**3. Amyloid Precursor Protein (APP)**

### **3.1. Overview of APP**

APP was identified as a cDNA cloned using a partial amino acid sequence of the Aβ frag‐ ment isolated from the amyloid plaque of AD brains [53]. This cDNA coded for an evolutio‐ narily conserved type 1 transmembrane protein. Fig. 2 shows schematic diagram of APP protein. Although APP is expressed in many tissues, this protein especially accumulates in the synapses of neurons. The human *APP* gene is about 240 kb in length containing at least 18 exons [54] and is localized on the long arm of chromosome 21 [53], an extra copy of which is present in patients with Down's syndrome (trisomy 21). Several alternative splicing iso‐ forms of APP have been found, which differ mainly in the absence (APP-695, predominantly expressed in neurons) or presence (APP-751 and APP-770) of the Kunitz protease inhibitor (KPI) domain toward the N-terminus of the protein [55].

*Drosophila* has two different Notch ligands, Delta [34] and Serrate [35]. In mammals, two families of Notch ligands, Delta-like protein family (Dll1, 3, and 4) [36-38] and Jagged family (Jagged 1 and 2) [39, 40], have been identified. The extracellular domains of all these ligands also contain variable number of EGF-like repeats, e.g., *Drosophila* Delta has nine, while most vertebrate Deltas have eight, and *Caenorhabditis elegans* Lag-2 has two repeats. All these li‐ gands share a single copy of a 2nd cysteine-rich conservative motif called the DSL (**D**elta:

As shown in Fig.1, in the canonical Notch signaling pathway, ligands bind to the extracellu‐ lar domain of Notch proteins on neighboring cells and trigger sequential proteolytic cleav‐ age reactions; this is called the regulated intramembrane proteolysis (RIP) mechanism [43]. Precise steps of Notch processing are mentioned in section 4.2 of this chapter. In brief, first, Notch is cleaved within the juxtamembrane (JM) domain by metalloproteases to remove most of the extracellular region [44, 45]. Next, the remaining protein stub is further cleaved by γ-secretase within the transmembrane (TM) domain and NICD is released from the membrane [46-48]. The released NICD translocates to the nucleus and controls the expres‐ sion of certain genes through binding to transcription factors. Thus, γ-secretase plays a cen‐

Members of the CSL transcription factor family (CBF1/RBP-jκ in mammals, Su(H) in *Droso‐ phila*, and Lag-1 in *C. elegans*) are major downstream targets of Notch signaling [19]. NICD binds to CSL transcription factors, and six tandem ankyrin-like repeats in NICD are essen‐ tial for binding to CSL transcription factors [49]. NICD also binds to Mastermind-like pro‐ teins (MAML family in mammals) [50], thus forming the CSL-NICD-MAML complex. The formation of these complexes results in removal of co-repressors from CSL and recruitment of co-activators, such as P/CAF and P300 [50, 51]. Through this process, the CSL complex is converted from a transcriptional repressor to an activator. Finally, these complexes bind to the *cis*-acting DNA sequences of target genes, such as Hes (Hairy/Enhancer of split in *Droso‐ phila*), which encode the basic helix-loop-helix (bHLH) transcription factors, and promote

APP was identified as a cDNA cloned using a partial amino acid sequence of the Aβ frag‐ ment isolated from the amyloid plaque of AD brains [53]. This cDNA coded for an evolutio‐ narily conserved type 1 transmembrane protein. Fig. 2 shows schematic diagram of APP protein. Although APP is expressed in many tissues, this protein especially accumulates in the synapses of neurons. The human *APP* gene is about 240 kb in length containing at least 18 exons [54] and is localized on the long arm of chromosome 21 [53], an extra copy of which is present in patients with Down's syndrome (trisomy 21). Several alternative splicing iso‐ forms of APP have been found, which differ mainly in the absence (APP-695, predominantly

**S**errate: **L**ag-2) domain [41], which is essential for binding to Notch [42].

tral regulatory role in Notch signaling.

**3. Amyloid Precursor Protein (APP)**

their transcription [52].

