2020年03月, アメリカ生化学・分子生物学会, 2020 ASBMB トラベルアワード
Implication of protein glycolsylation impairment in celluar cholesterol accumulation caused by Presenilin deficiency
Marietta Fabiano;Naoto Oikawa*;Dieter Lütjohann;Jochen Walter (*: corresponding author), 30423313

Presenilin Deficiency Results in Cellular Cholesterol Accumulation by Impairment of Protein Glycosylation and NPC1 Function
Marietta Fabiano, Naoto Oikawa, Anja Kerksiek, Jun-ichi Furukawa, Hirokazu Yagi, Koichi Kato, Ulrich Schweizer, Wim Annaert, Jongkyun Kang, Jie Shen, Dieter Lütjohann, Jochen Walter
International Journal of Molecular Sciences, 25, 10, 5417, 5417, MDPI AG, 2024年05月16日, ［査読有り］, ［責任著者］
研究論文（学術雑誌）, Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD., 30423313

Carboxy-terminal fragment of amyloid precursor protein mediates lipid droplet accumulation upon γ-secretase inhibition
Naoto Oikawa, Marietta Fabiano, Ulrike C. Müller, Jochen Walter
Biochemical and Biophysical Research Communications, 570, 137, 142, Elsevier BV, 2021年09月, ［査読有り］, ［筆頭著者, 責任著者］
研究論文（学術雑誌）, 30423313

Importance of γ-secretase in the regulation of liver X receptor and cellular lipid metabolism.
Esteban Gutierrez, Dieter Lütjohann, Anja Kerksiek, Marietta Fabiano, Naoto Oikawa, Lars Kuerschner, Christoph Thiele, Jochen Walter
Life science alliance, 3, 6, 2020年06月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Presenilins (PS) are the catalytic components of γ-secretase complexes that mediate intramembrane proteolysis. Mutations in the PS genes are a major cause of familial early-onset Alzheimer disease and affect the cleavage of the amyloid precursor protein, thereby altering the production of the amyloid β-peptide. However, multiple additional protein substrates have been identified, suggesting pleiotropic functions of γ-secretase. Here, we demonstrate that inhibition of γ-secretase causes dysregulation of cellular lipid homeostasis, including up-regulation of liver X receptors, and complex changes in the cellular lipid composition. Genetic and pharmacological inhibition of γsecretase leads to strong accumulation of cytoplasmic lipid droplets, associated with increased levels of acylglycerols, but lowered cholesteryl esters. Furthermore, accumulation of lipid droplets was augmented by increasing levels of amyloid precursor protein C-terminal fragments, indicating a critical involvement of this γ-secretase substrate. Together, these data provide a mechanism that functionally connects γ-secretase activity to cellular lipid metabolism. These effects were also observed in human astrocytic cells, indicating an important function of γ-secretase in cells critical for lipid homeostasis in the brain.

Presenilins and γ-Secretase in Membrane Proteostasis.
Naoto Oikawa, Jochen Walter
Cells, 8, 3, 2019年03月01日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The presenilin (PS) proteins exert a crucial role in the pathogenesis of Alzheimer disease (AD) by mediating the intramembranous cleavage of amyloid precursor protein (APP) and the generation of amyloid β-protein (Aβ). The two homologous proteins PS1 and PS2 represent the catalytic subunits of distinct γ-secretase complexes that mediate a variety of cellular processes, including membrane protein metabolism, signal transduction, and cell differentiation. While the intramembrane cleavage of select proteins by γ-secretase is critical in the regulation of intracellular signaling pathways, the plethora of identified protein substrates could also indicate an important role of these enzyme complexes in membrane protein homeostasis. In line with this notion, PS proteins and/or γ-secretase has also been implicated in autophagy, a fundamental process for the maintenance of cellular functions and homeostasis. Dysfunction in the clearance of proteins in the lysosome and during autophagy has been shown to contribute to neurodegeneration. This review summarizes the recent knowledge about the role of PS proteins and γ-secretase in membrane protein metabolism and trafficking, and the functional relation to lysosomal activity and autophagy.

