Jingshan Chen

Expression of the transcription factor |[Delta]|FosB in the brain controls sensitivity to cocaine

Acute exposure to cocaine transiently induces several Fos family transcription factors in the nucleus accumbens, a region of the brain that is important for addiction. In contrast, chronic exposure to cocaine does not induce these proteins, but instead causes the persistent expression of highly stable isoforms of ΔFosB. ΔFosB is also induced in the nucleus accumbens by repeated exposure to other drugs of abuse, including amphetamine, morphine, nicotine and phencyclidine. The sustained accumulation of ΔFosB in the nucleus accumbens indicates that this transcription factor may mediate some of the persistent neural and behavioural plasticity that accompanies chronic drug exposure. Using transgenic mice in which ΔFosB can be induced in adults in the subset of nucleus accumbens neurons in which cocaine induces the protein, we show that ΔFosB expression increases the responsiveness of an animal to the rewarding and locomotor-activating effects of cocaine. These effects of ΔFosB appear to be mediated partly by induction of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole) glutamate receptor subunit GluR2 in the nucleus accumbens. These results support a model in which ΔFosB, by altering gene expression, enhances sensitivity to cocaine and may thereby contribute to cocaine addiction.

Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5.

Cocaine enhances dopamine-mediated neurotransmission by blocking dopamine re-uptake at axon terminals. Most dopamine-containing nerve terminals innervate medium spiny neurons in the striatum of the brain. Cocaine addiction is thought to stem, in part, from neural adaptations that act to maintain equilibrium by countering the effects of repeated drug administration. Chronic exposure to cocaine upregulates several transcription factors that alter gene expression and which could mediate such compensatory neural and behavioural changes. One such transcription factor is ΔFosB, a protein that persists in striatum long after the end of cocaine exposure. Here we identify cyclin-dependent kinase 5 (Cdk5) as a downstream target gene of ΔFosB by use of DNA array analysis of striatal material from inducible transgenic mice. Overexpression of ΔFosB, or chronic cocaine administration, raised levels of Cdk5 messenger RNA, protein, and activity in the striatum. Moreover, injection of Cdk5 inhibitors into the striatum potentiated behavioural effects of repeated cocaine administration. Our results suggest that changes in Cdk5 levels mediated by ΔFosB, and resulting alterations in signalling involving D1 dopamine receptors, contribute to adaptive changes in the brain related to cocaine addiction.

Overexpression of ΔFosB transcription factor(s) increases boneformation and inhibits adipogenesis

Members of the AP-1 family of transcription factors participate in the regulation of bone cell proliferation and differentiation. We report here a potent AP-1-related regulator of osteoblast function: ΔFosB, a naturally occurring truncated form of FosB that arises from alternative splicing of the fosB transcript and is expressed in osteoblasts. Overexpression of ΔFosB in transgenic mice leads to increased bone formation throughout the skeleton and a continuous post-developmental increase in bone mass, leading to osteosclerosis. In contrast, ΔFosB inhibits adipogenesis both in vivo and in vitro, and downregulates the expression of early markers of adipocyte differentiation. Because osteoblasts and adipocytes are thought to share a common precursor, it is concluded that ΔFosB transcriptionally regulates osteoblastogenesis, possibly at the expense of adipogenesis.

ΔFosB: a molecular mediator of long-term neural and behavioral plasticity

DeltaFosB, a member of the Fos family of transcription factors, is derived from the fosB gene via alternative splicing. Just as c-Fos and many other Fos family members are induced rapidly and transiently in specific brain regions in response to many types of acute perturbations, novel isoforms of DeltaFosB accumulate in a region-specific manner in brain uniquely in response to many types of chronic perturbations, including repeated administration of drugs of abuse or of antidepressant or antipsychotic treatments. Importantly, once induced, these DeltaFosB isoforms persist in brain for relatively long periods due to their extraordinary stability. Mice lacking the fosB gene show abnormal biochemical and behavioral responses to chronic administration of drugs of abuse or antidepressant treatments, consistent with an important role for DeltaFosB in mediating long-term adaptations in the brain. More definitive evidence to support this hypothesis has recently been provided by inducible transgenic mice, wherein biochemical and behavioral changes, which mimic the chronic drug-treated state, are seen upon overexpression of DeltaFosB in specific brain regions. This evolving work supports the view that DeltaFosB functions as a type of molecular switch that gradually converts acute responses into relatively stable adaptations that underlie long-term neural and behavioral plasticity to repeated stimuli.

