Synaptic microRNAs Coordinately Regulate Synaptic mRNAs: Perturbation by Chronic Alcohol Consumption

Abstract

Local translation of mRNAs in the synapse has a major role in synaptic structure and function. Chronic alcohol use causes persistent changes in synaptic mRNA expression, possibly mediated by microRNAs localized in the synapse. We profiled the transcriptome of synaptoneurosomes (SN) obtained from the amygdala of mice that consumed 20% ethanol (alcohol) in a 30-day continuous two-bottle choice test to identify the microRNAs that target alcohol-induced mRNAs. SN are membrane vesicles containing pre-and post-synaptic compartments of neurons and astroglia and are a unique model for studying the synaptic transcriptome. We previously showed that chronic alcohol regulates mRNA expression in a coordinated manner. Here, we examine microRNAs and mRNAs from the same samples to define alcohol-responsive synaptic microRNAs and their predicted interactions with targeted mRNAs. The aim of the study was to identify the microRNA-mRNA synaptic interactions that are altered by alcohol. This was accomplished by comparing the effect of alcohol in SN and total homogenate preparations from the same samples. We used a combination of unbiased bioinformatic methods (differential expression, correlation, co-expression, microRNA-mRNA target prediction, co-targeting, and cell type-specific analyses) to identify key alcohol-sensitive microRNAs. Prediction analysis showed that a subset of alcohol-responsive microRNAs was predicted to target many alcohol-responsive mRNAs, providing a bidirectional analysis for identifying microRNA-mRNA interactions. We found microRNAs and mRNAs with overlapping patterns of expression that correlated with alcohol consumption. Cell type-specific analysis revealed that a significant number of alcohol-responsive mRNAs and microRNAs were unique to glutamate neurons and were predicted to target each other. Chronic alcohol consumption appears to perturb the coordinated microRNA regulation of mRNAs in SN, a mechanism that may explain the aberrations in synaptic plasticity affecting the alcoholic brain.

Emerging roles for ncRNAs in alcohol use disorders

“…It is likely that the small changes detected in miRNAs, as well as mRNA expression, in PFC of human alcoholics is due to an increased expression that is localized to a specific cellular compartment, such as the neuronal synapse. Such compartmentalized, enhanced differential expression has been demonstrated in an animal model where miRNAs were isolated from synaptoneurosomes when compared to total, unfractionated tissue (Most, Leiter, Blednov, Harris, & Mayfield, 2016). …”

“…Synaptoneurosomes (SNs) contain membrane vesicles of pre- and postsynaptic compartments of neurons and perisynaptic compartments of astrocytes and microglia (Raab-Graham, Haddick, Jan, & Jan, 2006), and provide a model for studying the synaptic transcriptome. Alcohol-responsive miRNAs have been examined in SNs prepared from amygdala of mice following chronic two-bottle choice (2BC) drinking (Most et al, 2016). This research was microarray-based and used different informatics approaches to identify key alcohol-sensitive miRNA-mRNA synaptic interactions.…”

“…These animal models are advantageous because select miRNAs can be upregulated or downregulated using viral vectors, mimics (molecules designed to simulate naturally occurring mature miRNAs), and antagomirs (artificial antisense oligonucleotides) to determine their effect on alcohol-related behaviors. Studies have demonstrated that alcohol-induced changes in miRNAs are associated with cellular tolerance to alcohol (Pietrzykowski et al, 2008), antianxiety effects (Teppen, Krishnan, Zhang, Sakharkar, & Pandey, 2015), cellular reward mechanisms (Li et al, 2013), regulation of alcohol consumption and preference (Bahi & Dreyer, 2013; Li et al, 2013; Most et al, 2016; Tapocik et al, 2014), episodes of binge drinking (Darcq et al, 2015; Nunez, Truitt, Gorini, Ponomarev, Harris, et al, 2013; Tian et al, 2016), dependence/withdrawal (Gorini, Nunez, & Mayfield, 2013; Tapocik et al, 2013, 2014), and alcohol-induced conditioned-place preference (Bahi & Dreyer, 2013). Recent research articles published since 2012 are listed in Table 1.…”

“…For example, whole-genome miRNA studies found many differentially regulated miRNAs in binge-alcohol models in adolescent rats (Prins, Przybycien-Szymanska, Rao, & Pak, 2014; Tian et al, 2016) and adult mice (Gorini, Nunez, et al, 2013; Most et al, 2016; Nunez, Truitt, Gorini, Ponomarev, Harris, et al, 2013). Using a 2BC-drinking in the dark (DID) paradigm in mice bred for high alcohol consumption (C57BL/6J × FVB/NJ hybrid), Nunez and colleagues (Nunez, Truitt, Gorini, Ponomarev, Harris, et al, 2013) found that miRNA expression was predominantly upregulated in the PFC of alcohol-drinking mice (52 miRNA families), a finding consistent with previous results from human alcoholic PFC (Lewohl et al, 2011).…”

Molecular mechanisms underlying alcohol-drinking behaviours

“…Notably, changes in miRNA profiling were detected in the mPFC of alcohol-dependent rats 157 , and the levels of miRNAs were elevated in the PFC in post-mortem tissue from humans with a severe AUD 158, 159 . Interestingly, although alcohol intake produces changes in the levels of numerous miRNAs, the animal studies discussed above indicate that manipulation of the function of a single miRNA is sufficient to produce robust behavioural changes.…”