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Statins Induce Insulin-Degrading Enzyme Secretion from Astrocytes Via an Autophagy-Based Unconventional Secretory Pathway

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Statins Induce Insulin-Degrading Enzyme Secretion from Astrocytes Via an Autophagy-Based Unconventional Secretory Pathway Son et al. Molecular Neurodegeneration (2015) 10:56 DOI 10.1186/s13024-015-0054-3 RESEARCH ARTICLE Open Access Statins induce insulin-degrading enzyme secretion from astrocytes via an autophagy-based unconventional secretory pathway Sung Min Son1,2, Seokjo Kang1, Heesun Choi1 and Inhee Mook-Jung1,2* Abstract Background: Insulin degrading enzyme (IDE) is a major protease of amyloid beta peptide (Aβ), a prominent toxic protein in Alzheimer’s disease (AD) pathogenesis. Previous studies suggested that statins promote IDE secretion; however, the underlying mechanism is unknown, as IDE has no signal sequence. Results: In this study, we found that simvastatin (0.2 μM for 12 h) induced the degradation of extracellular Aβ40, which depended on IDE secretion from primary astrocytes. In addition, simvastatin increased IDE secretion from astrocytes in a time- and dose-dependent manner. Moreover, simvastatin-mediated IDE secretion was mediated by an autophagy-based unconventional secretory pathway, and autophagic flux regulated simvastatin-mediated IDE secretion. Finally, simvastatin activated autophagy via the LKB1-AMPK-mTOR signaling pathway in astrocytes. Conclusions: These results demonstrate a novel pathway for statin-mediated IDE secretion from astrocytes. Modulation of this pathway could provide a potential therapeutic target for treatment of Aβ pathology by enhancing extracellular clearance of Aβ. Keywords: Statin, Insulin-degrading enzyme (IDE), Autophagy-based unconventional secretion, Amyloid-β (Aβ), Alzheimer’s disease (AD) Background insulin-degrading enzyme (IDE) [7–9]. NEP is located Alzheimer’s disease (AD) is the most common form of mainly in the plasma membrane, and its catalytic domain dementia; it is characterized by senile plaques, neurofibril- faces the extracellular space [7]. MMP-9 and IDE can be lary tangles, and neuronal cell death [1, 2]. Abnormally in- secreted extracellularly and degrade extracellular Aβ,des- creased levels of amyloid beta peptides (Aβ) lead to pite the fact that IDE has no signal sequence for secretion formation of extracellular senile plaques and are associ- through a conventional secretory pathway [10]. Many ated with neurodegeneration in AD [3, 4]. The Aβ levels studies have demonstrated that IDE is secreted [11]; how- in the brain are not only determined by the rate of ever, the mechanism of secretion is still elusive. production by amyloid precursor protein (APP) process- Macroautophagy (hereafter referred to as autophagy) ing [5, 6], but also by several clearance mechanisms. These is a fundamental biological process in eukaryotes and include proteolytic degradation of extracellular Aβ by cell has an impact on essential biological processes including surface-localized and/or secreted proteases such as nepri- aging, cancer, neurodegenerative diseases, and metabolic lysin (NEP), matrix metalloproteinase-9 (MMP-9), and disorders [12, 13]. Autophagy is currently best known as a degradative pathway that delivers cytoplasmic mate- * Correspondence: [email protected] rials and organelles to the lysosomes for degradation 1Department of Biochemistry & Biomedical Sciences, Seoul National [14]. All autophagy-related processes include the formation University College of Medicine, 103 Daehak-ro, Jongro-gu, Seoul 110-799, of double-membrane structures called autophagosomes Korea 2Neuroscience Research Institute, Seoul National University College of and are induced by the inhibition of the mammalian target Medicine, Seoul, Korea © 2015 Son et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Son et al. Molecular Neurodegeneration (2015) 10:56 Page 2 of 11 of rapamycin (mTOR) signaling pathway. Autophagosomes that IDE levels in the cells were reduced conversely and their contents undergo clearance upon fusion with an (Fig. 1a, b). Because statins are known to lower choles- endosome (amphisomes) or lysosome (autolysosomes) for terol levels, we checked whether statins regulate choles- degradation and recycling (autophagic flux) [12, 13, 15]. terol levels in astrocytes. By using a filipin assay, we However, recent studies show that autophagy also has a found that simvastatin reduced intracellular cholesterol role in non-autophagic processes, especially in the secretory levels (Additional file 1: Figure S1A,B). To determine pathway [16]. In eukaryotic cells, the autophagy-based whether