Mechanisms Of Testosterone Ameliorating Mitochondrial Biogenesis In Aged Male Rats

The brain is a highly differentiated organ and demands high energy.Mitochondrial oxidative phosphorylation generates cellular energy in the form of ATP,which supplies for the neurons to conduct action potential and release neurotransmitters,to ensure normal brain functions.During aging,mitochondrial oxidative phosphorylation capacity declines,ATP synthesis decreases,reactive oxygen species and oxidative damage to macromolecules increases.Meanwhile,the electron transport capacity of mitochondrial complex I,II,III and IV reduced,and the mitochondrial membrane potential decreased.Mitochondrial dysfunction induces oxidative stress and neuronal damage,which is a key hallmark of aging and age-related neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease.Hence,ensuring normal mitochondrial function is essential to delay aging and reduce the risk of age-related neurodegenerative diseases.Several mechanisms involve in maintaining normal function of mitochodria,including preservation of mitochondrial mass through mitochondrial biogenesis,clearance of damaged mitochondria through mitophagy,and optimization of energy efficiency through mitochondrial dynamics.However,abnomal mitochondrial biogenesis and mitophagy may disrupt mitochondrial homeostasis,and lead to mitochondrial dysfunction and cellular dysfunction.Therefore,it is urgent to discover efficient measures for improving mitochondrial dysfunction in the aging process.Serum testosterone（T）concentration in men declines gradually after reaching a peak in about thirty-year-old,which is associated with the onset and progression of age-related neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease.The incidence of Parkinson’s disease in elderly men was significantly higher than that in elderly women.Symptoms in men with AD can be aggravated by T deficiency,suggesting a potential role of T in neurodegenerative diseases.Although studies have shown that T supplementation improves cognitive function of men with Alzheimer’s disease and alleviates motor and non-motor symptoms of Parkinson’s disease,it is unkown whether T prevents or reverses neurodegeneration by improving mitochondrial dysfunction.It is known that T regulates the growth,differentiation,survival or death of neurons through both genomic and non-genomic signaling pathways.The classic genomic pathway is activated by androgen receptor binding to specific DNA response elements in target gene promoters,which subsequently regulates the transcription of mRNA and protein synthesis.Studies have shown that physiological concentrations of T can directly induce neuroprotective effects through androgen receptor in primary neuronal cultures.Moreover,in skeletal muscle cells,H₂O₂-induced oxidative stress reduces the expression of mitochondrial genes,while T increases the expression of mitochondrial genes and nuclear-encoded mitochondrial biogenesis-related genes,and may directly regulate mitochondrial transcription through androgen receptor.In a word,T may ameliorate mitochondrial dysfunction in the brain aging process through regulating mitochondria-related gene expression via androgen receptor.In this study,the effects of T on neuronal functional state,oxidative stress levels and mitochondrial function were analysed in the brain of aged male rats and in the SH-SY5Y cells subjected to oxidative stress,and the possible mechanisms of T regulating mitochondrial biogenesis in damaged neurons was investigated,looking forward to find key molecules and signaling pathways for the prevention or treatment of age-related neurodegenerative diseases.Part 1 Testosterone reduces oxidative damage in aged male rat brainObjective:To observe the effects of testosterone on neuronal functional state and oxidative stress levels in aged male rat brain.Methods:1.Male SD rats were divided into 