Comprehensive Proteome and Transcriptome Studies Reveal RNA Processing Dysfunction in Alzheimer's disease Open Access

Bai, Bing (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/t722h901h?locale=en
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Abstract

Alzheimer's disease (AD) is an age-related neurodegenerative disorder, currently with no effective cure. The major hypothesis posits that AD is caused by two hallmark proteins, A! and Tau. However, patients with similar A! and Tau pathology can demonstrate completely different types of dementia. In order to find more mechanistic proteins to better understand AD neurodegeneration, we comprehensively studied the insoluble proteome where A! and Tau were initially discovered. Proteins resistant to detergents were extracted from affected frontal cortex in postmortem brains of AD and non-demented controls, and then analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Among the 4,216 proteins identified, 36 are significantly increased in AD. These include A!, Tau and other proteins in pathways known to be disrupted in AD. In addition to these known proteins, novel proteins from RNA splicing pathway such as U1-70K and U1A were found to be significantly enriched in AD patients. Validation by western blotting showed specific, widespread and early-occurring accumulation of U1-70K and U1A insoluble in AD. Brain tissue immunohistochemical staining of U1-70K and U1A demonstrated cytoplasmic tangle-like structures in AD neurons, indicating possible aggregation and dysfunction. Aberrancy of these U1 spliceosomal proteins may lead to defective RNA splicing in AD. Analysis of the mRNA transcriptome by RNA-seq revealed several splicing alterations in AD: 1) accumulated pre-mRNA, where the ratio of exon reads to intron reads is reduced; 2) altered exon junctions; 3) increased premature cleavage and polyadenylation in AD, where more polyA reads were mapped to the 5' terminus of coding regions rather than the 3' UTR region. All these point to deficient RNA splicing in AD. In summary, two comprehensive analyses involving proteomics and transcriptomics both revealed RNA splicing disruption in AD, providing a novel mechanistic clue to AD neurodegeneration.

Table of Contents

Chapter 1: Introduction........................................................................................................................1

1. Neurodegeneration diseases and protein aggregation..............................................................................2 2. AD general facts..............................................................................................................................3 3. AD pathology..................................................................................................................................4 4. AD molecular research......................................................................................................................6 4.1 Toxic insults and disrupted pathways................................................................................................6 4.2 The genetics of Alzheimer's disease..................................................................................................18 4.3 Animal models of Alzheimer's disease................................................................................................19 4.4 Summarized AD model and opening questions.....................................................................................19 5. Introduction of Proteomics by LC-MS/MS............................................................................................22 5.1 Working principle of LC-MS/MS........................................................................................................22 5.2 LC-MS/MS application in neurodegenerative diseases..........................................................................24 6. Transcriptome analysis by RNA-seq...................................................................................................25 6.1 Working principle of RNA-seq..........................................................................................................25 6.2 RNA-seq application in neurodegenerative diseases............................................................................25 Chapter 2: U1 Small Nuclear Ribonucleoprotein Complex and RNA Splicing Alterations in Alzheimer's disease........27 Chapter 3: Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer's disease................................43 Chapter 4: General Discussion and Future Directions...............................................................................60 1. Summary of the dissertation work....................................................................................................61 2. Future directions..........................................................................................................................65 2.1 To determine if U1-70K can be causally involved in AD.......................................................................65 2.2 To study the mechanism of U1-70K pathology..................................................................................71 REFERENCES...................................................................................................................................73

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