Imbalance in fatty-acid-chain length of gangliosides triggers Alzheimer amyloid deposition in the precuneus.
Naoto Oikawa, Teruhiko Matsubara, Ryoto Fukuda, Hanaki Yasumori, Hiroyuki Hatsuta, Shigeo Murayama, Toshinori Sato, Akemi Suzuki, Katsuhiko Yanagisawa
PloS one, 10, 3, e0121356, 2015年, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Amyloid deposition, a crucial event of Alzheimer's disease (AD), emerges in distinct brain regions. A key question is what triggers the assembly of the monomeric amyloid ß-protein (Aß) into fibrils in the regions. On the basis of our previous findings that gangliosides facilitate the initiation of Aß assembly at presynaptic neuritic terminals, we investigated how lipids, including gangliosides, cholesterol and sphingomyelin, extracted from synaptic plasma membranes (SPMs) isolated from autopsy brains were involved in the Aß assembly. We focused on two regions of the cerebral cortex; precuneus and calcarine cortex, one of the most vulnerable and one of the most resistant regions to amyloid deposition, respectively. Here, we show that lipids extracted from SPMs isolated from the amyloid-bearing precuneus, but neither the amyloid-free precuneus nor the calcarine cortex, markedly accelerate the Aß assembly in vitro. Through liquid chromatography-mass spectrometry of the lipids, we identified an increase in the ratio of the level of GD1b-ganglioside containing C20:0 fatty acid to that containing C18:0 as a cause of the enhanced Aß assembly in the precuneus. Our results suggest that the local glycolipid environment play a critical role in the initiation of Alzheimer amyloid deposition.

Influence of APOE genotype and the presence of Alzheimer's pathology on synaptic membrane lipids of human brains.
Naoto Oikawa, Hiroyuki Hatsuta, Shigeo Murayama, Akemi Suzuki, Katsuhiko Yanagisawa
Journal of neuroscience research, 92, 5, 641, 50, 2014年05月, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, The APOE genotype is the major risk factor for Alzheimer's disease (AD); however, it remains unclarified how the ε4 allele accelerates whereas the ε2 allele suppresses AD development, compared with the more common ε3 allele. On the basis of the previous finding that the assembly of the amyloid-β protein (Aβ) into fibrils in the brain, an early and invariable pathological feature of AD, depends on the lipid environment, we determined the levels of synaptic membrane lipids in aged individuals of different APOE genotypes. In the comparison between amyloid-free ε2/ε3 and ε3/ε3 brains, the presence of the ε2 allele significantly decreased the level of cholesterol. Alternatively, in the comparison among ε3/ε3 brains, the presence of AD pathology substantially decreased the levels of cholesterol. This study suggests that the ε2 allele suppresses the initiation of AD development by lowering the cholesterol levels in synaptic membranes.

The γ-secretase inhibitor DAPT increases the levels of gangliosides at neuritic terminals of differentiating PC12 cells.
Naoto Oikawa, Miho Goto, Kazutaka Ikeda, Ryo Taguchi, Katsuhiko Yanagisawa
Neuroscience letters, 525, 1, 49, 53, 2012年09月06日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Mutations in presenilins are the major cause of early onset familial Alzheimer disease. It has recently been argued that clinical presenilin mutations work as loss-of-function but not toxic gain-of-function. To investigate whether presenilins are involved in the regulation of the distribution of neuronal membrane lipids, we treated neuronally differentiated PC12 cells with DAPT, an inhibitor of presenilin-dependent γ-secretase, and performed lipid analyses of neuritic terminals, which is an initial site of Aβ deposition in brains, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with multiple reaction monitoring (MRM). With DAPT treatment, levels of sphingomyelin, phosphatidylcholine, and cholesterol remained unchanged. However, DAPT treatment increased the ganglioside levels in PC12 neuritic terminals. Together with a previous finding that accumulation of gangliosides at neuritic terminals facilitates Aβ assembly and deposition, the present data suggest that the loss-of-function of presenilins, i.e., a decrease in γ-secretase activity, has an impact on neuronal membrane architecture in a way that eventually exacerbates Alzheimer pathology.

