IDENTIFICATION OF ALTERED RAS SIGNALING AND INTERMEDIATE FILAMENT ...

120 Pages·2015·4.37 MB·English

by  Kyle Martin

IDENTIFICATION OF ALTERED RAS SIGNALING AND INTERMEDIATE
FILAMENT HYPERPHOSPHORYLATION IN GIANT AXONAL NEUROPATHY

Kyle Benjamin Martin

Submitted to the faculty of the University Graduate School
in partial fulfillment of the requirements
for the degree
Doctor of Philosophy
in the Department of Medical and Molecular Genetics,
Indiana University

May 2015
Accepted by the Graduate Faculty, Indiana University, in partial
fulfillment of the requirements for the degree of Doctor of Philosophy.

___________________________
R. Mark Payne, M.D., Chair

___________________________
Theodore R. Cummins, Ph.D.

Doctoral Committee

___________________________
Nuria Morral, Ph.D.

December 17, 2014

___________________________
Ronald C. Wek, Ph.D.

ii
ACKNOWLEDGMENTS

First and foremost, I would like to thank my advisor Dr. R. Mark Payne for
his continued guidance and support. Although my project was not directly related
to his other research interests and outside his clinical expertise, Dr. Payne’s
patience and encouragement allowed this project to fully develop into what it is
today. I would also like to thank the members of my research committee, Dr.
Ronald Wek, Dr. Nuria Morral, and Dr. Theodore Cummins, for their help and
constructive criticism over the years. I am also grateful for the continual guidance
of Dr. Brittney-Shea Herbert, whose advice on academics and the Ph. D. process
have been invaluable to my matriculation through the program.
Next, I would like to thank all the current and former members of the
Payne Lab, of which I am particularly grateful to Sam Beard, Loren Nelson, and
Kelsey Loden for their contributions to this work. I also am especially thankful for
the help of Malgorzata "Gosia" Kamocka and Seth Winfree of the Indiana Center
for Biological Microscopy for their training and troubleshooting for all the
microscopy contained in this work.
I am also grateful to Lori and Matt Sames for organizing multiple Giant
Axonal Neuropathy conferences, which I attended with other researchers from
around the world. In addition to learning about other critical GAN research, these
conferences provided me a forum to meet the Sames family, whose daughter,
Hannah, suffers from GAN. Meeting the Sames family and experiencing their
iii
resolve was truly moving and provided me with more than enough motivation to
complete this research.
Finally, I would like to thank all my friends and family for their support
throughout my tenure in graduate school. In particular I would like to thank my
parents who, despite not understanding the science of what I was doing, were
always there to support me. Without them, none of this would have been
possible.

iv
Kyle Benjamin Martin

IDENTIFICATION OF ALTERED RAS SIGNALING AND INTERMEDIATE
FILAMENT HYPERPHOSPHORYLATION IN GIANT AXONAL NEUROPATHY

Giant axonal neuropathy (GAN) is a rare genetic disease that causes
progressive damage to the nervous system. Neurons in GAN patients develop an
abnormal organization of cytoskeletal proteins called intermediate filaments (IFs),
which normally provide strength and support for the overall cell structure. The
irregular IF structure in GAN patient neurons leads to a progressive loss of motor
skills in children and subsequent death in adolescence. GAN is caused by
reduced levels of the gigaxonin (Giga) protein. Giga functions to control the
degradation of other cellular proteins, and the loss of Giga in GAN cells results in
significantly elevated levels of the galectin-1 (Gal-1) protein. Gal-1 stabilizes the
active form of the Ras signaling protein, which functions as a molecular switch to
regulate the phosphorylation and subsequent organization of IFs. The connection
between these pathways led us to propose that Giga regulates IF
phosphorylation and structure by modulating Ras signaling through the
degradation of Gal-1. Using GAN patient cells, we demonstrated that restoring
Giga reduced Gal-1 protein levels, decreased IF phosphorylation, and
reestablished normal IF organization. Similar effects of reduced IF
phosphorylation and improved IF structure were also obtained in GAN cells by
directly decreasing the protein levels of either Gal-1, or downstream Ras
v
signaling proteins. Taken together, these results demonstrate that the loss of
Giga induces Gal-1 mediated activation of Ras signaling, thereby leading to the
increased IF phosphorylation and abnormal IF structure observed in GAN cells.
Identification of aberrant Ras signaling is significant because it is the first to
specify a mechanism by which the loss of Giga leads to the development of GAN
and provides targets for novel drug therapies for the treatment of this currently
immedicable genetic disease.