Production of phosphorylation state-specific antibodies.

Publisher Summary This chapter discusses the production of phosphorylation state-specific antibodies. The ability to detect and to quantitate changes in the state of phosphorylation of specific substrate proteins is of great utility in the study of their functional significance. Standard methods for measuring the state of protein phosphorylation in intact cell preparations utilize prelabeling with [ 32 P]P i or back phosphorylation. The degree of sensitivity and selectivity afforded by immunochemical methodology makes it an attractive alternative for detecting changes in the state of phosphorylation of specific proteins. The methods used for these proteins have general application in the production of phosphorylation state-specific antibodies to substrates whose sites of phosphorylation have been established.

Induction of nuclear factor‐κB in nucleus accumbens by chronic cocaine administration

ΔFosB is a Fos family transcription factor that is induced by chronic exposure to cocaine and other drugs of abuse in the nucleus accumbens and related striatal regions, brain regions that are important for the behavioral effects of these drugs. To better understand the mechanisms by which ΔFosB contributes to the effects of chronic drug treatment, we used DNA microarray analysis to identify genes that are regulated in the nucleus accumbens upon ΔFosB expression in inducible bitransgenic mice. One of the most highly regulated genes was that encoding a subunit of another transcription factor, nuclear factor‐κB (NF‐κB). Subsequent experiments confirmed the induction of NF‐κB in the nucleus accumbens of mice overexpressing ΔFosB as well as in wild‐type mice treated chronically, but not acutely, with cocaine. These results establish NF‐κB as a putative target for ΔFosB and implicate NF‐κB signaling pathways in the long‐term adaptations of nucleus accumbens neurons to cocaine.

Cell Type-Specific Regulation of CREB Gene Expression: Mutational Analysis of CREB Promoter Activity

Abstract: Previous studies have shown that activation of the cyclic AMP (cAMP) pathway down‐regulates CREB expression in CATH.a cells, an effect that appears to be mediated via inhibition of CREB gene transcription. In the current study, we compared this effect in CATH.a cells with regulation of CREB expression in another cell line, C6 glioma cells. In contrast to the findings in CATH.a cells, activation of the cAMP pathway up‐regulates CREB expression in C6 glioma cells. To determine whether these opposite effects can be explained by regulation of CREB promoter activity, chloramphenicol acetyltransferase (CAT) assays were performed in CATH.a and C6 glioma cells that were transiently transfected with a CREB promoter‐CAT fusion plasmid. Activation of the cAMP pathway decreased levels of CAT activity in transfected CATH.a cells but increased CAT activity in transfected C6 glioma cells. We next investigated the effect of mutations in the CREB promoter on such regulation in these two cell lines. Mutations of single CRE or Sp1 binding sites in the CREB promoter reduced basal levels of CAT activity but did not significantly attenuate regulation of the promoter in CATH.a or C6 glioma cells. However, mutation or deletion of two CRE sites in the CREB promoter completely abolished up‐regulation of CAT activity in the C6 glioma cells and abolished basal levels of CAT activity in CATH.a cells. CREB promoter activity was also studied in cultured SHSY5Y cells and in primary cultures of striatal neurons as further comparisons. Activation of the cAMP pathway was found to increase CAT activity in both cell types. In the striatal cultures, this effect was obliterated by mutation or deletion of either of the two CREs in the promoter. These findings demonstrate cell type‐specific effects of the cAMP pathway on CREB expression, which appear to be mediated via differential regulation of the CREB promoter.

