Armen N. Akopian - US grants

DESCRIPTION (provided by applicant): It is now recognized that inflammation or peripheral nerve injury induces functional and structural modifications in sensory neurons and thereby alters nociceptive transmission. The changes in gene expression triggered by chronic inflammation or nerve injury are thought to play an important role in the initiation, development and maintenance of these alterations. However, little is known about the mechanisms controlling such changes in gene expression, particularly with respect to the nuclear proteins that regulate this process. To address this question, we have hypothesized that expression patterns of certain ehromatinassociated Polycomb- (PcG) and trithorax-group (trxG) genes are changed in sensory neurons after nerve injury and/or inflammation. In Drosophila, as well as in mammals, these genes are part of the cellular memory system that controls stable expression patterns of target genes. We believe, this application is innovative because our preliminary data are the first, to our knowledge, to demonstrate that certain PcG and/or trxG genes undergo selective and differential regulation in sensory neurons in distinct models of neuropathic pain. Thus, the application has potential for high scientific impact, because regulated PcG and/or trxG could control persistent nociceptive neuronal plasticity during inflammatory and/or neuropathic pain states. Our specific aims will: 1) Determine the expression levels and cell-type distribution of PcG and trxG genes in adult rat dorsal root ganglia (DRG) and trigeminal ganglia (TG) under control conditions (i.e. uninjured and uninflamed). 2) Characterize changes in the expression levels and cell-type distribution of PcG and trxG genes in DRG and TG in animal models of chronic inflammatory and neuropathic pain. 3) Determine whether over/under-expression of certain PcG and/or trxG gene products elucidated in Specific Aim #2 modulates the secretion of nociceptive neurotransmitters in primary TG culture. The multimethodological approach outlined in this proposal should help to identify and characterize the chromatin-associated PcG and/or trxG nuclear factors regulated by chronic inflammation and/or peripheral nerve injury. The knowledge gained by these studies not only should open new avenues of research on the molecular and cellular mechanisms responsible for persistent nocieeptive neuronal plasticity during clinical pain states, but also may provide a novel strategy for modifying the activity of nociceptors using gene and/or drug therapy via the targeting of PcG and trxG nuclear factors.

DESCRIPTION (provided by applicant): The role of gender and pain remains a major health care problem, and in preliminary studies on this topic, we evaluated the long-term effects of estradiol on gene expression in trigeminal neurons. The results constituted an unexpected discovery that estradiol upregulates prolactin (PRL) more than 40 fold in sensory neurons. Follow-up studies demonstrated that PRL and the PRL receptors (PRL-R) are expressed in sensory neurons of both female and male rats, and that application of capsaicin evokes PRL release from trigeminal sensory neurons. Furthermore, application of exogenous PRL significantly and acutely increases nociceptor responsiveness to capsaicin as measured by inward currents, CGRP exocytosis, accumulation of intracellular calcium levels, and nocifensive behavior. These preliminary data provide strong initial support for a completely new hypothesis of nociceptor regulation by an autocrine/paracrine system containing PRL. Based upon this hypothesis, PRL may serve as a novel hyperalgesic agent in both females and in males. We believe that this discovery has substantial scientific and medical implications, and is highly innovative from a conceptual perspective. Therefore, this project will characterize the mechanisms mediating prolactin effects in female and male rats and will directly test the hypotheses that PRL evokes a rapid increase in the responsiveness sensory neurons to noxious stimuli such as capsaicin. Our specific aims will: Specific Aim 1: Determine the effects of exogenous PRL on capsaicin- and inflammation-induced hyperalgesia/allodynia. Specific Aim 2: Determine the mechanisms by which PRL rapidly increases the responsiveness of trigeminal neurons to noxious chemical and thermal stimuli. Specific Aim 3: Characterize the stimuli that evoke PRL release in trigeminal sensory neurons from in vitro cultures and from acutely isolated and superfused peripheral terminals. The discovery that trigeminal sensory neurons express both PRL and PRLR, and that application of exogenous PRL significantly and rapidly sensitizes trigeminal nociceptors to noxious stimuli such as capsaicin, provides strong initial support for a completely new and innovative hypothesis of nociceptor regulation by an autocrine/paracrine PRL system, and compounds that block the PRL-R may serve as a novel class of analgesic drugs in gender dependent pain.

