Characterization of the Teneurin C-Terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line

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Characterization of the Teneurin C-terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line

Thomas Dodsworth

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Cell and Systems Biology University of Toronto

Characterization of the Teneurin C-terminal Associated Peptide (TCAP) and Latrophilin Ligand-Receptor Pair in an Immortalized Skeletal Muscle Cell Line

Thomas Dodsworth

Master of Science

Graduate Department of Cell and Systems Biology University of Toronto

2019

Abstract

Teneurin C-terminal associated peptide (TCAP) is an ancient and conserved bioactive peptide that is evolutionarily related to corticotropin releasing factor (CRF). Recently, synthetic

TCAP-1 was shown to increase cellular energy availability and alter contractile performance in rodent skeletal muscle. However, the exact receptor signalling mechanism through which this occurs is unknown. Based on evidence of their interaction in vitro, we hypothesized that TCAP-1 signals through latrophilins—a family of Adhesion G-protein coupled receptors—to elicit its effects in muscle. To test this, I knocked-down and knocked-out latrophilins in the immortalized mouse myoblast C2C12 cell line by small interfering RNA (siRNA) and CRISPR/Cas9 gene editing methods, respectively, and examined the efficacy of TCAP-1 in knockdown and knockout cells. We determined that latrophilin-1 is necessary for TCAP-1-mediated increases in intracellular calcium, NADH turnover, and PGC-1a expression. This establishes, for the first time, that the

TCAP-latrophilin ligand-receptor pair has a functional role in skeletal muscle.

Acknowledgements

My completion of this degree would not have been possible without the support of these incredible individuals. First, I would like to thank my supervisor Dr. David Lovejoy for being an incredibly supportive mentor through this entire experience. You have instilled in me a passion for research that I never knew I had, and I am so grateful for the opportunity to be a part of your lab. I would like to thank the members of my thesis committee, Dr. Les Buck and Dr. Junchul Kim, for their support and feedback throughout this degree, and to Dr. Marius Locke for agreeing to serve as an examiner. To Dr. Dalia Barsyte-Lovejoy, who never ceases to amaze me with her knowledge of molecular biology—your help with this project has been immeasurable. I extend my gratitude to everyone in the Lovejoy lab who made this experience fulfilling. To Dr. David Hogg, thank you for your training and guidance, and for always breaking the silence on quiet days in the lab. To Dr. Andrea D’Aquila, thank you for passing along the reins of this project, and for always keeping me entertained in line for coffee. To Mia Husić, I would not have been able cross the finish line without your level-headed pep talks over lunch. To David Wosnick, I’m glad to have shared this experience with someone as resilient and persevering as you—I’ll see you on the other side. To Norzin Shrestha, thank you for brightening my day whenever we pass each other in the hall, and to my undergraduate student Fernando Jurado Soria, thank you for your assistance with this project and for always being a positive spirit in the lab. I would like to thank everyone in CSB who has lent a hand along the way. To the Buck, Mitchell and Chang labs, and to Dr. Pauline Wang at CAGEF—thank you for allowing me to use your equipment and for helping me troubleshoot. To Peggy Salmon, Chris Garside and everyone I had the pleasure of TA-ing with—thank you for making it a positive experience. To everyone who works in Ramsay Wright, especially those I see here late at night: I am inspired by your dedication, and it has been a pleasure to work alongside you. I thank my parents, my sisters and my friends in Toronto and abroad who have listened to me rant and rave about the ups and downs of this program for the past 2 years—I promise that I’ll be less annoying now. And lastly, I send my gratitude to the staff at Second Cup, Tim Hortons and the AC, who have kept me caffeinated for the past 2 years—you are the true unsung heroes of this degree.

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Table of Contents

Abstract ...... ii Acknowledgements ...... iii Table of Contents ...... iv List of Figures & Tables ...... vi List of Abbreviations ...... viii

Chapter 1: Introduction 1.1. Discovery, Structure and Expression of the Teneurins ...... 1 1.2. Discovery and Structure of TCAP ...... 3 1.3. Expression and Processing of TCAP ...... 5 1.4. Actions of Teneurin and TCAP in the Central Nervous System ...... 7 1.5. Actions of Teneurin and TCAP in Reproductive Tissues ...... 9 1.6. Actions of Teneurins and TCAP in Skeletal Muscle ...... 9 1.7. Evidence for Teneurin and TCAP Receptors ...... 10 1.8. Discovery, Structure and Expression of the Latrophilins ...... 11 1.9. Ligands and Signalling of Latrophilin ...... 14 1.10. Description of the Teneurin/TCAP-Latrophilin System ...... 15 1.11. Evolution of the Teneurin/TCAP-Latrophilin System ...... 17 1.12. Skeletal Muscle Cell Biology & Use of Immortalized Muscle Cell Lines ...... 19 1.13. Thesis Rationale & Research Aims ...... 20

Chapter 2: Methods 2.1. Cell Culture ...... 23 2.2. RNA Extraction ...... 24 2.3. Reverse Transcription and Polymerase Chain Reaction ...... 25 2.4. Cloning and Sequencing of Polymerase Chain Reaction Products ...... 27 2.5. Quantitative Reverse Transcription Polymerase Chain Reaction ...... 27 2.6. Protein Extraction & Western Blot ...... 28 2.7. Small Interfering RNA Transfection ...... 29

iv 2.8. T7 Endonuclease Assay ...... 29 2.9. Live-Cell Calcium Imaging ...... 30 2.10. Resazurin-Resorufin Fluorescence Assay ...... 30 2.11. Statistical Analysis ...... 31

Chapter 3: Results 3.1. Characterization of TCAP-3 mRNA in the Adult Mouse Brain by 5’RACE PCR ...... 32 3.2. Expression of Latrophilins in C2C12 cells ...... 34 3.3. Establishing Methods of Latrophilin-1 and 3 siRNA Knockdown ...... 35 3.4. Intracellular Calcium Dynamics in TCAP-1-treated Latrophilin-1 and 3 Knockdowns ...... 36 3.5. Development of Latrophilin-1 CRISPR/Cas9 Knockouts ...... 36 3.6. Intracellular Calcium Dynamics in TCAP-1-treated Latrophilin-1 Knockouts ...... 40 3.7. Resorufin Fluorescence in TCAP-1-treated Latrophilin-1 CRISPR/Cas9 Knockouts ...... 40 3.8. Myosin Heavy Chain Expression in TCAP-1-treated Tibialis Anterior Muscle Tissue ...... 44 3.9. Myosin Heavy Chain I and Peroxisome Proliferator-activated Receptor-g Coactivator 1a Expression in TCAP-1-treated C2C12 cells ...... 45 3.10. Peroxisome Proliferator-activated Receptor-g Coactivator 1a Expression in TCAP-1- treated Latrophilin-1 CRISPR/Cas9 Knockouts ...... 47

Chapter 4: Discussion 4.1. Independent Transcription of TCAP ...... 48 4.2. Small Interfering RNA Knockdown of Latrophilins ...... 50 4.3. CRISPR/Cas9 Knockout of Latrophilins ...... 51 4.4. The TCAP-Latrophilin Ligand Receptor Pair Regulates Intracellular Calcium ...... 53 4.5. The TCAP-Latrophilin Ligand Receptor Pair Regulates Cellular Metabolism ...... 56 4.6. The TCAP- Latrophilin Ligand Receptor Pair Regulates Muscle Gene Expression ...... 59 4.7. Future Directions ...... 63 4.8. Concluding Remarks ...... 65

References ...... 67 Appendix ...... 79

v List of Figures & Tables

Chapter 1: Introduction Figure 1.1. Schematic of the teneurin proteins ...... 2 Figure 1.2. Sequence alignment of the teneurin C-terminal associated peptides (TCAPs) ...... 4 Figure 1.3. Proposed mechanism for TCAP-1 processing ...... 6 Figure 1.4. Schematic of the latrophilin proteins ...... 13 Figure 1.5. Schematic of teneurin-latrophilin trans-synaptic complex ...... 16

Figure 1.6. Proposed mechanism for TCAP-latrophilin signalling through Gq/11-proteins ...... 17

Chapter 2: Methods Figure 2.1. Schematics of the LPHN-1 and 3 genes depicting sites targeted for CRISPR/Cas9 gene editing ...... 24 Table 2.1. Single guide RNA (sgRNA) sequences for CRISPR/Cas9 Gene Editing ...... 24 Table 2.2. Primers for Polymerase Chain Reaction ...... 26 Table 2.3. Antibodies for Western Blot ...... 29

Chapter 3: Results Figure 3.1. 5’-Rapid Amplification of cDNA Ends for Identification of TCAP-3-containing mRNAs ...... 33 Figure 3.2. Expression of latrophilins in C2C12 cells ...... 34 Figure 3.3. Expression of latrophilin-1 and 3 in siRNA knockdown C2C12 cells ...... 35 Figure 3.4. Fluo-4 intracellular calcium fluorescence in TCAP-1-treated latrophilin-1 and 3 siRNA knockdown C2C12 cells ...... 37 Figure 3.5. T7 endonuclease assay of heterogenous latrophilin-1 and 3 CRISPR/Cas9-transfected C2C12 cell pools ...... 38 Figure 3.6. Characterization of latrophilin-1 CRISPR/Cas9 knockout C2C12 clones ...... 39 Figure 3.7. Fluo-4 intracellular calcium fluorescence in TCAP-1-treated latrophilin-1 CRISPR/ Cas9 knockout C2C12 cells ...... 41 Figure 3.8. Resorufin fluorescence in TCAP-1-treated latrophilin-1 CRISPR/Cas9 knockout C2C12 cells ...... 42

vi Figure 3.9. Normalized resorufin fluorescence in latrophilin-1 CRISPR/Cas9 knockout C2C12 cells following 120 minutes of TCAP-1 treatment ...... 43 Figure 3.10. Expression of myosin heavy chain (MyHC) genes in tibialis anterior muscle of TCAP-1-treated rats ...... 44 Figure 3.11. Expression of myosin heavy chain I (MyHCI) and peroxisome proliferator-activated receptor-g coactivator 1a (PGC-1a) in TCAP-1-treated wild-type C2C12 cells ..... 45 Figure 3.12. Expression of PGC-1a in TCAP-1-treated LPHN-1 CRISRP/Cas9 knockout C2C12 cells ...... 46

Chapter 4: Discussion Figure 4.1. Schematic of the putative role of TCAP-1 in calcium regulation of actin-myosin contraction in skeletal muscle ...... 55 Figure 4.2. Schematic of the proposed relationship between the TCAP-latrophilin-induced intracellular calcium surge and TCAP-latrophilin-induced increase in NADH turnover ... 58 Figure 4.3. Schematic of the proposed relationship between the TCAP-latrophilin-induced intracellular calcium surge and TCAP-latrophilin-induced increase in PGC-1a expression ...... 62 Figure 4.4. Summary of latrophilin knockdown and knockout effects in TCAP-1-treated C2C12 cells ...... 66

Appendix Supplementary Figure 2.1. Sample images of C2C12 myotubes ...... 79 Supplementary Figure 3.1. Identification of candidate latrophilin-1 CRISPR/Cas9 knockout C2C12s from homogenous clone lines ...... 80

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List of Abbreviations

2-APB, 2-aminoethoxydiphenyl borate 5’RACE, 5’-rapid amplification of cDNA ends Acetyl-CoA, acetyl coenzyme A ACh, acetylcholine AChR, acetylcholine receptor ADHD, attention deficit hyperactivity disorder ADP, adenosine diphosphate AFU, arbitrary fluorescent unit AKT, phosphoinositide-3 kinase-protein kinase B AMP, adenosine monophosphate ANOVA, analysis of variance ATG, adenine-thymine-guanine ATP, adenosine triphosphate BCA, bicinchoninic acid bp, base pairs BSA, bovine serum albumin Ca2+, calcium ion

CaCl2, calcium chloride CaMKIV, calcium/calmodulin dependent protein kinase type IV cAMP, cyclic AMP CaN, calcineurin Cas9, CRISPR-associated protein 9 cDNA, complementary DNA CIRL, calcium-independent receptor for latrotoxin CNS, central nervous system

CO2, carbon dioxide gas CREB, cAMP response element binding protein CRF-R, corticotropin releasing factor receptor CRF, corticotropin releasing factor

viii CRISPR, clustered regularly interspaced short palindromic repeats CTF, C-terminal fragment CTRL, empty vector-transfected control DAG, diacylglycerol ddH2O, double-distilled water DHPR, dihydropyridine receptor DIC, differential interference contrast DMEM, Dulbecco’s Modified Eagle Media DMSO, dimethyl sulfoxide DNA, deoxyribonucleic acid DNAse, deoxyribonuclease dNTP, deoxy-nucleotide triphosphate DSB, double-stranded DNA break DTT, dithiothreitol E1, exon 1 E2, exon 2 E3, exon 3 E4, exon 4 E5D, exon 5 downstream E5U, exon 5 upstream ECL, enhanced chemiluminescence EDTA, ethylenediaminetetraacetic acid EGF, epidermal growth factor ER, endoplasmic reticulum ETC, Electron transport chain

FAD(H2), flavin adenine dinucleotide FBS, fetal bovine serum FCCP, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone FLRT, fibronectin-like domain containing leucin-rich transmembrane protein GAIN, GPCR autoproteolysis-inducing GAPD, glyceraldehyde-3-phosphate dehydrogenase GDP, guanosine diphosphate

ix GHH, glycine-histidine-histidine GKR, glycine-lysine-arginine GLUT, glucose transporter GPCR, G-protein coupled receptor GPS, GPCR proteolysis site GTP, guanosine triphosphate H+, hydrogen ion

H2O, water HBD, hormone binding domain HDAC, histone deacetylase HDR, homology directed repair HEK, human embryonic kidney HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HGT, horizontal gene transfer HNH, histidine-arginine-histidine HRP, horseradish peroxidase Ig, immunoglobulin Indel, insertion/deletion

IP1, inositol 3-phosphate

IP3, inositol 1,4,5-triphosphate

IP3R, inositol 1,4,5-triphosphate receptor KCl, potassium chloride KD, knockdown kDA, kilodalton KO, knockout Lasso, Latrophilin-1-associated synaptic surface organizer LB, Luria broth LEC, lectin-like domain LPHN, latrophilin LTXN4C, recombinant mutant a-latrotoxin MCU, mitochondrial calcium uniporters MEF2, myocyte enhancement factor 2

x MEK-ERK, mitogen-activated protein kinase-extracellular signal-regulated kinase

MgCl2, magnesium chloride mRNA, messenger RNA MyHC, myosin heavy chain NaCl, sodium chloride NAD(H), nicotinamide adenine dinucleotide

NaHCO3, sodium bicarbonate NFAT, nuclear factor of activated T-cell NHEJ, non-homologous end-joining NHL, NCL-1, HT2A, and Lin-41 NT, non-targeting NTF, N-terminal fragment

O2, oxygen gas OLF, olfactomedin-like domain PAM, protospacer adjacent motif PBS, phosphate buffered solution PBST, 0.3% PBS-Tween20 PC, prohormone convertase PCR, polymerase chain reaction PGC-1a, Peroxisome proliferator-activated receptor-g coactivator 1a

Pi, inorganic phosphate

PIP2, phosphatidylinositol 4,5-biphosphate PLC, phospholipase C PMSF, phenylmethane sulfonyl fluoride PPT, polymorphic proteinaceous toxin qPCR, quantitative polymerase chain reaction qRT-PCR, quantitative reverse transcription polymerase chain reaction RHS, recombinant hot-spot RIPA, radioimmunoprecipitation assay RISC, RNA-induced silencing complex RNA, ribonucleic acid RNAi, RNA interference

xi RNAse, ribonuclease ROI, region of interest rpm, rotations per minute RT-PCR, reverse transcription PCR RyR, ryanodine receptors SNP, single nucleotide polymorphisms S.O.C., super optimal broth with catabolite repression SC, subcutaneous injection SDS-PAGE, sodium dodecyl polyacrylamide gel electrophoresis SEM, standard error of mean SERCA, sarco/endoplasmic reticulum calcium ATPase sgRNA, single guided RNA siRNA, small interfering RNA SMRT, small molecule, real-time sequencing SOD1, copper-zinc superoxide dismutase 1 SR, sarcoplasmic reticulum T-tubules, transverse tubules TBE, Tris-borate-EDTA TBP, TATA box binding protein TCAP, teneurin C-terminal associated peptide Ten-a, tenascin-like protein accessory Ten-m, tenascin-like protein major TF, transcription factor TMR, transmembrane region TR, transcriptional regulator VDAC, voltage dependent anion channels WT, wild-type YD, tyrosine-aspartate

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Chapter 1

Introduction

1.1. Discovery, Structure and Expression of the Teneurins

The teneurin genes were discovered in Drosophila melanogaster as two paralogous forms: Ten-a and Ten-m (Baumgartner and Chiquet-Ehrismann, 1993; Baumgartner et al., 1994; Levine et al., 1994). Baumgartner and colleagues (1994) initially identified the teneurins in a screen for proteins related to the tenascins and named them ‘teneurins’ based on their high expression in the central nervous system (CNS). At the same time, Levine and colleagues (1994) identified a protein they called ‘odz’ (equivalent to Ten-m) in a screen for phosphotyrosine-containing proteins. The odz gene functioned as a late-acting pair rule gene in Drosophila melanogaster segmentation where odz mutants lost odd-numbered body segments. Teneurin genes were soon identified across numerous species. Invertebrates generally possess one teneurin gene, although genome duplication events subsequently produced two teneurin paralogues in insects and four in vertebrates (Minet and Chiquet-Ehrismann, 2000). The domain organization of the teneurins is generally conserved across all vertebrates and invertebrates, with most divergence occurring within the intracellular domains (Tucker and Chiquet-Ehrismann, 2006). There is about 58-72% sequence identity among vertebrate paralogues (Minet and Chiquet-Ehrismann, 2000). Teneurins are primarily expressed in the CNS where in situ hybridization experiments show that the four paralogues have mostly distinct but sometimes overlapping expression profiles in the adult mouse brain (Zhou et al., 2003). The teneurins also have vast and varying expression during embryogenesis in both vertebrates and invertebrates, including in cardiac cells, muscle attachment sites and other non-neuronal tissues of D. melanogaster (Zhou et al., 2003; Baumgartner and Chiquet-Ehrismann, 1993; Baumgartner et al., 1994). Structurally, the teneurins are 2500-2800 amino acids in length and are classified as type II transmembrane proteins. Each teneurin is comprised of an approximately 300-375 amino acid N-terminal intracellular region, a short transmembrane region, and an approximately 2400 amino acid C-terminal extracellular region (Tucker and Chiquet-Ehrismann, 2006; Figure 1.1). The

1 intracellular region is comprised of two proline-rich regions located close to the transmembrane region as well as two EF-hand like domains located closer to the N-terminus. The extracellular region contains several functional domains. Proximal to the transmembrane region, there is an epidermal growth factor (EGF) domain containing 8 highly conserved tenascin-type EGF-like repeats which allow dimerization of adjacent teneurins (Tucker and Chiquet-Ehrismann, 2006; Feng et al., 2002; Li et al., 2018). This is followed by a region containing 17 conserved cysteine residues, followed by a domain containing five NCL-1, HT2A, and Lin-41 (NHL) repeats that mediate homophilic interactions between teneurins (Tucker and Chiquet-Ehrismann, 2006; Beckmann et al., 2013). More distally, there is a YD (tyrosine and aspartate) domain containing 26 conserved YD repeats as well as a tyrosine-rich recombinant hot spot (RHS)-like domain (Minet and Chiquet-Ehrismann, 2000). The NHL, YD and RHS-like domains are typical of prokaryotic proteins, suggesting that the ancestral teneurin gene had a prokaryotic origin and subsequently became associated with a typical metazoan EGF-containing gene (discussed further in 1.11; Tucker et al., 2012; Woelfle et al., 2015). Lastly, at the C-terminus, there is a 40-41 amino acid functional bioactive peptide-like region named teneurin C-terminal associated peptide (TCAP; Qian et al., 2004; Wang et al., 2005).

