Nageotte nodules in human DRG reveal neurodegeneration in painful diabetic neuropathy

Abstract

Diabetic neuropathy is frequently accompanied by pain and loss of sensation attributed to axonal dieback. We recovered dorsal root ganglia (DRGs) from 90 organ donors, 19 of whom had medical indices for diabetic painful neuropathy (DPN). Nageotte nodules, dead sensory neurons engulfed by non-neuronal cells, were abundant in DPN DRGs and accounted for 25% of all neurons. Peripherin-and Nav1.7-positive dystrophic axons invaded Nageotte nodules, forming small neuroma-like structures. Using histology and spatial sequencing, we demonstrate that Nageotte nodules are mainly composed of satellite glia and non-myelinating Schwann cells that express SPP1 and are intertwined with sprouting sensory axons originating from neighboring neurons. Our findings solve a 100-year mystery of the nature of Nageotte nodules linking these pathological structures to pain and sensory loss in DPN.

Figure 1. Identification of Nageotte nodules in DRGs from diabetic organ donors.

A) Hematoxylin and eosin staining was performed on DRGs from organ donors and then each DRG was scored for prevalence of Nageotte nodules using a qualitative scoring system. B) After scoring, donors were grouped based on their medical history of diabetes, analgesic usage, or medical note of diabetic peripheral neuropathy. Diabetic donors had significantly higher Nageotte nodule scores compared to non-diabetic donors. In diabetics, Nageotte nodule content increased in severity in relation to painful diabetic neuropathy as indicated by analgesic usage, medical note of diabetic peripheral neuropathy, and/or diabetes-related amputation. C) Representative images of an L4 bi ganglia from a DPN donor immunostained for GFAP (red, satellite glial cells), peripherin (green, sensory neurons), and DAPI (blue, nuclei). Asterisks denote Nageotte nodules. D) The percentage of Nageotte nodules was significantly higher in the DPN DRGs (average: 25%) compared to non-diabetic DRGs (average: 8%). E) Confocal image of a peripherin-positive axon bundle intertwined with other cells at a Nageotte nodules. F) Image taken from Jean Nageotte’s original 1922 publication in which three Nageotte nodules (mid top, mid bottom, and left) contain axon bundles which sprout from a glomerulus (middle). G) Transmission electron microscopy (TEM) of a Nageotte nodule. Arrows point to unmyelinated axonal fibers. Statistical tests: B: One-way ANOVA with Bonferroni’s multiple comparisons test. D: Unpaired t-test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. Scale bars: A: 100 μm. C: Mosaic – 1mm and other panels - 50 μm. E: 10 μm. G: 2 μm.

Figure 2:. Nageotte nodule axons express nociceptive, but not sympathetic markers.

A) Tyrosine hydroxylase (TH, red) labeling in combination with peripherin (green) and DAPI (blue) revealed that Nageotte nodule axonal sprouts were not sympathetic in origin. Sample size: DPN n=9. B) TrpV1 fibers at a Nageotte nodule in a DPN DRG (white arrow). TrpV1 was only detected in Nageotte nodules from a single DPN donor. The TrpV1+ fibers at the Nageotte nodule appeared to arise from a glomerulus (magenta arrow). Sample size: DPN n=5. C) Nav1.7 (red) was detected in the axonal fibers intertwined with Nageotte nodules. Sample size: DPN n=6. Scale bars: A: 10 μm. B: 20 μm. C: 10 μm.

Figure 3:. Nageotte nodule axon bundles originate from axonal sprouts from local sensory neurons in situ and in vitro.

