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In response to mild ischemic stress, the brain elicits endogenous survival mechanisms to protect cells against a subsequent lethal ischemic stress, referred to as ischemic tolerance. The molecular signals that mediate this protection are thought to involve the expression and activation of multiple kinases, including protein kinase C (PKC). Here we demonstrate that εPKC mediates cerebral ischemic tolerance in vivo. Systemic delivery of ψεRACK, an εPKC-selective peptide activator, confers neuroprotection against a subsequent cerebral ischemic event when delivered immediately prior to stroke. In addition, activation of εPKC by ψεRACK treatment decreases vascular tone in vivo, as demonstrated by a reduction in microvascular cerebral blood flow. Here we demonstrate the role of acute and transient εPKC in early cerebral tolerance in vivo and suggest that extra-parenchymal mechanisms, such as vasoconstriction, may contribute to the conferred protection.
A wide network of intracellular signaling pathways are activated in response to mild stresses such as sublethal ischemia or hypothermia to reduce metabolic demand, maintain basic cellular function, and increase survival in the event of a period of severe oxygen and glucose starvation such as stroke [8, 13]. These endogenous neuroprotective events confer what is termed ischemic tolerance or preconditioning. Strategies to identify and harness the cellular and molecular mechanisms of this protection hold tremendous potential for therapeutic benefit.
Preconditioning has been observed in multiple tissues, including the heart and brain, suggesting that conserved signaling pathways may mediate ischemic insult [8, 23]. In response to a number of preconditioning-stimuli, changes in gene expression and protein activation initiate common signaling pathways including upregulation of free radical scavengers, opening of KATP channels, and activation of protein kinases including protein kinase C (PKC) [2, 5, 8, 32]. Protein kinase C is a widely-expressed family of serine/ threonine kinases. The role of εPKC has been well established in cardiac preconditioning , and is a point of signaling convergence following multiple types of cardiac preconditioning stimuli including transient sublethal ischemia, ethanol and adenosine . More recently, a role for εPKC in cerebral tolerance has been suggested using various in vitro models. These studies have demonstrated that εPKC is activated following a preconditioning stimulus such as application of adenosine or NMDA, and is required for preconditioning- induced protection [26, 31]. However, whether this protection correlates with the in vivo activity of εPKC, in which the role of the vasculature, inflammation and other extra-parenchymal mediators are important, has not been addressed.
Here we determined the role of εPKC in cerebral ischemia in vivo, using ψεRACK, an εPKC-specific activator peptide. Multiple reports have demonstrated that PKCs play an important role in mediating vascular tone [6, 19]. We therefore also examined the effects of εPKC activation on cerebrovascular function. A clear understanding of the mechanisms which govern preconditioning holds great promise for therapies against diseases such as stroke, as well as conditions associated with reduced blood flow to the brain, such as during cardiac bypass surgery. Here we demonstrate that εPKC may be an important target for acute neuroprotective therapies.
ψεRACK (εPKC activator peptide; εPKC85–92: C-HDAPIGYD) or εV1-2 (εPKC inhibitor peptide; εPKC14–21: C-EAVSLKPT) were synthesized and Tat, the protein transduction domain of the TAT protein (TAT47–57; CYGRKKRRQRRR), was conjugated to the PKC peptides via a cysteine S-S bond. The εPKC isozyme-selective activity of ψεRACK-Tat (ψεRACK) and εV1-2-Tat (εV1-2) has previously been demonstrated in both in vitro and in vivo models [3, 31], and in particular, has been shown to alter εPKC activity in the brain following systemic intraperitoneal delivery . Peptide dose (0.2mg/kg) was based on previous studies [3, 11, 31].
Transient ischemia (2hrs) was induced in male Sprague-Dawley rats (290–320g) using an intraluminal suture occluding the ostium of the middle cerebral artery (MCA) as previously described . Animals were maintained and monitored under isoflurane anesthesia during all surgical procedures. Tat (vehicle control) or ψεRACK peptides were delivered as a bolus dose intraperitoneally at the following time points; I) 30 mins prior to ischemia and 3 mins into ischemia, II) 30 mins prior to ischemia, III) 3 mins into ischemia, IV) 24 hrs prior to ischemia, V) 3 mins following ischemia, at the onset of reperfusion (all injections 0.2 mg/kg in 1 mL saline). εV1-2 peptide was delivered only at one time point; 30 mins prior to ischemia. Physiological parameters including body temperature (35°C–38°C) and respiration rate were monitored and maintained using a heat blanket and anesthetic adjustment. All animals were euthanized by anesthetic overdose. For PKC translocation assays, rats were sacrificed at 10 mins, 30 mins or 120 mins of ischemia (without removal of the suture to allow reperfusion). Sham control animals were treated with an identical protocol, however the suture was not advanced to occlude the MCA. The brain was sliced and striatum isolated and snap frozen for tissue homogenization. All animal protocols were approved by the Institutional Animal Care and Use Committee of Stanford University.
Animals were euthanized and brains were quickly removed and sliced into five, 3 mm coronal sections. Slices were stained using 3% triphenyl tetrazolium chloride (TTC) and both faces of each slice were photographed for infarct assessment. Approximately 15% of animals were excluded from the analysis due to unsuccessful induction of ischemia, with no observable infarct. Relative stroke area (ratio of the infarct size relative to the ipsilateral hemisphere, corrected for edema based on measurement of the contralateral hemisphere) was measured to assess infarct size in the central three slices (two faces each; six faces total). Results were expressed as the mean±SEM infarct size. All statistical analyses were performed using ANOVA followed by Bonferroni’s post-hoc test.
Brains were quickly isolated, sliced, and striatal regions dissected from each central slice and frozen. Tissue fractionation was performed to collect soluble (cytosolic) and particulate (membrane) fractions, as previously described . To compare εPKC concentration in each fraction, total protein concentration was assessed using Bradford reagent, and 10 µg of total lysate (40 µg from hippocampal slice lysate) from each fraction was subjected to gel electrophoresis (10% bisacrylamide gel) and transferred to nitrocellulose membrane. Blots were blocked in 3% milk TBST (Tris-Buffered Saline Tween), probed with an anti- εPKC antibody (Santa Cruz; 1:500) in 2% milk TBST and probed with an anti-rabbit secondary antibody (Amersham). All statistical analyses were performed using ANOVA followed by Fisher’s posthoc tests.
Male Sprague-Dawley rats (280–400g) were used. Animals were maintained under isoflurane anesthesia. Cerebral blood flow measurements were recorded using a Laser Doppler probe via a cranial burr hole (1mm posterior, 6 mm lateral of bregma on the left cortex). Flow measurements were taken for 20 mins to establish baseline flow, followed by intraperitoneal injection of ψεRACK, or Tat control peptide (0.2 mg/kg in 1ml saline), and flow was recorded for an additional 20–40 mins. Statistical analysis of cerebral blood flow was performed as follows; blood flow readings (at 1 minute intervals) for a period of 20–30 minutes were compared with blood flow measurements following delivery of ψεRACK, for a period of 20–40 mins. Statistical significance was assessed using Student’s t-test.
Protein Kinase C (PKC) intracellular translocation from the soluble to the membrane-enriched, particulate fraction is a standard method to measure activation state of the enzyme . Using a time course of ischemic injury, εPKC translocation was assessed in brain tissue homogenates. Within 10 mins of ischemia, the proportion of εPKC in the membrane-bound fraction was significantly higher than in non-ischemic sham animals (45% increase, p<0.05). This translocation was maintained at 30 mins into ischemia (41% increase, p<0.05). Translocation of εPKC was diminished by 120 mins of ischemia (Figure 1A, B).
