Cyclophilin D deficiency attenuates mitochondrial and neuronal perturbation and ameliorates learning and memory in Alzheimer's disease

Abstract

Cyclophilin D (CypD, encoded by Ppif) is an integral part of the mitochondrial permeability transition pore, whose opening leads to cell death. Here we show that interaction of CypD with mitochondrial amyloid-beta protein (Abeta) potentiates mitochondrial, neuronal and synaptic stress. The CypD-deficient cortical mitochondria are resistant to Abeta- and Ca(2+)-induced mitochondrial swelling and permeability transition. Additionally, they have an increased calcium buffering capacity and generate fewer mitochondrial reactive oxygen species. Furthermore, the absence of CypD protects neurons from Abeta- and oxidative stress-induced cell death. Notably, CypD deficiency substantially improves learning and memory and synaptic function in an Alzheimer's disease mouse model and alleviates Abeta-mediated reduction of long-term potentiation. Thus, the CypD-mediated mitochondrial permeability transition pore is directly linked to the cellular and synaptic perturbations observed in the pathogenesis of Alzheimer's disease. Blockade of CypD may be a therapeutic strategy in Alzheimer's disease.

Full text

Cyclophilin D deficiency attenuates mitochondrial and neuronal
perturbation and ameliorates learning and memory in Alzheimer's
disease
Heng Du1, Lan Guo1, Fang Fang1, Doris Chen1, Alexander A Sosunov2, Guy M McKhann2,
Yilin Yan3, Chunyu Wang3, Hong Zhang4,5, Jeffery D Molkentin6, Frank J Gunn-Moore7,
Jean Paul Vonsattel4, Ottavio Arancio4,5, John Xi Chen8, and Shi Du Yan1,4,5
1Department of Surgery, College of Physicians and Surgeons, Columbia University, 630 West 168th
Street, New York, New York 10032, USA 2Department of Neurosurgery, College of Physicians and
Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032, USA 3Biology
Department, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic
Institute, 110 8th Street, Troy, New York 12180-3590, USA 4Department of Pathology, College of
Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032,
USA 5Taub Institute for Research on Alzheimer's Disease and the Aging Brain, College of
Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, New York 10032,
USA 6Department of Pediatrics, University of Cincinnati, Children's Hospital Medical Center, 3333
Burnet Avenue, Cincinnati, Ohio 45229, USA 7School of Biology, 79 North Street, University of St.
Andrews, St. Andrews KY16 9TS, Scotland 8Department of Neurology, Memorial Sloan-Kettering
Cancer Center, Cornell University, 1275 York Avenue, New York, New York 10065, USA
Abstract
Cyclophilin D (CypD, encoded by Ppif) is an integral part of the mitochondrial permeability transition
pore, whose opening leads to cell death. Here we show that interaction of CypD with mitochondrial
amyloid-β protein (Aβ) potentiates mitochondrial, neuronal and synaptic stress. The CypD-deficient
cortical mitochondria are resistant to Aβ- and Ca2+-induced mitochondrial swelling and permeability
transition. Additionally, they have an increased calcium buffering capacity and generate fewer
mitochondrial reactive oxygen species. Furthermore, the absence of CypD protects neurons from
Aβ- and oxidative stress-induced cell death. Notably, CypD deficiency substantially improves
learning and memory and synaptic function in an Alzheimer's disease mouse model and alleviates
Aβ-mediated reduction of long-term potentiation. Thus, the CypD-mediated mitochondrial
permeability transition pore is directly linked to the cellular and synaptic perturbations observed in
the pathogenesis of Alzheimer's disease. Blockade of CypD may be a therapeutic strategy in
Alzheimer's disease.
© 2008 Natural Publishing Group
Correspondence should be addressed to S.D.Y. (sdy1@columbia.edu)..
AUTHOR CONTRIBUTIONS H.D. designed and did experiments and assisted with the preparation of the manuscript. L.G. contributed
to the study of in vitro cultured neurons. F.F. did quantitative real-time PCR experiments. L.G. and D.C. performed genotyping of
transgenic mice. A.A.S. and G.M.M. conducted electron microscopy studies. Y.Y. and C.W. performed surface plasmon resonance
experiments. F.J.G.-M. provided some suggestions. J.D.M. provided CypD-knockout mice. H.Z. and O.A. performed LTP experiments.
J.P.V. provided information of human brain tissues. J.X.C. provided suggestions for the experimental design and assisted with the
preparation of manuscript. S.D.Y. initiated, directed and supervised the research, designed and assisted experiments, analyzed data,
developed the concept and wrote the manuscript.
Note: Supplementary information is available on the Nature Medicine website.
NIH Public Access
Author Manuscript
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Published in final edited form as:
Nat Med. 2008 October ; 14(10): 1097–1105. doi:10.1038/nm.1868.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Mitochondrial dysfunction is a feature of the Alzheimer's disease brain1-5. Recent studies have
highlighted the role of mitochondrial Aβ in Alzheimer's disease pathogenesis6-15. Aβ species
have been found in the mitochondria of both Alzheimer's disease brain and transgenic mouse
models of Alzheimer's disease overexpressing Aβ6,7,9,13-16. Accumulation of Aβ in
mitochondria occurs before extracellular amyloid deposition and increases with age.
