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MtDNA From Senescent Astrocytes Contributes To The Microglia And Neurons’ Crosstalk In The Evolution Of Brain Aging To Cognitive Dysfunction

Posted on:2024-01-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:W Y ZhangFull Text:PDF
GTID:1524307064973929Subject:Pharmacology
Abstract/Summary:PDF Full Text Request
Aging is an inevitable natural process,in which senescent cells accumulate in various tissues and exhibit a series of degenerative changes in morphology and physiological function as human growth and development reach maturity.The nervous system is the most vulnerable target organ to aging.Studies have shown that organ and cell senescence have different synchronicity,and astrocytes in premature aging mice age earlier than neurons.In the longest-lived human brain,about 30%of astrocytes have already aged,while in AD patients,more than 40%of astrocytes have aged.Therefore,it is speculated that astrocyte senescence may be the basis in the evolution of aging towards AD.Senescent cells can accumulate and release a variety of damage-associated molecular patterns(DAMPs),which directly activate inflammatory responses or indirectly cause adjacent cells to undergo senescence through paracrine signaling.Furthermore,DAMPs can also play important roles in accelerating neurodegenerative processes by entering the cytoplasm and extracellular space,participating in cell-cell contacts required for inflammation and destruction of neuron-glia interactions.Thus,the DAMPs secreted by senescent astrocytes are important contributors to neurodegenerative diseases,and mitochondrial dysfunction,which is considered a hallmark of aging,underlies the physiological functions of astrocytes.mtDNA,an important factor released during mitochondrial dysfunction,is destabilized in aging animal models of mitochondrial dysfunction,causing the mitochondrial membrane pores to open and mtDNA to be released into the cytoplasm and extracellular space.Studies have shown that circulating mtDNA increases with age,is associated with the accumulation of senescent cells,and is positively correlated with the degree of chronic inflammation in the body.Other studies have shown that the release of mtDNA due to mitochondrial dysfunction in aging is a key factor in neurodegenerative disease progression,and is related to the occurrence and development of Parkinson’s disease.In addition,a significant increase in mtDNA copy number was found in the cerebrospinal fluid of AD patients.Therefore,we speculate that the release of mtDNA by senescent astrocytes may be the material basis for the transformation from aging to AD.However,the multidimensional molecular regulatory mechanism of mtDNA in promoting the evolution of aging to AD and whether it has different tendencies towards inflammatory signaling pathways in different target cells remain to be clarified.Currently,it has been discovered that in the case of mitochondrial dysfunction,mtDNA can be released into the cytoplasm or extracellular space,recognized by ①TLR9,②cGAS,or③NLRP3 receptors,and activate the MyD88-NF-κB,STING-I-IFN,or Caspase1 inflammatory signaling pathways,releasing a large number of pro-inflammatory cytokines and participating in the occurrence and development of numerous inflammatory-related diseases.As the main immune effector cells in the central nervous system,microglia play a dual role in neuroprotection and immune damage during the occurrence and development of AD.It is not clear whether cells in the brain will respond to free mtDNA in the brain during the aging process,participate in the brain’s aging process towards AD,and whether the above three DNA recognition pathways in microglia or neurons in the brain all recognize mtDNA and play a role.The research content of this paper is divided into three parts based on the background described above.Part 1:Investigation on the role of the senescent of astrocytes during the progression from brain aging to cognitive impairment.(1)The impact of age on cognitive function in miceMethods:A study was conducted to evaluate the relationship between cognitive impairment and age by constructing three different age nodes for mice,namely 4-month-old,18-month-old,and 26-month-old,to simulate the young,middle-aged,and elderly periods of human life.Water maze behavioral experiments were used to detect changes in cognitive impairment in mice.Results:The spatial exploration and navigation abilities of mice continuously declined with increasing age,with the most significant decline observed in 26-month-old mice.These findings indicate that age-related cognitive impairment is prone to occur in older mice.