In the United States, there are currently more than 40 million postmenopausal women. These women are faced with a variety of physiological changes including ovarian steroid withdrawal and alterations in hypothalamic neurons. Within the hypothalamic infundibular nucleus of postmenopausal women, there is neuronal hypertrophy and an increase in neurokinin B gene expression. Recent studies identified the kisspeptins and dynorphins as major regulators of reproduction. In our first experiment, we examined the location and alterations of KiSS-1 mRNA-expressing neurons in the hypothalami of pre and postmenopausal women. KiSS-1 neurons were largely confined to the infundibular nucleus, and in postmenopausal women, exhibited neuronal hypertrophy and increased gene expression. To determine if these changes could result from alterations in ovarian steroids, we investigated KiSS-1 gene expression in the hypothalamic infundibular nucleus of non-human primates. Similar to the findings in postmenopausal women, ovariectomy of monkeys resulted in neuronal hypertrophy and increased KiSS-1 gene expression within the infundibular nucleus. Further, estrogen treatment of ovariectomized monkeys yielded a dramatic decrease in KiSS-1 gene expression. Together, these findings suggest that the postmenopausal alterations in KiSS-1 neurons are secondary to ovarian failure.In a second study, we examined alterations in dynorphin gene expression in the hypothalami of pre and postmenopausal women. Dynorphin mRNA-expressing neurons were identified in multiple nuclei. Numbers of dynorphin neurons were decreased within the mPOA and infundibular nucleus of postmenopausal women. In the infundibular nucleus of postmenopausal women, dynorphin neurons were hypertrophied. To determine the contribution of ovarian steroids on dynorphin gene expression, we examined dynorphin mRNA in a monkey model of menopause. Young ovariectomized monkeys exhibited hypertrophy of dynorphin neurons, with no changes in dynorphin gene expression. Estrogen replacement yielded a decrease in neuronal size and an increase in dynorphin neuron number.In future studies, we will use Quantum Dot FISH to determine if NKB, KiSS-1, and dynorphin are colocalized in the hypertrophied neurons. These neuropeptides are involved in the regulation of GnRH and changes in their gene expression likely contribute to postmenopausal alterations in reproductive hormones. Our findings provide greater understanding of the postmenopausal condition and offer opportunities for pharmaceutical investigation and treatment.

The structural requirements for the binding of dynorphin to the kappa opioid receptor are of profound clinical interest in the search for a powerful non-addictive analgesic. These requirements are thought to be met by the membrane-mediated conformation of the opioid peptide dynorphin A-(1-13}, Tyr¹-Gly²-Gly³-Phe⁴-Leu⁵-Arg⁶-Arg⁷-Ile⁸-Arg⁹-Pro¹⁰-Lys¹¹-Leu¹²-Lys¹³. Schwyzer [𝘉𝘪𝘰𝘤𝘩𝘦𝘮𝘪𝘴𝘵𝘳𝘺 25: 4281-4286 (1986)] has proposed an essentially α-helical membrane-mediated conformation of the tridecapeptide. In the present study, the hydrophobic moment, the helix probability and a four -state secondary structure prediction were computed. They signified, in agreement with circular dichroism (CD) studies on phospholipid-bound dynorphin A-(1-13)-tridecapeptide, negligible helical content of the peptide. CD studies demonstrated that the aqueous-membraneous interphase can be mimicked by methanol. The 500 and 620 MHz ¹H nuclear magnetic resonance (NMR) spectra of dynorphin A-(1-13) in methanolic solution were sequence-specifically assigned with the aid of correlated spectroscopy (COSY), double-quantum filtered phase-sensitive COSY, relayed COSY (RELAY) and nuclear Overhauser enhancement spectroscopy (NOESY). 2-D CAMELSPIN/ROESY experiments indicated that at least the part of the molecule from Arg⁷ to Arg⁹ was in an extended or β-strand conformation, which was in line with deuterium exchange and temperature dependence studies of the amide protons. ¹³C_α spin-lattice relaxation rate constants indicated a non-rigid backbone conformation. Transferred nuclear Overhauser effect studies on aqueous systems containing dynorphin A-(1-13) in the presence of dimyristoyl-phosphatidylcholine bilayers indicated a folded conformation from Tyr¹ to Leu⁵. The findings were incorporated into a tentative molecular model, which also indicated a non-helical, non-extended conformation for the rest of the molecule in the presence of corresponding distance-restrained negative charges. / Thesis / Master of Science (MSc)

