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Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease.

Yu WH, Cuervo AM, Kumar A, Peterhoff CM, Schmidt SD, Lee JH, Mohan PS, Mercken M, Farmery MR, Tjernberg LO, Jiang Y, Duff K, Uchiyama Y, Näslund J, Mathews PM, Cataldo AM, Nixon RA - J. Cell Biol. (2005)

Bottom Line: Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity.Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production.Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

View Article: PubMed Central - PubMed

Affiliation: Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY 10962, USA.

ABSTRACT
Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

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Related in: MedlinePlus

Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B). (C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top). (D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.
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fig4: Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B). (C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top). (D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.

Mentions: In light of these observations, we further investigated the possibilities that macroautophagy is a pathway capable of producing Aβ and that AVs are a site of Aβ generation. We used murine fibroblast-like L cells that were stably transfected with APP695 (murine L cell type + wild-type human alkaline phosphatase [L/APP]), human (SH-SY5Y), and murine (N2a) neuroblastoma cell lines to determine the effect of macroautophagy on APP metabolism. In both nonneuronal (Fig. 4 A, top, L/APP) and neuronal (Fig. 4 A, bottom, SH-SY5Y; N2a, not depicted) cell models, inducing macroautophagy via the mTOR kinase pathway (Seglen et al., 1996; Petiot et al., 2000; Kadowaki and Kanazawa, 2003) by treating cells with rapamycin, which is a specific inhibitor of mTOR phosphorylation and an inducer of macroautophagy (Noda and Ohsumi, 1998), or by depriving them of Leu and/or His (Kanazawa et al., 2003) substantially increased the number of AVs (Fig. 4 A, right), including autophagosomes (Fig. 4 B) relative to that in cells cultured under baseline conditions (10% serum; Fig. 4 A, left). Monodansylcadaverine, a fluorescent compound that preferentially accumulates in multilayer membranous structures such as AVs (Biederbick et al., 1995), was localized in large vacuolar structures after the deprivation of Leu, His, or both (Fig. 4 C, bottom left). Similarly, as expected with macroautophagy induction, LC3 immunofluorescence redistributed from the cytoplasm of untreated cells (Fig. 4 C, top middle) to a population of large vacuoles in Leu/His-deprived L/APP cells (Fig. 4 C, bottom middle) and serum-deprived SH-SY5Y cells (Fig. 4 C, bottom right), confirming our ultrastructural evidence of autophagosome generation (Fig. 4, A and B). Cells that were cultured in complete medium are shown in the top panels (Fig. 4 C). Serum or specific amino acid (Leu or His) deprivation induce autophagy through the AMP kinase pathway (Kadowaki and Kanazawa, 2003). We found a similar induction of autophagy in all of the cell lines (L/APP, N2a, and SH-SY5Y) after Leu/His deprivation or 10 nM rapamycin treatment (Fig. 4 D). Both treatments elevated levels of LC3-II (Fig. 4 D) and reduced levels of phosphorylated mTOR but not total mTOR relative to the values measured in untreated or 3-methyladenine (3MA)–treated cells as expected (Shigemitsu et al., 1999).


Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease.

Yu WH, Cuervo AM, Kumar A, Peterhoff CM, Schmidt SD, Lee JH, Mohan PS, Mercken M, Farmery MR, Tjernberg LO, Jiang Y, Duff K, Uchiyama Y, Näslund J, Mathews PM, Cataldo AM, Nixon RA - J. Cell Biol. (2005)

Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B). (C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top). (D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.
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Related In: Results  -  Collection

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fig4: Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells. (A and B) EM images showing changes in the number of AVs (arrows) in L/APP-overexpressing APP695 (L/APP cells) grown in complete medium (A, top left) or in medium lacking Leu and His (A, top right) for 6 h and in SH-SY5Y cells grown in the presence (A, bottom left) or absence (A, bottom right) of serum. At higher magnification, early and late AVs with typical morphologies are seen in a Leu/His-deprived L/APP cell (B). (C) Fluorescent and immunofluorescent labeling of large vesicles by the AV marker monodansylcadaverine (0.5 μg/ml for 30 min; left) and LC3 antibody (middle, L/APP; right, SH-SY5Y) in macroautophagy-induced cells (bottom), which are much less abundant in cells grown in complete medium (top). (D) Western blots confirm the cytochemical evidence for increased LC3-II levels as well as phospho-mTOR (P-2481) but not total mTOR after macroautophagy induction by Leu and His deprivation or 10 nM rapamycin (Rap) and macroautophagy inhibition by 5 mM 3MA in L/APP, N2a, and SH-SY5Y cells. Immunoblots for LC3 in SH-SY5Y cells and P-2481 mTOR in L/APP cells have been spliced but are derived from the same blot.
Mentions: In light of these observations, we further investigated the possibilities that macroautophagy is a pathway capable of producing Aβ and that AVs are a site of Aβ generation. We used murine fibroblast-like L cells that were stably transfected with APP695 (murine L cell type + wild-type human alkaline phosphatase [L/APP]), human (SH-SY5Y), and murine (N2a) neuroblastoma cell lines to determine the effect of macroautophagy on APP metabolism. In both nonneuronal (Fig. 4 A, top, L/APP) and neuronal (Fig. 4 A, bottom, SH-SY5Y; N2a, not depicted) cell models, inducing macroautophagy via the mTOR kinase pathway (Seglen et al., 1996; Petiot et al., 2000; Kadowaki and Kanazawa, 2003) by treating cells with rapamycin, which is a specific inhibitor of mTOR phosphorylation and an inducer of macroautophagy (Noda and Ohsumi, 1998), or by depriving them of Leu and/or His (Kanazawa et al., 2003) substantially increased the number of AVs (Fig. 4 A, right), including autophagosomes (Fig. 4 B) relative to that in cells cultured under baseline conditions (10% serum; Fig. 4 A, left). Monodansylcadaverine, a fluorescent compound that preferentially accumulates in multilayer membranous structures such as AVs (Biederbick et al., 1995), was localized in large vacuolar structures after the deprivation of Leu, His, or both (Fig. 4 C, bottom left). Similarly, as expected with macroautophagy induction, LC3 immunofluorescence redistributed from the cytoplasm of untreated cells (Fig. 4 C, top middle) to a population of large vacuoles in Leu/His-deprived L/APP cells (Fig. 4 C, bottom middle) and serum-deprived SH-SY5Y cells (Fig. 4 C, bottom right), confirming our ultrastructural evidence of autophagosome generation (Fig. 4, A and B). Cells that were cultured in complete medium are shown in the top panels (Fig. 4 C). Serum or specific amino acid (Leu or His) deprivation induce autophagy through the AMP kinase pathway (Kadowaki and Kanazawa, 2003). We found a similar induction of autophagy in all of the cell lines (L/APP, N2a, and SH-SY5Y) after Leu/His deprivation or 10 nM rapamycin treatment (Fig. 4 D). Both treatments elevated levels of LC3-II (Fig. 4 D) and reduced levels of phosphorylated mTOR but not total mTOR relative to the values measured in untreated or 3-methyladenine (3MA)–treated cells as expected (Shigemitsu et al., 1999).

Bottom Line: Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity.Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production.Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

View Article: PubMed Central - PubMed

Affiliation: Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY 10962, USA.

ABSTRACT
Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Show MeSH
Related in: MedlinePlus