The Molecular Mechanism Of Cognitive Dysfunction Induced By NO₂ Exposure

NO₂ is an important air pollutant.Ambient NO₂ is mainly emitted from the combustion of fossil fuels,and vehicle exhaust is the principal outdoor source.On congested roadways,NO₂ concentration typically reaches up to0.4 ppm.The most important indoor sources include tobacco smoke and coal-or gas-burning appliances,and the level of NO₂ in indoor air often higher that in outdoors,with peak concentrations exceeding 2-4 ppm.Exposure to NO₂ has been considered as a potential risk factor for central nervous system（CNS）injury and disease,and is closely related to adult neurodegenerative diseases and neurodevelopmental disorders in children.Alzheimer’s disease（AD）is a typical neurodegenerative disease clinically featured by a slowly progressive memory loss and cognitive impairment.The pathological characteristics of AD include deposition ofβ-amyloid（Aβ）peptides and hyperphosphorylated tau,and mitochondrial dysfunction is its early symptoms.International researches demonstrated that exposure to traffic-related air pollution or atmospheric NO₂ contributed to tau hyperphosphorylation,Aβplaques deposition,neuroinflammatory factors up-regulation,neurodegenerative changes in the brain,and learning and memory dysfunction.However,these results are mostly depend on epidemiological studies which assess the health impact following exposure to the complex mix of air pollutants,lacking experimental evidence to support the association between single air pollutant NO₂ exposure and AD-like neurological damage,especially a detailed understanding of the molecular mechanisms that underpin its pathogenicity.Therefore,in this thesis we developed animal models to investigate the neurotoxical responses of NO₂inhalation and the molecular mechanism of NO₂-induced AD-like pathological lesions.We found that:（1）NO₂ exposure（5,10,20 mg/m³,5 h/day,7 days）significantly up-regulated ROS and MDA levels in rat cortex,the induced oxidative stress in turn caused mitochondrial ultra-structural changes（such as mitochondrial membranes damage and cristae break and loss）,and dose-dependently inhibited the activity of mitochondrial respiratory chain complexes II,IV,V,and decreased ATP content.Additionally,high level NO₂ exposure significantly reduced the protein content of mitochondrial biogenesis regulatory factors,including PGC-1α,NRF1,and TFAM.（2）5 mg/m³ NO₂ inhalation（5 h/day,4 weeks）caused spatial learning and memory dysfunction,aggravated Aβ₄₂2 accumulation,enhanced neuroinflammatoryresponse,andpromotedneuronaldegeneration.Meanwhile,NO₂ caused 5,487 genes significantly changed in APP/PS1mouse cortex.The KEGG pathway results showed that DEGs were enriched in synaptic function,learning and memory,Aβproduction and AA metabolism pathways.Furthermore,JZL184（8 mg/kg body weight）inhibited the activity of monoacylglycerol lipase（MAGL）that increasing endocannabinoid 2-arachidonoylglycerol（2-AG）and decreasing AA metabolites,couldeffectivelyrelievedtheneuroinflammation,neurodegeneration,Aβaccumulation and cognitive deficits caused by NO₂exposure.（3）5 mg/m³ NO₂ inhalation（5 h/day,4 weeks）significantly suppressed insulin signaling（IRS-1-AKT）in brain and therefore disrupted the function of glycogen synthase kinase-3β（GSK-3β）,which directly potentiated the phosphorylation of tau,and induced a decreased expression of glutamate receptors and a thinner postsynaptic density.In addition,NO₂ activated the MAPK/JNK pathway to disturb IRS-1-AKT insulin signaling in the brain,liver,and skeletal muscles,and induced systemic insulin resistance（IR）.The whole-body IR triggered a compensatory increase in serum insulin levels,which further attenuated the IRS-1/AKT/GSK-3βsignaling pathway in brain,leading to the hyperphosphorylation of tau.Taken together,NO₂ inhalation exposure contributed to AD-like pathological lesions,including mitochondrial structural and functional damage,excessive Aβ₄₂2 accumulation,hyperphosphorylation of tau,and cognitive impairment.The overproduction of ROS-elicited mitochondrial dysfunction axis forms a vicious cycle and further exacerbated oxidative stress,was the initial event of CNS damage.Following these,disturbances in AA metabolism and insulin signal transduction elevated the Aβ₄₂2 production and tau phosphorylation may be the important mechanisms in these processes.It has been well-known that air pollution exposure can lead to CNS damage.Recent studies suggested that atmospheric pollutant-induced neurological impairment and disease are not only related to direct exposure,but may also be related to neurodevelopmental toxicity in offspring caused by maternal exposure.Although several results indicated that maternal exposure to traffic-related or atmospheric NO₂ associated with adverse neonatal outcomes,disrupted fetal neurodevelopmental processes,and contributed to multiple neurobehavioural and neurocognitive disorders in childhood（e.g.autism,schizophrenia,developmental delay,and cognitive impairment）,however,these epidemiological reports combined neurological effects of multiple air pollutants,further exploration of mechanisms for neurotoxicity of individual NO₂ exposure was insufficient.Based on these,we establishied an animal model to detect the neurodevelopmental toxicity of NO₂ exposure by using the spatial learning and memory impairment as an endpoint,and then to investigate the molecular mechanisms based on the changes of gene expression profiles in the offspring brain.We found that prenatal NO₂inhalationcausedsex-specificgenetranscriptionalabnormalities,histopathological changes and neurocognitive dysfunction.On PND21,only male offspring exhibited DEGs-associated biological functions and cellular components enrichment,neuronal necrosis and apoptosis,and spatial learning and memory decline.Among the 14 persistently changing mRNAs,Apolipoprotein E（ApoE）,which highly expressed in males,was likely a key factor in the prenatal exposure-induced male-specific neurological dysfunction.In addition,prenatal NO₂ inhalation caused sex-dependent lncRNAsabnormallyexpressed.ConstructingalncRNA-mRNA co-expressed network to predict lncRNA’s potential functions according to the annotation of their co-expressed mRNAs,the subsequent KEGG pathway analyses indicated that AD and oxidative phosphorylation were the most highly represented biofunctional pathways activated by the prenatal NO₂exposureinthemaleoffspring.Moreover,ahublncRNA metastasis-associatedlungadenocarcinomatranscript1（Malat1）co-expressed with most coding genes in the lncRNA-mRNA co-expressed pairs,up-regulating ApoE expression through NF-κB activation contributed to cognitive dysfunction in males.