High cyclophilin D content of synaptic mitochondria results in increased vulnerability to permeability transition

Abstract

Mitochondria isolated from synaptosomes are more sensitive to Ca2+ overload and the resultant opening of the mitochondrial permeability transition pore (mPTP) than nonsynaptic mitochondria. To identify the mechanisms underlying these differences in Ca2+ dynamics, we examined relative levels of mPTP components in synaptic versus nonsynaptic mitochondria. Synaptic mitochondria had higher levels of cyclophilin D when compared with nonsynaptic mitochondria, whereas levels of the voltage-dependent anion channel and the adenine nucleotide translocase were similar in the two mitochondrial fractions. These differences in Ca2+ handling between synaptic and nonsynaptic mitochondria were greatly reduced in cyclophilin D null [Ppif-/- (peptidylprolyl isomerase F)] mice. Higher concentrations of cyclosporine A, which interacts with cyclophilin D to delay mPTP opening, were necessary to increase the Ca2+ uptake capacity of synaptic versus nonsynaptic mitochondria. To determine whether the differences in Ca2+ handling might reflect the relative abundance of neuronal and glial mitochondria in the two mitochondrial fractions, we compared cyclophilin D levels in primary cortical neurons and astrocytes. Primary rat cortical neurons possess higher cyclophilin D levels than do primary astrocytes. In the adult rat brain, cyclophilin D immunoreactivity was abundant in neurons but sparse in astrocytes. Together, these results demonstrate that the Ca2+ handling differences observed in synaptic versus nonsynaptic mitochondria are primarily the result of the high levels of cyclophilin D in synaptic mitochondria, reflecting the greater proportion of neuronal mitochondria in this fraction. The high levels of cyclophilin D in neuronal mitochondria result in their greater vulnerability to mPT and in higher levels of cyclosporine A being required to inhibit mPTP opening.

Figure 1.

Synaptic mitochondria contain a higher content of cyclophilin D than nonsynaptic mitochondria. Synaptic (S) and nonsynaptic (NS) mitochondrial fractions probed by Western blot for CypD and additional mitochondrial proteins, including VDAC, ANT, and mHSP70. Levels of cyclophilin D were significantly higher in synaptic when compared with nonsynaptic mitochondrial fractions, whereas levels of other mitochondrial proteins examined were similar in the two fractions. Quantitative data are presented in Results.

Figure 2.

Greater cyclosporine A concentrations are required to increase Ca²⁺ uptake capacity in synaptic versus nonsynaptic mitochondrial fractions. Isolated synaptic or nonsynaptic mitochondria, in the presence or absence of 1 or 5 μm CsA, were placed in a constantly stirred, temperature-controlled, cuvette. CaG5N fluorescence was monitored continuously. Malate and pyruvate (M/P) and ADP were provided. Oligomycin (O), an ATP synthase inhibitor, was then added to ensure that the mitochondria were at maximal ΔΨ_m. Ca²⁺ infusion began at 5 min (160 nmol Ca²⁺/mg protein per minute), causing a small, initial increase in CaG5N fluorescence until the mitochondria were able to sequester the added Ca²⁺. The subsequent sharp rise in CaG5N fluorescence signifies mitochondrial permeability transition and the release of Ca²⁺ from the mitochondria into the surrounding buffer. A, B, In the absence of cyclosporine, synaptic mitochondria (A) sequestered much less Ca²⁺ than nonsynaptic mitochondria (B) before undergoing permeability transition, as reported previously (Brown et al., 2006). AU, Arbitrary units. Arrows indicate the onset (On) and termination (Off) of CaCl₂ infusion. C, Quantitative estimates of the nanomoles of Ca²⁺ infused per milligram of mitochondrial protein before permeability transition; n = 4 per group. One micromolar cyclosporine A significantly increased the Ca²⁺ uptake capacity of nonsynaptic mitochondria before permeability transition but did not influence the Ca²⁺ uptake capacity of synaptic mitochondria. Increasing the cyclosporine A concentration to 5 μm significantly increased the Ca²⁺ uptake capacity of synaptic mitochondria compared with both 0 and 1 μm cyclosporine A. In contrast, the higher cyclosporine A concentration did not further improve the Ca²⁺ uptake capacity of nonsynaptic mitochondria compared with results obtained with 1 μm cyclosporine A. In the presence of 5 μm cyclosporine A, the Ca²⁺ uptake capacity of nonsynaptic mitochondria remained greater than that of synaptic mitochondria. *p < 0.05.

Figure 3.

In cyclophilin D null (Ppif^−/−) mice, the Ca²⁺ uptake capacity of synaptic mitochondria is increased. Isolated synaptic or nonsynaptic mitochondria from wild-type C57BL/6 or Ppif^−/− mice (Baines et al., 2005) were placed in a constantly stirred, temperature-controlled cuvette as in Figure 2. A, Representative traces of CaG5N fluorescence for synaptic mitochondria. B, Nonsynaptic mitochondria. AU, Arbitrary units. C, Quantitative results from five animals in each group (*p < 0.05). Synaptic and nonsynaptic mitochondria from Ppif^−/− mice were able to sequester more Ca²⁺ than wild-type C57BL/6 mice, before undergoing permeability transition indicated by the rapid rise in the CaG5N fluorescent signal. The Ca²⁺ uptake capacity of synaptic mitochondria from Ppif^−/− mice was similar to that of nonsynaptic mitochondrial from wild-type C57BL/6 mice. However, the Ca²⁺ uptake capacities of synaptic and nonsynaptic mitochondria were significantly different in the CypD-deficient Ppif^−/− mice.

Figure 4.

Primary rat cortical neurons contain higher levels of cyclophilin D than primary astrocytes. In lysates of primary rat cortical neurons and astrocyte cultures, prepared from E18 rats, 7 d in vitro, levels of mitochondrial proteins were evaluated by Western blot as in Figure 1. The predominance of neurons and astrocytes in the respective primary cultures was confirmed with antibodies against NeuN and GFAP. Western blots of the nuclear encoded COX IV indicate similar mitochondrial content in the two cellular homogenates. Representative Western blots are shown in A. Quantitative data are described in Results.

Figure 5.

Cyclophilin D is abundant in neurons in adult rat brain. A–C, Confocal images obtained from coronal sections in the hippocampal region of a male Sprague Dawley rats, aged 3 months. B and C are 2 μm optical sections. A, In the hippocampus, CypD immunoreactivity was evident in neuronal layers, including the dentate gyrus granule cell layer (DG) and CA1 stratum pyramidale (SP). Immunoreactivity was also prominent in the dentate gyrus molecular layer (ML) and stratum lacunosom-moleculare of CA1 (LM), in which it was difficult to associate immunoreactivity with dendrites, axons, or astrocytes. B, A higher-magnification image of CA1 reveals punctuate immunoreactivity surrounding pyramidal neurons and occasional neurons (arrowhead) exhibiting robust immunoreactivity. Cell nuclei are labeled with Hoechst 33258. Within these neurons, the punctuate immunoreactivity surrounding the nucleus is consistent with the mitochondrial localization of cyclophilin D. In stratum radiatum (SR) and stratum oriens (SO), immunoreactivity was relatively sparse but was evident in astrocytes double labeled for GFAP. C, This is more clearly observed in an enlarged view, in which the arrows identify cyclophilin D immunoreactivity associated with GFAP-labeled astrocytes. However, much of the cyclophilin D immunoreactivity in stratum radiatum and stratum oriens did not colocalize with GFAP and is therefore thought to represent axonal, dendritic, or synaptic localization. Scale bars: A, 100 μm; B, 50 μm; C, 25 μm.