Aging and injury drive neuronal senescence in the dorsal root ganglia

Abstract

Aging negatively impacts central nervous system function; however, there is limited information about the cellular impact of aging on peripheral nervous system function. Importantly, injury to vulnerable peripheral axons of dorsal root ganglion (DRG) neurons results in somatosensory dysfunction, such as pain, at higher rates in aged individuals. Cellular senescence is common to both aging and injury and contributes to the aged pro-inflammatory environment. We discovered DRG neuron senescence in the context of aging and pain-inducing peripheral nerve injury in young (~3 months) and aged (~24 months) male and female mice. Senescent neurons were dynamic and heterogeneous in their expression of multiple senescence markers, including pro-inflammatory factor IL6. Senescence marker-expressing neurons had nociceptor-like profiles, included high-firing phenotypes and displayed increased excitability after IL6 application. Furthermore, elimination of senescent cells resulted in improvement of nociceptive behaviors in nerve-injured mice. Finally, male and female post-mortem human DRG contained senescent neurons that increased with age (~32 years old versus 65 years old). Overall, we describe a susceptibility of the peripheral nervous system to neuronal senescence-a potential targetable mechanism to treat sensory dysfunction, such as chronic pain, particularly in aged populations.

Fig. 1. Senescent neurons accumulate with age in the mouse DRG.

a, Representative images of SA-β-gal activity staining (blue) in the lumbar DRG of young (11–16 weeks) and aged (20–24 months) mice. Percent SA-β-gal-positive pixels per DRG area (right) (n = 6 young, 5 aged mice; two-tailed unpaired t-test, P = 0.0153). Scale bar, 100 µm. b, Representative RNAscope images for senescence markers p21 and p16 with SASP factor IL6 in whole DRG section. Scale bar, 100 µm. c,d, Quantification of neuronal expression of each marker (c) or in combination (d) expressed as a percent of total DRG neurons (n = 5 young, 4 aged mice, two-tailed unpaired t-test, p21⁺, P = 0.0014; p16⁺, P = 0.006; IL6⁺, P = 0.0031; p21⁺IL6⁺, P = 0.0005; p16⁺IL6⁺, P = 0.3076; p21⁺p16⁺IL6⁺, P = 0.1560). e, Analysis of IL6-expressing DRG neuron population to show co-expression with senescence markers p21 and/or p16 in young and aged mice (n = 5 young, 4 aged mice). f, Quantification of IL6 protein levels by ELISA assay in young or aged plasma (n = 6 young, 5 aged mice, two-tailed unpaired t-test, P = 0.0474). All data are expressed as the mean ± s.e.m. NS, not significant. Source data

Fig. 2. DRG neurons express senescence markers and SASP factors after peripheral nerve injury.

a, Schematic of SNI and DRG tissue analysis timepoints (BioRender). b, qPCR from lumbar DRG in young (11–16 weeks) mice (n = 4 control; n = 4 SNI young mice; two-tailed unpaired t-test; Supplementary Table 1). c, Left, RNAscope image of DRG slice in young mice. Scale bar, 100 µm. Right, number of DRG neurons expressing p21 (upper) or p16 (lower) in young mice (n = 5 uninjured mice, n = 4, 7-day and 3-week post-SNI mice, n = 3, 7-week post-SNI mice; one-way ANOVA, p21: uninj versus 7 days or 3 weeks, P < 0.0001; uninj versus 7 weeks, P = 0.0002. p16: uninj versus 3 weeks, P = 0.0117; uninj versus 7 weeks, P = 0.0002; 7 days versus 7 weeks, P = 0.0085). d,e, Re-analysis of Renthal et al.. RNA-seq dataset using young adult mouse DRG. d, Percentage of p16 (Cdkn2a⁺)-expressing senescent cells relative to all DRG cells after ScNT. Cells are negative for Lmnb1 and Top2a to filter out any nonsenescent cells. Glia, satellite glia and Schwann cells; Immune & other cells, neutrophils, macrophages, B cells, fibroblasts, endothelial cells and pericytes; Neuron, all DRG neurons. Dot plot (e) of senescence marker gene expression by DRG neurons after ScNT. SenMayo genes are significant, at least one timepoint (Supplementary Table 2). f, RNAscope image of DRG slice in aged (20–24 months) mice. Scale bar, 100 µm. Number of DRG neurons expressing either p21 (right) or p16 (right) in aged mice (n = 4 uninjured mice, n = 3 post-SNI mice/timepoint; one-way ANOVA, p21: uninj versus 7 days, P = 0.0335; uninj versus 3 weeks, P = 0.0298. p16: uninj versus 7 days, P = 0.0182; uninj versus 3 weeks, P = 0.0009; uninj versus 7 weeks, P = 0.0008). g, p21⁺p16⁺ co-expressing DRG neurons (n = 5 young uninjured mice, n = 4 young 3-week post-SNI mice; n = 4 aged uninjured mice, n = 3 aged 3-week post-SNI mice; one-way ANOVA, young uninj versus SNI, P = 0.0002; aged uninj versus SNI, P < 0.0001; young SNI versus aged SNI, P = 0.0003). h, p21⁺p16⁺IL6⁺ (asterisk) neuron by RNAscope. Scale bar, 10 µm. i, IL6⁺ DRG neurons co-expressing p21 and/or p16 (n = 5 young uninjured mice, n = 4 aged uninjured mice, n = 4 young 3-week post-SNI mice, n = 3 aged 3-week post-SNI mice; one-way ANOVA; Supplementary Table 1). j, IL6⁺ DRG neurons co-express p21 and/or p16 at 3 weeks after SNI (n = 3 mice per group). All data are mean values ± s.e.m. Ag, aged; d, days; h, hours; NS, not significant; wk, weeks; Yg, young. Source data

