Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH-and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

The mitochondrial permeability transition (mPT) is increasingly implicated in neuronal cell death. In the present study, isolated respiring brain mitochondria were examined for their ability to undergo calcium-induced mPT and their sensitivity to mPT inhibition by cyclosporin A (CsA). Previous studies have suggested a heterogeneous response to calcium, a limitation of CsA inhibition, and a relative resistance in the ability of respiring brain mitochondria to undergo mPT. Using fluorometric and electron microscopic analyses, we found that virtually the whole population of respiring brain mitochondria readily undergo mPT and swell upon calcium exposure. Further, brain mitochondria were highly sensitive to CsA which potentiated morphological recovery after transient swelling as well as completely blocked mPT induction in the presence of a low concentration of ADP. Using flow cytometry, which allows analysis of individual mitochondria, we demonstrate that both brain and liver mitochondria display homogeneous responses to calcium-induced mPT. We conclude that the mPT is one likely target for the broad in vivo neuroprotective effects displayed by CsA when allowed to penetrate the blood-brain barrier, and that development of compounds inhibiting mPT may prove beneficial for the treatment of severe brain disease.

Minocycline has been shown to be neuroprotective in ischemic and neurodegenerative disease models and could potentially be relevant for clinical use. We revisited the hypothesis that minocycline acts through direct inhibition of calcium-induced mitochondrial permeability transition (mPT) resulting in reduced release of cytochrome c (cyt c). Minocycline, at high dosage, was found to prevent calcium-induced mitochondrial swelling under energized conditions similarly to the mPT inhibitor cyclosporin A (CsA) in rodent mitochondria derived from the CNS. In contrast to CsA, minocycline dose-dependently reduced mitochondrial calcium retention capacity (CRC) and respiratory control ratios and was ineffective in the de-energized mPT assay. Further, minocycline did not inhibit calcium-or tBid-induced cyt c release. We conclude that the neuroprotective mechanism of minocycline is likely not related to direct inhibition of mPT and propose that the mitochondrial effects of minocycline may contribute to toxicity rather than tissue protection at high dosing in animals and humans.

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The antibiotic minocycline exerts cytoprotection in animal disease models. One proposed mechanism is modulation of the mitochondrial permeability transition (mPT), a Ca 2ϩ -dependent pathogenic event leading to necrotic and/or apoptotic cell death. [1][2][3][4][5] A recent study in HEPATOLOGY by Theruvath et al., 6 investigating storage/ reperfusion injury following rat liver transplantation, concluded that minocycline prevented mPT and mitigated liver injury by decreasing mitochondrial Ca 2ϩ uptake without affecting mitochondrial respiration. Further, the authors argue that it could be consistent with clinical practice to (pre)treat stored livers and graft recipients with minocycline. The driving force for mitochondrial Ca 2ϩ transport is the mitochondrial membrane potential and the amount of Ca 2ϩ retained is dependent on the proton gradient and the matrix pH. 7 Respiratory inhibition will decrease Ca 2ϩ retention capacity and sensitize mitochondria toward mPT. 5,7 Further, endogenous inhibitors of mPT such as adenine nucleotides and Mg 2ϩ will influence the amount of Ca 2ϩ sequestered prior to mPT. In Theruvath et al., the effect of minocyline on mPT was determined in two classical assays, both using bolus additions of calcium chloride: (1) the swelling assay and (2) the Ca 2ϩ retention capacity assay. In both assays, the endpoint is Ca 2ϩ overload and induction of mPT. The authors found that minocycline prevented Ca 2ϩ -induced swelling and decreased Ca 2ϩ retention and interpreted this as a specific inhibitory effect on Ca 2ϩ uptake. They excluded respiratory inhibition as the explanation to their findings by determining the respiration of mitochondria exposed to minocycline with and without Ca 2ϩ addition. However, the buffer used in the respiration assay was different from the one used in the Ca 2ϩ bolus assays, with high Mg 2ϩ concentration (Mg 2ϩ is a known endogenous inhibitor of mPT) and with the presence of the potent pharmacological mPT inhibitor cyclosporin A during Ca 2ϩ addition. We argue that minocycline at moderate to high dosing, similar to what we have shown in brain mitochondria, prevents Ca 2ϩ -uptake and mPT-induced swelling by respiratory inhibition. 1,5 Further, depending on the buffer system used, the decreased Ca 2ϩ retention can be explained by minocycline-induced increase of mPT sensitivity related to (1) inhibited respiration 1,5 and (2) chelating of Mg 2ϩ , 8 or (3) direct activation of mPT (even during concurrent cyclosporin A treatment) by adding Ca 2ϩ or in Ca 2ϩ loaded mitochondria, as recently shown by Kupsch et al. 8 To stringently evaluate effects of minocycline during the process of Ca 2ϩ uptake, retention, and mPT, mitochondrial oxygen consumption can be monitored during a continuous Ca 2ϩ infusion (Fig. 1A,B). This assay provides information of the bioenergetic demand on mitochondria caused by Ca 2ϩ uptake as well as the respiratory inhibition triggered by mitochondrial Ca 2ϩ overload and mPT. 5,7 Alternatively, the effect of minocycline on isolated mitochondria can be di...

Strains of Propionibacterium acnes, isolated from different kinds of orthopaedic and biomaterial‐associated infections and from skin flora were shown to express binding of soluble as well as immobilized fibronectin. Among these 7 strains isolated from orthopaedic infections, 2 from breast prostheses, and 9 skin isolates, 2, 2, and 5 strains respectively bound immobilized fibronectin. The fibronectin binding was sensitive to protease and heat treatment, and was inhibited by a cell surface extract from one of the binding strains. In SDS‐PAGE and autoradiography of cell surface extracts, a band corresponding to a MW of about 80 kD reacted with fibronectin and the 150 kD fragment of fibronectin. Binding to fibronectin and the 150 kD fragment of fibronectin could be inhibited with heparin. We thus present a first Fn binding protein of P. acnes, a surface exposed protein of 80 kD. None of the strains bound soluble collagen, and only one strain expressed weak binding of vitronectin and bone sialoprotein II.