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Vol. 54, Issue 3, 469-525, September 2002

Amyloid Precursor Protein, Presenilins, and alpha -Synuclein: Molecular Pathogenesis and Pharmacological Applications in Alzheimer's Disease

Yoo-Hun Suh and Frederic Checler

Department of Pharmacology, College of Medicine, National Creative Research Initiative Center for Alzheimer's Dementia and Neuroscience Research Institute, MRC, Seoul National University, Seoul, South Korea (Y.-H.S.); and Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France (F.C.)

Abstract
I. Introduction
II. Amyloid Precursor Protein
    A. Structure of Amyloid Precursor Protein
    B. Trafficking and Proteolytic Processing of Amyloid Precursor Protein
        1. alpha -Secretase.
        2. beta -Secretase.
        3. gamma -Secretase.
        4. Caspases.
        5. Amyloid-Degrading Enzymes.
            a. Insulin-Degrading Enzyme.
            b. Neprilysin.
            c. Plasmin.
            d. Endothelin-Converting Enzyme.
            e. Other Candidate Proteases.
    C. Amyloid Cascade Hypothesis: Two Major Amyloid Precursor Protein Metabolites Involved in Alzheimer's Disease Pathogenesis
        1. Amyloid beta -Peptide Hypothesis.
            a. Neurotoxic Mechanisms of Amyloid beta -Peptide: Free Radical Accumulation, Altered Calcium Homeostasis, and Inflammatory Response.
               i. Free-Radical Accumulation.
               ii. Altered Calcium Homeostasis.
               iii. Inflammatory Response.
               iv. Activation of Signaling Pathways.
        2. C-Terminal Fragment Hypothesis.
            a. Neurotoxic Mechanisms of C-Terminal Fragment of beta -Amyloid Precursor Protein.
               i. In Vivo Generation of Amyloidogenic Carboxyl-Terminal Fragments of beta -Amyloid Precursor Protein.
               ii. Toxicity of Carboxyl-Terminal Fragments.
            b. The Involvement of Carboxyl-Terminal Fragments of Amyloid Precursor Protein in Gene Transactivation.
    D. Amyloid and Tau
    E. Transgenic Models of Amyloidogenesis
        1. APPswe Transgenic Mice (Tg2576).
        2. Amyloid Precursor Protein V717F Transgenic Mice (PDAPP Mice).
        3. APP-751swe/V717I Transgenic Mice.
        4. TgAPP23.
        5. C100/C104 Transgenic Mice.
III. Presenilin
    A. Preliminary Remarks
    B. Cell Biology of Presenilins
    C. Presenilins and Their Molecular Partners
    D. Physiological and Pathological Roles of Presenilins.
        1. Presenilins and the gamma -Secretase Cleavage of beta -Amyloid Precursor Protein.
        2. Presenilins and Notch Signaling.
        3. Presenilins and Programmed Cell Death.
        4. Presenilins and the Unfolded-Protein Response.
        5. Other Putative Functions of Presenilins.
            a. Presenilin As a Receptor/Channel.
            b. Presenilin in Cell Adhesion.
            c. Other Putative Functions.
    E. Concluding Remarks on Presenilin Physiology
IV. alpha -Synuclein
    A. Molecular and Cell Biology of alpha -Synuclein
    B. Putative Functions of alpha -Synuclein in Cell Death
    C. alpha -Synucleinopathies
    D. alpha -Synuclein: A Bridge between Parkinson's and Alzheimer's Pathologies
V. Therapeutic Targets for Alzheimer's Disease
    A. Agents Affecting Secretary Amyloid Precursor Protein-alpha
    B. Acetylcholinesterase Inhibitors
        1. Tacrine Hydrochloride (Cognex).
        2. Donepezil Hydrochloride (Aricept).
        3. Galantamine (Reminyl).
        4. Rivastigmine Tartrate (Exelon).
    C. Agents Inhibiting Aggregation of Amyloid Precursor Protein Metabolites
        1. Metal Chelators.
        2. beta -Sheet Breakers.
    D. Antioxidants
    E. Anti-Inflammatory Agents
    F. Estrogens
    G. Vaccines
    H. beta -Secretase Inhibitors
    I. gamma -Secretase Inhibitors
        1. Peptidic Inhibitors.
        2. Nonpeptidic Inhibitors.
            a. JLK Inhibitors.
Acknowledgments
References


