Bidirectional Modulation of Alcohol-Associated Memory Reconsolidation through Manipulation of Adrenergic Signaling.

Alcohol addiction is a problem of great societal concern, for which there is scope to improve current treatments. One potential new treatment for alcohol addiction is based on disrupting the reconsolidation of the maladaptive Pavlovian memories that can precipitate relapse to drug-seeking behavior. In alcohol self-administering rats, we investigated the effects of bidirectionally modulating adrenergic signaling on the strength of a Pavlovian cue-alcohol memory, using a behavioral procedure that isolates the specific contribution of one maladaptive Pavlovian memory to relapse, the acquisition of a new alcohol-seeking response for an alcohol-associated conditioned reinforcer. The β-adrenergic receptor antagonist propranolol, administered in conjunction with memory reactivation, persistently disrupted the memory that underlies the capacity of a previously alcohol-associated cue to act as a conditioned reinforcer. By contrast, enhancement of adrenergic signaling by administration of the adrenergic prodrug dipivefrin at reactivation increased the strength of the cue-alcohol memory and potentiated alcohol seeking. These data demonstrate the importance of adrenergic signaling in alcohol-associated memory reconsolidation, and suggest a pharmacological target for treatments aiming to prevent relapse through the disruption of maladaptive memories.This work was supported by a UK Medical Research Council Programme Grant (G1002231) to BJE and ALM and was conducted in the Behavioural and Clinical Neuroscience Institute (BCNI), an initiative jointly funded by the MRC and the Wellcome Trust. MJWS was supported by an MRC Doctoral Training Grant and the James Baird Fund at the Medical School of the University of Cambridge. ALM was partly supported by a BCNI lectureship and the Ferreras-Willetts Fellowship from Downing College, Cambridge.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/npp.2015.24

Depot medroxyprogesterone acetate use is associated with elevated innate immune effector molecules in cervicovaginal secretions of HIV-1-uninfected women

Objective: The effects of sex hormones on the immune defenses of the female genital mucosa and its susceptibility to infections are poorly understood. The injectable hormonal contraceptive depot medroxyprogesterone acetate (DMPA) may increase the risk for HIV-1 acquisition. We assessed the local concentration in the female genital mucosa of cationic polypeptides with reported antiviral activity in relation to DMPA use. Methods: HIV-1-uninfected women were recruited from among couples testing for HIV in Nairobi, Kenya. Cervicovaginal secretion samples were collected, and the concentrations of HNP1-3, LL-37, lactoferrin, HBD-2, and SLPI were measured by enzyme-linked immunosorbent assays. Levels of cationic polypeptides in cervicovaginal secretions were compared between women who were not using hormonal contraception and those using DMPA, oral, or implantable contraception. Results: Among 228 women, 165 (72%) reported not using hormonal contraception at enrollment, 41 (18%) used DMPA, 16 (7%) used an oral contraceptive, and 6 (3%) used a contraceptive implant. Compared with nonusers of hormonal contraception, DMPA users had significantly higher mean levels of HNP1-3 (2.38 vs. 2.04 log10 ng/mL; P = 0.024), LL-37 (0.81 vs. 0.40 log10 ng/mL; P = 0.027), and lactoferrin (3.03 vs. 2.60 log10 ng/mL; P = 0.002), whereas SLPI and HBD-2 were similar. Conclusions: Although all analyzed cationic polypeptides have intrinsic antiviral capacity, their interaction and cumulative effect on female genital mucosa susceptibility to infections in vivo has yet to be unraveled. This study suggests a potential mechanism underlying the effect of DMPA on the innate immune defenses, providing a rationale to investigate its effect on HIV-1 acquisition risk

A high affinity switch for cAMP in the HCN pacemaker channels

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramole- cular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity

Heterogeneous nuclear ribonucleoprotein K: altered pattern of expression associated with diagnosis and prognosis of prostate cancer

