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Gas1 Interferes with AβPP Trafficking by Facilitating the
Accumulation of Immature AβPP in Endoplasmic Reticulum-
Associated Raft Subdomains
Julien Chapuisa, Valérie Vingtdeuxa, Hemachander Capirallaa, Peter Daviesa,b, and Philippe
Marambauda,*
aLitwin-Zucker Research Center for the Study of Alzheimer’s Disease, The Feinstein Institute for
Medical Research, Manhasset, NY, USA
bDepartment of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA
Abstract
The amyloid-β protein precursor (AβPP) is a type I transmembrane protein that undergoes
maturation during trafficking in the secretory pathway. Proper maturation and trafficking of AβPP
are necessary prerequisites for AβPP processing to generate amyloid-β (Aβ), the core component
of Alzheimer’s disease senile plaques. Recently, we reported that the glycosylphosphatidylinositol
(GPI)-anchored protein growth arrest-specific 1 (Gas1) binds to and interferes with the maturation
and processing of AβPP. Gas1 expression led to a trafficking blockade of AβPP between the
endoplasmic reticulum (ER) and the Golgi. GPI-anchored proteins can exit the ER by transiting
through raft subdomains acting as specialized sorting platforms. Here, we show that Gas1 co-
partitioned and formed a complex with AβPP in raft fractions, wherein Gas1 overexpression
triggered immature AβPP accumulation. Pharmacological interference of ER to Golgi transport
increased immature AβPP accumulation upon Gas1 expression in these raft fractions, which were
found to be positive for the COPII protein complex component Sec31A, a specific marker for ER
exit sites. Furthermore, a Gas1 mutant lacking the GPI anchor that could not transit through rafts
was still able to form a complex with AβPP but did not lead to immature AβPP accumulation in
rafts. Together these data show that Gas1 interfered with AβPP trafficking by interacting with
AβPP to facilitate its translocation into specialized ER-associated rafts where immature AβPP
accumulated.
Keywords
AβPP; Alzheimer’s disease; (Gas1); lipid rafts; protein trafficking
INTRODUCTION
Alzheimer’s disease (AD) is the leading cause of dementia and is due to progressive
neurodegeneration in specific regions of the neocortex and hippocampus [1–3]. Two lesions
are invariably found in the AD brain: the neurofibrillary tangles formed by tau deposition
and senile plaques comprised of aggregated amyloid-β (Aβ) peptides [4, 5]. Aβ is produced
by cleavage of the amyloid-β protein precursor (AβPP) via the sequential action of two
© 2012 – IOS Press and the authors. All rights reserved
*Correspondence to: Philippe Marambaud, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New
York, NY 11030, USA. Fax: +(516) 562-0401; PMaramba@nshs.edu.
Authors’ disclosures available online (http://www.j-alz.com/disclosures/view.php?id=980).
NIH Public Access
Author Manuscript
J Alzheimers Dis. Author manuscript; available in PMC 2013 January 1.
Published in final edited form as:
J Alzheimers Dis
. 2012 January ; 28(1): 127–135. doi:10.3233/JAD-2011-110434.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
enzymes, β- and γ-secretases [6–9]. The identification of mutations in the AβPP gene
causing familial AD has underscored the central role played by AβPP in AD pathogenesis
[10, 11]. The trafficking of AβPP in the secretory pathway controls AβPP maturation in the
endoplasmic reticulum (ER) and the Golgi before AβPP transport to the cell surface [12,
13]. Proper AβPP trafficking and maturation, via O- and N-glycosylation for instance, are
necessary prerequisites for AβPP accessibility to the secretases at the cell surface or in the
endosomal system and thus for the production of Aβ [14]. In this context, the different
mechanisms controlling AβPP trafficking are under intense investigation.
To identify new genes potentially involved in AD pathogenesis, we previously used a tissue
expression profiling strategy [15, 16] to screen for genes preferentially expressed in the
hippocampus and located in AD chromosomal linkage regions. This approach identified the
CALHM1 (calcium homeostasis modulator 1) and Gas1 (growth arrest-specific 1) genes
[17]. We reported that CALHM1 codes for a novel calcium channel controlling cytosolic
calcium homeostasis and Aβ metabolism [17]. Some studies indicated that CALHM1 may
also control Aβ levels in human cerebrospinal fluid [18, 19] and influence AD age at onset
[17, 20–22]. These results indicated that tissue expression profiling is a valuable approach
for the identification of novel AD candidate genes.
