How to get from here to there: macrophage recruitment in Alzheimer's disease.

Alzheimer's disease (AD) is pathologically defined by presence of intracellular neurofibrillary tangles and extracellular amyloid plaques comprised of amyoid-ß (Aß) peptides. Despite local recruitment of brain microglia to sites of amyloid deposition, these mononuclear phagocytes ultimately fail at restricting ß-amyloid plaque formation. On the other hand, it is becoming increasingly clear that professional phagocytes from the periphery possess Aß clearance aptitude. Yet, in order to harness this beneficial innate immune response, effective strategies must be developed to coax monocytes/macrophages from the periphery into the brain. It has previously been suggested that Aß 'immunotherapy' clears cerebral Aß deposits via mononuclear phagocytes, and recent evidence suggests that targeting transforming growth factor-ß-Smad 2/3 signaling and chemokine pathways such as Ccr2 impacts blood-to-brain trafficking of these cells in transgenic mouse models of AD. It has also been shown that the fractalkine receptor (Cx3cr1) pathway plays a critical role in chemotaxis of mononuclear phagocytes toward neurons destined for death in AD model mice. In order to translate these basic science findings into AD treatments, a key challenge will be to develop a new generation of pharmacotherapeutics that safely and effectively promote recruitment of peripheral amyloid phagocytes into the AD brain.

SOLUBLE CD40 LIGAND IN DEMENTIA.

Some 15-20% of the population over the age of 65 years suffer from dementia, currently one of the leading causes of death behind cardiovascular diseases, cancer and cerebrovascular diseases. The major forms of dementia share in common overactivation of the CD40-CD40-L complex, leading to high levels of proinflammatory cytokine production by immune cells of the central nervous system (CNS), including microglia and astrocytes. Consequently, both neuronal survival and signaling are negatively affected, leading to the characteristic progressive loss of higher cortical functions. We have reviewed the literature concerning the involvement of this complex in the pathology of three major forms of dementia: Alzheimer's-type, HIV-associated and vascular dementia. This is followed by a discussion of current preclinical and clinical therapies that may influence this interaction, and thus point the way toward a future neuroimmunological approach to inhibiting the effects of CD40-CD40-L in neuropsychiatric disease.

CD40L disruption enhances Abeta vaccine-mediated reduction of cerebral amyloidosis while minimizing cerebral amyloid angiopathy and inflammation.

Amyloid-beta (Abeta) immunization efficiently reduces amyloid plaque load and memory impairment in transgenic mouse models of Alzheimer's disease (AD). Active Abeta immunization has also yielded favorable results in a subset of AD patients. However, a small percentage of patients developed severe aseptic meningoencephalitis associated with brain inflammation and infiltration of T-cells. We have shown that blocking the CD40-CD40 ligand (L) interaction mitigates Abeta-induced inflammatory responses and enhances Abeta clearance. Here, we utilized genetic and pharmacologic approaches to test whether CD40-CD40L blockade could enhance the efficacy of Abeta(1-42) immunization, while limiting potentially damaging inflammatory responses. We show that genetic or pharmacologic interruption of the CD40-CD40L interaction enhanced Abeta(1-42) immunization efficacy to reduce cerebral amyloidosis in the PSAPP and Tg2576 mouse models of AD. Potentially deleterious pro-inflammatory immune responses, cerebral amyloid angiopathy (CAA) and cerebral microhemorrhage were reduced or absent in these combined approaches. Pharmacologic blockade of CD40L decreased T-cell neurotoxicity to Abeta-producing neurons. Further reduction of cerebral amyloidosis in Abeta-immunized PSAPP mice completely deficient for CD40 occurred in the absence of Abeta immunoglobulin G (IgG) antibodies or efflux of Abeta from brain to blood, but was rather correlated with anti-inflammatory cytokine profiles and reduced plasma soluble CD40L. These results suggest CD40-CD40L blockade promotes anti-inflammatory cellular immune responses, likely resulting in promotion of microglial phagocytic activity and Abeta clearance without generation of neurotoxic Abeta-reactive T-cells. Thus, combined approaches of Abeta immunotherapy and CD40-CD40L blockade may provide for a safer and more effective Abeta vaccine.

