Kohichi Kawahara

IL-4-Induced Selective Clearance of Oligomeric β-Amyloid Peptide1–42 by Rat Primary Type 2 Microglia

A hallmark of immunopathology associated with Alzheimer’s disease is the presence of activated microglia (MG) surrounding senile plaque deposition of β-amyloid (Aβ) peptides. Aβ peptides are believed to be potent activators of MG, which leads to Alzheimer’s disease pathology, but the role of MG subtypes in Aβ clearance still remains unclear. In this study, we found that IL-4 treatment of rat primary-type 2 MG enhanced uptake and degradation of oligomeric Aβ1–42 (o-Aβ1–42). IL-4 treatment induced significant expression of the scavenger receptor CD36 and the Aβ-degrading enzymes neprilysin (NEP) and insulin-degrading enzyme (IDE) but reduced expression of certain other scavenger receptors. Of cytokines and stimulants tested, the anti-inflammatory cytokines IL-4 and IL-13 effectively enhanced CD36, NEP, and IDE. We demonstrated the CD36 contribution to IL-4-induced Aβ clearance: Chinese hamster ovary cells overexpressing CD36 exhibited marked, dose-dependent degradation of 125I-labeled o-Aβ1–42 compared with controls, the degradation being blocked by anti-CD36 Ab. Also, we found IL-4-induced clearance of o-Aβ1–42 in type 2 MG from CD36-expressing WKY/NCrj rats but not in cells from SHR/NCrj rats with dysfunctional CD36 expression. NEP and IDE also contributed to IL-4-induced degradation of Aβ1–42, because their inhibitors, thiorphan and insulin, respectively, significantly suppressed this activity. IL-4-stimulated uptake and degradation of o-Aβ1–42 were selectively enhanced in type 2, but not type 1 MG that express CD40, which suggests that the two MG types may play different neuroimmunomodulating roles in the Aβ-overproducing brain. Thus, selective o-Aβ1–42 clearance, which is induced by IL-4, may provide an additional focus for developing strategies to prevent and treat Alzheimer’s disease.

Induction of CHOP and apoptosis by nitric oxide in p53-deficient microglial cells

Excessive nitric oxide (NO) has been implicated in neurotoxicity after stresses such as ischemia. NO toxicity is generally thought to be mediated by the DNA damage–p53 pathway or mitochondrial dysfunction. We investigated the mechanism of NO toxicity by using murine microglial MG5 cells established from p53‐deficient mice. When MG5 cells were exposed to bacterial lipopolysaccharide plus interferon‐γ, mRNA and protein for inducible NO synthase (iNOS) were markedly induced, and apoptosis occurred. Under these conditions, we found that mRNA and protein for CHOP/GADD153, a C/EBP family transcription factor which is involved in endoplasmic reticulum (ER) stress‐induced apoptosis, are induced. iNOS mRNA was induced 2 h after treatment, whereas CHOP mRNA began to increase at 6 h with a time lag. CHOP mRNA was also induced by NO donors S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) or NOC18, or a peroxynitrite generator 3‐(4‐morpholinyl)‐sydnonimine hydrochloride (SIN‐1). Bip/GRP78, an ER chaperone which is known to be induced by ER stress, was also induced by SNAP or SIN‐1, indicating that NO causes ER stress. These results suggest that NO‐induced apoptosis in MG5 cells occurs through the ER stress pathway involving CHOP, but is independent of p53.

Co-induction of argininosuccinate synthetase, cationic amino acid transporter-2, and nitric oxide synthase in activated murine microglial cells.

Nitric oxide (NO) produced by activated microglia has been implicated in many pathophysiological events in the brain including neurodegenerative diseases. Cellular NO production depends absolutely on the availability of arginine, a substrate of NO synthase (NOS). Murine microglial MG5 cells were treated with bacterial lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma), and expression of inducible NO synthase (iNOS) and arginine-supplying enzymes was investigated by RNA blot analysis. iNOS mRNA was strongly induced after treatment and reached a maximum at 6-12 h. mRNA for argininosuccinate synthetase (AS), a citrulline-arginine recycling enzyme, increased at 6 h and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and AS proteins were also induced. In addition, mRNA encoding the cationic amino acid transporter-2 (CAT-2) was strongly induced shortly after treatment. Induction of mRNAs for iNOS, AS, and CAT-2 by LPS/IFN-gamma was also observed following stimulation of rat primary microglial cells. These results strongly suggest that both arginine transport by CAT-2 and citrulline-arginine recycling are important for high-output production of NO in activated microglial cells.