    Abstract
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Alzheimer's disease (AD) is the most common cause of dementia that arises on a neuropathological background of amyloid plaques containing beta -amyloid (Abeta ) derived from amyloid precursor protein (APP) and tau -rich neurofibrillary tangles. To date, the cause and progression of both familial and sporadic AD have not been fully elucidated. The autosomal-dominant inherited forms of early-onset Alzheimer's disease are caused by mutations in the genes encoding APP, presenilin-1 (chromosome 14), and presenilin-2 (chromosome 1). APP is processed by several different proteases such as secretases and/or caspases to yield Abeta and carboxyl-terminal fragments, which have been implicated in the pathogenesis of Alzheimer's disease. Alzheimer's disease and Parkinson's disease are associated with the cerebral accumulation of Abeta and alpha -synuclein, respectively. Some patients have clinical and pathological features of both diseases, raising the possibility of overlapping pathogenic pathways. Recent studies have strongly suggested the possible pathogenic interactions between Abeta , presenilins, and/or alpha -synuclein. Therefore, treatments that block the accumulation of Abeta and alpha -synuclein might benefit a broad spectrum of neurodegenerative disorders. This review covers the trafficking and processing of APP, amyloid cascade hypothesis in AD pathogenesis, physiological and pathological roles of presenilins, molecular characteristics of alpha -synuclein, their interactions, and therapeutic strategies for AD.


    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 beta -amyloid (Abeta ) 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 beta -amyloid (Abeta ), and tau -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 genes---the presenilin gene (PS1) on chromosome 14, the presenilin 2 gene (PS2) on chromosome 1, and the amyloid precursor protein gene (APP) on chromosome 21---all 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 epsilon 4 variant) (Poirier et al., 1996), alpha 2-macroglobulin (Blacker et al., 1998), a gene for a component of alpha -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 beta -secretase, gamma -secretase, and caspases generates Abeta 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 gamma -secretase cleavage of APP to increase the production of the amyloidogenic Abeta 42, 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 Abeta and Abeta 40 production without affecting Abeta 42 production, but it increased Abeta 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 Abeta and alpha -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 alpha -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 Abeta and alpha -synuclein suggest that drugs aimed at blocking the accumulation of Abeta or alpha -synuclein might benefit a broader spectrum of neurodegenerative disorders than previously anticipated.


    II. Amyloid Precursor Protein
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A. Structure of Amyloid Precursor Protein

A partial amino acid sequence of Abeta 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 Abeta 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).



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Fig. 1.   Structure of human beta -APP. beta -APP consists of a large extracellular domain, a hydrophobic transmembrane domain, and a short cytoplasmic carboxyl terminus. Some isoforms of beta -APP (APP751 and APP770) contain a KPI domain near the N terminus (Ponte et al., 1988). The Abeta sequence (amino acids 597-639 of APP695) lies partially outside the cell membrane (amino acids 1-17 of Abeta ; amino acids 597-613 of APP695). A heparin-binding domain lies within the NH2 terminal portion of Abeta (VHHQK) (Multhaup et al., 1994; Small et al., 1994). A heavily glycosylated region of APP lies NH2-terminal to the Abeta sequence. This sequence is highly conserved and is nearly identical in species from Drosophila to human. Amino acid sequences of human and rodent Abeta 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 Abeta .

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 Abeta sequence (Wasco et al., 1993; Slunt et al., 1994; Webster et al., 1995) (Fig. 1).

B. Trafficking and Proteolytic Processing of Amyloid Precursor Protein

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



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Fig. 2.   Processing of beta -APP and Notch. A, beta -APP is cleaved sequentially by beta -secretase (BACE) and gamma -secretase and/or caspases to generate Abeta , CT99, CT57-59 (AICD), and/or CT31. CTF might translocate to the nucleus and affect the transcription of target genes. B, alternatively, beta -APP can be cleaved by alpha -secretase and gamma -secretase to produce P3 and CT57-59 and/or CT31. C, upon ligand binding, Notch is also cleaved sequentially by alpha -secretase and gamma -secretase to produce NICD, which translocates to the nucleus and affects the transcription of target genes.

