| Background:Neurodevelopmental disorders（NDDs）are a group of heterogeneous disorders affecting brain development and function,including autism spectrum disorders（ASDs）,intellectual disabilities（ids）,developmental delays（DD）,attention deficit hyperactivity disorders（ADHD）and epilepsy.With an incidence of more than 1%worldwide and autism alone affecting nearly76 million people,NDDs imposes a heavy economic and health burden on society and individuals.Both genetic and environmental factors can lead to the occurrence of NDDs,among which genetic factors play a major role in the occurrence of NDDs,with a heritability of70%～80%.Therefore,the identification of pathogenic genes and the study of pathogenic mechanism of NDDs are of great significance for the diagnosis and prevention of NDDs disease.The positioning and cloning of disease-causing genes are traditionally dependent on a large family of diseases.However,in our present fertility situation,it is difficult to find a suitable system for location cloning by chain analysis,and neurodevelopmental disorders are less in the form of family of diseases.Therefore,using second-generation sequencing,chromosome chip and other high-throughput technologies is a new strategy to detect NDDs pathogenic genes.In previous clinical studies,we focused on a rare 14q11.2 deletion syndrome,in which patients mainly presented with mental retardation,craniofacial deformity and other NDDS-related phenotypes.However,the molecular basis of the neurodevelopmental phenotype of this syndrome remains unclear,and the specific pathogenic genes remain to be discovered.By polymeric analysis of the genomic region of previously reported case 14q11.2,we found that the minimum overlap region（SRO）contained only one complete protein-coding gene SUPT16H,which may be a novel NDDs candidate gene.The Supt16 gene encodes the large subunit of Facilitates chromatin transcription complex FACT,and the SPT16 protein and the small subunit SSRP1 co-constitute the dimer structure of FACT.As a histone chaperone protein,FACT is mainly involved in the regulation of chromatin homeostasis and affects biological processes such as DNA transcription,replication and damage repair.Suggs,B.Z.et al and Bina,R.et al.found that deletion or mutation of one of the two subunits of FACT can affect cell function or ontogeny.Supt16 inhibition resulted in chromosome separation disorders during mitosis,and Ssrp1 homozygous knockout mouse embryos died at E3.5 days of blastocyst stage.Supt16 deletion was also found in 14q11.2microdeletion syndrome,and Supt16 mutant was also found in NDDs patients.However,no studies have confirmed that Supt16 gene is the pathogenic gene of NDDs,and the relationship between Supt16 gene and NDDs and its pathogenic mechanism need to be further studied.In this study,the Supt16 gene heterozygous knockout mouse model and hiPSC model were constructed,phenotypic evaluation and cytomolecular level study,to explore the relationship between Supt16 gene and NDDs and the pathogenesis.Materials and methods:1.Supt16 heterozygous knockout(Supt16+/-)mice were constructed and bred using CRISPR/Cas9 technology,and the mouse genotypes were identified by polymerase chain reaction（PCR）,agarose gel electrophoresis and sanger sequencing.Protein immunoimprinting（WB）was used to identify SPT16 protein knockdown in Supt16+/-mice.The behavioral differences between Supt16+/-and wild-type（WT）mice were evaluated through a series of mouse behavioral experiments,including water maze experiment,Y maze experiment,new object recognition experiment,three-box social interaction experiment,and open field experiment.The number of neurons in the cortex and hippocampus of mice was observed by immunofluorescence.The length of neuronal processes and the density of dendritic spines in the cortex and hippocampus of mice were studied by Golgi staining.Electrophysiological experiments showed that Supt16 gene heterozygous deletion resulted in decreased brain basal field potential（LFP）oscillations,andθand High-γwave oscillations decreased significantly.2.Primary isolation of neonatal Mouse brain neural stem cells（mNSCs）was performed by pellet forming assay,Ed U incorporation assay,immunofluorescence assay and flow cytometry to evaluate the dry maintenance ability.The effect of Supt16 gene heterozygous knockout on mNSCs gene expression profile was evaluated by RNA-seq technique,and the dry maintenance related signaling pathway was enriched by KEGG.The differences in the chromatin enrichment region of SPT16 protein between the two groups of primary neural stem cells were analyzed by ChP-Se Q technique,and the correlation between the above low expression genes and the heterozygous knockout of Supt16 gene was evaluated.3.Based on wild-type hiPSCs cell line PGP1,which expressed endogenous Cas9 gene,Supt16 gene was targeted by plasmid electrotransfer gRNA expression plasmid,and the heterozygous knockout monoclonal cells(Supt16+/-)of the gene were selected and identified.Supt16+/-hiPSCs were differentiated into Human neural stem cells（hNSCs）by directed induction differentiation technique.The dry maintenance ability and autophagy level of Supt16+/-hiPSCs were evaluated by Ed U incorporation assay,immunofluorescence assay,flow cytometry and WB assay.The effect of Supt16 gene heterozygous knockout on hNSCs gene