神经细胞表面ATP合酶的鉴定和功能研究
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摘要
ATP合酶是合成ATP的关键酶,主要由F1和F0组成。以往的研究认为,ATP合酶定位于能量代谢的细胞器如线粒体以及植物的“叶绿体”等。但近年来的研究发现ATP合酶存在着异位分布,不仅存在于原核生物的细胞膜表面,还存在于真核生物的一些细胞膜表面,被认为可以转换ATP／ADP,并作为多配基的受体,参与许多生理病理过程。已有研究表明,在脑内神经元膜表面广泛存在着嘌呤类受体如P2X、P2Y受体,细胞外的ATP可以作为快速的神经递质和神经调质来调节神经元的兴奋性,在脑的生理以及病理生理中发挥重要的作用,而脑内神经细胞膜表面是否也存在着ATP合酶,还不清楚。
在2003年,Sergeant等人在AD脑内的神经元胞浆内发现有ATP合酶的α亚单位与神经纤维缠结(由高度磷酸化Tau组成)的积聚现象。提示在病理情况下,ATP合酶在胞浆内除线粒体之外存在着异位分布。我们课题组在前期实验中,曾用Aβ结合的Affi-gel研究大鼠脑细胞膜蛋白中的Aβ结合组份,质谱分析显示存在ATP合酶的α亚单位,初步提示Aβ与脑内异位分布的ATP合酶可以结合并可能参与脑内的病理过程。
因此本研究就脑内细胞,尤其是神经元是否也存在着ATP合酶在细胞表面的异位分布以及它的生理功能；Aβ与ATP合酶的结合作用及其效应进行了一系列研究。现将实验结果概括如下：
1神经细胞膜表面ATP合酶的鉴定
1.1脑组织细胞质膜ATP合酶的鉴定
用蔗糖梯度离心法提取大鼠脑细胞亚组份,电镜鉴定显示,分离的细胞膜与线粒体的纯度可以用于后续的研究,Western blot检测显示,作为线粒体标记的cytochrome c,主要分布在线粒体组份,在细胞膜分布很低；作为细胞膜标记的caveolin-1,则主要分布在细胞膜组份,在线粒体组份分布很低；在鼠脑中分离的细胞质膜中不仅有F1 ATP合酶的α以及β亚单位的分布,而且还分布有F0ATP合酶的c亚单位；结果提示,ATP合酶位于脑组织细胞质膜。
1.2脑组织细胞质膜ATP合酶在筏区的鉴定
用蔗糖梯度离心法分离脑细胞亚组份,从上至下共分为12组份,Westernblot检测显示,作为筏区标记的caveolin-1、flotillin-1主要分布于第3-5组份,ATP合酶不仅分布于9-12组份,在3-5组份也有分布,结果提示：ATP合酶不仅分布在线粒体,也分布在质膜的筏区。
1.3 ATP合酶位于脑组织细胞膜表面
免疫荧光标记结合流式细胞检测脑组织细胞膜ATP合酶的分布,结果显示,与阴性对照组(一抗用PBS代替)相比,实验组(用抗ATP合酶α亚单位抗体标记,不用Triton X-100透膜)平均荧光强度要显著高于对照组,但低于阳性对照组(用抗ATP合酶α抗体标记,用Triton X-100透膜)。结果进一步说明ATP合酶位于脑组织细胞质膜,且位于细胞膜表面。
2原代神经元质膜ATP合酶的鉴定及其功能研究
2.1原代神经元质膜ATP合酶的生化鉴定
将收集的培养细胞经蔗糖密度梯度离心分离出细胞膜,Western blot检测显示,F1 ATP合酶的α、β亚单位以及F0段的c亚单位分布在原代神经元细胞质膜。
为了进一步研究ATP合酶是否分布在神经元细胞膜表面,用生物素标记活的原代培养神经细胞膜表面蛋白,利用卵白素结合生物素的原理将生物素标记的膜蛋白分离。进行Western blot鉴定,结果检测,F1ATP合酶的α、β亚单位分布于原代神经元细胞膜上,但F0段的c亚单位并未检测到。结果进一步提示,ATP合酶位于原代神经元质膜,且F1段朝向细胞表面。
2.2 ATP合酶在原代神经元质膜分布的形态学观察
免疫荧光双标结果显示,在不使用透膜剂Triton X-100的情况下,可看到ATP合酶α与β亚单位在神经细胞质膜分布,且几乎完全共存,结果提示：ATP合酶α与β亚单位分布在细胞质膜。
进一步在原代培养神经元、神经母细胞瘤来源的细胞株SH-SY5Y上,用激光共聚焦显微镜分析荧光标记的ATP合酶α与Mitotracker Red或通过转染pDsRed-Mito标记的线粒体,结果显示,除线粒体之外有ATP合酶在膜上的点状分布。提示：ATP合酶不仅分布在线粒体,也分布于线粒体之外。
2.3 ATP合酶在原代神经元质膜筏区的分布
为明确神经细胞质膜ATP合酶与筏区的关系,用抗ATP合酶α的抗体与霍乱毒素B亚单位(CTB)标记的细胞质膜筏区双标,激光共聚焦显微镜检测结果显示：ATP合酶α亚单位与筏区标记CTB存在着部分共存,提示：ATP合酶至少是部分分布于质膜的筏区中。
3神经细胞膜表面ATP合酶的生理功能研究
3.1神经细胞膜表面ATP的合成分析
用生物发光法检测神经细胞膜表面ATP的合成,结果显示：在加入底物ADP和无机磷后,细胞外ATP开始增加,到50秒时,胞外的ATP量达到平台。在加入ATP合酶的抑制剂寡霉素以及抗ATP合酶的抗体的情况下,胞外的ATP的生成被抑制,并且呈现随寡霉素以及抗ATP合酶的抗体浓度增加剂量依赖性地降低。结果提示：神经细胞质膜ATP合酶具有合成ATP作用。
3.2神经细胞膜表面ATP合酶的水解功能分析
采用偶连NADH的方法分析ATP合酶的水解功能,结果显示,在加入含有ATP底物的混合液时,在检测时间300秒内,细胞外ADP的含量并未发生显著改变。结果提示：神经细胞膜质膜ATP合酶不参与水解ATP。
3.3神经细胞质膜ATP合酶调节细胞内pH的分析
当细胞外pH为6.0时,细胞内pH下降,同时加入抗ATP合酶α亚单位的抗体,可抑制细胞内的pH下降,当细胞外pH为8.0时,细胞内pH上升,加入抗ATP合酶α亚单位的抗体,同样可抑制细胞内pH的上升,提示：神经细胞质膜ATP合酶可以调节细胞内pH。
细胞载入荧光素calcein后,在细胞外pH8.0的条件下,可导致calcein释放增加,用抗ATP合酶抗体,可抑制细胞外碱性环境导致的calcein释放,结果提示：细胞质膜ATP合酶能调节细胞内pH,进而影响到细胞的活力状态。
4 Aβ对神经细胞膜表面ATP合酶的作用及其病理生理意义研究
4.1 Aβ与异位ATP合酶的结合作用
用与Affi-gel结合的Aβ对提取的大鼠脑膜蛋白提取液进行结合反应,接着将Aβ结合的膜蛋白用免疫印迹检测显示有ATP合酶的成份,进一步质谱结果显示,有ATP合酶α、β亚单位被检测到。在12月龄的转基因鼠(APP／PS1)脑片上,用ATP合酶的抗体与Aβ的抗体进行免疫荧光双标,结果显示,在转基因小鼠脑片上,有广泛的老年斑的分布,同时,在这些老年斑内,可看到Aβ与ATP合酶的共存。提示Aβ能特异性地与质膜ATP合酶蛋白结合。
4.2 Aβ对神经细胞质膜ATP合酶分布的影响
原代培养神经元,用单体Aβ(1μM)或纤维状Aβ(1μM)作用1h,用生物素标记细胞膜表面蛋白,Western blot检测显示：在单体Ap作用后,细胞表面生物素标记的膜蛋白中ATP合酶明显减少,而在纤维状Aβ作用下,尽管细胞表面ATP合酶也有减少,但不如单体Aβ的作用明显。提示：不同状态的Aβ对神经细胞质膜ATP合酶分布的影响有所差异。
4.3 Aβ对神经细胞质膜ATP合酶功能的影响
4.3.1 Aβ对神经细胞质膜ATP合酶合成ATP的影响
原代培养神经元在加入Aβ(fAβ),与ATP合成的底物ADP和无机磷后,细胞外的ATP合成较无Aβ对照组减少,以0.1μM和1μM的Aβ有显著性,而0.1μM和1μM的fAβ也有抑制ATP合成的作用。提示：Aβ可以影响神经细胞质膜ATP合酶合成ATP的功能。
4.3.2 Aβ对神经细胞质膜ATP合酶调节pH的影响
结果显示：当细胞外pH为6.0时,与对照组(正常组)相比,Aβ(0.01μM-1μM)有使细胞内pH进一步降低的趋势,当细胞外pH为8.0时,与对照组(正常组)相比,Aβ使细胞内pH降低,1μM Ap作用有显著性意义。提示：在细胞外碱性条件下,Aβ可以结合到神经细胞质膜ATP合酶进而调节细胞内pH。
当细胞外pH为8.0时,与对照组(正常组)相比,加入Aβ可以使细胞外LDH、calcein的释放减少。提示：在细胞外碱性条件下,Aβ影响到神经细胞质膜ATP合酶来调节细胞内pH进而影响到细胞的活力状态。
小结：本研究结果说明“线粒体蛋白”ATP合酶,存在于脑内细胞以及原代培养神经元细胞质膜上以及F1朝向细胞外表面,其中至少部分定位于细胞质膜的筏区中。研究还表明神经细胞质膜ATP合酶能够合成ATP,且能够通过转运质子来调节细胞内pH；Aβ能结合神经细胞质膜ATP合酶,影响其ATP合成和质子转运功能,另外Aβ还可影响ATP合酶在神经元表面的分布。
ATP synthase is an enzymatic complex responsible for ATP synthesis in mitochondria. The mitochondrial complex consists of a membrane-embedded F0 portion and a soluble F1 portion. Although this complex is generally believed to be strictly expressed in mitochondrial membrane of prokaryote and chloroplasts, a number of new observations suggesting that the enzyme is also located on the cell surface of numerous cell types. It is not only located on the plasma membrane of certain bacteria, but it also located on the plasma membrane of several types of eukaryotic cells, where they can switch ATP/ADP, and serve as receptors for multiple ligands and participate in diverse processes. In nerve system, there are many receptors for extracellular ATP and nucleotides, such as P2 receptors including ionotrophic P2X and G protein-coupled P2Y receptors. Extracellular ATP can act as a fast neurotransmitter, a modulator of neurotransmitter release and neuronal excitability in physiological and pathophysiological processes of brain. Therefore, it was valuable to study the ATP synthase on neuronal surface.
Sergeant et al found that the a chain of ATP synthase can be associated with neurofibrillary and accumulated in cytoplasm of neurons in 2003, which showed that ATP synthase have ectopic location in cytoplasm besides mitochondria under pathological condition. Our previous study showed that the Aβcan bind the ATP synthase from plasma membrane protein of rat brain, which implied that Aβcan bind to ectopic ATP synthase and participate in pathological process in brain.
The aim of this investigation is to study that the ectopic ATP synthase on the neural cells and its physiological function, Aβbind ATP synthase and its effect. Now we summarize the total results as follow described:
1 The identification of ATP synthase on the neural cell
1.1 The identification of ATP synthase on the neural cell from rat brain
