An Investigation of the Role of the Histone Demethylase LSD1, and Its Disordered Domain, in Neuronal Maintenance and Tau-Mediated Neurodegeneration Pubblico

Abstract

Over a century ago, Alois Alzheimer first investigated the brains of dementia patients. Since then, researchers have been investigating the mechanism of neurodegeneration. Alzheimer’s disease (AD) is a tauopathy, due to deposition of tau protein into filamentous and insoluble aggregates. In AD, the amount of pathological tau (pTau) highly correlates with the degree of dementia, implicating pTau in the mechanism. In this dissertation, we provide evidence that pTau causes neurodegeneration by interfering with the lysine specific histone demethylase 1 (LSD1/KDM1A). We show that deletion of Lsd1 in adult mice results in paralysis, hippocampal and cortical neurodegeneration, and genome-wide transcriptional changes that highly correlate with the those in the degenerating brain of AD cases. We find LSD1 aberrantly co-localized with pTau in the cytoplasm of AD cases. Additionally, in mice, we show that LSD1 is completely depleted from the nucleus of neurons with pTau. These data raise the possibility that tau contributes to neuronal cell death by sequestering LSD1 in the cytoplasm, which interferes with its required nuclear function. If LSD1 is the main target of pTau, then altering LSD1 levels should be sufficient to modulate the tauopathy phenotype. Strikingly, making tauopathy mice heterozygous for Lsd1 exacerbates the neurodegenerative phenotypes while overexpressing LSD1 in hippocampal neurons with pTau present can block the neurodegeneration. Additionally, Lsd1 heterozygosity exacerbates the expression changes induced by pTau, but does not induce new pathways. These data argue that pTau is functioning through LSD1 to induce neurodegeneration. This suggests that it may be possible to target the LSD1 pathway for therapeutic intervention. Therefore, we focused on the interaction between pTau and LSD1. Preliminarily, our data suggests that the N-terminal disordered domain of LSD1 is necessary for the interaction with pTau. This makes this domain a potential target for therapeutic intervention. However, utilizing a novel N-terminal deletion of Lsd1 mouse model, we find that this domain may have a required function. Our results provide direct functional evidence that pTau induces neurodegeneration through the sequestration of LSD1, likely by interacting with the N-terminal disordered domain. This novel mechanism provides a highly promising target for therapeutic intervention in tau-mediated neurodegeneration.

Table of Contents

CHAPTER 1:

1.    An Introduction to Neurodegeneration and Epigenetic Regulation .………………Page 1

1.1. Tau’s neuronal function and role in neurodegeneration……………………………Page 2

1.1.1.    Biological function of tau in neurons ……………………………………… Page 2

1.1.2.    The pathophysiology of tauopathies ………………………………………. Page 4

1.1.3.    Tau’s role in Alzheimer’s disease …………………………………………..Page 6

1.1.4.    Modeling tauopathy in animal models………………………………………Page 8

1.2. Epigenetic regulation in the nervous system .…………………………………….Page 10

1.2.1.    DNA methylation ………………………………………………………… Page 10

1.2.2.    Histone modifications.……………………………………………………. Page 13

1.2.3.    Noncoding RNAs that regulate chromatin…………………………………Page 16

1.3. Neuroepigenetic mechanisms of degeneration……………………………………Page 17

1.3.1.    Chromatin changes in the aging brain .…………………………………… Page 17

1.3.2.    Epigenetic component of Parkinson’s disease …………………………….Page 19

1.3.3.    Epigenetic component of Huntington’s disease……………………………Page 20

1.3.4.    Epigenetics in Alzheimer’s disease .……………………………………….Page 21

1.4. The lysine specific histone demethylase LSD1 .………………………………….Page 23

1.4.1.    LSD1 decommissions enhancers at stem cell genes ………………………Page 23

1.4.2.    LSD1 in neurons .………………………………………………………….Page 25

1.5. Outstanding questions and objectives .……………………………………………Page 27

CHAPTER 2:

