Noriaki Kinoshita

SOD1 (Copper/Zinc Superoxide Dismutase) Deficiency Drives Amyloid β Protein Oligomerization and Memory Loss in Mouse Model of Alzheimer Disease

Oxidative stress is closely linked to the pathogenesis of neurodegeneration. Soluble amyloid β (Aβ) oligomers cause cognitive impairment and synaptic dysfunction in Alzheimer disease (AD). However, the relationship between oligomers, oxidative stress, and their localization during disease progression is uncertain. Our previous study demonstrated that mice deficient in cytoplasmic copper/zinc superoxide dismutase (CuZn-SOD, SOD1) have features of drusen formation, a hallmark of age-related macular degeneration (Imamura, Y., Noda, S., Hashizume, K., Shinoda, K., Yamaguchi, M., Uchiyama, S., Shimizu, T., Mizushima, Y., Shirasawa, T., and Tsubota, K. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 11282–11287). Amyloid assembly has been implicated as a common mechanism of plaque and drusen formation. Here, we show that Sod1 deficiency in an amyloid precursor protein-overexpressing mouse model (AD mouse, Tg2576) accelerated Aβ oligomerization and memory impairment as compared with control AD mouse and that these phenomena were basically mediated by oxidative damage. The increased plaque and neuronal inflammation were accompanied by the generation of Nϵ-carboxymethyl lysine in advanced glycation end products, a rapid marker of oxidative damage, induced by Sod1 gene-dependent reduction. The Sod1 deletion also caused Tau phosphorylation and the lower levels of synaptophysin. Furthermore, the levels of SOD1 were significantly decreased in human AD patients rather than non-AD age-matched individuals, but mitochondrial SOD (Mn-SOD, SOD2) and extracellular SOD (CuZn-SOD, SOD3) were not. These findings suggest that cytoplasmic superoxide radical plays a critical role in the pathogenesis of AD. Activation of Sod1 may be a therapeutic strategy for the inhibition of AD progression.

Cytoplasmic and serum galectin-3 in diagnosis of thyroid malignancies

In order to address whether galectin-3 in the sera and fine needle aspirates serve as a diagnostic marker distinguishing between benign and malignant thyroid nodules, we developed an enzyme-linked immunosorbent assay. We quantified galectin-3 in fine needle aspirates from a series of 118 patients with thyroid nodules and serum galectin-3 from another series of 46 patients, which were compared with final histology after thyroidectomy. Relative galectin-3 value (ng/mg), defined as galectin-3 concentration (ng/ml) divided by total protein concentration (mg/ml) in fine needle aspirates, was significantly higher in papillary carcinoma than in the other thyroid entities. There was no significant difference in serum galectin-3 level among patients with thyroid nodules and healthy individuals. Accordingly, relative galectin-3 value allows a definitive diagnosis of papillary carcinoma independent of cellular morphology, whereas serum galectin-3 does not serve as a marker for papillary carcinoma.

Serum Mac‐2 binding protein levels as a novel diagnostic biomarker for prediction of disease severity and nonalcoholic steatohepatitis

Mac‐2 binding protein (Mac‐2bp) is one of the major fucosylated glycoproteins, which we identified with glycol‐proteomic analyses. We previously reported that fucosylated glycoproteins are secreted into bile, but scarcely secreted into sera in normal liver and hypothesized that the fucosylation‐based sorting machinery would be disrupted in ballooning hepatocytes due to the loss of cellular polarity. In the present study, we investigated the availability of Mac‐2bp for differential diagnosis of nonalcoholic steatohepatitis (NASH) from nonalcoholic fatty liver disease (NAFLD) as a biomarker.

Soluble Amyloid Precursor Protein 770 Is Released from Inflamed Endothelial Cells and Activated Platelets: A NOVEL BIOMARKER FOR ACUTE CORONARY SYNDROME*

