Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease.
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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ABSTRACT: The presence of senile plaques is one of the major pathologic hallmarks of the brain with Alzheimer's disease (AD). The plaques predominantly contain insoluble amyloid β-peptide, a cleavage product of the larger amyloid precursor protein (APP). Two enzymes, named β and γ secretase, generate the neurotoxic amyloid-β peptide from APP. Mature APP is also turned over endogenously by autophagy, more specifically by the endosomal-lysosomal pathway. A defective lysosomal system is known to be pathogenic in AD. Modulation of NF-E2 related factor 2 (Nrf2) has been shown in several neurodegenerative disorders, and Nrf2 has become a potential therapeutic target for various neurodegenerative disorders, including AD, Parkinson's disease, and amyotrophic lateral sclerosis. In the current study, we explored the effect of genetic ablation of Nrf2 on APP/Aβ processing and/or aggregation as well as changes in autophagic dysfunction in APP/PS1 mice. There was a significant increase in inflammatory response in APP/PS1 mice lacking Nrf2. This was accompanied by increased intracellular levels of APP, Aβ (1-42), and Aβ (1-40), without a change total full-length APP. There was a shift of APP and Aβ into the insoluble fraction, as well as increased poly-ubiquitin conjugated proteins in mice lacking Nrf2. APP/PS1-mediated autophagic dysfunction is also enhanced in Nrf2-deficient mice. Finally, neurons in the APP/PS1/Nrf2-/- mice had increased accumulation of multivesicular bodies, endosomes, and lysosomes. These outcomes provide a better understanding of the role of Nrf2 in modulating autophagy in an AD mouse model and may help design better Nrf2 targeted therapeutics that could be efficacious in the treatment of AD.Neurobiology of Aging 09/2014; · 4.85 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is a chronic progressive neurodegenerative disorder. As the most common form of dementia, it affects more than 35 million people worldwide and is increasing. Excessive extracellular deposition of amyloid-β peptide (Aβ) is a pathologic feature of AD. Accumulating evidence indicates that macroautophagy is involved in the pathogenesis of AD, but its exact role is still unclear. Although major findings on the molecular mechanisms have been reported, there are still no effective treatments to prevent, halt, or reverse Alzheimer's disease. In this study, we investigated whether Aβ25-35 could trigger an autophagy process and inhibit the growth of SH-SY5Y cells. Furthermore, we examined the effect of methyllycaconitine (MLA) on the cytotoxity of Aβ25-35. MLA had a protective effect against cytotoxity of Aβ, which may be related to its inhibition of Aβ-induced autophagy and the involvement of the mammalian target of rapamycin pathway. Moreover, MLA had a good safety profile. MLA treatment may be a promising therapeutic tool for AD.PLoS ONE 10/2014; 9(10):e111536. · 3.53 Impact Factor
- Frontiers in Bioscience 01/2008; 13(13):718. · 4.25 Impact Factor
T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 171, No. 1, October 10, 2005 87–98
The Rockefeller University Press$8.00
peptide-generating pathway activated in
W. Haung Yu,
Panaiyur S. Mohan,
Ana Maria Cuervo,
Mark R. Farmery,
Paul M. Mathews,
Corrinne M. Peterhoff,
Lars O. Tjernberg,
Anne M. Cataldo,
Stephen D. Schmidt,
and Ralph A. Nixon
Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY 10962
Department of Psychiatry and Department of Cell Biology, New York University School of Medicine, New York, NY 10016
Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461
Johnson and Johnson/Janssen Pharmaceutica, B-2340 Beerse, Belgium
Neurotec, Karolinska Institutet, Novum-KASPAC, SE-141 57 Huddinge, Sweden
Department of Cell Biology and Neuroscience, Osaka University Graduate School of Medicine, Suita 565-0871, Japan
McLean Hospital, Belmont, MA 02478
acroautophagy, 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 macro-
autophagy is induced early in Alzheimer’s disease (AD)
) deposits extracellularly in the
presenilin (PS) 1/A
precursor protein (APP) mouse model
-amyloidosis. Subsequently, autophagosomes and late
autophagic vacuoles (AVs) accumulate markedly in dystro-
phic dendrites, implying an impaired maturation of AVs to
lysosomes. Immunolabeling identifies AVs in the brain as a
major reservoir of intracellular A
-cleaved APP and are highly enriched in PS1,
nicastrin, and PS-dependent
or inhibiting macroautophagy in neuronal and nonneu-
ronal cells by modulating mammalian target of rapamycin
kinase elicits parallel changes in AV proliferation and A
production. Our results, therefore, link
and cell survival pathways through macroautophagy,
which is activated and is abnormal in AD.
. Purified AVs contain
-secretase activity. Inducing
Macroautophagy is a constitutive mechanism for the turnover
of cytoplasmic constituents that is activated under conditions
of trophic stress or nutritional deprivation (Mortimore and
Schworer, 1977). Its activation reduces the size of cells,
thereby decreasing their metabolic burden while generating
new substrates for energy and cellular remodeling (Mortimore
and Schworer, 1977; Seglen et al., 1986). The better under-
stood form of autophagy, macroautophagy, is initiated when an
“isolation” membrane is created under the direction of multi-
ple proteins, including microtubule-associated light chain 3-II
(LC3-II), that, together, orchestrate membrane elongation and
sequestration of a region of cytoplasm and organelles into a
double membrane–limited autophagic vacuole (AV) or auto-
phagosome (Asanuma et al., 2003). This sequestration process is
controlled by the mammalian target of rapamycin (mTOR) ki-
nase pathway, which is regulated by insulin via the PI3 kinase–
Akt (protein kinase B) pathway and by specific amino acids
(e.g., Leu or His) via AMP kinase (Petiot et al., 2000). Auto-
phagosomes mature to single membrane autophagolysosomes
(Dunn, 1990b) by fusing with lysosomes or other mature AVs,
whereupon they acidify and acquire proteolytic enzymes
(Meijer and Codogno, 2004). In addition, endocytosed constit-
uents can also enter the autophagic pathway when late endo-
somes fuse with autophagosomes to generate an amphisome
and may be retained in the cell without complete digestion of
the compartment’s contents (Gordon and Seglen, 1988).
As a system that is induced by nutritional stress or cell
injury, macroautophagy may be activated in pathologic states
(Rubinsztein et al., 2005). Macroautophagy may protect cells
from apoptosis (Boya et al., 2005), probably by eliminating
damaged mitochondria (Brunk and Terman, 2002), and is re-
Correspondence to Ralph A. Nixon: firstname.lastname@example.org
Abbreviations used in this paper: 3MA, 3-methyladenine; A
Alzheimer’s disease; APP, A
–carboxy-terminal fragment; IEM, immuno-EM; KO, knockout; L/APP,
murine L cell type
wild-type human alkaline phosphatase; LC3, light chain 3;
mTOR, mammalian target of rapamycin; NTg, nontransgenic; PS, presenilin.
