Gvido Cebers

A μ-receptor opioid agonist induces AP-1 and NF-κB transcription factor activity in primary cultures of rat cortical neurons

Abstract Activator protein 1 (AP-1) and nuclear factor κB (NF-κB) represent mammalian transcription factors which bind to distinct enhancer motifs. The specific μ-receptor opioid agonist, Tyr, d -Ala 2 , Gly, N -Me-Phe 4 , Gly-ol 5 (DAMGO), was found to increase AP-1 and NF-κB activity in primary cultures of neurons from rat cerebral cortex. Acute (2 h, 4 h) and long-term (72 h) treatment with DAMGO time-dependently increased the DNA-binding activity of both AP-1 and NF-κB and the stimulation could be abolished or inhibited by concurrent incubation with naloxone. However, acute naloxone-precipitated withdrawal did not significantly change AP-1 or NF-κB activity. These results indicate a μ-opioid receptor-related co-induction of AP-1 and NF-κB transcription factors in cultured cortical neurons.

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)

AMPA neurotoxicity in cultured cerebellar granule neurons : Mode of cell death

Various forms of cell death induced by the glutamate receptor agonist, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), were analyzed by determining the capacity of cultured cerebellar granule cells to metabolize 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan, by measuring the leakage of lactate dehydrogenase (LDH), by using confocal microscopy to visualize propidium iodide staining of apoptotic nuclei, and by using field inversion gel electrophoresis (FIGE) for the detection of AMPA-produced cleavage of DNA into high molecular-weight fragments (50 kbp). All these measures indicated that stimulation of AMPA receptors may be involved in the neurotoxic effects of glutamate, and that AMPA-induced neurotoxicity in cerebellar granule cells display morphologically distinct features of both necrotic and apoptotic modes of cell death. In agreement with previous observations, a blockade of AMPA receptor desensitization was necessary to unmask AMPA-induced functional responses in cultured cerebellar granule neurons in vitro. Microfluorimetric measurements of free cytoplasmic calcium concentrations ([Ca2+]i) in single cerebellar neurons revealed that AMPA neurotoxicity was accompanied by a pronounced elevation of [Ca2+]i. Our current results add further evidence to the notion that glutamate-induced neurotoxicity in cerebellar granule cells is mediated not only through NMDA receptors but also through a direct activation of AMPA receptor-regulated cation channels.

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.

Cortical and Striatal Neuronal Cultures of the Same Embryonic Origin Show Intrinsic Differences in Glutamate Receptor Expression and Vulnerability to Excitotoxicity

Cortical and striatal cultures were prepared from the same embryonic rat brains and maintained in identical culture conditions. In this way, the intrinsic, genetically imprinted differences determine the responses of cortical and striatal neurons in comparative studies. Cortical and striatal neurons differed in their sensitivity to glutamate receptor-mediated neurotoxicity as measured by the MTT cell viability assay. On the 8th day in vitro, striatal cultures were less sensitive to N-methyl-d-aspartate (NMDA)-induced toxicity than cortical, although both cultures were equally vulnerable to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)- or kainate-induced toxicity. The AMPA receptor-mediated cell death in cortical cultures, however, was much more dependent on preventing AMPA receptor desensitization than in striatal cultures. Furthermore, glutamate-induced neurotoxicity was primarily mediated by NMDA receptors in cortical cultures, while blockade of either NMDA or AMPA receptors gave almost complete protection against glutamate in striatal cultures. To elucidate the molecular mechanisms responsible for the observed differences, we analyzed the expression of NMDA receptor subunits (NR1, NR2A-C) at the mRNA and the protein level in cortical and striatal cultures as well as in standard cerebellar granule cell cultures. The lowest expression level of NMDA receptor subunits was found in striatal cultures, thereby providing a possible explanation for their lower sensitivity to NMDA. Remarkable differences were found between the relative rates of mRNA and protein expression for NR1 and NR2B in the three cultures, indicative of intrinsic differences in the posttranscriptional regulation of NMDA receptor subunit expression in cultures from various brain regions.

