The Role of the Neuronal Primary Cilium in the Modulation of G -Coupled Receptor Signaling

DISSERTATION

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

By

Andrew Ian Koemeter-Cox, BS

Biomedical Sciences Graduate Program

The Ohio State University

2014

Dissertation Committee:

Kirk Mykytyn, PhD, Advisor

R Thomas Boyd, PhD

Howard Gu, PhD

Denis Guttridge, PhD

Copyright by

Andrew Ian Koemeter-Cox

2014

Abstract

Primary cilia are based sensory present in mammals on nearly every type, including neurons. Defects in associated with the cilium result in a variety of , known as , with including cystic , obesity, blindness, anosmia, cognitive deficits, and hypogonadism. In a few tissues in the body, primary cilia have well defined functions, but the role that primary cilia play in neuronal function is still mostly unknown. These neuronal cilia are enriched for specific signaling molecules, suggesting that the cilia may serve an important role in signaling. Our laboratory identified a number of G protein-coupled receptors that contain a localization sequence necessary for targeting to cilia. This work focuses on Kisspeptin Receptor 1 (Kiss1r), Receptor D1 (D1R), and Melanin

Concentrating Hormone Receptor 1 (Mchr1). Our hypothesis is that the cilium is an important modulator of the signaling of receptors that are enriched in the .

Mutations in Kiss1r lead to reproductive deficits in mice and , similar to those seen in ciliopathies. Our work demonstrated that Kiss1r was located in the primary cilia of Gonadotropin-Releasing Hormone (GnRH) neurons, and that these neurons could posses multiple primary cilia, a rarity for central neurons. When cilia are lost on GnRH neurons, their ability to respond to stimulation of Kiss1r is reduced, indicating that the cilium is important to the functioning of Kiss1r. Previous work in our laboratory ii identified that D1R is enriched in the cilia of neurons when BBS protein function is lost.

BBS proteins are important for trafficking of receptors to and from the cilium. We created a mouse model where the BBS protein function is disrupted exclusively in D1R expressing cells. These mice display reduced levels of locomotor activity and increased weight, phenotypes that are present in other mouse models of reduced D1R signaling.

Work is currently underway to characterize D1R signaling in these mice. Mchr1, unlike

D1R, localizes to the cilia of neurons in the brains of wild-type mice, but is lost from the cilium when BBS protein function is lost. Mchr1 signaling in a brain region known as the has been shown to be a regulator of reward and anxiety. Mouse models with germline disruption of BBS proteins display increased feeding and anxiety, leading to our interest in the receptor. We employed injection of a virally expressed Cre recombinase into the Nucleus Accumbens in order to disrupt BBS protein function exclusively in this brain region. We were able to disrupt BBS protein function in the

Nucleus Accumbens utilizing this strategy and experiments with these mice are ongoing.

Taken together, the work on these receptors supports the hypothesis that in neurons, the primary cilium is a vital signaling organelle. In the realm of public health, this research has implications for the investigations in the fields of reproductive biology, obesity, and feeding behavior.

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Dedication

This document is dedicated to my family.

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Acknowledgments

I would like to thank my advisor, Dr. Kirk Mykytyn, for welcoming me into his lab over four years ago. Over the years, Kirk has given me the independence and responsibility to grow as a scientist, while providing guidance and assistance whenever it was needed.

None of this work would be possible without his support, and I will be forever grateful for all the opportunities he has provided for me.

Thank you to my committee members, Dr. Denis Guttridge, Dr. Howard Gu, and

Dr. R Thomas Boyd. I appreciate the time and effort you have taken in guiding me throughout the years.

I am grateful to everyone in the Mykytyn laboratory who has worked with me and assisted me on these projects. A big thank you to Jackie Domire, whose training and guidance when I first entered the lab were invaluable. Thank you to Dr. Jill Green, who in addition to providing great training, education, and insight, has become a great friend.

Thank you to everyone from other labs at OSU that have helped me during the course of my research. Dr. Candice Askwith and Dr. Tom Sherwood, thank you for your technical and non-technical expertise. Thank you to Dr. Howard Gu and his lab members

Dr. Keerthi Thirtamara-Rajamani, Dr. Brian O’Neill, and Dr. Bart Naughton for invaluable training and technical assistance.

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I would like to thank my friends, both here in Columbus and scattered across the east coast, who have always been there when I needed them.

I would not be where I am today without the love of my family. Thank you so much to my parents, Carol and Phil, for always supporting me. I cannot put into words how grateful I am that I have you. To my sister Emily, thank you for always making me laugh when I needed it.

And to my fiancée, Jessica: Thank you for your unconditional love and support.

I could not have done this without you. You are, quite simply, the best.

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Vita

March 1985 ...... Born-Valhalla, New York, USA

2007 ...... B.S. Biochemistry, University of Delaware

2009 to present ...... Graduate Research Associate, Biomedical

Science Graduate Program, The Ohio State

University Publications

1. Koemeter-Cox AI, Sherwood TW, Green JA, Steiner RA, Berbari NF, Yoder BK, Kauffman AS, Monsma PC, Brown A, Askwith CC, Mykytyn K. Primary Cilia Enhance Kisspeptin Receptor Signaling on Gonadotropin-Releasing Hormone Neurons, PNAS 2014 Jun 30 [epub ahead of print]

2. Johnson EA, Dao TT, Guignet MA, Geddes CE, Koemeter-Cox AI, Kan RK. Increased expression of the chemokines CXCL1 and MIP-1 alpha by resident brain cells precedes neutrophil infiltration in the brain following prolonged soman-induced status epilepticus in , Journal of Neuroinflammation 2011, 8:41

3. Cooper CR, Graves B, Pruitt F, Chaib H, Lynch JE, Koemeter-Cox AI, Sequeria L, van Golen KL, Evans A, Czymmek K, Bullard RS, Donald CD, Sol-Church K, Gendernalik JD, Weksler B, Farach-Carson MC, Macoska JA, Sikes RA, Pienta KJ Reticulocalbin-1 Surface Expression on Bone Endothelial Cells and in the LNcaP Prostate Cancer Progression Model, J Cell Biochem, 2008 June 17

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Fields of Study

Major Field: Biomedical Sciences Graduate Program

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Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments ...... v

Vita ...... vii

Publications ...... vii

Fields of Study ...... viii

Table of Contents ...... ix

List of Tables...... xii

List of Figures ...... xiii

List of Abbreviations ...... xv

Chapter 1 : Introduction to Primary Cilia ...... 1

Summary ...... 1

A Brief History of Early Primary Cilia Research ...... 2

The Structure and Formation of Cilia ...... 3

The Primary Cilium as a Signaling Organelle ...... 5

ix

Ciliopathies: The consequences of disruption of proper ciliary function ...... 7

Neuronal Primary Cilia ...... Error! Bookmark not defined.

Hypothesis and introduction to Chapters 2-4 ...... 11

Chapter 2 : The Primary Cilium Enhances Kiss1r Signaling on Gonadotropin-Releasing

Hormone Neurons ...... 20

Summary ...... 20

Introduction: ...... 21

Results ...... 24

Discussion: ...... 31

Materials and Methods: ...... 38

Chapter 3 : Disruption of BBS1 Function in Dopamine D1 Receptor Containing Cells

Results in Reduced Locomotor Activity and Obesity in Mice ...... 58

Summary ...... 58

Introduction: ...... 59

Results ...... 62

Discussion: ...... 69

Materials and Methods ...... 74

Chapter 4 : Targeted Disruption of Ciliary in The Nucleus Accumbens of Mice .. 94

Summary ...... 94

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Introduction: ...... 95

Results: ...... 97

Discussion: ...... 101

Materials and Methods: ...... 104

Chapter 5 : Conclusions and Discussions ...... 112

Kiss1r and the cilium: Implications and future directions ...... 112

D1R and the Cilium: Implications and future directions ...... 113

Mchr1 and the Cilium: Implications and future directions ...... 114

Conclusions and Discussion ...... 116

References ...... 118

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List of Tables

Table 1.1: Ciliary targeting sequences of known and putative ciliary GPCRs………….19

Table 2.1: Physiological Measures of Sexual Maturation in GnRHcilia+ and GnRHcilia- Mice……………………………………………………………………………...56

Table 3.1: Viability of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice………………..84

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List of Figures

Figure 1.1- The basic structure of the cilium is conserved throughout evolution……….15

Figure 1.2- Primary cilia function in a variety of distinct signaling roles……………….16

Figure 1.3-Primary cilia are present on central neurons and are enriched for signaling molecules………………………………………………………………………...17

Figure 1.4-Loss of the cilium structure or mislocalization of ciliary receptors could potentially alter signaling………………………………………………………...18

Figure 2.1- Kiss1r polyclonal antibody generated in rabbit specifically recognizes mouse Kiss1r in immunofluorescent and Western blot analysis………………...47

Figure 2.2- Kiss1r localizes to primary cilia in vitro and in vivo………………………..48

Figure 2.3- Multiple Kiss1r cilia can project from the same GnRH cell body………….49

Figure 2.4- Cilia on GnRH neurons do not label for canonical cilia markers but are primary cilia……………………………………………………………………...50

Figure 2.5- Quantification of Kiss1r cilia on GnRH neurons……………………………51

Figure 2.6- GnRH neurons in GnRHcilia- mice do not have Kiss1r cilia but still express Kiss1r…………………………………………………………………………….52

Figure 2.7- Intracerebroventricular injection of kisspeptin induces Fos expression in GnRHcilia+ and GnRHcilia- mice………………………………………………….53

Figure 2.8- Loss of Kiss1r-positive cilia primarily occurs prenatally in both male and GnRHcilia- mice and does not impact GnRH neuronal migration………..54

Figure 2.9- Loss of primary cilia on GnRH neurons does not result in reproductive Phenotypes……………………………………………………………………….55

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Figure 2.10 GnRH cilia are required for proper Kiss1r signaling……………………….57

Figure 3.1- D1R is expressed at the same levels in the brains D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ transgenic mice………………………………………………83

Figure 3.2- D1R::Cre/BBS1fl/Δ mice avoid some phenotypes present in BBS KO mice...85

Figure 3.3- D1R::Cre/BBS1fl/Δ are heavier compared to D1R::Cre/BBS1fl/wt mice……...86

Figure 3.4- Food consumption by D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice……87

Figure 3.5- Age and weight matched D1R::CRE BBS1fl/Δ mice have lower levels of activity compared to D1R::Cre/BBS1fl/wt counterparts………..………………...88

Figure 3.6 Age and weight matched D1R::Cre/BBS1fl/Δ do not have gross locomotor defects compared to D1R::Cre/BBS1fl/wt counterparts…………………………...89

Figure 3.7- D1R localization in D1R::Cre/BBS1fl/Δ mice recapitulates that seen in BBS1 Δ /Δ mice…………………………………………………………………….90

Figure 3.8- Mchr1 localization in D1R::Cre/BBS1fl/Δ mice is similar to that in control mice……………………………………………………………………………...91

Figure 3.9- D1R::Cre/BBS1fl/Δ mice respond to D1R stimulation the same as D1R::Cre/BBS1fl/wt counterparts…………………………………………………92

Figure 3.10- Neurons from D1R::Cre/BBS1fl/Δ mice respond to acute D1R stimulation similarly to neurons from D1R::Cre/BBS1fl/wt mice……………………………..93

Figure 4.1- Representative images of injections of AAV-CRE-GFP into the Nucleus Accumbens of IFT88fl/fl mice…………………………………………………...109

Figure 4.2- CRE-GFP expression in BBS1fl/Δ cells results in loss of ciliary Mchr1……110

Figure 4.3- Nucleus Accumbens mediated behaviors are unaltered in AAV-CRE-GFP injected BBS1fl/Δ Mice…………………………………………………………..111

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List of Abbreviations

5-HT6- Serotonin receptor subtype 6

AAV- Adeno-Associated Virus

AC3- type III aCSF- Artificial

AcTub- Acetylated

AHA- Anterior Hypothalamic Area

BBS- Bardet-Biedl Syndrome cAMP – Cyclic adenosine monophosphate cGMP- Cyclic guanosine monophosphate

D1R- D1

D5R- Dopamine Receptor D5

DAG- Diacylglycerol

DARPP-32- Dopamine and cAMP Regulated Neuronal Phosphoprotein

DMEM- Dulbecco’s Modified Eagle Medium

DNA- Deoxyribonucleic Acid

EGFP- Enhanced Green Fluorescent Protein

GFP- Green Fluorescent Protein

GnRH- Gonadotropin-Releasing Hormone

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GPCR- G protein-coupled receptor

GPR88- G Protein-Coupled Receptor 88

HEK293T- Human Embryonic Kidney

Hh- Hedgehog

IFT-

IMCD- Inner Medullary Collecting Duct

IP3- Inositol Tri-Phosphates

Kiss1r- Kisspeptin Receptor 1

KO- Knockout

KP-10- Kisspeptin 10

Mchr1- Melanin Concentrating Hormone Receptor 1

MKS- Meckel-Gruber Syndrome

MRI- Magnetic Resonance Imaging

MS- Medial Septumb

NAc- Nucleus Accumbens

OFD1- Oro-facio-digital syndrome 1

OVX- Ovariectomized

PBS- Phosphate Buffered Saline

PCR- Polymerase Chain Reaction

PKA- Protein Kinase A

PKC- Protein Kinase C

PLC- Phospho-Lipase C

xvi qRT-PCR- Quantitative real-time polymerase chain reaction

RNA- Ribonucleic Acid rPOA- Rostral Preoptic Area

Shh- Sonic Hedgehog

Sstr3- type 3

TRPC- Transient Receptor Potential Cation

WT- Wild-Type

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Chapter 1: Introduction to Primary Cilia

Summary

Primary cilia are microtubule based structures with an environment that is exclusive from the that are present on nearly every cell type in mammals.

While the existence of primary cilia has been known for almost 100 years, discerning the relevance to homeostasis and disease of these organelles is a relatively modern phenomenon in biological research. Research in models and genetic screens has revealed that a set of diseases with overlapping phenotypes are the result of dysfunction of proteins that are involved in the proper functioning of the primary cilium. These diseases, known collectively as ciliopathies, reveal through their pathologies an ever- growing catalogue of roles for the primary cilium in homeostasis and disease in many different organ systems. While defined functions of the primary cilium have been elucidated in olfaction, vision, and kidney function, the role of primary cilia in most cells in the body remains a mystery. With cognitive deficits, mood disorders, hyperphagia, obesity, and reproductive deficits being hallmarks of many ciliopathies, a few research programs have focused on the importance of the primary cilium in the proper function of neurons. This work focuses on a set of receptors that are known to be enriched in primary cilia, and the importance of the primary cilium in their signaling.

1

A Brief History of Early Primary Cilia Research

Some experts claim that the discovery of the cilium goes back to the beginning of microscopy itself. Antoni van Leeuwenhoek is said to have seen single celled organisms in his crude microscope with cilia-like projections emanating from them in the 17th century. Literature on cilia or flagella, and their point of origination in the cell, the , can be found as far back as the mid 19th century. Karl Whilhelm Zimmerman was one of the first researchers to identify primary cilia in a variety of tissues such as rabbit proximal tubules. Zimmerman also was the first to classify primary cilia, which were expressed at a frequency of one per cell, as a phenomenon distinct from the multitude of motile cilia observed on certain epithelial cells and single celled organisms [1]. Finally,

Zimmerman even postulated that these “central flagella” could perform sensory roles, over a century before this role was empirically demonstrated [2]. In the 1960s, some groups used the advent of electron microscopy and ultrathin sections to generate the first published accounts including detailed examinations of the structure of primary cilia [3,

4]. During this period it was also determined that most neurons in the brain possess primary cilia [5, 6]. Finally, while signaling roles for primary cilia had been proposed by researchers for almost a century, the first evidence of this phenomenon was published in 2001, less than two decades ago [7, 8].

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The Structure and Formation of Cilia

The structure of the cilium is very well conserved throughout evolution, from single celled to humans [9]. Cilia can be classified broadly into two groups, non- motile, or primary, and motile. Primary cilia are usually found at a frequency of one per cell, and are distinguished by their circular arrangement of 9 microtubule doublets (9+0)

[10]. Motile cilia have the same arrangement of 9 outer microtubule doublets with an additional inner 2 (9+2), connected to the outer doublets (Fig 1.1A). While motile cilia are generally considered to be generators of movement or fluid flow, they can perform sensory functions as well [11]. Primary cilia, on rare occasions, can generate fluid flow as well, such as the primary cilia of the embryonic node [12]. The primary cilia of olfactory neurons, which serve a sensory role, are non-motile but have a 9+2 arrangement of their microtubules [13].

Primary cilia arise from a structure known as the basal body, which is a highly modified mother [14]. Due to basal body’s centriolar origins, the primary cilium is disassembled and resorbed into the cell before cell division. The formation of the cilium begins when the basal body travels to the apical surface of the cell and anchors to the plasma membrane. The basal body is anchored to the base of the cilium by a set of structures known as the transition fibers, which when viewed from above form a pinwheel arrangement [15]. Associated with the transition fibers and the base of the cilium are proteins that form a diffusion barrier, that help regulate protein entry and exit from the cilium [16]. This “transition zone” is often a site of accumulation for ciliary transport proteins and their cargoes as they enter and exit the cell [17].

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A golgi-derived vesicle, which will eventually help form the ciliary membrane, attaches to the distal part of the basal body in the beginning stages of [18].

The ciliary membrane, even though it is continuous with the cell’s plasma membrane, has a different composition, which makes it biochemically unique when compared to the rest of the cell. The unique protein and lipid composition of the ciliary membrane help define its function [19]. The ciliary membrane is attached to the microtubule backbone near the base of the cilium by y-shaped linker structures, which forms a region known as the ciliary necklace [10].

The microtubule backbone of the cilium is known as the . The nine microtubule doublets that form the axoneme grow out of the basal body in a circular arrangement in both motile and primary cilia. The cilium grows by the continual addition of microtubule components to the distal end of the axoneme, which requires a process known as intraflagellar transport, or IFT. In the cilium, particles in IFT complex A are important for movement of cargoes towards the base of the cilium, known as retrograde movement. IFT complex B is responsible for movement of particles towards the tip of the cilium, known as anterograde movement. IFT is required not only for the formation, but the continued maintenance of cilia, and disruption of the system will result in loss of cilia in affected cells [17]. Also vital to the construction and movement of proteins within the cilium are the and microtubule-associated motors. Kinesin motors are required to move cargo towards the tip of the cilium, while dynein motors move cargoes from the tip of the cilium to the basal body [20].

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The Primary Cilium as a Signaling Organelle

Since primary cilia were first identified, it has been postulated that they are environmental sensors that can relay information about the outside world to the cell [1].

Research over the past two decades has revealed that the function of the primary cilium is often defined by the proteins that are located within the cilium. Primary cilia in different cells contain specific molecules and perform a multitude of tasks depending on the where they reside. Primary cilia have been observed to function as mechano-, photo-, and chemosenory organelles, and have been implicated in the regulation of many receptor-mediated signaling pathways as well [2].

One of the most studied functions of the cilium is its role as a mechanosensor in a variety of tissues. The primary cilium has been shown to act as a force transducer in the kidney, bone, embryonic node, and connective tissue [2],[21]. In the kidney, bending of the primary cilium induces a current in renal tubule cells in response to fluid flow, and abrogation of the cilium removes the ability of the cell to fluid flow [8,

22]. This is mediated by two proteins, polycystin-1 and polycystin-2, that localize to the primary cilia (Fig 1.2A). Polycystin-1 and 2 heterodimerize, and regulate calcium entry into the cell in response to the bending of the cilium. Mutations in these proteins are the cause of autosomal dominant polycystic kidney disease [23].

Polycystin-2 is also implicated in sensing fluid flow in the embryonic node, which helps determine correct left-right patterning in [24].

The proper functioning of primary cilia is vitally important to phototransduction in the retina. Photoreceptor cells consist of an inner segment, where proteins are

5 synthesized, and an outer segment, which is a modified primary cilium. In between these two segments is the connecting cilium, through which all the machinery necessary for the proper functioning of the cell is transported [25]. Transport of proteins through the connecting cilium is achieved through IFT and diffusion of proteins through the membrane [26]. is the light sensitive protein necessary for vision, and is transported in great quantities to the outer segment, where it couples with a heterotrimeric

G protein, transducin (Fig 1.2B). Transducin activates a signaling cascade that reduces cGMP levels, closing gated channels and hyperpolarizing the cell [27].

Olfactory neurons extend through the cribiform plate, and from these dendrites extend multiple long primary cilia that extend into the nasal .

Olfactory primary cilia are unique in that unlike most primary cilia, they have a 9+2 microtubule structure in their [28]. G protein-coupled receptors for odorants localize to these cilia. Binding of an odorant to its specific receptor results in the activation of adenylyl cyclase type 3 (AC3) in the cilium via a heterotrimeric G-protein,

Golf. This results in a cascade of activations by cAMP in the cilium, which creates a current that if strong enough can depolarize the entire neuron (Fig 1.2C) [29].

