Endoplasmic Reticulum Stress Mediates Amyloid β Neurotoxicity via Mitochondrial Cholesterol Trafficking

August 25, 2017 | Autor: Anna Fernández | Categoría: Membrane Proteins, Mitochondria, Endoplasmic Reticulum Stress, Humans, Cholesterol, Animals, Male, Glutathione, Endoplasmic Reticulum, Alzheimer Disease, Animals, Male, Glutathione, Endoplasmic Reticulum, Alzheimer Disease

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The American Journal of Pathology, Vol. -, No. -, - 2014

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Endoplasmic Reticulum Stress Mediates Amyloid b Neurotoxicity via Mitochondrial Cholesterol Trafﬁcking Q68

Elisabet Barbero-Camps,*y Anna Fernández,*y Anna Baulies,*y Laura Martinez,*y Jose C. Fernández-Checa,*yz and Anna Colell*y

Q2 Q3 From the Department of Cell Death and Proliferation,* Biomedical Research Institute of Barcelona - Higher Council for Scientiﬁc Research (IIBB-CSIC), Q4 Q5 y

Barcelona, Spain; the Liver Unit, Hospital Clinic, Institute of Biomedical Research August Pi i Sunyer (IDIBAPS), the Center for Biomedical Research in Hepatic and Digestive Diseases (CIBEREHD), Barcelona, Spain; and the Southern California Research Center for Alcoholic Liver and Pancreatic Diseases,z Q6 Keck School of Medicine, University of Southern California, Los Angeles, California Accepted for publication March 18, 2014.

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Address correspondence to: Anna Colell, Ph.D., or Jose C. Fernández-Checa, Ph.D., Department of Cell Death and Proliferation, Institut d’Investigacions Biomèdiques de Barcelona-CSIC, Rosselló 161, 08036 Barcelona, Spain. E-mail: [email protected]. es, josecarlos.fernandezcheca@ iibb.csic.es or checa229@ yahoo.com.

Disrupted cholesterol homeostasis has been reported in Alzheimer disease and is thought to contribute to disease progression by promoting amyloid b (Ab) accumulation. In particular, mitochondrial cholesterol enrichment has been shown to sensitize to Ab-induced neurotoxicity. However, the molecular mechanisms responsible for the increased cholesterol levels and its trafﬁcking to mitochondria in Alzheimer disease remain poorly understood. Here, we show that endoplasmic reticulum (ER) stress triggered by Ab promotes cholesterol synthesis and mitochondrial cholesterol inﬂux, resulting in mitochondrial glutathione (mGSH) depletion in older age amyloid precursor protein/presenilin-1 (APP/ PS1) mice. Mitochondrial cholesterol accumulation was associated with increased expression of mitochondrial-associated ER membrane proteins, which favor cholesterol translocation from ER to mitochondria along with speciﬁc cholesterol carriers, particularly the steroidogenic acute regulatory protein. In vivo treatment with the ER stress inhibitor 4-phenylbutyric acid prevented mitochondrial cholesterol loading and mGSH depletion, thereby protecting APP/PS1 mice against Ab-induced neurotoxicity. Similar protection was observed with GSH ethyl ester administration, which replenishes mGSH without affecting the unfolded protein response, thus positioning mGSH depletion downstream of ER stress. Overall, these results indicate that Ab-mediated ER stress and increased mitochondrial cholesterol trafﬁcking contribute to the pathologic progression observed in old APP/PS1 mice, and that ER stress inhibitors may be explored as therapeutic agents for Alzheimer disease. (Am J Pathol 2014, -: 1e16; http://dx.doi.org/10.1016/j.ajpath.2014.03.014)

Since the initial observation that feeding rabbits with a cholesterol-enriched diet leads to amyloid b (Ab) accumulation,1 the number of studies linking cholesterol and Ab metabolism have increased exponentially. Although the impact of cholesterol in clinical studies remains unsettled, cumulating evidence indicates increased cholesterol levels in brain from Alzheimer disease (AD) patients.2e5 Moreover, ﬁndings in animal and cultured-cell models demonstrate that cholesterol enrichment in lipid rafts fosters Ab production6e8 and aggregation,9 and inhibits the intracellular degradation of the peptide.10 By using mouse models of cholesterol loading, namely sterol regulatory element binding protein 2 (SREBP-2) transgenic mice and Niemann-Pick type C1 knockout mice, we have shown that the trafﬁcking of cholesterol to mitochondria depletes mitochondrial glutathione (mGSH), which in turn exacerbates Ab-induced

oxidative neuronal death.11 Furthermore, in the amyloid precursor protein/presenilin-1 (APP/PS1) transgenic mouse the overexpression of the endoplasmic reticulum (ER)-resident transcription factor SREBP-2 accelerated and worsened key pathologic manifestations of AD, correlating with mitochondrial cholesterol loading and mGSH depletion.12 Supported in part by Plan Nacional de Investigación Cientíﬁca, Desarrollo e Innovación Tecnológica grants SAF2010-15760 (A.C.) and SAF2009-11417 and SAF2012-34831 (J.C.F.-C.), the Fundació Marató TV3 (J.C.F.-C.), the Instituto de Salud Carlos III grant PI041804 (J.C.F.-C.), the Research Center for Alcoholic Liver and Pancreatic Diseases of the NIH/National Institute on Alcohol Abuse and Alcoholism grant P50AA011999-15 (J.C.F.-C.), the Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (A.C. and J.C.F.-C.), and Formación de Personal Investigador (FPI) fellowships from the Ministerio de Economía y Competitividad (E.B.-C., A.B. and L.M.).

Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.03.014

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Consequently, in vivo mGSH recovery in APP/PS1 mice signiﬁcantly reduced Tau pathology and Ab depositions.12 Interestingly, increased mitochondrial cholesterol levels, concomitant with reduced mGSH content, also were observed in old APP/PS1 mice after Ab accumulation,11 suggesting that Ab per se can regulate cellular cholesterol. However, although the role of APP and the amyloidogenic processing on cholesterol homeostasis disruption has been suggested previously,2,13e15 the molecular mechanisms remain largely unknown. Previous studies have reported that moderate ER stress can deregulate lipid metabolism.16 The cellular response to alleviate the ER stress is elicited by the stress transducers inositol-requiring protein 1 (IRE1), activating transcription factor 6 (ATF6), and protein kinase R (PRKR)-like endoplasmic reticulum kinase (PERK) that trigger a signaling pathway called the unfolded protein response (UPR). In particular, the engagement of the PERK pathway has been involved in the activation of SREBPs.17e19 Conversely, overexpression of the chaperone glucose regulated protein 78 (GRP78) has been described to inhibit the expression of SREBP-1c and SREBP-2 proteins.20 ER stress is an early event in AD and has been implicated indirectly as a mediator of Ab neurotoxicity,21e25 whereas caspase-12 and caspase-4 have been reported as intermediates of the ER stress-speciﬁc apoptosis triggered by Ab.26,27 Hence, these studies lead us to hypothesize that Ab-induced ER stress might be an additional mechanism contributing to the increased brain cholesterol content observed in AD, as a result of enhanced SREBP-2 processing. By using 2- to 15month-old APP/PS1 mice and the SH-SY5Y cell line we examined the regulatory role of ER stress on cholesterol and mGSH levels and further analyzed the impact of these alterations on neuronal death. In vivo treatment of APP/PS1 mice with ER stress inhibitors restored the mitochondrial cholesterol homeostasis, replenished mGSH, and blocked Ab-mediated cell death and neurotoxicity. A similar degree of protection was observed in APP/PS1 mice treated with GSH ethyl ester, which prevented mGSH depletion without affecting ER stress. These data provide evidence that ER stress contributes to Ab-induced neurotoxicity, at least in part by increasing mitochondrial cholesterol accumulation, which leads to impaired mitochondrial antioxidant defense.

Materials and Methods APP/PS1 and SREBP-2 Mice Breeding pairs of B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J (APP/PS1) and B6;SJLTg(rPEPCKSREBF2)788Reh/J Q16 Q17 (SREBP-2) mice were purchased from The Jackson LaboQ18 ratory (Bar Harbor, ME) and characterized as previously described.11 Brieﬂy, at the time of weaning (21 days), mice were identiﬁed genetically by PCR using DNA from ear-tips and following the genotyping protocols provided by the supplier. We observed an age-dependent increase of brain

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cholesterol levels in female wild-type (WT) mice compared with male mice (not shown). Therefore, although female APP/PS1 mice have a more pronounced AD phenotype, in the present study we focused on male transgenic and WT mice to minimize variability caused by sex differences. All procedures involving animals and their care were approved by the ethics committee of the University of Barcelona and were conducted in accordance with institutional guidelines in compliance with national and international laws and policies.

Subcellular Fractionation to Isolate Mitochondria, ER, and MAM Puriﬁcation Brains were removed of olfactory bulbs, midbrain, and cerebellum, and were homogenated in 210 mmol/L mannitol, 60 mmol/L sucrose, 10 mmol/L KCl, 10 mmol/L sodium succinate, 1 mmol/L ADP, 0.25 mmol/L dithiothreitol, 0.1 mmol/L EGTA, and 10 mmol/L HEPES, pH 7.4. Homogenates were centrifuged at 700 g for 10 minutes, and the supernatants were centrifuged at 10,000 g for 15 minutes. The resulting pellet (crude mitochondria) was suspended in 2 mL, loaded onto 8 mL of 30% (v/v) Percoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) Q19 gradient, and centrifuged at 95,000 g for 30 minutes. The mitochondrial pellet then was rinsed twice by centrifuging for 15 minutes at 10,000 g. To isolate mitochondria from cell culture, 5 107 SH-SY5Y cells were trypsinized and rinsed ﬁrst in PBS and then in hypotonic buffer (20 mmol/L HEPES, 5 mmol/L KCl, and 1.5 mmol/L MgCl2, pH 8.0). After centrifugation at 800 g for 2 minutes the resulting pellet was resuspended in three volumes of hypotonic buffer Q20 and incubated for 10 minutes on ice. Then, cells were disrupted by four aspirations through a 30-gauge needle. Mannitol-sucrose-HEPES buffer (2.5) (50 mmol/L Q21 HEPES, 525 mmol/L mannitol, 175 mmol/L sucrose, and 5 mmol/L EDTA, pH 8.0) was added to the lysates before centrifuging at 1500 g for 5 minutes. The postnuclear supernatant was separated further through a 14% Percoll density gradient and centrifuged at 12,000 g for 20 minutes. Loose mitochondrial pellets were rinsed in 210 mmol/ L mannitol, 60 mmol/L sucrose, 10 mmol/L KCl, 10 mmol/ L sodium succinate, 0.1 mmol/L EGTA, and 10 mmol/L HEPES, pH 7.4, by centrifuging for 10 minutes at 8000 g. In both cases (brain and SH-SY5Y cells), mitochondrial purity was examined for cross-contamination with ER and plasma membrane by Western blot analysis. ER and mitochondria-associated ER membrane (MAM) were isolated according to the Wieckowski et al28 protocol. Postnuclear supernatant (S1) was centrifuged at 9000 g for 10 minutes to further process supernatants (S2) and Q22 pellets (P2). The ER fraction was obtained by washing the S2 supernatant at 20,000 g for 15 minutes, followed by centrifugation at 100,000 g for 1 hour to collect the pellet. In parallel, P2 was centrifuged twice at 10,000 g before Q23 centrifugation on a 30% Percoll gradient to obtain the lower

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Ab, ER Stress, and Cholesterol Q1 band, which contains mitochondria, and the upper band, which contains MAM, which were collected and washed further to remove any Percoll contaminants.

exposure in the presence of the 0.75 mmol/L P450 side-chain cleavage enzyme (CYP11A1) inhibitor aminoglutethimide.

