Browsing by Author "Dussor, Gregory"
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Item 17-β-Estradiol Induces Spreading Depression and Pain Behavior in Alert Female Rats(Impact Journals LLC) Sandweiss, Alexander J.; Cottier, Karissa E.; McIntosh, Mary I.; Dussor, Gregory; Davis, Thomas P.; Vanderah, Todd W.; Largent-Milnes, Tally M.; Dussor, GregoryAims: Test the putative contribution of 17-β-estradiol in the development of spreading depression (SD) events and head pain in awake, non-restrained rats. Main Methods: Female, Sprague-Dawley rats were intact or underwent ovariectomy followed one week later by surgery to place electrodes onto the dura to detect epidural electroencephalographic activity (dEEG). dEEG activity was recorded two days later for 12 hours after systemic administration of 17-β-estradiol (180 μg/kg, i.p.). A separate set of rats were observed for changes in exploratory, ambulatory, fine, and rearing behaviors; periorbital allodynia was also assessed. Key Findings: A bolus of 17-β-estradiol significantly elevated serum estrogen levels, increased SD episodes over a 12-hour recording period and decreased rearing behaviors in ovariectomized rats. Pre-administration of ICI 182,780, an estrogen receptor antagonist, blocked 17-β-estradiol-evoked SD events and pain behaviors; similar results were observed when the antimigraine therapeutic sumatriptan was used. Significance: These data indicate that an estrogen receptor-mediated mechanism contributes to SD events in ovariectomized rats and pain behaviors in both ovariectomized -and intact-rats. This suggests that estrogen plays a different role in each phenomenon of migraine where intense fluctuations in concentration may influence SD susceptibility. This is the first study to relate estrogen peaks to SD development and pain behaviors in awake, freely moving female rats, establishing a framework for future preclinical migraine studies.Item Activation of the Integrated Stress Response in Nociceptors Drives Methylglyoxal-Induced Pain(Lippincott Williams & Wilkins, 2019-01) Barragan-Iglesias, Paulino; Kuhn, Jasper; Vidal-Cantu, Guadalupe C.; Belen Salinas-Abarca, Ana; Granados-Soto, Vinicio; Dussor, Gregory; Campbell, Zachary T.; Price, Theodore J.; 0000-0002-3768-6996 (Campbell, ZT); 0000-0002-6971-6221 (Price, TJ); Dussor, Gregory; Campbell, Zachary T.; Price, Theodore J.Methylglyoxal (MGO) is a reactive glycolytic metabolite associated with painful diabetic neuropathy at plasma concentrations between 500 nM and 5 μM. The mechanisms through which MGO causes neuropathic pain at these pathological concentrations are not known. Because MGO has been linked to diabetic neuropathic pain, which is prevalent and poorly treated, insight into this unsolved biomedical problem could lead to much needed therapeutics. Our experiments provide compelling evidence that ~ 1-μM concentrations of MGO activate the integrated stress response (ISR) in IB4-positive nociceptors in the dorsal root ganglion (DRG) of mice in vivo and in vitro. Blocking the integrated stress response with a specific inhibitor (ISRIB) strongly attenuates and reverses MGO-evoked pain. Moreover, ISRIB reduces neuropathic pain induced by diabetes in both mice and rats. Our work elucidates the mechanism of action of MGO in the production of pain at pathophysiologically relevant concentrations and suggests a new pharmacological avenue for the treatment of diabetic and other types of MGO-driven neuropathic pain.Item A Critical Role for Dopamine D5 Receptors in Pain Chronicity in Male Mice(Soc Neuroscience) Megat, Salim; Shiers, Stephanie; Moy, Jamie K.; Barragán-Iglesias, Paulino; Pradhan, Grishma; Megat, Salim; Dussor, Gregory; Price, Theodore J.; 0000-0003-2186-6770 (Megat, S); 0000-0002-9646-1850 (Shiers, S); 0000-0001-8579-5540 (Moy, JK); 0000-0003-3178-8606 (Barragán-Iglesias, P); 0000-0002-6971-6221 (Price, TJ); Megat, Salim; Shiers, Stephanie; Moy, Jamie K.; Barragan-Iglesias, Paulino; Pradhan, Grishma; Seal, Rebecca P.; Dussor, Gregory; Price, Theodore J.Dopaminergic modulation of spinal cord plasticity has long been recognized, but circuits affected by this system and the precise receptor subtypes involved in this modulation have not been defined. Dopaminergic modulation from the A11 nucleus of the hypothalamus contributes to plasticity in a model of chronic pain called hyperalgesic priming. Here we tested the hypothesis that the key receptor subtype mediating this effect is the D5 receptor (D5R). We find that a spinally directed lesion of dopaminergic neurons reverses hyperalgesic priming in both sexes and that a D1/D5 antagonist transiently inhibits neuropathic pain. We used mice lacking D5Rs (DRD5KO mice) to show that carrageenan, interleukin 6, as well as BDNF-induced hyperalgesia and priming are reduced specifically in male mice. These male DRD5KO mice also show reduced formalin pain responses and decreased heat pain. To characterize the subtypes of dorsal horn neurons engaged by dopamine signaling in the hyperalgesic priming model, we used c-fos labeling. We find that a mixed D1/D5 agonist given spinally to primed mice activates a subset of neurons in lamina III and IV of the dorsal horn that coexpress PAX2, a transcription factor for GABAergic interneurons. In line with