Characterization of Neuronal Primary Cilia in Cellular Homeostasis and Disease

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CHARACTERIZATION OF NEURONAL PRIMARY CILIA IN CELLULAR HOMEOSTASIS AND DISEASE

DISSERTATION

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

Jill Augusta Green, BS

Graduate Program in Integrated Biomedical Sciences

The Ohio State University

2012

Dissertation Committee:

Kirk Mykytyn, PhD, Advisor

Dawn Chandler, PhD

Jeff Kuret, PhD

Karl Obrietan, PhD

Jill Augusta Green

2012

ABSTRACT

On nearly every mammalian cell, a tiny hair-like organelle known as a primary cilium protrudes from the cell surface. These cellular appendages act as specialized antennae to survey the extracellular milieu and transmit signals into the cell that are essential for cellular homeostasis. Although primary cilia were discovered over a hundred years ago and were originally considered evolutionary remnants, interest in these organelles has increased dramatically over the last ten years due to the recognized link between primary cilia and human disease. Improper formation or function of primary cilia can result in a myriad of human diseases and genetic disorders that are collectively called ciliopathies. Due to the ubiquity of cilia, ciliopathies can affect multiple organ systems and tissues and ciliopathy patients present with a wide range of clinical features including cystic kidney disease, retinal degeneration, anosmia, obesity, polydactyly, hypogenitalism, brain malformations, and intellectual disabilities.

The pathophysiological consequences of primary cilia dysfunction highlight the important roles cilia play during development and in the normal function of most tissues.

Although great progress has been made in understanding the functions of primary cilia on some cell types, primary cilia function on most cell types is still not known. This is particularly true for primary cilia on central neurons in the mammalian brain. Historically, the functions of primary cilia have been defined by the complement of unique proteins that localize to them. The seminal discovery that the G protein-coupled receptor

(GPCR), Somatostatin receptor 3 (Sstr3), selectively localizes to neuronal cilia indicated that primary cilia on central neurons in the mammalian brain potentially function as specialized non-synaptic sensory and signaling organelles.

This work aims to further explore the hypothesis that neuronal cilia are signaling organelles affecting neuronal function. Determining which proteins localize to primary cilia as well as the regulatory mechanisms that dictate this specificity is vital to understanding neuronal ciliary function. We have shown that one of the proteins mutated in the human ciliopathy Bardet-Biedl syndrome (BBS) interacts with multiple ciliary GPCRs regulating the trafficking of these receptors into and out of the cilium. In addition, utilizing a proteomic approach we have discovered novel BBS protein- interacting proteins (Chapter 2). These findings highlight the role of the BBS proteins in not only GPCR trafficking but shed light on new and exciting roles BBS proteins may play in ciliary and cellular biology.

Although Sstr3 was discovered to localize to neuronal cilia more than a decade ago, it has yet to be determined whether the receptor is functional within the cilium and can generate a signal. We have discovered that upon ciliary GPCR activation, the localization of signaling machinery is dynamic (Chapter 3), suggesting a specialized ciliary signal is generated. As the field of ciliary biology is ever changing, we have discovered ciliary GPCRs can form heteromers within the mouse brain (Chapter 4). As

GPCR heteromerization can affect ligand binding properties and downstream signaling, these findings add a previously unrecognized layer of complexity to neuronal ciliary signaling. Taken together, this work presents novel trafficking mechanisms responsible for the tightly regulated localization of ciliary proteins and has highlighted how neuronal cilia function as specialized signaling organelles orchestrating signal transduction cascades important for neuronal function.

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DEDICATION

This document is dedicated to my husband.

ACKNOWLEDGMENTS

I would like to first acknowledge my advisor, Kirk Mykytyn. This work would have not been possible without your patience, guidance, and scientific wisdom. Thank you for the independence needed to complete this work, and the opportunities given to attend conferences where I was able to network and develop my career. I greatly appreciate it and I know with the experience and knowledge gained from your lab I will be able to make an impact in the lives of individuals with PKD.

I would also like to thank each of my committee members, Dr. Dawn Chandler,

Dr. Jeff Kuret, and Dr. Karl Obrietan, who have been extremely generous with their time and have provided invaluable advice.

I am grateful to the Integrated Biomedical Science Graduate Program for the opportunity to be part of the Systems and Integrative Biology Training Grant (T32

GM068412), a two-year funded fellowship.

I would like to thank members of the Mykytyn lab, past and present. I would like to especially thank Jackie Domire for her patience and guidance my first few years of graduate school and Andrew Koemeter-Cox for his camaraderie and invaluable scientific discussion.

I would like to thank my family and friends for all of the support over the years and the reminder that I chose a career in science so that I could make a difference. I would like to especially thank my parents, Ron and Barb Laisure, for always believing in me. Finally, I would like to thank my husband for all of the unending patience and

v encouragement during my graduate school career. Thank you for the endless love, keeping me grounded, and the motivation needed to complete this degree.

VITA

April 1st 1985 ...... Born - Fort Wayne, Indiana, USA

2006 ...... B.S. Microbiology, Bowling Green State University

2007 to present ...... Graduate Research Associate, Integrated Biomedical Sciences Program, The Ohio State University

PUBLICATIONS

1. Green, JA, Mykytyn, K. Neuronal ciliary signaling in homeostasis and disease. Cell Mol Life Sci. 2010 Oct; 67(19):3287-97.

2. Domire JS, Green JA, Lee KG, Johnson AD, Askwith CC, Mykytyn K. Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet- Biedl syndrome proteins. Cell Mol Life Sci. 2011 Sep; 68(17):2951-60.

3. Green JA, Mykytyn K. Primary cilia. AccessScience. McGraw-Hill Yearbook of Science & Technology, 2012.

4. Green, JA, Gu. C, Mykytyn, K. Heteromerization of ciliary G protein-coupled receptors in the mouse brain. PLoS ONE. 2012; 7(9):e46303. Epub 2012 Sep.

FIELDS OF STUDY

Major Field: Integrated Biomedical Science Program

Area of Research Emphasis: Biochemical and Molecular Basis of Disease

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TABLE OF CONTENTS

ABSTRACT ...... ii

DEDICATION ...... iv

ACKNOWLEDGMENTS ...... v

VITA ...... vii

Publications ...... vii

Fields of Study ...... vii

TABLE OF CONTENTS ...... viii

LIST OF TABLES ...... xi

LIST OF FIGURES ...... xii

LIST OF ABBREVIATIONS ...... xiv

CHAPTER 1: INTRODUCTION TO PRIMARY CILIA ...... 1

Summary ...... 1

Cilia Classification and Structure ...... 1

Primary Cilia Formation and Regulation ...... 2

Primary Cilia Function ...... 4

Ciliopathies and Bardet-Biedl Syndrome ...... 7

Neuronal Primary Cilia and Neuronal Ciliary Signaling Machinery ...... 8

Hypothesis and Chapter 2-4 Overview ...... 10

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CHAPTER 2: DISCOVERY OF INTERACTING PARTNERS WITH PROTEINS

MUTATED IN THE HUMAN CILIOPATHY, BARDET-BIEDL SYNDROME ...... 19

Summary ...... 19

Introduction ...... 20

Results ...... 22

Discussion ...... 25

Materials and Methods ...... 30

CHAPTER 3: DYNAMIC LOCALIZATION OF CILIARY SIGNALING MACHINERY...... 40

Summary ...... 40

Introduction ...... 41

Results ...... 43

Discussion ...... 47

Materials and Methods ...... 52

CHAPTER 4: HETEROMERIZATION OF CILIARY G PROTEIN-COUPLED

RECEPTORS IN THE MOUSE BRAIN ...... 63

Summary ...... 63

Introduction ...... 63

Results ...... 65

Discussion ...... 69

Materials and Methods ...... 72

Acknowledgments ...... 76

CHAPTER 5: CONCLUSIONS AND DISCUSSIONS ...... 85

Implications and future studies elucidating the function of BBS proteins ...... 85

Implications and future studies investigating dynamic localization of neuronal ciliary

signaling machinery ...... 86

Implications and future studies exploring ciliary GPCR heteromerization ...... 87

Implications of understanding the role neuronal cilia play in cell biology ...... 88

REFERENCES ...... 90

LIST OF TABLES

Table 1.1: Common Clinical Features Observed in Ciliopathies...... 18

Table 2.1: Bbs4 associated proteins identified through proteomic analysis of mouse striatal lysate ...... 37

Table 2.2: Bbs4 associated proteins identified through proteomic analysis of mouse kidney lysate...... 38

Table 4.1: Distribution of Mchr1 immunoreactive cilia in the central nervous system .....84

LIST OF FIGURES

Figure 1.1: Cilia structure and examples of motile and primary cilia ...... 13

Figure 1.2: Examples of ciliary chemo-, photo-, and mechanotransduction ...... 14

Figure 1.3: Model of ciliary signaling disruption ...... 15

Figure 1.4: Localization of somatostatin receptor subtype 3 (Sstr3) to neuronal cilia. ....16

Figure 1.5: Neuronal cilia trafficking model ...... 17

Figure 2.1: Dopamine receptor 1 (D1) and Bardet-Biedl syndrome 5 (Bbs5) interact ...... 35

Figure 2.2: Bbs5 can directly interact with the third intracellular loop of multiple ciliary

GPCRs ...... 36

Figure 2.3: Bbs4 associates with the kinase, Nek1 ...... 39

Figure 3.1: Sstr3 dynamically localizes in the neuronal ciliary membrane ...... 58

Figure 3.2: Somatostatin-mediated βarr2 recruitment into primary cilia on IMCD cells...59

Figure 3.3: Somatostatin-mediated βarr2 recruitment into neuronal cilia...... 60

Figure 3.4: Quantification of βarr2 ciliary localization mediated by phosphorylation mutants of Sstr3 in IMCD cells...... 61

Figure 3.5: Quantification of βarr2 ciliary localization mediated by Sstr3 WT or Sstr3

T357A in cultured neurons ...... 62

Figure 4.1: Melanin-concentrating hormone receptor 1 (Mchr1) localizes to neuronal cilia throughout the mouse brain ...... 77

Figure 4.2: Mchr1 and somatostatin receptor 3 (Sstr3) colocalize in a subset of hippocampal neuronal cilia ...... 78

Figure 4.3: Mchr1 and Sstr3 colocalize in a subset of piriform cortical neuronal cilia .....79

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Figure 4.4: Mchr1 and Sstr3 colocalize in a subset of hypothalamic neuronal cilia ...... 80

Figure 4.5: Mchr1 and Sstr3 proteins interact ...... 81

Figure 4.6: Mchr1 and Sstr3 interact in live cells ...... 82

Figure 4.7: Mchr1 and Sstr3 interact in mouse hippocampal lysate ...... 83

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LIST OF ABBREVIATIONS

ACVI: adenylyl cyclase type VI

ACIII: adenylyl cyclase type III

AQP2: aquaporin-2

BBS: Bardet-Biedl syndrome

BSA: bovine serum albumin cAMP: adenosine 3’, 5’-cyclic monophosphate

CFTR: cystic fibrosis transmembrane conductance regulator cGMP: cyclic guanosine monophosphate

CNG channels: cyclic-nucleotide gated channels

CTS: ciliary targeting sequences

D1: dopamine receptor 1

D1: dopamine receptor 2

FRET: fluorescence resonance energy transfer

Gapdh: glyceraldehyde-3-phosphate

GPCR: G protein-coupled receptor

GRK: G protein-coupled receptor kinase

GTPases: guanosine triphosphatases

HEK293T: Human Embryonic Kidney 293T cell line

Hh: Hedgehog

Htr6: serotonin receptor 6 i3 loop: third intracellular loop

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IB: immunoblot/western blot

IFT: Intraflagellar transport

IMCD: Inner medullary collecting duct cell line

INPP5E: phosphatidylinositol-4,5-bisphosphate 5-phosphatase

IP: immunoprecipitation

MCH: melanin-concentrating hormone

Mchr1: Melanin-concentrating hormone receptor 1

MEFs: mouse embryonic fibroblasts

Nek1: Never In Mitosis A (NIMA)-related kinase 1

Nek3: Never In Mitosis A (NIMA)-related kinase 3

NES: Nuclear export signal

NIH3T3: mouse embryonic fibroblast 3T3 cell line

NIMA: Never In Mitosis A

NLS: nuclear localization signal

OSN: olfactory sensory neuron

PBS: phosphate-buffered saline

PC1: polycystin-1

PC2: polycystin-2

PFA: paraformaldehyde

PH-like: pleckstrin homology-like

PKA: Protein Kinase A

PKC: Protein Kinase C

PKD: Polycystic Kidney Disease

PRKA: A kinase anchor protein 5

Ptc: Patched

RPE cell: retinal pigmented epithelium cell line

SEM: standard error of the mean

Ser: serine

Smo: smoothened

SST: somatostatin

Sstr1: somatostatin receptor 1

Sstr2a: somatostatin receptor 2a

Sstr2a: somatostatin receptor 2b

Sstr3: somatostatin receptor 3

Sstr4: somatostatin receptor 4

Sstr5: somatostatin receptor 5

T357A: threonine 357 to alanine mutant

T-Ag: T antigen

Thr: threonine

UT: untreated

V2R: type 2 vasopressin receptor

Varr: visual arrestin

Y2H: yeast two-hybrid

βarr2: beta-arrestin-2

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1 CHAPTER 1: INTRODUCTION TO PRIMARY CILIA

1 – Partial citation for this chapter: Green, JA, Mykytyn, K. Neuronal ciliary signaling in homeostasis and disease. Cell Mol Life Sci. 2010 Oct; 67(19):3287-97

Summary:

Nearly every mammalian cell type projects from its surface a primary cilium that is thought to provide important sensory and signaling functions during development and cellular homeostasis. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Interestingly, ciliopathies present with a wide range of clinical features, including cystic kidney disease, retinal degeneration, obesity, polydactyly, anosmia, intellectual disability, and brain malformations. Although significant progress has been made in elucidating the functions of primary cilia on some cell types, the precise functions of most primary cilia remain unknown. This is particularly true for primary cilia on neurons throughout the mammalian brain. This work investigates the hypothesis that neuronal cilia are specialized non-synaptic signaling organelles.

Understanding the specific complement of proteins that selectively localize to neuronal cilia and the regulatory mechanisms of these processes will provide insight into the function of these specialized organelles.

Cilia Classification and Structure:

Cilia within the mammalian body are generally classified as either motile or primary (Fig. 1.1A & B). Motile cilia are mainly responsible for generating flow or movement and include respiratory cilia, ependymal cilia, oviduct cilia, and sperm flagella.

Primary cilia are typically immotile and function principally as sensory organelles. These functional classifications are not mutually exclusive, as subsets of primary cilia on the embryonic node are motile and generate flow [1] and motile respiratory cilia also possess sensory functions [2, 3]. All cilia are comprised of a microtubule core called an axoneme that is nucleated by a basal body, which is a specialized centriole that has migrated to the ciliary assembly site and is linked to the plasma membrane by transition fibers (Fig. 1.1C). In general, the axonemal structure of motile cilia consists of nine outer microtubule doublets and two centrally located microtubule singlets (“9+2”; Fig. 1.1C).

The axonemal structure of primary cilia consists of only the nine outer microtubule doublets (“9+0”; Fig. 1.1C). However, there are exceptions, such as “9+0” motile cilia on the embryonic node and “9+2” immotile cilia on olfactory neurons.

Primary Cilia Formation and Regulation:

Proteins are not synthesized within the cilium. Therefore, the structural proteins required to build and maintain the cilium, as well as the signaling proteins required for cilia function, are synthesized in the cell body and transported into and out of the cilium.

This transport is mediated by a highly conserved mechanism known as intraflagellar transport (IFT) [4-6]. IFT is a bidirectional microtubule-based transport process in which complexes of proteins called IFT particles are transported by kinesin and dynein motors along the axonemal outer doublet microtubules from the base of the cilium to the distal tip (anterograde transport) and then back to the cell body (retrograde transport; Fig.

1.1C) [4-6]. IFT is required for the formation and maintenance of all mammalian cilia and defective IFT is associated with severe diseases and developmental defects [7-10].

As the axoneme is assembled, it projects from the cell and becomes ensheathed by a membrane that is continuous with the plasma membrane [11]. The transition fibers

2 act as a selective barrier allowing only specific proteins to localize within the cilium and on the ciliary membrane. It has been proposed that proteins destined for the cilium accumulate at the site where the transition fibers contact the membrane and then are assembled into IFT particles before transport into the cilium [5, 12].