Functional Domains: Proline-rich EGF-like regions repeats NHL repeats RHS-like domain

NH2- -COOH

EF hand-like Transmembrane region Conserved YD repeats TCAP domains cysteines Structural Domains: Ig-like b-propeller Barrel Tox-like

Figure 1.1. Schematic of the teneurin proteins. The intracellular region of the teneurins possesses two EF hand-like domains and two proline-rich regions. The extracellular region possesses 8 EGF-like repeats, a conserved cysteines region, 5 NHL repeats, 26 YD repeats, an RHS-like domain, and most distally, the teneurin C-terminal associated peptide (TCAP) region. From the recently elucidated 3D structure of the teneurin-2 extracellular region, there are four structural domains: an Ig-like domain, a b-propeller domain, a Barrel domain, and a Tox-like domain. Adapted from Tucker et al., 2012; Jackson et al., 2018; Li et al., 2018.

2 The recently elucidated 3-dimensional structure of human (Li et al., 2018) and chicken (Jackson et al., 2018) teneurin-2 reveals that the extracellular region of teneurin possesses a unique bacteria ‘Tc’ toxin-like organization. Bacterial Tc toxins are comprised of three proteins (A, B, C) where the ‘A’ protein allows binding to target cells and the ‘B’ and ‘C’ proteins form a shell that protects the carboxy-terminal toxic component from the host (Busby et al., 2013). In human and chicken teneurin-2, the EGF repeats form a flexible ‘stalk’ domain that constitutively cis-dimerizes proximal to the cell membrane. The YD repeats form a spiralling ‘Barrel’ domain made of up b- hairpins that act as a shell for a hydrophobic core. The barrel is sealed on the top by a spiral domain and on the bottom by an immunoglobulin ‘(Ig)-like’ domain. Perpendicular to the bottom seal, the NHL repeats form a ‘b-propeller’ domain that, along with the Ig-like domain, likely plays a role in ligand binding and trans-cellular interactions. The C-terminal 120 amino acids exit through a gap in the wall of the barrel region; this exposed region is called the ‘toxin-like’ or ‘Tox-GHH’ domain (discussed further in 1.2). There are some important differences between the structures of teneurin and bacterial Tc toxins. First, teneurins are much smaller than Tc toxins; second, the b- propeller acts as the barrel’s bottom seal in Tc toxins, whereas the Ig-like domain serves this function in teneurins; third, the C-terminal region of Tc toxins is fully encapsulated in the barrel, whereas the C-terminal region of teneurin is only partially encapsulated; and lastly, Tc toxins possess an active catalytic pocket in their barrel interior that can autoproteolytically cleave a toxic C-terminal peptide, whereas teneurins possess a dormant catalytic pocket that may become activated upon a significant conformational change to release a C-terminal peptide. Overall, the similarities between the teneurins and bacterial Tc toxins point to an interesting evolutionary history (discussed further in 1.11) that may explain the diverse biological actions of the teneurins and TCAPs as regulators of stress of metabolism across the metazoans.

1.2. Discovery and Structure of TCAP

The TCAP region of the teneurins was initially discovered in a screen of a rainbow trout hypothalamic cDNA library for genes related to the corticotropin releasing factor (CRF) family. In this study, Qian and colleagues (2004) used a hamster urocortin cDNA probe to identify a neuropeptide-like region representing the C-terminal 40 amino acids of rainbow trout teneurin-3 which they named ‘teneurin C-terminal associated peptide’ (TCAP). Vertebrates have four paralogous forms of this 40-41 amino acid peptide, with each paralogue found at the C-terminus

3 of one of the teneurin genes (Figure 1.2). They are named according to their association with each of the teneurin paralogues; hence TCAPs 1-4. There is 73-88% amino acid sequence identity among human TCAP paralogues and 71-87% identity among mouse paralogues (Wang et al., 2005). The TCAPs also share approximately 20% amino acid identity with CRF, indicating they may share a distant evolutionary ancestor (discussed further in 1.11). The TCAP sequence is located within the terminal exon of the teneurin mRNA and is flanked by a prohormone convertase (PC)-like cleavage signal at the 5’ end and a glycine-lysine-arginine (GKR) amidation motif at the 3’ end which precedes the stop codon and subsequent 3’ untranslated region (Qian et al., 2004; Wang et al., 2005). Additional basic/dibasic cleavage sites are located further upstream of TCAP, but only some are conserved (Chand et al., 2013). These sites indicate that TCAP may be enzymatically cleaved from the teneurin protein and may act on receptors in an autocrine or paracrine manner (discussed further in 1.3).

human TCAP-1 QQLLSTGRVQGYDGYFVLSVEQYLELSDSANNIHFMRQSEI-NH2 human TCAP-2 QQLLSTGRVQGYEGYYVLPVEQYPELADSSSNIQFLRQNEM-NH2 human TCAP-3 QLLSAGKVQGYDGYYVLSVEQYPELADSANNIQFLRQSEI-NH2 human TCAP-4 QQVLSTGRVQGYDGFFVISVEQYPELSDSANNIHFMRQSEM-NH2 Teneurin ● ● ● -COOH mouse TCAP-1 QQLLGTGRVQGYDGYFVLSVEQYLELSDSANNIHFMRQSEI-NH2 mouse TCAP-2 QQLLSTGRVQGYEGYYVLPVEQYPELADSSSNIQFLRQNEM-NH2 TCAP mouse TCAP-3 QLLSAGKVQGYDGYYVLSVEQYPELADSANNIQFLRQSEI-NH2 mouse TCAP-4 QQVLSTGRVQGYDGFFVTSVEQYPELSDSANNIHFMRQSEM-NH2

Figure 1.2. Sequence alignment of the teneurin C-terminal associated peptides (TCAPs). The TCAP region of the teneurins is highly conserved across paralogues and between mouse and human orthologues. Sequence identity is depicted in dark grey and functional substitutions are depicted in light grey. Adapted from Wang et al., 2005.

Based on the 3-dimensional structure of teneurin-2, TCAP-2 is part of the toxin-like domain, which exits through the wall of the barrel region allowing exposure to the extracellular environment (Li et al., 2018; Jackson et al., 2018). Typical bacterial Tc toxins possess a histidine- arginine-histidine (HNH) domain near their C-terminus that confers endonuclease activity (Zhang et al., 2012). Teneurin’s toxin-like domain possesses an inactive glycine-histidine-histidine (GHH) domain that includes only the arginine from the HNH motif as well as a single DNA-binding helix that could allow non-enzymatic interactions with DNA (Li et al., 2018). Recombinant versions of

4 the teneurin-1 and 2 toxin-like domains possess endonuclease activity in vitro (Ferralli et al., 2018). TCAP occupies the distal part of the a-helical DNA-binding region at its N-terminus and forms a b-hairpin loop structure at its C-terminus. Importantly, it is not predicted to have endonuclease activity like the recombinant toxin-like domains (Jackson et al., 2018; Ferralli et al., 2018). Unwinding of the helix may allow access to proteases to release soluble form of TCAP.

1.3. Expression and Processing of TCAP

Although the TCAP region is part of the teneurin protein, there is mounting evidence that some TCAP paralogues may be functionally processed as an individual peptide, either through cleavage from the full-length teneurin or through separate transcription and translation (Figure 1.3). In situ hybridization experiments have revealed some differences between the expression of TCAP-1 and teneurin-1 in the rodent brain; specifically, TCAP-1 has high expression in the diencephalon and limbic system, whereas teneurin-1 does not (Wang et al., 2005; Zhou et al., 2003). Similarly, immunocytochemical labelling has shown distinct cellular localization of teneurin-1 and TCAP-1 in mouse hippocampal E14 cells, where both co-localize on the plasma membrane, but TCAP-1 labelling alone is detected diffusely in the cytosol (Chand et al., 2013). As previously mentioned, conserved cleavage sites upstream of the 40-41 amino acid sequence indicate that TCAPs may be cleaved from teneurin and act on receptors in an autocrine or paracrine manner (Chand et al., 2013). Based on the criteria outlined by Seidah and Chretien (1997), the translated portion of the terminal exon of mouse teneurin-1 possesses five potential sites for cleavage by prohormone convertases, where three of these sites are conserved across all teneurins (Chand et al., 2013). TCAP is exteriorly oriented when teneurin is folded, and therefore the exposed helix at the N-terminus of TCAP could be accessed by proteases (Jackson et al., 2018; Li et al., 2018). This type of processing is observed in numerous other neuropeptides including CRF, which is cleaved in exocytotic vesicles as it is trafficked for extracellular release (Hook et al., 2008; Lovejoy et al., 2006). TCAP could also be cleaved at the plasma membrane via ectodomain shedding; this type of processing is characteristic of the tumour necrosis factor family of peptides (Garton et al., 2001; Lovejoy et al., 2006). Northern blot studies completed in our laboratory further suggest that the terminal exons of teneurin-1 and 3, which contain TCAP-1 and 3, can be transcribed independently from their full-length teneurin genes (Chand et al., 2013). Using 5’ rapid amplification of cDNA ends

A Full-length teneurin-1 mRNA Teneurin-1 protein

Short TCAP-1-containing mRNA TCAP-1 propeptide (13 kDa) TCAP-1 coding region = basic/dibasic cleavage site

B Proteolytic cleavage Neuron Teneurin-1 Secretory pathway

Teneurin-1 gene TCAP-1 propeptide Translation by free ribosomes Free soluble TCAP-1

Figure 1.3. Proposed mechanism for TCAP-1 processing. (A) TCAP-1 can be expressed as part of the full-length teneurin protein or as a short transcript which produces a 13 kDa TCAP-1 propeptide. (B) Teneurin-1 is trafficked to the plasma membrane through the secretory pathway. Extracellular proteases could cleave TCAP-1 from teneurin-1, releasing a soluble peptide. Alternatively, the short TCAP-1-containing transcript is putatively translated by free ribosomes and localized to the cytoplasm, where it may be released into the extracellular space upon cell stress or rupture. The propeptide may be further cleaved to release the 40-41 amino acid form. Adapted from Chand et al., 2013.

polymerase chain reaction (5’RACE PCR), a 485-base pair transcript that contains TCAP-1 was isolated from whole mouse brain RNA. The separate TCAP-1 mRNA produces a 13-kDa propeptide when hypothetically translated from the first ATG signal. In fact, Chand and colleagues did identify a 13-kDa TCAP-1-immunoreactive band by Western blot, but it is unclear if this band is the product of TCAP-1 mRNA translation or the product of teneurin cleavage at one of the prohormone convertase motifs. Regardless, the putative translation product of the TCAP-1 mRNA does not code for a signal peptide, which could explain the diffuse immunoreactivity of TCAP-1 in E14 cells as the propeptide would be translated by free ribosomes and remain in the cytosol. This contrasts with the teneurin-derived form, which enters the secretory pathway and becomes associated with the extracellular face of the plasma membrane due to the type II orientation of the teneurin protein. Given the distinction in cellular localizations, a separately transcribed form of TCAP-1 may have physiological functions that are distinct from the teneurin-1-derived form.

6 Specifically, because the separately transcribed form is hypothesized to be released upon cell rupture, it may act in a juxtacrine or paracrine manner to alert nearby cells of stress or injury. Although these conjectures may apply to TCAP-1, no equivalent studies have been performed on TCAP-3, and at present, it is unknown if TCAP-3 can be expressed as a separate mRNA and peptide. Interestingly, teneurin-3 is alternatively spliced within its terminal exon leading to deletions upstream of TCAP-3 (Elia and Lovejoy, unpublished observations). One splice variant identified in the mouse neuroblastoma Neuro2a cell line produces a truncated form of TCAP-3 when translated, whereas two splice variants identified in the embryonic mouse brain produce truncations that do not possess TCAP-3. This indicates that cells could differentially express full-length teneurin-3, TCAP-truncated forms of teneurin-3, and TCAP-3 alone, and that there may be a distinct functional importance to each of these isoforms.

1.4. Actions of Teneurin and TCAP in the Central Nervous System

The teneurins have a functional importance in development and maintenance of the nervous system. Specifically, they mediate process outgrowth, cell adhesion and synaptic organization in both vertebrates and invertebrates, especially in the developing brain (review: Mosca, 2015). Some paralogues have specific functions: for example, teneurin-3 is important for neuronal wiring in the developing visual system and hippocampus (Young and Leamey 2009; Berns et al., 2018). Although teneurins possess many functional domains, a comparison of the actions of teneurin and TCAP suggests that TCAP is especially important for teneurin neuromodulatory activity. For example, mouse Nb2a neuroblastoma cells overexpressing teneurin-2 experienced increased process outgrowth similar to that observed in immortalized mouse hypothalamic N38 cells treated with synthetic TCAP-1 (Rubin et al., 1999; Al Chawaf et al., 2007). TCAP has a number of biological actions at the cellular and organismal levels. In the immortalized gonadotropin-releasing hormone-expressing Gn11 cell line, recombinant rainbow trout TCAP-3 increased cyclic AMP (cAMP) at low concentrations and decreased cAMP at high concentrations, indicating an inverse dose-dependent relationship (Qian et al., 2004). Synthetic mouse TCAP-1 showed similar effects in Gn11 cells, but decreased cAMP accumulation in N38 cells (Wang et al., 2005). TCAP-1 treatment in these cells elicited increased process length but fewer processes per cell as well as increased b-actin and b-tubulin protein expression (Al Chawaf

7 et al., 2007). Similar increases in actin polymerization, expression of a-tubulin and b-tubulin, and process formation and length were observed in the immortalized embryonic mouse hippocampal E14 cell line (Chand et al., 2012). In vivo, TCAP-1 treatment increased dendritic spine density in the CA1 and CA3 regions of the rat hippocampus (Tan et al., 2011). Additionally, TCAP-1 protected against alkalosis-associated necrotic cell death in N38 cells by upregulating expression of Cu-Zn superoxide dismutase 1 (SOD1) and catalase enzymes (Trubiani et al., 2007). These results indicate that TCAP-1 regulates neuronal function in the vertebrate brain. However, because most of our studies have focused on the activity of TCAP-1, it remains unclear how other TCAP paralogues regulate this role. At the organismal level, TCAP-1 modulates stress- and anxiety-related behaviours in rodents. TCAP-1-treated rats with high baseline emotionality experienced a decrease in their acoustic startle response whereas rats with low baseline emotionality showed an increase, suggesting that TCAP-1 has a normalizing effect on anxiety (Wang et al., 2005). In elevated plus- maze experiments, TCAP-1-treated rats spent less time in the open arms, whereas rats treated with TCAP-1 and corticotrophin releasing factor (CRF) showed a decrease in stretch-attend posture compared to those treated with CRF alone (Tan et al., 2011; Al Chawaf et al., 2007). Similarly, TCAP-1 treatment significantly reduced CRF-induced expression of c-Fos in rat brain regions associated with anxiety (Tan et al., 2009). These results indicate that TCAP-1 may attenuate stress through antagonism of CRF actions. TCAP-1’s ability to modulate behaviour further delineates its importance CNS function. Recently, our laboratory has established a role for TCAP-1 in energy regulation in the brain. In vivo studies in rats showed that TCAP-1 increased uptake of radioactively-labelled fluorodeoxyglucose (18F-deoxyglucose) in the frontal cortex and subcortical regions of the brain, and this coincided with a decrease in serum glucose levels in both healthy Wistar rats and insulin- insensitive Goto-Kakizaki rats (Hogg et al., 2018). Increased glucose uptake was also observed in vitro in TCAP-1-treated N38 cells, where it was shown to be depolarization-independent, as altering extracellular potassium concentrations did not affect TCAP-1-mediated glucose uptake. This is unlike insulin, which requires depolarization to stimulate glucose uptake and prompted significantly higher glucose uptake under high extracellular potassium conditions (Uemura and Greenlee, 2006; Hogg et al., 2018). However, both TCAP-1 and insulin stimulated translocation of GLUT3 (the primary glucose transporter in the brain) to the plasma membrane, likely through activation of the phosphoinositide-3 kinase-protein kinase B (AKT) pathway and/or the mitogen-

8 activated protein kinase-extracellular signal-regulated kinase 1/2 (MEK-ERK 1/2) pathway (Hogg et al., 2018). These findings suggest that TCAP-1 modulates glucose via a mechanism that is similar to but independent from insulin. TCAP-1 also increased intracellular ATP concentrations and decreased intracellular pyruvate and lactate concentrations in N38 cells, indicating that glucose metabolism through aerobic pathways was also upregulated.

1.5. Actions of Teneurin and TCAP in Reproductive Tissues

Teneurin-1 and TCAP-1 also have a demonstrated regulatory role in the male reproductive system of mice. Immunohistochemical studies demonstrated strong teneurin-1 immunoreactivity within the seminiferous tubules and epididymis, where it co-localized with a-dystroglycan and a- smooth muscle actin (Chand et al., 2014). Contrastingly, TCAP-1 was localized to the spermatagonia and spermatocytes and co-localized with b-dystroglycan. Treatment with TCAP-1 modulated seminiferous tubule and caput-corda epididymis diameter, where a low-dose elicited a decrease in diameter and a high-dose caused an increase. At both high and low doses, TCAP-1 significantly increased testicular diameter and serum testosterone levels after 9 days of treatment. This study by Chand and colleagues (2014) was the first study to investigate the expression and functions of teneurins and TCAPs in adult mammalian reproductive tissues, indicating that TCAPs can have actions on tissues outside of the CNS.

1.6. Actions of Teneurins and TCAP in Skeletal Muscle

Expression of the teneurins and TCAPs was recently established in mammalian skeletal muscle, where teneurin-3 and 4, as well as all TCAP isoforms, were detected in mouse hind-limb muscle extracts by reverse transcription polymerase chain reaction (RT-PCR; D’Aquila et al., manuscript submitted). Teneurin-3 alone and all TCAPs were also identified in the immortalized mouse myoblast C2C12 cell line. Given that TCAP-1 had a compelling regulatory role on glucose uptake and metabolism in the brain, potential effects on glucose metabolism were also investigated in skeletal muscle, where immediate glucose uptake from the blood can be responsible for up to 40% of total oxidative metabolism under exercise conditions (Richter and Hargreaves, 2013). Similar to observations in the brain, TCAP-1 caused a significant increase in uptake of radioactively-labelled glucose in rat hind limb skeletal muscle (D’Aquila et al., manuscript

9 submitted). Tibialis anterior muscle extracts from rats treated with TCAP-1 over a short-term regimen (once per day for five days) demonstrated increased NADH turnover and improved contractile kinetics, including increased peak force of contraction, slowed contractile velocity, increased ½ relaxation rate, and reduced susceptibility to fatigue. Using a long-term regimen (once per week for three months), TCAP-1 lowered serum glucose levels and similarly slowed contractile velocity and increased ½ relaxation rate in the tibialis anterior. In vitro, TCAP-1 increased glucose uptake in C2C12 myotubes and increased translocation of GLUT4 (the primary glucose transporter in skeletal muscle) to the plasma membrane, likely through an insulin- independent mechanism. Intracellular ATP concentration, rate of NADH turnover and expression of the Kreb’s cycle enzyme succinyl dehydrogenase (SDH) were also increased in TCAP-1-treated C2C12 cells. Together, these findings indicate that TCAP-1 regulates glucose metabolism in mammalian skeletal muscle as well, implicating the TCAPs as conserved energetic modulators across numerous organisms and cell types.