Intracerebral microinjection of interleukin-4/interleukin-13 reduces β-amyloid accumulation in the ipsilateral side and improves cognitive deficits in young amyloid precursor protein 23 mice

We previously reported that the anti-inflammatory cytokine interleukin (IL)-4 induced selective clearance of oligomeric β-amyloid (Aβ(1-42)) in rat primary type 2 microglial cells. For the present study, we investigated whether IL-4 and IL-13 could activate microglial cells to induce Aβ clearance in vivo and improve cognitive deficits in APP23 mice, which are amyloid precursor protein transgenic mice. We administered an intracerebral microinjection of a mixture of IL-4 and IL-13 or of saline vehicle into one hemisphere of APP23 mice and their wild-type littermates, 4.5 and 9 months old, after which we evaluated the effects of these treatments on spatial learning and memory by Morris Water Maze test and on accumulated amounts of Aβ. The cytokine injection significantly improved memory deficits of 4.5-month-old APP23 mice, but did not do so in 9-month-old APP23 mice, even though similar Aβ reductions were observed in both age groups of APP23 mice in the ipsilateral neocortex. The cytokine injection improved memory impairment of 9-month-old wild-type (WT) mice in the probe trial. Immunohistochemical analysis of the 4.5-month-old APP23 mice revealed the presence of increased numbers of microglial cells at 2 days after the cytokine injection. In addition to induced CD36 expression in the activated microglia, increased expression of neprilysin, mainly in neurons, suggested that the cytokines improved the cognitive deficits via degradation and clearance of intra- and extraneuronal Aβ peptides, of buffer-extractable nonplaque form. Double immunostaining also revealed that most of the activated microglia had the M2-like phenotype. This unique mechanism of IL-4/IL-13-induced clearance of Aβ may provide an additional strategy to prevent and/or cure Alzheimers disease at early stage.

Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts

Cardiac ATP‐sensitive K+ (KATP) channels are proposed to contribute to cardio‐protection and ischemic preconditioning. Although mRNAs for all subunits of KATP channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti‐peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin‐solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2–SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)4–(SUR2A)4, suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac KATP channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.

Oxidized LDL and lysophosphatidylcholine stimulate plasminogen activator inhibitor-1 expression through reactive oxygen species generation and ERK1/2 activation in 3T3-L1 adipocytes

Plasminogen activator inhibitor-1 (PAI-1) is secreted from adipose tissue and is considered to be a risk factor for both atherosclerosis and insulin resistance. Here we report for the first time that PAI-1 expression is enhanced by oxidized low-density lipoprotein (OxLDL) and its lipid component lysophosphatidylcholine (LPC) in mouse 3T3-L1 adipocytes. In fully differentiated 3T3-L1 cells, OxLDL treatment increased the mRNA expression and protein secretion of PAI-1 in a dose- and time-dependent manner, whereas native LDL had no effect. The addition of an anti-CD36 antibody suppressed OxLDL-stimulated PAI-1 expression by 50%, suggesting that adipose-derived CD36 contributes to roughly half of the PAI-1 expression stimulated by OxLDL. In addition, pharmacological experiments showed that the OxLDL-stimulated enhancement in PAI-1 expression was mediated through the generation of reactive oxygen species (ROS) and phosphorylation of extracellular signal-regulated kinase 1/2. Furthermore, LPC, a major lipid component of OxLDL, was responsible for the enhanced expression of PAI-1 as phospholipase A(2)-treated acetyl LDL, which generates LPC, strongly stimulated PAI-1 expression, whereas acetyl LDL itself had no such activity. These data demonstrate that the uptake of OxLDL and, in particular, its lipid component LPC into adipocytes triggers aberrant ROS-mediated PAI-1 expression, which may be involved in the pathogenesis of metabolic syndrome.

Marked induction of inducible nitric oxide synthase and tumor necrosis factor-α in rat CD40+ microglia by comparison to CD40− microglia

There may be two subtypes of microglia (MG) at least in the CNS. We separated the two types from rat mixed glial culture. mRNAs and proteins for inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNFalpha) were more induced in CD40(+) MG than CD40(-) MG after LPS stimulation. Although the expression level of LPS receptors showed a little difference between the subtypes, LPS-induced degradation of phosphorylated IkappaBalpha was marked in CD40(+) MG. These results strongly suggest that CD40(+) MG produce larger amount of NO and TNFalpha to exhibit neurotoxic action under certain pathological conditions in brains.

