Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a

Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.     

Clock Genes Influence Gene Expression in Growth Plate and Endochondral Ossification in Mice

We have previously shown transient promotion by parathyroid hormone of Period-1 (Per1) expression in cultured chondrocytes. Here we show the modulation by clock genes of chondrogenic differentiation through gene transactivation of the master regulator of chondrogenesis Indian hedgehog (IHH) in chondrocytes of the growth plate. Several clock genes were expressed with oscillatory rhythmicity in cultured chondrocytes and rib growth plate in mice, whereas chondrogenesis was markedly inhibited in stable transfectants of Per1 in chondrocytic ATDC5 cells and in rib growth plate chondrocytes from mice deficient of brain and muscle aryl hydrocarbon receptor nuclear translocator-like (BMAL1). Ihh promoter activity was regulated by different clock gene products, with clear circadian rhythmicity in expression profiles of Ihh in the growth plate. In BMAL1-null mice, a predominant decrease was seen in Ihh expression in the growth plate with a smaller body size than in wild-type mice. BMAL1 deficit led to disruption of the rhythmic expression profiles of both Per1 and Ihh in the growth plate. A clear rhythmicity was seen with Ihh expression in ATDC5 cells exposed to dexamethasone. In young mice defective of BMAL1 exclusively in chondrocytes, similar abnormalities were found in bone growth and Ihh expression. These results suggest that endochondral ossification is under the regulation of particular clock gene products expressed in chondrocytes during postnatal skeletogenesis through a mechanism relevant to the rhythmic Ihh expression.     

A single nucleotide polymorphism in 3'-untranslated region contributes to the regulation of Toll-like receptor 4 translation

We have previously shown that a single nucleotide polymorphism rs11536889 in the 3'-untranslated region (UTR) of TLR4 was associated with periodontitis. In this study the effects of this single nucleotide polymorphism on Toll-like receptor (TLR) 4 expression were investigated. Monocytes from subjects with the C/C genotype expressed higher levels of TLR4 on their surfaces than those from subjects with the other genotypes. Peripheral blood mononuclear cells (PBMCs) from the C/C and G/C subjects secreted higher levels of IL-8 in response to lipopolysaccharide (LPS), a TLR4 ligand, than the cells from the G/G subjects. However, there was no significant difference in TLR4 mRNA levels in PBMCs from the subjects with each genotype. After stimulation with tripalmitoylated CSK(4) (Pam(3)CSK(4)), TLR4 mRNA levels increased in PBMCs from both the C/C and G/G subjects, whereas TLR4 protein levels increased in PBMCs from the C/C but not G/G subjects. Transient transfection of a series of chimeric luciferase constructs revealed that a fragment of 3'-UTR containing rs11536889 G allele, but not C allele, suppressed luciferase activity induced by LPS or IL-6. Two microRNAs, hsa-miR-1236 and hsa-miR-642a, were predicted to bind to rs11536889 G allele. Inhibition of these microRNAs reversed the suppressed luciferase activity. These microRNA inhibitors also up-regulated endogenous TLR4 protein on THP-1 cells (the G/G genotype) after LPS stimulation. Furthermore, mutant microRNAs that bind to the C allele inhibited the luciferase activity of the construct containing the C allele. These results indicate that genetic variation of rs11536889 contributes to translational regulation of TLR4, possibly by binding to microRNAs.     

Variation in Courtship Ultrasounds of Three Ostrinia Moths with Different Sex Pheromones

Moths use ultrasounds as well as pheromones for sexual communication. In closely related moth species, variations in ultrasounds and pheromones are likely to profoundly affect mate recognition, reproductive isolation, and speciation. The European corn borer, Ostrinia nubilalis, and its Asian congeners, Ostrinia furnacalis and Ostrinia scapulalis, exhibit within-species and between-species variation in their pheromone communication. Recently, we reported ultrasound communication in O. furnacalis; however, variations in ultrasounds in the three congeners have not been addressed to date. Here we investigated features of ultrasound production and hearing in O. nubilalis and O. scapulalis, and compared them with those of O. furnacalis. As in O. furnacalis, males of O. nubilalis and O. scapulalis produced ultrasounds during courtship by rubbing specialized scales on the wings against scales on the thorax. The covering of these scales with nail polish muffled the sounds and significantly reduced mating success in O. nubilalis, showing the importance of ultrasound signaling in mating. The ultrasounds produced by O. nubilalis and O. scapulalis were similar, consisting of long trains of pairs of pulses with a main energy at 40 kHz, but distinctly different from the ultrasound produced by O. furnacalis, consisting of groups of pulses peaking at 50 kHz and with substantially more energy up to 80 kHz. Despite overall similarities, temporal features and patterns of amplitude modulation differed significantly among the geographic populations of O. nubilalis and O. scapulalis, which differed in pheromone type. In contrast, no significant difference in hearing was found among the three species with regard to the most sensitive frequencies and hearing threshold levels. The patterns of variations in the songs and pheromones well reflected those of the phylogenetic relationships, implying that ultrasound and pheromone communications have diverged concordantly. Our results suggest that concordant evolution in sexual signals such as courtship ultrasounds and sex pheromones occurs in moths.     

