RNA-Seq analysis reveals SLC44A1 regulates oligodendrocyte differentiation and choline metabolism

We hypothesized that SLC44A1 deficiency in oligodendrocytes disrupts the transcriptional programs essential for cellular maturation and myelin membrane synthesis, with a particular impact on choline metabolism.To test this, we conducted RNA sequencing (RNA-seq) on primary rat oligodendrocytes following lentivirus-mediated knockdown of Slc44a1. Cells were cultured and induced to differentiate for 3 days post-infection. Total RNA was extracted from four biological replicates per group. Strand-specific RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina) and sequenced on an Illumina NovaSeq 6000 platform. Differential gene expression analysis was performed using DESeq2.Our data revealed that Slc44a1 knockdown significantly altered the expression of 1,430 genes. Notably, key myelination-related genes, including Mbp, Mag, Mog, Cnp, Aspa, and Enpp6, were markedly downregulated. Gene Set Enrichment Analysis (GSEA) confirmed that the downregulated genes were predominantly associated with biological processes of oligodendrocyte maturation and myelination. Gene Ontology (GO) enrichment analysis further supported this, showing significant enrichment in terms related to "myelination" and "myelin sheath." Concurrently, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted a significant enrichment of downregulated genes in metabolic pathways, most notably the choline metabolism pathway. Within this pathway, Pcyt1a, encoding the rate-limiting enzyme phosphatidylcholine cytidylyltransferase 1A (PCYT1A) in the Kennedy pathway for phosphatidylcholine synthesis, was identified as the most significantly downregulated choline metabolic gene following Slc44a1 knockdown. This transcriptional downregulation was validated at both the mRNA and protein levels.These transcriptomic findings demonstrate that SLC44A1 is essential for orchestrating the genetic programs that drive oligodendrocyte differentiation and myelin gene expression. Crucially, they mechanistically link SLC44A1 function to the regulation of choline metabolism, explaining the observed deficits in phosphatidylcholine biosynthesis and myelin lipid composition. The data establish SLC44A1 as a key upstream regulator connecting choline transport to the transcriptional activation of the myelination program.This RNA-seq dataset provides a comprehensive resource for understanding the transcriptomic landscape governed by SLC44A1 in oligodendrocytes. It is instrumental for identifying downstream effector genes and pathways implicated in hypomyelination disorders and offers a foundation for exploring metabolic interventions.