Epigenetic and Transcriptional Control of the Opioid Prodynorphine Gene: In-Depth Analysis in the Human Brain.

Neuropeptides serve as neurohormones and local paracrine regulators that control neural networks regulating behavior, endocrine system and sensorimotor functions. Their expression is characterized by exceptionally restricted profiles. Circuit-specific and adaptive expression of neuropeptide genes may be defined by transcriptional and epigenetic mechanisms controlled by cell type and subtype sequence-specific transcription factors, insulators and silencers. The opioid peptide dynorphins play a critical role in neurological and psychiatric disorders, pain processing and stress, while their mutations cause profound neurodegeneration in the human brain. In this review, we focus on the prodynorphin gene as a model for the in-depth epigenetic and transcriptional analysis of expression of the neuropeptide genes. Prodynorphin studies may provide a framework for analysis of mechanisms relevant for regulation of neuropeptide genes in normal and pathological human brain.

Neuronal Expression of Opioid Gene is Controlled by Dual Epigenetic and Transcriptional Mechanism in Human Brain.

Molecular mechanisms that define patterns of neuropeptide expression are essential for the formation and rewiring of neural circuits. The prodynorphin gene (PDYN) gives rise to dynorphin opioid peptides mediating depression and substance dependence. We here demonstrated that PDYN is expressed in neurons in human dorsolateral prefrontal cortex (dlPFC), and identified neuronal differentially methylated region in PDYN locus framed by CCCTC-binding factor binding sites. A short, nucleosome size human-specific promoter CpG island (CGI), a core of this region may serve as a regulatory module, which is hypomethylated in neurons, enriched in 5-hydroxymethylcytosine, and targeted by USF2, a methylation-sensitive E-box transcription factor (TF). USF2 activates PDYN transcription in model systems, and binds to nonmethylated CGI in dlPFC. USF2 and PDYN expression is correlated, and USF2 and PDYN proteins are co-localized in dlPFC. Segregation of activatory TF and repressive CGI methylation may ensure contrasting PDYN expression in neurons and glia in human brain.

Intra- and interregional coregulation of opioid genes: broken symmetry in spinal circuits.

Regulation of the formation and rewiring of neural circuits by neuropeptides may require coordinated production of these signaling molecules and their receptors that may be established at the transcriptional level. Here, we address this hypothesis by comparing absolute expression levels of opioid peptides with their receptors, the largest neuropeptide family, and by characterizing coexpression (transcriptionally coordinated) patterns of these genes. We demonstrated that expression patterns of opioid genes highly correlate within and across functionally and anatomically different areas. Opioid peptide genes, compared with their receptor genes, are transcribed at much greater absolute levels, which suggests formation of a neuropeptide cloud that covers the receptor-expressed circuits. Surprisingly, we found that both expression levels and the proportion of opioid receptors are strongly lateralized in the spinal cord, interregional coexpression patterns are side specific, and intraregional coexpression profiles are affected differently by left- and right-side unilateral body injury. We propose that opioid genes are regulated as interconnected components of the same molecular system distributed between distinct anatomic regions. The striking feature of this system is its asymmetric coexpression patterns, which suggest side-specific regulation of selective neural circuits by opioid neurohormones.-Kononenko, O., Galatenko, V., Andersson, M., Bazov, I., Watanabe, H., Zhou, X. W., Iatsyshyna, A., Mityakina, I., Yakovleva, T., Sarkisyan, D., Ponomarev, I., Krishtal, O., Marklund, N., Tonevitsky, A., Adkins, D. L., Bakalkin, G. Intra- and interregional coregulation of opioid genes: broken symmetry in spinal circuits.

Opioid precursor protein isoform is targeted to the cell nuclei in the human brain.

