Glycogen synthase kinase 3 (GSK‐3) dysregulation is implicated in the two Alzheimer's disease (AD) pathological hallmarks: β‐amyloid plaques and neurofibrillary tangles. GSK‐3 inhibitors may abrogate AD pathology by inhibiting amyloidogenic γ‐secretase cleavage of amyloid precursor protein (APP). Here, we report that the citrus bioflavonoid luteolin reduces amyloid‐β (Aβ) peptide generation in both human ‘Swedish’ mutant APP transgene‐bearing neuron‐like cells and primary neurons. We also find that luteolin induces changes consistent with GSK‐3 inhibition that ( i ) decrease amyloidogenic γ‐secretase APP processing, and ( ii ) promote presenilin‐1 (PS1) carboxyl‐terminal fragment (CTF) phosphorylation. Importantly, we find GSK‐3α activity is essential for both PS1 CTF phosphorylation and PS1‐APP interaction. As validation of these findings in vivo , we find that luteolin, when applied to the Tg2576 mouse model of AD, decreases soluble Aβ levels, reduces GSK‐3 activity, and disrupts PS1‐APP association. In addition, we find that Tg2576 mice treated with diosmin, a glycoside of a flavonoid structurally similar to luteolin, display significantly reduced Aβ pathology. We suggest that GSK‐3 inhibition is a viable therapeutic approach for AD by impacting PS1 phosphorylation‐dependent regulation of amyloidogenesis.

Background It is well known that most neurodegenerative diseases are associated with microglia-mediated inflammation. Our previous research demonstrates that the CD40 signaling is critically involved in microglia-related immune responses in the brain. For example, it is well known that the activation of the signal transducer and activator of transcription (STAT) signaling pathway plays a central role in interferon-gamma (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. Recent studies have shown that certain flavonoids possess anti-inflammatory and neuroprotective properties distinct from their well-known anti-oxidant effects. In particular, flavonoids, apigenin and luteolin have been found to be effective CD40 immunomodulators. Methods Cultured microglia, both N9 and primary derived lines, were treated with flavonoids in the presence of IFN-γ and/or CD40 ligation to assess any anti-inflammatory effects and/or mechanisms. CD40 expression on microglia was analyzed by fluorescence activated cell sorting (FACS). Anti-inflammatory effects and mechanisms were confirmed by ELISA for interlekin-6 (IL-6) and TNF-α, lactate dehydrogenase (LDH) assay, and STAT1 Western blotting. Results Apigenin and luteolin concentration-dependently suppressed IFN-γ-induced CD40 expression. Apigenin and luteolin also suppressed microglial TNF-α and IL-6 production stimulated by IFN-gamma challenge in the presence of CD40 ligation. In addition, apigenin and luteolin markedly inhibited IFN-γ-induced phosphorylation of STAT1 with little impact on cell survival. Conclusion Our findings provide further support for apigenin and luteolin's anti-inflammatory effects and suggest that these flavonoids may have neuroprotective/disease-modifying properties in various neurodegenerative disorders, including Alzheimer's disease (AD).

Alzheimer's disease (AD) immunotherapy accomplished by vaccination with beta-amyloid (A beta) peptide has proved efficacious in AD mouse models. However, "active" A beta vaccination strategies for the treatment of cerebral amyloidosis without concurrent induction of detrimental side effects are lacking. We have developed a transcutaneous (t.c.) A beta vaccination approach and evaluated efficacy and monitored for deleterious side effects, including meningoencephalitis and microhemorrhage, in WT mice and a transgenic mouse model of AD. We demonstrate that t.c. immunization of WT mice with aggregated A beta(1-42) plus the adjuvant cholera toxin (CT) results in high-titer A beta antibodies (mainly of the Ig G1 class) and A beta(1-42)-specific splenocyte immune responses. Confocal microscopy of the t.c. immunization site revealed Langerhans cells in areas of the skin containing the A beta(1-42) immunogen, suggesting that these unique innate immune cells participate in A beta(1-42) antigen processing. To evaluate the efficacy of t.c. immunization in reducing cerebral amyloidosis, transgenic PSAPP (APP(sw), PSEN1dE9) mice were immunized with aggregated A beta(1-42) peptide plus CT. Similar to WT mice, PSAPP mice showed high A beta antibody titers. Most importantly, t.c. immunization with A beta(1-42) plus CT resulted in significant decreases in cerebral A beta(1-40),42 levels coincident with increased circulating levels of A beta 1-40,42, suggesting brain-to-blood efflux of A beta. Reduction in cerebral amyloidosis was not associated with deleterious side effects, including brain T cell infiltration or cerebral microhemorrhage. Together, these data suggest that t.c. immunization constitutes an effective and potentially safe treatment strategy for AD.

Active and passive Aβ immunization efficiently reduces amyloid plaque load and memory impairment in transgenic mouse models of Alzheimer’s disease. Immunization has also yielded favorable results for many patients; but a significant percentage also developed severe meningioencephalitis. Prior studies by our group suggest CD40 receptor-CD40 ligand interaction between T-cells and microglia initiates release of proinflammatory factors, which drive phenotypic switching from a phagocytic to an antigen presenting cell phenotype thus impairing the clearance of Aβ. We therefore tested whether a CD40 blockade could enhance Aβ vaccination-mediated Aβ clearance in vivo. Importantly in our present study, Aβ vaccinated CD40 deficient heterozygous transgenic mice over-expressing mutant amyloid precursor protein and presenilin-1 demonstrated enhanced Aβ clearance; compared with vaccinated PSAPP mice. Similar results were obtained with Aβ vaccinated PSAPP mice passively immunized with CD40L antibody injections. Taken together with our previous studies these data suggest that CD40 blockade, in the context of active Aβ vaccination, acts to enhance CNS Aβ clearance by maintaining microglia in a phagocytic phenotype. These data also may provide the basis for a method whereby enhancing Aβ vaccination-mediated Aβ clearance strategies.

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.

Although the recent clinical trial of the ABeta42 peptide vaccine against Alzheimer's Disease (AD) has been halted due to adverse events, the apparent clinical utility of this approach underscores the need to further improve the safety of the vaccine, as well as to understand the potential immunological basis for complications. In this study, we examine both humoral and cellular immune responses elicited by immunization with peptide or DNA encoding wild-type and the Flemish and Dutch mutations of ABeta42 (i.e. the beta amyloid peptide spanning amino acids 1–42) in mice of different immune haplotypes as well as HLA Class II transgenic mice. The Flemish and Dutch mutations have been associated with cerebrovascular hemorrhages in affected individuals. These data allow determination of potential immunological responses that could mediate pathology observed with mutant forms of amyloid beta, as well as lead to the generation of safer vaccine preparations. Following peptide or plasmid immunization, antibody responses were measured against the different ABeta42 peptides in an ELISA assay, while T cell epitopes were analyzed through interferon gamma ELISPOT and lymphocyte proliferation assays. B cell mapping studies indicated that sera from all of the haplotype mice vaccinated with any of the ABeta42 peptides reacted specifically to the first 10 amino acids of ABeta42 with the ABeta42 mutants eliciting higher immune responses. ELISPOT analysis, which accessed cellular immune responses indicated that mice expressed differences in Class I epitopes dependent on the different immune haplotypes. These results may have implications for the design of future ABeta42 based vaccines against Alzheimer's Disease.

forms of APP (sAPPalpha) and C83, which might reduce neurotoxic A␤.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.

Background: Activated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD), multiple sclerosis (MS), and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB2).Methods: In this study, we investigated the effects of a cannabinoid agonist on CD40 expression and function by cultured microglial cells activated by IFN-gamma using RT-PCR, Western immunoblotting, flow cytometry, and anti-CB2 small interfering RNA (siRNA) analyses. Furthermore, we examined if the stimulation of CB2 could modulate the capacity of microglial cells to phagocytise A beta(1-42) peptide using a phagocytosis assay.Results: We found that the selective stimulation of cannabinoid receptor CB2 by JWH-015 suppressed IFN-gamma-induced CD40 expression. In addition, this CB2 agonist markedly inhibited IFN-gamma-induced phosphorylation of JAK/STAT1. Further, this stimulation was also able to suppress microglial TNF-alpha and nitric oxide production induced either by IFN-gamma or A beta peptide challenge in the presence of CD40 ligation. Finally, we showed that CB2 activation by JWH-015 markedly attenuated CD40-mediated inhibition of microglial phagocytosis of A beta(1-42) peptide. Taken together, these results provide mechanistic insight into beneficial effects provided by cannabinoid receptor CB2 modulation in neurodegenerative diseases, particularly AD.

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 Alzheimer's 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 lovastatin's 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 Alzheimer's disease. © 2004 Wiley‐Liss, Inc.