Comprehensive behavioural study of GluR4 knockout mice: implication in cognitive function

Fast excitatory transmission in the mammalian central nervous system is mediated by AMPA‐type glutamate receptors. The tetrameric AMPA receptor complexes are composed of four subunits, GluR1–4. The GluR4 subunit is highly expressed in the cerebellum and the early postnatal hippocampus and is thought to be involved in synaptic plasticity and the development of functional neural circuitry through the recruitment of other AMPA receptor subunits. Previously, we reported an association of the human GluR4 gene (GRIA4) with schizophrenia. To examine the role of the GluR4 subunit in the higher brain function, we generated GluR4 knockout mice and conducted electrophysiological and behavioural analyses. The mutant mice showed normal long‐term potentiation (LTP) in the CA1 region of the hippocampus. The GluR4 knockout mice showed mildly improved spatial working memory in the T‐maze test. Although the retention of spatial reference memory was intact in the mutant mice, the acquisition of spatial reference memory was impaired in the Barnes circular maze test. The GluR4 knockout mice showed impaired prepulse inhibition. These results suggest the involvement of the GluR4 subunit in cognitive function.     

Verification of the turn at positions 22 and 23 of the β-amyloid fibrils with Italian mutation using solid-state NMR

The aggregation of 42-mer amyloid β (Aβ42) plays a central role in the pathogenesis of Alzheimer’s disease. Our recent research on proline mutagenesis of Aβ42 suggested that the formation of a turn structure at positions 22 and 23 could play a crucial role in its aggregative ability and neurotoxicity. Since E22K-Aβ42 (Italian mutation) aggregated more rapidly and with more potent neurotoxicity than wild-type Aβ42, the tertiary structure at positions 21–24 of E22K-Aβ42 fibrils was analyzed by solid-state NMR using dipolar-assisted rotational resonance (DARR) to identify the ‘malignant’ conformation of Aβ42. Two sets of chemical shifts for Asp-23 were observed in a ratio of about 2.6:1. The 2D DARR spectra at the mixing time of 500 ms suggested that the side chains of Asp-23 and Val-24 in the major conformer, and those of Lys-22 and Asp-23 in the minor conformer could be located on the same side, respectively. These data support the presence of a turn structure at positions 22 and 23 in E22K-Aβ42 fibrils. The formation of a salt bridge between Lys-22 and Asp-23 in the minor conformer might be a reason why E22K-Aβ42 is more pathogenic than wild-type Aβ42.     

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background

In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed.

Principal Findings

Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7.

Conclusions/Significance

Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.     

Characterization of feline serum ferritin-binding proteins: the presence of a novel ferritin-binding protein as an inhibitory factor in feline ferritin immunoassay

Ferritin-binding proteins (FBPs) such as anti-ferritin antibody, α-2-macroglobulin, apolipoprotein B are expected to interact with circulating ferritin to eliminate it from circulation. However, we found that feline serum more strongly inhibits the detection of canine liver ferritin by immunoassay than its apoferritin; putative FBPs probably conceal ferritin epitopes detected by anti-ferritin antibodies. After complex formation between affinity-purified FBPs and canine liver ferritin, co-immunoprecipitates of the complex by anti-bovine spleen ferritin antibody were found to contain autoantibodies (IgG, IgM, and IgA) to ferritin by immunoblot analysis with antibodies specific for feline IgG, IgM, and IgA. On the other hand, affinity-purified samples did not show any inhibitory effect in the ferritin immunoassay. This result shows that feline serum has another FBP, which inhibits ferritin immunoassays, but not anti-ferritin autoantibody. A feline FBP was partially purified from feline serum by (NH₄)₂SO₄ fractionation (33–50%), gel filtration chromatography, and anion exchange chromatography. After binding of the partially purified sample with canine liver ferritin coupled-Sepharose gel, the FBP was separated and purified from complexes formed in a native-PAGE gel. SDS–PAGE analysis showed that the purified FBP is a homomultimer composed of 31 kDa monomeric subunits connected by intermolecular disulfide bonds. Detection of feline liver ferritin by immunoassay was inhibited by FBP in a dose-dependent manner. The purified protein molecules appeared to be conglomerate of pentraxin-like molecules by its electron micrographic appearance. These results demonstrate that feline serum contains a novel FBP as inhibitory factor of ferritin immunoassay with different molecular properties from those of other mammalian FBPs, in addition to auto-antibodies (IgG, IgM, and IgA) to ferritin.     

SAR-based optimization of 2-(1H-pyrazol-1-yl)-thiazole derivatives as highly potent EP1 receptor antagonists

We describe a medicinal chemistry approach for generating a series of 2-(1H-pyrazol-1-yl)thiazoles as EP₁ receptor antagonists. To improve the physicochemical properties of compound 1, we investigated its structure–activity relationships (SAR). Optimization of this lead compound provided small compound 25 which exhibited the best EP₁ receptor antagonist activity and a good SAR profile.     

Model mice for tissue-specific deletion of the manganese superoxide dismutase (MnSOD) gene

Manganese superoxide dismutase (MnSOD) is the enzyme that converts toxic O(2)(-) to H(2)O(2) in mitochondria. Previous reports showed that a deficiency of MnSOD in mice was neonatal lethal. Therefore, a model mouse was not available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type model mouse, we designed tissue-specific MnSOD conditional knockout mice using a Cre-loxp system. First, we crossbred MnSOD flox mice with transgenic mice expressing Cre recombinase under the control of the chicken actin promoter (CAG). We confirmed that CAG MnSOD knockout mice were completely deficient in MnSOD and died as neonates, validating the use of the Cre-loxp system. Next, we generated liver-specific MnSOD-deficient mice by crossbreeding with Alb-Cre transgenic mice. MnSOD activity and protein were both significantly downregulated in the liver of liver-specific MnSOD knockout mice. However, no obvious morphological abnormality was observed in the liver when biochemical alterations such as lipid peroxidation were not detectable, suggesting a redundant or less important physiological role for MnSOD in the liver than previously thought. In the present study, we successfully generated tissue-specific MnSOD conditional knockout mice that would provide a useful tool for the analysis of various age-associated diseases such as diabetes mellitus, Parkinson's disease, stroke, and heart disease, when crossbred with tissue-specific transgenic Cre mice.     

Endurance treadmill training in rats alters CRH activity in the hypothalamic paraventricular nucleus at rest and during acute running according to its period

Running training on the treadmill increases the resting hypothalamic corticotropin-releasing hormone (CRH) content in rats, though is still unknown whether and how it occurs in the parvocellular region of the hypothalamic paraventricular nucleus (PVN) where is a predominant region of pituitary-adrenal activity and where CRH and arginine vasopressin (AVP) are colocalized. We thus aimed at examining whether treadmill training would alter the CRH and AVP mRNA levels in the PVN at rest and during acute running with different lengths of a training regime. Male Wistar rats were subjected to treadmill running (approximately 25 m/min, 60 minutes/day, 5 times/week) for training regimes of 0, 1, 2 or 4 weeks. All training regimes induced an adrenal hypertrophy. Plasma corticosterone levels before acute running increased with lengthening the training period. Four weeks of training produced a significant increase in the resting CRH, but not AVP, mRNA levels in the PVN though relatively shorter training regimes did not. Acute responses of lactate and ACTH release were reduced after 2 and 4 weeks of training, respectively. The responsive PVN CRH mRNA level to acute running decreased with 4 weeks of training but increased with relatively shorter training regimes. These results indicate that running training changes the PVN CRH biosynthetic activity with the regime lasting for 4 weeks, which follows adaptive changes in adrenal functions. Thus, running training-induced changes in hypothalamic CRH activity would originate from the PVN and be induced according to the training period.     

Attenuating effect of angiotensin-(1-7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-A(y)/Ta mice

ANG-(1-7) is associated with vasodilation and nitric oxide synthase stimulation. However, the role of ANG-(1-7) in type 2 diabetes mellitus is unknown. In this study, we examined the hypothesis that ANG-(1-7) attenuates ANG II-induced reactive oxygen species stress (ROS)-mediated injury in type 2 diabetic nephropathy of KK-A(y)/Ta mice. KK-A(y)/Ta mice were divided into four groups: 1) a control group; 2) ANG II infusion group; 3) ANG II+ANG-(1-7) coinfusion group; and 4) ANG II+ANG-(1-7)+d-Ala(7)-ANG-(1-7) (A779) coinfusion group. In addition, primary mesangial cells were cultured and then stimulated with 25 mM glucose with or without ANG II, ANG-(1-7), and A779. The ANG II+ANG-(1-7) coinfusion group showed a lower urinary albumin/creatinine ratio increase than the ANG II group. ANG-(1-7) attenuated ANG II-mediated NAD(P)H oxidase activation and ROS production in diabetic glomeruli and mesangial cells. ANG II-induced NF-κB and MAPK signaling activation was also attenuated by ANG-(1-7) in the mesangial cells. These findings were related to improved mesangial expansion and to fibronectin and transforming growth factor-β1 production in response to ANG II and suggest that ANG-(1-7) may attenuate ANG II-stimulated ROS-mediated injury in type 2 diabetic nephropathy. The ACE2-ANG-(1-7)-Mas receptor axis should be investigated as a novel target for treatment of type 2 diabetic nephropathy.     

Genetic link between Cabeza, a Drosophila homologue of Fused in Sarcoma (FUS), and the EGFR signaling pathway

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila has Cabeza (Caz) as a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was here found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror suppressed the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescued the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila.