DUSP6 Deficiency Attenuates Neurodegeneration after Global Cerebral Ischemia.

Transient global cerebral ischemia (tGCI) resulting from cardiac arrest causes selective neurodegeneration in hippocampal CA1 neurons. Although the effect is clear, the underlying mechanisms directing this process remain unclear. Previous studies have shown that phosphorylation of Erk1/2 promotes cell survival in response to tGCI. DUSP6 (also named MKP3) serves as a cytosolic phosphatase that dephosphorylates Erk1/2, but the role of DUSP6 in tGCI has not been characterized. We found that DUSP6 was specifically induced in the cytoplasm of hippocampal CA1 neurons 4 to 24 h after tGCI. DUSP6-deficient mice showed normal spatial memory acquisition and retention in the Barnes maze. Impairment of spatial memory acquisition and retention after tGCI was attenuated in DUSP6-deficient mice. Neurodegeneration after tGCI, revealed by Fluoro-Jade C and H&E staining, was reduced in the hippocampus of DUSP6-deficient mice and DUSP6 deficiency enhanced the phosphorylation and nuclear translocation of Erk1/2 in the hippocampal CA1 region. These data support the role of DUSP6 as a negative regulator of Erk1/2 signaling and indicate the potential of DUSP6 inhibition as a novel therapeutic strategy to treat neurodegeneration after tGCI.

Lipocalin-2 mediates the rejection of neural transplants.

Lipocalin-2 (LCN2) has been implicated in promoting apoptosis and neuroinflammation in neurological disorders; however, its role in neural transplantation remains unknown. In this study, we cultured and differentiated Lund human mesencephalic (LUHMES) cells into human dopaminergic-like neurons and found that LCN2 mRNA was progressively induced in mouse brain after the intrastriatal transplantation of human dopaminergic-like neurons. The induction of LCN2 protein was detected in a subset of astrocytes and neutrophils infiltrating the core of the engrafted sites, but not in neurons and microglia. LCN2-immunoreactive astrocytes within the engrafted sites expressed lower levels of A1 and A2 astrocytic markers. Recruitment of microglia, neutrophils, and monocytes after transplantation was attenuated in LCN2 deficiency mice. The expression of M2 microglial markers was significantly elevated and survival of engrafted neurons was markedly improved after transplantation in LCN2 deficiency mice. Brain type organic cation transporter (BOCT), the cell surface receptor for LCN2, was induced in dopaminergic-like neurons after differentiation, and treatment with recombinant LCN2 protein directly induced apoptosis in dopaminergic-like neurons in a dose-dependent manner. Our results, therefore, suggested that LCN2 is a neurotoxic factor for the engrafted neurons and a modulator of neuroinflammation. LCN2 inhibition may be useful in reducing rejection after neural transplantation.

Splice-Site Variants in the Gene Encoding GABA-A Receptor Delta Subunit Are Associated with Amphetamine Use in Patients under Methadone Maintenance Treatment.

Chronic opioid use disorder patients often also use other substances such as amphetamines. The gene-based analysis method was applied in the genomic database obtained from our previous study with 343 methadone maintenance treatment (MMT) patients. We found that the gene encoding gamma-aminobutyric acid type A receptors (GABA-A receptor) delta subunit isoforms (GABRD) was associated with amphetamine use in heroin dependent patients under MMT in Taiwan. A total of 15% of the 343 MMT patients tested positive for amphetamine in the urine toxicology test. Two genetic variants in the GABRD, rs2889475 and rs2376805, were found to be associated with the positive urine amphetamine test. They are located in the exon 1 of the splice variant and altered amino acid compositions (T126I, C/T, for rs2889475, and R252Q, G/A, for rs2376805). The CC genotype carriers of rs2889475 showed a four times higher risk of amphetamine use than those with TT genotype. The GG genotype carriers of rs2376805 showed a three times higher risk of amphetamine use than the AA genotype carriers. To our knowledge, this is the first report that demonstrated an association of the delta splice variant isoform in the GABA-A receptor with an increased risk of amphetamine use in MMT patients. Our results suggest that rs2889475 and rs2376805 may be indicators for the functional role and risk of amphetamine use in MMT patients.

Protective effects of ischemic preconditioning against neuronal apoptosis and dendritic injury in the hippocampus are age-dependent.