1.7. Evidence for Teneurin and TCAP Receptors

Despite the demonstrated bioactivity of the teneurins and TCAPs, their mechanisms of receptor signalling remain poorly understood. Early studies indicated the teneurins are capable of forming homo- or hetero-dimers, and therefore could initiate signalling cascades upon dimerization (Oohashi et al., 1999; Feng et al., 2002; Rubin et al., 2002). Given the distinct expression patterns of teneurin paralogues in the vertebrate brain, this led to the theory that the teneurins dimerized across connected neurons to facilitate synapse formation, but the mechanism for this is unclear (Tucker and Chiquet-Ehrismann, 2006; Baumgartner and Wides, 2019). Proteolytic cleavage of the teneurins may be essential to some mechanisms of signalling. For example, cleavage of intracellular domains and translocation to the nucleus has been demonstrated for chicken (Gallus gallus) teneurins-1 and 2 (Bagutti et al., 2003; Nunes et al., 2005). For TCAP, given its homology to CRF and its ability to modulate cAMP, it was initially hypothesized to act as an antagonist on one or both of the CRF receptors (CRF-Rs) or another member of the Secretin family of G-protein coupled receptors (GPCRs). However, no such interactions have been found (Lovejoy, unpublished observations). Although TCAP-1 co-localizes with b-dystroglycan on the cell surface of E14 cells and may induce cytoskeletal rearrangement through the dystroglycan-associated MEK-ERK1/2 pathway, there is no direct evidence of a

10 ligand-receptor interaction between the two, indicating their co-localization may be part of a larger complex (Chand et al., 2012). Recently, the latrophilin family of Adhesion GPCRs have been identified as receptors for the teneurins and TCAPs. This putative ligand-receptor pair has generated significant interest in recent years for several reasons. First, the teneurins and latrophilins represent the only trans- synaptic protein pair conserved between vertebrates and invertebrates (Woelfle et al., 2015). Second, the latrophilins were previously considered ‘orphan receptors’ and have few known endogenous ligands (Silva and Ushkaryov, 2010). Third, the latrophilins are G-protein coupled receptors (GPCRs), which are clear drug-associated targets in human medicine. Thus, the teneurin/TCAP-latrophilin ligand receptor pair will be described in detail in the following sections.

1.8. Discovery, Structure and Expression of the Latrophilins

The latrophilins are members of the Adhesion family of GPCRs, which are a relatively unexplored class of GPCRs with unusually large N-terminal extracellular domains that possess adhesive properties (review: Bjarnadóttir et al., 2007). Latrophilins were initially discovered as proteins with high affinity for a-latrotoxin, which is the main vertebrate-affecting neurotoxin in black widow spider (Latrodectus tredecimguttatus) venom (Frontali et al., 1976). Before the discovery of its receptors, a-latrotoxin was shown to induce massive neurotransmitter release from nerve terminals through opening of a non-specific cation channel (Wanke et al., 1986). Interestingly, this neurotransmitter release also occurred when calcium was removed from the extracellular media, indicating that a-latrotoxin could also release calcium from intracellular stores. This led to the hypothesis that a-latrotoxin may act through two separate receptor signalling pathways: one that is calcium-dependent and another that is calcium-independent (Rosenthal et al., 1990). In 1990, Petrenko and colleagues isolated several protein subunits from solubilized bovine brain cortices that bound with high-affinity to immobilized a-latrotoxin in the presence of calcium. These calcium-dependent receptors for a-latrotoxin, termed ‘neurexins’, have demonstrated important roles in synapse formation and cognitive function (Ushkaryov et al., 1992; review: Südhof, 2008). Subsequently, Davletov and colleagues (1996) and Krasnoperov and colleagues (1996) both isolated proteins with affinity for a-latrotoxin in the absence of calcium. They named these proteins calcium-independent receptor for latrotoxin (CIRL) and latrophilin

11 (LPHN, now called LPHN-1), respectively. Several other naming conventions have since been adopted, but ‘latrophilin’ remains the most commonly used. Genomic analyses led to the discovery of two additional latrophilin paralogues in vertebrates (LPHN-2 and LPHN-3; Matsushita et al., 1999). Chickens and nematodes (Caenorhabditis elegans) possess 2 latrophilin paralogues, whereas insects possess one (Silva and Ushkaryov, 2010). There is 70-75% amino acid sequence similarity between bovine paralogues and 98% similarity between bovine and rat orthologues of LPHN-1, indicating that each paralogue may have considerably different functions within species and each function may be conserved across species (Matsushita et al., 1999; Silva and Ushkaryov, 2010). In humans, RNA-blotting studies indicated that LPHN-1 and 3 are primarily expressed in the brain, while LPHN-2 is expressed broadly in various tissues (Sugita et al., 1998). Recent findings, however, have challenged the supposition that LPHN-1 and 3 are specific to nervous tissue. In our laboratory, we observed expression of LPHN-1 and 3 in mouse skeletal muscle tissue (D’Aquila et al., manuscript submitted), and Rothë and colleagues (2019) recently reported broad expression of all three LPHN paralogues in mouse and human pancreatic islet cells and in immortalized pancreatic cell lines. The structure of latrophilin is typical of Adhesion GPCRs: they are type I-oriented with 7 looping transmembrane regions (TMRs), a long N-terminal extracellular domain, and a shorter C- terminal cytoplasmic tail (Figure 1.4). The latrophilins are approximately 1400 amino acids long and are constitutively cleaved at a GPCR proteolysis site (GPS) located 19 residues upstream of the first TMR, producing a 120 kDa N-terminal fragment (NTF) and an 85 kDa C-terminal fragment (CTF; Krasnoperov et al., 1997; Silva and Ushkaryov, 2010). The CTF and NTF are both membrane-bound and are isolated together in vitro; however, interaction between the fragments may be ligand-dependent in vivo (Volynski et al., 2004). The GPS is located within a novel domain unique to Adhesion GPCRs and polycystic kidney disease proteins termed the ‘GPCR autoproteolysis-inducing (GAIN) domain’ (Araç et al., 2012). The CTF includes part of the GAIN domain, as well as the 7 TMRs, which share significant homology with the TMRs of Secretin GPCRs, and the intracellular tail, which has few known functions and is highly variable between latrophilin homologues (Krasnoperov et al., 1997; Silva and Ushkaryov, 2010). The NTF possesses several functional domains: most distally, there is a lectin-like domain (LEC) followed by an olfactomedin-like domain (OLF), both of which appear to be involved in endogenous protein-protein interactions (Krasnoperov et al., 1997; Silva and Ushkaryov, 2010; Meza-Aguilar and Boucard, 2014). Between the LEC and OLF domain, there is an alternatively spliced region

NH2 | Lectin-like domain (LEC)

Alternatively spliced region

Olfactomedin-like domain (OLF)

Serine-threonine rich region Hormone-binding domain (HBD)

GAIN domain 7 transmembrane loops

Associated COOH- G-protein

Intracellular tail

Figure 1.4. Schematic of the latrophilin proteins. The latrophilin N-terminal extracellular region possesses a lectin-like domain (LEC), an alternatively spliced region, an olfactomedin-like domain (OLF), a serine-threonine-rich region, a hormone-binding domain (HBD), and GAIN domain. Like all GPCRs, latrophilin possesses 7 transmembrane-spanning domains. The intracellular C-terminal tail associates with G-proteins. Adapted from Meza-Aguilar and Boucard, 2014; Silva and Ushkaryov, 2010.

that regulates ligand interactions (discussed further in 1.10). Next, there is a serine-threonine rich region with unknown function, followed by a hormone-binding domain (HBD) which shares homology with CRF receptors and other Secretin family GPCRs, therefore implying a role in ligand binding (Holz and Habener, 1998; Silva and Ushkaryov, 2010; Meza-Aguilar and Boucard, 2014). Lastly, the NTF possesses part of the GAIN domain after cleavage at the GPS.

13 1.9. Ligands and Signalling of Latrophilin

The latrophilins interact with several ligands via their extracellular adhesive domains. The first discovered ligand was a-latrotoxin, which requires both the HBD and GAIN domain for binding (Krasnoperov et al., 1999; Silva and Ushkaryov, 2010). The neurexins, which act as receptors for a-latrotoxin in the presence of calcium, also act as ligands for LPHN-1; specifically, neurexin interacts with the OLF domain (Boucard et al., 2012). Fibronectin-like domain- containing leucine-rich transmembrane protein 3 (FLRT-3) similarly interacts with LPHN-1 and 3 at their OLF domains (O’Sullivan et al., 2012). Most importantly, the teneurins have been identified as ligands for latrophilin, indicating that TCAP may mediate its cellular actions through latrophilin signalling (discussed further in 1.10). Intracellularly, the latrophilins interact with heterotrimeric G-proteins. Using a-latrotoxin affinity chromatography, LPHN-1 co-purified with Gao and Gaq subunits in vitro (Rahman et al., 1999). Stimulation of LPHN-1-overexpressing cells with a-latrotoxin led to increased cAMP and inositol 1,4,5-triphosphate (IP3) production, and treatment with a phospholipase C (PLC) inhibitor eliminated a-latrotoxin-induced vesicular exocytosis (Lelianova et al., 1997; Davletov et al., 1998). Similarly, simian COS-7 cells overexpressing mouse LPHN-2 and certain splice variants of LPHN-3 experienced increases in IP1, the terminal metabolite of IP3, when stimulated with its self-derived ‘Stachel’ peptide (Rothë et al., 2019). This indicates a functional role for the Gq/11- protein signalling system, which works as follows: first, a ligand binds to a GPCR, causing a conformational change in the intracellular region of the GPCR which facilitates the exchange of

GDP for GTP on the associated Gaq subunit. When bound to GTP, Gaq loses affinity for Gbg, allowing the two complexes to act on downstream effectors independently. Both complexes activate PLC, an enzyme that catalyses the conversion of phosphatidylinositol 4,5-biphosphate

(PIP2) to diacylglycerol (DAG) and IP3, which act as secondary messengers. IP3 binds to the IP3 receptor (IP3R) on the endoplasmic reticulum (ER), which acts as a ligand-gated ion channel to increase cytosolic calcium (review: Foskett et al., 2007). In the case of latrophilin, this is likely the source of intracellular calcium that evokes vesicular exocytosis in response to a-latrotoxin

(Lelianova et al., 1997). Although most evidence suggests that Gq/11 is prevalent in latrophilin signalling, the Gi/o, Gs and G12/13 families should not be ruled out. In over-expression models with

Stachel treatment, functional associations have been shown between human LPHN-1 and Gi or

G12/13, between C. elegans LPHN-1 and Gas, and between a pancreas-specific splice variant of

LPHN-3 and Gai. (Nazarko et al., 2018; Müller et al., 2015; Röthe et al., 2019; discussed in 4.7).

14 1.10. Description of the Teneurin/TCAP-Latrophilin System

The first evidence for a teneurin-latrophilin ligand receptor interaction was exhibited by Silva and colleagues (2011), where teneurin-2 expressed on post-synaptic dendritic branches bound to LPHN-1 expressed on pre-synaptic nerve terminals to form a trans-synaptic complex. Similar trans-cellular interactions were observed between teneurins-2 and 4 and all three LPHNs (Boucard et al., 2014) and between teneurin-1 and LPHN-3 (O’Sullivan et al., 2014). The LEC and OLF domains are responsible for latrophilin’s interactions with teneurin; in fact, alternative splicing between the LEC and OLF domains directly regulates latrophilin’s ability to bind to teneurins but not to its other ligand, FLRT (O’Sullivan et al., 2014; Boucard et al., 2014; O’Sullivan et al., 2012). Recent studies have shown that the toxin-like region of the teneurin-2 (which contains TCAP-2) specifically binds to the LEC domain of LPHN-1 (Li et al., 2018). Given that the teneurins and latrophilins have many other endogenous binding partners in the brain, the teneurin-latrophilin pair is likely part of a larger trans-synaptic complex that includes the neurexins, FLRTs, and dystroglycans (Woelfle et al., 2015; Figure 1.5). The existence of a trans-cellular teneurin-latrophilin interaction suggests that the teneurins could transduce signals trans-synaptically through LPHN-associated G-proteins. A C-terminal fragment of teneurin-2, Lasso, which also contains TCAP-2, triggered an increase in cytosolic calcium in Nb2a cells overexpressing LPHN-1 and in pre-synaptic nerve terminals of hippocampal cells (Silva et al., 2011). The Lasso-mediated calcium surge increased the rate of neurotransmitter exocytosis in a manner similar to a-latrotoxin. This suggests that, like a-latrotoxin, Lasso signals through Gaq (Silva et al., 2011; Vysokov et al., 2018). Functionally, Lasso’s agonist activity has been implicated in neuronal signalling and axonal pathfinding in the developing hippocampus (Silva et al., 2011; Vysokov et al., 2018). These findings in teneurin-2 and Lasso support the hypothesis that latrophilins act as receptors for TCAPs. In addition, our laboratory has shown that human embryonic kidney 293 (HEK 293) cells overexpressing LPHN-1 demonstrate significantly higher co-localization with fluorescently-tagged TCAP-1 compared to wild-type cells, as well as greater cytoskeletal remodelling in response to TCAP-1 (Husić et al., 2019). Given LPHN’s homology to the CRF-Rs, we posit that latrophilin’s HBD is responsible for TCAP binding. In fact, cells transfected with a mutated LPHN-1 construct that does not contain the HBD showed minimal co-localization with TCAP-1, and TCAP-1 co-immunoprecipitated with isolated HBD constructs in vitro (Husić et al.,

Teneurin Post-synaptic cell

Neurexin a-dystroglycan LEC

FLRT OLF

b-dystroglycan HBD

Latrophilin TCAP

Pre-synaptic cell

Figure 1.5. Schematic of teneurin-latrophilin trans-synaptic complex. Teneurin and latrophilin form a trans-synaptic complex, where teneurin is post-synaptic and latrophilin is pre-synaptic. This interaction is stabilized by other proteins: FLRTs, neurexins, and dystroglycans. Adapted from Woelfle et al., 2015.

2019). Like a-latrotoxin and Lasso, TCAP-1 may utilize the Gq/11 signalling pathway (Figure 1.6). This is supported by findings in C2C12 cells that TCAP-1 treatment causes an increase in intracellular calcium, and that this increase is eliminated with the addition of either a PLC inhibitor or an IP3R antagonist (D’Aquila et al., manuscript submitted). However, TCAP-1 has also been shown to modulate intracellular cAMP, so Gai/o and Gas could also be involved (Qian et al., 2004; Wang et al., 2005). Given that there are 4 isoforms of TCAP in vertebrates and 3 of LPHN, and numerous splice variants and some interspecies variation within these genes, there are many possible combinations of TCAP-latrophilin interactions which could account for TCAP’s diverse actions.

TCAP HBD

Latrophilin

G DAG q/11 PLC

Ca2+ PIP2 IP R 2+ ER/ IP3 3 Ca SR Ca2+ GTP GDP Ca2+ Ca2+

GTP Figure 1.6. Proposed mechanism for TCAP-latrophilin signalling through Gq/11-proteins. TCAP binds to the HBD of latrophilin, which prompts a latrophilin conformational change that facilitates the exchange of GDP for GTP on the associated Gaq subunit. Gaq and Gbg subunits activate PLC, which catalyzes the conversion of the membrane phospholipid PIP2 to IP3 and DAG. IP3 triggers opening of the IP3R ligand-gated calcium channel, located on the endoplasmic reticulum (ER), or in muscle cells, the sarcoplasmic reticulum (SR). Calcium in the cytoplasm mediates downstream effects.

1.11. Evolution of the Teneurin/TCAP-Latrophilin System

The teneurins and TCAPs are evolutionarily ancient and conserved across metazoans. The teneurin genes likely evolved through the horizontal gene transfer (HGT) of a polymorphic proteinaceous toxin (PPT) gene from an aquatic prokaryote to a choanoflagellate, which is a unicellular ancestor to metazoans (Zhang et al., 2012; Tucker et al., 2012). Specifically, the cysteine-rich region, NHL repeats, YD repeats and RHS-like domain are all typical of prokaryotic proteins, suggesting that they were inserted downstream of a typical metazoan EGF-containing gene (Tucker et al., 2012). Functionally, the ancestral teneurin gene may have assisted choanoflagellates in food acquisition, given the adhesive properties of the teneurin extracellular domains (Zhang et al., 2012). Moreover, the presence of 4 teneurin genes in vertebrates supports the ‘2R’ hypothesis, which postulates that 2 genome duplication events occurred during the course of vertebrate evolution (Ohno, 1970; Lovejoy et al., 2006)

17 Like much of the C-terminal region of teneurin, TCAP appears to have a prokaryotic origin and possibly represents an inactive form of the toxin payload from a PPT (Chand et al., 2013). TCAP also shares homology with (but evolutionarily predates) the CRFs, calcitonins and Secretin family peptides, and has approximately 20% sequence similarity with a-latrotoxin (Lovejoy et al., 2006; D’Aquila and Lovejoy, unpublished observations). Thus, the ancestral toxin-like gene acquired by HGT could have given rise to all of these peptides. We postulate that in early metazoans, CRF-like and TCAP-like genes worked antagonistically as a rudimentary stress and metabolism control system. This is supported by findings in the chordate species, vase tunicate (Ciona intestinalis), where CRF/diuretic hormone-like peptide and TCAP oppositely modulated feeding behaviours (D’Aquila et al., 2017). The latrophilins are similarly omnipresent in the metazoans, indicating that like the teneurins, they were acquired early in evolutionary history. LPHN-2 is the closest relative to the ancestral latrophilin gene and is the most conserved across species, whereas LPHN-1 and 3 show more interspecies diversity (Gao et al., 2017). Upon discovery, latrophilins were initially classified as Secretin family GPCRs due to their possession of an HBD, but they have since been re-classified as members of the Adhesion family (Lelianova et al., 1997). Evolutionarily, the Adhesion family of GPCRs are among the oldest GPCRs, even appearing in unicellular fungi, though the extracellular adhesive domains associated with latrophilins, such as the LEC, OLF and HBD evolved later (Krishnan et al., 2012; Schöneberg and Prömel, 2019). The Adhesion GPCRs precede the Secretin GPCRs, and comparably, TCAP is a predecessor to the peptide family that binds to Secretin GPCRs (Nordström et al., 2009; Lovejoy et al., 2006). Thus, the ancestral teneurin/TCAP and latrophilins genes could have given rise to many of the peptide signalling systems present in modern organisms. Given the evolutionarily ancient history of the teneurins, TCAPs, and latrophilins, there is a high probability that this protein system is expressed and functional in a number of tissues across the metazoans. Since early organisms possessing these genes did not have complex nervous systems, there were likely some other evolutionary advantages distinct from its modern role in CNS maintenance and development. The adhesive properties of both teneurins and latrophilins could assist in the development of non-neuronal tissues that require precise organization and structure. Furthermore, the conserved energy regulating actions of TCAP would be particularly advantageous in tissues that have high energy demands, such as those involved in locomotion.

