Lysosomal Proteolysis and Autophagy
Require Presenilin 1 and Are Disrupted
by Alzheimer-Related PS1 Mutations
Ju-Hyun Lee,1,2W. Haung Yu,1,2,9Asok Kumar,1,3Sooyeon Lee,1,4Panaiyur S. Mohan,1,2Corrinne M. Peterhoff,1
Devin M. Wolfe,1Marta Martinez-Vicente,6,10Ashish C. Massey,6Guy Sovak,6,11Yasuo Uchiyama,7David Westaway,8
Ana Maria Cuervo,6and Ralph A. Nixon1,2,5,*
1Center for Dementia Research, Nathan S. Kline Institute, 140 Old Orangeburg Road, Orangeburg, NY 10962, USA
2Department of Psychiatry
3Department of Pathology
4Department of Neuroscience
5Department of Cell Biology
New York University Langone Medical Center, 550 First Avenue, New York, NY 10016, USA
6Department of Developmental and Molecular Biology, Institute for Aging Research, Albert Einstein College of Medicine, 1300 Morris Park
Avenue, Bronx, NY 10461, USA
8Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2B7, Canada
9Present address: Taub Institute, Columbia University, New York, NY 10032, USA
10Present address: Institute of Neuropathology, IDIBELL-Hospital Universitari de Bellvitge, Hospitalet de Llobregat, 80907 Barcelona, Spain
11Present address: Department of Anatomy, Canadian Memorial Chiropractic College, Toronto, Ontario M4G 3E6, Canada
Macroautophagy is a lysosomal degradative pathway
essential for neuron survival. Here, we show that
macroautophagy requires the Alzheimer’s disease
(AD)-related protein presenilin-1 (PS1). In PS1 null blas-
tocysts, neurons from mice hypomorphic for PS1 or
conditionally depleted of PS1, substrate proteolysis
and autophagosome clearance during macroautoph-
agy are prevented as a result of a selective impairment
of autolysosome acidification and cathepsin activation.
These deficits are caused by failed PS1-dependent
targeting of the v-ATPase V0a1 subunit to lysosomes.
N-glycosylation of the V0a1 subunit, essential for its
binding of PS1 holoprotein to the unglycosylated
subunit and the Sec61alpha/oligosaccharyltransferase
complex. PS1 mutations causing early-onset AD pro-
duce a similar lysosomal/autophagy phenotype in
fibroblasts from AD patients. PS1 is therefore essential
for v-ATPase targeting to lysosomes, lysosome acidifi-
cation, and proteolysis during autophagy. Defective
suggests previously unidentified therapeutic targets.
Macroautophagy, the major lysosomal degradative pathway
in cells, is responsible for degrading long-lived cytoplasmic
constituents and is the principal mechanism for turning over
cellular organelles and protein aggregates too large to be
degraded by the proteasome (Klionsky, 2007; Mizushima,
2007; Rubinsztein, 2006). Macroautophagy, hereafter referred
to as autophagy, involves the sequestration of a region of cyto-
plasm within an enveloping double-membrane structure to
form an autophagosome. Autophagosome formation is induced
by inhibition of mTOR (mammalian target of Rapamycin)
(Schmelzle and Hall, 2000) or AMP-activated protein kinase
(AMPK) (Samari and Seglen, 1998). Autophagosomes and their
contents are cleared upon fusing with late endosomes or lyso-
somes containing cathepsins, other acid hydrolases, and vacu-
olar [H+] ATPase (v-ATPase) (Yamamoto et al., 1998), a proton
pump that acidifies the newly created autolysosome. Acidifica-
tion of autolysosomes is crucial for activating cathepsins and
effecting proteolysis of substrates; however, these late digestive
steps of autophagy remain relatively uncharacterized.
Autophagic vacuoles (AVs), the general term for intermediate
et al., 2005; Ravikumar et al., 2004), but autophagy pathology
in Alzheimer’s disease (AD) is exceptionally robust. AVs, many
containing amyloid-b peptide, collect in massive numbers within
grossly distended portions of axons and dendrites of affected
neurons (Yu et al., 2005), likely reflecting defective AV clearance
(Boland et al., 2008). This lysosome-related pathology, along
common cause of FAD (Cataldo et al., 2004).
Presenilin 1 (PS1), a ubiquitous transmembrane protein, has
diverse putative biological roles in cell adhesion, apoptosis, neu-
rite outgrowth, calcium homeostasis, and synaptic plasticity
1146 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
(Kim and Tanzi, 1997; Shen and Kelleher, 2007). A portion of the
PS1 holoprotein, an ?45 kDa protein, is cleaved in the endo-
plasmic reticulum (ER) to create a two-chain form (Zhang et al.,
1998). Many known PS1 functions, but not all, involve the
cleaved form of PS1 as the catalytic subunit of the gamma (g)-
secretase enzyme complex, which mediates the intramembra-
nous cleavage of many type 1 membrane proteins, including
APP and Notch (Citron et al., 1997; De Strooper et al., 1998).
Although the pathogenic effects of PS1 mutations in AD are
commonly ascribed to increased generation of the neurotoxic
Ab peptide from APP, not all of the disease-causing PS1 muta-
tions have this effect (Shioi et al., 2007). Additional contributions
to AD pathogenesis may involve loss of one or more of the other
suspected biological functions of PS1 (Naruse et al., 1998).
In this report, we show that PS1 is required for lysosomal turn-
causes virtually complete loss of macroautophagy while having
minimal influence on nonlysosomal types of proteolysis. We
have identified the molecular basis for this requirement to be an
action of PS1 holoprotein in the ER as an ER chaperone to facili-
tate maturation and targeting of the v-ATPase V0a1 subunit to
lysosomes, which is essential for acidification, protease activa-
tion, and degradation of autophagic/lysosomal substrates. We
demonstrate defects in these processes in cells lacking PS1,
which are completely reversed by the introduction of wild-type
(WT) human PS1 into the cells. Similar autophagy pathology and
deficits in lysosomal acidification are demonstrated in neurons
of mice hypomorphic for PS1 or conditional PS knockout (KO)
mice. Of particular clinical significance, we further show that
mutations of PS1 that cause early-onset FAD disrupt the same
lysosomal/autophagic functions that are more severely affected
in PS1 KO cells. Our findings underscore the pathogenic impor-
tance of lysosomal proteolytic dysfunction seen in all forms of
AD (Nixon and Cataldo, 2006; Nixon et al., 2008) and provide
a basis for the accelerated autophagy dysfunction and defective
neuronal protein clearance seen in PS-FAD.
PS1 Gene Deletion Selectively Inhibits
Macroautophagic Turnover of Proteins
We investigated the competence of proteolytic systems in blas-
tocysts from WT mice and constitutive PS1 KO mice using
a well-established metabolic labeling procedure (Auteri et al.,
1983). Neither incorporation of [3H]-leucine into proteins, used
as a measurement of protein synthesis (Figure 1A), nor proteol-
ysis of short-lived proteins, reflective mainly of ubiquitin-protea-
some-dependent degradation (Figure 1B), was significantly
altered in PS1 KO cells. However, the turnover of long-lived
proteins was decreased in PS1 KO cells compared to WT under
Figure 1. Protein Turnover in PS1 KO Cells
(A) Incorporation of [3H]-leucine in blastocysts
from WT or PS1 KO mice.
(B) After labeling, the proteolysis of short-lived
proteins was measured after a chase period.
(C) Degradationof long-lived
measured in WT (left) and PS1 KO cells (right).
After incorporation of [3H]-leucine, cells were incu-
medium during the chase period (up to 20 hr)
(* for p < 0.05, n = 9).
(D) The increase in proteolysis at 12 hr after
removal of serum relative to serum-replete condi-
tions was determined for WT and PS1 KO cells
that were untreated (control) or treated with
NH4Cl or 3MA (** for p < 0.001, n = 9).
(E) Total p70S6K and phospho (Thr389)-p70S6K
levels quantified by densitometry, after growth in
the presence or absence of serum for 6 hr (* for
p < 0.05, n = 3).
(F) LC3 immunostaining after incubation in the
presence and absence of serum.
(G) Percentages of cell area occupied by LC3
puncta analyzed with ImageJ software (see the
Experimental Procedures) (* for p < 0.05 and **
for p < 0.001, n = 50).
(H) LC3-II and LC3-I immunoreactivity and LC3-II/
LC3-I ratios by western blot analysis with tubulin
used as a loading control (** for p < 0.001, n = 3).
All values are shown as the mean ± SEM. See also
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. 1147
serum-supplemented media(Figure1C). Whenautophagic/lyso-
somal degradation was induced through serum withdrawal,
proteolysis increased 15%–20% in WT cells (p < 0.05) but was
not significantly changed in PS1 KO cells (Figure 1C). Under
these induced conditions, NH4Cl (20 mM) entirely blocked the
increase of proteolysis in both cell types as expected
(Figure 1D). When macroautophagy was selectively inhibited
with 3-methyladenine (3MA, 10 mM) (Seglen and Gordon,
1982), only the increased proteolysis in WT cells was blocked
(Figure 1D), indicating that the residual increase in lysosome-
related degradation in response to serum removal in PS1 KO
cells is not due to macroautophagy. Together, these findings
demonstrate that PS1 deletion selectively affects macroauto-
phagic turnover of proteins.
We next assessed the competence of the nutrient-related
signaling pathway leading to mTOR-mediated induction of au-
tophagy. For this, we measured the phosphorylation state of
of both total p70S6K and its phosphor-epitope (Thr389),
rableinWTandPS1KO cellsgrown inserum-containingmedium
(Figure 1E). Moreover, phospho-p70S6K levels declined compa-
drawal for 6 hr, indicating that mTOR is inhibited normally in
response to nutrient deprivation in PS1 KO cells (Figure 1E).