The online version of this article contains supplemental material.
precursor protein; AV, autophagic vacuole;
JCB • VOLUME 171 • NUMBER 1 • 2005 88
quired for supranormal longevity in
(Melendez et al., 2003). Moreover, macroautophagy is in-
volved in degrading mutated and aggregated proteins that are
implicated in neurodegenerative diseases, including Parkinson’s
(Cuervo et al., 2004) and Huntington’s disease (Ravikumar et
al., 2004). Defective removal of these proteins has been linked
to disease progression in these disorders, and stimulating mac-
roautophagy in a model of Huntington’s disease reduced abnor-
mal protein aggregation and improved neurological function
(Ravikumar et al., 2004). Although macroautophagy appears to
be neuroprotective in these settings, sustained overactivity dur-
ing embryonic development (Clarke, 1990), response to ER
stress, exposure to toxins (Kanzawa et al., 2004), or dysfunc-
tion of the autophagic pathway in pathological states mediate a
caspase-independent form of cell death that shares certain fea-
tures with apoptosis (Bursch, 2001; Gozuacik and Kimchi,
2004). Despite the importance of the macroautophagy pathway
in cell survival and its connections to other protein-trafficking
pathways that are implicated in Alzheimer’s disease (AD;
Nixon et al., 2000), the involvement of macroautophagy in AD
has received limited attention. Recent immuno-EM (IEM)
analyses, however, have shown that autophagosomes and late
AVs appear in neurons in the AD brain and accumulate mark-
edly within dystrophic neurites, suggesting a progressive dys-
function of macroautophagy-mediated protein turnover (Nixon
et al., 2005).
In AD, extensive neuronal atrophy and loss is preceded
by the intraneuronal formation of neurofibrillary tangles, which
is composed mainly of aggregated tau protein, and by the extra-
cellular deposition of
predominantly as a 40–42–amino acid peptide from
-secretase cleavage of the A
precursor protein (APP; Cupers
et al., 2001). Both
-site APP cleaving enzyme and
which is a protein complex containing presenilin (PS), nicas-
trin, PEN2, and Aph-1 (Edbauer et al., 2003), reside in one or
more compartments of the central vacuolar apparatus, which
includes the ER–Golgi, plasma membrane, endosomes, and lyso-
somes (Cupers et al., 2001; Pasternak et al., 2003). We recently
identified APP, the
-cleaved APP product (
CTF]), PS1, and nicastrin in purified AVs from
livers of wild-type APP yeast artificial chromosome mice (Yu
et al., 2004), raising the possibility that AVs may also be able
to generate A
In this study, we demonstrate that macroautophagy is in-
duced in the brain at early stages of sporadic AD and in an ani-
mal model of AD pathology, the PS1/APP transgenic mouse
(Holcomb et al., 1998). In the PS1/APP model, macroautophagy
induction is evident in vulnerable neuronal populations before
the extracellular deposition of A
ing types accumulate abnormally within dystrophic neurites, in-
dicating a progressive impairment of AV maturation to lyso-
somes. We also show that AVs are highly enriched in both the
components and enzyme activity of the
contain APP and
CTF, and are a major source of intracellular
in the AD brain. Finally, we demonstrate in both neuronal
and nonneuronal cell lines that macroautophagy is a previously
unrecognized pathway for A
generation under conditions in
) peptide. A
, and, as in AD, AVs of vary-
which AVs accumulate. The early and persistent induction of
macroautophagy and the later pathological accumulation of AVs
in AD and related mouse models, therefore, potentially link
-amyloidogenic and neurodegenerative mechanisms in AD.
Macroautophagic induction and
pathological AV accumulation in AD and
PS1/APP mouse brain
AVs are rare in neurons of the normal adult brain (Nixon et al.,
2005). In AD, however, AVs appear in neocortical and hippo-
campal pyramidal neurons and accumulate markedly within the
dendritic arbors of these affected cells (Nixon et al., 2005). We
observed similar pathological AV accumulation in PS1/APP
animals, which is a mouse model of AD that expresses human
mutant PS1 and the Swedish variant of A
PS1/APP mice begin to deposit A
gressively develop many neuritic plaques that mainly consist of
and grossly swollen dendrites and axons (Holcomb et al.,
1998). AVs were rarely found in the neuropil of nontransgenic
(NTg) mice (Fig. 1 A). In contrast, the numbers of AVs were at
least 23-fold higher in the neurons of 9-mo-old PS1/APP mice
1.91 SEM) compared with age-matched NTg controls
0.08 SEM) based on ultrastructural morphometric
analyses of AV numbers in a series of 100 EM images. AV pa-
thology in 9-mo-old mice (Fig. 1, B–D), like that in AD brains
(Fig. 1 B, inset), ranged from small numbers of AVs in rela-
tively normal-appearing dendrites (Fig. 1 D) to striking patho-
logic accumulations of AVs within dystrophic neurites, where
AVs were usually the predominant organelles (Fig. 1, B and
C). A significant proportion of AVs met the morphologic crite-
ria for autophagosomes, including a size
a double limiting membrane, and the presence within a single
vacuole of multiple membranous organelle-derived structures
(Fig. 1 B; Dunn, 1990a). Other AVs included translucent and
dense multivesicular and multilamellar bodies with single or
double outer membranes (Fig. 1 C), reflecting later stages of
macroautophagy. In AD brain, we identified these AVs as au-
tophagolysosomes (“late” AVs; Dunn, 1990b), which contain
acid hydrolases but are distinct from lysosomal dense bodies
that are small (
m) and uniformly dense (Nixon et al.,
2005). Therefore, we documented a robust accumulation of
both early and late AVs in AD and PS1/APP brains, reflecting
marked macroautophagic induction, failed maturation of AVs
to lysosomes, or both (Nixon et al., 2005).
We also used an antibody against the AV marker protein,
LC3, to identify the specific subpopulation of autophagosomes,
which is the earliest compartment of macroautophagy. LC3-II,
a phosphatidylethanolamine-modified isoform of the microtu-
bule-associated protein LC3-I, is generated and translocated to
nascent autophagosomes when macroautophagy is induced, and
its presence, therefore, is a putative index of macroautophagy
induction (Mizushima et al., 2004). Very low levels of LC3-II
were detected by Western blot analysis in cortical gray matter of
normal control human brains. LC3-II levels were significantly
elevated at early stages of AD and, to a greater extent, at a mild/
(Duff et al., 1996).
after 10 wk of age and pro-
m in diameter,
MACROAUTOPHAGY, ALZHEIMER’S, AND A
GENERATION • YU ET AL.89
moderate stage of AD as compared with neuropathologically
normal age-matched elderly controls (P
respectively; Fig. 1 E). The level of LC3-I, which was present in
the brain at much higher amounts than LC3-II, rose modestly
but not significantly (Fig. 1 E). This biochemical change implied
an induction of macroautophagy in early AD.