18 1.12. Skeletal Muscle Cell Biology & Use of Immortalized Muscle Cell Lines

From the above descriptions, it is evident that skeletal muscle could be an appropriate non- neuronal model for investigating the functionality of the teneurin/TCAP-latrophilin protein complex. Given that synthetic TCAP-1 already has a demonstrated bioactivity in mammalian skeletal muscle cells, where it regulates both intracellular calcium and energy availability in the form of ATP (D’Aquila et al., manuscript submitted), this tissue would be ideal for targeted manipulations of latrophilins as a means to probe the functionality of the ligand-receptor pair. In order to fully understand the potential actions of teneurins, TCAPs and latrophilins in this tissue, it is necessary to first understand some key concepts in skeletal muscle physiology that are relevant to the present study. Skeletal muscle has several unique aspects to its cell biology. First, skeletal muscle cells possess parallel-arranged myofibrils, which are bundles of actin-myosin filaments that mediate muscle contraction. Myosin ‘thick filaments’ are composed of two intertwining heavy chains and four light chains. The heavy chains possess a ‘myosin head,’ each of which can bind to two light chains, one actin protein, and one molecule of ATP. Actin ‘thin filaments’ exist as two helical chains of actin subunits that bind to troponin and tropomyosin at rest. Second, skeletal muscle cells are multi-nucleated and have a large number of mitochondria, due their derivation from the fusion of multiple muscle precursor cells during development. Third, skeletal muscle cells possess a specialized smooth ER called the ‘sarcoplasmic reticulum’ (SR) that stores and releases cytoplasmic calcium in a highly regulated manner to coordinate actin-myosin contraction. Lastly, skeletal muscle is histologically grouped into two types: ‘slow-twitch’ (dark/red appearance) and ‘fast-twitch’ (light appearance). Biochemically, the distinction between slow-twitch and fast- twitch muscle fibres is dependent on expression of various myosin heavy chain (MyHC) gene isoforms (Jean et al., 1975; Schiaffino and Reggiani, 1994). Slow-twitch fibres express the MyHCI isoform, which has a slow ATPase activity. Performance-wise, these fibres are fatigue resistant and depend more on oxidative phosphorylation to generate ATP. Fast-twitch fibres express the MyHCIIb isoform, which has a fast ATPase activity. These fibres are more fatigable and depend more on glycolysis for ATP. There are also intermediate fibres, which express MyHCIIa or IIx isoforms and share characteristics of both slow-twitch and fast-twitch. Whole muscles are classified as either slow-twitch or fast-twitch based on whichever fibre-type is more predominant. Skeletal muscle contraction occurs through the following step-by-step process: acetylcholine (ACh) released from a motoneuron causes depolarization at specialized regions of

19 the skeletal muscle plasma membrane called transverse tubules (T-tubules). This depolarization causes a conformational change in dihydropyridine receptors (DHPRs), allowing them to physically interact with ryanodine receptors (RyRs) on the terminal cisternae of the SR. Opening of RyR calcium channels causes a massive efflux of calcium from the SR to the cytoplasm. Free calcium binds to troponin, which detaches tropomyosin from actin to unmask actin’s myosin- binding sites. This allows myosin to form cross-bridges with actin which are necessary for contraction; however, conformation constrictions only permit myosin to bind actin when also bound to ADP and inorganic phosphate (Pi). Removal of ADP and Pi causes a conformational change in the myosin head that pulls the actin and myosin filaments closer together, leading to shortening (contraction) of the filaments known as the ‘power stroke,’ and subsequent replacement with ATP causes detachment of the myosin head from the actin chain. When ATP is hydrolyzed to ADP and Pi, the myosin head regains its affinity for actin and can perform another power stroke. Thus, from this description, it is evident that calcium and ATP are two essential regulators of skeletal muscle contractile activity Homogenous immortalized cell lines are useful tools in cell biology, as they are easy to grow and maintain and offer better experimental consistency than primary cell cultures. The mouse myoblast C2C12 cell line has been widely used to study skeletal muscle cell biology since its invention by Yaffe and Saxel in 1977. These cells are unique in that they can be used duplicitously as myoblasts or myotubes. In high serum growth conditions, C2C12 cells exist as uninuclear myoblasts that can be grown and passaged as typical immortalized cells. In low serum growth conditions, C2C12 myoblasts fuse and differentiate into myotubules, which are multinucleated and possess myofibrils. Myotubes more closely resemble mature skeletal muscle fibres, making them a relevant model to study energetics and gene expression. C2C12 cells have a tetraploid karyotype, which makes targeted genomic manipulations more difficult; however, this is not a unique problem for the C2C12 cells, as most immortalized cell lines have an unusual ploidy (Chang et al., 2007).

1.13. Thesis Rationale & Research Aims

The TCAP region of the teneurins is highly bioactive as a regulator of stress and metabolism in a number of metazoan species. Although a physical interaction between TCAP-1 and latrophilin-1 has been established using over-expression models, it is not clear if receptor signalling through latrophilins is responsible for TCAP-1’s biological actions in mammalian

20 skeletal muscle. Thus, the primary aim of my project is to elucidate the potential role of latrophilin signalling in TCAP-1’s cellular actions. To do this, I will manipulate latrophilin expression in the mouse myoblast C2C12 cell line using two methods: small interfering RNA (siRNA) and CRISPR/Cas9 gene editing. Then, I will treat cells with TCAP-1 and perform in vitro assays to determine if TCAP-1’s actions are dependent on latrophilin expression. I hypothesize that the latrophilins act as receptors for TCAP-1 in skeletal muscle, and that knocking down or knocking out latrophilins in C2C12 cells will attenuate TCAP-1-mediated changes in intracellular calcium, cellular metabolism and gene expression. I will test my hypothesis within the scope of the following aims:

Aim 1. Determine which TCAP and latrophilin paralogues are ideal for studying the ligand- receptor system in C2C12 cells. § Characterize expression of TCAP-3 in the adult mouse brain by 5’ rapid amplification of cDNA ends polymerase chain reaction (5’RACE PCR) § Characterize expression of the latrophilins in C2C12 cells by RT-PCR.

Aim 2. Develop latrophilin gene manipulation methods using siRNA and CRISPR/Cas9 and examine the effects of TCAP-1 on intracellular calcium dynamics in knockdowns and knockouts. § Establish methods for knocking down LPHN-1 and 3 by siRNA in C2C12 cells and determine knockdown efficacy by quantitative reverse transcription PCR (qRT-PCR) and Western Blot. § Examine intracellular calcium dynamics in TCAP-1-treated LPHN-1 and 3 siRNA knockdown C2C12 cells by Fluo-4 live cell fluorescent imaging. § Generate LPHN-1 knockout C2C12 cell lines by CRISPR/Cas9 gene editing and determine knockout efficacy by qRT-PCR and Western Blot. § Examine intracellular calcium dynamics in TCAP-1-treated LPHN-1 CRISPR/Cas9 knockout C2C12 cells by Fluo-4 live cell fluorescent imaging.

Aim 3. Investigate the effects of TCAP-1 on cellular metabolism in latrophilin-1 CRISPR/Cas9 knockouts. § Examine NADH turnover in TCAP-1-treated LPHN-1 CRISPR/Cas9 knockouts by resazurin- resorufin fluorescence assay.

21 Aim 4. Investigate the effects of TCAP-1 on expression of skeletal muscle-regulating genes in tissue extracts and in cells. § Quantify myosin heavy chain (MyHC) mRNA expression in TCAP-1-treated rat tibialis anterior muscle by qRT-PCR. § Quantify MyHCI and peroxisome proliferator-activated receptor-g coactivator 1a (PGC-1a) expression in TCAP-1-treated wild-type C2C12 cells by qRT-PCR. § Quantify PGC-1a expression in TCAP-1-treated LPHN-1 CRISPR/Cas9 knockout C2C12 cells by qRT-PCR.

Chapter 2

Methods

2.1. Cell Culture

Mouse myoblast C2C12 cells were grown on 10-cm tissue culture dishes in 12 mL Dulbecco’s Modified Eagle Media (DMEM) supplemented with 20% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (hereon referred to as C2C12 growth media). Confluent cells were washed with 7 mL phosphate buffered solution (PBS), passaged with 3 mL 0.25% trypsin in PBS for 2 minutes and inactivated with 4 mL C2C12 growth media. Cells were then centrifuged at 1600 rotations per minute (rpm) for 3 minutes. The supernatant was aspirated and cells were resuspended in 5 mL C2C12 growth media. To differentiate C2C12 myoblasts into myotubes, cells were seeded at onto a 6-well plate and incubated in 2 mL C2C12 growth media/well for 2 days at 37˚C and 5% CO2 until 70-90% confluency. Then, C2C12 growth media was replaced with DMEM supplemented with 10% horse serum, 100 U/mL penicillin and 100 µg/mL streptomycin (hereon referred to as C2C12 differentiation media) and incubated for an additional 6 days at 37˚C and 5% CO2. To store, trypsinized cells were resuspended in 4.5 mL C2C12 growth media and 500 µL dimethyl sulfoxide (DMSO), transferred to 1 mL cryovials, and kept frozen at -80˚C. Heterogenous pools of C2C12 cells transfected with sgRNA oligomers and Cas9 enzyme expression vectors were provided by Dr. Dalia Barsyte-Lovejoy at the Structural Genomics Consortium at MaRS Discovery District (sgRNA sequences detailed in Figure 2.1 and Table 2.1). To isolate clones, cells were diluted to 0.5 cells/100 µL in C2C12 growth media and aliquoted into 96-well plates. Cells were monitored and media was changed every 2-3 days until 70-90% confluency was reached, at which point they were trypsinized and transferred into 12-well plates, and then into 10-cm tissue culture dishes. Following clone isolation, CRISPR/Cas9-transfected C2C12 cells were cultured as described for wild-type C2C12 cells.

Figure 2.1. Schematics of the LPHN-1 and 3 genes depicting sites targeted for CRISPR/Cas9 gene editing. Three targets each against the mRNA coding regions of LPHN-1 and 3 were designed by Dr. Dalia Barsyte-Lovejoy at the Structural Genomics Consortium at MaRS Discovery District (arrows). Target names are based on exon number (for example, ‘E4’ indicates exon 4). For the two LPHN-1 exon 5 targets, the more upstream target is termed ‘E5U’ and the more downstream target is termed ‘E5D.’

Table 2.1. Single guide RNA (sgRNA) sequences for CRISPR/Cas9 Gene Editing

LPHN-1 LPHN-3 E5U 5’-ACATTGTCAAATATGACCTG-3’ E2 5’-GAGCGCTCAACGGCTCATCG-3’ E4 5’-TGGAACCTACAAATACCTGG-3’ E1 5’-CACGATGCTTTTAGCACCTG-3’ E5D 5’-CGTGGACTATGCCTTCAACA-3’ E3 5’-TCGAGAGCGCCAACTACGGG-3’

2.2. RNA Extraction

For RNA extraction from cells, cells were grown to 70-90% confluency in 6-well plates as described in 2.1. Cells were washed twice with 1 mL PBS, then 1mL TriZOL reagent was added to each well, incubated for 30 seconds, and collected in 1.5 mL tubes. 200 µL chloroform was added to each sample, incubated at room temperature for 3 minutes, vortexed for 10 seconds, and centrifuged at 12,000 g for 15 minutes at 4˚C. The supernatant was collected and 500 µL chilled isopropanol was added to each sample. Following 10 minutes incubation at room temperature, samples were centrifuged at 12,000 g for 10 minutes at 4˚C. The supernatant was discarded and pellets were subsequently washed 3 times with 75% ethanol and centrifuged at 7,500 g for 5

24 minutes at 4˚C between washes. Pellets were air-dried for 10 minutes, resuspended in 20 µL DNAse/RNAse-free water and incubated at room temperature for 15 minutes to allow solubilization of the pellet. RNA concentration was measured using a Thermo Scientific NanoDrop 2000 spectrophotometer with DNAse/RNAse-free water as a blank. Samples with a 260/280 ratio outside the range 1.80-2.00 were discarded. RNA samples were stored at -80˚C. Studies involving rat tibialis anterior muscle were done in collaboration with Dr. Andrea D’Aquila, currently at the Department of Pediatrics at the University of Alabama at Birmingham. For a full description of treatment and tissue harvesting methods, see D’Aquila, 2018. Briefly, rats were subcutaneously injected with TCAP-1 (25 nmol/kg) or vehicle (0.9% saline) daily for 5 days in the short-term treatment group, or weekly for 3 months in the long-term treatment group. Tibialis anterior muscle was harvested and stored at -80˚C. For RNA extraction, 1mL of TriZOL reagent was combined with 1.0 mg frozen muscle and mixed thoroughly using a pestle. Chloroform extraction, precipitation and washing were performed as described above for cells. Pellets were resuspended in 50 µL DNAse/RNAse-free water. RNA concentration was measured as described above.

2.3. Reverse Transcription and Polymerase Chain Reaction

Master mix containing 1.5 µg RNA, random primers, deoxy-nucleotide triphosphates (dNTPs) and DNAse/RNAse-free water was incubated at 65˚C for 5 minutes, chilled on ice for 1 minute, then combined with first strand buffer (1x) and dithiothreitol (DTT, 0.01M) and incubated at room temperature for 2 minutes. SuperScript II reverse transcriptase was added and incubated at room temperature for 10 minutes, then 42˚C for 50 minutes, and lastly 70˚C for 15 minutes. The cDNA product was chilled on ice before use in PCR. PCR amplification of the cDNA product was performed using the Q5 Hot Start High- Fidelity Master Mix. For each reaction, 12.5 µL master mix (2X) was combined with 5 µL cDNA, 1.25 µL 10 µM forward and reverse primers (Table 2.2) and 5 µL DNAse/RNAse-free water. Sample were immediately thermal cycled using an Eppendorf Mastercycler Nexus Gradient under the following conditions: initial 30 seconds denaturation at 98˚C; 35 times repeated 10 second denaturation at 98˚C, 30 seconds annealing at 65-67˚C, and 30 seconds extension at 72˚C; and final 2 minutes extension phase at 72˚C. PCR products were visualized on a 1X TBE 3% agarose gel using ethidium bromide and a Bio-Rad Molecular Imager Gel Doc XR+ system.

25 5’RACE PCR was performed using the Invitrogen GeneRacer Kit Version L according to the manufacturer’s directions. 2.5 µg of whole mouse brain RNA was used as a template for mRNA de-capping, RNA ligation and reverse transcription reactions. TCAP-3 primers (Table 2.2) were used in conjunction with the kit-provided 5’-oligo specific ‘GeneRacer’ primer, as well as kit- provided b-actin primers. 5’RACE products were amplified using the Invitrogen Platinum Taq DNA Polymerase High Fidelity for PCR, as recommended by the manufacturer, using the following thermal cycler settings: initial 2 minutes denaturation at 94˚C; 35 times repeated 30 second denaturation at 94˚C, 30 seconds annealing at 63-64˚C, and 30 seconds extension at 68˚C; and final 10 minutes extension phase at 68˚C. PCR products were visualized as described above.

Table 2.2. Primers for Polymerase Chain Reaction

Size Experiment Gene Forward Sequence Reverse Sequence (bp) 5’RACE M. TCAP-3 5’-TCAACAACGCCTTCTACCTGGAGAAC-3’ 5’-TGTCGCAAGAACTGGATGTTGTTGGC-3’ 496 PCR M. TCAP-3 Nested 5’-TTCTACCTGGAGAACCTGCACTTCA-3’ 5’-AGAACTGGATGTTGTTGGCACTGTC-3’ 478 M. LPHN-1 5’-GCTCCCATTTGGGTTGATGC-3’ 5’-GGCACACGAAGATGTAAGGG-3’ 320 M. LPHN-2 5’-TTCAGCAGAGCAGCCTTACC-3’ 5’-TGCTCCATGTAAGGGACACA-3’ 329 RT-PCR M. LPHN-3 5’-TGCTCATAATGTCTCTGCTCCT-3’ 5’-ACGATCTGTTATGAGTAAACGTGAC-3’ 434 M. b-actin 5’-CAGCCATGTACGTAGCCATCCA-3’ 5’-ATGTCACGCACGATTTCCCTCT-3’ 247 M. LPHN-1 5’-CGACTGTGTCCCTTACATCTTCG-3’ 5’- AGATACGGTCACCTGCCTGC-3’ 131 M. LPHN-3 5’-ACTTCAGACCAGCCATTCCTCC-3’ 5’-CGCGGCTGAAAGCATGAACT-3’ 101 M. TBP 5’-CAGACCCCACAACTCTTCCATT-3’ 5’-TCTCAGAAGCTGGTGTGGCA-3’ 124 M. MyHCI 5’-CTCAAGCTGCTCAGCAATCTATTT-3’ 5’-GGAGCGCAAGTTTGTCATAAGT-3’ 153 M. PGC-1a 5’-GACCACAACGATGACCCTC-3’ 5’-ATGTTGCGACTGCGGTT-3’ 108 qPCR R. MyHCI 5’-GAATGGCAAGACGGTGACTGT-3’ 5’-GGAAGCGTACCTCTCCTTGAGA-3’ 142 R. MyHCIIa 5’-ATGACAACTCCTCTCGCTTTG-3’ 5’-TTAAGCTGGAAAGTGACCCGG-3’ 121 R. MyHCIIx 5’-GAACACGAAGCGTGTCATCCA-3’ 5’-AGGTTTCGATATCTGCGGAGG-3’ 245 R. MyHCIIb 5’-CCAATGAGACTAAGACGCCTGG-3’ 5’-GCTATCGATGAATTGTCCCTCG-3’ 191 R. b-actin 5’-AGCCATGTACGTAGCCA-3’ 5’-CTCTCAGCTGTGGTGGTGAA-3’ 228 M. LPHN-1 E5U 5’-CCACCCGGCCCAGTCTTC-3’ 5’-AGACAGAACGCAGCACATAGAG-3’ 598 M. LPHN-1 E4 5’-CAGAGGTGTACTGGTCTAGGGA-3’ 5’-GTTGTAGGAGTGTGGCTCTGAA-3’ 616 T7 Endo- M. LPHN-1 E5D 5’-ATCTATGCAACTGAGGGCAACA-3’ 5’-TTACCTCCTAATCCCCCAGAGG-3’ 470 nuclease Assay M. LPHN-3 E2 5’-AATAAGCAGGGGTCTGATGGTG-3’ 5’-ATGTGGAGAATTGCTTGGGACT-3’ 327 M. LPHN-3 E1 5’-TGCTCATAATGTCTCTGCTCCT-3’ 5’-ACGATCTGTTATGAGTAAACGTGAC-3’ 434 M. LPHN-3 E3 5’-CACTGGAAAAGCCAAAGTGTGT-3’ 5’-TAGGGGGAGTGATCTACAGTGG-3’ 419

Note: M. indicates Mus musclus; R. indicates Rattus norvegicus

26 2.4. Cloning and Sequencing of Polymerase Chain Reaction Products

To clone 5’RACE PCR products, amplicons were extracted from agarose gels and purified using S.N.A.P. Gel Purification columns. Purified PCR products were cloned into pCR 4-TOPO plasmids and transformed into OneShot TOP10 chemically competent Escherichia coli using the Invitrogen TOPO TA Cloning Kit according to manufacturer’s directions. Briefly, the purified PCR product was combined with DNAse/RNAse-free water, salt solution, and pCR 4-TOPO plasmids and incubated at room temperature for 5 minutes. The cloning mixture was added to one vial of TOP10 E. coli and incubated on ice for 20 minutes, then at 42˚C for 30 seconds, and finally on ice for an additional 5 minutes. Super optimal broth with catabolite repression (S.O.C.) media was added and vials were incubated on a 37˚C shaking incubator for 1 hour. E. coli were then plated onto Luria broth (LB) agar supplemented with 50 µg/mL kanamycin and incubated at 37˚C overnight. Successfully transformed clones were transferred into 10 mL LB media supplemented with 50 µg/mL kanamycin and incubated on a 37˚C shaking incubator overnight. Plasmids were extracted from E. coli cultures using the GeneJET Plasmid Miniprep kit according to manufacturer’s directions. Extracted plasmids were sequenced by ACGT Corp.

