Content uploaded by Ehud Cohen
Author content
All content in this area was uploaded by Ehud Cohen on Oct 08, 2015
Content may be subject to copyright.
Article
Alzheimer’s disease-causing proline substitutions
lead to presenilin 1aggregation and malfunction
Tziona Ben-Gedalya
1
, Lorna Moll
1
, Michal Bejerano-Sagie
1
, Samuel Frere
2
, Wayne A Cabral
3
, Dinorah
Friedmann-Morvinski
4
, Inna Slutsky
2
, Tal Burstyn-Cohen
5
, Joan C Marini
3
& Ehud Cohen
1,*
Abstract
Do different neurodegenerative maladies emanate from the failure
of a mutual protein folding mechanism? We have addressed this
question by comparing mutational patterns that are linked to the
manifestation of distinct neurodegenerative disorders and identi-
fied similar neurodegeneration-linked proline substitutions in the
prion protein and in presenilin 1that underlie the development of
a prion disorder and of familial Alzheimer’s disease (fAD), respec-
tively. These substitutions were found to prevent the endoplasmic
reticulum (ER)-resident chaperone, cyclophilin B, from assisting
presenilin 1to fold properly, leading to its aggregation, deposition
in the ER, reduction of c-secretase activity, and impaired mito-
chondrial distribution and function. Similarly, reduced quantities
of the processed, active presenilin 1were observed in brains of
cyclophilin B knockout mice. These discoveries imply that reduced
cyclophilin activity contributes to the development of distinct
neurodegenerative disorders, propose a novel mechanism for the
development of certain fAD cases, and support the emerging
theme that this disorder can stem from aberrant presenilin 1func-
tion. This study also points at ER chaperones as targets for the
development of counter-neurodegeneration therapies.
Keywords Alzheimer’s disease; cyclophilin B; presenilin 1; proteostasis
Subject Categories Molecular Biology of Disease; Neuroscience
DOI 10.15252/embj.201592042 | Received 13 May 2015 | Revised 21 August
2015 | Accepted 25 August 2015
Introduction
To mature properly, newly synthesized polypeptides undergo
complex folding and modification events that are assisted and
supervised by specialized chaperones (Kim et al, 2013). Despite
these chaperones’ activities, not all nascent proteins attain their
desired spatial conformations. Cellular quality control surveillance
mechanisms identify terminally misfolded molecules and designate
them for degradation by autophagy (Arias & Cuervo, 2011) or by
the ubiquitin–proteasome system (UPS) (Schrader et al, 2009).
However, in some cases, misfolded polypeptides escape degradation
and form insoluble aggregates that lead to the development of
diseases that were collectively termed “proteinopathies” (Walker
et al, 2006). Prion disorders (Aguzzi & Calella, 2009), fronto-
temporal dementia (FTD) (Roberson, 2012), Huntington’s disease
(HD), and Alzheimer’s (AD) disease (Selkoe, 2003) are late-onset
neurodegenerative disorders that emanate from toxic protein aggre-
gation (proteotoxicity) and consist a group of proteinopathies.
Most neurodegenerative disorders exhibit more than one
pattern of emergence. While the majority of AD and prion disease
cases onset sporadically during the patient’s seventh decade of
life or later, fewer cases manifest during the fifth or sixth decade
as familial, mutation-linked maladies [certain prion diseases can
also be infectious (Prusiner, 1998)]. This common temporal emer-
gence pattern defines aging as the major risk factor for the devel-
opment of neurodegeneration (Amaducci & Tesco, 1994) and
suggests that aging-associated decline in the efficiency of protein
quality control mechanisms underlies the etiology of these
illnesses. This theme is strongly supported by the finding that the
alteration of aging protects model worms (Morley et al, 2002;
Cohen et al, 2006) and mice (Cohen et al, 2009; Freude et al,
2009) from proteotoxicity.
The detailed molecular mechanisms that lead to the development
of AD, the most prevalent human neurodegenerative disorder, are
largely obscure. However, according to the amyloid hypothesis, the
highly aggregative family of Abpeptides plays a crucial role in the
development of the disease (Hardy & Higgins, 1992). Abpeptides
are released as a result of a dual proteolytic digestion of the type 1
transmembranal amyloid precursor protein (APP) by the b-secretase
(BACE) and c-secretase proteolytic complex which is composed of
presenilin 1, presenilin 2 (PS1 and PS2, respectively), presenilin
enhancer 2 (Pen-2), nicastrin, and the anterior pharynx defective 1
(APH-1) (Selkoe & Wolfe, 2007). Shortly after translation, PS1
undergoes a rapid auto-cleavage to generate the PS1 N-terminal
domain (NTF) and C-terminal domain (CTF) (Thinakaran et al,
1996). Mutations in PS1, a multi-spanning transmembranal aspartic
1Biochemistry and Molecular Biology, The Institute for Medical Research Israel –Canada (IMRIC), The Hebrew University Medical School, Jerusalem, Israel
2Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
3Bone and Extracellular Matrix Branch, NICHD, NIH, Bethesda, MD, USA
4Biochemistry and Molecular Biology, Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
5Institute for Dental Sciences, Faculty of Dental Medicine, Hebrew University –Hadassah, Jerusalem, Israel
*Corresponding author. Tel: +972 2 6757328; E-mail: ehudc@ekmd.huji.ac.il
ª2015 The Authors The EMBO Journal 1
Published online: October 5, 2015
protease which possesses the proteolytic activity of the c-secretase
complex (De Strooper et al, 1998), are accountable for the majority
of familial AD cases (Bertram & Tanzi, 2008). PS1 was shown to
play crucial roles in key biological functions including autophagy
(Lee et al, 2010), the mediation of correct interactions between the
ER and mitochondria (Area-Gomez et al, 2009), as well as in the
maintenance of calcium homeostasis (Bezprozvanny & Mattson,
2008).
Although rare, mutation-linked neurodegeneration cases provide
invaluable hints that can help decipher the mechanisms that
underlie the development of these maladies. Interestingly, many
AD-causing mutations in the sequence of PS1 do not elevate total
Ablevels (Chavez-Gutierrez et al, 2012), suggesting that non-
canonical mechanisms are accountable for the development of
certain AD cases by attenuating PS1 function. Moreover, while most
PS1 mutations cause familial AD (fAD), certain amino acid substa-
tions in the sequence of PS1 were reported to initiate FTD (Mendez
& McMurtray, 2006).
Similarly, mutations in the sequence of the highly aggregative
prion protein (PrP) are associated with the emergence of at least
three familial neurodegenerative disorders: Creutzfeldt–Jakob
disease (CJD), fatal familial insomnia (FFI), and Gerstmann–
Stra
¨ussler–Scheinker syndrome (GSS) (reviewed in Aguzzi &
Polymenidou, 2004). The indications that one protein can be
involved in the development of more than one neurodegenerative
malady and the common temporal emergence pattern of different
disorders have led us to hypothesize that a common mechanism
may underlie the emergence of distinct neurodegenerative diseases.
To test this hypothesis, we adopted a comparative approach,
searching for similar mutational patterns that are linked to the onset
of different neurodegenerative diseases. This comparison was based
on the assumption that the substitution of amino acids in a
sequence that serves as a recognition site for a folding chaperone
impedes the functional interaction between the chaperone and its
client, preventing the mutated protein from folding properly. The
misfolding and aggregation of the protein may initiate the etiological
process that eventually causes neurodegeneration. Our search
unveiled that similar proline substitutions in the sequences of
PrP and of PS1 underlie the development of GSS and of fAD,
respectively.
The amino acid proline is known to serve as an axis for the
isomerization of polypeptides from cis to trans and, thus, to play
key roles in protein folding. At least three groups of peptidyl prolyl
cis/trans isomerases, chaperones that catalyze this conformational
conversion, have been identified: cyclophilins, FK506-binding
proteins (FKBPs), and parvulins (Schiene-Fischer, 2014). Among
these, the cyclophilins are most abundant within different cellular
organelles. The drug cyclosporin-A (CsA) specifically and efficiently
inhibits the activity of cyclophilins (Handschumacher et al, 1984).
Here, we found that inhibiting the activity of the ER-resident chaper-
one cyclophilin B results in PS1 aggregation, deposition in an ER-
derived quality control compartment (ERQC) and loss or reduction
of its proteolytic activity as well as impairment of mitochondrial
distribution and function. Reduced levels of the processed, active
form of PS1 were also observed in the brains of mice that lack cyclo-
philin B, confirming that these findings are conserved in the
mammalian brain. Since cyclophilin activity is also needed for the
correct folding of PrP (Cohen & Taraboulos, 2003), our discoveries
indicate that the failure of one folding mechanism can underlie the
development of more than one neurodegenerative malady and
support the emerging notion that in some cases AD emanates from
the attenuation of PS1 activity.
Results
Similar mutational patterns underlie the development of familial
Alzheimer’s disease and of GSS
Speculating that analogous mutational patterns in distinct neurode-
generation-linked proteins can serve as hints for the identification
of failed chaperone–client interactions (Fig 1A), we searched for
common mutated motifs and found that both PrP and PS1 carry
disease-linked proline substitutions in the motif PXXP, residing in
basic regions (Fig 1B). While the substitution of either P102
(Hsiao et al, 1989) or P105 (Yamazaki et al, 1999) in the sequence
of PrP underlies the development of GSS, the replacement of either
proline P264 (Campion et al, 1995) or P267 (Hutton et al, 1996) in
the motif PXXP of PS1 with other amino acids causes early-onset
fAD. Previously, we found that the GSS-associated proline substi-
tutions prevent folding chaperones, members of the cyclophilin
family of peptidyl prolyl cis/trans isomerases (PPIase) from assist-
ing PrP to fold properly (Cohen & Taraboulos, 2003). Analogously,
the inhibition of cyclophilin activity by CsA leads to PrP misfold-
ing, aggregation, and deposition in cellular sites that were termed
“aggresomes” (Johnston et al, 1998) that serve as quality control
compartments (Ben-Gedalya et al, 2011). The nearly identical
disease-linked proline substitutions in the sequences of PrP and
PS1 suggested that cyclophilins are also required for the correct
folding of PS1.
▸
Figure 1. The inhibition of cyclophilin activity leads to the aggregation of presenilin 1.
A A conceptual model suggests that the abolishment of similar folding chaperone recognition sites in discrete neurodegeneration-linked proteins can lead to the
development of distinct maladies by analogous mechanisms.
B The prion protein (PrP) and presenilin 1(PS1) share similar mutational patterns. The substitution of proline in residue 102 or 105 in the sequence of PrP causes GSS,
while the replacement of proline in residue 264 or 267 leads to the development of familial AD.
C The inhibition of chaperones of the cyclophilin family by the drug cyclosporin-A (CsA) results in the aggregation of PS1in CHO cells that stably over-express wild-type
PS1. This phenomenon is concentration-dependent.
D The aggregation of the endogenous mouse PS1was observed by Western blot (WB) analysis in CsA-treated MEFs, indicating that the over-expression of PS1is not a
prerequisite for its aggregation when cyclophilin activity is inhibited by CsA.
E WB analysis indicates that CsA-induced PS1aggregates cross-react with antibodies toward the N- and C-terminal fragments of the protein (lanes 5and 11), implying
that the full-length protein forms aggregates prior to its self-cleavage. In contrast, proteasome inhibition by MG132 does not induce the aggregation of PS1(lanes 6
and 12).
