Beta Amyloid (Abeta) Blog~Alternative Pathways for Production of Beta-Amyloid Peptides of Alzheimer’s Disease

Alternative Pathways for Production of Beta-Amyloid Peptides of Alzheimer’s Disease

Vivian Hook,^1,2* Israel Schechter,³ Hans-Ulrich Demuth,⁴ and Gregory Hook⁵

¹Skaggs School of Pharmacy and Pharmaceutical Sciences, Univ. of Calif., San Diego, La Jolla, CA 92093 USA

²Depts. of Neuroscience, Pharmacology, and Medicine, School of Medicine, Univ. of Calif., San Diego, La Jolla, CA 92093 USA

³Dept. of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

⁴Probiodrug AG, Weinbergweg 22, Halle (Saale), Germany

⁵American Life Science Pharmaceuticals, Inc., San Diego, CA 92109 USA

^* To whom correspondence should be addressed: Dr. V. Hook, Skaggs School of Pharmacy and Pharmaceutical Sciences, Univ. of Calif., San Diego, 9500 Gilman Dr. MC 0744, La Jolla, CA 92093-0744, phone (858) 822-6682, email vhook@ucsd.edu

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Abstract

This highlight article describes three Alzheimer’s disease (AD) presentations made at the 5^th General Meeting of the International Proteolysis Society that address enzymatic mechanisms that produce neurotoxic beta-amyloid (Aβ) peptides. One group described the poor kinetic properties of the BACE 1 β-secretase for cleaving the wild-type β-secretase site in the APP found in most AD patients. They demonstrated that cathepsin D displays BACE 1-like specificity, is 280-fold more abundant in human brain than BACE 1, and pepstatin A inhibits cleavage of β-secretase site peptides by brain extracts and cathepsin D, but not by BACE 1. Nevertheless, as BACE 1 and cathepsin D show poor activity towards the wild type β-secretase site, they suggested continuing the search for additional β-secretase candidate(s). The second group reported that cathepsin B is such an alternative β-secretase candidate possessing excellent kinetic efficiency and specificity for cleaving the wild-type β-secretase site. Significantly, they demonstrated that inhibitors of cathepsin B improved memory function with reduced amyloid plaque neuropathology and decreased brain Aβ(40/42) and β-secretase activity in AD animal models expressing APP containing the wild-type β-secretase site. The third group addressed isoaspartate and pyroglutamate (pGlu) posttranslational modifications of Aβ that are present in AD brains, with evidence that cathepsin B, but not BACE 1, efficiently cleaves the wild-type β-secretase site containing isoaspartate. They also found that cyclization of N-terminal Glu by glutaminyl cyclase generates pGluAβ(3-40/42) peptides that are highly amyloidogenic. These presentations suggested that cathepsin B and glutaminyl cyclase are potential new AD therapeutic targets.

Keywords: Alzheimer’s disease, beta-amyloid, beta-secretase, cathepsins, glutaminyl cyclase, inhibitors

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Introduction

This article reports on presentations made by three independent groups led by Schechter, Hook, and Demuth on Alzheimer’s disease (AD) enzyme drug targets reported at the 5^th General Meeting of the International Proteolysis Society (IPS) held in Patras, Greece on October 20-24, 2007. Together these studies challenge the current hypothesis that BACE 1 is the predominant β-secretase responsible for the processing of the amyloid precursor protein. These presentations showed that (a) cathepsin D displays BACE 1-like specificity and is much more abundant than BACE 1 in the brain, yet since both enzymes cleave the wild-type (WT) β-site sequence very slowly it is necessary to search for additional β-secretase(s), (b) cathepsin B displays highly efficient processing of the WT β-secretase site of APP expressed in the majority of AD patients, and (c) the production by cathepsin B and glutaminyl cyclase of posttranslationally modified forms of Aβ consisting of isoAsp1-Aβ and the highly amyloidogenic pGluAβ(3-40/42). The authors of this paper represent each of the three research groups.

Beta-Amyloid of Alzheimer’s Disease

Abnormal accumulation of neurotoxic Aβ peptides is a significant factor in the development of AD, and thought to be the likely cause of memory and cognitive loss in this disease condition (Iversen et al., 1995; Sisodia, 1999; Selkoe, 2001; Gandy et al., 2003). Neuropathological examination of brains from AD patients reveals the accumulation of secreted Aβ peptides in extracellular amyloid plaques that are involved in neuronal loss in brain regions (hippocampus and cortex) responsible for memory and cognitive functions. The majority of AD patients are afflicted with sporadic AD that is not linked to genetic mutations (Blennow et al., 2006; Turner, 2006). A smaller portion of AD patients possess familial forms of AD with inherited genetic mutations, especially those mutations in APP and presenilins that result in increased Aβ production and memory deficit when overexpressed in transgenic mouse models of AD (Dodart et al., 2002; Masliah and Rockenstein, 2000; Price and Sisodia, 1998). Notably, abnormal accumulation of Aβ occurs in sporadic and familial AD patients and, therefore, Aβ represents a biochemical hallmark of the disease condition. Clearly, strategies to reduce Aβ production will facilitate development of therapeutic agents for this devastating disease for which currently there is no effective drug treatment.

β-Amyloid (Aβ) production by proteolytic processing of APP

Aβ peptides are generated in neuronal secretory vesicles by proteolytic cleavage of the amyloid precursor protein (APP) by proteases, called β-secretase and γ-secretase that cleave at the N-terminus and variant C-termini of Aβ within APP, respectively, resulting in Aβ of 40 or 42 amino acids (Aβ40 and Aβ42, respectively) (Figure 1). Because the N-termini of Aβ40 and Aβ42 are identical, development of inhibitors that reduce cleavage of APP at the β-secretase site are likely to be effective for reducing Aβ peptide forms with alleviation of neurodegeneration and memory deficit. The APP in the vast majority of AD patients possesses the wild-type β-secretase site sequence.

Figure 1 Aβ production from APP in neurons via regulated and constitutive secretory pathways

Production of β-amyloid peptides involves cleavage of the β-secretase site that is located in the soluble intracellular environment, while cleavage at the γ-secretase site occurs within a transmembrane domain of the APP precursor protein (Selkoe, 2004;Haass and Selkoe, 2007). Proteolytic processing of the γ-secretase site, within the transmembrane domain, has been demonstrated in detail by studies of the presenilin γ-secretase complex in membranes (Selkoe and Wolfe, 2007). Significantly, APP in the regulated secretory vesicles (RSV) APP is present as a fully soluble protein (Tezapsidis et al., 1998 ), with secretion of full-length APP from RSV (Efthimiopouloset al., 1996). These studies have in large part been conducted in studies of APP and β-amyloid production in the constitutive secretory pathway.

Importantly, the major portion of secreted, extracellular β-amyloid peptides is produced in the regulated secretory pathway (Tezapsidis et al., 1998). In the regulated secretory vesicles (RSV), APP is also present as a fully soluble protein. The full-length soluble APP, thus, has β- and γ-secretease sites accessible for proteolytic cleavage. The approach by the Hook laboratory for examining APP processing in the RSV for producing the majority of extracellular β-amyloid peptide is unique in the field (Hook et al., 2002, 2005), since most studies have examined β-amyloid peptide in the constitutive secretory pathway. The secretory vesicles of the regulated secretory pathway are distinct from those of the constitutive secretory pathway (Lodish et al., 1999). Clearly, in the RSV, the soluble and transmembrane orientation of APP allows its proteolytic processing by β- and γ-secretases.

According to the amyloid hypothesis, proteolytic processing of human brain APP (Selkoe, 2001), preferentially generates Aβ 1-40 and to a minor degree Aβ 1-42. Both peptides have been believed to initiate aggregation and later deposition in the neuritic plaques characteristic for AD (Findeis, 2007). In contrast several studies recently clarified that Aβ 1-40 has neuroprotective functions (McGowan et al, 2005; Kim et al., 2007). In addition, the post mortem analysis of brain samples from sporadic Alzheimer and Down syndrome patients revealed many more Aβ species whose profiles differ from that in commonly used AD animal models (Roher et al., 1993; Kuoet al., 1997; Saido 1998).

Most abundant among these APP-derived peptides are the isoAsp1-Aβ and the pGlu3-Aβ species (Roher et al., 1993; Saido 1998; Shimizu et al., 2000). The isoAsp-Aβ and also pGlu-Aβ can make up more than 50% of all amyloid, depending on the site of deposition or on the soluble Aβ fraction of the brain (Roher et al., 1993; Picciniet al., 2005). In contrast to all other Aβ species, pGlu-Aβ aggregates much faster (He and Barrow, 1999) and also seeds the oligomerization and fibrillation process of the full-length Aβ species (Schilling et al., 2006).

