Andreas Bosio

A Mammalian microRNA Expression Atlas Based on Small RNA Library Sequencing

MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence-complementary target mRNAs. In order to identify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were enriched in neuronal as well as normal and malignant hematopoietic cells and tissues. We present expression profiles derived from clone count data and provide computational tools for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubiquitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred transcriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses.

The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes

After being activated by antigen, helper T lymphocytes switch from a resting state to clonal expansion. This switch requires inactivation of the transcription factor Foxo1, a suppressor of proliferation expressed in resting helper T lymphocytes. In the early antigen-dependent phase of expansion, Foxo1 is inactivated by antigen receptor–mediated post-translational modifications. Here we show that in the late phase of expansion, Foxo1 was no longer post-translationally regulated but was inhibited post-transcriptionally by the interleukin 2 (IL-2)-induced microRNA miR-182. Specific inhibition of miR-182 in helper T lymphocytes limited their population expansion in vitro and in vivo. Our results demonstrate a central role for miR-182 in the physiological regulation of IL-2-driven helper T cell–mediated immune responses and open new therapeutic possibilities.

Absolute quantification of microRNAs by using a universal reference

MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/CD133(-) hematopoietic progenitor cells.

Myelin glycolipids and their functions

During myelination, oligodendrocytes in the CNS and Schwann cells in the PNS synthesise myelin-specific proteins and lipids for the assembly of the axon myelin sheath. A dominant class of lipids in the myelin bilayer are the glycolipids, which include galactocerebroside (GalC), galactosulfatide (sGalC) and galactodiglyceride (GalDG). A promising approach for unravelling the roles played by various lipids in the myelin membrane involves knocking out the genes encoding important enzymes in lipid biosynthesis. The recent ablation of the ceramide galactosyltransferase ( cgt) gene in mice is the first example. The cgt gene encodes a key enzyme in glycolipid biosynthesis. Its absence causes glycolipid deficiency in the lipid bilayer, breakdown of axon insulation and loss of saltatory conduction. Additional knock-out studies should provide important insights into the various functions of glycolipids in myelinogenesis and myelin structure.

Glial conversion of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain.

In the adult mouse forebrain, large numbers of neuronal precursors, destined to become GABA- and dopamine-producing interneurons of the olfactory bulb (OB), are generated in the subventricular zone (SVZ). Although this neurogenic system represents a potential reservoir of stem and progenitor cells for brain repair approaches, information about the survival and differentiation of SVZ-derived cells in ectopic brain regions is still fragmentary. We show here that ectopic grafting of SVZ tissue gave rise to two morphologically distinguishable cell types displaying oligodendrocytic or astrocytic characteristics. Since SVZ tissue contains neuronal and glial progenitors, we used magnetic cell sorting to deplete A2B5+ glial progenitors from the dissociated SVZ and to positively select cells that express PSA-NCAM. This procedure allowed the purification of neuronal precursors expressing TUJ1, DCX and GAD65/67. Transplantation of these cells led again to the generation of the same two glial cell types, showing that committed interneuron precursors undergo glial differentiation outside their normal environment.

Purification of neuronal precursors from the adult mouse brain: comprehensive gene expression analysis provides new insights into the control of cell migration, differentiation, and homeostasis.

The progeny of neural stem cells in the subventricular zone (SVZ) of the adult mammalian brain consists in polysialylated NCAM-expressing immature neurons (PSA(+) cells), which migrate to the olfactory bulb (OB) to differentiate into GABAergic interneurons. We purified murine PSA(+) cells directly from the adult brain by FACS and analyzed their gene expression profile by SAGE. Comparative analyses led to the identification of precursor-enriched genes, including Survivin, Sox-4, Meis2, Dishevelled-2, C3aR1 and Riken 3110003A17, and many so far uncharacterized transcripts. Cluster analysis showed that groups of genes involved in axon guidance and gene clusters implicated in chemotaxis are strongly upregulated, indicating a role of both cues in the control of cell migration in the adult brain. Furthermore, genes involved in apoptosis and cell proliferation are co-expressed, suggesting that the amount of precursors that is present in the adult brain is a result of an equilibrium of these processes.

