Robert M. Caudle - US grants

DESCRIPTION (Adapted from the Investigator's Abstract): Chronic or persistent pain is a major health problem in the United States, which results in medical care and productivity losses in the billions of dollars. Two symptoms of chronic or persistent pain, which result in the greatest number of complaints from sufferers, are allodynia (pain to normally non-painful stimuli) and hyperalgesia (enhanced sensation of pain in response to painful stimuli). Recent work with antagonists for the N-methyl-D-aspartate (NMDA) receptor demonstrates that a significant proportion of allodynia and hyperalgesia is mediated by spinal cord NMDA receptors. This work suggests that there is a functional change in the activity of the spinal cord NMDA receptors in response to persistent nociceptive stimuli. NMDA receptors are regulated by a number of factors, including phosphorylation. Phosphorylation of the receptors results in an increase in the activity of the receptors. The two subunits of the NMDA receptor (NR1 and NR2) have consensus sites for several kinases, and activity from some of these kinases in the spinal cord has been associated with allodynia and hyperalgesia. Thus, the major hypothesis of this project is that NMDA receptors are phosphorylated by persistent nociception. To test this hypothesis, however, the PI, who is a new investigator, needs to develop new techniques in his laboratory. Because of the need to develop new techniques, the scope of this application has been narrowed to testing the hypothesis that the NR1 subunit of the NMDA receptor is phosphorylated during persistent nociception. This small grant application will: 1) allow the PI to develop and test the specificity of immunoprecipitation techniques on NR1 subunits transfected into HEK-293 cells, 2) transfer these techniques to the study of NR1 subunits in vivo, and 3) evaluate changes in NR1 phosphorylation in response to nociceptive stimuli. A complete understanding of the dynamics of NMDA receptor phosphorylation in response to pain is the ultimate goal of this project. This knowledge should lead to valuable insights into spinal cord pain processing and, hopefully, to new effective therapies.

DESCRIPTION (provided by applicant): In a study conducted by the American Pain Society it was demonstrated that 9 percent of the United States population suffers from moderate to severe chronic pain. Current methods to treat chronic pain are in many instances not effective or produce a wide range of side effects that limit their utility. In this project we are going to take advantage of the recent discoveries that intrathecally administered cholera toxin blocks hyperalgesia and allodynia in rodent models of chronic pain, and that toxins can be directly targeted to nociceptive neurons in the spinal cord via the neurokinin 1 (NK1) receptor. We will conjugate the catalytic portion of cholera toxin to substance P in order to direct the cholera toxin to NK1 receptor expressing cells in the spinal cord. This conjugate will be tested for activity in NK1 expressing cell lines and in rodent models of chronic pain for its ability to stimulate cAMP production, suppress the expression of Gs g-proteins and to inhibit hyperalgesia and allodynia. The unique aspect of this conjugate is that cholera toxin will not kill the NK1 expressing cells in the spinal cord like saporin, diphtheria toxin and pseudomonas exotoxin, which have previously been used in NK1 receptor targeting strategies. Instead, cholera toxin will uncouple opioid receptors from Gs, thus enhancing their inhibitory actions, and will reduce the activity of Gs coupled receptor systems, which would further suppress nociceptive transmission. Thus, this project will be the first, to the best our knowledge, to produce a therapeutic effect by directly manipulating g-protein function in an identified population of neurons in vivo. If successful, this project will produce a novel agent for the control of pain and will open up an entire new dimension in therapeutics by making g-proteins in specific cells the pharmacological target.

DESCRIPTION (provided by applicant): Thousands of lives are devastated each year by spinal cord injury. A significant portion of the motor deficits and pain experienced by these patients could be prevented if therapies were devised to block posttrauma degradation of the surviving tissue. Trauma to the spinal cord initiates a cascade of biochemical events that exacerbate the injury. The extended damage leads to an additional decline in motor function and to sensory disturbances such as chronic pain. As part of these processes the opioid peptide dynorphin increases in concentration and is released in the spinal cord during the post-trauma period. Dynorphin was previously demonstrated to enhance N-methyl-D-aspartate (NMDA) receptor function to produce neuronal damage in the spinal cord. Thus, dynorphin is likely to be a significant participant in the post-insult degenerative events. This study will test the hypothesis that dynorphin binds to NR1 splice variants that do not possess the exon-5 coded region of the protein (NRla) to expose previously inactive or low activity NMDA receptors. A second hypothesis that will be tested is that dynorphin induces migration of internal stores of NMDA receptors to the plasma membrane. To test these hypotheses three aims are proposed. In the first aim Hek-293 cells will be transiently transfected with the 32 possible pairs of NR1 splice variants and NR2 subunits. The sensitivity of the various subunit combinations to dynorphin and NMDA induced excitotoxicity will be evaluated by measuring the release of lactate dehydrogenase into the culture media using a high throughput 96 well assay. In the second aim the combinations of NR1 and NR2 subunits that are most sensitive to dynorphin (determined from aim 1) will be examined using whole cell patch clamp and cell surface labeling techniques to determine how dynorphin influences the receptor's function. In the final aim the pairs of NR1 splice variants and NR2 subunits expressed in the spinal cord will be determined using co-immunoprecipitation, 2D-electrophoresis, immunohistochemistry and RT-PCR. These experiments will provide the subunit identity of the dynorphin enhanced NMDA receptors in the spinal cord and the mechanism for how dynorphin enhances NMDA receptor function. It is hoped that this information will provide a valuable pharmacological target for interrupting dynorphin's participation in post spinal trauma neurodegeneration.

