Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo: evidence for compartment-specific translation control

Protein synthesis underlying activity-dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor-2 (eEF2), during long-term potentiation (LTP) induced by local infusion of brain-derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF-induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked BDNF-LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho-eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of alpha-calcium/calmodulin-dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK-dependent regulation of the initiation and elongation steps of protein synthesis during BDNF-LTP in vivo. Furthermore, the results suggest a compartment-specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non-synaptic sites.     

Developmental and Activity-Dependent miRNA Expression Profiling in Primary Hippocampal Neuron Cultures

MicroRNAs (miRNAs) are evolutionarily conserved non-coding RNAs of ∼22 nucleotides that regulate gene expression at the level of translation and play vital roles in hippocampal neuron development, function and plasticity. Here, we performed a systematic and in-depth analysis of miRNA expression profiles in cultured hippocampal neurons during development and after induction of neuronal activity. MiRNA profiling of primary hippocampal cultures was carried out using locked nucleic-acid-based miRNA arrays. The expression of 264 different miRNAs was tested in young neurons, at various developmental stages (stage 2–4) and in mature fully differentiated neurons (stage 5) following the induction of neuronal activity using chemical stimulation protocols. We identified 210 miRNAs in mature hippocampal neurons; the expression of most neuronal miRNAs is low at early stages of development and steadily increases during neuronal differentiation. We found a specific subset of 14 miRNAs with reduced expression at stage 3 and showed that sustained expression of these miRNAs stimulates axonal outgrowth. Expression profiling following induction of neuronal activity demonstrates that 51 miRNAs, including miR-134, miR-146, miR-181, miR-185, miR-191 and miR-200a show altered patterns of expression after NMDA receptor-dependent plasticity, and 31 miRNAs, including miR-107, miR-134, miR-470 and miR-546 were upregulated by homeostatic plasticity protocols. Our results indicate that specific miRNA expression profiles correlate with changes in neuronal development and neuronal activity. Identification and characterization of miRNA targets may further elucidate translational control mechanisms involved in hippocampal development, differentiation and activity-depended processes.     

miR-132/212 Knockout Mice Reveal Roles for These miRNAs in Regulating Cortical Synaptic Transmission and Plasticity

miR-132 and miR-212 are two closely related miRNAs encoded in the same intron of a small non-coding gene, which have been suggested to play roles in both immune and neuronal function. We describe here the generation and initial characterisation of a miR-132/212 double knockout mouse. These mice were viable and fertile with no overt adverse phenotype. Analysis of innate immune responses, including TLR-induced cytokine production and IFNβ induction in response to viral infection of primary fibroblasts did not reveal any phenotype in the knockouts. In contrast, the loss of miR-132 and miR-212, while not overtly affecting neuronal morphology, did affect synaptic function. In both hippocampal and neocortical slices miR-132/212 knockout reduced basal synaptic transmission, without affecting paired-pulse facilitation. Hippocampal long-term potentiation (LTP) induced by tetanic stimulation was not affected by miR-132/212 deletion, whilst theta burst LTP was enhanced. In contrast, neocortical theta burst-induced LTP was inhibited by loss of miR-132/212. Together these results indicate that miR-132 and/or miR-212 play a significant role in synaptic function, possibly by regulating the number of postsynaptic AMPA receptors under basal conditions and during activity-dependent synaptic plasticity.     

MicroRNAs associated with mitogen-activated protein kinase in human pancreatic cancer

Aberrant expression of microRNAs (miRNA) is associated with phenotypes of various cancers, including pancreatic cancer. However, the mechanism of the aberrant expression is largely unknown. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in gene expression related to the malignant phenotype of pancreatic cancer. Hence, we studied the role of MAPK in the aberrant expression of miRNAs in pancreatic cancer cells. The alterations in expression of 183 miRNAs induced by activation or inactivation of MAPK were assayed in cultured pancreatic cancer cells and HEK293 cells by means of the quantitative real-time PCR method. We found that four miRNAs, namely, miR-7-3, miR-34a, miR-181d, and miR-193b, were preferentially associated with MAPK activity. Among these miRNAs, miR-7-3 was upregulated by active MAPK, whereas the others were downregulated. Promoter assays indicated that the promoter activities of the host genes of miR-7-3 and miR-34a were both downregulated by alteration in MAPK activity. Exogenous overexpression of the MAPK-associated miRNAs had the effect of inhibition of the proliferation of cultured pancreatic cancer cells; miR-193b was found to exhibit the most remarkable inhibition. A search for target genes of miR-193b led to identification of CCND1, NT5E, PLAU, STARD7, STMN1, and YWHAZ as the targets. Translational suppression of these genes by miR-193b was confirmed by reporter assay. These results indicate that activation of MAPK may play a significant role in aberrant expression of miRNAs and their associated phenotypes in pancreatic cancer.     

