Metabolism of amyloid-β peptide and Alzheimer's disease

Pharmacology & Therapeutics 108 (2005) 129 – 148 www.elsevier.com/locate/pharmthera

Associate editor: I. Kimura

Metabolism of amyloid-h peptide and Alzheimer’s disease Nobuhisa Iwata *, Makoto Higuchi, Takaomi C. Saido Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Wako-shi, Saitama 351-0198, Japan

Abstract The accumulation of amyloid-h peptide (Ah), a physiological peptide, in the brain is a triggering event leading to the pathological cascade of Alzheimer’s disease (AD) and appears to be caused by an increase in the anabolic activity, as seen in familial AD cases or by a decrease in catabolic activity. Neprilysin is a rate-limiting peptidase involved in the physiological degradation of Ah in the brain. As demonstrated by reverse genetics studies, disruption of the neprilysin gene causes elevation of endogenous Ah levels in mouse brain in a gene-dose-dependent manner. Thus, the reduction of neprilysin activity will contribute to Ah accumulation and consequently to AD development. Evidence that neprilysin in the hippocampus and cerebral cortex is down-regulated with aging and from an early stage of AD development supports a close association of neprilysin with the etiology and pathogenesis of AD. Therefore, the up-regulation of neprilysin represents a promising strategy for therapy and prevention. Recently, somatostatin, which acts via a G-protein-coupled receptor (GPCR), has been identified as a modulator that increases brain neprilysin activity, resulting in a decrease of Ah levels. Thus, it may be possible to pharmacologically control brain Ah levels with somatostatin receptor agonists. D 2005 Elsevier Inc. All rights reserved. Keywords: Alzheimer’s disease; Amyloid-h peptide; Aging; Neprilysin; Somatostatin Abbreviations: Ah, amyloid-h peptide; ACE, angiotensin-converting enzyme; AD, Alzheimer’s disease; AICD, APP intracellular domain; apoE, apolipoprotein E; APP, amyloid precursor protein; APP tg, familial AD-linked mutant APP tg; BSB, (E,E)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4hydroxy)styrylbenzene; CALLA, common acute lymphoblastic leukemia antigen; CD10, leukocyte cell surface antigen 10; DNP, dinucleotide repeat polymorphisms; ECE, endothelin-converting enzyme; ELISA, enzyme-linked immunosorbent assay; FSB, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4hydroxy); GABA, g-aminobutyric acid; GPCR, G-protein-coupled receptor; HNE, adduct of 4-hydroxynonenal; HPLC, high-pressure liquid chromatography; IDE, insulin-degrading enzyme; MCI, mild cognitive impairment; PS, presenilin; SNP, single nucleotide polymorphism; SSTP, somatostatin precursor protein; tg, transgenic.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Catabolic system of Ah in the brain . . . . . . . . . . . . . . . . 2.1. Candidate Ah-degrading enzymes . . . . . . . . . . . . . 2.2. In vivo analysis of the brain Ah catabolic system . . . . . 2.3. Studies of Ah-degrading enzyme candidates using reverse genetic techniques. . . . . . . . . . . . . . . . . . . . . . 2.4. Enzymatic properties of neprilysin . . . . . . . . . . . . . Pathological relevance of neprilysin to AD . . . . . . . . . . . . 3.1. Distribution of neprilysin in brain. . . . . . . . . . . . . . 3.2. Subcellular localization of neprilysin in neurons . . . . . . 3.3. Age-related decline in neprilysin . . . . . . . . . . . . . . 3.4. Expression levels of neprilysin in AD brains . . . . . . . .

* Corresponding author. Tel.: +81 48 462 1111x7614; fax: +81 48 467 9716. E-mail address: [email protected] (N. Iwata). 0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.03.010

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3.5. Polymorphism of human neprilysin gene . . . 3.6. Relationship with hereditary cerebral amyloid 4. Regulatory mechanisms of neprilysin . . . . . . . . 4.1. Tissue-specific regulation . . . . . . . . . . . 4.2. Region- and neuron type-specific regulation . 4.3. Ah fibril-mediated regulation . . . . . . . . . 4.4. Up-regulation by anti-oxidants . . . . . . . . 4.5. Regulation by neuropeptides . . . . . . . . . 5. Medical applications . . . . . . . . . . . . . . . . . 5.1. Gene therapeutic approach . . . . . . . . . . 5.2. Pharmacological approach . . . . . . . . . . 6. Conclusion and prospects . . . . . . . . . . . . . . Acknowledgments. . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder that robs patients of their memory and cognitive abilities, and even their personalities, and is the most common form of senile dementia. These changes are due to the progressive dysfunction and death of neurons that are responsible for learning and memory processes. AD is characterized by a variety of pathological features, such as extracellular senile plaques, intracellular neurofibrillary tangles, synaptic loss, and brain atrophy (Selkoe, 2001a; Hardy & Selkoe, 2002; Forman et al., 2004). The senile plaques are mainly composed of amyloid-h peptide (Ah) of 40 –43 amino acids (called Ah1 – 40, Ah1 – 42, or Ah1 – 43 according to the number of amino acid residues), and the neurofibrillary tangles consist of twisted filaments of hyperphosphorylated tau. In AD development, a decadeslong pathological cascade of Ah deposition, accumulation of hyperphosphorylated tau, dysfunction of neurons, and neuronal death leads to overt dementia (Selkoe, 2001a; Hardy & Selkoe, 2002; Forman et al., 2004). Ah deposition occurs as oligomeric, protofibrillar, amylospheroid, and fibrillar forms (Kuo et al., 1996; Lambert et al., 1998; Hartley et al., 1999; Walsh et al., 1999, 2002; Hoshi et al., 2003; Fig. 1). The cause-and-effect relationship between Ah deposition and AD development has been strongly supported by the consistent increased Ah (particularly Ah42) production phenotypes of early-onset familial AD-causing gene mutations, such as amyloid precursor protein (APP), presenilin (PS) 1, and PS2, observed in both in vitro and in vivo experiments (Scheuner et al., 1996; Selkoe, 2001a; Hardy & Selkoe, 2002). Ah42 is more hydrophobic and shows a higher potential for aggregation than Ah40 does, and it functions as the primary pathogenic agent. Importantly, Ah is a physiological peptide, which is constantly anabolized and catabolized in the brain (Seubert et al., 1993), so that the steady-state Ah levels are determined by the metabolic balance between anabolic and catabolic activities (Figs. 1 and 2; Selkoe, 2001b, Saido, 2003). Even

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subtle alterations in this metabolic balance over a long period of time could result in the appearance of pathogenic forms of Ah, influencing both the pathological progression and the incidence of the disease. For instance, just a 1.5-fold increase in Ah42, caused by most of the above mutations, results in aggressive presenile Ah pathology (Fig. 1b; Sherrington et al., 1995; Borchelt et al., 1996; Duff et al., 1996; Nakano et al., 1999). Therefore, in order to overcome AD, it is necessary to lower the Ah levels in the brain, and several therapeutic strategies (referred to as anti-Ah strategies), such as inhibition of production, promotion of degradation, inhibition of aggregation, and clearance of deposits, have been proposed (Hardy & Selkoe, 2002; Forman et al., 2004). In sporadic AD brains, where the elevation of anabolic activity seems to be rarely observed, a reduction in the catabolic activity towards Ah involving so-

Fig. 1. Metabolism of Ah. Ah is a physiological peptide that is constantly anabolized and catabolized in vivo (a). Either the up-regulation of anabolism (b) or the down-regulation of catabolism (c) accelerates aggregation/deposition through increasing the steady-state levels of Ah.

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Fig. 2. Schematic pathway of Ah metabolism and formation of Ah fibrils in the brain. Ah is produced from APP by sequential cleavage involving h-secretase (h-site APP cleaving enzyme 1) and g-secretase (presenilin complex), and released into the extracellular spaces and, in part, to the intravesicular side (Hardy & Selkoe, 2002; De Strooper, 2003; Koo & Kopan, 2004). The released Ah undergoes proteolytic degradation by the action of neprilysin, IDE, ECE, etc., but Ah that escapes degradation aggregates and forms fibrils via transition states, such as oligomeric forms, protofibrils, or amylospheroids. Oligomeric Ah, as well as monomeric Ah, degraded by neprilysin (Kanemitsu et al., 2003). There is a dynamic equilibrium between monomeric and oligomeric Ah and further between oligomeric and protofibrillar Ah (Walsh et al., 1999).

called Ah-degrading enzyme(s) has been a candidate cause for Ah accumulation associated with late-onset AD development (Fig. 2c). Either the up-regulation of the catabolic activity or down-regulation of the anabolic activity should prevent or reduce Ah deposition and thus be applicable to the prevention and therapy of AD. This review describes the current status of studies on Ah catabolism, focusing on the Ah-degrading enzyme neprilysin, and on therapeutic strategies targeting Ah catabolism.