BACKGROUND: Neuropeptide precursors are traditionally viewed as proteins giving rise to small neuropeptide molecules. Prodynorphin (PDYN) is the precursor protein to dynorphins, endogenous ligands for the κ-opioid receptor. Alternative mRNA splicing of neuropeptide genes may regulate cell- and tissue-specific neuropeptide expression and produce novel protein isoforms. We here searched for novel PDYN mRNA and their protein product in the human brain. METHODS: Novel PDYN transcripts were identified using nested PCR amplification of oligo(dT) selected full-length capped mRNA. Gene expression was analyzed by qRT-PCR, PDYN protein by western blotting and confocal imaging, dynorphin peptides by radioimmunoassay. Neuronal nuclei were isolated using fluorescence-activated nuclei sorting (FANS) from postmortem human striatal tissue. Immunofluorescence staining and confocal microscopy was performed for human caudate nucleus. RESULTS: Two novel human PDYN mRNA splicing variants were identified. Expression of one of them was confined to the striatum where its levels constituted up to 30% of total PDYN mRNA. This transcript may be translated into ∆SP-PDYN protein lacking 13 N-terminal amino acids, a fragment of signal peptide (SP). ∆SP-PDYN was not processed to mature dynorphins and surprisingly, was targeted to the cell nuclei in a model cellular system. The endogenous PDYN protein was identified in the cell nuclei in human striatum by western blotting of isolated neuronal nuclei, and by confocal imaging. CONCLUSIONS AND GENERAL SIGNIFICANCE: High levels of alternatively spliced ∆SP-PDYN mRNA and nuclear localization of PDYN protein suggests a nuclear function for this isoform of the opioid peptide precursor in human striatum.

PDYN, a gene implicated in brain/mental disorders, is targeted by REST in the adult human brain.

The dynorphin κ-opioid receptor system is implicated in mental health and brain/mental disorders. However, despite accumulating evidence that PDYN and/or dynorphin peptide expression is altered in the brain of individuals with brain/mental disorders, little is known about transcriptional control of PDYN in humans. In the present study, we show that PDYN is targeted by the transcription factor REST in human neuroblastoma SH-SY5Y cells and that that interfering with REST activity increases PDYN expression in these cells. We also show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Our results suggest that REST represses PDYN expression in SH-SY5Y cells and the adult human brain and may have implications for mental health and brain/mental disorders.

Identification and validation of a blood- based diagnostic lipidomic signature of pediatric inflammatory bowel disease.

Improved biomarkers are needed for pediatric inflammatory bowel disease. Here we identify a diagnostic lipidomic signature for pediatric inflammatory bowel disease by analyzing blood samples from a discovery cohort of incident treatment-naïve pediatric patients and validating findings in an independent inception cohort. The lipidomic signature comprising of only lactosyl ceramide (d18:1/16:0) and phosphatidylcholine (18:0p/22:6) improves the diagnostic prediction compared with high-sensitivity C-reactive protein. Adding high-sensitivity C-reactive protein to the signature does not improve its performance. In patients providing a stool sample, the diagnostic performance of the lipidomic signature and fecal calprotectin, a marker of gastrointestinal inflammation, does not substantially differ. Upon investigation in a third pediatric cohort, the findings of increased lactosyl ceramide (d18:1/16:0) and decreased phosphatidylcholine (18:0p/22:6) absolute concentrations are confirmed. Translation of the lipidomic signature into a scalable diagnostic blood test for pediatric inflammatory bowel disease has the potential to support clinical decision making.

Prodynorphin mutations cause the neurodegenerative disorder spinocerebellar ataxia type 23.

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (∼0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.

Association of the PDYN gene with alcohol dependence and the propensity to drink in negative emotional states.

Synthetic κ-opioid receptor (KOR) agonists induce dysphoric and pro-depressive effects and variations in the KOR (OPRK1) and prodynorphin (PDYN) genes have been shown to be associated with alcohol dependence. We genotyped 23 single nucleotide polymorphisms (SNPs) in the PDYN and OPRK1 genes in 816 alcohol-dependent subjects and investigated their association with: (1) negative craving measured by a subscale of the Inventory of Drug Taking Situations; (2) a self-reported history of depression; (3) the intensity of depressive symptoms measured by the Beck Depression Inventory-II. In addition, 13 of the 23 PDYN and OPRK1 SNPs, which were previously genotyped in a set of 1248 controls, were used to evaluate association with alcohol dependence. SNP and haplotype tests of association were performed. Analysis of a haplotype spanning the PDYN gene (rs6045784, rs910080, rs2235751, rs2281285) revealed significant association with alcohol dependence (p = 0.00079) and with negative craving (p = 0.0499). A candidate haplotype containing the PDYN rs2281285-rs1997794 SNPs that was previously associated with alcohol dependence was also associated with negative craving (p = 0.024) and alcohol dependence (p = 0.0008) in this study. A trend for association between depression severity and PDYN variation was detected. No associations of OPRK1 gene variation with alcohol dependence or other studied phenotypes were found. These findings support the hypothesis that sequence variation in the PDYN gene contributes to both alcohol dependence and the induction of negative craving in alcohol-dependent subjects.

The endogenous opioid system in human alcoholics: molecular adaptations in brain areas involved in cognitive control of addiction.