2.5. Quantitative Reverse Transcription Polymerase Chain Reaction

Reverse transcription was performed as described in 2.3 with the addition of a final incubation with 1 U RNAse H for 20 minutes. cDNA concentrations were determined using a Thermo Scientific NanoDrop 2000 spectrophotometer and diluted to working concentrations in DNAse/RNAse-free water. PCR was performed in 384-well plates containing 5 µL SYBR Select Master Mix (contains SYBR GreenER dye), 0.1 µL 100 µM forward and reverse primer (Table 2.2), 3.3 µL DNAse/RNAse-free water and 1.5 µL cDNA per well. cDNA pools containing 1:4 serial dilutions of cDNA starting at 500 ng/µL were prepared as standards to ensure a linear relationship between cDNA concentration and cycle threshold (Ct) for each primer pair used. 13.3 ng/µL cDNA was used in samples for myosin heavy chain expression studies and 33.3 ng/µL cDNA was used for latrophilin and PGC-1a expression studies. Following master mix and cDNA aliquotation, plates were sealed and centrifuged at 1000 rpm for 2 minutes. Plates were thermal cycled in a Bio-Rad C1000 thermal cycler under the following conditions: initial 2 minutes enzyme activation at 50˚C;

27 initial 2 minutes denaturation at 95˚C; and 39 times repeated 15 seconds denaturation at 95˚C followed by 1 minute annealing and extension at 60˚C. Melting curves were established using a step-wise gradient cycle from 60-90˚C. Fluorescence of the SYBR GreenER dye was detected using the Bio-Rad CFX384 Real-Time System.

2.6. Protein Extraction & Western Blot

Cells were grown and differentiated as described in 2.1. Cells were lysed with 500 µL radioimmunoprecipitation assay (RIPA) buffer supplemented with 1% phenylmethane sulfonyl fluoride (PMSF) protease inhibitor, agitated for 5 minutes on ice, and centrifuged for 20 minutes at 14,000 g. The protein-containing supernatant was collected and protein concentration was determined using the Pierce Bicinchoninic Acid (BCA) Protein Assay according to kit directions: 25 µL albumin standards (concentration range: 25 to 2000 µg/mL) and cell lysate samples were aliquoted in duplicate onto a 96-well plate; then, 200 µL BCA ‘working reagent’ was added to each well, mixed thoroughly for 30 seconds, and incubated for 30 minutes at 37˚C. The plate was cooled to room temperature for 5 minutes, then absorbance was measured at 562 nm using the SpectramaxPlus 384 spectrophotometer. Standard curves were constructed and sample protein concentrations were calculated using SoftMax Pro software. Cell lysates were diluted in RIPA buffer to equal protein concentrations. For each sample, 20 µg protein was combined with Tris sample loading buffer/beta-mercaptoethanol and heated to 95˚C for 5 minutes. Samples were loaded into a combination 5% acrylamide stacking gel/12% acrylamide running gel and resolved by sodium dodecyl polyacrylamide gel electrophoresis (SDS- PAGE) at 100V for 1 hour and 30 minutes. Separated proteins were then electro-transferred onto a nitrocellulose blotting membrane at 100V for 2 hours. Membranes were washed three times in PBS for 5 minutes and blocked in 5% bovine serum albumin-0.3% PBS-Tween 20 (5% BSA- PBST) for 1 hour at room temperature. Membranes were then incubated with primary antibody (1:1000; Table 2.3) in 1% BSA-PBST at 4˚C overnight. Membranes were washed three times in PBST for 10 minutes, incubated with secondary antibody (1:5000; Table 2.3) in 1% skim milk powder-PBST for 1 hour, and washed an additional three times in PBST for 10 minutes at room temperature. To visualize antibody-conjugated protein, membranes were incubated with 1 mL enhanced chemiluminescence (ECL) reagent for 1 minute and imaged using a Chemiluminescent HiRes protocol on the Bio-Rad Molecular Imager Gel Doc XR+ system.

28 Table 2.3. Antibodies for Western Blot

Size Gene Primary Antibody Secondary Antibody (kDa) Goat anti-LPHN-1 polyclonal Donkey anti-goat HRP-linked M. LPHN-1 130 IgG (Santa Cruz) (Santa Cruz)

Goat anti-TBP polyclonal IgG Donkey anti-goat HRP-linked M. TBP 40 (Sigma-Aldrich) (Santa Cruz)

2.7. Small Interfering RNA Transfection

C2C12 cells were grown and differentiated on poly-D-lysine coated 25 mm round No. 1 glass coverslips. Transfection was performed after 4 days of differentiation in C2C12 differentiation media. Dharmacon SmartPOOL siRNA against LPHN-1, LPHN-3, glyceraldehyde-3-phosphate dehydrogenase (GAPD) and a non-targeting control were resuspended in 1X siRNA buffer to 20 µM stocks. Stocks were diluted in serum-free and antibiotic-free DMEM to 250 nM. 7.5 µL Mirus TransIT-X2 transfection reagent was diluted in 200 µL serum-free and antibiotic-free DMEM, combined with 200 µL siRNA solution, and incubated at room temperature for 30 min. The complete transfection mixture was added to C2C12s in differentiation media to a final siRNA concentration of 25 nM. Cells were differentiated in siRNA-containing media for an additional 2 days for a total of 6 days of differentiation before use in experiments.

2.8. T7 Endonuclease Assay

CRISPR/Cas9-transfected C2C12 cells (either heterogenous pools or clones) were trypsinized and pelleted for DNA extraction. Genomic DNA was extracted using Lucigen QuickExtract DNA Extraction Solution according to manufacturer’s directions. The LPHN-1 and 3 genes were amplified by PCR as described in 2.3, reannealed and digested by T7 endonuclease using the EnGen Mutation Detection Kit according to directions in combination with custom primers that flank the appropriate CRISPR/Cas9-targeting region (Table 2.2). Fragments were visualized as described previously.

29 2.9. Live-Cell Calcium Imaging

For visualization of intracellular calcium, day 6-differentiated C2C12 cells on coverslips were incubated in 2 mL C2C12 differentiation media pre-loaded with 10 µM Fluo-4 AM (diluted from a 1 mM stock in DMSO) for 30 minutes at 37˚C and 5% CO2. Cells were then washed in 1 mL Locke’s Buffer (154 mM NaCl, 4 mM NaHCO3, 5 mM KCl, 2.3 mM CaCl2, 1 mM MgCl2, 5 mM glucose and 10 mM HEPES; set to pH=7.5 and osmolarity=305-315 mOsm/L) for 15 minutes at room temperature. Cells were visualized with a Zeiss Axio Observer ZI inverted microscope equipped with a 40x oil immersion objective. For TCAP-1 treatment, cells were bulk perfused with Locke’s buffer for 30 seconds, then 100 nM TCAP-1 in Locke’s buffer for 5 minutes, then Locke’s buffer for another 30 seconds, and lastly 8 mM caffeine for 2 minutes as a positive control. For vehicle treatment, cells were bulk perfused with Locke’s buffer for 6 minutes with appropriate vehicle switches, followed by 8 mM caffeine for 2 minutes. During perfusion, Fluo-4 was excited at 480 nm wavelength for 100 milliseconds every 3 seconds using an X-Cite 120 fluorescence illumination system and emission was captured at 516 nm wavelength using an Orca-EW Hamamatsu camera. Emissions were recorded and analyzed using the Volocity 4.0 imaging software. All experiments took place at room temperature with minimal exposure to ambient light. To analyze, five regions of interest (ROIs) were selected from 1-2 myotubes identified by their large size and multiple nuclei (Supplementary Figure 2.1 in Appendix). Pixel intensity was assessed at each ROI using the Volocity 4.0 imaging software and plotted against time to produce fluorescent traces. The five traces were averaged and normalized to 1.0 arbitrary fluorescent units (AFUs) at time 0; each of these averaged traces represents n=1.

2.10. Resazurin-Resorufin Fluorescence Assay

C2C12 myoblasts were seeded at 10,000 cells/well into a white-walled/clear-bottom 96- well plate and grown for 12-18 hours. Resazurin sodium salt (0.025 mg/well from 10X stock in PBS) was added to each well and mixed for 1 minute. Fluorescence was measured using the Tecan Infinite M1000 Pro plate reader, where each well was excited at 530 nm and emission was captured at 590 nm. Measurements were taken every 10 minutes from 0 to 30 minutes to establish a pre- treatment baseline. Then, vehicle (distilled water), TCAP-1 (100 nM) or Carbonyl cyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 1 µM; all from 10X stocks in PBS) was added to each well and mixed for 1 minute. Measurements were taken every 30 minutes for 150 minutes.

30 To analyze, background fluorescence measurements from wells containing no cells were subtracted from sample fluorescence measurements. The background-subtracted fluorescence at time 0 was subtracted from each group, and data was normalized to mean the vehicle response observed after 150 minutes of treatment. Each well represents n=1.

2.11. Statistical Analysis

Graphical data is presented as mean ± standard error of mean (SEM). All data was analyzed using t-test or one-way analysis of variance (ANOVA), as described in figure captions. Dunnett’s post-hoc test was used to determine significance in one-way ANOVA analyses. An a priori hypothesis states that asterisks represent the following: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. GraphPad Prism 8 software was used for all graphing and statistical testing.

Chapter 3

Results

The following chapter details the results obtained over the course of this research program, with respect to each experiment objective outlined in the Introduction (Chapter 1.13).

Aim 1. Determine which TCAP and latrophilin paralogues are ideal for studying the ligand- receptor system in C2C12 cells.

3.1. Characterization of TCAP-3 mRNA in the Adult Mouse Brain by 5’RACE PCR

Previous Northern blot studies have identified short (<600 bp) mRNAs in the mouse brain that hybridize to TCAP-1 and 3 probes, indicating that these paralogues may be transcribed separately from their full-length teneurins. These TCAP paralogues are particularly interesting for our study of the ligand-receptor relationship because they could be independently synthesized and secreted from one tissue (for example, the brain) and act on receptors in distant tissues (for example, skeletal muscle). A short TCAP-1-containing mRNA was previously isolated and sequenced by 5’RACE PCR (Chand et al., 2013), but TCAP-3 has not been studied beyond the Northern blot. Thus, the existence of short TCAP-3-containing mRNAs was investigated by 5’RACE PCR. In the adult mouse brain, a short TCAP-3-containing mRNA by 5’RACE PCR could not be detected by 5’RACE PCR (Figure 3.1). An amplicon of approximately 800 bp length was detected following gel electrophoresis of our 5’RACE PCR product, but cloning and sequencing of this band indicated it was non-specific. This ~800 bp band was also observed using the kit- provided b-actin primers and when the kit-provided GeneRacer primer was used alone. Additionally, the intensity of this band was reduced when nested PCR was performed. Together, these results suggest this band was a non-specific by-product of the 5’RACE PCR reaction. Some

32 smaller (<800 bp) amplicons were faintly detected in the 5’RACE product, but were eliminated following nested PCR, indicating they too are non-specific. Three splice variants of TCAP-3 were detected in our nested PCR positive control, as evidenced by the presence of triplicate amplicons (Figure 3.1, fourth lane). Given the amplicon sizes, these are likely the same variants previously identified in the embryonic mouse brain and in Neuro2a cells (Elia and Lovejoy, unpublished observations). Because short TCAP-3-containing transcripts could not be detected by 5’RACE PCR, our investigation of the TCAP-latrophilin ligand-receptor system in C2C12 cells was continued using synthetic TCAP-1 treatments alone.

TCAP-3 TCAP-3 Nested β-Actin Additional Controls

GeneRacer F + R GeneRacer F + R GeneRacer F + R GeneRacer No primers + R + R + R alone

1500 1000 700 500 400

300

200

Figure 3.1. 5’-Rapid Amplification of cDNA Ends for Identification of TCAP-3-containing mRNAs. The kit-provided GeneRacer primer was used in combination with a TCAP-3 reverse primer to detect TCAP-3-containing mRNAs by 5’RACE PCR (first lane). TCAP-3 forward and reverse primers were used in conjunction as a positive control (second lane). Nested PCR was performed on the TCAP-3 5’RACE PCR product (third lane) and TCAP-3 positive control (fourth lane). 5’RACE PCR was performed for b-actin with a corresponding positive control (fifth and sixth lanes). Additional controls where PCR was performed with the GeneRacer primer alone and with no primers was performed (seventh and eight lanes). Expected band sizes: TCAP-3 F+R=496 bp; TCAP-3 Nested F+R=478 bp; b-actin GeneRacer+R=~900 bp; b-actin F+R=748 bp; additional controls=no band expected.

33 3.2. Expression of Latrophilins in C2C12 cells

To determine which latrophilin paralogues could act as receptors for TCAP-1, latrophilin expression was investigated in the C2C12 cell line. In both the undifferentiated myoblast form and day 6-differentiated myotube form, C2C12 cells strongly expressed LPHN-1 and 3, but showed minimal expression of LPHN-2, as determined by RT-PCR (Figure 3.2). Thus, the LPHN-1 and 3 paralogues were selected for targeting by siRNA and CRISPR/Cas9 methods.

Undifferentiated Myoblasts Day 6-Differentiated Myotubes

LPHN-1 LPHN-2 LPHN-3 b-Actin - LPHN-1 LPHN-2 LPHN-3 b-Actin -

700 600 500 400

300

200

100

Figure 3.2. Expression of latrophilins in C2C12 cells. RT-PCR analysis of the three mouse latrophilin paralogues was performed using cDNA from undifferentiated C2C12 myoblasts and day 6-differentiated C2C12 myotubes. Expression of b-actin was used as a positive control, and PCR containing no primers was used as a negative control. Expected band sizes: LPHN-1=320 bp; LPHN-2=329 bp; LPHN-3=434 bp; b-actin=247 bp; negative control=no band expected.

34 Aim 2. Develop latrophilin gene manipulation methods using siRNA and CRISPR/Cas9 and examine the effects of TCAP-1 on intracellular calcium dynamics in knockdowns and knockouts.

3.3. Establishing Methods of Latrophilin-1 and 3 siRNA Knockdown

Commercially available siRNA oligomers were used in combination with TransIT-X2 transfection reagent to establish a knockdown regimen for LPHN-1 and 3. This regimen significantly reduced (p<0.01; t-test) LPHN-1 and 3 mRNA expression by 75% and 60% compared to wild-type cells, as quantified by qRT-PCR (Figure 3.3). Transfection with a non-targeting siRNA control did not significantly alter either paralogue’s expression. Furthermore, LPHN-1- targeting siRNA did not affect LPHN-3 expression, and vice versa. Thus, this knockdown regimen was deemed successful for use in assays with TCAP-1 treatment.

A B

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5 ** (Fold Change from WT) (Fold Change from WT) **

Relative LPHN-3 mRNA Expression Relative LPHN-1 mRNA Expression 0.0 0.0

NT NT

WT C2C12LPHN-1 KDLPHN-3 KD WT C2C12LPHN-1 KDLPHN-3 KD

Figure 3.3. Expression of latrophilin-1 and 3 in siRNA knockdown C2C12 cells. (A) Cells transfected with LPHN-1-targetting siRNA (LPHN-1 KD) showed a significant reduction in LPHN-1 mRNA expression compared to wild-type (WT), as determined by qRT-PCR. (B) Cells transfected with LPHN-3-targetting siRNA (LPHN-3 KD) showed a significant reduction in LPHN-3 mRNA expression compared to WT. Transfection with non-targeting (NT) siRNA did not affect expression. All data were normalized to the housekeeper gene, TATA box binding protein (TBP). Mean ± SEM; n=7-9; **p<0.01; unpaired one-tailed t-test; comparison to WT.

35 3.4. Intracellular Calcium Dynamics in TCAP-1-treated Latrophilin-1 and 3 siRNA Knockdowns

Previous studies in our laboratory using the Fluo-4 calcium indicator dye have determined that TCAP-1 causes an immediate surge in intracellular calcium in wild-type C2C12 cells (D’Aquila et al., manuscript submitted). To determine if this intracellular calcium surge requires expression of latrophilins, Fluo-4 experiments with TCAP-1 were performed in LPHN-1 and 3 siRNA knockdown C2C12 cells, as well as in the non-targeting control cells. LPHN-1 and 3 siRNA knockdowns did not experience an intracellular calcium surge in response to TCAP-1, whereas non-targeting control cells experienced a significant increase (p<0.0001; one-way ANOVA; F=53.25; df=18) comparable to that observed in wild-type cells (Figure 3.4).

3.5. Development of Latrophilin-1 CRISPR/Cas9 Knockouts

CRISPR/Cas9 gene editing was employed to generate stable permanent knockouts of LPHN-1 and 3. Heterogenous C2C12 cell pools transfected with LPHN-1 or 3-targeting sgRNA sequences and Cas9 plasmid constructs were provided by Dr. Dalia Barsyte-Lovejoy (Structural Genomics Consortium, MaRS Discovery District). T7 endonuclease assay revealed that transfection with the E5U and E5D sgRNA sequences against LPHN-1 as well as the E2 and E1 sequences against LPHN-3 yielded mutants, as indicated by the presence of multiple bands upon gel electrophoresis of the assay product (Figure 3.5). This indicates that some full and/or partial knockouts are present in these heterogenous cell pools. Isolation of single cells and growth of homogenous clonal lines was performed on the 4 successful heterogenous pools, and selected clones showing growth and morphology indistinguishable from wild-type were tested for the presence of mutations using T7 endonuclease assay (Supplementary Figure 3.1 in Appendix). Any clone that showed reduced intensity of a wild-type amplicon or multiple amplicons was considered a ‘candidate knockout.’ Several candidate knockouts were identified for LPHN-1, but very few clones grew from the LPHN-3 pools. Thus, CRISPR/Cas9 knockout development was continued for LPHN-1 only.

A 1.3 Treatment (Vehicle or TCAP-1)

F ΔF 1.2 0

1.1

1.0

Normalized Fluo-4 (AFU)

0.9 0 20 40 60 80 100 120 Time (seconds) B

15 **** Vehicle Vehicle (Locke’s Buffer) **** 10 TCAP-1 (100 nM) (%)

0 LPHN-1 KD + TCAP-1

F/F 5 LPHN-3 KD + TCAP1 Δ NT + TCAP-1 0

-5

Figure 3.4. Fluo-4 intracellular calcium fluorescence in TCAP-1-treated latrophilin-1 and 3 siRNA knockdown C2C12 cells. (A) Bulk perfusion of either vehicle (Locke’s buffer; black) or TCAP-1 (100 nM) was performed on wild-type cells (red), non-targeting siRNA-transfected cells (NT; green), LPHN-1 knockdowns (LPHN-1 KD; orange), and LPHN-3 knockdowns (LPHN-3 KD; purple). Data were normalized to pre-treatment baselines (0-30 seconds). (B) Wild-type and NT cells experienced a significant increase in normalized Fluo-4 fluorescence upon treatment with TCAP-1, whereas LPHN-1 KD and LPHN-3 KD cells do not. DF/F0 (%) represents the average normalized Fluo-4 fluorescence from 100-130s (DF) compared to the pre-treatment baseline (0-30 seconds, F0). Mean ± SEM; n=4-5; ****p<0.0001; one-way ANOVA with Dunnett’s post-hoc test; comparison to vehicle.