Accordingly, Aβ is linked to the mitochondrial malfunction observed in the Alzheimer's disease
brain and mouse models of Alzheimer's disease6,7,17. For instance, increased expression of
amyloid-binding alcohol dehydrogenase, an intracellular Aβ-binding protein, exacerbates
Aβ-mediated mitochondrial and neuronal stress8,9. Aβ can also directly disrupt mitochondrial
function, and such disruption causes oxidative stress4,18. However, the essential intracellular
mechanisms underlying Aβ-mediated mitochondrial malfunction have yet to be elucidated.
The mitochondrial permeability transition pore (mPTP) has a central role in both necrotic and
apoptotic neuronal cell death. Opening of the mPTP collapses the membrane potential and
amplifies apoptotic mechanisms by releasing proteins with apoptogenic potential from the
inner membrane space19-21. The mPTP is thought to involve the voltage-dependent anion
channel in the outer membrane, the adenine nucleotide translocase in the inner membrane and
CypD in the mitochondrial matrix20,22-25. CypD, a peptidylprolyl isomerase F, resides in the
mitochondrial matrix and associates with the inner mitochondrial membrane during the
opening of the mPTP. Oxidative and other cellular stresses promote CypD translocation to the
inner membrane26-31, and this translocation acts as a key factor to trigger the opening of the
mPTP. Moreover, recent studies show that a genetic deficiency in CypD protects from Ca2+-
and oxidative stress-induced cell death and that CypD functions as a necessary component of
the mPTP30,32-34. The observations that Aβ progressively accumulates in brain mitochondria
from individuals with Alzheimer's disease and Alzheimer's disease model mice and that
oxidative stress is enhanced in an Aβ-rich environment led us to explore the mechanisms
underlying Aβ-mediated mitochondrial dysfunction. Our present study offers new insights into
the mechanism underlying CypD-dependent mPTP opening and synaptic function during the
pathogenesis of Alzheimer's disease.
RESULTS
Interaction of CypD with mitochondrial Aβ
In view of the increased expression of CypD associated with amyloid pathology in addition to
aging (Supplementary Fig. 1a-i online), we explored whether CypD serves as a mitochondrial
target potentiating Aβ-induced cellular perturbation. We first examined the interaction of CypD
with Aβ by surface plasmon resonance (SPR)35,36. Recombinant human CypD protein
(Supplementary Fig. 2a online) bound Aβ in a dose-dependent manner (Fig. 1a-f). The CypD-
Aβ interaction was specific, because reversed-sequence Aβ peptide showed no binding with
CypD (Fig. 1c,f), and antibodies against either Aβ or CypD inhibited binding (data not shown).
The equilibrium dissociation constants (Kd) for Aβ40 (Aβ peptide residues 1-40), Aβ42 (Aβ
peptide residues 1-42), oligomeric Aβ40 and oligomeric Aβ42 were 1.7 μM, 164 nM, 227 nM
and 4 nM, respectively. Therefore, Aβ oligomers and Aβ42 have higher affinity for binding to
CypD.
To determine whether CypD and Aβ actually interact in pathophysiologically relevant settings,
we subjected mitochondrial proteins to immunoprecipitation with an antibody to CypD
followed by immunoblotting with an antibody to Aβ. CypD-Aβ complexes, corresponding to
Aβ-immunoreactive bands, were detected in the cortical mitochondria of Alzheimer's disease
brains (Fig. 1g) but not (or very little) in those of non-Alzheimer's disease control brains (Fig.
1g). Aβ-immunoreactive bands disappeared when the antibody to CypD was replaced by
preimmune IgG (Fig. 1g). Densitometry of all immunoreactive bands combined revealed that
CypD-Aβ complexes were increased by 10-13-fold in Alzheimer's disease cortical
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mitochondria compared to non-Alzheimer's disease cortical mitochondria (Fig. 1h). In parallel,
mitochondrial Aβ was increased by nine- to tenfold in Alzheimer's disease brain
(Supplementary Fig. 2b), indicating an association between CypD-Aβ complex and the
presence of mitochondrial Aβ. Furthermore, CypD-Aβ complex was also found in the cortical
mitochondria of transgenic mAPP mice overexpressing a mutant form of human amyloid
precursor protein (APP) and Aβ, but not in mitochondria from CypD-deficient mAPP mice
(mAPP-Ppif-/-), CypD-null (Ppif-/-) or nontransgenic mice (Fig. 1i-j). Transgenic CypD-null
mice are described in Supplementary Figure 2c-g. These results indicate that the CypD-Aβ
interaction occurs in Alzheimer's disease brain and transgenic mice with Alzheimer's-like
pathology.
Confocal and electron microscopic studies confirmed colocalization of CypD and Aβ in
mitochondria (Fig. 2a-d). In the cerebral cortices of people with Alzheimer's disease (Fig. 2a)
and mAPP mice (Fig. 2b), Aβ and CypD colocalized extensively (Fig. 2a,b). In the absence of
antibodies to Aβ and CypD (Fig. 2a,b) or after neutralization of the antibodies with their
antigens (Aβ42 and CypD protein; Fig. 2a and data not shown), staining was lost. Immunogold
electron microscopy with gold-conjugated antibodies to Aβ42 (18-nm gold particles) and
CypD (12-nm gold particles) revealed that the two different sizes of gold particles were
colocalized in the Alzheimer's disease (Fig. 2c and Supplementary Fig. 3a online) and mAPP
brain mitochondria (Fig. 2d). Two gold particles that did not overlap but were extremely close
to each other may also be indicative of CypD-Aβ colocalization because of the intercenter
distance of the two gold particles37. As a positive control, we looked for Aβ in the plaques of
mAPP mice (Supplementary Fig. 3g). The gold particle labeling disappeared when antibodies
to Aβ42 and CypD were absent, replaced by preimmune IgG or preadsorbed with the respective
antigens (Aβ42 or CypD) (Supplementary Fig. 3c-f,h,i).