Fig. 3. ATF3⁺ injured and neighboring noninjured DRG neurons express senescence markers after nerve injury.

a, Quantification of number of ATF3⁺ neurons as a percent of total L3/4 DRG neurons in uninjured and multiple post-SNI timepoints in young (11–16 weeks) and aged (20–24 months) mice (n = 3 young, n = 5 aged uninjured mice; n = 3 7-day post-SNI mice/age group; n = 5 young, n = 3 aged 3-week post-SNI mice; n = 3 7-week post-SNI mice/age group, one-way ANOVA; see Supplementary Table 1 for all P values). Data are mean values ± s.e.m. b, Representative images of dual immunohistochemistry/RNAscope labeling ATF3⁺ injured neurons (nuclear-localized protein) and RNA puncta of p21 and p16 at 3 weeks after SNI. Co-expression of ATF3 with p21 and or p16 (arrows). Asterisks represent ATF3⁻ cells that express p21 and/or p16 senescence markers. Scale bar, 100 µm. Inset scale bar, 20 µm. c,d, Quantification of ATF3⁺ neuron population that co-express p21 and/or p16 at multiple timepoints after injury in young and aged DRG (young: n = 3–5 mice per timepoint per group: 7-day: n = 1,421 ATF3⁺ neurons, 3-week: n = 1,056 ATF3⁺ neurons; 7-week: n = 523 ATF3⁺ neurons; aged: n = 3 mice per timepoint: 7-day: n = 1,004 ATF3⁺ neurons; 3-week: n = 983 ATF3⁺ neurons; 7-week: n = 722 ATF3⁺ neurons). e, Quantification of ATF3⁻ population co-expressing senescence marker p21 at multiple timepoints after injury in young and aged DRG (n = 3 mice per group per timepoint, one-way ANOVA, young, P = 0.0323; aged, P = 0.1761). Data are mean values ± s.e.m. f, Quantification of ATF3⁻ population co-expressing senescence marker p16 at multiple timepoints after injury in young and aged DRG (n = 3 mice per group per timepoint, one-way ANOVA, young, P = 0.0397; aged, P = 0.0188). All data are expressed as the mean ± s.e.m. g, Re-analysis of Renthal et al.. RNA-seq dataset from young adult mouse DRG. Dot plot demonstrates the timecourse of senescence marker gene expression after ScNT by either ATF3⁻ (top) or ATF3⁺ (bottom) DRG neurons (Supplementary Table 3). NS, not significant; wk, week. Source data

Fig. 4. Trpv1⁺ nociceptors express senescence markers after nerve injury.

