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Keywords:

  • amyloid β-peptide;
  • angiotensin-converting enzyme;
  • endothelin-converting enzyme;
  • insulin-degrading enzyme;
  • neprilysin;
  • valproate

Abstract

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

J. Neurochem. (2012) 120 (Suppl. 1), 167–185.

Abstract

The amyloid cascade hypothesis of Alzheimer’s disease envisages that the initial elevation of amyloid β-peptide (Aβ) levels, especially of Aβ1-42, is the primary trigger for the neuronal cell death specific to onset of Alzheimer’s disease. There is now substantial evidence that brain amyloid levels are manipulable because of a dynamic equilibrium between their synthesis from the amyloid precursor protein and their removal by amyloid-degrading enzymes (ADEs) providing a potential therapeutic strategy. Since the initial reports over a decade ago that two zinc metallopeptidases, insulin-degrading enzyme and neprilysin (NEP), contributed to amyloid degradation in the brain, there is now an embarras de richesses in relation to this category of enzymes, which currently number almost 20. These now include serine and cysteine proteinases, as well as numerous zinc peptidases. The experimental validation for each of these enzymes, and which to target, varies enormously but up-regulation of several of them individually in mouse models of Alzheimer’s disease has proved effective in amyloid and plaque clearance, as well as cognitive enhancement. The relative status of each of these enzymes will be critically evaluated. NEP and its homologues, as well as insulin-degrading enzyme, remain as principal ADEs and recently discovered mechanisms of epigenetic regulation of NEP expression potentially open new avenues in manipulation of AD-related genes, including ADEs.


Abbreviations used

amyloid β-peptide

ACE

angiotensin-converting enzyme

AD

Alzheimer’s disease

ADAM

A disintegrin and metalloprotease

ADE

amyloid-degrading enzyme

AICD

APP intracellular domain

AP-A

aminopeptidase A

APH

acyl peptide hydrolase

APLP

APP-like protein

APP

amyloid precursor protein

CatB

cathepsin B

CD

cluster differentiation (antigen)

CysC

cystatin C

ECE

endothelin-converting enzyme

GCP

glutamate carboxypeptidase

HDAC

histone deacetylase

IDE

insulin-degrading enzyme

MMP

matrix metalloproteinase

NEP

neprilysin

PAI-1

plasminogen activator inhibitor-1

PKC

protein kinase C

PPAR

peroxisome proliferator-activated receptor

PreP

pre-sequence protease or peptidasome

SEP

secreted endopeptidase

SNP

single nucleotide polymorphism

TIMP

tissue inhibitor of metalloproteinase

VA

valproic acid

Introduction: the dynamic nature of amyloid

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

It is 20 years since the amyloid cascade hypothesis of Alzheimer’s disease (AD) was originally proposed (Selkoe 1991; Hardy and Higgins 1992) and its underlying message, namely that the accumulation of the amyloid β-peptide (Aβ), especially Aβ1-42, is the initial trigger for neurodegeneration, has held sway during all this period, although not without its detractors. Various re-evaluations of the initial hypothesis have taken place (e.g.Hardy 2009; Pimplikar 2009; Castellani and Smith 2011) and, over time, the precise nature of the toxic Aβ species has been elusive, ranging from plaques and fibrils to monomers and then oligomers: ‘invisible molecules targeting invisible structures’ in the words of Castellani and Smith (2011). The failure of the amyloid hypothesis to lead to the delivery of any clinically successful drugs to date has put pressure on amyloid-based therapeutic approaches but the underlying genetics still strongly places Aβ as a favoured target. A key conceptual change has been the realisation that amyloid levels represent a dynamic equilibrium between biosynthesis and removal rather than an irreversible pathway to cell death. Just as the biosynthetic enzymes (β- and γ-secretases) represent prime targets, so too do the catabolic enzymes, if Aβ accumulation is to be prevented or reversed.

Complete elimination of Aβ is not desirable because it probably has a normal physiological role as a regulatory peptide (see e.g. Pearson and Peers 2006; Hardy 2007), and even as a transcription factor (Ohyagi et al. 2005; Bailey et al. 2011), with its steady-state concentration being tightly controlled by amyloid-degrading proteolytic enzymes and by perivascular drainage (Weller et al. 2002; Nalivaeva et al. 2008; Hawkes et al. 2011), mechanisms which become impaired with aging and disease. This review will focus on recent advances in understanding the nature, diversity and regulation of these degrading enzymes, critically evaluating candidates and highlighting new approaches to the manipulation of their expression and activity.

Amyloid-degrading enzymes: the key players and strategies for manipulation

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

Although most of the potential amyloid-degrading enzymes (ADEs) that have been identified fall into the class of zinc metalloproteinases, especially the M13 neprilysin family, both serine (plasmin) and cysteine (cathepsin B) proteases have also been implicated. Their relative merits as therapeutic targets will be critically assessed below alongside some of the catalytic and regulatory aspects of this diverse collection of enzymes (Table 1, Fig. 1). Until recently, only the M13 peptidases neprilysin (NEP), endothelin-converting enzyme (ECE)-1, ECE-2 and NEP-2, alongside insulin-degrading enzyme (IDE), plasmin, angiotensin-converting enzyme (ACE) and matrix metalloproteinase (MMP)-9 had been shown to control Aβ levels in experimental animal models as discussed below. However, the catalogue of Aβ-degrading enzymes continues to grow and, as summarised in this review, some recently identified candidates could form the basis of novel therapeutic targets. Diverse strategies for up-regulation of ADEs are being developed ranging from viral gene delivery to pharmacological modulation at the transcriptional or protein level. A potential complication is that, like most proteinases, they hydrolyse a range of physiological substrates and therefore manipulation of their activity could lead to unintended side-effects. However, despite this complication, many proteinases have been successfully targeted by drugs in widespread clinical use, e.g. ACE inhibitors for hypertension. Moreover, if AD is attributable to reduced activity of a particular ADE, then restoring it to normal levels pharmacologically would restore homeostasis to the system.

Table 1.   List of proposed amyloid-degrading enzymes, their specificity and locations
NameCleavage site within Aβ1-40 or Aβ1-42Forms of Aβ hydrolysed and locationModels of studyReferences
  1. The references listed are indicative of the action of the various ADEs and fuller details and further references can be found in the text. Note for ACE that different studies have reported different sites of cleavage, and some studies have used Aβ fragments rather than intact Aβ indicated by * (see text for fuller details). Endo/lyso, endososomal/lysosomal location; MMP, matrix metalloproteinase; PreP, mitochondrial peptidasome; MBP, myelin basic protein; PAI, plasminogen activator inhibitor.

Neprilysin (NEP)A2-E3, E3-F4, R5-H6, G9-Y10, V12-H13, F19-F20, K28-G29, G29-A30, A30-I31, G33-L34Monomeric, oligomeric; extracellular (ectoenzyme)Cell-based, transgenic animals, NEP inhibitor, Drosophila modelHowell et al. 1995; Leissring et al. 2003b
Insulin-degrading enzyme (IDE)V12-H13, H13-H14, H14-Q15, V18-F19, F19-F20, F20-A21, K28-G29Monomeric; extracellular (secreted), cytosol, mitochondriaCell-based, transgenic animalsChesneau et al. 2000; Farris et al. 2003; Leissring et al. 2003b; Morelli et al. 2005
Endothelin-converting enzyme (ECE-1/ECE-2)K16-L17, L17-V18, F19-F20, (sites identified for ECE-1)Monomeric; extracellular (ectoenzyme), secreted, endo/lysoCell-based, transgenic animalsEckman et al. 2001, 2003, 2006
Angiotensin-converting enzyme (ACE)D7-S8, E11-V12*, H13-H14*, V18-F19, E22-D23, S26-N27, L34-M35, V40-I41 (see legend)Monomeric, *Aβ fragments; extracellular (ectoenzyme), secreted (shed)Cell-based, transgenic animalsHu et al. 2001; Oba et al. 2005; Zou et al. 2007; Sun et al. 2008b
NEP2K16-L17, L17-V18Monomeric; secreted, endo/lysoCell-based, transgenic animalsHuang et al. 2008; Hafez et al. 2011
PlasminR5-H6, K16-L17,K28-G29monomeric, oligomeric, fibrillar; extracellularCell-based, transgenic animals (PAI-1 and neuroserpin knockouts), PAI-1 inhibitorTucker et al. 2000; Jacobsen et al. 2008; Fabbro et al. 2011; Liu et al. 2011
Cathepsin BG38-V39, V40-I41Monomeric, oligomeric, fibrillar; extracellular (secreted), endo/lyso cell-based, transgenic animalsMueller-Steiner et al. 2006
Aminopeptidase AD1-A2Monomeric; extracellular (ectoenzyme)Cell-basedSevalle et al. 2009
Acyl peptide hydrolaseH13-H14, H14-Q15, F19-F20Monomeric, oligomeric; secretedCell-basedYamin et al. 2009
Glutamate carboxypeptidase IIH14-Q15, V18-F19, M35-V36Monomeric, soluble Oligomers, fibrillar; extracellular (ectoenzyme, secreted?)Cell-based, transgenic animalsKim et al. 2010
MMP-2K16-L17, L34-M35, M35-V36Monomeric, fibrillar; extracellularCell-based, transgenic animalsRoher et al. 1994; Yin et al. 2006
MMP-9K16-L17, F20-A21, D23-V24, A30-I31, D33-L34, L34-M35Monomeric, fibrillar; extracellularCell-based, transgenic animalsBackstrom et al. 1996; Yan et al. 2006
PrePQ15-K16, K16-L17, F19-F20, F20-A21, A30-I31, G33-L34, L34-M35Monomeric; mitochondriaIn vitro studies, cell-based, human brain studiesFalkevall et al. 2006; Alikhani et al. 2011
MBPF4-R5, E11-V12*, Q15-K16, D23-V24, V24-G25, G25-S26*, K28-G29*, I32-G33*, V39-V40, I41-A42*Monomeric, fibrillar (*); extracellularIn vitro studies, cell-basedLiao et al. 2009
image

Figure 1.  Main cleavage sites of amyloid-β peptide by amyloid-degrading enzymes. See also Table 1 for more detail and key references.

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Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

NEP

Neprilysin is one of seven members of the human M13 family of zinc-dependent endopeptidases utilising the HEXXH zinc-binding (zincin) active site motif. It has undergone a variety of nomenclature changes in its history, also being known as neutral endopeptidase (NEP), endopeptidase-24.11, CD10 or CALLA. It was originally identified in the renal brush border but subsequently rediscovered in the brain as a key neuropeptide-degrading enzyme (‘enkephalinase’, ‘substance P-degrading enzyme’), although expressed at much lower levels than in the kidney (Roques et al. 1980; Fulcher et al. 1982; Matsas et al. 1983). NEP is a cell-surface ectoenzyme with a large extracellular domain containing the catalytic site localised in a cavity allowing access to peptides containing up to 50 amino acids (Turner and Tanzawa 1997). It cleaves a rather wide spectrum of regulatory peptides preferentially on the N-terminal side of hydrophobic amino acid residues (Kerr and Kenny 1973), which makes Aβ a competitive substrate for NEP. Pre- and post-synaptic localization of NEP in the nervous system further emphasized its important role in neuronal function (Barnes et al. 1992). Over the years, most attention has been focused on the physiological and pathological implications of its ability to degrade the Aβ peptide in vitro (Howell et al. 1995) and in vivo (Shirotani et al. 2001), although it does not affect the amyloid precursor protein (APP) holoprotein itself. Importantly, it was demonstrated that NEP is a major Aβ-degrading enzyme in the brain (Shirotani et al. 2001). To establish that NEP is capable of reducing amyloid deposits in vivo, Saido and colleagues employed a targeted gene delivery technique using Sindbis viral vector and demonstrated that expression of wild-type NEP in murine cortical primary neurones reduced Aβ1-40 and Aβ1-42 burden in the culture medium (Hama et al. 2001). Later, it was shown that lentiviral delivery of the NEP gene to the brain of AD transgenic mice reduced human amyloid pathology in the brain (Marr et al. 2003) and a number of subsequent studies (e.g. Hemming et al. 2007a) have supported this observation.

By examining the levels of NEP expression in brain structures related to AD pathology it was found that, in rodents, NEP levels decrease with age in the cortex and hippocampus and that NEP expression is also reduced in the AD brain compared with age-matched control patients (Iwata et al. 2002; Nalivaeva et al. 2004). In the AD brain, the decreased levels of NEP expression were also observed along the vasculature suggesting its role in the development of cerebral amyloid angiopathy in AD patients (Carpentier et al. 2002). However, along with the age-related decrease of NEP expression seen in neuronal cells, Apelt and colleagues have reported an up-regulation of this enzyme in reactive astrocytes surrounding amyloid plaques in AD transgenic mice (Apelt et al. 2003). These observations stimulated a cascade of studies on possible mechanisms of NEP up-regulation as a strategy for AD treatment through stimulating clearance of Aβ peptides and/or their various aggregated forms. In line with the attempts at transgenic over-expression of NEP in neuronal cells which were shown to reduce brain Aβ levels, retard amyloid plaque formation and delay AD pathology (Leissring et al. 2003a), the optimal timing of NEP over-expression has also been examined suggesting that earlier up-regulation of NEP levels was more beneficial in alleviating symptoms in a mouse model of AD (El-Amouri et al. 2008).

To dissect the mechanisms of action of NEP on the Aβ peptide and its neuroprotective role, various model systems have been employed allowing analysis of the effects of NEP over-expression on organism and brain development. Of particular note, in a Drosophila model expressing human Aβ1-42, over-expression of human NEP successfully suppressed formation of intraneuronal Aβ1-42 deposits and neuron loss but caused age-dependent axon degeneration and shortened the fly lifespan (Iijima-Ando et al. 2008). The authors related the latter with possible NEP effects on other substrates, for example fly neuropeptides, and further suggested that the natural decline in NEP expression in flies and mammals may represent an evolutionarily conserved protective mechanism which, however, could pre-dispose to late onset AD in humans with increased longevity.

More detailed studies on transgenic mice have shown that, while NEP over-expression in AD transgenic mice inhibited plaque formation, it failed to reduce the more pathogenic Aβ oligomers and associated cognitive deficits observed (Meilandt et al. 2009) but this might be linked to persistent NEP over-expression during mouse development and a deficit of other neuropeptides degraded by NEP. However, another study using over-expression of NEP in transgenic mice, but at a lower level, demonstrated that the products of cleavage of the NEP substrate, neuropeptide Y, were neuroprotective and ameliorated the neurodegenerative pathology in this model (Rose et al. 2009). Hence, interpretation of such transgenic over-expression systems, depends critically on the type of model, expression and delivery systems used, timing and extent of delivery, and also even the nature of the APP isoforms expressed in any over-expression studies (Belyaev et al. 2010).

Apart from the importance of the timing of NEP up-regulation in the brain in relation to the AD pathology, several studies have also attempted to investigate the ability of peripherally delivered NEP to reduce brain Aβ levels, because this would provide an alternative, more convenient and readily monitored delivery system. Because brain and plasma Aβ are in equilibrium through transport mechanisms (Deane et al. 2004), Hersh and colleagues, in a series of papers, have developed strategies to examine whether enhancing peripheral degradation of Aβ by NEP could reduce brain levels and associated pathology. They found that in AD transgenic mice over-expressing NEP in erythrocytes or leukocytes there was reduced Aβ burden in the brain (Liu et al. 2007; Guan et al. 2009). Similar results were observed when NEP was over-expressed in skeletal muscle (Liu et al. 2009). An alternative strategy of expressing a secreted, soluble form of NEP in plasma through an adenovirus construct was also effective in clearing brain Aβ yet did not affect plasma levels of other peptide substrates of NEP such as bradykinin or substance P (Liu et al. 2010). Expressing NEP in plasma in this way also provides a simple system in which to monitor its long-term activity.

The means of delivery of NEP from the periphery to the brain has also been addressed and it was demonstrated that intra-peritoneal injections of a lentivirus vector-expressing NEP fused with the ApoB transport domain reduced levels of Aβ, number of plaques and increased synaptic density in the CNS of AD transgenic mice (Spencer et al. 2011) suggesting a non-invasive therapeutic approach for potential treatment in patients with AD. NEP delivery to peripheral tissues has also proved effective in reducing the amyloid polypeptide deposits which contribute to type 2 diabetes. Thus, adenoviral delivery of NEP to the pancreas reduced these amyloid deposits and cell death in the islets (Zraika et al. 2010). Surprisingly, however, in this case, NEP did not degrade the amyloid peptide but appeared to act by inhibiting the amyloid fibril formation through protein–protein interactions involving the enzyme active site, rather than by hydrolysing the peptide.

Apart from genetic manipulations of NEP expression, a number of studies have aimed at pharmacological up-regulation of the enzyme which have demonstrated that NEP activity can be increased by, among other compounds, a component of green-tea extract, Epigallocatechin-3-gallate (EGCG) (Melzig and Janka 2003) and other plant extracts and polyphenols (e.g. Kiss et al. 2006). Saido and colleagues tested the hypothesis that elevated levels of neuropeptide substrates of NEP could themselves up-regulate NEP as a feedback control mechanism. They hence screened a wide range of NEP substrates demonstrating that, of those tested, only somatostatin was capable of up-regulating NEP expression in primary neuronal cells in a mechanism possibly mediated through somatostatin receptor sub-types 2 or 4 (Saito et al. 2005). Another potential strategy for pharmacological up-regulation of NEP was reported in a study analysing the effects of a selective peroxisome proliferator activated receptor-δ (PPARδ) agonist, GW742, which activated the NEP promoter driving luciferase expression in transfected HEK293 cells (Kalinin et al. 2009).

A completely new turn in development of strategies for modulation of NEP expression and activity was made by Checler and colleagues, who demonstrated that the C-terminal APP intracellular domain (AICD) was able to up-regulate NEP transcription and to reduce accumulation of Aβ (Pardossi-Piquard et al. 2005), although some authors have failed to observe an AICD effect on NEP or other gene expression (e.g. Hébert et al. 2006; Chen and Selkoe 2007; Waldron et al. 2008). These discrepancies could be explained by recent observations that factors such as cell type, cell aging, nature of constructs used and even the particular isoform of APP (specifically APP695) affect the production of transcriptionally active AICD via the β-secretase pathway (Goodger et al. 2009; Belyaev et al. 2010; Bauer et al. 2011; Hong et al. 2011; Xu et al. 2011).

Since the first report of the transcriptional activity of the C-terminal fragment released from APP cleavage by γ-secretase (Cao and Südhof 2001), the transcriptional activity of AICD has been intensively studied (for review, see Konietzko 2011; Pardossi-Piquard and Checler 2011) and the number of genes suggested to be regulated by AICD is steadily increasing (Aydin et al. 2011). However, to date, only the NEP gene has unequivocally been shown to be functionally regulated by AICD (Pardossi-Piquard et al. 2005; Belyaev et al. 2009, 2010) and this has been shown to involve the retrograde transport of AICD formed from APP into nuclear transcription factories (Goodger et al. 2009; Konietzko et al. 2010) promoting the binding of AICD to the mediator 12 unit of the mediator RNA polymerase II complex (Xu et al. 2011). This finding provides a missing functional link between nuclear AICD binding and its transcriptional activity (Turner et al. 2011) and allowed the validation of additional AICD-dependent genes such as aquaporin-1, MICAL2 and Fibronectin-1 (Xu et al. 2011).

Following these initial observations (Pardossi-Piquard et al. 2005) further attempts were made to up-regulate NEP expression using this AICD-dependent mechanism and it was reported that the tyrosine kinase inhibitor, Gleevec (imatinib, STI-571), could elevate AICD levels and, in turn, increase NEP mRNA and protein levels and decrease Aβ secretion (Eisele et al. 2007) subsequently supported, in part, by Bauer et al. (2011), although a separate study failed to observe this (Vázquez et al. 2009). However, the work by Bauer et al. (2011) clearly demonstrated that the Gleevec-induced NEP increase was abolished by depletion of APP or its homologues, APP-like protein (APLP)1 and APLP2, which supports a direct role for AICD in this effect. It is still uncertain whether the Gleevec-induced NEP up-regulation is mediated through its tyrosine kinase inhibitory activity or by binding to the γ-secretase activating protein (GSAP), blocking its interaction with the APP C-terminal fragment (He et al. 2010).

While investigating the epigenetic mechanisms of NEP regulation by AICD it was observed that the NEP promoter is competitively regulated by AICD and the histone deacetylase histone deacetylase (HDAC)1 and that HDAC inhibitors such as trichostatin A and valproic acid (VA) could up-regulate NEP at the mRNA, protein and activity levels (Belyaev et al. 2009). VA is chiefly known as a highly successful and widely used antiepileptic drug but its efficacy in neurodegenerative disease has been much less studied (Nalivaeva et al. 2009), although there are several recent reports of its beneficial effects in reducing amyloid accumulation and improving cognitive function in AD mice (e.g. Qing et al. 2008), and amyloid clearance via microglial phagocytosis (Smith et al. 2010). VA also up-regulates NEP in the cortex and hippocampus and restores memory deficit caused by hypoxia in rats (Nalivaeva et al. 2011). The potential of VA and other HDAC inhibitors in the treatment of neurodegeneration has been further strengthened by the recent report that inhibitors of class 1 HDACs reverse contextual memory deficits in an AD mouse model (Kilgore et al. 2010), a process termed ‘epigenetic memory rescue’. Others have also supported the potential value of valproate in AD as a mechanism to ‘untie tangles’ coupled with its ability to induce neurogenesis of neural progenitor/stem cells both in vitro and in vivo via multiple signalling pathways (Nalivaeva et al. 2009; Tariot and Aisen 2009; Zhang et al. 2010). There is clearly scope for fuller clinical trials on HDAC inhibitors in AD, particularly at earlier stages in disease progression. Given the prolonged and successful use of valproate clinically in the treatment of epilepsy, there are grounds for more detailed retrospective epidemiological surveys in relation to AD incidence in valproate-users and control patients.