LPHN-1 LPHN-3 Controls E5U E4 E5D CTRL WT E2 E1 E3 CTRL WT + -

700 600 500 400 300 200

100

Figure 3.5. T7 endonuclease assay of heterogenous latrophilin-1 and 3 CRISPR/Cas9- transfected C2C12 cell pools. T7 endonuclease assay produced multiple bands (including the expected wild-type bands) in cells transfected with LPHN-1 E5U and E5D targets, as well as LPHN-3 E2 and E1 targets. This suggests there are full and/or partial knockouts present in these heterogenous pools. LPHN-1 E4 produced only the expected wild-type band, indicating an absence of mutations in these cells. LPHN-3 E3 did not produce a band. A kit-provided positive control and PCR containing no primers were also assayed. Expected band sizes: LPHN-1 E5U=598 bp; E4=616 bp; E5D=470 bp; LPHN-1 CTRL and WT=598 bp; LPHN-3 E2=327 bp; E1=434 bp; E3=419 bp; LPHN-3 CTRL and WT=327 bp; kit-provided positive control=~600, 400 and 200 bp; negative control=no band expected.

After screening LPHN-1 mRNA expression in candidate knockouts by qRT-PCR, two clones (E5U7 and E5D3) were selected which showed significant reductions (p<0.01; t-test) in LPHN-1 mRNA expression, as well as a comparable reduction (p<0.05; t-test) in protein expression by Western blot. These clones were selected for use in further assays with TCAP-1 treatment.

A B 2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5 (Fold Change from WT) (Fold Change from WT) * ** * ** Relative LPHN-1 mRNA Expression 0.0 Relative LPHN-1 Protein Expression 0.0

CTRL CTRL WT C2C12 WT C2C12 LPHN1-E5U7LPHN1-E5D3 LPHN1-E5U7LPHN1-E5D3 C LPHN1-E5U7 LPHN1-E5D3 CTRL WT C2C12

135 kDa

35 kDa

Figure 3.6. Characterization of successful latrophilin-1 CRISPR/Cas9 knockout C2C12 clones. (A) Two clones (E5U7 and E5D3) showed significantly reduced LPHN-1 mRNA expression compared to WT, as determined by qRT-PCR. CTRL cells did not experience significant expression changes. (B, C) These clones also showed significantly reduced LPHN-1 protein expression, as determined by Western blot. Expected band sizes: LPHN-1, 130 kDa; TBP, 40 kDa. All data were normalized to the housekeeper gene, TBP. Mean ± SEM; n=3-4; *p<0.05; **p<0.01; unpaired one-tailed t-test; comparison to WT.

39 3.6. Intracellular Calcium Dynamics in TCAP-1-treated Latrophilin-1 CRISPR/Cas9 Knockouts

To determine if the LPHN-1 CRISPR/Cas9 knockouts demonstrate reduced sensitivity to TCAP-1, Fluo-4 experiments were performed as described for siRNA knockdowns. LPHN-1 E5U7 and E5D3 knockouts did not experience an intracellular calcium surge in response to TCAP- 1, whereas empty vector-transfected control cells experienced a significant (p<0.0001; one-way ANOVA; F=18.14; df=18) increase comparable to that observed in wild-type cells (Figure 3.7).

Aim 3. Investigate the effects of TCAP-1 on cellular metabolism in latrophilin-1 CRISPR/Cas9 knockouts.

3.7. Resorufin Fluorescence in TCAP-1-treated Latrophilin-1 CRISPR/Cas9 Knockouts

The resazurin-resorufin fluorescence assay is an established method for measuring NADH turnover as an indicator of metabolic rate for a pool of cells. As NADH is produced in a cell, resazurin is irreversibly reduced to the resorufin, which can be fluorescently measured. Using this assay, TCAP-1 has been shown to increase resorufin fluorescence compared to vehicle in both larval and adult zebrafish (Reid et al., manuscript in preparation) and wild-type C2C12 cells (D’Aquila et al., manuscript submitted). Therefore, to determine if the TCAP-1-mediated increase in NADH turnover in C2C12 cells is dependent on latrophilin expression, resorufin fluorescence was measured in TCAP-1-treated LPHN-1 CRISPR/Cas9 knockouts. From 30 to 150 minutes, TCAP-1-treated wild-type C2C12 cells experienced a small but statistically significant increase (p<0.01 or p<0.05; t-test) in resorufin fluorescence compared to vehicle (Figure 3.8). LPHN-1 E5U7 and E5D3 knockouts did not show an increase in resorufin fluorescence with TCAP-1 treatment, whereas empty vector-transfected control cells experienced a significant increase (p<0.05; t-test) after 120 minutes only, though there was a trend towards significance at other time points. FCCP treatment caused a significant increase (p<0.001 or p<0.0001; t-test) in resorufin fluorescence across all cell types.

A 1.3 Treatment (Vehicle or TCAP-1)

F ΔF 1.2 0

1.1

1.0

Normalized Fluo-4 (AFU)

0.9 0 20 40 60 80 100 120 Time (seconds)

B 20 **** 15 **** Vehicle (Locke’s Buffer)

TCAP-1 (100 nM) 10 (%)

0 LPHN-1 E5U7 + TCAP-1

F/F 5

Δ LPHN-1 E5D3 + TCAP-1

0 CTRL + TCAP-1

-5

Figure 3.7. Fluo-4 intracellular calcium fluorescence in TCAP-1-treated latrophilin-1 CRISPR/Cas9 knockout C2C12 cells. (A) Bulk perfusion of either vehicle (Locke’s buffer; black) or TCAP-1 (100 nM) was performed on wild-type cells (red), empty vector control cells (CTRL; blue), LPHN-1 E5U7 knockouts (white), and LPHN-1 E5D3 knockouts (grey). Data were normalized to pre-treatment baselines (0-30 seconds). (B) Wild-type and CTRL cells experienced a significant increase in normalized Fluo-4 fluorescence upon treatment with TCAP-1, whereas LPHN-1 CRISPR/Cas9 knockouts did not. DF/F0 (%) represents the average normalized Fluo-4 fluorescence from 100-130s (DF) compared to the pre-treatment baseline (0-30 seconds, F0). Mean ± SEM; n=4-5; ****p<0.0001; one-way ANOVA with Dunnett’s post-hoc test; comparison to vehicle.

A B **** WT C2C12 **** LPHN-1 E5U7 120 120 **** **** * 100 ** 100 **** **** ** 80 **** 80 ** ****

60 60 **** * ! 40 40 *** Vehicle (ddH2O) Resorufin 590 nm fluorescence Resorufin 590 nm fluorescence (% maximum vehicle response) (% maximum vehicle response) Δ 20 TCAP-1 (100nM) Δ 20 FCCP (1 µM) 0 0 0 30 60 90 120 150 0 30 60 90 120 150

Time (min) Time (min)

C D LPHN-1 E5D3 CTRL **** 120 **** 120 *** **** p=0.09 100 100 *** * **** p=0.07 80 80 *** **** p=0.06 ! 60 60 ** p=0.06 ! 40 **** 40

Resorufin 590 nm fluorescence Resorufin 590 nm fluorescence (% maximum vehicle response) (% maximum vehicle response) Δ Δ 20 20

0 0 0 30 60 90 120 150 0 30 60 90 120 150 Time (min) Time (min)

Figure 3.8. Resorufin fluorescence in TCAP-1-treated latrophilin-1 CRISPR/Cas9 knockout C2C12 cells. Resorufin fluorescence was measured in wild-type cells (WT; A), LPHN-1 E5U7 knockouts (B), LPHN-1 E5D3 knockouts (C), and empty vector controls cells (CTRL; D). Cells were treated for 150 minutes with vehicle (ddH2O; black), TCAP-1 (100 nM; white), or FCCP (1 µM; blue) as a positive control. Data were presented as a percentage of the mean change in fluorescence following 150 minutes of vehicle treatment. Mean ± SEM; n=10-12; *p<0.05, **

42 FCCP is an ionophore that uncouples oxidative phosphorylation by disrupting the proton gradient that drives ATP synthesis, therefore causing maximal oxidation of NADH as cells attempt to re-establish this proton gradient (Blacker and Duchen, 2016). In order to compare data across cell types, the TCAP-1 response was calculated as a percentage of each type’s maximum FCCP response (FFCCP; Figure 3.9). After 120 minutes, TCAP-1 elicited a significant increase (p<0.05; one-way ANOVA; F=5.346; df=49) in resorufin fluorescence to approximately 30% of the FCCP response in wild-type and empty vector-transfected control cells, but did not have a significant effect on either LPHN-1 E5U7 or E5D3 knockouts.

100 * * Vehicle (ddH2O) 50 TCAP-1 (100 nM) (%) LPHN-1 E5U7 + TCAP-1

FCCP LPHN-1 E5D3 + TCAP-1

F/F CTRL + TCAP-1

Δ 0

-50

Figure 3.9. Normalized resorufin fluorescence in latrophilin-1 CRISPR/Cas9 knockout C2C12 cells following 120 minutes of TCAP-1 treatment. Wild-type cells (red) and empty vector control cells (CTRL; blue) experienced a significant increase in normalized resorufin fluorescence following 120 minutes of TCAP-1 treatment, whereas LPHN-1 E5U7 knockouts and LPHN-1 E5D3 knockouts did not. DF/FFCCP (%) represents the change in resorufin fluorescence following 120 minutes of vehicle or TCAP-1 treatment (DF) as a percentage of the mean change in fluorescence following 120 minutes of FCCP treatment (FFCCP). Mean ± SEM; n=10-12; *p<0.05; one-way ANOVA with Dunnett’s post-hoc test; comparison to vehicle.

Aim 4. Investigate the effects of TCAP-1 on expression of skeletal muscle-regulating genes in tissue extracts and in cells.

3.8. Myosin Heavy Chain Expression in TCAP-1-treated Tibialis Anterior Muscle Tissue

Previous in vivo studies showed that TCAP-1 treatment substantially altered contractile kinetics in rat tibialis anterior muscle, without inducing any changes to muscle mass, indicating possible changes to the muscle’s underlying histological architecture (D’Aquila et al., manuscript submitted). Specifically, both short-term and long-term TCAP-1-treated muscle showed slowed contractile velocity and increased ½ relaxation, which are indicative of a more slow-twitch phenotype. Therefore, in collaboration with Dr. Andrea D’Aquila (Department of Pediatrics, University of Alabama at Birmingham), expression of the myosin heavy chain (MyHC) genes, which primarily dictate muscle fibre-type, was examined in tibialis anterior muscle extracts from TCAP-1-treated rats. In short-term treated rats, TCAP-1 significantly increased (p<0.01; t-test) MyHCI mRNA expression by 180% compared to vehicle, as quantified by qRT-PCR (Figure 3.10). The increase in MyHCI corresponded with a reduction in MyHCIIa, MyHCIIx and MyHCIIb mRNA expression, as expected. Similarly, in long-term treated rats, TCAP-1 significantly increased (p<0.01; t-test) MyHCI mRNA expression by 250% with a corresponding reduction in MyHCIIb.

A B

Vehicle ** Vehicle TCAP-1 TCAP-1 4 4 **

3 3

2 2

Relative mRNA Expression Relative mRNA Expression (Fold Change from Vehicle) 1 * (Fold Change from Vehicle) 1 * ** **

0 0

MyHCI MyHCI MyHCIIa MyHCIIx MyHCIIb MyHCIIa MyHCIIx MyHCIIb

Figure 3.10. Expression of myosin heavy chain (MyHC) genes in tibialis anterior muscle of TCAP-1-treated rats. (A) Using a short-term treatment regimen of daily subcutaneous (SC) injection for 5 days, MyHCI mRNA expression significantly increased in TCAP-1 (25 nmol/kg) treated rats compared to vehicle (0.9% saline), as quantified by qRT-PCR. MyHCIIa, IIx and IIb decreased in the TCAP-1 group compared to vehicle. (B) Using a long-term regimen of weekly SC injection for 3 months, MyHCI mRNA expression significantly increased in TCAP-1-treated rats, whereas MyHCIIb decreased. All data were normalized to the housekeeper gene, b-actin. Mean ± SEM; n=7-9; *p<0.05; **p<0.01; unpaired two-tailed t-test per gene; comparison to vehicle.

3.9. Myosin Heavy Chain I and Peroxisome Proliferator-activated Receptor-g Coactivator 1a Expression in TCAP-1-treated C2C12 cells

Given that TCAP-1 may modulate other hormones and signalling factors in vivo, it is unclear if the purported fibre-type switch observed in the tibialis anterior muscle is a direct result of gene regulation by TCAP-1. In order to eliminate these confounding factors, MyHCI expression

45 was examined in TCAP-1-treated C2C12 cells. Using a daily treatment regimen from day 3 to day 6 of differentiation, no changes in MyHCI mRNA expression were detected in TCAP-1-treated C2C12 cells compared to vehicle (Figure 3.11). However, TCAP-1-treated cells did experience a significant (p<0.05; t-test) 50% increase in mRNA expression of peroxisome proliferator-activated receptor-g coactivator 1a (PGC-1a), a related transcription factor known to directly upregulate MyHCI expression (review: Liang & Ward, 2006).

2.0 Vehicle TCAP-1

* 1.5

1.0

0.5 Relative mRNA Expression (Fold Change from Vehicle)

0.0 α MyHCI PGC-1

Figure 3.11. Expression of myosin heavy chain I (MyHCI) and peroxisome proliferator- activated receptor-g coactivator 1a (PGC-1a) in TCAP-1-treated wild-type C2C12 cells. Cells were treated with either vehicle (distilled water) or TCAP-1 (100 nM) daily from day 3 to day 6 of differentiation. MyHCI expression did not change in the TCAP-1-treated cells compared to vehicle, as quantified by qRT-PCR. PGC-1a mRNA expression significantly increased in TCAP- 1-treated cells compared to vehicle. All data were normalized to the housekeeper gene, TBP. Mean ± SEM; n=6; *p<0.05; unpaired two-tailed t-test; comparison to vehicle.

46 3.10. Peroxisome Proliferator-activated Receptor-g Coactivator 1a Expression in TCAP-1- treated Latrophilin-1 CRISPR/Cas9 Knockouts

To determine if the TCAP-1-mediated increase in PGC-1a in C2C12 cells is dependent on expression of LPHN-1, PGC-1a mRNA levels were measured in TCAP-1-treated LPHN-1 CRISPR/Cas9 knockouts. Using the same daily treatment regimen from day 3 to day 6, no significant changes in PGC-1a expression were observed in LPHN-1 E5U7 or LPHN-1 E5D3 knockout cells, whereas empty vector-transfected control cells experienced a significant increase (p<0.05; t-test) similar to that observed in wild-type cells (Figure 3.12).

2.0 Vehicle TCAP-1 * *

1.5

mRNA Expression 1.0 α

0.5 (Fold Change from Vehicle)

Relative PGC-1

0.0

CTRL WT C2C12 LPHN-1 E5U7 LPHN-1 E5D3

Figure 3.12. Expression of PGC-1a in TCAP-1-treated LPHN-1 CRISRP/Cas9 knockout C2C12 cells. Cells were treated with either vehicle (distilled water) or TCAP-1 (100 nM) daily from day 3 to day 6 of differentiation. PGC-1a mRNA expression significantly increased in TCAP-1-treated wild-type (WT) and empty vector control (CTRL) cells compared to vehicle, as determined by qRT-PCR. PGC-1a mRNA expression did not change with TCAP-1 treatment in LPHN-1 E5U7 and E5D3 knockouts. All data were normalized to the housekeeper gene, TBP. Mean ± SEM; n=6; *p<0.05; unpaired two-tailed t-test; comparison to vehicle.

Chapter 4

Discussion

The following chapter discusses the importance and implications of the results presented in this research program. To briefly summarize the results: I established, for the first time, that latrophilins acts as receptors for TCAP-1 in the mouse skeletal muscle C2C12 cell line. TCAP-1 was selected as a model ligand for studying the TCAP-latrophilin ligand-receptor pair, as TCAP- 3 did not appear to be separately transcribed as a propeptide. LPHN-1 and 3 were selected for receptor knockdown and knockout based on their high expression in C2C12 cells. siRNA knockdown was successful for LPHN-1 and 3, and CRISPR/Cas9-based knockout was successful for LPHN-1. Both knockdown and knockout C2C12 cells did not demonstrate an intracellular calcium surge in response to TCAP-1. Following TCAP-1 treatment, LPHN-1 knockouts did not exhibit increased NADH turnover nor increased PGC-1a mRNA expression, which is a gene associated with the demonstrated in vivo fibre-type switching. These results establish that the TCAP-latrophilin ligand-receptor pair is biologically functional in this skeletal muscle cell line, and that this protein system has a regulatory role in the metabolic function and histological architecture of skeletal muscle.

4.1. Independent Transcription of TCAP

It is intriguing to consider the possibility that some TCAP paralogues are transcribed and translated independently from their full length teneurin genes, as this would suggest that TCAPs could be synthesized and released in a regulated manner, as well as possess functional activities that are distinct from the teneurins. Northern blot studies have identified short (<600 bp) mRNAs in the mouse brain that hybridize to TCAP-1 and 3 probes, indicating that these paralogues can be transcribed separately from their full length teneurins. A short TCAP-1-containing mRNA was isolated and sequenced by 5’RACE PCR, and its putative translation product would give rise to a 13 kDa TCAP-1 propeptide that may be further cleaved into the 41-amino acid form (Chand et al.,

48 2013). Given the vast evidence of synthetic mouse TCAP-1’s bioactivity in a number of mammalian tissues, this separately transcribed form of TCAP-1 likely has some functional importance when produced endogenously, therefore making TCAP-1 a relevant paralogue for studying TCAP-latrophilin ligand-receptor relationships. Synthetic rainbow trout TCAP-3 has similar biological actions to mouse TCAP-1 in neuronal cell lines (Qian et al., 2004; Wang et al., 2005). Moreover, given the evidence for a short TCAP-3 containing mRNA by Northern blot, TCAP-3 may be endogenously expressed as a separate transcript similar to TCAP-1. In this present study, similar methods to those described by Chand and colleagues (2013) were utilized in an attempt to identify short TCAP-3-containing mRNAs. This ultimately proved to be unsuccessful, as no short TCAP-3-containing mRNAs were detected by 5’RACE PCR. There are several possible explanations for the difficulty in isolating a short TCAP-3-containing mRNA: first, given the prevalence of non-specific amplicons in the 5’RACE PCR products, it is possible that the 5’RACE PCR kit used was ineffective or not compatible with the template or reverse primers used. Second, the actual short TCAP-3-containing mRNA could be too large to be amplified by this method; although 5’RACE PCR is typically effective for mRNAs up to 1000 bp, it is possible that the initial Northern blot underestimated the length of the short TCAP-3-containing mRNA. Lastly, it could be that the original TCAP-3- containing mRNA observed by Northern blot was a nonspecific artifact, and that no short TCAP- 3-containing mRNA exists in the mouse brain. If it is true that TCAP-3 is not separately transcribed, then this would not be an ideal paralogue for studying the endogenous TCAP- latrophilin ligand-receptor relationship, as there would be no ability for TCAP-3 to be synthesized and released in a regulated manner independent from the full-length teneurin-3. Interestingly, teneurin-3 is alternatively spliced within its terminal exon leading to deletions upstream of TCAP-3 (Elia and Lovejoy, unpublished observations). One splice variant identified in the mouse neuroblastoma Neuro2a cell line produced a truncated form of TCAP-3 when translated, whereas two splice variants identified in the embryonic mouse brain produced truncations that do not possess TCAP-3. We detected these same variants in the adult mouse brain, as indicated by the presence of triplicate amplicons in the nested TCAP-3 PCR product. The existence of these splice variants supports our hypothesis that there is a functional distinction between the activities of teneurin-3 and TCAP-3 in the brain, as cells would be able to express forms of teneurin-3 that do not contain TCAP-3. If TCAP-3 is endogenously cleaved and processed from the full-length teneurin-3, then perhaps these truncated splice variants evolved as a method

49 for cells to regulate their production of soluble TCAP-3 without altering their expression of the rest of the teneurin-3 protein. That is, cells could upregulate soluble TCAP-3 by favouring expression of the full length teneurin-3 or downregulate soluble TCAP-3 by favouring expression of the truncated version; in either case, expression of all other teneurin-3 domains would remain unaffected. Future studies should investigate the transcriptional regulation and anatomical distribution of these splice variants, as this would offer insight into what conditions or signals regulate their expression.

