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Alzheimer's disease (AD) is the major neurodegenerative disease of the aging brain and, whereas the underlying pathology remains extremely complex and poorly understood, poses an ever-expanding burden on health services in the context of an aging population. By 2010, it is estimated that there will be half a million AD sufferers in the UK, while currently there are greater than 12 million sufferers worldwide. AD is characterized by a decline in cognitive function that progresses slowly, leaving patients in the later stages of the illness bedridden, incontinent, and dependent on custodial care, with death occurring, on average, 9 years after diagnosis. Although there are currently a few drugs used to help manage the cognitive effects of AD, namely the acetylcholinesterase inhibitors and the N-methyl-D-aspartate receptor antagonist memantine, there is presently no available therapy to arrest or modify the progress of the disease (Vardy, Catto, & Hooper, 2005). [ABSTRACT FROM AUTHOR]

Notch proteins are evolutionary conserved transmembrane receptors used by metazoans to direct cell fate decisions, proliferation, differentiation and cell death at all stages of development, including self-renewing adult tissues. Notch signaling is a well-established example of a pathway that is mediated by Regulated Intramembrane Proteolysis (RIP). Upon binding of ligand, the Notch receptor undergoes successive proteolytic cleavages - an ectodomain shedding cleavage followed by intramembrane proteolysis by γ-secretase. This process releases the Notch intracellular domain, which translocates to the nucleus to activate its target genes. Deciphering the proteolytic mechanism for Notch activation relied on the convergence of previously independent fields of research, revealing that the Notch receptor resembled another Type I membrane protein, the amyloid-γ precursor protein, in that both are proteolytically cleaved within their transmembrane domains (TMDs) by the same protease, γ-secretase, whose catalytic center resided in the protein Presenilin. Intramembrane proteolysis has continued to emerge as an exciting research area in cell biology. Recent studies on γ-secretase function have begun to reveal the molecular details involved in ectodomain shedding and intramembrane cleavage events as well as the importance of endocytosis and endosomal sorting as key regulators of γ-secretase cleavage of Notch [ABSTRACT FROM AUTHOR]

Deposition of the amyloid β-protein is a defining pathological characteristic of Alzheimer's disease, and this small protein is proteolytically produced from the amyloid β-protein precursor. ,-Secretase is responsible for the second cut, which forms the C-terminus of amyloid-β and determines how much of the transmembrane domain is included in this aggregation-prone protein. This intramembrane aspartyl protease is a complex of four different integral membrane proteins: presenilin, nicastrin, Aph-1 and Pen-2. During assembly and maturation of the protease complex, presenilin is endoproteolyzed into two subunits, each of which contributes one aspartate to the active site. A model of successive proteolysis may explain how Alzheimer-causing mutations in presenilin can both decrease enzyme activity and increase the proportion of longer, more aggregation-prone forms of amyloid-β. Substrate apparently interacts with an initial docking site before passing in whole or in part between the two presenilin subunits to the internal water-containing active site. The ectodomain of nicastrin also interacts with the N-terminus of the substrate as an essential step in substrate recognition and processing. Inhibitors and allosteric modulators of γ-secretase activity are under investigation as potential Alzheimer therapeutics. Elucidation of detailed structural features of γ-secretase is the next logical step toward understanding how this enzyme carries out intramembrane proteolysis and will set the stage for structure-based drug design [ABSTRACT FROM AUTHOR]

Rhomboid proteins are a class of serine proteases conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. In yeast, two members of the rhomboid family are known, Rbd2 and Pcp1. Rbd2 is associated with the Golgi apparatus, but its function and its substrates are still unknown. The rhomboid protease Pcp1, located in the mitochondrial inner membrane, catalyses the second step in the proteolytic processing of cytochrome $c$ peroxidase, a mitochondrial enzyme that acts as a peroxide scavenger. Pcp1 also affects the morphology of mitochondria by acting on, Mgm1, a dynamin-related GTPase. Mgm1 is present in short and long forms, and both isoforms are required for fusion of mitochondria and the maintenance of mitochondrial DNA. The proteolytic conversion of the long to the short form is catalysed by Pcp1. The cleavage sites in their substrates are not typical transmembrane domains but hydrophobic regions that must be actively translocated into the inner mitochondrial membrane by an ATP-consuming process to make them accessible to cleavage by the rhomboid protease [ABSTRACT FROM AUTHOR]