Both CT99 and CT83 are substrates for gamma -secretase, which performs an unusual proteolysis in the middle of the transmembrane domain to produce the 4-kDa Abeta and CT57-59 [amyloid intracellular domain (AICD)] from CT99, and a 3-kDa peptide called p3 and CT57-59 from CT83. Proteolysis by gamma -secretase is heterogeneous: Most of the full-length Abeta species produced is a 40-residue peptide (Abeta 40), whereas a small proportion is a 42-residue COOH-terminal variant (Abeta 42). The longer and more hydrophobic Abeta 42 is much more prone to fibril formation than is Abeta 40 (Jarrett et al., 1993), and even though Abeta 42 is a minor form of Abeta , it is the major Abeta species found in cerebral plaques (Iwatsubo et al., 1994), Moreover, AD-causing mutations in APP near the beta - and gamma -secretase cleavage sites all increase Abeta 42, those near beta -secretase cleavage site augment beta -site proteolysis, leading to the elevation of both Abeta 40 and Abeta 42 (Citron et al., 1992; Cai et al., 1993), whereas those near the gamma -site specifically increase production of Abeta 42 (Suzuki et al., 1994). Taken together, these findings implicated Abeta in the pathogenesis of AD and spurred AD researchers to identify the Abeta -releasing proteases.

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

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

Two members of a disintegrin and metalloprotease (ADAM) family, tumor necrosis factor-alpha (TNF-alpha )-converting enzyme (TACE or ADAM-17) and ADAM-10, are candidate alpha -secretases (Fig. 2). TACE cleaves pro-TNF-alpha , releasing the extracellular domain (TNF-alpha ) in a manner similar to that of APP. TACE apparently processes a spectrum of type 1 membrane glycoproteins, including TNF-alpha , the p75 TNF receptor, L-selectin adhesion molecule, and TGF-alpha .

The inhibition or knockout of TACE decreases the release of the alpha -cleaved product salpha -APP (Buxbaum et al., 1998b). Mice lacking TACE die in utero, emphasizing the importance of ectodomain shedding during embryonic development (Peschon et al., 1998). However, cells deficient in TACE still have a residual alpha -secretase activity that cannot be increased by phorbol esters (Buxbaum et al., 1998b). Thus, TACE may play a role in regulated PKC-dependent alpha -secretion.

TACE also seems to process Notch receptor. Upon ligand activation, Notch is processed by TACE (Brou et al., 2000; Mumm et al., 2000) (Fig. 2C) The membrane-associated carboxyl terminus is then cut by a PS-dependent r-secretase to produce the Notch intracellular domain (NICD), which translocates to the nucleus in which it interacts with and activates the CSL family of transcription factors (Schroeter et al., 1998). Such signaling is essential for cell fate determinations and tissue patterning during embryonic development.

Another metalloprotease, ADAM-10, also seems to process APP in an alpha -secretase-like manner (Lammich et al., 1999; Lopez-Perez et al., 2001). Overexpression of ADAM-10 increased both basal and phorbal ester-inducible alpha -secretase activity (Lammich et al., 1999). A dominant-negative form of ADAM-10 with a point mutation in the zinc-binding site was found to inhibit basal and inducible alpha -secretase activity, but it did not totally abolish salpha -APP production (Lammich et al., 1999). ADAM-10 exists in a proenzyme form in the Golgi, but it becomes activated at the plasma membrane (Lammich et al., 1999)

ADAM-10 is also implicated in the Notch signaling pathway (Wen et al., 1997). Thus, TACE (ADAM-17) and ADAM-10 may both be alpha -secretases, which have very similar roles with respect to APP and Notch processing. Lopez-Perez et al. (1999, 2001) showed evidence for a role of the prohormone convertase PC7 in the constitutive alpha -secretase pathway. Definitive proof that they are alpha -secretases and whether other proteases also contribute to alpha -secretases activity remain to be determined. Because it is likely that several proteases contribute to alpha -secretase activity, it may be difficult to regulate APP processing pharmacologically through this pathway.