To evaluate autophagosome formation, we used immunofluo-
rescence labeling with antibodies to LC3. LC3-positive vesicular
profiles of sizes 0.5–2.0 mm were significantly more numerous in
PS1 KO cells than in WT cells grown in serum and were slightly
increasedafter serum withdrawal
Figure S1 available online). Consistent with immunocytochem-
ical findings, LC3 western blot analyses showed that ratios of
LC3-II to LC3-I, or LC3-II levels alone, were more than 2-fold
higher (p < 0.001) in PS1 KO cells than WT cells grown in the
presence of serum. Serum withdrawal resulted in higher LC3-II
levels in both WT cells and PS1 KO cells, although in the latter
cells the proportional increase over the already elevated level
of LC3-II was less than in WT cells (Figure 1H). Induction of au-
results (data not shown).
their size and morphology (Figure S1B). Autophagosomes and
early autolysosomes were more numerous in PS1 KO cells,
whereas most AVs in WT cells were late autolysosomes. Despite
abnormally high baseline AV numbers, PS1 KO cells exhibited
(Figure S1C). AVs and lysosomes isolated from cells on metriza-
mide gradients confirmed that engulfed materials were less
degraded in PS1 KO cells than in WT cells, where most materials
were extensively digested, and lysosomes were mainly small and
protein degradation was impaired after fusion with lysosomes.
(Figures1F and 1G;
Defective Clearance of Autophagic Vacuoles
in PS1 KO Blastocysts
Further analyses of autolysosome maturation showed that
clearance of LC3 after fusion, a measure of autophagy degra-
dative competence, was greatly impaired in PS1 KO cells.
In WT cells, acute autophagy induction with rapamycin
elevated LC3-II levels by immunoblot analysis. These levels re-
turned to pretreatment baseline levels within 6 hr after removal
of rapamycin from the medium; however, in PS1 KO cells, LC3-
II levels remained significantly elevated (Figure 2A). Double-
immunofluorescence labeling with LC3 and LAMP-2 antibodies
confirmed that LC3 accumulated in LAMP-2-positive vesicles
after rapamycin exposure of WT (Figure 2B) and PS1 KO cells
(Figure 2D), but 6 hr after rapamycin was removed, LC3
remained only in LAMP vesicles of PS1 KO cells (Figures 2C
and 2E). Despite this evidence that autophagosomes can
Figure 2. Impaired Clearance of LC3-II from Autolysosomes in PS1
(A) Immunoblot analysis of LC3-I and -II levels in cells under conditions of no
treatment (Ctrl), serum starvation (-Ser), rapamycin (Rap), rapamycin treat-
ment followed by rapamycin removal (Rap/RC), and 3MA.
(B–E)WT(Band C)and PS1KOcells (Dand E)analyzed by double-immunoflu-
removal of rapamycin (C and E). Right panels depict enlarged images of the
boxed areas seen in the left panels. The scale bar represents 10 mm. ** for
p < 0.001.
All values are shown as the mean ± SEM. See also Figure S2.
1148 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
fuse with lysosomes, inspection at higher magnification
revealed that LC3 distributed more peripherally along the
membrane of the fused vesicles in PS1 KO cells compared
with those in WT cells, suggesting that the handling of LC3 after
autophagosome-lysosome fusion is impaired (compare Figures
2B and 2D).
Using an alternative approach to investigate autophago-
some clearance, we observed that LC3 levels remained abnor-
mally high in PS1 KO cells even when autophagosome
formation was blocked for 6 hr with 3MA (Figure 2A;
Figure S1E). Moreover, treatment with leupeptin (0.3 mM, 6
hr) to inhibit cysteine proteases in autolysosomes significantly
elevated LC3-II levels (Figure S2A) and LC3-positive puncta in
WT cells (Figure S2C), whereas in PS1 KO cells, this treatment
did not increase LC3 levels beyond the elevated baseline
evident in these cells before leupeptin addition (Figures S2D
and S2E). As an alternative assessment of LC3-II turnover,
p62/SQSTM1 degradation may also be used to evaluate
impairments of autophagic protein degradation (Bjørkøy
et al., 2005). In addition to LC3-II accumulation, p62 levels
also increased in PS1 KO cells (Figure S2F). Each of these
three lines of evidence consistently supports a defect in auto-
phagic vacuole clearance.
Proteolysis Deficits in Autolysosomes
of PS1 KO Blastocysts
We investigated further the basis for delayed clearance of LC3
and other autophagy substrate proteins by examining the acti-
vation of cathepsins in PS1 KO cells. Western blot analyses of
Cathepsin D (CatD), the major aspartyl protease of lysosomes,
showed slightly elevated total Cat D immunoreactivity levels
but a more rapid migration of the mature single chain enzyme
on gels than the WT enzyme (46 kDa) and decreased proteo-
lytic generation of 31 and 14 kDa forms of the mature enzyme
in PS1 KO cells (Figure 3A). This deficit was similar to that seen
in WT cells when lysosomal pH was neutralized by treatment
(Figure S3A). Metabolic labeling data confirmed that Cat D
maturation was impaired in PS1 KO cells (Figure S3B). To
assess Cat D activation within lysosomes, we incubated cells
with Bodipy-FL-pepstatin A, which binds selectively to active
Cat D (Chen et al., 2000). Dual fluorescence analyses of WT
cells with Cat D antibodies showed strong double labeling of
compartments with Cat D antibodies and Bodipy-FL-pepstatin
A, which was nearly completely abolished when lysosomal pH
was neutralized by treatment with NH4Cl. By contrast, in PS1
KO cells, Bodipy-FL-pepstatin A labeling within Cat D-positive
vesicles was markedly reduced despite normal numbers of
these compartments (Figure 3B). To analyze Cat B activity
in situ, we used MR-Cat B, a cresyl-violet conjugated (Arg-
Arg)2peptide that fluoresces only after it is cleaved by Cat B
in an acidic environment. We confirmed that, in PS1 KO cells,
MR-CatB signal was dramatically reduced compared to the
WT and was similar to levels seen in WT cells in which lyso-
somal acidification was inhibited with NH4Cl (Figure S3D).
In vitro assays of Cat D activity in PS1 KO cells in the absence
or presence of rapamycin confirmed a markedly reduced
proteolytic activity relative to WT cells (Figure 3C). The activities
of the cysteine proteases Cat B and Cat L were also similarly
lowered in PS1 KO cells under these conditions (Figures S3E
and S3F). Similar or more severe reductions in cathepsins B,
D, and L activities were achieved in WT cells by incubating
them in the presence of NH4Cl (Figure 3C; Figures S3E
Defective Lysosome Acidification in PS1 KO Blastocysts
The possibility that lysosome acidification may be impaired,
raised by the foregoing observations, was further investigated
by evaluation of another process requiring lysosome acidifica-
tion, namely the dissociation of the cation-independent
mannose-6-phosphate receptor (CI-MPR) from cathepsins
after their delivery to late endosomes. Using double-immuno-
fluorescence labeling with antibodies to Cat D and CI-MPR,
we observed that most Cat D-positive vesicles were CI-MPR
negative in WT cells, but were nearly all CI-MPR positive in
PS1 KO cells (Figure S3C), indicating that dissociation of CI-
MPR from Cat D was impaired. CI-MPR coimmunoprecipitation
with Cat D revealed more Cat D bound to CI-MPR in PS1 KO
cells, a phenomena reversed by human PS1 reintroduction
(Figure S3C, bottom). To assess lysosome acidification directly,
we used LysoTracker. In WT cells, LysoTracker demonstrated
strong fluorescence in virtually all Cat D-positive vesicles
(Figure 3D), whereas in PS1 KO cells, fewer than 20% of Cat
(Figure 3F). Compared to that in WT cells, LysoTracker signal
in PS1 KO cells remained low after inducing autophagy with ra-
pamycin (Figures 3E, 3G, and 3H). Measurement of average
lysosomal pH with LysoSensor yellow/blue DND-160-Dextran
(Diwu et al., 1999) confirmed that WT cells display an average
lysosomal pH of 4.7 ± 0.08, consistent with previously pub-
lished studies (Ohkuma and Poole, 1978; Ramachandran
et al., 2009). By contrast, PS1KO cells displayed a substantially
elevated lysosomal pH of 5.4 ± 0.04 (p < 0.001) (Figure 3I). We
show that lysosomal acidification is normal in mouse fibroblasts
lacking Nicastrin (Nct), a g-secretase complex component
required for secretase activity (Figure S3H). In addition, g-sec-
retase inhibitor (L685,458) has no effect on lysosomal acidifica-
tion, Cat B activity and Cat D processing (Figures S3I and S3J).
This suggests that the effect of PS1 on autophagy is not
dependent on g-secretase.
