Indiana University-Purdue University Indianapolis (IUPUI) / Synaptic transmission is coordinated by a litany of protein-protein interactions that rely on the proper localization and function of pre- and post-synaptic Ca2+ channels. The axonal guidance/specification collapsin response mediator protein-2 (CRMP-2) was identified as a potential partner of the pre-synaptic N-type voltage-gated Ca2+ channel (CaV2.2). CRMP-2 bound directly to CaV2.2 in two regions; the channel domain I-II intracellular loop and the distal C-terminus. Both proteins co-localized within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to EGFP caused a significant increase in Ca2+ channel current density whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2–overexpressing neurons. Both activity- and CRMP-2-phosphoryation altered the interaction between CaV2.2 and CRMP-2. I identified a CRMP-2-derived peptide (called CBD3) that bound CaV2.2 and effectively disrupted the interaction between CaV2.2 and CRMP-2. CBD3 peptide fused to the HIV TAT protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, and reversed neuropathic hypersensitivity produced by an antiretroviral drug. Unchecked Ca2+ influx via N-methyl-D-aspartate receptors (NMDARs) has been linked to activation of neurotoxic cascades culminating in cell death (i.e. excitotoxicity). CRMP-2 was suggested to affect NMDAR trafficking and possibly involved in neuronal survival following excitotoxicity. Based upon these studies, I hypothesized that a peptide from CRMP2 could preserve neurons in the face of excitotoxic challenges. Lentiviral–mediated CRMP2 knockdown or treatment with TAT-CBD3 blocked neuronal death following glutamate exposure likely via blunting toxicity from NMDAR-mediated delayed calcium deregulation. TAT-CBD3 induced internalization of the NMDAR subunit NR2B in dendritic spines without altering somal surface expression. TAT-CBD3 reduced NMDA-mediated Ca2+-influx and currents in cultured neurons. The presented work validates CRMP-2 as a novel modulator of pre- and post-synaptic Ca2+ channels and provides evidence that the TAT-CBD3 peptide could be useful as a potential therapeutic for both chronic neuropathic pain and excitotoxicity following stroke or other neuronal insults.

Indiana University-Purdue University Indianapolis (IUPUI) / Aromatase inhibitors (AIs) are the current standard of care for the treatment of hormone receptor positive breast cancer in postmenopausal women. Nearly one-half of patients receiving AI therapy develop musculoskeletal toxicity that is characterized by joint and/or muscle pain and approximately one-fourth of patients discontinue their therapy as a result of musculoskeletal pain. Since there are no effective strategies for prevention or treatment, insight into the mechanisms of AI-induced pain is critical to improve treatment. However, there are few studies of AI effects in animal models of nociception. To determine whether AIs produce hypersensitivity in animal models of pain, I examined the effects of AI administration on mechanical, thermal, and chemical sensitivity in rats. The results demonstrate that (1) repeated injection of 5 mg/kg letrozole in male rats produces mechanical, but not thermal, hypersensitivity that extinguishes when drug dosing is stopped; (2) administering a single dose of 1 or 5 mg/kg letrozole in ovariectomized (OVX) rats also induces mechanical hypersensitivity, without altering thermal sensitivity and (3) a single dose of 5 mg/kg letrozole or daily dosing of letrozole or exemestane in male rats augments flinching behavior induced by intraplantar ATP injection. To determine whether the effects of AIs on nociceptive behaviors are mediated by activation or sensitization of peptidergic sensory neurons, I determined whether letrozole exposure alters release of calcitonin gene-related peptide (CGRP) from isolated rat sensory neurons and from sensory nerve endings in rat spinal cord slices. No changes in basal, capsaicin-evoked or high extracellular potassium-evoked CGRP release were observed in sensory neuronal cultures acutely or chronically exposed to letrozole. Furthermore, letrozole exposure did not alter the ability of ATP to augment CGRP release from sensory neurons in culture. Finally, chronic letrozole treatment did not augment neuropeptide release from spinal cord slices. Taken together, these results do not support altered release of this neuropeptide into the spinal cord as mediator of letrozole-induced mechanical hypersensitivity and suggest the involvement of other mechanisms. Results from this dissertation provide a new experimental model for AI-induced hypersensitivity that could be beneficial in delineating mechanisms mediating pain during AI therapy.