Alzheimer-type tau pathology in advanced aged nonhuman primate brains harboring substantial amyloid deposition.
Naoto Oikawa, Nobuyuki Kimura, Katsuhiko Yanagisawa
Brain research, 1315, 137, 49, 2010年02月22日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, We elucidated how Alzheimer-type pathologies of amyloid beta-protein (Abeta) and tau spatiotemporally emerge in brains of nontransgenic nonhuman primate, cynomolgus monkey, in the present study. To examine the accumulation of deposited Abeta, phosphorylated tau accumulation, intracellular tau accumulation, and neurofibrillary tangle formation, the brains, mainly temporal cortex and hippocampus, of 34 cynomolgus monkeys aged 6 to 36 years were studied by biochemical and histochemical analyses. Biochemically, first, the accumulation of insoluble Abeta was detected in the neocortical (temporal and frontal) and hippocampal regions of animals as young as mid-20s and their levels were extremely high in those of advanced age. The accumulation of phosphorylated tau in the same regions occurred before the age of 20 with poor correlation to the levels of insoluble Abeta. Histologically, intraneuronal and intraoligodendroglial tau accumulation was observed in temporal cortex and hippocampus of animals before the age of 20. In an advanced aged 36-year-old individual, argyrophilic tangles and tau-accumulated dystrophic neurites were markedly observed in the medial temporal area contiguous to limbic structures. Notably, these tau pathologies also emerged, to a lesser extent, in the temporal cortices of advanced aged animals harboring significant amounts of insoluble Abeta. These results suggest that the cynomolgus monkey can be used to elucidate the age-dependent sequence of Abeta and tau pathologies.

Gender effect on the accumulation of hyperphosphorylated tau in the brain of locus-ceruleus-injured APP-transgenic mouse.
Naoto Oikawa, Koichi Ogino, Takumi Masumoto, Haruyasu Yamaguchi, Katsuhiko Yanagisawa
Neuroscience letters, 468, 3, 243, 7, 2010年01月14日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Locus ceruleus (LC) neurons are preferentially and initially affected in Alzheimer disease (AD); however, the impact of the loss of LC neurons on the pathological sequence of AD, including amyloid beta-protein (Abeta) deposition and neurofibrillary tangle formation, has not been elucidated. In this study, we chemically injured LC neurons of the brains of familial AD-related amyloid precursor protein (APP)-transgenic mice using the LC-noradrenergic neuron-selective neurotoxin, N-(2-chloroethyl)-N-ethyl-bromo-benzylamine (DSP4). The levels of noradrenaline significantly decreased in the cerebral cortices of DSP4-treated mice. The deposition of amyloid fibrils was biochemically observed in the APP-transgenic mouse brains; however, those levels were not significantly altered following DSP4 treatment. In contrast, the levels of accumulated hyperphosphorylated tau markedly increased in the cerebral cortices of DSP4-treated female but not male APP-transgenic mice. Our results suggest that innervation from LC neurons and testosterone secretion are potent and mutually independent suppressors of amyloid-related accumulation of hyperphosphorylated tau in the brain.

Gangliosides determine the amyloid pathology of Alzheimer's disease.
Naoto Oikawa, Haruyasu Yamaguchi, Koichi Ogino, Takao Taki, Kohei Yuyama, Naoki Yamamoto, Ryong-Woon Shin, Koichi Furukawa, Katsuhiko Yanagisawa
Neuroreport, 20, 12, 1043, 6, 2009年08月05日, ［査読有り］, ［国際誌］
英語, 研究論文（学術雑誌）, Gangliosides, GM3 and GM1, are suggested to accelerate the deposition of the amyloid beta-protein as amyloid angiopathy and senile plaques, respectively, in the Alzheimer brain. We investigated the profile of amyloid deposition in the brains of transgenic mice expressing a mutant amyloid precursor protein with a disrupted GM2 synthase gene, in which GM3 accumulates whereas GM1 is lacking. These mice showed a significantly increased level of deposited amyloid beta-protein in the vascular tissues. Furthermore, formation of severe dyshoric-form amyloid angiopathy, in which amyloid extended from the blood vessel walls deeply into the surrounding parenchyma was observed. Our results indicate that the expression of gangliosides is a critical determinant for the amyloid pathology in the Alzheimer brain.

