Docosahexaenoic acid intake decreases proliferation, increases apoptosis and decreases the invasive potential of the human breast carcinoma cell line MDA-MB-231

International audienceBreast cancer is the most common cancer in women in industrialized countries. Environmental factors, such as differences in diet are likely to have an important influence on cancer emergence. Among these factors, n-3 polyunsaturated-fatty acids, such as docosahexaenoic acid (DHA), are good candidates for preventing breast cancer. Here we investigate the effect of DHA on the human breast cancer cell line MDA-MB-231 and show that DHA incorpo- ration i) has an anti-proliferative effect, ii) induces apoptosis via a transient increase in caspase-3 activity and the promotion of nuclear condensation, and iii) reduces the invasive potential of MDA-MB-231 cells. To conclude, DHA may have beneficial effects as a result of slowing the proliferation of tumor cells, and minimizing their metastatic potential

Increase of KCC2 in hippocampal synaptic plasticity disturbances after perinatal ethanol exposure

International audienceLow to moderate perinatal ethanol exposure (PEE) may have disastrous consequences for the central nervous system resulting notably in permanent cognitive deficits. Learning and memory are mediated in the hippocampus by long-term potentiation (LTP) and long term depression (LTD), two forms of synaptic plasticity. PEE decreases LTP but also abnormally facilitates LTD (Kervern et al. 2015) through a presently unknown mechanism. We studied in rat hippocampus slice, the involvement of the chloride co-transporters NKCC1 and KCC2, in the role of GABA(A) inhibitions in facilitated LTD after moderate PEE. After PEE and in contrast to control slices, facilitated LTD in CA1 field was reduced by the GABA(A) receptor antagonist bicuculline with no changes in sensitivity to bicuculline and in GABA and benzodiazepine binding sites. Also, sensitivity to diazepam was unaltered, whereas aberrant LTD was blocked. Immunohistochemistry and protein analysis demonstrated an increase in KCC2 protein level at cell membrane in CA1 after PEE with no change in NKCC1 expression. Specifically, both monomeric and dimeric forms of KCC2 were increased in CA1. Bumetanide (10100 mu M), a dose-dependent blocker of NKCC1 and KCC2, or VU0240551 (10 mu M) a specific antagonist of KCC2, corrected the enhanced LTD and interestingly bumetanide also restored the lower LTP after PEE. These results demonstrate for the first time an upregulation of the KCC2 co-transporter expression after moderate PEE associated with disturbances in GABAergic neurotransmission modulating bidirectional synaptic plasticity in the hippocampus. Importantly, bumetanide compensated deficits in both LTP and LTD, revealing its potential therapeutic properties

Resistance to ethanol sensitization is associated with a loss of synaptic plasticity in the hippocampus

International audienceBehavioral sensitization to repeated ethanol (EtOH) exposure induces an increase in locomotor activity in mice. However, not all animals express such sensitization. Although the literature indicated that the hippocampus may play a role in EtOH sensitization, it is not known whether behavioral sensitization to EtOH is associated with preferential changes in bidirectional synaptic plasticity, i.e., LTP and LTD, two markers of learning capabilities that have also been shown to be involved in addictive behavior. In the present study, we examined whether the vulnerability to develop and express behavioral sensitization to EtOH is associated with altered bidirectional synaptic plasticity in the CA1 area of the dorsal hippocampus. For this purpose, we analyzed both LTP and LTD in resistant and sensitized mice during the expression phase, i.e., 7 days after 10 days of repeated EtOH i.p. administration. We found that resistant mice showed a lack of LTD without changes in LTP. The lack of LTD was associated with an increase in GluN2A protein level and was not due to an altered level of neuronal activity, since no difference was observed between the number of c-FOS positive neurons in sensitized and resistant mice. Given that both types of synaptic plasticity signals may have distinct roles in specific learning and behaviors, our results suggest that resistant mice could exhibit different phenotypes in terms of learning/memory and addictive behaviors compared to sensitized ones. Synapse 71:e21899, 2017. (c) 2016 Wiley Periodicals, Inc

Ethanol (EtOH)-Related Behaviors in alpha-Synuclein Mutant Mice and Association of SNCA SNPs with Anxiety in EtOH-Dependent Patients