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
2
Published online: October 5, 2015
C
E
AB
D
Figure 1.
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
3
Published online: October 5, 2015
Aggregated wild-type PS1accumulates in CsA-treated cells
To test whether cyclophilins assist the folding of PS1, we created
Chinese hamster ovary cells (CHO) that stably over-express moderate
amounts of the human wild-type PS1 (Appendix Fig S1A) (CHO-PS1
cells). CHO-PS1 cells were treated for 16 h with increasing CsA
concentrations, and aggregated proteins were isolated from soluble
forms of the protein by high-speed centrifugation. Using Western blot
analysis (WB) and a PS1 antibody that reacts with the protein’s CTF,
we discovered that PS1 forms aggregates in cells that were treated
with 60 lg/ml or more CsA (Fig 1C). Thus, this concentration was
used for cyclophilin inhibition in the experiments described below.
To examine whether this phenotype emanates from PS1 over-
expression, we used wild-type mouse embryonic fibroblasts (MEFs)
and tested whether endogenous PS1 forms aggregates in response to
CsA treatment. The cells were treated either with ethanol [the vehi-
cle of CsA (Ve)] or with 60 lg/ml CsA, lysed, and subjected to high-
speed centrifugation. Supernatants and pellets were analyzed by
WB. Our results (Fig 1D) show that endogenous PS1 aggregates
accumulate in pellets of CsA-treated cells, indicating that PS1 aggre-
gation in cells in which cyclophilin activities were inhibited is not a
result of over-expression.
To ascertain whether CsA induces the aggregation of PS1 in an
early processing stage, presumably prior to its self-cleavage, or in a
later maturation step, we used antibodies that react with the
protein’s NTF or CTF. We also assessed the possibility that aggre-
gated PS1 accumulates due to proteasome overtaxing emanating
from the misfolding of other cyclophilin substrates and not from the
requirement of cyclophilins for PS1 maturation. CHO-PS1 cells were
treated for 16 h with CsA or for 4 h with the proteasome inhibitor
MG132 and subjected to high-speed sedimentation, and PS1 was
probed in the supernatants and pellets by NTF and CTF antibodies.
The absence of aggregated PS1 in the supernatants and pellets of
MG132-treated cells indicated that proteasome malfunction
(Appendix Fig S1B) is not sufficient for the accumulation of PS1
aggregates (Fig 1E and Appendix Fig S1C).
The observation that aggregated PS1 reacts with both PS1 NTF
and CTF antibodies demonstrates that CsA induces the aggregation
of the full-length wild-type PS1 prior to its self-cleavage, implying
that PS1 forms aggregates in an early maturation stage within
the ER.
Aggregated PS1accumulates in the ER quality control
compartment (ERQC) of CsA-treated cells
In order to determine where aggregated PS1 accumulates within
CsA-treated cells, we used immunofluorescence. CHO-PS1 cells
were either treated for 16 h with CsA or for 5 h with MG132 or left
untreated, and PS1 was labeled with NTF (green) and CTF (red)
antibodies. While both PS1 antibodies indicated that the protein is
distributed throughout untreated and MG132-treated cells (Fig 2A),
PS1 accumulated in a juxta-nuclear ringlike shape in cells that were
exposed to CsA (Figs 2A and EV1A, CsA arrows). Similar ringlike
shapes were seen in untransfected, CsA-treated NIH 3T3 cells
(Fig EV1B), indicating that this phenomenon is not cell line-specific,
and further show that it does not emanate from PS1 over-
expression. The observation that both PS1 antibodies label the
ringlike deposition site suggests that this structure contains the
full-length PS1 molecules, corroborating the theme that PS1 forms
aggregates within the ER, prior to the self-cleavage event.
Using a battery of antibodies, we characterized the PS1-
containing ringlike structure. First, we examined whether it is
caged by collapsed vimentin filaments, a hallmark of aggresomes
(Johnston et al, 1998), and could not identify a distinct caging
(Fig EV1C). We also found that the ringlike structure does not
contain ubiquitinated proteins (Fig EV1D) but overlaps with the
ER membrane-integral chaperone calnexin (Brodsky & Skach,
2011) in CsA-treated cells (Fig 2B). This observation proposes that
aggregated PS1 accumulates in the ER-derived quality control
compartment (ERQC), a sub-organelle that was previously shown
to contain misfolded ER-resident proteins (Kamhi-Nesher et al,
2001). To test this hypothesis, we transiently expressed in CHO-
PS1 cells a fluorescently tagged chimera of the secreted form of
the asialoglycoprotein receptor H2a (H2a-RFP), a well-established
ERQC marker (Kamhi-Nesher et al, 2001). The cells were treated
for 16 h with CsA, and PS1 was labeled using the PS1-NTF anti-
body. Our results revealed co-localization of PS1 and H2a-RFP
[Fig 2C, no such H2a-RFP-containing structure was seen in
untreated cells (Fig EV1E)].
To examine whether the PS1-containing ringlike shape is present
at a pericentriolar localization, a known feature of the ERQC
(Kondratyev et al, 2007), CHO-PS1 cells were treated with CsA as
described above and PS1 and c-tubulin (a marker of the centriole)
were labeled by specific antibodies. Our results revealed that
the PS1-containing ringlike structures (Fig 2D, green) are located
around the centriole (red). Finally, we found that the PS1-containing
structure is segregated from the non-ERQC-resident chaperone BiP
(Kondratyev et al, 2007) (Fig 2E).
While the inhibition of autophagy by the drugs pepstatin A and
E64 (Yu et al, 2010) resulted in elevated quantities of the endoge-
nous PS1 in naive 3T3 cells, the protein did not accumulate in the
ERQC in response to this treatment (Appendix Fig S2), suggesting
that PS1 molecules that fail to fold properly within the ER are
directed for proteasomal degradation.
▸
Figure 2. Aggregated PS1accumulates in the ER quality control compartment (ERQC) of CsA-treated cells.
A Fluorescent immunocytochemistry using PS1NTF and CTF antibodies performed on CHO cells over-expressing wild-type PS1and treated with CsA (60 lM)
uncovers PS1accumulation in a juxta-nuclear, ringlike structure (arrows). This phenomenon was not observed in cells that were treated with 10 lMMG132 (for
5h) or with the vehicle of CsA (EtOH). Scale bar, 5lm.
B–D Fluorescent immunocytochemistry using PS1NTF and calnexin or c-tubulin antibodies or the expression of fluorescent asialoglycoprotein receptor H2a(H2a-RFP)
performed on CHO cells expressing wild-type PS1and treated with CsA. The CsA-induced, PS1-containing ringlike structure overlaps with the ER-residing chaperone
calnexin (B), co-localizes with the ERQC marker H2a-RFP (C), and organizes around the microtubule-organizing center (“Ce,”arrows) (D). Scale bar, 5lm.
EPS1signal does not overlap with BiP, a chaperone that is known not to reside within the ERQC.
F Fluorescence microscopy unveiled that GFP-PS1accumulates in ERQC of CHO cells over-expressing wild-type PS1that were treated with siRNA toward the ER-
resident cyclophilin B (arrows). No such phenomenon was seen in cells that were treated with siRNA toward cyclophilin A.
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
4
Published online: October 5, 2015
CsAMG132 Untreated (Ve)
DIC Merge (+DAPI)
PS1-NTF PS1-CTF
A
CsA
Calnexin
H2a
Ce
Ce
Ɣ tubulin
B
CsACsA
C
D
Ce
CsA
BiP
Red Channel Merge (+DAPI)PS1-NTFDIC Inset
E
RISC free Cyp-B siRNA
Luciferase siRNA Cyp-A siRNA
DICGFP-PS1
F
Figure 2.
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
5
Published online: October 5, 2015
The observation that PS1 accumulates within the ER predicts that
cyclophilin B, an ER-resident member of this family, is the chaperone
that is critically required for the correct folding of PS1. To scrutinize
this hypothesis, we fused the green fluorescent protein (GFP) to PS1
and expressed this construct in CHO cells (CHO-GFP-PS1 cells). The
cells were treated for 48 h with small interfering RNA (siRNA)
toward either cyclophilin B or the cytosolic family member, cyclo-
philin A (48 h of treatment is sufficient to notably reduce the levels
of cyclophilin A and B, Fig EV1F). Visualization by fluorescence
microscopy indicated that GFP-PS1 accumulates in ERQC of cells that
were treated with cyclophilin B-targeting siRNA (red) but not in cells
that were transfected with siRNA toward cyclophilin A (Fig 2F).
These results indicate that cyclophilin B is the chaperone that is func-
tionally required for the correct folding of nascent PS1 molecules.
Collectively, our results clearly show that the inhibition of cyclo-
philin B by CsA results in the misfolding and aggregation of full-
length PS1 and in its deposition in the ERQC.
P264L and P267SPS1accumulate in ERQC upon
proteasome inhibition
If cyclophilin B assists the maturation of PS1 by promoting cis/trans
isomerization that is based on proline 264, 267 or both, it is expected
that the fAD-linked substitution of these prolines will result in the
accumulation of aggregated, mutated PS1 in the ERQC. To test this
hypothesis, we created mutated human PS1 constructs that carry
either one of these mutations: P264L, P267S, or both [double mutant
(DM)]; and expressed them in CHO cells (CHO-PS1-P264L, CHO-
PS1-P267S, and CHO-PS1-DM, respectively). First, we examined the
effects of CsA on DM PS1 molecules expressed in these cells and
found that the inhibition of cyclophilins induces their aggregation as
tested by a high-speed sedimentation assay (Fig EV2A). Next, we
examined the effect of CsA treatment on the cellular distribution of
P264L, P267S, and the DM PS1 and found that the inhibition of
cyclophilins leads to their accumulation in the ERQC (Fig EV2B).
We also asked whether proteasome inhibition leads to the deposition
of the mutated PS1 molecules in the ERQC. CHO cells expressing
either the wild-type PS1 or one of the aforementioned mutants were
treated for 5 h with either vehicle or 10 lM MG132, to inhibit
proteasomes (as demonstrated in Appendix Fig S1B), and the cellu-
lar distribution of PS1 was visualized. While proteasome inhibition
led to the accumulation of wild-type PS1 in a reticular pattern
throughout the cell but not in its deposition in the ERQC (Fig 3A),
MG132 treatment directed P264L and P267S PS1 mutants to the
ERQC (Fig 3B and C, arrows) in ~10% of the cells. Similarly, protea-
some inhibition induced the aggregation (Fig EV2A) and accumula-
tion of DM PS1 in the ERQC (Fig EV2C), but neither the inactive
D257A PS1 (Wolfe et al, 1999) (Fig EV2D) nor the A246E fAD-
associated PS1 mutant (Sherrington et al, 1995) accumulated in this
structure (Fig EV2E). In addition, CsA does not induce the accumu-
lation of the YFP-fused transmembrane dopamine transporter in the
ERQC of CsA-treated cells (Fig EV2F), indicating that not all multi-
transmembrane proteins are directed to the ERQC upon cyclophilin
inhibition. Finally, ERQC formation could not be seen in CHO
cells expressing the wild-type or mutated PS1 in resting conditions
(vehicle treatment) as displayed by the H2a RFP reticular appear-
ance (Appendix Fig S3). This result indicates that these mutants are
not deposited in the ERQC when proteasomes are active.
