Developmental genetic profiles of glutamate receptor system,neuromodulator system,protector of normal tissue and mitochondria,and reelin in marmoset cortex: Potential molecular mechanisms of pruning phase of spines in primate synaptic formation process during the end of infancy and prepuberty (II)

This is the second report of a series paper, which reports molecular mechanisms underlying the occurrence of pruning spine phase after rapid spinogenesis phase in neonates and young infant in the primate brain. We performed microarray analysis between the peak of spine numbers [postnatal 3 months (M)] and spine pruning (postnatal 6 M) in prefrontal, inferior temporal, and primary visual cortices of the common marmoset (Callithrix jacchus). The pruning phase is not clearly defined in rodents but is in primates including the marmoset. The differentially expressed genes between 3 M and 6 M in all three cortical areas were selected by two-way analysis of variance. The list of selected genes was analyzed by canonical pathway analysis using “Ingenuity Pathway Analysis of complex omics data” (IPA; Ingenuity Systems, Qiagen, Hilden, Germany). In this report, we discuss these lists of genes for the glutamate receptor system, G-protein-coupled neuromodulator system, protector of normal tissue and mitochondria, and reelin. (1) Glutamate is a common neurotransmitter. Its receptors AMPA1, GRIK1, and their scaffold protein DLG4 decreased as spine numbers decreased. Instead, GRIN3 (NMDA receptor) increased, suggesting that strong NMDA excitatory currents may be required for a single neuron to receive sufficient net synaptic activity in order to compensate for the decrease in synapse. (2) Most of the G protein-coupled receptor genes (e.g., ADRA1D, HTR2A, HTR4, and DRD1) in the selected list were upregulated at 6 M. The downstream gene ROCK2 in these receptor systems plays a role of decreasing synapses, and ROCK2 decreased at 6 M. (3) Synaptic phagosytosis by microglia with complement and other cytokines could cause damage to normal tissue and mitochondria. SOD1, XIAP, CD46, and CD55, which play protective roles in normal tissue and mitochondria, showed higher expression at 6 M than at 3 M, suggesting that normal brain tissue is more protected at 6 M. (4) Reelin has an important role in cortical layer formation. In addition, RELN and three different pathways of reelin were expressed at 6 M, suggesting that new synapse formation decreased at that age. Moreover, if new synapses were formed, their positions were free and probably dependent on activity.     

Developmental expression profiles of axon guidance signaling and the immune system in the marmoset cortex: Potential molecular mechanisms of pruning of dendritic spines during primate synapse formation in late infancy and prepuberty (I)

The synapse number and the related dendritic spine number in the cerebral cortex of primates shows a rapid increase after birth. Depending on the brain region and species, the number of synapses reaches a peak before adulthood, and pruning takes place after this peak (overshoot-type synaptic formation). Human mental disorders, such as autism and schizophrenia, are hypothesized to be a result of either too weak or excessive pruning after the peak is reached. Thus, it is important to study the molecular mechanisms underlying overshoot-type synaptic formation, particularly the pruning phase.     

Relating body size to the role of aquatic subsidies for the riparian spider <Emphasis Type="Italic">Nephila clavata</Emphasis>

We examined the relationship between body size of the riparian spider Nephila clavata and the contribution of allochthonous (aquatic insects) and autochthonous (terrestrial insects) sources to its diet using stable isotope analysis. During the study period from July to September, the body size of the females increased remarkably (about 60-fold) but that of males remained small. The biomass of both aquatic and terrestrial insects trapped on the spider webs increased with spider size, with the biomass of the former ranging between 30 and 70% of that of the terrestrial insects. The average relative contribution of aquatic insects to the diet of the spiders, calculated from δ¹³C values, was 40–50% in spiders in the early juvenile and juvenile stages, 35% in adult males and 4% in adult females. There was a significant negative relationship between the relative contribution of aquatic insects and body size of the female spiders. We conclude that aquatic insects might be an important seasonal dietary subsidy for small spiders and that these allochthonous subsidies may facilitate the growth of riparian spiders, which may in turn enable the spiders to feed on larger prey.     

O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-Fut1), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-Fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, here we generated a knock-in mutant of O-fut1 (O-fut1^{R245A knock-in}), which expresses a mutant protein that lacks O-fucosyltransferase activity but maintains the N-specific chaperon activity. Using O-fut1^{R245A knock-in} and other gene mutations that abolish the O-fucosylation of N, we found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. We also showed that the redundant function of these two modifications is responsible for the presence of N at the cell surface. Our findings elucidate how different forms of glycosylation on a protein can influence the protein''s functions.     

Dual oscillator model of the respiratory neuronal network generating quantal slowing of respiratory rhythm