this, we show that gabazine, a GABA-A receptor antagonist, is antihyperalgesic in primed mice exposed to spinal administration of a D1/D5 agonist. Therefore, the D5R, in males, and the D1R, in females, exert a powerful influence over spinal cord circuitry in pathological pain likely via modulation of deep dorsal horn GABAergic neurons.Item Dural Calcitonin Gene-Related Peptide Produces Female-Specific Responses in Rodent Migraine Models(Society for Neuroscience, 2019-05-29) Avona, Amanda; Burgos-Vega, Carolina; Burton, Michael D.; Akopian, A. N.; Price, Theodore J.; Dussor, Gregory; 0000-0002-6971-6221 (Price, TJ); 0000-0002-0628-824X (Burton, MD); Avona, Amanda; Burgos-Vega, Carolina; Burton, Michael D.; Price, Theodore J.; Dussor, GregoryMigraine is the second leading cause for disability worldwide and the most common neurological disorder. It is also three times more common in women; reasons for this sex difference are not known. Using preclinical behavioral models of migraine, we show that application of calcitonin gene-related peptide (CGRP) to the rat dura mater produces cutaneous periorbital hypersensitivity. Surprisingly, this response was observed only in females; dural CGRP at doses from 1 pg to 3.8 μg produce no responses in males. In females, dural CGRP causes priming to a pH 7.0 solution after animals recover from the initial CGRP-induced allodynia. Dural application of interleukin-6 causes acute responses in males and females but only causes priming to subthreshold dural CGRP (0.1 pg) in females. Intracisternal application of BDNF also causes similar acute hypersensitivity responses in males and females but only priming to subthreshold dural CGRP (0.1 pg) in females. Females were additionally primed to a subthreshold dose of the NO-donor sodium nitroprusside (0.1 mg/kg) following dural CGRP. Finally, the sexually dimorphic responses to dural CGRP were not specific to rats as similar female-specific hypersensitivity responses were seen in mice, where increased grimace responses were also observed. These data are the first to demonstrate that CGRP-induced headache-like behavioral responses at doses up to 3.8 μg are female-specific both acutely and following central and peripheral priming. These data further implicate dural CGRP signaling in the pathophysiology of migraine and propose a model where dural CGRP-based mechanisms contribute to the sexual disparity of this female-biased disorder.SIGNIFICANCE STATEMENT Calcitonin gene-related peptide (CGRP) has long been implicated in the pathophysiology of migraine, and CGRP-based therapeutics are efficacious for the treatment of migraine in humans. However, the location of action for CGRP in migraine remains unclear. We show here that application of CGRP to the cranial meninges causes behavioral responses consistent with headache in preclinical rodent models. Surprisingly, however, these responses are only observed in females. Acute responses to meningeal CGRP are female-specific and sensitization to CGRP after two distinct stimuli are also female-specific. These data implicate the dura mater as a primary location of action for CGRP in migraine and suggest that female-specific mechanisms downstream of CGRP receptor activation contribute to the higher prevalence of migraine in women. Copyright © 2019 the authors.Item eIF4E Phosphorylation Influences BDNF mRNA Translation in Mouse Dorsal Root Ganglion Neurons(Frontiers Media SA) Moy, Jamie K.; Khoutorsky, Arkady; Asiedu, Marina N.; Dussor, Gregory; Price, Theodore J.; 0000-0002-6971-6221 (Price, TJ); Moy, Jamie K.; Asiedu, Marina N.; Dussor, Gregory; Price, Theodore J.Plasticity in dorsal root ganglion (DRG) neurons that promotes pain requires activity-dependent mRNA translation. Protein synthesis inhibitors block the ability of many pain-promoting molecules to enhance excitability in DRG neurons and attenuate behavioral signs of pain plasticity. In line with this, we have recently shown that phosphorylation of the 5' cap-binding protein, eIF4E, plays a pivotal role in plasticity of DRG nociceptors in models of hyperalgesic priming. However, mRNA targets of eIF4E phosphorylation have not been elucidated in the DRG. Brain-derived neurotrophic factor (BDNF) signaling from nociceptors in the DRG to spinal dorsal horn neurons is an important mediator of hyperalgesic priming. Regulatory mechanisms that promote pain plasticity via controlling BDNF expression that is involved in promoting pain plasticity have not been identified. We show that phosphorylation of eIF4E is paramount for BDNF mRNA translation in the DRG. BDNF mRNA translation is reduced in mice lacking eIF4E phosphorylation (eIF4E^(S209A)) and pro-nociceptive factors fail to increase BDNF protein levels in the DRGs of these mice despite robust upregulation of BDNF-201 mRNA levels. Importantly, bypassing the DRG by giving intrathecal injection of BDNF in eIF4E^(S209A) mice creates a strong hyperalgesic priming response that is normally absent or reduced in these mice. We conclude that eIF4E phosphorylation-mediated translational control of BDNF expression is a key mechanism for nociceptor plasticity leading to hyperalgesic priming.Item Emerging Neurotechnology for Antinoceptive Mechanisms and Therapeutics Discovery(Elsevier Advanced Technology, 