Amyloid precursor protein mediates proinflammatory activation of monocytic lineage cells

Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular deposition of beta-amyloid (Abeta) peptide containing neuritic plaques. Abeta peptides are proteolytically derived from the membrane-bound amyloid precursor protein (APP). Although the function of APP is not entirely clear, previous studies demonstrate that neuronal APP colocalizes with beta(1) integrin receptors at sites of focal adhesion, suggesting that APP is involved in mediating neuronal process adhesion. Integrin-dependent adhesion is also a well-characterized component of immune cell proinflammatory activation. Using primary mouse microglia and the human monocytic cell line, THP-1, we have begun investigating the role of APP in integrin-dependent activation. Co-immunoprecipitation studies demonstrate that APP is recruited into a multi-receptor signaling complex during beta(1) integrin-mediated adhesion of monocytes. Stimulation induces a subsequent, specific recruitment of tyrosine phosphorylated proteins to APP, including Lyn and Syk. Antibody cross-linking of cell surface APP leads to a similar response characterized by activation and recruitment of tyrosine kinases to APP as well as subsequent activation of mitogen-activated protein kinases and increased proinflammatory protein levels. These data demonstrate that APP can act as a proinflammatory receptor in monocytic lineage cells and provide insight into the contribution of this protein to the inflammatory conditions described in Alzheimer's disease.     

The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.     

The β-Amyloid Precursor Protein (APP) and Alzheimer's Disease: Does the Tail Wag the Dog?

The β-amyloid precursor protein has been the focus of much attention from the Alzheimer's disease community for the past decade and a half. The β-amyloid precursor protein holds a pivotal position in Alzheimer's disease research because it is the precursor to the amyloid β-protein which many believe plays a central role in Alzheimer's disease pathogenesis. It was also the first gene in which mutations associated with inherited Alzheimer's disease were found. Although the molecular details of the generation of amyloid β-protein from β-amyloid precursor protein are being unraveled, the actual physiological functions of β-amyloid precursor protein are far from clear. This situation is changing as accumulating new evidence suggests that the C-terminal cytosolic tail of β-amyloid precursor protein may have multiple biological activities, ranging from axonal transport to nuclear signaling. This article reviews the current state of knowledge about the biological functions of β-amyloid precursor protein .     

Amyloid beta and amylin fibrils induce increases in proinflammatory cytokine and chemokine production by THP-1 cells and murine microglia

Activated microglia surrounding amyloid beta-containing senile plaques synthesize interleukin-1, an inflammatory cytokine that has been postulated to contribute to Alzheimer's disease pathology. Studies have demonstrated that amyloid beta treatment causes increased cytokine release in microglia and related cell cultures. The present work evaluates the specificity of this cellular response by comparing the effects of amyloid beta to that of amylin, another amyloidotic peptide. Both lipopolysaccharide-treated THP-1 monocytes and mouse microglia showed significant increases in mature interleukin-1beta release 48 h following amyloid beta or human amylin treatment, whereas nonfibrillar rat amylin had no effect on interleukin-1beta production by THP-1 cells. Lipopolysaccharide-stimulated THP-1 cells treated with amyloid beta or amylin also showed increased release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6, as well as the chemokines interleukin-8 and macrophage inflammatory protein-1alpha and -1beta. THP-1 cells incubated with fibrillar amyloid beta or amylin in the absence of lipopolysaccharide also showed significant increases of both interleukin-1beta and tumor necrosis factor-alpha mRNA. Furthermore, treatment of THP-1 cells with amyloid fibrils resulted in an elevated expression of the immediate-early genes c-fos and junB. These studies provide further evidence that fibrillar amyloid peptides can induce signal transduction pathways that initiate an inflammatory response that is likely to contribute to Alzheimer's disease pathology.     