3 groups:adult 6-month-old（6Mon）group,elderly 24-month-old（24Mon）group and 24-month-old treated with testosterone propionate（TP,24Mon-TP）group.2.Exploration behaviors and coordinated motor behaviors of animals were detected by open field test,cylinder test and tapered beam walking test.3.The mean optical density of the immune responses of tyrosine hydroxylase and dopamine transporter in the substantia nigra,as well as3-nitrotyrosine in the hippocampus was detected by immunohistochemistry,and the nucleus pyknosis was explored in neurons in the hippocampal CA1region stained with hematoxylin-eosin.4.Oxidative stress parameters,such as MDA,GSH,GSSG,GSH-PX,CAT,Cu Zn-SOD and Mn-SOD levels in substantia nigra and hippocampus were detected by spectrophotometry methods.5.The expression of tyrosine hydroxylase and dopamine transporter in substantia nigra,as well as GSH-PX,CAT and Mn-SOD in both substantia nigra and hippocampus was detected by Western Blot.6.The mRNA expression of GSH-PX,CAT and Mn-SOD in substantia nigra and hippocampus were quantified by qPCR.5.Serum T level was determined by T radioimmunoassay kit.Results:1.Compared with 6Mon or 24Mon-TP rats,the serum T level decreased in 24Mon rats.2.Exploration behaviors such as walking,climbing,standing（corrected P<0.0167）,and sniffing（P<0.01）were significantly reduced in 24Mon rats compared with 6Mon rats（P<0.01）.Compared with 24Mon rats,the climbing,standing（corrected P<0.0167）and sniffing（P<0.01）behaviors were significantly increased in 24Mon-TP rats.3.In the cylinder test,the number of times of both forelimbs touching the wall in 24Mon rats was lower than that in 6Mon rats（corrected P<0.0167）,and 24Mon-TP rats had higher forelimb contact times than 24Mon rats（corrected P<0.0167）.The error scores of bilateral hindlimb in tapered beam walking test of 24Mon rats were higher than those of 6Mon rats（P<0.01）,and the error scores of 24Mon-TP rats were lower than those of 24Mon rats（P<0.01）.4.Western Blot and immunohistochemical results revealed that tyrosine hydroxylase and dopamine transporter expression levels in the substantia nigra of 24Mon rats were lower than that in 6Mon rats（P<0.05）,which were significantly higher in 24Mon-TP rats than that in 24Mon rats（P<0.05）.5.The hematoxylin-eosin staining results showed that neurons in the pyramidal layer of the hippocampal CA1 region of 6Mon rats displayed large and clear nuclei,but obvious nuclear pyknosis was observed in a fraction of CA1 neurons in 24Mon rats,which was significantly reduced in CA1 samples from 24Mon-TP rats（corrected P<0.0167）.6.Immunohistochemical results revealed that 3-nitrotyrosine in the hippocampus of 24Mon rats were lower than that in 6Mon and 24Mon-TP rats（corrected P<0.0167）.7.Compared with 6Mon rats,the MDA levels in the substantia nigra and hippocampus of 24Mon rats were increased（P<0.01）,and TP supplementation decreased MDA levels in the two brain regions of aged rats（substantia nigra,P<0.01;hippocampus,P<0.05）.8 Compared with 6Mon rats,the ratio of GSH/GSSG and the activities of GSH-PX,CAT and Mn-SOD were decreased in the two brain regions of24Mon rats（P<0.01）,but were increased in 24Mon-TP rats relative to 24Mon rats（P<0.05）.In turn,the expression of mRNA and protein of GSH-PX,CAT and Mn-SOD was higher in the two brain regions of 24Mon-TP rats than that in 24Mon rats（P<0.05）.Summary:1.TP supplementation increases serum T level in aged male rats.2.TP supplementation improves exploratory behavioral deficits and coordinated motor behaviors disorder in aged male rats.3.TP supplementation ameliorates neuronal functional state in aged male rat brain.4.TP supplementation increases antioxidant capacity in the substantia nigra and hippocampus of aged male rats.Part 2 Ameliorative effects of testosterone on mitochondrial function in aged male rat brain by promoting mitochondrial biogenesisObjective:To explore the effects of T on