The Secretogranin II Gene Is a Signal Integrator of Glutamate and Dopamine Inputs

Cooperative gene regulation by different neurotransmitters likely underlies the long-term forms of associative learning and memory, but this mechanism largely remains to be elucidated. Following cDNA microarray analysis for genes regulated by Ca(2+) or cAMP, we found that the secretogranin II gene (Scg2) was cooperatively activated by glutamate and dopamine in primary cultured mouse hippocampal neurons. The Ca(2+) chelator BAPTA-AM and the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 prevented Scg2 activation by glutamate or dopamine; thus, the Ca(2+) /MEK pathway is predicted to include a convergence point(s) of glutamatergic and dopaminergic signaling. Unexpectedly, the protein kinase A (PKA) inhibitor KT5720 enhanced Scg2 activation by dopamine. The protein-synthesis inhibitor cycloheximide also enhanced Scg2 activation, and the proteasome inhibitor ZLLLH diminished the KT5720-mediated augmentation of Scg2 activation. These results are concordant with the notion that dopaminergic input leads to accumulation of a KT5720-sensitive transcriptional repressor, which is short-lived because of rapid degradation by proteasomes. This repression pathway may effectively limit the time window permissive to Scg2 activation by in-phase glutamate and dopamine inputs via the Ca(2+) /MEK pathway. We propose that the regulatory system of Scg2 expression is equipped with machinery that is refined for the signal integration of in-phase synaptic inputs. This article is protected by copyright. All rights reserved

GADD34 activates p53 and may have utility as a marker of atherosclerosis

We previously identified growth arrest and DNA-damage-inducible gene 34 (GADD34) as a marker of ischemic stroke. In the present study, serum levels of anti-GADD34 antibodies were found to be significantly higher in patients with acute ischemic stroke or chronic kidney disease compared to healthy donors. We then examined the biological function of GADD34 by transfection into U2OS human osteosarcoma and U87 human glioblastoma cells. Knockdown of GADD34 by siRNA resulted in enhanced cell proliferation, which was reversed by co-knockdown of MDM2. Luciferase reporter assays revealed that the transactivation ability of p53 enhanced by genotoxic anticancer drugs such as camptothecin and etoposide was further potentiated by enforced expression of GADD34 but attenuated by co-transfection with p53 shRNA expression plasmids. Western blotting demonstrated increased p53 protein levels after treatment with camptothecin, which was also potentiated by GADD34 but suppressed by GADD34 siRNA, ATM siRNA, and ATM inhibitor wortmannin. GADD34 levels also increased in response to treatment with camptothecin or adriamycin, and this increase was attenuated by MDM2 siRNA. Immunoprecipitation with anti-GADD34 antibody followed by Western blotting with anti-MDM2 antibodies indicated ubiquitination of GADD34 is mediated by MDM2. Accordingly, GADD34 may function as a ubiquitination decoy to reduce p53 ubiquitination and increase p53 protein levels. Increased neuronal cell death due to activation of p53 by GADD34 may account for the elevated serum levels of anti-GADD34 antibodies observed in patients with acute ischemic stroke

Subtraction efficiency evaluated by cDNA microarray analysis.

<p>An in-house microarray containing cDNA clones derived from the control cells (gray bars, 97 spots in total), LPS/IFNγ-stimulated cells (white bars, 105 spots), and subtracted products (black bars, 124 spots) was prepared, and analyzed for changes in the mRNA levels in response to LPS and IFNγ. The percentages of the clones that had altered mRNA levels indicated in the horizontal axis are plotted for each clone group.</p

Monitoring subtraction processes.

<p>PCR-amplified cDNA mixtures (0.2 µg) derived from the control cells (C), cells stimulated by LPS and IFNγ (L/I), first-round subtracted products (S1), and second-round subtracted products (S2) were electrophoresed, stained (top panel), and subjected to Southern analysis to detect the cDNAs of interferon gamma inducible protein 47 (IFI47) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).</p

Chromosomal loci of putative non-coding RNAs.

<p>The chromosomal loci of the clone Nos. 42 (A), 61 (B), and 74 (C) of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079236#pone-0079236-t001" target="_blank">Table 1</a> are shown. The span and direction of the RNAs are shown by red arrows below the screen shots from the UCSC Genome Browser on Mouse July 2007 (NCBI37/mm9) Assembly.</p

Genes activated by LPS and IFNγ.

a<p>Genes obtained by subtractive cloning are marked “S”.</p

Schematic illustration of cDNA amplification (A) and subsequent subtraction (B).

<p>See the text for explanation.</p

Time course of changes in mRNA levels of several genes in response to LPS and IFNγ.

<p>Total RNAs were prepared from primary-cultured neuronal/glial cells stimulated by IFNγ (I), LPS (L), or both (I/L) for indicated periods. RNAs (0.5 µg per lane) were electrophoresed and subjected to Northern analysis for the indicated mRNAs and for GAPDH mRNA as a control. Below the chemiluminogram, the densitometrically quantified band intensities are shown. “−” indicates that the band intensity was below the detectable level.</p

Efficient Subtractive Cloning of Genes Activated by Lipopolysaccharide and Interferon γ in Primary-Cultured Cortical Cells of Newborn Mice.

Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders