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Comparative Study
. 2005 Aug 23;102(34):12005-10.
doi: 10.1073/pnas.0505294102. Epub 2005 Aug 15.

Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia

Anna C Schinzel  1 , Osamu Takeuchi, Zhihong Huang, Jill K Fisher, Zhipeng Zhou, Jeffery Rubens, Claudio Hetz, Nika N Danial, Michael A Moskowitz, Stanley J Korsmeyer
Affiliations
Comparative Study

Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia

Anna C Schinzel et al. Proc Natl Acad Sci U S A. .

Abstract

Mitochondrial permeability transition (PT) is a phenomenon induced by high levels of matrix calcium and is characterized by the opening of the PT pore (PTP). Activation of the PTP results in loss of mitochondrial membrane potential, expansion of the matrix, and rupture of the mitochondrial outer membrane. Consequently, PT has been implicated in both apoptotic and necrotic cell death. Cyclophilin D (CypD) appears to be a critical component of the PTP. To investigate the role of CypD in cell death, we created a CypD-deficient mouse. In vitro, CypD-deficient mitochondria showed an increased capacity to retain calcium and were no longer susceptible to PT induced by the addition of calcium. CypD-deficient primary mouse embryonic fibroblasts (MEFs) were as susceptible to classical apoptotic stimuli as the WT, suggesting that CypD is not a central component of cell death in response to these specific death stimuli. However, CypD-deficient MEFs were significantly less susceptible than their WT counterparts to cell death induced by hydrogen peroxide, implicating CypD in oxidative stress-induced cell death. Importantly, CypD-deficient mice displayed a dramatic reduction in brain infarct size after acute middle cerebral artery occlusion and reperfusion, strongly supporting an essential role for CypD in an ischemic injury model in which calcium overload and oxidative stress have been implicated.

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Figures

Fig. 1.
Fig. 1.
Deletion of CypD. (A) Schematic of the CypD locus. PCR primers distinguishing WT and null alleles are designated. wt for, WT forward; wt rev, WT reverse; null rev, null reverse. (B) Genotyping of CypDWT/WT using WT forward and WT reverse primers generated a 490-bp product in the WT allele. Genotyping of CypDnull/null mice using the null primer set WT forward and null reverse generated a 700-bp product. (C) Western blots of mitochondria purified from CypDWT/WT and CypDnull/null mice using a CypD-specific antibody.
Fig. 2.
Fig. 2.
Induction of PT in CypD-deficient and WT mitochondria. (A) Mitochondrial swelling induced by treatment with 100 μM CaCl2 was measured by monitoring mitochondrial size at an absorbance of 540 nm. Mitochondria isolated from WT (Left) and CypD-deficient (Right) mice were resuspended at a concentration of 0.5 mg/ml in EB for 5 min. Where indicated (arrow), 100 μM CaCl2 was added. Mitochondria were preincubated with 1 μM CsA or buffer alone for 5 min. (B) Transmission electron microscopy of liver mitochondria taken from WT and CypD-deficient mitochondria. Mitochondria were treated with 100 μM CaCl2 for 200 s and fixed in 1.25% gluteraldehyde. (Scale bars, 200 nm.) (C) Loss of CaCl2 buffering capacity in liver mitochondria isolated from WT and CypD-deficient mice resuspended at 0.5 mg/ml in EB. Extramitochondrial Ca2+ was measured fluorometrically with the calcium indicator Calcium Green 5N. Pulses of 10 μM CaCl2 were added at intervals of 100 s.
Fig. 3.
Fig. 3.
Susceptibility of WT and CypD-deficient MEFs to apoptotic stimuli. (A) Susceptibility of MEFs to apoptotic death by mitochondria-dependent intrinsic signals. WT and CypD KO MEFs were treated with 1 μM staurosporine or 100 μM etoposide for 24 and 48 h. (B) Susceptibility to apoptotic cell death induced by ER stresses. WT and CypD KO MEFs were treated with thapsigargin (2 μM), tunicamycin (5 μg/μl), or brefeldin A (2 μM) for 24 and 48 h. (C) Susceptibility to apoptotic death by mitochondria-independent extrinsic stimuli. WT and CypD KO MEFs were treated with TNFα (1 ng/ml) or TNF-related apoptosis-inducing ligand (TRAIL) (1 μg/μl) in the presence of actinomycin D (2 μg/μl) for 12 and 16 h. (D) Susceptibility of WT and CypD-deficient MEFs to cell death induced by H2O2 (1 mM) for 16 and 20 h (*, P < 0.05). All error bars show SD.
Fig. 4.
Fig. 4.
Ischemia/reperfusion-mediated cell death in WT and CypD-deficient mice. (A) Infarct volume was reduced significantly (by 37%) in heterozygous (Het) mice and reduced by 62% in CypD KO mice compared with WT mice (n = 9 per group; P < 0.05) after 2 h of MCAo and 24 h of reperfusion. All error bars show SEM. (B) Protection in CypD KO mice (coronal sections 3, 4, and 5; P < 0.05) and heterozygous mice (coronal sections 4 and 5; P < 0.05) was significant in comparison with WT controls with a trend of infarct size difference at other brain section levels. (C) Ischemic tissue damage in CypD KO and WT mice. As shown by representative triphenyltetrazolium chloride-stained coronal brain sections, CypD KO mice had significantly smaller infarcts after 2 h of MCAo and 24 h of reperfusion (A) compared with WT mice.
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