Cytoplasmic Polyadenylation Element Binding Proteins CPEB1 and CPEB3 Regulate the Translation of FosB and Are Required for Maintaining Addiction-Like Behaviors Induced by Cocaine.

A recurrent and devastating feature of addiction to a drug of abuse is its persistence, which is mediated by maladaptive long-term memories of the highly pleasurable experience initially associated with the consumption of the drug. We have recently found that members of the CPEB family of proteins (Cytoplasmic Polyadenylation Element-Binding Proteins) are involved in the maintenance of spatial memory. However, their possible role in the maintenance of memories that sustain addictive behavior has yet to be explored. Little is known about any of the mechanisms for maintaining memories for addictive behavior. To address the mechanisms whereby addictive behavior is maintained over time, we utilized a conditional transgenic mouse model expressing a dominant-negative version of CPEB1 that abolishes the activity in the forebrain of two of the four CPEB isoforms (CPEB1 and CPEB3). We found that, following cocaine administration, these dominant-negative (DN) CPEB mice showed a significant decrease, when compared to wild type (WT) mice, in both locomotor sensitizations and conditioned place preference (CPP), two indices of addictive behavior. Supporting these behavioral results, we also found a difference between WT and DN-CPEB1-3 mice in the cocaine-induced synaptic depression in the core of the Nucleus Accumbens (NAc). Finally, we found that (1) CPEB is reduced in transgenic mice following cocaine injections and that (2) FosB, known for its contribution to establishing the addictive phenotype, when its expression in the striatum is increased by drug administration, is a novel target of CPEBs molecules. Thus, our study highlights how CPEB1 and CPEB3 act on target mRNAs to build the neuroadaptative implicit memory responses that lead to the development of the cocaine addictive phenotypes in mammals.

The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3.

Consolidation of long-term memories depends on de novo protein synthesis. Several translational regulators have been identified, and their contribution to the formation of memory has been assessed in the mouse hippocampus. None of them, however, has been implicated in the persistence of memory. Although persistence is a key feature of long-term memory, how this occurs, despite the rapid turnover of its molecular substrates, is poorly understood. Here we find that both memory storage and its underlying synaptic plasticity are mediated by the increase in level and in the aggregation of the prion-like translational regulator CPEB3 (cytoplasmic polyadenylation element-binding protein). Genetic ablation of CPEB3 impairs the maintenance of both hippocampal long-term potentiation and hippocampus-dependent spatial memory. We propose a model whereby persistence of long-term memory results from the assembly of CPEB3 into aggregates. These aggregates serve as functional prions and regulate local protein synthesis necessary for the maintenance of long-term memory.

SUMOylation Is an Inhibitory Constraint that Regulates the Prion-like Aggregation and Activity of CPEB3.

Protein synthesis is crucial for the maintenance of long-term-memory-related synaptic plasticity. The prion-like cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. Here, we provide evidence that the prion-like aggregation and activity of CPEB3 is controlled by SUMOylation. In the basal state, CPEB3 is a repressor and is soluble. Under these circumstances, CPEB3 is SUMOylated in hippocampal neurons both in vitro and in vivo. Following neuronal stimulation, CPEB3 is converted into an active form that promotes the translation of target mRNAs, and this is associated with a decrease of SUMOylation and an increase of aggregation. A chimeric CPEB3 protein fused to SUMO cannot form aggregates and cannot activate the translation of target mRNAs. These findings suggest a model whereby SUMO regulates translation of mRNAs and structural synaptic plasticity by modulating the aggregation of the prion-like protein CPEB3.

D1/D5 receptors and histone deacetylation mediate the Gateway Effect of LTP in hippocampal dentate gyrus.

The dentate gyrus (DG) of the hippocampus is critical for spatial memory and is also thought to be involved in the formation of drug-related associative memory. Here, we attempt to test an aspect of the Gateway Hypothesis, by studying the effect of consecutive exposure to nicotine and cocaine on long-term synaptic potentiation (LTP) in the DG. We find that a single injection of cocaine does not alter LTP. However, pretreatment with nicotine followed by a single injection of cocaine causes a substantial enhancement of LTP. This priming effect of nicotine is unidirectional: There is no enhancement of LTP if cocaine is administrated prior to nicotine. The facilitation induced by nicotine and cocaine can be blocked by oral administration of the dopamine D1/D5 receptor antagonist (SKF 83566) and enhanced by the D1/D5 agonist (SKF 38393). Application of the histone deacetylation inhibitor suberoylanilide hydroxamic acid (SAHA) simulates the priming effect of nicotine on cocaine. By contrast, the priming effect of nicotine on cocaine is blocked in genetically modified mice that are haploinsufficient for the CREB-binding protein (CBP) and possess only one functional CBP allele and therefore exhibit a reduction in histone acetylation. These results demonstrate that the DG of the hippocampus is an important brain region contributing to the priming effect of nicotine on cocaine. Moreover, both activation of dopamine-D1 receptor/PKA signaling pathway and histone deacetylation/CBP mediated transcription are required for the nicotine priming effect in the DG.

Molecular mechanism for a gateway drug: epigenetic changes initiated by nicotine prime gene expression by cocaine.

In human populations, cigarettes and alcohol generally serve as gateway drugs, which people use first before progressing to marijuana, cocaine, or other illicit substances. To understand the biological basis of the gateway sequence of drug use, we developed an animal model in mice and used it to study the effects of nicotine on subsequent responses to cocaine. We found that pretreatment of mice with nicotine increased the response to cocaine, as assessed by addiction-related behaviors and synaptic plasticity in the striatum, a brain region critical for addiction-related reward. Locomotor sensitization was increased by 98%, conditioned place preference was increased by 78%, and cocaine-induced reduction in long-term potentiation (LTP) was enhanced by 24%. The responses to cocaine were altered only when nicotine was administered first, and nicotine and cocaine were then administered concurrently. Reversing the order of drug administration was ineffective; cocaine had no effect on nicotine-induced behaviors and synaptic plasticity. Nicotine primed the response to cocaine by enhancing its ability to induce transcriptional activation of the FosB gene through inhibition of histone deacetylase, which caused global histone acetylation in the striatum. We tested this conclusion further and found that a histone deacetylase inhibitor simulated the actions of nicotine by priming the response to cocaine and enhancing FosB gene expression and LTP depression in the nucleus accumbens. Conversely, in a genetic mouse model characterized by reduced histone acetylation, the effects of cocaine on LTP were diminished. We achieved a similar effect by infusing a low dose of theophylline, an activator of histone deacetylase, into the nucleus accumbens. These results from mice prompted an analysis of epidemiological data, which indicated that most cocaine users initiate cocaine use after the onset of smoking and while actively still smoking, and that initiating cocaine use after smoking increases the risk of becoming dependent on cocaine, consistent with our data from mice. If our findings in mice apply to humans, a decrease in smoking rates in young people would be expected to lead to a decrease in cocaine addiction.

The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections.

The cellular prion protein, PrP(C), is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP ('DeltaPrP'). This paradigm has facilitated mapping of activity determinants in PrP(C) and implicated a cryptic PrP(C)-like protein, 'pi'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrP(C), but is low in cerebellar granular neurons (CGNs) containing PrP(C) and high in PrP(C)-deficient dendritic processes. In Prnp(0/0) CGNs, Sho transgenes were PrP(C)-like in their ability to counteract neurotoxic effects of either Doppel or DeltaPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for pi, and since it engenders a PrP(C)-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology.

Genetic mapping of activity determinants within cellular prion proteins: N-terminal modules in PrPC offset pro-apoptotic activity of the Doppel helix B/B' region.

The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.

In vivo reduction of amyloid-beta by a mutant copper transporter.

Cu ions have been suggested to enhance the assembly and pathogenic potential of the Alzheimer's disease amyloid-beta (Abeta) peptide. To explore this relationship in vivo, toxic-milk (txJ) mice with a mutant ATPase7b transporter favoring elevated Cu levels were analyzed in combination with the transgenic (Tg) CRND8 amyloid precursor protein mice exhibiting robust Abeta deposition. Unexpectedly, TgCRND8 mice homozygous for the recessive txJ mutation examined at 6 months of age exhibited a reduced number of amyloid plaques and diminished plasma Abeta levels. In addition, homozygosity for txJ increased survival of young TgCRND8 mice and lowered endogenous CNS Abeta at times before detectable increases in Cu in the CNS. These data suggest that the beneficial effect of the txJ mutation on CNS Abeta burden may proceed by a previously undescribed mechanism, likely involving increased clearance of peripheral pools of Abeta peptide.

Molecular distinction between pathogenic and infectious properties of the prion protein.

Tg(PG14) mice express a prion protein (PrP) with a nine-octapeptide insertion associated with a human familial prion disease. These animals spontaneously develop a fatal neurodegenerative disorder characterized by ataxia, neuronal apoptosis, and accumulation in the brain of an aggregated and weakly protease-resistant form of mutant PrP (designated PG14(spon)). Brain homogenates from Tg(PG14) mice fail to transmit disease after intracerebral inoculation into recipient mice, indicating that PG14(spon), although pathogenic, is distinct from PrP(Sc), the infectious form of PrP. In contrast, inoculation of Tg(PG14) mice with exogenous prions of the RML strain induces accumulation of PG14(RML), a PrP(Sc) form of the mutant protein that is infectious and highly protease resistant. Like PrP(Sc), both PG14(spon) and PG14(RML) display conformationally masked epitopes in the central and octapeptide repeat regions. However, these two forms differ profoundly in their oligomeric states, with PG14(RML) aggregates being much larger and more resistant to dissociation. Our analysis provides new molecular insight into an emerging puzzle in prion biology, the discrepancy between the infectious and neurotoxic properties of PrP.

A murine model of a familial prion disease.

We have produced a mouse model of a familial prion disorder by introduction of a transgene that encodes the moPrP homolog of a nine-octapeptide insertional mutant associated with an inherited form of CJD in humans. These mice develop progressive neurologic symptoms, display neuropathologic changes, and accumulate a form of mutant PrP in their brains and peripheral tissues that displays some of the biochemical properties of PrPSc. These mice have been extremely valuable for analyzing the cellular and biochemical mechanisms involved in inherited prion disorders and correlating the appearance of the PrPSc-like form with clinical and neuropathologic findings. Because the mutant protein in the mice is highly neurotoxic but appears to lack infectivity, further analysis of its properties promises to shed new light on the molecular distinction between pathogenic and infectious forms of PrP.

Mutant PrP is delayed in its exit from the endoplasmic reticulum, but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation.

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.