Impaired Glycosylation and Targeting of the v-ATPase
V0a1 Subunit in PS1 KO Cells
To further understand the basis for the acidification defect in
PS1 KO cells, we investigated the v-ATPase V0a1 subunit as
a marker of proton pump function in lysosomes. Double-immu-
nofluorescence labeling analysis revealed strong colocalization
of v-ATPase with LAMP-2-positive compartments in WT cells
(Figure 4A). In PS1 KO cells, however, v-ATPase immunolabel-
ing was concentrated in a perinuclear region remote from
most of the peripherally distributed LAMP-2-positive compart-
ments (Figure 4B). v-ATPase immunoreactivity strongly colocal-
ized with calnexin, an ER-integral protein, in PS1KO cells
(Figure 4F) but minimally in WT cells (Figure 4E). As expected,
antigen-1) was nearly absent in early endosomes of both WT
EEA1 (early endosome
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. 1149
and PS1KO cells (Figures 4C and 4D). Quantitative analyses of
marker colocalization showed a significantly greater extent of
association of v-ATPase with ER than with lysosomes in
PS1KO cells, converse to the pattern seen in WT cells
(Figure 4G). Subcellular fractionation studies confirmed the
immunocytochemical results showing that v-ATPase was re-
tained in the ER-rich (calnexin-positive) fraction but was mark-
edly depleted from the lysosomal (LAMP2) fraction in PS1 KO
cells, whereas in WT cells, v-ATPase was enriched in both ER
and lysosomal fractions (Figure 4H).
In subcellular fractions from PS1 KO cells, we also observed
that the v-ATPase V0a1 subunit located in ER-enriched fractions
exists as a single 100 kDa band, whereas in WT cells it was
present as a double band (120/100 kDa) in the ER-enriched frac-
tion and as asingle 120 kDaband in the lysosome-enriched frac-
tion. In cell lysates treated with either PNGase F or O-glycanase
(Figure 4I), PNGase F converted the mature 120 kDa v-ATPase
form in WT cells to a 100 kDa form, suggesting that the 100 kD
protein is an unglycosylated core protein. Both 100 and 120
kDa bands were insensitive to Endo H (Figure 4J). Because
nicastrin glycosylation patterns are well established in WT and
PS1 KO cells (Herreman et al., 2003), we compared nicastrin
as a positive control, confirming these previously published
data and the competence of Endo H used in our experiments.
Figure 3. Cathepsin Processing and Activity Impairment in PS1 KO Cells
(A) Cat D immunoblots show reduced generation of the mature two-chain (31 kDa, 14kDa) form in PS1 KO cells, similar to NH4Cl treated (20 mM; 6 hr) WT cells.
(B) In vivo Cat D activity assays with Bodipy-FL-pepstatin A. After Bodipy-FL-pepstatin A treatment, cells were immunolabeled with Cat D antibody. Bodipy-FL-
pepstatin A binds to active Cat D of WT blastocysts and colocalize, but minimal colocalization is shown in PS1 KO cells or WT cells treated with NH4Cl. The scale
bar represents 50 mm.
(C) In vitro assays of Cat D enzyme activities in WT and PS1 KO cells with or without rapamycin or NH4Cl. ** for p < 0.001.
(D–H) Cells with or without rapamycin (10 nM; 6 hr) were preincubated with LysoTracker and immunolabeled with Cat D antibody (D–G). Cat D-positive compart-
ments were LysoTracker-positive in WT cells (D and E) but LysoTracker-negative in PS1 KO cells (F and G). Scale bars represent 50 or 10 mm.
(H) Quantitative analysis of LysoTracker and Cat D-positive compartments. ** for p < 0.001.
(I) Lysosomal pH values were measured ratiometrically using LysoSensor yellow/blue DND-160–Dextran. ** for p < 0.001.
Values are shown as means ± SEM. See also Figure S3.
1150 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
The results showed that v-ATPase maturation follows a different
pattern than that of nicastrin. Nicastrin maturation involves
a glycosylated Endo H-sensitive intermediate, which is the
precursor to a fully mature glycosylated Endo H insensitive
form. By comparison, the Endo H and PNGase F digestions indi-
cated that the 100 kDa v-ATPase V0a1 subunit is unglycosylated
and that the 120 kD form is a complex glycosylated mature
Figure 4. Lysosomal Targeting of v-ATPase Is Impaired in PS1 KO Blastocysts
(A–F) Double-immunofluorescence labeling shows strong colocalization of v-ATPase (V0a1 subunit) and LAMP-2 inWT cells (A) but minimal colocalization inPS1
KO cells (B). v-ATPase V0a1 and early endosomal marker, EEA1, show little colocalization in WT (C) and PS1 KO cells (D). v-ATPase V0a1 and the ER marker,
calnexin, strongly colocalized in PS1KO cells (F) but minimally colocalized in WT cells (E). Scale bars represent 20 or 10 mm.
(G) Quantitation analysis of v-ATPase V0a1 association with organelle markers (**for p < 0.001).
(H) Immunoblots of v-ATPase V0a1 subunit distribution in subcellular fractions of WT and PS1KO cells. Calnexin primarily localizes in fraction 12 and LAMP2
mainly in fraction 22. In WT cells, the v-ATPase V0a1 subunit, detected as 100 and 120 kDa bands was present in fraction 12 (120 kDa and 100 kDa) and fraction
22 (only in its 120 kDa form), but was primarily detected in the ER-rich fraction (only as a 100 kDa protein) of PS1KO cells.
(I)WTandPS1KOcelllysatestreatedwithPNGaseForO-glycanase.TheN-glycosylated formofv-ATPaseV0a1subunit (120kDa)wasdeglycosylated(100kDa)
after treatment with PNGase F but not with O-glycanase in WT cells. The v-ATPase V0a1 subunit in PS1 KO cells was not N-glycosylated, with the 100 kDa form
unchanged by treatment.
(J) Insensitivity to Endo H of 100 and 120 kDa bands in WT lysates. Both mature and immature glycosylated forms of nicastrin, serving as a positive control, are
(K) Cells incubated 24 hr withtunicamycin to block glycoprotein synthesisin the ER display reduced levels of 120 kDav-ATPase V0 subunit inWT cells but hadno
effect on 100 kDa subunit. Nicastrin immunoblot analysis under the same conditions is shown.
(L) After cell lysates were incubated with Con A beads, glycoproteins were eluted. Con A binds 120 kDa but not 100 kDa v-ATPase V0a1, and both mature and
partially glycosylated nicastrin species. Rab7, a negative control, was not bound.
Values are shown as means ± SEM.
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. 1151
To further confirm the glycosylation state of v-ATPase
variants, cells were incubated with tunicamycin and the mobility
of the v-ATPase V0a1 subunit expressed in the absence
or presence of tunicamycin was compared by western blot.
Tunicamycin treatment of WT cells caused a substantial loss
of the 120 kDa protein and increased levels of the 100 kDa
form. By contrast, tunicamycin had no effect on the mobility of
the 100 kDa form in either WT cells or in the PS1 KO cells where
this form was the only detectable variant (Figure 4K). Both
mature and partially glycosylated immature nicastrin were mark-
edly reduced, yielding small amounts of unglycosylated nicastrin
present in both WT and PS1 KO cells. As further confirmation of
the Endo H results, we also performed lectin affinity binding
studies using concanavalin A (Con A). As expected, Con A
bound the 120 kDa v-ATPase V0a1 species in WT cells, and
the mature and partially glycosylated forms of nicastrin, but
none of the 100 kDa v-ATPase species in PS1 KO cells, even
though this form was present in amounts comparable to the
mature forms of v-ATPase V0a1 subunit and nicastrin in WT
cells. Rab7, a negative control, did not bind Con A (Figure 4L).
These lines of evidence confirm that the 100 kDa protein is not
glycosylated and that the 120 kDa protein is the complex glyco-
sylated mature v-ATPase V0a1 subunit.
To investigate a possible role of PS1 in v-ATPase V0a1
assays with the endogenous proteins from WT cells and mouse
brain. Precipitation of endogenous v-ATPase V0a1 subunit led
to coprecipitation of full-length PS1 but not its more abundant
N- or C-terminal cleaved forms, indicating that only full-length
PS1 can bind to the v-ATPase V0a1 subunit (Figure 5A, top).
Importantly, PS1 preferentially coprecipitated with the immature
100 kDa form of v-ATPase V0a1 subunit (Figure 5A, bottom). In
WT mouse brain, only unglycosylated immature v-ATPase
bound to PS1, even though most of the v-ATPase V0a1 subunit
pool in this tissue was N-glycosylated (Figure 5B). These data
indicated that uncleaved PS1 binds to immature v-ATPase
V0a1 to modulate its maturation in the ER and affect its delivery
The v-ATPase is a multicomplex molecule composed of a
and is only active when both are assembled in the lysosomal
membrane. We next examined the v-ATPase multicomplex
assembly status using membrane fractionation. Relatively small
amounts of the v-ATPase V1B subunit were present in the
membrane fraction and negligible amounts were detected in the
cytosolic fraction in PS1 KO cells relative to WT cells (Figure 5C).
This observation shows that PS1 deletion impairs v-ATPase
assembly and that the unassembled V1 subunit is depleted and
presumably degraded at greater rates in PS1 KO cells.
We suspected that PS1 may be involved in N-glycan transfer
from the oligosaccharyltransferase (OST) to the v-ATPase V0a1
subunit after its translation and translocation via the translocon
(Figure 5D). Supporting this mechanism, coimmunoprecipitation
data showed that endogenous full-length PS1 preferentially
coprecipitates with Sec61a (translocon subunit) and STT3B
(OST subunit) (Figure 5E) and vice versa; however, other ER
proteins, such as GRP94 and PDI, did not interact (data not
shown). Collectively, these data support a mechanism in which
full-length PS1 holds the v-ATPase V0a1 subunit close to the
OST complex, thereby facilitating posttranslational N-glycosyla-
tion of the subunit (Figure 5D), as described previously for
several other membrane proteins (Bolt et al., 2005; Ruiz-Canada
et al., 2009).