age-related brain mitochondrial dysfunction in male rats.Methods:1.Male SD rats were grouped according to the first part.2.ATP levels and activity of citrate synthase and mitochondrial complex I-V in substantia nigra and hippocampus were detected by spectrophotometry methods.3.Mitochondrial membrane potential in substantia nigra and hippocampus was detected by flow cytometry.4.The mRNA expression of PGC-1α,NRF-1,TFAM,ND1,COX1,ATP6and AMPK,as well as the mitochondrial DNA copy number in substantia nigra and hippocampus were quantified by qPCR.5.Protein expression of PGC-1α,NRF-1,TFAM,ND1,COX1,ATP6,AMPK and p-AMPK was detected by Western Blot analysis in substantia nigra and hippocampus.Results:1.Compared with the 6Mon rats,the mitochondrial membrane potential in the substantia nigra and hippocampus cells of the 24Mon rats was decreased（P<0.01）,and TP supplementation increased the mitochondrial membrane potential in the substantia nigra and hippocampus cells of aged rats（P<0.05）.2.ATP levels in substantia nigra（corrected P<0.0167）and hippocampus（P<0.01）of 24Mon rats were lower than those of 6Mon rats,and TP supplementation increased ATP levels in substantia nigra（corrected P<0.0167）and hippocampus（P<0.01）of aged rats.3.Compared with 6Mon rats,the activities of complexes I and V（P<0.01）in the substantia nigra and complex V（corrected P<0.0167）in the hippocampus were decreased in 24Mon rats,and TP supplementation increased the activities of complex I（P<0.01）and V（P<0.05）in substantia nigra and complex V（corrected P<0.0167）in hippocampus of 24Mon-TP rats.4.Compared with 6Mon and 24Mon-TP rats,the mRNA and protein levels of mitochondrial biogenesis-related genes PGC-1α,NRF-1 and TFAM in the substantia nigra and hippocampus of 24Mon rats were decreased（P<0.05）.5.Compared with 6Mon rats,the citrate synthase activity and mitochondrial DNA copy number reduced in the substantia nigra and hippocampus of 24Mon male rats（P<0.01）.In contrast,in 24Mon-TP rats,these variables increased in both brain regions（P<0.05）.Likewise,increased expression of ND1,COX1 and ATP6 was detected in the substantia nigra and hippocampus of 24Mon-TP rats relative to 24Mon controls（P<0.05）.6.AMPK mRNA levels were decreased in the substantia nigra and hippocampus of 24Mon rats compared with 6Mon rats（P<0.01）.24Mon-TP rats had higher mRNA levels of AMPK in the substantia nigra（P<0.01）and hippocampus（P<0.05）than 24Mon rats,and the ratio of p-AMPK/AMPK in the substantia nigra and hippocampus of 24Mon-TP rats was increased（P<0.01）.Summary:1.TP supplementation increases mitochondrial membrane potential,ATP content and mitochondrial complex enzyme activities in substantia nigra and hippocampus of aged male rats,and improves mitochondrial dysfunction.2.TP supplementation promotes mitochondrial biogenesis in the substantia nigra and hippocampus of aged male rats.3.Mitochondrial content increases in the substantia nigra and hippocampus of aged male rats after TP supplementation.4.In the brain of aged male rats,the AMPK pathway is activated by TP supplementation.Part 3 Testosterone promotes mitochondrial biogenesis via AMPK and TFAMObjective:To investigate the possible pathway of testosterone regulating mitochondrial biogenesis and its locus of action on TFAM gene.Methods:1.Human neuroblastoma SH-SY5Y cells were administrated with different concentrations of T,H₂O₂,and androgen receptor antagonist flutamide（F）,AMPK inhibitor or small RNA interference（si RNA）of TFAM gene.2.Cell viability,oxidative stress parameter GSH/GSSG,ATP content and citrate synthase activity were detected by spectrophotometry.3.Oxygen consumption rate of cells was detected by Seahorse Bioscience XF24 Analyser,and mitochondrial basic respiratory function,ATP production,maximum respiration and spare respiration capacity were analysed.4.Mitochondrial membrane potential,mitochondrial mass and mitochondrial fragmentation were observed