RNA-Binding Proteins, Neural Development and the Addictions

Genes, Brain and Behavior (2016) 15: 169 – 186 doi: 10.1111/gbb.12273

Review

RNA-binding proteins, neural development and the addictions

C. D. Bryant∗ and N. Yazdani RNA-binding proteins (RBPs) bind both RNAs and proteins to regulate all aspects of mRNA biogenesis and Laboratory of Addiction Genetics, Department of Pharmacology metabolism, ‘from the cradle (transcription) to the grave and Experimental Therapeutics and Psychiatry, Boston (decay)’ (Doyle & Kiebler 2012). They bind and package University School of Medicine, Boston, MA, USA specific pre-mRNAs and proteins into unique and highly *Corresponding author: C. D. Bryant, Laboratory of Addiction dynamic ribonucleoprotein (RNP) complexes to regulate Genetics, Department of Pharmacology and Experimental Ther- splicing, editing, polyadenylation, nuclear export, localization, apeutics and Psychiatry, Boston University School of Medicine, translation and stability (Glisovic et al. 2008). More than 1000 72 E. Concord St., L-606C, Boston, MA 02118, USA. E-mail: cam- mammalian genes code for RBPs and 20% of all protein [email protected] products represent RBPs (Gerstberger et al. 2014), reflect- ing the extensive splicing and diversity of RBP function. RNA-binding proteins possess modular RNA-binding motifs Transcriptional and post-transcriptional regulation of that cooperatively determine target specificity as well as gene expression defines the neurobiological mecha- auxiliary domains that mediate protein–protein interactions nisms that bridge genetic and environmental risk factors and post-translational modifications that can modify RNA with neurobehavioral dysfunction underlying the addic- binding, transport and localization of RBPs (Glisovic et al. tions. More than 1000 genes in the eukaryotic genome 2008). Importantly, post-translational modification of RBP code for multifunctional RNA-binding proteins (RBPs) signaling is potentially a useful strategy to prevent and treat that can regulate all levels of RNA biogenesis. More a variety of disease states (Kim et al. 2014; Wang et al. than 50% of these RBPs are expressed in the brain 2009). where they regulate alternative splicing, transport, Dozens of RBPs have established roles in neurodevelop- localization, stability and translation of RNAs during development and adulthood. Dysfunction of RBPs can ment and synaptic plasticity (Doxakis 2014) and a large lit- exert global effects on their targetomes that underlie erature documents the contribution of RBPs to neurode- neurodegenerative disorders such as Alzheimer’s and generative disorders (Romano & Buratti 2013) and neurode- Parkinson’s diseases as well as neurodevelopmental velopmental disorders such as autism and schizophrenia disorders, including autism and schizophrenia. Here, we (Bill et al. 2013; Fernandez et al. 2013). However, much less consider the evidence that RBPs influence key molecu- is known regarding RBPs and neuropsychiatric disorders lar targets, neurodevelopment, synaptic plasticity and such as the addictions. There are several reasons to sus- neurobehavioral dysfunction underlying the addictions. pect that RBPs play a crucial role in the addictions. First, Increasingly well-powered genome-wide association alternative splicing – one of the key nuclear functions of studies in humans and mammalian model organisms RBPs – is highly prevalent within the central nervous system combined with ever more precise transcriptomic and pro- (CNS) and is associated with several psychiatric disorders teomic approaches will continue to uncover novel and (Glatt et al. 2011). Human genome-wide association studies possibly selective roles for RBPs in the addictions. Key (GWAS) have yet to uncover statistically significant associ- challenges include identifying the biological functions ations between RBPs and the addictions; however, several of the dynamic RBP targetomes from specific cell types genome-wide significant intronic variants for psychiatric dis- throughout subcellular space (e.g. the nuclear spliceome orders have been identified within RBP targets that affect vs. the synaptic translatome) and time and manipulating RBP binding and splicing (Glatt et al. 2011). Second, sev- RBP programs through post-transcriptional modifica- eral RBPs play a critical role in neurodevelopment and, thus, tions to prevent or reverse aberrant neurodevelopment could mediate transcriptomic programs that are activated and plasticity underlying the addictions. following encounter with stressors during critical develop- Keywords: Amphetamine, dopamine, genetic, hnRNP, neu- mental periods and increase risk for the addictions (Ander- rodevelopmental, opioid, psychostimulant, RNA-binding pro- sen & Teicher 2009). Third, drug-induced synaptic plasticity tein, substance abuse, substance use disorder is an important component throughout all stages of addic- Received 9 September 2015, revised 30 October 2015, tion, and dozens of RBPs have been identified that exhibit accepted for publication 9 November 2015 cytoplasmic function in transporting, localizing and translating mRNAs in synaptic plasticity (Thomas et al. 2014; Tolino et al. 2012).

Considering the addictions as neuropsychiatric protein (FMRP) and heterogeneous nuclear ribonucleopro- disorders that have a neurodevelopmental teins (hnRNPs)]. Toward the end, we discuss those RBPs for component which there is less, yet accumulating evidence [RNA-binding protein, fox-1 homolog (RBFOX) and CUG-BP, ELAV-like factor (CELF) proteins]. Finally, we include a section on Genetic and fluctuating environmental risk factors affect neu- RNA editing and the addictions, as this exciting new area rodevelopment and the later neurobiological responses to of research comprises distinct molecular mechanisms from external stimuli (van Loo & Martens 2007). The addictions are the remainder of the review. Because multiple RBPs fre- gene × environment (G × E) disorders that require drug expo- quently coordinate in large RNP complexes to coordinate sure to manifest. Both genetic and environmental risk factors post-transcriptional regulation of mRNAs, we have attempted likely interact to affect neurodevelopment and neuropharma- to draw links between various discussed RBPs and their fam- cological sensitivity to reinforcing stimuli, including drugs and ilies whenever relevant, while keeping in mind that it would associated cues (Andersen & Teicher 2009; Leyton & Vezina be beyond the scope of the review to document all possible 2014). Severe childhood adversity is a key environmental risk connections between the large number of RBPs that are factor that greatly increases susceptibility to the addictions discussed. (Kendler et al. 2000) and early-life stress causes structural changes that affect mesolimbic reward function, including reduced hippocampal and prefrontal cortical development that may heighten dopamine release in the nucleus accum- FMRP bens (Andersen & Teicher 2009). We posit that a subset of RBPs orchestrate neurodevelopmental plasticity induced Fragile X mental retardation protein (FMRP) is a by environmental risk factors that increases susceptibility polyribosome-associated neuronal RBP that targets and to the addictions. The mesocorticolimbic dopaminergic translationally represses mRNAs associated with synaptic circuitry is involved in reward/aversion processing and pos- plasticity and has been implicated in autism, affective disor- itive/negative reinforcement learning in the addictions and ders, attention-deficit hyperactivity disorder, bipolar disorder, includes midbrain dopaminergic neuron projections from the schizophrenia and the addictions (Fernandez et al. 2013; ventral tegmental area to the medial prefrontal cortex and Smith et al. 2014). It is expressed throughout neurodevelop- nucleus accumbens and glutamatergic projections from the ment and is necessary for proper differentiation, migration, prefrontal cortex to the nucleus accumbens (Volman et al. axon formation, refinement and stabilization, synapse for- 2013). Mesocorticolimbic dysfunction is common in numer- mation and circuit wiring of neocortical layers (Till 2010). It ous neurodevelopmental and neuropsychiatric disorders, inhibits ribosomal translocation of mRNAs that is relieved especially the addictions (Dichter et al. 2012). Understanding following activity-dependent signaling to permit cytoskeletal the potential contribution of RBPs in mesocorticolimbic remodeling that underlies synaptic plasticity (Darnell & Klann development, environmental risk factor-induced plasticity 2013; Darnell et al. 2011). The N-terminal region contains and drug-induced plasticity could improve our understanding two Tudor domains that bind non-coding RNAs followed by of the heritable basis of addictive disorders, especially within a nuclear localization signal (NLS). The middle of the protein the context of G × E interactions (Wermter et al. 2010). contains two hnRNP K homology (KH) domains that further As an example of a hypothesized role for RBPs in G × E specify RNA and protein interactions. The C-terminal region interactions in neurodevelopment and plasticity underlying contains a nuclear export signal (NES) followed by an RGG psychiatric traits, a genetic variant in the gene coding for box that directly binds to mRNA targets (Fernandez et al. brain-derived neurotrophic factor (BDNF; the Val66/Met 2013). allele) that decreases activity-dependent secretion of Signaling by several different receptor types modulates BDNF is associated with phenotypic variation in psychi- FMRP activity, including mGluRs, AMPA, GABA, NMDA, atric endophenotypes (e.g. fear/aversion learning) during TrkB, dopamine and cannabinoid receptors (Fernandez et al. childhood vs. adolescence (Casey et al. 2009). Furthermore, 2013). Accordingly, FMRP targets hundreds of neurodevelop- individuals carrying the Val66/Met BDNF variant that also mental and neuroplasticity proteins involved in cytoskeletal underwent early childhood adversity (institutionalization) remodeling; many of these targets have been associated showed a decrease in cortical volume, an increase in amyg- with the addictions, including mGluR1, mGluR5, PSD-95, dala volume, an increase in behavioral anxiety and an increase CYFIP1/2, GABA-A receptor subunits, NR2A/2B NMDA in cortisol stress response (Casey et al. 2009). Accumulating receptor subunits, Homer1, neuroligins, CREB-binding pro- evidence indicates that RBPs can regulate translation of tein and D1 dopamine receptor-coupled GRK2 (Darnell & BDNF (Allen et al. 2013; Vanevski & Xu 2015) and that an Klann 2013; Wang et al. 2008). The FMRP also targets elon- increase in BDNF signaling can increase translation and gation factor-1 and -2 (EF-1, EF-2), argonaute 1/2 (Ago1/2) and synaptic localization of RBPs (Castren et al. 2002). In this Dicer, which are ubiquitously involved in protein translation review, we summarize recent examples of RBPs that affect and miRNA processing, respectively (Darnell & Klann 2013). primary molecular targets and cellular, neurodevelopmental Thus, FMRP can also exert widespread, indirect regulation and neurobehavioral functions relevant to the addictions of the translatome. (Table 1). We begin by discussing examples that contain the With regard to drugs of abuse, deletion of FMRP most direct experimental and circumstantial evidence that perturbs midbrain dopaminergic neuron development, draws a link to the addictions [fragile X mental retardation amphetamine-induced dopamine release in the striatum and

