Investigating the Effect of scyllo-Inositol Treatment on the Brain in a Mouse Model of Alzheimer’s Disease by Qingda Hu A thesis submitted in conformity with the requirements for the degree of Master’s of Science Department of Laboratory Medicine and Pathobiology University of Toronto ©Copyright by Qingda Hu 2016 Investigating the Effect of scyllo-Inositol Treatment on the Brain in a Mouse Model of Alzheimer’s Disease Qingda Hu Master’s of Science Department of Laboratory Medicine and Pathobiology University of Toronto 2016 Abstract scyllo-Inositol is a potential therapeutic for the treatment of agitation and aggression in patients with Alzheimer’s disease. scyllo-Inositol reduces aggregation of beta-amyloid peptide, reduces amyloid plaques, and reduces myo-inositol concentration in the brain. The change in gene expression in the brain of TgCRND8 mice after scyllo-inositol treatment has not been previously investigated. I hypothesized that scyllo-inositol treatment would cause changes in expression of genes involved in neurodegenerative diseases and neuropsychiatric disorders. Affymetrix gene expression microarrays were used to analyze changes in the hippocampus and cerebral cortex of TgCRND8 mice as well as in non-transgenic littermates after 30 days of scyllo-inositol treatment. Analysis of the data with the Database for Annotation, Visualization and Integrated Discovery and Ingenuity Pathways Analysis program showed gene expression changes in synaptic function, calcium, dopamine receptor, glutamate receptor, and myo-inositol signalling pathways. In light of these results, I concluded that my hypothesis was correct. ii Acknowledgments I would like to thank Dr. JoAnne McLaurin for her guidance and support throughout my Master’s degree. This project would not have been possible without the assistance of Mary Brown. I would also like to thank all the other members of the McLaurin laboratory for their feedback and discussion. I would like to thank the members of my graduate advisory committee, Dr. Isabelle Aubert and Dr. Krista Lanctôt, for their assistance and feedback. iii Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Abbreviations ..................................................................................................................... ix Chapter 1 Introduction .....................................................................................................................1 1.1 Alzheimer’s disease .............................................................................................................1 1.2 Neuropsychiatric symptoms - Agitation and aggression .....................................................3 1.3 scyllo-Inositol .......................................................................................................................6 Chapter 2 Rationale, Objectives, and Hypothesis ..........................................................................11 2.1 Rationale ............................................................................................................................11 2.2 Hypothesis..........................................................................................................................11 2.3 Specific aims ......................................................................................................................12 Chapter 3 Materials and Methods ..................................................................................................13 3.1 Animals ..............................................................................................................................13 3.2 RNA isolation ....................................................................................................................13 3.3 Affymetrix microarray .......................................................................................................14 3.4 Functional clustering and pathway analysis ......................................................................15 3.5 RT-qPCR............................................................................................................................16 3.6 Primer design .....................................................................................................................17 3.7 Analysis of qPCR data .......................................................................................................21 Chapter 4 Results ...........................................................................................................................23 4.1 Effect of scyllo-inositol treatment in TgCRND8 mouse model of Alzheimer’s disease ...23 iv 4.1.1 Hippocampal microarray comparison between scyllo-inositol treated and untreated 100 day old TgCRND8 mice .................................................................25 4.1.2 Cortical microarray comparison between scyllo-inositol treated and untreated 100 day old TgCRND8 mice .................................................................................39 4.1.3 Primer design, quality control, and choosing internal control for qPCR ...............45 4.1.4 qPCR comparison between scyllo-inositol treated and untreated 100 day old TgCRND8 mice .....................................................................................................52 4.1.5 qPCR comparison between scyllo-inositol treated and untreated 150 day old TgCRND8 mice .....................................................................................................55 4.1.6 Hippocampal microarray comparison between scyllo-inositol treated and untreated 200 day old TgCRND8 mice .................................................................60 4.1.7 Cortical microarray comparison between scyllo-inositol treated and untreated 200 day old TgCRND8 mice .................................................................................69 4.2 Investigating the Aβ dependent and Aβ independent effects of scyllo-inositol treatment ............................................................................................................................71 4.2.1 Hippocampal microarray comparison between 200 day old TgCRND8 mice and non-transgenic littermates ...............................................................................72 4.2.2 Cortical microarray comparison between 200 day old TgCRND8 mice and non-transgenic littermates ......................................................................................80 4.2.3 Hippocampal microarray comparison between scyllo-inositol treated and untreated 200 day old non-transgenic littermates ..................................................89 4.2.4 Cortical microarray comparison between scyllo-inositol treated and untreated 200 day old non-transgenic littermates ..................................................................95 Chapter 5 Discussion ...................................................................................................................101 Chapter 6 Conclusion and future directions.................................................................................106 6.1 Conclusion .......................................................................................................................106 6.2 Future directions ..............................................................................................................106 References or Bibliography .........................................................................................................108 v List of Tables Table 3.1 The default run method for standard comparative Ct using SYBR Green 17 detection with a melt curve Table 3.2 List of all the primers that were used in this thesis 18 Table 4.1 List of top enriched clusters identified by DAVID in the comparison of 30 hippocampal samples from 100 day old TgCRND8 mice treated and untreated with scyllo-inositol Table 4.2 Top upstream regulators identified in the IPA core analysis for hippocampal 39 comparison of treated and untreated 100 day old TgCRND8 mice Table 4.3 List of top enriched clusters identified by DAVID in the cortical comparison 42 of treated and untreated 100 day old TgCRND8 samples Table 4.4 Top upstream regulators identified in the IPA core analysis for cortical 44 comparison of treated and untreated 100 day old TgCRND8 mice. Table 4.5 The efficiency and melt curve peak temperature for all of the primers used 49 Table 4.6 Sample average Ct values from rt-qPCR of all 8 control genes of 10 samples 51 Table 4.7 Analysis by BestKeeper of the data from Table 4.6 51 Table 4.8 Clustering of annotation from DAVID of hippocampal comparison of 66 treated and untreated 200 day old TgCRND8 Table 4.9 The 19 probe sets and extra information for the cortical comparison of 200 70 day TgCRND8 mice Table 4.10 List of top enriched clusters identified by DAVID in the hippocampal 76 comparison of 200 day old TgCRND8 mice and non-transgenic littermates Table 4.11 List of top enriched clusters identified by DAVID for