Western Blot Analysis Cell Culture and Treatments Q24

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The SH-SY5Y human neuroblastoma cell line was obtained from the European Collection of Cell Cultures (SigmaAldrich, St. Louis, MO) and grown in Dulbecco’s modiﬁed Eagle’s medium/Ham’s nutrient mixture F12 (Invitrogen, Life Technologies, Carlsbad, CA), containing 10% fetal bovine serum and 2 mmol/L glutamine. Cells were plated at a density of 0.5 106 cells/well. Twenty-four hours after plating, cells were treated with 10 mmol/L oligomeric Ab (Bachem AG, Bubendorf, Switzerland), 2 nmol/L thapsigargin (Sigma-Aldrich), 5 mmol/L 4-phenylbutyric acid (PBA) (Enzo Life Sciences, Farmingdale, NY), 50 mmol/L salubrinal (Calbiochem, Merck Millipore, Billerica, MA), 100 mmol/L tauroursodeoxycholic acid (Calbiochem, Merck Millipore), or 1 nmol/L gonadotropin-releasing hormone (Sigma-Aldrich). In some cases, cells were fractionated into cytosol or mitochondria on digitonin permeabilization of the plasma membrane as described previously.29

Preparation of Ab Peptides Human Ab1-42 was dissolved to 1 mmol/L in hexaﬂuoroisopropanol and aliquoted into microcentrifuge tubes, then the hexaﬂuoroisopropanol was evaporated and the peptides were stored at 20 C until use. For oligomeric assembly, concentrated peptides were resuspended in 5 mmol/L dimethyl sulfoxide and then diluted to 100 mmol/L in phenol redefree media and incubated at 4 C for 24 hours. Oligomeric forms of Ab were conﬁrmed by Western blot as previously described.11

GSH and Cholesterol Measurements

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GSH levels in homogenates and mitochondria were analyzed by the recycling method.30 For cholesterol determination from brain, samples were extracted with alcoholic KOH, distilled water, and hexane (1:1:2, v/v/v). Appropriate aliquots of the hexane layer were evaporated and used for cholesterol measurement.31 High-performance liquid chromatography analysis was performed using a mBondapak C18 10 mm reverse-phase column (30 cm 4 mm inner diameter; Waters Corp., Milford, MA), with a mobile phase of 2-propanol/ acetonitrile/distilled water (6:3:1, v/v/v) at a ﬂow rate of 1 mL/ min. The amount of cholesterol was calculated from standard curves and the identity of the peaks was conﬁrmed by spiking the sample with known standards. Cholesterol levels in membrane fractions from ER and MAM were quantiﬁed by the Ampex Red cholesterol assay (Invitrogen). Samples were resuspended in the homogenization buffer containing 5% Triton X-100 (Sigma-Aldrich) and brieﬂy sonicated. Mitochondrial cholesterol levels in cells were determined after Ab

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Samples (30 to 80 mg of protein per lane) were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with the antibodies listed in Table 1. After overnight incubation at 4 C, bound antibodies were visualized using horseradish-peroxidaseecoupled secondary antibodies and an ECL developing kit (GE Healthcare BioScience AB, Uppsala, Sweden). Densitometry of the bands was measured with QuantityOne software (Bio-Rad Laboratories, Hercules, CA) and values were normalized to b-actin or glyceraldehyde-3-phosphate dehydrogenase.

Immunohistochemistry Parafﬁn-embedded blocks were prepared by sequential dehydration in graded ethanol and inﬁltration in parafﬁn before embedding. Blocks were sectioned serially at a thickness of 5 mm from 1.2 mm through 2.4 mm from Bregma. Sections were processed according to the avidine biotineperoxidase staining method (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). After antigen retrieval, the endogenous peroxidase was blocked by exposure to 3% H2O2 in methanol for 30 minutes. Samples then were treated with the avidin/biotin blocking kit (Vector Laboratories) according to the manufacturer’s instructions and incubated overnight at 4 C with mouse monoclonal antiATF6 (1:500; Acris, San Diego, CA) and mouse monoclonal antieAb1-42 (1:300; Sigma-Aldrich). After washing with PBS, sections were incubated for 1 hour with a biotinylated secondary antibody (1:400), followed by ABC staining for 1 hour. The immunoreaction was visualized with diaminobenzidine using a diaminobenzidine-enhanced liquid substrate system (Sigma-Aldrich). Sections were counterstained with hematoxylin (Dako, Glostrup, Denmark). TUNEL labeling was performed using the in situ Cell Death Detection Kit (Roche Applied Science, Penzberg, Germany).

Immunoﬂuorescence and Laser Confocal Imaging Dewaxed hippocampal sections were ﬁrst boiled in citrate buffer (10 mmol/L sodium citrate, pH 6.0) and incubated with 0.1 mol/L glycine/PBS for 20 minutes to reduce autoﬂuorescence. The sections were incubated overnight at 4 C with rabbit polyclonal anti-steroidogenic acute regulatory protein (StAR; 1:500; Abcam, Cambridge, UK), mouse monoclonal anti-microtubule-associated protein 2 (MAP2; 1:500; Sigma-Aldrich), and rat monoclonal antieglial ﬁbrillary acidic protein (GFAP, 1:500; Calbiochem Merck Millipore). After washing in PBS, antibody staining was visualized using Alexa Fluor 488 and 594 secondary antibodies (1:400; Invitrogen, Life Technologies). Confocal images were collected using a Leica TCS SPE laser scanning confocal

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311 312 313 314 315 316 317 318 ½T1 319 320 321 Q31 322 323 324 Q32 325 326 327 328 329 330 331 332 333 334 335 Q33 336 337 Q34 338 339 340 Q35 341 342 343 344 Q36 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 Q37 364 365 Q38 Q39 366 367 368 Q40 369 370 371 Q41 372

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Barbero-Camps et al Table 1

Details of the Primary Antibodies Used in This Study

Antibody

Source and type

Company

Dilution

ABCA1 Calregulin (H-170) Caspase-3 Caspase-12 CHOP COX IV Cytochrome c (clone 7H8.2C12) Phospho-eIF2a eIF2a GAPDH (6C5) GRP78 HXK I (G-1) HMG-CoA reductase INSIG-1 MAP2 MFN2 StARD3 Naþ,KþATPase a1 PACS-2 Phospho-PERK (Thr980) PERK Rab5A Rab7 Sigma receptor (B-5) SREBP-2 StAR VDAC b-actin

Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal Rat monoclonal Mouse monoclonal Rabbit polyclonal Mouse monoclonal Rabbit polyclonal Mouse monoclonal Mouse monoclonal Rabbit polyclonal Mouse monoclonal Goat polyclonal Rabbit polyclonal Mouse monoclonal Mouse monoclonal Rabbit polyclonal Mouse monoclonal Goat polyclonal Rabbit polyclonal Rabbit polyclonal Rabbit polyclonal Goat polyclonal Mouse monoclonal Rabbit polyclonal Mouse monoclonal Rabbit polyclonal Mouse monoclonal

Novus Biologicals (Littleton, CO) Santa Cruz Biotech Cell Signaling (Danvers, MA) Sigma-Aldrich Cell Signaling Cell Signaling Santa Cruz Biotech Invitrogen Abcam Santa Cruz Biotech Stressgen (Enzo Life Science) Santa Cruz Biotech Santa Cruz Biotech Abcam Sigma-Aldrich Abcam Abcam Santa Cruz Biotech Santa Cruz Biotech Cell Signaling Sigma-Aldrich Santa Cruz Biotech Santa Cruz Biotech Santa Cruz Biotech Abcam Abcam Calbiochem (EMD Millipore) Sigma-Aldrich

1:1000 1:500 1:1000 1:500 1:1000 1:1000 1:500 1:1000 1:1000 1:5000 1:1000 1:200 1:500 1:1000 1:500 1:1000 1:1000 1:500 1:200 1:1000 1:1000 1:250 1:250 1:200 1:1000 1:1000 1:500 1:10,000

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GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MFN2, mitofusin-2; PACS-2, phosphofurin acidic cluster sorting protein 2; VDAC, voltage-dependent anion-selective channel protein.

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microscope equipped with UV excitation, an argon laser, a 63/ 1.32 OIL PH3 CS objective, and a confocal pinhole set at 1 Airy unit (Leica Microsystems GmbH, Wetzlar, Germany). All of the confocal images shown were single optical sections. Immunoﬂuorescence intensities and colocalization coefﬁcients were determined using MBF ImageJ software version 1.43m (NIH, Bethesda, MD).

Real-Time RT-PCR Q43

Real-time RT-PCR ampliﬁcation was performed using the iScript one-step RT-PCR kit (Bio-Rad Laboratories) with Table 2 Name

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GRP78 CHOP SREBP2 STAR Stard4 Stard5 Xbp1

SYBR Green (Molecular Probes, Life Technologies). The primer sequences used are listed in Table 2. PCRs were run in ½T2 duplicate for each sample. The relative gene expression was quantiﬁed by the DDCt method.

