Aluminum Hydroxide Exposure Caused Development Deficits In Human Embryonic Stem Cell-derived Cerebral Organoids And Social And Cognitive Impairments In Mice

Background:Environmental toxic metal exposure has a critical effluence on neurological development,and abnormal neurological development can lead to neurological developmental disorders,such as intellectual disability,autism spectrum disorder,and attention deficit disorder.Because the fetal and neonatal brains are abnormally sensitive to toxic substances,disruption of normal brain development processes may lead to neurological developmental deficits and related developmental disorders in childhood,affecting their cognitive and behavioral functions in adulthood.Aluminum is one of the neurotoxic metals widely found in the natural environment,and numerous studies have linked aluminum exposure to a variety of neurological disorders,such as Alzheimer’s disease（AD）,Parkinson’s disease（PD）,and autism spectrum disorder（ASD）.Aluminum can penetrate the blood-brain barrier and accumulate in the brain for long periods,thus causing neurotoxicity.In population epidemiological surveys,occupational aluminum exposure has been found to impair cognitive functions such as orientation,reaction time,memory,and attention,leading to cognitive dysfunction.Meta-analyses have also shown a dose-response relationship between occupational aluminum exposure and cognitive impairment.In addition,analysis of human biological samples showed that maternal blood aluminum levels were nearly identical to neonatal blood aluminum levels,which impaired brain development in premature infants given long-term intravenous aluminum-containing solutions.Aluminum exposure comes in a variety of forms and routes,of which the primary source of aluminum exposure during childhood development is vaccine adjuvants,with aluminum hydroxide（Al（OH）₃）being one of the most used vaccine adjuvants.Although a single vaccine may contain only a small amount of aluminum（typically less than 0.5 mg of the aluminum adjuvant compound）,multiple vaccines containing aluminum adjuvants can greatly increase the overall aluminum burden on the body.Childhood vaccination programs in many countries indicate that children will receive 23-32 vaccines from birth to 6 years of age,many of which contain aluminum-based adjuvants,such as hepatitis B and diphtheria vaccines.In animal model tests,neonatal mice exposed to 100μg Al/kg of vaccine adjuvant after birth showed a range of behavioral abnormalities and associated motor neuron death.In line with this,sheep have shown neuroinflammatory/degenerative syndromes after repeated injections of aluminum-containing vaccines.An association between aluminum in vaccines and an increased incidence of ASD has been noted in recent years.The adverse effects of aluminum adjuvants on the developing human central nervous system require attention when the high levels of aluminum that children are often exposed to from vaccines.However,the adverse effects of aluminum exposure on human neurological development are unclear due to strict ethical restrictions and practical issues.Brain-like models are ideal in vitro cellular models derived from human embryonic stem cells（h ESCs）or human induced pluripotent stem cells（hi PSCs）that largely recapitulate the molecular,cellular,and anatomical features of early human brain development.With the development of brain-like cultivation techniques,brain-like models are now widely used to study human neurodevelopmental disorders and in vitro experimental models of neurodevelopmental toxicity induced by various drugs or environmental pollutants.Because its cortical-like structure formed in vitro can reenact the dynamic developmental process of the human cerebral cortex,and the o SVZ region containing outward radial glial cells（o RGCs）can be detected,thus providing a good model for studying the involvement of Al（OH）₃ exposure in the developmental disorders of the nervous system.Methods:1.In this study,h ESCs were spontaneously formed into a cortical brain-like model with a typical cortical-like structure in vitro based on the method of dorsal cortex induction combined with the 3D suspension culture technique.Immunofluorescence staining was used to clarify the tissue characteristics of cerebral organoids at different time points.To establish a model of chronic Al（OH）₃ exposure in early development and chronic exposure in late development of Al（OH）₃,and to clarify the effects of different doses of Al（OH）₃ exposure on the morphology and size of cerebral organoids at different developmental stages using morphological measurements.The expression of RGCs marker（SOX2）in the D28 cerebral organoids was detected by immunofluorescence staining,and the