Investigation Of Impacts Of DARS2 Gene Variants On LBSL Cerebral Organoids Using Single-Cell Rna Sequencing

Background: Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation(LBSL)is a rare autosomal recessive neurological disorder,which is caused by mutations in DARS2 gene.DARS2 encodes mitochondrial aminoacyl-t RNA synthetase(mt-Asp RS)that specifically charges aspartate to its corresponding t RNA,facilitating the protein synthesis in mitochondria.To date,the researches based on human neural cells of LBSL patients have been very rare.The development of in vitro differentiation of neural tissues/cells from induced pluripotent cells(iPSCs)has provided the unprecedented technical platform for studying these rare neurological disorders.Objective:(1)To investigate the effects of DARS2 gene variants on the gene and transcript expression of human cerebral organoids(h COs)that were derived from iPSCs of LBSL patients at the single-cell level using SMART-seq2 sequencing technique.(2)To explore the DARS2 protein expression level,exon 3 skipping pattern,as well as live changes of early neuronal differentiation using iPSCs and iPSC-derived neurons(i Ns)as supplementary models.Methods:(1)Clinical records,including sex,age of onset,DARS2 gene variants,as well as scales of ataxia,gross motor function and manual ability,were collected retrospectively from seven LBSL patients who got diagnosed at Kennedy Krieger Institute of Johns Hopkins Medical Institutions.(2)h COs were generated from iPSCs of seven LBSL patients and three healthy controls using unguided protocol.Seventy-day-old h COs were subject to SMART-seq2 single-cell RNA sequencing(sc RNA-seq).sc RNA-seq data were mapped to human reference genome using Rsubread package.Seurat package was used to perform data quality control,integration and cluster analysis.(3)Differentially gene expression(DGE)analysis was performed between LBSL and control cells.Gene set enrichment analysis(GESA)was done by Panther Classification System.(4)sc RNA-seq data were mapped to human reference transcriptome to obtain the transcript expression matrix of each cell using kallisto software.DTUrtle package was used for differential transcript usage(DTU)analysis;exon-level analysis was done by BRIE2 software.(5)Lentivirus particles containing Ngn2 gene were transduced into iPSCs.After puromycin selection and Doxycycline exposure,Neurogenin 2 was induced to promote neuronal differentiation.Polymerase chain reaction(PCR),quantitative reverse transcription PCR(RT-q PCR)and Western Blot were used to explore the skipping of exon 3 in DARS2 transcripts and the expression pattern of DARS2 protein in iPSCs and i Ns.Live cell imaging system was used to study the early differentiation of i Ns.Results:(1)According to their genotypes,these patients were subset into two groups: Group 1 patients carrying only one or no splice site mutation and Group 2 patients carrying two splice site mutations simultaneously.The average age of onset of two groups was 2.56±0.94 and 1.94±1.55 years old,respectively.The average time interval between onset and follow up was 4.56±1.60 and 7.06±3.65 years.The average SARA scale of two groups for ataxia was 12.83±1.20 and 5.50±1.00 and average MRI Loes scale was 13.00±3.24 and 5.25±2.25,respectively.The GMFCS and MACS for all patients ranged I to II level.(2)A total of 809 cells passed the quality control and were used for downstream analysis.Nine major cell clusters were identified,including neuroepithelial cells,radial glial cells,cortical neurons,choroid plexus(Ch P)epithelial cells and so on.DARS2 was predominately expressed in highly proliferative neuroepithelial cells but downregulated in other differentiated cell clusters.(3)Through DGE analysis of sc RNA-seq data,we identified 736(333 up and 403 down)and 490(198 up and 292 down)DEGs in Group 1 and Group 2 LBSL cells,respectively.Inflammation and stress related genes were upregulated mainly in Group 1 cells,whereas translation and oxidative phosphorylation related genes were upregulated in Group 2cells.Multiple RNA binding protein(RBP)genes and neuronal development related genes were dysregulated in LBSL cells with exacerbation in neuronal cells.(4)DTU analysis of sc RNA-seq data identified 950 and 710 DTU events in Group 1 and Group2 cells,respectively.About 80% of DTU genes failed to be detected by DGE analysis.GSEA of DTU genes of Group 1and Group 2 showed similar clusters of enriched GO terms related to RNA binding,protein binding and metabolic process.(5)Differentially spliced exon(DSE)analysis of sc RNA-seq by BRIE2 software found 194 DSE events,one of which was exon 3 of DARS2(FDR = 7.99E-06).Some of the LBSL cells only expressed transcripts lacking exon 3(PSI = 0).(6)Live cell imaging verified that the neurite outgrowth of LBSL i Ns was significantly decreased compared to control i Ns(P < 0.0001).(7)Western Blot experiments verified that DARS2 protein expression levels were downregulated in both control(P.adj < 0.0001)and LBSL i Ns(P.adj = 0.0174),compared to corresponding iPSCs.(8)Using primers annealing to exon 3-4 and 2-4 junctions,RT-q PCR results demonstrated the expression of transcripts containing exon 3decreased in LBSL iPSCs(P.adj < 0.0001)and the level of transcripts lacking exon 3 in LBSL iPSCs was 20-fold higher than that in control iPSCs(P.adj = 0.0003).When iPSCs were differentiated into i Ns,the expression of transcripts lacking exon 3 significantly increased.Conclusions:(1)Multiple RNA binding protein genes were dysregulated with a certain extent of genotype-and cell-type-specificity,and the dysregulation was exacerbated in neuronal cells of LBSL h COs.(2)Pervasive differential transcript usage and exon splicing events were detected in LBSL h COs,mainly affecting the translation and metabolism of protein,which is one of the pathophysiological features of LBSL.(3)Not all LBSL cells could benefit from the leaky mechanism of splice site mutations.(4)LBSL i Ns showed deficits in neurite outgrowth,which might be the primary culprit of LBSL.(5)DARS2 is predominantly expressed in stem cells but downregulated in other differentiated post-mitotic cells,which is mainly regulated by exon3 skipping.