Transcriptomic analysis of human sensory neurons in painful diabetic neuropathy reveals inflammation and neuronal loss

Pathological sensations caused by peripheral painful neuropathy occurring in Type 2 diabetes mellitus (T2DM) are often described as ‘sharp’ and ‘burning’ and are commonly spontaneous in origin. Proposed etiologies implicate dysfunction of nociceptive sensory neurons in dorsal root ganglia (DRG) induced by generation of reactive oxygen species, microvascular defects, and ongoing axonal degeneration and regeneration. To investigate the molecular mechanisms contributing to diabetic pain, DRGs were acquired postmortem from patients who had been experiencing painful diabetic peripheral neuropathy (DPN) and subjected to transcriptome analyses to identify genes contributing to pathological processes and neuropathic pain. DPN occurs in distal extremities resulting in the characteristic “glove and stocking” pattern. Accordingly, the L4 and L5 DRGs, which contain the perikarya of primary afferent neurons innervating the foot, were analyzed from five DPN patients and compared with seven controls. Transcriptome analyses identified 844 differentially expressed genes. We observed increases in levels of inflammation-associated transcripts from macrophages in DPN patients that may contribute to pain hypersensitivity and, conversely, there were frequent decreases in neuronally-related genes. The elevated inflammatory gene profile and the accompanying downregulation of multiple neuronal genes provide new insights into intraganglionic pathology and mechanisms causing neuropathic pain in DPN patients with T2DM.


Supplemental Figure 1
Nonsignificant differences between group demographics of interest: No significant difference was found in A) age (Mann-Whitney Test, p = 0.0530), B) BMI (Mann-Whitney Test, p = 0.4596), or C) sex distribution (Fisher's Exact Test, p = 0.9999) between diabetic and non-diabetic groups.

Supplemental Figure 2
Principal component analysis (PCA) of human DPN transcriptome data: PCA plot A) before and B) after including sex as a covariate as part of the DESEQ2 analysis. PC1 is the first principal component direction where the most variance is occurring, and PC2 is the second most one that is orthogonal to PC1. With sex as a covariate, the DPN donors and the controls then separate into two independent groups.

Supplemental Figure 3
Ingenuity pathway analysis (IPA) of transcriptomic data: The IPA report using all 844 dysregulated genes predominantly centered on the immunological functions occurring in the DRG of the DPN individuals. Thereby, further assessment of the DEGs was separated between A) the upregulated largely inflammatory gene responses and B) the downregulated gene expression changes, where synaptogenesis was appears to be affected by decreases in gene expression.

Supplemental Figure 4
Interaction network of neuronally related genes: 62 genes out of our DEG list were considered to perform as a cellular component of a neuron (GO:0097458). Most genes were downregulated (n=51) while a few were upregulated (n=11) in the DPN donors. To further determine how these dysregulated genes might impact neuronal function, all 62 gene were separately evaluated using STRING (https://string-db.org/) for additional enrichment analysis. A) About 66% of the neuronal genes (n=41) were synaptically related (blue -GO:0045202), while 39% (n=24) were associated with the neuron cell body (green -GO:0043025). B). In terms of biological function, a few genes in red were qualified as being associated with neurotransmitter secretion (GO:0007269).

Supplemental Figure 5
Interaction network of immune responses: 89 genes from our DEG list were registered as immune response related genes (GO:0006955). The immune response genes were subsequently reentered into STRING to identify the nature of the inflammatory reactions and to determine possible protein network interactions. Gene changes related to both adaptive and innate immune responses were recognized in our gene list. 38 genes (red) are considered part of an innate immune response (GO:0045087), while 17 genes (blue) are associated with a humoral immune response (GO:0006959) and 12 genes (green) are involved in T cell activation (GO:0042110).

Supplemental Table 1
DRGs used in this study were acquired post-mortem from the cadaveric donors. As organ donors, information about their medical history was provided through an extensive interview with a family member by a trained interviewer. Included in the table is a list of medications taken by the donors and available data on the duration of DPN.

Supplemental Table 2
Hematoxylin and eosin-stained slides from 5 controls and 5 DPN donors were scored by a pathologist in a blinded fashion on a scale of 0-3 (0 being no ganglionic cell loss/within normal limits and 3 being severe cell loss).

Supplemental Table 3
Significant genes (adjusted p-value cutoff of 0.05 by Benjamini Hochberg's False Discovery Rate).

Supplemental Table 4
A list of all genes, including base mean, log2 fold change, and adjusted p-values (padj).

Supplemental Table 5
Normalized data (DESEQ2 normalized counts) Supplemental Table 6 Table of 71 dysregulated immunoglobulin genes including IGHG1-4, IGHA1-2, and IGHM. Table 7 Further gene enrichment was conducted using ToppGene Suite (https://toppgene.cchmc.org). In the DisGeNET database of gene-disease associations, 79 dysregulated genes were listed as involved in pain (C0030193). Interaction network of neuronally related genes: 62 genes out of our DEG list were considered to perform as a cellular component of a neuron (GO:0097458). Most genes were downregulated (n=51) while a few were upregulated (n=11) in the DPN donors. To further determine how these dysregulated genes might impact neuronal function, all 62 gene were separately evaluated using STRING (https://stringdb.org/) for additional enrichment analysis. A) About 66% of the neuronal genes (n=41) were synaptically related (blue -GO:0045202), while 39% (n=24) were associated with the neuron cell body (green -GO:0043025). B). In terms of biological function, a few genes in red were qualified as being associated with neurotransmitter secretion (GO:0007269).