Altered mTOR signaling in Huntington's Disease

Huntington’s Disease (HD) is caused by a polyglutamine tract expansion in huntingtin (HTT). Despite HTTs ubiquitous expression, there is selective vulnerability in a specific brain region known as the striatum, the cause of which is poorly understood. Here, we provide evidence that impaired striatal mTORC1 activity underlies varied metabolic and degenerative phenotypes in striatal tissues from HD mouse models and patients, and show that further mTORC1 impairment in mouse models, achieved through the knockdown of Rhes, a striatum-enriched mTORC1 activator, exacerbates disease phenotypes. In contrast, exogenous addition of Rhes or the constitutively active form of the mTORC1 regulator, Rheb, into HD mouse brain, alleviates mitochondrial dysfunction, aberrant cholesterol homeostasis, striatal atrophy, and elicits increased autophagy, and reverses impaired dopamine signaling. Furthermore, while HD has been considered primarily a neurological disease, organs with high metabolic demand, such as heart, are also severely affected. The mechanism by which mHTT disrupts cardiac function remains unknown. I provide evidence that mTORC1 is impaired in HD mouse model hearts, causing hyperactive FoxO1 signaling which may render HD hearts vulnerable to stress induced cardiomyopathy. In sum, my combined work indicates impaired mTORC1 signaling as a primary mechanism underlying the neurodegenerative and heart-related disease phenotypes in HD, and thus presents a rational therapeutic target. TABLE OF CONTENTS LIST OF TABLES . viii LIST OF FIGURES . ix LIST of ABBREVIATIONS. xi CHAPTER I: INTRODUCTION . 1 Huntington’s Disease . 1 Overview . 1 Neurological symptoms . 2 Peripheral symptoms . 3 HD mouse models . 3 N-terminal fragment HD mouse models . 3 Full-length HD mouse models . 5 Knockin HD mouse models . 6 Mechanism . 7 Mitochondrial dysfunction . 7 Cholesterol dysregulation . 9 Post-translational modification of mHTT . 10 Rhes and striatal degeneration in HD . 12 Significance and Focus of Dissertation. 13 Contributions to work presented in this Thesis. 14 Chapter II: ENHANCED MTORC1 SIGNALING IMPROVES HUNTINGTON’S DISEASE PHENOTYPES . 15 Introduction . 15 Results . 16 mTORC1 activity is reduced in the striatum of HD human and mouse brains . 16 mTOR increases expression of genes in the antioxidant pathway in HD . 17 mTORC1 controls lipogenic gene expression in HD brains . 18 mTORC1 enhances pathways implicated in mHTT clearance . 18 mTORC1 activity rescued striatal atrophy and improved motor phenotypes in HD mice . 20 Discussion . 21 Materials and Methods . 23 Animals . 23 Plasmids and AAV vectors production . 24 Stereotaxic Injection . 24 Cell culture . 25 Mouse Brain Isolation . 25 Immunohistochemical staining and cell morphology quantification . 25 Stereology . 26 Electron microscopy . 27 Western blot analysis . 27 Human postmortem brain . 28 Quantitative real-time PCR (QPCR). 29 Amphetamine induced rotational assay . 29 Statistical analyses . 29 Chapter III: INVESTIGATING THE IN VIVO ROLE OF RHES IN HUNTINGTON’S DISEASE MOUSE MODELS . 45 Introduction . 45 Results . 46 Rhes overexpression rescues mHTT-associated disease phenotypes in HD mice . 46 Inhibit Rhes in vivo . 47 Suppression of Rhes exacerbates disease phenotypes in HD transgenic mice . 49 Discussion . 50 Materials and Methods . 52 Animals . 52 Plasmids and AAV vectors production . 52 Hypothyroidism induction . 53 Injection . 53 Cell culture and transfections. 54 Mouse Brain Isolation . 54 Immunohistochemical staining and cell morphology quantification . 54 Western blot analysis . 55 Quantitative real-time PCR (QPCR) . 56 Accelerating Rotarod . 56 Activity chamber. 57 Magnetic Resonance Imaging (MRI) Acquisition . 57 Statistical analyses . 58 Chapter IV: ALTERED MTORC1 SIGNALING IN HUNTINGTON’S DISEASE HEARTS . 7