2. beta -Secretase. In 1999, beta -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). beta -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 beta -secretase does not proteolytically cleave itself. Instead, the responsible protease seems to be a furin-like protease (Bennett et al., 2000). beta -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 Abeta is normally produced by many cell types and in accordance with that expected for beta -secretase (Haass et al., 1992; Seubert et al., 1992; Shoji et al., 1992; Busciglio et al., 1993). The intracellular localization of beta -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 beta -secretase is located on chromosome 11, but no AD-causing mutation in this gene has been identified so far (Saunders et al., 1999). A beta -secretase homolog, BACE-2, maps to chromosome 21, raising the possibility that BACE-2 contributes to Down syndrome. Down syndrome patients secrete more Abeta 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 beta -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.

BACE-2 is strongly expressed in heart, kidney, and placenta, suggesting that it may be important in highly vascularized systemic tissues (Farzan et al., 2000). It will be critical to develop drugs that selectively block BACE but not BACE-2. BACE knockout mice seemed to abolish Abeta production totally and to develop normally, healthy, and fertile (Luo et al., 2001; Roberds et al., 2001), showing that the therapeutic of BACE for treatment of AD may be free of mechanism-based toxicity.

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

Proteins that undergo RIP include APP; Notch, a receptor involved in fate decisions during embryonic development; sterol regulatory element binding proteins (SREBPs), transmembrane proteins of the endoplasmic reticulum that regulate lipid metabolism (Brown et al., 2000); and ErbB-4, a member of the epidermal growth factor tyrosine kinase receptor family (Ni et al., 2001). How hydrolysis takes place in what is otherwise a water-excluded environment is unclear.

There is firm evidence that the intracellular portions of Notch and SREBP modulate gene transcription (Brown et al., 2000). Notch interacts with the coactivator p300 (Oswald et al., 2001), and SREBPs contain well-characterized DNA binding and transactivating domains that interact with a number of other transcription factors and coactivators (Edwards and Ericsson, 1999). Furthermore, the intracellular portion of APP forms a complex with the nuclear adaptor protein Fe65 and with Tip60, which has histone acetyltransferase activity like p300, and stimulates transcription when fused to the DNA binding domains of the heterologous transcription factors Gal4 or LexA (Cao and Sudhof, 2001; Kimberly et al., 2001). However, the term RIP may be misleading, because proteolytic cleavage within a membrane has never been demonstrated directly. For example, it is possible that alpha - or beta -secretase cleavage of APP results in movement of the C-terminal protein and exposure of the gamma -secretase sites to the aqueous environment (Nunan and Small, 2000).

The enzyme that catalyzes the secondary cleavage of APP, Notch, ErbB-4, and SREBPs is gamma -secretase. This gamma -secretase has pharmacological characteristics of an aspartyl protease and remarkably loose sequence specificity for its substrate because many mutations in APP near the gamma -secretase site still allow Abeta production in transfected cells (Maruyama et al., 1996; Tischer and Cordell, 1996; Lichtenthaler et al., 1997, 1999; Wolfe et al., 1999a-c) and seem to be a multiprotein complex, making its identification through expression cloning unlikely to succeed.

A crucial question is how protein cleavage itself is regulated. It seems that the triggering event is the removal of most of the extracytoplasmic part of the protein. This seems to be a prerequisite for the intramembrane cleavage by gamma -secretase (Heldin and Ericsson, 2001).

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 Abeta causes neurons in culture to die by apoptosis.

Researchers found many more terminal deoxynucleotidyl transferase dUTP nick-end labeling-stained nerve cells in Alzheimer's patients' brains than in those from people who had died of other causes. The problem, however, was that the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-stained neurons was so large that it was not consistent with the time course of the disease.

Several investigators have looked at some of the 14 caspases so far identified. Stadelmann et al. (1999) showed that brain tissue from Alzheimer's patients had more nerve cells with activated caspase-3 than did samples from people who died of other causes, and the number of apoptotic neurons with the active enzyme was small---only approximately 1 in 1100 to 5000 neurons was affected---to be consistent with the slow course of AD.