Analysis of Xbp1 mRNA Splicing

RNA was reverse-transcribed using Xbp1-speciﬁc primers, described in Table 2, that ampliﬁed a 600-bp cDNA product encompassing the IRE1 cleavage sites.32 This fragment was digested further by PstI (New England BioLabs, Q45 Ipswich, MA) to show a restriction site that is lost after

Details of the Primers Used in This Study Host H H H H M M M

Forward sequence 0

Reverse sequence 0

5 -AATGACCAGAATCGCCTGAC-3 50 -GCGCATGAAGGAGAAAGAAC-30 50 -TGCCATTGGCCGTTTGTGTC-3 50 -ATCTTTTCCGATCTTCTGC-30 50 -GAGAGATGGCTGACCCTGAGA-30 50 -GACGCGTCGGGCTGG-30 50 -GGATCTCTAAAACTAGAGGCTTGGTG-3

50 -CGCTCCTTGAGCTTTTTGTC-30 50 -ACCATTCGGTCAATCAGAGC-30 50 -CCCTTCAGTGCAACGGTCATTCAC-30 50 -CTTGGGCATCCTTAGCAAC-30 50 -TCAGACAGTCCTTCCAGTTTGATC-30 50 -AACTCCTCAGATGGCCTCCA-30 50 -AAACAGAGTAGCAGCGCAGACTGC-30

H, human; M, Mus musculus.

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Ab, ER Stress, and Cholesterol IRE1-mediated cleavage and mRNA splicing. The PCR products were resolved on a 2% agarose gel.

Pregnenolone Determination

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To assess pregnenolone production, cells were plated in Dulbecco’s modiﬁed Eagle’s medium phenol-free media containing 5% fetal bovine serum and 2 mmol/L glutamine. Cells were exposed to 10 mmol/L Ab with or without pretreatment (1 hour) with ER stress inhibitors. After 48 hours of incubation, new media containing 10 mmol/L of trilostane (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to prevent further processing of pregnenolone, and cells were left to accumulate it for 48 hours. Media then was recovered and levels of the steroid hormone precursor were determined using a human pregnenolone enzyme-linked immunosorbent assay kit (Diagnostics Biochem Canada, Inc., Dorchester, ON, Canada), according to the manufacturer’s instructions. Data were normalized to total protein values.

Statistics Results are expressed as means SD of the number of experiments. Statistical signiﬁcance was examined using the unpaired, two-tailed Student’s t-test or one-way analysis of variance with the Dunnett or Bonferroni post hoc multiple comparisons test when required. P < 0.05 value was considered statistically signiﬁcant.

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559 560 561 Enhanced SREBP-2 Processing and Decreased 562 ATP-Binding Cassette, Sub-Family A, Member 1 563 Transporter Expression Correlate with Increased Brain 564 Mitochondrial Cholesterol Levels in APP/PS1 Mice 565 566 We ﬁrst monitored the age-dependent changes in brain 567 cholesterol levels of APP/PS1 mice. Cholesterol content 568 in homogenate or isolated mitochondria from brains of 569 APP/PS1 mice signiﬁcantly increased at 10 months of age 570 571 (Supplemental Figure S1A), consistent with previous ob572 servations.11 The increment in the sterol levels was 573 observed in hippocampus samples from 7-month-old APP/ PS1 mice (Figure 1A), whereas cholesterol levels in cere- ½F1 574 575 bellum, a brain area relatively unaffected in AD, remained 576 unchanged at 10 months of age (Figure 1A). These ﬁndings 577 were accompanied by a signiﬁcant depletion of GSH in 578 brain mitochondria of 10-month-old APP/PS1 mice 579 (Supplemental Figure S1B). Western blot analysis in the 580 ﬁnal mitochondrial fraction from 10-month-old WT and 581 APP/PS1 mice showed an absence of plasma membrane and 582 583 endosome markers, as well as a negligible presence of 584 PERK and calreticulin compared with ER fraction. 585 Thus, the contribution from cross-contamination of other 586 organellar fractions to the higher level of mitochondrial 587 cholesterol seen in aged APP/PS1 mice was minimized 588 (Supplemental Figure S2). 589 590 591 592 593 594 595 596 Figure 1 Expression levels of cholesterol 597 biosynthesis-related proteins. A: Cholesterol 598 levels. B: Western blot analysis of SREBP-2 protein Q57 599 levels in brain extracts from WT mice and APP/PS1 600 (A/P) mice at the indicated ages. C: Western blot 601 analysis of SREBP-2 and HMG-CoA reductase 602 (HMGCR) proteins in brain extracts from 10603 month-old WT mice and APP/PS1 mice at 604 different ages. D: Representative immunoblots of 605 INSIG-1 and ABCA1 presence in brain extracts 606 from WT mice and APP/PS1 mice at the indicated ages. Relative intensity values are densitometric 607 values of the bands representing the speciﬁc 608 protein immunoreactivity normalized with the 609 values of the corresponding b-actin bands. 610 *P < 0.05, **P < 0.01 versus WT mice values by 611 unpaired, two-tailed Student’s t-test (A, B, and D) 612 and one-way analysis of variance with the Dunnett 613 post hoc test (C) (n Z 4 to 6). mth, month. 614 615 616 617 618 619 620

Results

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To relate these observations with pathways leading to cholesterol synthesis, we analyzed the processing of mature SREBP-2. The increase in the levels of the transcriptionally active fragment of SREBP-2 (68 kDa) was ﬁrst detectable at 4 months of age (Figure 1, B and C) and correlated with an enhanced expression of its target gene encoding the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of the cholesterol synthesis pathway (Figure 1C). The expression of SREBP-2 and HMG-CoA reductase further increased in older APP/PS1 mice (Figure 1C), whereas the levels of both peptides remained unchanged in WT mice at all ages analyzed (Supplemental Figure S3A). Because SREBP activation is tightly regulated by levels of insulin-induced gene-1 (INSIG1)17,33 we next analyzed its expression in APP/PS1 mice. As shown, transgenic mice compared with WT mice displayed decreased levels of INSIG-1 at all ages analyzed (Figure 1D). Moreover, because of the time lag between the early SREBP2 processing (age, 4 mo) and the late increase in cholesterol levels (age, 10 mo) observed in APP/PS1 mice, we considered the contribution of alternative mechanisms in regulating cholesterol homeostasis. For instance, decreased levels of the ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) have been described in brains of 11-month-old APP/PS1 mice, with accumulation of cholesterol in cultured astrocytes resulting from Ab-induced down-regulation of ABCA1 expression.34 In turn, data from mouse models and in vitro experiments have suggested that ABCA1 may affect Ab deposition and clearance by regulating apolipoprotein E lipidation and brain cholesterol homeostasis.35 Furthermore, despite the fact that the expression levels of this protein in AD still are under debate, with studies showing high levels of ABCA1 in hippocampal tissues from AD patients,36,37 several reports have linked ABCA1 polymorphisms to an increased risk for AD.35 Interestingly, when the levels of the protein were analyzed in APP/PS1 mice (Figure 1D), we observed an increase in the expression at 4 and 7 months of age that decreased to WT levels in 10-month-old mice. The existence of an impaired cholesterol efﬂux resulting from low ABCA1 levels associated with an enhanced synthesis may explain in part the cholesterol increase in old APP/PS1 mice.

Increased Expression of Mitochondrial Cholesterol Carriers in APP/PS1 Mice

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Mitochondria are cholesterol-poor organelles. However, cholesterol transport to the inner mitochondrial membrane (IMM) plays a physiological role and is a highly regulated process.38 Cholesterol trafﬁcking to IMMs is controlled chieﬂy by StAR, which moves cholesterol from the outer mitochondrial membrane to the IMM for metabolism. StAR belongs to a family of proteins that contain StAR-related lipid transfer domains (StAR-related lipid transfer proteins), of which StARD3 (MLN64) and StARD4 also have been described to allow delivery of cholesterol to mitochondria. In WT mice the levels of StAR and StARD3 were maintained steadily during aging

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(Supplemental Figure S3B). By contrast, APP/PS1 mice displayed high levels of StARD3 at all ages analyzed compared with WT mice (Figure 2A), whereas StAR increased in 10- ½F2 month-old transgenic mice (Figure 2A), concomitant to the increase of mitochondrial cholesterol observed at this age (Supplemental Figure S1A). The mRNA expression of StARD4 also was increased in 10-month-old APP/PS1 mice, whereas StARD5, which is involved in the transport of cholesterol between Golgi, ER, and plasma membrane, remained unchanged (Figure 2B). Immunohistochemical analysis by confocal microscopy conﬁrmed the increase of StAR in the hippocampus of 10-month-old APP/PS1 mice (Figure 2C). The double-labeling showed colocalization between StAR and the neuronal marker anti-MAP2, but not with the astrocyte marker glial ﬁbrillary acidic protein (Figure 2, C and D). Similarly, signiﬁcant up-regulation of StAR and StARD3 protein levels was observed in SREBP-2 transgenic mice (Figure 2E), establishing a link between induced cholesterol synthesis and mitochondrial cholesterol trafﬁcking. Furthermore, differences in the time expression of both cholesterol carriers between APP/PS1 and TgeSREBP-2 mice highly suggest that although StARD3 may be regulated by the transcription factor SREBP-2, the induction of StAR expression requires high levels of cholesterol, but is not directly dependent on SREBP-2 activation. In addition to StAR-related lipid transfer proteins as mediators of mitochondrial cholesterol transport, recent studies have suggested the involvement of MAMs. MAMs are molecular hubs that regulate several aspects of mitochondrial biology, ranging from calcium transmission to lipid metabolism,39 including cholesterol homeostasis.40,41 Moreover, data from experimental AD models have shown increased MAM function as determined by cholesteryl ester and phospholipid synthesis,42 accompanied by high levels of MAMassociated proteins, including the sigma-1 receptor,43 and Q49 suggest that these interaction sites may contribute to AD pathology. To evaluate the role of MAMs on the enhanced mitochondrial cholesterol transport observed in old APP/PS1 mice, we ﬁrst tested whether cholesterol enrichment in isolated MAMs was similar to that of mitochondria. As previously reported, cholesterol levels were higher in MAM fractions compared with the total content in the ER (Figure 2F). However, we did not ﬁnd signiﬁcant differences between WT and APP/PS1 mice in either ER or in the MAM subdomains (Figure 2F). As indicators of increased area of apposition between ER and mitochondria we next evaluated the levels of phosphofurin acidic cluster sorting protein 2 and mitofusin-2, both are proteins that are required to regulate the contact sites between the two organelles.44,45 As shown, Western blot analysis showed similar levels of both peptides in MAMs isolated from brain extract of WT and APP/PS1 mice (Figure 2G). Conversely, the levels of the sigma-1 receptor and voltage-dependent anion-selective channel protein were increased signiﬁcantly in MAMs from APP/PS1 mice (Figure 2G), suggesting that these MAM-associated proteins, previously proposed to facilitate cholesterol translocation from

