The role of the histone demethylase LSD1 in hippocampal neuron survival and tau-mediated neurodegeneration. 公开

Abstract

We inducibly deleted the histone demethylase LSD1 in adult mice initially to investiagte mechanisms that maintained cell fate fully differentiated cells. We found that the loss of LSD1 led to transcriptional changes such as the reactivation of stem cell genes. Furthermore, we oberved transcriptional changes in common neurodegenerative pathways and substantial neurodegeneration of the hippocampus and cortex. These mice also displayed significant defects in learning and memory tasks. In adddition to this, all Lsd1CAGG mice eventually develop progressive hind-limb paralysis and expire prematurely. Here we have chosen to focus on hippocampal neuronal cell death and the potential link to human neurodegenerative disease. In addition to transcriptional changes that implicated potentially shared neurodegenerative pathways with AD and FTD, we find that LSD1 is mislocalized to pathological protein aggregates in these human cases. This raised the possibility that pathological protein aggregates such as hyperphosphorlated TAU could compromise the functions of LSD1. To investiagte this possibility, we utilized the P301S tauopathy mouse model to demonstrate that pathological tau can exclude LSD1 from the nucleus in neurons. Furthermore, we performed experiments in which we both reduced and increased the expression of LSD1 in P301S mice. The reduction of LSD1 highly exacerbated tau-mediated neurodegeneration and overexpression was sufficient to significantly delay neurodegeneration, even after the formation of TAU pathology. Taken together, these data suggest that the colocalization of LSD1 and TAU protein contributes to tau-mediated neurodegeneration via nuclear sequestration of LSD1. 

Table of Contents

CHAPTER 1. INTRODUCTION AND BACKGROUND .............................................................................................. 1 

1.1 EPIGENETIC TRANSCRIPTIONAL REGULATION................................................................................................................... 2 

1.2 LYSINE SPECIFIC DEMETHYLASE 1 (KDM1A/LSD1) ........................................................................................................ 3 

1.2.1 Structure/Domains ...................................................................................................................................... 4 

1.2.2 Biological roles ............................................................................................................................................. 6 

1.2.3 Germline ...................................................................................................................................................... 6 

1.2.4 Neural stem cells.......................................................................................................................................... 7 

1.2.5 Alternative splicing ...................................................................................................................................... 8 

1.3 LSD1 AND NEURODEGENERATION. .............................................................................................................................. 9 

1.4 REFERENCES .......................................................................................................................................................... 12 

CHAPTER 2. LSD1 PROTECTS AGAINST HIPPOCAMPAL AND CORTICAL NEURODEGENERATION .......................... 17 

2.1 ABSTRACT ............................................................................................................................................................. 18 

2.2 INTRODUCTION ...................................................................................................................................................... 19 

2.3 RESULTS................................................................................................................................................................ 20 

2.3.1 LSD1 is continuously required to prevent neurodegeneration .................................................................. 20 

2.3.2 Loss of LSD1 results in learning and memory defects................................................................................ 25 

2.3.3 LSD1 inhibits reactivation of stem cell transcription ................................................................................. 26 

2.3.4 Loss of LSD1 induces common neurodegeneration pathways .................................................................. 28 

2.3.5 Lsd1CAGG expression changes overlap with AD and FTD cases ................................................................... 29 

2.3.6 LSD1 is mislocalized in human dementias ................................................................................................. 32 

2.3.7 Lsd1CAGG mice do not have protein aggregates ......................................................................................... 34 

2.3.8 Increased stem cell gene expression in AD and FTD patients.................................................................... 34 

2.4 DISCUSSION ........................................................................................................................................................... 35 

2.5 METHODS ............................................................................................................................................................. 37 

2.5.1 Tamoxifen injections .................................................................................................................................. 38 

2.5.2 Mouse Tissue Fixation ............................................................................................................................... 38 

2.5.3 Histology .................................................................................................................................................... 38 

2.5.4 Mouse Immunofluorescence ..................................................................................................................... 38 

2.5.5 Mouse Immunohistochemistry .................................................................................................................. 39 

2.5.6 Human Immunohistochemistry ................................................................................................................. 39 

2.5.7 Human Immunofluorescence ..................................................................................................................... 40 

2.5.8 Immunohistochemistry with Peptide block ............................................................................................... 40 

2.5.9 TUNEL Assay .............................................................................................................................................. 40 

2.5.10 Neuromuscular Junctions ........................................................................................................................ 40 

2.5.11 Quantification of LSD1 Colocalization with pTau and pTDP-43 .............................................................. 41 

2.5.12 Motor Neuron Counting .......................................................................................................................... 41 

2.5.13 CA1 Nuclei Counting ................................................................................................................................ 41 

2.5.14 Lsd1 Deletion Quantification ................................................................................................................... 42 

2.5.15 Morris Water Maze ................................................................................................................................. 42 

2.5.16 Fear Conditioning .................................................................................................................................... 42 

2.5.17 RNA Sequencing ....................................................................................................................................... 43 

2.5.18 RNA-seq Analysis ..................................................................................................................................... 43 

2.5.19 Comparison to Human Gene Expression Data......................................................................................... 44 

2.6 REFERENCES .......................................................................................................................................................... 45 

2.7 ACKNOWLEDGMENTS .............................................................................................................................................. 48 

2.8 FIGURES AND TABLES .............................................................................................................................................. 50 

CHAPTER 3. THE INHIBITION OF LSD1 VIA SEQUESTRATION CONTRIBUTES TO TAU-MEDIATED NEURODEGENERATION .................................................................................................................................. 93 