a, Analysis of cell diameter (µm) of p21⁺IL6⁺, p16⁺IL6⁺ or p21⁺p16⁺IL6⁺ co-positive neurons in the DRG at 3 weeks after nerve injury in young (11–16 weeks) and aged (20–24 months) mice (young: n = 215 p21⁺IL6⁺ neurons; n = 51 p16⁺IL6⁺ neurons; n = 102 p21⁺p16⁺IL6⁺ neurons; aged: n = 155 p21⁺IL6⁺ neurons; n = 21 p16⁺IL6⁺ neurons; n = 46 p21⁺p16⁺IL6⁺ neurons). b, Representative RNAscope images of young or aged DRG co-labeled for the ion channel Trpv1, senescence marker p21 and SASP factor/cytokine IL6. Merged images also have DAPI overlay (gray). For IL6 signal, intense puncta signal with white center are positive neurons, and fainter/dull blue is background. Arrows: Trpv1⁺ senescent neurons; asterisks: Trpv1⁻ senescent neurons. Scale bars, 100 µm and 20 µm (insets). c,d, Quantification of Trpv1 neuron population and its co-expression with p21 and/or IL6 in young (c) and aged (d) L3/4 DRG of uninjured (controls) and 3 weeks after SNI (n = 3 uninjured young mice, n = 972 Trpv1⁺ neurons; n = 3 SNI young mice, n = 1,548 Trpv1⁺ neurons; n = 4 uninjured aged mice, n = 1,056 Trpv1⁺ neurons; n = 3 SNI aged mice, n = 1,292 Trpv1⁺ neurons). wk, weeks. Source data

Fig. 5. DRG neurons expressing senescence and SASP markers include high-firing and nociceptor-like phenotypes, and the SASP factor IL6 increases excitability in these populations.

a, Representative traces from p16-expressing neurons demonstrating repetitive firing (left), hyperpolarization-activated current (Ih) presence (middle) and the firing parameters rheobase and AP latency (right). b, Clusters identified with the hierarchical density-based algorithm HDBSCAN after UMAP alignment of individual neurons constructed with diameter (range, 14–41 µm), firing properties and intrinsic currents. Discrete clusters^– are identified by color (n = 82 recorded DRG neurons from young (11–16 weeks) and aged (20–24 months) mice). UMAP highlighting senescence marker p16 (orange) (c), p21 (pink) (d) and the SASP factor IL6 (blue) (e). f, Heatmap depicting parameters from left to right as follows: clusters (cool gradient), gene expression (black, no expression; light teal, expression), diameter and physiology parameters (warm gradient; higher normalized values are lighter and lower values are darker). Senescence marker p16 and SASP factor IL6 groups contain neurons with high-firing phenotypes (>100 total APs fired during current steps), which is outlined over increasing depolarizing current steps (lower left panel). Ih current amplitude was also measured at decreasing hyperpolarizing steps (lower right panel). g, Senescent neurons (p21 in magenta, p16 in orange and IL6 in blue) display the DRG nociceptor-associated property of wide APs (half-width above 0.5 ms, gray dotted line). h, IL6 application increases evoked firing in senescence marker-expressing neurons in monolayer culture (155.5 ± 18 APs, n = 26 cells from 11 mice for control; 209.7 ± 19.06 APs, n = 27 cells from 13 mice for IL6 application; U = 234.5, P = 0.038, two-sided, Mann–Whitney test; neuronal expression of p21 in magenta, p16 in orange, p21 and p16 in green and p21 and IL6 in blue). All data are mean values ± s.e.m. Source data

Fig. 6. In vivo elimination of senescent neurons using senolytics alleviates pain behaviors after nerve injury.