Gas1 is a gene involved in the central nervous system development and in cell cycle control
[23]. Gas1 codes for a glycosylphosphatidylinositol (GPI)-anchored protein required for
normal postnatal proliferation in regions of the brain, such as the cerebellum [24–27]. Gas1
has also been associated with neuronal death in different models of excitotoxicity [28]. GPI-
anchored proteins can exit the ER by transiting through specific coat protein complex II
(COPII)-dependent raft subdomains acting as specialized sorting platforms at ER exit sites
[29, 30]. These ER exit sites express specific protein markers, such as the COPII component
Sec31A, and like plasma membrane lipid rafts, they are characterized by their resistance to
non-ionic detergents. The detergent-resistant property of these subdomains allows their
biochemical isolation in buffers containing Triton X-100 [29, 31].
We reported recently that, although Gas1 did not appear to represent an independent genetic
determinant of AD risk from the available genome-wide association studies, Gas1 can form
a complex with AβPP to control AβPP maturation and trafficking [32]. Gas1 expression was
found to inhibit AβPP full glycosylation and to interfere with AβPP routing to the cell
surface by leading to a trafficking blockade of AβPP between the ER and the Golgi.
Consequently, Gas1 overexpression led to a reduction of Aβ production and conversely,
Gas1 silencing in cells expressing endogenously Gas1 increased Aβ levels [32]. Here, we
show that Gas1 interacted with AβPP in specific raft fractions, wherein Gas1 overexpression
significantly facilitated the accumulation of immature AβPP. Using brefeldin A (BFA)
treatments, we found that interference of ER to Golgi transport increased immature AβPP
accumulation upon Gas1 expression in these raft fractions, which were found to be positive
for the specific ER exit site marker, Sec31A. Furthermore, a GPI-free C-terminally truncated
Gas1 mutant that could not transit through rafts was still able to form a complex with AβPP
but did not lead to immature AβPP accumulation in rafts. Together these data confirm that
Gas1 blocked AβPP trafficking between the ER and the Golgi and further show that Gas1
acted by interacting with AβPP to facilitate its translocation into specialized ER-associated
raft subdomains where immature AβPP accumulated.
MATERIALS AND METHODS
Materials and antibodies
Methyl-β-cyclodextrin (MCD) was purchased from Sigma-Aldrich. BFA was from
Epicentre Technologies. Anti-AβPP-(1–200) LN27 antibody was from Zymed Laboratories
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and anti-Aβ-(17–24) 4G8 antibody was from Signet. The anti-AβPP C-terminal domain R1
antibody was provided by Dr. P.D. Mehta (Institute for Basic Research in Developmental
Disabilities, Staten Island, NY). Antibodies directed against Gas1 were purchased from
Santa Cruz Biotechnology (C-17, sc-9585) and Genetex Inc. (GTX101732). Anti-actin, anti-
flotillin-1, and anti-Sec31A antibodies were from BD Transduction Laboratories. Anti-
calreticulin (CRT) antibody was from ABR-Affinity Bio Reagents.
Cell culture, transfections, and western blot (WB) analyses
N2a cells stably transfected with human AβPP695 harboring the Swedish double mutation
(AβPP-N2a) and HEK293 cells stably transfected with human wild type AβPP695 (AβPP-
HEK293) were maintained in 1 : 1 Dulbecco’s modified Eagle’s medium (DMEM)/Opti-
MEM supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a
humidified atmosphere with 5% CO2. All cell lines were tested negative for mycoplasma
contaminant [33]. Prior to transfection, cells were plated at a density of ~50%. Transient
transfection (24 h) of Gas1 cDNA (cloned into pCMV6 expression vector, Origene) was
performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
recommendations. For WB, cells were washed with PBS and solubilized in ice-cold lysis
buffer (25 mM HEPES, pH 7.4; 150 mM NaCl; 1% SDS; 1× Complete protease inhibitor
mixture, Roche Applied Sciences). Cell extracts (5–20 μg) were analyzed by SDS-PAGE
using the antibodies listed above. A standard ECL detection procedure was then used.
