Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.excitotoxicity ͉ poly(ADP-ribose) glycohydrolase ͉ poly(ADP-ribose) polymerase ͉ stroke P oly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nuclear protein that is involved in the DNA base excision repair system, where it is potently activated by DNA strand nicks and breaks (1, 2). Using NAD ϩ as a substrate, PARP-1 builds up homopolymers of ADP ribose units on various nuclear proteins including histones, DNA polymerases, topoisomerases, DNA ligase-2, transcription factors (3, 4), and PARP-1 itself (5, 6). Although the exact physiologic function of PARP-1 is not completely understood, in some tissues it plays an important role in DNA repair and genomic stability (5,7,8). Poly(ADP-ribose) (PAR) catabolism and metabolism is a dynamic process, with PAR glycohydrolase (PARG) playing the major role in the degradation of the polymer (9).Recent studies using pharmacologic inhibition of PARP or genetic KO of PARP-1 indicate that PARP-1 plays a dramatic and significant role in cellular injury after stroke, trauma, ischemiareperfusion of the heart, spleen, skeletal muscle, and retina, arthritis, ␤-islet cytotoxicity͞diabetes mellitus, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease, experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, endotoxic shock, multiple-system organ failure, and liver damage (for review, see refs. 1 and 10). PARP-1 activation also plays a prominent role in NMDA excitotoxicity, because PARP-1 KO mice are remarkably resistant both in vitro and in vivo to the excitotoxic effects of glutamate and NMDA (11,12). A cell-suicide hypothesis has been proposed (1,2,13,14) to explain the actions of PARP-1 in mediating cell death. However, studies in mice lacking PARG suggest that PAR polymer formed during the activation of PARP-1 might play a role in PARP-1-dependent cell death. PARG KO mice die at embryoni...

The water channel AQP4 is concentrated in perivascular and subpial membrane domains of brain astrocytes. These membranes form the interface between the neuropil and extracerebral liquid spaces. AQP4 is anchored at these membranes by its carboxyl terminus to ␣-syntrophin, an adapter protein associated with dystrophin. To test functions of the perivascular AQP4 pool, we studied mice homozygous for targeted disruption of the gene encoding ␣-syntrophin (␣-Syn ؊/؊ ). These animals show a marked loss of AQP4 from perivascular and subpial membranes but no decrease in other membrane domains, as judged by quantitative immunogold electron microscopy. In the basal state, perivascular and subpial astroglial end-feet were swollen in brains of ␣-Syn ؊/؊ mice compared to WT mice, suggesting reduced clearance of water generated by brain metabolism. When stressed by transient cerebral ischemia, brain edema was attenuated in ␣-Syn ؊/؊ mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but ␣-syntrophin was retained in perivascular astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus ␣-syntrophin-dependent anchoring of AQP4 is sensitive to ischemia, and loss of AQP4 from this site may retard the dissipation of postischemic brain edema. These studies identify a specific, syntrophin-dependent AQP4 pool that is expressed at distinct membrane domains and which mediates bidirectional transport of water across the brain-blood interface. The anchoring of AQP4 to ␣-syntrophin may be a target for treatment of brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule. C erebral edema is essentially a loss of water homeostasis entailing a net increase of water flux into the brain. The route of water influx in this life-threatening condition is unknown, and no efficient therapy exists. We have previously shown that the brain expresses a water channel molecule, AQP4, that is strongly enriched in those astrocyte membrane domains forming the interface between brain neuropil and extracerebral spaces filled with blood or cerebrospinal fluid (1-3). To determine whether the pools of AQP4 in these specialized membrane domains are responsible for the fast influx of water that occurs during the development of brain edema, one must specifically eliminate the perivascular and subpial pools of AQP4 while leaving other pools of AQP4 intact. This can be achieved by deletion of ␣-syntrophin (␣-syn), an adapter protein in the dystrophin-associated protein complex that is required for anchoring AQP4 at these specialized membrane domains (4). Mice homozygous for targeted disruption of the gene encoding ␣-syntrophin (␣-Syn Ϫ/Ϫ ) exhibit a marked reduction of AQP4 in perivascular and subpial membranes but not in other locations in brain, because total brain AQP4 protein content is not reduced (4).The first aim of the present study was to use ␣-Syn Ϫ/Ϫ mice to investigate whether a selective depletion of the perivascular AQP4 pool reduces the vol...

