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Bioinformatics, Computational and Systems Biology

Bioinformatics, Computational and Systems Biology - Poster Session D

Poster M11 - Alzheimer’s Disease-Associated Variants Impair TREM2 Multimerization

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Rory Greer (he/him/his)

Graduate Student
University of Alabama at Birmingham
Birmingham, Alabama, United States

Co-Author(s)

Hunter B. Dean, PhD (he/him/his)

MD/PhD Student
University of Alabama at Birmingham, United States
SY

Shan-Zhong Yang

Scientist
University of Alabama at Birmingham, United States
DE

Daniel Elston

MD Student
University of Alabama at Birmingham, United States
TB

Thomas Brett

Associate Professor of Medicine
Washington University School of Medicine in St. Louis, United States
ER

Erik Roberson

Professor of Neurology
University of Alabama at Birmingham, United States

Last Author(s)

Yuhua Song, PhD (she/her/hers)

Professor in the Department of Biomedical Engineering
University of Alabama at Birmingham
Birmingham, Alabama, United States

Introduction: Triggering Receptor Expression on Myeloid Cells 2 (TREM2) is an innate immune receptor expressed on microglia and is one of the strongest genetic risk factors for Alzheimer’s Disease (AD). X-ray crystallography studies have described potential common variant (CV) TREM2 multimers in the past, including a loosely packed TREM2 dimer in a tail-to-tail orientation as part of a larger TREM2 hexamer (a core trimer surrounded by three loosely associated subunits). Notably, crystal structures of TREM2 multimers fail to form in the presence of the AD-associated variant R47H. Additionally, studies developing antibodies that can divalently engage TREM2 have shown these antibodies can induce downstream signaling while monovalent engagement of TREM2 cannot. Further, antibodies engineered with four variable domains can induce TREM2-acitivation at 100-fold lower concentration than divalent antibodies, suggesting multimerization may play a role in TREM2 function. However, the importance of TREM2 multimerization for signaling and regulating TREM2-dependent functions, as well as the effect AD-associated variants have on the TREM2 multimers remains poorly understood.

Materials and

Methods: Using a combination of classical all-atom molecular dynamics (MD) simulations along with binding free energy analysis, we studied the effects of AD-associated TREM2 variants on the multimerization of TREM2. To further analyze TREM2 multimerization we utilized steered MD (SMD) simulations to determine if AD-associated TREM2 variants affect the forces required to dissociate a TREM2 trimer. To validate our computational findings on TREM2 multimerization, we utilized co-immunoprecipitation (co-IP) assays to determine if AD-associated variants could reduce or inhibit TREM2 multimerization.

Results, Conclusions, and Discussions: Our MD simulations show CV TREM2 trimers remain stably bound through an interaction primarily driven by a salt bridge between residues D87 and R76 at the interface of the TREM2 trimer. We further show AD-associated variants R47H and R98W both reduce binding affinity of the trimer, while D87N causes the dissociation of the trimer in our simulations. Notably, these AD-associated variants weaken the trimer’s interactions by disrupting the R76-D87 salt bridge. To further determine the effects of AD-associated variants on the TREM2 trimers we ran SMD simulations, showing R47H and D87N variants require similar levels of forces to dissociate the trimer. Our computational experiments additionally predict acidic environments weaken the trimer’s binding affinity, while basic environments and variants engineered to introduce a second salt bridge to the binding interface strengthen the trimer’s binding affinity. In our experimental validation, we show TREM2 can exists as a monomer as well as multimers in our native PAGE experiments. Additionally, through our co-IP assays, we show both AD-associated variants R47H and D87N decrease TREM2 multimerization. Further, through additional co-IP assays we show reducing the pH environment from 7.4 to 6.4 does reduce multimerization while increasing the pH to 8.4 increases multimerization.

Through using an integrated computational prediction and experimental validation approach, we have shown TREM2 multimers do exist and are affected by AD-associated TREM2 variants R47H and D87N. Additionally, we show a salt bridge between residues R76 and D87 plays an important role for the formation of these multimers, and AD-associated TREM2 variants weaken this interaction. Further, we have shown introducing a second engineered salt bridge between TREM2 subunits and altering the environmental pH can affect binding affinity of the TREM2 multimer. This work offers important insight for therapeutic strategies targeting TREM2, as strategies meant to monovalently engage TREM2 may not be as effective as treatments meant to target TREM2 multimers, and may fail to replicate multimerization effects displayed in TREM2-targeting antibody therapies. Moving forward, we plan to test if small molecule TREM2 ligands we have identified can regulate TREM2 multimerization and counteract lost multimerization due to AD-associated variants.

Acknowledgements (Optional): This work was supported by the National Institutes of Health (T32GM008361, T32NS095775, T32EB023872, R01AG068395, R01AG081228, and P20AG068024), Alzheimer's of Central Alabama, the Alzheimer's Drug Discovery Foundation, Alzheimer's Association (AARG-16-441560), and BrightFocus Foundation (A2022032S).