ψεRACK peptide was delivered by intraperitoneal-dose at various time points prior, during and following ischemia (0.2 mg/kg in 1 mL) (Figure 2A). Infarct size was assessed following 24 hrs of reperfusion. When delivered twice, both 30 mins prior to ischemia and 3 mins into ischemia, a significant reduction in infarct size was observed compared to Tat-carrier-treated controls. When ψεRACK was delivered at 30 mins prior to ischemia only, infarct size was also significantly reduced. When ψεRACK was delivered at 3 mins into ischemia, protection was observed, as seen by a reduction in infarct size; this reduction however did not reach statistical significance (Figure 2B and 2C; Tat-control treated (all groups combined); 27+/−3%, 30 mins prior and 3 mins intra-ischemia (n=9); 14+/−5% p<0.05, 30 mins prior; (n=14); 15+/−4% p<0.05, 3 mins intra (n=11); 18+/−4% p=0.07). We next tested whether increasing εPKC activity mediates long term protection against cerebral ischemic injury (delayed tolerance), by delivering ψεRACK peptide 24 hrs prior to stroke injury. In this protocol, ψεRACK treatment did not result in observable protection against ischemic injury (Figure 2C; n=9). ψεRACK peptide was also delivered immediately following 2 hours of ischemia, at the onset of reperfusion, to determine whether εPKC activity mediates delayed injury processes. In these animals, ψεRACK did not alter infarct size compared to Tat-vehicle treated animals (Figure 2D; Tat vehicle control 33+/−5%, ψεRACK post- 33+/−6%, n=8 per group).
To determine whether inhibiting εPKC activity is detrimental, εV1-2 or Tat carrier was delivered 30 mins prior to the onset of transient focal ischemia. No significant difference in infarct size was observed (data not shown).
We next examined whether delivery of ψεRACK alters vascular tone by measuring microvascular cerebral blood flow using laser Doppler flowmetry. Delivery of ψεRACK peptide (0.2 mg/kg) significantly decreased flow in all animals tested by an average of 15+/−6% (Figure 3; n=10, p<0.05). Delivery of Tat peptide alone did not significantly alter cerebral blood flow (data not shown.) The onset of flow reduction following ψεRACK delivery was variable between animals, from 5–30 minutes. Importantly, delivery of ψεRACK peptide does not alter mean arterial blood pressure, blood oxygen or carbon dioxide (data not shown).
The role of εPKC in cerebral ischemia in vivo has been unclear, in part due to conflicting reports on the expression and activity of εPKC following stroke . We demonstrate here that εPKC is activated during cerebral ischemia, and may play an important role in mediating the early cellular response to ischemic stress.
Following ischemic stress, ion channel dysregulation and influx of calcium into the cell leads to the activation of multiple protein kinases including PKC. We recognize that individual PKC isozymes play unique roles in mediating both survival and cell death pathways in the brain. However, until now, we have had only a limited understanding of the activation pattern and activity of individual PKC isozymes.
Conflicting reports exist on εPKC activity during and following ischemic injury. Some studies suggest that εPKC is activated following oxygen/ glucose deprivation, or in response to kainic acid (a glutamate analog) treatment in in vitro and ex vivo models [20, 28]. Other reports suggest that εPKC does not respond to ischemia or ischemia-like insults [9, 14, 30]. Differences in the insult duration and severity in these models, cell types involved, and time points selected for assessing εPKC activity, may have contributed to these conflicting findings. Importantly, many of these studies assess εPKC activity during the period of reperfusion, and not early during ischemia, or acutely during or following preconditioning.
Here, we show that εPKC is activated during transient focal ischemia. Within 10 minutes of ischemia, an approximately 40% increase in membrane translocation of εPKC was observed. εPKC activation at early timepoints during ischemia suggests that this isozyme is activated following even short periods of ischemia, similar to a tolerizing ischemic period. Indeed, in models of focal preconditioning, 10–15 minutes of transient MCA occlusion is sufficient to induce tolerance . These results are consistent with ex vivo studies, in which εPKC is activated within 1 hour following a preconditioning stress . Therefore, it is likely that εPKC is activated, and may mediate the initial cellular response to brief or mild ischemia such as preconditioning stress, in vivo. Sustained εPKC activation at 30 minutes of ischemia also implies that εPKC activity contributes to the ischemic response under more severe ischemic insults.