170 Genes, Brain and Behavior (2016) 15: 169 – 186 RNA-binding proteins, neural development and the addictions et al. 2015) 2013; 2014; 2009; 2014; 2015 2014; 2012; 2012; 2013; et al. et al. 2011; et al. 2011; 2011) 2011; Fish et al. et al. et al. 2012) et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. 2013; Fulks et al. 2010; Kumar 2013; Pilaz & Silver 2015; Smith 2014) Wang Dougherty Maloney Watanabe Zaniewska et al. Dracheva Satterlee Schmidt Halgren Hart Tiruchinapalli 2008) (Johnson (Dougherty 2015; (Bhogal (Akubuiro (Comuzzie (Pascale result References psychostimulant- induced changes in expression; co-regulated with Nurr1 reward amphetamine neurobehavioral plasticity, synaptic and structural plasticity eat in a competing reward environment, novelty-induced locomotor activity, alcohol drinking, cocaine abstinence and seeking, nicotine withdrawal in humans, hyperactivity, hyperphagia- associated obesity, food-related obsessions changes in expression and phosphorylation Cocaine-conditioned Cocaine and Addiction-associated Overeating, motivation to Amphetamine response circuitry tegmental area, locus coeruleus, prefrontal and hippocampal cortices, hypothalamus circuitry, hippocampus hypothalamus, striatum, mesocorticolimbic circuitry, dorsal raphe nucleus, forebrain, cortex, hippocampus, diencephalon hippocampus, cortex Basal forebrain, ventral Mesocorticolimbic Addiction-relevant Ubiquitous expression, Ubiquitous expression, Neocortex, hippocampus Cocaine-induced neurodevelopment, beginning at E14 maintenance, neurite, axonal and dendritic outgrowth, glutamate receptor trafﬁcking, dendritic spine morphology induction and excitation, corticothalamic development, synaptic transmission and function, predicted synaptic plasticity maintenance, maturation, axon growth, dendritogenesis, synaptic plasticity Neurodevelopment, Neurogenesis Ubiquitous expression Opioid and Expressed during Cell fate, progenitor neuroplastic function Neural differentiation and Neuronal differentiation Neuronal differentiation, UTR binding UTR binding, ′ ′ translation, export, stability repression repression via stalling of ribosomal translocation, stability binding and miRNA biogenesis and mRNA stability, translation, localization translation, polyadenylation, stability, protein synthesis, transport Splicing, 3 Splice enhancement and Transport, translational RNA editing, pri-miRNA targets RNA function Nova1, GABA-A receptor, glycine alpha-2 receptor MAPT mGluR1, mGluR5, ADAR, PSD-95, Homer1, CREB BP, GRK2 receptor, GABA-A receptor Addiction-relevant RBPs associated with the addictions ELAVL cfos, BDNF,GAP-43, CELF4 NMDA receptors, MAPT Splicing, 3 HNRNPA1 NMDA receptors Splicing, spliceosome, CELF6 Serotonergic neurons, FMRP Cyﬁp1/2, AMPA receptor, Ta b l e 1 : RBP ADAR1/2 5-HT2C receptor, AMPA

Genes, Brain and Behavior (2016) 15: 169 – 186 171 Bryant and Yazdani 2014; 2011; 2011) 2012; Xu 2015; 2007, 2008; Du 2011) 2014; Fogel 2011) 2011) et al. et al. et al. et al. et al. 2012; Zhong 2015) 1998) 2014; Yazdani 2015) et al. et al. et al. et al. et al. et al. Choi 2008, 2009; Pilaz & Silver 2015) et al. et al. Choi et al. et al. et al. Song (Park (Sasabe (Park (Wang (Boschi (Feng (Banerjee (Johnson Nurr1 result References dopamine receptor splicing dopamine receptor psychostimulant- induced change in expression changes in TH mRNA reward, nicotine and alcohol dependence transcription, promotes SERT translation, activates transcription of beta2 subunit of the neuronal nicotinic acetylcholine receptor splicing, methamphetamine stimulant response, association with expression Splicing of D2 Nicotine-induced Cocaine-conditioned Activates TH and MOR Addiction-associated circuitry (Nova1), hindbrain, motoneurons and ventral spinal cord (Nova2) midbrain, locus coeruleus forebrain, neocortex, hippocampus, nucleus accumbens circuitry, hippocampus, ventral midbrain Neocortex and midbrain Cortex, hippocampus D2 dopamine receptor TH-positive cells of the Ubiquitous, basal Mesocorticolimbic Addiction-relevant neuronal migration of cortical and purkinje neurons, neuronal inhibition synapse maturation and stability, plasticity axonogenesis membrane excitability plasticity, dendritic spine morphology, ﬁlopodia formation, synapse maturation, axon development Neurodevelopment, Synaptogenesis, Neuronal differentiation, Corticogenesis, Unknown Ubiquitous expression Heroin addiction, MOR Transmission, Hippocampal synaptic neuroplastic function UTR ′ UTR UTR ′ ′ UTR binding ′ polyadenylation splicing, 3 polyadenylation, nonsense-mediated decay, transport, stability, translation initiation splicing activity, polyadenylation and cleavage, 5 binding and stability, translational co-activation and repression translational suppression and stability, polyadenylation binding and stability, translation Transcription, 3 Splicing, 3 Repression of splicing, Transcription, silencing, Splicing , Splicing, regulation of targets RNA function fosB, PSD-95, PTBP, MOR HNRNPH1, PACAP receptor, GABA-A receptor, NMDA receptor, Grip1, Csnk1d, PTBP GABA-A and GABA-B receptors, NMDA receptor, nicotinic acetylcholine receptor, GIRK2 nicotinic acetylcholine receptor beta2 Δ GABA-A receptor, Grip1, glycine receptor alpha2 receptor, Per2 Tyrosine hydroxylase, Cell adhesion molecules, D2 dopamine receptor, Addiction-relevant Continued (HNRNPE1–4) (A2BP1) (HNRNPI) PCBP 1–4 RBFOX1 Nova1/2 D2 dopamine receptor, HNRNPK TH, MOR, SERT, neuronal HNRNPM1 D2 dopamine receptor Unknown Unknown Unknown Splicing of D2 PTBP1–2 HNRNPH2 Splicing Unknown Low expression Opioid and Ta b l e 1 : RBP HNRNPH1 OPRM1, RBFOX, NMDA