Confirmation of association between D10S583 and Alzheimer's disease in a case--control sample.

Several independent studies have reported that loci on chromosome 10 are associated/linked with Alzheimer's disease (AD), including a family-based study demonstrating an association between the marker D10S583 and AD. We have examined the D10S583 polymorphic marker and apolipoprotein E (APOE) gene in a case-control study. We observed the expected association of the APOE allele varepsilon4 with AD, and an inverse association between the D10S583 allele 209 and AD. These data support the original findings that suggest the presence of a candidate gene for AD in this region of chromosome 10. The nearby insulin degrading enzyme gene has been previously proposed as a candidate gene; however, a number of other putative candidate genes are also located in this region. The ongoing investigation of the genetic source of association and linkage in this region is clearly warranted.

A functional polymorphism within the mu-opioid receptor gene and risk for abuse of alcohol and other substances.

Genetic association studies investigating the role of the +118A allele of the human mu-opioid receptor gene in risk for alcohol dependency have produced inconsistent findings, possibly because of the failure to recognize sampling methodology difficulties inherent in association studies of polygenic disorders. We examined the frequency of the AA genotype and A allele in several groups of substance-dependent cases, unrestricted controls, and super controls screened for the use of alcohol and cigarettes. Our findings and analyses suggest that the OPRM1 +118 polymorphism is a general risk gene for substance dependence, but is not specific to a particular substance. The nature of the conferred risk is likely to be in use of multiple substances, but it is not yet determined if the risk could be expressed in severity of use of any particular substance. The contribution of the gene to risk for substance dependence is small, and is detected most easily in studies that use control samples that are screened for all forms of substance dependence.

CD40 signaling and Alzheimer's disease pathogenesis.

The interaction between CD40 and its cognate ligand, CD40 ligand, is a primary regulator of the peripheral immune response, including modulation of T lymphocyte activation, B lymphocyte differentiation and antibody secretion, and innate immune cell activation, maturation, and survival. Recently, we and others have identified CD40 expression on a variety of CNS cells, including endothelial cells, smooth muscle cells, astroglia and microglia, and have found that, on many of these cells, CD40 expression is enhanced by pro-inflammatory stimuli. Importantly, the CD40-CD40 ligand interaction on microglia triggers a series of intracellular signaling events that are discussed, beginning with Src-family kinase activation and culminating in microglial activation as evidenced by tumor necrosis factor-alpha secretion. Based on the involvement of microglial activation and brain inflammation in Alzheimer's disease pathogenesis, we have investigated co-stimulation of microglia, smooth muscle, and endothelial cells with CD40 ligand in the presence of low doses of freshly solubilized amyloid-beta peptides. Data reviewed herein show that CD40 ligand and amyloid-beta act synergistically to promote pro-inflammatory responses by these cells, including secretion of interleukin-1 beta by endothelial cells and tumor necrosis factor-alpha by microglia. As these cytokines have been implicated in neuronal injury, a comprehensive model of pro-inflammatory CD40 ligand and amyloid-beta initiated Alzheimer's disease pathogenesis (mediated by multiple CNS cells) is proposed.

Cholesterol accumulates in senile plaques of Alzheimer disease patients and in transgenic APP(SW) mice.

Mounting evidence suggests that cholesterol may contribute to the pathogenesis of Alzheimer disease (AD). We examined whether cholesterol might be present in senile plaques, a hallmark neuropathological feature of AD. We employed 2 different fluorometric-staining techniques (filipin staining and an enzymatic technique) for the determination of cholesterol in brains of postmortem confirmed AD patients and in nondemented, age-matched histopathologically normal controls. AD patient brains showed abnormal accumulation of cholesterol in congophilic/birefringent dense cores of senile plaques that was essentially absent in histopathologically normal controls. To determine whether increased senile plaque-associated cholesterol occurred generally in all plaques or was restricted to a specific subset, quantitative analysis was performed. Data indicate abnormal accumulation of cholesterol in cores of mature plaques but not in diffuse or immature plaques. Additionally, transgenic mice that overexpress the "Swedish" amyloid precursor protein (Tg APP(SW), line 2576) exhibited a similar pattern of abnormal cholesterol accumulation in mature, congophilic amyloid plaques at 24 months of age that was absent in their control littermates or in 8-month-old Tg APP(SW) mice (an age prior to amyloid deposition). Taken together, our results imply a link between cholesterol and AD pathogenesis and suggest that cholesterol plays an important role in the formation and/or progression of senile plaques.