Two non-tissue specific pathways that have been thoroughly studied in the context of primary cilia are the Hedgehog (Hh) and Wnt pathways. Like most ciliary signaling pathways, the importance of the cilium to the proper execution of these pathways is only appreciated when the function of the cilium is disrupted or absent. The involvement of the cilium in the Hh pathway was revealed when screens for Shh pathway phenotypes found several mutations in IFT genes [30]. The transmembrane protein

6 patched, the Hh receptor, localizes to the primary cilium and represses the activity of . In the presence of Hh ligand, patched leaves the cilium, and smoothened translocates into the cilium [31]. This releases repression of a set of transcription factors that are important in cell fate designation in development [31]. The role of the cilium in

Wnt signaling was initially uncovered in cells derived from mice with mutations in vital ciliary proteins [32]. Many proteins in the Wnt signaling cascade localize to the basal body and primary cilium, although the exact role that these structures play in regulating the pathway remains unclear [33].

Ciliopathies: The consequences of disruption of proper ciliary function

The cilium is an intricate structure, with an exclusive proteome of hundreds of proteins [34]. Mutations in proteins that compose structures vital to the cilium’s function, transport of cargoes to the cilium, or even peripherally associated with the basal body can cause a set of diseases known as ciliopathies. So far over 40 genes have been identified as causative factors in ciliopathies. However, since the protein products of these genes often have closely related functions in the cilium, the phenotypes of ciliopathies often overlap, and genes associated with one have been identified as causative factors in others [35]. The primary features all ciliopathies usually have in common are retinal degeneration, renal anomalies, and development and function deficits. Ciliopathies also present with a common host of secondary phenotypes that vary in penetrance and severity even between patients with the same mutations in certain genes.

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Joubert syndrome occurs at about a frequency of 1 in 90,000 live births in the general population, although it is more prevalent in isolated populations where causative mutations are common. The hallmark symptom of Joubert’s is the “molar tooth sign,” a brain malformation that is visible by MRI in patients. Alongside this ,

Joubert’s usually presents with developmental disabilities and poor muscle and motor control. A range of other phenotypes including facial malformations, renal deficits, and retinopathies can be present as well, sometimes complicating the diagnosis of the disease as Joubert’s [36]. Over 21 different genes have been identified so faras causative in

Joubert’s, and many have overlap with other ciliopathies. Meckel-Gruber syndrome

(MKS) shares many clinical features with Joubert’s and, indeed, many causative genes

[37]. Senior-Løken syndrome also shares many phenotypes and identified disease alleles with MKS, and both are classified as Related Diseases [35].

Mutations in a known as OFD1, the protein product of which localizes to the basal body, result in a disease known as Orofaciodigital syndrome type 1 (OFD1)

[38]. As the name would suggest, patients present with incorrectly formed faces and digits, in addition to brain malformations and polycystic kidney disease [39]. These phenotypes can be attributed to the dysregulation of the sonic hedgehog (Shh) and Wnt signaling pathways when OFD1 function is perturbed.

Bardet-Biedl Syndrome (BBS) is a heterogeneous ciliopathy that shares a number of phenotypes with the above-mentioned ciliopathies. These include retinal degeneration, polydactyly, brain malformations, obesity, cognitive deficits, and renal dysfunction [13].

As of this writing, 19 causative genes have been identified, which account for about 80%

8 of known cases, so more genes remain to be identified [40]. BBS genes were initially identified in inbred families with a large proportion of affected members via linkage analysis. While affected individuals within families share mutations that mapped to the same areas on the , the heterogenetic nature of BBS quickly became apparent when multiple, unrelated families with affected individuals were analyzed [147].

Seven BBS proteins, BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9 make up a complex of proteins known as the BBSome, which is vital for trafficking certain membrane proteins to and possibly out of the cilium [41],[42].

Neuronal Primary Cilia

Although neurons were identified as having primary cilia over half a century ago, the structure and function of these organelles in the central nervous system has only been a focus of concerted research efforts for the past 20 years [3, 5, 43]. Almost every neuron in the mammalian brain possesses a single primary cilium, which can vary in length depending on the location of the neuron and changes in the extracellular milieu (Fig

1.3A-F) [44, 45]. The importance of primary cilia for the proper function of olfactory neurons and photoreceptor cells is well known and has been well characterized, but the function of the primary cilium on most central neurons remains to be determined [13].

The physical abnormalities in the central nervous system that present in ciliopathies give hints as to the vital role that cilia play in the proper development and placement of neurons in the brain. The relatively recent discovery that signaling molecules and

9 receptors for a variety of neuromodulators associate with the cilium hints that these organelles could play a vital post-developmental role in the neuron [46].

The Hedgehog signaling pathway is important in proper development of the neural tube, and disruptions to the system can result in patterning defects in the brain and spinal cord [47]. It is also important in the survival and proliferation of certain neuronal precursors [48]. Therefore, it should come as no surprise that many ciliopathies present with varying levels of defects to the formation of the central nervous system. On the level of the individual neuron, proper function of the primary cilium is also important in regulating the migration and synaptic integration of neurons [49].

While the obvious physical manifestations in the brain of ciliary dysfunction can be attributed to the localization of components of the Hh and Wnt pathways to cilia, the etiology of subtler cognitive phenotypes is still unclear. The first hint that primary cilia were not merely vestigial organs in neurons, but could potentially mediate signaling, came at the turn of the 21st century. It was reported that the G protein-coupled receptor

(GPCR) somatostatin receptor type 3 (Sstr3) localized to the surface of the neuronal primary cilium, suggesting that the receptor could respond to ligand in the cilium [50]. A little over a year later, another group reported that another GPCR, serotonin receptor subtype 6 (5-HT6), localized to primary cilia in neurons [51]. The enzyme adenylyl cyclase type III (AC3) was found to localize to the primary cilia of olfactory neurons, where it helps execute the transduction of odorants into electrical signals in the cilium

[52]. Work from our laboratory determined that AC3 is present on neuronal cilia throughout the mammalian brain [44]. Further strengthening the idea that these ciliary

10 signaling molecules could contribute to phenotypes seen in ciliopathies is the relatively recent work showing that Sstr3-null and AC3-null mice have cognitive deficits [53, 54].

Both Sstr3 and 5-HT6 are part of families of somatostatin and serotonin receptors, respectively [55, 56]. However, out of the five known somatostatin receptors and 14 known serotonin receptors, Sstr3 and 5-HT6 are the only ones that localize to cilia.

Using chimeras of ciliary and non-ciliary receptors, our laboratory identified conserved sequences that are necessary for the targeting of certain GPCRs to the primary cilium.

This sequence, located in the third intracellular loop of the receptors, was used to screen a library of GPCRs, and identified a host of known and putative ciliary receptors. Melanin- concentrating hormone receptor 1 (Mchr1) was a identified as a ciliary receptor in this screen, and in vitro and in vivo work confirmed its enrichment in AC3 positive neuronal primary cilia [57]. Dopamine Receptor subtype 1 (D1R) and Kisspeptin Receptor 1

(Kiss1r) were also identified in this screen as ciliary receptors (Table 1.1) [42].

Hypothesis and introduction to Chapters 2-4

While researchers have been aware of the existence of primary cilia on neurons in the mammalian brain for over half a century, only in the last 15 years have scientists started to probe the function of these organelles in neurons. With the conservation of many of the vital structural proteins in cilia from algae up to humans, great progress has been made in elucidating the ultrastructure of the cilium and its basal body. The sensory roles of flagella in these algae and other organs in the mammalian body are fairly well understood, but the role of the cilium as a signaling center in neurons remains mostly a

11 mystery. Two relatively recent developments have sparked interest in the primary cilia of neurons as vital signal transducers in the central nervous system: 1) The identification of numerous GPCRs and other signaling molecules that are enriched within primary cilia of neurons, and 2) The genetic and biochemical work linking mutations in genes associated with the cilium and ciliopathies with idiopathic cognitive phenotypes.

This work aims to advance the field of cilia biology and GPCR signaling by examining the role the primary cilium plays in the signaling of a set of ciliary GPCRs.

We hypothesize that since only certain receptors and signaling molecules are targeted to the cilium, and cognitive phenotypes related to these signaling molecules are present in ciliopathies, the localization of certain GPCRs to the primary cilium must be vital to their proper signaling. We this hypothesis in this work by examining the signaling of the putative ciliary receptor Kiss1r and the known ciliary receptors D1R and Mchr1.

In chapter 2, we build on previous work that our laboratory had done identifying

Kiss1r as a receptor that contains ciliary localization sequences. Using in vitro and in vivo immunofluorescence, we demonstrate that Kiss1r localizes to the primary cilium of gonadotropin-releasing hormone (GnRH) neurons in the mouse. Both Kiss1r and GnRH neurons are vital to sexual maturation and function, which can be disrupted in ciliopathies. Using a promoter-driven, conditional knockout approach, we disrupt the formation of primary cilia exclusively in GnRH neurons. While the mutant mice are fully reproductively competent, the GnRH neurons in these mice fail to respond to Kiss1r stimulation with the same increase in firing rate that their control counterparts show.

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These findings strengthen the overarching idea in the field that the cilium can be an important modulator of GPCR signaling.

Chapter 3 focuses on D1R, a receptor that our lab identified and confirmed as a ciliary receptor. While other ciliary receptors that our laboratory has studied fail to localize to the cilium in the absence of Bardet-Biedl Syndrome (BBS) proteins, D1R behaves in the opposite manner. D1R is enriched in the primary cilia of neurons in the absence of BBS proteins. In order to examine the physiological effects of altered D1R localization in mice without the host of confounding phenotypes associated with BBS, we used a promoter-driven conditional knockout approach to disrupt BBS function exclusively in D1R expressing neurons. These mice become obese, and have lower levels of locomotor activity than controls. The phenotype seen in these mice mirrors that seen in mouse models with reduced levels of D1R signaling. This leads us to hypothesize that the aberrant enrichment of D1R in the cilium leads a decrease in the receptors signaling.

Chapter 4 presents pilot studies performed on Mchr1, a GPCR that our laboratory identified as ciliary. Mchr1 is highly expressed in the Nucleus Accumbens, a brain region where it has been shown to regulate reward and other behaviors. We used stereotactic injection of an Adeno-Associated virus (AAV) expressing Cre recombinase into mice expressing conditional alleles of a BBS protein to disrupt BBS expression in a temporally and regionally specific manner. Disruption of the BBS proteins in these mice was successful and behavioral and physiological analysis of the mutant mice was performed.

13

The following work on these receptors supports the hypothesis that the proper localization of ciliary GPCRs is vital to efficient transduction of their signaling (Fig

1.4A-D). We show that the cilium is vital to the proper signaling of Kiss1r, and that the disruption of BBS protein function in D1R expressing cells can lead to physiological phenotypes.

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Figure 1.1- The basic structure of the cilium is conserved throughout evolution. (A) Simplified diagram of the cilium and major components. The insets show either motile or primary cilia from a top down perspective, highlighting the different arrangement of microtubule doublets between the two types. IFT stands for “Intra-Flagellar Transport” Modified with permission from Green JA, et al 2010.

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Figure 1.2- Primary cilia function in a variety of distinct signaling roles. (A) In the kidney, PC1 and PC2 localize to the cilium and transform the organelle into a flow sensor by allowing calcium entry into the cell in response to fluid flow bending the cilium. (B) The outer segment of photoreceptor cells is a highly modified primary cilium where visual proteins localize and transduce light into chemical signals in the cell. (C) Olfactory neurons extend multiple primary cilia from their dendritic knobs. Olfactory GPCRs and associated signaling machinery localize to these primary cilia. When odorants bind to olfactory GPCRs in the cilium, signaling cascades are initiated and propagated in the cilium and eventually spread to the rest of the neuron. Modified with permission from Green JA, et al 2010.

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Figure 1.3-Primary cilia are present on central neurons and are enriched for signaling molecules. (A- C) Neurons from the cortex of an adult mouse. (A) Immunofluorescent labeling of brain tissue for the ciliary marker AC3 (red) reveals primary cilia in the cortex. (B). Staining of the neuron for the basal body marker Pericentrin (green). (C) In the merged image, AC3 positive cilia emerge from Pericentrin positive basal bodies, DRAQ-5 (blue) labels the nuclei. Scale bar = 5 microns (D-F) A neuron cultured from the of a newborn mouse. (D) Labeling for the ciliary marker AC3 (red) reveals a cilium emerging from the cell body of the neuron. (E) Labeling for Mchr1(green) shows the receptor emerging from the cell body. (F) Merged image shows co-labeling of AC3 and Mchr1, indicating the receptor localizes to the cilium. The neuron express GFP (blue) throughout the cell body. Scale bar = 10 microns.

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Fig 1.4-Loss of the cilium structure or mislocalization of ciliary receptors could potentially alter signaling. (A) In our model, normal GPCR signaling could occur in the exclusive environment of the cilium, resulting in modulation or changing of the signaling of the receptor. (B) When the cilium is lost, the GPCR is trafficked to the plasma membrane or the cytoplasm, and signaling of the GPCR is reduced or altered. (C) Normally, the BBSome is responsible for the trafficking of GPCRs into and out of the cilium. (D) When any of the components of the BBSome are lost, the complex fails to properly function, resulting in the altered localization of certain ciliary GPCRs. Modified with permission from Green JA, et al 2010.

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Table 1.1: Ciliary targeting sequences of known and putative ciliary GPCRs

i3 C-tail Localization G protein-coupled receptor sequence sequence(s)

VLP/LPP/LL AARKQ Ciliary Serotonin receptor 6 (5-HT6) P

Somatostatin receptor 3 (Sstr3) APSCQ LRP/LLP Ciliary

Kisspeptin receptor 1 (Kiss1r) ALQGQ VCP ?

LCP/LIP/LS AKNCQ Ciliary Dopamine receptor 1 (D1R) P

Melanin-concentrating hormone APASQ VKP Ciliary receptor 1 (Mchr1)

Table 1.1- Conserved sequences in the third intracellular loop and c-terminal tail of known ciliary GPCRs were used to predict putative ciliary GPCRs. Conserved sequences in the third intracellular loop (i3 sequence) that are necessary for the localization of known ciliary receptors to the cilium were identified, and from these sequences more ciliary GPCRs were identified.

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Chapter 2: The Primary Cilium Enhances Kiss1r Signaling on Gonadotropin-Releasing Hormone Neurons Summary

Primary cilia are microtubule based sensory organelles present in mammals on nearly every cell type, including neurons. Defects in proteins associated with the cilium result in a variety of diseases known as ciliopathies. Phenotypes of ciliopathies include cystic kidney disease, obesity, blindness, anosmia, cognitive deficits, and hypogonadism.

The functions of these neuronal primary cilia are still mostly unknown, but they are enriched for specific signaling molecules, hinting that the cilia may serve an important role in signaling. Our laboratory identified a number of G protein-coupled receptors that contain a localization sequence necessary for targeting to cilia. One of the identified receptors is kisspeptin receptor 1 (Kiss1r), which piqued our interest because Kiss1r knockout mice suffer from stunted development of the reproductive system, as do humans with mutations in the Kiss1r gene. We determined that Kiss1r does indeed localize to the primary cilia of a specific set of neurons known as gonadotropin-releasing hormone (GnRH) neurons in the . GnRH neurons are a small subset of neurons located in the that play a critical role in sexual maturation in mammals. Stimulation of Kiss1r with its natural ligand kisspeptin results in an increase

20 in the firing rate of GnRH neurons that express the receptor. This increase in firing of the neurons results in a release of the hormone GnRH, initiating hormone cascades necessary for proper sexual maturation and reproductive function. We hypothesized that localization of Kiss1r to primary cilia on GnRH neurons is vital to the proper signaling of the receptor, and its modulation of sexual maturity and function. To test this hypothesis, we utilized a promoter-driven, conditional knockout approach in mice to delete a gene vital for cilium construction and maintenance specifically in GnRH neurons. Measures of physiological parameters related to appear to be unaffected in these mice.

In electrophysiological patch clamp recordings from GnRH neurons, it is observed that when treated with kisspeptin, GnRH neurons with disrupted cilia have a significantly lower increase in their rate of firing when compared to those with intact cilia. This result suggests that the cilium plays a vital role in the proper signaling of Kiss1r.

The evidence that disruption of the cilium can disrupt the signaling of a ciliary

GPCR, and consequently the electrophysiological properties of a neuron, is incredibly novel. These findings hint that ciliary localization of certain GPCRs across the central nervous system is vital to the proper transduction of their signals. The ciliary localization of Kiss1r, and its importance in proper signaling of the receptor, could have implications for future research in the field of reproductive biology as well.

Introduction:

Primary cilia are known to facilitate in the kidney and bone, chemosensation in olfactory neurons, and phototransduction in the retina [13]. The existence of primary cilia on central neurons has been known for over 50 years, but their 21 function, for the most part, remains a mystery[5]. The discovery of an increasing number of signaling proteins that are enriched in the primary cilium, coupled with the behavioral and cognitive phenotypes displayed in many ciliopathies, leads to the hypothesis that the primary cilium is a vital extra-synaptic signaling center in neurons [2].

Somatostatin receptor subtype 3 (Sstr3) and serotonin receptor subtype 6 (5-HT6) were both identified as G protein-coupled receptors (GPCRs) that localized to the ciliary membrane around 15 years ago [50, 51]. In 2007 our laboratory identified that the type 3 adenylyl cyclase (AC3) localized to the primary cilia of neurons in the mouse and brain [44]. Since AC3 had already been identified as a vital signaling component in olfactory cilia, this further hinted that cilia on central neurons could be involved in signaling. A year later, our laboratory identified a sequence that was conserved in the third intracellular loop of GPCRs that were enriched in primary cilia. Further in vitro work determined that this sequence was sufficient to target GPCRs to the primary cilium

[57]. A screen of GPCRs with this sequence in the third intracellular loop turned up a number of candidates, one of which was known as GPR54, or kisspeptin receptor 1

(Kiss1r) [42].

Despite the obvious connection, the name “Kiss1r” was assigned to the receptor also known as GPR54 before its vital role in reproduction was revealed. The ligand for

Kiss1r, kisspeptin, was discovered in the mid-1990’s, and originally investigated as a metastasis suppressor. Since the researchers were based in Hershey, Pennsylvania, they added “Ki” to the front of “ss,” which stood for “suppressor sequence” [58]. Known to cancer researchers as “metastatin,” kisspeptin was identified as the ligand for the

22 formerly orphan GPCR GPR54 in 2001 [59]. Two years later, studies of human families with null Kiss1r mutations, and creation of a Kiss1r knockout mouse, revealed that the receptor was vital to the proper development of the reproductive system and fertility [60].

Soon after, it was determined that treatment of mice with kisspeptin induced GnRH release, and could even induce early onset puberty in mice if given early in life [61, 62].

Expression studies utilizing in situ hybridization and β-galactosidase staining revealed that Kiss1r was expressed on GnRH neurons in the hypothalamus [63, 64].

Electrophysiological studies also revealed that stimulation of the GnRH neurons with kisspeptin resulted in a large increase in the rate of their firing, which resulted in the release of GnRH [63]. This release of GnRH stimulates release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which helps control puberty and the estrous cycle in [65].

Kiss1r became the focus of our research efforts over other putative ciliary GPCRs as one of the main clinical features of the model ciliopathy used in our lab, Bardet-Biedl syndrome (BBS), is a set of reproductive deficits. Humans and mice with mutations in

BBS proteins suffer from hypogonadism, which are small reproductive organs, and males also fail to form mature [13]. Interestingly, humans and mice with null mutations in Kiss1r also suffer from hypogonadism, and males fail to produce mature sperm [66].

After identifying Kiss1r as a putative ciliary receptor, our hypothesis was that disruption of the receptors function on the cilium of GnRH neurons in BBS was responsible for the reproductive phenotypes observed in the ciliopathy. In order to test this hypothesis, we first confirmed ciliary localization of the receptor in vitro and in vivo

23 using immunofluorescence. Interestingly, while around 75% of GnRH neurons displayed a Kiss1r cilium, a proportion of these neurons displayed multiple Kiss1r cilia, which is extremely rare in normal central neurons. Next, we used a promoter-driven, conditional knockout approach to disrupt primary cilia exclusively in GnRH neurons in order to test our hypothesis that ciliary localization is vital to the proper functioning of the receptor.

Disruption of the primary cilium on GnRH neurons in mice was complete, but these mice did not display any reproductive or physiological phenotypes. Electrophysiological analysis of GnRH neurons from these mice revealed that while they fire at the same basal rate as their control counterparts, their response to kisspeptin treatment is reduced. This is a novel finding, and the first of its kind in the context of central neurons

Results

Confirmation of the specificity of the antibody towards Kiss1r

Our laboratory had previously identified Kiss1r, also known as GPR54, as containing sequences in its third intracellular loop necessary for localization to the cilium [42]. In order to study this receptor in vivo, we commissioned Strategic Diagnostics to produce a custom rabbit polyclonal antibody against Kiss1r. Kiss1r was cloned from mouse cDNA and fused to eGFP on its C-terminus to create a Kiss1r::EGFP fusion protein. This

Kiss1r::EGFP construct was transfected into inner medullary collecting duct (IMCD) cells, a ciliated cell line our laboratory has used in the past to study ciliary GPCRs.

