Inhibition of NF-kB potentiates amyloid b-mediated neuronal apoptosis (IkB-aytumor necrosis factoryoxidative stressyneuroprotectionypreconditioning)

One mechanism leading to neurodegeneration during Alzheimer’s disease (AD) is amyloid b peptide (Ab) neurotoxicity. Ab elicits in cultured central nervous system neurons a biphasic response: a low-dose neurotrophic response and a high-dose neurotoxic response. Previously we reported that NF-kB is activated by low doses of Ab only. Here we show that NF-kB activation leads to neuroprotection. In primary neurons we found that a pretreatment with 0.1 mM Ab-(1–40) protects against neuronal death induced with 10 mM Ab-(1–40). As a known neuroprotective agent we next analyzed the effect of tumor necrosis factor a (TNF-a). Maximal activation of NF-kB was found with 2 ngyml TNF-a. Pretreatment with TNF-a protected cerebellar granule cells from cell death induced by 10 mM Ab-(1–40). This protection is described by an inverted U-shaped dose response and is maximal with a NF-kB-activating dose. The molecular specificity of this protective effect was analyzed by specific blockade of NF-kB activation. Overexpression of a transdominant negative IkB-a blocks NF-kB activation and potentiates Abmediated neuronal apoptosis. Our findings show that activation of NF-kB is the underlying mechanism of the neuroprotective effect of low-dose Ab and TNF-a. In accordance with these in vitro data we find that nuclear NF-kB immunoreactivity around various plaque stages of AD patients is reduced in comparison to age-matched controls. Taken together these data suggest that pharmacological NF-kB activation may be a useful approach in the treatment of AD and related neurodegenerative disorders. Alzheimer’s disease (AD) is characterized by plaques within many brain regions, including the cerebellum (1). The major component of plaques is an amyloid peptide named bA4 or Ab (2). Ab is a proteolytic product of the larger amyloid precursor protein (APP; ref. 3). Further pathological criteria are the formation of neurofibrillary tangles (4) or increased advanced glycation end products (5), which also can activate NF-kB (6). Mutations in the presenilin genes 1 and 2 (for review see ref. 7) were described, which can lead to increased production of Ab in transgenic mice (8). Mutant presenilin proteins interact directly with APP (9). Activation of NF-kB protects against the proapoptotic action of mutated presenilin-1 (10). Knockout of APP results in agenesis of corpus callosum, memory defects, and reactive gliosis (11, 12). An important mechanism leading to neurodegeneration during AD is Ab neurotoxicity (13). Two dose-dependent effects of Ab were described: a low neurotrophic dose and a high neurotoxic dose (13). Previously we reported that NF-kB is activated only by low doses of Ab in cerebellar granule cells (14). Similarly Ab could activate NF-kB in neuroblastoma cells (15), and Ab-resistant cells have constitutive NF-kB activity (16). To date five mammalian NF-kB DNA-binding subunits are known: p50, p52, p65 (RelA), c-Rel, and RelB (17). Inhibitory proteins are IkB-a, IkB-b, IkB-g(p105), IkB-d(p100), and IkB-« (18, 19). Frequently NF-kB in the nervous system is composed from the DNA-binding subunits p50 and p65 (RelA) and an inhibitory subunit IkB-a (20). In neurons NF-kB can be activated either by glutamate (21, 22) or nerve growth factor (23) or is constitutively activated (24, 25). In glia it can be activated by various proinflammatory cytokines (20) or by nerve growth factor via p75NTR (26). Activation of NF-kB by external stimuli involves IkB kinases (for review see ref. 27), which are present in a large complex with the kinase Nemo (28), also called Ikkg (29). This kinase complex is assembled with the help of the scaffold protein IKAP (30). Expanding on our previous study (14), we have investigated here whether a preincubation with low amounts of Ab leads to neuronal protection against a toxic dose of Ab. We found that intracellular protection of neurons against neurotoxic stimuli is feasible and relies on activated NF-kB. MATERIALS AND METHODS Primary Culture. Primary rat cerebellar granule cells were prepared as described (31). Human recombinant tumor necrosis factor a (TNF-a) was from Boehringer-Mannheim. Ab-(1–40) (lot nos. 506773 and 510598) and Ab-(40–1) (lot no. H-2972) (Bachem) were used as described (14). Immunofluorescence and Cell Survival Analysis. Cells were fixed for 2 min in ethanol and for 5 min in 3.7% formaldehyde and immunostained with an a-p65 mAb (BoehringerMannheim) detected with a biotinylated a-mouse antibody decorated with streptavidin Cy3. Analysis of neuronal survival in parallel to immunostaining was performed with the fluorescent nuclear dye 49,6-diamidino-2-phenylindole (DAPI) (Boehringer-Mannheim), and images were digitally mounted (14) or digitally captured with a Princeton Instruments (Trenton, NJ) MicroMax camera. Nuclear chromatin morphology was analyzed with a 403 objective. Nonviable neurons were recognized by nuclear condensation andyor fragmented chromatin. The number of viable and nonviable neurons was counted in five fixed fieldsychamber of up to five separate cultures. Biolistic Transfection. One microgram DNAy1 mg Gold was used to coat 0.6-mm Gold particles (Bio-Rad) as described by the manufacturer. Granule cells were seeded in 4-well slides 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. PNAS is available online at www.pnas.org. Abbreviations: Ab, amyloid b peptide; TNF-a, tumor necrosis factor a; AD, Alzheimer’s disease; DAPI, 49,6-diamidino-2-phenylindole; NBB, Netherlands Brain Bank; ROI, reactive oxygen intermediate; PDTC, pyrrolidine dithiocarbamate. ‡To whom reprint requests should be addressed. E-mail: kaltschm@ ruf.uni-freiburg.de.