To further test the conclusion that proline-substituted PS1
accumulates within the ER as a result of endoplasmic reticulum
associated protein degradation (ERAD) impairment, we blocked the
retro-translocation of proteins from the ER to the cytosol using
the potent ERAD inhibitor Eeyarestatin I (Wang et al, 2008). Our
observations showed that similar to MG132, this treatment induced
the accumulation of P264L PS1 but not of wild-type PS1 in the ring-
like structure, supporting the theme that misfolded, P264L PS1
molecules are retained within the ER and accumulate within the
ERQC (Fig 3D).
These findings imply that PS1 molecules that harbor the fAD-
linked proline substitutions are degraded by proteasomes. However,
when proteasome activity is impaired, these molecules accumulate
within the ERQC. The similar cell biological features seen in CsA-
treated CHO-PS1 cells and in cells which express the mutated PS1
molecules suggest that P264 and P267 are critical for the correct
folding of PS1 and that the abolishment of the cyclophilin recogni-
tion site plays a key role in the development of fAD in individuals
who carry these mutations. The possible role of the ERQC in the
etiology of AD has prompted us to further characterize the physical
properties of PS1 species that reside in this structure.
ERQC-trapped PS1molecules are immobile
At least two types of cellular deposition sites have been described:
dynamic quality control compartments in which resident molecules
exhibit high mobility, and structures that accumulate terminally
aggregated, immobile proteins (Kaganovich et al, 2008). To investi-
gate the rate of mobility of ERQC-resident PS1 molecules, we used
CHO-GFP-PS1 cells which were treated with CsA and subjected to a
fluorescence recovery after photobleaching (FRAP) assay. This tech-
nique is based on a high-power laser pulse which bleaches the fluo-
rescent signal of tagged molecules in a limited area within the
examined compartment, followed by a kinetic analysis of the signal
recovery (Lippincott-Schwartz et al, 2003). High mobility rate
enables rapid recovery of the fluorescent signal in the affected area
while immobility results in slow recovery. While the fluorescent
signal in vehicle-treated cells recovered quickly (from ~55% after
bleach to ~93% at 34 s) (Appendix Fig S4A and B), almost no recov-
ery was seen in the bleached area of PS1 ERQC 15 min after the
bleach (from ~55% after bleach to ~65% at 34 s) (Fig 4A and C),
indicating that ERQC-resident PS1 is terminally aggregated.
Utilizing the fluorescence loss in photobleaching (FLIP) tech-
nique (Lippincott-Schwartz et al, 2003), we measured the rate of
molecular exchange between the ERQC and its surrounding. This
method is based on continuous bleaching of a small area outside of
the examined cellular structure by a laser beam. High rate of
exchange results in a rapid decline in the deposit’s fluorescence over
time, while the outcome of low rate of exchange is a stable fluores-
cence level. The fluorescent signal seen in the PS1 ERQC exhibited
only a marginal decline over time (Fig 4B and D), demonstrating a
very low level of molecular exchange between this deposition site
and its vicinity.
Together, the analyses of GFP-PS1 dynamics in live cells and the
sedimentation experiments (Fig 1E) propose that highly aggregated
PS1 is trapped within the ERQC upon cyclophilin B inhibition. These
results raise the question of whether the aggregation and deposition
of PS1 results in the attenuation of c-secretase activity.
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
6
Published online: October 5, 2015
P264L (vehicle)
P264L MG132
BDIC Merge with DAPI
PS1-NTF
P267S (vehicle)
P267S MG132 WT (vehicle)WT MG132
ADIC Merge with DAPIPS1-NTF
CDIC Merge with DAPIPS1-NTF
DWild-Type PS1 P264L PS1
MG132Eeyarestatin I
PS1 (CTF) PS1 (CTF)PS1/tubulin/DAPI PS1/tubulin/DAPI
Vehicle
Figure 3. Proteasome inhibition directs mutated PS1carrying either P264LorP267S substitution to the ERQC.
A–C Fluorescent immunocytochemistry using PS1NTF antibody (green) and PS1CTF (red in merge) shows that the inhibition of proteasome activity by 10 lMMG132
(5h) did not induce the accumulation of wild-type PS1in the ERQC of CHO cells (A), but directed PS1carrying the fAD-linked P264L (B) or P267 S (C) mutation to
this structure (arrows). These results imply that in unstressed cells, proteasomes degrade PS1molecules that misfold due to failed interaction with cyclophilin B.
D Similar to MG132 treatment, the inhibition of ER-associated degradation (ERAD) by Eeyarestatin I (1h, 10 lM) directs P264 LPS1but not wild-type protein to the
ERQC (arrows).
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
7
Published online: October 5, 2015
Reduced c-secretase activity in cells expressing mutated P264L
or P267SPS1
To address this question, we tested whether the fAD-associated
proline substitutions affect c-secretase maturation and activity.
Auto-cleavage of PS1 to generate the NTF and CTF fragments is
an early maturation step of the c-secretase complex (Xia, 2008). We
stably expressed the human wild-type PS1 or either one of the fAD-
linked proline substituted PS1, P264L or P267S or the double mutant
DM P264L-P267S in mouse embryonic fibroblasts (MEFs) that were
+15 min+3minPost bleachPre bleachDIC
0
20
40
60
80
100
1.8
12.6
23.4
34.2
45
55.8
66.6
77.4
88.2
99
109.8
120.6
131.4
142.2
153
163.8
174.6
Bleach
n=6
CsA
+15 min+6minPost bleachPre bleachDIC
CsA
Inset
0
20
40
60
80
100
120
3
24
45
66
87
108
129
150
171
192
213
234
255
276
297
318
339
360
% recovery
Bleach
% Recovery
% Recovery
n=6
Time (seconds) Time (seconds)
A
B
CD
Bleaching
area
Figure 4. ERQC-resident PS1-GFP molecules are immobile.
A Fluorescence recovery after photobleaching (FRAP) experiment performed on CHO cells over-expressing GFP-PS1shows slow recovery of the fluorescent signal in a
bleached area within the ERQC. This result indicates a low rate of mobility of PS1-GFP molecules within the ERQC, implying that they are aggregated.
B Fluorescence loss in photobleaching (FLIP) experiment indicates low rate of molecular exchange between the cytosol and the ERQC in GFP-PS1-over-expressing
CHO cells.
C, D Quantitative analysis of (C) 6FRAP experiments as in (A), and (D) 6FLIP experiments as in (B). Signal was normalized to the initial signal prior to bleaching. Error
bars represent SEM of 6independent experiments.
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
8
Published online: October 5, 2015
derived from psen 1 knockout mice (PS1-KO MEF, Fig EV3A), and
thus lacking endogenous PS1 activity (Herreman et al, 1999, 2003).
As a negative control, we expressed in PS1-KO MEF cells the human
PS1 gene that harbors the artificial D257A mutation which abolishes
the proteolytic activity of the c-secretase complex (Wolfe et al,
1999). To examine the levels of the full-length uncleaved PS1 in
relation to the NTF fragment, we used WB analysis (Fig 5A) and
found that while the P264L mutation reduces the efficiency of
endo-cleavage compared to the wild-type PS1 (lanes 2 and 3,
respectively), the introduction of the P267S mutation results in the
disappearance of PS1 (lane 4). Similarly to the D257A PS1 (lane 6),
no auto-cleavage could be detected in cells that expressed the DM
PS1 (lane 5) implying loss of c-secretase activity.
To examine whether cellular degradation mechanisms are
accountable for the disappearance of the P267L PS1 mutant, we
used MEF cells, which lack both endogenous PS1 and PS2 (PS1/2-KO
MEF) (Herreman et al, 1999, 2003), and transiently expressed
either the wild-type PS1 gene or the P267L PS1 mutant. The cells
were exposed to the proteasome inhibitor MG132 (20 lM, 6 h) or to
the combination of autophagy inhibitors E64 and pepstatin A
(20 lg/ml each, 2 h), and PS1 quantities in the cells were compared
by WB using the NTF PS1 antibody. Our results (Fig 5B) clearly
show that MG132 stabilized the full-length wild-type PS1. No signifi-
cant stabilization of transiently expressed PS1 was observed when
the cells were treated with the autophagy inhibitors. Interestingly,
MG132 stabilized the NTF fragment of the P267S PS1 mutant but
not the full-length protein, suggesting that this mutant undergoes
self-cleavage prior to proteasomal degradation.
We further tested the effect of the fAD-linked proline substitu-
tions on c-secretase activity by stably expressing the human wild-
type and mutated PS1 constructs described above, in PS1/2-KO MEF
cells. The cells were transiently transfected with myc-tagged APP
C99, and the level of c-secretase activity was assessed by measuring
the amounts of its cleaved product, the APP intracellular domain
(AICD) (Hecimovic et al, 2004). Using WB and a myc antibody, we
detected a prominent band, corresponding to the AICD, in cells
expressing the wild-type PS1 but a band of ~50% lower intensity in
cells expressing P264L PS1. Similar to PS1/2-KO MEF cells
(Fig EV3B), no c-secretase activity could be detected in cells
expressing P267S PS1, DM PS1, or the D257A inactive PS1 (Fig 5C
and D). Similar results were obtained by transient transfection of
both the APP C99-myc and PS1 constructs into the PS1/2-KO cells
(Fig EV3C and D). A reciprocal experiment indicated that the inhibi-
tion of cyclophilin activity by CsA reduces c-secretase activity in
cells over-expressing wild-type PS1 (Fig EV3E).
As a complementary technique, we used an in vitro c-secretase
activity assay based on a C-terminal b-APP-fluorescent peptide. In
this assay, the proteolysis of the internally quenched peptide at the
Ab40-, Ab42-, and Ab43-generating cleavage sites results in
enhanced fluorescence. A calibration experiment using purified
membranes containing c-secretase complex (Sato et al, 2007)
showed a dose-dependent increase in fluorescence (Fig EV3F). A
direct comparison of fluorescence levels generated by membrane
fractions of wild-type cells versus either PS1- or PS2-deficient cells
indicated a reduction in PS1 activity of 49 and 23%, respectively
(Fig EV3G). Comparison of c-secretase activity of the PS1 KO cells
stably expressing the different human constructs showed that
membrane extracts of cells that expressed the P264L PS1 displayed
~25% reduction in activity compared to extracts of cell expressing
the wild-type PS1, while extracts of cells expressing the P267S
mutant showed 33% reduced activity level (Fig 5E).
To further test c-secretase activity in cells that express the
proline-substituted PS1 mutants, we performed ELISAs and
measured the amounts of Abin media collected from PS1/2-KO
MEF cells in which mutated PS1 constructs together with myc-
APP-C99 were expressed. While the amounts of Ab
1–40
were ~70%
Figure 5.P264L and P267SPS1mutations reduce c-secretase activity.
APS1self-cleavage was examined by WB analysis of the myc-conjugated APP intracellular domain (AICD), using MEFs that were derived from a PS1/2knockout
mouse and stably infected with the indicated PS1constructs, carried by the pBABE retroviral vector. APP C99-myc was transiently expressed in these cells. While
wild-type PS1exhibited complete self-cleavage (lane 2), P264LPS1showed partial digestion (lane 3), and P267S (lane 4) disappeared. The DM PS1and D257A
mutant exhibited no activity (lanes 5and 6).
