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.

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.

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.

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.

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.

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.

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.

Human CRB2 inhibits gamma-secretase cleavage of amyloid precursor protein by binding to the presenilin complex.

Drosophila Crumbs has been reported to attenuate Notch signaling by inhibition of gamma-secretase cleavage at the wing margins. gamma-Secretase is an intramembrane protease that is responsible for the generation of amyloid-beta (Abeta) peptides from the beta-amyloid precursor protein (APP). Here, we re-examined gamma-secretase inhibition by human CRB2, which is the most abundant Crumbs ortholog in the brain. Transfected CRB2 inhibited proteolytic production of Abeta and APP intracellular domains from APP C-terminal fragments in HEK293 and SH-SY5Y cells. Conversely, knockdown of endogenous CRB2 increased gamma-secretase cleavage products in SH-SY5Y cells. CRB2 inhibition of gamma-cleavage was also detected in cell-free assays. CRB2 interacted with the gamma-secretase complex, but was not a competitive substrate for gamma-cleavage. The transmembrane domain of CRB2 was indispensable for inhibition of Abeta generation and mediated CRB2 binding with the gamma-secretase complex. In addition, the cytoplasmic domain appeared to play a supportive role in gamma-secretase inhibition, whereas mutational disruption of the two protein-binding motifs involved in the formation of cell adhesion complexes did not affect gamma-secretase inhibition. Co-overexpression of presenilin-1 or APH-1 abrogated gamma-secretase inhibition probably through prevention of the incorporation of CRB2 into the gamma-secretase complex. Our results suggest that CRB2 functions as an inhibitory binding protein that is involved in the formation of a mature but inactive pool of the gamma-secretase complex.

Sigma-1Rs are upregulated via PERK/eIF2α/ATF4 pathway and execute protective function in ER stress.

Sigma-1 receptors (Sig-1Rs) are the ER resident proteins. Sig-1Rs in the brain have been reported to be significantly reduced in patients with schizophrenia. The impediment of regulating Sig-1Rs expression levels increases the risk for schizophrenia. Thus elucidating the mechanism regulating Sig-1Rs expression might provide the strategy to prevent mental disorders. In this study, we have demonstrated that Sig-1Rs were transcriptionally upregulated by ATF4 in ER stress. Moreover, ATF4 directly bounds to the 5' flanking region of Sig-1R gene. The reporter activities using this region were enhanced in ER stress, or by ATF4 alone. The reporter activities with the pathogenic polymorphisms (GC-241-240TT, T-485A) were reduced. In addition, the processing of Caspase-4 was inhibited by Sig-1Rs. These results indicate that Sig-1Rs are transcriptionally upregulated via the PERK/eIF2α/ATF4 pathway and ameliolate cell death signaling. This study is the first report identifying the transcription factor regulating Sig-1Rs expression.

Protein kinase C stabilizes X-linked inhibitor of apoptosis protein (XIAP) through phosphorylation at Ser(87) to suppress apoptotic cell death.

BACKGROUND: Multiple protein kinases have been shown to be involved in the apoptotic neuronal loss of Alzheimer's disease (AD). Although some studies support the role of protein kinase C (PKC) in amyloid precursor protein processing as well as in tau phosphorylation, a direct role for PKC in apoptotic neuronal death remains to be clarified. In the present study, we report on the possible role of PKC in cell survival during conditions of stress through phosphorylation of the X-linked inhibitor of apoptosis protein (XIAP). METHODS: Phosphorylation of XIAP at Ser87 was confirmed by western blot analysis employing phosphorylation dependent anti-XIAP antibody after incubation of recombinant XIAP with active PKC in vitro. And increased phosphorylation of XIAP at the site was also confirmed in SH-SY5Y cells treated with PKC activator, phorbol 12-myristate 13-acetate (PMA). A mutant XIAP construct in which Ser87 was substituted by Ala, was prepared, and transfected to cells. After the transfection of wild or mutant XIAP, cells viability was evaluated by counting living and dead cells treated with PMA during etoposide-induced apoptosis. RESULTS: Recombinant XIAP was phosphorylated at Ser(87) by PKC in vitro and treatment of XIAP-transfected SH-SY5Y cells with a PKC activator, phorbol 12-myristate 13-acetate (PMA) induced phosphorylation of XIAP at Ser(87) . Pulse chase experiments revealed that, when phosphorylated at Ser(87) , wild-type XIAP is more stable than XIAP with a Ser87Ala substitution, which is degraded faster. Importantly, the phosphorylation of XIAP at the site by PKC significantly increased cell survival up to approximately 2.5 times under the condition of apoptosis induced by 25 µg/ml etoposide. CONCLUSION: The findings of the present study indicate a role for PKC, through phosphorylation of XIAP at Ser(87) and its stabilization, in cell survival under conditions of stress and lend strength to the idea that PKC is crucial in regulating neuronal homeostasis, which may be impaired in AD.

