Kirk P. Townsend

Green Tea Epigallocatechin-3-Gallate (EGCG) Modulates Amyloid Precursor Protein Cleavage and Reduces Cerebral Amyloidosis in Alzheimer Transgenic Mice

Alzheimers disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of β-amyloid (Aβ) peptides as senile plaques in the brain. Recent studies suggest that green tea flavonoids may be used for the prevention and treatment of a variety of neurodegenerative diseases. Here, we report that (-)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, reduces Aβ generation in both murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP) and in primary neurons derived from Swedish mutant APP-overexpressing mice (Tg APPsw line 2576). In concert with these observations, we find that EGCG markedly promotes cleavage of the α-C-terminal fragment of APP and elevates the N-terminal APP cleavage product, soluble APP-α. These cleavage events are associated with elevated α-secretase activity and enhanced hydrolysis of tumor necrosis factor α-converting enzyme, a primary candidate α-secretase. As a validation of these findings in vivo, we treated Tg APPsw transgenic mice overproducing Aβ with EGCG and found decreased Aβ levels and plaques associated with promotion of the nonamyloidogenic α-secretase proteolytic pathway. These data raise the possibility that EGCG dietary supplementation may provide effective prophylaxis for AD.

CD40 signaling regulates innate and adaptive activation of microglia in response to amyloid β-peptide

Although deposition of amyloid β‐peptide (Aβ) as Aβ plaques involves activation of microglia‐mediated inflammatory responses, activated microglia ultimately fail to clear Aβ plaques in the brains of either Alzheimers disease (AD) patients or AD mouse models. Mounting evidence suggests that chronic microglia‐mediated immune response during Aβ deposition etiologically contributes to AD pathogenesis by promoting Aβ plaque formation. However, the mechanisms that govern microglia response in the context of cerebral Aβ/β‐amyloid pathology are not well understood. We show that ligation of CD40 by CD40L modulates Aβ‐induced innate immune responses in microglia, including decreased microglia phagocytosis of exogenous Aβ1–42 and increased production of pro‐inflammatory cytokines. CD40 ligation in the presence of Aβ1–42 leads to adaptive activation of microglia, as evidenced by increased co‐localization of MHC class II with Aβ. To assess their antigen‐presenting cell (APC) function, cultured microglia were pulsed with Aβ1–42 in the presence of CD40L and co‐cultured with CD4+ T cells. Under these conditions, microglia stimulate T cell‐derived IFN‐γ and IL‐2 production, suggesting that CD40 signaling promotes the APC phenotype. These data provide a mechanistic explanation for our previous work showing decreased microgliosis associated with diminished cerebral Aβ/β‐amyloid pathology when blocking CD40 signaling in transgenic Alzheimers mice.

Galantamine and nicotine have a synergistic effect on inhibition of microglial activation induced by HIV-1 gp120

Chronic brain inflammation is the common final pathway in the majority of neurodegenerative diseases and central to this phenomenon is the immunological activation of brain mononuclear phagocyte cells, called microglia. This inflammatory mechanism is a central component of HIV-associated dementia (HAD). In the healthy state, there are endogenous signals from neurons and astrocytes, which limit excessive central nervous system (CNS) inflammation. However, the signals controlling this process have not been fully elucidated. Studies on the peripheral nervous system suggest that a cholinergic anti-inflammatory pathway regulates systemic inflammatory response by way of acetylcholine acting at the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) found on blood-borne macrophages. Recent data from our laboratory indicates that cultured microglial cells also express this same receptor and that microglial anti-inflammatory properties are mediated through it and the p44/42 mitogen-activated protein kinase (MAPK) system. Here we report for the first time the creation of an in vitro model of HAD composed of cultured microglial cells synergistically activated by the addition of IFN-gamma and the HIV-1 coat glycoprotein, gp120. Furthermore, this activation, as measured by TNF-alpha and nitric oxide (NO) release, is synergistically attenuated through the alpha7 nAChR and p44/42 MAPK system by pretreatment with nicotine, and the cholinesterase inhibitor, galantamine. Our findings suggest a novel therapeutic combination to treat or prevent the onset of HAD through this modulation of the microglia inflammatory mechanism.

Lovastatin modulation of microglial activation via suppression of functional CD40 expression

Recent studies have shown that the 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins) possess antiinflammatory and immunomodulatory properties, distinct from their action of lowering serum lipid levels. Moreover, results of epidemiological studies suggest that long‐term use of statins is associated with a decreased risk for Alzheimers disease (AD). Interestingly, lovastatin (one of the most commonly used anticholesterol drugs) treatment of vascular‐derived cells has been reported to antagonize activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, and it is well known that the JAK/STAT pathway plays a central role in interferon‐γ (IFN‐γ)‐induced microglial CD40 expression. We and others have previously reported that microglial CD40 expression is significantly induced by IFN‐γ and amyloid‐β (Aβ) peptide. Moreover, it has been shown that CD40 signaling is critically involved in microglia‐related immune responses in the CNS. In this study, we examined the putative role of lovastatin in modulation of CD40 expression and its signaling in cultured microglia. RT‐PCR, Western immunoblotting, and flow cytometry data show that lovastatin suppresses IFN‐γ‐induced CD40 expression. Additionally, lovastatin markedly inhibits IFN‐γ‐induced phosphorylation of JAK/STAT1. Furthermore, lovastatin is able to suppress microglial tumor necrosis factor‐α, interleukin (IL)‐β1 and IL‐6 production promoted either by IFN‐γ or by Aβ peptide challenge in the presence of CD40 cross‐linking. To characterize further lovastatins effect on microglial function, we examined microglial phagocytic capability following CD40 cross‐linking. Data reveal that lovastatin markedly attenuates CD40‐mediated inhibition of microglial phagocytosis of Aβ. These results provide an insight into the mechanism of the beneficial effects of lovastatin in neurodegenerative disorders, particularly Alzheimers disease.

