Research On The Roles Of Mitochondrial Unfolded Protein Response And NAD+ In The Aging Process Of Mouse Spinal Cord

BackgroundThe spinal cord is the bridge between brain and the outside world.Spinal cord sensory neurons transmit external information to the brain,and spinal cord motor neurons control muscles to produce corresponding behavioral responses.Studies have shown that during the aging process of the nervous system,20–40%of cortical,hippocampal,and peripheral neurons have aging-like properties such as increased DNA damage and increased?-galactosidase activity.We have always paid attention to the aging phenomenon of spinal cord tissues,and studied the mechanism of spinal cord tissue?neuronal?during aging.However,little is known about aging changes in spinal cord tissue and spinal motor neurons.Recent studies have found that"mitochondrial unfolded protein response?mito UPR?"plays an important role in the aging process.mito UPR refers to the response mechanism that cells will initiate when mitochondrial function is impaired or a large amount of unfolded proteins are accumulated in the mitochondria,that is,the expression of molecular chaperones HSP60,HSP10,and protein degrading enzymes CLPP and LONP1 are rapidly up-regulated to assist protein folding.At the same time,the misfolded proteins are degraded to promote protein homeostasis and cell survival of damaged mitochondria.Experimental data in C.elegans,Drosophila and mice have shown that activation of mito UPR can promote the up-regulation of mito UPR marker HSP60 protein expression,which plays a significant role in promoting body health and delaying aging.These results give us an important hint that mito UPR may be involved in the aging process of spinal cord tissue.When looking for a possible upstream signaling pathway that regulates mito UPR,we obtained important clues.The signal pathway initiated by amide adenine dinucleotide?NAD⁺?regulates C.elegans lifespan,possibly by activating mito UPR.This leads us to consider NAD⁺in conjunction with mito UPR.NAD⁺is an enzyme involved in cell energy metabolism and a cofactor for the cell's adaptive response to oxidative stress.Recent research shows that NAD⁺content in the human brain decreases with age,while increased NAD⁺content and improved body function in aging mice.In the field of aging research,NAD⁺plays a key regulatory role in cell signaling pathways,and most attention is on its involvement in regulating the enzymatic response of the sirtuin family?SIRTs?.Among them,SIRT3 is a unique member of the SIRTs family.SIRT3 is localized to mitochondria and translocates to the nucleus in response to cellular stress,regulating mitochondrial homeostasis.Compared with other members,there is more and more evidence to show that SIRT3 is more closely related to human lifespan and is an essential factor in delaying the aging of cells and tissues.Moreover,SIRT3 participates in the homeostasis regulation of mitochondrial proteins and exhibits neuroprotective effects in neurodegenerative diseases.These studies suggest us to focus on the role of the NAD⁺-SIRT3 signaling pathway in spinal aging and to explore its downstream regulation.In summary,mito UPR and NAD⁺play an important role in the aging process of the body.How NAD⁺and mito UPR are related in aging spinal cord tissue is not clear.We proposed the following hypothesis:the degree of aging of spinal cord tissue is closely related to the decline of age-dependent NAD⁺content.Decreased NAD⁺content in aging spinal cord tissue inhibits the function of mitochondrial SIRT3;and further leads to weakened mito UPR activation and participates in the aging process.We intraperitoneally injected young and old mice with NAMPT inhibitor?FK866?to observe the relationship between the NAD⁺-SIRT3 signaling pathway and mito UPR;We established a cell senescence model in motor neurons?NSC-34?in vitro to observe the changes of NAD⁺-SIRT3 signal pathway and mito UPR;Moreover,we treated motor neurons with FK866to reduce their NAD⁺content,block NAD⁺-SIRT3 signal pathway,inhibit mito UPR,which resulted in promotion of neuron senescence;Finally,we added NAD⁺precursor nicotinamide mononuclotide?NMN?to evaluate the effect of NMN on anti-senescence in motor neurons.ObjectiveThrough this study,we tried to deeply reveal the pathophysiology of spinal cord during aging,to clarify the role of mito UPR and NAD⁺in the spinal cord,and to preliminarily explore the potential anti-aging mechanism in order to provide new ideas for clinical application.Method?1?In vivo experiments:2 and 19-month-old SPF male C57BL/6 mice were injected intraperitoneally with FK866?0.5 mg/kg?every other day.The control group was injected with the corresponding dose of vehicle.The mice were divided into four groups:?1?3 months+vehicle group?3M+Ve,n=10?;?2?3 months+FK866 group?3M+FK866,n=10?;?3?20 months+vehicle group?20M+Ve,n=10?;?4?20 months+FK866 group?20M+FK866,n=10?.One month later,the entire spinal cord was isolated after anesthetizing the mice and used for NAD⁺detection and Western blot assay to detect the protein expression of NAMPT,SIRT3,HSP60,HSP10,CLPP,LONP1 and P53.In addition,the spinal cord of 6-month-old and 12-month-old SPF male C57BL/6mice were used to detect the expression of HSP60,HSP10,CLPP and LONP1 by Western blot assay.?2?In vitro experiments:Mouse spinal cord motor neuron cell line NSC-34 were cultured,and CCK-8 was used to detect the effects of FK866 and NMN on cell proliferation.H₂O₂-induced NSC-34 cell senescence model was established.Motor neurons were divided into following groups:Control,H₂O₂,FK866,NMN,NMN+FK866.SA-?-galactosidase staining was used to detect cell senescence.NAD⁺kit was used to detect NAD⁺content.Western blot assay was used to detect the protein expression of NAMPT,SIRT3,HSP60 and P53.Immunofluorescence was used to detect the expression of HSP60.Result?1?In vivo experiments:Western blot showed that the expression levels of LONP1,HSP60,CLPP,and HSP10 were significantly reduced in 20M group as compared with the values in 3M group;The expression levels of LONP1,HSP60,CLPP and HSP10 protein and the content of NAD⁺was significantly reduced in 3M+FK866group than that in 3M+Ve group,but the expression level of P53 was significantly higher than that in in 3M+Ve group.20M+FK866 group also showed similar results tendency when compared with 20M+Ve group.?2?In vitro experiments:?1?H₂O₂ was used to induce senescence in NSC-34 cells;The number of SA-?-galactosidase positive cells in H₂O₂ group was significantly higher than that in control group.P53 protein expression level was increased in H₂O₂ group than that in control group;NAD⁺content and NAMPT,SIRT3 protein expression levels were significantly reduced;The expression levels of mito UPR related protein HSP60 were down-regulated.?2?Treatment of NSC-34 cells with FK866 significantly increased the number of SA-?-galactosidase-positive cells,and increased the expression level of P53protein,which indicating that FK866 accelerated senescence in NSC-34 cells;NAD⁺content and the expression levels of NAMPT and SIRT3 protein in the FK866 group were significantly reduced and the mito UPR related protein HSP60 expression was also down-regulated;However,pretreated with NMN,restored the above parameters returned to normal levels.Conclusion?1?As age increases,mito UPR was weaken,and NAD⁺-SIRT3 signal pathway was impaired in the aging process of spinal cord?motor neurons?in mice;?2?FK866treatment further reduced NAD⁺levels,further inhibited mito UPR,aggravated the aging process of spinal cord tissue?motor neurons?;?3?NMN treatment increased NAD⁺levels and showed the anti-senescence effect in motor neurons.