Although this process is not well understood, several recent discoveries lend significant insight into the molecular mechanisms that control the specificity of ciliary protein localization. Identification of ciliary targeting sequences (CTS) that appear to mediate ciliary localization of specific proteins and the identification of proteins that may act as gate keepers at the base of the cilium demonstrate the high degree of regulation involved in keeping primary cilia distinct and compartmentalized organelles.

Strikingly, there is not one particular targeting sequence that directs protein localization to the cilium and multiple ciliary targeting sequences have been discovered

[13-18]. Some CTSs contain sites for acylation like myristoylation and palmitoylation, suggesting targeting to lipids and perhaps lipid rafts is required for ciliary localization of some proteins [16]. Identification of a CTS in the kinesin motor protein KIF17 revealed similarity to a nuclear localization signal, suggesting cilia possess an entry pathway for proteins that is analogous to nuclear entry of proteins [19]. In agreement with this hypothesis, the trafficking molecules importin-β2 and the GTPase Ran, which are necessary for classical nuclear import, were found to play a critical role in ciliary import

[19, 20].

The observation that access to the cilium is restricted, led to the discovery of selective barriers at the base of the cilium that allow only specific proteins to localize within the ciliary compartment. Proteins mutated in the human disease,

Nephronophthisis, appear to localize to the ciliary transition zone and act as ciliary gatekeepers to regulate entry into the cilium [21]. There are also mechanisms for retaining proteins within the cilium. Septin 2, a member of the septin family of guanosine

3 triphosphatases (GTPases) that forms a diffusion barrier in budding yeast, forms a barrier at the base of the ciliary membrane to restrict the diffusion of ciliary membrane proteins between the ciliary and plasma membrane [22]. This tightly regulated process of ciliary localization highlights the importance of proper targeting and retention of proteins to the ciliary compartment for normal ciliary function in development and cellular homeostasis.

Primary Cilia Function:

The functions of cilia are defined by the signaling proteins enriched within the ciliary compartment. Primary cilia mediate numerous signaling pathways throughout the mammalian body in response to a diverse set of sensory stimuli. These organelles are able to sense and respond to their cellular environment due to the particular complement of proteins localizing to cilia on different cell types. In general, cilia mediate chemo-, photo-, and mechanotransduction. Olfactory cilia on olfactory sensory neurons (OSNs) are an example of chemosensory cilia that directly sense odorants from the environment

(Fig. 1.2A). Each OSN possesses 10-30 olfactory cilia that protrude through the olfactory epithelium into the nasal cavity and contain the signaling molecules and downstream effectors needed for olfaction [23, 24]. The olfactory signal transduction cascade begins with an odorant binding to an olfactory G protein-coupled receptor

(GPCR) on the ciliary membrane, which triggers the activation of the stimulatory G protein (Gαolf). The G protein activates type III adenylyl cyclase (ACIII), which increases adenosine 3’, 5’-cyclic monophosphate (cAMP) within the cilium. An increase in cAMP levels leads to the activation and opening of cyclic-nucleotide gated (CNG) channels, allowing for the influx of calcium ions. The increase in intracellular calcium leads to the activation and opening of Ca2+-gated chloride channels, resulting in an efflux of chloride

4 ions further depolarizing the neuron [25, 26]. There is also evidence that renal cilia mediate chemosensation (Fig. 1.2C). Recently, the type 2 vasopressin receptor (V2R) was found to localize to cilia on renal epithelial cells [27]. V2R is a GPCR that regulates

Na+ and water reabsorption in the mammalian nephron. Interestingly, V2R functionally couples with type V/VI adenylyl cyclase in the cilium and vasopressin treatment of isolated renal cilia results in localized production of cAMP and cAMP-dependent activation of cation-selective channel activity [27]. Thus, in response to vasopressin, renal cilia mediate a cAMP-signaling pathway that targets ciliary channel function.

Phototransduction in the eye is mediated in the outer segments of photoreceptors [28] (Fig. 1.2B). The outer segment is a highly modified cilium packed with membrane disks full of visual pigments that are composed of a vitamin A-based chromophore and the GPCR, opsin. Upon light activation, opsin activates the G protein transducin, which then stimulates a phosphodiesterase that hydrolyzes cyclic GMP

(cGMP) to GMP. This light-induced reduction in cGMP levels causes cGMP-gated channels to close, thereby hyperpolarizing the cell.

Mechanosensation is mediated by primary cilia on a variety of cell types, including renal epithelial cells [29, 30], embryonic nodal cells [31], endothelial cells [32,

33], cholangiocytes [34], chondrocytes [35], and smooth muscle cells [36]. In the kidney, bending of renal epithelial cilia by fluid flow generates an intracellular calcium signal [29].

The calcium signal is facilitated by polycystin-1 (PC1) and polycystin-2 (PC2), which localize to primary cilia and form a mechanosensitive Ca2+ channel [37] (Fig. 1.2C). A conserved mechanism utilizing these same signaling proteins has been observed in the liver, where cholangiocytes selectively localize PC1, PC2, and type VI adenylyl cyclase

(ACVI) to their primary cilia. This allows the cells to sense and respond to changes in luminal fluid flow and the generation of Ca2+ and cAMP signaling [34].

In addition to the three general functions in differentiated cells, primary cilia also play a critical role in mammalian development by orchestrating numerous transduction pathways. This is no more apparent than in the case of the well-studied and characterized Hedgehog (Hh) signaling pathway [38, 39], which is essential for normal patterning of multicellular embryos and the development of numerous tissues and organs [40]. Disruptions in Hh signaling can cause severe developmental abnormalities in mice and humans, including neural tube defects, polydactyly, holoprosencephaly, craniofacial defects, and skeletal malformations [41]. Hh signaling is regulated by

Patched (Ptc), the 12-transmembrane Hh receptor, which localizes to primary cilia in the absence of Hh stimulation and inhibits the 7-transmembrane GPCR, Smoothened (Smo) by preventing it from accumulating in cilia. Upon Hh activation, Ptc traffics out of the cilium and allows for Smo to be trafficked into the cilium [42, 43]. Smo then activates signaling at the distal tip of the cilium where the Gli transcription factors are localized

[44]. Thus, cilia are required to coordinate the signaling components of the Hh pathway.

There are important conclusions that can be drawn from these examples of cilia functions and the signaling pathways involved. It is clear cilia coordinate specialized signaling and their functions are determined in large part by the specific signaling proteins that are enriched in the ciliary compartment (Fig. 1.3A). Loss of ciliary signaling proteins or cilia structure disrupts ciliary signaling and can cause diseases (Fig. 1.3B &

C). Finally, although there is great diversity in the functions of different cilia and the stimuli they detect, there is apparent conservation between different ciliary signaling pathways, including the utilization of Ca2+, GPCR, cyclic nucleotide, and ionic signaling, enabling for the detection of the extracellular environment and transmitting signals back to the cell. This conservation may provide great insights into the ciliary signaling pathways mediated by cilia on other cell types, such as post-mitotic neurons in the brain.

Ciliopathies and Bardet-Biedl Syndrome:

Scientific intrigue and research surrounding primary cilia has increased in the past 10-15 years due to the link between primary cilia dysfunction and human disease.

Improper formation or function of primary cilia has been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies.

Due to the presence of primary cilia on numerous cell types in nearly every organ system, ciliopathy patients present with a wide range of clinical features including cystic kidney disease, retinal degeneration, anosmia, obesity, hepatic dysfunction, polydactyly, hypogonadism, defective left-right patterning, brain malformations, and intellectual disability (Table 1.1).

To further elucidate the function of primary cilia, mouse models have been developed of the human ciliopathy, Bardet-Biedl syndrome (BBS). BBS is a rare autosomal recessive genetic disorder only occurring in approximately 1:120,000 live births in North America and Europe [45]. However in small isolated communities, BBS can occur as high as 1:13,500-1:17,500 live births [46]. Similar to many ciliopathies,

BBS is heterogeneous and at least 16 causative genes have been identified [46]. The primary sequences and predicted structures of these 16 gene products have not provided much insight into the role BBS proteins play in BBS pathogenesis. Many BBS proteins contain domains important for protein-protein interactions. Uniquely, BBS5 contains two Pleckstrin Homology-like (PH-like) and binds to specific phosphoinositides which have been implicated in vesicular transport, actin cytoskeleton remodeling, and signal transduction. Other BBS proteins show sequence homology with chaperonins suggesting BBS proteins may regulate the assembly and maturation of proteins or protein complexes [45, 47].

Although little is known about individual BBS proteins, it appears that a group of

BBS proteins may be involved in a conserved cellular pathway regarding protein trafficking. Seven, highly conserved BBS proteins form a stable complex known as the

BBSome. The BBSome, which includes the BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9 proteins, localizes to centriolar satellites in the cytoplasm, travels into the cilium, and associates with the ciliary membrane [47]. Furthermore, the BBSome can form a coat-like complex that may be involved in docking and fusion of post-Golgi vesicles at the base of the cilium [48]. These findings led to the hypothesis that BBS results from global ciliary dysfunction due to defective transport of ciliary proteins.

The cardinal features of the pleiotropic disorder BBS are retinopathy, obesity, cognitive deficits, hypogonadism, and polydactlyl [46, 49]. However this disease can also manifest with other phenotypes such as renal cysts, diabetes, anosmia, hearing loss, hypertension [50]. Although BBS is rare, it can serve as a valuable model to investigate the pathophysiology of ciliary dysfunction due to its multi-systematic nature.

Many observed phenotypes in BBS can be explained by cilia dysfunction in distinct organ systems such as the eye and kidney, this work however focuses on phenotypes of the disease that might be explained by cilia dysfunction within the brain.

Neuronal Primary Cilia and Neuronal Ciliary Signaling Machinery:

It has been nearly fifty years since primary cilia were first discovered on central neurons in the mammalian brain. At the time, it was hypothesized that neuronal cilia were vestigial organelles and were formed simply because of an innate tendency of centrioles to form cilia [51]. Not until the discovery that somatostatin receptor subtype 3

(Sstr3) selectively localizes to neuronal cilia throughout the rat brain, were neuronal cilia considered to be functional organelles [52]. The presence of a particular receptor

8 enriched on the neuronal ciliary membrane suggested at least some neuronal cilia function as chemosensors of the extracellular milieu. Indeed, this was further supported by the subsequent findings that serotonin receptor 6 [53, 54], melanin-concentrating hormone receptor 1 [17, 55], and dopamine receptor 1 [56] are enriched on neuronal cilia.

Although it has been known for more than ten years that Sstr3 localizes to neuronal cilia, remarkably we still do not know whether Sstr3 signals on the ciliary membrane and how this affects neuronal function. However, recent studies utilizing

Sstr3 knockout mice have begun to elucidate potential ciliary somatostatin signaling.

Specifically, an investigation of the role of Sstr3 signaling in learning and memory revealed that Sstr3 is critical for object recognition memory but not spatial memory [57].

Furthermore, the authors found that regulation of cAMP signaling is disrupted in Sstr3 knockout mice or WT mice treated with an Sstr3 antagonist. Given that Sstr3 is highly enriched in cilia (Fig. 1.4A), the authors suggest Sstr3 mediates cAMP signaling within cilia and impacts cognition. However, further studies are necessary to confirm that Sstr3 modulates cAMP levels within the cilium and whether loss of cilia on Sstr3-expressing neurons similarly impacts object recognition memory and cAMP signaling.

In addition to receptors selectively localizing to neuronal cilia, further studies revealed a large proportion of neuronal cilia are enriched for the signaling protein type III adenylyl cyclase (ACIII). ACIII colocalizes with Sstr3 (Fig. 1.4B-D) [55, 58] and Mchr1

[17, 55] on subsets of cilia, suggesting neuronal cilia can sense the extracellular milieu by localizing receptors to the ciliary membrane and also mediate cAMP signaling by coupling to the adenylyl cyclase cascade. ACIII is one of ten known mammalian isoforms of adenylyl cyclase that function to convert ATP to cyclic cAMP in response to activation by a variety of hormones, neurotransmitters, and other regulatory molecules [59]. Cyclic

AMP, in turn, activates several other target molecules to control a broad range of

9 intracellular processes. As discussed earlier, ACIII is enriched in cilia of olfactory sensory neurons [60] where it couples activation of odorant GPCRs to increased cAMP levels in olfactory cilia [61]. It is possible that ACIII in neuronal cilia acts analogously to

ACIII in olfactory cilia to affect membrane potential and alter neuronal firing rates.

Studies utilizing ACIII knockout mice have further supported the notion that neuronal cilia are signaling organelles. Similarly to Sstr3 knockout mice, ACIII knockout mice display learning and memory deficits [62]. Given that ACIII predominantly localizes to cilia, the authors suggest neuronal cilia generate a unique cAMP signal via ACIII which is needed for proper neuronal function that affects such physiological processes as learning and memory. However, like the Sstr3 findings, further studies are needed to confirm signals are generated within the ciliary membrane and neuronal cilia can be classified as a non-synaptic signaling organelle.

Hypothesis and Chapter 2-4 Overview:

The recognition that neuronal cilia are widespread and enriched for specific receptors, and combined with the discovered link between cilia dysfunction and human disease, sparked a renewed interest in understanding the functions of these organelles in the brain. This document presents a collection of work that aims to elucidate the function of neuronal cilia in cellular homeostasis and disease. As the functions of primary cilia are defined by the complement of proteins that selectively localize to them, this work aims to understand which proteins specifically localize to neuronal cilia and how they are regulated.

Throughout this work, experiments were designed to test the hypothesis that neuronal cilia act as specialized non-synaptic signaling organelles due to the observation that distinct G protein-coupled receptors (GPCRs) and signaling cascade

10 components (ACIII) selectively localize to neuronal cilia. Utilizing mouse models of the human ciliopathy Bardet-Biedl syndrome (BBS), this work provides strong evidence that indeed neuronal cilia are signaling organelles with functional receptors localizing to them and provides evidence that BBS proteins are directly involved in regulating ciliary GPCR localization.

Chapter 2 determines the role BBS proteins play in ciliary GPCR trafficking.

Interestingly, it appears that a particular BBS protein, Bbs5, can directly interact with ciliary GPCRs and mediate their trafficking into and out of the cilium. This work is highlighted in a Cellular and Molecular Life Sciences publication. Furthermore in

Chapter 2, utilizing a proteomic approach we were able to determine novel BBS protein- interacting partners. These findings have laid the groundwork for future studies determining BBS protein function in cellular processes other than ciliary GPCR trafficking.

Chapter 3 focuses on the dynamic process of protein localization within neuronal cilia. Somatostatin receptor 3 (Sstr3) selectively localizes to neuronal cilia however this work reveals upon activation of the receptor, Sstr3 is also trafficked out of the cilium.

Classically upon GPCR activation the receptor becomes phosphorylated allowing for the recruitment of arrestin proteins to desensitize the receptor and facilitate internalization.

Interestingly this work shows, upon Sstr3 activation, beta-arrestin-2 (βarr2) is recruited into the cilium where it colocalizes with the activated receptor. Furthermore this trafficking of βarr2 into the cilium upon receptor activation can be blocked by the expression of an Sstr3 phosphorylation mutant. Taken together, Chapter 3 focuses on signals generated by functional ciliary GPCRs and how neuronal cilia orchestrate the dynamic localization of many proteins involved in signaling cascades.

Neuronal ciliary signaling appears to be a very elaborate and intricate process.

Findings presented in Chapter 4 illustrate that different ciliary GPCRs can colocalize

11 within the same cilium in distinct brain regions and can form heteromers. As GPCR heteromerization can affect ligand binding properties and downstream signaling, these findings published in PLoS ONE bring to light an unrecognized layer of complexity about neuronal ciliary signaling.

In conclusion, this work provides solid evidence that indeed neuronal cilia are signaling organelles. Our working model suggests improper dynamic localization of receptors and signaling machinery leading to neuronal cilia dysfunction could explain phenotypes such as obesity and cognitive deficits observed in BBS patients and other ciliopathies (Fig. 1.5).

