The Mechanism Of Maternal Exposure To Benzophenone On Hippocampal Development And Cognitive Impairment In Offspring

Benzophenones（BPs）are widely used in sunscreen and other personal care products owing to their good UV absorption.The parental structure of BP comprises two aromatic rings and a carbonyl group,based on which a family of compounds has been derived,including 4-hydroxybenzophenone（4HBP）,2-hydroxy-4-methoxy BP（BP-3）and so on.BP-3 can be also quickly metabolized into 4-hydroxybenzophenone（4HBP）in body.The detection rate of 4HBP was over 80% in population samples,including pregnant women and children.Of note,4HBP has been shown to cross the placenta barrier,yet little is known about the health risks of exposure during pregnancy.Recent birth cohort studies have found a significantly negative correlation between maternal 4HBP exposure and children’s neurocognitive development.However,the toxicological effects and underlying molecular mechanisms remain to be elucidated.The fetal brain goes through a complex developmental process that ultimately shape the functional architecture of the adult brain.Higher brain functions,such as learning memory function,are primarily established between the second trimester and early childhood,wherein the proliferation and differentiation of neural stem cells（NSCs）play a fundamental role.Exposure to environmental insults during this period of time may impair NSCs function,resulting in profound and long-lasting adverse effects on neurodevelopment.Endoplasmic reticulum（ER）is the place of protein and lipid synthesis and processing in eukaryotic cells and exquisitely sensitive to environmental insults.Changes in tissue microenvironment can cause imbalance of ER homeostasis（e.g.,protein and redox homeostasis）,induce ER stress,and activate unfolded protein response（UPR）.Classical UPR consists of three signaling pathways,one of which is mediated by protein kinase Rlike ER kinase（PERK）.Phosphorylated PERK phosphorylates the α subunit of eukaryotic initiation factor 2（eIF2α）.This transiently halts global translation,at the same time allows for translation of a small subset of m RNAs,such as ATF4.ATF4 is an important transcription factor which elicits adaptive response by regulating the expression of genes involved in protein folding,autophagy and redox homeostasis,while it also transactivates CHOP under chronic ER stress and triggers apoptosis.It has been reported that activation of PERK pathway not only inhibits the translation of important synaptic proteins in neurons and promotes neurodegeneration,but also activates CHOP to induce neuronal apoptosis.Therefore,PERK-ATF4 signal transduction plays an important role in neurological diseases.Based on the above background and scientific questions,this study first established the primary hippocampus-derived NSCs and maternal 4HBP exposure models.The effects of4 HBP on the function and cognitive development of NSCs in the hippocampal area of offspring mice and the dose-response relationship were determined.Furthermore,mechanistic studies have shown that ER stress PERK-ATF4 signals mediate 4HBP-induced hippocampal developmental toxicity by interacting with NFk B-p65 signals.Finally,the inhibition of PERK-ATF4 signal has a good protective effect in both pregnant mice and human brain organoids 4HBP-infection models.Part 1 Maternal exposure to 4HBP impaired hippocampal NSCs function and cognitive development in offspring mice Objectives: The effects of 4HBP exposure on the function and cognitive development of NSCs in the hippocampus of offspring mice were determined by established primary hippocampus-derived NSCs and maternal 4HBP exposure models.Methods: Cell experiments: Primary NSCs were isolated from the hippocamus of E14.5fetal mice.The effects of 4HBP（0、1、10、102、103 n M）on the viability,proliferation,apoptosis and differentiation of NSCs in vitro were observed by neurosphere dynamics,CCK-8,immunofluorescence staining and Western blot assay.Animal experiments:pregnant mice were exposed to a concentration gradient of 4HBP(0、0.01、0.1、1 mg kg^-1 day^-1).Neonatal mice were fed under normal conditions until postnatal day 56（P56）to detect cognitive function