**5. Regulation of APP CTF translocation to lipid rafts by Thr668 phosphorylation**

Because similar amounts of nCTFs and pCTFs were found in mouse brain (Figure 2C), gen‐ eration of similar levels of the APP intracellular cytoplasmic domain fragments, nonphos‐ phorylated AICD (nAICD) and phosphorylated AICD (pAICD), is expected if γ-secretase cleaves nCTFs and pCTFs equivalently. However, membrane prepared from mouse brain generated higher levels of nAICD than pAICD in an *in vitro* γ-secretase assay (Figure 7A). Incubation of membrane preparations demonstrated a time-dependent, nearly linear in‐ crease in the generation of nAICD and pAICD during the 0–2 h time period, and the reac‐ tion essentially reached a plateau in the 2–4 h period (Figure 7B and C). Dephosphorylation and degradation of pAICD did not occur in this assay. Importantly, the ratio of pAICD to AICD generation was constant throughout the incubation time (1–4 h) with the relative ratio (amount of pAICD/amount of nAICD) measuring 0.35 ± 0.10 at the 2 h point (Figure 7D). Taken together, these *in vitro* analyses indicate that both phosphorylated and nonphos‐ phorylated CTFs are kinetically equivalent as a substrate for γ-secretase, but the results also show that the generation of pAICD was significantly lower when compared to that of nAICD. These observations suggest that pCTFs are located at a distance from active γ-secre‐ tase in the membrane, while nCTFs are positioned nearer to the active enzyme.

**Figure 7.** *In vitro* kinetic analysis of phosphorylated and nonphosphorylated CTF cleavage by γ-secretase. (A) *in vitro* γsecretase assay with membrane preparations from wild-type mouse brain. (B) and (C) kinetic analysis of AICD generat‐ ed by incubation of membrane preparations. (D) the production ratio of pAICD to nAICD (pAICD/nAICD) at the indicated times are shown. Both phosphorylated and nonphosphorylated CTFs are kinetically equivalent as a substrate for γ-secretase, but the results also show that the generation of pAICD was significantly lower when compared to that of nAICD.

**Figure 6. Possible role of X11 proteins in regulating the DRM association and β-site cleavage of APP.** X11s asso‐ ciate with APP outside of DRMs and prevent translocation of APP into DRM. When X11L dissociates from APP, the APP translocates into DRMs, and that fraction of APP molecules is cleaved by BACE which is active in DRM (upper panel). In the absence of X11s, APP molecules are not anchored outside of DRMs, and more APP translocates into DRMs, result‐

Because similar amounts of nCTFs and pCTFs were found in mouse brain (Figure 2C), gen‐ eration of similar levels of the APP intracellular cytoplasmic domain fragments, nonphos‐ phorylated AICD (nAICD) and phosphorylated AICD (pAICD), is expected if γ-secretase cleaves nCTFs and pCTFs equivalently. However, membrane prepared from mouse brain generated higher levels of nAICD than pAICD in an *in vitro* γ-secretase assay (Figure 7A). Incubation of membrane preparations demonstrated a time-dependent, nearly linear in‐ crease in the generation of nAICD and pAICD during the 0–2 h time period, and the reac‐ tion essentially reached a plateau in the 2–4 h period (Figure 7B and C). Dephosphorylation and degradation of pAICD did not occur in this assay. Importantly, the ratio of pAICD to AICD generation was constant throughout the incubation time (1–4 h) with the relative ratio

ing in increased β-site cleavage of APP (lower panel). The arrows indicate translocation direction of APP.

