<em>Saccharomyces cerevisiae</em> Models of Alzheimer's Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by &#947;-Secretase

Eugene Futai; Mio Shiina; Ying Dai; Khadija Daoudi; Masafumi Hidaka; Tomohisa Ogawa

doi:10.3791/67733

Saccharomyces cerevisiae Modèles de la maladie d’Alzheimer pour cribler les gènes, les m...

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JoVE Journal Neuroscience

Saccharomyces cerevisiae Models of Alzheimer’s Disease to Screen Genes, Mutations, and Chemicals Affecting Amyloid Beta Production by γ-Secretase

Saccharomyces cerevisiae Modèles de la maladie d’Alzheimer pour cribler les gènes, les mutations et les produits chimiques affectant la production de bêta-amyloïde par la γ-sécrétase

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11:57 min

June 24, 2025

DOI: 10.3791/67733-v

Eugene Futai¹, Mio Shiina¹, Ying Dai¹, Khadija Daoudi¹, Masafumi Hidaka¹, Tomohisa Ogawa¹

¹Department of Agricultural Chemistry, Graduate School of Agricultural Science,Tohoku University

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Overview

This study reconstitutes gamma-secretase in yeast to explore the impact of familial Alzheimer's disease mutations on its activity. The research aims to identify suppressor mutations and chemical modulators that can restore gamma-secretase function and reduce pathogenic Abeta 42 levels.

Key Study Components

Area of Science

Neuroscience
Biochemistry
Genetics

Background

Gamma-secretase is crucial for the production of Abeta peptides.
Familial Alzheimer's disease (FAD) mutations can impair gamma-secretase activity.
Understanding gamma-secretase modulation can lead to therapeutic strategies.
Yeast systems provide a simplified model for studying human proteins.

Purpose of Study

To investigate how FAD-linked mutations affect gamma-secretase activity.
To identify suppressor mutations that restore gamma-secretase function.
To screen for chemical modulators that reduce Abeta 42 levels.

Methods Used

Reconstitution of gamma-secretase in yeast.
Functional analysis using both mammalian cells and yeast systems.
Structural analysis via cryo-electron microscopy (cryo-EM).
Screening for genetic and chemical modulators of gamma-secretase.

Main Results

Identified suppressor mutations that restore gamma-secretase activity.
Demonstrated the ability to reduce Abeta 42 levels in yeast.
Established a rapid screening protocol for gamma-secretase activity.
Expanded the understanding of human intramembrane proteolysis.

Conclusions

The yeast system is effective for studying gamma-secretase modulation.
New genetic and chemical modulators can be identified through this approach.
This research contributes to potential therapeutic strategies for Alzheimer's disease.

Frequently Asked Questions

What is gamma-secretase?

Gamma-secretase is an enzyme complex that plays a key role in the cleavage of amyloid precursor protein, leading to the production of Abeta peptides.

How does the yeast system help in studying gamma-secretase?

The yeast system simplifies the study of human proteins, allowing for functional analysis and screening of mutations and modulators without complex mammalian systems.

What are familial Alzheimer's disease mutations?

These are genetic mutations linked to early-onset Alzheimer's disease that can disrupt normal gamma-secretase function.

What are gamma-secretase modulators?

Gamma-secretase modulators are compounds that can enhance or inhibit the activity of gamma-secretase, potentially altering Abeta peptide production.

What is the significance of reducing Abeta 42 levels?

Reducing Abeta 42 levels is important as its accumulation is associated with the development of Alzheimer's disease pathology.

What techniques were used to analyze gamma-secretase?

The study utilized functional analysis, structural analysis via cryo-EM, and a yeast-based reconstitution system.

Nous décrivons ici un système de levure reconstituant la γ-sécrétase humaine. Ce système permet d’identifier et d’étudier les mutations affectant l’activité et de cribler les inhibiteurs de la γ-sécrétase (GSI). En utilisant les propriétés de perte de fonction des mutants familiaux de la maladie d’Alzheimer, il est possible de dépister les modulateurs de la γ-sécrétase (GSM).

Nous reconstituons la gamma-sécrétase chez la levure afin d’étudier comment les mutations liées à la FAD impactent son activité et comment les mutations suppressrices ou modulateurs chimiques restaurent sa fonction et réduisent les niveaux pathogènes d’Abeta 42. Nous combinons l’analyse fonctionnelle utilisant le système cellulaire et de levure de mammifères avec l’analyse de structure via cryo-EM pour étudier le mécanisme de modulation gamma-sécrétase. Nous avons analysé les mutations de la FAD à l’aide d’un système de reconstitution gamma-sécrétase chez la levure et identifié les suppresseurs capables de restaurer l’activité des mutations de la FAD gamma-sécrétase, ainsi que de réduire le niveau d’Abeta 42, imitant l’effet des modulateurs gamma-sécrétase.

Notre protocole permet un dépistage fonctionnel rapide et évolutif de l’activité gamma-sécrétase chez la levure, permettant l’analyse des mutations humaines et des modulateurs sans dépendre de systèmes mammifères complexes. Nous nous concentrerons sur l’identification de nouveaux modulateurs génétiques ou chimiques et inhibiteurs de la gamma-sécrétase A, illustrant le mécanisme de production d’Abeta 42 et l’expansion des systèmes de reconstruction à base de levure dans un ensemble sous forme de protéolyse intramembranaire humaine. Pour commencer, transformez les plasmides en souche de levure PJ69-4A en utilisant la méthode standard de l’acétate de lithium pour exprimer la gamma-sécrétase et son substrat.

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