Investigating the therapeutic role of EP2 antagonism in two-hit mouse model of Alzheimer’s disease Open Access

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder, characterized by loss of neurons, the formation of pathological proteins like amyloid-beta (Aβ), and activation of glial cells in the brain leading to progressive cognitive decline and dementia. So far, there is no drug available to treat the underlying pathology of AD. In our laboratory, we have demonstrated that the prostaglandin E2 receptor (EP2) has a role in neuroinflammation in mouse models of neurodegeneration and small molecule EP2 inhibitors attenuate the robust inflammatory bursts following status epilepticus in rodents. The overall goal of this study is to further investigate the therapeutic efficacy of our EP2 antagonist in ameliorating AD pathology and inflammation. Here, we aim to use a two-hit model of Alzheimer’s disease where transgenic 5xFAD mice are subjected to an external stimulus of lipopolysaccharide (LPS) to induce an additional level of inflammation in the AD brain. Subsequently, cohorts of mice will be treated with a potent and selective EP2 antagonist (TG11-77.HCl) added to the drinking water and their brains will be investigated for amyloid pathology and associated neuroinflammation by qRT-PCR as well as immunohistological staining and quantification. The results show that administration of LPS to 5xFAD mice results in a more robust neuroinflammation compared to non-LPS treated mice, which was similar to results that were found in previous studies in our laboratory. Furthermore, we demonstrate attenuation of neuroinflammation, gliosis and amyloid pathology by selective inhibition of the EP2 receptor with the TG11-77.HCl compound. This study strengthens the candidacy of the EP2 receptor as a therapeutic target for combating AD pathology.

Table of Contents

Table of Contents 

INTRODUCTION ........................................................................................................................................................ 1 

BACKGROUND ............................................................................................................................................................ 1 

PROINFLAMMATORY MEDIATORS .............................................................................................................................. 3 

FIGURE 1 .................................................................................................................................................................... 6 

CYTOKINES IN AD...................................................................................................................................................... 7 

CHEMOKINES IN AD ................................................................................................................................................... 9 

GLIOSIS IN AD ......................................................................................................................................................... 11 

LIPOPOLYSACCHARIDE AND AD .............................................................................................................................. 12 

RATIONALE .............................................................................................................................................................. 13 

HYPOTHESIS ............................................................................................................................................................. 14 

MATERIALS AND METHODS .............................................................................................................................. 15 

ANIMALS .................................................................................................................................................................. 15 

STUDY DESIGN ......................................................................................................................................................... 15 

RNA EXTRACTION ................................................................................................................................................... 16 

CDNA SYNTHESIS .................................................................................................................................................... 17 

QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QRT‑PCR) ................................................................ 17 

TISSUE SECTIONING .................................................................................................................................................. 18 

FREE FLOATING GFAP STAIN .................................................................................................................................. 18 

CONGO RED STAINING .............................................................................................................................................. 19 

IMAGE QUANTIFICATION .......................................................................................................................................... 20 

STATISTICAL ANALYSIS ............................................................................................................................................ 20 

RESULTS.................................................................................................................................................................... 21 

EXPRESSION OF PROINFLAMMATORY MEDIATORS .................................................................................................. 22 

UPREGULATION OF SELECT CYTOKINES AND CHEMOKINES .................................................................................... 24 

GLIOSIS .................................................................................................................................................................... 27 

CONGO RED STAINING ............................................................................................................................................. 29 

GFAP STAINING ...................................................................................................................................................... 32 

DISCUSSION ............................................................................................................................................................. 33 

CONCLUSION ........................................................................................................................................................... 37 

FUTURE DIRECTIONS ........................................................................................................................................... 38 

LIMITATIONS .......................................................................................................................................................... 38 

APPENDIX ................................................................................................................................................................. 40 

TABLE 1A. ................................................................................................................................................................ 40 

TABLE 1B. ................................................................................................................................................................ 40 

TABLE 1C. ................................................................................................................................................................ 41 

TABLE 1D ................................................................................................................................................................. 41 

TABLE 1E. ................................................................................................................................................................ 41 

TABLE 2A. ................................................................................................................................................................ 42 

TABLE 2B. ................................................................................................................................................................ 42 

TABLE 2C. ................................................................................................................................................................ 43 

TABLE 2D. ................................................................................................................................................................ 43 

TABLE 2E ................................................................................................................................................................. 44 

TABLE 3A. ................................................................................................................................................................ 44 

TABLE 3B. ................................................................................................................................................................ 44 

TABLE 3C. ................................................................................................................................................................ 45 

TABLE 3D. ................................................................................................................................................................ 45 

TABLE 3E. ................................................................................................................................................................ 45 

TABLE 4. .................................................................................................................................................................. 46 

REFERENCES ........................................................................................................................................................... 47 

About this Honors Thesis

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School	Emory College
Department	Human Health
Degree	B.A.
Submission	Honors Thesis
Language	English
Research Field	Health Sciences, Pharmacology
Keyword	Alzheimer's Disease EP2 Antagonist
Committee Chair / Thesis Advisor	Ganesh, Thota, Emory University
Committee Members	Cole, Megan, Emory University Frenzel, Kristen, Emory University Freeman, Amanda, Emory University

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