ECE-1 and ECE-2

ECE-1 and ECE-2 are close homologues of NEP which participate in peptide processing with ECE-1 being the enzyme primarily responsible for the biosynthesis of the potent vasoconstrictor peptide, endothelin-1 (Turner and Tanzawa 1997). ECE-1 also hydrolyses some other biogically active peptides, including bradykinin (Hoang and Turner 1997). ECE-1 is localized mainly in vascular endothelial cells (Palmer et al. 2010) but is also detectable in both neuronal and glial populations in the brain (Barnes and Turner 1997). ECE-1 mRNAs have generally been observed in neuronal populations known to express the pre-proendothelin-1 gene, confirming the primary function of this metallopeptidase in endothelin-1 generation (Facchinetti et al. 2003).

Like NEP, ECE-1 is able to degrade Aβ efficiently both in vitro and in cell-based models, and transgenic studies have suggested a role for the enzyme as a physiologically relevant ADE (Eckman et al. 2001, 2003). Evidence reviewing the role of ECE-1 in amyloid metabolism and in AD has been assessed in several reviews and will not be addressed in further detail here (Eckman and Eckman 2005; Leissring 2008; Miners et al. 2008; Nalivaeva et al. 2008). It is noteworthy that a more recent study has, however, failed to observe any difference in ECE-1 levels in AD or in vascular dementia patients compared with controls (Palmer et al. 2010). In addition to degrading Aβ, ECE-1 controls neuropeptide signalling by degrading certain neuropeptides within endosomes hence regulating receptor recycling (Roosterman et al. 2007; Cottrell et al. 2009). Four isoforms of human ECE-1 (1a, 1b, 1c and 1d), generated from a single gene via the use of alternative promoters, have been identified (Valdenaire et al. 1995, 1999; Schweizer et al. 1997). These four isoforms have identical extracellular domains, differing only in their cytoplasmic tails. Amino acid sequences within the cytoplasmic tails determine whether the isoforms are targeted to the cell surface or to intracellular compartments such as the trans-Golgi network. Hence, ECE-1 could potentially metabolise Aβ in multiple cellular compartments, although the precise isoform localization of ECE-1 in neuronal populations is still not clarified.

Relatively, little is known about the regulation of ECE-1 expression although, in endothelial cells, treatment with phorbol 12-myristate 13-acetate, which activates protein kinase C (PKC), has been shown to cause an increase in ECE-1 expression and activity at the cell surface (Smith et al. 2006; Kuruppu et al. 2010). In endothelial cells, high glucose levels, which caused an increase in total PKC activity, also caused an increase in ECE-1c isoform mRNA and protein expression (Keynan et al. 2004). Over-expression of PKCδ increased ECE-1 protein expression (Khamaisi et al. 2009) and PKCε over-expression increased enzyme activity (Choi et al. 2006). Activators of this PKC isoform reduced cellular Aβ principally through ECE-1 activation rather than α-secretase activation (Nelson et al. 2009). PKC induction using phorbol 12-myristate 13-acetate increased ECE-1a mRNA in endothelial cells via the Ets-1 transcription factor (Orzechowski et al. 2001). Hence, PKC activation both promotes α-secretase activity and production of the neuroprotective soluble APP ectodomain while simultaneously activating the ADE, ECE-1, in both cases probably via activation of the MAPK pathway. The roles of PKC in both the production and clearance of Aβ have recently been reviewed in (Kim et al. 2011).

The much less studied homologue ECE-2, originally identified by Emoto and Yanagisawa (1995) is attracting recent attention in relation to amyloid metabolism given that it is principally located in the brain, is increased in AD and is up-regulated by Aβ itself (Palmer et al. 2009). ECE-2 is localized in secretory compartments and operates optimally at acidic pH values, is most likely to be involved in protein processing events (Mzhavia et al. 2003) and increased concentrations of both Aβ1-40 and Aβ1-42 are found in the brains of ECE-2 knockout mice (Eckman et al. 2006). An unbiased microarray study of gene expression patterns in AD (Weeraratna et al. 2007) identified ECE-2 as the most significantly down-regulated gene in the AD sample, but not in other types of dementia, and no other protease gene was identified. ECE-2 levels are also elevated in AD brain (Palmer et al. 2009) and further studies on the role of ECE-2 in AD progression and its potential as a therapeutic target are merited.

NEP2

Neprilysin-2 (NEP2) was originally identified in ECE-1 knockout mice in a search for novel NEP/ECE-like genes that may participate in endothelin metabolism (Ikeda et al. 1999). Although the closest homologue to NEP itself, it displays a number of key differences particularly in cellular localization. Whereas NEP is exclusively an ectoenzyme, NEP2 exists in two alternatively spliced forms, one being a soluble secreted form, hence its original nomenclature as soluble, secreted endopeptidase (SEP) (Ikeda et al. 1999). Similar strategies developed independently led to the characterization of the same gene product from mouse testis (neprilysin-like 1) (Ghaddar et al. 2000) and from rat brain (NEP II) (Ouimet et al. 2000). Targeted disruption of the mouse neprilysin-like 1 gene produced viable mice that developed normally but which showed reduced fertility in male mice, consistent with the high expression of the gene in testis and suggesting a major role in sperm function (Carpentier et al. 2004). NEP2 expression has been detected in a number of rodent tissues although at much lower levels than in the testis. In particular, it is localized to specific neuronal populations in the CNS and spinal cord, including cortex and hippocampus where its distribution is correlated inversely with NEP localization (Ouimet et al. 2000; Facchinetti et al. 2003). Rodent NEP2 is able both to produce endothelin-1 from its precursor big endothelin-1 and to degrade the mature peptide; hence, it is unlikely to act as a physiological endothelin-converting enzyme (Ikeda et al. 1999). Originally, NEP2 was reported to have a broad repertoire of substrates, similar to those for NEP itself (Ikeda et al. 1999), although its physiological role apart from in male fertility, remains unresolved. However, most of the studies on NEP2 have been performed on the rodent enzyme, which turns out to display a much broader substrate specificity than human NEP2, consistent with the somewhat restricted sequence identity of the two enzymes (77.5% between mouse and human cDNA sequences) (Whyteside and Turner 2008). Hence, caution needs to be displayed in extrapolating physiological or pathological data from NEP2 knock-out mice to the human situation. Of a variety of neuropeptides tested, only substance P and angiotensin I were hydrolysed at comparable rates by human NEP and NEP2 (Whyteside and Turner 2008). There are also differences between NEP and NEP2 in terms of sensitivity to the two inhibitors, phosphoramidon and thiorphan, in particular thiorphan being far more effective against NEP than NEP2 (Rose et al. 2002; Whyteside and Turner 2008). A three-dimensional model of the active site of NEP2 compared with NEP has highlighted potential critical residues involved in specificity differences between these two enzyme (Voisin et al. 2004).