2. Catabolic system of AB in the brain

system in the brain in vivo. Almost any peptidase would be capable of proteolyzing Ah to some extent under optimal conditions in vitro. In tissue culture, cells usually exist in a monolayer in the presence of a large excess (more than 1000 times the cell volume) of medium. In contrast, cells in brain tissue are in close proximity in a specific extracellular matrix, with relatively small volumes of extracellular fluid. Furthermore, proteolytic systems are often altered when cells are transferred from an organ to culture. Therefore, it is problematic to use immortalized cell lines derived from neurons or nonneuronal cells for identifying the key protease(s). The critical difference between in vivo and in vitro environments has greatly hindered identification of

2.1. Candidate Ab-degrading enzymes Many peptidases have been proposed as Ah-degrading enzymes: gelatinase A (Yamada et al., 1995), gelatinase B (Backstrom et al., 1996), neprilysin (Howell et al., 1995), insulin-degrading enzyme (IDE; Kurochkin & Goto, 1994; McDermott & Gibson, 1997; Qiu et al., 1998; Chesneau et al., 2000; Vekrellis et al., 2000), cathepsin D (Hamazaki, 1996), metalloendopeptidase (Carvalho et al., 1997), coagulation factor XIa (Saporito-Irwin & Van Nostrand, 1995), plasmin (Van Nostrand & Porter, 1999), endothelin-converting enzyme (ECE)-1 (Eckman et al., 2001), and angiotensin-converting enzyme (ACE; Hu et al., 2001; Fig. 3). There are also other possible candidates, if all the potential cleavage sites in Ah sequences are taken into consideration. However, it was not clear which candidate peptidase plays the predominant role in the Ah catabolic

Fig. 3. Possible cleavage sites in the Ah sequence by candidate Ahdegrading enzymes. Most of the cleavage sites were determined using Ah1 – 42 or Ah1 – 40 as a substrate (a – m) and the others were assessed based on the enzymatic specificities of peptidases (n – r). (a) Gelatinase A (MMP2); (b) gelatinase B (MMP-9); (c) neprilysin; (d) insulin-degrading enzyme; (e) cathepsin D; (f) coagulation factor XIa; (g) plasmin; (h) carboxypeptidase; (i) endothelin-converting enzyme-1; (j) angiotensin-converting enzyme; (k) metalloendopeptidase (unidentified); (l) serine protease (unidentified); (m) metallopeptidase (unidentified); (n) aminopeptidase; (o) dipeptidyl peptidase; (p) chymotrypsin-like endopeptidase; (q) trypsinlike endopeptidase; (r) peptidyl dipeptidase.

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potent Ah-degrading peptidases participating in in vivo Ah catabolism. 2.2. In vivo analysis of the brain Ab catabolic system We developed a new experimental paradigm to clarify the rate-limiting process of Ah degradation in vivo (Iwata et al., 2000b). Because Ah1 42 has several possible cleavage sites that can be attacked by various peptidases as described above, we chemically synthesized Ah1 – 42 multiply radiolabeled with 3H and 14C at appropriate amino acid residues (Fig. 3). Conventional biochemical labeling techniques, such as addition of 125I to a tyrosine residue (the tenth residue in the case of Ah) or conjugation of a fluorescent dye to the amino terminal or carboxyl terminal of the peptide, are not sufficient to elucidate in detail the mechanism of the in vivo degradation, because the modification may influence or even prevent proteolytic events by altering the conformation of the peptide. We injected the multiply labeled peptide into the rat hippocampus for in vivo degradation. The resultant fragments were analyzed by high-pressure liquid chromatography (HPLC) equipped with a flow scintillation monitor. We found that 3H/14C-Ah1 – 42 underwent proteolytic degradation in the hippocampus with a half life of 17.5 min, and the degradation proceeded via Ah10 – 37 as the major catabolic intermediate. Because almost all the radioactivity derived from the injected 3H/14C-Ah1 – 42 could be recovered from the hippocampal tissue for at least 1 hr after the injection, Ah clearance may depend predominantly on the proteolytic system in the brain parenchymal cells, rather than transport across the blood –brain barrier into the blood or cerebrospinal fluid in this experimental paradigm (Iwata et al., 2000a, 2000b). However, 125I-labeled Ah40 injected into the mouse brain was more readily transferred to blood, compared with Ah42 (Ji et al., 2001), so the relative contributions of degradation and transport to Ah clearance appear to be different between Ah40 and Ah42. To determine what kind of peptidase acts on Ah1 – 42 in the brain parenchymal cells, we tested the inhibitory effects of various peptidase inhibitors that were coinjected with 3 H/14C-Ah1 – 42. Only neutral endopeptidase inhibitors, phosphoramidon and thiorphan, effectively suppressed the in vivo degradation of 3H/14C-Ah1 – 42 and the appearance of the catabolic intermediate. In addition, to demonstrate that neutral endopeptidase-dependent Ah degradation is not an artifact due to injection of 3H/14C-Ah1 – 42, we infused thiorphan into rat hippocampus. Chronic infusion of thiorphan into rat hippocampus for 3 days elevated endogenous Ah levels, and infusion for 30 days resulted in the accumulation of endogenous Ah and the formation of amyloid plaques. Recently, this result has been confirmed by another research group, which examined the effect of apolipoprotein E (apoE) 4 on Ah accumulation in apoE transgenic (tg) or knockout mice with a nonfamilial ADlinked mutant APP tg (APP tg) background (Dolev &

Michaelson, 2004). These observations indicated that a neutral endopeptidase sensitive to both phosphoramidon and thiorphan plays a major and rate-limiting role in Ah catabolism. Despite this breakthrough, the exact molecular identity of the endopeptidase remained unclear, because at least 4 independent-gene-derived endopeptidases, neprilysin, phosphate-regulating gene with homologies to endopeptidases on the X chromosome, damage-induced neuronal endopeptidase, and neprilysin-like peptidase a, h, and g, with similar biochemical properties are present in the brain (Shirotani et al., 2001). When we transfected cDNAs encoding these peptidases into HEK 293 cells and compared the Ah-degrading activities in cell lysates of the transfectants using 2 different methods (HPLC and enzymelinked immunosorbent assay [ELISA]), with both synthetic and cell-secreted Ah, we found that neprilysin most rapidly and efficiently degraded both Ah1 – 42 and Ah1 – 40 (Shirotani et al., 2001). In addition, Ah-degrading activity in the membrane fraction extracted from brain was almost entirely absorbed with anti-neprilysin antibody (Takaki et al., 2000). Neprilysin thus accounts for the majority of the inhibitorsensitive endopeptidase activity in the brain and exhibits the most potent Ah-degrading activity among the above candidates. We hypothesized that neprilysin deficiency would influence the steady-state Ah levels in the brain by altering Ah metabolism and further investigated the Ah catabolic system in brain using neprilysin-knockout mice. 2.3. Studies of Ab-degrading enzyme candidates using reverse genetic techniques To clarify the contribution of neprilysin to in vivo Ah catabolic system, we first analyzed the degradation of 3 14 H/ C-Ah1 – 42 in neprilysin gene-knockout mouse brain using the in vivo experimental paradigm (Iwata et al., 2001). Wild-type mice exhibited a complete degradation of 3H/14CAh1 – 42 30 min after the intrahippocampal injection. This degradation was markedly inhibited by coinjection of 3 14 H/ C-Ah1 – 42 with a neprilysin inhibitor, thiorphan, as in the case of rats. In contrast, most of the Ah1 – 42 injected into neprilysin-knockout ( / ) mouse hippocampus remained intact, even in the absence of thiorphan. Ah degradation was also decelerated, although to a lesser extent, in heterozygous knockout (+/ ) mice. If neprilysin is the major Ah-degrading enzyme in vivo, neprilysin deficiency should result in the elevation of endogenous Ah levels in the brain. We next analyzed changes in endogenous Ah40 and Ah42 levels in mouse brain using an ELISA identical to the one employed to examine the effect of PS mutations in tg mice (Duff et al., 1996; Nakano et al., 1999). As a positive control, we analyzed mice carrying a familial AD-causing PS 1 gene mutation (Nakano et al., 1999) and confirmed a selective 1.5-fold increase in Ah42 content, as previously established (Sherrington et al., 1995; Borchelt et al., 1996; Duff et al., 1996). The levels of Ah40 and Ah42 were significantly