MicroRNA-322 Cluster Promotes Tau Phosphorylation via Targeting Brain-Derived Neurotrophic Factor

Brain-derived neurotrophic factor (BDNF) is a crucial regulator to support synaptic plasticity and neuronal survival, its significant decrease is a pathophysiological hallmark in Alzheimer’s disease (AD) brains and accounts for poor prognosis. MicroRNAs (miRNAs) interfere with the translation of target mRNAs and control a variety of physiological and pathological processes. MiR-322 is the rodent homologue of human miR-424, it is involved in the modulation of cell differentiation, proliferation, apoptosis and metabolic activities in diverse tissues and organs. However, the roles and potential mechanisms of miR-322 remain elusive in AD pathogenesis. Here we observed miR-322 is significantly increased along with BDNF decrease in AD mouse brain. Bioinformatics prediction implicated that BDNF 3′-untranslated region (3′-UTR) possesses the putative target sequence of miR-322. Luciferase reporter assay identified that miR-322 can directly conjugate to BDNF 3′-UTR. The functional research showed that MiR-322 input deregulates BDNF expression at either mRNA or protein levels, whereas miR-322 silence restores BDNF expression in vitro. Furthermore, we found miR-322 promotes Tau phosphorylation via negatively controlling BDNF–TrkB receptor activation, otherwise MiR-322 silence restores TrkB activation and attenuates tau phosphorylation. Collectively, this study demonstrated a novel miRNA-dependent manner of BDNF degradation in AD pathogenesis, it may drive a miRNAs- or BDNF based therapeutic strategies against Alzheimer’s disease.     

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes.     

The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.

Members of the Eph family of receptor tyrosine kinases control many aspects of cellular interactions during development, including axon guidance. Here, we demonstrate that EphB2 also regulates postnatal synaptic function in the mammalian CNS. Mice lacking the EphB2 intracellular kinase domain showed wild-type levels of LTP, whereas mice lacking the entire EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. Synaptic NMDA-mediated current was reduced in dentate granule neurons in EphB2 null mice, as was synaptically localized NR1 as revealed by immunogold localization. Finally, we show that EphB2 is upregulated in hippocampal pyramidal neurons in vitro and in vivo by stimuli known to induce changes in synaptic structure. Together, these data demonstrate that EphB2 plays an important role in regulating synaptic function.     

Timing specific requirement of microRNA function is essential for embryonic and postnatal hippocampal development

The adult hippocampus consists of the dentate gyrus (DG) and the CA1, CA2 and CA3 regions and is essential for learning and memory functions. During embryonic development, hippocampal neurons are derived from hippocampal neuroepithelial cells and dentate granular progenitors. The molecular mechanisms that control hippocampal progenitor proliferation and differentiation are not well understood. Here we show that noncoding microRNAs (miRNAs) are essential for early hippocampal development in mice. Conditionally ablating the RNAase III enzyme Dicer at different embryonic time points utilizing three Cre mouse lines causes abnormal hippocampal morphology and affects the number of hippocampal progenitors due to altered proliferation and increased apoptosis. Lack of miRNAs at earlier stages causes early differentiation of hippocampal neurons, in particular in the CA1 and DG regions. Lack of miRNAs at a later stage specifically affects neuronal production in the CA3 region. Our results reveal a timing requirement of miRNAs for the formation of specific hippocampal regions, with the CA1 and DG developmentally hindered by an early loss of miRNAs and the CA3 region to a late loss of miRNAs. Collectively, our studies indicate the importance of the Dicer-mediated miRNA pathway in hippocampal development and functions.     

Relationship Between Downregulation of miRNAs and Increase of Oxidative Stress in the Development of Diabetic Cardiac Dysfunction: Junctin as a Target Protein of miR-1

Oxidative stress is involved in the etiology of diabetes-induced cardiac dysfunction while microRNAs (miRNAs) are known as regulators for genes involved in cardiac remodeling. However, a functional link between miRNAs and diabetes-induced cardiac dysfunction remains to be investigated. Here, we aimed to identify whether the expression levels of miRNAs are associated with oxidative stress/diabetic heart and if proteins responsible from contractile activity during diabetes might be directly modulated by miRNAs. Diabetic cardiomyopathy developed with streptozotocin, is characterized with marked changes in sarcomere and mitochondria, depressed left ventricular developed pressure, and a massive oxidative stress that is particularly evident in the heart. miRNA profiling was performed in freshly isolated left ventricular cells from diabetic rats. Using microarray analysis, we identified marked changes in the expression of 43 miRNAs (37 of them were downregulated while 6 miRNAs were upregulated) out of examined total of 351 miRNAs. Among them, 6 miRNAs were further validated by real-time PCR. The expression levels of miR-1, miR-499, miR-133a, and miR-133b were markedly depressed in the diabetic cardiomyocytes while miR-21 level increased and miR-16 level was unchanged. Notably, normalization of cardiac function and oxidant/antioxidant level after N-acetylcysteine (NAC)-treatment of diabetic rats resulted with a significant restoration in the expression levels of miR-499, miR-1, miR-133a, and miR-133b in the myocardium. Since changes in the level of muscle-specific miR-1 has been implicated in cardiac diseases and its specific molecular targets involved in its action, in part, associated with oxidative stress are limited, we first examined the protein levels of some SR-associated proteins such as junctin and triadin. Junctin but not triadin is markedly overexpressed in diabetic cardiomyocytes while its level was normalized in NAC-treated diabetics. Luciferase reporter assay showed that junctin is targetted by miR-1. Taken together, our data demonstrates that intervention with an antioxidant treatment for 4-week leads to significant cardioprotection against diabetes-induced injury, controlling oxidant/antioxidant level, which may directly control the levels of some miRNAs including miR-1 and its target protein junctin, which is involved in the development of diabetic cardiomyopathy.     

Expression profiles of microRNAs in oxidized low-density lipoprotein-stimulated RAW 264.7 cells

Macrophage-derived foam cells were one of the hallmarks of atherosclerosis, and microRNAs played an important role in the formation of foam cells. In order to explore the roles of miRNA in the formation of foam cells, we investigated miRNA expression profiles in foam cells through high-throughput sequencing technology. A total of 84 miRNAs were differentially expressed between RAW 264.7 macrophages and foam cells induced by ox-LDL. Thirty miRNAs were upregulated and 54 miRNAs were downregulated. GO terms and KEGG pathways analysis revealed that the target genes of most of DE miRNAs were mainly enriched in “cell differentiation,” “endocytosis,” “MAPK signaling pathway,” and “FoxO signaling pathway.” The target genes of some DE miRNAs were enriched in “Insulin signaling pathway,” “Hippo signaling pathway,” “TNF signaling pathway,” “NF-kappa B signaling pathway,” and “cell death.” Using bioinformatics analyses and dual-luciferase reporter assays, we found that miR-28a-5p and miR-30c-1-3p directly inhibited LRAD3 and LOX-1 mRNA expression through targeting the 3’UTR of LRAD3 and LOX-1 mRNA, respectively. Our study indicates that miRNAs are extensively involved in the formation of foam cells, and provides a valuable resource for further study the role of miRNAs in atherosclerosis.     

应用双荧光报告系统检测斑马鱼microRNA的动态表达

microRNA(miRNA)是一类细胞内源表达的小分子非编码RNA, 主要通过降解靶基因的mRNA或者抑制靶基因的翻译, 在动植物的发育以及其他重要的生理过程中起调控作用。miRNA的功能跟它的表达位置与时间密切相关, 但是目前尚缺乏一个能够在活体与个体水平稳定、持续地实时观察miRNA动态表达的方法。文章以斑马鱼为模式, 建立了一个双荧光报告系统(我们称之为miRNA Tracer), 用于在斑马鱼整体胚胎中追踪特定miRNA的表达谱及动态变化过程。该系统以Tol2转座子为基础, 采用来自斑马鱼hsp70基因的热激启动子分别驱动eGFP和mRFP1荧光报告基因, 同时在其中一个报告基因的3′-UTR区连接待测miRNA的互补序列, 构成Tracer质粒。该互补序列与斑马鱼胚胎中相应的内源miRNA结合后能够使对应报告基因的荧光信号强度减弱, 通过比较两个报告基因在表达谱上的差异辨别miRNA的表达区域, 检测斑马鱼胚胎中miRNA起作用的位置和时间。文章选择在肌肉系统特异表达的miR-206以及在神经系统特异表达的miR-219, 分别在显微注射瞬时表达和转基因稳定整合等两个层次上验证了上述Tracer系统。结果表明, 所用的方法能够如实地在单细胞水平和整体水平检测到目标miRNA的时空表达动态变化。miRNA Tracer系统为在斑马鱼发育过程中对miRNA进行活体、实时的时空定位提供了一个独特而有效的方法, 也为对miRNA进行功能与作用机制等更深入的研究奠定了基础。

补充资料

s219mRFP1-dF转基因胚胎的3-D图像 [视频]