2018-11-13) Black, Bryan J.; Atmaramani, Rahul; Plagens, Sarah; Campbell, Zachary T.; Dussor, Gregory; Price, Theodore J.; Pancrazio, Joseph J.; 0000-0002-3768-6996 (Campbell, ZT); 0000-0002-6971-6221 (Price, TJ); 0000-0001-8276-3690 (Pancrazio, JJ); Black, Bryan J.; Atmaramani, Rahul; Plagens, Sarah; Campbell, Zachary T.; Dussor, Gregory; Price, Theodore J.; Pancrazio, Joseph J.The tolerance, abuse, and potential exacerbation associated with classical chronic pain medications such as opioids creates a need for alternative therapeutics. Phenotypic screening provides a complementary approach to traditional target-based drug discovery. Profiling cellular phenotypes enables quantification of physiologically relevant traits central to a disease pathology without prior identification of a specific drug target. For complex disorders such as chronic pain, which likely involves many molecular targets, this approach may identify novel treatments. Sensory neurons, termed nociceptors, are derived from dorsal root ganglia (DRG) and can undergo changes in membrane excitability during chronic pain. In this review, we describe phenotypic screening paradigms that make use of nociceptor electrophysiology. The purpose of this paper is to review the bioelectrical behavior of DRG neurons, signaling complexity in sensory neurons, various sensory neuron models, assays for bioelectrical behavior, and emerging efforts to leverage microfabrication and microfluidics for assay development. We discuss limitations and advantages of these various approaches and offer perspectives on opportunities for future development.Item Haptic Stroke Testbed for Pharmacological Evaluation of Dynamic Allodynia in Mouse Models(IEEE Computer Society) Lee, Jin; Atwood, Brian J.; Megat, Salim; Dussor, Gregory; Price, Theodore J.; Fey, Ann Majewicz; Lee, Jin; Atwood, Brian J.; Megat, Salim; Dussor, Gregory; Price, Theodore J.; Fey, Ann MajewiczDynamic mechanical allodynia is an aggravating neuropathological condition in which light, physical touch leads to pain. Developing pharmaceutical agents to treat this condition requires extensive animal trials using a mouse model, and a laborious process of manually stroking inflicted mouse paws, with a brush or cotton swab, while recording responses to that stimulus. In this paper, we developed an autonomous testing mechanism to create repeatable stroking sensations for mice during dynamic allodynia testing. The chamber consists of a belt driven brush mechanism and light and dark chambers. Additionally, we conducted a human subjects study to determine the baseline variability in human-performed dynamic allodynia testing. Our tactile stoke display is capable of stroking a mouse paw between 1-5 mm/s with a repeatable force. In our human subject experiments, the user applied force ranged from 0.1-9.0 gF with a maximum standard deviation of 4.13 gF. In contrast, our device is capable of producing repeatable brush strokes at 0.69 gF (SD = 0.13 gF) and 1.78 gF (SD = 0.16 gF) for two brushes. Preliminary animal studies show that normal mice are not disturbed by the stroking sensation; however, mice afflicted with allodynia move away from it. On average the injured mice spent 90% of their time in a bright, adverse environment to avoid the brush, whereas normal mice only spent 40% of their time in the bright environment.Item Investigating the Cell-specific Mechanisms That Drive Sex Differences During Neuropathic Pain Development(2022-08-01T05:00:00.000Z) Szabo-Pardi, Thomas Alexander; Burton, Michael D; O'Toole, Alice; Price, Theodore; Dussor, Gregory; Rincón-Cortés, MillieChronic pain patients often suffer from a decline in quality of life due to a lack of efficacious long- term therapeutics. Moreover, the prevalence of chronic pain conditions is on the rise, with an average increase of nearly 10 percent in patients per decade. This, coupled with the devastating impact of the opioid crisis, highlights the need for novel pain therapeutics. An extensive literature has placed microglia, the resident immune cells of the central nervous system, at the forefront of male-specific mechanisms that mediate chronic pain plasticity. Recently, efforts have been made to design studies aimed at dissecting female specific mechanisms in pain plasticity. Evidence suggests that immune-related components of nociceptors are heavily dysregulated following insult and are more directly responsible for changes in female-specific sensitization. Despite advances in the field of pain neurobiology, there remains a clear disconnect between the cellular mechanisms that underlie maladaptive chronic pain in males and females. Moreover, a lack of studies directed towards interventions during early neuropathic pain development make it difficult to assess how functional changes in cellular phenotypes following injury can be manipulated to prevent maladaptive pain plasticity from taking place. The goal of our research was to use an innovative approach to identify nociceptor and immune cell-specific mechanisms in both the peripheral and central nervous systems that mediate sex differences during neuropathic pain development. Our findings suggest that the initial phase of neuropathic development is sexually dimorphic, characterized by nociceptor-specific