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Δ-Protocadherin Function: from Molecular Adhesion Properties to Brain Circuitry

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δ-Protocadherin Function: From Molecular Adhesion Properties to Brain Circuitry

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University

By
Sharon Rose Cooper
Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University
2017

Dissertation Committee: Dr. James Jontes, Advisor
Dr. Marcos Sotomayor, Co-advisor
Dr. Heithem El-Hodiri Dr. Sharon Amacher
Copyrighted by
Sharon Rose Cooper
2017

Abstract

Selective cell-to-cell adhesion is essential for normal development of the vertebrate brain, contributing to coordinated cell movements, regional partitioning and synapse formation. Members of the cadherin superfamily mediate calcium-dependent cell adhesion, and selective adhesion by various family members is thought to

contribute to the development of neural circuitry. Members of the δ-protocadherin

subfamily of cadherins are differentially expressed in the vertebrate nervous system and have been implicated in a range of neurodevelopmental disorders: schizophrenia,

mental retardation, and epilepsy. However, little is known about how the δ-

protocadherins contribute to the development of the nervous system, nor how this development is disrupted in the disease state.
Here I focus on one member of the δ-protocadherin family, protocadherin-19
(pcdh19), since it has the clearest link to a neurodevelopmental disease, being the second most clinically relevant gene in epilepsy. Using pcdh19 transgenic zebrafish, we observed columnar modules of pcdh19-expresing cells in the optic tectum. In the absence of Pcdh19, the columnar organization is disrupted and visually guided behaviors are impaired. Furthermore, similar columns were observed in pcdh1a transgenic zebrafish, located both in the tectum and in other brain regions. This suggests protocadherin defined columns may be a theme of neural development. ii
Our X-ray crystal structure of Pcdh19 reveals the adhesion interface for Pcdh19 and infers the molecular consequences of epilepsy causing mutations. We found several epilepsy causing mutations were located at the interface and disrupted adhesion, which further validated the interface and revealed a possible biochemical cause of Pcdh19

dysfunction. Furthermore, sequence alignments of other δ-protocadherins with Pcdh19 suggest that this interface may be relevant to the entire δ-protocadherin subfamily.

We used the information gained about Pcdh19 to design PCDH19-FE mutations in the genome of zebrafish for comparing the circuitry of embryos with wild-type

pcdh19, non-adhesive pcdh19 or without pcdh19. The combination of in vitro adhesion

studies and in vivo brain imaging analysis provides a more comprehensive

understanding of protocadherin-19 function, and suggests a broader role for the δ-

protocadherin family in differential adhesion during brain development. iii

This work is dedicated to my family for their unconditional love and support.

iv

Acknowledgments

First, I would like to thank both of my advisors, Dr. James Jontes and Dr. Marcos
Sotomayor, for their mentorship. I appreciate their sincere investment in my research projects and my development as a scientist. I am also thankful to my committee members for their guidance.
Thank you to all the members of the Jontes and Sotomayor labs for their comradery and scientific insights. A special thank you goes to Dr. Michelle Emond, who has taught me many valuable research techniques. I would also like to thank Deepanshu Choudhary and Avinash Jaiganesh for their advice on differential scanning fluorimetry, and Dr. Raul Araya-Secchi for his assistance with structural biology software.
Additionally, I would like to thank our collaborator, Marc Wolman, for performing behavioral analysis on the pcdh19 mutant zebrafish. I am also grateful for technical assistance and advice from Dr. Min An, Dr. Jared Talbot, Dr. Michael Berberoglu, Dr. Hao Le, and Phan Duy.
I would like to acknowledge the Jeffery J. Seilhamer Cancer foundation for its financial support during my final years of graduate school. Thank you for the honor and privilege of being a Seilhamer fellow.
Last but not least, I would like to thank my family for their constant encouragement, particularly my husband for his understanding and support through the ups-and downs of pursuing my doctorate.

v

Vita

2009 ...............................................................Research Intern, University of Arkansas for
Medical Sciences
2010 ...............................................................Research Intern, Princeton University 2010 ...............................................................B.S. Molecular and Cellular Biology,
Cedarville University
2011 to present .............................................Graduate Research Associate,
Department of Neuroscience, and Department of Chemistry and Biochemistry, The Ohio State University

