Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes.
Mutations that cause familial Alzheimer's disease (FAD) are found in amyloid precursor protein (APP) and presenilin, the catalytic component of γ-secretase, that together produce amyloid β-peptide (Aβ). Nevertheless, whether Aβ is the primary disease driver remains controversial. We report here that FAD mutations disrupt initial proteolytic events in the multistep processing of APP substrate C99 by γ-secretase. Cryoelectron microscopy reveals that a substrate mimetic traps γ-secretase during the transition state, and this structure aligns with activated enzyme-substrate complex captured by molecular dynamics simulations. In silico simulations and in cellulo fluorescence microscopy support stabilization of enzyme-substrate complexes by FAD mutations. Neuronal expression of C99 and/or presenilin-1 in Caenorhabditis elegans leads to synaptic loss only with FAD-mutant transgenes. Designed mutations that stabilize the enzyme-substrate complex and block Aβ production likewise led to synaptic loss. Collectively, these findings implicate the stalled process-not the products-of γ-secretase cleavage of substrates in FAD pathogenesis.
Pubmed ID: 38349793
Research resources used in this publication
Additional research tools detected in this publication
- ATCC (RRID:SCR_001672)
- Cell Signaling Technology (RRID:SCR_002071)
- Fiji (RRID:SCR_002285)
- SigmaPlot (RRID:SCR_003210)
- Novus Biologicals (RRID:SCR_004286)
- HPA (RRID:SCR_006710)
- Assisted Model Building with Energy Refinement (AMBER) (RRID:SCR_014230)
- Microsoft Excel (RRID:SCR_016137)
- ThermoFisher Scientific EVOS M5000 Imaging System (RRID:SCR_023650)
Antibodies used in this publication
- Monoclonal ANTI-FLAG® M2 antibody produced in mouse (RRID:AB_262044)
- ANTI-FLAG(R) M2 Affinity Gel (RRID:AB_10063035)
- Purified (azide-free) anti-Presenilin 1 (NH2 terminus) (RRID:AB_2564868)
- Presenilin 1 antibody [EP2000Y] (RRID:AB_1310605)
- β-Amyloid (D54D2) XP® Rabbit mAb (RRID:AB_2797642)
- Purified (azide-free) anti-beta-Amyloid, 1-16 (RRID:AB_2564653)
- Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Cyanine3 (RRID:AB_2534029)
- Mouse Anti-GAPDH Monoclonal Antibody, Unconjugated, Clone D4C6R (RRID:AB_2756824)
- Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (RRID:AB_2633275)
Associated grants
- Agency: NIA NIH HHS, United States
Id: P30 AG072973 - Agency: NIA NIH HHS, United States
Id: R01 AG066986 - Agency: NIGMS NIH HHS, United States
Id: R01 GM122894 - Agency: NIA NIH HHS, United States
Id: RF1 AG079569
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ATCC (tool)
RRID:SCR_001672
Global nonprofit biological resource center (BRC) and research organization that provides biological products, technical services and educational programs to private industry, government and academic organizations. Its mission is to acquire, authenticate, preserve, develop and distribute biological materials, information, technology, intellectual property and standards for the advancement and application of scientific knowledge. The primary purpose of ATCC is to use its resources and experience as a BRC to become the world leader in standard biological reference materials management, intellectual property resource management and translational research as applied to biomaterial development, standardization and certification. ATCC characterizes cell lines, bacteria, viruses, fungi and protozoa, as well as develops and evaluates assays and techniques for validating research resources and preserving and distributing biological materials to the public and private sector research communities.
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RRID:SCR_002071
Privately held company that develops and produces antibodies, ELISA kits, ChIP kits, proteomic kits, and other related reagents used to study cell signaling pathways that impact human health.
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RRID:SCR_002285
Software package as distribution of ImageJ and ImageJ2 together with Java, Java3D and plugins organized into coherent menu structure. Used to assist research in life sciences.
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RRID:SCR_003210
Statistical analysis and scientific graphing software for Windows OS.
View all literature mentionsNovus Biologicals (tool)
RRID:SCR_004286
Commercial antibody vendor which supplies antibodies and other products to life science researchers.