The management of inflammatory pain represents a major scientific and health care challenge and many currently used analgesics provide inadequate pain relief. A mechanistic-based approach to pain management might contribute to the development of valid and novel hypotheses and improve target selection, assist the development and analysis of animal pain models, and eventually inform the design of clinical trials. Inflammatory pain has peripheral and central components. Research into peripheral mechanisms of inflammatory pain is of particular interest since it offers the potential for developing analgesics with minimal CNS side effects. This is an area in which there have been rapid advances over the past decade, although our understanding of peripheral mechanisms of inflammatory pain still remains incomplete. In this application, we propose to test the primary hypothesis that a functional interaction between TRPA1 and TRPV1 channels plays a key role in the integration of inflammation-induced stimuli by sensory neurons. According to this hypothesis, activation and sensitization of the TRPA1 channel by inflammatory mediators are directly controlled by the TRPV1 channel within a complex that fulfills these actions by serving as a modulator to the TRPA1 channel. Our specific aims will: Specific Aim #1: Determine whether TRPA1 and TRPV1 channels are a part of a receptor complex in sensory neurons and whether this complex exhibits a novel phenotype. Specific Aim #2: Determine whether TRPV1 controls the sensitization of the TRPA1-mediated responses by protein kinase C (PKC) and an inflammatory mediator bradykinin in sensory neurons. Specific Aim #3: Determine whether inflammation-induced diacylglycerol and 2-arachydonic glycerol responses mediated by TRPA1 and controlled by TRPV1 in sensory neurons This conceptually innovative hypothesis which proposes distinct mechanisms of integration of inflammatory stimuli by sensory neurons has strong potential for scientific and medical implications.

DESCRIPTION (provided by applicant): Asthma is the most common chronic inflammatory disease of the airways. It affects approximately 34.1 million Americans throughout their lifetime, and the number of people with asthma continues to grow. Standard treatment using an inhaled short-acting beta-2 agonist is only effective against acute symptoms. Though avoiding allergens and irritants while utilizing inhaled corticosteroids may help some patients, these preventatives are still ineffective in completely deterring asthma. Furthermore, cases of asthma will have varying responses to the standard treatments available. Accordingly, finding the specific mechanisms for the defined subgroups of asthma that respond well to various types of treatments is a current critical goal of asthma research. Allergens and environmental irritants play a key role in initiation and maintenance of asthma in children and adults. It is well accepted that airway inflammation plays an important role in the development of airway hyperresponsiveness (AHR) in asthmatic patients. Recent evidence demonstrates airway inflammation during asthma in animals and humans may be at least partially neurogenic in nature. This neurogenic inflammation is induced by neuropeptides released from airway innervating C-fibers of sensory neurons with nodose (ND), jugular, and dorsal root (T1-T6) ganglia (DRG) origin. However, the molecular and physiological mechanisms involving allergens and a combination of allergens and environmental irritants inducing neurogenic inflammation of airways are largely unknown. One of the possibilities is that neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), which trigger neurogenic inflammation, could be released from sensory nerve terminals innervating airways upon stimulation by certain allergens. According to this mechanism, certain allergens would be able to promote neurogenic acute and/or chronic inflammation of the airways. To address this critical question of how exposure to certain allergens results in initiation of neurogenic inflammation of the airways, we hypothesize that certain asthma-inducing mold and house dust mite allergens - DerP1, DerP3&9 and PenC13 - belonging to the protease family initiate inflammation of the airways by activating the sensory neurons innervating these airways and by sensitizing the effects of acrolein and carvacrol (potent tobacco smoke and environmental irritants) on sensory neurons. This conceptually innovative hypothesis which proposes distinct mechanisms of airway inflammation by mold and house dust mite allergens has a strong potential for scientific and medical developments. PUBLIC HEALTH RELEVANCE: The management of asthma represents a major scientific and health care challenge as many of the currently used medications are ineffective in completely deterring asthma or may produce debilitating side effects. Here we propose a novel hypothesis for the initiation of neurogenic inflammation of airways by certain house dust mite and mold asthma-inducing allergens, and by a combination of these allergens and environmental irritants from cigarette smoke, smog and pollution. This conceptually innovative hypothesis has implications for both the scientific and medical fields by expanding our understanding of the role of the peripheral nervous system in initiation of airway inflammation by certain allergens, and by creating a foundation for novel therapeutic strategies aiming to correct the underlying causes of the initiation of asthma by allergens and environmental irritants.

