Splicing Repression Is a Major Function of Tdp-43 in Motor Neurons

SPLICING REPRESSION IS A MAJOR FUNCTION OF TDP-43 IN MOTOR NEURONS: THERAPEUTIC IMPLICATIONS FOR AMYOTROPHIC LATERAL SCLEROSIS by Aneesh N. Donde A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland May, 2018 Title: Splicing Repression is a Major Function of Tdp-43 in Motor Neurons Ph.D Dissertator: Aneesh N. Donde Ph.D Advisor: Philip C. Wong, Ph.D Abstract Nuclear depletion of TDP-43, an RNA binding protein which serves to protect the transcriptome by repressing aberrant splicing, may underlie neurodegeneration in amyotrophic lateral sclerosis (ALS). As multiple functions have been ascribed to TDP-43, whether splicing repression is its major role in motor neurons – that may be compromised in ALS – remains to be established. Here, we show that TDP-43 mediated splicing repression is central to the physiology of motor neurons. To validate TDP-43 mediated splicing repression as a therapeutic target, an AAV9-mediated gene delivery approach was employed to deliver a chimeric protein comprised of the N-terminal RNA recognition domain of TDP-43 fused to an unrelated splicing repressor (RAVER1) to mice lacking TDP-43 in motor neurons. This strategy allowed long-term expression of the repressor without any untoward effects, delayed the onset and slowed the progression of disease, and extended survival. In treated mice, evidence of aberrant splicing was markedly decreased and accompanied by amelioration of motor neuron loss. These findings establish that splicing repression is a principal role of TDP-43 in motor neurons and support the idea that loss of TDP-43-mediated splicing repression represents a key pathogenic mechanism underling motor neuron loss, validating a novel mechanism-based therapeutic strategy for ALS. Dissertation Committee: Philip C. Wong, Ph.D. ii Charlotte J. Sumner, M.D. Lee Martin, Ph.D. Tom Lloyd, M.D., Ph.D. iii Acknowledgements First and foremost, I would like to thank Philip Wong for being an exceptional mentor and thesis advisor to me during my tenure at Johns Hopkins. His unwavering support and guidance has carried me through the difficult times, and I am extremely grateful for the opportunity to work with and learn from him. As my role model, Phil has played an instrumental role in shaping me in becoming the scientist and person that I am today, and one day, I hope to be like him and pass on the same lessons he has taught me. I would also like to thank the members of my thesis committee: Charlotte Sumner, Lee Martin, and Tom Lloyd, for their thoughtful and insightful discussions and comments regarding my thesis project. I appreciate all the input they have provided during my thesis meetings and individual discussions. I would also like to thank Liam Chen for collaborating with me on several projects and providing me the opportunity to write a review article on gene therapy in CNS neurodegenerative disease. The departments of Neuroscience and Pathology at Johns Hopkins have provided invaluable resources that have made my thesis project possible, and for that, I am deeply grateful. In particular, I would like to thank Rita Ragan and Beth Wood-Roig of the Neuroscience department, and Elaine Delman, Argie Laffoon, and Sherry Reed of the Pathology department for their administrative assistance. Lastly, I would like to thank the current and past members of my lab, my friends in the graduate program, and my family for their unwavering support and companionship during my journey. iv Table of Contents Abstract.............................................................................................................................................i Acknowledgements ........................................................................................................................ iii List of Figures and Tables ................................................................................................................ v Chapter 1: Introduction .................................................................................................................. 1 Gain and Loss of Function of TDP-43 Models ............................................................................. 4 TDP-43 Represses Aberrant Splicing ........................................................................................... 6 Chapter 2: Methods ....................................................................................................................... 9 Chapter 3: Results ........................................................................................................................ 17 Conditional Deletion of Tdp-43 Results in Age-dependent Neurodegeneration...................... 18 A Chimeric Protein Restores Splicing Repression in Mice lacking Tardbp in Motor Neurons .. 