Involvement Of VPS4B A Dentin Defects ⅠCausing Gene In The Pathogenesis Of Cognitive Impairments In Vps4b Knockout Mice Study On An Missense Mutation In AMBN Gene Causes Amelogenesis Imperfecta And Dentin Disorders

BackgroundAlzheimer’s disease(AD)is a common progressive neurodegenerative disease.The main symptom of this disease is irreversible memory impairment of the whole brain.It is recognized as an intractable disease which is related to the growth of age.The main pathogenesis of AD is the deposition of extracellular β-amyloid(Aβ)and excessive phosphorylation of Tau proteins to form neurofibrillary tangles(NFTs)intracellular,resulting in synaptic dysfunction and neuronal loss.Vesicle translocation is a crucial process in the cell.Once the vesicle transport system fails to work properly,it causes the accumulation of inimical proteins intracellular,and the disorder of lysosome dependent proteindegradation pathway,eventually leading to neurodegenerative diseases such as cognitive impairment.ObjectivesCRISPR/cas 9 technology was used to construct a Vps4b+/-knockout mouse model of C57BL/6J.By the behavioral experiments in mice,to evaluate the changes in the emotional and cognitive abilities of knockout mice;Experiments to determine the association with AD-related pathways after gene knockout.Validation of changes in AD-related protein expression in brain tissue of mice,and studies on neuronal and synaptic plasticity,to elucidate the role of Vps4b in cognitive impairment in mice.Materials and MethodsTo explore changes in mouse emotions and cognitive ability by mouse behavioral experiments.The iTRAQ proteomics experiment was used to detect the differential expression of protein in brain tissue of Vps4b+/-mice.Western blot was used to verify differentially expressed proteins.Immunofluorescence and immunoblot experiments were used to investigate the differential expression of protein in brain tissue of aged mice.The number of neurons and changes in synaptic plasticity were explored by Golgi staining and Nissl’s staining.Bioinformatics was used to predict the interacted regions of VPS4B chromatin.Transmission electron microscopy was used to observe the changes in the structure of multivesicular bodies,autophagosomes,and lysosomes.ResultsOpen field experiments,light-dark transtition test,and elevated plus-maze showed that the emotions of Vps4b+/-were more anxious than normal mice;3-chambered social test,novel object recognition test,and Morris water maze test showed Vps4b+/-mice were impaired of cognitive capacity.The results of iTRAQ showed that the expression of AD-associated pathway proteins in Vps4b+/-mice was significantly increased.The results of WB experiments confirmed the experimental results;Immunofluorescence and WB experimental showed that the expressions of App,Aβ40,Aβ42,P-Tau in the brain tissue of Vps4b+/-mice were increased,the expressions of Map2,Syp and Bcl2 were decreased;Pathological results showed that the number of neurons in Vps4b+/-mice was decreased,neuronal branches and dendritic spines were significantly reduced compared with normal mice;Bioinformatics showed VPS4B and BCL2 Chromatin areas may interact with each other in human cerebral cortex.Transmission electron microscopy revealed that vesicular trafficking was impaired in the brain of Vps4b+/-mice,and autophagosomes and lysosomes increased significantly.ConclusionThe cognitive function of Vps4b+/-knockout heterozygous mice were impaired.The pathogenic mechanism is that Vps4b knockdown causes App,Aβ40,Aβ42,P-Tau expression increaseed,Map2,Syp expression decreased,resulting in the number and structure of the nerves were impaired in mouse brain tissue.At the same time,the vesicle transport in the mouse brain is impaired,which leads to the accumulation of harmful substances in the cells and can also lead to neurodegenerative diseases.Background and ObjectivesAmeloblastin(AMBN)is involved in enamel formation and participates in the formation of dentin in the early stages of tooth development through epithelial mesenchymal interactions.AMBN is the second most abundant enamel matrix protein and plays an important role in enamel formation.AMBN mutations can cause inherited enamel hypoplasia,but AMBN mutations have led to dentinal abnormalities that have not yet been reported.Materials and MethodsTake the family with abnormal teeth development in Zhanjiang,Guangdong,China as a research object,collected their peripheral blood and clinical examination data.Using MICRO-CT and scanning electron microscopy to analyze the ultrastructure of shed teeth.To extract peripheral blood DNA of family members,adopt full-exome sequencing,Sanger sequencing technology and family co-segregation and other methods to identify the causative genes and mutation sites and conduct population mutation screening.Polyphen-2 and I-TASSER were used to predict the conservation of the AMBN mutation site and the three-dimensional conformational change of the mutant protein.Transfect wild-type and mutant of AMBN by eukaryotic vectors into human HEK-293T cells and compare the difference in the subcellular localization of wild-type and mutant proteins in the cells.To evaluated expression levels of mRNA and AMBN protein of mutant and wild-type AMBN in HGF and HEK-293T cells.ResultsAccording to the patient’s condition in the family,we mapped the family lineage and diagnosed that it was an autosomal dominant genetic enamel hypoplasia and dentin dysplasia disease,which can affect both permanent and deciduous teeth.Micro-CT results showed that the density of enamel and dentine was lower than that of normal teeth,and high-density calcified myeloids were seen in the pulp cavity.The SEM results showed that the enamel prisms was sparsely arranged,and occasionally there was calcification area of no structural,and the enamel-dentinal junction lacked continuous conchoidal curves,showing a straight line with widened gaps,the dentinal tubules were uneven distribution and sizes.The exon sequencing,Sanger sequencing,and STR linkage analysis of family members revealed a mutation of exon 13 of the AMBN gene(c.1069C>T)in the family members,and the mutations were absent in populations.The analysis results showed that the 357 locus of AMBN protein was a conserved site in many species.After the mutation,the three-dimensional structure of the protein changed and was a harmful change.Subcellular localization results showed that wild-type and mutant AMBN proteins were located in the cytoplasm.The results of qRT-PCR showed that the relative mRNA expression level of mutant AMBN gene was not different from that of wild type,but the expression level of the mutant protein was significantly higher than that of wild type protein(P<0.001).ConclusionFor the first time,we found that AMBN(c.1069 C>T)mutations resulted in tooth enamel dysplasia with abnormal dentin development.The ultrastructural of teeth was changed.The new mutation site is highly conserved in the AMBN protein sequence,resulting in a change in the three-dimensional conformation of the protein,which is a deleterious mutation and the expression of the protein was increased after the mutation.This study showed that AMBN plays an important role in the development of enamel and dentin.