Ischemic preconditioning with non-lethal ischemia can be protective against lethal forebrain ischemia. We hypothesized that aging may aggravate ischemic susceptibility and reduce brain plasticity against preconditioning. Magnetic resonance diffusion tensor imaging (DTI) is a sensitive tool to detect brain integrity and white matter architecture. This study used DTI and histopathology to investigate the effect of aging on ischemic preconditioning. In this study, adult and middle-aged male Mongolian gerbils were subjected to non-lethal 5-min forebrain ischemia (ischemic preconditioning) or sham-operation, followed by 3 days of reperfusion, and then lethal 15-min forebrain ischemia. A 9.4-Tesla MR imaging system was used to study DTI indices, namely fractional anisotropy (FA), mean diffusivity (MD), and intervoxel coherence (IC) in the hippocampal CA1 and dentate gyrus (DG) areas. In situ expressions of microtubule-associated protein 2 (MAP2, dendritic marker protein) and apoptosis were also examined. The 5-min ischemia did not cause dendritic and neuronal injury and any significant change in DTI indices and MAP2 in adult and middle-aged gerbils. The 15-min ischemia-induced significant delayed neuronal apoptosis and early dendritic injury evidenced by DTI and MAP2 studies in both CA1 and DG areas with more severe injury in middle-aged gerbils than adult gerbils. Ischemic preconditioning could improve neuronal apoptosis in CA1 area and dendritic integrity in both CA1 and DG areas with better improvement in adult gerbils than middle-aged gerbils. This study thus suggests an age-dependent protective effect of ischemic preconditioning against both neuronal apoptosis and dendritic injury in hippocampus after forebrain ischemia.

Neutralization of Lipocalin-2 Diminishes Stroke-Reperfusion Injury.

Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the ß3 and ß4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.

Genetic inhibition of PKCε attenuates neurodegeneration after global cerebral ischemia in male mice.

Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCε) is a member of the novel PKC subfamily and plays a vital role in ischemic preconditioning. Pharmacological activation of PKCε before cerebral ischemia confers neuroprotection. The role of endogenous PKCε after cerebral ischemia remains elusive. Here we used male PKCε-null mice to assess the effects of PKCε deficiency on neurodegeneration after transient global cerebral ischemia (tGCI). We found that the cerebral vasculature, blood flow, and the expression of other PKC isozymes were not altered in the PKCε-null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCε-null mice as compared to male wild-type controls. A significant reduction in Fluoro-Jade C labeling and mitochondrial release of cytochrome C in the hippocampus was found in male PKCε-null mice after tGCI. Male PKCε-null mice expressed increased levels of PKCδ in the mitochondria, which may prevent the translocation of PKCδ from the cytosol to the mitochondria after tGCI. Our results demonstrate the neuroprotective effects of PKCε deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCε-null mice.

PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration.

BACKGROUND: Global cerebral ischemia triggers neurodegeneration in the hippocampal CA1 region, but the mechanism of neuronal death remains elusive. The epsilon isoform of protein kinase C (PKCε) has recently been identified as a master switch that controls the nucleocytoplasmic trafficking of ATF2 and the survival of melanoma cells. It is of interest to assess the role of PKCε-ATF2 signaling in neurodegeneration. RESULTS: Phosphorylation of ATF2 at Thr-52 was reduced in the hippocampus of PKCε null mice, suggesting that ATF2 is a phosphorylation substrate of PKCε. PKCε protein concentrations were significantly reduced 4, 24, 48 and 72 h after transient global cerebral ischemia, resulting in translocation of nuclear ATF2 to the mitochondria. Degenerating neurons staining positively with Fluoro-Jade C exhibited cytoplasmic ATF2. CONCLUSIONS: Our results support the hypothesis that PKCε regulates phosphorylation and nuclear sequestration of ATF2 in hippocampal neurons during ischemia-induced neurodegeneration.

Generation and characterization of ATP analog-specific protein kinase Cδ.