4.2. Small Interfering RNA Knockdown of Latrophilins

In the present study, LPHN-1 and 3 were targeted for siRNA knockdown in C2C12 myotubes. siRNA transiently silences gene expression using the existing biological machinery associated with RNA interference (RNAi, also referred to as post-transcriptional gene silencing, PTGS) in eukaryotic cells. Synthetic siRNAs are 20-25 bp double-stranded oligomers where one strand is complementary to the mRNA sequence of the gene targeted for knockdown—in this case, LPHN-1 and 3. Upon delivery to cells by transfection, these oligomers associate with proteins to form an RNA-Induced Silencing Complex (RISC), which unwinds the double-stranded helix and integrates a single strand into the active complex (review: Dana et al., 2017). Single stranded siRNA then complementarily binds to mRNAs targeted for silencing, forming a double-stranded siRNA-mRNA hybrid. Then, the argonaute family enzyme Ago2 within the active RISC cleaves the mRNA rendering it untranslatable, and finally the siRNA-RISC is released and reused for further rounds of gene silencing. The main advantage of siRNA for targeted gene silencing is the availability of commercially validated oligomer products designed to target virtually every gene in the human, rat and mouse genomes. The main disadvantage of siRNA is the transient nature of the knockdown effect. Immortalized cells transfected with siRNA typically experience a knockdown effect for 24-72 hours, which means these knockdowns are not suitable for assays that involve long periods of treatment or data collection. Here, an siRNA transfection protocol was established in C2C12 myotubes that elicited a 75% reduction in mRNA expression for LPHN-1 and a 60% reduction for LPHN-3, 48 hours post- transfection. The expected range for mRNA reduction indicated by the product manufacturer is 70-90%, so these results are within range for LPHN-1 and slightly below range for LPHN-3. A similar protocol to what is described here was recently reported for siRNA knockdown of LPHN-

50 1 and 3 in the immortalized mouse pancreatic b-islet MIN6 cell line (Rothë et al., 2019). C2C12 knockdowns were used in Fluo-4 calcium fluorescence assays, which typically last approximately one hour and are therefore compatible with the knockdown timeline for siRNA. In both LPHN-1 and 3 siRNA knockdown C2C12 myotubes, the TCAP-1-mediated intracellular calcium surge was ablated. This demonstrates, for the first time, that LPHN-1 or 3 expression is required for an intracellular effect of TCAP-1, and therefore confirms our hypothesis that latrophilins function as receptors for TCAP-1 in C2C12 cells. Interestingly, knocking down just one paralogue was sufficient to entirely eliminate the calcium surge, which suggests that both LPHN-1 and 3 paralogues function as receptors for TCAP-1, but expression of one alone was not sufficient to transduce intracellular signals. It is possible that there is still a LPHN-1-mediated calcium response in the LPHN-3 knockdowns (and vice versa), but the response could be too small to be detected by Fluo-4. Alternatively, LPHN-1 and 3 may work together to transduce signals upon binding TCAP-1, perhaps through cis-association upon ligand binding. Homophilic and heterophilic oligomerization of GPCRs upon ligand binding is well-characterized phenomenon in molecular pharmacology, but this occurrence has not yet been studied for any member of the Adhesion GPCR family (Milligan, 2004; Meza-Aguilar and Boucard, 2014). Moreover, Volynski and colleagues (2004) have demonstrated that the CTF and NTF of LPHN-1 are constitutively cleaved in vivo and functionally re-associate upon stimulation with a recombinant a-latrotoxin mutant ligand, LTXN4C. Although interaction between the NTFs and CTFs of different latrophilin paralogues has not been explored, such an interaction could be possible, especially given the high degree of conservation between the latrophilin GAIN domains which are responsible for the physical re-association of NTFs and CTFs (Araç et al., 2012). Thus, in addition to the possibility of oligomerization, TCAP-1 could cause re-association of some specific combination of LPHN-1 and 3 NTFs and CTFs to activate intracellular signalling. This supposition requires further investigation of potential interactions between latrophilin paralogues upon ligand-binding.

4.3. CRISPR/Cas9 Knockout of Latrophilins

Given the limitations of siRNA knockdown, LPHN-1 and 3 were also targeted for permanent knockout in C2C12 cells using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 is a method of targeted genetic manipulation that applies the biological machinery of the bacterial adaptive immune system to eukaryotic cells (review: Ran et al., 2013). CRISPR/Cas9 works as

51 follows: cells are co-transfected with a Cas9 enzyme expression vector and a 20-nucleotide single guided RNA (sgRNA) oligomer that is complementary to the DNA sequence targeted for knockout and is directly upstream of a 5’-NGG protospacer adjacent motif (PAM; where ‘N’ is any nucleotide). Subsequently, Cas9 recognizes the sgRNA-associated PAM and catalyzes a double- stranded DNA break (DSB) approximately 3 nucleotides upstream of the first PAM nucleotide. Cells then attempt to repair the DSB using one of two mechanisms: homology directed repair (HDR) or non-homologous end-joining (NHEJ). HDR is typically leveraged during CRISPR/Cas9 gene editing if precise manipulation is desired (for example, generation of single nucleotide polymorphisms, SNPs), as this process requires an additional transfection with a mutant DNA template. With NHEJ, the DNA ends where the DSB occurred are degraded and re-ligated, leading to non-specific insertion/deletion (‘indel’) mutations at the break site. If an indel mutation produces a premature stop codon within a gene’s mRNA coding region, then the gene is effectively knocked out. The main advantage of CRISPR/Cas9 gene editing compared to siRNA is the permanency of the knockout effect, as it is passed on to daughter cells following division. The main disadvantage is the greater risk of having ineffective targets or off-target effects sgRNA oligomers must be custom synthesized and therefore cannot are not pre-validated for efficacy or specificity by commercial vendors. In the present study, the CRISPR/Cas9 gene editing system to generate indel mutations within the LPHN-1 and 3 genes. In total, six sgRNA oligomers were designed and synthesized, where four produced indel mutations in the heterogenous transfected cell pools, as determined by T7 endonuclease assay. It is unclear why two sgRNA oligomers failed to produce mutations, though this is not an uncommon issue faced with CRISPR/Cas9. A recent report estimated that CRISPR/Cas9 gene editing has an approximately 15% fail rate (Clarke et al., 2018). Sometimes, these failures are caused by sgRNA oligomers stabilizing the Cas9 enzyme at the site of the DSB, which blocks the HDR or NHEJ repair machinery from accessing the DSB (Clarke et al., 2018). From the two successful LPHN-1-targeting heterogenous transfected pools (E5U and E5D), two knockout clone lines were cultured (LPHN-1 E5U7 and E5D3) which showed >85% reductions in LPHN-1 mRNA expression and >75% reductions in protein expression, while still possessing growth and morphology indistinguishable from wild-type cells. It is advantageous to possess two clones that are mutated at different sites within the LPHN-1 mRNA coding region, as this reduces the likelihood of interpreting off-target effects as a LPHN-1 knockout effects due to the very low probability of two different target sites eliciting identical off-target effects. In prior

52 in vivo studies, deletion of the LPHN-1 gene in mice was embryonically lethal, although successfully bred full knockouts displayed a lack of maternal behaviour and increased aggression, as well as phenotypes associated with schizophrenia (Silva and Ushkaryov, 2010). No abnormalities in skeletal muscle development were reported for these LPHN-1 knockout mice, which agrees with the present evidence that knocking out LPHN-1 in C2C12 cells had no immediately noticeable impact on cell health or viability. In Fluo-4 calcium fluorescence assays identical to those performed in siRNA knockdowns, both LPHN-1 CRISPR/Cas9 knockout clones did not show a calcium surge in response to TCAP-1. This agreement between the siRNA and CRISPR/Cas9 data provides further proof of successful receptor knockdown and knockout. Manipulation of LPHN-3 proved to be more difficult using CRISPR/Cas9 methods. Although there appeared to be mutants present in the E1 and E2 heterogenous transfected pools based on T7 endonuclease assay, few candidate knockouts were identified following isolation and growth of clones. This could mean that clones possessing LPHN-3 mutations were unviable or had a poor rate of proliferation. Also, although the LPHN-3 siRNA knockdowns were viable and did attenuate the effect of TCAP-1 on intracellular calcium, the siRNA-mediated reduction in LPHN- 3 mRNA levels was less efficacious than expected. Taken together, these results suggest that LPHN-3 expression may be necessary for C2C12 cell heath and survival, possibly due to some endogenous role unrelated to its TCAP-1 receptor function. A previous study reported an increase in reward motivation, which is associated with attention deficit hyperactivity disorder (ADHD) and addiction phenotypes, in LPHN-3 knockout mice, but effects on skeletal muscle health were not reported (Orsini et al., 2016). It would be interesting to generate skeletal muscle-specific knockouts of LPHN-3 in live rodent models to determine if expression of this paralogue is required for proper skeletal muscle health and function in vivo.

4.4. The TCAP-Latrophilin Ligand Receptor Pair Regulates Intracellular Calcium

Our laboratory has previously established that C2C12 myotubes experience an increase in intracellular calcium upon treatment with TCAP-1, as shown by Fluo-4 fluorescence (D’Aquila et al., manuscript submitted). Moreover, pre-treatment with either a PLC inhibitor, U73122, or an

IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB), eliminated the TCAP-1-mediated calcium surge. Given that PLC and IP3R activity are critical to the Gq/11-protein signalling pathway, TCAP-

1 appears to elicit this calcium response through activation of some Gaq-associated GPCR. The

53 present study, for the first time, determined that latrophilin expression is required for the TCAP- 1-mediated increase in intracellular calcium. siRNA knockdown of LPHN-1 or 3 and CRISPR/Cas9 knockout of LPHN-1 at two sites effectively eliminated the calcium surge in TCAP- 1-treated C2C12 myotubes. This finding, in combination with evidence of an in vitro association between TCAP-1 and the LPHN-1 HBD as well as between latrophilins and Gaq protein subunits, implies that the TCAP-1-mediated calcium surge is a direct result of latrophilin GPCR signalling through the Gq/11-protein pathway (Husić et al., 2019; Rahman et al., 1999).

There is substantial evidence of latrophilins signalling through the Gq/11-proteins when stimulated with a-latrotoxin or LTXN4C (Lelianova et al., 1997; Davletov et al., 1998). Thus, it is unsurprising that TCAP-1 apparently uses this signalling pathway in C2C12 cells. However, there is also evidence that TCAP-1 modulates intracellular cAMP levels in immortalized neuronal cell lines, which would be indicative of GPCR signalling through Gs or Gi/o-protein pathways (Qian et al., 2004; Wang et al., 2005). In fact, over-expressed latrophilins have been shown to associate in vivo with cAMP-regulating G-proteins upon stimulation with the self-derived Stachel peptide agonist (discussed further in 4.8; Müller et al., 2015; Röthe et al., 2019; Nazarko et al., 2018). TCAP-1’s effects on intracellular cAMP in C2C12 cells were not investigated, so it could be possible that such signalling pathways are active here as well. In N38 cells, a decrease in intracellular calcium was observed upon treatment with TCAP-1, which refutes the likelihood of a TCAP-latrophilin-Gq/11-protein signalling mechanism in this cell line (Hogg and Lovejoy, unpublished observations). Thus, there could be tissue-specific differences in latrophilin-G-protein associations, where the Gq/11-protein pathway is favoured in skeletal muscle but not in neurons. Tissue-level differences in latrophilin-G-protein associations could be regulated by the relative expression of paralogues, or through alternative splicing. In fact, splice variation within LPHN-3 regulated association with either Gai of Gaq in pancreatic cell lines following stimulation with a Stachel peptide (Röthe et al., 2019). C2C12 cells could express splice variants of LPHN-1 and 3 that favour association with the Gq/11 pathway upon stimulation with TCAP-1, whereas other cells might express variants that favour association with the Gi/o or Gs pathways.

The TCAP-latrophilin-Gq/11-protein signalling pathway has especially important implications in skeletal muscle, as calcium is a critical regulator of skeletal muscle function. Cytosolic calcium directly regulates actin-myosin contraction by binding to troponin and inducing a conformational change that detaches tropomyosin from actin, therefore unmasking the actin binding site for myosin. Strength of contraction depends on the number of actin-myosin cross

54 bridges formed, which increases when more calcium is available in the cytoplasm (review: Berchtold et al., 2000). Given our observation of an intracellular calcium surge in TCAP-1-treated C2C12 cells, as well as increased contractile force in isolated skeletal muscle tissue from TCAP- 1-treated rats, the TCAP-latrophilin calcium response may directly influence contractile kinetics by increasing the availability of cytosolic calcium for actin-myosin cross bridge formation (Figure 4.1). However, this hypothesis is complicated by the duplicitous nature of calcium regulation in skeletal muscle. In adult skeletal muscle, intracellular calcium is stored in the SR at rest and released into the cytosol predominately through RyR calcium channels following depolarization of the muscle cell. TCAP-1 appears to cause SR calcium release through IP3Rs, which are known to regulate calcium-mediated contraction in smooth muscle and potentially cardiac muscle, but

Motoneuron

TCAP HBD ACh

Latrophilin D Voltage

AChR DHPR

Ca2+ 2+ Gq/11 signalling Ca2+ Ca Ca2+ Ca2+ 2+ Ca2+ Ca IP3R SR RyR 2+ Ca IP3 Ca2+ Ca2+ GTP GDP Actin-myosin coupling

Skeletal muscle cell

Figure 4.1. Schematic of the putative role of TCAP-1 in calcium regulation of actin-myosin contraction in skeletal muscle. Motoneuron innervation of skeletal muscle results in calcium release from the SR through RyR calcium channels. Contrastingly, TCAP-1 signalling through latrophilin results in calcium release from the SR through IP3R calcium channel, which could strengthen contraction by increasing available intracellular calcium. Calcium promotes actin- myosin cross-bridge formation by inducing detachment of tropomyosin from actin, which unmasks binding sites.

55 there is conflicting evidence regarding their role in skeletal muscle (review: Santulli et al., 2017).

Using three different methods of IP3R stimulation, Blaauw and colleagues (2012) observed an increase in intracellular calcium in C2C12 myotubes, but not in isolated adult mouse fibres. They concluded that IP3R expression is too low and its kinetics are too slow for IP3R-mediated SR calcium release to have any meaningful impact on contractile function in adult fibres. In contrast,

Díaz-Vegas and colleagues (2018) showed that IP3R-mediated SR calcium release does occur in adult fibres; however, they did not investigate if this calcium surge was responsible for changes in contractile strength via actin-myosin cross bridge formation. Given the disagreement in the field, further experiments are required to determine if the TCAP-1-mediated calcium surge could directly regulate actin-myosin cross bridge formation. In addition to facilitating contraction via actin-myosin cross bridge formation, intracellular calcium regulates mitochondrial function and gene expression in skeletal muscle cells. Therefore, the TCAP-latrophilin calcium surge could indirectly modulate contractile function by altering metabolic processes and/or genetic expression without any direct effects on actin-myosin coupling. These possibilities are discussed further in the next two sections.

4.5. The TCAP-Latrophilin Ligand Receptor Pair Regulates Cellular Metabolism

TCAP-1 has an established role in the regulation of cellular metabolism in both neuronal and muscular immortalized cell lines and tissues (Hogg et al., 2018; D’Aquila et al., manuscript submitted). In C2C12 cells, TCAP-1 increased glucose uptake, translocation of GLUT4 to the plasma membrane, intracellular ATP concentration, and, most pertinent to the present study, NADH turnover (D’Aquila et al., manuscript submitted). Here, both wild-type and empty vector- transfected control C2C12 myoblasts showed a significant increase in NADH turnover over 2 hours, whereas LPHN-1 E5U7 and E5D3 knockouts did not. This indicates that LPHN-1 expression in C2C12 cells is necessary for the TCAP-1-mediated increase in NADH turnover and that, like the intracellular calcium surge, TCAP-1’s regulatory actions on cellular metabolism depend on LPHN-1 receptor signalling. NADH is an electron carrier involved in both aerobic and anaerobic metabolism. In glycolysis, two NAD+ molecules are reduced to NADH as one molecule of glucose is converted to two pyruvates. Under anaerobic conditions, this NADH is subsequently oxidized to NAD+ as pyruvate is reduced to lactate. Under aerobic conditions, another NAD+ molecule is reduced to

56 NADH as pyruvate is converted to acetyl-CoA, and then three more are produced in one round of the Kreb’s cycle. NADH is recycled to NAD+ during oxidative phosphorylation, where it donates its electrons to Complex I of the electron transport chain (ETC). As these electrons are carried through the ETC, they drive the formation of an H+ gradient across the inner mitochondrial membrane which ultimately drives ATP synthesis, and finally reduce O2 to H2O. In total, anaerobic metabolism of one glucose molecule involves redox turnover of 2 NADH molecules, whereas aerobic metabolism of one glucose uses 10 NADH molecules. Thus, from this description, it is evident that indirectly measuring the rate of NADH oxidation to NAD+ via resazurin-resorufin fluorescence assay is a viable proxy for metabolic rate within a population of cells. Additionally, the resazurin-resorufin fluorescence assay has several methodological advantages over other assays that measure metabolic rate: first, it does not require specialized equipment unlike, for example, O2 consumption assays which require electrodes. Second, in contrast to luminescent intracellular ATP assays which require cell lysis, resazurin is compatible with live cells and is relatively non-toxic. Given that TCAP-1-treated LPHN-1 CRISPR/Cas9 knockouts did not experience an increase in NADH turnover and did not experience a surge in intracellular calcium, this may indicate that the TCAP-latrophilin calcium response has a direct role in the regulation of cellular metabolism. This supposition is supported by evidence that the activities of several enzymes associated with oxidative metabolism are directly upregulated by intracellular calcium (review: Denton, 2009). FAD-glycerol phosphate dehydrogenase, which effectively shuttles electrons transferred to NADH during glycolysis into the ETC, is activated by low micromolar levels of cytosolic calcium (Hansford and Chappell, 1967; Denton, 2009). Pyruvate dehydrogenase phosphatase, an enzyme involved in the conversion of pyruvate to acetyl-CoA, as well as isocitrate dehydrogenase and oxoglutarate dehydrogenase, which are Kreb’s cycle enzymes, are all activated by mitochondrial matrix calcium (Denton, 2009). In addition to increasing intracellular calcium by Fluo-4 fluorescence, TCAP-1 also specifically increased mitochondrial calcium fluorescence in wild-type C2C12 myotubes by Rhodamine-2 fluorescence (D’Aquila et al., manuscript submitted). Thus, calcium released from the SR through the latrophilin-Gq/11-protein signalling pathway could upregulate the activity of mitochondrial enzymes either directly in the cytoplasm or via shuttling to the mitochondrial matrix through voltage dependent anion channels (VDAC) and mitochondrial calcium uniporters (MCU; Figure 4.2; Giorgi et al., 2018).

