Xiaolu A. Cambronne

microRNA-132 regulates dendritic growth and arborization of newborn neurons in the adult hippocampus

Newborn neurons in the dentate gyrus of the adult hippocampus rely upon cAMP response element binding protein (CREB) signaling for their differentiation into mature granule cells and their integration into the dentate network. Among its many targets, the transcription factor CREB activates expression of a gene locus that produces two microRNAs, miR-132 and miR-212. In cultured cortical and hippocampal neurons, miR-132 functions downstream from CREB to mediate activity-dependent dendritic growth and spine formation in response to a variety of signaling pathways. To investigate whether miR-132 and/or miR-212 contribute to the maturation of dendrites in newborn neurons in the adult hippocampus, we inserted LoxP sites surrounding the miR-212/132 locus and specifically targeted its deletion by stereotactically injecting a retrovirus expressing Cre recombinase. Deletion of the miR-212/132 locus caused a dramatic decrease in dendrite length, arborization, and spine density. The miR-212/132 locus may express up to four distinct microRNAs, miR-132 and -212 and their reverse strands miR-132* and -212*. Using ratiometric microRNA sensors, we determined that miR-132 is the predominantly active product in hippocampal neurons. We conclude that miR-132 is required for normal dendrite maturation in newborn neurons in the adult hippocampus and suggest that this microRNA also may participate in other examples of CREB-mediated signaling.

Biosensor reveals multiple sources for mitochondrial NAD

A fluorescent sensor for NAD+ in living cells Roles of cellular nicotinamide adenine dinucleotide (NAD+) in metabolism, aging, and disease have garnered much interest, but methods have been lacking to measure the amounts of NAD+ in living cells. Cambronne et al. developed a genetically encoded biosensor that can be used to monitor concentrations of free NAD+ in various compartments of a cell (see the Perspective by Guarente). Such concentrations of NAD+ appear to be important in regulating the activity of NAD+-consuming enzymes such as sirtuins and ADP-ribosyltransferases. The authors used the sensor to demonstrate that NAD+ concentrations in mitochondria of cultured human cells can be controlled by multiple mechanisms. Science, this issue p. 1474; see also p. 1396 A fluorescent sensor allows measurement of NAD+ concentrations in cultured mammalian cells. Nicotinamide adenine dinucleotide (NAD+) is an essential substrate for sirtuins and poly(adenosine diphosphate–ribose) polymerases (PARPs), which are NAD+-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD+ concentrations within these subcellular compartments are thought to regulate the activity of NAD+-consuming enzymes; however, the challenge in measuring compartmentalized NAD+ in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD+ concentrations in subcellular compartments. We found that the concentrations of free NAD+ in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD+ biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD+ concentrations.

MicroRNA pathways in neural development and plasticity

MicroRNAs contribute significantly to the development, survival, function, and plasticity of neurons. They silence expression of target genes by reducing mRNA stability and translation. Production of microRNAs is controlled via developmental and environmental cues and these small molecules, in concert with classical transcriptional regulators, amplify changes in neuronal maturation, dendrite morphogenesis, and synaptogenesis. Neurons compartmentalize mRNAs and microRNAs within specific subcellular domains to facilitate control of local protein synthesis in response to neuronal stimuli and to modulate synaptic plasticity. This review addresses issues relevant to microRNA function in neurons, in particular, their ability to reinforce developmental decisions and promote synaptic plasticity.

Novel primate miRNAs coevolved with ancient target genes in germinal zone-specific expression patterns

Major nonprimate-primate differences in cortico-genesis include the dimensions, precursor lineages, and developmental timing of the germinal zones (GZs). microRNAs (miRNAs) of laser-dissected GZ compartments and cortical plate (CP) from embryonic E80 macaque visual cortex were deep sequenced. The CP and the GZ including ventricular zone (VZ) and outer and inner subcompartments of the outer subventricular zone (OSVZ) in area 17 displayed unique miRNA profiles. miRNAs present in primate, but absent in rodent, contributed disproportionately to the differential expression between GZ subregions. Prominent among the validated targets of these miRNAs were cell-cycle and neurogenesis regulators. Coevolution between the emergent miRNAs and their targets suggested that novel miRNAs became integrated into ancient gene circuitry to exert additional control over proliferation. We conclude that multiple cell-cycle regulatory events contribute to the emergence of primate-specific cortical features, including the OSVZ, generated enlarged supragranular layers, largely responsible for the increased primate cortex computational abilities.

Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach

Identifying targets is critical for understanding the biological effects of microRNA (miRNA) expression. The challenge lies in characterizing the cohort of targets for a specific miRNA, especially when targets are being actively down-regulated in miRNA– RNA-induced silencing complex (RISC)–messengerRNA (mRNA) complexes. We have developed a robust and versatile strategy called RISCtrap to stabilize and purify targets from this transient interaction. Its utility was demonstrated by determining specific high-confidence target datasets for miR-124, miR-132, and miR-181 that contained known and previously unknown transcripts. Two previously unknown miR-132 targets identified with RISCtrap, adaptor protein CT10 regulator of kinase 1 (CRK1) and tight junction-associated protein 1 (TJAP1), were shown to be endogenously regulated by miR-132 in adult mouse forebrain. The datasets, moreover, differed in the number of targets and in the types and frequency of microRNA recognition element (MRE) motifs, thus revealing a previously underappreciated level of specificity in the target sets regulated by individual miRNAs.

miR-218 is essential to establish motor neuron fate as a downstream effector of Isl1-Lhx3

While microRNAs have emerged as an important component of gene regulatory networks, it remains unclear how microRNAs collaborate with transcription factors in the gene networks that determines neuronal cell fate. Here, we show that in the developing spinal cord, the expression of miR-218 is directly upregulated by the Isl1-Lhx3 complex, which drives motor neuron fate. Inhibition of miR-218 suppresses the generation of motor neurons in both chick neural tube and mouse embryonic stem cells, suggesting that miR-218 plays a crucial role in motor neuron differentiation. Results from unbiased RISC-trap screens, in vivo reporter assays, and overexpression studies indicated that miR-218 directly represses transcripts that promote developmental programs for interneurons. Additionally, we found that miR-218 activity is required for Isl1-Lhx3 to effectively induce motor neurons and suppress interneuron fates. Together, our results reveal an essential role of miR-218 as a downstream effector of the Isl1-Lhx3 complex in establishing motor neuron identity.

AML suppresses hematopoiesis by releasing exosomes that contain microRNAs targeting c-MYB

Exosomes shed from acute myeloid leukemia cells suppress the expression of a hematopoietic transcription factor in the bone marrow. AML dispatches micromanagers In patients with acute myeloid leukemia (AML), the production of healthy blood cells from hematopoietic stem cells in the bone marrow (a process called hematopoiesis) is suppressed, prompting the need for bone marrow transplants. AML cells shed extracellular vesicles called exosomes that contain molecules that suppress hematopoiesis by reprogramming the stem cell niche. Hornick et al. discovered another way that AML exosomes block this critical process by delivering microRNAs to hematopoietic stem cells. The AML-derived exosomes contained two mature microRNAs that target the mRNA encoding c-MYB, a transcription factor involved in hematopoiesis. Other targets of these AML-derived exosomal microRNAs reveal interconnected networks targeting transcripts that produce proteins that control the cell cycle. The findings suggest that disrupting this mode of intercellular communication might enhance hematopoiesis in AML patients. Exosomes are paracrine regulators of the tumor microenvironment and contain complex cargo. We previously reported that exosomes released from acute myeloid leukemia (AML) cells can suppress residual hematopoietic stem and progenitor cell (HSPC) function indirectly through stromal reprogramming of niche retention factors. We found that the systemic loss of hematopoietic function is also in part a consequence of AML exosome–directed microRNA (miRNA) trafficking to HSPCs. Exosomes isolated from cultured AML or the plasma from mice bearing AML xenografts exhibited enrichment of miR-150 and miR-155. HSPCs cocultured with either of these exosomes exhibited impaired clonogenicity, through the miR-150– and miR-155–mediated suppression of the translation of transcripts encoding c-MYB, a transcription factor involved in HSPC differentiation and proliferation. To discover additional miRNA targets, we captured miR-155 and its target transcripts by coimmunoprecipitation with an attenuated RNA-induced silencing complex (RISC)–trap, followed by high-throughput sequencing. This approach identified known and previously unknown miR-155 target transcripts. Integration of the miR-155 targets with information from the protein interaction database STRING revealed proteins indirectly affected by AML exosome–derived miRNA. Our findings indicate a direct effect of AML exosomes on HSPCs that, through a stroma-independent mechanism, compromises hematopoiesis. Furthermore, combining miRNA target data with protein-protein interaction data may be a broadly applicable strategy to define the effects of exosome-mediated trafficking of regulatory molecules within the tumor microenvironment.

MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9

Significance Most brain regions comprise numerous neural cell types that are distinct in function and molecular characteristics. We examined microRNA-134 (miR-134) activity in cortical cultures using a microRNA sensor and discovered that miR-134 was induced in response to neuronal activity in somatostatin- and calretinin-expressing interneurons but not in pyramidal neurons. We identified the palmitoylation enzyme DHHC9 as a direct target of miR-134 and showed that it contributed to the regulation of H-Ras in somatostatin interneurons. H-Ras is a signaling molecule crucial for neuronal development and function. This study uncovered an activity-dependent miR-134 regulatory pathway in cortical interneurons that can potentially affect membrane localization of multiple signaling molecules. MicroRNA-134 (miR-134) serves as a widely accepted model for microRNA function in synaptic plasticity. In this model, synaptic activity stimulates miR-134 expression, which then regulates dendrite growth and spine formation. By using a ratiometric microRNA sensor, we found, unexpectedly, that miR-134 activity in cortical neurons was restricted to interneurons. Using an assay designed to trap microRNA–mRNA complexes, we determined that miR-134 interacted directly with the mRNA encoding the palmitoylation enzyme, DHHC9. This enzyme is known to palmitoylate H-Ras, a modification required for proper membrane trafficking. Treatment with bicuculline, a GABAA receptor antagonist, decreased DHHC9 expression in somatostatin-positive interneurons and membrane localization of an H-Ras reporter in a manner that depended on miR-134. Thus, although miR-134 has been proposed to affect all types of neurons, we showed that functionally active miR-134 is produced in only a selected population of neurons where it influences the expression of targets, such as DHHC9, that regulate membrane targeting of critical signaling molecules.

Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection

Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments and microtubules are important for F. novicida entry into the host cells. In addition, we observed differential phosphorylation of tristetraprolin, a key component of the mRNA-degrading machinery that controls the expression of a variety of genes including many cytokines. Infection with the delta-lpcC mutant induced the hyper-phosphorylation and inhibition of tristetraprolin, leading to the production of cytokines such as IL-1beta and TNF-alpha that may kill the host cells by triggering apoptosis. Together, our data provide new insights for Francisella invasion and a post-transcriptional mechanism that prevents the expression of host immune response factors that control infection by this pathogen.

Corrigendum: miR-218 is essential to establish motor neuron fate as a downstream effector of Isl1-Lhx3.

Corrigendum: miR-218 is essential to establish motor neuron fate as a downstream effector of Isl1–Lhx3