a, Schematic of treatment paradigm (BioRender). Young (11–16 weeks) and aged (20–24 months) mice were treated with senolytic (ABT263, 100 mg kg⁻¹) or vehicle for 10 days by oral gavage, starting at 3 weeks after SNI. Mechanical allodynia and weight bearing were assessed during and after treatment. b, Quantification of CC3⁺ neurons in the DRG after treatment with vehicle or ABT263 for five consecutive days (n = 3 male, n = 3 female aged mice per treatment group, two-tailed unpaired t-test, P = 0.0034). c, CC3⁺ neurons analyzed for their co-expression with p21 and/or p16 senescence markers. Categories are mutually exclusive (n = 3 male, n = 3 female aged mice per treatment group; data show minimum to maximum, all points). d, Aged mice treated with ABT263 or vehicle (light blue indicates treatment window) and their mechanical allodynia thresholds were assessed (n = 12 female, n =10 male vehicle-treated mice; n = 15 female, n = 13 male ABT263-treated mice, mixed effects analysis, two-sided, Sidakʼs multiple comparisons test, day 19, P = 0.0309; day 29, P = 0.0033; day 39, P < 0.001). e, Aged mice treated with ABT263 displayed improved weight bearing on injured limb compared to vehicle-treated mice at both day 16 (n = 10 female, n = 8 male vehicle-treated mice; n = 9 female, n = 9 male ABT263-treated mice, two-tailed unpaired t-test, P = 0.0002) and day 39 (n = 9 female, n = 7 male vehicle-treated mice; n = 9 female, n = 9 male ABT263-treated mice, two-tailed unpaired t-test, P < 0.0001) after treatment start. f, Young mice were treated with ABT263 or vehicle (light blue indicates treatment window), and their mechanical allodynia thresholds were assessed (n = 12 male vehicle-treated mice, n = 12 male ABT263-treated mice, two-way ANOVA, two-sided, Sidak’s multiple comparisons test, day 12, P = 0.0018). g, Young mice treated with ABT263 displayed improved weight bearing on injured limb compared to vehicle-treated mice at both day 16 (n = 11 vehicle-treated, n = 13 ABT263-treated male mice, two-tailed unpaired t-test, day 16, P < 0.0001) and day 29 (n = 5 vehicle-treated, n = 5 ABT263-treated male mice, two-tailed unpaired t-test; day 29, P = 0.002) after treatment start. All data are mean values ± s.e.m. BL, baseline. Source data

Fig. 7. Human DRG neurons express senescence markers and SASP factor IL6 with age.

a,b, Representative RNAscope images from young or aged human L4 DRG showing expression of p21 and p16 senescence markers (enlarged left images with DAPI; scale bar, 100 µm). The large globular signal present in both channels is autofluorescent lipofuscin and not RNAscope signal (small puncta). c, Quantification of p21⁺ and p16⁺ neurons in the young and aged human DRG as a percent of total DRG neurons (n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). d, Quantification of IL6-expressing neurons as a percent of total DRG neurons (n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). e, Analysis of IL6⁺ neuron population and quantification of the co-expression of senescence markers p21 and/or p16. f, Quantification of neurons co-expressing senescence markers p21 and/or p16 with IL6 as a percent of total DRG neurons (n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). g, Percent of DRG neurons that are ATF3⁺ in young and aged human DRG (n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). h, Example image depicting a single human neuron positive for ATF3 (nuclear-localized, immunohistochemistry) and p21 (RNAscope, puncta). Scale bars, 20 µm. Analysis of ATF3⁺ neuron population and quantification of the co-expression with p21 in young and aged human DRG (right, donuts) (n = 64 young ATF3⁺ DRG neurons, n = 54 aged ATF3⁺ DRG neurons). i, Total percentage of TRPV1⁺ neurons as a percent of total DRG neurons in young and aged human DRG. Quantification of the subsets of TRPV1⁺ neurons that co-express either p21 or p16 by RNAscope (boxed right) (n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG). j, Single representative human neurons (quantified in k) showing co-expression of TRPV1 with p21 and/or p16. DAPI in gray. Scale bars, 20 µm. k, Venn diagram of human DRG neurons that express TRPV1, p16 and p21. Aged DRG display a greater overlapping fraction of TRPV1⁺ neurons expressing either or both senescence markers p21 and p16 compared to young neurons. n = 2 young female (32-year-old and 33-year-old); n = 2 aged male/female (65-year-old) DRG. All data are mean values ± s.e.m. Source data

Extended Data Fig. 1. Confirmation of p16^INK4A-specific RNA expression in the DRG.

RNAscope using RNA probes spanning exons encoding both p16^INK4A and p19^ARF protein (Cdkn2a-tv1). Cdkn2a-tv2 RNA probe spans exons specific to p16^INK4A protein. Individual primary sensory neurons outlined in inset images. Complete cellular co-localization of the two Cdkn2a variant probes in mouse lumbar DRG sections confirm p16-specific expression in mouse lumbar DRG neurons. Scale bar of upper panels are 25 µm. Scale bars of inset are 15 µm. Source data

Extended Data Fig. 2. Senescence marker gene expression patterns in DRG neurons post nerve-injury.

