Role of calcineurin in calcium-mediated hypoxic injury to white matter☆☆☆

Introduction 

It has been shown that calcium influx plays a key role in the pathogenesis of primary neural injury to traumatic or hypoxic injury. Past evidence includes studies of glutamate cytotoxicity in neuronal cultures 1, 2, 3, 4, hypoxic white matter injury 5, 6, ultrastructural analysis of calcium accumulation in axons after spinal cord trauma, confocal imaging of postinjury axonal and glial calcium level changes in optic nerve and spinal cord and posttraumatic spinal cord blood flow alterations. Although the mechanisms of calcium-mediated damage to white matter have not been elucidated to the degree that injury to neurons have, recent studies in our laboratory have shown a similar trend in the role that calcium plays in perpetuating injury. Specifically, it has been shown that blocking ryanodine-sensitive receptors (RyRs) or inositol (1,4,5)-triphosphate receptors with high-affinity antagonists has a protective effect on compound action potential (CAP) recovery after compressive injury [7]. This study also showed that calcium chelation also showed a protective effect and caffeine-exacerbated injury. More recently, it was shown that inhibition of the Na+-Ca2+ exchanger (NCX), which is involved in the regulation of intracellular Ca2+ concentration, with antagonists has a protective effect on CAP recovery [8].

The pharmacological action of immunosuppressant drugs, such as CsA (CsA), FK-506 and rapamycin, has been elucidated in the past and are complex and varied. Both FK-506 and rapamycin bind to the immunophilin FK binding protein 12 (FKBP12). The FK506-FKBP12 complex inhibits calcineurin, a Ca2+/calmodulin-dependant protein kinase. CsA binds to a cyclophilin and also inhibits calcineurin. Both FK-506 and CsA provide immunosuppression by blocking the Ca2+-dependant activation of antigen-reactive T cells. It has been demonstrated that FK-506 and CsA, when bound to their respective binding proteins FKBP 12 and cyclophilin A, are specific inhibitors of calcineurin and modulate the calcineurin-calmodulin complex [9]. Shibasaki and McKeon [10] in 1995 and Wang et al. [11] in 1999 reported that calcineurin functions in Ca2+ activated apoptosis and cell death through dephosphorylation. It has been shown that immunophilins mediate neuroprotective effects of FK-506 and CsA in rat focal and forebrain ischemic models, respectively 12, 13. Immunophilins have interactions with many other proteins, one of which is the RyR, a tetrameric Ca2+ channel. This interaction serves to stabilize the Ca2+-release channel, increases the threshold for channel opening and optimizes Ca2+ release once it is activated. The neuroprotective effects of various immunosuppressants on dorsal column white matter injury are the subjects of this study.

CsA binds to numerous proteins belonging to the cyclophilin family, and forms a complex with each of them. Similarly, FK-506 binds to immunophilins, the most important of which is FK-506 binding protein 12 (FKBP12), although rapamycin binds more strongly to FKBP12 than does FK-506. FK-506 does not bind to cyclophilins. CsA and FK-506, bound to their immunophilins, inhibit calcineurin, a Ca/calmodulin-activated protein kinase. In the present study we examined the role of Cyclosporin A (CsA) and FK-506 in the neuroprotection of spinal cord white matter during hypoxic conditions. We also aimed to assess ultrastructural signs of axonal and mitochondrial dysfunction during hypoxia and whether treatment with CsA or FK-506 prevents ultrastructural damage during hypoxic damage to spinal cord white matter.

Methods 

Results 

Effect of Cyclosporin A after hypoxic injury to white matter 

Dorsal columns exposed to 60 minutes of hypoxia while perfused with Ringers (no drug added) demonstrated mean CAP reduction to 53.8±7.6% of control readings (no hypoxia) after 2 hours of reoxygenation. Exposure of the dorsal column to Ringers plus CsA (1 μM and 10 μM) during the period of hypoxia significantly improved recovery of CAP amplitude after reoxygenation (Fig. 1). After 2 hours of reoxygenation, mean CAP amplitude recovered to 76.8±5.2% of control in CsA (1 μM) and 72.0±13.2% of control in CsA-treated (10 μM) segments, (p<.05). This suggests that calcineurin may partially modulate white matter injury during hypoxia/ischemia. The graph demonstrating CAP versus time with CsA-treated dorsal columns and control readings are shown in Fig. 1.