? DESCRIPTION (provided by applicant): Despite recent advances in our understanding of pain mechanisms, there has been little-to-no overall improvement in the clinical management of postoperative pain. It is now recognized that effective postoperative pain management depends on key predictors, especially patient sex. Preclinical and clinical data indicate that, while postoperative pain levels show little-to-no sex-dependent dimorphisms, there are significant sex-based differences in analgesic efficacy, safety profile and abuse/addiction potential of current drugs used to treat postoperative pain. Hence, there is an urgent need to customize postoperative pain management schemes as based on sex-specific pain pharmacology. Our long-term goal is to define sex-dependent postoperative pain mechanisms, and utilize this knowledge to provide more effective postoperative pain management schemes. Our recent published and preliminary data show that a preclinical model of post-operative surgical incision pain leads to a sex-dependent up-regulation of prolactin (PRL) via extra-pituitary mechanisms at surgical sites and in the spinal cord. The PRL receptor (Prlr) is more responsive in female sensory neurons than in males. Moreover, administration of a Prlr antagonist at surgical sites, and especially in the spinal cord, suppresses postoperative hypersensitivity only in females and across all pain modalities. The objective of this proposal is to define peripheral and spinal mechanisms responsible for female-specific regulation of postoperative pain by the PRL system (i.e. PRL and Prlr). Our central hypothesis is that extra-pituitary PRL regulates postoperative pain in a female-specific manner via both peripheral and spinal mechanisms. The rationale is that understanding mechanisms contributing to the female-specific effects of PRL and Prlr on postoperative pain will 1) greatly expand knowledge of sex differences in pain mechanisms; and 2) provide translational potential for the development of novel therapeutic strategies specifically tailored for postoperative pain management in females. Our hypothesis is tested by interconnected yet independent aims. Aim 1 evaluates surgery-induced PRL and Prlr plasticity in peripheral terminals and innate immune cells at surgical sites, and central terminals and glia in spinal cord of female and males. Aim 2 defines the influences of sensory neurons, innate immune cells and glia in peripheral and spinal mechanisms of PRL- mediated regulation of postoperative hypersensitivity and ongoing pain in females and males. Aim 3 examines the regulation of nociceptor excitability by PRL in naïve and operated females and males. The proposed study is innovative since if defines the sex-dependent role of extra-pituitary PRL in regulating postoperative pain, and pathways for specific regulation of postoperative pain in females. The proposed research is significant as it advances our knowledge of sex differences in postoperative pain mechanisms, and has substantial translational potential for new sex-based postoperative pain management strategies.