a, Re-analysis of Wang et al.. RNA-sequencing dataset from young adult mouse DRG. Dot plot displays changes in senescence marker gene expression following spared nerve injury (SNI). Genes under SenMayo subgroup are all significantly increased post-injury (at least one time point) (see Supplementary Table 3). b, Re-analysis of Renthal et al.. RNA-sequencing dataset from young adult mouse DRG. Heatmap represents changes in SenMayo gene expression in DRG neuronal subtypes following sciatic nerve transection (ScNT). (see Supplementary Table 4). Source data

Extended Data Fig. 3. Senolytic treatment induces senescent neuron apoptosis and does not alter normal sensory function.

a, Immunohistochemistry showing co-localization of cleaved caspase-3 (CC3) apoptotic marker with neuronal marker (CGRP) and nuclei (DAPI) in the DRG post-ABT263 treatment. Scale bars 20 µm. b, Co-localization of CC3 (immunohistochemistry) with p21 and p16 (RNAscope) in DRG neurons post-ABT263 treatment. Positive neurons outlined. Top panel row represent neuron co-positive for CC3 and p16, while bottom to panel rows show examples of triple positive neurons. Scale bars 20 µm. c,d, The mechanical threshold of the contralateral (uninjured) hindlimb was measured after application of senolytic ABT263 or vehicle control in aged (c) and young (d) mice at Day19 post-treatment (n = 21 aged vehicle-treated mice, n = 24 aged ABT263-treated mice; n = 9 young vehicle-treated mice, n = 11 young ABT263-treated mice; two-tailed unpaired t-test, p = 0.8562 (aged mice), p = 0.5858 (young mice)). e, Open field analysis of aged mice treated with or without ABT263. (n = 10 vehicle-treated, 13 ABT263-treated aged mice, two-tailed unpaired t-test, p = 0.2819). All data are mean values ± SEM. Source data

Extended Data Fig. 4. Increased percentage of human DRG neurons filled with lipofuscin with age.

a, Representative neurons in aged (65yo) human DRG with accumulated lipofuscin, a marker of senescence. Example of neuron either mostly filled (left arrow) or completely filled (right arrow). Scale bar is 10 µm. b, Quantification of DRG neurons whose cell bodies were greater than 75% occluded by lipofuscin as a percentage of all DRG neurons in young and aged human DRG (n = 2 young (32 & 33yo female); n = 1 aged male and n = 1 aged female (each 65yo) DRG. Lipofuscin signal is defined by strong autofluorescence signal across all channels (488 nm, 550 nm, 647 nm) which presents as a bright yellow/white signal in overlay. Data are mean values ± SEM. Source data

Extended Data Fig. 5. Confirmation of human DRG senescence with age and in painful conditions using existing RNA-sequencing datasets.

a-c, Re-analysis of Yu et al. single-soma human DRG RNAseq dataset. Bar graphs represent the percent positive p16 (CDKN2A) cells (a) and p21 (CDKN1A) cells (b) of all DRG cells, which are also negative for LMNB1, MKI67, TOP2A to filter out any proliferating and otherwise non-senescent cells. c, Percent of ATF3+ neurons which are co-positive for either p21 and/or p16 senescence markers. d,e, Re-analysis of North et al. human DRG bulk RNAseq dataset for expression of p16 (CDKN2A)(d) or SenMayo gene set (e) across multiple ages using DRG samples taken from patients either with associated pain (red) or without pain (black). Significant correlation found for CDKN2A expression with age (Spearman correlation, coefficient= 0.612, p = 0.00321). Significant association of SenMayo gene expression in pain DRG samples (t-test, p = 0.0182, see Supplementary Table 1). Source data

Extended Data Fig. 6. SASP-expressing senescent neuron diameters in young and aged human DRG.

Cell diameters (µm) of human DRG neurons co-expressing either p21 + IL6 + , p16 + IL6 + , or p21 + p16 + IL6 + , as a percent of total neurons counted in each population in young (a) and aged (b) DRG (n = 2 young female (33yo and 32yo) DRG: n = 19 p21 + IL6 + DRG neurons, n = 96 p16 + IL6 + DRG neurons, n = 71 p16 + p21 + IL6 + DRG neurons; n = 2 aged male and female (65yo) DRG: n = 126 p21 + IL6 + DRG neurons, n = 84 p16 + IL6 + DRG neurons, n = 271 p16 + p21 + IL6 + DRG neurons). Source data