Ribavirin Treatment for Severe Schizophrenia with Anti-Borna Disease Virus 1 Antibodies 30 Years after Onset.

Objective: Borna disease virus 1 (BoDV-1) was proven to cause fatal encephalitis in humans in 2018. However, the effects of persistent infections remain unclear. Here, we present the case of a 50-year-old woman with a 30-year history of severe schizophrenia, who was exposed to fleas from stray cats prior to disease onset, suggesting the possibility of zoonosis including BoDV-1 infection. The patient had experienced significant social impairment, thought deterioration, delusions, and hallucinations for more than 20 years. Method: A radioligand assay was used to test the patient for IgG and IgM antibodies against BoDV-1 nucleoprotein (N) and phosphoprotein (P). Based on the protocol for hepatitis C, we treated the patient with 400 mg/day ribavirin, which was later increased to 600 mg/day. Results: The serological examination revealed anti-BoDV-1 N IgG. Although only subtle changes were observed over the 24 weeks of treatment, the family noticed that the patient's Cotard delusions had disappeared 7 months after completing the treatment, accompanied by some improvements in the relationship with the family. Conclusion: Though definite proof was not obtained, this presumed suppression of BoDV-1 by ribavirin leading to improvements in Cotard syndrome-like symptoms suggests that intractable schizophrenia might be one of the BoDV-1 infection phenotypes. Further studies are needed to clarify the effect of persistent BoDV-1 infections in humans.

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.

Plasma NfL is associated with mild cognitive decline in patients with diabetes.

BACKGROUND: Patients with diabetes are at a higher risk for cognitive decline. Thus, biomarkers that can provide early and simple detection of cognitive decline are required. Neurofilament light chain (NfL) is a cytoskeletal protein that constitutes neural axons. Plasma NfL levels are elevated when neurodegeneration occurs. Here, we investigated whether plasma NfL levels were associated with cognitive decline in patients with type 2 diabetes. METHOD: This study included 183 patients with type 2 diabetes who visited Osaka University Hospital. All participants were tested for cognitive function using the Mini-Mental State Examination (MMSE) and the Rivermead Behavioural Memory Test (RBMT). NfL levels were analysed in the plasma and the relationship between NfL and cognitive function was examined. RESULTS: Lower RBMT-standardized profile scores (SPS) or MMSE scores correlated with higher plasma NfL levels (one-way analysis of variance: MMSE, P = 0.0237; RBMT-SPS, P = 0.0001). Furthermore, plasma NfL levels (ß = -0.34, P = 0.0005) and age (ß = -0.19, P = 0.016) were significantly associated with the RBMT score after multivariable regression adjustment. CONCLUSIONS: Plasma NfL levels were correlated with mild cognitive decline which is detected by the RBMT but not the MMSE in patients with type 2 diabetes. This suggests that plasma NfL levels may provide a valuable clinical tool for identifying mild cognitive decline in patients with diabetes.

Association of weekday-to-weekend sleep differences and stress response among a Japanese working population: a cross-sectional study.

OBJECTIVES: To investigate the relationship between differences in weekday-to-weekend sleep habits and stress responses in a working population. METHODS: This cross-sectional study used data from university workers on sleep habits, differences in sleep duration between weekdays and weekends, and each midpoint of the sleep phase on weekdays and weekends. Social jetlag was defined as the difference in the midpoint of the sleep phase between weekdays and weekends. In addition, the Brief Job Stress Questionnaire assessed stress responses and stress-related factors. To examine sleep-related factors affecting stress responses, regression analysis was performed with adjustments for age, sex, and stress-related factors. RESULTS: Analyzed were 2,739 participants. Sleep duration differences obtained by subtracting sleep duration on weekdays from that on weekends, social jetlag, and weekday sleep duration were significantly associated with an increased risk of stress responses in a univariate linear regression model. Adjusting for age, sex, job stressors, and stressor buffering factors did not change this trend. However, when additionally adjusting for all sleep parameters, only sleep duration differences and weekday sleep duration were significantly associated with stress responses (ß 0.67 [95% CI 0.24, 1.10], p = 0.002), (-0.66 [-1.20, -0.13], p = 0.015). CONCLUSIONS: This study provided further evidence that weekday sleep duration and weekday-to-weekend sleep duration differences were independently associated with stress responses even when considering stress-related factors. However, social jetlag was not clearly associated with stress responses. Our findings highlighted the necessity of securing sufficient sleep for stress management and mental health promotion in a working population.

Renal function is associated with blood neurofilament light chain level in older adults.

Neurofilament light chain (NfL) is a novel biomarker of neurodegenerative diseases. It is detectable in the peripheral blood, allowing low-invasive assessment of early signs of neurodegeneration. The level of NfL gradually increases with age; however, what other factors affect it remains unclear. The present study examined the association between blood NfL level and renal function among healthy participants undergoing a health check (n = 43, serum NfL) and patients with diabetes mellitus (n = 188, plasma NfL). All participants were 60 years of age or older; none were diagnosed with dementia. In each group, levels of blood NfL and serum creatinine significantly correlated (coefficient r = 0.50, 0.56). These associations remained statistically significant even after adjustment for age, sex, and body mass index. These findings indicate that blood NfL level might be partially affected by renal function. We recommend measuring renal function for a more precise evaluation of neuroaxonal damage, in particular, among older adults.

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.

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.

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.

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.

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.

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.

[Surrogate marker for ABeta42].

Currently, therapeutic intervention for Alzheimer disease (AD) after the disease onset is not very effective because progressive neuronal death precedes clinical symptoms. Available medicines such as AchE inhibitors transiently slow the progression of the symptoms, but they do not inhibit the pathological process. On the other hand, most of the next generation anti-AD drugs under development in pharmaceutical companies are to function to inhibit the progress of the pathological process and, thus, establishment of highly probable prediction of AD onset is necessary. AD is diagnosed using clinical criteria coupled with brain imaging systems such as SPECT and PET. To diagnose AD cases before the onset, it will be necessary to develop methods for detecting the pathological accumulation of proteins (e.g., Abeta in the brain, or develop biomarkers for predicting Abeta/tau accumulation in the brain. Our recent discovery of APL1beta28, a possible biomarker of AD, may contribute to early detection of AD.

Destruxin E decreases Beta-amyloid generation by reducing colocalization of beta-amyloid-cleaving enzyme 1 and beta-amyloid protein precursor.

Alzheimer-disease-associated beta-amyloid (Abeta) is produced by sequential endoproteolysis of beta-amyloid protein precursor (betaAPP): the extracellular portion is shed by cleavage in the juxtamembrane region by beta-amyloid-cleaving enzyme (BACE)/beta-secretase, after which it is cleaved by presenilin (PS)/gamma-secretase near the middle of the transmembrane domain. Thus, inhibition of either of the secretases reduces Abeta generation and is a fundamental strategy for the development of drugs to prevent Alzheimer disease. However, it is not clear how small compounds reduce Abeta production without inhibition of the secretases. Such compounds are expected to avoid some of the side effects of secretase inhibitors. Here, we report that destruxin E (Dx-E), a natural cyclic hexadepsipeptide, reduces Abeta generation without affecting BACE or PS/gamma-secretase activity. In agreement with this, Dx-E did not inhibit Notch signaling. We found that Dx-E decreases colocalization of BACE1 and betaAPP, which reduces beta-cleavage of betaAPP. Therefore, the data demonstrate that Dx-E represents a novel Abeta-reducing process which could have fewer side effects than secretase inhibitors.

The 28-amino acid form of an APLP1-derived Abeta-like peptide is a surrogate marker for Abeta42 production in the central nervous system.