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Ab, ER Stress, and Cholesterol

Figure 2

Expression levels of cholesterol-transporting polypeptides. A: Representative immunoblots of StARD3 and StAR showing an increased presence of these cholesterol carriers in brain samples from APP/PS1 mice compared with 10-month-old WT mice. B: mRNA levels of StARD4 and StARD5 in brain samples from WT and APP/PS1 mice at 10 months of age analyzed by quantitative RT-PCR. Absolute mRNA values were determined, normalized to 18S rRNA, and reported as relative levels compared to the expression in WT mice. C: Representative confocal images of StAR (red) and MAP2 (green) immunoﬂuorescence labeling of hippocampal sections from 10-month-old WT mice and APP/PS1 mice. D: Representative confocal images of hippocampal sections from 10-monthold APP/PS1 mice immunostained with anti-StAR (red) and anti-MAP2 or antieglial ﬁbrillary acidic protein (GFAP; green). The extent of colocalization was quantiﬁed using the linear Pearson correlation coefﬁcient (rp); the resulting scatterplot images are shown on the right. A value of 1.0 indicates complete colocalization of two ﬂuorescent signals. Note that StAR preferentially colocalizes with neurons labeled with anti-MAP2 compared with astrocytes labeled with anti-GFAP. E: Western blot analysis of StARD3 and StAR expression in brain from 10-month-old WT mice and TgeSREBP-2 mice at the indicated ages. F: Total cholesterol in ER and MAMs from brain extracts of 10-month-old WT and APP/PS1 mice. G: Representative immunoblots showing the expression of calreticulin (calregulin), the sigma-1 receptor (s1R), phosphofurin acidic cluster sorting protein 2 (PACS-2), and the mitochondrial proteins cyclooxygenase 4 (COX IV), voltage-dependent anion-selective channel protein (VDAC), and mitofusin 2 (MFN2) in ER, MAMs, and mitochondria from 10-month-old WT and APP/PS1 (A/P) mice. In all Western blot analyses the densitometric values of the bands representing the speciﬁc protein immunoreactivity were normalized with the values of the corresponding b-actin bands or Ponceau Staining. *P < 0.05, **P < 0.01 versus WT mice values by one-way analysis of variance with the Dunnett post hoc test (A and E) and the unpaired, two-tailed Student’s t-test (B, C, and G) (n Z 3 to 6). Scale bars: 15 mm (C and D). mito., mitochondria; mth, month.

ER to mitochondria,41,46 may contribute to mitochondrial cholesterol content in APP/PS1 mice.

Early ER Stress Precedes Mitochondrial Cholesterol Accumulation in APP/PS1 Mice Given the cause-and-effect relationship between increased cholesterol synthesis and enhanced mitochondrial cholesterol

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trafﬁcking and mGSH regulation, we aimed to analyze further the mechanism responsible for these changes. Because previous studies had shown that moderate ER stress modulates the cellular lipid content,16 we explored whether the Ab-induced ER stress can affect cholesterol homeostasis. Although no signs of ER stress were detectable in WT mice at all of the ages analyzed (Supplemental Figure S4A), the UPR was activated in APP/PS1 mice, with markers showing age-

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869 870 APP/PS1 871 WT 4-mth 7-mth 10-mth 872 GRP78 (78 kDa) Figure 3 APP/PS1 mice display early ER stress. 873 CHOP A: Representative immunoblots showing the 874 GRP78 (27 kDa) expression of the chaperonic protein GRP78 and CHOP 875 p-PERK 6 p-PERK (170 kDa) * the ER stress signaling pathway proteins PERK, 876 p-eIF2α * PERK * phospho-PERK, eIF2a, phospho-eIF2a, and CHOP * * (125 kDa) 877 * 4 in brain extracts from 10-month-old WT mice and p-eIF2α * * 878 * (40 kDa) APP/PS1 mice at the indicated ages. B: Immuno* 879 eIF2α 2 histochemical staining of ATF6. Representative (40 kDa) 880 photomicrographs of CA1 hippocampus showing β-actin 881 0 nuclear localization of ATF6 in 7-month-old APP/ WT 4-mth 7-mth 10-mth 882 PS1 mice (n Z 3). C: Analysis of Xbp1 mRNA APP/PS1 splicing. Total RNAs from brain of 7-month-old WT 883 and APP/PS1 mice were subjected to RT-PCR as 884 WT APP/PS1 described in Materials and Methods. After diges885 TUN tion with PstI, the PCR products were resolved in a ATF6 886 S 2% agarose gel electrophoresis. The PCR products WT APP/PS1 887 of Xbp1 mRNA spliced (S) remained intact (454 U 888 bp), whereas the unspliced products (U) were cut 889 PstI into two fragments of 289 and 191 bp, as indi80 Unspliced (480 bp) 890 cated by the arrows. mRNA from liver of WT mice Spliced (454 bp) 60 891 treated i.p. with 1 mg/kg 24 hours tunicamycin 40 892 (TUN) was used as a positive control (n Z 3). D: Q63 Western blot analysis of GRP78, eIF2a, and 20 893 phospho-eIF2a in brain extracts from WT mice and 894 0 WT APP/PS1 TUN SREBP-2 transgenic mice. In all Western blot an895 alyses, densitometric values of the bands repre- Q64 896 WT Tg-SREBP-2 senting the speciﬁc protein immunoreactivity were 897 normalized with the values of the corresponding 7-mth 10-mth 7-mth 10-mth 3 898 * GRP78 GRP78 b-actin bands. *P < 0.05 versus WT mice values by (78 kDa) p-eIF2α 899 one-way analysis of variance with the Dunnett 2 * p-eIF2α * 900 post hoc test (A), and the unpaired two-tailed (40 kDa) 901 Student’s t-test (D) (n Z 6). Scale bar Z 50 eIF2α 1 (40 kDa) 902 mmol/L (B). mth, month. 903 β-actin 0 904 7-mth 10-mth 7-mth 10-mth 905 WT Tg-SREBP-2 906 907 ½F3 dependent changes (Figure 3A). GRP78 and phospho-PERK Thus, APP/PS1 mice showed an early UPR response that 908 were the earliest markers up-regulated at 4 months of age preceded the increase of cholesterol levels, the increase in the 909 (Figure 3A and Supplemental Figure S4B), whereas eukaryexpression of mitochondrial cholesterol transporting poly910 Q50 otic initiation factor (eIF)-2a phosphorylation and C/EBP peptides, particularly StAR, and mGSH depletion. 911 homologous protein (CHOP) increase were detected in older 912 APP/PS1 mice (Figure 3A). Moreover, Ab loading paralleled Ab Induces ER Stress in SH-SY5Y Cells, Resulting in 913 ER stress in APP/PS1 mice, with hippocampal amyloid 914 Increased Mitochondrial Cholesterol Inﬂux and mGSH 915 accumulation in intracellular compartments of 4-month-old Depletion 916 APP/PS1 mice (Supplemental Figure S5). APP/PS1 mice also 917 showed activation of the ATF6-dependent branch of UPR. Once we established that ER stress precedes the disruption in 918 ATF6 is processed by the same metalloproteases that activate cholesterol homeostasis in APP/PS1 mice, we analyzed the 919 SREBPs47 and although previous reports suggested that link between ER stress and mitochondrial cholesterol loading 920 by Ab in vitro. We used soluble oligomeric forms of Ab1-42, ATF6 antagonizes the activity of SREBP-2,48 we found 921 nuclear ATF6 in hippocampal neurons from 7-month-old previously conﬁrmed by Western blot.11 As shown, Ab1-42 922 APP/PS1 mice (Figure 3B). In contrast, compared with exposure increased the expression of GRP78 and CHOP at the 923 tunicamycin-treated mice, IRE1 phosphorylation and Xbp1 mRNA (Figure 4A) and protein levels (Figure 4B) over time ½F4 924 splicing were not observed in APP/PS1 mice (data not shown in SH-SY5Y cells. These ﬁndings were accompanied by 925 and Figure 3C). The correlation between ER stress and eIF2a phosphorylation (Figure 4B), conﬁrming the involve926 927 cholesterol accumulation also was noticed in SREPB-2 ment of the PERK branch observed in APP/PS1 mice. 928 transgenic mice, which showed increased GRP78 levels and This outcome preceded the increased expression of tran929 eIF2a phosphorylation compared with WT mice (Figure 3D). scription factor SREBP-2 and StAR by Ab both at the 930

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Ab, ER Stress, and Cholesterol

Figure 4 ER stress induced by oligomeric Ab1-42 promotes mitochondrial cholesterol transport and mGSH depletion in SH-SY5Y cells. GRP78, p-eIF2a, and CHOP expression levels in cells treated with 10 mmol/L Ab at the indicated time points, analyzed by quantitative RT-PCR (A) and Western blot (B). SREBP-2 and StAR expression levels after exposure to 10 mmol/L Ab at the indicated time points, analyzed by quantitative RT-PCR (C) and Western blot (D). E: mRNA levels of SREBP-2 and StAR after incubation with 2 mmol/L thapsighargin (TG) at the indicated time points, analyzed by quantitative RT-PCR. mRNA values were normalized to 18S rRNA and reported as relative levels compared to the expression in WT mice. F: Mitochondrial cholesterol levels after incubation with 10 mmol/L Ab for 48 hours, and 0.75 mmol/L inhibitor aminoglutethimide was added for the last 24 hours. G: Quantiﬁcation of pregnenolone delivered to media as indicator of mitochondrial cholesterol loading. Cells were exposed to 10 mmol/L Ab with or without 5 mmol/L PBA, 50 mmol/L salubrinal (SAL), or 100 mmol/L tauroursodeoxycholic acid (TUDCA) pretreatment for 1 hour. After 48 hours of incubation new media containing 20 mmol/L of trilostane was replaced and cells were left to accumulate the steroid hormone for 48 hours. Pregnenolone levels in media were determined as described in Materials and Methods. As a positive control, cells were treated with 1 nmol/L gonadotropinreleasing hormone (GnRH) for 48 hours. Western blot analysis of the mitochondrial cholesterol carriers StAR and StARD3 in cellular extracts (H) and mitochondrial GSH levels after 4 days of 10 mmol/L Ab incubation with or without 5 mmol/L PBA, 50 mmol/L SAL, or 100 mmol/L TUDCA pretreatment for 1 hour (I). In all of the Western blot analyses densitometric values of the bands representing the speciﬁc protein immunoreactivity were normalized with the values of the corresponding b-actin bands. *P < 0.05 versus control values; yP < 0.05, yyP < 0.01 versus Ab treatment values by one-way analysis of variance with the Dunnett post hoc test (AeE), the unpaired, two-tailed Student’s t-test (F), and one-way analysis of variance with the Bonferroni multiple comparisons test (G-I) (n Z 3 to 6).

