Tatjana Yakovleva

NF-κB-like factors in the murine brain. Developmentally-regulated and tissue-specific expression

Abstract NF-κB and related factors are important transducers of external signals to the cell nucleus. They are abundant in the brain, where they may be significant for the regulation of gene transcription in plasticity-related processes for instance, via activation of protein kinase C. The subunit composition and levels of these factors in the mouse and rat brain and other tissues, using an assay based on gel retardation of the oligonucleotides corresponding to the κB DNA-element, are reported here. Three major κB-binding factors were observed. Factors I and II were activated by the dissociating agent deoxycholate. DNA protein cross-linking and antibody neutralization experiments suggest that factor I is a heterodimer of c-Rel and p65; factor II is a heterodimer of p50 and p65 (authentic NF-κB), and of p50 and c-Rel; factor III is the p50 homodimer (KBF1). All three factors were generally expressed in the 17-day-old rat embryo and 5-day-old pup, whereas in the adult rat, expression was more limited and showed certain tissue specificity. Factor II was the most generally expressed and the only factor observed in adult brain. Factor I was only detected in the adult testis whereas factor III was observed in the adult spleen and, in small amounts, in the liver and lung. Two minor κB-specific factors (A and B), distinctive to the brain and spleen, respectively, showed very slow gel mobility. Their estimated molecular weights were about 125 kDa and 95 kDa, respectively. Expression of factor A was stable in the rat brain during development. Factor A may be identical to a previously described brain-specific factor, BETA (Korner et al., Neuron , 3 (1989) 563–572). Thus, the expression pattern of κB-binding activities is apparently developmentally regulated and tissue-specific particularly in the adult. In the adult mouse and rat brain, only factors II (probably NF-κB and p50/c-Rel heterodimer) and A (probably BETA) could be observed.

Effects of the Circadian Rhythm Gene Period 1 (Per1) on Psychosocial Stress-Induced Alcohol Drinking

OBJECTIVE Circadian and stress-response systems mediate environmental changes that affect alcohol drinking. Psychosocial stress is an environmental risk factor for alcohol abuse. Circadian rhythm gene period 1 (Per1) is targeted by stress hormones and is transcriptionally activated in corticotropin releasing factor-expressing cells. The authors hypothesized that Per1 is involved in integrating stress response and circadian rhythmicity and explored its relevance to alcohol drinking. METHOD In mice, the effects of stress on ethanol intake in mPer1-mutant and wild-type mice were assessed. In humans, single nucleotide polymorphisms (SNPs) in hPer1 were tested for association with alcohol drinking behavior in 273 adolescents and an adult case-control sample of 1,006 alcohol-dependent patients and 1,178 comparison subjects. In vitro experiments were conducted to measure genotype-specific expression and transcription factor binding to hPer1. RESULTS The mPer1-mutant mice showed enhanced alcohol consumption in response to social defeat stress relative to their wild-type littermates. An association with the frequency of heavy drinking in adolescents with the hPer1 promoter SNP rs3027172 and with psychosocial adversity was found. There was significant interaction between the rs3027172 genotype and psychosocial adversity on this drinking measure. In a confirmatory analysis, association of hPer1 rs3027172 with alcohol dependence was shown. Cortisol-induced transcriptional activation of hPer1 was reduced in human B-lymphoblastoid cells carrying the risk genotype of rs3027172. Binding affinity of the transcription factor Snail1 to the risk allele of the hPer1 SNP rs3027172 was also reduced. CONCLUSIONS The findings indicate that the hPer1 gene regulates alcohol drinking behavior during stressful conditions and provide evidence for underlying neurobiological mechanisms.

Study of molecular events in cells by fluorescence correlation spectroscopy

Abstract.To understand processes in a living cell, sophisticated and creative approaches are required that can be used for gathering quantitative information about large number of components interacting across temporal and spatial scales without major disruption of the integral network of processes. A physical method of analysis that can meet these requirements is fluorescence correlation spectroscopy (FCS), which is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution. Since its introduction about 3 decades ago, this until recently emerging technology has reached maturity. As commercially built equipment is now available, FCS is extensively applied for extracting biological information from living cells unattainable by other methods, and new biological concepts are formulated based on findings by FCS. In this review, we focus on examples in the field of molecular cellular biology. The versatility of the technique in this field is illustrated in studies of single-molecule dynamics and conformational flexibility of proteins, and the relevance of conformational flexibility for biological functions regarding the multispecificity of antibodies, modulation of activity of C5a receptors in clathrin-mediated endocytosis and multiplicity of functional responses mediated by the p53 tumor suppressor protein; quantitative characterization of physicochemical properties of the cellular interior; protein trafficking; and ligand-receptor interactions. FCS can also be used to study cell-to-cell communication, here exemplified by clustering of apoptotic cells via bystander killing by hydrogen peroxide.

