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
About this Dissertation
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