Senior Honors Thesis Proposal: "Investigating LSD1 interaction with tau aggregates by examining LSD1 in rare tauopathy cases"

Background/Introduction and Research Objectives 

Alzheimer’s Disease (AD), as the most prevalent form of dementia, was shown to be the only cause of death in America’s top 10 causes of death that cannot be prevented or cured (alz.org, 2014). Tauopathy, as a subgroup of dementia, is characterized by the deposition of pathological tau in the brain. The spectrum of tau pathologies generally includes disease forms, such as AD, Pick disease, progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) (Kovacs, 2017).Tau is a microtubule binding protein that plays a fundamental role in stabilizing microtubules in the axons and neurons (Wang, 2016). A disruption of this microtubule network, which can be caused by tau loss of function, is the bridge to tauopathies (Barbier, 2019). When the tau loses its function, it becomes hyperphosphorylated and accumulates abnormally in the somata and dendrites of affected cells and forms pathological filamentous inclusions (paired helical filaments, or PHFs), which aggregate to form insoluble neurofibrillary tangles (Hall, 1997). Pathological tau has been associated with neuronal cell death, on top of synaptic loss and increased inflammation (Gendron, 2009), but how pathological tau causes/contributes to neurodegeneration is not well understood. Thus, the objective of this project is to investigate the molecular mechanisms underlying tau-mediated neurodegeneration, which could be used as a potential therapeutic target.

Lysine-specific histone demethylase, LSD1, represses transcription by removing mono- or di-methyl groups from lysine 4 histone 3 (H3K4me1/2) (Shi et al., 2004). Previously, our lab has shown that the inhibition of LSD1 in adult mice induces cortical and hippocampal neurodegeneration, learning and memory deficits, and transcription alternations that match human AD cases (Christopher et al., 2017). To study the functional interaction between LSD1 and pathological tau, our lab utilized PS19 tau mice, a tauopathy mice model which expresses P301S mutated form of tau from human familial frontotemporal dementia patients throughout the central nerve system (Engstrom et al., 2019). We found that reduction of LSD1 in PS19 tau mice exacerbates neurodegeneration while overexpression of LSD1 rescues hippocampal neurodegeneration. Most importantly, LSD1 colocalizes with cytoplasmic pathological tau in P301S tau mice and AD cases (Figure 1 and 2) (Christopher et al., 2017, Engstrom et al., 2019). Based on this, we hypothesize that after translation, LSD1 interacts with pathological tau in the cytoplasm. This process will prevent LSD1 from entering the nucleus and cause neurodegeneration. 

Figure 1. LSD1 is colocalized with pathological tau in human AD cases. Figure 1A-C are immunofluorescence images of LSD1 (red), pathological tau(green), and merged immunofluorescence in AD cases (Christopher et al., 2017).

Figure 2. This set of nine images displays the LSD1 sequestration and tau accumulation in the presence of pathological tau. The images 2A, B, and C are representative immunofluorescence of 12-month-old control wild-type mice showing the nuclear marker DAPI (4',6-diamidino-2-phenylindole) (2A), LSD1 (2B), and merged (2C) in the cerebral cortex where LSD1 is localized specifically to DAPI-positive nuclei. The images 2D, E, and F are representative images of the cerebral cortex in 12-mo-old PS19 Tau mice. Staining for DAPI (2D), LSD1 (2E), and merged (2F) shows that LSD1 is localized outside the nucleus and depleted from the DAPI-positive nucleus. Arrows denote cells where LSD1 is localized outside of the nucleus, and asterisks denote LSD1 localized specifically to the nucleus. The images 2G, H, and I are representative immunofluorescence of 12-mo-old PS19 Tau mouse with staining for DAPI (2G), AT8-positive hyperphosphorylated tau (2H), and merge (2I) where hyperphosphorylated tau accumulates in the cytoplasm of the cell body. Arrowheads denote hyperphosphorylated tau (Engstrom et al., 2019).

AD and other types of rare tauopathy cases, such as CBS, PSP, Pick’s disease, and Frontotemporal dementia with parkinsonism-17 cases have varying tau pathology and symptoms. AD expresses both tau isoforms containing 3 microtubule binding repeats (3R) and 4 microtubule binding repeats (4R), The tau pathology of AD follows neuroanatomical pathways and can reflect transmission of abnormal tau proteins from cell to cell in a “prion-like” manner (Ando, 2021). At the early stage, pathological tau is associated with neurodegeneration in hippocampus and entorhinal cortex, leading to learning and memory deficits (Wolfe, 2012). Then at the late stage, pathological tau is transmitted to cortical regions (Vogel et al., 2020). The disease symptoms are also related to deficits in language, reasoning, and social behavior (Kumar et al., 2021). 

One of the most prominent examples of 3R tauopathy is Pick’s disease, which is unique in that it is a form of cortical atrophy that has neuronal lesions that are balloon-shaped, hence giving the name “Pick bodies.” Commonly, the Pick-body-like inclusions can be detected in oligodendrocytes in affected white matter. This disease features similar phenotypes to FTLD-tau in patients, such as deterioration of language, personality and memory (Chung et al., 2020). On the other hand, PSP and CBD both tend to have prominent accumulation of 4R tau in neurons and glial cells (Chung et al., 2020). CBD and PSP are alike in that they exhibit symptoms of parkinsonism and postural instability (Chung et al., 2020). However, these two diseases vary in locations of pathological tau. While cerebral cortex and basal ganglia are preferentially affected by the CBD, the PSP tends to show neuronal loss in globus pallidus, subthalamic nucleus, and substantia nigra, along with astrocytes (Chung et al., 2020). Because these rare tauopathies are so different in terms of pathology, it provides an unique opportunity to interrogate the specificity of LSD1’s interaction with pathological tau. Thus, my project is to investigate how the tau aggregates interact with LSD1 to inhibit the required function of LSD1 in these rare tauopathies.