Characterization of murine immunoglobulin G antibodies against human amyloid-beta1-42.

It has been demonstrated that immunization of transgenic mouse models of Alzheimer's disease (AD) with amyloid-beta1-42 peptide (Abeta1-42) results in amelioration of AD-like pathology, including reduced soluble and deposited beta-amyloid and decreased cognitive impairment. Based on the proposed importance of immunoglobulin G (IgG) anti-Abeta antibodies (Abs) in these effects, we sought to characterize these Abs in splenocytes from mice immunized with Abeta1-42. Data show that a more aggregated preparation of Abeta1-42 gives a robust IgG anti-Abeta Ab response, while these Abs are almost undetectable when a less aggregated preparation of Abeta1-42 is used as the immunogen. Importantly, IgG anti-Abeta Ab production is detected after just 12 weeks of Abeta1-42 treatment. Analysis of anti-Abeta Ab IgG isotypes reveals that the majority of these Abs are IgG1, with significantly fewer Abs of the IgG2a or IgG2b isotypes (IgG1>IgG2a>IgG2b), suggesting a T lymphocyte helper type II response after Abeta1-42 immunization. To determine the epitope of Abeta recognized by IgG anti-Abeta Abs, intact Abeta and Abeta peptide fragments were analyzed for their ability to bind these Abs. Data show that these Abs specifically recognize an amino-terminal epitope of Abeta between amino acids one and twelve, with higher affinity for a more soluble preparation of Abeta1-42. These data further indicate the immunogenic potential of Abeta1-42 and offer insight into the nature of the IgG anti-Abeta Ab response.

Intracisternal increase of superoxide anion production in a canine subarachnoid hemorrhage model.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) are thought to be primary in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). However, as direct evidence of ROS has not yet been demonstrated in cerebral vasospasm, we sought to substantiate superoxide anion (.O(2)(-)) generation in the subarachnoid space after SAH using a modification of Karnovsky's manganese/diaminobenzidine (Mn(2+)/DAB) technique. METHODS: SAH or sham operation was induced according to a 2-hemorrhage model in a total of 24 beagle dogs. On day 2 or 7 after SAH or sham operation, dogs were intrathecally infused with buffer containing Mn(2+) and DAB, and the brain stem was prepared for light and electron microscopy. Possible colocalization of ferrous (Fe(2+)) or ferric (Fe(3+)) iron ions with.O(2)(-) was also examined with the use of Turnbull blue or Berlin blue staining, respectively. RESULTS: Light microscopy revealed amorphous, amber deposits within the subarachnoid hematoma, the periarterial space, and the tunica adventitia of the basilar artery on days 2 and 7 after SAH.O(2)(-) deposits were eliminated by addition of superoxide dismutase or exclusion of either Mn(2+) or DAB from the perfusate, confirming the specificity of the reaction. These deposits were colocalized with blue reaction deposits indicating Fe(2+) and Fe(3+). Within the subarachnoid space,.O(2)(-) indicating electron-dense fine granules were preferentially located around degenerated erythrocytes and, secondarily, infiltrating macrophages and neutrophils. CONCLUSIONS: We show direct evidence for enhanced production of.O(2)(-) and Fe(2+)/Fe(3+) iron ions in the subarachnoid space after SAH, lending further support to the pathogenic role of ROS in cerebral vasospasm after SAH.

CD45 opposes beta-amyloid peptide-induced microglial activation via inhibition of p44/42 mitogen-activated protein kinase.

