Kouhei Nishitomi

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.

BACE1 inhibition reduces endogenous Abeta and alters APP processing in wild-type mice.

Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimers disease (AD). Inhibition of beta‐site amyloid precursor protein (APP)‐cleaving enzyme‐1 (BACE1), the enzyme that initiates Abeta production, and other Abeta‐lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta‐lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non‐transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full‐length Abeta. A newly developed ELISA detected a significant reduction of full‐length soluble Abeta 1–40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x‐40 Abeta was moderately reduced due to detection of non‐full‐length Abeta and compensatory activation of alpha‐secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non‐transgenic mice provide more accurate evaluation of Abeta‐reducing strategies than was previously feasible.

Avenolide, a Streptomyces hormone controlling antibiotic production in Streptomyces avermitilis

Gram-positive bacteria of the genus Streptomyces are industrially important microorganisms, producing >70% of commercially important antibiotics. The production of these compounds is often regulated by low-molecular-weight bacterial hormones called autoregulators. Although 60% of Streptomyces strains may use γ-butyrolactone–type molecules as autoregulators and some use furan-type molecules, little is known about the signaling molecules used to regulate antibiotic production in many other members of this genus. Here, we purified a signaling molecule (avenolide) from Streptomyces avermitilis—the producer of the important anthelmintic agent avermectin with annual world sales of

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

850 million—and determined its structure, including stereochemistry, by spectroscopic analysis and chemical synthesis as (4S,10R)-10-hydroxy-10-methyl-9-oxo-dodec-2-en-1,4-olide, a class of Streptomyces autoregulator. Avenolide is essential for eliciting avermectin production and is effective at nanomolar concentrations with a minimum effective concentration of 4 nM. The aco gene of S. avermitilis, which encodes an acyl-CoA oxidase, is required for avenolide biosynthesis, and homologs are also present in Streptomyces fradiae, Streptomyces ghanaensis, and Streptomyces griseoauranticus, suggesting that butenolide-type autoregulators may represent a widespread and another class of Streptomyces autoregulator involved in regulating antibiotic production.

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

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.

Deglycosylated anti‐amyloid beta antibodies reduce microglial phagocytosis and cytokine production while retaining the capacity to induce amyloid beta sequestration

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.

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

Accumulation of amyloid beta (Abeta) is a pathological hallmark of Alzheimers disease, and lowering Abeta is a promising therapeutic approach. Intact anti‐Abeta antibodies reduce brain Abeta through two pathways: enhanced microglial phagocytosis and Abeta transfer from the brain to the periphery (Abeta sequestration). While activation of microglia, which is essential for microglial phagocytosis, is necessarily accompanied by undesired neuroinflammatory events, the capacity for sequestration does not seem to be linked to such effects. We and other groups have found that simple Abeta binding agents are sufficient to reduce brain Abeta through the sequestration pathway. In this study, we aimed to eliminate potentially deleterious immune activation from antibodies without affecting the ability to induce sequestration. The glycan portion of immunoglobulin is critically involved in interactions with immune effectors including the Fc receptor and complement c1q; deglycosylation eliminates these interactions, while antigen (Abeta)‐binding affinity is maintained. In this study, we investigated whether deglycosylated anti‐Abeta antibodies reduce microglial phagocytosis and neuroinflammation without altering the capacity to induce Abeta sequestration. Deglycosylated antibodies maintained Abeta binding affinity. Deglycosylated antibodies did not enhance Abeta phagocytosis or cytokine release in primary cultured microglia, whereas intact antibodies did so significantly. Intravenous injection of deglycosylated antibodies elevated plasma Abeta levels and induced Abeta sequestration to a similar or greater degree compared with intact antibodies in an Alzheimers transgenic mouse model without or with Abeta plaque pathology. We conclude that deglycosylated antibodies effectively induced Abeta sequestration without provoking neuroinflammation; thus, these deglycosylated antibodies may be optimal for sequestration therapy for Alzheimers disease.

Macrophage colony stimulating factor is associated with excretion of amyloid‐β peptides from cerebrospinal fluid to peripheral blood

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.

PRECLINICAL MULTI-SPECIES PHARMACOKINETIC/PHARMACODYNAMIC ANALYSIS OF THE ORAL BACE INHIBITOR JNJ-54861911

Background:  The process of aggregation of brain amyloid‐β peptides (Aβ) is thought to be associated with the pathogenesis of Alzheimers disease (AD). Amyloid‐β peptides are produced by sequential endoproteolysis by β‐site amyloid‐β protein precursor‐cleaving enzyme (BACE) followed by presenilin (PS)/γ‐secretase. There are several species of Aβ due to cleavage diversity of PS/γ‐secretase. The predominant species in human cerebrospinal fluid (CSF) or plasma is Aβ40, whereas Aβ42 is much more aggregatable and accumulated in senile plaques. The level of Aβ in the brain is determined by the balance between the generation and clearance of Aβ, including transport across the brain–blood barrier (BBB). Although the processes of Aβ generation and degradation have been studied in some detail, knowledge of the Aβ transport process across the BBB is limited. So far, low‐density lipoprotein receptor‐related protein (LRP1), P‐glycoprotein (P‐gp), and insulin‐like growth factor‐1 (IGF‐1) have been identified to modify the excretion of brain Aβ to the blood.

P2-366: Inhibition of Aβ production without perturbing Notch and Neuregulin signaling

analysis of cognition collaboration’ (OA-Cog) aims to identify the most efficient cognitive measurement and analysis technique for cognition data and dementia in randomised controlled trials including patients with or at risk of vascular dementia or Alzheimer’s disease. Methods: Chief investigators of randomised controlled trials with cognitive outcome assessments are asked to share individual patient data from their trials. Variables requested include baseline prognostic factors, treatment group, cognitive measures (e.g. Mini Mental State Examination (MMSE), Alzheimer’s Disease Assessment Scale cognitive sub-score (ADAS-cog)) and other outcome measures (e.g. death, dementia). Shared trial data are merged into a single dataset and analysed using various endpoints (e.g. mean MMSE score at end of trial, MMSE score as a gradient over time) and statistical methods (e.g. Wilcoxon rank-sum test, repeated measures ANOVA) in order to identify which is the most efficient approach. Methods for dealing with missing data and, in particular, the case of missing data due to death will be addressed; currently, such patients are often ignored from analyses. Results: As of 23 December 2016, data from 32 clinical trials have been shared with the collaboration. Some of these trials have more than two treatment arms, so 50 datasets are available with a total of 120,576 participants. Conclusions:Optimising the design and analysis of cognition trials will allow future trials to detect smaller but still clinically important effects, and/or have smaller sample sizes than current trials.