These cells transfected with the Kiss1r::EGFP construct were fixed and incubated with the antibody against Kiss1r, and confocal microscopy revealed co-labeling of the

Kiss1r::EGFP construct and the Kiss1r antibody (Fig 2.1A-C). To further confirm the 24 specificity of the antibody, HEK293T cells were transfected with either a Kiss1r::myc fusion protein or empty vector, and protein collected from the cells was immunoblotted for Kiss1r or myc (Fig 2.1D). Immunoblotting for both Kiss1r and myc revealed bands in the 43 kDa range, the predicted size of Kiss1r, and no staining in the empty vector control. Wild-type (WT) and Kiss1r knockout (Kiss1rKO) tissue was co-labeled for

GnRH and Kiss1r (Fig 2.1E,F). Kiss1r is known to be expressed on GnRH neurons in

WT tissue [64]. Kiss1r positive projections were seen on GnRH neurons in the WT tissue, but not in Kiss1rKO tissue, further confirming the specificity of the antibody for

Kiss1r (Fig2.1G). Finally, both the WT and Kiss1rKO tissue stained equally well for

AC3, a common ciliary marker used in our lab, indicated that tissue quality was the same between the two (Fig 2.1H-I)

Kiss1r localizes to cilia on GnRH neurons

Cilia-like structures were seen both in vitro and in vivo during confirmation of the specificity of the Kiss1r antibody, but further work was needed to support our hypothesis that Kiss1r is a ciliary receptor. The Kiss1r::EGFP construct was transfected into IMCD cells, and the cells were labeled for acetylated-tubulin (AcTub), a canonical marker of cilia (Fig 2.2A-C). In this in vitro model, the Kiss1r::EGFP construct co-localized very strongly with AcTub, indicating that the Kiss1r::EGFP construct localizes to cilia.

In order to determine if Kiss1r localizes to cilia in vivo, specifically on GnRH neurons, we stained brain tissue from mice expressing GFP under the control of the

GnRH promoter (GnRH::GFP) [67] with the antibody towards Kiss1r (Fig 2.2D-F).

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This staining revealed that a majority of GnRH neurons expressed long Kiss1r-positive projections emanating from their cell body. A significant percentage of these GnRH neurons expressed multiple Kiss1r-positive projections, which is striking, since most central neurons display only a single primary cilium [5, 68, 69]. Three-Dimensional (3D) reconstruction of confocal images of GnRH neurons confirmed that the multiple structures seen were originating from a single GFP positive cell body (Fig 2.2G). 3D reconstruction of additional representative confocal images of GnRH neurons with

Kiss1r-positive projections revealed that GnRH neurons can express multiple Kiss1r- positive primary cilia (Fig 2.3A,B). Since the phenomenon of multiple cilia on neurons in the brain is so rare, another experiment was performed to ensure that these multiple cilia were projecting from the same cell body. Brain tissue from GnRH::GFP mice was stained for Rootletin, a protein that is enriched at the base of cilia, and Kiss1r [70]. In two representative images, it is clear that the Kiss1r containing cilia originate from the same Rootletin positive structure in the cell body of the GnRH neuron (Fig 2.3C,D).

Kiss1r contains conserved sequences necessary for localization to primary cilia and heterologously expressed Kiss1r localizes to primary cilia in vitro. In vivo, Kiss1r staining appears to show long, cilia-like projections emanating from GnRH neurons, and

GnRH neurons have previously been shown to display cilia-like projections [71, 72].

However, besides rootletin, no canonical markers of cilia or the basal body appeared to label GnRH neurons (Fig 2.4A). In order to confirm that these Kiss1r containing structures on GnRH neurons are indeed cilia, we obtained brain tissue from mice that express an Sstr3::GFP fusion construct (CiliaGFP) in all cell types, eliminating the need to

26 label for ciliary markers [73]. When this tissue was labeled for an antibody against

GnRH, we observed GFP+ cilia originating from GnRH labeled cell bodies (Fig 2.4B-D).

Labeling this tissue for Kiss1r revealed that in the hypothalamus, a small number of

GFP+ cilia were labeled for Kiss1r (Fig 2.4E-G). These results indicate that GnRH neurons do indeed possess cilia, and these cilia are enriched for Kiss1r.

Previous expression studies have shown that around 75% of GnRH neurons express Kiss1r [74]. In order to determine if a similar percentage of GnRH neurons displayed Kiss1r positive cilia, we stained brain tissue from newborn (P0) to adult (P60)

GnRH::GFP mice with Kiss1r and quantified the number of GnRH neurons that expressed one or more Kiss1r positive cilia (Fig 2.5A). The percentage of GnRH neurons expressing one or more Kiss1r primary cilia was about 75% and did not differ significantly by age or sex from P0 through P60. Since the phenomenon of multiple cilia on central neurons is so novel, we also quantified the percentage of ciliated GnRH neurons that had multiple Kiss1r primary cilia (Fig 2.5B). The percentage of ciliated

GnRH neurons with multiple cilia started at around 10% at birth and increased to 40% in males and 35% in females by adulthood. This suggests that multiciliation of GnRH neurons occurs in parallel with sexual maturation in mice, and may be involved in the process.

Primary cilia on GnRH neurons are not essential for sexual maturation and function

Since we firmly established the presence of Kiss1r containing cilia on GnRH neurons, we hypothesized that the receptors localization to the cilium was vital for its

27 proper function. The stimulation of Kiss1r on GnRH neurons is a critical step in initiating the cascade of hormone release necessary to activate the neuroendocrine reproductive circuit [60, 61, 64, 75]. In order to test our hypothesis, we used a conditional knockout approach in mice to disrupt cilia on GnRH neurons while preserving cilia in all other cells. We acquired transgenic mice that contain conditional alleles of the gene Ift88 (Ift88fl/fl), which is essential for the construction and maintenance of cilia [76]. We crossbred these animals with mice expressing Cre recombinase under the control of the GnRH promoter (GnRH::Cre) [77]. We also bred these mice with our

GnRH::GFP mice in order to more easily visualize GnRH neurons in these mice. After all the transgenes were bred onto the same line of mice, we produced animals of the Ift88fl/wt GnRH::Cre GnRH::GFP (GnRHcilia+) and Ift88fl/Δ GnRH::Cre

GnRH::GFP (GnRHcilia-). We used this breeding scheme as one allele of Ift88 is sufficient for normal function of the gene, and also increases efficiency of the Cre- mediated in GnRH neurons [76]. Initial examination of GnRH neurons in our GnRHcilia+ mice revealed that they expressed Kiss1r positive cilia, while GnRH neurons in GnRHcilia- failed to display Kiss1r positive cilia (Fig 2.6A-F).

While no Kiss1r staining was visible on GnRHcilia- neurons, quantitative real-time

PCR (qRT-PCR) of mRNA isolated from the hypothalamus of GnRHcilia+ and GnRHcilia- mice revealed that there was no significant difference in Kiss1r mRNA levels between the two genotypes (Fig 2.6G). qRT-PCR for GnRH mRNA ensured that an equivalent number of GnRH neurons were isolated from animals of each genotype (Fig 2.6H). To ensure that Kiss1r protein was still expressed and functional in GnRH neurons in our

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GnRHcilia- mice, we examined Fos expression in GnRH neurons after treatment with kisspeptin-10 (KP-10), the ligand for Kiss1r. KP-10 has been shown to induce expression of Fos in GnRH neurons, and GnRH neurons that do not express Kiss1r do not express Fos after KP-10 treatment [75]. Intracerebroventricular injection of KP-10 was able to induce Fos expression in GnRH neurons both in GnRHcilia+ and GnRHcilia- mice, indicating the presence of functional Kiss1r protein in both genotypes (Fig 2.7A,B).

Quantification of the number of GnRH neurons expressing Fos before and after KP-10 treatment revealed no significant difference between the genotypes (Fig 2.7C).

GnRH expression can be detected as early as 11 days after conception in mice.

Since the Cre-mediated recombination process does not happen instantaneously in cells, we stained the brains of newborn (P0) GnRHcilia+ and GnRHcilia- mice with our antibody towards Kiss1r. We quantified the number of GnRH neurons displaying Kiss1r positive cilia in both genotypes and sexes, and found that cilia had been ablated on most neurons in GnRHcilia- mice at P0 (Fig 2.8A). When we examined adult animals, we found that no

GnRH neurons in GnRHcilia- mice had Kiss1r positive cilia, while GnRHcilia+ mice had the same percentage of GnRH neurons with Kiss1r positive cilia as their GnRH::GFP counterparts (Fig 2.8B). It has been recently reported that disruption of the function of primary cilia on certain neurons can affect their migration [49]. GnRH neurons are born in the nasal placode and migrate to their final locations in the hypothalamus [78]. We assessed the migration of GnRH neurons at P0 and adulthood in order to determine if disruption of primary cilia function on the neurons affected their migration (Fig 2.8C,D).

There was no statistically significant difference in the number of GnRH neurons detected

29 in different brain regions at either age between the two genotypes, indicating that the migration of these neurons is unaffected by the disruption of their primary cilium.

Disruption of the function of the Kiss1r/GnRH system can lead to profound reproductive deficits and disruption of certain functions of the reproductive system in adults [60, 62]. A reduction or lack of Kiss1r signaling in mice and humans can lead to hypogonadism, which is a reduction in the size of the sex organs [60]. Examination of the testes of males and the of females revealed no significant difference in the weight of the sex organs (Fig 2.9A, Table 2.1). Time of vaginal opening, an indicator of puberty onset in females, showed no significant difference between GnRHcilia+ and

GnRHcilia- mice (Table 2.1). Microscopic examination of the testes of GnRHcilia+ and

GnRHcilia- male mice revealed the presence of mature sperm (Fig 2.9B,C). Examination of the ovaries of females also revealed follicles in all stages of development, indicating that the sex organs of both genotypes were fully functional (Fig 2.9D,E). We also examined the estrus cycle, which is regulated by the GnRH neuron network, in adult female GnRHcilia+ and GnRHcilia- mice, and noticed no significant difference between the two genotypes (Fig2.9 F) [79]. Body weight was also unaffected by disruption of cilia on GnRH neurons (Table 2.1).

Disruption of primary cilia on GnRH neurons reduces their responsiveness to Kiss1r stimulation

Stimulation of Kiss1r on GnRH neurons by its ligand kisspeptin results in an increase in the rate of action potentials in GnRH neurons, resulting in increased GnRH

30 release [64, 67]. We used patch clamp electrophysiology to study the properties of

GnRH neurons in GnRHcilia+ and GnRHcilia- mice of both sexes. GnRH neurons fire at a basal rate even when unstimulated, and observation of samples from GnRHcilia+ and

GnRHcilia- mice revealed no difference in the basal firing rate between the two genotypes in males or females (Fig 2.10A-C). After addition of KP-10, GnRH neurons in both genotypes showed an increase in their firing rates (Fig 2.10A). Quantification of the changes in firing rates revealed that in males, neurons from GnRHcilia- animals responded to Kiss1r stimulation at a significantly lower rate than GnRHcilia+ counterparts (Fig 2D).

A similar effect of genotype was observed in ovariectomized females, however, the difference was not statistically significant (Fig 2E). These results reveal that the primary cilium is critical to the proper signaling of Kiss1r on GnRH neurons in the mouse.

Discussion:

This works confirms that the predicted ciliary receptor, Kiss1r, does indeed localize to the cilium of GnRH neurons in mice. Kiss1r cilia on GnRH neurons are interesting, in that they do not appear to express AC3 or other common ciliary markers that are localized to primary cilia throughout the central nervous system [44]. The Kiss1r containing primary cilia on GnRH neurons appear to be unique among cilia in central neurons in that they are longer than most (10-15 microns), and that there can be multiple cilia on a neuron. Another potential reason that Kiss1r positive cilia do not stain positively for AC3 could be due to the properties of Kiss1r itself. Other identified ciliary receptors, such as serotonin receptor 6 (5-HT6), Sstr3, Mchr1 and D1R are all coupled to

G proteins that propagate their signaling via adenylyl cyclases. Kiss1r is coupled to Gq, 31 which signals via a second messenger system independent of adynylyl cyclases [80].

AC3 may not be trafficked to or retained in Kiss1r containing cilia since it may not be needed.

As stated above, it is extremely unusual for central neurons to possess more than one primary cilium. Our results are striking in that not all GnRH neurons possess multiple cilia, while some can possess three and rarely, four. Interestingly, GnRH neurons are first observed, and assumed to be born in, the olfactory placode, which gives rise to the [78]. Olfactory neurons, which originate from the same tissue, project multiple primary cilia which contain G protein-coupled receptors [29]. It is unknown when exactly GnRH neurons diverge in development from the rest of the olfactory epithelium, but a shared lineage with olfactory neurons could explain why

GnRH neurons can display multiple primary cilia, unlike other central neurons [81].

Evidence from previous reports, combined with our results, suggest that nearly every

GnRH neuron that expresses the receptor trafficks it to the cilium [63, 64, 74, 75]. Our results fail to explain, however, why a portion of these GnRH neurons express multiple

Kiss1r positive primary cilia. This phenomenon of multi-ciliation is not random, however, as the percentage of multi-ciliated GnRH neurons starts at around 10% in newborn mice and reaches close to 40% in adult mice. That this increase in multi- ciliation mirrors sexual maturation in mice suggests that the development of multiple

Kiss1r positive cilia on GnRH neurons is somehow linked to sexual development. GnRH neurons become more sensitive to kisspeptin, the ligand for Kiss1r, over postnatal development, while the level of expression of Kiss1r does not change [63]. As our

32 results above indicate, the cilium increases the sensitivity of GnRH neurons to kisspeptin stimulation, so perhaps GnRH neurons extend additional Kiss1r positive cilia in order to increase their sensitivity to the ligand. Conversely, increased kisspeptin stimulation between birth and adulthood could induce GnRH neurons to extend additional cilia.

Kisspeptin neuron fiber projection to GnRH neurons increases across postnatal development, as does the total number of Kisspeptin neurons [82]. The triggers and mechanisms for multi-ciliation in GnRH neurons could be interesting programs of research that could impact the fields of reproduction and fertility.

It stands to reason that when Kiss1r is trafficked to the cilium in most, if not all the GnRH neurons where it is expressed, the cilium must serve some important role in the signaling of the receptor. In order to explore our hypothesis that the cilium is a vital regulator of the signaling of Kiss1r, we disrupted the function of a gene vital to the construction and maintenance of cilia in GnRH neurons (GnRHcilia-). Kiss1r positive cilia were completely absent in GnRHcilia- neurons. Kiss1r staining was completely absent, but qRT-PCR for the receptor showed that the receptor was still expressed at equal levels in

GnRHcilia+ and GnRHcilia- mice. By labeling for Fos expression in GnRH neurons after kisspeptin treatment, we were able to show that functional Kiss1r protein is present in

GnRHcilia- neurons, even though it is not detectable via immunofluorescence. Kisspeptin cannot induce Fos expression in GnRH neurons in the absence of Kiss1r [75]. The most likely scenario is that in GnRHcilia- neurons, since the receptor is no longer concentrated in the cilium, it spreads throughout the plasma membrane of the cell, and is too diffuse to

33 detect by immunofluorescence. Kiss1r has been shown to localize to the plasma membrane in non-ciliated cells [83].

Disruption of primary cilia in other neurons has been shown to disrupt migration and integration into neuronal networks, however this does not appear to be the case with

GnRH neurons [49]. In both newborn and adult animals, quantification of GnRH neurons in different brain regions showed no difference in the number of neurons that migrated to those areas.

We chose to study Kiss1r over other putative ciliary receptors that our lab identified due to the phenotype of hypogonadic hypogonadism that is present in human patients with mutations in the BBS proteins or Kiss1r [13] [84]. When we created the

GnRHcilia- mice and observed the loss of Kiss1r positive cilia on GnRH neurons, we expected to see disruption of some aspects of reproductive function in the mice.

Interestingly, according to several commonly used measures of puberty onset and reproductive fitness, GnRHcilia- mice were indistinguishable from GnRHcilia+ mice.

Recent studies have revealed that the Kiss1r/GnRH system is incredibly redundant, and can correctly initiate puberty and normal sexual function even after a loss of a large number of GnRH neurons [82] [85]. Therefore, it is not unexpected that even with the statistically significant, but modest decrease in signaling seen in our GnRHcilia- neurons, that GnRHcilia- mice retain full reproductive competence. This suggests that the hypogonadotropic phenotype observed in BBS patients and mouse models are due to defects in other components of the neuro-endocrine reproductive circuit.

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The most novel finding in this work is the reduction in the response of GnRH neurons to kisspeptin in the absence of the cilium in male mice. A similar trend was observed in ovariectomized (OVX) female mice, but was not statistically significant.

There are several factors that could account for this difference. Firstly, from our examination of the basal firing rates of the GnRH neurons in both genotypes in males and females, we see that the basal firing rate of GnRH neurons in females of both genotypes is much lower than that of the male animals. It has also been previously reported that

GnRH neurons in females are less sensitive to stimulation by kisspeptin than those in male animals [63]. This could indicate that GnRH neurons in OVX females have a lower capacity to fire action potentials, decreasing their measured response to kisspeptin stimulation and preventing us from being able to measure a difference between the two genotypes. Our results also indicate that in GnRHcilia+ neurons, there is a high degree of variability in the response of the neurons to kisspeptin, which limited our ability to find a statistically significant difference between the two genotypes. If the experiment were repeated with more animals, this could potentially be overcome.

Previous reports have shown that, at the cellular level, disruption of the cilium can lead to dysregulation of a few signaling pathways [2]. Our laboratory and other groups have identified a number of GPCRs that localize to the primary cilium. This is the first study to demonstrate that the loss of the cilium on a central neuron can acutely impact the signaling of a ciliary GPCR.

There are a few different possible ways the cilium could promote Kiss1r signaling. Kiss1r, a GPCR, exerts its effects through coupling to Gq. Gq activation

35 results in the formation of diacylglycerol (DAG) and inositol tri-phosphates (IP3). DAG activates protein kinase C (PKC), which is important for gene regulation in the neuron, while IP3 initiates intracellular calcium release, resulting in the depolarization and firing of the GnRH neuron [86]. Olfactory neurons, which we have already compared GnRH neurons to, are known to increase intra-ciliary IP3 levels in response to the activation of odorant GPCRs [87, 88]. The cilium, as a compartment of the cell exclusive from the cytoplasm, could propagate IP3 formation in response to Kiss1r stimulation. This environment conducive to IP3 signaling could be lost when the formation of the cilium is disrupted and Kiss1r is localized to the plasma membrane, as in our GnRHcilia- neurons.

Calcium signaling is also an important part of the Kiss1r signaling pathway, and the primary cilium has been implicated as a organelle in the cell [89, 90].

The loss of the cilium as a tightly regulated calcium signaling compartment could affect

GnRH signaling independently of Kiss1r stimulation, however, our examinations of the basal firing rates of these neurons argues against that. Transient Receptor Potential

Cation (TRPC) channels are calcium and permeable ion channels that are activated by PLC and are critical to the excitation of GnRH neurons by Kiss1r [91]. At least one TRPC channel that is known to be expressed in GnRH neurons has been detected via immunofluroscence in the cilium [92]. Other ion channels are known to localize to primary cilia, and their function is disrupted in the absence of cilia, suggesting that loss of cilia in GnRH neurons could disrupt the association of Kiss1r with important ion channels. Finally, work in our laboratory has shown that β-arrestin-2 localizes to the base of primary cilia, and is recruited into the primary cilium upon activation of a GPCR

36 within the cilium. β-arrestin-2 is responsible for de-sensitizing GPCRs, along with β- arrestin-1, which does not localize to the cilium upon GPCR stimulation (Green, JA unpublished data). β-arrestin-2 has been shown to rapidly associate with Kiss1r upon activation of the receptor [83]. With the loss of the cilium on GnRHcilia- neurons, Kiss1r may be desensitized more rapidly on the plasma membrane than in the cilium due to increased availability of β-arrestins in the cytoplasm as compared to the cilium. With the establishment of the localization of Kiss1r to the cilium in this work, further studies can examine the signaling of the receptor in the context of the cilium and hopefully answer some of these questions.

In summary, the work in this chapter showed that Kiss1r localized to primary cilia on GnRH neurons, these neurons can be multiciliated, and that the disruption of cilia on these neurons reduces their responsiveness to Kiss1r. As the first example of disruption of cilia on central neurons affecting the signaling of ciliary GPCR, this work is extremely novel and should further convince researchers from outside the cilia field of the importance of these underappreciated organelles. As Kiss1r and GnRH neurons are vital components of the neuroendocrine circuit governing the reproductive system, further research on the ciliary signaling mechanisms of this receptor could have implications for the field of reproductive biology.

37

Materials and Methods:

Ethics Statement:

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Ohio

State University (Animal Welfare Assurance #A3261-01).

Plasmid Construction

The coding sequence for Kiss1r was amplified from cDNA generated from reverse- transcribed mouse whole brain RNA using the Superscript First-Strand Synthesis RT-

PCR kit (Invitrogen). The coding sequence was then cloned into the TA cloning vector pSTBlue-1 (Novagen), with primers at the C and N-terminal regions designed for directional cloning. The Kiss1r construct was then subcloned into pEGFP-N (Clontech).

All DNA constructs were sequence verified at the Nucleic Acid Shared Resource at Ohio

State’s Comprehensive Cancer Center.

Cell Culture and Transient Transfections

IMCD-3 cells (ATCC) were maintained in DMEM:F12 media supplemented with 10%

FBS, 1.2 g/l of sodium bicarbonate, and 0.5 mM sodium pyruvate (Invitrogen). Cells (n =

5 x 106) were electroporated with 10 µg DNA and plated at high density on glass coverslips. Cells were refed 16-18 h after transfection and harvested at 48 h after transfection by fixation in 4% paraformaldehyde.

38

Protein Isolation and Immunoblotting

HEK293T cells (ATCC) were maintained in DMEM supplemented with 10% FBS and

1.5 g/l of sodium bicarbonate (Invitrogen). pcDNA3.1 encoding Kiss1r::myc or nothing was transfected by electroporation into HEK293T cells. After 48 hours, proteins were isolated and immunoblotted, as previously described [42]}

Mice and Tissue Preparation

All animal procedures described are in accordance with institutional guidelines based on

National Institutes of Health Standards, and were performed with Institutional Animal

Care and Use Committee approval at the Ohio State University and the University of

Alabama at Birmingham. All animals were maintained in a temperature and humidity controlled vivarium with 12 hr light/dark cycle and given access to food and water ad libitum. Littermates were group housed by sex, no more than 5 to a cage, after weaning.

CiliaGFP mice expressed Sstr3::EGFP systemically [73]. Generation of the IFT88 conditional mice has been described previously [93]. GnRH::GFP mice were a gift of

Suzanne M. Moenter (University of Michigan). GnRH::Cre mice were a gift of Catherine

Dulac (Harvard University). P0 and P5 mice were anesthetized via isoflurane vapors and then decapitated. The brains were fixed 16-18h at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer and cryoprotected in 30% sucrose at 4°C for at least 24 hours. Brains from P20 and older mice were isolated and processed as previously described [57, 94], with the exception that the mice were perfused and tissue was fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. All brains were sectioned on a freezing microtome at 60 microns. Ovaries and testes were collected from animals after perfusion,

39 post-fixed overnight at 4°C in 4% paraformaldehyde, and cryoprotected in 30% sucrose.

Testes and ovaries were weighed after cryoprotection. Testes were sectioned at 5 microns and ovaries were sectioned at 10 microns on a cryostat and mounted on Superfrost Plus slides (Fisher Scientific).

Immunofluorescence

The Kiss1r antibody, an affinity purified rabbit polyclonal generated against amino acids

348-396 of mouse Kiss1r (Strategic Diagnostics, Inc.), was used at 1:5000. Mouse monoclonal anti-acetylated α-tubulin (T-6793; Sigma-Aldrich) was used at 1:1000.

Rabbit polyclonal anti-GnRH (Thermo Scientific) was used at 1:1000. Rabbit anti- adenylyl cyclase III (C-20; Santa Cruz Biotechnology) was used at 1:350. Secondary antibodies included; Alexa Fluor 546-conjugated goat anti-mouse IgG and Alexa Fluor

546-conjugated goat anti-rabbit IgG (Invitrogen). Nucleic acids were stained with

DRAQ5 (Axxora). Immunofluorescence procedures have been previously described

[95]}.The Kiss1r knockout (KO) mice have been described previously [75]. To label sections from Kiss1r WT and KO mice with rabbit anti-GnRH and anti-Kiss1r simultaneously, we used a modified double labeling protocol. Following tissue blocking and permeabilization, sections were incubated with anti-Kiss1r at 1:5000 overnight at

4°C, washed with PBS, and incubated with Alexa Fluor 546-conjugated goat anti-rabbit

IgG for 1h at room temperature. Sections were washed with PBS and incubated with 5% normal rabbit serum in PBS for 1h at room temperature to saturate open binding sites on the first secondary, followed by washing with PBS and incubation with 20 g/mL goat anti-rabbit Fab fragments (Jackson ImmunoResearch) in PBS for 2h at room temperature

40 to block any exposed rabbit IgG binding sites. Sections were then washed with PBS and incubated with anti-GnRH at 1:1000 in PBS overnight at 4°C. Sections were washed with

PBS, incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG in PBS, washed with PBS again, and mounted. GnRH::GFP tissue was labeled with chicken anti-rootletin

[96] at 1:1000 and anti-Kiss1r at 1:5000. Secondary antibodies were donkey anti-chicken

Cy3 (Jackson ImmunoResearch) and Alexa Fluor 647-conjugated donkey anti-rabbit IgG

(Invitrogen). Samples were imaged either on a Zeiss LSM 510 laser scanning confocal microscope at the Hunt-Curtis Imaging Facility in the Department of Neuroscience at

OSU or an Andor Revolution WD spinning disk confocal imaging system at the OSU

Neuroscience Center Imaging Core. On the laser scanning confocal, z-stacks were acquired using 40x/1.3NA and 63x/1.3NA Oil DIC objectives and a step size of 0.43 µm.

On the spinning disk confocal, z-stacks were acquired using a 100x/1.4 NA Plan Apo VC objective and an iXon Ultra 897 back-illuminated EMCCD camera (512 x 512 pixel array; 160nm x 160 nm pixels) and a step size of 0.2 µm. 3D volume rendering of the image stacks was performed in MetaMorph software using the 4D viewer. The raw images were first processed using a 3 x 3 low pass “denoising” filter.

Neuron and Cilia Analysis

Coronal brain sections from P20, P30 and adult animals were matched to the Franklin and Paxinos mouse atlas [97]. GFP-positive (GFP+) neurons were examined at the medial septum (MS) on plate 22, rostral preoptic area (rPOA) on plate 27, and anterior hypothalamic area (AHA) on plate 32. Sections for P0 and P5 animals were matched to plates 9 and 10 of the coronal gestation day (GD) 18 atlas from the Schambra, Lauder

41 and Silver prenatal mouse brain atlas [98]. For each animal, two sections corresponding to the MS, rPOA and AHA were analyzed. The number of GFP+ neurons in each region was averaged across animals to calculate the mean and standard error. The number of

GFP+ neurons in a particular region was determined by examination under the 20x objective. Once this number was recorded, the same region was examined under the 40x objective. Each GFP+ neuron was centered under the objective, and the filter set was switched to the 546 wavelength. If a Kiss1r-positive cilium emanated from the same area as the GFP+ cell body, it was considered positive for a Kiss1r cilium. If more than one cilium emanated from a single GFP+ cell body, the neuron was considered multiciliated.

The number of ciliated GFP+ neurons was counted using a manual cell counter. The percentage of ciliated GnRH neurons was calculated by dividing the number of GFP+ neurons possessing one or more cilium by the total number of GFP+ neurons and multiplying by 100. The percentage of multiciliated GnRH neurons was calculated by dividing the number of GFP+ neurons possessing multiple cilia by the number of ciliated

GFP+ neurons and multiplying by 100. Statistical analysis was performed using

Graphpad Prism (Graphpad Software). Analysis of the percentages of GnRH neurons possessing Kiss1r-positive cilia across age and sex was performed using ANOVA with post hoc tukey test for multiple comparisons. Analysis of the percentages of GnRH neurons possessing Kiss1r-positive cilia between genotypes was performed using

Student’s t test. A “p” value < 0.05 was considered significant. For P0 neuron migration analysis, sections for P0 animals were matched to plate 13 of the coronal gestation day

(GD) 18 atlas from the Schambra, Lauder and Silver prenatal mouse brain atlas [98]. For

42 each animal, four sections containing the paraventricular hypothalamic nucleus (pvh) were analyzed. Reductions in the number of GnRH neurons present in this caudal region at P0 is an indication of aberrant GnRH neuronal migration [99]. The number of GFP+ neurons in this particular region was determined by examination under the 20x objective.

The numbers counted in the four sections were added together to obtain the number for that animal. The numbers from each animal were then averaged to calculate the mean and standard error by genotype.

Histology

Hematoxylin/eosin staining of testes and ovaries sections was performed according to the manufacturer’s instructions (BBC Biochemical) kit directions. Stained sections were mounted with Permount (Fisher Scientific) and imaged on a Zeiss LSM 510 laser scanning confocal microscope using an Axiocam and Axiocam software.

Vaginal Opening and Estrous Cycle Determination

Time of vaginal opening was determined by daily inspection of mice between 9-11:00 am from P20 onward. Estrous cycle samples were collected daily between 9-11:00 am from group housed animals via vaginal flush with 1x PBS. Samples were deposited on

Superfrost Plus slides, allowed to dry and stained with a Kwik-Diff kit (Thermo

Scientific) according to the manufacturer’s instructions. Slides were allowed to dry and examined on a Zeiss Axioskop 2 MOT light microscope by an individual blinded to the genotypes of the animals. Criteria for the different phases of the estrous cycle have been described previously [100].

43

Slice Preparation for Electrophysiology

Procedures were adapted from a previous study [91]. Briefly, adult mice (age 6-7 weeks) were euthanized by decapitation, and whole brains were removed and immediately submerged in ice cold high-sucrose extracellular solution for 3 min (in mM: 208 sucrose,

2 KCl, 26 NaHCO3, 10 glucose, 1.25 NaH2PO4, 2 MgSO4, 1 MgCl2, 10 Hepes, oxygenated (95% O2, 5% CO2), pH 7.4). Cerebellar tissue was then removed, and 250

M sagittal slices were cut from the tissue block containing the diagonal band of the pre- optical area (DB-POA) using a manual advance vibroslicer (World Precision

Instruments) in ice cold high-sucrose extracellular solution (oxygenated). Slices were immediately transferred upon isolation to an auxiliary chamber containing artificial CSF

(aCSF) at 25oC for 1.5-3.5 hours before use in electrophysiology experiments (aCSF in mM: 124 NaCl, 5 KCl, 2.6 NaH2PO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, 10 Hepes, 10 glucose, oxygenated (95% O2, 5% CO2), pH 7.4).

Electrophysiology Recording and Data Analysis

Individual GnRH neurons in tissue slices were visually selected for recording by positive detection of GFP expression (Nikon eclipseE600FN epifluorescence microscope with

40X water-immersed objective). Loose cell-attached patch configuration in current-clamp mode (I=0) was used to record extracellular firing of the selected GnRH neurons. Recording pipettes with 2.5 – 4.0 MΩ (bath resistance) were filled with aCSF.

At time of recording, seal resistance was between 18-100 MΩ. Recordings were obtained using an EPC10 amplifier (HEKA) with Patchmaster software (HEKA); the sampling

o frequency was 5 kHZ. aCSF (25 C) was oxygenated with 95% O2, 5% CO2 and perfused

44 over slices at rate of 1.4 ml/min with a peristaltic pump (Peri-Star ProTM World Precision

Instruments). Basal firing rate was recorded for at least 3 minutes before treatment with

100 nM KiSS (112-121) Amide (Phoenix Pharmaceuticals) in aCSF for 4.5 minutes. Data was analyzed with Clampfit 9.2 software (Axon), and average firing rate was determined from the action potential events per time for each condition. “% Change in Firing Rate” was determined from the difference in average firing rates with normalized to the average basal firing rate for each cell. Statistical significance was assessed with a 2-tailed

T-test (unpaired data), a “p” value < 0.05 was considered significant.

Real-Time PCR

Total RNA was made from the hypothalami of adult GnRHcilia+ and GnRHcilia- mice (n =

4 animals of each genotype) using the RNeasy Miniprep Kit (Qiagen), according to manufacturer’s instructions. The extracted RNA was treated with DNase using the

Ambion DNA-free Kit (Ambion, Austin, TX) to eliminate genomic DNA contamination.

Oligo(dT)12-18 primed cDNA was made from purified RNA using Invitrogen SuperScript

III Reverse Transcriptase (Invitrogen, Carlsbad, CA). For each sample, 300 ng of RNA was used in the cDNA synthesis reaction. Control “No RT” reactions lacking reverse transcriptase were also performed to test for genomic DNA contamination. cDNA was quantified by qPCR, using the POWER SYBR Green Master Mix (Applied Biosystems) on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The primers used for β were forward 5’-tacagcttcaccaccacagc-3’ and reverse 5’- tctccagggaggaagaggat-3’ to produce a 121 product. The primers used for Kiss1r were forward: 5’-ttcaccgcactcctctaccc-3’ and reverse 5’-cacataccagcggtccacac-3’ to

45 produce a 138 base pair product. The primers used for GnRH were forward: 5’- cactggtcctatgggttgcg-3’ and reverse: 5’-gctggggttctgccatttga-3’ for a 106 base pair product. The efficiency of each primer pair was tested. Reactions were set up in triplicate.

Per well, 20 μL reactions consisted of: 10 μL 2X Power SYBR Green Master Mix, 7.8 μL

DEPC treated DNase, RNase free water (Invitrogen), 0.6 μL of each forward and reverse primer at 10 μM concentration (Integrated DNA Technologies) and 1 μL diluted cDNA.

Data were normalized to mouse β actin expression and was analyzed to determine ΔCt of

Kiss1r and GnRH expression. Cycle threshold was taken at 0.2 ΔRn. Melt curve analysis indicated single products.

Fos Labeling

Gonad intact adult male GnRHcilia+ and GnRHcilia- mice were anesthetized with isofluorane and then injected ICV with either aCSF (vehicle) or aCSF containing 200 pmol KiSS (112-121) Amide. Two hours post injection, mice were transcardially perfused with 4% PFA and brains were collected for immunofluorescence. Sections were labeled with Rabbit anti C-Fos (sc-253; Santa Cruz) and Alexa Fluor 546-conjugated goat anti-rabbit IgG, along with DRAQ-5 as a nuclear marker. Five sections containing GFP+ neurons from each animal were evaluated. The total number of GFP+ neurons with C-Fos positive nuclei was divided by the total number of GFP+ neurons counted in each animal and multiplied by 100 to obtain the percentage of C-Fos positive GnRH neurons in each animal. At least 50 GFP+ neurons were evaluated per animal.

46

Figure 2.1- Kiss1r polyclonal antibody generated in rabbit specifically recognizes mouse Kiss1r in immunofluorescent and Western blot analysis. (A-C) Representative image of transiently transfected IMCD cells expressing Kiss1r fused at the carboxy-terminus to EGFP. (A) EGFP fluorescence (green) shows the expression of the Kiss1r::EGFP construct. (B) Kiss1r polyclonal antibody (red) marks Kiss1r. (C) Merged images confirming anti-Kiss1r labels Kiss1r::EGFP. Scale bar represents 10 µm. (D) Extracts from HEK293T cells expressing myc-tagged Kiss1r (Kiss1r::myc) analyzed by Western blotting (IB) with anti-Kiss1r (left panel) and anti-myc (right panel). Extracts from HEK293T cells expressing empty vector (EV) are included as a negative control. Note the presence of a band around 43 kDa, which is the predicted molecular weight of Kiss1r::myc, specifically in extracts from cells expressing Kiss1r::myc. (E and F) Representative images of the medial hypothalamus in adult Kiss1r (E) wildtype (WT) and (F) knockout (KO) mice (n=3 animals of each genotype) colabeled with anti-GnRH (green) and anti-Kiss1r (red). Corresponding insets (a and b) show higher magnification images of the boxed regions containing GnRH- positive neurons. Note that GnRH neurons in the WT section possess Kiss1r-positive cilia while GnRH neurons in the KO section do not. Scale bars represent 50 µm (main images) and 10 µm (insets). (G) The majority of GnRH neurons in sections from Kiss1r WT mice possessed Kiss1r-positive cilia (24/36 GnRH neurons). Kiss1r-positive cilia were not detected (0/35 GnRH neurons) in Kiss1r KO sections. (H and I) Representative images of the medial hypothalamus in adult Kiss1r (H) wildtype (WT) and (I) knockout (KO) mice (n=3 animals of each genotype) labeled with anti-AC3 (green). Note the presence of AC3-positive cilia in both genotypes. Nuclei were stained with DRAQ5. Scale bar represents 10 µm. 47

Figure 2.2- Kiss1r localizes to primary cilia in vitro and in vivo. (A-C) Representative image of transiently transfected IMCD cells expressing Kiss1r::EGFP. (A) EGFP fluorescence (green) shows expression of Kiss1r. (B) Acetylated α-tubulin (red) marks the cilia. (C) Merged image. (D-F) Representative image of the medial hypothalamus in adult GnRH::GFP mice. (D) GFP fluorescence (green) indicates a GnRH neuron. (E) Labeling for Kiss1r (red) shows the presence of multiple Kiss1r-positive cilia. (F) Merged image. (G) 3D rendering of the same GnRH neuron showing that not only cilia, but multiple cilia, project from a single GFP+ cell body. Nuclei are stained with DRAQ5 (blue). Scale bars represent 10 µm.

48

Figure 2.3- Multiple Kiss1r cilia can project from the same GnRH cell body (A and B) Representative images of the medial hypothalamus in adult GnRH::GFP mice. GFP fluorescence (green) indicates a GnRH neuron. Labeling for Kiss1r (red) shows the presence of multiple Kiss1r-positive cilia. 2D projections (left panels) and corresponding 3D renderings (right panels) are shown. Nuclei are stained with DRAQ5 (blue). Scale bar represents 10 µm. (C and D) Representative images of the medial hypothalamus in adult GnRH::GFP mice. GFP fluorescence (green) indicates a GnRH neuron. Labeling for Kiss1r (red) shows the presence of multiple Kiss1r-positive cilia. Labeling for Rootletin (white) marks the base of each cilium and confirms the cilia are projecting from the same neuron. The cell bodies are displayed as maximum projections and the cilia and rootlets are isosurfaced. Note in panel C the presence of two cilia that project from the cell in parallel and diverge at their tips (indicated by an arrow). Scale bars represent 5 µm.

49

Figure 2.4- Cilia on GnRH neurons do not label for canonical cilia markers but are primary cilia. Representative images of the medial hypothalamus in adult (A) GnRHcilia+ and (B) GnRHcilia- mice. EGFP fluorescence (green) indicates a GnRH neuron. Labeling for AC3 (red) confirms cilia on non-GnRH neurons are positive for AC3 in both GnRHcilia+ and GnRHcilia- mice. Note the lack of AC3-positive cilia on GnRH neurons. Nuclei are stained with DRAQ5 (blue). Scale bars represent 50 µm (main images) and 5 µm (insets). (C-E) Representative image of the medial hypothalamus in adult CiliaGFP mice. (C) EGFP fluorescence (green) shows Sstr3::GFP expression and ciliary localization. (D) Labeling for GnRH (red) indicates GnRH neurons. (E) Merged image confirms GnRH neurons are ciliated. (F-H) Representative image of the medial hypothalamus in adult CiliaGFP mice. (F) EGFP fluorescence (green) shows Sstr3 expression and ciliary localization. (G) Labeling for Kiss1r (red) confirms Kiss1r ciliary localization. (H) Merged image. Nuclei are stained with DRAQ5 (blue). Scale bars represent 10 µm.

50

Figure 2.5- Quantification of Kiss1r cilia on GnRH neurons. (A) Percentage of GnRH neurons with one or more Kiss1r-positive cilia in the medial hypothalamus of P0-P60 male and female GnRH::GFP mice (n = 4 animals of each sex at all ages, with the exception of P0 (n = 3)). Note the percentages do not vary significantly between ages or sexes. (B) Percentage of ciliated GnRH neurons with more than one Kiss1r- positive cilium in the medial hypothalamus of P0-P60 male and female GnRH::GFP mice (n = 4 animals of each sex at all ages, with the exception of P0 (n = 3)). Note the proportion of multiciliated GnRH neurons increases significantly between P0 and P60. (aSignificantly different from P0 (p < 0.01), bSignificantly different from P0 (p < 0.001), cSignificantly different from P5 (p < 0.01), dSignificantly different from P5 (p < 0.001))

51

Figure 2.6- GnRH neurons in GnRHcilia- mice do not have Kiss1r cilia but still express Kiss1r (A-G) Representative images of the medial hypothalamus in adult (A-C) GnRHcilia+ and (D-F) GnRHcilia- mice. (A- C) Representative image of the medial hypothalamus in adult GnRHcilia+ mice. (A) GFP fluorescence (green) indicates a GnRH neuron. (B) Labeling for Kiss1r (red) shows the presence of multiple Kiss1r- positive cilia. (C) Merged image. (D-F) Representative image of the medial hypothalamus in adult GnRHcilia- mice. (D) GFP fluorescence (green) indicates a GnRH neuron. (E) Labeling for Kiss1r (red) shows the lack of any Kiss1r-positive cilia or staining. (F) Merged image. Scale bar represents 5 µm. Real- time PCR data representing the relative level of Kiss1r (G) and GnRH (H) mRNA expression compared to β actin (Kiss1r or GnRH mRNA/ β actin mRNA) as determined by quantitative real time PCR using the SYBR green method. Note the relative level of Kiss1r mRNA in the hypothalamus of GnRHcilia- mice is not significantly different from GnRHcilia+ mice (p = 0.384). RNA was extracted from hypothalamic tissue from adult male GnRHcilia+ and GnRHcilia- mice (n = 4 animals for each genotype). Values are expressed as mean ± SEM.