If PS1 is solely responsible for these deficits in autolysosome
function, exogenously reintroduced PS1 should be able to
rescue the PS1 KO phenotype. Stable transfection of human
PS1 into PS1/2 KO cells completely restored vesicular compart-
ment acidification, Cat D maturation, v-ATPase V0a1 glycosyla-
tion, and macroautophagy responses (Figure S4). These data
strongly support the conclusion that, under conditions of PS1
ablation, the v-ATPase V0a1 subunit is not N-glycosylated and
therefore is retained in the ER, thereby preventing acidification
of lysosome-related compartments and activation of proteases
in autolysosomes during autophagy.
Defective Vesicle Acidification and Autophagic
Pathology in Neurons of PS1 Hypomorphic
and PS cKO Mice
To extend these observations to neurons in vivo, we examined
whether AVs accumulate in association with defective lysosome
acidification in mouse models of PS1 hypofunction. PS1 hypo-
morphic mice, which express very low levels of PS1 protein
(1%) that are still sufficient for brain development and normal
tive compartments in cortical and hippocampal neurons
(Figure 6A) and 6-fold more immature AVs (p < 0.001), as quan-
tified by electron microscopy (EM) morphometry, compared to
WT mice (Figures 6B and 6C). To assess lysosome acidification
in vivo in these mice, we performed intraventricular injections of
DAMP, a probe sensitive to changes in vesicular pH and whose
abundance reflects the degree of acidification (Anderson et al.,
1984), followed by double-immunogold EM using antibodies to
CatD to identify lysosome-related vesicles and dinitrophenol to
detect the presence of DAMP. These data showed that vesicle
acidification was much less in Cat D-positive compartments in
neuronsof PS1hypomorph micethaninWTcontrols(Figure6D).
Similarly, PS cKO mice, under conditions of PS1 conditional
knockdown, exhibited abnormally increased numbers of LC3-
positive compartments and AV in brain (Figures S5A and S5B)
and significantly decreased acidification of lysosomal compart-
ments compared to controls (p < 0.001) (Figures S5C and
S5D). These results confirm that the PS1-dependent lysosomal
acidification defect observed in the cell culture system also
occurs in vivo in the brains of PS1-deficient mice.
PS1 Mutations Impair Macroautophagy and v-ATPase
Targeting in Fibroblasts from Patients with Familial AD
PS1-FAD and control human fibroblasts exhibited comparable
rates of [3H]-leucine incorporation into proteins (Figure 7A),
degradation of short-lived proteins (Figure 7B), and proteolysis
of long-lived proteins under autophagy-suppressed conditions
(Figure 7C). By contrast, when macroautophagy was induced
by serum withdrawal, PS1-FAD fibroblasts exhibited a minimal
rise in proteolysis compared to control fibroblasts (Figure 7C).
NH4Cl treatment eliminated the difference in proteolytic rates
between the two cell groups (Figure 7D), confirming that
1152 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
proteolysis impairment selectively involved the lysosomal
system in PS1-FAD fibroblasts. In an expanded analysis
including a total of 16 different PS1-FAD lines subclassified by
the specific PS1 mutation (A246E, M233T, H163Y, M146L,
L392V), autophagic protein degradation was lowered compared
to that in control cells (Figure 7E). The LC3-II/LC3-I ratio and also
total LC3-II levels in PS1-FAD fibroblasts were also compara-
tively high even in serum supplemented conditions and stayed
elevated compared to control cells after serum removal
(Figure S6A). To assess autophagosome formation, we immuno-
stained control and PS1-FAD fibroblasts with LC3 antibody,
which revealed increased LC3 vesicular puncta in PS1-FAD
compared to control fibroblasts after autophagy was induced
by serum withdrawal (Figure S6B). Morphometric ultrastructural
analyses revealed that immature AVs were more numerous in
PS1-FAD cells than in control fibroblasts where the AVs were
Figure 5. PS1 Directly Binds to the v-ATPase V0a Subunit Affecting Its Maturation and Assembly of the v-ATPase Complex
(A) Coimmunoprecipitation of endogenous PS1 with anti-v-ATPase V0a1 antibody and v-ATPase V0a1 with anti-PS1-NTF antibody. Precipitated proteins were
detected by immunoblot with either anti-PS1 (Ab14) or anti-v-ATPase V0a1. M, marker lane.
(B) Lysate from WT mouse brain was treated with PNGase F or O-glycanase. The v-ATPase V0a1 subunit is highly glycosylated in the mouse brain. The
N-glycosylated form of v-ATPase V0a1 subunit (120 kDa) is deglycosylated (i.e., MW shift to 100 kDa after treatment with PNGase F but not with O-glycanase).
The v-ATPase V0a1 subunit was immunoprecipitated with anti-PS1-NTF antibody and detected by anti-v-ATPaseV0a1 antibody. Only unglycosylated v-ATPase
V0a1 subunit coprecipitates with PS1.
(C) The top panel is a model of v-ATPase assembly. The immunoblot shows v-ATPase V1B1 subunit distributes between the membrane and cytosolic fractions.
(D)The diagram shows ahypothetical model of N-glycosylation of thev-ATPaseV0a1 subunit via PS1. PS1binding totransloconand OST complex facilitates the
presentation of the v-ATPase V0a1 subunit to the OST complex.
and STT3B were also coimmunoprecipitated each other. * represents a nonspecific band.
See also Figure S4.
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. 1153
of autophagic degradation (Figure S6C). We also confirmed with
LysoTracker that the level of acidification of the lysosomal
compartments (highlighted with LAMP-1) in PS1-FAD was mark-
edly lower (Figure 7F).
Toinvestigate themechanism underlying impairedautophagic
protein turnover in PS1-FAD fibroblasts, we used a double-
tagged mRFP-GFP-LC3 construct, enabling us to assess lyso-
some acidification in vitro. Almost all of the RFP and GFP signal
colocalized in PS1 FAD fibroblasts under serum starved condi-
tions (Figure S7A), indicating that acidification of lysosomes is
port of mRFP-GFP-LC3 to acidic compartments is delayed.
By contrast, in control fibroblasts, only 10% GFP colocalized
with RFP, indicating that formation of acidified autolysosomes
isefficient. Accumulation of p62 immunoreactivity before or after
serum starvation was disproportionately high in PS1-FAD fibro-
blasts by western blot analysis (Figure S7B, left) or by ICC anal-
ysis of p62 localization within LC3-positive compartments
(Figure S7B, right), consistent with delayed proteolytic autolyso-
somal clearance of this autophagy-selective substrate. Double-
immunofluorescence labeling with V0a1 antibodies established
that the v-ATPase was localized with CatD-positive compart-
ments in control fibroblasts, but only a small proportion of
v-ATPase colocalized with Cat D in PS1 FAD fibroblasts (Fig-
ures S7C and S7F); however, most v-ATPase immunoreactivity
Figure 6. Defective Autophagosome Accumulation and Acidification in PS1 Hypomorphic Mice
(A) LC3 immunohistochemistry ofPS1hypomorph brain showsgreaterLC3staining (arrow) inthePS1-deficient mouse compared tothe WT.Thescalebar repre-
sents 20 mm.
(B) EM of AVs and dystrophic neurite-like structures in brains of PS1 hypomorph mice compared to littermate controls. The scale bar represents 500 nm.
(C) Quantitation of AVs per EM field. ** for p < 0.001.
(D) DAMP,amarkerwhichlocalizes toacidic compartments,wasinfused intraventricularly intothebrainsofmiceand analyzed byimmuno-EMwithdinitrophenol
(DNP; 10 nm-gold, arrowheads) and CatD (6 nm-gold, arrows)antibodies. Graphs show quantitation of immunogold labeling for DAMP and CatD. ** for p < 0.001.
See also Figure S5. All values are means ± SEM.
1154 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
colocalized with PDI in PS1-FAD fibroblasts but not in control
fibroblasts (Figures S7D and S7F). Additional double-label anal-
yses showed that the v-ATPase V0a1subunit also colocalized
less with V1B1 subunit in PS1 FAD fibroblasts compared to
control cells (Figures S7E and S7F). Levels of v-ATPase were
Our results identify an essential role of PS1 in lysosomal-depen-
dent proteolysis directly relevant to the mechanism by which
PS1 mutations accelerate the pathogenesis of AD. PS1 deletion
prevented macroautophagic protein turnover while minimally
affecting nonlysosomal turnover of short- and long-lived
proteins. We have traced this defect in autophagy-dependent
proteolysis to inadequate autolysosome/lysosome acidification
resulting from a failure of the V0a1 subunit of v-ATPase to
become N-glycosylated in the ER and subsequently delivered
to autolysosomes/lysosomes. This acidification defect explains
the many other abnormalities of AV dynamics and autolysosome
maturation/digestion that we and others have observed in PS1
KO cells (Esselens et al., 2004; Wilson et al., 2004). Furthermore,
we also demonstrate that neurons in the brains of mouse models
of PS1 hypofunction and cells from patients with AD caused by
PS1 mutations show similar autolysosome maturation defects
as in PS1 KO cells. Lysosomal acidification is necessary to
dissociate CI-MPR from cathepsins, complete the proteolytic
maturation of cathepsin D, and activate cathepsins (Kokkonen
etal., 2004). Allof these functions were impaired in PS1 KO cells,
resulting in delayed proteolytic clearance of autophagic
substrates and their accumulation in autophagic vacuoles.