Su et al. (2001) reported a similar percentage of neurons with active caspase-3 in brain with Alzheimer's disease and that the enzyme tends to be located in and around the amyloid plaques and neurofibrillary tangles. Neurons with caspase-3 were found in brains of mouse models of Alzheimer's or nerve cells in culture.

Yuan and Yankner (1999) showed that cortical neurons taken from the brains of mice in which the caspase-12 gene had been knocked out resist Abeta apoptosis-inducing effects. Caspase-12 is located in the membrane of endoplasmic reticulum, which regulates cellular responses to stresses such as protein misfolding and aggregation, free radicals, and the high concentrations of calcium ions and chemical toxins. Yuan and Yankner (1999) and Troy et al. (2001) found that hippocampal neurons from mice with an inactivated caspase-2 gene were completely resistant to apoptosis when exposed to Abeta .

Raina et al. (2001) showed that upstream caspases such as caspase-8 and -9 are activated in the brains of patients with AD, but they did not find activation of downstream caspases such as caspase-3. Therefore, they proposed that although apoptosis may be initiated in the neurons, it is aborted before it can kill them. Gervais et al. (1999) have evidence, from both cultured cells and examination of Alzheimer's brains, that caspases cut APP releasing Abeta (Fig. 3).



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Fig. 3.   Probable processing of APP by caspases.

In addition, Lu et al. (2000) found that caspase cleavage of APP releases a second apoptosis-promoting peptide called CT31, because it contains 31 amino acids from APP's carboxyl end. If brain caspases attack APP to release the toxic products Abeta and CT31, then there may be a vicious cycle in which Abeta , by triggering caspase activation, fosters its own production, and thus further caspase activation and cell death. However, Soriano et al. (2001) found that caspase cleavage of APP actually decreases Abeta secretion by cells because it removes a signal sequence that would direct the peptide into the cell's secretory pathway.

The researchers found that the antibody which recognizes a caspase-3-cleaved fragment of fodrin, a major component of the fibers that form the cell skeleton of neurons, stained many more neurons in brains from Alzheimer's patients than in control brains. Dumanchin-Njock et al. (2001) showed that overexpression of CT31 triggers selective increase of Abeta 42 but not Abeta 40 production and elicits a concomitant cell toxicity that is caspase-independent. Rohn et al. (2001) reported that as the disease progressed, the brains received more of the fodrin staining and the tangles. de Boer et al. (2000) show that actin, another prominent protein of the cytoskeleton, undergoes caspase cleavage in brain with Alzheimer's disease. The researchers localized the caspase-cleaved actin to the degenerating nerve terminals. Mattson et al. (1998) showed that staining for caspases activated by Abeta can be found in the dendrites.

Several of the researchers suggested that Alzheimer's begins with such nerve terminal degeneration, consistent with findings that patients' degree of dementia is more highly correlated with the loss of nerve terminals in their brains than with other pathological features, such as plaque formation. Ultimately, as a nerve cell loses more and more of its terminals, it will die.

A big question concerns how Abeta might trigger caspase activation. Troy et al. (2001) report that in cultured neurons, Abeta seems to act through c-Jun NH2-terminal kinase (JNK). JNK, as well as the caspases, would be a potential target for drugs aimed at stopping or at least slowing Alzheimer's development. However, developing such drugs will not be easy, given the fact that both the caspases and the kinases play key roles in cell regulation throughout the body. For example, by helping eliminate cells with damaged DNA, the caspases protect against cancer. Caspase or kinase inhibitors aimed at stopping apoptosis in Alzheimer's could have unacceptable side effects. Antiapoptosis drugs by themselves may not be sufficient.

5. Amyloid-Degrading Enzymes. The most important question yet to be answered is why Abeta is deposited in sporadic AD, which accounts for more than 90% of all AD cases (Saido, 2000). Abeta 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 Abeta deposition is primarily a function of the steady-state level of Abeta in brain, the focus should be placed on how Abeta is metabolized in vivo.

