Reactive microglia associated with the ␤-amyloid plaques in Alzheimer's disease (AD) brains initiate a sequence of inflammatory events integral to the disease process. We have observed that fibrillar ␤-amyloid peptides activate a tyrosine kinase-based signaling response in primary mouse microglia and the human monocytic cell line, THP-1, resulting in production of neurotoxic secretory products, proinflammatory cytokines, and reactive oxygen species. We report that most of the amyloid-induced tyrosine kinase activity was stimulated after activation of Src family members such as Lyn. However, transduction of the signaling response required for increased production of the cytokines TNF␣ and IL1-␤ was mediated by the nonreceptor tyrosine kinase, Syk. Additionally, ␤-amyloid stimulated an NFB-dependent pathway in parallel that was required for cytokine production. Importantly, TNF␣ generated by the monocytes and microglia was responsible for the majority of the neuorotoxic activity secreted by these cells after ␤-amyloid stimulation but must act in concert with other factors elaborated by microglia to elicit neuronal death. Moreover, we observed that the neuronal loss was apoptotic in nature and involved increased neuronal expression of inducible nitric oxide synthase and subsequent peroxynitrite production. Selective inhibitors of inducible nitric oxide synthase effectively protected cells from toxicity associated with the microglial and monocytic secretory products. This study demonstrates a functional linkage between ␤-amyloid-dependent activation of microglia and several characteristic markers of neuronal death occurring in Alzheimer's disease brains.Key words: Alzheimer's disease; ␤-amyloid; microglia; THP-1 monocytes; signal transduction; tyrosine kinase; Lyn; Syk; inflammation; neurotoxicity; apoptosis; nitric oxide; nitrotyrosine; peroxynitrite; TNF␣; cytokines Alzheimer's disease (AD) is characterized by an accumulation of extracellular deposits of ␤-amyloid and abundant neurofibrillary tangles in the brain that is correlated with a progressive dementia and neuron loss (Berg et al., 1993;Braak and Braak, 1997). There is extensive and compelling evidence that amyloid deposition provokes a microglial-mediated inflammatory response that contributes significantly to the cell loss and cognitive decline that is characteristic of this disease (Akiyama et al., 2000). Significantly, both epidemiological and clinical trial data have demonstrated the value of anti-inflammatory therapies for lowering the incidence, slowing the progression, and reducing the symptomatic severity of AD (McGeer and Rogers, 1992;Rogers et al., 1993;Rich et al., 1995;McGeer et al., 1996;Aisen, 1997;Stewart et al., 1997;Mackenzie and Munoz, 1998).It is now well documented that fibrillar forms of ␤-amyloid serve as an inflammatory stimulus for microglial lineage cells, and the signal transduction cascades mediating the effects of the amyloid peptides have been identified (Del Bo et al

Amyloid-β peptide (Aβ) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the β-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by α-secretase. The production of Aβ is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Aβ generation.

In PC12 cells, epidermal growth factor (EGF) transiently stimulates the mitogen-activated protein (MAP) kinases, ERK1 and ERK2, and provokes cellular proliferation. In contrast, nerve growth factor (NGF) stimulation leads to the sustained activation of the MAPKs and subsequently to neuronal differentiation. It has been shown that both the magnitude and longevity of MAPK activation governs the nature of the cellular response. The activations of MAPKs are dependent upon two distinct small G-proteins, Ras and Rap1, that link the growth factor receptors to the MAPK cascade by activating c-Raf and B-Raf, respectively. We found that Ras was transiently stimulated upon both EGF and NGF treatment of PC12 cells. However, EGF transiently activated Rap1, whereas NGF stimulated prolonged Rap1 activation. The activation of the ERKs was due almost exclusively (>90%) to the action of B-Raf. The transient activation of the MAPKs by EGF was a consequence of the formation of a short lived complex assembling on the EGF receptor itself, composed of Crk, C3G, Rap1, and B-Raf. In contrast, NGF stimulation of the cells resulted in the phosphorylation of FRS2. FRS2 scaffolded the assembly of a stable complex of Crk, C3G, Rap1, and B-Raf resulting in the prolonged activation of the MAPKs. Together, these data provide a signaling link between growth factor receptors and MAPK activation and a mechanistic explanation of the differential MAPK kinetics exhibited by these growth factors.The MAP 1 kinase cascade is one of the principal intracellular signaling pathways linking activation of cell surface receptors to cytoplasmic and nuclear effectors. The MAP kinases (MAPKs), ERK1 and ERK2, have been shown to be essential for cellular proliferation as well as for acquisition and maintenance of a differentiated phenotype (1). One of the best studied models employed to examine how the MAPKs act to regulate cellular phenotypes is the rat pheochromocytoma cell line, PC12 cells. These cells respond to epidermal growth factor (EGF) treatment by an increase in mitotic rates (2). In contrast, nerve growth factor (NGF) stimulation of the PC12 cells results in their differentiation into a sympathetic neuron-like phenotype (3). There is compelling evidence that the longevity of MAPK activation governs whether these cells are stimulated either to proliferate or to withdraw from the cell cycle and differentiate into a neuronal phenotype. EGF and other mitogens provoke the evanescent activation of the MAPKs, whereas NGF treatment results in the sustained activation of this signaling pathway (4 -6). The question of how different growth factors elicit distinct biological outcomes through common signal transduction elements has provoked considerable interest resulting in a complex and controversial literature.The MAPK cascade transduces signals from receptor tyrosine kinases to two members of the Ras family of small Gproteins, Ras and Rap1, which then stimulates the sequential activation of Raf serine/threonine kinases (c-Raf and B-Raf), MEK, and the MAPKs (ERK1...