**4. γ-Secretase**

**3.2. Proposed APP functions**

66 Understanding Alzheimer's Disease

hance cell–cell adhesion [62].

extracellular domain of APP [65].

**3.3. Aβ amyloid**

neurodegeneration [66].

90% of AD patients [70].

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

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‐

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

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

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

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 insulin-

without remarkable neuronal loss [61]. This evidence also supports this idea.

#### **4.1. Overview of γ-secretase**

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‐ sis in the AD brain.

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

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


**4.2. Some γ-secretase substrates share a common proteolytic process**

expressed through binding to transcription factors.

Fig.3 shows the proteolytic processes of Notch, APP, and CD44. There are surprising simi‐ larities between these processes and all of these processes follow the RIP mechanism. For ex‐ ample, in the canonical Notch signaling pathway, ligands bind to the extracellular domain of Notch on neighboring cells and trigger sequential proteolytic cleavage reactions (the RIP mechanism) and shedding at the S2 site by TACE or ADAM protease making the truncated Notch [44, 45]. Truncated Notch is further cleaved by γ-secretase in at least two sites within the TM domain [46-48], i.e., at the S3 site to release NICD and at the S4 site to release the remaining small peptide (Nβ). As mentioned above, NICD, which is released from the cell membrane to the cytoplasm by γ-secretase, translocates to the nucleus where its activity is

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69

The proteolytic process of APP is very similar to that of Notch and also follows the RIP mechanism. Cleavage of APP by α- secretase [80] or β-secretase [81] at the α- or β-site, re‐ spectively, within the JM region results in shedding of almost the entire extracellular do‐ main and generates membrane-tethered α- or β-carboxy terminal fragments (CTFs). Several zinc metalloproteinases, such as TACE and ADAM [82, 83], and the aspartyl protease BACE2 [84] can cleave APP at the α-site, while BACE1 (β-site APP cleaving enzyme) cleaves APP at the β-site [81]. Once the extracellular domain has been shed, the remaining stub is further cleaved at least twice by γ-secretase within the TM domain at γ- and ε-sites resulting in production of either non-amyloidogenic p3 peptide (in combination with α-secretase) or amyloidogenic Aβ (in combination with BACE1), respectively, and AICD [11, 12]. As dis‐ cussed in the next paragraph, although a large proportion of AICD is rapidly degraded in the cytoplasm, a small amount of the remaining AICD may translocate to the nucleus.

It has been reported that several other type 1 membrane proteins also follow the RIP mecha‐ nism and their ICDs are released from the cell membrane [13, 14, 22]. For example, as shown in Fig.3, the process of sequential proteolytic cleavage of CD44, which is important for im‐ mune system function, is very similar to those of Notch and APP [22]. In addition, the ICD

As discussed here, several γ-secretase substrates follow the RIP mechanism. The ICDs of these substrates are released from the cell membrane by γ-secretase, and these ICDs translo‐ cate to the nucleus. These processes are very similar to those involved in Notch signaling. Therefore, the observations that the common enzyme, γ-secretase, modulates proteolysis and the turnover of possible signaling molecules led to the attractive idea, the signaling hy‐ pothesis, which suggests that mechanisms similar to those occurring in the Notch signaling

Actually, Dll1, a major ligand of Notch, is cleaved sequentially by metalloproteases and γsecretase, and ICD of Dll1 (Dll1IC) is released from the cell membrane and then translocates to the nucleus [85, 86]. Furthermore, we have shown that Dll1IC then binds to Smad 2 and 3, which are transcription factors involved in the TGF-β/activin signaling pathway, and may alter transcription of specific genes that are involved in neuronal differentiation [87]. These

pathway may contribute widely to γ-secretase–regulated signaling mechanisms.

results suggest that Dll1 also has a signaling mechanism similar to that of Notch.

of this protein (CD44ICD) also translocates to the nucleus (Fig.3).

**Table 1.** Substrates for γ-secretase

#### **4.2. Some γ-secretase substrates share a common proteolytic process**

**Substrate Function PS or ICD function**

properties, axonal transport?

CD43 Signal transduction Signaling molecule?