2.    Materials and Methods………………………………………………………………Page 28

2.1. Solutions and buffers .…………………………………………………………….Page 29

2.2. Mouse work ………………………………………………………………………Page 30

2.2.1.    Mouse lines .……………………………………………………………….Page 30

2.2.2.    Mouse genotyping by PCR .……………………………………………….Page 31

2.2.3.    Euthanasia and tissue fixation .…………………………………………….Page 32

2.2.4.    Quantitative analysis of paralysis: Rotarod and Grid Performance .………Page 33

2.2.5.    Mouse magnetic resonance imagining (MRI) .…………………………….Page 33

2.2.6.    Stereotaxic surgery and viral infusion .…………………………………….Page 34

2.2.7.    Tamoxifen injections and quantification of Lsd1 deletion…………………Page 34

2.2.8.    Learning and memory testing: Morris water maze and fear conditioning…Page 35

2.3. Staining……………………………………………………………………………Page 36

2.3.1.    Mouse histology and histological quantification .…………………………Page 36

2.3.2.    Immunofluorescence and Immunohistochemistry- Mouse .……………….Page 37

2.3.3.    Quantification of tau accumulation .……………………………………….Page 38

2.3.4.    Immunofluorescence and Immunohistochemistry- Human .………………Page 38

2.3.5.    Quantification of LSD1 colocalization with aggregates .………………….Page 40

2.3.6.    Protein quantification………………………………………………………Page 40

2.3.7.    TUNEL assay………………………………………………………………Page 41

2.4. RNA sequencing .…………………………………………………………………Page 41

2.4.1.    RNA isolation………………………………………………………………Page 41

2.4.2.    RNA sequencing analysis …………………………………………………Page 42

2.4.3.    Comparison to human gene expression data ………………………………Page 44

CHAPTER 3:

3.    LSD1 Protects Against Hippocampal and Cortical Degeneration .……………….Page 48

3.1. Abstract……………………………………………………………………………Page 49

3.2. Introduction .………………………………………………………………………Page 49

3.3. Results .……………………………………………………………………………Page 51

3.3.1.    LSD1 is continuously required to prevent neurodegeneration .……………Page 51

3.3.2.    Loss of LSD1 results in learning and memory defects ……………………Page 56

3.3.3.    LSD1 inhibits reactivation of stem cell transcription……………………...Page 57

3.3.4.    Loss of LSD1 induces common neurodegenerative pathways ……………Page 59

3.3.5.    Lsd1^CAGG gene expression changes overlap with AD and FTD cases .……Page 60

3.3.6.    LSD1 is mislocalized in human dementias .……………………………….Page 63

3.3.7.    Lsd1^CAGG mice do not have protein aggregates ……………………………Page 64

3.3.8.    Increased stem cell gene expression in AD and FTD patients .……………Page 65

3.4. Discussion…………………………………………………………………………Page 65

3.5. Figures .……………………………………………………………………………Page 69

CHAPTER 4:

4.    The Inhibition of LSD1 via Sequestration Contributes to Tau-Mediated Neurodegeneration……………………………………………………………………Page 98

4.1. Abstract……………………………………………………………………………Page 99

4.2. Introduction .………………………………………………………………………Page 99

4.3. Results……………………………………………………………………………Page 101

4.3.1.    Tau pathology depletes LSd1 from the nucleus in the PS19 Tau mouse…Page 101

4.3.2.    Reduction of LSD1 increases the mouse tauopathy phenotype .…………Page 102

4.3.3.    Reduction of LSD1 exacerbates PS19 Tau neurodegeneration .…………Page 105

4.3.4.    Tau pathology is not effected by change in LSD1 levels…………………Page 106

4.3.5.    The interaction between tau pathology and LSD1 inhibition is specific…Page 107

4.3.6.    Overexpression of LSD1 rescues neurodegeneration in PS19 Tau mice…Page 108

4.4. Discussion .………………………………………………………………………Page 111

4.5. Figures……………………………………………………………………………Page 116

CHAPTER 5:

5.     The Interaction of pathological tau and LSD1 through the N-terminal

disordered domain .………………………………………………………………………………Page 141

5.1. Introduction………………………………………………………………………Page 142

5.2. Results……………………………………………………………………………Page 145

5.2.1.    Overexpression of LSD1ΔN rescues neurodegeneration more completely

than full length LSD1 ………………………………………………………Page 145

5.2.2.    Removal of the N-terminal domain aids in LSD1 nuclear localization in the

presence of pathological tau .……………………………………………… Page 148

5.2.3.    Heterozygous LSD1ΔN modulates the PS19 Tau phenotype ……………Page 148

5.2.4.    Homozygous loss of the N-terminal domain of LSD1……………………Page 151

5.3. Discussion .………………………………………………………………………Page 152

5.4. Figures……………………………………………………………………………Page 158

CHAPTER 6:

6.     Discussion……………………………………………………………………………Page 167

6.1. LSD1 is continuously required for neuronal maintenance .….………………….Page 168

6.2. LSD1 represses the stem cell program in terminally differentiated cells .………Page 173

6.3. LSD1 interacts with specific pathological aggregates .………………………….Page 175

6.4. Reduction of LSD1 specifically exacerbates the tauopathy disease pathway .….Page 180

6.5. Intervention via LSD1 overexpression or disruption of LSD1-tau interactions…Page 183

6.6. Investigating the function of the N-terminal domain of LSD1 in vivo .…………Page 187

6.7. Conclusions………………………………………………………………………Page 192

REFERENCES .…………………………………………………………………………Page 195

LIST OF TABLES

Table 2-1        Genotyping Primers .………………………………………………………Page 45

Table 2-2        Primary Antibodies .……………………………………………………….Page 46

LIST OF FIGURES

Figure 3-1      LSD1 expression in adult murine hippocampal and cortical neurons .…….Page 69

Figure 3-2      LSD1 expression in adult murine hippocampal and cortical astrocytes .….Page 70

