Substance P-Botulinum Mediates Long-term Silencing of Pain Pathways that can be Re-instated with a Second Injection of the Construct in Mice.

Chronic pain presents an enormous personal and economic burden and there is an urgent need for effective treatments. In a mouse model of chronic neuropathic pain, selective silencing of key neurons in spinal pain signalling networks with botulinum constructs resulted in a reduction of pain behaviours associated with the peripheral nerve. However, to establish clinical relevance it was important to know how long this silencing period lasted. Now, we show that neuronal silencing and the concomitant reduction of neuropathic mechanical and thermal hypersensitivity lasts for up to 120d following a single injection of botulinum construct. Crucially, we show that silencing and analgesia can then be reinstated with a second injection of the botulinum conjugate. Here we demonstrate that single doses of botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing and pain relief. PERSPECTIVE: This research demonstrates that botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing without toxicity following a single injection of the conjugate and have the potential for repeated dosing when silencing reverses.

A DNA methylation signature in the stress driver gene Fkbp5 indicates a neuropathic component in chronic pain.

BACKGROUND: Epigenetic changes can bring insight into gene regulatory mechanisms associated with disease pathogenicity, including chronicity and increased vulnerability. To date, we are yet to identify genes sensitive to epigenetic regulation that contribute to the maintenance of chronic pain and with an epigenetic landscape indicative of the susceptibility to persistent pain. Such genes would provide a novel opportunity for better pain management, as their epigenetic profile could be targeted for the treatment of chronic pain or used as an indication of vulnerability for prevention strategies. Here, we investigated the epigenetic profile of the gene Fkbp5 for this potential, using targeted bisulphite sequencing in rodent pre-clinical models of chronic and latent hypersensitive states. RESULTS: The Fkbp5 promoter DNA methylation (DNAm) signature in the CNS was significantly different between models of persistent pain, and there was a significant correlation between CNS and peripheral blood Fkbp5 DNAm, indicating that further exploration of Fkbp5 promoter DNAm as an indicator of chronic pain pathogenic origin is warranted. We also found that maternal separation, which promotes the persistency of inflammatory pain in adulthood, was accompanied by long-lasting reduction in Fkbp5 DNAm, suggesting that Fkbp5 DNAm profile may indicate the increased vulnerability to chronic pain in individuals exposed to trauma in early life. CONCLUSIONS: Overall, our data demonstrate that the Fkbp5 promoter DNAm landscape brings novel insight into the differing pathogenic origins of chronic pain, may be able to stratify patients and predict the susceptibility to chronic pain.

New botulinum neurotoxin constructs for treatment of chronic pain.

Chronic pain affects one in five people across human societies, with few therapeutic options available. Botulinum neurotoxin (BoNT) can provide long-lasting pain relief by inhibiting local release of neuropeptides and neurotransmitters, but its highly paralytic nature has limited its analgesic potential. Recent advances in protein engineering have raised the possibility of synthesising non-paralysing botulinum molecules for translation to pain sufferers. However, the synthesis of these molecules, via several synthetic steps, has been challenging. Here, we describe a simple platform for safe production of botulinum molecules for treating nerve injury-induced pain. We produced two versions of isopeptide-bonded BoNT from separate botulinum parts using an isopeptide bonding system. Although both molecules cleaved their natural substrate, SNAP25, in sensory neurons, the structurally elongated iBoNT did not cause motor deficit in rats. We show that the non-paralytic elongated iBoNT targets specific cutaneous nerve fibres and provides sustained pain relief in a rat nerve injury model. Our results demonstrate that novel botulinum molecules can be produced in a simple and safe manner and be useful for treating neuropathic pain.

Selective neuronal silencing using synthetic botulinum molecules alleviates chronic pain in mice.

Chronic pain is a widespread debilitating condition affecting millions of people worldwide. Although several pharmacological treatments for relieving chronic pain have been developed, they require frequent chronic administration and are often associated with severe adverse events, including overdose and addiction. Persistent increased sensitization of neuronal subpopulations of the peripheral and central nervous system has been recognized as a central mechanism mediating chronic pain, suggesting that inhibition of specific neuronal subpopulations might produce antinociceptive effects. We leveraged the neurotoxic properties of the botulinum toxin to specifically silence key pain-processing neurons in the spinal cords of mice. We show that a single intrathecal injection of botulinum toxin conjugates produced long-lasting pain relief in mouse models of inflammatory and neuropathic pain without toxic side effects. Our results suggest that this strategy might be a safe and effective approach for relieving chronic pain while avoiding the adverse events associated with repeated chronic drug administration.

