We investigated mRNA-lncRNA co-expression patterns in a cellular model system of non-small cell lung cancer (NSCLC) sensitive and resistant to the epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) erlotinib/gefitinib. The aim of this study was to unveil insights into the complex mechanisms of NSCLC targeted therapy resistance and epithelial-to-mesenchymal transition (EMT). Genome-wide RNA expression was quantified for weighted gene co-expression network analysis (WGCNA) to correlate the expression levels of mRNAs and lncRNAs. Functional enrichment analysis and identification of lncRNAs were conducted on modules associated with the EGFR-TKI response and/or intermediate EMT phenotypes. We constructed lncRNA-mRNA co-expression networks and identified key modules and their enriched biological functions. Processes enriched in the selected modules included RHO (A, B, C) GTPase and regulatory signaling pathways, apoptosis, inflammatory and interleukin signaling pathways, cell adhesion, cell migration, cell and extracellular matrix organization, metabolism, and lipid metabolism. Interestingly, several lncRNAs, already shown to be dysregulated in cancer, are connected to a small number of mRNAs, and several lncRNAs are interlinked with each other in the co-expression network.

RNA-binding proteins (RBPs) play important roles in modulating miRNA-mediated mRNA target repression. Argonaute2 (Ago2) is an essential component of the RNA-induced silencing complex (RISC) that plays a central role in silencing mechanisms via small non-coding RNA molecules known as siRNAs and miRNAs. Small RNAs loaded into Argonaute proteins catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and mRNA target destabilization. In previous studies we have shown that KCC2, a neuronal Cl (−) extruding K (+) Cl (−) co-transporter 2, is regulated by miR-92 in neuronal cells. Searching for Ago2 partners by immunoprecipitation and LC-MS/MS analysis, we isolated among other proteins the Serpine mRNA binding protein 1 (SERBP1) from SH-SY5Y neuroblastoma cells. Exploring the role of SERBP1 in miRNA-mediated gene silencing in SH-SY5Y cells and primary hippocampal neurons, we demonstrated that SERBP1 silencing regulates KCC2 expression through the 3′ untranslated region (UTR). In addition, we found that SERBP1 as well as Ago2/miR-92 complex bind to KCC2 3′UTR. Finally, we demonstrated the attenuation of miR-92-mediated repression of KCC2 3′UTR by SERBP1 silencing. These findings advance our knowledge regarding the miR-92-mediated modulation of KCC2 translation in neuronal cells and highlight SERBP1 as a key component of this gene regulation.

Alzheimer's disease (AD), one of the most common causes of dementia in elderly people, is characterized by progressive impairment in cognitive function, early degeneration of basal forebrain cholinergic neurons (BFCNs), abnormal metabolism of the amyloid precursor protein (APP), amyloid beta-peptide (A beta) depositions, and neurofibrillary tangles. According to the cholinergic hypothesis, dysfunction of acetylcholine-containing neurons in the basal forebrain contributes markedly to the cognitive decline observed in AD. In addition, the neurotrophic factor hypothesis posits that the loss nerve growth factor (NGF) signalling in AD may account for the vulnerability to atrophy of BFCNs and consequent impairment of cholinergic functions. Though acetylcholinesterase inhibitors provide only partial and symptomatic relief to AD patients, emerging data from in vivo magnetic resonance imaging (MRI) and positron emission tomography (PET) studies in mild cognitive impairment (MCI) and AD patients highlight the early involvement of BFCNs in MCI and the early phase of AD. These data support the cholinergic and neurotrophic hypotheses of AD and suggest new targets for AD therapy. Different mechanisms account for selective vulnerability of BFCNs to AD pathology, with regard to altered metabolism of APP and tau. In this review, we provide a general overview of the current knowledge of NGF and APP interplay, focusing on the role of APP in regulating NGF receptors trafficking/signalling and on the involvement of NGF in modulating phosphorylation of APP, which in turn controls APP intracellular trafficking and processing. Moreover, we highlight the consequences of APP interaction with p75NTR and TrkA receptor, which share the same binding site within the APP juxtamembrane domain. We underline the importance of insulin dysmetabolism in AD pathology, in the light of our recent data showing that overlapping intracellular signalling pathways stimulated by NGF or insulin can be compensatory. In particular, NGF-based signalling is able to ameliorates deficiencies in insulin signalling in the medial septum of 3xTg-AD mice. Finally, we present an overview of NGF-regulated microRNAs (miRNAs). These small non-coding RNAs are involved in post-transcriptional regulation of gene expression, and we focus on a subset that are specifically deregulated in AD and thus potentially contribute to its pathology.