64 Understanding Alzheimer's Disease

**3.1. Overview of APP**

As described below, APP undergoes sequential proteolytic cleavage reactions to generate the extracellular fragment, intracellular fragment (AICD), and Aβ fragment that is located in the membrane-spanning region. Note that both the extracellular fragment and AICD are generated at the same time as Aβ. Extensive post-translational modifications of APP, such as glycosylation, phosphorylation, and tyrosine sulfation, have been observed.

Mammals have two other members of APP family called APP-like protein 1 (APLP1) and 2 (APLP2) [56]. APLP1 expression is restricted to neurons. On the other hand, expression of APLP2 is detected in many tissues, although it is highly enriched in the brain. These APP family proteins share conserved domains, such as the E1 and E2, in the extracellular region. The E1 domain contains several subdomains, such as a growth factor-like domain and a metal-binding motif [57]. The E2 domain has a coiled coil dimerization motif and may bind proteoglycans in the extracellular matrix [58].

Interestingly, the amino acid sequence of the Aβ fragment is not highly conserved and is unique to APP; on the other hand, the highest degree of sequence conservation is found in the ICD not only within the APP homologues [29] but also within the APP family [9]. This strong sequence conservation most likely reflects functional importance of the ICDs in the APP family proteins.

**Figure 2.** Schematic domain structure of APP. APP protein family shares the conserved E1 and E2 domains in their extracellular region. The E1 domain contains N-terminal growth factor-like domain (GFLD) and copper-binding do‐ main (CuBD). The E1 domain is linked via acidic domain to the carbohydrate domain including E2 domain, which con‐ sists of RERMS sequence and central APP domain (CAPPD). E2 domain is followed by the Aβ region, and the intracellular domain (AICD) which is the most conserved region. Although the Kunitz protease inhibitor (KPI) domain is present at the indicated site in APP-751 and APP-770, APP-695 lacks this domain.

#### **3.2. Proposed APP functions**

Although the physiological functions of APP are not clear, several possibilities have been proposed. The most considerable functions are synapse formation and repair [59, 60]. In‐ deed, APP expression is upregulated after neural injury as well as during neuronal dif‐ ferentiation [59, 60]. After translation in the soma, APP is transported in an anterograde manner to the synaptic region, where the amount of APP is correlated with synaptogene‐ sis. APP knockout mice show impaired long-term potentiation and declined memory without remarkable neuronal loss [61]. This evidence also supports this idea.

degrading enzyme (IDE) are expressed in neurons as well as within the vasculature and the levels of both these enzymes are reduced in AD [71]; therefore, these enzymes have been well studied in relation to AD. Interestingly, it has been reported that *APOE* e4, which is the most-established genetic risk factor for the onset of AD and CAA, is associ‐ ated with reduced levels of both enzymes [72, 73]. Furthermore, other candidates for Aβ degradation enzymes have been proposed, including endothelin-converting enzymes 1 and 2 (ECE-1 and ECE-2) [74] and angiotensin-converting enzyme (ACE) [75]. The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 have also been shown

γ-Secretase–Regulated Signaling and Alzheimer's Disease

http://dx.doi.org/10.5772/54230

67

γ-Secretase was first identified as a protease that cleaves APP within the TM domain and produces Aβ peptides [10], which is thought to be a major cause of the pathogene‐

γ-Secretase is a complicated complex composed of PS, nicastrin (NCT), anterior pharynx defective-1 (Aph-1), and PS enhancer-2 protein (Pen-2) [8, 11, 12]. Two PS genes, *PS1* lo‐ cated on chromosome 14 [5] and *PS2* located on chromosome 1 [6, 7], have been identi‐ fied by genetic linkage analyses as the genes responsible for early-onset FAD. The *PS1* and *PS2* genes encode proteins with eight or nine transmembrane domains of 467 and 448 amino acids, respectively, with about 65% sequence identity between the two pro‐ teins. Both proteins are the catalytic components of the γ-secretase complex. Although both PS1 and PS2 are expressed ubiquitously in the brain and peripheral tissues of adult mammals, PS1 expression level is significantly higher than that of PS2 [76]. NCT is a sin‐ gle-pass membrane protein and may recognize the substrate proteins of γ-secretase [77]. Indeed, the extracellular domain of NCT resembles an aminopeptidase, but lacks catalyt‐ ic residues. Thus, this domain can interact with the free N-terminal of stubs of γ-secre‐ tase substrates generated by ectodomain shedding [78]; hence, shedding of γ-secretase substrates may be essential for the production of free N-termini of these proteins re‐ tained in the membrane to be recognized by NCT. Aph-1 may act as a scaffold during the process of γ-secretase complex assembly, and Pen-2 may act as a trigger for the pro‐