The stress regulator FKBP51: a novel and promising druggable target for the treatment of persistent pain states across sexes.

It is well established that FKBP51 regulates the stress system by modulating the sensitivity of the glucocorticoid receptor to stress hormones. Recently, we have demonstrated that FKBP51 also drives long-term inflammatory pain states in male mice by modulating glucocorticoid signalling at spinal cord level. Here, we explored the potential of FKBP51 as a new pharmacological target for the treatment of persistent pain across the sexes. First, we demonstrated that FKBP51 regulates long-term pain states of different aetiologies independently of sex. Deletion of FKBP51 reduced the mechanical hypersensitivity seen in joint inflammatory and neuropathic pain states in female and male mice. Furthermore, FKBP51 deletion also reduced the hypersensitivity seen in a translational model of chemotherapy-induced pain. Interestingly, these 3 pain states were associated with changes in glucocorticoid signalling, as indicated by the increased expression, at spinal cord level, of the glucocorticoid receptor isoform associated with glucocorticoid resistance, GRß, and increased levels of plasma corticosterone. These pain states were also accompanied by an upregulation of interleukin-6 in the spinal cord. Crucially, we were able to pharmacologically reduce the severity of the mechanical hypersensitivity seen in these 3 models of persistent pain with the unique FKBP51 ligand SAFit2. When SAFit2 was combined with a state-of-the-art vesicular phospholipid gel formulation for slow release, a single injection of SAFit2 offered pain relief for at least 7 days. We therefore propose the pharmacological blockade of FKBP51 as a new approach for the treatment of persistent pain across sexes, likely in humans as well as rodents.

The mitogen and stress-activated protein kinase 1 regulates the rapid epigenetic tagging of dorsal horn neurons and nocifensive behaviour.

Phosphorylation of histone H3 at serine 10 (p-H3S10) is a marker of active gene transcription. Using cognitive models of neural plasticity, p-H3S10 was shown to be downstream of extracellular signal-regulated kinase (ERK) signalling in the hippocampus. In this study, we show that nociceptive signalling after peripheral formalin injection increased p-H3S10 expression in the ipsilateral dorsal horn. This increase was maximal 30 minutes after formalin injection and occurred mainly within p-ERK-positive neurons. Spinal p-H3S10-enhanced expression was also observed in neurokinin 1 receptor (NK1R), c-Fos, and Zif268 positive neurons and was inhibited by ablation of serotonergic descending controls. The mitogen and stress-activated protein kinase 1 (MSK1) is downstream of ERK and can induce p-H3S10. We found that, after formalin injection, most phospho-MSK1 (p-MSK1)-positive cells (87% ± 3%) expressed p-ERK and the majority of p-H3S10-positive cells (85% ± 5%) expressed p-MSK1. Inhibition of ERK activity with the MEK inhibitor SL327 reduced formalin-induced p-ERK, p-MSK1, and p-H3S10, demonstrating that spinal p-MSK1 and p-H3S10 were at least partly downstream of ERK signalling. Crucially, pharmacological blockade of spinal MSK1 activity with the novel MSK1 inhibitor SB727651A inhibited formalin-induced spinal p-H3S10 and nocifensive behaviour. These findings are the first to establish the involvement of p-H3S10 and its main kinase, MSK1, in ERK regulation of nociception. Given the general importance of ERK signalling in pain processing, our results suggest that p-H3S10 could play a role in the response to injury.

Complex regulation of the regulator of synaptic plasticity histone deacetylase 2 in the rodent dorsal horn after peripheral injury.

