| Background:Autism spectrum disorder(ASD)is a complex,highly genetically heterogeneous neurodevelopmental disorder.A variety of factors,including genetic factors and environmental stimuli,are risk factors for the development of ASD.Tetraspanin 7(TSPAN7)is a cell surface glycoprotein encoded by the Tspan7 gene located on the X chromosome and is highly expressed in brain tissue.Under physiological conditions,TSPAN7 is involved in synaptic transmission,neuronal morphogenesis,dendritic cells(DCs)and osteoblast morphogenesis.Molecular studies have shown that copy number variation in Tspan7 is closely associated with the development and progression of human ASD.However,the underlying mechanisms of TSPAN7 at the synaptic and synaptic integrity levels of ASD remain unclear.To explore the role of TSPAN7 in autism,we constructed a Tspan7 knockout rat strain using CRISPR/Cas9 technology with a view to establishing a Tspan7 knockout rat model of ASD and using this model to explore the mechanisms of TSPAN7 in autism.Methods:(1)Bioinformatics analysis,PCR,Western blot(WB)and immunofluorescence were used to detect Tspan7 sequence features and expression profiles.(2)A Tspan 7 knockout rat strain(Tspan 7-/-)was constructed by CRISPR/Cas9 technology,and genotyped and protein expression was identified using PCR and WB.(3)The behavioural phenotypes of autism in the four areas of stereotypic behaviour,pleasure deficit,social activity and learning memory were analysed in Wild Type(WT)rats and Tspan7-/-rats by the open field,sugar-water preference,three-box socialization,Ymaze and Morris water-maze experiments.(4)Magnetic resonance imaging(MRI),haematoxylin-eosin staining(HE)and Nissl staining were applied to analyze the alterations in the overall brain structure and the number of neurons at the microscopic level in Tspan7-/-rats.(5)The expression of ASD-related genes and changes in the number of neuronal branches and dendritic spines in the brain of Tspan 7-/-rats were observed by qRT-PCR and GolgiCox staining.Immunofluorescence(IF)was used to detect synaptic integrity of neurons in Tspan7-/-rat brains.(6)WB and IF were used to analyse the changes in expression of signalling molecules caused by TSPAN7 deletion.Results:(1)Under physiological conditions,the protein sequence of TSPAN7 is highly conserved in mammals,with highest expression in rat brain tissue.At the same time,TSPAN7 protein expression is low at birth and increases significantly from 0.5 months of age to 4 months of age TSPAN7 expression.subcellular localization of TSPAN7 is mainly highly expressed in neuronal cell membranes.Under pathological conditions,TSPAN7 showed a trend towards low expression in brain tissue from patients with ASD,Huntington’s disease(HD),Parkinson’s disease(PD)and Alzheimer’s disease(AD)This suggests that TSPAN7 plays a key role in the development of neurological diseases.(2)Tspan7 knockout rats were successfully constructed,and the results of the open field,sugar-water preference,three-box socialization,Y-maze and Morris water-maze experiments showed that Tspan7-/-rats exhibited typical behavioural phenotypes of ASD such as increased stereotypic behaviour,reduced pleasure,reduced socialization and impaired learning memory.(3)Imaging results showed that Tspan7-/-rats exhibited structural brain changes at the global level with reduced hippocampal and cortical volumes.(4)Histomorphology showed that the number of neurons in the hippocampus of Tspan7-/rats was reduced and the neuronal dendrites and axonal branches,neuronal complexity,and dendritic spines in the apical and basal dendrites of neurons were significantly reduced in the hippocampus and cerebral cortex of Tspan7-/-rats.(5)TSPAN7 deletion resulted in a significant reduction in the expression of synapseassociated proteins such as PSD95 and SYN and overlapping sites of PSD95immunolabeled postsynaptic and SYN-immunolabeled presynaptic membranes in the hippocampus and cortex of the rat brain.(6)TSPAN7 deletion inhibited the activation of the Integrinβ1/FAK/SRC signaling pathway,which in turn downregulated the expression of synapse-associated proteins such as PSD95,SYN and GluRl/2.(7)In Tspan7-/-rat primary neurons,reactivation of Proto-Oncogene Tyrosine-Protein Kinase Src(SRC)restored the expression and synaptic integrity of synapse-associated proteins such as PSD95,SYN and GluR1/2.Conclusions:The protein sequence of TSPAN7 is highly conserved in mammals and plays a key role in the development of neurological disorders.We successfully constructed Tspan7-/-rats,which showed behavioral changes such as increased stereotypic behavior,decreased pleasure and reduced socialization,reproducing the behavioral phenotype of human ASD;neuronal dendrites and axonal branches,neuronal complexity,and dendritic spines of apical and basal dendrites of neurons were significantly reduced in the hippocampus and cortex of Tspan7-/-rats,similar to the brain histology of human ASD.Pathological alterations were similar;Tspan7-/-rats could be used as an animal model for human ASD.Using the model,TSPAN7 deficiency was found to be an important pathogenetic mechanism leading to ASD by inhibiting the activation of the Integrinβ1/FAK/SRC signalling pathway,causing a decrease in synapse-associated proteins and loss of synaptic integrity.Background:Alzheimer’s disease(AD)is a neurodegenerative disease characterised