Sage Journals: Discover world-class research

Abstract

Protein phosphorylation and dephosphorylation mediated by protein kinases and protein phosphatases, respectively, represent essential steps in a variety of vital neuronal processes that could affect susceptibility to ischemic stroke. In this study, the role of the neuron-specific γ isoform of protein kinase C (γPKC) in reversible focal ischemia was examined using mutant mice in which the gene for γPKC was knockedout (γPKC-KO). A period of 150 minutes of unilateral middle cerebral artery and common carotid artery (MCA/CCA) occlusion followed by 21.5 hours of reperfusion resulted in significantly larger (P < 0.005) infarct volumes (n = 10; 31.1 ± 4.2 mm³) in γPKC-KO than in wild-type (WT) animals (n = 12; 22.6 ± 7.4 mm³). To control for possible differences related to genetic background, the authors analyzed Balb/cJ, C57BL/6J, and 129SVJ WT in the MCA/CCA model of focal ischemia. No significant differences in stroke volume were detected between these WT strains. Impaired substrate phosphorylation as a consequence of γPKC-KO might be corrected by inhibition of protein dephosphorylation. To test this possibility, γPKC-KO mice were treated with the protein phosphatase 2B (calcineurin) inhibitor, FK-506, before ischemia. FK-506 reduced (P < 0.008) the infarct volume in γPKC-KO mice (n = 7; 24.6 ± 4.6 mm³), but at this dose in this model, had no effect on the infarct volume in WT mice (n = 7; 20.5 ± 10.7 mm³). These results indicate that γPKC plays some neuroprotective role in reversible focal ischemia.

Keywords

Reversible focal cerebral ischemia γ-Protein kinase C-knockout mice Calcineurin FK-506 Infarct volume Phosphorylation

During ischemia and reperfusion, increases in intracellular Ca²⁺ occur at least in part in response to excitotoxicity (Choi, 1995). Ca²⁺ is an essential second messenger with a pivotal role in ischemic disease, and Ca²⁺ regulates the phosphorylation of many intracellular proteins (Hanson and Schulman, 1992). Signal transduction through Ca²⁺ is mediated in part by the activation of protein kinases, including protein kinase C (PKC), and protein phosphatases, including Ca²⁺/calmodulin (CaM)-dependent protein phosphatase 2B (calcineurin). Postranslational modification through protein phosphorylation plays an essential role in regulating various enzymes, ion channels, receptors, and cytoskeletal elements (Hanson and Schulman, 1992). Dysfunctional phosphorylation may, therefore, lead to loss of intracellular homeostasis and consequent collapse of cell integrity (Saitoh et al., 1991).

A member of the family of serine/threonine kinases, PKC is one of the most important multifunctional protein kinases in the brain. It is implicated in many vital aspects of CNS physiologic function, including synaptic plasticity, excitability, growth, proliferation, gene expression, and apoptosis (Tanaka and Nishizuka, 1994; Zhang et al., 1995). Protein kinase C consists of a family of closely related isoforms, most of which are detectable in the brain and are differentially activated by phospholipids and Ca²⁺. In response to cerebral ischemia, the catalytic activity of PKC in brain is greatly inhibited, and the enzyme is proteolyzed (Louis et al., 1988; Kochhar et al., 1989; Wieloch et al., 1991; Aronowski et al., 1992a; Cardell and Wieloch 1993). The γ isoform of PKC (γPKC) is highly enriched in neurons and is particularly susceptible to proteolytic degradation after ischemia (Huang et al., 1988; Wieloch et al., 1991; Zablocka et al., 1998). Irreversible inactivation and translocation of PKC always precedes neuronal damage, and is blocked by treatments that protect neurons from ischemic damage (Cardell et al., 1991; Aronowski et al., 1992b), implying a positive correlation between sustained activity of PKC and neuroprotection. On the other hand, Sieber et al. (1998) demonstrated that after global ischemia, expression and activity of PKC increases before delayed cell death in brain regions that are most susceptible to ischemic damage. Finally, results suggesting either a protective or deleterious role of PKC were obtained when ischemic damage was probed with inhibitors of protein kinases, staurosporine or 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride (H-7), which also affect PKC activity. Hara et al (1990), injecting animals with staurosporine, observed neuroprotection after global ischemia, whereas Madden et al. (1991), using staurosporine or H-7, produced augmentation of ischemic damage using a model of spinal cord ischemia. There are many reasons for these discrepancies; however, the lack of these inhibitor's efficacy or specificity and different ischemic paradigms may be a factor.

