Caratterizzazione del ruolo fisiopatologico e funzionale del canali Transient Receptor Potential (TRP) nelle patologie dolorose di origine infiammatoria e neuropatica
Year: 2019
Type: dissertation
Abstract: Summary \nThe TRPA1 receptor has been identified as a pivotal molecular entity in sensory biology, \nespecially as a sensor of chemical species/irritants present in foods, atmospheric pollutants and \ntoxicants. Rapid progress is being made in characterizing the function of TRPA1 in various tissues \nand organs under normal and pathophysiological conditions. TRPA1 receptor is an excitatory ion \nchannel expressed by a subpopulation of primary afferent somatosensory neurons that contain SP and \nCGRP. Environmental irritants (mustard oil, allicin, acrolein) can activate TRPA1, causing acute \npain, neuropeptide release, and neurogenic inflammation. Genetic studies indicate that TRPA1 is also \nactivated downstream of one or more proalgesic agents that stimulate phospholipase C signaling \npathways, thereby implicating this channel in peripheral mechanisms controlling pain \nhypersensitivity. \nThe commonly accepted paradigm is that TRPA1 expressed in primary sensory neurons is \ndirectly activated by ROS/RNS/RCS, thus signalling pain from the PNS to the CNS. While direct \nstimulation of nociceptor TRPA1 mediates acute spontaneous pain, recent findings from our \nlaboratory also, have shifted the focus to the TRPA1 expressed in non-neuronal cells as the critical \nfactor to sustain allodynia and chronic pain. In our study we aim at identifying the role of TRPA1 in \ndifferent model of pain ranging from inflammatory to neuropathic pain. \nThe first aim of the present study was to identify the role of TRPA1 in a mouse model of \ntrigeminal neuropathic pain produced by the constriction of the infraorbital nerve (CION) and to \nexplore the molecular and cellular pathways that, from the initial nerve injury, result in channel \nengagement. The monocyte chemoattractant protein 1 (MCP-1), also known as chemoattractant \nchemokine (C-C motif) ligand 2 (CCL2), by binding to the chemotactic cytokine receptor 2 (CCR- \n2), promotes monocyte transendothelial migration to the site of nerve injury. In various paradigms of \nperipheral nerve injury, CCL2 inhibition and CCR-2 genetic ablation abrogates mechanical allodynia. \nIn addition, antioxidants have been reported to attenuate neural hypersensitivity in various models of \nneuropathic pain, such as sciatic chronic constriction injury and spinal nerve ligation. Thus, the \ncontribution of monocyte/macrophage infiltration and the ensuing oxidative stress in the TRPA1- \nmediated pain-like behaviors was investigated in the CION model. Results propose that CCL2-driven \nmonocyte/macrophage accumulation within the injured nerve and the neighboring tissue generates \noxidative burst that, by TRPA1 targeting, promotes and maintains CION-evoked pain-like behaviors. \nHowever, while exploring a similar model (partial sciatic nerve ligation, pSNL), we \nsurprisingly observed that mice with genetic deletion or pharmacological blockade of TRPA1 not \nonly showed the expected reduced mechanical allodynia, but also exhibited a marked reduction in \n2 \nmacrophages infiltration and H2O2 generation in the injured nerve trunk. Thus, by a series of genetic \nand pharmacological interventions and, more importantly, by generating mice with conditional \nTRPA1 deletion in different cells, we identified the expression of TRPA1 in Schwann cells and \nshowed a role of the receptor in molecular events that sustain allodynia after a damaged of the nerve \ntrunk. Our results reveal distinct kinetics of monocyte/macrophage accumulation by CCL2 and the \nTRPA1/oxidative stress pathways. The most parsimonious explanation of the present results is that \noxidative stress generated by Schwann cell TRPA1/NOX1 has bidirectional effects. The inwardly \nreleased H2O2 targets TRPA1 on adjacent nociceptor nerve fibers in a paracrine fashion to sustain \nallodynia. The outwardly released H2O2 promotes the final part (about 200 μm) of the journey of \nmacrophages, which, deriving from the blood stream, slowly accumulate into the perineural space \nfollowing the CCL2 gradient. Thereafter, following the Schwann cell derived oxidative stress \ngradient, macrophages rapidly pass across the perineurium to enter the damaged nerve trunk. TRPA1 \nhas been identified in oligodendrocytes, with possible detrimental roles in ischemia and \nneurodegeneration (Hamilton et al., 2016). Herein, we extend this observation to Schwann cells, the \nperipheral analogues of oligodendrocytes, which, via TRPA1, orchestrate neuroinflammation and \nensuing neuropathic pain. Amelioration of neuropathic pain by currently developed TRPA1 \nantagonists may derive from their ability to attenuate macrophage-dependent neuroinflammation. \nSince