During their lifespan, immature cells normally pass through sequential transitions to a differentiated state and eventually undergo cell death. This progression is aberrant in cancer, although the transition to differentiation can be reestablished in inducible leukemia cell lines. This report describes a gene, MCLl, that we isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Our results demonstrate that expression ofMCLI increases early in the induction, or "programming," of differentiation in ML-1 (at 1-3 hr), before the appearance of differentiation markers and mature morphology (at 1-3 days). They further show that MCLI has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(l4;18) chromosome translocation. MCLI and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the association of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-associated genes, expression of MCLI and BCL2 relates to the programming of differentiation and cell viability/death. The discovery of MCLI broadens our perspective on an emerging MCLI/BCL2 gene family and will allow further comparison with oncogene families.

Progranulin (PGRN) is a widely expressed protein involved in diverse biological processes. Haploinsufficiency of PGRN in the human causes tau-negative, ubiquitin-positive frontotemporal dementia (FTD). However, the mechanisms are unknown. To explore the role of PGRN in vivo, we generated PGRN-deficient mice. Macrophages from these mice released less interleukin-10 and more inflammatory cytokines than wild type (WT) when exposed to bacterial lipopolysaccharide. PGRN-deficient mice failed to clear Listeria monocytogenes infection as quickly as WT and allowed bacteria to proliferate in the brain, with correspondingly greater inflammation than in WT. PGRN-deficient macrophages and microglia were cytotoxic to hippocampal cells in vitro, and PGRN-deficient hippocampal slices were hypersusceptible to deprivation of oxygen and glucose. With age, brains of PGRN-deficient mice displayed greater activation of microglia and astrocytes than WT, and their hippocampal and thalamic neurons accumulated cytosolic phosphorylated transactivation response element DNA binding protein–43. Thus, PGRN is a key regulator of inflammation and plays critical roles in both host defense and neuronal integrity. FTD associated with PGRN insufficiency may result from many years of reduced neutrotrophic support together with cumulative damage in association with dysregulated inflammation.

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.

Alterations in cerebrovascular regulation related to vascular oxidative stress have been implicated in the mechanisms of Alzheimer's disease (AD), but their role in the amyloid deposition and cognitive impairment associated with AD remains unclear. We used mice overexpressing the Swedish mutation of the amyloid precursor protein (Tg2576) as a model of AD to examine the role of reactive oxygen species produced by NADPH oxidase in the cerebrovascular alterations, amyloid deposition, and behavioral deficits observed in these mice. We found that 12-to 15-month-old Tg2576 mice lacking the catalytic subunit Nox2 of NADPH oxidase do not develop oxidative stress, cerebrovascular dysfunction, or behavioral deficits. These improvements occurred without reductions in brain amyloid-␤ peptide (A␤) levels or amyloid plaques. The findings unveil a previously unrecognized role of Nox2-derived radicals in the behavioral deficits of Tg2576 mice and provide a link between the neurovascular dysfunction and cognitive decline associated with amyloid pathology.Alzheimer's disease ͉ cerebral blood flow ͉ tg2576 T he amyloid-␤ peptide (A␤) is central to the pathogenesis of Alzheimer's disease (AD), the most common form of dementia in the elderly (1). A␤ peptides are cleaved from the amyloid precursor protein (APP) by two aspartyl proteases, termed ␤-and ␥-secretases, and form deposits in the brain parenchyma (amyloid plaques) and around blood vessels (amyloid angiopathy) (2). The mechanisms by which A␤ leads to cognitive impairment have not been completely elucidated, although recent evidence suggests that small aggregates of A␤ may be key pathogenic factors by disrupting synaptic function and inducing neuronal death (2).However, A␤ also exerts powerful effects on cerebral blood vessels (3). In vitro and in vivo studies have demonstrated that A␤ enhances vasoconstriction, impairs responses to vasodilators, and reduces cerebral blood flow (CBF) (4, 5). In addition, transgenic mice overexpressing APP and A␤ have major alterations in resting CBF and in key cerebrovascular control mechanisms (5-9). For example, the increase in CBF induced by neural activity (functional hyperemia), a response that matches the brain's energy demands with its blood supply, and the ability of cerebral endothelial cells to regulate CBF are profoundly impaired in mice overexpressing APP (7, 10). The vasoconstriction induced by A␤ may underlie the marked reductions in CBF observed in the early stages of AD (11). The harmful cerebrovascular effects of A␤, in concert with epidemiological and pathological findings linking AD with cerebrovascular diseases (12-16), have suggested that A␤ has deleterious actions both on neurons and cerebral blood vessels, which may act synergistically to induce brain dysfunction in AD (3,17).The cerebrovascular alterations observed in mice overexpressing APP are associated with vascular oxidative stress and are counteracted by free radical scavengers (6,18,19), implicating reactive oxygen species (ROS) in the dysfunction. A major source...