MicroRNAs are shaping the hematopoietic landscape

Hematopoiesis is regulated by microRNAs (miRNAs). These small regulatory RNAs are master regulators of developmental processes that modulate expression of several target genes post-transcriptionally. Various miRNAs are up-regulated at specific stages during hematopoietic development and the functional relevance of miRNAs has been proven at many different stages of lineage specification. Knockout of specific miRNAs can produce dramatic phenotypes leading to severe hematopoietic defects. Furthermore, several studies demonstrated that specific miRNAs are differentially expressed in hematopoietic stem cells. However, the emerging picture is extremely complex due to differences between species, cell type dependent variation in miRNA expression and differential expression of diverse target genes that are involved in various regulatory networks. There is also evidence that miRNAs play a role in cellular aging or in the inter-cellular crosstalk between hematopoietic cells and their microenvironment. The field is rapidly evolving due to new profiling tools and deep sequencing technology. The expression profiles of miRNAs are of diagnostic relevance for classification of different diseases. Recent reports on the generation of induced pluripotent stem cells with miRNAs have fuelled the hope that specific miRNAs and culture conditions facilitate directed differentiation or culture expansion of the hematopoietic stem cell pool. This review summarizes our current knowledge about miRNA expression in hematopoietic stem and progenitor cells, and their role in the hematopoietic stem cell niche.

Identification of RELMγ, a novel resistin-like molecule with a distinct expression pattern☆ ☆

We have identified RELMgamma, a novel member of the resistin-like molecule/found in inflammatory zone (RELM/FIZZ) family in mice and rats. Microarray and real-time RT-PCR experiments revealed a repression of RELMgamma mRNA in nasal respiratory epithelium of cigarette smoke-exposed versus untreated rats. The analysis of the physiological tissue-specific expression revealed highest expression in hematopoietic tissues, suggesting a cytokine-like role for RELMgamma. RELMgamma is most closely related to RELMalpha/FIZZ1. Despite the high similarity, the expression properties of the two genes are clearly distinct. While RELMgamma (approved symbol retnlg) is expressed in rat white adipose tissue, minute to no expression of RELMalpha was detected in that system. Thus, previous reports analyzing RELMalpha expression in rat adipose tissue might have been influenced by cross-hybridization with RELMgamma. Finally we could demonstrate that white adipose tissue of mice shows strong RELMalpha expression but only low levels of RELMgamma, indicating a species-specific gene regulation.

GALACTOSPHINGOLIPIDS AND AXONO-GLIAL INTERACTION IN MYELIN OF THE CENTRAL NERVOUS SYSTEM

Abstract The myelin of central and peripheral nervous system of UDP-galactose-ceramide galactosyltransferase deficient mice (cgt-/-) is completely depleted of its major lipid constituents, galactocerebrosides and sulfatides. The deficiency of these glycolipids affects the biophysical properties of the myelin sheath and causes the loss of the rapid saltatory conduction velocity of myelinated axons. With the onset of myelination, null mutant cgt-/- mice develop fatal neurological defects. CNS and PNS analysis of cgt-/- mice revealed (1) hypomyelination of axons of the spinal cord and optic nerves, but no apoptosis of oligodendrocytes, (2) redundant myelin in younger mice leading to vacuolated nerve fibers in cgt-/- mice, (3) the occurrence of multiple myelinated CNS axons, and (4) severely distorted lateral loops in CNS paranodes. The loss of saltatory conduction is not associated with a randomization of voltage-gated sodium channels in the axolemma of PNS fibers. We conclude that cerebrosides (GalC) and sulfatides (sGalC) play a major role in CNS axono-glial interaction. A close axono-glial contact is not a prerequisite for the spiraling and compaction process of myelin. Axonal sodium channels remain clustered at the nodes of Ranvier independent of the change in the physical properties of myelin membrane devoid of galactosphingolipids. Increased intracellular concentrations of free ceramides do not trigger apoptosis of oligodendrocytes.

Isolation of functional pure mitochondria by superparamagnetic microbeads

Isolation of mitochondria by current methods relies mainly on their physicochemical properties. Here we describe an alternative approach to obtain functional mitochondria from human cells in a fast, reproducible, and standardized procedure. The new approach is based on superparamagnetic microbeads conjugated to anti-TOM22 antibody. The bead conjugates label the cytoplasmic part of the human mitochondrial membrane protein TOM22 and, thus, allow for a gentle isolation of mitochondria in a high gradient magnetic field. By comparing the MACS (magnetic cell separation) approach with mitochondria isolation methods using differential centrifugation and ultracentrifugation we demonstrate that the MACS approach provides the highest yield of isolated mitochondria. The quality, enrichment, and purity of mitochondria isolated with this protocol are comparable to mitochondria obtained using the ultracentrifuge method, and a typical separation procedure takes only approximately 1 to 2h from initial cell homogenization. Mitochondria isolated with the new approach are sufficient for protein import, blue native gel electrophoresis, and other mitochondrial assays.