Figure 1.1: Cilia structure and examples of motile and primary cilia. (A) Scanning electron micrograph showing numerous motile cilia protruding from epithelial cells of a mouse trachea. Scale bar = 5µm. (B) Scanning electron micrograph showing solitary primary cilia projecting from mouse renal epithelial cells lining the nephron. Scale bar = 5µm. (C) Membrane and cytosolic proteins destined for the ciliary compartment are transported in Golgi-derived vesicles and exocytosed at the base of the cilium where they associate with intraflagellar transport (IFT) particles. The transition fibers form a selective barrier to the ciliary compartment and only proteins containing specific ciliary targeting motifs are allowed access. Following entry into the cilium, proteins are transported along the axoneme. Cross-sections show the typical microtubule structures of motile and primary cilia.

Figure 1.2: Examples of ciliary chemo-, photo-, and mechanotransduction. (A) Schematic of a single olfactory sensory neuron. Boxed region of interest is magnified and illustrates the ciliary signaling pathway. Odorant activation of olfactory G protein- coupled receptors (GPCRs) results in an increase in cAMP levels, which is mediated by type III adenylyl cyclase (ACIII). This results in activation of cyclic nucleotide-gated (CNG) channels leading to an increase in Ca2+ levels, subsequent activation of chloride channels, and depolarization of the neuron. (B) Schematic of a photoreceptor, which is comprised of an inner and outer segment that are connected by a “9+0” connecting cilium. Proteins are synthesized in the inner segment and transported by IFT across the connecting cilium to the outer segment, which is a highly modified cilium, where they mediate phototransduction. (C) Schematic of a renal cilium demonstrating Ca2+ signaling mediated by the ciliary proteins polycystin-1 (PC1) and polycystin-2 (PC2) in response to bending of the cilium by fluid flow. Renal cilia may also act as chemosensors by mediating vasopressin activation of the type 2 vasopressin receptor (V2R) on the ciliary membrane, which in turn modulates cAMP signaling.

Figure 1.3: Model of ciliary signaling disruption. (A) Signaling proteins are enriched in the cilium where they coordinate cilia-specific signaling that is transmitted to the cell. Two examples of ciliary signaling pathways are illustrated; PC1- and PC2-mediated Ca2+ signaling and GPCR-mediated cAMP signaling. (B) Disruption in the trafficking of signaling proteins (indicated by X’s) into the ciliary compartment leads to loss of a cilia-specific signal. Ciliary receptors that are not trafficked to the cilium may accumulate in intracellular vesicles, mislocalize on the cell membrane, or be degraded resulting in a loss or gain of signal. (C) Defects in cilia structure prevents proper localization of ciliary signaling proteins (indicated by X’s) and leads to loss of a cilia-specific signal. Again, ciliary receptors that are not trafficked to the cilium may accumulate in intracellular vesicles, mislocalize on the cell membrane, or be degraded, resulting in a loss or gain of signal.

A B C D

Sstr3 Sstr3 ACIII Merge

Figure 1.4: Localization of somatostatin receptor subtype 3 (Sstr3) to neuronal cilia. (A) Adult mouse brain section corresponding to the CA3 region of the hippocampus immunolabeled with an antibody to Sstr3 (green). Note the abundance of Sstr3-positive cilia. Nuclei were stained with DRAQ5 (blue). (B-C) Images of a day 7 mouse hippocampal neuron immunolabeled with antibodies to Sstr3 (green) and type III adenylyl cyclase (ACIII; red). (D) Merged image demonstrating colocalization of Sstr3 and ACIII to neuronal cilia. Nuclei were stained with DRAQ5 (blue) and the cilium is indicated with arrows. Scale bar = 5 µm.

Figure 1.5: Neuronal cilia trafficking model. During cellular homeostasis receptors and necessary signaling machinery (GPCRs, G proteins, second messengers, βarr2, etc.) are trafficked into and out of the neuronal cilium. One mechanism for transporting ciliary GPCRs into and out of cilia is through interactions with the BBSome subunit, Bbs5. Certain ciliary GPCRs require BBS proteins to properly localize to cilia (red). In contrast, upon agonist binding, other ciliary GPCRs require BBS proteins to be properly trafficked out of the cilium (blue). This dynamic process is necessary to elicit a proper ciliary signal. In disease neurons, receptors and signaling machinery cannot properly traffic into and out of cilia resulting in a loss or gain of ciliary signaling. Ultimately, altered neuronal ciliary signaling leads to phenotypes observed in ciliopathy patients.

Table 1.1: COMMON CLINICAL FEATURES OBSERVED IN CILIOPATHIES

Disease Clinical Features

Autosomal Dominant Polycystic Kidney Disease Cystic kidneys, hepatic cysts (ADPKD)

Autosomal Recessive Polycystic Kidney Disease Cystic kidneys, hepatic cysts (ARPKD) Cystic kidneys, hepatic dysfunction, left-right Nephronophthisis (NPHP) asymmetry defects CNS malformations, retinal degeneration, Bardet-Biedl Syndrome polydactyly, truncal obesity, intellectual disabilities, (BBS) hypogonadism, cystic kidneys, left-right asymmetry defects CNS malformations, cystic kidneys, hepatic Joubert’s Syndrome dysfunction, intellectual disabilities, polydactyly, (JBTS) retinal degeneration, left-right asymmetry defects CNS malformations, cystic kidneys, gonadal Meckel’s Syndrome malformations, hepatic dysfunction, intellectual (MKS) disabilities, polydactyly, left-right asymmetry defects Leber congenital Retinal degeneration, intellectual disabilities amaurosis (LCA) Senior-Løken Syndrome Cystic kidneys, hepatic dysfunction, retinal (SLNS) degeneration, left-right asymmetry defects Alstrom Syndrome CNS malformations, cystic kidneys, hepatic (ALMS) dysfunction, obesity, retinal degeneration Skeletal defects, CNS malformations, cystic kidneys, Oral-facial-digital hepatic dysfunction, intellectual disabilities, syndrome type 1 (OFD 1) polydactyly Skeletal defects, CNS malformations, cystic kidneys, Jeune asphyxiating hepatic dysfunction, intellectual disabilities, thoracic dystrophy (JATD) polydactyly, retinal degeneration, left-right asymmetry defects Ellis-van Creveld Skeletal defects, CNS malformations, gonadal syndrome (EVC) malformations, intellectual disabilities, polydactyly

Table 1.1: Common Clinical Features Observed in Ciliopathies.

Various ciliopathies are listed with their associated phenotypes. Note, due to the nearly ubiquitous presence of primary cilia throughout the body, ciliopathies can affect multiple organ systems and tissues.

CHAPTER 2: DISCOVERY OF INTERACTING PARTNERS WITH PROTEINS MUTATED IN THE HUMAN CILIOPATHY, BARDET-BIEDL 2 SYNDROME

2 – Partial citation for this chapter: Domire JS, Green JA, Lee KG, Johnson AD, Askwith CC, Mykytyn K. Dopamine receptor 1 localizes to neuronal cilia in a dynamic process that requires the Bardet-Biedl syndrome proteins. Cell Mol Life Sci. 2011 Sep; 68(17):2951-60.

Summary:

Although discovered over a hundred years ago, a renewed interest in primary cilia has emerged due to the link between their dysfunction and human disease. Indeed, defects in the formation or function of primary cilia can lead to a myriad of diseases collectively termed ciliopathies. The human genetic disease, Bardet-Biedl syndrome

(BBS), is a pleiotropic disorder that has been linked to dysfunction of primary cilia.

Recently it has been shown that disruption of BBS proteins results in the mislocalization of G protein-coupled receptors (GPCRs) potentially altering ciliary function.

Interestingly, seven BBS proteins form a complex called the BBSome that localizes to the base of and within primary cilia. We hypothesized interactions between GPCRs and the BBSome are required for proper ciliary localization. To test this hypothesis we have used several biochemical approaches to identify the proteins mediating GPCR ciliary localization. Our data suggest Bbs5, a BBSome subunit, interacts directly with the third intracellular loop of multiple ciliary GPCRs, supporting our model that BBS proteins mediate the localization of GPCRs into and out of cilia. Furthermore, by utilizing a proteomic approach we have identified novel BBS protein-interacting partners. These results will lay the ground work for future studies focused on elucidating the function of

BBS proteins in cellular processes other than GPCR trafficking and provide insight into primary cilia function.

Introduction:

Primary cilia are able to sense and respond to the extracellular milieu and relay signals back to the cell due to proteins that selectively localize to the subcellular compartment. For example, olfactory cilia present on olfactory sensory neurons (OSNs) can sense and respond to odorants from the environment. Olfactory cilia are capable of sensing the extracellular environment by localizing the necessary signaling machinery for olfaction [23, 24]. Specifically olfactory cilia localize G protein-coupled receptors

(GPCRs), G proteins, adenylyl cyclase, cyclic-nucleotide gated (CNG) channels, and

Ca2+-gated chloride channels to the ciliary compartment enabling the neuron to sense and respond to odorants. Activation of the GPCRs triggers a signaling cascade which eventually leads to depolarization of the olfactory sensory neurons [25, 26].

Interestingly, only a small collection of proteins present within the cell can selectively localize to primary cilia. For example in neurons, there are five somatostatin receptor genes (Sstr1, Sstr2, Sstr3, Sstr4, and Sstr5) that can encode six different receptors, due to alternative splicing of the Sstr2 gene. All of these receptors are members of the GPCR superfamily of cell surface receptors that couple to heterotrimeric

G proteins to regulate numerous signaling cascades [63, 64]. All GPCRs are seven transmembrane-spanning receptors and share a common structure with an extracellular amino (N)-terminus, three extracellular loops, three intracellular loops, and a carboxyl

(C)-terminus inside the cell [65]. Although all six somatostatin receptors share similar sequence and structure homology, interestingly only Sstr3 selectively localizes to primary cilia on central neurons throughout the brain [52]. This finding suggested there

20 are unique sequences present within Sstr3 that mediate its selective localization to neuronal cilia.

Because ciliary proteins are synthesized in the cell body and need to be trafficked to the cilium, a great deal of interest has surrounded the sequences and mechanisms responsible for the sorting and trafficking of proteins. Various ciliary targeting sequences have been identified however it appears that there is not just one particular sequence that dictates ciliary localization [13-18]. Furthermore, even more mystery surrounds the trafficking mechanisms responsible for localizing proteins destined for the primary cilium [16]. It was recently discovered in our lab that neurons lacking proteins mutated in the human ciliary disease, Bardet-Biedl syndrome (BBS), fail to properly localize GPCRs into and out of neuronal cilia [55, 56]. Bardet-Biedl syndrome is a pleiotropic disease and appears to result from ciliary dysfunction in multiple organ systems. BBS patients can present with a wide range of clinical features including retinopathy, obesity, cognitive deficits, hypogonadism, and polydactyly, renal cysts, anosmia, hearing loss, diabetes, and hypertension [46, 49, 50]. Little is known about individual proteins mutated in BBS; however it appears that a group of BBS proteins are involved in a conserved cellular pathway. Seven, highly conserved BBS proteins form a stable complex known as the BBSome. The BBSome, which includes the BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9 proteins, localizes to centriolar satellites in the cytoplasm, travels into the cilium, and associates with the ciliary membrane [47]. Furthermore, the BBSome can form a coat-like complex that may be involved in docking and fusion of post-Golgi vesicles at the base of the cilium [48].

These findings and the knowledge that ciliary GPCRs mislocalize in BBS neurons led to the following hypotheses: 1) BBS results from global ciliary dysfunction and 2) ciliary dysfunction is a result of defective transport of ciliary proteins.

Many observed BBS phenotypes can be explained by defective ciliary transport in easily identifiable organ systems, such as the eye or kidney, however here we focus on BBS phenotypes that might be explained by ciliary dysfunction within the brain such as obesity and intellectual disability. We propose that obesity and intellectual disability may arise from altered trafficking and signaling of ciliary GPCRs in BBS neurons. Our results conclude that a major function of BBS proteins is to mediate the localization of

GPCRs that traffic into and out of the cilium. Specifically, we show that a subunit of the

BBSome, Bbs5, directly interacts with the ciliary GPCRs dopamine receptor 1 (D1), somatostatin receptor subtype 3 (Sstr3), and melanin-concentrating hormone receptor 1

(Mchr1). To further understand BBS protein function and how they contribute to BBS pathology we set out to determine novel BBS protein functions. By utilizing a proteomics approach, we have identified novel putative Bbs4-interacting proteins which provide important new avenues to pursue in understanding the underlying mechanism of BBS pathology and potentially ciliary dysfunction.

Results:

Bbs5 Interacts with Dopamine Receptor 1

Recently, our laboratory discovered that the third intracellular loop (i3 loop) of certain GPCRs contain a putative ciliary targeting sequence that is important for ciliary localization [17]. Furthermore, by taking a bioinformatics approach and screening a human GPCR library for the presence of the i3 loop putative ciliary targeting sequence, we were able to discover that dopamine receptor 1 (D1) is a ciliary GPCR that localizes to neuronal cilia. Additionally, data from our lab shows the BBS proteins are involved in

D1 ciliary distribution [56]. As the BBSome is known to localize to cilia [47], we tested the hypothesis that subunits of the BBSome directly interact with D1 to regulate its ciliary

22 localization. We first screened the i3 loop of D1 against the seven BBSome subunits using yeast two-hybrid analysis and found that the i3 loop of D1 specifically interacts with Bbs5 and none of the other BBSome subunits analyzed (Fig. 2.1A).

We then confirmed the interaction of D1 and Bbs5 by co-immunoprecipitation of transiently transfected epitope-tagged full-length proteins. Lysates of HEK293T cells expressing HA-tagged Bbs5 and myc-tagged D1 or myc-tagged glyceraldehyde-3- phosphate dehydrogenase (Gapdh), as a negative control, were immunoprecipitated with an anti-myc monoclonal antibody followed by immunoblotting with anti-HA. Bbs5 was detected in the D1 immunoprecipitate, but not the Gapdh immunoprecipitate, indicating that D1 and Bbs5 interact (Fig. 2.1B). These results suggest the BBSome regulates D1 ciliary localization through an interaction that is mediated by Bbs5.

Bbs5 interacts with other ciliary GPCRs

To determine whether Bbs5 is involved in a conserved ciliary GPCR trafficking mechanism we tested whether Bbs5 could interact with other ciliary GPCRs. We screened the i3 loop of somatostatin receptor subtype 3 (Sstr3) and melanin- concentrating hormone receptor (Mchr1) against Bbs5 using yeast two-hybrid analysis and found that indeed Bbs5 directly interacts with other known ciliary GPCRs (Fig 2.2).

Interestingly, it has been shown Mchr1 and Sstr3 require BBS proteins to properly localize to neuronal cilia [55]. In the case of D1, the receptor traffics to the cilium in a

BBSome-independent manner. However, the BBS proteins are required to traffic D1 out of the cilium upon D1-agonist treatment [56]. Therefore, these results suggest the

BBSome regulates the trafficking of multiple ciliary GPCRs into and out of the cilium by direct interaction between Bbs5 and the ciliary GPCR.

Identification of novel BBSome-interacting proteins

Often protein-protein interactions provide insight into protein function and cellular processes. In addition to trafficking cargo needed for ciliary signaling, it has recently been proposed that BBS proteins have other cellular functions [66]. To further investigate the functions of BBS proteins we utilized a proteomic approach to identify novel BBSome-interacting proteins. Striatal and kidney lysate was isolated from Bbs4+/+ and Bbs4-/- mice and subjected to immunoprecipitation with magnetic beads coupled to an anti-Bbs4 antibody. Finally, any Bbs4-associated proteins were subjected to tandem mass spectrometry and protein identification. Because Bbs4 has been shown to be part of the BBSome complex, this approach allowed for the detection of novel protein-protein interactions with Bbs4 and/or the BBSome. Furthermore, utilizing lysate from Bbs4-/- mice as a negative control allowed for easy detection of non-specific interacting proteins.

For the first time ever, immunoprecipitation results utilizing striatal lysate allowed for the detection of endogenous BBSome (Table 2.1). This exciting result validated the proteomics approach, using the mentioned parameters, to detect Bbs4/BBSome- interacting proteins. Furthermore, by utilizing kidney lysate we were able to detect a partial BBSome (Bbs1, Bbs2, Bbs5, and Bbs9) and pericentriolar material-1, which is a known Bbs4-interacting protein (Table 2.2). Again, these results validated our approach. Excitingly, using this method we were able to detect putative Bbs4/BBSome- interacting proteins. Our results show A kinase (PRKA) anchor protein 5 and diacylglycerol kinase iota were immunoprecipitated from striatal lysate and phosphatidylinositol (4, 5) bisphosphate 5-phosphatase was immunoprecipitated from kidney lysate. In addition, our proteomic analysis revealed that Bbs4 or the BBSome associates with the kinase, Nek1, in both striatal and kidney lysate (Tables 2.1 & 2).