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elevated in the neprilysin-knockout mice in a gene-dosedependent manner (Table 1). The increase in Ah42 caused by the heterozygous deficiency is comparable to that seen in mutant PS tg or knockin mice. Most importantly, the inverse correlation between the neprilysin gene dose and Ah level (Table 1) suggests that even a partial down-regulation of neprilysin activity will promote Ah deposition in the brain. Recently, in addition to neprilysin, several peptidases, such as ECE1, ECE2, and IDE, have been identified as Ahdegrading enzymes by means of reverse genetics studies (Eckman et al., 2003; Farris et al., 2003; Miller et al., 2003). The brain Ah-elevating effect of neprilysin deficiency is greater than that of a deficiency of any other Ah-degrading enzyme candidate (Table 1). In a rat model of type 2 diabetes mellitus with partial loss-of-function mutations in the IDE gene, involving a 15 –30% decrease in the catalytic efficiency for the degradation of both insulin and Ah, the steady-state levels of Ah in the brain are unchanged, although endogenously secreted Ah is significantly increased in primary-cultured neurons from the animals (Farris et al., 2004). Thus, the partial deficit in IDE is compensated for by other Ah-degrading enzymes. Therefore, these candidate peptidases are likely to contribute to overall Ah clearance in the brain by complementing each other in a subcellular, cell type-, and/or brain region-specific manner. Because of the differences in their enzymatic and cellular properties, neprilysin and ECEs are likely to play complementary roles in distinct subcellular compartments; the former degrades Ah inside secretory vesicles and on the extracellular surface, while the latter does so in acidic compartments represented by the trans-Golgi network (Eckman et al., 2001; Saido & Nakahara, 2003). IDE, which is primarily a cytosolic and peroxisomal enzyme (Duckworth et al., 1998), can be, in part, detected in the plasma membrane (Vekrellis et al., 2000). IDE is also secreted from the microglial cell line, BV-2, and degrades extracellular Ah (Qiu et al., 1998). In fact, the deceleration

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of amyloid deposition and its associated inflammatory pathology is observed in the brain of familial AD-linked mutant APP tg (APP tg) mice crossbred with IDE tg mice, in which expression is controlled in neurons by using a calmodulin-dependent kinase IIa promoter, as well as the case of crossbreeding with neprilysin tg mice (Leissring et al., 2003). IDE may also be involved in the degradation of soluble Ah in particular compartments (Morelli et al., 2003; Schmitz et al., 2004), in which neprilysin does not play a major role. Since the level of APP intracellular domain (AICD) is markedly increased in IDE-knockout mouse brains (Farris et al., 2003; Miller et al., 2003), IDE may be involved in degradation of the APP intracellular domain, rather than Ah (Edbauer et al., 2002). 2.4. Enzymatic properties of neprilysin Neprilysin (EC 3.4.24.11) is a type II membrane metalloendopeptidase composed of ¨ 750 residues with an active site containing a zinc-binding motif (HEXXH) at the extracellular carboxyl terminal domain and exists on plasma membrane as a noncovalently associated homodimer (Fig. 4; Turner, 2004). Neprilysin, functioning as an ectoenzyme at the cell surface, acts mainly on peptides smaller than 5 kDa (5– 40 amino acid residues in length) based on the size of known substrate peptides and cleaves a hydrophobic residue in the P1V position of substrate peptides. Although ˚ ) suggests that the the size of the catalytic cavity (20 A neprilysin active site is not accessible to peptides of more than 3 kDa (Oefner et al., 2000), the secondary and tertiary structures of the substrate also need to be taken into account. As regards polypeptide length, Ah1 – 42 is the longest known neprilysin substrate. Neprilysin is also capable of degrading oligomeric forms of Ah (Kanemitsu et al., 2003; see Fig. 1), although IDE cannot degrade the oligomers (Walsh et al., 2002; Morelli et al., 2003). The active site of neprilysin faces the extracellular side, where Ah should be released,

Table 1 Ah-degrading enzyme candidates studied by using reverse genetic techniques and comparison of Ah-elevating effects in the brain KO or KI mice

Ah42

Ah40

References

Neprilysin-KO ( / ) Neprilysin-KO (+/ ) ECE 2-KO ( / ) ECE 2-KO (+/ ) ECE 1-KO (+/ ) IDE-KO ( / ) tPA-KO ( / ) uPA-KO ( / ) ACE-KO (+/ ) Plasmin ( / ) Familial AD presenilin 1-KI (positive control)

2-fold 1.5-fold 1.3-fold 1.2-fold 1.3-fold 1.4-fold Not significant* Not significant* Not significant* Not significant* 1.5-fold increase

2-fold 1.5-fold 1.3-fold 1.2-fold 1.3-fold 1.2- to 1.6-fold Not significant* Not significant* Not significant* Not significant* Not significant*

Iwata et al., 2001 Iwata et al., 2001 Eckman et al., 2003 Eckman et al., 2003 Eckman et al., 2003 Farris et al., 2003; Miller et al., 2003 Unpublished data by Iwata et al. Ertekin-Taner et al., 2005 Unpublished data by Takaki et al. Tucker et al., 2004 Nakano et al., 1999

KO: (gene) knock-out; KI: (gene) knock-in; tPA: tissue-type plasminogen activator; uPA: urokinase-type plasminogen activator; ACE: angiotensin-converting enzyme. *Not significant is defined as a difference compared to control mice of <10%. Plasminogen-KO mice were examined as plasmin-KO mice, because plasmin is activated by processing from plasminogen. The data from mutant presenilin 1-KI mice, which show typical pathogenic alterations in the Ah42 level, leading to accelerated Ah accumulation in the brain, are presented as a positive control. Quantification was performed using an identical ELISA (Suzuki et al., 1994). The mice were from 8 to 10 weeks of age.

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Fig. 4. Diagrams of neprilysin gene, transcripts, and protein. The human neprilysin gene spans more than 80 kb and is composed of 24 exons. Three single nucleotide repeat polymorphisms (SNPs) and 1 dinucleotide repeat polymorphism (DNP), which are reported to be associated with susceptibility to sporadic AD, are shown. Four types of mRNA transcripts with different structures at the 5V-noncoding region are generated from the neprilysin gene in a tissue-specific manner by alternative splicing. From these transcripts, neprilysin protein of the same amino acid sequence is translated. Neprilysin is a type II membrane metalloendopeptidase with an active site containing a zinc-binding motif (HEXXH) at the extracellular carboxyl-terminal domain.

indicating that it has access to physiological Ah in vivo. Indeed, the overexpression of neprilysin led to significant decreases in secreted and membrane-associated Ah in primary cortical neurons (Hama et al., 2001). Neprilysin may play a role in degrading a variety of neuropeptides, such as enkephalin, somatostatin, atrial natriuretic peptide, substance P, neurokinins, cholecystokinin, nociceptin, and corticotropin-releasing factor, based on in vitro experiments (Roques et al., 1993; Barnes et al., 1995; O’Cuinn et al., 1995; Johnson et al., 1999; Sakurada et al., 2002; Turner, 2004). However, whether these neuropeptides are in vivo substrates is unclear. For instance, the levels of enkephalins were not increased in the brains of the neprilysin-knockout mice (Saria et al., 1997), despite the fact that neprilysin is a potent enkephalin degrader previously termed ‘‘enkephalinase’’, indicating that neprilysin deficiency is compensated for by other peptidases, such as aminopeptidases, in the case of enkephalin metabolism. In addition, neprilysin deficiency does not seem to alter the levels of somatostatin, cholecystokinin, and substance P in the hippocampal formation and cerebral cortex (Iwata and Saido, unpublished observation), indicating the presence of redundant mechanisms or pathways to metabolize these peptides. Although the possibility that the gene expression of these neuropeptides (more precisely, their prepropeptides) is innately up-regulated in neprilysin-knockout mouse brains cannot be excluded, Ah is the only physiological

substrate peptide for which degradation in the brain has been proven to be regulated by neprilysin, so far. Neprilysin requires maturation by glycosylation for full activity (Lafrance et al., 1994), though the possible involvement of other post-translational modifications, such as shedding, phosphorylation, and acetylation, in the activation of neprilysin and any requirement for auxiliary proteins to activate or modify neprilysin have not been clarified. Because the optimal pH of neprilysin is neutral, neprilysin should be translocated to the plasma membrane (presynaptic membrane, in the case of neurons) after synthesis to exhibit full activity. Thiorphan and phosphoramidon are widely used as neprilysin inhibitors (Roques et al., 1993; Turner, 2004). As thiorphan inhibits neutral endopeptidase activity to a similar level to that observed in neprilysin-knockout mice, thiorphan is regarded a potent and specific inhibitor of neprilysin. The neprilysin-dependent neutral endopeptidase activity in brain extract can be spectrophotometrically or fluorometrically assayed using Z-Ala-Ala-Leu-p-nitroanilide or succinyl-Ala-Ala-PheMCA as a substrate, and determined based on the decrease in the rate of digestion caused by thiorphan (Iwata et al., 2002, 2004). Neprilysin is also commonly referred to as neutral endopeptidase-24.11, enkephalinase, or NEP and is identical with leukocyte cell surface antigen 10 (CD10) or the common acute lymphoblastic leukemia antigen (CALLA).