MicroRNAs have emerged as regulators of brain development and function. Reduction of miR-101 expression has been reported in rodent hippocampus during ageing, in the brain of Alzheimer's disease (AD) patients and in AD animal models. In this study, we investigated the behavioral and molecular consequences of inhibition of endogenous miR-101 in 4–5-month-old C57BL/6J mice, infused with lentiviral particles expressing a miR-101 sponge (pLSyn-miR-101 sponge) in the CA1 field of the hippocampus. The sponge-infected mouse model showed cognitive impairment. The pLSyn-miR-101 sponge-infected mice were unable to discriminate either a novel object location or a novel object as assessed by object place recognition (OPR) and novel object recognition (NOR) tasks, respectively. Moreover, the sponge-infected mice evaluated for contextual memory in inhibitory avoidance task showed shorter retention latency compared to control pLSyn mice. These cognitive impairment features were associated with increased hippocampal expression of relevant miR-101 target genes, amyloid precursor protein (APP), RanBP9 and Rab5 and overproduction of amyloid beta (Aβ) 42 levels, the more toxic species of Aβ peptide. Notably, phosphorylation-dependent AMP-activated protein kinase (AMPK) hyperactivation is associated with AD pathology and age-dependent memory decline, and we found AMPK hyperphosphorylation in the hippocampus of pLSyn-miR-101 sponge mice. This study demonstrates that mimicking age-associated loss of miR-101 in hippocampal neurons induces cognitive decline and modulation of AD-related genes in mice.

MicroRNAs (miRNAs) are highly expressed in the mammalian nervous system and regulate neuronal gene expression during neurogenesis, neurodevelopment, differentiation, dendritic morphogenesis, synaptic plasticity, learning, and memory. MiRNAs are spatially and temporally modulated in the nervous system and they exhibit context-dependent functions. MiRNAs often act through regulatory networks in specific cellular contexts and at specific times to ensure the progression through each biological state. MiRNAs are involved in local protein synthesis and contribute to synaptic plasticity by modulating dendritic mRNA translation at dendritic spines. Studies in animal models showed that RNA-induced silencing complexes and specific miRNAs might be recruited in synaptic plasticity processes supporting learning, memory, and cognition. Significant progress has been made in our understanding of miRNAs in the nervous system and it provides an encouraging starting point to investigate miRNA pathway involvement in the development and progression of neurological and psychiatric diseases and to search future therapeutic applications. Here, we describe recent molecular and cellular neurobiological findings 4that highlight the role of miRNAs in the neuronal regulatory networks, which represent the exploration of a new frontier of miRNAs biology.

Individual microRNAs and/or microRNA signatures were associated with Alzheimer's disease (AD). We report here the recent advances brought to the identification of microRNA changes in AD brain and their biological function in the molecular pathways associated with the disease. This field represents a fertile route to understand the pathogenic mechanisms underlying Alzheimer's disease. In addition we review recent studies aimed to discover promising biomarkers for AD diagnosis by microRNA expression profiles of biofluids from AD patients.