Aging is associated with cerebrovascular dysregulation, which may underlie the increased susceptibility to ischemic stroke and vascular cognitive impairment occurring in the elder individuals. Although it has long been known that oxidative stress is responsible for the cerebrovascular dysfunction, the enzymatic system(s) generating the reactive oxygen species (ROS) have not been identified. In this study, we investigated whether the superoxide-producing enzyme NADPH oxidase is involved in alterations of neurovascular regulation induced by aging. Cerebral blood flow (CBF) was recorded by laser-Doppler flowmetry in anesthetized C57BL/6 mice equipped with a cranial window (age = 3, 12, and 24 months). In 12-month-old mice, the CBF increases evoked by whisker stimulation or by the endothelium-dependent vasodilators acetylcholine and bradykinin were attenuated by 42, 36, and 53%, respectively (P < 0.05). In contrast, responses to the nitric oxide donor S-nitroso-D-penicillamine or adenosine were not attenuated (P > 0.05). These cerebrovascular effects were associated with increased production of ROS in neurons and cerebral blood vessels, assessed by hydroethidine microfluorography. The cerebrovascular impairment present in 12-month-old mice was reversed by the ROS scavenger Mn (III) tetrakis (4-benzoic acid) porphyrin chloride or by the NADPH oxidase peptide inhibitor gp91ds-tat, and was not observed in mice lacking the Nox2 subunit of NADPH oxidase. These findings establish Nox2 as a critical source of the neurovascular oxidative stress mediating the deleterious cerebrovascular effects associated with increasing age.