only simvastatin induced IDE secretion from as- secretory pathway regulates the unconventional secretion trocytes, one of the other known statins, fluvastatin, was of several cytosolic proteins or factors such as IL (interleu- applied to astrocytes; we found that fluvastatin also in- kin)-1β, IL-18, High-mobility Group Box 1 (HMGB1), ATP creased IDE secretion (Additional file 2: Figure S2A,B). (adenosine triphosphate), Aβ, and von Willebrand factor To examine whether the IDE secreted by statin treat- [17–20]. These proteins share important features, including ment functions as insulysin (having an insulin-degrading the lack of a signal sequence for conventional secretion, function), we utilized an IDE enzymatic activity assay. and the contribution of autophagy-related (Atg) protein to We detected an increased in fluorescent intensity gener- their secretion. ated by the cleavage of fluorometric IDE substrates in Several studies report that increased cholesterol levels the statin-treated astrocyte-conditioned media (ACM) might be related to AD [21, 22], and that statin-mediated (Fig. 1d). In an alternative approach, we performed an inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A Aβ degradation assay. When the statin-treated ACM (HMG-CoA) reductase decreases cholesterol levels; thus, was incubated with the recombinant Aβ40 peptide, the reducing Aβ levels [23, 24]. However, this is controversial level of the remaining Aβ40 peptide was reduced (Fig. 1e; [22]. Several studies have demonstrated that statins can lane 1 vs. lane 2). Furthermore, the reduced Aβ40 levels decrease the generation of Aβ by enhancing non- after incubation with the statin-treated ACM were amyloidogenic processing of APP [25, 26]. In addition, sta- restored when bacitracin A, an IDE inhibitor, was added tins also impair the generation of isoprenoids, which play (Fig. 1e; lane 2 vs. lane 6). The inhibitors of other known important roles in the post-translational modification of Aβ degrading enzymes including thiorphan, a NEP proteins in the Rho and Rab families [26, 27]. Isoprenoids inhibitor, or GM6001, a MMP inhibitor, did not restore regulate the localization and biological function of Rho the reduced Aβ40 levels by the statin. These data demon- and Rab proteins, and affect Aβ generation by modulating strate that statins induce IDE secretion from astrocytes, APP processing [27]. Previous studies have also shown and statin-induced IDE functions as a protease to de- that statins promote Aβ degradation by microglia via IDE grade the Aβ peptide. secretion [24]. However, the molecular mechanisms by which statins could offer protection against AD have not Statin-mediated IDE secretion from astrocytes is been studied extensively. associated with an autophagy-based unconventional In this study, we found that IDE secretion from pri- secretory pathway mary astrocytes was increased by statins in a time- and To examine the mechanism of statin-induced IDE secre- concentration-dependent manner, and statin-induced tion, transcript levels of IDE after statin treatment were IDE secretion was associated with autophagy-based un- measured by quantitative real-time PCR (qPCR). Simva- conventional secretion. Additionally, we found that au- statin did not alter ide mRNA levels in astrocytes tophagic flux is important in IDE secretion and that (Fig. 2a). Because IDE has no signal sequence for secre- statin activates autophagy in astrocytes via the LKB1- tion, we further investigated the secretory mechanism of AMPK-mTOR signal pathway. These results indicate IDE. Previous studies showed that statins could stimu- that IDE is secreted from astrocytes via an autophagy- late secretion of IDE proteins via an unconventional based secretory pathway, and that regulation of autoph- pathway in association with exosomes [24]. To determine agy is a potential therapeutic target in Aβ pathology. whether statin-induced IDE secretion is associated with exosomes in astrocytes, both exosomes and non-exosome Results fractions were isolated from vehicle- or statin-treated Statins induce extracellular secretion of functional IDE ACM. We found that simvastatin increased secreted IDE from astrocytes levels both in the exosomes and non-exosome fractions Previous studies have shown that astrocytes are the main (Additional file 3: Figure S3), indicating that the secretory source for IDE in AD pathology [28]. Therefore, we first pathway for IDE is mediated by both exosome- and non- determined whether statins regulate IDE levels in the exosome-associated pathways. To determine the exact extracellular space of astrocytes. We found that simva- mechanisms of IDE secretion
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