52

Figure 2.7- Intracerebroventricular injection of kisspeptin induces Fos expression in GnRHcilia+ and GnRHcilia- mice. GnRH neurons (green) from (A) GnRHcilia+ and (B) GnRHcilia- gonad intact male mice (n = 3 animals for each genotype) treated with aCSF (vehicle; upper rows) show little Fos expression (red). GnRH neurons from GnRHcilia+ and GnRHcilia- mice (n = 3 animals for each genotype) treated with 200 pmol kisspeptin (KP-10; lower rows) show induction of Fos expression (red). (C) Percentages of GnRH neurons positive for Fos labeling in GnRHcilia+ and GnRHcilia- mice in response to aCSF and kisspeptin injection. The percentages of GnRH neurons positive for Fos labeling is low in response to aCSF injection but is significantly increased in response to kisspeptin injection in both GnRHcilia+ and GnRHcilia- mice. There was no significant difference in Fos expression between genotypes in response to aCSF or kisspeptin injection. Values are expressed as mean ± SEM. *Significantly different from aCSF injection (p < 0.05).

53

Figure 2.8- Loss of Kiss1r-positive cilia primarily occurs prenatally in both male and female GnRHcilia- mice and does not impact GnRH neuronal migration. (A) Percentages of GnRH neurons with a Kiss1r-positive cilium in the medial hypothalamus of P0 male and female GnRHcilia+ and GnRHcilia- mice (n = 3 animals for each sex and genotype). There is no significant difference between the percentage of GnRH neurons with cilia in male and female GnRHcilia+ mice or male and female GnRHcilia- mice. (B) Percentage of GnRH neurons with one or more Kiss1r-positive cilia in the medial hypothalamus of P60 male GnRH::GFP, GnRHcilia+, and GnRHcilia- mice (n = 3-4 animals for each genotype). Note there is no difference in the percentage of Kiss1r-positive cilia between GnRH::GFP and GnRHcilia+ mice, but Kiss1r- positive cilia are completely lacking in GnRHcilia- mice. (C) Number of GnRH neurons in the paraventricular hypothalamic nucleus of P0 male GnRHcilia+ and GnRHcilia- mice (n = 3 animals for each genotype). Note there is no significant difference in the number of GnRH neurons in this region between GnRHcilia+ and GnRHcilia- mice. Values are expressed as mean ± SEM. (D) Number of GnRH neurons throughout the medial septum (MS), rostral pre-optic area (rPOA), and anterior hypothalamic area (AHA) of P60 male GnRHcilia+ and GnRHcilia- mice (n = 3 animals for each genotype). Note there is no significant difference in the number of GnRH neurons in any region between GnRHcilia+ and GnRHcilia- mice. Values are expressed as mean ± SEM.

54

Figure 2.9- Loss of primary cilia on GnRH neurons does not result in reproductive phenotypes. (A) Reproductive organ weights of P60 GnRHcilia+ and GnRHcilia- mice are identical. Ovaries and testes isolated from female and male adult animals, respectively, showed no difference in weight between GnRHcilia+ and GnRHcilia- mice. (n=8-10 animals per sex per genotype). (B and C) Representative testis (left panel) and seminiferous tubule (right panel) sections from P60 GnRHcilia+ (B) and GnRHcilia- (C) mice (n = 3 animals for each genotype) shows the presence of mature sperm in both genotypes. (D and E) Representative section from P60 GnRHcilia+ (D) and GnRHcilia- (E) mice (n = 3 animals for each genotype) shows the presence of follicles at all stages of development. Scale bars represent 100 µm. (F) Vaginal cytology of P60 GnRHcilia+ and GnRHcilia- mice show all stages of estrous cyclicity. C, cornified (estrous); N, nucleated (proestrous); L, leukocytic (metestrous and diestrous).

55

Table 2.1: Physiological Measures of Sexual Maturation in GnRHcilia+ and GnRHcilia- Mice Male Female

GnRHcilia+ GnRHcilia- GnRHcilia+ GnRHcilia- 20.3 ± Body Weight (g) 22.6 ± 0.85 23.8 ± .087 19.8 ± 1.03 0.97 Sex Organ Weight 153.4 ± 9.1 146.4 ± 9.5 7.6 ± 0.8 7.5 ± 0.7 (mg) Time of Vaginal NA NA 28.5 ± 0.6 28.0 ± 0.6 Opening (day)

Table 2.1- Gross physiological measures do not differ between GnRHcilia+ and GnRHcilia- mice. Physiological measurements from GnRHcilia+ and GnRHcilia- animals ages 2.5-4 months (n=8-12). Sex organ refers to both testis for each male and both ovaries for each female. Values are expressed as the mean ± SEM.

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Figure 2.10 GnRH cilia are required for proper Kiss1r signaling. (A) Representative traces of the action potentials from basal and kisspeptin (KP-10) stimulated GnRH neurons from GnRHcilia+ and GnRHcilia- mice. Quantification of the basal firing rates of GnRH neurons from adult male (B) and ovariectomized female (C) GnRHcilia+ and GnRHcilia- mice. Note there is no significant difference in the basal firing rates between GnRHcilia+ and GnRHcilia- mice. Quantification of the percentage increase in the firing rate of GnRH neurons after kisspeptin treatment in adult male (D) and ovariectomized female (E) GnRHcilia+ and GnRHcilia- mice. Note the increase in firing rate is significantly lower in male GnRHcilia- mice compared to GnRHcilia+ mice. Values are expressed as mean ± SEM. For males n = 10-13 neurons from 4-7 animals of each genotype. For females n = 9 neurons from 4-5 animals of each genotype. *Significantly different from GnRHcilia+ percentage (p = 0.02).

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Chapter 3: Disruption of BBS1 Function in Dopamine D1 Receptor Containing Cells Results in Reduced Locomotor Activity and Obesity in Mice Summary

Neurons throughout the mammalian brain possess primary cilia, antenna-like sensory organelles, the functions of which are still mostly unknown. Defects in proteins associated with the cilium result in a variety of diseases known as ciliopathies, with phenotypes including obesity, blindness, anosmia, cognitive deficits, cystic kidney disease, and hypogonadism. G protein-coupled receptors (GPCRs) are important signaling molecules in the brain, and there are several GPCRs that localize to neuronal primary cilia. Proteins that are mutated in the ciliopathy Bardet-Biedl Syndrome (BBS) are critical in the trafficking of certain ciliary GPCRs to and from the cilium.

Our laboratory has determined that one of the GPCRs for Dopamine, Dopamine

Receptor 1 (D1R), localizes to primary cilia of neurons. This localization is dynamic, and levels of D1R in the cilium can vary due to receptor stimulation and induction of downstream secondary messengers. When BBS protein function is disrupted, D1R levels in the cilium increase, and dynamic localization of the receptor is lost. D1R is involved in cognition, motor function, and feeding, all of which are altered in BBS. Our hypothesis is that mislocalization of the receptor alters its signaling, resulting in some of the

58 physiological and behavioral phenotypes seen in BBS patients and mouse models of the disease. To test this hypothesis, we crossbred mice expressing a D1R promoter driven

Cre recombinase and mice carrying conditional alleles of a BBS gene. Interestingly, we found mice with the BBS gene disrupted in D1R expressing cells are significantly heavier than age and sex matched controls from 10 weeks of age onward. These mutant mice show a significantly lower activity level than age and weight matched controls. Based on previous reports, this suggest a deficit in D1R signaling. Further behavioral and biochemical characterization of these mice should elucidate how D1R signaling is altered in the absence of proper BBS function.

Introduction:

Primary cilia are present on most neurons in the mammalian brain, and many of these cilia are enriched for various signaling molecules. The function of these microtubule based organelles that have their own exclusive proteome have been implicated in sensory roles in other tissues, but their roles in the central nervous system remains a mystery for the most part [13]. Cognitive and behavioral phenotypes present in diseases where the function of the cilium is perturbed hint that the primary cilium could be a vital signaling center in neurons [35]. Strengthening this hypothesis is the identification of a number of signaling molecules as being enriched in the primary cilia of neurons [44, 50, 51, 57].

Work in our laboratory identified a conserved sequence in the third intracellular loop of some GPCRs that was necessary for their targeting to the primary 59 cilium[57]. This sequence was found to be present in a number of GPCRS, one of which is the (D1R). In wild-type (WT) mouse brain tissue the receptor is not detected in the cilium of neurons. However, D1R is highly enriched in the primary cilia of neurons in BBS4-/- and BBS2-/- mice [42]. This makes D1R fairly unique among ciliary receptors, as other GPCRs fail to localize to the cilium in the absence of BBS protein function [94]. A small percentage of cultured WT neurons display D1R in their cilium. A significantly higher percentage of neurons cultured from BBS4-/- mice display

D1R positive cilia. Interestingly, when stimulated with an , the percentage of WT neurons displaying D1R positive cilia in vitro decreases, while the same treatment has no effect in BBS4-/- neurons. It was also determined that BBS5, a subunit of the BBSome, interacts with the third intracellular loop of D1R. The BBSome is necessary for the trafficking of Mchr1 and Sstr3 to the cilium. The observation that D1R does not leave the cilium in response to agonist treatment in neurons where the function of the BBSome is disrupted led to the hypothesis that the BBSome is necessary for the trafficking of D1R from the cilium. In cultured WT neurons, induction of cAMP production can increase the levels of D1R in the cilium [42]. This change of localization in a mouse model of a ciliopathy and dynamic localization in response to agonist treatment make D1R an interesting topic of study.

D1R belongs to the D1-type class of dopamine receptors, which also includes

Dopamine receptor D5, or D5R. The other three dopamine receptors, D2, D3, and D4, are known as the D2-type receptors. D1R and D5R stimulate cAMP production through

Gαolf/s, while the D2-type receptors inhibit cAMP production through coupling with Gαi

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[101]. In the brain, D1R is highly expressed in the , nucleus accumbens, and olfactory tubercule. D1R is also expressed in the medial pre-frontal cortex, the , the substantia nigra, and the arcuate nucleus.

D1R signaling in the striatum is heavily involved in locomotor control, and human ciliopathy patients suffer from a litany of movement disorders, and BBS patients have lower levels of activity when compared to weight-matched controls [35, 102, 103].

BBS patients and mice also suffer from increased levels of anxiety [104, 105]. D1R activity in the amygdala has been implicated in the generation of anxiety [106]. D1R is also highly expressed in the nucleus accumbens, a region of the brain known to regulate food consumption and reward behaviors [107] [108]. One of the most persistent phenotypes present in human patients with BBS and the mouse model is hyperphagia induced obesity [109]. Our hypothesis is that at least some of the behavioral phenotypes seen in BBS human patients and mice are due to aberrant signaling of D1R due to its altered localization in the absence of functional BBS proteins. This hypothesis is supported by recent work showing that a slight reduction in D1R signaling is sufficient to produce lower locomotor activity and obesity in a mouse model [110].

In order to test this hypothesis without the litany of confounding phenotypes present in germline BBS protein knockout mice [111], we decided to use a conditional knockout approach. We cross-bred mice expressing Cre recombinase under the control of the D1R promoter with mice expressing a conditional allele of BBS1. BBS1 is one of the seven proteins that is a vital component of the BBSome [41, 112]. The offspring of this cross expressed D1R at the same levels as controls, and were born at the same ratios

61 as their control littermates. These mutant mice also did not suffer from the enlarged ventricles present in mouse models of disrupted BBS1 function [113]. D1R localization was altered as expected in neurons with loss of BBS protein function, but localization of other ciliary receptors in non-D1R expressing neurons was unaffected. Interestingly, mice with a loss of BBS1 activity in D1R neurons become overweight when compared to controls around 8-10 weeks of age, and have lower levels of locomotor activity that precedes the onset of obesity. Although these mice also suffer from retinal degeneration, they retain a functional level of visual acuity at least until 8-10 weeks of age, suggesting that lower levels of locomotor activity are not due simply to an inability to navigate the environment.

Results

Conditional disruption of BBS1 in D1R::Cre expressing cells

Previous work in our laboratory showed that disruption of BBSome function, by way of knockout of certain BBS proteins, changed the pattern of localization of D1R

[42]. With the confounding phenotypes present in BBS mouse models, and mislocalization of other ciliary receptors in the brain, it would be challenging to pinpoint the consequences of mislocalized D1R in these mouse models [13]. In order to isolate the disruption of BBS proteins to D1R-expressing cells, we cross-bred transgenic mice expressing Cre recombinase under the D1R promoter (D1R::Cre) with mice containing conditional alleles of Bbs1 (BBS1fl/fl). We also bred a BBS null (BBS1Δ) allele into these mice in order to ensure efficient knockdown of BBS1 in our experimental animals. The

62 final products of our breeding pairs were mice of the genotype D1R::Cre/BBS1fl/wt

(control animals) and D1R::Cre/BBS1fl/Δ (experimental animals).

Even though the loss of BBS protein function in mice will disrupt the proper localization of certain receptors, including D1R, it should not affect the level of their expression [42, 94]. In order to confirm this, we isolated protein from the striatum of

D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice and immunoblotted the membrane enriched fraction with antibodies against D1R and actin (Fig 3.1A). Immunoblotting of protein from the brains of 4 adult mice of each genotype revealed that there was no significant difference in the expression levels of D1R between D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice (Fig 3.1B). BBS null mice are not born at the same rate as their wild-type littermates and have higher perinatal mortality rates [105]. D1R::Cre/BBS1fl/Δ mice were born and survived to adulthood at the same rate as their D1R::Cre/BBS1fl/wt counterparts (Table 3.1).

D1R::Cre/BBS1fl/Δ mice display retinal degeneration but avoid enlarged ventricles and infertility present in BBSKO mice

One of our goals in creating the D1R::Cre/BBS1fl/Δ mouse line was to expand on the work begun by our lab on D1R while avoiding the confounding phenotypes present in mouse models of BBS. When we started characterizing these mice, we observed that we avoided some, but not all of the potentially confounding phenotypes present in BBSKO mice. Mouse models of BBS, especially those with disrupted BBS1 function, suffer from enlarged ventricles in the brain [114]. Examination of the brains of D1R::Cre/BBS1fl/wt

63 and D1R::Cre/BBS1fl/Δ mice alongside that of a wild-type (WT) and BBS1 null

(BBS1KO) mouse reveal that ventricles in the brain of the BBS1KO mouse are enlarged, while the D1R::Cre/BBS1fl/Δ mouse has ventricles indistinguishable from WT and

D1R::Cre/BBS1fl/wt (Fig 3.2A). One of the phenotypes that almost all ciliopathies share is that of retinal degeneration, and BBS is no exception [35]. Retinas isolated from 6 week old D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice and stained for the visual proteins and Rhodopsin show normal retinal structure in D1R::Cre/BBS1fl/wt mice and improper trafficking in D1R::Cre/BBS1fl/Δ mice (Fig 3.2B). In order to test if these mice still maintained some visual acuity, we ran 6-8 week old mice through the visual cliff paradigm. The visual cliff paradigm measures practical vision function in mice by forcing mice to choose between what appears to be a “cliff” and solid ground[115].

When D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice were subjected to the paradigm, there was no difference in their performance, and both performed at a level that indicated they possessed functional visual acuity (Fig 3.2C). Finally, male D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice produce fully mature sperm, indicating no reproductive deficit in

D1R::Cre/BBS1fl/Δ mice (Fig 3.2D).

D1R::Cre/BBS1fl/Δ mice are obese compared to D1R::Cre/BBS1fl/wt counterparts, but are not hyperphagic

One of the cardinal phenotypes present in human patients and mouse models of

BBS is obesity [105]. One of the first noticeable phenotypes present in adult

D1R::Cre/BBS1fl/Δ mice when compared to sex and age-matched D1R::Cre/BBS1fl/wt mice 64 is increased body mass (Fig 3.3A). The weights of D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice aged from 4 weeks to 20 weeks were measured at 2 week intervals. Male D1R::Cre/BBS1fl/Δ mice are significantly heavier than their

D1R::Cre/BBS1fl/wt counterparts starting at 10 weeks of age, while the difference became statistically significant starting at 8 weeks in females (Fig 3.3B,C). While obesity in

BBS is attributed, at least partially, to hyperphagia, our D1R::Cre/BBS1fl/Δ mice do not appear to consume more food than D1R::Cre/BBS1fl/wt controls (Fig 3.4A).

D1R::Cre/BBS1fl/Δ mice have lower levels of activity compared to D1R::Cre/BBS1fl/wt counterparts

BBS human patients have lower levels of locomotor activity than weight matched controls [103]. Lower levels of locomotor activity have been reported in several different mouse models of BBS as well, but these studies did not control for the potentially confounding effects obesity can have on locomotor activity [116, 117]. We measured the locomotor activity of 6-8 week old male and female mice for a 24 hour period using the

Columbus Instruments Laboratory Animal Monitoring System (CLAMS). The age of 6-8 weeks was chosen so the animals could be measured in a pre-obese state. Over the course of 24 hours, D1R::Cre/BBS1fl/Δ mice had lower levels of locomotor activity than their age matched D1R::Cre/BBS1fl/wt controls (Fig 3.5A). This change in activity held true when broken down into the dark period when mice are typically more active and the light period, when they are typically less active (Fig 3.5B,C). Measurement of the oxygen usage by the CLAMS of a subset of mice used in the activity monitoring 65 experiment revealed no difference in metabolic activity between the two genotypes (Fig

3.5D).

D1R is highly expressed in the striatum, and interruption of striatal function can affect locomotor coordination and function [118]. In order to evaluate locomotor coordination and strength in mice, we subjected male and female D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice to the rotating rod (RotaRod) paradigm and the fore and hindlimb grip strength test. D1R::Cre/BBS1fl/Δ mice performed just as well as

D1R::Cre/BBS1fl/wt mice in these paradigms, providing evidence that the reduced activity seen is not due to a defect in coordination or strength (Fig 3.6A,B).

Localization of GPCRs in the brains of D1R::Cre/BBS1fl/Δ and D1R::Cre/BBS1fl/wt mice

One of our goals in creating the D1R::Cre/BBS1fl/Δ mouse line was to alter the ciliary localization of D1R while preserving BBS function in cells that do not express

D1R. Immunofluorescent labeling of brain tissue from D1R::Cre/BBS1fl/wt controls shows no detectable D1R in the cilium, consistent with staining seen in WT tissue (Fig

3.7A-C) [42]. Labeling of D1R::Cre/BBS1fl/Δ mice, on the other hand, reveals the accumulation of D1R in AC3 positive cilia (Fig3.7D-F). Labeling of brain tissue from a

BBS1KO animal also reveals an accumulation of D1R in AC3 positive cilia, confirming that knockout of BBS1 has the same consequences as knockout of BBS4 for D1R localization (Fig 3.7G-I) [42].

Melanin concentrating hormone receptor 1 (Mchr1), which our laboratory has identified as a ciliary receptor, fails to localize to primary cilia in the absence of BBSome

66 function [57, 94]. Mchr1 is highly expressed in the nucleus accumbens (NAc), where

D1R expression is also high [119]. Labeling for Mchr1 in the NAc in D1R::Cre/BBS1fl/wt mice reveals ciliary localization of the receptor, consistent with WT tissue (Fig 3.8A-C)

[94]. Labeling of D1R::Cre/BBS1fl/Δ mice shows that Mchr1 co-localizes with AC3 positive cilia in the NAc, indicating that BBS1 function is preserved in non-D1R expressing cells (Fig 3.8D-F). Finally, labeling of tissue from a BBS1KO mouse reveals a complete lack of Mchr1 in cilia, confirming that BBS1 is required for ciliary localization of Mchr1 (Fig 3.8H-I). Taken together, the staining for D1R and Mchr1 in the NAc indicate successful knockout of BBS1 in D1R expressing neurons, leading to its enrichment in cilia, while BBS1 function is preserved in non D1R expressing cells.

Stimulation of D1R induces equivalent locomotor response in D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice

Stimulation of D1R, specifically in the NAc, has been shown to produce an increase in locomotor activity in mice [102]. We hypothesized that if the change in localization of the D1R receptor was affecting its signaling, stimulation of the receptor with the D1R agonist SKF81207 would produce different levels of locomotor activity in

D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice were allowed to acclimate to 20 cm x 20 cm testing chambers for 30 minutes, and then injected intraperitonealy (i.p.) with 1.5 mg/kg SKF81297. After injection of the agonist, the mice were monitored for 90 more minutes (Fig 3.9A). Both genotypes showed an increase in locomotion after injection of the agonist and there was

67 no significant difference in the distance travelled in the 30 minutes immediately after the injection (Fig 3.9C). This experiment was repeated with a new set of mice, except a higher concentration of SKF81297 (5.0 mg/kg) was used. The mice displayed a robust locomotor response to D1R stimulation, but there was no significant difference in response between the genotypes (Fig 3.9B,D).