CypD deficiency attenuates Aβ-induced mitochondrial stress
First, we assessed the capacity of cortical mitochondria for Ca2+ uptake by measuring the
disappearance of extramitochondrial free Ca2+ from the medium after the addition of CaCl2
pulses. The capacity for calcium uptake changed in an age-dependent manner in both
nontransgenic and mAPP mice. Compared to mitochondria from mice at 3-6 months of age,
nontransgenic mitochondria from 12-month-old mice showed a trend toward a reduction of the
capacity for Ca2+ uptake (9% reduction, 235.7 ± 10.08 nmoles Ca2+ per milligram protein at
12 months versus 260 ± 10.03 nmoles Ca2+ per milligram protein at 3 months; Fig. 3a). mAPP
mitochondria showed an even poorer calcium capacity compared to the nontransgenic
mitochondria; impaired Ca2+ uptake capacity started at 6 months and progressively decreased
in 12-month-old mAPP mice (reduction of 18% (220 ± 10.79 nmoles Ca2+ per milligram
protein) and 50% (133.3 ± 16.67 nmoles Ca2+ per milligram protein) for 6 and 12 months,
respectively, versus 3-month-old mAPP mice (267 ± 16.67 nmoles Ca2+ per milligram protein);
Fig. 3a). Notably, mAPP-Ppif-/- cortical mitochondria were able to take up more Ca2+ (591.7
± 11.1 nmoles Ca2+ per milligram protein and 333.3 ± 10.5 nmoles Ca2+ per milligram protein
for 6 and 12 months, respectively) than mAPP mitochondria. Similarly, the addition of
cyclosporine A, an inhibitor of CypD, to mAPP cortical mitochondria showed a higher
buffering capacity of Ca2+ (Fig. 3b,c). Nontransgenic cortical mitochondria buffered against
CaCl2 uptake (242.9 ± 13 nmoles Ca2+ per milligram protein), and this capacity was
significantly increased after preincubation with cyclosporine A (Supplementary Fig. 4a,b
online). The Ppif-/- cortical mitochondria took up CaCl2 (900 ± 25.8 nmoles Ca2+ per milligram
protein) with a similar capacity to the cyclosporine A-treated nontransgenic mitochondria
(Supplementary Fig. 4a,b).
To determine the function of the mPTP, we measured mitochondrial swelling in response to
Ca2+. Cortical mitochondria from transgenic and nontransgenic mice showed swelling in
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response to Ca2+, and mAPP mitochondria showed a greater swelling at 12 months of age than
did nontransgenic mitochondria, though cortical mitochondria of both nontransgenic and
mAPP mice showed an age-dependent increase in swelling in response to Ca2+ (Fig. 3d,e).
Notably, mAPP-Ppif-/- cortical mitochondria were more resistant to swelling and permeability
transition induced by Ca2+ than were mAPP mitochondria (Fig. 3d,f). The addition of
cyclosporine A to mAPP mitochondria also attenuated swelling in response to Ca2+ (Fig. 3d,f).
To assess the inner mitochondrial membrane potential in brain in situ, we loaded brain slices
from transgenic mice with tetramethylrhodamine methyl ester (TMRM), a fluorescent probe
to monitor the mitochondrial membrane potential. This indicator dye is a lipophilic cation
accumulated by mitochondria in proportion to the membrane potential. Mitochondrial
depolarization (disrupting or decreasing membrane potential) results in a loss of dye from the
mitochondria and a decrease in mitochondrial fluorescence intensity. The intensity of TMRM
staining was significantly decreased in the cerebral cortex and hippocampus of mAPP mice
compared to other groups of mice (Fig. 3g). However, mAPP-Ppif-/- mice had mitochondria
that were largely resistant to the loss of inner membrane potential, showing higher TMRM
staining intensity than mAPP mice (Fig. 3g). Thus, mitochondria lacking CypD were protected
from Aβ-mediated swelling and opening of the membrane permeability transition pore.
To evaluate mitochondrial reactive oxygen species (ROS) generation, we gave transgenic mice
MitoSox Red, a unique fluorogenic dye used for highly selective detection of superoxide
production in the mitochondria of live cells. The percentage of area occupied by MitoSox Red
staining was considerably increased in the cerebral cortices and hippocampi (hippocampal
regions CA1 to CA3) of mAPP mice by two- to threefold compared to other groups of mice,
whereas mAPP-Ppif-/- mice showed much less MitoSox staining (Fig. 4a and Supplementary
Fig. 4c,d). These data indicate that the absence of CypD attenuates Aβ-mediated mitochondrial
ROS generation.
Further, mAPP mice showed an age-dependent increase in CypD translocation to the
mitochondrial inner membrane (Fig. 4b). The CypD-Aβ complex was also present in the
mitochondrial inner membrane of mAPP mice (Fig. 4c).