Reactive microglia have been suggested to play a role in the Alzheimer's disease (AD) process, and previous studies have shown that expression of CD45, a membrane-bound protein-tyrosine phosphatase (PTP), is elevated in microglia in AD brain compared with controls. To investigate the possible role of CD45 in microglial responsiveness to beta-amyloid (Abeta) peptides, we first co-treated primary cultured microglia with a tyrosine phosphatase inhibitor [potassium bisperoxo (1,10-phenanthroline) oxovanadate (phen), 5 micrometer] and freshly solubilized Abeta peptides (1000 nm). Data show synergistic induction of microglial activation as evidenced by tumor necrosis factor alpha (TNF-alpha) production and nitric oxide (NO) release, both of which we show to be dependent on activation of p44/42 mitogen-activated protein kinase (MAPK). Furthermore, co-treatment with phen and Abeta peptides results in microglia-induced neuronal cell injury. Stimulation of microglial CD45 by anti-CD45 antibody markedly inhibits these effects via inhibition of p44/42 MAPK, suggesting that CD45 is a negative regulator of microglial activation. Accordingly, primary cultured microglia from CD45-deficient mice demonstrate hyper-responsiveness to Abeta, as evidenced by TNF-alpha release, NO production, and neuronal injury after stimulation with Abeta peptides. As a validation of these findings in vivo, brains from a transgenic mouse model of AD [transgenic Swedish APP-overexpressing (Tg APP(sw)) mice] deficient for CD45 demonstrate markedly increased production of TNF-alpha compared with Tg APP(sw) mice. Taken together, these results suggest that therapeutic agents that stimulate the CD45 PTP signaling pathway may be effective in suppressing microglial activation associated with AD.

CD45 inhibits CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK pathway.

It has been reported that ligation of CD40 with CD40 ligand (CD40L) results in microglial activation as evidenced by p44/42 mitogen-activated protein kinase (MAPK) dependent tumor necrosis factor alpha (TNF-alpha) production. Previous studies have shown that CD45, a functional transmembrane protein-tyrosine phosphatase, is constitutively expressed at moderate levels on microglial cells and this expression is greatly elevated on activated microglia. To investigate the possibility that CD45 might modulate CD40L-induced microglial activation, we treated primary cultured microglial cells with CD40L and anti-CD45 antibody. Data show that cross-linking of CD45 markedly inhibits CD40L-induced activity of the Src family kinases Lck and Lyn. Further, co-treatment of microglia with CD40L and anti-CD45 antibody results in significant inhibition of microglial TNF-alpha production through inhibition of p44/42 MAPK activity, a downstream signaling event resulting from Src activation. Accordingly, primary cultured microglial cells from mice deficient in CD45 demonstrate hyper-responsiveness to ligation of CD40, as evidenced by increased p44/42 MAPK activation and TNF-alpha production. Taken together, these results show that CD45 plays a novel role in suppressing CD40L-induced microglial activation via negative regulation of the Src/p44/42 MAPK cascade.

Cholesterol modulates vascular reactivity to endothelin-1 by stimulating a pro-inflammatory pathway.

Hypercholesterolemia (HC) is associated with coronary endothelial dysfunction and increased circulating levels of endothelin-1. We show that pre-treatment of intact rat aortic rings with cholesterol synergistically enhances the vasoconstriction induced by endothelin-1 suggesting that elevated levels of cholesterol may predispose to hypertension by modulating the vascular reactivity to endogenous vasoconstrictors. Moreover, we report that SB202190, a selective inhibitor of p38 MAPK, and PD98059 an inhibitor of MEK1/2 are able to abolish the vasoactive properties of cholesterol. MK-886, an inhibitor of 5-lipoxygenase is inefficient at blocking the vasoactive properties of cholesterol whereas NS-398, a selective inhibitor of cyclooxygenase-2 (COX-2) completely abolishes cholesterol-induced vasoconstriction. In intact rat aortae, cholesterol stimulates prostaglandin E(2) and prostaglandin F(2 alpha) production, an effect that can be completely prevented by inhibiting p38 MAPK, or COX-2. In vitro, cholesterol appears to stimulate a similar pro-inflammatory pathway in human cerebrovascular smooth muscle cells. Disruption of the MAPK/COX-2 pathway may represent a valuable therapy to block the hypertension associated with HC, as well as the development of atherosclerosis.