We developed a dual oscillator model to facilitate the understanding of dynamic interactions between the parafacial respiratory group (pFRG) and the preBötzinger complex (preBötC) neurons in the respiratory rhythm generation. Both neuronal groups were modeled as groups of 81 interconnected pacemaker neurons; the bursting cell model described by Butera and others [model 1 in Butera et al. (J Neurophysiol 81:382–397, 1999a)] were used to model the pacemaker neurons. We assumed (1) both pFRG and preBötC networks are rhythm generators, (2) preBötC receives excitatory inputs from pFRG, and pFRG receives inhibitory inputs from preBötC, and (3) persistent Na⁺ current conductance and synaptic current conductances are randomly distributed within each population. Our model could reproduce 1:1 coupling of bursting rhythms between pFRG and preBötC with the characteristic biphasic firing pattern of pFRG neurons, i.e., firings during pre-inspiratory and post-inspiratory phases. Compatible with experimental results, the model predicted the changes in firing pattern of pFRG neurons from biphasic expiratory to monophasic inspiratory, synchronous with preBötC neurons. Quantal slowing, a phenomena of prolonged respiratory period that jumps non-deterministically to integer multiples of the control period, was observed when the excitability of preBötC network decreased while strengths of synaptic connections between the two groups remained unchanged, suggesting that, in contrast to the earlier suggestions (Mellen et al., Neuron 37:821–826, 2003; Wittmeier et al., Proc Natl Acad Sci USA 105(46):18000–18005, 2008), quantal slowing could occur without suppressed or stochastic excitatory synaptic transmission. With a reduced excitability of preBötC network, the breakdown of synchronous bursting of preBötC neurons was predicted by simulation. We suggest that quantal slowing could result from a breakdown of synchronized bursting within the preBötC.     

Resveratrol improves cognitive function in mice by increasing production of insulin-like growth factor-I in the hippocampus

We examined whether resveratrol increases insulin-like growth factor-I (IGF-I) production in the hippocampus by stimulating sensory neurons in the gastrointestinal tract, thereby improving cognitive function in mice. Resveratrol increased calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) neurons isolated from wild-type (WT) mice. Increases in tissue levels of CGRP, IGF-I, and IGF-I mRNA and immunohistochemical expression of IGF-I were observed in the hippocampus at 3 weeks after oral administration of resveratrol in WT mice. Significant enhancement of angiogenesis and neurogenesis was observed in the dentate gyrus of the hippocampus in these animals (P<.01). Improvement of spatial learning in the Morris water maze was observed in WT mice after administration of resveratrol. None of the effects of resveratrol observed in WT mice were seen after resveratrol administration in CGRP-knockout (CGRP^−/−) mice. Although red wine containing 20 mg/L of resveratrol produced effects similar to those of resveratrol administrationl in WT mice, neither red wine containing 3.1 mg/L of resveratrol nor white wine exhibited such effects in WT mice. Resveratrol was undetectable in the hippocampus of WT mice administered resveratrol and red wine containing 20 mg/L of resveratrol. These observations strongly suggest that resveratrol increases hippocampal IGF-I production via sensory neuron stimulation in the gastrointestinal tract, thereby improving cognitive function in mice.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

Optimization of a coagulation factor VIIa inhibitor found in factor Xa inhibitor library

An inhibitor of the complex of factor VIIa and tissue factor (fVIIa/TF), 2-substituted-4-amidinophenylpyruvic acid 1a, was structurally modified with the aim of increasing its potency and selectivity. The lead compound 1a was originally found in our factor Xa (fXa) inhibitor library on the basis of structural similarity of the primary binding sites of fVIIa and fXa. The design was based on computational docking studies using the extracted active site of fVIIa. Compound 1j was found to inhibit factor VIIa/TF at nanomolar concentration with improved selectivity versus fXa and thrombin and it preferentially prolonged the clotting time in the TF-dependent extrinsic pathway.     

Sphingosine 1-phosphate inhibits migration and RANTES production in human bronchial smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, has been shown to be increased in bronchoalveolar lavage fluid after allergen challenge in asthmatic patients. Here, we examined S1P actions and their intracellular signalings in cultured human bronchial smooth muscle cells (BSMCs). Expression of mRNAs of three subtypes of S1P receptors, including S1P(1), S1P(2), and S1P(3), was detected in BSMCs, and exposure of the cells to S1P inhibited platelet-derived growth factor (PDGF)-induced migration and tumor necrosis factor-alpha-induced RANTES production. S1P also inhibited PDGF-induced Rac1 activation, and dominant negative Rac1 inhibited PDGF-induced migration. On the other hand, dominant negative Galpha(q) attenuated the S1P-induced inhibition of RANTES production. Finally, an S1P(2)-selective antagonist, JTE-013, suppressed the S1P-induced inhibition of migration response and RANTES production. These results suggest that S1P attenuates cell migration by inhibiting a Rac1-dependent signaling pathway and decreases RANTES production by stimulating a Galpha(q)-dependent mechanism both possibly through the S1P(2) receptors.     

Oxidized but not acetylated low-density lipoprotein reduces preproinsulin mRNA expression and secretion of insulin from HIT-T15 cells

We examined the effect of oxidized low-density lipoprotein (oxLDL) on the insulin secretion in the culture of HIT-T15 cell line, an islet beta-cell line derived from a hamster pancreatic tumor. In order to check the uptake of modified LDL by HIT-T15 cells, we prepared DiI-labeled native LDL (nLDL), acetylated LDL (AcLDL), and oxLDL. After the addition of each LDL into the cultures of HIT-T15 cells, fluorescence microscopic study was done. It was suggested that AcLDL and oxLDL were taken up by HIT-T15 cells, as well as nLDL. mRNA expression of the LDL receptor, CD36, and SR-B1 was detected in HIT-T15 by RT-PCR. The medium insulin level was measured in the culture of HIT-T15 cells with each LDL. oxLDL significantly reduced the insulin secretion stimulated by various concentrations of glucose, the intracellular content of insulin, and the expression of preproinsulin mRNA compared to the control cultures without LDL addition. In contrast, nLDL and AcLDL had no effect on the insulin secretion, the intracellular insulin level, or the expression of preproinsulin mRNA. MTT assay findings (reflecting cell numbers) were not different between cultures with and without LDLs. These results indicated that oxLDL disturbed the insulin metabolism of HIT-T15 cells.