Surrogate markers for the Alzheimer disease (AD)-associated 42-amino acid form of amyloid-beta (Abeta42) have been sought because they may aid in the diagnosis of AD and for clarification of disease pathogenesis. Here, we demonstrate that human cerebrospinal fluid (CSF) contains three APLP1-derived Abeta-like peptides (APL1beta) that are generated by beta- and gamma-cleavages at a concentration of approximately 4.5 nM. These novel peptides, APL1beta25, APL1beta27 and APL1beta28, were not deposited in AD brains. Interestingly, most gamma-secretase modulators (GSMs) and familial AD-associated presenilin1 mutants that up-regulate the relative production of Abeta42 cause a parallel increase in the production of APL1beta28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1beta28 levels are higher than in non-AD controls, while the relative Abeta42 levels are unchanged or lower. Most strikingly, the relative APL1beta28 levels are higher in CSF from sporadic AD patients (regardless of whether they are at mild cognitive impairment or AD stage), than those of non-AD controls. Based on these results, we propose the relative level of APL1beta28 in the CSF as a candidate surrogate marker for the relative level of Abeta42 production in the brain.

Processes of beta-amyloid and intracellular cytoplasmic domain generation by presenilin/gamma-secretase.

BACKGROUND/AIMS: Following extracellular shedding, transmembrane domains (TMs) of beta-amyloid precursor protein (betaAPP) and Notch-1 undergo proteolysis by presenilin (PS)/gamma-secretase at least at two sites, near the middle of the TM (gamma-/S4 cleavage) and at the interface between cytosol and the TM (epsilon-/S3 cleavage), releasing Alzheimer disease (AD)-associated beta-amyloid (Abeta)/Notch-1beta (Nbeta) and betaAPP intracellular cytoplasmic domain (AICD)/Notch-1 intracellular cytoplasmic domain (NICD). Inhibiting PS/gamma-secretase activity is an essential approach to AD treatment, but it also decreases NICD production, which may cause severe side effects. Therefore, it is important to investigate the differences between the cleavages at the two sites. Gamma-/S4 and epsilon-cleavages have diversity, and produce a number of Abeta/Nbeta and AICD species. S3 cleavage diversity has been recently identified. It is significant that each cleavage occurs with strict precision, not randomly. METHODS: Biochemical analysis of cultured cells was performed to explore the processing mechanisms. RESULTS: Familial AD-associated PS1 mutations as well as a subset of nonsteroidal anti-inflammatory drugs cause similar changes in gamma-/S4 cleavage precision, suggesting a common process for these cleavages near the middle of the TM. While the precision of the epsilon-cleavage is drastically affected by physiological factors, that of epsilon-/S3 cleavage is not. CONCLUSION: The processes of the two cleavages occurring in different portions of TMs may be diverse, thus representing possible targets for anti-AD therapeutics to selectively reduce Abeta.

Regulation of Notch signaling by dynamic changes in the precision of S3 cleavage of Notch-1.

Intramembrane proteolysis by presenilin-dependent gamma-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-beta. Here, we show that upon Notch signaling the intracellular domain of Notch-1 is cleaved into two distinct types of NICD species due to diversity in the site of S3 cleavage. Consistent with the N-end rule, the S3-V cleavage produces stable NICD with Val at the N terminus, whereas the S3-S/S3-L cleavage generates unstable NICD with Ser/Leu at the N terminus. Moreover, intracellular Notch signal transmission with unstable NICDs is much weaker than that with stable NICD. Importantly, the extent of endocytosis in target cells affects the relative production ratio of the two types of NICD, which changes in parallel with Notch signaling. Surprisingly, substantial amounts of unstable NICD species are generated from the Val-->Gly and the Lys-->Arg mutants, which have been reported to decrease S3 cleavage efficiency in cultured cells. Thus, we suggest that the existence of two distinct types of NICD points to a novel aspect of the intracellular signaling and that changes in the precision of S3 cleavage play an important role in the process of conversion from extracellular to intracellular Notch signaling.