We showed previously that cells expressing wild-type (WT) -amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49 -99 and 50 -99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49 -99) than AICD-(50 -99). In addition, the expression of amyloid -protein (A) 49 in cells resulted in predominant production of A40 and that of A48 leads to preferential production of A42. These observations suggest that -cleavage and ␥-cleavage are interrelated. To determine the stoichiometry between A and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized ␥-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of A and AICD are produced from -carboxyl-terminal fragment (C99) by ␥-secretase, irrespective of WT or MTAPP and PS1/2. Although various A species, including A40, A42, A43, A45, A48, and A49, are generated, only two species of AICD, AICD-(49 -99) and AICD-(50 -99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49 -99), and only one for its counterpart, A48, in contrast to WT and other MTPS1s. These strongly suggest that -cleavage is the primary event, and the produced A48 and A49 rapidly undergo ␥-cleavage, resulting in generation of various A species.Amyloid -protein (A) 4 is the major component of senile plaques, one of the neuropathological hallmarks of Alzheimer disease (AD). Current data indicate that A forms large fibrous aggregates that may not be toxic to the cells, but its intermediates, diffusible oligomers, exhibit neuronal toxicity, supporting the view that A is a real culprit for AD. This 38 -43-amino acid residue, a small protein, is derived from -amyloid precursor protein (APP) through successive cleavages by -and ␥-secretases (1). -Secretase is a membrane-bound aspartyl protease and produces a carboxyl-terminal fragment (CTF or C99) of APP by cleaving its luminal portion (2). Cumulative evidence indicates that ␥-secretase is also an aspartyl protease, a high molecular mass protein complex composed of four different membrane proteins, Aph-1, nicastrin, Pen-2, and presenilin (PS) 1/2 (3-5). ␥-Secretase cleaves C99 in the middle of its transmembrane domain (␥-cleavage), leading to release of A. The mechanism of A production is controversial, mainly because the hydrolytic event is postulated to occur in the very hydrophobic environment of the lipid bilayer.Besides ␥-cleavage sites, we and other groups identified novel cleavage sites close to the membrane/cytoplasmic boundary of APP (-cleavage) (6 -8). -Cleavage sites of C99 are analogous to the ␥-secretasemediated Notch S3 cleavage site (9). As in Notch signaling, -cleavage results in release of the APP intracellular domains (AICD) 49 -99 and 50 -99, which translocate to the nucleus an...
A novel cleavage of -amyloid precursor protein (APP), referred to as ⑀-cleavage, occurs downstream of the ␥-cleavage and generates predominantly a C-terminal fragment (CTF␥) that begins at Val-50, according to amyloid -protein (A) numbering. Whether this cleavage occurs independently of, or is coordinated with, ␥-cleavage is unknown. Using a cell-free system, we show here that, although A40 and CTF␥ 50 -99 were the predominant species produced by membranes prepared from cells overexpressing wild-type (wt) APP and wt presenilin (PS) 1 or 2, the production of CTF␥ 49 -99, which begins at Leu-49, was remarkably enhanced in membranes from cells overexpressing mutant (mt) APP or mtPS1/2 that increases the production of A42. Furthermore, a ␥-secretase inhibitor, which suppresses A40 production and paradoxically enhances A42 production at low concentrations, caused the proportion of CTF␥ 50 -99 to decrease and that of CTF␥ 49 -99 to increase significantly. These results strongly suggest a link between the production of A42 and CTF␥ 49 -99 and provide an important insight into the mechanisms of altered ␥-cleavage caused by mtAPP and mtPS1/2.Senile plaques, one of the neuropathological hallmarks of Alzheimer's disease (AD), 1 are composed primarily of amyloid -protein (A) (1). Two major A species consisting of 40 and 42 residues are generated mainly in neurons and constitutively secreted. A shorter one, A40, is predominant, and a longer one, A42, is a minor species (Ͻ10%) among secreted A species. A is produced from -amyloid precursor protein (APP), through sequential cleavage by proteases referred to as -and ␥-secretases. -Secretase was identified as a type I membrane aspartic protease -site APP-cleaving enzyme (BACE) (2), but the identity of ␥-secretase has remained unknown. ␥-Secretase cleaves APP in the middle of the transmembrane domain, releasing A and its counterpart, C-terminal fragment ␥ of APP (CTF␥). Most recent studies have shown that ␥-secretase forms a large complex composed of presenilin (PS) 1 or 2, nicastrin, PEN-2, and APH-1, and the activity of ␥-secretase is now known to depend on these proteins (3-7).One of the A species, A42, has a much higher aggregation potential (8, 9) and is believed to be initially deposited in senile plaques (10). It is reasonable to postulate that A42 accumulation in the brain is the very initial event in the development of AD including sporadic AD. Indeed, all mutations of PS1/2 and some mutations of APP that cause familial AD result in increased A42 production (11).Recently, we and other groups found that APP is cleaved by PS-dependent ␥-secretase, not only in the middle of the transmembrane domain (␥-cleavage) but also near the cytoplasmic membrane boundary (⑀-cleavage) (12-15). The major product of the latter process is a CTF␥ of APP that begins at Val-50. This cleavage site is a few residues inside the membrane from the cytoplasmic/membrane boundary and is similar to site 3 cleavage of Notch (16). Since production of CTF␥ is inhibited by a dominan...
