Current Alzheimer Research - Volume 5, Issue 2, 2008

Alois Alzheimer's described his seminal observation more than one hundred years ago. Yet, the main component of one of the principal cerebral lesions, the senile plaque, was only identified in 1984 by Glenner and Wong. This discovery now appears as a temporal frontier between important initial research that was mainly descriptive in nature and a new modern area in which the biology of the disease was investigated and the quest for causal determinants was initiated. An important step towards the understanding of the etiology of Alzheimer's disease (AD) was achieved when the genes harboring autosomal dominant mutations were identified as those encoding the β-amyloid precursor protein (APP) and presenilins. Thus, even if these mutations overall account for a low percentage of AD cases, they proved extremely useful to delineate some of the perturbations occurring in AD brain. Particularly interesting was the observation that although APP and presenilins are distinct entities, mutations on these proteins all lead to a modification of APP processing and altered production of amyloid-β (Aβ) peptides. Therefore, Aβ (or more likely a set of Aβ peptides) can be seen as a common molecular denominator for various “types” of AD [1]. Does it mean that Aβ is, stricto sensu, the only etiological determinant of the disease? Likely not, and we all agree that environmental and risk factors among others could drastically influence the course of the disease, as supported by the fact that patients harboring identical mutations on presenilins could develop the disease with a strikingly variable age of onset. On the other hand, it is hard to unquestionably deem that Aβ-like peptides (including their various biophysical states) have nothing to do with the neurodegenerative process. However, even if the majority is not always right [in science], the fact remains that the so-called amyloid cascade postulating a key role of Aβ in AD pathogenesis is one of the main hypothesis studied worldwide. On the whole, much attention has been paid to the mechanisms by which Aβ peptides are produced and cleared off. Hence, some of the molecular actors were identified a dozen or so years ago and an immense amount of information has been gathered. However, in this regard it is again remarkable that some of the data are not consensual and sometimes hardly discussed. This explains why a special issue of the Current Alzheimer's Research has been dedicated to the various catalytic events and putative enzymes involved in the proteolytic conversion of APP. In the field, there are the “good” and the “bad”. We can identify the bad as the enzymes that produce Aβ peptides: β- and ??-secretases. It is obviously somewhat caricatural because, Aβ remains a catabolic product of physiological APP processing and potential “toxic” or “deleterious” effects are drastically linked to the concentration of Aβ monomers, the biophysical state of Aβ (fibrils, protofibrils, aggregates) and the nature and ratio of Aβ-like peptides (Aβ40/42, Nterminally truncated, C-terminally extended). It stands that aggregation processes or secondary cleavages yielding Aβ- like species harboring enhanced toxicity remains dependent upon the initial cleavages yielding “genuine” Aβ. As a corollary, the proteases involved in these cleavages are of great importance. The enzyme β-secretase liberates the N-terminal moiety of Aβ and has been consensually identified as an aspartyl protease referred to as BACE1 (β-site APP cleaving enzyme), memapsin 2 or ASP2 by several independent groups. Cole and Vassar with Tang and colleagues, two of the discoverers of the enzyme, describe here (pp. 100-131) the biology of β-secretase and survey the recent potential pharmacological blockers of the enzyme as putative therapeutic probes. γ-secretase is one of the stars in the field. Thus, the enzyme is particularly important as it conditions the nature of the C-terminal part of Aβ peptides, which drives their toxic potential. There likely exist several γ-secretases, dependent or independent of presenilins, but the former has been the center of a significant amount of data leading to the discovery of a high molecular weight complex, the biologically active structure of which still remains debated. The biology of the complex, i.e its assembly and proteolytic maturation is currently a rapidly “moving” field. Therefore, the state of the art description of the γ-secretase complex (Dries and Yu, pp. 132-146), the revisited survey on presenilin structure and function (Steiner, pp. 147-157) and the late developments concerning the design of presenilin-directed γ-secretase inhibitors (Wolfe, pp. 158-164) are included in this issue. Of particular interest, (Kametani, pp. 165-171) describe recent advances concerning an additional “epsilon” cleavage site on APP that has been the center of many recent studies, while Xia overviews (pp. 172-178) the data available on presenilinase, an activity that harbors the potential of cleaving presenilins, thereby yielding the catalytically active γ- secretase complex.