Women consistently report higher headache-related disabilities, higher relapse rate, more frequent, longer lasting, and more severe headaches than men. Hence, there is an urgent need to customize migraine management schemes based on sex-specific pain mechanisms. Accordingly, our long-term goal is to define sex-specific mechanisms of migraine, and utilize this knowledge to provide more effective sex- based personalized migraine management schemes. It is well accepted that the pathogenesis of headache syndromes, especially migraine, are sex-dependent due to important contributions of gonadal hormones (GnH). First, some reports show that migraine attacks in female and males are accompanied by a rise in plasma levels of prolactin (PRL). Second, we and others demonstrated that PRL responsiveness in pain pathways is sex-dependent and strictly controlled by estrogen. There is a critical gap in knowledge pertaining to whether and how the PRL system sex- dependently regulates migraine. The objective of this proposal is to identify mechanisms linking the PRL system to stress- and sex-dependent regulation of certain types of migraine. Our preliminary data demonstrate that PRL applied to cranial dura induces long-lasting facial allodynia in females, but not males. PRL also sensitizes mustard oil-evoked CGRP release from female, but not male dura. Finally, to further link the PRL system to migraine, we showed that a PRL receptor (Prlr) antagonist blocks CGRP- induced migraine behavior in females. Thus, our central hypothesis is that PRL acting through the Prlr on dural-innervating sensory neurons mediates female-specific mechanisms contributing to migraine. The rationale for the proposed study is that it 1) greatly expands our knowledge of sex differences in migraine mechanisms; and 2) provides translational potential by offering therapeutic targets for sex-based migraine management. Our hypothesis is tested by interconnected yet independent aims. Aim 1 examines sex-specific expression and regulation of PRL and Prlr in the trigemino-vascular system. Aim 2 determines how PRL and Prlr sex-specifically modulate the activity of dural afferents and migraine- like behavior in stress-induced migraine models. Aim 3 assesses a link between CGRP-induced migraine behavioral responses and the dural PRL system. The proposed study is innovative since it defines conceptually novel sex-specific regulatory mechanisms for certain migraine models based on PRL signaling. The proposed research is significant as it advances our understanding of sex differences in migraine mechanisms ? an understudied area where increasing basic science knowledge has the potential to lead to better sex-based personalized therapeutics.

Despite recent advances in our understanding of pain mechanisms, there has been little-to-no overall improvement in the clinical management of chronic pain. It is now recognized that effective chronic pain management depends on key biological variables, especially patient sex. Hence, there is an urgent need to customize chronic pain management schemes based on sex-specific pain mechanisms. Accordingly, our long-term goal is to define sex-dependent mechanisms controlling pain chronicity, and utilize this knowledge to develop sex-specific therapeutics for more effective chronic pain management. Gonadal hormones (GnH) play a key role in sex-dependent regulation of pain mechanisms. There is a gap in knowledge pertaining to how GnH regulate pain chronicity. The objective of this proposal is to identify regulatory mechanisms recruited by GnH for sex-dependent control of pain chronicity. Based on the existing literature and our preliminary data, we propose an entirely novel regulatory mechanism for sexual dimorphisms in chronic pain plasticity wherein the transition from acute to chronic pain is governed by remote control of gene function via GnH-dependent local translation. Our preliminary data suggests that the prolactin receptor (Prlr) may be a linchpin in this mechanism. Prlr is locally translated in females but not males in nociceptor terminals where it contributes strongly to pain plasticity exclusively in females. For instance, sensory neuronal specific Prlr ablation leads to a suppression of IL-6-induced hypersensitivity only in females. Moreover, Prolactin (PRL)-induced hyperalgesic priming, which models the transition from acute to chronic pain, is dramatically enhanced in females compared to males. Therefore, our central hypothesis is that sex- specific regulation of the transition to chronic pain occurs via continuous local translation in nociceptor terminals of mRNAs such as Prlr, and this is fundamentally controlled by gonadal hormones. The proposed study will: 1) greatly expand our knowledge of mechanisms controlling chronicity of pain conditions in females; and 2) provide translational potential by offering therapeutic targets for sex- based chronic pain management. Our hypothesis is tested by interconnected yet independent aims. Aim 1 defines the contribution of local translation in nociceptive terminals, GnH and Prlr in sex-dependent regulation of hyperalgesic priming. Aim 2 examines the involvement of GnH in sex-specific local translation. Aim 3 identifies Prlr sequence motifs controlling sex-specific local translation in nociceptor terminals. The proposed study is innovative because it defines the conceptually novel sex-specific regulatory mechanisms for neuronal plasticity underlying chronic pain in females with technically innovative mouse genetics. The proposed research is significant as it advances our understanding of sex differences in chronic pain mechanisms ? an understudied area where increasing basic science knowledge has the potential to lead to better therapeutics.