172 Genes, Brain and Behavior (2016) 15: 169 – 186 RNA-binding proteins, neural development and the addictions

prefrontal cortex, psychostimulant-induced locomotor activity, stereotypy and drug reward (Fish et al. 2013; Fulks et al. 2010;

2012) 2010). The FMRP deletion also disrupts cocaine-induced neu-

2012; robehavioral plasticity in the nucleus accumbens, including et al. et al. 2013) decreased locomotor sensitization, enhanced stereotypy,

et al. perturbed dendritic morphology, changes in AMPA/NMDA

et al. receptor ratios and glutamatergic transmission and reduced Lapidus Li cocaine reward that is associated with increased mGluR5 (Bryant (Benderska activation (Smith et al. 2014). In prefrontal cortical neurons, D1 dopamine receptor activation is sufficient to induce FMRP phosphorylation and synthesis of synaptic proteins involved 𝛽 in glutamate receptor trafficking and plasticity (Wang et al. 2008, 2010). In another example that indirectly implicates FMRP in the addictions, cytoplasmic FMRP-interacting result References protein 1 (CYFIP1) inhibits elf4E-mediated cap-dependent mRNA translation of proteins involved in actin cytoskeleton reward, methamphetamine stimulant response splicing of RGS4; DARPP-32 induces splicing of Tra2 remodeling and dendritic spine maturation (De Rubeis et al. Cocaine-conditioned Morphine-induced Addiction-associated 2013; Napoli et al. 2008) and mutations in the closely related Cyfip2 gene in mice modulate psychostimulant-induced locomotor activity and sensitization, dendritic morphology and AMPA receptor neurotransmission (Kumar et al. 2013). In addition to an mGluR5 mechanisms underlying neurobehavioral dysfunction in the absence of FMRP (Bear et al. 2004; Smith et al. 2014), neuronal activity and BDNF/TrkB signaling circuitry can regulate expression of FMRP in the hippocampus (Cas- forebrain, hippocampus cerebral cortex Ubiquitous expression, Addiction-relevant Locus coeruleus, tren et al. 2002) and FMRP deletion perturbs both BDNF and TrkB spatiotemporal expression and signaling in neurodevelopment and neuroplasticity (Castren & Castren 2014). Thus, both BDNF/TrkB and mGluR5 signaling could converge on FMRP mechanisms of psychostimulant neurobehavioral plasticity (Kumar et al. 2013; Smith et al. 2014). Because FMRP deletion increases protein translation and disrupts structural and synaptic plasticity induced by gluta- outgrowth and branching, dendritic development and morphology, hippocampal development and plasticity, corticogenesis, axonogenesis, synaptogenesis, regeneration differentiation, cortical neurogenesis mate receptor signaling (e.g. mGluR-LTD), an important ques- Neurodevelopment, Axon guidance, neuroplastic function tion is whether or not fine-tuning glutamatergic signaling by the use of pharmaceuticals targeting NMDA receptors and mGluR can improve the outcome of neurodevelopmental and neuropsychiatric disorders associated with FMRP dysfunction (Bear et al. 2004; Michalon et al. 2012), including the addictions (Cleva et al. 2010). The FMRP also undergoes post-translational modifications, including mGluR-mediated dephosphorylation and relief from translational repression UTR binding and stability, translational repression, axonal mRNA transport and localization

′ as well as mTOR-mediated dephosphorylation by protein 3 phosphatase 2A (PP2A) and re-establishment of translational repression via PP2A suppression (Ceman et al. 2003; Narayanan et al. 2008), presenting additional opportunities for perturbing FMRP mechanisms of neurodevelopmental and neuropsychiatric dysfunction.

hnRNPs targets RNA function many other predicted targets Addiction-relevant RGS4, glutamate receptors Splicing Neuronal survival, Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large and diverse group of nucleoplasmic-localized multifunctional RBPs that form RNP complexes and can reg-

Continued ulate splicing, export, localization, translation and stability (Dreyfuss et al. 2002; Han et al. 2010). Rapidly accumulat- 𝛽 ing studies are identifying new contributions of hnRNPs to

Ta b l e 1 : RBP Tra2 ZBP1 B-actin, GAP-43, Homer1, neurodevelopment (Liu & Szaro 2011; Sinnamon et al. 2012)

