Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers

Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.     

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 precursor protein cross-linking stimulates beta amyloid production and pro-inflammatory cytokine release in monocytic lineage cells

Beta amyloid peptide-containing neuritic plaques are a defining feature of Alzheimer's disease pathology. Beta amyloid are 38-43 residue peptides derived by proteolytic cleavage of amyloid precursor protein. Although much attention has focused on the proteolytic events leading to beta amyloid generation, the function of amyloid precursor protein remains poorly described. Previously, we reported that amyloid precursor protein functions as a pro-inflammatory receptor on monocytic lineage cells and defined a role for amyloid precursor protein in adhesion by demonstrating that beta(1) integrin-mediated pro-inflammatory activation of monocytes is amyloid precursor protein dependent. We demonstrated that antibody-induced cross-linking of amyloid precursor protein in human THP-1 monocytes and primary mouse microglia stimulates a tyrosine kinase-based pro-inflammatory signaling response leading to acquisition of a reactive phenotype. Here, we have identified pro-inflammatory mediators released upon amyloid precursor protein-dependent activation of monocytes and microglia. We show that amyloid precursor protein cross-linking stimulated tyrosine kinase-dependent increases in pro-inflammatory cytokine release and a tyrosine kinase-independent increase in beta amyloid 1-42 generation. These data provide much needed insight into the function of amyloid precursor protein and provide potential therapeutic targets to limit inflammatory changes associated with the progression of Alzheimer's disease.     

BAG-1M is up-regulated in hippocampus of Alzheimer's disease patients and associates with tau and APP proteins

The accumulation of tau and amyloid beta proteins is the major molecular pathology of Alzheimer's disease (AD). The mechanisms leading to the accumulation of these proteins are not completely clear. Hsc-70/Hsp-70, a chaperone protein, has been shown to bind both these proteins and regulate their degradation. We have previously shown that the co-chaperone protein BAG-1 can inhibit the degradation of tau by forming a complex with Hsc-70 and tau. In this current work, we show that there is an increase in the BAG-1M isoform in the hippocampus of AD patients. In addition, BAG-1 binds to both tau and amyloid precursor protein physically, and is found highly expressed in the same neurons that contain intracellular tau or amyloid in hippocampal sections from AD patients. Over-expression of BAG-1M in cell culture also induced an increase in both tau and amyloid precursor protein levels. In conclusion, we report a specific increase of BAG-1M in human AD patients, which is both physically and functionally associated to the two major molecular markers of AD.     

Interaction of Alzheimer's presenilin-1 and presenilin-2 with Bcl-X(L). A potential role in modulating the threshold of cell death.

The familial Alzheimer's disease gene products, presenilin-1 and presenilin-2, have been reported to be functionally involved in amyloid precursor protein processing, notch receptor signaling, and programmed cell death or apoptosis. However, the molecular mechanisms by which presenilins regulate these processes remain unknown. With regard to the latter, we describe a molecular link between presenilins and the apoptotic pathway. Bcl-X(L), an anti-apoptotic member of the Bcl-2 family was shown to interact with the carboxyl-terminal fragments of PS1 and PS2 by the yeast two-hybrid system. In vivo interaction analysis revealed that both PS2 and its naturally occurring carboxyl-terminal products, PS2short and PS2Ccas, associated with Bcl-X(L), whereas the caspase-3-generated amino-terminal PS2Ncas fragment did not. This interaction was corroborated by demonstrating that Bcl-X(L) and PS2 partially co-localized to sites of the vesicular transport system. Functional analysis revealed that presenilins can influence mitochondrial-dependent apoptotic activities, such as cytochrome c release and Bax-mediated apoptosis. Together, these data support a possible role of the Alzheimer's presenilins in modulating the anti-apoptotic effects of Bcl-X(L).     

Proteolytic mechanisms in amyloid-beta metabolism: therapeutic implications for Alzheimer's disease

The accumulation of the amyloid-beta peptide, the main constituent of the "amyloid plaque", is widely considered to be the key pathological event in Alzheimer's disease. Amyloid-beta is produced from the amyloid precursor protein through the action of the proteases beta-secretase and gamma-secretase. Alternative cleavage of amyloid precursor protein by the enzyme alpha-secretase precludes amyloid-beta production. In addition, several proteases are involved in the degradation of amyloid-beta. This review focuses on the proteolytic mechanisms of amyloid-beta metabolism. An increasingly detailed understanding of proteolysis in both amyloid-beta deposition and clearance has identified some of these proteases as potential therapeutic targets for Alzheimer's disease. A more complex knowledge of these proteases takes us one step closer to developing "disease-modifying" therapies, but these advances also emphasize that significant challenges must be overcome before clinically effective drugs to treat Alzheimer's disease become a reality.     

Regulation of gamma-secretase activity in Alzheimer's disease

The gamma-secretase complex is an intramembrane aspartyl protease that cleaves its substrates along their transmembrane regions. Sequential proteolytic processing of amyloid precursor protein by beta- and gamma-secretase produces amyloid beta-peptides, which are the major components of amyloid plaques in the brains of Alzheimer's disease patients. The gamma-secretase complex is therefore believed to be critical in the pathogenesis of Alzheimer's disease. Here we review the range of factors found to affect the nature and degree of gamma-secretase complex activity; these include gamma-secretase complex assembly and activation, the integral regulatory subunit CD147, transient or weak binding partners, the levels of cholesterol and sphingolipids in cell membranes, and inflammatory cytokines. Integrated knowledge of the molecular mechanisms supporting the actions of these factors is expected to lead to a comprehensive understanding of the functional regulation of the gamma-secretase complex, and this, in turn, should facilitate the development of novel therapeutic strategies for the treatment of Alzheimer's disease.     

Presenilin 1 regulates pharmacologically distinct gamma -secretase activities. Implications for the role of presenilin in gamma -secretase cleavage

Presenilins (PSs) are polytopic membrane proteins that have been implicated as potential therapeutic targets in Alzheimer's disease because of their role in regulating the gamma-secretase cleavage that generates the amyloid beta protein (Abeta). It is not clear how PSs regulate gamma-secretase cleavage, but there is evidence that PSs could be either essential cofactors in the gamma-secretase cleavage, gamma-secretase themselves, or regulators of intracellular trafficking that indirectly influence gamma-secretase cleavage. Using presenilin 1 (PS1) mutants that inhibit Abeta production in conjunction with transmembrane domain mutants of the amyloid protein precursor that are cleaved by pharmacologically distinct gamma-secretases, we show that PS1 regulates multiple pharmacologically distinct gamma-secretase activities as well as inducible alpha-secretase activity. It is likely that PS1 acts indirectly to regulate these activities (as in a trafficking or chaperone role), because these data indicate that for PS1 to be gamma-secretase it must either have multiple active sites or exist in a variety of catalytically active forms that are altered to an equivalent extent by the mutations we have studied.     

Recent work on Alzheimer''s disease transgenics

The most significant feature of the current transgenic models of Alzheimer's disease continues to be the amyloid phenotype. In the past year, mice have been more extensively characterized in terms of the effect of amyloid accumulation on downstream events, such as neurodegeneration and behavioral changes, but the results have been complex. Genetic crosses have shown that apolipoprotein E and TGF-β1 influence the deposition event and that the presenilins act synergistically with the amyloid precursor protein in pathology development. The mice have great utility in amyloid modulation studies but are still not complete models of Alzheimer's disease.     

Presenilin mutations and calcium signaling defects in the nervous and immune systems

Presenilin-1 (PS1) is thought to regulate cell differentiation and survival by modulating the Notch signaling pathway. Mutations in PS1 have been shown to cause early-onset inherited forms of Alzheimer's disease (AD) by a gain-of-function mechanism that alters proteolytic processing of the amyloid precursor protein (APP) resulting in increased production of neurotoxic forms of amyloid beta-peptide. The present article considers a second pathogenic mode of action of PS1 mutations, a defect in cellular calcium signaling characterized by overfilling of endoplasmic reticulum (ER) calcium stores and altered capacitive calcium entry; this abnormality may impair synaptic plasticity and sensitize neurons to apoptosis and excitotoxicity. The calcium signaling defect has also been documented in lymphocytes, suggesting a contribution of immune dysfunction to the pathogenesis of AD. A better understanding of the calcium signaling defect resulting from PS1 mutations may lead to the development of novel preventative and therapeutic strategies for disorders of the nervous and immune systems.     

A RIP tide in neuronal signal transduction

The generation of nuclear signaling proteins by regulated intramembrane proteolysis (RIP) is a new paradigm of signal transduction. Mammalian proteins that are processed by RIP include SREBP-1, Notch-1, amyloid precursor protein (APP), and ErbB-4. Intramembranous gamma-secretase cleavage of APP plays a central role in Alzheimer's disease by generating the amyloid beta protein. An intriguing possibility is that the cognate C-terminal fragment generated by gamma-secretase cleavage could also play a role through the regulation of nuclear signaling events. Thus, RIP may contribute to both brain development and degeneration and may provide unexpected diversity to the signaling repertoire of a cell.     

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.     

Alzheimer's disease untangled.

The last year has seen major advances in the study of Alzheimer's disease (AD). Four mutations involving amino acid substitutions in exons 16 and 17 of the amyloid precursor protein (APP) gene, have been identified which co-segregate with the disease in some families multiply affected by early onset Alzheimer's disease. These mutations are strongly suggestive of a causative role for the amyloid precursor protein in Alzheimer's disease. Despite their rarity, these mutations are important because they represent the first known cause of Alzheimer's disease. Processing of APP must be central to the pathogenesis of the disease although the precise effects of these amino acid substitutions are not understood. Work is now being undertaken to characterise the processing pathways of APP and to identify other causes of AD. The development of models of AD using the APP mutations offers the possibility of identifying drug targets and developing more effective treatments than are presently available.     

Are amyloid-degrading enzymes viable therapeutic targets in Alzheimer's disease?

: The amyloid cascade hypothesis of Alzheimer's disease envisages that the initial elevation of amyloid β-peptide (Aβ) levels, especially of Aβ(1-42) , is the primary trigger for the neuronal cell death specific to onset of Alzheimer's disease. There is now substantial evidence that brain amyloid levels are manipulable because of a dynamic equilibrium between their synthesis from the amyloid precursor protein and their removal by amyloid-degrading enzymes (ADEs) providing a potential therapeutic strategy. Since the initial reports over a decade ago that two zinc metallopeptidases, insulin-degrading enzyme and neprilysin (NEP), contributed to amyloid degradation in the brain, there is now an embarras de richesses in relation to this category of enzymes, which currently number almost 20. These now include serine and cysteine proteinases, as well as numerous zinc peptidases. The experimental validation for each of these enzymes, and which to target, varies enormously but up-regulation of several of them individually in mouse models of Alzheimer's disease has proved effective in amyloid and plaque clearance, as well as cognitive enhancement. The relative status of each of these enzymes will be critically evaluated. NEP and its homologues, as well as insulin-degrading enzyme, remain as principal ADEs and recently discovered mechanisms of epigenetic regulation of NEP expression potentially open new avenues in manipulation of AD-related genes, including ADEs.     

The PreA4(695) precursor protein of Alzheimer''s disease A4 amyloid is encoded by 16 exons.

Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4(695] of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.     

Bilobalide regulates soluble amyloid precursor protein release via phosphatidyl inositol 3 kinase-dependent pathway

Bilobalide (BB) is a sesquiterpenoid extracted from Ginkgo biloba leaves. An increasing number of studies have demonstrated its neuroprotective effects. The neuroprotective mechanisms may be associated with modulation of intracellular signaling cascades such as the phosphatidyl inositol 3-kinase (PI3K) pathway. Using differentiated SH-SY5Y cells, this study investigated whether BB modulation of intracellular signaling pathways, such as the protein kinase C (PKC) and PI3K pathways, contributes to amyloid precursor protein (APP) metabolism, a key event in the pathogenesis of Alzheimer’s disease (AD). We demonstrated in this study that BB enhanced the secretion of α-secretase-cleaved soluble amyloid precursor protein (sAPPα, a by-product of non-amyloidogenic processing of APP) and decreased the β amyloid protein (Aβ, a by-product of amyloidogenic processing of APP) via PI3K-dependent pathway. The PI3K pathway mediated the rapid effect of BB on APP processing possibly via regulation of intracellular APP trafficking. After longer time BB incubation (12 h), this effect was reinforced by PI3K pathway-mediated up-regulation of disintegrin and metalloproteinase domain-containing protein 10 (ADAM10, an α-secretase candidate). Given the strong association between APP metabolism and AD pathogenesis, the ability of BB to regulate APP processing suggests its potential use in AD prevention.     

Ginkgo biloba extract (Egb 761) inhibits beta-amyloid production by lowering free cholesterol levels

Ginkgo biloba extract (EGb 761) can improve cognitive function in patients with Alzheimer's disease, but the molecular mechanisms underlying this effect remain undefined. Because free cholesterol may be involved in the production of β-amyloid precursor protein and amyloid β-peptide, key events in the development of Alzheimer's disease, we examined EGb 761 in relation to cholesterol and amyloidogenesis. In aging rats, EGb 761 treatment lowered circulating free cholesterol and inhibited the production of brain β-amyloid precursor protein and amyloid β-peptide. Exposure of PC12 cells to EGb 761 decreased the processing of β-amyloid precursor protein and abolished cholesterol-induced overproduction of this protein. Exposure of human NT2 cells to EGb 761 decreased free cholesterol influx and increased free cholesterol efflux. Our findings indicate that free circulating and intracellular cholesterol levels affect the processing of β-amyloid precursor protein and amyloidogenesis. Our findings also provide the first demonstration that EGb 761 can influence these mechanisms.     

Amyloid-beta precursor protein processing in neurodegeneration

The amyloid-beta precursor protein is proteolytically cleaved by secretases, resulting in a series of fragments, including the amyloid-beta peptide of Alzheimer's disease. The amyloid precursor protein, when membrane anchored, could operate as a receptor. After cleavage, the soluble ectodomain exerts a trophic function in the subventricular zone. The amyloid-beta peptide itself has a depressant role in synaptic transmission, with both physiological and pathological implications. During the past two years, much time has been invested in determining the molecular pathways that regulate the processing and the signal transduction of the amyloid precursor protein. However, the absence of consistent and informative phenotypes in different loss of function animal models make elucidating the molecular actions of the amyloid-beta precursor protein an ongoing challenge.     

Isoform-Specific Modulation by Apolipoprotein E of the Activities of Secreted β-Amyloid Precursor Protein

Abstract: The genes for both the β-amyloid precursor protein and apolipoprotein E (ApoE) have been linked to Alzheimer's disease. This connection suggests the possibility that these proteins interact physically or functionally. To explore this idea, we focused on the neuroprotective activity of secreted amyloid precursor protein (sAPP) and related signal transduction events. After coincubation with ApoE, sAPP exhibited an enhanced [Ca²⁺]_i-lowering activity and enhanced protection against excitotoxicity in rat primary hippocampal neurons. In contrast, the stimulation of phosphoinositide production by sAPP was inhibited by ApoE. Kinetic analyses and coimmunoprecipitation experiments indicated that these actions result from formation of a heteromeric complex between ApoE and sAPP. Furthermore, the ApoE4 isoform, which seems to accelerate the onset of Alzheimer's disease, was less potent than ApoE3 in modifying each activity of sAPP. These data suggest that sAPP-dependent neuroprotective mechanisms would be compromised in individuals expressing ApoE4, a scenario that may contribute to the development of Alzheimer's disease.