Neuronal dysfunction in Fragile X spectrum disorders by Abigail Josephine Renoux A dissertation ...

151 Pages·2015·8.82 MB·English

by  Trash Cat

Neuronal dysfunction in Fragile X spectrum disorders
by
Abigail Josephine Renoux

A dissertation submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
(Molecular and Integrative Physiology)
in The University of Michigan
2015

Doctoral Committee:

Associate Professor Michael M. A. Sutton, Co-Chair
Assistant Professor Peter K. Todd, Co-Chair
Professor Lori L. Isom
Associate Professor Geoffrey G. Murphy
Professor Henry L. Paulson
Dedication

For Mr. Alberto Real
(Mystery Al),
who taught me to be curious,
and how to learn,
and to love science.

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Acknowledgements

This dissertation work is the result of a very successful collaboration between
Drs. Peter Todd and Mike Sutton and me. I was fortunate enough to begin work on
these projects at the beginning of the collaboration, and as a result serve as a unique tie
between the labs. Peter and Mike have very complimentary approaches to problems,
and together make an excellent team. Both Mike and Peter have also gone out of their
way for me many times, to ensure I had the most successful graduate career possible. I
am eternally grateful for the opportunity to have worked with both of them, and I hope
they continue the fruitful collaboration they’ve begun.
Both the Sutton and Todd labs have adopted me as their own, and have always
been willing to offer help when needed. Dr. Adam Iliff is my co-author, and has been
instrumental in showing me how to be a hardworking, efficient, and diligent scientist. I
was incredibly fortunate to write a paper with Dr. Iliff, which put me in very good stead
for everything else that came along. Adam is also a good friend, and made long days in
lab infinitely more manageable. Dr. Amber McCartney taught me everything I know
about electrophysiology, and I am incredibly grateful for her time and patience teaching
me how to record. She is another incredible example of diligence and productivity, and
was always an exceptional example for me to try to follow.
As a member of two labs, I had the unfair advantage of three exceptional
technicians/lab managers who have been critical to this work. Amy Krans is always
willing to lend a helping hand, and has spoiled me with her cloning know-how, and
ability to wield a homogenizer to make many, many synaptoneurosomes. Cynthia
Carruthers is an outstanding and meticulous scientist who taught me how to culture
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correctly, and instilled me with many good habits. Christian Althaus is another superb
scientist, who can always be counted on for assistance, and good conversation. I have
benefit immensely from working with all three of these researchers.
I have also been fortunate enough to take advantage of the other exceptional
labs at the University of Michigan. Dr. Henry Paulson, Dr. Geoff Murphy, and Dr. Bill
Dauer have all been in close proximity to my respective labs, and are always willing to
share resources, or lend another set of eyes to a problem. Dr. Paulson and Dr. Murphy
also served as members of my committee, along with Dr. Lori Isom, and all have given
me incredible help and insight into my work.
I must thank Dr. Daniel Michele, who took a chance on me out of undergrad, and
hired me as a technician in his lab. It was there under his mentorship that I developed
many of the basic technical skills I still use regularly, and an eye for critical thinking.
The Michele lab was an incredibly fun, productive environment, and I owe a lot to the
friends I made there: Dr. Jessica Gumerson, Dr. Matthew Campbell, Dr. Zhyldyz
Kabaeva, and Dr. Joel McDade. Dan was also the driving force for applying for
graduate school here in the department of Physiology, so in the end he is culpable for
all this (along with one torn ACL from an excellent pass up-field I couldn’t help but try to
take).
I’ve been fortunate to call the department of Molecular and Integrative Physiology
home, as they’re incredibly committed to graduate education, and would move
mountains for us. The grad chairs throughout my time in the department have all been
outstanding: Dr. Ormond MacDougald, Dr. Scott Pletcher, and Dr. Sue Moenter.
Michele Boggs, the graduate coordinator, made every transition and step along the way
run smoothly.
I am grateful for all of the funding I was awarded during my career here: the
Systems and Integrative Physiology training grant (T32GM008322) from the NIH, the
Ruth L. Kirschstein Institutional National Research Service Award (F31MH097451) from
the NIHM at NIH, and the generous support from Rackham in the form of a predoctoral
award, and multiple travel grants.
Finally, I would like to thank my friends and family for their unwavering support.
My parents, Clare and Eddie, sister Kaylee, and brother-in-law TJ, have always been
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my best cheerleaders. My father’s (at the time) oppressive television rule growing up
that I could only watch PBS after 7pm, was in retrospect a boon which exposed me to
science in the form of Nature and American Scientific Frontiers.
Dr. Henry Colburn has been instrumental in my progress. His own application to
graduate school helped me decide to follow suit, and he has been nothing but
supportive and proud of me. Henry is the reason I could be on the microscope for 12
hours at a time, as he would make sure I ate, and bring me coffee, and keep me
company, all of which made sure I got my work done. His incredible work ethic is
infectious; a fact which I took advantage of many, many times. He also helped maintain
my work-life balance, and ensured I got plenty of exercise playing racquetball and biking
and hiking with me.
Drs. Lauren Kett and Ania Dabrowski were a constant resource for me, both as
scientific ears to bounce ideas off of, and as friends to relax and have fun with. Lauren
is one of the smartest people I know, and I hope some of that rubbed off on me by
proximity. I count myself very lucky to have found both of them.