Following our finding that εPKC is activated in response to ischemic insult, we examined whether activation or inhibition of εPKC altered outcome following ischemic stroke. Delivery of ψεRACK activator peptide prior to ischemia conferred a significant reduction in infarct size compared to Tat control. Activation of εPKC did not confer protection against a delayed ischemic insult when ψεRACK was delivered 24 hrs prior to transient focal ischemia, indicating that εPKC activity may be involved only in acute protection paradigms. Importantly, delivery of ψεRACK during reperfusion resulted in no change in lesion size at 24 hrs. This suggests that εPKC specifically mediates protective signaling prior to, or early during ischemia, however, may not be involved in promoting cell survival during the reperfusion period (Figure 2). There was no significant difference in the infarct size between εV1-2 treated- and carrier-control treated animals (data not shown), suggesting inhibiting εPKC activity does not worsen ischemic injury. Significant progress has been made in understanding εPKC function in ischemic tissue. Most recent work has demonstrated the involvement of εPKC in regulation of heat shock protein signaling, preservation of mitochondrial function, and reduction of free radical generation [7, 16, 22, 25]. εPKC signaling in ischemic tissue has been the subject of a number of reviews [4, 22].
Multiple studies have shown that PKC enzymes are important in mediating vascular tone in a variety of models, in in vitro primary endothelial and smooth muscle cells, isolated arteries, and in vivo cerebrovascular responses . Inhibition of PKC isozymes with pan-specific PKC inhibitors such as staurosporine and calphostin C induce vasodilation, whereas activation of PKC using phorbol esters such as PMA causes potent vasoconstriction [1, 13]. However, the specific PKC isozymes which mediate these responses are largely unknown.
Here we show that acute delivery of ψεRACK causes a mild, but significant reduction of cerebral blood flow, by approximately 15%, suggesting εPKC activity promotes microvascular vasoconstriction. Interestingly, this reduction in flow occurs in the absence of any ischemic stimulus, (however measurements were made while the animal was under isoflurane anesthesia, a known vasodilator). This vasoconstriction was maintained for the duration of the study (up to 60 minutes post-ψεRACK delivery).
An εPKC-mediated reduction in cerebral blood flow is an unexpected finding. Indeed, agents that induce cerebral vasodilation, such as arginine (via nitric oxide production) or adenosine, confer neuroprotection during stroke by increasing flow to ‘at-risk’ areas via collateral supply arteries [21, 33]. It is possible that a decrease in blood flow may cause a preconditioning-like effect by mildly reducing the amount of oxygen and glucose supplied to tissue and suppressing metabolic rate, similar to a mild ischemic event. Indeed, hypothermia, a well known mechanism to protect the brain against damage under ischemic conditions, confers protection in part by suppressing metabolic demand in tissue . Interestingly, it has recently been shown that hypothermia may preserve εPKC activity in the brain after stroke , suggesting a link between these two neuroprotective pathways.
The role of εPKC in mediating CBF is unknown; ψεRACK may have direct effects on vascular cell function, or may play an indirect role on neuronal regulation of cerebral blood flow. Interestingly, recent work has demonstrated that inhibition of PKC using Ro-32-0432 suppresses vascular smooth muscle cell endothelin-B receptor upregulation in response to ischemia, and contractile response in arterial segments , suggesting the direct role of εPKC on vascular cell contractile function. However, previous studies have demonstrated that delivery of ψεRACK in in vitro and ex vivo cerebral ischemia models, in which blood flow does not play a role, confers neuroprotection [26, 31]. The neuroprotection observed following delivery of ψεRACK peptide may not therefore be solely or directly attributable to the reduction in cerebral blood flow. Whether this reduction in blood flow in the observed neuroprotective effect of ψεRACK contributes to preconditioning-like effects observed in vivo is a subject of future studies.
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Conflict of interest statement:
Daria Mochly-Rosen is a founder of KAI Pharmaceuticals, whose goal is to bring peptide regulators of PKC to the clinic. However, the research described in this study was carried out in her lab with sole support from NIH grants to her university activities.