Parkinson's Disease and Mechanisms of Fast Axonal Transport BY RAMONA MIANA PUFAN AA ...

166 Pages·2011·11.03 MB·English

by  Ramona Pufan

Parkinson’s Disease and Mechanisms of Fast Axonal Transport

BY

RAMONA MIANA PUFAN
A.A., Diablo Valley College, Pleasant Hill, California 2000
B.A., University of California Berkeley, Berkeley, California 2002

THESIS

Submitted as partial fulfillment of the requirements
for the degree of Doctor of Philosophy in Anatomy and Cell Biology
in the Graduate College of the
University of Illinois at Chicago, 2011

Chicago, Illinois

Defense Committee:

Jonathan J. Art, Chair
Scott T. Brady, Advisor
Grardo Morfini
Brian Kay, Biological Sciences
Daniel Corcos, Kinesiology and Nutrition
Martha Bohn, Northwestern University
This thesis is dedicated to my husband, Arzhang Zereshki and my aunt Nicole Szilagyi,
for without them I would have not made it here.
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ACKNOWLEDGEMENTS
I would like to thank my advisor, Dr. Scott Brady, who made earning this degree
possible. I thank him for his patience and kindness, and for allowing me the
independence to understand myself, to discover what I can do, and to grow as a
scientist. Second, I am deeply grateful to my thesis committee: Dr. Jonathan Art, Dr.
Martha Bohn, Dr. Daniel Corcos, Dr. Brian Kay and Dr. Gerardo Morfini for their
constant support, time and invaluable insight. I thank Dr. Art for everything from help
with experiments to always having his door open regardless of need. It is rare to come
across someone who is so dedicated to teaching and is as strong a champion of
students. I thank Dr. Bohn for giving me the perspective of a woman in science. I thank
her for all the encouragement and extra care. I thank Dr. Corcos for his straight talk and
direct questions. I have appreciated his guidance about procedures and for always
making sure I took everything into account. I thank Dr. Kay, for all his expert advice and
counsel, especially about presentations. He has always given me the extra needed
boost of confidence. I thank Dr. Morfini for introducing me to the project and his
extraordinary attention to detail.
I thank all the laboratory members, past and present, for their support and
making work fun: Bin Wang, Sarah Pollema, Natalia Marangoni, Yuka Atagi, Sarah
Berth, Tulika Sarma, Gustavo Pigino, Hanwu Liu, Hugo Caicedo, Sara Aspengren,
Robert Gould, Yuyu Song, and Scott deBoer. I am deeply grateful to Bin for always
getting anything we needed and taking care of me. I thank Yuka and Sarah Berth for
being the best of office mates. I thank Tulika, for always being cheerful and for teaching

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ACKNOWLEDGEMENTS (continued)
me about cells and how to image them. I thank Gustavo for all the discussions about
science and otherwise.
I would be remiss if I failed to thank the others whose help and advice was
invaluable to the progress of my project: Tamie Chilcote of Elan, Nikki LaPointe of
UCSB, the La Du laboratory, Yee-Kin Ho, Bao-Shiang Lee, Lasanthi Jayathilaka and
Larry Helseth of UIC.
Particular thanks go to the staff of the Anatomy & Cell Biology office who always
made sure that all things administrative were taken care of: Rory Sullivan, for all the
friendly banter and oversight; Maria Carlstrom, Paula Caicedo, Sydelle Bautista and Lea
Smuker for their help with grants and all the issues that arose with payroll; and finally to
the truest, kindest of friends, Mike Driscoll for everything – starting with room
reservations – and Linda Johnston, for fixing anything.
Thanks also goes to the members of the department – an incredible group of
people – in particular to Dr. Anderson, who was my first contact with UIC and on whom,
throughout the years, I could always rely on for kindness and words of encouragement.
Last but not least, I would like to thank my family. First, I thank my husband,
Arzhang, for being the epitome of patience and understanding, and for always being my
home. I thank my aunt Nicole, who showed me what strength really is. It is through her
I learned that one could survive anything, pick up and keep going. I thank my family in
Romania, for always being patient, and for encouraging me to stay strong even when
years passed without seeing each other. I thank my childhood friends, Ana and Laura –

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ACKNOWLEDGEMENTS (continued)
no matter where life took us, and how good or bad things got – we managed to stick it
through and keep in touch.
Second, I thank my US family, without whom I could not have made it. My
mentors and friends in California, in particular, Dr. Jarmila Jancarick, now retired from
Lawrence Berkeley laboratory, who took me under her wing, and not only taught me the
art of making protein crystals, but also advised me that, especially as a woman and
regardless of circumstance, I should always stand on my own two feet. It is she that
encouraged me to go to graduate school. And finally, my friends in Chicago, with whom
I shared the last years, all the highs and the lows: Sarah Pollema for being a source of
inspiration, Natalia for bringing color to my life and giving me a home, and Jamie who
made me believe that I could do anything, anytime, even when I felt I was at my worst.
Thank you everyone for your love, patience and support.

