Switched Aß43 generation in familial Alzheimer's disease with presenilin 1 mutation.

Presenilin (PS) with a genetic mutation generates abundant ß-amyloid protein (Aß) 43. Senile plaques are formed by Aß43 in the cerebral parenchyma together with Aß42 at middle ages. These brains cause the early onset of Alzheimer's disease (AD), which is known as familial Alzheimer's disease (FAD). Based on the stepwise processing model of Aß generation by γ-secretase, we reassessed the levels of Aßs in the cerebrospinal fluid (CSF) of FAD participants. While low levels of Aß38, Aß40, and Aß42 were generated in the CSF of FAD participants, the levels of Aß43 were unchanged in some of them compared with other participants. We sought to investigate why the level of Aß43 was unchanged in FAD participants. These characteristics of Aß generation were observed in the γ-secretase assay in vitro using cells, which express FAD mutations in PS1. Aß38 and Aß40 generation from their precursors, Aß42 and Aß43, was decreased in PS1 mutants compared with wild-type (WT) PS1, as observed in the CSF. Both the ratios of Aß38/Aß42 and Aß40/Aß43 in PS1 mutants were lower than those in the WT. However, the ratio of Aß43/amyloid precursor protein intracellular domain (AICD) increased in the PS1 mutants in an onset age dependency, while other Aß/AICD ratios were decreased or unchanged. Importantly, liquid chromatography-mass spectrometry found that the generation of Aß43 was stimulated from Aß48 in PS1 mutants. This result indicates that PS1 mutants switched the Aß43 generating line, which reflects the level of Aß43 in the CSF and forming senile plaques.

Semagacestat Is a Pseudo-Inhibitor of γ-Secretase.

γ-secretase inhibitors (GSI) are drugs developed to decrease amyloid-ß peptide (Aß) production by inhibiting intramembranous cleavage of ß-amyloid protein precursor (ßAPP). However, a large phase 3 trial of semagacestat, a potential non-transition state analog (non-TSA) GSI, in patients with Alzheimer's disease (AD) was terminated due to unexpected aggravation of cognitive deficits and side effects. Here, we show that some semagacestat effects are clearly different from a phenotype caused by a loss of function of presenilins, core proteins in the γ-secretase complex. Semagacestat increases intracellular byproduct peptides, produced along with Aß through serial γ-cleavage of ßAPP, as well as intracellular long Aß species, in cell-based and in vivo studies of AD model mice. Other potential non-TSA GSIs, but not L685,458, a TSA GSI, have similar effects. Furthermore, semagacestat inhibits release of de novo intramembranous γ-byproducts to the soluble space. Thus, semagacestat is a pseudo-GSI, and therefore, the semagacestat clinical trial did not truly test the Aß hypothesis.

Identification of Small Peptides in Human Cerebrospinal Fluid upon Amyloid-ß Degradation.

BACKGROUND: Amyloid-ß (Aß) degradation in brains of Alzheimer disease patients is a crucial focus for the clarification of disease pathogenesis. Nevertheless, the mechanisms underlying Aß degradation in the human brain remain unclear. OBJECTIVE: This study aimed to quantify the levels of small C-terminal Aß fragments generated upon Aß degradation in human cerebrospinal fluid (CSF). METHODS: A fraction containing small peptides was isolated and purified from human CSF by high-pressure liquid chromatography. Degradation products of Aß C termini were identified and measured by liquid chromatography-tandem mass spectrometry. The C-terminal fragments of Aß in the conditioned medium of cultured cells transfected with the Swedish variant of ßAPP (sw ßAPP) were analyzed. These fragments in brains of PS1 I213T knock-in transgenic mice, overexpressing sw ßAPP, were also analyzed. RESULTS: The peptide fragments GGVV and GVV, produced by the cleavage of Aß40, were identified in human CSF as well as in the brains of the transgenic mice and in the conditioned medium of the cultured cells. Relative to Aß40 levels, GGVV and GVV levels were 7.6 ± 0.81 and 1.5 ± 0.18%, respectively, in human CSF. Levels of the GGVV fragment did not increase by the introduction of genes encoding neprilysin and insulin-degrading enzyme to the cultured cells. CONCLUSION: Our results indicate that a substantial amount of Aß40 in human brains is degraded via a neprilysin- or insulin-degrading enzyme-independent pathway.

γ-Secretase associated with lipid rafts: multiple interactive pathways in the stepwise processing of ß-carboxyl-terminal fragment.

