Kanta Yanagida

Regulation of Notch Signaling by Dynamic Changes in the Precision of S3 Cleavage of Notch-1†

ABSTRACT Intramembrane proteolysis by presenilin-dependent γ-secretase produces the Notch intracellular cytoplasmic domain (NCID) and Alzheimer disease-associated amyloid-β. 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.

γ-Secretase Modulators and Presenilin 1 Mutants Act Differently on Presenilin/γ-Secretase Function to Cleave Aβ42 and Aβ43

Deciphering the mechanism by which the relative Aβ42(43) to total Aβ ratio is regulated is central to understanding Alzheimer disease (AD) etiology; however, the mechanisms underlying changes in the Aβ42(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 Aβ42 into Aβ38, and Aβ43 into Aβ40 or Aβ38. Approximately 40% of Aβ38 is derived from Aβ43. Aβ42(43) cleavage is involved in the regulation of the Aβ42(43) ratio in living cells. GSMs increase the cleavage of PS/γ-secretase-bound Aβ42 (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 Aβ42(43) production process and propose how GSMs act, and we suggest that a loss of PS/γ-secretase function to cleave Aβ42(43) may initiate AD and might represent a therapeutic target.

The 28‐amino acid form of an APLP1‐derived Aβ‐like peptide is a surrogate marker for Aβ42 production in the central nervous system

Surrogate markers for the Alzheimer disease (AD)‐associated 42‐amino acid form of amyloid‐β (Aβ42) 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 Aβ‐like peptides (APL1β) that are generated by β‐ and γ‐cleavages at a concentration of ∼4.5 nM. These novel peptides, APL1β25, APL1β27 and APL1β28, were not deposited in AD brains. Interestingly, most γ‐secretase modulators (GSMs) and familial AD‐associated presenilin1 mutants that up‐regulate the relative production of Aβ42 cause a parallel increase in the production of APL1β28 in cultured cells. Moreover, in CSF from patients with pathological mutations in presenilin1 gene, the relative APL1β28 levels are higher than in non‐AD controls, while the relative Aβ42 levels are unchanged or lower. Most strikingly, the relative APL1β28 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 APL1β28 in the CSF as a candidate surrogate marker for the relative level of Aβ42 production in the brain.

Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor.

We recently demonstrated that endoplasmic reticulum (ER) stress induces sigma-1 receptor (Sig-1R) expression through the PERK pathway, which is one of the cell’s responses to ER stress. In addition, it has been demonstrated that induction of Sig-1R can repress cell death signaling. Fluvoxamine (Flv) is a selective serotonin reuptake inhibitor (SSRI) with a high affinity for Sig-1R. In the present study, we show that treatment of neuroblastoma cells with Flv induces Sig-1R expression by increasing ATF4 translation directly, through its own activation, without involvement of the PERK pathway. The Flv-mediated induction of Sig-1R prevents neuronal cell death resulting from ER stress. Moreover, Flv-induced ER stress resistance reduces the infarct area in mice after focal cerebral ischemia. Thus, Flv, which is used frequently in clinical practice, can alleviate ER stress. This suggests that Flv could be a feasible therapy for cerebral diseases caused by ER stress.

Introduction of a new scale into reversed-phase high-performance liquid chromatography of pyridylamino sugar chains for structural assignment

Addition of a monosaccharide residue to a pyridylaminated (PA)-N-linked sugar chain results in an increment or decrement in the elution time on reversed-phase HPLC, the difference being defined as the partial elution time of the residue. Based on this principle, an empirical rule was deduced, which states that the elution time is roughly equal to the sum of the partial elution times of the component sugar residues [Anal. Biochem., 167 (1987) 321-326]. In practice, however, some partial elution times obtained from different pairs of mother PA-sugar chains are found to deviate, and consequently the closeness of the elution times of PA-sugar chains calculated therefrom to the observed times is reduced in such cases. To improve the reliability of the additivity rule and to generalize elution times so that they are less dependent on minor alterations in the elution conditions, we have devised a new scale for elution time, which we have named a reversed-phase scale. The elution times on the reversed-phase scale (the R values) are read from a conversion curve constructed using the elution times of eight selected standard PA-sugar chains. The partial elution times on the reversed-phase scale of 22 monosaccharide residues were calculated from the R values of 93 PA-sugar chains. The R values obtained by summing the partial elution times of all the component monosaccharide residues became much closer to the R values obtained from the reversed-phase scale, compared to the results obtained using the previous method. In addition, the R values were less influenced by minor change in the elution conditions. These features of the new scale allow more accurate structural assignment of sugar chains.