**5. Regulation of APP CTF translocation to lipid rafts by Thr668**

**phosphorylation**

30 Understanding Alzheimer's Disease

Thr668 phosphorylation could regulate APP CTF translocation to the lipid raft microdo‐ main. To examine this hypothesis, γ-secretase-enriched lipid raft-like membrane microdo‐ mains were prepared as DRMs using CHAPSO. Application of CHAPSO is preferable for the isolation of DRMs, including active γ-secretase complexes, compared to procedures us‐ ing other detergents such as Triton X-100 [23, 24]. Components of the active γ-secretase com‐ plex, both PS1 N- and C-terminal fragments and PEN2, were predominantly recovered in the DRM fraction along with a small amount of APP CTFs (~20% measured) [7]. Phosphory‐ lation levels of APP CTFs in the DRM and non-DRM fractions were examined, and the re‐ spective nCTFs and pCTFs were compared as a relative ratio in which pC99 in the DRM was set to 1.0 (Figure 8).

**Figure 8.** Quantification of pCTFs and nCTFs in DRM and non-DRM fractions. (A) Identification of APP CTFs in DRM and non-DRM fractions. (B) CTFs levels in DRM and non-DRM fractions. Significantly higher levels of the phosphorylat‐ ed species pC99, pC89, and pC83 were found in the non-DRM fractions.

Significantly higher levels of the phosphorylated species pC99, pC89, and pC83 were found in the non-DRM fractions compared to the DRM fractions. Additionally, the phosphoryla‐ tion level of total APP CTFs in DRM was significantly lower than that in non-DRM. These results indicate that phosphorylated CTFs are preferentially localized outside of the DRM/ lipid raft-like membrane microdomain and thus prevented from cleavage by γ-secretase.

**Figure 9.** Liposome-binding ability of APP cytoplasmic domain and ist localization in mouse brain. (A) The binding ability of the phosphorylated APP cytoplasmic domain peptide with liposomes composed of lipids from mouse brain membranes. (B) distribution of AICD endogenously generated in mouse brain. Nonphosphorylated nC47 and AICD

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

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33

Therefore, the nonphosphorylated forms of APP CTFs and AICD tend to bind membrane lipids, mediated by their C-termini, and phosphorylation of APP CTFs and AICD at Thr668 functions to prevent direct membrane association, apparently by changing the conformation of their cytoplasmic regions. In addition to these observations, pCTFβ levels were signifi‐ cantly decreased with age in cynomolgus monkey brains [7], indicating that the preservation

To conclude this section, first, almost equal amounts of pCTFs and nCTFs are present in mouse brain, while lower amounts of pAICD are generated compared to nAICD. Second, both pAICD and nAICD are kinetically equivalent substrates for γ-secretase. These observa‐ tions suggest that pCTFs are sequestered away from the membrane region where γ-secre‐ tase is active (DRM/lipid raft-like membrane microdomain) [15], and that pCTFs are located outside of the DRM/lipid raft-like membrane microdomain due to a change in the conforma‐ tion of their cytoplasmic tail, to which the membrane lipids bind. Thus, the pCTFs can quickly disperse from the DRM/lipid raft-like membrane microdomain with their increased

of APP CTF phosphorylation levels correlates with the suppression of γ-cleavage.

bound strongly to the liposome and membrane fraction.

mobility in the membrane (Figure 10).

How does phosphorylation of Thr668 regulate the localization of APP CTFs between DRM and non-DRM? A recent structural analysis revealed that the cytoplasmic domain tail of APP can interact with membrane lipids [25]. Since phosphorylation of APP at Thr668 indu‐ ces a significant change in its cytoplasmic domain conformation (Figure 2) [9, 10, 26], phos‐ phorylation of the APP cytoplasmic domain at Thr668 can influence the association of the APP cytoplasmic tail with membrane lipids.