Plasma membrane and mitochondria were prepared from the cortices of rat brains. The isolated plasma membrane and mitochondria fractions were confirmed by electron microscopy. Fractions of plasma membrane and mitochondria were analyzed by immunoblotting. Cytochrome c was mainly found in mitochondria fraction whereas caveolin-1 was only detected in the plasma membrane. In the plasma membrane, subunitsαandβof ATP synthase F1, and c subunit of ATP synthase Fo could be detected.
1.2 ATP synthase on lipid rafts in brain
Detergent-resistant lipid rafts in brain were isolated by sucrose gradient ultracentrifugation. The sucrose gradient was isolated 12 fractions from the top to the bottom. Immunoblots showed that fractions 3-5 contained typical raft marker proteins caveolin-1、flotillin-1. ATP synthase a was detectable in lipid raft fractions containing caveolin-1 and flotillin-1, indicating that ATP synthase complex is partially present in the detergent-resistant microdomains of plasma membrane in rat brain.
1.3 ATP synthase located on the cell surface of neural cells
Acute separated cells from cortices were analyzed by fluorescence-assisted flow cytometry. Under impermeabilization condition, the fluorescence intensity of cells labeled by monoclonal antibody against ATP synthase a was significantly higher than that omitting the primary antibody. Under permeabilization condition, the fluorescence intensity of ATP synthase a labeling obviously increased. This result indicated ATP synthase a might be endogenously present on the surface of neural cells.
2 The identification of plasma membrane ATP synthase in cultured neurons and its function
2.1 The identification of ATP synthase by biochemistry assay
The plasma membrane and mitochondria prepared from the primary cultured neurons were analyzed by immunoblot. Results showed that the F1-α,-βand F0-c subunits of ATP synthase were detected in fraction of plasma membrane.
In order to confirm the surface localization of ATP synthase complex in neurons, primary cultured neurons were labeled with membrane impermeable biotin. The biotinylated surface proteins were precipitated with avidin-conjugated agarose beads, and analyzed by immunoblot. Both ATP synthaseαandβ, as well as a transmembrane protein APP (amyloid precursor protein), but not cytochrome c, were detected in biotinylated surface proteins, although, F0-c subunit was not detected in biotinylated proteins. These results demonstrated that F1-ATP synthase is indeed present on the neuronal surface.
2.2 Morphological study for ATP synthase on the surface of primary cultured neurons
In morphological studies, double labeling with antibodies againstαandβsubunits omitting permeabilization step, showed the colocalization of them on neurons.
