Ratan V. Bhat

Expression of the APC tumor suppressor protein in oligodendroglia.

Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to familial polyposis, an inherited predisposition to colon cancer, and a high percentage of sporadic colon adenomas. Although this gene is best known for its role in development of bowel neoplasms, in recent studies we have found that APC mRNA levels are greatly enriched in brain compared with peripheral tissues. To help define its role in the nervous system, in this study we have determined its cellular localization immunohistochemically in adult rat brain sections and have detected intense APC immunoreactivity in oligodendrocytes. Since prominent APC immunostaining is detected in cell bodies of mature oligodendrocytes, these antibodies may provide a useful addition to available oligodendrocyte markers. Although the cellular function of APC remains undefined, previous biochemical studies have demonstrated that APC is associated with catenins, cytoplasmic proteins involved in regulating cell‐cell adhesion. We propose that, in addition to its critical role in ensuring normal maturation of colonic epithelial cells, the APC tumor suppressor protein also regulates the adhesive properties of oligodendrocytes.

D1 dopamine receptor activation of multiple transcription factor genes in rat striatum

Abstract: Recent studies have shown that dopamine receptor agonists induce expression of Fos‐like immunoreactivity in rat striatal neurons. The protooncogene c‐fos belongs to a family of immediate early genes that are rapidly induced in fibroblasts by growth factors. In light of previous findings that several immediate early gene mRNAs that encode proven or putative transcription factors are differentially regulated by neuronal stimulation in vivo, we have examined the effect of dopaminergic agents on mRNA levels of several such genes using in situ hybridization and northern blot analysis. d‐Amphetamine (2.5‐10 mg/kg i.p.) causes a rapid but transient dose‐dependent increase in zif268 and jun‐B mRNA levels in striatum that was abolished by striatal 6‐hydroxydopamine lesions or by pretreatment with the specific D1 receptor antagonist SCH‐23390 but not by specific D2 receptor antagonists. Apomorphine, a dopamine agonist that acts at both D1 and D2 receptors, and SKF‐38393, a specific D1 receptor agonist, produce similar mRNA changes in rats pretreated with either 6‐hydroxydopamine or reserpine, whereas LY‐171,555, a specific D2 receptor agonist, has no effect. Direct dopamine agonist effects on these immediate early gene mRNA levels are also blocked by D1 but not by D2 antagonists. We observed similar, although less robust, changes in c‐fos and fos‐B mRNA levels. These results demonstrate that striatal D1 dopamine receptors are coupled to activation of multiple transcription factor genes, including zif268 and jun‐B as well as members of the fos family.

Activation of arc, a Putative “Effector” Immediate Early Gene, by Cocaine in Rat Brain

Abstract: As immediate early genes (IEGs) are thought to play a critical role in mediating stimulus‐induced neuronal plasticity, several laboratories have characterized the IEG response induced by cocaine to help define the changes in gene expression that may underlie its long‐lasting behavioral effects. Although activation of several transcription factor IEGs has been described, little is known about which “effector” IEGs, if any, are also induced. In the present study, we have examined whether cocaine administration affects expression of a recently identified “effector” IEG, referred to as arc (activity‐regulated, cytoskeleton‐associated). This IEG encodes a protein with homology to spectrin that appears to be associated with the actin cytoskeleton. Using in situ hybridization, we have found that systemic cocaine administration elicits a robust, transient rise in arc mRNA levels in striatum, which is suppressed by D1 dopamine receptor blockade, reserpine treatment, or striatal 6‐hydroxydopamine lesions. D2 receptor antagonists triggered arc expression when administered alone. Immunohistochemical studies indicated that Arc protein induced by cocaine is expressed in neuronal cell bodies and dendrites. As Arc appears to be a component of the neuronal cytoskeleton, it may be involved in structural alterations underlying neuronal plasticity triggered by cocaine.

Region-Specific Targets of p42/p44MAPK Signaling in Rat Brain

Abstract: In vitro studies indicate that p42/p44MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT‐induced p42/p44MAPK activation by the extracellular signal‐regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule‐associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c‐Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos‐like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44MAPK in vivo. However, the functional targets of p42/p44MAPK signaling depend on the precise location of p42/p44MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44MAPK‐mediated signal transduction within different brain regions in vivo.