Liposomes prepared with endogenous lipids from the membrane fractions of mouse brain have been used as a model for neural membranes [27]. Synthetic cytoplasmic APP 648–695 pep‐ tide with (pC47) or without (nC47) a phosphate group at residue Thr668 was incubated with the liposomes, and the liposome-bound peptides were recovered and analyzed by immuno‐ blotting. Notably, nonphosphorylated APP cytoplasmic peptide (nC47) bound strongly to the liposomes, while phosphorylated peptide (pC47) demonstrated no detectable association (Fig‐ ure 9A) [7]. This trend was also confirmed by examining the AICD, which lacks the transmem‐ brane domain due to ε-cleavage by γ-secretase [28, 29]. Most nAICD was recovered in the brain membrane fraction (~75%) rather than in the soluble cytoplasmic fraction (~25%), while com‐ paratively more pAICD was found in the cytoplasmic fraction (~45%) (Figure 9B).

Mechanism of Alzheimer Amyloid β-Protein Precursor Localization to Membrane Lipid Rafts http://dx.doi.org/10.5772/54096 33

**Figure 9.** Liposome-binding ability of APP cytoplasmic domain and ist localization in mouse brain. (A) The binding ability of the phosphorylated APP cytoplasmic domain peptide with liposomes composed of lipids from mouse brain membranes. (B) distribution of AICD endogenously generated in mouse brain. Nonphosphorylated nC47 and AICD bound strongly to the liposome and membrane fraction.

**Figure 8.** Quantification of pCTFs and nCTFs in DRM and non-DRM fractions. (A) Identification of APP CTFs in DRM and non-DRM fractions. (B) CTFs levels in DRM and non-DRM fractions. Significantly higher levels of the phosphorylat‐

Significantly higher levels of the phosphorylated species pC99, pC89, and pC83 were found in the non-DRM fractions compared to the DRM fractions. Additionally, the phosphoryla‐ tion level of total APP CTFs in DRM was significantly lower than that in non-DRM. These results indicate that phosphorylated CTFs are preferentially localized outside of the DRM/ lipid raft-like membrane microdomain and thus prevented from cleavage by γ-secretase.

How does phosphorylation of Thr668 regulate the localization of APP CTFs between DRM and non-DRM? A recent structural analysis revealed that the cytoplasmic domain tail of APP can interact with membrane lipids [25]. Since phosphorylation of APP at Thr668 indu‐ ces a significant change in its cytoplasmic domain conformation (Figure 2) [9, 10, 26], phos‐ phorylation of the APP cytoplasmic domain at Thr668 can influence the association of the

Liposomes prepared with endogenous lipids from the membrane fractions of mouse brain have been used as a model for neural membranes [27]. Synthetic cytoplasmic APP 648–695 pep‐ tide with (pC47) or without (nC47) a phosphate group at residue Thr668 was incubated with the liposomes, and the liposome-bound peptides were recovered and analyzed by immuno‐ blotting. Notably, nonphosphorylated APP cytoplasmic peptide (nC47) bound strongly to the liposomes, while phosphorylated peptide (pC47) demonstrated no detectable association (Fig‐ ure 9A) [7]. This trend was also confirmed by examining the AICD, which lacks the transmem‐ brane domain due to ε-cleavage by γ-secretase [28, 29]. Most nAICD was recovered in the brain membrane fraction (~75%) rather than in the soluble cytoplasmic fraction (~25%), while com‐

paratively more pAICD was found in the cytoplasmic fraction (~45%) (Figure 9B).

ed species pC99, pC89, and pC83 were found in the non-DRM fractions.

32 Understanding Alzheimer's Disease

APP cytoplasmic tail with membrane lipids.

Therefore, the nonphosphorylated forms of APP CTFs and AICD tend to bind membrane lipids, mediated by their C-termini, and phosphorylation of APP CTFs and AICD at Thr668 functions to prevent direct membrane association, apparently by changing the conformation of their cytoplasmic regions. In addition to these observations, pCTFβ levels were signifi‐ cantly decreased with age in cynomolgus monkey brains [7], indicating that the preservation of APP CTF phosphorylation levels correlates with the suppression of γ-cleavage.