The mitochondria were pre-labeled with MitoTracker Red or a mitochondrial matrix-targeted red fluorescent protein by transfecting with pDsRed-Mito vector, were immunostained with anti-ATP synthase a antibody. We demonstrated that ATP synthase a on neurons were not fully colocalized with mitochondrial markers by using confocal microscopy, and presented on the subcellular region including cell surface except mitochondria.
2.3 ATP synthase located on the lipid rafts on the cultured neurons
Moreover, to analyze the relationship between cell surface ATP synthase and lipid rafts, the primary cultured neurons were inserted the fluorescent cholera toxin subunit B that is used as lipid raft landmarker and then immunostained with anti-ATP synthase a antibody omitting permeabilization step. The result showed that ATP synthase a presented within CTB labeled structures on neurons partially. It indicated that ATP synthase is presented on the lipid rafts partially.
3 The physiological function of neural surface ATP synthase
3.1 ATP synthesis on neuronal surface
ATP contents in neuronal cultured media were analyzed by bioluminescent method. Extracellular ATP content greatly increased within 50 seconds after adding ADP and Pi and reached a plateau level. The extracellular ATP synthesis was dose dependently inhibited by ATP synthase inhibitor oligomycin and anti-ATP synthase-αantibody. These results presumably indicated that the ATP synthase on neuronal surface has the ability of ATP synthesis.
3.2 The enzymatic assay of neural surface ATP hydrolysis
ADP contents were analyzed by coupled with NADH methods. The result showed that ADP has no significant changes after adding ATP mixtures within 300s. The result indicated that the ATP synthase of neural surface do not participate in hydrolysis of ATP.
3.3 ATP synthase on plasma membrane regulates pHi and calcein release under alkaline condition
When the cells were challenged by acidic media (pH 6.0), the intracellular pH decrease; Adding the antibody of ATP synthase a could inhibit this decrease. When the cells were challenged by alkaline media (pH 8.0), the intracellular pH increase; Adding the antibody of ATP synthase a could inhibit this increase. The result showed that ATP synthase can regulate intracellular pH.
After the cells loaded with calcein, the treatment with alkaline media for 8h caused a significant release of calcein. However, the antibody againstα-subunit of ATP synthase was able to significantly inhibit the calcein release caused by alkaline media. The result inferred that the ATP synthase on neuronal surface might have proton-transporting activity, which mediates neuronal injury under alkaline circumstance.
4 Aβregulates the activity of neural surface ATP synthase and its pathphysiological
function
4.1 Aβcan bind with ectopic ATP synthase
We used Aβlinked affi-gel to study the Aβbinding protein in isolated plasma membrane fraction. Immunoblot showed ATP synthase a in plasma membrane fraction can be pulled down by Aβlinked affi-gel. And the ingredient of ATP synthase in Aβbinding fracton also confirmed by Mass spectrum which showed that ATP synthaseαandβchain can be detected. The brain slices from the AD transgenic mice at age 12 months, were immunofluorescent stained by double-labeling with antibodies against ATP synthase and Aβ; the result showed that there are many senile plaques in transgenic mice, and ATP synthase can be partially colocalized with Aβin senile plaques.
4.2 Aβcan influence the distribution of ATP synthase on the neural surface
After the primary cultured neuron exposed to 1μMAβor 1μM fAβfor 1 h, the cells were labeled with membrane impermeable biotin. Immunoblot showed that the biotinylated surface proteins-ATP synthase a decreased after Aβtreating. Although the biotinylated surface proteins-ATP synthase a chain has decrease after fAβtreating, it did not change significantly. The results indicated that different states of Aβhave different influence on the distribution of ATP synthase on neuronl surface.
4.3 Aβhas effect on the function of neural surface ATP synthase
4.3.1 Aβhas effect on the ATP synthesis of neural surface ATP synthase
After the primary cultured neuron exposed to Aβ(fAβ) for 1 h, the extracellular ATP content decreased, especially 0.1μM and 1μM Aβhad significant influence. Meanwhile, the fAβhad the same effect. The result showed that Aβcan influence the ATP synthesis.
4.3.2 Aβcan regulate intracellular pH
When the cells were challenged with acidic media (pH 6.0), Aβ(0.01μM-1μM) make the primary cultured neurons slightly more acidification. When the cells were challenged with alkaline media (pH 8.0), Aβcan decrease intracellular pH, especially Aβhad statistical significant. The result showed that ATP synthase can affect intracellular pH under alkaline condition.
When the cells were challenged with alkaline media (pH 8.0), Aβmake the release of LDH and calcein by alkaline media decreased. The result showed that Aβcan influence the function of neural surface ATP synthase and the cell vital under extracelluar alkaline condition.