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Fig. 1. Effect of Cyclosporin A (CsA) on compound action potential (CAP) recovery after hypoxic injury. Graph of normalized CAP amplitude versus time. At time zero (arrow), hypoxic conditions were continued for 60 minutes (length of horizontal line) and incubated with Ringers solution alone (controls) and compared with (A) Ringers solution plus CsA and (B) CsA (10 μM). Also included in graph is (C) the comparison of CAP amplitude recovery between the tissue treated with two doses of CsA.

Effect of FK-506 after hypoxic injury to white matter 

Exposure of the dorsal column to Ringers plus FK-506 (0.1 μM) during the period of hypoxia also showed significantly improved recovery of CAP amplitude after reoxygenation (Fig. 2, A). After 2 hours of reoxygenation, mean CAP amplitude recovered to 82.6±5.0% of control in FK-506-treated (0.1 μM) dorsal columns (p<.05). This also suggests that calcineurin inhibition may be responsible for the protective effect seen in the first two sets of experiments. The graph for the FK-506-treated dorsal columns are shown in Fig. 2.

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Fig. 2. Effect of FK-506 alone and with rapamycin on compound action potential (CAP) recovery after hypoxic injury. At time zero (arrow), hypoxic conditions were continued for 60 minutes (length of horizontal line) and incubated with Ringers solution alone and compared with (A) Ringers solution plus FK-506 (0.1 μM) and (C) Ringers solution plus FK-506 (0.1 μM) and rapamycin (1 μM). Also included in graph is (B) the comparison of CAP amplitude recovery between the FK-506-treated tissue and FK-506/rapamycin-treated tissue.

Electron microscopic appearance after hypoxia/ischemia 

Electron microscopy was used to examine the appearance of dorsal columns in control (oxygenated) dorsal columns and compared with segments that had undergone hypoxic conditions with subsequent treatment with no drug, CsA and FK-506. The results are shown in Fig. 3. As described in a previous correspondence [8], there appears to be granular dissolution of the axoplasm in dorsal columns injured with hypoxia without drug treatment when compared with noninjured controls (Fig. 3, E). CsA (Fig. 3, H) and, to a greater extent, FK-506 (Fig. 3, K) prevent dissolution of the axon within the myelin sheaths. Higher-power magnification reveals mitochondrial cisternae disruption and mitochondrial swelling in axons exposed to hypoxic conditions (Fig. 3, F), whereas the mitochondrial membranes remain intact when the axons are inoculated with CsA (Fig. 3, I) or FK-506 (Fig. 3, L).

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Fig. 3. (Previous page). Effects of hypoxia on the visual appearance of dorsal column segments under various conditions. Electron micrograph of dorsal column segments incubated for 60 minutes in (A, B, C) oxygenated Ringers solution, (D, E, F) hypoxic Ringers solution, (G, H, I) hypoxic Ringers solution with Cyclosporin A and (J, K, L) hypoxic Ringers solution with FK-506. (A, D, G, J) Cross-sections show dozens of axons surrounded by their myelin sheath. Arrowheads show myelin sheaths surrounding axons. (B, E,H, K) Higher power shows separation of myelin sheath and dissolution of axon in hypoxic conditions with (E) Ringers alone, and (H) relative sparing of these changes with Cyclosporin A and (K) FK-506. Arrowheads show amount of axon dissolution and retraction. (C, F, I, L) Appearance of mitochondria in axons of dorsal column segments. The cisternae of the mitochondria appear to be disrupted and the mitochondria are (F) swollen with hypoxic Ringers solution alone when compared with (C) oxygenated Ringers solution alone. These changes do not appear to occur in the presence of (I) hypoxic Cyclosporin A or (L) FK-506. Arrowheads indicated mitochondria at high magnification. (A, D, G, J) Scale bars equal 20 &mulm, (B, E, H, K) 4 μm and (C,F, I, L) 200 nm.