Background: Separate monitoring of the cleavage products of different amyloid β precursor protein (APP) variants may provide useful information. Results: We found that soluble APP770 (sAPP770) is released from inflamed endothelial cells and activated platelets as judged by ELISA. Conclusion: sAPP770 is an indicator for endothelial and platelet dysfunctions. Significance: How sAPP770 is released in vivo has been shown. Most Alzheimer disease (AD) patients show deposition of amyloid β (Aβ) peptide in blood vessels as well as the brain parenchyma. We previously found that vascular endothelial cells express amyloid β precursor protein (APP) 770, a different APP isoform from neuronal APP695, and produce Aβ. Since the soluble APP cleavage product, sAPP, is considered to be a possible marker for AD diagnosis, sAPP has been widely measured as a mixture of these variants. We hypothesized that measurement of the endothelial APP770 cleavage product in patients separately from that of neuronal APP695 would enable discrimination between endothelial and neurological dysfunctions. Using our newly developed ELISA system for sAPP770, we observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells. Furthermore, we unexpectedly found that sAPP770 was rapidly released from activated platelets. We also found that cerebrospinal fluid mainly contained sAPP695, while serum mostly contained sAPP770. Finally, to test our hypothesis that sAPP770 could be an indicator for endothelial dysfunction, we applied our APP770 ELISA to patients with acute coronary syndrome (ACS), in which endothelial injury and platelet activation lead to fibrous plaque disruption and thrombus formation. Development of a biomarker is essential to facilitate ACS diagnosis in clinical practice. The results revealed that ACS patients had significantly higher plasma sAPP770 levels. Furthermore, in myocardial infarction model rats, an increase in plasma sAPP preceded the release of cardiac enzymes, currently used markers for acute myocardial infarction. These findings raise the possibility that sAPP770 can be a useful biomarker for ACS.

Glycosylation at the Fab Portion of Myeloma Immunoglobulin G and Increased Fucosylated Biantennary Sugar Chains: Structural Analysis by High-Performance Liquid Chromatography and Antibody-Lectin Enzyme Immunoassay Using Lens culinaris Agglutinin

An antibody-lectin enzyme immunoassay technique which had been developed for the analysis of sugar chains of alpha-fetoprotein (N. Kinoshita et al., Clin. Chim. Acta, 179: 143-152, 1989) was used for analysis of sugar chains of myeloma immunoglobulin G (IgG). The IgG sugar chains of four of nine patients with myeloma were found to be highly reactive to Lens culinaris agglutinin as compared with those of six normal controls and 177 patients without myeloma. This reflected a high L. culinaris agglutinin/concanavalin A ratio. The IgGs of these patients were found to have highly sialylated, fucosylated, and bisected biantennary sugar chains at Fab portions as judged by the lectin-blotting technique as well as by high-performance liquid chromatography analysis. These results indicate that some of the myeloma IgG proteins undergo unusual glycosylation processes.

Methylene blue modulates β-secretase, reverses cerebral amyloidosis, and improves cognition in transgenic mice.

Background: Recent focus has been given to anti-amyloidogenic approaches for Alzheimer disease therapy. Results: Methylene blue prevented behavioral impairment and mitigated Alzheimer disease-like pathology. Conclusion: Methylene blue may be prophylactic for Alzheimer disease by inhibiting β-secretase activity and mitigating brain pathology. Significance: Oral methylene blue treatment modulates β-secretase. Amyloid precursor protein (APP) proteolysis is required for production of amyloid-β (Aβ) peptides that comprise β-amyloid plaques in the brains of patients with Alzheimer disease (AD). Here, we tested whether the experimental agent methylene blue (MB), used for treatment of methemoglobinemia, might improve AD-like pathology and behavioral deficits. We orally administered MB to the aged transgenic PSAPP mouse model of cerebral amyloidosis and evaluated cognitive function and cerebral amyloid pathology. Beginning at 15 months of age, animals were gavaged with MB (3 mg/kg) or vehicle once daily for 3 months. MB treatment significantly prevented transgene-associated behavioral impairment, including hyperactivity, decreased object recognition, and defective spatial working and reference memory, but it did not alter nontransgenic mouse behavior. Moreover, brain parenchymal and cerebral vascular β-amyloid deposits as well as levels of various Aβ species, including oligomers, were mitigated in MB-treated PSAPP mice. These effects occurred with inhibition of amyloidogenic APP proteolysis. Specifically, β-carboxyl-terminal APP fragment and β-site APP cleaving enzyme 1 protein expression and activity were attenuated. Additionally, treatment of Chinese hamster ovary cells overexpressing human wild-type APP with MB significantly decreased Aβ production and amyloidogenic APP proteolysis. These results underscore the potential for oral MB treatment against AD-related cerebral amyloidosis by modulating the amyloidogenic pathway.

Reevaluation of a lectin antibody ELISA kit for measuring fucosylated haptoglobin in various conditions.