DESCRIPTION (provided by applicant): Opioids are a mainstay of modern pain therapy, yet opioid abuse has a significant negative impact on the abuser and society. It is estimated that 21% of currently incarcerated individuals are in prison because of drug related crimes. The cost to incarcerate these individuals is tens of billions of dollars per year. Since only a small percentage of opioid use results in abuse it is likely that there are some distinguishing molecular factors that may predispose an individual to opioid abuse. This project will investigate morphine induced alterations in N-methyl-D-aspartate (NMDA) receptors as one potential mechanism for predisposition to opioid abuse. NMDA receptors are intimately entwined in the processes of tolerance, withdrawal and addiction to opioids. NMDA antagonists can suppress these adverse effects of opioids. However, a single dose of an opioid is insufficient to induce significant tolerance, withdrawal or addiction indicating that time is required for some change in the NMDA receptors or neuronal circuitry to occur in order to observe these sequelae. We have found that the NR1 and NR2 subunits of NMDA receptors are highly susceptible to alterations as a result of changes in neuronal activity and that these changes in NMDA receptors may persist for an extended period of time. The altered NMDA receptors have a profound effect on behavior. Thus we hypothesize that there are changes in the splicing of NR1 subunits and changes in NR2 subunits that are induced in various regions of the CNS by morphine or morphine withdrawal. We further hypothesize that some individuals may retain these altered or "disease state" NMDA receptors long after withdrawal of the opioid, which may result in prolonged tolerance or withdrawal signs and increased susceptibility to opioid abuse. In this project we will evaluate the effects of morphine and morphine withdrawal on NR1 splice variants and NR2 subunits in various regions of the rat CNS. We will also determine if these changes persist for up to 16 weeks following the withdrawal of morphine. The NR1 and NR2 proteins will be evaluated by western blots and two-dimensional western blots and the data will be correlated to behavior in two nociceptive assays. If the data indicate that some individuals retain the disease state NMDA receptors techniques could be devised to reverse the NMDA receptor changes in an attempt to reduce the abuse susceptibility of these individuals. PUBLIC HEALTH RELEVANCE: N-methyl-D-aspartate (NMDA) receptor antagonists suppress tolerance and addiction to opioids. This project will determine if morphine treatment alters NMDA receptor subunit expression within the CNS and determine if these changes persist in a subgroup of rats. These data could provide clues to novel treatments or novel methods to prevent opioid abuse.

Abstract Chronic pain is a significant public health problem that costs society billions of dollars per year and causes great suffering in countless individuals. Opioid-based medications are among the most prescribed for various forms of chronic pain contributing to the current opioid epidemic. Recently, cannabis and cannabinoid compounds (e.g., ?9-tetrahydrocannabinol (THC) and cannabidiol (CBD)) have been described as having pain-alleviating properties. While these cannabinoids, particularly the less psychoactive variant, CBD, may offer alternatives to opioid treatments for pain, few well-controlled studies demonstrate analgesic efficacy, especially for CBD. While it is still unclear if cannabinoids are good stand-alone options for treating pain, cannabinoids may act as useful opioid-sparing drugs, given the substantial overlap between opioid and cannabinoid receptors in reward- and pain-related pathways. Our proposed project will focus on a heuristic approach that incorporates fundamental pharmacology, novel operant behavioral assays of pain, and functional neuroimaging. The long-term goal of this research program is to establish novel approaches to treat chronic pain by maximizing analgesic efficacy and minimizing abuse liability. The objective of this proposal, which embodies the first step toward this long- term goal, is to determine how CBD modifies the effects of oxycodone (OXY), a commonly prescribed opioid analgesic, in the contexts of chronic pain and opioid self-administration. Our overarching hypothesis is that CBD and OXY will act synergistically to yield enhanced analgesic effects, and that CBD will attenuate the effects of pain on OXY self-administration. Two major specific aims will be investigated: (1) to determine how CBD interacts with OXY to reduce chronic operant pain behaviors; and (2) to determine the interacting effects of chronic pain, OXY self-administration, and CBD on analgesia, reinforcement, and dependence. We will assess the effects of preexisting pain on OXY self-administration, as well as the effects of preexisting OXY self- administration on pain. The latter goal is a particularly innovative aspect of this proposal. CBD-modulatory effects on pain and OXY self-administration will be evaluated under both conditions. Neuroimaging will be used across experiments to map and quantify changes in neural connectivity across reward and pain centers of the brain following the various drug treatments (CBD, OXY) and pain states (acute, chronic). Further, we will use clinically important and innovative pain-depressed behavioral assessments that accurately model pain in human subjects. The rationale for completing these studies is that by determining how CBD and OXY interact to affect pain and substance use, we will establish the necessary foundation for future efforts to develop effective analgesics with reduced abuse liability. We believe we are particularly well suited to undertake this project because we have a unified (and already collaborating) multidisciplinary team with complementary expertise in behavioral neuroscience, pharmacology, and neuroimaging.