University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2016 Increased Expression of Frontotemporal Dementia Risk Factor Tmem106b Alters Lysosomal and Autophagosomal Pathways Johanna Irene Busch University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Cell Biology Commons, and the Neuroscience and Neurobiology Commons Recommended Citation Busch, Johanna Irene, "Increased Expression of Frontotemporal Dementia Risk Factor Tmem106b Alters Lysosomal and Autophagosomal Pathways" (2016). Publicly Accessible Penn Dissertations. 1630. https://repository.upenn.edu/edissertations/1630 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1630 For more information, please contact [email protected]. Increased Expression of Frontotemporal Dementia Risk Factor Tmem106b Alters Lysosomal and Autophagosomal Pathways Abstract Frontotemporal lobar degeneration (FTLD) is an important cause of dementia in individuals under age 65. Common variants in the TMEM106B gene were previously discovered by genome-wide association (GWAS) to confer genetic risk for FTLD-TDP, the largest neuropathological subset of FTLD (p=1x10-11, OR=1.6). Prior to its discovery in the GWAS, TMEM106B, or Transmembrane Protein 106B, was uncharacterized. To further understand the role of TMEM106B in disease pathogenesis, we used immortalized as well as primary neurons to assess the cell biological effects of disease-relevant levels of TMEM106B overexpression and the interaction of TMEM106B with additional disease-associated proteins. We also employed immunostaining to assess its expression pattern in human brain from controls and FTLD cases. We discovered that TMEM106B is a highly glycosylated, Type II late endosomal/lysosomal transmembrane protein. We found that it is expressed by neurons, glia, and peri-vascular cells in disease- affected and unaffected regions of human brain from normal controls in a cytoplasmic, perikaryal distribution. In brain from FTLD patients, we discovered a striking loss of subcellular localization with highly disordered TMEM106B immunostaining patterns in a subset of FTLD-TDP cases. Evidence suggests that TMEM106B variants increase risk for developing FTLD-TDP by increasing TMEM106B mRNA and protein expression levels. We therefore investigated the cell biological effects of increased TMEM106B expression. Increased TMEM106B results in a decrease in the average number of late endosomes/lysosomes per cell, loss of lysosomal acidification, and impaired lysosomal degradation. In addition, lysosomal deficits are accompanied by the appearance of enlarged organelles (>2-3μm) demonstrating ultrastructural characteristics of late autophagic vacuoles (autolysosomes/amphisomes). We observed these effects in both immortalized cell lines and in primary neurons overexpressing TMEM106B. Furthermore, we show that the effects of increased TMEM106B expression can be abrogated by (1) a single point mutation to a lysosomal sorting motif in TMEM106B newly identified here, or (2) knockdown of C9orf72 protein. In sum, our results suggest that TMEM106B exerts its effects on FTLD-TDP disease risk through alterations of lysosomal and autophagic pathways and that TMEM106B and C9orf72 may interact in disease pathophysiology. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Alice Chen-Plotkin Keywords C9orf72, Frontotemporal dementia, FTD, FTLD, Lysosome, TMEM106B Subject Categories Cell Biology | Neuroscience and Neurobiology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1630 INCREASED EXPRESSION OF FRONTOTEMPORAL DEMENTIA RISK FACTOR TMEM106B ALTERS LYSOSOMAL AND AUTOPHAGOSOMAL PATHWAYS Johanna I. Busch A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation _________________ Alice Chen-Plotkin, M.D. Assistant Professor of Neurology Graduate Group Chairperson _________________ Daniel S. Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Erika L. F. Holzbaur, Ph.D., Professor of Physiology (Chair) Robert W. Doms, M.D., Ph.D., Professor of Pathology and Laboratory Medicine Virginia M.