FMRP Mediates Chronic Ethanol‐Induced Changes in NMDA, Kv4.2, and KChIP3 Expression in the Hippocampus

Abstract

Background Exposure to chronic ethanol results in changes in expression of proteins that regulate neuronal excitability. The present study examined whether chronic ethanol alters the hippocampal expression and function of Fragile-X mental retardation protein (FMRP), and the role of FMRP in the modulation of chronic ethanol-induced changes in expression of NMDA receptors and Kv4.2 channels. Methods For in-vivo studies, C57Bl6/J mice underwent a chronic intermittent ethanol (CIE) vapor exposure procedure. After CIE, hippocampal tissue was collected and subjected to immunoblot blot analysis of NMDA receptor subunits (GluN1, GluN2B), Kv4.2 and its accessory protein KChIP3. For in-vitro studies, hippocampal slice cultures were exposed to 75 mM ethanol for 8 days. Following ethanol exposure, mRNAs bound to FMRP was measured. In a separate set of studies, cultures were exposed to an inhibitor of S6K1 (PF-4708671, 6 μM) in order to assess whether ethanol-induced homeostatic changes in protein expression depend upon changes in FMRP activity. Results Immunoblot blot analysis revealed increases in GluN1 and GluN2B but reductions in Kv4.2 and KChIP3. Analysis of mRNAs bound to FMRP revealed a similar bidirectional change observed as reduction of GluN2B and increase in Kv4.2 and KChiP3 mRNA transcripts. Analysis of FMRP further revealed that while chronic ethanol did not alter the expression of FMRP, it significantly increased phosphorylation of FMRP at the S499 residue that is known to critically regulate its activity. Inhibition of S6K1 prevented the chronic ethanol-induced increase in phospho-FMRP and changes in NMDA subunits, Kv4.2 and KChiP3. In contrast, PF-4708671 had no effect in the absence of alcohol, indicating it was specific for the chronic ethanol-induce changes. Conclusions These findings demonstrate that chronic ethanol exposure enhances translational control of plasticity related proteins by FMRP, and that S6K1 and FMRP activity are required for expression of chronic ethanol-induced homeostatic plasticity at glutamatergic synapses in the hippocampus.

Acute Ethanol Produces Ataxia and Induces Fmr1 Expression via Histone Modifications in the Rat Cerebellum

“…Adolescent rats exposed to alcohol showed dendritic spine density deficits, increased Fmr1 mRNA expression, and increased H3K27 acetylation occupancy of the Fmr1 gene in the hippocampus (Mulholland et al., ). This followed the previous reports of altered hippocampal FMRP expression in mice following chronic EtOH vapor treatment (Spencer et al., ). One other recent study has elucidated that EtOH‐induced translation of the GABA type B receptor protein in the hippocampus requires FMRP, as shown in wild‐type mice compared with Fmr1 ‐knockout mice (Wolfe et al., ).…”

“…The present findings expand upon past research which has linked alcohol to alterations in the molecular and epigenetic landscape in the cerebellum of postmortem AUD subjects (Gatta et al., ) and in the cerebellum of rodents exposed to perinatal EtOH (Guo et al., ; Qi et al., ) or chronic adult EtOH exposure (Auta et al., ). They also expand upon limited recent research illustrating a role for Fmr1 following EtOH exposure, albeit one that was, until now, exclusive to the hippocampus (Mulholland et al., ; Spencer et al., ; Wolfe et al., ). Fmr1 is a dynamic epigenetic target that can alter expression of hundreds of transcripts, but that itself may also be regulated by microRNAs (Kenny et al., ; Liu et al., ; Tan et al., ) or its own noncoding antisense RNA Fxr4 (Pastori et al., ; Peschansky et al., ).…”

Differential potassium channel gene regulation in BXD mice reveals novel targets for pharmacogenetic therapies to reduce heavy alcohol drinking

“…Prolonged withdrawal from intermittent ethanol exposure enhanced A-type K + currents in medium spiny neurons in the NAc (Marty & Spigelman, 2012). Our previous studies showed that chronic ethanol reduced A-type K + currents and K V 4.2 channel surface expression in hippocampus, (Mulholland et al, 2015; Spencer et al, In Press), demonstrating differential effects of chronic ethanol on K V 4.2 channels in across brain structures. Interestingly, two SNPs in KCND2 (rs728115 and rs17142876) were recently identified as a risk factor for developing alcohol and nicotine co-dependence (Buhler et al, 2015; Zuo et al, 2012), and decreased expression of KCND2 was reported in the frontal cortex and amygdala (central and basolateral nuclei) of alcoholics (Ponomarev, Wang, Zhang, Harris, & Mayfield, 2012).…”

“…We previously reported that chronic ethanol exposure reduces expression and function of K Ca 2 ( Kcnn ) and K V 4.2 ( Kcnd2 ) channels in the NAc and hippocampus (Mulholland et al, 2015; Padula et al, 2015; Spencer et al, In Press). To identify novel K + channel genes in the NAc and PFC that are responsive to ethanol dependence, we utilized the Significance-score (S-score) algorithm (Wolen et al, 2012; Zhang et al, 2002) for probe-level analysis of each strain’s K + channel transcriptional response to CIE exposure.…”

“…Originally implicated for their role in epilepsy (Maljevic & Lerche, 2014; Smets et al, 2015), studies show that calcium-activated (K Ca ), voltage-dependent (K V ), and G protein-coupled inwardly-rectifying (K ir ) K + channels are targets for both acute and chronic effects of ethanol (Hopf et al, 2011; Mayfield, Blednov, & Harris, 2015; Mulholland, 2012; Mulholland, Hopf, et al, 2009). Additionally, preclinical studies have demonstrated that chronic ethanol reduces the function and trafficking of K Ca 2 ( Kcnn ), K V 4.2 ( Kcnd2 ), and K V 7.2 ( Kcnq2 ) channels in the nucleus accumbens (NAc) and hippocampus (Hopf et al, 2010; McGuier et al, 2015; Mulholland, Spencer, Hu, Kroener, & Chandler, 2015; Padula et al, 2015; Spencer, Mulholland, & Chandler, In Press), and pharmacologically enhancing K Ca 2 and K V 7 channel function attenuated voluntary drinking in rodents (Hopf et al, 2011; Knapp, O’Malley, Datta, & Ciraulo, 2014; McGuier et al, 2015; Padula et al, 2013). Ethanol actions on voltage- and calcium-dependent K Ca 1.1 ( Kcnma1 ) channels are involved in acute ethanol tolerance, dependence, and heavy ethanol consumption (Bukiya et al, 2014; Ghezzi, Pohl, Wang, & Atkinson, 2010; Kreifeldt, Le, Treistman, Koob, & Contet, 2013; Treistman & Martin, 2009), and Kcnma1 and Kcnq5 were identified as major hub genes for the acute actions of ethanol (Wolen et al, 2012).…”