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

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‐

**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].

crease the generation of Aβ [5].

24 Understanding Alzheimer's Disease

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

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 phosphorylated CTFα, pC83, demonstrated a trend toward decreased levels in comparison to nonphosphorylated CTFα, nC83 (Figure 2B). The relative ratio of total phosphorylated CTFs was equivalent to that of nonphosphorylated CTFs (Figure 2C), although phosphory‐ lated CTFβ and CTFβ' were predominant compared to their nonphosphorylated forms. These observations indicate that pCTFs and nCTFs are present at equal levels in the brain as potential substrates for γ-secretase.

**Figure 3.** Circular dichroism (CD) spectra of APP cytoplasmic peptides (A) and schematic of changes to the APP cyto‐ plasmic domain dependent on Thr668 residue modification (B). The substitution of Asp for Thr668 did not alter the carboxyl terminal helix state as remarkably as phosphorylation of Thr668. By contrast, substitution of Thr668 with Ala668 in APP has been found to mimic effectively the nonphosphorylated state in the helix structure of the APP cyto‐

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

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

27

Dynamic and highly ordered membrane microdomains, termed lipid rafts, are rich in cho‐ lesterol and sphingolipids such as seramide, gangliosides, glycerophospholipids, and ster‐ ols. The average diameter of lipid rafts has been estimated at 50 nm. However, several classes of lipid rafts that vary in size and duration can exist in a cell [11]. Lipid rafts are formed in the Golgi and transported to the plasma membrane [12], where they serve as plat‐ forms for cell signaling, pathogen entry, cell adhesion, and protein sorting. Lipid rafts are biochemically defined as the detergent-resistant membrane (DRM) fraction [12]. Aβ genera‐ tion and aggregation occur in lipid rafts, suggesting that lipid rafts play an important role in APP processing and subsequent AD pathogenesis. A growing body of evidence indicates that active β- and γ-secretases are located in membrane microdomains [13-15]. *S*-Palmitoyla‐ tion of BACE1 at residues Cys474/478/482/485 is essential for the localization of BACE1 to lipid rafts [13,14]. *S*-Palmitoylation of nicastrin at Cys689 and of APH1 at Cys182 and Cys245 contributed to their stability and the lipid raft association of these nascent subunits, but did not affect the lipid raft localization of PS1 and PEN2 or the assembly of γ-secretase complex [15]. Taken together, lipid raft localization of secretases involved in amyloidogenic APP cleavage is regulated by their post-translational modification. However, the factors that

plasmic domain.

**3. Lipid rafts and Alzheimer's disease**

determine lipid raft localization of APP remain unclear.

**Figure 2.** Level of CTF species in brain membrane fractions. (A) CTF species in brain membrane preparations. (B) and (C) Levels of CTF species in brain membrane preparations. Levels of the phosphorylated CTFβ species (pC99 and pC89) were significantly higher than those of their nonphosphorylated forms, nC99 and nC89.

The 667-VTPEER-672 motif, including the phosphorable amino acid Thr668, forms a type I β-turn and N-terminal helix-capping box structure to stabilize its C-terminal helix structure [9]. Therefore, phosphorylation of Thr668 induces significant conformational changes in the cytoplasmic region of APP (Figure 3) that affect its interaction with FE65, a neuronal adaptor protein [10]. The usual procedure to explore the function of a protein phosphorylation site is to mimic the phosphorylation state by amino acid substitutions of Asp or Glu for the appro‐ priate Thr and Ser residues. However, this strategy may not be suitable in the case of APP phosphorylation, as the substitution of Asp for Thr668 did not alter the carboxyl terminal helix state as remarkably as phosphorylation of Thr668 (Figure 3A). By contrast, substitution of Thr668 with Ala668 in APP has been found to mimic effectively the nonphosphorylated state in the helix structure of the APP cytoplasmic domain. Figure 3B presents a schematic illustration of the Thr668-dependent conformational changes. Thr668Ala mutation mimics the nonphosphorylated state of APP, but Thr668Asp mutation did not completely mimic the phosphorylation structure of APP. Therefore, to reveal the role of APP phosphorylation at Thr668, careful analysis for the phosphorylation state of both APP and the APP metabolic fragments in the brain are described here.