Soluble beta-amyloid peptides mediate vasoactivity via activation of a pro-inflammatory pathway.

Freshly solubilized beta-amyloid (Abeta) peptides display vasoactive properties, increasing both the magnitude and the duration of endothelin-1-induced vasoconstriction. We show that Abeta vasoactivity is mediated by the stimulation of a pro-inflammatory pathway involving activation of secretory phospholipase A(2) (PLA(2)), mitogen activated protein kinase (MAPK) kinase (MEK1/2), p38 MAPK, cytosolic PLA(2), and the release of arachidonic acid. Ultimately, arachidonic acid is metabolized into proinflammatory eicosanoids via the 5-lipoxygenase and cyclooxygenase-2 (COX-2) enzymes, both of which we show to be required for A beta vasoactivity. Accordingly, p38 MAPK activity is higher in the brains of transgenic mice that overproduce A beta, and COX-2 immunoreactivity is increased in the cerebrovasculature of these transgenic animals. Taken together, our data show that freshly solubilized A beta peptides can trigger a pro-inflammatory reaction in the vasculature that can be blocked by inhibiting specific target molecules, providing the basis for novel therapeutic intervention.

A beta vasoactivity: an inflammatory reaction.

Mounting evidence from in vitro and in vivo studies in transgenic mice overproducing beta-amyloid peptides (A beta) suggests that A beta can induce vasoconstriction and decrease cerebral blood flow. In this report, we describe the vasoactive properties of A beta, in particular the enhancement of endothelin-1-induced vasoconstriction and A beta's induction of a long-lasting vasoconstrictive event. Furthermore, we show that low doses (as low as 50 nM) of freshly solubilized A beta similar to those observed in the plasma of patients suffering from Alzheimer's disease are vasoactive. By using various inhibitors and activators of the phospholipase A2 (PLA2)/arachidonic acid (AA) cascade, we demonstrate that A beta vasoactivity is dependent on activation of this intracellular signaling pathway, resulting in stimulation of downstream cyclooxygenase-2 and 5-lipoxygenase, which mediate production of proinflammatory eicosanoids. Taken together, our data show that A beta directly activates an intracellular proinflammatory pathway, which is responsible for its vasoactive properties.

Novel strategies for opposing murine microglial activation.

Pathologic microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially neurotoxic agents, such as pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha). Using cultured N9 microglial cells, we have examined the regulation of TNF-alpha following endotoxic insult with lipopolysacharide (LPS), focusing on the role of the pro-inflammatory phospholipase A2/mitogen activated protein kinase/arachidonic acid/cyclo-oxygenase-2 cascade and the nitric oxide/cGMP pathway. Data show that various inhibitors of the PLA2 cascade markedly inhibit LPS-induced TNF-alpha release, supporting a key role of this pathway in the regulation of microglial activation. We also investigated the putative effects of cGMP-elevating agents on blocking microglial activation induced by LPS. Data show that each member of this class of cGMP-elevating compounds that we employed opposed microglial TNF-alpha release, suggesting that strengthening intracellular cGMP signaling mitigates against microglial activation. Taken together, our results suggest novel strategies for reducing microglial activation.

The opioid receptor system and alcoholism: a genetic perspective.

Over the past decade, mounting evidence has implicated the endogenous opioid receptor system as a central player in the etiology of alcohol drinking behavior in animals and alcoholism in humans. Much of this work is a product of a pharmacological approach, where differences in opioid receptor pharmacology have been found to predict drinking behavior in animal models of alcoholism, including rats and mice selectively bred for alcohol preference and avoidance. This review considers the opioid receptor system and alcoholism from a genetic standpoint, and discusses investigation into opioid receptor pharmacology in animal models of alcoholism as work that paved the way for the more recent molecular genetic studies implicating the delta-, and particularly, the mu opioid receptors as genetically linked to alcoholism-associated phenotypes in animal models of the disease. These genetic studies are set within the broader context of the candidate gene approach for alcoholism, where opioid receptor genes are taken to be partial, rather than complete, risk factors for alcoholism. Building upon these findings, the recent genetic association between alcoholism and the mu opioid receptor gene in humans is discussed. Finally, the translation of such genetic association studies between opioid receptor genes and alcoholism to a pharmacogenetic approach, allowing for the evaluation of putative relationships between genotype and pharmacological response profiles, is suggested to address the etiological question of what the molecular mechanism is underlying opioid receptor genetic risk for alcoholism phenotypes.