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Barbero-Camps et al mRNA and protein levels (Figure 4, C and D). Similar ﬁndings were observed when SH-SY5Y cells were exposed to the ER stressor thapsigargin (Figure 4E). Consistent with the function of StAR in mitochondrial cholesterol trafﬁcking, there was a signiﬁcant increase in cholesterol levels in isolated mitochondria from SH-SY5Y cells treated with Ab for 48 hours (Figure 4F), pointing to the amyloid peptide as responsible for the mitochondrial cholesterol enrichment observed in APP/PS1 mice. To further assess the involvement of the Ab-induced ER stress we tested the effects of ER stress inhibitors. In steroidogenic cells, cholesterol is metabolized in the IMM by P450 side-chain cleavage enzyme (CYP11A) and converted into the steroid precursor pregnenolone. Mitochondrial cholesterol availability is the rate-limiting step in the steroid biosynthesis and, therefore, pregnenolone levels can be used as a gauge of the rate of mitochondrial cholesterol inﬂux.49 In agreement with the increased mitochondrial cholesterol levels observed in isolated mitochondria, exposure to 10 mmol/L Ab1-42 resulted in a signiﬁcant increase in pregnenolone levels (Figure 4G). This increase was similar to that caused by gonadotropin-releasing hormone (Figure 4G), which has been described previously to modulate steroidogenesis in SH-SY5Y cells.50 Interestingly, pre-incubation with different chemical inhibitors of ER stress, including 3 mmol/L PBA, 50 mmol/L salubrinal, or 100 mmol/L tauroursodeoxycholic acid, signiﬁcantly decreased the levels of StARD3 and StAR protein induced by Ab (Figure 4H) and prevented the increase of pregnenolone in Ab-exposed cells (Figure 4G). As expected for mitochondrial cholesterol loading, Ab challenge depleted mGSH levels (Figure 4I), and this effect was prevented by PBA. Thus, these ﬁndings establish a cause-andeffect relationship between ER stress triggered by Ab and mGSH depletion.

ER Stress Contributes to in Vivo Neurotoxicity in APP/ PS1 Mice due to mGSH Depletion Although UPR is considered a protective mechanism, it can initiate an apoptotic cascade if the response persists over time. To analyze the contribution of ER stress to neuronal death we tested the role of PBA treatment in vivo (500 mg/kg per day), administered i.p. every 12 hours for 2 weeks, in 15month-old APP/PS1 mice, when neurodegeneration is fully detectable. Treatment with the chaperone signiﬁcantly reduced the levels of the ER stress markers GRP78 and CHOP in APP/PS1 mice (Figure 5A). Moreover, the proteolytic processing of caspase-12 and caspase-3 to their active fragments was blocked by PBA treatment (Figure 5B), resulting in reduced apoptosis in the hippocampus of APP/ PS1 mice (Figure 5C). The use of the chaperone-like molecule also prevented the expression of SREBP-2 as well as the increase of the mitochondrial cholesterol carriers StAR and StARD3 observed in APP/PS1 mice (Figure 5D). Paralleling these effects, mitochondrial cholesterol loading (Figure 5E) and mGSH depletion (Figure 5F) in APP/PS1 mice were

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1179 abolished by PBA therapy, further validating the link be1180 tween ER stress and mGSH regulation. 1181 We previously showed that the mGSH content is critical in 1182 regulating the cellular response to different apoptotic stimuli 1183 51 including tumor necrosis factor-a, hypoxia, or Ab peptides. 1184 Therefore, it is conceivable to speculate that the protective 1185 effect exerted by PBA may be owing, at least in part, to the 1186 recovery of the pool of mGSH. To further test this hypothesis 1187 mice were administered 1.25 mmol/kg/d GSH ethyl ester 1188 1189 (GSHee) i.p. every 12 hours for 2 weeks. As shown, GSHee replenished the pool of mGSH (Figure 6A). The expression ½F6 1190 1191 of the cholesterol-related proteins SREBP-2 and StARD3, as 1192 well as the increased levels of the ER stress markers GRP78, 1193 CHOP, and cleaved caspase-12, observed in APP/PS1 mice 1194 remained unchanged after GSHee treatment (Figure 6, B and 1195 C). However, GSHee signiﬁcantly reduced the cleavage of 1196 caspase-3 (Figure 6D) and prevented brain apoptosis in APP/ 1197 PS1 mice to a similar extent as observed with PBA 1198 (Figure 5C). Overall, these ﬁndings suggest that ER stress 1199 contributes to in vivo Ab neurotoxicity in APP/PS1 mice, at 1200 1201 least in part by depletion of mGSH.

Discussion A recent lipidomic analysis described a complex landscape of lipid alterations in speciﬁc brain regions in AD.52 Among these, disruption of cholesterol homeostasis has been described in AD,2e5 and our recent studies advanced a role for mitochondrial cholesterol in Ab-induced neuroinﬂammation and neurotoxicity.11,12 Here, we analyzed the role of ER stress in cholesterol homeostasis, mitochondrial cholesterol loading, and mGSH depletion in APP/PS1 mice (Figure 6E). First, we observed that APP/PS1 mice show early ER stress and increased levels of active SREBP-2 associated with decreased expression of INSIG-1. INSIGs exert their action in the ER by binding SREBP cleavage-activating protein and preventing it from escorting SREBPs to the Golgi apparatus where the SREBPs are processed to their active forms. Interestingly, INSIG-1 is a short-lived protein, and inhibition of protein translation processes as a response to ER stress has been shown to reduce its levels severely,17 thus raising the possibility that a similar mechanism may take place in APP/PS1 mice, likely contributing to enhanced processing of SREBP-2. Despite the presence of active SREBP-2 since 4 months of age, cholesterol accumulates only in old transgenic mice, and is ﬁrst detectable in the hippocampus. A potential factor that accounts for this age-dependent difference may be ABCA1 expression, which increases in young transgenic mice but decreases in old mice. Although in-depth research would be needed to discern the exact mechanism, chronic inﬂammatory conditions41,42 or changes in cerebral oxysterols53,54 might account for this age-dependent decrease of ABCA1 expression. A key feature of our ﬁndings is the increased mitochondrial cholesterol ﬂux that correlates with depleted mGSH content. The evidence for this outcome was generated in a

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Ab, ER Stress, and Cholesterol

Figure 5 Treatment with the chemical chaperone PBA restores cholesterol homeostasis and provides protection against ER stresseinduced cell death in APP/PS1 mice. Fifteen-month-old mice were treated i.p. with 500 mg/kg/d PBA for 2 weeks (n Z 6 mice per group) and levels of ER stress markers, caspase activation, and cholesterol-related proteins were analyzed by Western blot. A: Representative immunoblots of GRP78 and CHOP presence in brain extracts. B: Representative immunoblots of caspase-12 and caspase-3 cleavage; arrows show fragmented caspase-12 (Csp12) (w42 kDa) and cleaved caspase-3 active fragment (Csp3) (17 kDa). C: Representative images of apoptotic cells in hippocampus from 15-month-old WT and APP/PS1 mice with or without PBA or GSHee (i.p. 1.25 mmol/kg/d for 2 weeks) assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). D: Representative immunoblots showing a signiﬁcant reduction of SREBP-2, StARD3, and StAR in brain samples from APP/PS1 mice after PBA treatment. Mitochondrial cholesterol content (E) and mGSH levels (F). In all Western blot analyses densitometric values of the bands representing the speciﬁc protein immunoreactivity were normalized with the values of the corresponding b-actin bands. *P < 0.05, **P < 0.01 versus WT values; yP < 0.05, yyP < 0.01 versus APP/PS1 mice without treatment by one-way analysis of variance with the Bonferroni multiple comparisons test (n Z 3 to 6). Scale bar Z 50 mmol/L (C).

dual fashion. First, the increase in mitochondrial cholesterol parallels that of pregnenolone in human neuroblastoma cells challenged with Ab peptide. Because mitochondrial cholesterol availability is the rate-limiting step in steroidogenesis,49,55 pregnenolone determination is a surrogate of mitochondrial cholesterol trafﬁcking to IMM. Second, we

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examined the expression of different protein carriers that potentially involved in cholesterol transport to mitochondria. Unlike StARD3, changes in the expression of StAR were detectable in 10-month-old APP/PS1 mice, paralleling the increase of cholesterol in mitochondria, which positions StAR as most likely responsible for mitochondrial

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In vivo GSH ethyl ester treatment. WT and APP/PS1 mice were treated i.p. with 1.25 mmol/kg/d GSHee for 2 weeks (n Z 6 mice per group). A: mGSH levels. B: Representative immunoblots showing the expression levels of SREBP-2 protein and the mitochondrial cholesterol carrier StARD3 in brain extracts after GSHee treatment. C: Western blots analysis of ER stress markers GRP78 and CHOP levels and the presence of cleaved caspase-12 active fragment (42 kDa) in brain extracts after GSHee treatment. D: Representative immunoblots of caspase-3 cleavage; arrow shows cleaved active fragment (17 kDa). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were analyzed as a loading control. In all Western blot analyses densitometric, values of the bands representing the speciﬁc protein immunoreactivity were normalized with the values of the corresponding GAPDH bands. E: Scheme illustrating the proposed model by which Ab-induced ER stress would contribute to neuronal death through promoting cholesterol synthesis and translocation to mitochondria. This cascade of events leading to neuronal death could be counteracted by inhibiting ER stress or by restoring the mGSH levels with GSHee therapy. *P < 0.05, **P < 0.01 versus WT mice values; yP < 0.05 versus APP/PS1 mice values by one-way analysis of variance with the Bonferroni multiple comparisons test; n Z (3 to 6). CHOL, cholesterol; Mito., mitochondrial; MMP, mitochondrial membrane permeabilization.

