Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the γ-secretase subunit presenilin.

The γ-secretase complex catalyzes the intramembrane cleavage of C99, a carboxy-terminal fragment of the amyloid precursor protein. Two paralogs of its catalytic subunit presenilin (PS1 and PS2) are expressed which are autocatalytically cleaved into an N-terminal and a C-terminal fragment during maturation of γ-secretase. In this study, we compared the efficiency and specificity of C99 cleavage by PS1- and PS2-containing γ-secretases. Mass spectrometric analysis of cleavage products obtained in cell-free and cell-based assays revealed that the previously described lower amyloid-ß (Aß)38 generation by PS2 is accompanied by a reciprocal increase in Aß37 production. We further found PS1 and PS2 to show different preferences in the choice of the initial cleavage site of C99. However, the differences in Aß38 and Aß37 generation appear to mainly result from altered subsequent stepwise cleavage of Aß peptides. Apart from these differences in cleavage specificity, we confirmed a lower efficiency of initial C99 cleavage by PS2 using a detergent-solubilized γ-secretase system. By investigating chimeric PS1/2 molecules, we show that the membrane-embedded, nonconserved residues of the N-terminal fragment mainly account for the differential cleavage efficiency and specificity of both presenilins. At the level of individual transmembrane domains (TMDs), TMD3 was identified as a major modulator of initial cleavage site specificity. The efficiency of endoproteolysis strongly depends on nonconserved TMD6 residues at the interface to TMD2, i.e., at a putative gate of substrate entry. Taken together, our results highlight the role of individual presenilin TMDs in the cleavage of C99 and the generation of Aß peptides.

APLP2 is predominantly cleaved by ß-secretase and γ-secretase in the human brain.

BACKGROUND: Amyloid-ß peptide is well-known as a pathogen of Alzheimer's disease, but its precursor, amyloid-beta precursor protein (APP), remains unexplained 30 years after its discovery. APP has two homologues called amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2), and shares a similar structural organisation with them and has partially overlapping functions. APP family proteins are essential for survival, shown by the crossbreeding analysis of knockout mice of APP family molecules, including APLP1 and APLP2. APLP2 is known to play the most important role among them, but the molecular metabolism of APLP2 is only partially understood. Here, we analysed ectodomain shedding and γ-secretase cleavage of APLP2 by molecular biological and biochemical techniques. METHOD: We analysed the culture supernatant of HEK293 cells overexpressing APLP2 and human cerebrospinal fluid. For the analysis of secreted APLP2 fragments, we raised the OA603 antibody that reacts with the juxtamembrane domain of APLP2. Substrate cleavage sites were identified by matrix assisted laser desorption/ionisation mass spectrometry. RESULTS: By overexpressing in HEK293 cells, APLP2 undergoes ectodomain shedding at three sites in the extracellular region by α- and ß-secretase-like activity and then is intramembranously cleaved at three sites by γ-secretase. In particular, in shedding, α-secretase-like activity was dominant in HEK cells. Surprisingly, in human cerebrospinal fluid, APLP2-derived metabolic fragments were mainly cleaved by ß-secretase-like activity, not by α-secretase-like activity. Because APP is also mainly cleaved by beta-site amyloid precursor protein cleaving enzyme 1 in neurons and APLP1 is expressed exclusively in neurons, these findings suggest that APP family proteins may play a common role via ß-secretase-like cleavage in the central nerve system. CONCLUSIONS: Thus, these findings may contribute to a better understanding of the role of APP family proteins in Alzheimer's disease.

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.

Successive cleavage of ß-amyloid precursor protein by γ-secretase.

γ-Secretase is a multimeric aspartyl protease that cleaves the membrane-spanning region of the ß-carboxyl terminal fragment (ßCTF) generated from ß-amyloid precursor protein. γ-Secretase defines the generated molecular species of amyloid ß-protein (Aß), a critical molecule in the pathogenesis of Alzheimer's disease (AD). Many therapeutic trials for AD have targeted γ-secretase. However, in contrast to the great efforts in drug discovery, the enzymatic features and cleavage mechanism of γ-secretase are poorly understood. Here we review our protein-chemical analyses of the cleavage products generated from ßCTF by γ-secretase, which revealed that Aß was produced by γ-secretase through successive cleavages of ßCTF, mainly at three-residue intervals. Two representative product lines were identified. ε-Cleavages occur first at Leu49-Val50 and Thr48-Leu49 of ßCTF (in accordance with Aß numbering). Longer generated Aßs, Aß49 and Aß48, are precursors to the majority of Aß40 and Aß42, concomitantly releasing the tripeptides, ITL, VIV, and IAT; and VIT and TVI, respectively. A portion of Aß42 is processed further to Aß38, releasing a tetrapeptide, VVIA. The presence of additional multiple minor pathways may reflect labile cleavage activities derived from the conformational flexibility of γ-secretase through molecular interactions. Because these peptide byproducts are not secreted and remain within the cells, they may serve as an indicator that reflects γ-secretase activity more directly than secreted Aß.