Similarly, adult (18–22 mo old) PS1/APP mice also dis-
played nearly twofold higher levels of LC3-II but not LC3-I
(Fig. 1 F, top) in the frontal cortex as compared with age-
matched NTg mice (
3 each; P
by immunofluorescence labeling, total LC3 was higher in the
dendrites relative to the perikarya of neocortical pyramidal
neurons in PS1/APP mice (Fig. 1, G, H, K, and L) as compared
with NTg mice (Fig. 1, I and J). LC3 in PS1/APP dendrites was
frequently vesicular (Fig. 1, K and L), indicating the presence
of LC3-II, which is associated with nascent AVs (Fig. 1, G, K,
and L). LC3-positive vesicles were numerous in the dystrophic
portions of dendrites, particularly those at the periphery of
plaques (Fig. 1 H). Dystrophic neurites close to A
contained high numbers of AVs (Fig. 1 H), but these were
mostly LC3 negative (Fig. 1 H, arrowheads), reflecting a later
maturational state of AV populations in these presumably older
0.05 and P
0.01; Fig. 1 F). Moreover,
Macroautophagy is induced very early in
AD and PS1/APP mice
Elevation of LC3-II in AD brains at preclinical stages of AD
(Fig. 1 E) suggested that macroautophagic induction is an early
response in the disease process. To investigate early induction
in PS1/APP mice, we also analyzed LC3-II levels in these mice
at an age (8–9 wk) that precedes known neuropathology, in-
deposition (“predepositing” mice; Matsuoka et al.,
2001). AVs were visible in the cell bodies and neurites of pre-
depositing PS1/APP mice (Fig. 2, A and B) but were signifi-
cantly less frequent in NTg animals (Fig. 2 C). Based on the
EM fields that were surveyed from NTg and PS1/APP mice at
95% of images from NTg mice had zero or one AV
80% of EM images from PS1/APP mice
had at least one AV per field (Fig. 2 C). Quantitative ultrastruc-
tural analyses confirmed an increased incidence of AVs in the
neocortex of these predepositing PS1/APP mice, where AVs
were fivefold more numerous (
age-matched NTg mice (Fig. 2 D). LC3-II levels in predeposit-
ing PS1/APP brains, as determined by Western blot analysis,
were modestly yet significantly elevated (
the absence of a significant change in LC3-I levels (Fig. 2 E).
Furthermore, LC3 immunoreactivity, which was present pre-
dominantly in neuronal cell bodies in NTg mice (Fig. 2, F and
I), was distributed to both hippocampal cell bodies and den-
drites in 9-mo- (Fig. 2, G and H) and 9-wk-old (Fig. 2, J and K)
PS1/APP mice. The staining pattern for LC3 in many dendrites
was more frequently punctate in PS1/APP than in NTg mice
(Fig. 2, I and J), although less so than in older PS1/APP mice
(Fig. 2, compare H with K). Collectively, these observations
demonstrate that macroautophagy is induced at a prepathological
stage of disease in PS1/APP mice and that, in addition, different
subtypes of AVs accumulate pathologically as neuritic dystro-
phy develops in older mice, as in AD.
3 each; P
0.001) than in
PS1/APP mice and human brains. (A–D) EM
images of cortical neuropil show an absence
of AVs and normal neurite profile in 9-mo-old
NTg mouse brains (A, arrowheads outline nor-
mal neurites) and a marked accumulation of
AVs within enlarged or dystrophic neurites in
PS1/APP mice (B, arrowheads outline dystro-
phic neurite profiles in C) and biopsied brain
material from an AD patient (B, inset). At
higher magnification (C), AVs include auto-
phagosomes (arrows) and multilamellar bod-
ies (arrowhead). In normal dendrites of PS1/
APP mice, multiple AVs are frequently seen (D,
arrows). (E and F) LC3 quantification analyzed
from immunoblots of LC3-I and LC3-II (top) in
prefrontal cortical homogenates from cases of
nonaffected (Cont), early stage (preclinical)
AD (AD-ES), and moderate AD (AD-MS; E),
and from brains of 18–22-mo-old PS1/APP
(PA) mice (n ? 3; F) compared with nontrans-
genic (NTg) controls (n ? 3; *, P ? 0.01). Error
bars represent SEM. (G–L) LC3 immunofluores-
cence in 9-mo-old PS1/APP mice can be seen
mainly as puncta in dystrophic dendrites of
the cortex (G, arrows) and along adjacent
dendrites. LC3 (H, arrows) is strong in dystro-
phic neurites in the periphery (asterisks) of a
thioflavin S–labeled plaque core (H, inset) but
is less so in neurites closest (H, arrowheads) to
the A? deposit. LC3 is diffuse and uniform in
neurons of NTg mice (I and J) but is predomi-
nantly vesicular and distributed more to the
dendrites (arrows) than the cell soma (arrow-
heads) in 9-mo-old PS1/APP cortex (K and L).
Increased macroautophagy in
JCB • VOLUME 171 • NUMBER 1 • 2005 90
Modulation of macroautophagy markedly
Multiple tubulovesicular compartments that were implicated in
APP metabolism, such as the ER–Golgi and endosomes, be-
come components of AVs either as substrates or by contri-
buting to the limiting membrane of autophagosomes (Dunn,
1990a). Moreover, APP,
-site APP cleaving enzyme, and PS1
are reported to be abundant within neuritic plaques (Leuba et
al., 2005), raising the possibility that these components are lo-
calized in AVs. We confirmed that neuritic plaques of PS1/
APP mice are intensely labeled with a monoclonal antibody to
PS1 (Fig. 3, A and B). Based on IEM, AVs were the principle
immunoreactive structures within the dystrophic neurite, ac-
90% of PS1 immunolabeling (Fig. 3, C–F). In
the brains of both PS1/APP mice (Fig. 3, C and D) and AD pa-
tients (Fig. 3, E and F), PS1 antibody predominantly decorated
the double-limiting membranes of AVs or single or double
membranes within AVs (Fig. 3, arrows). Tubulovesicular mem-
branes in the adjacent normal neurites were the only other im-
munolabeled structures that were detected in the neuropil (Fig.
3 D, arrowheads), which is consistent with the expected contri-
bution of PS1-rich smooth ER to AV formation (Culvenor et al.,
1997). Mitochondria, plasma membranes, small vesicles, and
cytoplasm were essentially devoid of gold, underscoring the
specificity of immunolocalization.
In light of these observations, we further investigated
the possibilities that macroautophagy is a pathway capable of
and that AVs are a site of A
used murine fibroblast-like L cells that were stably transfected
(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 macroau-
tophagy 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 phosphory-
lation 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 struc-
tures 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 mac-
roautophagy 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 pan-
els (Fig. 4 C). Serum or specific amino acid (Leu or His) dep-
rivation induce autophagy through the AMP kinase pathway
(Kadowaki and Kanazawa, 2003). We found a similar induc-
tion of autophagy in all of the cell lines (L/APP, N2a, and
SH-SY5Y) after Leu/His deprivation or 10 nM rapamycin
campus of predepositing PS1/APP mice. Ultrastructural in-
spection of brain tissue from PS1/APP mice (A–D) shows
that AVs (A and B, arrows) are five times more frequent in
the dendrites of 8-wk-old PS1/APP than in those of age-
matched NTg mice. The frequency of AVs per EM field
(C) and mean number of AVs per EM field (D) within the
hippocampal molecular layer (n ? 3) are shown. LC3 im-
munoblot and analysis (E) and immunofluorescent label-
ing (F–K) of the hippocampal dendrites (brackets) in 8–9-
wk-old PS1/APP and NTg mice show LC3-II elevation
(P ? 0.05) in 8-wk-old PS1/APP compared with NTg
mice (E). (D) *, P ? 0.001. (E) *, P ? 0.05. Error bars
represent SEM. LC3 immunoreactivity in pyramidal cell
dendrites is increased in 9-mo-old (F–H) and 9-wk-old
(I–K) PS1/APP mice and frequently exhibits a punctate
labeling pattern, which is more evident at 9 mo than at
9 wk (H and K, arrows) and is uncommon in NTg mice
(F and I). Bars (F, G, I, and J), 20 ?m; (H and K), 10 ?m.