To better study the role of PKCδ in normal function and disease, we developed an ATP analog-specific (AS) PKCδ that is sensitive to specific kinase inhibitors and can be used to identify PKCδ substrates. AS PKCδ showed nearly 200 times higher affinity (Km) and 150 times higher efficiency (kcat/Km) than wild type (WT) PKCδ toward N(6)-(benzyl)-ATP. AS PKCδ was uniquely inhibited by 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NA-PP1) and 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2MB-PP1) but not by other 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) analogs tested, whereas WT PKCδ was insensitive to all PP1 analogs. To understand the mechanisms for specificity and affinity of these analogs, we created in silico WT and AS PKCδ homology models based on the crystal structure of PKCι. N(6)-(Benzyl)-ATP and ATP showed similar positioning within the purine binding pocket of AS PKCδ, whereas N(6)-(benzyl)-ATP was displaced from the pocket of WT PKCδ and was unable to interact with the glycine-rich loop that is required for phosphoryl transfer. The adenine rings of 1NA-PP1 and 2MB-PP1 matched the adenine ring of ATP when docked in AS PKCδ, and this interaction prevented the potential interaction of ATP with Lys-378, Glu-428, Leu-430, and Phe-633 residues. 1NA-PP1 failed to effectively dock within WT PKCδ. Other PP1 analogs failed to interact with either AS PKCδ or WT PKCδ. These results provide a structural basis for the ability of AS PKCδ to efficiently and specifically utilize N(6)-(benzyl)-ATP as a phosphate donor and for its selective inhibition by 1NA-PP1 and 2MB-PP1. Such homology modeling could prove useful in designing molecules to target PKCδ and other kinases to understand their function in cell signaling and to identify unique substrates.

Lipocalin-2 released in response to cerebral ischaemia mediates reperfusion injury in mice.

Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin-2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose-dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke-reperfusion injury.

Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils.

BACKGROUND: PKCδ expressed in neutrophils is implicated in promoting reperfusion injury after ischemic stroke. To understand the molecular and cellular actions of PKCδ, we employed a chemical-genetics approach to identify PKCδ substrates in neutrophils. RESULTS: We recently generated knock-in mice endogenously expressing analog-specific PKCδ (AS-PKCδ) that can utilize ATP analogs as phosphate donors. Using neutrophils isolated from the knock-in mice, we identified several PKCδ substrates, one of which was lipocalin-2 (LCN2), which is an iron-binding protein that can trigger apoptosis by reducing intracellular iron concentrations. We found that PKCδ phosphorylated LCN2 at T115 and this phosphorylation was reduced in Prkcd (-/-) mice. PKCδ colocalized with LCN2 in resting and stimulated neutrophils. LCN2 release from neutrophils after cerebral ischemia was reduced in PKCδ null mice. CONCLUSIONS: These findings suggest that PKCδ phosphorylates LCN2 and mediates its release from neutrophils during ischemia-reperfusion injury.

MMP-9 upregulation may predict hemorrhagic transformation after endovascular thrombectomy.

Background: Hemorrhagic transformation (HT) is a serious complication after endovascular thrombectomy (EVT) for patients with acute ischemic stroke (AIS). We analyzed the plasma levels of MMP-9 before and after EVT and assessed the temporal changes of MMP-9 that may be associated with, and therefore predict, HT after EVT. Methods: We enrolled 30 AIS patients who received EVT, and 16 (53.3%) developed HT. The levels of MMP-9 in plasma collected from the arteries of AIS patients before and immediately after EVT were measured using ELISA. The percent change in MMP-9 after EVT (after/before) was calculated and compared between patients with and without HT. Results: The median age of the AIS patients was 70 years, and 13 patients (43.3%) were men. The median National Institutes of Health Stroke Scale (NIHSS) scores of patients with HT were 18 on admission and 18 after EVT. The median NIHSS scores of patients without HT were 17 on admission and 11 after EVT. Patients with HT demonstrated significantly greater percentage increases in arterial MMP-9 levels after EVT. Conclusion: Patients with AIS who developed HT had significantly increased arterial MMP-9 levels after EVT, suggesting that the upregulation of MMP-9 following EVT could serve as a predictive biomarker for HT.

GABAA receptor trafficking is regulated by protein kinase C(epsilon) and the N-ethylmaleimide-sensitive factor.

Disturbances in GABA(A) receptor trafficking contribute to several neurological and psychiatric disorders by altering inhibitory neurotransmission. Identifying mechanisms that regulate GABA(A) receptor trafficking could lead to better understanding of disease pathogenesis and treatment. Here, we show that protein kinase Cε (PKCε) regulates the N-ethylmaleimide-sensitive factor (NSF), an ATPase critical for membrane fusion events, and thereby promotes the trafficking of GABA(A) receptors. Activation of PKCε decreased cell surface expression of GABA(A) receptors and attenuated GABA(A) currents. Activated PKCε associated with NSF, phosphorylated NSF at serine 460 and threonine 461, and increased NSF ATPase activity, which was required for GABA(A) receptor downregulation. These findings identify new roles for NSF and PKCε in regulating synaptic inhibition through downregulation of GABA(A) receptors. Reducing NSF activity by inhibiting PKCε could help restore synaptic inhibition in disease states in which it is impaired.