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3. Pathological relevance of neprilysin to AD Since neprilysin appears to be the major Ah-degrading enzyme, as described above, it is likely to be associated with Ah pathology in AD brains. The regional Ah levels in the wild-type mouse brain were in the order of hippocampus > cortex > thalamus/striatum > cerebellum, and this tendency is exaggerated in neprilysin-knockout mice (Iwata et al., 2001). The order correlates well with the regional severity of Ah pathology in AD brains (Braak & Braak, 1991; Yasojima et al., 2001a, 2001b). Nevertheless, proof of the pathological relevance of neprilysin to AD requires detailed evidence, such as evaluation of the precise localization of neprilysin in the brain, the relationship with AD risk factors, and changes in expression in AD brain. Similar logic is, of course, applicable to dissecting the pathological relevance of other Ah-degrading enzymes, such as IDE and ECE. 3.1. Distribution of neprilysin in brain Neprilysin is widely distributed in mammalian brains; it is abundant in the caudate putamen, olfactory tubercle, globus pallidus, substantia nigra, and choroid plexus, and less so in the hippocampal formation, neocortex, superior colliculus, inferior colliculus, amygdala, and central gray matter of the mesencephalon, as observed by autoradiography with radiolabeled anti-neprilysin antibodies or radiolabeled neprilysin inhibitors and by activity staining of the enzyme (Waksman et al., 1986a, 1986b; Back & Gorenstein, 1989; Pollard et al., 1989). In the hippocampus, neprilysin is most abundant in the stratum lacunosum-moleculare of the CA1 –CA3 field and the molecular layer of the dentate gyrus, followed by the stratum pyramidale of the CA1 –

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CA3 field, the granular and polymorphic layers of the dentate gyrus, and the entorhinal cortex (Fukami et al., 2002; Fig. 5). The stratum oriens, the stratum radiata, and the subiculum give very weak signals. In the neocortex, layers II/III and V give somewhat more intense signals than other layers. In the dentate gyrus molecular layer, neprilysin appears in a laminar pattern with clear boundaries among the inner, middle, and outer molecular layers. The middle molecular layer shows a more intense signal than the outer and inner layers. It has been observed that Ah deposition correlates inversely with neprilysin localization in the hippocampus of APP tg mice (TgCRND8; Fukami et al., 2002). 3.2. Subcellular localization of neprilysin in neurons In the substantia nigra of the pig brain, neprilysin is localized in the plasma membrane of axons, axonal terminal boutons, and some dendrites, as determined by subcellular fractionation studies and electron microscopic immunocytochemistry (Barnes et al., 1988, 1992). In accordance with this, synaptic and axonal localization of neprilysin was observed in hippocampal and neocortical neurons by means of double immunofluorescence analyses using confocal microscopy (Fukami et al., 2002); the immunoreactivities of the presynaptic marker proteins synaptic vesicle protein 2 and synaptophysin and the axonal marker protein tau merged with that of neprilysin, whereas a postsynaptic marker, microtubule associated protein 2, was rarely colocalized with neprilysin. The synaptic and axonal localization of neprilysin suggests that after synthesis in the soma, neprilysin, a membrane-integrated protein, is axonally transported to the presynaptic terminals, where Ah degradation is likely to

Fig. 5. Immunostaining pattern of neprilysin (a) and diagram of the physiological localization and aging-associated reduction of neprilysin (b) in the limbic region of mice. Abbreviations: Or, stratum oriens; Py, stratum pyramidale; Ra, stratum radiata; Lm, stratum lacunosum-moleculare; Omo, outer molecular layer; Mmo, middle molecular layer; Imo, inner molecular layer; Gr, granule cell layer; Po, polymorphic cell layer; Lu, stratum lucidum; LEA, lateral entorhinal area; MEA, medial entorhinal area.

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take place. This idea is reinforced by the following observations. For instance, when neprilysin was expressed in the unilateral dentate gyrus of neprilysin-knockout mice by neprilysin gene transfer with recombinant adeno-associated viral (AAV) vector, signals of neprilysin were observed from the bilateral hippocampal formations (Iwata et al., 2004). It is noteworthy that the localization of neprilysin in the contralateral side of the hippocampal formation was similar to the pattern of afferent projection from the ipsilateral neurons, that is, the commissural, associational, and associational/commissural projections (Witter & Amaral, 2004). Furthermore, the localization of neprilysin after the injection of rAAV-neprilysin vector into the lateral entorhinal cortex was observed in the outer molecular layer of the dentate gyrus, which is consistent with the projection of the perforant path (Witter & Amaral, 2004). Thus, neprilysin expressed by rAAV vector-mediated gene transfer appears to be axonally transported to presynaptic sites through afferent projections of neuronal circuits. Next, although earlier studies found that dystrophic neurites in senile plaques were immunostained for neprilysin in AD brains and in brain of APP tg mouse (Akiyama et al., 2001; Fukami et al., 2002), neprilysin gene transfer resulted in clearer visualization of dystrophic neurites and amyloid plaques containing immunoreactive neprilysin. APP also undergoes axonal transport to presynaptic terminals, while Ah is generated through sequential cleavages by h- and g-secretases and released to extracellular space (Price et al., 2000; Selkoe, 2001a, 2001b). Ah released from presynaptic sites contributes to extracellular amyloid deposits, as demonstrated by in vivo experiments (Lazarov et al., 2002; Sheng et al., 2002). The role of the relatively high expression of neprilysin in the molecular layer of the dentate gyrus, which is the terminal of the perforant pathway, may be to counterbalance the active transport of APP in this region (Buxbaum et al., 1998; Phinney et al., 1999) and the resultant generation of Ah (Shukla & Bridges, 2001). Degradation of Ah by neprilysin is likely to take place at or near synapses and may also proceed inside the secretory vesicles during axonal transport if both APP and neprilysin are colocalized in the same vesicles. Thus, the sites for release and degradation of Ah seem to be closely related to each other, and synaptic localization of neprilysin is of particular importance in supporting a link with AD pathology. 3.3. Age-related decline in neprilysin The accumulation of Ah42 in the human brain occurs from the age of 40 years old, and the Ah42 level in normal brain becomes close to that of clinically AD-affected patients with progression of aging (Funato et al., 1998). Thus, aging is regarded as the most potent risk factor for sporadic AD. If the down-regulation of neprilysin is actually implicated in the Ah deposition observed in human brains during normal aging and in AD develop-