by morris water maze,T maze and step-through test.The levels of4 HBP in maternal serum,placental tissue and neonatal brain tissue were determined by liquid chromatography-tandem mass spectrometry（LC-MS/MS）at the dose that induced behavioral changes.In additional,the hippocampal tissues of P1 and P56 offspring were collected for histological staining to observe hippocampal volume.Immunofluorescence staining and Western blot were used to detect the expression of proliferation and apoptosisrelated protein in hippocampus.Results:（1）4HBP inhibited the proliferation and differentiation of NSCs in vitro: 4HBP in1 μM dose inhibited neurosphere diameter,primary NSCs activity and proliferation,and promoted NSCs apoptosis.In addition,1 μM 4HBP significantly reduced the length of total dendrite,terminal branch and morphological complexity of neurons formed by NSCs differentiation,and promoted differentiation into astrocytes.（2）Maternal 1 mg kg^-1 day^-14 HBP exposure impairs cognitive function of offspring: Compared with the control group,spatial learning test of Morris water maze showed that 1 mg kg^-1 day^-1 4HBP treatment significantly increased the time spent in searching for the underwater platform.Spatial memory test showed that 1 mg kg^-1 day^-1 4HBP exposure reduced offspring swimming time in the target quadrant and the times of platform crossing.The spontaneous exploration in the T-maze test was significantly reduced in 1 mg kg^-1 day^-1 4HBP-treatment group.In stepthrough test,1 mg kg^-1 day^-1 4HBP exposure significantly increased the time spent in the dark cage.（3）Maternal 4HBP exposure impairs hippocampus development of offspring:Histological and immunofluorescence staining showed that maternal 1 mg kg^-1 day^-1 4HBP exposure reduced the volume of hippocampus,decreased the proliferation and promoted apoptosis of hippocampal NSCs,inhibited the differentiation of hippocampal NSCs into neurons and promoted differentiation into astrocytes in P1 and P56 offspring.Conclusion: Maternal exposure to 4HBP impairs hippocampal-derived NSCs function and cognitive development in offspring mice.Part 2 Molecular mechanism of endoplasmic reticulum stress PERK-ATF4 signal mediates neurodevelopmental toxicity of 4HBP Objectives: To explore the molecular mechanism of ER stress PERK-ATF4 signal mediated hippocampal developmental impairment induced by 4HBP.Methods:（1）The cultured NSCs were exposed to 1 μM 4HBP under proliferation and differentiation conditions for 7 days.Then,the cultured cells were collected for transcriptome sequencing and bioinformatics analysis,mainly including GSEA and HALLMARK gene enrichment analysis.（2）Transmission electron microscopy was used to observe the subcellular ultrastructural of newborn hippocampus and primary NSCs.（3）Hippocampal-derived NSCs exposed to 4HBP for 7 days in vitro and hippocampus of P1 and P56 offspring exposed to 4HBP during pregnancy were collected,and the effects of4 HBP on ER stress PERK-ATF4,inflammation and NFk B signal pathway were investigated by Western blot and RT-q PCR.（4）In primary hippocampal NSCs,RNA interference,Chromatin Immunoprecipitation and Dual Luciferase Reporter Assay were used to reveal the molecular mechanism of ER stress PERK-ATF4 signal activates NFk Bp65 inflammatory signals.Results:（1）Transcriptome sequencing and bioinformatics analysis showed that there were644 differentially expressed genes under proliferation conditions,but only 152 differentially expressed genes under differentiation conditions,suggesting that 4HBP mainly affected the proliferation function of NSCs.Gene enrichment analysis was conducted on all genes under proliferation conditions according to their expression levels.It was found that 4HBP mainly activated endoplasmic reticulum stress,NFk B mediated inflammatory and apoptosis signaling pathways.（2）The results of transmission electron microscopy showed,compared to control that ER boundaries were irregular and enlarged,and the organelles（such as ribosomes）around the ER were significantly increased in the 4HBP exposed offspring’s hippocampus and NSCs.