Identification of macroautophagy in the hippo-
MACROAUTOPHAGY, ALZHEIMER’S, AND A
GENERATION • YU ET AL. 91
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 (Shige-
mitsu et al., 1999).
A relationship between macroautophagy induction and
A? generation (Fig. 5, A and B) was established when we ob-
served that suppressing macroautophagy in L/APP cells for 6 h
with either 5 mM 3MA or 4:1 mM Leu/His decreased A?40
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, re-
spectively, as measured by sandwich ELISA and standardized
to total protein. Conversely, inducing macroautophagy by de-
priving L/APP cells of Leu or His stimulated A?40 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 macro-
autophagy suppression by amino acid supplementation and re-
duction in A? levels (A?40, P ? 0.01; A?42, P ? 0.001) and
between macroautophagy induction by Leu/His deprivation
and increased A? production (A?40, P ? 0.01; A?42, P ?
0.0001). These results were confirmed with a second method of
macroautophagy induction, rapamycin, which also effectively
increased A? production nearly twofold in L/APP cells. Effects
on A?40 levels were similar (Fig. 5 B), although there was a
nonsignificant trend toward greater effects on A?42 produc-
tion, as indicated by an increase in the ratio of A?42 to A?40.
Modulating other amino acids, such as Gly or Val, in the me-
dium had no effect on A? 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 ?CTF and full-length APP levels were not
significantly altered by these macroautophagy modulations
(Fig. 5, C and D). We also examined the effect of macro-
neurites in AD and PS1/APP mice. Cingulate cortex from 9-mo-old PS1/
APP mice immunolabeled with PS1 antibody and NT1 showed that PS1 lo-
calized to plaques (A). At higher magnification, anti-PS1 antibodies
strongly labeled neuritic profiles that were distributed within the plaque co-
rona (B). PS1 immunoreactivity is identified by IEM in AVs within dystro-
phic neurites of PS1/APP animals (C and D) and human brain (E and F)
by IEM. Arrowheads identify tubulovesicular membrane labeling. PS1 (C–F,
arrows) was localized on the outer limiting membrane of the AV but not in
mitochondria (Mito) or on plasma membranes (PM). IEM followed by silver
stain enhancement for PS1 was performed on a human brain that was
diagnosed for AD (F).
Immunolocalization of PS1 in plaques and AVs within dystrophic
(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 mor-
phologies are seen in a Leu/His-deprived L/APP cell (B). (C) Fluorescent and
immunofluorescent labeling of large vesicles by the AV marker monodansyl-
cadaverine (0.5 ?g/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 induc-
tion by Leu and His deprivation or 10 nM rapamycin (Rap) and macro-
autophagy 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.
Induction of macroautophagy in L/APP, SH-SY5Y, and N2a cells.
JCB • VOLUME 171 • NUMBER 1 • 200592
autophagy modulators on A?40 and A?42 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 A?40 in both cell lines
(52–132%; P ? 0.01; Fig. 5, E and G) and A?42 in N2a cells
(98%; P ? 0.05; Fig. 5 H), whereas suppressing macroautoph-
agy with 3MA or Leu and His supplementation decreased A?
levels by 12–54% (P ? 0.05; Fig. 5 H).
Ultrastructural localization of A? and
?-secretase complex components
Using immunogold labeling and EM in Leu- and His-deprived
L/APP cells, we observed that A? and ?-secretase components
were preferentially localized to AVs in situ and, as previously
described (Cupers et al., 2001), were localized to tubulove-
sicular compartments corresponding morphologically to ER/
Golgi/endosomes. Immunolabeling for A?40 or A?42 using
COOH-terminal–specific antibodies (Mathews et al., 2002)
preferentially decorated the same compartments (Fig. 6, A
and B, respectively). PS1 and nicastrin antibodies directed
against endogenously expressed proteins also principally deco-
rated both internal and limiting membrane components of AVs
in L/APP cells (Fig. 6, C and D, respectively). Quantitative
analysis of gold particles showed high proportions of immu-
nogold staining for A?40, A?42, PS1, and nicastrin that were
associated with AVs (43, 35, 48, and 35%, respectively) and tu-
bulovesicular compartments (31, 37, 33, and 35%, respectively;
Fig. 6 F). We found that the AV and tubulovesicular compart-
ments comprise 27.0 ? 11.0 and 19.2 ? 7.2%, respectively, of
the total cell area, indicating that PS1 signal in these organelles
was five times higher than in other subcellular compartments
and was similar to the organellar distribution in the PS1/APP
brain (Fig. 3, C and D). Immunolabeling of brains from 9-mo-
old PS1/APP mice with A?40 antibody also indicated a signifi-
cant localization of A?40 in AVs (Fig. 6, G and H).
Enrichment of ?-secretase activity and
protein components in isolated AVs
In further studies, we assayed for the protein components and the
?-secretase activity that are needed for A? generation in highly
purified fractions of AVs from serum-deprived L/APP cells. The
AV and lysosome fractions were isolated, and the identity and
purity of AV and lysosome fractions were confirmed by EM
A?40 (A), A?42 (B), ?CTF (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, A?40 and A?42 levels from SH-SY5Y (E and F) and
N2a cells (G and H) after various treatments. Error bars represent SEM.
A? generation in cells after autophagic modulation. Levels of
from L/APP cells and PS1/APP brains and
?-secretase components (PS1 and nicastrin)
in L/APP cells. Immunogold localization of
A?40 (A), A?42 (B), PS1 (C), nicastrin (D),
and in the absence of primary antibody (E) in
L/APP cells grown for 6 h in the absence of
Leu and His. (F) Quantification of gold particle
frequency in AV or tubulovesicular compart-
ments (TBV), which comprise 27.0 ? 11.0
and 19.2 ? 7.2%, respectively, of the total
cell area. Error bars represent SEM. (G and
H) IEM followed by silver stain enhancement
for A?40 was performed in 9-mo-old PS1/
Immunolocalization of A? in AVs
MACROAUTOPHAGY, ALZHEIMER’S, AND A? GENERATION • YU ET AL.93
(Fig. 7 A) and by immunoblotting using antibodies to LC3-II and
rab24, which is a ras-related GTPase that is associated with mac-
roautophagy (Fig. 7 B; Munafo and Colombo, 2002). Consistent
with the immunogold studies of L/APP cells in Fig. 6, AV frac-
tions were highly enriched in levels of PS1 and nicastrin relative
to the other subcellular fractions. Purified AVs also contained
significant levels of APP and ?CTF (Fig. 7 B). APP was en-
riched in the ER/endosome/Golgi fraction as well as the AV iso-
lates in cells that were treated with media lacking Leu and His
and cells grown in serum. In uninduced cells, the total amount of
AVs recovered was considerably lower. PS1 was most abundant
in the AV (A2) fraction after macroautophagic induction by se-
rum deprivation, with lesser amounts in the A1 and lysosome
fractions and in the ER/endosome/Golgi.