Next, we assessed mitochondrial function by measuring oxygen consumption, the activity of
cytochrome c oxidase (COX IV) and ATP abundance in transgenic mouse brains. Compared
to Ppif-/- and nontransgenic mice, mAPP mice showed a reduction in ADP-induced respiration
control rate (RCR) at 6 and 12 months of age, whereas mAPP-Ppif-/- mice had a diminished
reduction in RCR (Fig. 4d). Because mitochondria from mAPP mice had an impaired calcium
capacity, we determined the effect of calcium on RCR. Calcium-induced RCR was decreased
in 12-month-old mAPP cortical mitochondria but not in mAPP-Ppif-/- mitochondria as
compared with nontransgenic mitochondria (Fig. 4e). Additionally, mAPPmice had a reduced
COX IV activity (Fig. 4f) and ATP abundance (Fig. 4g), whereas mAPP-Ppif-/- mice had
markedly increased mitochondrial enzyme activity and ATP abundance (Fig. 4f,g). The COX
IV activity and ATP abundance in nontransgenic mice were comparable to those in the
Ppif-/- mice (Fig. 4f,g). Similarly, CypD-deficient mitochondria were also resistant to
exogenous Aβ-mediated impairment in calcium capacity, swelling, CypD translocation and
cytochrome c release (Supplementary Fig. 5 online). These data indicate that CypD deficiency
attenuates or protects against Aβ-mediated mitochondrial dysfunction.
CypD-Aβ interaction induces neuronal death
To directly determine the effects of CypD deficiency on Aβ- and oxidative stress-induced
neuronal death, we examined cultured cortical neurons from nontransgenic and Ppif-/- mice
(Supplementary Fig. 6a online). The CypD-Aβ complex was detected in nontransgenic
cortical neurons but not in Ppif-/- neurons exposed to Aβ (Supplementary Fig. 6b). Incubation
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of Aβ42 with nontransgenic cortical neurons reduced mitochondrial membrane potential, as
shown by TMRM staining, in a time- and dose-dependent manner, whereas Ppif-/- neurons had
attenuated Aβ-induced reduction of the mitochondrial membrane potential (Fig. 5a,b).
Consequently, Aβ-treated nontransgenic cortical neurons showed increased cytochrome c
release as compared to Aβ-treated Ppif-/- and vehicle-treated neurons (Fig. 5c). The addition
of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a mitochondrial
uncoupler, dissipated membrane potential and increased cytochrome c release in nontransgenic
and Ppif-/- neurons. The absence of CypD attenuated Aβ-induced apoptosis, as shown by a
reduction in the number of TUNEL-positive cells in Ppif-/- neurons exposed to Aβ (Fig. 5d).
The addition of cyclosporine A had a similar effect on Aβ-induced apoptosis (Fig. 5d).
Because CypD deficiency attenuated ROS generation (Fig. 4a) in mAPP mice, we evaluated
the direct effect of CypD deficiency on oxidative stress-induced mitochondrial and neuronal
toxicity. Flow cytometry analysis of fluorescently labeled cells showed a marked dose-
dependent reduction in the number of TMRM-positive cells in both nontransgenic and Ppif-/-
neurons exposed to increasing concentrations of H2O2. However, Ppif-/- neurons were more
resistant to H2O2-induced loss of membrane potential than were nontransgenic neurons, as
shown by a higher percentage of TMRM-positive cells amongst H2O2-treated Ppif-/- neurons
(31.8%) compared to H2O2- treated nontransgenic neurons (5.5%; Fig. 5e,f). A protective effect
of CypD deficiency on H2O2-mediated reduction in membrane potential was further evaluated
by measuring the percentage of TMRM-labeled living cells with fluorescent microscopy
(Supplementary Fig. 6c). FACS analysis revealed significant increases in propidium iodide-
(Fig. 5g,h) and annexin V- (Fig. 5i,j) positive cells after H2O2 treatment in nontransgenic
neurons, whereas Ppif-/- neurons were protected from H2O2-induced cell death (Fig. 5g-j).
CypD deficiency improves behavioral and synaptic function
The radial arm water maze test was used to assess the spatial learning and memory of transgenic
mice. At 6 and 12 months of age, Ppif-/- and nontransgenic mice showed a strong learning and
memory capacity (Fig. 6a,b), whereas mAPP mice showed impaired spatial learning and
memory for the platform location during trial 4 and retention test (average of about five or six
errors by trial 4 and the retention test). The mAPP-Ppif-/- mice had substantially improved
spatial learning and memory (approximately two or three errors by trial 4 and the retention
test; Fig. 6a,b), indicating that the absence of CypD improves learning and memory in mice
with Alzheimer's-like disease.
Given that mAPP-Ppif-/- mice showed an improvement in learning and memory, we examined
whether these mice also had an improvement in long-term potentiation (LTP), a form of
synaptic plasticity that is widely studied as a cellular model for learning and memory. Slices
from 12-13-month-old mAPP mice showed a reduction in LTP compared to slices from
nontransgenic littermates (140.99 ± 11.81% at 120 min after the tetanus versus 218.52 ±
24.38%; n = 10-12, P < 0.05; Fig. 6c). Slices from mAPP-Ppif-/- littermates, in turn, showed
normal LTP (199.32 ± 20.01%; n = 13, P < 0.05 compared to mAPP mice and P > 0.05
compared to nontransgenic mice; Fig. 6c) and improved basal synaptic transmission compared
to mAPP slices (Supplementary Fig. 6d). The Ppif-/- slices also showed a normal LTP (184.70
± 16.47%; n = 10, P > 0.05 compared to nontransgenic slices). To test a direct effect of CypD
deficiency on Aβ-mediated reduction of LTP, we recorded LTP in hippocampal slices from
Ppif-/- and nontransgenic mice treated with Aβ. We found similar amounts of potentiation in
CypD-deficient slices compared to nontransgenic slices in the presence of vehicle (230.06 ±
24.71% versus 209.39 ± 15.77%, n = 6 or 7, P > 0.05; Fig. 6d). However, CypD deficiency
protected hippocampal slices against a reduction of LTP by 200 nM oligomeric Aβ42 (206.42
± 17.35% in Aβ-treated Ppif-/- slices versus 163.91 ± 17.36% in Aβ-treated nontransgenic
slices; n = 7-9, P < 0.05; Fig. 6d). Basal synaptic transmission was not affected in the Ppif-/-
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mice. The addition of cyclosporine A (1 μM) rescued Aβ-induced reduction of LTP in
nontransgenic hippocampal slices (219.61 ± 30.27% after treatment with cyclosporine A and
Aβ versus 145.96 ± 13.09% after Aβ treatment; n = 7 or 8, P < 0.05; Fig. 6e). Cyclosporine A
alone did not alter LTP (232.43 ± 23.19% in cyclosporine A-treated slices versus 227.57 ±
24.16% in vehicle-treated nontransgenic slices; n = 6 or 7, P > 0.05; Fig. 6e). These results
confirm previous data showing that Aβ impairs LTP38. Most notably, they indicate that CypD
deficiency may protect against the deleterious effects of Aβ soluble oligomers on synaptic
function.
