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Mutations in the parkin gene are a major cause of autosomal recessive Parkinson's disease. Here we show that the E3 ubiquitin ligase parkin activates signaling through the IkappaB kinase (IKK)/nuclear factor kappaB (NF-kappaB) pathway. Our analysis revealed that activation of this signaling cascade is causally linked to the neuroprotective potential of parkin. Inhibition of NF-kappaB activation by an IkappaB super-repressor or a kinase-inactive IKKbeta interferes with the neuroprotective activity of parkin. Furthermore, pathogenic parkin mutants with an impaired neuroprotective capacity show a reduced ability to stimulate NF-kappaB-dependent transcription. Finally, we present evidence that parkin interacts with and promotes degradation-independent ubiquitylation of IKKgamma/NEMO (NF-kappaB essential modifier) and TRAF2 [TNF (tumor necrosis factor) receptor-associated factor 2], two critical components of the NF-kappaB pathway. Thus, our results support a direct link between the neuroprotective activity of parkin and ubiquitin signaling in the IKK/NF-kappaB pathway.

The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 precedes apoptosis in many cell types. Controversial reports exist on the role of the transcription factor nuclear factor-kappaB (NF-kappaB) in p53-mediated apoptosis, depending on the cell type and experimental conditions. Therefore, we sought to elucidate the role of NF-kappaB in p53-mediated neuron death. In cultured neurons DNA damaging compounds induced activation of p53, whereas NF-kappaB activity declined significantly. The p53 inhibitor pifithrin-alpha (PFT) preserved NF-kappaB activity and protected neurons against apoptosis. Immunoprecipitation experiments revealed enhanced p53 binding to the transcriptional cofactor p300 after induction of DNA damage, whereas binding of p300 to NF-kappaB was reduced. In contrast, PFT blocked the interaction of p53 with the cofactor, whereas NF-kappaB binding to p300 was enhanced. Most interestingly, similar results were observed after oxygen glucose deprivation in cultured neurons and in ischemic brain tissue. Ischemia-induced repression of NF-kappaB activity was prevented and brain damage was reduced by the p53 inhibitor PFT in a dose-dependent manner. It is concluded that a balanced competitive interaction of p53 and NF-kappaB with the transcriptional cofactor p300 exists in neurons. Exposure of neurons to lethal stress activates p53 and disrupts NF-kappaB binding to p300, thereby blocking NF-kappaB-mediated survival signaling. Inhibitors of p53 provide pronounced neuroprotective effects because they block p53-mediated induction of cell death and concomitantly enhance NF-kappaB-induced survival signaling.

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.

Despite the characterization of neuroprotection by transforming growth factor-beta1 (TGF-beta1), the signaling pathway mediating its protective effect is unclear. Bad is a proapoptotic member of the Bcl-2 family and is inactivated on phosphorylation via mitogen-activated protein kinase (MAPK). This study attempted to address whether MAPK signaling and Bad phosphorylation were influenced by TGF-beta1 and, furthermore, whether these two events were involved in the antiapoptotic effect of TGF-beta1. We found a gradual activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and MAPK-activated protein kinase-1 (also called Rsk1) and a concomitant increase in Bad phosphorylation at Ser(112) in mouse brains after adenovirus-mediated TGF-beta1 transduction under nonischemic and ischemic conditions induced by transient middle cerebral artery occlusion. Consistent with these effects, the ischemia-induced increase in Bad protein level and caspase-3 activation were suppressed in TGF-beta1-transduced brain. Consequently, DNA fragmentation, ischemic lesions, and neurological deficiency were significantly reduced. In cultured rat hippocampal cells, TGF-beta1 inhibited the increase in Bad expression caused by staurosporine. TGF-beta1 concentration- and time-dependently activated Erk1/2 and Rsk1 accompanied by an increase in Bad phosphorylation. These effects were blocked by U0126, a mitogen-activated protein kinase/Erk kinase 1/2 inhibitor, suggesting an association between Bad phosphorylation and MAPK activation. Notably, U0126 and a Rsk1 inhibitor (Ro318220) abolished the neuroprotective activity of TGF-beta1 in staurosporine-induced apoptosis, indicating that activation of MAPK is necessary for the antiapoptotic effect of TGF-beta1 in cultured hippocampal cells. Together, we demonstrate that TGF-beta1 suppresses Bad expression under lesion conditions, increases Bad phosphorylation, and activates the MAPK/Erk pathway, which may contribute to its neuroprotective activity.

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.

Elevated plasma levels of the sulfur-containing amino acid homocysteine increase the risk for atherosclerosis, stroke, and possibly Alzheimer's disease, but the underlying mechanisms are unknown. We now report that homocysteine induces apoptosis in rat hippocampal neurons. DNA strand breaks and associated activation of poly-ADP-ribose polymerase (PARP) and NAD depletion occur rapidly after exposure to homocysteine and precede mitochondrial dysfunction, oxidative stress, and caspase activation. The PARP inhibitor 3-aminobenzamide (3AB) protects neurons against homocysteine-induced NAD depletion, loss of mitochondrial transmembrane potential, and cell death, demonstrating a requirement for PARP activation and/or NAD depletion in homocysteine-induced apoptosis. Caspase inhibition accelerates the loss of mitochondrial potential and shifts the mode of cell death to necrosis; inhibition of PARP with 3AB attenuates this effect of caspase inhibition. Homocysteine markedly increases the vulnerability of hippocampal neurons to excitotoxic and oxidative injury in cell culture and in vivo, suggesting a mechanism by which homocysteine may contribute to the pathogenesis of neurodegenerative disorders.

A short ischemic period induced by the transient occlusion of major brain arteries induces neuronal damage in selectively vulnerable regions of the hippocampus. Adenosine is considered to be one of the major neuroprotective substances produced in the ischemic brain. It can be released from damaged cells, but it also could be generated extracellularly from released ATP via a surface-located enzyme chain. Using the rat model of global forebrain ischemia, we applied a short (10 min) transient interruption of blood flow and studied the distribution of ectonucleotidase activities in the hippocampus. Northern hybridization of mRNA isolated from hippocampi of sham-operated and ischemic animals revealed an upregulation of ectoapyrase (capable of hydrolyzing nucleoside 5'-tri- and diphosphates) and ecto-5'-nucleotidase (capable of hydrolyzing nucleoside 5'-monophosphates). A histochemical analysis that used ATP, UTP, ADP, or AMP as substrates revealed a strong and selective increase in enzyme activity in the injured areas of the hippocampus. Enhanced staining could be observed first at 2 d. Staining increased within the next days and persisted at 28 d after ischemia. The spatiotemporal development of catalytic activities was identical for all substrates. It was most pronounced in the CA1 subfield and also could be detected in the dentate hilus and to a marginal extent in CA3. The histochemical staining corresponded closely to the development of markers for reactive glia, in particular of microglia. The upregulation of ectonucleotidase activities implies increased nucleotide release from the damaged tissue and could play a role in the postischemic control of nucleotide-mediated cellular responses.