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Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2+]i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca2+]i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (KCa2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of KCa2.2 channels within 3 h after the onset of glutamate exposure. Activation of KCa2 channels preserved KCa2 expression and significantly reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for KCa2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders.

Neuroprotective effects of the lipophilic beta(2)-adrenoceptor agonist clenbuterol have been established in neuronal cultures and in various rodent models of stroke. In previous studies, however, clenbuterol was always applied as a racemate, while it has not been established whether the enantiomers differ in their neuroprotective activities. Here, we demonstrate that R,S-clenbuterol and S(+)-clenbuterol, but not the R(-)-enantiomer protect cultured neurons against glutamate-mediated excitotoxicity and staurosporine-induced apoptosis. Similar to previous findings with clenbuterol racemate, the neuroprotective effect of S(+)-clenbuterol correlated well with morphological changes of astrocytes which transformed into dense stellate cells with dendritic processes indicating beta(2)-adrenoceptor-mediated activation. Most importantly, the S(+)-enantiomer but not R(-)-clenbuterol reduced ischemic brain damage similar to the effect of the racemate. The selective beta(2)-adrenoceptor antagonist butoxamine blocked this neuroprotective effect of S(+)-clenbuterol. In addition, S(+)-clenbuterol significantly reduced blood pressure, enhanced blood glucose levels and increased glucocorticoid levels compared to vehicle-or R(-)-clenbuterol-treated controls. These results clearly demonstrate that S(+)-clenbuterol is the eutomer that mediates neuroprotective effects of the beta(2)-adrenoceptor agonist but also according changes of physiological parameters.

Delayed neuronal cell death occurring hours after reperfusion is a hallmark of ischemic stroke and a primary target for neuroprotective strategies. In the present study, we investigated whether apoptosis-inducing factor (AIF), a caspase-independent proapoptotic protein, is responsible for neuronal cell death after glutamate toxicity and oxygen-glucose deprivation (OGD) in vitro and after experimental stroke in vivo. AIF translocated to the nucleus in which it colocalized with DNA fragmentation and nuclear apoptotic morphology after exposure to glutamate or OGD in cultured neurons or after transient middle cerebral artery occlusion (MCAo) in mice. Small inhibitory RNA-mediated downregulation of AIF reduced glutamate- and OGD-induced neuronal apoptosis by 37 and 60%, respectively (p < 0.01). Moreover, Harlequin mutant mice, which express AIF at low levels (approximately 20% of wild-type mice), displayed smaller infarct volumes (-43%; p < 0.03) and showed dramatically reduced cell death in the ischemic penumbra after 45 min of MCAo compared with wild-type littermates. Inhibition of poly(ADP-ribose) polymerase and Bid reduced nuclear AIF translocation. These results provide the first evidence for a causal role of AIF in ischemic neuronal cell death. Therefore, caspase-independent cell death signaling may provide a promising novel target for therapeutic interventions in cerebrovascular diseases.

Background and Purpose: Although excitotoxic overactivation of glutamate receptors has been identified as a major mechanism of ischemic brain damage, glutamate receptor antagonists failed in stroke trials, in most cases because of limited therapeutic windows or severe adverse effects. Therefore, we chose memantine and clenbuterol, both approved safe and efficient in their respective therapeutical categories, and examined combinations of these neuroprotectants for possible therapeutic interactions in ischemic stroke., Methods: Combinations of the N-methyl-D-aspartate (NMDA) receptor antagonist memantine (20 mg/kg) with the beta2-adrenoceptor agonist clenbuterol (0.3 to 3 mg/kg) were tested in a mouse model of permanent focal cerebral ischemia. In addition, combinations of memantine (1 to 10 nmol/L) and clenbuterol (1 to 10 nmol/L) were examined in cultured hippocampal neurons exposed to glutamate (500 micromol/L) or staurosporine (200 nmol/L)., Results: The infarct size was further reduced by combination therapy as compared with effects of the respective neuroprotectants alone. Of note, in combination with memantine, the therapeutic window of clenbuterol was significantly prolonged up to 2 hours after ischemia. Experiments in postnatal cultures of rat hippocampal neurons exposed to glutamate or staurosporine confirmed that neuroprotection by combinations of memantine and clenbuterol exceeded the effects of the individual compounds., Conclusions: Combinations of memantine with clenbuterol extend the respective therapeutic window and provide synergistic cerebroprotective effects after stroke.