The large percentage of familial or sporadic AD are not currently explained by abnormalities of Abeta production, so efforts to identify a candidate for Abeta -degrading proteases are important. Because an increase of only 50% in the production of a particular form of Abeta , caused by the majority of familial AD cases, leads to aggressive presenile Abeta pathology (Hardy, 1997; Selkoe, 1998), subtle alterations in this metabolic balance over a long period of time are likely to influence not only the pathological progression but also the incidence of the disease.

Reduced catabolism may account for the unresolved mechanism of late-onset AD development (Saido, 2000). Studies in mice show that newly generated Abeta is rapidly turned over in the brain (Savage et al., 1998), suggesting that Abeta -degrading proteases regulate its levels. However, the mechanism of Abeta catabolism has been less well understood than that of anabolism.

There are numerous proteases in the brain that could potentially participate in Abeta turnover. From the tube and tissue culture paradigms, Abeta degrader candidates include cathepsin D and E, gelatinase A and B, trypsin- or chymotrypsin-like endopeptidase, aminopeptidase, neprilysin (enkephalinase), serine protease complexed with alpha 2-macroglobulin, and insulin-degrading enzyme (Saido, 2000). However, a single, dominant protease that is responsible for Abeta degradation has not yet been found.

It is necessary to distinguish between proteases that can degrade Abeta only in its monomer state and those that can degrade oligomeric and/or highly aggregated fibrillar forms of Abeta . Among the former class, neprilysin and insulin-degrading enzyme (IDE) have been focused on to date. Very few proteases belonging to the latter class have been documented because Abeta becomes resistant to proteases as a result of structural changes associated with its polymerization into fibrils. Biochemical experiments in which purified proteases are tested on synthetic Abeta peptides are of limited value. The ability of a particular protease to degrade naturally produced Abeta species at physiological concentrations of enzyme and substrate is important. Each candidate protease will need to be tested in transgenic and knockout mice to determine its effects on normal Abeta clearing and deposits. Human brain tissue should also be studied, taking into account in which subcellular locus and under which conditions a protease is expected to cleave Abeta . Pharmacologically up-regulating certain Abeta -degrading proteases or interfering with the production or processing of their natural inhibitors could have great therapeutic potential.

a. Insulin-Degrading Enzyme. The enzyme occurs principally in a soluble form in the cytoplasm and is also present on intracellular membranes (Vekrellis et al., 2000). It occurs abundantly in a soluble, extracellular form in the nervous system as documented in human CSF and neuronal and microglial culture media (Qiu et al.,1998; Vekrellis et al., 2000). The existence of a membrane-anchored form of the protease suggests that it may regulate insulin-signaling at the plasma membrane and can participate in the degradation of both soluble and membrane-associated forms of Abeta . IDE degrades insulin, glucagon, atrial naturetic peptide, TGF-alpha , amylin and Abeta (Bennett et al., 2000). IDE has been shown to degrade rat and human amylin peptides similarly, despite the fact that only human amylin can form amyloid fibrils, suggesting that the motif recognized by IDE is not the beta -pleated sheet region per se, but it is a conformation of the monomer in a preamyloid state (Bennett et al., 2000).

The cleavage products of Abeta by IDE are not neurotoxic and are not prone to depositing amyloid plaques, and recombinant IDE reduces Abeta toxicity in cortical neuronal cultures (Mukherjee et al., 2000). Endogenous IDE has been shown to degrade synthetic Abeta monomers in homogenates and membrane fractions of human brain (Perez et al., 2000; K. Vekrellis and D. J. Selkoe, unpublished data).

Naturally occurring oligomers of secreted Abeta in culture medium are resistant to IDE, whereas Abeta monomers are avidly degraded by the enzyme (Qiu et al., 1998; Vekrellis et al., 2000). These findings suggest that IDEs mediate much of the degradation of soluble monomeric Abeta but have less ability to degrade Abeta once it becomes insoluble and/or oligomeric (Selkoe, 2001b).