Alternative splicing of the NEP2 gene produces two mature products, NEP2 and a shorter form, NEP2Δ (originally termed SEP and SEPΔ by Ikeda et al. 1999). The NEP2 and NEP2Δ cDNAs differ only by a 69 bp exon immediately after the transmembrane domain which is unique to NEP2 (Raharjo et al. 2001) and which includes a potential prohormone convertase site. This difference leads to quite distinct locations for the two forms: a membrane-bound form as well as a secreted and soluble form (NEP2), and for NEP2Δ, only a membrane-bound form especially in early secretory compartments including the endoplasmic reticulum. With the mouse and human enzymes, secreted NEP2 was detected and NEP2Δ was exclusively membrane-bound but detectable both in the endoplasmic reticulum and also at the plasma membrane (Oh-hashi et al. 2008; Whyteside and Turner 2008) where it could possibly degrade synaptic peptides.

Given the role of NEP in amyloid degradation, NEP2 was soon examined for potential involvement in Aβ metabolism and hence as a potential therapeutic target. Shirotani et al. (2001) compared the ability of several NEP family members to degrade Aβ1-40 and Aβ1-42 and deduced that NEP was the most potent Aβ-degrading enzyme, although Aβ was a substrate in vitro for NEP2 (termed NEP-like peptidase [NEPLP] in that publication) as subsequently confirmed in (Huang et al. 2008). However, as NEP2 is more abundantly expressed in brain regions relevant to AD pathology (Facchinetti et al. 2003), we previously suggested that it may exert a significant role in vivo and that transgenic studies would be needed to test that hypothesis (Nalivaeva et al. 2008). This has now been achieved by Hafez et al. (2011) who have shown that NEP2 is a contributor to Aβ degradation both in vitro and in vivo using gene knockout and transgenic studies. Mice lacking NEP2 showed a 1.5-fold increase in total Aβ and a substantially greater effect in knockout mice cross-bred with APP transgenic mice. Aβ levels were elevated approx 2-fold in NEP/NEP2 double knockout mice and when these mice were treated with intracerebroventricular phosphoramidon even greater increases were seen implicating other phosphoramidon-sensitive enzymes in Aβ degradation, probably including ECE-1 but perhaps other NEP-like peptidases. Intranasal administration of phosphoramidon is also apparently sufficient to increase Aβ levels in wild-type and NEP/NEP2-deficient mice (Hanson et al. 2011). A common strategy to increase, even relatively modestly, this cohort of enzymes could bring about very significant attenuation of Aβ levels and associated cognitive deficits. Unfortunately at present, apart from NEP, very little is known about regulation of expression and activity of these phosphoramidon-sensitive enzymes.

Insulin-degrading enzyme (insulysin, IDE)

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

Insulin-degrading enzyme (IDE, insulysin) is a zinc endopeptidase but distinct from the NEP family both in subcellular localization and in catalytic mechanism, possessing an unusual tertiary structure and an inverted zinc-binding motif (HxxEH). As its name suggests, IDE was originally isolated as an enzyme implicated in insulin metabolism and therefore of potential significance in diabetes mellitus. IDE was the first ADE to be identified (Kurochkin and Goto 1994; Qiu et al. 1998) and a wide range of evidence now supports its role in this regard from studies in vitro through cell-based and animal models (e.g. Chesneau et al. 2000; Farris et al. 2003) reviewed elsewhere (Leissring 2008; Nalivaeva et al. 2008). IDE additionally degrades in vitro the amyloid peptides associated with British and Danish familial dementia (Morelli et al. 2005). In parallel, studies of IDE structure and mechanism have advanced considerably in recent years. IDE operates primarily as a dimer and exhibits unusual allosteric kinetic behaviour with small peptide substrates for which the structural basis has recently been elucidated (Song et al. 2003; Noinaj et al. 2011). Overall, the structure of IDE reveals a clamshell-like arrangement composed of four homologous domains, which enclose a large central chamber engulfing the substrate (Shen et al. 2006) and ATP binding was shown to induce the transition from the closed state to the open conformation (Im et al. 2007). Studies of IDE and peptide metabolism have been much hindered, however, by the lack of suitable potent and specific inhibitors but recent significant advances in this area generating novel compounds using a rational design approach have subsequently confirmed the role of IDE in attenuating insulin signalling and should allow evaluation of their benefit in type 2 diabetes (Leissring et al. 2010; Abdul-Hay et al. 2011). IDE has important substrates other than insulin and Aβ and, in particular, is the principal proteinase degrading cytosolic AICD (Edbauer et al. 2002). Another physiological substrate for IDE is somatostatin, a neuropeptide that declines in aging and AD, which is also able to bind to and enhance IDE activity towards Aβ through an allosteric action (Ciaccio et al. 2009). Although it is primarily located in the cytosol and therefore not appropriately placed to degrade Aβ, IDE is found in lesser amounts in mitochondria where Aβ can also be found (Leissring et al. 2004) and, surprisingly given that it lacks any known secretory signal, a small but significant proportion of IDE is secreted from cells through an unconventional protein secretion pathway (Zhao et al. 2009). This secreted IDE is the component that is functional in Aβ degradation and it appears to be routed via detergent-resistant membrane complexes into exosomes for secretion, along with Aβ (Bulloj et al. 2010).

Agonists of the PPARγ pathway have been shown to enhance Aβ clearance by a mechanism that appeared to implicate IDE, for example, its metalloprotease profile and its inhibition by insulin and glucagon. However, there were no changes in IDE transcription nor did knock-down of IDE influence the PPARγ effect perhaps implicating a novel IDE-like protease in this process (Espuny-Camacho et al. 2010). Nevertheless, further studies of PPAR involvement (both γ and δ) in amyloid clearance would appear to be worthwhile. The Notch signalling pathway may also regulate IDE expression through binding of Notch signalling proteins to the IDE promoter repressing its transcription and activity (Leal et al. 2011).

The data establishing an involvement of IDE in Aβ degradation in vivo are very strong (Farris et al. 2003; Leissring et al. 2003a) and hence, from a therapeutic point of view, up-regulation of IDE activity remains a viable prospect. This is especially important because, with age or after some insults, levels of IDE in the brain, like NEP, reduce significantly (Cook et al. 2003). The recently developed McGill-Thyl-APP transgenic mouse model of AD demonstrates significant down-regulation of IDE at the pre-clinical early stage of AD pathology which could make it a valuable model for exploring mechanisms to restore enzyme activity (Ferretti et al. 2011), such as by β2-adrenergic receptor activation (Kong et al. 2010). Using another transgenic model [APPswe/PSEN1(A246E)] it was shown that IDE can be up-regulated by Aβ1-40 and Aβ1-42 (Vepsäläinen et al. 2008) but the molecular mechanisms of such up-regulation have not been addressed.