ment, neprilysin activity should be decreased with aging in the AD-vulnerable regions of the brain. Quantitative immunohistochemical analysis of the brains of aged laboratory mice (C57BL/6) revealed that neprilysin levels are reduced by 20 –40% at 2.5 years of age in specific regions, such as the outer molecular layer and polymorphic layer of the dentate gyrus and the stratum lucidum of the hippocampus, indicating that neprilysin is selectively decreased at the terminal zones and on axons of the lateral perforant path and the mossy fibers (Fig. 5b; Iwata et al., 2002). This decrease in neprilysin expression is not due to a simple loss of neurons or of presynapses, because the immunoreactivities to presynaptic marker proteins remain unchanged in mice of this age. If such an agingdependent decrease of neprilysin level occurs in the human brain as well, the down-regulation of neprilysin is likely to be related to the Ah deposition associated with normal aging in humans and ultimately to AD pathology, and differences in the velocity of the decline would probably differentiate the pathological progression to AD among different individuals. It is of particular importance that a prominent local decline in neprilysin appears first in the areas corresponding to the presynaptic terminal zones of the perforant path and mossy fibers originally projecting from the entorhinal cortex, where the initial neurodegeneration takes place in the early stages of the onset/progression of AD (Braak & Braak, 1991, Go´mez-Isla et al., 1996). The hippocampal formation is composed of major trisynaptic neuronal circuits, such as the perforant pathway (the entorhinaldentate projection), mossy fibers (the granule cell-CA3 pyramidal cell projection), and Schaffer collaterals (pyramidal cell projection from the CA3 to CA1 field). Information and signals from the association cortex are inputted into the dentate gyrus via the entorhinal cortex, integrated in the hippocampus, and outputted to the cerebral cortex. Neuronal dysfunction caused by a local increase in Ah via down-regulation of neprilysin in the first neuronal circuit of the hippocampal formation could result in impaired overall function of the hippocampal formation. In AD pathology and normal aging, the perforant pathway is known to be one of the most vulnerable sites (Morrison & Hof, 1997). Numerous senile plaques occur in the entorhinal cortex, the presubiculum, the hippocampus, and the dentate gyrus as well as in the frontal, temporal, and parietal neocortices (Braak & Braak, 1991). In APP tg mice, Ah tends to be deposited in similarly circumscribed areas of the brain, including the neocortex and hippocampal formation, in which the heaviest deposition occurs in the molecular layer of the dentate gyrus and the subiculum. Even within the molecular layer of the dentate gyrus, Ah preferentially deposits in the outer layer, with the middle and inner layers being almost devoid of Ah accumulation. Such accelerated Ah deposition with aging at the outer molecular layer of the dentate gyrus is

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observed consistently among APP tg mouse strains regardless of the use of different neuron-specific promoters and familial AD-mutant APP constructs (Games et al., 1995; Borchelt et al., 1997; Irizarry et al., 1997; Johnson-Wood et al., 1997; Sturchler-Pierrat et al., 1997; Chapman et al., 1999; Chishti et al., 2001) and could be accounted for by a selective reduction of neprilysin level. In addition, in the subiculum, where neprilysin levels are relatively low, Ah appears to be readily deposited from the first. 3.4. Expression levels of neprilysin in AD brains Is neprilysin expression changed in the brains of AD patients? Reports from McGeer’s research group clearly show that neprilysin mRNA and protein levels, particularly in high plaque regions such as the hippocampus and temporal gyrus, were significantly lower (50%) even in the early stages of sporadic AD progression (Braak stage II) than in age-matched controls, while this was not the case in other brain areas or peripheral organs (Yasojima et al., 2001a, 2001b). Because the expression levels of 2 control genes encoding cyclophilin and microtubule-associated protein 2, which are neuronal marker proteins, were not significantly different in the 2 groups, the down-regulation of neprilysin does not seem to be a simple consequence of neurodegeneration, but occurs selectively in the regions susceptible to AD pathology. Wang et al. (2003) confirmed the above observation and found that the ratio of oxidized form (adduct of 4hydroxynonenal; HNE) to total neprilysin protein is greater in AD patients’ brains than age-matched control brains, although neprilysin in both AD and controls showed modification with HNE. If neprilysin undergoes inactivation by such oxidative modification, in addition to downregulation, age-related cerebral amyloidosis would be further exacerbated in AD. Thus, in human cases, there is substantial evidence for a causal relationship between the down-regulation of neprilysin and amyloid deposition. 3.5. Polymorphism of human neprilysin gene The human neprilysin gene (MME) is located in chromosome 3, 3q25.1 –q25.2, spans more than 80 kb and is composed of 24 exons. If gene mutations linked to familial AD or polymorphisms associated with susceptibility to AD were to be found on the neprilysin gene, they might be either a risk or an anti-risk factor(s), depending on whether the consequence is down- or up-regulation of neprilysin expression, respectively. Up to the present, no gene mutation in the human neprilysin gene linked to familial AD has been reported. Many single nucleotide polymorphisms (SNP) of the human neprilysin gene with > 10% allele frequency have been found, but no ADassociated SNP has been identified in the coding region of

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the neprilysin gene. One SNP (C/C genotype) on the 3Vuntranslated region associated with AD in Spanish patients under the age of 75 years (Clarimo´n et al., 2003), and 2 SNPs (T/T and AA genotypes, respectively) on intron 1 and intron 19 associated with AD in Finnish patients (Helisalmi et al., 2004) have been identified (Fig. 4), but it is not established how these polymorphisms affect the activity or gene expression of neprilysin. If similar results could be obtained for a greater number of subjects, populations of other areas, or other ethnic populations, the relationship between the appearance of the SNPs and sporadic AD would be much clearer. On the other hand, 2 dinucleotide repeat polymorphisms (DNPs; CA repeats ranging from 12 to 19, with the 16 repeat being most frequent; GT repeats ranging from 18 to 25, with the 22 repeat being most frequent; Fig. 4), which may alter DNA – protein interaction via conformational changes in the DNA helix, are present in the enhancer and promoter regions upstream of exon 1. These polymorphisms may influence the transcription of the neprilysin gene to type 1 mRNA in neurons (see Section 4.1). The association of GT DNP with susceptibility to AD is currently controversial; Sakai et al. (2004) reported a positive association, but 3 other groups found no association (Sodeyama et al., 2001; Oda et al., 2002; Lilius et al., 2003). 3.6. Relationship with hereditary cerebral amyloid angiopathy A significant role of Ah in AD pathogenesis has been definitively confirmed by the identification of APP and presenilin genes as early-onset familial AD-causal genes and by evidence that the familial AD-linked mutations of these genes promote Ah production, resulting in Ah accumulation. Although familial AD is rare, these findings represent strong evidence of a significant role of Ah in the etiology and pathogenesis of AD, because familial AD cases show autosomal dominant inheritance and exhibit the same phenotype as sporadic AD cases. Although most familial AD-linked gene mutations act to increase Ah anabolic activity, gene mutations in the intra-Ah sequence of the APP gene have been suggested to affect the physical properties, such as the aggregation of Ah, by in vitro experiments. We investigated the sensitivity of familial AD-linked mutant Ah to neprilysin-catalyzed degradation and found that Dutch, Flemish, Arctic, and Italian mutations in the intra-Ah sequence showed resistance to neprilysin-dependent proteolysis, compared with wild-type human Ah (Tsubuki et al., 2003). This finding indicates that deposition of Ah with intra-Ah mutation is due not only to its intrinsic aggregation properties, but also to its resistance to degradation. Thus, the resistance to proteolytic degradation also leads to a prolonged life span of Ah and may contribute to hereditary cerebral amyloid angiopathy induced by Dutch, Flemish, and Italian mutations of the APP gene.

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4. Regulatory mechanisms of neprilysin 4.1. Tissue-specific regulation The expression of neprilysin is transcriptionally regulated in a tissue-specific manner in human and rat (D’Adamio et al., 1989; Li et al., 1995; Li & Hersh, 1998). The 5V-untranslated region including from exon 1 to 3 is alternatively spliced, resulting in 4 types of mRNA transcripts (D’Adamio et al., 1989; Li et al., 1995; Fig. 4). The coding region, which begins in exon 4, is not affected by the alternative splicing, and the transcript variants share the same protein sequence of neprilysin. The major transcripts with distinct tissue distributions vary in their 5Vuntranslated regions (Li et al., 1995). Neurons predominantly express the type 1 transcript containing exon 1, and oligodendrocytes express the type 3 transcript starting at exon 3, while type 2a and type 2b are major forms in peripheral tissues. The sequences in alternatively spliced 5Vuntranslated regions might affect the mRNA translation rate or stability and, ultimately, the protein expression level, as shown in the cases of basic transcription element binding protein and APP gene expression (Imataka et al., 1994; Rogers et al., 1999). The enhancer and promoter regions upstream of exon 1 are likely to selectively regulate the expression of neprilysin in neurons. Indeed, there are several clusters of possible transcription factor binding sites and 2 dinucleotide repeats (AC or CA and GT or TG) in the upstream region (Ishimaru & Shipp, 1995; Ishimaru et al., 1997; Iwata et al., 2001; Sezaki et al., 2003). Therefore, it is possible that at least some of the mutations or polymorphisms in these regions could influence the expression of neprilysin in a neuron-specific manner and consequently alter the Ah levels in the brain as described in Section 3.5. 4.2. Region- and neuron type-specific regulation As described in Section 3.3, a dramatic decline in neprilysin is observed in the specific region of the hippocampal formation. When we compared age-dependent changes in neprilysin level in several regions of the brain, such as the hippocampal formation, cerebral cortex, cerebellum, and striatum/thalamus, age-dependent reduction of neprilysin level (estimated as protein amount) was observed in the bulk of the hippocampal formation and cerebral cortex, whereas in the cerebellum and the striatum/ thalamus, no significant change was observed (Iwata et al., 2002). This observation indicates the presence of regionspecific regulatory mechanisms of neprilysin gene expression in the brain. For instance, in the hippocampal formation, neprilysin is expressed in some GABAergic neurons, but not in catecholaminergic or cholinergic neurons (Fukami et al., 2002), implying the existence of interneuron-specific regulation of neprilysin. Interneurons are associated with various neuropeptides (Freund & Buzsaki, 1996), which are regarded as neprilysin substrates

