Thinking about RNA? MicroRNAs in the brain

MicroRNAs (miRNAs) are a recently discovered class of small RNA molecules implicated in a wide range of diverse gene regulatory mechanisms. Interestingly, numerous miRNAs are expressed in a spatially and temporally controlled manner in the nervous system. This suggests that gene regulation networks based on miRNA activities may be particularly relevant in neurons. Recent studies show the involvement of RNA-mediated gene silencing in neurogenesis, neural differentiation, synaptic plasticity, and neurologic and psychiatric diseases. This review focuses on the roles of miRNAs in the gene regulation of the nervous system.     

Paired Siglec receptors generate opposite inflammatory responses to a human-specific pathogen

Paired immune receptors display near-identical extracellular ligand-binding regions but have intracellular sequences with opposing signaling functions. While inhibitory receptors dampen cellular activation by recognizing self-associated molecules, the functions of activating counterparts are less clear. Here, we studied the inhibitory receptor Siglec-11 that shows uniquely human expression in brain microglia and engages endogenous polysialic acid to suppress inflammation. We demonstrated that the human-specific pathogen Escherichia coli K1 uses its polysialic acid capsule as a molecular mimic to engage Siglec-11 and escape killing. In contrast, engagement of the activating counterpart Siglec-16 increases elimination of bacteria. Since mice do not have paired Siglec receptors, we generated a model by replacing the inhibitory domain of mouse Siglec-E with the activating module of Siglec-16. Siglec-E16 enhanced proinflammatory cytokine expression and bacterial killing in macrophages and boosted protection against intravenous bacterial challenge. These data elucidate uniquely human interactions of a pathogen with Siglecs and support the long-standing hypothesis that activating counterparts of paired immune receptors evolved as a response to pathogen molecular mimicry of host ligands for inhibitory receptors.     

In Vivo Identification of Nuclear Factors Interacting with the Conserved Elements of Box C/D Small Nucleolar RNAs

The U16 small nucleolar RNA (snoRNA) is encoded by the third intron of the L1 (L4, according to the novel nomenclature) ribosomal protein gene of Xenopus laevis and originates from processing of the pre-mRNA in which it resides. The U16 snoRNA belongs to the box C/D snoRNA family, whose members are known to assemble in ribonucleoprotein particles (snoRNPs) containing the protein fibrillarin. We have utilized U16 snoRNA in order to characterize the factors that interact with the conserved elements common to the other members of the box C/D class. In this study, we have analyzed the in vivo assembly of U16 snoRNP particles in X. laevis oocytes and identified the proteins which interact with the RNA by label transfer after UV cross-linking. This analysis revealed two proteins, of 40- and 68-kDa apparent molecular size, which require intact boxes C and D together with the conserved 5′,3′-terminal stem for binding. Immunoprecipitation experiments showed that the p40 protein corresponds to fibrillarin, indicating that this protein is intimately associated with the RNA. We propose that fibrillarin and p68 represent the RNA-binding factors common to box C/D snoRNPs and that both proteins are essential for the assembly of snoRNP particles and the stabilization of the snoRNA.One of the most interesting recent findings related to ribosome biogenesis has been the identification of a large number of small RNAs localized in the nucleolus (snoRNAs). So far, more than 60 snoRNAs have been identified in vertebrates (17), and more than 30 have been identified in yeast (2). The total number of snoRNAs is not known, but it is likely to be close to 200 (33, 38). These snoRNAs, with the exception of the mitochondrial RNA processing (MRP) species (38), can be grouped into two major families on the basis of conserved structural and sequence elements. The first group includes molecules referred to as box C/D snoRNAs, whereas the second one comprises the species belonging to the box H/ACA family (2, 15).The two families differ in many aspects. The box C/D snoRNAs are functionally heterogeneous. Most of them function as antisense RNAs in site-specific ribose methylation of the pre-rRNA (1, 10, 17, 26); a minority have been shown to play a direct role in pre-rRNA processing in both yeast and metazoan cells (11, 21). The box C/D snoRNAs play their role by means of unusually long (up to 21 contiguous nucleotides) regions of complementarity to highly conserved sequences of 28S and 18S rRNAs (1). In contrast, several members of the H/ACA RNA family have been shown to direct site-specific isomerization of uridines into pseudouridines and to display shorter regions of complementarity to rRNA (14, 24). Mutational analysis suggests that H/ACA snoRNAs can also play a role as antisense RNAs by base pairing with complementary regions on rRNA (15, 24).Another difference between the two families can be seen by comparison of secondary structures. A Y-shaped motif, where a 5′,3′-terminal stem adjoins the C and D conserved elements, has been proposed for many box C/D snoRNAs (16, 26, 40, 42), whereas box H/ACA snoRNAs have been proposed to fold into two conserved hairpin structures connected by a single-stranded hinge region, followed by a short 3′ tail (15).Despite these differences, analogies have been found in the roles played by the conserved box elements. Mutational analysis and competition experiments indicated that C/D and H/ACA boxes are required both for processing and stable accumulation of the mature snoRNA, suggesting that they represent binding sites for specific trans-acting factors (2, 3, 8, 15, 16, 28, 36, 41).All snoRNAs are associated with proteins to form specific ribonucleoparticles (snoRNPs). The study of these particles began only recently, and so far, very few aspects of their structure and biosynthesis have been clarified. The only detailed analysis performed was on the mammalian U3 (19) and the yeast snR30 (20) snoRNPs. Of the identified components, a few appear to be more general factors: fibrillarin, which was shown to be associated with C/D snoRNPs (3, 4, 8, 13, 28, 31, 39), and the nucleolar protein GAR1, which was found associated with H/ACA snoRNAs in yeast (20). Just as the study of small nuclear RNP (snRNP) particles was crucial to the understanding of the splicing process, a detailed structural and functional analysis of snoRNP particles will be essential to elucidate the complex process of ribosome biosynthesis.In this study, we have analyzed the snoRNP assembly of wild-type and mutant U16 snoRNAs by following the kinetics of complex formation in the in vivo system of the Xenopus laevis oocyte. By a UV cross-linking technique, we have identified two proteins, of 40- and 68-kDa apparent molecular mass, which require intact boxes C and D together with the terminal stem for their binding. The 40-kDa species is specifically recognized by fibrillarin antibodies, indicating that this protein is intimately associated with the RNA.     

