Mutations of the neurofibromin 1 gene cause neurofibromatosis type 1, a disease in which learning and behavioral abnormalities are common. The disease is completely penetrant but shows variable phenotypic expression in patients. The repertoire of regulatory interactions utilized by neurons to control neurofibromin 1 expression is poorly understood. Here, we examined the contribution of microRNAs into this regulatory network. Using reporter assays, we provided evidence that miR-128 and to a lesser extent miR-137 and miR-103 reduced neurofibromin 1 reporter levels through specific binding to Nf1 3′-UTR. Mutations in all three predicted binding sites eliminated the reporter response. MiR-128 and miR-137, unlike miR-103 that showed a more ubiquitous expression, were predominantly expressed in brain with a distribution that resembled neurofibromin 1 expression in different tissues as well as during the course of neuronal development. In the nervous system, all three microRNAs showed highest expression in neurons and least in Schwann cells and astrocytes. Overexpression of miR-128 alone or with miR-103 and miR-137 significantly reduced endogenous neurofibromin 1 protein levels, while antisense inhibition of these microRNAs enhanced translation of endogenous neurofibromin 1 and reporter in primary cultures of hippocampal neurons. These findings revealed a significant additional mechanism by which neurofibromin 1 is regulated in neurons and implicated new candidates for the treatment of multifarious neurofibromatosis type 1 cognitive symptoms.

Differential RNA localization and local protein synthesis regulate synapse function and plasticity in neurons. MicroRNAs are a conserved class of regulatory RNAs that control mRNA stability and translation in tissues. They are abundant in the brain but the extent into which they are involved in synaptic mRNA regulation is poorly known. Herein, a computational analysis of the coding and 3′UTR regions of 242 presynaptic and 304 postsynaptic proteins revealed that 91% of them are predicted to be microRNA targets. Analysis of the longest 3′UTR isoform of synaptic transcripts showed that presynaptic mRNAs have significantly longer 3′UTR than control and postsynaptic mRNAs. In contrast, the shortest 3′UTR isoform of postsynaptic mRNAs is significantly shorter than control and presynaptic mRNAs, indicating they avert microRNA regulation under specific conditions. Examination of microRNA binding site density of synaptic 3′UTRs revealed that they are twice as dense as the rest of protein-coding transcripts and that approximately 50% of synaptic transcripts are predicted to have more than five different microRNA sites. An interaction map exploring the association of microRNAs and their targets revealed that a small set of ten microRNAs is predicted to regulate 77% and 80% of presynaptic and postsynaptic transcripts, respectively. Intriguingly, many of these microRNAs have yet to be identified outside primate mammals, implicating them in cognition differences observed between high-level primates and non-primate mammals. Importantly, the identified miRNAs have been previously associated with psychotic disorders that are characterized by neural circuitry dysfunction, such as schizophrenia. Finally, molecular dissection of their KEGG pathways showed enrichment for neuronal and synaptic processes. Adding on current knowledge, this investigation revealed the extent of miRNA regulation at the synapse and predicted critical microRNAs that would aid future research on the control of neuronal plasticity and etiology of psychiatric diseases.

The TREK family of leak potassium channels has been found to play critical roles in nociception, sensitivity to general anaesthetics, neuroprotection, and memory. The three members of the family, TREK1, TREK2 and TRAAK establish the resting potential and modify the duration, frequency and amplitude of action potentials. Despite their apparent importance, the repertoire of regulatory interactions utilized by cells to control their expression is poorly understood. Herein, the contribution of miRNAs in the regulation of their posttranscriptional gene expression has been examined. Using different assays, miR-124 and to a lesser extent miR-128 and miR-183 were found to reduce TREK1 and TREK2 levels through specific binding to their 3ʹUTRs. In contrast, miR-9 which was predicted to bind to TRAAK 3ʹUTR, did not alter its expression. Expression of miR-124, miR-128 and miR-183 was found to mirror that of Trek1 and Trek2 mRNAs during brain development. Moreover, application of proinflammatory mediators in dorsal root ganglion (DRG) neurons revealed an inverse correlation between miR-124 and Trek1 and Trek2 mRNA expression. Voltage clamp recordings of TREK2-mediated currents showed that miR-124 reduced the sensitivity of TREK2-expressing cells to nonaversive warmth stimulation. Overall, these findings reveal a significant regulatory mechanism by which TREK1 and TREK2 expression and hence activity are controlled in neurons and uncover new druggable targets for analgesia and neuroprotection.Abbreviations: microRNA: miRNA; UTR: untranslated region; K 2p channels: two-pore domain K + channels; DRG: dorsal root ganglion; CNS: central nervous system; FBS: fetal bovine serum; TuD: Tough Decoy; TREK: tandem P-domain weak inward rectifying K + (TWIK)-related K + channel 1; TRAAK: TWIK-related arachidonic acid K + .

The two crucial cellular insults that take place during cerebral ischemia are the loss of oxygen and loss of glucose, which can both activate a cascade of events leading to neuronal death. In addition, the toxic overactivation of neuronal excitatory receptors, leading to Ca2+ overload, may contribute to ischemic neuronal injury. Brain ischemia can be simulated in vitro by oxygen/glucose deprivation, which can be reversible by the re-establishment of physiological conditions. Accordingly, we examined the effects of glucose deprivation on the PI3K/Akt survival signaling pathway and its crosstalk with HIF-1α and Ca2+ homeostasis in SH-SY5Y human neuroblastoma cells. It was found that glucose withdrawal decreased HIF-1α protein levels even in the presence of the ischemia-mimicking CoCl2. On the contrary, and despite neuronal death, we identified a strong activation of the master pro-survival kinase Akt, a finding that was also confirmed by the increased phosphorylation of GSK3, a direct target of p-Akt. Remarkably, the elevated Ca2+ influx recorded was found to promptly trigger the activation of Akt, while a re-addition of glucose resulted in rapid restoration of both Ca2+ entry and p-Akt levels, highlighting the plasticity of neurons to respond to ischemic challenges and the important role of glucose homeostasis for multiple neurological disorders.