Non-cell autonomous downregulation of the purinergic receptor P2Y1R promotes neuroprotection after ischemic injury.

Cell Survival through Trk Neurotrophin Receptors Is Differentially Regulated by Ubiquitination

Extracellular ATP, which is released from damaged cells after ischemia, activates P2 receptors. P2Y1 receptors (P2Y1R) have received considerable attention, especially in astrocytes, because their activation plays a central role in the regulation of neuron-to-glia communication. However, the functions or even existence of P2Y1R in microglia remain unknown, despite the fact that many microglial P2 receptors are involved in several brain diseases. Herein, we demonstrate the presence and functional capability of microglial P2Y1R to provide neuroprotective effects following ischemic stress. Cerebral ischemia resulted in increased microglial P2Y1R expression. The number of injured hippocampal neurons was significantly higher in P2Y1 R knockout (KO) mice than wildtype mice after forebrain ischemia. Propidium iodide (PI) uptake, a marker for dying cells, was significantly higher in P2Y1R KO hippocampal slices compared with wildtype hippocampal slices at 48 h after 40-min oxygen–glucose deprivation (OGD). Furthermore, increased PI uptake following OGD was rescued by ectopic overexpression of P2Y1R in microglia. In summary, these data suggest that microglial P2Y1R mediate neuroprotective effects against ischemic stress and OGD insult.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that acts through G protein-coupled receptors, exerts neuroprotective effects upon many neuronal populations. However, the intracellular signaling mechanisms that account for PACAP's trophic effects are not well characterized. Here we have tested the possibility that PACAP uses neurotrophin signaling pathways. We have found that PACAP treatment resulted in an increase in TrkA tyrosine kinase activity in PC12 cells and TrkB activity in hippocampal neurons. The activation of TrkA receptors by PACAP required at least 1 h of treatment and did not involve binding to nerve growth factor. Moreover, PACAP induced an increase in activated Akt through a Trk-dependent mechanism that resulted in increased cell survival after trophic factor withdrawal. The increases in Trk and Akt were blocked by K252a, an inhibitor of Trk receptor activity. In addition, transactivation of TrkA receptors by PACAP could be inhibited with PP1, an inhibitor of Src family kinases or BAPTA/AM, (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester), an intracellular calcium chelator. Therefore, PACAP can exert trophic effects through a mechanism involving Trk receptors and utilization of tyrosine kinase signaling. This ability may explain several neuroprotective actions of PACAP upon neuronal populations after injury, nerve lesion, or neurotrophin deprivation.

Primary rat microglia stimulated with either ATP or 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) release copious amounts of superoxide (O(2)(-)*). ATP and BzATP stimulate O(2)(-)* production through purinergic receptors, primarily the P2X(7) receptor. O(2)(-)* is produced through the activation of the NADPH oxidase. Although both p42/44 MAPK and p38 MAPK were activated rapidly in cells stimulated with BzATP, only pharmacological inhibition of p38 MAPK attenuated O(2)(-)* production. Furthermore, an inhibitor of phosphatidylinositol 3-kinase attenuated O(2)(-)* production to a greater extent than an inhibitor of p38 MAPK. Both ATP and BzATP stimulated microglia-induced cortical cell death indicating this pathway may contribute to neurodegeneration. Consistent with this hypothesis, P2X(7) receptor was specifically up-regulated around beta-amyloid plaques in a mouse model of Alzheimer's disease (Tg2576).