We next determined whether Aβ-mediated reduction of LTP can be prevented by ROS
scavenging. The addition of 100 U ml-1 superoxide dismutase (SOD, a scavenger of superoxide,
converting it into oxygen and hydrogen peroxide) plus 260 U ml-1 catalase (to prevent
inhibition of LTP by H2O2 through its conversion into oxygen and water39,40) blocked Aβ-
induced inhibition of LTP in nontransgenic hippocampal slices (220.89 ± 30.97% in SOD-,
catalase- and Aβ-treated slices versus 145.37 ± 12.24% in Aβ alone-treated nontransgenic
slices; n = 7 or 8, P < 0.05; Fig. 6f). SOD plus catalase did not alter LTP (205.05 ± 11.79% in
SOD- and catalase-treated slices versus 219.30 ± 24.42% in vehicle-treated nontransgenic
slices; n = 6-8, P > 0.05; Fig. 6f). These experiments suggest a role for ROS in Aβ-mediated
impairment of LTP.
DISCUSSION
Our data show that the expression of CypD is associated with amyloid pathology and aging in
the brain. The increased expression of CypD could be an explanation for the observed aging-
and Aβ-related impairment of mitochondrial function, as CypD is a key component of the
mPTP, and its abundance is associated with the vulnerability of the mPTP to Ca2+ (refs. 41,42).
Our studies indicate that the genetic removal of this Aβ binding partner, within Aβ-containing
mitochondria, improves mitochondrial, neuronal and synaptic function. Therefore, it will be
useful to understand the structural basis of the CypD-Aβ interaction, and further investigation
by crystallization and mutational analysis is required to identify the amino acid sequences of
CypD responsible for its binding to Aβ.
Although Aβ can directly disrupt mitochondrial function and cause oxidative stress18,43,44, the
interaction of mitochondrial Aβ with CypD significantly enhances the accumulation and
production of mitochondrial ROS, which is a strong inducer for the recruitment of CypD to
the mitochondrial inner membrane. In addition, other stimuli such as ROS, directly produced
by Aβ itself or by the interaction of Aβ with mitochondrial amyloid-binding alcohol
dehydrogenase8,9, could result in CypD recruitment, leading to mPTP opening, loss of
membrane potential and, eventually, cell death. The excessive ROS will exaggerate oxidative
damage and mitochondrial malfunction, including the collapse of the membrane potential9,
45,46. This is evident in CypD-deficient mAPP mice, which had a reduction in the accumulation
of mitochondrial ROS in conjunction with a higher mitochondrial polarization.
Finally, deficiency of CypD significantly improved cognitive and synaptic function in a mouse
model of Alzheimer's disease. The addition of ROS-scavenging enzymes alleviated Aβ-
mediated reduction of LTP. These results, combined with the evidence that lack of CypD
attenuated ROS generation and protected neurons from Aβ- and oxidative stress-induced
injury, indicate that oxidative damage induced by the CypD-Aβ interaction may be a
mechanism underlying the impairments in synaptic plasticity and memory in Alzheimer's
disease47-49. Mitochondria can also become severely dysfunctional through the permeability
transition induced by the synergistic effects of oxidative stress and dysregulation of cytosolic
free Ca2+. Therefore, synaptic and memory dysfunction mediated by Aβ binding to CypD may
involve other mechanisms such as Ca2+-regulated signaling pathways, oxidative stress-
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mediated kinase systems and activation of transcription factors38,50-53. Further investigations
are required to elucidate these alternative mechanisms.
Taken together, our studies have clearly shown that CypD and Aβ directly interact with each
other in the mitochondria of Alzheimer's disease brain and in a transgenic mouse model of
Alzheimer's disease. This CypD-Aβ interaction promotes ROS generation and CypD
recruitment to the mitochondrial inner membrane, leading to the formation of the mPTP. CypD-
mediated mPTP formation has a crucial role in regulating mitochondrial-induced cell death in
an Aβ-rich environment, although the openings in the outer mitochondrial membrane
associated with activation of members of the Bcl-2 family, either proapoptotic (Bax, BAD and
Bak among others) or antiapoptotic (Bcl-2, Bcl-xL and Bcl-w, among others) proteins (such
as the accumulation of Bax at the outer mitochondrial membrane induced by intracellular
Aβ), may also contribute to neurotoxicity54. Our studies provide new insights into the
mechanism underlying Aβ-mediated mitochondrial stress through an interaction with CypD.