(Section 2.4). Therefore, the decrease in some neuropeptides, such as somatostatin, substance P, corticotropinreleasing hormone, vasopressin, and neuropeptide Y, in sporadic AD brains (Davies et al., 1980; Rossor et al., 1980; Rossor et al., 1981; Crystal & Davies, 1982; Bissette et al., 1985, 1998; Fujiyoshi et al., 1987; Beal et al., 1986; ChanPalay, 1988; Bierer et al., 1995, Schindler et al., 1996) may be closely associated with Ah deposition through the downregulation of neprilysin expression (see Section 4.5). Neprilysin appeared not to be present in astrocytes or microglial cells, as far as we investigated in young laboratory mice. However, Apelt et al. (2003) observed the expression of neprilysin in reactive astrocyes surrounding amyloid plaques of aged APP tg mice and suggested a possible role of plaque-associated astrogliosis in Ah degradation via up-regulation of neprilysin. 4.3. Ab fibril-mediated regulation Mohajeri et al. (2002, 2004) reported that a single injection of fibrillar Ah into mouse brain transcriptionally increased the neprilysin level in neurons of hippocampus and cerebral cortex, as confirmed by consistent changes in activity, protein amount, and mRNA for neprilysin, whereas such induction was not observed for IDE, ACE, or endopeptidase 24.15. In addition, amyloid plaque formation decelerated 20 weeks after the injection into the brains of young APP tg mice (11 weeks old), whereas this was not the case for aged APP tg mice. It is noteworthy that the injection of a peptide with the reverse sequence of Ah, as well as those of bovine serum albumin and vehicle solution, also induced neprilysin gene expression for 1 week, whereas Ah fibrils maintained the increased level (4 –5 fold) of neprilysin for over 20 weeks after the injection. In addition, increased neprilysin expression throughout the brain, for example, on the contralateral side to the injection side, was observed in this experimental system. Tachykinins and bradykinin receptors are also up-regulated contralaterally to the lesion site in response to unilateral injury (Henken et al., 1990; Eckert et al., 1999), so they proposed that the increased neprilysin expression may be caused by the injection-induced damage in the presence of the injected Ah fibrils in the brain. Since chronic up-regulation of neuronal neprilysin is not observed in aged APP tg mouse brains, where a number of amyloid plaques are present, the increased neprilysin expression may be a synergic effect of surgical stress, such as the injection stimulus, and Ah fibrils. 4.4. Up-regulation by anti-oxidants In human neuroblastoma SK-N-SH cell culture, antioxidants, such as quercetin, and a green tea extract (EFLA 85942), of which the major components are polyphenols such as ( )-epigallocatechin-3-gallate and ( )-epicatechin3-gallate, up-regulate neprilysin activity (Melzig & Escher, 2002; Melzig & Janka, 2003). Although long-term admin-

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istration of vitamin E reduced the amyloid burden in the brains of APP tg mice (Sung et al., 2004), it is not clear whether this is related to neprilysin up-regulation. 4.5. Regulation by neuropeptides Neuronal neprilysin is regulated by the selective expression of type 1 mRNA in brain tissues through alternative splicing (Li et al., 1995), the interneuron-specific expression of neprilysin, and age-dependent decline in neprilysin level (Iwata et al., 2002), as described above. Endogenous peptidic or nonpeptidic ligands may be involved in the neuronal regulatory mechanism via specific receptors. At least 3 cell type-specific ligands capable of up-regulating neprilysin activity have been identified so far: opioid for neutrophils (Wang et al., 1997), calcitonin for osteoblastlike cells (Howell et al., 1993), and substance P for fibroblast (Bae et al., 2002) and bone marrow cells (Joshi et al., 2001). Because these ligands are known to act via specific G-protein-coupled receptors (GPCRs), the possibility arises that we may be able to pharmacologically control brain Ah levels by modulating neprilysin activity (Saito et al., 2003). To search for potential ligands, we screened nearly 50 candidates, such as neuropeptides, neurotransmitters, growth factors, cytokines, and so on, using primary cortical neurons and a neprilysin activity-based staining method. We found out that only somatostatin significantly elevated neuronal neprilysin activity (Saito et al., 2005). Interestingly, somatostatin treatment resulted in a selective and significant reduction of Ah42, but not Ah40, in the culture medium of primary neurons. When we analyzed somatostatin precursor protein (SSTP; because somatostatin is liberated from its precursor protein by processing)-knockout mice to confirm these observations in vivo, neprilysin activity in the hippocampus of SSTP-knockout mice was significantly lower than that in wild-type mice, in agreement with the above in vitro observations (Fig. 6). Consistently, a selective 50% increase in Ah42, but not in Ah40, was observed in the brain of SSTP-knockout mice. Thus, the effect of somatostatin deficiency was selective for Ah42 in this brain region (Fig. 6; Saito et al., 2005). However, somatostatin deficiency does not decrease neprilysin mRNA and its protein amount as much as it decreases neprilysin activity. Thus, somatostatin may affect post-translational processes such as protein turnover and cellular localization of neprilysin, rather than transcription. In fact, somatostatin promotes presynaptic localization of neprilysin, as demonstrated by a double immunofluorescence study of neprilysin with a presynaptic marker protein vesicular g-aminobutyric acid (GABA) transporter, or an axonal marker protein tau, and by biochemical methods, such as a subcellular fractionation (Saito et al., 2005). The appearance of neprilysin activity on the cell surface, probably the outer surface of the presynaptic membrane, may have a selective effect on Ah42. This is because a

Fig. 6. Neprilysin level and Ah levels in wild-type (grey bars) and SSTPknockout (black bars) mouse brains. (a) Neprilysin-dependent endopeptidase activities in Triton-X-solubilized membrane fractions of mouse brains. (b) Brain Ah levels. Ah40 and Ah42(43) were extracted from mouse brains with guanidine hydrochloride and quantified using an ELISA. The antibodies for the ELISA were generously provided by Takeda Chemical Industries, Ltd. Each value represents the mean T SE (n = 10 – 14 mouse brains). *P < 0.05. Somatostatin deficiency does not affect the expression level and metabolism of APP in the hippocampus as evaluated by Western blot analyses of full-length APP and the N-terminal and C-terminal fragments of APP (data not shown).

genetic deficiency of neprilysin resulted in increases in both Ah40 and Ah42 in an identical manner (Iwata et al., 2001), whereas infusion of the cell-impermeable neprilysin inhibitor, thiorphan, into the brain elevated Ah42 but not Ah40 (Iwata et al., 2000b), suggesting that Ah40 is mainly degraded intracellularly, whereas Ah42 degradation takes place on or near the cell surface in vivo. This notion is further supported by the results of experiments targeting neprilysin to different cellular compartments in primary cortical neurons (Hama et al., 2004) and evaluating differences in neprilysin-dependent degradation of Ah40 and Ah42 under different pH conditions (the internal pH of secretory vesicles is about 5, and the pH in the extracellular space is about 7; Miesenbock et al., 1998): Ah40 was degraded by neprilysin in a similar manner under these different pH conditions, but the degradation of Ah42 was reduced to ¨ 1/10 at pH 5.0 as compared with that at pH 7.2 (Saito et al., 2005) (Fig. 7).