Conclusion:
Here, we demonstrated that "mitochondria protein" ATP synthase, located on the brain cell surface, especially located on the neural surface with F1 expose extracellullar space. And located on the lipid rafts partially; In addition, the ATP synthase on the plasma membrane can synthesis extracellular ATP and regulate intracellular pH through transport proton. Aβcan binding to the neural surface ATP synthase and influenced ATP synthesis and proton transporting, meanwhile, Aβcan influence the distribution of the ATP synthase on plasma membrane of neurons.
在2003年,Sergeant等人在AD脑内的神经元胞浆内发现有ATP合酶的α亚单位与神经纤维缠结(由高度磷酸化Tau组成)的积聚现象。提示在病理情况下,ATP合酶在胞浆内除线粒体之外存在着异位分布。我们课题组在前期实验中,曾用Aβ结合的Affi-gel研究大鼠脑细胞膜蛋白中的Aβ结合组份,质谱分析显示存在ATP合酶的α亚单位,初步提示Aβ与脑内异位分布的ATP合酶可以结合并可能参与脑内的病理过程。
因此本研究就脑内细胞,尤其是神经元是否也存在着ATP合酶在细胞表面的异位分布以及它的生理功能；Aβ与ATP合酶的结合作用及其效应进行了一系列研究。现将实验结果概括如下：
1神经细胞膜表面ATP合酶的鉴定
1.1脑组织细胞质膜ATP合酶的鉴定
用蔗糖梯度离心法提取大鼠脑细胞亚组份,电镜鉴定显示,分离的细胞膜与线粒体的纯度可以用于后续的研究,Western blot检测显示,作为线粒体标记的cytochrome c,主要分布在线粒体组份,在细胞膜分布很低；作为细胞膜标记的caveolin-1,则主要分布在细胞膜组份,在线粒体组份分布很低；在鼠脑中分离的细胞质膜中不仅有F1 ATP合酶的α以及β亚单位的分布,而且还分布有F0ATP合酶的c亚单位；结果提示,ATP合酶位于脑组织细胞质膜。
1.2脑组织细胞质膜ATP合酶在筏区的鉴定
用蔗糖梯度离心法分离脑细胞亚组份,从上至下共分为12组份,Westernblot检测显示,作为筏区标记的caveolin-1、flotillin-1主要分布于第3-5组份,ATP合酶不仅分布于9-12组份,在3-5组份也有分布,结果提示：ATP合酶不仅分布在线粒体,也分布在质膜的筏区。
1.3 ATP合酶位于脑组织细胞膜表面
免疫荧光标记结合流式细胞检测脑组织细胞膜ATP合酶的分布,结果显示,与阴性对照组(一抗用PBS代替)相比,实验组(用抗ATP合酶α亚单位抗体标记,不用Triton X-100透膜)平均荧光强度要显著高于对照组,但低于阳性对照组(用抗ATP合酶α抗体标记,用Triton X-100透膜)。结果进一步说明ATP合酶位于脑组织细胞质膜,且位于细胞膜表面。
2原代神经元质膜ATP合酶的鉴定及其功能研究
2.1原代神经元质膜ATP合酶的生化鉴定
将收集的培养细胞经蔗糖密度梯度离心分离出细胞膜,Western blot检测显示,F1 ATP合酶的α、β亚单位以及F0段的c亚单位分布在原代神经元细胞质膜。
为了进一步研究ATP合酶是否分布在神经元细胞膜表面,用生物素标记活的原代培养神经细胞膜表面蛋白,利用卵白素结合生物素的原理将生物素标记的膜蛋白分离。进行Western blot鉴定,结果检测,F1ATP合酶的α、β亚单位分布于原代神经元细胞膜上,但F0段的c亚单位并未检测到。结果进一步提示,ATP合酶位于原代神经元质膜,且F1段朝向细胞表面。
2.2 ATP合酶在原代神经元质膜分布的形态学观察
免疫荧光双标结果显示,在不使用透膜剂Triton X-100的情况下,可看到ATP合酶α与β亚单位在神经细胞质膜分布,且几乎完全共存,结果提示：ATP合酶α与β亚单位分布在细胞质膜。
进一步在原代培养神经元、神经母细胞瘤来源的细胞株SH-SY5Y上,用激光共聚焦显微镜分析荧光标记的ATP合酶α与Mitotracker Red或通过转染pDsRed-Mito标记的线粒体,结果显示,除线粒体之外有ATP合酶在膜上的点状分布。提示：ATP合酶不仅分布在线粒体,也分布于线粒体之外。
2.3 ATP合酶在原代神经元质膜筏区的分布
为明确神经细胞质膜ATP合酶与筏区的关系,用抗ATP合酶α的抗体与霍乱毒素B亚单位(CTB)标记的细胞质膜筏区双标,激光共聚焦显微镜检测结果显示：ATP合酶α亚单位与筏区标记CTB存在着部分共存,提示：ATP合酶至少是部分分布于质膜的筏区中。
3神经细胞膜表面ATP合酶的生理功能研究
3.1神经细胞膜表面ATP的合成分析
用生物发光法检测神经细胞膜表面ATP的合成,结果显示：在加入底物ADP和无机磷后,细胞外ATP开始增加,到50秒时,胞外的ATP量达到平台。在加入ATP合酶的抑制剂寡霉素以及抗ATP合酶的抗体的情况下,胞外的ATP的生成被抑制,并且呈现随寡霉素以及抗ATP合酶的抗体浓度增加剂量依赖性地降低。结果提示：神经细胞质膜ATP合酶具有合成ATP作用。
3.2神经细胞膜表面ATP合酶的水解功能分析
采用偶连NADH的方法分析ATP合酶的水解功能,结果显示,在加入含有ATP底物的混合液时,在检测时间300秒内,细胞外ADP的含量并未发生显著改变。结果提示：神经细胞膜质膜ATP合酶不参与水解ATP。
