The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular ...

218 Pages·2017·18.81 MB·English

The Prion Protein Controls Polysialylation of Neural Cell
Adhesion Molecule 1 during Cellular Morphogenesis
by
Mohadeseh Mehrabian
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Department of Laboratory Medicine and Pathobiology
University of Toronto
© Copyright by Mohadeseh Mehrabian 2017
The Prion Protein Controls Polysialylation of Neural Cell Adhesion
Molecule 1 during Cellular Morphogenesis
Mohadeseh Mehrabian
Doctor of Philosophy
Department of Laboratory Medicine and Pathobiology
University of Toronto
2017
Abstract
The cellular prion protein (PrPC) is one of the most studied mammalian proteins. Despite the
ongoing efforts and the ubiquitous expression of PrPC in vertebrate cells, the main function of
this protein has remained enigmatic to date. Several lines of evidence pointed towards the
involvement of PrPC in the morphogenetic reprogramming underlying the epithelial-to-
mesenchymal transition (EMT). To address this, we applied CRISPR-Cas9 genome engineering
technique to establish multiple PrPC knockout cell models (Chapter 2). The follow-up studies
uncovered a critical role of PrPC in EMT and showed that PrPC controls the polysialic acid
(polySia) modification of neural cell adhesion molecule 1 (NCAM1). Surprisingly, this effect of
PrPC on NCAM1 polysialylation relies on a novel signaling loop that modulates expression of
ST8SIA2, one of the two polysialyltransferase enzymes responsible for adding polySia on
NCAM1 (Chapter 3). Intriguingly, a comparative analysis revealed that PrPC levels correlate
directly or inversely with ST8SIA2 levels in different cell models. Deep global proteome
analyses of multiple PrP-deficient models not only uncovered surprising cell model-specific
proteome shifts but also showed robust changes in the levels of MARCKS and BASP protein
families at the plasma membrane. Further investigation of their involvement in a biology that
controls NCAM1 polysialylation revealed that reduced levels of MARCKSL1 also led to a
significant reduction of polySia-modified NCAM1 (Chapter 4). Data from this study emphasize a
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thus far underappreciated coordinated biology of PrP and NCAM1. A close comparison of
knockout phenotypes in mice is consistent with a model, whereby several of the known
phenotypes in PrP-deficient mice might be explained by the contribution of PrPC to NCAM1
polysialylation. Attempts to validate this model and to assert its significance for determining if
perturbed PrPC signaling contributes to toxicity in prion diseases are ongoing and have not been
included in this thesis at this time.
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Acknowledgments
I would like to express my greatest and most sincere thanks to my supervisor, Dr. Gerold
Schmitt-Ulms for the excellent guidance, supervision and training on scientific thinking for
tackling biological questions. I owe a lot of my increasing passion for science to his scientific
character, genuine dedication to teaching and the overall very positive experience of my doctoral
training.
I would also like to thank members of my Ph.D. advisory committee, Dr. Karim Mekhail and Dr.
Emil F. Pai and the Department of Laboratory Medicine and Pathobiology’s Graduate
Coordinator Dr. Harry P. Elsholtz and the Graduate Administrators Ferzeen Sammy and Rama
Ponda for their continued support and guidance. Thank you also to Dr. Ilia V. Baskakov, Dr.
Lorraine Kalia and Dr. Joel Watts for evaluating my thesis.
This project had started with the initial discovery of Prion-ZIP molecules’ ancestral connection,
the accomplishment of a previous lab member, Dr. Sepehr Ehsani. I would like to acknowledge
his many contributions to this project. Also, I would like to extend my thanks to the previous and
current laboratory members especially the members I was fortunate to collaborate with on this
project: Dylan Brethour, Dr. Cosmin L. Pocanschi, Hezhen Ren and Dr. Hansen Wang. These
discoveries would have not been possible without the valuable help and expertise of Dr. Declan
Williams in mass spectrometry technology.
Work on this project has been feasible through the generous donation of Arnold Irwin that
facilitated our laboratory with the cutting-edge technology in proteomics. The research is
supported by the Ontario Trillium Scholarship, the University of Toronto Fellowship program,
the Weston Brain Institute, the Canadian Institute of Health Research and PrioNet Canada.
I would like to dedicate this thesis to my parents.
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Table of Contents