Abundant accumulation of Aβ species also occurs in brains of cognitive normal elderly patients, without signs of neuronal dysfunction. However, the most prominent species found in these people are the amyloid peptides Aβ1-40 and Aβ1-42 but not the pGlu3-Aβ species (Piccini et al., 2005). The proposal that the BACE 1 product Aβ1-40 is neuroprotective, combined with the high abundance of N-truncated and pyroglutamated Aβ peptides, which correlate to deposition markers and the severity of dementia, supports the hypothesis that there may be different pathways of APP turnover which trigger formation of different Aβ peptide species (Kim et al., 2007;Maeda et al., 2007).

For these reasons, the β-secretase activity that cleaves the wild-type β-secretase site in APP must be determined for effective β-secretase inhibitors to be developed for the majority of the AD population.

Enzymatic and kinetic properties of proteases for cleaving the wild-type β-secretase site of APP: cleavage-site specificity of proteases

The specificity for peptide substrate cleavage by proteases involves a large active site divided into subsites, using the model and nomenclature of Schechter & Berger (Schechter and Berger, 1967). This model describes interactions of the protease with the substrate amino acids flanking the cleavage site (Figure 2a). The amino acids forming the cleaved peptide bond are termed P1-↓P1’, with residues at the N-terminal and C-terminal sides of the cleavage site indicated as P1, P2…. and P1’, P2’…., respectively. The protease active site is viewed as a series of subsites (S1, S2… and S1’, S2’…) each accommodating one amino acid residue of the substrate. Protease subsites interact with the polypeptide backbone and with side chains of amino acids of the substrate.

Figure 2 Model of protease and peptide substrate interactions: application to the wild-type β-secretase cleavage site

Multiple points of interactions in several subsites are essential for obtaining the high association constants necessary for efficient biological function. Within the large active site certain subsites (close or remote from the catalytic site) play a major role in determining specificity (Schechter and Berger, 1967; Schechter, 2005). For example, the S1 subsite of trypsin has a clear preference for binding to basic P1 residues consisting of lysine or arginine (Halfon et al., 2004). In papain (Schechter and Berger, 1968) and the cysteine cathepsins (Rawlings and Barrett, 2004), specificity is determined mainly by the hydrophobic S2 subsite that prefers binding to hydrophobic P2 residues. The specificity and biological activity of caspases is determined by both S1 and S4 remote from the catalytic site (Nicholson and Thornberry, 1997). Generally, the P1 to P3 residues are important for protease selectivity for cleavage sites, based on interaction with S1 to S3 subsites of the protease. Therefore, cleavage site-specific assays often incorporate the native P1 to P3 residues, or longer extensions of the peptide substrate.

Evaluation of the P2-P1 residues at the Wild-type and Swedish mutant β-secretase cleavage sites

At the wild-type β-secretase site of APP, the -P3-P2-P1-↓P1’-P2’-P3’- residues are Val-Lys-Met-↓Asp-Ala-Glu- (Figure 2b). This wild-type β-secretase site is expressed by the majority of AD patients, and, therefore, represents a key cleavage event for production of neurotoxic Aβ. Interestingly, a mutant β-secretase site expressed in an extended Swedish family (Citron et al., 1992) results in elevated production of Aβ in these patients, and transgenic mice expressing the human Swedish mutant APP show elevated levels of Aβ (Hsiao et al., 1996). The Swedish mutant P2-P1 residues consist of Asn-Leu that substitute for the wild-type P2-P1 residues of Lys-Met. It is important to realize that the amino acids at the Swedish mutant compared to the wild-type β-secretases site differ in chemical properties. A change in the chemical properties of the amino acid side chains may define distinct substrate preferences by the protease.

Notably, the wild-type Lys (lysine) residue at the P2 position is a positively charged hydrophilic residue, but the Swedish mutant Asn (asparagine) residue is an uncharged amino acid. The charged and uncharged properties of Lys and Asn, respectively, at the P2 position may be recognized by different proteases. The P2 residue that interacts with the S2 subsite of proteases is known to be an important determinant of substrate specificity for many proteases, such as the cysteine cathepsins (Rawlings and Barrett, 2004). Based on the different charged property of the P2 residue of the wild-type site compared to uncharged P2 residue of the Swedish mutant β-secretase site, it is possible that different proteases may cleave the wild-type β-secretase site compared to the Swedish mutant β-secretase site.

Kinetic properties of protease enzymes for polypeptide substrates

Kinetic properties of enzymes are studied by measuring their catalytic efficiency (Berg et al., 2002; Voet and Voet, 2004). The ratio k_cat/K_m represents the rate of hydrolysis at infinite dilution of the substrate (Schechter, 1970). In general, higher values of k_cat/K_m indicate greater enzymatic performance towards the substrate (Berg et al., 2002; Voet and Voet, 2004). Active proteases usually have k_cat/K_mvalues that range between tens of thousands to a few millions.

These kinetic properties should be assessed in studies of proteases with β-secretase activity to identify those that possess the greatest efficiency for β-secretase cleaving activity. Because cleavage of a peptide bond may occur by several proteases, inhibition of the protease with the highest catalytic efficiency is likely to be most effective for reducing cellular Aβ.

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Overview of the Presentations

The presentations by the Schechter, Hook, and Demuth groups together implicate new β-secretase mechanisms and posttranslational modifying enzymatic mechanisms for the production of Aβ peptide forms. The Schechter group demonstrates that human cathepsin D displays BACE 1-like β-secretase specificity, that cathepsin D is more abundant than BACE 1 in the brain, and that β-secretase activity observed in brain extracts at pH 3.8 is mainly due to cathepsin D, which indicates the need to search for additional β-secretase candidate(s) (Schechter and Ziv, 2008).

The Hook group showed that cathepsin B efficiently cleaves the wild-type β-secretase site (Hook et al., 2005), and cathepsin B is colocalized with Aβ peptides in regulated secretory vesicles (Hook et al., 2005) that provide the majority of extracellular Aβ that accumulates in AD. Moreover, inhibitors of cathepsin B improve memory, with reduction of brain Aβ and amyloid plaques, in the London APP mouse model of AD expressing the wild-type β-secretase site of APP (Hook et al., 2008). These inhibitors also reduced Aβ in the guinea pig animal model that expresses APP containing the wild-type β-secretase site (Hook et al., 2007b; Hook et al., 2007a). These findings support cathepsin B as a new β-secretase target for drugs to reduce Aβ that contributes to memory deficit in AD.

The Demuth group discussed the production of posttranslationally modified Aβ forms in AD brain, consisting of isoaspartate (isoAsp) Aβ, isoAβ(40/42), and N-terminal truncated peptides including the pyroglutamate (pGlu) modified form of truncated Aβ, pGluAβ(3/11-40/42). Evidence was provided that cathepsin B, but not BACE 1, efficiently cleaves the wild-type β-secretase site containing isoAsp and that glutaminyl cyclase catalyzes the formation of pGlu to produce pGluAβ(3/11-40/42). These findings independently confirmed cathepsin B as a possible drug target to reduce Aβ production, and also suggests glutaminyl cyclase as a new drug target for inhibiting the production of pGluAβ(3/11-40/42) peptide forms.

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Properties of BACE 1 and Cathepsin D Indicate the Need to Search for Additional β-Secretase Candidate(s) (presented by I. Schechter)

BACE 1 fulfills many criteria for the β-secretase that cleaves at the β-site of APP (Linet al., 2000; Vassar et al., 1999; Wolfe, 2006), yet it has some problems that are described below (Schechter and Ziv, 2008).

Poor kinetic properties of BACE 1 for the wild-type β-secretase site

BACE 1 cleaves the Swedish mutant β-secretase site sequence of APP much better than the wild type β-secretase sequence present in the worldwide AD population. BACE 1 activity for the wild-type β-site is extremely low, cleaving it with very lowk_cat/K_m values reported as 40 M^-1s^-1 (Lin et al., 2000) and 62 M^-1s^-1 (Shi et al., 2001). Proteases acting on relevant substrates usually have k_cat/K_m values that range between tens of thousands to a few millions (Dunn and Hung, 2000).