These findings suggest that Bbs4 or the BBSome complex interacts with and mediates the trafficking of other signaling proteins. However, further studies are needed to

24 confirm these interactions and determine any role they might play in ciliary signaling.

Taken together, the proteomic analysis of Bbs4/BBSome-interacting proteins has resulted in a large data set which guides future studies focused on elucidating the function of BBS proteins.

The BBSome protein, Bbs4, associates with Nek1

Interestingly disruption of Nek1 in mice can cause ciliopathy-associated phenotypes including polycystic kidney disease, facial dysmorphism, dwarfing, male sterility, and cystic choroid plexus [67]. In addition, Nek1 has been implicated in ciliogenesis [68, 69] therefore we felt it was important to pursue the target, Nek1, and validate the Nek1-Bbs4 interaction. To confirm the association between Bbs4 and Nek1 we performed co-immunoprecipitation experiments on transiently transfected full-length proteins. HEK293T cells were transiently transfected with constructs expressing Bbs4 and myc-tagged Nek1 or myc-tagged Gapdh, as a negative control. HEK293T cell lysates were immunoprecipitated with an anti-Bbs4 antibody followed by immunoblotting with an anti-myc antibody. Bbs4 immunoprecipitated Nek1-myc but did not immunoprecipitate Gapdh-myc (Fig 2.3), indicating that Bbs4 specifically associates with

Nek1. These results validate the previous proteomics approach and suggest that Bbs4, either alone or when associated with the BBSome, interacts with the kinase, Nek1.

Discussion:

Our results show through yeast two-hybrid analysis that the BBSome subunit,

Bbs5, can directly interact with the third intracellular loop of the ciliary GPCR, dopamine receptor 1 (D1). Furthermore, we confirmed that full length D1 interacts with Bbs5 via co-immunoprecipitation. It has been shown that D1 localizes to neuronal cilia through a

BBSome-independent mechanism however treatment with a D1-specific agonist leads to the receptor being trafficked out of the cilium. In Bbs4-/- neurons, after D1 activation the

D1 receptor is retained in the cilium suggesting that D1’s translocation from the cilium upon activation requires BBS proteins [56]. Taken together, our results suggest that the

BBSome subunit, Bbs5, directly interacts with the ciliary GPCR, D1 and traffics the receptor out of the cilium upon activation.

Interestingly, D1 requires BBS proteins to be trafficked out of the cilium which is contrary to previous results showing two other ciliary GPCRs, Sstr3 and Mchr1, requiring

BBS proteins to be trafficked to neuronal cilia [55]. We hypothesized that Bbs5 could also interact with Sstr3 and Mchr1 and mediate their ciliary localization. Indeed, through yeast two-hybrid analysis we show that the BBSome subunit, Bbs5, interacts with the third intracellular loop of not only D1 but also Sstr3 and Mchr1. Therefore, in neuronal cilia the BBSome appears to mediate the import and export of ciliary GCPRs through direct interaction with Bbs5.

Interestingly, a loose consensus sequence in the third intracellular loop of

GPCRs was discovered by our lab which has been implicated as a ciliary targeting sequence [17]. Strikingly, Sstr3, Mchr1, and D1 all contain this sequence. However this observation begs the question how the BBSome determines which GPCRs need to be trafficked into the cilium and which GPCRs need to be trafficked out. Perhaps, upon D1- agonist binding there is a conformational change in the receptor allowing the BBSome to interact and mediate trafficking. This hypothesis suggests that in the inactive state, cis- or trans-acting elements may prevent the BBSome from interacting with D1. The C- terminus tail of D1 (cis) may sterically hinder an interaction with a large protein complex, such as the BBSome, at the third intracellular loop. Alternatively, D1-interacting proteins

(trans) may block BBSome binding and possibly only upon D1 activation these D1- interacting proteins dissociate allowing for BBSome binding and trafficking of the

26 receptor out of the cilium. Irrespective of the precise mechanism we have shown that the BBSome subunit, Bbs5, can directly interact with certain GPCRs and mediate their ciliary localization. We propose that one major function of BBS proteins is to mediate the localization of ciliary GPCRs. Furthermore, due to the ubiquitous expression of BBS proteins and the presence of primary cilia on nearly every cell, it is tempting to speculate that a general function of BBS proteins is to transport ciliary signaling proteins (other than GPCRs) into and out of cilia.

To reveal novel BBS protein functions, we proposed that identifying proteins that interact with Bbs4, a BBSome subunit, could provide insight into BBS protein function and the cellular processes the BBS proteins are involved in. We report that by utilizing a proteomic approach and a specific Bbs4 antibody, we have successfully immunoprecipitated the BBSome from mouse striatal lysate. This is the first time that the entire endogenous BBSome has been isolated from mouse tissue and in addition validated our approach to discovering novel Bbs4/BBSome-interacting proteins. Indeed, with this approach we were able to identify numerous putative Bbs4/BBSome-interacting proteins that implicate BBS protein function in various cellular processes.

As previously mentioned we identified that BBS proteins play an important role in

GPCR trafficking. Unfortunately, we were unable to immunoprecipitate any novel ciliary

GPCRs with our proteomics pull down approach. However, we were able to immunoprecipitate diacylglycerol kinase iota from mouse striatal lysate and a phosphatidylinositol (4,5) bisphosphate 5-phosphatase from mouse kidney lysate, suggesting Bbs4 or the BBSome may regulate phospholipid signaling.

Phosphatidylinositol and the phosphorylated derivatives (phosphoinositides) are important signaling molecules that have been implicated in numerous cellular processes such as protein synthesis, vesicular trafficking, and cytoskeletal dynamics [70]. The composition and levels of phospholipids in the cell is a very tightly regulated process

27 mediated by kinases and phosphatases [71] including phosphatidylinositol (4,5) bisphosphate 5-phosphatase and diacylglycerol kinase. Furthermore the phosphatidylinositol (4,5) bisphosphate 5-phosphatase, INPP5E, has been shown to localize to primary cilia and is mutated in the human ciliopathy, Jourbert Syndrome suggesting phospholipid signaling in primary cilia [72, 73]. Taken together, our proteomics data suggest Bbs4 or in general the BBSome may be involved in trafficking signaling machinery including phospholipid signaling molecules needed for ciliary signaling. However, further studies are needed to confirm these interactions which would implicate BBS proteins in trafficking signaling components other than GPCRs into and out of cilia.

Excitingly, we identified for the first time and validated a novel Bbs4-interacting protein, Nek1. Our proteomics analysis shows that Bbs4 immunoprecipitated Nek1 from both mouse striatal and kidney lysate. This interaction was confirmed by heterologously expressing full length Bbs4 and Nek1 in HEK293T cells and performing a co- immunoprecipitation. Nek1 is a member of a large family of serine/threonine kinases known as Never In Mitosis A (NIMA)-related kinases (Neks) that have critical roles in coordinating microtubules during mitotic progression. However, more recently it has been discovered that Neks can be involved in ciliogenesis and microtubule stability in axonal projections influencing neuronal morphology [68, 69, 74]. Recently, both Nek1 and Bbs4 have been shown to cycle through the nucleus. Interestingly, Nek1 contains two nuclear localization signals (NLS) however lacks a confirmed nuclear export signal

(NES) [75]. In contrast, Bbs4 contains a predicted NES but lacks any sequences that would predict a NLS. These results echoed observations of another BBSome subunit,

Bbs7, which contains a validated NES but lacks a proper NLS [66]. Whether Nek1 and

Bbs4 cycling through the nucleus is physiologically relevant remains to be determined, however the interaction between these two proteins may be mutually beneficial. One

28 hypothesis is that signals received at the cilium by Bbs4 and Nek1 allows for translocation of the two proteins to the nucleus. Entry of the protein complex into the nucleus relies on the NLSs present in Nek1. Upon gene regulation, subsequent trafficking out of the nucleus relies on the NES present in Bbs4. The proposed “cilium- to-nucleus” signaling mechanism is similar to one utilized by other ciliary proteins including polycystin-2 and fibrocystin, where mechanical or Ca2+ stimulation received at the ciliary membrane, causes proteolytic cleavage of these proteins, allowing for the translocation of the C-terminus to the nucleus to regulate gene expression [76, 77].

On the other hand, Nek1 and Bbs4 might functionally interact to regulate microtubule stability throughout the cell or specifically within the cilium. It has been shown that overexpression of Nek1 in the ciliated inner medullary collecting duct (IMCD) cell line, disrupts ciliogenesis [68]. In addition, it has been shown that Nek3 can affect microtubule acetylation and stability in neurons [74]. Interestingly, both neuronal axons and primary cilia are microtubule-rich structures within the cell. Perhaps, by interacting with Bbs4, Nek1 is targeted to specific subcellular locations, such as the cilium, where it regulates acetylation and stability of microtubules. Due to the fact that mutations in

Bbs4 lead to the human ciliopathy, BBS, and mutations in Nek1 have been shown to cause polycystic kidney disease (a well-studied ciliopathy) in mice, understanding the interplay between BBS proteins and Nek kinases may provide insight into BBS protein function but also confirm the link between Nek1 and cilia dysfunction.

In summary, our results provide new clues into the functions of BBS proteins. In the context of neuronal cilia, we have shown that Bbs5, a subunit of the stable BBSome protein complex, can directly bind to ciliary GPCRs and mediate their trafficking into and out of the cilium. Furthermore, by optimizing a new proteomics tool we have begun to identify novel BBS protein-interacting proteins. By pursuing data obtained from these

29 experiments, future studies will reveal new and exciting functions of the BBS proteins and provide insight into their role in ciliary biology, cellular homeostasis, and disease.

Materials and Methods:

Ethics statement and animals used

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). The animals used for proteomic experiments were 6 week old Bbs4 +/+ and Bbs4 -/- littermates.

Plasmid Construction

Coding sequences of the D1 receptor, BBSome subunits and Gapdh were amplified from reverse-transcribed mouse whole brain RNA using the Superscript First-

Strand Synthesis RT-PCR Kit (Life Technologies/Invitrogen, Grand Island, NY, USA) and cloned into a TA cloning vector (pSTBlue-1; Novagen, San Diego, CA, USA). Primers corresponding to the N-terminal and C-terminal regions of the receptors were designed with 5' restriction sites for directional cloning. Primers were used to add an HA tag to the

C-terminus of the Bbs5 protein. All amplifications were performed with Accuprime Pfx

Taq Polymerase (Life Technologies/Invitrogen). The final PCR products were cloned into the pcDNA 3.1 (-), pcDNA3.1 myc/His (Life Technologies/Invitrogen), pGBKT7, or pGADT7 (Clontech). The pcDNA 3.1 myc/His Nek1 construct was a kind gift from Dr.

Lynne Quarmby. All DNA sequences were verified at the Nuclei Acid Shared Resource at The Ohio State University.

Antibodies

Antibodies used for immunoprecipitations and immunoblotting are as follows: anti-HA (3F10; Roche), anti-myc (9E10; Santa Cruz), custom made affinity purified anti-

Bbs4 against the CVEASPTEASEQKKEK peptide (Open Biosystems, Inc, Huntsville,

AL, USA).

Yeast two-hybrid analysis

The analysis utilized the Yeastmaker Yeast Transformation System 2 (Clontech).

The third intracellular loop (i3 loop) of mouse D1 (nucleotides 652 -819), Sstr3

(nucleotides 691-798), and Mchr1 (nucleotides 694-759) was cloned into the yeast bait vector pGBKT7 and transformed into the yeast mating strain AH109. The prey vector pGADT7 containing each BBSome subunit was individually transformed into the yeast mating strain Y187. AH109 and Y187 yeast expressing the respective constructs were mated and plated. Growth was assessed on -Leu/-Trp and -Ade/-His/-Leu/-Trp (X--gal) selective media.

Protein Isolation from transfected HEK293T cells

HEK293T cells (ATCC) were maintained in DMEM supplemented with 10% FBS and 1.5g/L of sodium bicarbonate (Life Technologies/Invitrogen). Bbs5-HA and D1-myc or Gapdh-myc constructs were co-transfected by electroporation into HEK293T cells.

Alternatively, Bbs4 and Nek1-myc or Gapdh-myc were co-transfected. After 48 hours, cells were lysed in solubilization buffer (20mM Tris pH 8.0, 150mM NaCl, 2mM EDTA,

10% glycerol, 1% NP-40) supplemented with sodium orthovanadate and protease inhibitor cocktail (Roche, Indianapolis, IN, USA). Following an one hour incubation at

4°C with rotation, cell debris and insoluble material was cleared by centrifugation for 20 minutes at 15,000 x g at 4°C. The resulting supernatant containing soluble protein was

31 collected and the concentration was determined by the Bradford assay (Bio-Rad,

Richmond, CA, USA).

Co-Immunoprecipitation

Soluble protein was precleared at 4°C with rotation for 1 hour with protein A- sepharose beads (GE Healthcare, Piscataway, NJ, USA) pre-equilibrated in detergent buffer (1mM Tris pH 7.5, 5mM NaCl, 1mM KCl, 1mM MgCl2, 1% NP-40) supplemented with protease inhibitor cocktail (Roche). Precleared protein was incubated overnight at

4°C with anti-myc antibody (9E10; Santa Cruz Biotechnology) or anti-Bbs4 antibody

(Open Biosystems) immobilized to protein A-sepharose beads. Samples were centrifuged and washed three times with 1X PBS supplemented with protease inhibitor cocktail. Immunoprecipitated proteins were subjected to 60°C heat for 15 minutes in

SDS sample buffer to elute purified proteins. Purified proteins were then analyzed by immunoblotting.

Immunoblotting

Protein samples were run on a denaturing 4-15% gradient polyacrylamide gel

(Bio-Rad) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA).

Membranes were blocked in TBS-T (10mM Tris-HCl (pH 7.5), 150mM NaCl, 0.1%

Tween 20) with 5% milk and incubated overnight at 4°C with appropriate antibodies diluted in TBS-T with 5% milk. Membranes were probed with horseradish peroxidase- conjugated secondary antibodies diluted in TBS-T with 5% milk for 1 hour at room temperature. The secondary antibody was detected using SuperSignal West Pico

Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and bands were visualized using Blue Ultra Autorad Film (ISC Bioexpress, Kaysville, UT, USA).

Protein isolation from mouse tissue

The striatum or one kidney was dissected in cold 1X PBS from 6 week old

Bbs4+/+ and Bbs4-/- littermates and further separated into smaller pieces. Tissue fragments were placed in lysis buffer (20mM Tris pH 8.0, 150mM NaCl, 2mM EDTA,

10% glycerol, 1% NP-40) supplemented with sodium orthovanadate and protease inhibitor cocktail (Roche). Cell lysis was obtained by brief sonication. Cell debris was cleared by centrifugation at 5,000 x g for 10 minute. Supernatant contained soluble protein. The isolation process was at least two times with cell debris pellet to ensure a maximal amount of protein was acquired from lysed cells.

Dynabead immunoprecipitation

The Bbs4 antibody was purified by removing BSA and other additives via Pierce

Antibody Clean-up Kit (Pierce/Thermo Scientific) and concentrated using a Pierce

Protein Concentrator (Pierce/Thermo Scientific). Purified Bbs4 antibody was coupled to

Dynabeads M-270 Epoxy utilizing the Dynabead Co-Immunoprecipitation Kit and following manufacturer’s guidelines (Life Technologies/Invitrogen). Next, immunoprecipitation utilizing antibody-coupled dynabeads and striatum or kidney cell lysate was performed utilizing the Dynabead Co-Immunoprecipitation Kit and following manufacturer’s guidelines (Life Technologies/Invitrogen) except proteins were not eluted off of the beads. Instead, after the last wash antibody-coupled dynabeads with interacting proteins bound were resuspended in 15μl of Last Wash Buffer.

Proteomics

Antibody-coupled dynabeads with interacting proteins bound were processed by the Proteomics Shared Resource at The Ohio State University. Proteins bound to antibody-coupled dynabeads were not eluted and separated on a polyacrylamide gel.

Instead, a polyacrylamide gel solution was applied directly to the sample and once solidified protein digestion was performed. Nano LC-MS/MS analysis of the peptides was performed using an LTQ Orbitrap mass spectrometer.