The chemoattractant stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) are key modulators of immune function. In the developing brain, SDF-1 is crucial for neuronal guidance; however, cerebral functions of SDF-1/CXCR4 in adulthood are unclear. Here, we examine the cellular expression of SDF-1 isoforms and CXCR4 in the brain of mice receiving systemic lipopolysaccharide (LPS) or permanent focal cerebral ischemia. CXCR4 mRNA was constitutively expressed in cortical and hippocampal neurons and ependymal cells. Hippocampal neurons targeted the CXCR4 receptor to their somatodendritic and axonal compartments. In cortex and hippocampus, CXCR4-expressing neurons exhibited an overlapping distribution with neurons expressing SDF-1 transcripts. Although neurons synthesized SDF-1alpha mRNA, the SDF-1beta isoform was selectively expressed by endothelial cells of cerebral microvessels. LPS stimulation dramatically decreased endothelial SDF-1beta mRNA expression throughout the forebrain but did not affect neuronal SDF-1alpha. After focal cerebral ischemia, SDF-1beta expression was selectively increased in endothelial cells of penumbral blood vessels and decreased in endothelial cells of nonlesioned brain areas. In the penumbra, SDF-1beta upregulation was associated with a concomitant infiltration of CXCR4-expressing peripheral blood cells, including macrophages. Neuronal SDF-1alpha was transiently downregulated and neuronal CXCR4 was transiently upregulated in the nonlesioned cerebral cortex in response to ischemia. Although endothelial SDF-1beta may control cerebral infiltration of CXCR4-carrying leukocytes during cerebral ischemia, the neuronal SDF-1alpha/CXCR4 system may contribute to ischemia-induced neuronal plasticity. Thus, the isoform-specific regulation of SDF-1 expression modulates neurotransmission and cerebral infiltration via distinct CXCR4-dependent pathways.

The tumor suppressor protein p53 is essential for neuronal death in several experimental settings and may participate in human neurodegenerative disorders. Based upon recent studies characterizing chemical inhibitors of p53 in preclinical studies in the cancer therapy field, we synthesized the compound pifithrin-alpha and evaluated its potential neuroprotective properties in experimental models relevant to the pathogenesis of stroke and neurodegenerative disorders. Pifithrin-alpha protected neurons against apoptosis induced by DNA-damaging agents, amyloid beta-peptide and glutamate. Protection by pifithrin-alpha was correlated with decreased p53 DNA-binding activity, decreased expression of the p53 target gene BAX and suppression of mitochondrial dysfunction and caspase activation. Mice given pifithrin-alpha exhibited increased resistance of cortical and striatal neurons to focal ischemic injury and of hippocampal neurons to excitotoxic damage. These preclinical studies demonstrate the efficacy of a p53 inhibitor in models of stroke and neurodegenerative disorders, and suggest that drugs that inhibit p53 may reduce the extent of brain damage in related human neurodegenerative conditions.

To test the hypothesis of an involvement of tachykinins in destabilization and hyperexcitation of neuronal circuits, gliosis, and neuroinflammation during cerebral ischemia, we investigated cell-specific expressional changes of the genes encoding substance P (SP), neurokinin B (NKB), and the tachykinin/neurokinin receptors (NK1, NK2, and NK3) after middle cerebral artery occlusion (MCAO) in the rat. Our analysis by quantitative in situ hybridization, immunohistochemistry, and confocal microscopy was concentrated on cerebrocortical areas that survive primary infarction but undergo secondary damage. Here, SP-encoding preprotachykinin-A and NK1 mRNA levels and SP-like immunoreactivity were transiently increased in GABAergic interneurons at 2 d after MCAO. Coincidently, MCAO caused a marked expression of SP and NK1 in a subpopulation of glutamatergic pyramidal cells, and in some neurons SP and NK1 mRNAs were coinduced. Elevated levels of the NKB-encoding preprotachykinin-B mRNA and of NKB-like immunoreactivity at 2 and 7 d after MCAO were confined to GABAergic interneurons. In parallel, the expression of NK3 was markedly downregulated in pyramidal neurons. MCAO caused transient NK1 expression in activated cerebrovenular endothelium within and adjacent to the infarct. NK1 expression was absent from activated astroglia or microglia. The differential ischemia-induced plasticity of the tachykinin system in distinct inhibitory and excitatory cerebrocortical circuits suggests that it may be involved in the balance of endogenous neuroprotection and neurotoxicity by enhancing GABAergic inhibitory circuits or by facilitating glutamate-mediated hyperexcitability. The transient induction of NK1 in cerebrovenular endothelium may contribute to ischemia-induced edema and leukocyte diapedesis. Brain tachykinin receptors are proposed as potential drug targets in stroke.

