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Autophagy-Related Protein 7 Deficiency in Amyloid β (Aβ) Precursor Protein Transgenic Mice Decreases Aβ in the Multivesicular Bodies and Induces Aβ Accumulation in the Golgi

2014; Elsevier BV; Volume: 185; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2014.10.011

1525-2191

Per Nilsson, Misaki Sekiguchi, Takumi Akagi, Shinichi Izumi, Toshihisa Komori, Kelvin K. Hui, Karin Sörgjerd, Motomasa Tanaka, Takashi Saito, Nobuhisa Iwata, Takaomi C. Saido,

Parkinson's Disease Mechanisms and Treatments

Alzheimer disease (AD) is biochemically characterized by increased levels of amyloid β (Aβ) peptide, which aggregates into extracellular Aβ plaques in AD brains. Before plaque formation, Aβ accumulates intracellularly in both AD brains and in the brains of AD model mice, which may contribute to disease progression. Autophagy, which is impaired in AD, clears cellular protein aggregates and participates in Aβ metabolism. In addition to a degradative role of autophagy in Aβ metabolism we recently showed that Aβ secretion is inhibited in mice lacking autophagy-related gene 7 (Atg7) in excitatory neurons in the mouse forebrain. This inhibition of Aβ secretion leads to intracellular accumulation of Aβ. Here, we used fluorescence and immunoelectron microscopy to elucidate the subcellular localization of the intracellular Aβ accumulation which accumulates in Aβ precursor protein mice lacking Atg7. Autophagy deficiency causes accumulation of p62+ aggregates, but these aggregates do not contain Aβ. However, knockdown of Atg7 induced Aβ accumulation in the Golgi and a concomitant reduction of Aβ in the multivesicular bodies. This indicates that Atg7 influences the transport of Aβ possibly derived from Golgi to multivesicular bodies. Alzheimer disease (AD) is biochemically characterized by increased levels of amyloid β (Aβ) peptide, which aggregates into extracellular Aβ plaques in AD brains. Before plaque formation, Aβ accumulates intracellularly in both AD brains and in the brains of AD model mice, which may contribute to disease progression. Autophagy, which is impaired in AD, clears cellular protein aggregates and participates in Aβ metabolism. In addition to a degradative role of autophagy in Aβ metabolism we recently showed that Aβ secretion is inhibited in mice lacking autophagy-related gene 7 (Atg7) in excitatory neurons in the mouse forebrain. This inhibition of Aβ secretion leads to intracellular accumulation of Aβ. Here, we used fluorescence and immunoelectron microscopy to elucidate the subcellular localization of the intracellular Aβ accumulation which accumulates in Aβ precursor protein mice lacking Atg7. Autophagy deficiency causes accumulation of p62+ aggregates, but these aggregates do not contain Aβ. However, knockdown of Atg7 induced Aβ accumulation in the Golgi and a concomitant reduction of Aβ in the multivesicular bodies. This indicates that Atg7 influences the transport of Aβ possibly derived from Golgi to multivesicular bodies. Alzheimer disease (AD) is the most common form of dementia in the elderly. Biochemical hallmarks of AD include aggregation of intracellular Tau protein in neurofibrillary tangles and extracellular plaques that consist of amyloid β (Aβ) peptide. Aβ is generated by sequential cleavage of Aβ precursor protein (APP) by β- and γ-secretase. Mutations in APP or the in the genes encoding the enzymatic components of γ-secretase, PSEN1 and PSEN2, lead to increased levels of Aβ, especially the aggregation-prone Aβ42 and Aβ43, and cause aggressive early-onset familial AD.1Haass C. Kaether C. Thinakaran G. Sisodia S. Trafficking and Proteolytic Processing of APP.Cold Spring Harb Perspect Med. 2012; 2: a006270Crossref Scopus (685) Google Scholar, 2Saito T. Suemoto T. Brouwers N. Sleegers K. Funamoto S. Mihira N. Matsuba Y. Yamada K. Nilsson P. Takano J. Nishimura M. Iwata N. Van Broeckhoven C. Ihara Y. Saido T.C. Potent amyloidogenicity and pathogenicity of Aβ43.Nat Neurosci. 2011; 14: 1023-1032Crossref PubMed Scopus (204) Google Scholar Before extracellular Aβ plaque formation, Aβ accumulates intracellularly in AD brains and in the brains of AD model mice, which may contribute to disease progression.3LaFerla F.M. Green K.N. Oddo S. Intracellular Aβ in Alzheimer's disease.Nat Rev Neurosci. 