Alzheimer''s disease (AD), a progressive neurodegenerative disorder that is the most common cause of dementia in the elderly, is characterized by the accumulation of amyloid-
β (A
β) plaques and neurofibrillary tangles, as well as a progressive loss of synapses and neurons in the brain. The major pertinacious component of amyloid plaques is A
β, a variably sized peptide derived from the integral membrane protein amyloid precursor protein (APP). The A
β region of APP locates partly within its ecto- and trans-membrane domains. APP is cleaved by three proteases, designated as
α-,
β-, and
γ-secretases. Processing by
β- and
γ-secretase cleaves the N- and C-terminal ends of the A
β region, respectively, releasing A
β, whereas
α-secretase cleaves within the A
β sequence, releasing soluble APP
α (sAPP
α). The
γ-secretase cleaves at several adjacent sites to yield A
β species containing 39–43 amino acid residues. Both
α- and
β-cleavage sites of human wild-type APP are located in APP
672–699 region (ectodomain of
β-C-terminal fragment, ED-
β-CTF or ED-C99). Therefore, the amino acid residues within or near this region are definitely pivotal for human wild-type APP function and processing. Here, we report that one ED-C99-specific monoclonal antibody (mAb
ED-C99) blocks human wild-type APP endocytosis and shifts its processing from
α- to
β-cleavage, as evidenced by elevated accumulation of cell surface full-length APP and
β-CTF together with reduced sAPP
α and
α-CTF levels. Moreover, mAb
ED-C99 enhances the interactions of APP with cholesterol. Consistently, intracerebroventricular injection of mAb
ED-C99 to human wild-type APP transgenic mice markedly increases membrane-associated
β-CTF. All these findings suggest that APP
672–699 region is critical for human wild-type APP processing and may provide new clues for the pathogenesis of sporadic AD.Abnormal functioning and/or processing of amyloid precursor protein (APP), a type I membrane protein, has a pivotal role in the pathogenesis of Alzheimer''s disease (AD).
1, 2, 3 APP is cleaved by three proteases, designated as
α-,
β-, and
γ-secretases (
Supplementary Figure S1). The major fraction (>90%) of wild-type APP is proteolyzed by
α-secretase that cleaves wild-type APP between residues APP
687 and APP
688 within the amyloid-
β (A
β) sequence, releasing soluble APP
α (sAPP
α) and
α-C-terminal fragment (
α-CTF, C83). Only a minority (<10%) of all wild-type APP molecules undergo
β-cleavage at the
β-cleavage site (between residues APP
671 and APP
672) generating sAPP
β and
β-CTF (C99), the latter of which is subsequently processed by
γ-secretase complex to generate a mixture of A
β peptides primarily 40 or 42 residues in length (A
β1-40/42).
4, 5 The
β-secretase cleaves APP in addition at a
β′-site (between residues APP
681 and APP
682) to generate C89 that is further processed by
γ-secretase to produce truncated A
β11–40/42 species.
6Both
α- and
β-cleavage sites of wild-type APP are located in APP
672–699 region (the ectodomain of
β-CTF, ED-
β-CTF, or ED-C99;
Supplementary Figure S1). Therefore, the amino acid residues within or near this region are definitely pivotal for wild-type APP function and processing. Previous studies have identified that mutation in ED-C99 region can affect the physiological processing of APP and contribute to pathological features of familial AD (fAD). For example, Swedish APP carrying APP
670/671 mutation (KM→NL) is cleaved by
β-secretase over 50-fold more efficiently than wild-type APP.
7 APP
673 mutation (A→V) and APP
693 mutation (E→G) can enhance A
β production and accelerate formation of amyloid fibrils.
8, 9, 10 APP
682 mutation (E→K) blocks APP
β′-site and shifts cleavage to
β-site, thus increasing A
β1–40/42 production.
6 Although sporadic AD (sAD), the more common type of AD comprising 90 to 95% of all AD cases, lacks mutations in the
APP gene, region-specific protein modifications within the ED-C99 region may affect wild-type APP processing similarly to APP gene mutations. For example, phosphorylation of ED-C99 at the threonine 687 (of APP
770 isoform, or corresponding threonine 668 of APP
751 isoform;
Supplementary Figure S1) facilitates APP processing by
γ-secretase.
11 Therefore, the elucidation of potential influences of region-specific modifications, induced by either endogenous or exogenous molecules, on wild-type APP processing would be especially critical for clarifying the mechanisms underlying the pathogenesis of sAD.To confirm this hypothesis, we used one mouse monoclonal antibody specifically recognizing ED-C99 (mAb
ED-C99) with its epitope at APP
674–679 (
Supplementary Figure S1). The influences of mAb
ED-C99 binding on human wild-type APP processing were evaluated
in vitro using Chinese hamster ovary cells expressing human wild-type APP (CHO/APP
wt cells) and cortical neurons derived from human wild-type APP transgenic (TgAPP
wt) mice. The
in vitro effects of ED-C99 binding with mAb
ED-C99 on wild-type APP processing were further evaluated and confirmed
in vivo using TgAPP
wt mice and 5 × FAD transgenic mice (Tg6799 line).
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