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Qt0p78229s.Pdf UC Irvine ICTS Publications Title Altered Expression of Diabetes-Related Genes in Alzheimer's Disease Brains: The Hisayama Study Permalink https://escholarship.org/uc/item/0p78229s Journal Cerebral Cortex, 24(9) ISSN 1460-2199 1047-3211 Authors Hokama, Masaaki Oka, Sugako Leon, Julio et al. Publication Date 2014-09-01 DOI 10.1093/cercor/bht101 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Cerebral Cortex September 2014;24:2476–2488 doi:10.1093/cercor/bht101 Advance Access publication April 17, 2013 Altered Expression of Diabetes-Related Genes in Alzheimer’s Disease Brains: The Hisayama Study Masaaki Hokama1,2, Sugako Oka1,3, Julio Leon1, Toshiharu Ninomiya4, Hiroyuki Honda5, Kensuke Sasaki5, Toru Iwaki5, Tomoyuki Ohara6, Tomio Sasaki2, Frank M. LaFerla8, Yutaka Kiyohara7 and Yusaku Nakabeppu1,3 1Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, 2Department of Neurosurgery, Graduate School of Medical Sciences, 3Research Center for Nucleotide Pool, 4Department of Medicine and Clinical Science, Graduate School of Medical Sciences, 5Department of Neuropathology, Graduate School of Medical Sciences, 6Department of Neuropsychiatry, Graduate School of Medical Sciences, 7Department of Environmental Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan and 8Department of Neurobiology and Behavior, University of California, Irvine, CA 92697, USA Address correspondence to Prof. Yusaku Nakabeppu, Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Email: [email protected] Diabetes mellitus (DM) is considered to be a risk factor for dementia To identify molecular pathological alterations in AD brains, including Alzheimer’s disease (AD). However, the molecular mechan- we performed interspecies comparative microarray analyses ism underlying this risk is not well understood. We examined gene using RNA prepared from postmortem human brain tissues expression profiles in postmortem human brains donated for the Hi- donated for the Hisayama study (Katsuki 1966; Matsuzaki sayama study. Three-way analysis of variance of microarray data et al. 2010; Sekita et al. 2010), and hippocampal RNAs from from frontal cortex, temporal cortex, and hippocampus was per- the triple-transgenic mouse model of AD (3xTg-AD) (Oddo formed with the presence/absence of AD and vascular dementia, et al. 2003). We found altered expression profiles of diabetes and sex, as factors. Comparative analyses of expression changes in mellitus (DM)-related genes in AD brains, which were inde- the brains of AD patients and a mouse model of AD were also per- pendent of peripheral DM-related abnormalities. formed. Relevant changes in gene expression identified by microarray analysis were validated by quantitative real-time reverse-transcription Materials and Methods polymerase chain reaction and western blotting. The hippocampi of AD brains showed the most significant alteration in gene expression Postmortem Brain Tissues profile. Genes involved in noninsulin-dependent DM and obesity were We examined 88 autopsy samples from Hisayama residents obtained significantly altered in both AD brains and the AD mouse model, as between 15 December 2008 and 24 February 2011. Clinical data related were genes related to psychiatric disorders and AD. The alterations to DM or prediabetes were collected as described (Ohara et al. 2011). in the expression profiles of DM-related genes in AD brains were The study was approved by the Ethics Committee of the Faculty of Medicine, Kyushu University. Written informed consent for all subjects independent of peripheral DM-related abnormalities. These results was obtained from their families. Neuropathologic changes were exam- indicate that altered expression of genes related to DM in AD brains ined as described previously (Matsuzaki et al. 2010). Sections were rou- is a result of AD pathology, which may thereby be exacerbated by tinely stained using hematoxylin–eosin, Klüver-Barrera stain, and a peripheral insulin resistance or DM. modified Bielschowsky method. Specimens from each subject were immunostained using antibodies against phosphorylated microtubule- Keywords: animal model, hippocampus, insulin, microarray, postmortem brains associated protein tau (MAPT) (AT8, mouse monoclonal, 1:500; Inno- genetics, Belgium) and the assessment of AD pathology was conducted ’ Introduction according to the Consortium to Establish a Registry for Alzheimer s Disease (CERAD) guidelines (Mirra et al. 1991) and the