DCC Axon guidance, tumor suppressor Activates nuclear reporter Delta Notch ligand Transcriptional regulation

IFN-αR2 Subunit of type I IFN-α receptor Transcriptional regulation Insulin receptor Receptor tyrosine kinase Accumulates in nucleus

LAR Receptor tyrosine phosphatase Accumulates in nucleus

LRP Scavenger and signaling receptor Activates nuclear reporter

N-cadherin Cell adhesion Promotes CBP degradation

Notch1-4 Signaling receptor Transcriptional regulation

Telencephalin Cell adhesion Turnover of telencephalin

adherens junctions

CSF1-R Protein tyrosine kinase Unknown CXCL16 & CX3CL1 Membrane chemokine ligands Unknown

HLA-A2 MHC class I molecule Unknown

IGIF-R Receptor tyrosine kinase Unknown IL-1RI Cytokine receptor Unknown IL-1RII Cytokine receptor Unknown

LDLR Lipoprotein receptor Unknown

dependence receptor

Vasorin TGF-β inhibitor Unknown

differentiation

of voltage-gated Na channel

Na channel β-subunit Cell adhesion, an auxiliary subunit

Nectin-1α Adherens junction, synapse receptor

P75NTR Neurotrophin co-receptor,

γ-Protocadherin Cell adhesion, neuronal

Tyrosinase,Tyrosinaserelated protein 1/2

**Table 1.** Substrates for γ-secretase

Syndecan-3 Cell surface proteoglycan coreceptor

APLP1/2 Cell adhesion? Forms complex with Fe65 and Tip60

CD44 Cell adhesion Activates TRE-mediated nuclear transcription

E-cadherin Cell adhesion Promotes disassembly of adhesion complex ERBB4 Receptor tyrosine kinase Regulates heregulin-induced growth inhibition

Jagged Notch ligand Modulates AP-1-mediated transcription

NRADD Apoptosis in neuronal cells Modulates glycosylation/maturation of NRADD

Activates nuclear reporter

Ab generation, release of ICD, Complex with Fe65/Tip60, Cell death?

Facilitates phosphorylation

Alters cell adhesion and migration

Modulates p75-TrkA complex? Nuclear signaling?

Regulation of membrane-targeting of CASK

Remodeling of cell junctions?

Regulation of gene transcription?

Pigment synthesis Intracellular transport of Post-Golgi Tyr-containing vesicles

ApoER2 Lipoprotein receptor, neuronal migration

68 Understanding Alzheimer's Disease

APP Precursor to Aβ, adhesion, trophic

β-Catenin Transduce Wnt signals stabilize

Fig.3 shows the proteolytic processes of Notch, APP, and CD44. There are surprising simi‐ larities between these processes and all of these processes follow the RIP mechanism. For ex‐ ample, in the canonical Notch signaling pathway, ligands bind to the extracellular domain of Notch on neighboring cells and trigger sequential proteolytic cleavage reactions (the RIP mechanism) and shedding at the S2 site by TACE or ADAM protease making the truncated Notch [44, 45]. Truncated Notch is further cleaved by γ-secretase in at least two sites within the TM domain [46-48], i.e., at the S3 site to release NICD and at the S4 site to release the remaining small peptide (Nβ). As mentioned above, NICD, which is released from the cell membrane to the cytoplasm by γ-secretase, translocates to the nucleus where its activity is expressed through binding to transcription factors.

The proteolytic process of APP is very similar to that of Notch and also follows the RIP mechanism. Cleavage of APP by α- secretase [80] or β-secretase [81] at the α- or β-site, re‐ spectively, within the JM region results in shedding of almost the entire extracellular do‐ main and generates membrane-tethered α- or β-carboxy terminal fragments (CTFs). Several zinc metalloproteinases, such as TACE and ADAM [82, 83], and the aspartyl protease BACE2 [84] can cleave APP at the α-site, while BACE1 (β-site APP cleaving enzyme) cleaves APP at the β-site [81]. Once the extracellular domain has been shed, the remaining stub is further cleaved at least twice by γ-secretase within the TM domain at γ- and ε-sites resulting in production of either non-amyloidogenic p3 peptide (in combination with α-secretase) or amyloidogenic Aβ (in combination with BACE1), respectively, and AICD [11, 12]. As dis‐ cussed in the next paragraph, although a large proportion of AICD is rapidly degraded in the cytoplasm, a small amount of the remaining AICD may translocate to the nucleus.

It has been reported that several other type 1 membrane proteins also follow the RIP mecha‐ nism and their ICDs are released from the cell membrane [13, 14, 22]. For example, as shown in Fig.3, the process of sequential proteolytic cleavage of CD44, which is important for im‐ mune system function, is very similar to those of Notch and APP [22]. In addition, the ICD of this protein (CD44ICD) also translocates to the nucleus (Fig.3).

As discussed here, several γ-secretase substrates follow the RIP mechanism. The ICDs of these substrates are released from the cell membrane by γ-secretase, and these ICDs translo‐ cate to the nucleus. These processes are very similar to those involved in Notch signaling. Therefore, the observations that the common enzyme, γ-secretase, modulates proteolysis and the turnover of possible signaling molecules led to the attractive idea, the signaling hy‐ pothesis, which suggests that mechanisms similar to those occurring in the Notch signaling pathway may contribute widely to γ-secretase–regulated signaling mechanisms.

