Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostanoid synthesis, has been implicated in the neurotoxicity resulting from hypoxia-ischemia, and its inhibition has therapeutic potential for ischemic stroke. However, COX-2 inhibitors increase the risk of cardiovascular complications. We therefore sought to identify the downstream effectors of COX-2 neurotoxicity, and found that prostaglandin E(2) EP1 receptors are essential for the neurotoxicity mediated by COX-2-derived prostaglandin E(2). EP1 receptors disrupt Ca(2+) homeostasis by impairing Na(+)-Ca(2+) exchange, a key mechanism by which neurons cope with excess Ca(2+) accumulation after an excitotoxic insult. Thus, EP1 receptors contribute to neurotoxicity by augmenting the Ca(2+) dysregulation underlying excitotoxic neuronal death. Pharmacological inhibition or gene inactivation of EP1 receptors ameliorates brain injury induced by excitotoxicity, oxygen glucose deprivation and middle cerebral artery (MCA) occlusion. An EP1 receptor inhibitor reduces brain injury when administered 6 hours after MCA occlusion, suggesting that EP1 receptor inhibition may be a viable therapeutic option in ischemic stroke.

The class B scavenger receptor CD36 is involved in the cytotoxicity associated with inflammation, but its role in the inflammatory reaction that accompanies cerebral ischemia has not been examined. In this study, we investigated whether CD36 contributes to the brain damage produced by cerebral ischemia. The middle cerebral artery was transiently occluded in wild-type mice and in mice deficient in CD36. In wild-type mice, CD36 protein expression was increased in the ischemic brain, such that it was located predominantly in cells expressing the microglia/macrophage marker CD11b. The infarct produced by middle cerebral artery occlusion was 49% smaller in CD36-null mice than in wild-type controls, an effect associated with improved neurological function. The attenuation in brain injury in CD36 nulls could not be attributed to differences in cerebral blood flow during ischemia-reperfusion. However, the increase in reactive oxygen species (ROS) produced by cerebral ischemia was markedly attenuated in CD36-null mice in the early stage after reperfusion. The data unveil a previously unrecognized role of CD36 in ischemia-induced ROS production and brain injury. Modulation of CD36 signaling may provide a new strategy for the treatment of ischemic stroke.

Monocytes/macrophages (MMs), mononuclear phagocytes, have been implicated in stroke-induced inflammation and injury. However, the presence of pro-inflammatory Ly-6Chigh and antiinflammatory Ly-6Clow monocyte subsets raises uncertainty regarding their role in stroke pathologic assessment. With recent identification of the spleen as an immediate reservoir of MMs, this current study addresses whether the spleen-derived MMs are required for stroke pathologic assessment. We observed that the spleen was contracted in poststroke animals and the contraction was accompanied by decreased number of Ly-6Chigh and Ly-6Clow subsets in the spleen. The deployment of these subsets from the spleen temporally coincided with respective increases in the ischemic brain. Compared to mice with the spleen, mice receiving a splenectomy just before the stroke displayed less accumulation of Ly-6Chigh and Ly-6Clow MMs in the brain. Despite the reduced accumulation of both subsets, infarct size and swelling were not reduced in the asplenic mice. The dissociative findings of infarct size and extent of MM infiltration in the postischemic brain indicate minimal involvement of spleen-derived total MMs in acute infarct development. Selective Ly-6Chigh or Ly-6Clow MM targeting is suggested to address the contribution of the individual subset to acute stroke pathologic assessment.

The major pathological lesion of Parkinson's disease (PD) is the selective cell death of dopaminergic (DA) neurons in substantia nigra (SN). Although the initial cause and subsequent molecular signaling mechanisms leading to DA cell death underlying the PD process remain elusive, brain-derived neurotrophic factor (BDNF) is thought to exert neuroprotective as well as neurotrophic roles for the survival and differentiation of DA neurons in SN. Addressing molecular mechanisms of BDNF action in both primary embryonic mesencephalic cultures and in vivo animal models has been technically difficult because DA neurons in SN are relatively rare and present with many heterogeneous cell populations in midbrain. We have developed and characterized a DA neuronal cell line of embryonic SN origin that is more accessible to molecular analysis and can be used as an in vitro model system for studying SN DA neurons. A clonal SN DA neuronal progenitor cell line SN4741, arrested at an early DA developmental stage, was established from transgenic mouse embryos containing the targeted expression of the thermolabile SV40Tag in SN DA neurons. The phenotypic and morphological differentiation of the SN4741 cells could be manipulated by environmental cues in vitro. Exogenous BDNF treatment produced significant neuroprotection against 1-methyl-4-phenylpyridinium, glutamate, and nitric oxide-induced neurotoxicity in the SN4741 cells. Simultaneous phosphorylation of receptor tyrosine kinase B accompanied the neuroprotection. This SN DA neuronal cell line provides a unique model system to circumvent the limitations associated with primary mesencephalic cultures for the elucidation of molecular mechanisms of BDNF action on DA neurons of the SN. Key words: neuroprotection; BDNF; substantia nigra; dopaminergic neuron; Parkinson's disease; transgenic mice; neuronal differentiation; conditional immortalizationThe neuropathological symptoms of Parkinson's disease (PD) result from the greater than normal selective degeneration of substantia nigra (SN) dopaminergic (DA) neurons during aging. In PD the initial cause or causes and molecular mechanisms leading to the DA cell death are unknown. Treatment to prevent DA cell loss in PD is not available. Because of the paucity of DA neurons and the presence of numerous other cell populations in SN, it has not been possible to definitively address the molecular mechanisms of both DA neuronal survival and differentiation. Thus, it is crucial to acquire an abundant source of homogeneous DA neurons in vitro for the molecular dissection of SN DA neurons. To generate a consistent and abundant source of SN DA neurons in vitro, several approaches, using developmental, anatomical, somatic, or genetic manipulations, have been investigated. First, Hynes et al. (1995) tried to recapitulate the ontogeny of SN DA neuronal differentiation in explant cultures. Sonic hedgehog can induce effectively DA neurons in rat embryonic forebrain /midbrain explant. But, to practically manipulate the DA neuronal differentiation in vitro...