Figure 3-3      LSD1 expression in adult hippocampal and cortical oligodendrocytes……Page 71

Figure 3-4      LSD1 is not expressed in adult hippocampal and cortical microglia………Page 73

Figure 3-5      Neurodegeneration in Lsd1^CAGG mice .…………………………………….Page 74    

Figure 3-6      Absence of spinal cord motor neuron & muscle defects in Lsd1^CAGG mice..Page 76

Figure 3-7      Neurodegeneration in Lsd1^CAGG mice .…………………………………….Page 78

Figure 3-8      LSD1 in different cell types .………………………………………………Page 80

Figure 3-9      Absence of neurodegeneration in Lsd1^CAGG cerebellum .………………….Page 81

Figure 3-10    LSD1 is not required for kidney and liver cell viability .………………….Page 82

Figure 3-11    Loss of LSD1 results in learning and memory deficits ……………………Page 83

Figure 3-12    Lsd1^CAGG mice have learning and memory deficits .………………………Page 84

Figure 3-13    Differential expression of genes in Lsd1^CAGG hippocampus ………………Page 85

Figure 3-14    Ectopic activation of stem cell genes Lsd1^CAGG mice .…………………….Page 87

Figure 3-15    Neuronal stem cell gene expression in Lsd1^CAGG mice ……………………Page 88

Figure 3-16    Loss of LSD1 induces common neurodegeneration pathways ……………Page 90

Figure 3-17    Expression changes in Lsd1^CAGG mice correlate with AD and FTD ………Page 92

Figure 3-18    LSD1 co-localization with pTau and pTDP-43 aggregates .………………Page 93

Figure 3-19    LSD1 mislocalization is specific to AD and FTD…………………………Page 95

Figure 3-20    Absence of pathological protein aggregates in Lsd1^CAGG mice……………Page 96

Figure 3-21    Stem cell gene expression in human dementia ……………………………Page 97

Figure 4-1      LSD1 sequestration and tau accumulation in PS19 Tau mice……………Page 116

Figure 4-2      Sequestration of LSD1 in PS19 Tau mice .……………………………….Page 117

Figure 4-3      Generation of PS19 Tau mice with reduced levels of LSD1 .……………Page 119

Figure 4-4      Reduction of Lsd1exacerbates the PS19 Tau mouse paralysis phenotype..Page 121

Figure 4-5      Reduction of Lsd1 affects spinal cord in PS19 Tau mice .……………….Page 122

Figure 4-6      There is no exacerbation of neurodegeneration in PS19 Tau mice with reduced Lsd1 until 10 months of age………………………………………………Page 124

Figure 4-7      Reduction of Lsd1 exacerbates neurodegeneration in PS19 Tau mice .….Page 125

Figure 4-8      Increased neurodegeneration throughout the hippocampus and cortex of 12 months old mice .…………………………………………………………Page 127

Figure 4-9      Reduction of Lsd1 does not affect AT8 positive tau pathology .…………Page 128

Figure 4-10    Reduction of Lsd1 does not affect PHF1 positive tau pathology…………Page 130

Figure 4-11    Differential expression in 9 month old Lsd1^Δ/+, PS19 Tau, and PS19;Lsd1^Δ/+ hippocampus .…………………………………………………………….Page 131

Figure 4-12    Molecular overlap between loss of LSD1 function and tauopathy ………Page 133

Figure 4-13    LSD1 overexpression in hippocampal neurons of PS19 Tau mice ………Page 135

Figure 4-14    LSD1 overexpression rescues the neurodegenerative phenotype in the hippocampus of 11 month old PS19 Tau mice .………………………….Page 137

Figure 4-15    LSD1 overexpression reduces the gliosis in PS19 Tau mice .……………Page 139

Figure 5-1      LSD1 contains a partially conserved intrinsically disordered domain……Page 158

Figure 5-2      N-terminal domain of LSD1 is not required for neuronal survival of

PS19 Tau mice……………………………………………………………………Page 160

Figure 5-3      Removal of the N-temrinal domain of LSD1 rescues nuclear blebbing …Page 162

Figure 5-4      LSD1ΔN virus remains nuclear at the timepoint when the full length LSD1

           is sequestered .……………………………………………………………………Page 163

Figure 5-5      Tauopathy mice heterozygous for the exon 1 deletion Lsd1 ..……………Page 164

Figure 5-6      Lsd1^ΔN/+ allele has a hypomorphic-like effect on survival and paralysis…Page 165

Figure 5-7      Generation of tauopathy mice homozygous for exon 1 deletion of Lsd1...Page 166

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Genetics Biology, Neuroscience
Parola chiave	Tauopahty Alzheimer's disease Epigenetics
Committee Chair / Thesis Advisor	David Katz, Emory University
Committee Members	Nicholas Seyfried, Emory University Allan Levey, Emory University Gary Bassell, Emory University Victor Corces, Emory University Dorothy Lerit, Emory University

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