Neuroadaptations in human chronic alcoholics : dysregulation of the NF-κB system

Background Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-κB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics. Methods and Findings Analysis of DNA-binding of NF-κB (p65/p50 heterodimer) and the p50 homodimer as well as NF-κB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant κB binding factor in analyzed tissues. NF-κB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-κB target DNA sites, κB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with κB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex. Conclusions We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-κB, when repeated over years downregulate RELA expression and NF-κB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of κB regulated genes. Alterations in expression of p50 homodimer/NF-κB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.

Big dynorphin as a putative endogenous ligand for the κ‐opioid receptor

The diversity of peptide ligands for a particular receptor may provide a greater dynamic range of functional responses, while maintaining selectivity in receptor activation. Dynorphin A (Dyn A), and dynorphin B (Dyn B) are endogenous opioid peptides that activate the κ‐opioid receptor (KOR). Here, we characterized interactions of big dynorphin (Big Dyn), a 32‐amino acid prodynorphin‐derived peptide consisting of Dyn A and Dyn B, with human KOR, μ‐ (hMOR) and δ‐ (hDOR) opioid receptors and opioid receptor‐like receptor 1 (hORL1) expressed in cells transfected with respective cDNA. Big Dyn and Dyn A demonstrated roughly similar affinity for binding to hKOR that was higher than that of Dyn B. Dyn A was more selective for hKOR over hMOR, hDOR and hORL1 than Big Dyn, while Dyn B demonstrated low selectivity. In contrast, Big Dyn activated G proteins through KOR with much greater potency, efficacy and selectivity than other dynorphins. There was no correlation between the rank order of the potency for the KOR‐mediated activation of G proteins and the binding affinity of dynorphins for KOR. The rank of the selectivity for the activation of G proteins through hKOR and of the binding to this receptor also differed. Immunoreactive Big Dyn was detected using the combination of radioimmunoassay (RIA) and HPLC in the human nucleus accumbens, caudate nucleus, hippocampus and cerebrospinal fluid (CSF) with the ratio of Big Dyn and Dyn B being approximately 1 : 3. The presence in the brain implies that Big Dyn, along with other dynorphins, is processed from prodynorphin and secreted from neurons. Collectively, the high potency and efficacy and the relative abundance suggest that Big Dyn may play a role in the KOR‐mediated activation of G proteins.

Dysregulation of dynorphins in Alzheimer disease

The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.

Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation

Intrathecal (i.t.) administration into mice of N‐ethylmaleimide (NEM), a cysteine protease inhibitor, produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank. The behavior induced by NEM was inhibited by the intraperitoneal injection of morphine. We have recently reported that dynorphin A and, more potently big dynorphin, consisting of dynorphins A and B, produce the same type of nociceptive response whereas dynorphin B does not [Tan‐No K, Esashi A, Nakagawasai O, Niijima F, Tadano T, Sakurada C, Sakurada T, Bakalkin G, Terenius L, Kisara K. Intrathecally administered big dynorphin, a prodynorphin‐derived peptide, produces nociceptive behavior through an N‐methyl‐D‐aspartate receptor mechanism. Brain Res 2002;952:7–14]. The NEM‐induced nociceptive behavior was inhibited by pretreatment with dynorphin A‐ or dynorphin B‐antiserum and each antiserum also reduced the nociceptive effects of i.t.‐injected synthetic big dynorphin. The characteristic NEM‐evoked response was not observed in prodynorphin knockout mice. Naloxone, an opioid receptor antagonist, had no effects on the NEM‐induced behavior. Ifenprodil, arcaine and agmatine, antagonists at the polyamine recognition site on the N‐methyl‐D‐aspartate (NMDA) receptor ion‐channel complex, and MK‐801, an NMDA ion‐channel blocker inhibited the NEM‐induced effects. Ro25‐6981, an antagonist of the NMDA receptor subtype containing NR2B subunit was not active. NEM completely inhibited degradation of dynorphin A by soluble and particulate fractions of mouse spinal cord. Collectively, the results demonstrate that endogenous prodynorphin‐derived peptides are pronociceptive in uninjured animals, and required for the NEM‐induced behavior. The NEM effects may be mediated through inhibition of the degradation of endogenous dynorphins, presumably big dynorphin that in turn activates the NMDA receptor ion‐channel complex by acting on the polyamine recognition site.