Transfection of WT hPS1 into PS1/2 KO cells rescued all of these
deficits. We observed similar autophagic/lysosomal deficits in
neurons of PS1 deficient mice, indicating a similar role for PS1
Multiple lines of evidence in our study established that defec-
tive autophagy in PS1 KO cells principally reflects a failure to
degrade autophagy substrates and clear AVs as a result of
a specific defect in lysosome/autolysosome acidification. This
evidence in PS1 KO cells includes abnormally elevated levels
of autophagy substrates (p62, LC3), a failure to clear AVs formed
after rapamycin-mediated autophagy induction, the minimal
effect of a cathepsin inhibitor (leupeptin) on LC3-II accumulation
in PS1 KO cells, impaired maturation of cathepsin D, reduced
specific activities of multiple cathepsins in vitro and in situ within
lysosomes, and impaired dissociation of MPR from cathepsins.
Impaired degradation of autophagy substrates in PS1 KO cells
is also indicated by the high proportions of early autolysosomes
compared to electron-dense late autolysosomes in morpho-
metric analyses and the low recovery of dense lysosomes in
Figure 7. Defective Autophagy in PS1-FAD
(A) [3H]-leucine incorporation into fibroblasts from
five different PS1-FAD patients and age-matched
(B) After [3H]-leucine labeling, proteolysis of short-
lived proteins was measured after the chase
(C) Degradationof long-lived
measured after incorporation of [3H]-leucine fol-
lowed by incubation in serum-supplemented or
-deprived medium during the chase period (up to
20 hr) (* for p < 0.05, n = 15).
(D) The increase in proteolysis at 12 hr after serum
removal relative to serum-replete conditions was
determined for control and PS1-FAD fibroblasts
cells treated with NH4Cl (20 mM) or 3MA (10 mM)
(** for p < 0.001, n = 15), or left untreated.
(E) Increases in degradation of long-lived proteins
after serum removal were compared in fibroblasts
from control (n = 11) and PS1-FAD patients
carrying different PS1 mutations (as labeled).
(F) Control or PS1-FAD fibroblasts treated in the
LysoTracker and immunolabeled for LAMP-1.
LAMP-1-positive compartments colocalized with
LysoTracker control cells, but not in PS1-FAD, as
verified by quantitative analysis. ** for p < 0.001.
Values are shown as means ± SEM. See also
Figures S6 and S7.
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. 1155
subcellular fractionations. All of these effects are expected
outcomes of a defect in the acidification of autolysosomes/lyso-
somes, which was demonstrated directly by the marked
decrease of LysoTracker fluorescence, ratiometric assays of
lysosomal pH, DAMP labeling, and the very low abundance of
the V0a1 subunit of v-ATPase in lysosome-related compart-
ments. Similar types of deficits and lysosomal pH increases of
a similar magnitude have been observed when v-ATPase is in-
hibited with bafilomycin (Yoshimori et al., 1991).
In contrast to these striking effects on autophagic proteolysis,
PS1 deletion did not detectably alter major upstream aspects of
macroautophagy, including nutrient-dependent regulation of
mTOR, a protein kinase-signaling pathway that utilizes AKT-
PI3K (Sarbassov et al., 2005) and is modulated in part by PS2
(Kang et al., 2005). In response to autophagy induction,
p70S6K was dephosphorylated normally and LC3-positive
puncta and AVs increased modestly but significantly above an
already elevated level in PS1 KO cells, indicating preservation
of an ability to form autophagosomes. The accumulation of
p62, a known autophagy substrate (Bjørkøy et al., 2005), indi-
cated that PS1 KO cells can sequester substrates despite the
subsequent impairment of degradation. The presence of LC3/
LAMP2-positive profiles in PS1 KO cells is evidence of autopha-
gosome-late endosome fusion as seen by Wilson et al. (2004),
but not by Esselens et al. (2004), and is consistent with other
evidence that autophagosome-lysosome fusion is not depen-
dent on lysosomal acidification (Jahreiss et al., 2008). Other
key aspects of lysosomal biogenesis and function were also
not detectably altered in PS1 KO cells, such as the delivery of
cathepsins and LAMPs to late endosome/lysosomes and the
distribution and levels of rab7 required for lysosome maturation
(Bucci et al., 2000). Thus, the failure of v-ATPase targeting to
lysosomes is a relatively selective effect of PS1 deletion on
proteolytic steps in the autophagy pathway, and, as expected,
the endocytic pathway.
The v-ATPasesare multisubunit
composed of a membrane-bound subcomplex V0 and a cyto-
solic V1 subcomplex (Forgac, 2007). We established by Endo
H digestion, tunicamycin treatment, and lectin affinity binding
that the 100 kDa immature form of v-ATPase V0a1 subunit is un-
glycosylated. Its physical interaction with PS1 enables the
N-glycosylation required for this subunit to be efficiently deliv-
ered to lysosomes (Gillespie et al., 1991; Nishi and Forgac,
2002). This is not a generalized effect on the N-glycosylation
mechanism itself since LAMP-2 was normally glycosylated in
PS1KO cells. Most commonly, N-glycosylation occurs cotrans-
lationally (Kelleher and Gilmore, 2006); however, for some
proteins, glycosylation occurs after the entire polypeptide has
been translocated into the ER lumen (Bolt et al., 2005) and
may preferentially involve the STT3B catalytic subunit of the
OST (Ruiz-Canada et al., 2009). Consistent with a posttransla-
tional glycosylation of the v-ATPase V0a1 subunit, our coimmu-
noprecipitation studies showed that PS1 binds to STT3B. Given
this context, our evidence that the 100 kDa v-ATPase is unglyco-
sylated supports a model (Figure 5D) of posttranslational
N-glycosylation of v-ATPase V0a1 subunit, a function lost
when PS1 is deleted. Our findings implicating PS1 holoprotein
provide an explanation for the observation (Esselens et al.,
complexes that are
2004) confirmed here that PS1 effects on autophagy are not
dependent on g-secretase activity.
maturation and trafficking of the v-ATPase responsible for lyso-
some acidification. We have demonstrated the adverse conse-
quences of ablating this function for the normal turnover of
proteins and organelles by autophagy. It is likely that a failure
of PS1-dependent v-ATPase trafficking would have a range of
additional effects on functions of other compartments that rely
on this proton pump to acidify the intralumenal environment.
Finally, we have demonstrated the clinical relevance of this
PS1 function by showing that a range of mutations of PS1
causing early onset FAD also display a similar loss of lysosomal
function as in PS1 KO, which can account for the marked accel-
associated with PS1-FAD. Because Ab is both generated and
degraded duringautophagy (Yu etal., 2005),impaired lysosomal
FAD and that developing with a later onset in sporadic AD
suggests that lysosomal dysfunction is also a pathogenic mech-
anism in the common sporadic form of AD (Nixon et al., 2008).
Cell Lines, Mouse, and Reagents
Murine blastocysts with different PS1 genotypes (WT, BD6; PS1 KO BD15;
PS1/2 KO, BD8), previously characterized by Lai et al. (2003), were used in
this study. In addition, human PS1 WT was stably transfected into the BD8
line (Laudon et al., 2004). Human fibroblasts lines, acquired from the Coriell
Institute (Camden, NJ), Karolinska Institute (Upsala, Sweden), University di
Firenze (Italy), and University of Western Australia (Perth, Australia). PS1 hypo-
morph (Rozmahel et al., 2002) and PS cKO mice (Saura et al., 2004) were
studied at 13 month and 2–3 months, respectively, together with age-matched
Intracellular Protein Degradation Measurements
Confluent cells were labeled with [3H]-leucine (2 mCi/ml) for 48 hr at 37?C in
ordertopreferentially labellong-lived proteins.After labeling, cells wereexten-
sively washed and maintained in complete medium (Dulbecco’s Modified
plemented with unlabeled 2.8 mM leucine to prevent [3H]-leucine reincorpora-
tion into newly synthesized proteins. Total protein degradation measured by
pulse-chase was performed as described previously (Auteri et al., 1983).
Isolation and Subcellular Fractionation
identified using previous morphological criteria (Yu et al., 2005), were classi-
fied and counted on electron micrographs (79003 print magnification, of 20
EM images from each experimental group). AVs were isolated by centrifuga-
tion in a discontinuous metrizamide density gradient (Marzella et al., 1982)
for each cell. For subcellular fractionation, homogenate (0.5 ml) was layered
on top of an Optiprep (Sigma) step gradient (10%, 15%, 20%, 25%, and
30%, 2.3 ml each) into polyallomer tubes (Beckman) and centrifuged in
a SW-40Ti rotor with a model L8-80M Beckman ultracentrifuge (100,000 g,
16 hr, 4?C).
Gel Electrophoresis, Immunoblotting, and Deglycosylation
Immunoblotting was performed as previously described (Herreman et al.,
2003). In brief, cells used for western blot analyses were lysed in buffer con-
taining 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM ethylenediaminetetraacetic
acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Triton X-100,
1156 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
and 0.5% Tween-20 with protease and phosphatase inhibitors. After electro-
phoresis on 4%?20% gradient gel (Invitrogen), proteins were transferred onto
0.45 mm polyvinylidene fluoride (PVDF) membranes (Millipore) and then incu-
bated overnight in primary antibody followed by HRP-conjugated secondary
antibody. Blots were developed by ECL-kit (GE Healthcare).
Cathepsin B and L activities were assayed as described previously (Nakanishi
et al., 1994), and Cathepsin D activity was assayed with [14C]methemoglobin
as previously described by Dottavio-Martin and Ravel (1978).
Thequantitative colocalizationanalyses wereperformed withImageJ software
(NIH Image) with colocalization analysis plugins (Wright Cell Imaging Facility).
The values shown represent Pearson’s coefficient. Statistical analyses were
calculated by two-tailed paired student t test with GraphPad InStat (GraphPad
Software). Error bars represent standard error of the mean (±SEM).
figures, and one table and can be found withthis article online atdoi:10.1016/j.
