| Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by deposition of extracellular senile plaques formed by accumulatedβ-amyloid proteins (AP), accumulation of intracellular neurofibrillary tangles formed by abnormal phosphorylated tau, neuronal dystrophy and neuronal cell loss. P-amyloid precursor protein (APP), a transmembrane protein, is important for the pathophysiology of AD. Aβpeptide is generated by proteolysis of APP. Most of previous attentions have been focused on the neurotoxicity of Aβ.The F0F1-ATP synthase is the enzyme complex responsible for ATP synthesis driven by oxidative phosphorylation. The complex is a molecular motor that normally operates as an ATP synthase in the mitochondrial inner membrane in which ADP phosphorylation is driven by the movement of protons along the electrochemical potential gradient established by respiration. During conditions of reduced membrane, the ability of the F0F1-ATP synthase to reverse by hydrolyzing ATP and generating an electrochemical proton gradient across a membrane to support locomotion or nutrient uptake and act as an ATPase potential has been appreciated for many years.Many reports from several laboratories in recent years have concerned the location and function of the ATP synthase complex or its component subunits on the external surface of the plasma membrane of various mammalian cell types, including vascular endothelial cells, hepatocytes, adipocytes and tumor cells. F0F1 components, especialy a subunit, have been identified as cell-surface receptors for multiple ligands in studies of angiogenesis, tumor proliferation/toxicity, lipid/lipoprotein metabolism, immune recognition of tumors, hypertension, the synthesis of ATP, the regulation of intercellular pH, and the cytolytic pathway of tumor cells.Currently, mounting evidences indicate ATP synthase a may play a role in neurodegenerative diseases, including AD and PD. For example, increased ATP synthase levels were found in the frontal cortex in PD. ATP synthase has a twofold higher abundance in PD specimens. ATP synthase a can bind the extracellular domain of APP and Aβ. A study showed that Aβ25-35 can induce the expression of ATP synthaseα-chain in primary rat cortical neurons. An interesting research showed that ATP synthase a-chain associated with neurofibrillary degeneration accumulate in the cytosol of Alzheimer degenerating neurons in AD. However, little is understood about the role of ATP synthase a in cell death and neurodegeneration.Recent reports have also implicated that the nuclear coded F0F1-ATP synthase a physically interacts with HSP60, which is known to stabilize numerous mitochondrial proteins and HSP90. We studied the role of ATP synthase a in mhtt aggregate formation in SH-SY5Y cells. The studies reporte demonstrated that ATP synthas areduced aggregate formation and neurotoxicity in cultured neuronal cells expressing htt protein with an expanded polyglutamine tract. We previously studied the role of ATP synthase a in mhtt aggregate formation in SH-SY5Y cells. Results demonstrated that ATP synthase a reduced aggregate formation and neurotoxicity in cultured neuronal cells expressing htt protein with an expanded polyglutamine tract.These and other results have stimulated the search for pharmacological compounds capable of up-regulatinga subunit of ATP synthase expression, thus such compounds would theoretically have potential as treatments for these and other neurologic disorders. In this light very promising, in this study, we investigated whether ATP synthase a protecting the SH-SY5Y cells from Aβ25-35-induced oxidative cell death, and explored the underlying protective mechanisms. The results were summarized as follows:1. In this study, we cloned ATP syn a cDNA, and constructed pDsRed-N 1-ATP syn-αplasmid. After treansfected into SH-SY5Y cells, westen blot detected DsRed-ATP syna bands, and showed DsRed-ATP syn a in mitochrondia marker TOM or TIM labeled subcellular structures.2. First, we showed that Aβcould induced an increase of ATP syn-a in SH-SY5Y cell. The overexpression of a subunit of ATP synthase protected beta-amyloid-induced cytotoxicity and regulated the expression of Bcl-2 family proteins and to decrease the activation of caspase-3. The a subunit of ATP synthase ameliorated Aβ25-35-induced cell injury. The overexpression of a subunit of ATP synthase protected SH-SY5Y cells against Aβ25-35-induced cell apoptosis and death showed by MTT assay. WST assay also showed that the overexpression of a subunit of ATP synthase inhibited Aβ25-35-induced cell injury in SH-SY5Y cells transfected with pDsRed-Nl-ATP syn compared that transfected with pDsRed-N 1 DNA.3. The overexpression of a subunit of ATP synthase inhibited Aβ25-35-induced dissipation of the mitochondrial membrane potential (MMP) monitored using the Rh123 in SH-SY5Y cells transfected with pDsRed-Nl-ATP syn compared to transfected with pDsRed-N1 DNA. After Aβtreatment, the overexpression ofαsubunit of ATP synthase increased the Bcl-2/Bax ratio in SH-SY5Y cells transfected with pDsRed-N1-ATP compared to the cells pDsRed-N1 DNA detected by Western blot. The overexpression ofαsubunit of ATP synthase also downregulated the caspase-3 activation in SH-SY5Y cells transfected with pDsRed-N1-ATP syn compared to that transfected with pDsRed-N1 DNA.4. In this study, we constructed pGEX-4T2-ATP-syn-αplasmid, and expressed and purified ATP-syn-α. With turbid assay and Thioflavin T measurement, experiments showed thatαsubunit of ATP synthase counteract the the aggregation of amyloid Aβin vitro and the anti-aggregation activity ofαsubunit of ATP synthase was enhanced by ATP. These effects are not only for Aβ25-35 but also for Aβ1-40 in vitro5. With turbid assay and Thioflavin T measurement, we showed thatαsubunit of ATP synthase did not disassemble existing fibrils Aβ. ATP synthaseαincubated with Aβ25-35 and Aβ1-40 did not disassemble existing fibrils Aβ25-35 and Aβ1-40. Addition of ATP did not induce the function of ATP synthaseαfor Aβdisassembly.5. By immunostaining and Thioflavin S staining, we observed that in amyloid plaques of APPswe/PS1ΔE9 AD mice, there are many amyloid plaques with high level ofαsubunit of ATP synthase, but notβsubunit of ATP synthase.In summary, we described that amyloid could induced the increase of ATP synthaseαexpression, the increased ATP synthaseαcould inhibited amyloid toxicity. Moreover, ATP synthaseαcould inhibited amyloid aggregation but did not disassemble aggregated amyloid. In addition, ATP synthaseαin amyloid plaques of AD transgenic mice, imply that ATP synthaseαmight try to inhibit amyloid aggregation. |