(2)The effect of age on brain cell senescence in miceMethods:In order to explore possible causes of cognitive impairment,a study conducted immunofluorescent double staining of natural aging mice’s astrocytes(GFAP+)and neurons(MAP2+,NEUN+)co-expressing with senescent markers(P16INK4a+,P21WAF1+),as well as detection of neuronal degeneration(HE,Nissl staining),dendritic changes(MAP2+),neuronal degradation level(FJC staining),and expression levels of synaptic pre-and post-membrane functional proteins(SYN+,PSD95+).Additionally,mitochondrial function changes and oxidative level changes in the brain were examined,and electron microscopy was used to observe changes in mitochondrial structure in astrocytes,microglia,and neurons of mice of different ages.Furthermore,the activation of microglia(Iba1+)and expression levels of inflammatory factors(TNF-α,IL-6,IL-1β)in the brain were also detected.Results:① With increasing age of mice,activation of hippocampal astrocytes(GFAP+)increased,and astrocytes and neurons expressed cellular senescent markers P16INK4a and P21WAF1 extensively at 18 months of age;②neuronal senescent marker P16INK4a was also extensively expressed in 18-month-old mice,but P21WAF1 expression was only detected in the hippocampal DG area at 26 months of age;③neuronal morphology was loose,the number of neurons decreased,dendrites decreased,and expression levels of synaptic pre-and post-membrane functional proteins(SYN,PSD95)decreased,accompanied by neuronal degeneration;④in the hippocampal area of mice,mitochondrial function(membrane potential,ATP)decreased and oxidative level(MDA,protein carbonyl content)increased,and changes in mitochondrial cristae structure were found in astrocytes;and⑤microglia were activated(IBA1+)and hippocampal inflammatory factors(TNF-α,IL-6,IL-1β)were released.The study results indicate that with increasing age,astrocytic mitochondrial structure in mice’s brains is abnormal and aging phenotypes appear,microglia are activated,and neurons are damaged.(3)The impact of replicative senescent model of astrocytes on microglia and neurons.Methods:In order to further investigate whether senescent astrocytes could have detrimental effects on oligodendrocytes or neurons,a replicative senescence model of astrocytes was established using serial passaging methods to observe their interactions with microglia and neurons.Changes in the expression of senescence-associated proteins P53,P21,and P16,as well as secretion levels of senescence-associated phenotype IL-6,were detected in replicative senescent astrocytes.In addition,changes in mitochondrial membrane potential,ATP,and ROS were also measured.Furthermore,the effects of the senescent astrocyte-conditioned medium on the proliferation of neurons and secretion of inflammatory factors(TNF-α,IL-6,IL-1β)in microglia-conditioned medium were evaluated using MTT assays.Results:① Replicative senescent astrocytes exhibited increased expression of senescence-associated proteins P53,P21,and P16,as well as elevated secretion of senescence-associated phenotype IL-6,accompanied by a decrease in mitochondrial membrane potential and ATP and an increase in intracellular ROS levels;②when microglia were exposed to senescent astrocyte-conditioned medium,the secretion of TNF-α and IL-6 from microglia increased,and the survival rate of neurons significantly decreased;③after exposure to senescent astrocyte-conditioned medium,the survival rate of neurons also decreased.These results suggest that replicative senescent astrocytes can directly cause neuronal damage or indirectly induce neuronal damage by activating microglia in vitro.(4)The impact of oxidative stress-induced astrocytes senescent model on microglia and neurons.Methods:An acute stress-induced cellular senescence model was constructed to investigate the impact of senescence in oxidative stress-induced astrocytes on other cells in the brain.The changes in the expression of senescence-related proteins,including P53,P21,and P16,and the secretion of the senescence-related phenotype IL-6 were detected in oxidative senescent astrocytes.Additionally,changes in mitochondrial membrane potential,ATP,ROS,and the expression of mitochondrial fission and fusion proteins(Drpl,Fis1,Opal)were also examined.Furthermore,the proliferation of neurons and the secretion of inflammatory factors(TNF-α,IL-6,IL-1β)in the supernatant of microglia were detected using the MTT assay after direct or indirect exposure to senescent astrocytes.Results:① The expression of senescence-related proteins P53,P21,and P16 increased in oxidative senescent astrocytes,accompanied by an increase in the secretion of senescence-related phenotype IL-6.The mitochondrial membrane potential and ATP levels rapidly decreased,and intracellular ROS levels significantly increased.The expression of mitochondrial fission protein Drp-1 also increased.②Oxidative senescent astrocytes can cause neuronal damage through direct or indirect effects on neurons by activating microglia to secrete inflammatory factors(TNF-α,IL-6).These findings suggest that oxidative senescent astrocytes can cause neuronal damage through direct or indirect pathways.(5)Investigation on the neuroprotective effects of enhanced mitochondrial function in senescent astrocytesMethods:Berberine has been demonstrated to protect mitochondria and improve mitochondrial function,so we intervened in astrocytes induced by a cellular senescent model as mitochondrial protectants and investigated whether they could protect astrocytes from senescence and reduce their ability to directly or indirectly damage neurons.The study examined the levels of senescence-related proteins P53,P21,and P16 and IL-6 secretion in oxidative astrocytes after intervention with berberine,as well as changes in mitochondrial membrane potential and expression of fission and fusion proteins(Drp1,Fis1,Opal)in astrocytes.In addition,we tested the effects of replicative and oxidative senescent astrocytes on mitochondrial membrane potential,ATP,LDH