2007; 8: 499-509Crossref PubMed Scopus (1567) Google Scholar, 4Wirths O. Bayer T.A. Intraneuronal Aβ accumulation and neurodegeneration: lessons from transgenic models.Life Sci. 2012; 91: 1148-1152Crossref PubMed Scopus (65) Google Scholar Transgene expression in mice of human ApoE4, which is a risk factor for AD, increases intraneuronal Aβ.5Zhao W. Dumanis S.B. Tamboli I.Y. Rodriguez G.A. Jo LaDu M. Moussa C.E. William Rebeck G. Human APOE genotype affects intraneuronal Aβ1–42 accumulation in a lentiviral gene transfer model.Hum Mol Genet. 2013; 23: 1365-1375Crossref PubMed Scopus (24) Google Scholar Targeting Aβ42 to the endoplasmic reticulum (ER) induces neurodegeneration, suggesting that intraneuronal Aβ may be toxic.6Abramowski D. Rabe S. Upadhaya A.R. Reichwald J. Danner S. Staab D. Capetillo-Zarate E. Yamaguchi H. Saido T.C. Wiederhold K.H. Thal D.R. Staufenbiel M. Transgenic expression of intraneuronal Aβ42 but not Aβ40 leads to cellular Aβ lesions, degeneration, and functional impairment without typical Alzheimer's disease pathology.J Neurosci. 2012; 32: 1273-1283Crossref PubMed Scopus (35) Google Scholar However, the mechanistic link between Aβ pathology and the extensive loss of synapses and neurons that occurs in AD remains largely unresolved. AD brains also exhibit an accumulation of autophagosomes in the dystrophic neurites, indicating that autophagy is deregulated in AD.7Nixon R.A. Wegiel J. Kumar A. Yu W.H. Peterhoff C. Cataldo A. Cuervo A.M. Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study.J Neuropathol Exp Neurol. 2005; 64: 113-122Crossref PubMed Scopus (1145) Google Scholar Macroautophagy (herein referred to as autophagy) is a cellular quality control system that degrades aberrant organelles and potentially toxic protein aggregates by delivering them to the lysosomes for degradation, reviewed by Nixon.8Nixon R.A. The role of autophagy in neurodegenerative disease.Nat Med. 2013; 19: 983-997Crossref PubMed Scopus (1334) Google Scholar The increased levels and abnormal distribution of lysosomal hydrolases in AD brains and in the brains of AD mouse models indicate that the end stage of autophagosomal clearance is impaired.9Cataldo A.M. Paskevich P.A. Kominami E. Nixon R.A. Lysosomal hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease.Proc Natl Acad Sci U S A. 1991; 88: 10998-11002Crossref PubMed Scopus (243) Google Scholar, 10Yang D.S. Stavrides P. Mohan P.S. Kaushik S. Kumar A. Ohno M. Schmidt S.D. Wesson D. Bandyopadhyay U. Jiang Y. Pawlik M. Peterhoff C.M. Yang A.J. Wilson D.A. St George-Hyslop P. Westaway D. Mathews P.M. Levy E. Cuervo A.M. Nixon R.A. Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits.Brain. 2011; 134: 258-277Crossref PubMed Scopus (353) Google Scholar Consistent with these reports, familial AD-associated mutations in PSEN1 impair lysosomal proteolysis,11Lee J.H. Yu W.H. Kumar A. Lee S. Mohan P.S. Peterhoff C.M. Wolfe D.M. Martinez-Vicente M. Massey A.C. Sovak G. Uchiyama Y. Westaway D. Cuervo A.M. Nixon R.A. Lysosomal proteolysis and autophagy require presenilin 1 and are disrupted by Alzheimer-related PS1 mutations.Cell. 2010; 141: 1146-1158Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar and pharmacologic inhibition of lysosomes in cultured neurons causes an AD-like accumulation of autophagosomes.12Boland B. Kumar A. Lee S. Platt F.M. Wegiel J. Yu W.H. Nixon R.A. Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease.J Neurosci. 2008; 28: 6926-6937Crossref PubMed Scopus (862) Google Scholar Autophagosomes contain Aβ-generating enzymes and Aβ,13Yu W.H. Cuervo A.M. Kumar A. Peterhoff C.M. Schmidt S.D. Lee J.H. Mohan P.S. Mercken M. Farmery M.R. Tjernberg L.O. Jiang Y. Duff K. Uchiyama Y. Näslund J. Mathews P.M. Cataldo A.M. Nixon R.A. Macroautophagy—a novel β-amyloid peptide-generating pathway activated in Alzheimer's disease.J Cell Biol. 2005; 171: 87-98Crossref PubMed Scopus (818) Google Scholar thus linking autophagy to Aβ metabolism.14Boland B. Smith D.A. Mooney D. Jung S.S. Walsh D.M. Platt F.M. Macroautophagy is not directly involved in the metabolism of amyloid precursor protein.J Biol Chem. 2010; 285: 37415-37426Crossref PubMed Scopus (77) Google Scholar, 15Jaeger P.A. Pickford F. Sun C.H. Lucin K.M. Masliah E. Wyss-Coray T. Regulation of amyloid precursor protein processing by the Beclin 1 complex.PLoS One. 2010; 5: e11102Crossref PubMed Scopus (168) Google Scholar Rapamycin treatment induces autophagy that decreases intracellular Aβ and extracellular Aβ plaque load and improves cognition in AD model mice.16Caccamo A. Majumder S. Richardson A. Strong R. Oddo S. Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-β, and Tau: effects on cognitive impairments.J Biol Chem. 2010; 285: 13107-13120Crossref PubMed Scopus (691) Google Scholar, 17Majumder S. Richardson A. Strong R. Oddo S. Inducing autophagy by rapamycin before, but not after, the formation of plaques and tangles ameliorates cognitive deficits.PLoS One. 2011; 6: e25416Crossref PubMed Scopus (305) Google Scholar Conversely, heterozygous deletion of autophagy-initiating Beclin1 increases intracellular and extracellular Aβ.18Pickford F. Masliah E. Britschgi M. Lucin K. Narasimhan R. Jaeger P.A. Small S. Spencer B. Rockenstein E. Levine B. Wyss-Coray T. The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid β accumulation in mice.J Clin Invest. 2008; 118: 2190-2199PubMed Google Scholar, 19Salminen A. Kaarniranta K. Kauppinen A. Ojala J. Haapasalo A. Soininen H. Hiltunen M. Impaired autophagy and APP processing in Alzheimer's disease: the potential role of Beclin 1 interactome.Prog Neurobiol. 2013; 106–107: 33-54Crossref PubMed Scopus (242) Google Scholar Autophagy also influences the secretion of Aβ via a nondegradative route shown by genetic deletion of autophagy-related protein 7 (Atg7) in APP transgenic mice.20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar The inhibition of Aβ secretion leads to drastically decreased extracellular Aβ deposits.20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar Concomitantly, Aβ accumulates intracellularly which exacerbates the neurodegeneration caused by impaired autophagy and severely impairs the memory.20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar This finding adds Aβ to a growing number of proteins, the secretion of which is mediated by autophagy.21Nilsson P. Saido T.C. Dual roles for autophagy: degradation and secretion of Alzheimer's disease Aβ peptide.Bioessays. 2014; 36: 570-578Crossref PubMed Scopus (127) Google Scholar Here, we investigated by immunofluorescence and immunoelectron microscopy (IEM) the secretory pathway of Aβ by tracing the intracellular Aβ that accumulates in Atg7-deficient APP transgenic mice. We found that genetic deletion of Atg7 decreased Aβ in the multivesicular bodies (MVBs) and induced Aβ accumulation in the Golgi, suggesting that autophagy may mediate the trafficking of Aβ between the Golgi and the MVBs and that Aβ secretion to some extent occurs through the MVBs. Atg7flox/flox mice22Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. Ezaki J. Mizushima N. Ohsumi Y. Uchiyama Y. Kominami E. Tanaka K. Chiba T. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1911) Google Scholar were provided by Keiji Tanaka (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), and calcium/calmodulin-dependent protein kinase II (CaMKII)-Cre mice23Tsien J.Z. Chen D.F. Gerber D. Tom C. Mercer E.H. Anderson D.J. Mayford M. Kandel E.R. Tonegawa S. Subregion- and cell type-restricted gene knockout in mouse brain.Cell. 1996; 87: 1317-1326Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar were provided by Shigeyoshi Itohara (RIKEN Brain Science Institute, Wako, Japan). APP23 mice have been described previously.24Sturchler-Pierrat C. Abramowski D. Duke M. Wiederhold K.H. Mistl C. Rothacher S. Ledermann B. Bürki K. Frey P. Paganetti P.A. Waridel C. Calhoun M.E. Jucker M. Probst A. Staufenbiel M. Sommer B. Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology.Proc Natl Acad Sci U S A. 1997; 94: 13287-13292Crossref PubMed Scopus (1248) Google Scholar All mice were on C57BL/6 background. Atg7flox/flox × APP mice and Atg7flox/flox;CaMKII-Cre × APP mice used in the experiments were littermates. All animal experiments were conducted according to the guidelines of RIKEN Brain Science Institute. Paraffin-embedded brain sections were cut to 4 μm and immunostained with antibodies against N1D-Aβ,25Saido T.C. Yokota M. Maruyama K. Yamao-Harigaya W. Tani E. Ihara Y. Kawashima S. Spatial resolution of the primary beta-amyloidogenic process induced in postischemic hippocampus.J Biol Chem. 1994; 269: 15253-15257Abstract Full Text PDF PubMed Google Scholar Aβ40 (no. 44348A; Invitrogen, Carlsbad, CA), APP–C-terminal fragment (CTF; no. A8717; Sigma-Aldrich, St. Louis, MO), p62 (no. GP62-C; Cell Signaling Technology Inc., Danvers, MA), vimentin (developed by Arturo Alvarez-Buylla and obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by Department of Biology, The University of Iowa, Iowa City, IA), and cleaved caspase 3 (no. 9961; Cell Signaling Technology Inc.). For Aβ immunostaining, tissue sections were treated with 90% formic acid for 5 minutes. Quantification was performed with MetaMorph imaging software version 7.7.3.0 (Universal Imaging, Bedford Hills, NY). Cortical/hippocampal neurons were prepared from mouse embryos (embryonic day 17 to 18) as previously described.26Hama E. Shirotani K. Masumoto H. Sekine-Aizawa Y. Aizawa H. Saido T.C. Clearance of extracellular and cell-associated amyloid β peptide through viral expression of neprilysin in primary neurons.J Biochem. 2001; 130: 721-726Crossref PubMed Scopus (80) Google Scholar Embryos were separately genotyped. Vital cells (1.8 × 105) were plated in 24-well plates with poly-l-lysine–coated coverslips. Primary neurons were infected with Semliki Forest virus-APP for 24 hours, fixed for 10 minutes in cold 4% paraformaldehyde (PFA) and 0.1 mol/L phosphate buffer, washed with phosphate-buffered saline, blocked with 5% normal goat serum and 1% saponin, and incubated with antibodies N1D-Aβ,25Saido T.C. Yokota M. Maruyama K. Yamao-Harigaya W. Tani E. Ihara Y. Kawashima S. Spatial resolution of the primary beta-amyloidogenic process induced in postischemic hippocampus.J Biol Chem. 1994; 269: 15253-15257Abstract Full Text PDF PubMed Google Scholar early endosome antigen 1 (no. 7659; Sigma-Aldrich), or microtubule-associated protein 2 (no. 5622; Millipore, Billerica, MA) overnight wash buffer and antibody dilutions contained 1% saponin. Aβ40 (100 ng) and 10 μg of delipidated brain homogenates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with N1D-Aβ,25Saido T.C. Yokota M. Maruyama K. Yamao-Harigaya W. Tani E. Ihara Y. Kawashima S. Spatial resolution of the primary beta-amyloidogenic process induced in postischemic hippocampus.J Biol Chem. 1994; 269: 15253-15257Abstract Full Text PDF PubMed Google Scholar Aβ40 (no. 44348A; Invitrogen), 6E10 (no. SIG-39320; Covance Inc., Princeton, NJ), and APP-CTF (no. A8717; Sigma-Aldrich) antibodies. For visualization of Golgi-localized Aβ, brains were perfused, fixed for 2 hours in 4% PFA and 0.25% glutaraldehyde, and cut into 200-μm sections. Brain sections were incubated with 10%, 20%, and 30% sucrose; frozen; and thawed three times in liquid N2. The brain sections were blocked in 10% bovine serum albumin, 10% normal goat serum, and 0.1% gelatin; incubated with N1D-Aβ25Saido T.C. Yokota M. Maruyama K. Yamao-Harigaya W. Tani E. Ihara Y. Kawashima S. Spatial resolution of the primary beta-amyloidogenic process induced in postischemic hippocampus.J Biol Chem. 1994; 269: 15253-15257Abstract Full Text PDF PubMed Google Scholar antibody overnight; washed with phosphate-buffered saline that contained 0.1% gelatin; and incubated with 10 nm of nano-gold–conjugated secondary antibodies. After immunostaining, the sections were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer for 1 hour at room temperature, washed with distilled H2O, and intensified with the GoldEnhance EM (Nanoprobes, Yaphank, NY) to visualize the nano-gold labeling. After the intensification, the sections were osmicated with 1% OsO4 and 1.5% potassium ferrocyanide in 0.1 mol/L phosphate buffer for 1 hour at room temperature, dehydrated through a gradient series of ethanol concentrations, and embedded in epoxy resin (Epon 812; TAAB Laboratories Equipment Ltd., Berkshire, UK) by polymerization. Ultrathin sections (80 nm) were cut with an ultramicrotome (Ultracut UCT; Leica Microsystems, Wetzlar, Germany) and collected on copper grids. After counterstaining with uranyl acetate and lead citrate, the sections were examined by electron microscopy (Tecnai 12; FEI, Eindhoven, the Netherlands). For visualization of Aβ in MVBs, the procedure was performed as described