Daniela Maftei

Controlling the activation of the Bv8/prokineticin system reduces neuroinflammation and abolishes thermal and tactile hyperalgesia in neuropathic animals

Chemokines are involved in neuroinflammation and contribute to chronic pain processing. The new chemokine prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2) have a role in inflammatory pain and immunomodulation. In the present study, we investigated the involvement of PROK2 and its receptors in neuropathic pain.

Prokineticin 2 Upregulation in the Peripheral Nervous System Has a Major Role in Triggering and Maintaining Neuropathic Pain in the Chronic Constriction Injury Model

The new chemokine Prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2) have a role in inflammatory pain and immunomodulation. Here we identified PROK2 as a critical mediator of neuropathic pain in the chronic constriction injury (CCI) of the sciatic nerve in mice and demonstrated that blocking the prokineticin receptors with two PKR1-preferring antagonists (PC1 and PC7) reduces pain and nerve damage. PROK2 mRNA expression was upregulated in the injured nerve since day 3 post injury (dpi) and in the ipsilateral DRG since 6 dpi. PROK2 protein overexpression was evident in Schwann Cells, infiltrating macrophages and axons in the peripheral nerve and in the neuronal bodies and some satellite cells in the DRG. Therapeutic treatment of neuropathic mice with the PKR-antagonist, PC1, impaired the PROK2 upregulation and signalling. This fact, besides alleviating pain, brought down the burden of proinflammatory cytokines in the damaged nerve and prompted an anti-inflammatory repair program. Such a treatment also reduced intraneural oedema and axon degeneration as demonstrated by the physiological skin innervation and thickness conserved in CCI-PC1 mice. These findings suggest that PROK2 plays a crucial role in neuropathic pain and might represent a novel target of treatment for this disease.

Bv8/prokineticin 2 is involved in Aβ-induced neurotoxicity

Bv8/Prokineticin 2 (PROK2) is a bioactive peptide initially discovered as a regulator of gastrointestinal motility. Among multiple biological roles demonstrated for PROK2, it was recently established that PROK2 is an insult-inducible endangering mediator for cerebral damage. Aim of the present study was to evaluate the PROK2 and its receptors’ potential involvement in amyloid beta (Aβ) neurotoxicity, a hallmark of Alzheimer’s disease (AD) and various forms of traumatic brain injury (TBI). Analyzing primary cortical cultures (CNs) and cortex and hippocampus from Aβ treated rats, we found that PROK2 and its receptors PKR1 and PKR2 mRNA are up-regulated by Aβ, suggesting their potential involvement in AD. Hence we evaluated if impairing the prokineticin system activation might have protective effect against neuronal death induced by Aβ. We found that a PKR antagonist concentration-dependently protects CNs against Aβ1–42-induced neurotoxicity, by reducing the Aβ-induced PROK2 neuronal up-regulation. Moreover, the antagonist completely rescued LTP impairment in hippocampal slices from 6 month-old Tg2576 AD mice without affecting basal synaptic transmission and paired pulse-facilitation paradigms. These results indicate that PROK2 plays a role in cerebral amyloidosis and that PROK2 antagonists may represent a new approach for ameliorating the defining pathology of AD.

PC1, a non-peptide PKR1-preferring antagonist, reduces pain behavior and spinal neuronal sensitization in neuropathic mice.

Peripheral neuropathy is characterized by abnormal pain responses triggered by the release of several mediators and neuronal hyperexcitability at the spinal cord level. Emerging evidence indicates that the enhanced activity of dorsal horn neurons requires communication with glia and microglia, cells that are physiologically involved in neuronal wellbeing. Prokineticins (PKs), which include PK1 and PK2, represent a novel family of chemokines characterized by a unique structural motif comprising five disulfide bonds. They are expressed in the peripheral and central nervous system. PKs bind two G protein coupled receptors, PKR1 and PKR2, and participate in the regulation of several biological processes, including pain sensation. This study aimed to investigate the anti-nociceptive effect of PC1, a non-peptide PKR1-preferring antagonist, in a mouse model of neuropathic pain. To do this, we assessed the activity of spinal cord nociceptive neurons as well as astrocyte and microglia phenotypes after repeated administration of PC1 in vivo. PC1 treatment strongly delayed the development of thermal hyperalgesia and tactile and mechanical allodynia. It also reduced spinal microglial and glial activation 8 days post injury in spared nerve injury (SNI) mice. Neuropathic mice showed an increased level of PK2 protein in the spinal cord, mostly in astrocytes. PC1 treatment completely reversed the increased responsiveness to mechanical stimuli, the decreased threshold of neuronal activation, and the increased spontaneous activity that were observed in nociceptive specific (NS) neurons of SNI mice.

