The Effects Of Mysm1 On Astrocytic Metabolism Processes And Its Function On The Control Of Cognition

【Background】The most fundamental mental process in humans is cognition,which is the process of learning or using knowledge or processing information.Sensation,perception,memory,thought,imagination,and language are all part of it.The process by which the human brain processes information from the outside environment into internal mental activity that in turn control behavior is known as information processing and is sometimes referred to as cognitive processing.According to research,troops who spend a lot of time in unique locations like plateaus or submarines will experience some degree of cognitive impairment.For instance,when it comes to shooting,a military skill that is embodied in the skills and tactics of officers and soldiers,cognitive function has a significant impact on both speed and time depth fit.When plateau officers and soldiers are compared to the lower altitude group,their reaction times are significantly longer,their target tracking speed is significantly slower,and as a result,their overall level of shooting performance declines.The level of combat effectiveness and fighting capabilities of military troops can be improved by improving their cognitive dysfunction.Alzheimer’s disease is an example of a neurodegenerative disease that frequently comes with varied degrees of cognitive impairment.The incidence of chronic diseases,such as neurodegenerative diseases,has recently increased due to the rapid rise in the elderly population and rising life expectancy,endangering the physical and mental health of the elderly and negatively impacting their quality of life.These diseases also cause excruciating pain for patients and place a significant burden on families and society,making them a serious social issue.To improve the combat efficiency of officers and troops and lessen the suffering of patients with neurodegenerative disorders,it is crucial to understand the mechanisms of cognitive impairment and devise intervention approaches.Astrocytes are the largest glial cells in the mammalian brain and make up 20%–40%of all glial cells in the brain.They are crucial for the growth and operation of the central nervous system.In contrast to neurons,astrocytes have several properties that are yet unknown because there aren’t any precise markers or manipulative methods available.Recent research has demonstrated that astrocytes play a variety of roles in the central nervous system（CNS）,including helping to form synapse,preserving a constant ion concentration both inside and outside the cell,transmitting neurotransmitters,and establishing and maintaining the blood-brain barrier.In order to form"tripartite synapses"and participate actively in synaptic processing,astrocytes can interact with synapses and neurons.However,under pathological circumstances,astrocyte-neuron interactions may be significantly altered,having a significant impact on the brain circuits that support memory formation and cognitive function.Many investigations have established that astrocytes are really involved in cognitive functions,despite the fact that the mechanism of astrocyte action is still not entirely understood.Using Alzheimer’s disease（AD）as an example,analysis of postmortem AD brain tissue has shown that reactive astrocytes accumulate around Aβdeposits.Similar to microglia,upon exposure to Aβ,astrocytes polarize to the A1 state and subsequently release cytokines（e.g.,IL-1βor IL-6）,nitric oxide,reactive oxygen species,and excess glutamate.This series of neurotoxicity or excitotoxicity eventually evolves into neuronal loss and neurodegeneration.In addition,BACE1 expression was found in astrocytes,suggesting that astrocytes may be involved in Aβaggregation.To determine the source of Aβin astrocytes,Veeraraghavalu et al.isolated primary astrocytes from the brains of neonatal PS1ΔE9flox or APPswe mice and 8-week APPswe/PS1ΔE9flo mice,and they detected the expression of astrocyte-secreted Aβin the culture medium.Mysm1,belonging to the metalloproteinase family,has deubiquitinating enzyme catalytic activity.It has three structural domains,SANT,SWIRN,and MPN.Mysm1has important roles in hematopoietic stem cells,lymphocytes,and mature blood cells.2011:Jiang et al.found that Mysm1 plays a very important role in early B-cell development and that Mysm1 deficiency leads to blocked early B-cell lineage-directed differentiation and gene expression of EBF1 and other B-lymphocytes.In2013,Nandakumar et al.found that Mysm1 plays a large role in the maturation of NK cells and that Mysm1^-/-NK cells are phenotypically immature,but NK cell lineage direction is normal,i.e.,NK cell differentiation is not affected.In 2014,Won et al.found that Mysm1 plays an important role in the growth and development of dendritic cells（DCs）,differentiation of bone marrow progenitor cells into dendritic