signaling mechanisms in females and immune cell mediated sensitization in males which may be modulated by genetic and pharmacological manipulation of toll-like receptor 4 signaling.Item Mechanisms Underlying Migraine Headache Pathophysiology: Novel Insights From Preclinical Models(2021-12-01T06:00:00.000Z) Lackovic, Jacob Edward; Dussor, Gregory; Katz, William; McIntyre Rodriguez, Christa; Price, Theodore; Burton, MichaelMigraine is a highly prevalent and complex disorder characterized by severe, unilateral, pulsating headaches associated with photophobia, phonophobia, nausea, and, in some cases, auras. Headaches are the most disabling component of the condition and, while treatments have improved over the last few decades, the complexity of migraine pathophysiology has made it extremely challenging to develop highly efficacious therapeutics. Patients are particularly susceptible to attacks following exposure to normally innocuous stimuli and mounting clinical and preclinical evidence suggests that this may be due to maladaptive sensitization of the trigeminal sensory system. Although it is widely accepted that the trigeminovascular system is responsible for the pain associated with migraine, the mechanisms by which dura-projecting trigeminal ganglia (TG) nociceptors become activated and sensitized remain poorly understood. In other preclinical pain models, reactive nitroxidative species such as nitric oxide (NO), but particularly peroxynitrite (PN), have been implicated in establishing long-lasting hypersensitivity and targeting these molecules has achieved antinociceptive efficacy. Despite NO donors being one of the most consistent triggers of headache, little is known about the role of nitroxidative species in migraine mechanisms. Similarly, other mechanisms that have been shown to contribute to nociceptor activation and sensitization in preclinical pain models, such as translational dysregulation of mRNA, have not been studied in the context of migraine. Thus, the goal of our research was to utilize pharmacological techniques and transgenic animals in our novel preclinical migraine models to further understand the mechanisms that contribute to the development and persistence of migraine headache. The first part of our work highlights a novel, critical role for PN formation in mediating long-lasting hypersensitivity in preclinical models of migraine while the second part of our work defines MNK regulation of eIF4E phosphorylation as a key target for migraine therapeutics.Item Meningeal Afferent Signaling and the Pathophysiology of Migraine(2017-05) Burgos Vega, Carolina Cristina; Dussor, Gregory; Price, Theodore; Thompson, Tres; McIntyre, ChristaMigraine is the most common neurological disorder. Attacks are complex and consist of multiple phases but are most commonly characterized by intense, unilateral, throbbing headache. The pathophysiology contributing to migraine is poorly understood and the disorder is not well managed with currently available therapeutics, often rendering patients disabled during attacks. The mechanisms most likely to contribute to the pain phase of migraine require activation of trigeminal afferent signaling from the cranial meninges and subsequent relay of nociceptive information into the central nervous system in a region of the dorsal brainstem known as the trigeminal nucleus caudalis (TNC). A three-fold higher prevalence of migraine in females implies sex-dependent mechanisms are involved in migraine. Yet, sex-dependent mechanisms of migraine remain unknown and have yet to be investigated thoroughly. Events leading to activation of meningeal afferents are unclear but nerve endings within this tissue are mechanosensitive and also express a variety of ion channels including acid-sensing (ASIC) and transient receptor-potential (TRP) channels. These properties may provide clues into the pathophysiology of migraine by suggesting that decreased extracellular pH and environmental irritant exposure in the meninges contributes to headache. Neuroplasticity is also likely to play a role in migraine given that attacks are triggered by routine events that are typically non-noxious in healthy patients and clear evidence of sensitization occurs during an attack. Where and how plasticity develops is also not clear but may include events directly on the afferents and/or within the TNC. Among the mediators potentially contributing to plasticity, calcitonin gene-related peptide (CGRP) has received the most attention within the migraine field but other mechanisms may also contribute. Ultimately, greater understanding of the molecules and mechanisms contributing to migraine will undoubtedly lead to better therapeutics and relief for the large numbers of patients across the globe who suffer from this highly disabling neurological disorder.Item The MNK–eIF4E Signaling Axis Contributes to Injury-Induced Nociceptive Plasticity and the Development of Chronic Pain(Society for Neuroscience) Moy, Jamie K.; Khoutorsky, A.; Black, Brian J.; Kuhn, Jasper L.; Barragán-Iglesias, Paulino; Megat, Salim; Burton, Michael D.; Burgos-Vega, Carolina C.; Melemedjian, O. K.; Boitano, S.; Vagner, J.; Gkogkas, C. G.; Pancrazio, Joseph J.; Mogil, J. S.; Dussor, Gregory; Sonenberg, N.; Price, Theodore J.; 0000 0001 3721 