Mechanism of Alzheimer Amyloid β-Protein Precursor Localization to Membrane Lipid Rafts http://dx.doi.org/10.5772/54096 27

**Figure 3.** Circular dichroism (CD) spectra of APP cytoplasmic peptides (A) and schematic of changes to the APP cyto‐ plasmic domain dependent on Thr668 residue modification (B). The substitution of Asp for Thr668 did not alter the carboxyl terminal helix state as remarkably as phosphorylation of Thr668. By contrast, substitution of Thr668 with Ala668 in APP has been found to mimic effectively the nonphosphorylated state in the helix structure of the APP cyto‐ plasmic domain.

#### **3. Lipid rafts and Alzheimer's disease**

phosphorylated CTFα, pC83, demonstrated a trend toward decreased levels in comparison to nonphosphorylated CTFα, nC83 (Figure 2B). The relative ratio of total phosphorylated CTFs was equivalent to that of nonphosphorylated CTFs (Figure 2C), although phosphory‐ lated CTFβ and CTFβ' were predominant compared to their nonphosphorylated forms. These observations indicate that pCTFs and nCTFs are present at equal levels in the brain as

**Figure 2.** Level of CTF species in brain membrane fractions. (A) CTF species in brain membrane preparations. (B) and (C) Levels of CTF species in brain membrane preparations. Levels of the phosphorylated CTFβ species (pC99 and pC89)

The 667-VTPEER-672 motif, including the phosphorable amino acid Thr668, forms a type I β-turn and N-terminal helix-capping box structure to stabilize its C-terminal helix structure [9]. Therefore, phosphorylation of Thr668 induces significant conformational changes in the cytoplasmic region of APP (Figure 3) that affect its interaction with FE65, a neuronal adaptor protein [10]. The usual procedure to explore the function of a protein phosphorylation site is to mimic the phosphorylation state by amino acid substitutions of Asp or Glu for the appro‐ priate Thr and Ser residues. However, this strategy may not be suitable in the case of APP phosphorylation, as the substitution of Asp for Thr668 did not alter the carboxyl terminal helix state as remarkably as phosphorylation of Thr668 (Figure 3A). By contrast, substitution of Thr668 with Ala668 in APP has been found to mimic effectively the nonphosphorylated state in the helix structure of the APP cytoplasmic domain. Figure 3B presents a schematic illustration of the Thr668-dependent conformational changes. Thr668Ala mutation mimics the nonphosphorylated state of APP, but Thr668Asp mutation did not completely mimic the phosphorylation structure of APP. Therefore, to reveal the role of APP phosphorylation at Thr668, careful analysis for the phosphorylation state of both APP and the APP metabolic

were significantly higher than those of their nonphosphorylated forms, nC99 and nC89.

fragments in the brain are described here.

potential substrates for γ-secretase.

26 Understanding Alzheimer's Disease

Dynamic and highly ordered membrane microdomains, termed lipid rafts, are rich in cho‐ lesterol and sphingolipids such as seramide, gangliosides, glycerophospholipids, and ster‐ ols. The average diameter of lipid rafts has been estimated at 50 nm. However, several classes of lipid rafts that vary in size and duration can exist in a cell [11]. Lipid rafts are formed in the Golgi and transported to the plasma membrane [12], where they serve as plat‐ forms for cell signaling, pathogen entry, cell adhesion, and protein sorting. Lipid rafts are biochemically defined as the detergent-resistant membrane (DRM) fraction [12]. Aβ genera‐ tion and aggregation occur in lipid rafts, suggesting that lipid rafts play an important role in APP processing and subsequent AD pathogenesis. A growing body of evidence indicates that active β- and γ-secretases are located in membrane microdomains [13-15]. *S*-Palmitoyla‐ tion of BACE1 at residues Cys474/478/482/485 is essential for the localization of BACE1 to lipid rafts [13,14]. *S*-Palmitoylation of nicastrin at Cys689 and of APH1 at Cys182 and Cys245 contributed to their stability and the lipid raft association of these nascent subunits, but did not affect the lipid raft localization of PS1 and PEN2 or the assembly of γ-secretase complex [15]. Taken together, lipid raft localization of secretases involved in amyloidogenic APP cleavage is regulated by their post-translational modification. However, the factors that determine lipid raft localization of APP remain unclear.