Genes, Brain and Behavior (2016) 15: 169 – 186 173 Bryant and Yazdani and synaptic plasticity (Folci et al. 2014; Leal et al. 2014; synaptic/neuronal function, transmission and signaling Sinnamon & Czaplinski 2011; Zhang et al. 2012). Heteroge- (Wang et al. 2011). We are currently evaluating the potential neous nuclear ribonucleoproteins contain highly conserved, role of Hnrnph1 in the rewarding and reinforcing proper- modular RNA recognition motifs (RRMs) that determine ties of psychostimulants and opioids and changes in the sequence binding specificity and affinity and intervening transcriptome and spliceome associated with Hnrnph1 regions that localize and permit generalized and specific roles dysfunction. in post-transcriptional processing. The hnRNPs were origi- Hnrnph1 is highly and ubiquitously expressed through- nally classified based on their co-immunopurification in RNA out the mouse brain (Lein et al. 2007). It contains three complexes with monoclonal antibodies against the founding quasi-RRMs that mediate binding to poly-(G) tracts to member hnRNP C, thus identifying hnRNP A-U (Dreyfuss either enhance or silence splicing (Han et al. 2010). hnRNP et al. 1993). Although hnRNPs can be highly similar in protein H protein is primarily localized to the nucleus in primary structure and function, their nomenclature is not consistent rat cortical neurons and depolarization via KCl application with their sequence homology, suggesting independent evo- increased the intensity of nuclear immunocytochemical lution and expansion of several RRMs. As examples, some staining (Fig. 1), suggesting activity-dependent nuclear func- hnRNPs share greater sequence similarity with other RBP tion that is consistent with its role in alternative splicing and classes such as hnRNP A1 vs. ELAVL4/CELF1 or hnRNP C polyadenylation (Katz et al. 2010). Nevertheless, hnRNP H1 vs. the SR protein transformer-2𝛽 (Tra2𝛽)(Tanget al. 2012). also contains three, intervening glycine-rich domains and the Furthermore, other hnRNPs such as E/K, I/L, U, I (PTBP1) and central domain contains an NLS that permits bidirectional hnRNP F/H contain separate, quasi-RRMs that do not share transport between the nucleus and cytoplasm (Van Dusen sequence homology with canonical RRMs of hnRNPs (Tang et al. 2010). These modular glycine-rich domains are also et al. 2012). Thus, discussing hnRNPs as a separate class necessary for regulation of splicing (Wang et al. 2012) and is somewhat arbitrary but for ease of reference, here we likely mediate interaction with other proteins, including will discuss addiction-relevant RBPs that have been named complexing and cooperating with hnRNP A1, RBFOX2 and hnRNPs and in the immediately following sections, we will hnRNP F to enhance or suppress exon inclusion (Fisette et al. discuss hnRNPs that have alias names. 2010; Mauger et al. 2008). hnRNP H1 can also cooperate The mu opioid receptor (MOR) is a primary molecular tar- with hnRNP I (see PCBP-1 below) to form a splicer enhancer get for the addictive properties of opioids and other drugs of complex in neurons (Chou et al. 1999). abuse (Contet et al. 2004) and undergoes alternative splic- To summarize, increasing evidence indicates that hnRNPs ing by hnRNP H1 binding to the intronic AGGG sequence and such as hnRNP H1 may be involved in establishing multiple recruiting hnRNP A1, A2B1, AB, C, H3 and U, which results in addictions. The role of hnRNP H1 in activity-dependent exclusion of exon 2 and decreased expression of Oprm1 (mu synaptic plasticity is not known although intriguingly, opioid receptor; MOR) (Xu et al. 2014). The MOR intronic SNP Hnrnph1 was identified as one of the most significantly rs9479757 decreased binding of hnRNP H1, resulting in exon upregulated transcripts in transcriptome analysis of corti- 2 skipping and increased MOR expression that was associ- cal brain tissue following experimental traumatic brain injury ated with increased severity of heroin dependence (Xu et al. (Kobori et al. 2002), which could indicate a role in regenerative 2014). hnRNP H1 and hnRNP F can also post-transcriptionally plasticity. Available evidence from our laboratory indicates regulate MOR expression by repressing translation at the 5′ that neuronal stimulation induces an increase in hnRNP H UTR (Song et al. 2012), providing a second level of MOR reg- staining that is localized to the nucleus (Fig. 1), suggesting ulation by hnRNP H1. that the contribution of hnRNP H1 to neuroplasticity is either We used fine-scale gene mapping with interval-specific indirect (e.g. via nuclear splicing or nuclear polyadenylation congenic mouse lines to identify a 206-kb region on chro- of synaptic targets) or that dendritic localization of hnRNP mosome 11 containing Hnrnph1 (the gene coding for hnRNP H requires signaling through specific receptors. We are H1) and Rufy1 that was necessary for reduced sensitivity currently testing the latter hypothesis in the context of the methamphetamine-induced locomotor activity. Replicate addictions by treating neurons with dopamine receptor ago- mouse lines harboring transcription activator-like effector nists and examining changes in hnRNP H staining. Because nucleases (TALENs)-induced frameshift deletions in Hnrnph1 hnRNP H1 can undergo several post-translational modifica- recapitulated the quantitative trait locus (QTL) phenotype, tions such as phosphorylation, methylation and sumoylation thus identifying Hnrnph1 as the quantitative trait gene (Yaz- (Chaudhury et al. 2010), there is potential to fine-tune hnRNP dani et al. 2015). Inheritance of this QTL caused a decrease H1 signaling to prevent or normalize plasticity associated in expression of Bdnf, Elavl2, Elavl4 and Nurr1 (nuclear with the addictions. receptor-related 1 protein) (Yazdani et al. 2015), a transcription factor that is crucial for the development and function of midbrain dopaminergic neurons (Campos-Melo et al. 2013). PCBPs (including hnRNP K) hnRNP H1 (along with hnRNP A1, K and M) has been shown to be co-regulated with Nurr1 expression across neuronal cell Polycytosine-binding proteins (PCBPs) 1–4 (hnRNP E1–E4) lines (Johnson et al. 2011) and a recent review of proteomic and hnRNP K bind with high affinity to poly(C) DNA and RNA studies of neuronal and brain expression with drugs of abuse sequences to regulate transcription and post-transcriptional identified psychostimulant- and opioid-induced changes in processing via three, modular hnRNP KH RNA-binding hnRNP H2 and A1 expression that could regulate gene domains (Han et al. 2010). They also contain an intervening networks enriched for protein modification/degradation, sequence between the second and third KH domains with

174 Genes, Brain and Behavior (2016) 15: 169 – 186 RNA-binding proteins, neural development and the addictions

(A) No Tx 1 h 20mM KCl 2 h 20mM KCl

(B) 1200 No Tx * ** 1000 1h 2h 800

600

400

200 Intensity/frame (arb.units)

DAPI hnRNPH

Figure 1: Immunocytochemical staining of hnRNP H in rat cortical neurons following KCl-induced depolarization. (a) Primary neocortical neurons were dissected from E18 Sprague-Dawley rat embryos (Charles River Laboratories, Newton, MA). Dissociated neurons were cultured neurons for 1 week. For the control, no treatment (No Tx) group, 1 ml of conditioned media was replaced with 1 ml of neurobasal media. For the 1-h and 2-h Tx groups, 1 ml of conditioned media was replaced with 1 ml of 20 mM KCl-enriched neurobasal media. Treated neurons were then washed, fixed, permeabilized, blocked and incubated with primary hnRNP H antibody (1:500 rabbit polyclonal, Bethyl Labs Montgomery, TX) in 1% bovine serum albumin (BSA) overnight at 4∘C. Twelve hours later, neurons were washed and incubated with an Alexa Fluor 594 antibody (1:500 donkey anti-rabbit, Life Technologies, Carlsbad, CA) in 1% BSA. Processed coverslips were then stained with DAPI (blue) and mounted onto glass slides. Images were collected using a Zeiss AxioObserver microscope (Carl Zeiss Inc., Jena, Germany) under uniform settings for all three groups. Twenty serial images (frames) were captured per condition and fluorescence was quantified using ImageJ 1.49 under a uniform threshold range. Note both an increase in the number of H1-stained neurons following 1–2 h of KCl Tx as well as an increase in the fluorescent staining intensity after KCl treatment. (b) Semiquantification of fluorescence staining intensity. One-way ANOVA indicated a main effect of genotype (F 2,57 = 8.4; P = 0.0006). *P = 0.01; **P < 0.001 (unpaired t-tests vs. No Tx).

NLSs (PCBP1 and 2) or an hnRNP K-speciﬁc nuclear shuttling neurodevelopment, transport, localization and cytoskeleton, domain to permit bidirectional transport. hnRNP K also has including GAP-43 (Liu & Szaro 2011; Liu et al. 2008). hnRNP a SH-3 (KI)-binding domain and both hnRNP K and PCBP4 K exhibits early developmental expression in several compo- contain an NLS in the third KH domain (Choi et al. 2009). nents of the mesocorticolimbic circuitry and subsequently a hnRNP K, PCBP1 and PCBP2 are located primarily in the more restricted expression to the hippocampus (Blanchette nucleus whereas PCBP3 and 4 are located primarily in the et al. 2006). hnRNP K can bind to the TH promoter to acti- cytoplasm (Chaudhury et al. 2010). vate transcription (Banerjee et al. 2014) and co-localizes with In neurodevelopment, PCBPs regulate axonogenesis TH in the ventral midbrain. Thus, hnRNP K could regu- (Thyagarajan & Szaro 2004) and corticogenesis (Pilaz & Sil- late catecholamine synthesis during midbrain dopaminergic ver 2015). PCBP1–3 are expressed in the adrenal medulla, neuron development, maintenance and synaptic plasticity midbrain and locus coeruleus, respectively, where they in adulthood (Folci et al. 2014). hnRNPK also binds to the bind the 3′ UTR of tyrosine hydroxylase (TH, the enzyme polyadenylation sequence within the 3′ UTR of the serotonin necessary for synthesis of dopamine and norepinephrine) transporter (SERT), a primary molecular target for cocaine in the cytoplasm to increase stability and protein translation reward (Sora et al. 2001), to increase SERT protein levels (Boschi et al. 2015; Czyzyk-Krzeska & Beresh 1996). In addi- by preventing miR-16-mediated inhibition of translation (Yoon tion to regulating catecholamine synthesis, hnRNP K, PCBP1 et al. 2013). In addition to its link to catecholamines and and PCBP2 can all bind to the MOR promoter to activate monoamines, hnRNP K can bind to the MOR promoter and to MOR transcription whereas PCBP3 acts as a transcriptional the promoter of the beta-2 subunit of the neuronal nicotinic repressor (Choi et al. 2007, 2008, 2009). acetylcholine receptor to activate transcription (Choi et al. hnRNP K interacts with ELAVL2 to control neuronal dif- 2008; Du et al. 1998). Finally, morphine can stimulate hnRNP ferentiation (Yano et al. 2005) and regulates axonogenesis K translation in multiple brain regions independent from tran- via post-transcriptional interaction with genes involved in scription (Lee et al. 2014).