The endogenous opioid system (EOS) plays a critical role in addictive processes. Molecular dysregulations in this system may be specific for different stages of addiction cycle and neurocircuitries involved and therefore may differentially contribute to the initiation and maintenance of addiction. Here we evaluated whether the EOS is altered in brain areas involved in cognitive control of addiction including the dorsolateral prefrontal cortex (dl-PFC), orbitofrontal cortex (OFC) and hippocampus in human alcohol-dependent subjects. Levels of EOS mRNAs were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and levels of dynorphins by radioimmunoassay (RIA) in post-mortem specimens obtained from 14 alcoholics and 14 controls. Prodynorphin mRNA and dynorphins in dl-PFC, κ-opioid receptor mRNA in OFC and dynorphins in hippocampus were up-regulated in alcoholics. No significant changes in expression of proenkephalin, and µ- and δ-opioid receptors were evident; pro-opiomelanocortin mRNA levels were below the detection limit. Activation of the κ-opioid receptor by up-regulated dynorphins in alcoholics may underlie in part neurocognitive dysfunctions relevant for addiction and disrupted inhibitory control.

Transcriptional control of maladaptive and protective responses in alcoholics: a role of the NF-κB system.

Alcohol dependence and associated cognitive impairment appear to result from maladaptive neuroplasticity in response to chronic alcohol consumption, neuroinflammation and neurodegeneration. The inherent stability of behavioral alterations associated with the addicted state suggests that transcriptional and epigenetic mechanisms are operative. NF-κB transcription factors are regulators of synaptic plasticity and inflammation, and responsive to a variety of stimuli including alcohol. These factors are abundant in the brain where they have diverse functions that depend on the composition of the NF-κB complex and cellular context. In neuron cell bodies, NF-κB is constitutively active, and involved in neuronal injury and neuroprotection. However, at the synapse, NF-κB is present in a latent form and upon activation is transported to the cell nucleus. In glia, NF-κB is inducible and regulates inflammatory processes that exacerbate alcohol-induced neurodegeneration. Animal studies demonstrate that acute alcohol exposure transiently activates NF-κB, which induces neuroinflammatory responses and neurodegeneration. Postmortem studies of brains of human alcoholics suggest that repeated cycles of alcohol consumption and withdrawal cause adaptive changes in the NF-κB system that may permit the system to better tolerate excessive stimulation. This type of tolerance, ensuring a low degree of responsiveness to applied stimuli, apparently differs from that in the immune system, and may represent a compensatory response that protects brain cells against alcohol neurotoxicity. This view is supported by findings showing preferential downregulation of pro-apoptotic gene expression in the affected brain areas in human alcoholics. Although further verification is needed, we speculate that NF-κB-driven neuroinflammation and disruption to neuroplasticity play a significant role in regulating alcohol dependence and cognitive impairment.

Prodynorphin CpG-SNPs associated with alcohol dependence: elevated methylation in the brain of human alcoholics.

The genetic, epigenetic and environmental factors may influence the risk for neuropsychiatric disease through their effects on gene transcription. Mechanistically, these effects may be integrated through regulation of methylation of CpG dinucleotides overlapping with single-nucleotide polymorphisms (SNPs) associated with a disorder. We addressed this hypothesis by analyzing methylation of prodynorphin (PDYN) CpG-SNPs associated with alcohol dependence, in human alcoholics. Postmortem specimens of the dorsolateral prefrontal cortex (dl-PFC) involved in cognitive control of addictive behavior were obtained from 14 alcohol-dependent and 14 control subjects. Methylation was measured by pyrosequencing after bisulfite treatment of DNA. DNA binding proteins were analyzed by electromobility shift assay. Three PDYN CpG-SNPs associated with alcoholism were found to be differently methylated in the human brain. In the dl-PFC of alcoholics, methylation levels of the C, non-risk variant of 3'-untranslated region (3'-UTR) SNP (rs2235749; C > T) were increased, and positively correlated with dynorphins. A DNA-binding factor that differentially targeted the T, risk allele and methylated and unmethylated C allele of this SNP was identified in the brain. The findings suggest a causal link between alcoholism-associated PDYN 3'-UTR CpG-SNP methylation, activation of PDYN transcription and vulnerability of individuals with the C, non-risk allele(s) to develop alcohol dependence.

Left-right side-specific endocrine signaling complements neural pathways to mediate acute asymmetric effects of brain injury.