We are very grateful to Alan Bernstein (Global HIV Vaccine Enterprise, Seattle)
for blastocysts, Tamotsu Yoshimori (Osaka University) for the tandem-tagged
LC3 construct, Samuel E. Gandy (Mount Sinai Medical Center) for PS1 anti-
body, Satoshi Sato (Kyoto University) for the rabbit pAb against 116 kDa
subunit of the v-ATPase, Jean Gruenberg (Universite de Geneve) for anti-
LBPA antibody, and Richard Cowburn (Karolinska Institute), Ralph Martins
(Cornell University), Sandro Sorbi (Universita degli studi di Firenze) for PS1-
FAD human fibroblasts, and Sangram S. Sisodia (University of Chicago) for
PS cKO mice. We also thank Gert Kreibich (New York University Langone
Medical Center) and members of the Nixon laboratory for valuable discussions
and Nicole Piorkowski for assistance with manuscript preparation. This work
was supported by National Institutes of Health grant number P01AG017617
(R.A.N) and the Alzheimer’s Association (R.A.N).
Received: October 21, 2008
Revised: March 17, 2010
Accepted: April 21, 2010
Published online: June 10, 2010
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1158 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
EXTENDED EXPERIMENTAL PROCEDURES
Cell Lines and Mouse
Murine blastocysts with different presenilin (PS1) genotypes (WT, BD6; PS1 KO BD15; PS1/2 KO, BD8) previously characterized by
Lai et al. (2003), were used in this study. In addition, human PS1 wt was stably transfected into the BD8 line (Laudon et al., 2004). The
cells were grown in 35 mm dishes in DMEM supplemented with penicillin/streptomycin (Invitrogen), 15% fetal bovine serum (Hy-
clone), and b-mercaptoethanol (Sigma). Human fibroblast lines (see Table S1), acquired from the Coriell Institute (Camden NJ), Kar-
olinska Institute (Upsala, Sweden), University di Firenze (Italy) and University of Western Australia (Perth), were maintained in MEM
(Invitrogen, Carlsbad CA) with 15% FBS (Hyclone, Logan, UT) at 37?C and 5% CO2. PS1 hypomorph (Rozmahel et al., 2002) and PS
cKO mice (Saura et al., 2004) were studied at 13 month and 2-3 month, respectively, together with age-matched controls. All animal
experiments were performed according to ‘‘Principles of Animal Care’’ (U.S. Office of Science and Technology Policy, 1985) and
approved by the Institutional Animal Care and Use Committee at the NKI.
Antibodies and Reagents
Rabbit pAb to LC3 (1/500) (Koike et al., 2005) and LC3 (1/200, Novus) were used for cell and mouse brain, respectively, immunoflu-
orescence studies and a polyclonal LC3 (generated in house, 1/1000) (Yu et al., 2005) was used for immunoblotting, anti-murine
LAMP (LAMP-2: ABL-93, 1/200 and LAMP-1: 1D4B, 1/5 or H4A3, 1/200) mAb was purchased from Developmental Studies
Hybridoma Bank. Rabbit anti-Cathepsin D pAb (1/1000) was purchased from Scripps Laboratories for MeOH fixed cell ICC, Rabbit
anti-Cathepsin D pAb (1/5000) was generated in house for 4% PFA fixed ICC and Western blot, and rabbit polyclonal antibody to Cat
D (1:50, IEM) was purchased from DAKO. The Anti-PDI mouse mAb (1/5000) and Anti-GRP94 rat mAb (9G10, 1/10000) were
purchased from Assay designs. Anti-Calnexin mouse mAb (1/1000) was from Affinity Bioreagent. The mouse monoclonal anti-
LBPA antibody (1/2) was a generous gift from Dr. Jean Gruenberg. Anti-EEA1 mouse mAb (clone 14, 1/1000) was purchased
from BD Bioscience. The mouse monoclonal anti-CI-MPR (clone 2G11, 1/500) and mouse monoclonal anti-rab7 were from Abcam.
Guinea pig polyclonal anti-p62 (GP62-C, 1/2000) was from Progen Biotechni and mouse monoclonal anti-human p62 (1/1000) was
ml), DAMP (30 mM stock), and mouse monoclonal antibody to DNP (1:50) were from Invitrogen. Total p70S6K (#9292, 1/1000) and
phospho-p70S6K (#9206, 1/1000) were purchased from Cell Signaling. Anti-PS1 rabbit pAb (Ab14) was a generous gift from Dr.
Sam Gandy, anti-PS1-NTF rabbit pAb (34-4600, 1/1000) was purchased from Zymed. Anti-PS1 loop mouse mAb (MAB5232,
1/1000) and anti-nicastrin mouse mAb (MAB5556, 1/1000) were purchased from Chemicon. b-tubulin (clone 2-28-33, 1/5000),
anti-actin (clone AC-40, 1/5000), and rabbit polyclonal anti-GAPDH (1/5000) were purchased from Sigma. A rabbit pAb against
mouse v-ATPase V0a1 (W249, 1/5000) and mouse pAb against human v-ATPase V0a1 (Osw2, 1/500) were generous gift from Dr.
Satoshi Sato and other rabbit pAb against V0a1 subunit of the vacuolar proton pump (1/500) was purchased from Synaptic Systems.
The v-ATPase V1 B1 antibody (H-180, 1/200) was purchased from Santa Cruz Biotech. Following reagents were used for autophagy
modulation experiments. Rapamycin (Rapa, final 10 nM), ammonium chloride (NH4Cl, final 20 mM), bafilomycin A1 (final 0.2 mM) and
3-methyladenine (3MA, final 10 mM) were from Sigma and leupeptin (final 0.3 mM) was from Peptide Institute Inc. The g-secretase
inhibitor (L685,458) was purchased from Sigma.
Gel Electrophoresis, Immunoblotting, and Deglycosylation
Immunoblotting was performed as previously described (Yu et al., 2005). Briefly, cells used for Western blot analysis were lysed in
buffer containing 50 mM Tris (pH = 7.4), 150 mM NaCl, 1mM EDTA, 1 mM EGTA, 1% Triton X-100 and 0.5% Tween-20 with protease
and phosphatase inhibitors. Following electrophoresis on 4 ?20% gradient gel (Invitrogen), proteins were transferred onto 0.45 mm
PVDF membranes (Millipore) and the membrane was incubated overnight in primary antibody then incubated with HRP conjugated
secondary antibody. The blot was developed byECL-kit (GE Healthcare). To assessv-ATPase V0a subunit glycosylation, lysate from
WT and PS1KO cells were either treated for 24 hr at 37?C with PNGase F or O-glycanase using an enzymatic deglycosylation kit ac-
cording to the manufacturer’s instructions (PROzyme) or with Endo H (New England Biolabs) for 24 hr at 37?C. Cells were treated for
24 hr at 37?C with tunicamycin (5 mg/ml). Total glycoproteins were isolated using Glycoprotein Isolation Kit, ConA according to the
manufacturer’s instructions (Thermo Scientific).
Homogenate (0.5 ml) was layered on the top layer of 10, 15, 20, 25, and 30% Optiprep (Sigma) step gradient, 2.3 ml each, into poly-
allomer tubes (Beckman) and centrifuged in a SW-40Ti rotor with a model L8-80M Beckman ultracentrifuge (100,000 g, 16 hr, 4?C).
After centrifugation, the gradients were fractionated into 0.5 ml fractions. 40 ml of each collected fraction was mixed with an equal
volume of sample buffer and then loaded onto gels. The homogenates were fractionated into cytosolic and membrane fractions
by high speed centrifugation (150,000 x g, 60 min) and equal proteins was loaded on gel following 55?C for 10 min incubation
with 2x urea sample buffer. All data represent an average at least three independent experiments.
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. S1
Autophagic Vacuole Isolation
AVs were isolated bycentrifugation in a discontinuous metrizamide densitygradient (modified from Marzella et al.,1982) for each cell
line. Cells (5 3 108) were serum-deprived overnight to induce autophagic activity prior to AV isolation. The cells were collected, dis-
rupted by nitrogen cavitation then homogenized in 3 volume of 0.25 M sucrose in a glass homogenizer with Teflon pestle for 10
at 17,000 x g for 12 min to yield a pellet and a supernatant which was spun again at 100,000 x g for 1 hr to yield a pellet containing ER
and a supernatant containing cytosol. The pellet from the 17,000 x g centrifugation was resuspended in the same volume of 0.25 M
sucrose and spun again at 17,000 x g for 12 min. The pellet was resuspended in 1.9 ml of 0.25 M sucrose and 2.8 ml of 85.6% met-
rizamide (Mtz). This mixture (2.4 ml volume) was layered on top with a 26% (4 ml), 24% (2 ml), 20% (2 ml) and 15% (2 ml) Mtz step
gradient matrix. The sample on the Mtz gradient was centrifuged at 247,000 rpm for 3 hr in an ultracentrifuge using an SW41 rotor.
Each gradient interface was collected and diluted in 0.25 M sucrose. The samples were then pelleted at 24,000 x g for 10 min. Light
26% interface, and mitochondria were located in the 26% Mtz area. Fractions were pelleted and immersed in a cacodylate fixation
buffer for EM analysis or analyzed directly by Western blot or enzyme assay. All data represent an average at least three independent
experiments, unless otherwise indicated.
Ultrastructural and Morphometric Analyses
Following treatments, cells were prepared for EM as previously described and AVs, identified using previous morphological criteria
in each experimental group.