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.

KIBRA genetic polymorphism influences episodic memory in Alzheimer's disease, but does not show association with disease in a Japanese cohort.

BACKGROUND/AIMS: A single-nucleotide polymorphism (SNP) in the KIBRA gene, rs17070145, was reported to be significantly associated with episodic memory in cognitively normal cohorts. This observation has expanded genetic studies on KIBRA to Alzheimer's disease (AD). Importantly, the association between KIBRA and episodic memory in AD has never been addressed. In this study, we investigated whether the KIBRA rs17070145 SNP influences AD episodic memory and the disease in a Japanese cohort. METHODS: Blood samples from 346 AD patients and 375 normal cognitive controls were collected and genotyped for rs17070145. Episodic memory was measured in 32 AD patients, diagnosed for the first time, by use of the Rivermead Behavioral Memory Test (RBMT). RESULTS: We found that KIBRA C allele carriers scored significantly lower than KIBRA non-C carriers on both RBMT total profile score (p = 0.042, effect size = 0.84) and RBMT total screening score (p < 0.001, effect size = 1.42). The KIBRA gene did not show association with AD in our Japanese cohort. CONCLUSION: Our results evidence a strong association between the KIBRA gene and episodic memory impairment in AD, but show no influence on AD in our Japanese cohort. We propose that KIBRA might have an effect similar to cognitive reserve.

Apolipoprotein E and central nervous system disorders: reviews of clinical findings.

Dementia is a major health problem in developed countries with over 25 million people affected worldwide and probably over 75 million people at risk during the next 20 years. Alzheimer's disease (AD) is the most frequent cause of dementia (50-70%), followed by vascular dementia (30-40%), and mixed dementia (15-20%). AD pathogenesis is still to be elucidated but it is believed to be the complex interaction between genetic and environmental factors in later life. Three causative genes for familial AD have been identified: amyloid precursor protein, presenilin-1, and presenilin-2. There are 150 genes involved with increased neuronal vulnerability to premature death in the AD brain. Among these susceptibility genes, the apolipoprotein E (ApoE) gene is the most prevalent as a risk for AD pathogenic process in which complex interactions between genetic and environmental factors are involved, leading to a cascade of pathogenic events converging in final pathways to premature neuronal death. Some of these mechanisms are common to several neurodegenerative disorders that differ depending upon the genes affected and the involvement of environmental conditions. ApoE is a key lipoprotein in lipid and cholesterol metabolism and it is also the major risk gene for AD and many other central nervous system disorders. The pathogenic role of ApoE-4 is still to be clarified; however, diverse evidence suggests that ApoE may play pleiotropic functions in dementia and central nervous system disorders.

Laughter and humor as complementary and alternative medicines for dementia patients.

