Proteomic profiling of the cerebrospinal fluid of African Americans and Caucasians reveals common and unique biomarkers of Alzheimer’s disease Open Access
Modeste, Erica (Summer 2024)
Abstract
Despite being twice as likely to get Alzheimer’s disease (AD), African Americans have been grossly underrepresented in AD research. While emerging evidence indicates that African Americans with AD have lower cerebrospinal fluid (CSF) levels of Tau compared to Caucasians, other differences in AD CSF biomarkers have not been fully elucidated. In this thesis, we performed unbiased proteomic profiling of CSF from African Americans and Caucasians with and without AD to identify both common and divergent AD CSF biomarkers. Multiplex tandem mass tag-based mass spectrometry (TMT-MS) quantified 1,840 proteins from 105 control and 98 AD patients of which 100 identified as Caucasian and 103 identified as African American. Differential protein expression and co-expression approaches were then utilized to assess how changes in the CSF proteome were related to race and AD. Co-expression network analysis organized the CSF proteome into 14 modules associated with brain cell-types and biological pathways. Consistent with previous findings, the increase of Tau levels in AD was greater in Caucasians than in African Americans by both immunoassay and TMT-MS measurements. Similarly, modules enriched with proteins involved with glycolysis and neuronal/cytoskeletal proteins were more increased in Caucasians than in African Americans with AD. In contrast, a module enriched with synaptic proteins including VGF, SCG2, and NPTX2 was significantly lower in African Americans than Caucasians with AD. CSF modules which included 14-3-3 proteins (YWHAZ and YWHAG) demonstrated equivalent disease-related elevations in both African Americans and Caucasians with AD. A targeted mass spectrometry method, selected reaction monitoring (SRM), with heavy labeled internal standards was then used to measure a subset of CSF module proteins and a receiver operating characteristic (ROC) curve analysis assessed the performance of each protein biomarker in differentiating controls and AD by race. Following SRM and ROC analysis, VGF, SCG2, and NPTX2 were significantly better at classifying African Americans than Caucasians with AD. In total, these findings provide insight into additional protein biomarkers and pathways reflecting underlying brain pathology that are shared or differ by race.
Table of Contents
TABLE OF CONTENTS
LIST OF FIGURES………………………………………………………………………………………..9
LIST OF TABLES……………………………………………………………………………………….10
LIST OF ABBREVIATIONS……………………………………………………………………………11
LIST OF PROTEINS…………………………………………………………………………………….13
CHAPTER 1: INTRODUCTION………………………………………………………………………..18
1.1 The increasing burden of Alzheimer’s disease (AD)………..………………...........19
1.2 Monitoring memory loss in those with AD...…...……………………………………..22
1.3 The initial discovery of AD…...………………………………………………………….27
1.4 Neuropathological hallmarks of AD……………………...……………………………28
1.4.1 Amyloid cascade…………………………………………...……………...........28
1.4.2 Pathologic Tau…………………………………………………………………...31
1.5 The ATN network for staging AD progression………………………………..……...35
1.6 Early-onset AD versus late-onset AD………………………………………………….36
1.6.1 Early-onset AD……………..……………………………………………...........36
1.6.2 Late-onset AD……………...…………………………………………………….36
1.7. Risk factors for AD……………………………………………………………………….37
1.7.1. Age…………………...…………………………………………………………..37
1.7.2 Sex…………………...……………………………………………………..........38
1.8 Modifiable risk factors for AD…………………..……………………………………….38
1.8.1 Role of modifiable risk factors in AD……………………...……………………38
1.8.2 Cardiovascular health…...………………………………………………………39
1.8.3 Smoking / physical activity / diet……………………….………………............40
1.9 Social determinants of health…………………………..………………………...........40
1.9.1 Education………………...………………………………………………………40
1.9.2 Employment……………..……………………………………………………….41
1.9.3 Environment………………..……………………………………………...........42
1.9.4 Stress……………………...………………………………………………..........43
1.9.5 Discrimination and social exclusion…………………...……………………….44
1.9.6 Final conclusions………………………………..………………………...........44
1.10 Therapeutic attempts to slow the progression of AD…………………………...…45
1.11 Cerebrospinal fluid (CSF) as a gateway to neuropathological changes in AD..46
1.12 African Americans: the most at risk racial group for AD……………...….............51
1.13 The utility of mass spectrometry (MS)-based proteomics in identifying novel
protein signatures in AD.…………………..…….……………………………………52
1.13.1 Strategies for MS-based quantification of proteomes….……………….…….52
1.13.2 Fundamentals of network construction and module identification………..…53
1.13.3 Why prioritize the study of the proteome over the genome?..........................54
1.13.4 Core modules of the AD brain network proteome……..….…………………...55
1.13.5 The CSF proteome as a reflection of AD brain changes…….…….………….56
1.14 Summary………………………………………………………………………………….56
CHAPTER 2: MATERIALS AND METHODS………………………………………………….……..58
2.1 CSF samples…………………………….…………………………………………………59
2.2 Protein digestion of CSF………………………………………………………………...60
2.3 Tandem mass tag labeling of CSF peptides…………………………………………60
2.4 High-pH fractionation……………………………………..……………………………...61
2.5 Mass spectrometry analysis and data acquisition……………………………...…..62
2.6 Database search and protein quantification………………………………...……….62
2.7 Adjustment for batch and other sources of variance……………………………….63
2.8 Differential expression analysis…………………………………………………..……66
2.9 Weighted Gene Co-expression Network Analysis……………………...…………...66
2.10 Gene ontology and cell type enrichment analysis……………….………………...66
2.11 Selected Reaction Monitoring…………………………………………………………67
CHAPTER 3: RESULTS………………………………………….……………...,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,69
3.1 CSF Cohort characteristics……………………………………………………………..70
3.2 Discovery tandem mass spectrometry analysis of CSF from African Americans and Caucasians reveals unique and shared changes in Alzheimer’s disease…………………………………………………………………..……………………...72
3.2.1 Correlation analysis uncovers a strong relationship between mass spectrometry and immunoassay measurements of Tau……………………………72
3.2.2 Differential expression analysis of African American and Caucasian CSF proteome reveals unique and shared changes in AD……………………………….72
3.2.3 Network analysis of the CSF proteome reveals modules related to pathways and brain cell-types…………………………………………………………………….75
3.2.4 CSF protein modules correlate to race and clinicopathological phenotypes of
AD……………………………………………………………………………………….81
3.3 Selected reaction monitoring validates protein alterations across Alzheimer's disease and race………………………………………………………………………………89
CHAPTER 4: DISCUSSION…………………………………………………………………………...98
4.1 Protein co-expression between the brain and CSF reflects the crucial role of CSF in brain function and health………………………………………………………………….99
4.2 CSF network analysis indicated differences in endothelial markers across race, irrespective of disease, yet there is insufficient evidence to indicate that these differences stem from variations in endothelial damage……………………………..101
4.3 Unveiling the interplay between neuronal alterations in AD and the role of the CSF in mirroring cognitive decline………………………………………………………..103
4.4 Future directions………………………………………………………………………...107
CHAPTER 5: REFERENCES………………………………………………………………………...110
CHAPTER 6: APPENDIX……………………………………………………………………………..126
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