by cognitive impairment.Multiple risk factors,including genetic and environmental factors,make treatment and drug development difficult,and the limited number of drugs currently available have limitations such as adverse effects and rebound.Therefore,AD drug development is urgently needed.With a better understanding of the components of Chinese herbal medicine,research has revealed that evodiamine(Evo)can improve learning cognitive dysfunction in AD model mice.However,Evo has high cytotoxicity and poor biological activity,and it is not feasible to use Evo directly for the treatment of clinical AD.Therefore,in this study,a series of Evo derivatives were designed and synthesized through heterocyclic substitution and amide introduction,from which compound 4c with lower cytotoxicity and higher biological activity was screened and its mechanism of action for the treatment of AD was investigated.Methods:(1)The derivatives of Evo were synthesized by heterocyclic substitution using 1,2-dimethoxybenzene in place of the indole ring of Evo,and their structures were identified and analysed by NMR and high-resolution mass spectrometry.(2)The SH-SY5Y and HepaG2 cell lines were intervened with the evodiamine derivatives and their cytotoxicity,anti-H2O2 and AβOs activities were assayed using CCK-8,CalceinAM/PI staining and xCELLigence real-time cell analysis.(3)The screened compound 4c was subjected to in vivo experiments.Compound 4c was injected intraperitoneally into two AD models,3×Tg and APP/PS1 mice,and the therapeutic effects of compound 4c on learning memory impairment in the two AD models were examined using Y-maze and Morris water-maze.(4)The effects of compound 4c on Tau hyperphosphorylation,amyloid aggregation and neuroglial cell proliferation levels in the brains of 3×Tg and APP/PS1 mice were evaluated by immunofluorescence,immunohistochemistry and immunoblotting.(5)The effect of compound 4c on mRNA expression in the posterior hippocampal tissue of 3×Tg model mice was analyzed by transcriptome sequencing and its differential genes were analyzed by KEGG and PPI networks.(6)The differential genes were validated by qRT-PCR,immunoblotting and immunofluorescence,and the potential mechanism of action of compound 4c in the treatment of AD was analysed.Results:(1)A total of 21 Evo derivatives were synthesized by heterocyclic substitution using wogonianine as the prototype.Among them,compound 4c showed lower cytotoxicity and higher anti-H2O2 activity.(2)Compound 4c exhibited higher LD50,anti-H2O2 and AβOs compared to Evo.(3)In the Y-maze experiment,the correct spontaneous alternation rate of 3×Tg and APP/PS1 model mice in the compound 4c intervention group was significantly increased compared with the model control group;the results of Morris water-maze experiment showed that the latency exploration time of hidden platform in the 3×Tg model mice in the compound 4c intervention group was significantly reduced compared with the model control group,and the number of platform crossing and target quadrant of mice duration were significantly increased.It was suggested that compound 4c significantly improved the working memory and spatial learning memory of the AD model mice.(4)The levels of Tau phosphorylation at Ser202/Thr205(AT8),Thr231(T231)and Thr181(T181)phosphorylation sites in the brain tissue of 3×Tg model mice were decreased in the compound 4c intervention group.(5)Aβ monomers,oligomers and amyloid aggregation were significantly less in the hippocampus and cortex of APP/PS1 mice in the Compound 4c intervention group.(6)The aggregation of microglia and astrocytes around Aβ plaques in the hippocampus and cortex of APP/PS1 mice in the compound 4c intervention group was significantly reduced.(7)The expression of insulin signaling,AMP-activated protein kinase(AMPK)and other AD-related signaling pathway-related mRNAs in the brain tissue of 3×Tg model mice was significantly decreased in the compound 4c intervention group.(8)The phosphorylation levels of PI3K and AKT in the brain tissues of 3×Tg model mice significantly increased and the phosphorylation levels of downstream GSK3β protein significantly decreased.The results suggested that compound 4c could improve the impairment of PI3K/AKT/GSK3β signaling pathway in the brain tissues of AD model mice.(9)The results of cellular experiments showed that compound 4c ameliorated the Aβinduced PI3K/AKT/GSK3β/Tau dysfunction in SH-SY5Y cells.Conclusion:In this work,we designed and synthesized a series of derivatives of Evo by heterocyclic substitution and amide introduction,from which compound 4c was screened.In vivo studies demonstrated that compound 4c significantly improved behavioral impairment,reduced β-amyloid aggregation,Tau hyperphosphorylation and glial cell proliferation in AD mice.At the same time,the LD50 of compound 4c was increased by 2fold and the effective dose of in vivo treatment was reduced by approximately 500-fold compared to Evo,making compound 4c an improved and successful lead compound for the treatment of AD.In addition,to investigate the mechanism of action of compound 4c,RNA-seq and pathway enrichment analysis revealed that compound 4c was able to affect the expression of AD-related genes,AMPK and insulin signalling pathway-related genes.In vivo and in vitro experiments confirmed that compound 4c could improve PI3K/AKT/GSK3β/Tau dysfunction. |
