Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.

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Stroke leads to brain damage with subsequent slow and incomplete recovery of lost brain functions. Enriched housing of stroke-injured rats provides multi-modal sensorimotor stimulation, which improves recovery, although the specific mechanisms involved have not been identified. In rats housed in an enriched environment for two weeks after permanent middle cerebral artery occlusion, we found increased sigma-1 receptor expression in peri-infarct areas. Treatment of rats subjected to permanent or transient middle cerebral artery occlusion with 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride, an agonist of the sigma-1 receptor, starting two days after injury, enhanced the recovery of lost sensorimotor function without decreasing infarct size. The sigma-1 receptor was found in the galactocerebroside enriched membrane microdomains of reactive astrocytes and in neurons. Sigma-1 receptor activation increased the levels of the synaptic protein neurabin and neurexin in membrane rafts in the peri-infarct area, while sigma-1 receptor silencing prevented sigma-1 receptor-mediated neurite outgrowth in primary cortical neuronal cultures. In astrocytic cultures, oxygen and glucose deprivation induced sigma-1 receptor expression and actin dependent membrane raft formation, the latter blocked by sigma-1 receptor small interfering RNA silencing and pharmacological inhibition. We conclude that sigma-1 receptor activation stimulates recovery after stroke by enhancing cellular transport of biomolecules required for brain repair, thereby stimulating brain plasticity. Pharmacological targeting of the sigma-1 receptor provides new opportunities for stroke treatment beyond the therapeutic window of neuroprotection.

expressed on the leading edge of membrane filopodia and colocalizes with a-actinin. Purified recombinant GPR56 extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morphology and cell rounding. These results indicate that the extracellular domain may compete for unidentified ligand(s), and block the normal function of GPR56 in cell attachment. In reporter assays, overexpression of GPR56 activates the NF-jB, PAI-1 and TCF transcriptional response elements. These pathways have been implicated in cytoskeletal signaling, adhesion and tumor biology. The above results indicate that GPR56 serves as an adhesion GPCR and is involved in adhesion signaling.

Death-associated protein kinase (DAPK) is a calcium calmodulin-regulated serine/threonine protein kinase involved in ischemic neuronal death. In situ hybridization experiments show that DAPK mRNA expression is up-regulated in brain following a global ischemic insult and down-regulated in ischemic tissues after focal ischemia. DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo. A similar dephosphorylation pattern is detected in primary cortical neurons subjected to oxygen glucose deprivation or N-methyl-D-aspartate (NMDA)-induced toxicity. Both a calcineurin inhibitor, FK506, and a selective NMDA receptor antagonist, MK-801, inhibit the dephosphorylation of DAPK after in vitro ischemia. This indicates that DAPK could be activated by NMDA receptor-mediated calcium flux, activation of calcineurin, and subsequent DAPK dephosphorylation. Moreover, concomitantly to dephosphorylation, DAPK is proteolytically processed by cathepsin after ischemia. Furthermore, a selective DAPK inhibitor is neuroprotective in both in vitro and in vivo ischemic models. These results indicate that DAPK plays a key role in mediating ischemic neuronal injury.Ischemic stroke is characterized by apoptotic and necrotic cell death leading to neuronal loss (1). To date, there is no effective neuroprotective drug in clinical use for the treatment of acute ischemic neuronal damage (2). Several biological processes have been proposed to contribute to ischemic cell death, including excitotoxicity, ionic imbalance, oxidative stress, and apoptosis (3). These pathological events in the brain trigger aberrant cell signaling (4) and subsequent gene expression (5), which have been studied to understand the pathophysiology of stroke. We have identified DAPK 2 as a protein regulated by ischemic conditions both in vitro and in vivo.DAPK is a Ca 2ϩ /calmodulin-regulated serine/threonine kinase that acts as a positive mediator of apoptotic pathways, including those involved in neuronal cell death. It is involved in Fas-, INF-␥-, tumor necrosis factor-(6), ceramide- (7), and p53-mediated apoptosis (8), as well as in the disruption of matrix survival signals and suppression of integrin-mediated cell adhesion (9). DAPK appears to function early in the apoptotic pathway prior to the commitment of the cells to apoptosis. DAPK has also been extensively implicated as a tumor suppressor whose inactivation predicts malignancy, as DAPK expression is frequently lost in tumors due to hypermethylation of the DAPK gene (10, 11).DAPK has a unique multidomain structure containing a kinase domain, a calmodulin regulatory segment, eight ankyrin repeats, a cytoskeleton binding region, and a death domain (12). DAPK is negatively regulated by autophosphorylation on serine 308 in the Ca 2ϩ /CaM regulatory domain. This autoregulation of DAPK serves as an inhibitory mechanism in the basal state (13). Dephosphorylation relieves autoinhibition and stim...