Methodology 

To examine the co-localization of LSD1 with pathological tau in tau-mediated neurodegeneration, we perform LSD1 immunohistochemistry and LSD1-pathological tau dual immunofluorescence in tauopathy and age matched non-demented cases. We analyze the human brain tissue from various brain regions, including frontal cortex, basal ganglia, and hippocampus, which are highly affected by different tauopathies. The brain samples are provided by the Emory University Brain Bank.

1. LSD1 Immunohistochemistry 

Immunohistochemistry is a method commonly used in research, yet we have developed a revised protocol that best works with our brain tissue slides. This two-day staining protocol begins with the deparaffinization of the brain tissue slides utilizing xylenes. Once the antigen is retrieved through microwaving in the citrate buffer, the edges of the tissue in each slide is marked with a PAP Pen, which is a pen that creates a thin film-like hydrophobic barrier when a circle is drawn around a specimen on a slide. This helps the amount of staining solutions to remain on the slides, maximizing the interaction of the tissues with the solutions. Then, hydrogen peroxide solution is applied to the slides to block the endogenous peroxidase activity. In the following step, Triton X is used to increase the lipid membrane permeabilization which will improve the antibody-antigen bonding. Once the permeabilization is done, the blocking solution made with animal serum will prevent non-specific binding. As the last part of the first day of staining, the slides are incubated with the primary antibody against LSD1 overnight at 4 degrees Celsius.

The second day of staining begins with  applying the biotinylated secondary antibody which will specifically bind with the primary antibody. Solution created with Vectastain ABC HRP Kit is applied afterwards to form the avidin-biotin complex. Once the incubation is done, the DAB working solution (Vector SK-4110 -Impact DAB) is applied to generate color. The slides are then dried, and coverslip is then placed on top of the slides for imaging analysis. 

2. LSD1-pathological tau dual immunofluorescence

The immunofluorescence is a lab technique using fluorescent-tagged antibodies to label the antigen presence in tissues. This process is similar to the immunohistochemistry process in that the first day of staining is almost the same. Beginning with deparaffinization of the tissue slides, the antigen is retrieved and the lipid membrane is permeabilized. After blocking brain tissue slides are incubated with primary antibodies against pathological tau and LSD1. 

The second day is where the process is divergent from immunohistochemistry. Pathological tau is labeled by a fluorescent-tagged secondary antibody, and LSD1 is labeled with a HRP secondary antibody followed by TSA amplification to enhance fluorescent signal. Then the true black solution is used to suppress the autofluorescence background in human tissue. Coverslip is applied and the slides are dried before they are transferred to the fluorescent microscope for imaging and analysis.

3. Data Analysis

We will quantify the abnormal tangle shaped LSD1 from immunohistochemistry staining and LSD1-pathological tau colocalization from Immunofluorescence staining. Analysis is performed using analyses of variance (ANOVA) with post hoc Sidak’s multiple comparison test on cases of the rare tauopathies (N=5 for each tauopathy), along with age matched non-demented patients as controls (N=7). Importantly, this will be performed blind to the patient status between experimental cases and non-demented controls. 

Expected Results and Implications 

After the analysis of slides,  if LSD1 co-localizes with all forms of pathological tau in different tauopathies cases, we can conclude that there is no specificity to the colocalization between LSD1 and pathological tau in terms of cell type, aggregated structure or particular isoform. Accordingly, we could follow up with the method of fractionation to study the co-localization in closer detail. From here, if LSD1 co-localizes with pathological tau in all of these rare tauopathies, it would indicate that any outstanding therapeutic intervention targeted to the LSD1 pathway could also potentially be effective in various tauopathies in general. On the contrary, we can determine that there is some specificity to the colocalization that is dependent on cell type, aggregated structure or particular tau isoform if LSD1 co-localizes with a subset or none of the rare tauopathies. From our preliminary data, we recapitulated the pathological tau tangles and the threads in AD cases. In addition, we observed some abnormally shaped LSD1 staining in control and some rare tauopathies cases. To get a better understanding, however, more research must be done on the cases. Once we have a further clarification of the staining, our next steps will include determining how LSD1 is interacting with pathological tau in each of the rare tauopathy cases.  

I firmly believe that my research will make a significant difference by showing light to what causes neurodegeneration in humans. Using my research, the field of medicine can unearth the potential pathways of neurodegenerative disease and accordingly find effective cures or treatments for patients struggling from the pathology. The research particularly excites me because I am making a personal effort and contributing to something that can possibly aid my grandmother; I hope in the future this research can be a crucial step in developing a remedy. By undertaking this research project, I aspire to discover more about the pathway that leads to neurodegeneration and most importantly how we can target this mechanism to stop neurons from dying. This work will provide the basis of the mechanism, which could untimely inform a future drug screen as a future project.

References

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