Clustering of apoptotic cells via bystander killing by peroxides

Clustering of apoptotic cells is a characteristic of many developing or renewing systems, suggesting that apoptotic cells kill bystanders. Bystander killing can be triggered experimentally by inducing apoptosis in single cells and may be based on the exchange of as yet unidentified chemical cell death signals between nearby cells without the need for cell‐to‐cell communication via gap junctions. Here we demonstrate that apoptotic cell clusters occurred spontaneously, after serum deprivation or p53 transfection in cell monolayers in vitro. Clustering was apparently induced through bystander killing by primary apoptotic cells. Catalase, a peroxide scavenger, suppressed bystander killing, suggesting that hydrogen peroxide generated by apoptotic cells is the death signal. Although p53 expression increased the number of apoptoses, clustering was found to be similar around apoptotic cells whether or not p53 was expressed, indicating that there is no specific p53 contribution to bystander killing. Bystander killing through peroxides emitted by apoptotic cells may propagate tissue injury in different pathological situations and be relevant in chemo‐, γ‐ray, and gene therapy of cancer.—Reznikov, K., Kolesnikova, L., Pramanik, A., Tan‐No, K., Gileva, I., Yakovleva, T., Rigler, R., Terenius, L., Bakalkin, G. Clustering of apoptotic cells via bystander killing by peroxides. FASEB J. 14, 1754–1764 (2000)

Prodynorphin gene expression relates to NF-κB factors

Abstract The prodynorphin gene contains several κB motifs, suggesting that κB-specific DNA-binding factors may regulate its expression. Prodynorphin is known to be expressed in human tumor cell lines [Geijer et al.,Regul. Peptides, 34 (1991) 181–188] and we report here that several DNA-binding factors of the NF-κB/c-Rel-family are present in the same cells. Three main κB-specific factors, presumably a p50 homodimer, NF-κB which is a p50/p65 heterodimer and a p65/c-Rel heterodimer were identified using an electromobility shift assay (EMSA), immunoabsorption and UV cross-linking experiments. Minor factors consisting of a novel κB-specific protein of about 125 kDa (p125) or being hetero-oligomeric, composed of p125 and either of three other subunits, namely p50, p65 and c-Rel, were also identified. The homo-oligomer of p125 may be identical to the κB-specific factor BETA, previously found only in brain [Korner et al.,Neuron, 3 (1989) 563–572]. Comparison of prodynorphin mRNA levels with levels of the κB-specific DNA-binding factors revealed a negative correlation with the level of p50 homodimer, and a positive correlation with the ratio of the levels of p65/c-Rel to NF-κB. No association was found with proenkephalin mRNA levels which were significant in only one cell line. The p50 homodimer, but not p65/c-Rel and NF-κB, bound specifically to a DNA-motif within the dynorphin A-encoding gene sequence. This sequence is located in exon 4 and similar to the consensus κB-sequence. The dynorphin A-encoding sequence may represent an intragenic target for the p50 homodimer, which when bound to the sequence suppresses transcription.

Autoantigen Ku in the brain. Developmentally regulated expression and subcellular localization.

A double-stranded DNA end-binding factor with high levels of expression in brain and testis of adult mice was identified as the Ku protein, earlier described as an autoantigen in connective tissue diseases and found to be essential for recombination of the immunoglobulin genes and DNA repair. High Ku levels were found in the cerebellum and pituitary gland, lower levels in the hippocampus, hypothalamus and white matter structures. Ku levels were much higher in embryonic rat brain than in the adult brain, suggesting a role of the Ku protein in brain development. In embryonic rat brain, Ku was associated with cell nuclei, but was predominantly located in the cytosol in the adult rat cerebellum and hippocampus. The abundant expression of Ku in the brain suggests the involvement of Ku autoantibodies in the pathogenesis of neuropsychiatric complications in connective tissue diseases.