Analyzing the localization of LSD1 in Tau transfected HEK293 cells: a novel method for exploring biological mechanisms of tauopathy and potential therapeutics Restricted; Files Only

Gadomski, Christopher (Summer 2023)

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Background: Alzheimer’s Disease (AD) as a secondary tauopathy is characterized by the presence of amyloid beta plaques and neurofibrillary tangles of phosphorylated tau. However, despite decades of research efforts, there is a lack of significant clinical treatments of AD and other tauopathies. Further research is required to explore the complexities of these diseases and create treatments that target the etiological mechanisms of these pathologies. In this study, we present a brief review of the evidence for the role of LSD1 in tau mediated neurodegeneration and demonstrate further evidence of an interaction between LSD1 and pathological tau: a novel method for future drug discovery pursuits.

Methods: HEK 293 cells were transfected with tau and stained for LSD1. Subsequently, CRISPR-modified HEK293 cells with endogenous Green Fluorescent Protein (eGFP)-tagged LSD1 were transfected with various tau mutations, and DNA was stained with NucRed 647. Cell cycle was synchronized via thymidine block, and cells were imaged periodically to determine LSD1 localization in relation to tau. HEK cells were transfected with tau and LSD1 and proteins were analyzed by western blot.

Results: Tau transfection is incredibly cytotoxic to cells and causes LSD1 mislocalization disrupting normal cell cycle. Despite the colocalization of LSD1 and tau, they fail to appear together in pulldown experiments.

Conclusion: These findings demonstrate that tau interferes with the nuclear localization of LSD1 in HEK cells. This observation provides further support that tau prevents LSD1 nuclear localization by blocking the nuclear localization signal (NLS) and can be tested in this system in future experiments. These results offer a novel target for the treatment of AD and other tauopathies. These protocols are promising first steps for drug discovery processes aimed to prevent LSD1-mediated neurodegeneration in AD and other tauopathies. 

Table of Contents

Table of Contents

Chapter I 1 Introduction 1

1.1 Despite increasing prevalence Alzheimer’s Disease lacks effective treatment 2

1.2: Significance of tau in neurodegeneration 3

1.3: Lysine-specific demethylase 1 is a potential therapeutic target of AD/tauopathies

4 Chapter II 6 Methodology 6

2.1: Protocols to explore LSD1-tau interaction in vitro 7

2.1A: Cell Culture 7

2.1B: Transfection 7

2.1C: Immunostaining 8

2.2: Live imagining of fluorophores to create time lapse movies 9

2.3 GFP-Trap and Western blot 9

Chapter III 10

Results 10

3.1 In vitro experiments offer mechanistic insight and development of potential therapeutics 11

3.2 Time intensive experiments require continual optimization 11

3.3 Summary of results 12

Figure 1. Initial LSD1 staining and live imaging shows LSD1 losing nuclear enrichment13

Figure 2. Mock Transfection and DNA staining demonstrates LSD1 to be nuclear(Control) 15

Figure 3. Tau transfection does not immediately colocalize to LSD1 17

Figure 4. LSD1 loses nuclear enrichment 21 hours after transfection 20

Figure 5. Tau transfection causes altered LSD1 morphology 22

Figure 6. Increased tau expression causes LSD1 to lose distinct nuclear enrichment more globally 24

Figure 7. Tau- LSD1 interaction is not strong enough to appear via immunoprecipitation 26

Chapter IV 27 Discussion and Future Directions 27

Chapter V 31 References 31 

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