miR-200b precursor can ameliorate renal tubulointerstitial fibrosis

Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.     

Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development

Background

Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.

Methodology/Principal Findings

Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca²⁺ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.

Conclusions/Significance

These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.     

Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K_d of ∼82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4–8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.     

Establishment of induced pluripotent stem cells from human neonatal tissues

Following the success in establishing human induced pluripotent stem (iPS) cells, research into various applications of the cells derived from human iPS cells has begun in earnest. The use of iPS cell-derived cells in clinical therapies is one of the most exciting of the possible applications. However, the risk of tumorigenicity is the biggest potential obstacle to use iPS cell derivatives in the clinic. It should be noted that the human cells used to generate iPS cell lines may have acquired genetic mutations and these might influence the tumorigenicity of the cells. In particular, the cells of older people have a higher risk of genetic mutations than those of younger people. Here, we show that iPS cells could be derived from short-term cultures of neonatal tissues. The established human iPS cells expressed various markers of undifferentiated cells and formed teratoma in immunodeficient mice. The human iPS cells derived from neonatal tissues may represent a clinical material possessing less tumorigenicity.     

Multicenter evaluation of prototype real‐time PCR assays for Epstein‐Barr virus and cytomegalovirus DNA in whole blood samples from transplant recipients

Quantitative PCR is becoming widespread for diagnosing and monitoring post‐transplantation diseases associated with EBV and CMV. These assays need to be standardized to manage patients in different facilities. Five independent laboratories in Japan compared home‐brew assays and a prototype assay system to establish a standard quantitative procedure for measuring EBV and CMV. Reference standards and a total of 816 (642 EBV and 174 CMV) whole blood samples from post‐transplantation recipients were used for this multicenter evaluation. The prototype reference standard for EBV was compared to a panel of samples, with a theoretical expected value made using EBV‐positive cells containing two virus genome copies per cell. The mean ratio of the reference standard at each site to the standard of the prototype assay was ≤4.15 for EBV among three different sites and ≤3.0 for CMV between two laboratories. The mean of the theoretical expected number of the EBV genome: prototype reference was close to 1.0. The correlation coefficients between the viral copy numbers determined using the prototype assay and those using each home‐brew assay were high (EBV, 0.73–0.83, median = 0.78; CMV, 0.54–0.60, median = 0.57). The dynamics of the EBV and CMV loads in transplant recipients were similar between the assay types. There was an inter‐laboratory difference among the quantification results, indicating that a unified protocol and kit are favorable for standardizing the quantification of EBV and CMV. Such standardization will help to standardize the diagnosis and monitoring of diseases associated with EBV and CMV.     

Biaryl modification of the 5'-terminus of one strand of a microRNA duplex induces strand specificity

MicroRNAs (miRNAs) are single-stranded non-coding RNAs composed of 20-23 nucleotides. They are initially transcribed in the nucleus as pri-miRNAs. After processing, one strand from the miRNA duplex (miR-5p/miR-3p duplex) is loaded onto the RNA-induced silencing complex (RISC) to produce a functional, mature miRNA that inhibits the expression of multiple target genes. In the case of some miRNAs, both strands can be equally incorporated into the RISC as single strands, and both strands can function as mature miRNAs. Thus, a technique for selective expression of miR-5p and miR-3p strands is required to identify distinct targets of miRNAs. In this Letter, we report the synthesis and properties of miRNA duplexes carrying biaryl units at the 5'-terminus of one strand. We found that incorporation of biaryl units at the 5'-terminus of one strand of miRNA duplexes induced strand specificity in these duplexes. Further, we succeeded in identifying endogenous mRNA targets for each strand of the duplex by using the biaryl-modified miRNA duplexes.