Changes in intracellular Ca²⁺ also regulate dephosphorylation of intracellular substrates through activation of a ubiquitous Ca²⁺/CaM-dependent serine/threonine protein phosphatase, calcineurin. Calcineurin's effects are either by direct dephosphorylation of phosphosubstrates or by activation of protein phosphatase 1 in a reaction that involves dephosphorylation of a protein phosphatase 1 inhibitory protein (Klee, 1991; Mulkey et al., 1994). An inhibitor of calcineurin, FK506, has been shown to reduce ischemic damage after global (Drake et al., 1996) and focal (Sharkey and Butcher., 1994; Butcher et al., 1997) ischemia, suggesting that calcineurin substrates could be involved in ischemic disease.

Recently, γPKC knock-out mice (γPKC-KO) were used to characterize the role of this enzyme in synaptic plasticity. The γPKC-KO mice are fully viable, although they display abnormal gaits and mild deficits in spatial and contextual learning when subjected to behavioral testing (Abeliovich et al., 1993b). Mutant mice have little detectable difference in baseline synaptic transmission, exhibit normal long-term depression and paired-pulse facilitation, although they have diminished long-term potentiation (Abeliovich et al., 1993a).

We hypothesized that decreases in PKC phosphorylation resulting from ischemia may cause phosphorylation imbalances in favor of protein dephosphorylation, mediated at least in part by calcineurin, leading to increased damage. To test this hypothesis, we used γPKC-KO mice and demonstrated their elevated susceptibility to focal ischemia. Interestingly, the increased damage was reversed by FK-506 treatment, an inhibitor of calcineurin activity.

METHODS

Animals

The production of mice genetically deficient in the γ isoform of PKC was described previously (Abeliovich et al., 1993a). Briefly, a homologous recombination vector was constructed containing γPKC sequences harboring 2.5 kb deletion. Integration of this vector also containing neo resulted in a loss of an exon containing the nucleotide-binding domain required for catalytic activity. The vector was transfected into E14ES cells, and clones containing the desired homologous integration were identified by G418 selection and Southern blot hybridization. Chimeric males initially were mated to C57BL/6J females and were the same animals used in the study by Abeliovich et al. (1993a, 1993b). Finally, to improve the efficiency of breeding, mice were back-crossed eight times into the Balb/cJ background. Homozygous animals were bred to produce offspring (γPKC-KO) that were subjected to the experimental protocols described later. Mice were typed by polymerase chain reaction analysis with a set of primers as described by Abeliovich et al. (1993a). The average weight of γPKC-KO mice was 18.8 ± 1.4 g. Western blotting of brain extracts with an antibody specific to the γ isoform of PKC verified the absence of γPKC in these animals. Balb/cJ (18.6 ± 1.5 g), C57BL/6J (18.7 ± 2.0 g), and 129SVJ (19.2 ± 1.9 g) were obtained from Jackson Laboratories and housed in the institutional animal care facility for at least 72 hours before surgery. Both male and female mice were used. Each experimental group was built out of an equal number of mice of the same sex. In no group were the differences between males and females greater than 1. All animals were kept in a 12:12-hour light-dark cycle with free access to food and water. All procedures were in compliance with the National Institute of Health guidelines for the humane care of animals.

Focal cerebral ischemia

Unilateral middle cerebral artery (MCA) and common carotid artery (CCA) occlusion was performed exactly as described previously (Waxham et al., 1996). Briefly, mice starved overnight between 5 and 7 weeks of age were anesthetized with chloral hydrate (0.35 g/kg), the skull was exposed, and a small burr hole was produced over the MCA. A 0.003-cm (0.005-inch) diameter stainless steel wire (Small Parts, Inc., Miami, FL, U.S.A.) was placed underneath the left MCA rostral to the rhinal fissure, proximal to the major bifurcation of the MCA, and distal to the lenticulostriate arteries. The artery then was lifted and the wire rotated clockwise. The left CCA then was occluded using atraumatic Heifetz aneurysm clips. Reperfusion was established after 150 minutes of occlusion by first removing the aneurysm clips from the CCA, then rotating the wire counterclockwise, and removing it from beneath the MCA. This paradigm resulted in reproducible slightly submaximal infarct volume in our previous studies (Waxham et al., 1996). Interruption of flow through the MCA was inspected under the microscope and verified by cerebral perfusion measurement using a laser Doppler flowmeter (model BPM2, Vasamedics, Inc., St. Paul, MN, U.S.A.). To analyze animals with a similar level of ischemia, only mice that displayed reduction of cerebral perfusion after ischemia to less than 15% of their preischemic value in the center of the ischemic zone were included in the study. Core body temperature was monitored and maintained at 36.5 ± 0.35°C during ischemia and for the first hour of reperfusion using a feed-forward temperature controller (YSI Model 72, Yellow Springs, OH, U.S.A.) coupled to a heating lamp and warming blanket.

