Biology subjects

Singleton, S.

Publications and source records attributed to Singleton, S..

5 recordsLinked to original sources

bioRxiv2026

Pre-injury subchronic stress confers sex-specific protection against pain-associated symptoms in osteoarthritic mice

Exposure to stress in adulthood alters the manifestation of persistent pain, yet its role in chronic pain vulnerability remains unclear. Here, we report that sub-chronic restraint stress experienced two weeks before unilateral osteoarthritis (OA) induction via intra-articular mono-iodoacetate (MIA) promoted the emergence of a resilient-like phenotype in adult male mice. This was evidenced by decreased MIA-induced mechanical hypersensitivity, improved gait dynamics and lower anxiety-like behaviour. In contrast, stress exposed females exhibited augmented pain-associated symptoms. In males, sub-chronic stress mitigated several MIA-induced molecular changes, including reduced adult hippocampal neurogenesis, increased glucocorticoid receptor levels in the hypothalamic paraventricular nucleus (PVN) and elevated c-Fos expression in deep spinal laminae, periaqueductal gray (PAG) and PVN. RNA sequencing suggested that restraint stress in males primed the spinal cord for an exacerbated GABAergic response post-MIA and pre-empted some pro-nociceptive MIA-induced transcriptional changes. However, the combination of stress and injury disrupted longevity-associated programs and shifted neurons toward a stress-vulnerable state. These findings reveal that prior stress exposure can modulate the long-term pain experience in osteoarthritis, with sexually dimorphic outcomes. Importantly, these results challenge the notion of stress as inherently maladaptive and underscore its potential to foster resilience to chronic pain.

neuroscience↗

bioRxiv2025

The c-Src inhibitor eCF506 diminishes opioid tolerance creating bias against β-arrestin2 recruitment

Opioids reduce severe pain, but persistent use is compromised by tolerance, attenuated by either {beta}-arrestin2 depletion, prompting development of biased opioids limited by partial efficacy, or c-Src kinase inhibitors, potentially acting through off-target effects. We tested eCF506, a conformationally selective c-Src inhibitor, on morphine antinociception and examined its effect on receptor signaling and {beta}-arrestin2 recruitment. Oral eCF506 inhibited morphine tolerance in C57BL/6J mice. Exposure of PathHunter CHO cells to eCF506 did not affect inhibition of cAMP accumulation by the agonist, DAMGO, but reduced {beta}-arrestin2 recruitment. This effect, mimicked by targeted degradation of c-Src, occurred through inhibition of c-Src catalytic function as evidenced by its diminution by the catalytically inactive Src250-536(K298M) construct. This mutant also restricted the effect of c-Src inhibitors on {beta}-arrestin2 recruitment. eCF506 additionally increased surface expression of receptors and limited their internalization by endomorphin-2 but did not alter DAMGO-evoked GRK-mediated receptor phosphorylation. These findings suggest that eCF506 prolongs opioid antinociception by inducing signalling bias, diminishing {beta}-arrestin2-mediated receptor regulation.

neuroscience↗

bioRxiv2025

Stress, Epigenetic Remodeling and FKBP51: Pathways to Chronic Pain Vulnerability

Stress is thought to contribute to the persistence of pain and comorbid anxiety, yet the underlying mechanisms remain unclear. In our pre-clinical model, sub-chronic stress exacerbated subsequently induced inflammatory pain and accelerated the development of comorbid anxiety. DNA methylation analysis of spinal cord tissue after stress exposure revealed hypomethylation in the Fkbp5 promoter site for the canonical FKBP51 transcript and other stress-related genes. However, most epigenetic changes in key regulatory regions did not correlate with changes in gene expression assessed by RNA sequencing, suggesting that stress exposure had remodeled the epigenome without altering gene activity and primed genes for hyper-responsiveness to future challenges. FKBP51 inhibition during stress exposure reduced the exacerbation of inflammatory pain by stress and reversed several stress-induced DNA methylation changes in promoter regions of genes associated with stress and nociception, including Rtn4, Cdk5 and Nrxn1, but not Fkbp5. These results indicate that sub-chronic stress leads to the hypomethylation of Fkbp5 and increased susceptibility to chronic pain driven by FKBP51, but reversing Fkbp5 hypomethylation is not necessary to prevent chronic pain vulnerability, which is likely driven by complex epigenetic regulation of multiple stress-regulated genes.