Transcriptional regulation of CREB (cyclic AMP response element-binding protein) expression in CATH.a cells

Abstract: We have recently demonstrated that mRNA expression of cyclic AMP (cAMP) response element‐binding protein (CREB) is down‐regulated in CATH.a cells (a neural‐derived cell line) by activation of the cAMP pathway. We now demonstrate that this down‐regulation can be accounted for by a decrease in the rate of CREB gene transcription. It was found that cycloheximide, a protein synthesis inhibitor, prevented the forskolin‐induced decrease in CREB mRNA levels in CATH.a cells. Nuclear run‐on assays demonstrated that forskolin decreased the rate of CREB transcription by close to 50%. Moreover, forskolin decreased chloramphenicol acetyltransferase (CAT) activity in CATH.a cells transiently transfected with a construct containing 1,240 bp of CREB promoter fused to a CAT reporter plasmid. Possible mechanisms by which activation of the cAMP pathway leads to a decrease in CREB gene transcription are discussed.

Regulating ΔFosB expression in adult tet-off-ΔFosB transgenic mice alters bone formation and bone mass

Abstract The ΔFosB isoforms are naturally occurring AP-1 family members that increase bone volume via a cell-autonomous effect on osteoblastic bone formation. Mice overexpressing ΔFosB demonstrate a very high level of bone formation, resulting in a progressive osteosclerosis. Despite the linkage of bone formation and resorption in physiological systems, no alteration in bone resorption was detected in mice overexpressing ΔFosB. To determine whether altering ΔFosB expression can regulate bone formation independently of bone resorption in adult mice, we used the Tet-Off-inducible transgene system to induce or block transgenic ΔFosB overexpression and thereby regulate bone formation in vivo. Overexpression of ΔFosB after skeletal maturity increased trabecular bone volume by increasing bone formation, again without altering bone resorption, indicating that developmental ΔFosB overexpression is not required for the osteosclerotic phenotype. Similarly, switching off ΔFosB overexpression after osteosclerosis had developed led to a marked decrease in bone formation and loss of bone mass such that trabecular bone volume approached normal levels. Despite this dramatic reduction, no alteration in bone resorption was detected. These results clearly demonstrate that ΔFosB regulates bone formation and bone mass in adult mice with no effect on bone resorption.

Propagation of γPKC translocation along the dendrites of Purkinje cell in γPKC‐GFP transgenic mice

To elucidate spatial and temporal profiles of the protein kinase C (PKC) activation in relation to neuronal functions including synaptic plasticity, we tried to detect PKC translocation in living brain slices. We first developed brain region‐specific and inducible γPKC‐GFP transgenic mice using a tetracycline (tet)‐regulated system. In the transgenic mice, the expression of γPKC‐GFP was region‐specifically regulated by the promoter and abolished by the administration of doxycycline. Cerebellar slices from the mice were utilized for intracellular recording and fluorescence imaging of γPKC‐GFP in Purkinje cells. GFP fluorescence was uniformly distributed from soma to dendritic arbor. When mGluR agonists were applied, the intensity was transiently increased at the edge of the dendrite and concomitantly decreased in the cytoplasm, indicating that γPKC translocated to the plasma membrane. This transient change in the pattern of GFP fluorescence simultaneously occurred throughout the Purkinje cell dendrites by agonist stimulation. Translocation of γPKC‐GFP was also induced by electrical stimulation of parallel fibres. However, the event was not restricted at the distal dendrites, propagated forwardly along the dendritic tree and reached to the proximal trunk close to the soma. Time course of the propagation was slower than the electrical signal and Ca2+ waves and faster than conveying molecules through microtubules. The present results indicate that PKC signals activated locally by parallel fibre input could propagate to the soma through dendrites in living Purkinje neurones. The findings may provide us with a new insight for understanding molecular mechanisms of the synaptic plasticity including cerebellar long‐term depression.