BACE 1 is a membrane-bound enzyme and the kinetic studies described above (Linet al., 2000, Shi et al., 2001) were performed in vitro using soluble BACE 1. It has been argued that enzymes anchored in the cell membrane might exhibit reduced activity when freed in solution. TACE is a membrane-bound enzyme like BACE 1, and in vivo it cleaves the precursor TNF-α that is a membrane-bound protein like APP. Yet in vitro TACE cleaves a peptide spanning the cleavage site of the precursor TNF-α with k_cat/K_m of 1.8 × 10⁵ M⁻¹s⁻¹ (Moss et al., 1997), which shows that solubilized TACE retains its enzymatic efficiency. Furthermore, mapping of the active site of BACE 1 led to synthesis of an excellent peptide substrate cleaved by BACE 1 with k_cat/K_m of 3.42 × 10⁵ M^-1s^-1 (Turner et al., 2001). This peptide differs from the wild-type β- secretase site octapeptide sequence (P4-P4’) in 7 out of 8 amino acid residues. These findings suggest that BACE 1 acts on yet unknown substrate(s), and that its cleavage of the wild-type β-secretase site of APP may be a physiological aberration.

Gene knockout experiments in rodents and corresponding genetic defects in human may reveal different phenotype(s)

Several BACE 1 knockout studies conducted in mice expressing human APP show that the absence of BACE 1 reduces Aβ formation (Cai et al., 2001; Luo et al., 2001,Roberds et al., 2001). These important findings support BACE 1 as a β-secretase. Yet studies in other systems show that gene knockout experiments in rodents may show different phenotypes compared to the corresponding genetic phenotype in the human disease condition (Jacks, 1996). For example, nine different tumor suppressor genes associated with human tumors were mutated in mouse and rat. Two mutated genes represented the human phenotype, five mutated genes showed a different spectrum of tumors, and two mutated genes did not show any tumors in rodents (Jacks, 1996). These studies show that genetic phenotypes in animal models may or may not represent the human disease phenotype.

Considering the above issues, regarding the poor activity of BACE 1 and knockout of BACE 1 in the context of knockout of other genes, we searched for other β-secretase candidate(s) in the brain.

Cathepsin D displays BACE 1-like specificity as β-secretase: their properties indicate the need to search for additional β-secretase candidate(s)

The Schechter group searched for β-secretase in brain utilizing assays of β-secretase activity with peptide substrates containing the wild-type and mutant Swedish β-secretase site sequences. Beta-secretase activity was purified from brain extracts (bovine) and identified as cathepsin D. Although early studies implicated cathepsin D in Alzheimer’s disease (Ladror et al., 1994; Cataldo et al., 1995), the finding that knockout of the cathepsin D gene in mice did not affect Aβ formation (Saftig et al., 1996) abolished interest in cathepsin D. However, in consideration of the relevance or irrelevance of animal gene knockout studies to the human genetic disease phenotype, further studies of cathepsin D were warranted. Thus, we studied the properties of cathepsin D compared to BACE 1 that were not examined in prior studies (Schechter and Ziv, 2008).

The kinetic constants k_cat/K_m for cleaving β-secretase sites of peptides with the Swedish mutant and wild-type sequences by cathepsin D and BACE 1 were determined (Table 1). Results showed that human BACE 1 and cathepsin D have similar kinetic constants, cleaving the Swedish mutant β-secretase site more efficiently than the wild-type sequence. Gel filtration experiments of brain extracts revealed β-secretase activity at the position of cathepsin D (42 kDa) but not at that of BACE 1 (72 kDa). Pepstatin A at 20 nM completely inhibits the cleavage of peptide substrates with the Swedish mutant β-site by both cathepsin D and brain extracts, while BACE 1 activity on the same peptides is not inhibited by one thousand fold higher concentration of pepstatin A (20 μM). Quantitative Western blots show that in human brain cathepsin D is about 280-fold more abundant than BACE 1. Therefore, it is conceivable that β-secretase activity observed in brain extracts may be primarily due to cathepsin D and not BACE 1.

Table 1 The kcat/Km values for cleavage of the β-secretase sites of Swedish mutant (SW) and wild-type (WT) peptide substrates by human cathepsin D and human BACE 1

Nevertheless, since both BACE 1 and cathepsin D show poor kinetic activity towards the wild-type β-secretase site sequence (Table 1), it is necessary to continue the search for additional β-secretase candidate(s) for cleaving the wild-type β-secretase site (Schechter and Ziv, 2008).

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Cathepsin B as a β-Secretase of the Wild-Type β-Secretase Site and Cathepsin B Inhibitors as Potential Alzheimer’s Disease Therapeutics (presented by V. & G. Hook)

The Hook group reported that cathepsin B may represent such a novel β-secretase for wild-type β-secretase site cleavage. Moreover, they showed that inhibitors of cathepsin B are efficacious to improve memory in animal models expressing APP containing the wild-type β-secretase site and suggest that such inhibitors may have potential as AD therapeutics.

Aβ production in the regulated secretory pathway of neurons

Neurons possess two distinct secretory pathways consisting of the regulated secretory pathway for activity-dependent secretion, and the constitutive secretory pathway for continuous basal secretion (Thomas, 2002; Fugere and Day, 2005;Seidah et al., 1994; Zhou et al., 1999) (Figure 1). Different proteolytic mechanisms occur in the two secretory pathways (Gensberg et al., 1998; Lodish et al., 1999;Gumbiner and Kelly, 1982; Docherty and Steiner, 1982; Gainer et al., 1985;Benjannet et al., 1997; Hook et al., 2004; Thomas, 2002) and thus, it is important to define the primary secretory pathway(s) involved in Aβ production for elucidation of the major proteases that generate secreted Aβ, which accumulates in amyloid plaques of AD brains. Such studies indicate that regulated secretion of Aβ from brain neurons in vivo produces most of the secreted Aβ (Farber et al., 1995; Cirrito et al., 2005; Efthimiopoulos et al., 1996; Hook et al., 2002; Nitsch et al., 1993; Nitsch et al., 1992; Hook et al., 2005; Jolly-Tornetta et al., 1998). Therefore, β-secretase was studied in regulated secretory vesicles that contain Aβ.

Cleavage site specific assay for cleaving the wild-type β-secretase site leads to identification of cathepsin B in regulated secretory vesicles for Aβ production

In search of endogenous protease activity for cleaving the wild-type β-secretase site, a cleavage site-specific assay was developed with the Z-Val-Lys-Met-↓MCA substrate that mimics the P1 to P3 residues of the wild-type β-secretase site. This peptide-MCA substrate monitors β-secretase activity by proteolytic production of fluorescent MCA (methylcoumarinamine). The Z-Val-Lys-Met-↓MCA cleaving activity in regulated secretory vesicles, purified from neuronal-like chromaffin cells, showed a pH optimum of approximately pH 5.5 to 6.0 that is consistent with the acidic intravesicular environment (Hook et al., 2002). Moreover, its activity required reducing conditions, which mimics the in vivo reducing environment of secretory vesicles (Hook et al., 2002). The Z-Val-Lys-Met-↓MCA cleaving activity was represented by cysteine protease activity, since it was inhibited by E64c (Hook et al., 2002).

The Z-Val-Lys-Met-↓MCA cleaving activity was purified from the regulated secretory vesicles (RSV) by a series of chromatographic purification steps. Identification of the enzyme was facilitated by use of the activity-based probe, DCG-04, that detects active cysteine proteases by irreversible binding to the active site (detected by avidin-biotin) (Hook et al., 2005). Peptide sequencing of the purified enzyme revealed its identity as the cysteine protease cathepsin B (Hook et al., 2005). Immunoelectron microscopy confirmed the localization of cathepsin B within regulated secretory vesicles (Hook et al., 2005). In contrast with the known function of cathepsin B in lysosomes for protein degradation (Mort, 2004), these findings indicate the secretory vesicle as a new subcellular site for a biological function of cathepsin B.

Notably, inhibition of cathepsin B with cysteine protease inhibitors substantially reduced Aβ production in the isolated RSV (Hook et al., 2005). Aβ40 production was reduced by the highly selective cathepsin B inhibitor CA074 (Towatari et al., 1991), as well as by the general cysteine protease inhibitor E64c (Towatari et al., 1991). Treatment of chromaffin cells in primary culture with CA074Me, the-cell permeable form of CA074, also resulted in reduced amounts of Aβ produced in the regulated secretory pathway (but not the constitutive secretory pathway) (Hook et al., 2005). In addition, the inhibitor lowered cellular levels of CTFβ derived from APP cleavage by β-secretase, suggesting that β-secretase activity was inhibited. These findings indicate cathepsin B as a novel β-secretase in the regulated secretory pathway for production of secreted Aβ.