Publications

1. Cooper, S. R., Emond, M. R., Duy, P. Q., Liebau, B. G., Wolman, M. A., & Jontes, J. D.
(2015). Protocadherins control the modular assembly of neuronal columns in the zebrafish optic tectum. The Journal of Cell Biology, 211(4), 807–814. http://doi.org/10.1083/jcb.201507108

2. Cooper, S. R., Jontes, J. D., & Sotomayor, M. (2016). Structural determinants of adhesion by Protocadherin-19 and implications for its role in epilepsy. eLife, 5. http://doi.org/10.7554/eLife.18529

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology vi

Table of Contents

Abstract................................................................................................................................ii Acknowledgments................................................................................................................v Vita......................................................................................................................................vi List of Tables ........................................................................................................................x List of Figures ......................................................................................................................xi Chapter 1: Introduction ...................................................................................................... 1
The Cadherin Superfamily and Adhesion.............................................................. 3 Neural Circuitry, Neurodevelopmental Disease, and the δ-Protocadherins ...... 19 Pcdh19 Female Epilepsy...................................................................................... 25 Zebrafish as a Neurodevelopmental Model........................................................ 30 Multidisciplinary Approach to a Fundamental Question.................................... 34
Chapter 2: Protocadherins Control the Modular Assembly of Neuronal Columns in the Zebrafish Optic Tectum..................................................................................................... 40
Abstract ............................................................................................................... 40 Introduction......................................................................................................... 41 Results and Discussion ........................................................................................ 42 Materials and Methods....................................................................................... 48

vii
Chapter 3: Structural Determinants of Adhesion by Protocadherin-19 and Implications for its Role in Epilepsy....................................................................................................... 65
Abstract ................................................................................................................ 65 Introduction......................................................................................................... 66 Results ................................................................................................................. 69 Discussion and Conclusions................................................................................. 83 Materials and Methods....................................................................................... 87
Chapter 4: Creating PCDH19 Female Epilepsy Mutations in the Endogenous Zebrafish pcdh19 Gene by CRISPR Genome Editing....................................................................... 115
Introduction....................................................................................................... 115 Results ............................................................................................................... 117 Conclusions and Future Directions.................................................................... 119 Materials and Methods..................................................................................... 120
Chapter 5: Anatomy of Protocadherin1a Expressing Cells Reveals a Theme of Columnar Development in the Zebrafish Brain............................................................................... 129
Introduction....................................................................................................... 129 Results ............................................................................................................... 130 Discussion.......................................................................................................... 134 Material and methods....................................................................................... 135
Chapter 6: Conclusion..................................................................................................... 144
Columnar Development of the Nervous System .............................................. 144 Adhesion Mechanism of δ-Protocadherins....................................................... 145 viii
Implications for PCDH19 Female Epilepsy ........................................................ 147
References ...................................................................................................................... 149 Appendix A: Pcdh19-FE Mutations Found in Literature Search ..................................... 171 Appendix B: Quantification of Aggregation Assays ........................................................ 176 Appendix C: Sequences Used for Conservation Analysis of Protocadherin-19.............. 180 Appendix D: Sequences Used for Conservation Analysis between Protocadherin Family Members......................................................................................................................... 194

ix

List of Tables

Table 1.1. δ-protocadherin members association with brain circuitry functions and neurodevelopmental disease. .......................................................................................... 39 Table 3.1. X-ray diffraction statistics for drPcdh19EC1-4 and drPcdh19EC3-4. ............. 114 Table A.1. Pcdh19-FE mutations found in literature search........................................... 172 Table B.1. Quantification of aggregation assays – time point 0 minutes (t=0) .............. 176 Table B.2. Quantification of aggregation assays – time point 60 minutes (t=60) .......... 177 Table B.3. Quantification of aggregation assays – time point 1 minute rocking (t=R1). 178 Table B.4. Quantification of aggregation assays – time point 2 minute rocking (t=R2). 179

