Effect Of Orexin-A On Alzheimer's Disease And Its Related Mechanism

Background and ObjectiveAlzheimer's disease(AD)is characterized by progressive cognitive dysfunction and memory impairment.The pathological hallmarks of AD include extracellular senile plaques(deposition of amyloid-?(A?)peptide)and intracellular neurofibrillary tangles.Orexin,also known as hypocretin,is produced in the hypothalamus and includes Orexin-A and Orexin-B.The orexinergic neurons project into several brain areas.By activating its receptor,Orexin-A participates in many physiological processes,such as sleep/wake regulation,modulation of energy metabolism,feeding,learning,and memory.It is now believed that Orexin-A is involved in the pathogenesis of ADMitochondria play an important role in the central nervous system,which provide sufficient energy to meet the demand of the brain.Mitochondrial impairment has been found in AD animal and cell model,and it is an important mechanism that contributes to the progression of AD.Since Orexin-A has been found to regulate mitochondrial function,this study aimed to investigate the effect of Orexin-A on AD model and whether this process is related to the effect of Orexin-A on mitochondria.Methods1.Seven months old male APP/PS1 double transgenic mice were selected as AD animal model,and age matched wild type(WT)C57BL/6 J mice were used as normal controls.The experimental animals were divided into WT mice,APP/PS1 mice,Orexin-A-treated APP/PS1 mice(n=12 per group).After the cannula was placed in the lateral ventricle,Orexin-A was dissolved in the artificial cerebrospinal fluid and injected continuously for 21 days.The other two groups were injected with the same volume of artificial cerebrospinal fluid.Morris water maze was used to detect behavioral performance.After sacrificed,thioflavine-S staining was used to test the expression of senile plaques under fluorescence microscope,A?1-40 and A?1-42 level was quantified by ELISA2.To explore the effect of Orexin-A on mitochondria in AD animal model,transmission electron microscopy was used to test the morphology of mitochondria in mouse neurons,test of ATP level,cytochrome c oxidase(CCO)activity detected by kit and mitochondrial DNA(mtDNA)copy number detected by real-time quantitative PCR was used to evaluate mitochondrial function,western blotting was used to test the expression of mitochondrial biogenesis factor PGC1-?,NRF1,NRF2 and TFAM.3.In this study,two kinds of AD cell model were used,namely SH-S Y5Y cells treated with A? and SH-SY5Y cells transfected with Swedish mutant amyloid precursor protein(APP).In order to study the effect of Orexin-A on cell growth in AD cell model,CCK-8 was used to test cell viability,ELISA BrdU was used to test cell proliferation,A?1-40 and A?1-42 level in cell medium was quantified by ELISA.4.To explore the effect of Orexin-A on mitochondria in AD cell model,transmission electron microscopy was used to test the morphology of mitochondria in cells,test of ATP level,CCO activity detected by kit and mtDNA copy number detected by real-time quantitative PCR was used to evaluate mitochondrial function,western blotting was used to test the expression of mitochondrial biogenesis factor PGC1-?,NRF1,NRF2 and TFAM,confocal microscope was used to assay the expression of TMRM for mitochondrial membrane potential(MMP),fluorescence microscope was used to test the expression of reactive oxygen species(ROS),test of GPx and SOD detected by kit was used to evaluate the activity of antioxidants.Results1.Orexin-A aggravates the symptom and biomarker expression in AD animal model1.1 Orexin-A aggravates cognitive deficit in APP/PS1 miceIn Morris water maze,APP/PS1 mice exhibited a significantly increased escape latency,Orexin-A significantly increased escape latency in APP/PS1 mice.The number of platform crossing and the time in target quadrant were significantly decreased in APP/PS1 mice.Orexin-A significantly decreased these parameters in APP/PS1 mice.1.2 Orexin-A aggravates the accumulation of senile plaques in APP/PS1 miceIn fluorescence staining of thioflavine-S,APP/PS1 mice exhibited obvious accumulation of senile plaques.Orexin-A significantly increased the accumulation of senile plaques in APP/PS1 mice.1.3 Orexin-A increases A? level in APP/PS1 miceIn ELISA test,APP/PS1 mice exhibited higher level of A?1-40 and A?1-42-Orexin-A significantly increased the level of A?1-40 and A?1-42 in APP/PS1 mice.2.Orexin-A aggravates mitochondrial impairment in AD animal model2.1 Orexin-A aggravates mitochondrial morphologic impairment