The absence of CypD protects neurons from Aβ- and oxidative stress-induced cell death,
impaired learning and memory and synaptic dysfunction. Therefore, CypD is a key
mitochondrial target for Aβ-induced mitochondrial and synaptic dysfunction. Blockade of
CypD may be a potential therapeutic approach for halting Alzheimer's disease.
METHODS
Mice
Mouse studies were approved by the Animal Care and Use Committee of Columbia University
in accordance with the US National Institutes of Health guidelines for animal care. We crossed
transgenic mice overexpressing a mutant human form of amyloid precursor protein (mAPP,
J-20 line)9,50 with Ppif-/- mice30 to generate CypD-deficient mAPP mice (mAPP-Ppif-/-).
Human tissues
We obtained human brain tissues of temporal cortex (temporal pole, including Brodmann area
38, which is the apparent rostral origin of the superior, middle and inferior temporal gyri) and
hippocampus from individuals with Alzheimer's disease and age-matched, non-Alzheimer's
disease controls from New York Brain Bank at Columbia University. Detailed information for
each of the cases studied is shown in Supplementary Table 1 online. We obtained informed
consent from all subjects. The study was approved by the Institutional Review Board of
Columbia University.
Isolation of mitochondria
We isolated mitochondria from the cortices of Alzheimer's disease brains or cortices of mouse
brains as previously described6,9. We used the highly purified mitochondria for
immunoblotting and immunoprecipitation. For the mitochondrial function assay, mitochondria
were isolated by centrifuging brain homogenates at 1,500g for 5 min at 4 °C. We adjusted the
supernatant to 10% Percoll and recentrifuged at 12,000g for 10 min. We resuspended the
mitochondrial pellet in the isolation buffer (225 mM D-mannitol, 75 mM sucrose, 2 mM
K2HPO4, 5 mM HEPES, pH 7.2) containing 0.01% digitonin and recentrifuged at 6,000g for
10 min. We isolated cell mitochondria with a mitochondrial fractionation kit (Active Motif).
We determined protein concentration by the Bio-Rad DC protein assay (BioRad).
Immunoblotting analysis
We subjected mitochondrial proteins to immunoblotting with antibody to CypD (1 μg ml-1,
generated in our laboratory) followed by goat antibody to rabbit IgG conjugated with
peroxidase to determine the expression level of CypD. We used chemiluminescent substrate
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(Roche) to detect the CypD immunoreactive band. We reprobed the membrane with antibody
to COX IV (to human COX IV, Invitrogen; or to mouse COX IV, Abcam).
Immunoprecipitation and immunoblotting for detection of CypD Aβ complex
We resuspended mitochondria from cerebral cortices of transgenic mice or human subjects in
buffer (500 μg ml-1, 50 mM Tris, 150 mM NaCl, 1 mM EDTA, protease inhibitors
(Calbiochem, set V, EDTA free), 0.1% NP-40, pH 7.5) and subjected them to five freeze-thaw
cycles, followed by centrifugation at 14,000g for 5 min at 4 °C. We immunoprecipitated the
resulting supernatant with rabbit antibody to CypD (1:500 dilution) at 4 ° overnight, followed
by a second incubation with protein A/G (Pierce) for 2 h at 20 °. We subjected the resultant
immunoprecipitant to immunoblotting with antibody to Aβ (6E10,1:3,000, Signat).
Surface plasmon resonance study of CypD Aβ interaction
See Supplementary Methods online.
Immunostaining for confocal and electron microscopy study
See Supplementary Methods online.
Mitochondrial function assays
We monitored mitochondrial oxygen consumption with a Clark oxygen electrode (Oxytherm,
Hansatech) as previously described6.
We performed the mitochondrial swelling assay according to a previously published
method34 with modifications (Supplementary Methods).
We measured mitochondrial calcium retention capacity with the fluorescent indicator Calcium
Green 5N (Invitrogen). We suspended mitochondria (100 μg) at 20 ° in 1 ml respiration buffer
(150mM KCl, 5mM HEPES, 2mM K2HPO4, pH 7.2) containing 1 μM Calcium Green 5N with
pulsate injection of calcium into the cuvette. We monitored fluorescence with an excitation of
506 nm and an emission of 531 nm with a FluoroMax-2 spectrophotometer (Jobin Yvon-Spex
Instruments).
We measured cytochrome c release, activity of COX IV and ATP abundance in brain as
described in the Supplementary Methods.
In situ detection of mitochondrial reactive oxygen species and membrane potential
We performed in situ measurements of ROS in brain slices as previously described55 with
modifications. We injected MitoSox Red (1mg kg-1, Invitrogen) intravenously via the tail vein.
After 30 min, we anesthetized the mice with ketamine (100 mg kg-1) and xylazine (10 mg
kg-1) and killed them by transcardial perfusion with cold saline (5 min) and then cold, freshly
prepared 3.7% paraformaldehyde (3 min). We quickly removed the brain and froze it in 2-
methyl butane with dry ice. We immediately cut coronal frozen brain sections (6 μm) and
mounted them with DAPI-containing mounting medium (Vector Laboratories). We examined
sections under a fluorescence microscope immediately after the mounting. We analyzed the
area occupied by MitoSox Red staining with the Universal image program. Brain sections from
mice were blindly coded and processed in parallel. Codes were broken after the analysis was
complete.