Production of BBF2H7-derived small peptide fragments via endoplasmic reticulum stress-dependent regulated intramembrane proteolysis.

Intramembrane cleavage of transmembrane proteins is a fundamental cellular process to produce important signals that elicit biological responses. These proteolytic events are known as regulated intramembrane proteolysis (RIP). ATF6 and BBF2H7 are transmembrane basic leucine zipper transcription factors and are subjected to RIP by site-1 protease (S1P) and site-2 protease (S2P) sequentially in response to endoplasmic reticulum (ER) stress. However, the detailed mechanisms responsible for RIP of the transcription factors, including the precise cutting sites, are still unknown. In this study, we demonstrated that S1P cleaves BBF2H7 just before the RXXL S1P recognition motif. Conversely, S2P cut at least three different sites in the membrane (next to Leu380, Met381, and Leu385), indicating that S2P cleaves the substrates at variable sites or via a multistep process. Interestingly, we found BBF2H7-derived small peptide (BSP) fragments located between the S1P and S2P cleavage sites in cells exposed to ER stress. Major type of BSP fragments was composed of 45 amino acid including partial transmembrane and luminal regions and easily aggregates like amyloid ß (Aß) protein. These results advance the understanding of poorly characterized ER stress-dependent RIP. Furthermore, the aggregable peptides produced by ER stress could link to the pathophysiology of neurodegenerative disorders.

Presenilin 1 Regulates NF-κB Activation via Association with Breakpoint Cluster Region and Casein Kinase II.

We recently reported that NF-κB-mediated inflammation caused by breakpoint cluster region (BCR) is dependent on the α subunit of casein kinase II (CK2α) complex. In the current study, we demonstrate that presenilin 1 (Psen1), which is a catalytic component of the γ-secretase complex and the mutations of which are known to cause familial Alzheimer disease, acts as a scaffold of the BCR-CK2α-p65 complex to induce NF-κB activation. Indeed, Psen1 deficiency in mouse endothelial cells showed a significant reduction of NF-κB p65 recruitment to target gene promoters. Conversely, Psen1 overexpression enhanced reporter activation under NF-κB responsive elements and IL-6 promoter. Furthermore, the transcription of NF-κB target genes was not inhibited by a γ-secretase inhibitor, suggesting that Psen1 regulates NF-κB activation in a manner independent of γ-secretase activity. Mechanistically, Psen1 associated with the BCR-CK2α complex, which is required for phosphorylation of p65 at serine 529. Consistently, TNF-α-induced phosphorylation of p65 at serine 529 was significantly decreased in Psen1-deficient cells. The association of the BCR-CK2α-p65 complex was perturbed in the absence of Psen1. These results suggest that Psen1 functions as a scaffold of the BCR-CK2α-p65 complex and that this signaling cascade could be a novel therapeutic target for various chronic inflammation conditions, including those in Alzheimer disease.

Making the final cut: pathogenic amyloid-ß peptide generation by γ-secretase.

Alzheimer´s disease (AD) is a devastating neurodegenerative disease of the elderly population. Genetic evidence strongly suggests that aberrant generation and/or clearance of the neurotoxic amyloid-ß peptide (Aß) is triggering the disease. Aß is generated from the amyloid precursor protein (APP) by the sequential cleavages of ß- and γ-secretase. The latter cleavage by γ-secretase, a unique and fascinating four-component protease complex, occurs in the APP transmembrane domain thereby releasing Aß species of 37-43 amino acids in length including the longer, highly pathogenic peptides Aß42 and Aß43. The lack of a precise understanding of Aß generation as well as of the functions of other γ-secretase substrates has been one factor underlying the disappointing failure of γ-secretase inhibitors in clinical trials, but on the other side also been a major driving force for structural and in depth mechanistic studies on this key AD drug target in the past few years. Here we review recent breakthroughs in our understanding of how the γ-secretase complex recognizes substrates, of how it binds and processes ß-secretase cleaved APP into different Aß species, as well as the progress made on a question of outstanding interest, namely how clinical AD mutations in the catalytic subunit presenilin and the γ-secretase cleavage region of APP lead to relative increases of Aß42/43. Finally, we discuss how the knowledge emerging from these studies could be used to therapeutically target this enzyme in a safe way.

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.

[Cerebrospinal Fluid and Blood Biomarkers in Alzheimer's Disease].