previously.27Goto S. Kawakatsu M. Izumi S. Urata Y. Kageyama K. Ihara Y. Koji T. Kondo T. Glutathione S-transferase π localizes in mitochondria and protects against oxidative stress.Free Radic Biol Med. 2009; 46: 1392-1403Crossref PubMed Scopus (65) Google Scholar Briefly, brains were perfused with 4% PFA, incubated overnight, and cut into 200-μm thick sections. The sections were embedded in LR white resin, cut into ultrathin sections, mounted on nickel grids, and incubated with anti–N1D-Aβ25Saido T.C. Yokota M. Maruyama K. Yamao-Harigaya W. Tani E. Ihara Y. Kawashima S. Spatial resolution of the primary beta-amyloidogenic process induced in postischemic hippocampus.J Biol Chem. 1994; 269: 15253-15257Abstract Full Text PDF PubMed Google Scholar antibody, followed by colloidal gold-conjugated (10 nm) goat anti-rabbit IgG. The sections were viewed in transmission electron image mode (JEOL 1200EX; JEOL Ltd, Tokyo, Japan). Data were analyzed by Student's t-test and expressed as means ± SEMs. We recently reported that autophagy influences the extracellular delivery of Aβ.20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar This was shown by using autophagy-deficient APP transgenic mice generated by crossbreeding Atg7flox/flox mice22Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. Ezaki J. Mizushima N. Ohsumi Y. Uchiyama Y. Kominami E. Tanaka K. Chiba T. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1911) Google Scholar with CaMKII-Cre transgenic mice, which were further crossbred with APP23 mice. Lack of Atg7 inhibited Aβ secretion which led to a drastically decreased Aβ plaque formation (Figure 1E).20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar In addition, immunohistochemical analysis found that Aβ accumulates intracellularly in cornu ammonis 1 (CA1) pyramidal neurons of 4-month-old autophagy-deficient Atg7flox/flox;CaMKII-Cre × APP mice compared with Atg7flox/flox × APP mice (P < 0.05) (Figure 1A). The accumulation further increases by age, as shown by immunohistochemical experiments of 9-month-old mice with the use of two different Aβ-specific antibodies (N1D-Aβ25Saido T.C. Yokota M. Maruyama K. Yamao-Harigaya W. Tani E. Ihara Y. Kawashima S. Spatial resolution of the primary beta-amyloidogenic process induced in postischemic hippocampus.J Biol Chem. 1994; 269: 15253-15257Abstract Full Text PDF PubMed Google Scholar that recognizes the N1D structure of N-terminus of monomeric and aggregated Aβ and anti-Aβ40 antibody that recognizes the C-terminus of Aβ40 of monomeric and aggregated Aβ40) (P < 0.05) (Figure 1, B–E). In contrast, APP-CTF+ staining, APP levels, and APP-CTF levels were unchanged, although the APP-CTFβ/APP-CTFα ratio was slightly decreased in Atg7flox/flox;CaMKII-Cre × APP mice (Figure 1, F and G). In agreement with previous studies,28Inoue K. Rispoli J. Kaphzan H. Klann E. Chen E.I. Kim J. Komatsu M. Abeliovich A. Macroautophagy deficiency mediates age-dependent neurodegeneration through a phospho-tau pathway.Mol Neurodegener. 2012; 7: 48-60Crossref PubMed Scopus (121) Google Scholar autophagy deficiency in the CA1 neurons also leads to accumulation of protein aggregates that contain autophagic adaptor protein p62 (Figure 2A). Coimmunostaining experiments found that the p62+ aggregates contain vimentin, suggesting that these structures are aggresomes (Figure 2, A and B). In contrast, the p62+ aggregates did not colocalize with Aβ, indicating that the site of intracellular Aβ buildup is not associated with these intracellular aggregates (Figure 2, C and D). Furthermore, cleavage of caspase 3 is detected in Atg7flox/flox;CaMKII-Cre mice, which is exacerbated in the Atg7flox/flox;CaMKII-Cre × APP mice (Figure 2E). The presence of cleaved caspase 3 suggests an onset of apoptotic processes, in agreement with previous data which indicated a significant neurodegeneration in 15-month-old Atg7flox/flox;CaMKII-Cre and Atg7flox/flox;CaMKII-Cre × APP mice.20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar To get an insight into how the Aβ secretory pathway is affected in the autophagy-deficient Atg7flox/flox;CaMKII-Cre × APP mice, we investigated the subcellular localization of the intracellular Aβ accumulation that occurs in the brains of 9-month-old mice by IEM. To establish conditions for IEM