Dynorphin has a well-established role in feeding and gustation. Alterations in taste perception and feeding behavior are common with age. We hypothesized that proDynorphin gene expression in brain areas involved in taste and feeding declines with age. Male Sprague-Dawley rats were housed individually with ad libitum access to food and water. Brain punches of the selected regions were dissected out in groups of rats aged 4-6, 12-14 and 18-21 months. ProDynorphin mRNA (measured using a cDNA probe) decreased significantly with age in arcuate nucleus and amygdala; increased significantly with age in hippocampus; and was not significantly affected in nucleus of the solitary tract, cortex, caudate putamen or hypothalamic paraventricular nucleus. These data suggest an age-related decrease in the synthesis of dynorphin in two brain regions strongly associated with feeding behavior, and an increase in dynorphin synthesis in a brain region associated with learning and memory.

Neuropeptides are special in their expression profiles restricted to neuronal subpopulations and low tissue mRNA levels. Genetic, epigenetic and transcriptional mechanisms that define spatiotemporal expression of the neuropeptide genes have utmost importance for the formation and functions of neural circuits in normal and pathological human brain. This thesis focuses on regulation of transcription of the opioid/nociceptin genes, the largest neuropeptide family, and on identification of adaptive changes in these mechanisms associated with alcoholism as model human pathology. Two epigenetic mechanisms, the common for most cells in the dorsolateral prefrontal cortex (dlPFC) and the neuron-subpopulation specific that may orchestrate prodynorphin (PDYN) transcription in the human dlPFC have been uncovered. The first, repressive mechanism may operate through control of DNA methylation/demethylation in a short, nucleosome size promoter CpG island (CGI). The second mechanism may involve USF2, the sequence–specific methylation–sensitive transcription factor which interaction with its target element in the CpG island results in USF2 and PDYN co-expression in the same neurons. The short PDYN promoter CGI may function as a chromatin element that integrates cellular and environmental signals through changes in methylation and transcription factor binding. Alterations in USF2–dependent PDYN transcription are affected by the promoter SNP (rs1997794: T>C) under transition to pathological state, i.e. in the alcoholic brain. This and two other PDYN SNPs that are most significantly associated with alcoholism represent CpG-SNPs, which are differentially methylated in the human dlPFC. The T, low risk allele of the promoter SNP forms a noncanonical AP-1–binding element. JUND and FOSB proteins, which may form homo- or heterodimers have been identified as dominant constituents of AP-1 complex. The C, non-risk variant of the PDYN 3′ UTR SNP (rs2235749 SNP: C>T) demonstrated significantly higher methylation in alcoholics compared to controls. PDYN mRNA and dynorphin levels significantly and positively correlated with methylation of the PDYN 3′ UTR CpG-SNP suggesting its involvement in PDYN regulation. A DNA–binding factor with differential binding affinity for the T allele and methylated and unmethylated C alleles of the PDYN 3′ UTR SNP (the T allele specific binding factor, Ta-BF) has been discovered, which may function as a regulator of PDYN transcription. These findings emphasize the complexity of PDYN regulation that determines its expression in specific neuronal subpopulations and suggest previously unknown integration of epigenetic, transcriptional and genetic mechanisms that orchestrate alcohol–induced molecular adaptations in the human brain. Given the important role of PDYN in addictive behavior, the findings provide a new insight into fundamental molecular mechanisms of human brain disorder. In addition to PDYN in the dlPFC, the PNOC gene in the hippocampus and OPRL1 gene in central amygdala that were downregulated in alcoholics may contribute to impairment of cognitive control over alcohol seeking and taking behaviour.