BACKGROUND: The number of dementia patients has increased worldwide, with an estimated 13.7 million dementia patients in the Asia Pacific region alone. This number is expected to increase to 64.6 million by the year 2050. DISCUSSION: As a result of advances in research, there several pharmacological therapies available for the treatment of dementia patients. However, current treatments do not suppress the disease process and cannot prevent dementia, and it will be some time before these goals are realized. In the meantime, complementary and alternative medicine (CAM) is an important aspect in the treatment of dementia patients to improve their quality of life throughout the long course of the disease. Considering the individuality of dementia patients, applicability of laughter and humor therapy is discussed. Even though there are many things that need to be elucidated regarding the mechanisms underlying the beneficial effects of laughter and humor, both may be good CAM for dementia patients if they are applied carefully and properly. SUMMARY: In this debate article, the physiological basis and actual application of laughter and humor in the treatment of dementia patients are presented for discussion on the applicability to dementia patients.

Analysis of APL1beta28, a surrogate marker for Alzheimer Abeta42, indicates altered precision of gamma-cleavage in the brains of Alzheimer disease patients.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly. Currently, therapeutic intervention after the disease onset is difficult because progressive neuronal death precedes clinical symptoms. Available medicines for AD, such as AchE inhibitors, transiently slow the progression of the dementia symptoms, but they do not inhibit the pathological process. At present, next generation anti-AD drugs are in development in many pharmaceutical companies. Importantly, most of them are to inhibit the progress of the pathological process and, thus, at the same time, the establishment of a highly probable prediction of future AD onset is inseparable. AD is now diagnosed using clinical criteria coupled with brain imaging systems such as SPECT and PET. To diagnose AD cases before the appearance of clinical symptoms, it will be necessary to (a) establish new, more sensitive clinical criteria, (b) develop methods for detecting the pathological accumulation of proteins (e.g. Abeta) in the brain, or (c) develop biomarkers for predicting the accumulation of Abeta/tau in the brain. Our recent discovery of APL1beta28, a possible biomarker of AD, may help in the development of early detection methods for AD.

The production ratios of AICDε51 and Aß42 by intramembrane proteolysis of ßAPP do not always change in parallel.

BACKGROUND: During intramembrane proteolysis of ß-amyloid protein precursor (ßAPP) by presenilin (PS)/γ-secretase, ε-cleavages at the membrane-cytoplasmic border precede γ-cleavages at the middle of the transmembrane domain. Generation ratios of Aß42, a critical molecule for Alzheimer's disease (AD) pathogenesis, and the major Aß40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aß42 production have been investigated by many pharmaceutical companies. Therefore, the ε-cleavages, rather than the γ-cleavage, might be more effective upstream targets for decreasing the relative generation of Aß42. Thus, one might evaluate compounds by analyzing the generation ratio of the ßAPP intracellular domain (AICD) species (ε-cleavage-derived), instead of that of Aß42. METHODS: Cell-free γ-secretase assays were carried out to observe de novo AICD production. Immunoprecipitation/MALDI-TOF MS analysis was carried out to detect the N-termini of AICD species. Aß and AICD species were measured by ELISA and immunoblotting techniques. RESULTS: Effects on the ε-cleavage by AD-associated pathological mutations around the ε-cleavage sites (i.e., ßAPP V642I, L648P and K649N) were analyzed. The V642I and L648P mutations caused an increase in the relative ratio of ε48 cleavage, as expected from previous reports. Cells expressing the K649N mutant, however, underwent a major ε-cleavage at the ε51 site. These results suggest that ε51, as well as ε48 cleavage, is associated with Aß42 production. Only AICDε51, though, and not Aß42 production, dramatically changed with modifications to the cell-free assay conditions. Interestingly, the increase in the relative ratio of the ε51 cleavage by the K649N mutation was not cancelled by these changes. CONCLUSION: Our current data show that the generation ratio of AICDε51 and Aß42 do not always change in parallel. Thus, to identify compounds that decrease the relative ratio of Aß42 generation, measurement of the relative level of Aß42-related AICD species (i.e., AICDε48 and AICDε51) might not be useful. Further studies to reveal how the ε-cleavage precision is decided are necessary before it will be possible to develop drugs targeting ε-cleavage as a means for decreasing Aß42 production.