by confocal microscope in cells stained with JC-1 and Mito Tracker Red CMXRos.5.mRNA expression of PGC-1α,NRF-1,TFAM,ND1,COX1 and ATP6,as well as mitochondrial DNA copy number were quantified by qPCR.6.Western Blot analysis was used to determine protein expression of3-NT,PGC-1α,TFAM,AMPK and p-AMPK.7.In the TFAM si RNA transfection（si-TFAM）group,TFAM gene was silenced by si RNA transfection,and the si RNA negative control（NC）group was set up.8.Ch IP-qPCR was used to verify the androgen receptor binding site in the promoter region of TFAM gene.Results:1.10 nM T pretreatment for 24h+200μM H₂O₂treatment for 24h group significantly increased cell viability compared with 200μM H₂O₂treatment alone for 24h group（P<0.01）.Administration of 10μM androgen receptor antagonist F for 1 h before T pretreatment inhibited the ameliorative effect of10 n M T on 200μM H₂O₂-induced decreased cell viability of SH-SY5Y（P<0.01）.2.Compared with control group,GSH decreased,GSSG increased and GSH/GSSG ratio decreased in H₂O₂treatment group,（P<0.01）.T pretreatment increased GSH（P<0.01）,decreased GSSG（P<0.01）,and increased the ratio of GSH/GSSG（P<0.01）,which was inhibited when F was given before T pretreatment（P<0.01）.3.Compared with control group,the protein expression of 3-NT,a biomarker of oxidative stress,was increased in the H₂O₂treatment group（P<0.01）.T pretreatment reduced the expression of 3-NT protein（P<0.01）,whereas administration of F before T pretreatment inhibited the reducing effect of T pretreatment on the 3-NT expression（P<0.01）.4.The basal respiratory function,ATP production,maximal respiration and spare respiration capacity were decreased in the H₂O₂treatment group relative to the control group（P<0.01）.T pretreatment increased basal respiratory function,ATP production,maximal respiration and spare respiration capacity compared with H₂O₂treatment group（P<0.01）.Administration of F before T pretreatment inhibited the ameliorating effect of T pretreatment on H₂O₂-induced mitochondrial bioenergetics defects（P<0.01）.5.The mitochondrial membrane potential in H₂O₂treated group was lower than that in control group（P<0.05）,and T pretreatment increased the membrane potential of H₂O₂treated group（P<0.05）and reached the level of the control group.Administration of F before T pretreatment inhibited the ameliorative effect of T pretreatment on H₂O₂-induced mitochondrial membrane potential depolarization in SH-SY5Y cells（P<0.01）.6.Compared with control group,ATP content in the H₂O₂treatment group decreased（P<0.01）.T pretreatment before H₂O₂treatment increased ATP content（P<0.01）.Administration of F before T pretreatment inhibited the ameliorating effect of T pretreatment on the reduction of ATP content in SH-SY5Y cells induced by H₂O₂（P<0.01）.7.The mitochondrial mass in H₂O₂treatment group was lower than that in control group（P<0.01）,and T pretreatment before H₂O₂treatment increased the mitochondrial mass（P<0.05）.Administration of F before T pretreatment inhibited the enhancing effect of T pretreatment on the reduction of mitochondrial mass in SH-SY5Y cell lines induced by H₂O₂（P<0.05）.8.H₂O₂treatment decreased the mitochondrial copy number（P<0.01）,while T pretreatment increased the mitochondrial copy number（P<0.01）.Administration of F before T pretreatment inhibited the increasing effect of T pretreatment on H₂O₂-induced mitochondrial copy number reduction in SH-SY5Y cell lines（P<0.01）.9.Compared with control group,the mRNA expressions of mitochondrial biogenesis-related genes PGC-1α,NRF-1 and TFAM were decreased in H₂O₂-treated group（P<0.01）,which were increased by T pretreatment（P<0.01）.Administration of F before T pretreatment weakened the effect of T pretreatment on the reduction of PGC-1α,NRF1 and TFAM gene expression in SH-SY5Y cells induced by H₂O₂（P<0.05）.10.Compared with control group,the mRNA expression of mitochondrial dynamics-related gene Drp1 increased and