R. Mark Payne, M.D., Chair

vi
TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................... x
LIST OF ABBREVIATIONS ................................................................................. xii
CHAPTER I: INTRODUCTION ............................................................................. 1
Giant Axonal Neuropathy: description and clinical overview ............................. 1
Function and dynamics of intermediate filaments ............................................. 4
Generalized IF dysfunction in GAN ................................................................... 9
The GAN gene and encoded gigaxonin protein .............................................. 12
Regulation of the ubiquitin-proteasome system .............................................. 14
Gigaxonin functions as a substrate adaptor .................................................... 18
Galectin-1 and Ras stabilization ...................................................................... 23
Role of intermediate filament phosphorylation ................................................ 29
Summary and hypothesis ................................................................................ 34
CHAPTER II: MATERIALS AND METHODS ...................................................... 37
Cell culture ...................................................................................................... 37
Immunofluorescence and image acquisition ................................................... 38
GAN phenotypic scoring .................................................................................. 39
Western blot analysis ...................................................................................... 41
Lentiviral production and transduction ............................................................. 42
vii
TAT-Giga expression, purification, and treatment ........................................... 43
Proteasome inhibition ...................................................................................... 45
Small interfering RNA (siRNA) inhibition ......................................................... 45
Statistics .......................................................................................................... 46
CHAPTER III: RESULTS .................................................................................... 47
GAN cells have altered vimentin distribution in low serum .............................. 47
GAN cells have elevated Gal-1 and phosphorylated vimentin in low serum .... 51
Giga controls the proteasome mediated degradation of Gal-1 ........................ 54
Restoration of Giga corrects GAN phenotype ................................................. 57
Gal-1 knockdown decreases vimentin phosphorylation and restores vimentin
distribution ....................................................................................................... 61
H-Ras siRNA decreases vimentin phosphorylation and improves vimentin
distribution ....................................................................................................... 63
MEK1/2 knockdown reduces vimentin phosphorylation and improves vimentin
distribution ....................................................................................................... 67
CHAPTER IV: DISCUSSION .............................................................................. 70
GAN cells have altered vimentin distribution in low serum .............................. 71
GAN cells have elevated Gal-1 and phosphorylated vimentin in low serum .... 72
Giga controls the proteasome mediated degradation of Gal-1 ........................ 75

viii
Gal-1 knockdown decreases vimentin phosphorylation and restores vimentin
distribution ....................................................................................................... 76
H-Ras siRNA decreases vimentin phosphorylation and improves vimentin
distribution ....................................................................................................... 78
Future directions ............................................................................................. 79
REFERENCES ................................................................................................... 85
CURRICULUM VITAE

ix
LIST OF FIGURES

Figure 1: Giga functions as a substrate adaptor for an E3 ubiquitin ligase ......... 18
Figure 2: Gal-1 stabilizes GTP bound H-Ras and forms activated Ras
nanoclusters ....................................................................................................... 28
Figure 3: Map of identified vimentin phosphorylation sites ................................. 30
Figure 4: Schematic of the proposed Giga-mediated signaling pathway ............ 35
Figure 5: Illustration of the proposed signaling pathway in GAN ........................ 36
Figure 6: Generation of threshold images to obtain tubulin and vimentin areas
for quantification of the VFA ............................................................................... 40
Figure 7: Vimentin distribution in normal serum (10% FBS) conditions .............. 48
Figure 8: Vimentin distribution in low serum (0.1% FBS) conditions ................... 49
Figure 9: Quantification of the VFA in normal and low serum ............................. 51
Figure 10: GAN cells have increased levels of Gal-1 and phosphorylated
vimentin in low serum ......................................................................................... 52
Figure 11: Restoring the wild-type GAN gene decreases Gal-1 and reverses
vimentin hyperphosphorylation in GAN cells ...................................................... 55
Figure 12: Replacing wild-type Giga protein into GAN cells decreases Gal-1
protein levels ...................................................................................................... 56
Figure 13: Giga controls the proteasome mediated degradation of Gal-1 .......... 57
Figure 14: Restoring wild-type GAN gene corrects the GAN phenotype ............ 59
Figure 15: Restoring Giga corrects the GAN phenotype .................................... 60
Figure 16: Gal-1 knockdown reduces vimentin phosphorylation ........................ 61
x