x

List of Figures

Figure 1.1. Extracellular cadherin (EC) repeat architecture. ............................................ 36 Figure 1.2. The cadherin superfamily. .............................................................................. 37 Figure 1.3. Known adhesion mechanism of the cadherin superfamily. ........................... 38 Figure 2.1. δ-pcdhs define neuronal columns in the zebrafish optic tectum................... 58 Figure 2.2. Pcdh19+ neuronal columns are clonal............................................................ 59 Figure 2.3. Partitioning of the optic tectum by δ-pcdh expression.................................. 60 Figure 2.4. Loss of pcdh19 disrupts the columnar organization of the optic tectum. ..... 61 Figure 2.5. Cell autonomous and non-cell autonomous requirement for Pcdh19. ......... 62 Figure 2.6. Increased proliferation in the optic tectum of pcdh19-/- mutants. ................ 63 Figure 2.7. pcdh19-/- mutants exhibit impaired visually-guided behaviors...................... 64 Figure 2.8. pcdh19-/- mutants show normal motor behavior in phototaxis assay. .......... 64 Figure 3.1. Electron density maps for the EC3-4 linker. ................................................... 96 Figure 3.2 Pcdh19 EC1-4 structure reveals location of PCDH19-FE missense mutations. 97 Figure 3.3. Sequence alignment of zebrafish, mouse, and human Pcdh19 EC repeats. .. 98 Figure 3.4. Predicted structural consequences of PCDH19-FE mutations. ...................... 99 Figure 3.5. Two states for Pcdh19 EC1-4 in solution. ..................................................... 100

xi
Figure 3.6. A crystallographic Pcdh19 antiparallel interface involves fully overlapped EC1-4 repeats.................................................................................................................. 100 Figure 3.7. Alternate crystallographic antiparallel interface involves EC1 to EC5 repeats. ......................................................................................................................................... 101 Figure 3.8. Pcdh19 dimer interfaces and predicted glycosylation and glycation sites. . 102 Figure 3.9. Modified bead aggregation assays can detect calcium-dependent homophilic Pcdh19 interactions. ....................................................................................................... 103 Figure 3.10. Minimal adhesive Pcdh19 fragment includes repeats EC1-4. .................... 104 Figure 3.11. PCDH19-FE mutations at Pcdh19-I1 antiparallel interface impair Pcdh19- mediated bead aggregation............................................................................................ 105 Figure 3.12. PCDH19-FE mutations at Pcdh19-I1 impair bead aggregation even in the presence of N-cadherin................................................................................................... 106 Figure 3.13. PCDH19-FE mutations at Pcdh19-I1 do not abolish the interaction between the extracellular domains of Pcdh19 and N-cadherin.................................................... 106 Figure 3.14. Pcdh19-I1 antiparallel EC1-4 dimer interface involves charged, hydrophilic, and hydrophobic residues. ............................................................................................. 107 Figure 3.15. A common binding mechanism with sequence-diverse interfaces for  and clustered protocadherins................................................................................................ 108 Figure 3.16. Sequence alignment of Pcdh19 EC1-4........................................................ 109 Figure 3.17. Sequence alignment of selected protocadherins (next page).................... 110 Figure 3.18. Structural comparison of Pcdh19-I1 EC1-4 dimer to clustered-protocadherin dimers. ............................................................................................................................ 112 xii
Figure 3.19. Structural comparison of protocadherin 1 and 2 EC3 repeats............... 113 Figure 4.1. Schematic of the CRISP-Cas9 system (modified from Ramalingam, Annaluru, & Chandrasegaran, 2013). .............................................................................................. 123 Figure 4.2. PCDH19-FE missense mutation at Pcdh19 adhesion interface. ................... 124 Figure 4.3. Design of CRISPR induced recombination. ................................................... 125 Figure 4.4. Screen of T147 CRISPR injected embryos by restriction digest.................... 126 Figure 4.5. Screen of E314 CRISPR injected embryos by high resolution melt analysis (HRMA)............................................................................................................................ 127 Figure 4.6. Screening for highly efficient CRISPR. ......................................................... 128