Neurodegeneration associated with amyloid β (Aβ) peptide accumulation, synaptic loss, and memory impairment are pathophysiological features of Alzheimer's disease (AD). Numerous microRNAs regulate amyloid precursor protein (APP) expression and metabolism. We previously reported that miR-101 is a negative regulator of APP expression in cultured hippocampal neurons. In this study, a search for predicted APP metabolism-associated miR-101 targets led to the identification of a conserved miR-101 binding site within the 3’ untranslated region (UTR) of the mRNA encoding Ran-binding protein 9 (RanBP9). RanBP9 increases APP processing by β-amyloid converting enzyme 1 (BACE1), secretion of soluble APPβ (sAPPβ), and generation of Aβ. MiR-101 significantly reduced reporter gene expression when co-transfected with a RanBP9 3'-UTR reporter construct, while site-directed mutagenesis of the predicted miR-101 target site eliminated the reporter response. To investigate the effect of stable inhibition of miR-101 both in vitro and in vivo, a microRNA sponge was developed to bind miR-101 and derepress its targets. Four tandem bulged miR-101 responsive elements (REs), located downstream of the enhanced green fluorescence protein (EGFP) open reading frame and driven by the synapsin promoter, were placed in a lentiviral vector to create the pLSyn-miR-101 sponge. Delivery of the sponge to primary hippocampal neurons significantly increased both APP and RanBP9 expression, as well as sAPPβ levels in the conditioned medium. Importantly, silencing of endogenous RanBP9 reduced sAPPβ levels in miR-101 sponge-containing hippocampal cultures, indicating that miR-101 inhibition may increase amyloidogenic processing of APP by RanBP9. Lastly, the impact of miR-101 on its targets was demonstrated in vivo by intrahippocampal injection of the pLSyn-miR-101 sponge into C57BL6 mice. This study thus provides the basis for studying the consequences of long-term miR-101 inhibition on the pathology of AD.

ABSTRACTPost‐transcriptional gene regulation mediated by microRNAs (miRNAs) is implicated in memory formation; however, the function of miR‐92 in this regulation is uncharacterized. The present study shows that training mice in contextual fear conditioning produces a transient increase in miR‐92 levels in the hippocampus and decreases several miR‐92 gene targets, including: (i) the neuronal Cl‐ extruding K+Cl‐ co‐transporter 2 (KCC2) protein; (ii) the cytoplasmic polyadenylation protein (CPEB3), an RNA‐binding protein regulator of protein synthesis in neurons; and (iii) the transcription factor myocyte enhancer factor 2D (MEF2D), one of the MEF2 genes which negatively regulates memory‐induced structural plasticity. Selective inhibition of endogenous miR‐92 in CA1 hippocampal neurons, by a sponge lentiviral vector expressing multiple sequences imperfectly complementary to mature miR‐92 under the control of the neuronal specific synapsin promoter, leads to up‐regulation of KCC2, CPEB3 and MEF2D, impairs contextual fear conditioning, and prevents a memory‐induced increase in the spine density. Taken together, the results indicate that neuronal‐expressed miR‐92 is an endogenous fine regulator of contextual fear memory in mice. © 2014 Wiley Periodicals, Inc.

In the past few years, the understanding of microRNA (miRNA) biogenesis, the molecular mechanisms by which miRNAs regulate gene expression, and the functional roles of miRNAs has been expanded. Interestingly, numerous miRNAs are expressed in a spatially and temporally controlled manner in the nervous system, suggesting that their post-transcriptional regulation may be particularly relevant in neural development and function. miRNA studies in neurobiology have shown their involvement in synaptic plasticity and brain diseases. Approaches for manipulating miRNA levels in neuronal cells in vitro and in vivo are described here. Recent applications of miRNA antisense oligonucleotides, miRNA gene knockout and miRNA sponges in neuronal cells are reviewed. Finally, miRNA-based therapies for neurological pathologies related to alterations in miRNA functions are discussed.