BPS1/2-KO MEFs were transiently transfected with plasmids carrying either the wild-type or P267SPS1constructs. The cells were treated with either MG132 (20 lM
for 6h) to inhibit proteasomes or with E64 + pepstatin A (20 lg/ml each for 2h) to inhibit autophagy. PS1levels were analyzed by WB using the NTF PS1
antibody. Inhibition of proteasomes results in the accumulation of wild-type and mutated PS1(lanes 2and 5respectively). No PS1accumulation was observed
when autophagy was inhibited (lanes 3and 6) in MEF cells lacking endogenous PS1and transiently over-expressing PS1. These results imply no role for this
mechanism in the digestion of PS1.
C, D Western blot analysis of myc-tagged APP (C) unveils that while the expression of wild-type PS1in PS1/2knockout MEF cells efficiently restores APP digestion
(AICD-myc, lane 3, arrow), the expression of P264LPS1in these cells (lane 4) exhibits reduced level of APP digestion. P267S (lane 5), DM (lane 6), or D257APS1
expression in the cells results in no detectable c-secretase activity (D). Error bars represent SEM of 4independent experiments, *P<0.01 (Student’st-test).
E Membranes purified from MEFs of PS1KO mice that stably express either P264LorP267S human PS1exhibit reduced c-secretase activity relative to wild-type PS1
as detected using the fluorogenic c-secretase substrate NMA-Gly-Gly-Val-Val-Ile-Ala-Thr-Val-Lys(DNP)-D-Arg-D-Arg-D-Arg-NH2. Bars indicate relative levels of
fluorescence SEM, *P<0.01 (Student’s t-test).
FPS1/2-KO MEF cells were transiently transfected with the indicated PS1constructs as well as with APP. The levels of Ab1–40 in the cell media were measured
using a peptide-specific ELISA kit. While ~70% reduction in the levels of both peptides was detected in media of cells that express the P264LPS1mutant compared
to the amounts detected in media of cells that express the wild-type PS1construct, a 90% reduction was seen in the media of cells that express the P267S or the
DM PS1mutants. These Abamounts were similar to the background level (broken line) that was observed in media of cells that were transfected with the empty
vector or with the inactive D257APS1. Error bars represent SEM of three independent experiments, *P<0.01 (Student’st-test).
G The relative amounts of Ab1–42 in the cell media were measured by a specific ELISA kit as in (F). While the Ab1–42 signals that were observed in the media of cells
that express the P267S or the DM PS1mutants are indistinguishable from the background level (broken line), merely 33% reduction was detected in the media of
P264LPS1-expressing cells compared to the level observed in media of cells that expressed wild-type PS1(this difference was not significant). Error bars represent
SEM of three independent experiments, *P<0.01 (Student’st-test).
H The expression of P264LPS1elevates the relative ratio of Ab1–42/Ab1–40 as measured by ELISA. This observation shows that the P264L mutation affects the
AD-related Abform ratio differently than the P267S mutation.
▸
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
9
Published online: October 5, 2015
A
NTF
fragment
0
20
40
60
80
100
120
P264L P267S
*
*
*P<0.01
D
Full length
PS1
Actin
55 kDa
25 kDa
Lane 1234567
PS1 NTF Ab
E
0
0.2
0.4
0.6
0.8
1
1.2
Empty P264L P267S DM D257A
Normilized arbitrary Units
PS1 mutant
PS1 mutant
*
*
*P<0.01
**
*
PS1/2-KO MEFs
Normalized fluorescence
PS1-KO MEFs
Relative AICD band intensities
Relative fluorescence of cleaved substrate
*P<0.01
PS1
mutant
25 kDa
35 kDa C99-myc
AICD-myc
C83-myc
C
Lane 123456
None-
specific band
Actin
PS1
mutant
WT
Relative amounts of Aβ1–42 (ELISA)
0
20
40
60
80
100
120
Empty P264L P267S DM D257A
Aβ levels normalized to WT
PS1 mutant
Relative amounts of Aβ1–40 (ELISA)
*
*
**
*
*P<0.01
0
20
40
60
80
100
120
Empty P264L P267S DM D257A
Aβ levels normalized to WT
PS1 mutant
G
F
WT
WT
WT
*
ns
**
*
PS1/2-KO MEFs
PS1/2-KO MEFs
H
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
Empty P264L P267S DM D257A
PS1 mutant
N=4
N=3
N=3
N=3
Relative ratio of Aβ1–42 / Aβ1– 40
RAU
X2.23
X1.04X1.04 X1.08
NTF
fragment
Full length
PS1
Actin
Lane 123456
B
55 kDa
25 kDa
Wild type PS1 P267S PS1
Treatment
Figure 5.
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
10
Published online: October 5, 2015
lower in media of cells that expressed the P264L PS1 mutant
compared to WT, the replacement of P267 or of both prolines (DM
mutant) reduced the amounts of this peptide by nearly 90%
(Fig 5F). These levels were similar to the background level (broken
line) seen in media of cells that expressed the empty plasmid or the
inactive D257A PS1 mutant. These results were consistent with
the observations that P264L-mutated PS1 exhibits only residual
c-secretase activity and P267S mutation leads to nearly complete
loss of function (Fig 5A–E).
We also used an ELISA kit to measure the relative amounts of
Ab
1–42
in media of PS1/2-KO MEF cells as described above and
found that the reduction in the amounts of this aggregative peptide
in the media of cells that express the P264L mutant was merely
33% and not significantly different from the levels detected in the
media of cells that were transfected with wild-type PS1 (Fig 5G).
The levels of Ab
1–42
in media of cells that expressed the P267S or
the double-mutated PS1 (DM) were similar to background level and
significantly lower than those detected in media of wild-type PS1-
expressing cells.
Besides modulating the proteolytic activity of the c-secretase
complex, AD-causing mutations in the sequence of PS1 have been
shown to change the ratio of Ab
1–42
to Ab
1–40
, a feature which is
believed to be associated with the development of the disease
(Chavez-Gutierrez et al, 2012). A comparison of the Ab
1–40
/Ab
1–42
ratio (as measured by the ELISAs) revealed that this AD-associated
parameter is increased in PS1/2-KO MEFs that express the P264L
PS1 mutant but not in those which express either the P267S, DM, or
D257A PS1 mutants (Fig 5H).
Collectively, our experiments revealed that PS1 carrying the
fAD-linked proline substitution P264L exhibits notable reduction in
c-secretase activity and increases the ratio of Ab
1–42
to Ab
1–40
, while
the P267S mutation appears to cause a near-complete loss of PS1
function.
PXXP mutations in PS1impair mitochondrial distribution
and function
The involvement of PS1 in the formation of mitochondria-associated
ER membranes (MAMs) (Area-Gomez et al, 2009) and the observa-
tions that PS1 accumulates in the ER (Fig 2) and exhibits attenuated
function (Fig 5) have led us to ask whether mitochondrial distribu-
tion and function are impaired in cells that possess PS1-containing
ERQC. We simultaneously followed the localization of GFP-PS1 and
mitochondria in living CsA-treated CHO-GFP-PS1 cells and observed
concurrent accumulation of GFP-PS1 in the ERQC (Fig 6A, green
channel) and clustering of the mitochondria around this structure
(red channel and Movies EV1 and EV2). Since presenilin 2 (PS2) is
also present and functions in MAMs (Area-Gomez et al, 2012) and
contains a PXXP motif (at positions 270–273), we asked whether
this protein also accumulates in the ERQC upon CsA treatment.
CHO cells expressing GFP-tagged PS2 were exposed to CsA for 16 h,
fixed, and visualized. Similar to PS1, PS2 accumulates in the ERQC
of CsA-treated CHO and NIH3T3 cells (Fig EV4A and B, respec-
tively). To test whether PS2-containing ERQC and mitochondria also
co-localize, we recorded the localization of GFP-PS2 (Fig 6B, green)
and mitochondria (Fig 6B, red channel, and Movies EV3 and EV4)
as described above and found that they redistribute to create nearly
identical patterns within the cell.
To further examine whether the accumulation of PS1 in the
ERQC leads to impaired mitochondrial distribution and to directly
test whether the fAD-linked proline substitutions in PS1 are associ-
ated with this phenomenon, we treated CHO-PS1 and CHO-PS1-DM
cells for 5 h with 10 lM MG132 and examined them by transmis-
sion electron microscopy (TEM). Our results unveiled that while the
mitochondria of untreated (Fig 6C) and MG132-treated (Fig 6E)
CHO-PS1 cells were evenly distributed, the mitochondria of CHO-
PS1-DM cells accumulated in a juxta-nuclear localization upon
proteasome inhibition (Fig 6D).
These observations imply that the accumulation of mutated PS1
in the ERQC leads to impaired mitochondrial distribution due to PS1-
induced change of MAMs, which may be related to a phenomenon
seen previously in cells derived from patients with AD (Area-Gomez
et al, 2012). Thus, we next tested whether P264L-mutated PS1 also
affects mitochondrial function. PS1-KO MEF cells were stably
infected with retroviruses carrying wild-type human PS1, or the
P264L PS1 mutant or the double mutant (DM). These cells were
stained by MitoTracker Green to label total mitochondria (Fig 7A,
green) and tetramethylrhodamine methyl ester perchlorate (TMRM)
(red) to stain active mitochondria (Petronilli et al, 2001). The cells
were visualized by confocal microscopy, and the signals were quan-
tified (Fig 7B). Our results show that the lack of PS1 resulted in
lower rate of mitochondrial activity as judged by low TMRM signal.
While the expression of wild-type human PS1 restored mitochon-
drial function and fragmentation to levels nearly double than seen in
MEFs of wild-type mouse, no change in TMRM signal could be
detected in cells expressing the P264L or the DM PS1.
As an additional approach to assess whether the expression of
mutated PS1 impedes mitochondria activity, we measured ATP
production in PS1-KO MEFs stably expressing the wild-type PS1 or
either one of the mutated forms of the protein, P264L, P267S or DM,
using the ATPlite luminescence assay. We found (Fig 7C) that while
the restoration of wild-type PS1 expression elevated the rates of
ATP compared to cells infected with the empty viral vector, the
expression of the mutated PS1 proteins had remarkably lower
effects on the levels of ATP. Together, the reduced TMRM signal
and lower ATP levels indicate that mitochondria are not only
aberrantly distributed but also malfunction in cells expressing PS1
that carries the AD-associated proline substitutions.
