Amyloid b-peptide stimulates nitric oxide production in astrocytes through an NFkB-dependent mechanism

The major pathological features of Alzheimer’s disease (AD) include amyloid plaques composed primarily of the b-amyloid (Ab) peptide, degenerating neurons and neurofibrillary tangles, and the presence of numerous activated astrocytes and microglia. Although extensive genetic data implicate Ab in the neurodegenerative cascade of AD, the molecular mechanisms underlying its effects on neurons and glia and the relationship between glial activation and neuronal death are not well defined. Ab has been shown to induce glial activation, and a growing body of evidence suggests that activated glia contribute to neurotoxicity through generation of inf lammatory cytokines and neurotoxic free radicals, such as nitric oxide (NO), potent sources of oxidative stress known to occur in AD. It is therefore crucial to identify specific Ab-induced molecular pathways mediating these responses in activated glia. We report that Ab stimulates the activation of the transcription factor NFkB in rat astrocytes, that NFkB activation occurs selectively from p65 transactivation domain 2, and that Ab-induced NO synthase expression and NO production occur through an NFkB-dependent mechanism. This demonstration of how Ab couples an intracellular signal transduction pathway involving NFkB to a potentially neurotoxic response provides a key mechanistic link between Ab and the generation of oxidative damage. Our results also suggest possible molecular targets upon which to focus future drug discovery efforts for AD. Alzheimer’s disease (AD) is a neurodegenerative disorder resulting in progressive neuronal death and memory loss. Neuropathologically, the disease is characterized by neurofibrillary tangles and neuritic plaques composed of aggregates of b-amyloid (Ab) protein, a 40–43 amino acid proteolytic fragment derived from the amyloid precursor protein. The importance of Ab in AD has been shown by means of several transgenic animal studies. The overexpression of mutant amyloid precursor protein results in neuritic plaque formation and synapse loss (1) and correlative memory deficits, as well as behavioral and pathological abnormalities similar to those found in AD (2). Neuritic plaques in AD are densely surrounded by reactive astrocytes (3, 4). These reactive astrocytes participate in the inflammatory response observed in AD by their production of proinflammatory cytokines such as interleukin 1b (5), and by their expression of inducible nitric oxide synthase (iNOS) (6, 7). iNOS generates nitric oxide (NO) and NO-derived reactive nitrogen species such as peroxynitrite. One possible pathology of AD therefore can be viewed as the accumulation of such free radicals during inflammation resulting in lipid peroxidation, tyrosine nitrosylation, DNA oxidative damage, and ultimately neuronal destruction within the brain (8–11). Understanding the expression of iNOS in the AD brain is therefore critical. NOS immunoreactivity has been observed near Ab neuritic plaques (7), and iNOS expression can be stimulated in cultured astrocytes or microglia by Ab (6, 12–15). This Ab stimulation of iNOS can result in the production of excessive amounts of diffusible NO, which when converted to peroxynitrite, becomes a powerfully detrimental oxidant with direct cytopathological consequences (16). Relatively little is known about the molecular mechanisms governing Ab stimulation of astrocyte iNOS activity. One candidate pathway involves the transcription factor NFkB (17). NFkB is a heterodimeric transcription factor composed of subunits from the Rel family of proteins. It is located in the cytoplasm as an inactive complex when associated with its inhibitor IkB, which masks the NFkB nuclear localization signal. Upon stimulation by cytokines or cellular stress, NFkB can be rapidly activated by the phosphorylation of IkB at serine residues, which direct IkB for proteosome-mediated degradation (18). The activated NFkB heterodimer is then free to translocate into the nucleus and bind to specific 10-bp response elements of target genes, typically found in inflammationresponsive genes. The iNOS promoter contains at least one NFkB response element (19), and activated NFkB is an important transcription factor in iNOS gene expression in response to cytokines or cellular stress (20). Recently, NFkB was observed immunohistochemically in postmortem AD brain (21). We report here that Ab activates NFkB in cultured rat astrocytes and demonstrate that Ab stimulation of iNOS expression and NO production occurs through an NFkBdependent mechanism. These data define a specific molecular pathway that links Ab activation of glia to a potentially neurotoxic oxidative stress response, and support the concept that Ab-induced oxidative damage is neuropathogenic in AD. MATERIALS AND METHODS Cell Culture and Amyloid b 1–42 Peptide (Ab42) Preparation. Cultured rat cortical astrocytes were prepared and tertiary cultures made as described (22). Cells were maintained in aMEM supplemented with 10% fetal bovine serum (FBS) (HyClone) and antibiotics [100 units/ml penicilliny100 mg/ml streptomycin (GIBCOyBRL)]. Twenty-four hours before stimulation, astrocyte medium was removed, cells were washed once with prewarmed PBS, and then serum-free aMEM containing N2 media supplement (GIBCOyBRL) was added to the cultures. Ab peptides [Ab 1–42 or scrambled 1–42 sequence (Ab42scr) KVKGLIDGAHIGDLVYEFMDSNSAIFREGVGAGHVHVAQVEF] were either purchased The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y955795-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: Ab42, amyloid b 1–42 peptide; Ab42scr, scrambled Ab1–42 sequence; AD, Alzheimer’s disease; EMSA, electrophoretic mobility-shift assay; iNOS, inducible nitric oxide synthase; TAD, transactivating domain; RLU, relative light unit. ¶To whom reprint requests should be addressed at: Northwestern University Medical School, W-129, 303 East Chicago Avenue, Chicago, IL 60611-3008. e-mail: [email protected].