Degeneration and death of neurons is the fundamental process responsible for the clinical manifestations of many different neurological disorders of aging, incuding Alzheimer's disease, Parkinson's disease and stroke. The death of neurons in such disorders involves apoptotic biochemical cascades involving upstream effectors (Par-4, p53 and pro-apoptotic Bcl-2 family members), mitochondrial alterations and caspase activation. Both genetic and environmental factors, and the aging process itself, contribute to intiation of such neuronal apoptosis. For example, mutations in the amyloid precursor protein and presenilin genes can cause Alzheimer's disease, while head injury is a risk factor for both Alzheimer's and Parkinson's diseases. At the cellular level, neuronal apoptosis in neurodegenerative disorders may be triggered by oxidative stress, metabolic compromise and disruption of calcium homeostasis. Neuroprotective (antiapoptotic) signaling pathways involving neurotrophic factors, cytokines and "conditioning responses" can counteract the effects of aging and genetic predisposition in experimental models of neurodegenerative disorders. A better understanding of the molecular underpinnings of neuronal death is leading directly to novel preventative and therapeutic approaches to neurodegenerative disorders.

Estrogens have been suggested for the treatment of neurodegenerative disorders, including stroke, because of their neuroprotective activities against various neurotoxic stimuli such as glutamate, glucose deprivation, iron, or beta-amyloid. Here, the authors report that 17beta-estradiol (0.3 to 30 mg/kg) and 2-OH-estradiol (0.003 to 30 mg/kg) reduced brain tissue damage after permanent occlusion of the middle cerebral artery in male NMRI mice. In vitro, 17beta-estradiol (1 to 10 micromol/L) and 2-OH-estradiol (0.01 to 1 micromol/L) reduced the percentage of damaged chick embryonic neurons treated with FeSO4. In these primary neurons exposed to FeSO4, the authors also found reactive oxygen species to be diminished after treatment with 17beta-estradiol (1 to 10 micromol/L) or 2-OH-estradiol (0.01 to 10 micromol/L), suggesting a strong antioxidant activity of the estrogens that were used. Neither the neuroprotective effect nor the free radical scavenging properties of the estrogens were influenced by the estrogen receptor antagonist tamoxifen. The authors conclude that estrogens protect neurons against damage by radical scavenging rather than through estrogen receptor activation.

Angiotensin-converting enzyme inhibitors have been demonstrated to protect spontaneously hypertensive rats from cerebral ischemia. The present study investigated the protective effect of enalapril and moexipril in models of permanent focal cerebral ischemia in normotensive mice and rats. To elucidate the mechanism of neuroprotection the influence of these angiotensin-converting enzyme inhibitors on glutamate-, staurosporine- or Fe2+/3+-induced generation of reactive oxygen species and neuronal cell death in primary cultures from chick embryo telencephalons was studied. Treatment with moexipril or enalapril dose-dependently reduced the percentage of damaged neurons, as well as mitochondrial reactive oxygen species generation induced by glutamate, staurosporine or Fe2+/3+. Furthermore, moexipril and enalapril attenuated staurosporine-induced neuronal apoptosis as determined by nuclear staining with Hoechst 33258. In mice, 1 h pretreatment with enalapril (0.03 mg/kg) or moexipril (0.3 mg/kg) significantly reduced brain damage after focal ischemia as compared to control animals. Additionally, moexipril (0.01 mg/kg) was able to reduce the infarct volume in the rat model after focal cerebral ischemia. The results of the present study indicate that the angiotensin-converting enzyme inhibitors enalapril and moexipril promote neuronal survival due to radical scavenging properties.

Previously reported effects of lubeluzole, such as inhibition of glutamate release, inhibition of nitric oxide (NO) synthesis and blockage of voltage-gated Na+- and Ca2+-ion channels, suggest a neuroprotective action of this drug. Here we report about the effects of lubeluzole and its R-isomer on glutamate-induced neuronal cell death in mixed hippocampal cultures. In addition, we studied the effect of lubeluzole in focal cerebral ischemia models in mice and rats. In hippocampal cultures exposed to 500 nM glutamate for 1 h, lubeluzole (0.1-100 nM), but not the R-isomer (1-100 nM), reduced the percentage of damaged neurons from 42 +/- 8% to 18 +/- 7% (P < 0.01). In mice and rats, lubeluzole reduced ischemic brain damage, when administered immediately after middle cerebral artery occlusion. Interestingly, the protective effect (reduction of the infarct volume in rats to 77% of control; P < 0.01) was also found when the lubeluzole treatment (2.5 mg/kg) was started 3 h after ischemia. Especially this latter effect suggests that lubeluzole will be a useful drug for stroke therapy.