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Table of Contents

Dedication ........................................................................................................................ii
Acknowledgements ......................................................................................................... iii
List of Figures ................................................................................................................ viii
List of Appendices ........................................................................................................... x
Chapter 1: Introduction .................................................................................................... 1
Abstract ........................................................................................................................ 1
Neurodegeneration in FXTAS ...................................................................................... 3
FMRP expression in FXTAS ...................................................................................... 12
Neuronal function of FMRP ........................................................................................ 14
Hypotheses and addressable questions .................................................................... 17
Chapter 2: Impaired activity-dependent FMRP translation and enhanced mGluR-
dependent LTD in Fragile X premutation mice .............................................................. 20
Abstract ...................................................................................................................... 20
Introduction ................................................................................................................ 21
Results ....................................................................................................................... 24
Discussion.................................................................................................................. 35
Materials and Methods ............................................................................................... 42
Acknowledgements .................................................................................................... 47
Chapter 3: Fragile X mental retardation protein expression in Alzheimer’s disease ...... 48
Abstract ...................................................................................................................... 48
Introduction ................................................................................................................ 49
Results ....................................................................................................................... 51
Discussion.................................................................................................................. 62
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Materials and Methods ............................................................................................... 65
Acknowledgements .................................................................................................... 69
Chapter 4: Impaired sensorimotor gating in Fmr1 knock out and Fragile X premutation
model mice .................................................................................................................... 70
Abstract ...................................................................................................................... 70
Introduction ................................................................................................................ 71
Results ....................................................................................................................... 72
Discussion.................................................................................................................. 78
Acknowledgements .................................................................................................... 82
Chapter 5: Axonal protein expression and homeostatic plasticity are altered in Fragile X
premutation model mice ................................................................................................ 84
Abstract ...................................................................................................................... 84
Introduction ................................................................................................................ 85
Results ....................................................................................................................... 88
Discussion................................................................................................................ 101
Materials and Methods ............................................................................................. 104
Acknowledgements .................................................................................................. 107
Chapter 6: Conclusions and Future Directions ............................................................ 108
Summary and significance ....................................................................................... 108
Outstanding questions ............................................................................................. 111
Appendices ................................................................................................................. 115
References .................................................................................................................. 123

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List of Figures

Figure 1.1: Molecular hallmarks of Fragile X family disorders. ........................................ 3
Figure 1.2: Mechanisms underlying RNA mediated neurodegeneration in nucleotide
repeat disorders. ........................................................................................................... 10
Figure 2.1: Elevated cortical Fmr1 mRNA and decreased FMRP in the fragile X
premutation mouse........................................................................................................ 25
Figure 2.2: Reduced FMRP is distributed throughout dendrites in cultured CGG KI
neurons. ........................................................................................................................ 27
Figure 2.3: CGG KI synaptoneurosomes do not respond to mGluR stimulation. .......... 28
Figure 2.4: CGG KI/XGFP heterozygous cultures reveal selective DHPG induction of
FMRP in WT neurons. ................................................................................................... 32
Figure 2.5: Basal syanptic function is unchanged in CGG KI mice. .............................. 33
Figure 2.6: Exaggerated mGluR-LTD in CGG KI mice is protein synthesis dependent. 34
Figure 2.7: A working model of mGluR-LTD in WT, KO and CGG KI mice. .................. 39
Figure 3.1: FMRP antibody specificity. .......................................................................... 52
Figure 3.2: FMRP expression in AD model APP/PS1 mice. .......................................... 56
Figure 3.3: FMRP expression in Alzheimer's Disease cortex and cerebellum. ............. 58
Figure 3.4: Cortical and cerebellar FMRP expression in FXTAS patients. .................... 59
Figure 3.5: APP expression in FXTAS patients and mouse models. ............................. 61
Figure 4.1: Auditory startle response and reduced pre-pule inhibition in Fmr1 KO
animals. ......................................................................................................................... 74
Figure 4.2: Auditory startle response and age-dependent pre-pulse inhibition in CGG KI
mice. .............................................................................................................................. 76
Figure 4.3: Auditory startle response and increased pre-pulse inhibition with fenobam
administration. ............................................................................................................... 78
Figure 5.1: AIS protein expression in XGFP/CGG KI neurons ...................................... 90
Figure 5.2: Axonal protein expression in XGFP/Fmr1 KO cultures ............................... 91
Figure 5.3: Intrinsic excitability in CGG KI and Fmr1 KO neurons ................................. 94
Figure 5.4: mGluR-LTD in cultured neurons .................................................................. 96
Figure 5.5: Homeostatic plasticity in CGG KI and Fmr1 KO neurons ............................ 99
Figure 5.6: Homeostatic plasticity in CGG-expressing neurons .................................. 101
Figure A.1: Arc expression in CGG KI neurons ........................................................... 116
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Figure A.2: PSD95 in XGFP/CGG KI neurons ............................................................ 118
Figure A.3: UBE3A levels in CGG KI mice .................................................................. 120
Figure A.4: Shank 3 levels in CGG KI mice ................................................................. 122

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List of Appendices

Appendix A: Arc expression in CGG KI hippocampal neurons .................................... 115
Appendix B: PSD95 expression in XGFP/CGG KI neurons ........................................ 117
Appendix C: UBE3A expression in CGG KI mice ........................................................ 119
Appendix D: Shank 3 expression in CGG KI mice ...................................................... 121

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