The Neurodevelopmental Mechanism Of Cognitive Impairment In Male Offspring Induced By Prenatal PM_2.5 Exposure

Ambient particulate matter pollution is directly linked to human health.Particulate matter less than 2.5μm in aerodynamic diameter(PM_2.5)can stay in the air for a long time.PM_2.5 can enter the lung deeply and reach other target organs via blood circulation,thereby posing a great threat to human health.The development of the central nervous system（CNS）begins in the fetal period.However,fetal organs are not mature and highly susceptible to external environmental factors.Epidemiological studies have confirmed that prenatal PM_2.5exposure can cause adverse effects on the fetal nervous system,such as an increased risk of cognitive impairment,autism spectrum disorder（ASD）,depression and anxiety disorders in children.However,it is still unclear about the precise molecular mechanism of CNS disorders in offspring induced by prenatal PM_2.5 exposure,and there is also a lack of continuous monitoring of neurodevelopmental outcomes during critical developmental windows.Therefore,based on regional characteristics of PM_2.5 pollution,we established a PM_2.5exposure model（3 mg/kg b.w.）in C57BL/6 mice during pregnancy to investigate the effects of PM_2.5on the birth indices in offspring.Furthermore,we elucidated the molecular mechanisms of PM_2.5-induced neurodevelopmental disorders in offspring in terms of functional changes,morphogenesis,gene expression profiles,mitochondrial function and so on.1.Physicochemical properties of PM_2.5 vary greatly in different regions due to different sources,indicating that PM_2.5 pollution has regional characteristics.Prenatal PM_2.5exposure may also change birth indices in offspring.First,we analyzed the physicochemical properties of PM_2.5 sampled at different sites.The morphology and hydrodynamic diameters of PM_2.5 were examined using transmission electron microscopy（TEM）and a Malvern Zetasizer Nano ZS,respectively.The elements and polycyclic aromatic hydrocarbons（PAHs）in PM_2.5 samples were determined using inductively coupled plasma mass spectrometry（ICP-MS）and gas chromatography-mass spectrometry（GC-MS）,respectively.The results showed that PM_2.5 exhibited high diversity in their morphology and consisted of agglomerated soot aggregates,regular and irregular mineral particles,spherical particles,etc.The average particle size of PM_2.5was 600.1±30.25 nm,with a polydispersity index（PDI）of 0.785±0.044 and a zeta potential of-33.03±0.61 m V.The average concentration of the elements in PM_2.5was 168.98 ng/m³,and the top four abundant heavy metals included Fe,Zn,Cu,and Pb.The average concentration of PAHs was 7.13 ng/m³,and the top three abundant PAHs included benzo（b）fluoranthene,fluoranthene,and chrysene.The species and concentrations of the elements and PAHs in PM_2.5 were similar for three consecutive years.Next,we established a mouse model of PM_2.5 exposure during pregnancy to reflect its potential adverse health effects by examining the birth indices of offspring.The results showed that there were no significant differences in the embryonic period,litter sizes,sex ratio,eye-opening time,brain-to-body ratio,body length,and tail length between the offspring in the control and PM_2.5-exposed groups.However,the body weight of male offspring in the PM_2.5-exposed group significantly decreased compared to that in the control group at postnatal days 1（PND1）and 7（PND7）.The above results suggested that PM_2.5collected in sampling locations were regionally specific and stable,and male offspring in the PM_2.5-exposed group had significantly low birth weights compared to those in the control group.2.Numerous studies reveal that there was an association between low birth weight and neurodevelopmental disorders（NDDs）.However,it remains unclear whether low birth weight in male offspring due to prenatal PM_2.5 exposure induces sex-specific neurocognitive outcomes,as well as the underlying mechanisms.In the present study,the Morris water maze test was used to assess the cognitive function in weanling offspring.Neuronal morphology was observed in vivo and in vitro by brain tissue sections of offspring at different developmental windows and primary culture neurons from newborn mice,respectively.The cortex tissues from male offspring were collected on postnatal days（PNDs）1,7,and 21 for messenger RNA sequencing（m RNA-Seq）analysis to explore the possible mechanisms.The results showed that prenatal PM_2.5 exposure induced neurocognitive deficits in weanling male offspring,but not female offspring.The sex-specific neurocognitive outcomes were associated with abnormal cortical neuronal development and altered m RNA expression profiles in male offspring cortex during postnatal critical windows.The annotations in Gene Ontology（GO）of differentially expressed genes（DEGs）in male offspring revealed that prenatal PM_2.5 exposure persistently disrupted the expression of genes involved in the morphological development of axons,dendrites and synapses.Consistently,prenatal PM_2.5 exposure impaired axonal growth and reduced dendritic complexity.Importantly,prenatal PM_2.5 exposure significantly decreased the expression of Homeobox A5（Hoxa5）in the cortex of male offspring on PNDs 1,7,and 21.Hoxa5 is a critical transcription factor that can regulate the expression of hub genes associated with the development