For evaluation of membrane potential, we perfused anesthetized mice with cold saline for 3
min. We incubated frozen brain sections with TMRM (50 nM, Invitrogen) in PBS for 15 min.
We quantified the intensity of TMRM staining with the Universal image program.
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CypD translocation
We examined CypD translocation to the inner membrane as previously described28,56
(Supplementary Methods).
Determination of cytochrome c release, membrane potential and apoptosis in cultured
neurons
We incubated primary cultured neurons with Aβ (2-5 μM) for 12-24 h or with H2O2 (0.5, 2
and 5 mM) for 1 h and then treated them with TMRM (100 nM) for 30 min. We observed the
cells under a microscope or trypsinized them for detection of TMRM by flow cytometry
(Becton Dickinson FACS Calibur). We analyzed flow cytometric data by Flowjo 7 (Tree Star).
We assessed cytochrome c release by immunoblotting with antibody to cytochrome c
(Invitrogen). We determined apoptosis by detecting TUNEL-positive cells using an in situ cell
death detection kit (Roche). We analyzed propidium iodide- or annexin V-labeled cells by flow
cytometry.
Behavioral and electrophysiological studies
We performed behavioral studies to assess spatial learning and memory in the radial arm water
maze as previously described9,50. We performed the retention tests 30 min after the fourth test.
The four groups of mice in the behavioral studies were littermates and gender-matched to
enhance the reproducibility and reliability of our results in the radial arm water maze.
Investigators were unaware of mouse genotypes until the behavioral tests were finished.
We performed electrophysiological recordings on transverse hippocampal slices (400 μm in
thickness) as previously described38 (Supplementary Methods).
Statistical analyses
We performed statistical analyses with Student's t-test and one-way analysis of variance using
the Statview statistics software. P < 0.05 was considered significant. All data are expressed as
means ± s.e.m.
Acknowledgments
This work was supported by the US Public Health Service Commissioned Corps (PO1 AG17490, PO50 AG08702)
and the Alzheimer's Association. We thank S. Katz for assistance with performing the behavioral experiments.
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Figure 1.
Interaction of CypD with Aβ. (a-f) Surface plasmon resonance (SPR) analysis of CypD-Aβ
interaction. Globally fit data (black lines) were overlaid with experimental data (red lines).
(a,b) Sensorgram of Aβ40 (a) or Aβ42 (b) interaction with CypD immobilized on the CM5
chip. (c) CypD interaction with different types of Aβ. Aβ42 (20 μM), Aβ40 (60 μM) and
sequence-reversed Aβ40 (60 μM) bind to CypD immobilized on the CM5 chip. (d,e)
Sensorgram of CypD interaction with oligomers of Aβ40 (d) or Aβ42 (e) immobilized on the
CM5 chip. (f) CypD interaction with different types of oligomeric and reversed Aβ
immobilized on the CM5 chip. (g-j) Coimmunoprecipitation of CypD and Aβ in brain
mitochondria from human subjects with Alzheimer's disease and transgenic mice.
Representative immunoblots show the presence of CypD-Aβ complex in temporal cortical
mitochondria of subjects with Alzheimer's disease (AD) or subjects without Alzheimer's
disease (ND) (g) and in the cortical mitochondria of transgenic mice at 12 months of age (i).
Lower panels of g and i indicate immunoblotting of the same preparations of mitochondria
with antibody to COX IV, showing equal amount of mitochondrial protein used in the
experiment. Lane 4 in panel g is an immunoblot for Aβ40 peptide (5 ng). (h,j) Densitometry
of all immunoreactive bands generated from coimmunoprecipitation results (AD, n = 9; ND,
n = 6; transgenic mice, n = 4-6 per group). *P < 0.0001 compared to ND or other groups of
mice. NonTg, nontransgenic.
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Figure 2.
Colocalization of CypD and Aβ in mitochondria. (a,b) Confocal microscopy showed the
staining of Aβ (red) and CypD (green) in the of human Alzheimer's disease brain (a) and in
the hippocampus of 12-month-old transgenic mAPP mice (b). Colocalization of CypD-Aβ is
shown in the overlay images (yellow). The specific staining pattern disappeared when the
primary antibodies to CypD and Aβ were omitted (control) or preadsorbed with their antigens
(CypD protein and Aβ peptide; in a). Scale bars, 10 μM for a and 5 μM for b. (c,d) Electron
microscopy with the double immunogold staining of CypD (12-nm gold particle) and Aβ (18-
nm gold particle) showing colocalization of CypD and Aβ in mitochondria of the brains from
people with Alzheimer's disease (c) and mAPP mice (d). Age-matched ND controls show only
immunogold particles for CypD (12 nm). Black arrow indicates mitochondria, and white
arrowheads denote colocalization of gold particles labeling both CypD and Aβ. Scale bars, 180
nm for c and 100 nm for d.
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Figure 3.