RMP

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TABLE OF CONTENTS
CHAPTER PAGE
I. INTRODUCTION..........................................................................................................1
A. Parkinson’s disease ...............................................................................................1
1. History of Parkinson’s disease............................................................................1
2. Clinical Symptoms and Pathology in Parkinson Disease....................................2
3. Causes of PD: sporadic versus familial forms. Genetic mutations.....................6
B. The α- Synuclein Protein........................................................................................8
1. Discovery ............................................................................................................8
2. Role and function................................................................................................9
3. Structure............................................................................................................11
4. Lewy Bodies......................................................................................................13
C. Pathogenic Mechanisms of Parkinson’s Disease and α-Synuclein......................15
1. Role of aggregation...........................................................................................15
2. Cell Specific Vulnerability..................................................................................18
3. Disruption of Axonal Transport..........................................................................19
D. Statement of hypothesis.......................................................................................21

II. PATHOGENIC EFFECTS OF α-SYNUCLEIN DOMAINS ON AXONAL TRANSPORT
........................................................................................................................................23
A. Summary..............................................................................................................23
B. Introduction...........................................................................................................24
C. Materials and Methods.........................................................................................38
1. Materials............................................................................................................38
2. Generation of α-synuclein constructs................................................................39
3. Expression and purification of α-synuclein protein............................................40
4. Generation of α-synuclein fibrils........................................................................41
5. Assessment of fibrillization................................................................................41
6. Squid axoplasm perfusions...............................................................................42
7. Gel electrophoresis, Coomassie blue staining and western blotting.................42
D. RESULTS.............................................................................................................43
1. Generation of full-length and C-terminus truncated forms of wild type, A30P and
A53T mutated forms of α-synuclein........................................................................43
2. Generation of full-length and C-terminus truncated forms of wild type, A30P and
A53T mutated forms of α-synuclein fibrils...............................................................49
3. Effects of C-terminus truncated forms of wild type, A30P and A53T mutated
forms of α-synuclein in soluble and fibril form on axonal transport.........................50
E. Discussion............................................................................................................53

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TABLE OF CONTENTS (continued)
CHAPTER PAGE

III. EFFECTS OF α-SYNUCLEIN EXPRESSION ON AXONAL TRANSPORT IN CELLS
........................................................................................................................................57
A. Summary..............................................................................................................57
B. Introduction...........................................................................................................59
C. Materials and methods.........................................................................................61
1. Materials............................................................................................................61
2. General cell handling procedures .....................................................................64
3. Poly-lysine coating............................................................................................65
4. Cell differentiation protocol................................................................................65
5. Stable cell line generation.................................................................................66
6. Gel Electrophoresis, Coomassie blue staining and western blotting ................67
7. Immunocytochemistry.......................................................................................67
8. Polymerization of tubulin for microtubule binding assay...................................68
9. Microtubule binding assay.................................................................................69
10. Cellular Fractionation ......................................................................................70
11. Immunoprecipitation assays............................................................................71
12. Mass spectrometry..........................................................................................72
13. Statistical analysis...........................................................................................73
D. Results.................................................................................................................73
1. Screening and selection of neuroblastoma cell lines........................................73
2. Development and characterization of stable cell lines expressing wild type,
A30P and A53T forms of α-synuclein .....................................................................78
3. Characterization of molecular motors: expression of kinesin and dynein.........86
4. Characterization of molecular motors: microtubule binding assays..................88
5. Characterization of molecular motors: membrane association assay...............93
6. Evaluation of PKCµ and identification of a protein dependent on α-synuclein
expression...............................................................................................................97
E. DISCUSSION.....................................................................................................111

IV. FUTURE DIRECTIONS.........................................................................................118

CITED LITERATURE.............................................................................................122

VITA........................................................................................................................148

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LIST OF TABLES

TABLE PAGE

I. ANTIBODIES USED............................................................................................62

II. LIST OF CELL LINES CONSIDERED.................................................................75

III. SUMMARY OF PROTEINS DETECTED...........................................................108

IV. PKCµ ANTIBODIES USED ...............................................................................114


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LIST OF FIGURES

FIGURE PAGE
Figure 1: Motility assay of fast axonal transport in isolated squid axoplams.......29

Figure 2: Effects of kinases on axonal transport..................................................31

Figure 3: Effect of various forms of α-synuclein associated with PD on FAT......34

Figure 4: Coperfusion of pathogenic α-synuclein and kinase inhibitors in squid
axoplasm.............................................................................................................35

Figure 5: C-terminus truncation of α-synuclein....................................................45

Figure 6: α-synuclein expression in SG13009 E. coli strains..............................46

Figure 7: Expression and purification α-synuclein constructs .............................48

Figure 8: Light scattering of α-synuclein fibrlils ...................................................51

Figure 9: Effects of α-synuclein on axonal transport...........................................52

Figure 10: Model of α-synuclein pathology in FAT..............................................56

Figure 11: Effects of α-synuclein in NB69 cells...................................................77

Figure 12: Expression of α-synuclein in clones of SK-N-SH...............................79

Figure 13: Expression of α-synuclein in stably transfected SK-N-SH cells.........81

Figure 14: Expression of α-synuclein in clones of SH-SY5Y...............................83

Figure 15: Expression of α-synuclein in stably transfected SH-SY5Y cells.........85

Figure 16: Expression of molecular motors in stable SH-SY5Y clones...............87

Figure 17: Overview of microtubule binding assay..............................................89

Figure 18: α-Synuclein expression does not affect kinesin binding to microtubules
.............................................................................................................................90

Figure 19: Microtubule binding assay, evaluation of dynein................................92

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LIST OF FIGURES (continued)

Figure 20: Overview of membrane association assay protocol...........................95

Figure 21: Molecular motor partitioning within cellular fractions..........................96

Figure 22: α-synuclein dependent changes in protein recognized by PKCµ
antibodies............................................................................................................99

Figure 23: Sample comparison for PCKµ identification.....................................100

Figure 24: Molecular weight estimation of the unknown protein recognized by
PKCµ antibodies................................................................................................102

Figure 25: Effects of PMA treatment on SH-SY5Y cells....................................104

Figure 26: Immunoprecipitation assays.............................................................107


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