3.1 ABSTRACT .......................................................................................................................................................... 94 

3.2 INTRODUCTION ................................................................................................................................................. 95 

3.3 RESULTS ............................................................................................................................................................. 97 

3.3.1 Reduction of LSD1 increases the mouse tauopathy phenotype ................................................................ 98 

3.3.2 Reduction of LSD1 exacerbates PS19 Tau neurodegeneration................................................................ 100 

3.3.3 Tau pathology is not affected by change in LSD1 levels .......................................................................... 101 

3.3.4 The functional interaction between tau pathology and LSD1 inhibition is specific ................................ 102 

3.3.5 Overexpression of LSD1 rescues neurodegeneration in the hippocampus of PS19 Tau mice ................. 104 

3.4 DISCUSSION ..................................................................................................................................................... 106 

3.5 MATERIALS AND METHODS ............................................................................................................................. 110 

3.5.1 Mouse tissue fixation ............................................................................................................................... 110 

3.5.2 Histology and histological studies ........................................................................................................... 110 

3.5.3 Immunohistochemistry and immunofluorescence .................................................................................. 111 

3.5.4 Protein Quantification ............................................................................................................................. 112 

3.5.5 Quantitative analysis of paralysis............................................................................................................ 112 

3.5.6 MRI of brain atrophy ............................................................................................................................... 113 

3.5.7 RNA sequencing ....................................................................................................................................... 113 

3.5.8 RNA sequencing analysis ......................................................................................................................... 114 

3.5.9 Stereotaxic surgery and viral infusion ..................................................................................................... 115 

3.6 REFERENCES ..................................................................................................................................................... 116 

3.7 ACKNOWLEDGMENTS ..................................................................................................................................... 118 

3.8 FIGURES AND TABLES....................................................................................................................................... 121 

CHAPTER 4. DISCUSSION .............................................................................................................................. 151 

4.1 SUMMARY OF FINDINGS AND SIGNIFICANCE............................................................................................................... 152 

4.2 FUTURE DIRECTIONS ............................................................................................................................................. 154 

4.4 REFERENCES ........................................................................................................................................................ 156 

CHAPTER 5. EXTENDED MATERIALS AND METHODS....................................................................................... 158 

5.1 RNA SEQUENCING ANALYSIS .................................................................................................................................. 159 

5.1.1 Quality Control ......................................................................................................................................... 159 

5.1.2 Sequence Mapping .................................................................................................................................. 159 

5.1.3 Sequence Mapping Quality Control ......................................................................................................... 160 

5.1.4 Differential Expression ............................................................................................................................. 160 

5.2 RNA SEQUENCING DATA PROCESSING ..................................................................................................................... 162 

5.2.1 Transcripts per million reads (TPM) expression values ........................................................................... 162 

5.2.2 Identification of differentially expressed genes (DEGs) ........................................................................... 163 

5.2.3 Convert Ensembl IDs to Gene Symbols .................................................................................................... 163 

5.2.4 Heatmaps................................................................................................................................................. 164 

5.2.5 Volcano plots ........................................................................................................................................... 165 

5.2.6 Gene Set Enrichment Analysis (GSEA) ..................................................................................................... 166 

5.3 DATA VISUALIZATION ............................................................................................................................................ 168 

5.3.1 Comparison to Human Gene Expression Data......................................................................................... 168 

5.3.2 Bar Plot .................................................................................................................................................... 173 

5.3.3 Scatterplot ............................................................................................................................................... 174 

APPENDIX A. KDM1A/LSD1 REGULATES THE DIFFERENTIATION AND MAINTENANCE OF SPERMATOGONIA IN MICE............................................................................................................................................................ 176 

A1. ABSTRACT ........................................................................................................................................................... 177 

A2. INTRODUCTION .................................................................................................................................................... 178 

A3. RESULTS ............................................................................................................................................................. 180 

A3.1 KDM1A is dynamically expressed in the murine testis ............................................................................. 180 

A3.2 Loss of KDM1A causes defects in the maintenance and differentiation of spermatogonia .................... 180 

A3.3 Loss of KDM1A does not derepress Line1 and IAP retrotransposons ....................................................... 184 

A3.4 KDM1A binds to the Oct4 locus in the adult testis ................................................................................... 186 

A3.5 KDM1A is required for the repression of Oct4 ......................................................................................... 186 

A3.6 H3K4me2 at the Oct4 locus ...................................................................................................................... 187 

A4. DISCUSSION ........................................................................................................................................................ 188 

A5. MATERIALS AND METHODS .................................................................................................................................... 191 

A5.1 Ethics Statement....................................................................................................................................... 191 

A5.2 Mice .......................................................................................................................................................... 191 

A5.3 Generation of KDM1A conditional mutant mice ...................................................................................... 191 

A5.4 Histological Methods ................................................................................................................................ 192 

A5.5 TUNEL Assay ............................................................................................................................................. 193 

A5.6 In Situ Hybridization ................................................................................................................................. 193 

A5.7 Bisulfite Analysis ....................................................................................................................................... 194 

A5.8 Chromatin Immunoprecipitation .............................................................................................................. 194 

A6. REFERENCES ........................................................................................................................................................ 196 

A7. ACKNOWLEDGEMENTS .......................................................................................................................................... 198 

A8. FIGURES AND TABLES ............................................................................................................................................ 199 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Neuroscience
关键词	LSD1
Committee Chair / Thesis Advisor	Katz, David, Emory University
Committee Members	Bassell, Gary, Emory University Corces, Victor, Emory University Lah, Jim, Emory University Faundez, Victor, Emory University

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