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 5' 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 (eIF4E(S209A)). 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.

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<p> Osteoarthritis (OA) is the most common arthritis, and the second most common diagnosis leading to disability. While loss of joint function is disabling, patients report that the greatest disabler of OA is the pain. Unfortunately, OA pain remains an unmet medical need. Numerous mechanisms have been proposed for the pathogenesis of OA pain. However, none of these mechanisms has led to satisfactory evidence-based treatment for OA pain. There is a critical need to address the mechanisms for OA pain due to the aging demographics and the prevalence of OA in older adults. This thesis project was aimed to study neural mechanisms for OA pain. The general hypothesis was that the pain of OA arises as a result of phenotypic changes in primary sensory neurons, especially in larger diameter A-fiber neurons. In vivo intracellular recordings were used to determine changes in specific populations of DRG neuron in a surgical knee derangement model of OA in the rat. It was found that AB-fiber low threshold mechanoreceptors, particularly muscle spindle afferents underwent significant changes (including changes in action potential configurations and in responses to repetitive stimulation) one month following the model induction when histopathological changes of the knee joint and the nocifensive behaviors of the affected lower limb favor OA. Nociceptors, including C-, As- and AB-fiber neurons remained largely unchanged at one month OA. AB-fiber high threshold mechanoreceptors exhibited significant changes at two month OA, a later phase during the progression of OA. The data demonstrate that distinct populations of dorsal root ganglia neuron are altered during the progression of OA, which might be the neuronal basis for clinical presentations of sensory deficit in OA including pain and loss of proprioception. The data also suggest that the pain in OA might be a form of neuropathic pain. </p> / Thesis / Doctor of Philosophy (PhD)

Primary sensory axons fail to regenerate into the spinal cord following dorsal root injury leading to permanent sensory deficits. Re-entry is prevented at the dorsal root entry zone (DREZ), the CNS-PNS interface. Current approaches for promoting DR regeneration across the DREZ have had some success, but sustained, long-distance regeneration, particularly of large-diameter myelinated axons, still remains a formidable challenge. Our lab has previously shown that induced expression of constitutively active B-RAF (kaBRAF) enhanced the regenerative competence of injured DRG neurons in adult mice. In this study, I investigated whether robust intraspinal regeneration can be achieved by selective expression of kaBRAF alone or in combination with deletion of the myelin-associated inhibitors or neuron-intrinsic growth suppressors (PTEN or SOCS3). To this end, I used LSL-kaBRAF: brn3a-CreERT2 transgenic mice in which kaBRAF can be induced selectively in sensory neurons. I have also bred LSL-kaBRAF: brn3a-CreERT2 mice with triple knock-out mice lacking Nogo, Mag and OMgp or mouse lines carrying floxed alleles of PTEN or SOCS3. Single, double, and triple conditional mice were subjected to cervical DR crush and AAV2-eGFP vectors were used to selectively label regenerating axons of large-diameter neurons. I compared the extent of regeneration at 3 weeks or 2 months after DR injury using conventional anatomical and behavioral analyses. I found that kaBRAF alone promoted axon regeneration across the DREZ but did not produce significant functional recovery by two months. Supplementary deletion of Nogo, MAG, and OMgp did not improve kaBRAF-induced regeneration. Deletion of PTEN or SOCS3 individually or in combination failed to promote axon regeneration across the DREZ. In marked contrast, simultaneous deletion of PTEN, but not SOCS3, dramatically enhanced kaBRAF-mediated regeneration enabling many more axons to penetrate the DREZ and grow deep into the spinal cord. This study shows that dual activation of BRAF-MEK-ERK and PI3K-Akt signaling is an effective strategy to stimulate robust intraspinal DR regeneration and may lead to recovery of sensory function after DR injury. / Biomedical Sciences

Studies have suggested the possibility that there is sensory (SS) afferent signaling from white adipose tissue (WAT) to the brain, which may play an important role in communication with the brain sympathetic nervous system (SNS) outflow to WAT. Therefore, we tested whether the SNS-SS feedback loop between the subcutaneous inguinal WAT (IWAT) and the epididymal WAT (EWAT) exists. These fat pads were chosen due to 1) their divergent role in manifestation of metabolic disorders with the IWAT being beneficial and the EWAT being detrimental, as well as 2) different lipolytic response to glucoprivic 2-deoxyglucose (2DG). By using retrograde tract tracers Fast Blue (FB) and Fluorogold (FG), we found that the IWAT is more innervated than EWAT by both the SS and SNS ganglia (T13-L3). Surprisingly, we found ~12-17% of double-labeled cells in the SNS and SS ganglia innervating fat depots, implying SNS-SS crosstalk loops between the IWAT and EWAT. Increased neuronal activation by 2DG was observed in the SNS ganglia to both IWAT and EWAT but not in the SS dorsal root ganglia. In addition, 2DG induced lipolysis in both fat pads with greater lipolytic properties in the IWAT as a result of higher density of the SNS-SS fibers. Collectively, our results show neuroanatomical reality of the IWAT and EWAT SNS-SS neural crosstalk with a coordinated control of lipolytic function.

Macrophages and other cells of the innate immune system recognize foreign particles that could be potentially dangerous and respond by initiating an inflammatory response. The biologically active chemical mediators of this response called pro-inflammatory cytokines are produced in various myeloid derived immune cells and can affect other cells of the body. Interleukin-1β, a pro-inflammatory cytokine, has been shown to have direct effects on dorsal root ganglion (DRG) cell bodies including the upregulation and direct release of a nociceptive neurotransmitter called substance P (SP). Using a zymosan-induced model of systemic inflammation, we hypothesized that murine DRG neurons and the nerve processes associated with them in the dorsal horn of the spinal cord (SC) at the L1 level will show an upregulation of SP expression in response to inflammation in the peritoneum. Experimental mice were treated with a zymosan suspension (500mg/kg, intraperitoneal injection), and control mice received sterile filtered solution (intraperitoneal injection). Both DRG and SC specimens were collected after in situ fixation and subjected to immunofluorescence staining to label SP. Using confocal microscopy, fluorescence microscopy, and image analysis software this expression of SP was quantified and compared. In both tissue specimen groups, an increase in SP expression was discovered in zymosan treated mice. The exact cause of this increase was not specifically determined in this experiment. This experiment provided valuable insight about how a systemic inflammatory response can affect sensory nerve function. Successful methods for further experimentation were identified and information about the zymosan model of inflammation was obtained

Several factors contribute to the failure of the central nervous system (CNS) to regenerate after injury. These include inhibition of axonal growth by myelin and glial scar associated molecules, as well as the intrinsic inability of adult CNS neurons to grow long axons in environments that are permissive for younger neurons. Neurons in the peripheral nervous system (PNS) display a much higher capacity to regenerate after injury than CNS neurons, as shown by conditioning lesion experiments and by microtransplantation of dorsal root ganglia neurons into CNS white matter tracts. Our central hypothesis is that neurons of the PNS express specific regeneration associated genes that mediate their enhanced growth response after injury. We have employed a combination of subtractive hybridization, microarray comparison and promoter analysis to probe for genes specific to neurons of the dorsal root ganglia (DRG), using cerebellar granule neurons (CGN) as a reference. We have identified over a thousand different genes, many of whose products form interaction networks and signaling pathways. Moreover, we have identified several dozen transcription factors that may play a role in establishing DRG neuron identity and shape their responses after injury. One of these transcription factors is Signal Transducer and Activator of Transcription 3 (STAT3), previously known to be upregulated in the PNS after a conditioning lesion but not known to be specific to the PNS. Using a real time PCR and immunochemical approaches we have shown that STAT3 is constitutively expressed and selectively active in DRG neurons both in culture and in vivo. We show that the overexpression of wild type STAT3 in cerebellar granule neurons leads to the formation of supernumerary neurites, whereas the overexpression of constitutively active STAT3-C leads to a 20% increase in total neurite outgrowth. It is hoped that the genetic delivery of STAT3-C, potentially combined with co-activators of transcription, will improve functional regeneration of CNS axons in vivo.

The structure of afferent neurons is pseudounipolar. Studies suggest that they relay action potentials (APs) to both directions of the T-junctions to reach the cell body and the spinal cord. Moreover, the somata are electrically excitable and shown to be able to transmit the signals to associated satellite cells. Our study demonstrates that this transmission can go further and pass onto passive neighbouring somata, if they are in direct contact with same satellite cells. The neurons activate the satellite cells by releasing ATP. This triggers the satellite cells to exocytose acetylcholine to the neighbouring neurons. In addition, the ATP inhibits the nicotinic receptors of the neurons by activating P2Y receptors and initiating the G-protein-mediated pathway, thus reducing the signals that return to the neurons that initiated the signals. This “sandwich synapse” represents a unique pathway by the ectopic release between the somata and the satellite cells.

The structure of afferent neurons is pseudounipolar. Studies suggest that they relay action potentials (APs) to both directions of the T-junctions to reach the cell body and the spinal cord. Moreover, the somata are electrically excitable and shown to be able to transmit the signals to associated satellite cells. Our study demonstrates that this transmission can go further and pass onto passive neighbouring somata, if they are in direct contact with same satellite cells. The neurons activate the satellite cells by releasing ATP. This triggers the satellite cells to exocytose acetylcholine to the neighbouring neurons. In addition, the ATP inhibits the nicotinic receptors of the neurons by activating P2Y receptors and initiating the G-protein-mediated pathway, thus reducing the signals that return to the neurons that initiated the signals. This “sandwich synapse” represents a unique pathway by the ectopic release between the somata and the satellite cells.