Genes, Brain and Behavior (2016) 15: 169 – 186 175 Bryant and Yazdani

To summarize, PCBPs and hnRNP K are associated with proliferation, actin cytoskeleton and neuronal differentiation neurodevelopment, catecholaminergic, monoaminergic, opi- (Licatalosi et al. 2012). oidergic and cholinergic signaling and, thus, are prime sus- With regard to dopamine receptors and the addictions, the pects in regulating neurotransmitter signaling underlying the short and long splice forms of the D2 dopamine receptor addictions. Importantly, post-translational modifications can (D2L and D2S) are highly conserved and have different signal- also significantly impact cell function of PCBPs. A notable ing properties and physiological function (Picetti et al. 1997) example is ERK-induced phosphorylation of hnRNP K, which that have been associated with the addictions (Levran et al. translocates hnRNP K to the cytoplasm where it can then 2015; Smith et al. 2002). Both PTBP1 and Nova1 promote bind to the 3′ UTR of mRNAs to regulate protein translation exon 6 inclusion whereas hnRNP M inhibits exon 6 inclusion, (Habelhah et al. 2001). Therefore, therapeutic modulation of thus bidirectionally regulating D2L vs. D2S expression (Park hnRNP K signaling could be a future treatment avenue in the et al. 2011; Sasabe et al. 2011). Overexpression of PTBP1 addictions. in vitro was associated with a decrease in transcription of ΔfosB, a stable splice variant of the transcription factor fosB that accumulates in the nucleus accumbens following PTBPs (including PTBP-1; a.k.a. hnRNP I) chronic administration of drugs of abuse and is associated with sustained drug-induced synaptic plasticity (Alibhai et al. 2007; Nestler et al. 2001). Thus, perhaps post-translationally Polypyrimidine tract-binding proteins (PTBPs) bind to both modifying PTBP1, e.g. via phosphorylation and cyto- intronic and exonic polypyrimidine tract sequences to repress plasmic translocation (Xie et al. 2003), could modulate exon inclusion. They contain four RRMs, an NLS and an ΔfosB-mediated neuroplasticity associated with the NES in the N-terminus that permit alternative splicing, shut- addictions. tling, polyadenylation, transport, localization, stabilization and translation (Keppetipola et al. 2012; Romanelli et al. 2013). RRM-1 and RRM-2 are separated by flexible linkers that per- ELAVL mit independent conformations whereas RRM-3 and -4 form a globular structure and binds to nearby, intervening pyrim- The neuronal-specific mammalian embryonic lethal, abnor- idine tracts to induce RNA looping (Romanelli et al. 2013). mal vision-like (ELAVL)2, 3 and 4 RBPs are an RBP family Alternatively spliced isoforms of PTBP1 can contain longer based on homology to ELAV protein in Drosophila (Robi- intervening domains or isoforms that lack RRM-1 and -2, now et al. 1988) and regulate the transport, stabilization, thus diversifying PTBP targets and function within the con- localization and translation of mRNAs. They contain three text of an RNP complex. Splicing repression by PTBP can RRMs that mediate binding to intronic targets for splicing as depend on its proximity to the splice site, its antagonism well as binding to highly conserved AU-rich element (ARE) by other RBP splicing co-factors such as CELF proteins (Gro- sequences of 3′ UTR targets to regulate stability (Colombrita mak et al. 2003; Spellman et al. 2005) and neuro-oncological et al. 2013), including Nova1 (Ratti et al. 2008). ELAVL4 ventral antigen1 (Nova1) (Polydorides et al. 2000), and its can also enhance cap-dependent translation via structural interaction with co-repressors such as the Raver proteins unwinding of 5′ UTRs (Fukao et al. 2009). ELAVL RBPs (Henneberg et al. 2010). In addition to splicing, PTBP1 can contribute to all stages of neuronal differentiation, mainte- ′ bind directly to 3 UTRs to regulate cleavage in polyadenyla- nance, synaptogenesis and activity-dependent synaptic plas- ′ tion, 3 UTR exon inclusion and mRNA stability (Sawicka et al. ticity (Perrone-Bizzozero & Bolognani 2002). The localization 2008). Finally, PTBPs interact with PCBPs and act at the inter- of ELAVL RBPs in the neocortex and hippocampus (Okano & nal ribosomal entry site to initiate translation (Bushell et al. Darnell 1997) suggests potential involvement in neurodevel- 2006). opmental and neuroanatomical risk for the addictions (Ander- During neurodevelopment, reciprocal changes in PTBP1 sen & Teicher 2009). RNA-binding protein target analysis and 2 expression coordinate differential splicing of PSD-95 of ELAVL of mouse forebrain tissue identified a spliceome to control the timing of neuronal differentiation (Zheng enriched for axonal and synaptic cytoskeleton dynamics and et al. 2012). PTBP1 is expressed in neural progenitors and 3′ UTR-regulated genes involved in amino acid synthesis. Of decreases during differentiation and is restricted to glia during note, ELAVL regulates the splicing and half-life of glutami- adulthood. In contrast, expression of the gene paralog PTBP2 nase, indicating an essential role in excitatory neurotransmis- increases during neuronal differentiation, decreases during sion (Ince-Dunn et al. 2012). cortical maturation and shows moderate neuronal expression Acute cocaine treatment caused a decrease in whole-brain during adulthood (Keppetipola et al. 2012). Notably, PTBP1 FMRP expression and an increase in ELAVL expression inhibits the expression of PSD-95 in neural progenitors that was associated with an increase in expression of whereby it represses the inclusion of a coding exon which genes enriched for dendritic synaptic plasticity (Tiruchina- results in nonsense-mediated decay and delays excitatory palli et al. 2008). Combining repeated cocaine administra- synapse stabilization and maturation (Keppetipola et al. 2012; tion with swim stress increased ELAVL4 phosphorylation Zheng et al. 2012). A similar mechanism is used by PTBPs and translation in the hippocampus as well as expression to inhibit their own expression (Boutz et al. 2007). PTBP2 is of its canonical cytoskeletal target, growth-associated pro- essential for postnatal survival and genome-wide RNA target tein (GAP)-43 (Pascale et al. 2011), which could contribute analysis of the developing mouse brain revealed that PTBP2 to structural plasticity and drug-associative learning (Bolog- inhibits a splicing program involved in neuronal cell cycle, nani et al. 2007). Importantly, ELAVL4 selectively binds to