neuroscience↗

bioRxiv2024

Acute and early stress axis modulation in joint disease permanently reduces pain and emotional comorbidities

Chronic pain affects 20-30% of the population and imposes a significant socio-economic burden as it is often accompanied by substantial emotional comorbidities such as anxiety and depression. Yet, the mechanisms underlying the interactions between the sensory and emotional aspects of chronic pain remain poorly understood. Here, we investigated the role of FKBP51, a regulator of the stress response, in mediating both sensory and emotional symptoms of chronic pain. Inhibition of FKBP51, via genetic deletion or pharmacological blockade, in persistent joint pain reduced fast-onset sensory, functional and activity-related symptoms, as well as late anxio-depressive comorbidities. FKBP51 inhibition after the establishment of the hypersensitive state provided only temporary symptoms relief, while acute inhibition at disease onset protected from the full development of sensory and anxio-depressive symptoms for up to 6 months. Our results also indicated that early pain symptoms could predict the late sensory and emotional outcomes of chronic pain. RNA sequencing of spinal cord tissue revealed that late FKBP51 inhibition transiently altered nociceptive genes associated with mechanical hypersensitivity. In contrast, early inhibition persistently downregulated the Naaa gene, a key regulator of the transition to chronic pain, and reorganized spinal cilia. Our results indicate that early FKBP51 inhibition after injury can persistently reduce chronic pain and prevent the onset of associated emotional comorbidities by modulating critical spinal neurobiological pathways that play pivotal roles in the transition to chronic pain. Significance statementOur study reveals that early inhibition of FKBP51, a modulator in the stress axis, at the onset of joint damage provides sustained pain relief and significantly delays or prevents emotional comorbidities in a sex-dependent manner. In contrast, FKBP51 inhibition initiated after chronic pain is established results in only temporary symptoms improvement. These findings highlight a critical therapeutic window during which timely intervention can prevent the transition from acute to chronic pain. By establishing a predictive link between early therapeutic response and long-term outcomes, this work has important clinical implications for proactive and personalized chronic pain management.

neuroscience↗

bioRxiv2022

Morphine-induced mechanical hypersensitivity in mice requires delta receptors, beta-arrestin2 and c-Src activity

BackgroundMorphine diminishes acute pain, but long-term use is compromised by tolerance and hyperalgesia. Studies implicate {delta} receptors, {beta}-arrestin2 and Src kinase in tolerance. We examined whether these proteins are also involved in morphine-induced hypersensitivity (MIH). A common pathway for tolerance and hypersensitivity may provide a single target to guide improved analgesic approaches. MethodsWe examined mechanical sensitivity using automated von Frey in wild type (WT) and transgenic male and female C57Bl/6 mice before and after hind paw inflammation by complete Freunds adjuvant (CFA). We explored the expression of opioid genes in the spinal cord using quantitative RT-PCR. ResultsCFA-evoked hypersensitivity ceased on day 7 in WT mice but persisted in -/- mice. Recovery was delayed until day 13 in {delta}-/- mice. Restoration to basal sensitivity in WT mice occurred with increased {delta} expression. By contrast, {kappa} expression was reduced, while remained unchanged. Daily morphine reduced hypersensitivity in WT mice on day 3 compared to controls, however hypersensitivity recurred on day 9 and beyond. By contrast, WT mice had no recurrence of hypersensitivity in the absence of daily morphine. We used {beta}-arrestin2-/-, {delta}-/- and Src inhibition by dasatinib in WT mice to establish whether these approaches, which diminish tolerance, also attenuate MIH. While none of these approaches affected CFA-evoked inflammation or acute hypersensitivity, all caused sustained morphine anti-hypersensitivity, abolishing MIH. ConclusionsLike morphine tolerance, MIH in this model requires {delta} receptors, {beta}-arrestin2 and Src activity. Our findings suggest that MIH is caused by a tolerance-induced reduction in endogenous opioid signalling.

physiology↗