Figure 6

cholesterol loading. Interestingly, the immunohistologic analyses show that the increase in StAR is limited to hippocampal neurons; although we cannot exclude that an enhanced cholesterol synthesis also may occur in astrocytes,

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which are known to be the primary site for cholesterol loading in adult brain. The relevance of the mitochondrial cholesterol loading described in APP/PS1 mice is reinforced further by the reported presence of high StAR levels in affected

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Ab, ER Stress, and Cholesterol

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brains of AD patients.56 In these studies, the enrichment in StAR was observed both in hippocampal pyramidal neurons and astrocytes. Moreover, although recent studies indicate that StARD3 regulates mitochondrial cholesterol in the absence of Niemann-Pick type C1,49 it is likely that the differential localization of StARD3 (endolysosomal) versus StAR (mitochondria) determines their contribution to mitochondrial cholesterol regulation. In addition, posttranslational modiﬁcations of StAR, including its phosphorylation at serine residues, have been described to modulate steroidogenesis.57 The impact of these factors in promoting mitochondrial cholesterol accumulation in the APP/PS1 mouse model remains to be established. Mitochondrial cholesterol trafﬁcking implies two events: cholesterol transfer to the outer mitochondrial membrane from extramitochondrial sources and intramitochondrial cholesterol movement from the outer mitochondrial membrane to IMM for metabolism. StAR plays an essential role in the latter. Indeed, germline StAR deﬁciency in mice results in lethal congenital adrenal lipoid hyperplasia,58 meaning that other StAR-related lipid transfer members cannot replace StAR deﬁciency. A remaining question, however, is how does cholesterol transfer to the outer mitochondrial membrane to be available for StAR? We found increased levels of the sigma-1 receptor and voltagedependent anion-selective channel protein, both MAMassociated proteins, which have been shown to interact and collaborate with StAR in the transfer of cholesterol from ER to mitochondria.41,46 The responsiveness of MAM to ER stress has been described in Ca2þ-dependent mitochondrial apoptosis59; in particular, the sigma-1 receptor has been reported to regulate ER/mitochondrial Ca2þ mobilization under ER stress conditions.60 Moreover, in addition to these possibilities, caveolin-1 has recently been shown to regulate mitochondrial cholesterol homeostasis.55 However, its putative contribution to Ab-mediated mitochondrial cholesterol loading remains to be investigated. On the other hand, overexpression of StARD4 in mouse hepatocytes also has been reported to stimulate bile acid synthesis,61 supporting its role of cholesterol transport to mitochondria. Furthermore, by using cholesterol-containing liposomes as donors, a recent study62 showed that recombinant StARD4 accelerates cholesterol transfer to isolated liver mitochondria. Consistent with these studies, we found a 1.5- to 2-fold increase of Stard4 mRNA levels in 10-month-old APP/PS1 mice whereas Stard5 levels remained unchanged, consistent with its inability to transfer cholesterol to mitochondria.63,64 Previous data have indicated that StARD5 expression was up-regulated by ER stress63; however, the transcriptional induction of StARD5 is mediated by XBP1,65 a signal transducer that was unprocessed and therefore inactive in the ER stress response displayed by APP/PS1 mice. We observed an early activation of the UPR, coinciding with the burst of Ab generation in 4-month-old APP/PS1 mice, and the induction of cholesterol synthesis, indicating that Ab-induced ER stress can play a role in this alteration. In support of this link, we observed that Ab-induced ER

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stress in SH-SY5Y cells stimulates the expression of SREBP-2 and StAR. The ER stressor thapsigargin reproduced these ﬁndings, conﬁrming and extending the initial observations describing a link between ER stress and cholesterol synthesis.16,66 Moreover, Ab exposure resulted in accumulation of mitochondrial cholesterol and pregnenolone, indicating that Ab promotes cholesterol ﬂux to mitochondria. The efﬁcacy of different ER stress inhibitors in normalizing the expression of StARD3 and StAR and reducing the hormone levels, further proved that this effect was mediated via ER stress. Consistent with these data, we recently provided compelling evidence in hepatocytes that StAR is an ER stress target gene whose expression, induced by tunicamycin, is prevented by tauroursodeoxycholic acid.67 Finally, we observed that the Ab-enhanced mitochondrial cholesterol inﬂux is accompanied by signiﬁcant mGSH depletion. The link between these events, namely increased cholesterol and mGSH depletion, is owing to the sensitivity of the mGSH carrier to changes in membrane dynamics induced by cholesterol accumulation in mitochondrial membranes.68 The reduction of mGSH levels was prevented by the chemical chaperone PBA, suggesting a key role of Ab-induced ER stress in controlling the mitochondrial antioxidant defense. APP/PS1 mice display speciﬁc UPR signaling, characterized by selective PERK and ATF6 activation, without IRE1a phosphorylation and Xbp1 processing. Although the underlying mechanisms contributing to this particular UPR branch activation are unknown, initial observations have described that mutations in PS1 increased the vulnerability to ER stress owing to a down-regulation of the UPR, caused by disturbed function of the IRE1a.69 Whether this particular UPR signaling is a speciﬁc feature of APP/PS1 mice Q54 has yet to be conﬁrmed. ER stressemediated cell death has been linked consistently to Ab neurotoxicity.21,23,24 Consistent with these ﬁndings, APP/PS1 mice showed increased processing of caspase-12 and caspase-3 associated with neuronal death, which was reduced signiﬁcantly by inhibiting ER stress. Moreover, in vivo treatment with PBA also normalized the expression of SREBP-2 and that of StAR and StARD3 in APP/PS1 mice, preventing mitochondrial cholesterol accumulation. Recent studies in Tg2576 and APP/PS1 mice show that chronic administration of PBA is accompanied by clearance of Ab, reduced Tau pathology, and reversal of learning deﬁcits.70e72 In addition to its established role as an anti-ER stress agent, PBA has been shown to inhibit histone deacetylases, which may contribute to PBA-mediated effects by restoring the transcription of proteins involved in synaptic plasticity and structural remodeling. Histone deacetylase inhibitors also have been described to modulate cholesterol homeostasis, favoring cholesterol trafﬁcking and degradation.73e75 However, the fact that other ER stress inhibitors such as salubrinal and tauroursodeoxycholic acid can modulate cholesterol homeostasis minimizes the potential contribution of histone deacetylase inhibition by PBA, further supporting an ER stressedependent mechanism.

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Barbero-Camps et al We observed a signiﬁcant recovery of the mGSH content in cells and APP/PS1 mice that was associated with PBAinduced cholesterol normalization. Given the key role of mGSH in controlling the oxidative stress generated in mitochondria,51 we inferred that part of the ER stressemediated cell death in the transgenic mice resulted from a compromised mitochondrial antioxidant defense that sensitizes cells to Ab insult. To conﬁrm this hypothesis, mice were administered GSHee, which signiﬁcantly recovered the mitochondrial pool of GSH in APP/PS1 mice. As expected, the treatment did not modify the levels of ER stress or the increased brain cholesterol content displayed by the transgenic mice, but signiﬁcantly reduced caspase-3 cleavage and neuronal death, to the same extent as PBA treatment, highlighting the key role of mGSH in regulating ER stressemediated neuronal death. It is worth noting that TgeSREBP-2 mice also showed ER stress associated with enhanced mitochondrial cholesterol loading and depleted mGSH levels,11 whereas no signs of apoptosis or Ab accumulation were observed in these mice during aging,12 indicating that altered cholesterol homeostasis per se is not sufﬁcient to initiate the disease. Taken together, our ﬁndings point to ER stress as a link between Ab and cholesterol up-regulation and subsequent mitochondrial trafﬁcking (Figure 6E). Moreover, although the relative contribution of neuronal and glial cells has to be established, the histochemical analyses show an increased presence of StAR and nuclear ATF6 in the hippocampal pyramidal cell layer of transgenic mice, indicating that deregulated cholesterol homeostasis and ER stress is present in neurons. In addition, the data uncover mGSH depletion as a key mechanism of ER stress-mediated neurotoxicity in APP/ PS1 mice. This view is of particular relevance given recent ﬁndings suggesting a feed-forward loop between ER stress and Ab processing in AD,76 with the expected consequences of mitochondrial cholesterol loading and mGSH depletion contributing to Ab-mediated neurotoxicity. Thus, therapeutic strategies targeting ER stress and resulting in the restoration of mGSH may be of signiﬁcance in the treatment of AD.

Acknowledgments

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We thank Drs. Carmen Garcia-Ruiz, Montserrat Mari, and Albert Morales for critical comments and suggestions and Susana Nuñez for her help with mice care. This work was developed, in part, at the Esther Koplowitz Center (Barcelona, Spain).

Supplemental Data

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References 1. Sparks DL, Scheff SW, Hunsaker JC 3rd, Liu H, Landers T, Gross DR: Induction of Alzheimer-like beta-amyloid immunoreactivity in the

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2.