Kinetic properties of cathepsin B demonstrate its preference for the wild-type β-secretase site, rather than the Swedish mutant β-secretase site

Kinetic studies demonstrated the high activity and preference of cathepsin B for cleaving the wild-type β-secretase site, but not the Swedish mutant β-secretase site (Table 2) (Hook et al., 2008). The k_cat/K_m value for cathepsin B cleavage of the wild-type β-secretase substrate is 3.17 × 10⁵ M^-1sec^-1, indicating that cathepsin B efficiently cleaves the wild-type β-secretase substrate with high turnover of substrate to product.

Table 2 Kinetic properties of cathepsin B and BACE 1 for cleaving the wild-type β-secretase site of APP.

However, cathepsin B showed very low activity with the Swedish mutant β-secretase substrate Z-Val-Asn-Leu-↓MCA (mutant residues underlined). This finding is in marked contrast to BACE 1, which shows a preference for the Swedish mutant and not the wild-type β-secretase substrate. These biochemical kinetic studies demonstrate the high preference of cathepsin B for the wild-type β-secretase site, rather than the Swedish mutant site of APP. Significantly, the wild-type β-secretase site is relevant to the majority of the AD population, which expresses wild-type APP.

Animal models that express the wild-type β-secretase site of APP for inhibitor studies

The guinea pig and a transgenic mouse model of AD expressing human APP with the wild-type were used to evaluate the effects of cathepsin B inhibitors on Aβ production. The guinea pig (gp) expresses APP with the wild-type β-secretase site that generates Aβ, which is identical to the primary sequence of human Aβ (Johnstone et al., 1991; Beck et al., 2003; Beck et al., 1997). Transgenic AD mice expressing human APP containing the wild-type β-secretase site, with the London mutation near the γ-secretase site (London APP mice), overproduce Aβ in amyloid plaques and develop memory deficit (Moechars et al., 1999; Dewachter et al., 2000). The preference of cathepsin B for cleaving the wild-type β-secretase site predicted that cathepsin B inhibitors would be effective in these animal models expressing APP containing the wild-type β-secretase site, but not those expressing APP with the Swedish mutant β-secretase site. This prediction was tested with animal models expressing wild-type and Swedish mutant β-secretase sites of APP (Hook et al., 2007b; Hook et al., 2007a; Hook et al., 2008).

Inhibitors of cathepsin B reduce Aβ in guinea pig brains

In vivo studies in guinea pigs demonstrated significant reduction of Aβ in brain by inhibitors of cathepsin B consisting of CA074Me (prodrug form of the selective cathepsin B inhibitor, CA074), E64d (the ester prodrug of its biologically active acid form, E64c) (Tamai et al., 1986), and acetyl-L-leucyl-L-valyl-L-lysinal (Ac-LVK-CHO) (McConnell et al., 1993). Administration (icv) of E64d or CA074Me resulted in substantial reductions of Aβ40 and Aβ42 by 50-70% after 7 or 30 days of treatment (Figure 3), and similar reductions of these Aβ peptides resulted after treatment with Ac-LVK-CHO (Hook et al., 2007b; Hook et al., 2007a). The inhibitor treatments also reduced CTFβ, suggesting that the inhibitors reduced β-secretase activity. Moreover, the inhibitor treatment reduced Aβ levels in brain synaptosomes, which are enriched with RSV, thus, indicating inhibition of Aβ production in the regulated secretory pathway of brain neurons (Hook et al., 2007b). These data demonstrate the in vivoeffectiveness of cathepsin B inhibitors to reduce brain Aβ levels produced by cleavage of the wild-type β-secretase site of APP in the regulated secretory pathway.

Figure 3 In vivo administration of CA074Me, E64d or Ac-LVK-CHO reduces brain Aβ and CTFβ in the guinea pig

Inhibitors of cathepsin B improve memory and reduce Aβ in transgenic AD mice that express the wild-type, but not the Swedish mutant, β-secretase site of APP

In the London APP transgenic mouse model of AD that expresses human APP with the wild-type β-secretase site, administration of E64d or CA074Me for 30 days significantly improved the memory deficit (measured by the Morris water maze test) and reduced brain amyloid plaque (Hook et al., 2008). These inhibitors improved memory by about 50%, which approached the normal level of memory function in non-transgenic age matched controls (Figure 4a). Each inhibitor treatment also reduced brain amyloid plaque load by about 50% and reduced brain Aβ by about 40-50% (Figure 4b). Furthermore, the reduction of CTFβ by these inhibitors indicated inhibition of β-secretase activity.

Figure 4 In vivo administration of CA074Me or E64d improves memory and reduces brain plaque in transgenic mice expressing human APP containing the wild-type β-secretase site

In contrast, inhibitors of cathepsin B had no effect in a transgenic AD mouse model expressing the Swedish mutant β-secretase site of APP (Hook et al., 2008). In London APP mice that also express the Swedish mutation (Swedish/London APP), treatment with E64d or CA074Me had no effects on memory, brain amyloid plaque, brain Aβ, or CTFβ. The in vivo effects of these cathepsin B inhibitors mirrored their in vitro cleavage specificity for the wild-type β-seretase site compared to the Swedish mutant β-secretase site. These data support the hypothesis that cathepsin B possesses selectivity for cleaving the wild-type β-secretase site, rather than the Swedish mutant site of APP, for production of neurotoxic Aβ peptides. Importantly, cathepsin B cleavage of the wild-type β-secretase site is relevant to the majority of the AD population that express wild-type APP.

The function of cathepsin B as β-secretase is not precluded by BACE 1 knockout mouse results

Results presented by the Hook group, combined with knowledge in the field, suggest that cathepsin B and BACE 1 both function as β-secretase enzymes. While BACE 1 has been proposed to be the major β-secretase based on BACE 1 knockout experiments (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001; Vassar, 2004), detailed assessment of the BACE 1 knockout studies indicate that they do not preclude cathepsin B as a β-secretase for the wild-type β-secretase site in the regulated secretory pathway.

Roberds et al. describes that BACE 1 knockout mice lack β-secretase activity but the assays in which the β-secretase activity was assayed contained E64, a potent inhibitor of cysteine proteases, which precluded detection of cathepsin B β-secretase activity in the assay (Roberds et al., 2001). Thus, Roberds et al. does not rule out cathepsin B as a β-secretase.

Luo et al. found that knocking out BACE 1 in mice expressing human APP containing the Swedish mutant β-secretase site resulted in no Aβ or C99 production (CTFβ) (Luo et al., 2001). However, this study did not evaluate wild-type β-secretase site cleavage. Thus, Luo et al. does not rule out cathepsin B cleavage of APP containing the wild-type β-secretase site.

Cai et al. found that ‘conditioned media’ lacked Aβ from embryonic brain neurons of BACE 1 knockout animals expressing APP containing the wild-type β-secretase site (Cai et al., 2001). However, the ‘conditioned media’ reflected only constitutive secretion because the neurons were not stimulated with a triggering agent and, therefore, no regulated secretion occurred. Thus, Cai et al. does not exclude cathepsin B β-secretase activity in the regulated secretory pathway.

Taken together, the knockout BACE 1 experiments do not address cysteine protease β-secretase cleavage of APP containing the wild-type β-secretase site in the regulated secretory pathway. Therefore, cathepsin B β-secretase activity can be reconciled with BACE 1 knockout data because that data does not rule out cathepsin B as a possible β-secretase.

Interestingly, a recent study examined the role of BACE 1 on endogenous in vivolevels of Aβ in brain to address mechanisms for Aβ production in sporadic AD, where there is no overexpression of APP (Hirata-Fukae et al., 2008). In adult mice, overexpression of BACE 1, by approximately 12-fold greater than that expressed in wild-type mice, did not result in elevated endogenous Aβ. Thus, factors other than BACE 1 must be involved in Aβ production in adult and aging brain, and such factors may provide therapeutic targets. These findings are consistent with the proposal for cathepsin B as a candidate β-secretase for production of Aβ in adult conditions (Hook et al., 2007a, b; Hook et al., 2008).

Inhibitors of cathepsin B as potential therapeutic agents for Alzheimer’s disease

Key findings from studies of cathepsin B in regulated secretory vesicles for the cleavage of the wild-type β-secretase site of APP indicate cathepsin B as a newly identified drug target for AD. The notable efficacy of the CA074Me and E64d inhibitors to improve memory and reduce Aβ in an AD animal model expressing the wild-type β-secretase APP site present in the majority of AD patients provides support for these inhibitors as potential AD therapeutic agents.

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β-secretase and Glutaminyl Cyclase Generation of Post-translationally modified IsoAspartate and Pyroglutamate Forms of Aβ (presented by H.-U. Demuth)

Analyses of the peptide forms of Aβ in amyloid plaques of AD brains have identified post-translationally modified forms of Aβ. These modified Aβ peptides consist of Aβ with isoaspartate instead of aspartate as the N-terminal residue (IsoAsp-Aβ(1-40/42), and the truncated peptide with pyroglumate (pGlu) of the pGluAβ(3/11-40/42) peptides. The Demuth group elucidated the cathepsin B β-secretase and glutaminyl pathway utilized to generate modified Aβ peptides.