3.3神经细胞质膜ATP合酶调节细胞内pH的分析
当细胞外pH为6.0时,细胞内pH下降,同时加入抗ATP合酶α亚单位的抗体,可抑制细胞内的pH下降,当细胞外pH为8.0时,细胞内pH上升,加入抗ATP合酶α亚单位的抗体,同样可抑制细胞内pH的上升,提示：神经细胞质膜ATP合酶可以调节细胞内pH。
细胞载入荧光素calcein后,在细胞外pH8.0的条件下,可导致calcein释放增加,用抗ATP合酶抗体,可抑制细胞外碱性环境导致的calcein释放,结果提示：细胞质膜ATP合酶能调节细胞内pH,进而影响到细胞的活力状态。
4 Aβ对神经细胞膜表面ATP合酶的作用及其病理生理意义研究
4.1 Aβ与异位ATP合酶的结合作用
用与Affi-gel结合的Aβ对提取的大鼠脑膜蛋白提取液进行结合反应,接着将Aβ结合的膜蛋白用免疫印迹检测显示有ATP合酶的成份,进一步质谱结果显示,有ATP合酶α、β亚单位被检测到。在12月龄的转基因鼠(APP／PS1)脑片上,用ATP合酶的抗体与Aβ的抗体进行免疫荧光双标,结果显示,在转基因小鼠脑片上,有广泛的老年斑的分布,同时,在这些老年斑内,可看到Aβ与ATP合酶的共存。提示Aβ能特异性地与质膜ATP合酶蛋白结合。
4.2 Aβ对神经细胞质膜ATP合酶分布的影响
原代培养神经元,用单体Aβ(1μM)或纤维状Aβ(1μM)作用1h,用生物素标记细胞膜表面蛋白,Western blot检测显示：在单体Ap作用后,细胞表面生物素标记的膜蛋白中ATP合酶明显减少,而在纤维状Aβ作用下,尽管细胞表面ATP合酶也有减少,但不如单体Aβ的作用明显。提示：不同状态的Aβ对神经细胞质膜ATP合酶分布的影响有所差异。
4.3 Aβ对神经细胞质膜ATP合酶功能的影响
4.3.1 Aβ对神经细胞质膜ATP合酶合成ATP的影响
原代培养神经元在加入Aβ(fAβ),与ATP合成的底物ADP和无机磷后,细胞外的ATP合成较无Aβ对照组减少,以0.1μM和1μM的Aβ有显著性,而0.1μM和1μM的fAβ也有抑制ATP合成的作用。提示：Aβ可以影响神经细胞质膜ATP合酶合成ATP的功能。
4.3.2 Aβ对神经细胞质膜ATP合酶调节pH的影响
结果显示：当细胞外pH为6.0时,与对照组(正常组)相比,Aβ(0.01μM-1μM)有使细胞内pH进一步降低的趋势,当细胞外pH为8.0时,与对照组(正常组)相比,Aβ使细胞内pH降低,1μM Ap作用有显著性意义。提示：在细胞外碱性条件下,Aβ可以结合到神经细胞质膜ATP合酶进而调节细胞内pH。
当细胞外pH为8.0时,与对照组(正常组)相比,加入Aβ可以使细胞外LDH、calcein的释放减少。提示：在细胞外碱性条件下,Aβ影响到神经细胞质膜ATP合酶来调节细胞内pH进而影响到细胞的活力状态。
小结：本研究结果说明“线粒体蛋白”ATP合酶,存在于脑内细胞以及原代培养神经元细胞质膜上以及F1朝向细胞外表面,其中至少部分定位于细胞质膜的筏区中。研究还表明神经细胞质膜ATP合酶能够合成ATP,且能够通过转运质子来调节细胞内pH；Aβ能结合神经细胞质膜ATP合酶,影响其ATP合成和质子转运功能,另外Aβ还可影响ATP合酶在神经元表面的分布。
ATP synthase is an enzymatic complex responsible for ATP synthesis in mitochondria. The mitochondrial complex consists of a membrane-embedded F0 portion and a soluble F1 portion. Although this complex is generally believed to be strictly expressed in mitochondrial membrane of prokaryote and chloroplasts, a number of new observations suggesting that the enzyme is also located on the cell surface of numerous cell types. It is not only located on the plasma membrane of certain bacteria, but it also located on the plasma membrane of several types of eukaryotic cells, where they can switch ATP/ADP, and serve as receptors for multiple ligands and participate in diverse processes. In nerve system, there are many receptors for extracellular ATP and nucleotides, such as P2 receptors including ionotrophic P2X and G protein-coupled P2Y receptors. Extracellular ATP can act as a fast neurotransmitter, a modulator of neurotransmitter release and neuronal excitability in physiological and pathophysiological processes of brain. Therefore, it was valuable to study the ATP synthase on neuronal surface.
