Characterization of clinical, pathological, and proteomic differences along the Amyotrophic lateral sclerosis and Frontotemporal dementia disease spectrum Pubblico

Abstract

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two neurodegenerative diseases that share genetic, clinical, and pathological overlap. This overlap has been strengthened by the identification of the C9orf72 genetic expansion mutation. This hexanucleotide repeat (GGGGCC) expansion within the Chromosome 9 open reading frame 72 (C9orf72) gene was identified in 2011 and is recognized as the most common genetic cause of both ALS and FTD. The goal of this dissertation project was to increase our understanding of the clinical, pathological, and proteomic differences in patients with and without this expansion mutation as well as to gain a better understanding of the ALS-FTD disease spectrum. Using DNA samples from an entire ALS clinic population spanning 14 years, demographic, clinical, and survival differences were compared in ALS patients with and without the expansion to understand the clinical consequences of the C9orf72 expansion. Post mortem tissue from patients along the clinical spectrum of ALS and FTD were analyzed to gain a better understanding of the disease pathobiology. Specifically, mass spectrometry was used to analyze frontal cortex tissue samples to investigate differential protein expression within the insoluble proteome across the ALS-FTD disease spectrum and relate differences to the hallmark TDP-43 pathology present in patient brains. Finally, mass spectrometry was also used to investigate the total proteome across the clinical continuum and to generate a protein co-expression network that allows us to better understand the pathways implicated across these diseases and the contribution of the C9orf72 genetic mutation. Our results show that there are very few demographic and clinical differences in the C9orf72 ALS population, but there is reduced survival in this group and an increased presence of comorbid frontotemporal dementia when compared to the general ALS population not carrying the C9orf72 mutation. Proteomic analysis showed differences in protein co-expression along the disease spectrum and a distinct difference in C9orf72 ALS cases compared to sporadic ALS. These differences were validated by comparing our network to externally generated networks and using immunohistochemistry and immunoblotting techniques. Overall, these studies provide important new information regarding the clinical differences conferred by the c9orf72 genetic expansion and the molecular underpinnings of the ALS-FTD disease spectrum. The unbiased proteomic data from these clinical populations deliver new avenues for future investigation, including potential targets for understanding pathogenesis and designing therapeutic interventions.

Table of Contents

TABLE OF CONTENTS

Abstract .........................................................................................................................................IV

Acknowledgements .....................................................................................................................VII

Table of Contents………………………………………………………………………...………IX

List of Figures ....................................................................................................................…….XII

List of Tables.................................................................................................................……….XIV

CHAPTER 1: INTRODUCTION…………………………………………………...…………..1

• Clinical description of ALS, FTD, and overlap syndrome………………………..2
• Historical overview………………………………………………………………..5
• Epidemiological overview & prognosis…………………………………………...7
• Pathological overview……………………………………………………………10
• Genetic landscape………………………………………………………………..11
• C9orf72 overview………………………………………………………………..15
• Mass spectrometry based proteomics for neurodegenerative disease research….19
• Proposed research………………………………………………………………..24
• Research overview……………………………………………………………….25

CHAPTER 2: MATERIALS AND METHODS….……………………………………….….28

• Case materials……………………………………………………………………29
  • Samples for clinical analyses…………………………………………….29
  • Samples for pathological analyses………………………………………29
  • Acquisition of clinical and demographic information from database……38
• DNA extraction…………………………………………………………………..38
• Repeat primed PCR for identification of C9orf72 expansion…………………..39
• Family history investigation……………………………………………………..40
• Antibodies………………………………………………………………………..44
• Immunocytochemistry…………………………………………………………...44
• Western blotting………………………………………………………………….45
• Proteomics sample preparation………………………………………………….48
  • Insoluble homogenate……………………………………………………48
  • Total Homogenate……………………………………………………….54
• Label Free Mass spectrometry- Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)………………………………..55
• Statistics …………………………………………………………………………57
  • Statistics for clinical analyses……………………………………………57
  • Statistics for pathological analyses………………………………………59
  • Statistics for proteomic analyses………………………………………..59
• Rationale…………………………………………………………………………67

CHAPTER 3: COMPARATIVE ANALYSIS OF C9ORF72 AND SPORADIC DISEASE IN AN ALS CLINIC POPULATION……..……………...……………..72

· Introduction………………………………………………………………………73

· Results……………………………………………………………………………78

· Discussion……………………………………………………………………...89

CHAPTER 4: TDP-43 INTERACTOME IN THE INSOLUBLE PROTEOME ALONG THE ALS-FTD DISEASE SPECTRUM ……………………………………..97

· Introduction……………………………………………………………………..98

· Results…………………………………………………………………………..103

· Discussion………………………………………………………………………119

CHAPTER 5: INTEGRATED NETWORK APPROACH ACROSS ALS-FTD FRONTAL CORTEX TOTAL HOMOGENATE PROTEOME………….…………….124

· Introduction……………………………………………………………………..125

· Results…………………………………………………………………………..127

· Discussion………………………………………………………………………156

CHAPTER 6: SUMMARY AND FUTURE DIRECTIONS………………….…………….160

• Summary of the work…………………………………………………………...161

• Therapeutic implications……………………………………………………….164

• Future directions……………………………………………………………….165

• Conclusions……………………………………………………………………..169

REFERENCES………………….…………………………………………………………...170

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Parola chiave	Amyotrophic lateral sclerosis Neurodegeneration Proteomics
Committee Chair / Thesis Advisor	Glass, Jonathan D, Emory University
Committee Members	Wichmann, Thomas, Emory University Li, Lian, Emory University Gearing, Marla, Emory University Bassell, Gary, Emory University Seyfried, Nicholas, Emory University

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