International audienceBackground Data have shown a role of alpha-synuclein in anxiety and also in addiction, particularly in alcohol use disorders (AUD). Since the comorbidity between AUD and anxiety is very high and because anxiety is an important factor in ethanol (EtOH) relapse, the aim of the present study was to investigate the role of alpha-synuclein in moderating EtOH intake, the anxiolytic effects of EtOH, and EtOH withdrawal-induced anxiety and convulsions in mice. The study aimed to determine whether SNCA variants moderated anxiety in EtOH-dependent patients. Methods We analyzed the moderator effect of 3 SNCA Tag-single nucleotide polymorphisms (Tag-SNPs) rs356200, rs356219, and rs2119787 on the anxiety symptoms in 128 EtOH-dependent patients. We used the C57BL/6JOlaHsd Snca mutant mice to assess EtOH intake; sensitivity to the anxiolytic effects of EtOH in a test battery comprising the open field, the light-dark box, and the elevated plus maze; and both anxiety and convulsions induced by EtOH withdrawal. Results Our results demonstrated a reduction in both EtOH intake and preference and also a lack of sensitivity to the anxiolytic effects of EtOH in alpha-synuclein mutant mice. Results on anxiety-like behavior were mixed, but mutant mice displayed increased anxiety when exposed to a low anxiogenic environment. Mutant mice also displayed an increase in handling-induced convulsion scores during withdrawal after EtOH inhalation, but did not differ in terms of EtOH withdrawal-induced anxiety. In humans, we found a significant association of the rs356219 SNP with a high level of anxiety (Beck Anxiety Inventory score >15) and the rs356200 SNP with a positive familial history of AUD. Conclusions Our translational study highlights a significant role of alpha-synuclein in components of AUD

Patch-Clamp Recording of Low Frequency Stimulation-induced Long-Term Synaptic Depression in Rat Hippocampus Slices During Early and Late Neurodevelopment

International audienceBackground Studying synaptic plasticity in the rat hippocampus slice is a well-established way to analyze cellular mechanisms related to learning and memory. Different modes of recording can be used, such as extracellular field excitatory post-synaptic potential (EPSP) and diverse patch-clamp methods. However, most studies using these methods have examined only up to the juvenile stage of brain maturation, which is known to terminate during late adolescence/early adulthood. Moreover, several animal models of human diseases have been developed at this late stage of brain development. To study the vulnerability of adolescent rat to the cognitive impairment of alcohol, we developed a model of binge-like exposure in which ethanol selectively abolishes low frequency stimulation (LFS)-induced, field EPSP long-term depression (LTD) in the rat hippocampus slice. Methods In the present study, we sought to use whole-cell patch-clamp recording in the voltage-clamp mode to further investigate the mechanisms involved in the abolition of LFS-induced LTD in our model of binge-like exposure in adolescent rat hippocampus slices. In addition, we investigated LFS-induced NMDAR-LTD and mGluR-LTD at different ages and changed several parameters to improve the recordings. Results Using patch-clamp recording, LFS-induced NMDAR-LTD and mGluR-LTD could be measured until 4 weeks of age, but not in older animals. Similarly, chemical mGluR-LTD and a combined LFS-LTD involving both N-Methyl-D-Aspartate Receptor (NMDAR) and mGluR were not measured in older animals. The absence of LFS-LTD was not due to the loss of a diffusible intracellular agent nor the voltage mode of recording or intracellular blockade of either sodium or potassium currents. In contrast to voltage-clamp recordings, LFS-induced LTD tested with field recordings was measured at all ages and the effects of EtOH were visible in all cases. Conclusions We concluded that whole-cell patch-clamp recordings are not suitable for studying synaptic LFS-induced LTD in rats older than 4 weeks of age and therefore cannot be used to explore electrophysiological disturbances, such as those induced by alcohol binge drinking during adolescence, which constitutes a late period of brain maturation

Proteomics Exploration Reveals That Actin Is a Signaling Target of the Kinase Akt*

International audienceThe serine/threonine kinase Akt is a key mediator of cell survival and cell growth that is activated by most growth factors, but its downstream signaling largely remains to be elucidated. To identify signaling partners of Akt, we analyzed proteins co-immunoprecipitated with Akt in MCF-7 breast cancer cells. Mass spectrometry analysis (MALDI-TOF and MS-MS) of SDS-PAGE-separated Akt co-immunoprecipitates allowed the identification of 10 proteins: ␣-actinin, valosin-containing protein, inhibitor B kinase, mortalin, tubulin ␤, cytokeratin 8, actin, 14-3-3, proliferating cell nuclear antigen, and heat shock protein HSP27. The identification of these putative Akt binding partners were validated with specific antibodies. Interestingly, the major protein band observed in Akt coimmunoprecipitates was found to be the cytoskeleton protein actin for which a 14-fold increase was observed in Akt-activated compared with non-activated conditions. The interaction between Akt and actin was further confirmed by reverse immunoprecipitation, and confocal microscopy demonstrated a co-localization specifically induced under growth factor stimulation. The use of wortmannin indicated a dependence on the phosphatidylinositol 3-kinase pathway. Using a phospho-Akt substrate antibody, the phosphorylation of actin on an Akt consensus site was detected upon growth factor stimulation, both in cellulo and in vitro, suggesting that actin is a substrate of Akt kinase activity. Interestingly, cortical remodeling of actin associated with cell migration was reversed by small interfering RNA directed against Akt, indicating the involvement of Akt in the dynamic reorganization of actin cytoskeleton germane to breast cancer cell migration. Together these data identify actin as a new functional target of Akt signaling. Molecular & Cellular Proteomics 6:114-124, 2007

