| Alzheimer's disease(AD)is the most common type of age-associated dementia,which has a long incubation period and a slow progression of symptoms.And dementia symptoms due to AD seriously affect the patients' daily activities and quality of life.The pathogenic mechanism of AD is complated,including misfolding and aggregation of proteins,aggregation of amyloid-?(A?)peptide,metabolic and mitochondrial dysfunction,excitotoxicity,Ca2+ dysregulation,microglia dysfunction,neuroinflammation,and oxidative stress.As a result,neuroinflammation and oxidative stress are thought to play a dominant role in the pathological process of AD.The exact pathogenesis and etiology of AD,especially the various pathological changes that selectively contribute to the decline in cognitive domains require further elucidation,.Cells have different defense systems to protect themselves against oxidative stress,and regulate oxidative stress-induced damage.The second antioxidant response is considered to be the most important defense system which is regulated by Nrf2.The transcription factor,Nrf2 is widely considered the master regulator of oxidative stress that regulates the expression of many proteins of the body's resistance to internal and external oxidation or chemical stimulation.Under physiological conditions,Nrf2 is bound to Keap1 in the cytoplasm,which maintains Nrf2 at low levels.Under stress conditions,Nrf2 is activated and dissociated from KEAP1,then Nrf2 translocates from the cytoplasm to the nucleus,and binds with antioxidant response element(ARE)to form Nrf2/ARE signaling pathway.The activation of Nrf2/ARE signaling pathway can induce electrophile-responsive element(EpRE)-regulated gene expression,the target genes of Nrf2 include proteins involved in the regulationof the synthesis of glutathione(GSH),antioxidant proteins/enzymes,drug-metabolizing enzymes or drug transporters,proteasome subunits,pentose phosphate pathway enzymes,and enzymes involved in nucleotide synthesis,such as heme oxygenase-1(HO-1),glutathione-S-transferase(GST),Q10,quinone oxidoreductase-1(NQO1),thioredoxins(Trxs),superoxide dismutase-1(SOD1),glutathione peroxidase(GPx)and phase II and phase III enzymes.The Nrf2/ARE signaling pathway can protect cells against oxidative damage and exogenous toxic substances,maintain the steady state of the cellular redox homeostasis.The Nrf2/ARE signaling pathway plays an important role in protecting the cell from oxidative damage.Nrf2 can play a role in autoimmunity through the regulation of the anti-inflammatory and pro-inflammatory cytokines.And Nrf2 can also enhance autophagy through p62/SQSTM1 pathway.Recently,one new function of Nrf2 was discovered.Nrf2 can regulate mitochondrial membrane potential and the availability of substrates for respiration and ATP synthesis.Therefore,Nrf2 is considered to be a master regulator of inflammation,autophagy,mitochondrial function and oxidative stress which is going to be a potential therapeutic target in the field of neuroprotection research.Senescence accelerated mouse prone 8(SAMP8)is an age-related spontaneous deterioration model characterized by early-onset memory and behavioral impairment at 4 months of age.The 4-8 months age in SAMP8 mice are an important period to respond to their decline in learning and memory abilities with aging.Memory and cognitive impairments are severely impaired with increased oxidative stress,and activated microglia are found in brain,especially in the hippocampal region.SAMP8 mice may be a useful tool to study early pathological changes and neuroinflammation processes of aging.SAMR1 mice,in contrast,are aging-resistant mice without above pathological changes.Due to the multi-target neuroprotective effects of the transcription factor(Nrf2)on regulating autophagy,maintaining cell detoxification,improving mitochon-drial function by targeting multiple pathophysiological mechanismsof AD,Nrf2 has becomed a promising drug in the field of neurodegenerative disease.However,prior researches mainly focused on the role and effect of Nrf2 on their downstream products,to our knowledge,few studies have examined the direct effect of Nrf2 on cognitive function in AD patients.It is still not clear whether NRF2 is a key factor that affecting cognitive function in AD.The detailed relationship between NRF2 and AD,and the underlying mechanisms need to study deeper exploration.In this study,we injected Nrf2-ShRNA into the hippocampus of SAMP8 mice and SAMR1 mice and used lentivirus-mediated RNA interference technology to downregulate expression of target gene Nrf2.Based on the important period at 9 months of age that the cognitive deficits in SAMP8 mice,the main objective of this