that preserve the ultrastructure of the tissue but partially maintain the antigenicity of Aβ, the effect of different concentrations of glutaraldehyde and different time of tissue fixation was investigated. The ultrastructure of the brain tissue was well preserved in a fixation method that was based on 4% PFA and 0.25% glutaraldehyde (see Materials and Methods), which allowed structural identification of the cellular organelles, including the aggregates in the perinuclear region (Figure 3A). With the use of this fixation protocol, we localized Aβ by immunolabeling brain tissue from an APP knockout mouse and Atg7flox/flox × APP and Atg7flox/flox;CaMKII-Cre × APP littermates with Aβ antibodies and gold-labeled secondary antibodies and analyzed the samples by IEM. Even though the gold labeling was sparse, signal was detected in the Golgi and the MVBs in the pyramidal cells in CA1 (Figure 3B). By studying numerous CA1 pyramidal cells, a significant increase of gold-labeled Aβ in the Golgi was observed in the autophagy-deficient CA1 neurons of Atg7flox/flox;CaMKII-Cre × APP mice compared with Atg7flox/flox × APP mice (n > 40; P < 0.005) (Figure 3C). Immunogold-labeled Aβ was found at the cis- and trans-face of the Golgi and was associated with both the membrane of formed vesicles in the late Golgi apparatus and the cisternae. Consistent with the immunofluorescence data (Figure 2, C and D), no Aβ was detected in the fibrillary aggregates in the Atg7flox/flox;CaMKII-Cre × APP mice (Figure 3A). In an attempt to increase the antigenicity of Aβ, and thereby the IEM signal, we applied a milder fixation protocol that was based on only 4% PFA. Although this protocol did not fully preserve the ultrastructure of the ER and the Golgi, as in the glutaraldehyde-based fixation method, some structures within neurites were indeed preserved, including the MVBs (Figure 4A). Previous reports have found that the MVBs contain significant amounts of Aβ.29Takahashi R.H. Milner T.A. Li F. Nam E.E. Edgar M.A. Yamaguchi H. Beal M.F. Xu H. Greengard P. Gouras G.K. Intraneuronal Alzheimer aβ42 accumulates in multivesicular bodies and is associated with synaptic pathology.Am J Pathol. 2002; 161: 1869-1879Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar Therefore, we used this fixation method and gold-labeled Aβ in brain tissue from Atg7flox/flox × APP and Atg7flox/flox;CaMKII-Cre × APP littermates and subjected the samples to IEM. In agreement with earlier studies, gold-labeled Aβ decorated the membranes of internalized vesicles in the MVBs in neurites of Atg7flox/flox × APP mice (Figure 4, A and B). In contrast, the Aβ+ signal in the MVBs in the neurites of the autophagy-deficient Atg7flox/flox;CaMKII-Cre × APP mice was significantly decreased (P < 0.05) (Figure 4, A and B). To further study the effect of Atg7 deficiency on Aβ localization, primary neurons derived from Atg7flox/flox and Atg7flox/flox;Nes-Cre mice were infected with Semliki Forest virus that expressed wild-type APP and subsequently stained for Aβ. Interestingly, Atg7 deficiency induced accumulation of Aβ in the perinuclear region, whereas less Aβ localized to the neurites (Figure 4, C and D). The punctuate Aβ staining in the neurites of autophagy-competent neurons was identified as endosomes, the Aβ content of which was decreased in the Atg7-deficient neurons (Figure 4E). Together, these data indicate that Atg7 influences the sorting of Aβ in the cell and that autophagosomes may be involved in the transport of Aβ between the Golgi and the MVBs. The accumulation of autophagosomes in the dystrophic neurites of AD brains shows that autophagy is dysregulated in AD, although whether it is because of increased initiation of autophagy or decreased lysosomal clearance remains to be established. In addition, a number of studies have associated autophagy with two of the biochemical hallmarks of AD: Aβ and Tau.30Lee M.J. Lee J.H. Rubinsztein D.C. Tau degradation: the ubiquitin–proteasome system versus the autophagy-lysosome system.Prog Neurobiol. 