Reduced quantities and elevated PS1aggregation in brains of
cyclophilin B knockout mice
To evaluate the relevance of our findings to the mechanism that
underlies the development of fAD in individuals who carry the 264
or 267 proline substitutions (and perhaps in some sporadic cases),
we sought to test whether cyclophilin B is required for the correct
folding of PS1 in the mammalian brain. Thus, we examined the
levels and distribution of the endogenous PS1 in brain of mice that
lack cyclophilin B (CyPB KO mice) (Cabral et al, 2014). Brains of
four CyPB KO mice and of four matched genetic background wild-
type mice were homogenized and cleared by low-speed centrifuga-
tion, and soluble proteins were separated from aggregated proteins
by high-speed centrifugation. The levels of PS1 in both the soluble
and insoluble fractions were compared using WB analysis and the
CTF PS1 antibody. Our results clearly show lower levels of
processed PS1 in the soluble fractions of brains that were obtained
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
11
Published online: October 5, 2015
5 h 8.5 h 9.5 h 11 h 15.5 h
GFP-PS1
DsRed-Mito
A
1.5 h 9.5 h 12.15 h 15 h 17.45 h
GFP-PS2MitoTracker
B
C
ii
ii
ii
iii
iii
CHO WT-PS1 Vehicle
CHO DM-PS1 MG132
D
MTOC
2μm
5μm
ECHO WT-PS1 MG132
Figure 6. The accumulation of aggregated PS1in the ERQC impairs mitochondrial distribution.
A, B Live CHO cells expressing either GFP-PS1(A) or GFP-PS2(B) were treated for 16 h with CsA and visualized overnight by a confocal microscope. Our results indicate
that both GFP-tagged presenilins 1and 2were deposited in the ERQC (arrows) following the inhibition of cyclophilins by CsA. This phenomenon was accompanied
by the clustering of mitochondria (tagged by mito-DsRed expression or stained by MitoTracker, arrows) around this structure (scale bars, 5lm).
C–E Transmission electron microscopy (TEM) shows an even distribution of mitochondria in an untreated (vehicle) CHO cell over-expressing the wild-type PS1(C). In
contrast, MG132 treatment (10 lM, 5h) causes mitochondria clustering in cells over-expressing the DM PS1(D), but not in wild-type PS1-expressing cells (E).
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
12
Published online: October 5, 2015
0
20
40
60
80
100
120
empty DM
Luminesc ence (% of wt)
PS1 mutant
P264L P267S
WT
MEFs of wt mouse
Empty virus H P264L PS1Human wt PS1 H DM PS1
MitoTrackerTMRMMerged
Uninfected
MEFs of PS1 knockout mouse
Infection:
A
Relative ATP quantities
Insets
ns
*
*
Relative fraction of active mitochondria
*P<0.01
*
BC
Figure 7. The inhibition of cyclophilins impairs mitochondrial function.
A, B PS1-KO MEFs were stably infected with an empty virus or with a virus carrying either human wild-type, P264L, or DM PS1. Total mitochondria were stained with
MitoTracker (green), and active mitochondria were labeled using tetramethylrhodamine methyl ester perchlorate (TMRM). The cells were visualized by confocal
microscopy (A), and the signals were quantified and normalized to the wild-type mouse cells expressing only the endogenous mouse PS1. Error bars represent SEM
of three independent experiments, *P<0.01 (Student’st-test) (B). While the expression of wild-type PS1restored mitochondria activity to higher levels than those
of MEFs of wild-type mice (left panels), the expression of P264LorDMPS1did not restore mitochondria activity levels.
C A luminescence-based ATP assay performed on PS1-KO MEFs expressing the indicated PS1constructs displays lower ATP levels in cells expressing the proline-
substituted PS1compared to their wild-type-expressing counterparts. This result shows that the substitution of prolines of the PXXP motif in PS1impairs
mitochondrial activity. Error bars represent SEM of four independent experiments.
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
13
Published online: October 5, 2015
from CyPB KO mice compared to the amounts detected in brains
of control WT animals (Fig 8A and C). In contrast, no reduction
in PS1 levels was observed in the insoluble fractions (pellets)
(Fig 8B and D).
Using immunohistochemistry (IHC) and the CTF PS1 antibody,
we further compared the PS1 levels in brains of CyPB KO animals
and of their wild-type counterparts. Since PS1 is highly expressed in
the hippocampus (Quarteronet et al, 1996), we focused on this brain
structure and visualized the dentate gyrus (DG). The levels of PS1
in the DG of CyPB KO animals were remarkably lower than those
seen in DG of wild-type animals (Figs 8E and EV5A). A similar
phenomenon of reduced PS1 levels was detected when PS1 levels
were compared in the cortices of CyPB KO and wild-type mice
(Fig EV5B).
To examine whether the absence of cyclophilin B affects
c-secretase activity, we purified membranes of wild-type and CyPB
KO mouse brains and utilized the C-terminal b-APP-fluorescent
peptide-based assay as described above. Our results (Fig 8F) indi-
cated that the activity of the endogenous c-secretase is significantly
reduced (P<0.02) in brains of CyPB KO animals compared to the
activity levels seen in their WT counterparts. The observed reduc-
tion of ~20% in fluorescence level was analogous to that seen in
cells that express the P264L or P267S PS1 (Fig 5E).
Together, these results indicate that cyclophilin B activity is
crucially required for the proper maturation of PS1 and activity of
the c-secretase complex in the mouse brain. They also suggest that
a misfolded PS1 subpopulation escapes degradation and forms
aggregates in the brain.
P264LPS1forms aggregates in the hippocampus of mice
We also asked whether the fAD-linked proline 264 substitution
affects PS1 distribution in the mouse brain. To address this ques-
tion, we created GFP-labeled lentiviral vectors that drive the expres-
sion of either the human wild-type or P264L PS1 and injected them
into the hippocampi of young naı
¨ve mice (strain BALB/c). The
animals’ brains were harvested 5 weeks after injection and human
PS1 was labeled using the NTF antibody (Figs 8G and EV5C, red),
nuclei were stained with Hoechst (blue), and GFP was enhanced by
an antibody (green). While human wild-type PS1 was diffused
throughout the cell, the P264L-mutated protein accumulated in foci
(Figs 8G and EV5C, insets (arrows)), suggesting that this mutation
induces the aggregation of PS1 within the brain. To compare the
number and average area of PS1 deposits in brains of mice injected
with the mutated or wild-type PS1-expressing virus, we used image
processing software (ImageJ). Our results indicated that mice
injected with the virus that drives the expression of P264L PS1 had
much more PS1 foci than their counterparts that expressed the wild-
type PS1 (average of 34.8 and 8.8 foci/slice, respectively). Further-
more, the PS1 foci observed in P264L PS1-expressing mice were
much smaller in size (Figs 8H and EV5D). These results are consis-
tent with the observation that PS1 accumulates in foci in the brain
of humans that carry the P264L, fAD-linked mutation (Martikainen
et al, 2010).
Discussion
The common temporal emergence patterns of different neurode-
generative maladies (Amaducci & Tesco, 1994) and the involvement
of certain aggregative proteins in the development of more than one
disorder have led us to speculate that in some cases, one mecha-
nism underlies the manifestation of distinct neurodegenerative
diseases. Here, we show that similar to its key role in the correct
folding of PrP (Cohen & Taraboulos, 2003; Ben-Gedalya et al, 2011),
cyclophilin B activity is critically required for the correct folding and
processing (Fig 9) of PS1. The inhibition of cyclophilin activity
results in PS1 misfolding (III), aggregation (IV), and deposition in
the ERQC (V). Similarly, the substitution of proline 264 or 267
abolishes a cyclophilin recognition site which is vital for the proper
maturation of PS1 (VI) leading to its digestion by the proteasome or
aggregation (VII). Interestingly, our results indicate that while
P264L PS1 mutant is preferably deposited in the ERQC, molecules
that carry the P267S substitution are highly prone to proteasomal
degradation. The attenuation of PS1 proteolytic activity impairs
mitochondrial distribution and function, plausibly impedes addi-
tional PS1 functions, and initiates the pathological process that
underlies the development of fAD (VIII).
Our discoveries point at the comparison of mutational patterns
as a valid approach for investigating mechanisms that trigger the
manifestation of neurodegeneration, support the concept that
distinct disorders can emanate from common mechanisms, and
strengthen the emerging idea that in some cases, the attenuation
of PS1 activity is accountable for the emergence of AD (Shen &
Kelleher, 2007; Xia et al, 2015).
How misfold polypeptides are sorted to be deposited in different
cellular sites is largely unknown. While PrP accumulates in cyto-
solic aggresomes following CsA treatment (Cohen & Taraboulos,
▸
Figure 8. Cyclophilin B is required for the maturation and integrity of PS1in the mouse brain.
A–D Brains of cyclophilin B knockout (KO) mice and of their wild-type siblings were homogenized and subjected to high-speed sedimentation assay. WB analysis using
aPS1CTF antibody revealed reduced levels of soluble PS1in the supernatants of cyclophilin B KO animals compared to supernatants of control animals (A, C). In
contrast, PS1quantities in the pellets of wild-type and cyclophilin B KO mouse brains were similar (B, D). Error bars represent SEM of 4mice for each genotype,
*P<0.013 (Student’st-test).
E Similar results were obtained by immunohistochemistry (IHC) and visualization of PS1in the paraffin-embedded hippocampi of wild-type and cyclophilin B KO
animals using the CTF PS1antibody.
F A C-terminal b-APP-fluorescence-based activity assay shows reduced c-secretase activity in membranes of brains of cyclophilin B KO mice compared to
membranes isolated from brains of wild-type animals. Error bars represent SEM of three mice for each genotype, *P<0.02 (Student’st-test).
G Wild-type or P264L human PS1was expressed in the hippocampi of BALB/c mice by GFP-labeled lentiviral vectors (green channel). The mice were sacrificed
5weeks after the injection of the viruses, and brain slices were visualized by confocal microscopy. While wild-type human PS1exhibits diffuse cellular pattern
(upper panels, red), the human P264L mutant accumulates in foci throughout the dentate gyrus (DG) (lower panels, arrows). Scale bar, 20 lm.
H Image analysis indicated that PS1foci in brains of mice that were injected with P264LPS1virus occupy smaller areas than those seen in brains of their
counterparts that express wild-type PS1(a total of 5brain slices per genotype were analyzed; error bars represent SEM).
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
14
Published online: October 5, 2015
AE
Wild-type Cyclophilin B KO
B
Cyp B
Cyp B
Pellets
Supernatants
0
10
20
30
40
50
WT KO
Supernatants
0
5
10
15
20
25
WT KO
Pellets
ns
*P=0.013
Processed
PS1 (CTF)
Processed
PS1 (CTF)
High MW
PS1
CD
Actin
Actin
Relative band intensities
Relative band intensities
Dentate gyrus – PS1 CTF Ab
High MW
PS1 Wild-type
Cyclophilin B KO
F
G
H
GFP (Virus) /
Hoechst / PS1 Hoechst / PS1
DG
P264L human PS1
DG
Wild-type human PS1
Insets
0
0.1
0.2
0.3
0.4
0.5
0.6
WT P264L
Average foci area (μm2)
*P=7.48E-7
0
20
40
60
80
100
120
WT KO
Normalized fluorescence
*P<0.02
Rate of γsecretase activity
Relative size of PS1 foci
PS1 in brains of cyclophilin B KO and WT mice
Figure 8.
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
15
Published online: October 5, 2015
2003), PS1 is deposited in the ERQC as a result of the same treat-
ment. Moreover, while proteasome inhibition directs PS1 molecules
that carry the P264L and/or P267S substitutions to the ERQC, the
same treatment sends PS1 molecules that carry the fAD-linked
A264E mutation to aggresomes (Johnston et al, 1998). These find-
ings raise the question of what determines the fate of a specific
misfolded PS1 conformer and directs it to the adequate deposition
site. It is plausible that the accumulation of an aggregative protein
in an aggresome, where it can be recycled (Ben-Gedalya et al,
2011), is preferred compared to its deposition in the ERQC where it
is trapped and may impair ER function. Thus, it is probable that a
rapid and robust aggregation averts the retro-translocation of P264L
and P267S PS1 to the cytosol rendering its deposition in the ERQC.