study was to investigate the effects of Nrf2 on cognitive function,synaptic plasticity changes,inflammatory factors,and neuronal ultrastructure in 9-month-old SAMP8 mice and SAMR1 mice and the underlying neurobiological mechanism.Part one Effects of down-regulated Nrf2 gene expression in the hippocampus on the cognitive function in SAMP8 miceObjective: Three behavioral experiments were performed to investigate the effects of down-regulated Nrf2 gene expression in the hippocampus on different cognitive domains,including spatial learning ability,episodic memory and emotionfunction of 9-month-old SAMR1 and SAMP8 mice and identify the effects of Nrf2 on the Cognitive impairment in SAMP8 mice.Methods:1.Nrf2-ShRNA primer was designed.Lentiviral Nrf2-ShRNA plasmids were constructed through plasmid vector digestion and virus packaging by Genechen(Shanghai,China).The interference efficiency of the Nrf2 ShRNA in hippocampal CA1 region was evaluated by real-time PCR test.2.Lentivirus NRF2-ShRNA or lenti-GFP were injected into bilaterally the hippocampus of four-month-old SAMR1 and SAMP8 mice.Low expression of target gene Nrf2 was maintained in the injection region.Allmice were reared until nearly nine months of age.Control virus GFP did not carry the target gene,virus titer and packaging were identical to the positive virus,expressed only Green Fluorescent Protein.The positive virus Nrf2 ShRNA contain the plasmid vector that interferes with Nrf2 gene,which also expressed green fluorescence.These mice were then randomly divided into four groups,i.e.,R1+GFP group(SAMR1 mice receiving green fluorescent protein-labeled control lentivirus injection),R1+Nrf2Sh RNA group(SAMR1 mice receiving Nrf2shRNA-lentivirus injection),P8+GFP(SAMP8 mice receiving green fluorescent protein-labeled control lentivirus injection),and P8+Nrf2ShRNA(SAMR8 mice receiving Nrf2ShRNA-lentivirus injection).3.Five months after lentivirus injection,behavioral experiments were performed to investigate the cognitive function of 9-month-old SAMP8 and SAMR1 mice.Novel object recognition task,Morris Water Maze task and passive avoidance task were performed in order.4.After behavioral tests,we determined the stability of lentivirusmediated transfer in the hippocampus of 9-month-old mice by immunofluorescence staining.These results proved the stability and efficiency of Nrf2 knockdown in hippocampus.Fluorescence microscopy and confocal microscopy were performed to detect microtubule-associated protein 2(MAP2)and Nrf2 expression in the hippocampus.Results:1.In the novel object test,9-month-old SAMP8 mice were worse at distinguishing the novel object compared with the SAMR1 mice(P<0.05),showing the impaired situational memory at the age of 9 months in SAMP8 mice.There was no significant difference(0.32±0.12 vs 0.41 ± 0.14,P>0.05)in the recognition index in the P8+Nrf2 shRNA group after 1 h,but this became worse after 24 h(P8+Nrf2 shRNA group vs P8+GFP group:0.36±0.15 vs 0.53±0.24).There was no significant difference in the recognition index in R1+Nrf2 shRNA group and R1++GFP group after 1h and 24 h(P >0.05).2.Morris water maze task results showed that compared with the R1 groups,escape latency was significantly prolonged in the P8 groups(P < 0.05).There was no significant difference in escape latency between R1+GFP and R1+Nrf2-ShRNA groups(P>0.05).Down-regulating Nrf2 expression further prolonged the escape latency(P<0.05,P8+GFP group vs.P8+Nrf2-ShRNA group).In the spatial probe test,the platform was removed.The number of times test animals crossed the platform was not significantly different between the R1+GFP(6.2±1.04)and R1+Nrf2-Sh RNA groups(6.6±0.88)(P>0.05).Compared with R1 groups,the number of times mice crossed the platform was significantly less in the P8 groups(P<0.05),suggesting that SAMP8 mice have impaired spatial memory.The number of times mice crossed the platform was further reduced in the P8+Nrf2-ShRNA group(P<0.05,P8+Nrf2-ShRNA group 1.6±0.72 vs P8+GFP group 3.8±0.64).The above results indicate that aging in SAMP8 mice resulted in decreased cognition,whereas Nrf2 knockdown further aggravated cognitive impairment in SAMP8 mice,but Nrf2 knockdown did not have an obvious effect on cognitive function in SAMR1 mice.3.The passive avoidance task further suggested the effect of Nrf2 on emotional changes in SAMP8 mice.The times of the electric shock in all9-month-old SAMP8 mice deteriorated modestly compared with the SAMR1 group,and the passive avoidance latency of P8 group mice also decrease.Compared with P8+GFP group,the times of the electric shock increased significantly in the P8+Nrf2-ShRNA group(3.2±1.2 vs 2.7 ± 0.99,Fig.6B).However,passive avoidance latency in the