Lysosomal pH Measurement
Quantification of lysosomal pH was determined using Dextran conjugated Lysosensor Yellow/Blue DND-160 (Invitrogen). Wild-Type
and PS1KO blastocysts were grown in High Glucose DMEM + 15% FBS with antibiotics to ?90% confluency. Cells were then trypsi-
nized, harvested (1 X 106cells/ml) and loaded with 1mg/ml of Lysosensor-dextran for 1 hr at 37?C with 5% CO2. The cells were then
washed 3X in HBSS and aliquoted at 100 ml into a black 96-well microplate. pH calibration was performed according to the protocol
established by Diwu et al. (Diwu et al., 1999). Briefly, wild-type and PS1KO blastocysts were treated with 10 mM monensin and 10mM
nigericin in MES buffer (5 mM NaCl, 115 mM KCL, 1.3 mM MgSO4, 25 mM MES), with the pH adjusted to a range from 3.5-7.0. The
calculated for each sample. The pHvalues were determined fromthe linear standard curvegenerated via the pHcalibration samples.
In Vivo Vesicle Acidification Study
The mouse was anaesthetized with a 1% body weight IP injection of chloral hydrate, (400 mg/kg at a concentration of 50 mg/ml, 26 g
needle, and volume less than 200 ul) and allowed sufficient time to go down. Under sterile conditions, the subject was then shaved,
and cleaned at the site of the surgery, in this case the scalp. The subject was placed in position in a stereotaxic holder with drill
(BenchMark). The position of the drill/burr arm was located in right ventricle (?0.22 mm from bregma; L, ?1 mm; D/V, ?2.5 mm),
the coordinates relative to the bregma, were determined by using The Mouse Brain (Keith B. J. Franklin and George Paxinos,
Mouse was injected with DAMP (20 ml of 30 mM stock solution prepared in PBS) by intra-ventricular method with 1.5 ml/min speed.
After 4hrs, animals were anesthetized and perfused with a fixative containing 0.1% glutaraldehyde and 4% paraformaldehyde in
sodium cacodylate buffer (Electron Microscopy Sciences). Brains were dissected and immersed in the same fixative for 4 hr and
then 40 mm sagital sections were made using a vibratome. The sections were processed routinely for electron microscopy and
subsequently washed with PBS and incubated for 2 hr in room temperature with secondary antibody coupled with 10 nm gold.
Sections were washed with PBS and were stained briefly with uranyl acetate and lead citrate. Sections were examined and photo-
graphed with a Philips CM10 electron microscope.
Intracellular Protein Degradation Measurements
Total protein degradation in cultured cells was measured by pulse-chase experiments (Auteri et al., 1983). Confluent cells were
labeled with [3H]-leucine (2 mCi/ml) for 48 hr at 37?C in order to preferentially label long-lived proteins. Following labeling, cells
is suppressed, or in serum-deprived medium, where autophagy is induced. Under both conditions, after washing the cells, the
medium was supplemented with unlabeled 2.8 mM leucine to prevent [3H]-leucine reincorporation into newly synthesized proteins.
Aliquots of the medium taken at different time-points were precipitated with 10% TCA, filtered using a 0.22 mm pore membrane and
radioactivity in the flow-through was measured. Proteolysis is expressed as the percentage of the initial acid-precipitable radioac-
tivity (protein) transformed to acid-soluble radioactivity (amino acids and small peptides) over time. To inhibit autophagy in this
system, 20 mM NH4Cl or 10 mM 3MA was added immediately after the labeling period and maintained at that concentration
S2 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
throughout the chase. NH4Cl blocks lysosomal degradation, so this procedure permits one to estimate the relative contributions of
somes and is used to block macroautophagic contributions to proteolysis. Degradation rates of short-half life proteins were deter-
mined by the same procedure but after a labeling period of 30 min at 37?C. Nearly all turnover of short-lived proteins is due to pro-
teasomal activity. Protein synthesis was determined as the incorporation of [3H]leucine into acid-insoluble material in the presence of
an excess (2.8 mM) unlabeled leucine in the medium. Under these conditions incorporation of radioactivity into protein accurately
reflects rates of proteins synthesis and minimizes differences due to alteration of amino acid transport and/or intracellular amino
acid pool sizes.
Confocal Laser Scanning Microscopy
Immunocytochemistry was performed as previously described (Yu et al., 2005). Secondary antibodies used were from Invitrogen:
mat 40x or 100x/1.4 oil DIC objective lens on the laser scanning confocal microscope, LSM 510 META, with LSM software v3.5 (Carl
Zeiss MicroImaging Inc). Images were analyses using ImageJ program (NIH). Organelles with low internal pH were labeled by Lyso-
Tracker DND-99 dye (Invitrogen) at the final 100 nM for 30 min. Following 4% PFA fixation, cells were further immunolabeled with Cat
D, LAMP1, or LC3 antibody for 4 hr then visualized with Alexa Fluor 488 conjugated secondary antibody. Active Cat D was labeled by
counter stained with Cat D antibody for 4 hr and visualized with Alexa Fluor 568 conjugated secondary antibody. For assessing Cat B
activation, MagicRed–Cathepsin B (Immunochemistry Technologies) for active Cat B was added to the cells at the concentration
suggested by company (1:260). Cells were incubated for 30 min with MR-Cathepsin B prior to mounting them under the confocal
microscope. To block v-ATPase proton pump activity, ammonium chloride (NH4Cl) was added directly to the medium at a final
concentration of 20 mM for 6 hr prior to LysoTracker addition.
Wild-type mouse blastocysts cells were lysed with lysis buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 100 mM EDTA, and protease
inhibitor cocktail) with 1% digitonin. Immunoprecipitations of complex proteins with anti-PS1 pAb, anti-V-ATPase V0a1 subunit pAb,
anti-Sec61a, or anti-STT3B were performed using the Seize Primary immunoprecipitation kit (Pierce Biotechnology) (Hiesinger et al.,
Degradation of HRP Internalized by Fluid Phase Endocytosis
HRP degradation was evaluated in cells that were incubated for 30 min at 37?C with medium containing 5 mg/ml horseradish perox-
idase (HRP) (HRP type II; Sigma) and washed three times with phosphate-buffered saline then chased 37?C for 0, 1, and 6 hr with
fresh media. Following fixation, the cells were immunolabeled with rhodamine-conjugated anti-HRP antibody (Jackson lab).
Cathepsin B and L activity were assayed as described previously (Nakanishi et al., 1994) and Cathepsin D activity was assayed using
100 ml of 0.4 M acetate buffer (pH 3.2) containing 10 mg/ml [14C]methemoglobin. Cathepsin D activity was expressed in terms of
methemoglobin degrading activity; one unit of enzyme activity is defined as the capacity to degrade one nanomol of methemoglobin
Cathepsin D Metabolic Labeling
Cells were grown in a 3.5 cm dish to 5 3 105cells/dish, after which they were metabolically labeled for 30 min with 100 mCi/ml
[35S]methionine/cysteine (EXPRE35S35S Protein Labeling Mix, NEG0702 14MC: Perkin Elmer) and chased with normal medium
forspecific periods.Thelabeledcellswerelysedandsubsequentlyprocessed forimmunoprecipitationwith anti-ratcathepsin Danti-
body and protein G-agarose bead (Santa Cruz Biocech). The immunoprecipitates were analyzed by 10% SDS-PAGE.
Transfection of Plasmid
amine 2000 (Invitrogen) according to manufacture’s instruction. Following transfection fibroblasts were incubated for 24 hr and
before imaging incubated for another 6 hr with serum or without serum medium.
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elles of live cells. Chem. Biol. 6, 411–418.
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Dottavio-Martin, D., and Ravel, J.M. (1978). Radiolabeling of proteins by reductive alkylation with [14C]formaldehyde and sodium cyanoborohydride. Anal. Bio-
chem. 87, 562–565.
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Koike, M., Shibata, M., Waguri, S., Yoshimura, K., Tanida, I., Kominami, E., Gotow, T., Peters, C., von Figura, K., Mizushima, N., et al. (2005). Participation of
autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease). Am. J. Pathol. 167, 1713–1728.
Lai, M.T., Chen, E., Crouthamel, M.C., DiMuzio-Mower, J., Xu, M., Huang, Q., Price, E., Register, R.B., Shi, X.P., Donoviel, D.B., et al. (2003). Presenilin-1 and
presenilin-2 exhibit distinct yet overlapping gamma-secretase activities. J. Biol. Chem. 278, 22475–22481.
Laudon, H., Mathews, P.M., Karlstro ¨m, H., Bergman, A., Farmery, M.R., Nixon, R.A., Winblad, B., Gandy, S.E., Lendahl, U., Lundkvist, J., and Na ¨slund, J. (2004).
Co-expressed presenilin 1 NTF and CTF form functional gamma-secretasecomplexes in cells devoid of full-length protein. J. Neurochem. 89, 44–53.
Nakanishi, H., Tominaga, K., Amano, T., Hirotsu, I., Inoue, T., and Yamamoto, K. (1994). Age-related changes in activities and localizations of cathepsins D, E, B,
and L in the rat brain tissues. Exp. Neurol. 126, 119–128.
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PS1 hypomorphic mice with markedly reduced g-secretase cleavage of betaAPP. Neurobiol. Aging 23, 187–194.
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S4 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
Figure S1. Ultrastructural Morphometry of AVs in WT and PS1 KO Cells, Related to Figure 1
(A) AV ultrastructure in WT and PS1 KO cells grown in the presence (control) or absence (-serum) of serum. Scale bar represents 500 nm.