Prolonged inhibition of glutamate reuptake down-regulates NMDA receptor functions in cultured cerebellar granule cells.

Abstract: In the present study, we have examined the effects of prolonged (up to 72 h) inhibition of high‐affinity glutamate reuptake by L‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC; 100 γM) on glutamate receptor functions in primary cultures of rat cerebellar granule neurons. This was done by comparing the effects of various glutamate receptor agonists on neuronal 45Ca2+ uptake, free cytoplasmic Ca2+ concentration ([Ca2+]i), and cell viability. We also determined the parameters of [3H]MK‐801 binding as well as the expression of the NMDAR1 subunit protein in control and PDC‐exposed cultures. The blockade of glutamate reuptake by PDC led to a gradual increase of ambient glutamate to concentrations that are neurotoxic when applied acutely to control cells. In PDC‐exposed cells, however, the acute glutamate‐induced NMDA receptor‐mediated calcium fluxes were strongly diminished and no toxicity was observed. The down‐regulation of the functional effects of glutamate was dependent on the duration of PDC exposure and was accompanied by a reduced NMDAR1 subunit expression and decreased [3H]MK‐801 binding, indicative of a pronounced structural rearrangement of NMDA receptors. The possibility that the decrease of NMDA glutamate receptor sensitivity can be explained on the basis of a reduced density or altered subunit composition of NMDA receptors is discussed.

Reduced HO-1 protein expression is associated with more severe neurodegeneration after transient ischemia induced by cortical compression in diabetic Goto-Kakizaki rats

Pronounced hyperglycemia provoked by extradural compression (EC) of the sensorimotor cortex was recently described in the non-insulin dependent Goto—Kakizaki (GK) diabetic rat. Compared with control Wistar rats, GK rats exhibited more extensive brain damage after cortical ischemia at 48 h of reperfusion (Moreira et al, 2007). We hypothesized that the enhanced brain injury in GK rats could be caused by differential regulation of the heme degrading enzyme heme oxygenase (HO)-1, known to interact with the expression of other target genes implicated in antioxidant defense, inflammation and neurodegeneration, such as superoxide dismutase (SOD)-1, −2, inducible nitric oxide synthase (iNOS), and tumor necrosis factor-α (TNFα). At 48 h after ischemia, relative mRNA expression of such target genes was compared between ipsilateral (compressed) and contralateral (uncompressed) hemispheres of GK rats, along with baseline comparison of sham, uncompressed GK and Wistar rats. Immunohistochemistry was performed to detect cellular and regional localization of HO-1 at this time point. Baseline expression of HO-1, iNOS, and TNFα mRNA was increased in the cortex of sham GK rats. GK rats showed pronounced hyperglycemia during EC and transient attenuation of regional cerebral blood flow recovery. At 48 h after reperfusion, HO-1 mRNA expression was 7- to 8-fold higher in the ischemic cortex of both strains, being the most upregulated gene under study. Heme oxygenase-1 protein expression was significantly reduced in diabetic rats and was found in perilesional astrocytes and rare microglial cells, in both strains.

Prolonged labour associated with lower expression of syndecan 3 and connexin 43 in human uterine tissue

