Changes Of Golgi Morphous In Skin Fibroblasts And Establishment Of IPSCs In Patients With SCA3

BackgroundSpinocerebellar Ataxia Type 3(SCA3),also known as Machado-Joseph disease(MJD),is the most common subtypes of autosomal dominant hereditary spinocerebellar ataxia.The prevalence in our country is about 3 to 5 per 100,000.This disease is caused by abnormal repeated amplification of the CAG trinucleotide of ATXN3 gene located on 14q32.1.It is one of the 9 diseases which caused by repeated amplification of CAG trinucleotide.The main clinical manifestations of the disease are progressive aggravation of cerebellar ataxia,unstable gait,accompanied by nystagmus,awkward movement,dysphagia,and sensory abnormalities.Currently,there is no effective treatment.The exact pathogenesis of SCA3 remains unclear.Previous studies have shown the formation of inclusion bodies,abnormal function of protein quality control systems,impaired axonal transport of nerve cells,dysfunction of cellular oxidative stress,impaired cellular autophagy and apoptosis,mitochondrial structure and function disorders may participate in the pathogenesis of SCA3.The Golgi apparatus is an important organelle in eukaryotic cells and is mainly involved in the processing,modification,classification,packaging,and transportation of partially lipid and endoplasmic reticulum synthesis proteins.The Golgi apparatus plays an important role in the transport of intracellular substances,especially nerve cells.Studies have shown that the Golgi apparatus is involved in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis(ALS),Parkinson's disease(PD),Alzheimer's disease(AD),and spinocerebellar ataxia type 2(SCA2).Golgi fragmentation may be an irreversible pathological manifestation of apoptosis.At present,there are few reports about the changes of Golgi morphology and the involvement of Golgi apparatus in the pathogenesis of SCA3.Induced pluripotent stem cells(iPSCs)are a group of cells with similar morphological and biological functions to embryonic stem cells.They are cell types established by Japanese scientist Yamanaka in 2006.Similar to embryonic stem cells,induced pluripotent stem cells can be further differentiated into various types of mature functional somatic cells in vitro,which provides an ideal cell model for the study of disease pathogenesis,drug screening,gene editing,and cell transplantation therapy.In this study,we examined the expression of mutant ataxin-3 protein and Golgi's structural protein GM130 in dermal fibroblasts derived from SCA3 patients,and preliminary explored the changes in the morphology of Golgi apparatus of dermal fibroblasts derived from SCA3 patient.Successful construction of patient-derived induced pluripotent stem cells(iPSCs)has laid the foundation for the subsequent differentiation of patient-derived neural cells for further exploration of the pathogenesis of the disease and drug screening.ObjectiveTo observe the morphological changes of Golgi in skin fibroblasts of SCA3patient,and to construct patient-derived induced pluripotent stem cells(iPSCs).Methods1.Tissue mass culture was used to establish skin fibroblasts derived from SCA3 patients and healthy volunteers.2.Cell viability was detected by CCK-8 assay and apoptosis was detected by flow cytometry,paired t test and two independent samples t test were used for data analysis.3.Skin fibroblast Golgi protein GM130 was labeled by immunofluorescence to observe Golgi morphology,the number of Golgi fragmented cells were counted,?2test was used for data analysis;4.Western blot was used for the detection of protein expression of ataxin-3,GM130 in skin fibroblasts,t test was used for data analysis;5.Transmission Electron Microscopy verifies the changes of Golgi morphology in skin fibroblasts of SCA3 patients;6.To construct patient-derived induced pluripotent stem cells(iPSCs)by Sendai virus transfection;7.Identification of patient induced pluripotent stem cells(iPSCs)by alkaline phosphatase staining,karyotyping,immunofluorescence,flow cytometry,teratoma formation;8.Capillary electrophoresis was used to detect the expression of ATXN3 gene in iPSCs derived from patients.Results1.Successfully established skin fibroblasts of SCA3 patient and healthy volunteers by tissue culture method.2.The patient-derived dermal fibroblasts expressed mutant ataxin-3 protein with reduced cell viability(t=2.889,p=0.045),increased apoptosis(t=-5.159,p=0.007),fragmentation of the Golgi apparatus(?~2=16.61,p<0.01)and decreased expression of GM130(t=43.658,p<0.01).3.Patient-derived induced pluripotent stem cells(iPSCs)were positive for alkaline phosphatase staining with normal diploid karyotypes and highly expressed stem cell markers OCT4,Nanog and TRA-1-60,and successfully differentiated into teratomas with three germ layers in NOD-SCID mice.4.Patient-derived induced pluripotent stem cells(iPSCs)still express the pathogenic ATXN3 gene,and the number of CAG trinucleotide repeats did not change.Conclusions1.Fragmentation of Golgi apparatus occurs in patient-derived skin fibroblasts,Golgi fragmentation may be involved in the pathogenesis of SCA3,providing a new way for the study of the pathogenesis of SCA3..2.Induced pluripotent stem cells(iPSCs)derived from SCA3 patient may provide an ideal cell model for further exploring the pathogenesis of SCA3.