Yoko H. Ohnishi

[Delta]FosB in brain reward circuits mediates resilience to stress and antidepressant responses

In contrast with the many studies of stress effects on the brain, relatively little is known about the molecular mechanisms of resilience, the ability of some individuals to escape the deleterious effects of stress. We found that the transcription factor ΔFosB mediates an essential mechanism of resilience in mice. Induction of ΔFosB in the nucleus accumbens, an important brain reward-associated region, in response to chronic social defeat stress was both necessary and sufficient for resilience. ΔFosB induction was also required for the standard antidepressant fluoxetine to reverse behavioral pathology induced by social defeat. ΔFosB produced these effects through induction of the GluR2 AMPA glutamate receptor subunit, which decreased the responsiveness of nucleus accumbens neurons to glutamate, and through other synaptic proteins. Together, these findings establish a previously unknown molecular pathway underlying both resilience and antidepressant action.

Rac1 is essential in cocaine-induced structural plasticity of nucleus accumbens neurons.

Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.

BDNF Is a Negative Modulator of Morphine Action

Regulating Opioid Responses Different drugs of abuse are thought to highjack similar reward systems in the brain using common mechanisms. However, Koo et al. (p. 124) now observe that some of the neural mechanisms that regulate opiate reward can be both different and even opposite to those that regulate reward by stimulant drugs. While knockdown of brain-derived neurotrophic factor (BDNF) in the ventral tegmental area in mice antagonized the response to cocaine, the same manipulation strengthened the potential of opiates to increase dopamine neuron excitability. Optogenetic stimulation of dopaminergic terminals in the nucleus accumbens could counteract the effects of BDNF on morphine reward blockade. Morphine reward is modulated by ventral tegmental area brain-derived neurotrophic factor in a way that is opposite to its modulation of cocaine reward. Brain-derived neurotrophic factor (BDNF) is a key positive regulator of neural plasticity, promoting, for example, the actions of stimulant drugs of abuse such as cocaine. We discovered a surprising opposite role for BDNF in countering responses to chronic morphine exposure. The suppression of BDNF in the ventral tegmental area (VTA) enhanced the ability of morphine to increase dopamine (DA) neuron excitability and promote reward. In contrast, optical stimulation of VTA DA terminals in nucleus accumbens (NAc) completely reversed the suppressive effect of BDNF on morphine reward. Furthermore, we identified numerous genes in the NAc, a major target region of VTA DA neurons, whose regulation by BDNF in the context of chronic morphine exposure mediated this counteractive function. These findings provide insight into the molecular basis of morphine-induced neuroadaptations in the brain’s reward circuitry.

fosB-Null Mice Display Impaired Adult Hippocampal Neurogenesis and Spontaneous Epilepsy with Depressive Behavior

Patients with epilepsy are at high risk for major depression relative to the general population, and both disorders are associated with changes in adult hippocampal neurogenesis, although the mechanisms underlying disease onset remain unknown. The expression of fosB, an immediate early gene encoding FosB and ΔFosB/Δ2ΔFosB by alternative splicing and translation initiation, is known to be induced in neural progenitor cells within the subventricular zone of the lateral ventricles and subgranular zone of the hippocampus, following transient forebrain ischemia in the rat brain. Moreover, adenovirus-mediated expression of fosB gene products can promote neural stem cell proliferation. We recently found that fosB-null mice show increased depressive behavior, suggesting impaired neurogenesis in fosB-null mice. In the current study, we analyzed neurogenesis in the hippocampal dentate gyrus of fosB-null and fosBd/d mice that express ΔFosB/Δ2ΔFosB but not FosB, in comparison with wild-type mice, alongside neuropathology, behaviors, and gene expression profiles. fosB-null but not fosBd/d mice displayed impaired neurogenesis in the adult hippocampus and spontaneous epilepsy. Microarray analysis revealed that genes related to neurogenesis, depression, and epilepsy were altered in the hippocampus of fosB-null mice. Thus, we conclude that the fosB-null mouse is the first animal model to provide a genetic and molecular basis for the comorbidity between depression and epilepsy with abnormal neurogenesis, all of which are caused by loss of a single gene, fosB.

FosB is essential for the enhancement of stress tolerance and antagonizes locomotor sensitization by ΔfosB

BACKGROUND Molecular mechanisms underlying stress tolerance and vulnerability are incompletely understood. The fosB gene is an attractive candidate for regulating stress responses, because ΔFosB, an alternative splice product of the fosB gene, accumulates after repeated stress or antidepressant treatments. On the other hand, FosB, the other alternative splice product of the fosB gene, expresses more transiently than ΔFosB but exerts higher transcriptional activity. However, the functional differences of these two fosB products remain unclear. METHODS We established various mouse lines carrying three different types of fosB allele, wild-type (fosB(+)), fosB-null (fosB(G)), and fosB(d) allele, which encodes ΔFosB but not FosB, and analyzed them in stress-related behavioral tests. RESULTS Because fosB(+/d) mice show enhanced ΔFosB levels in the presence of FosB and fosB(d/d) mice show more enhanced ΔFosB levels in the absence of FosB, the function of FosB can be inferred from differences observed between these lines. The fosB(+/d) and fosB(d/d) mice showed increased locomotor activity and elevated Akt phosphorylation, whereas only fosB(+/d) mice showed antidepressive-like behaviors and increased E-cadherin expression in striatum compared with wild-type mice. In contrast, fosB-null mice showed increased depression-like behavior and lower E-cadherin expression. CONCLUSIONS These findings indicate that FosB is essential for stress tolerance mediated by ΔFosB. These data suggest that fosB gene products have a potential to regulate mood disorder-related behaviors.

