Aging and injury drive neuronal senescence in the dorsal root ganglia

Abstract

Aging negatively impacts central nervous system function; however, the cellular impact of aging in the peripheral nervous system remains poorly understood. Aged individuals are more likely to experience increased pain and slower recovery after trauma. Such injury can damage vulnerable peripheral axons of dorsal root ganglion (DRG) neurons resulting in somatosensory dysfunction. One cellular mechanism common to both aging and injury is cellular senescence, a complex cell state that can contribute to the aged pro-inflammatory environment. We uncovered, for the first time, DRG neuron senescence in the context of aging and pain-inducing peripheral nerve injury in young and aged mice. Aged DRG neurons displayed multiple markers of senescence (SA-β-gal, p21, p16, IL6) when compared to young DRG neurons. Peripheral nerve injury triggered a further accumulation of senescent DRG neurons over time post-injury in young and aged DRG. These senescent neurons were dynamic and heterogeneous in their expression of senescence markers, p16, p21, and senescence-associated secretory phenotype (SASP) expression of IL6, which was influenced by age. An electrophysiological characterization of senescence marker-expressing neurons revealed high-firing and nociceptor-like phenotypes within these populations. In addition, we observed improvement in nociceptive behaviors in young and aged nerve-injured mice after treatment with a senolytic agent that eliminates senescent cells. Finally, we confirmed in human post-mortem DRG samples that neuronal senescence is present and increases with age. Overall, we describe a susceptibility of the peripheral nervous system to neuronal senescence with age or injury that may be a targetable mechanism to treat sensory dysfunction, such as chronic pain, particularly in aged populations.

Keywords: Neuron; chronic pain; human; mouse; spared nerve injury.

Figure 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 and aged mice. Positive pixels detected were quantified (right) (n= 5–6 mice per group; two-tailed unpaired t-test, *p<0.05.). B. Representative RNAscope images for senescence markers p21 and p16 with SASP factor IL6 in whole DRG section. C. Quantification of neuronal expression of each marker or in combination expressed as a percent of total DRG neurons (n=4–5 mice per group, two-tailed unpaired t-test, *p<0.05, **p<0.01, ***p<0.001). D. Quantification of IL6 protein levels by ELISA assay in young or aged plasma (n=5–6 mice per group, two-tailed unpaired t-test, *p<0.05). Data are expressed as the mean ± SEM.

Figure 2.. DRG neurons express senescence markers and SASP factors following peripheral nerve injury.

A. Schematic of spared nerve injury (SNI) model in mice. Timeline over which DRG tissues were analyzed following SNI. B. qPCR of a panel of senescence markers and SASP factor gene expression from whole lumbar DRG in controls or following SNI (3-weeks post) in young mice (n=4 control; n=4 SNI young mice; two-tailed unpaired t-test, *p<0.05, **p<0.01, ***p<0.001). C. Representative RNAscope image of whole DRG section from uninjured or 3-weeks post-SNI in young mice. Scale bar 100μm. D. Quantification of the number of L3/4 DRG neurons expressing either p21 (upper graph) or p16 (lower graph) in young mice. (n= 3–5 mice per group/time-point; One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). E. Representative RNAscope image of whole DRG section from uninjured or 3-weeks post-SNI in aged mice. Scale bar 100μm. F. Quantification of the number of L3/4 DRG neurons expressing either p21 (upper graph) or p16 (lower graph) in aged mice. (n= 3–4 mice per group/time-point; One-way ANOVA, *p<0.05, **p<0.01, ***p<0.001). G. Quantification of p21+p16+ co-expressing DRG neurons as a percent of total DRG neurons in uninjured and 3-weeks post-injury in young and aged mice. (n= 3–5 mice per group/time-point; One-way ANOVA, ***p<0.001, ****p<0.0001. H. Representative RNAscope images showing co-expressing senescence markers (p21 and p16) with SASP factor (IL6). Scale bar 10μm. I. Quantification of p21+IL6+ co-expressing DRG neurons as a percent of total DRG neurons in uninjured and 3-weeks post-injury in young and aged mice. (n=3–5 mice per group; One-way ANOVA, **p<0.01, ****p<0.0001. J. Quantification of p21+p16+IL6+ triple positive DRG neurons as a percent of total DRG neurons in uninjured and 3-weeks post-injury in young and aged mice (n=3–5 mice per group; One-way ANOVA, *p<0.05, **p<0.01). K. Analysis of IL6-expressing DRG neuron population that co-express senescence markers p21 and/or p16 at 3-weeks post-injury in young and aged mice (n=3 mice per group). Data are expressed as the mean ± SEM.