Histone deacetylases (HDACs), HDAC2 in particular, have been shown to regulate various forms of learning and memory. Since cognitive processes share mechanisms with spinal nociceptive signalling, we decided to investigate the HDAC2 expression in the dorsal horn after peripheral injury. Using immunohistochemistry, we found that spinal HDAC2 was mainly seen in neurons and astrocytes, with neuronal expression in naïve tissue 2.6 times greater than that in astrocytes. Cysteine (S)-nitrosylation of HDAC2 releases HDAC2 gene silencing and is controlled by nitric oxide (NO). A duration of 48 h after intraplantar injection of complete Freund's adjuvant, there was an ipsilateral increase in the most important NO-producing enzyme in pain states, nitric oxide synthase (nNOS), accompanied by an increase in HDAC2 S-nitrosylation. Moreover, a subset of nNOS-positive neurons expressed cFos, a known target of HDAC2, suggesting that derepression of cFos expression following HDAC2 S-nitrosylation might occur after noxious stimulation. We saw no change in global HDAC2 expression in both short- and long-term pain states. However, HDAC2 was increased in astrocytes 7 days after neuropathic injury suggesting that HDAC2 might inhibit astrocytic gene expression in neuropathic pain states. All together, our results indicate that the epigenetic regulation of transcriptional programmes in the dorsal horn after injury is cell specific. Moreover, the prominent role of NO in persistent pain states suggests that HDAC2 S-nitrosylation could play a crucial role in the regulation of gene expression leading to hypersensitivity. Our manuscript describes for the first time the regulation of the memory regulator histone deacetylase 2 (HDAC2) in the superficial dorsal horn of adult rats following peripheral injury. Our cell-specific approach has revealed a complex pattern of expression of spinal HDAC2 that depends on the injury and the cell type, suggesting a sophisticated regulation of gene expression by HDAC2.

The stress regulator FKBP51 drives chronic pain by modulating spinal glucocorticoid signaling.

Polymorphisms in FKBP51 are associated with stress-related psychiatric disorders and influence the severity of pain symptoms experienced after trauma. We report that FKBP51 (FK506 binding protein 51) is crucial for the full development and maintenance of long-term pain states. Indeed, FKBP51 knockout mice, as well as mice in which silencing of FKBP51 is restricted to the spinal cord, showed reduced hypersensitivity in several persistent pain models in rodents. FKBP51 deletion did not compromise the detection of acute painful stimuli, a critical protective mechanism. Moreover, the intrathecal administration of the specific FKBP51 inhibitor SAFit2 reduced the severity of an established pain state, confirming the crucial role of spinal FKBP51 in nociceptive processing. Finally, glucocorticoid signaling, which is known to modulate persistent pain states in rodents, was impaired in FKBP51 knockout mice. This finding suggested that FKBP51 regulates chronic pain by modulation of glucocorticoid signaling. Thus, FKBP51 is a central mediator of chronic pain, likely in humans as well as rodents, and is a new pharmacologically tractable target for the treatment of long-term pain states.

Nonparalytic botulinum molecules for the control of pain.

Local injections of botulinum toxins have been reported to be useful not only for the treatment of peripheral neuropathic pain and migraine but also to cause long-lasting muscle paralysis, a potentially serious side effect. Recently, a botulinum A-based molecule ("BiTox") has been synthesized that retains neuronal silencing capacity without triggering muscle paralysis. In this study, we examined whether BiTox delivered peripherally was able to reduce or prevent the increased nociceptive sensitivity found in animal models of inflammatory, surgical, and neuropathic pain. Plasma extravasation and edema were also measured as well as keratinocyte proliferation. No motor deficits were seen and acute thermal and mechanical nociceptive thresholds were unimpaired by BiTox injections. We found reduced plasma extravasation and inflammatory edema as well as lower levels of keratinocyte proliferation in cutaneous tissue after local BiTox injection. However, we found no evidence that BiTox was transported to the dorsal root ganglia or dorsal horn and no deficits in formalin-elicited behaviors or capsaicin or formalin-induced c-Fos expression within the dorsal horn. In contrast, Bitox treatment strongly reduced A-nociceptor-mediated secondary mechanical hyperalgesia associated with either complete Freund's adjuvant (CFA)-induced joint inflammation or capsaicin injection and the hypersensitivity associated with spared nerve injury. These results imply that although local release of neuromodulators from C-fibers was inhibited by BiTox injection, C-nociceptive signaling function was not impaired. Taken together with recent clinical data the results suggest that BiTox should be considered for treatment of pain conditions in which A-nociceptors are thought to play a significant role.

Short-term anesthesia inhibits formalin-induced extracellular signal-regulated kinase (ERK) activation in the rostral anterior cingulate cortex but not in the spinal cord.