4764 (Dussor, G); 0000-0001-8579-5540 (Moy, JK); 0000-0001-8571-6486 (Black, B); 0000-0001-6524-9411 (Kuhn JL); 0000-0003-3178-8606 (Barragán-Iglesias, P); 0000-0002-6971-6221 (Price, TJ); Moy, Jamie K.; Asiedu, Marina N.; Black, Brian J.; Kuhn, Jasper L.; Barragán-Iglesias, Paulino; Megat, Salim; Burton, Michael D.; Burgos-Vega, Carolina C.; Pancrazio, Joseph J.; Dussor, Gregory; Price, Theodore J.Injury-induced sensitization of nociceptors contributes to pain states and the development of chronic pain. Inhibiting activity-dependent mRNA translation through mechanistic target of rapamycin and mitogen-activated protein kinase (MAPK) pathways blocks the development of nociceptor sensitization. These pathways convergently signal to the eukaryotic translation initiation factor (eIF) 4F complex to regulate the sensitization of nociceptors, but the details of this process are ill defined. Here we investigated the hypothesis that phosphorylation of the 5β cap-binding protein eIF4E by its specific kinase MAPK interacting kinases (MNKs) 1/2 is a key factor in nociceptor sensitization and the development of chronic pain. Phosphorylation of ser209 on eIF4E regulates the translation of a subset of mRNAs. We show that pronociceptive and inflammatory factors, such as nerve growth factor (NGF), interleukin-6 (IL-6), and carrageenan, produce decreased mechanical and thermal hypersensitivity, decreased affective pain behaviors, and strongly reduced hyperalgesic priming in mice lacking eIF4E phosphorylation (eIF4ES209A). Tests were done in both sexes, and no sex differences were found. Moreover, in patch-clamp electrophysiology and Ca2+ imaging experiments on dorsal root ganglion neurons, NGF- and IL-6-induced increases in excitability were attenuated in neurons from eIF4ES209A mice. These effects were recapitulated in Mnk1/2-/- mice and with the MNK1/2 inhibitor cercosporamide. We also find that cold hypersensitivity induced by peripheral nerve injury is reduced in eIF4ES209A and Mnk1/2-/- mice and following cercosporamide treatment. Our findings demonstrate that the MNK1/2–eIF4E signaling axis is an important contributing factor to mechanisms of nociceptor plasticity and the development of chronic pain.Item Neuroimmune and Endocrine Interactions Driving Female-biased Mechanisms in Reproductive Physiology and Pain(August 2023) Lenert, Melissa Elizabeth 1995-; Burton, Michael; Shoup, Angela; Dussor, Gregory; Merriwether, Ericka; Thompson, LucienSex and gender disparities in healthcare have a profound negative impact on women’s health. Until recently, most preclinical neuroscience research has relied almost entirely on male animals to the exclusion of females due to perceived confounds by hormonal cycling. The usage of female animals in preclinical settings in recent years has opened a realm of possibility in neuroscience research as many groups have demonstrated sex biases in neuroimmune and endocrine crosstalk. Several disorders exhibit female-biased prevalence, including many chronic pain disorders and reproductive system disorders. Additionally, treatments for these disorders are often less effective in women and come with ill-tolerated side effects. Thus, there is a strong need to study female- biased mechanisms in these disorders to improve therapeutics and patient outcomes. This work focuses the role of metabolic stress, both cellular and whole-body, in the regulation of female fertility. First, we induced whole-body metabolic stress via consumption of a high-fat diet by young female mice and measured changes in estrous cycling and serum progesterone. We found that a high-fat diet induces transient shifts in estrous cycling and progesterone levels prior to overt weight gain. Next, we utilized a transgenic mouse model with conditional removal of LKB1 in peripheral sensory neurons to model neuronal metabolic stress. Females with LKB1 deletion have greatly enhanced fertility compared to wild-type mice, with no effect of LKB1 removal in male mice. Further, LKB1 in sensory neurons promotes ovarian innervation. We then utilized a preclinical model of chronic muscle pain to validate a battery of pain and functional assessments. Those measurements were directly compared to pain and functional assessments performed in a clinical trial with women with FM. Finally, we assessed changes in adaptive immune cell phenotypes before and after treatment with IL-5, a cytokine previously demonstrated to play a unique role in chronic muscle pain and women with FM. Overall, this work highlights female biased mechanisms that modulate neuroendocrine communication and fertility and neuroimmune crosstalk during chronic muscle pain.Item Nociceptor Translational Profiling Reveals the Ragulator-Rag GTPase Complex as a Critical Generator of Neuropathic Pain(Soc Neuroscience, 2019-01-16) Megat, Salim; Ray, Pradipta R.; Moy, Jamie K.; Lou, Tzu-Fang; Barragan-Iglesias, Paulino; Li, Yan; Pradhan, Grishma; Wanghzou, Andi; Ahmad, Ayesha; Burton, Michael D.; North, Robert Y.; Dougherty, Patrick M.; Khoutorsky, Arkady; Sonenberg, Nahum; Webster, Nevin R.; Dussor, Gregory; Campbell, Zachary T.; Price, Theodore J.; 0000-0003-4281-3985 (Pradhan, G); 0000-0002-0628-824X (Burton, MD); 0000-0002-3768-6996 (Campbell, ZT); 0000-0002-6971-6221 (Price, TJ); Megat, Salim; Ray, Pradipta R.; Moy, Jamie K.; Lou, Tzu-Fang; Barragan-Iglesias, Paulino; Pradhan, Grishma; Wanghzou, Andi; Ahmad, Ayesha; Burton, Michael D.; Dussor, Gregory; Campbell, Zachary T.; Price, Theodore J.Nociceptors, sensory neurons in the DRG that detect damaging or potentially damaging stimuli, are key drivers of neuropathic pain. Injury to these neurons causes activation of translation regulation signaling, including the mechanistic target of rapamycin complex 1 (mTORC1) and mitogen-activated protein kinase interacting kinase(MNK) eukaryotic initiation factor (eIF) 4E pathways. This is a mechanism driving changes in excitability of nociceptors that is critical for the generation of chronic pain states; however, the mRNAs that are translated to lead to this plasticity have not been elucidated. To address this gap in knowledge, we used translating ribosome affinity purification in male and female mice to comprehensively characterize mRNA translation in Scn10a-positive nociceptors in chemotherapy-induced neuropathic pain (CIPN) caused by paclitaxel treatment. This unbiased method creates a new resource for the field, confirms many findings in the CIPN literature and also find extensive evidence for new target mechanisms that may cause CIPN. We provide evidence that an underlying mechanism of CIPN is sustained mTORC1 activation driven by MNK1-eIF4E signaling. RagA, aGTPase controlling mTORC1 activity, is identified as a novel target of MNK1-eIF4E signaling. This demonstrates a novel translation regulation signaling circuit wherein MNK1-eIF4E activity drives mTORC1 via control of RagA translation. CIPN and RagA translation are strongly attenuated by genetic ablation of eIF4E phosphorylation, MNK1 elimination or treatment with the MNK inhibitor eFT508. We identify a novel translational circuit for the genesis of neuropathic pain caused by chemotherapy with important implications for therapeutics.Item Pharmacological Regulation of Protein Translation in Fragile X Syndrome(2022-12-01T06:00:00.000Z) Shukla, Tarjani; Dussor, Gregory; Price, Theodore; Delk, Nikki; Palmer, Kelli; Sapkota, DarshanThe behavioral hallmarks of Autism Spectrum Disorder (ASD) are driven by molecular mechanisms that remain largely unknown. One model of ASD involves the deletion of the Fmr1 gene. Mutations in Fmr1 cause Fragile X Syndrome (FXS) and are the most common monogenetic source of intellectual disability in humans. The Fmr1 gene encodes for Fragile X Mental Retardation Protein (FMRP). FMRP is a conserved RNA-binding protein that binds the ribosome and attenuates translation. Loss of FMRP results in aberrant protein translation. This genetic lesion results in widespread cognitive deficiencies, specifically with learning, memory, and social interaction. A deeper understanding of the mechanism driving FMRP-dependent synaptic plasticity enables identification of highly specific therapeutics that ameliorate core neurological deficits associated with the disorder. In FXS, eIF4E is hyperphosphorylated and has emerged as a therapeutic target. Yet, specific inhibitors of Mitogen-Activated Protein Kinase Interacting Protein Kinase (MNK) have not been examined in the context of Fmr1 -/y mice. The goal of this research is to understand if compensation of FMRP loss can be achieved through manipulation of translation by the MNK-eIF4E regulatory axis. The results of this dissertation outline the viability of a novel therapeutic for the reversal of behaviors associated with Fragile X Syndrome, for which there are no FDA-approved treatments.Item Protease Activated Receptor 2 (PAR2) Activation Causes Migraine-Like Pain Behaviors in Mice(Sage Publications Ltd, 2018-05-31) Hassler, Shayne N.; Ahmad, Fatima B.; Burgos-Vega, Carolina C.; Boitano, Scott; Vagner, Josef; Price, Theodore J.; Dussor, Gregory; 0000-0002-6971-6221 (Price, TJ); Hassler, Shayne N.; Ahmad, Fatima B.; Burgos-Vega, Carolina C.; Price, Theodore J.; Dussor, GregoryBackground Pain is the most debilitating symptom of migraine. The cause of migraine pain likely requires activation of meningeal nociceptors. Mast cell degranulation, with subsequent meningeal nociceptor activation, has been implicated in migraine pathophysiology. Degranulating mast cells release serine proteases that can cleave and activate protease activated receptors. The purpose of these studies was to investigate whether protease activated receptor 2 is a potential generator of nociceptive input from the meninges by using selective pharmacological agents and knockout mice. Methods Ratiometric Ca⁺⁺ imaging was performed on primary trigeminal and dural cell cultures after application of 2at-LIGRL-NH₂, a specific protease activated receptor 2 agonist. Cutaneous hypersensitivity and facial grimace was measured in wild-type and protease activated receptor 2⁻ᐟ⁻ mice after dural application of 2at-LIGRL-NH₂ or compound 48-80, a mast cell degranulator. Behavioral experiments were also conducted in mice after dural application of 2at-LIGRL-NH₂ (2AT) in the presence of either C391, a selective protease activated receptor 2 antagonist, or sumatriptan. Results 2at-LIGRL-NH₂ evoked Ca²⁺ signaling in mouse trigeminal neurons, dural fibroblasts and in meningeal afferents. Dural application of 2at-LIGRL-NH₂ or 48-80 caused