Abstract-Angiotensin II (Ang II) exerts detrimental effects on cerebral circulation, the mechanisms of which have not been elucidated. In particular, Ang II impairs the increase in cerebral blood flow (CBF) produced by neural activity, a critical mechanism that matches substrate delivery with energy demands in brain. We investigated whether Ang II exerts its deleterious actions by activating Ang II type 1 (AT 1 ) receptors on cerebral blood vessels and producing reactive oxygen species (ROS) through NADPH oxidase. Somatosensory cortex CBF was monitored in anesthetized mice by laser-Doppler flowmetry. Ang II (0.25 g/kg per minute IV) attenuated the CBF increase produced by mechanical stimulation of the vibrissae. The effect was blocked by the AT 1 antagonist losartan and by ROS scavenger superoxide dismutase or tiron and was not observed in mice lacking the gp91 phox subunit of NADPH oxidase or in wild-type mice treated with the NADPH oxidase peptide inhibitor gp91ds-tat. Ang II increased ROS production in cerebral microvessels, an effect blocked by the ROS scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin and by the NADPH oxidase assembly inhibitor apocynin. Ang II did not increase ROS production in gp91-null mice. Double-label immunoelectron microscopy demonstrated that AT 1 and gp91phox immunoreactivities were present in endothelium and adventitia of neocortical arterioles. Collectively, these findings suggest that Ang II impairs functional hyperemia by activating AT 1 receptors and inducing ROS production via a gp91 phox containing NADPH oxidase. The data provide the mechanistic basis for the cerebrovascular dysregulation induced by Ang II and suggest novel therapeutic strategies to counteract the effects of hypertension on the brain. T he functional and structural integrity of the brain depends on a continuous blood supply commensurate to its changing energy needs. 1 Thus, if a brain region is activated, cerebral blood flow (CBF) to that region increases to match the increased energy demands and to remove potentially deleterious byproducts of cellular metabolism. 2 This phenomenon, termed functional hyperemia, is crucial to maintain the homeostasis of the cerebral microenvironment, and its alteration leads to brain dysfunction and disease. 3 Hypertension has profound effects on the brain and its circulation. 4 Whereas hypertension alters the structure of cerebral blood vessels, it also disrupts regulation of CBF. 5 These alterations are believed to underlie the cognitive impairment and brain damage associated with hypertension. 6,7 Angiotensin II (Ang II) has emerged as a critical factor in the deleterious cerebrovascular effects of hypertension. 6 Ang II produces cerebrovascular remodeling, promotes vascular inflammation, and impairs CBF regulation. 8 -11 Importantly, Ang II attenuates the CBF increase produced by activation of the mouse somatosensory cortex. 12 Such impairment in functional hyperemia is not related to the associated elevation in arterial pressure (AP) or to actions of Ang II on neural ...

Progranulin (PGRN) is a widely expressed secreted protein that is linked to inflammation. In humans, PGRN haploinsufficiency is a major inherited cause of frontotemporal dementia (FTD), but how PGRN deficiency causes neurodegeneration is unknown. Here we show that loss of PGRN results in increased neuron loss in response to injury in the CNS. When exposed acutely to 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydrophine (MPTP), mice lacking PGRN (Grn -/-) showed more neuron loss and increased microgliosis compared with wild-type mice. The exacerbated neuron loss was due not to selective vulnerability of Grn -/-neurons to MPTP, but rather to an increased microglial inflammatory response. Consistent with this, conditional mutants lacking PGRN in microglia exhibited MPTP-induced phenotypes similar to Grn -/-mice. Selective depletion of PGRN from microglia in mixed cortical cultures resulted in increased death of wild-type neurons in the absence of injury. Furthermore, Grn -/-microglia treated with LPS/IFN-γ exhibited an amplified inflammatory response, and conditioned media from these microglia promoted death of cultured neurons. Our results indicate that PGRN deficiency leads to dysregulated microglial activation and thereby contributes to increased neuron loss with injury. These findings suggest that PGRN deficiency may cause increased neuron loss in other forms of CNS injury accompanied by neuroinflammation.

Overproduction of the amyloid ␤ (A␤) peptide is a key factor in the pathogenesis of Alzheimer's disease (AD), but the mechanisms of its pathogenic effects have not been defined. Patients with AD have cerebrovascular alterations attributable to the deleterious effects of A␤ on cerebral blood vessels. We report here that NADPH oxidase, the major source of free radicals in blood vessels, is responsible for the cerebrovascular dysregulation induced by A␤. Thus, the free-radical production and the associated alterations in vasoregulation induced by A␤ are abrogated by the NADPH oxidase peptide inhibitor gp91ds-tat and are not observed in mice lacking the catalytic subunit of NADPH oxidase (gp91 phox ). Furthermore, oxidative stress and cerebrovascular dysfunction do not occur in transgenic mice overexpressing the amyloid precursor protein but lacking gp91 phox . The mechanisms by which NADPH oxidase-derived radicals mediate the cerebrovascular dysfunction involve reduced bioavailability of nitric oxide. Thus, a gp91 phox -containing NADPH oxidase is the critical link between A␤ and cerebrovascular dysfunction, which may underlie the alteration in cerebral blood flow regulation observed in AD patients.