Ligation of microglial CD40 results in p44/42 mitogen-activated protein kinase-dependent TNF-alpha production that is opposed by TGF-beta 1 and IL-10.

Recently, it has been demonstrated that the CD40 receptor is constitutively expressed on cultured microglia at low levels. Ligation of CD40 by CD40 ligand on these cells results in microglial activation, as measured by TNF-alpha production and neuronal injury. However, the intracellular events mediating this effect have yet to be investigated. We report that ligation of microglial CD40 triggers activation of p44/42 mitogen-activated protein kinase (MAPK). This effect is evident 30 min posttreatment, and progressively declines thereafter (from 30 to 240 min). Phosphorylated p38 MAPK is not observed in response to ligation of microglial CD40 across the time course examined. Inhibition of the upstream activator of p44/42 MAPK, mitogen-activated protein/extracellular signal-related kinase kinase 1/2, with PD98059, decreases phosphorylation of p44/42 MAPK and significantly reduces TNF-alpha release following ligation of microglial CD40. Furthermore, cotreatment of microglial cells with CD40 ligand and TGF-beta1 or IL-10, or both, inhibits CD40-mediated activation of p44/42 MAPK and production of TNF-alpha in a statistically interactive manner. Taken together, these data show that ligation of microglial CD40 triggers TNF-alpha release through the p44/42 MAPK pathway, an effect that can be opposed by TGF-beta1 and IL-10.

Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation.

Alzheimer's disease (AD) has a substantial inflammatory component, and activated microglia may play a central role in neuronal degeneration. CD40 expression was increased on cultured microglia treated with freshly solublized amyloid-beta (Abeta, 500 nanomolar) and on microglia from a transgenic murine model of AD (Tg APPsw). Increased tumor necrosis factor alpha production and induction of neuronal injury occurred when Abeta-stimulated microglia were treated with CD40 ligand (CD40L). Microglia from Tg APPsw mice deficient for CD40L demonstrated reduction in activation, suggesting that the CD40-CD40L interaction is necessary for Abeta-induced microglial activation. Finally, abnormal tau phosphorylation was reduced in Tg APPsw animals deficient for CD40L, suggesting that the CD40-CD40L interaction is an early event in AD pathogenesis.

Bcl-X(L) inhibits apoptosis and necrosis produced by Alzheimer's beta-amyloid1-40 peptide in PC12 cells.

Recent studies have shown that neuronal apoptosis induced by the Alzheimer's disease (AD) beta-amyloid peptide (Abeta) is related to alteration of the Bax/Bcl-2 ratio. It has been demonstrated that Bcl-X(L) (Bcl-X(L) = protein, bcl-X(L) = gene), a Bcl-2-related protein, prevents apoptosis in mammalian cells. Additionally, TGF-beta1 is able to protect cultured neuronal cells from Abeta-induced apoptosis via upregulation of bcl-X(L) and bcl-2 gene expression. We show that Abeta treatment (500 nM, freshly solubilized) results in apoptosis and necrosis in differentiated PC12 cells maintained with a low dose of NGF-beta (1 ng/ml). To investigate whether transfection of PC12 cells with bcl-X(L) could block Abeta-induced apoptosis, we transfected these cells with a bcl-X(L) construct (pcDNA-bcl-X(L)). Data show that bcl-X(L) significantly inhibits both early-stage apoptosis and late-stage apoptosis/necrosis produced by Abeta treatment (1000 nM) in pcDNA3-bcl-X(L)-transfected PC12 cells as compared with pcDNA3 vector-transfected PC12 cells. These results suggest that Bcl-X(L) exhibits both anti-necrotic as well as anti-apoptotic roles in Abeta-challenged PC12 cells.