BackgroundPerinatal brain injury results in neurodevelopmental disabilities (neuroDDs) that include cerebral palsy, autism, attention deficit disorder, epilepsy, learning disabilities and others. Commonly, injury occurs when placental circulation, that is responsible for transporting nutrients and oxygen to the fetus, is compromised. Placental insufficiency (PI) is a reduced supply of blood and oxygen to the fetus and results in a hypoxic-ischemic (HI) environment. A significant HI state in-utero leads to perinatal compromise, characterized by fetal growth restriction and brain injury. Given that over 80% of perinatal brain injuries that result in neuroDDs occur during gestation, prior to birth, preventive approaches are needed to reduce or eliminate the potential for injury and subsequent neuroDDs. Sulforaphane (SFA) derived from cruciferous vegetables such as broccoli sprouts (BrSps) is a phase-II enzyme inducer that acts via cytoplasmic Nrf2 to enhance the production of anti-oxidants in the brain through the glutathione pathway. We have previously shown a profound in vivo neuro-protective effect of BrSps/SFA as a dietary supplement in pregnant rat models of both PI and fetal inflammation. Strong evidence also points to a role for SFA as treatment for various cancers. Paradoxically, then SFA has the ability to enhance cell survival, and with conditions of cancer, enhance cell death. Given our findings of the benefit of SFA/Broccoli Sprouts as a dietary supplement during pregnancy, with improvement to the fetus, it is important to determine the beneficial and toxic dosing range of SFA. We therefore explored, in vitro, the dosing range of SFA for neuronal and glial protection and toxicity in normal and oxygen/glucose deprived (OGD) cell cultures.MethodsOGD simulates, in vitro, the condition experienced by the fetal brain due to PI. We developed a cell culture model of primary cortical neuronal, astrocyte and combined brain cell co-cultures from newborn rodent brains. The cultures were exposed to an OGD environment for various durations of time to determine the LD50 (duration of OGD required for 50% cell death). Using the LD50 as the time point, we evaluated the efficacy of varying doses of SFA for neuroprotective and neurotoxicity effects. Control cultures were exposed to normal media without OGD, and cytotoxicity of varying doses of SFA was also evaluated. Immunofluorescence (IF) and Western blot analysis of cell specific markers were used for culture characterization, and quantification of LD50. Efficacy and toxicity effect of SFA was assessed by IF/high content microscopy and by AlamarBlue viability assay, respectively.ResultsWe determined the LD50 to be 2 hours for neurons, 8 hours for astrocytes, and 10 hours for co-cultures. The protective effect of SFA was noticeable at 2.5 μM and 5 μM for neurons, although it was not significant. There was a significant protective effect of SFA at 2.5 μM (p<0.05) for astrocytes and co-cultures. Significant toxicity ranges were also confirmed in OGD cultures as ≥ 100 μM (p<0.05) for astrocytes, ≥ 50 μM (p<0.01) for co-cultures, but not toxic in neurons; and toxic in control cultures as ≥ 100 μM (p<0.01) for neurons, and ≥ 50 μM (p<0.01) for astrocytes and co-cultures. One Way ANOVA and Dunnett’s Multiple Comparison Test were used for statistical analysis.ConclusionsOur results indicate that cell death shows a trend to reduction in neuronal and astrocyte cultures, and is significantly reduced in co-cultures treated with low doses of SFA exposed to OGD. Doses of SFA that were 10 times higher were toxic, not only under conditions of OGD, but in normal control cultures as well. The findings suggest that: 1. SFA shows promise as a preventative agent for fetal ischemic brain injury, and 2. Because the fetus is a rapidly growing organism with profound cell multiplication, dosing parameters must be established to insure safety within efficacious ranges. This study will influence the development of innovative therapies for the prevention of childhood neuroDD.

Small ubiquitin-like modifier (SUMO)2/3 but not SUMO1 conjugation is activated after transient cerebral ischemia. To investigate its function, we blocked neuronal SUMO2/3 translation through lentiviral microRNA delivery in primary cortical neurons. Viability was unaffected by SUMO2/3 silencing unless neurons were stressed by transient oxygen–glucose deprivation (OGD). Both 15 and 45 minutes of OGD were tolerated by control microRNA-expressing neurons but damaged >60% of neurons expressing SUMO2/3 microRNA. Damaging OGD (75 minutes) increased neuronal loss to 54% (control microRNA) and to 99% (SUMO2/3 microRNA). This suggests that activation of SUMO2/3 conjugation is an endogenous neuroprotective stress response.

Microglial cells are the primary immune effector cells in the brain. Extracellular ATP, e.g., released after brain injury, may initiate microglial activation via stimulation of purinergic receptors. In the rat nucleus accumbens (NAc), the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated. A stab wound in the NAc increased immunoreactivity (IR) for P2X1,2,4,7 and P2Y1,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy. A prominent immunolabeling of P2X7 receptors with antibodies directed against the ecto- or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells. Additionally, the P2X7 receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt. Four days after local application of the agonists α,βmeATP, ADPβS, 2MeSATP, and BzATP, an increase in OX 42- and G. simplicifolia isolectin-IR was observed around the stab wound, quantified both densitometrically and by counting the number of ramified and activated microglial cells, whereas UTPγS appeared to be ineffective. The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects. Further, the intra-accumbally applied P2X7 receptor agonist BzATP induced an increase in the number of caspase-3-positive cells. These results indicate that ATP, acting via different P2X and P2Y receptors, is a signaling molecule in microglial cell activation after injury in vivo. The up-regulation of P2X7-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects.

4-Hydroxyderricin attenuates ischemic brain injury and neuroinflammation by upregulating haptoglobin expression in microglia.