Mfn1 and Opa1decreased in H₂O₂-treated group（P<0.01）.T pretreatment decreased the mRNA levels of Drp1 and increased the mRNA levels of Mfn1 and Opa1 after H₂O₂treatment（P<0.01）.Administration of F before T pretreatment inhibited the effect of T pretreatment on reducing Drp1（P<0.01）and increasing Mfn1（P<0.01）and Opa1（P<0.05）gene expression in SH-SY5Y cells.11.The number of cells with mitochondrial fragmentation in H₂O₂treatment group was more than that in control group（P<0.01）.T pretreatment decreased the number of cells with mitochondrial fragmentation after H₂O₂treatment（P<0.05）.Administration of F before T pretreatment abrogated the reducing effect of T pretreatment on mitochondrial fragmentation in SH-SY5Y cells（P<0.05）.12.T pretreatment increased the expression of PGC-1α,NRF-1 and TFAM after H₂O₂treatment（P<0.01）.Administration of AMPK-specific inhibitor before T pretreatment decreased the expressions of PGC-1α,NRF-1and TFAM compared with T+H₂O₂group（P<0.05）,but did not completely inhibit the up-regulation effect of T on mitochondrial biogenesis-related genes（P<0.05）.13.Compared with NC group,the mitochondrial membrane potential increased in NC+T group（P<0.01）.After TFAM knockdown,the mitochondrial membrane potential of si-TFAM group decreased compared with NC group（P<0.01）,and the mitochondrial membrane potential of si-TFAM+T group was decreased compared with NC+T group（P<0.01）.14.Compared with NC group,ATP level increased in NC+T group（P<0.05）and decreased in si-TFAM group（P<0.01）.The ATP level of si-TFAM+T group was lower than that in NC+T group（P<0.01）.15.Compared with NC group,the citrate synthase activity increased in NC+T group（P<0.01）and decreased in si-TFAM group（P<0.01）.Compared with NC+T group,the activity of citrate synthase decreased in si-TFAM+T group（P<0.01）.16.Compared with NC group,the mitochondrial DNA copy number in the NC+T group was increased（P<0.01）.After TFAM silencing,the mitochondrial DNA copy number in the si-TFAM and si-TFAM+T groups was decreased compared with NC group（P<0.01）,and the mitochondrial DNA copy number in the si-TFAM+T group was decreased compared with NC+T group（P<0.01）.17.Compared with NC group,the expressions of mitochondrial genes ND1,COX1 and ATP6 were up-regulated in NC+T group（P<0.01）and down-regulated in si-TFAM group and si-TFAM+T group（P<0.05）.Compared with NC+T group,the expressions of ND1,COX1 and ATP6 were down-regulated in si-TFAM+T group（P<0.01）.18.The putative androgen receptor binding site is on the upstream promoter region 1285-1299 of TFAM gene.Ch IP-qPCR results showed that the specific amplification in AR group was higher than that in Ig G group（P<0.01）.Agarose gel electrophoresis showed that the bands were brighter in androgen receptor group than in Ig G group.Summary:1.T protects against H₂O₂-induced oxidative damage and cell death in SH-SY5Y cells via androgen receptors.2.T ameliorates mitochondrial dysfunction via androgen receptors.3.T activates androgen receptors and promotes mitochondrial biogenesis and mitochondrial integrity.4.AMPK-specific inhibitors blocks the activation of AMPK signaling pathway,and partially inhibits the up-regulation effect of T on mitochondrial biogenesis-related genes.5.Silencing TFAM gene inhibits the promoting effect of T on mitochondrial biogenesis and mitochondrial function.6.There is an androgen receptor binding site in the TFAM promoter region.Conclusion:1.Testosterone activates the AMPK-PGC-1αpathway and its effector molecules,increases the expression of TFAM,and then promotes mitochondrial biogenesis.2.Androgen receptors activated by testosterone directly bind to the promoter region of TFAM gene and mitochondrial biogenesis is promoted.3.Testosterone supplementation improved neuronal functional status,and ameliorated mitochondrial dysfunction by enhancing both mitochondrial antioxidative capacity and mitochondrial biogenesis of aged male rats.