Tatiana Yakovleva

Morphine Exacerbates HIV-1 Tat-Induced Cytokine Production in Astrocytes through Convergent Effects on [Ca2+]i, NF-κB Trafficking and Transcription

Astroglia are key cellular sites where opiate drug signals converge with the proinflammatory effects of HIV-1 Tat signals to exacerbate HIV encephalitis. Despite this understanding, the molecular sites of convergence driving opiate-accelerated neuropathogenesis have not been deciphered. We therefore explored potential points of interaction between the signaling pathways initiated by HIV-1 Tat and opioids in striatal astrocytes. Profiling studies screening 152 transcription factors indicated that the nuclear factor-kappa B (NF-κB) subunit, c-Rel, was a likely candidate for Tat or Tat plus opiate-induced increases in cytokine and chemokine production by astrocytes. Pretreatment with the NF-κB inhibitor parthenolide provided evidence that Tat±morphine-induced release of MCP-1, IL-6 and TNF-α by astrocytes is NF-κB dependent. The nuclear export inhibitor, leptomycin B, blocked the nucleocytoplasmic shuttling of NF-κB; causing p65 (RelA) accumulation in the nucleus, and significantly attenuated cytokine production in Tat±morphine exposed astrocytes. Similarly, chelating intracellular calcium ([Ca2+]i) blocked Tat±morphine-evoked MCP-1 and IL-6 release, while artificially increasing the concentration of extracellular Ca2+ reversed this effect. Taken together, these results demonstrate that: 1) exposure to Tat±morphine is sufficient to activate NF-κB and cytokine production, 2) the release of MCP-1 and IL-6 by Tat±morphine are highly Ca2+-dependent, while TNF-α appears to be less affected by the changes in [Ca2+]i, and 3) in the presence of Tat, exposure to opiates augments Tat-induced NF-κB activation and cytokine release through a Ca2+-dependent pathway.

PATHOBIOLOGY OF DYNORPHINS IN TRAUMA AND DISEASE

Dynorphins, endogenous opioid neuropeptides derived from the prodynorphin gene, are involved in a variety of normative physiologic functions including antinociception and neuroendocrine signaling, and may be protective to neurons and oligodendroglia via their opioid receptor-mediated effects. However, under experimental or pathophysiological conditions in which dynorphin levels are substantially elevated, these peptides are excitotoxic largely through actions at glutamate receptors. Because the excitotoxic actions of dynorphins require supraphysiological concentrations or prolonged tissue exposure, there has likely been little evolutionary pressure to ameliorate the maladaptive, non-opioid receptor mediated consequences of dynorphins. Thus, dynorphins can have protective and/or proapoptotic actions in neurons and glia, and the net effect may depend upon the distribution of receptors in a particular region and the amount of dynorphin released. Increased prodynorphin gene expression is observed in several disease states and disruptions in dynorphin processing can accompany pathophysiological situations. Aberrant processing may contribute to the net negative effects of dysregulated dynorphin production by tilting the balance towards dynorphin derivatives that are toxic to neurons and/or oligodendroglia. Evidence outlined in this review suggests that a variety of CNS pathologies alter dynorphin biogenesis. Such alterations are likely maladaptive and contribute to secondary injury and the pathogenesis of disease.

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. —Yakovleva, T., Bazov, I., Cebers, G., Marinova, Z., Hara, H., Ahmed, A., Vlaskovska, M., Johansson, B., Hochgeschwender, U., Singh, I. N., Bruce‐Keller, A. J., Hurd, Y. L., Kaneko, T., Terenius, L., Ekström, T. J., Hauser, K. F., Pickel, V. M., Bakalkin, G. Prodynorphin storage and processing in axon terminals and dendrites FASEB J. 20, E1430 –E1440 (2006)

Attenuation of pain and inflammation in adjuvant-induced arthritis by the proteasome inhibitor MG132.

OBJECTIVE In rheumatoid arthritis (RA), pain and joint destruction are initiated and propagated by the production of proinflammatory mediators. Synthesis of these mediators is regulated by the transcription factor NF-kappaB, which is controlled by the ubiquitin proteasome system (UPS). The present study explored the effects of the proteasome inhibitor MG132 on inflammation, pain, joint destruction, and expression of sensory neuropeptides as markers of neuronal response in a rat model of arthritis. METHODS Arthritis was induced in rats by injection of heat-killed Mycobacterium butyricum. Arthritis severity was scored, and nociception was evaluated by mechanical pressure applied to the hind paw. Joint destruction was assessed by radiologic and histologic analyses. NF-kappaB DNA-binding activity was analyzed by electromobility shift assay, and changes in the expression of the p50 NF-kappaB subunit and the proinflammatory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) were detected by immunohistochemistry. RESULTS Arthritic rats treated with MG132 demonstrated a marked reduction in inflammation, pain, and joint destruction. The elevated DNA-binding activity of the NF-kappaB/p50 homodimer and p50, as well as the neuronal expression of SP and CGRP, observed in the ankle joints of arthritic rats were normalized after treatment with MG132. CONCLUSION In arthritic rats, inhibition of proteasome reduced the severity of arthritis and reversed the pain behavior associated with joint inflammation. These effects may be mediated through the inhibition of NF-kappaB activation and may possibly involve the peripheral nervous system. New generations of nontoxic proteasome inhibitors may represent a novel pharmacotherapy for RA.

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.

Dynorphin A (1–17) induces apoptosis in striatal neurons in vitro through α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome C release and caspase-3 activation

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

p53 latency – out of the blind alley

A crucial mechanism by which p53 transcription function is regulated appears to operate through control of its DNA-binding properties. Allosteric/conformational and steric mechanisms of latency and activation of p53 DNA-binding function have been proposed. Both are inconsistent with recent data obtained by NMR and fluorescence correlation spectroscopy. This article presents the two-binding sites mechanism, which postulates that p53 is not a latent DNA-binding factor but is involved in multiple interactions with DNA via its core- and C-terminal domains, and that in the p53 tetramer–DNA complexes, binding of the C-terminal-domain to DNA prevents interactions of the core domain with target DNA elements but not with nonspecific DNA sequences. We also discuss whether the conformations assumed by p53 when bound to structurally diverse target DNAs are crucial determinants of interaction with co-activators and co-repressors of transcription.

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 endogenous opioid peptides in the dorsal striatum of human alcoholics

The endogenous opioid peptides dynorphins and enkephalins may be involved in brain-area specific synaptic adaptations relevant for different stages of an addiction cycle. We compared the levels of prodynorphin (PDYN) and proenkephalin (PENK) mRNAs (by qRT-PCR), and dynorphins and enkephalins (by radioimmunoassay) in the caudate nucleus and putamen between alcoholics and control subjects. We also evaluated whether PDYN promoter variant rs1997794 associated with alcoholism affects PDYN expression. Postmortem specimens obtained from 24 alcoholics and 26 controls were included in final statistical analysis. PDYN mRNA and Met-enkephalin-Arg-Phe, a marker of PENK were downregulated in the caudate of alcoholics, while PDYN mRNA and Leu-enkephalin-Arg, a marker of PDYN were decreased in the putamen of alcoholics carrying high risk rs1997794 C allele. Downregulation of opioid peptides in the dorsal striatum may contribute to development of alcoholism including changes in goal directed behavior and formation of a compulsive habit in alcoholics.

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.