Loss of the Ca2+/calmodulin-dependent protein kinase type IV in dopaminoceptive neurons enhances behavioral effects of cocaine

The persistent nature of addiction has been associated with activity-induced plasticity of neurons within the striatum and nucleus accumbens (NAc). To identify the molecular processes leading to these adaptations, we performed Cre/loxP-mediated genetic ablations of two key regulators of gene expression in response to activity, the Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) and its postulated main target, the cAMP-responsive element binding protein (CREB). We found that acute cocaine-induced gene expression in the striatum was largely unaffected by the loss of CaMKIV. On the behavioral level, mice lacking CaMKIV in dopaminoceptive neurons displayed increased sensitivity to cocaine as evidenced by augmented expression of locomotor sensitization and enhanced conditioned place preference and reinstatement after extinction. However, the loss of CREB in the forebrain had no effect on either of these behaviors, even though it robustly blunted acute cocaine-induced transcription. To test the relevance of these observations for addiction in humans, we performed an association study of CAMK4 and CREB promoter polymorphisms with cocaine addiction in a large sample of addicts. We found that a single nucleotide polymorphism in the CAMK4 promoter was significantly associated with cocaine addiction, whereas variations in the CREB promoter regions did not correlate with drug abuse. These findings reveal a critical role for CaMKIV in the development and persistence of cocaine-induced behaviors, through mechanisms dissociated from acute effects on gene expression and CREB-dependent transcription.

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by > 60%, but also the level of myosin heavy chains (MHCs) by > 40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.

Chronic administration of harmine elicits antidepressant-like effects and increases BDNF levels in rat hippocampus

A growing body of evidence has pointed to the beta-carboline harmine as a potential therapeutic target for the treatment of major depression. The present study was aimed to evaluate behavioural and molecular effects of the chronic treatment with harmine and imipramine in rats. To this aim, rats were treated for 14 days once a day with harmine (5, 10 and 15 mg/kg) and imipramine (10, 20 and 30 mg/kg) and then subjected to the forced swimming and open-field tests. Harmine and imipramine, at all doses tested, reduced immobility time of rats compared with the saline group. Imipramine increased the swimming time at 20 and 30 mg/kg and harmine increased swimming time at all doses. The climbing time increased in rats treated with imipramine (10 and 30 mg/kg) and harmine (5 and 10 mg/kg), without affecting spontaneous locomotor activity. Brain-derived neurotrophic factor (BDNF) hippocampal levels were assessed in imipramine and harmine-treated rats by ELISA sandwich assay. Interestingly, chronic administration of harmine at the higher doses (10 and 15 mg/kg), but not imipramine, increased BDNF protein levels in rat hippocampus. Finally, these findings further support the hypothesis that harmine could bring about behavior and molecular effects, similar to antidepressants drugs.

Degeneration of dystrophic or injured skeletal muscles induces high expression of Galectin-1

Muscle degenerative diseases such as Duchenne Muscular Dystrophy are incurable and treatment options are still restrained. Understanding the mechanisms and factors responsible for muscle degeneration and regeneration will facilitate the development of novel therapeutics. Several recent studies have demonstrated that Galectin-1 (Gal-1), a carbohydrate-binding protein, induces myoblast differentiation and fusion in vitro, suggesting a potential role for this mammalian lectin in muscle regenerative processes in vivo. However, the expression and localization of Gal-1 in vivo during muscle injury and repair are unclear. We report the expression and localization of Gal-1 during degenerative-regenerative processes in vivo using two models of muscular dystrophy and muscle injury. Gal-1 expression increased significantly during muscle degeneration in the murine mdx and in the canine Golden Retriever Muscular Dystrophy animal models. Compulsory exercise of mdx mouse, which intensifies degeneration, also resulted in sustained Gal-1 levels. Furthermore, muscle injury of wild-type C57BL/6 mice, induced by BaCl(2) treatment, also resulted in a marked increase in Gal-1 levels. Increased Gal-1 levels appeared to localize both inside and outside the muscle fibers with significant extracellular Gal-1 colocalized with infiltrating CD45(+) leukocytes. By contrast, regenerating muscle tissue showed a marked decrease in Gal-1 to baseline levels. These results demonstrate significant regulation of Gal-1 expression in vivo and suggest a potential role for Gal-1 in muscle homeostasis and repair.