Two Binges of Ethanol a Day Keep the Memory Away in Adolescent Rats: Key Role for GLUN2B Subunit

International audienceBackground: Binge drinking is common in adolescents, but the impact of only a few binges on learning and memory appears underestimated. Many studies have tested the effects of long and intermittent ethanol exposure on long-term synaptic potentiation, and whether long-term synaptic depression is affected remains unknown. Methods: We studied the effects of one (3 g/kg, i.p.; blood ethanol content of 197.5 +/- 19 mg/dL) or 2 alcohol intoxications (given 9 hours apart) on adolescent rat's memory and synaptic plasticity in hippocampus slice after different delay. Results: Animals treated with 2 ethanol intoxications 48 hours before training phase in the novel object recognition task failed during test phase. As learning is related to NMDA-dependent mechanisms, we tested ketamine and found the same effect as ethanol, whereas D-serine prevented learning deficit. In hippocampus slice, NMDA-dependent long-term synaptic depression was abolished 48 hours after ethanol or ketamine but prevented after D-serine or in a low-Mg2+ recording medium. Long-term synaptic depression abolition was not observed 8 days after treatment. An i.p. treatment with MK-801, tetrahydroisoxazolopyridine, or muscimol was ineffective, and long-term synaptic potentiation, intrinsic excitability, and glutamate release remained unaffected. The input/ouput curve for NMDA-fEPSPs was shifted to the left 48 hours after the binges with a stronger contribution of GluN2B subunit, leading to a leftward shift of the Bienenstock-Cooper-Munro relationship. Interestingly, there were no cellular effects after only one ethanol injection. Conclusion: Two ethanol ``binges'' in adolescent rats are sufficient to reversibly abolish long-term synaptic depression and to evoke cognitive deficits via a short-lasting, repeated blockade of NMDA receptors only, inducing a change in the receptor subunit composition. Furthermore, ethanol effects developed over a 48-hour period of abstinence, indicating an important role of intermittence during a repeated long-duration binge behavior

Two Binges of Ethanol a Day Keep the Memory Away in Adolescent Rats: Key Role for GLUN2B Subunit

BACKGROUND: Binge drinking is common in adolescents, but the impact of only a few binges on learning and memory appears underestimated. Many studies have tested the effects of long and intermittent ethanol exposure on long-term synaptic potentiation, and whether long-term synaptic depression is affected remains unknown. METHODS: We studied the effects of one (3g/kg, i.p.; blood ethanol content of 197.5±19mg/dL) or 2 alcohol intoxications (given 9 hours apart) on adolescent rat’s memory and synaptic plasticity in hippocampus slice after different delay. RESULTS: Animals treated with 2 ethanol intoxications 48 hours before training phase in the novel object recognition task failed during test phase. As learning is related to NMDA-dependent mechanisms, we tested ketamine and found the same effect as ethanol, whereas D-serine prevented learning deficit. In hippocampus slice, NMDA-dependent long-term synaptic depression was abolished 48 hours after ethanol or ketamine but prevented after D-serine or in a low-Mg(2+) recording medium. Long-term synaptic depression abolition was not observed 8 days after treatment. An i.p. treatment with MK-801, tetrahydroisoxazolopyridine, or muscimol was ineffective, and long-term synaptic potentiation, intrinsic excitability, and glutamate release remained unaffected. The input/ouput curve for NMDA-fEPSPs was shifted to the left 48 hours after the binges with a stronger contribution of GluN2B subunit, leading to a leftward shift of the Bienenstock-Cooper-Munro relationship. Interestingly, there were no cellular effects after only one ethanol injection. CONCLUSION: Two ethanol “binges” in adolescent rats are sufficient to reversibly abolish long-term synaptic depression and to evoke cognitive deficits via a short-lasting, repeated blockade of NMDA receptors only, inducing a change in the receptor subunit composition. Furthermore, ethanol effects developed over a 48-hour period of abstinence, indicating an important role of intermittence during a repeated long-duration binge behavior