Tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate differentially modulate cytotoxic effect of nitric oxide generated by serum deprivation in neuronal PC12 cells

Nitric oxide (NO) is a signaling molecule that mediates several physiological processes in a range of cell and tissue types. Here we investigated the effect of serum deprivation in the absence or presence of phorbol 12-myristate 1 3-acetate (PMA) or tumor necrosis factor-alpha (TNFalpha) on cell viability, NO formation, inducible NO synthase (iNOS) induction, and activation of mitogen-activated protein kinase in neuronal PC12 cells. Within 24 h of serum deprivation, apoptosis occurred in up to 65-70% of the cells, and significant levels of NO were generated. When PMA was added in serum-free medium, NO formation and cell death were decreased. In contrast, addition of TNFalpha in serum-free medium increased the levels of NO formation and apoptosis compared with those in serum-deprived cells. We have demonstrated that differential generation of NO levels by PMA or TNFalpha under conditions of serum deprivation is mediated by the same pattern of iNOS induction. NO formation via iNOS induction resulted in the activation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. From this study it is suggested that the differential formation of cytotoxic NO by serum deprivation plus PMA or TNFalpha is primarily mediated by the induction of iNOS enzymes in neuronal PC12 cells and that its action is mediated by the activation of JNK.ope

Basic fibroblast growth factor-induced activation of novel CREB kinase during the differentiation of immortalized hippocampal cells

Growth factors bind to their specific receptors on the responsive cell surface and thereby initiate dramatic changes in the proliferation, differentiation, and survival of their target cells. In the present study we have examined the mechanism by which growth factor-induced signals are propagated to the nucleus, leading to the activation of transcription factor, cis-acting cAMP response element (CRE)-binding protein (CREB), in immortalized hippocampal progenitor cells (H19-7). During the differentiation of H19-7 cells by basic fibroblast growth factor (bFGF) a critical regulatory Ser(133) residue of CREB was phosphorylated followed by an increase of CRE-mediated gene transcription. Expression of S133A CREB mutants blocked the differentiation of H19-7 cells by bFGF. Although the kinetics of CREB phosphorylation by EGF was transient, bFGF induced a prolonged pattern of CREB phosphorylation. Interestingly, bFGF-induced CREB phosphorylation and subsequent CRE-mediated gene transcription is not likely to be mediated by any of previously known signaling pathways that lead to phosphorylation of CREB, such as mitogen-activated protein kinases, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase-p70(S6K), calcium/calmodulin dependent protein kinase, and casein kinase 2. By using in vitro in gel kinase assay the presence of a novel 120-kDa bFGF-inducible CREB kinase was identified. These findings identify a new growth factor-activated signaling pathway that regulates gene expression at the CRE.ope

Protein Kinase Dyrk1 Activates cAMP Response Element-binding Protein during Neuronal Differentiation in Hippocampal Progenitor Cells

Dyrk is a dual specific protein kinase thought to be involved in normal embryo neurogenesis and brain development. Defects/imperfections in this kinase have been suggested to play an important role in the mental retardation of patients with Down's syndrome. The transcriptional factor cAMP response element-binding protein (CREB) has been implicated in the formation of many types of synaptic plasticity, such as learning and memory. In the present study we show that Dyrk1 activity is markedly induced during the differentiation of immortalized hippocampal progenitor (H19-7) cells. The addition of a neurogenic factor, basic fibroblast growth factor, to the H19-7 cells results in an increased specific binding of Dyrk1 to active CREB. In addition, Dyrk1 directly phosphorylates CREB, leading to the stimulation of subsequent CRE-mediated gene transcription during the neuronal differentiation in H19-7 cells. Blockade of Dyrk1 activation significantly inhibits the neurite outgrowth as well as CREB phosphorylation induced by basic fibroblast growth factor. These findings suggest that Dyrk1 activation and subsequent CREB phosphorylation is important in the neuronal differentiation of central nervous system hippocampal cells.ope

A systemic administration of NMDA induces immediate early gene pip92 in the hippocampus.

In the mammalian CNS, aspartate and glutamate are major excitatory amino acids, and their receptors are believed to mediate a wide range of physiological and pathological processes, including neurotransmission, plasticity, excitotoxicity, and various forms of neurodegeneration. The immediate early gene pip92 has been identified in serum‐stimulated BALB/c 3T3 fibroblasts, activated T lymphocytes treated with cycloheximide, and fibroblast growth factor‐stimulated hippocampal cells during neuronal differentiation. In this study we have demonstrated that pip92 is expressed in the mouse brain after a single intraperitoneal injection of NMDA. The distribution of pip92 mRNA levels in the NMDA‐treated mouse brain was investigated using in situ RT‐PCR. The region‐specific activation of pip92 in the CNS was observed 3 h after NMDA injection, and high levels of pip92 mRNA were detected in the hippocampal dentate gyrus and piriform cortex regions. In addition, the activation of pip92 by NMDA was mediated by activation of mitogen‐activated protein kinases (MAPKs), such as c‐Jun N‐terminal kinase (JNK) and p38 kinase, but not extracellular signal‐regulated kinase (ERK) in the mouse hippocampus and immortalized rat hippocampal progenitor cells. This study suggests that pip92 is likely to play an important role in neuronal cell death induced by excitotoxic NMDA injury in the CNS.ope