DARPP-32 is unchanged between D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice

We sought to test whether the intracellular signaling of D1R was affected.

Dopamine and cAMP Regulated Neuronal Phosphoprotein (DARPP-32) is a well characterized downstream target of D1R signaling [120]. Stimulation of D1R results in activation of Protein Kinase A (PKA) which phosphorylates threonine-34 of DARPP-32

(pDARPP-32). Measurement of pDARPP-32 levels is commonly used as an indicator of

PKA activity [121,122]. We isolated striatal slices from the brains of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice, and treated them with vehicle, 10 µM Forskolin, 1 µM

SKF81297, or 10 µM SKF81927 for 5 minutes. Forskolin stimulates PKA activity via direct stimulation of adenylyl cyclases and was used as a positive control. Protein was collected from the striatal samples after treatment, and was immunblotted for pDARPP-

32 and total DARPP-32 (Fig 3.10A). The levels of pDARPP-32 were normalized to total

DARPP-32 for each sample. These levels were then normalized to the value obtained for the vehicle treatment of D1R::Cre/BBS1fl/wt tissue (Fig 3.10B). While Forskolin, 1 µM

SKF81297, and 10 µM SKF81927 were able to induce significant amounts of pDARPP-

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32 with respect to the vehicle treatments for their respective tissues, there was no significant difference in induction of pDARPP-32 between treatments of different genotypes.

Discussion:

In this chapter we successfully used a conditional knockout approach to disrupt

BBS1 in a subpopulation of neurons that expressed the ciliary receptor D1R. These mice recapitulated the phenotypes of obesity, lower activity, and retinopathy present in mice with germline mutations in BBS1, but did not display reproductive phenotypes and brain malformations [113]. Stimulation of D1R both in vitro and in vivo failed to produce detectable differences between our experimental and control mice, but different or more sensitive assays may be needed.

While the expression levels of GPCRs are unaffected in other models of BBS dysfunction, we felt it necessary to confirm equal D1R expression in our newly generated mutant mice [42, 94]. Equivalent expression of D1R in D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice can allow us to interpret our results in the context that both genotypes express the receptor at the same rate.

Viability of Bbs2 and Bbs4 KO neonates is reduced compared to their heterozyogous and wild-type littermates [105, 123]. Mice with a missense mutation in

BBS1 are born at normal Mendelian ratios [113]. However, in our experience, BBS1KO mice are rarely viable, and are born at a rate that is drastically lower than the expected

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Mendelian ratio. Therefore, the evidence that D1R::Cre/BBS1fl/Δ mice are viable at the same rate as their D1R::Cre/BBS1fl/wt littermates suggests that disruption of the BBS1 protein in D1R expressing cells is not a hindrance in pre- and perinatal development.

Germline disruption of BBS1 in mice results in enlarged ventricles in the brain, which has been at least partially attributed to the dysfunction of a set of neural progenitor cells in the periventricular area [114]. D1R::Cre/BBS1fl/Δ mice appear to avoid this phenotype, further suggesting that central nervous system malformations in BBS animals can be attributed to certain sets of cells. This is also beneficial, as it could allow us to perform cognitive and behavioral assays on our mutant mice without the potential confounding factor of hydrocephaly.

One of the objectives behind breeding the D1R::Cre/BBS1fl/Δ mouse was to perform behavioral assays related to D1R on mice without the confounding phenotype of retinopathy present in mouse models of BBS. The retinas of D1R::Cre/BBS1fl/Δ mice show loss of proper localization of visual proteins similar to that seen in germline BBS knockout animals [27]. However, the performance of the mice on the visual cliff paradigm suggests that they retain enough visual acuity to navigate their environment, making the performance of behavioral assays on these mice feasible. Finally, one of the hallmark phenotypes of BBS is infertility, and the inability of males to form mature sperm[111, 113]. D1R::Cre/BBS1fl/Δ mice do not suffer from this phenotype.

One of the most striking and common phenotypes present in human patients and mouse models of BBS is obesity [103][116]. In human patients, obesity onset can occur

70 as early as one year of life, but occurs relatively later in life in mice, and can vary with the particular BBS gene that is disrupted or even the particular mouse model [105, 116].

Mouse models with null or disrupted forms of other BBS proteins have been created, and all of them develop obesity [111-113, 123-126]. Mouse models with disruptions of

BBSome proteins seem to follow a pattern of obesity development with differences in weight becoming evident at around 8-12 weeks, similar to our D1R::Cre/BBS1fl/Δ mice

[111-113, 123, 126]. The exact similarity of the weight gain between our mice and previously published BBSome disruption models is difficult to determine, as each study measured weight gain at slightly different time points, and some studies combined the data from male and female mice. However, the broad pattern of weight gain between all these BBSome disruption models is constant. That this pattern is maintained in

D1R::Cre/BBS1fl/Δ mice should help to begin to narrow the focus of research that is attempting to determine the etiology of obesity in BBS.

The obesity phenotype in human patients and mice has been mostly attributed to hyperphagia, or increased eating, but lower levels of activity compared to weight matched controls has been noted as well [103],[116]. In our studies we did not see any evidence of hyperphagia in adult D1R::Cre/BBS1fl/Δ mice, but we did see reduced levels of locomotor activity when compared to D1R::Cre/BBS1fl/wt controls. Obesity onset with reduced activity and a lack of hyperphagia is not a novel finding in a mouse model. Mice with mutations in the hypothalamic transcription factors Nhlh2 and Tubby have reduced pre-obesity locomotor activity and a lack of hyperphagia, and become obese at similar rates to our D1R::Cre/BBS1fl/Δ mice [127, 128]. Since the activity of D1R expressing 71 neurons in the striatum can affect motor control, we tested coordination, balance, endurance and strength in D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. The lack of a difference in basic locomotor skills between the genotypes suggests that the lower level of activity in D1R::Cre/BBS1fl/Δ mice is voluntary, and not the product of impaired movement capability. Oxygen usage is a measure of metabolic activity in mice, and there was no significant difference between our genotypes in metabolic activity. Even with the reduced activity of D1R::Cre/BBS1fl/Δ mice, this is not unexpected, as small changes in activity can leave basal metabolic activity unaffected [129].

Our hypothesis is that mislocalization of the receptor due to loss of BBSome function results in altered signaling of the receptor when it is enriched in the cilium.

D1R is known to be heavily involved in stimulating locomotion, so our phenotype of reduced basal locomotion is unsurprising. We sought to extend these results by stimulating D1R in D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice with an agonist and observing the mice for differences in induced locomotion. While a moderate dose (1.5 mg/kg) and a high dose (5.0 mg/kg) of the D1R agonist SKF81297 were able to induce locomotion in D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice, we were unable to observe any significant differences. This demonstrates that D1R receptor is functional and can stimulate locomotion even when its localization is altered. However, our hypothesis that D1R signaling is reduced predicts that we should see a reduced amount of locomotion in the D1R::Cre/BBS1fl/Δ mice when compared to D1R::Cre/BBS1fl/wt mice.

The possibility remains that differences could be seen by using a different concentration of SKF81297, or another behavioral paradigm. Stimulating D1R well beyond its normal 72 thresholds with the exogenous agonist might mask subtle signaling defects that are present when the receptor is stimulated by endogenous concentrations of dopamine.

Measurement of D1R signaling via phosphorylation of the downstream signaling effector DARPP-32 failed to show differences between our genotypes. The reason for this could be similar to those outlined in the discussion on the D1R agonist induced locomotion experiments above. The signaling defect could be too small to measure in our assay, or might be masked by the over-stimulation of the system by artificial means.

Also, while DARPP-32 phosphorylation at threonine 34 in response to D1R stimulation is a good way to measure acute D1R stimulation, the difference in D1R signaling that may lead to our phenotype of reduced activity may be a more chronic defect that cannot be detected by the assay we chose.

Previous studies have shown that slight reductions in D1R activity are sufficient to induce obesity and reduced activity in a mouse model [110]. Due to the similar phenotype seen in our D1R::Cre/BBS1fl/Δ mice, we hypothesized that D1R’s enrichment in the cilium leads to a decrease in signaling. How could the change in D1R’s localization to the cilium be affecting its signaling? Previous studies in our laboratory have shown that D1R interacts with BBS5, a subunit of the BBS1 containing BBSome.

The BBSome appears to be responsible for trafficking the receptor out of the cilium after it is stimulated with agonist [42]. The BBSome has been shown to resemble complexes that are responsible for the formation of endocytic vesicles, and the endocytosis of D1R has been shown to enhance its signaling [41, 130]. Therefore, when BBSsome function is disrupted by the loss of any of its component proteins, endocytic enhancement of D1R 73 signaling is lost. Another possible cause of reduced D1R signaling due to enrichment in the cilium is the orphan G protein-couple receptor GPR88. GPR88 is highly expressed in the striatum, in the same medium-spiny neurons that express D1R [131]. In an in vitro system, GPR88 that localizes to the cilium has been shown to reduce the signaling of ciliary D1R, while leaving the signaling of a plasma-membrane localized receptor unaffected [132]. Potentially, in wild-type neurons, stimulation of D1R causes it to leave the cilium, escaping the suppression of its signaling by GPR88. However, when D1R is unable to be trafficked out of the cilium due to lack of BBSome function, its signaling could be suppressed by GPR88. Much work remains to be done to determine if this is a viable hypothesis.

Materials and Methods

Ethics Statement:

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Ohio

State University (Animal Welfare Assurance #A3261-01).

Generation of D1R::CRE BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice

All animal procedures described are in accordance with institutional guidelines based on

National Institutes of Health Standards, and were performed with Institutional Animal

Care and Use Committee approval at the Ohio State University and the University of

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Alabama at Birmingham. All animals were maintained in a temperature and humidity controlled vivarium with 12 hr light/dark cycle and given access to food and water ad libitum. Littermates were group housed by sex, no more than 5 to a cage, after weaning.

Mice containing the floxed BBS1 allele (BBS1fl/fl) were a gift from Val Sheffield

(University of Iowa). The BBS1Δ allele was generated by crossing BBS1fl/fl mice with

B6.C-Tg(CMV-cre)1Cgn/J (CMV-Cre) mice (The Jackson Laboratory, Bar Harbor,

Maine, US). CMV-Cre/BBS1Δ/wt mice were crossed with each other to generate BBS1Δ/wt mice. These BBS1Δ/wt mice were then crossed with mice expressing Cre recombinase under control of the Dopamine Receptor D1R promotor (D1R::CRE) (The UC Davis

Mouse Resource Center) to generate D1R::CRE BBS1Δ/wt mice. Finally,

D1R::Cre/BBS1Δ/wt male mice were crossed with BBS1fl/fl female mice to generate

D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice for use in experiments.

Mouse Weight Capture and Image

Photograph of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice was captured using the

Camera application on an iPhone 5c (Apple, Cupertino, CA, USA). Male and female mice were weighed once every two weeks on a balance (Bioexpress, Kaysville, UT,

USA) starting at four weeks of age and continuing through twenty weeks of age.

Isolation of Membrane Enriched Protein Fraction

Isolation of membrane enriched protein from mouse brain has been previously described

[42]. Briefly, dissected tissue was sonicated in ice cold PBS. Cell debris was pelleted by centrifugation at 5,000 x g for 15 minutes at 4°C. The supernatant was collected and centrifuged for 1 hour at 120,000 x g at 4°C to pellet the membrane. The supernatant

75 from this step was stored at -80°C and considered cytosolic enriched protein. The resultant membrane pellet was solubilized at 4°C overnight in membrane lysis buffer

(20mM Tris pH 8.0, 150mM NaCl, 2mM EDTA, 10% glycerol, 1% NP40, 0.1% SDS, and 0.25% DOC) supplemented with sodium orthovanadate and protease inhibitor cocktail (Thermo Scientific). Unsolubilized material was cleared by centrifugation for 1 hour at 120,000 x g at 4°C and the resulting solubilized protein was collected. Protein concentrations were determined by Bradford assay. Protein was stored at -80°C until use.

Immunoblotting

Membrane enriched striatal protein samples were run on a Ready Gel© denaturing 4–15% gradient polyacrylamide gel (Bio-Rad), while protein isolated from slices in ex vivo signaling experiments were run on a Mini-PROTEAN® denaturing 4–15% gradient polyacrylamide gel (Bio-Rad). For membrane enriched striatal protein samples, 50 µg of protein was loaded per sample. For protein isolated from slices used in ex vivo signaling experiments, 30 µg of protein was loaded per sample. Proteins were transferred to a

PVDF-FL membrane (Millipore, Billerica, MA, USA). Membranes were blocked in

Odyssey Blocking Buffer (LiCor Biosciences, Lincoln, Nebraska USA) for 1 hour and incubated overnight at 4°C with appropriate antibodies diluted in Odyssey Blocking buffer with 0.2% Tween-20. For immunoblotting of membrane enriched striatal protein fractions, the primary antibodies used were mouse monoclonal antibody against D1R

(Santa Cruz Biotechnology) at 1:250 and mouse monoclonal antibody against α-Actin (C-

20) (Santa Cruz Biotechnology) at 1:250. For immunoblotting of protein isolated from ex vivo signaling experiments, primary antibodies used were mouse antibody against

76

DARPP-32 (Santa Cruz Biotechnology) at 1:250 and rabbit antibody against phosphor- threonine-34 DARPP-32 (Cell Signaling, Danvers, MA, USA) at 1:1000. Membranes were then washed in PBS-T. Membranes were probed with Goat anti-Mouse IRDye

680LT and Goat anti-Rabbit IRDye 800LT (LiCor) secondary antibodies diluted

1:15,000 in Odyssey Blocking buffer with 0.2% Tween-20 and 0.01% SDS for 1 h at room temperature. Membranes were washed in PBS-T, PBS, and then dried for a minimum of 3 hours before being visualized. Proteins were visualized using the Odyssey

Infrared Imager (84 μm resolution, 0 mm offset with high quality) and the Image Studio

2.0 program.

Quantification of Protein Levels

Western bands visualized with the Odyssey Imager and Image Studio 2.0 program were highlighted using the manual western band selection tool in the Image Studio 2.0 program. Once selected, the program calculated the intensity of the signal in each channel for each band.

Tissue Isolation

Brains were isolated and processed as previously described [57, 94], with the exception that the mice were perfused and tissue was fixed with a 2% paraformaldehyde/50%

HistoChoice MB Tissue Fixative (Amresco) in 0.1 M phosphate buffer. All brains were sectioned on a freezing microtome at 60 microns. Testes were collected from animals after perfusion, post-fixed overnight at 4°C in 4% paraformaldehyde, and cryoprotected in 30% sucrose. Testes were weighed after cryoprotection. Testes were sectioned at 5 microns on a cryostat and mounted on Superfrost Plus slides (Fisher Scientific). Eyes

77 were removed from perfused mice, and a hole was poked in the outer layer to allow fixative to penetrate the eyeball. Eyes were incubated in 4% PFA for one hour before washing in PBS. Eyes were then cryoprotected in 20% sucrose, and then mounted in

OCT and sectioned on a cryostat at 10 microns and mounted on Superfrost Plus Slides.

Immunofluorescence

Mouse monoclonal antibody against D1R (Santa Cruz Biotechnology) was used at

1:250. Goat polyclonal antibody against Mchr1 (Santa Cruz Biotechnology) was used at

1:250. Rabbit anti-adenylyl cyclase III (C-20; Santa Cruz Biotechnology) was used at

1:350. Mouse antibody against Rhodopsin, from the laboratory of Dr. R Molday

(University of British Columbia) was used at 1:1000. Rabbit anti-Opsin (Millipore) was used at 1:200. Secondary antibodies included; Alexa Fluor 488-conjugated goat anti- mouse IgG, Alexa Fluor 546-conjugated goat anti-rabbit IgG, Alexa Fluor 488- conjugated donkey anti-mouse IgG, and Alexa Fluor 546-conjugated donkey anti-goat

IgG (Invitrogen). Nucleic acids were stained with DRAQ5 (Axxora).

Immunofluorescence procedures have been previously described [95]. Samples were imaged on a Zeiss LSM 510 laser scanning confocal microscope at the Hunt-Curtis

Imaging Facility in the Department of Neuroscience at OSU. Z-stacks were acquired using 40x/1.3NA and 63x/1.3NA Oil DIC objectives and a step size of 0.43 µm. Images were processed using LSM software.

Histology

Hematoxylin/eosin staining of testes and retina sections was performed according to the manufacturer’s instructions (BBC Biochemical) kit directions. Stained sections were

78 mounted with Permount (Fisher Scientific) and imaged on a Zeiss LSM 510 laser scanning confocal microscope using an Axiocam and Axiocam software.

D1 Agonist Induced Locomotion

Assays were performed in 20 x 20 x 20 cm clear acrylic plastic boxes, with one animal per box. Animals were placed in the box, and basal movement was recorded for 30 minutes. After 30 minutes, animals were given an intraperitoneal injection of SKF-81297

(either 1.5 mg/kg or 5.0 mg/kg), and locomotion was recorded for 90 more minutes.

Animal movement was recorded and tracked by Anymaze software (Stoelting, Wood

Dale, IL, USA). All movement data was binned in 5 minute increments, and extracted from videos by the ANY-maze software.

Activity and VO2 Tracking

Tracking of the locomotor activity of mice over the course of 24 hours was performed using the Comprehensive Lab Animal Monitoring System (CLAMS) (Columbus

Instruments, Columbus, OH USA). Mice were placed in cages in the CLAMS and allowed to acclimate to the cages for at least one full dark cycle before the start of monitoring. The physical activity of each mouse was monitored using a multidimensional infrared light detection system placed on bottom and top levels of each individual cage of the CLAMS. Activity measurements were added for the 12 hour light period, 12 hour dark period, and 24 hour total period. Additionally, oxygen consumption was measured by the CLAMS via indirect calorimetry every 20 minutes during the monitored activity period. Mice were weighed prior to being placed in the CLAMS and oxygen consumption was normalized to weight for each animal by the system.

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Rotating Rod and Grip Strength Assays

The rotating rod (Rotarod) paradigm was performed by placing the mice on an accelerating rod (4–40 rpm over the course of a 300 second trial). A Five Station Mouse

Rota-Rod (Med Associates Inc, St. Albans, Vermont) was used in these experiments.

Latency to fall was automatically recorded by the apparatus, and mice that did not fall were scored at 300 seconds. Mice were subjected to three trials per day, with an inter- trial interval of 15 minutes. Experiments were performed three days in succession. Fore- and hindlimb grip strength tests were performed using a Grip Strength Meter (Columbus

Instruments, Columbus, Ohio) by an experimenter blinded to the genotypes of the animals. Animals were held by the tail, and lightly pulled along the wired mesh of the device until they obtained a grip on the mesh with all four paws. Then the mice were pulled from the apparatus until their grip failed. This process was repeated 3 times per animal in succession.

Feeding assessment

Adult mice of the same genotype (D1R::Cre/BBS1fl/wt or D1R::Cre/BBS1fl/Δ) were doubly housed in normal cages. An excess of normal mouse chow was weighed and placed in the wire tops of the cages. The amount of food in the tops of the cages was measured once weekly, and subtracted from the previous weeks amount to determine the amount of food consumed by the animals in the cage. This amount was then divided by 2 to determine the average amount eaten per animal in the cage. During each food weighing period, the bottom of the cage was inspected for any small pieces of food that may have

80 slipped through the wire cage top, and these pieces were combined with cage top food and weighed.

Preparation of striatal slices for ex vivo slicing

Ex vivo slices of the striatum were prepared from adult D1R::Cre/BBS1fl/wt and

D1R::Cre/BBS1fl/Δ mice. Mice were euthanized by decapitation, and whole brains were removed and immediately submerged in ice cold high-sucrose extracellular solution for 3 min (in mM: 208 sucrose, 2 KCl, 26 NaHCO3, 10 glucose, 1.25 NaH2PO4, 2 MgSO4, 1

MgCl2, 10 Hepes, oxygenated (95% O2, 5% CO2), pH 7.4). Cerebellar tissue was then removed, and 400 M coronal slices were cut from the tissue block containing the striatum using a manual advance vibroslicer (World Precision Instruments) in ice cold high-sucrose extracellular solution (oxygenated). The striatum was dissected out of these coronal slices. Striatal slices were then immediately transferred upon isolation to an auxiliary chamber containing artificial CSF (aCSF) supplemented with 10 µg/mL

Adenosine Deaminase (AD) at 30oC for 30 minutes (aCSF in mM: 124 NaCl, 5 KCl, 2.6

NaH2PO4, 2 MgCl2, 2 CaCl2, 26 NaHCO3, 10 Hepes, 10 glucose, oxygenated (95% O2,

5% CO2), pH 7.4). After 30 minutes, the AD supplemented aCSF was removed from the chamber and replaced with fresh warmed, oxygenated aCSF for another 30 minutes.