Sucrose gradient centrifugation
AβPP-HEK293 cells in a 100-mm dish were washed and harvested in ice-cold PBS, and
pelleted by centrifugation for 5 min at 1000 × g at 4°C. Cell pellets were lysed in 1.7 ml of
MBS buffer (25 mM MES, pH 6.5; 150 mM NaCl; 1% CHAPS; 1× Complete protease
inhibitor mixture), disrupted with 10 strokes in a glass Dounce homogenizer with a Teflon
pestle, and sonicated 3 times for 5 s. The resulting cell lysates were mixed with 2.3 ml of
MBS buffer containing 72% sucrose and added to the bottom of ultracentrifuge tubes. MBS
buffer containing 35% sucrose (4 ml) was laid over the cell lysates, followed by 4 ml of
MBS buffer containing 5% sucrose. Samples were centrifuged at 4°C for 16 h at 190,000 ×
g. Twelve fractions of 1 ml each were collected from the top to bottom of the ultracentrifuge
tubes. An aliquot of each fraction was subjected to SDS-PAGE and WB analyses, as
described above.
Preparation of Triton X-100-resistant membranes
Triton X-100-resistant membrane preparations were performed as described before [32].
Briefly, cells were lysed with Triton X-100 lysis buffer (TBS, 1% Triton X-100, 500 μM
sodium orthovanadate, 1× Complete protease inhibitor mixture) and under gentle shaking
for 20 min at 4°C. The crude lysates were then centrifuged at 16,000 × g at 4°C for 10 min.
The supernatants (Triton X-100-soluble fractions) were separated from the pellets and
placed into separate tubes. The pellets were washed with ice-cold Triton X-100 lysis buffer
and centrifuged again. Pellets (Triton X-100-resistant fractions) and Triton X-100-soluble
fractions were then analyzed by WB.
Co-immunoprecipitation assays
Twenty-four hours post-transfection with pCMV6-Gas1 or empty vector, cells were washed
with PBS and Triton X-100-resistant membranes were isolated as described above. Triton
X-100-resistant membranes were resuspended in ice-cold lysis buffer (50 mM Tris-HCl, pH
7.4; 150 mM NaCl; 1 mM EDTA; 0.25% deoxycholic acid; 1% NP-40; 1% Triton X-100; 1
× Complete protease inhibitor mixture) and disrupted with 10 strokes in a glass Dounce
homogenizer with a Teflon pestle and centrifuged at 16,000 × g for 5 min at 4°C. The
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supernatants were pre-cleared with 50 μl protein-G for 2 h at 4°C. After centrifugation at
1,500 × g for 30 s, supernatants were incubated overnight at 4°C with the indicated
antibodies. The following day, antibodies were precipitated by incubation with protein-G for
2 h at 4°C. Precipitates were washed 3 times with lysis buffer and then were resuspended in
loading buffer (50 mM Tris, pH 6.8; 5% β-mercaptoethanol; 2% SDS; 0.1% bromophenol
blue; 10% glycerol) and resolved by SDS-PAGE.
Statistical analysis
The results are presented as the mean ± standard deviation (S.D.). Significant differences
between groups were determined using the unpaired Student’s t-test. For all analyses, p <
0.05 was considered statistically significant.
