The Function Of EMC3 Gene In The Pathogenesis Of Cerebellar And Retinal Neurodegenerative Diseases

Neurodegenerative diseases are a group of diseases characterized by the progressive degeneration and apoptosis of the central nervous system?neurons?and/or peripheral nervous system?myelin?as a result of the progressive degradation of neurons.With the increasing aging population,neurodegenerative diseases have become one of the most serious diseases that endanger human health in the world.The pathogenesis of neurodegenerative diseases is extremely complex and cannot be fully explained yet.And so far,there is a lack of effective diagnostic and diagnostic methods.Objective:Among neurodegenerative diseases,neurodegenerative lesions of the brain and retina are one of the current research hotspots.The pathological lesions caused by such diseases are irreversible damage of neuronal cells,and there is currently no effective treatment for this in clinical practice.The molecular mechanism of neuronal cell damage is not completely understood.Retinal dystrophy is a type of heterogeneous genetic disease caused by the loss of photoreceptor function.In the world,especially industrialized countries,retinal dystrophy is one of the important causes of blindness.The clinical phenotypic complexity and allelic heterogeneity of these diseases increase the difficulty of correct diagnosis of the disease.The endoplasmic reticulum membrane protein complex?EMC?was first identified in yeast as a 6-subunit transmembrane protein complex required for protein folding in the endoplasmic reticulum.The loss of EMC subunits in yeast leads to accumulation of misfolded membrane proteins and induction of unfolded protein response?UPR?.These results showed that mutations in the EMC1 gene can cause intellectual disability and cerebellar developmental degeneration syndrome.In eukaryotes,d Pob/EMC3 localizes to the ER and associates with EMC1 and calnexin.In addition,EMC is required for the stable expression of other multi-pass transmembrane proteins such as rhodopsin and Na⁺K⁺-ATPase.Moreover,it was also found that the d Pob/EMC3 deficiency induces rhabdomere degeneration of Drosophila.These results collectively indicate that the endoplasmic reticulum membrane protein complex?EMC?is a key factor in the biogenesis of multi-pass transmembrane proteins,including rhodopsin 1,and its loss causes retinal degeneration.EMC composed of EMC1 and EMC3 participates in the ER-associated degradation?ERAD?pathway,indicating that there is a close relationship between EMC and the ERAD pathway.Disorders of endoplasmic reticulum structure and function have been widely observed in neurodegenerative diseases,However,whether endoplasmic reticulum dysfunction is the cause of neurodegenerative disease lesions or is only the manifestation of neuronal death remains unclear.This thesis focuses on the function of EMC3 in cerebellum and retina.Methods:EMC3,EMC1 and other expression plasmids?including wild-type and mutant?were generated by standard techniques,respectively.Cells were transfected with the above-mentioned plasmids.Immunohistochemistry was performed using high-resolution laser confocal microscopy to analyze its expression and location in cells Circumstances,to analyze whether EMC1 mutations cause changes in localization in cells.Expression and localization of EMC3 and other subunits of endoplasmic reticulum protein complex were analyzed.In previous work,we found that knockout of the mouse Emc3 gene caused early embryonic death.Using Pcp2-cre transgenic mice and Emc3conditional knockout mice,we established an animal model that specifically knocks out Emc3 in cerebellar Purkinje cells and retinal bipolar cells.We plan to study the function of Emc3 in retinal bipolar cells and cerebellar Purkinje cells in detail using molecular biology,cell biology and immunohistochemistry methods.Tissue-specific markers in bipolar cells and Purkinje cells were used to immunostain frozen sections of the retina and cerebellum of mice with Emc3 deletion and the high-resolution laser confocal microscope was used to analyze the transportation and localization of these key proteins in detail.Results:EMC3 is encoded by the mouse Tmem111 gene and is a subunit of the highly conserved endoplasmic reticulum protein complex?EMC?.In our study,targeted deletion of Emc3 resulted in a severe neurodegenerative phenotype in mice.We found that Emc3^{lox P/lox P};Pcp2-Cre mice can survive up to 18 months and are fertile,but behaviorally exhibits a phenotype of cerebellar ataxia.Compared with wild-type mice,the dendritic development of Purkinje cells in the cerebellum of three-month-old Emc3mutant mice was significantly reduced.Purkinje cell loss and ataxia initially appeared around two weeks after birth,but gradually deteriorated with age.We further found that selective deletion of Emc3 in Purkinje cells resulted in the accumulation of misfolded membrane proteins in the endoplasmic reticulum,which hindered the secretory transport of Purkinje cells and caused dendritic atrophy.These phenotypes are characterized by a decrease in the size of the cerebellum and a decrease in the number of Purkinje cells,leading to ataxia.The deletion of Emc3 resulted in increased expression of glial fibrillary acidic protein?GFAP?and hyperplasia of astrocytes in the Purkinje cell-deficient region.Elevated C/EBP homologous protein?CHOP?and glucose-regulated protein78?GRP78/BIP?expression levels indicate that Purkinje cells exhibit endoplasmic reticulum stress?ER Stress?before visible cell loss.TUNEL?Td T-mediated d UTP Nick-End Labeling?analysis showed apoptosis in cerebellar Purkinje cells.Our data indicate that the deletion of Emc3 in Purkinje cells leads to defects in protein folding and transport,significantly reduced dendritic density,astrocyte proliferation and Purkinje cell death.In addition,we found that the retinal rod bipolar cells of Emc3 mutant mice exhibited progressive lesions compared with wild-type mice,suggesting that Emc3 may play an important role in maintaining the retina structure and function of mice.Conclusion:Our results show that Emc3 plays an important role in the development and function maintenance of Purkinje cells and retinal bipolar cells in mice.Our study reveals the important role of EMC3 protein in cerebellar Purkinje cells and retina,and provides new directions and ideas for the study of human neurodegenerative diseases,especially cerebellar ataxia and retinal neurodegenerative diseases.