Identification And Functional Studies Of The Nerve Cell Surface Atp Synthase

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 cell1.1 The identification of ATP synthase on the neural cell from rat brainPlasma 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 brainDetergent-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 cellsAcute 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 function2.1 The identification of ATP synthase by biochemistry assayThe 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 neuronsIn 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 neuronsMoreover, 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 synthase3.1 ATP synthesis on neuronal surfaceATP 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 hydrolysisADP 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 conditionWhen 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 pathphysiologicalfunction4.1 Aβcan bind with ectopic ATP synthaseWe 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 surfaceAfter 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 synthase4.3.1 Aβhas effect on the ATP synthesis of neural surface ATP synthaseAfter 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 pHWhen 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.