Intranasal Delivery of RGD Motif-Containing Osteopontin Icosamer Confers Neuroprotection in the Postischemic Brain via alpha(v)beta(3) Integrin Binding

Osteopontin (OPN) is a phosphorylated glycoprotein possessing an arginine-glycine-aspartate (RGD)-motif, which binds to several cell surface integrins and mediates a wide range of cellular processes. Inductions of OPN have been reported in the postischemic brain, and the neuroprotective effects of OPN have been demonstrated in animal models of stroke. In the present study, we showed a robust neuroprotective effect of RGD-containing icosamer OPN peptide (OPNpt20) in a rat model of focal cerebral ischemia (middle cerebral artery occlusion, MCAO). Intranasally administered OPNpt20 reduced mean infarct volume by 79.7 % compared to the treatment-na < ve MCAO control animals and markedly ameliorated neurological deficits. In addition, OPNpt20 significantly suppressed the inductions of iNOS and of inflammatory markers in postischemic brains and in primary microglial cultures, demonstrating anti-inflammatory effects. Administration of a mutant peptide, in which RGD was replaced by arginine-alanine-alanine (RAA), failed to suppress infarct volumes in MCAO animals and co-administration of OPNpt20 with anti-alpha(v)beta(3) integrin antibody failed to suppress iNOS induction in primary microglia culture, indicating that the RGD motif in OPNpt20 and endogenous alpha(v)beta(3) integrin play critical roles. Furthermore, pull-down assay revealed a direct binding between OPNpt20 and alpha(v)beta(3) integrin in primary microglia culture. Together, these results indicate that RGD-containing OPN icosamer has therapeutic potential in the postischemic brain and alpha(v)beta(3) integrin-mediated anti-inflammatory effect might be an underlying mechanism

Administration of the p38 MAPK inhibitor SB203580 affords brain protection with a wide therapeutic window against focal ischemic insult

We have reported previously the delayed and differential induction of p38α and p38β mitogen-activated protein kinases (MAPKs) in microglia and astrocytes, respectively, in brain after transient global ischemia. We report here the sustained induction and activation of p38α MAPK in activating microglia in rat brain after transient middle cerebral artery occlusion (MCAO). The intraventricular administration of SB203580, a p38 MAPK inhibitor, 30 min before MCAO reduced the infarct volume to 50% of the control, which was accompanied by the significant improvement of neurological deficits. More interestingly, the infarct volume was reduced to 72% and 77% when SB203580 was administered 6 hr and 12 hr after MCAO, respectively. The induction of various factors involved in inflammatory processes, such as inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and cyclooxygenase-2 (COX-2), was suppressed by the administration of SB203580 at 6 hr after MCAO. These results suggest that sustained activation of p38 MAPK pathway and p38 MAPK-associated inflammatory processes play a crucial role in postischemic brain. © 2003 Wiley-Liss, Inc

Zinc-triggered induction of tissue plasminogen activator by brain-derived neurotrophic factor and metalloproteinases

Tissue plasminogen activator (tPA) is necessary for hippocampal long-term potentiation. Synaptically released zinc also contributes to long-term potentiation, especially in the hippocampal CA3 region. Using cortical cultures, we examined whether zinc increased the concentration and/or activity of tPA. Two hours after a 10-min exposure to 300 μM zinc, expression of tPA and its substrate, plasminogen, were significantly increased, as was the proteolytic activity of tPA. In contrast, increasing extracellular or intracellular calcium levels did not affect the expression or secretion of tPA. Changing zinc influx or chelating intracellular zinc also failed to alter tPA/plasminogen induction by zinc, indicating that zinc acts extracellularly. Zinc-mediated extracellular activation of matrix metalloproteinase (MMP) underlies the up-regulation of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase (Trk) signaling. Consistent with these findings, co-treatment with a neutralizing antibody against BDNF or specific inhibitors of MMPs or Trk largely reversed tPA/plasminogen induction by zinc. Treatment of cortical cultures with p-aminophenylmercuric acetate, an MMP activator, MMP-2, or BDNF alone induced tPA/plasminogen expression. BDNF mRNA and protein expression was also increased by zinc and mediated by MMPs. Thus, an extracellular zinc-dependent, MMP- and BDNF-mediated synaptic mechanism may regulate the levels and activity of tPA. © 2011 The Authors. Journal of Neurochemistry