Neuropathic pain is a prevalent disease with no effective, safe treatments and limited knowledge on the mechanisms involved. One target for neuropathic pain treatment may be the blockade of Dynorphin A (Dyn A). Dyn A is a unique endogenous ligand that possesses well-known neuroinhibitory effects via opioid receptors and neuroexcitatory effects that are mediated through the bradykinin 2 receptors (B2Rs). Extensive SAR was carried out to develop a ligand for the blockade of the excitatory actions of Dyn A at the B2R. A lead ligand was able to block Dyn A-induced hyperalgesia in naïve animals and was effective in a neuropathic pain model. However, the ligand was susceptible to enzymatic degradation. In an effort to increase the stability, modifications of the ligand using non-natural amino acids were performed. Analogues substituted at or near the N-terminus with a D-isomer retained binding at the receptor as well as provided a large increase in stability. These ligands were also found to be non-toxic in a cell toxicity assay. Dyn A has been found to not activate the classical signaling of the B2R, PI hydrolysis or Ca²⁺ mobilization. In an effort to determine Dyn A's signaling, a study was done examining up-regulation of phosphorylated proteins. It was found that Dyn A did not activate; pERK, 7 PKC isoforms or PKA. A well known B2R antagonist, HOE140, was found to have low affinity at rat and guinea pig brain B2Rs but high affinity in the guinea pig ileum. Further examination revealed that this discrepancy in binding may arise from a different isoform of the B2R that has not been previously examined. To date, we have discovered Dyn A analogues that have high affinity for the B2R, are very stable, and have low toxicity. The signaling pathway is still not fully understood, but further studies are underway. Also, there is evidence that the B2R in which the analogues are interacting at may be a different form than what has previously been described. Targeting this different isoform of the B2R with our current stable ligands may provide beneficial therapeutics for the treatment of neuropathic pain without the cardiovascular liabilities.

The neuropeptides dynorphin and cholecystokinin (CCK), and their associated pronociceptive effects were investigated in the RVM. Utilizing a nerve-injury model (SNL), RT-PCR analysis revealed increases (p<0.05) of prodynorphin mRNA, and bradyinkin, B1- and B2-receptor mRNA, post-SNL, 14-days, 2-days, and 14-days, respectively. Administration of dynorphin into the RVM produced both acute and long-lasting (>30-days) tactile hypersensitivity. Administration of the B1-antagonist, DALBK and the B2-antagonist, Hoe-140, into the RVM significantly attenuated dynorphin-induced tactile hypersensitivity. Nerve-injury induced tactile hypersensitivity was significantly reversed by RVM administration of dynorphin antiserum or the B2-antagonist, Hoe-140. These data suggest that dynorphin is up-regulated in the RVM in nerve-injury, and via the activation of bradykinin receptors in the RVM, produces abnormal pain. Like dynorphin, CCK is up-regulated in the RVM in nerve-injury, with studies suggesting that elevated levels of CCK in the RVM mediate pronociceptive activity through CCK2 receptor activation, resulting in enhanced spinal nociceptive transmission. At present, it is unknown what key neurotransmitters are mediating this RVM CCK-driven effect at the level of the spinal cord. Here, spinal cerebrospinal fluid (CSF) levels of serotonin (5-HT) and prostaglandin E2 (PGE2) were measured in the lumbar spinal cord in naïve rats following CCK administration into the RVM. Following RVM CCK microinjection, an approximate 5-fold increase in spinal (CSF) PGE2 levels was observed, as compared to baseline controls. PGE2 levels showed a progressive increase with peak levels observed at the 80-minute post-CCK injection timepoint, whereas 5-HT levels in the spinal CSF remained unchanged following CCK administration into the RVM. This release of PGE2 coincided with the timecourse for CCK-induced mechanical hypersensitivity. Administration of the CCK2-antagonist YM022 prior to CCK into the RVM, significantly attenuated (>50%) the release of PGE2 in the spinal cord. The non-selective COX-inhibitor naproxen and the 5-HT3 antagonist ondansetron, both administered intrathecally, significantly attenuated RVM CCK-induced hindpaw tactile hypersensitivity. In summary, these data suggest a bradykinin- or CCK2-receptor antagonist could be used alone or in conjunction with current therapies in the treatment of chronic pain.

