**1. Introduction**

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22 Understanding Alzheimer's Disease

Alzheimer's disease (AD) is a group of common neurodegenerative diseases associated with progressive dementia with aging. The principal pathological hallmarks of AD are senile pla‐ ques and neurofibrillary tangles in the brain, which are found at significantly higher fre‐ quencies in AD patients than age-matched healthy (non-AD) subjects [1]. Senile plaques consist mainly of 39–43 amino-acid amyloid-β (Aβ) peptide, which is generated by sequen‐ tial proteolytic processing of amyloid β-protein precursor (APP) (Figure 1) [2]. Common Aβ species generated in the human and murine brain are Aβ40 and Aβ42. Mutations in *APP* and *Presenilin*, which have been identified as familial AD-causative genes, result in in‐ creased Aβ production and/or an increased ratio of neurotoxic Aβ42.

Aβ is generated by sequential processing of APP with β- and γ-secretase, the catalytic unit of which is presenilin. Findings reported during the late 1980s and early 1990s led to the proposal of the "Aβ cascade hypothesis" of AD onset, which states that Aβ generation is a primary cause of AD [3]. Several lines of evidence indicate that the amyloidogenic process‐ ing of APP, including Aβ generation, occurs in membrane microdomains termed lipid rafts [4]. However, the molecular mechanisms underlying APP translocation to lipid rafts remain unclear. In this chapter, regulatory mechanisms for lipid raft translocation of APP and APP C-terminal fragments (APP CTFs) generated primarily by the cleavage of APP are described.

Membrane lipid rafts are known as sites of amyloidogenic processing of APP and enriched with active β-secretase, while non-amyloidogenic cleavage of APP by α-secretase is per‐ formed outside lipid rafts. Neural adaptor protein X11-like (X11L) regulates the transloca‐ tion of mature APP (mAPP), which is the *N*- and *O*-glycosylated form and real substrate of

© 2013 Saito et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

secretases in the late protein secretory pathway, to lipid rafts. APP bound to X11L preferen‐ tially localizes to sites outside of lipid rafts and escapes from active β-secretase [5]. Dissocia‐ tion of the APP-X11L complex leads to APP entry into lipid rafts, suggesting that dysfunction of X11L in its interaction with APP may recruit more APP to lipid rafts and in‐ crease the generation of Aβ [5].

A recent study found that the phosphorylation level of APP CTFs was much higher than that of full-length APP, and phosphorylated CTFs (pCTFs), but not nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like mem‐ brane microdomains, indicating that Thr668 phosphorylation appears to function on the APP CTF rather than full-length APP [7]. Recent analysis revealed that pCTFs are relatively movable within the membrane as integral membrane proteins, while nCTFs are susceptible to being anchored to a lipid raft by direct binding of the C-terminal tail to membrane lipids. Once in lipid rafts, nCTFs can be preferentially captured and cleaved by γ-secretase. Inter‐ estingly, phosphorylation levels of amyloidogenic CTFβ were significantly decreased in aged brain [7]. Two molecular mechanisms of APP and APP CTF translocation to ripid rafts

Mechanism of Alzheimer Amyloid β-Protein Precursor Localization to Membrane Lipid Rafts

http://dx.doi.org/10.5772/54096

25

APP, which is a type I membrane protein, is subjected to *N*-glycosylation at the endo‐ plasmic reticulum (ER) to form immature APP (imAPP) followed by *O*-glycosylation at the *medial-/trans*-Golgi apparatus to form mature APP (mAPP) (Figure 1). mAPP is then transported through the *trans*-Golgi network to the plasma membrane, where it enters the late secretory pathway and is metabolized by either amyloidogenic or amyloidolytic (non-amyloiodgenic) processing [6, 8]. In the amyloidogenic pathway, APP is cleaved in sequential proteolytic events mediated by β-secretase (β-site APP cleaving enzyme 1 or BACE1) and the γ-secretase complex comprised of four core subunits, presenilins (PS1 or PS2), anterior pharynx defective 1 (APH-1), presenilin enhancer 2 (PEN2), and nicastrin. β-secretase primarily cleaves APP in the luminal domain to generate soluble APPβ (sAPPβ) and membrane-associated APP carboxyl terminal fragments (CTFβ/C99 and CTFβ′/C89). C99 contains an intact Aβ sequence (Figure 1). γ-secretase complex mediates the cleavage of CTFβ at ε, ζ, and γ-sites to generate Aβ and APP intracellular domain (AICD) peptides. Non-amyloidogenic cleavage of APP is mediated by α-site APP cleav‐ ing enzyme (α-secretase, including ADAM9, ADAM10, and ADAM17) to generate sAPPα and CTFα (C83), which contains only the carboxyl half of Aβ peptide. CTFα