View all literature mentionsHPA (tool)
RRID:SCR_006710
Public database with millions of high-resolution images showing the spatial distribution of proteins in different normal human tissues and cancer types, as well as different human cell lines. The data is released together with application-specific validation performed for each antibody, including immunohistochemisty, Western blot analysis and, for a large fraction, a protein array assay and immunofluorescent based confocal microscopy. The database has been developed in a gene-centric manner with the inclusion of all human genes predicted from genome efforts. Search functionalities allow for complex queries regarding protein expression profiles, protein classes and chromosome location. Antibodies included have been analyzed using a standardized protocol in a single attempt without further efforts to optimize the procedure and therefore it cannot be excluded that certain observed binding properties are due to technical rather than biological reasons and that further optimization could result in a different outcome. Submission of antibodies: The Swedish Human Proteome Atlas (HPA) program, invites submission of antibodies from both academic and commercial sources to be included in the human protein atlas. All antibodies will be validated by the HPA-program by a standard procedure and antibodies that are accepted will be use in the tissue- profiling program to generate high-resolution immunohistochemistry images representing a wide spectrum of normal tissues and cancer types.
View all literature mentionsAssisted Model Building with Energy Refinement (AMBER) (tool)
RRID:SCR_014230
Software package of molecular simulation programs. It is distributed into AmberTools15 and Amber14. AmberTools15 is a software package which can carry out complete molecular dynamics simulations with either explicit water or generalized Born solvent models. It is distributed in source code format and must be compiled in order to be used. Amber14 builds on AmberTools15 by adding the pmemd program, which provides better performance on multiple CPUs and dramatic speed improvements on GPUs compared to sander (molecular dynamics). GPU info, manuals, and tutorials are available on the website.
View all literature mentionsMicrosoft Excel (tool)
RRID:SCR_016137
Software application with data analysis tools and spreadsheet templates to track and visualize data. It is used to manage and process data.
View all literature mentionsThermoFisher Scientific EVOS M5000 Imaging System (tool)
RRID:SCR_023650
Microsoft system includes integrated digital inverted benchtop microscope for four-color fluorescence, transmitted-light, and color imaging. Provides interchangeable optics with autofocus, single-click multi-channel image acquisition.3.2 Megapixels, monochrome, CMOS camera. Offers software for acquisition, analysis, and automated cell counting.
View all literature mentionsMonoclonal ANTI-FLAG® M2 antibody produced in mouse (antibody)
RRID:AB_262044
This monoclonal targets FLAG
View all literature mentionsANTI-FLAG(R) M2 Affinity Gel (antibody)
RRID:AB_10063035
This unknown targets ANTI-FLAG(R) M2 Affinity Gel
View all literature mentionsPurified (azide-free) anti-Presenilin 1 (NH2 terminus) (antibody)
RRID:AB_2564868
This monoclonal targets Presenilin 1 NH2 terminus
View all literature mentionsPresenilin 1 antibody [EP2000Y] (antibody)
RRID:AB_1310605
This monoclonal targets Presenilin 1 antibody [EP2000Y]
View all literature mentionsβ-Amyloid (D54D2) XP® Rabbit mAb (antibody)
RRID:AB_2797642
This monoclonal targets APP
View all literature mentionsPurified (azide-free) anti-beta-Amyloid, 1-16 (antibody)
RRID:AB_2564653
This monoclonal targets beta-Amyloid 1-16
View all literature mentionsGoat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Cyanine3 (antibody)
RRID:AB_2534029
This polyclonal secondary targets IgG (H+L)
View all literature mentionsMouse Anti-GAPDH Monoclonal Antibody, Unconjugated, Clone D4C6R (antibody)
RRID:AB_2756824
This monoclonal targets GAPDH
View all literature mentionsGoat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (antibody)
RRID:AB_2633275
This polyclonal secondary targets IgG (H+L)
View all literature mentionsHEK293 (cell line)
RRID:CVCL_0045
Cell line HEK293 is a Transformed cell line with a species of origin Homo sapiens (Human)
View all literature mentionsUniversity of Kansas Microscopy and Analytical Imaging Research Resource Core Laboratory Core Facility (service resource)
RRID:SCR_021801
Core laboratory in Room 1043 in Haworth Hall at the University of Kansas Lawrence campus.Provides life and physical sciences and engineering users with wide spectrum of sample preparation tools and techniques and imaging technologies.Provides training for undergraduate, graduate students, postdoctoral researchers, faculty, staff, and industry collaborators.
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