Activation of the zinc finger encoding gene krox-20 in adult rat brain: comparison with zif268

Zif268 and krox-20 are transcription regulatory factors that contain highly homologous zinc finger DNA-binding domains. Recent studies have demonstrated that zif268 expression is rapidly regulated in brain by neuronal stimulation. We now report that, like zif268, krox-20 is rapidly and transiently activated by electroconvulsive shock treatment (ECT), D1 dopamine receptor activation, and opiate withdrawal. These studies indicate that, as found for the leucine zipper family of transcription factors, multiple members of the zinc finger family of transcription factors are induced by neuronal stimulation.

Rapid Increases in Peptide Processing Enzyme Expression in Hippocampal Neurons

Abstract: Recent studies have demonstrated that seizure activity causes a dramatic increase in neuropeptide expression in specific regions of the rat hippocampus. In this study we investigated the effect of electroconvulsive treatment (ECT) on the expression of three posttranslational processing enzymes involved in the production of many bioactive peptides from their inactive precursors. Peptidylglycine α‐amidating monooxygenase (PAM) converts peptidylglycine substrates into α‐amidated products and prohormone convertases 1 and 2 perform the tissue‐specific endoproteolytic cleavage of many prohormones. After a single ECT, in situ hybridization demonstrated a rapid increase in the level of PAM mRNA in the dentate granule cells of the hippocampus, reaching peak levels between 1 and 4 h and then returning to near baseline levels within 24 h. Northern blot analysis confirmed the changes in PAM mRNA expression seen by using in situ hybridization. Similar rapid changes in PAM mRNA expression were seen after repeated ECT, suggesting that chronic ECT did not affect the regulation of PAM expression in the hippocampus. Immunohistochemical staining demonstrated an increase in PAM protein in the molecular layer of the dentate gyrus at 4 and 8 h after a single ECT. Based on in situ hybridization, levels of mRNA for the prohormone convertases 1 and 2 were also increased in dentate granule cells after a single ECT. Prohormone convertase 2 mRNA levels exhibited a slower response to ECT, not reaching maximal levels until 8 h after ECT. The response of the dentate granule cells of the hippocampus to ECT provides a model system for studying the rapid, coordinate regulation of peptide‐processing enzymes.

High basal expression of zif268 in cortex is dependent on intact noradrenergic system

The transcription factor Zif268 displays high basal levels of expression in cortex that appear to be dependent on physiological synaptic activity. We report that selective lesions of the noradrenergic system induced by DSP4 markedly suppress basal zif268 mRNA levels in cortex. Accordingly, the noradrenergic system which projects extensively to the cortex and is tonically active may play a key role in maintaining normal patterns of gene expression in target neurons.

Interactions between GSK3β and caspase signalling pathways during NGF deprivation induced cell death

Withdrawal of NGF (NGF-W) in PC12 cells leads to caspase and GSK3beta activation which results in cell death. Our recent findings suggest that inhibition of GSK3beta promotes PC12 cell survival after NGF-W. To determine whether these pathways interact from a signalling perspective, we compared the effects of BAF (a general caspase inhibitor), Li+ (a GSK3beta inhibitor) and insulin on NGF-W induced PC12 cell death. Maximal increase in DNA fragmentation was observed 3 h after NGF-W and was inhibited by BAF (7.5 microM), Li+ (IC(50) = 2 mM) and insulin (IC(50) = 100 nM). BAF inhibited caspase-3 activity and delayed cell death up to 6 h after NGF-W indicating that caspase inhibition is sufficient to prevent apoptosis. BAF had no major effect on GSK3betaactive site phosphorylation or activity suggesting the caspase pathway does not regulate GSK3beta activity. Conversely, Li+ inhibited caspase activity by only 20% but promoted cell survival for 24 h after NGF-W. Overexpression of dominant negative mutants of GSK3beta also inhibited apoptosis, but had only a minor effect on caspase activity after NGF-W. Taken together, these results suggest that GSK3beta is upstream of caspase signalling, and exerts a small effect on the caspase pathway.