IDE gene is located on chromosome 10q, and different sets of late-onset AD pedigrees have shown linkage to DNA markers in the vicinity of this gene (Tanzi and Bertram, 2001). In the National Institute of Mental Health registry of 435 families with late-onset AD, genetic linkage to markers near l0q23-q25 and an allelic association of one of these markers with AD have been documented (Tanzi and Bertram, 2001). However, any association of the AD phenotype with polymorphisms in the IDE gene itself remains to be examined. Missense mutations in IDE that decrease its ability to degrade insulin in muscle have been discovered in the inbred Goto-Kakizaki diabetic rat, a compelling model of type 2 diabetes mellitus (Fakhrai-Rad et al., 2000).

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 Abeta 1-42 catabolism (Iwata et al., 2001c). Neprilysin occurs almost exclusively in a membrane-anchored form and hydrolyzes several circulating peptides, such as enkephalin, atrial natriuretic peptide, endothelin, and substance P, and has wide tissue distribution and substrate specificity (Turner and Tanzawa, 1997). The intracerebral injection of synthetic Abeta peptides provided evidence that neprilysin is a major Abeta 42-degrading protease in rat brain, although the enzyme did not mediate Abeta 40 degradation in this paradigms (Iwata et al., 2001c).

Degradation of Abeta in the soluble fraction of brain seems not to be decreased by inhibition or deletion of neprilysin (Iwata et al., 2001c), whereas degradation of Abeta in the membrane fraction of brain is decreased ~25% to 35% by neprilysin inhibitors and ~70% by IDE inhibitors (K. Vekrellis and D. J. Selkoe, unpublished data), suggesting that neprilysin has little role in degrading soluble Abeta but can degrade buffer-insoluble, SDS-extractable Abeta associated with membranes (Selkoe, 2001b).

Steady-state levels of endogenous Abeta are elevated in the brains of young neprilysin-deficient mice (Iwata et al., 2001c), but the increase was not large and plaque formation was not observed. Given the rapid turnover of Abeta in brain (Savage et al., 1998), if neprilysin were the major degrader of Abeta , its deletion should produce an even greater accumulation. Therefore, other proteases may compensate in part for the loss of neprilysin. Long-term thiorphan infusion, which should inhibit several proteases, led to actual plaque formation in rats (Iwata et al., 2001c), a more robust effect than that of deleting neprilysin (Selkoe, 2001b).

The regional levels of Abeta in the neprilysin-deficient mouse brain were in the distinct order of hippocampus, cortex, thalamus/striatum, and cerebellum, with hippocampus having the highest level and cerebellum the lowest, correlating with the vulnerability to Abeta deposition in brains of humans with AD (Iwata et al., 2001c). Neprilysin mRNA levels were lowest in the hippocampus and temporal gyrus of brains with AD, which are vulnerable to senile plaque development, but levels were highest in the caudate and peripheral organs, which are resistant to senile plaque development (Yasojima et al., 2001). Work is underway to find evidence of linkage between AD and markers on chromosome 3, where the neprilysin gene is found.

c. Plasmin. The plasmin proteolytic cascade, known to be crucial for fibrinolysis and cell migration, has recently been implicated in Abeta 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 Abeta aggregates to generate plasmin (Tucker et al., 2000).

Plasmin can significantly decrease the amount of neuronal injury induced by aggregated Abeta (10-30 µM) (Tucker et al., 2000). In vitro biochemical assays indicate that plasmin can proteolyze fibrillar Abeta , although at an efficacy that is approximately 100-fold less than that for freshly dissolved (largely monomeric) Abeta . The reaction was approximately 20-fold less efficient than that involving aggregated fibrin (Tucker et al., 2000). Moreover, in vivo evidence for a role of plasmin in regulating Abeta monomer or polymer levels has not yet appeared. uPA gene is mapped to a position near the center of the linkage region near 10q23-q25 (Tanzi and Bertram, 2001).

d. Endothelin-Converting Enzyme. This integral membrane zinc metalloprotease, with its active site located in the lumen and extracellularly, can cleave the endothelin precursors, bradykinin, substance P, and the oxidized insulin B chain (Eckman et al., 2001). Cellular overexpression of ECE-1 leads to a marked reduction in the levels of naturally secreted Abeta 40 and Abeta 42 in CHO cells. The enzyme directly proteolyzes both synthetic peptides in vitro (Eckman et al., 2001). Whether this protease can alter Abeta levels in vivo remains to be determined.