cells,and has a selective role in the formation of early hematopoietic precursor DCs induced by FLT3L;in 2015,Gekara et al.found that Mysm1,a key regulator of natural immunity,can prevent excessive immune responses by inhibiting the pattern recognition receptor（PRR）pathway,thereby preventing the inflammatory response.In 2016,Li et al.found that Mysm1 plays a crucial role in the maintenance and differentiation of MSC.Mysm1 may serve as a potential therapeutic target for regulating MSC spectrum differentiation and may be used for the treatment of metabolic bone diseases such as osteoporosis.In 2017,F?rster et al.found that Mysm1 plays an important role in the regulation of cell activation,proliferation,cytokine production,and apoptosis of T cells and maintains the number of peripheral CD8+T cells.In 2020,Tian et al.found that Mysm1 suppresses innate immunity and autoimmunity through the c GAS-STING pathway and can suppress systemic lupus erythematosus.In 2021,Tian et al.showed that Mysm1 can delay aging by promoting DNA repair.Astrocytes and Mysm1 co-localized significantly in the previous study’s findings,and Mysm1 expression was increased in the hippocampus of mice with cognitive impairment brought on by neuroinflammation.Meanwhile,knocking down Mysm1could lessen the mice’s depressive-like behavior by controlling the expression of ATP.Consequently,the focus of this work will be on Mysm1’s impact on astrocyte metabolic activity and its involvement in cognitive regulation.【Purpose】Investigating the effect of Mysm1 on astrocyte metabolic function and the role it plays in cognitive regulation.【Method】1.Mysm1 expression was detected in the human brain using immunohistochemical staining,in the brain tissue of mice of various ages using immunofluorescence staining,in the young and old mice using Western blot and real-time fluorescence quantitative PCR,and in the expression of aging genes,glycolytic genes,and related genes involved in the control of oxidative stress using real-time fluorescence quantitative PCR.2.In vitro inflammation and aging models were constructed,and mitochondrial membrane potential was detected by the Mitochondrial Membrane Potential Assay Kit;cellular oxygen consumption rate was detected by the O2K Energy Metabolism Analyzer.Cellular ATP content was detected by an ATP assay kit;oxidative stress level was detected by an oxidative stress assay kit;ROS content was detected by a superoxide anion fluorescence probe;astrocytes transferred to control（sh Ctrl）and knockdown Mysm1 viruses（sh Mysm1）were stimulated with hydrogen peroxide,and ROS content was detected by a superoxide anion fluorescence probe;ROS content was detected by transmission electron microscopy to observe the morphology,structure,and number of mitochondria.3.Astrocytes transferred into control（sh Ctrl）and knockdown Mysm1 virus（sh Mysm1）were subjected to transcriptomic,proteomic and metabolomic analyses,and the effects of knockdown Mysm1 on astrocytes were analyzed by sequencing results;Western blot was performed to detect the signaling pathways that Mysm1 may regulate in astrocytes and to verify its role by inhibitors of key pathway molecules;Co-IP was performed to detect proteins that interact with Mysm1 and to clarify the mechanisms by which Mysm1 regulates the metabolic functions of astrocytes.【Results】1.Elevated Mysm1 expression in the hippocampus in cognitive impairment models.Immunohistochemical staining showed that Mysm1 expression was significantly elevated in the hippocampus of elderly and AD patients;immunofluorescence staining showed that Mysm1 expression was elevated in aged mice and AD model mice.The results of real-time fluorescence quantitative PCR and Western blot showed that the expression level of Mysm1 was significantly higher in hippocampal tissues of 8-week-old and 48-week-old mice than in 48-week-old mice.2.Cognitively impaired mice exhibit metabolic disturbances.Behavioral evaluation of young and old mice showed that there was no significant difference between young and old mice in terms of functional capacity in the open field experiment;the results of the water maze experiment showed that old mice took longer to find the hidden platform during the learning period and traversed the hidden platform less frequently during the testing period.The results of real-time fluorescence quantitative PCR showed that the expression of senescence genes,inflammatory factors,and genes related to key enzymes of glycolysis were elevated in aged mice.3.Knockdown of Mysm1 improves cognitive impairment in aged miceReal-time fluorescence quantitative PCR and Western blot were used to detect the efficiency of viral knockdown;the results of open field experiment showed that there was no significant difference in motor ability between the control group（AAV-CON group）and knockdown Mysm1 group（AAV-GFAP-Cre group）;the water maze experiment showed that mice in the