Antagonism of the Prokineticin System Prevents and Reverses Allodynia and Inflammation in a Mouse Model of Diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.

The prokineticin Bv8 sensitizes cutaneous terminals of female mice to heat.

Injection of the noxious peptide Bv8 has previously been shown to induce a biphasic thermal hyperalgesia in rodents, the first peak presumably due to peripheral sensitization. This hypothesis has never been directly confirmed. We have assessed whether Bv8 can indeed sensitize peripheral nerve fibres in the mouse to heat.

Halogenated triazinediones behave as antagonists of PKR1: in vitro and in vivo pharmacological characterization

Different prokineticin receptor antagonists, based on the triazinedione scaffold, were synthesized by a new efficient method. Here we demonstrated that 5-benzyl triazinediones substituted in position para of the benzyl group with halogens provide compounds endowed with interesting selectivity for the Prokineticin receptor 1 (PKR1). BRET technology indicates that such substitution results in increased affinity for the PKR1.The affinity for PKR2, always in M range, was never significantly affected by the parahalogen-benzyl pharmacophores. The analog bearing a para-bromobenzyl pharmacophore (PC-25) displayed the highest affinity for PKR1 (~18 times higher than the reference PC-1 that bears a para-ethyl benzyl group) and the highest selectivity (~300 times). The other halogen substituted analogs (PC-7, PC-18 and PC-35), showed selectivity for PKR1 more than 100 times higher than for PKR2. Using transgenic mice lacking one of the two PKRs we demonstrated that all these compounds were able to abolish the Bv8-induced hyperalgesia in mice still expressing the PKR1 at doses lower than those necessary to abolish hyperalgesia in mice expressing only the PKR2. The dose ratio reflected the invitro evaluated receptor selectivity. INTRODUCTION: The prokineticins (among them the mammalian molecules named Prokineticin 1, PROK1, prokineticin 2, PROK2, and their Amphibian homologous, Bv8) make up a new family of chemokines 1, 2 which, in mammals, activate two G-protein linked receptors (prokineticin receptor 1 and 2, PKR1 and PKR2). QUICK RESPONSE CODE DOI: 10.13040/IJPSR.0975-8232.5(11).5066-74 Article can be accessed online on: www.ijpsr.com DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.5(11).5066-74 Intensive research of the prokineticin system over the past decade has revealed a dazzling array of physiological functions 3 . In addition, the disruption of prokineticin system has been implicated in several pathological conditions, including cancer 4 , immunological response 5, 6 and persistent pain 3 . In animal models of inflammatory 7 and neuropathic 8 pain, in which the prokineticin system is highly activated, we already demonstrated that the prokineticin receptor antagonist PC-1 9 not only abolishes pain, hindering the nociceptor sensitization, but also reduces the over expression of the endogenous