Ion channel hypothesis for Alzheimer amyloid peptide neurotoxicity

Summary 1. Alzheimer's disease (AD) is a chronic dementia and neurodegenerative disorder affecting the oldest portions of the population. Brains of AD patients accumulate large amount of the AP peptide in amyloid plaques.2. The AP[1–40] peptide is derived by proteolytic processing from a much larger amyloid precursor protein (APP), and has been circumstantially identified as the toxic principle causing cell damage in the disease.4. The AP[1–40] peptide is able to form quite characteristic calcium channels in planar lipid bilayers. These channels have conductances in the nS range, and can dissipate ion gradients quickly. The peptide can also cause equivalent cation conductances in cells.5. We suggest that amyloid channel blocking agents might be therapeutically useful in Alzheimer's Disease, and have constructed molecular models of the channels to aid in the design of such compounds.     

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts

Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.     

Implications of the kynurenine pathway and quinolinic acid in Alzheimer's disease

The kynurenine pathway (KP) is a major route of L-tryptophan catabolism leading to production of a number of biologically active molecules. Among them, the neurotoxin quinolinic acid (QUIN), is considered to be involved in the pathogenesis of a number of inflammatory neurological diseases. Alzheimer's disease is the major dementing disorder of the elderly that affects over 20 million peoples world-wide. Most of the approaches to explain the pathogenesis of Alzheimer's disease focus on the accumulation of amyloid beta peptide (A beta), in the form of insoluble deposits leading to formation of senile plaques, and on the formation of neurofibrillary tangles composed of hyperphosphorylated Tau protein. Accumulation of A beta is believed to be an early and critical step in the neuropathogenesis of Alzheimer's disease. There is now evidence for the KP being associated with Alzheimer's disease. Disturbances of the KP have already been described in Alzheimer's disease. Recently, we demonstrated that A beta 1-42, a cleavage product of amyloid precursor protein, induces production of QUIN, in neurotoxic concentrations, by macrophages and, more importantly, microglia. Senile plaques in Alzheimer's disease are associated with evidence of chronic local inflammation (especially activated microglia) A major aspect of QUIN toxicity is lipid peroxidation and markers of lipid peroxidation are found in Alzheimer's disease. Together, these data imply that QUIN may be one of the critical factors in the pathogenesis of neuronal damage in Alzheimer's disease. This review describes the multiple correlations between the KP and the neuropathogenesis of Alzheimer's disease and highlights more particularly the aspects of QUIN neurotoxicity, emphasizing its roles in lipid peroxidation and the amplification of the local inflammation.     

Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases beta-cleavage of amyloid precursor protein and amyloid-beta production in vivo

Generation and deposition of the amyloid beta (Abeta) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and gamma-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits beta-cleavage of APP and reduces levels of secreted and intracellular Abeta in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Abeta in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases beta-cleavage of APP and results in a significant reduction in the level of Abeta40 and Abeta42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Abeta. This pivotal first report of central Abeta lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.     

Amyloid beta protein activates PKC-delta and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

The increased accumulation of activated microglia containing amyloid beta protein (Abeta) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Abeta. We investigated intracellular signaling in response to Abeta stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-delta but not that of PKC-alpha or -epsilon is increased by stimulation of microglia with Abeta, with a striking tyrosine phosphorylation of PKC-delta. In microglia stimulated with Abeta, tyrosine phosphorylation of PKC-delta was evident at the membrane fraction without an overt translocation of PKC-delta. PKC-delta co-immunoprecipitated with MARCKS from microglia stimulated with Abeta. Abeta induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Abeta. Taken together with our previous observations that Abeta-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Abeta induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-delta signaling pathway in microglia.     

How chronic inflammation can affect the brain and support the development of Alzheimer's disease in old age: the role of microglia and astrocytes

A huge amount of evidence has implicated amyloid beta (A beta) peptides and other derivatives of the amyloid precursor protein (beta APP) as central to the pathogenesis of Alzheimer's disease (AD). It is also widely recognized that age is the most important risk factor for AD and that the innate immune system plays a role in the development of neurodegeneration. Little is known, however, about the molecular mechanisms that underlie age-related changes of innate immunity and how they affect brain pathology. Aging is characteristically accompanied by a shift within innate immunity towards a pro-inflammatory status. Pro-inflammatory mediators such as tumour necrosis factor-alpha or interleukin-1 beta can then in combination with interferon-gamma be toxic on neurons and affect the metabolism of beta APP such that increased concentrations of amyloidogenic peptides are produced by neuronal cells as well as by astrocytes. A disturbed balance between the production and the degradation of A beta can trigger chronic inflammatory processes in microglial cells and astrocytes and thus initiate a vicious circle. This leads to a perpetuation of the disease.     