Cooperative therapeutic action of retinoic acid receptor and retinoid x receptor agonists in a mouse model of Alzheimer's disease.

Alzheimers disease (AD) is a neurodegenerative process involving amyloid-β (Aβ) peptide deposition, neuroinflammation, and progressive memory loss. Here, we evaluated whether oral administration of retinoic acid receptor (RAR)α,β agonist Am80 (tamibarotene) or specific retinoid X receptor (RXR) pan agonist HX630 or their combination could improve deficits in an AD model, 8.5-month-old amyloid-β protein precursor 23 (AβPP23) mice. Co-administration of Am80 (0.5 mg/kg) and HX630 (5 mg/kg) for 17 days significantly improved memory deficits (Morris water maze) in AβPP23 mice, whereas administration of either agent alone produced no effect. Only co-administration significantly reduced the level of insoluble Aβ peptide in the brain. These results thus indicate that effective memory improvement via reduction of insoluble Aβ peptide in 8.5-month-old AβPP23 mice requires co-activation of RARα,β and RXRs. RARα-positive microglia accumulated Aβ plaques in the AβPP23 mice. Rat primary microglia co-treated with Am80/HX630 showed increased degradation activity towards 125I-labeled oligomeric Aβ1-42 peptide in an insulin-degrading enzyme (IDE)-dependent manner. The co-administration increased mRNA for IDE and membrane-associated IDE protein in vivo, suggesting that IDE contributes to Aβ clearance in Am80/HX630-treated AβPP23 mice. Am80/HX630 also increased IL-4Rα expression in microglial MG5 cells. The improvement in memory of Am80/HX630-treated AβPP23 mice was correlated with the levels and signaling of hippocampal interleukin-4 (IL-4). Therefore, Am80/HX630 may promote differentiation of IL-4-responsive M2-like microglia and increase their activity for clearance of oligomeric Aβ peptides by restoring impaired IL-4 signaling in AβPP23 mice. Combination treatment with RAR and RXR agonists may be an effective approach for AD therapy.

Subdomain IIIA of dog albumin contains a binding site similar to site II of human albumin.

Dog albumin contains a specific drug-binding site that binds most of the site II ligands of human albumin. This study was undertaken to elucidate the structural configuration of this binding site using a photoaffinity labeling technique. Dog albumin and albumins of other animal species were photolabeled with [14C]ketoprofen. The photolabeled albumins were cleaved with cyanogen bromide (CNBr) and analyzed autoradiographically after electrophoretic separation. A 11.6-kDa CNBr fragment of the photolabeled dog albumin was found to have incorporated most of the radioactivity. Site II ligands of human albumin inhibited photoincorporation of radioactivity to this fragment. The binding constants of human and dog albumins ranged from 10 to 12 × 105 M-1, at least twice as high as those of rat, rabbit, and bovine albumins. Edman degradation was performed to elucidate the amino acid sequence of the photolabeled peptide derived from further digestion of the dog 11.6-kDa CNBr fragment with lysyl endopeptidase. The sequence was XXSESLVXRX, which corresponds to Cys476-Arg485 of dog albumin. Dog albumin contains a binding site that may have a binding microenvironment similar to that of site II on human albumin. Therefore, dog may be a better experimental animal for data extrapolation from animal to human with regard to site II drug-drug interactions.

Nitric oxide inhibits the proliferation of murine microglial MG5 cells by a mechanism involving p21 but independent of p53 and cyclic guanosine monophosphate.

We investigated the effect of nitric oxide (NO) on the proliferation of microglial MG5 cells established from p53-deficient mice. Cells were treated with bacterial lipopolysaccharide and interferon-gamma, and expression of inducible NO synthase (iNOS) and p21/waf1, a cyclin-dependent kinase inhibitor protein which is a critical downstream effector of p53, was investigated by RNA blot and immunoblot analyses. iNOS mRNA was induced 2 h after treatment and increased with time up to 24 h. p21 mRNA was expressed at a low level in untreated cells and increased with a kinetics similar to that for iNOS mRNA. iNOS and p21 proteins were also induced. An NO donor SNAP induced p21 mRNA and protein. SNAP inhibited incorporation of [(3)H]thymidine in MG5 cells in a dose-dependent manner. 8-Bromo-cGMP neither induced p21 mRNA nor inhibited [(3)H]thymidine incorporation. These results suggest that NO inhibits the proliferation of MG5 cells by induction of p21, which occurs independent of p53 and cGMP.