sAPPα rescues deficits in amyloid precursor protein knockout mice following focal traumatic brain injury

The amyloid precursor protein (APP) is thought to be neuroprotective following traumatic brain injury (TBI), although definitive evidence at moderate to severe levels of injury is lacking. In the current study, we investigated histological and functional outcomes in APP-/- mice compared with APP+/+ mice following a moderate focal injury, and whether administration of sAPPα restored the outcomes in knockout animals back to the wildtype state. Following moderate controlled cortical impact injury, APP-/- mice demonstrated greater impairment in motor and cognitive outcome as determined by the ledged beam and Barnes Maze tests respectively (p < 0.05). This corresponded with the degree of neuronal damage, with APP-/- mice having significantly greater lesion volume (25.0 ± 1.6 vs. 20.3 ± 1.6%, p < 0.01) and hippocampal damage, with less remaining CA neurons (839 ± 245 vs. 1353 ± 142 and 1401 ± 263). This was also associated with an impaired neuroreparative response, with decreased GAP-43 immunoreactivity within the cortex around the lesion edge compared with APP+/+ mice. The deficits observed in the APP-/- mice related to a lack of sAPPα, as treatment with exogenously added sAPPα post-injury improved APP-/- mice histological and functional outcome to the point that they were no longer significantly different to APP+/+ mice (p < 0.05). This study shows that endogenous APP is potentially protective at moderate levels of TBI, and that this neuroprotective activity is related to the presence of sAPPα. Importantly, it indicates that the mechanism of action of exogenously added sAPPα is independent of the presence of endogenous APP.     

Natural and Vaccine-Mediated Immunity to Salmonella Typhimurium is Impaired by the Helminth Nippostrongylus brasiliensis

Background

The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined.

Methodology/Principal Findings

Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection.

Conclusion

These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization.     