（3）Maternal 4HBP exposure induced increased expression of Pe IF2 a,ATF4,and CHOP in the hippocampal tissues of P1 and P56 offspring.Meanwhile,NFk B inflammatory signaling was also activated,in which p65 protein and m RNA increased,Ik Ba protein and m RNA levels decreased.（4）NSCs exposed to 4HBP in vitro induced activation of PERK-ATF4 signaling pathway,and activation of NFk B inflammatory signaling pathway,in which the protein and m RNA levels of p65 increased,and Ik Ba protein and m RNA levels decreased.In addition,the total newly generated proteins of NSCs were significantly reduced after 4HBP exposure in vitro.（5）ATF4knockdown mitigated the upregulation of p65 protein and m RNA and nuclear p65 induced by 4HBP exposure in NSCs.In additional,ATF4 knockdown alleviated the reduced proliferation,increased apoptosis,declined cell viability and neurosphere growth restriction induced by 4HBP exposure.Importantly,Chromatin Immunoprecipitation and Dual Luciferase Reporter Assay confirmed that ATF4 bound to the p65 promoter to activate p65 gene,thereby promoting NFk B inflammatory signaling.Conclusion: ER stress PERK-ATF4 signaling mediated neurodevelopmental toxicity of4 HBP by activating NFk B-p65 inflammatory signaling.Part 3 PERK inhibition ameliorate 4HBP-induced neurodevelopment damage Objectives: The protective effect of small molecular compounds on hippocampal development and cognitive impairment was verified by inhibiting activated PERK-ATF4 signals.Methods:（1）Primary NSCs: NSCs were cultured and treated with 4HBP for 7 days under proliferation conditions,and added 0.5 μM PERK inhibitor（PERKi）.The protective effect of PERKi on 4HBP-induced NSCs toxicity was investigated by neurosphere dynamics,immunofluorescence staining,Western blot and RT-q PCR.（2）Pregnancy mice: Pregnant mice were divided into four groups: 0.1% anhydrous ethanol control group,1 mg kg^-1 day^-1 4HBP exposure group,50 mg kg^-1 day^-1 PERKi control group and PERKi intervention group.After delivery,offspring were continued to be fed until P56 under normal conditions for cognitive behavior experiments.Further immunofluorescence staining,Western blot and RT-q PCR were used to investigate the effects of PERKi on alleviating neurodevelopmental toxicity induced by 4HBP exposure during pregnancy.（3）Human brain organoids: Human induced pluripotent stem cells were induced and cultured in a rotating bioreactor containing extracellular matrix and specific growth factors for 60 days to form brain organoids.During the cultured periods,4HBP treatment and PERKi intervention were performed.The brain organoids were divided into 0.1% anhydrous ethanol control group,0.1 μM 4HBP exposure group,0.1 μM PERKi control group and PERKi intervention group.Subsequently,the protective effects of PERKi on 4HBP-induced apoptosis and p65 activation in brain organoids were investigated by brain organoid dynamics and immunofluorescence staining experiment.Results:（1）4HBP-induced NSCs apoptosis,cell viability decrease,as well as limited neurosphere growth were ameliorated by PERK signal inhibition.Meanwhile,4HBPinduced activation of PERK-ATF4 and NFk B-p65 signaling pathways and imbalance of protein synthesis homeostasis were significantly alleviated after PERKi treatment in hippocampus-derived NSCs.（2）Spatial learning test of Morris water maze showed that4HBP-induced increase of the time spent in searching for the underwater platform was alleviated by PERKi.Memory tests further showed that PERKi also improved the decrease of swimming time and crossing platform times in the quadrant of platform caused by 4HBP exposure.In the T-maze,PERKi significantly alleviated 4HBP-induced reduction of spontaneous alternation in offspring.（3）PERKi attenuated hyperactivated PERK-ATF4 and NFk B-p65 signals in 4HBP exposed offspring hippocampus and NSCs.（4）Compared with the control group,4HBP induced marked necrosis in the internal area,decreased proliferation,increased apoptosis of NSCs,and increased expression of p65 protein and nuclear localization in human brain organoids.PERKi treatment significantly improved the apoptosis of human brain organoids induced by 4HBP,and inhibited the activation and nuclear localization of p65.Conclusion: PERK-ATF4 inhibition ameliorates 4HBP-induced neurodevelopmental toxicity.