176 Genes, Brain and Behavior (2016) 15: 169 – 186 RNA-binding proteins, neural development and the addictions the unique ARE sequence of the long 3′ UTR but not the stress (Pascale et al. 2011), in vivo axonal injury induced an short 3′ UTR of BDNF mRNA to increase BDNF stability and increased in the expression of GAP-43 that interacts with activity-dependent translation (Allen et al. 2013). The increase ELAVL4 as well as ZBP1 in a complex to induce axonal out- in BDNF translation is mediated by PKC-induced phosphory- growth and branching (Yoo et al. 2013). It was recently shown lation of ELAVL4, which disinhibits 3′ UTR-mediated trans- that although both ZBP1 and ELAVL4 interact with beta-actin, lational repression of the long 3′ UTR BDNF mRNA in hip- ELAVL4 binds specifically to the ARE sequence in the 3′ UTR pocampal neuronal dendrites (Vanevski & Xu 2015). Selec- whereas ZBP1 requires a specific secondary structure (Kim tive regulation of the long 3′ UTR of BDNF is important et al. 2015) that could permit its ability to form a EVAL/ZBP because it is responsible for activity-dependent neuronal complex in neuroplasticity underlying axonal regeneration translation of BDNF (Lau et al. 2010) and synaptic matu- and possibly cocaine neuroplasticity. In further support ration in the dendrites (An et al. 2008). Psychostimulant of a link between ZBP1 and cocaine, transgenic, ectopic administration increases the expression of BDNF (Russo expression of ZBP1 in the striatum during adulthood blocked et al. 2009) and in turn, BDNF can regulate neuronal expres- cocaine-induced conditioned place preference that was res- sion of FMRP and synaptic protein translation (Castren cued by eliminating ZBP1 expression. Direct, experimental & Castren 2014). Thus, an ELAVL-mediated increase in target analysis identified nearly 200 transcripts involved in activity-dependent translation of BDNF and other mRNA tar- synaptic plasticity that could be responsible for the effect gets may work in parallel with FMRP to induce neurobehav- of ZBP1 on cocaine reward, including the scaffolding gene ioral plasticity in response to drugs of abuse (Smith et al. Homer1 that negatively regulates cocaine reward (Szumlin- 2014). Interestingly, an increase in BDNF expression may ski et al. 2004) as well as cadherins, transcription factors, serve as a biomarker for severity of psychostimulant addiction kinases, ion channels and Ras members (Lapidus et al. 2012). and vulnerability to relapse in recently abstinent individuals Interestingly, we previously identified both a behavioral QTL (Sinha 2011). and a cis-acting eQTL from striatal tissue on chromosome 11 for Igf2bp1 (insulin growth factor 2 mRNA-binding protein 1; the gene coding for ZBP1 protein) that was causally associated with reduced methamphetamine-induced locomotor ZBP1 activity and increased Igf2bp1 expression. In light of the recent ZBP1 findings discussed above, Igf2bp1 could repre- Zipcode-binding protein-1 (ZBP1) is a cytoplasmic protein that sent a quantitative trait gene underlying methamphetamine shuttles between the nucleus and cytoplasm via its NES and stimulant sensitivity (Bryant et al. 2012). NLS sequence. ZBP2 is a second, homologous RBP that is An intriguing possibility is that native ZBP1 expression localized to the nucleus and cooperates with ZBP1 to shuttle could be reawakened in the mature brain following chronic beta-actin mRNA into the cytoplasm (Gu et al. 2002; Pan exposure to drugs of abuse and regulate a neurodevel- et al. 2007). ZBPs share significant homology with PCBPs opmental program that underlies certain addictions such and contain two RRMs and four KH-type RNA-binding motifs. as cocaine (Dong & Nestler 2014). Furthermore, based KH3 and KH4 domains of ZBP1 form a pseudodimer that on the combined evidence described above, ZBP1 could recognizes a 54-nucleotide zipcode sequence on the 3′ UTR promote BDNF-mediated structural recovery of damaged of beta-actin mRNA for transport and translation in dendrites catecholaminergic and monoaminergic axons following (Doyle & Kiebler 2012; Farina et al. 2003; Gu et al. 2002; administration of neurotoxic drugs such as metham- Huttelmaier et al. 2005; Ross et al. 1997). phetamine and MDMA (Adori et al. 2010; Ares-Santos ZBP1 represses beta-actin translation in the cytoplasm that et al. 2014). is relieved upon Src phosphorylation at Tyr396 (Huttelmaier et al. 2005). Both BDNF and netrin-1 stimulate phosphorylation of ZBP1 at Tyr396 and increased protein synthesis RBFOX1 (A2BP1) of beta-actin in a model of axon guidance in cortical neurons (Sasaki et al. 2010; Welshhans & Bassell 2011). Neu- RBFOX (RNA-binding protein, fox-1 homolog) proteins are rodevelopmental studies demonstrate ZBP1 transport and neuronal splicing factors that promote both exon inclusion localization of beta-actin in synaptogenesis of dendritic filopo- and skipping that depends on the position of the canon- dia (Eom et al. 2003) and neurotrophin-induced growth cone ical UGCAUG binding motif near the exon (Underwood motility (Zhang et al. 2001). Furthermore, ZBP1 is necessary et al. 2005; Zhang et al. 2008). RBFOX proteins contain a for NMDA receptor-dependent targeting of beta-actin mRNA highly conserved, identical RRM that is responsible for bind- to the hippocampal dendrites (Tiruchinapalli et al. 2003) as ing RNAs and less conserved regions in the N-terminus well as dendritic arborization induced by Src-induced ZBP1 and NLS-containing C-terminus that also dictate splicing of phosphorylation and relief of translational repression (Perycz RBFOX proteins themselves and other proteins that can gov- et al. 2011). In addition to dendritic plasticity, axonal regen- ern subcellular localization and increase the functional diver- eration in severed sensory neurons involves ZBP1-mediated sity of these RBPs (Kuroyanagi 2009). transport of both beta-actin and GAP-43 mRNAs to localize Genome-wide target analysis of RBFOX2 in human embry- protein synthesis and increase axonal growth and branching, onic stem cells identified several RBP splicing factors as tar- respectively (Donnelly et al. 2011, 2013). gets, including hnRNP A2/B1, H1, H2 and PTBP (Yeo et al. Similar to ELAVL-associated increased expression of 2009), which emphasizes the high degree of network con- GAP-43 following in vivo administration of cocaine and nectivity in genomic coordination of RBP splicing (Huelga

