THE KRUPPEL LIKE FACTORS IN AGING AND AGING ASSOCIATED PATHOLOGY By PAISHIUN NELSON HSIEH Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology CASE WESTERN RESERVE UNIVERSITY May, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Paishiun Nelson Hsieh candidate for the Ph.D. degree *. (signed) George R. Dubyak, Ph.D. (chair of the committee) Mukesh K. Jain, M.D. Jeff Coller, M.D. Goutham Narla, M.D., Ph.D. Clive R. Hamlin, Ph.D. 03/29/2018 (date) *We also certify that written approval has been obtained for any proprietary material contained therein. 2 CONTENTS LIST OF TABLES 6 LIST OF FIGURES 7 ACKNOWLEDGEMENTS 10 ABSTRACT 12 CHAPTER 1: Introduction to aging 14 Evolutionary perspectives on aging .......................................................................... 14 General strategies for studying aging ...................................................................... 16 Determinants of longevity, or the “Hallmarks of Aging” ...................................... 19 Signaling pathways in aging ....................................................................................... 28 Chronic diseases in aging and extension of healthspan .................................... 31 The Kruppel like factors in aging and aging associated diseases ................... 33 Anti-aging interventions ................................................................................................ 46 CHAPTER 2: Methods 48 CHAPTER 3: Longevity and healthy aging converge on a conserved Kruppel-like Factor-autophagy pathway 61 Authors: ............................................................................................................................. 61 Summary ........................................................................................................................... 61 Introduction ....................................................................................................................... 62 Results ............................................................................................................................... 65 3 KLF requirement for lifespan extension in multiple pathways ...................... 65 klf-3 overexpression enhances health and lifespan in C. elegans .............. 79 KLF-mediated lifespan extension is dependent on regulation of autophagy ............................................................................................ 87 Mammalian KLF4 regulates autophagy ............................................................. 108 Endothelial restricted KLF4 overexpression delays vessel aging and enhances autophagy ....................................................................................... 116 Discussion ...................................................................................................................... 126 CHAPTER 4: A complement protein mediates neuroprotection in a model of Parkinson’s disease via a gut-neuron axis 134 Authors ............................................................................................................................ 134 Summary ......................................................................................................................... 134 Introduction ..................................................................................................................... 136 Results ............................................................................................................................. 137 Overexpression of klf-3 delays neurodegeneration in a C. elegans model of Parkinson’s Disease .............................................................................. 137 Intestinal specific overexpression of klf-3 delays neurodegeneration ................................................................................................... 143 Intestinal clec-186 is required for intestinal klf-3 mediated neuroprotection ........................................................................................................ 147 Discussion ...................................................................................................................... 155 4 CHAPTER 5: Conclusions and discussion 157 Conclusions .................................................................................................................... 157 Discussion ...................................................................................................................... 158 Closing Thoughts .......................................................................................................... 159 REFERENCES 163 5 LIST OF TABLES Suppl. Table 3-1 Summary table of combinatorial lifespan analysis of 67 double KLF loss of function Suppl. Table 3-2 Combinatorial lifespan analysis of C. elegans animals 69 with reduced klf-1, klf-2 or klf-3 levels Suppl. Table 3-3 Lifespan analysis of C. elegans mutant animals with 76 reduced klf-1 levels Suppl. Table 3-4 Lifespan analysis of C. elegans with klf-3 over-expression 81 (o/e) Suppl. Table 3-5 In silico search identifies presence of KLF response 96 elements GA/GCCC within 1000 base pairs upstream and 200 base pairs downstream of start codon in autophagy genes Suppl. Table 3-6 Lifespan analysis of C. elegans klf-3 and klf-1 o/e animals 107 with reduced beclin-1, lgg-3, atg-13 or atg-7 levels Suppl. Table 3-7 KLF4 manipulation alters expression of a broad spectrum 115 of genes in the autophagy pathway. 6 LIST OF FIGURES Figure 1 Kruppel like factor regulation of aging 45 Suppl. Figure 3-1 Dual loss of function of the Kruppel-like factors reduces 70 C. elegans lifespan Suppl. Figure 3-2 Single RNAi depletion of either klf-1 or klf-3 is specific and 72 does not alter expression of another klf, while mutant worms demonstrate compensatory induction of klfs Figure 3-1 KLFs are required for long lifespan in multiple longevity 77 paradigms Suppl. Figure 3-3 Double loss of function of klf-1 and klf-3 suppresses 78 enhanced longevity in multiple longevity paradigms Figure 3-2 Klf-3 overexpression extends healthspan in C. elegans 82 Suppl. Figure 3-4 Overexpression of klf-3 does not strongly change total 85 number of hatched eggs laid. Suppl. Figure 3-5 Overexpression of klf-3 does not significantly alter 86 pharyngeal pumping rates Figure 3-3 KLF-mediated lifespan extension is dependent on 89 autophagy Suppl. Figure 3-6 Both klf-1 and klf-3 are induced in eat-2 animals and by 91 inhibition of TOR at age Day 12 Suppl. Figure 3-7 Loss of DNA binding region abolishes klf-3 mediated 92 enhancement of autophagy gene expression Suppl. Figure 3-8 Klf-3 mutants do not strongly suppress autophagy gene 93 expression, and concurrent RNAi inactivation of klf-1 in klf-3 mutants weakly reduces expression Suppl. Figure 3-9 Klf-1 overexpression driven by ges-1 promoter 94 Suppl. Figure 3-10 Klf-1 overexpression driven by ges-1 promoter increases 95 autophagy gene expression Suppl. Figure 3-11 Little to no autophagic-like vesicles presence in wild-type 100 and klf-3 RNAi klf-1 animals by TEM at Day 5 or Day 9 (post-fertile period) 7 Suppl. Figure 3-12 KLF-mediated lifespan extension is autophagy dependent 102 Suppl. Figure 3-13 Autophagy is decreased in daf-2 or eat-2 animals with 103 single or compound deficiency of klf-1 and klf-3 Figure 3-4 KLF regulation of autophagy is conserved in mammalian 109 cells Figure 3-5 KLF4 regulates autophagy and ageing in vasculature and 118 decreases with age Suppl. Figure 3-14 VO2max and KLF4 levels decrease with age in humans 120 Suppl. Figure 3-15 Structural wall components are unchanged in aged WT 122 versus aged ECK4TG mice Suppl. Figure 3-16 KLF4 is induced in HUVECs by serum starvation and 124 rapamycin Suppl. Figure 3-17 Flow-induced eNOS expression is autophagy dependent 125 Figure 4-1 Klf-3 delays neurodegeneration in a C. elegans model of 140 Parkinson’s Disease Figure 4-2 Klf-3 alters dopaminergic neuron dependent functions 141 Figure 4-3 Klf-3 does not impact motor deficit in αSyn animals 142 Figure 4-4 Intestinal klf-3 delays dopaminergic neurodegeneration 144 Figure 4-5 Intestinal klf-3 overexpression does not extend lifespan 145 Figure 4-6 RNA-seq analysis of putative secreted genes in klf-3 o/e 146 animals Figure 4-7 Intestinal klf-3 mediated dopaminergic neuroprotection 149 requires systemic clec-186 Figure 4-8 Intestinal klf-3 mediated dopaminergic neuroprotection 150 does not require systemic nlp-24 Figure 4-9 Whole animal RNAi depletion of clec-186 abrogates 151 intestinal klf-3 mediated dopaminergic neuroprotection Figure 4-10 Scheme for generation of intestine and dopaminergic 152 neuron specific RNAi models 8 Figure 4-11 Intestinal klf-3 mediated dopaminergic neuroprotection 153 requires intestinal clec-186 Figure 4-12 Intestinal klf-3 mediated dopaminergic neuroprotection 154 does not require dopaminergic neuron clec-186 9 ACKNOWLEDGEMENTS I have many people to thank who have contributed to my professional and personal growth. First, I want to acknowledge my thesis advisor, Mukesh Jain, for his mentorship and faith in me. From his example I learned how to conduct rigorous science, work collaboratively, and conduct myself with integrity. Many of my lab colleagues, senior and junior, have imparted valuable lessons to me. Specifically, thanks go to Tony Prosdocimo, Yuan Lu, Xudong Liao, Lilei Zhang, Lalitha Nayak, Rongli Zhang, and Guangjin Zhou, who have taught me everything I know about benchwork, scientific