Actually, Dll1, a major ligand of Notch, is cleaved sequentially by metalloproteases and γsecretase, and ICD of Dll1 (Dll1IC) is released from the cell membrane and then translocates to the nucleus [85, 86]. Furthermore, we have shown that Dll1IC then binds to Smad 2 and 3, which are transcription factors involved in the TGF-β/activin signaling pathway, and may alter transcription of specific genes that are involved in neuronal differentiation [87]. These results suggest that Dll1 also has a signaling mechanism similar to that of Notch.

**4.3. Is γ-secretase a proteasome of the membrane?**

brane proteins has been proposed [11, 12].

degradation.

play roles in signaling.

As mentioned above, more than five dozen γ-secretase substrates, most of which are type 1 membrane proteins, have been reported. This raises the simple question against the signaling hypothesis, why so many membrane proteins can transmit signals to the nucleus. In reply to this question, another possibility that γ-secretase acts as a protea‐ some of the membrane has been proposed [11, 12]. Indeed, as the ICDs of these sub‐ strates including AICD, which are released by γ-secretase, are rapidly degraded [24, 88], it is usually difficult to detect their ICDs by western blotting. Furthermore, ectodo‐ main shedding seems to be constitutive for some substrates, and ligand binding has been reported to enhance only cleavage of Notch [47], Delta [87], Syndecan-3 [89], and ERBB4 [90]. In addition, much evidence supporting the signaling hypothesis was ob‐ tained in overexpression assays that differ somewhat from normal physiological condi‐ tions. Based on these observations, the proteasome hypothesis suggesting that the primary function of γ-secretase is to facilitate the selective disposal of type 1 mem‐

γ-Secretase–Regulated Signaling and Alzheimer's Disease

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

71

Although the proteasome hypothesis for γ-secretase is reasonable and potent, there is no doubt that the certain signaling mechanisms regulated by γ-secretase, such as Notch sig‐ naling, exist. Therefore, it is likely that different functions of γ-secretases reflect their var‐ iant complexes in different combinations with multiple components, such as Aph-1, Pen2, and/or PS isoforms, with different cellular functions, such as roles in signaling or

In addition, it seems that a small proportion of ICDs of these substrates that are re‐ leased by γ-secretase are sufficient for signaling mechanisms. Generally, γ-secretase substrates like APP are considerably more abundant than transcription factors, which are usually rare molecules. Although a large proportion of ICDs of these substrates are rapidly degraded, a small amount of the remaining ICDs may be sufficient for their signaling functions with small quantities of transcription factors. Thus, the majori‐ ty of ICDs of these substrates may be degraded, and only a small proportion may

In relation to this issue, an attractive idea has been proposed in which a certain stimulus controls APP signaling through phosphorylation and dephosphorylation of AICD. Since AICD is stabilized [91] and translocated into the nucleus by Fe65 [26], it is thought that Fe65 is essential for the signaling function of AICD. Non-phosphorylated AICD can bind to Fe65 and form a complex; thus, this complex is stabilized and immediately translo‐ cates to the nucleus, where it mediates the expression of target genes in association with Tip60. On the other hand, phosphorylated AICD cannot bind to Fe65. Therefore, phos‐ phorylated AICD without Fe65 cannot translocate to the nucleus. Phosphorylated AICD that remains in the cytosol is rapidly degraded by degradation enzymes such as the pro‐ teasome and/or IDE. Indeed, it has been reported that phosphorylation at Thr668 in the

APP-695 isoform strongly inhibited binding to Fe65 [92, 93].

**Figure 3.** Similarities in the proteolytic processes among Notch, APP, and CD44. (A) In response to ligand binding, Notch undergoes shedding due to metalloprotease cleavage at the S2 site within the juxtamembrane domain. After shedding the extracellular domain, the remaining Notch stub is further cleaved by γ-secretase at S3 and S4 sites within the trans‐ membrane domain. This sequential proteolysis produces NICD and Nβ fragment. (B) Cleavage of APP by α-secretase or βsecretase at the α-site or β-site, respectively, within the juxtamembrane domain results in shedding of almost the entire extracellular domain and generates membrane-tethered α- or β-carboxy terminal fragments (CTFs). Several zinc metallo‐ proteinases and BACE2 can cleave APP at the α-site, while BACE1 cleaves APP at the β-site. After shedding the extracellular domain, the remaining stub is further cleaved at least twice within the transmembrane domain at γ- and ε-sites by γ-secre‐ tase, producing either p3 peptide (in combination with α-secretase) or Aβ (in combination with BACE1), respectively, and AICD. (C) Several stimuli, such as PKC activation and Ca2+ influx, trigger ectodomain cleavage of CD44 by a metalloprotease at the site within the juxtamembrane domain, resulting in the secretion of soluble CD44 (sCD44). After shedding the ex‐ tracellular domain, the remaining CD44 stub is further cleaved by γ-secretase at two sites within the transmembrane do‐ main. This sequential proteolysis produces the CD44ICD and CD44β, an Aβ-like peptide.

