Investigating Mechanisms Regulating the In Vivo Actions of Delta Opioid Receptor Ligands

Chronic pain and depression are widespread and debilitating diseases that, for many people, cannot be adequately addressed with current treatment options. Delta opioid receptor (DOR) agonists have been proposed as novel treatments for these disorders. DOR is a member of the opioid receptor family of G protein-coupled receptors (GPCRs). DOR signals through inhibitory Gαi/o proteins that are negatively regulated by regulator of G protein signaling (RGS) proteins. Activation of DOR induces antihyperalgesia and antidepressant-like effects in animal models without the constipation, respiratory depression, and abuse liability associated with mu opioid receptor agonists such as morphine. Unfortunately, some DOR agonists cause convulsions, hindering their development as therapeutics in humans. The experiments described in this thesis sought to further characterize the intracellular signaling pathways and mechanisms underlying DOR-mediated behaviors. Specifically, these studies used a number of mouse models to determine differences in the regulation of DOR-mediated convulsions relative to the antihyperalgesic and antidepressant-like effects of DOR agonists. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. Antidepressant-like effects were evaluated in the forced swim and tail suspension tests. Mice were also observed for convulsive activity post-agonist treatment. To assess the role of G protein signaling in DOR-mediated behaviors, we compared behaviors induced by the DOR agonist SNC80 in RGS4 knockout, Gαo RGS-insensitive (RGSi) knock-in, and Gαo knockout mice. SNC80-induced antihyperalgesia was enhanced in RGS4 knockout and Gαo RGSi mice. SNC80-induced antidepressant-like effects were also potentiated in both RGS4 knockout and Gαo RGSi mice. However, SNC80-induced convulsions were not changed in either strain. In Gαo heterozygous knockout mice, SNC80-induced antihyperalgesia was abolished while the antidepressant-like effects and convulsions were unaltered. Taken together, these data demonstrate that DOR-mediated antihyperalgesia and antidepressant-like effects, but not convulsions, are regulated by Gαo and RGS4. To further characterize the pharmacological properties mediating behavioral outcomes of DOR agonists, we compared the behavioral effects of SNC80 with those of the DOR partial agonist BU48. BU48 produced convulsions with similar potency to SNC80. BU48 also produced antidepressant-like effects with reduced potency relative to SNC80 and failed to elicit antihyperalgesia. These results suggest that the efficacy requirement for DOR-mediated convulsions may be low relative to other DOR-mediated behaviors. The efficacy requirements for DOR-mediated behaviors were further evaluated by comparing the shifts in the SNC80 dose response curves for each of these behaviors following decreases in DOR receptor reserve. Decreases in receptor reserve were produced using DOR heterozygous knockout mice as well as by treating mice with the DOR irreversible antagonist naltrindole-5’-isothiocyanate (5’-NTII). SNC80-induced antihyperalgesia displayed the largest potency shift in DOR heterozygous and 5’-NTII treated mice. Antidepressant-like effects displayed the next largest shift followed by convulsions. These findings suggest that DOR-mediated behaviors display the following rank order of efficacy requirement: convulsions < antidepressant like effects < antihyperalgesia. Furthermore, the DOR competitive antagonist naltrindole differentially shifted the SNC80 dose response curves of these behaviors, suggesting that different DOR receptor populations may mediate these behaviors. Overall, the work presented in this thesis suggests that DOR-mediated behaviors are generated by distinct signaling mechanisms and receptor populations. The possibility of pharmacologically targeting receptor populations or signaling pathways responsible for DOR-mediated analgesic and antidepressant-like effects without activating receptors that mediated convulsions should greatly aid the clinical viability of DOR agonists.PHDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138549/1/drippsis_1.pd

Investigating the relationship between repeated DNA and gene silencing in the model organism Neurospora crassa

