Identification and characterization of disease-associated proteins in frontotemporal lobar degeneration Público

Abstract

Frontotemporal lobar degeneration (FTLD) is the second most common cause of presenile dementia, accounting for up to 20% of cases in this age group. Characterized by circumscribed degeneration of the prefrontal and anterior temporal lobes, FTLD results in profound personality changes, altered social behavior, language disturbances, and deterioration of executive function. The most common pathological subtype of FTLD is defined by the presence of neuronal ubiquitin-immunoreactive inclusions in affected brain regions, and is thus known as FTLD-ubiquitinated (FTLD-U). The pathophysiological mechanisms underlying neurodegeneration in FTLD-U are poorly understood, and little is known about the primary aggregating proteins in this disorder. We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U. Using these approaches, we proteomically profiled detergent-insoluble protein extracts prepared from frontal cortex of FTLD-U cases, unaffected controls, or neurologic controls (i.e. Alzheimer's disease; AD). Demonstrating the validity of our strategies, examination of the proteins specifically enriched in the AD samples revealed the presence of both microtubule-associated protein tau and amyloid β, two well-established AD-linked proteins, as well as several novel targets. Among the proteins altered specifically in FTLD-U, we identified TAR DNA binding protein-43 (TDP-43), a known component of ubiquitinated inclusions. Finally, we identified additional proteins enriched in detergent-resistant fractions in FTLD-U, and characterized one of them, septin 11 (SEPT11), in detail. Using highly sensitive targeted proteomics approaches, we confirmed the enrichment of SEPT11 in FTLD-U extracts. We further showed that SEPT11 is proteolytically cleaved and, in addition to its prominent glial localization in normal brain, accumulates in thread-like pathology in affected cortex of patients. Moreover, we identified a significant association of an intronic single nucleotide polymorphism in the SEPT11 gene with sporadic FTLD in a clinical cohort. This association was close to significance in a second cohort from Mayo Clinic, but was not replicated in a third cohort from UCLA. In sum, we have developed and validated a novel proteomics strategy that successfully identifies disease-specific proteins in neurodegeneration, and used this method to discover SEPT11 as a new protein with altered abundance, unique pathology, and potential genetic linkage to FTLD-U.

Table of Contents

Table of Contents

Abstract IV

Acknowledgements VII

Table of Contents VIII

List of Figures XII

List of Tables XIV

1. Introduction 1

1.1 Frontotemporal Lobar Degeneration 1

1.2 History and Clinical Description of FTLD 2

1.3 Classification of FTLD: Clinical 3

1.4 Classification of FTLD: Pathologic 8

1.5 Motor Neuron Disease (MND) and FTLD-U 14

1.6 The Genetics of FTLD-U 15

1.7 The Genetics of FTLD-U 18

1.7.1 Identification of TDP-43 18

1.7.2 Biology of TDP-43 20

1.7.3 Molecular Signature in FTLD-U 20

1.7.4 Genetic Variation 23

1.7.5 Disease Specificity of TDP-43 24

1.8 Proteomics in Neurodegenerative Diseases 25

1.8.1 Proteomics Platforms 25

1.8.2 Proteomics in Alzheimer's Disease 28

1.8.3 Proteomics in FTLD 35

1.9 Proposed Research 37

2. Materials and methods 39

2.1 Case Materials 39

2.2 Antibodies 40

2.3 DNA Constructs 41

2.4 Laser Capture Microdissection (LCM) 42

2.4.1 Preparation of Tissue for LCM 42

2.4.2 LCM 42

2.4.3 Protein Extraction from LCM Caps 44

2.5 Sequential Biochemical Fractionation 45

2.5.1 Human Frontal Cortex 45

2.5.2 Mammalian Cells 48

2.6 Proteomic Analysis 48

2.6.1 Stable Isotope Labeling With Amino Acids in Cell Culture (SILAC) 48

2.6.2 Analysis and Protein Identification by Mass Spectrometry 49

2.6.3 Label-Free Quantification: Extracted Ion Current 51

2.6.4 Label-Free Quantification: Spectral Counts 53

2.6.5 SILAC Quantification and Bioinformatics Analysis 54

2.6.6 Quantitative Proteomics with Culture Derived Isotopic Tags (CDIT) 55

2.6.7 Quantitative Analysis of Polyubiquitin Chains and Targeted Proteins by LC/MRM 56

2.6.8 Quantitative Analysis of SEPT11 by Targeted Proteomics and LC/MRM 57

2.7 Immunohistochemistry (IHC) 57

2.7.1 Human Free Floating Sections 57

2.7.2 Human Paraffin-Embedded Tissue 59

2.7.3 Blinded Scoring of SEPT11 Immunoreactivity 59

2.8 Primary Neuronal Cultures 60

2.9 Cell Culture and Immunocytochemistry (ICC) 61

2.10 Western Blotting 62

2.11 Biochemical Assays 63

2.12 Genotyping and Sequencing Assays 63

2.12.1 SNPstream Genotyping 63

2.12.2 Taqman SNP Assay 64

2.12.3 Septin 11 Sequencing 65

3. Proteomic analysis of ubiquitin-positive inclusions in frontotemporal lobar degeneration: application of laser capture technology 67

3.1 Introduction 67

3.2 Results 70

3.2.1 Identification of proteins enriched in FTLD-U dentate granule cells by LC-MS/MS 70

3.2.2 Relative quantification of identified proteins by the label-free strategy 74

3.2.3 Validation of selected FTLD-U enriched components 77

3.3 Discussion 81

4. proteomics analysis reveals novel components in the detergent-insoluble subproteome in alzheimer's disease 86

4.1 Introduction 86

4.2 Results 90

4.2.1 Protein identification in urea samples by LC-MS/MS 90

4.2.2 Relative quantification of proteins in urea samples by the label-free strategy 94

4.2.3 Validation of selected AD-specific, detergent-insoluble proteins 101

4.3 Discussion 107

5. Multiplex silac analysis of a cellular tdp-43 proteinopathy model reveals protein inclusions associated with sumoylation and diverse polyubiquitin chains 114

5.1 Introduction 114

5.2 Results 117

5.2.1 Expression, localization, and biochemical properties of recombinant TDP-43 and TDP-S6 117

5.2.2 Quantitative analysis of the insoluble TDP-43 and TDP-S6 proteome using multiplex SILAC 129

5.2.3 Validation and subcellular colocalization of SUMO-2/3 and ubiquitin 133

5.3 Discussion 145

6. aberrant Septin 11 is associated with sporadic frontotemporal lobar degeneration 151

6.1 Introduction 151

6.2 Results 154

6.2.1 Discovery of altered proteins in FTLD-U by LC-MS/MS 154

6.2.2 Validation of SEPT11 enrichment in FTLD-U 166

6.2.3 Genetic associations of SEPT11 with FTLD 180

6.3 Discussion 184

7. Summary and future directions 186

8. references 199

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Palavra-chave	Ubiquitin Alzheimer's Disease Neurodegeneration Proteomics Mass Spectrometry Frontotemporal Dementia Detergent-Insoluble Aggregates
Committee Chair / Thesis Advisor	Lah, James J, Emory University Levey, Allan I, Emory University
Committee Members	Wilkinson, Keith D, Emory University Walker, Lary, Emory University Peng, Junmin, Emory University

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