Acknowledgments .................................................................................................................. iv
Table of Contents .................................................................................................................... v
List of Tables .......................................................................................................................... ix
List of Figures .......................................................................................................................... x
List of Appendices ................................................................................................................. xii
Abbreviations ....................................................................................................................... xiv
Chapter 1 An Emerging Role of the Cellular Prion Protein as a Modulator of the
Morphogenetic Program Underlying Epithelial-to-Mesenchymal Transition: An Introduction . 1
1.1 Preamble .................................................................................................................................... 1
1.2 The physiological role of a protein- phenotypic change vs. function ............................................ 1
1.3 EMT, yet another phenotypic change the prion protein may contribute to- but not its function . 3
1.3.1 Evidence for a role of PrPC in EMT in the cancer literature ........................................................ 4
1.3.2 Zebrafish PrP knockdown models ............................................................................................... 5
1.3.3 Protein-protein interactions of PrPC with a known EMT connection .......................................... 5
1.3.4 Function of closest evolutionary relatives of PrPC ...................................................................... 6
1.4 Model of PrP/ZIP E-cadherin modulation .................................................................................... 7
1.5 Advanced methodology .............................................................................................................. 9
1.5.1 Genome editing using CRISPR-Cas9 ............................................................................................ 9
1.5.2 Relative quantitation of proteins during mass spectrometry analyses .................................... 10
1.6 Conclusions .............................................................................................................................. 11
Chapter 2 Development of CRISPR-Cas9-Based Knockout Models of the Prion Protein and Its
Effect on the Proteome ......................................................................................................... 12
2.1 Introduction ............................................................................................................................. 12
2.2 Materials and methods ............................................................................................................. 14
2.2.1 Generation of gRNA Expression Vectors .................................................................................. 14
2.2.2 Cell Culture and Transfection ................................................................................................... 15
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2.2.3 Generation of stable knockdown cell clones ............................................................................ 15
2.2.4 Genetic analysis ........................................................................................................................ 15
2.2.5 Western blot analyses .............................................................................................................. 16
2.2.6 Sample preparation for comparative global proteomics analysis ............................................ 16
2.2.7 Nanospray ionization tandem mass spectrometry ................................................................... 17
2.2.8 Post-acquisition analyses .......................................................................................................... 17
2.3 Results ...................................................................................................................................... 18
2.3.1 Strategy of CRISPR-Cas9-based PrP knockout in three mouse cell lines .................................. 18
2.3.2 Validation and characterization of PrP knockout cell clones .................................................... 20
2.3.3 Workflow of global proteome comparison of PrP knockout (or knockdown) and wild-type
NMuMG cells ..................................................................................................................................... 22
2.3.4 The global proteome of PrP-deficient NMuMG cells ................................................................ 24
2.4 Discussion ................................................................................................................................ 30
2.5 Conclusions .............................................................................................................................. 32
Chapter 3 The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1
During Cellular Morphogenesis ............................................................................................. 34
3.1 Introduction ............................................................................................................................. 34
3.2 Materials and methods ............................................................................................................. 36
3.2.1 Inhibitors, proteins, plasmids and antibodies .......................................................................... 36
3.2.2 Cell lines and culture conditions and transfections .................................................................. 37
3.2.3 CRISPR-Cas9 mediated knockout clones and transient or stable knockdown of PrP ............... 37
3.2.4 Western blot analyses .............................................................................................................. 37
3.2.5 Enzymatic characterization of post-translational modifications of NCAM1 ............................. 38
3.2.6 RT-PCR analyses ........................................................................................................................ 38
3.2.7 Co-immunofluorescence analyses ............................................................................................ 39
3.2.8 Sample preparation for comparative global proteomic analysis .............................................. 39
3.2.9 Global proteome analyses ........................................................................................................ 39
3.2.10 Post-acquisition and statistical analyses of global proteome datasets .................................. 40
3.3 Results ...................................................................................................................................... 41
3.3.1 PrP contributes to morphogenetic reprogramming of cells during EMT .................................. 41
3.3.2 Comparative proteome analyses identify NCAM1 as a candidate for mediating PrP’s influence
on EMT ............................................................................................................................................... 43
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3.3.3 PrP is critical for EMT-dependent polysialylation of NCAM1 ................................................... 47
3.3.4 PrP regulates NCAM1 polysialylation at the level of polyST transcription ............................... 49
3.3.5 β-catenin contributes to differential ST8SIA2 expression ........................................................ 52
3.4 Discussion ................................................................................................................................ 55
3.4.1 The PrP-ST8SIA2-NCAM1 signaling loop ................................................................................... 56
3.4.2 The search for the physiological function of PrP ...................................................................... 57
3.5 Conclusions .............................................................................................................................. 58
Chapter 4 Prion Protein Deficiency Causes Diverse Proteome Shifts in Cell Models That Escape
Detection in Bain Tissue ........................................................................................................ 59
4.1 Introduction ............................................................................................................................. 59
4.2 Materials and methods ............................................................................................................. 60
4.2.1 Antibodies ................................................................................................................................. 60
4.2.2 Mouse brain and cell models .................................................................................................... 61
4.2.3 Generation of PrP-deficiency .................................................................................................... 61
4.2.4 Sample preparation for global proteome analyses .................................................................. 62
4.2.5 Quantitative mass spectrometry .............................................................................................. 62
4.2.6 Gene ontology analysis ............................................................................................................. 63
4.2.7 Cluster analysis ......................................................................................................................... 64
4.2.8 Multiple alignment ................................................................................................................... 64
4.2.9 SDS-PAGE and western blot ...................................................................................................... 64
4.2.10 RT-PCR methodology .............................................................................................................. 65
4.2.11 Statistics .................................................................................................................................. 65
4.3 Results ...................................................................................................................................... 66
4.3.1 Experimental design of global proteome analyses of PrP-deficient mouse models ................. 66
4.3.2 At similar depths of proteome coverage, global proteomes reflect differences in cellular
origins of mouse models suited ......................................................................................................... 68
4.3.3 Hierarchical clustering reveals surprising disparity in the consequences of PrP-deficiency on
global proteomes ............................................................................................................................... 71
4.3.4 PrP-deficiency causes consistent but opposite changes of steady-state levels of MRACKS and
its paralog MARCKSL1 in all cell models ............................................................................................ 74
4.3.5 The effect of PrP on nerve ending signal hub proteins extends to members of the BASP
protein family .................................................................................................................................... 77
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4.3.6 MARCKSL1 is a downstream partner in PrP-dependent NCAM1 polysialylation ...................... 82
4.4 Discussion ................................................................................................................................ 86
4.4.1 General comments on global proteomics datasets .................................................................. 86
4.4.2 Influence of PrP on steady-state levels of MARCKS family proteins ......................................... 88
4.4.3 MARCKS family proteins influence biology of PrP-dependent NCAM1 polysialylation and
cation-selective channels .................................................................................................................. 89
4.5 Conclusions .............................................................................................................................. 90
Chapter 5 Future Directions of the Role of PrP in Controlling the EMT Program and
Polysialylation on NCAM1 ..................................................................................................... 91
5.1 Introduction ............................................................................................................................. 91
5.2 Protein-protein interactions ..................................................................................................... 95
5.3 Phenotypes of polySia-NCAM1 or PrPC deficient models ........................................................... 97
5.3.1 EMT and gastrulation ................................................................................................................ 98
5.3.2 Subventricular zone, rostral migratory system and olfactory bulb .......................................... 99
5.3.3 Hippocampal formation .......................................................................................................... 101
5.3.4 Hematopoietic stem cells and their derivatives ..................................................................... 105
5.3.5 Myelin repair and maintenance ............................................................................................. 107
5.3.6 Circadian Rhythm .................................................................................................................... 108
5.3.7 Glutamatergic signaling and long-term potentiation ............................................................. 110
5.4 Evolutionary context .............................................................................................................. 111
5.6 Conclusions ............................................................................................................................ 117
Chapter 6 : Perspective ........................................................................................................ 119
References .......................................................................................................................... 122
Appendices ......................................................................................................................... 145
Copyright Acknowledgements ............................................................................................. 149