Genes, Brain and Behavior (2016) 15: 169 – 186 177 Bryant and Yazdani et al. 2012). Enrichment analysis of the predicted RBFOX that are associated with neurodevelopmental connectivity, spliceome revealed sets of genes involved in neuromus- plasticity and genetic variation underlying the addictions cular, cytoskeleton, ion channel and phosphorylation func- (Zhong et al. 2015). tions (Zhang et al. 2008). Experimental identification of direct RBFOX targets in the mouse brain identified intronic splicing targets and 3′ UTR targets that could regulate alter- CELF4 and CELF6 native polyadenylation and mRNA stability. RBFOX targets were enriched for cytoskeleton anchoring, scaffolding and CELF (CUG-BP, ELAV-like factor) is a family of highly signaling, and neuronal projections. Increased expression of expressed multifunctional RBPs in the brain that have RBFOX1 and RBFOX3 and decreased expression of RBFOX2 both nuclear splicing and cytoplasmic functions in RNA pro- were associated with a change in the RBFOX spliceome cessing. CELF and ELAV can cooperatively promote splicing programs from E17 to adulthood (Weyn-Vanhentenryck et al. in mammalian neurons and a recent study in Caenorhab- 2014). ditis elegans indicates that they co-regulate overlapping RBFOX1 is a neurodevelopmental splicing RBP whose and distinct splicing networks to determine cholinergic vs. dysfunction is associated with autism, intellectual disability, GABAergic neuronal cell type (Norris et al. 2014). CELF ADHD, bipolar disorder and schizophrenia (Bill et al. 2013; RBPs bind to pyrimidine-rich sequences and compete with Fogel et al. 2012). CNS-specific knockout of Rbfox1 in mice PTBPs to activate splicing (Gromak et al. 2003; Spellman resulted in enhanced hippocampal neuronal excitability and et al. 2005). Notably, the high sequence similarity between susceptibility to seizures, demonstrating a role for RBFOX1 hnRNP A1 vs. CELF1 suggests shared functions (Tang et al. in neuronal excitation and synaptic transmission (Gehman 2012). CELF RBPs contain highly conserved RNA recognition et al. 2011). RBFOX1 is expressed throughout development motifs (RRM)-1 and -2 at the N-terminus and a third RRM in the mouse and human basal forebrain, neocortex and at the C-terminus (Dasgupta & Ladd 2012). RRM-1 and -2 hippocampus (Fogel et al. 2012; Hammock & Levitt 2011). are separated from RRM-3 by a non-conserved divergent Spliceome and transcriptome analysis of differentiated pri- linker domain that differentiates CELF1-2 from CELF 3–6 in mary human neural progenitor cells following RBFOX1 knock- determining RNA–protein and protein–protein interactions down identified parallel networks of transcription factors, in forming target-specific RNP complexes (Dasgupta & splicing factors and synaptic proteins involved in neurogene- Ladd 2012; Gallo & Spickett 2010). The RRMs contain RNP sis, neurodevelopment, maintenance, cytoskeletal organiza- motifs that typically bind to introns, 3′ UTRs and 5′ UTRs tion and cell adhesion, projection, proliferation and synapse of mRNAs to regulate splicing, poly(A)-specific ribonuclease function. Notably, differential splicing was observed for sev- recruitment and deadenylation [e.g. with c-fos (Moraes eral genes coding for RBPs, including HNRNPD, HNRNPA1, et al. 2006)], polyadenylation, mRNA stability and translation ELAVL2 and HNRNPH1 (Fogel et al. 2012),whichinturnwas (Dasgupta & Ladd 2012). Both the divergent domain and associated with a perturbation in their predicted splicing pro- C-terminus contain the signals that determine nuclear vs. grams based on their RNA-binding motifs. For example, 205 cytosolic localization of CELF1 and CELF2 (Fujimura et al. of the total 996 alternative splicing events that were identified 2008; Ladd & Cooper 2004). Importantly, CELF proteins con- following RBFOX1 knockdown contained the binding site for tain multiple phosphorylation sites that regulate protein and hnRNP H1, suggesting that RBFOX1 regulates the splicing RNA interactions to influence protein stability, localization of HNRNPH1 (Fogel et al. 2012). Conversely, hnRNP H1 reg- and translation of CELF targets (Dasgupta & Ladd 2012). ulates the splicing activity of RBFOX1/2 by interacting with CELF RBPs are linked to several neurological and neu- the C-terminal domain (Sun et al. 2012). In addition to splic- rodegenerative disorders, as well as neurodevelopmental and ing HNRNPH1, RBFOX2 can form a complex with hnRNP H1 neuropsychiatric disorders, including social communication and F to silence splicing of other genes (Mauger et al. 2008). problems in autism (CELF4, CELF6), epilepsy (CELF4), bipo- Thus, hnRNP H1 and RBFOX proteins could coordinate splic- lar disorder (CELF5) and schizophrenia (CELF5) (Dougherty ing in affecting methamphetamine stimulant behavior (Yaz- et al. 2013; Ladd 2013; Welter et al. 2014). Recent evidence dani et al. 2015) and heroin addiction (Xu et al. 2014). implicates both CELF4 and CELF6 in the addictions. CELF4 is Recent, direct evidence implicating RBFOX in the nucleus a brain-specific isoform that is expressed throughout devel- accumbens in the addictions comes from a genome-wide opment and is highly expressed in the hippocampus, amyg- transcriptomic and epigenomic study of chronic cocaine dala and cortex. CELF4 loss of function in mice causes administration in mice that identified a translocation of seizures (Yang et al. 2007) and a functional deficit in excita- RBFOX1 to the nucleus which was associated with an tory synaptic transmission in cortical and hippocampal neu- increase in splicing events that coincided with the loca- rons (Wagnon et al. 2011). The complex seizure phenotype tion of histone modifications. Furthermore, site-specific implicates a role for CELF4 in corticothalamic development Cre-mediated knockdown in the nucleus accumbens of (Wagnon et al. 2011). RNA target analysis of CELF4 in corti- floxed RBFOX1 mice blocked cocaine reward (Feng et al. cal and hippocampal tissue identified several 3′ UTR-targeted 2014), which together suggests that that RBFOX1 coor- mRNAs involved in synaptic transmission (Wagnon et al. dinates an adaptive splicing program underlying cocaine 2012). Of potential relevance to the addictions, genetic vari- dependence. Interestingly, RBFOX1 variants have been ation in CELF4 showed a nominal association (P = 3–5 × nominally associated with nicotine dependence, alcohol 10−6) with the subjective amphetamine response (Hart et al. dependence and cocaine reward and regulate the splicing 2012) and has been linked to hyperphagia-related obesity of cell adhesion molecule genes in dopaminergic neurons (Comuzzie et al. 2012; Halgren et al. 2012).