This theme is supported by the finding that ERQC-resident PS1
molecules are immobile (Fig 4), while PrP in aggresomes is dynamic
(Ben-Gedalya et al, 2011).
The nature of the aggregating protein may also be a determining
factor in the triage to different quality control compartments. While
the PrP is a GPI anchored protein, PS1 is a multi-pass transmem-
brane protein, a feature that may present a challenge to the cellular
degradation machinery. Recently, an alternative route involving
intermembrane proteolysis was suggested (Fleig et al, 2012), thus
allowing dislocation of clipped products.
Another interesting question is why P264L PS1 exhibits partial
activity (Fig 5A and C–E) while P267S PS1 appears to undergo
degradation (Fig 5A and B), thereby showing no c-secretase activity
(Fig 5C and D). It is possible that the substitutions of prolines 264
and 267 in the sequence of PS1 differentially affect the spatial struc-
ture of the protein. This explanation proposes that P264L PS1 mole-
cules attain a three-dimensional structure which is more similar to
the wild-type conformation than that of the P267S-mutated PS1.
Thus, P264L PS1 molecules escape degradation and exhibit partial
c-secretase activity, while PS1 molecules that carry the P267S muta-
tion are directed for digestion by the proteasome. Structural analy-
ses are required to examine this idea.
The widely accepted amyloid hypothesis suggests that AD stems
from the over-production and aggregation of Abpeptides which lead
to synaptic malfunction (Selkoe, 2011). Our study strongly suggests
that the substitution of proline 264 or 267 in the sequence of PS1
leads to AD by an alternative, non-canonical mechanism. The
reduced c-secretase activity and mitochondrial aberrant distribution
and function support the notion that the failure of vital cellular func-
tions that emanate from PS1 aberrant folding can cause AD late in
life (Shen & Kelleher, 2007; Chavez-Gutierrez et al, 2012). A recent
study strongly supports this idea by showing that certain fAD-
causing mutations in the sequence of PS1 lead to the abolishment of
c-secretase activity (Xia et al, 2015). Moreover, the aggregation of
PS1 itself may cause ER stress and an additional burden on the
proteasome, leading to cell death and disease. This concept is
strengthened by the reduced levels of PS1 observed in brains of
Attenuation
or loss of
PS1
functions
Translation
Cyclosporin-A / Aging
Properly folded PS1
γ secretase activity
Autophagy
Mitochondria-
ER interaction
Calcium
homeostasis
ERQC
Functional
PS1
Modifications,
endo-cleavage
and assembly
Aberrantly
folded PS1 Familial
Alzheimer’s
disease
Proteasome-mediated
degradation
ER
Cytosol
I
II
III IV
V
VI
VII
VIII
Figure 9. A model for the development of fAD in individuals that carry the P264LorP267L/S mutations.
To attain functionality, nascent PS1molecules require the assistance of cyclophilin B (I) in order to fold properly and undergo additional modifications (II). Due to aging-
associated decline in the efficiency of cyclophilin activity or as a result of inhibition by CsA, PS1molecules fail to fold properly (III), aggregate (IV), and are deposited
at the ERQC (V). Similarly, cyclophilins cannot support the folding of mutated PS1molecules which carry the P264LorP267L/S substitutions (VI). These molecules are
designated to proteasomal degradation (VII) or deposited at the ERQC when proteasomes are overtaxed (V). The aggregation and deposition in the ERQC lead to
attenuated PS1function and to the development of Alzheimer’s disease (VIII).
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
16
Published online: October 5, 2015
cyclophilin B knockout mice (Fig 8A–E), the findings that PS1-
containing cellular inclusions are present in brains of mice that
express the P264L PS1 (Fig 8G) and of individuals carrying this
mutated protein (Martikainen et al, 2010) as well as in brain
sections of patients who had sporadic AD (Busciglio et al, 1997;
Chui et al, 1998).
Why fAD which stems from the proline substitutions in PS1
onsets late in life is another key enigma. One possibility suggests
that early in life, sufficient efficiencies of two cellular mechanisms
prevent AD from emerging. First, a small fraction of the nascent
mutated PS1 molecules that are formed in cis conformation exhibit
sufficient PS1 activity, and second, an increased degradation capac-
ity clears the mutated PS1 molecules that are synthesized in trans
conformation. According to this model, aging-associated decline in
the competence of protein degradation mechanisms exposes the
aged organism to proteotoxicity and disease. This hypothesis is rein-
forced by the findings that the alteration of aging by the inhibition
of IGF1 signaling protects mice (Cohen et al, 2009; Freude et al,
2009) and worms (Cohen et al, 2006; Teixeira-Castro et al, 2011;
El-Ami et al, 2014) from proteotoxicity. Furthermore, cyclophilins
were found to be modifiers of proteostasis (Silva et al, 2011) that
aggregate and sediment in aged worms (David et al, 2010; Kirstein-
Miles et al, 2013).
The requirement of cyclophilins for the correct maturation of PrP
and PS1 highlights the key roles of proline cis/trans isomerization
for the maintenance of proteostasis and the prevention of proteino-
pathies. For instance, the activity of the prolyl isomerase Pin1
restores functionality of microtubule-associated protein TAU (Lu
et al, 1999). Our study points at ER-resident cyclophilin activity as
critical for the correct folding of PS1. In contrast, FKBP51 was
reported to enhance proteotoxicity and to be more abundant in
brains of old individuals and patients with AD than in those
of healthy controls (Blair et al, 2013). Similarly, cyclophilin D
deficiency was found to attenuate mitochondrial perturbation and
alleviate learning deficiencies of AD-model mice (Du et al, 2008),
implying that elevated activity of PPIase chaperones is not always
beneficial.
Finally, it is also possible that an aging-associated decline in the
integrity of deposition sites leads to the release of toxic species,
transforming these structures from protective entities to sources of
toxicity in late life stages (Ben-Gedalya & Cohen, 2012).
In conclusion, our study highlights the complexity of mecha-
nisms that lead to the development of neurodegenerative disorders,
indicating that while one mechanism underlies the development of
two distinct maladies, fAD may emanate from different types of
proteostasis failures. It also emphasizes the key role of the aging
process in enabling the manifestation of neurodegeneration late
in life.
Materials and Methods
Materials
Cell culture reagents were purchased from Biological Industries
(Beit Haemek, Israel). Protein concentration was determined using
BCA kit (Pierce 23223). Cyclosporin-A (CsA) (C1832 and MG132
(C2211) and all other reagents were from Sigma.
Cell cultures
Cells were grown at 37°C in DMEM supplemented with 10% fetal
calf serum. Transfections were achieved with the reagent TransIT-
LT1 (Mirus MC-MIR-2300) according to the manufacturer’s instruc-
tions. CHO cells stably expressing moderate levels of wild-type
human PS1 were selected by G418 (1 mg/ml). To generate stable
cell lines of PS1 constructs on PS1 or PS1 and PS2 null background,
presenilin KO cells (gift from Dr. Bart De Strooper; Herreman et al,
1999, 2003) were infected with the pBABE-puro retroviral vectors
expressing either empty, wild-type PS1 P264L, P267S, P264L/P267S,
or D257A (all based on the human wild-type PS1). Media was
replaced 24 h after infection, and 24 h later, infected cells were
selected with 2 lg/ml puromycin for 72 h.
Antibodies and dyes
PS1 NTF and PS1 CTF antibodies were purchased from Chemicon
(MAB1563 and MAB5232, respectively) and used for both immune
fluorescence assays and WB. PS2 antibody (Abcam ab51249) was
used to detect PS2 CTF by WB. BML-Pw8810-0500 for mono- and
poly-ubiquitinylated conjugates (FK2) was from Enzo. Calnexin
C-20, sc-6465, was from Santa Cruz. c-tubulin (T6557) and vimentin
(V6630) antibodies were from Sigma. MitoTracker Red CMXRos
M7512 was purchased from Invitrogen (San Diego, CA). DAPI
staining was achieved by Vectashield-DAPI mounting media
(VE-H-1200). BiP antibody was purchased from Abcam (ab21685).
c-secretase activity assays
Myc-tagged substrate method
PS1 and PS2 knockout MEFs (a generous gift of Bart De Strooper,
Leuven, Belgium) were maintained in DMEM F12 (Invitrogen,
Carlsbad, CA) supplemented with 10% HIFCS and penicillin–
streptomycin. PS1 constructs were expressed either transiently or by
retroviral infection producing stable clones. The c-secretase
substrate APP C99-myc (4°C) (kindly provided by A. Goate, Wash-
ington University) was expressed by transient transfection using
TransIT X2
TM
(Mirus Bio LLC, Madison, WI). Cells were harvested
20–22 h post-transfection and lysed in modified buffer containing
1% CHAPSO, 50 mM Hepes pH 7.2, 150 mM NaCl, 1% TX-100, and
protease inhibitor cocktail (Calbiochem #539134). Samples were
spun at 16,000 ×gfor 10 min, and supernatant was tested for
protein concentration by BCA assay. Cell lysates were subjected to
SDS–PAGE and then transferred to nitrocellulose membranes.
Membranes were blocked with TBST/5% milk and probed with
mouse anti-myc antibody (Sigma, clone 9E10) for the AICD and C99.
Fluorescent substrate method
Membrane fraction was isolated by homogenizing the cells in
50 mM Hepes (pH 7), 250 mM sucrose, 5 mM EDTA, and complete
protease inhibitor (Roche) (which does not contain pepstatin A—an
aspartyl protease inhibitor). Homogenates were centrifuged at
3,000 gfor 10 min to remove debris and nuclei. The supernatants
were then centrifuged at 100,000 gfor 1 h at 4°C. To extract the
dissolved proteins from the crude membranes, the pellets were
dissolved by shaking for 90 min in 100 ll of the same buffer supple-
mented with 1% CHAPSO at 4°C. The supernatants were collected
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
17
Published online: October 5, 2015
after an additional 100,000 gcentrifugation for 1 h at 4°C. Aliquots
were removed for protein concentration, and CHAPSO was diluted
to 0.25%. To measure the c-secretase activity in vitro, samples
containing 10 lg protein were incubated with 8 lM of a fluores-
cence-conjugated peptide c-secretase substrate (NMA-GGVVIATVK
(DNP)-DRDRDR-NH2). The proteolysis at the Ab40-, Ab42-, and
Ab43-generating cleavage sites results in enhanced fluorescence
(excitation 320 nm, emission 460 nm).
Quantification of soluble Ab42/40 using sandwich ELISA
Millipore (Temecula, CA USA) high-sensitivity human amyloid b42
(EZHS42) and b40 (EZHS40) ELISA kits were used according to the
manufacturer’s instructions. Samples were prepared from media
collected from PS1/2-KO MEF cells expressing both the specified
PS1 construct and APP C99-myc.