TRPA1 is involved in multiple painful condition, we also investigate the role of TRPA1 \nin pain symptoms associated with the treatment of the third-generation AIs, which include the \nsteroidal agent exemestane (Aromasin) the triazoles anastrozole (Arimidex) and letrozole (Femara), \ncurrently recommended for adjuvant endocrine treatment as primary, sequential, or extended therapy \nwith tamoxifen, for postmenopausal women diagnosed with estrogen receptor-positive breast cancer. \nAmong these, the AI-associated musculoskeletal symptoms (AIMSS) are characterized by morning \nstiffness and pain of the hands, knees, hips, lower back, and shoulders. In addition to musculoskeletal \npain, pain symptoms associated with AIs have recently been more accurately described with the \ninclusion of neuropathic, diffused, and mixed pain (Laroche et al., 2014). The chemical structure of \nexemestane includes a system of highly electrophilic conjugated Michael acceptor groups, which \nmight react with the thiol groups of reactive cysteine residues. Michael addition reaction with specific \ncysteine residues is a major mechanism that results in TRPA1 activation by a large variety of \nelectrophilic compounds. In addition, aliphatic and aromatic nitriles can react with cysteine to form \nthiazoline derivatives and accordingly the tear gas 2-chlorobenzylidene malononitrile (CS) has been \nidentified as a TRPA1 agonist. We noticed that both letrozole and anastrozole possess nitrile moieties. \nThus, we hypothesized that exemestane, letrozole and anastrozole may produce neurogenic \ninflammation, nociception and hyperalgesia by targeting TRPA1. The ability to gate TRPA1 in vitro \n3 \nwas confirmed in vivo by the observation that the pain-like behaviors evoked by AIs in mice are \nabrogated by genetic deletion or pharmacological blockade of the channel. However, AI \nconcentrations required for TRPA1 gating in vitro are 1-2 order of magnitude higher than those found \nin patient plasma. In addition, an important proportion (30-40%), but not all, of treated patients \ndevelop the painful condition. These observations suggest that exposure to AIs is necessary, but not \nsufficient, to produce AIMSS, and that additional factors should cooperate with AIs to promote pain \nsymptoms. Aromatase inhibition, while reducing downstream production of estrogens, moderately \nincreases upstream plasma concentrations of androgens, including androstenedione (ASD) \n(Gallicchio et al., 2011). Exemestane, a false aromatase substrate, blocks enzymatic activity by \naccommodating in the binding pocket that snugly encloses ASD. We showed that ASD, which retains \nsome of the reactive chemical features of exemestane, such as the α,β-carbonyl moiety of the A ring \nand the ketone group at the 17 position, targets TRPA1, thus evoking pain-like responses and \nneurogenic inflammation, supported the hypothesis that channel activation in peptidergic nociceptors \npromotes AIMSS. \nAnother painful condition is represented by migraine pain. Occupational exposure to, or \ntreatment with, organic nitrates has long been known to provoke headaches. These observations have \nled to the clinical use of glyceryl trinitrate (GTN) as a reliable provocation test for migraine attacks \n(Iversen et al., 1989; Olesen, 2008; Sicuteri et al., 1987). In most subjects, including healthy controls, \nGTN administration causes a mild headache that develops rapidly and is short-lived. However, after \na remarkable time lag (hours) from GTN exposure, migraineurs develop severe headaches that fulfill \nthe criteria of a typical migraine attack (Iversen et al., 1989; Olesen, 2008; Sicuteri et al., 1987). \nGTN administration to rodents and humans produces a delayed and prolonged (hours) \nhyperalgesia that temporally correlates with GTN-induced migraine-like attacks in humans (Ferrari \net al., 2016; Tassorelli et al., 2003). Several mechanisms have been proposed to explain the \nmechanism, including degranulation of meningeal mast cells (Ferrari et al., 2016), delayed meningeal \ninflammation sustained by induction of NO synthase and prolonged NO generation, and the release \nof CGRP (Ramachandran et al., 2014), a primary migraine neuropeptide (Edvinsson, 2015), but the \nprecise mechanisms was not identified. In our study we identified a pivotal role of TRPA1 expressed \nin trigeminal ganglion neurons in sustaining the delayed mechanical allodynia induced by GTN \nadministration, which recapitulate the migraine attach. Our data clearly showed that TRPA1 receptor \ncan be considered as a novel and major general pain mechanism and TRPA1 antagonists may \nrepresent novel analgesic for a long-waited advancement for the treatment of various types of pain.
Language: Italian
Topic: Ion Channels and Receptors
Subfield: Sensory Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2967963617