Angiotensin converting-enzyme

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

Early indications that the ACE gene may have some relevance to AD came from demonstrations that ACE activity is increased in the AD brain, especially hippocampus and frontal cortex (Arregui et al. 1982), although the underlying mechanisms were not identified. Subsequently, two independent studies reported that the relatively common insertion/deletion (I/D) polymorphism in the ACE gene was associated with late-onset AD in a number of population studies (Hu et al. 1999; Kehoe et al. 1999), observations supported by a subsequent large meta-analysis (Lehmann et al. 2005), although some studies have failed to find any correlation (Lendon et al. 2002). The original observation of Hu et al. (1999) prompted these authors to explore the mechanism involved and, to their surprise, they observed that ACE inhibited Aβ aggregation and this effect was blocked by an ACE inhibitor (Hu et al. 2001). The explanation was that ACE was able to hydrolyse Aβ1-40 at the Asp7-Ser8 bond, although this was a cleavage inconsistent with the preferred carboxydipeptidase specificity of ACE. When each of the two catalytic domains was expressed separately, this activity was apparently contributed primarily by the N-terminal domain of ACE (Oba et al. 2005) but Hemming and Selkoe (2005) showed that, in the intact two-domain enzyme, both active sites could contribute to Aβ hydrolysis. The ability of both active sites to act on Aβ has also been observed by Sun et al. (2008b) although they failed to detect cleavage at the Asp7–Ser8 bond and identified a number of other endoproteolytic sites of cleavage (Table 1). Further confusion regarding the specificity of ACE has come from a study by Zou et al. (2007) who have shown that ACE can convert Aβ1-42 to Aβ1-40, which is entirely consistent with the primary specificity of ACE as a carboxydipeptidase and would remove the more toxic Aβ1-42 species. This study also showed that treatment of Tg2576 mice with an ACE inhibitor increases Aβ1-42 deposition in the brain. The Aβ1-42 to Aβ1-40 converting activity of ACE was again attributed to the N-domain (Zou et al. 2009).

The potential involvement of ACE in amyloid clearance has raised the concern that widely used brain penetrant ACE inhibitors (e.g. captopril, perindopril) could elevate Aβ levels adversely, much as similar concerns have been highlighted regarding the use of NEP (vasopeptidase) inhibitors for cardiovascular disorders (Hemming and Selkoe 2005; Eckman et al. 2006). These concerns are heightened by the development of ‘triple inhibitors’ of ACE, ECE and NEP as a novel class of antihypertensive drug (Battistini et al. 2005), which could potentially exacerbate amyloid accumulation even further. However, there is some epidemiological evidence indicating that brain-penetrant ACE inhibitors pose no risk for the development of AD, or may even slow the risk of cognitive decline (Ohrui et al. 2004; Hajjar et al. 2008). The beneficial effects on brain cognition are potentially because of direct effects on the brain renin-angiotensin system and vascular homeostasis; brain ACE inhibition (and hence reduction of brain angiotensin II levels) exerts a beneficial effect on cognition because of increased cholinergic function (Ohrui et al. 2004), because Ang II inhibits acetylcholine release. Studies on the effects of ACE inhibitor treatment on Aβ levels in mouse models of AD have also failed to show any increased Aβ accumulation in vivo (Eckman et al. 2006; Hemming et al. 2007b; Ferrington et al. 2011), whereas Aβ levels were significantly increased after treatment with NEP or ECE inhibitors, or in mice deficient in either of these enzymes (Eckman et al. 2006). Combined reductions in ECE and NEP activities led to additive increases in Aβ levels consistent with their distinct regional and subcellular localizations in the brain. Rationalising all these contrary observations on the contribution of ACE to Aβ clearance is problematic but the efficiency of hydrolysis of Aβ by ACE is much inferior to NEP, IDE and ECE and hence correlations between ACE levels and AD may relate more to effects on the renin-angiotensin system and brain vascular changes than to a major contribution to Aβ clearance. Higher activity of peripheral blood ACE also appears to be associated with later onset of AD (Akatsu et al. 2011) consistent with another study which has shown a lower level of plasma ACE in AD patients compared with controls (Vardy et al. 2009). Thus, current data provide conflicting information on whether activation or inhibition of ACE could be beneficial in AD.

Matrix metalloproteinases

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

Any neuronal (or possibly glial) ectopeptidase (i.e. a cell surface peptidase with the active site located in the ectodomain) could potentially be involved in Aβ degradation. A major class of such enzymes are the metzincin proteinases, which include the MMPs and the adamalysin (A Disintegrin And Metalloproteinase; ADAM) families (for reviews see Rivera et al. 2010; van Goor et al. 2009). To date, no ADAM family member has been identified as a potential Aβ-degrading enzyme. Rather, they function as α-secretases in the non-amyloidogenic pathway of APP processing, particularly ADAM10 in primary neurons (Kuhn et al. 2010), and in the processing of APLP2 (Hogl et al. 2011). Most of the 24 human MMPs are either secreted or exist as integral membrane proteins and, of these, MMP-2, -3, -6 and -9 have been reported to degrade Aβ1-40, although most attention has focused on MMP-2 (gelatinase A) (Roher et al. 1994) and MMP-9 (gelatinase B) (Backstrom et al. 1996), which can also degrade the fibrillar form of Aβ, as well as compact plaques (Yan et al. 2006). Aβ itself appears to up-regulate expression of several MMPs (Roher et al. 1994; Deb and Gottschall 1996), and indeed of NEP (Mohajeri et al. 2002), implying a corrective feedback mechanism which could be exploited therapeutically. For example, the levels of Aβ in the medium of neuroblastoma cells over-expressing APP can be reduced by addition of the metal ligand, clioquinol, together with Cu2+, which act to up-regulate MMP-2 and MMP-3 (White et al. 2006). Transgenic mice lacking either MMP-2 or MMP-9 have elevated levels of brain Aβ and inhibition of these enzymes causes increases in brain Aβ levels (Yin et al. 2006). The tissue inhibitors of metalloproteinases (TIMPs) provide natural endogenous inhibitors of MMPs in the brain and they have been reported to be elevated in neurodegenerative conditions (Lorenzl et al. 2003), which could be a cause of decreased Aβ clearance. However, relatively little is known of the regulation of TIMP expression in the CNS, although mice lacking TIMP-1 exhibit impaired learning (Chaillan et al. 2006). This is an area that merits more detailed exploration.

Serine proteases: plasmin and acyl peptide hydrolase

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

The thrombolytic serine protease, plasmin, plays important roles in the brain including in long-tern potentiation and cognition (Nakagami et al. 2000). Links of the plasmin proteolytic cascade with AD came from studies revealing reductions in plasmin activity in the serum of AD patients and in AD brain (Aoyagi et al. 1992; Ledesma et al. 2000). These observations correlated with an ability of plasmin to cleave Aβ, both monomer and fibrils (Van Nostrand and Porter 1999; Ledesma et al. 2000; Tucker et al. 2000) which, in turn, induce the plasmin system (Tucker et al. 2000; Lee et al. 2007). Pharmacological inhibition of a key regulatory component of the plasmin activation cascade (plasminogen activator inhibitor-1, PAI-1) has been shown to increase the activity of hippocampal tissue plasminogen activator and plasmin and to lower significantly both plasma and brain Aβ. Cognitive deficits in a transgenic AD mouse model were reversed on administration of the small molecule inhibitor of PAI-1 (designated PAZ-417) (Jacobsen et al. 2008). Further validation of the plasmin cascade as a potential therapeutic target in AD has come from more recent studies showing that knockout of PAI-1 in APP/PS1 mice reduces Aβ burden (Liu et al. 2011). The main endogenous inhibitor of tissue plasminogen activator in brain is the protein, neuroserpin, which is synthesized by neurons, and this protein is up-regulated in AD brain resulting in reduced plasmin activity (Fabbro and Seeds 2009). Knockout of neuroserpin in human APP-J20 mice causes rapid clearance of Aβ1-42 and retention of cognitive abilities lost in the transgenic J20 animals alone (Fabbro et al. 2011). Whereas the various studies considered above have apparently strongly implicated the plasmin system in Aβ clearance, one recent study has failed to corroborate significant changes in plasminogen mRNA or protein levels, and of plasmin activity, in AD (Barker et al. 2010). Additionally, Lu et al. (2009) failed to find an association between PAI-1 promoter polymorphisms and sporadic AD. The jury remains out, however, on the potential of chronic PAI antagonism in disease therapy as succinctly summarised by Vaughan (2010).