A Y2H B Co-IP pGBKT7 pGADT7 Growth Cotransfected D1- Gapdh- with Bbs5-HA myc myc kDa p53 large T + 37 IP: Myc laminin C large T IP - IB: HA D1 i3 Bbs1 - 25 37 D1 i3 Bbs2 - IB: HA D1 i3 Bbs4 - 25 D1 i3 Bbs5 + 100 Input D1 i3 Bbs7 - 75 IB: Myc D1 i3 Bbs8 - 50 D1 i3 Bbs9 - 37 laminin C Bbs5 -

Figure 2.1:6Dopamine receptor 1 (D1) and Bardet-Biedl syndrome 5 (Bbs5) interact. (A) Results of yeast matings between cells expressing the i3 loop of mouse D1, p53 (positive control), or lamin C (negative control) in the bait vector (pGBKT7) and cells expressing each of the seven BBSome subunits or large T antigen (T-Ag) in the prey vector (pGADT7). Note that only D1 i3 loop plus Bbs5 mating and the positive control p53 plus T-Ag mating show yeast growth (indicated as +) on selective growth media. (B) HA-tagged Bbs5 (Bbs5-HA) was coexpressed with myc-tagged D1 (D1-myc) or myc- tagged glyceraldehyde-3-phosphate dehydrogenase (Gapdh-myc) in HEK293T cells. Cell extracts were immunoprecipitated (IP) with an anti-myc antibody. Immunoprecipitates were analyzed by Western blotting (IB) with an anti-HA antibody (upper panel). Note that Bbs5 is immunoprecipitated with D1 but not Gapdh. The input, confirming expression of each protein, is also shown (middle and bottom panels).

Y2H

pGBKT7 pGADT7 Growth D1 i3 Bbs5 + Sstr3 i3 Bbs5 + Mchr1 i3 Bbs5 +

7 Figure 2.2: Bbs5 can directly interact with the third intracellular loop of multiple ciliary GPCRs. Results of yeast matings between cells expressing the i3 loop of mouse D1, Sstr3, or Mchr1 in the bait vector (pGBKT7) and cells expressing the BBSome subunit, Bbs5 in the prey vector (pGADT7). Note that all three conditions resulted in yeast growth (indicated as +) on selective growth media suggesting Bbs5 can directly interact with the i3 loop of multiple ciliary GPCRs.

TABLE 2.1: BBS4 ASSOCIATED PROTEINS FROM MOUSE STRIATUM

2 Table 2.1: Bbs4 associated proteins identified through proteomic analysis of mouse striatal lysate. Peptide sequences and proteins identified through proteomic analysis utilizing an anti-Bbs4 antibody and mouse striatal lysate. Note the entire endogenous BBSome (Bbs1, Bbs2, Bbs4, Bbs5, Bbs7, Bbs8, and Bbs9) was immunoprecipitated with endogenous Bbs4. Novel putative Bbs4/BBSome-interacting proteins were also identified.

TABLE 2.2: BBS4 ASSOCIATED PROTEINS FROM MOUSE KIDNEY

3 Table 2.2: Bbs4 associated proteins identified through proteomic analysis of mouse kidney lysate. Peptide sequences and proteins identified through proteomic analysis utilizing an anti-Bbs4 antibody and mouse kidney lysate. Note some known Bbs4-interacting proteins were immunoprecipitated (Bbs2, Bbs1, pericentriolar material- 1, Bbs9, and Bbs5). Proteins previously associated with centrosome or cilia function and novel putative Bbs4/BBSome-interacting proteins were also identified.

Co-IP Input Cotransfected Nek1- Gapdh- Cotransfected Nek1- Gapdh- with Bbs4 myc myc with Bbs4 myc myc

200 200 150 150 100 100 IP: Bbs4 75 IB: Myc IP IB: Myc 75 50 Input 50 37 37

100 75 IB: Bbs4 50 37

8 Figure 2.3: Bbs4 associates with the kinase, Nek1. Constructs expressing Bbs4 and myc-tagged Nek1 (Nek1-myc) or myc-tagged Gapdh (Gapdh-myc) were transiently transfected into HEK293T cells. Cell extracts were immunoprecipitated (IP) with an anti-Bbs4 antibody and then analyzed by western blotting (IB) with an anti-myc antibody (left panel). Note only Nek1-myc was immunoprecipitated with Bbs4 not Gapdh, suggesting a true and specific interaction. The input, confirming expression of each protein is also shown (right panel).

CHAPTER 3: DYNAMIC LOCALIZATION OF CILIARY SIGNALING MACHINERY

Summary:

Although a great deal has been discovered about the function of primary cilia on certain cell types, little is known about the function of primary cilia on central neurons in the mammalian brain. Recently it was discovered that certain G protein-coupled receptors (GPCRs) and the secondary messenger type III adenylyl cyclase (ACIII) selectively localize to neuronal cilia. Due to the ciliary localization of signaling cascade components, we hypothesized that neuronal cilia are specialized non-synaptic signaling organelles. To test this hypothesis we investigated the dynamic localization of components of the somatostatin pathway. Somatostatin receptor subtype 3 (Sstr3) selectively localizes to neuronal cilia throughout the rodent brain. We have shown for the first time that components of the Sstr3 signaling pathway dynamically localize to cilia.

Upon Sstr3 activation, the receptor is trafficked out of the cilium. Classically, upon

GPCR activation phosphorylation of the receptor leads to recruitment of arrestin proteins to facilitate desensitization and internalization. We have shown that upon Sstr3 activation a cellular response is elicited that allows for beta-arrestin-2 (βarr2) recruitment and localization into the cilium. Furthermore, this dynamic localization of βarr2 can be blocked by the expression of an Sstr3 phosphorylation mutant, suggesting phosphorylation of the receptor is critical for downstream ciliary signaling. Improper neuronal cilia formation or improper trafficking of ciliary signaling machinery could result in altered neuronal signaling and disease phenotypes.

Introduction:

Very few proteins that selectively localize to neuronal cilia have been identified therefore little is known about the role cilia play in neuronal function. To determine the function of neuronal cilia it is important to understand which proteins localize there and which signaling pathways are regulated. By elucidating the signaling pathways that neuronal cilia coordinate we can then understand how neuronal cilia dysfunction can lead to disease. Precedence has been set with a seminal discovery that was made a little over ten years ago that linked primary cilia and human disease. Mice with a hypomorphic mutation in a gene that encodes for a protein critical for the formation and maintenance of primary cilia, develop polycystic kidney disease (PKD) - one of the most common genetic diseases in humans [78, 79]. This connection between primary cilia dysfunction and human disease set off a renewed interest in primary cilia and an entire field of research has been devoted to understanding the function of this specialized organelle.

Further studies trying to link ciliary dysfunction and PKD led to the discovery that renal primary cilia can sense and respond to fluid flow in the tubule lumen due to the ciliary localization of the mechanosensor formed by polycystin-1 (PC1) and polycystin-2

(PC2) [37]. Defects in the formation of renal cilia or mutations in PC1 or PC2, can lead to altered Ca2+ signaling which can ultimately lead to PKD pathology [37, 80]. Although a great deal has been discovered about the function of primary cilia on certain cell types, little is known about the function of primary cilia on central neurons in the mammalian brain [81]. The first indication that neuronal primary cilia may act as sensory and signaling organelles was the discovery that the G protein-coupled receptor (GPCR), somatostatin receptor subtype 3 (Sstr3), selectively localizes to neuronal cilia throughout the rodent brain [52]. Furthermore, type III adenylyl cyclase (ACIII), an important

41 signaling molecule involved in cAMP signaling, has also been shown by our laboratory to selectively localize to primary cilia on central neurons and colocalize with a subset of

Sstr3-positive cilia [82].

Remarkably, although it is known that signaling machinery (Sstr3 and ACIII) are enriched in primary cilia on central neurons; it still has not been determined whether these signaling proteins are functional within the ciliary membrane and neuronal cilia are indeed signaling organelles. Recent work utilizing Sstr3 and ACIII knockout mice revealed that mice lacking either component of the signaling pathway results in impaired memory [62, 83]. Because both Sstr3 and ACIII are enriched in neuronal cilia these findings suggest neuronal cilia are signaling organelles and dysfunctional neuronal ciliary signaling can result in disease phenotypes. However, further studies are needed to confirm Sstr3 is activated within the neuronal ciliary membrane and to determine the precise function of neuronal cilia.

To provide evidence that Sstr3 is functional within the ciliary membrane we examined the dynamic localization of somatostatin signaling elements upon Sstr3 activation. Interestingly, our laboratory has shown that the ciliary GPCR, dopamine receptor 1 (D1) dynamically localizes to neuronal cilia upon particular signaling cues

[56], suggesting signals are generated by cellular processes that determine the localization of the receptor. We tested whether Sstr3 has dynamic localization and can be trafficked out of the cilium upon receptor activation. Similar to D1, we show for the first time that upon activation, Sstr3 is trafficked out of the cilium.

Classically the pathway for GPCR activation and internalization begins with agonist binding to the GPCR resulting in a conformational change in the receptor. This allows for the activation and dissociation of G proteins, which regulate numerous signal transduction pathways [84]. Activated GPCRs are then phosphorylated at specific residues on their intracellular domains predominantly by G protein-coupled receptor

42 kinases (GRKs) [85], but also second-messenger activated kinases such as PKA or PKC

[86]. The phosphorylated receptors now have an increased affinity for arrestin proteins, leading to arrestin recruitment to the plasma membrane. Arrestins facilitate desensitization by preventing further G protein coupling and activation. Furthermore, arrestin proteins can facilitate internalization of the receptor by promoting clathrin- mediated endocytosis [87]. Once the receptors are internalized they can be either degraded through lysosomes or recycled back to the plasma membrane for additional signaling [84]. Determining whether the same regulatory mechanisms are utilized for ciliary GPCRs will help illustrate GPCRs trafficked to neuronal cilia are functional and can generate signals critical for ciliary signaling. Additionally these findings will further confirm the role of neuronal cilia as specialized signaling organelles.

Strikingly, we show for the first time that upon Sstr3 activation beta-arrestin-2

(βarr2), a known modulator of GPCR signaling, is recruited into the cilium and colocalizes with Sstr3. Furthermore, the recruitment of βarr2 into the ciliary compartment can be inhibited by expressing phosphorylation mutants of Sstr3.

Specifically, threonine 357 in the C-terminus of Sstr3 is a critical residue needed for the dynamic localization of βarr2 into primary cilia. Taken together, we have provided evidence that neuronal cilia are signaling organelles and that signals generated within the cilium allows for the dynamic localization of the ciliary GPCR, Sstr3, and signaling machinery, βarr2.

Results:

Somatostatin receptor subtype 3 dynamically localizes to cilia

To test whether somatostatin receptor subtype 3 (Sstr3) dynamically localizes within the ciliary membrane, hippocampal neuronal cultures were assessed before and

43 after Sstr3-agonist treatment. Hippocampal cultures were generated from newborn WT mice and after 7 days in culture, neurons were either treated with 10μM SST for 0, 15, or

30 minutes. Neurons were then colabeled with antibodies to Sstr3 and the neuronal ciliary marker, type III adenylyl cyclase (ACIII; Fig 3.1A & B). The percentage of Sstr3- positive cilia was then quantified (Fig 3.1C). Under basal conditions 50.3% of ACIII- positive cilia colocalize Sstr3. However the amount of Sstr3 localizing to neuronal cilia decreases upon increasing SST exposure. After 15 minutes of SST exposure, only

40.5% of ACIII-positive cilia colocalize Sstr3. Furthermore, after 30 minutes of SST exposure a significant decrease of Sstr3 ciliary localization is observed with only 26.4% of ACIII-positive cilia colocalizing Sstr3. These findings suggest that upon agonist exposure, the ciliary G protein-coupled receptor (GPCR) Sstr3 is trafficked out of neuronal cilia.

βarr2 dynamically localizes to cilia in response to Sstr3 activation in a ciliated cell line.

Classically upon GPCR activation, arrestin proteins are recruited to the receptor to facilitate desensitization and internalization [87]. To test whether arrestin proteins are recruited to ciliary GPCRs upon activation, we first assessed localization of βarr2 upon

Sstr3 activation in the ciliated cell line, inner medullary collecting duct (IMCD) cells.

IMCD cells were transiently co-transfected with dsRed-tagged Sstr3 and gfp-tagged

βarr2. Cells were either treated with 10μM SST or vehicle as a negative control. After

20 minutes of SST treatment, the percentage of cilia colocalizing Sstr3 and βarr2 was then quantified. As previously reported under basal conditions, Sstr3 selectively localizes to the ciliary membrane (Fig 3.2B & C). In contrast, βarr2 localizes throughout the cytoplasm with detectable accumulation of βarr2 at the base of the cilium.

Importantly, under basal conditions it was observed that βarr2 is excluded from the cilium (Fig 3.2A & C). Interestingly, upon Sstr3 agonist exposure it appears that βarr2 is

44 recruited into the cilium and colocalizes with Sstr3 (Fig 3.2D-F). Figure 3.2G shows under basal conditions there is no ciliary localization of βarr2 (0%). However upon SST treatment, a significant increase of Sstr3 and βarr2 ciliary colocalization is observed

(81.8%). These findings suggest that upon SST exposure the ciliary GPCR, Sstr3, is activated and a signal is generated within the cell allowing for the recruitment and ciliary localization of βarr2.

βarr2 dynamically localizes to neuronal cilia in response to Sstr3 activation

To further investigate the dynamic ciliary localization of βarr2 and test whether a conserved trafficking mechanism is utilized in neuronal cilia, we assessed βarr2 recruitment in cultured neurons. Hippocampal neuronal cultures were generated from newborn WT mice and after 6 days in culture were transiently co-transfected with dsRed-tagged Sstr3 and gfp-tagged βarr2. Neurons were then treated with 10μM SST or vehicle 24 hours post-transfection and the percentage of neuronal cilia colocalizing

Sstr3 and βarr2 was quantified. As previously described, Sstr3 localized to neuronal cilia under basal conditions (Fig 3.3B & C). βarr2 localized within the cytoplasm and accumulated at the base of neuronal cilia (Fig 3.3A & C). Similar to the mechanism utilized by IMCD cells, upon SST treatment βarr2 is recruited into neuronal cilia and colocalizes with Sstr3 (Fig 3.3D-F), suggesting a conserved trafficking mechanism is utilized by IMCD cells and central neurons to traffic βarr2 into the cilium upon Sstr3 activation.

Additionally, to examine the activated-GPCR dependent ciliary localization of

βarr2 in real time we utilized live-cell imaging techniques. Again, hippocampal neuronal cultures were generated from newborn WT mice and after 6 days in culture were transiently co-transfected with dsRed-tagged Sstr3 and gfp-tagged βarr2. 24-48 hours post transfection, neurons were evaluated for Sstr3-agonist dependent βarr2 ciliary

45 localization at various time points. In basal conditions, live-cell imaging data revealed

βarr2 predominantly localizes to the cytoplasm (Fig 3.3G-I). However upon SST treatment, βarr2 is trafficked into neuronal cilia and colocalizes with Sstr3 in a time- dependent manner (Fig 3.3J-L). Taken together, we have shown for the first time that

βarr2, a known modulator of GPCR signaling, is trafficked into cilia on central neurons in an activated-ciliary GPCR dependent mechanism.

Sstr3’s threonine 357 is a critical residue in the dynamic ciliary localization of βarr2.

Although little is known about the regulatory mechanisms influencing agonist- dependent trafficking of Sstr3, it has been shown that phosphorylation of four residues

(Ser341, Ser346, Ser351, Thr357) in the C-terminus of Sstr3 play a critical role in proper desensitization and internalization of the receptor [88]. To test whether any of these four residues play an essential role in the recruitment of βarrr2 into cilia, we tested the ability of various Sstr3 phosphorylation mutants to recruit βarr2 into cilia upon activation.

Through site directed mutagenesis, various phosphorylation mutants were generated including a triple serine mutant (Ser341, Ser346, and Ser351 mutated to alanines), a quadruple mutant (Ser341, Ser346, Ser351, and Thr357 mutated to alanines), and a threonine only mutant (Thr357 mutated to alanine).

IMCD cells were co-transfected with a gfp-tagged βarr2 and either a myc-tagged

WT Sstr3 or a myc-tagged phosphorylation mutant Sstr3. 48 hours post-transfection, cells were either treated with vehicle or 10μM SST and labeled with an anti-myc antibody to detect Sstr3 localization. βarr2 recruitment into the cilium was assessed by quantitating the percentage of Sstr3 (WT or mutant)-positive cilia colocalizaing βarr2.