thickness of the VZ area where SOX2 was located was counted.The proliferation and apoptosis of SOX2 in the VZ area of the cerebral organoids were detected by Ki67,Ed U,and TUNEL staining to clarify the effect of early Al（OH）₃ exposure on the development of the cerebral organoids.The distribution of SOX2,IPCs marker（TBR2）,deep neuronal markers（CTIP2）,and mature neuronal markers（Neu N）in the D56 cerebral organoids were detected by immunofluorescence staining and the thickness of VZ,SVZ,and CP regions were counted.Quantitative real time polymerase chain reaction（q RT-PCR）was used to detect the NESTIN,SOX2,Ctip2 and TBR2 in the D56 cerebral organoids.The detection of m RNA expression was used to clarify the effect of Al（OH）₃ exposure on further neuronal differentiation in the cerebral organoids.The expression of SOX2,o RGCs marker（HOPX）,a neuronal marker（MAP2）,and an astrocyte marker（S100β）in the D96cerebral organoids was detected by immunofluorescence staining to clarify the effect of chronic Al（OH）₃ exposure on the differentiation of o RGCs and astrocytes in the cerebral organoids during late development.2.We used transcriptome sequencing technology to sequence the brain-like model of Al（OH）₃ exposure on day 28,screened the differential genes by DEseq2 method,and performed Gene ontology（GO）pathway enrichment,KEGG enrichment.Protein-Protein Interaction Networks（PPI）analysis was performed to identify the potential mechanism of Al（OH）₃exposure leading to abnormalities in brain-like neurogenesis.The expression and distribution of YAP in Al（OH）₃-exposed cortical brain were detected by immunofluorescence technique,and the protein expression levels of YAP and LATS1/2 were detected by immunoblotting technique,and the abnormal inhibition of Hippo Signaling way in cerebral organoids caused by Al（OH）₃ exposure was identified as the main pathway leading to the depletion of neural progenitor cell pool and promoting neural cell differentiation.3.To examine the locomotor ability and anxiety-like behaviors of Al（OH）₃exposed mice in adulthood using the open field experiment,elevated cross-maze experiment,and bright and dark box experiments;to examine the social and repetitive stereotype-like behaviors of mice using the three-box social experiment,grooming experiment,and bead burial experiment;to examine the short-term memory ability and hippocampal function of mice using the Y-maze experiment,new object recognition experiment,and nesting experiment.The spatial learning memory ability of mice was examined using the Morris water maze experiment;the expression of neural stem cells,neurons,microglia,and astrocytes within the hippocampal structure of P14mice was examined using immunofluorescence methods to clarify the behavioral and morphological phenotypes of aluminum hydroxide exposure in mice.Results:1.Successful construction of human embryonic stem cell-derived cortical brain-like model and aluminum hydroxide brain-like exposure model（1）h ESCs were induced to construct a brain-like model using the three-dimensional culture suspension technique,and the dorsal forebrain-specific marker PAX6 was expressed in the cerebral organoids of D28 as detected by immunofluorescence,indicating that we successfully established a dorsal cerebral organoid model.（2）The immunostaining results of the cortical organoid at different developmental stages（D28,D56,D96）showed that the dorsal cortical organoid had developmental specificity and continuity and could mimic the key events of neurocortical development in a spatial and temporal order,including cell proliferation,differentiation,migration and maturation,generating mature astrocytes at D96 and showing a more typical laminar structure.（3）Constructing h ESC-derived cerebral organoid models of early and late developmental exposure of Al（OH）₃,we found that Al（OH）₃ exposure limits normal brain-like growth in a dose-dependent manner.In the early-developmental-exposure model,Al（OH）₃exposure inhibited the proliferation and promoted the apoptosis of neural progenitor cells in D28;in the D56 model,early Al（OH）₃ exposure still inhibited the proliferation of neural progenitor cells,but at the same time promoted the differentiation of different neurons and disrupted the normal differentiation process.In the late developmentally exposed h ESC-derived cerebral organoid model,the D96-day cerebral organoid showed a decrease in o RGCs after Al（OH）₃ exposure,but an increase in astrocyte differentiation and insignificant changes in neuronal differentiation.2.Exploration of the potential mechanism of development deficits in cerebral organoid caused by Al（OH）₃ exposure（1）We measured the aluminum content in the D28-day brain tissue by ICP-MS and found that the aluminum content in the brain was significantly higher after Al（OH）₃ exposure than in the control group,and showed a concentration-dose dependence.