Activity‐dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA‐binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p‐glycogen synthase kinase 3β (GSK3β) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIα, vascular early response gene, GAP‐43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3β signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3β, p‐Akt, and β‐catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile‐X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.     

HuD Interacts with Bdnf mRNA and Is Essential for Activity-Induced BDNF Synthesis in Dendrites

Highly specific activity-dependent neuronal responses are necessary for modulating synapses to facilitate learning and memory. We present evidence linking a number of important processes involved in regulating synaptic plasticity, suggesting a mechanistic pathway whereby activity-dependent signaling, likely through protein kinase C (PKC)-mediated phosphorylation of HuD, can relieve basal repression of Bdnf mRNA translation in dendrites, allowing for increased TrkB signaling and synaptic remodeling. We demonstrate that the neuronal ELAV family of RNA binding proteins associates in vivo with several Bdnf mRNA isoforms present in the adult brain in an activity-dependent manner, and that one member, HuD, interacts directly with sequences in the long Bdnf 3'' untranslated region (3''UTR) and co-localizes with Bdnf mRNA in dendrites of hippocampal neurons. Activation of PKC leads to increased dendritic translation of mRNAs containing the long Bdnf 3''UTR, a process that is dependent on the presence of HuD and its phosphorylation at threonine residues 149 and/or 165. Thus, we found a direct effect of HuD on regulating translation of dendritic Bdnf mRNAs to mediate local and activity-dependent increases in dendritic BDNF synthesis.     

Calmodulin-dependent protein kinase II

One of the most important mechanisms for regulating neuronal functions is through second messenger cascades that control protein kinases and the subsequent phosphorylation of substrate proteins. Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) is the most abundant protein kinase in mammalian brain tissues, and the alpha-subunit of this kinase is the major protein and enzymatic molecule of synaptic junctions in many brain regions. CaM-kinase II regulates itself through a complex autophosphorylation mechanism whereby it becomes calcium-independent following its initial activation. This property has implicated CaM-kinase II as a potential molecular switch at central nervous system (CNS) synapses. Recent studies have suggested that CaM-kinase II is involved in many diverse phenomena such as epilepsy, sensory deprivation, ischemia, synapse formation, synaptic transmission, long-term potentiation, learning, and memory. During brain development, the expression of CaM-kinase II at both protein and mRNA levels coincides with the active periods of synapse formation and, therefore, factors regulating the genes encoding kinase subunits may play a role in the cell-to-cell recognition events that underlie neuronal differentiation and the establishment of mature synaptic functions. Recent findings have demonstrated that the mRNA encoding the alpha-subunit of CaM-kinase II is localized in neuronal dendrites. Current speculation suggests that the localized translation of dendritic mRNAs encoding specific synaptic proteins may be responsible for producing synapse-specific changes associated with the processing, storage, and retrieval of information in neural networks.     

HuD Regulates the cpg15 Expression Via the 3′-UTR and AU-Rich Element

The candidate plasticity related gene 15 (cpg15) plays important roles in neural development and plasticity. In the present study, we studied the role of the cpg15 3′-untranslated region (UTR) in regulating the expression of the gene. The results showed that the presence of the 3′-UTR significantly decreases, while loss of a putative AU-rich element (ARE) in the 3′-UTR increases the cpg15 expression, indicating that the 3′-UTR and ARE may be essential for regulation of cpg15 expression. In addition, HuD, a neural-specific RNA binding protein, increased the cpg15 expression, which depends on the presence of the 3′-UTR and ARE. RNA-binding protein immunoprecipitation (RIP) assay demonstrated that HuD forms a complex with cpg15 mRNA in the cells of rat hippocampus. Deletion of HuD domains RRM1 plus RRM2 or Hinge region plus RRM3 attenuates the function of HuD in enhancing the cpg15 expression. The results suggest that HuD regulates the cpg15 expression via the 3′-UTR-mediated mechanism, which requires the presence of the ARE.     

Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain

Nitric oxide (NO) is believed to act as an intercellular signal that regulates synaptic plasticity in mature neurons. We now report that NO also regulates the proliferation and differentiation of mouse brain neural progenitor cells (NPCs). Treatment of dissociated mouse cortical neuroepithelial cluster cell cultures with the NO synthase inhibitor L-NAME or the NO scavenger hemoglobin increased cell proliferation and decreased differentiation of the NPCs into neurons, whereas the NO donor sodium nitroprusside inhibited NPC proliferation and increased neuronal differentiation. Brain-derived neurotrophic factor (BDNF) reduced NPC proliferation and increased the expression of neuronal NO synthase (nNOS) in differentiating neurons. The stimulatory effect of BDNF on neuronal differentation of NPC was blocked by L-NAME and hemoglobin, suggesting that NO produced by the latter cells inhibited proliferation and induced neuronal differentiation of neighboring NPCs. A similar role for NO in regulating the switch of neural stem cells from proliferation to differentiation in the adult brain is suggested by data showing that NO synthase inhibition enhances NPC proliferation and inhibits neuronal differentiation in the subventricular zone of adult mice. These findings identify NO as a paracrine messenger stimulated by neurotrophin signaling in newly generated neurons to control the proliferation and differentiation of NPC, a novel mechanism for the regulation of developmental and adult neurogenesis.     

Neuronal RNA‐binding protein HuD regulates addiction‐related gene expression and behavior

The neuronal RNA‐binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD‐mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuD_OE). When this set was further limited to genes in the knowledgebase of addiction‐related genes database (KARG) that contains predicted HuD‐binding sites in their 3′ untranslated regions (3′UTRs), we found that HuD regulates networks that have been associated with addiction‐like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction‐related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild‐type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuD_OE and wild‐type mice in CPP and found that HuD_OE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction‐related behaviors such as cocaine conditioning and seeking, through increased plasticity‐related gene expression.     

HuD binds to three AU-rich sequences in the 3'-UTR of neuroserpin mRNA and promotes the accumulation of neuroserpin mRNA and protein

Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3′-untranslated region (3′-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3′-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3′-UTR, which prolongs the mRNA lifetime and increases protein level.