Presenilin deficiency increases cholesterol by protein glycosylation impairment
Naoto Oikawa, Marietta Fabiano, Jun-Ichi Furukawa, Hirokazu Yagi, Koichi Kato, Wim Annaer, Jongkyun Kang, Jie Shen, Dieter Luetjohann, Jochen Walter, Dementia Japan, 36, 4, 2022年10月, ［責任著者］
英語, 研究発表ペーパー・要旨（全国大会，その他学術会議）, 45305365

Implication of protein glycosylation impairment in cellular cholesterol accumulation caused by Presenilin deficiency
Marietta Fabiano, Naoto Oikawa, Dieter Lütjohann, Jochen Walter, The FASEB Journal, 34, S1, 1, 1, 2020年04月16日, ［責任著者］
Background

Major genetic causes of familial Alzheimer disease (FAD) are mutations in genes encoding presenilin (PS) 1 and 2, respectively. PS1 and PS2 are transmembrane proteins known to constitute the catalytic subunits of the γ‐secretase complex, a membrane protease involved in proteolytic processing of amyloid precursor protein (APP) that gives rise to amyloid β‐protein (Aβ) secretion. In addiction, PSs are known to be involved in cellular cholesterol metabolism, which is fundamental for physiological cellular functions. Alterations in cholesterol metabolism could be implicated in AD development, indicated by the genetic risk factor apolipoprotein E (APOE). However, the molecular mechanisms how PSs play a role in cellular cholesterol metabolism is still not fully clarified. In this study, we characterized cholesterol metabolism in the cells deficient in either PS1 or PS2.

Methods

We have studied PS1‐ or PS2‐knock out (KO) MEFs by mass spectrometry (MS) for sterols including cholesterol and by microscopy with a cholesterol binding compound, filipin. Expression level of proteins involved in cellular cholesterol metabolism was analyzed by Western blotting (WB).

Results

Cellular level of cholesterol as well as its precursors, desmosterol and lathosterol, were elevated in PS1‐ and PS2‐KO as compared to WT MEFs. Fluorescence microscopy also revealed intracellular cholesterol accumulation in PS1 and PS2 KO cells. Interestingly, the subcellular distribution of cholesterol was different between PS1‐ and PS2‐KO MEFs. We further analyzed expression of proteins involved in cellular cholesterol metabolism, including NPC1, an intracellular cholesterol transporter whose deficiency leads to lysosomal cholesterol storage and neurodegeneration in Niemann‐Pick disease type C. The deficiency of PS1‐ and PS2‐KO MEFs was associated with altered glycosylation of NPC1. The glycosylation and expression of other membrane proteins, such as N‐cadherin, LAMP2, and LRP1 was also dependent on the PS genotype. To investigate the role of protein glycosylation in cellular cholesterol metabolism, we chronically treated wild‐type MEFs with several inhibitors of protein glycosylation targeting different steps of glycosylation processes. The inhibitors differentially affected protein glycosylation pattern and subcellular cholesterol distribution. Importantly, some of the inhibitors partially mimicked the cellular cholesterol accumulation as well as the protein glycosylation pattern observed in PS1‐ and PS2‐KO MEFs.

Discussion/Conclusions

These results suggest that deficiency of PS1 or PS2 differentially affect cellular cholesterol metabolism via impairment of protein glycolsylation. The exact mechanisms of altered expression of NPC1 as well as γ‐secretase activity in the impairment of cholesterol metabolism and protein glycosylation remain to be elucidated. It will be interesting to further dissect the role of protein glycosylation and cholesterol mismetabolism in the pathogenesis of AD.

