Functional proteome analysis of age associated PrPC knockout mice liver along with regulatory response of cytoskeleton associated tau protein and fatty liver disease. Dissertation For the award of the degree “Doctor rerum naturalium (Dr. rer. nat.)” in the Molecular Medicine Study Program at the Georg-August-University Göttingen Submitted by Amandeep Singh Arora From SAS Nagar (Punjab - India) Göttingen, 2015 i Members of the Thesis Committee: Supervisor: Name, Institute: Prof. Dr. Mathias Bähr, Department of Neurology, University Medical Center, Georg-August University, Göttingen Second member of the thesis committee: Name, Institute: Prof. Dr. Karsten Hanisch, Department of Neuropathology, University Medical Center, Georg-August University, Göttingen Third member of the thesis committee: Name, Institute: Prof. Dr. Mikael Simons, Max Planck Institute for Experimental Medicine, Göttingen Date of Disputation: 14-04-2015 ii DECLARATION Here I declare that my doctoral thesis entitled “Functional proteome analysis of age associated PrPC knockout mice liver along with regulatory response of cytoskeleton associated tau protein and fatty liver disease” has been written independently with no other sources and aids than quoted. Amandeep Singh Arora Göttingen, January 2015 iii Acknowledgments A long journey of more than three years has been interesting and adventurous but it would not have been possible to complete it without the blessings and support of my father Sr. Parlad singh and my mother Smt. Surinder kaur. So, I dedicate my PhD degree to my parents. Firstly, I would like to thanks my direct supervisor prof. Dr. Inga Zerr for giving me opportunity to do PhD in the prion research group and for her scientific and moral support during failures and successful of experiments. Secondly, I thanks to Dr. Saima Zafar for her support in this challenging project which finally gave an interesting and a new outcome. It was a great experience of learning new innovative techniques like CE and executes new challenging ideas, which had not been possible without the support of Dr. Matthias Schmitz. I thanks to Dr. Franc Llorenz, from whom I learnt how to be more productive and efficient during performing of experiments. It was a great help of Prof. Dr. Sabine Mihm from the department of gastroenterology in analyzing the liver disease samples. I would also like to acknowledge the names of Prof. Dr. kollmar and Dr. Katrin Eckermann for their helping contribution in my PhD project. I thanks to my family of labmates and friends, Waqas, Maria, Mohsin, Nadine, Tobias, Neelam, katrin dittmar, Ulla, Siri and our new Portuguese girls, Susana and Angela for their kind help and support during this journey of up and downs. I specially thanks to the funniest creature of our lab and my friend Thüne for making the cool and easy environment for research over the last year. It was a great experience to learn the lab work by our senior lab technician Barbara and I have no words to thanks for the excellent administrative help that I got from our secretaries, Maja and Jolanthe. It was a great help of all kinds from Dr. Prateek kumar, especially during the challenging phases of PhD. I also thank to my friends Dr. Ashish Arora, Dr. Bharat and Dr. Anirban dutta for helping me in taking the right decisions during this journey. There has been a lot of contributions from the long list of my best friends including Rose, Simar, Ashish Rajput, katharina luellmann, Svetlana, Yves, Ali, Irmi Spiess, Jeoffery, Anurag Sankhayan, Gaurav patharia, Johannes, Ranjai sir, Pantalis, Arpit, Nonu and my brother monu, my cousins, Arun, Dipu and Kittu. iv Table of contents 1 Abstract .............................................................................................................. 1 2 Introduction ........................................................................................................ 3 2.1 PrPC and prion .............................................................................................. 3 2.1 Structural features and expression of PrPC .................................................. 3 2.2 Phenotypic interpretation of PrPC knockout mouse models. ......................... 4 2.3 PrPC function and significance of sequence ................................................. 6 2.4 Role of PrPC in aging and oxidative stress ................................................... 6 2.5 Change in behavioral phenotypes during aging ............................................ 8 2.6 Change in biochemical properties of PrPC in aging ...................................... 8 2.6.1 Glycosylation patterns ............................................................................. 8 2.6.2 Localization ............................................................................................. 9 2.7 Role of PrPC in age related neurodegenerative diseases ............................. 9 2.8 Role of PrPC in peripheral tissues ................................................................. 9 2.8.1 Spleen ................................................................................................... 10 2.8.2 Liver ...................................................................................................... 10 2.8.3 Muscle .................................................................................................. 10 2.8.4 Intestine ................................................................................................ 10 2.9 Liver: A model tissue to study PrPC function ............................................... 11 2.10 PrPC knockout mouse models: Importance of Zurich I ................................ 12 2.11 Proteome/genome wide functional analysis of PrPC ................................... 14 2.12 Hypothesis and objectives ........................................................................... 16 3 Materials and Methods .................................................................................... 17 3.1 Animals ........................................................................................................ 17 3.2 2 Dimensional (D) gel electrophoresis ......................................................... 17 3.2.1 Sample preparation. .............................................................................. 17 3.2.2 2D gel electrophoresis, staining and Image Analysis ............................ 17 3.3 In gel digestion and mass spectrometry identification ................................. 18 3.4 Screening of mass spectrometry data ......................................................... 18 3.5 Western blotting. .......................................................................................... 19 3.5.1 Preparation of mice liver samples ......................................................... 19 v 3.5.2 Liver homogenate ................................................................................. 19 3.6 Immunofluorescence ................................................................................... 20 3.7 Measurement of fat in liver tissue ................................................................ 20 3.7.1 Sudan III staining .................................................................................. 21 3.7.2 Triglyceride content ............................................................................... 21 3.8 Capillary Electrophoresis ............................................................................. 21 3.8.1 Sample preparation and capillary immunoassay ................................... 22 3.8.2 Calculations (Expression of PrPC): ....................................................... 22 3.9 Quantitative Real-Time PCR (qPCR) .......................................................... 23 3.10 IPA Analysis ................................................................................................ 25 4 Results .............................................................................................................. 26 4.1 Age and gender specific PrPC expression in mice liver .............................. 26 4.1.1 mRNA expression of PrPC ................................................................... 26 4.1.2 Protein expression of PrPC................................................................... .27 4.1.3 Analysis of PrPC expression by Capillary electrophoresis (CE) ............ 27 4.1.4 Analysis of PrPC expression by Western blot ....................................... 27 4.2 Age and gender specific differential proteome analysis in PrPC knockout mice liver ............................................................................................................... 29 4.2.1 2D gel electrophoresis .......................................................................... 29 4.3 Bio-informatics based functional network analysis ...................................... 37 4.3.1 IPA analysis (Liver as a reference tissue) ............................................. 38 4.3.1.1 Triglyceride content: .......................................................................... 39 4.3.1.2 Fat globules in PrPC knockout mice liver .......................................... 40 4.3.1.3 Liver pathology: ................................................................................. 40 4.3.2 IPA analysis (Brain as a reference tissue) ............................................ 43 4.3.2.1 Altered Tau/Ptau expression in mice liver by Western blot ................ 44 4.3.2.2 Altered Tau/Ptau expression in mice liver by immunofluorescence ... 45 4.3.2.3 Cyclin