Alzheimer's disease is characterized by the presence of two types of lesions in brain: neurofibrillary tangles and senile plaques. Intraneuronal neurofibrillary tangles are made of paired helical filaments containing hyperphosphorylated microtubule associated protein tau. Extracellular senile plaques contain a core of beta-amyloid peptide (Aβ), which is produced by cleavage of the Amyloid Precursor Protein (APP). Among the two catabolic pathways of APP, the amyloidogenic pathway producing Aβ peptides was intensively studied in different cellular models expressing human APP. Differences in APP processing and in toxicity resulting from Aβ accumulation can be observed from one cell type to another. In particular, primary cultures of neurons process APP differently compared with other cultured cells including neuronal cell lines. Neurons accumulate intraneuronal Aβ, which is neurotoxic, and in these cells, APP can be phosphorylated at specific residues. Recent studies suggest that APP phosphorylation can play an important role in its amyloidogenic processing. In addition, protein kinases that phosphorylate APP are also able to phosphorylate the neuronal protein tau. Biochemical analysis of these two proteins in primary cultures of neurons show that phosphorylation of both APP and tau can be a factor linking the two characteristic lesions of Alzheimer's disease.

Amyloid plaques, hallmark neuropathological lesions in Alzheimer's disease (AD) brain, are composed of the β-amyloid peptide (Aβ). Much evidence suggests that Aβ is central to the pathophysiology of AD and is likely to play an early role in this intractable neurodegenerative disorder. Given the strong correlation between Aβ and AD, therapeutic strategies to lower cerebral Aβ levels should prove beneficial for AD treatment. Aβ is derived from amyloid precursor protein (APP) via cleavage by two proteases, β- and γ-secretase. The β-secretase has been identified as a novel aspartic protease named BACE1 (β-site APP Cleaving Enzyme 1) that initiates Aβ formation. Importantly, BACE1 appears to be dysregulated in AD. As the rate-limiting enzyme in Aβ generation, BACE1, in principle, is an excellent therapeutic target for strategies to reduce the production of Aβ in AD. While BACE1 knockout (BACE1-/-) mice have been instrumental in validating BACE1 as the authentic β-secretase in vivo, data indicates that complete abolishment of BACE1 may be associated with specific behavioral and physiological alterations. Recently a number of non-APP BACE1 substrates have been identified. It is plausible that failure to process certain BACE1 substrates may underlie some of the reported abnormalities in the BACE1-/- mice. Here we review the basic biology of BACE1, focusing on the regulation, structure and function of this enzyme. We pay special attention to the putative function of BACE1 during normal conditions and discuss in detail the relationship that exists between key risk factors for AD and the pathogenic alterations in BACE1 that are observed in the diseased state.

Memapsin 2 (β-secretase, BACE 1) processing of β-amyloid precursor protein is the first step in the pathway leading to the production of amyloid-β, thus, it is a major target for the development of inhibitor drug for the treatment of Alzheimers's Disease. Although there are distinctive advantages of this protease as a drug target, the development of drug-like memapsin 2 inhibitors has been somewhat slow since the cloning of the protease seven years ago. Here we review the progress of memapsin 2 inhibitor development using crystal structure-based design cycles. Recent progress has evolved the inhibitors into sizes sufficiently small to penetrate cell membranes and the blood-brain barrier yet retain potency for the inhibition of Aβ production in cultured cells and experimental animals. Such progress lends optimism that clinically useful memapsin 2 inhibitors will eventually be developed.

In this review, we discuss the biology of γ-secretase, an enigmatic enzyme complex that is responsible for the generation of the amyloid-β peptide that constitutes the amyloid plaques of Alzheimer's disease. We begin with a brief review on the processing of the amyloid precursor protein and a brief discussion on the family of enzymes involved in regulated intramembrane proteolysis, of which γ-secretase is a member. We then identify the four major components of the γ- secretase complex - presenilin, nicastrin, Aph-1, and Pen-2 - with a focus on the identification of each and the role that each plays in the maturation and activity of the complex. We also discuss two new proteins that may play a role in modulating the assembly and activity of the γ-secretase complex. Next, we summarize the known subcellular locations of each γ-secretase component and the sites of γ-secretase activity, as defined by the production of Aβ. Finally, we close by synthesizing all of the included topics into an overarching model for the assembly and trafficking of the γ-secretase complex, which serves as a launching point for further questions into the biology and function of γ-secretase in Alzheimer's disease.

Mutations in the presenilin 1 (PS1) gene are the major cause of familial Alzheimer's disease (AD). They effect an increased production of the highly neurotoxic 42 amino acid variant of the amyloid-β peptide (Aβ), which is believed to initiate the disease. Aβ is the product of two consecutive cleavages of the β-amyloid precursor protein (APP) by two proteases, β-secretase and γ-secretase. The latter enzyme has been identified as an intramembrane-cleaving multiprotein complex that apart from APP cleaves a large number of other type I transmembrane proteins. PS1 and its homologue PS2 are essential for γ-secretase cleavage and more than a decade after their discovery it is now firmly established that they function as catalytic subunits of γ-secretase. This review recapitulates the findings that led to this conclusion as well as the further progress made on the function of PS as γ-secretase since then.

γ-Secretase is a multi-protein complex that proteolyzes the transmembrane region of the amyloid β-peptide (Aβ) precursor (APP), producing the Aβ peptide implicated in the pathogenesis of Alzheimer's disease (AD). This protease has been a top target for AD, and various inhibitors have been identified, including transition-state analogue inhibitors that interact with the active site, helical peptides that interact with the initial substrate docking site, and other less peptidelike, more drug-like compounds. Although one γ-secretase inhibitor has advanced into late-phase clinical trials, concerns about inhibiting this protease remain. The protease complex cleaves a number of other substrates, and in vivo toxicities observed with γ-secretase inhibitors are apparently due to blocking one particularly important substrate, the Notch receptor. Thus, the potential of γ-secretase as therapeutic target likely depends on the ability to selectively inhibit Aβ production without hindering Notch proteolysis (i.e., modulation rather than inhibition). The discovery of γ-secretase modulators has revived γ-secretase as an attractive target and has so far resulted in one compound in late-phase clinical trials. The identification of other modulators in a variety of structural classes raise the hope that more promising agents will soon be in the pipeline.

The accumulation and deposition of fibrillar Aβ is thought the primary cause of Alzheimer's disease (AD). Aβ is generated by sequential proteolytic processing involving β- and γ-secretase on Amyloid β protein precursor (APP). Recently, γ-secretase was shown to cleave near the cytoplasmic membrane boundary of APP, called η-site cleavage, as well as in the middle of the membrane domain, called γ-site cleavage. Recent findings indicate that γ- and η-site cleavage are regulated independently. In this review, the reduction of η-site cleavage in AD and the importance of η-site cleavage are discussed.

Mutations in two genes, presenilin 1 (PS1) and its homologue presenilin 2 (PS2), account for a majority of early onset familial Alzheimer disease cases which are characterized by intracellular neurofibrillary tangles and extracellular amyloid fibrils composed of the amyloid β protein (Aβ). Aß is derived from sequential cleavages of Amyloid Precursor Protein (APP) by ß-secretase and γ-secretase, the latter is composed of four components, PS1, nicastrin (NCT), presenilin enhancer 2 (PEN-2), and anterior pharynx defective (APH-1). These components not only maintain the stability of the γ-secretase complex but also regulate the activity of presenilinase, the protease responsible for the cleavage of full length PS1 into N-terminal and C-terminal fragments (NTF/CTF). We have previously shown that endoproteolysis of PS1 into NTF/CTF by presenilinase requires two critical aspartate residues, suggesting that PS1 may undergo autoproteolysis; full length PS1 complexes with NCT, PEN-2, APH-1 and forms the presenilinase. While these two aspartate residues are necessary for the endoproteolysis of full length PS1, they are equally critical for the γ-secretase cleavage of multiple substrates, and it is hypothesized that the full length PS1/presenilinase is the zymogen of γ-secretase. The inhibition profiles of presenilinase and γ-secretase are illustrated by their biochemical similarity but are pharmacologically distinct. Since the uncleaved PS1 loop may obstruct the entry of γ-secretase substrates to the docking site of the γ-secretase complex, investigation of presenilinase inhibitors interfering with substrate-docking may facilitate a novel approach to identify APP specific γ-secretase inhibitors.

Accumulation of amyloid β-peptides (Aβ) in the brain is believed to contribute to the development of Alzheimer disease (AD). Aβ, a 40-42 amino acid-comprising proteolytical fragment of the amyloid precursor protein (APP), is released from APP by sequential cleavages via β- and γ-secretases. However, the predominant route of APP processing consists of successive cleavages by α- and γ-secretases. Alpha-secretase attacks APP inside the Aβ sequence, and therefore prevents formation of neurotoxic Aβ. After cleavage by α-secretase, the soluble N-terminal domain of APP, which possesses neurotrophic and neuroprotective properties, is released. In AD patients, a decrease in α-secretase processing of APP has been found and therefore, strategies to improve α-secretase activity are obvious. Several years after descriptive reports on α-secretase, the responsible enzymes have been identified to belong to the family of A Disintegrin And Metalloproteinase (ADAM). Three of these membrane-anchored zinc-dependent metalloproteinases, ADAM10 as well as ADAM17 and presumably also ADAM9 display α-secretase activity. Since the individual knock-out of these proteinases in neither case completely prevented α-secretase processing of APP, it seems likely that different ADAMs are compensating mutually, and under different conditions may contribute to α-secretase cleavage of APP. In addition to ADAMs, perhaps other membrane-associated metalloproteinases contribute to the shedding of APP. Stimulation of α-secretase activities can be achieved via several signaling cascades including phospholipase C, phosphatidylinositol 3-kinase and serine/threonine-specific kinases such as protein kinases C, and mitogen activated protein kinases. Direct activation of protein kinase C and stimulation of distinct G protein-coupled receptors are known to increase α-secretase processing of APP. Agonists for M1 muscarinic acetylcholine receptors and serotonin 5-HT4 receptors are currently in clinical trials to test their efficiency in the treatment of AD.

Disintegrin metalloproteases of the ADAM family form a large (at present > 40 members in mammals) family of multidomain membrane proteins that in their ectodomain combine a cystein-rich, disintegrin and a zinc metalloprotease domain. Via their metalloprotease domain, ADAMs are often implicated in ectodomain shedding, either to release e.g. growth factors or to initiate further intracellular signalling via regulated intramembrane proteolysis. Mainly based upon overexpression studies in vehicle cells, three of them, ADAMs 9, 10 and 17, have been proposed to act as α-secretases for amyloid precursor protein (APP). It is striking thereby that this role has since then remained somewhat ill-defined, as APP processing in ADAM9 deficient neurons is unaltered, and also ADAM10 deficient murine embryonic fibroblasts exhibit at best a highly variable reduction in α-secretase activity. However, during the past years, numerous other substrates have been linked to all three sheddases, the cleavage of which in some cases appears to be strikingly more important for the organism than APP processing. Most notably, the perinatally lethal phenotype of ADAM17 knockout mice is dominated by a loss of growth factor shedding, while the even earlier fatal effects of ADAM10 deficiency exhibit key features of disabled Notch signalling and possibly also cadherin processing defects. In this review, we will summarize the published data on the “non-APP” functions of all three ADAMs, the further evaluation of which may be crucial when attempting to treat Alzheimer's Disease by increasing their expression and/or activity. As the knockouts of ADAM10 and ADAM17 are only informative for their roles in (early) development, while a number of recently assigned new substrates play crucial roles in the normal and/or diseased adult organism as well, work on conditional knockout models will be crucial to fully characterize both the full functional portfolio of (candidate) α-secretases as well as their clinical relevance, which may go way beyond Alzheimer's Disease.

Alzheimer's disease (AD) is by far the most common form of dementia in the elderly and concerns one out of three individuals over 85. Like other neurodegenerative disorders such as Parkinson, Hungtington or prion diseases, AD is characterized by the formation of amyloid plaques in the central nervous system. In the brain of AD patients, the main component of these abnormal deposits is an aggregated form of the so-called amyloid β-peptide (Aβ), which is produced from a large trans-membrane type-1 protein, the β-amyloid precursor protein (βAPP), by the sequential action of the β- and γ-secretases. Beside these two amyloidogenic proteolytic attacks, βAPP is targeted by a third enzyme termed α- secretase. Of utmost importance, this cleavage, which can be of constitutive or regulated origin, occurs right in the middle of the Aβ sequence, thus precluding its production. For this reason, and because the sAPPα secreted fragment derived from this cleavage displays beneficial effects, tremendous efforts have been made recently in order to both identify the proteases involved and the way they are regulated. More recently, it emerged that α-secretase was also responsible for the physiological processing of the cellular prion protein (PrPc) in the middle of its toxic 106-126 sequence. This review will focus on the recent advances in the α-secretase pathways regulation and will discuss the putative therapeutic approaches that could be envisioned concerning the treatment of two apparently distinct diseases that share common denominators according to their metabolism.

The steady state concentration of the Alzheimer's amyloid-β peptide in the brain represents a balance between its biosynthesis from the transmembrane amyloid precursor protein (APP), its oligomerisation into neurotoxic and stable species and its degradation by a variety of amyloid-degrading enzymes, principally metallopeptidases. These include, among others, neprilysin (NEP) and its homologue endothelin-converting enzyme (ECE), insulysin (IDE), angiotensinconverting enzyme (ACE) and matrix metalloproteinase-9 (MMP-9). In addition, the serine proteinase, plasmin, may participate in extracellular metabolism of the amyloid peptide under regulation of the plasminogen-activator inhibitor. These various amyloid-degrading enzymes have distinct subcellular localizations, and differential responses to aging, oxidative stress and pharmacological agents and their upregulation may provide a novel and viable therapeutic strategy for prevention and treatment of Alzheimer's disease. Potential approaches to manipulate expression levels of the key amyloiddegrading enzymes are highlighted.

Neprilysin is a zinc metalloendopeptidase with relatively broad substrate specificity. The enzyme is localized to the plasma membrane of cells where it can function to degrade extracellular peptides. Structural studies show that neprilysin preferentially cleaves peptides on the amino side of hydrophobic amino acids. Neprilysin has been implicated in the catabolism of amyloid β peptides in the brain and as such has received considerable attention, particularly as a therapeutic target for Alzheimer's disease. An inverse relationship between neprilysin levels and amyloid β peptide levels and between neprilysin levels and amyloid plaque formation has been observed in human brain. Neprilysin levels decline with aging in the temporal and frontal cortex possibly contributing to higher amyloid β peptide levels. A number of studies have shown that increasing neprilysin levels in the brain leads to a decrease in brain amyloid β peptide levels. Most recently a potential relationship between amyloid β peptide synthesis from the amyloid precursor protein and neprilysin activity has been proposed.