brains of rabbits with dietary cholesterol. Exp Neurol 1994, 126: 88e94 Cutler RG, Kelly J, Storie K, Pedersen WA, Tammara A, Hatanpaa K, Troncoso JC, Mattson MP: Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer’s disease. Proc Natl Acad Sci U S A 2004, 101:2070e2075 Bandaru VV, Troncoso J, Wheeler D, Pletnikova O, Wang J, Conant K, Haughey NJ: ApoE4 disrupts sterol and sphingolipid metabolism in Alzheimer’s but not normal brain. Neurobiol Aging 2009, 30:591e599 Xiong H, Callaghan D, Jones A, Walker DG, Lue LF, Beach TG, Sue LI, Woulfe J, Xu H, Stanimirovic DB, Zhang W: Cholesterol retention in Alzheimer’s brain is responsible for high beta- and gamma-secretase activities and Abeta production. Neurobiol Dis 2008, 29:422e437 Panchal M, Loeper J, Cossec JC, Perruchini C, Lazar A, Pompon D, Duyckaerts C: Enrichment of cholesterol in microdissected Alzheimer’s disease senile plaques as assessed by mass spectrometry. J Lipid Res 2010, 51:598e605 Osenkowski P, Ye W, Wang R, Wolfe MS, Selkoe DJ: Direct and potent regulation of gamma-secretase by its lipid microenvironment. J Biol Chem 2008, 283:22529e22540 Kalvodova L, Kahya N, Schwille P, Ehehalt R, Verkade P, Drechsel D, Simons K: Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro. J Biol Chem 2005, 280:36815e36823 Ehehalt R, Keller P, Haass C, Thiele C, Simons K: Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J Cell Biol 2003, 160:113e123 Yip CM, Elton EA, Darabie AA, Morrison MR, McLaurin J: Cholesterol, a modulator of membrane-associated Abeta-ﬁbrillogenesis and neurotoxicity. J Mol Biol 2001, 311:723e734 Lee CY, Tse W, Smith JD, Landreth GE: Apolipoprotein E promotes beta-amyloid trafﬁcking and degradation by modulating microglial cholesterol levels. J Biol Chem 2012, 287:2032e2044 Fernandez A, Llacuna L, Fernandez-Checa JC, Colell A: Mitochondrial cholesterol loading exacerbates amyloid beta peptide-induced inﬂammation and neurotoxicity. J Neurosci 2009, 29:6394e6405 Barbero-Camps E, Fernandez A, Martinez L, Fernandez-Checa JC, Colell A: APP/PS1 mice overexpressing SREBP-2 exhibit combined Abeta accumulation and tau pathology underlying Alzheimer’s disease. Hum Mol Genet 2013, 22:3460e3476 Grimm MO, Grimm HS, Patzold AJ, Zinser EG, Halonen R, Duering M, Tschape JA, De Strooper B, Muller U, Shen J, Hartmann T: Regulation of cholesterol and sphingomyelin metabolism by amyloidbeta and presenilin. Nat Cell Biol 2005, 7:1118e1123 Liu Q, Zerbinatti CV, Zhang J, Hoe HS, Wang B, Cole SL, Herz J, Muglia L, Bu G: Amyloid precursor protein regulates brain apolipoprotein E and cholesterol metabolism through lipoprotein receptor LRP1. Neuron 2007, 56:66e78 Pierrot N, Tyteca D, D’Auria L, Dewachter I, Gailly P, Hendrickx A, Tasiaux B, Haylani LE, Muls N, N’Kuli F, Laquerriere A, Demoulin JB, Campion D, Brion JP, Courtoy PJ, KienlenCampard P, Octave JN: Amyloid precursor protein controls cholesterol turnover needed for neuronal activity. EMBO Mol Med 2013, 5: 608e625 Colgan SM, Hashimi AA, Austin RC: Endoplasmic reticulum stress and lipid dysregulation. Expert Rev Mol Med 2011, 13:e4 Lee JN, Ye J: Proteolytic activation of sterol regulatory elementbinding protein induced by cellular stress through depletion of Insig-1. J Biol Chem 2004, 279:45257e45265 Bobrovnikova-Marjon E, Hatzivassiliou G, Grigoriadou C, Romero M, Cavener DR, Thompson CB, Diehl JA: PERK-dependent regulation of lipogenesis during mouse mammary gland development and adipocyte differentiation. Proc Natl Acad Sci U S A 2008, 105: 16314e16319

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Ab, ER Stress, and Cholesterol 19. Colgan SM, Tang D, Werstuck GH, Austin RC: Endoplasmic reticulum stress causes the activation of sterol regulatory element binding protein-2. Int J Biochem Cell Biol 2007, 39:1843e1851 20. Kammoun HL, Chabanon H, Hainault I, Luquet S, Magnan C, Koike T, Ferre P, Foufelle F: GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice. J Clin Invest 2009, 119:1201e1215 21. Lee JH, Won SM, Suh J, Son SJ, Moon GJ, Park UJ, Gwag BJ: Induction of the unfolded protein response and cell death pathway in Alzheimer’s disease, but not in aged Tg2576 mice. Exp Mol Med 2010, 42:386e394 22. Hoozemans JJ, van Haastert ES, Nijholt DA, Rozemuller AJ, Scheper W: Activation of the unfolded protein response is an early event in Alzheimer’s and Parkinson’s disease. Neurodegener Dis 2012, 10:212e215 23. Ferreiro E, Resende R, Costa R, Oliveira CR, Pereira CM: An endoplasmic-reticulum-speciﬁc apoptotic pathway is involved in prion and amyloid-beta peptides neurotoxicity. Neurobiol Dis 2006, 23:669e678 24. Umeda T, Tomiyama T, Sakama N, Tanaka S, Lambert MP, Klein WL, Mori H: Intraneuronal amyloid beta oligomers cause cell death via endoplasmic reticulum stress, endosomal/lysosomal leakage, and mitochondrial dysfunction in vivo. J Neurosci Res 2011, 89: 1031e1042 25. Chafekar SM, Hoozemans JJ, Zwart R, Baas F, Scheper W: Abeta 142 induces mild endoplasmic reticulum stress in an aggregation statedependent manner. Antioxid Redox Signal 2007, 9:2245e2254 26. Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, Yuan J: Caspase-12 mediates endoplasmic-reticulum-speciﬁc apoptosis and cytotoxicity by amyloid-beta. Nature 2000, 403:98e103 27. Hitomi J, Katayama T, Eguchi Y, Kudo T, Taniguchi M, Koyama Y, Manabe T, Yamagishi S, Bando Y, Imaizumi K, Tsujimoto Y, Tohyama M: Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J Cell Biol 2004, 165:347e356 28. Wieckowski MR, Giorgi C, Lebiedzinska M, Duszynski J, Pinton P: Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat Protoc 2009, 4:1582e1590 29. Fernandez-Checa JC, Ookhtens M, Kaplowitz N: Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, efﬂux, and response to incubation with ethanol. J Clin Invest 1987, 80:57e62 30. Tietze F: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 1969, 27: 502e522 31. Duncan IW, Culbreth PH, Burtis CA: Determination of free, total, and esteriﬁed cholesterol by high-performance liquid chromatography. J Chromatogr 1979, 162:281e292 32. Calfon M, Zeng H, Urano F, Till JH, Hubbard SR, Harding HP, Clark SG, Ron D: IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 2002, 415:92e96 33. Engelking LJ, Kuriyama H, Hammer RE, Horton JD, Brown MS, Goldstein JL, Liang G: Overexpression of Insig-1 in the livers of transgenic mice inhibits SREBP processing and reduces insulinstimulated lipogenesis. J Clin Invest 2004, 113:1168e1175 34. Canepa E, Borghi R, Vina J, Traverso N, Gambini J, Domenicotti C, Marinari UM, Poli G, Pronzato MA, Ricciarelli R: Cholesterol and amyloid-beta: evidence for a cross-talk between astrocytes and neuronal cells. J Alzheimers Dis 2011, 25:645e653 35. Wolf A, Bauer B, Hartz AM: ABC transporters and the Alzheimer’s disease enigma. Front Psychiatry 2012, 3:54 36. Akram A, Schmeidler J, Katsel P, Hof PR, Haroutunian V: Increased expression of cholesterol transporter ABCA1 is highly correlated with severity of dementia in AD hippocampus. Brain Res 2010, 1318: 167e177

The American Journal of Pathology

-

37. Kim WS, Bhatia S, Elliott DA, Agholme L, Kagedal K, McCann H, Halliday GM, Barnham KJ, Garner B: Increased ATP-binding cassette transporter A1 expression in Alzheimer’s disease hippocampal neurons. J Alzheimers Dis 2010, 21:193e205 38. Rone MB, Fan J, Papadopoulos V: Cholesterol transport in steroid biosynthesis: role of protein-protein interactions and implications in disease states. Biochim Biophys Acta 2009, 1791:646e658 39. Hayashi T, Rizzuto R, Hajnoczky G, Su TP: MAM: more than just a housekeeper. Trends Cell Biol 2009, 19:81e88 40. Hayashi T, Fujimoto M: Detergent-resistant microdomains determine the localization of sigma-1 receptors to the endoplasmic reticulummitochondria junction. Mol Pharmacol 2010, 77:517e528 41. Marriott KS, Prasad M, Thapliyal V, Bose HS: Sigma-1 receptor at the mitochondrial-associated endoplasmic reticulum membrane is responsible for mitochondrial metabolic regulation. J Pharmacol Exp Ther 2012, 343:578e586 42. Area-Gomez E, Del Carmen Lara Castillo M, Tambini MD, GuardiaLaguarta C, de Groof AJ, Madra M, Ikenouchi J, Umeda M, Bird TD, Sturley SL, Schon EA: Upregulated function of mitochondriaassociated ER membranes in Alzheimer disease. EMBO J 2012, 31: 4106e4123 43. Hedskog L, Pinho CM, Filadi R, Ronnback A, Hertwig L, Wiehager B, Larssen P, Gellhaar S, Sandebring A, Westerlund M, Graff C, Winblad B, Galter D, Behbahani H, Pizzo P, Glaser E, Ankarcrona M: Modulation of the endoplasmic reticulummitochondria interface in Alzheimer’s disease and related models. Proc Natl Acad Sci U S A 2013, 110:7916e7921 44. Simmen T, Aslan JE, Blagoveshchenskaya AD, Thomas L, Wan L, Xiang Y, Feliciangeli SF, Hung CH, Crump CM, Thomas G: PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis. EMBO J 2005, 24:717e729 45. de Brito OM, Scorrano L: Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 2008, 456:605e610 46. Campbell AM, Chan SH: The voltage dependent anion channel affects mitochondrial cholesterol distribution and function. Arch Biochem Biophys 2007, 466:203e210 47. Ye J, Rawson RB, Komuro R, Chen X, Dave UP, Prywes R, Brown MS, Goldstein JL: ER stress induces cleavage of membranebound ATF6 by the same proteases that process SREBPs. Mol Cell 2000, 6:1355e1364 48. Zeng L, Lu M, Mori K, Luo S, Lee AS, Zhu Y, Shyy JY: ATF6 modulates SREBP2-mediated lipogenesis. EMBO J 2004, 23: 950e958 49. Charman M, Kennedy BE, Osborne N, Karten B: MLN64 mediates egress of cholesterol from endosomes to mitochondria in the absence of functional Niemann-Pick Type C1 protein. J Lipid Res 2010, 51: 1023e1034 50. Rosati F, Sturli N, Cungi MC, Morello M, Villanelli F, Bartolucci G, Finocchi C, Peri A, Serio M, Danza G: Gonadotropin-releasing hormone modulates cholesterol synthesis and steroidogenesis in SHSY5Y cells. J Steroid Biochem Mol Biol 2011, 124:77e83 51. Mari M, Morales A, Colell A, Garcia-Ruiz C, Fernandez-Checa JC: Mitochondrial glutathione, a key survival antioxidant. Antioxid Redox Signal 2009, 11:2685e2700 52. Chan RB, Oliveira TG, Cortes EP, Honig LS, Duff KE, Small SA, Wenk MR, Shui G, Di Paolo G: Comparative lipidomic analysis of mouse and human brain with Alzheimer disease. J Biol Chem 2012, 287:2678e2688 53. Brown J 3rd, Theisler C, Silberman S, Magnuson D, GottardiLittell N, Lee JM, Yager D, Crowley J, Sambamurti K, Rahman MM, Reiss AB, Eckman CB, Wolozin B: Differential expression of cholesterol hydroxylases in Alzheimer’s disease. J Biol Chem 2004, 279:34674e34681 54. Heverin M, Bogdanovic N, Lutjohann D, Bayer T, Pikuleva I, Bretillon L, Diczfalusy U, Winblad B, Bjorkhem I: Changes in the levels of cerebral and extracerebral sterols in the brain of patients with Alzheimer’s disease. J Lipid Res 2004, 45:186e193