Sergeant et al found that the a chain of ATP synthase can be associated with neurofibrillary and accumulated in cytoplasm of neurons in 2003, which showed that ATP synthase have ectopic location in cytoplasm besides mitochondria under pathological condition. Our previous study showed that the Aβcan bind the ATP synthase from plasma membrane protein of rat brain, which implied that Aβcan bind to ectopic ATP synthase and participate in pathological process in brain.
The aim of this investigation is to study that the ectopic ATP synthase on the neural cells and its physiological function, Aβbind ATP synthase and its effect. Now we summarize the total results as follow described:
1 The identification of ATP synthase on the neural cell
1.1 The identification of ATP synthase on the neural cell from rat brain
Plasma membrane and mitochondria were prepared from the cortices of rat brains. The isolated plasma membrane and mitochondria fractions were confirmed by electron microscopy. Fractions of plasma membrane and mitochondria were analyzed by immunoblotting. Cytochrome c was mainly found in mitochondria fraction whereas caveolin-1 was only detected in the plasma membrane. In the plasma membrane, subunitsαandβof ATP synthase F1, and c subunit of ATP synthase Fo could be detected.
1.2 ATP synthase on lipid rafts in brain
Detergent-resistant lipid rafts in brain were isolated by sucrose gradient ultracentrifugation. The sucrose gradient was isolated 12 fractions from the top to the bottom. Immunoblots showed that fractions 3-5 contained typical raft marker proteins caveolin-1、flotillin-1. ATP synthase a was detectable in lipid raft fractions containing caveolin-1 and flotillin-1, indicating that ATP synthase complex is partially present in the detergent-resistant microdomains of plasma membrane in rat brain.
1.3 ATP synthase located on the cell surface of neural cells
Acute separated cells from cortices were analyzed by fluorescence-assisted flow cytometry. Under impermeabilization condition, the fluorescence intensity of cells labeled by monoclonal antibody against ATP synthase a was significantly higher than that omitting the primary antibody. Under permeabilization condition, the fluorescence intensity of ATP synthase a labeling obviously increased. This result indicated ATP synthase a might be endogenously present on the surface of neural cells.
2 The identification of plasma membrane ATP synthase in cultured neurons and its function
2.1 The identification of ATP synthase by biochemistry assay
The plasma membrane and mitochondria prepared from the primary cultured neurons were analyzed by immunoblot. Results showed that the F1-α,-βand F0-c subunits of ATP synthase were detected in fraction of plasma membrane.
In order to confirm the surface localization of ATP synthase complex in neurons, primary cultured neurons were labeled with membrane impermeable biotin. The biotinylated surface proteins were precipitated with avidin-conjugated agarose beads, and analyzed by immunoblot. Both ATP synthaseαandβ, as well as a transmembrane protein APP (amyloid precursor protein), but not cytochrome c, were detected in biotinylated surface proteins, although, F0-c subunit was not detected in biotinylated proteins. These results demonstrated that F1-ATP synthase is indeed present on the neuronal surface.
2.2 Morphological study for ATP synthase on the surface of primary cultured neurons
In morphological studies, double labeling with antibodies againstαandβsubunits omitting permeabilization step, showed the colocalization of them on neurons.
The mitochondria were pre-labeled with MitoTracker Red or a mitochondrial matrix-targeted red fluorescent protein by transfecting with pDsRed-Mito vector, were immunostained with anti-ATP synthase a antibody. We demonstrated that ATP synthase a on neurons were not fully colocalized with mitochondrial markers by using confocal microscopy, and presented on the subcellular region including cell surface except mitochondria.