Ca2+ Pyruvate Acetyl-CoA

NAD+ NADH Ca2+

Ca2+ MCU

2+ 3X NAD+ Ca Ca2+

2+ Kreb’s Ca Cycle 3X NADH Ca2+

2+ VDAC Ca H+ + H + H+ H ATP Ca2+ NAD+ ADP NADH + Pi

III C IV I Q ATP synthase

Electron Transport Chain

Figure 4.2. Schematic of the proposed relationship between the TCAP-latrophilin-induced intracellular calcium surge and TCAP-latrophilin-induced increase in NADH turnover. Intracellular calcium may be shuttled to mitochondrial matrix through VDACs and MCUs. In the matrix, calcium stimulates the activity on enzymes associated with pyruvate conversion to acetyl- CoA and the Kreb’s cycle (yellow arrows; Denton, 2009). This leads to elevated NADH production. Electrons from NADH are transferred to Complex I of the electron transport chain, where they are used to generate an H+ gradient across the inner mitochondrial membrane that ultimately drives ATP synthesis.

These finding in C2C12 cells provide important context to previous in vivo findings with TCAP-1 treatment. TCAP-1-treated rat tibialis anterior muscle showed increased nitro tetrazolium blue (NTB) staining, indicating increased NADH turnover, predominately in small and medium- sized fibres (D’Aquila et al., manuscript submitted). Interestingly, immunohistochemical staining

58 of LPHN-1 expression was especially high in small and medium-sized fibres as well. Given this observation, the relationship between TCAP-1, LPHN-1 and NADH described here in C2C12 cells is probably translated to the mature skeletal muscle tissue. Moreover, a recent calcium fluorescence study showed that SR calcium released through IP3Rs was directed to mitochondria in adult skeletal muscle, suggesting that the purported role for calcium as a mitochondrial enzyme stimulant could also be conserved in vivo (Díaz-Vegas et al., 2018). Increased NADH turnover is indicative of increased ATP synthesis, given that electrons donated by NADH are used in the ETC to facilitate ATP synthase activity. As previously mentioned, ATP cycling during the power stroke is critical to actin-myosin contraction. Skeletal muscle also requires ATP to facilitate re-uptake of calcium into the SR by sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps when contraction is complete (review: Periasamy et al., 2017). Thus, a TCAP-latrophilin-mediated increase in ATP availability would explain the increased force of contraction and reduced fatigability observed in isolated tibialis anterior muscle tissue.

4.6. The TCAP- Latrophilin Ligand Receptor Pair Regulates Skeletal Muscle Gene Expression

Though prior studies in C2C12 cells and rat skeletal muscle tissue have investigated TCAP- 1’s ability to modulate signalling factors and levels of metabolic substrates, the present study is the first to thoroughly investigate TCAP-1’s regulatory actions on gene expression in this tissue. Here, TCAP-1-treated rat tibialis anterior muscle showed a significant increase in MyHCI mRNA expression, whereas TCAP-1-treated C2C12 myotubes showed a significant an increase in PGC- 1a mRNA expression. Both of these genes are associated with a slow-twitch muscle phenotype. Furthermore, TCAP-1 did not elicit an increase in PGC-1a mRNA expression in either of the LPHN-1 CRISPR/Cas9 knockouts, indicating that TCAP-1 requires signalling through LPHN-1 in order to upregulate this transcription factor. The rat tibialis anterior muscle is a predominately fast-twitch muscle and typically possesses only 5% slow-twitch fibres (Armstrong & Phelps, 1984). Thus, these findings suggest that the percentage of MyHCI-expressing fibres increased from 5% to 14% in the short-term treatment group and to 18% in the long-term treatment group, assuming the total number of fibres did not change during treatment. MyHCI-expressing skeletal muscle cells possess slow myosin ATPase activity and high mitochondrial content, which confers a slow-twitch phenotype where contraction velocity is slowed and fatigue resistance is improved (Schiaffino and Reggiani, 1994).

59 In contractile kinetics experiments using the same muscle, significant reductions in contractile velocity and fatigability were observed in the TCAP-1-treated group compared to vehicle (D’Aquila et al., manuscript submitted). Thus, TCAP-1 appears to modulate tibialis anterior muscle contractile performance by altering MyHC expression, which fundamentally shifts the histological makeup of the muscle towards a more slow-twitch phenotype. Remarkably, slow- twitch fibres typically possess a smaller cross-sectional diameter than fast-twitch fibres, and as was previously noted, small and moderate-sized fibres demonstrated strong LPHN-1 immunoreactivity and prominent NADH staining following treatment with TCAP-1 (D’Aquila et al., manuscript submitted). Given our lack of a LPHN-1 knockout live animal model, it is not possible to directly test if LPHN-1 expression is required for TCAP-1-mediated regulation of MyHC expression, but the correlation between LPHN-1 expression, NADH turnover and phenotypic indicators of slow-twitch muscle in the adult rat skeletal muscle would suggest that this ligand-receptor relationship is present and functional. In C2C12 myotubes, TCAP-1 did not cause an increase in MyHCI mRNA expression compared to vehicle. There are several possible explanations for this discrepancy between the in vivo data and the findings in C2C12 cells. First, the 3-day TCAP-1 treatment regimen may not have been long enough or potent enough to induce changes to MyHCI expression. Second, MyHC expression is highly regulated over the course of C2C12 differentiation, and therefore may not be influenced by TCAP-1. In early differentiation (days 1-4), MyHCI is predominantly co-expressed with embryonic and neonatal MyHC isoforms which are not found in adult skeletal muscle, whereas from day 5 onwards, MyHCII isoforms are prevalent (Brown et al., 2012). The intracellular factors regulating this transition are unclear, but a similar pattern is conserved in the developing fast-twitch muscle of mouse embryos (Agbulut et al., 2003). Thus, given that MyHC expression in differentiating C2C12 cells appears to have a more important role in development rather than twitch kinetics, TCAP-1 may not possess any relevant bioactivity under these circumstances. Third, TCAP-1’s effects on MyHC expression could be indirect, where instead of having latrophilin agonist activity in skeletal muscle cells, TCAP-1 modulates levels of other in vivo signalling factors which subsequently act on skeletal muscle to alter MyHC expression. These factors might not be present in C2C12 cell culture, which would explain why no change in MyHCI was observed in cells alone. TCAP-1 did, however, elicit a 50% increase in PGC-1a mRNA expression in C2C12 cells. PGC-1a is a multifunctional transcription factor that acts as a ‘master regulator’ for mitochondrial

60 biogenesis (reviews: Liang and Ward, 2006; Chan and Arnay, 2014). Elevated PGC-1a expression is associated with slow-twitch muscle, as it was shown to directly upregulate MyHCI transcription in primary muscle cultures (Lin et al., 2002). Given the observation of increased MyHCI mRNA expression in TCAP-1-treated rat tibialis anterior muscle, it is expected that PGC-1a is elevated in this muscle as well, though this requires confirmation by qRT-PCR or Western blot. PGC-1a is highly expressed through the course of C2C12 differentiation and is especially elevated from days 4-6 (Lin et al., 2014). In day 4-differentiated C2C12 cells, over-expression of PGC-1a correlated with elevated MyHCI expression, whereas siRNA knockdown of PGC-1a effectively eliminated MyHCI expression (Lin et al., 2014). Given the demonstrated relationship between these two genes, it is surprising that there was an increase in PGC-1a but not MyHCI in TCAP-1-treated C2C12 myotubes. However, in the over-expression study, PGC-1a was increased 80-fold over endogenous levels, whereas TCAP-1-mediated only a 50% increase, which may not be substantial enough to induce MyHCI changes after only 3 days of treatment. Instead, the present findings could represent an initiation phase of fibre-type switching, where PGC-1a is upregulated but increases in MyHCI are not yet detectable. LPHN-1 CRISPR/Cas9 knockouts did not experience an increase in PGC-1a mRNA expression following TCAP-1 treatment, indicating that TCAP-1-mediated upregulation of PGC- 1a expression depends on LPHN-1 signalling. PGC-1a transcription is associated with a number of transcriptional regulators that are activated upon binding to intracellular calcium, including calcium/calmodulin dependent protein kinase type IV (CaMKIV) and calcineurin (CaN; Figure 4.3; Handschin et al., 2003; Ventura-Clapier et al., 2008; Kuo and Ehrlich, 2015). CaMKIV upregulates PGC-1a via a cAMP response element binding protein (CREB), whereas CaN activates myocyte enhancement factor 2 (MEF2), potentially by dephosphorylating and activating the associated nuclear factor of activated T-cell (NFAT) transcription factors (Handschin et al., 2003; Potthoff et al., 2007). MEF2 and PGC-1a function in tandem to promote fibre-type switching towards a slow-twitch phenotype. PGC-1a promotes MEF2-mediated transcription of itself through an apparent positive feedback loop (Handschin et al., 2003) Moreover, CaMKIV and CaN promote nuclear exportation and proteasome-mediated degradation of class II histone deacetylases (HDAC), which inhibit MEF2 activity (Potthoff et al., 2007). In skeletal muscle, CaMKIV and CaN are activated by elevated intracellular calcium associated with prolonged periods of contractile activity (Handschin et al., 2003; Ventura-Clapier et al., 2008). However, in

Ca2+ Ca2+

Ca2+ 2+ CaN --Ca2+ Ca -- CaMKIV

Degradation Pi Pi HDAC NFAT HDAC

NFAT CREB PGC-1a MEF2

Nucleus

Figure 4.3. Schematic of the proposed relationship between the TCAP-latrophilin-induced intracellular calcium surge and TCAP-latrophilin-induced increase in PGC-1a expression. The transcriptional regulators CaMKIV and CaN are activated by intracellular calcium. CaN promotes dephosphorylation of NFAT, which activates MEF2, as well as degradation of HDAC, a MEF2 repressor. CaMKIV activates CREB, phosphorylates HDAC to prevent its shuttling into the nucleus, and promotes degradation of HDAC. MEF2 promotes transcription of PGC-1a, which induces a positive feedback loop where PGC-1a activates MEF2 to promote its own transcription. Adapted from Potthoff et al., 2007; Kuo and Ehrlich, 2015.

cardiac muscle, Gq/11-protein-associated IP3R-mediated calcium release is a potent activator of these factors (Heineke and Molkentin, 2006). Thus, the calcium surge induced by Gq/11 during TCAP-latrophilin signalling could activate CaMKIV and CaN transcriptional regulators to promote transcription of PGC-1a. Moreover, PGC-1a is a master regulator of mitochondrial biogenesis, and an increase in mitochondrial content would prompt a higher rate of NADH turnover. Elevated PGC-1a expression could explain the TCAP-latrophilin-dependent increase in resorufin fluorescence described in the previous section. It should be noted, however, that the timelines of these two experiments were considerably different, as PGC-1a expression was examined following 3 days of TCAP-1 treatment, whereas NADH turnover was measured only for 2.5 hours post-treatment.

62 The in vivo evidence for fibre-type switching is complicated by TCAP-1’s regulatory actions on serum testosterone levels. Chand and colleagues (2014) demonstrated elevated serum testosterone in TCAP-1-treated mice over 9 days. Testosterone treatment was also shown to increase protein expression of PGC-1a and other type I fibre-associated genes in the mouse gastrocnemius muscle over 4 weeks (Usui et al., 2014). It is therefore possible that TCAP-1 induced fibre-type switching in the rat tibialis anterior muscle by increasing circulating testosterone, rather than through a direct TCAP-latrophilin ligand-receptor mechanism in skeletal muscle. Given that no increase in MyHCI was detected in the C2C12 cells, the in vivo MyHCI upregulation may have required testosterone, which could not be elevated in C2C12 cell culture. However, the expression data from C2C12 cells somewhat challenges this possibility, as PGC- 1a expression was upregulated upon TCAP-1 treatment. Furthermore, testosterone treatment is typically associated with an increase in muscle mass, which was not observed in the TCAP-1 group compared to vehicle (D’Aquila et al., manuscript submitted). The fidelity of the testosterone-fibre- type relationship is also questionable, as numerous clinical studies in humans have shown that testosterone treatment does not significantly alter muscle fibre-type composition or PGC-1a expression (Petersson et al., 2014; Sinha-Hikim et al., 2002; Sinha-Hikim et al., 2006). To further eliminate the testosterone variable, similar experiments could be performed in castrated rodents.

4.7. Future Directions

Future experiments to complement the present study could employ RNA sequencing as an alternative to 5’RACE PCR to examine transcript variants among the teneurins and TCAPs. Recent advancements in small molecule, real-time sequencing (SMRT) allow resolution of full-length mRNA sequences in whole transcriptomes (Wang et al., 2016). This technology could be applied to the adult mouse brain transcriptome or to other tissue types where the teneurins, TCAPs and latrophilins are present. Given the recent advances in CRISPR/Cas9 technology, it may soon be relatively easy and inexpensive to knockout or genetically edit the teneurin, TCAP and latrophilin genes in rodent models. This would provide tremendous insights into the endogenous functions of this ligand-receptor pair in vivo. Latrophilin knockdown and knockout studies could also be performed in other immortalized cell lines where TCAP-1 has demonstrated bioactivity (for example, N38 cells) in order to establish latrophilin receptor dynamics across tissues. Other TCAP- 1 actions in the C2C12 cell line could be probed for their latrophilin dependency, such as the

63 TCAP-1-mediated increase in glucose uptake (D’Aquila et al., manuscript submitted). In neurons, TCAP-1-mediated glucose uptake occurred through an insulin-independent mechanism, so it is intriguing to consider the possibility that latrophilin shares some functional overlap with insulin receptors, as this would suggest that latrophilin agonists like TCAP-1 could have therapeutic potential in the treatment of diabetes (Hogg et al., 2018). Additionally, potential TCAP-1-mediated changes to the activities of other skeletal muscle-regulating transcription factors, such as MEF2 and HDAC, could be investigated in C2C12 cells to provide a fuller understanding of TCAP-1- mediated fibre-type switching. Such investigations might be difficult though, given that MEF2 has numerous splice variants and HDAC activity is regulated at the post-transcriptional level (Black and Olson, 1998; Potthoff et al., 2007). The promiscuity of latrophilin-G-protein associations also requires further investigation. Recently, a number of studies have investigated latrophilin-G-protein associations upon receptor stimulation with a recombinant Stachel peptide (Müller et al., 2015; Röthe et al., 2019; Nazarko et al., 2018). The Stachel sequence represents the N-terminus of the latrophilin CTF following cleavage at the GAIN domain, and is a highly conserved feature of Adhesion GPCRs (Liebscher et al., 2014). In the above listed studies, intracellular indicators of G-protein signalling were measured in recombinant Stachel-treated cells over-expressing various latrophilin paralogues. There appears to be considerable differences between the latrophilin GPCR signalling properties of Stachel and those of a-latrotoxin, as Stachel showed remarkably diverse G-protein associations across paralogues, whereas a-latrotoxin favours Gq/11 signalling through LPHN-1 alone (Lelianova et al., 1997; Davletov et al., 1998; Rahman et al., 1999). It is interesting to consider where the TCAP agonists fall on this spectrum: that is, do TCAPs signal through a wide variety of G-proteins like Stachel, or is TCAP more selective like a-latrotoxin? The present evidence would indicate that TCAP-1 could have some diversity in its G-protein coupling patterns, as here it was shown to associate with Gq/11, but it also modulated cAMP in neuronal cell lines (Qian et al., 2004; Wang et al., 2005). Contrastingly, TCAP and a-latrotoxin do share 20% sequence similarity, and like a- latrotoxin, TCAP-1 binds to the latrophilin-1 HBD, indicating the two may share signalling properties (D’Aquila and Lovejoy; unpublished observations; Husić et al., 2019; Krasnoperov et al., 1999; Silva and Ushkaryov, 2010). Latrophilin over-expression studies similar to those described above but using TCAP-1 as an agonist instead of Stachel would certainly complement the present knockdown and knockout study, and could identify the full array of TCAP-latrophilin- G-protein associations.

64 4.8. Concluding Remarks

My hypothesis that the latrophilins act as receptors for TCAP-1 in skeletal muscle was supported by the results of this research program. The present study is the first to establish the necessity of latrophilin expression for TCAP-1’s intracellular actions in the C2C12 cell line. Despite some difficulty in characterizing TCAP-3 expression and manipulating LPHN-3, I ultimately provided substantial evidence that TCAP-1 and LPHN-1 form a functional ligand- receptor pair that influences multiple aspects of skeletal muscle cellular function (Figure 4.4). Calcium likely acts as the primary intracellular effector for TCAP-1’s actions in this cell type. There are at least three mechanisms of calcium regulation in skeletal muscle function where TCAP-1 could have modulatory actions. First, calcium increases the formation of actin-myosin cross bridges to strengthen contraction. Second, calcium upregulates enzymatic activity in the mitochondria to increase oxidative metabolism. And third, calcium modulates the activity of transcription factors involved in fibre-type switching. TCAP-1’s diverse actions both in vivo and in vitro could be explained by its ability to upregulate one or more of these calcium-dependent processes through latrophilin GPCR signalling. Furthermore, this study belongs to a small but growing body of work that considers the functionality of teneurins, TCAPs and latrophilins outside of the CNS. Given the ancient evolutionary history of the teneurin/TCAP-latrophilin system, it is unsurprising that these proteins are functional across a number of tissue types. There is potential for vast ligand-receptor signalling diversity among the teneurins, TCAPs and latrophilins due to paralogue combinatorics, alternative splicing, and differential expression patterns among tissues and between species. Here, we have established the functionality of one TCAP-latrophilin ligand-receptor signalling pair as a critical regulator of skeletal muscle function—there could be countless more waiting to be discovered.

TCAP HBD

Latrophilin

G DAG Skeletal muscle cell q/11 PLC

Ca2+ PIP2 IP R 2+ IP3 3 Ca SR

Ca2+ GTP GDP Ca2+ Ca2+ TR Ca2+

Ca2+

TF PGC-1a expression Kreb’s Cycle NADH turnover

Nucleus Mitochondria

Figure 4.4. Summary of latrophilin knockdown and knockout effects in TCAP-1-treated C2C12 cells. Knocking-down or knocking-out latrophilins eliminated the TCAP-1-mediated intracellular calcium surge. In LPHN-1 knockouts, an increase in PGC-1a was not observed upon TCAP-1 treatment, likely because the TCAP-1-mediated calcium surge is required to activate transcription regulators (TR) that promote transcription factor (TF) binding upstream of the PGC- 1a gene (Kuo and Ehrlich, 2015). Similarly, TCAP-1 did not increase NADH turnover in LPHN- 1 knockouts, possibly because the TCAP-1-mediated calcium surge is necessary for upregulated Kreb’s cycle enzyme activity (Denton, 2009). Effects where LPHN-1 knockout eliminated TCAP- 1 effects are indicated by red ‘X’.

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Appendix

A B

DIC Fluo-4

Supplementary Figure 2.1. Sample images of C2C12 myotubes. (A) Differential interference contrast (DIC) image of day 6-differentiated C2C12 myotubes. (B) Fluo-4 fluorescent image of day 6-differentiated C2C12 myotubes.

LPHN-1 E5U (598 bp) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 CTRL WT

LPHN-1 E5D (470 bp) Controls 1 2 3 4 5 6 7 8 9 10 11 12 CTRL WT - +

Supplementary Figure 3.1. Identification of candidate latrophilin-1 CRISPR/Cas9 knockout C2C12s from homogenous clone lines. All clones from the LPHN-1 E5U and E5D groups appear to produce a wildtype band; therefore, there are no immediately identifiable full knockouts. However, select clones show multiple bands or reduced intensity of a wildtype band, indicating the presence of some mutated copies of the LPHN-1 gene. These clones, deemed ‘candidate knockouts,’ include clones 1, 2, 4, 5, 7, 8, 9, and 10 from the E5U group and clones 1, 2, 3, and 11 from the E5D group. Expected band sizes: LPHN-1 E5U=598 bp; E5D=470 bp; kit-provided positive control=600, 400 and 200 bp; negative control (no DNA)=no band expected.