Small molecule screening identifies regulators of the transcription factor ΔFosB.

ΔFosB protein accumulates in the striatum in response to chronic administration of drugs of abuse, L-DOPA, or stress, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, abnormal involuntary movements (dyskinesia), and depression. ΔFosB binds AP-1 DNA consensus sequences found in promoters of many genes and can both repress or activate gene transcription. In the striatum, ΔFosB is thought to dimerize with JunD to form a functional transcription factor, though strikingly JunD does not accumulate in parallel. One explanation is that ΔFosB can recruit different partners, including itself, depending on the neuron type in which it is induced and the chronic stimulus, generating protein complexes with different effects on gene transcription. To develop chemical probes to study ΔFosB, a high-throughput screen was carried out to identify small molecules that modulate ΔFosB function. Two compounds with low micromolar activity, termed C2 and C6, disrupt the binding of ΔFosB to DNA via different mechanisms, and in in vitro assays stimulate ΔFosB-mediated transcription. In cocaine-treated mice, C2 significantly elevates mRNA levels of the AMPA glutamate receptor GluR2 subunit with specificity, a known target gene of ΔFosB that plays a role in drug addiction and endogenous resilience mechanisms. C2 and C6 show different activities against ΔFosB homodimers compared to ΔFosB/JunD heterodimers, suggesting that these compounds can be used as probes to study the contribution of different ΔFosB-containing complexes on the regulation of gene transcription in biological systems and to assess the utility of ΔFosB as a therapeutic target.

Antagonistic Regulation of Cell-Matrix Adhesion by FosB and ΔFosB/Δ2ΔFosB Encoded by Alternatively Spliced Forms of fosB Transcripts

Among fos family genes encoding components of activator protein-1 complex, only the fosB gene produces two forms of mature transcripts, namely fosB and DeltafosB mRNAs, by alternative splicing of an exonic intron. The former encodes full-length FosB. The latter encodes DeltaFosB and Delta2DeltaFosB by alternative translation initiation, and both of these lack the C-terminal transactivation domain of FosB. We established two mutant mouse embryonic stem (ES) cell lines carrying homozygous fosB-null alleles and fosB(d) alleles, the latter exclusively encoding DeltaFosB/Delta2DeltaFosB. Comparison of their gene expression profiles with that of the wild type revealed that more than 200 genes were up-regulated, whereas 19 genes were down-regulated in a DeltaFosB/Delta2DeltaFosB-dependent manner. We furthermore found that mRNAs for basement membrane proteins were significantly up-regulated in fosB(d/d) but not fosB-null mutant cells, whereas genes involved in the TGF-beta1 signaling pathway were up-regulated in both mutants. Cell-matrix adhesion was remarkably augmented in fosB(d/d) ES cells and to some extent in fosB-null cells. By analyzing ES cell lines carrying homozygous fosB(FN) alleles, which exclusively encode FosB, we confirmed that FosB negatively regulates cell-matrix adhesion and the TGF-beta1 signaling pathway. We thus concluded that FosB and DeltaFosB/Delta2DeltaFosB use this pathway to antagonistically regulate cell matrix adhesion.

FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices of human alcoholics

The transcription factor ΔFosB is accumulated in the addiction circuitry, including the orbitofrontal and medial prefrontal cortices of rodents chronically exposed to ethanol or other drugs of abuse, and has been suggested to play a direct role in addiction maintenance. To address this hypothesis in the context of substance dependence in humans, we compared the immunoreactivities of FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices (OFC and DLPFC respectively) between controls and alcoholics using semiquantitative immunoblotting. In both structures, we detected three forms of FOSB, one of which was ΔFOSB, but in neither case did their immunoreactivities differ between the groups. Our results indicate that the ΔFOSB immunoreactivity in the human brain is very low, and that it is not accumulated in the OFC and DLPFC of human alcoholics, suggesting that it may not be directly involved in addiction maintenance, at least not in ethanol dependence.

Functional Role of the N-Terminal Domain of ΔFosB in Response to Stress and Drugs of Abuse

Previous work has implicated the transcription factor, ΔFosB, acting in the nucleus accumbens, in mediating the pro-rewarding effects of drugs of abuse such as cocaine as well as in mediating resilience to chronic social stress. However, the transgenic and viral gene transfer models used to establish these ΔFosB phenotypes express, in addition to ΔFosB, an alternative translation product of ΔFosB mRNA, termed Δ2ΔFosB, which lacks the N-terminal 78 aa present in ΔFosB. To study the possible contribution of Δ2ΔFosB to these drug and stress phenotypes, we prepared a viral vector that overexpresses a point mutant form of ΔFosB mRNA which cannot undergo alternative translation as well as a vector that overexpresses Δ2ΔFosB alone. Our results show that the mutant form of ΔFosB, when overexpressed in the nucleus accumbens, reproduces the enhancement of reward and of resilience seen with our earlier models, with no effects seen for Δ2ΔFosB. Overexpression of full length FosB, the other major product of the FosB gene, also has no effect. These findings confirm the unique role of ΔFosB in the nucleus accumbens in controlling responses to drugs of abuse and stress.

PSMC5, a 19S Proteasomal ATPase, Regulates Cocaine Action in the Nucleus Accumbens

ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain’s reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5—also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex—as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity.