I. Introduction

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Aging is a major risk factor for neurodegenerative disorders, such as Alzheimer's disease (AD¹) and Parkinson's disease (PD), and the number of people with these conditions is increasing rapidly. Alzheimer's disease is the most common cause of dementia. United Nation population projections estimate that the number of people older than 80 years will approach 370 million by the year 2050. Currently, it is estimated that 50% of people older than age 85 years are afflicted with AD. Therefore, if these statistics hold true, in 50 years, more than 100 million people worldwide will suffer from dementia. The vast number of people requiring constant care and other services will severely strain medical, monetary, and human resources.

First described by Alois Alzheimer in 1906, the disease that bears his name largely remained an enigma until the twilight of the 20th century. Along with descriptions of progressive loss of memory and general cognitive decline, Alzheimer noted the presence of intraneuronal tangles and extracellular "amyloid" plaques in the disease-damaged brain, but he could not decipher whether the tangles or plaques were causative or merely markers of the disease. In 1991, the search for genetic linkages yielded a major clue: missense mutations in APP caused autosomal-dominant, early-onset (familial) AD, and these mutations occurred in and around the -amyloid (A) region of the precursor protein (Chartier-Harlin et al., 1991; Goate et al., 1991; Murrell et al., 1991; Hardy and Higgins, 1992)

The pathological hallmark of AD includes widespread neuronal degeneration, neuritic plaques containing -amyloid (A), and -rich neurofibrillary tangles (NFT) (Glenner and Wong, 1984). By the fourth decade of life, individuals with Down's syndrome display many of the same neuropathological features as do individuals with AD, and many of these individuals develop dementia early in life (Casanova et al., 1985; Wisniewski et al., 1985; Mann and Esiri, 1989; Sendera et al., 2000; Head et al., 2001). AD is multifactorial, with both genetic and environmental factors implicated in its pathogenesis. To date, mutations in three genesthe presenilin gene (PS1) on chromosome 14, the presenilin 2 gene (PS2) on chromosome 1, and the amyloid precursor protein gene (APP) on chromosome 21all serve to transmit AD via autosomal-dominant inheritance. This form of AD is referred to as familial Alzheimer's disease (FAD) and is characterized by earlier onset of symptoms. There are other genes that are considered susceptibility or risk factors for AD. These include apolipoprotein E (ApoE 4 variant) (Poirier et al., 1996), 2-macroglobulin (Blacker et al., 1998), a gene for a component of -ketoglutarate dehydrogenase (Ali et al., 1994), the K-variant of butyryl-cholinesterase (Lehmann et al., 1997a), and several mitochondrial genes (Law et al., 2001). Epidemiological studies have demonstrated risk factors for AD that include age, gender (females are at greater risk), previous head injury, and cardiovascular disease (Law et al., 2001). Much work remains to be done to fully elucidate environmental factors that can influence both the onset and the progression of AD.

To date, the cause and progression of both familial and sporadic (late-onset) AD have not been fully elucidated. Proteolytic processing of APP by -secretase, -secretase, and caspases generates A peptide and carboxyl-terminal fragments (CTF) of APP, which have been implicated in the pathogenesis of Alzheimer's disease (Checler, 1995; Suh, 1997; Selkoe, 1999). The missense mutations in the gene encoding APP, as well as those in the genes encoding PS1 and PS2, share the common feature that they alter the -secretase cleavage of APP to increase the production of the amyloidogenic A₄₂, a primary component of amyloid plaques in both familial and sporadic AD. All but one mutation triggers this phenotype. Ancolio et al. (1999) reported that V715M-APP significantly reduced total A and A₄₀ production without affecting A₄₂ production, but it increased A_X-42.

For the last decade, two major hypotheses on the cause of AD have been proposed: the "amyloid cascade hypothesis", which states that the neurodegenerative process is a series of events triggered by the abnormal processing of the amyloid precursor protein (Hardy and Higgins, 1992), and the "neuronal cytoskeletal degeneration hypothesis" (Braak and Braak, 1991), which proposes that cytoskeletal changes are the triggering events.

The most frequent sporadic forms of AD and PD are associated with an abnormal accumulation of A and -synuclein, respectively (Spillantini et al., 1997; Takeda et al., 1998, Selkoe, 2001). Human cases with clinical and neuropathological features of both AD and PD raise the possibility that these diseases involve overlapping pathways. Approximately 25% of patients with AD develop frank PD (Galasko et al., 1994), and -synuclein-immunoreactive Lewy-body-like inclusions develop in most cases of sporadic AD and FAD, as well as in Down syndrome (Lippa et al., 1999; Hamilton, 2000). Moreover, Lewy bodies contain APP (Arai et al., 1991; Van Gool et al., 1995; Halliday et al., 1997). The possible pathogenic interactions between A and -synuclein suggest that drugs aimed at blocking the accumulation of A or -synuclein might benefit a broader spectrum of neurodegenerative disorders than previously anticipated.

	II. Amyloid Precursor Protein

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A partial amino acid sequence of A was used to clone a cDNA encoding a protein now referred to as the APP, which has features of an integral type I transmembrane glycoprotein (Kang et al., 1987). The APP gene contains 18 exons spanning more than 170 kb (Yoshikai et al., 1990). The region encoding the A sequence comprises part of exons 16 and 17 and contains between 40- and 43-amino acid residues that extend from the ectodomain into the transmembrane domain of the protein (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Structure of human -APP. -APP consists of a large extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic carboxyl terminus. Some isoforms of -APP (APP751 and APP770) contain a KPI domain near the N terminus (Ponte et al., 1988). The A sequence (amino acids 597-639 of APP695) lies partially outside the cell membrane (amino acids 1-17 of A; amino acids 597-613 of APP695). A heparin-binding domain lies within the NH2 terminal portion of A (VHHQK) (Multhaup et al., 1994; Small et al., 1994). A heavily glycosylated region of APP lies NH2-terminal to the A sequence. This sequence is highly conserved and is nearly identical in species from Drosophila to human. Amino acid sequences of human and rodent A differ by three amino acids located in the NH2-terminal portion of peptide: amino acids 5 (human R, rodent G), 10 (human Y, rodent F), and 13 (human H and rodent R) of A.

Several APP mRNAs arise from alternative splicing and encode forms that differ mainly by the absence (APP-695) or presence (APP-751 and APP-770) of a Kunitz protease inhibitor (KPI) domain located toward the NH₂ terminus of the protein (Kitaguchi et al., 1988; Tanzi et al., 1988; De Sauvage and Octave, 1989; Oltersdorf et al., 1990; Sinha et al., 1990; Konig et al., 1992). There also exists a set of proteins called APLPs with structure similar to APPs (including forms containing or lacking a KPI domain), except that APLPs lack the A sequence (Wasco et al., 1993; Slunt et al., 1994; Webster et al., 1995) (Fig. 1).

The APP is an integral membrane protein processed by the three proteases -, -, and -secretase, which have been implicated in the cause of AD (Fig. 2). -Secretase generates the NH₂ teminus of A, cleaving APP to produce a soluble version of APP (-APPs) and a 99-residue COOH-terminal fragment (CT₉₉) that remains membrane-bound. In contrast, -secretase cuts within the A region to produce APPs, an 83-residue COOH-terminal fragment (CT₈₃).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Processing of -APP and Notch. A, -APP is cleaved sequentially by -secretase (BACE) and -secretase and/or caspases to generate A, CT99, CT57-59 (AICD), and/or CT31. CTF might translocate to the nucleus and affect the transcription of target genes. B, alternatively, -APP can be cleaved by -secretase and -secretase to produce P3 and CT57-59 and/or CT31. C, upon ligand binding, Notch is also cleaved sequentially by -secretase and -secretase to produce NICD, which translocates to the nucleus and affects the transcription of target genes.

Both CT₉₉ and CT₈₃ are substrates for -secretase, which performs an unusual proteolysis in the middle of the transmembrane domain to produce the 4-kDa A and CT_57-59 [amyloid intracellular domain (AICD)] from CT₉₉, and a 3-kDa peptide called p3 and CT_57-59 from CT₈₃. Proteolysis by -secretase is heterogeneous: Most of the full-length A species produced is a 40-residue peptide (A₄₀), whereas a small proportion is a 42-residue COOH-terminal variant (A₄₂). The longer and more hydrophobic A₄₂ is much more prone to fibril formation than is A₄₀ (Jarrett et al., 1993), and even though A₄₂ is a minor form of A, it is the major A species found in cerebral plaques (Iwatsubo et al., 1994), Moreover, AD-causing mutations in APP near the - and -secretase cleavage sites all increase A₄₂, those near -secretase cleavage site augment -site proteolysis, leading to the elevation of both A₄₀ and A₄₂ (Citron et al., 1992; Cai et al., 1993), whereas those near the -site specifically increase production of A₄₂ (Suzuki et al., 1994). Taken together, these findings implicated A in the pathogenesis of AD and spurred AD researchers to identify the A-releasing proteases.

-Secretase displays characteristics of certain membrane-tethered metalloproteases, and -secretase is a membrane-anchored protein with clear homology to soluble aspartyl proteases. The identification of the -, -, and -secretases provides potential targets for designing new drugs to treat AD.

1. -Secretase. A major route of APP processing is via the -secretase pathway, which cleaves on the C-terminal side of residue 16 of the A sequence, generating an 83-residue C-terminal fragment (CT₈₃) (Figs. 1 and 2) (Esch et al., 1990). -Secretase activity has both constitutive and inducible components. The constitutive activity has not yet been identified, but inducible -secretase activity seems to be under the control of protein kinase C (PKC).

2. -Secretase. In 1999, -secretase was identified as a protein with homology to the pepsin family of aspartyl proteases (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000). -Secretase contains a single transmembrane domain near the COOH terminus, a signal sequence and propeptide region at the NH₂ terminus, and two aspartates in its ectodmain, Asp₉₃ and Asp₂₈₉, that are required for activity. Mutation of either aspartate does not affect removal of the propeptide region, indicating that -secretase does not proteolytically cleave itself. Instead, the responsible protease seems to be a furin-like protease (Bennett et al., 2000). -Secretase RNA is highly expressed in the brain and is also found in a variety of human tissues (Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000), consistent with the finding that A is normally produced by many cell types and in accordance with that expected for -secretase (Haass et al., 1992; Seubert et al., 1992; Shoji et al., 1992; Busciglio et al., 1993). The intracellular localization of -secretase protein is expressed primarily in the Golgi and in endosomes, whereas only a small amount of it can be detected in endoplasmic reticulum, lysosomes, and the plasma membrane (Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000). BACE is phosphorylated within its cytoplasmic domain at serine residue 498 by casein kinase 1, and the phosphorylation exclusively occurs after full maturation of BACE by propeptide cleavage and N-glycosylation and drives the localization of BACE to Golgi compartments and endosome (Walter et al., 2001). The gene for -secretase is located on chromosome 11, but no AD-causing mutation in this gene has been identified so far (Saunders et al., 1999). A -secretase homolog, BACE-2, maps to chromosome 21, raising the possibility that BACE-2 contributes to Down syndrome. Down syndrome patients secrete more A from birth and invariably develop AD by age 50 years (Saunders et al., 1999). A1though BACE-2 cleaves APP and short-model peptides in a -secretase-like manner (Farzan et al., 2000), there is very little of this protease in the brain, suggesting that it may play little if any role in the formation of cerebral plaques seen in AD. Instead, the AD associated with Down syndrome is probably caused by the presence of an extra copy of the APP gene, which is also located on chromosome 21.

3. -Secretase. After either - or -secretase releases the bulk of APP, the remaining carboxyl-terminal fragments, CT₈₃ and CT₉₉, undergo proteolysis within their plasma membrane domainregulated intramembrane proteolysis (RIP)and the intracellular portion moves to the nucleus where it may affect the transcription of target genes (Fig. 2).

4. Caspases. Not everyone agrees that nerve cells die by apoptosis in AD, but if the findings are confirmed, they could provide new targets for drugs aimed at slowing the progression of the disease. Reports that apoptosis might be involved in AD began emerging in the early to mid-1990s. Ivins et al. (1998) and Forloni et al. (1996) showed that A causes neurons in culture to die by apoptosis.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Probable processing of APP by caspases.

5. Amyloid-Degrading Enzymes. The most important question yet to be answered is why A is deposited in sporadic AD, which accounts for more than 90% of all AD cases (Saido, 2000). A is a physiological peptide, the steady-state levels of which are determined by the balance between the anabolic and catabolic activities (Selkoe, 1993; Saido, 1998). Because the rate of A deposition is primarily a function of the steady-state level of A in brain, the focus should be placed on how A is metabolized in vivo.

b. Neprilysin. Neprilysin, a type 2 membrane protein on the cell surface and a neutral endopeptidase sensitive to both phosphoramidon and thiorphan, plays a major rate-limiting role in A_1-42 catabolism (Iwata et al., 2001c

c. Plasmin. The plasmin proteolytic cascade, known to be crucial for fibrinolysis and cell migration, has recently been implicated in A clearance. In this cascade, tissue-type plasminogen activator or urokinase-type plasminogen activator (uPA) can be activated by binding to fibrin aggregates and then cleave plasminogen to yield the active serine protease, plasmin, which proteolyses fibrin and other substrates. Tissue-type plasminogen activator and uPA can be activated by A aggregates to generate plasmin (Tucker et al., 2000

e. Other Candidate Proteases. Other proteases that have been reported to digest synthetic A under in vitro conditions include matrix metalloproteinase-9 (Backstrom et al., 1996

The amyloid cascade hypothesis was first formulated more than a decade ago and centers around the A peptide that is the main component of plaques (Glenner and Wong, 1984). There is a wealth of evidence to support this hypothesis (Selkoe, 1994; Checler, 1995; Mudher and Lovestone, 2002). The amyloid proteins involved in the pathogenesis of AD are A- and CT- (carboxyl terminal peptides of APP) peptides (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   The amyloid cascade hypothesis.

Classic A is the marker for AD, and it has been linked to the accompanying neurodegeneration (Sisodia et al., 1990). Several lines of evidence suggest that the overexpression of -APP and the subsequent production of A could be linked to the genesis of AD (Checler, 1995). Furthermore, studies of plasma and fibroblasts from subjects with mutations in the genes encoding -APP have established that they all alter -APP processing, which normally leads to the secretion of A [relative molecular mass, 4,000; M_r, 4 K; ~90% A_1-40, 10% A_1-42(43)], so that the extracellular concentration of A_1-42(43) is increased (Suzuki et al., 1994). These results indicate that the -APP mutations probably cause AD through an increase of A_1-42(43) in the brain.

Most obviously, mutations in APP are a rare cause of early-onset familial AD with all of the neuropathological and clinical features of AD. All but one of these mutations result in increased A_1-42 generation in cell and animal models and in fibroblasts from affected families (Ancolio et al., 1999).

Trisomy 21 (Down syndrome), which leads to an overproduction of APP and A, invariably leads to the early emergence of AD neuropathology (Selkoe, 1994). In addition, the E4 allele of ApoE promotes the precipitation of A into insoluble plaques (Yankner, 1996). Even more persuasively, a locus on chromosome 10 associated with late-onset AD (Myers et al., 2000) is associated with increased A generation (Ertekin-Taner et al., 2000).

In line with this are those studies showing that A is neurotoxic to cultured cells and, at least in some conditions, induces tau phosphorylation (Takashima et al., 1993, 1998a; Alvarez et al., 1999). Amyloid vaccine (both passive and active immunization against A) arrests and even reverses both plaque pathology and behavioral phenotypes in the transgenic animals (Schenk et al., 1999; Bard et al., 2000; Morgan et al., 2000).

Increasingly, attention is turning away from the deposits of extracellular insoluble aggregated amyloid in plaques and toward soluble, oligomeric and even intracellular A_1-42 (Wilson et al., 1999; Klein et al., 2001). There are also some puzzling observations which hint that this hypothesis is not complete. For example, whereas transgenic mouse models bearing the FAD mutations do not show evidence of significant neuronal loss (Hsiao et al., 1995; Irizarry et al., 1997; Holcomb et al., 1998), little tau phosphorylation, and no tangle formation (Games et al., 1995). A relatively high concentration (two or three orders of magnitude) of A was needed to exert toxicity, and some studies still failed to demonstrate A toxicity in vivo (Clemens and Stephenson, 1992; Games et al., 1992; Stein-Behrens et al., 1992; Podlisny et al., 1993). Furthermore, it was reported that under certain culture conditions, A promoted neurite outgrowth (Yankner et al., 1990; Koo et al., 1993) instead of exerting toxic action. Most important is that A deposition has been observed in various brain areas without accompanying neurodegeneration (Joachim et al., 1989; Gearing et al., 1993; Einstein et al., 1994), whereas neurodegeneration can occur in areas with no A deposition (Cochran et al., 1991).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   The tau and tangle hypothesis.

It is also possible that the critical factor is not A itself but that A is a marker for proteolytic cleavage of APP, and it is the transcriptionally active carboxyl terminal of APP itself that is involved in the pathogenesis (Cao and Sudhof, 2001). Thus, A may not be the sole active fragment in AD, and some other factor could be involved in inducing neuronal loss. The recent concentration on other potentially amyloidogenic products of -APP has produced interesting candidates, the most promising of which are the amyloidogenic CT fragments of -APP. The transgenic mice expressing -APP and presenilins were not, however, examined for the presence of CT₁₀₀.

First, CT peptides have been found not only in various cultured cells (Maruyama et al., 1990; Wolf et al., 1990; Dyrks et al., 1992; Estus et al., 1992; Gandy et al., 1992a,b; Golde et al., 1992; Haass et al., 1992) but also in paired helical filaments (Caputo et al., 1992), in senile plaques (Selkoe et al., 1988), in microvessels (Tamaoka et al., 1992), in choroid plexus from human brain (Tokuda et al., 1995), and in human platelets (Gardella et al., 1992) (Table 2). CT fragments with molecular masses of 12 to 16 kDa have also been found in media and cytosol of lymphoblastoid cells obtained from patients with early- or late-onset FAD (Matsumoto, 1994) and Down syndrome (Kametani et al., 1994). Finally, several transfection studies have correlated production of the A-bearing CT fragment with neurotoxicity (Yankner et al., 1989; Fukuchi et al., 1992a,b, 1993; Hayashi et al., 1992; Neve et al., 1992; Yoshikawa et al., 1992), whereas recent transgenic animal experiments using CT₁₀₀ peptide have linked CT fragment production with neurodegeneration (Kammesheidt et al., 1992; Howland et al., 1995; Oster-Granite et al., 1996; Nalbantoglu et al., 1997).

View this table: [in this window] [in a new window]   TABLE 1 Neurotoxic mechanisms of A

In addition, this amyloidogenic CT peptide is not only expressed in the extracellular fluid of some FAD and Down syndrome cells, it is also secreted in the media of PC12 cells transfected with CT₁₀₄ and human mixed brain-cell cultures (Yankner et al., 1989; Seubert et al., 1993; Matsumoto, 1994; Matsumoto and Matsumoto, 1994; Kim and Suh, 1996) (Table 2).

It has been recently demonstrated that either extracellular or intracellular application of CT₁₀₅ elicited strong nonselective inward currents and toxic effects in Xenopus oocytes (Fraser et al., 1996), in rat Purkinje neurons (Hartell and Suh, 2000), and in PC12 and cultured rat cortical cells (Kim and Suh, 1996). The channel-inducing and toxic activity of CT₁₀₅ was much more potent than that of any A fragments (Table 3).

View this table: [in this window] [in a new window]   TABLE 3 Summary of various effects of A and CT

The synthetic peptide APP_713-730 of APP₇₇₀ is highly fibrillogenic and interacts with tau in vitro and causes apoptotic neuronal death, suggesting that APP sequences other than A may play a role in nerve-cell degeneration in AD (Marcon et al., 1999). Taken together, these lines of evidence postulate that the CT fragment is an alternative toxic element important in the generation of the symptoms common to AD. If clearing amyloid neither reverses dementia nor affects tangles, then the hypothesis needs considerable revision. Overall, however, the A amyloid cascade hypothesis has fared remarkably well and has had few serious challenges.

1. Amyloid -Peptide Hypothesis. a. Neurotoxic Mechanisms of Amyloid -Peptide: Free Radical Accumulation, Altered Calcium Homeostasis, and Inflammatory Response. AD researchers have mainly focused on determining the mechanisms underlying the toxicity associated with A proteins. A is a normal physiological product of APP processing (Estus et al., 1992; Golde et al., 1992) and a soluble component of the plasma and the cerebrospinal fluid (Seubert et al., 1992). The aggregation of soluble A peptide into fibrillar cross- pleated-sheet conformation is generally considered to be a critical event in the pathology of AD (Dumery et al., 2001). A peptides may begin their toxic actions even before fibril formation. Increasing evidence suggests that soluble A levels, and not A plaques, are the best A correlates of cognitive dysfunction in AD (McLean et al., 1999).

View larger version (33K): [in this window] [in a new window]   Fig. 6.   Summarized neurotoxic mechanisms of A. A is the main component of neuritic plaques found in brains with AD. A exert neurotoxicity via several mechanisms: the alteration of calcium ion homeostasis, free-radical accumulation, inflammatory response, and activation of signaling pathways. Free radicals, generated by the interactions between the cytosolic membrane and A, may lead to cellular dysfunctions via the inhibition of various enzyme activities, disruption of signaling pathways, and an activation of nuclear transcription factors. A can disrupt cellular ion homeostasis by the potentiation of calcium channels and formation of ion pore. Inflammatory responses may be triggered by glial cells activated by A, releasing a variety of proinflammatory cytokines, chemokines, and NO, exerting toxi