Brain injuries can interrupt descending neural pathways that convey motor commands from the cortex to spinal motoneurons. Here, we demonstrate that a unilateral injury of the hindlimb sensorimotor cortex of rats with completely transected thoracic spinal cord produces hindlimb postural asymmetry with contralateral flexion and asymmetric hindlimb withdrawal reflexes within 3 hr, as well as asymmetry in gene expression patterns in the lumbar spinal cord. The injury-induced postural effects were abolished by hypophysectomy and were mimicked by transfusion of serum from animals with brain injury. Administration of the pituitary neurohormones ß-endorphin or Arg-vasopressin-induced side-specific hindlimb responses in naive animals, while antagonists of the opioid and vasopressin receptors blocked hindlimb postural asymmetry in rats with brain injury. Thus, in addition to the well-established involvement of motor pathways descending from the brain to spinal circuits, the side-specific humoral signaling may also add to postural and reflex asymmetries seen after brain injury.

Brain trauma or a stroke often lead to severe problems in posture and movement. These injuries frequently occur only on one side, causing asymmetrical motor changes: damage to the left brain hemisphere triggers abnormal contractions of the right limbs, and vice-versa. The injuries can disrupt neural tracts between the brain and the spinal cord, the structure that conveys electric messages to muscles. However, research has also shed light on new actors: the hormones released into the bloodstream by the pituitary gland. Similar to the effects of brain lesions, several of these molecules cause asymmetric posture in healthy rats. In fact, a group of hormones can trigger muscle contraction of the left back leg, and another of the right one. Could pituitary hormones mediate the asymmetric effects of brain injuries? To investigate this question, Lukoyanov, Watanabe, Carvalho, Kononenko, Sarkisyan et al. focused on rats in which the connection between the brain and the spinal cord segments that control the hindlimbs had been surgically removed. This stopped transmission of electric messages from the brain to muscles in the back legs. Strikingly, lesions on one side of the brain in these animals still led to asymmetric posture, with contraction of the leg on the opposite side of the body. These effects were abolished when the pituitary gland was excised. Postural asymmetry also emerged when blood serum from injured rats was injected into healthy animals. The findings suggest that hormones play an essential role in signalling from the brain to the spinal cord. Further experiments identified that two pituitary hormones, ß-endorphin and Arg-vasopressin, induced contraction of the right but not the left hindlimb of healthy animals. In addition, small molecules that inhibit these hormones could block the deficits seen on the right side after an injury on the left hemisphere of the brain. Taken together, these results show that neurons in the spinal cord are not just controlled by the neural tracts that descend from the brain, but also by hormones which have left-right side-specific actions. This unique signalling could be a part of a previously unknown hormonal mechanism that selectively targets either the left or the right side of the body. This knowledge could help to design side-specific treatments for stroke and brain trauma.

Left-Right Side-Specific Neuropeptide Mechanism Mediates Contralateral Responses to a Unilateral Brain Injury.

Neuropeptides are implicated in control of lateralized processes in the brain. A unilateral brain injury (UBI) causes the contralesional sensorimotor deficits. To examine whether opioid neuropeptides mediate UBI induced asymmetric processes we compared effects of opioid antagonists on the contralesional and ipsilesional hindlimb responses to the left-sided and right-sided injury in rats. UBI induced hindlimb postural asymmetry (HL-PA) with the contralesional hindlimb flexion, and activated contralesional withdrawal reflex of extensor digitorum longus (EDL) evoked by electrical stimulation and recorded with EMG technique. No effects on the interossei (Int) and peroneaus longus (PL) were evident. The general opioid antagonist naloxone blocked postural effects, did not change EDL asymmetry while uncovered cryptic asymmetry in the PL and Int reflexes induced by UBI. Thus, the spinal opioid system may either mediate or counteract the injury effects. Strikingly, effects of selective opioid antagonists were the injury side-specific. The µ-antagonist ß-funaltrexamine (FNA) and κ-antagonist nor-binaltorphimine (BNI) reduced postural asymmetry after the right but not left UBI. In contrast, the δ-antagonist naltrindole (NTI) inhibited HL-PA after the left but not right-side brain injury. The opioid gene expression and opioid peptides were lateralized in the lumbar spinal cord, and coordination between expression of the opioid and neuroplasticity-related genes was impaired by UBI that together may underlie the side-specific effects of the antagonists. We suggest that mirror-symmetric neural circuits that mediate effects of left and right brain injury on the contralesional hindlimbs are differentially controlled by the lateralized opioid system.

Control of chronic pain by the ubiquitin proteasome system in the spinal cord.

