Resolving Alzheimer’s Disease Heterogeneity Through Proteome-Based Subtyping Restricted; Files Only

Abstract

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, yet our comprehension predominantly relies on studies within non-Hispanic White (NHW) populations. Here we provide an extensive survey of the proteomic landscape of AD using large-scale proteomic datasets from racially and ethnically diverse postmortem brain samples to investigate molecular disparities in AD and define biologically relevant disease subtypes.

First, we performed tandem mass tag - mass spectrometry (TMT-MS) on 1105 dorsolateral prefrontal cortex (DLPFC) samples from 998 individuals and 280 superior temporal gyrus (STG) samples from 244 individuals, representing one of the largest racially diverse AD brain proteome cohorts to date. After rigorous quality control, we quantified over 9000 proteins per region and analyzed differential abundance across race and diagnosis. Our results showed while amyloid precursor protein and the microtubule-associated protein tau demonstrated higher abundance in AD brains, no significant race-related differences were observed. Further proteome-wide and focused analyses (specific amyloid beta [Aβ] species and the tau domains) supported the absence of racial differences in these AD pathologies within the brain proteome. Our findings indicate that the racial differences in AD risk and clinical presentation are not underpinned by dramatically divergent patterns in the brain proteome, suggesting that other determinants account for these clinical disparities. Next, we used a separate cohort from ROSMAP (Religious Orders Study and Memory and Aging Project), we applied co-expression network analysis and unsupervised clustering to 1017 DLPFC samples to identify molecular subtypes of AD. This revealed four reproducible proteomic subtypes. Notably, subtype C was enriched for glial proteins and showed lower synaptic markers, while subtype D showed the opposite pattern, low glial proteins, high synaptic markers, with a steeper slope of cognitive decline. These subtypes showed only modest demographic differences and were reproducible using the racially diverse cohort in the first study.

Together, these findings reveal both shared and distinct molecular features of AD across racial groups and define biologically meaningful subtypes. This work lays the foundation for more inclusive, precise biomarker development and therapeutic targeting in AD.

Table of Contents

1.       Introduction: The Heterogeneity of Alzheimer’s Disease Across Demographics and Pathologies

1.1.        Cognitive Impairment and Alzheimer’s Disease

1.2.        Cognitive Impairment, Definition and Causes

1.3.        Dementia: Definition, Etiology and Pathology

1.3.1.          Definition

1.3.2.          Etiology

1.3.3.          Pathophysiology

1.4.        Alzheimer’s Disease

1.4.1.          Pathogenesis of Alzheimer’s Disease

1.4.2.          Risk Factors for Alzheimer’s Disease and Related Dementia

1.4.3.          Diagnosis and Treatment

1.5.        Heterogeneity in Pathology of Alzheimer’s and Other Dementia

1.6.        Research Objectives and Significance

1.6.1.          Research Aims

1.6.2.          Significance

2.       Proteomic Signatures of Alzheimer’s Disease Across Racial Groups

2.1. Background

2.2. Methods:

2.2.1. Brain Tissue Collection

2.2.2. Tissue homogenization, protein digestion, TMT peptide labeling, and (LC)- MS/ MS

2.2.3. Database Searches and Protein Quantification

2.2.4. Data Analysis and QC

2.2.5. Variance Partition Analysis:

2.2.6. Data QC for APP and MAPT domains and age and sex regression

2.2.7. Differential Abundance Analysis

2.2.8. Shiny APP

2.3.        Results:

2.3.1.          Cohort Characteristics

2.3.2.          Proteomics Data Quality Control in Frontal and Temporal cortices

2.3.3.          Variance of Protein Abundance in Frontal and Temporal Cortex Explained by Individual traits

2.3.4.          Association between APOE4 Genotype and APOE4 Protein Abundance in Human Brain Proteome

2.3.5.          Correlation of Amyloid and Tau Abundance in Human Brain Proteome with AD Neuropathology and Other Proteins

2.3.6.          TMT-MS Quantification of APP, MAPT, Revealed No Racial Difference in AD

2.3.7.          Differential Protein Abundance across Race in AD Brain Proteomes Reveals a Convergence of Predominantly Shared Changes.

2.4.        Discussion

2.4.1. Network analysis

2.4.2. Mapping post-translational modifications

2.4.3. Proteogenomics

2.4.4. Limitations and Future Directions

2.5.        Conclusions:

2.6.        Figures

2.6.1.          Fig 1.

2.6.2.          Fig 2.

2.6.3.          Fig 3.

2.6.4.          Fig 4.

2.6.5.          Fig 5.

2.6.6.          Fig 6.

2.6.7.          Fig 7.

2.6.8.          Fig. 8.

2.6.9.          Fig. 9.

2.6.10.       Fig. 10.

3.       Heterogeneity of Proteomic Subtypes of Alzheimer’s Disease

3.1.        Background

3.2.        Methods

3.2.1.          Brain Tissue Collection

3.2.2.          Preparation of Samples and Protein Digestion

3.2.3.          Tandem Mass Tag (TMT) Labeling

3.2.4.          High-pH Fractionation

3.2.5.          LC-MS/MS Analysis

3.2.6.          Database Searches and Protein Quantification

3.2.7.          Data Analysis and QC

3.2.8.          WGCNA and MONET M1 Clustering

3.2.9.          SHAP (SHapley Additive exPlanations) Analysis

3.2.10.       Uniform manifold approximation and projection (UMAP)

3.3. Results

3.3.1. Proteomic Profiling of ROSMAP Brain Cohorts

3.3.2. Proteomic Co-Expression Network Analysis

3.3.3. Unbiased Clustering Reveals Four Distinct Proteomic Subtypes with Minor Demographic Differences

3.3.4. Proteomic Differences Between Subtypes C and D

3.3.5. Proteomic Subtypes Are Reproducible in an Independent Racially Diverse Cohort

3.3.6. Modules Associated with Proteomic Subtypes in the Diversity Cohort Mirror ROSMAP Cohort Findings

3.4.        Discussion

3.4.1.          Brain Co-Expression Network Analysis

3.4.2.          Synaptic Markers, Inflammation, and Cognitive Decline

3.4.3.          Race, and Sex Distribution in Brain Proteomic Subtypes

3.4.4.          Genetic Variability and Disease Stage

3.4.5.          Limitations of the Current Study

3.4.6.          Implications for Future Studies

3.5.        Conclusion:

3.6. Figures:

3.6.1. Fig 1.

3.6.2. Fig 2.

3.6.3. Fig 3.

3.6.4. Fig. 4.

3.6.5. Figure 5.

3.6.6. Figure 6.

4.       Discussion and Future Directions

4.1. Summary of the Aims

4.1.1. Molecular Differences Across Racial and Ethnic Groups in AD

4.1.2. Molecular Subtypes of AD and Their Reproducibility Across Different Racial Groups

4.2. Implications for AD Research and Clinical Translation

4.2.1. Racial and Ethnic Disparities in AD Biomarkers and the Role of the BBB

4.2.2. Precision Medicine and Molecular Subtypes of AD

4.2.3. The Dual Role of Neuroinflammation in AD

4.3. Limitations and Future Directions

4.3.1. AD progression and Stages

4.3.2. Limitations of Studying Postmortem Brain Tissues

4.3.3. Postmortem Interval (PMI) Effects

4.3.4. Multi-Omics Studies and Post-Translational Modifications (PTMs)

4.3.5. Lack of Direct BBB Assessments

5. References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Chemistry, Biochemistry Biology, Neuroscience
Parola chiave	Proteomics Alzheimer's disease Biomarkers Precision medicine
Committee Chair / Thesis Advisor	Nicholas Seyfried, Emory University
Committee Members	Anita Corbett, Emory University James Lah, Emory University Steven Sloan, Emory University Victor Faundez, Emory University

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