| BackgroundsAlzheimer's disease(AD)is one of the most common age-related diseases,also known as senile dementia,which occurs in the elderly and preseniles,and is characterized by progressive cognitive dysfunction and behavioral damage.It seriously affects the patient's life and quality of life,and brings a heavy social and economic burden.In 2015,there were 9.5 million people with dementia in China,accounting for one fifth of the world's total,which seriously affected patients' life expectancy and quality of life,and brought serious social and economic burdens.So far,the etiology and pathogenesis of AD is still unclear,and there is no specific treatment for AD.Therefore,further research on the etiology and pathogenesis of AD is of great significance for exploring treatment methods for the prevention and treatment of AD,and is a major problem that needs to be urgently addressed.Mitochondria plays important role in eukaryotic cells by taking part in biological oxidation and energy conversion,and being involvied in various biological processes such as intracellular homeostasis,proliferation,aging,and cell death.Asymmetric division of mitochondria can lead to weakening or disappear of mitochondrial membrane potential or disappearance,that is,depolarization of mitochondria;a variety of stimuli,both in vivo and in vitro,such as A? oligomers,can also cause pathological depolarization of mitochondria.These depolarized mitochondria can be recycled through mitochondrial fusion or through a selective autophagy,which is called mitophagy.PINK1 is a serine/threonine kinase on the mitochondrial membrane,and Parkin is an E3 ubiquitin protein ligase in the cytoplasm.When the mitochondrial membrane is damaged,PINK1 recruits Parkin to the mitochondria and promotes mitophagy.This reduction in mitochondrial clearance can lead to the accumulation of damaged mitochondria,cause mitochondrial dysfunction,and ultimately lead to the onset of neurodegenerative diseases.It is already known that mitophagy is involved in the pathogenesis of AD and the number of normal mitochondria in the hippocampal neurons of AD patients was significantly reduced..Researches showed that impaired mitochondrial autophagy in AD was associated with abnormal lysosomal function.Transcription factor EB(TFEB)is a transcription factor found in recent years that is closely related to autophagy.It plays an important role in the normal function of lysosomes.TFEB plays a critical role in maintaining autophagy/mitophagy.Studies have found that over-expressing TFEB in APP/PS1 transgenic mice can effectively reduce APP protein production,reduce soluble A?levels and reduce amyloid plaque formation,and reduce intellectual damage in APP/PS1 transgenic mice,indicating that TFEB playing a protective role in ADPattern-recognition receptors(PRRs),expressed by innate immune effector cells,have the capacity to recognize foreign stimuli and senses damage-specific proteins from the body.NLR(NOD-like receptor)is a PRR that is localized in the cell and can recruit precursors of pro-caspase-1 directly or through apoptosis-associated speck-like protein containing a CARD(ASC)to forms a protein complex called the inflammasome.The assembly of inflammasome can activate pro-caspase-1 into mature Caspase-1,which,in turn,converts pro-interleukin-1?(IL-1?)and interleukin-18(IL-18)to mature IL-1? and IL-18.The best characterized inflammasome is the NLRP3 inflammasome.Mitochondria was essential for NLRP3 inflammasome activation and mitochondrial dysfunction-derived signals,such as reactive oxygen species(mtROS),oxidized mtDNA,or externalization of phospholipid cardiolipin,have been suggested to mediate NLRP3 activation.Strong upregulation of NLRP3 inflammasome pathway was found in the brain of both AD patients and APP/PS1 transgenic mice.Specific NLRP3 inflammasome blocker MCC950 was found to promote the clearance of amyloid protein P(A?)in APP/PS1 transgenic mice and improve cognitive function and mitophagy possessed a significant negative regulatory effect on NLRP3 inflammasome activation.Melatonin has been found to improve cognitive decline as well as reduce A? and tau oligomers levels in APP/PS1 transgenic mice by improving mitochondrial function.Are there age related changes of mitophagy,NLRP3 inflammasomes and their related regulatory proteins in the brain of APP/PS1 transgenic mice?What is the effect and mechanism of mitophagy on disorders of APP/PS1 transgenic mice by regulating NLRP3 inflammasome and caspase-1 activity?Is the protective mechanism of melatonin in AD related