Background and Purpose-Cytochrome P450 epoxygenase metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs). EETs are produced in the brain and perform important biological functions, including vasodilation and neuroprotection. However, EETs are rapidly metabolized via soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). We tested the hypothesis that sEH gene deletion is protective against focal cerebral ischemia through enhanced collateral blood flow. Methods-sEH knockout (sEHKO) mice with and without EETs antagonist 14, 15 epoxyeicosa-5(Z)-enoic acid (EEZE) were subjected to 2-hour middle cerebral artery occlusion (MCAO), and infarct size was measured at 24 hours of reperfusion and compared to wild-type (WT) mice. Local CBF rates were measured at the end of MCAO using iodoantipyrine (IAP) autoradiography, sEH protein was analyzed by Western blot and immunohistochemistry, and hydrolase activity and levels of EETs/DHETs were measured in brain and plasma using LC-MS/MS and ELISA, respectively. Results-sEH immunoreactivity was detected in WT, but not sEHKO mouse brain, and was localized to vascular and nonvascular cells. 14,15-DHET was abundantly present in WT, but virtually absent in sEHKO mouse plasma. However, hydrolase activity and free 14,15-EET in brain tissue were not different between WT and sEHKO mice. Infarct size was significantly smaller, whereas regional cerebral blood flow rates were significantly higher in sEHKO compared to WT mice. Infarct size reduction was recapitulated by 14,15-EET infusion. However, 14,15-EEZE did not alter infarct size in sEHKO mice. Conclusions-sEH gene deletion is protective against ischemic stroke by a vascular mechanism linked to reduced hydration of circulating EETs.

The aquaporin-4 (AQP4) pool in the perivascular astrocyte membranes has been shown to be critically involved in the formation and dissolution of brain edema. Cerebral edema is a major cause of morbidity and mortality in stroke. It is therefore essential to know whether the perivascular pool of AQP4 is up-or down-regulated after an ischemic insult, because such changes would determine the time course of edema formation. Here we demonstrate by quantitative immunogold cytochemistry that the ischemic striatum and neocortex show distinct patterns of AQP4 expression in the reperfusion phase after 90 min of middle cerebral artery occlusion. The striatal core displays a loss of perivascular AQP4 at 24 hr of reperfusion with no sign of subsequent recovery. The most affected part of the cortex also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the striatal core, but it shows a partial recovery toward 72 hr of reperfusion. By freeze fracture we show that the loss of perivascular AQP4 is associated with the disappearance of the square lattices of particles that normally are distinct features of the perivascular astrocyte membrane. The cortical border zone differs from the central part of the ischemic lesion by showing no loss of perivascular AQP4 at 24 hr of reperfusion but rather a slight increase. These data indicate that the size of the AQP4 pool that controls the exchange of fluid between brain and blood during edema formation and dissolution is subject to large and region-specific changes in the reperfusion phase.astrocytes ͉ brain edema ͉ ischemia ͉ stroke ͉ water channels S troke is invariably associated with a brain edema that accounts for much of the morbidity and mortality of this condition. The brain edema is often long lasting and therapyresistant and thus poses a major challenge in the clinic. A better understanding is needed of the molecular mechanisms that promote water flux across the brain-blood interface in the build-up phase and resolution phase of cerebral edema.Aquaporin-4 (AQP4) water channels are strongly enriched in the astrocyte plasma membrane domains that ensheathe the cerebral microvessels (1, 2). It was hypothesized (1) that this perivascular pool of AQP4 could become rate-limiting for water flux in pathophysiological conditions, such as in the reperfusion phase after an ischemic insult. This hypothesis was tested in a model that took advantage of the fact that the perivascular AQP4 pool is anchored through the dystrophin complex (comprising the brain dystrophin isoform DP-71 and ␣-syntrophin) (3). Mice with targeted deletion of ␣-syntrophin displayed a dramatic loss of perivascular AQP4 and a concomitant reduction in the extent of postischemic edema (4). These findings [and experiments in mdx mice (5)] support the idea that the perivascular pool of AQP4 facilitates water flux across the brain-blood interface and offer a mechanistic explanation for the reduction in brain edema formation and dissolution observed in AQP4 Ϫ/Ϫ animals (6, 7) The implication of a sp...

Increases in COX-2 enzymatic activity and prostaglandin production have been associated with neuronal injury in both acute and age-related degenerative neurological diseases. In this study, we tested the effects of increased COX-2 activity in a model of transient focal ischemia using a transgenic mouse model in which human COX-2 is constitutively expressed selectively in neurons of the striatum, cerebral cortex, and hippocampus. These COX-2 transgenic mice harbor elevated levels of PGE(2) that are 10-fold higher than nontransgenic levels. A significant increase in infarct volume was observed after middle cerebral artery occlusion with 4 days of reperfusion in COX-2 transgenic mice as compared with nontransgenic littermates. Pretreatment of nontransgenic mice with the selective COX-2 inhibitor SC58236 resulted in a significant reduction of infarct volume in nontransgenic mice, consistent with previous pharmacological studies. However, transgenic COX-2 mice treated with SC58236 did not show a significant reduction. This suggests that chronic increases in COX-2 expression and enzymatic activity, which can occur in aging and in pathological states characterized by oxidative stress and chronic inflammatory processes, can lead to downstream cellular changes that have a negative impact on neuronal survival in cerebrovascular disease.