#### **4.3. Is γ-secretase a proteasome of the membrane?**

**Figure 3.** Similarities in the proteolytic processes among Notch, APP, and CD44. (A) In response to ligand binding, Notch undergoes shedding due to metalloprotease cleavage at the S2 site within the juxtamembrane domain. After shedding the extracellular domain, the remaining Notch stub is further cleaved by γ-secretase at S3 and S4 sites within the trans‐ membrane domain. This sequential proteolysis produces NICD and Nβ fragment. (B) Cleavage of APP by α-secretase or βsecretase at the α-site or β-site, respectively, within the juxtamembrane domain results in shedding of almost the entire extracellular domain and generates membrane-tethered α- or β-carboxy terminal fragments (CTFs). Several zinc metallo‐ proteinases and BACE2 can cleave APP at the α-site, while BACE1 cleaves APP at the β-site. After shedding the extracellular domain, the remaining stub is further cleaved at least twice within the transmembrane domain at γ- and ε-sites by γ-secre‐ tase, producing either p3 peptide (in combination with α-secretase) or Aβ (in combination with BACE1), respectively, and AICD. (C) Several stimuli, such as PKC activation and Ca2+ influx, trigger ectodomain cleavage of CD44 by a metalloprotease at the site within the juxtamembrane domain, resulting in the secretion of soluble CD44 (sCD44). After shedding the ex‐ tracellular domain, the remaining CD44 stub is further cleaved by γ-secretase at two sites within the transmembrane do‐

main. This sequential proteolysis produces the CD44ICD and CD44β, an Aβ-like peptide.

70 Understanding Alzheimer's Disease

As mentioned above, more than five dozen γ-secretase substrates, most of which are type 1 membrane proteins, have been reported. This raises the simple question against the signaling hypothesis, why so many membrane proteins can transmit signals to the nucleus. In reply to this question, another possibility that γ-secretase acts as a protea‐ some of the membrane has been proposed [11, 12]. Indeed, as the ICDs of these sub‐ strates including AICD, which are released by γ-secretase, are rapidly degraded [24, 88], it is usually difficult to detect their ICDs by western blotting. Furthermore, ectodo‐ main shedding seems to be constitutive for some substrates, and ligand binding has been reported to enhance only cleavage of Notch [47], Delta [87], Syndecan-3 [89], and ERBB4 [90]. In addition, much evidence supporting the signaling hypothesis was ob‐ tained in overexpression assays that differ somewhat from normal physiological condi‐ tions. Based on these observations, the proteasome hypothesis suggesting that the primary function of γ-secretase is to facilitate the selective disposal of type 1 mem‐ brane proteins has been proposed [11, 12].

Although the proteasome hypothesis for γ-secretase is reasonable and potent, there is no doubt that the certain signaling mechanisms regulated by γ-secretase, such as Notch sig‐ naling, exist. Therefore, it is likely that different functions of γ-secretases reflect their var‐ iant complexes in different combinations with multiple components, such as Aph-1, Pen2, and/or PS isoforms, with different cellular functions, such as roles in signaling or degradation.

In addition, it seems that a small proportion of ICDs of these substrates that are re‐ leased by γ-secretase are sufficient for signaling mechanisms. Generally, γ-secretase substrates like APP are considerably more abundant than transcription factors, which are usually rare molecules. Although a large proportion of ICDs of these substrates are rapidly degraded, a small amount of the remaining ICDs may be sufficient for their signaling functions with small quantities of transcription factors. Thus, the majori‐ ty of ICDs of these substrates may be degraded, and only a small proportion may play roles in signaling.

In relation to this issue, an attractive idea has been proposed in which a certain stimulus controls APP signaling through phosphorylation and dephosphorylation of AICD. Since AICD is stabilized [91] and translocated into the nucleus by Fe65 [26], it is thought that Fe65 is essential for the signaling function of AICD. Non-phosphorylated AICD can bind to Fe65 and form a complex; thus, this complex is stabilized and immediately translo‐ cates to the nucleus, where it mediates the expression of target genes in association with Tip60. On the other hand, phosphorylated AICD cannot bind to Fe65. Therefore, phos‐ phorylated AICD without Fe65 cannot translocate to the nucleus. Phosphorylated AICD that remains in the cytosol is rapidly degraded by degradation enzymes such as the pro‐ teasome and/or IDE. Indeed, it has been reported that phosphorylation at Thr668 in the APP-695 isoform strongly inhibited binding to Fe65 [92, 93].