dose-dependent grimace behavior and mechanical allodynia that were attenuated by either local or systemic application of C391 as well as in protease activated receptor 2⁻ᐟ⁻ mice. Nociceptive behavior after dural injection of 2at-LIGRL-NH₂ was also attenuated by sumatriptan. Conclusions Functional protease activated receptor 2 receptors are expressed on both dural afferents and fibroblasts and activation of dural protease activated receptor 2 produces migraine-like behavioral responses. Protease activated receptor 2 may link resident immune cells to meningeal nociceptor activation, driving migraine-like pain and implicating protease activated receptor 2 as a therapeutic target for migraine in humans.Item Sex Differences in the Expression of Calcitonin Gene-Related Peptide Receptor Components in the Spinal Trigeminal Nucleus(Elsevier B.V., 2019-04-24) Ji, Y.; Rizk, A.; Voulalas, P.; Aljohani, H.; Akerman, S.; Dussor, Gregory; Keller, A.; Masri, R.; Dussor, GregoryBackground and purpose: Calcitonin gene-related peptide (CGRP) plays an important role in migraine pathophysiology. CGRP acts primarily by activating a receptor composed of 3 proteins: calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), and receptor component protein (RCP). We tested the hypothesis that sex differences exist in protein levels of two key components of this CGRP receptor: CLR and RCP. Methods: We used specific antibodies to assess baseline protein levels of CLR and RCP in the spinal trigeminal nucleus caudalis (SpVc) and upper cervical spinal cord of both male and female rats. We also tested if manipulations that knock-down the expression of RCP in SpVc, using locally-mediated gene transfer of short hairpin RNA (shRNA), ameliorate pain in an animal model of intracranial migraine-like pain induced by chemical noxious stimulation of the meninges. To assess pain, we used tests of ongoing pain (rat face grimace test and freezing behavior)and tests of facial mechanical hypersensitivity and allodynia. Results: There was no difference in CLR levels between male and female animals (p > 0.11) in SpVc and the upper cervical cord. However, female animals exhibited greater baseline levels of RCP (up to 3-fold higher) compared to males (p < 0.002). The knock-down of RCP expression in SpVc attenuated mechanical facial allodynia induced by chemical noxious stimulation of the meninges, but had little effect on ongoing pain behaviors in female and male animals. Conclusions: RCP is an integral component of the CGRP receptor and may play a key role in mediating CGRP induced central sensitization after noxious stimulation of the meninges. RCP expression in the SpVc and upper cervical cord is sexually dimorphic, with higher levels of expression in females. This dimorphism may be related to the increased incidence of migraines in females–a hypothesis that should be tested in the future. ©2019 The AuthorsItem Spinal Inhibition of P2XR or p38 Signaling Disrupts Hyperalgesic Priming in Male, but not Female, Mice(Elsevier Ltd) Paige, Candler; Maruthy, Gayathri B.; Mejia, Galo; Dussor, Gregory; Price, Theodore J.; 0000 0001 3721 4764 (Dussor, G); 0000-0002-6971-6221 (Price, TJ); Paige, Candler; Maruthy, Gayathri B.; Mejia, Galo; Dussor, Gregory; Price, Theodore J.Recent studies have demonstrated sexual dimorphisms in the mechanisms contributing to the development of chronic pain. Here we tested the hypothesis that microglia might preferentially regulate hyperalgesic priming in male mice. We based this hypothesis on evidence that microglia preferentially contribute to neuropathic pain in male mice via ionotropic purinergic receptor (P2XR) or p38 mitogen-activated protein kinase (p38) signaling. Mice given a single-priming injection of the soluble human interleukin-6 receptor (IL-6r) and then a second injection of prostaglandin E2 (PGE2), which unmasks hyperalgesic priming, shows a significant increase in levels of activated microglia at 3 h following the PGE2 injection in both male and female mice. There was no change in microglia following PGE2. Intrathecal injection of the P2X3/4 inhibitor TNP-ATP blocked the initial response to IL-6r in both males and females, but only blocked hyperalgesic priming in male mice. Intrathecally applied p38 inhibitor, skepinone, had no effect on the initial response to IL-6r but attenuated hyperalgesic priming in males only. Neither TNP-ATP nor skepinone could reverse priming once it had already been established in male mice suggesting that these pathways must be inhibited early in the development of hyperalgesic priming to have an effect. Our work is consistent with previous findings that P2XR and p38 inhibition can lead to male-specific effects on pain behaviors in mice. However, given that we did not observe microglial activation at time points where these drugs were effective, our work also questions whether these effects can be completely attributed to microglia. © 2018 IBROItem Targeted Acid-Sensing Ion Channel Therapies for Migraine(Springer) Karsan, Nazia; Gonzales, Eric B.; Dussor, Gregory; 0000 0001 3721 4764 (Dussor, G); Dussor, GregoryAcid-sensing ion channels (ASICs) are a family of ion channels, consisting of four members; ASIC1 to 4. These channels are sensitive to changes in pH and are expressed throughout