1These authors contributed equally to this study.Abbreviations used: BCCAO, bilateral common carotid arteries occlusion; COX-2, cyclooxygenase-2; EAAT, excitatory amino acid transporter; IjB alpha, inhibitory kappa B alpha; iNOS, inducible nitric oxide synthase; IPC, ischemic preconditioning; IT, ischemic tolerance; LPS, lipopolysaccharide; MCA, middle cerebral artery; NF-jB, nuclear factor kappa B; pMCAO, permanent middle cerebral artery occlusion; TACE, TNF-alpha converting enzyme; TLR, toll-like receptor; TNF-a, tumor necrosis factor alpha. AbstractIt has been demonstrated that a short ischemic event (ischemic preconditioning, IPC) results in a subsequent resistance to severe ischemia (ischemic tolerance, IT). We have recently demonstrated the role of innate immunity and in particular of toll-like receptor (TLR) 4 in brain ischemia. Several evidences suggest that TLR4 might also be involved in IT. Therefore, we have now used an in vivo model of IPC to investigate whether TLR4 is involved in IT. A 6-min temporary bilateral common carotid arteries occlusion was used for focal IPC and it was performed on TLR4-deficient mice (C57BL/ 10ScNJ) and animals that express TLR4 normally (C57BL/ 10ScSn). To assess the ability of IPC to induce IT, permanent middle cerebral artery occlusion was performed 48 h after IPC. Stroke outcome was evaluated by determination of infarct volume and assessment of neurological scores. IPC caused neuroprotection as shown by a reduction in infarct volume and better outcome in mice expressing TLR4 normally. TLR4-deficient mice showed less IPC-induced neuroprotection than wild-type animals. Western blot analysis of tumor necrosis factor alpha (TNF-a), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) showed an up-regulation in the expression of these proteins in both substrains of mice measured 18, 24 and 48 h after IPC, being higher in mice with TLR4. Similarly, nuclear factor-kappa B (NF-jB) activation was observed 18, 24 and 48 h after IPC, being more intense in TLR4-expressing mice. These data demonstrate that TLR4 signalling is involved in brain tolerance as shown by the difference in the percentage of neuroprotection produced by IPC between ScSn and ScNJ (60% vs. 18%). The higher expression of TNF-a, iNOS and cyclooxygenase-2 and NF-jB activation in mice expressing TLR4 is likely to participate in this endogenous neuroprotective effect.

Functional and protein interactions between the N-methyl-D-aspartate type of glutamate receptor (NMDAR) and neurotrophin or ephrin receptors play essential roles in neuronal survival and differentiation. A shared downstream effector for neurotrophin- and ephrin-receptor signaling is kinase D-interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS). Because this molecule is obligatory for neurotrophin-induced differentiation, we investigated whether Kidins220/ARMS and NMDAR functions were related. Here, we identify an association between these proteins and discover that excitotoxicity, a specific form of neuronal death induced by NMDAR overstimulation, dramatically decreases Kidins220/ARMS levels in cortical neurons and in a model of cerebral ischemia. Kidins220/ARMS downregulation is triggered by overactivation of NMDARs containing NR2B subunits and subsequent Ca2+ influx, and involves a dual mechanism: rapid cleavage by the Ca2+-dependent protease calpain and calpain-independent silencing of Kidins220/Arms gene transcription. Additionally, Kidins220/ARMS knockdown decreases ERK activation and basal neuronal viability, and enhances neuronal death under excitotoxic conditions. Our results demonstrate Kidins220/ARMS participation in neuronal life and death pathways, and constitute the first report of its regulation under pathological conditions.

The N-methyl-D-aspartate receptor (NMDAR) is central to physiological and pathological functioning of neurons. Although promising results are beginning to be obtained in the treatment of dementias, clinical trials with NMDAR antagonists for stroke, trauma and neurodegenerative disorders, such as Hungtinton's disease, have failed before. In order to design effective therapies to prevent excitotoxic neuronal death, it is critical to characterize the consequences of excessive NMDAR activation on its expression and function. Previous data have reported partial downregulation of the NR1 and NR2B receptor subunits in response to excitotoxicity and cerebral ischemia. However, the effect of NMDAR overactivation on NR2A, a subunit fundamental to synaptic transmission and neuronal survival, is still elusive. In this study, we report the rapid and extensive proteolytic processing of NR2A, together with the scaffolding protein postsynaptic density-95 (PSD-95), induced by excitotoxic stimulation of cortical neurons in vitro and by transient focal cerebral ischemia. Processing of the C terminus of NR2A is irreversibly induced by brief agonist exposure of NR2B-containing receptors, and requires calcium influx and the activity of calpain, also responsible for PSD-95 cleavage. The outcome is a truncated NR2A subunit that is stable and capable to interact with NR1 at the surface of neurons, but lacking the structural domains required for association with scaffolding, downstream signaling and cytoskeletal proteins. Therefore, a rapid and significant uncoupling of synaptic NMDARs from downstream survival pathways is expected to occur during ischemia. This novel mechanism induced by excitotoxicity helps to explain the failure of most therapies based on NMDAR antagonists.