Immunofluorescence microscopy and live imaging
To detect PS1, cells were grown on poly-D-lysine-coated chamber
slides (Nunc, #155411), fixed (10% formalin in PBS, 30 min, RT),
quenched with cold 1% NH
4
Cl in PBS, permeabilized (0.1% TX-100
in PBS, 2 min, RT), and blocked with 2% BSA (30 min, RT). The
cells were then incubated overnight with the primary antibody (in
1% BSA, 4°C) and rinsed, and the secondary antibody conjugated to
fluorescent probe as mentioned (diluted 1:200 in 1% BSA) was
added for 1 h (RT). The labeled cells were mounted using Vecta-
shield-DAPI mounting media VE-H-1200 and viewed with a Zeiss
LSM 710 Axio Observer.Z1 laser scanning confocal microscope.
For time-lapse microscopy and FRAP and FLIP experiments, cells
were plated on poly-L-lysine (Sigma)-coated glass-bottom plates
(MatTek Corp., #P35GC-1.0-14-C) or on a chambered cover glass
system (Nunc, #177445). Confocal microscopy was conducted using
Zeiss LSM 710 scanning microscope with a 63×oil for FRAP, FLIP,
and immunofluorescence, and an LD Plan-Neofluar 40×/0.6 Corr
M27 objective for time-lapse microscopy. For time-lapse experi-
ments, Z-stack series of 1-lm scans were collected in 10-min inter-
vals for 16 h. For FRAP, a region of interest was bleached using a
488-nm laser for 2 s at full laser power, and single-scan images
were collected every 1 s for 1 min following the bleach. Fluores-
cence of the bleached region of interest (F) was calculated as
F=(Ii–Ib)/(Ir–Ib), where “Ii” is fluorescence intensity in the region
of interest, “Ir” is intensity in a reference area, and “Ib” is back-
ground intensity (outside all cells). Intensity data were recorded
using Zeiss ZEN software. Reported values are the average of at
least three data points. Zeiss ZEN and ImageJ software were used
for processing and quantification. For FLIP experiments, a 2 ×2lm
area of the cytosol was bleached continuously (with each scan),
while the fluorescence of the inclusion and a control cytosolic
region was measured.
Transmission electron microscopy (TEM)
Cells were seeded on poly-D-lysine-coated chamber slides (Nunc,
#155411) and treated with either vehicle or MG132 (10 lM for 5 h).
Cells were rinsed 3 times with PBS and fixed by incubation in 2.5%
paraformaldehyde and 0.1 M PB buffer (22 mM NaH
2
PO
4
,78mM
Na
2
HPO
4
) for 1 h at RT. After three rinses with cold PB buffer
containing 1%NH
4
Cl, cells were permeabilized with 0.05% TX-100
for 3 min at RT followed by three rinses with PB buffer and blocking
with 4% BSA in PBS for 30 min at RT. The cell monolayer was
fixed, dehydrated, embedded, and cut into thin sections as described
previously (Cohen & Taraboulos, 2003).
The analysis of PS1in the brain of cyclophilin B knockout mice
CyPB KO mice (Cabral et al, 2014) and their wild-type siblings
(strain C57BL/6, which were bred from Het X Hat mating) were
sacrificed at the age of 2 months, and brains were removed. One
hemisphere was snap-frozen and used for biochemical assays, while
the other half was fixed in 3% PFA for 24 h, then transferred to 1%
PFA, dehydrated, cleared with xylene, and embedded in paraffin.
PS1 immunohistochemistry was performed on 8-micron-thick paraf-
fin sections, precleared for endogenous peroxidase activity by incu-
bation in 3% H
2
O
2
. Antigen retrieval was performed by boiling in
pressure cooker 110°C for 5 min in 10 mM citrate buffer. Brain
sections were blocked by CAS block (00-8120; Invitrogen San Diego,
CA). Immunostaining was performed with the PS1 CTF antibody
(MAB5232; Millipore, MA USA). Anti-mouse Ig peroxidase
ImmPRESS reagent (MP-7402) was used as a secondary antibody,
and immunoreactivity was visualized using DAB substrate kit (SK-
4100), both from Vector laboratories (Burlingame, CA USA). Slides
were developed in parallel and then stained with Mayer’s hema-
toxylin, rinsed, and mounted prior to visualization.
Image analysis brain sections
Images of brain slices were taken using a Zeiss LSM 710 confocal
microscope. PS1 foci were counted and measured using ImageJ
software (using the “analyze particles” option, threshold was set to
0–60).
Expanded View for this article is available online:
http://emboj.embopress.org
Acknowledgements
This study was generously supported by the Rosalinde and Arthur Gilbert
Foundation (AFAR), the European Research Council (ERC, 281010), the National
Institute for Psychobiology in Israel (NIPI), and the Israel Science Foundation
(ISF, 671/11). This research was also partially supported by The Israel Science
Foundation (ISF 1764/12 awarded to TBC). We thank Dr. Gerardo Lederkremer
(Tel Aviv University) for providing us with the H2a-RFP plasmid, Dr. Alison
Goate (Mount Sinai Hospital) for the APP-Myc expression vector, Dr. Jonathan
Javitch (Colombia University) for the YFP-DAT plasmid, and Dr. Bart De
Strooper (K.U. Leuven) for presenilin 1/2-KO MEF cells. We thank Mrs. Naomi
Feinstein for expert assistance with EM experiments and Dr. Denes Agoston for
assisting mouse procedures. We also thank Ms. Filipa Carvalhal Marques for
critical reading of the manuscript.
Author contributions
EC and TBG initiated and designed the study and wrote the manuscript. TBG
performed cloning procedures, WB, IF, EM, FRAP and FLIP experiments as well
as in vitro assays and mouse brain analyses. LM performed WB experiments,
and MBS created mutated PS1plasmids. JCM and WAC created cyclophilin B
knockout mice. DFM created PS1-expressing lentiviruses, and IS and SF
injected mouse brains with the lentiviruses. TBC performed mouse brain
sectioning and IHC experiments.
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
18
Published online: October 5, 2015
Conflict of interest
The authors declare that they have no conflict of interest.
References
Aguzzi A, Polymenidou M (2004) Mammalian prion biology: one century of
evolving concepts. Cell 116:313 –327
Aguzzi A, Calella AM (2009) Prions: protein aggregation and infectious
diseases. Physiol Rev 89:1105 –1152
Amaducci L, Tesco G (1994) Aging as a major risk for degenerative diseases of
the central nervous system. Curr Opin Neurol 7:283 –286
Area-Gomez E, de Groof AJ, Boldogh I, Bird TD, Gibson GE, Koehler CM, Yu
WH, Duff KE, Yaffe MP, Pon LA, Schon EA (2009) Presenilins are enriched
in endoplasmic reticulum membranes associated with mitochondria. Am J
Pathol 175:1810 –1816
Area-Gomez E, Del Carmen Lara Castillo M, Tambini MD, Guardia-Laguarta C,
de Groof AJ, Madra M, Ikenouchi J, Umeda M, Bird TD, Sturley SL, Schon
EA (2012) Upregulated function of mitochondria-associated ER
membranes in Alzheimer disease. EMBO J 31:4106 –4123
Arias E, Cuervo AM (2011) Chaperone-mediated autophagy in protein quality
control. Curr Opin Cell Biol 23:184 –189
Ben-Gedalya T, Lyakhovetsky R, Yedidia Y, Bejerano-Sagie M, Kogan NM,
Karpuj MV, Kaganovich D, Cohen E (2011) Cyclosporin-A-induced prion
protein aggresomes are dynamic quality-control cellular compartments.
J Cell Sci 124:1891 –1902
Ben-Gedalya T, Cohen E (2012) Quality control compartments coming of age.
Traffic 13:635 –642
Bertram L, Tanzi RE (2008) Thirty years of Alzheimer’s disease genetics:
the implications of systematic meta-analyses. Nat Rev Neurosci 9:
768 –778
Bezprozvanny I, Mattson MP (2008) Neuronal calcium mishandling and the
pathogenesis of Alzheimer’s disease. Trends Neurosci 31:454 –463
Blair LJ, Nordhues BA, Hill SE, Scaglione KM, O’Leary JC III, Fontaine SN,
Breydo L, Zhang B, Li P, Wang L, Cotman C, Paulson HL, Muschol M,
Uversky VN, Klengel T, Binder EB, Kayed R, Golde TE, Berchtold N, Dickey
CA (2013) Accelerated neurodegeneration through chaperone-mediated
oligomerization of tau. J Clin Invest 123:4158 –4169
Brodsky JL, Skach WR (2011) Protein folding and quality control in the
endoplasmic reticulum: recent lessons from yeast and mammalian cell
systems. Curr Opin Cell Biol 23:464 –475
Busciglio J, Hartmann H, Lorenzo A, Wong C, Baumann K, Sommer B,
Staufenbiel M, Yankner BA (1997) Neuronal localization of presenilin-1
and association with amyloid plaques and neurofibrillary tangles in
Alzheimer’s disease. J Neurosci 17:5101 –5107
Cabral WA, Perdivara I, Weis M, Terajima M, Blissett AR, Chang W, Perosky JE,
Makareeva EN, Mertz EL, Leikin S, Tomer KB, Kozloff KM, Eyre DR,
Yamauchi M, Marini JC (2014) Abnormal type I collagen post-translational
modification and crosslinking in a cyclophilin B KO mouse model of
recessive osteogenesis imperfecta. PLoS Genet 10:e1004465
Campion D, Flaman J-M, Brice A, Hannequin D, Dubois B, Martin C, Moreau V,
Charbonnier F, Didierjean O, Tardieu S, Penet C, Puel M, Pasquier F, Le
Doze F, Bellis G, Calenda A, Heilig R, Martinez M, Mallet J, Bellis M et al
(1995) Mutations of the presenilin I gene in families with early-onset
Alzheimer’s disease. Hum Mol Genet 4:2373–2377
Chavez-Gutierrez L, Bammens L, Benilova I, Vandersteen A, Benurwar M,
Borgers M, Lismont S, Zhou L, Van Cleynenbreugel S, Esselmann H,
Wiltfang J, Serneels L, Karran E, Gijsen H, Schymkowitz J, Rousseau F,
Broersen K, De Strooper B (2012) The mechanism of gamma-Secretase
dysfunction in familial Alzheimer disease. EMBO J 31:2261 –2274
Chui DH, Shirotani K, Tanahashi H, Akiyama H, Ozawa K, Kunishita T,
Takahashi K, Makifuchi T, Tabira T (1998) Both N-terminal and C-terminal
fragments of presenilin 1colocalize with neurofibrillary tangles in
neurons and dystrophic neurites of senile plaques in Alzheimer’s disease.
J Neurosci Res 53:99 –106
Cohen E, Taraboulos A (2003) Scrapie-like prion protein accumulates in
aggresomes of cyclosporin A-treated cells. EMBO J 22:404 –417
Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A (2006) Opposing
activities protect against age-onset proteotoxicity. Science 313:
1604 –1610
Cohen E, Paulsson JF, Blinder P, Burstyn-Cohen T, Du D, Estepa G, Adame A,
Pham HM, Holzenberger M, Kelly JW, Masliah E, Dillin A (2009) Reduced
IGF-1signaling delays age-associated proteotoxicity in mice. Cell 139:
1157 –1169
David DC, Ollikainen N, Trinidad JC, Cary MP, Burlingame AL, Kenyon C (2010)
Widespread protein aggregation as an inherent part of aging in C. elegans.