2.3 ATP synthase located on the lipid rafts on the cultured neurons
Moreover, to analyze the relationship between cell surface ATP synthase and lipid rafts, the primary cultured neurons were inserted the fluorescent cholera toxin subunit B that is used as lipid raft landmarker and then immunostained with anti-ATP synthase a antibody omitting permeabilization step. The result showed that ATP synthase a presented within CTB labeled structures on neurons partially. It indicated that ATP synthase is presented on the lipid rafts partially.
3 The physiological function of neural surface ATP synthase
3.1 ATP synthesis on neuronal surface
ATP contents in neuronal cultured media were analyzed by bioluminescent method. Extracellular ATP content greatly increased within 50 seconds after adding ADP and Pi and reached a plateau level. The extracellular ATP synthesis was dose dependently inhibited by ATP synthase inhibitor oligomycin and anti-ATP synthase-αantibody. These results presumably indicated that the ATP synthase on neuronal surface has the ability of ATP synthesis.
3.2 The enzymatic assay of neural surface ATP hydrolysis
ADP contents were analyzed by coupled with NADH methods. The result showed that ADP has no significant changes after adding ATP mixtures within 300s. The result indicated that the ATP synthase of neural surface do not participate in hydrolysis of ATP.
3.3 ATP synthase on plasma membrane regulates pHi and calcein release under alkaline condition
When the cells were challenged by acidic media (pH 6.0), the intracellular pH decrease; Adding the antibody of ATP synthase a could inhibit this decrease. When the cells were challenged by alkaline media (pH 8.0), the intracellular pH increase; Adding the antibody of ATP synthase a could inhibit this increase. The result showed that ATP synthase can regulate intracellular pH.
After the cells loaded with calcein, the treatment with alkaline media for 8h caused a significant release of calcein. However, the antibody againstα-subunit of ATP synthase was able to significantly inhibit the calcein release caused by alkaline media. The result inferred that the ATP synthase on neuronal surface might have proton-transporting activity, which mediates neuronal injury under alkaline circumstance.
4 Aβregulates the activity of neural surface ATP synthase and its pathphysiological
function
4.1 Aβcan bind with ectopic ATP synthase
We used Aβlinked affi-gel to study the Aβbinding protein in isolated plasma membrane fraction. Immunoblot showed ATP synthase a in plasma membrane fraction can be pulled down by Aβlinked affi-gel. And the ingredient of ATP synthase in Aβbinding fracton also confirmed by Mass spectrum which showed that ATP synthaseαandβchain can be detected. The brain slices from the AD transgenic mice at age 12 months, were immunofluorescent stained by double-labeling with antibodies against ATP synthase and Aβ; the result showed that there are many senile plaques in transgenic mice, and ATP synthase can be partially colocalized with Aβin senile plaques.
4.2 Aβcan influence the distribution of ATP synthase on the neural surface
After the primary cultured neuron exposed to 1μMAβor 1μM fAβfor 1 h, the cells were labeled with membrane impermeable biotin. Immunoblot showed that the biotinylated surface proteins-ATP synthase a decreased after Aβtreating. Although the biotinylated surface proteins-ATP synthase a chain has decrease after fAβtreating, it did not change significantly. The results indicated that different states of Aβhave different influence on the distribution of ATP synthase on neuronl surface.
4.3 Aβhas effect on the function of neural surface ATP synthase
4.3.1 Aβhas effect on the ATP synthesis of neural surface ATP synthase
After the primary cultured neuron exposed to Aβ(fAβ) for 1 h, the extracellular ATP content decreased, especially 0.1μM and 1μM Aβhad significant influence. Meanwhile, the fAβhad the same effect. The result showed that Aβcan influence the ATP synthesis.
4.3.2 Aβcan regulate intracellular pH
When the cells were challenged with acidic media (pH 6.0), Aβ(0.01μM-1μM) make the primary cultured neurons slightly more acidification. When the cells were challenged with alkaline media (pH 8.0), Aβcan decrease intracellular pH, especially Aβhad statistical significant. The result showed that ATP synthase can affect intracellular pH under alkaline condition.
When the cells were challenged with alkaline media (pH 8.0), Aβmake the release of LDH and calcein by alkaline media decreased. The result showed that Aβcan influence the function of neural surface ATP synthase and the cell vital under extracelluar alkaline condition.
Conclusion:
Here, we demonstrated that "mitochondria protein" ATP synthase, located on the brain cell surface, especially located on the neural surface with F1 expose extracellullar space. And located on the lipid rafts partially; In addition, the ATP synthase on the plasma membrane can synthesis extracellular ATP and regulate intracellular pH through transport proton. Aβcan binding to the neural surface ATP synthase and influenced ATP synthesis and proton transporting, meanwhile, Aβcan influence the distribution of the ATP synthase on plasma membrane of neurons.
引文
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