Formation of IsoAsp-Aβ(1-40/42) involves cathepsin B but not BACE 1

Aβ peptides containing the modified aspartate residue, isoaspartate (isoAsp), at the N-terminal amino acid of Aβ(1-40/42) are highly abundant in AD amyloid depositions. They can make up over 50% of all Aβ found, depending on the particular tissue (Shimizu et al., 2005). The formation of isoAsp within a peptide chain is a chemically spontaneously occurring process during aging of proteins (for a recent review seeShimizu et al., 2005). Aspartic acid (Asp) or asparagine (Asn) residues within a peptide chain can undergo intramolecular cyclization with release of water or ammonia, respectively (Figure 5). The generated metastable succinimide spontaneously decomposes to yield L-isoAsp, L-Asp and D-isoAsp and D-Asp in an approximate ratio of 70:20:5:5 (Vigneswara et al., 2006). The tendency to form the intramolecular succinimide depends markedly on the amino acids located at the C-terminal side of the Asp/Asn residue. It was found that the velocity of isoAsp formation is much faster if small amino acids (e. g. glycine, histidine, serine or alanine) are located at this position (Galletti et al., 1995). The formation of isoAsp introduces an additional methylene group in the peptide backbone, which could alter the structure and function of proteins. Moreover, this change in the peptide backbone could affect the susceptibility of the proteins for cleavage by proteases as demonstrated for prolyl endopeptidase or the proteasome (Tarcsa et al., 2000).

Figure 5 Scheme of Peptidyl-isoAspartyl-Peptide formation

To address the question of what β-secretease may cleave the isoAsp β-secretase site of APP, internally quenched fluorescent peptide substrates containing isoAsp at the wild-type and Swedish mutant β-secretase site (RE(Edans)EVKM(or NL) D(orisoD) AEFK(Dabcyl)R-NH₂) were assessed for cleavage by BACE 1 and cathepsin B. Interestingly, cathepsin B showed excellent catalytic efficiency to cleave in vitrothe isoAsp wild-type β-secretase peptide with a k_cat/K_m value of 1.83 × 10⁴ M^-1sec^-1, but, in contrast, BACE 1 did not cleave that substrate at all (Table 3). Moreover, the cathepsin B inhibitor, Ac-LVK-CHO, inhibited cleavage of the wild-type β-secretase substrate by cell extracts, whereas the BACE 1 inhibitor KTEEISEVN(Statine)VAEF had no effect. These data suggest the possibility that cathepsin B, rather than BACE 1, produces isoAsp-Aβ in AD patients by cleaving the wild-type β-secretase site containing isoAsp (Böhme et al., 2008).

Table 3 Kinetic parameters of BACE 1 and cathepsin B hydrolysis of peptides containing the wild-type (WT) β-secretase cleavage site, with and without the chemical isoAsp modification (WTiso)

Further evaluation of β-secretase activities utilized longer, more structured peptides, consisting of unlabelled 27 amino acid long peptides Ac-EEISEVKM(NL)D(isoD)AEFRHDSGYEVHHQKLVF-NH₂ covering the β cleavage site from amino acid −8 to 19 (Aβ numbering, corresponding to APP 664-690) were synthesized. Hydrolysis of these peptides by BACE-1, cathepsin B and SH-SY5Y cell extracts was analyzed by MALDI-TOF mass spectrometry (Demuth et. al., 2007;Böhme et al., 2008). The incubation of the peptides with BACE-1 for up to 24 hours supported the findings obtained with the 12 amino acids long internally quenched fluorescent peptide substrates. A cleavage pattern characteristic for BACE-1 (prior to Asp1) could only be observed in the case of the SW peptide (−L↓D-). This hydrolysis was abolished during coincubation of peptide, BACE-1 and the specific BACE-1 inhibitor. However, the isoAsp-containing 27 amino acid long peptides are not cleaved by BACE-1 but in contrast, cathepsin B hydrolyses the isoAsp containing substrates even more readily than the L-Asp containing variants. Incubating the peptides with neuroblastoma cell extract resulted in a cleavage pattern that was basically the same as with the purified enzymes, and substrate cleavage could be blocked by the cathepsin B inhibitor Ac-LVK-CHO (Böhme et al., 2008). Similar as with the 12 amino acid long peptides, cathepsin B cleavage occurred within the longer peptides preferentially after position P2 of the substrates which corresponds to penultimate amino acid of the Asp or isoAsp-linkage (i.e. in WT at -K↓MDA- or -K↓MisoDA- and in the Swedish sequences at -N↓LDA- or -N↓LisoDA-). This cleavage pattern clearly differentiates the cathepsin B mediated hydrolysis of APP-like substrates from the known BACE-1 mediated APP hydrolysis. The data clearly corroborate the results obtained by Hook et al., 2005 and by Schechter and Ziv, 2008. However, in contrast to BACE-1 and cathepsin D, the turnover of longer substrates by cathepsin B takes place not at the common APP 672 cleavage site. Moreover, posttranslational modifications such as the isoAsp formation, which is highly abundant in APP as well as in deposited Aβ in AD, even augment the unexpected cathepsin B cleavage pattern. In fact, this could be one reason for the different metabolic fate of APP leading to further N-terminal truncated and modified, neurotoxic Aβ peptide species such as pyroGluAβ (Russo et al., 2002; Cynis et al., 2006; Cynis et al., 2007).

Truncated pyroGluAβ(3-40/42) peptide forms in senile plaques of AD brains

Initial studies of Aβ peptide forms in AD brains identified Aβ(1-40) and the C-terminally extended Aβ(1-42) peptide forms of β-amyloid in amyloid plaques (Saido et al., 1996; Saido et al., 1995). In addition to differences in C-termini of Aβ forms, N-terminal peptide heterogeneity represents a significant portion of Aβ in AD brains. Interestingly, a greater number of plaques positive with antibodies directed to pGluAβ(3-40/42) was observed in AD compared to normal age-matched brains. These data suggest that the deposition of pGluAβ(3-40/42) may precede that of Aβ(1-40/42).

Notable results were obtained from analyses of the seeding of Aβ peptides to form oligomers, showing that the pGlu-modified Aβ peptides accelerated initial acceleration of aggregate formation by up to 250-fold compared to unmodified Aβ (Schilling et al., 2006). Furthermore, results suggested that pGlu-modified Aβ peptides promote seeding of mixed Aβ peptide aggregates. These biophysical characteristics of pGlu-amyloid peptides may be crucial for the initial development of AD.

Glutaminyl cyclase (QC) mediates N-terminal pyroglutamate cyclization to generate pGluAβ(3-40/42)

N-terminal pyroglutamate (pGlu) modification of Aβ(3-40/42) by glutaminyl cyclase (QC) is predicted to generate pGluAβ(3-40/42) (Figure 6). Inhibition of glutaminyl cyclase reduces formation of pGluAβ(3-42), thus, revealing the importance of QC activity for maturation of cellular pGlu-containing Aβ peptides (Schilling et al., 2004;Cynis et al., 2006).

Figure 6 Pyroglutamate modification of Aβ peptides by glutaminyl cyclase

Recent in vivo studies showed that administration of a QC inhibitor prevented the formation of pGlu-containing Aβ peptides in brains of rats, as well as in a transgenic mouse model of AD. Importantly, the QC inhibitor improved memory during reduction of pGluAβ peptides. These findings implicate the importance of QC in catalyzing the production of neurotoxic pGlu-modified Aβ peptides (Schilling et al., 2007; Schilling et al., 2008).

Evidence that formation of pGlu-Aβ peptides prefers utilization of the β-secretase pathway derived from wild-type APP, rather than the Swedish mutant APP

Because formation of N-terminally truncated pGluAβ(3-42) may be a key event in the development of AD, it is important to understand cellular mechanisms that generate these forms of Aβ from APP. Recent experiments have compared the formation of pGluAβ(3-40/42) and Aβ(1-40/42) from wild-type and Swedish mutant APP forms by expression in cells (HEK293 cell line). Results showed that a larger amount of pGluAβ(3-40/42) was produced from wild-type human APP and human London APP, both of which express the wild-type β-secretase site. However, a larger amount of Aβ(1-40/42) was generated from Swedish mutant APP or Swedish/London APP, both of which possess the Swedish mutant β-secretase site. Furthermore, production of pGluAβ(3-40/42) from wild-type APP was not reduced by an inhibitor of BACE 1. These findings suggest that a β-secretase other than BACE 1 participates in the formation of pGluAβ(3-40/42) from wild-type APP. Based on the studies of this article describing a role for cathepsin B in Aβ production utilizing the wild-type β-secretase site of APP, it will be of interest in future studies to examine the possibility that cathepsin B may be involved in the production of pGlu forms of Aβ (Cynis et. al., 2007; Cynis et al., 2008).