Cross-tolerance to otherwise lethal N-methyl- D-aspartate and oxygen–glucose deprivation in preconditioned cortical cultures

Axonal injury and dysfunction are responsible for much of the disability observed following a stroke. Human brain comprises equal proportions of gray matter and white matter and white matter is injured in most strokes. Casein kinase 2 (CK2) is a protein kinase expressed in brain, including white matter, and is regulated by ischemia. We therefore hypothesized that transient CK2 inhibition would protect white matter from ischemic injury. To assess the impact of CK2 inhibition on axonal electrical activity following oxygen glucose deprivation (OGD), mouse optic nerves (MONs), a pure white matter track, from C57BL/6J were subjected to OGD (1h) while eliciting compound action potentials (CAPs) and exposed to CX-4945, a selective CK2 inhibitor, or control artificial cerebrospinal fluid (ACSF). We observed that CX-4945 preserved CAPs when applied either before or after OGD. Then to determine the impact of CK2 inhibition on glial cell survival following OGD, MONs exposed to OGD that were treated with either CX-4945 or control ACSF were processed for immunohistochemistry. We observed that CX-4945 treatment protected oligodendrocytes from OGD. And finally, to determine if CK2 inhibition protected mitochondrial from OGD, MONs from Thy-1 mito-CFP mice were similarly subjected to OGD in the presence of either CX-4945 or control ACSF. We observed that CX-4945 maintained Thy-1 mito-CFP fluorescence following OGD. In conclusion, our results suggest that CK2 inhibition preserves axonal function by preserving oligodendrocytes and mitochondrial function following ischemic injury. We propose that CK2 inhibitors, which are currently in phase II-III clinical trials for cancer therapy could be repurposed and provide a novel therapeutic target to protect white matter against ischemic injury, reducing mortality and morbidity and improving recovery following stroke.

Using a mild insult to induce endogenous neuroprotective mechanisms is known as preconditioning. When followed by a severe insult, or challenge, preconditioning results in a state of tolerance in which the injury inflicted by the challenge is mitigated. Tolerance is observed in ischemia, seizure, and infection and can be induced when the severe insult is preceded by a mild insult of the same or different type. Classical tolerance requires new protein synthesis and confers delayed and temporary neuroprotection, taking hours to develop, peaking over the course of 1–3 days, and then abating (Barone et al., 1998). In rodents and humans, transient ischemic attacks (TIAs) attenuate the injury sustained from an ensuing stroke (Chen et al., 1996; Weih et al., 1999). We used microarray analysis to profile the transcriptional changes that occur in the mouse cortex after ischemic preconditioning [15-min middle cerebral artery occlusion (MCAO)], ischemic challenge (60-min MCAO), and ischemic tolerance (15-min MCAO followed 72 h later by 60-min MCAO). The analysis showed that the genes regulated by preconditioning and the genes regulated by challenge were different. Moreover, genes regulated in tolerance were distinct from those regulated by either preconditioning or challenge. Accordingly, we propose that preconditioning reprograms the brain’s response to challenge, altering the transcriptional response, and thereby producing a neuroprotected phenotype (Stenzel-Poore et al., 2003). The reprogrammed response to ischemic challenge induced by ischemic preconditioning begins to elucidate putative mechanisms of endogenous neuroprotection. In ischemic tolerance, gene expression is predominantly suppressed. Ion channels, transporters, and metabolic pathways are particularly influenced. This is reminiscent of the endogenous neuroprotective mechanisms that allow hibernating animals to survive periods of prolonged oxygen and glucose deprivation (Storey & Storey, 1990; Hochachka et al., 1996). The endotoxin lipopolysaccharide (LPS) is a surface component of bacteria that triggers an inflammatory response through its interaction with toll-like receptor 4 (TLR4). Exposure to a low dose of LPS preconditions the brain and can produce ischemic tolerance (Tasaki et al., 1997). We used microarray analysis to profile the mouse cortex after endotoxin preconditioning (5-μg LPS), ischemic challenge (60-min MCAO), and LPS–ischemic cross-tolerance (5-μg LPS followed 72 h later by 60-min MCAO). The results support our conclusion that preconditioning achieves neuroprotection by reprogramming the transcriptional response to a severe insult. A majority of genes regulated in LPS-induced ischemic tolerance were not regulated in ischemic challenge (Stenzel-Poore et al., 2007). The nature of the reprogrammed response to ischemic challenge induced by LPS preconditioning offers further insight into endogenous neuroprotection. Whereas ischemic preconditioning suppresses expression of genes influencing metabolism and transport, LPS preconditioning modulates inflammatory mediators, inducing neuroprotective cytokines and repressing proinflammatory molecules. This suggests that the preconditioning stimulus provides neuroprotection by reprogramming particular aspects of the response to challenge, specifically those intrinsic to the preconditioning stimulus (Davis & Patel, 2003; Stenzel-Poore et al., 2007). Cell damage and death caused by a prolonged seizure diminishes when preceded by a brief, mild seizure. Various stimuli, including kindling, bicuculline, kainic acid, and electroshock effectively precondition the brain against status epilepticus (Kelly & McIntyre, 1994; Sasahira et al., 1995; Najm et al., 1998; Plamondon et al., 1999; Andre et al., 2000; Kondratyev et al., 2001; Borges et al., 2007; Hatazaki et al., 2007). We have used microarray analysis to profile the transcriptional changes that occur in the CA3 subfield of the mouse hippocampus after epileptic challenge (intraamygdala administration of kainic acid) and after epileptic tolerance (systemic administration of kainic acid followed 24 h later by intraamygdala administration of kainic acid). In contrast to ischemia, many of the same genes were regulated by both epileptic challenge and epileptic tolerance. However, a substantial subset of genes was regulated only by epileptic tolerance, and, notably, these differentially regulated genes were predominantly suppressed (Jimenez-Mateos et al., 2008). Prominent among the suppressed genes were those whose products participate in calcium signaling, synaptic function, long-term potentiation, and excitatory neurotransmission. We conclude that reprogramming, albeit to a less-pronounced degree, underlies the neuroprotection provided by seizure preconditioning. Moreover, seizure preconditioning specifically promotes an anti-excitotoxic phenotype, particularly apposite to the inducing stimulus, as an antiinflammatory phenotype is to LPS and a hypometabolic phenotype is to ischemia. We note that these phenotypes are appropriate to the nature of the preconditioning stimulus and not to the nature of the challenging stimulus. As an endogenous neuroprotective mechanism this can be understood as a first insult priming the brain to respond advantageously in the likelihood of a second insult of the same kind. Yet the brain appears also to respond advantageously to a second insult of a different kind. The basis for this is not yet clear. Our microarray studies make apparent that the response to any brain challenge is complex, engaging numerous and diverse pathways. Therefore, modulating metabolism, inflammation, or excitoxicity could provide a measure of protection against diverse insults that disrupt these functions to greater and lesser degrees. Our studies show that seizure preconditioning alters the expression of inflammatory mediators after epileptic challenge in a manner similar to LPS preconditioning (M.B. Johnson and R.P. Simon, unpublished results). Consequently, different preconditioning stimuli may activate common neuroprotective pathways. There may also be shared neuroprotective mechanisms not detectable at the transcriptional level. The ongoing task is to apply our understanding of endogenous neuroprotection to therapy while continuing to elucidate mechanisms. The authors confirm that they have read the Journal’s position on issues involved in ethical publication and affirm that this paper is consistent with those guidelines. Disclosure: The authors have no conflicts of interest to disclose.

