Transcriptomic profiling of sciatic nerves and dorsal root ganglia reveals site-specific effects of prediabetic neuropathy

Abstract

Peripheral neuropathy (PN) is a severe and frequent complication of obesity, prediabetes, and type 2 diabetes characterized by progressive distal-to-proximal peripheral nerve degeneration. However, a comprehensive understanding of the mechanisms underlying PN, and whether these mechanisms change during PN progression, is currently lacking. Here, gene expression data were obtained from distal (sciatic nerve; SCN) and proximal (dorsal root ganglia; DRG) injury sites of a high-fat diet (HFD)-induced mouse model of obesity/prediabetes at early and late disease stages. Self-organizing map and differentially expressed gene analyses followed by pathway enrichment analysis identified genes and pathways altered across disease stage and injury site. Pathways related to immune response, inflammation, and glucose and lipid metabolism were consistently dysregulated with HFD-induced PN, irrespective of injury site. However, regulation of oxidative stress was unique to the SCN while dysregulated Hippo and Notch signaling were only observed in the DRG. The role of the immune system and inflammation in disease progression was supported by an increase in the percentage of immune cells in the SCN with PN progression. Finally, when comparing these data to transcriptomic signatures from human patients with PN, we observed conserved pathways related to metabolic dysregulation across species, highlighting the translational relevance of our mouse data. Our findings demonstrate that PN is associated with distinct site-specific molecular re-programming in the peripheral nervous system, identifying novel, clinically relevant therapeutic targets.

Keywords: Differentially expressed genes; Dorsal root ganglia; High-fat diet; Peripheral neuropathy; Prediabetes; RNA-seq; Sciatic nerve; Self-organizing map.

Figure 1.. Study overview and analysis pipeline.

C57BL/6J mice were fed a 45% high-fat diet (HFD) starting at 3 weeks of age. Metabolic and peripheral neuropathy (PN) phenotypic assessment was performed at 12 weeks and 36 weeks of age (study conclusion). After phenotypic assessment, mice were euthanized, and the sciatic nerve (SCN) and dorsal root ganglia (DRG) collected. Gene expression in SCN and DRG were assessed using RNA-seq. Processed data were then analyzed in parallel using self-organizing map (SOM) or differentially expressed gene (DEG) analysis. Functional enrichment and canonical molecular pathways associated with altered gene expression were identified using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Gene expression data was analyzed with CIBERSORT to determine changes in cell-type abundance within tissues. Finally, mouse DEGs were cross-referenced against human sural nerve RNA-seq data.

Figure 2.. Self-organizing map (SOM) analysis.

(A, B) SOM analysis was used to identify patterns of gene expression in the sciatic nerve (SCN, A) and the dorsal root ganglia (DRG, B) at 12 and 36 weeks of age from high-fat diet (HFD) and standard diet (SD) mice. All gene expression patterns were projected onto a 5 x 5 module and genes with similar expression patterns were organized into modules. The number of genes within modules represented by a color scale from 0 (white) to 700 (red). Modules were grouped into clusters based on similar changes in gene expression between groups (12 wk SD; 12 wk HFD; 36 wk SD; 36 wk HFD). Clusters are identified as groups of modules within bold blue lines.

Figure 3.. Functional enrichment analysis of sciatic nerve (SCN) clusters.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed on self-organizing (SOM) clusters in SCN. A heat map was generated using the top 10 most significantly enriched pathways in the SOM clusters, as determined by richR. Color indicates significance (Benjamini-Hochberg (BH)-adjusted p-value) on a −log₁₀ scale from less (white) to most (red) significant.

Figure 4.. Functional enrichment analysis of dorsal root ganglia (DRG) clusters.

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed on self-organizing (SOM) clusters in DRG. A heat map was generated using the top 10 most significantly enriched pathways in the SOM clusters, as determined by richR. Color indicates significance (Benjamini-Hochberg (BH)-adjusted p-value) on a −log₁₀ scale from less (white) to most (red) significant.

Figure 5.. Differential gene expression analysis.

Gene expression from sciatic nerve (SCN; A, C) and dorsal root ganglia (DRG; B, D) were analyzed using differential expression analysis. (A, B) Significantly up- or down-regulated genes were detected in high-fat diet (HFD) versus standard diet (SD) mice at 12 and 36 weeks of age in both tissue types. (C, D) Venn diagram of the number of shared and unique differentially expressed genes (DEGs) between 12- and 36-week time points for SD and HFD-fed mice. (E) Venn diagram of the number of shared and unique DEGs with disease progression between SCN and DRG tissues. DEGs selected for pathway analysis are indicated in bold with underlining.

Figure 6.. Functional enrichment analysis.

Functional enrichment of unique differentially expressed genes (DEGs) from the sciatic nerve (SCN; A) or dorsal root ganglia (DRG; B) from mice fed a standard diet (SD) or high-fat diet (HFD) at 12 and 36 weeks of age was performed by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The top 10 significantly enriched canonical pathways for each comparison are illustrated. Comparisons are the DEGs unique to: early disease stage (SD vs. HFD at 12 weeks of age; SCN only) and disease progression (HFD 12 vs. HFD 36 weeks of age; SCN and DRG). Color indicates significance (Benjamini-Hochberg (BH)-adjusted p-value) on a −log₁₀ scale from less (white) to most (red) significant.

Figure 7.. Cell composition analysis.

Composition and phenotype of cells in the sciatic nerve (SCN, A) and dorsal root ganglia (DRG, B) of mice fed a standard diet (SD) or high-fat diet (HFD) at 12 or 36 weeks of age. The fraction of each cell type is depicted. Cell types include B cells (BC), DRG, vascular endothelial cells (EC1), blood nerve barrier endothelial cells (EC2), fibroblasts (fibro), lymphatic endothelial cells (lymph), myeloid lineage cells (MC), macrophages (MP), myelinating Schwann cells (mySC), nonmyelinating Schwann cells (nmSC), pericytes (PC), T cells (TC), and vascular smooth muscle cells (vSMCs).

Figure 8.. Peripheral neuropathy gene expression overlap between mouse and human.

Kyoto Encyclopedia of Genes and Genomes pathway analysis was performed on the differentially expressed genes (DEGs) shared by both the current mouse study and sural nerve biopsies from human participants with diabetic peripheral neuropathy. The significantly enriched canonical pathways are illustrated as dot plots. Node size refers to the number of DEGs within each term and node color indicates significance level (p-value) shown on a −log₁₀ scale.