By using a fluorogenic synthetic polypeptide substrate to
measure ?-secretase activity after inducing macroautophagy in
L/APP cells (serum or Leu ? His deprivation), we found that
in L/APP cells, AV-enriched fractions contained the highest
?-secretase activity of all fractions analyzed on a per protein basis
(Fig. 7 C). Lysosome, Golgi, and microsome (E: ER/endosome/
Golgi) fractions also contained significant ?-secretase activity
relative to the low activities in the mitochondrial, post nuclear
pellet/unbroken cells, and cytosol fractions (Fig. 7 C). Based on
the ?-secretase activity per total recovered amount of each com-
partment, AVs accounted for 20–25% of the total ?-secretase
activity recovered from L/APP cells that were deprived of se-
rum (Fig. 7 D). Microsomes (ER and endosome) and Golgi frac-
tions showed less (per protein basis) ?-secretase than AVs
(19%), but, because these organelles are more abundant in the
cell, they accounted for slightly higher proportions (28 and
20%, respectively) of the total recovered ?-secretase activity in
these cells. Similar findings were made in L/APP cells that were
grown in ?Leu/?His media (Fig. 7 E). In contrast to conditions
of macroautophagy induction, total AV yields in noninduced
L/APP cells were much lower but exhibited similar properties
to those seen in induced cells (unpublished data). In noninduced
cells, the ?-secretase activity in AVs was ?10% of the total cell
activity, whereas that in the mixed microsome fraction in-
creased to ?60% (Fig. 7 E). In both induced and noninduced
conditions, tubulovesicular compartments and AV fractions
contained 60–70% of the total ?-secretase activity, suggesting
that this activity shifts from tubulovesicular compartments to
AVs when macroautophagy is induced.
Mass spectroscopic analysis indicated that the predomi-
nant cleavage of the model fluorogenic substrate in AVs oc-
curred at the A?42 site (Fig. 7 F), reflecting similar findings of
preference for A?42 cleavage over A?40 with this substrate in
other compartments. This A?-specific cleavage product did not
undergo further cleavage in AVs even after incubating the sub-
strate in AV isolates for 24 h, indicating that the product is not
a transient proteolytic intermediate. This is in contrast to the
A?-specific cleavage product in lysosomal fractions, where the
?-cleaved product was further cleaved to smaller products
when the incubation time was extended or when the pH was
lowered (Table S1, available at http://www.jcb.org/cgi/content/
? Cleavage in AVs is PS dependent
To confirm that fluorogenic substrate cleavage was PS1 spe-
cific, we assayed its activity in AVs that were isolated from
PS1-dependent ?-secretase activity in AVs.
(A and B) Ultrastructure of AVs (A1 and A2)
and lysosomes in subcellular fractions from se-
rum-deprived L/APP cells (A) and Western blot
analysis (B) for LC3-II, rab24, APP, ?CTF, PS1
(PS1 amino-terminal fragment), and nicastrin
(NCT) in L/APP subcellular fractions. AVs, A1
and A2; L, lysosomes; E, tubulovesicular com-
partments (Golgi/ER/endosomes); P, postnu-
clear pellet; C, cytosol; M, mitochondria.
Empty lanes in the original blot have been re-
moved from the figure and noted with a white
line. (C) Rates of cleavage of the fluorogenic
substrate in subcellular fractions from L/APP
cells grown in ?serum media (left) or ?Leu/
?His media (right). PNP, postnuclear pellet.
(D and E) Proportions of the total recovered
cell ?-secretase activity in different subcellular
fractions after serum deprivation (D) or in unin-
duced (?serum) or induced (?Leu/?His) cells
(E). Comparison of ?serum versus ?Leu and
?His conditions shows the redistribution of
?-secretase activity from the tubulovesicular to
AV fractions after macroautophagic induction.
G, Golgi; A, AV. (F) Liquid chromatography
mass spectrometry analysis of the fluorogenic
substrate after incubation with purified AVs.
Selected ion chromatogram corresponding to
Evidence for the enrichment of
different cleavage products is displayed. Numbers in graph refer to the cleavage/amino acid site from the A? peptide. (G) ?-Secretase activity in AV and
lysosome fractions from mice blastocysts in which the PS1 and PS2 genes were deleted (PS KO; BD8) or in which human PS1 was stably transfected into
the PS KO blastocysts (hPS1; BD8/hPS1). Numbers on x axis are in minutes. (H) A?40 and A?42 levels in medium from these cells as detected by
sandwich ELISA. Values are given as means ? SEM (error bars).
JCB • VOLUME 171 • NUMBER 1 • 200594
PS1 ? PS2 knockout (PS KO) blastocysts (BD8; Lai et al.,
2003) and BD8 blastocysts that were transfected with human
PS1 (BD8/hPS1). Cleavage of the ? substrate was 60–65%
lower in AVs from the BD8 cells compared with AVs from
PS1-rescued BD8/hPS1 cells (Fig. 7 G), which is comparable
with studies in human brain showing that 50% of total substrate
activity is inhibited by using specific ?-secretase inhibitors.
Although there was some residual cleavage of the substrate in
the BD8 double PS KO blastocysts, these cells generated no
detectable murine A?, whereas the BD8/hPS1 cells produced
abundant A?40 and A?42 (Fig. 7 H). Consistent with these re-
sults, a 5-?M IC50 concentration of the selective ?-secretase
inhibitor L685,458 inhibited 29.6–66.6% of the secretase activ-
ity in AV fractions of hPS1-rescued cells (unpublished data).
The lower levels of ?-secretase cleavage that were measured in
lysosomal fractions were much less affected by PS deletion
(25%) than were the AV fractions, indicating that lysosomes
(and to a lesser extent AVs) contain PS-independent protease
activity that is capable of ? cleavage of the model substrate.
This activity, however, does not support A? generation in in-
tact cells based on the negligible A? production in double PS
Our data provide strong evidence that A? is generated in AVs
during macroautophagy. Moreover, macroautophagy is both
induced and impaired in AD brain and PS1/APP mice, leading
to the pathological accumulation of A?-containing AVs within
affected neurons. Macroautophagy induction was evidenced by
an appearance of autophagosomes, which are only rarely de-
tectable in normal brain (Nixon et al., 2005), and by elevated
levels and cytosol-to-vesicle translocation of LC3-II, which is a
specific marker of autophagosome formation. These changes
are evident at the earliest stage of AD and in 8–9-wk-old PS1/
APP mice before A? deposition, which suggests that macroau-
tophagy induction is an early response in disease development,
although it is not necessarily independent of A? influences.