γ-Secretase generates amyloid ß-protein (Aß), a pathogenic molecule in Alzheimer disease, through the intramembrane cleavage of the ß-carboxyl-terminal fragment (ßCTF) of ß-amyloid precursor protein. We previously showed the framework of the γ-secretase cleavage, i.e. the stepwise successive processing of ßCTF at every three (or four) amino acids. However, the membrane integrity of γ-secretase was not taken into consideration because of the use of the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized reconstituted γ-secretase system. Here, we sought to address how the membrane-integrated γ-secretase cleaves ßCTF by using γ-secretase associated with lipid rafts. Quantitative analyses using liquid chromatography-tandem mass spectrometry of the ßCTF transmembrane domain-derived peptides released along with Aß generation revealed that the raft-associated γ-secretase cleaves ßCTF in a stepwise sequential manner, but novel penta- and hexapeptides as well as tri- and tetrapeptides are released. The cropping of these peptides links the two major tripeptide-cleaving pathways generating Aß40 and Aß42 at several points, implying that there are multiple interactive pathways for the stepwise cleavages of ßCTF. It should be noted that Aß38 and Aß43 are generated through three routes, and γ-secretase modulator 1 enhances all the three routes generating Aß38, which results in decreases in Aß42 and Aß43 and an increase in Aß38. These observations indicate that multiple interactive pathways for stepwise successive processing by γ-secretase define the species and quantity of Aß produced.

γ-secretase modulators and presenilin 1 mutants act differently on presenilin/γ-secretase function to cleave Aß42 and Aß43.

Deciphering the mechanism by which the relative Aß42(43) to total Aß ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aß42(43) ratio caused by familial mutations and γ-secretase modulators (GSMs) are unclear. Here, we show in vitro and in living cells that presenilin (PS)/γ-secretase cleaves Aß42 into Aß38, and Aß43 into Aß40 or Aß38. Approximately 40% of Aß38 is derived from Aß43. Aß42(43) cleavage is involved in the regulation of the Aß42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aß42 (increase k(cat)) and slow its dissociation from the enzyme (decrease k(b)), whereas PS1 mutants and inverse GSMs show the opposite effects. Therefore, we suggest a concept to describe the Aß42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aß42(43) may initiate AD and might represent a therapeutic target.

Altered γ-secretase activity in mild cognitive impairment and Alzheimer's disease.

We investigated why the cerebrospinal fluid (CSF) concentrations of Aß42 are lower in mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients. Because Aß38/42 and Aß40/43 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, Aß42 and 43, in MCI/AD CSF tended to accompany increases in the levels of the products, Aß38 and 40, respectively. The ratios Aß40/43 versus Aß38/42 in CSF (each representing cleavage efficiency of Aß43 or Aß42) 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 Aß43 and 42 to Aß40 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.

γ-Secretase-Dependent Proteolysis of Transmembrane Domain of Amyloid Precursor Protein: Successive Tri- and Tetrapeptide Release in Amyloid ß-Protein Production.

γ-Secretase cleaves the carboxyl-terminal fragment (ßCTF) of APP not only in the middle of the transmembrane domain (γ-cleavage), but also at sites close to the membrane/cytoplasm boundary (ε-cleavage), to produce the amyloid ß protein (Aß) and the APP intracellular domain (AICD), respectively. The AICD49-99 and AICD50-99 species were identified as counterparts of the long Aß species Aß48 and Aß49, respectively. We found that Aß40 and AICD50-99 were the predominant species in cells expressing wild-type APP and presenilin, whereas the production of Aß42 and AICD49-99 was enhanced in cells expressing familial Alzheimer's disease mutants of APP and presenilin. These long Aß species were identified in cell lysates and mouse brain extracts, which suggests that ε-cleavage is the first cleavage of ßCTF to produce Aß by γ-secretase. Here, we review the progress of research on the mechanism underlying the proteolysis of the APP transmembrane domain based on tri- and tetrapeptide release.

gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment.

Amyloid beta protein (Abeta), a pathogenic molecule associated with Alzheimer's disease, is produced by gamma-secretase, which cleaves the beta-carboxyl terminal fragment (betaCTF) of beta-amyloid precursor protein in the middle of its transmembrane domain. How the cleavage proceeds within the membrane has long been enigmatic. We hypothesized previously that betaCTF is cleaved first at the membrane-cytoplasm boundary, producing two long Abetas, Abeta(48) and Abeta(49), which are processed further by releasing three residues at each step to produce Abeta(42) and Abeta(40), respectively. To test this hypothesis, we used liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify the specific tripeptides that are postulated to be released. Using CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxyl-1-propanesulfonate)-reconstituted gamma-secretase system, we confirmed that Abeta(49) is converted to Abeta(43/40) by successively releasing two or three tripeptides and that Abeta(48) is converted to Abeta(42/38) by successively releasing two tripeptides or these plus an additional tetrapeptide. Most unexpectedly, LC-MS/MS quantification revealed an induction period, 3-4 min, in the generation of peptides. When extrapolated, each time line for each tripeptide appears to intercept the same point on the x-axis. According to numerical simulation based on the successive reaction kinetics, the induction period exists. These results strongly suggest that Abeta is generated through the stepwise processing of betaCTF by gamma-secretase.

Equimolar production of amyloid beta-protein and amyloid precursor protein intracellular domain from beta-carboxyl-terminal fragment by gamma-secretase.

We showed previously that cells expressing wild-type (WT) beta-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 beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, 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, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.