Mutating the four residues critical for phosphorylation, did not affect the localization of

Sstr3 and all phosphorylation mutants tested properly localized to primary cilia.

Interestingly, although it has been shown that mutating all three serines in the C-

46 terminus to alanines (triple serine mutant) results in decreased desensitization and internalization of the receptor, it did not significantly affect the recruitment of βarr2 into the cilium upon SST exposure compared to WT Sstr3, 79.2% vs 90% respectively, (Fig

3.4).

In contrast, when all four critical residues were mutated to alanines (quadruple mutant), βarr2 was not recruited into the cilium upon SST treatment (0%). Furthermore, expression of the threonine only mutant (T357A) completely inhibited βarr2 recruitment into the cilium upon SST treatment (0%, Fig 3.4). These findings suggest that phosphorylation of threonine 357 in the C-terminus of Sstr3 is required for the recruitment of βarr2 into the cilium. Moreover, these findings hold true in the context of cilia on central neurons. Hippocampal neurons were cultured and co-transfected with either WT or threonine only mutant (T357A) Sstr3 and βarr2. In neurons expressing WT

Sstr3, βarr2 is recruited into neuronal cilia and robustly colocalizes with the ciliary receptor upon SST treatment (100%, Fig 3.5A-F, M). Strikingly, upon SST treatment,

βarr2 is not recruited into neuronal cilia expressing the Sstr3 T357A mutant and does not colocalize with the ciliary receptor (0%, Fig 3.5G-L, M).

Discussion:

Our results show that the ciliary GPCR, Sstr3, dynamically localizes into and out of the cilium. Specifically, our findings reveal that upon SST treatment, the natural ligand for Sstr3, endogenous Sstr3 is trafficked out of neuronal cilia. We hypothesize that ciliary GPCRs generate signals within the ciliary membrane and dynamic localization into and out of the cilium is a mechanism for regulating ciliary GPCR signaling. Although numerous studies have been conducted and much has been discovered about the desensitization and internalization of plasma membrane receptors,

47 nothing is known about the regulatory mechanisms that determine the fate of ciliary

GPCRs and the consequences on ciliary GPCR signaling. Early studies performed in non-ciliated cells suggested the vast majority of activated Sstr3 is endocytosed into recycling vesicles and is recycled back to the plasma membrane [89]. In contrast, more recent studies show that activated Sstr3 localizes to large endocytic vesicles that colocalize with recycling endosomal components. However, upon increased SST exposure Sstr3 undergoes ubiquitin-dependent lysosomal degradation [63].

Interestingly, we observed that upon increasing SST exposure, Sstr3 appears to accumulate in punctate vesicles within the cell body of the neuron. It is tempting to speculate that upon activation, Sstr3 is trafficked out of the cilium and localizes to endocytic vesicles to be degraded or recycled back to the ciliary membrane. However, further colocalization studies with endosomal and lysosomal markers are needed to determine whether Sstr3 that has been trafficked out of the cilium is recycled or degraded.

Classically, upon GPCR activation there is a conformational change allowing for

G protein dissociation and initiation of appropriate signaling cascades. Furthermore, the receptor is phosphorylated allowing for the association with arrestin proteins to facilitate desensitization and internalization of the receptor. Our data reveal that in the ciliated

IMCD cell line, βarr2, a regulator of GPCR signaling, localizes throughout the cytoplasm and at the base of the cilium. This is in contrast to other reports utilizing other ciliated cell lines (NIH3T3 cells, MEFs, RPE cells) which showed βarr2 localizing throughout the ciliary compartment [90, 91]. A stark contrast between the other ciliated cell lines referenced and IMCD cells is that serum starvation is necessary for ciliogenesis in

NIH3T3 cells, MEFs, and RPE cells and is not necessary for ciliogenesis in IMCD cells.

Due to this fact, we feel that IMCD cells closer recapitulate physiological conditions compared to ciliated cell lines that require serum starvation. Moreover, our results

48 reveal that βarr2 can be robustly recruited into the ciliary compartment by activation of the ciliary GPCR, Sstr3.

Likewise, this novel ciliary GPCR regulatory mechanism is conserved in primary cilia found on central neurons. Our data illustrate βarr2 predominantly localizes to the cytoplasm in cultured central neurons and remains excluded from cilia. This is in contrast to reports that demonstrated βarr2 localization within ciliary compartment in other neuronal cell types such olfactory sensory neurons and photoreceptors [92, 93].

One possibility for this discrepancy is the level of detection of small amounts of βarr2 cycling through the cilium. This was evident in the olfactory sensory neuronal cilia findings. The authors showed βarr2 is abundantly detected in the dendritic knob region of the olfactory sensory neuron, a region adjacent to the proximal end of olfactory sensory neuronal cilia by immunofluorescence. The accumulation of βarr2 at the dendritic knob in olfactory sensory neurons could be analogous to our observations that

βarr2 localizes at the base of the cilium on central neurons. In contrast, the authors found βarr2 is not detected in olfactory sensory neuronal cilia using immunofluorescence techniques, however it is detected via immunoblot from isolated cilia lysate [92]. The authors suggest a small amount of βarr2 is present within olfactory sensory neuronal cilia however the majority of detectable βarr2 localizes to the dendritic knob.

Dynamic localization of proteins is often a cellular response to extracellular stimuli. Similar to IMCD cells we have shown that upon SST treatment and Sstr3 activation, βarr2 is robustly trafficked into cilia on central neurons where it colocalizes with Sstr3. These findings are in agreement with the trafficking mechanism of visual arrestin (varr) in photoreceptors. In dark-adapted cells, it has been shown that the majority of varr is located in the inner segment as well as the connecting cilium, part of a modified primary cilium in photoreceptors [93]. The outer segment of photoreceptors is filled with the light-sensing GPCR, rhodopsin. Studies revealed that upon increasing

49 light exposure, rhodopsin is activated and varr is transported from the inner segment, across the connecting cilium, and into the outer segment of the photoreceptor to regulate desensitization of rhodopsin [94, 95]. Taken together, our data suggest that neuronal cilia are able to sense and respond to neuromodulators due to functional ligand receptors that localize to them. In addition, βarr2 may be utilized by neuronal ciliary

GPCRs to regulate their signaling.

Historically it has been shown that βarr2 can be recruited to an activated receptor localized to the plasma membrane after the receptor has been phosphorylated by particular kinases [85, 86]. Phosphorylation of four residues in the C-terminus is critical for the proper desensitization and internalization of Sstr3 [88]. Our data show that phosphorylation of one of these residues, threonine 357, is necessary for βarr2 recruitment into the cilium. One question our studies did not address was which kinase is responsible for phosphorylation of Sstr3 in the ciliary membrane. Based on sequences surrounding the threonine 357, it is predicted that a GRK mediates the phosphorylation of the residue (http://KinasePhos2.mbc.nctu.edu.tw). There are seven known GRKs [96] that could be facilitating the phosphorylation of ciliary Sstr3; however no GRK has ever been shown to be active within primary cilia on central neurons.

These exciting future studies will further elucidate the mechanisms regulating ciliary

GPCR signaling and provide a novel role for GRK regulation.

Taken together, we have provided evidence that Sstr3 is functional on the ciliary membrane. We theorize that Sstr3 binds agonist at the ciliary membrane and elicits a specialized ciliary signal. The signal is regulated by phosphorylation of Sstr3 by an undetermined kinase. Phosphorylation of Sstr3 generates a signal allowing for the trafficking of βarr2 into the cilium to potentially facilitate desensitization and/or internalization of the ciliary GPCR. As the functions of primary cilia are defined by the complement of proteins that localize to them, we have provided evidence that indeed

50 neuronal cilia are specialized non-synaptic organelles that sense and respond to the extracellular milieu by orchestrating the dynamic localization of various neuromodulator receptors and signaling machinery.

Importantly, these findings provide a link between neuronal ciliary signaling and cellular homeostasis. Furthermore, these findings may provide new therapeutic targets in disease. SST is a neuropeptide expressed broadly throughout the brain and its signaling has been implicated in a wide variety of physiological processes including inhibition of growth hormone secretion, inhibition of cancer cell proliferation, and neuromodulation in the central nervous system [97]. SST elicits a response by binding to six known receptors: Sstr1, Sstr2a, Sstr2b, Sstr3, Sstr4, and Sstr5 [98]. Of these six receptors, only Sstr3 has been shown to localize to the privileged ciliary compartment

[52]. The role of SST in cognition gained interest due to the fact that SST levels are decreased in the brains of Alzheimer’s patients [99]. Interestingly, treatment with octreotide which stimulates SST signaling, results in memory improvements in

Alzheimer’s patients [100]. In addition, aberrant SST levels have been associated with other neurological disorders such as Huntington’s disease, schizophrenia, and epilepsy

[101, 102]. Therefore a clear link between SST signaling and cognition has been established however the exact mechanism remains to be determined. Interestingly, many ciliopathy patients present with intellectual disabilities suggesting neuronal cilia play a critical role in neuronal function necessary for proper cognition [103]. Any component of the SST-ciliary signaling cascade (including the Sstr3, βarr2, and unidentified kinases) could offer a target for future research in diseases that have been associated with improper SST signaling and further link neuronal ciliary dysfunction with human disease.

In conclusion, we propose neuronal cilia are signaling organelles in the central nervous system responsible for sensing neuromodulators, such as SST, due to the

51 specific and unique subcellular localization of GPCRs. We believe these ciliary GPCRs are functional within the ciliary membrane and once activated by their appropriate ligand transmit a signal affecting neuronal function. Improper formation or function of neuronal cilia, including improper localization of signaling pathway components, could be an underlying mechanism in the pathophysiology of human diseases.

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).

Neuronal cell culturing

Culturing of hippocampal neurons were previously described [58, 104]. Briefly, hippocampi were dissected from FVB WT mouse pups on P0 and placed in a pre- warmed (37°C), sterile solution of Leibovitz’s L-15 medium (Life Technologies/Invitrogen,

Grand Island, NY) supplemented with 0.25mg/ml bovine serum albumin (BSA). The hippocampal tissue was freed from any extraneous tissue and then dissected further into small pieces. This tissue was then transferred into a solution of L-15/BSA containing

0.375mg/ml papain (Sigma-Aldrich, St. Louis, MO) and incubated for 15 minutes at 37°C with 95% O2/5% CO2 blowing gently over the surface of the solution. The tissue was then washed three times in pre-warmed M5-5 medium (Earle's minimal essential medium with 5% fetal bovine serum, 5% horse serum, 0.4mM L-glutamine, 16.7mM glucose, 5,000U/l penicillin, 50mg/l streptomycin, 2.5mg/l insulin, 16nM selenite, and

1.4mg/l transferrin). The tissue was then triturated in M5-5 medium with a series of

Pasteur pipettes of decreasing diameters. Dissociated neurons were pelleted by centrifugation and resuspended in pre-warmed Neurobasal-A medium containing B-27 supplement, glutamine, insulin/selenite/transferrin, and gentamycin (Life

Technologies/Invitrogen). Hippocampal neurons were plated onto poly-D-lysine

(Sigma-Aldrich) coated coverslips. After 2-3 days, glial replication was prevented by addition of cytosine arabinofuranoside to a final concentration of 10μm (ARA-C; Sigma).

Processing and immunofluorescence procedures for neuronal cultures

Seven days post plating neuronal cultures were fixed and processed for immunofluorescence as previously described [55]. Briefly day 7 neurons were fixed with a solution of 4% (weight/vol) paraformaldehyde and 10% (weight/vol) sucrose for 10 min at room temperature, followed by a 5-min PBS wash. Neurons were then postfixed with cold MeOH at -20°C for 15 min and permeabilized with 0.3% Triton X-100 in PBS with

4% donkey serum, 0.02% sodium azide, and 10mg/ml bovine serum albumin (BSA) for 6 min. After permeabilization, the cells were put in a blocking solution of PBS with 4% donkey serum, 0.02% sodium azide, and 10 mg/ml BSA for ~1 h at room temperature.

Immunofluorescent procedures have been previously described [17]. Briefly, cultured neurons were permeabilized with 0.3% Triton X-100 in PBS with 4% donkey serum, 0.02% sodium azide, and 10mg/ml bovine serum albumin (BSA). Primary antibody incubations were carried out for 16-24 h at 4°C and secondary antibody incubations were carried out for 1 h at room temperature. Neurons were washed three times for five minutes with PBS containing 4% donkey serum, 0.02% sodium azide, and

10mg/ml BSA after primary and secondary antibody incubations. Primary antibodies included rabbit anti-adenylyl cyclase III (C-20; Santa Cruz Biotechnology, Santa Cruz,

CA) and goat anti-somatostatin receptor 3 (M-18; Santa Cruz Biotechnology).

Secondary antibodies included Alexa Fluor 488-conjugated donkey anti-goat IgG and

Alexa Fluor 546-conjugated donkey anti-rabbit IgG (Life Technologies/Molecular Probes,

Grand Island, NY). Nucleic acids were stained with DRAQ5 (Cell Signaling, Danvers,

MA). Coverslips containing neurons were mounted using Immuno-Mount (Thermo

Scientific, Pittsburgh, PA). All samples were imaged on a Zeiss LSM 510 laser scanning confocal microscope at the Hunt-Curtis Imaging Facility in the Department of

Neuroscience at The Ohio State University. Multiple consecutive focal planes (Z-stack), spaced at approximately 0.43μm intervals, were captured. For all collected images, the brightness and contrast of each channel were adjusted using the Zeiss LSM Image

Browser program.

Quantification and Statistical Analysis of neuronal cilia

Quantification of somatostatin receptor subtype 3 (Sstr3)-positive cilia was performed on at least two independent coverslips. For each coverslip, at least five fields were imaged. The number of Sstr3-positive and ACIII-positive cilia in each image was counted by an individual blinded to the treatment conditions. The results were expressed as the percentage of ACIII-positive cilia showing Sstr3 ciliary colocalization and statistical analysis was displayed as mean ± standard error of the mean (SEM).

Transient transfections for IMCD cell line and cultured neurons

IMCD cells (ATCC, Manassas, VA) were maintained in DMEM:F12 medium supplemented with 10% FBS, 1.2g/L of sodium bicarbonate, and 0.5mM sodium pyruvate (Life Technologies/Invitrogen). IMCDs cells were electroporated with 10μg of

DNA and plated onto glass coverslips. Cells were harvested at 48 hours post- transfection for immunofluorescence processing.

Cultured hippocampal neurons were transfected using Lipofectamine LTX & Plus

Reagent methods (Life Technologies/Invitrogen) with 1μg of DNA, 6 days post plating.

Neurons were processed for immunofluorescence 24 hours post-transfection.

Processing and immunofluorescence procedures for transiently transfected cell lines and neurons

IMCD cells and cultured neurons were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100 in PBS with 4% serum, 0.02% sodium azide, and 10mg/ml bovine serum albumin (BSA) for 10 min. To visualize the myc-tagged

Sstr3 receptors, IMCD cells were labeled with anti-myc (9E10; Santa Cruz

Biotechnology, Santa Cruz, CA), followed by incubation with Alexa Fluor 546-conjugated goat anti-mouse IgG (Life Technologies/Molecular Probes). Nuclei were visualized by

DRAQ5 staining. Primary antibody incubations were carried out for 16-24 hours at 4°C and secondary antibody incubations were carried out for 1 hour at room temperature.

Coverslips containing IMCD cells or neurons were washed with three times for five minutes with PBS containing 4% serum, 0.02% sodium azide, and 10mg/ml BSA after primary and secondary antibody incubations. Coverslips containing neurons were mounted using Immuno-Mount (Thermo Scientific). All samples were imaged on a Zeiss

LSM 510 laser scanning confocal microscope at the Hunt-Curtis Imaging Facility in the

Department of Neuroscience at The Ohio State University. Multiple consecutive focal planes (Z-stack), spaced at approximately 0.43μm intervals, were captured. For all collected images, the brightness and contrast of each channel were adjusted using the

Zeiss LSM Image Browser program.

Quantification and Statistical Analysis of Beta-arrestin-2 recruitment

Quantification of beta-arrestin-2 (βarr2) recruitment into the cilium was performed on three independent coverslips of either IMCD cells or cultured neurons. For each coverslip, 4-10 fields were imaged. The number of Sstr3-positive and βarr2-positive cilia was quantitated. The results were expressed as the percentage of Sstr3-positive cilia displaying βarr2 ciliary colocalization. The data was expressed as mean ± standard error of the mean (SEM).