To cope with an aging society, development of disease-modifying drugs for Alzheimer's disease (AD) is essential. Currently, only symptomatic treatments that suppress clinical manifestations are available. Amyloid-ß42 (Aß42) is an AD-related pathogenic molecule that triggers development of AD pathology; thus, decreasing Aß42 in the brain is a promising candidate for AD therapy. Numerous pharmaceutical companies have developed therapeutic drugs against Aß42, such as ß-secretase inhibitors, γ-secretase inhibitors, and anti-Aß monoclonal antibodies, but in clinical trials for patients with mild to moderate AD, these drugs did not meet the expected endpoints. These results suggest that earlier administration of these drugs to individuals who have not yet developed cognitive decline, but have AD pathological changes in the brain or high risk of developing these changes, may be beneficial. To enable such early treatment, preclinical AD biomarkers are required. In this review, we comment on current AD biomarkers in cerebrospinal fluid and in blood. We also explain CSF/blood APL1ß, which is a candidate surrogate marker for Aß42 in the brain.

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.

TRC8-dependent degradation of hepatitis C virus immature core protein regulates viral propagation and pathogenesis.

Signal-peptide peptidase (SPP) is an intramembrane protease that participates in the production of the mature core protein of hepatitis C virus (HCV). Here we show that SPP inhibition reduces the production of infectious HCV particles and pathogenesis. The immature core protein produced in SPP-knockout cells or by treatment with an SPP inhibitor is quickly degraded by the ubiquitin-proteasome pathway. Oral administration of the SPP inhibitor to transgenic mice expressing HCV core protein (CoreTg) reduces the expression of core protein and ameliorates insulin resistance and liver steatosis. Moreover, the haploinsufficiency of SPP in CoreTg has similar effects. TRC8, an E3 ubiquitin ligase, is required for the degradation of the immature core protein. The expression of the HCV core protein alters endoplasmic reticulum (ER) distribution and induces ER stress in SPP/TRC8 double-knockout cells. These data suggest that HCV utilizes SPP cleavage to circumvent the induction of ER stress in host cells.

Transcriptome analysis of distinct mouse strains reveals kinesin light chain-1 splicing as an amyloid-ß accumulation modifier.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß). The genes that govern this process, however, have remained elusive. To this end, we combined distinct mouse strains with transcriptomics to directly identify disease-relevant genes. We show that AD model mice (APP-Tg) with DBA/2 genetic backgrounds have significantly lower levels of Aß accumulation compared with SJL and C57BL/6 mice. We then applied brain transcriptomics to reveal the genes in DBA/2 that suppress Aß accumulation. To avoid detecting secondarily affected genes by Aß, we used non-Tg mice in the absence of Aß pathology and selected candidate genes differently expressed in DBA/2 mice. Additional transcriptome analysis of APP-Tg mice with mixed genetic backgrounds revealed kinesin light chain-1 (Klc1) as an Aß modifier, indicating a role for intracellular trafficking in Aß accumulation. Aß levels correlated with the expression levels of Klc1 splice variant E and the genotype of Klc1 in these APP-Tg mice. In humans, the expression levels of KLC1 variant E in brain and lymphocyte were significantly higher in AD patients compared with unaffected individuals. Finally, functional analysis using neuroblastoma cells showed that overexpression or knockdown of KLC1 variant E increases or decreases the production of Aß, respectively. The identification of KLC1 variant E suggests that the dysfunction of intracellular trafficking is a causative factor of Aß pathology. This unique combination of distinct mouse strains and model mice with transcriptomics is expected to be useful for the study of genetic mechanisms of other complex diseases.

Absolute quantitation of low abundance plasma APL1ß peptides at sub-fmol/mL Level by SRM/MRM without immunoaffinity enrichment.

Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived Aß-like peptides (APL1ß), a surrogate marker for Alzheimer's disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C18-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1ß were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1ß peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM.

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.

Relative ratio and level of amyloid-ß 42 surrogate in cerebrospinal fluid of familial Alzheimer disease patients with presenilin 1 mutations.

BACKGROUND: Presenilin 1 (PS1) mutations associated with familial Alzheimer disease (FAD) generally increase the amyloid-ß 42 (Aß42) to Aß40 ratio secreted in cultured cells. Some of these mutants reduce the secretion of Aß40 rather than increase that of Aß42. Since it has been difficult to estimate Aß42 secretion in brains of PS1-FAD patients due to substantial Aß42 accumulation, it remains unknown whether the enhanced Aß42 to Aß40 ratio in brains of FAD patients is caused by elevated Aß42 secretion or by reduced secretion of Aß40. OBJECTIVE/METHODS: Cerebrospinal fluids (CSF) of PS1-FAD patients and neurological control patients (controls) were collected. Levels of CSF amyloid precursor-like protein-1-derived Aß-like peptide (APL1ß), including APL1ß28, an Aß42 surrogate marker, were quantified by liquid chromatography tandem mass spectrometry, and Aß42 secretion in the brain was estimated. RESULTS: The relative ratio of CSF APL1ß28 to total APL1ß was higher in PS1-FAD patients than in controls. Importantly, CSF APL1ß28 was not significantly higher. However, C-terminally shorter CSF APL1ß25 and APL1ß27 were significantly lower in PS1-FAD patients and, as expected, so were CSF Aß40 and Aß42. CONCLUSION: A higher relative ratio of the CSF Aß42 surrogate in PS1-FAD patients is not due to its increase in CSF, suggesting that massive Aß42 accumulation in the PS1-FAD brain occurs without an apparent increase in Aß42 secretion.

