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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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Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least). In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.
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fig5: Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least). In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.

Mentions: A relationship between macroautophagy induction and Aβ generation (Fig. 5, A and B) was established when we observed that suppressing macroautophagy in L/APP cells for 6 h with either 5 mM 3MA or 4:1 mM Leu/His decreased Aβ40 secretion into the medium by 39 (n = 6 each; P < 0.01) and 26–41% (n = 6 each; P < 0.05) relative to untreated cells, respectively, as measured by sandwich ELISA and standardized to total protein. Conversely, inducing macroautophagy by depriving L/APP cells of Leu or His stimulated Aβ40 secretion by 22 and 39%, respectively (n = 6 each; P < 0.05) compared with untreated cells (Fig. 5 A) and 70–90% over the levels in macroautophagy-suppressed cells. An analysis of covariance revealed a highly significant relationship between macroautophagy suppression by amino acid supplementation and reduction in Aβ levels (Aβ40, P < 0.01; Aβ42, P < 0.001) and between macroautophagy induction by Leu/His deprivation and increased Aβ production (Aβ40, P < 0.01; Aβ42, P < 0.0001). These results were confirmed with a second method of macroautophagy induction, rapamycin, which also effectively increased Aβ production nearly twofold in L/APP cells. Effects on Aβ40 levels were similar (Fig. 5 B), although there was a nonsignificant trend toward greater effects on Aβ42 production, as indicated by an increase in the ratio of Aβ42 to Aβ40. Modulating other amino acids, such as Gly or Val, in the medium had no effect on Aβ levels (Fig. 5, A and B); this finding is consistent with the known negligible influence of these amino acids on macroautophagy (Kadowaki and Kanazawa, 2003). The levels of βCTF and full-length APP levels were not significantly altered by these macroautophagy modulations (Fig. 5, C and D). We also examined the effect of macroautophagy modulators on Aβ40 and Aβ42 generation in the neuronal cell lines SH-SY5Y (Fig. 5, E and F) and N2a (Fig. 5, G and H). Inducing macroautophagy with rapamycin or Leu/His deprivation significantly increased Aβ40 in both cell lines (52–132%; P < 0.01; Fig. 5, E and G) and Aβ42 in N2a cells (98%; P < 0.05; Fig. 5 H), whereas suppressing macroautophagy with 3MA or Leu and His supplementation decreased Aβ levels by 12–54% (P < 0.05; Fig. 5 H).


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)

Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least). In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.
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Related In: Results  -  Collection

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fig5: Aβ generation in cells after autophagic modulation. Levels of Aβ40 (A), Aβ42 (B), βCTF (C), and APP (D) measured by sandwich ELISA after the incubation of L/APP cells (6 h) in conditions that block autophagy (+Leu, +His, +Leu/+His, and 5 mM 3MA), activate macroautophagy (−Leu, −His, −Leu/−His, and rapamycin), or do not affect autophagy (complete media and enrichment of the deprivation of Gly or Val). Values reported as percent difference of control ± SEM; *, P < 0.05 (at least). In similar experiments, Aβ40 and Aβ42 levels from SH-SY5Y (E and F) and N2a cells (G and H) after various treatments. Error bars represent SEM.
Mentions: A relationship between macroautophagy induction and Aβ generation (Fig. 5, A and B) was established when we observed that suppressing macroautophagy in L/APP cells for 6 h with either 5 mM 3MA or 4:1 mM Leu/His decreased Aβ40 secretion into the medium by 39 (n = 6 each; P < 0.01) and 26–41% (n = 6 each; P < 0.05) relative to untreated cells, respectively, as measured by sandwich ELISA and standardized to total protein. Conversely, inducing macroautophagy by depriving L/APP cells of Leu or His stimulated Aβ40 secretion by 22 and 39%, respectively (n = 6 each; P < 0.05) compared with untreated cells (Fig. 5 A) and 70–90% over the levels in macroautophagy-suppressed cells. An analysis of covariance revealed a highly significant relationship between macroautophagy suppression by amino acid supplementation and reduction in Aβ levels (Aβ40, P < 0.01; Aβ42, P < 0.001) and between macroautophagy induction by Leu/His deprivation and increased Aβ production (Aβ40, P < 0.01; Aβ42, P < 0.0001). These results were confirmed with a second method of macroautophagy induction, rapamycin, which also effectively increased Aβ production nearly twofold in L/APP cells. Effects on Aβ40 levels were similar (Fig. 5 B), although there was a nonsignificant trend toward greater effects on Aβ42 production, as indicated by an increase in the ratio of Aβ42 to Aβ40. Modulating other amino acids, such as Gly or Val, in the medium had no effect on Aβ levels (Fig. 5, A and B); this finding is consistent with the known negligible influence of these amino acids on macroautophagy (Kadowaki and Kanazawa, 2003). The levels of βCTF and full-length APP levels were not significantly altered by these macroautophagy modulations (Fig. 5, C and D). We also examined the effect of macroautophagy modulators on Aβ40 and Aβ42 generation in the neuronal cell lines SH-SY5Y (Fig. 5, E and F) and N2a (Fig. 5, G and H). Inducing macroautophagy with rapamycin or Leu/His deprivation significantly increased Aβ40 in both cell lines (52–132%; P < 0.01; Fig. 5, E and G) and Aβ42 in N2a cells (98%; P < 0.05; Fig. 5 H), whereas suppressing macroautophagy with 3MA or Leu and His supplementation decreased Aβ levels by 12–54% (P < 0.05; Fig. 5 H).

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