Background: Recent focus has been given to anti-amyloidogenic naturally occurring polyphenols known as flavonoids. Results:The polyphenol tannic acid prevented behavioral impairment and mitigated Alzheimer disease-like pathology. Conclusion: Tannic acid may be prophylactic for Alzheimer disease by inhibiting -secretase activity and mitigating brain pathology. Significance: This nutraceutical approach offers a new class of drug for inhibiting -secretase with few if any side effects.
Amyloid β (Aβ) deposition in the brain is an early and invariable feature of Alzheimer’s disease (AD). The Aβ peptides are composed of about 40 amino acids and are generated from amyloid precursor proteins (APP), by β- and γ-secretases. The distribution of individual Aβ peptides in the brains of aged people, and those suffering from AD and cerebral amyloid angiopathy (CAA), is not fully characterized. We employed the matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) to illustrate the spatial distribution of a broad range of Aβ species in human autopsied brains. With technical advancements such as formic acid pretreatment of frozen autopsied brain samples, we have: i) demonstrated that Aβ1–42 and Aβ1–43 were selectively deposited in senile plaques while full-length Aβ peptides such as Aβ1–36, 1–37, 1–38, 1–39, 1–40, and Aβ1–41 were deposited in leptomeningeal blood vessels. ii) Visualized distinct depositions of N-terminal truncated Aβ40 and Aβ42, including pyroglutamate modified at Glu-3 (N3pE), only with IMS for the first time. iii) Demonstrated that one single amino acid alteration at the C-terminus between Aβ1–42 and Aβ1–41 results in profound changes in their distribution pattern. In vitro, this can be attributed to the difference in the self-aggregation ability amongst Aβ1–40, Aβ1–41, and Aβ1–42. These observations were further confirmed with immunohistochemistry (IHC), using the newly developed anti-Aβ1–41 antibody. Here, distinct depositions of truncated and/or modified C- and N-terminal fragments of Aβs in AD and CAA brains with MALDI-IMS were visualized in a spacio-temporal specific manner. Specifically, Aβ1–41 was detected both with MALDI-IMS and IHC suggesting that a single amino acid alteration at the C-terminus of Aβ results in drastic distribution changes. These results suggest that MALDI-IMS could be used as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD and CAA.Electronic supplementary materialThe online version of this article (10.1186/s40478-017-0477-x) contains supplementary material, which is available to authorized users.
We investigated why the cerebrospinal fluid (CSF) concentrations of Aβ42 are lower in mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients. Because Aβ38/42 and Aβ40/43 are distinct product/precursor pairs, these four species in the CSF together should faithfully reflect the status of brain γ-secretase activity, and were quantified by specific enzyme-linked immunosorbent assays in the CSF from controls and MCI/AD patients. Decreases in the levels of the precursors, Aβ42 and 43, in MCI/AD CSF tended to accompany increases in the levels of the products, Aβ38 and 40, respectively. The ratios Aβ40/43 versus Aβ38/42 in CSF (each representing cleavage efficiency of Aβ43 or Aβ42) were largely proportional to each other but generally higher in MCI/AD patients compared to control subjects. These data suggest that γ-secretase activity in MCI/AD patients is enhanced at the conversion of Aβ43 and 42 to Aβ40 and 38, respectively. Consequently, we measured the in vitro activity of raft-associated γ-secretase isolated from control as well as MCI/AD brains and found the same, significant alterations in the γ-secretase activity in MCI/AD brains.
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