Peroxisome proliferator-activated receptors gamma (PPAR␥) are nuclear receptors with essential roles as transcriptional regulators of glucose and lipid homeostasis. PPAR␥ are also potent anti-inflammatory receptors, a property that contributes to the neuroprotective effects of PPAR␥ agonists in experimental stroke. The mechanism of these beneficial actions, however, is not fully elucidated. Therefore, we have explored further the actions of the PPAR␥ agonist rosiglitazone in experimental stroke induced by permanent middle cerebral artery occlusion (MCAO) in rodents. Rosiglitazone induced brain 5-lipoxygenase (5-LO) expression in ischemic rat brain, concomitantly with neuroprotection. Rosiglitazone also increased cerebral lipoxin A 4 (LXA 4 ) levels and inhibited MCAO-induced production of leukotriene B4 (LTB 4 ). Furthermore, pharmacological inhibition and/or genetic deletion of 5-LO inhibited rosiglitazone-induced neuroprotection and downregulation of inflammatory gene expression, LXA 4 synthesis and PPAR␥ transcriptional activity in rodents. Finally, LXA 4 caused neuroprotection, which was partly inhibited by the PPAR␥ antagonist T0070907, and increased PPAR␥ transcriptional activity in isolated nuclei, showing for the first time that LXA 4 has PPAR␥ agonistic actions. Altogether, our data illustrate that some effects of rosiglitazone are attributable to de novo synthesis of 5-LO, able to induce a switch from the synthesis of proinflammatory LTB 4 to the synthesis of the proresolving LXA 4 . Our study suggests novel lines of study suchastheinterestoflipoxin-likeanti-inflammatorydrugsortheuseofthesemoleculesasprognosticand/ordiagnosticmarkersforpathologies in which inflammation is involved, such as stroke.

A better understanding of the mechanisms underlying neuronal death in cerebral ischemia is required for the development of stroke therapies. Here we analyze the contribution of the tropomyosin-related kinase B (TrkB) neurotrophin receptor to excitotoxicity, a primary pathological mechanism in ischemia, which is induced by overstimulation of glutamate receptors of the N-methyl-D-aspartate type. We demonstrate a significant modification of TrkB expression that is strongly associated with neurodegeneration in models of ischemia and in vitro excitotoxicity. Two mechanisms cooperate for TrkB dysregulation: (1) calpain-processing of full-length TrkB (TrkB-FL), high-affinity receptor for brain-derived neurotrophic factor, which produces a truncated protein lacking the tyrosine-kinase domain and strikingly similar to the inactive TrkB-T1 isoform and (2) reverse regulation of the mRNA of these isoforms. Collectively, excitotoxicity results in a decrease of TrkB-FL, the production of truncated TrkB-FL and the upregulation of TrkB-T1. A similar neuro-specific increase of the TrkB-T1 isoform is also observed in stroke patients. A lentivirus designed for both neuro-specific TrkB-T1 interference and increased TrkB-FL expression allows recovery of the TrkB-FL/TrkB-T1 balance and protects neurons from excitotoxic death. These data implicate a combination of TrkB-FL downregulation and TrkB-T1 upregulation as significant causes of neuronal death in excitotoxicity, and reveal novel targets for the design of stroke therapies.