BACKGROUND: The rostral anterior cingulate cortex (rACC) has been implicated in the negative affective response to injury, and importantly, it has been shown that activation of extracellular signal-regulated kinase (ERK) signaling in the rACC contributes to the full expression of the affective component of pain in rodents. In this study, we investigated whether administration of anesthesia at the time of injury could reduce phosphorylated-ERK (PERK) expression in the rACC, which might eliminate the negative affective component of noxious stimulation. Intraplantar hindpaw formalin stimulation, an aversive event in the awake animal, was given with or without general isoflurane anesthesia, and PERK expression was subsequently quantified in the rACC using immunohistochemistry. Furthermore, as numerous studies have demonstrated the importance of spinal ERK signaling in the regulation of nociceptive behaviour, we also examined PERK in the superficial dorsal horn of the spinal cord. FINDINGS: Formalin injection with and without short-term (<10 min) general isoflurane anesthesia induced the same level of PERK expression in spinal cord laminae I-II. However, PERK expression was significantly inhibited across all laminae of the rACC in animals anesthetized during formalin injection. The effect of anesthesia was such that levels of PERK were the same in formalin and sham treated anesthesized animals. CONCLUSIONS: This study is the first to demonstrate that isoflurane anesthesia can inhibit formalin-induced PERK in the rACC and therefore might eliminate the unpleasantness of restraint associated with awake hindpaw injection.

Spinal autophagy is differently modulated in distinct mouse models of neuropathic pain.

BACKGROUND: Autophagy is a homeostatic degradative process essential for basal turnover of long-lived proteins and organelles as well as for removal of dysfunctional cellular components. Dysregulation of the autophagic machinery has been recently associated to several conditions including neurodegenerative diseases and cancer, but only very few studies have investigated its role in pain processing. RESULTS: We previously described autophagy impairment at the spinal cord in the experimental model of neuropathic pain induced by spinal nerve ligation (SNL). In this study, we characterized the main autophagic markers in two other common experimental models of neuropathic pain, the chronic constriction injury (CCI) and the spared nerve injury (SNI). The different modulation of LC3-I, Beclin 1 and p62 suggested that autophagy is differentially affected in the spinal dorsal horn depending on the type of peripheral injury. Confocal analysis of p62 distribution in the spinal dorsal horn indicated its presence mainly in NeuN-positive cell bodies and occasionally in glial processes, thus suggesting a predominant expression in the neuronal compartment. Finally, we investigated the consequences of autophagy impairment on pain behaviour by using the autophagy blocker cloroquine. Intrathecal chloroquine injection in naïve mice induced spinal accumulation of LC3 and p62 paralleled by significant mechanical hypersensitivity thus confirming the block in autophagosome clearance and suggesting the participation of the autophagic process in spinal mechanisms of pain processing. Altogether, our data indicate that spinal autophagy is differentially altered in different experimental pain models of neuropathic pain and that this process may be relevant for pain control.

Autophagy impairment in a mouse model of neuropathic pain.

Autophagy is an intracellular membrane trafficking pathway controlling the delivery of cytoplasmic material to the lysosomes for degradation. It plays an important role in cell homeostasis in both normal settings and abnormal, stressful conditions. It is now recognised that an imbalance in the autophagic process can impact basal cell functions and this has recently been implicated in several human diseases, including neurodegeneration and cancer.Here, we investigated the consequences of nerve injury on the autophagic process in a commonly used model of neuropathic pain. The expression and modulation of the main autophagic marker, the microtubule-associated protein 1 light chain 3 (LC3), was evaluated in the L4-L5 cord segment seven days after spinal nerve ligation (SNL). Levels of LC3-II, the autophagosome-associated LC3 form, were markedly higher in the spinal cord ipsilateral to the ligation side, appeared to correlate with the upregulation of the calcium channel subunit α2δ-1 and were not present in mice that underwent sham surgery. However, LC3-I and Beclin 1 expression were only slightly increased. On the contrary, SNL promoted the accumulation of the ubiquitin- and LC3-binding protein p62, which inversely correlates with autophagic activity, thus pointing to a block of autophagosome turnover.Our data showed for the first time that basal autophagy is disrupted in a model of neuropathic pain.