secretion,neuronal degradation,and the expression of synaptic membrane function proteins(SYN,PSD95)in neurons directly or indirectly affected by astrocytes.Results:Berberine could down-regulate the expression of senescence-related proteins in oxidative astrocytes,improve mitochondrial membrane potential levels and stable mitochondrial fission ability,and reduce the expression of Drp1.Berberine could also improve the damage caused by the conditioned medium of replicative and oxidative senescent astrocytes to neuronal mitochondrial membrane potential and ATP,significantly decrease neuronal LDH secretion,improve neuronal degradation,and enhance the expression levels of synaptic membrane function proteins SYN and PSD95 in neurons.These results suggest that berberine can alleviate the senescent phenotype of astrocytes,improve mitochondrial function in senescent astrocytes,reduce neuronal damage,and enhance synaptic function.Part 2:Exploring the release of mtDNA from senescent astrocytes as a central molecule in the cell interaction underlying cognitive decline during brain aging.(1)Study on the extracellular release of mtDNA from senescent astrocytesMethods:In this study,a dsDNA detection kit was used to detect the levels of dsDNA in the supernatants of two different astrocyte senescent models after pre-treatment with BBR.Taqman probes were used to detect the copy number of mtDNA in the cerebrospinal fluid,cytoplasm of brain cells,and serum of mice of different ages.Results:① The levels of free mtDNA in the supernatants of replicative and oxidative senescent astrocytes were significantly increased,and BBR could reduce the extracellular release of mtDNA by protecting mitochondria;② With increasing age,the levels of free mtDNA in the cytoplasm of brain cells and hippocampal cells of mice significantly increased,while the levels of free mtDNA in the serum significantly decreased.These findings suggest that astrocytes may undergo senescence and release mtDNA extracellularly with age.(2)Study on the impact of mtDNA analog injection into lateral ventricle on cognitive functionMethods:To investigate whether mtDNA is involved in the development of cognitive dysfunction in mice,different concentrations of mtDNA analogues were injected into the lateral ventricle of young mice.Results:① After 7,14,and 28 days of mtDNA analogue injection,mice in the Y-maze behavioral test exhibited decreased interest in exploring novel objects;②in the water maze behavioral test,mice showed cognitive dysfunction after 14 and 28 days of mtDNA analogue injection,with the CpG-1826 20μg injection group showing the most significant impairment.These results indicate that injection of mtDNA analogues into the lateral ventricle could induce cognitive dysfunction in young mice.(3)Study on the activation of microglia induced by mtDNA analog injection into lateral ventriclesMethods:To investigate whether mtDNA induces activation of microglia and production of inflammatory cytokines in mouse brain,the study examined the levels of microglial activation(Iba1+)and expression of inflammatory cytokines(TNF-α,IL-6,IL-β)in the brains of mice after intracerebroventricular injection of mtDNA analog.Results:Intracerebroventricular injection of mtDNA analog for 14 and 28 days significantly increased the number of activated microglia in the mouse brain,and the degree of activation was positively correlated with the dose of mtDNA analog.In addition,the expression of the inflammatory cytokines TNF-α,IL-6,and IL-1β in the brain was also increased.These findings suggest that intracerebroventricular injection of mtDNA analog induces activation of microglia and production of inflammatory cytokines in the mouse brain,and the degree of activation is positively correlated with the dose of mtDNA analog.(4)Study on neuronal damage after mtDNA analog injection into the lateral ventricleMethods:In order to investigate whether mtDNA could cause changes in brain neuronal structure and function,immunohistochemical analysis was performed to detect the expression levels of dendritic marker protein MAP2,synaptic membrane functional proteins(SYN,PSD95)in the brains of mice after mtDNA analog injection into the lateral ventricle.The changes in neuronal structure and number were observed by HE and Nissl staining,and the alterations in dendrite length,number,branching,and spine density of hippocampal neurons were analyzed by Golgi silver staining.Results:① The expression of MAP2 in the cortex and CA1 area of neurons decreased significantly,as well as the expression of synaptic membrane functional proteins,after 28 days of mtDNA analog injection into the lateral ventricle;②after 28 days of mtDNA analog injection,the dendrite length,number,branching,and spine density of hippocampal neurons decreased significantly,and the degree of decrease was more significant with the increase of dosage;③after 14 and 28 days of mtDNA analog injection,the cytoplasm of pyramidal and granular neurons appeared to be deeply stained,similar to that in naturally aging mice,but no significant effect was found on the number of neurons.The results of this study indicate that mtDNA analog injection into the lateral ventricle for 28 days can cause neuronal damage in mice,and the degree of damage is positively correlated with the dosage of mtDNA analog administered.