To conclude this section, first, almost equal amounts of pCTFs and nCTFs are present in mouse brain, while lower amounts of pAICD are generated compared to nAICD. Second, both pAICD and nAICD are kinetically equivalent substrates for γ-secretase. These observa‐ tions suggest that pCTFs are sequestered away from the membrane region where γ-secre‐ tase is active (DRM/lipid raft-like membrane microdomain) [15], and that pCTFs are located outside of the DRM/lipid raft-like membrane microdomain due to a change in the conforma‐ tion of their cytoplasmic tail, to which the membrane lipids bind. Thus, the pCTFs can quickly disperse from the DRM/lipid raft-like membrane microdomain with their increased mobility in the membrane (Figure 10).

**Abbrevations**

APP, X11L; X11-like.

**Author details**

sity, Sapporo, Japan

741-766.

**References**

Yuhki Saito, Takahide Matsushima and Toshiharu Suzuki

Neuropathol. 1991;82(4): 239-259.

ence 1992; 256: 184-185.

ADAM: a disintegrin and metalloprotease domain, APH-1: anterior pharynx defective 1, AICD: APP intracellular domain, APP: amyloid precursor protein, APP CTFs: APP C-termi‐ nal fragments, BACE1: β-site APP cleaving enzyme 1/β-secretase, CDK5: cyclin-dependent kinase-5, CD spectra: Circular dichroism spectra, DRM: detergent-resistant membrane, GSK3:βglycogen synthase kinase-3β, JNK: c-Jun N-terminal kinase, imAPP: immature APP, mAPP: mature APP, pAICD; nAICD; nonphosphorylated AICD, nCTFs; nonphosphorylated CTFs, phosphorylated AICD, pCTFs; phosphorylated CTFs, PS: presenilins, PEN2; preseni‐ lin enhancer 2, PTB/PI domain; phosphotyrosine binding/interaction domain; sAPP; soluble

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

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35

Laboratory of Neuroscience, Graduate School of Pharmaceutical Sciences, Hokkaido Univer‐

[1] Braak H, Braak E. Neuropathological stageing of Alzheimer-related changes. Acta.

[2] Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 2001;81(2):

[3] Hardy JA and Higgins GA. Alzheimer's disease: the amyloid cascade hypothesis. Sci‐

[4] Vetrivel KS and Thinakaran G. Membrane rafys in Alzheimer's disease beta-amyloid

[5] Saito Y, Sano Y, Vassar R, Gandy S, Nakaya T, Yamamoto T. and Suzuki T. X11 pro‐ teins regulate the translocation of amyloid beta-protein precursor (APP) into deter‐ gent-resistant membrane and suppress the amyloidogenic cleavage of APP by beta-

[6] Suzuki T and Nakaya T. Regulation of amyloid beta-protein precursor by phosphor‐

[7] Matsushima T, Saito Y, Elliott JI, Iijima-Ando K, Nishimura M, Kimura N, Hata S, Yamamoto T, Nakaya T, Suzuki T. Membrane-microdomain Localization of Amyloid

production. Biochem. Biophys. Acta. 2010; 1801: 860-867.

site-cleaving enzyme in brain. J. Biol. Chem. 2008;283(51): 35763–71.

ylation and protein interactions. J Biol Chem 2008;31(44): 29633-37.

**Figure 10.** Possible role of APP CTF phosphorylation at Thr668 in regulating its fluidity within the membrane and its cleavage by γ-secretase.

#### **6. Conclusions**

X11L abundantly present in non-DRM traps APP outside of the DRM and prevents contact between APP and the β-secretases located within the DRM. Phosphorylation of APP at Thr668 induces conformational changes to the APP cytoplasmic domain and reduces the af‐ finity of the APP C terminal to lipids. This change alters APP CTF fluidity and decreases the probability of APP CTF presence in lipid rafts, in which contact between APP CTFs and γsecretase occurs. In conclusion, translocation of APP and APP CTFs to lipid rafts is regulat‐ ed by neuronal adaptor protein X11L and Thr668 phosphorylation of APP CTFs.