2013; 105: 49-59Crossref PubMed Scopus (239) Google Scholar The link between Aβ and autophagy is supported by the direct observation that the autophagosomes contain Aβ and that manipulation of autophagy by genetic or pharmacologic means affects the Aβ pathology of AD model mice. The role of autophagy in Aβ metabolism was interpreted to be degradation of Aβ within the autophagosomal-lysosomal network. However, we recently extended the knowledge about the role of autophagy in Aβ metabolism by showing that Aβ secretion, and hence Aβ plaque formation, depends on autophagy.20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar Therefore, autophagy may target Aβ for either degradation or secretion. Aβ is produced from APP in two major pathways; APP translocated from the ER is processed within the Golgi from where secretory vesicles transport Aβ to the plasma membrane where Aβ is secreted or unprocessed APP located at the plasma membrane is endocytosed and Aβ is cleaved in late endosomes/MVBs.31O'Brien R.J. Wong P.C. Amyloid precursor protein processing and Alzheimer's disease.Annu Rev Neurosci. 2011; 34: 185-204Crossref PubMed Scopus (1113) Google Scholar Here, we investigated how Aβ transport and secretion were affected on genetic deletion of Atg7 by using IEM to determine the subcellular localization of intracellular Aβ that accumulates in the autophagy-deficient Atg7flox/flox;CaMKII-Cre × APP mice (Figure 1, Figure 2). Interestingly, we found that Atg7 deficiency leads to Aβ accumulation in the Golgi, whereas the levels of Aβ in the MVBs are significantly decreased (Figure 3, Figure 4). Whether this redistribution of Aβ is caused by a lack of autophagosomes per se or is because of the absence of Atg7 protein, and therefore independent of autophagosomes, remains to be elucidated. A role for autophagy in protein secretion has recently emerged (reviewed in Deretic et al32Deretic V. Jiang S. Dupont N. Autophagy intersections with conventional and unconventional secretion in tissue development, remodeling and inflammation.Trends Cell Biol. 2012; 22: 397-406Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar and Zhang and Schekman33Zhang M. Schekman R. Cell biology. Unconventional secretion, unconventional solutions.Science. 2013; 340: 559-561Crossref PubMed Scopus (127) Google Scholar). Autophagy regulates protein secretion by intersecting with conventional constitutive, unconventional, and regulated secretory pathways. Autophagy-mediated conventional secretion of interleukin 6 and 8, for example, is initiated at the TOR (target of rapamycin)-autophagy spatial coupling compartment. This compartment is closely located to the Golgi and is sensitive to Brefeldin A, which indicates a close connection of this secretory pathway to the classic constitutive ER-Golgi-plasma membrane secretory pathway.34Narita M. Young A.R. Arakawa S. Samarajiwa S.A. Nakashima T. Yoshida S. Hong S. Berry L.S. Reichelt S. Ferreira M. Tavaré S. Inoki K. Shimizu S. Narita M. Spatial coupling of mTOR and autophagy augments secretory phenotypes.Science. 2011; 332: 966-970Crossref PubMed Scopus (397) Google Scholar Autophagy also mediates the unconventional secretion of a number of cytoplasmic proteins that lack a transport signal peptide, including acetyl-coenzyme binding protein 1.35Duran J.M. Anjard C. Stefan C. Loomis W.F. Malhotra V. Unconventional secretion of Acb1 is mediated by autophagosomes.J Cell Biol. 2010; 188: 527-536Crossref PubMed Scopus (322) Google Scholar, 36Manjithaya R. Anjard C. Loomis W.F. Subramani S. Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation.J Cell Biol. 2010; 188: 537-546Crossref PubMed Scopus (267) Google Scholar Unconventional protein secretion is initiated from a recently identified structure termed omegasome. Although the current knowledge about the omegasome is limited, it is described as a membranous structure found on the ER, and its function depends on the Golgi reassembly and stacking protein. The Aβ accumulation in the Golgi induced by Atg7 deficiency indicates that the Golgi or Golgi-associated structures are involved in Aβ secretion influenced by Atg7. Indeed, it was found that Aβ is generated in the Golgi after retromer transport of endocytosed APP, a process that depends on factors involved in the formation of MVBs.37Choy R.W. Cheng Z. Schekman R. Amyloid precursor protein (APP) traffics from the cell surface via endosomes for amyloid β (Aβ) production in the trans-Golgi network.Proc Natl Acad Sci U S A. 2012; 109: E2077-E2082Crossref PubMed Scopus (170) Google Scholar Interestingly, mutations in genes involved in retromer transport, including Rab7A, have been identified in some AD patients.38Vardarajan B.N. Bruesegem S.Y. Harbour M.E. Inzelberg R. Friedland R. St George-Hyslop P.S. Seaman M.N. Farrer L.A. Identification of Alzheimer disease-associated variants in genes that regulate retromer function.Neurobiol Aging. 