**4. X11 protein regulation of APP localization to lipid rafts**

X11 family proteins (X11s), consisting of X11/X11α/Mint1, X11L/X11β/Mint2, and X11L2/ X11γ/Mint3, are encoded by separate genes on human chromosomes 9, 15, and 19 and mouse chromosomes 19, 7, and 10, respectively. X11s contain an evolutionarily conserved central phosphotyrosine binding/interaction (PTB/PI) domain and two C-terminal PDZ do‐ mains [16]. The PTB/PI and PDZ domains are well-characterized protein-protein interaction domains, and X11 proteins interact with various types of proteins, including APP, alcadein, apoER2, munc18, KIF17, kalirin, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, and Arfs, through their PTB/PI and PDZ domains. Interaction of X11L with APP can stabilize APP metabolism and intracellular trafficking, which induce the suppression of Aβ generation [16-18]. Metabolic analysis of APP in X11 and/or X11L knockout mice con‐ firmed that X11s modulated APP metabolism and suppressed Aβ generation as an endophe‐ notype *in vivo* [5, 19, 20]. X11 or X11L transgenic mice crossed to commonly used AD model mice (APPswe transgenic mice) demonstrated reduced amyloid deposition along with de‐ creased levels of Aβ40 and Aβ42 in the brain compared to APPswe transgenic mice [21, 22].

The molecular mechanisms underlying the suppression of APP amyloidogenic metabolism by X11 and X11L have been addressed in a recent analysis. In the brains of mice lacking X11 and/or X11L, levels of CTFβ and Aβ were increased relative to wild-type animals (Figure 4) [5].

**Figure 4.** Quantification of APP CTFs in the hippocampus of wild-type, X11-deficient, X11L-deficient, and X11/X11L doubly deficient mice. Levels of CTFβ and Aβ were increased in X11s deficient mice, indicating that amyloidogenic me‐ tabolism of APP was enhanced in X11s deficient mice.

**Figure 5.** Quantification of APP, APP CTFs, BACE, and PS1 in (A) DRM and (B) non-DRM fractions from wild-type, X11 deficient, X11L-deficient, and X11/X11L doubly deficient mouse cortex. Higher levels of mAPP and CTFβ were recov‐ ered in DRM of the X11L-deficient and the X11/X11L doubly deficient mouse brain. (C) Localization of membraneattached X11 proteins to DRM and non-DRM fractions. X11s were recovered in membrane fractions, and they largely

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The Dysfunction of X11s in aged neurons may thus contribute to sporadic AD etiology. The dysfunction of X11s could lead to a weakening of the association between X11s and APP, resulting in greater translocation of APP to DRMs. Alteration in the lipid composition of membranes may enlarge lipid raft areas or increase the number of lipid rafts, which could also enhance APP translocation to DRMs. These qualitative alterations in X11s and/or lipid metabolism could result in increased β-cleavage of APP even if β-secretase itself is not enzy‐

localized to non-DRMs but not DRMs.

mopathic.

The absence of X11s resulted in more APP and APP CTF translocation to DRMs and en‐ hanced colocalization of APP or APP CTFs with BACE1 in DRMs but not in non-DRMs (Fig‐ ure 5A and B) [5]. Interestingly, X11s were recovered in membrane fractions, and they largely localized to non-DRMs but not DRMs (Figure 5C), indicating that APP can associate exclusively with X11s outside of DRMs to prevent APP translocation to lipid rafts, where amyloidogenic metabolism of APP occurs (Figure 6).

**4. X11 protein regulation of APP localization to lipid rafts**

28 Understanding Alzheimer's Disease

X11 family proteins (X11s), consisting of X11/X11α/Mint1, X11L/X11β/Mint2, and X11L2/ X11γ/Mint3, are encoded by separate genes on human chromosomes 9, 15, and 19 and mouse chromosomes 19, 7, and 10, respectively. X11s contain an evolutionarily conserved central phosphotyrosine binding/interaction (PTB/PI) domain and two C-terminal PDZ do‐ mains [16]. The PTB/PI and PDZ domains are well-characterized protein-protein interaction domains, and X11 proteins interact with various types of proteins, including APP, alcadein, apoER2, munc18, KIF17, kalirin, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, and Arfs, through their PTB/PI and PDZ domains. Interaction of X11L with APP can stabilize APP metabolism and intracellular trafficking, which induce the suppression of Aβ generation [16-18]. Metabolic analysis of APP in X11 and/or X11L knockout mice con‐ firmed that X11s modulated APP metabolism and suppressed Aβ generation as an endophe‐ notype *in vivo* [5, 19, 20]. X11 or X11L transgenic mice crossed to commonly used AD model mice (APPswe transgenic mice) demonstrated reduced amyloid deposition along with de‐ creased levels of Aβ40 and Aβ42 in the brain compared to APPswe transgenic mice [21, 22].

The molecular mechanisms underlying the suppression of APP amyloidogenic metabolism by X11 and X11L have been addressed in a recent analysis. In the brains of mice lacking X11 and/or

**Figure 4.** Quantification of APP CTFs in the hippocampus of wild-type, X11-deficient, X11L-deficient, and X11/X11L doubly deficient mice. Levels of CTFβ and Aβ were increased in X11s deficient mice, indicating that amyloidogenic me‐

The absence of X11s resulted in more APP and APP CTF translocation to DRMs and en‐ hanced colocalization of APP or APP CTFs with BACE1 in DRMs but not in non-DRMs (Fig‐ ure 5A and B) [5]. Interestingly, X11s were recovered in membrane fractions, and they largely localized to non-DRMs but not DRMs (Figure 5C), indicating that APP can associate exclusively with X11s outside of DRMs to prevent APP translocation to lipid rafts, where

tabolism of APP was enhanced in X11s deficient mice.

amyloidogenic metabolism of APP occurs (Figure 6).

X11L, levels of CTFβ and Aβ were increased relative to wild-type animals (Figure 4) [5].

**Figure 5.** Quantification of APP, APP CTFs, BACE, and PS1 in (A) DRM and (B) non-DRM fractions from wild-type, X11 deficient, X11L-deficient, and X11/X11L doubly deficient mouse cortex. Higher levels of mAPP and CTFβ were recov‐ ered in DRM of the X11L-deficient and the X11/X11L doubly deficient mouse brain. (C) Localization of membraneattached X11 proteins to DRM and non-DRM fractions. X11s were recovered in membrane fractions, and they largely localized to non-DRMs but not DRMs.

The Dysfunction of X11s in aged neurons may thus contribute to sporadic AD etiology. The dysfunction of X11s could lead to a weakening of the association between X11s and APP, resulting in greater translocation of APP to DRMs. Alteration in the lipid composition of membranes may enlarge lipid raft areas or increase the number of lipid rafts, which could also enhance APP translocation to DRMs. These qualitative alterations in X11s and/or lipid metabolism could result in increased β-cleavage of APP even if β-secretase itself is not enzy‐ mopathic.

(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‐

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**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

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

of nAICD.

set to 1.0 (Figure 8).

tase in the membrane, while nCTFs are positioned nearer to the active enzyme.

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