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.

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.

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.

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.

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.

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 H₂O_2- 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).

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.