Figure 5.1: TgBAC(pcdh1a:Gal4-VP16, USA:lifeact-GFP) recapitulates endogenous

pcdh1a expression. ......................................................................................................... 139 Figure 5.2: pcdh1a expression in the zebrafish midbrain............................................... 140 Figure 5.3: pcdh1a expression in the zebrafish forebrain. ............................................. 141 Figure 5.4: pcdh1a expression in the zebrafish hindbrain.............................................. 142 Figure 5.5. A variety of motor neurons, sensory neurons and interneurons express pcdh1a in the spinal cord................................................................................................ 143

xiii

Chapter 1: Introduction

During early embryonic development, cells must differentially adhere to one another to form the diverse tissues and organs of the body. In concept, the very existence of a multicellular organism necessitates associations between cells, as the absence of all such interactions would make each cell an independent unicellular organism by definition. Additionally, cells of multicellular organisms must sort into distinct tissue layers during embryonic development, which caused early researchers to wonder at the mechanisms of the morphogenic process. The inherent ability of dispersed cells of an organism to reassemble into organized tissue layers was first observed in spongi and later in amphibians, leading to the differential adhesion hypothesis, which proposed that cells have varying strengthens of adhesion causing them to sort according to thermodynamic principles. (Steinberg, 2007; Townes & Holtfreter, 1955; Wilson, 1907).
Several families of cell adhesion molecules have been discovered that influence the sorting of cells during morphogenesis, including the immunoglobulins (Ig), integrins, and the cadherins (Bock, 1991; McMillen & Holley, 2015; Niessen, Leckband, & Yap, 2011; Shimono, Rikitake, Mandai, Mori, & Takai, 2012). Each of these cell surface proteins physically interacts with molecules on adjacent cells to form stable links, and this process is called cell adhesion. The cadherin superfamily consists of over one
1hundred calcium-dependent cell adhesion molecule and is divided into multiple subfamilies, including classical cadherins, clustered protocadherins and non-clustered δ- protocadherins. These cadherins have various functions during embryo morphogenesis and some members of the cadherin superfamily are important for the segregation of cell types (Bock, 1991; Niessen, Leckband, & Yap, 2011; Takeichi, 1990). For example, E- cadherin and N-cadherin function in neural tube formation and C-cadherin and paraxial protocadherin (PAPC) play a role in cell sorting during gastrulation (Chen & Gumbiner, 2006; Hatta, Takagi, Fujisawa, & Takeichi, 1987).
Cell-to-cell recognition is particularly critical during neurodevelopment for partitioning brain regions and forming intricate brain circuitry within and between brain regions. Neural circuits require numerous precise connections throughout the brain, with each neuron making hundreds of synaptic connections. Furthermore, disruption of brain wiring processes (e.g. axon guidance and synaptogenesis) are increasingly recognized for their involvement in neuropsychiatric disorders (Guilmatre et al., 2009; Mitchell, 2011; Nugent, Kolpak, & Engle, 2012). Although only a small percent of psychiatric disorder cases have a known genetic cause, many members of the cadherin superfamily are associated with increased susceptibly for these diseases (Mitchell, 2011; Redies, Hertel, & Hübner, 2012; Sakurai, 2016). Moreover, one adhesion molecule in particular, Protocadherin-19 (PCDH19), presents a clear causal relationship between genetic mutations in this cadherin gene and epilepsy, with PCDH19 being the second most clinically relevant gene in epilepsy (Depienne & LeGuern, 2012; Dibbens et al., 2008).
2
Since many neurological disorders are thought to originate from aberrant embryonic development of the brain and have been linked to many cell adhesion molecules, it is critical to understand how these cell adhesion molecules function at the molecular level and how they contribute to brain development. This chapter will review the cadherin superfamily, specifically as it relates to their adhesion properties. In addition, the chapter will address principles of neural circuitry development, along with the role of δ-protocadherins in these processes, and specifically the involvement of Pcdh19 in epilepsy. Finally, amenable features of zebrafish for the study of neurodevelopment will be presented along with this model’s limitations.