Pro-inflammatory effect of freshly solubilized β-amyloid peptides in the brain

Abstract It has recently been shown that the level of soluble β-amyloid (Aβ) peptides correlates well with the severity of synaptic loss and the density of neurofibrillary tangles observed in Alzheimer’s disease (AD) brain. However, the biological activity of soluble forms of Aβ peptides in the brain remains to be determined. We have investigated ex vivo the effect of freshly solubilized Aβ 1–40 peptides (fsAβ) on prostaglandin E 2 (PGE 2 ) production in rat brain slices. PGE 2 levels increased rapidly following treatment with fsAβ, an effect that was prevented by SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), and by NS-398, which preferentially inhibits cyclooxygenase-2 (COX-2) compared to COX-1. In an attempt to determine the cellular systems of the brain responsible for prostaglandin production in response to fsAβ, the effect of fsAβ was tested on isolated brain microvessels, primary cultures of brain smooth muscle cells/pericytes and endothelial cells, and a human neuron-like cell line (IMR32). Our data show that fsAβ ex vivo can stimulate prostaglandin accumulation in incubates of isolated rat brain microvessels. In addition, fsAβ appears to cause a concentration-dependent enhancement of prostaglandin accumulation in primary cultures of brain microvessel-derived smooth muscle cells/pericytes but not of brain endothelial cells. Finally, fsAβ also stimulated PGF 2α accumulation in cultures of differentiated IMR32 cells, but to a lesser extent than in brain smooth muscle cell/pericyte cultures. Deposition of aggregated forms of Aβ in the brain has been thought to trigger an inflammatory response which accompanies the neuropathologic events of AD. Our data provide evidence that fsAβ triggers a pro-inflammatory reaction in rat brain, and suggest that the cerebrovasculature may constitute an important source of pro-inflammatory eicosanoids.

Statins inhibit Aβ-neurotoxicity in vitro and Aβ-induced vasoconstriction and inflammation in rat aortae

Abstract Freshly solubilized Aβ peptides synergistically increase the magnitude of the constriction induced by endothelin-1 (ET-1), via the activation of a pro-inflammatory pathway. We report that mevinolin and mevastatin, two inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase are able to completely abolish the vasoactive properties of Aβ in rat aortae. Mevinolin also appears to oppose the increased vascular reactivity to ET-1 induced by interleukin 1-β and phospholipase A 2 suggesting that statins display some anti-inflammatory properties. We show that freshly solubilized Aβ stimulates prostaglandin E 2 and F 2α production (by 6 and 3.6 times, respectively) in isolated rat aortae and that mevinolin completely antagonizes this effect confirming the anti-inflammatory action of mevinolin ex vivo in rat aortae. In addition, we observed that Aβ vasoactivity is not mediated nor modulated by mevalonic acid suggesting that the anti-inflammatory action of the statins are not related to an inhibition of HMG-CoA reductase activity. Differentiated human neuroblastoma cells (IMR32) were used to assess the neurotoxic effect of pre-aggregated Aβ by quantifying the release of lactate dehydrogenase (LDH) in the cell culture medium. Aβ appears to enhance LDH release by 30% in IMR32 cells, an effect that can be completely opposed by mevastatin. Taken together these data show that statins can antagonize the effect of Aβ in different assays and provide new clues to understand the prophylactic action of the statins against Alzheimers disease.

CD45 isoform RB as a molecular target to oppose lipopolysaccharide-induced microglial activation in mice.

CD45 is a membrane-bound protein tyrosine phosphatase expressed on all hemopoietic cells with multiple splice variants, including RA, RB, RC and RO. Our previous studies have shown that cross-linking of CD45 with an anti-CD45 antibody markedly inhibits LPS-induced microglia activation. In order to determine which of the CD45 isoforms may be responsible for these effects, we have investigated the expression of CD45 isoforms on cultured microglial cells using flow cytometric analysis. Data reveal that CD45RB is the predominant isoform expressed in murine primary cultured microglial cells. Furthermore, incubation of these cultured cells with anti-CD45RB antibody results in a reduction of microglial activation induced by LPS as evidenced by TNF-alpha production. As a validation of these findings in vivo, brain homogenates from anti-CD45RB antibody (MG23G2)-injected animals that had been treated with LPS demonstrate a significant decrease in TNF-alpha levels compared to control mice treated with LPS plus vehicle. Taken together, these findings suggest that therapeutic agents that specifically stimulate the microglial CD45RB signaling pathway may be effective in suppressing microglial activation associated with several neurodegenerative disorders.