UNIVERSITY of CALIFORNIA, SAN DIEGO the Effects of Cytoskeletal

UNIVERSITY OF CALIFORNIA, SAN DIEGO The Effects of Cytoskeletal Perturbation on Neuronal Structure, Biomechanics, and Function A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Bioengineering by Brian Geoffrey Bober Committee in Charge: Professor Sameer Shah, Chair Professor Adam Engler, Co-Chair Professor Alex Groisman Professor Andrew McCulloch Professor Samuel Ward 2015 Copyright Brian Geoffrey Bober, 2015 All rights reserved. The Dissertation of Brian Geoffrey Bober is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________ _______________________________________________________________ ________________________________________________________________ ________________________________________________________________ Co-Chair ________________________________________________________________ Chair University of California, San Diego 2015 iii TABLE OF CONTENTS SIGNATURE PAGE .............................................................................................. iii TABLE OF CONTENTS .......................................................................................iv LIST OF FIGURES .............................................................................................. vii ACKNOWLEDGMENTS ....................................................................................... x VITA ................................................................................................................... xiii ABSTRACT OF THE DISSERTATION ............................................................... xiv CHAPTER 1: INTRODUCTION ............................................................................ 1 1.1 Peripheral Nervous System Anatomy ................................................................... 1 1.2 Mechanical Loading in the Peripheral Nervous system ......................................... 3 1.3 Peripheral Nerve Structure and Biomechaics ........................................................ 4 1.4 Neuronal Structure and the Cytoskeleton ............................................................. 6 1.5 Axonal Transport .................................................................................................11 1.6 Paclitaxel .............................................................................................................13 1.7 Paclitaxel, Mechanisms of Disease, and Axonal Transport ..................................14 1.8 Mechanisms of Paclitaxel Induced Microtubule Bundling .....................................18 1.9 The Interaction Between Neuronal Mechanics and Axonal Transport ..................20 1.10 Thesis Scope .....................................................................................................21 CHAPTER 2: THE COMBINATORIAL INFLUENCES OF PACLITAXEL AND STRAIN ON AXONAL TRANSPORT ................................................................. 25 iv 2.1 Summary .............................................................................................................25 2.2 Introduction ..........................................................................................................26 2.3 Methods ...............................................................................................................28 2.4 Results ................................................................................................................32 2.5 Discussion ...........................................................................................................44 2.6 Conclusions and Future Directions ......................................................................47 2.7 Acknowledgements ..............................................................................................48 CHAPTER 3: PACLITAXEL ALTERS SENSORY NERVE BIOMECHANICAL PROPERTIES .................................................................................................... 50 3.1 Summary .............................................................................................................50 3.2 Introduction ..........................................................................................................51 3.3 Methods ...............................................................................................................52 3.4 Results ................................................................................................................57 3.5 Discussion ...........................................................................................................66 3.6 Conclusions .........................................................................................................71 3.7 Acknowledgements ..............................................................................................71 CHAPTER 4: TAU INFLUENCES PACLITAXEL-INDUCED MICROTUBULE BUNDLING IN NEURONS ................................................................................. 73 4.1 Summary .............................................................................................................73 4.2 Introduction ..........................................................................................................74 4.3 Methods ...............................................................................................................76 v 4.4 Results ................................................................................................................81 4.5 Discussion ...........................................................................................................92 4.6 Acknowledgements ..............................................................................................96 CHAPTER 5: ACTIN–MYOSIN NETWORK INFLUENCES MORPHOLOGICAL RESPONSE OF NEURONAL CELLS TO ALTERED OSMOLARITY ................. 97 5.1 Summary .............................................................................................................97 5.2 Introduction ..........................................................................................................98 5.3 Results .............................................................................................................. 100 5.4 Discussion ......................................................................................................... 118 5.5 Conclusion ......................................................................................................... 123 5.6 Materials and Methods....................................................................................... 124 5.7 Acknowledgements ............................................................................................ 129 CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS .......................... 130 6.1 Summary of Findings ......................................................................................... 130 6.2 Significance ....................................................................................................... 131 6.3 Future Directions ............................................................................................... 133 REFERENCES ............................................................................................................ 135 vi LIST OF FIGURES AND TABLES Figure 1.1 – Central vs. peripheral nervous system ............................................. 2 Figure 1.2 – Axonal undulations ........................................................................... 4 Figure 1.3 – Architecture of peripheral nerve ....................................................... 5 Figure 1.4 – Neuron morphology .......................................................................... 7 Figure 1.5 – Axonal microtubules ....................................................................... 10 Figure 1.6 – Paclitaxel induced microtubule bundles .......................................... 16 Figure 2.1 – Dorsal root ganglia cells cultured on flexible substrates ................ 29 Table 2.1 – Mitochondrial transport parameters ................................................ 33 Figure 2.2 – Mitochondrial transport observed with and without paclitaxel treatment and stretch .................................................................................. 34 Table 2.2 – Synaptophysin transport parameters .............................................. 37 Figure 2.3 – Synaptophysin transport observed with and without paclitaxel treatment and stretch .................................................................................. 38 Table 2.3 – Actin transport parameters ............................................................. 41 Figure 2.4 – Actin transport observed with and without paclitaxel treatment and stretch.......................................................................................................... 42 Figure 3.1 – Mechanical testing of sural nerves ................................................. 53 Figure 3.2 – Paclitaxel treatment alters whole nerve mechancial properties ..... 58 Figure 3.3 – Sural nerve histology ...................................................................... 61 Figure 3.4 – Peeled epineurium testing .............................................................. 62 vii Figure 3.5 – Model of paclitaxel induced stiffening of cells ................................. 63 Figure 3.6 – Paclitaxel causes bundling of microtubules in sural nerves ............ 64

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