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1861 1862 1863 1864 1865 1866 1867 1868 1869 1870 1871 1872 1873 1874 1875 1876 1877 1878 1879 1880 1881 1882 1883 1884 1885 1886 1887 1888 1889 1890 1891 1892 1893 1894 1895 1896 1897 1898 1899 1900 1901 1902 1903 1904 1905 1906 1907 1908 1909 1910 1911 1912 1913 1914 1915 1916 1917 1918 1919 1920 1921 1922

Barbero-Camps et al 55. Bosch M, Mari M, Herms A, Fernandez A, Fajardo A, Kassan A, Giralt A, Colell A, Balgoma D, Barbero E, Gonzalez-Moreno E, Matias N, Tebar F, Balsinde J, Camps M, Enrich C, Gross SP, GarciaRuiz C, Perez-Navarro E, Fernandez-Checa JC, Pol A: Caveolin-1 deﬁciency causes cholesterol-dependent mitochondrial dysfunction and apoptotic susceptibility. Curr Biol 2011, 21:681e686 56. Webber KM, Stocco DM, Casadesus G, Bowen RL, Atwood CS, Previll LA, Harris PL, Zhu X, Perry G, Smith MA: Steroidogenic acute regulatory protein (StAR): evidence of gonadotropin-induced steroidogenesis in Alzheimer disease. Mol Neurodegener 2006, 1:14 57. Kil IS, Lee SK, Ryu KW, Woo HA, Hu MC, Bae SH, Rhee SG: Feedback control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in mitochondria. Mol Cell 2012, 46:584e594 58. Caron KM, Soo SC, Wetsel WC, Stocco DM, Clark BJ, Parker KL: Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia. Proc Natl Acad Sci U S A 1997, 94:11540e11545 59. Sano R, Annunziata I, Patterson A, Moshiach S, Gomero E, Opferman J, Forte M, d’Azzo A: GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2þ)-dependent mitochondrial apoptosis. Mol Cell 2009, 36: 500e511 60. Shioda N, Ishikawa K, Tagashira H, Ishizuka T, Yawo H, Fukunaga K: Expression of a truncated form of the endoplasmic reticulum chaperone protein, sigma1 receptor, promotes mitochondrial energy depletion and apoptosis. J Biol Chem 2012, 287: 23318e23331 61. Rodriguez-Agudo D, Ren S, Wong E, Marques D, Redford K, Gil G, Hylemon P, Pandak WM: Intracellular cholesterol transporter StarD4 binds free cholesterol and increases cholesteryl ester formation. J Lipid Res 2008, 49:1409e1419 62. Korytowski W, Rodriguez-Agudo D, Pilat A, Girotti AW: StarD4mediated translocation of 7-hydroperoxycholesterol to isolated mitochondria: deleterious effects and implications for steroidogenesis under oxidative stress conditions. Biochem Biophys Res Commun 2010, 392:58e62 63. Soccio RE, Adams RM, Maxwell KN, Breslow JL: Differential gene regulation of StarD4 and StarD5 cholesterol transfer proteins. Activation of StarD4 by sterol regulatory element-binding protein-2 and StarD5 by endoplasmic reticulum stress. J Biol Chem 2005, 280: 19410e19418 64. Rodriguez-Agudo D, Ren S, Hylemon PB, Redford K, Natarajan R, Del Castillo A, Gil G, Pandak WM: Human StarD5, a cytosolic StAR-related lipid binding protein. J Lipid Res 2005, 46:1615e1623 65. Rodriguez-Agudo D, Calderon-Dominguez M, Medina MA, Ren S, Gil G, Pandak WM: ER stress increases StarD5 expression by

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66. 67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

stabilizing its mRNA and leads to relocalization of its protein from the nucleus to the membranes. J Lipid Res 2012, 53:2708e2715 Zheng Z, Zhang C, Zhang K: Role of unfolded protein response in lipogenesis. World J Hepatol 2010, 2:203e207 Fernandez A, Matias N, Fucho R, Ribas V, Von Montfort C, Nuno N, Baulies A, Martinez L, Tarrats N, Mari M, Colell A, Morales A, Dubuquoy L, Mathurin P, Bataller R, Caballeria J, Elena M, Balsinde J, Kaplowitz N, Garcia-Ruiz C, Fernandez-Checa JC: ASMase is required for chronic alcohol induced hepatic endoplasmic reticulum stress and mitochondrial cholesterol loading. J Hepatol 2013, 59:805e813 Mari M, Morales A, Colell A, Garcia-Ruiz C, Kaplowitz N, Fernandez-Checa JC: Mitochondrial glutathione: features, regulation and role in disease. Biochim Biophys Acta 2013, 1830:3317e3328 Katayama T, Imaizumi K, Sato N, Miyoshi K, Kudo T, Hitomi J, Morihara T, Yoneda T, Gomi F, Mori Y, Nakano Y, Takeda J, Tsuda T, Itoyama Y, Murayama O, Takashima A, St GeorgeHyslop P, Takeda M, Tohyama M: Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response. Nat Cell Biol 1999, 1:479e485 Ricobaraza A, Cuadrado-Tejedor M, Marco S, Perez-Otano I, GarciaOsta A: Phenylbutyrate rescues dendritic spine loss associated with memory deﬁcits in a mouse model of Alzheimer disease. Hippocampus 2012, 22:1040e1050 Ricobaraza A, Cuadrado-Tejedor M, Perez-Mediavilla A, Frechilla D, Del Rio J, Garcia-Osta A: Phenylbutyrate ameliorates cognitive deﬁcit and reduces tau pathology in an Alzheimer’s disease mouse model. Neuropsychopharmacology 2009, 34:1721e1732 Wiley JC, Pettan-Brewer C, Ladiges WC: Phenylbutyric acid reduces amyloid plaques and rescues cognitive behavior in AD transgenic mice. Aging Cell 2011, 10:418e428 Shafaati M, O’Driscoll R, Bjorkhem I, Meaney S: Transcriptional regulation of cholesterol 24-hydroxylase by histone deacetylase inhibitors. Biochem Biophys Res Commun 2009, 378:689e694 Pipalia NH, Cosner CC, Huang A, Chatterjee A, Bourbon P, Farley N, Helquist P, Wiest O, Maxﬁeld FR: Histone deacetylase inhibitor treatment dramatically reduces cholesterol accumulation in Niemann-Pick type C1 mutant human ﬁbroblasts. Proc Natl Acad Sci U S A 2011, 108:5620e5625 Kim SJ, Lee BH, Lee YS, Kang KS: Defective cholesterol trafﬁc and neuronal differentiation in neural stem cells of Niemann-Pick type C disease improved by valproic acid, a histone deacetylase inhibitor. Biochem Biophys Res Commun 2007, 360:593e599 Yoon SO, Park DJ, Ryu JC, Ozer HG, Tep C, Shin YJ, Lim TH, Pastorino L, Kunwar AJ, Walton JC, Nagahara AH, Lu KP, Nelson RJ, Tuszynski MH, Huang K: JNK3 perpetuates metabolic stress induced by Abeta peptides. Neuron 2012, 75:824e837

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Supplemental Figure S1

Increased cholesterol levels and depleted mGSH content in 10-month-old APP/PS1 mice. Total cholesterol (A) and GSH (B) levels of brain homogenate and isolated mitochondria from WT and APP/PS1 transgenic mice at the indicated ages. **P < 0.01 versus WT mice values by unpaired two-tailed Student’s t-test (n Z 6 to 8). mth, month.

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Supplemental Figure S2 Mitochondria isolated from brains of WT and APP/PS1 mice. After cellular subfraction, the purity of mitochondria was conﬁrmed by determining in homogenate (Homo.), ER, or mitochondria (Mito.) the distribution of speciﬁc markers such as PERK and calreticulin (ER), Naþ/KþATPase a1 (plasma membrane), Rab5a (early endosomes), Rab7 (late endosomes/lysosomes), and cyclooxygenase 4 (COX-IV; mitochondria) using immunoblot analysis. The densitometric values of the bands representing the speciﬁc antibody immunoreactivity were expressed as relative intensity compared with homogenate values (n Z 3). Supplemental Figure S3 Stable expression levels of cholesterol-related proteins in WT mice. Representative immunoblots of SREBP-2 and HMG-CoA reductase (HMGCR) (A) and the cholsterol carriers StAR and StARD3 (B) in brain extracts from WT and APP/PS1 mice at the indicated ages. The densitometric values of the bands representing the speciﬁc antibody immunoreactivity were normalized to the values of the corresponding b-actin band and expressed as relative intensity compared with 4-month-old WT mice values. **P < 0.01 versus 4-month-old WT mice values by one-way analysis of variance with the Dunnett post hoc test (n Z 3). mth, month. Supplemental Figure S4 Undetectable signs of UPR response in WT mice. A and B: Representative immunoblots showing the expression of the chaperonic protein GRP78 and the ER stress signaling pathway proteins PERK, phospho-PERK, eIF2a, phospho-eIF2a, and CHOP in brain extracts from WT and APP/PS1 (A/P) mice at the indicated ages. The densitometric values of the bands representing the speciﬁc antibody immunoreactivity were normalized to the values of the corresponding b-actin band and expressed as relative intensity compared with WT mice values. *P < 0.05 versus 4-month-old mice values; y P < 0.05 versus WT mice values by one-way analysis of variance with the Dunnett post hoc test (n Z 3). mth, month. Supplemental Figure S5 Age-dependent Ab accumulation in APP/PS1 mice. Representative photomicrographs of hippocampus labeled with human antieAb1-42 and counterstained with hematoxylin (n Z 3). Scale bar Z 100 mm.

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Endoplasmic Reticulum Stress Mediates Amyloid β Neurotoxicity via Mitochondrial Cholesterol Trafficking

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