The Cadherin Superfamily and Adhesion

The cadherin superfamily was originally discovered as a group of calciumdependent cell adhesion molecules (Hyafil, Babinet, and Jacob 1981; Kemler et al. 1977; Takeichi 1988; Takeichi 2004). Each of the family members is a transmembrane glycoprotein containing extracellular repeat domains that mediate adhesion and a disordered intracellular domain that anchors some cadherins to the cytoskeleton (Aberle, Schwartz, & Kemler, 1996; Klezovitch & Vasioukhin, 2015). The extracellular

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    Structural basis of adhesive binding by desmocollins and desmogleins Oliver J. Harrisona,b,1, Julia Braschc,1, Gorka Lassoa,d, Phinikoula S. Katsambaa,b, Goran Ahlsena,b, Barry Honiga,b,c,d,e,f,2, and Lawrence Shapiroa,c,f,2 aDepartment of Systems Biology, Columbia University, New York, NY 10032; bHoward Hughes Medical Institute, Columbia University, New York, NY 10032; cDepartment of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032; dCenter for Computational Biology and Bioinformatics, Columbia University, New York, NY 10032; eDepartment of Medicine, Columbia University, New York, NY 10032; and fZuckerman Mind Brain Behavior Institute, Columbia University, New York, NY 10032 Contributed by Barry Honig, April 23, 2016 (sent for review January 21, 2016; reviewed by Steven C. Almo and Dimitar B. Nikolov) Desmosomes are intercellular adhesive junctions that impart dense midline, consistent with a strand-swap mode of interaction first strength to vertebrate tissues. Their dense, ordered intercellular characterized for classical cadherins (19, 20). Nevertheless, the attachments are formed by desmogleins (Dsgs) and desmocollins identity of Dscs and Dsgs in these tomographic reconstructions could (Dscs), but the nature of trans-cellular interactions between these not be determined, and atomic-resolution structures of desmosomal specialized cadherins is unclear. Here, using solution biophysics and cadherins have not been available, with the exception of an NMR coated-bead aggregation experiments, we demonstrate family-wise structure of a monomeric EC1 fragment of mouse Dsg2 with an heterophilic specificity: All Dsgs form adhesive dimers with all Dscs, artificially extended N terminus (PDB ID code 2YQG). In addition, a with affinities characteristic of each Dsg:Dsc pair.
  • Functional Role of the Overexpression of the Myelin and Lymphocyte

    Functional Role of the Overexpression of the Myelin and Lymphocyte

    Functional role of the overexpression of the myelin and lymphocyte protein MAL in Schwann cells Inauguraldissertation Zur Erlangung der Würde eines Doktors der Philosophie Vorgelegt der Philosophisch‐Naturwissenschaftlichen Fakultät der Universität Basel von Daniela Schmid aus Ramsen (SH) Basel, 2013 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von: Prof. M.A. Rüegg (Fakultätsverantwortlicher) Prof. N. Schaeren-Wiemers (Dissertationsleiterin) Prof. J. Kapfhammer (Korreferent) Basel, den 18. Juni 2013 Prof. J. Schibler (Dekan) To Michael 1. Acknowledgments 1. ACKNOWLEDGMENTS ................................................................................................................................ 6 2. ABBREVIATIONS ......................................................................................................................................... 7 3. SUMMARY ............................................................................................................................................... 10 4. INTRODUCTION ........................................................................................................................................ 11 4.1. THE NERVOUS SYSTEM AND MYELIN SHEATH COMPOSITION ..................................................................................... 11 4.2. SCHWANN CELL ORIGIN AND LINEAGE ................................................................................................................. 12 4.3. THE FUNCTIONAL ROLE OF THE BASAL LAMINA .....................................................................................................
  • Frequent Silencing of Protocadherin 8 by Promoter Methylation, a Candidate Tumor Suppressor for Human Gastric Cancer

    Frequent Silencing of Protocadherin 8 by Promoter Methylation, a Candidate Tumor Suppressor for Human Gastric Cancer

