Studying The Effects Of KIF1A Missense Mutations On Neural Stem Cells Using IPSCs Model

BACKGROUND:Hereditary spastic paraplegia(HSP)is a group of neurological monogenic disorders with a high degree of clinical and genetic heterogeneity,characterized by progressive muscle weakness and increased muscle tone in both lower limbs.The main pathological changes are predominantly axonal degeneration.It most often involves the longest downstream corticospinal tract axons and distal upstream sensory axons(thin tracts)in the body,showing retrograde mortality progression.Eighty-seven HSP-causing genes have been identified,respectively named SPG1-87,of which SPG30 is caused by a mutation in the KIF1A gene,but its pathogenesis is not yet clear.It has been shown that KIF1A mutations impede substance transport within neuronal axons,such as prominent vesicular precursors,dense core vesicles,etc.This affects neuronal complexity and leads to abnormal early neurogenesis in mice.However,due to the vast differences between species,mouse models do not reproduce well the developmental profile and disease characteristics of the human nervous system.The previous group identified a family with SPG30 caused by the KIF1A c.773C>T(p.T258M)mutation in Binzhou,Shandong Province.Induced pluripotent stem cells(iPSCs)have been obtained from normal human and patient-specific stem cells in home lines using peripheral blood mononuclear cell(PBMC)reprogramming.In this study,we will use patient-specific iPSCs to induce neural stem cells(NSCs)and investigate the effect of KIF1A mutations on NSCs in early neurological development.METHODS:First,in this study,isogenic control iPSCs with identical genetic background were obtained and characterized using CRISPR-Cas9-mediated homologous recombination repair technology to correct the KIF1A c.773C>T(p.T258M)mutation in patient iPSCs.Subsequently,in this study,normal control iPSCs(WT),patient iPSCs(KIF1AM/+ iPSCs)and isogenic control iPSCs(KIF1AC/+ iPSCs)in the family line were induced to differentiate into neural stem cells in a neural direction,respectively.The three different genotypes of neural stem cells were tested for their proliferation and differentiation ability by EdU assay and immunofluorescent staining,and for their in vitro migration ability by scratch assay.Finally,a transcriptome analysis of NSCs of different genotypes was performed and preliminary validation was carried out to explore the potential mechanisms by which the KIF1A mutation causes a range of phenotypes in NSCs.RESULTS:First,this study used CRISPR-Cas9 gene editing technology to correct mutations in KIF1AM/+ iPSCs,and KIF1AC+ iPSCs with successful mutation correction were screened using Sanger sequencing.It was also determined by immunofluorescence staining and RT-qPCR that all KIFIAC/+ iPSCs were highly expressed in pluripotency genes and had the potential to differentiate towards the trichoblast.By G-banded karyotype analysis,it was determined that the KIF1AC/+iPSCs did not undergo chromosomal mutations after gene editing and still had a normal karyotype.To determine the origin of KIF1AC/+iPSCs,this study proved that KIFIAC/+iPSCs were derived from SPG30 patients and were not contaminated with other cells by STR locus analysis.Subsequently,an experimental protocol for induced differentiation from iPSCs to NSCs was established in this study to obtain high purity NSCs.The following results have been obtained from the study of three genotypes of NSCs:(1)The KIF1A c.773C>T(p.T258M)mutation causes accelerated proliferation of neural stem cells,a decrease in cells in G0/G1 phase and an increase in cells in S phase,and a decrease in asymmetric division of NSCs and a decrease in the ability of NSCs to exit the cell cycle.(2)KIF1A mutations cause reduced migration of NSCs in vitro.(3)KIF1A mutations result in reduced neuronal differentiation and shorter axon lengths in neurons.In addition,there was an imbalance in the number of glutamatergic and dopaminergic neurons and GABA neurons obtained by differentiation.Finally,450 differentially expressed genes were screened by RNA-seq sequencing of NSCs derived from KIF1AC/+ iPSCs and KIF1AM/+ iPSCs,GO enrichment analysis revealed differences in the expression of several genes associated with the proliferation and differentiation of NSCs.KEGG enrichment analysis and subsequent validation identified significant changes in several genes of the Wnt signaling pathway at the mRNA level and a significant decrease in the expression of β-catenin as well as the GSK3β protein.CONCLUSION:The KIF1A c.773C>T(p.T258M)mutation leads to accelerated proliferation of NSCs,reduced asymmetric division of NSCs and reduced migration capacity in SPG30 patients.The mutation also resulted in a reduced ability of NSCs to differentiate into neurons.The transcriptome sequencing results suggest that these phenomena may be related to the down-regulation of the Wnt signaling pathway.