to promoting mito autophagy regulating NLRP3 inflammasome and caspase-1 activity?Is the mechanism of melatonin promoting mitophagy related to TFEB?Based on the above background,this study first observed the dynamic changes of mitochondrial autophagy,NLRP3 inflammasome and related regulatory proteins with increasing age in the brain of APP/PS1 transgenic mouse models;AP oligomers were used to in vitro culture in SH-SY5Y cells,observe changes in mitophagy,NLRP3 inflammasome and related regulatory proteins,and then pre-treated with mitophagy promoters and NLRP3 inflammasome inhibitors respectively,and observe the effects on the above changes again.Observe the effects of melatonin on the cognitive function improvement of APP/PS1 transgenic mouse models,mitochondrial autophagy,NLRP3 inflammasome and their related regulatory proteins,and the effects of melatonin on TFEB were described.Contents1.Age related changes of mitochondrial autophagy,NLRP3 inflammasome activation and related proteins in APP/PS1 transgenic mouse models.2.Mitophagy reduces cell toxicity of A? by inhibiting NLRP3 inflammasome activation in vitro3.The mechanism of melatonin improving animal cognitive function by promoting mitophagy and inhibiting NLRP3 inflammasome activation in the brain of APP/PS1 transgenic mice.4.Melatonin promotes mitophagy through induction of TFEB nuclear translocationMethods1.Age related changes of mitophagy,NLRP3 inflammasome and related proteins in APP/PS1 transgenic mouse models.APP/PS1 transgenic mice and control group(C57/BL6J mice)were killed at the age of 6 months,9 months,and 12 months.Hippocampus,frontal and temporal cortex were used for the following studies:(1)Age related changes of mitophagy-related proteins PINK1/Parkin and p62/LC3;(2)Mitochondrial related ROS and mitochondrial ultrastructure changes;(3)Expression of NLRP3 inflammasome pathway related proteins ASC,caspase-1,and concentrations of inflammatory factors IL-1?,IL-18;(4)A? deposition and soluble AP40,A?42 concentrations;(5)Cognitive function of animals tested by Morris water maze.2.Mitophagy protect the cells from the cytotoxicity of A? 42 through activation of NLRP3 inflammasome.A? 42 oligomer was added into the culture of SH-SY5Y cells to observe(1)expression of mitophagy-related proteins,(2)mitochondria related ROS and ultrastructure of mitochondria,and(3)changes in NLRP3 inflammasome related proteins and inflammatory cytokines.Then,SH-SY5Y cells were pre-treated with mitophagy inducer MMN or with mitophagy inhibitor MCC950 before being incubated with A? to explore whether induction of mitophagy have protective effects by testing all the parameters mentioned above.3.The mechanism of melatonin improving cognitive function by promoting mitophagy in the brain of APP/PS1 transgenic mice.APP/PS1 transgenic mice were given melatonin in drinking water for 3 month.Compared with mice without melatonin treatment,the following parameters were observed,including(1)changes in cognitive function and(2)mitophagy-related proteins in the brain,(3)mitochondrial ROS and ultrastructure(4)NLRP3 inflammasome and related proteins and concentration of inflammatory cytokines,(5)A? deposition and concentration of soluble A?40 and A?42.4.Melatonin promotes mitophagy in AD by inducing TFEB nuclear translocation.The expression of TFEB in cytoplasm and nucleus were tested in APP/PS1 transgenic mice aged 6 months,9 months,and 12 months,which is pre-treated with melatonin or with distilled water to determine the effect of melatonin on TFEB translocation.TFEB was then overexpressed in SH-SY5Y cells to observe changes in A? induced mitophagy and mtROSResults1.Expression of mitophagy,NLRP3 inflammasome and related proteins changes with increasing age in APP/PS1 transgenic mouse models.(1)Compared with C57/BL6J mice of the same age,expression of PINK-1 and LC3-II was lower in the hippocampus and cortex of APP/PS1 transgenic mice of different age groups,while the level of Parkin and P62 was higher in AD mice,indicating dynamic changes of mitophagy in MD mouse models.