Chronic pain is maintained in part by long-lasting neuroplastic changes in synapses and several proteins critical for synaptic plasticity are degraded by the ubiquitin-proteasome system (UPS). Here, we show that proteasome inhibitors administered intrathecally or subcutaneously prevented the development and reversed nerve injury-induced pain behavior. They also blocked pathological pain induced by sustained administration of morphine or spinal injection of dynorphin A, an endogenous mediator of chronic pain. Proteasome inhibitors blocked mechanical allodynia and thermal hyperalgesia in all three pain models although they did not modify responses to mechanical stimuli, but partially inhibited responses to thermal stimuli in control rats. In the spinal cord, these compounds abolished the enhanced capsaicin-evoked calcitonin gene-related peptide (CGRP) release and dynorphin A upregulation, both elicited by nerve injury. Model experiments demonstrated that the inhibitors may act directly on dynorphin-producing cells, blocking dynorphin secretion. Thus, the effects of proteasome inhibitors on chronic pain were apparently mediated through several cellular mechanisms indispensable for chronic pain, including those of dynorphin A release and postsynaptic actions, and of CGRP secretion. Levels of several UPS proteins were reduced in animals with neuropathic pain, suggesting that UPS downregulation, like effects of proteasome inhibitors, counteracts the development of chronic pain. The inhibitors did not produce marked or disabling motor disturbances at doses that were used to modify chronic pain. These results suggest that the UPS is a critical intracellular regulator of pathological pain, and that UPS-mediated protein degradation is required for maintenance of chronic pain and nociceptive, but not non-nociceptive responses in normal animals.

Downregulation of the neuronal opioid gene expression concomitantly with neuronal decline in dorsolateral prefrontal cortex of human alcoholics.

Molecular changes in cortical areas of addicted brain may underlie cognitive impairment and loss of control over intake of addictive substances and alcohol. Prodynorphin (PDYN) gives rise to dynorphin (DYNs) opioid peptides which target kappa-opioid receptor (KOR). DYNs mediate alcohol-induced impairment of learning and memory, while KOR antagonists block excessive, compulsive-like drug and alcohol self-administration in animal models. In human brain, the DYN/KOR system may undergo adaptive changes, which along with neuronal loss, may contribute to alcohol-associated cognitive deficit. We addressed this hypothesis by comparing the expression levels and co-expression (transcriptionally coordinated) patterns of PDYN and KOR (OPRK1) genes in dorsolateral prefrontal cortex (dlPFC) between human alcoholics and controls. Postmortem brain specimens of 53 alcoholics and 55 controls were analyzed. PDYN was found to be downregulated in dlPFC of alcoholics, while OPRK1 transcription was not altered. PDYN downregulation was confined to subgroup of subjects carrying C, a high-risk allele of PDYN promoter SNP rs1997794 associated with alcoholism. Changes in PDYN expression did not depend on the decline in neuronal proportion in alcoholics, and thereby may be attributed to transcriptional adaptations in alcoholic brain. Absolute expression levels of PDYN were lower compared to those of OPRK1, suggesting that PDYN expression is a limiting factor in the DYN/KOR signaling, and that the PDYN downregulation diminishes efficacy of DYN/KOR signaling in dlPFC of human alcoholics. The overall outcome of the DYN/KOR downregulation may be disinhibition of neurotransmission, which when overactivated could contribute to formation of alcohol-related behavior.

Dynorphin and κ-Opioid Receptor Dysregulation in the Dopaminergic Reward System of Human Alcoholics.

Molecular changes induced by excessive alcohol consumption may underlie formation of dysphoric state during acute and protracted alcohol withdrawal which leads to craving and relapse. A main molecular addiction hypothesis is that the upregulation of the dynorphin (DYN)/κ-opioid receptor (KOR) system in the nucleus accumbens (NAc) of alcohol-dependent individuals causes the imbalance in activity of D1- and D2 dopamine receptor (DR) expressing neural circuits that results in dysphoria. We here analyzed post-mortem NAc samples of human alcoholics to assess changes in prodynorphin (PDYN) and KOR (OPRK1) gene expression and co-expression (transcriptionally coordinated) patterns. To address alterations in D1- and D2-receptor circuits, we studied the regulatory interactions between these pathways and the DYN/KOR system. No significant differences in PDYN and OPRK1 gene expression levels between alcoholics and controls were evident. However, PDYN and OPRK1 showed transcriptionally coordinated pattern that was significantly different between alcoholics and controls. A downregulation of DRD1 but not DRD2 expression was seen in alcoholics. Expression of DRD1 and DRD2 strongly correlated with that of PDYN and OPRK1 suggesting high levels of transcriptional coordination between these gene clusters. The differences in expression and co-expression patterns were not due to the decline in neuronal proportion in alcoholic brain and thereby represent transcriptional phenomena. Dysregulation of DYN/KOR system and dopamine signaling through both alterations in co-expression patterns of opioid genes and decreased DRD1 gene expression may contribute to imbalance in the activity of D1- and D2-containing pathways which may lead to the negative affective state in human alcoholics.