RESULTS
Gas1 induces the accumulation of immature AβPP in rafts
Recently, we reported that Gas1 inhibited AβPP maturation and processing by leading to a
trafficking blockade of AβPP between the ER and the Golgi [32]. Because Gas1, like many
other GPI-anchored proteins, is known to localize in rafts, we asked whether Gas1
expression controls AβPP levels in rafts. Using sucrose gradient centrifugation, we observed
a significant shift of AβPP into the flotillin-positive raft fraction in Gas1-transfected cells, as
compared to control cells (Fig. 1A, fraction 5, and Fig. 1B). The effect of Gas1 expression
on AβPP translocation into rafts was confirmed by another method using Triton X-100-
resistant membrane preparations [34]. Similar to the observation using sucrose gradient
centrifugation, Gas1 expression resulted in a robust accumulation of AβPP in flotillin-
positive detergent-resistant raft fractions (Fig. 1C). Protein analysis using higher resolution
WB showed that low-molecular-weight immature AβPP specifically accumulated in raft
fractions upon Gas1 expression, whereas mature AβPP and AβPP C-terminal fragments,
CTFα and CTFβ, were detected at low levels in these fractions and were not significantly
affected by Gas1 expression (Fig. 1D and E). Of note, Gas1 interfered with AβPP trafficking
both in cells expressing wild type (AβPP-HEK293, Fig. 1A) and Swedish (AβPP-N2a, Fig.
1C) AβPP, suggesting that the Swedish mutation on AβPP did not impact Gas1 control of
AβPP trafficking. Rafts and detergent-resistant membrane fractions are enriched in
cholesterol and sphingolipids, two critical structural components for raft formation.
Cholesterol depletion using methyl-β-cyclodextrin was found to lower the accumulation of
both Gas1 and immature AβPP in detergent-resistant fractions (Fig. 1F and G), confirming
that immature AβPP accumulated and co-partitioned with Gas1 in rafts in Gas1-transfected
cells.
Gas1 and AβPP form a complex in rafts
Co-immunoprecipitation experiments previously revealed that Gas1 and AβPP can form a
complex in cells [32]. Here, we asked whether this interaction takes place in rafts by
performing reciprocal immunoprecipitations with antibodies directed against Gas1 or AβPP
in detergent-resistant raft fractions resuspended in lysis buffer containing raft disrupting
detergents. We found that Gas1 co-immunoprecipitated with AβPP in cells expressing the
two proteins (Fig. 2A). These results indicated that Gas1/AβPP interaction can occur in
rafts. We also observed that Gas1 preferentially co-precipitated with immature AβPP,
suggesting that Gas1 interacted with AβPP early in the secretory pathway prior to AβPP
maturation.
To confirm that Gas1 controls AβPP translocation into rafts, we asked whether inhibition of
Gas1 trafficking in rafts affects AβPP accumulation in rafts upon Gas1 expression. Because
incorporation of GPI-anchored proteins into rafts is selectively driven by their GPI anchor, a
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Gas1 construct was generated in which the GPI consensus sequence was removed (Fig. 2B).
We confirmed the inability of this GPI-free soluble Gas1 mutant (sGas1) to translocate into
rafts (Fig. 2C). We found that GPI-free sGas1 was still able to form a complex with AβPP
(Fig. 2D) but failed to trigger the accumulation of immature AβPP in rafts (Fig. 2E),
showing that Gas1 translocation into rafts is required for its effect on AβPP accumulation in
rafts. These results further demonstrate that the GPI anchor is dispensable for the Gas1/
AβPP interaction.
Gas1 controls immature AβPP translocation into ER-associated rafts
Gas1 expression led to the accumulation of immature AβPP in rafts (Figs. 1 and 2),
suggesting that the two proteins interacted early in the secretory pathway in a compartment
close to the ER. Because GPI-anchored proteins can exit the ER by transiting through
specific ER sorting platforms [29, 30], we next asked whether immature AβPP accumulates
and co-partitions with Gas1 in ER-associated rafts. We previously reported that while the
Triton X-100-soluble fractions are immunoreactive for calreticulin and therefore contained
most of the ER, the Triton X-100-resistant fractions were found to be strongly
immunoreactive for Sec31A and thus are enriched in ER exit sites, indicating that Gas1 may
facilitate AβPP accumulation at these specific ER-associated subdomains [32]. To go
further, we investigated whether interference of ER to Golgi transport prevents the effect of
Gas1 expression on AβPP translocation into rafts. Treatments with BFA, an agent that
blocks protein transport from the ER to the Golgi by disassembling the Golgi complex [35],
failed to inhibit immature AβPP accumulation in raft fractions, which were found to be still
positive for the ER exit site marker Sec31A and negative for the ER marker calreticulin
(Fig. 3A and B). Instead, we found that BFA treatments slightly but significantly
strengthened the effect of Gas1 expression on immature AβPP accumulation in rafts (Fig.