Orofacial chronic pain mismanagement substantially contributes to opioid overuse, overdose related deaths and cardiovascular, renal and neurological complications at epidemic proportions. To combat this problem, it is necessary to elucidate a critical gap in knowledge by identifying and vigorously validating novel therapeutic targets controlling the development and maintenance of chronic orofacial pain. The current paradigm implies that orofacial conditions, such as temporomandibular joint and muscle disorders (TMJD) and oral cancer, could trigger maladaptation of the immune system and cell plasticity supporting persistent inflammation, which influences the development and maintenance of orofacial chronic pain. LIGHT (TNFSF14) and Lymphotoxin-beta (LT?), members of the tumor necrosis factor superfamily, are critical components controlling a delicate balance between protective immunity and immunopathology during chronic inflammatory diseases. The objectives of this proposal are: first, to rigorously validate whether local blockade of LIGHT and LT? signaling via LT? receptor (LT?R) or Herpes Virus Entry Mediator (HVEM; TNFRSF14) prevent the development and/or inhibit maintenance of chronic pain in several models of TMJD and oral cancer; and second, to identify LIGHT and LT? signaling-induced plasticity of immune, stromal and tumor cells in masseter muscle and tongue, as well as of sensory neurons in trigeminal ganglia (TG), leading to orofacial chronic pain. Based on the existing literature and our preliminary data, our central hypothesis is that targeting LIGHT and LT? signaling will prevent the development and inhibit maintenance of chronic pain produced by TMJD and oral cancer via peripheral mechanisms involving plasticity of immune, stromal and tumor cells as well as sensory neurons. Our hypothesis will be tested by three relevant yet independent aims. Aim 1 validates whether local LIGHT and LT? inhibition in masseter muscle prevents the development and blocks maintenance of chronic pain in TMJD models. Aim 2 defines contribution of LIGHT and LT? to immune, stromal and neuronal cell plasticity during TMJD. Aim 3 determines whether LIGHT and LT? signaling contribute to the development and maintenance of chronic pain in oral cancer models via regulation of cell plasticity in tongue. The proposed study is innovative because it validates novel targets to facilitate the development of orofacial chronic pain therapeutics; and proposes conceptually novel peripheral regulatory mechanisms involving LIGHT and LT? signaling that control the development and maintenance of TMJD and oral cancer chronic pain. The proposed research is significant as it advances our understanding of mechanisms regulating the development and maintenance of orofacial pain; and offers targets and an immunotherapeutic approach for preventing and blocking chronic pain during TMJD and oral cancer.