Ex vivo signaling on striatal slices

An hour after isolation, striatal slices were transferred to eppendorf tubes. Slices were then treated with DMSO (vehicle), 10 µM Forskolin, 1 µM SKF81297, or 10 µM

SKF81927 dissolved in aCSF for 5 minutes at 30 oC. Each experiment included a treatment of slices from one D1R::Cre/BBS1fl/wt mouse and one D1R::Cre/BBS1fl/Δ mouse

81 side by side. After 5 minutes, treatment solution was removed, and slices were immediately frozen on dry ice and stored at -80°C until protein isolation. Frozen slices were re-suspended in boiling 1% SDS in PBS supplemented with protein inhibitor cocktail, sonicated briefly, and boiled for 10 minutes. Protein concentration was determined on a Nano-Drop.

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Figure 3.1- D1R is expressed at the same levels in the brains of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ transgenic mice. (A) Representative Western blot of the membrane enriched fraction of striatal tissue isolated from adult D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. Membranes were probed with antibodies towards D1R and Actin. Bands are present at ~75kDa and ~43 kDA, which are the predicted sizes of D1R and actin respectively. (B) Quantification of protein levels from immunoblotting performed on tissue from adult D1R::Cre/BBS1fl/wt and D1R::CRE BBS1fl/Δ mice (n=4 animals per genotype). Intensity of D1R bands was normalized to intensity of Actin bands within each animal. Values are expressed as mean + SEM.

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Table 3.1: Viability of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice

GENOTYPE % total pups

D1R::CRE BBS1fl/wt 21%

D1R::CRE BBS1fl/Δ 24%

Table 3.1- Disruption of BBS proteins in D1R expressing neurons does not affect viability. We examined the number of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice born to our breeding pairs. Since the D1R::Cre gene is only present in one allele on the male breeders in this line, only approximately half of all animals produced are D1R::Cre positive. n=242 animals total.

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Figure 3.2- D1R::Cre/BBS1fl/Δ mice avoid some phenotypes present in BBS KO mice. (A) Wide-field images of coronal sections from the brains of wild-type (WT), BBS1Δ/Δ (BBS1KO), D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. Compared to WT littermates, BBS1KO display enlarged ventricles in the brain, a phenotype that does not appear to be present in either D1R::Cre/BBS1fl/wt or D1R::Cre/BBS1fl/Δ mice. (B) Immunofluorescent (top) and hematoxylin and eosin (H&E) (bottom) staining of retinas from 8 week old D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. Staining for Opsin and Rhodospin, two visual proteins, reveals normal trafficking to the outer segment in D1R::Cre/BBS1fl/wt mice. Trafficking of Opsin to the outer segment appears disrupted, while Rhodopsin staining in the outer segment is completely absent in D1R::Cre/BBS1fl/Δ mice. H&E staining does not reveal any gross morphological defects in the retinas of either genotype. Scale bars = 50 μm (C) H&E staining of the seminiferous tubules of the testes from male D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. Males of both genotypes appear to produce fully mature sperm, an ability absent in BBS KO mice. (D) Visual Cliff test performed on 8 week old D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice reveals intact visual function in animals of both genotypes. Both D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ animals chose the “ground” side of the apparatus with around 70% frequency. The 95% confidence interval for both animals lies above 50%, indicating some functional acuity remaining in D1R::Cre/BBS1fl/Δ mice (n=8 animals per genotype).

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Figure 3.3- D1R::Cre/BBS1fl/Δ are heavier compared to D1R::Cre/BBS1fl/wt mice. (A) Image of a female D1R::Cre/BBS1fl/wt mouse and a littermate D1R::Cre/BBS1fl/Δ female mouse at 20 weeks of age. The D1R::Cre/BBS1fl/Δ is noticeably heavier than the control. (B) Weight tracking of male D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice (n=8-12 animals per genotype). D1R::Cre/BBS1fl/Δ mice are significantly heavier than D1R::Cre/BBS1fl/wt controls starting at around 10 weeks of age, and this difference persists through 20 weeks of age. (C) Weight tracking of female D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice (n=8-12 animals per genotype). D1R::Cre/BBS1fl/Δ mice are significantly heavier than D1R::Cre/BBS1fl/wt controls starting at around 8 weeks of age, and this difference persists through 20 weeks of age. *Significantly different from D1R::Cre/BBS1fl/wt weight (p<0.05). All numbers are mean + SEM.

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Figure 3.4- Food consumption by D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. (A) Food consumption was tracked in group housed adult male and female D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. (n= 4 cages of animals per genotype).All numbers are mean + SEM.

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Fi gure 3.5- Age and weight matched D1R::CRE BBS1fl/Δ mice have lower levels of activity than D1R::Cre/BBS1fl/wt counterparts. (A) Metabolic cage activity tracking over 24 hours of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ male and female 6-8 week old mice revealed that D1R::Cre/BBS1fl/Δ mice have lower levels of activity than D1R::Cre/BBS1fl/wt counterparts (n=14-15 animals per genotype) ( * p<0.005). (B) D1R::Cre/BBS1fl/Δ mice have lower levels of activity than D1R::Cre/BBS1fl/wt counterparts during the 12 hour “lights on” period of the day. (n=14-15 animals per genotype) ( * p<0.005). (C) D1R::Cre/BBS1fl/Δ mice have lower levels of activity than D1R::Cre/BBS1fl/wt counterparts during the 12 hour “lights off” period of the day. (n=14-15 animals per genotype) ( * p<0.005). (D) Measures of oxygen usage during the 24 hour activity tracking period do not differ between D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice (n=6-9 animals per genotype). All numbers are mean + SEM.

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Figure 3.6 Age and weight matched D1R::Cre/BBS1fl/Δ do not have gross locomotor defects compared to D1R::Cre/BBS1fl/wt counterparts. Testing of basic locomotor coordination and strength in D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice. (A) D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ 6-8 week old male and female mice were subjected to the Rotarod test. Mice were placed on the rotarod, and the device increased in speed from 4-40 rpm over the course of 300 seconds for three trials per day. No significant difference was found in the performance on the task between genotypes (n=6-7 animals per genotype). (B) Combined forelimb and hindlimb strength was tested in D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ 6-8 week old male and female mice. Three trials were performed per mouse. No significant difference was detected between the genotypes (p=0.0758) (n=6-7 animals per genotype). All numbers are mean + SEM.

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Figure 3.7- D1R localization in D1R::Cre/BBS1fl/Δ mice recapitulates that seen in BBS1 Δ /Δ mice. Immunofluorescent staining of the striatum in brain tissue from D1R::Cre/BBS1fl/wt, D1R::Cre/BBS1fl/Δ, and BBS1Δ/Δ mice. (A-C) Staining for D1R (A) and adenylyl cyclase type III (AC3) (B) in the striatum of D1R::Cre/BBS1fl/wt mice reveals that D1R is not enriched in AC3 positive cilia (C) merged image. (D-F) Staining for D1R (D) and AC3 (E) in the striatum of D1R::Cre/BBS1fl/Δ mice shows D1R enriched in AC3 positive cilia in (F), the merged image. (G-I) Staining for D1R (G) and AC3 (H) in the striatum of a BBS1Δ/Δ mouse reveals enrichment of D1R in the AC3 positive cilia (I). Scale bar equals 10 microns.

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Figure 3.8- Mchr1 localization in D1R::Cre/BBS1fl/Δ mice is similar to that in control mice. Immunofluorescent staining of the striatum in brain tissue from D1R::Cre/BBS1fl/wt, D1R::Cre/BBS1fl/Δ, and BBS1Δ/Δ mice. (A-C) Staining for Mchr1 (A) and (AC3) (B) in the striatum of D1R::Cre/BBS1fl/wt mice reveals that Mchr1 is enriched in AC3 positive cilia (C) merged image. (D-F) Staining for Mchr1 (D) and AC3 (E) in the striatum of D1R::Cre/BBS1fl/Δ mice shows Mchr1 enriched in AC3 positive cilia in (F), the merged image. (G-I) Staining for Mchr1 (G) and AC3 (H) in the striatum of a BBS1Δ/Δ mouse reveals failure of Mchr1 to localize to the AC3 positive cilia (I). Scale bar equals 10 microns.

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Figure 3.9- D1R::Cre/BBS1fl/Δ mice respond to D1R stimulation the same as D1R::Cre/BBS1fl/wt counterparts. (A) Activity plots of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice, binned in 5 minute intervals. Recording started when the mice were placed in the box at time 0. At minute 30, mice were given a 1.5 mg/kg i.p. injection of the selective D1R antagonist SKF81297. There was no significant difference in locomotion between the two genotypes before or after SKF8127 injection (n=8 animals per genotype). (B) Activity plots of D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice, binned in 5 minute intervals. Recording started when the mice were placed in the box at time 0. At minute 30, mice were given a 5.0 mg/kg i.p. injection of the selective D1R antagonist SKF81297. There was no significant difference in locomotion between the two genotypes before or after SKF8127 injection (n=8 animals per genotype). (C) Quantification of the total distance travelled of each genotype 30 minutes before (UT) or after (SKF81297) 1.5 mg/kg SKF81297 injection. (D) Quantification of the total distance travelled of each genotype 30 minutes before (UT) or after (SKF81297) 5.0 mg/kg SKF81297 injection.

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Figure 3.10- Neurons from D1R::Cre/BBS1fl/Δ mice respond to acute D1R stimulation at the same rate as D1R::Cre/BBS1fl/wt mice. Ex vivo slices of the striatum from adult D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice were treated with vehicle, Forskolin, or SKF81297, and DARPP-32 phosphorylation was measured. (A) 400 micron thick Striatal slices from D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice were treated with vehicle, 10 µM Forskolin, 1 µM SKF81297, or 10 µM SKF 81297 for 5 minutes. Protein was isolated from the striatal slices, and immunoblotted for DARPP-32 phosphorylated at threonine 34 (pDARPP-32) and total DARPP-32. Representative immunoblots developed with the LiCor Odyssey imaging system are shown. (B) Quantification of results from ex vivo signaling experiments. The intensity of bands from immunoblotting for pDARPP-32 was normalized to the intensity of the bands from immunoblotting for total DARPP-32 for each experiment, and all values were normalized to the D1R::Cre/BBS1fl/wt vehicle within each experiment. (*= significantly different from vehicle treatment within genotype, p<0.05). (n=8 slices per vehicle, 10 µM Forskolin, and 1 µM SKF81297 experiments. n=5 slices per 10 µM SKF 81297 experiment). There was no statistically significant difference within treatments between genotypes. All numbers are mean + SEM.

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Chapter 4: Targeted Disruption of Ciliary Genes in the Nucleus Accumbens of Mice Summary

Nearly every neuron in the central nervous system has at least one primary cilium. However, little is known about the role that this ubiquitous organelle can play in modulating behavior and cognition in the fully developed central nervous system. The ciliopathy Bardet-Biedl Syndrome (BBS) is a heterogeneous disorder. The BBS proteins form a complex that is vital to the proper trafficking of certain membrane proteins to and from the cilium. Our laboratory has identified a number of G protein-coupled receptors

(GPCRs) that localize predominantly to the cilia of neurons in wild type animals, but not in mouse models of BBS. One of these is the receptor for Melanin Concentrating

Hormone (Mchr1). Mchr1 is known to be involved in behaviors that are altered in BBS, including feeding behavior and anxiety. Our hypothesis is that localization of Mchr1 to the cilium is vital to the proper regulation of its signaling and modulation of behavior.

We believe that mislocalization of Mchr1 leads to a loss of modulation in its downstream signaling resulting in changes in behaviors associated with the receptor. This chapter will discuss a project designed to examine the role of primary cilia in the Nucleus Accumbens

(NAc), a discrete brain region involved in the modulation of feeding, reward behavior, and anxiety. Mchr1 expression is highly enriched in neurons of the NAc, and the receptor localizes to the cilia of the neurons in this region. We used delivery of a virally 94 expressed Cre recombinase into the NAc of BBS1 conditional mice in order to confine disruption of BBS function to our region of interest. We successfully disrupted the ciliary localization of Mchr1 in the NAc in these mice, but failed to see a significant difference when the mice were subjected to behavioral paradigms.

Introduction:

Almost every neuron in the central nervous system projects a primary cilium, an exclusive compartment that has a proteome unique from the rest of the cell [13]. Primary cilia are nearly ubiquitous on cells throughout the rest of the mammalian body as well, and have been revealed to play vital roles in environmental sensing and tissue homeostasis [2]. While the basic structure of the cilium is conserved throughout different cell types in the body, and indeed, different organisms, its function can be defined by the sensory and signaling molecules that are localized to the cilium in any particular cell or tissue type [10, 13].

The importance of cilia in the proper development and function of the body and numerous organ systems has become a research focus over the last decade due to the identification of a class of diseases known as ciliopathies. Ciliopathies are united in the fact that they share a broad spectrum of phenotypes, and causative mutations have been mapped to genes that encode proteins crucial to the proper functioning of the cilium[35].

Identification of vital signaling proteins that localize to the cilium in photoreceptor cells, olfactory neurons, and kidney cells has helped determine the etiology of certain phenotypes present in ciliopathies [13]. 95

Bardet-Biedl Syndrome (BBS) is a model ciliopathy used by our lab to study the signaling properties and function of cilia. BBS in humans commonly presents with hyperphagia-induced obesity, cognitive deficits, retinal degeneration, renal dysfunction, polydactyly, and central nervous system abnormalities. As of this writing, 19 causative genes have been identified, 7 of which make up a conserved structure known as the

BBSome [133], [41]. The BBSome has been shown to be vital for the trafficking of proteins to and from the cilium, including G protein-coupled receptors [41, 42, 94].

Certain GPCRs selectively localize to primary cilia on neurons throughout the brain [50],[51, 57]. One of these, Mchr1, localizes to the cilia of neurons in the hippocampus, nucleus accumbens, , hypothalamus, and other regions [95].

The nucleus accumbens (NAc) is a discrete brain region located in the ventral striatum that is involved in motivation, reward, motor function and learning [134]. Genetic and pharmacological studies have shown that Mchr1 and its ligand play a critical role in the ability of the NAc to regulate feeding behavior, depression, and reward. MCH and Mchr1 knockout animals are lean, while animals that overexpress the ligand are hyperphagic and obese [135]. Stimulation of the Mchr1 system in the NAc is known to stimulate feeding behavior, while blockade of the system in the same region blocks feeding behavior in mice [119],[108]. Mchr1 activity in the NAc increases depressive like behavior in rodents, while antagonism of the system decreases it [119]. As the NAc is a vital component of the reward pathways, it is interesting that Mchr1KO mice are resistant to the rewarding properties of cocaine [108]. The phenotypes of anxious behavior and hyperphagia in BBS mice, who completely lack ciliary Mchr1, suggest that ciliary

96 localization of the receptor could be important in the proper modulation of its signaling in the NAc [35, 105].

The goal of this chapter is to examine the behavioral, physiological, and signaling consequences of knockdown of BBS proteins in the nucleus accumbens. Using AAV-

Cre injection in BBS1 conditional mice, we disrupted the expression of BBS1 in the nucleus accumbens while avoiding the developmental and confounding phenotypes present in germline BBS knockout animals. Disruption of the ciliary localization of

Mchr1 in the NAc did not appear to affect behavior or physiology, however the study was performed on a small group of animals. Experimental animals also did not gain weight after disruption of BBS proteins in the NAc. This study helps establish the feasibility of this approach for examining the role the primary cilium plays in the modulation of behavior in a discrete brain region. With the enrichment of Mchr1 in the NAc, further work on this project could also provide clues on how disruption of the ciliary localization of the receptor can affect its modulation of behavior and signaling.

Results:

Specific spatial and temporal disruption of ciliary genes

Loss of BBS protein function in animals and humans results in mis-localization of certain GPCRs and a host of phenotypes. The litany of physiological and cognitive phenotypes in BBS makes studying the etiology of a single phenotype in BBS mouse models challenging. Therefore, our strategy is to focus on receptors and brain regions of interest we believe could be driving these phenotypes. Mchr1 is highly expressed in the 97

NAc, and stimulation of the receptor in the NAc is known to be sufficient to produce behavioral responses and phenotypes [119]. Using mice with conditional alleles for

BBS1 (BBS1fl/fl), we could confine disruption of Mchr1’s ciliary localization both spatially and temporally, and avoid affecting any other brain regions.

Adeno-Associated Viruses (AAV) are commonly used as vectors to introduce exogenous genes into the central nervous system of rodents [136]. The Cre/loxP system is widely used to disrupt genes of interest in a temporal, spatial, and/or cell-type specific manner [137]. Together, AAV is often used to introduce Cre recombinase into specific brain regions in mice expressing “floxed” genes in order to temporally and spatially confine gene knockout [136]. With this knowledge, we decided to use AAV expressing

Cre recombinase fused with a GFP construct in our experiments (AAV-CRE-GFP) in order to disrupt floxed ciliary genes in mice. Expression of GFP alongside Cre is helpful in determining the effectiveness of the injection and Cre expression. To test the efficacy of this approach, we unilaterally injected an AAV-CRE-GFP construct, a gift of the

Kaspar Lab (AAV-CRE-GFPBK), into the NAc of IFT88fl/fl mice. IFT88 is a gene that is essential for the construction and maintenance of primary cilia in cells [76]. The coordinates for stereotactic injection of the virus were determined using the Paxinos and

Watson mouse atlas, in order to target the injection to the center of the NAc [97]. Mice were injected with AAV-CRE-GFP, and brain tissue was collected 4 weeks later, in order to allow time for virus transduction and maximum expression of the transgene [136]. A wide field image of the NAc from an animal injected with AAV-CRE-GFPBK shows a high level of Cre-GFP expression throughout the NAc (Fig 4.1A-C). The targeting and

98 confining of the Cre-GFP expression to the NAc shows that AAV-CRE-GFP can be effectively targeted to the NAc in high amounts, with minimum infection of nearby brain regions. Staining of this tissue revealed that in the injected side of the brain, recombination of IFT88fl/fl resulted in the loss of the majority of AC3 positive cilia in a zoomed in field of CRE-GFP positive cells. (Fig 4.1D). The equivalent, un-injected side of the brain revealed a large number of AC3 positive cilia (Fig 4.1E). These results indicate that AAV-CRE-GFP injection into the NAc in mice is effective in disrupting the expression of conditional ciliary genes.

Disruption of ciliary localization of Mchr1 after knockout of BBS1 in the Nucleus

Accumbens

We wanted to preserve the structure of the cilium while studying the effects of mis-localized Mchr1, so we decided to use BBS1fl/Δ mice for our experiments instead of

IFT88fl/fl. BBS1fl/wt mice were designated as controls for these experiments. BBS1fl/wt and

BBS1fl/Δ mice were injected bi-laterally in the NAc with AAV-CRE-GFP acquired from the UNC viral vector core (AAV-CRE-GFPUNC). Evaluation of the brains of BBS1fl/wt and BBS1fl/Δ mice 9 weeks post injection revealed effective targeting of the NAc in all injected animals. High magnification of tissue from the animals stained for Mchr1 and

AC3 reveals that in BBS1fl/wt mice, Mchr1 is still enriched in the cilium, showing that

Mchr1 ciliary localization is not affected by Cre-GFP expression and that a single BBS1 allele is sufficient for proper function (Fig 4.2A-3). In BBS1fl/Δ mice, staining for AC3 reveals that while AC3 cilia are still present in Cre-GFP expressing cells, Mchr1 staining

99 is absent from the cilium (Fig 4.2D-F). These results show that using AAV-CRE-GFP injection into the NAc of BBS1fl/Δ mice, we can effectively disrupt BBS1 expression.

Behavioral Evaluation of AAV-CRE-GFP injected mice

BBS1fl/wt and BBS1fl/Δ mice were allowed to recover for 4 weeks after AAV-CRE-

GFPUNC injection in order to allow time for transduction of cells, expression of Cre-GFP, and recombination of the BBS1fl allele [136]. Since manipulation of Mchr1 activity in the

NAc has been shown to affect feeding activity, we tracked the weight of BBS1fl/wt and

BBS1fl/Δ mice from the day of injection to 9 weeks post-injection (Fig 4.3A). The weights did not significantly differ between the two genotypes, even at 9 weeks post- injection. Mchr1 signaling in the NAc has also been shown to modulate anxiety in rodent models [138]. We exposed the BBS1fl/wt and BBS1fl/Δ mice injected with AAV-CRE-

GFPUNC to the Open Field and Elevated Plus Maze paradigms, which measure anxious- like behavior in mice. There was no significant difference between the genotypes in these two behavioral paradigms (Fig 4.3B,C). Mchr1 is highly expressed in the NAc, which is considered the reward center of the brain, and Mchr1 has been shown to modulate the rewarding effects of cocaine [108]. We subjected our BBS1fl/wt and BBS1fl/Δ mice injected with AAV-CRE-GFPUNC to the Conditioned Place Preference (CPP) paradigm, which measures the susceptibility of animals to the rewarding or aversive properties of drugs or other stimuli [139]. Unstimulated and cocaine-stimulated locomotion over the course of 30 minutes was measured in the mice during the first two days of the CPP training paradigm, and no significant difference was seen between the

100 genotypes (Fig 4.3D,E). Finally, the CPP “score” indicates the strength of the preference of the animals for the drug or being tested in the particular paradigm. While we were able to show a preference for cocaine in both BBS1fl/wt and BBS1fl/Δ mice injected with AAV-CRE-GFPUNC, there was no statistically significant difference in the CPP scores between the two genotypes (Fig 4.3F).