(B) AVs were subcategorized based on morphology (AP: autophagosome - double membrane and/or undigested organelles, EAL: early autolysosome - single
membrane with relatively undigested material, LAL: late autolysosome - single membrane with amorphous electron-dense material. Double arrow heads repre-
sent double membranes and single arrow heads represent single membranes. Scale bar represents 500 nm.
(C) Quantitative analysis of the relative proportions of vacuoles (categorized base on B) in WT and PS1 KO cells (** for p < 0.001, n = 20 EM images). All values are
reported as mean ± SEM.
(D) Ultrastructure of lighter and heavier AVs (AV10, AV20, respectively) and lysosomes purified from serum-starved (6 hr) WT and PS1 KO cells. Long arrows
highlight the more abundant immature AV forms in PS1 KO fractions compared to those in WT fractions. Scale bar represents 500 nm.
(E) Immunofluorescence labeling using antibodies to LC3 in WT and PS1 KO cells under conditions of no treatment (Ctrl), serum starvation (w/o serum, 6 hr),
rapamycin (Rap, 10 nM; 6 hr), rapamycin treatment followed by rapamycin removal (Rap/RC) for 6 hr, and 3MA (10 mM; 6 hr). Scale bar represents 10 mm.
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. S5
Figure S2. Endogenous and exogenous protein degradation impairment in PS1 KO cells, Related to Figure 2
(A) Immunoblot analysis of LC3-I and –II levels in cells following leupeptin treatment.
(B–E) Double-immunofluorescence analysis of WT (B,C) and PS1 KO cells (D,E) with LC3 and LAMP-2 antibodies after treatment with leupeptin (0.3 mM) (C,E).
Quantitative immunoblot analysis of data in (A) is presented graphically as mean ± SEM for 3 different experiments. Scale bar represents 10 mm. ** for p < 0.001.
(F) Levels of p62, an intracellular autophagy substrate, are increased in PS1KO cells.
rodamine-conjugated anti-HRP antibody. Internalized HRP is rapidly degraded within 1 hr in WT cells but remains for 6 hr in PS1 KO cells, indicating impaired
lysosomal proteolysis in PS1 KO cells. Scale bar represents 50 mm and 10 mm (enlarged panel).
S6 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
Figure S3. Cathepsin D Maturation Is Delayed in PS1 KO Cells and Lysosome Acidification Is g-secretase Independent, Related to Figure 3
(A) Cat D immunoblots show reduced generation of the mature two-chain (31 kDa; 14 kDa) form in PS1 KO cells as in bafilomycin A1 (Baf, 100 nM) treated WT
(B) WT and PS1 KO cells were metabolically labeled for 30 min and chased for the indicated time periods. The generation of the mature form (31 kDa) of Cat D is
impaired in PS1 KO cells.
(C) Double-immunofluorescence labeling with antibodies to Cat D and CI-MPR shows strong co-localization in PS1 KO cells in contrast to untreated WT cells but
similar to the pattern in WT cells treated with NH4Cl (20 mM; 6 hr). CI-MPR co-precipitated with anti-Cat D antibody and immunoblotted with antibodies against
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. S7
CI-MPR. Compared to WT cells, larger amounts of Cat D were bound to CI-MPR in PS1 KO cells and this pattern was reversed by human PS1 transfection (**p <
0.001, n = 3).
(D) MR-Cat B signal is abundantly evident in WT cells but is minimal in PS1 KO cells, similar to that in NH4Cl treated WT cells. DIC images are depicted to aid in
visualizing cells. Scale bar represents 50 mm.
(E and F) In vitro assays of Cat B (E), and L (F) enzyme activities in WT and PS1 KO cells in the absence or presence of rapamycin or NH4Cl.
(G) Nicastrin (Nct) gene knockout mouse fibroblasts were immunostained with anti-nicastrin antibody.
(H) WT or Nct KO fibroblasts were incubated with 100 nM LysoTracker for 1 hr and immunolabeled with polyclonal antibody against cathepsin D. Cat D-positive
compartments are LysoTracker-positive in both WT and Nct KO cells. Scale bar represents 50 mm.
treated cells but is minimal after leupeptin treatment.
(J) WT blastocysts were incubated with 100 nM LysoTracker for 1 hr followed by L685,458 (final 10 nM, 18 hr) treatment and immunolabeling. DIC images are
depicted to aid in visualizing cells. Scale bar represents 50 mm. Quantitative analysis of data is presented as mean ± SEM for 3 different experiments. ** for
p < 0.001.
S8 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
Figure S4. The hPS1 Introduction Restored Autophagic/Lysosomal System, Related to Figure 5
(A) Lysosome acidification is restored after human wild-type PS1 is stably transfected into PS KO (PS1/2 KO) cells. Scale bars represent 50 mm and 20 mm
(B) PS1 introduction rescues v-ATPase V0a1 subunit and Cat D maturation (31 kDa form).
(C) Top panel depicts Western blot of PS1 and middle panel shows, in the same cell types, serum deprivation-dependent increases in proteolysis based on
measurements of proteolysis in the absence of serum relative to those in the presence of serum. The increases in proteolysis at 12 hr after serum removal
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. S9
were determined for WT, PS1 KO, PS KO (PS1/2 KO) and hPS1 incubated in the presence or absence of serum. Bottom panel shows the percentage of 3MA-
sensitive proteolysis determined for WT, PS1 KO, PS KO and hPS1 incubated with and without 3MA (10 mM) for 12 hr in the absence of serum (** for p < 0.001,
n = 9).
(D) LC3-IIWestern blots and graphs depicting results of densitometricanalyses of LC3-II and LC3-I immunoreactivity and expressionas LC3-II/LC3-I ratios using
tubulin as a loading control. Values are the mean ± SEM. ** for p < 0.001, n = 3).
(E) Morphometric ultrastructural analyses of electron micrographs (EM) show that engulfed materials by autophagy were sufficiently digested in serum starved
conditions in hPS1 cells.
S10 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
Figure S5. Defective autophagosome accumulation and acidification in PS cKO mice, Related to Figure 6
(A) LC3 immunohistochemistry of brains of PS cKO mice show agreater level of LC3 positive staining in the PS1 altered models compared to their controls. Scale
bar 50 and 10 mm, respectively.
(B) Electron micrographs show an increase of AV and dystrophic neurite-like structures in PS cKO (arrowheads) compared to their littermate controls. Scale bars
equal 500 nm.
(C) DAMP, amarker which localizes to acidic compartments, was infused intraventricularly into the brains of mice, which were then analyzed by immuno-electron
microscopy using DNP (arrowheads) and CatD (arrows) antibodies.
(D) Graphs show quantitative results of immunogold labeling for DAMP and CatD. **p < 0.001.
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. S11
Figure S6. Defective autophagy in PS1-FAD human fibroblasts, Related to Figure 7
(A) LC3-II Western blot and graph depicts results of densitometric analyses of LC3-II and LC3-I immunoreactivities and expression as LC3-II/LC3-I ratios using
tubulin as a loading control (** for p < 0.001, n = 3). All values are the mean ± SEM.
(B) Immunostaining for LC3 of control and PS1 FAD fibroblast following incubation in the presence or absence of serum. Scale bar represents 10 mm.
(C) Electron micrographs of human fibroblasts from non-affected and PS1-FAD human fibroblasts were examined following treatment in FBS (+FBS) and 12 hr in
FBS-free media (-FBS). AVs with relatively undigested material and amorphous electron-dense digested materials were identified ultrastructurally in PS1 FAD
fibroblasts. Scale bar represents 500 nm.** for p < 0.001. All values are reported as mean ± SEM of n = 30.
S12 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.
Figure S7. Defective Acidification and Lysosomal Targeting of v-ATPase Was Impaired in PS1 FAD Fibroblasts, Related to Figure 7
(A) control (7621) and PS1-FAD (6840) fibroblasts were grown in the absence of serum (6 hr) followed by transfection of the mRFP-GFP-LC3 construct and
live confocal imaging in an incubation chamber. Bottom panel shows the quantitative analysis of GFP and RFP-positive compartments. Scale bar represents
20 mm. ** for p < 0.001.
(B) p62 immunoblot shows increased p62 protein levels in PS1-FAD fibroblasts in either the presence or absence of serum (6 hr). Double-immunostaining shows
LC3/p62 positive compartment size is increased in PS-FAD fibroblasts. * for p < 0.01. Scale bar represent 50 and 10 mm.
(C) Double-immunofluorescence labeling with antibodies to v-ATPase (V0a1 subunit) and CatD showing strong co-localization in control fibroblast but signifi-
cantly reduced co-localization in PS1 FAD fibroblasts.
Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc. S13
(D) Double immunostaining with antibodies to v-ATPase (V0a1 subunit) and PDI, ER marker shows strong co-localization in PS1 FAD fibroblasts but minimal co- Download full-text
localization in control fibroblast. All values are reported as mean ± SEM for n = 30 cells.
(E)v-ATPasecomplexformationwasimpairedinPS1FADfibroblast. Double-immunofluorescencelabelingwithantibodiestov-ATPase(V0a1subunit)and v-AT-
Pase (V1B1 subunit) shows strong co-localization in control fibroblast but significantly reduced co-localization in PS1 FAD fibroblasts.
(F) Quantitative analysis of v-ATPase V0a1 subunit association with organelle markers. Values are the mean ± SEM of n = 30 cells. ** for p < 0.001. (G) v-ATPase
V0a1 subunit Western blots and graphs depicting results of densitometric analyses of v-ATPase V0a1 immunoreactivity and expression as total amount using
GAPDH as a loading control (* for p < 0.05).
S14 Cell 141, 1146–1158, June 25, 2010 ª2010 Elsevier Inc.