Infarct volume measurement

Twenty-four hours after the onset of ischemia, under chloral hydrate anesthesia, mice were perfused with 30 mL of saline through the ascending aorta. The brain was removed, sectioned into 2-mm thick sections, and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate-buffered saline for 30 minutes at room temperature before fixing in 10% buffered formalin. Morphometric determination of infarct size was achieved using a computer-based image analyzer operated by “Brain” software (Drexel University, Philadelphia, PA, U.S.A.) as previously described (Waxham et al., 1996). Infarcts were restricted to cortical tissue. The infarct volume (in cubic millimeters) was calculated from the difference between the volume of contralateral cortex and the volume of the TTC-stained portion (nonischemic) of ipsilateral cortex of each mouse. This indirect measure of infarct volume, based on the assumption that the volume of the ipsilateral and contralateral cortex are the same before ischemia, corrects the total infarct volume for the edema component (Swanson et al., 1990). Statistical significance was determined by analysis of variance and the Newman-Keuls test.

Physiologic parameters

Body temperature in our study was controlled and sustained during ischemia and the initial hour of reperfusion. In a recent report, FK506 at the same dose used in this study (1 mg/kg) had no effect on brain and body temperature in experimental stroke in rats (Sharkey and Butcher, 1994; Butcher et al., 1997). In the current study, we did not measure PO₂, PCO₂, pH, and blood pressure. However, recent studies using rats and the same dose of FK506 showed no effect on blood gases, pH, and arterial blood pressure (Sharkey and Butcher, 1994; Butcher et al., 1997). Finally, using laser Doppler flowmeter to measure cerebral perfusion in the core of the infarction before and 15 minutes after ischemia, we did not find statistical cross-strain (including γPKC-KO) differences or treatment effect on cerebral perfusion (Table 1).

TABLE 1.

Cerebral perfusion

	Preocclusion	Occlusion
BALB/cJ-	81.66 ± 28.33	3.48 ± 1.31
C57BL/6J	82.35 ± 23.22	4.23 ± 1.54
129SVJ	71.67 ± 23.96	3.44 ± 1.56
γPKC-KO	86.01 ± 24.17	3.18 ± 1.00

Other methods

FK506 (Fujisawa Pharmaceutical, Osaka, Japan) was dissolved in 100% ethanol, then diluted in saline (0.1% final concentration of ethanol) and administered at 1 mg/kg in 200 μL intraperitoneally 1 hour before vessel occlusion. This dose has been shown to produce protection from damage in the rat model of focal ischemia (Sharkey and Butcher., 1994). Because of the negligible amount of ethanol (equivalent to 0.2 μL of 100% ethanol) used during FK-506 injection, we used untreated animals as controls for FK-506.

Western blot analysis was performed on ipsilateral and contralateral cortex homogenates obtained from Balb/cJ and γPKC-KO mice as described previously (Waxham et al., 1996). Immunostaining of Western blots, as described previously (Aronowski et al., 1996), was performed using anti-γPKC-specific rabbit polyclonal antibody (C-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.), anti-α-subunit of CaM-KII monoclonal antibody (2D5), and a monoclonal antibody specific to all isoforms of actin (supplied by Dr. Jim Lessard). Immunopositive bands were visualized with alkaline phosphatase-labeled secondary antibody followed by 5-bromo-1-chloro-indolyl phosphate and nitro-blue tetrazolium (Promega, Madison, WI, U.S.A.).

Both CaM-KII and PKC catalytic activity were assayed using 3 μg of crude brain homogenate protein exactly as described previously (Aronowski et al., 1992a) using either synthetic peptide autocamtide-3 (KKALHRQETVDAL) (Aronowski et al., 1996) for CaM-KII or peptide NG_(28-43) (AAAKIQASFRGHMAR) (Klann et al., 1998) for PKC.