The physiological functions of γ-secretase have not been clarified. However, this protease can cleave a surprisingly large number of transmembrane proteins [79]. Indeed, more than five dozen proteins, most of which are type 1 membrane proteins, have been reported as γsecretase substrates [21]. Interestingly, these substrates have a wide range of biological func‐

to be reduced in the AD brain [71].

teolytic cleavage of PS in order to activate it [11, 12].

tions. Representative γ-secretase substrates are shown in Table 1.

**4. γ-Secretase**

sis in the AD brain.

**4.1. Overview of γ-secretase**

It has also been suggested that APP acts as a cell adhesion molecule and plays a role in cell– cell interaction. Indeed, the E1 and E2 domains can interact with extracellular matrix pro‐ teins and heparan sulfate proteoglycans [57, 58]. In addition, it has also been shown that ex‐ tracellular domains of APP family proteins can interact with each other *in trans*. Therefore, APP family proteins may bind to each other in a homophilic or heterophilic manner to en‐ hance cell–cell adhesion [62].

As APP may have a signaling mechanism, as described in detail below, the idea that APP is a cell-surface receptor is interesting. Indeed, several candidates of ligand for APP have been proposed. For example, F-spondin [63] and Nogo-66 [64] receptor bind to the extracellular domain of APP and regulate the production of Aβ. In addition, Aβ itself can also bind to the extracellular domain of APP [65].

#### **3.3. Aβ amyloid**

Aβ is the main constituent of an amyloid plaque, which is thought to be the hallmark and a major cause of AD pathogenesis in the brain. Thus, the amyloid hypothesis is generally ac‐ cepted as the mechanism of the onset and progression of AD. The traditional amyloid hy‐ pothesis is that overproduced Aβ forms insoluble amyloid plaques, which are commonly observed in the AD brain and are believed to be the toxic form of APP and responsible for neurodegeneration [66].

As detailed in section 4.2., Aβ is generated after sequential cleavage of APP by β- and γ-sec‐ retases. Although these fragments range from 36 to 43 amino acid residues in length, Aβ40 and Aβ42 are the most common isoforms. Aβ40 is predominant over Aβ42, but Aβ42 is more amyloidogenic [67] and is, therefore, thought to be closely associated with AD. Fur‐ thermore, similar amyloid plaques are found in particular variants of Lewy body dementia [68] and in the muscle disease inclusion body myositis [69]. Aβ also forms aggregates that coat cerebral blood vessels in cerebral amyloid angiopathy (CAA), which is observed in over 90% of AD patients [70].

Deposition of Aβ in the AD brain is thought to be formed due to imbalances between the production of Aβ and its removal from the brain through various clearance mecha‐ nisms, including enzyme-mediated degradation [71]. Therefore, mechanisms of not only production but also degradation of Aβ have been studied extensively. As a result, sever‐ al candidates for Aβ degradation enzymes are proposed. Neprilysin (NEP) and insulindegrading enzyme (IDE) are expressed in neurons as well as within the vasculature and the levels of both these enzymes are reduced in AD [71]; therefore, these enzymes have been well studied in relation to AD. Interestingly, it has been reported that *APOE* e4, which is the most-established genetic risk factor for the onset of AD and CAA, is associ‐ ated with reduced levels of both enzymes [72, 73]. Furthermore, other candidates for Aβ degradation enzymes have been proposed, including endothelin-converting enzymes 1 and 2 (ECE-1 and ECE-2) [74] and angiotensin-converting enzyme (ACE) [75]. The levels of plasmin and plasminogen activators (uPA and tPA) and ECE-2 have also been shown to be reduced in the AD brain [71].