（2）Transcriptome sequencing RNA-seq revealed that there were some differences in gene expression patterns between D28 groups.Compared with the control group,there are 535differential genes in 5μg/ml Al（OH）3 group,of which 290 genes are down regulated and 235genes are up regulated;Μeanwhile,compared with the control group,there are 934 differential genes were found in the 25μg/m L Al（OH）₃ group,of which 708 genes are down regulated and226 genes are up regulated.（3）The two groups differentially expressed genes were enriched for GO pathway,and the results showed that the top ranked pathways enriched for forebrain development,embryonic development,nervous system development,neuronal differentiation,etc.（4）Two groups of differentially expressed genes were enriched for the KEGG pathway,and the results showed that the Hippo signaling way was present and ranked high in both groups of differential pathways.The expression of YAP in the three groups was examined by immunofluorescence,q RT-PCR and immunoblotting experiments,and it was found that the expression of YAP in the Al（OH）₃ group showed a decreasing trend compared with the control group,and the expression of YAP in the 25μg/m L Al（OH）3 group was significantly lower,indicating that Hippo signaling way may be the role of Al（OH）3 exposure in causing abnormal neurogenesis.3.Phenotypes of social abnormalities and cognitive dysfunction in adult mice caused by neonatal Al（OH）₃ exposure（1）We examined the motor function and anxiety-like behaviors of neonatal Al（OH）₃exposed mice in adulthood by open field experiment,elevated cross-maze experiment,and light-dark box experiment,and the results showed that the mice in the Al（OH）₃ group showed normal motor function and no significant anxiety-like behaviors compared with the control group.（2）We examined the social behavior and repetitive stereotyped behavior of neonatal Al（OH）₃ exposed mice in adulthood through the three-box socialization experiment,hair grooming experiment and bead burial experiment.（3）The short-term memory and cognitive performance of neonatal Al（OH）₃ exposed mice were examined in the new object recognition test,Y-maze test and nesting test.In the nesting experiment,the nesting scores of mice were significantly lower than those of the normal control group,indicating that the mice had abnormal cognitive functions.（4）The spatial learning memory ability of neonatal Al（OH）₃ exposed mice in adulthood was examined by Morris water maze experiment,and the results showed that:in the positioning cruising stage,there was no difference in the platform finding latency time between the two groups of mice,and the platform finding latency time was gradually shortened as the number of training days increased,indicating that there was no difference in the learning ability of the two groups of mice;in the spatial exploration experiment part,the mice in the Al（OH）₃ group took significantly more time to find the platform imagination than normal control mice,indicating that Al（OH）₃ exposure affected the spatial memory ability of mice in adulthood.4.Neonatal Al（OH）₃ exposure caused abnormal hippocampal structure development in P14 mice（1）Sox2 and Sox2 double labeled with GFAP were used to label NPCs and RGCs within the hippocampal dentate gyrus,respectively,and it was found that the number of NPCs and RGCs in the Al（OH）₃ group was significantly reduced compared with control group.Meanwhile,compared with the control group,the number of proliferative cells in the group Al（OH）₃ group showed a downward trend by PCNA staining.（2）Neu N was used to stain the granular cell layer and it was found that the thickness of the hippocampal granular layer was significantly thinner in the Al（OH）₃ group than control group.Iba1 was used to stain microglia and S100βwas used to stain mature astrocytes,compared with control group,the number of S100βin the hippocampus was significantly increased in the Al（OH）₃ group but the number of microglia did not change.Conclusion:The present study demonstrates that exposure to Al（OH）₃ during brain development impairs normal neurological development in cerebral organoids and leads to behavioral and cognitive abnormalities in mice.In vitro,we successfully established a 3D dorsal cerebral organoids model derived from h ESCs and demonstrated that Al（OH）₃ exposure causes abnormal neurogenesis and inhibits the production of o RGCs by affecting the Hippo signaling way,thus disrupting laminar structure development.In vivo,it was verified that neonatal Al（OH）₃ exposure damaged hippocampal structures in mice,which in turn possibly caused social and cognitive impairment in adulthood.