3A and B), confirming that AβPP accumulated in a compartment localized between the ER
and the Golgi. Altogether these data show that Gas1 expression led to immature AβPP
accumulation in ER-associated rafts.
DISCUSSION
Strong evidence indicates that lipid rafts are involved in AβPP amyloidogenic processing
[36, 37]. Lipid rafts are cholesterol- and sphingolipid-rich membrane microdomains and
studies in cell cultures and animal models have shown that alteration in cholesterol and
sphingolipid distribution or metabolism can significantly impact AβPP processing and Aβ
production [37, 38]. Both BACE1/β-secretase and γ-secretase are enriched in lipid rafts and
can target AβPP for Aβ production in these microdomains. γ-Secretase is involved in
regulated intramembranous proteolysis of multiple type I transmembrane proteins beyond
AβPP, such as Notch or N- and E-cadherins [8, 39]. γ-Secretase in the adult brain, however,
appears to preferentially target AβPP in lipid rafts [40]. Together, these results motivate
further studies aimed at determining whether targeting raft-dependent amyloidogenic
processing of AβPP is therapeutically relevant.
Lipid raft biogenesis and cellular trafficking is complex. Most rafts are found at the plasma
membrane where they follow the endocytotic pathway to recycle back to the plasma
membrane or to return to the Golgi. Rafts found at the plasma membrane are mostly
generated at the Golgi and use the anterograde pathway to traffic to the plasma membrane
[41–43]. Some rafts, however, can be generated from the ER membrane and GPI-anchored
proteins can use these rafts to exit the ER. These specific ER rafts are COPII-dependent
subdomains acting as specialized sorting platforms [29, 30, 44]. Like plasma membrane
lipid rafts, rafts at ER exit sites are characterized by their resistance to Triton X-100 at 4°C
[29, 31]. At steady state, it is estimated that about 10% of AβPP is targeted to lipid rafts and
that raft-localized AβPP is mostly regulated by endocytosis [45]. Here, we reveal another
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level of complexity in AβPP trafficking in rafts. Using Triton X-100-resistant membrane
preparations, we observed that Gas1 physically interacted with AβPP to co-partition in rafts
at ER exit sites. It is important to note that the use of non-ionic detergents to isolate rafts and
study protein trafficking in these microdomains has raised some concerns [46]. In this
context, we also used a GPI-free Gas1 mutant that lost its properties to traffic through rafts
to show that Gas1 translocation into rafts was required for its effect on AβPP accumulation
in rafts. Thus, the current work shows that AβPP can also accumulate in specialized ER-
associated rafts. Although further studies will be required to determine the relative amounts
at steady state of AβPP transiting through these rafts and what might be the contribution of
this pathway to constitutive AβPP processing, this work suggests that approaches aimed at
increasing AβPP translocation in these ER-associated subdomains (e.g., by increasing Gas1
expression) can significantly influence AβPP maturation and processing, and thus Aβ
production [32].
In summary, we provide strong evidence that Gas1 negatively controlled AβPP maturation
by inhibiting AβPP trafficking between the ER and the Golgi and this by physically
interacting with AβPP to trigger its translocation into specialized ER-associated raft
subdomains where immature AβPP accumulated. This study reveals a new facet of AβPP
trafficking and further increases our understanding of Gas1 function.
Acknowledgments
We thank Dr. G. Thinakaran (University of Chicago, Chicago, IL, USA) for kindly providing us with AβPP-N2a
cells, Dr. L. D’Adamio (Albert Einstein College of Medicine, Bronx, NY, USA) for AβPP-HEK293 cells; Dr P.D.
Mehta (Institute for Basic Research in Developmental Disabilities, Staten Island, NY, USA) for anti-AβPP R1
antibody. J.C. is the recipient of a fellowship from the Philippe Foundation (New York, NY, USA).
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Fig. 1.
