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Open Cai Thesis Edited.Pdf THE PENNSYLVANIA STATE UNIVERSITY SCHREYER HONORS COLLEGE DEPARTMENT OF BIOLOGY REPROGRAMMING ASTROCYTES INTO FUNCTIONAL NEURONS FOR SPINAL CORD REPAIR ALICE CAI SPRING 2017 A thesis submitted in partial fulfillment of the requirements for baccalaureate degrees in Biochemistry and Molecular Biology and Immunology and Infectious Disease with honors in Biology Reviewed and approved* by the following: Gong Chen Professor of Biology Thesis Supervisor Timothy Jegla Associate Professor of Biology Faculty Reader * Signatures are on file in the Schreyer Honors College. i ABSTRACT Spinal cord injury (SCI) and Amyotrophic Lateral Sclerosis (ALS), commonly known as Lou Gehrig’s disease, are both highly debilitating afflictions of the spinal cord with limited available therapies. Pathology is comparable to that of other central nervous system (CNS) injury and disease, involving neuron degeneration and reactive gliosis, which prevents axonal recovery and results in a glial scar. Since the invention of induced pluripotent stem cell technology, many studies have demonstrated direct trans-differentiation across cell lineages through the upregulation of developmental transcription factors. Previous work, including ours, has shown that upregulation of neural transcription factor NeuroD1 can directly convert astroglial cells into neurons in the brain. Through cell culture and mouse models for SCI and ALS, this technology is applied to the spinal cord to regenerate spinal cord neurons from the glial scar. This thesis aims to demonstrate the potential of improved functionality and quality of life after injury by reprogramming reactive astrocytes in the spinal cord into neurons. ii TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................... iii LIST OF TABLES ....................................................................................................... iv ACKNOWLEDGEMENTS ......................................................................................... v Chapter 1 Introduction ................................................................................................. 1 Spinal Cord Injury ............................................................................................................ 1 Amyotrophic Lateral Sclerosis ......................................................................................... 2 Reprogramming Astrocytes to Regenerate Neurons ........................................................ 4 Chapter 2 Materials and Methods ................................................................................ 7 Plasmid Constructs and Transfection ............................................................................... 7 Cell Culture of Mouse Astrocytes .................................................................................... 8 Patch-Clamp Recording of Cell Cultures ......................................................................... 9 Mouse Models and Stereotaxic Viral Injection ................................................................ 10 Sample Preparation and Immunocytochemistry .............................................................. 12 Chapter 3 Results ......................................................................................................... 15 Characterization of Mouse Spinal Cord Cell Cultures ..................................................... 15 Ngn2 and NeuroD1 Can Convert Spinal Cord Glia into Neurons in Vitro ...................... 16 Transcription Factors Drive Differentiation into Specific Neuronal Subtypes ................ 20 Characterization of the Spinal Cord Stab Injury Model ................................................... 26 Characterization of the SOD1 Mouse Model ................................................................... 28 Stereotaxic Injection of AAV9 in SOD1 Mice ................................................................ 30 Chapter 4 Discussion ................................................................................................... 34 Reprogramming Spinal Cord Glia into Neurons in vitro ................................................. 34 Efficacy of in vivo Models of Spinal Cord Injury and ALS ............................................. 36 Future Work and Conclusive Remarks ............................................................................ 37 BIBLIOGRAPHY ........................................................................................................ 40 iii LIST OF FIGURES Figure 1. Plasmid constructs encoding GFP and neurogenic factors for retroviral packaging.7 Figure 2. Characterization of primary cultures of mouse spinal cord glia. .............................. 16 Figure 3. Live time-course images of morphological changes of primary spinal cord cell cultures transduced with retrovirus. ............................................................................................... 17 Figure 4. Direct conversion of mouse spinal cord astrocytes into neurons in vitro. ................ 20 Figure 5. Converted neurons express neuronal subtype markers after 3 weeks. ..................... 22 Figure 6. Co-expression of Ngn2, Lhx3, and Isl1 may promote differentiation of mouse spinal cord glia into a cholinergic fate. ....................................................................................... 23 Figure 7. NeuroD1 and Dlx2 co-expression most efficiently reprograms mouse spinal cord glia into GAD65+ neurons. ..................................................................................................... 25 Figure 8. Characterization of the in vivo model for spinal cord injury. ................................... 27 Figure 9. Characterization of SOD1 mice shows motor neuron loss and reactive astrocytes. 29 Figure 10. Rotarod performance of SOD1 and age-matched wild-type mice. ......................... 30 Figure 11. NeuroD1 expression through AAV9 vector successfully upregulates NeuroD1. ... 31 Figure 12. mCherry is expressed in some OPCs and astrocytes. ............................................. 33 iv LIST OF TABLES Table 1. Behavioral Scale for SOD1 Mouse Model ................................................................ 12 v ACKNOWLEDGEMENTS First, I would like to acknowledge Dr. Gong Chen for welcoming me into his research lab for the duration of my undergraduate education, and for supervising the work that has culminated in this honors thesis. He has been an incredibly inspiring mentor by always aiming high and showing his students that anything can be achieved with hard work and ambition. I would also like to thank Dr. Hedong Li for his mentorship and leadership in our research on the spinal cord. He not only brought his expertise and knowledge of the spinal cord, but was an encouraging teacher in guiding me through the start of a new project. Thank you also to postdoctoral researcher Dr. Lei Zhang for providing me mentorship when I entered the lab as a freshman. She patiently taught me various experimental techniques, scientific thinking, and great appreciation for research by involving me in all parts of the research process. I would like to thank the spinal cord group - Dr. Yuan Liu, Matt Keefe, Xiaoyun Ding, Brendan Puls, Yan Ding, Austin Redilla, and Michael Lai - for being great friends and research partners. I also wish to thank Dr. Zifei Pei for producing AAV9 viruses and providing cells for transformation, and Yuting Bai for supporting all of us in the lab. Thank you to the rest of Chen Lab for their endless support and companionship, and my family and roommates for their care and support. Finally, I would like to acknowledge Dr. Timothy Jegla and Dr. Gong Chen for reading this honors thesis and providing feedback. I would also like to note that while I conducted the cell culture work, which includes all work described in Chapter 2 unless otherwise noted, I have included others’ data generated from the cell cultures for a more complete telling of the results, including RT-PCR data collected by Matt Keefe, and electrophysiology data collected by Lei Zhang. Dr. Hedong Li performed the vi stereotaxic injections described in the mouse models. I also worked side by side with Xiaoyun Ding to characterize the mouse models, and have included results we generated together. 1 Chapter 1 Introduction Spinal Cord Injury The spinal cord is a major component of the central nervous system and is responsible for muscle reflexes and communicating information from the brain to the body. Spinal cord injury is permanent and can be highly debilitating with limited available therapies. Worldwide incidence of spinal cord injury is not well reported, but in the United States, about 282,000 people were living with para- or tetraplegia in 2016, and about 17,000 new cases occur each year, not including those who die shortly after injury (National Spinal Cord Injury Statistical Center, 2016 Data Sheet). Acute trauma of the spinal cord causes a primary and a secondary injury; primary injury is characterized by ischemia, blood vessel damage, and cell death due to mechanical disruption and swelling of the spinal cord, and secondary injury is characterized by inflammatory damage, glutamate excitotoxicity, and associated neurotoxicity and massive cell death (Vercelli et al., 2015, McDonald et al., 2002). The loss of neurons is partnered with hypertrophy and proliferation of glial cells, also known as reactive gliosis, and results in a glial scar. Reactive gliosis is a common pathological response to
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