From epigenetic memory to memory loss: How tau prevents LSD1 from regulating transcription and induces cell death in Alzheimer’s disease 公开
Walker, Alicia (Spring 2020)
Published
Abstract
During development, the histone demethylase LSD1 functions as an eraser of epigenetic memory, removing ‘bookmarks’ of active transcription to induce cellular reprogramming. However, LSD1’s role in terminally differentiated cells is unknown. Previously, our lab implicated LSD1 in tau-induced neurodegeneration by showing that LSD1 localizes to pathological tau aggregates in Alzheimer's disease (AD) cases, and that it is continuously required for the survival of hippocampal and cortical neurons in mice. Here, we utilize the P301S tauopathy mouse model to demonstrate that pathological tau can exclude LSD1 from the nucleus in neurons. In addition, we show that reducing LSD1 in these mice is sufficient to highly exacerbate the tauopathy phenotype. We find that overexpressing LSD1 in the hippocampus of tauopathy mice, even after pathology has formed, is sufficient to significantly delay neurodegeneration. These results suggest that inhibiting LSD1 via sequestration contributes to tau-mediated neurodegeneration. In considering the mechanism of the interaction between tau and LSD1, we recognized that both proteins have disordered regions. Tau has been shown to interact with disordered regions of other proteins, therefore we hypothesized that tau might similarly interact with LSD1 in this way. Here, we show preliminary evidence that loss of the N-terminal disordered domain reduces the sequestration of LSD1 by tau aggregates. Additionally, we further conceive that this disordered domain may be partially required for LSD1 functionality in vivo. Taken together, this work provides evidence that LSD1, specifically its N-terminal disordered domain, is a promising therapeutic target for tauopathies such as AD.
Table of Contents
1. Chapter I – Introduction…………………………………………………………………. 1
2. Chapter II – Materials and Methods……………………………………………………... 5
A. Solutions and Buffers……………………………………………………………. 6
B. Mouse work……………………………………………………………………… 8
i. Mouse lines………………………………………………………………. 8
ii. Mouse genotyping by PCR………………………………………………. 9
iii. Euthanasia and tissue fixation…………………………………………... 10
iv. Quantitative analysis of paralysis: rotarod and grid performance……… 10
v. Mouse magnetic resonance imaging (MRI)…………………………….. 11
vi. Stereotaxic surgery and viral infusion………………………………….. 11
C. Staining…………………………………………………………………………. 12
i. Histology and histological quantification………………………………. 12
ii. Immunohistochemistry and immunofluorescence……………………… 12
iii. Quantification of tau accumulation……………………………………... 13
iv. Protein quantification…………………………………………………… 14
D. RNA Sequencing……………………………………………………………….. 14
i. RNA isolation…………………………………………………………... 14
ii. RNA Sequencing analysis………………………………………………. 15
iii. Comparison to human gene expression data……………………………. 17
E. Table 2-1 Genotyping primers………………………………………………….. 18
F. Table 2-2 Primary antibodies…………………………………………………… 18
3. Chapter III – The inhibition of LSD1 via sequestration contributes to tau-mediated neurodegeneration……………………………………………………………………… 19
A. Introduction…………………………………………………………………….. 20
B. Results………………………………………………………………………….. 22
i. Tau pathology depletes LSD1 from the nucleus in the PS19 Tau mouse model…………………………………………………………………… 22
ii. Reduction of LSD1 increases the mouse tauopathy phenotype………… 23
iii. Reduction of LSD1 exacerbates PS19 Tau neurodegeneration………… 26
iv. Tau pathology is not affected by change in LSD1 levels………………. 27
v. The functional interaction between tau pathology and LSD1 inhibition is specific………………………………………………………………….. 28
vi. Overexpression of LSD1 rescues neurodegeneration in the hippocampus of PS19 Tau mice……………………………………………………….. 29
C. Discussion………………………………………………………………………. 32
D. Figures…………………………………………………………………………... 35
i. Fig. 3.1: LSD1 sequestration and tau accumulation in PS19 Tau mice… 36
ii. Fig. 3.2: Reduction of Lsd1 exacerbates the PS19 Tau mouse paralysis phenotype……………………………………………………………….. 37