of axons,dendrites,and synapses.In addition,both inorganic and organic components of PM_2.5 had adverse impacts on neuronal morphogenesis,with organic components exhibiting stronger inhibition.In conclusion,prenatal PM_2.5exposure affected spatial learning and memory in male offspring by disrupting Hoxa5-mediated neuronal morphogenesis,whereas organic components,including polycyclic aromatic hydrocarbons（PAHs）,exerted more adverse effects than inorganic components.3.The above results showed that prenatal PM_2.5 exposure caused low birth weight and NDDs in male offspring.However,because postnatal neurodevelopment in mice occurs across the entire lactation period and long non-coding RNAs（lnc RNAs）are closely associated with neurodevelopmental diseases,it is necessary to perform a time-series analysis of m RNA expression profiles in the cortex of male offspring during lactation to explore the potential regulatory role of lnc RNAs in abnormal neurodevelopment caused by prenatal PM_2.5 exposure.Therefore,this study is conducted based on the m RNA-seq results described in Section 2.Firstly,the two-factor analysis of variance(“PM_2.5 exposure”and“developmental windows”)was applied to analyze the m RNA transcriptional profile,and then the weighted gene co-expression network analysis（WGCNA）was combined to determine the neurodevelopmental processes that were most significantly affected.The results from GO enrichment and Encyclopedia of Genes and Genomes（KEGG）enrichment analyses showed that prenatal PM_2.5 exposure significantly affected biological processes（such as substrate adhesion-dependent cell spreading,myelination,ensheathment of neurons,etc.）and KEGG pathways（such as tight junction and axon guidance）.Furthermore,prenatal PM_2.5 exposure significantly affected the expression of myelination-related genes during the postnatal development of male offspring,and caused damage to myelin ultrastructure in male offspring on PNDs 14 and 21,such as a decrease in myelin thickness of the optic nerve and a mild loss of myelin in the corpus callosum.In addition,lnc RNA sequencing（lnc RNA-seq）was performed on the cerebral cortex of weanling male offspring on PND21,and the results showed that differentially expressed lnc RNAs in their lnc RNA expression profiles could potentially regulate the development of axons,dendrites,synapses,glial cells,myelin,and the corpus callosum.Importantly,lnc RNA NONMMUT058932.2 and NONMMUT029203.2 played key roles in PM_2.5-induced abnormal myelination by regulating the expression of myelination-related genes（Fa2h,Mal,Tppp,Trf,and Sh3tc2）through the transcription factor Ctcf.4.The above results confirmed that prenatal PM_2.5 exposure caused neurodevelopmental disorders in male offspring,suggesting a correlation between prenatal PM_2.5 exposure and impaired neuronal and myelin development.Mitochondria are important organelles that provide energy for CNS development and play a critical role in mediating normal neuronal and myelin development.Mitochondrial dysfunction and structural damage are associated with adverse neurodevelopmental outcomes.However,it is still unclear whether prenatal PM_2.5 exposure causes mitochondrial dysfunction in the cortex of male offspring.In this part of the study,we collected cortex tissues from male offspring on PNDs 1,7,and 21 to explore the correlation between prenatal PM_2.5 exposure and impaired mitochondrial function.The results indicated that prenatal PM_2.5 exposure induced mitochondrial dysfunction in the cortex of postnatal male offspring,as evidenced by decreased mitochondrial ATP production,abnormal mitochondrial ultrastructure,significantly reduced the expression of rate-limiting enzymes（CS,IDH2,and FH）in the tricarboxylic acid cycle and the subunits of mitochondrial complex IV and V（CO1,CO4,ATP6,and ATP8）,and excessive generation of ROS.Given that mitochondrial dysfunction is an important factor in inducing abnormal neuronal and myelin development.Therefore,the above results imply that mitochondrial dysfunction in the cortex of male offspring triggered by prenatal PM_2.5 exposure may be a key cause of induced neurodevelopmental impairment in male offspring.In this work,we first analyzed the regional pollution characteristics of PM_2.5 based on its physicochemical properties in sampling regions.Then,we established a prenatal PM_2.5exposure model of mice to examine the effects of prenatal PM_2.5 exposure on birth outcomes in offspring.Based on these results,m RNA-seq analysis was used to elucidate the molecular regulatory mechanisms of cognitive dysfunction in male offspring.In addition,the time-series-based m RNA-seq analysis was performed to explore the association between impaired myelination and neurodevelopmental disorders in male offspring caused by prenatal PM_2.5 exposure.Furthermore,in combination with lnc RNA-seq analysis in weanling male offspring,we unraveled the molecular regulatory mechanisms.Finally,we demonstrated that mitochondrial dysfunction may be a key cause of neurodevelopmental impairment in male offspring following prenatal PM_2.5 exposure.In summary,our study revealed the correlation between abnormal neuronal development and cognitive impairment in male offspring caused by prenatal PM_2.5 exposure,and provides an experimental basis for regional PM_2.5 pollution control and neurodevelopmental health protection in offspring.