Live cell imaging

Cultured hippocampal neurons were transfected using Lipofectamine LTX & Plus

Reagent methods (Life Technologies/Invitrogen) with 1μg of DNA, 6 days post plating.

Neurons were processed for live-cell imaging 24-48 hours post-transfection. Prior to live-cell imaging, neuronal media was replaced with Hibernate A low fluorescence medium (BrainBits, Springfield, IL, USA) supplemented with B-27 supplement, glutamine, and insulin/selenite/transferrin to allow for manipulation and survival of neurons at ambient CO2. A field of view was chosen containing a neuron expressing dsRed-tagged Sstr3 and gfp-tagged βarr2. An image was captured (t0) using the 100x oil objective from a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar,

Germany). Neurons were then treated with SST for a final concentration of 10μM.

Consecutive images were taken at 10 and 20 minutes post SST treatment (t10, t20) of the same neuron. Openlab software (PerkinElmer, Waltham, MA, USA) was used for image acquisition and Image J software was used to adjust brightness and contrast levels.

Plasmid Construction

The coding sequence of mouse somatostatin receptor subtype 3 (Sstr3) was previously amplified from mouse genomic DNA [17]. The coding sequence was further subcloned into the pDsRed-N vector (Clontech, Mountain View, CA, USA) and pcDNA

3.1/myc-His (Life Technologies/Invitrogen) . The coding sequence of mouse beta- arrestin-2 (βarr2) was a kind gift from Dr. Laura Bohn. The coding sequence was further subcloned into pEGFP-N (Clontech). Sstr3 plasmids containing mutated phosphorylation sites were constructed by utilizing the QuikChange Site Directed

Mutagenesis Kit (Stratagene, La Jolla, CA). Briefly, pcDNA 3.1/myc-His Sstr3 was used for the PCR template. Primers were designed to mutate serine 341, serine 346, serine

351, or threonine 357 to alanine. All DNA sequences were verified at the Nuclei Acid

Shared Resource at The Ohio State University.

9 Figure 3.1: Sstr3 dynamically localizes in the neuronal ciliary membrane. (A-B) Representative images of day 7 hippocampal neurons from WT mice, labeled with antibodies to Sstr3 (green) and ACIII (red). Nuclei were stained with DRAQ5. Neurons were either under basal conditions (UT = untreated; A) or treated with 10μM somatostatin (SST; B). Insets in the top right corner are zoomed in images of the cell body and cilium from a particular neuron. The amount of ciliary Sstr3 localization was quantified under basal conditions (0 min), 15 min and 30 min after SST exposure (C). Note the percentage of Sstr3-positive cilia is significantly decreased upon 30 minutes of SST exposure compared to basal conditions (p < 0.05). Values are expressed as mean ± SEM.

10 Figure 3.2: Somatostatin-mediated βarr2 recruitment into primary cilia on IMCD cells. (A-F) Representative images of IMCD cells co-transfected with dsRed-tagged Sstr3 and gfp-tagged βarr2 under basal conditions (UT = untreated; A-C) or after 10μM somatostatin (SST) exposure for 20 minutes (D-F). Nuclei were stained with DRAQ5. βarr2-gfp localizes throughout the cytoplasm and a small portion localizes at the base of the cilium (arrow) under untreated conditions (A). Sstr3 selectively localizes throughout the ciliary membrane (B). However upon treatment, βarr2 is recruited into the ciliary compartment (D) and colocalizes with Sstr3 (E&F). Quantification of SST-mediated βarr2 ciliary localization was performed (G). Three independent experiments were performed and values are expressed as mean ± SEM.

A B C

UT arr2-gfp Sstr3-dsRed merge D E F

SST arr2-gfp Sstr3-dsRed merge G H I

UT arr2-gfp Sstr3-dsRed merge J K L

SST arr2-gfp Sstr3-dsRed merge 11 Figure 3.3: Somatostatin-mediated βarr2 recruitment into neuronal cilia. (A-F) Representative images of fixed neurons co-transfected with dsRed-tagged Sstr3 and gfp-tagged βarr2 under basal conditions (UT = untreated; A-C) or after 10μM somatostatin (SST) exposure for 20 minutes (D-F). Nuclei were stained with DRAQ5. Insets are zoomed in images of neuronal cilia. Similar to IMCD cells, βarr2-gfp localizes throughout the cytoplasm and is excluded from the primary cilium (A). Sstr3 selectively localizes throughout the ciliary membrane (B). However upon treatment, βarr2 is recruited into the neuronal ciliary compartment (D) and colocalizes with Sstr3 (E&F). (G- L) Images of neurons from live-cell imaging data collection co-transfected with dsRed- tagged Sstr3 and gfp-tagged βarr2 under basal conditions (UT = untreated; A-C) or after 10μM SST exposure for 20 minutes (D-F). Insets are zoomed in images of neuronal cilia. Under basal conditions, βarr2-gfp localizes throughout the cytoplasm and is excluded from the primary cilium (G). In contrast, Sstr3 selectively localizes throughout the ciliary membrane (H). Upon SST exposure, βarrr2 is recruited into the ciliary compartment where it colocalizes with Sstr3 in a time-dependent manner.

12 Figure 3.4: Quantification of βarr2 ciliary localization mediated by phosphorylation mutants of Sstr3 in IMCD cells. IMCD cells co-transfected with myc-tagged Sstr3 WT or phosphorylation mutant and gfp- tagged βarr2 were assessed for SST-mediated ciliary recruitment of βarr2. In transfected cells, the number of cilia colocalizing Sstr3 and βarr2 in the ciliary compartment were quantified. In all transfection combinations tested, during basal conditions (UT = untreated) no βarr2 was detected colocalizing with Sstr3 in the ciliary compartment. When βarr2-gfp was co-transfected with WT or the Sstr3 triple serine mutant and treated with 10μM SST for 20 min, 90% and 79.2% (respectively) of Sstr3- positive cilia colocalized βarr2. When βarr2-gfp was co-transfected with the Sstr3 quadruple mutant or threonine only mutant (T357A) and treated with SST, βarr2 was not recruited into the cilium (0%). Three independent experiments were performed and n ranged from 24-49 cilia per condition. Values are expressed as mean ± SEM.

13 Figure 3.5: Quantification of βarr2 ciliary localization mediated by Sstr3 WT or Sstr3 T357A in cultured neurons. (A-L) Representative images of neurons co-transfected with either dsRed-tagged Sstr3 and gfp-tagged βarr2 (A-F) or dsRed-tagged Sstr3 T357A and gfp-tagged βarr2 (G-L) under basal conditions (UT = untreated; A-C & G-I) or after 10μM somatostatin (SST) exposure for 20 minutes (D-F & J-L). Nuclei were stained with DRAQ5. Insets are zoomed in images of neuronal cilia. βarr2 neuronal ciliary localization was quantified (M). For each condition, 20-30 Sstr3-positive cilia were assessed for βarr2 ciliary localization. Upon SST exposure all Sstr3 WT-positive cilia colocalized βarr2. In contrast, upon SST exposure, no Sstr3 T357A-positive cilia colocalized βarr2. Values are expressed as mean ± SEM.

CHAPTER 4: HETEROMERIZATION OF CILIARY G PROTEIN-COUPLED RECEPTORS IN THE MOUSE BRAIN

3 – Partial citation for this chapter: Green, JA, Gu. C, Mykytyn, K. Heteromerization of ciliary G protein- coupled receptors in the mouse brain. PLoS ONE. 2012; 7(9):e46303. Epub 2012 Sep.

Summary:

Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and type III adenylyl cyclase, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling.

Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin- concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling.

Introduction:

Primary cilia are organelles that have been shown to coordinate specialized signaling [105-107]. Loss of cilia or ciliary signaling proteins is associated with a group of

63 diseases termed ciliopathies [108]. Due to the ubiquity of cilia and their critical roles in numerous signaling pathways, ciliopathies present with a wide range of clinical features.

Although the precise functions of most primary cilia are unknown, it is clear their functions are defined by the proteins that are targeted to and retained in the ciliary compartment [81]. Thus, identification of the proteins enriched within cilia can lend important insight into their functions.

Most neurons in the mammalian brain possess primary cilia that are enriched for specific signaling proteins, including type III adenylyl cyclase (ACIII) [109], which converts ATP to cAMP, and the G protein-coupled receptors (GPCRs) somatostatin receptor 3 (Sstr3) [52], serotonin receptor 6 (Htr6) [53], melanin-concentrating hormone receptor 1 (Mchr1) [17], and dopamine receptor 1 (D1) [56]. The colocalization of

GPCRs with ACIII in neuronal cilia suggests they mediate cAMP signaling in response to

GPCR activation. Yet, the precise functions of GPCRs on neuronal cilia and how they impact neuronal function is unknown. Studies using conditional knockout mice have implicated neuronal cilia in the regulation of feeding behavior. Disruption of cilia specifically on POMC-expressing neurons in the hypothalamus, a region of the brain involved in the regulation of appetite, causes hyperphagia-induced obesity in mice [110].

Mchr1, which is a key regulator of feeding behavior, localizes to neuronal cilia in the hypothalamus of wildtype mice but fails to localize to cilia on neurons lacking proteins mutated in the human obesity syndrome Bardet-Biedl syndrome (BBS) [55]. Neuronal cilia have also been linked to learning and memory. Mice lacking either Sstr3 or ACIII display defective novel object recognition [62, 83]. Although knockout mouse models do not distinguish the contribution of ciliary signaling from non-ciliary signaling, the fact that

Sstr3 and ACIII are enriched together in neuronal cilia and disruption of either one gives a similar learning deficit strongly supports a role for neuronal ciliary signaling in learning

64 and memory. Interestingly, BBS proteins are required for Sstr3 ciliary localization and

BBS patients commonly display cognitive deficits [55].

Identification of GPCRs that are enriched in neuronal cilia and characterization of their distribution throughout the brain can provide insight into the functions of these specialized organelles. Here we report, for the first time, two different ciliary GPCRs,

Mchr1 and Sstr3, colocalize within the same cilia in multiple brain regions of mice. In addition to colocalizing, we show that Mchr1 and Sstr3 can interact to form heteromers.

As GPCR heteromerization can affect ligand binding properties and downstream signaling, our findings add a previously unrecognized layer of complexity to neuronal ciliary signaling.

Results:

Melanin-concentrating hormone receptor 1 (Mchr1) localizes to neuronal cilia throughout the mouse brain

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide that has been implicated in a variety of physiological processes such as food intake and energy balance, anxiety, depression, sleep, reward, and cognitive function. In rodents, the physiological functions of MCH are mediated by a single receptor, Mchr1, which shows the highest homology to the somatostatin receptor family [111]. We previously demonstrated Mchr1 ciliary localization in several regions of the mouse brain, including the hypothalamus, striatum, and nucleus accumbens [17, 55, 112]. To better understand

Mchr1’s function and provide insight into the prevalence of Mchr1 ciliary signaling, we performed a thorough analysis of Mchr1 ciliary localization throughout the mouse brain.

Wild type brain slices were colabeled with antibodies to type III adenylyl cyclase (ACIII), the canonical neuronal ciliary marker [109], and Mchr1. In addition to the previously

65 described regions, we detected Mchr1-positive cilia in the hippocampus, amygdala, piriform cortex, and fasciolar gyrus (Fig. 4.1). Overall, Mchr1 ciliary localization is widespread throughout the mouse brain (Table 4.1), which correlates with the widespread expression pattern of Mchr1 mRNA in the mouse brain [113].

Mchr1 and somatostatin receptor 3 (Sstr3) colocalize to a subset of neuronal cilia in specific brain regions

In the hippocampus, Mchr1 ciliary localization was limited to the CA fields.

Mchr1-positive cilia were most abundant in the CA1 region with reduced numbers in the

CA2 region and very few in the CA3 region. As somatostatin receptor 3 (Sstr3) localizes to cilia throughout the hippocampus, including the CA1 and CA2 regions [52], we tested whether Mchr1 and Sstr3 ever localize within the same cilium. Colabeling brain sections with anti-Mchr1 and anti-Sstr3 antibodies revealed numerous cilia within the hippocampus that were positive for both Mchr1 and Sstr3 (Fig. 4.2A-F), with the majority of Mchr1/Sstr3 colocalization located in the CA1 and CA2 regions. This is the first time the presence of different GPCRs within the same cilium has been reported.

To quantify Mchr1/Sstr3 colocalization, we prepared primary neuronal cultures from the hippocampus of P0 mice. After 7 days in culture the neurons were fixed and colabeled with antibodies against Mchr1 and ACIII or Sstr3 and ACIII. In hippocampal neuronal cultures, approximately 25% of the ACIII-positive cilia were Mchr1-positive

(Fig. 4.2J). Sstr3 ciliary localization was much more frequent with approximately 61% of

ACIII-positive cilia positive for Sstr3 (Fig. 4.2J). We then colabeled the hippocampal neuronal cultures with antibodies against Mchr1 and Sstr3 to quantify the number of cilia positive for both ciliary GPCRs (Fig. 4.2G-I). Approximately 54% of Mchr1-positive cilia were also positive for Sstr3 (Fig. 4.2K). Thus, Mchr1 and Sstr3 colocalize to the majority of Mchr1-positive cilia in hippocampal cultures and this finding reflects what was

66 observed in colabeled brain slices. Overall, our results indicate there are at least three different populations of neuronal cilia in the hippocampus; cilia that are positive for Sstr3, cilia that are positive for Mchr1, and cilia that are positive for both Sstr3 and Mchr1.

Due to the extensive distribution of Mchr1- and Sstr3-positive cilia throughout the mouse brain, we then asked whether colocalization of these ciliary GPCRs was restricted to the hippocampus or whether it occurred in other brain regions. Examination of brain slices colabeled for Mchr1 and Sstr3 revealed colocalization of Mchr1 and Sstr3 in a subset of neuronal cilia in the piriform cortex (Fig. 4.3) and hypothalamus (Fig. 4.4), regions of the brain that play important roles in olfaction and feeding behavior.

To quantify Mchr1/Sstr3 colocalization in the piriform cortex and hypothalamus, we prepared neuronal cultures from these regions and colabeled them with antibodies against Mchr1 and ACIII or Sstr3 and ACIII. In piriform cortical cultures approximately

55% of ACIII-positive cilia were positive for Mchr1 (Fig. 4.3G). In contrast, only 14% of

ACIII-positive cilia were positive for Sstr3 (Fig. 4.3G). It should be noted that the abundance of Sstr3-positive cilia appeared to be higher in the adult brain slices compared to the neuronal cultures, which may reflect changes in Sstr3 ciliary localization throughout the brain during development [114]. In hypothalamic cultures, approximately

63% of ACIII-positive cilia were positive for Mchr1 and 33% of ACIII-positive cilia were positive for Sstr3 (Fig. 4.4G). To quantify the number of cilia positive for both ciliary

GPCRs, cultured neurons from the piriform cortex and hypothalamus were colabeled with anti-Mchr1 and anti-Sstr3. Approximately 23% of Mchr1-positive cilia in the piriform cortical neuronal cultures and 20% of Mchr1-positive cilia in the hypothalamic neuronal cultures were also positive for Sstr3 (Figs. 4.3H and 4.4H). Thus, similar to the hippocampus, our results indicate there are at least three different populations of neuronal cilia in the piriform cortex and hypothalamus.

Mchr1 and Sstr3 interact

Mchr1 and Sstr3 on the same cilium may signal independently of one another or they may interact and form heteromers that could generate a signal unique from their individual components. To begin to address this question, we tested whether Mchr1 and

Sst3 interact and form a heteromer. HEK293T cells were transiently cotransfected with constructs encoding Sstr3 and either myc-tagged Mchr1 or myc-tagged Gapdh, as a negative control. Lysates were precipitated with an anti-myc antibody and immunoblotted with an anti-Sstr3 antibody. We found that Sstr3 co-immunoprecipitated with Mchr1 but not Gapdh (Fig. 4.5A). Performing the reverse experiment confirmed

Mchr1 is co-immunoprecipitated with Sstr3 but not Gapdh (Fig. 4.5B). Together, these results indicate the interaction between the two ciliary GPCRs is specific.

