Characterization of Mouse Models of Aging and Alzheimer’s Disease using Protein Stability Measurements

Abstract

A recently developed suite of mass spectrometry-based proteomic methods allows for the large-scale evaluation of protein folding stability. These techniques employ chemical or thermal denaturation approaches (such as SPROX and TPP) as well as proteolysis strategies (including DARTS, LiP, and PP) to assess protein stability. While these methods have been proven effective for protein target discovery, there is still limited understanding of the relative strengths and weaknesses of each technique when it comes to characterizing biological phenotypes. Therefore, in chapter 2 of this dissertation, it presents a comparative study of SPROX, TPP, and LiP in characterizing aging in mice model. By assessing proteins in brain tissue cell lysates, the study highlights the complementarity of these methods. This work also introduces a novel peptide-level analysis of TPP data, which could help for accurate interpretation of phenotype analyses. The comparative approach underscores the need for multiple techniques to fully capture protein stability changes.With the knowledge of that using multiple techniques could comprehensively profile a phenotype and reduce false positives rate, chapter 3 describes these three methods (SPROX, TPP, and LiP) in profiling the proteins in hippocampal tissue cell lysates to study the development and progression of AD in the mouse model. In these series of experiments, each technique detected few hundreds of protein hits with AD-related stability changes at different age points, highlighting the distinct advantages of these methods over traditional protein expression analysis. Key findings include the identification of 25 high-value conformation biomarkers, the consistent behavior of Clusterin in AD between mouse brain samples and human CSF samples, and subunit- and age-specific proteasome stability changes, which were linked to dysfunction at both early and late stages of AD. Chapter 4 extends this research to male mice, aiming to explore the similarities and differences in AD progression across genders. The results show that males exhibit a less pronounced phenotype than females, with fewer hits identified at early time points. Additionally, the 48 overlapping protein hits between males and females are enriched in AD pathway, and many of these don’t have expression level changes, indicating that stability measurements are sensitive to subtle protein folding changes. Proteasome behavior differs between males and females at early stages, but becomes consistent at later stages, with activity assay results aligning with the observed stability differences. Furthermore, this work focuses on establishing a connection between protein folding stability and function. In chapter 5, the stability and function of glutamate decarboxylase 2 (Gad2), a protein exhibits age-related destabilization in older mice, were examined. The observed stability change was attributed to one proteoform of Gad2 having significantly higher thermal stability compared to the other. Overall, this dissertation explores the power of integrated protein stability profiling techniques in identifying biomarkers and potential drug targets, as well as elucidating molecular mechanisms underlying stability change.