Figure 3.. ATF3+ injured and neighboring non-injured 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 time points in young and aged mice (n=3–5 mice per group/time-point). B. Representative images of dual immunohistochemistry/RNAscope labeling ATF3+ injured neurons (nuclear localized protein) and RNA puncta of p21 and p16 at 3-weeks post-SNI. Co-expression of ATF3 with p21 and or p16 (arrows). Asterisks represent ATF3-negative cells that express p21 and/or p16 senescence markers. C & D. Quantification of ATF3-positive neuron population that co-express p21 and/or p16 at multiple time points post-injury in young and aged DRG (Young: n=3–5 mice per time-point 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 time-point: 7-day: n=1,004 ATF3+ neurons; 3-week: n=983 ATF3+ neurons; 7-week: n=722 ATF3+ neurons). E. Quantification of ATF3-negative population co-expressing senescence marker p21 at multiple time points post-injury in young and aged DRG (n=3 mice per group per time-point). E. Quantification of ATF3-negative population co-expressing senescence marker p16 at multiple time points post-injury in young and aged DRG (n=3–5 mice per group per time-point). Data are expressed as the mean ± SEM.

Figure 4.. Trpv1+ nociceptors express senescence markers following 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 post-nerve injury in young and aged 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 (grey). For IL6-signal, intense puncta signal with white center are positive neurons, while fainter/dull blue is background. Arrows: Trpv1+ senescent neurons, Asterisks: Trpv1-negative senescent neurons. Scale bars 100μm and 20μm (insets). C. Quantification of Trpv1 neuron population and its co-expression with p21 and/or IL6 in young and (D) aged L3/4 DRG of uninjured (controls) and 3-weeks post-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).

Figure 5.. DRG neurons expressing senescence and SASP markers have unique functional profiles that include high-firing and nociceptor-like phenotypes.

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: 14–41μm), firing properties and intrinsic currents. Discrete clusters are identified by color (1–5). (n= 82 recorded DRG neurons). C. The senescence marker p16 (Orange) and D. the SASP factor IL6 (blue) are mainly found in cluster five. E. The senescence marker p21 (pink) is widely distributed throughout the clusters. F. Heatmap depicting parameters from left to right as follows: Clusters (cool gradient), gene expression (black=no expression and white=expression), diameter and physiology parameters (warm gradient= higher normalized values in lighter reds and lower values in darker reds). Senescence marker p16 and SASP factor IL6 groups contain neurons with high-firing phenotypes (>100 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).

Figure 6.. in vivo elimination of senescent neurons using senolytics alleviates pain behaviors after nerve injury.

A. Schematic of treatment paradigm. Young and aged mice were treated with senolytic (ABT263, 100mg/kg) or vehicle for 10 days by oral gavage, starting at 3-weeks post-spared nerve injury (SNI). Mechanical allodynia and weight bearing were assessed during and after treatment. B. Senolytics induce apoptosis in the DRG. Representative image of CC3-immunohistochemistry to capture apoptotic cells after 5-day treatment with ABT263 (white arrows = CC3-positive neurons, other red fluorescent signal is background lipofuscin. Quantification of cleaved-caspase-3 (CC3) positive neurons in the DRG following treatment with vehicle or ABT263 for 5 consecutive days (n=3 male, n=3 female aged mice per treatment group, two-tailed unpaired t-test, **p<0.01). C. Aged mice were treated with ABT263 or vehicle (light blue indicates treatment window) and their mechanical allodynia thresholds were assessed (n=13 female, n=9 male vehicle-treated mice; n=14 female, n=13 male ABT263-treated mice, mixed-effects analysis, Šídák’s multiple comparisons test, *p<0.05, **p<0.01, ***p<0.001). D. 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<0.001) and Day 39 post treatment start (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). E. 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, mixed-effects analysis, Šídák’s multiple comparisons test, **p<0.01). F. 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, **p<0.01, ****p<0.0001) and Day 29 post treatment start (n=5 vehicle-treated, n=5 ABT263-treated male mice, two-tailed unpaired t-test **p<0.01). Data are expressed as the mean ± SEM.

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

A & B. Representative RNAscope images from young (33yr) or aged (65yr) human L4 DRG showing expression of p21 and p16 senescence markers (enlarged left images with DAPI, scale bar 100μm). The large globular signal present in both channels is auto fluorescent lipofuscin and not RNAscope signal (small puncta). C. Quantification of p21+, p16+, and co-positive p21+p16+ neurons in the young and aged human DRG as a percent of total DRG neurons. D. Quantification of IL6-expressing neurons as a percent of total DRG neurons. E. Analysis of IL6-positive 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. G. Percent of DRG neurons that are ATF3-positive in young and aged human DRG. H. Example image depicting a single human neuron positive for ATF3 (nuclear-localized, immunohistochemistry) and p21 (RNAscope). Scale bars 20μm. Analysis of ATF3-positive 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. Boxed right, Quantification of the subsets of TRPV1+ neurons that co-express either p21 or p16 by RNAscope. J. Single human neurons showing co-expression of TRPV1 with p21 and/or p16. DAPI in grey. Scale bars 20μm. K. Venn diagram of human DRG neurons representing total numbers of 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.