Our data indicate that we are able to effectively target the NAc with AAV-CRE-

GFP and obtain successful knockout of conditional ciliary genes. As a result, we can effectively disrupt ciliary localization of Mchr1 in BBS1fl/Δ mice with AAV-CRE-

GFPUNC, while preserving proper Mchr1 localization in BBS1fl/wt mice. The results from our initial behavioral characterization of these mice fail to show a significant difference between the genotypes, however, the number of animals used in these experiments was very low. More animals are needed to increase the statistical power of this study, and more experiments are needed to uncover any potential effects of BBS1 disruption in the

NAc.

Discussion:

While the behavioral paradigms performed on mice from this pilot study failed to show a statistically significant difference between genotypes, we were able to demonstrate that injection of AAV-CRE-GFP into discrete brain regions is a viable strategy to disrupt ciliary localization of GPCRs. It is difficult to draw many conclusions from the data due to the small number of animals used in the experiments. However, this pilot study still raises some interesting questions and points to consider. 101

Mchr1 is known to modulate behaviors that are altered in BBS, making it an ideal target of investigation after our laboratory identified that it was enriched in cilia normally and absent in mouse models of BBS [57, 94, 111, 140]. As in the other projects outlined in this work, we sought to use a conditional knockout of ciliary proteins to study the consequences of the altered localization of Mchr1 and minimizing the contribution of phenotypes resulting from the dysfunction of other ciliary proteins. Mchr1 is strongly expressed in a discrete brain region, the Nucleus Accumbens, and modulation of the receptor’s activity in this region has been shown to induce behavioral phenotypes [119,

134]. The NAc has been successfully targeted with stereotactic injections of AAV in numerous other studies, so we settled on this approach for this project.

One of the causes of the obesity phenotype in BBS patients and model mice is believed to be hyperphagia [116, 141]. Stimulation of Mchr1 in the Nucleus Accumbens has a potent effect on feeding behavior, leading us to hypothesize that Mchr1 activity could be increased in BBS knockout mice [119]. After injection of AAV-CRE-GFP into the NAc of our BBS1fl/Δ mice, we expected they would gain weight if disruption of

Mchr1’s ciliary localization caused an increase in the signaling of the receptor. We were not able to measure food intake of the animals, as they were group housed. Other studies have been able to demonstrate weight gain after a genetic disruption causes mice to develop hyperphagia [76]. We were unable to demonstrate a difference in weight or weight gain of the mice 9 weeks post injections. This could mean that our mice did not develop a feeding behavior phenotype, or developed a subtle one that we were unable to

102 detect by simply measuring the weight of the mice. In future studies on these mice, individual food intake could be monitored.

Stimulation of the Mchr1 system is anxiogenic, which is why we subjected mice to the open field and elevated plus maze paradigms [119]. Mchr1 has also been shown to modulate the rewarding effects of cocaine in the mouse, so we subjected our mice to the

Conditioned Place Preference paradigm [108]. We saw no significant difference in the performance of the mice in any of the behavioral paradigms. While it is difficult to detect a significant difference with the number of animals used, we did not see a non- significant trend either. Assuming that our hypothesis that Mchr1 signaling is disrupted when it is not localized to the cilium is correct, our inability to detect a difference could be due to a number of factors. While the stereotactic AAV-CRE-GFP injection may be able to infect a large amount of neurons, we still may not be disrupting BBS1 expression in enough Mchr1 expressing neurons to cause a detectable difference in behavior. This could be remedied by injecting more virus into the NAc, or utilizing a cre recombinase linked to the Mchr1 promoter. Finally, as the results in Chapter 2 demonstrated, sometimes a change in signaling at the cellular level cannot be detected by physiological or behavioral assays. In the future, signaling experiments on Mchr1 expressing neurons with disruptions to the BBS proteins should enable us to better test our hypothesis.

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Materials and Methods:

Ethics Statement:

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the Ohio

State University (Animal Welfare Assurance #A3261-01).

Transgenic Mouse Breeding

All animal procedures described are in accordance with institutional guidelines based on

National Institutes of Health Standards, and were performed with Institutional Animal

Care and Use Committee approval at the Ohio State University. All animals were maintained in a temperature and humidity controlled vivarium with 12 hr light/dark cycle and given access to food and water ad libitum. Littermates were group housed by sex, no more than 5 to a cage, after weaning. Mice containing the floxed BBS1 allele (BBS1fl/fl) were a gift from Val Sheffield (University of Iowa). The BBS1Δ allele was generated by crossing BBS1fl/fl mice with B6.C-Tg(CMV-cre)1Cgn/J (CMV-CRE) mice (The Jackson

Laboratory, Bar Harbor, Maine, US). CMV-Cre::BBS1Δ/wt mice were crossed with each other to generate BBS1Δ/wt mice. BBS1Δ/wt male mice were then crossed with BBS1fl/fl female mice to generate BBS1fl/wt and BBS1fl/Δ male mice for use in experiments.

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Stereotactic injections of AAV-CRE-GFP

Stereotactic injections of AAV-CRE-GFP vectors have been previously described [142].

All surgical procedures were performed using aseptic technique. Briefly, mice were anaesthetized with a mixture of 100 mg/kg ketamine and 15 mg/kg xylazine (Sigma-

Aldrich). Once mice were determined to be in a surgical plane of anesthesia by way of toe pinch, they were secured in a stereotaxic frame (Stoelting Co, Wood Dale, Illinois,

USA). A small incision was made in the skin above the skull, and bregma was determined using landmarks on the skull. The coordinates used for NAc injections were

+1.30mm Anterior/Posterior, 1.00mm lateral, and -4.60mm dorsal/ventral. After a hole was drilled in the skull at the correct anterior/posterior and lateral coordinates, the point of the needle of the injection apparatus was lowered to correct coordinates for injection.

A Hamilton syringe and tubing system, connected to a 33 gage injector cannula (Plastics

One, Roanoke, Virginia, USA) was used to inject the virus. An injection rate of 0.1

μl/min was maintained using a syringe pump. 1.5μl of virus was injected per side. After the virus was injected, the needle was kept in place for 5 minutes, and then raised very slowly from the injection site. The incisions in the heads of the animals were sutured after the surgery, and animals were given post-operative care. Four weeks were allowed for virus expression, as this has been shown to be sufficient for maximal expression in the ventral striatum [136]. Injections were performed bilaterally for all behavioral experiments.

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Tissue Collection

Brains were isolated and processed as previously described [57, 94], with the exception that the mice were perfused and tissue was fixed with a 2% paraformaldehyde/50%

HistoChoice MB Tissue Fixative (Amresco) in 0.1 M phosphate buffer. All brains were sectioned on a freezing microtome at 60 microns.

Immunofluorescence

Goat polyclonal antibody against Mchr1 (Santa Cruz Biotechnology) was used at 1:250.

Rabbit anti-adenylyl cyclase III (C-20; Santa Cruz Biotechnology) was used at 1:350.

Alexa Fluor 546-conjugated goat anti-rabbit IgG, Alexa Fluor 546-conjugated donkey anti-goat IgG, and Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Invitrogen).

Nucleic acids were stained with DRAQ5 (Axxora). Immunofluorescence procedures have been previously described [95]. Samples were imaged on a Zeiss LSM 510 laser scanning confocal microscope at the Hunt-Curtis Imaging Facility in the Department of

Neuroscience at OSU. Z-stacks were acquired using 10x/1.3NA Air and 40x/1.3NA Oil

DIC objectives and a step size of 0.43 µm. Images were processed using LSM software

Weight Tracking of Pre and Post-Injected Mice

Mice were weighed right before injection of virus and once every three weeks post injection, up until sacrifice. Mice were weighed on a balance (Bioexpress, Kaysville,

UT, USA).

Behavioral Assays

The open-field paradigm is a measure of anxiety in mice. Mice were placed in 40cm x

40cm x 40cm boxes in dimly lit chambers for 30 minutes, and their movements were

106 recorded by Any-Maze software via a camera system set up above the testing box. In the software, a virtual 20cm x 20cm box was drawn in the middle of the testing area, and the number of entries and time spent in this central box was recorded. Time spent in the outer area and total distance travelled were recorded as well. The elevated plus maze is another a measure of anxiety in mice [143]. Mice were placed on the elevated plus maze apparatus, which consists of an “X” shaped set of platforms elevated 90cm off the ground. Two “arms” of the maze have 15cm walls enclosing them, while the other two arms are completely open. Mice were placed in the center of the apparatus and allowed to explore freely for 5 minutes while all activity was monitored by the Any-Maze system.

Total locomotion, and time spend in the “open” and “closed” arms was recorded.

Conditioned Place Preference (CPP) is a paradigm that tests the susceptibility of mice to the rewarding or aversive affects of different stimuli, such as food or drugs [139]. The stimulus used in this case was 10 mg/kg cocaine, injected i.p, since Mchr1 has been shown to modulate the rewarding effects of cocaine in mice [108]. Before the mice are subjected to any time in the apparatus, they are handled each day for three days before the paradigm begins. CPP is an 8 day paradigm from start to finish, but each day of training and testing takes place in the same 12.5cm x 42.5cm acrylic box that can be subdivided into three parts. The three parts include a small (12.5cm x 7.5cm) central chamber, and two larger (12.5cm x 17.5cm) chambers on either end of the apparatus.

The larger chambers are fitted with one of two different patterns, with a different floor surface associated with each pattern. The mice were in the apparatus for 30 minutes during each day of each phase. On the first (pre-test) day, the mice are placed in the

107 apparatus and allowed free access to all compartments, and the time they spend in each compartment is recorded. Any pre-existing biases for one compartment or another that the animals displayed were balanced by assigning some animals to be paired with drug in compartments they initially favored, while other animals were assigned to be paired with drug in the compartment they avoided. This approach, when applied to an entire group of animals, eliminates the bias of the group for one compartment or another. For the next six days, animals are given saline or 10 mg/kg cocaine (the stimulus) on alternate days, known as the conditioning phase. For each animal, saline is always paired with one pattern, while cocaine is paired with the other pattern. During the conditioning phase, the entire apparatus was decorated with the flooring and pattern that was assigned to be paired with the appropriate stimulus, so the locomotor activity of animals could be assessed. On the last day of the paradigm, (the post-test), the apparatus was configured as in the pre-test, with the three compartments. Animals were placed in the apparatus, and the time spent in each compartment was recorded. The time spent in the cocaine- paired compartment in the pre-test was subtracted from the time spent in the compartment during the post-test to obtain the CPP score. The score is a measure of how rewarding cocaine was to the particular animal.

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Figure 4.1- Representative images of injections of AAV-CRE-GFP into the Nucleus Accumbens of IFT88fl/fl mice. (A) Low magnification immunofluorescence imaging of a coronal section from an IFT88fl/fl mouse injected with AAV-CRE-GFP. The Nucleus Accumbens Shell (NAcSh) and Anterior Commissure (ACo) are noted as neuroanatomical landmarks. Nuclei are in blue and CRE-GFP expressing cells are green. Scale bar = 100 microns. (B) Image from a coronal brain atlas highlighting the stereotactic coordinates used to target injections of AAV-CRE-GFP to the Nucleus Accumbens. (C) Zoomed in image from (B), showing the neuroanatomy of the targeted injection site. (D) Zoomed in image of the NAc from an IFT88fl/fl mouse after injection with AAV-CRE-GFP. The GFP channel is in the inset. Staining for AC3 reveals a lack of AC3 positive cilia 4 weeks after AAV-CRE-GFP injection into IFT88fl/fl cells. (E) Image from the uninjected nucleus accumbens of the same animal reveals an abundance of AC3 positive cilia. Scale bars = 50 microns. 109

Fi gure 4.2- CRE-GFP expression in BBS1fl/Δ cells results in loss of ciliary Mchr1. (A-C) Immunofluorescence image from the NAc of a BBS1fl/wt mouse injected with AAV-CRE-GFP 4 weeks prior. (A) Cells expressing CRE-GFP (green) express Mchr1 (red) in the cilium. (B) Numerous cells expressing CRE-GFP and un-labeled cells express AC3 (blue) positive cilia. (C) Merged image shows that Mchr1 staining co-localizes with AC3 staining, confirming that BBS1 expression is preserved in BBS1fl/wt cells that express CRE-GFP. (D-F) Immunofluorescence image from the NAc of a BBS1fl/Δ mouse injected with AAV-CRE-GFP 4 weeks prior. (D) Cells expressing CRE-GFP (green) do not appear to express Mchr1 (red) in the cilium. (E) Numerous cells expressing CRE-GFP and un-labeled cells express AC3 (blue) positive cilia. (C) Merged image shows that there is no co-localization of Mchr1 and AC3 staining, confirming that BBS1 expression is disrupted in BBS1fl/Δ cells that express CRE-GFP. Scale bars = 10 microns.

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Figure 4.3- Nucleus Accumbens mediated behaviors are unaltered in AAV-CRE-GFP injected BBS1fl/Δ mice. (A) The weights of BBS1fl/Δ mice do not increase significantly compared to BBS1fl/wt mice after injection of AAV-CRE-GFP into the NAc. (B) The time spent in the center of the apparatus does not differ when AAV-CRE-GFP injected BBS1fl/wt and BBS1fl/Δ mice are subjected to the Open Field paradigm. (C) Time spent in the closed arms of the Elevated Plus Maze does not differ between AAV-CRE-GFP injected BBS1fl/wt and BBS1fl/Δ mice. (D-F) Results from the Conditioned Place Preference (CPP) paradigm. (D) Basal locomotion does not differ between BBS1fl/wt and BBS1fl/Δ mice injected with AAV-CRE-GFP. (E) After treatment with 10mg/kg cocaine, locomotion of BBS1fl/wt and BBS1fl/Δ mice injected with AAV- CRE-GFP does not differ. (F) The CPP score for 10mg/kg Cocaine does not differ between BBS1fl/wt and BBS1fl/Δ mice injected with AAV-CRE-GFP. (n= 3 BBS1fl/wt, 5 BBS1fl/Δ animals for each experiment). All data is mean + SEM.

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Chapter 5: Conclusions and Discussions Kiss1r and the cilium: Implications and future directions

Our findings that Kiss1r localizes to the primary cilia on GnRH neurons, and that the loss of cilia on these neurons affects their response to kisspeptin could have broad ramifications for researchers in the fields of reproductive endocrinology and primary cilia. Since the discovery that the stimulation of Kiss1r on GnRH neurons is vital for regulation of the reproductive circuit, researchers have uncovered that the final output of

GnRH neurons is regulated by a myriad of factors [144]. With the demonstration that the cilium enhances Kiss1r signaling, researchers have another avenue to explore in dissecting Kiss1r’s modulation of GnRH neuron activity.

For investigations in the field of primary cilia, our work on this receptor raises many interesting points. If disruption of Kiss1r signaling due to dysfunctional cilia is not sufficient to cause the reproductive phenotypes seen in some ciliopathies, what is the etiology of these phenotypes? When the Kiss1r and GnRH system is disrupted very early in development, but not in adults, unknown compensatory mechanisms arise to rescue reproductive function in mice [85]. A worthwhile investigation could involve disrupting cilia in GnRH neurons on adult mice, and assessing the reproductive status of the mice.

The localization of other ciliary GPCRs is dynamic, and can change in response to agonism of the receptor or downstream signaling components {Green, unpublished data}

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[42]. These receptors also depend on the BBSome to traffic them into or out of the cilium, which does not seem to be the case for Kiss1r [42, 94]. Recent published reports have used live-cell imaging to study second-messenger signaling in the cilium [89, 132].

With the proliferation of intracellular sensors for IP3 and calcium, and the ability to target these sensors to the cilium, work could focus on determining where in the GnRH neuron Kiss1r generates its signals, and how the cilium modulates them. A great obstacle to many of these proposed directions is that GnRH neurons are a small, scattered population of neurons in the mammalian brain that are very difficult to isolate and study in culture. However, if this issue can be overcome, dissection of the mechanisms of

Kiss1r signaling in the cilium could have profound impacts in the fields of reproductive and cilia biology.

D1R and the Cilium: Implications and future directions

We have shown, in the second chapter of this work, that disruption of BBS1 function exclusively in cells expressing the dopamine receptor D1R leads to a phenotype of lowered activity and adult onset obesity. BBS1 is an essential component of the seven-protein complex known as the BBSome that is essential for the trafficking of certain GPCRs to the cilium, and D1R out of the cilium [42, 94]. Our hypothesis is that the inability of D1R to be trafficked out of the cilium alters its downstream signaling, resulting in the phenotypes observed.

Obesity and lower activity levels are on the rise among the world’s population, which makes the results of this work relevant from a public health perspective. While studies in mouse models of BBS have shown that one allele can be sufficient for proper 113 function of the gene, human studies have revealed that the presence of even one copy of a mutated BBS gene increases the risk of the carrier developing obesity [141]. The identification of mutations in obese human patients that perturb D1R localization could lead to a new understanding of and interventions for obesity. D1R is also known to be involved in the regulation of locomotion, reward, and cognition, and further investigation into the behavioral consequences of the mis-localization of the receptor could yield new insights into disorders where D1R mediated behaviors are altered [101].

Future work on this project should place a priority on elucidating the pathology of the reduced basal locomotion in D1R::Cre/BBS1fl/Δ mice. Further characterization could be performed on the D1R expressing neurons that are known to modulate locomotion in these mice to ensure that they develop normally when BBS1 is disrupted. Measurement of the response of post-synaptic neurons in D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice after D1R stimulation of the pre-synaptic neurons could help answer this question.

While we have examined acute signaling in D1R::Cre/BBS1fl/wt and D1R::Cre/BBS1fl/Δ mice, D1R stimulation can induce long term signaling in neurons as well [101].

Dissection of these pathways in our mutant mice could reveal changes in signaling that our acute assays have failed to detect.

Mchr1 and the Cilium: Implications and future directions

In our pilot study in which we disrupted the ciliary localization of Mchr1 in the nucleus accumbens of mice, we did not observe a significant difference in several behavioral measures between our control and experimental mice. However, our study did 114 demonstrate that we can effectively target discrete regions of the brain with stereotactic injections and disrupt the function of BBS proteins.

As mentioned previously, obesity is a massive public health crisis in the developed world, and a large component in the development of obesity currently is increased food intake. With the knowledge that Mchr1 is a potent regulator of food intake, and that is localization is disrupted in a mouse model of a disease known to involve hyperphagia, the study of the cilium’s role in the signaling of Mchr1 could be very relevant to public health.

The methods proposed in this study could be built upon by other cilia researchers to begin to dissect the idiopathic cognitive and behavioral phenotypes present in BBS.

While a few reports have examined the consequences of knocking out ciliary signaling molecules on behavior in mouse models, these studies do not address the importance of the cilium in the regulation of signaling and behavior [53, 54]. The stereotactic injection of Cre recombinase expressing viral vectors into discrete brain regions of mice containing conditional genes has the advantages of the researcher being able to very precisely choose the temporal and spatial coordinates of the disruption of their gene of interest.

This strategy can avoid developmental or off-target effects present in promoter driven

Cre recombinases, and allow study of acute effects of loss of genes.

Another strategy to determine the importance of the ciliary localization of Mchr1, and other ciliary receptors, could be rescue experiments in BBS KO mouse models. Our laboratory was previously able to restore localization of ciliary receptors in BBS KO neurons with endogenous expression of BBS proteins [94]. A group was able to restore

115 olfactory function in mice with a mutation in a ciliary gene with gene therapy [145]. In other areas of investigation, researchers have been able to rescue the behavioral responses of mice to certain stimuli by restoring the expression of a lost gene in a particular brain region of a knockout mouse [102, 146]. A novel approach could involve the restoration of BBS to different regions of the brain in BBS knockout mice, and assessment of these mice to determine if certain behavioral and cognitive phenotypes can be rescued using this method. These experiments could also serve to elucidate whether certain phenotypes are due to developmental defects or acute signaling defects caused by lack of BBSome function.

Conclusions and Discussion

This work provides strong evidence that the primary cilium in neurons is a critical regulator of the signaling of a certain subset of G protein-coupled receptors that localize to the organelle. While it has been demonstrated that primary cilia are vital signaling organelles in other organ systems in the body, the role of the primary cilium in the central nervous system is still relatively unknown. The organelle has been implicated in the regulation of developmental pathways that control neuronal migration and synaptic integration, but until now its modulation of the acute signaling of neurons in the brain has not been examined. We have demonstrated that the loss of a cilium can adversely affect the signaling of a receptor in a central neuron, and the alteration of the localization of a ciliary receptor correlates with behavioral and physiological phenotypes. The ciliary receptors we examine in this work are involved in reproduction, locomotion, energy balance, and food consumption, processes vitally important for the survival of any 116 species. While basic survival is not a concern with the human population in modern times, these processes are still at the core of many issues in the realm of public health.

By providing evidence that the cilium of the neuron is a vital regulator of the signaling of receptors that are deeply involved in clinically relevant conditions, we hope to spark research programs that will further examine and dissect the role the cilium plays in the function of central neurons.

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