β‐Amyloid catabolism: roles for neprilysin (NEP) and other metallopeptidases?

The steady-state level of amyloid beta-peptide (Abeta) represents a balance between its biosynthesis from the amyloid precursor protein (APP) through the action of the beta- and gamma-secretases and its catabolism by a variety of proteolytic enzymes. Recent attention has focused on members of the neprilysin (NEP) family of zinc metalloproteinases in amyloid metabolism. NEP itself degrades both Abeta(1-40) and Abeta(1-42) in vitro and in vivo, and this metabolism is prevented by NEP inhibitors. Other NEP family members, for example endothelin-converting enzyme, may contribute to amyloid catabolism and may also play a role in neuroprotection. Another metalloproteinase, insulysin (insulin-degrading enzyme) has also been advocated as an amyloid-degrading enzyme and may contribute more generally to metabolism of amyloid-forming peptides. Other candidate enzymes proposed include angiotensin-converting enzyme, some matrix metalloproteinases, plasmin and, indirectly, thimet oligopeptidase (endopeptidase-24.15). This review critically evaluates the evidence relating to proteinases implicated in amyloid catabolism. Therapeutic strategies aimed at promoting A,beta degradation may provide a novel approach to the therapy of Alzheimer's disease.     

Amyloid precursor protein-processing products affect mononuclear phagocyte activation: pathways for sAPP- and Abeta-mediated neurotoxicity

Increasing evidence strongly supports the role of glial immunity in the pathogenesis of Alzheimer's disease (AD). To investigate such events we have developed cell systems mimicking the interactions between beta-amyloid precursor protein (APP)-expressing neurons and brain mononuclear phagocytes (MP; macrophages and microglia). MP were co-cultured with neuronal cells expressing wild type APP or familial AD-linked APP mutants. The latter was derived from recombinant adenoviral constructs. Neuronal APP processing products induced MP activation, reactive oxygen species, and neurotoxic activities. These occurred without the addition of pro-inflammatory cytokines and were reversed by depletion of amyloid beta-peptide (Abeta) and secreted APP (sAPP). Neurotoxic activities were diminished by superoxide dismutase mimetics and NMDA receptor inhibitors. Microglial glutamate secretion was suppressed by the cystine-glutamate antiporter inhibitor and its levels paralleled the depletion of sAPP and Abeta from conditioned media prepared from APP-expressing neurons. The excitotoxins from activated MP were potent enough to evoke recombinant NMDA receptor-mediated inward currents expressed in vitro in the Xenopus oocytes. These results demonstrate that neuronal APP-processing products can induce oxidative neurotoxicity through microglial activation.     

Notch and presenilin: a proteolytic mechanism emerges.

Presenilins are needed for proteolytic processing of transmembrane proteins of the Notch/Lin-12 family and for cleavage of the amyloid precursor protein. Accumulating evidence now strongly implicates Presenilin as the catalytic core of a multiprotein complex that executes an unusual intramembranous cleavage of its substrates. In the case of amyloid precursor protein, this cleavage contributes to the generation of small, toxic amyloid peptides that trigger the pathological development of Alzheimer's disease. In the Notch/Lin-12 pathway, Presenilin-mediated cleavage of the receptor is a crucial feature of ligand-induced receptor activation and signal transduction. In this pathway, the Presenilins perform a regulated cleavage event that follows additional processing steps during receptor maturation and ligand-induced ectodomain removal.     