Hypertensive encephalopathy and the blood-brain barrier: is deltaPKC a gatekeeper?

Hypertensive encephalopathy is a life-threatening condition due to elevation of cerebral perfusion pressure beyond the limits of autoregulation. Breakdown of the blood-brain barrier (BBB) leads to cerebral edema and reduced blood flow. In this issue of the JCI, Mochly-Rosen and colleagues demonstrate a novel molecular strategy for preserving the BBB in a model of hypertension-induced encephalopathy (see the related article beginning on page 173). Using a rationally designed peptide inhibitor of deltaPKC, they stabilized the BBB and improved mortality in hypertensive rats. This study highlights the therapeutic potential of deltaPKC inhibitors in hypertensive encephalopathy and provides incentive to elucidate deltaPKC signaling pathways that mediate BBB dysfunction in other disease states.

rhomboid mediates specification of blue- and green-sensitive R8 photoreceptor cells in Drosophila.

Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila there is growing evidence that the color sensitivity of the R8 cell within an individual ommatidium is regulated by an inductive signal from the adjacent R7 cell. We previously examined the retinal patterning defect in Scutoid mutants, which results from a disruption of rhomboid expression. Here we show that loss of rhomboid blocks the induction of Rh5 expression and misexpression of rhomboid leads to the inappropriate induction of Rh5. These effects are specific to rhomboid, because its paralogue roughoid is neither required nor sufficient for the induction of Rh5 expression. We show that rhomboid is required cell-autonomously within the R8 photoreceptor cells and nonautonomously elsewhere in the eye for Rh5 induction. Interestingly, we found that the Epidermal growth factor receptor is also required for Rh5 induction, and its activation is sufficient to rescue the loss of Rh5 induction in a rhomboid mutant. This suggests that rhomboid may function in R8 cells to activate Epidermal growth factor receptor signaling in R7 cells and promote their differentiation to a signaling competent state.

Drosophila R8 photoreceptor cell subtype specification requires hibris.

Cell differentiation and cell fate determination in sensory systems are essential for stimulus discrimination and coding of environmental stimuli. Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila melanogaster, there is evidence that the color sensitivity of different photoreceptors in the compound eye is regulated by inductive signals between cells, but the exact nature of these signals and how they are propagated remains unknown. We conducted a genetic screen to identify additional regulators of this process and identified a novel mutation in the hibris gene, which encodes an irre cell recognition module protein (IRM). These immunoglobulin super family cell adhesion molecules include human KIRREL and nephrin (NPHS1). hibris is expressed dynamically in the developing Drosophila melanogaster eye and loss-of-function mutations give rise to a diverse range of mutant phenotypes including disruption of the specification of R8 photoreceptor cell diversity. We demonstrate that hibris is required within the retina, and that hibris over-expression is sufficient to disrupt normal photoreceptor cell patterning. These findings suggest an additional layer of complexity in the signaling process that produces paired expression of opsin genes in adjacent R7 and R8 photoreceptor cells.

Serum Level and Activity of Butylcholinesterase: A Biomarker for Post-Stroke Dementia.

Cholinergic neurotransmission regulates the immune response and inhibits cytokine release after stroke. The changes in the level/activity of blood cholinesterase (ChE) in patients with post-stroke dementia (PSD) are less known. This study aimed to examine post-stroke plasma acetylcholinesterase (AChE) and butylcholinesterase (BChE) and determine whether they are biomarkers for PSD. Thirty patients with PSD, 87 post-stroke patients without dementia (PSNoD), and 117 age- and gender-matched healthy controls were recruited. Missense genetic variants AChE rs1799806 and BChE rs1803274 were genotyped. The plasma AChE level did not differ between the PSD and PSNoD groups. However, BChE levels were significantly lower in the PSD than in the PSNoD group (3300.66 ± 515.35 vs 3855.74 ± 677.60 ng/mL, respectively; p = 0.0033). The activities of total ChE, BChE, and AChE were all lower in the PSD group (19,563.33 ± 4366.03, 7650.17 ± 1912.29, 11,913.17 ± 2992.42 mU/mL, respectively) than in the PSNoD group (23,579.08 ± 5251.55, 9077.72 ± 1727.28, and 14,501.36 ± 4197.17 mU/mL, respectively). When further adjusting for age and sex, significance remained in BChE level and activity and in total ChE activity. BChE rs1803274 was associated with reduced BChE activity, while AChE rs1799806 did not influence AChE activity. The level and activity of BChE, but not of AChE, were decreased in PSD patients and may therefore aid in PSD diagnosis.