Intramembrane proteolysis catalyzed by rhomboid proteases plays key roles in such diverse cell communication events as receptor tyrosine kinase signalling during animal development, and quorum sensing during bacterial growth. In these contexts, rhomboid proteins act in the signal-sending cell to activate signal precursor proteins and initiate the signalling event. Recent biochemical advances have culminated in the first high-resolution crystal structures of an intramembrane protease, and a pure enzyme reconstitution system for studying rhomboid activity. Functional studies have expanded the cellular role of rhomboid proteins to broad biological processes, including host-cell invasion by malaria parasites, which is the first implication of these enzymes as possible therapeutic targets in human disease [ABSTRACT FROM AUTHOR]

Among the known intramembrane-cleaving proteases (I-CLiPs), the aspartate proteases are unique. Unlike I-CLiPs of the serine- and metalloprotease-type, which share their respective active site motifs with their classical counterparts, the aspartate protease I-CLiPs acquired a novel characteristic GxGD active site motif during evolution. These so-called GxGD-type proteases include the presenilin (PS), signal peptide peptidase (SPP), SPP-like protease (SPPL) families and the related type IV prepilin peptidase family, bacterial leader peptidases, which share the same active site motif, but which cleave their substrates directly at, rather than within, the membrane. PS, SPP and SPPLs adopt a similar, but inverted membrane topology with respect to their active site orientation. PS is the founding member of the GxGD-type I-CLiPs and has been identified as the catalytic subunit of Γ-secretase. The major function of this protease complex appears to be the clearance of the remnants of a large number of type I membrane proteins that have undergone shedding of their ectodomains. For some substrates of Γ-secretase, most prominently for the cell surface receptor Notch, Γ-secretase cleavage is coupled with signaling by the release of a nuclear-targeted intracellular domain (ICD). In the case of Notch, the ICD functions in the nucleus as a key transcriptional regulator for cell differentiation in development and adulthood. In addition, Γ-secretase is a pivotal enzyme in Alzheimer's disease (AD), responsible for the liberation of the AD-causing amyloid β-peptide from its precursor protein. SPP and SPPLs exert similar functions, which, however, use type II membrane proteins as substrates consistent with their opposite topologies compared to PS. Thus, the major function of SPP is likely to be to clear the ER membrane of signal peptides of secretory proteins, whereas SPPL2a and b have recently been shown to cleave tumor necrosis factor UPalpha to release an ICD that triggers interleukin-12 signaling. Despite the similarities in their overall biological functions, the major difference is that PS requires partner proteins for its proteolytic function, whereas SPP and probably also the SPPLs do not [ABSTRACT FROM AUTHOR]

Signal peptide peptidases (SPPs) are the most recently identified members of a protease family of integral membrane proteins that includes the intensively studied presenilin 1 (PS1) and presenilin (PS2) proteins. There are 5 human genes encoding SPPs which can be divided into two branches based on homology and initial functional studies. One branch, which is the focus of this chapter, consists of the SPP and SPPL3 proteins. The second branch will be the focus of a subsequent chapter, and consists of the three SPPL2 proteins (SPPL2a, b, and c). The SPP proteins are conserved through evolution with family members found in fungi, archaea and plants. Presenilins (PSs) and SPPs cleave substrate polypeptides within a transmembrane region, but differ in that PSs cleave type 1 membrane proteins whereas SPPs cleave type 2 membrane proteins. SPPs and PSs have low overall sequence homology, yet exhibit considerable structural similarity as well as strict conservation of several small motifs. They are both multipass membrane proteins that contain two conserved active site motifs YD and GxGD in adjacent membrane-spanning domains and a conserved PAL motif of unknown function near their C-termini. They differ in that the active site topology of SPPs is inverted relative to PSs. Moreover, SPP and SPPL3 appear to function as proteases without the need for additional cofactors. In contrast, PSs function as the UPgamma-secretase protease only when complexed with three accessory proteins. Although the biological roles of PSs are reasonably well understood, the biological roles of SPP are largely unknown, and only a few endogenous substrates for SPP have been identified. SPP and possibly SPPL3 appear to cleave a number of endogenous type 2 signal peptides and these genes are essential genes in the development of several model organisms. In addition, in many human parasites, there is only a single SPP gene that is most closely related to the human SPP. Thus, SPPs may be novel antiviral drug targets in humans and represent a novel drug target for major human pathogens such as malaria [ABSTRACT FROM AUTHOR]

The site-2 protease (S2P) is a highly hydrophobic integral membrane protean required for cleavage of various mambrane-bound transcription factors within a membrane-spanning helix. S2P was the first intramembrane-cleaving protease to be recognized but more has been learned about other such proteins. Fundamental questions about the role and function of S2P remain unanswered. S2P plays a crucial role in mammalian lipid metabolism and the unfolded protein response. Thus, finding the answers has implications for our understanding of human health and disease. Recent advances with rhomboid proteins and gamma secretase indicate that the technical challenges to getting the answers can be overcome [ABSTRACT FROM AUTHOR]

There has been gratifying progress in the development of drugs for Alzheimer's disease (AD). Even though the current generation of medications, the cholinesterase inhibitors (CEIs), has produced only modest benefits, our concept of an "effective" therapy has matured considerably over this time. A less visible but equally important advance has been a quantum leap in expertise in clinical trial methodology. This chapter reviews the methodological underpinnings of clinical trials in AD: patient selection issues, key design issues, and an overview of currently available agents and the prospects for drugs of the future. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which abnormal filamentous inclusions accumulate in dystrophic and dying nerve cells. These inclusions have been described as neurofibrillary tangles (NFTs) of which paired helical filaments (PHFs) are the primary constituents (1-3). The PHFs primarily are composed of the microtubule-associated protein tau, which has undergone posttranslational modification such as phosphorylation (4,5), glycation (6-9), and crosslinking by transglutaminase (TGase) (10-16). Crosslinking of proteins catalyzed by TGase results in the deposition of these proteins into insoluble matrices that are resistant to proteolytic digestion and chaotropic denaturation (for review see ref. 17). In this regard, TGase has been demonstrated to be associated with NFTs from the Alzheimer brain (13,14) and to exhibit elevated activity in the AD brain as compared with normal aged-matched control subjects (16). Here we discuss important aspects of TGase and in vitro experimental approaches that address its ability to catalyze the tau protein into insoluble complexes exhibiting biophysical and immuno-logical properties similar to those of the Alzheimer PHFs and NFTs. [ABSTRACT FROM AUTHOR]

The role of Aβ accumulation in the pathogenesis of Alzheimer's disease (AD) is supported by genetic studies showing that mutations in the amyloid-β precursor protein (APP) that alter Aβ production are linked to a subset of familial AD (FAD) cases with autosomal penetrance (reviewed in ref. 1). Several of these FAD-associated APP mutations, as well as FAD-associated mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes, lead to an increase in the production of Aβ1-42 relative to Aβ1_40. This, combined with the observation that these peptides are differentially deposited in senile plaques (SPs) in vivo, suggests that differential production of Aβ1-40 and Aβ1_42 may be crucially important in the pathogenesis of AD. Thus, it is important to use techniques that not only quantitate Aβ production, but also specifically differentiate between these two peptides in a variety of experimental paradigms. Here we describe the use of a highly sensitive sandwich-ELISA (enzyme-linked immunosorbent assay) to quantitate both Aβ1-40 and Aβ1-42 in soluble pools, after secretion by cultured cells into the medium or in human cerebrospinal fluid (CSF) samples, as well as in insoluble pools, as found intracellularly in cultured cells, or deposited in the brain parenchyma. [ABSTRACT FROM AUTHOR]

Tau was originally isolated from brain microtubules and shown to be a microtubule-associated protein (MAP) that promoted tubulin polymerization (1). It is largely confined to axons, where it is the major MAP. It promotes microtubule nucleation, elongation, and bundling, and stabilizes microtubules by inhibiting depolymerization. [ABSTRACT FROM AUTHOR]