in APP/PS1 miceAs shown by electron microscopy,mitochondrial morphologic impairment was mild in APP/PS1 mice.In Orexin-A-treated APP/PS1 mice,mitochondrial morphologic impairment was severe.2.2 Orexin-A aggravates mitochondrial dysfunction in APP/PS1 miceThe mitochondrial function index ATP level,CCO activity and mtDNA copy number was lower in APP/PS1 mice.Orexin-A decreased ATP level,CCO activity and mtDNA copy number in APP/PS1 mice.2.3 Orexin-A aggravates mitochondrial biogenesis impairment in APP/PS1 miceAs shown by western blotting,APP/PS 1 mice showed decreased expression of mitochondrial biogenesis factor PGC1-?,NRF1,NRF2 and TFAM.Orexin-A decreased the expression of PGC1-?,NRF1,NRF2 and TFAM in APP/PS 1 mice.3.Orexin-A has damaging effect on cell growth in AD cell model3.1 Orexin-A inhibits cell viability and cell proliferation in A?-treated SH-SY5Y cellsThe result of the CCK-8 test demonstrated that A? significantly decreased cell viability in SH-SY5Y cells.Orexin-A had no significant effect on cell viability in SH-SY5Y cells.In A?-treated SH-SY5Y cells,with the increased dose of Orexin-A,cell viability of A?-treated SH-SY5Y cells decreased gradually.As shown by ELISA BrdU assay,A? significantly decreased cell proliferation in SH-SY5Y cells,Orexin-A reduced cell proliferation in A?-treated SH-SY5Y cells further.3.2 Orexin-A inhibits cell viability and cell proliferation in APP cellsIn CCK-8 test,cell viability of APP cells decreased significantly.After treated with Orexin-A,cell viability of empty vector cells did not change significantly.With the increasing dose of Orexin-A,cell viability of APP cells decreased accordingly.As shown by ELISA BrdU assay,APP cells showed reduced cell proliferation,Orexin-A reduced cell proliferation in APP cells further.3.3 Orexin-A increases A? level in APP cellsAs shown by ELISA assay,the level of A?1-40 and A?1-42 was higher in APP cells.When APP cells were treated with Orexin-A,the level of A?1-40 and A?1-42 increased further.4.Orexin-A aggravates mitochondrial impairment in AD cell model4.1 Orexin-A aggravates mitochondrial morphologic impairment in A?-treated SH-SY5Y cells and APP cellsAs shown by electron microscopy,mitochondrial morphologic impairment was mild in A?-treated SH-SY5Y cells and APP cells.After treatment with Orexin-A,mitochondrial morphologic impairment was severe in A?-treated SH-SY5Y cells and APP cells.4.2 Orexin-A aggravates mitochondrial dysfunction in A?-treated SH-SY5Y cells and APP cellsThe mitochondrial function index ATP level,CCO activity and mtDNA copy number was lower in A?-treated SH-SY5Y cells and APP cells.Orexin-A decreased ATP level,CCO activity and mtDNA copy number in APP cells.Orexin-A decreased ATP level,CCO activity in A?-treated SH-SY5Y cells,but did not change mtDNA copy number.4.3 Orexin-A aggravates mitochondrial biogenesis impairment in A?-treated SH-SY5Y cells and APP cellsAs shown by western blotting,the expression of mitochondrial biogenesis factor PGC1-?,NRF1,NRF2 and TFAM was lower in A?-treated SH-SY5Y cells and APP cells.Orexin-A decreased the expression of PGC1-?,NRF1,NRF2 and TFAM in A?-treated SH-SY5Y cells and APP cells.4.4 Orexin-A decreases MMP in A?-treated SH-SY5Y cells and APP cellsAs shown by confocal microscopy,TMRM expression was lower in A?-treated SH-SY5Y cells and APP cells.Orexin-A decreased TMRM expression in A?-treated SH-SY5Y cells and APP cells.4.5 Orexin-A increases ROS expression in A?-treated SH-SY5Y cells and APP cellsAs shown by fluorescence microscopy,ROS expression was higher in A?-treated SH-SY5Y cells and APP cells.Orexin-A increased ROS expression in A?-treated SH-SY5Y cells and APP cells.4.6 Orexin-A decreases antioxidants activity in A?-treated SH-SY5Y cells and APP cellsThe activity of GPx and SOD was lower in A?-treated SH-SY5Y cells and APP cells.Orexin-A decreased the activity of GPx and SOD in A?-treated SH-SY5Y cells and APP cells.Conclusion1.Orexin-A aggravates the symptom and biomarker expression in AD animal model2.Orexin-A has damaging effect on cell growth in AD cell model3.Orexin-A aggravates mitochondrial impairment in AD animal and cell model in the aspects of mitochondrial morphology,mitochondrial function and mitochondrial biogenesis.4.Orexin-A accelerates the development of AD possibly through inducing mitochondrial impairment.SignificanceThis research confirms that Orexin-A participates in the progression of AD and explores its specific mechanism,which can better understand the role of Orexin-A in AD and provide a new treatment target for AD.