RESULTS

Genetic background and susceptibility to ischemic damage

Gene knock-out approaches in mice have provided important new tools to investigate the genetic and molecular basis of brain damage after ischemia. However, an important concern in interpreting results from this approach is that of background genetic heterogeneity. This heterogeneity comes from three sources: (1) the embryonic stem cells used for “knocking out” the gene are from 129 mouse; (2) the founder mouse is typically C57BL/6; and (3) in the case of the current study, the strain used for generating mice for experimental analysis is BALB/c. Despite attempts to minimize heterogeneity by extensive back-crossing into the BALB/cJ strain, eliminating the possibility that the observed differences are caused by heterogeneity from either 129SV or C57BL/6 genetic carryover is difficult. This is particularly salient in studies of focal ischemia in mice, since genetic background has been shown to play a significant role in altering the extent of damage after the insult (Barone et al., 1993; Fujii et al., 1997; Maeda et al., 1999). To establish a database for the current and future work, we undertook a study to define the extent of damage after focal ischemia in each of the three mouse strains Balb/cJ, C57BL/6J, and 129/SVJ. Earlier work (Waxham et al., 1996) showed that 150 minutes of focal ischemia, followed by 21.5 hours of reperfusion, produced just submaximal infarct volume in the wild-type C57BL/6J strain of mice. A similar protocol was applied to evaluate the contribution of genetic background to infarct volume. These data are summarized in Fig. 1 and show that, overall, there is no statistical difference in infarct volume between the three strains of mice using this protocol. The average infarct volumes were 16.7 ± 4.9 mm³, 20.9 ± 6.3 mm³, and 22.6 ± 7.4 mm³ for 129/SVJ, C57BL/6J, and BALB/cJ, respectively. The infarct volume of commercially available C57BL/6J (20.9 ± 6.3 mm³) is nearly identical to wild-type litter mates bred from heterozygous α-subunit CaM-KII (αCaM-KII) knock-out mice in the C57BL/6 background (21.9 ± 6.4 mm³) (Waxham et al., 1996). Thus, the protocol we use for producing infarcts is consistent, and, overall, the genetic background contributes little to differences in infarct volume under the conditions used in our experiments.

FIG. 1.

Infarct volume after 150 minutes of ischemia and 21.5 hours of reperfusion in 129/SVJ, C57BL/6J, and Balb/cJ mice. Statistical analysis was determined by analysis of variance and the Newman-Keuls test and showed no difference between the groups.

Genetic knock-out of γPKC increases susceptibility to ischemic damage

Because, as mentioned earlier, γPKC-KO mice were back-crossed into the BALB/c background, all remaining analyses were performed using BALB/cJ mice as controls. As anticipated, probing extracts from BALB/cJ and γPKC-KO brains, using antibody to γPKC, we detected an immunopositive band at approximately 87 kd in BALB/cJ, whereas no immunoreactivity was detected in γPKC-KO animals (Fig. 2). Probing the same blot with antibody to αCaM-KII and actin showed no difference in the quantity of these proteins between BALB/cJ and γPKC-KO animals (Fig. 2). Similar to the results of Abeliovich et al. (1993), γPKC-KO brain extracts displayed 74% of PKC activity relative to BALB/cJ. In contrast, there was no difference in CaM-KII activity between γPKC-KO and BALB/cJ mice (Table 2).

TABLE 2.

Kinase activity (nmol · mg⁻¹ · min⁻¹)

PKC		CaM-KII
BALB/cJ	γPKC-KO	BALB/cJ	γPKC-KO
0.31 ± 0.01	0.23 ± 0.01	1.55 ± 0.05	1.58 ± 0.21

FIG. 2.

Immunoblot of γ isoform of protein kinase C (γPKC), α-subunit of Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII), and actin. Forty micrograms of cortical homogenate protein from γPKC-KO and BALB/CJ mice were analyzed. Notice the absence of immunostaining of γPKC in γPKC-KO mice and no changes in immunostaining of CaM-KII and actin. Two representative γPKC-KO and BALB/CJ mice are shown.

To assess PKC's role in the pathobiologic mechanism of ischemia, we analyzed the susceptibility of γPKC-KO animals to damage produced by focal ischemia. Occlusion of the MCA/CCA in γPKC-KO mice resulted in infarct volumes of 31.1 ± 4.2 mm³ (n = 10) (Figs. 3 and 4), which were significantly larger than infarct volumes in BALB/cJ mice 22.6 ± 7.4 mm³ (n = 12) (P < 0.003) (Figs. 3 and 4). In general, the infarct volume between animals was consistent; however, one γPKC-KO animal developed whole ipsilateral hemisphere infarction (56.5 mm³). This type of infarction has been detected in only 4 of 300 mice subjected to this protocol, and the cause is not clear. We excluded this animal from the analysis to avoid overinflating the average infarct volume in the γPKC-KO group. We conclude that mice deficient in the γ isoform of PKC exhibit increased damage after focal ischemia.

FIG. 3.

Reversible MCA/CCA occlusion resulted in larger infarct volumes in γPKC-KO mice. After 150 minutes of ischemia and 21.5 hours of reperfusion, animals were killed and infarct volume analyzed with triphenyltetrazolium chloride. Distribution of the infarcted areas is shown at different rostrocaudal levels in BALB/cJ and γPKC-KO mice. Infarct area is different (*P < 0.05) at the first two, but not the third, level as determined by the unpaired t-test.

FIG. 4.