Antibodies to the microtubule-associated protein tau have been used for more than a decade, both in studies of the role of tau neuronal function, and in examination of the neurofibrillary pathology of Alzheimer's disease (AD). The vast majority of the available antibodies have been produced with preparations obtained from the brains of patients with AD, although a few antibodies have been generated with tau purified from bovine brain. This chapter restricts discussion to the production and characterization of monoclonal antibodies, although some investigators continue to use affinity-purified polyclonal antibodies. The opinions expressed in this chapter are based on the author's experience in the production of several series of monoclonal antibodies to tau and paired helical filaments (PHF-tau) over the last 10 yr. There is no doubt that modifications in the procedures described can be developed for specific purposes, but the discussion is confined to those methods that we have found to be reliable and informative. [ABSTRACT FROM AUTHOR]

Until recently, the detergent insolubility of certain membrane-associated proteins was singularly attributed to an association with the cytoskeleton. However, in 1988 we observed that a number of glycosyl-phosphatidylinositol (GPI)-anchored proteins were resistant to solubilization by nonionic detergents such as Triton X-100 (1). This detergent insolubility is acquired as the proteins pass through the endoplasmic reticulum and on to the Golgi apparatus (2), and arises not from a direct interaction of the GPI-anchored proteins with cytoskeletal elements but as a result of the specific lipid composition of the membrane domains with which these proteins associate (3,4). Mammalian cell membranes contain hundreds of individual lipid species which can be grouped under several major headings (e.g., glycerophospholipids, sphingomyelins, ceramides, glycosphingolipids, and cholesterol) (2,5,6). Glycerophospholipids, such as phosphatidylcholine and phosphatidylethanolamine, predominate in the membrane milieu. Consequently, the bulk of the cell membrane is fluid and in a continual state of flux. However, the membrane domains with which GPI-anchored proteins associate are enriched with sphingolipids and cholesterol, making them less fluid than the membrane milieu (2,4). Such membrane domains have been referred to as "lipid rafts" (7) and there has been some controversy as to whether they exist in vivo or whether they form as an artefact of the procedures employed in their isolation (8). However, recent studies in both artificial lipid bilayers and living cell membranes using such techniques. [ABSTRACT FROM AUTHOR]

The genes encoding presenilin-1 (PS1) and presenilin-2 (PS2) were identified as the genes that harbour mutations that cause more than 60% of early onset familial Alzheimer's disease cases (FAD) (1-3). So far, more than 40 missense mutations have been described for presenilin-1 and two have been found in the gene coding for presenilin-2 (reviewed in refs. 4 and 5). Carriers of mutated presenilin genes develop in their brain neuropathological changes characteristic of Alzheimer's disease including the deposition of amyloid Aβ peptide. The latter is released from its cognate amyloid precursor protein (APP) by a two-step proteolytic conversion: first, proteolysis of APP by β-secretase, which releases the N-terminus of Aβ, and second, conversion of the remaining fragment by γ-secretase, which cleaves within the predicted transmembrane region of APP. This releases the C-terminus of Aβ, which may end either at position 40 or, to a lesser extent, at position 42 (reviewed in ref. 6). The latter species, Aβ1-42, is more prone to aggregation and deposition than Aβ1-40 and is produced at higher levels in the brains and primary fibroblasts of FAD patients carrying PS missense mutations (7). The same result was obtained when cultured cells transfected with mutated PS1 orPS2, or transgenic mice harboring missense PS1 were analyzed for the production of Aβ1-42: in every case increased amounts of the longer Aβ1-42 species were observed (8-10). The mechanisms by which mutations in the PS genes affect the proteolytic processing of APP by γ-secretase have not been resolved in detail. There are two possibilities by which the normal processing of APP may be disturbed: either mutations in the presenilins affect APP metabolism in an indirect way by modulation of proteases or interaction with proteins involved in APP intracellular routing, or presenilins may modulate APP processing directly through physical interactions with APP. Such a direct interaction between presenilins and APP was first demonstrated by us for PS2 (11). Later on, formation of stable complexes with APP was reported not only for PS2 but also for PS1 (12,13,13a). [ABSTRACT FROM AUTHOR]

The majority of familial Alzheimer's disease (AD) cases are linked to mutations of the presenilin 1 and 2 (PS1, PS2) genes on chromosomes 14 and 1, respectively (1-3). PS1 and PS2 are about 67% identical in amino acid sequence. Based on hydrophobicity analysis, the presenilins are predicted to have multiple transmembrane domains. Structural analysis (seeChapter 19}) suggest that presenilins are 6-8 transmembrane proteins which are located in the endoplasmic reticulum (ER) and Golgi. The N- and C-termini and the large hydrophilic loop region are oriented to the cytoplasm (4,5). More than 40 AD-causing mutations have been identified in PS1, whereas only two mutations have been identified in PS2. The disease-causing mutations span most domains of the protein, with clusters of mutations in the second transmembrane domain and the large hydrophilic loop region Fig. 1). [ABSTRACT FROM AUTHOR]

The phosphorylation of presenilin (PS) proteins was initially analyzed in cultured cells overexpressing the respective proteins. These studies revealed that the homologous PS proteins are differentially phosphorylated in vivo. Fulllength PS2 was found to be constitutively phosphorylated on serine residues (1,2). In contrast, very little if any (1) or a variable phosphorylation (2) was observed for PS1. The familial Alzheimer's disease (FAD) mutations tested, the A246E mutation of PS1 and the N141I mutation (volga german) of PS2, apparently have no effect on the differential phosphorylation of PS1 and PS2 (1). Because both PS proteins appeared to reside predominantly within the endoplasmic reticulum (1-4) differential phosphorylation is not due to distinct subcellular localizations of these proteins. Instead, the differential phosphorylation seems to be determined by structural differences between PS1 and PS2. The phosphorylation of full-length PS2 was localized to its N-terminal domain preceding the first transmembrane region (1). Although both PS proteins are highly homologous (5-7), their N-terminal domains differ in the primary structure. PS2 contains a stretch of acidic residues (amino acids 1-20), which is lacking in PS1 Fig. 1). This acidic domain of PS2 contains three consensus sites for casein kinases (CK), one site for CK-1 (serine 19) and two for CK-2 (serines 7 and 9; Fig. 1). Mutagenesis analyzes demonstrated that all three serine residues (serines 7, 9, and 19) are phosphorylated in cultured cells overexpressing PS2 (1). Moreover, in vitro phosphorylation demonstrated that the N-terminal domain of PS2 can be phosphorylated by both CK-1 and CK-2 (1). Thus, it is likely that full-length PS2 is phosphorylated by CK-1 and CK-2 in vivo within its N-terminal domain. The phosphorylated residues within the acidic region of PS2 precedes a PEST motif (8,9), which is lacking in PS1. PEST sequences have been shown to be implicated in the regulation of protein turnover, e.g., the degradation of proteins containing a PEST motif is enhanced (9). It will be of great interest to test whether the phosphorylation of PS2 influences its turnover. [ABSTRACT FROM AUTHOR]

Familial Alzheimer's disease (FAD) is a genetically heterogeneous disorder that is caused by defects in at least three early onset genes (age of onset:<60 yr.): presenilin 2 (PS2) on chromosome 1 (1), presenilin 1 (PS1) on chromosome 14 (2), and amyloid protein precursor (APP) on chromosome 21 (3,4). Mutations within the APP gene are responsible for only a small portion (<2%) of reported cases of FAD (5), whereas up to half of all early onset FAD cases are caused by mutations in the PSEN1 and PSEN2 genes (6,7). [ABSTRACT FROM AUTHOR]

Mutations in two related genes, PS1 (1) and PS2 (2,3) located on chromosomes 14 and 1, respectively, account for the majority of early onset cases of familial Alzheimer's disease (FAD). PS1 and PS2 are predominantly localized in the endoplasmic reticulum and Golgi (4-7). PS1 is a 467 amino acid peptide predicted to contain between seven and nine transmembrane helices based on hydrophobicity profiles (1,8). The protein topology of PS1 and its C. elegans homologues, SEL-12 and HOP-1, have been examined by several investigators (7,9-13). This chapter describes two approaches we utilized to determine the topological orientation of the PS1 N-terminal, and C-terminal domains, and a hydrophilic "loop" region encompassing amino acids 263-407. The first approach is based on the proteolytic sensitivity of amyloid precursor protein (APP) protein chimeras to endoproteolytic cleavage by β-secretase in the lumen of the Golgi. The second approach is based on selective permeabilization of the plasma membrane using a bacterial pore-forming toxin, streptolysin-O (SLO), and subsequent immunocytochemical probing for cytosolic epitopes using specific antibodies. Both of these methods can be easily adapted to determine the topology of other membrane proteins. [ABSTRACT FROM AUTHOR]

It has been well established that β-amyloid peptide is the principal protein component of extracellular cerebral amyloid deposits in patients with Alzheimer's disease (1,2). β-Amyloid is derived from a large precursor protein, amyloid precursor protein (APP), which is an integral membrane protein, with a receptor-like structure (3). APP is a member of a gene family which encodes extremely well-conserved membrane proteins. APP/APP-like genes have been isolated from various species including fly (4), nematode (5), and fish (6). In mammals, two APP-like genes, amyloid precursor-like protein 1 (APLP1) and 2 (APLP2), have been isolated (7,8). The amino acid sequences of these APP family proteins are highly conserved, especially in the cytoplasmic domain, except that unlike APP, APP-like proteins lack the β-amyloid sequence. It has been thought that APP and APLP2 have a similar physiological function (9). In contrast, APLP1 is believed to differ functionally from APP and APLP2, although the physiological functions of these APP family proteins have not yet been well analyzed. [ABSTRACT FROM AUTHOR]

Amyloid precursor protein (APP) and Alzheimer's disease are irrevocably linked, as APP is cleaved to form the Aβ peptides that are the major components of amyloid plaques. One of the most resilient hypotheses about the cause of AD centers on the Aβ peptide; all genetic causes and risk factors can be fitted into a general "amyloid cascade hypothesis," which maintains that all pathology is initiated by an abnormal accumulation of Aβ amyloid (1). [ABSTRACT FROM AUTHOR]

A large body of evidence suggests a causative role of β-amyloid (Aβ) in the pathogenesis of Alzheimer's disease (reviewed in refs. 1 and 2). Aβ is neurotoxic and toxicity requires the formation of amyloid fibrils similar to those found in senile plaques (3). Autosomal dominant mutations linked to Alzheimer's disease were identified in three different genes (4 ,5). All mutations apparently alter amyloid precursor protein (APP) metabolism to increase the generation of Aβ peptides terminating at amino acid Ala-42. Due to the tendency of the longer Aβ peptides to more readily form fibrils (7), these may accelerate Aβ deposition, which ultimately leads to more aggressive, early onset forms of Alzheimer's disease (8). With the transgenic expression of APP in mice this was explored further (9). Whereas a twofold overexpression of APP did not lead to Aβ deposition, the same quantitative expression of APP with a mutation at codon 717 known to increase the formation of Aβ42 led to the appearance of Aβ deposits at the age of 18 mo. These data suggest that the Aβ load in the brain as well as the amyloidogenic properties of the Aβ isoforms directly regulate deposition and senile plaque formation. [ABSTRACT FROM AUTHOR]

When the amyloid precursor protein (APP) was cloned, Aβ was found to be part of the large APP molecule and it became obvious that at least two endoproteolytic cleavage events are required to release Aβ from its large precursor (1). More than 10 yr later nobody has published definitive identification of either of the proteases, although they are prime therapeutic targets for an antiamyloid therapy for Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Human genetic studies suggest decreasing amyloid peptide (Aβ) levels in the brain could alter the course of Alzheimer's disease (AD) (1 -4). Proteolytic cleavages govern the level of Aβ generated from the amyloid precursor protein (APP). β- and γ-cleavages at the amino and carboxyl termini of Aβ produce amyloidogenic peptides; in contrast, α-cleavage within the Aβ domain destroys the amyloidogenic potential of APP. The proteases responsible for these cleavages have not been identified. [ABSTRACT FROM AUTHOR]

A detailed understanding of the biochemical events leading to the proteolytic excision of the β-amyloid peptide (A β) from the amyloid precursor protein (APP) has eluded many researchers. This is largely because the measurement of the various APP processing products is technically challenging owing to their low levels of production in in vitro and in vivo test systems. Sequence analysis of products in cell cultures, cerebrospinal fluid (CSF), and amyloid plaques has been used to predict the major cleavage sites resulting from the β- and γ-secretase proteolytic activities that release the Aβ peptide from APP (1 -3). More routine identification of the secretase activities has relied on the specificity and sensitivity of antibodies raised to the predicted cleavage products and has been impeded by the difficulties associated with the generation of such reagents. [ABSTRACT FROM AUTHOR]

The amyloid precursor protein (APP) is cleaved by at least three proteinases termed the α-, β-, and γ-secretases. Cleavage of APP at the N-terminus of the β-amyloid (Aβ) peptide by β-secretase and at the C-terminus by one or more γ-secretases constitutes the amyloidogenic pathway. In the nonamyloidogenic pathway, α-secretase cleaves APP within the Aβ peptide between Lys16 and Leu17 (numbering from the N-terminus of the Aβ peptide) (1), thereby preventing deposition of intact Aβ peptide. The α-secretase cleavage site lies some 12 amino acid residues on the extracellular side of the membrane, releasing the large ectodomain of APP (sAPPα), which has neuroprotective properties (2 ,3). The identification and characterization of the APP secretases is important for the development of therapeutic strategies to control the buildup of Aβ in the brain and the subsequent pathological effects of Alzheimer's disease. Regulation of the balance of APP processing by the amyloidogenic and nonamyloidogenic pathways through either selective inhibition of β- and γ-secretases or activation of α-secretase can all be considered as potential therapeutic approaches. As a first step towards isolating the APP secretases, we have investigated the effect of protease inhibitors on the activities of α- and β-secretase. From these studies we have identified low molecular weight inhibitors of α-secretase. [ABSTRACT FROM AUTHOR]

Many studies have demonstrated the importance of amyloid precurser protein (APP) in the pathogenesis of Alzheimer's disease. Nonetheless, the exact mechanism by which APP contributes to the pathogenesis of Alzheimer's disease is still not clear. Because APP is a glycoprotein, and because glycosylation can be important in the cell biology of individual glycoproteins (for review, seerefs. 1 and 2), it is possible that changes in APP glycosylation during development and aging are important in APP biosynthesis, proteolysis, and degradation. However, few studies have addressed this issue (3 -8). This chapter provides methods for analyzing the glycosylation of APP that is actively synthesized by living cells in tissue culture. These methods can be applied to primary cultures, continuous cell lines, and transfected cell lines expressing recombinant APP. [ABSTRACT FROM AUTHOR]

The amyloid precursor protein (APP) is a type I transmembrane protein with a large ectodomain, a single transmembrane domain and a small cytoplasmic tail (1). Translation of APP occurs at the endoplasmic reticulum (ER) and the protein is translocated into the ER lumen. The N-terminal domain of APP is directed towards the luminal compartment of the ER, whereas the C-terminal domain faces the cytoplasm. After synthesis, APP passes from the ER to the Golgi compartment. APP can then be transported in secretory vesicles to the cell surface, where the large ectodomain faces the extracellular milieu. Cell surface APP can be reinternalized into endosomes and lysosomes (for review seerefs. 2 and 3). During its passage through the secretory pathway, APP is subjected to a variety of posttranslational modifications, including proteolytic processing, glycosylation, sulfation, and phosphorylation. Immediately on translocation into the ER, the signal peptide of APP is removed from the N-terminus by signal peptidase. APP is then modified cotranslationally by N-glycosylation on NH2-groups of asparagine residues. After passage into the Golgi compartment, the ectodomain of APP is subjected to O-glycosylation. In late Golgi compartments, e.g., the trans Golgi network, APP is subjected to sulfation on tyrosine residues within its ectodomain (4). [ABSTRACT FROM AUTHOR]

Since the first description of Alzheimer's disease (AD) at the beginning of the century until relatively recently, it was customary to define Alzheimer's disease as occurring in the presenium. The same neuropathological changes occurring in brains over the age of 65 were called "senile dementia." Because there have been no clinical or pathological features to separate the two groups, this somewhat arbitrary distinction has been abandoned. Although AD is currently considered to be a heterogeneous disease, the most consistent risk factor to be implicated other than advancing age is the presence of a positive family history. This potential genetic vulnerability to AD has been recognized for some time. Some of the earliest evidence suggestive of a genetic contribution to AD came from Kallmann's 1956 study (1) demonstrating a higher concordance rate in monozygotic twins for "parenchymatous senile dementia" compared with dizygotic twins and siblings. This monozygotic excess has been confirmed in studies applying more rigorous diagnostic criteria although there may be widely disparate ages of onset between twins (2). The most convincing evidence for a genetic contribution to AD has come form the study of pedigrees in which the pattern of disease segregation can be clearly defined. Thus, the abandonment of the early and late-onset dichotomy has occurred at a time when, at the genetic level, important differences have been identified through the discovery of specific gene defects in early onset cases. [ABSTRACT FROM AUTHOR]

The accumulation of insoluble Aβ peptide aggregates in the brain is the diagnostic feature of Alzheimer's disease. Identical deposits are seen in the elderly who are at risk for this disease. The formation of the approx 4 kDa Aβ peptide is implicated as a key component in the development of Alzheimer's disease pathology. Genetic evidence strongly supports this contention (1,2), as well as a number of demonstrated relevant biological activities of the Aβ peptide such as its neurotoxicity (3) and proinflammatory properties (4). A great deal of attention has been focused on the processes involved in the generation of Aβ peptide. In contrast, the fate of this peptide once it has been released from the cell is less well understood. Recently, this situation has been changing as studies on the clearance of Aβ peptide are being published. The identification of Aβ-degrading enzymes produced in the brain, their class, and selectivity, as well as their cellular origin, are important unresolved questions. One key issue of Aβ peptide clearance is whether the brain may be limited in its capacity to degrade this protein, as all cells produce Aβ, yet it is seen to accumulate only in brain tissue. Because alterations in Aβ peptide clearance may potentially contribute to increased levels and to the development of insoluble Aβ deposits in the brains of afflicted individuals, this chapter focuses on specific approaches to clarifying Aβ peptide-clearance mechanisms. [ABSTRACT FROM AUTHOR]

Several lines of evidence suggest a role for membrane ion channels in the neurotoxic effects of the β-amyloid peptide (Aβ). This chapter describes the electrophysiological techniques that can be employed to isolate and record specific membrane conductances that may be altered by Aβ. In general, an increase in conductances that cause depolarization of the cell membrane may be considered excitotoxic since they will: (1) increase Ca2+influx through voltage-gated Ca2+channels and (2) reduce Mg2+-dependent block of ionotropic glutamate receptors, thereby increasing Ca2+influx through N-methyl-D-aspartate (NMDA) receptor channels. Conversely, an increase in conductances that cause membrane hyperpolarization might be considered to have a protective effect. This is a simplistic view, as it has been shown that for certain forms of apoptosis an increase in hyperpolarizing K+currents may be involved (1). It is, therefore, important to consider the functional effects of any changes in membrane conductances or ion channel currents induced by Aβ in the light of neurotoxic effects of the peptide. [ABSTRACT FROM AUTHOR]

As noted in the introductory chapters of this book, neuritic plaques composed of accumulated amyloid β (Aβ) peptide are a hallmark pathological feature of the Alzheimer's disease (AD) brain. Compelling genetic data now implicate these plaques as key causative agents in AD onset, as all known mutations that lead to early onset familial AD (1-6) result in an increased production of the amyloidogenic Aβ1-42 isoform (7-11). Although it appears likely that the deposition of multimeric Aβ fibrils into plaques is a necessary step in AD onset, there is still uncertainty as to how Aβ and neuritic plaques might cause the neuropathology that leads to the dementia that is characteristic of this disease. [ABSTRACT FROM AUTHOR]