-Y. Lee, Ph.D., John H. Ware 3rd Endowed Professor in Alzheimer’s Research, Professor of Pathology and Laboratory Medicine Michael S. Marks, Ph.D., Professor of Pathology and Laboratory Medicine ACKNOWLEDGMENT I would like to thank my mentor, Dr. Alice Chen-Plotkin, for giving me the opportunity to study the exciting new world of TMEM106B and for providing me with advice, mentorship, and scientific discourse throughout the past few years. I would also like to thank the members of the lab and the members of my thesis committee for their insights and support throughout. I also thank my family and those who are like family to me for their emotional support and for the fun, both inside and outside the lab, we have had over the past years. I thank Margie Maronski and the "Neurons R Us" core facility for providing primary murine neurons, the Electron Microscopy Resource Laboratory for processing and assistance with electron microscopy, Dr. Fenghua Hu for providing the GFP-TMEM106B construct, Robert Arauz Perez, Emily Bill, Nimansha Jain, Jordan Mak, Tyler Skrinak, and Travis Unger for their assistance, and Dr. Mickey Marks for his input and advice regarding lysosomal targeting motifs. Finally, I would like to thank the patients and their families, whose contributions and dedication to understanding this disease make this work possible. ii ABSTRACT INCREASED EXPRESSION OF FRONTOTEMPORAL DEMENTIA RISK FACTOR TMEM106B ALTERS LYSOSOMAL AND AUTOPHAGOSOMAL PATHWAY Johanna Busch Dr. Alice Chen-Plotkin Frontotemporal lobar degeneration (FTLD) is an important cause of dementia in individuals under age 65. Common variants in the TMEM106B gene were previously discovered by genome-wide association (GWAS) to confer genetic risk for FTLD-TDP, the largest neuropathological subset of FTLD (p=1x10-11, OR=1.6). Prior to its discovery in the GWAS, TMEM106B, or Transmembrane Protein 106B, was uncharacterized. To further understand the role of TMEM106B in disease pathogenesis, we used immortalized as well as primary neurons to assess the cell biological effects of disease-relevant levels of TMEM106B overexpression and the interaction of TMEM106B with additional disease-associated proteins. We also employed immunostaining to assess its expression pattern in human brain from controls and FTLD cases. We discovered that TMEM106B is a highly glycosylated, Type II late endosomal/lysosomal transmembrane protein. We found that it is expressed by neurons, glia, and peri-vascular cells in disease-affected and unaffected regions of human brain from normal controls in a cytoplasmic, perikaryal distribution. In brain from FTLD patients, we discovered a striking loss of subcellular localization with highly disordered TMEM106B immunostaining patterns in a subset of FTLD-TDP cases. Evidence suggests that TMEM106B variants increase risk for developing FTLD-TDP by increasing TMEM106B mRNA and protein expression levels. We therefore investigated the cell biological effects of increased TMEM106B expression. Increased TMEM106B results in a decrease in the average number of late endosomes/lysosomes per cell, loss of lysosomal acidification, and impaired lysosomal degradation. In addition, lysosomal deficits are accompanied by the appearance of enlarged organelles (>2-3μm) demonstrating ultrastructural characteristics of late autophagic vacuoles iii (autolysosomes/amphisomes). We observed these effects in both immortalized cell lines and in primary neurons overexpressing TMEM106B. Furthermore, we show that the effects of increased TMEM106B expression can be abrogated by (1) a single point mutation to a lysosomal sorting motif in TMEM106B newly identified here, or (2) knockdown of C9orf72 protein. In sum, our results suggest that TMEM106B exerts its effects on FTLD-TDP disease risk through alterations of lysosomal and autophagic pathways and that TMEM106B and C9orf72 may interact in disease pathophysiology. iv TABLE OF CONTENTS ACKNOWLEDGMENT .................................................................................................................... II ABSTRACT .................................................................................................................................... III TABLE OF CONTENTS .................................................................................................................. V LIST OF TABLES .......................................................................................................................... VII LIST OF ILLUSTRATIONS........................................................................................................... VIII CHAPTER 1: INTRODUCTION ....................................................................................................... 1 1.1 PART I: Frontotemporal dementia and disease-associated mutations ........................... 1 1.1.1 Frontotemporal Lobar Degeneration ........................................................................ 1 1.1.2 Epidemiology ...........................................................................................................