Doug Shytle

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.

Cholinergic modulation of microglial activation via alpha-7 nicotinic receptors

Almost all degenerative diseases of the CNS are associated with chronic inflammation. A central step in this process is the activation of brain mononuclear phagocyte cells, called microglia. While it is recognized that healthy neurons and astrocytes regulate the magnitude of microglia‐mediated innate immune responses and limit excessive CNS inflammation, the endogenous signals governing this process are not fully understood. In the peripheral nervous system, recent studies suggest that an endogenous ‘cholinergic anti‐inflammatory pathway’ regulates systemic inflammatory responses via α7 nicotinic acetylcholinergic receptors (nAChR) found on blood‐borne macrophages. These data led us to investigate whether a similar cholinergic pathway exists in the brain that could regulate microglial activation. Here we report for the first time that cultured microglial cells express α7 nAChR subunit as determined by RT–PCR, western blot, immunofluorescent, and immunochistochemistry analyses. Acetylcholine and nicotine pre‐treatment inhibit lipopolysaccharide (LPS)‐induced TNF‐α release in murine‐derived microglial cells, an effect attenuated by α7 selective nicotinic antagonist, α‐bungarotoxin. Furthermore, this inhibition appears to be mediated by a reduction in phosphorylation of p44/42 and p38 mitogen‐activated protein kinase (MAPK). Though preliminary, our findings suggest the existence of a brain cholinergic pathway that regulates microglial activation through α7 nicotinic receptors. Negative regulation of microglia activation may also represent additional mechanism underlying nicotines reported neuroprotective properties.

ADAM10 Activation Is Required for Green Tea (–)-Epigallocatechin-3-gallate-induced α-Secretase Cleavage of Amyloid Precursor Protein

Recently, we have shown that green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG) exerts a beneficial role on reducing brain Aβ levels, resulting in mitigation of cerebral amyloidosis in a mouse model of Alzheimer disease. EGCG seems to accomplish this by modulating amyloid precursor protein (APP) processing, resulting in enhanced cleavage of the α-COOH-terminal fragment (α-CTF) of APP and corresponding elevation of the NH2-terminal APP product, soluble APP-α (sAPP-α). These beneficial effects were associated with increased α-secretase cleavage activity, but no significant alteration in β-or γ-secretase activities. To gain insight into the molecular mechanism whereby EGCG modulates APP processing, we evaluated the involvement of three candidateα-secretase enzymes, a-disintegrin and metalloprotease (ADAM) 9, 10, or 17, in EGCG-induced non-amyloidogenic APP metabolism. Results show that EGCG treatment of N2a cells stably transfected with “Swedish” mutant human APP (SweAPP N2a cells) leads to markedly elevated active (∼60 kDa mature form) ADAM10 protein. Elevation of active ADAM10 correlates with increased α-CTF cleavage, and elevated sAPP-α. To specifically test the contribution of ADAM10 to non-amyloidogenic APP metabolism, small interfering RNA knockdown of ADAM9, -10, or -17 mRNA was employed. Results show that ADAM10 (but not ADAM9 or -17) is critical for EGCG-mediated α-secretase cleavage activity. In summary, ADAM10 activation is necessary for EGCG promotion of non-amyloidogenic (α-secretase cleavage) APP processing. Thus, ADAM10 represents an important pharmacotherapeutic target for the treatment of cerebral amyloidosis in Alzheimer disease.

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.

GFAP expression and social deficits in transgenic mice overexpressing human sAPPα

Autistic individuals display impaired social interactions and language, and restricted, stereotyped behaviors. Elevated levels of secreted amyloid precursor protein‐alpha (sAPPα), the product of α‐secretase cleavage of APP, are found in the plasma of some individuals with autism. The sAPPα protein is neurotrophic and neuroprotective and recently showed a correlation to glial differentiation in human neural stem cells (NSCs) via the IL‐6 pathway. Considering evidence of gliosis in postmortem autistic brains, we hypothesized that subsets of patients with autism would exhibit elevations in CNS sAPPα and mice generated to mimic this observation would display markers suggestive of gliosis and autism‐like behavior. Elevations in sAPPα levels were observed in brains of autistic patients compared to controls. Transgenic mice engineered to overexpress human sAPPα (TgsAPPα mice) displayed hypoactivity, impaired sociability, increased brain glial fibrillary acidic protein (GFAP) expression, and altered Notch1 and IL‐6 levels. NSCs isolated from TgsAPPα mice, and those derived from wild‐type mice treated with sAPPα, displayed suppressed β‐tubulin III and elevated GFAP expression. These results suggest that elevations in brain sAPPα levels are observed in subsets of individuals with autism and TgsAPPα mice display signs suggestive of gliosis and behavioral impairment.

P4-267: ADAM10 activation is required for green tea EGCG-induced alpha-secretase cleavage of amyloid precursor protein

Objective(s): We investigated, therefore, the effect of the nicotinic acetylcholine receptor (nAChR) stimulation on A -related neuronal death, using nicotine or acetylcholinesterase inhibitors (AChEIs) which could augment acetylcholine levels. Effects of nicotine or AChEIs on alphasecretase activities were also examined. Methods: Cultured rat cortical neurons were prepared. The number of neurons was evaluated by counting neurons stained with anti-MAP2 antibody. Neurotoxicity in each experiment was defined as a reduction in the survival rate. Akt phosphorylation was evaluated by immunoblot using anti-phosphorylated Akt antibodies. The levels of sAPPalpha, C83 or A were analyzed using immunoblot or ELISA. Results: Nicotine (10 M, 24 hr pretreatment) protected neurons against A and glutamate-induced neurotoxicity, which was blocked by an alpha7 nicotinic receptor antagonist (alpha-bungarotoxin), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002 and wortmannin), and a Src inhibitor (PP2). Levels of phosphorylated Akt, an effector of PI3K, and Bcl-2 were increased by nicotine. AChEIs, such as donepezil (10 M, 24 hr pre-treatment) and galantamine (10 M, 24 hr pre-treatment), also protected neurons against the toxicity. Akt phosphorylation was also induced by AChEIs, indicating the involvement of PI3K. We next examined the effect on the secretase activities. Nicotine or AChEI treatment significantly increased the C83 level. Furthermore, sAPPalpha released into the medium was also increased by the administration of nicotine (10 M, 24 hr), donepezil (10 M, 24 hr) or galantamine (10 M, 24 hr). On the contrary, the levels of A 42 and A 40 in culture medium were significantly decreased. Conclusions: Stimulation of the nAChR exerts neuroprotective effects, and shifts the amyloidogenic pathway of APP metabolism to the non-amyloidogenic one. These effects might play, at least in part, key roles on AD treatment.