Dynamic expression of p38β MAPK in neurons and astrocytes after transient focal ischemia

Here we report the dynamically regulated expression of p38β MAPK isoform in specific subsets of cells in postischemic brain. The activity of p38β MAPK in the postischemic brain revealed biphasic induction at 30 min and 4 days after 1 h MCAO. During the early surge period, p38β MAPK was preferentially localized in the nucleus and dendrites of neurons in the future infarction area, while during the delayed surge p38β MAPK was heavily induced in reactive astrocytes in penumbra. The temporally and spatially regulated pattern of p38β MAPK expression in the postischemic brain suggests distinct roles of p38β MAPK in neuronal death and in the astrocyte activation. © 2003 Elsevier Science B.V. All rights reserved

The dorsal striatum expressing adenylyl cyclase-5 controls behavioral sensitivity of the righting reflex to high-dose ethanol

High-dose ethanol inflicts sedation and loss of righting reflex (LORR). Recently, it was reported that AC5 knockout (AC5-/-) mice consumed more ethanol and showed reduced sensitivity to high-dose ethanol compared to wild-type mice. As an extension of the previous study, in the present study we examined the signaling mechanism regulating altered behavioral sensitivity of LORR in AC5-/- mice. AC5-/- mice had enhanced phosphorylation of the NR2B subunit of NMDA receptors in the dorsal striatum and a partial reduction of MK801 (NMDA receptor antagonist)/ethanol-induced LORR. AC5-/- mice showed increased levels of phospho-CaMKIIα, phospho-CREB, and BDNF in the dorsal striatum. CaMKIIα+/- or BDNF+/- mice displayed enhanced LORR, a behavioral phenotype opposite to that displayed by AC5-/- mice. Consistently with these results, stereotaxic infusion of KN62 (CaMKII inhibitor), siRNA-CaMKIIα, or siRNA-BDNF, within the dorsal striatum was sufficient to prolong LORR. These results suggest that neural mechanism is important for regulating behavioral sensitivity of LORR and that the signaling pathway(s) interplayed by AC5, CaMKIIα and BDNF within the dorsal striatum is important for regulating the duration of ethanol-induced LORR. © 2012 Elsevier B.V

Impaired D2 dopamine receptor function in mice lacking type 5 adenylyl cyclase

Dopamine receptor subtypes D1 and D2, and many other seven-transmembrane receptors including adenosine receptor A2A, are colocalized in striatum of brain. These receptors stimulate or inhibit adenylyl cyclases (ACs) to produce distinct physiological and pharmacological responses and interact with each other synergistically or antagonistically at various levels. The identity of the AC isoform that is coupled to each of these receptors, however, remains unknown, To investigate the in vivo role of the type 5 adenylyl cyclase (AC5), which is preferentially expressed in striatum, mice deficient for the AC5 gene were generated. The genetic ablation of the AC5 gene eliminated <80% of forskolin-induced AC activity and 85-90% of AC activity stimulated by either D1 or A2A receptor agonists in striatum. However, D1- or A2A-specific pharmaco-behaviors were basically preserved, whereas the signal cascade from D2 to AC was completely abolished in AC5-/-, and motor activity of AC5-/- was not suppressed by treatment of cataleptic doses of the antipsychotic drugs haloperidol and sulpiride. Interestingly, both haloperidol and clozapine at low doses remarkably increased the locomotion of AC5-/- in the open field test that was produced in part by a common mechanism that involved the increased activation of D1 dopamine receptors. Together, these results suggest that AC5 is the principal AC integrating signals from multiple receptors including D1, D2, and A2A in striatum and the cascade involving AC5 among diverse D2 signaling pathways is essential for neuroleptic effects of antipsychotic drugs

Expression of the plant viral protease NIa in the brain of a mouse model of Alzheimer's disease mitigates Aβ pathology and improves cognitive function