The specific pathologic events that induce macroautophagy in
predepositing PS1/APP mice are not known, but sources of ox-
idative stress, including intracellular forms of A? (Billings et
al., 2005), are possible factors contributing to macroautophagy
induction or to later dysfunction of the pathway. The induction
of macroautophagy at early stages of AD is consistent with the
expected need for increased protein/organelle turnover in in-
jured and regenerating neurites as well as for protection against
apoptotic stimuli, such as damaged mitochondria, that are
turned over by macroautophagy (Brunk and Terman, 2002).
The most striking feature of the macroautophagy-related
pathology in PS1/APP mouse brains was a grossly abnormal
accumulation of autophagosomes and other AV subtypes in
dystrophic neurites of the cortex and hippocampus, as also seen
in AD brain (Nixon et al., 2005). Lysosome-related multilamel-
lar and dense bodies have been described previously in neuro-
logical disease states (Suzuki and Terry, 1967; Masliah et al.,
1993) and, in part, represent late stages of macroautophagic ac-
tivity and lysosomal digestion (Nixon et al., 2005). In contrast,
we found that dystrophic neurites contain very high propor-
tions of autophagosomes and other immature AVs, implying
impairment in the normal maturation of these nascent AVs to
lysosomes. During normal neurite outgrowth or regeneration,
immature AVs move retrogradely and fuse with lysosomes in
the neurite or are more likely near or within the cell body (Hol-
lenbeck, 1993). After this fusion event, the contents of the AV
are rapidly degraded, and the AV becomes a lysosome (Overly
and Hollenbeck, 1996). This process is normally highly effi-
cient, with little evidence of AV buildup. In dystrophic neu-
rites, however, the striking buildup of autophagosomes and late
AVs implies a defect in AV transport, maturation to lysosomes,
or both, which is likely to impede lysosomal degradation
through this pathway (Nixon et al., 2005).
Our data provide strong evidence that A? is generated in
one or more subtypes of AVs that build up abnormally in af-
fected neurons. AVs not only contain immunoreactive A? and
?CTF but are also enriched in PS-dependent ?-secretase activ-
ity. Senile plaques in AD and PS1/APP mouse brains are also
abundantly immunoreactive for these components. Our data
confirm findings that PS1 and nicastrin are enriched in lyso-
somes (Pasternak et al., 2003) and indicate that AVs, which
were not distinguished from lysosomes in these earlier studies,
are a more concentrated source of these ?-secretase compo-
nents. Our findings also explain and are supported by observa-
tions that inclusion body myositis, which is the only known
condition in which A? deposits occur outside the nervous sys-
tem, involves the accumulation of macroautophagy-related
“rimmed” vacuoles containing elevated APP, A?, and PS1
(Askanas et al., 1998). Previous studies have identified intra-
cellular A? accumulation in endosomes (Cataldo et al., 2004a)
and multivesicular bodies (Takahashi et al., 2002) in AD and
Down syndrome (Gyure et al., 2001). Macroautophagy could
substantially increase pools of intracellular A? and contribute
to the formation of A? oligomers and protofibrils, which is a
process that is promoted in the acidic environment of lyso-
some-related organelles (Vassar and Citron, 2000).
We have also demonstrated, for the first time, that A? is
generated during macroautophagy. A? production rises when
macroautophagy is acutely stimulated, and AVs proliferate and
fall when macroautophagy is inhibited. AVs are depleted by
blocking either of the two independent signaling pathways
for macroautophagy that converge on mTOR kinase activity:
amino acid–mediated signaling and the PI3-kinase–dependent
pathway. Conditions that either stimulate AV production and
delay or impair maturation of AVs to lysosomes might be ex-
pected to increase the number of AVs and raise intracellular
A? levels (Fig. 8). A? is believed to be generated at several
sites within neurons, including endosomes, Golgi, and ER
(Cataldo et al., 2004a), and this multiplicity of APP processing
routes would account for our observations that considerable
A? is still secreted from L/APP, SH-SY5Y, and N2a cells
when macroautophagy is inhibited. Endocytic and autophagic
pathways communicate extensively, and both Golgi and ER are
turned over by macroautophagy (Dunn, 1990a), raising the
possibility that each of these organelles could contribute to A?
generation, in part, via macroautophagy. This communication
MACROAUTOPHAGY, ALZHEIMER’S, AND A? GENERATION • YU ET AL. 95
between the macroautophagy and endosomal systems also pro-
vides one possible avenue for A? that is generated during mac-
roautophagy to be released from cells, because late endosomes
also communicate with endocytic recycling compartments (for
review see Luzio et al., 2005). Extracellular release of some
A? from AVs is also possible from exosomes, which is a
mechanism proposed for prion release (Fevrier et al., 2005), or
from the direct fusion of AVs with the plasma membrane
(Jackson et al., 2005). It is worth noting, however, that the inef-
ficient extracellular elimination of autophagy-generated A?
may imply greater pathogenicity of this pool than the A? that is
normally secreted because intracellular A? appears to be more
cytotoxic than extracellular A?.
Based on the low number of AVs that were detected in
normal brain, macroautophagy may play a relatively minor role
in constitutive A? generation (Fig. 8). At low levels of mac-
roautophagy induction, A? that was generated in AVs would
be subsequently degraded within lysosomes, which contain the
necessary proteases (Bendiske and Bahr, 2003). In damaged or
regenerating neurites, however, where more APP-rich sub-
strates are diverted into the macroautophagy pathway and AV–
lysosome fusion may be delayed, intracellular A? is generated
within specific subtypes of AVs that accumulate (Fig. 8 B). AV
accumulation that is associated with any significant neuritic in-
jury could stimulate local A? production, as seen, for example,
in traumatic brain injury (Smith et al., 2003). Moreover, in AD,
in which large numbers of AVs accumulate and persist without
maturing within dystrophic neurites, macroautophagy could
contribute substantially to ?-amyloidogenesis and especially to
intracellular A? accumulation. In addition, several risk factors
for AD, including aging (Cuervo and Dice, 2000) and PS muta-
tions (unpublished data), impair AV maturation to lysosomes.
This accounts, in part, for the aging-related accentuation of au-
tophagic–lysosomal system pathology and ?-amyloidogenesis
in familial forms of AD that are caused by PS mutations and in
PS/APP mice relative to mice overexpressing mutant APP
alone (Cataldo et al., 2004b).
Collectively, these studies identify AVs as A?-generat-
ing compartments that accumulate pathologically in AD brain
(Nixon et al., 2005), accounting for a significant source of intra-
cellular A? in AD. Macroautophagy, as a pathway for A? gener-
ation and a mediator of both cell survival and degenerative phe-
nomena (Nixon et al., 2001), represents a new direction for
investigations into the pathogenesis and possible therapy of AD.
Materials and methods
We examined biopsy specimens from the temporal or frontal cortices of
10 patients, which were collected to confirm the suspected diagnosis of
encephalopathy. Seven of these cases (aged 71–86 yr) were later found
to meet the neuropathological criteria of AD (Braak and Braak, 1991;
Mirra et al., 1991, 1993; Newell et al., 1999), and the remaining three
specimens (aged 67–72 yr) were found to be neuropathologically normal
(Wegiel et al., 2000). Clinicopathologic characteristics of these cases
have been described previously (Nixon et al., 2005). Tissue was fixed in
3% PFA and 1% glutaraldehyde/0.1 M phosphate buffer, pH 7.4, and
were postfixed in 1% osmium tetroxide in Sorensen’s phosphate buffer.
