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 (). The CypD-Aβ interaction was specific, because reversed-sequence Aβ peptide showed no binding with CypD (), 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.
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 (more ...)
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 () but not (or very little) in those of non-Alzheimer's disease control brains (). Aβ-immunoreactive bands disappeared when the antibody to CypD was replaced by preimmune IgG (). Densitometry of all immunoreactive bands combined revealed that CypD-Aβ complexes were increased by 10-13-fold in Alzheimer's disease cortical mitochondria compared to non-Alzheimer's disease cortical mitochondria (). 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 (). 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 (). In the cerebral cortices of people with Alzheimer's disease () and mAPP mice (), Aβ and CypD colocalized extensively (). In the absence of antibodies to Aβ and CypD () or after neutralization of the antibodies with their antigens (Aβ42 and CypD protein; 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 ( and Supplementary Fig. 3a
online) and mAPP brain mitochondria (). 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
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 (more ...)
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; ). 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); ). 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+ (). 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).
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 (more ...)
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 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+ (). Notably, mAPP-Ppif-/- cortical mitochondria were more resistant to swelling and permeability transition induced by Ca2+ than were mAPP mitochondria (). The addition of cyclosporine A to mAPP mitochondria also attenuated swelling in response to Ca2+ ().
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 (). 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 (). 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 ( and Supplementary Fig. 4c,d). These data indicate that the absence of CypD attenuates Aβ-mediated mitochondrial ROS generation.
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 (more ...)
Further, mAPP mice showed an age-dependent increase in CypD translocation to the mitochondrial inner membrane (). The CypD-Aβ complex was also present in the mitochondrial inner membrane of mAPP mice ().
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 (). 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 (). Additionally, mAPPmice had a reduced COX IV activity () and ATP abundance (), whereas mAPP-Ppif-/- mice had markedly increased mitochondrial enzyme activity and ATP abundance (). The COX IV activity and ATP abundance in nontransgenic mice were comparable to those in the Ppif-/- mice (). 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 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 (). Consequently, Aβ-treated nontransgenic cortical neurons showed increased cytochrome c release as compared to Aβ-treated Ppif-/- and vehicle-treated neurons (). 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 (). The addition of cyclosporine A had a similar effect on Aβ-induced apoptosis ().
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 (more ...)
Because CypD deficiency attenuated ROS generation () 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%; ). 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-() and annexin V- () positive cells after H2O2 treatment in nontransgenic neurons, whereas Ppif-/- neurons were protected from H2O2-induced cell death ().
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 (), 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; ), indicating that the absence of CypD improves learning and memory in mice with Alzheimer's-like disease.
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 (more ...)
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; ). 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; ) 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; ). 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; ). Basal synaptic transmission was not affected in the Ppif-/-
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; ). 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; ). 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 H2
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; ). 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; ). These experiments suggest a role for ROS in Aβ-mediated impairment of LTP.