Discussion 

In the present study, the dorsal column white matter was exposed to hypoxic conditions for 60 minutes, and the measured CAP decreased to 20% of baseline readings. CAP amplitudes returned to 53.8±7.6% of baseline readings after 2 hours of reoxygenation. In contrast to control hypoxia readings, the addition of the immunosuppressants CsA and FK-506 improved CAP amplitude recovery to 76.8±5.2% and 82.6±5.0% of baseline oxygenated dorsal column readings, respectively. This demonstrates that CsA and FK-506 both improve CAP recovery after hypoxic conditions in the dorsal column of a rat when compared with controls. The improvement was more remarkable in FK-506-treated tissues. The neuroprotection to segments by FK-506 was abolished by concurrent incubation with rapamycin during hypoxia. This was seen as CAP amplitude recovery that returned to 56.9±6.7% of baseline, similar to control hypoxia readings.

Electron microscopy showed remarkable changes in the white matter after 1 hour of hypoxia in untreated sections. There was granular dissolution of the axoplasm, which manifested as retraction of the axon away from the relatively intact myelin sheath on photos. If the tissues were perfused with CsA or FK-506 during hypoxia, this dissolution was decreased significantly and was almost nonexistent with FK-506. Under higher magnification, mitochondria appeared to lose their membranous structure with hypoxia. CsA and FK-506 also attenuated these changes. This suggests that organelle breakdown is an indication of severe injury to the axon and may contribute to its demise by means of release of massive amounts of Ca2+.

In prior experiments, we have elaborated on several possible mechanisms and therapeutic treatments of receptor-mediated injury to spinal cord axons. These include the Na+-K+-ATPase and the Na+-H+ exchanger 14, 15, the Na+-Ca2+ exchanger 8, 13, N-methyl-D-aspartic acid (NMDA) and non-NMDA glutamate receptors [1], sodium channel blockade [16], L- and N-type calcium channels [17] and RyRs and IP3 receptors [7]. In this study we attempted to elucidate the role that immune suppressants have in the protection of spinal cord axons, and if it is the binding of immunophilins or the blockade of calcineurin that confer this protection.

Calcium homeostasis is integral to the normal function of all cells. The presence of calcium during injury is necessary for cell death to occur in many models [18]. Schanne et al. [18] termed this process the “final common pathway of cell death,” noting that calcium dysregulation is important in different pathways that all contribute to cell injury and death. Calcium overload has been implicated in overstimulation of such enzymes as phospholipases [19], proteases 20, 21, protein kinases 22, 23, guanylate cyclase [24] and nitric oxide synthetase; calcineurins and endonucleases [25]. The consequences, which occur as a result of this activation, include free radicals, lethal alteration of cytoskeletal organization and possibly activation of genetic signals leading to cell death [26].

Many mechanisms of calcium flux have been described and include calcium influx, calcium buffering, internal calcium storage and calcium efflux [26]. A possible target for intervention includes Ca2+-release channel receptors, such as the RyR and IP3 receptors. The RyR copurifies with the immunophilin FKBP12 27, 28, as does the IP3 receptor [28]. Each RyR subtype is a homomeric tetramer [29], and an FKBP12 molecule interacts with each of the four RyR subunits in the tetrameric RyR [30]. FKBP12 stabilizes the ryanodine receptor in the natural state and increases the threshold for Ca2+ channel opening [27]. FKBP12 dissociation from the RyR receptor by addition of FK-506 or rapamycin abolished this effect, suggesting the action on FKBP12 as the mediating effect rather than calcineurin.

[Ca2+]i oscillations in adenosine 5′-triphosphate (ATP)-stimulated cow tracheal cells were decreased when treated with FK-506 or rapamycin, but not with CsA. They postulated that displacing FKBP12 from Ca2+-release channel receptors increased the leakiness of these receptors to Ca2+, with immediate inhibition of oscillations, and a delayed depletion for Ca2+ from intracellular stores [31]. Several studies have shown that FKBP12 copurifies with both RyRs and IP3 receptors in Ca2+-mediated injury. Both receptor types have structural similarities but a recent affinity chromatography study showed only the RyR1 and RyR3 bound FKBP12, whereas the IP3R1 and IP3R3 did not bind at all [32]. In the current study, rapamycin did not confer protection of the segments, and FK-506 and CsA did. If FKBP12 binding were responsible for the protection that we demonstrated, both rapamycin and FK-506 would have shown similar results. This suggests that FKBP12 modulation probably is not the mechanism of protection in our study.

