Amyloid beta…first the making…then the breaking…

Although the pathway of Aß production has been extensively studied since the discovery of Aß, only recently the catabolism of Aß has started to gain attention. Understanding the mechanisms behind Aß degradation and the enzymes involved, could be an important therapeutic tool in the future for eliminating Aß levels in brains of AD patients.

A few enzymes have been reported with the capability to break down Aß, the first to be identified was the “insulin degrading enzyme” [IDE]. A year later neprilysin [NEP] was reported to metabolize Aß in vitro, and later on in vivo.

Other members of the NEP family such as endothelin-converting enzyme [ECE] were examined for their ability to degrade Aß and proved their efficacy.

The biological features of proteases known to cleave Aß are summarized in opiod peptides, atrial natriuretic peptides, bombesin-like peptides, chemotatic peptides, adrenocorticotropin

Cytosol, cellular, and intracellular membrane extracellular space

Insulin, glucagon, atrial natriuretic factor, β-endorphin amylin, APP intracellular domain

TGFα

Endothelin-converting enzyme

EC 3.4.24.71

ECE M Trans-Golgi network

Cell surface

Big endothelin, substance P, bradykinin, oxidized insulin B chain

Biological features of Aβ degrading enzymes

Summary of the major enzymes known to cleave Aβ peptide (Wang et al. 2006)

1.1.7.1 Neprilysin [NEP]

NEP gene is located on chromosome 3q21-q27, and is composed of 750 amino acids with an approximate molecular weight of 86 kDa (Malfroy et al.

1988). It consists of a short N-terminal cytoplasmic tail, a membrane spanning domain, and a large c-terminal extracellular catalytic domain. NEP is expressed in a variety of tissues including the brain. In the brain NEP is mainly expressed in areas susceptible to Aß deposition such as the hippocampus.

In 1995, NEP was linked to Aß degradation by Howell et al (Howell et al.

1995). Further investigations proved the in vivo capability of Aß cleavage by

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degraded by NEP in the hippocampus of rats, this process was blocked by NEP inhibitor leading to accumulation of Aß and plaque formation (Iwata et al. 2000). Subsequently, the same authors reported that the levels of Aß 40 and Aß 42 were elevated in NEP knockout mice (Iwata et al. 2001).

Others were able to demonstrate the ability of NEP to degrade not only monomeric form of Aß but also the oligomeric form (Kanemitsu et al. 2003).

Quantitative analysis showed that NEP mRNA was significantly lower in AD (Caccamo et al. 2005;Yasojima et al. 2001), and an inverse relationship was observed with both Aß plaques and Aß levels (Wang et al. 2006).

From the aforementioned evidence NEP appears to contribute to the normal metabolism and accumulation of Aß in AD.

1.1.7.2 Insulin degrading enzyme IDE

IDE gene was mapped to chromosome 10q23-q25, consisting of 1019 amino acids. The Aß degrading property of IDE was first described in 1994 by Kurochekin et al (Kurochkin and Goto 1994). IDE is expressed in several tissues, such as liver, skeletal muscles and brain, and is primarily located in the cytosol (Wang et al. 2006). Anatomical data was first provided in suggesting that IDE is associated with neuropathlogical hallmarks of AD (Bernstein et al. 1999). Immunostaining revealed the presence of IDE in cortical and sub cortical neurons, senile plaques and microvessels (Morelli et al. 2004). Reduced mRNA levels and activity of IDE in the hippocampus of cases at high risk of developing AD and in APOE4 carriers (Zhao et al.

2007;Cook et al. 2003) supported the hypothesis that IDE activity may contribute to Aß accumulation in AD patients.

Additionally, animal studies suggest a role of IDE in Aß degradation.

Transgenic mice over expressing IDE demonstrated reduced levels of Aß accumulation and prevented amyloid plaque formation (Leissring et al. 2003).

On the other hand IDE knockout mice demonstrated increased Aß load and

the AICD (Miller et al. 2003;Farris et al. 2003). Another study also reported the ability of IDE to degrade AICD, suggesting that IDE is not specific to insulin and Aß only (Edbauer et al. 2002).

Although the above evidence suggests the involvement of IDE in Aß metabolism, however the proof for a genetic association remains controversial. As an example, one of the most recent studies concluded that there is no association of IDE haplotypes with the risk of developing dementia (Marlowe et al. 2006). Nevertheless, one can’t overlook the strong in vivo data present linking IDE activity and Aß, and from a therapeutic point of view up-regulation or increasing activity of IDE remains a viable prospect.

1.1.7.3 Endothelin converting enzyme [ECE]

ECE is a transmembrane metalloprotease that catalyzes the conversion of the inactive precursor pro-endothelin to its potent vasoactive peptide endothelin.

The most abundant form, ECE-1 is encoded by the gene located on the chromosome 1p36, and consists of 758 amino acids. In addition to pro-endothelin, ECE has been reported to hydrolyze other peptides such as bradykinin, substance P and neurotensin in vitro [see table 1.1]. ECE-1 is a member of the NEP family, and has shown 37 % homology to NEP (Sansom et al. 1998). Given this information and the fact that ECE-1 is non-specific in its substrate repertoire as mentioned above, it is not surprising that ECE-1 was examined as a potential Aß-degrading enzyme. The ability of ECE-1 to cleave Aß was first noticed by Eckmann et al, when they observed that phosphoramidon caused an increase of Aß accumulation in a cell line that expressed ECE, but not in another cell model devoid of ECE (Eckman et al.

2001). Recombinant soluble ECE-1 was demonstrated to hydrolyze Aß 40 and Aß 42 in vitro at multiple cleavage sites [see diagram 1-11]. Since ECE-1 devoid mice do not survive, heterozygous mice showing a 27 % decrease in ECE-1 activity were investigated for their Aß levels and they exhibited higher

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ECE-2 which is relatively less studied isoform, has recently gained attention as an Aß degrading enzyme. ECE-2 is mainly localized in the brain; however its overall expression is only 1-2 % as much as the more abundant form ECE-1 (Wang et al. 2006). ECE-2 knockout mice develop increased amounts of Aß 40 and Aß 42 (Eckman et al. 2006). Interestingly, a recent microarray study of gene expression patterns demonstrated that ECE-2 was down-regulated in AD patients (Weeraratna et al. 2007). Therefore, it seems that ECEs play a role in Aß metabolism, ECE-2 role is less apparent, but this could be due to the fact that it was not extensively studied like ECE-1, owing to its scarcity.

Figure 1-11

Aß cleavage sites by NEP, ECE-1 and IDE