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List of Tables
Table 2.1: Subset of proteins observed in PrP 'ko' and 'kd' NMuMG global proteomes at levels
that deviated from 'wt' levels
Table 5.1: Similarities amongst PrP- and polySia-NCAM1-related phenotypes

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List of Figures
Figure 1.1: Schematic outlining key morphological and molecular changes that accompany
EMT.
Figure 1.2: Schematic summarizing evidence consistent with a role of PrPC in EMT.
Figure 2.1: Strategy for generation of mouse PrP knockout clones based on CRISPR/Cas9-
system.
Figure 2.2: Generation of Prnp knockout clones in three different mouse cell lines.
Figure 2.3: Flow-chart depicting experimental strategy for comparative analyses of the global
proteomes of Prnp knockout (or knockdown) and wild-type NMuMG epithelial cell clones.
Figure 2.4: PrP deficiency generated by CRISPR/Cas9-mediated gene knockout or stable
shRNA-mediated knockdown manifests in highly reproducible changes to the expression of
more than hundred proteins in NMuMG cell model.
Figure 3.1: PrPC expression is transcriptionally upregulated during EMT.
Figure 3.2: Quantitative mass spectrometry identifies perturbed ‘response to metal ions’ and
EMT markers, including NCAM1, affected in PrP-deficient cells.
Figure 3.3: PrP-deficiency affects expression of a subset of proteins undergoing pronounced
expression levels changes during EMT.
Figure 3.4: Stable PrP-deficiency prevents EMT-dependent polysialylation of NCAM1.
Figure 3.5: PrP deficiency prevents EMT-dependent NCAM1 polysialylation by inhibiting
transcriptional activation of ST8SIA2 gene.
Figure 3.6: Inhibition of CTNNB1-dependent transcription phenocopies loss of PSA in NMuMG
cells.
Figure 4.1: Study design for global proteome comparison of PrP-deficient mouse models.
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