Dynorphins, endogenous peptide neurotransmitters expressed in the central nervous system, have been implicated in diverse pathophysiological processes, including excitotoxicity, chronic inflammation, traumatic injury, cognitive impairment, and motor dysfunction, with significant changes with aging or age-related disease processes. This has led to the hypothesis that the suppression of dynorphin expression would produce beneficial effects on learning and memory and motor function. To assess the phenotypic manifestations of chronic suppression of endogenous dynorphin, knockout (KO) mice lacking the coding exons of the gene encoding the prodynorphin (Pdyn) precursor protein, were tested in a series of behavioral, biochemical, and molecular biological studies. Moderately aged Pdyn KO perform comparatively better than similarly aged wild-type (WT) mice in the water maze task, although no Pdyn effect was seen among young adult mice. In addition, young adult Pdyn KO mice show mildly improved performance on a passive avoidance task. Minimal baseline differences were noted in spontaneous locomotor activity in an open-field assay, but Pdyn deletion produced a relative sparing of motor dysfunction induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To investigate the relationship between aging and brain dynorphin expression in mice, we examined dynorphin peptide levels at varying ages in hippocampus, striatum, and frontal cortex of WT mice by quantitative radioimmunoassay. While aging produces progressive decline in Dyn B in striatum and frontal cortex, Dyn A shows an upward trend in frontal cortex without significant change in striatum. Systemic MPTP produces significant short-term elevations in dynorphin peptides that regress to below baseline by 7 days. HPLC analysis of striatal dopamine shows an age-dependent increase in basal dopamine levels in Pdyn KO mice, an effect that is abolished after MPTP. Western blotting experiments demonstrate that Pdyn deletion is associated with greater phosphorylation at the serine-40 site of tyrosine hydroxylase (TH) despite relatively less total TH immunoreactivity, suggesting a suppressive effect of dynorphins on dopamine synthesis. Microarray analysis of hippocampal tissue from young and aged WT and Pdyn KO mice reveals a number of functional groups of genes demonstrating altered expression. The results of this dissertation support a role of endogenous dynorphins in age-associated cognitive and motor dysfunction.

The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA. The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event. With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge. Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.</p>

Lactation is the final stage of reproduction in mammals and is characterized by chronically elevated prolactin and suppressed luteinizing hormone. The neuroendocrine regulation of prolactin and luteinizing hormone during lactation are not fully understood. In the hypothalamic arcuate nucleus is a population of neurons known as KNDy neurons because they co-express the neuropeptides Kisspeptin, Neurokinin B and Dynorphin. These neurons are known to project to gonadotropin-releasing hormone cell bodes in the preoptic area and nerve terminals in the median eminence, which regulate the secretion of luteinizing hormone, and to dopaminergic tuberoinfundibular neurons in the arcuate nucleus, which are known to regulate prolactin. Because KNDy neurons project to neuronal populations known to regulate both prolactin and luteinizing hormone, the general hypothesis for these studies is that neuropeptides Kisspeptin, Neurokinin B and Dynorphin play a role in regulating these hormones or are regulated by these hormones during lactation. In a model of lactating rats deprived of their pups for 24 hours, intracerebroventricular injection of an endogenous Kisspeptin receptor ligand, Kp-10, modestly increased prolactin secretion and markedly increased luteinizing hormone secretion. Neither Neurokinin B nor the Neurokinin B receptor agonist, Senktide, had a significant effect on either hormone in this rat model. Dynorphin and U-50,488, a kappa opioid receptor agonist, robustly increased prolactin although no changes were measured in luteinizing hormone levels. In this model of 24-hour pup-deprived lactating rats, prolactin was responsive to kappa opioid receptor agonists and luteinizing hormone was responsive to Kisspeptin receptor agonists. In a second set of experiments, sense and anti-sense in situ hybridization probes were developed for Kiss1, the Kisspeptin gene, and Tac2, the gene encoding Neurokinin B. It was confirmed that the cDNA sequences cloned for these mRNAs were correct and were highly homologous to published rat mRNA sequences. In situ hybridization was performed using the Kiss1 and Tac2 probes, as well as a probe for Pdyn, which encodes Dynorphin. No specific cytoplasmic signal was observed using any of the three sense probes. With the anti-sense probes, clusters of reduced silver grains representing Kiss1, Tac2 and Pdyn mRNAs were observed in the arcuate nucleus, lateral to the third ventricle and superior to the median eminence. These expression patterns were consistent with the published literature. Also, the expression patterns for all three neuropeptides were similar to each other, suggesting that many of the arcuate nucleus neurons lateral to the third ventricle and superior to the median eminence are KNDy neurons.

No description available.