5. Medical applications Neprilysin is considered to be the most important Ahdegrading enzyme for the following reasons. (1) Neprilysin is the only peptidase known to be capable of degrading soluble oligomeric Ah, as well as monomeric Ah. (2) The disruption of neprilysin causes an elevation of endogenous Ah levels in mouse brain in a gene-dose-dependent manner and has a greater Ah-elevating effect in brain than the disruption of any other known Ah-degrading enzyme candidate gene does. (3) Neprilysin, a membrane-bound zinc metallopeptidase with its active site at the lumen side, is implicated in degradation of both extracellular and intracellular (membrane-associated)

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Ah, but plays a larger role in the degradation of Ah after secretion, as demonstrated by the neprilysin-dependent degradation of exogenously administered synthetic Ah1 – 42 peptide in vivo. (4) The localization at the presynapses and on axons indicates that neprilysin may play a key role in Ah catabolism at and around neuronal synapses. Therefore, a reduction of neprilysin activity may cause a local increase in the concentration of Ah at these regions. (5) An agingdependent decline in neprilysin level occurs naturally in regions which are generally affected in AD. (6) A decreased level of neprilysin gene expression is observed in regions vulnerable to Ah pathology from the early stage in sporadic AD patients. (7) The modulation of neprilysin activity by somatostatin may cause a selective decrease in the primary pathogenic agent Ah42 through promoting the presentation of neprilysin at the plasma membrane. These results suggest that an aging-induced decline of neprilysin, which may occur at different rates among different individuals, is a causal event for some cases of late-onset sporadic AD. If this is so, a potential therapeutic strategy to prevent the abnormal accumulation of Ah would be to restore neprilysin activity in the brain. Therefore, the up-regulation of neprilysin activity in the brain to lower Ah levels appears to be a promising strategy for the prevention and therapy of AD. There are several advantages in adopting the strategy of utilizing enhanced neprilysin activity for Ah degradation. (1) This approach may have fewer side effects than the inhibition of Ah production, because neprilysin activity does not influence the processing of APP (Howell et al., 1995) or degradation of APP fragments, such as secreted forms of APP and AICD. (2) Neprilysin is capable of degrading large amounts of Ah, as demonstrated by in vivo experiments in which Ah was injected into the hippocampus (Iwata et al., 2000b). (3) The Ah contents in presymptomatic and symptomatic AD brains are 1000- to 10,000-fold greater than those in young normal controls (Funato et al., 1998). Because neprilysin exhibits similar affinity for Ah and for its other known substrates (Table 2; Matsas et al., 1984; Shirotani et al., 2001), neprilysin would preferentially degrade Ah present in greatest abundance in AD brains and might have little impact on the metabolism of other neuropeptides. (4) The promotion of degradation would lead to a shortened life span of Ah and thus decrease the opportunity for Ah to undergo amyloidogenic structural transition. Table 2 Affinity of neprilysin for Ah and neuropeptides Substrate

K m (AM)

References

Ah1-40 Ah1-42 Met-enkephalin Leu-enkephalin Substance P Bradykinin Neurotensin Cholecystokinin-8

11 7 62 86 32 92 78 67

Shirotani et al., 2001 Shirotani et al., 2001 Matsas et al., 1984 Matsas et al., 1984 Matsas et al., 1984 Matsas et al., 1984 Matsas et al., 1984 Matsas et al., 1984

K m: Michaelis constant.

Besides the points outlined above, the most important advantage of utilizing neprilysin activity is apparent from the connection with AD pathology. Synaptic dysfunction is indeed a typical and early function-related event in the AD cascade and is associated with a locally enhanced concentration of Ah at the presynaptic terminals and inside synaptic vesicles in the AD brain (Terry et al., 1991; Sze et al., 1997; Hardy & Selkoe, 2002; Selkoe, 2002). An increase in soluble Ah levels, rather than Ah plaque load, appears to cause such synaptotoxicity. In fact, levels of soluble Ah, rather than insoluble Ah, are well correlated with synaptic changes and neuronal loss in AD brains (Lue et al., 1999; McLean et al., 1999) and in several lines of APP tg mice (Hsia et al., 1999; Mucke et al., 2000; Buttini et al., 2002). Recent studies have indicated that particular forms of soluble Ah, such as oligomeric forms, cause dysfunction or modification of synaptic transmission and/or long-term potentiation in vivo, as well as abnormal behaviors (Walsh et al., 2002; Wang et al., 2002, Cleary et al., 2005). The oligomeric forms of Ah are detected by immunoelectron microscopy with higher density at the axons and axon terminals in AD brains than at those locations in unaffected age-matched control brains (Kokubo et al., 2005). The presence of neprilysin at the presynaptic sites is of particular importance, because neprilysin is capable of degrading not only monomeric Ah, but also oligomeric Ah. Neprilysin would contribute to the removal of a toxic form of Ah in the extracellular space close to synapses and should thereby decelerate AD pathology. However, neprilysin is likely to have several shortcomings with regard to its substrate specificity. Neprilysin cannot efficiently degrade Ah variants with the familial ADlinked Flemish-, Arctic-, Italian-, and Dutch-type mutations in the peptide sequence. On the other hand, IDE is capable of degrading these Ah variants, as well as Iowa-type mutant Ah, although its activity towards Arctic- and Dutch-type mutants is low (Morelli et al., 2003). Therefore, IDE rather than neprilysin would play a key role in the removal of such hereditary variants of Ah from cerebral vessel walls or brain parenchyma. Furthermore, degrading ability of neprilysin for fibrillar Ah has not been observed yet. The catalytic cavity of the neprilysin active site is so small that neprilysin may not be able to act on tightly fibrilized Ah, such as cored amyloid plaques, although the secondary and tertiary structures of the substrate should be taken into account. In this respect, phagocytosis by microglia (Bard et al., 2000; Nicoll et al., 2003) and intake by astrocytes (Wyss-Coray et al., 2003; Koistinaho et al., 2004) appear to be critically involved in the clearance of fibrillar Ah from brain parenchyma. 5.1. Gene therapeutic approach Neprilysin gene transfer using a lentiviral or AAV vector induces long-term expression of neprilysin in the brain and successfully decelerated Ah pathology in aged APP tg

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mouse brains (Marr et al., 2003; Iwata et al., 2004). Interestingly, a difference in the subcellular localization of neprilysin exogenously expressed by gene transfer is observed, depending on the viral vector used for gene transfer. Although neprilysin expressed in neurons infected by a lentiviral vector was detected in the cell bodies in most cases (Marr et al., 2003), the use of AAV resulted in localization at the presynaptic sites as a result of axonal transport, but not in the cell bodies. The former case might mainly promote the intracellular degradation of Ah by neprilysin, while the latter might promote the degradation of synaptic cleft-associated, presynaptic membrane-associated, and extracellular Ah by neprilysin. In both cases, efficient clearance of Ah is observed, suggesting that neprilysin is involved in the degradation of both intracellular and extracellular Ah, in addition to synaptic cleft-associated and presynaptic membrane-associated Ah. Presynaptic localization of neprilysin by AAV-mediated neprilysin gene transfer results in efficient clearance of Ah and should protect synapses from the synaptic toxicity of Ah. Thus, gene therapy with neprilysin may have potential as a treatment for both familial and sporadic AD. The possibility exists that overexpressed neprilysin may be involved in the catabolism of various neuropeptides. However, Ah is the only substrate so far known, of which the level is elevated by neprilysin deficiency. Further, as discussed above, neprilysin exogenously expressed in AD brains by gene transfer should participate primarily in Ah degradation, and might have little impact on the metabolism of other neuropeptides. Neprilysin gene transfer might be particularly suitable to protect the synaptic function of certain highly vulnerable neuronal circuits, for example, the entorhinal-dentate projection, from Ah pathology. This approach may be therapeutically efficient. Thus, the results of experimental gene therapy indeed indicate that the up-regulation of neprilysin activity would be a promising strategy for therapy and prevention of AD. 5.2. Pharmacological approach An approach pharmacologically targeting a ligand – receptor system selectively expressed in neurons may be useful (Garcia-Jimenez et al., 2002). The possibility of upregulating neprilysin through somatostatin receptors, which are GPCRs, has been suggested, as described in Section 4.5. The up-regulation of neprilysin would have to be restricted to within the brain, because neprilysin is involved in the regulation of blood pressure via atrial natriuretic peptide degradation in the peripheral tissue. In fact, the blood pressure of neprilysin-knockout mice is lower than that of wild-type mice (Lu et al., 1997). Furthermore, the up-regulation of neprilysin should also be regionally restricted in the brain, because neprilysin (originally termed enkephalinase) is thought to play an important role in the catabolism of various neuropeptides in the brain tissue (except hippocampal formation and cerebral cortex), such as

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the caudate putamen, olfactory tubercle, globus pallidus, substantia nigra, and choroid plexus. So far, 5 somatostatin receptor subtypes have been identified, all of which are GPCRs (Schindler et al., 1996; Csaba & Dournaud, 2001; Moller et al., 2003). Although these receptor subtypes are distributed in distinct, but overlapping patterns in the brain, subtypes 4 and 2 may be primary candidate targets, because they are relatively highly expressed in neocortex and hippocampus (Bruno et al., 1992; Moller et al., 2003). Therefore, a blood – brain barrier-permeable nonpeptidic agonist specific for the type 4 or 2 somatostatin receptor should act selectively on the ligand– receptor system only in these brain regions and might therefore have minimal systemic effects. Cortistatin, an endogenous somatostatin homologue, binds to somatostatin receptor(s) with a similar affinity to that of somatostatin, although the affinity between cortistatin and somatostatin receptor(s) varies depending on the somatostatin receptor subtype, so that cortistatin appears to act redundantly with somatostatin via somatostatin receptor(s) (Spier & de Lecea, 2000; Moller et al., 2003). It remains a possibility that cortistatin may regulate neprilysin gene expression via somatostatin receptor(s) or its own receptor(s).