Regulation of N-methyl-D-aspartate (NMDA) receptors is critical for the normal functioning of the central nervous system. There must be precise mechanisms to allow for changes in receptor function required for learning and normal synaptic transmission, but within tight constraints to prevent pathology. Tyrosine phosphorylation is a major means by which NMDA receptors are regulated through the equilibrium between activity of Src family kinases and tyrosine phosphatases. Identification of NMDA receptor phosphatases has been difficult, the best candidate being striatal-enriched tyrosine phosphatase (STEP). Here we demonstrate that STEP is a critical regulator of NMDA receptors and reveal that the action of this tyrosine phosphatase controls the constitutive trafficking of NMDA receptors and leads to changes in NMDA receptor activity at the neuronal surface. We show that STEP binds directly to NMDA receptors in the absence of other synaptic proteins. The activity of STEP selectively affects the expression of NMDA receptors at the neuronal plasma membrane. The result of STEP's action upon the NMDA receptor affects the functional properties of the receptor and its downstream signaling. These effects are evident when STEP levels are chronically reduced, indicating that there is no redundancy amongst phosphatases to compensate for altered STEP function in the CNS. STEP may have evolved specifically to fill a pivotal role as the NMDA receptor phosphatase, having a distinct and restricted localization and compartmentalization, and unique activity towards the NMDA receptor and its signaling pathway.

Alzheimer's disease is a progressive neurodegenerative disease characterized by senile plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. Activated microglia are found to be intimately associated with senile plaques and may play a central role in mediating chronic inflammatory conditions in Alzheimer's disease. Activation of cultured murine microglial BV2 cells by freshly sonicated A␤42 results in the secretion of neurotoxic factors that kill primary cultured neurons. To understand molecular pathways underlying A␤-induced microglial activation, we analyzed the expression levels of transcripts isolated from A␤42-activated BV2 cells using high density filter arrays. The analysis of these arrays identified 554 genes that are transcriptionally up-regulated by A␤42 in a statistically significant manner. Quantitative reverse transcription-PCR was used to confirm the regulation of a subset of genes, including cysteine proteases cathepsin B and cathepsin L, tissue inhibitor of matrix metalloproteinase 2, cytochrome c oxidase, and allograft inflammatory factor 1. Small interfering RNA-mediated silencing of the cathepsin B gene in A␤-activated BV2 cells diminished the microglial activation-mediated neurotoxicity. Moreover, CA-074, a specific cathepsin B inhibitor, also abolished the neurotoxic effects caused by A␤42-activated BV2 cells. Our results suggest an essential role for secreted cathepsin B in neuronal death mediated by A␤-activated inflammatory response.

Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix (ECM), disruption of ECM components, and subsequent cell penetration into adjacent brain structures. Discoidin domain receptor 1 (DDR1) tyrosine kinases constitute a novel family of receptors characterized by a unique structure in the ectodomain (discoidin-I domain). These cell surface receptors bind to several collagens and facilitate cell adhesion. Little is known about DDR1 expression and function in glioblastoma multiforme. In this study we demonstrate that DDR1 is overexpressed in glioma tissues using cDNA arrays, immunohistochemistry and Western blot analysis. Functional comparison of two splice variants of DDR1 (DDR1a and DDR1b) reveal novel differences in cell based glioma models. Overexpression of either DDR1a or DDR1b caused increased cell attachment. However, glioma cells overexpressing DDR1a display enhanced invasion and migration. We also detect increased levels of matrix metalloproteinase-2 in DDR1a overexpressing cells as measured by zymography. Inhibition of MMP activity using MMP inhibitor suppressed DDR1a stimulated cell-invasion. Similarly, an antibody against DDR1 reduced DDR1a mediated invasion as well as the enhanced adhesion of DDR1a and DDR1b overexpressing cells. These results suggest that DDR1a plays a critical role in inducing tumor cell adhesion and invasion, and this invasive phenotype is caused by activation of matrix metalloproteinase-2.