Development of alpha-keto-based inhibitors of cruzain, a cysteine protease implicated in Chagas disease

Trypanosoma cruzi, a protozoan parasite, is the causative agent of Chagas disease, a major cause of cardiovascular disease in many Latin American countries. There is an urgent need to develop an improved therapy due to the toxicity of existing drugs and emerging drug resistance. Cruzain, the primary cysteine protease of T. cruzi, is essential for the survival of the parasite in host cells and therefore is an important target for the development of inhibitors as potential therapeutics. A novel series of alpha-ketoamide-, alpha-ketoacid-, alpha-ketoester-, and aldehyde-based inhibitors of cruzain has been developed. The inhibitors were identified by screening protease targeted small molecule libraries and systematically optimizing the P1, P2, P3, and P1' residues using specific structure-guided methods. A total of 20 compounds displayed picomolar potency in in vitro assays and three inhibitors representing different alpha-keto-based inhibitor scaffolds demonstrated anti-trypanosomal activity in cell culture. A 2.3A crystallographic structure of cruzain bound with one of the alpha-ketoester analogs is also reported. The structure and kinetic assay data illustrate the covalent binding, reversible inhibition mechanism of the inhibitor. Information on the compounds reported here will be useful in the development of new lead compounds as potential therapeutic agents for the treatment of Chagas disease and as biological probes to study the role that cruzain plays in the pathology. This study also demonstrates the validity of structure-guided approaches to focused library design and lead compound optimization.     

Jamip1 (marlin-1) defines a family of proteins interacting with janus kinases and microtubules

Jamip1 (Jak and microtubule interacting protein), an alias of Marlin-1, was identified for its ability to bind to the FERM (band 4.1 ezrin/radixin/moesin) homology domain of Tyk2, a member of the Janus kinase (Jak) family of non-receptor tyrosine kinases that are central elements of cytokine signaling cascades. Jamip1 belongs to a family of three genes conserved in vertebrates and is predominantly expressed in neural tissues and lymphoid organs. Jamip proteins lack known domains and are extremely rich in predicted coiled coils that mediate dimerization. In our initial characterization of Jamip1 (73 kDa), we found that it comprises an N-terminal region that targets the protein to microtubule polymers and, when overexpressed in fibroblasts, profoundly perturbs the microtubule network, inducing the formation of tight and stable bundles. Jamip1 was shown to associate with two Jak family members, Tyk2 and Jak1, in Jurkat T cells via its C-terminal region. The restricted expression of Jamip1 and its ability to associate to and modify microtubule polymers suggest a specialized function of these proteins in dynamic processes, e.g. cell polarization, segregation of signaling complexes, and vesicle traffic, some of which may involve Jak tyrosine kinases.     

Acute regulation of Na/H exchanger NHE3 by adenosine A(1) receptors is mediated by calcineurin homologous protein

Adenosine is an autacoid that regulates renal Na(+) transport. Activation of adenosine A(1) receptor (A(1)R) by N(6)-cyclopentidyladenosine (CPA) inhibits the Na(+)/H(+) exchanger 3 (NHE3) via phospholipase C/Ca(2+)/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A(1)R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10(-6) M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3(C terminus); prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3(C terminus) (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3(delta552)) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3(462-552)) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3(Full-length) interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A(1)R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.     

An anthocyanin/polyphenolic-rich fruit juice reduces oxidative DNA damage and increases glutathione level in healthy probands

Oxidative cell damage is involved in the pathogenesis of atherosclerosis, cancer, diabetes and other diseases. Uptake of fruit juice with especially high content of antioxidant flavonoids/polyphenols, might reduce oxidative cell damage. Therefore, an intervention study was performed with a red mixed berry juice [trolox equivalent antioxidative capacity (TEAC): 19.1 mmol/L trolox] and a corresponding polyphenol-depleted juice (polyphenols largely removed, TEAC 2.4 mmol/L trolox), serving as control. After a 3-week run-in period, 18 male probands daily consumed 700 mL juice, and 9 consumed control juice, in a 4-week intervention, followed by a 3-week wash-out. Samples were collected weekly to analyze DNA damage (comet assay), lipid peroxidation (plasma malondialdehyde: HPLC/fluorescence; urinary isoprostanes: GC-MS), blood glutathione (photometrically), DNA-binding activity of nuclear factor-kappaB (ELISA) and plasma carotenoid/alpha-tocopherol levels (HPLC-DAD). During intervention with the fruit juice, a decrease of oxidative DNA damage (p<5x10(-4)) and an increase of reduced glutathione (p<5x10(-4)) and of glutathione status (p<0.05) were observed, which returned to the run-in levels in the subsequent wash-out phase. The other biomarkers were not significantly modulated by the juice supplement. Intervention with the control juice did not result in reduction of oxidative damage. In conclusion, the fruit juice clearly reduces oxidative cell damage in healthy probands.