Effect of CypD deficiency on mitochondrial function in mAPP mice. (a-c) Calcium buffering
capacity. (a) Calcium uptake at the indicated age of mAPP mice and nontransgenic littermates
(n = 5 or 6 mice per group). *P < 0.05 versus mAPP cortical mitochondria. (b) Analysis of
calcium buffering capacity of the cortical mitochondria from the indicated transgenic mice at
12 months of age and of mAPP mitochondria treated with cyclosporine A (CSA; n = 5 or 6
mice per group). *P < 0.01 versus other groups of mice. (c) Representative results of calcium
uptake in cortical mitochondria from the indicated transgenic mice and in CSA (1μM)-treated
mAPP mitochondria. (d-f) Mitochondrial swelling induced by Ca2+. Ca2+ (500 μM)-induced
cortical mitochondrial swelling was measured in the indicated mice at 3, 6 and 12 months of
age, expressed as percentage decrease in the initial optical density (OD) at an absorbance of
540 nm (d). Representative results of swelling from the indicated mouse cortical mitochondria
(12 months old) or in CSA (1 μM)-treated mAPP mitochondria (e,f). Data are shown as the
percentage change relative to the initial OD at an absorbance of 540 nm. *P < 0.05 versus
mAPP mitochondria and #P < 0.05 versus nontransgenic mitochondria. (g) The quantification
of the intensity of TMRM staining in the indicated mouse brain slices (n = 4-6 per group, 12
months old). *P < 0.01 versus nontransgenic and mAPP-Ppif-/- mice.
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Figure 4.
Effect of CypD deficiency on ROS production and mitochondrial function in mAPP mice.
(a) MitoSox Red staining in mouse brains at 12 months of age. The percentage of area occupied
by MitoSox Red staining in the cerebral cortex and hippocampus (CA1 to CA3 regions; n = 3
or 4 mice per group, *P < 0.001 versus other groups of mice). (b) Immunoblotting of the
mitochondrial inner membranes from the indicated mice for CypD. The graph shows
densitometry of CypD intensity from all immunoreactive CypD bands combined from the
indicated mice. The bottom shows the representative immunoblotting for CypD and COX IV
from 12-month-old mice. COX IV served as a control, indicating equal amounts of
mitochondrial protein used for the experiment. (c) Immunoprecipitation with antibody to CypD
followed by antibody to Aβ (6E10) detected an immunoreactive Aβ band in the mitochondrial
inner membranes of the indicated mice. The Aβ-immunoreactive band disappeared when
antibody to CypD was replaced by the preimmune IgG (lane 1). (d-g) Respiratory control rate
(RCR) in response to ADP (d), respiratory control rate in response to Ca2+ (e), COX IV activity
(f) and ATP abundance (g) in cortices of the indicated mice (12 months old, n = 8-10 mice per
group). *P < 0.05 versus nontransgenic and mAPP-Ppif-/- mice.
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Figure 5.
Aβ- and H2O2-induced mitochondrial and neuronal dysfunction in cultured neurons. (a,b)
Fluorescence intensity of TMRM in cultured neurons treated with 5 μM Aβ42 at either the
indicated times (a) or the indicated doses of Aβ42 given for 24 h (b). *P < 0.01 versus Aβ-
treated Ppif-/- neurons. #P < 0.001 in FCCP-treated (5 μM) neurons compared to other groups
of neurons. (c) Immunoblotting for cytochrome c in cytosolic and membrane fractions from
nontransgenic and Ppif-/- cultured cortical neurons treated with Aβ (2 μM) or FCCP (5 μM)
for 24 h. (d) The percentage of TUNEL-positive neurons quantified in nonTg and Ppif-/-
neurons treated with Aβ (2 μM) or Aβ plus CSA (1 μM) for the indicated times. (e-j) Effect
of CypD on H2O2- induced cell death. (e-f) Mitochondrial inner membrane potential changes.
NonTg and Ppif-/- neurons were treated with increasing concentrations of H2O2 for one hour
and then analyzed by FACS for TMRM staining. (e) Representative FACS analysis of TMRM-
positive cells. (f,h,j) The percentage of TMRM- (f), propidium iodide (PI)- (h) and annexin
V- (j) positive cells combined from three or four independent experiments. *P < 0.001 versus
vehicle-treated neurons or H2O2-treated Ppif-/- neurons. #P < 0.01 versus vehicle-treated
neurons. (g-j) FACS analysis of PI (g,h) and annexin V (i,j) staining in nontransgenic and
Ppif-/- neurons treated with H2O2 for one hour. The percentage of PI- or annexin V-positive
cells is indicated as number with underline. Representative histograms for FACS analysis of
PI (g) and Annexin V (i).
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Figure 6.
Effect of CypD deficiency on spatial learning and memory and on Aβ-induced LTP. (a,b)
Radial water maze test in mice at 6 (a) and 12 months (b) of age. *P < 0.01 versus other groups
of mice (n = 8-10 mice per group). R represents the retention test. (c) LTP in the indicated
transgenic mice at 12-13 months of age. P < 0.05 comparing mAPP mice to other groups of
mice. (d) LTP in the indicated mouse brain slices treated with vehicle or Aβ. P < 0.05 comparing
Aβ-treated nonTg slices to Aβ-treated Ppif-/- slices and vehicle-treated nonTg or Ppif-/- slices.
The horizontal bar indicates the period during which Aβ42 was added to the bath solution in
this and in the experiments shown in the other graphs. (e) Effect of CSA (1 μM) on Aβ-induced
LTP in nonTg hippocamal slices. P < 0.05 comparing Aβ-induced LTP to LTP in the slices
treated with CSA plus Aβ, vehicle or CSA alone. P > 0.05 comparing CSA alone to vehicle-
treated slices. (f) Effect of scavenging superoxide through perfusion with SOD (100 U ml-1)
plus catalase (260 U ml-1) on Aβ-induced LTP in nonTg hippocampal slice. P < 0.05 comparing
Aβ-treated slices to the slices treated with SOD + catalase + Aβ or with vehicle. P > 0.05
comparing vehicle-treated slices to the slices treated with SOD and catalase alone.
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