Gas1 induces the accumulation of immature AβPP in rafts. A) AβPP-HEK293 cells
transiently transfected with Gas1 cDNA or empty vector were lysed in 1% CHAPS buffer
and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. All
fractions (1–12) were analyzed by WB using antibodies directed against AβPP, Gas1,
calreticulin (CRT, ER resident protein), and flotillin-1 (lipid raft resident protein). B) Shown
are densitometric analysis and quantification of AβPP immunoreactivity in fraction 5 (raft
AβPP) in three independent experiments performed as in A. C) AβPP-N2a transiently
transfected with Gas1 cDNA (+) or empty vector (−) were lysed in 1% Triton X-100 buffer
at 4°C and Triton X-100-soluble and Triton X-100-resistant fractions (see Materials and
Methods) were analyzed by WB for the indicated proteins. D) WB analysis of protein
extracts from cells treated as in C showing the accumulation of immature AβPP in Triton
X-100-resistant fractions upon Gas1 transfection. ma. AβPP, mature AβPP; im. AβPP,
immature AβPP. E) Shown are densitometric analysis and quantification of immature and
mature AβPP levels in three independent experiments performed as in D. F) AβPP-HEK293
cells transiently transfected with Gas1 cDNA (+) or empty vector (−) were treated (+) or not
(−) with methyl-β-cyclodextrin (MCD, 10 mM, 1 h). Triton X-100-soluble and Triton
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X-100-resistant fractions were then analyzed by WB using antibodies directed against the
indicated proteins. Note that the effect of MCD on raft integrity was confirmed by the partial
translocation of flotillin in the Triton X-100-soluble fractions (3rd and 4th lanes). G) Shown
are densitometric analysis and quantification of immature AβPP levels in Triton X-100-
resistant fractions in three independent experiments performed as in F. Histograms in B, E,
and G indicate the mean ± S.D. a.u., arbitrary units. *p < 0.05, **p < 0.01 (Student’s t-test).
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Fig. 2.
Gas1 and AβPP form a complex in rafts. A) Triton X-100 resistant fraction homogenates
from AβPP-HEK293 cells transiently transfected with Gas1 cDNA (+) or empty vector (−)
were incubated in the absence (CTRL) or presence of anti-AβPP (4G8) or anti-Gas1 (C-17)
antibodies. Total extracts from Triton X-100 resistant fractions (Input) and
immunoprecipitated (IP) proteins were analyzed by WB using antibodies directed against
AβPP (LN27) and Gas1 (GTX101732). B) Map of the constructs generated to express Gas1
full-length and soluble Gas1 (sGas1) lacking the GPI consensus sequence. C) AβPP-N2a
cells transiently transfected (+) or not (−) with Gas1 or sGas1 cDNAs were lysed in 1%
Triton X-100 buffer at 4°C and Triton X-100-soluble and Triton X-100-resistant fractions
were analyzed by WB for the indicated proteins. D) AβPP-HEK293 cells transiently
transfected with sGas1 cDNA (+) or empty vector (−) were IP with anti-AβPP (4G8)
antibody. Total extract (Input) and precipitated proteins were analyzed by WB using
antibodies directed against AβPP (LN27), Gas1 (GTX101732), and actin. E) AβPP-HEK293
cells transiently transfected with empty vector, Gas1 cDNA, or sGas1 cDNA were lysed in
1% Triton X-100 buffer at 4°C. Triton X-100-soluble and Triton X-100-resistant fractions
were then analyzed by WB for the indicated proteins.
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Fig. 3.
Gas1 controls immature AβPP translocation into ER-associated rafts. A) AβPP-N2a cells
transiently transfected with Gas1 (+) or empty vector (−) were treated with brefeldin A
(BFA, 5 μg/ml) for the indicted times. Cells were then lysed in 1% Triton X-100 buffer at
4°C and Triton X-100-soluble and Triton X-100-resistant fractions were analyzed by WB
for the indicated proteins. B) Shown are densitometric analysis and quantification of
immature and mature AβPP levels in three independent experiments performed as in A.
Histogram indicates the mean ± S.D. a.u., arbitrary units. *p < 0.05, **p < 0.01 (Student’s t-
test).
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