BackgroundProlonged labour is associated with greater morbidity and mortality for mother and child. Connexin 43 is a major myometrial gap junction protein found in human myometrium. Syndecan 3 seems to prevail in the human uterus among heparan sulphate proteoglycans, showing the most significant increase during labour. The aims of the present study were to investigate syndecan 3 and connexin 43 mRNA expressions and protein distributions in human uterine tissue during normal and prolonged labour.MethodsUterine isthmic biopsies were collected from non-pregnant (n = 7), term pregnant women not in labour (n = 14), in normal labour (n = 7) and in prolonged labour (n = 7). mRNA levels of syndecan 3 and connexin 43 were determined by real time RT-PCR. The localization and expression were demonstrated by immunohistochemistry and confocal microscopy.ResultsIn women with prolonged labour, the mRNA expressions of syndecan 3 and Connexin 43 were considerably lower than the expression level at normal labour (p < 0.05). In term-pregnant tissue, the expression of syndecan 3 and connexin 43 did not differ significantly compared to non-pregnant and normal labour. The immunoreactivity of syndecan 3 was strong at normal labour, in contrast to prolonged labour, where both a weaker expression and an irregular distribution were detected. The immunoreactivity of connexin 43 increased until term and further stronger staining occurred at normal labour. At prolonged labour, the immunoreactivity was weaker and more unevenly distributed. At labour, a co-localization of syndecan 3 and connexin 43 could be demonstrated in the smooth muscle by confocal microscopy.ConclusionThe high expression of syndecan 3 and connexin 43 and their co-localization to the smooth muscle bundles during normal labour, together with the significant reduction in prolonged labour, may indicate a role for these proteins in the co-ordination of myometrial contractility.

Increased ambient glutamate concentration alters the expression of NMDA receptor subunits in cerebellar granule neurons

Effects of prolonged (48 h) inhibition of glutamate reuptake on the relative abundance of mRNAs coding for N-methyl-D-aspartate (NMDA) receptor subunits, and the expression of corresponding proteins were investigated in primary cultures of rat cerebellar granule neurons. In cells exposed to the glutamate transport blocker, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), the expression of the C1 exon-positive NR1 mRNA variant was reduced by about 40% whereas, the expression of C1-negative mRNA was increased leading to significant reduction of the +C1/-C1 ratio. The expression of the N1-negative NR1 variants was slightly reduced following exposure to PDC, indicating that increased medium levels of glutamate changed the relative abundance of NR1 splice-variant expression but did not reduce the overall NR1 transcription. Expression of NR2A and NR2B mRNAs was 40-50% lower in PDC-treated cells as compared to control. Immunoblot experiments revealed that PDC exposure reduced the expression of NR1 and all NR2 proteins with NR2A and NR2B proteins being reduced to a greater extent than NR1. The overall decrease in NMDA receptor subunit protein expression was considerably more pronounced than the reduction of their corresponding mRNAs, suggesting involvement of a post-transcriptional regulation. Our data support the hypothesis that functional activity and number of NMDA receptors are regulated by strength of the glutamatergic input. Thus, reduced glutamate uptake resulting in increased concentration of ambient glutamate initiate a series of adaptive responses manifested as a gradual down-regulation of the functional activity and expression of NMDA receptors.

Metabolic inhibition potentiates AMPA-induced Ca2+ fluxes and neurotoxicity in rat cerebellar granule cells

The effects of partial metabolic inhibition (induced by 2 h exposure to low concentrations of cyanide (NaCN)) on the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-induced excitotoxicity and elevation of free cytoplasmic Ca2+ levels ([Ca2+]i) were studied in glucose-deprived primary cultures of cerebellar granule cells. Co-application of AMPA plus NaCN caused a marked increase of cell death, with morphological features of both necrotic and apoptotic cell death as estimated by the capacity of cultured cerebellar granule cells to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan (MTT method), and by measuring the amount of DNA fragmentation in neurons using an ELISA test for histone-bound DNA fragments, respectively. Cell morphology was assessed by confocal microscopy of propidium iodide-stained cultures. No toxic effects were observed when AMPA or a low concentration of NaCN (0.1-0.3 mM; in the presence of NMDA receptor antagonist MK-801; 10 microM) were applied alone. The neurotoxic actions induced by AMPA plus NaCN were preceded and accompanied by a significant elevation of [Ca2+]i, as well as by depletion of neuronal ATP stores. The marked enhancement in the functional responsiveness of AMPA receptors in energetically compromised neurons suggests that at least under certain conditions AMPA receptors may play an important role in excitotoxic processes which might be of relevance for the slowly developing neuronal death seen in several neurodegenerative diseases.