Neuroprotection studies exploring the efficacy of FK-506 to reduce infarct volume. Infarct volume (cubic millimeters, determined using triphenyltetrazolium choloride) after transient 150 minutes of left middle cerebral artery/common carotid artery (MCA/CCA) occlusion and 21.5 hours of reperfusion in γPKC-KO and BALB/cJ mice is illustrated. Mice were injected intraperitoneally with 1 mg/kg of FK506 1 hour before vessel occlusion. The number of animals in each group is indicated in parenthesis above each bar. Average infarct volume ± SD for animals are shown. Infarct volume from individual animals are indicated as dots within each group. Level of statistical significance was determined by analysis of variance and the Newman-Keuls test.

Selective attenuation of infarct volume in γPKC-KO animals by FK506

Because reduced PKC activity represents the likely explanation for increased susceptibility to ischemia in γPKC-KO mice, we explored the possibility that inhibition of protein dephosphorylation in these animals might be capable of attenuating their elevated ischemic vulnerability. Calcineurin is highly enriched in neurons and is postulated to reverse the phosphorylation of numerous PKC substrates, suggesting functional interplay between these two enzymes. To address this issue, the well-characterized brain-permeable calcineurin inhibitor FK506 was used. Treatment of γPKC-KO mice with FK506 1 hour before stroke resulted in significant (P < 0.01) reductions in infarct volume from 31.1 ± 4.2 (n = 10) to 24.6 ± 4.6 mm³ (n = 7). Similar treatment of wild-type BALB/cJ mice with FK506 did not significantly reduce ischemic infarct volume (Fig. 4). These results indicate that this dose of FK-506 can preferentially rescue neurons in mice missing the γ isoform of PKC. We propose that inhibition of calcineurin-mediated dephosphorylation is capable of compensating for impaired phosphorylation in γPKC-KO mice. These results also suggest that interplay between γPKC and calcineurin activity regulate ischemic vulnerability.

DISCUSSION

In the current study, we demonstrated that animals lacking the γ isoform of PKC displayed significantly greater susceptibility to brain damage produced by reversible focal ischemia, and this increase in damage was reversed by an inhibitor of calcineurin, FK506.

We were concerned with the genetic heterogeneity of these animals because strain differences in damage after ischemia have been documented in mice (Barone et al., 1993; Fujii et al., 1997). Our analysis showed different susceptibility to ischemia, although statistically insignificant, among BALB/cJ, C57BL/6J, and 129/SVJ, with BALB/cJ being most vulnerable and 129/SVJ being the least vulnerable. The gradient of strain vulnerability to ischemia was similar to that described in previous work (Barone et al., 1993; Fujii et al., 1997). When we compared susceptibility of all these mice to that of γPKC-KO, the γPKC-KO mice displayed significantly greater vulnerability to ischemia, strongly suggesting that the absence of functional γPKC contributed to the increased damage. This implies that γPKC plays an as yet undefined role in protecting neurons after an ischemic insult.

As mentioned earlier, studies on the role of PKC in ischemia have not established its relevance to ischemic damage. Both activation and inactivation of the enzyme by ischemia have been demonstrated, and both protective and detrimental effects of PKC have been postulated ((Louis et al., 1988; Kochhar et al., 1989; Wieloch et al., 1991; Aronowski et al., 1992; Cardell and Wieloch, 1993; Cardell et al., 1991; Aronowski et al., 1992b; Sieber et al., 1998; Hara et al., 1990; Madden et al., 1991). Protein kinase C is ubiquitously distributed throughout a variety of cell types, and in the brain PKC is present in neurons, glia, smooth muscles, endothelial cells, and other cells (Tanaka and Nishizuka, 1994). Protein kinase C consists of a family of at least 12 isoforms, with 4 isoforms (α, βI, βII, and γ) that require Ca²⁺ for their activation. Each isoform can be activated by a distinct subset of second messengers, can be expressed in distinct cell types, and has different intracellular localization (Tanaka and Nishizuka, 1994; Krizbai et al., 1995; Van der Zee et al., 1995; Erbrugger et al., 1997). Because each PKC isoform is likely to modulate distinct, potentially opposing functions, it could be misleading to draw conclusions about the role of PKC enzymes based on analysis of their (all isoforms together) generalized activity changes in whole brain. Additionally, compounds that inhibit all PKC isoform could lead to a different outcome than the selected loss of one isoform produced by gene knock-out.