BACKGROUND Fucosylated haptoglobin (Fuc-Hpt) is a novel cancer biomarker in a variety of pathological conditions. We previously found that the level of Fuc-Hpt is increased in the sera of patients with pancreatic cancer, and established a lectin antibody ELISA using Aleuria aurantia lectin, which specifically binds to fucosylated residues on oligosaccharides. METHODS To apply this assay system to the clinical detection of several diseases, several assay conditions such as serum dilutions and inhibitory factors were investigated. The Fuc-Hpt kit was available for 25-625 fold serum dilution. RESULTS While the values of Fuc-Hpt assay using sera and plasma were different, they showed positive correlation. The addition of bilirubin and formagine did not influence on Fuc-Hpt assay, but hemoglobin inhibited this assay in a dose-dependent manner. CONCLUSIONS We reevaluated this lectin antibody ELISA kit for measuring fucosylated haptoglobin in various conditions in this study.

Development of an antibody-lectin enzyme immunoassay for fucosylated α-fetoprotein ☆

BACKGROUND Fucosylation is one of the most important types of glycosylations related to cancer. Our previous studies of the enzymatic basis and structural studies of α-fetoprotein (AFP) samples from liver cancer patients indicated that core-fucosylation by α1,6-fucosyltransferase (FUT8) resulted in the production of fucosylated AFP, and in fact fucosylated AFP allowed differential diagnosis in some types of liver cancer from liver cirrhosis. This served as a predictive biomarker for the development of liver cancer 3 to 18 months before it could be detected using imaging techniques. Fucosylated AFP is currently measured by means of a liquid-phase binding assay (LBA) or by an electrokinetic analyte transport assay (EATA). However, these methods require special instrumentation that is currently available only in major medical laboratories. To overcome this problem, we attempted to develop an enzyme immunoassay (EIA) based on the sandwich technique with specific antibody and lectin. RESULTS Dilute solutions of highly fucosylated AFP in human sera were assayed using a microtiter plate coated with a periodate-oxidized anti-AFP antibody, a fucose-specific biotinylated Aleuria aurantia lectin (AAL), a peroxidase-conjugated streptoavidin, and a chemiluminescent detection system. The technique was able to measure highly fucosylated AFP diluted to 5 to 80ng/ml in human sera using the developed antibody-lectin EIA in combination with the enrichment of AFP. CONCLUSION A simple method using an antibody-lectin EIA for quantifying fucosylated AFP that does not require special instrumentation was developed. GENERAL SIGNIFICANCE The method can be generally applied to the quantitative measurement of various fucosylated glycoproteins using specific antibodies. This article is part of a Special Issue entitled Glycoproteomics.

Glycation of copper/zinc superoxide dismutase and its inactivation: identification of glycated sites.

Publisher Summary This chapter discusses the glycation of lysine residues located in an active ligand-binding loop of the Cu–Zn superoxide dismutase. The chapter also describes the identification of glycated Cu-Zn superoxide dismutase and procedures for detecting glucose adducts to lysine residues in human erythrocyte Cu-Zn superoxide dismutase. The in vitro glycated form of Cu–Zn superoxide dismutase undergoes gradual inactivation. Therefore, to determine the true amount of the glycated enzyme on a Glyco-Gel B column, it is essential to quantitate the immunoreactive Cu–Zn superoxide dismutase by enzyme-linked immunosorbent assay (ELISA) using a specific antibody raised against Cu–Zn superoxide dismutase. In the case of diabetic patients, however, the immunoreactive Cu–Zn superoxide dismutase content of the glycated form ( in vivo glycated form) is well correlated with the enzyme activity.

ELEVATION OF IMMUNOREACTIVE SERUM Mn-SUPEROXIDE DISMUTASE IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION

Serum Mn-superoxide dismutase (Mn-SOD) levels were determined in patients with acute myocardial infarction by an enzyme-linked immunosorbent assay (ELISA). The serial determinations performed on 29 patients with acute myocardial infarction indicated that Mn-SOD appears in serum in a biphasic manner; an early small elevation was observed at approximately 16.2 (+/- 7.3) h and a high elevation at approximately 108 (+/- 20.6) h after the onset of symptoms. A negative correlation was found between the maximum value of the late elevation of Mn-SOD and left ventricular function. Plasma levels of tumor necrosis factor-alpha (TNF-alpha) were also elevated in acute myocardial infarction. These facts suggest that TNF-alpha secretion from the infarcted area resulted in the production of Mn-SOD and that the induced Mn-SOD was released from myocytes into serum through damaged membranes, resulting in the late elevation.