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.

The AKT1 gene is associated with attention and brain morphology in schizophrenia.

OBJECTIVES: A meta-analysis of the associations between genetic variants in the AKT1 gene and schizophrenia found that a single nucleotide polymorphism (SNP5; rs2494732) was associated with schizophrenia in Asian populations. METHODS: In this study, we investigated the effects of this SNP on memory and attentional performance and brain structure using magnetic resonance imaging in a Japanese population (117 patients with schizophrenia and 189 healthy subjects). RESULTS: The memory performance, particularly attention/concentration score, measured by the Wechsler Memory Scale-Revised in A carriers of SNP5, which was found to be enriched in patients with schizophrenia, was lower than that in individuals with the G/G genotype. We confirmed the association of the SNP with attentional performance using the Continuous Performance Test, which assessed sustained attention and vigilance of attentional function. Patients with A allele demonstrated lower attentional performance than patients with the G/G genotype. Patients with the A allele had smaller gray matter volumes in the right inferior parietal lobule related to attentional processes and in the frontostriatal region related to different SNPs in AKT1 than patients with the G/G genotype. CONCLUSIONS: Our results suggest that a genetic variant of AKT1 might be associated with attentional deficits and brain morphological vulnerability in patients with schizophrenia.

Involvement of endoplasmic reticulum stress in tauopathy.

Tauopathy is a pathological condition with an abnormal intracellular accumulation of tau protein in neurons and glias, which is a feature of Alzheimer's disease (AD) as well as frontotemporal lobar degenerations (FTLD). Recent reports showed that tauopathy occupies an important position for pathological process of dementia generally. Previously, we reported that endoplasmic reticulum (ER) stress has an influence on the onset of AD. In addition, some reports on brain autopsy findings suggest that ER stress is associated with AD and tauopathy. However, the mechanism underlying the association between ER stress and tauopathy is still unknown. Here, we show that ER stress, induced by glucose deprivation or chemicals, increases total endogenous tau protein in cultured neurons and primary cultured neurons. Under ER stress, no significant differences were observed in the transcription of tau, and no differences were observed in the translation of tau with or without the 5'-untranslated region (5'UTR) of tau. In contrast, the degradation rate of tau was decreased by 20% under ER stress. ER stress reduced the binding between tau and carboxyl terminus of Hsc70-interacting protein (CHIP), ubiquitin E3 ligase for tau. These results suggest that ER stress increases total tau protein and its mechanism is due to the decrease in the binding between tau and CHIP, which delays the degradation of tau protein through the ubiquitin-proteasome pathway. This mechanism may provide clue to treatment for tauopathy.

Differential regulation of amyloid precursor protein/presenilin 1 interaction during Aß40/42 [corrected] production detected using fusion constructs.

Beta amyloid peptides (Aß) play a key role in the pathogenesis of Alzheimer disease (AD). Presenilins (PS) function as the catalytic subunits of γ-secretase, the enzyme that releases Aß from ectodomain cleaved amyloid precursor protein (APP) by intramembrane proteolysis. Familial Alzheimer disease (FAD)-linked PSEN mutations alter APP processing in a manner that increases the relative abundance of longer Aß42 peptides to that of Aß40 peptides. The mechanisms by which Aß40 and Aß42 peptides are produced in a ratio of ten to one by wild type presenilin (PS) and by which Aß42 is overproduced by FAD-linked PS variants are not completely understood. We generated chimeras of the amyloid precursor protein C-terminal fragment (C99) and PS to address this issue. We found a chimeric protein where C99 is fused to the PS1 N-terminus undergoes in cis processing to produce Aß and that a fusion protein harboring FAD-linked PS1 mutations overproduced Aß42. To change the molecular interactions within the C99-PS1 fusion protein, we made sequential deletions of the junction between C99 and PS1. We found differential effects of deletion in C99-PS1 on Aß40 and 42 production. Deletion of the junction between APP CTF and PS1 in the fusion protein decreased Aß40, while it did not decrease Aß42 production in the presence or absence of FAD-linked PS1 mutation. These results are consistent with the idea that the APP/PS interaction is differentially regulated during Aß40 and 42 production.