2004. 145(12): p. 5807-19.
60. Woronowicz, A., et al., Absence of carboxypeptidase E leads to adult hippocampal neuronal degeneration and memory deficits. Hippocampus, 2008. 18(10): p. 1051-63.
1.Arancio,O.and M.V.Chao,Neurotrophins,synaptic plasticity and dementia.Curr Opin Neurobiol,2007.17(3):p.325-30.
2.Huang,E.J.and L.F.Reichardt,Neurotrophins:roles in neuronal development and function.Annu Rev Neurosci,2001.24:p.677-736.
3.Lu,B.,BDNF and activity-dependent synaptic modulation.Learn Mem,2003.10(2):p.86-98.
4.Bramham,C.R.and E.Messaoudi,BDNF function in adult synaptic plasticity:the synaptic consolidation hypothesis.Prog Neurobiol,2005.76(2):p.99-125.
5.Cohen,S.and M.E.Greenberg,Communication between the synapse and the nucleus in neuronal development,plasticity,and disease.Annu Rev Cell Dev Biol,2008.24:p.183-209.
6.Nagappan,G.and B.Lu,Activity-dependent modulation of the BDNF receptor TrkB:mechanisms and implications.Trends Neurosci,2005.28(9):p.464-71.
7.Bonhoeffer,T.,Neurotrophins and activity-dependent development of the neocortex.Curr Opin Neurobiol,1996.6(1):p.119-26.
8.McAllister,A.K.,L.C.Katz,and D.C.Lo,Neurotrophins and synaptic plasticity.Annu Rev Neurosci,1999.22:p.295-318.
9.Poo,M.M.,Neurotrophins as synaptic modulators.Nat Rev Neurosci,2001.2(1):p.24-32.
10.Korte,M.,et al.,Hippocampal long-term potentiation is impaired in mice lacking brain-derived neurotrophic factor.Proc Natl Acad Sci U S A,1995.92(19):p.8856-60.
11.Patterson,S.L.,et al.,Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron,1996.16(6):p.1137-45.
12.Martinowich,K.,H.Manji,and B.Lu,New insights into BDNF function in depression and anxiety.Nat Neurosci,2007.10(9):p.1089-93.
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