Proteases and lipoprotein receptors in Alzheimer's disease

Alzheimer's disease (AD) is the leading cause of senile dementia, and is a complex disorder. The pathological hallmarks of AD were discovered by Dr. Alois Alzheimer in 1907, and include deposits of amyloid or senile plaques and neurofibrillar tangles. Plaques are composed of a peptide, termed the Abeta peptide, that is derived by proteolytic processing of the amyloid precursor protein (APP), while neurofibrillar tangles result from a hyperphosphorylation of the tau protein. Mechanisms associated with the formation of plaques and neurofibrillar tangles and their respective contributions to the disease process have been intensely investigated. Proteolytic processing of APP that results in the generation of the Abeta peptide is now well understood and is influenced by several proteins. Recent evidence suggests that the Abeta levels are carefully regulated, and several proteases play an important role in removing the Abeta peptide. Finally, it is becoming apparent that several members of the LDL receptor family play important roles in the brain, and may modulate the course of AD.     

Amyloid beta-peptide interactions with neuronal and glial cell plasma membrane: binding sites and implications for Alzheimer's disease.

The extracellular accumulation of amyloid-beta (Abeta) in neuritic plaques is one of the characteristic hallmarks of Alzheimer's disease (AD), a progressive dementing neurodegenerative disorder of the elderly. By virtue of its structure, Abeta is able to bind to a variety of biomolecules, including lipids, proteins and proteoglycans. The binding of the various forms of Abeta (soluble or fibrillar) to plasma membranes has been studied with regard to the direct toxicity of Abeta to neurons, and the activation of a local inflammation phase involving microglia. The binding of Abeta to membrane lipids facilitates Abeta fibrillation, which in turn disturbs the structure and function of the membranes, such as membrane fluidity or the formation of ion channels. A subset of membrane proteins binds Abeta. The serpin-enzyme complex receptor (SEC-R) and the insulin receptor can bind the monomeric form of Abeta. The alpha7nicotinic acetylcholine receptor (alpha7nAChR), integrins, RAGE (receptor for advanced glycosylation end-products) and FPRL1 (formyl peptide receptor-like 1) are able to bind the monomeric and fibrillar forms of Abeta. In addition, APP (amyloid precursor protein), the NMDA-R (N-methyl-D-aspartate receptor), the P75 neurotrophin receptor (P75NTR), the CLAC-P/collagen type XXV (collagen-like Alzheimer amyloid plaque component precursor/collagen XXV), the scavenger receptors A, BI (SR-A, SR-BI) and CD36, a complex involving CD36, alpha6beta1-integrin and CD47 have been reported to bind the fibrillar form of Abeta. Heparan sulfate proteoglycans have also been described as cell-surface binding sites for Abeta. The various effects of Abeta binding to these membrane molecules are discussed.     

Frame-shifted amyloid precursor protein found in Alzheimer's disease and Down's syndrome increases levels of secreted amyloid beta40

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.     

Neuronal localization of amyloid beta protein precursor mRNA in normal human brain and in Alzheimer''s disease.

Clones for the amyloid beta protein precursor gene were isolated from a cDNA library prepared from the frontal cortex of a patient who had died with a histologically confirmed diagnosis of Alzheimer's disease; they were used to investigate the tissue and cellular distribution of amyloid beta protein precursor mRNA in brain tissues from control patients and from Alzheimer's disease patients. Amyloid beta protein precursor mRNA was expressed in similar amounts in all control human brain regions examined, but a reduction of the mRNA level was observed in the frontal cortex from patients with Alzheimer's disease. By in situ hybridization amyloid beta protein precursor mRNA was present in granule and pyramidal cell bodies in the hippocampal formation and in pyramidal cell bodies in the cerebral cortex. No specific labelling of glial cells or endothelial cells was found. The same qualitative distribution was observed in tissues from control patients and from patients with Alzheimer's disease. Senile plaque amyloid thus probably derives from neurones. The tissue distribution of amyloid beta protein precursor mRNA and its cellular localization demonstrate that its expression is not confined to the brain regions and cells that exhibit the selective neuronal death characteristic of Alzheimer's disease.