Mismanagement of chronic orofacial pain substantially contributes to opioid misuse and opioid related deaths as well as to cardiovascular, renal and neurological complications at epidemic proportions. There is a critical gap in knowledge about the management of chronic orofacial pain which can be addressed by identifying and vigorously validating novel therapeutic targets controlling its development and maintenance. We have identified such targets - LIGHT and Lymphotoxin-beta (LT?), and have been awarded a R01 DE029187 grant within the HEAL Initiative program FOA RFA-NS-18-043 (title: Discovery and Validation of Novel Targets for Safe and Effective Pain Treatment) to vigorously validate these novel therapeutic targets in control of development and maintenance of chronic orofacial pain. This parent grant aims to test central hypothesis that targeting LIGHT and LT? signaling prevents the development of and inhibits maintenance of chronic pain produced by temporomandibular muscle and joint disorders (TMJD) and oral cancer via peripheral mechanisms involving plasticity of immune, stromal and tumor cells as well as sensory neurons. The objectives of this current proposal, which is submitted in response to opportunity ?Research Supplement to Promote Diversity in Health-Related Research (PA-20-222)?, are: first, to promote diversity in health-related research by training Ms. Karen Lindquist, a PhD student from a background underrepresented in bio-medical sciences, and second, to enhance a basic science aspect of the parent application by testing the central hypothesis that LIGHT and LT? modulate TMJD-induced excitability of specific populations of sensory neurons innervating the masseter muscle and the TMJ. Our hypothesis will be tested by two related yet independent aims. Aim 1 identifies and characterizes sensory neuron types innervating the masseter muscle and the TMJ in naïve mice. Aim 2 defines the contribution of LIGHT and LT? to TMJD-induced excitability of different groups of trigeminal sensory neurons innervating the masseter muscle and the TMJ. The proposed study will promote diversity in health-related research, since a PhD student from a background underrepresented in bio-medical sciences will be one of main beneficiaries of this study. This study provides an outstanding training opportunity, since it contains almost all aspects of a multi-level research training program, including a multi-disciplinary approach to research, data analysis and correlation to the literature, presentation and publication of research findings, development of research collaborations and project management. It is highly innovative and significant because it will generate fundamental data on sensory neuron type-dependency from target tissues in the trigeminal system. The proposal also advances our understanding of the mechanisms regulating excitability of different sensory neuron types by LIGHT and LT?.

Chronic pain mismanagement has led to opioid overuse, overdose related deaths and cardiovascular, renal and neurological complications at epidemic proportions. To combat these problems, it is essential to elucidate critical gaps in knowledge pertaining to the underlying mechanisms controlling the processes of initiation and maintenance of chronic pain conditions. The current paradigm implies that tissue or nerve damage triggers protective immune response that should be resolved as soon as its function is fulfilled. If inflammation is not resolved, then transition from the acute to chronic pain could occur. We propose that critical regulators of a delicate balance between protective immunity and immunopathology could be good candidates for controlling a sustained inflammatory response after tissue or nerve damage; and subsequently, regulating the process of development of chronic pain. One of such critical regulators is lymphotoxin-beta receptor (LT?R), a member of the tumor necrosis factor receptor family. The objective of this proposal is to elucidate whether and how peripheral LT?R signaling regulates the process of the initiation and maintenance of pain in inflammatory and chemotherapy-induced peripheral neuropathy (CIPN) models. Based on the existing literature and our preliminary data, we propose an entirely novel regulatory mechanism for the initiation and maintenance of inflammatory as well as CIPN pain wherein peripheral LT?R signaling controls these processes by regulating the network of transcriptional and cellular plasticity in hindpaw and DRG cells. Accordingly, our central hypothesis is that peripheral LT?R signaling controls the processes of initiation and maintenance of inflammatory and CIPN pain via governing the network of transcriptional and cellular plasticity mediating communication between peripheral cells and sensory neurons. Our hypothesis will be tested by three interconnected yet independent aims. Aim 1 defines the impact of peripheral LT?R signaling on the initiation and maintenance of inflammatory and CIPN pain in male and female mice. Aim 2 determines the cellular basis of LT?R signaling at the periphery during inflammatory and CIPN pain. Aim 3 examines the impact of peripheral LT?R signaling on inflammation- and CIPN-induced sensory neuronal and non-neuronal transcriptional and cell plasticity in paw and DRG. The proposed study is innovative because it describes conceptually novel peripheral regulatory mechanism controlling the processes of initiation and maintenance of chronic pain, which are regulated by LT?R. The proposed research is significant as it (1) advances our understanding of mechanisms regulating the transition from acute to chronic pain; and (2) offers LT?R signaling antagonists as potential therapeutic targets for prevention and full and sustained reversal of CIPN chronic pain, as well as effective and long-lasting management of inflammatory pain.