The mitochondrial calcium regulator cyclophilin D is an essential component of oestrogen-mediated neuroprotection in amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder that is more prevalent in males than in females. A similar gender difference has been reported in some strains of transgenic mouse models of familial amyotrophic lateral sclerosis harbouring the G93A mutation in CuZn superoxide dismutase. Mitochondrial damage caused by pathological alterations in Ca(2+) accumulation is frequently involved in neurodegenerative diseases, including CuZn superoxide dismutase-related amyotrophic lateral sclerosis, but its association with gender is not firmly established. In this study, we examined the effects of genetic ablation of cyclophilin D on gender differences in mice expressing G93A mutant CuZn superoxide dismutase. Cyclophilin D is a mitochondrial protein that promotes mitochondrial damage from accumulated Ca(2+). As anticipated, we found that cyclophilin D ablation markedly increased Ca(2+) retention in brain mitochondria of both males and females. Surprisingly, cyclophilin D ablation completely abolished the phenotypic advantage of G93A females, with no effect on disease in males. We also found that the 17β-oestradiol decreased Ca(2+) retention in brain mitochondria, and that cyclophilin D ablation abolished this effect. Furthermore, 17β-oestradiol protected G93A cortical neurons and spinal cord motor neurons against glutamate toxicity, but the protection was lost in neurons lacking cyclophilin D. Taken together, these results identify a novel mechanism of oestrogen-mediated neuroprotection in CuZn superoxide dismutase-related amyotrophic lateral sclerosis, whereby Ca(2+) overload and mitochondrial damage are prevented in a cyclophilin D-dependent manner. Such a protective mechanism may contribute to the lower incidence and later onset of amyotrophic lateral sclerosis, and perhaps other chronic neurodegenerative diseases, in females.

Figure 1

Genetic and molecular characterization of CypDKO and CypDKO–G93A mice. (A) PCR banding pattern corresponding to wild-type (WT), heterozygote CypDKO (Het) and homozygote CypDKO (DKO) mice (left panel); PCR banding pattern of mice containing the human SOD1 G93A transgene and interleukin 2 gene (non-Tg) as internal positive control (right panel). (B) Immunoblots of purified brain mitochondria using anti-CypD and anti-SOD1 antibodies. CypD was undetectable in the mitochondria of CypDKO and CypDKO–G93A mice. Human transgenic SOD1 was detected in the mitochondria of G93A, CypDHet–G93A and CypDKO–G93A mice. A monoclonal antibody against the inner membrane protein Tim23 was used as a loading control for mitochondria. mSOD1 = mouse SOD1; hSOD1 = human SOD1.

Figure 2

CypD ablation accelerates disease in female G93A mice. (A) Kaplan–Meier survival curve and (B) mean survival of G93A (males: n = 42; females: n = 40) and CypDKO–G93A (males: n = 33; females: n = 25) mice. CypDKO significantly shortened survival of female G93A mice. (C) Rotarod performance expressed as seconds spent on the accelerating rod (2 rpm/s) for G93A (males: n = 5; females: n = 7) and CypDKO–G93A (males: n = 9; females: n = 10) mice. (D) Muscle strength in G93A (males: n = 5; females: n = 6) and CypDKO–G93A (males n = 9, females n = 9) mice expressed as hanging time in seconds. CypDKO accelerated motor performance impairment and muscle weakness in female G93A mice. Data are presented as mean ± SEM. **P < 0.01, *P < 0.05.

Figure 3

CypD ablation abolishes gender differences in brain mitochondrial Ca²⁺ uptake and Ca²⁺-induced mitochondrial depolarization. (A) Kinetics of Ca²⁺ uptake in brain mitochondria measured by monitoring the change of Fura-6F fluorescence ratio (340/380 nm excitation, 510 nm emission) on Ca²⁺ loading (250 nmol of Ca²⁺/mg protein in each addition, indicated by arrows). The fluorescence peak corresponds to increase in extra-mitochonrial Ca²⁺, whereas the decrease in fluorescence reflects mitochondrial Ca²⁺ uptake. The sustained increase at the end of trace shows that mitochondria are unable to further accumulate Ca²⁺. In the example, control (WT) mitochondria took up six Ca²⁺ additions. However, G93A mice mitochondria only took five Ca²⁺ additions. CypDKO and CypDKO–G93A mice mitochondria took up significantly more Ca²⁺ than their WT and G93A counterparts, but CypDKO–G93A mice mitochondria took up less Ca²⁺ than CypDKO mice mitochondria. (B) Average brain mitochondrial Ca²⁺ uptake threshold in nmol Ca²⁺/mg of mitochondria protein (n = 6 mice per group). (C) ΔΨm response to 75 nmol Ca²⁺ load (750 nmol of Ca²⁺/mg protein) calculated as (ΔΨm 75 nmol Ca²⁺/ΔΨm-ini) × 100%. Mitochondria from males had higher Ca²⁺ uptake threshold and less ΔΨm sensitivity to Ca²⁺ loads than that from females, both in WT and G93A mice (n = 5 mice per group). CypDKO abolished the gender differences for both parameters. Error bars indicate mean ± SEM. *P < 0.01: statistically significant differences between male and female mice; #P < 0.01: statistically significant differences between non-transgenic and G93A mice; n.s = not significant.

Figure 4

Effect of CypD ablation on spinal cord mitochondrial Ca²⁺ uptake. Kinetics of Ca²⁺ uptake in spinal cord mitochondria assayed as in Fig. 3A. Experiments were performed on mitochondria isolated from a pool of three spinal cords for each genotype.

Figure 5

CypD ablation abolishes oestrogen effect on mitochondrial Ca²⁺ handling. Kinetics of Ca²⁺ uptake in brain mitochondria in 140-day-old mice was performed on purified brain mitochondria treated with 10 µM 17β-oestradiol (E2β), as in the experiment in Fig. 3A. 17β-Oestradiol was added to mitochondria for 100 s before Ca²⁺ loading. Averaged absolute Ca²⁺ uptake thresholds (nmol Ca²⁺/mg of protein) are shown (n = 5 mice for each genotype, using mitochondria preparation from independent animals; *P < 0.01).

Figure 6

Effect of CypD ablation on glutamate toxicity in cultured spinal cord motor neurons and cortical neurons. Embryonic motor neurons (E12.5) and cortical neurons (E18) were cultured in vitro for 2 weeks. Where indicated, neurons were pretreated with 10 µM 17β-oestradiol (E2β) for 1 h, followed by 100 μM glutamate (Glu) exposure for 24 h in the presence of 17β-oestradiol. (A) Phase contrast images of spinal cord motor neurons. Viable neurons (filled arrows) had intact long neuritic processes, whereas dead or dying neurons (unfilled arrows) had shorter neuritis with beaded appearance. Cell viability was assessed using an MTT-based assay (cell survival) in motor neurons (B) and cortical neurons. (C) 17β-Oestradiol attenuated the loss of cell viability caused by glutamate in G93A neurons, but not in CypDKO–G93A neurons. Data are presented as mean ± SEM (n = 3 for motor neurons; n = 9 for cortical neurons, *P < 0.01).