Cerebrovascular tone has a direct impact on ischemic stroke outcome. Vasoconstriction resulting in reduced CBF can produce augmentation of ischemia and subsequent damage. Recently, activation of vascular PKC was demonstrated to produce vasoconstriction linked to the pathogenesis of cerebral vasospasm (Ohta et al., 1995). In addition, inhibition of PKC by the protein kinase inhibitor, staurosporine, was reported to inhibit vascular smooth muscle contractility (Asano et al., 1995), providing a possible explanation for the protective mechanism of staurosporine in ischemic models (Hara et al., 1990). A role for γPKC in regulating vascular contractility is unknown, and although absent from blood vessels (Mattila et al., 1994; Krizbai et al., 1995; Erdbrugger et al., 1997), γPKC could affect vascular tone through its innervation. γPKC is highly enriched in neurons, where it is postulated to regulate synaptic activity or gene expression in response to signal activating Ca²⁺ entry (Hata et al., 1993; Abeliovich et al., 1993a; Sakai et al., 1997). It is, therefore, logical to consider that increased vulnerability to ischemia in γPKC-KO animals, observed in the current study, results from γPKC mutation-induced neuronal dysfunction.

Neuronal PKC, similar to calcineurin, is involved in regulation of diverse aspects of signal transduction including neurotransmitter release, receptors, ion channels, enzyme regulation, and gene expression (Klee et al., 1988; Yakel, 1997; Ryves et al., 1996; Tanaka and Nishizuka, 1994). Calcineurin and γPKC share common substrates. Some of these include dynamin I, GAP-43, MARCKS, IP₃ receptor, and nitric oxide synthase (Robinson et al., 1994; Robinson, 1991; Klee, 1991; Bredt et al., 1992; Dawson et al., 1993; Cameron, 1995; Mahoney et al., 1995). Deficient substrate phosphorylation by PKC, as that likely present in γPKC-KO mice, might, therefore, be corrected by inhibiting substrate dephosphorylation, including that by calcineurin.

One of the cell-permeable calcineurin inhibitors is the immunosuppressant drug FK506 (Liu, 1991). Similar to the effect of FK506 on infarct volume in γPKC-KO animals, FK506 was shown to protect rat brain from damage after reversible focal ischemia (Sharkey and Butcher, 1994; Butcher et al., 1997). This neuroprotective mechanism of FK506 was postulated to involve inhibition of calcineurin with subsequent inhibition of nitric oxide synthase and reduction of cytotoxic NO production (Bredt et al., 1992; Dawson et al., 1993). We find it surprising that the neuroprotective effect of FK506 was significant in γPKC-KO animals, whereas no effect in BALB/cJ mice was observed. It is generally accepted that the portion of ischemic brain most likely to be salvaged by therapeutic intervention is the ischemic penumbra, due to presence of collateral flow resulting in better perfusion than in the infarct core. Because γPKC-KO mice develop larger infarctions, it is possible that this is caused by lesser resistance of the γPKC mutated neurons to the level of hypoperfusion present in the penumbral region. The efficacy of FK506 in γPKC-KO may be linked to the possibility that there is a larger ischemic penumbra in these mice. The dose of FK506 used in the current study is the same as that which is optimal in producing immunosuppression and in protecting brain from focal ischemia in rats (Sharkey and Butcher, 1994). However, there is a difference in FK506 pharmacokinetics between rats and mice. The 1-mg/kg dose of FK506 used to produce immunosuppression may be suboptimal in mice (Iwata et al., 1993), since a three- to five-times larger dose is required to produce optimal immunosuppression and possibly neuroprotection in wild-type mice compared with rats (Bekersky I, personal communication, 1998). Because γPKC-KO mice may have impaired phosphorylation compared with wild-type animals, it is possible that a lower dose of FK506 is sufficient to correct deficient phosphorylation produced by the γPKC mutation.

In summary, γPKC-deficient mice are more susceptible to ischemia. This vulnerability may be mediated by imbalances between impaired PKC phosphorylation and unimpaired calcineurin-mediated protein dephosphorylation in the regulation of neuronal enzymes essential for cell homeostasis and survival.

Footnotes

Abbreviations used

References

Abeliovich

Paylor

Chen

Kim

Wahner

Tonegawa

(1993a) γPKC mutant mice exhibit mild deficits in spatial and contextual learnig. Cell 75:1263–1271

Abeliovich

Chen

Goda

Silva

Stevens

Tonegawa

(1993b) Modified hippocampal long-term potentiation in PKC gamma-mutant mice. Cell 75:1253–1262

Aronowski

Grotta

Waxham

(1992a) Ischemia-induced translocation of Ca²⁺/calmodulin-dependent protein kinase II: potential role in neuronal degeneration. J Neurochem 58:1743–1753.