A) Nageotte nodule (white asterisks), two sensory neuron cell bodies (yellow asterisks), and dystrophic axons (magenta arrows) in a 60X z-stack projection image of a DRG section stained for peripherin (green) and DAPI (blue) from donor 6. In the Trace panel, axonal filaments stemming from the neuronal soma of the middle sensory neuron were traced in Imaris (pink filament trace) which mainly connected to dystrophic axons (multi-colored axonal blebs). The Nageotte nodule axonal bundle (blue filament trace) were mainly spooling fibers that traced back to dystrophic axons. In some cases, dystrophic axons were interconnected to one another (yellow filament traces). B) A representative 20X z-stack projection image of human DRG sensory neurons that were cultured in vitro for 3 days and then stained for peripherin (green), and DAPI (blue). Human sensory neurons display a multipolar phenotype in which multiple axonal branches sprout from the neuronal soma (yellow arrows), form dystrophic axons (magenta arrows), and intertwine with structures resembling Nageotte nodules (white arrows). A digitally zoomed-in view of the outlined area (cyan) exemplifies a multipolar sensory neuron sprouting fibers into a Nageotte nodule. C) Jean Nageotte described collateral sprouting in 1922 (I-III; original illustration by Jean Nageotte, 1922) in which I) a normal ganglion cell with a T-bifurcated axon is II) deprived of the radicular branch of the axon resulting in III) neurite outgrowth from the soma and glomerulus which are equipped with encapsulated growth balls. Jean Nageotte as well as our imaging of DPN DRGs noted IV) the formation of non-sympathetic pericellular nests that formed around sensory neurons with intact somata and those with shrunken/misshapen somata likely in the process of dying. V) Neurites sprout from the dystrophic axons forming arborizations at Nageotte nodules. Scale bars: A: 15 μm. B: 50 μm and zoomed-in view panel: 10 μm.

Figure 4:. Spatial transcriptomics of Nageotte nodules identifies non-myelinating Schwann cells and satellite glia as prominent cell types.

A) Spatial transcriptomics was conducted on DRGs from 6 DPN donors. Barcodes touching Nageotte nodules (1094) and nearby neurons (1087) were selected for downstream analysis. B) Key gene ontology themes were related to neurodegeneration. C) RNAscope in situ hybridization for SOX10 (green, satellite glia and Schwann cells), FABP7 (red, satellite glia), CD68 (purple, macrophages) and DAPI (blue) in a DPN DRG. Confocal, 40X. Sample size: DPN n=6. D)Digitally zoomed overlay images of Nageotte nodule 1 (cyan arrow in panel C), and Nageotte nodule 2 (yellow arrow in panel C). Nageotte nodule 1 had higher content of FABP7+ nuclei, while Nageotte nodule 2 had little-to-no FABP7signal, indicating that there are differences in the composition of cell types between Nageotte nodules. E) Deconvolution using single-nuclear sequencing datasets revealed that the sources of the majority of mRNA transcripts in Nageotte nodules come from Satellite glial cells and non-myelinating (NM) Schwann cells. The remaining percentage of transcripts in Nageotte nodules arise from neurons, likely axonally trafficked mRNAs. F) Clustering analysis of Nageotte nodule barcodes identified 5 subclusters. G)Deconvolution reveals differences in the distribution of mRNA sources between clusters. Scale bars: C: 50 μm. D: 10 μm.

Figure 5:. Ligand-receptor interactions between Nageotte nodules and nearby neurons identifies osteopontin (SPP1) and CD44.

A) Differentially expressed ligands per Nageotte nodule cluster and corresponding receptors expressed in nearby neurons. B)RNAscope in situ hybridization for SCN10A (green, Nav1.8), SPP1(red, osteopontin), CD44 (blue), and DAPI (cyan) in a DPN DRG. White outline denotes the digitally zoomed-in image of a single Nageotte nodule shown in the bottom panel. Confocal, 40X. Sample size: DPN n=5. C) Top 20 expressed cytokines in Nageotte nodules. D) Immunohistochemistry for phosphorylated eukaryotic translation initiation factor (red, p-eIF4E) and DAPI (blue) in a non-diabetic and DPN DRG. E) p-eIF4E was significantly elevated in the soma of sensory neurons in the DPN DRGs. Sample size: Non-diabetic n=5, DPN n=5. F) p-eIF4E was also detected at Nageotte nodules in the DPN DRGs. Statistical tests: E: Unpaired t-test. ***p<0.001. Scale bars: B: top panel - 50 μm and bottom panel - 10 μm. D: 200 μm. F: 50 μm.