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Summary

This paper summarizes the work presented by three independent groups at the 5^thGeneral Meeting of the IPS on potential AD drug targets. The combined data support the existence of β-secretases other than BACE 1 and provide evidence for novel enzymatic pathways mediated by cathepsin B as β-secretase and glutaminyl cyclase production of pGlu-modified forms of Aβ. BACE 1 and the more abundant cathepsin D cleave the wild-type β-secretase site with low efficacy. Notably, cathepsin B specifically and efficiently cleaves the wild-type β-secretase site present in the majority of AD patients. Subsequent to such a cathepsin B β-secretase step, glutaminyl cyclase modification of truncated Aβ (3-40/42) can generate modified pGluAβ(3-40/42). These findings provide evidence for cathepsin B and glutaminyl cyclase as potential new drug targets to inhibit production of multiple Aβ peptide forms in Alzheimer’s disease.

Acknowledgments

This work was supported by a grant from the National Institutes of Health, National Institute of Aging, USA to American Life Science Pharmaceuticals (ALSP, Inc.). Dr. G. Hook is employed by and holds equity in ALSP, Inc. Dr. V. Hook holds equity in ALSP and serves on the advisory board of ALSP, Inc. The terms of this arrangement have been approved by the University of California, San Diego, in accordance with its conflict of interest policies. Part of the work presented by Dr. Demuth was supported by the German Department of Science and Technology (BMBF grant #3013185 to HUD). Prof. H.-U. Demuth serves as CSO of Probiodrug AG and holds stock of the company.