Synergistic neuroprotection by epicatechin and quercetin: Activation of convergent mitochondrial signaling pathways

All-trans-retinoic acid (ATRA) is a vitamin A derivative that is important in neuronal patterning, survival, and neurite outgrowth. Neuroprotective effects of ATRA in ischemia have been demonstrated but its effects on glial swelling are not known. We investigated the relatively acute effects of ATRA on cell swelling in ischemic injury and on key features hypothesized to contribute to cell swelling including increased reactive oxygen species/reactive nitrogen species (ROS/RNS), depolarization of the inner mitochondrial membrane potential (ΔΨm), and increased intracellular calcium ([Ca2+]i). C6 glial cultures were subjected to 5 hr oxygen-glucose deprivation (OGD). ATRA was added to separate groups after the end of OGD. OGD increased cell volume by 43%, determined at 90 min after the end of OGD, but this increase was significantly attenuated by ATRA. OGD induced an increase in ROS/RNS production in the whole cell and mitochondria, as assessed by the fluorescent dyes CM-H2DCFDA and MitoTracker CM-H2-XROS at the end of OGD. The increase in mitochondrial ROS, but not cellular ROS, was significantly attenuated by ATRA. OGD also induced a 67% decline in mitochondrial ΔΨm but this decline was significantly attenuated by ATRA. OGD-induced increase in [Ca2+]i was also significantly attenuated by ATRA. Taken together with our previous results where calcium channel blockers reduced cell swelling, the effects of ATRA in attenuating swelling are possibly mediated through its effects in regulating [Ca2+]i. Considering the paucity of agents in attenuating brain edema in ischemia, ATRA has the potential to reduce brain edema and associated neural damage in ischemic injury.

Downregulation of miR-338-3p alleviates neuronal ischemic injury by decreasing cPKCγ-Mediated autophagy through the Akt/mTOR pathway

Combination of xenon and isoflurane produces a synergistic protective effect against oxygen-glucose deprivation injury in a neuronal-glial co-culture model.