iii. Fig. 3.3: Reduction of Lsd1 exacerbates neurodegeneration in PS19 Tau mice……………………………………………………………………... 38
iv. Fig. 3.4: Molecular overlap between loss of LSD1 function and tauopathy………………………………………………………………... 40
v. Fig. 3.5: LSD1 overexpression rescues the neurodegenerative phenotype in the hippocampus of 11 month old PS19 Tau mice…………………... 41
E. Supplemental Data……………………………………………………………… 42
i. Fig. S1: Sequestration of LSD1 in PS19 Tau mice……………………... 43
ii. Fig. S2: Generation of PS19 Tau mice with reduced levels of LSD1….. 45
iii. Fig. S3: Reduction of Lsd1 affects spinal cord in PS19 Tau mice……... 47
iv. Fig. S4: There is no exacerbation of neurodegeneration in PS19 Tau mice with reduced Lsd1 until 10 months of age……………………………… 48
v. Fig. S5: Increased neurodegeneration throughout the hippocampus and cortex of 12 month old mice……………………………………………. 49
vi. Fig. S6: Reduction of Lsd1 does not affect AT8 positive tau pathology.. 51
vii. Fig. S7: Reduction of Lsd1 does not affect PHF1 positive tau pathology………………………………………………………………... 53
viii. Fig. S8: Differential expression in 9 month old Lsd1-/+, PS19 Tau, and PS19;Lsd1-/+ hippocampus……………………………………………… 55
ix. Fig. S9: LSD1 overexpression in hippocampal neurons of PS19 Tau mice……………………………………………………………………... 57
x. Fig. S10: LSD1 overexpression reduces the gliosis in PS19 Tau mice… 59
xi. Movie S1 (description): Reduction of LSD1 in PS19 Tau mice exacerbates paralysis………………………………………………………………… 60
xii. Movie S2 (description): Magnetic Resonance Imaging of hippocampal atrophy throughout the brain when LSD1 is reduced in PS19 Tau mice.. 60
xiii. Movie S3 (description): Hippocampal injection of viral LSD1 did not affect development of paralysis in PS19 mice………………………….. 61
xiv. Movie S4 (description): Pathological Tau Induces Neurodegeneration by sequestering and inhibiting LSD1………………………………………. 61
xv. Data S1- separate file (description): Expression changes in 9 month old Lsd1-/+, PS19 Tau, PS19;Lsd1-/+ mice………………………………….. 61
4. Chapter IV – Investigating the interaction between pathological tau and the N-terminal disordered domain of LSD1…………………………………………………………….. 62
A. Introduction……………………………………………………………………... 63
B. Results…………………………………………………………………………... 66
i. Overexpression of LSD1ΔN rescues as well as full length LSD1 until one year of age………………………………………………………………. 66
ii. Injection with truncated LSD1 does not result in neuronal blebbing…... 67
iii. Overexpressed LSD1ΔN localizes to the nucleus more consistently than full length LSD1………………………………………………………... 68
iv. PS19;Lsd1ΔN/+ mice show an intermediate survival phenotype to PS19 and PS19;Lsd1-/+ mice………………………………………………………. 69
v. PS19;Lsd1ΔN/+ mice show a bimodal paralysis phenotype……………... 70
vi. Generation of Lsd1ΔN/ΔN animals will rule out the N-terminal disordered domain as necessary for LSD1 function………………………………... 70
C. Discussion………………………………………………………………………. 71
D. Figures…………………………………………………………………………... 74
i. Fig. 4.1: Tau and LSD1 both have disordered regions…………………. 75
ii. Fig. 4.2: LSD1 homologs share conserved structure…………………… 76
iii. Fig. 4.3: N-terminally truncated LSD1 constructs……………………… 76
iv. Fig. 4.4: Overexpression of LSD1ΔN rescues as well as full length…… 77
v. Fig. 4.5: Only mice injected with the full length LSD1 virus show blebbing in the CA1…………………………………………………….. 78
vi. Fig. 4.6: LSD1ΔN virus remains nuclear even after sequestration in age-matched PS19- LSD1 inj……………………………………………….. 79
vii. Fig. 4.7: Generation of mice heterozygous for the exon 1 deletion of Lsd1 in the tau background…………………………………………………… 80
viii. Fig. 4.8: PS19;Lsd1ΔN/+ mice show a survival phenotype intermediate of PS19 and PS19;Lsd1-/+ mice……………………………………………. 80
ix. Figure 4.9: Some PS19;Lsd1ΔN/+ mice have no paralysis despite old age………………………………………………………………………. 81
x. Fig. 4.10: Generation of mice homozygous for the exon 1 deletion of Lsd1 in the tau background…………………………………………………… 82
5. Chapter V – Conclusions and Future Directions……………………………………….. 83
6. Chapter VI – References………………………………………………………………... 86
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