Background:The ␥-secretase complex is a drug target in the treatment of Alzheimer disease (AD). Results: Two novel second generation ␥-secretase modulators (GSMs) modulate both N␤ and A␤ but not Notch intracellular domain (NICD) production. Conclusion: Second generation and NSAID-based GSMs have different modes of action regarding Notch processing. Significance: GSMs that do not affect NICD signaling are essential for the development of tolerable AD therapeutics.

IntroductionUnhealthy lifestyle behaviours continue to be highly prevalent, including alcohol consumption, unhealthy diets, insufficient physical activity and smoking. There is a lack of effective interventions which have a large enough reach into the community to improve public health. Additionally, the common co-occurrence of multiple unhealthy behaviours demands investigation of efforts which address more than single behaviours.Methods and analysisThe effects of six components of a novel digital multiple health behaviour change intervention on alcohol consumption, diet, physical activity and smoking (coprimary outcomes) will be estimated in a factorial randomised trial. The components are designed to facilitate behaviour change, for example, through goal setting or increasing motivation, and are either present or absent depending on allocation (ie, six factors with two levels each). The study population will be those seeking help online, recruited through search engines, social media and lifestyle-related websites. Included will be those who are at least 18 years of age and have at least one unhealthy behaviour. An adaptive design will be used to periodically make decisions to continue or stop recruitment, with simulations suggesting a final sample size between 1500 and 2500 participants. Multilevel regression models will be used to analyse behavioural outcomes collected at 2 months and 4 months postrandomisation.Ethics and disseminationApproved by the Swedish Ethical Review Authority on 2021-08-11 (Dnr 2021-02855). Since participation is likely motivated by gaining access to novel support, the main concern is demotivation and opportunity cost if the intervention is found to only exert small effects. Recruitment began on 19 October 2021, with an anticipated recruitment period of 12 months.Trial registration numberISRCTN16420548.

Summary We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome—a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.

Background: g-Secretase plays a crucial role in the development of Alzheimer's disease since it participates in the generation of the toxic amyloid ß-peptide (Aß) from the amyloid precursor protein (APP). g-Secretase is a membrane bound protein complex consisting of four components; presenilin (PS), nicastrin, Aph-1, and Pen-2. While the catalytic core of the g-secretase complex is strongly associated with PS, less is known about the other components. The function of nicastrin has been debated during the last years. Despite several studies of the nicastrin protein, it is still unclear whether it is involved in substrate selectivity or has a more general role in the stabilization and maturation of the g-secretase complex. An artificial double mutant in PS1, F411Y/S438P, has previously been reported to be independent of nicastrin for g-secretase activity. Here, we focus on exploring the mechanism behind this in order to better understand the role of nicastrin for the g-secretase complex's assembly and activity. Methods: We are using co-immunoprecipitation and affinity capturing methods as well as a luciferase reporter gene assay to analyze the composition of the g-secretase complex and its activity on APP and Notch processing. All experiments are based on nicastrin deficient cells. We have also investigated the insertion efficiency of the transmembrane domains with and without the PS1 double mutant by using an assay based on N-linked glycosylation. Results: We can confirm that the PS1 double mutant F411Y/S438P is able to process both APP and Notch without nicastrin but with reduced activity. Moreover, we have found that the membrane integration is not changed compared to wild type PS1 and results regarding the changed conformation of the catalytic site will be presented. Conclusions: By understanding how the PS1 double mutant F411Y/S438P can form active gsecretase complexes in the absence of nicastrin, we will get more insight into nicastrin's function within the complex. P2-315BACE1 ACTIVITY IS MODULATED BY CELL-ASSOCIATED SPHINGOSINE-1-PHOSPHATE Background: BACE1 is a major beta-secretase for production of amyloidbeta peptide (Abeta). BACE1 resides in the lipid raft, a membrane microdomain enriched in cholesterol and sphingolipids, and a significant role of lipids and microdomain is implicated in the regulation of the beta-cleavage. In this study, we focused on a biologically active lipid metabolite, sphingosine-1-phosphate (S1P) in the regulation of BACE1 activity. Methods: We analyzed the impact of cellular S1P levels on BACE1 activity in neuronal cells as well as animal model using small compounds and RNAi knockdown. Moreover, we analyzed the brains of patients with sporadic AD. Results: We show that modulation of sphingosine kinases and S1P degrading enzymes, which are responsible enzymes for S1P production and catabolism, respectively, altered the Abeta generation by regulating the beta-cleavage. We found that BACE1 protein was specifically pulled down by S1P-coated beads, suggesting that modulatory action ...