The N-methyl-D-aspartate (NMDA) type of glutamate receptor (NMDAR) plays central roles in normal and pathological neuronal functioning. We have examined the regulation of the NR1 subunit of the NMDAR in response to excessive activation of this receptor in in vitro and in vivo models of excitotoxicity. NR1 protein expression in cultured cortical neurons was specifically reduced by stimulation with 100 M NMDA or glutamate. NMDA decreased NR1 protein amounts by 71% after 8 h. Low NMDA concentrations (<10 M) had no effect. NR1 down-regulation was inhibited by the general NMDAR antagonist DL-AP5 and also by ifenprodil, which specifically antagonizes NMDARs containing NR2B subunits. Arrest of NMDAR signaling with DL-AP5 after brief exposure to NMDA did not prevent subsequent NR1 decrease. Down-regulation of NR1 did not involve calpain cleavage but resulted from a decrease in de novo synthesis consequence of reduced mRNA amounts. In contrast, NMDA did not alter the expression of NR2A mRNA or newly synthesized protein. In neurons transiently transfected with an NR1 promoter/luciferase reporter construct, promoter activity was reduced by 68% after 2 h of stimulation with NMDA, and its inhibition required extracellular calcium. A similar mechanism of autoregulation of the receptor probably operates during cerebral ischemia, because NR1 mRNA and protein were strongly decreased at early stages of blood reperfusion in the infarcted brains of rats subjected to occlusion of the middle cerebral artery. Because NR1 is the obligatory subunit of NMDARs, this regulatory mechanism will be fundamental to NMDAR functioning.5 type of glutamate receptor (NMDAR) plays key roles in neuronal plasticity, learning, and memory in the central nervous system, most of which are related to its high permeability to Ca 2ϩ (1). However, excessive activation of NMDARs induces excitotoxic cell death and contributes to neuronal degeneration in hypoxia, ischemia, and several neurodegenerative pathologies (2). Functional NMDARs are hetero-oligomeric proteins composed of an obligatory NR1 subunit (3-7) and NR2 subunits, denoted A-D (3,4,8,9). It is these NR2 subunits that confer functional variability to the receptor. In the post-synaptic membrane, NMDARs form large and dynamic signaling complexes by association with additional proteins (10), although there are also extrasynaptic NMDARs, which trigger different responses (11).NMDARs are subjected to multiple levels of regulation, affecting subunit expression, subcellular location, and the assembly of functional receptors and their signaling complexes (12-17). The NR1 gene is expressed in virtually all neurons, whereas NR2 transcripts display developmental and regional patterns (5, 18). The NR1 gene is transcriptionally up-regulated during neuronal differentiation, mostly by promoter de-repression (19), although positive mechanisms are also required. Post-transcriptional mechanisms also contribute to NR1 regulation in brain development, and two pools of mRNA, with different translational activities, have b...

Background and Purpose-Thrombolytic treatment with tissue plasminogen activator (tPA) improves outcome of patients with stroke who can be treated within 3 hours of symptom onset. However, delayed treatment with tPA leads to increased risk of hemorrhagic transformation and can result in enhanced brain injury. The purpose of this study is to validate a reproducible mouse model of hemorrhagic transformation associated with delayed administration of tPA. Methods-Mice were anesthetized and thrombin was injected into the middle cerebral artery to induce the formation of a clot as described by Orset et al. To induce reperfusion, tPA (10 mg/kg) was intravenously administered 20 minutes or 3 hours after thrombin injection. Results-Thrombin produced a clot in 83.1% of the animals, which caused focal ischemia determined 24 hours after the injection. Different degrees of bleeding were found in the middle cerebral artery occlusion group, including hemorrhagic infarction type 1 (HI-1) in 46.2%, hemorrhagic infarction type 2 (HI-2) in 30.8% and parenchymal hemorrhage type 1 in 23.0%. Administration of tPA 20 minutes after the occlusion produced an effective reperfusion in 62.5% of the animals and reduced both infarct volume and appearance of severe hemorrhage (10% nonhemorrhage, 80% HI-1 and 10% HI-2). However, administration of tPA 3 hours after the occlusion led to effective reperfusion in 47.1% of the animals, did not reduce infarct volume, caused hemorrhagic transformation (25% HI-1, 37.5% HI-2, and 37.5% parenchymal hemorrhage type 1), and increased hemorrhage and brain swelling.

PPARγ-achieved neuroprotection in experimental stroke has been explained by the inhibition of inflammatory genes, an action in which 5-LO, Alox5, is involved. In addition, PPARγ is known to promote the expression of CD36, a scavenger receptor that binds lipoproteins and mediates bacterial recognition and also phagocytosis. As phagocytic clearance of neutrophils is a requisite for resolution of the inflammatory response, PPARγ-induced CD36 expression might help to limit inflammatory tissue injury in stroke, an effect in which 5-LO might also be involved. Homogenates, sections, and cellular suspensions were prepared from brains of WT and Alox5(-/-) mice exposed to distal pMCAO. BMMs were obtained from Lys-M Cre(+) PPARγ(f/f) and Lys-M Cre(-) PPARγ(f/f) mice. Stereological counting of double-immunofluorescence-labeled brain sections and FACS analysis of cell suspensions was performed. In vivo and in vitro phagocytosis of neutrophils by microglia/macrophages was analyzed. PPARγ activation with RSG induced CD36 expression in resident microglia. This process was mediated by the 5-LO gene, which is induced in neurons by PPARγ activation and at least by one of its products--LXA4--which induced CD36 independently of PPARγ. Moreover, CD36 expression helped resolution of inflammation through phagocytosis, concomitantly to neuroprotection. Based on these findings, in addition to a direct modulation by PPARγ, we propose in brain a paracrine model by which products generated by neuronal 5-LO, such as LXA4, increase the microglial expression of CD36 and promote tissue repair in pathologies with an inflammatory component, such as stroke.