Differential effects of left and right neuropathy on opioid gene expression in lumbar spinal cord.

The endogenous opioid system (EOS) controls the processing of nociceptive stimuli and is a pharmacological target for opioids. Alterations in expression of the EOS genes under neuropathic pain condition may account for low efficacy of opioid drugs. We here examined whether EOS expression patterns are altered in the lumbar spinal cord of the rats with spinal nerve ligation (SNL) as a neuropathic pain model. Effects of the left- and right-side SNL on expression of EOS genes in the ipsi- and contralateral spinal domains were analysed. The SNL-induced changes were complex and different between the genes; between the dorsal and ventral spinal domains; and between the left and right sides of the spinal cord. Prodynorphin (Pdyn) expression was upregulated in the ipsilateral dorsal domains by each the left and right-side SNL, while changes in expression of µ-opioid receptor (Oprm1) and proenkephalin (Penk) genes were dependent on the SNL side. Changes in expression of the Pdyn and κ-opioid receptor (Oprk1) genes were coordinated between the ipsi- and contralateral sides. Withdrawal response thresholds, indicators of mechanical allodynia correlated negatively with Pdyn expression in the right ventral domain after right side SNL. These findings suggest multiple roles of the EOS gene products in spinal sensitization and changes in motor reflexes, which may differ between the left and right sides.

Prodynorphin storage and processing in axon terminals and dendrites.

The classical view postulates that neuropeptide precursors in neurons are processed into mature neuropeptides in the somatic trans-Golgi network (TGN) and in secretory vesicles during axonal transport. Here we show that prodynorphin (PDYN), precursor to dynorphin opioid peptides, is predominantly located in axon terminals and dendrites in hippocampal and striatal neurons. The molar content of unprocessed PDYN was much greater than that of dynorphin peptides in axon terminals of PDYN-containing neurons projecting to the CA3 region of the hippocampus and in the striatal projections to the ventral tegmental area. Electron microscopy showed coexistence of PDYN and dynorphins in the same axon terminals with occasional codistribution in individual dense core vesicles. Thus, the precursor protein is apparently stored at presynaptic sites. In comparison with the hippocampus and striatum, PDYN and dynorphins were more equally distributed between neuronal somata and processes in the amygdala and cerebral cortex, suggesting regional differences in the regulation of trafficking and processing of the precursor protein. Potassium-induced depolarization activated PDYN processing and secretion of opioid peptides in neuronal cultures and in a model cell line. Regulation of PDYN storage and processing at synapses by neuronal activity or extracellular stimuli may provide a local mechanism for regulation of synaptic transmission.

Prodynorphin transcripts and proteins differentially expressed and regulated in the adult human brain.

Transcription from multiple promoters along with alternative mRNA splicing constitutes the basis for cell-specific gene expression and mRNA and protein diversity. The prodynorphin gene (PDYN) gives rise to prodynorphin (PDYN), precursor to dynorphin opioid peptides that regulate diverse physiological functions and are implicated in various neuropsychiatric disorders. Here, we characterized PDYN transcripts and proteins in the adult human brain and studied PDYN processing and intracellular localization in model cell lines. Seven PDYN mRNAs were identified in the human brain; two of the transcripts, FL1 and FL2, encode the full-length PDYN. The dominant, FL1 transcript shows high expression in limbic-related structures such as the nucleus accumbens and amygdala. The second, FL2 transcript is only expressed in few brain structures such as the claustrum and hypothalamus. FL-PDYN was identified for the first time in the brain as the dominant PDYN protein product. Three novel PDYNs expressed from spliced or truncated PDYN transcripts either lack a central segment but are still processed into dynorphins, or are translated into N-terminally truncated proteins. One truncated PDYN is located in the cell nucleus, suggesting a novel nonopioid function for this protein. The complexity of PDYN expression and diversity of its protein products may be relevant for diverse levels of plasticity in adaptive responses for the dynorphin system.