Using proteomic analysis of the nuclear matrix (NM), we found that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the hnRNP family with pleiotropic functions, was differentially expressed in prostate cancer (PCa) tissues. This study aimed to characterise the expression of hnRNP K and its subcellular localisation in PCa, utilising immunohistochemical and quantitative western blot techniques. Furthermore, the hnRNP K expression was studied in human PCa cell lines in order to determine its modulation by bicalutamide, the anti-androgen widely used in PCa therapy. Immunohistochemical staining of paraffin-embedded tissues showed that hnRNP K was overexpressed in PCa, where it was localised both in the cytoplasm and in the nucleus. Staining of non-tumour tissues showed exclusively nuclear localisation and a less intense or absent signal. Immunoblot analysis demonstrated that the hnRNP K level within the NM was higher in PCa compared with non-tumour tissues and closely correlated with Gleason score (P=0.008). Higher expression within the NM was significantly (P=0.032) associated with poor prognosis. In two-dimensional western blot analysis hnRNP K presented several isoforms; the one with pI 5.1 was the most differently expressed between non-tumour and PCa tissues. Preliminary results indicate that hnRNP K can be modulated in vitro by a non-steroidal anti-androgen. Taken together, our findings suggest that hnRNP K has potential implications at the diagnostic, prognostic and therapeutic levels in PCa

Track A Basic Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138319/1/jia218438.pd

DESIGN OF CHIMERIC ION CHANNELS TO MONITOR CAMP-INDUCED CONFORMATIONAL CHANGES AND DYNAMICS

Hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are the molecular correlate of Ih (or If) current, which plays a key role in the control of neuronal and cardiac rhythmicity. HCNs are activated by the hyperpolarization of the membrane potential and further regulated by the direct binding of cyclic AMP (cAMP) to the cytoplasmic Cyclic Nucleotide Binding Domain (CNBD). cAMP binding determines the removal of the autoinhibitory action exerted by the CNBD on the pore opening and causes conformational changes that propagate from the CNBD to the pore through the C-linker, increasing channel open probability and speeding activation kinetics. cAMP modulation of HCN controls rhythmicity and excitability at cardiac and neuronal level and pain perception. Thus, in order to understand several physiological functions, as well as diseases affecting both the cardiac and neuronal system, a detailed description of the cAMP-induced conformational changes is required. Moreover, functional studies indicate that cAMP binding to the CNBD induces the transition of the C-terminal domains, from dimer of dimer to tetramer and that this transition is driven by the movements of the C-linker. The goal of this work is to describe the propagation of the movements through the C-linker to the pore. To this end, I have applied spectroscopic techniques, such as Electron Paramagnetic Resonance (EPR) and Double Electron-Electron Resonance (DEER) to study dynamic changes in the structure of the purified and labelled protein. First, I constructed a chimeric channel by fusing the C-linker/CNBD of human HCN4 to the prokaryotic pore domain of KcsA. This protein has the advantage that can be produced and easily purified in E. coli. Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC) measurements demonstrated that the purified chimera is able to bind cAMP. Particularly, ITC gave a Kd value of 1.7 \u3bcM, which agrees with the one previously published for the isolated C-linker/CNBD fragment of HCN4. Moreover, the rescue of a K+-uptake deficient E. coli strain demonstrated that the chimeric channel is able to conduct a K+ ions flow. EPR-DEER experiments performed on the chimera revealed that the binding of cAMP to the CNBD domain causes clear conformational changes in the C-terminal region, which transits from a dimer of dimers conformation to a 4-fold symmetrical gating ring. These data confirm previous biochemical and functional indication obtained on the full-length channel and give further details on the direction and the extent of subunit displacement occurring during the conformational transition. The transition of the channel from a dimer of dimer to a tetrameric configuration of the cytosolic domain might reflect a pre-conditioning state for cAMP action on channel gating and partly explains the agonist activity exerted by cAMP on channel kinetics and open probability