Background and Purpose-Social interaction can have a profound effect on health. The purpose of the present study was to determine whether affiliative social interactions before and after stroke improve ischemic outcomes as assessed through histological analysis and behavioral assays. Methods-Male and female C57BL/6 mice were housed individually or with an ovariectomized female. Behavioral assessments were made 24 hours before 60 or 90 minutes of transient intraluminal middle cerebral artery occlusion (MCAO) or SHAM surgery and after 7 days of reperfusion. Two hours after behavioral testing on day 7, infarct size was determined by 2,3,5-triphenyltetrazolium histology, and blood samples were collected for assessment of corticosterone and C-reactive protein (CRP) concentrations. Results-Pair housing significantly decreased infarct size and improved contralateral paw use in 60-minute MCAO males and 90-minute MCAO females compared with socially isolated cohorts. Housing condition had no significant effect on infarct size in females that underwent 60 minutes of MCAO, but pair housing was associated with improved contralateral paw use relative to socially isolated mice. In a separate cohort of males, intraischemic CRP concentration was significantly reduced in pair-housed males relative to isolated males. Conclusions-Affiliative interaction during the peri-ischemic period reduces intraischemic CRP concentration, decreases ischemic damage in male and female mice, and improves behavioral outcome.

Background: Japanese cedar (JC) pollinosis is the most common seasonal allergic rhinitis in Japan. Standardized JC pollen extract is available for subcutaneous immunotherapy, but this treatment is limited by potentially serious side effects. The aim of this double-blind, randomized comparative study was to evaluate the efficacy and safety of standardized JC pollen extract in a new oral formulation (CEDARTOLEN®, Torii Pharmaceutical Co., Ltd., Tokyo, Japan) for sublingual immunotherapy (SLIT) for JC pollinosis. Methods: A total of 531 subjects with JC pollinosis were randomized into 2 groups at a ratio of 1:1 to receive daily sublingual administration of standardized JC pollen extract with a maintenance dose of 2,000 Japanese allergy units (JAU) or placebo for 2 consecutive pollen seasons. The efficacy was evaluated using the total nasal symptom and medication score (TNSMS) as the primary end point. Secondary end points included the total ocular symptom and medication score (TOSMS) and scores for individual symptoms and medication. Results: The TNSMS was significantly lower (p < 0.0001) in the SLIT group than in the placebo group in the peak symptom period by 18 and 30% in the first and second seasons, respectively. All secondary end points were also significantly lower in the SLIT group in both seasons. No systemic anaphylaxis occurred. Conclusions: SLIT with daily administration of standardized JC pollen extract was effective for improving nasal and ocular symptoms of JC pollinosis and reducing the use of relief medication. The JC pollen extract was well tolerated with only local adverse events.

Objective. This study was undertaken to identify characteristics of follicular regulatory T (Tfr) cells and elucidate the mechanisms by which follicular helper T (Tfh) cells convert to Tfr cells. We probed the phenotype of T helper cells in patients with systemic lupus erythematosus (SLE) and underlying transcriptional regulation using cytokine-induced STAT family factors. Methods. Peripheral blood mononuclear cells from 41 patients with SLE and 26 healthy donors were used to sort out the memory Tfh cell subset, and Tfh cells were cultured under various conditions. The phenotype of T helper cells and underlying mechanisms of transcriptional regulation were probed using flow cytometry and quantitative polymerase chain reaction analyses. These analyses evaluated the expression of characteristic markers and phosphorylation of STATs. Chromatin immunoprecipitation was used to evaluate histone modifications. Results. In patients with SLE, the proportion of CD4+CXCR5+FoxP3-PD-1 high Tfh cells was increased (P < 0.01), whereas the proportion of CD4+CXCR5+CD45RA-FoxP3 high activated Tfr cells was decreased (P < 0.05). Serum interleukin-2 (IL-2) levels were also reduced in patients with SLE. IL-2 induced conversion of memory Tfh cells to functional Tfr cells, which was characterized by CXCR5+Bcl-6+FoxP3 high pSTAT3+pSTAT5+ cells. The loci of FOXP3 and BCL6 at STAT binding sites were marked by bivalent histone modifications. Following IL-2 stimulation, STAT3 and STAT5 selectively bound to FOXP3 and BCL6 gene loci accompanied by suppression of H3K27me3. Finally, IL-2 stimulation suppressed the generation of CD38+CD27 high plasmablasts in Tfh and B cell coculture assays ex vivo. Conclusion. Impaired function of Tfr cells might be attributed to defective IL-2 production. Exogenous IL-2 restores the function of Tfr cells through the conversion of Tfh cells to Tfr cells in patients with SLE. Thus, restoring balance between Tfh and Tfr cells may provide new therapeutic approaches in SLE.