The plant viral protease, NIa, has a strict substrate specificity for the consensus sequence of Val-Xaa-His-Gln, with a scissoring property after Gln. We recently reported that NIa efficiently cleaved the amyloid-β (Aβ) peptide, which contains the sequence Val-His-His-Gln in the vicinity of the cleavage site by α-secretase, and that the expression of NIa using a lentiviral system in the brain of AD mouse model reduced plaque deposition levels. In the present study, we investigated whether exogenous expression of NIa in the brain of AD mouse model is beneficial to the improvement of cognitive deficits. To address this question, Lenti-NIa was intracerebrally injected into the brain of Tg-APPswe/ PS1dE9 (Tg-APP/PS1) mice at 7 months of age and behavioral tests were performed 15-30 days afterwards. The results of the water maze test indicated that Tg-APP/PS1 mice which had been injected with Lenti-GFP showed an increased latency in finding the hidden-platform and markedly enhanced navigation near the maze-wall, and that such behavioral deficits were significantly reversed in Tg-APP/PS1 mice injected with Lenti-NIa. In the passive avoidance test, Tg-APP/PS1 mice exhibited a severe deficit in their contextual memory retention, which was reversed by NIa expression. In the marble burying test, Tg-APP/PS1 mice buried marbles fewer than non-transgenic mice, which was also significantly improved by NIa. After behavioral tests, it was verified that the Tg-APP/PS1 mice with Lenti-NIa injection had reduced Aβ levels and plaque deposition when compared to Tg-APP/PS1 mice. These results showed that the plant viral protease, NIa, not only reduces Aβ pathology, but also improves behavioral deficits. © 2012 by the Korean Society for Biochemistry and Molecular Biology

Ethyl pyruvate attenuates kainic acid-induced neuronal cell death in the mouse hippocampus

Recent studies have shown that ethyl pyruvate (EP) acts as an anti-inflammatory molecule in several cell lines including RAW264.7 macrophages. However, the potential therapeutic value of EP for the treatment of the pathologic brain has not been investigated fully. In the present study, we examined whether EP has a beneficial effect on KA-induced neuronal cell death. Intracerebroventricular (i.c.v.) injection of 0.94 nmol (0.2 μg) of KA produced typical neuronal cell death in the CA1 and CA3 pyramidal layers of the hippocampus, and the systemic administration of EP significantly attenuated KA-induced neuronal cell death in these regions. Ethyl pyruvate was found to exert a protective effect when it was injected as late as 12 hr after KA-injection. Moreover, this EP-induced neuroprotection was accompanied by reduced levels of reactive gliosis and COX-2, IL-1β, and TNF-α in the hippocampus. In addition, in passive avoidance tests, KA-induced memory impairment was improved markedly by EP. These results suggest that EP has a therapeutic potential for suppressing KA-induced pathogenesis in the brain. © 2006 Wiley-Liss, Inc

Optimization of chronic stress paradigms using anxiety- and depression-like behavioral parameters

Chronic stress is a risk factor for psychiatric illnesses, such as anxiety and depression disorders. To understand the underlying mechanism regarding how chronic stress triggers such psychiatric dysfunctions, restraint-based chronic stress models have been attempted in the past. However, total durations of repeated restraint stress and the evaluation time points used after the last restraint application vary from experiment to experiment. One reason for these methodological heterogeneities is related to considerable ambiguity concerning the definition of chronic stress, particularly in animal models. In the present study, we used behavioral traits, anxiety and depression, as stress-assessment parameters that meet operationally useful requirements for the definition of the chronic stress state. We demonstrate that restraint treatment for 2 or 8 hr daily for 14 days is enough to produce anxiety- and depression-like behaviors, whereas a 2 hr-10 days restraint was marginally effective. cDNA microarray analysis identified 34 genes in the hippocampus and 72 genes in the amygdala with expression levels that were up- or down-regulated by >2.0-fold. Among the wide range of genes identified in this analysis, genes required for energy metabolism, signal transduction, transcription, synaptic plasticity, and remodeling of the brain architecture were notable. Our results suggest that the psychiatric criteria of anxiety and depression can be used as chronic stress-assessment parameters and that a restraint stress paradigm consisting of restraint treatment for 2 or 8 hr daily for 14 days could be used as a prototype paradigm for chronic stress studies. © 2006 Wiley-Liss, Inc