e. Other Candidate Proteases. Other proteases that have been reported to digest synthetic Abeta under in vitro conditions include matrix metalloproteinase-9 (Backstrom et al., 1996) and cathepsin D (McDermott and Gibson, 1996). The metalloendopeptidase 24.15 has been reported to indirectly regulate Abeta degradation. Whereas this enzyme does not proteolyze synthetic Abeta , decreasing its activity via antisense treatment leads to increased Abeta levels in cell culture (Yamin et al., 1999), suggesting that MP 24.15 processes a zymogen of an Abeta protease or degrades its endogenous inhibitor (Selkoe, 2001). Chevallier et al. (1997) showed that Abeta production or degradation is not affected by very selective inhibitors of endopeptidase 24.15, indicating that this enzyme is not involved in the genesis and degradation of Abeta . alpha 1-Antichymotrypsin, the serine protease inhibitor, can increase Abeta deposition in APP transgenic mice (Mucke et al., 2000; Nilsson et al., 2001)

C. Amyloid Cascade Hypothesis: Two Major Amyloid Precursor Protein Metabolites Involved in Alzheimer's Disease Pathogenesis

The amyloid cascade hypothesis was first formulated more than a decade ago and centers around the Abeta 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 Abeta - and CT- (carboxyl terminal peptides of APP) peptides (Fig. 4).



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Fig. 4.   The amyloid cascade hypothesis.

Classic Abeta 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 beta -APP and the subsequent production of Abeta could be linked to the genesis of AD (Checler, 1995). Furthermore, studies of plasma and fibroblasts from subjects with mutations in the genes encoding beta -APP have established that they all alter beta -APP processing, which normally leads to the secretion of Abeta [relative molecular mass, 4,000; Mr, 4 K; ~90% Abeta 1-40, 10% Abeta 1-42(43)], so that the extracellular concentration of Abeta 1-42(43) is increased (Suzuki et al., 1994). These results indicate that the beta -APP mutations probably cause AD through an increase of Abeta 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 Abeta 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 Abeta , invariably leads to the early emergence of AD neuropathology (Selkoe, 1994). In addition, the E4 allele of ApoE promotes the precipitation of Abeta 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 Abeta generation (Ertekin-Taner et al., 2000).

In line with this are those studies showing that Abeta 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 Abeta ) 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 Abeta 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 Abeta was needed to exert toxicity, and some studies still failed to demonstrate Abeta 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, Abeta promoted neurite outgrowth (Yankner et al., 1990; Koo et al., 1993) instead of exerting toxic action. Most important is that Abeta 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 Abeta deposition (Cochran et al., 1991).



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Fig. 5.   The tau and tangle hypothesis.

It is also possible that the critical factor is not Abeta itself but that Abeta 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, Abeta 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 beta -APP has produced interesting candidates, the most promising of which are the amyloidogenic CT fragments of beta -APP. The transgenic mice expressing beta -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 Abeta -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).


                              
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TABLE 1
Neurotoxic mechanisms of Abeta


                              
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TABLE 2
In vivo evidence of amyloidogenic CT fragment generation

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 Abeta fragments (Table 3).


                              
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TABLE 3
Summary of various effects of Abeta and CT

The synthetic peptide APP713-730 of APP770 is highly fibrillogenic and interacts with tau in vitro and causes apoptotic neuronal death, suggesting that APP sequences other than Abeta 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 Abeta amyloid cascade hypothesis has fared remarkably well and has had few serious challenges.

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



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Fig. 6.   Summarized neurotoxic mechanisms of Abeta . Abeta is the main component of neuritic plaques found in brains with AD. Abeta 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 Abeta , may lead to cellular dysfunctions via the inhibition of various enzyme activities, disruption of signaling pathways, and an activation of nuclear transcription factors. Abeta 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 Abeta , releasing a variety of proinflammatory cytokines, chemokines, and NO, exerting toxi