Establishment And Pathological Mechanism Study Of Rat Models With Gordon Holmes Syndrome

BackgroundHereditary ataxia is a common class of neurodegenerative diseases,the main clinical features of hereditary ataxia are cerebellar ataxia and cerebellar atrophy,although after years of research,the pathogenesis is unclearly so far,and the treatment for hereditary ataxia is extremely limited nowadays.Clinical pathology and animal experiments found that the death of Purkinje cells in the cerebellum is an important pathological feature.Studying the pathogenesis of hereditary ataxia has great significance especially for the clinical treatment of these diseases.Gordon Holmes syndrome is a rare subtype of hereditary ataxia,characterized by ataxia compared with hypogonadism with an autosomal recessive inheritance model.Although the clinical understanding of this disease has been more than 100 years,until 2013,its first and second pathogenic genes RNF216 and OTUD4 were identified.In our previous work,we collected the first Gordon Holmes syndrome pedigree in mainland China.And the third pathogenicity gene STUB1 in Gordon Holmes syndrome was identified by whole exon sequencing,the mutation site was T246M.STUB1,which with ubiquitin E3 ligase activity and can interacts with Hsp70,Hsp90 and other chaperones,plays a critical role in regulating protein quality control,loss of function of STUB1 has long been associated with protein misfolding and aggregation.Mutations in STUB1 have been identified in many autosomal recessive cerebellar ataxia diseases.Further studies have shown that STUB1 can also participates in the pathogenesis of many other nervous system diseases by regulating other pathways such as necropoptosis.However,the mechanism that STUB1~T246M246M mutation causes Gordon Holmes syndrome is still unclear,which needs further study.To provide a suitable animal model,we applied CRISPR/Cas9 system to generate transgenic rats expressing STUB1 with a T247M mutation.This disease model displays weight loss,short-lifetime,decreased motor coordination,impaired memory,concomitant with Purkinje cell death.While the genetic cause is known,we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in STUB1 mutant rat cerebellum.Using iTRAQ,143differentially expressed proteins were identified,of which 80 were up-regulated and63 were down-regulated.The biological functions of these differentially expressed proteins mainly involve in molecular binding,catalytic reactivity,transporter function,activity of nucleic acid-binding transcription factor,protein-binding transcription factor activity,enzyme activity regulation,receptor activity,electron transporter activity,etc.Protein component mainly involves in cytoplasm,organelles,cell membranes,macromolecular complexes,and extracellular regions.The involved biological processes are cellular processes,single-cell organism processes,metabolic processes,stimulatory response processes,biological regulation processes,multicellular organism processes,and signal transduction,etc..KEGG pathway analysis showed that the differences mainly focused on immune,endocrine and metabolism.According to current researches,we thought that PDE9A,tau,PHLPP1,?-syn,etc.may be involved in the pathogenicity of Gordon Holmes syndrome.This novel rat model faithfully simulates the clinical phenotype of human Gordon Holmes syndrome disease and will be used to further investigate the pathogenic mechanisms of STUB1-related neurodegenerative diseases.The results of proteomics provide us with many clues for further research.This study provides an initial report of dysregulated proteins in Gordon Holmes syndrome which will assist with further investigation of Gordon Holmes syndrome pathology and the study of potential therapeutic strategies.Objective1.To construct STUB1 gene mutation rat disease models of Gordon Holmes syndrome.2.To analysis that whether STUB1 mutation rat disease models can simulate the clinical phenotype of Gordon Holmes syndrome through examining the behaviour,STUB1 protein expression,the pathological characteristics of cerebrum and reproductive capacity.3.Using differential proteomics to study the possible pathogenic mechanism of Gordon Holmes syndrome and provide evidence for further research and the exploration of therapeutic strategies.Methods1.Using Cas9/sg RNA and microinjection technology to construct Gordon Holmes syndrome rat disease models and identify the genetype by sanger sequencing.2.The rotorad test,gait analysis experiment and morris water maze were used to test the behavior of the rats.The expression of STUB1 were studied by western blot,and immunohistochemistry.The pathological of cerebellum and hippocampus were performed by using immunofluorescence and Nissl staining.The radioimmunoassay was used to determine the sex hormone level in female rats.3.Proteomics and bioinformatics analysis were carried out to explore the differentially expressed proteins.Results1.STUB1 mutation rat disease model of Gordon Holmes syndrome was successfully constructed.The genotype was identified by PCR and Sanger sequencing.2.In the behavioral experiment,since the 12th week,the drop latency of homozygous rats was significantly shortened(P<0.05);since the 16th week,the gait analysis also showed statistical difference(P<0.05).From the early disease stage,homozygous rats showed significant reduction in learning and memory(P<0.05).In addition,from the eighth week on,homozygous rats were significantly reduced in body weight(P<0.05);survival time was significantly shorter(P<0.05).Among the above index,there was no significant difference between heterozygous and wild-type.3.The expression of STUB1 protein in the brain,cerebellum,and testis of the homozygous rats was significantly reduced by western blot(P<0.05),and the decrease in the high-week age was more obvious,and the immunohistochemistry results were consistent.The number of Purkinje cells in the cerebellum of the homozygous rat was significantly reduced(P<0.05).At the same time,the hippocampus of the homozygous rat showed obvious abnormal cell morphology and disordered arrangement.4.The serum levels in rats showed that the level of estradiol was reduced in homozygous STUB1 point-mutant rats(P<0.05),the levels of follicle stimulating hormone and luteinizing hormone were increased(P<0.05).There is no obvious difference between wild-type and heterozygous ones.5.Compared with the wild-type,a total of 143 differential proteins in the homozygous rat disease models were obtained,of which 80 were up-regulation proteins and 63 were down-regulation.6.Though GO and KEGG analysis,the biological function of these differentially expressed protein mainly involves in molecular binding,catalytic reaction activity,transporter function,nucleic acid binding transcription factor activity,protein binding transcription factor activity,enzyme activity regulation,receptor activity,charge transporter activity ect.The cellular components mainly include cytoplasm,organelles,cell membranes,macromolecular complexes,and extracellular regions.The biological processes mainly include cellular processes,single-cell organism processes,metabolic processes,stimulatory response processes,and biological regulation processes,multicellular organism processes,signal transduction processes,and developmental processes.The KEGG pathway showed that the differential proteins are mainly focus on immunity,endocrine and metabolism.7.According to these results,combined with current researches,we thought some proteins may be related to the pathogenesis of Gordon Holmes syndrome,such as PDE9A,tau,PHLPP1,?-syn,etc.,and they have been verified initially.Conclusions1.Through the application of CRISPR/Cas9 and microinjection technologies,the STUB1 mutation rat model of Gordon Holmes syndrome was successfully constructed.2.Compared with the wild-type and heterozygous ones,homozygous rats showed that their balance ability and motor coordination were significantly reduced,meanwhile learning and memory function was markedly reduced.STUB1 expression and the number of cerebellar Purkinje cells in homozygous rats were obviously decreased,and the rats also showed a low reproductive phenotype.All these indicate that the disease model was constructed successfully,which laid a foundation for further study on the pathogenesis and treatment strategy of STUB1-related neurodegenerative diseases.3.Through proteomics and bioinformatics analysis,143 differential expression proteins were identified.These results may provide us with new ideas for further study and provide us with new whip points for treatment.