After dehydration in ethyl alcohol, the tissue was embedded in Epon resin.
After routine histological inspection, the tissue blocks containing senile
plaques were cut serially into ultrathin (0.06 ?m) sections, and semithin
(0.5 ?m) sections were cut every 10th section. Ultrathin sections were
placed on formvar-coated grids and were poststained with uranyl acetate
and lead citrate.
In addition, we used postmortem fixed and frozen brain tissue (Brod-
mann areas 8 and 10) that was obtained from 43 elderly individuals of
both sexes ranging in age from 41 to 100 yr. Postmortem intervals for all
cases that were used were 17 h or less. Using the Consortium to Establish a
Registry for AD and Reagan guidelines (Mirra et al., 1991; Newell et al.,
1999), Braak staging (Braak and Braak, 1991), and the criteria proposed
by Mirra et al. (1993), 14 nondemented individuals from this group were
evaluated and diagnosed with early stage AD neuropathology (the neocor-
tex was devoid of plaques, and transentorhinal, entorhinal/hippocampal
cortices contained sparse plaques; Braak stages I–II). A second group con-
sisting of 16 age-matched cases met the criteria for mild/moderate stage
AD (moderate plaque numbers in the transentorhinal, entorhinal/hippocam-
pal cortices and slightly fewer in the neocortex; Braak stages III–V). A third
group consisting of 13 age-matched cases that were determined to be neu-
ropathologically normal (isocortex and entorhinal cortex/hippocampus
were devoid of plaques and neurofibrillary tangles) were used as controls.
The tissues were obtained from the Bronx Veteran’s Administration Medical
Center and the Harvard Brain Tissue Resource Center at McLean Hospital.
The magnitude of neuropathology was confirmed by histopathological in-
spection using Nissl stain, hematoxylin and eosin staining, Bielschowsky sil-
ver stain, and thioflavin S histofluorescence. Transgenic mice expressing the
Swedish mutation of human APP (APPK670M/N671L) and mutant human PS1
(PS1M146L; Duff et al., 1996) were studied at 8–9 wk, 9 mo, and 18–22 mo
of age together with age-matched controls.
Antibodies and reagents
The antibodies used for A? ELISAs have been previously described
(Mathews et al., 2002) and were a gift from Johnson and Johnson Phar-
maceutical Research and Development/Janssen Pharmaceutica (Beerse,
Belgium). NT1 was used to identify PS1 in PS1/APP mice. Ab14, which is
a pAb recognizing the amino terminus of murine PS1, was used for IEM
localization of PS1 in L/APP cells and was a gift from S. Gandy (Thomas
Jefferson University, Philadelphia, PA; Petanceska et al., 2000). An antini-
castrin pAb was obtained from P. Fraser and P. St George-Hyslop (Univer-
sity of Toronto, Toronto, Canada). mAbs, 4G8 recognizing residues 17–24
of the A? peptide, 6E10 recognizing residues 1–16 of the A? peptide,
?CTF, and APP were purchased from Signet. mTOR antibodies (total and
P-2481) were purchased from Cell Signaling Technology, and rab24 was
obtained from BD Biosciences. Organelle immunomarkers included anti-
rab5 affinity-purified rabbit pAb (Santa Cruz Biotechnology, Inc.) and
a polyclonal EEA1 (gift from S. Corvero, University of Massachusetts,
Worcester, MA) for endosomes, a mAb clone p58K-9 for Golgi (Sigma-
Aldrich), a pAb calnexin (StressGen Biotechnologies) for ER, and an anti-
cathepsin D polyclonal for lysosomes (Cataldo et al., 1996). A pAb to
LC3 was made that recognized native (LC3-I) and postautophagic-induced
levels. The schematic of macroautophagy depicts (A) the usual progres-
sion from autophagosomes (AP) to autophagolysosomes (APL) to lyso-
somes (L). Conditions that result in AV buildup (B and C) are expected to
promote A? generation and accumulation, including impaired or de-
layed maturation of autophagosomes to lysosomes (B) or acute maximum
induction of macroautophagy (C). Within neurons, AVs normally
progress to lysosomes efficiently and are rarely seen in neurons (D). In
AD, the disrupted retrograde transport of AVs in dendrites represents one
of several possible mechanisms that impede the maturation of AVs to
lysosomes, leading to A? generation in AVs and its delayed degradation
in lysosomes (E).
Proposed models of AV accumulation leading to elevated A?
JCB • VOLUME 171 • NUMBER 1 • 2005 96
protein (LC3-II; Kabeya et al., 2000, 2004; Mizushima et al., 2004).
500 ?g/ml monodansylcadaverine was added to living cells for 30 min
to accumulate in AVs (Biederbick et al., 1995).
Modulation of macroautophagy
Cell maintenance and human wild-type APP695 expression in L/APP cells
have been previously described (Mathews et al., 2002). Upon the induc-
tion of APP expression, L/APP cells were treated with various agents to
modulate macroautophagy for 6 h. SH-SY5Y and N2a cells were main-
tained in DME ? 10% FBS. PS-null and human PS1–transfected PS-null
blastocysts (a gift from A. Bernstein, Mount Sinai Hospital, Toronto, Can-
ada) were cultured in DME ? 15% FBS with 10 nM ?-mercaptoethanol.
Media were formulated as listed by Invitrogen for DME and was supple-
mented with sodium pyruvate, vitamins, and 10% FBS dialyzed at 10 kD
(all were obtained from Invitrogen). In some experiments, the media either
lacked one amino acid of interest or contained fivefold excess of this
amino acid from Sigma-Aldrich. 10 nM rapamycin was used to induce
macroautophagy, whereas 5 mM 3MA (Sigma-Aldrich) was used to inhibit
AV formation (Seglen and Gordon, 1984).
After treatment, cells that were used for immunofluorescence analy-
sis were fixed in 2% PFA (Electron Microscopy Sciences) in PBS, pH 7.4,
and were washed with PBS. Cells intended for EM were fixed in 4% PFA/
1% glutaraldehyde in 0.1 M sodium cacodylate buffer.
Immunofluorescence microscopy, sandwich ELISA for APP and its metab-
olites, ?CTF, A?40, A?42, and Western blot analyses on cells were per-
formed as previously described (Mathews et al., 2002; Grbovic et al.,
2003). Western blot analysis was also performed on L/APP, N2a, and
SH-SY5Y cell lysates probing for LC3-I/-II, phospho-mTOR (P-2481), and
total mTOR. Analysis was also performed on AV isolates from L/APP
cells, and cells were probed for LC3, rab24, APP, ?CTF, PS1 amino-ter-
minal fragment, and nicastrin. Immunofluorescence was visualized on ei-
ther a microscope (Axiovert 200; Carl Zeiss MicroImaging, Inc.) with a
100? objective equipped with a digital camera (AxioCam HRC; Carl
Zeiss MicroImaging, Inc.) and Axiovision 4.2 software (Carl Zeiss Micro-
Imaging, Inc.) or on a laser scanning confocal microscope (model TCSNT;
Leica) using a 100? objective and was analyzed using Leica Confocal
Software. Western blots were imaged on film, and the film was scanned
with a scanner (Duoscan T1200; Agfa). Denstitometry analysis was per-
formed using a National Institutes of Health image (version 1.59) with
samples that were within the linear range of the software. In some cases,
lanes from the same Western blot were rearranged or, if not relevant,
were eliminated to enable alignments of multiple gels in a composite
figure. Splicing within a given blot is indicated by a white line between
Mouse brains were harvested after intracardiac perfusion with 1%
PFA and were sucrose embedded overnight, frozen, and cryostat sec-
tioned at 40 ?m of thickness before immunolabeling with LC3. Fluorescent
labeling was identified using the laser scanning confocal microscope
(Leica) equipped with a 100? objective.