expression profile was evaluated by RNA-seq technique,through KEGG enrichment differential signaling pathway.Autophagy phenotype induced by Supt16 gene heterozygote knockout was reconstructed and partially saved using autophagy activator Rapamycin and autophagy inhibitor MHY1485.Results:1.Supt16+/-mice have learning and memory impairment,social impairment and increased anxietySupt16 gene heterozygous knockout mice were successfully obtained using CRISPR/Cas9technology,and the expression of SPT16 protein in knockout mice was significantly decreased.The results of the water maze experiment showed that Supt16+/-mice took longer time to find the platform,and the number of crossing the platform location and platform quadrant increased significantly.The three-box social experiment found that Supt16+/-mice showed no differences in the propensity to interact between different social mice.The results of the new object recognition experiment showed that Supt16+/-mice showed no difference in sniffing time between the old and new objects.Open field experiments showed that Supt16+/-mice spent longer time in the peripheral area than in the central area,showing anxiety-like emotions.2.The number of neurons in cortex and hippocampus of Supt16+/-mice decreased,and the frequency of field potential oscillation decreasedThe brain shape and weight of Supt16+/-mice did not differ significantly from those of wild-type mice.Immunofluorescence detection of brain tissue showed that the number of neurons in CA1 and DG regions of cortex and hippocampus of Supt16+/-mice was significantly decreased,and the number of neural stem cells in SVZ region was significantly decreased.Golgi staining of brain tissue revealed that the heterozygotic deletion of Supt16 resulted in decreased dendrite density in cortical neurons and shortened neuronal processes in hippocampus.In vivo electrophysiological experiment results showed that the frequency ofα-wave,β-wave,θwave,Low-γwave and High-γwave which are related to learning and memory ability in Supt16+/-mouse brain decreased significantly.3.Heterozygous knockout of Supt16 gene leads to dry maintenance impairment of mNSCs by inhibiting the activity of MAPK pathwayPrimary mouse neural stem cells were isolated from the brain of newborn mice.Pellet formation experiments showed that the ball diameter of Supt16+/-mNSCs was smaller than that of wild-type mNSCs during culture.Ed U inclusion experiments and immunofluorescence experiments showed that the proliferation capacity of Supt16+/-mNSCs was decreased.Flow cytometry results showed that Supt16+/-mNSCs cell cycle arrest at G1/S phase test site,the level of apoptosis increased significantly.The results of RNA-seq experiments showed that Supt16 heterozygotic knockout resulted in the change of mNSCs gene expression pattern,and the expression of proliferation-related genes was significantly decreased,while the expression of apoptosis-related genes was increased.KEGG pathway enrichment found that MAPK pathway related genes（including Map2k4,Map2k7,Max,Atf2,Rac1.）were significantly underexpressed.In the ChIP-seq assay,low expression of Supt16 significantly weakened its binding to targeted regulatory genomic regions.However,the binding activity of SPT16 protein,a gene associated with neural stem cell apoptosis（Casp9）,was increased.4.By inhibiting PI3K/AKT/mTOR pathway,Supt16 hybrid knockdown up-regulates autophagy,leading to dry maintenance disorders of hNSCshiPSCs with Supt16 gene heterozygous knockout were successfully obtained by CRISPR/Cas9 technology,and the cells were directed to induce and differentiate into hNSCs.Ed U incorporation assay and immunofluorescence assay showed that the proliferation ability of Supt16+/-hNSCs was decreased.Flow cytometry assay showed that the cell cycle arrest of Supt16+/-hNSCs was at the G1/S phase test point,and the apoptosis level of Supt16+/-HNSCS was significantly increased.The results of WB experiment showed that the autophagy level of Supt16+/-hNSCs increased significantly.The results of RNA-seq experiments showed that Supt16 hybrid knockout significantly changed the gene expression pattern of hNSCs,autophagy related genes were abnormally increased,and the enrichment of KEGG pathway showed that the expression of PI3K/AKT/mTOR pathway related genes was significantly decreased.Treatment of wild-type hNSCs with autophagy activator Rapamycin was able to reproduce the phenotypes of Supt16 heterozygous knockout resulting in dysproliferative ability and increased autophagy and apoptosis levels,which could be partially restored by the autophagy inhibitor MHY1485.Conclusions:The different experimental models in this study showed that Supt16 gene was involved in neural development,and there was a pathogenesis of NDDs caused by haplodose insufficiency.Heterozygous deletion of Supt16 leads to dry maintenance impairment of mNSCs by inhibiting the activity of RAS/MAPK pathway,resulting in reduced number of neurons in the cerebral cortex and hippocampus,abnormal development of dendritic spines,and eventually cognitive and social impairment in mice.In hiPSCs-derived Supt16 heterozygotic knockouts of hNSCs,low expression of Supt16 gene up-regulates autophagy by inhibiting PI3K/AKT/mTOR pathway,leading to dry maintenance impairment of hNSCs. |
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