Study On The Pathogenic Mechanism Of SCA Caused By SMEK1 Mutation And Generation Of The HiPSCs From The HSP Patient Carrying KIF1A Mutation

Neurodegenerative diseases are a type of neurological disorders characterized by the irreversibly progressive loss of the structure or function of specific neuron,which mainly including Parkinson's disease(PD),Alzheimer's disease(AD),different types of spinocerebellar ataxia(SCA),hereditary spastic paraplegia(HSP),etc.Limited to symptomatic treatment,there is stil no effective treatment and drugs to prevent the development of the disease.Neurodegenerative diseases affect millions of people around the world,bringing great pain and pressure to patients and medical staff,and at the same time causing a very large economic burden to our societies.We collected two families of neurodegenerative diseases in Shandong Province,namely spinal cerebellar ataxia(SCA)and hereditary spastic paraplegia(HSP),and identified the SCA pathogenic gene SMEK1 and HSP pathogenic gene KIF1A.For the two pathogenic genes,we conducted research on their function and pathogenic mechanism.(1)Research on the SCA pathogenic mechanism of sMEK1 K572T missense mutationOur laboratory identified SCA new pathogenic gene SMEK1 through linkage analysis and whole exome sequencing.In this family,all the patients carried SMEK1 c.1715A>C missense mutation,which resulted in the substitution of 572th lysine in the SMEK1 protein by threonine.It is reported that the SMEK1(Suppressor of MEK1)gene plays an important role in the early neural development process,mainly responsible for regulating the proliferation and differentiation of neural stem cells.In order to investigate whether the SMEK1 K572T missense mutation affected the early neural development process,we generated the Smek1K572T knock-in mouse model and obtained three genotypes of fetal neural stem cells.When the neural stem cells were cultured in vitro,we found that homozygous(Smeklmut/mut)and heterozygous(Smek1wt/mut)neural stem cells had higher self-renewal and proliferation ability.From the neural differentiation experiments in vitro,we found that the Smek1K572T missense mutation prevented the differentiation of neurons and promoted the differentiation of glial cells.According to the literatures,the Notch signaling pathway is involved in the neurogenesis process in mammalian development,and it also plays an important role in the maintenance,differentiation and fate determination of neural stem cells.Therefore,we used three genotypes of neural stem cells and SMEK1WT/Mut high-expression cell lines to detect the expression of key molecules in the Notch signaling pathway.Protein expression results showed that after Smek1K57lT missense mutation,the expression of NlCD in neural stem cells was up-regulated,resulting in the expressions of HES1 and HES5 were significantly increased.This was consistent with the detected phenotype results.In summary,the Smek1K572T missense mutation affects the early neural development process through abnormal activation of the Notch signaling pathway.In addition,we found that the Smek1 K572T missense mutation causes the accumulation of SMEK1 protein,which leads to endoplasmic reticulum dysfunction,causing endoplasmic reticulum stress and activating the PERK pathway of the unfolded protein response(UPR).The mRNA and protein levels of key molecules in the PERK pathway were significantly up-regulated.When SMEK1WT and SME1lMut were overexpressed,negative feedback regulation occured in the PERK pathway.The endoplasmic reticulum and mitochondria form a tight physical connection through a special domain,mitochondria-associated endoplasmic reticulum membranes(MAM),which is a functional platform for the interaction of two organelles.When the endoplasmic reticulum morphology changes,it will increase the association between endoplasmic reticulum and mitochondria,thereby changing the mitochondrial morphology.Therefore,we further investigated whether the SmeklK721 missense mutation affects the structure and function of mitochondria.The transmission electron microscopy results showed that the mitochondria in Smeklmut/mut MEF cells were obviously swollen,the intramembrane matrix was lightened,and the crest disappeared.In some severe cases,the matrix was dissolved with vacuolization.Mitochondrial staining results showed that abnormal mitochondrial fission and fusion occurred in Smeklmut/mut MEF cells,and abnormal expression of proteins related to mitochondrial fission and fusion was observed.In conclusion,we determined that the SMEK1K572T missense mutation abnormally activated the Notch signaling pathway,resulting in hindering the early neurogenesis process.It was also observed that the SMEK1K572T missense mutation caused abnormal accumulation of background protein,activated the PERK pathway of the unfolded protein response,and caused abnormalities in mitochondrial structure and function,resulting in the occurrence of SCA.The above results provide ideas for further research on the function of SMEK1 and the mechanism of SMEK1K572T missense mutation leading to SCA.(2)Establishment of hiPSCs from patients with HSP KIF1AT258M missense mutation andexploring the conditions of hiPSCs neural induction differentiationMost neurogenic diseases are genetically and clinically heterogeneous.Pathological characterizations caused by different mutation patterns of the same gene are quite different.Therefore,neither the in vitro cell model nor the animal model can specifically study the pathogenic gene function and its pathogenic mechanism.But disease-specific human induced pluripotent stem cells(iPSCs)have a full set of genetic material of patients,it is more illustrative to use it to study molecular and cellular changes of diseases.For the collected HSP families,our group has identified the disease-causing gene KIF1A through the whole exome sequencing technology.The missense mutation(p.T258M)resulting in the development of HSP.After the approval of the ethics committee and the informed consent of the family members,we obtained peripheral blood and isolate peripheral blood mononuclear cells(PBMCs).Then,we generated the HSP-specific hiPSCs by reprogramming PBMCs with non-integrative vectors.The obtained hiPSCs have larger nucleolus,higher nucleus-to-cytoplasm ratio,and expressed pluripotency markers.After the obtained hiPSCs were passed to 10th generation,exogenous genes were turned off,the endogenous genes were turned on,the hiPSCs could be differentiated into three germ layers in vitro and there is no chromosomal variation and mycoplasma contamination.After the identification of hiPSCs,we explored the conditions of hiPSCs neural induction differentiation in vitro.We successfully achieved EB formation and induced hiPSCs into neural stem cells.Subsequently,early neural induction differentiation and mature neural induction differentiation were carried out,and neuronal cells and glial cells were induced.In summary,we have successfully constructed human induced pluripotent stem cells derived from HSP KIF1AT258M patients and induced them into nerve cells,providing the cell model for further research on the function of KIF1A and the pathogenic mechanism of KIF1AT258M,which can also be used for drug screening and clinical treatment research.