PLoS Biol 8:e1000450
De Strooper B, Saftig P, Craessaerts K, Vanderstichele H, Guhde G, Annaert W,
Von Figura K, Van Leuven F (1998) Deficiency of presenilin-1inhibits the
normal cleavage of amyloid precursor protein. Nature 391:387 –390
Du H, Guo L, Fang F, Chen D, Sosunov AA, McKhann GM, Yan Y, Wang C,
Zhang H, Molkentin JD, Gunn-Moore FJ, Vonsattel JP, Arancio O, Chen JX,
Yan SD (2008) Cyclophilin D deficiency attenuates mitochondrial and
neuronal perturbation and ameliorates learning and memory in
Alzheimer’s disease. Nat Med 14:1097 –1105
El-Ami T, Moll L, Carvalhal Marques F, Volovik Y, Reuveni H, Cohen E
(2014) A novel inhibitor of the insulin/IGF signaling pathway protects
from age-onset, neurodegeneration-linked proteotoxicity. Aging Cell 13:
165 –174
Fleig L, Bergbold N, Sahasrabudhe P, Geiger B, Kaltak L, Lemberg MK (2012)
Ubiquitin-dependent intramembrane rhomboid protease promotes ERAD
of membrane proteins. Mol Cell 47:558 –569
Freude S, Hettich MM, Schumann C, Stohr O, Koch L, Kohler C, Udelhoven M,
Leeser U, Muller M, Kubota N, Kadowaki T, Krone W, Schroder H, Bruning
JC, Schubert M (2009) Neuronal IGF-1resistance reduces Abeta
accumulation and protects against premature death in a model of
Alzheimer’s disease. Faseb J 23:3315 –3324
Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW (1984)
Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science
226:544 –547
Hardy JA, Higgins GA (1992) Alzheimer’s disease: the amyloid cascade
hypothesis. Science 256:184 –185
Hecimovic S, Wang J, Dolios G, Martinez M, Wang R, Goate AM (2004 )
Mutations in APP have independent effects on Abeta and CTFgamma
generation. Neurobiol Dis 17:205 –218
Herreman A, Hartmann D, Annaert W, Saftig P, Craessaerts K, Serneels L,
Umans L, Schrijvers V, Checler F, Vanderstichele H, Baekelandt V, Dressel
R, Cupers P, Huylebroeck D, Zwijsen A, Van Leuven F, De Strooper B (1999)
Presenilin 2deficiency causes a mild pulmonary phenotype and no
changes in amyloid precursor protein processing but enhances the
embryonic lethal phenotype of presenilin 1deficiency. Proc Natl Acad Sci
USA 96:11872 –11877
Herreman A, Van Gassen G, Bentahir M, Nyabi O, Craessaerts K, Mueller U,
Annaert W, De Strooper B (2003) Gamma-Secretase activity requires the
presenilin-dependent trafficking of nicastrin through the Golgi apparatus
but not its complex glycosylation. J Cell Sci 116:1127 –1136
ª2015 The Authors The EMBO Journal
Tziona Ben-Gedalya et al Cyclophilin inhibition leads to PS1aggregation The EMBO Journal
19
Published online: October 5, 2015
Hsiao K, Baker HF, Crow TJ, Poulter M, Owen F, Terwilliger JD, Westaway D,
Ott J, Prusiner SB (1989) Linkage of a prion protein missense variant to
Gerstmann-Sträussler syndrome. Nature 338:342 –345
Hutton M, Busfield F, Wragg M, Crook R, Perez-Tur J, Clark RF, Prihar G,
Talbot C, Phillips H, Wright K, Baker M, Lendon C, Duff K, Martinez A,
Houlden H, Nichols A, Karran E, Roberts G, Roques P, Rossor M et al
(1996) Complete analysis of the presenilin 1gene in early onset
Alzheimer’s disease. NeuroReport 7:801 –805
Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to
misfolded proteins. J Cell Biol 143:1883 –1898
Kaganovich D, Kopito R, Frydman J (2008) Misfolded proteins partition between
two distinct quality control compartments. Nature 454:1088 –1095
Kamhi-Nesher S, Shenkman M, Tolchinsky S, Fromm SV, Ehrlich R,
Lederkremer GZ (2001) A novel quality control compartment derived from
the endoplasmic reticulum. Mol Biol Cell 12:1711 –1723
Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU (2013) Molecular
chaperone functions in protein folding and proteostasis. Annu Rev Biochem
82:323 –355
Kirstein-Miles J, Scior A, Deuerling E, Morimoto RI (2013) The nascent
polypeptide-associated complex is a key regulator of proteostasis. EMBO J
32:1451 –1468
Kondratyev M, Avezov E, Shenkman M, Groisman B, Lederkremer GZ (2007)
PERK-dependent compartmentalization of ERAD and unfolded protein
response machineries during ER stress. Exp Cell Res 313:3395 –3407
Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, Peterhoff CM, Wolfe DM,
Martinez-Vicente M, Massey AC, Sovak G, Uchiyama Y, Westaway D,
Cuervo AM, Nixon RA (2010) Lysosomal proteolysis and autophagy require
presenilin 1and are disrupted by Alzheimer-related PS1mutations. Cell
141:1146 –1158
Lippincott-Schwartz J, Altan-Bonnet N, Patterson GH (2003) Photobleaching
and photoactivation: following protein dynamics in living cells. Nat Cell
Biol 5:S7–S14
Lu PJ, Wulf G, Zhou XZ, Davies P, Lu KP (1999) The prolyl isomerase Pin1
restores the function of Alzheimer-associated phosphorylated tau protein.
Nature 399:784 –788
Martikainen P, Pikkarainen M, Pontynen K, Hiltunen M, Lehtovirta M, Tuisku
S, Soininen H, Alafuzoff I (2010) Brain pathology in three subjects from
the same pedigree with presenilin-1(PSEN1)P264L mutation. Neuropathol
Appl Neurobiol 36:41 –54
Mendez MF, McMurtray A (2006) Frontotemporal dementia-like phenotypes
associated with presenilin-1mutations. Am J Alzheimers Dis Other Demen
21:281 –286
Morley JF, Brignull HR, Weyers JJ, Morimoto RI (2002) The threshold for
polyglutamine-expansion protein aggregation and cellular toxicity is
dynamic and influenced by aging in Caenorhabditis elegans. Proc Natl
Acad Sci USA 99:10417 –10422
Petronilli V, Penzo D, Scorrano L, Bernardi P, Di Lisa F (2001) The
mitochondrial permeability transition, release of cytochrome c and cell
death. Correlation with the duration of pore openings in situ.J Biol Chem
276:12030 –12034
Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95:13363 –13383
Quarteronet D, Pradier L, Czech C, Delalonde L, Burgevin MC, Doble A, Petitet
F(1996) Localization of presenilin-1mRNA in rat brain. NeuroReport 7:
2587 –2591
Roberson ED (2012) Mouse models of frontotemporal dementia. Ann Neurol
72:837 –849
Sato T, Diehl TS, Narayanan S, Funamoto S, Ihara Y, De Strooper B, Steiner H,
Haass C, Wolfe MS (2007) Active gamma-secretase complexes contain
only one of each component. J Biol Chem 282:33985 –33993
Schiene-Fischer C (2014) Multidomain peptidyl prolyl cis/trans Isomerases.
Biochim Biophys Acta 1850:2005 –2016
Schrader EK, Harstad KG, Matouschek A (2009) Targeting proteins for
degradation. Nat Chem Biol 5:815 –822
Selkoe DJ (2003) Folding proteins in fatal ways. Nature 426:900 –904
Selkoe DJ, Wolfe MS (2007) Presenilin: running with scissors in the
membrane. Cell 131:215 –221
Selkoe DJ (2011) Alzheimer’s disease. Cold Spring Harb Perspect Biol 3: pii:
a004457
Shen J, Kelleher RJ III (2007) The presenilin hypothesis of Alzheimer’s disease:
evidence for a loss-of-function pathogenic mechanism. Proc Natl Acad Sci
USA 104:403 –409
Sherrington R, Rogaev EI, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H,
Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin JF, Bruni AC, Montesi MP,
Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I et al (1995) Cloning of a
gene bearing missense mutations in early-onset familial Alzheimer’s
disease. Nature 375:754 –760
Silva MC, Fox S, Beam M, Thakkar H, Amaral MD, Morimoto RI (2011)A
genetic screening strategy identifies novel regulators of the proteostasis
network. PLoS Genet 7:e1002438
Teixeira-Castro A, Ailion M, Jalles A, Brignull HR, Vilaca JL, Dias N, Rodrigues
P, Oliveira JF, Neves-Carvalho A, Morimoto RI, Maciel P (2011) Neuron-
specific proteotoxicity of mutant ataxin-3in C. elegans: rescue by the
DAF-16 and HSF-1pathways. Hum Mol Genet 20:2996 –3009
Thinakaran G, Borchelt DR, Lee MK, Slunt HH, Spitzer L, Kim G, Ratovitsky T,
Davenport F, Nordstedt C, Seeger M, Hardy J, Levey AI, Gandy SE,
Jenkins NA, Copeland NG, Price DL, Sisodia SS (1996) Endoproteolysis of
presenilin 1and accumulation of processed derivatives in vivo.Neuron 17:
181 –190
Walker LC, Levine H III, Mattson MP, Jucker M (2006) Inducible proteopathies.
Trends Neurosci 29:438 –443
Wang Q, Li L, Ye Y (2008) Inhibition of p97-dependent protein degradation by
Eeyarestatin I. J Biol Chem 283:7445 –7454
Wolfe MS, Xia W, Ostaszewski BL, Diehl TS, Kimberly WT, Selkoe DJ (1999)
Two transmembrane aspartates in presenilin-1required for presenilin
endoproteolysis and gamma-secretase activity. Nature 398:513 –517
Xia W (2008) From presenilinase to gamma-secretase, cleave to capacitate.
Curr Alzheimer Res 5:172 –178
Xia D, Watanabe H, Wu B, Lee SH, Li Y, Tsvetkov E, Bolshakov VY, Shen J,
Kelleher RJ III (2015) Presenilin-1knockin mice reveal loss-of-function
mechanism for familial Alzheimer’s disease. Neuron 85:967 –981
Yamazaki M, Oyanagi K, Mori O, Kitamura S, Ohyama M, Terashi A, Kitamoto
T, Katayama Y (1999) Variant Gerstmann-Sträussler syndrome with the
P105L prion gene mutation: an unusual case with nigral degeneration and
widespread neurofibrillary tangles. Acta Neuropathol 98:506 –511
Yu L, McPhee CK, Zheng L, Mardones GA, Rong Y, Peng J, Mi N, Zhao Y, Liu Z,
Wan F, Hailey DW, Oorschot V, Klumperman J, Baehrecke EH, Lenardo MJ
(2010) Termination of autophagy and reformation of lysosomes regulated
by mTOR. Nature 465:942 –946
The EMBO Journal ª2015 The Authors
The EMBO Journal Cyclophilin inhibition leads to PS1aggregation Tziona Ben-Gedalya et al
20
Published online: October 5, 2015