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References

• Beck M, Bigl V, Rossner S. Guinea pigs as a nontransgenic model for APP processing in vitro and in vivo. Neurochem Res. 2003;28:637–644. [PubMed]
• Beck M, Muller D, Bigl V. Amyloid precursor protein in guinea pigs-complete cDNA sequence and alternative splicing. Biochim Biophys Acta. 1997;1351:17–21. [PubMed]
• Benjannet S, Savaria D, Laslop A, Munzer JS, Chretien M, Marcinkiewicz M, Seidah NG. Alpha1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway. J Biol Chem.1997;272:26210–26218. [PubMed]
• Berg JM, Tymoczko JL, Stryer L. Biochemistry. W.H Freeman and Company; New York: 2002. pp. 203–204.
• Blennow K, de Leon MJ, Zetterberg H. Alzheimer’s disease. Lancet. 2006;368:387–403.[PubMed]
• Böhme L, Hoffmann T, Manhart S, Wolf R, Demuth H-U. Isoaspartate containing amyloid precursor protein derived peptides alter efficacy and specificity of potential β-secretases. Biol Chem. 2008 revision submitted.
• Cai H, Wang Y, McCarthy D, Wen H, Borchelt DR, Price DL, Wong PC. BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci. 2001;4:233–234.[PubMed]
• Cataldo AM, Barnett JL, Berman SA, Li J, Quarless S, Bursztajn S, Lippa C, Nixon RA. Gene expression and cellular content of cathepsin D in Alzheimer’s disease brain: evidence for early up-regulation of the endosomal-lysosomal system. Neuron. 1995;14:671–680.[PubMed]
• Cirrito JR, Yamada KA, Finn MB, Sloviter RS, Bales KR, May PC, Schoepp DD, Paul SM, Mennerick S, Holtzman DM. Synaptic Activity Regulates Interstitial Fluid Amyloid-ß Levels In Vivo. Neuron. 2005;48:913–922. [PubMed]
• Citron M, Oltersdorf T, Haass C, McConlogue L, Hung AY, Seubert P, Vigo-Pelfrey C, Lieberburg I, Selkoe DJ. Mutation of the beta-amyloid precursor protein in familial Alzheimer’s disease increases beta-protein production. Nature. 1992;360:672–674. [PubMed]
• Clarke S. Aging as war between chemical and biochemical processes: protein methylation and the recognition of age-damaged proteins for repair. Ageing Res Rev. 2003;2:263–285.[PubMed]
• Cynis H, Schilling S, Bodnar M, Hoffmann T, Heiser U, Saido TC, Demuth H-U. Inhibition of Glutaminyl Cyclase Alters Pyroglutamate Formation in Mammalian Cells. Biochim Biophys Acta - Proteins and Proteomics. 2006;1764:1618–1625.
• Cynis H, Schilling S, Demuth H-U. Generation of N-Terminally Truncated AŸ Species in HEK293 Cells. Neurodegen Dis. 2007;4(Suppl 1):217.
• Cynis H, Scheel E, Saido TC, Schilling S, Demuth H-U. Amyloidogenic Processing of APP: Evidence for a Pivotal Role of Glutaminyl Cyclase for Generation of Pyroglutamate-Modified Amyloid-β Biochemistry. 2008 in press.
• Demuth H-U, Böhme L, Cynis H, Schilling S, Hoffmann T. Pathways Leading to the Neurotoxic Pyroglutamated A -Peptides Differ in Wildtype APP- and Mutant APP-Expressing Systems.Neurodegen Dis. 2007;4(Suppl 1):193.
• Dewachter I, van Dorpe J, Spittaels K, Tesseur I, Van Den Haute C, Moechars D, Van Leuven F. Modeling Alzheimer’s disease in transgenic mice: effect of age and of presenilin1 on amyloid biochemistry and pathology in APP/London mice. Exp Gerontol. 2000;35:831–841.[PubMed]
• Docherty K, Steiner DF. Post-translational proteolysis in polypeptide hormone biosynthesis.Annu Rev Physiol. 1982;44:625–638. [PubMed]
• Dodart JC, Mathis C, Bales KR, Paul SM. Does my mouse have Alzheimer’s disease? Genes Brain Behav. 2002;1:142–155. [PubMed]
• Dunn BM, Hung S. The two sides of enzyme-substrate specificity: lessons from the aspartic proteinases. Biochim Biophys Acta. 2000;1477:231–240. [PubMed]
• Efthimiopoulos S, Vassilacopoulou D, Ripellino JA, Tezapsidis N, Robakis NK. Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells. Proc Natl Acad Sci U S A. 1996;93:8046–8050. [PMC free article][PubMed]
• Farber SA, Nitsch RM, Schulz JG, Wurtman RJ. Regulated secretion of beta-amyloid precursor protein in rat brain. J Neurosci. 1995;15:7442–7451. [PubMed]
• Findeis MA. The role of amyloid ß peptide 42 in Alzheimer’s disease Pharmacol. Therapeut.2007;116:266–286.
• Fugere M, Day R. Cutting back on pro-protein convertases: the latest approaches to pharmacological inhibition. Trends Pharmacol Sci. 2005;26:294–301. [PubMed]
• Gainer H, Russell JT, Loh YP. The enzymology and intracellular organization of peptide precursor processing: the secretory vesicle hypothesis. Neuroendocrinology. 1985;40:171–184. [PubMed]
• Galletti P, Ingrosso D, Manna C, Clemente G, Zappia V. Protein damage and methylation-mediated repair in the erythrocyte. Biochem J. 1995;306(Pt 2):313–325. [PMC free article][PubMed]
• Gandy S, Martins RN, Buxbaum J. Molecular and cellular basis for anti-amyloid therapy in Alzheimer disease. Alzheimer Dis Assoc Disord. 2003;17:259–266. [PubMed]
• Gensberg K, Jan S, Matthews GM. Subtilisin-related serine proteases in the mammalian constitutive secretory pathway. Semin Cell Dev Biol. 1998;9:11–17. [PubMed]
• Gumbiner B, Kelly RB. Two distinct intracellular pathways transport secretory and membrane glycoproteins to the surface of pituitary tumor cells. Cell. 1982;28:51–59. [PubMed]
• Halfon S, Baird TT, Craik CS. Trypsin. In: Barrett AJ, Rawings ND, Woesnner JF, editors.Handbook of Proteolytic Enzymes. Elsevier Academic Press; San Diego: 2004. pp. 1483–1488.
• Haass C, Selkoe DF. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid beta-peptide. Nature Rev Molec Cell Biol. 2007;8:101–112. [PubMed]
• He W, Barrow CJ. The A(3-Pyroglutamyl and 11-Pyroglutamyl Peptides Found in Senile Plaque Have Greater (β-Sheet Forming and Aggregation Propensities in vitro than Full-Length Aβ Biochemistry. 1999;38:10871–10877. [PubMed]
• Hirata-Fukae C, Sidahmed EHY, Gooskens TP, Aisen PS, Dewachter I, Devijver H, Van Leeuven FV, Matsuoka Y. Beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1)-mediated changes of endogenous amyloid beta in wild-type and transgenic mice in vivo.Neursosci Letters. 2008;435:186–189.
• Hook VYH, Toneff T, Aaron W, Yasothornsrikul S, Bundey R, Reisine T. ß-Amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for β-secretase activity in chromaffin vesicles. J Neurochem.2002;81:237–256. [PubMed]
• Hook V, Yasothornsrikul S, Greenbaum D, Medzihradszky KF, Troutner K, Toneff T, Bundey R, Logrinova A, Reinheckel T, Peters C, Bogyo M. Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones. Biol Chem. 2004;385:473–480. [PubMed]
• Hook V, Toneff T, Bogyo M, Medzihradszky KF, Nevenu J, Lane W, Hook G, Reisine T. Inhibition of cathepsin B reduces ß-amyloid production in regulated secretory vesicles of neuronal chromaffin cells: evidence for cathepsin B as a candidate ß-secretase of Alzheimer’s disease. Biological Chemistry. 2005;386:931–940. [PubMed]
• Hook G, Hook VY, Kindy M. Cysteine protease inhibitors reduce brain beta-amyloid and beta-secretase activity in vivo and are potential Alzheimer’s disease therapeutics. Biol Chem.2007a;388:979–983. [PubMed]
• Hook V, Kindy M, Hook G. Cysteine protease inhibitors effectively reduce in vivo levels of brain beta-amyloid related to Alzheimer’s disease. Biol Chem. 2007b;388:247–252. [PubMed]
• Hook VY, Kindy M, Hook G. Inhibitors of cathepsin B improve memory and reduce Abeta in transgenic Alzheimer’s Disease mice expressing the wild-type, but not the Swedish mutant, beta β-secretase APP site. J Biol Chem. 2008 in press.
• Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice.Science. 1996;274:99–102. [PubMed]
• Iversen LL, Mortishire-Smith RJ, Pollack SJ, Shearman MS. The toxicity in vitro of beta-amyloid protein. Biochem J. 1995;311(Pt 1):1–16. [PMC free article] [PubMed]
• Jacks T. Tumor suppressor gene mutations in mice. Annu Rev Genet. 1996;30:603–636.[PubMed]
• Johnstone EM, Chaney MO, Norris FH, Pascual R, Little SP. Conservation of the sequence of the Alzheimer’s disease amyloid peptide in dog, polar bear and five other mammals by cross-species polymerase chain reaction analysis. Brain Res Mol Brain Res. 1991;10:299–305.[PubMed]
• Jolly-Tornetta C, Gao ZY, Lee VM, Wolf BA. Regulation of amyloid precursor protein secretion by glutamate receptors in human Ntera 2 neurons. J Biol Chem. 1998;273:14015–14021.[PubMed]
• Kim J, Onstead L, Randle S, Price R, Smithson L, Zwizinski C, Dickson DW, Golde T, McGowan E. Abeta40 inhibits amyloid deposition in vivo. J Neurosci. 2007;17:627–633.[PubMed]
• Kuo YM, Emmerling MR, Woods AS, Cotter RJ, Roher AE. Isolation, chemical characterization, and quantitation of A beta 3-pyroglutamyl peptide from neuritic plaques and vascular amyloid deposits. Biochem Biophys Res Commun. 1997;237:188–191. [PubMed]
• Ladror US, Snyder SW, Wang GT, Holzman TF, Krafft GA. Cleavage at the amino and carboxyl termini of Alzheimer’s amyloid-beta by cathepsin D. J Biol Chem. 1994;269:18422–18428.[PubMed]
• Laird FM, Cai H, Savonenko AV, Farah MH, He K, Melnikova T, Wen H, Chiang HC, Xu G, Koliatsos VE, Borchelt DR, Price DL, Lee HK, Wong PC. BACE1, a major determinant of selective vulnerability of the brain to amyloid-beta amyloidogenesis, is essential for cognitive, emotional, and synaptic functions. J Neurosci. 2005;25:11693–11709. [PMC free article][PubMed]
• Lin X, Koelsch G, Wu S, Downs D, Dashti A, Tang J. Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A.2000;97:1456–1460. [PMC free article] [PubMed]
• Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Danell J. In: Molecular Cell Biology.Freeman WH, editor. New York: 1999. pp. 691–726.
• Luo Y, Bolon B, Kahn S, Bennett BD, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis JC, Yan Q, Richards WG, Citron M, Vassar R. Mice deficient in BACE1, the Alzheimer’s beta-secretase, have normal phenotype and abolished beta-amyloid generation.Nat Neurosci. 2001;4:231–232. [PubMed]
• Luo Y, Bolon B, Damore MA, Fitzpatrick D, Liu H, Zhang J, Yan Q, Vassar R, Citron M. BACE1 (beta-secretase) knockout mice do not acquire compensatory gene expression changes or develop neural lesions over time. Neurobiol Dis. 2003;14:81–88. [PubMed]