Calcineurin, a Ca2+-calmodulin serine/threonine phosphatase, is a common target of the cyclophilin-CsA complex and FKBP12-FK506 complex [9]. In addition, these complexes inhibit calcineurin. One of the actions of calcineurin is the dephosphorylation of the transcription factor NF-AT, which allows it to enter the nucleus and activate T cells. CsA and FK-506 abolish this step, and this is the mechanism for their immunosuppression [9]. This is a delayed response, and therefore it is unlikely that this is the mechanism of protection in this study. Calcineurin also regulates neuronal nitric oxide synthase (nNOS). In the presence of Ca2+/calmodulin, calcineurin is in its active state and dephosphorylates several enzymes. Dephosphorylated, nNOS is activated and increases the release of nitric oxide, which leads to neurotoxicity and neurotransmitter release [33]. FK-506 has been shown to keep nNOS in its phosphorylated, inactive state, presumably through inhibition of calcineurin [34]. Further actions of nitric oxide include elevating cyclic GMP levels, and these levels have also been shown to be lower during NMDA treatment of cortical neurons in the presence of low nanomolar concentrations of FK-506 or CsA [34]. This suggests that calcineurin inhibition protects axonal damage through several significant pathways of neurotoxicity.

Secondary phenomena that occur in the cell from Ca2+ overload include auto-oxidation of macromolecules and activation of phopholipase A2, calmodulin, proteases and endonucleases [26]. This results in damage to proteins, nucleic acids, lipids and cell membrane. Mitochondrial dysfunction is a common event in cell injury as well. The mitochondrial permeability transition (MPT) pore is a Ca2+-activated channel that is indiscriminately permeable to solutes of a certain size [35]. Activated mitochondria release factors that activate caspases, which lead to cell death. Mitochondria serve as energy generators of the cell as well as buffer intracellular Ca2+. Hypoxia abolishes the transmitochondrial membrane potential, leading to impairment of ATP. This provides two insults. First, the Ca2+-buffering capabilities are abolished and, secondly, the lack of ATP inhibits many other transmembrane ATP-dependent enzymes from maintaining ionic gradients. Thus, severe mitochondrial injury contributes to the death of the cell. Although this study did not directly measure the effect of CsA and FK-506 on its function, electron microscopy demonstrated severe injury sustained by mitochondria during hypoxia and relatively good structural integrity in tissue segments treated with CsA and FK-506.

This study was designed to elucidate the role of calcineurin inhibitors in axonal protection during hypoxia. Several pathways lead to Ca2+ overload, Ca2+-dependent enzyme activation and subsequent cellular demise (Fig. 4). The clinical application of using immunosuppressants to control human spinal cord injury is an exciting prospect. Recently, FK-506–mediated calcineurin blockade was shown to prevent dephosphorylation of the proapoptotic protein BAD, and thus activation of caspase-3, which within hours promotes apoptosis in spinal cord injury [36]. This suggests that early treatment after injury may provide a means to control damage to spinal cord–injured patients. Postinjury and in vivo studies may reveal if these compounds are useful in living systems, and possibly in human subjects.

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Fig. 4. This schema illustrates how, during white matter spinal cord injury or ischemia, several receptors contribute to an increase in intracellular Ca2+, which activates calcineurin causing it move into the cytoplasm. In turn, calcineurin induces mictochondrial membrane rupture and release of Ca2+, a step involved in Ca2+-mediated apoptosis. Calcineurin-mediated dephosphorylation of enzymes leads to apoptosis and necrosis, which ultimately leads to cell death. ADP = adenosine 5′-diphosphate; AMPA = L-amino-3 hydroxy-5-methyl-4-isoxazolepionic acid; ATP = adenosine 5′-triphosphate; CsA = Cyclosporin A; GP = G-proteins; NMDA = N-methyl-D-aspartic acid; PI = phosphate inisitol; PLC = phospholipase-C.