the central and peripheral nervous systems-including brain, spinal cord, and sensory ganglia. They have been implicated in a number of neurological conditions such as stroke and cerebral ischemia, traumatic brain injury, and epilepsy, and more recently in migraine. Their expression within areas of interest in the brain in migraine, such as the hypothalamus and PAG, their demonstrated involvement in preclinical models of meningeal afferent signaling, and their role in cortical spreading depression (the electrophysiological correlate of migraine aura), has enhanced research interest into these channels as potential therapeutic targets in migraine. Migraine is a disorder with a paucity of both acute and preventive therapies available, in which at best 50% of patients respond to available medications, and these medications often have intolerable side effects. There is therefore a great need for therapeutic development for this disabling condition. This review will summarize the understanding of the structure and CNS expression of ASICs, the mechanisms for their potential role in nociception, recent work in migraine, and areas for future research and drug development.Item Targeting T Cells and a Novel Neurotrophic Factor to Treat Paclitaxel-induced Peripheral Neuropathy in Mice(August 2022) Sankaranarayanan, Ishwarya; Price, Theodore J; Rennaker II, Robert L; Dussor, Gregory; McIntyre Rodriguez, Christa; Burton, MichaelChemotherapy-induced peripheral neuropathy (CIPN) is a challenging condition to treat and arises due to the severe, dose-limiting toxicity of chemotherapeutic drugs such as paclitaxel. Thirty-70% of patients are affected by CIPN. Neurotoxicity associated with CIPN often results in damage to the peripheral nerves and dorsal root ganglia (DRG) and causes debilitating sensory deficits. Presently, there are no effective treatments to prevent or reduce CIPN. Here we sought to investigate two new approaches to treat CIPN. In the first approach we took advantage of immune cell interactions that occur in CIPN. Immune cells have been associated in the development and progression of the disease and disease resolution. We investigated the potential role of Inducible co- stimulatory molecule (ICOS) in the resolution of paclitaxel-induced peripheral neuropathy in mice. ICOS is an immune checkpoint molecule that is expressed on the surface of activated T cells and promotes proliferation and differentiation of T cells. In the second approach we investigated the potential antinociceptive effects of recombinant mouse Meteorin (rmMeteorin) using the paclitaxel-induced peripheral neuropathy model in male and female mice. Meteorin, a novel neurotrophic factor with an uncharacterized receptor, has been shown to mediate the reversal of neuropathic pain injured model. Our findings indicate that both approaches produce a robust antinociceptive effect in paclitaxel- induced peripheral neuropathy in mice. Modulating T cells into an anti-inflammatory phenotype, using ICOS, and using neurotrophic factors such as Meteorin could provide novel and effective therapeutics that are urgently needed for CIPN.Item The Antidiabetic Drug Metformin Prevents and Reverses Neuropathic Pain and Spinal Cord Microglial Activation in Male but not Female Mice(Academic Press Ltd- Elsevier Science Ltd, 2018-11-01) Inyang, Kufreobong E.; Szabo-Pardi, Thomas; Wentworth, Emma; McDougal, Timothy A.; Dussor, Gregory; Burton, Michael D.; Price, Theodore J.; 0000-0002-6971-6221 (Price, TJ); Dussor, Gregory; Price, Theodore J.; Inyang, Kufreobong E.; Szabo-Pardi, Thomas; Wentworth, Emma; McDougal, Timothy A.Metformin is a widely prescribed drug used in the treatment of type II diabetes. While the drug has many mechanisms of action, most of these converge on AMP activated protein kinase (AMPK), which metformin activates. AMPK is a multifunctional kinase that is a negative regulator of mechanistic target of rapamycin (mTOR) and mitogen activated protein kinase (MAPK) signaling. Activation of AMPK decreases the excitability of dorsal root ganglion neurons and AMPK activators are effective in reducing chronic pain in inflammatory, post-surgical and neuropathic rodent models. We have previously shown that metformin leads to an enduring resolution of neuropathic pain in the spared nerve injury (SNI) model in male mice and rats. The precise mechanism underlying this long-lasting effect is not known. We conducted experiments to investigate the effects of metformin on SNI-induced microglial activation, a process implicated in the maintenance of neuropathic pain that has recently been shown to be sexually dimorphic. We find that metformin is effective at inhibiting development of neuropathic pain when treatment is given around the time of injury and that metformin is likewise effective at reversing neuropathic mechanical hypersensitivity when treatment is initiation weeks after injury. This effect is linked to decreased Iba-1 staining in the dorsal horn, a marker of microglial activation. Importantly, these positive behavioral and microglia effects of metformin were only observed in male mice. We conclude that the neuropathic pain modifying effects of metformin are sex-specific supporting a differential role for microglial activation in male and female mice.