• Masliah E, Rockenstein E. Genetically altered transgenic models of Alzheimer’s disease. J Neural Transm Suppl. 2000;59:175–183. [PubMed]
• Maeda J, Ji B, Irie T, Tomiyama T, Maruyama M, Okauchi T, Staufenbiel M, Iwata N, Ono M, Saido TC, Suzuki K, Mori H, Higuchi M, Suhara T. Longitudinal, quantitative assessment of amyloid, neuroinflammation, and anti-amyloid treatment in a living mouse model of Alzheimer’s disease enabled by positron emission tomography. J Neurosci. 2007;27:10957–10968. [PubMed]
• McConnell RM, York JL, Frizzell D, Ezell C. Inhibition studies of some serine and thiol proteinases by new leupeptin analogues. J Med Chem. 1993;36:1084–1089. [PubMed]
• McGowan E, Pickford F, Kim J, Onstead L, Eriksen J, Yu C, Skipper L, Murphy MP, Beard J, Das P, Jansen K, Delucia M, Lin WL, Dolios G, Wang R, Eckman CB, Dickson DW, Hutton M, Hardy J, Golde T. Abeta42 is essential for parenchymal and vascular amyloid deposition in mice. Neuron. 2005;47:191–199. [PMC free article] [PubMed]
• Moechars D, Dewachter I, Lorent K, Reverse D, Baekelandt V, Naidu A, Tesseur I, Spittaels K, Haute CV, Checler F, Godaux E, Cordell B, Van Leuven F. Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain. J Biol Chem. 1999;274:6483–6492. [PubMed]
• Mort JS. 333. Cathepsin B. In: Barrett AJ, Rawlings ND, Woessner JF, editors. Handbook of Proteolytic Enzymes. Vol. 2. Elsevier Academic Press; Amsterdam: 2004. pp. 1079–1086.
• Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, Chen WJ, Clay WC, Didsbury JR, Hassler D, Hoffman CR, Kost TA, Lambert MH, Leesnitzer MA, McCauley P, McGeehan G, Mitchell J, Moyer M, Pahel G, Rocque W, Overton LK, Schoenen F, Seaton T, Su JL, Becherer JD, et al. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature. 1997;385:733–736. [PubMed]
• Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci.1997;22:299–306. [PubMed]
• Nitsch RM, Slack BE, Wurtman RJ, Growdon JH. Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science.1992;258:304–307. [PubMed]
• Nitsch RM, Farber SA, Growdon JH, Wurtman RJ. Release of amyloid beta-protein precursor derivatives by electrical depolarization of rat hippocampal slices. Proc Natl Acad Sci U S A.1993;90:5191–5193. [PMC free article] [PubMed]
• Piccini A, Russo C, Gliozzi A, Relini A, Vitali A, Borghi R, Giliberto L, Armirotti A, D’Arrigo C, Bachi A, Cattaneo A, Canale C, Torrassa S, Saido TC, Markesbery W, Gambetti P, Tabaton M. {beta}-Amyloid Is Different in Normal Aging and in Alzheimer Disease. J Biol Chem.2005;280:34186–34192. [PubMed]
• Price DL, Sisodia SS. Mutant genes in familial Alzheimer’s disease and transgenic models.Annu Rev Neurosci. 1998;21:479–505. [PubMed]
• Rawlings ND, Barrett AJ. Introduction: The clans and families of cysteine proteases. In: Barrett AJ, Rawlings ND, Woesnner JF, editors. Handbook of Proteolytic Enzymes. Elsevier Academic Press; San Diego: 2004. pp. 1051–1057.
• Roberds SL, Anderson J, Basi G, Bienkowski MJ, Branstetter DG, Chen KS, Freedman SB, Frigon NL, Games D, Hu K, Johnson-Wood K, Kappenman KE, Kawabe TT, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols NF, Power M, Robertson DW, Schenk D, Schoor M, Shopp GM, Shuck ME, Sinha S, Svensson KA, Tatsuno G, Tintrup H, Wijsman J, Wright S, McConlogue L. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer’s disease therapeutics. Hum Mol Genet. 2001;10:1317–1324.[PubMed]
• Roher AE, Lowenson JD, Clarke S, Wolkow C, Wang R, Cotter RJ, Reardon IM, Ziircher-Neely HA, Heinrikson RL, Ball MJ, Greenberg BD. Structural Alterations in the Peptide Backbone of β-Amyloid Core Protein May Account for Its Deposition and Stability in Alzheimer’s Disease. J Biol Chem. 1993;286:3072–3083. [PubMed]
• Russo C, Violani E, Salis S, Venezia V, Dolcini V, Damonte G, Benatti U, D’Arrigo C, Patrone E, Carlo P, Schettini G. Pyroglutamate-modified amyloid beta-peptides-AbetaN3(pE)-strongly affect cultured neuron and astrocyte survival. J Neurochem. 2002;82:1480–1489. [PubMed]
• Saftig P, Peters C, von Figura K, Craessaerts K, Van Leuven F, De Strooper B. Amyloidogenic processing of human amyloid precursor protein in hippocampal neurons devoid of cathepsin D. J Biol Chem. 1996;271:27241–27244. [PubMed]
• Saido TC, Iwatsubo T, Mann DM, Shimada H, Ihara Y, Kawashima S. Dominant and differential deposition of distinct beta-amyloid peptide species, A beta N3(pE), in senile plaques. Neuron. 1995;14:457–466. [PubMed]
• Saido TC, Yamao-Harigaya W, Iwatsubo T, Kawashima S. Amino- and carboxyl-terminal heterogeneity of beta-amyloid peptides deposited in human brain. Neurosci Lett.1996;215:173–176. [PubMed]
• Saido TC. Alzheimer’s disease as proteolytic disorders: Anabolism and catabolism of beta-amyloid. Neurobiol Aging. 1998;19:69–75.
• Schechter I. On the active sites of proteases. Cleavage of peptide bonds involving D-alanine residues by carboxypeptidase A. Eur J Biochem. 1970;14:516–520. [PubMed]
• Schechter I. Mapping of the active site of proteases in the 1960s and rational design of inhibitors/drugs in the 1990s. Curr Protein Pept Sci. 2005;6:501–512. [PubMed]
• Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967;27:157–162. [PubMed]
• Schechter I, Berger A. On the active site of proteases III. Mapping the active site of papain; Specific peptide inhibitors of papain. Biochem Biophys Res Comm. 1968;32:898–902.[PubMed]
• Schechter I, Ziv E. Kinetic properties of cathepsin D and BACE 1 indicate the need to search for additional beta-secretase candidate(s) Biol Chem. 2008;389:313–320. [PubMed]
• Schilling S, Hoffmann T, Manhart S, Hoffmann M, Demuth H-U. Glutaminyl cyclases unfold glutamyl cyclase activity under mild acid conditions. FEBS Lett. 2004;563:191–196. [PubMed]
• Schilling S, Lauber T, Schaupp M, Manhart S, Scheel E, Bohm G, Demuth HU. On the seeding and oligomerization of pGlu-amyloid peptides (in vitro) Biochemistry. 2006;45:12393–12399.[PubMed]
• Schilling S, Cynis H, Demuth H-U. Pyroglutamated Aβ Peptides - N-Terminally Modified Aβ Variants Generated by Glutam(in)yl Cyclase. Neurodegen Dis. 2007;4(Suppl1):192–193.
• Schilling S, Wasternack C, Demuth H-U. Glutaminyl Cyclases from Animals and Plants: A Case of Functionally Convergent Protein Evolution. Biol Chem. 2008 in press.
• Seidah NG, Chretien M, Day R. The family of subtilisin/kexin like pro-protein and prohormone convertases: divergent or shared functions. Biochimie. 1994;76:197–209. [PubMed]
• Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev. 2001;81:741–766. [PubMed]
• Selkoe DF. Cell biology of protein misfolding: the examples of Alzheimer’s and Parkinson’s diseases. Nature Cell Biol. 2004;6:1054–1061. [PubMed]
• Selkoe DF, Wolfe M. Presenilin: running with scissors in the membrane. Cell. 2007;131:215–221. [PubMed]
• Shi XP, Chen E, Yin KC, Na S, Garsky VM, Lai MT, Li YM, Platchek M, Register RB, Sardana MK, Tang MJ, Thiebeau J, Wood T, Shafer JA, Gardell SJ. The pro domain of beta-secretase does not confer strict zymogen-like properties but does assist proper folding of the protease domain. J Biol Chem. 2001;276:10366–10373. [PubMed]
• Shimizu T, Watanabe A, Ogawara M, Mori H, Shirasawa T. Isoaspartate Formation and Neurodegeneration in Alzheimer’s Disease. Arch Biochem Biophys. 2000;381:225–234.[PubMed]
• Shimizu T, Matsuoka Y, Shirasawa T. Biological significance of isoaspartate and its repair system. Biol Pharm Bull. 2005;28:1590–1596. [PubMed]
• Sisodia SS. Alzheimer’s disease: perspectives for the new millennium. J Clin Invest.1999;104:1169–1170. [PMC free article] [PubMed]
• Tamai M, Matsumoto K, Omura S, Koyama I, Ozawa Y, Hanada K. In vitro and in vivo inhibition of cysteine proteinases by EST, a new analog of E-64. J Pharmacobiodyn. 1986;9:672–677.[PubMed]
• Tarcsa E, Szymanska G, Lecker S, O’Connor CM, Goldberg AL. Ca2+-free calmodulin and calmodulin damaged by in vitro aging are selectively degraded by 26 S proteasomes without ubiquitination. J Biol Chem. 2000;275:20295–20301. [PubMed]
• Tezapsidis N, Li H-G, Ripellino JA, Efthimiopoulos S, Vassilacopoulou D, Sambamurti K, Toneff T, Yasothornsrikul S, Hook VYH, Robakis NK. Release of nontransmembrane full-length Alzheimer’s amyloid precursor protein from the lumenar surface of chromaffin granule membranes. Biochemistry. 1998;37:1274–1282. [PubMed]
• Thomas G. Furin at the cutting edge: from protein traffic to embryogenesis and disease. Nat Rev Mol Cell Biol. 2002;3:753–766. [PMC free article] [PubMed]
• Towatari T, Nikawa T, Murata M, Yokoo C, Tamai M, Hanada K, Katunuma N. Novel epoxysuccinyl peptides. A selective inhibitor of cathepsin B, in vivo. FEBS Lett. 1991;280:311–315. [PubMed]
• Turner RS. Alzheimer’s disease. Semin Neurol. 2006;26:499–506. [PubMed]
• Turner RT, 3rd, Koelsch G, Hong L, Castanheira P, Ermolieff J, Ghosh AK, Tang J. Subsite specificity of memapsin 2 (beta-secretase): implications for inhibitor design. Biochemistry.2001;40:10001–10006. [PubMed]
• Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz EA, Denis P, Teplow DB, Ross S, Amarante P, Loeloff R, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski MA, Biere AL, Curran E, Burgess T, Louis JC, Collins F, Treanor J, Rogers G, Citron M. Beta-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science. 1999;286:735–741. [PubMed]
• Vassar R. BACE1: the beta-secretase enzyme in Alzheimer’s disease. J Mol Neurosci.2004;23:105–114. [PubMed]
• Vigneswara V, Lowenson JD, Powell CD, Thakur M, Bailey K, Clarke S, Ray DE, Carter WG. Proteomic identification of novel substrates of a protein isoaspartyl methyltransferase repair enzyme. J Biol Chem. 2006;281:32619–32629. [PubMed]
• Voet D, Voet JG. Biochemistry. John Wiley & Sons; New York: 2004. pp. 480–481.
• Wolfe MS. Shutting down Alzheimer’s. Sci Am. 2006;294:72–79. [PubMed]
• Zhou A, Webb G, Zhu X, Steiner DF. Proteolytic processing in the secretory pathway. J Biol Chem. 1999;274:20745–20748. [PubMed]