178 Genes, Brain and Behavior (2016) 15: 169 – 186 RNA-binding proteins, neural development and the addictions

Translational profiling of ribosome-bound mRNAs from nucleus accumbens shell. Overexpression of ADAR2 pre- mouse serotonergic neurons combined with analysis of vented relapse and the concomitant increase in surface human genetic variants identified CELF6 as a candidate gene receptor expression of AMPAR (Schmidt et al. 2014). Thus, associated with autism. Celf6 knockouts exhibit reduced unedited AMPAR receptors are associated with a model brain serotonin, deficits in ultrasonic vocalizations in neona- of drug relapse that could be mitigated by restoration of tal pups and behavioral resistance to change (Dougherty et al. surface-edited AMPA receptors. 2013). CELF6 is expressed in both the nucleus and cytoplasm In addition to AMPA receptor editing, ADAR proteins also throughout development and exhibits localized expression in edit the 5-HT2C receptor [a promising drug target for treating monoaminergic and catecholaminergic cells in the basal fore- addictive disorders (Higgins & Fletcher 2015)] at five differ- brain, ventral tegmental area, substantia nigra, raphe nuclei ent sites within the second intracellular loop, yielding up to and locus coeruleus (Maloney et al. 2015). Thus, Celf6 could 24 brain region-specific isoforms that could alter pre-mRNA influence neurodevelopment and function of the mesocorti- splicing, ligand affinity, ligand-induced g-protein signaling, colimbic reward circuitry. In support, Celf6 knockouts show ligand-induced blockade of constitutive activity and brain a disruption of conditioned cocaine reward (Dougherty 2015). region-specific functions, including modulation of dopamine CELF6 can promote both exon inclusion and skipping (Ladd release (Burns et al. 1997; Werry et al. 2008). Interestingly, et al. 2004). It will be important to determine the direct rats categorized as high vs. low novelty seeking (a trait that nuclear and cytoplasmic targets of CELF6, which will pro- correlates with future drug use) show differences in 5-HT2C vide insight into the subcellular mechanisms of behavioral receptor editing in the mesocorticolimbic circuitry, in particu- and psychiatric dysfunction. lar the nucleus accumbens shell that could affect dopamine transmission (Dracheva et al. 2009). An increase in alcohol drinking in different mouse strains was associated with an ADAR1/2 increase in anxiety as well as an increase in ADAR1/2 expression and 5-HT2C editing in the nucleus accumbens and dorsal RNA editing is yet another type of distinct and adaptive raphe nucleus (Watanabe et al. 2014). Finally, chronic nico- pre-mRNA processing mechanism that can increase pro- tine decreased editing of the 5-HT2C in the hippocampus that teome diversity to regulate neurodevelopment, plasticity was associated with depressive-like behavior during nicotine and human disease, including neurological and psychi- withdrawal (Zaniewska et al. 2010, 2015). Thus, differential atric disorders such as schizophrenia, bipolar disorder and 5-HT2C receptor editing could potentially predispose individu- depression (Li & Church 2013). Adenosine deaminase als to addiction risk as well as mediate the negative emotional acting on RNA (ADAR)-1, 2 and 3 are nuclear, enzymatic states that support addictive behaviors. RBPs that contain double-stranded RNA-binding motifs and An increase in 5-HT2C receptor editing is associated with destabilize pre-mRNA to ‘edit’ adenosine (A) nucleotides hyperphagia and obesity in patients with Prader–Willi syn- to guanosine-mimicking RNA inosine (I) nucleotides. A–I drome and in mice expressing the fully edited 5-HT2C editing changes the complementary nucleotide to cytosine, receptor (Kawahara et al. 2008). Furthermore, mice that dif- thus modulating RNA base pairing and potentially splice ferentially express the wild-type or catalytically inactive ver- site, transport, ribosome binding, translational efficiency and sion of ADAR2 show hyperphagia and obesity (Singh et al. amino acid sequence (Li & Church 2013; Slotkin & Nishikura 2007) and increased preference for high fat food over run- 2013). Compared to extensive editing of non-coding RNA, ning (Akubuiro et al. 2013). These physiological and behav- recoding of coding exons is rare in mammals and these sites ioral changes indicate a food ‘addiction’ propensity that is are enriched for proteins involved in neuronal function, including neuronal excitability, vesicular release and cytoskeleton supported by increased mRNA expression of ADAR2, D1 architecture (Rosenthal 2015). The expansion of RNA editing and D2 dopamine receptors, MOR, 5-HT2C long and short from rodents to non-human primates to humans suggests an splice variants in the hypothalamus as well as increased important role in brain evolution and cognition (Li & Church mRNA expression of D1 dopamine receptors in the stria- 2013). Although the regulation of ADAR in RNA editing is tum. Positron emission tomography imaging in mice using 18 poorly understood, it is relevant to note that FMRP interacts a tail vein injection of [ F]-fluorodeoxyglucose indicated an with ADAR in Drosophila to modulate enzymatic activity and increase in glucose metabolism in the mesolimbic reward cir- editing of mRNA transcripts that affect synaptic morphology cuitry, hypothalamus and hippocampus. The combined obser- (Bassell 2011; Bhogal et al. 2011). This observation highlights vations implicate hyperactive food-directed reward process- an additional function of FMRP and suggests that FMRP ing in ADAR2 transgenics (Akubuiro et al. 2013; Singh et al. and ADAR could work together in neurodevelopment and 2011); however, direct evidence linking RNA editing to food neuroplasticity in the addictions. or drug addictive behaviors is still lacking. ADAR2 has an established role in editing of the GluA2 To summarize, changes in AMPA receptor and 5-HT recep- subunit of the AMPA receptor at Q/R site 607 which reduces tor editing are associated with exposure to abused sub- calcium permeability (Geiger et al. 1995) and may pro- stances and could contribute to susceptibility and synap- tect against neuronal excitotoxicity in vivo (Higuchi et al. tic plasticity underlying the addictions. Future studies will 2000). With regard to the addictions, cocaine abstinence likely involve the use of advanced genome editing approaches and cocaine-primed reinstatement of self-administration in to directly test the causal, spatiotemporal role of recoded rats have been associated with decreased ADAR2 expres- proteins as well as non-coding RNAs in the brain in the estab- sion and decreased GluA2 editing of the Q/R site in the lishment and maintenance of addictive behaviors.

Genes, Brain and Behavior (2016) 15: 169 – 186 179 Bryant and Yazdani

Figure 2: RNA-binding proteins implicated in the addictions. Many RBPs have the capability to shuttle between the nucleus and cytoplasm to regulate all levels of RNA post-transcriptional processing. Here, we illustrate the location of action of the main examples that are discussed and some of their well-characterized targets. For the RBPs that are illustrated, we have also indicated whether they contribute to axon development by listing them in the axon terminal (bottom). The yellow rectangles denote dendrites. The blue spikes indicate an association with structural changes in dendritic morphology.

Summary interactive exposure to both? How do these programs change across neurodevelopment, across repeated drug exposure as This review highlights the diversity of RBP functions in regu- addiction progress and across recovery during abstinence? In lating transcription, RNA metabolism, neurodevelopment and addition to splicing, post-translational modifications of RBPs, neuroplasticity relevant to the addictions (Fig. 2). Although including phosphorylation, ubiquitination, sumoylation and beyond the scope of this review, RBPs also interact with methylation, can further regulate the transport, stabilization, non-coding RNAs and there are several new and emerg- degradation and binding of RBPs to their RNA targets and to ing RNA modifications that could be of relevance to the other proteins (Chaudhury et al. 2010), thus adding additional addictions (Satterlee et al. 2014). Identifying the key cell layers of regulation in RBP function. This complexity may one type-specific splice forms and unique functions of RBP splice day be harnessed to perturb RBP signaling in preventing and variants and their alternatively spliced targets will be critical treating the addictions as has recently been demonstrated in to yielding novel, biologically relevant discoveries in the addic- other disease models (Kim et al. 2014; Wang et al. 2009). tions. Do a specific set of RBPs become recruited during envi- We limited our discussion to RBPs where multiple lines ronmental stress exposure or drug exposure that influence of evidence implicate a potential importance in the addic- addiction risk? What are the transcriptomes, spliceomes and tions. However, additional RBPs are certainly going to be translatomes (King & Gerber 2014) that these RBPs gov- uncovered in the addictive process from neurodevelopment ern in response to risk exposure, drug exposure and the to drug-induced neuroplasticity. For instance, cytoplasmic

180 Genes, Brain and Behavior (2016) 15: 169 – 186 RNA-binding proteins, neural development and the addictions polyadenylation element-binding proteins (CPEBs) play an Blanchette, A.R., Fuentes Medel, Y.F. & Gardner, P.D. (2006) important role in synaptic plasticity (Ivshina et al. 2014). Cell-type-specific and developmental regulation of heteroge- Second, the SR protein Tra2𝛽 can be spliced by the dopamine neous nuclear ribonucleoprotein K mRNA in the rat nervous system. Gene Expr Patterns 6, 596–606. signaling molecule DARPP-32 (Benderska et al. 2010) and can Bolognani, F.,Qiu, S., Tanner, D.C., Paik, J., Perrone-Bizzozero, N.I. & promote splicing of RGS proteins (regulators of g-protein Weeber, E.J. (2007) Associative and spatial learning and memory signaling) in response to morphine (Li et al. 2013; Traynor deficits in transgenic mice overexpressing the RNA-binding protein 2010). Third, KH-type Nova proteins also regulate splicing of HuD. Neurobiol Learn Mem 87, 635–643. the D2 dopamine receptor and neocortical synaptic proteins Boschi, N.M., Takeuchi, K., Sterling, C. & Tank, A.W. (2015) Differential involved in neurotransmitter release and signaling, receptor expression of polycytosine-binding protein isoforms in adrenal gland, locus coeruleus and midbrain. Neuroscience 286,1–12. localization, synaptogenesis, axonogenesis, actin organiza- Boutz, P.L., Stoilov, P., Li, Q., Lin, C.H., Chawla, G., Ostrow, K., Shiue, tion, cell adhesion and extracellular matrix organization (Park L., Ares, M. Jr. & Black, D.L. (2007) A post-transcriptional regulatory et al. 2011; Ule et al. 2005). switch in polypyrimidine tract-binding proteins reprograms alterna- The topic of RBPs and the addictions is clearly a wide tive splicing in developing neurons. 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