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Genetic and Pharmacological Approaches to Preventing Neurodegeneration

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Genetic and Pharmacological Approaches to Preventing Neurodegeneration University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 Genetic and Pharmacological Approaches to Preventing Neurodegeneration Marco Boccitto University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Neuroscience and Neurobiology Commons Recommended Citation Boccitto, Marco, "Genetic and Pharmacological Approaches to Preventing Neurodegeneration" (2012). Publicly Accessible Penn Dissertations. 494. https://repository.upenn.edu/edissertations/494 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/494 For more information, please contact [email protected]. Genetic and Pharmacological Approaches to Preventing Neurodegeneration Abstract The Insulin/Insulin-like Growth Factor 1 Signaling (IIS) pathway was first identified as a major modifier of aging in C.elegans. It has since become clear that the ability of this pathway to modify aging is phylogenetically conserved. Aging is a major risk factor for a variety of neurodegenerative diseases including the motor neuron disease, Amyotrophic Lateral Sclerosis (ALS). This raises the possibility that the IIS pathway might have therapeutic potential to modify the disease progression of ALS. In a C. elegans model of ALS we found that decreased IIS had a beneficial effect on ALS pathology in this model. This beneficial effect was dependent on activation of the transcription factor daf-16. To further validate IIS as a potential therapeutic target for treatment of ALS, manipulations of IIS in mammalian cells were investigated for neuroprotective activity. Genetic manipulations that increase the activity of the mammalian ortholog of daf-16, FOXO3, were found to be neuroprotective in a series of in vitro models of ALS toxicity. The small molecule Psammaplysene A (PA) is known to increase the nuclear abundance of FOXO3. PA was also found to be protective in mammalian in vitro models of ALS toxicity as well as a fly and worm model of neurodegeneration. Due to the wide variety of neurodegenerative diseases that share aging as a risk factor, a small molecule modifier of FOXO/daf-16 such as PA could hold great therapeutic potential. Most clinically viable drugs have certain physico-chemical properties that fall within a well-defined set of values, which unfortunately PA does not share. Due to its poor "drug-likness", an investigation into the mechanism of action of PA was undertaken in order to potentially identify more "drug-like" compounds with similar activities. This investigation revealed the heterogeneous nuclear ribonucleoprotein K (HNRNPK) is a direct physical target of PA. PA modifies the ability of HNRNPK ot stabilize rRNA but does not affect many of HNRNPK's other functions. How changes in rRNA stability modify IIS or whether these changes definitively underlie PA's neuroprotective mechanism remains to be determined. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Neuroscience First Advisor Robert G. Kalb Subject Categories Neuroscience and Neurobiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/494 GENETIC AND PHARMACOLOGICAL APPROACHES TO PREVENTING NEURODEGENERATION Marco Boccitto A DISSERTATION in Neuroscience Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation ______________ Robert Gordon Kalb, MD Professor of Neurology Graduate Group Chairperson _________________ Joshua I. Gold, PhD Associate Professor of Neuroscience Dissertation Committee Todd Lamitina, PhD Assistant Professor of Physiology Amita Sehgal, PhD John Herr Musser Professor of Neuroscience Harry Ischiropoulos, PhD Research Professor of Pediatrics Diane E. Merry, PhD Associate Professor of Biochemistry and Molecular Biology GENETIC AND PHARMACOLOGICAL APPROACHES TO PREVENTING NEURODEGENERATION COPYRIGHT 2012 Marco Elio Boccitto This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/ To My wife, Baley for her loving support And My Parents for supporting my academic ambitions iii ABSTRACT: GENETIC AND PHARMACOLOGICAL APPROACHES TO PREVENTING NEURODEGENERATION Marco Boccitto Robert G. Kalb The Insulin/Insulin-like Growth Factor 1 Signaling (IIS) pathway was first identified as a major modifier of aging in C.elegans. It has since become clear that the ability of this pathway to modify aging is phylogenetically conserved. Aging is a major risk factor for a variety of neurodegenerative diseases including the motor neuron disease, Amyotrophic Lateral Sclerosis (ALS). This raises the possibility that the IIS pathway might have therapeutic potential to modify the disease progression of ALS. In a C. elegans model of ALS we found that decreased IIS had a beneficial effect on ALS pathology in this model. This beneficial effect was dependent on activation of the transcription factor daf-16. To further validate IIS as a potential therapeutic target for treatment of ALS, manipulations of IIS in mammalian cells were investigated for neuroprotective activity. Genetic manipulations that increase the activity of the mammalian ortholog of daf-16, FOXO3, were found to be neuroprotective in a series of in vitro models of ALS toxicity. The small molecule Psammaplysene A (PA) is known to increase the nuclear abundance of FOXO3. PA was also found to be protective in mammalian in vitro models of ALS toxicity as well as a fly and worm model of neurodegeneration. Due to the wide variety of neurodegenerative diseases that share aging as a risk factor, a small molecule modifier of FOXO/daf-16 such as PA could hold great therapeutic potential. Most clinically viable drugs have certain physico-chemical properties that fall within a well-defined set of values, which iv unfortunately PA does not share. Due to its poor “drug-likness”, an investigation into the mechanism of action of PA was undertaken in order to potentially identify more “drug-like” compounds with similar activities. This investigation revealed the heterogeneous nuclear ribonucleoprotein K (HNRNPK) is a direct physical target of PA. PA modifies the ability of HNRNPK to stabilize rRNA but does not affect many of HNRNPK’s other functions. How changes in rRNA stability modify IIS or whether these changes definitively underlie PA’s neuroprotective mechanism remains to be determined. v TABLE OF CONTENTS ABSTRACT:................................................................................................................................. IV LIST OF TABLES .................................................................................................................... IX LIST OF FIGURES ................................................................................................................... X CHAPTER 1 ................................................................................................................................ 1 BACKGROUND......................................................................................................................... 1 Introduction........................................................................................................................................... 1 Insulin Signaling Pathways: ...................................................................................................................... 1 Amyotrophic Lateral Sclerosis .................................................................................................................. 6 Heterogeneous Nuclear Ribonucleoprotein K ........................................................................................ 11 CHAPTER 2 ................................................................................................................................ 15 DAF-2 SIGNALING MODIFIES MUTANT SOD1 TOXICITY IN C. ELEGANS ............... 15 Abstract: ................................................................................................................................................ 15 Introduction: .......................................................................................................................................... 16 Results: .................................................................................................................................................. 17 Discussion: ............................................................................................................................................. 28 Materials and Methods: ......................................................................................................................... 32 Acknowledgments: ................................................................................................................................ 34 Author Contributions: ............................................................................................................................ 34 CHAPTER 3 ................................................................................................................................ 35 vi THE SMALL MOLECULE PSAMMAPLYSENE A IS NEUROPROTECTIVE IN MULTIPLE MODELS OF MOTOR NEURON DISEASE ..................................................... 35 Abstract: ...............................................................................................................................................
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