Development And Characterization Of A Rat Model Of Cockayne Syndrome Neurological Disease

Research BackgroundCurrently,the World Health Organization confirmed that the rare disease had exceeded more than 7,000 types,and 80%of which were inherited diseases that caused by genetic defects.In China,the number of patients with rare disease is estimated to exceed10 million,and more than 200,000 cases are newly diagnosed every year.However,the basic research in this field is relatively weak,and the research and development of"orphan drugs"remains a large part of blank.Compared with the common sporadic diseases without clear genetic clue,monogenetic genetic diseases are of great significance to study major problems in life science.Cockayne’s Syndrome is a rare developmental disorder of the nervous system in children.Also,it is an autosomal recessive disorder caused by a single gene(CSA or CSB)mutation,and often occurs in infants or childhood.Its primary symptoms include pronounced hypersensitivity to sunlight,severe growth failure,neurologic degeneration,as well as developmental abnormalities of multiple organ systems.The CS patients can be divided into three subgroups according to the onset time and severity of clinical features,that is type I(known as classic type)with symptoms appearing within two years after birth;type II with more severe symptoms than type I;and type III with relatively mild symptoms and later onset.The developmental disorders of the nervous system in Cockayne syndrome mainly manifested as developmental delay,neurological deterioration and cognitive decline;while in pathological,these disorders manifested as significant brain atrophy,myelin sheath reduction,calcification,and neuronal degeneration.Cockayne syndrome is primarily caused by the mutations in the ERCC8(CSA)or ERCC6(CSB)genes.The proteins encoded by CSA and CSB are involved in the transcription-coupled subpathway of nucleotide excision DNA repair(TC-NER).And the defects in TC-NER result in the skin photosensitivity of CS patients and defective recovery of RNA synthesis after UV light exposure.Therefore,the conventional views consider CS as a transcription-coupled nucleotide excision repair defect disease.This standpoint can explain the photoallergic phenotype of CS but failed to expound the severe neurological symptoms and premature aging of various systems.So,the pathogenesis of Cockayne syndrome is still unclear at present.So far,the mouse models studying CS consist of mice with CSA or CSB gene defects.These mice generally exhibit sensitivity to UV,defects in transcription coupled repair,defective recovery of RNA synthesis after UV irradiation,mild neurological symptoms and growth disorders,but did not develop into serious neurological disorders or premature aging.Since the existing mouse model is not sufficient to manipulate the neuropathologic features of patients with CS,optimizing the mouse model or generating new rat model is of great significance for studying the pathogenesis of CS and verifying its new molecular mechanism.Research AimsGenerating a rat model of Cockayne syndrome disease,and evaluating its phenotype in DNA damage repair and nervous system development.Research MethodsPart one:We use CRISPR/Cas9 technology to construct CSB knockout rats,and perform genotyping of the rats by PCR and Sanger sequencing.And then CSB gene expression is verified at the protein level and transcription level via western blot and q PCR method.Finally,the on-target and off-target conditions are investigated by the whole-genome sequencing.Part two:We evaluate the DNA damage and repair ability of CSB knockout rat fibroblasts through a series of experiments:using UV-irradiated growth curve experiments and clone formation experiments to evaluate photosensitivity,performing CPD and Ed U experiments to assess the excision repair ability of transcription coupled nucleotide and genome-wide nucleotide respectively,evaluating transcription recovery and base excision repair ability through EU and KBr O3 clone formation experiment,and verifying the damage of mitochondria via exploring the expression of mitochondrial DNA and mitochondrial DNA polymerase POLG1 by q PCR experiment..Part three:We evaluate the nervous system development of CSB knockout rats through a series of experiments:evaluating the overall development of the brain and cerebellum through HE staining of brain tissue,and evaluating whether there is neuron loss by labeling Purkinje cells with immunohistochemical Calbidin staining of cerebellar cortex.The expressions of neurofilament and MBP of the cerebellar white matter are detected by immunofluorescence to evaluate the axon growth and myelination.The HE staining and immunofluorescence Collagen IV staining of brain tissue are used to assess whether calcification occurs.The brain tissue immunofluorescence experiment is used to detect whether there is calcification.The DNA damage is detected by labelingγH2AX.Research ResultsPart one:The CSB knockout rat model was successfully constructed using CRISPR/Cas9 technology.The results suggested that the rat CSB gene was barely detected at the transcription level and protein level,and the whole genome sequencing did not show any off-target phenomenon.Part two:The growth curve experiment and clone formation experiment of UV irradiation showed that,comparing with the control group,the survival rate of CSB knockout rat fibroblasts dropped to less than 10%after 5 J/m~2UV irradiation.Meanwhile,the clone formation was also impaired.The results suggested a high sensitivity of CSB knockout rat fibroblasts to ultraviolet;CPD staining showed that CSB knockout rats were unable to repair the DNA lesions formed after UV irradiation,indicating that the TC-NER pathway of CSB knockout rats were defective;Ed U staining showed that the UDS(unscheduled DNA synthesis)level of CSB knockout rat fibroblasts was not significantly different from that of the control group,suggesting that CSB depletion has no effect on the genome-wide nucleotide excision repair subpathway;EU incorporation showed that the RNA synthesis of CSB knockout rats did not recover after UV irradiation;The results of KBr O3 clone formation experiment showed that CSB knockout rats could not form clones even at a low concentration of 5m M,indicating that their base excision repair ability was also impaired;The q PCR experiments showed that the expression of mitochondrial DNA and mitochondrial DNA polymerase POLG1 in CSB knockout rats decreased in varying degrees,indicating that their mitochondria were damaged to some extent.Part three:HE staining experiment results showed that,comparing with the control group,the CSB knockout rat cerebellum was significantly atrophy,the brain lobe defects appeared,and the brain hippocampus dentate gyrus appeared developmental malformations;The immunohistochemical results showed that the number and density of Purkinje cells in CSB knockout rats were significantly reduced,indicating that CSB knockout rats have a certain degree of neuron loss;Immunofluorescence experiment results showed that,CSB knockout rats neurofilament(neurofilament)expression level decreased significantly,the amount of myelin basic protein(MBP)dropped and the arrangement was disordered,indicating that the number of neuronal axons in CSB knockout rats decreased and degenerated,and myelin formation was impaired;HE staining experiment of brain tissue of CSB knockout rats showed that there were no calcified small crystals in the brain and cerebellum,and immunofluorescence experiments showed that there was no expression of type IV collagen around the cerebral blood vessels,which indicated that the brain tissue of CSB knockout rats did not appear calcification;The immunofluorescence experiments of CSB knockout rats showed the absence ofγH2AX fluorescence,indicating that CSB knockout rats did not have cumulative DNA damage.Research Conclusion1)A Cockayne syndrome rat model with Csb gene mutation was successfully developed using CRISPR/Cas9 technology.2)The rat model of Cockayne syndrome showed DNA damage repair defects.3)The rat model of Cockayne syndrome showed neurodevelopmental disorders.4)The rat model of Cockayne syndrome could well simulate some of the clinical symptoms of CS patients,especially the neuropathological characteristics,in that provides an ideal and new animal model for the clinical diagnosis and treatment of Cockayne syndrome.