Residue Thr668 of the APP cytoplasmic region is located within the 667-VTPEER-672 motif and is phosphorylated (number corresponding to the APP695 isoform) in the late secretory pathway in neurons. Protein kinases such as GSK3β (glycogen synthase kinase-3β), CDK5 (cyclin-dependent kinase-5), CDK1/CDC2, and JNK (c-Jun N-terminal kinase) are thought to mediate this phosphorylation of APP [6]. APP CTFs are also phosphorylated at Thr668 and detected as phosphopeptide pC99, pC89, and pC83 by western blot analysis using a phos‐ phorylation-state-specific anti-APP Thr668 antibody or pAPP antibody (Figure 2A). Typical APP CTF species in the brain appear as five bands: pC99, nC99, pC89, a mixture of nC89 plus pC83, and nC83. Treatment of CTFs with phosphatase is effective for the identification of the respective species. Levels of the phosphorylated CTFβ species pC99 and pC89 were significantly higher than those of their nonphosphorylated forms, nC99 and nC89, while

**2. Metabolism and post-translational modification of APP**

cleavage by γ-secretase complex then generates p3 peptide and AICD.

are described in the following section.

**Figure 1. The schema of APP metabolism and post-translational modification of APP.** APP is subjected to *N*-glyco‐ sylation at ER to form imAPP followed by *O*-glycosylation at the *medial-/trans*-Golgi apparatus to form mAPP. Residue Thr668 of mAPP is specifically phosphorylated in brain. mAPP is cleaved in sequential proteolytic events mediated by β-secretase or α-site APP cleaving enzyme, and the γ-secretase complex. β-secretase primarily cleaves APP in the lumi‐ nal domain to generate sAPPβ and CTFβ (C99 and C89). C99 contains an intact Aβ sequence. γ-secretase complex me‐ diates the cleavage of CTFβ at ε, ζ, and γ-sites to generate Aβ and AICD peptides. α-site APP cleaving enzyme generates sAPPα and CTFα (C83). CTFα cleavage by γ-secretase complex then generates p3 peptide and AICD.

In contrast to APP, APP CTF translocation to lipid rafts seems to involve another regulatory system that also includes active γ-secretase to cleave APP CTFs. The translocation of CTFs to lipid rafts is regulated by APP phosphorylation. The cytoplasmic region of APP is well known to demonstrate neuron-specific phosphorylation at Thr668 (numbering for the APP695 isoform). However, the maximum phosphorylation level of APP is 10–20% in the brain, and its physiological function is not clear [6].

A recent study found that the phosphorylation level of APP CTFs was much higher than that of full-length APP, and phosphorylated CTFs (pCTFs), but not nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like mem‐ brane microdomains, indicating that Thr668 phosphorylation appears to function on the APP CTF rather than full-length APP [7]. Recent analysis revealed that pCTFs are relatively movable within the membrane as integral membrane proteins, while nCTFs are susceptible to being anchored to a lipid raft by direct binding of the C-terminal tail to membrane lipids. Once in lipid rafts, nCTFs can be preferentially captured and cleaved by γ-secretase. Inter‐ estingly, phosphorylation levels of amyloidogenic CTFβ were significantly decreased in aged brain [7]. Two molecular mechanisms of APP and APP CTF translocation to ripid rafts are described in the following section.