(5)study on the role of senescent astrocytes releasing mtDNA in causing neuronal damageMethods:To investigate whether mtDNA can cause direct or indirect damage to neurons,the study examined the effects of conditioned media from replicative and oxidative senescent astrocytes on mitochondrial membrane potential,ATP production,LDH secretion,and neuronal degeneration and the expression of synaptic pre-and postsynaptic functional proteins(SYN,PSD95)in neurons.Results:mtDNA in the conditioned media of senescent astrocytes could directly or indirectly cause a decrease in neuronal membrane potential and ATP production,as well as an increase in LDH secretion and neuronal degeneration.The fluorescence intensity of synaptic functional proteins in neurons also decreased.However,treatment with DNAse I to degrade mtDNA prior to exposure to the conditioned media significantly improved the direct or indirect damage to neurons induced by the senescent astrocyte-conditioned media.These findings suggest that mtDNA released from senescent astrocytes can cause direct or indirect damage to neurons.Part 3:Activation of TLR9,cGAS,and NLRP3 Inflammatory Signaling Pathways by mtDNA Promotes Neuronal Damage.(1)Study on the activation of TLR9,cGAS,and NLRP3 Inflammatory Signaling Pathways in microglia and neurons.Methods:In this study,the expression levels of the mtDNA recognition receptors TLR9,cGAS,and NLRP3 were detected by immunofluorescence co-staining and protein expression experiments in naturally aged mice,mice injected with mtDNA analog into the lateral ventricle of the brain,and in vitro cultured microglia and neurons.The aim was to investigate whether mtDNA could activate the three DNA recognition receptors in microglia and neurons and to detect the levels of pro-inflammatory cytokines in the cell supernatant after the mtDNA analogues were applied to microglia and neurons in vitro.Results:① The co-localization of TLR9,cGAS,and NLRP3 in glial cells and neurons in the brains of naturally aged mice significantly increased with age.②The co-localization of TLR9,cGAS,and NLRP3 in microglia and neurons in the brains of mice injected with mtDNA analogues into the lateral ventricle of the brain increased with the dose of mtDNA analogues.③In vitro intervention of microglia and neurons with mtDNA analogues could increase the expression of TLR9,cGAS,and NLRP3 protein pathways in both types of cells.④After mtDNA analogues intervention,the secretion levels of TNF-α and IL-6 in the supernatant of microglia increased,and the secretion levels of IL-1β in neurons increased.These results indicate that mtDNA can activate the protein expression of TLR9,cGAS,and NLRP3,three DNA recognition receptors in microglia and neurons,and can induce the release of pro-inflammatory cytokines.(2)mtDNA analogs lead to neuronal damage through the TLR9,cGAS,and NLRP3 signaling pathwaysMethods:The secretion of IL-1β in the brains of aging mice and in mice injected with mtDNA analogues into the lateral ventricle is significantly increased.In vitro,after exposure to conditioned media from senescent astrocytes,some Neuro-2a cells exhibit cell body swelling.Therefore,does the release of mtDNA from senescent astrocytes act as an exogenous DAMP to mediate the occurrence of pyroptosis in neuronal cells?In this study,the expression levels of GSDMD-N in neurons were detected after direct and indirect effects of replicative and oxidative senescent astrocytes on neurons.Furthermore,the expression of GSDMD-N in neurons,as well as changes in neuronal degeneration and synaptic functional protein expression,were further verified after direct or indirect effects of mtDNA analogues on neurons.Results:① Conditioned media from replicative and oxidative senescent astrocytes caused an increase in GSDMD-N expression in neurons;② direct exposure to mtDNA analogues also caused an increase in GSDMD-N expression in neurons,while neuronal degeneration levels increased and synaptic functional protein expression decreased;③conditioned media from senescent astrocytes caused an increase in GSDMD-N expression in neurons through indirect effects between astrocytes and neurons;④Indirect exposure to mtDNA analogues through astrocytes also caused an increase in GSDMD-N expression in neurons,while neuronal degeneration levels increased and synaptic functional protein expression decreased.These study results indicate that the activation of TLR9,cGAS,and NLRP3 signaling mediated by mtDNA can induce pyroptosis in neurons,leading to neuronal degeneration and synaptic functional impairment.In summary,this study draws the following conclusions:(1)During the progression of brain aging to cognitive impairment,astrocytes undergo senescence and mitochondrial dysfunction,leading to the release of mtDNA into the cytoplasm or extracellular space.(2)mtDNA is a key molecule in the cell interactions involved in the progression of brain aging to cognitive impairment.(3)The released mtDNA can activate inflammatory signaling pathways via DNA pattern recognition receptors(TLR9,cGAS,NLRP3),amplifying the inflammatory response by activating microglia,accelerating neuronal damage,and inducing pyroptosis in neurons directly.
Keywords/Search Tags:Astrocyte, Cell senescence, Cognitive dysfunction, Cell interaction, mtDNA, TLR9, cGAS, NLRP3
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