2012; 33: 2231.e15-2231.e30Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar This indicates that autophagy-mediated transport between the Golgi and the endosomes/MVBs may play a role in Aβ secretion. In this context, it is worth noting that the membrane source for the autophagosomes may include the ER, the Golgi, mitochondria, and the plasma membrane (reviewed in Rubinsztein et al39Rubinsztein D.C. Shpilka T. Elazar Z. Mechanisms of autophagosome biogenesis.Curr Biol. 2012; 22: R29-R34Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar) and that APP, which is a membrane-bound protein, could potentially be recruited along with the membrane on autophagosome formation. Unconventional secretion of acetyl-coenzyme binding protein 1 additionally depends on factors important for the generation of the MVBs, which indicates that the amphisome is involved in the secretion of acetyl-coenzyme binding protein 1. We found that the Aβ content in the MVBs is decreased on genetic deletion of Atg7, suggesting that Aβ is delivered to the MVBs by autophagy and may be exocytosed via amphisomes. Indeed, Aβ is released, at least to some extent, to the extracellular space in exosomes.40Rajendran L. Honsho M. Zahn T.R. Keller P. Geiger K.D. Verkade P. Simons K. Alzheimer's disease β-amyloid peptides are released in association with exosomes.Proc Natl Acad Sci U S A. 2006; 103: 11172-11177Crossref PubMed Scopus (948) Google Scholar However, autophagy also participates in regulated secretion from lysosomes, and Aβ secretion is decreased as secretion from lysosomes is decreased.41Annunziata I. Patterson A. Helton D. Hu H. Moshiach S. Gomero E. Nixon R. d'Azzo A. Lysosomal NEU1 deficiency affects amyloid precursor protein levels and amyloid-β secretion via deregulated lysosomal exocytosis.Nat Commun. 2013; 4: 2734Crossref PubMed Scopus (94) Google Scholar Further research is needed to elucidate the contribution of each of these secretory pathways to total Aβ secretion and in which pathway autophagy is involved. Interestingly, recent research has established that Tau protein is secreted, at least partially, in exosomes.42Gendreau K.L. Hall G.F. Tangles, toxicity, and tau secretion in AD – new approaches to a vexing problem.Front Neurol. 2013; 4: 160Crossref PubMed Google Scholar Moreover, Tau protein is metabolized by autophagy30Lee M.J. Lee J.H. Rubinsztein D.C. Tau degradation: the ubiquitin–proteasome system versus the autophagy-lysosome system.Prog Neurobiol. 2013; 105: 49-59Crossref PubMed Scopus (239) Google Scholar; therefore, a role for autophagy in Tau secretion cannot be excluded. Intracellular Aβ accumulation in the late endosomes, which are closely related to the MVBs, has been found both in AD brain and in the brains of AD model mice, but whether this is directly linked to increased autophagy remains to be clarified. Nevertheless, increasing evidence indicates that intracellular Aβ accumulation is toxic, a conclusion that is supported by our previous work showing that Aβ amyloidosis (including intracellular Aβ) exacerbates the neurodegeneration associated with autophagy deficiency.20Nilsson P. Loganathan K. Sekiguchi M. Matsuba Y. Hui K. Tsubuki S. Tanaka M. Iwata N. Saito T. Saido T.C. Aβ secretion and plaque formation depend on autophagy.Cell Rep. 2013; 5: 61-69Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar Therefore, understanding the Aβ secretory pathway and its interplay with autophagy is of uttermost importance. We thank Keiji Tanaka and Shigeyoshi Itohara for generously providing Atg7flox/flox and CaMKII-Cre mice, respectively, Takashi Suematsu for help with electron microscopy measurements and the following members of the PNS laboratory: Jiro Takano, Satoshi Tsubuki, Ko Sato, Kenichi Nagata, Naomasa Kakiya, Shoko Hashimoto, Hayato Isshiki, Kaori Tsukakoshi, Yukio Matsuba, Emi Hosoki, Ryo Fujioka, Naomi Yamazaki, Yuya Tomita, and Yukiko Nagai.