A Post-hoc Study of D-Amino Acid Oxidase in Blood as an Indicator of Post-stroke Dementia.

Stroke is an important risk factor for dementia. Epidemiological studies have indicated a high incidence of dementia in stroke patients. There is currently no effective biomarker for the diagnosis of post-stroke dementia (PSD). D-amino acid oxidase (DAO) is a flavin-dependent enzyme widely distributed in the central nervous system. DAO oxidizes D-amino acids, a process which generates neurotoxic hydrogen peroxide and leads to neurodegeneration. This study aimed to examine post-stroke plasma DAO levels as a biomarker for PSD. In total, 53 patients with PSD, 20 post-stroke patients without dementia (PSNoD), and 71 age- and gender-matched normal controls were recruited. Cognitive function was evaluated at more than 30 days post-stroke. Plasma DAO was measured using the enzyme-linked immunosorbent assay. White matter hyperintensity (WMH), a neuroimaging biomarker of cerebral small vessel diseases, was determined by magnetic resonance imaging. We found that plasma DAO levels were independently higher in PSD subjects than in PSNoD subjects or the controls and were correlated with the WMH load in stroke patients. Using an area under the curve (AUC)/receiver operating characteristic analysis, plasma DAO levels were significantly reliable for the diagnosis of PSD. The sensitivity and specificity of the optimal cut-off value of 321 ng/ml of plasma DAO for the diagnosis of PSD were 75 and 88.7%, respectively. In conclusion, our data support that plasma DAO levels were increased in PSD patients and correlated with brain WMH, independent of age, gender, hypertension, and renal function. Plasma DAO levels may therefore aid in PSD diagnosis.

Neutrophil protein kinase Cdelta as a mediator of stroke-reperfusion injury.

Thrombolysis is widely used to intervene in acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Here we describe studies with mice lacking protein kinase Cdelta (PKCdelta) showing that absence of this enzyme markedly reduces reperfusion injury following transient ischemia. This was associated with reduced infiltration of peripheral blood neutrophils into infarcted tissue and with impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. Total body irradiation followed by transplantation with bone marrow from PKCdelta-null mice donors reduced infarct size and improved neurological outcome in WT mice, whereas marrow transplantation from WT donors increased infarction and worsened neurological scores in PKCdelta-null mice. These results indicate an important role for neutrophil PKCdelta in reperfusion injury and strongly suggest that PKCdelta inhibitors could prove useful in the treatment of stroke.

Acute functional tolerance to ethanol mediated by protein kinase Cepsilon.

A low level of response to ethanol is associated with increased risk of alcoholism. A major determinant of the level of response is the capacity to develop acute functional tolerance (AFT) to ethanol during a single drinking session. Mice lacking protein kinase C epsilon (PKCepsilon) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motor-impairing effects of ethanol is reduced in PKCepsilon (-/-) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCepsilon and resistance of GABA(A) receptors to ethanol. In contrast, in PKCepsilon (-/-) mice, GABA(A) receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCepsilon (-/-) and PKCepsilon (+/+) mice developed robust chronic tolerance to ethanol, but the presence of chronic tolerance did not change ethanol preference drinking. These findings suggest that ethanol activates a PKCepsilon signaling pathway that contributes to GABA(A) receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCepsilon and does not alter PKCepsilon modulation of ethanol preference.

Protein kinase C isozymes in stroke.

Stroke is a devastating neurologic disease and a leading cause of death and disability worldwide. Thrombolytic agents have been used to re-establish circulation in thromboembolic stroke, but their utility is limited by hemorrhage and reperfusion injury. Studies with experimental stroke models, mouse genetics, and selective peptide inhibitors and activators have implicated protein kinase C (PKC) epsilon in ischemic preconditioning and PKCdelta and gamma in tissue injury. PKCdelta, resident both in neutrophils and in the brain, appears particularly essential for reperfusion injury, and recent work using PKCdelta-specific peptide inhibitors suggests that PKCdelta inhibitors could prove useful in attenuating reperfusion injury and improving outcome following thrombolysis.