Acyl peptide hydrolase (APH) is a member of the prolyl oligopeptidase family whose primary role, being predominantly a cytosolic enzyme, is thought to be in the catabolism of intracellular N-terminally acetylated proteins by removal of the N-acyl amino acid (Perrier et al. 2005) and/or in removal of oxidized proteins (Shimizu et al. 2004). However, like IDE, APH is secreted in small amounts from cells by an unknown mechanism and has been isolated from the conditioned medium of neuroblastoma cells where it can degrade Aβ (Yamin et al. 2007). Hydrolysis of the peptide occurs after amino acids 13, 14 and 19 and the enzyme can degrade monomeric and oligomeric Aβ (Yamin et al. 2009). APH was also found to be present at significantly lower levels in AD brain than in age-matched controls. Substantive in vivo data are, however, currently lacking to substantiate any physiological role for APH in amyloid clearance.

Cathepsin B

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

A somewhat confusing picture has emerged of the potential involvement of the lysosomal cysteine protease, cathepsin B, in APP and Aβ metabolism. Cathepsin B has been proposed as an alternative β-secretase, particularly acting on wild-type rather than mutant APP and cathepsin B inhibitors have been shown to improve memory deficits and reduce Aβ levels in some AD mouse models (Hook et al. 2010), apparently consistent with this hypothesis. However, other studies have reported an anti-amyloidogenic and neuroprotective action of cathepsin B attributable to the ability of cathepsin B to reduce Aβ levels, particularly of Aβ1-42 (Mueller-Steiner et al. 2006) and this has been experimentally confirmed in cathepsin B-null mice also over-expressing human APP with familial AD-linked mutations where increased plaque load, levels of Aβ1-42, and neuronal deficits were seen (Mueller-Steiner et al. 2006). Recently, cathepsin B was shown to degrade APP C-terminal fragments (CTFs and AICD) which might suggest a role in endosomal-lysosomal control of APP metabolism (Asai et al. 2011). The action of cathepsin B on Aβ1-42 resembles that of ACE in acting as a carboxydipeptidase first forming Aβ1-40, and then Aβ1-38, hence reducing toxicity (Mueller-Steiner et al. 2006). Aβ1-33 is also formed through cathepsin B endopeptidase activity. Cathepsin B also has catalytic activity against preformed Aβ oligomers and fibrils, and is able to act both intracellularly in endosomal-lysosomal populations as well as extracellularly because it is actively secreted, especially from microglia. Thus, it has been argued that enhancing cathepsin B activity in the brain could be a useful therapeutic strategy. One way suggested to enhance cathepsin B activity is to reduce levels of its endogenous inhibitor, cystatin C (Turk et al. 1995). Knockout of the cystatin 3 gene (CST3) in hAPP-J20 transgenic mice significantly lowered soluble Aβ levels, the relative abundance of Aβ1-42, and plaque load. Cystatin C (CysC) removal also attenuated Aβ-associated cognitive deficits and behavioural abnormalities and restored synaptic plasticity in the hippocampus. These beneficial effects of CysC reduction were not seen on a cathepsin B null background, apparently supporting an effect via inhibition of cathepsin B and Aβ degradation (Sun et al. 2008a). Conversely and perversely, over-expression of human CysC in hAPPJ20 mice reduces plaque load through a different mechanism: by its binding to Aβ extracellularly and thereby inhibiting Aβ fibril formation (Kaeser et al. 2007; Mi et al. 2007). Genetic linkage studies have shown an association between a cystatin C polymorphism, which diminishes its secretion, and increased risk of late-onset AD, being the first autosomal recessive risk allele in late-onset AD (Crawford et al. 2000), observations subsequently supported by systematic meta-analysis (Bertram et al. 2007). The studies on cathepsin B, cystatin C and AD emphasize the complexities of unravelling the significance of individual proteases and their endogenous inhibitors to Aβ metabolism and its regulation, and in identifying the critical enzymes to target while minimising side-effects caused by the promiscuity of most proteases.

Other potential ADEs

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

Several unexpected candidates as ADEs have recently appeared, perhaps the most surprising being myelin basic protein (Liao et al. 2009), which is claimed to possess endogenous serine protease activity leading to autocatalytic cleavage mediated by an active site serine residue within the protein. Myelin basic protein had previously been shown to bind Aβ peptide avidly preventing its assembly into amyloid fibrils (Hoos et al. 2009).

The zinc ectopeptidase, aminopeptidase A (AP-A). which has a specificity for removal of N-terminal acidic residues, is known to play a key role in the central regulation of blood pressure through its conversion of angiotensin II to angiotensin III (Marc and Llorens-Cortes 2011) and AP-A selective inhibitors hence have potential as novel anti-hypertensive agents (Bodineau et al. 2008). N-terminally truncated and modified versions of Aβ are found in brain and may lie on the pathway to neurotoxicity, with Aβ2-42, for example, being increased in both sporadic and familial forms of AD (Wiltfang et al. 2001). Given that the N-terminus of full length Aβ is an aspartyl residue, Checler and colleagues speculated that it may be susceptible to the action of AP-A and showed that this was indeed the case in both cell-free and cellular models (Sevalle et al. 2009). AP-A did not degrade the peptide further, consistent with the subsequent residue being alanine whose removal may be effected by an aminopeptidase N-like activity revealing a free N-terminal glutamyl residue that can subsequently be cyclised by glutaminyl cyclase (Schilling et al. 2008). Inhibitors of AP-A therefore have potential neuroprotective activity, inhibiting the initial step in the pathway to cyclisation and subsequent toxicity, as shown in cell-based systems (Sevalle et al. 2009).

Glutamate carboxypeptidase II (GCP II) is a widely expressed and multifunctional zinc ectopeptidase which, in the brain, cleaves the abundant neurotransmitter dipeptide N-acetyaspartylglutamate and has been implicated in a number of neurological conditions and inhibition of the enzyme is neuroprotective (Neale et al. (2011). In the human brain the enzyme is primarily astrocyte-localized (Šácha et al. 2007). Like NEP, in addition to acting as a neuropeptidase, GCP II also plays an important role in prostate function being an important prostate cancer marker protein. Additionally, it hydrolyzes folylpoly-γ-glutamate in the intestine, facilitating the absorption of dietary folate. GCP II is now reported also to cleave Aβ (both 1-40 and 1-42) sequentially from the C-terminus producing Aβ1-14, Aβ1-18 and Aβ1-35 as major products which are no longer toxic. A specific GCPII inhibitor partially, but not completely, inhibited these cleavages. The relevance of GCP II to amyloid metabolism in vivo was demonstrated in a transgenic AD mouse model when treatment of 8-month old mice for 1 month with a specific GCP II inhibitor increased brain Aβ levels. Over-expression of GCP II in neuronal or glial cells also reduced the level of secreted Aβ and its toxicity. Hence, potentially GCP II could be considered another in vivo functional Aβ-degrading enzyme. In particular, it could degrade not only monomeric Aβ but also soluble oligomers and fibrils and reduced plaque size in brain sections in a plaque degradation assay (Kim et al. 2010). Independent validation of GCP II as a potential ADE is now required.

Intracellular Aβ may be an important component of toxicity and the occurrence of the peptide in mitochondria has suggested that it may contribute to the known mitochondrial dysfunction in AD because it accumulates in mitochondria of AD patients (Lustbader et al. 2004). Within mitochondria Aβ is bound to two proteins, the amyloid-binding alcohol dehydrogenase and the peptidylprolylisomerase, cyclophilin D, both mitochondrial matrix proteins and the binding of Aβ to these proteins can cause increased mitochondrial oxidative stress (for review, see Muirhead et al. 2010). Within mitochondria the levels of Aβ are controlled by the thiol-sensitive pre-sequence protease or peptidasome, PreP, which is a pitrilysin family member with an inverted zinc motif like IDE (Falkevall et al. 2006; Glaser and Alikhani 2010), and possibly by the mitochondrial form of IDE. The normal role of PreP is in degrading mitochondrial targeting peptide sequences. Unlike IDE, insulin is not a substrate for PreP. The structure of PreP reveals that, like IDE and NEP, it contains a catalytic chamber or ‘crypt’ that engulfs the Aβ peptide substrate for catalysis and this group of zinc peptidases have therefore been referred to as cryptidases (Malito et al. 2008). To date, no genetic association between any of the identified single nucleotide polymorphisms (SNPs) in PreP and the risk for AD has been found and its validity as a significant therapeutic target is therefore debatable at present (Pinho et al. 2010), although decreased proteolytic activity of PreP has been detected in brain mitochondria from AD patients compared to age-matched controls (Alikhani et al. 2011).

Genetic links of ADEs with AD

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

Taking into account the importance of ADEs in Aβ-clearing mechanisms and AD pathology, several genetic association studies have been attempted to find a link between polymorphisms of ADE genes and the progression of AD. With regard to the NEP gene the data reported by various groups are rather contradictory. Some studies have reported a link between the polymorphism in a GT-repeat in the NEP promoter region (Sakai et al. 2004) and SNPs in the NEP gene (Helisalmi et al. 2004) with a susceptibility to late-onset AD in a Japanese and Finnish cohort of patients. However, the most recent study in three independent northern European case–control series failed to confirm any detectable impact of SNP polymorphism in the NEP gene on frequency of AD (Blomqvist et al. 2010). Similarly, two studies in Chinese and Italian populations reported a lack of the suggested earlier protective effect of the C-338A polymorphism within the ECE1 promoter region on development of late onset AD (Scacchi et al. 2008; Jin et al. 2009). On the contrary, a recent study on IDE polymorphisms identified three SNPs that associated significantly with expression levels that provide strong evidence that IDE is a late-onset AD-related gene (Zou et al. 2010).

Several SNPs across the ACE gene also appear to contribute to AD pathology, the most significant being an SNP in the ACE promoter (Kehoe et al. 2003), with several affecting the age of onset (Kehoe et al. 2004). As mentioned above, this was supported later by a meta-analysis performed by two independent groups (Lehmann et al. 2005; Bertram et al. 2007). A further study has correlated different cognitive phenotypes in AD with genetic variants in ACE and IDE (Vardy et al. 2011). A modest protective effect of SNPs in the MMP-3 gene was shown among non-bearers of the apolipoprotein E ε4 allele (Helbecque et al. 2007). Further analysis has revealed some variation in the MMP-3 gene strongly associated with changes in Aβ in humans supporting a potential influence of this MMP on accumulation of Aβ in the brain (Reitz et al. 2010).

Future perspectives

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References

What is perhaps surprising is the sheer number of ADEs that have been identified existing in multiple cell types and brain regions acting co-ordinately to regulate Aβ levels, whether monomeric, oligomeric or fibrillar, and both intra- and extra-cellular (summarised in Fig. 2). However, an ability to degrade one or more Aβ species in vitro is a long way from identifying a physiologically significant ADE. Among a number of other factors, co-location of peptide substrate and degrading enzyme is critical. Which of these enzymes are the more critical to target, the easiest to up-regulate and with least potential off-target effects are key questions in the field, alongside the reasons for the failure of these mechanisms to cope in aging and in both sporadic and familial AD. Based on current evidence, up-regulation of ADEs in brain still appears to represent a viable strategy to pursue. Animal studies have demonstrated that amyloid burden can be partly cleared and cognitive function improved in transgenic mice by up-regulating NEP, ECE-1, IDE, plasmin, and other ADEs individually but these animal models in themselves have limitations to representing the human disease.

image

Figure 2.  Schematic presentation of main cellular and subcellular localisations of the key amyloid-degrading enzymes described in the text. Enzyme are indicated as follows: NEP, red symbols; ACE, green; IDE, yellow; ECE, E; cathepsin B, catB; matrix metalloproteinases, MMPs; Aβ forms (monomers, oligomers, fibrils) indicated by black wavy lines.

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As the various ADEs have different cellular and subcellular locations in the CNS, modest up-regulation of several of these co-ordinately may be a more effective strategy than individual modulation, given the diverse locations of Aβ in its various guises. However, each group in the field has generally tended to focus on its own ‘pet’ ADE rather than consider a more holistic approach, ourselves included! The links between ADEs and other pathological events that cause cognitive deficits and AD, and the alternative targets recently being considered in the field, remain to be evaluated (Seabrook et al. 2007). Taking into account that AD pathology develops over many years, early manipulation of ADEs might be essential to prevent amyloid deposition and disease progression much as is the case in the timing of other clearance mechanisms, e.g. amyloid immunisation. However, the efficacy of any preventive treatment will be strongly driven by the development of early diagnosis and pre-clinical markers of AD. Pharmacological approaches to enzyme up-regulation (allosteric modulators, activators, epigenetic regulators, etc.) might provide more economic approaches than gene delivery strategies but targeted delivery may be important given the alternative substrates for some of these enzymes. In any case, it is important to bear in mind that only a relatively small but prolonged increase in the activity of any ADEs could result in a positive shift of amyloid balance towards its clearance. A single focus for treatment of AD is unlikely to emerge so a combination therapy including targeting other links in the chain of pathological events leading to AD is likely to be optimal. But all depends on the primacy of the amyloid cascade hypothesis. Twenty years of research may not have produced the long-awaited anti-amyloid ‘magic bullet’ but the search continues.

References

  1. Top of page
  2. Abstract
  3. Introduction: the dynamic nature of amyloid
  4. Amyloid-degrading enzymes: the key players and strategies for manipulation
  5. Neprilysin and related peptidases (NEP, ECE-1, ECE-2, NEP2)
  6. Insulin-degrading enzyme (insulysin, IDE)
  7. Angiotensin converting-enzyme
  8. Matrix metalloproteinases
  9. Serine proteases: plasmin and acyl peptide hydrolase
  10. Cathepsin B
  11. Other potential ADEs
  12. Genetic links of ADEs with AD
  13. Future perspectives
  14. Acknowledgements
  15. Conflict of interests
  16. References
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