    ONCOLOGY REPORTS 28: 1785-1791, 2012 Frequent silencing of protocadherin 8 by promoter methylation, a candidate tumor suppressor for human gastric cancer DANJIE ZHANG, WEI ZHAO, XINHUA LIAO, TIEQIANG BI, HAIJUN LI and XIANGMING CHE Department of General Surgery, First Affiliated Hospital of Medical College of Xi'an JiaoTong University, Xi'an 710061, P.R. China Received May 10, 2012; Accepted July 30, 2012 DOI: 10.3892/or.2012.1997 Abstract. The cadherins are a family of cell surface glycopro- These abnormalities can also define biological characteristics of teins responsible for cell adhesion which play an important role gastric cancer, which can play a role in therapy (3,4). Although in cell morphology, contact inhibition and signal transduction genetic abnormalities which include gene mutation and dele- during tumorigenesis. Protocadherin 8 (PCDH8), a member tion are remarkable in causing oncogene activation and tumor of the cadherin family, has been reported to act as a tumor suppressor gene inactivation, epigenetic silence of tumor suppressor involved in oncogenesis in breast cancer. In this study, suppressor genes through aberrant promoter hypermethylation we aimed to investigate the epigenetic inactivation of PCDH8 have also been confirmed to be frequent in gastric carcinoma and its tumor suppressor function in gastric cancer. The expres- (5,6). Gene silencing by promoter hypermethylation has been sion of PCDH8 was markedly reduced or silenced in gastric affirmed in several genes in gastric cancer, including CDH1, cancer cell lines compared with normal gastric cells or tissues. which is involved in cell adhesion, and hMLH1, which is associ- Methylation of the PCDH8 gene promoter was observed in 100% ated with DNA mismatch repair and the cell cycle regulator p16.
  • Supplementary Table S5

    Supplementary Table S5

    Supplementary Table S5 - Genes upregulated by anti-MYCN PNA ProbeID Symbol Description RH30ctrl_SignalRH30mut__SignalRH30pna_Signalpna vs mut pna vs ctrl 206884_s_at SCEL sciellin 2.4 3.3 51.4 3.961 4.421 208266_at C8orf17 chromosome 8 open reading frame 17 16.9 4.1 52.1 3.668 1.624 217462_at C11orf9 chromosome 11 open reading frame 9 2.9 10.9 108.9 3.321 5.231 202175_at CHPF 215 123.9 939 2.922 2.127 210271_at NEUROD2 neurogenic differentiation 2 2.4 13.1 84.6 2.691 5.140 216058_s_at CYP2C19 cytochrome P450, family 2, subfamily C, polypeptide 19 4.8 12.8 78.2 2.611 4.026 205556_at MSX2 msh homeobox homolog 2 (Drosophila) 18.7 9 52.6 2.547 1.492 217500_at TIAL1 TIA1 cytotoxic granule-associated RNA binding protein-like 1 15.3 16.2 91.3 2.495 2.577 217124_at IQCE IQ motif containing E 25.8 22.3 116.7 2.388 2.177 213929_at 8.3 14 66.5 2.248 3.002 221159_at 33.4 19.1 81.5 2.093 1.287 207498_s_at CYP2D6 cytochrome P450, family 2, subfamily D, polypeptide 6 60.8 28.7 110.9 1.950 0.867 210881_s_at IGF2 insulin-like growth factor 2 (somatomedin A) 1556.7 1187.4 4274.8 1.848 1.457 219315_s_at C16orf30 chromosome 16 open reading frame 30 83 61.2 213 1.799 1.360 202410_x_at IGF2 insulin-like growth factor 2 (somatomedin A) 2587.4 1916.7 6376.5 1.734 1.301 220748_s_at ZNF580 zinc finger protein 580 298.5 218.7 722.9 1.725 1.276 221063_x_at RNF123 ring finger protein 123 148.5 84.9 277.3 1.708 0.901 210280_at MPZ myelin protein zero (Charcot-Marie-Tooth neuropathy 1B) 48.7 49.5 160.5 1.697 1.721 205236_x_at SOD3 superoxide dismutase 3, extracellular