MicroRNA-mediated gene silencingMicroRNAs are non-coding single stranded RNA molecules, 18-to 25 nucleotides in lenght, encoded in the genomes of invertebrates, plants and vertebrates.MicroRNA genes represent about 1-2% of the known eukaryotic genomes.The first two miRNAs to be discovered were lin-4 and let-7 in Caenorhabditis elegans, which regulate expression of lin-14 and lin-28 mRNAs, respectively, and are required for larval developmental timing (Lee et al., 1993;

Analyses of knockout and mutant transgenic mice as well as in vitro studies demonstrated a complex role of FADD in the regulation of cell fate. FADD is involved in death receptor induced apoptosis, cell cycle progression and cell proliferation. In a search for mechanisms that might regulate FADD functions, we identified, upon the screening of a lambda-phage cDNA library, calmodulin (CaM) as a novel FADD interacting protein. CaM is a key mediator of signals by the secondary messenger calcium and it is an essential regulator of cell cycle progression and cell survival. Here, we describe the identification and characterization of two calcium dependent CaM binding sites in the alpha helices 8-9 and 10-11 of FADD. Phosphorylation of human FADD at the C-terminal serine 194, by casein kinase I alpha (CKIalpha), has been shown to regulate FADD-dependent non-apoptotic activities. Remarkably, we showed that both FADD and CaM are CKIalpha substrates and that in synchronized HeLa cells, FADD, CaM and CKIalpha co-localize at the mitotic spindle in metaphase and anaphase. Moreover, complementation experiments in Jurkat FADD-/- T cells indicated that: a) cells expressing FADD mutants in the CaM binding sites are protected from Taxol-induced G2/M cell cycle arrest; b) FADD/CaM interaction is not required for Fas receptor-mediated apoptosis although Fas and CaM might compete for binding to FADD. We suggest that the interplay of FADD, CaM and CKIalpha may have an important role in the regulation of cell fate.

The amyloid precursor protein (APP) and its proteolytic product amyloid beta (Aβ) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3′-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and Aβ load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1β (IL-lβ). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of Aβ, suggesting a possible role for miR-101 in neuropathological conditions.

Amyloid Precursor Protein (APP) and its proteolytic product amyloid beta (Aβ) are critical in the pathogenesis of Alzheimer's Disease (AD). APP gene duplication and transcriptional upregulation are linked to AD. In addition, normal levels of APP appear to be required for some physiological functions in the developing brain. Several studies in mammalian cell lines and primary neuron cultures indicate that RNA binding proteins and microRNAs interacting with regulatory regions of the APP mRNA modulate expression of APP post-transcriptionally. However, when the various mechanisms of APP post-transcriptional regulation are recruited and which of them are acting in a synergistic fashion to balance APP protein levels, is unclear. Recent studies suggest that further investigation of the molecules and pathways involved in APP post-transcriptional regulation are warranted.

J. Neurochem. (2010) 113, 591–600.AbstractMicroRNAs have been associated to fine‐tuning spatial and temporal control of gene expression during neuronal development. The neuronal Cl(−) extruding, K(+)Cl(−) co‐transporter 2 (KCC2) is known to play an important role in neuronal Cl(−) homeostasis and in determining the physiological response to activation of anion selective GABA receptors. Here we show that microRNA‐92 is developmentally down‐regulated during maturation of rat cerebellar granule neurons (CGNs) in vitro. Computational predictions suggest several high‐ranking targets for microRNA‐92 including the KCC2 gene. Consistently, the KCC2 protein levels were up‐regulated in mature CGN in vitro and a functional association between microRNA‐92 and KCC2 3′ untranslated region was established using luciferase assays. The generation of an inward directed Cl(−) electrochemical gradient, necessary for the hyperpolarizing effect of GABA, requires robust KCC2 expression in several neuronal types. Here we show that lentiviral‐mediated microRNA‐92 over‐expression reduced KCC2 protein levels and positively shifted reversal potential of GABA induced Cl(−) currents in CGNs. In addition KCC2 re‐expression reversed microRNA‐92 electrophysiological phenotype. Consistently microRNA‐92 inhibition induced both an increase of the level of KCC2 and a negative shift in GABA reversal potential. These findings introduce a new player in the developmental change of GABA from depolarization to hyperpolarization.