Support or Funding Information

German Research Foundation (DFG) (Project number: 348207315, Naoto Oikawa), Wiley, 英語, 研究発表ペーパー・要旨（国際会議）, 30423313

ヒト脳由来脂質を用いた生体膜モデルにおけるAβ凝集メカニズムの解析
安盛花季, 松原輝彦, 福田竜統, 及川尚人, 初田裕幸, 村山繁雄, 柳澤勝彦, 佐藤智典, 日本化学会講演予稿集, 95th, 3, 2015年

脳内アミロイド形成に先行する神経細胞膜脂質組成変化の探索
及川尚人, 松原輝彦, 福田竜統, 安盛花季, 初田裕幸, 村山繁雄, 佐藤智典, 鈴木明身, 柳澤勝彦, Dementia Japan, 28, 4, 2014年

γセクレターゼ活性低下による神経突起末端部におけるガングリオシド量の増加　　　　　　　　　　　　　　　
及川 尚人, 後藤 美保, 池田 和貴, 田口 良, 柳澤 勝彦, Dementia Japan, 26, 4, 514, 514, 2012年10月
(一社)日本認知症学会, 日本語

Suppression of gamma-secretase activity increases ganglioside levels at neuritic terminals of differentiated PC12
Naoto Oikawa, Miho Goto, Kazutaka Ikeda, Ryo Taguchi, Katsuhiko Yanagisawa, FASEB JOURNAL, 26, 2012年04月
FEDERATION AMER SOC EXP BIOL, 英語, 研究発表ペーパー・要旨（国際会議）

Presenilin deficiency increases cholesterol by protein glycosylation impairment　　　　　　　　　　　　　　　
Naoto Oikawa, Marietta Fabiano, Jun-Ichi Furukawa, Hirokazu Yagi, Koichi Kato, Wim Annaer, Jongkyun Kang, Jie Shen, Dieter Luetjohann, Jochen Walter
第40回日本認知症学会学術集会, 2021年11月, 英語, ポスター発表

Gamma-secretase inhibition induces lipid droplet accumulation via APP-CTF accumulation
Naoto Oikawa, Ulrike C. Müller, Jochen Walter
Alzheimer's Association International Conference 2018, 2018年07月, 英語, ポスター発表
30423313

γ-Secretase in the regulation of autophagy and cellular cholesterol metabolism　　　　　　　　　　　　　　　
Naoto Oikawa, Esteban Gutierrez, Nguyen T Tien, Jochen Walter
Termination symposium, The Collaborative Research Center SFB 645 “Regulation and manipulation of information flow within dynamic protein and lipid environments”, 2016年11月09日, 英語, 口頭発表（一般）
2016年11月09日 - 2016年11月10日

Hsp70 rescues mutant tau-induced neurodegeneration in C. elegans　　　　　　　　　　　　　　　
及川 尚人, 宮坂 知宏, 坂 彰子, 三谷 昌平, 井原 康夫
第４９回日本神経化学会大会, 2006年, 日本語, 口頭発表（一般）

分子神経生物学実験実習　　　　　　　　　　　　　　　
ボン大学
2016年 - 2020年

アルツハイマー病プレセニリン機能障害のヒト神経コレステロール代謝への影響
科学研究費助成事業
2022年04月01日 - 2025年03月31日
及川 尚人
日本学術振興会, 基盤研究(C), 北海道大学, 22K07364

細胞コレステロール代謝におけるγ-セクレターゼの役割
科学研究費助成事業
2017年05月 - 2021年09月
及川 尚人, Dieter Lütjohann, Oliver Brüstle, Ronald Jabs, 中嶋 和紀, 舟本 聡, 小林 俊秀
ドイツ研究振興協会, Eigene Stelle, ボン大学, 研究代表者

家族性アルツハイマー病脳内アミロイド形成におけるγセクレターゼ機能障害の意義
科学研究費助成事業 研究活動スタート支援
2010年 - 2011年
及川 尚人
プレセニリン遺伝子に変異を有する家族性アルツハイマー病における脳内アミロイド形成機序を解明すべく、プレセニリン/γセクレターゼの機能障害及び神経細胞膜脂質(ガングリオシド)のアミロイド形成誘導能に着目し、γセクレターゼ活性低下による神経細胞膜脂質組成の変化の有無について検討した。その結果、神経系培養細胞のγセクレターゼ活性低下により、アミロイド形成起始部である突起末端部においてガングリオシド量の増加が観察された。このことから、プレセニリン遺伝子変異に起因するγセクレターゼ機能障害が、膜脂質環境の変化を介して脳内アミロイド形成を誘導している可能性が示された。
日本学術振興会, 研究活動スタート支援, 独立行政法人国立長寿医療研究センター, 22890251