P8+Nrf2-ShRNA group vs the P8+GFP group(168 ±62 s vs198 ± 36s)showed a modest decrease(168 ±62 s vs198 ± 36s).4.The expression of Nrf2 and MAP2 in the hippocampus detected by immunofluorescence microscopy showed that the expression of Nrf2(green fluorescence)was mainly distributed in the cytoplasm,but partially in the nucleus.These results proved the stability and efficiency of Nrf2 knockdownin hippocampus.The expression of MAP-2 protein was marked with red fluorescence,and the number of MAP-2 positive neurons in the hippocampus region of SAMP8 mice was significantly reduced compared with that of SAMR1 mice,indicating that aging has affected the cytoskeleton function.In the P8+Nrf2ShRNA group,the expression intensity of MAP2 and the MAP2 positive neurons in the hippocampus CA1 region was significantly reduced compared with P8+GFP group,indicating that MAP2 dependent neuronal structural integrity was significantly reduced after down-regulated Nrf2.Part two: Effects of down-regulated Nrf2 expression in the hippoc-ampus on synaptic plasticity and neuronal ultrastructure in SAMP8 miceObjective: Based on the correlation between synaptic plasticity and cognitive function,this study was performed to investigate the effect of down-regulating Nrf2 expression in the hippocampus on synaptic plasticity-related marker protein expression and neuronal ultrastructure in SAMP8 mice and then investigate the underlying mechanism by which a decrease in Nrf2 results in cognitive impairment.Methods:1.Animals and grouping were the same as in Part I.2.Five months after lentivirus injection,western blot assay was performed to detect the expression of Nrf2 and that of downstream HO-1product p65 protein in 9-month-old SAMR1(n=5)and SAMP8 mice(n=5).3.Western blot assay was performed to detect PSD95,SYN protein expression in the hippocampus of 9-month-old SAMR1 and SAMP8 mice.4.Immunofluorescence staining was used to detect the expression of SYN in hippocampal CA1 region of SAMR1 and SAMP8 mice(n=3).5.Changes in the structure of neurons and synapses were observed by transmission electron microscope.Results:1.Nrf2,HO-1 and p65 protein level in the 9-month-old SAMR1 and SAMP8 mice Total Nrf2 protein level and nuclear Nrf2 protein level were both decreased significantly in the P8+Nrf2-ShRNA and R1+Nrf2ShRNA groups than in the R1+GFP and P8+GFP groups.This suggests that lentivirus stably downregulated Nrf2 expression.Total Nrf2 protein level and nuclear Nrf2 protein level in the P8+Nrf2-ShRNA group were significantly decreased than in the R1+Nrf2-ShRNA group(P<0.05).HO-1 also exhibited similar expression tendency.The expression of NF-?B subunit p65 was significantly increased in the hippocampus of SAMP8 mice.This suggests that NF-?B activation associated inflammatory responses existed in the 9-month-old SAMP8 mice.P65 expression significantly increased in the P8+Nrf2-ShRNA group than in the P8+GFP group(P<0.05).2.Expression of synaptic plasticity markers PSD and SYN protein in the hippocampus.3.PSD and SYN protein expression in the hippocampus of SAMP8 mice was significantly decreased than in the SAMR1 mice(P<0.05).PSD and SYN protein expression in the P8+Nrf2 ShRNA group was more significantly decreased than in the P8+GFP group(P<0.05).4.Confocal microscopy observation showed that SYN expression in the hippocampal CA1 region was significantly decreased in the P8 group than that in the R1 groups.The decrease in SYN expression was more obvious in the P8+Nrf2 Sh RNA group than that in P8+GFP group.Similarly,western blot assay revealed that PSD and SYN expression in the P8+GFP and P8+Nrf2ShRNA groups was significantly decreased than in the R1+GFP and R1+Nrf2ShRNA groups,and the decrease in PSD and syn expression was more obvious in the P8+Nrf2 Sh RNA group.5.ltrastructural observation on neurons and synapse under ransmission electron microscopeMorphological changes of neurons: In the R1+GFP group,the organellestructure in hippocampal CA1 region was clear and complete with normal morphology,the cell membranes were intact,cytoplasmic matrix and ribosome were uniformly distributed.Mitochondrial membrane and mitochondrial cristae was distinct and complete.The rough endoplasmic reticulum presented lamellar-like or tubulovesicular-like structures.The Golgi complex composed of a stack of 4-8 flattened cisternal membranes that are aligned in parallel,nucleoplasm distribution was uniform.There was no obvious changes in the organelle morphology in the R1+Nrf2 ShRNA group compared with the R1+GFP group;lipid droplets and lipofuscin were occasionally visible within the cell matrix.In the P8+GFP group,cell membrane of hippocampal neurons and the organelle structure was