Targeting the Prokineticin System to Control Chronic Pain and Inflammation

Prokineticin1 and prokineticin2 belong to a new family of chemokines identified in several species including mammals and characterized by the presence of five disulfide bridges. These proteins signal through two G-coupled receptors (prokineticin-receptor1 and prokineticin- receptor2) widely expressed in all tissues and involved in a large spectrum of biological activities, including angiogenesis, hematopoiesis, immune processes, inflammation and nociceptive transmission. Prokineticin2 is overexpressed in inflamed tissues and has a crucial role in neutrophil dependent inflammation and hypernociception. Following tissue inflammation, peripheral nerve injury, cancer, bone metastasis the expression of prokineticin2 and of the prokineticin-receptor2 is increased also within dorsal root ganglia and spinal cord. Prokineticin receptors, highly expressed in nociceptor endings and dorsal root ganglia, exert a tonic activation of TRPV1 and TRPA1 contributing to peripheral sensitization. Prokineticin2-induces activation of the prokineticin receptors in the spinal dorsal horn and in activated astrocytes contributes to central sensitization and maintains chronic and neuropathic pain. Prokineticin2, acting on prokineticin receptors on monocytes, macrophages and dendritic cells, induces chemotaxis and release of inflammatory and pronociceptive cytokines. Hence, the prokineticin system represents a novel therapeutic target in chronic pain conditions. Evaluation of the mechanism of action of prokineticin2 and the potential effectiveness of its inhibition is discussed.

PK2β ligand, a splice variant of prokineticin 2, is able to modulate and drive signaling through PKR1 receptor

Prokineticin-2 (PK2) is a secreted bioactive peptide that signals through two GPCRs, the prokineticin receptors (PKRs), and regulates a variety of biological processes including angiogenesis, immunity and nociception. The PK2 primary transcript has two alternative splice variants, PK2 and PK2L (a Long form) which is cleaved in an active peptide, named PK2β that preferentially binds to PKR1 receptor. The aim of this study was to characterize the PK2β. Using different Saccharomyces cerevisiae strains, we examined the specificity of PKR1 and PKR2 G-protein coupling following PK2β binding. Data obtained in yeast confirmed that PK2 binds both receptors, inducing a comparable response throughout a promiscuous coupling of G protein subtypes. Conversely, we demonstrated, for the first time, that PK2β preferentially binding to PKR1, activates a signaling cascade that not depends on Gαi/o coupling. The binding specificity of PK2β for PKR1 was evaluated by the analysis of PKR mutant in yeast and GST pull-down experiments, suggesting an important role of PKR1 amino-terminal region. We also evaluated the ability of PK2β to differentially activate PKR1 and/or PKR2 by in vivo nociceptive experiments and we showed that PK2β induces intense sensitization of peripheral nociceptors to painful stimuli through the activation of PKR1. To analyze PK2β-induced signal transduction, we demonstrated the inability of PK2β to induce STAT3 protein phosphorylation in organotypic primary explants from mice Dorsal Root Ganglion (DRG), an important pain station. The control of the concentration ratio between PK2β and PK2 could be one of the keys to allow the specificity of the cell response of prokineticin signaling pathway.

[EXPRESS] The multimodal antidepressant vortioxetine causes analgesia in a mouse model of chronic neuropathic pain

Vortioxetine is a multimodal antidepressant that potently antagonizes 5-HT3 serotonin receptors, inhibits the high-affinity serotonin transporter, activates 5-HT1A and 5-HT1B receptors, and antagonizes 5-HT1D and 5-HT7 receptors. 5-HT3 receptors largely mediate the hyperalgesic activity of serotonin that occurs in response to nerve injury. Activation of 5-HT3 receptors contributes to explain why selective serotonin reuptake inhibitors, such as fluoxetine, are not indicated in the treatment of neuropathic pain. Here, we studied the analgesic action of vortioxetine in the chronic constriction injury model of neuropathic pain in mice. Vortioxetine was injected once a day for 27 days at doses (10 mg/kg, intraperitoneally) that determine >90% 5-HT3 receptor occupancy in the central nervous system. The action of vortioxetine was compared to the action of equal doses of the serotonin-noradrenaline reuptake inhibitor, venlafaxine (one of the gold standard drugs in the treatment of neuropathic pain), and fluoxetine. Vortioxetine caused a robust analgesia in chronic constriction injury mice, and its effect was identical to that produced by venlafaxine. In contrast, fluoxetine was inactive in chronic constriction injury mice. Vortioxetine enhanced mechanical pain thresholds in chronic constriction injury mice without changing motor activity, as assessed by the open-field and horizontal bar tests. None of the three antidepressants caused analgesia in the complete Freund’s adjuvant model of chronic inflammatory pain. These findings raise the attractive possibility that vortioxetine can be effective in the treatment of neuropathic pain, particularly in patients with comorbid depression and cognitive dysfunction.