Ultrathin sections from Epon-embedded blocks were placed on copper
grids for structural analysis or on nickel grids for immunogold labeling
and were air-dried and etched briefly with 1% sodium metaperiodate in
PBS followed by washing in filtered double distilled water and were incu-
bated in 1% BSA in PBS for 2 h. Sections were incubated overnight in pri-
mary antibody (calnexin or protein disulfide isomerase; both were ob-
tained from Stressgen) in a humidified chamber at 4?C, spin washed
several times in PBS, and incubated in 5–20 nm gold-conjugated second-
ary antibody (anti–mouse or anti–rabbit IgG; GE Healthcare) for 2 h at
RT. Silver enhancement (GE Healthcare) of PS1 in human brain and A?42
in mouse brain was performed after IEM. In negative control experiments,
primary antibody was substituted with normal rabbit or mouse serum (1:20)
depending on the primary antibody used (polyclonal or monoclonal) and
was incubated in a humidified chamber at 4?C. The grids were extensively
washed with PBS and incubated for 2 h at RT with 10 nm gold-labeled
secondary antibody (GE Healthcare). Grids were washed again and
briefly stained with uranyl acetate and lead citrate before being examined
with an electron microscope (model CM 10; Philips). Images were cap-
tured on a camera (model C4742-95; Hamamatsu) and on Advantage
CCD Camera System software (Advanced Microscopy Techniques Corpo-
ration). Some EM images (Fig. 4, A and B; L/APP cells) were captured
on film, printed on Kodak glossy paper, and scanned with a scanner
(StudioScan II; Agfa) at 600 dpi.
Quantitative analysis of AVs from EM images
For 9-wk-old PS1/APP and NTg mice images (n ? 3 each), 30 randomly
selected EM images per animal were captured at a final magnification of
10,500?, and the number of AVs in each captured field was counted by
visual inspection using criteria for identification that were previously estab-
lished (Dunn, 1990b; Nixon et al., 2005). For 9-mo-old mice, the same
procedure was applied, but 50 fields/mouse with two mice per group
Preparation of AVs
For each cell line (L/APP, PS KO–BD8, or hPS1–BD8/hPS1), 500 million
cells were serum deprived overnight to induce autophagic activity (Fuertes
et al., 2003). In additional experiments, AVs were isolated from L/APP
cells with normal serum and media lacking Leu and His. By using a proto-
col that was modified from Marzella et al. (1982), the cells were har-
vested, disrupted by nitrogen cavitation, homogenized, and separated by
differential centrifugation to first produce a (low speed) pellet containing
the nuclear fraction and (up to 30%) unbroken cells (postnuclear pellet)
and a second (supernatant in higher speed) pellet that was enriched in
AVs, lysosomes, and mitochondria. From the AV/lysosome/mitochondrial
fraction, lysosomes were separated from the two AV fractions by using a
discontinuous metrizamide gradient (A1, 10% metrizamide; A2, 20% me-
trizamide). A cytosol fraction was obtained by centrifuging the superna-
tant from the AV/lysosome/mitochondrial fraction at 100,000? g for 1 h
at 4?C. The pellet from this centrifugation yielded an enriched microsome
fraction that predominantly contained ER but also contained small propor-
tions of endosomes. Fractions were pelleted and either immersed in a ca-
codylate fixation buffer for EM or analyzed directly by Western blot or en-
zyme assay as described below. To isolate liver AVs, three C57BL/6 mice
were starved for 12 h and injected intraperitoneally with 5 mg/100 g of
body weight of vinblastine in normal saline (0.9% NaCl) 3 h before killing
(Marzella et al., 1982). Livers were harvested, minced, and homogenized
using a polytron Teflon homogenizer before differential and density gradi-
ent centrifugation as described above for the isolation of AVs from individ-
ual cell lines.
?-Secretase assay and mass spectrometry
A 0.5-?g aliquot of protein from each subcellular fraction was plated onto
an opaque 96-well microplate. Samples were diluted to a final concentra-
tion of 50 mM Tris, pH 6.5, 2 mM EDTA, and 0.25% CHAPS (Sigma-
Aldrich). In tandem, another set of samples was pretreated with 5 ?M of
the selective ?-secretase inhibitor L685,458 (Bachem) for 15 min. A fluo-
rogenic ?-secretase substrate (Nma-GGVVIATVK[DNP]-rrr-NH2; Farmery et
al., 2003) was added at a 10-?M final concentration to the plate, and the
samples were read in a fluorescent plate reader (Wallac Victor2; Perkin-
Elmer) using a 355-nm excitation filter and a 440-nm emission filter over a
period of 4 h. ?-Secretase activity was measured as increased fluores-
cence, which is achieved only when the fluorogen is cleaved (Farmery et
al., 2003). Using liquid chromatography mass spectrometry, samples
from the secretase assay containing solubilized membranes and peptide
probe were injected onto a liquid chromatography packings reverse
phase column (model 75 ID C18; Dionex). Samples were eluted over 30
min with distilled water/0.2% formic acid, and 0.2% formic acid/acetoni-
trile (10–40% gradient) was used as the mobile phase. The column was
coupled online to an electrospray ion trap mass spectrometer (model
1100 SL; Agilent Technologies). As a control, samples were also pre-
pared in the absence of incubation.
Online supplemental material
Table S1 details the percentage of peptide product for original peptide,
non-A?, and A?-specific cleavage as determined by mass spectroscopy
after incubation of the fluorogenic peptide in autophagic and lysosomal
isolates at pH 6.8 and 4.5 for 1 or 24 h or in the presence of the ?-secre-
tase inhibitor L-685,458. Online supplemental material is available at
We would like to thank J.N. Peterson, N.B. Terio, and O. Grbovic for techni-
cal assistance and L. Goldberg, H. McAuliff, H. Braunstein, and G. Lardi for
This research was supported by the Alzheimer Association (grant TLL-99-
1877), National Institutes of Health (grants AG17617-05 and AG021904 to
A.M. Cuervo), the Howard Hughes Medical Institutes (grant to A.M. Cuervo),
and the Canadian Institute of Health Research (grant to W.H. Yu).
Submitted: 16 May 2005
Accepted: 1 September 2005
MACROAUTOPHAGY, ALZHEIMER’S, AND A? GENERATION • YU ET AL.97
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