Braak stage More than 20 million people worldwide suffer from dementia, (Braak and Braak 1991). During autopsy dissection, parts of the frontal and this number is expected to exceed 80 million by 2040 cortex, temporal cortex, and hippocampus were cut out from each because of the rapid increase in the numbers of elderly (Ferri brain and preserved at −80 °C until RNA preparation. et al. 2005). The prevalences of all-cause dementia and Alzheimer’s disease (AD) in the general population of Japa- Animals nese elderly have increased significantly over the past 3xTg-AD-H mice harboring a homozygous Psen1M146V mutation and 20 years, especially among subjects aged ≧75 years (Sekita homozygous mutant transgenes for APPSwe and MAPTP301L, 3xTg-AD-h mice harboring a homozygous Psen1 mutation and et al. 2010). Thus, it is important to establish effective preven- M146V hemizygous APPSwe and MAPTP301L transgenes, and nontransgenic tion strategies for dementia, and particularly for AD. To reach control mice (non-Tg) (Oddo et al. 2003) were used in this study. At this goal, it is essential to understand the risk factors for de- age 14 months, brains were removed (N = 3 male mice of each type) veloping dementia, including AD, in the elderly population. under pentobarbital anesthesia (i.p.), with perfusion of 40 mL of Several recent studies have indicated effects of insulin and saline via the left ventricle. Hippocampi were isolated and preserved glucose metabolism on the risk of developing dementia, at −80 °C until RNA preparation. The handling and killing of all especially AD (Kuusisto et al. 1997; de la Monte and Wands animals was performed in accordance with the national prescribed guidelines, and ethical approval for the study was granted by the 2008; Schrijvers et al. 2010). The results of the Hisayama study Animal Experiment Committee of Kyushu University. suggested that hyperinsulinemia and hyperglycemia caused by insulin resistance accelerate the formation of neuritic plaques Gene Expression Profiling with Microarray Analyses (NPs) in combination with the effect of the APOE ε4 allele, a Total RNA was isolated using a combination of Isogen (Nippon Gene, majorriskfactorforAD(Matsuzaki et al. 2010). Tokyo, Japan) and the RNeasy Mini Kit (Qiagen, Tokyo, Japan), © The Author 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals. [email protected] according to the manufacturers’ instructions. RNA concentration was room temperature. Sections were blocked with a solution containing determined by the measurement of UV absorbance spectra, and the 1 × Block Ace (Dainippon Pharmaceutical, Osaka, Japan) for 30 min total RNA profile was analyzed using an Agilent 2100 Bioanalyzer at room temperature, incubated with anti-PCSK1 (sc-100578, 1:50, (Agilent Technologies Japan, Tokyo, Japan) to determine RNA integ- Santa Cruz Biotechnology, Santa Cruz, CA, USA) and antineuron- rity number (RIN). Expression profiles of the RNA samples with an specific nuclear protein (NeuN) (ABN78, 1:1000, Merck Japan, Tokyo, RIN ≧6.9 were determined using Affymetrix Human or Mouse Gene Japan) antibodies in 10% Block Ace at 4 °C overnight, and then incu- 1.0ST arrays (Affymetrix Japan, Tokyo, Japan) according to the manu- bated with an Alexa Fluor-labeled second antibody (Invitrogen Japan, facturer’s instructions. The Ambion WT Expression Kit (Life Technol- Tokyo, Japan) for 45 min at room temperature. Confocal images were ogies Japan, Tokyo, Japan) and the GeneChip WT Terminal Labeling acquired using an LSM510 META Confocal Microscope System (Carl and Controls Kit (Affymetrix Japan) were used to generate amplified Zeiss MicroImaging, Tokyo, Japan). and biotinylated sense-strand DNA targets from expressed transcripts (100 ng of total RNA). Manufacturer’s instructions were followed for hybridization, washing, and scanning steps, and CEL files were gener- Western Blot Analysis ated. CEL files were imported into Partek Genomics Suite software Frozen hippocampus samples were homogenized in buffer containing (Partek, St Louis, MO, USA), and both exon-level and gene-level esti- 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium mates were obtained for all transcript clusters. The gene-level esti- dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 8.0) with 2% protease mates were subjected to statistical analysis and hierarchical and inhibitor, and a 5% phosphatase inhibitor cocktail (Nacalai Tesque, partitioning clustering by the software.
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