Abstract only availableIn order to maintain genomic integrity, organisms must possess the capability to combat unwanted elements such as retroviruses and transposons. In the filamentous fungus Neurospora crassa, the expression from repeated genetic elements is turned off, or silenced, in a post-transcriptional manner. It is thus possible to induce gene silencing in N. crassa by inserting tandem copies of a transgene into its genome. However, the number and arrangement of transgenes required to activate this process, known as quelling, is unclear. Additionally, silenced strains tend to revert to an unsilenced phenotype after several generations. With the goal of creating a genetically-defined and stably quelled strain, we are examining the effect of various tandem transgene repeats on N. crassa. We have designed plasmid vectors containing 1 to 11 ~1kb fragments of the N. crassa caratenoid pigment producing gene, albino-1 (or al-1). Depending on the number of these constructs in a transformant, the al-1 gene could remain unsilenced (orange conidia), or be partially to completely silenced (light orange to white conidia). Transformants are currently being assayed for their spore color as well as the presence or absence of the transgene repeats. Although this work is still in progress, we have identified a transformant with a putative 5-repeat al-1 transgene that displays a slight quelling phenotype (yellow-light orange conidia). These data suggest that quelling requires the introduction of at least 5 copies of a transgene.NSF-REU Program in Biological Sciences & Biochemistr

Tolerance to highâ internalizing δ opioid receptor agonist is critically mediated by arrestin 2

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/144602/1/bph14353.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/144602/2/bph14353_am.pd

Gene Network Dysregulation in the Trigeminal Ganglia and Nucleus Accumbens of a Model of Chronic Migraine-Associated Hyperalgesia

The pharmacological agent nitroglycerin (NTG) elicits hyperalgesia and allodynia in mice. This model has been used to study the neurological disorder of trigeminovascular pain or migraine, a debilitating form of hyperalgesia. The present study validates hyperalgesia in an established mouse model of chronic migraine triggered by NTG and advances the understanding of the associated molecular mechanisms. The RNA-seq profiles of two nervous system regions associated with pain, the trigeminal ganglia (TG) and the nucleus accumbens (NAc), were compared in mice receiving chronic NTG treatment relative to control (CON) mice. Among the 109 genes that exhibited an NTG treatment-by-region interaction, solute carrier family 32 (GABA vesicular transporter) member 1 (Slc32a1) and preproenkephalin (Penk) exhibited reversal of expression patterns between the NTG and CON groups. Erb-b2 receptor tyrosine kinase 4 (Erbb4) and solute carrier family 1 (glial high affinity glutamate transporter) member 2 (Slc1a2) exhibited consistent differential expression between treatments across regions albeit at different magnitude. Period circadian clock 1 (Per1) was among the 165 genes that exhibited significant NTG treatment effect. Biological processes disrupted by NTG in a region-specific manner included adaptive and innate immune responses; whereas glutamatergic and dopaminergic synapses and rhythmic process were disrupted in both regions. Regulatory network reconstruction highlighted the widespread role of several transcription factors (including Snrnp70, Smad1, Pax6, Cebpa, and Smpx) among the NTG-disrupted target genes. These results advance the understanding of the molecular mechanisms of hyperalgesia that can be applied to therapies to ameliorate chronic pain and migraine

Differential medication overuse risk of novel anti-migraine therapeutics

Medication overuse headache is estimated to affect 2% of the population, and is ranked in the top 20 most disabling disorders due to its high level of disability. Several therapies used in the treatment of acute migraine are thought to be associated with medication overuse headache, including opioids and triptans. With limited treatment options, it is critical to determine the risk profile of novel therapies prior to their widespread use. The current study explores the potential medication overuse risk of two novel therapeutic drug classes, namely the ditans: 5-HT(1F) receptor agonists, and the gepants: calcitonin gene-related peptide receptor antagonists, in a preclinical model of medication overuse. Persistent exposure of mice to the 5-HT(1F) agonist LY344864, but not olcegepant produced a significant reduction in hind paw and orofacial mechanical withdrawal thresholds as a surrogate readout of allodynia. In agreement, only LY344864 induced neuroplastic changes in trigeminal sensory afferents, increasing calcitonin gene-related peptide expression and basal trigeminal nociception. Our data highlight a differential medication overuse headache risk profile for the ditan and gepant classes of drugs that has important implications for their clinical use and patient education to help reduce the burden of medication overuse headache