Ronald P. Hart

Multiple knockout mouse models reveal lincRNAs are required for life and brain development

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc–Brn1b and linc–Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr−/− neonates, whereas linc–Brn1b−/− mutants displayed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001

Toll‐like receptor (TLR)‐2 and TLR‐4 regulate inflammation, gliosis, and myelin sparing after spinal cord injury

Activation of macrophages via toll‐like receptors (TLRs) is important for inflammation and host defense against pathogens. Recent data suggest that non‐pathogenic molecules released by trauma also can trigger inflammation via TLR2 and TLR4. Here, we tested whether TLRs are regulated after sterile spinal cord injury (SCI) and examined their effects on functional and anatomical recovery. We show that mRNA for TLR1, 2, 4, 5, and 7 are increased after SCI as are molecules associated with TLR signaling (e.g. MyD88, NFκB). The significance of in vivo TLR2 and TLR4 signaling was evident in SCI TLR4 mutant (C3H/HeJ) and TLR2 knockout (TLR2−/−) mice. In C3H/HeJ mice, sustained locomotor deficits were observed relative to SCI wild‐type control mice and were associated with increased demyelination, astrogliosis, and macrophage activation. These changes were preceded by reduced intraspinal expression of interleukin‐1β mRNA. In TLR2−/− mice, locomotor recovery also was impaired relative to SCI wild‐type controls and novel patterns of myelin pathology existed within ventromedial white matter – an area important for overground locomotion. Together, these data suggest that in the absence of pathogens, TLR2 and TLR4 are important for coordinating post‐injury sequelae and perhaps in regulating inflammation and gliosis after SCI.

Protective autoimmunity: interferon-γ enables microglia to remove glutamate without evoking inflammatory mediators

Glutamate in excessive amounts is a major contributor to neuronal degeneration, and its removal is attributed mainly to astrocytes. Traumatic injury to the central nervous system (CNS) is often accompanied by disappearance of astrocytes from the lesion site and failure of the remaining cells to withstand the ensuing toxicity. Microglia that repopulate the lesion site are the usual suspects for causing redox imbalance and inflammation and thus further exacerbating the neurotoxicity. However, our group recently demonstrated that early post‐injury activation of microglia as antigen‐presenting cells correlates with an ability to withstand injurious conditions. Moreover, we found that T cells reactive to CNS‐specific self‐antigens protected neurons against glutamate toxicity. Here, we show that antigen‐specific autoimmune T cells, by tailoring the microglial phenotype, can increase the ability of microglia‐enriched cultures to remove glutamate. This T‐cell‐mediated effect could not be achieved by the potent microglia‐activating agent lipopolysaccharide (LPS), but was dose‐dependently reproduced by the Th1 cytokine interferon (IFN)‐γ and significantly reduced by neutralizing anti‐IFN‐γ antibodies. Under the same conditions, IFN‐γ had no effect on cultured astrocytes. Up‐regulation of glutamate uptake induced by IFN‐γ activation was not accompanied by the acute inflammatory response seen in LPS‐activated cultures. These findings suggest that T cells or their cytokines can cause microglia to adopt a phenotype that facilitates rather than impairs glutamate clearance, possibly contributing to restoration of homeostasis.

Cytokine activity contributes to induction of inflammatory cytokine mRNAs in spinal cord following contusion

Injury of the spinal cord leads to an inflammatory tissue response, probably mediated in part by cytokines. Because a common therapy for acute spinal cord injury is the use of an antiinflammatory synthetic glucocorticoid (methylprednisolone), we sought to determine mechanisms contributing to inflammation shortly after acute injury. Cytokine mRNAs [interleukin (IL)‐1α, IL‐1β, tumor necrosis factor (TNF)‐α, and IL‐6] were increased during the first 2 hr following weight‐drop compression injury by RNase protection assay, prior to the reported appearance of circulating lymphocytes. This immediate pattern of cytokine mRNA induction could be replicated in cultured, explanted spinal cord slices but not in whole blood of injured animals, which is consistent with a tissue source of cytokine mRNAs. Western blotting detected IL‐1β‐like immunoreactivity released into culture medium following explantation and pro‐IL‐1β‐like immunoreactivity in freshly dissected spinal cord tissue. Pharmacologically blocking IL‐1 and TNF‐α receptors significantly reduced expression of IL‐1α, IL‐1β, and TNF‐α mRNAs. Finally, mice lacking both IL‐1 and TNF‐α receptors exhibited diminished induction of TNF‐α, IL‐6, and IL‐1ra mRNAs following injury. Therefore, we conclude that contusion injury induces an immediate release of cytokines, which then contributes to the induction of cytokine mRNAs.

Genome-wide analysis reveals methyl-CpG–binding protein 2–dependent regulation of microRNAs in a mouse model of Rett syndrome

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function as posttranscriptional regulators of gene expression. Many miRNAs are expressed in the developing brain and regulate multiple aspects of neural development, including neurogenesis, dendritogenesis, and synapse formation. Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG–binding protein 2 (MECP2). Although Mecp2 is known to act as a global transcriptional regulator, miRNAs that are directly regulated by Mecp2 in the brain are not known. Using massively parallel sequencing methods, we have identified miRNAs whose expression is altered in cerebella of Mecp2-null mice before and after the onset of severe neurological symptoms. In vivo genome-wide analyses indicate that promoter regions of a significant fraction of dysregulated miRNA transcripts, including a large polycistronic cluster of brain-specific miRNAs, are DNA-methylated and are bound directly by Mecp2. Functional analysis demonstrates that the 3′ UTR of messenger RNA encoding Brain-derived neurotrophic factor (Bdnf) can be targeted by multiple miRNAs aberrantly up-regulated in the absence of Mecp2. Taken together, these results suggest that dysregulation of miRNAs may contribute to RTT pathoetiology and also may provide a valuable resource for further investigations of the role of miRNAs in RTT.

Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia telangiectasia

Ataxia telangiectasia is a neurodegenerative disease caused by mutation of the Atm gene. Here we report that ataxia telangiectasia mutated (ATM) deficiency causes nuclear accumulation of histone deacetylase 4 (HDAC4) in neurons and promotes neurodegeneration. Nuclear HDAC4 binds to chromatin, as well as to myocyte enhancer factor 2A (MEF2A) and cAMP-responsive element binding protein (CREB), leading to histone deacetylation and altered neuronal gene expression. Blocking either HDAC4 activity or its nuclear accumulation blunts these neurodegenerative changes and rescues several behavioral abnormalities of ATM-deficient mice. Full rescue of the neurodegeneration, however, also requires the presence of HDAC4 in the cytoplasm, suggesting that the ataxia telangiectasia phenotype results both from a loss of cytoplasmic HDAC4 as well as its nuclear accumulation. To remain cytoplasmic, HDAC4 must be phosphorylated. The activity of the HDAC4 phosphatase, protein phosphatase 2A (PP2A), is downregulated by ATM-mediated phosphorylation. In ATM deficiency, enhanced PP2A activity leads to HDAC4 dephosphorylation and the nuclear accumulation of HDAC4. Our results define a crucial role of the cellular localization of HDAC4 in the events leading to ataxia telangiectasia neurodegeneration.

Concise Review: MicroRNA Expression in Multipotent Mesenchymal Stromal Cells

Mesenchymal stem cells, or multipotent mesenchymal stromal cells (MSC), isolated from various adult tissue sources have the capacities to self‐renew and to differentiate into multiple lineages. Both of these processes are tightly regulated by genetic and epigenetic mechanisms. Emerging evidence indicates that the class of single‐stranded noncoding RNAs known as microRNAs also plays a critical role in this process. First described in nematodes and plants, microRNAs have been shown to modulate major regulatory mechanisms in eukaryotic cells involved in a broad array of cellular functions. Studies with various types of embryonic as well as adult stem cells indicate an intricate network of microRNAs regulating key transcription factors and other genes, which in turn determine cell fate. In addition, expression of unique microRNAs in specific cell types serves as a useful diagnostic marker to define a particular cell type. MicroRNAs are also found to be regulated by extracellular signaling pathways that are important for differentiation into specific tissues, suggesting that they play a role in specifying tissue identity. In this review, we describe the importance of microRNAs in stem cells, focusing on our current understanding of microRNAs in MSC and their derivatives.

Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors

Background MicroRNAs are required for maintenance of pluripotency as well as differentiation, but since more microRNAs have been computationally predicted in genome than have been found, there are likely to be undiscovered microRNAs expressed early in stem cell differentiation. Methodology/Principal Findings SOLiD ultra-deep sequencing identified >107 unique small RNAs from human embryonic stem cells (hESC) and neural-restricted precursors that were fit to a model of microRNA biogenesis to computationally predict 818 new microRNA genes. These predicted genomic loci are associated with chromatin patterns of modified histones that are predictive of regulated gene expression. 146 of the predicted microRNAs were enriched in Ago2-containing complexes along with 609 known microRNAs, demonstrating association with a functional RISC complex. This Ago2 IP-selected subset was consistently expressed in four independent hESC lines and exhibited complex patterns of regulation over development similar to previously-known microRNAs, including pluripotency-specific expression in both hESC and iPS cells. More than 30% of the Ago2 IP-enriched predicted microRNAs are new members of existing families since they share seed sequences with known microRNAs. Conclusions/Significance Extending the classic definition of microRNAs, this large number of new microRNA genes, the majority of which are less conserved than their canonical counterparts, likely represent evolutionarily recent regulators of early differentiation. The enrichment in Ago2 containing complexes, the presence of chromatin marks indicative of regulated gene expression, and differential expression over development all support the identification of 146 new microRNAs active during early hESC differentiation.

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish.

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR‐133b during zebrafish spinal cord regeneration and show upregulation of miR‐133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR‐133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR‐133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR‐133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth‐related molecules. Our results indicate that miR‐133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR‐133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.

Intrinsic Differences in Brain and Spinal Cord Mitochondria: Implication for Therapeutic Interventions

It is well known that regions of the CNS differentially respond to insults. After brain injury, cyclosporine A reduces damage but is ineffective following spinal cord injury. We address this disparity by assessing several parameters of mitochondrial physiology in the normal neocortex and spinal cord. In situ measurements of O  2– · production, lipid peroxidation, and mitochondrial DNA oxidation revealed significantly higher levels in spinal cord vs. neocortical neurons. Real‐time PCR demonstrated differences in mitochondrial transcripts coupled with decreases in complex I enzyme activity and respiration in spinal cord mitochondria. The threshold for calcium‐induced mitochondrial permeability transition was substantially reduced in spinal cord vs. neocortex and modulated by lipid peroxidation. These intrinsic differences may provide a pivotal target for strategies to ameliorate neuronal damage following injury, and this imbalance in oxidative stress may contribute to the susceptibility of spinal cord motor neurons in neuropathologies such as amyotrophic lateral sclerosis. J. Comp. Neurol. 474:524–534, 2004.