Aronowski

Waxham

Grotta

(1992b) Neuronal protection and preservation of calcium/calmodulin-dependent protein kinase II and protein kinase C activity by dextrorphan treatment in global ischemia. J Cereb Blood Flow Metab 13:550–557

Aronowski

Grotta

(1996) Calcium/calmodulin-dependent protein kinase II in forebrain postsynaptic densities after reversible global ischemia in rats. Brain Res 709:103–109

Asano

Matsunaga

Miura

Ito

Seto

Sakurada

Nagumo

Sasaki

Ito

(1995) Selectivity of action of staurosporine on Ca²⁺ movements and contractions in vascular smooth muscles. Eur J Pharmacol 294:693–701

Barone

Knudsen

Nelson

Feuerstein

Willette

(1993) Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow Metab 13:683–692

Bredt

Ferris

Snyder

(1992) Nitric oxide synthase regulatory sites: phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase. Identification of flavin and calmodulin binding sites. J Biol Chem 267:10976–10981

Butcher

Henshall

Teramura

Iwasaki

Sharkey

(1997) Neuroprotective actions of FK506 in experimental stroke: in vivo evidence against an antiexcitotoxic mechanism. J Neurosci 17:6939–6946

10.

Cameron

Steiner

Roskams

Ali

Ronnett

Snyder

(1995) Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca²⁺ flux. Cell 83:463–472

11.

Cardell

Boris-Moller

Wieloch

(1991) Hypothermia prevents the ischemia-induced translocation and inhibition of protein kinase C in the rat striatum. J Neurochem 57:1814–1817

12.

Cardell

Wieloch

(1993) Time course of the translocation and inhibition of protein kinase C during complete cerebral ischemia in the rat. J Neurochem 61:1308–1314

13.

Choi

(1995) Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci 18:58–60

14.

Dawson

Steiner

Dawson

Dinerman

Uhl

Snyder

(1993) Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc Natl Acad Sci USA 90:9808–9812

15.

Drake

Friberg

Boris-Moller

Sakata

Wieloch

(1996) The immunosuppressant FK506 ameliorates ischaemic damage in the rat brain. Acta Physiol Scand 158:155–159

16.

Erdbrugger

Keffel

Knocks

Otto

Philipp

Michel

(1997) Protein kinase C isoenzymes in rat and human cardiovascular tissues. Br J Pharmacol 120:177–186

17.

Fujii

Hara

Meng

Vonsattel

Huang

Moskowitz

(1997) Strain-related differences in susceptibility to transient forebrain ischemia in SV-129 and C57black/6 mice. Stroke 28:1805–1810

18.

Griffin

Watson

(1988) Axonal transport in neurological disease. Ann Neurol 23:3–13

19.

Hanson

Schulman

(1992) Neuronal Ca²⁺/calmodulin-dependent protein kinases. Annu Rev Biochem 61:559–601

20.

Hanson

Grotta

Waxham

Aronowski

Ostrow

(1994) Calcium/calmodulin-dependent protein kinase II activity in focal ischemia with reperfusion in rats. Stroke 25:466–473

21.

Hara

Onodera

Yoshidomi

Matsuda

Kogure

(1990) Staurosporine, a novel protein kinase C inhibitor, prevents postischemic neuronal damage in the gerbil and rat. J Cereb Blood Flow Metab 10:646–653

22.

Hata

Akita

Suzuki

Ohno

(1993) Functional divergence of protein kinase C (PKC) family members: PKC gamma differs from PKC alpha and beta II and nPKC epsilon in its competence to mediate 12-O-tetradecanoyl phorbol 13-acetate (TPA)-responsive transcriptional activation through a TPA-response element. J Biol Chem 268:9122–9129

23.

Huang

Yoshida

Nakabayashi

Woung

III Huang

(1988) Immunocytochemical localization of protein kinase C isozymes in rat brain. J Neurosci 8:4734–4744

24.

Iwata

Kitagawa

Sato

Kosugi

Hirose

Hamaoka

Shearer

Fujiwara

(1993) Suppression of allograft responses by combining donor alloantigen-specific intravenous presensitization with suboptimal doses of FK506. Transplantation 56:173–180

25.

Klann

Roberson

Knapp

Sweatt

(1998) A role for superoxide in protein kinase C activation and induction of long-term potentiation. J Biol Chem 273:4516–4522

26.

Klee

Draetta

Hubbard

(1988) Calcineurin. Adv Enzymol Relat Areas Mol Biol 61:149–200.

27.

Klee

(1991) Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin. Neurochem Res 16:1059–1065

28.

Kochhar

Saitoh

Zivin

(1989) Reduced protein kinase C activity in ischemic spinal cord. J Neurochem 53:946–952

29.

Krizbai

Szabo

Deli

Maderspach

Lehel

Olah

Wolff

Joo

(1995) Expression of protein kinase C family members in the cerebral endothelial cells. J Neurochem 65:459–462

30.

Liu

Farmer

Jr Lane

Friedman

Weissman

Schreiber

(1991) Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66:807–815

31.

Louis

Magal

Yavin

(1988) Protein kinase C alterations in the fetal rat brain after global ischemia. J Biol Chem 263:19282–19285

32.