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

Significance Genetic studies of common complex human diseases, including Alzheimers disease (AD), are extremely resource-intensive and have struggled to identify genes that are causal in disease. Combined with the costs of studies and the inability to identify the missing heritability, particularly in AD, alternate strategies warrant consideration. We devised a unique strategy that combines distinct mouse strains that vary naturally in amyloid-β production with transcriptomics to identify kinesin light chain-1 (Klc1) splice variant E as a modifier of amyloid-β accumulation, a causative factor of AD. In AD patients, the expression levels of KLC1 variant E in brain were significantly higher compared with levels in unaffected individuals. The identification of KLC1 variant E suggests that dysfunction of intracellular trafficking is causative in AD. Alzheimer’s disease (AD) is characterized by the accumulation of amyloid-β (Aβ). 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 Aβ accumulation compared with SJL and C57BL/6 mice. We then applied brain transcriptomics to reveal the genes in DBA/2 that suppress Aβ accumulation. To avoid detecting secondarily affected genes by Aβ, we used non-Tg mice in the absence of Aβ 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 Aβ modifier, indicating a role for intracellular trafficking in Aβ accumulation. Aβ 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 Aβ, respectively. The identification of KLC1 variant E suggests that the dysfunction of intracellular trafficking is a causative factor of Aβ 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.

Processes of β-Amyloid and Intracellular Cytoplasmic Domain Generation by Presenilin/γ-Secretase

Background/Aims: Following extracellular shedding, transmembrane domains (TMs) of β-amyloid precursor protein (βAPP) and Notch-1 undergo proteolysis by presenilin (PS)/γ-secretase at least at two sites, near the middle of the TM (γ-/S4 cleavage) and at the interface between cytosol and the TM (Ε-/S3 cleavage), releasing Alzheimer disease (AD)-associated β-amyloid (Aβ)/Notch-1β (Nβ) and βAPP intracellular cytoplasmic domain (AICD)/Notch-1 intracellular cytoplasmic domain (NICD). Inhibiting PS/γ-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. γ-/S4 and Ε-cleavages have diversity, and produce a number of Aβ/Nβ 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 γ-/S4 cleavage precision, suggesting a common process for these cleavages near the middle of the TM. While the precision of the Ε-cleavage is drastically affected by physiological factors, that of Ε-/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 Aβ.

The production ratios of AICDε51 and Aβ42 by intramembrane proteolysis of βAPP do not always change in parallel

Background:  During intramembrane proteolysis of β‐amyloid protein precursor (βAPP) 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 Aβ42, a critical molecule for Alzheimers disease (AD) pathogenesis, and the major Aβ40 species might be associated with ε48 and ε49 cleavages, respectively. Medicines to downregulate Aβ42 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 Aβ42. Thus, one might evaluate compounds by analyzing the generation ratio of the βAPP intracellular domain (AICD) species (ε‐cleavage‐derived), instead of that of Aβ42.

Relative Ratio and Level of Amyloid-β 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-β 42 (Aβ42) to Aβ40 ratio secreted in cultured cells. Some of these mutants reduce the secretion of Aβ40 rather than increase that of Aβ42. Since it has been difficult to estimate Aβ42 secretion in brains of PS1-FAD patients due to substantial Aβ42 accumulation, it remains unknown whether the enhanced Aβ42 to Aβ40 ratio in brains of FAD patients is caused by elevated Aβ42 secretion or by reduced secretion of Aβ40. 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 Aβ-like peptide (APL1β), including APL1β28, an Aβ42 surrogate marker, were quantified by liquid chromatography tandem mass spectrometry, and Aβ42 secretion in the brain was estimated. Results: The relative ratio of CSF APL1β28 to total APL1β was higher in PS1-FAD patients than in controls. Importantly, CSF APL1β28 was not significantly higher. However, C-terminally shorter CSF APL1β25 and APL1β27 were significantly lower in PS1-FAD patients and, as expected, so were CSF Aβ40 and Aβ42. Conclusion: A higher relative ratio of the CSF Aβ42 surrogate in PS1-FAD patients is not due to its increase in CSF, suggesting that massive Aβ42 accumulation in the PS1-FAD brain occurs without an apparent increase in Aβ42 secretion.

Destruxin E Decreases Beta-Amyloid Generation by Reducing Colocalization of Beta-Amyloid-Cleaving Enzyme 1 and Beta-Amyloid Protein Precursor

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