(2)In the hippocampal neuron of control C57/BL6J mice,electron microscopic study showed that the mitochondrial membrane was intact and mitochondrial ridges were clearly visible.In the brain of 6-month-old APP/PS1 transgenic mice,a portion of swelling mitochondria,mitochondria with disarrangement of cristae and mitophagy were observed.In the brain of 9-month-old APP/PS1 transgenic mice,morphological disorder of mitochondria was more apparent,with increased number of and more severely damaged mitochondria.Dissolution of bilateral membrane structures and destruction of mitochondrial ridges can be seen,and medullary corpuscle formed by degenerating mitochondria that have deepened and condensed in the axons.In the hippocampus of APP/PS1 transgenic mice at 12 months of age,a large number of medullary corpuscle can be seen.These results indicate that morphological damage of mitochondria increases with age in APP/PS1 mice.Compared with control group,the production of mitochondria ROS increased in APP/PS1 mice,and dynamically increased with increasing age.(3)Compared with C57/BL6J mice of the same age,the expression of ASC?caspase-1 were significantly increased,and the concentrations of inflammatory factors IL-1? and IL-18 was higher in APP/PS1 transgenic mice,and there is a dynamic change with increasing age;(4)Compared with C57/BL6J mice of the same age,APP/PS1 transgenic mice had more senile plaque and higher level of soluble A?40 and A?342,which also increased with aging.;(5)Morris water maze test showed that the escape latency,target quadrant time,and number of platform crossings of the APP/PS1 transgenic mice were significantly longer than those of the control group,and there parameters lengthened as the age increased..2..Mitophagy reduces cell toxicity of A? by inhibiting NLRP3 inflammasome activation in vitro.(1)Pre-treatment with mitophagy inducer NMN and specific NLRP3 inflammasome inhibitor MCC950 can reduce mitochondrial damage caused by A?25-35 in SH-SY5Y cells.After pre-treatment with NMN and MCC950,the number of damaged mitochondria was reduced,the morphologically change of damaged mitochondria ameliorated and the production of mitochondrial ROS decreased in A? 25-35 treated SH-SY5Y cells,.suggesting that promoting mitophagy or inhibiting NLRP3 inflammasome activation can reduce the cytotoxicity of A?(2)Promoting mitophagy can inhibits NLRP3 inflammasome activation,while inhibiting NLRP3 inflammasome activation showed on effect on mitopahgy.After NMN pretreatment,the levels of mitophagy-related proteins PINK1 was significantly increased,Parkin and p62 levels were significantly reduced,and caspase-1 and the level of IL-1? and IL-18 concentration were decreased.In MCC950 pre-treated group,the express of caspase-1 was and production of IL-1? and IL-18 were decreased.The colocalization of PINK1 and LC3 increased.However,no significant changes were observed in the expression of Parkin and p62.And no changes were detected in the colocalization of PINK1 and LC3 by immunofluorescent staining.3.Melatonin improved cognitive function by promoting mitophagy and inhibiting NLRP3 inflammasome iactivation in the brain of APP/PS1 transgenic mice.Melatonin was administered to APP/PS1 transgenic mice through drinking water for 3 months(from 9 to 12 months,0.5 mg melatonin daily).Compared with APP/PS1 transgenic mice treated with placebo,the Morris water maze tests showed shortened escape latency,longer time spent in target quadrant,and increased number of crossing platforms.Pathologically,the number of senile plaque and concentration of soluble A?40 and AP42 also reduced,indicating that melatonin exhibited protective role in the pathogenesis of AD.Furthermore,we found decreased production of mitochondrial ROS and less morphologically damaged mitochondria in melatonin treated group.The expression of mitophagy-related proteins PINK1 were significantly increased,and Parkin and p62 were significantly reduced.NLRP3 inflammasome activity was inhibit and the concentrations of IL-1? and IL-18 were significantly reduced.4.Melatonin promotes mitophagy through induction of TFEB nuclear translocation.The content of TFEB in nucleus of neuron,as detected by Western Blot and immunofluorescence assay,was decrease in APP/PS1 transgenic mouse decreased,and was decreasing with age.Melatonin intervention can increase the content of TFEB in the nucleus in animal models.Overexpressing TFEB in SH-SY5Y cells increased colocalization of PINK1 and LC3,and reduced the production of mitochondrial ROS,suggesting that TFEB can promote mitophagy and antagonize the cytotoxic effect of Ap.Conclusion1.Mitophagy,NLRP3 inflammasome and related proteins changes with increasing age in the brain of APP/PS1 transgenic mice.2.Mitophagy protect the cells from the cytotoxicity of A? 42 through activation of NLRP3 inflammasome in AD cell model.3.Melatonin promotes mitophagy by inducing TFEB nuclear translocation,up-regulates NLRP3 inflammasome activation and exerts protective effects in AD. |
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