Madden

Clark

Kochhar

Zivin

(1991) Effect of protein kinase C modulation on outcome of experimental CNS ischemia. Brain Res 547:193–198

33.

Maeda

Hata

Hossmann

(1999) Regional metabolic disturbances and cerebrovascular anatomy after permanent middle cerebral artery occlusion in C57black/6 and SV129 mice. Neurobiol Dis 2:101–108

34.

Mahoney

Seki

Huang

(1995) Phosphorylation of MARCKS, neuromodulin, and neurogranin by protein kinase C exhibits differential responses to diacylglycerols. Cell Signal 7:679–685

35.

Mattila

Majuri

Tiisala

Renkonen

(1994) Expression of six protein kinase C isotypes in endothelial cells. Life Sci 55:1253–1260

36.

Mulkey

Endo

Shenolikar

Malenka

(1994) Involvement of a calcineurin/inhibitor-1 phosphatase cascade in hippocampal long-term depression. Nature 369:486–488

37.

Ohta

Nishihara

Oka

Todo

Kumon

Sakaki

(1995) Possible mechanism to induce protein kinase C-dependent arterial smooth muscle contraction after subarachnoid haemorrhage. Acta Neurochir 137:217–225

38.

Robinson

(1991) The role of protein kinase C and its neuronal substrates dephosphin, B-50, and MARCKS in neurotransmitter release. Mol Neurobiol 5:87–130

39.

Robinson

Liu

J-P

Powell

Fykse

Sudhof

(1994) Phosphorylation of dynamin I and synaptic-vesicle recycling. TiNS 17:348–353

40.

Ryves

Dekker

Brammer

Campbell

(1996) PKC in rat cortical synaptosomes: effects of depolarization. Neuroreport 8:323–327

41.

Saitoh

Masliah

Jin

Cole

Wieloch

Shapiro

(1991) Protein kinases and phosphorylation in neurologic disorders and cell death. Lab Invest 64:596–616

42.

Sakai

Sasaki

Ikegaki

Shirai

Ono

Saito

(1997) Direct visualization of the translocation of the gamma-subspecies of protein kinase C in living cells using fusion proteins with green fluorescent protein. J Cell Biol 139:1465–1476

43.

Sharkey

Butcher

(1994) Immunophilins mediate the neuroprotective effects of FK506 in focal cerebral ischaemia. Nature 371:336–339

44.

Sieber

Traysman

Brown

Martin

(1998) Protein kinase C expression and activation after global incomplete cerebral ischemia in dogs. Stroke 29:1445–1453

45.

Swanson

Morton

Tsao-Wu

Savalos

Davidson

Sharp

(1990) A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10:290–293

46.

Tanaka

Nishizuka

(1994) The protein kinase C family for neuronal signaling. Annu Rev Neurosci 17:551–567

47.

Van der Zee

Bolhuis

Solomonia

Horn

Luiten

(1995) Differential distribution of protein kinase C (PKC alpha beta and PKC gamma) isoenzyme immunoreactivity in the chick brain. Brain Res 676:41–52

48.

Waxham

Grotta

Silva

Strong

Aronowski

(1996) Ischemia-induced neuronal damage: a role for calcium/calmodulin-dependent protein kinase II. J Cereb Blood Flow Metab 16:1–6

49.

Wieloch

Cardell

Bingren

Zivin

Saitoh

(1991) Changes in the activity of protein kinase C and the differential subcellular redistribution of its isozymes in the rat striatum during and following transient forebrain ischemia. J Neurochem 56:1227–1235

50.

Yakel

(1997) Calcineurin regulation of synaptic function: from ion channels to transmitter release and gene transcription. TiPS 18:124–134

51.

Zablocka

Maternicka

Zalewska

Domanska-Janik

(1998) Expression of Ca²⁺-dependent (classical) PKC mRNA isoforms after transient cerebral ischemia in gerbil hippocampus. Brain Res 779:254–258

52.

Zhang

Lawa

Hintona

Couldwell

(1995) Growth inhibition and apoptosis in human neuroblastoma SK-N-SH cells induced by hypericin, a potent inhibitor of protein kinase C. Cancer Lett 96:31–35

Interplay Between the Gamma Isoform of PKC and Calcineurin in Regulation of Vulnerability to Focal Cerebral Ischemia

Abstract

Keywords

METHODS

Animals

Focal cerebral ischemia

Infarct volume measurement

Physiologic parameters

Other methods

RESULTS

Genetic background and susceptibility to ischemic damage

Genetic knock-out of γPKC increases susceptibility to ischemic damage

Selective attenuation of infarct volume in γPKC-KO animals by FK506

DISCUSSION

Footnotes

Abbreviations used

References