LSD1 Continuously Protects the Differentiated State of Neurons Público
Christopher, Michael Anthony (2016)
Published
Abstract
Epigenetic regulation of transcription is carried out by a plethora of enzymes that read and modify the state of chromatin. One facet of this regulation takes place by the addition and removal of posttranslational modifications on the tails of the histone proteins to which the DNA is wrapped around. One of the many types of modifications is methylation of different lysine residues. Histone methylation can be associated with either active or repressed transcription, and the specificity is determined by which histone, and lysine residue it is found on. These modifications are dynamic and regulated by methyltransferases and demethylases. One of these is lysine specific demethylase 1 (LSD1/KDM1a). LSD1 has primarily been studied in the context of cell fate transitions, and little is known about its role in differentiated cells, specifically neurons of the brain. To investigate the role of LSD1 in differentiated cells types, we inducibly deleted Lsd1 in adult mice and assayed various tissues for detrimental effects. We find that ubiquitous deletion of Lsd1 in adult mice results in paralysis accompanied by hippocampal and cortical neurodegeneration. Further, we find that genome-wide transcriptional changes that occur in human dementia take place in mice in the absence of LSD1. These data suggest that deletion of Lsd1 is sufficient to recapitulate human neurodegeneration in mice. In addition, degenerating neurons lacking LSD1 display ectopic reactivation of stem cell genes. This suggests a new paradigm, where differentiated cells are continuously required to repress transcription of other cell fates and may not be fully committed to the differentiated fate. Finally, the mechanism by which the accumulation of pathological protein aggregates in human dementias leads to neuronal cell death is unknown. We find that LSD1 is mislocalized with cytoplasmic pTau in Alzheimer's Disease and pTDP-43 in Frontotemporal Dementia, suggesting its nuclear function could be compromised. Together with the observation that loss of LSD1 in adult mice is sufficient to cause neurodegeneration, my findings suggest a model of human neurodegeneration where LSD1 nuclear activity is inhibited by its sequestration in the cytoplasm by pathological protein aggregates, leading to neuronal cell death by misregulation LSD1 transcriptional targets.
Table of Contents
CHAPTER 1: INTRODUCTION
1.1 Introduction to epigenetics. 2
1.2 Epigenetics modifications and their regulators. 3
1.2.1 DNA Methylation. 3
1.2.2 Histone Modifications. 6
1.2.3 RNAs that regulate chromatin. 7
1.3 MeCP2 regulates long gene expression in neurons. 8
1.4 REST colocalizes with protein aggregates in neurodegenerative diseases. 11
1.5 FMR1 mRNA induces epigenetic silencing of the locus. 15
1.6 A possible role for LSD1 in neurological disease. 16
Scope of the dissertation. 19
1.7 Figures. 20
CHAPTER 2: METHODS. 22
2.1 Solutions and Buffers. 23
2.2 Mouse work. 24
2.2.1 Mouse lines. 24
2.2.2 Mouse genotyping by PCR.. 25
2.2.3 Tamoxifen injections. 25
2.2.4 Quantification of Lsd1 deletion. 26
2.2.5 Morris Water Maze. 26
2.2.6 Fear Conditioning. 27
2.2.7 Mouse Tissue Fixation. 27
2.3 Staining. 28
2.3.1 Immunofluorescence - Mouse. 28
2.3.2 Immunohistochemistry - Mouse. 28
2.3.3 Immunohistochemistry - Human. 29
2.3.4 Immunofluorescence - Human. 30
2.3.5 Quantification of LSD1 Colocalization with pTau and pTDP-43. 31
2.3.6 TUNEL Assay. 31
2.4 RNA Sequencing. 32
2.4.1 RNA isolation. 32
2.4.2 Sequencing library preparation. 32
2.4.3 RNA sequencing Analysis. 32
2.4.4 Comparison to Human Gene Expression Data. 33
CHAPTER 3: LSD1 PROTECTS AGAINST HIPPOCAMPAL AND CORTICAL NEURODEGENERATION 36
3.1 Abstract 37
3.2 Introduction. 37
3.3 Results. 39
3.3.1 LSD1 is continuously required to prevent neurodegeneration. 39
3.3.2 Loss of LSD1 results in learning and memory defects. 43
3.3.3 LSD1 inhibits reactivation of stem cell transcription. 44
3.3.4 Loss of LSD1 induces common neurodegeneration pathways. 45
3.3.5 Lsd1CAGG gene expression changes correlate with expression changes in AD and FTD cases 46
3.3.7 LSD1 is mislocalized in human dementias. 49
3.3.8 Lsd1CAGG mice do not have protein aggregates. 50
3.3.9 Increased stem cell gene expression in AD and FTD patients. 51
3.4 Discussion. 51
3.5 Figures. 54
CHAPTER 4: DISCUSSION.. 88
4.1 LSD1 is Neuroprotective. 89
4.2 Interaction of LSD1 and pTau. 94
4.3 LSD1 Continuously Maintains the Differentiated State of Neurons. 99
4.4 Conclusions and Future Directions. 102
4.5 Figures. 104
REFERENCES. 108
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