Genes Required for Wallerian Degeneration Also Govern Dendrite Degeneration

170 Pages·2017·3.45 MB·English

University of Massachusetts Medical School
eScholarship@UMMS
GSBS Dissertations and Theses Graduate School of Biomedical Sciences
4-3-2015
Genes Required for Wallerian Degeneration Also
Govern Dendrite Degeneration: A Dissertation
Timothy M. Rooney
University of Massachusetts Medical School
Follow this and additional works at:http://escholarship.umassmed.edu/gsbs_diss
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Recommended Citation
Rooney, TM. Genes Required for Wallerian Degeneration Also Govern Dendrite Degeneration: A Dissertation. (2015). University of
Massachusetts Medical School.GSBS Dissertations and Theses.Paper 775. DOI: 10.13028/M2WG69.
http://escholarship.umassmed.edu/gsbs_diss/775
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"GENES REQUIRED FOR WALLERIAN DEGENERATION ALSO GOVERN
DENDRITE DEGENERATION"

A Dissertation Presented
By

Timothy Michael Rooney

Submitted to the Faculty of the
University of Massachusetts Graduate School of Biomedical Sciences, Worcester
in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

April 3, 2015

MD/PhD Program in Biomedical Sciences/Neurobiology
"GENES REQUIRED FOR WALLERIAN DEGENERATION ALSO GOVERN
DENDRITE DEGENERATION"

A Dissertation Presented
By

Timothy Michael Rooney

The signatures of the Dissertation Defense Committee signify
completion and approval as to the style and content of the Dissertation

______________________________________________
Marc Freeman, Ph.D., Thesis Advisor

______________________________________________
Claire Bénard, PhD., Member of Committee

______________________________________________
Lawrence Hayward, MD/PhD., Member of Committee

______________________________________________
Neal Silverman, PhD., Member of Committee

______________________________________________
Christopher Gabel, PhD., Member of Committee

The signature of the Chair of the Committee signifies that the written dissertation
meets the requirements of the Dissertation Committee

______________________________________________
Eric Baehrecke, Ph.D., Chair of Committee

The signature of the Dean of the Graduate School of Biomedical Sciences
signifies that the student has met all the requirements of the school

__________________________________________
Anthony Carruthers, Ph.D.,
Dean of the Graduate School of Biomedical Sciences

MD/PhD Program in Biomedical Sciences/Neurobiology
April 3, 2015
iv

Dedication

This thesis is dedicated to my parents, Bob and Elaine Rooney.
v

Acknowledgements

I would first like to thank my advisor and mentor, Marc Freeman, for taking me in
and providing an excellent environment for learning to be a good scientist.

I would like to thank my family for their love and support, especially my mother
who also keeps me well fed. Mostest!

A big thanks to Freeman lab members past and present. Our shenanigans and
shared times are a highlight of my time in lab.

Thank you to my TRAC and Defense Committees for their guidance and support.

I would like to thank the Department of Defense Breast Cancer Research
Program for a pre-doctoral fellowship.

I would also like to thank the Gaffin family for having me as the Gaffin Family
Fellow.
vi

Abstract

Neurons comprise the main information processing cells of the nervous
system. To integrate and transmit information, neurons elaborate dendritic
structures to receive input and axons to relay that information to other cells. Due
to their intricate structures, dendrites and axons are susceptible to damage
whether by physical means or via disease mechanisms. Studying responses to
axon injury, called Wallerian degeneration, in the neuronal processes of
Drosophila melanogaster has allowed the identification of genes that are required
for injury responses. Screens in Drosophila have identified dsarm and highwire
as two genes required for axon degeneration; when these genes are mutated
axons fail to degenerate after injury, even when completely cut off from the
neuronal cell body. We found that these genes are also required for dendrite
degeneration after injury in vivo. Further, we reveal differences between axon
and dendrite injury responses using in vivo timelapse recordings and GCaMP
indicators of intracellular and mitochondrial calcium transients. These data
provide insights into the neuronal responses to injury, and better define novel
targets for the treatment of neurodegenerative diseases.
vii

TABLE OF CONTENTS

Title ii
Signature Page iii
Dedication iv
Acknowledgements v
Abstract vi
Table of Contents vii
List of Tables xi
List of Figures xii
List of Third Party Copyrighted Material xiv

CHAPTER I: Introduction 1
The Nervous System 1
Dendrites vs. Axons 2
The Drosophila Nervous System 4
Nervous System Dysfunction and Injury 5
Slow Wallerian Degeneration 7
Drosophila Models of Wallerian Degeneration 11
dSarm/SARM1 13
Hiw/PHR1 15

CHAPTER II: Screens to Identify Novel Regulators 18
of Wallerian Degeneration
Introduction 18
viii

Candidates Identified in the Literature 20
Materials and Methods 20
Results 22
Candidates Identified by Mouse Proteomics 34
Materials and Methods 34
Drosophila degeneration screen 34
Results 36
Unbiased Screening of the Drosophila 2nd Chromosome 46
MARCM Analysis of WD Using Chromosome 48
2L Deficiency Lines
Materials and Methods 48
Results 49
A Forward-Genetic Screen for Wallerian Degeneration 52
Mutants
Materials and Methods 52
Results 60
Discussion 63
Candidates from the Literature 63
Candidates Identified by Mouse Proteomics 66
MARCM Screening of EMS-generated Mutants 67
on the Drosophila 2nd Chromosome

CHAPTER III: Genes Required for Wallerian Degeneration 70
Also Govern Dendrite Degeneration After Injury
Introduction 70
Results 73
Genes required for Wallerian degeneration also govern 73
ix

dendrite degeneration after injury

Injury induces intra-dendritic calcium transients 76

Dendritic calcium transients upon injury in 77
Wallerian degeneration mutants

Mitochondrial calcium transients upon injury in 78
Wallerian degeneration mutants

hiw mutants reveal cell-autonomous branch- 79
specific degeneration of dendrites

Discussion 81
Materials and Methods 87
Figures 90

CHAPTER IV: Discussion 106
Endogenous regulators of Wallerian degeneration 106
Dendrites 113
Towards an inhibitor of SARM1 proteins 118
Remaining questions 121
Concluding remarks 125

Appendix I: Towards and Inhibitor of SARM1 127
x

Appendix II: dSARM RNAi in Glia 136

Appendix III: Ablation of Tracheal Clones 137

Bibliography 139