Serotonin Discharge Regulation by Additional Neurotransmitters of Rat Hippocampus Associated With the Continence Central Circuit

Purpose: The lower urinary tract is believed to be centrally regulated with the involvement of a range of neurotransmitters. The parasympathetic excitatory input to the urinary bladder is suppressed when the serotonergic system is activated, and thereby voiding is blocked. In healthy people, continence is usually underpinned by hippocampal formation (circuit 3). In order to advance knowledge of how serotoninergic neurons and additional nerve fibers were correlated, the purpose of the present work was to research how the discharge of serotonin from hippocampal slices was affected by different neurotransmitters in rat models. Methods: The adopted procedure involved administration of the central neurotransmitters acetylcholine, norepinephrine, dopamine, N-methyl-D-aspartate (NMDA), gamma-aminobutyric acid (GABA), glycine, and neuropeptide Y as well as monitoring of the alterations in the discharge of [3H]5-hydroxytryptamine (5-HT). Furthermore, to determine whether the effect of the neurotransmitters was influenced by interneuron, tetrodotoxin was also employed. Results: Acetylcholine (10-5M) did not alter [3H]5-HT discharge, whereas more 5-HT was discharged from the hippocampal slices of rats under stimulation by norepinephrine (10-5M) as well as dopamine (10-5M) and tetrodotoxin (10-6M) did not inhibit the discharge. By contrast, tetrodotoxin inhibited the discharge of [3H]5-HT that was exacerbated by NMDA (10-4M). Meanwhile, compared to control, GABA (10-5M), glycine (10-5M), or neuropeptide Y (10-6M) did not alter the [3H]5-HT discharge. Conclusion: From the research findings, it can be concluded that 5-HT discharge from rat hippocampus is enhanced by norepinephrine and dopamine through direct effect on the 5-HT neuronal terminal. By contrast, 5-HT discharge is intensified by NMDA by activating interneurons.ope

Liberation of Serotonin Is Not Unaffected by Acetylcholine in Rat Hippocampus

Purpose: Raised cerebral titers of acetylcholine have notable links with storage symptomatology related to lower urinary tract symptoms. The hippocampus contributes to the normal control of continence in the majority of instances (circuit 3). Owing to synaptic connections with other nerve cells, acetylcholine affects the micturition pathway via the liberation of additional cerebral neurotransmitters. Despite the fact that cerebral serotonin is a key inhibitor of reflex bladder muscle contractions, the influence of acetylcholine on its liberation is poorly delineated. The current research was conducted in order to explore the role of acetylcholine in serotonin liberation from sections of rat hippocampus in order to improve the comprehension of the relationship between cholinergic and serotonergic neurons. Methods: Hippocampal sections from 6 mature male Sprague-Dawley rats were equilibrated over a 30-minute period in standard incubation medium so as to facilitate [3H]5-hydroxytryptamine (5-HT) uptake. The cerebral neurotransmitter, acetylcholine, was applied to the sections. Aliquots of drained medium solution were utilized in order to quantify the radioactivity associated with [3H]5-HT liberation; any alterations in this parameter were noted. Results: When judged against the controls, [3H]5-HT liberation from the hippocampal sections remained unaltered following the administration of acetylcholine, implying that this agent has no inhibitory action on this process. Conclusion: Serotonin liberation from murine hippocampal sections is unaffected by acetylcholine. It is postulated that the bladder micturition reflex responds to acetylcholine through its immediate cholinergic activity rather than by its influence on serotonin release. These pathways are a promising target for the design of de novo therapeutic agents.ope

DYRK1A-mediated hyperphosphorylation of Tau. A functional link between Down syndrome and Alzheimer disease

Most individuals with Down syndrome show early onset of Alzheimer disease (AD), resulting from the extra copy of chromosome 21. Located on this chromosome is a gene that encodes the dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). One of the pathological hallmarks in AD is the presence of neurofibrillary tangles (NFTs), which are insoluble deposits that consist of abnormally hyperphosphorylated Tau. Previously it was reported that Tau at the Thr-212 residue was phosphorylated by Dyrk1A in vitro. To determine the physiological significance of this phosphorylation, an analysis was made of the amount of phospho-Thr-212-Tau (pT212) in the brains of transgenic mice that overexpress the human DYRK1A protein (DYRK1A TG mice) that we recently generated. A significant increase in the amount of pT212 was found in the brains of DYRK1A transgenic mice when compared with age-matched littermate controls. We further examined whether Dyrk1A phosphorylates other Tau residues that are implicated in NFTs. We found that Dyrk1A also phosphorylates Tau at Ser-202 and Ser-404 in vitro. Phosphorylation by Dyrk1A strongly inhibited the ability of Tau to promote microtubule assembly. Following this, using mammalian cells and DYRK1A TG mouse brains, it was demonstrated that the amounts of phospho-Ser-202-Tau and phospho-Ser-404-Tau are enhanced when DYRK1A amounts are high. These results provide the first in vivo evidence for a physiological role of DYRK1A in the hyperphosphorylation of Tau and suggest that the extra copy of the DYRK1A gene contributes to the early onset of ADope