is sufficient for neuroprotection, it is not necessary for HDAC inhibitor neuroprotection, because these agents can completely protect neurons cultured from p21 waf1/cip1 -null mice. Together these findings demonstrate (1) that pulse inhibition of HDACs in cortical neurons can induce neuroprotection without apparent toxicity; (2) that p21 waf1/cip1 is sufficient but not necessary to mimic the protective effects of HDAC inhibition; and (3) that oxidative stress in this model induces neuronal cell death via cell cycle-independent pathways that can be inhibited by a cytosolic, noncanonical action of p21 waf1/cip1 .

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Increasing evidence indicates that cerebrovascular dysfunction plays a pathogenic role in Alzheimer's dementia (AD). Amyloid-β (Aβ), a peptide central to the pathogenesis of AD, has profound vascular effects mediated, for the most part, by reactive oxygen species produced by the enzyme NADPH oxidase. The mechanisms linking Aβ to NADPH oxidase-dependent vascular oxidative stress have not been identified, however. We report that the scavenger receptor CD36, a membrane glycoprotein that binds Aβ, is essential for the vascular oxidative stress and neurovascular dysfunction induced by Aβ 1-40 . Thus, topical application of Aβ 1-40 onto the somatosensory cortex attenuates the increase in cerebral blood flow elicited by neural activity or by endothelium-dependent vasodilators in WT mice but not in CD36-null mice (CD36 0/0 ). The cerebrovascular effects of infusion of Aβ 1-40 into cerebral arteries are not observed in mice pretreated with CD36 blocking antibodies or in CD36 0/0 mice. Furthermore, CD36 deficiency prevents the neurovascular dysfunction observed in transgenic mice overexpressing the Swedish mutation of the amyloid precursor protein Tg2576 despite elevated levels of brain Aβ 1-40 . CD36 is also required for the vascular oxidative stress induced by exogenous Aβ 1-40 or observed in Tg2576 mice. These observations establish CD36 as a key link between Aβ 1-40 and the NADPH oxidase-dependent vascular oxidative stress underlying the neurovascular dysfunction and suggest that CD36 is a potential therapeutical target to counteract the cerebrovascular dysfunction associated with Aβ.Alzheimer's disease | functional hyperemia | Nox2 | receptor for advanced glycation end-products T here is increasing evidence that vascular factors play a significant role in the pathogenesis of Alzheimer's dementia (AD) (1, 2). Whereas the structure of cerebral blood vessels is altered in AD, cerebral blood flow (CBF) is reduced even in the early stages of the disease (3). Epidemiological studies have shown that cerebrovascular diseases and AD share similar risk factors, suggesting a pathogenic commonality (4). Furthermore, small ischemic lesions amplify the cognitive dysfunction linked to amyloid plaques and neurofibrillary tangles, the neuropathological hallmarks of AD (5, 6). These observations have suggested an interaction between vascular insufficiency and the clinical expression of AD, raising the possibility that cerebrovascular dysfunction promotes the disease process underlying the dementia (1, 4, 7).Amyloid-β (Aβ), the amyloid precursor protein (APP)-derived peptide central to AD pathogenesis, has profound cerebrovascular effects that disrupt the brain's blood supply and render the brain more vulnerable to injury (3). The survival of the brain depends on continuous and well-regulated delivery of blood to ensure that its energy requirements are met and that deleterious metabolic byproducts are cleared from the neuronal microenvironment (8). Consequently, sophisticated vascular control mechanisms ensure that the brain receives a...

Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier-permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.