**2. γ-Secretase controls Notch signaling**

Notch is a family of evolutionarily conserved type 1 membrane proteins with a mass of about 300 kDa [31] that mediates fates of numerous cells in both invertebrates and verte‐ brates [16, 17]. For example, cells expressing the major ligand Delta inhibit the neural differ‐ entiation of neighboring Notch-expressing cells during neurogenesis. Disruption of or disorder in Notch signaling leads to developmental defects or cancer in mammals [18].

While *Drosophila* has only one Notch gene, four Notch isoforms (Notch1 to 4) have been identified in mammals. The typical Notch protein contains 36 tandem epidermal growth fac‐ tor (EGF)-like repeats in its extracellular domain, and six tandem ankyrin-like (CDC10) re‐ peats, a nuclear localization signal, and a PEST sequence in its intracellular domain [31]. The 11th and 12th EGF-like repeats are essential for binding to its ligands [32]. Notch is cleaved in the trans-Golgi network, apparently by furin-like covertase, and is expressed on the cell surface as a heterodimer [33].

**Figure 1.** Notch signaling pathway. Notch proteins are expressed on the cell surface as heterodimers after cleavage at the S1 site by furin. The binding of Notch to the ligand triggers sequential proteolytic cleavage of the regulated intra‐ membrane proteolysis (RIP). When Notch binds to the ligand, Notch is cleaved at the S2 site in the juxtamembrane region by TACE or ADAM protease. Next, the remaining protein stub is further cleaved by γ-secretase at the S3 and S4 sites within the transmembrane domain and NICD is released from the membrane. Then, NICD translocates into the nucleus and binds to the CSL together with MAML. The resultant CSL–NICD–MAML complex removes co-repressors (Co-R) from CSL transcription factor and recruits a co-activator (Co-A), resulting in conversion from repressor to activa‐

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tor. Finally, the complexes of CSL-NICD-MAML-Co-activators promote transcription of the target genes.

on neighboring cells and trigger sequential proteolytic cleavage. Finally, the intracellular do‐ main (ICD) of Notch (NICD) is released from the cell membrane by γ-secretase; NICD then translocates into the nucleus where it modulates gene expression through binding to transcrip‐

Recently, more than five dozen type 1 transmembrane proteins, including Notch and APP, have been reported as substrates for γ-secretase [21]. The ICDs of these proteins are also re‐ leased from the cell membrane [13-15, 22]. Furthermore, it has been shown that some of these ICDs exist in the nucleus. These processes are very similar to those involved in Notch signaling. Thus, the common enzyme γ-secretase modulates the proteolysis and turnover of putative signaling molecules; this suggests that mechanisms similar to the Notch signaling pathway may widely contribute to γ-secretase–regulated signaling [13-15, 23]. Indeed, it has been shown that the ICD of APP (AICD), which is released from the cell membrane by γsecretase, also translocates to the nucleus [24-26] and may function as a transcriptional regu‐

To test the hypothesis that APP has a signaling mechanism similar to that of Notch, we es‐ tablished embryonic carcinoma P19 cell lines that overexpressed AICD [29], which may mimic signaling mechanisms. Although neurons differentiated from these cell lines, AICD expression induced dynamic changes in gene expression profile and neuron-specific apopto‐ sis [30]. These results suggest that APP also has a signaling mechanism, which may be close‐

In this chapter, we first summarize current research progress regarding Notch, APP, and γsecretase. We also focus on the signaling hypothesis; γ-secretase–regulated mechanisms similar to Notch signaling may widely play roles in signaling events involving type 1 trans‐ membrane proteins, including APP. Next, we review recent evidence supporting the exis‐ tence of APP signaling. Furthermore, we discuss the possibility that APP signaling is

Notch is a family of evolutionarily conserved type 1 membrane proteins with a mass of about 300 kDa [31] that mediates fates of numerous cells in both invertebrates and verte‐ brates [16, 17]. For example, cells expressing the major ligand Delta inhibit the neural differ‐ entiation of neighboring Notch-expressing cells during neurogenesis. Disruption of or disorder in Notch signaling leads to developmental defects or cancer in mammals [18].

While *Drosophila* has only one Notch gene, four Notch isoforms (Notch1 to 4) have been identified in mammals. The typical Notch protein contains 36 tandem epidermal growth fac‐ tor (EGF)-like repeats in its extracellular domain, and six tandem ankyrin-like (CDC10) re‐ peats, a nuclear localization signal, and a PEST sequence in its intracellular domain [31]. The 11th and 12th EGF-like repeats are essential for binding to its ligands [32]. Notch is cleaved in the trans-Golgi network, apparently by furin-like covertase, and is expressed on the cell

tion factors. Therefore, γ-secretase plays a central regulatory role in Notch signaling.

lator [27, 28]. These observations suggest the existence of APP signaling.

ly related to AD.

62 Understanding Alzheimer's Disease

involved in the onset and progression of AD.

surface as a heterodimer [33].

**2. γ-Secretase controls Notch signaling**

**Figure 1.** Notch signaling pathway. Notch proteins are expressed on the cell surface as heterodimers after cleavage at the S1 site by furin. The binding of Notch to the ligand triggers sequential proteolytic cleavage of the regulated intra‐ membrane proteolysis (RIP). When Notch binds to the ligand, Notch is cleaved at the S2 site in the juxtamembrane region by TACE or ADAM protease. Next, the remaining protein stub is further cleaved by γ-secretase at the S3 and S4 sites within the transmembrane domain and NICD is released from the membrane. Then, NICD translocates into the nucleus and binds to the CSL together with MAML. The resultant CSL–NICD–MAML complex removes co-repressors (Co-R) from CSL transcription factor and recruits a co-activator (Co-A), resulting in conversion from repressor to activa‐ tor. Finally, the complexes of CSL-NICD-MAML-Co-activators promote transcription of the target genes.

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

expressed in neurons) or presence (APP-751 and APP-770) of the Kunitz protease inhibitor

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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

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

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

**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.

glycosylation, phosphorylation, and tyrosine sulfation, have been observed.

(KPI) domain toward the N-terminus of the protein [55].

proteoglycans in the extracellular matrix [58].

APP family proteins.

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‐ tral regulatory role in Notch signaling.

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 their transcription [52].