Zinc Inhibits Amyloid beta Production from Alzheimer's Amyloid Precursor Protein in SH-SY5Y Cells

Zinc released from excited glutamatergic neurons accelerates amyloid beta (Abeta) aggregation, underscoring the therapeutic potential of zinc chelation for the treatment of Alzheimer's disease (AD). Zinc can also alter Abeta concentration by affecting its degradation. In order to elucidate the possible role of zinc influx in secretase-processed Abeta production, SH-SY5Y cells stably expressing amyloid precursor protein (APP) were treated with pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, and the resultant changes in APP processing were examined. PDTC decreased Abeta40 and Abeta42 concentrations in culture media bathing APP-expressing SH-SY5Y cells. Measuring the levels of a series of C-terminal APP fragments generated by enzymatic cutting at different APP-cleavage sites showed that both beta- and alpha-cleavage of APP were inhibited by zinc influx. PDTC also interfered with the maturation of APP. PDTC, however, paradoxically increased the intracellular levels of Abeta40. These results indicate that inhibition of secretase-mediated APP cleavage accounts -at least in part- for zinc inhibition of Abeta secretion.ope

Intracellular calcium mobilization induces immediate early gene pip92 via Src and mitogen-activated protein kinase in immortalized hippocampal cells

Regulation of intracellular calcium levels plays a central role in cell survival, proliferation, and differentiation. A cell-permeable, tumor-promoting thapsigargin elevates the intracellular calcium levels by inhibiting endoplasmic reticulum Ca(2+)-ATPase. The Src-tyrosine kinase family is involved in a broad range of cellular responses ranging from cell growth and cytoskeletal rearrangement to differentiation. The immediate early gene pip92 is induced in neuronal cell death as well as cell growth and differentiation. To resolve the molecular mechanism of cell growth by intracellular calcium mobilization, we have examined the effect of thapsigargin and subsequent intracellular calcium influx on pip92 expression in immortalized rat hippocampal H19-7 cells. An increase of intracellular calcium ion levels induced by thapsigargin stimulated the expression of pip92 in H19-7 cells. Transient transfection of the cells with kinase-inactive mitogen-activated protein kinase kinase (MEK) and Src kinase or pretreatment with the chemical MEK inhibitor PD98059 significantly inhibited pip92 expression induced by thapsigargin. When constitutively active v-Src or MEK was overexpressed, the transcriptional activity of the pip92 gene was markedly increased. Dominant inhibitory Raf-1 blocked the transcriptional activity of pip92 induced by thapsigargin. The transcription factor Elk1 is activated during thapsigargin-induced pip92 expression. Taken together, these results suggest that an increase of intracellular calcium ion levels by thapsigargin stimulates the pip92 expression via Raf-MEK-extracellular signal-regulated protein kinase- as well as Src kinase-dependent signaling pathways.ope

Polymorphism of the Hepatocyte Nuclear Factor-1α Gene in the Early-onset of Type 2 Diabetes Mellitus with a Strong Family History in Korea

Background: Maturity-onset diabetes of the young (MODY) is a genetically heterogenous subtype of type 2 diabetes characterized by an early onset, usually before 25 years of age, autosomal dominant inheritance and a primary defect in insulin secretion. Mutation of the hepatocyte nuclear factor-1α (HNF-1α) gene is known to be a cause of MODY3. This study was carried out to reveal whether HNF-1α gene polymorphism is a common cause of early-onset type 2 diabetes and MODY in the Korean population. Methods: Members of 12 pedigrees families with MODY and early-onset of type 2 diabetes were selected for the mutation detection. All of the families involved had at least two members with type 2 diabetes diagnosed before the age of 40 years, where the diabetes was inherited as an autosomal dominant trait, with at least 3 generations of diabetic subjects. Genomic DNA was extracted from whole-blood samples. The 10 exons and the promotor of the HNF-1α gene were sequenced. Results: In codon 17 of exon 1, 2 of the 10 control subjects and 5 of the 12 patients had nucleotide replacement where the CTC nucleotide was replaced by the CTG (p=0.381). This is a silent mutation where both the CTC and CTG code have the same amino acid leucine. In codon 27 of exon 1, 5 patients had a silent mutation, where the codon ATC is replaced by CTC and the amino acid changes from isoleucine to leucine, but no mutation was found in the control group (p=0.040). In codon 459 of exon 7, 2 of the controls and 3 of the patient group had a silent mutation (CTG → TTG) that were both codon code leucine (p=1.000). Another missense mutation was observed in codon 487 of exon 7. Nucleotide AGC (serine) was replaced by AAC (asparagines). This mutation was observed in 5 control subjects and 10 patients (p=0.172). Conclusion: This study did not reveal a new HNF-1α gene polymorphism. We conclude that the HNF-1α gene polymorphism does not play a major role in the early-onset of type 2 diabetes with a strong family history in Korea.ope