AAV-sh Mysm1 group found the hidden platform in a shorter time during the learning period and at the same time found the platform more often during the testing period.The water maze experiment showed that the mice in the AAV-sh Mysm1 group found the hidden platform for a shorter period of time during the learning period and more times during the testing period,indicating that the mice had improved cognitive function.The results of real-time fluorescence quantitative PCR showed that the expression of oxidative stress-related genes was reduced in the AAV-GFAP-Cre group.4.In vitro construction of aging and inflammation models leads to metabolic disorders in astrocytesIn vitro,primary astrocytes were isolated and stimulated with 50μM H₂O₂for 2hours in P3,followed by 48 hours of incubation to construct a senescence model;40ng/m L TNF-α+10 ng/m L IL-1βwas administered to the cells for 24 hours to construct an inflammation model.The mitochondrial membrane potential assay kit revealed that the mitochondrial membrane potential of astrocytes decreased after stimulation;the oxygen consumption rate of the cells was significantly reduced after stimulation.5.Enhanced mitochondrial function in astrocytes after knockdown of Mysm1The results of the ATP assay showed that the intracellular ATP content was higher in the knockdown Mysm1 group（sh Mysm1 group）compared with the control group（sh Ctrl group）;meanwhile,the results of the intracellular lactate assay showed that the lactate content was lower in the sh Mysm1 group,while the expression of key enzymes involved in glycolysis（GLUT1,G6PD,PKM2,LDHA,and MCT4）was significantly reduced in the sh Mysm1 group by real-time fluorescence quantitative PCR and Western blot showed that the expression of most key enzymes was significantly lower in the sh Mysm1 group.G6PD,PKM2,LDHA,and MCT4),the sh Mysm1 group showed that the expression of most key enzymes(GLUT The intracellular CS content was measured by a citrate synthase（CS）assay kit and was found to be somewhat elevated in the sh Mysm1 group.Western blot analysis revealed that the sh Mysm1 group had higher levels of Tomm20 and COX IV expression,and Mito-tracker fluorescence labeling was brighter,indicating a higher mitochondrial content.This suggests that Mysm1 may play a role in regulating mitochondria in astrocytes.6.Knockdown of Mysm1 ameliorated the elevated levels of oxidative stress and mitochondrial damage caused by external stimuli.The SOD,GSSG,and MDA contents in the sh Ctrl and sh Mysm1 groups were detected,and the SOD and GSSG contents in the sh Mysm1 group were increased while the MDA content was decreased.The level of oxidative stress in astrocytes was reduced after the knockdown of Mysm1.The superoxide anion of astrocytes was detected after H₂O₂stimulation of the cells,and it was found that the sh Mysm1+H₂O₂superoxide anion content was lower than that of the sh Ctrl+H₂O₂group.After inflammatory stimulation,electron microscopy was used to examine the number and morphology of mitochondria,and it was discovered that the sh Mysm1 group had significantly better mitochondrial structure and morphology than the sh Ctrl group.7.Histological results show changes in astrocyte metabolism-related molecules after knockdown of Mysm1.Transcriptomics,proteomics,and metabolomics were performed on cells in the sh Ctrl and sh Mysm1 groups.Transcriptomics showed that knockdown of Mysm1increased the expression of genes involved in the composition of the mitochondrial respiratory chain;proteomic results showed a significant increase in proteins related to the respiratory chain complex in the sh Mysm1 group,while molecular changes were mainly in oxidative phosphorylation,the TCA cycle,and AMPK.The metabolomic results showed that the content of key molecules of glycolysis was decreased and the content of molecules related to the TCA cycle was increased in the sh Mysm1 group,while the changes in metabolic molecules involving glycolysis and the TCA cycle were obvious.8.Mysm1 and p53 interact to activate the p53-AMPK pathway to promote mitochondrial genesis.Western blot assay of AMPK and its upstream and downstream molecules showed that p-AMPK expression was elevated,along with its upstream p-p53 and downstream Sirt1 and PGC1αexpression.Subsequently,a p53 inhibitor and an AMPK inhibitor were added to the cells,and the results showed that both inhibitors could inhibit the ATP elevation caused by Mysm1 knockdown;the Co-IP assay showed that Mysm1 and p53 interacted.【Conclusion】（1）Mysm1 expression was elevated in the hippocampus of aged and aged mice,whereas knockdown of Mysm1 in aged mice decreased oxidative stress levels and improved cognitive impairment.（2）Knockdown of Mysm1 enhances mitochondrial function in astrocytes and reduces mitochondrial damage and elevated levels of oxidative stress caused by external stimuli.（3）Mysm1 regulates the p53-AMPK pathway by interacting with p53.