19 Treatment with CTR Attenuates Deficits in Mice lacking Tardbp in Motor Neurons ............... 22 Chapter 4: Discussion ................................................................................................................... 24 Figures and Tables ........................................................................................................................ 29 References .................................................................................................................................... 63 C.V ................................................................................................................................................. 90 v List of Figures and Tables: Table 1: List of motor neuron diseases Table 2: Genetics of ALS Table 3: Sample size for CTR rescue experiments Table 4: Gene therapy delivery vehicles for preclinical and clinical CNS disease therapeutic trials Figure 1: Deletion of Tdp-43 in lower motor neurons of mice Figure 2: Loss of Tdp-43 in lower motor neurons leads to age-dependent neurodegeneration Figure 3: Inducible deletion of Tdp-43 in excitatory forebrain neurons in mice Figure 4: Neuronal loss in inducible forebrain Tdp-43 knockout mice Figure 5: Behavioral deficits in inducible forebrain Tdp-43 knockout mice Figure 6: Cryptic exon incorporation in inducible forebrain neuron Tdp-43 knockout mice Figure 7: Robust CTR expression in motor neurons of mice Figure 8: CTR expression delays onset and progression of symptoms and prolongs survival Figure 9: Differences in bodyweight for males and females Figure 10: Attenuation of pathological deficits in p90 CTR-treated knockout mice Figure 11: Representative dorsal and ventral roots of p90 mice Figure 12: Restoration of normal splicing repression Figure 13: N-terminal fragment alone has no therapeutic benefit vi Chapter 1: Introduction 1 Introduction Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is the most common motor neuron disease (Table 1) characterized by the death of upper and lower motor neurons(Cleveland, Rothstein, Cleveland, & Rothstein, 2001; del Aguila, Jr, McGuire, Koepsell, & van Belle, 2003). Patients with ALS show progressively worsening weakness, spasticity, muscle atrophy, and an eventual loss of voluntary muscle control(Cleveland et al., 2001; del Aguila et al., 2003). With an incidence rate of 2 per 100,000 people, the average life expectancy following diagnosis of this incurable disease is only 2-5 years, with most deaths occurring due to respiratory failure(Cleveland et al., 2001; del Aguila et al., 2003; Julien, 2001). The genetic underpinnings of ALS are complex, with only about 5-10% of ALS cases showing a recognizable family history of disease, termed ‘familial’ ALS (fALS), and the remaining 90-95% of cases termed ‘sporadic’ ALS (sALS). While the pathophysiology of ALS was described by Jean-Martin Charcot in 1874, to this day, the molecular mechanisms underlying motor neuron death in ALS still remain unknown. Nevertheless, important work over the last several decades has provided valuable insights into the causes and progression of this currently incurable disease. In 1993, it was discovered that mutations in the Cu-Zn superoxide dismutase (SOD1) gene were associated with ~20% of fALS, representing ~2% of all ALS cases(Jones, Brock, Chancellor, Warlow, & Swingler, 1993; Rosen et al., 1993). While the exact mechanisms through which mutant SOD1 affects motor neuron health are not fully elucidated to this day, this discovery allowed for the generation of the first transgenic mouse models (SOD1G93A mice), allowing researchers new opportunities to explore disease mechanisms and potential treatment options in a vertebrate model system. In the following years, mutations in several 2 other genes, such as ALS2, OPTN, VCP, TBK1, and UBQLN, among others, were associated with ALS and consequently have also been studied in animal models (Table 2). However, as these genetic mutations, taken together, only represent a small fraction of total ALS patients, our understanding of the molecular underpinnings of most cases of ALS still remained minimal. Furthermore, many of these genes affected diverse pathways and cellular functions, complicating efforts at identifying a unifying downstream mechanism of motor neuron death in ALS that could be targeted for therapeutic intervention. One major breakthrough in the ALS field was made in 2006, when it was discovered that in neurons and oligodendrocytes of patients with sALS, Tar DNA-binding protein 43 (TDP-43, encoded by the gene TARDBP), an essential, highly-conserved RNA binding protein(Sephton et al., 2010; L. S. Wu et al., 2010), unusually depletes from the

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