6. Conclusion and prospects Davies et al. (1980) discovered that somatostatin levels were significantly reduced in the brains of AD patients, concurrently with loss of acetylcholine-positive neurons (Davies & Maloney, 1976). Although this observation has been confirmed repeatedly by others (Ichai et al., 1994; Bierer et al., 1995; Gabriel et al., 1996; Bissette et al., 1998; De Bree, 2001; van de Nes et al., 2002), its pathogenic significance remains uncertain. Recently, Lu et al. (2004) showed that about 4% of ¨ 11,000 human genes exhibit aging-dependent changes in expression (1.5-fold or more). One of them is indeed the somatostatin gene, of which the expression level is reduced 2- to 3-fold from the age of 40 years old. A similar aging-dependent decrease in somatostatin expression level is observed in primate brains (Hayashi et al., 1997). Because the aging-dependent accumulation of Ah means that the onset of sporadic AD can be regarded as an extension of normal brain aging (Fig. 7, gray arrows), the observation by Davies and colleagues appears to support, after several decades, our proposal that somatostation controls brain Ah levels through modulating the activity of the major Ah-degrading enzyme, neprilysin. These observations have led us to draw a possible scenario for the development of late-onset sporadic AD (Fig. 7, black arrows). Our hypothesis is that the down-regulation of somatostatin expression in the human brain at the early stage of aging initiates a gradual decline in neprilysin activity, resulting in a corresponding elevation in the steady-state levels of Ah, over a decade or

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Fig. 7. Differences in nepilysin-mediated degradation of Ah40 and Ah42 at different pH values. Neprilysin degrades Ah40 in a similar manner at both pH 5 and 7.2 (a), but its degrading activity towards Ah42 is reduced to nearly one-tenth at pH 5.0 (b), compared with that at pH 7.2 (Saito et al., 2005). Ah is produced from APP, which undergoes axonal transport, by proteolytic cleavages, and is released into the extracellular spaces and, in part, to the intravesicular side. Neprilysin, a membrane-integrated protein, is taken into secretory vesicles after synthesis in the soma and axonally transported to the presynaptic terminals, where it degrades Ah. The internal pH of secretory/synaptic vesicles is about 5, and the pH of the extracellular space is about 7 (Miesenbock et al., 1998), thus, the deficiency or inhibition of axonal transport of neprilysin appears to cause a local increase in Ah42 at the presynaptic sites.

more, and this causes Ah accumulation that triggers the AD pathological cascade. This cascade causes progressive neuronal dysfunction and neurodegeneration over a long period, generating a vicious cycle of positive feedback that further lowers the somatostatin level and neprilysin activity, elevates the Ah levels, and accelerates AD development. The up-regulation of brain neprilysin by blood –brain barrier-permeable nonpeptidic agonists selective to somatostatin receptor(s) localized in AD-affected regions might break the vicious cycle through lowering Ah, particularly Ah42, levels. Utilizing somatostatin receptor agonists might also compensate for the decreased levels of somatostatin in aging or AD-affected brains. This could be important because somatostatin, which was originally found as a somatotropin release-inhibitory factor in the anterior pituitary, has recently been shown to improve long-term potentiation, an indicator of synaptic plasticity, in hippocampal slices, and to play an important role in memory and learning functions. Thus, a key target of current research would be to pharmacologically control brain Ah levels by modulating neprilysin activity, with the

aim of maintaining memory and learning functions at acceptable levels until the late stage of life (Fig. 8). The strategy of catabolic up-regulation would be complementary to that of anabolic down-regulation, such as inhibition of h- and g-secretases. These anti-Ah strategies should be combined with other strategies, such as targeting inflammation, oxidative stress, tauopathy, and so on (Forman et al., 2004), to achieve a maximum effect in overcoming AD by minimizing potential side effects of each strategy. Lastly, a fundamental problem for anti-Ah therapy is that AD is only recognized clinically after the amyloid burdens in the brain has reached a high level. Treatments at this stage may be too late to achieve a cure in view of the massive neurodegeneration that has already occurred. However, if we could pick up incipient AD at the early stages by using a combination of several specific, sensitive, and simple diagnostic examinations, we would be able to identify potentially treatable individuals. Recent research has led to the concept of mild cognitive impairment (MCI) as a transitional state present between the cognitive changes accompanying normal aging and AD (Petersen et al., 2001).

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Fig. 8. Hypothetical scenario for late-onset sporadic AD etiology. Ah is accumulated in human brains with the progression of aging, and differences in the velocity of Ah accumulation owing to unknown factors differentiate the onset of sporadic AD pathogenesis among different individuals, as shown with gray arrows. Based on the down-regulation of somatostatin and neprilysin upon aging and in the process of AD development, and our finding that somatostatin influences brain Ah42 level by modulating the activity of the major Ah-degrading enzyme, neprilysin, a possible scenario for the development of late-onset sporadic AD is illustrated with black arrows. The down-regulation of somatostatin expression in the human brain at the early stage of aging initiates a gradual decline in neprilysin activity, resulting in a corresponding elevation in the steady-state levels of Ah, over a decade or more, and this causes Ah accumulation that triggers the AD pathological cascade. In this case, differences in the velocity of the decline in somatostatin and neprilysin would probably differentiate the pathological progression to AD among different individuals. This cascade causes progressive neuronal dysfunction and neurodegeneration over a long period, generating a vicious cycle of positive feedback that further lowers the somatostatin level and neprilysin activity, elevates Ah levels, and accelerates AD development.

If it becomes possible to prediagnose the MCI phase of AD, then presymptomatic intervention could be initiated. However, the clinical and biochemical changes involved in MCI are not fully understood at present. Therefore, research should be also directed towards clarifying the nature of the MCI state, in addition to designing the therapeutic strategies for AD. On the other hand, we have synthesized a magnetic resonance-compatible 19F-containing amyloidophilic Congo Red-type compound, (E,E)-1-fluoro-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), in which the bromine of (E,E)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4hydroxy)styrylbenzene (BSB) is replaced with fluorine, and found that it has a higher amyloidophilic potential than that of BSB (Sato et al., 2004). Recently, we have successfully visualized the amyloid pathology in the brains of living APP tg mice by magnetic resonance imaging after the intravenous administration of FSB (Higuchi et al., 2005). Our findings provide a new approach for specific noninvasive amyloid imaging. Such noninvasive detection of amyloid plaques should be valuable for presymptomatic diagnosis, in addition to MCI, and thus should contribute to the establishment of early preventive treatment strategies.

Acknowledgments The authors thank all the present and past members of the Laboratory for Proteolytic Neuroscience, RIKEN Brain

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Science Institute, for their participation in the work described herein. In particular, Satoshi Tsubuki, Yoshie Takaki, Misaki Sekiguchi, Kaori Watanabe, Emi Hosoki, and Yukio Matsuba have made indispensable contributions to this research from the founding of our laboratory. We also thank our collaborators, including Maho MorishimaKawashima and Yasuo Ihara (University of Tokyo School of Medicine), Craig Gerard and his colleagues (Harvard Medical School), Masatoshi Takeda and his colleagues (University of Osaka, School of Medicine), Takashi Iwatsubo and his colleagues (University of Tokyo, Graduate School of Pharmaceutical Sciences), Hiroshi Kiyama and his colleagues (Osaka City University, Graduate School of Medicine), Keiya Ozawa and his colleagues (Jichi Medical School), and Ute Hochgeschwenger (Oklahoma Medical Research Foundation). Our work have been supported by many research grants from RIKEN BSI, the Ministry of Education, Culture, Sports, Science and Technology, Special Coordination Funds for promoting Science and Technology from the STA, the Ministry of Health, Labor and Welfare of Japan, and Takeda Chemical Industries.

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