still intact,cytoplasmic matrix was evenly distributed.However,much lipofuscin was observed in the cell matrix which associated with aging,the number of ribosomes was reduced,the membrane of mitochondrial cristae was partially fused.Rough endoplasmic reticulum was mildly swollen.Some Golgi complex membranes were fuzzy.Nucleus and nuclear membrane were intact,pyknosis appeared.In the P8+Nrf2 ShRNA group,SAMP8 mice showed accelerated aging,much lipofuscin and autophagosomes were seen in the cell matrix,autophagosomes were visible,mitochondrial cristae were broke and fused,suggesting that the mitochondrial function was reduced.The membrane of hippocampal neurons was blurred.Cytoplasmic vacuolization,pale,and uneven matrix were observed.The number of mitochondria,endoplasmic reticulum,and Golgi complex were reduced.Synaptic changes:There was no significant change in the structure of synapses between SAMR1+GFP and SAMR1+Nrf2ShRNA groups,the number of synapses was high,the synaptic glomerulus,presynaptic membrane,postsynaptic membrane,synaptic cleft,and links between synapses were distinct with large number of synaptic vesicles.In SAMP8+GFP group,the number of synapses was decreased,the synaptic structure was unclear with small number of synaptic vesicles,the thickness of post synaptic density(PSD)was increased,the synaptic cleft was decreased,and the presynaptic terminalswelling was occasionally observed.In the P8+NRF2 ShRNA group,the presynaptic membranes were swollen and destroyed,the postsynaptic membranes were thickened,the synaptic cleft had disappeared,suggesting that downregulated Nrf2 reduced the synaptic function in the SAMP8 mice.Part three Effects of down-regulated Nrf2 expression on neuroinflam mation in the hippocampus of SAMP8 miceObjective: To detect inflammatory factor level after down-regulated Nrf2 expression in the hippocampus of SAMR1 and SAMP8 mice,investigate the molecular biological mechanism of Nrf2 on neuroinflammation,and identify the reason why cognitive function declines in SAMP8 mice.Methods:1.Animal grouping and model preparation were the same as in Part I.2.Immunohistochemical staining was performed to determine the number of Iba1-positive area and GFAP-positive area in the hippocampal CA1 region in SAMP8 mice.3.The interleukin-6(IL-6),and tumor necrosis factor-alpha(TNF-?)levels in the hippocampus of rats in each group were detected by western blot assay.Results:1.The microglial protrusions in the Iba-1-positive cell aggregates in the hippocampal CA1 region of SAMP8 mice were more thick and larger and greater in numbers,the number of Iba-1-positive cells in the unit area of the hippocampal tissue was more greatly increased(P<0.01)than in the hippocampal CA1 region of SAMR1 mice.In addition,the GFAP immunoreactivity was increased and exhibited abnormal morphology in SAMP8 mice.2.Results of western blot assay showed that IL-6 and TNF-? levels were significantly increased in SAMP8 mice than in SAMR1 mice(P<0.05).IL-6and TNF-? levels were significantly increased in the P8+Nrf2 ShRNA group than in the P8+GFP group(P<0.05).The results indicated that downregulatedNrf2 can increase the expression of inflammatory factors and induce neuroinflammation in the hippocampus.Conclusions:1.Behavioral tests revealed that cognitive domains,including spatial learning ability,contextual memory and fear memory were impaired in9-month-old SAMP8 mice compared to SAMR1 mice at the same age.Lentivirus-mediated ShRNA injection into the hippocampus to down-regulate Nrf2 level did not affect the cognitive function of SAMR1 mice.Downregulating Nrf2 expression in the hippocampus caused impaired spatial learning and emotion function of SAMP8 mice.These findings suggest that Nrf2 worsens the cognitive deficit in 9-month-old SAMP8 mice,Nrf2 expression in the hippocampus is a key factor affecting cognitive function.2.Synapse-related protein,SYN and PSD expression in the hippocampus was decreased in P8+Nrf2 ShRNA group than in the P8+GFP group.In addition,hippocampal neurons and synapse were greatly damaged in the P8+Nrf2 ShRNA group.These suggest that hippocampal synaptic plasticity is likely to be one of neurobiological mechanisms underlying cognitive deficit in SAMP8 mice after down-regulation of Nrf2 expression.3.Down-regulation of Nrf2 resulted in the activation of microglial cells and astrocytes in hippocampal CA1 region of SAMP8 mice,and increased the level of inflammatory factors such as TNF-?,IL-6,which in turn increased neuroinflammation.This suggests that down-regulation of Nrf2 exacerbates cognitive deficit in SAMP8 mice through a neuroinflammation-synapse pathway. |
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