To further validate the interaction between Mchr1 and Sstr3, we performed fluorescence resonance energy transfer (FRET) analysis in live HEK293T cells expressing pECFP-Mchr1 and pEYFP-Sstr3. Some HEK293T cells possessed a cilium upon which the receptors colocalized. However, we excluded the cilium from our FRET analysis as it is possible that colocalization of the receptors within the confines of a cilium could generate an artefactual FRET signal. The two GPCRs highly colocalized

(Fig. 4.6A-C) and produced significant FRET signals (Fig. 4.6D, E) throughout the plasma membrane as well as some intracellular compartments (30 out of 40 cells). The colocalization and FRET signal at the plasma membrane of HEK293T cells is an indication that Mchr1 and Sstr3 can interact and potentially heteromerize in the ciliary membrane. The observed colocalization and FRET signal in intracellular compartments may be due to intracellular accumulation of overexpressed proteins. Alternatively, this may suggest the two GPCRs are trafficked together in the secretory pathway or recycled together in the endocytic pathway. Taken together with our co-immunoprecipitation data

68 these results suggest heterologously expressed Mchr1 and Sstr3 can heteromerize in

HEK293T cells.

To determine if endogenous Mchr1 and Sstr3 interact within the mouse brain, we performed a co-immunoprecipitation from hippocampal cell lysate that was enriched for membrane proteins (Fig. 4.7). Cell lysate was immunoprecipitated with goat anti-Mchr1 or goat IgG, as a negative control, and then immunoblotted with rabbit anti-Sstr3. We found that Sstr3 specifically co-immunoprecipitated with Mchr1, indicating endogenous

Mchr1 and Sstr3 can form heteromers within the brains of adult mice.

Discussion:

Our results show Mchr1 localizes to neuronal cilia throughout the mouse brain, including the hypothalamus, striatum, hippocampus, amygdala, piriform cortex, fasciolar gyrus and nucleus accumbens. In addition, subsets of Mchr1-positive cilia in the hippocampus, piriform cortex, and hypothalamus are also positive for Sstr3. This is the first time two different GPCRs have been shown to colocalize within the same cilium.

We further demonstrate Mchr1 and Sstr3 can interact via co-immunoprecipitation and FRET analysis, suggesting they form heteromers. This is a significant finding as heteromerization can have important functional consequences on GPCR signaling, including changes in ligand binding properties, G protein coupling, and/or receptor desensitization and internalization [115]. Heteromerization can have synergistic or antagonistic effects on signaling. D3 heteromerization with D1 increases the affinity of dopamine for D1 and potentiates D1 stimulation of adenylyl cyclase [116, 117].

Conversely, the heterodimer formed between Sstr2a and Sstr3 retains Sstr2a signaling while inactivating the Sstr3 subunit, thereby resulting in diminished Sstr3-like activity

[118]. Importantly, signaling modulation is not restricted to GPCRs within the same

69 family. In the context of the D2-Sstr5 heteromer, agonist occupation of D2 increases

Sstr5’s affinity for SST and potentiates signaling [119]. GPCR heteromers may also generate a completely unique signal due to changes in G protein coupling. Dopamine

Receptor 1 (D1) can couple to Gαs/olf and stimulate the production of cAMP through

AC. In contrast, Dopamine Receptor 2 (D2) inhibits cAMP production by coupling to Gαi.

However, D1-D2 heteromers couple to Gαq to elicit a Ca2+ signal [120, 121]. Further studies are required to determine how Mchr1-Sstr3 heteromerization affects ligand binding, G protein coupling, and/or desensitization and internalization.

It is intriguing that there is significant colocalization of Mchr1 and Sstr3 in cilia in the hippocampus. The MCH and SST systems have both been implicated in learning and memory. Specifically, MCH infusion into the rat hippocampus modulates memory

[122] and Mchr1 knockout mice demonstrate impaired long-term synaptic plasticity in hippocampal neurons [123, 124]. Sstr3 ciliary localization is also abundant in the hippocampus and Sstr3 is required for novel object recognition [83]. Together, these results implicate hippocampal ciliary signaling in learning and memory and it is tempting to speculate that altered ciliary signaling in the hippocampus may be the basis of cognitive deficits in some ciliopathies. Further studies are required to precisely determine the effects of Mchr1/Sstr3 colocalization on ciliary signaling in the hippocampus and their effects on learning and memory.

Mchr1/Sstr3 ciliary colocalization was also detected in the piriform cortex and hypothalamus. These two brain regions are thought to play an important role in olfaction and feeding behavior. MCH is an important regulator of feeding behavior and elicits an orexigenic effect [125]. Somatostatin signaling has also been shown to affect food intake. Selective central activation of Sstr2 increases food intake in rats [126] and pretreatment of rats with an Sstr3-specific agonist significantly reduces leptin-mediated suppression of food intake [127]. It will be interesting to determine whether Mchr1/Sstr3

70 colocalization correlates with appetite regulating neurons within the hypothalamus, suggesting a direct role for Mchr1-Sstr3 heteromers in feeding.

Quantification of Mchr1/Sstr3 colocalization in vitro reveals differences across regions. In the hippocampus, the majority of Mchr1-positive cilia are positive for Sstr3, whereas in the piriform cortex and hypothalamus the minority of Mchr1-positive cilia are positive for Sstr3. However, our quantification was performed on day 7 neurons. In the case of Sstr3, it is known that the relative abundance of ciliary localization is dynamic and varies with the age of the animal [114]. Thus, it is likely the ratios of Mchr1 and Sstr3 ciliary colocalization change during development and aging. A similar phenomenon has been observed with D1 and D2 heteromers. Specifically, the relative levels of signaling from D1-D2 heteromers increases in mice with increasing age [121]. It is also likely other

GPCRs colocalize with Mchr1 and/or Sstr3 in these and other regions, which would increase the complexity of ciliary signaling even further. Although D1 is a likely candidate, D1 ciliary localization is rarely observed in WT mouse brains, presumably due to high levels of signals stimulating D1 translocation from cilia [56]. D1 ciliary localization is commonly observed on cultured WT neurons and in preliminary studies we have observed ciliary colocalization of D1 and Mchr1 in neuronal cultures (J.G. and K.M. unpublished observations). Thus, Mchr1 and D1 may form heteromers in subsets of neuronal cilia.

Taken together, our findings suggest there may be functional interactions between different ciliary GPCRs within the same neuronal cilium, possibly allowing a specialized signal to be generated. Understanding the functional relevance of ciliary

GPCR heteromerization could elucidate the roles of neuronal cilia in non-synaptic signaling as well as provide new therapeutic avenues for ciliopathies.

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).

Brain tissue processing

The mice used in this study were on a FVB background. Five to six week old animals were anesthetized by 0.2ml/10g intraperitoneal injection of 2.5% tribromoethanol (Sigma-Aldrich, St. Louis, MO) to prevent pain and suffering, killed by cardiac puncture, and perfused with phosphate-buffered saline (PBS) followed by a 1:1 mixture of 4% paraformaldehyde (PFA) and Histochoice (Amresco, Solon, OH). The brains were then subjected to a 2 hour post fix in the 2% PFA/Histochoice mixture at

4°C. The brains were cryoprotected in 30% sucrose in PBS for 16-24 hours and sectioned on a freezing microtome at a thickness of 40μm.

Cultured neurons and processing

Primary hippocampal, piriform cortex, and hypothalamic neurons were cultured as previously described [58]. Seven days post plating neuronal cultures were fixed and processed for immunofluorescence as previously described [55].

Immunofluorescence procedures

Immunofluorescent procedures have been previously described [17]. Briefly, brain tissue and cultured neurons were permeabilized with 0.3% Triton X-100 in PBS

72 with 4% donkey serum, 0.02% sodium azide, and 10mg/ml bovine serum albumin (BSA).

Primary antibody incubations were carried out for 16-24 h at 4°C and secondary antibody incubations were carried out for 1 h at room temperature. Brain tissue was washed six times for ten minutes and cultured neurons were washed three times for five minutes with PBS containing 4% donkey serum, 0.02% sodium azide, and 10mg/ml BSA after primary and secondary antibody incubations. Primary antibodies included rabbit anti-adenylyl cyclase III (C-20; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti- melanin-concentrating hormone receptor 1 (C-17; Santa Cruz Biotechnology), goat anti- somatostatin receptor 3 (M-18; Santa Cruz Biotechnology), and rabbit anti-somatostatin receptor 3 (7986; [52]). Secondary antibodies included Alexa Fluor 488-conjucated donkey anti-goat IgG and Alexa Fluor 546-conjucated donkey anti-rabbit IgG (Life

Technologies/Molecular Probes, Grand Island, NY). Nucleic acids were stained with

DRAQ5 (Cell Signaling, Danvers, MA). Tissue or coverslips containing neurons were mounted using Immuno-Mount (Thermo Scientific, Pittsburgh, PA). All samples were imaged on a Zeiss LSM 510 laser scanning confocal microscope at the Hunt-Curtis

Imaging Facility in the Department of Neuroscience at The Ohio State University.

Multiple consecutive focal planes (Z-stack), spaced at approximately 0.43μm intervals, were captured. For all collected images, the brightness and contrast of each channel was adjusted using the Zeiss LSM Image Browser program.

Quantification of neuronal populations

Quantitative analysis of neuronal populations was performed on at least three independent coverslips generated from four different animals. For each coverslip, at least five fields were imaged. The number of Mchr1- or Sstr3-positive cilia and the number of ACIII-positive cilia in each image were counted by an individual blinded to the labeling condition. The results were expressed as the percentage of ACIII-positive cilia

73 showing either Mchr1 or Sstr3 ciliary colocalization. For Mchr1 and Sstr3 colocalization quantification, the number of Mchr1-positive and Sstr3-positive cilia was counted. The results were expressed as the percentage of cilia colocalizing Mchr1 and Sstr3, normalized to the number of Mchr1 positive cilia within the field. All data are expressed as mean ± standard error of the mean (SEM).

Cell culture and protein isolation

HEK293T cells (ATCC) were maintained in DMEM supplemented with 10% FBS and 1.5 g/L of sodium biocarbonate (Life Technologies/Invitrogen). Sstr3 and Mchr1-myc

(Sstr3 and Gapdh-myc for negative control) were cotransfected by electroporation into

HEK293T cells. After 48 h, cells were lysed in solubilization buffer (20mM Tris pH 8.0,

150mM NaCl, 2mM EDTA, 10% glycerol, 1% NP-40) supplemented with sodium orthovanadate and protease inhibitor cocktail (Roche, Indianapolis, IN). Following a 1 h incubation at 4°C with end-over-end rotation, cell debris and insoluble material were cleared by centrifugation for 20 min at 15,000 x g at 4°C. The resulting supernatant containing soluble protein was collected and the concentration was determined by the

Bradford assay (Bio-Rad, Richmond, CA, USA).

Co-immunoprecipitation and western blotting

Soluble protein was precleared at 4°C with rotation for 1 h with protein A- sepharose beads (GE Healthcare, Piscataway, NJ, USA) pre-equilibrated in detergent buffer (1mMTris pH 7.5, 5mM NaCl, 1mM KCl, 1mM MgCl2, 1% NP-40) supplemented with protease inhibitor cocktail (Roche). For HEK293T co-immunoprecipitation experiments, precleared protein was incubated overnight at 4°C with anti-myc (9E10;

Santa Cruz Biotechnology) or goat anti-Sstr3 immobilized to protein A-sepharose beads.

For endogenous co-immunoprecipitation experiments, precleared protein was incubated

74 overnight at 4°C with anti-Mchr1. Beads were washed three times with 1X PBS supplemented with protease inhibitor cocktail (Roche). Immunoprecipitated proteins were subjected to 60°C heat for 15 min in SDS sample buffer to elute purified proteins.

Purified proteins were analyzed by previously described immuoblotting procedures [56].

Briefly, for HEK293T co-immunoprecipitation experiments membranes were subjected to

SDS-PAGE, probed for Sstr3 using goat anti-Sstr3 followed by detection using horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology).

Alternatively, membranes were probed for myc using Mouse anti-myc, followed by detection using horseradish peroxidase-conjugated donkey anti-mouse IgG (Santa Cruz

Biotechnology). For endogenous co-immunoprecipitation experiments membranes were subjected to SDS-PAGE, probed with rabbit anti-Sstr3 or goat anti-Mchr1, followed by detection by using horseradish peroxidase-conjugated donkey anti-rabbit IgG or donkey anti-goat IgG (Santa Cruz Biotechnology), respectively.

Fluorescence resonance energy transfer imaging and quantification

Sensitized emission was the strategy used to perform fluorescence resonance energy transfer (FRET) in living HEK293T cells, as previously described [128, 129].

Briefly, HEK293T cells were transiently co-transfected with constructs containing Mchr1 fused at the C-terminus to ECFP and Sstr3 fused at the C-terminus to EYFP. After 48 hours, cells were imaged using three filter sets: (1) CFP filter set (excitation 430/25 nm; emission 470/30 nm); (2) YFP filter set (excitation 500/20 nm; emission 535/20 nm); (3)

FRET filter set (excitation 430/25 nm; emission 535/30nm). A single dichroic mirror

(86004BS; Chroma Technology) was used with all three filter channels. The amount of cross-bleeding of the CFP and YFP channels into the FRET filter set was previously determined. The corrected FRET (FRETcorrected = FRETraw - (0.65  CFP) - (0.015 

YFP)) was calculated by MetaMorph software (n = 40). As a positive control, CFP-Kv1.2

75 and YFP-Kvβ2 produced robust FRET signals. As a negative control, CFP-Kv1.2 and

YFP-Kvβ2K235E produced no significant FRET signal [128, 129].

Isolation of membrane enriched protein

Isolation of membrane enriched protein from mouse brain has been previously described [56]. Briefly, dissected tissue was sonicated in ice cold 1× PBS. Cell debris was pelleted by centrifugation at 5,000 x g at 4°C. The supernatant was collected and centrifuged at 120,000 x g at 4°C to pellet the membrane. 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. 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 quantification was determined by a NanoDrop (Thermo).

Acknowledgments:

We are grateful to Melanie Tallent for providing the rabbit anti-Sstr3 antibody.

A Hippocampus B C

Mchr1 Mchr1 ACIII ACIII D Amygdala E F

Mchr1 Mchr1 ACIII ACIII G Piriform Cortex H I

Mchr1 Mchr1 ACIII J K L

Mchr1 ACIII 14 Figure 4.1: Melanin-concentrating hormone receptor 1 (Mchr1) localizes to neuronal cilia throughout the mouse brain. (A-L) Representative images of multiple brain regions in 5-6 week old wild type mice showing colabeling for Mchr1 (green) and type III adenylyl cyclase (ACIII; red). Nuclei are stained with DRAQ5 (blue). Labeling for ACIII reveals numerous primary cilia present throughout the CA1 region of the hippocampus (B), amygdala (E), piriform cortex (H), and fasciolar gyrus (K). Labeling for Mchr1 (A, D, G, & J) reveals abundant Mchr1 ciliary localization in each region. Merged images (C, F, I, L) showing colocalization between Mchr1 and ACIII. Scale bars represent 10µm.

A B C

Mchr1 Mchr1 Sstr3 Sstr3 D E F

Mchr1 Mchr1 Sstr3 Sstr3 G H I

Mchr1 Mchr1 Sstr3 Sstr3 J K

15 Figure 4.2: Mchr1 and somatostatin receptor 3 (Sstr3) colocalize in a subset of hippocampal neuronal cilia. (A-F) Representative image of the CA1 region of the hippocampus from an adult wild type (WT) mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the hippocampus. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D-F) reveals a subset of neuronal cilia that are positive for Mchr1 only (arrow) and a subset that are positive for both Mchr1 and Sstr3 (arrowhead). (G-I) Colabeling of day 7 WT hippocampal neurons with antibodies to Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Merged image (I) shows Mchr1 and Sstr3 colocalization within the same cilium in vitro. (J) Quantification of day 7 WT hippocampal neurons colabeled for Mchr1 and ACIII or Sstr3 and ACIII reveals that Mchr1localizes to 25.73 ± 3.53% (n = 239) of ACIII-positive cilia and Sstr3 localizes to 61.46 ± 4.12% (n = 221) of ACIII-positive cilia. (K) Graphical representation of the quantification of day 7 WT hippocampal neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 54.43 ± 5.78% (n = 67) of Mchr1-positive cilia. Scale bars represent 10µm.

A B C

Mchr1 Mchr1 Sstr3 Sstr3 D E F

Mchr1 Mchr1 Sstr3 Sstr3 G H

16 Figure 4.3: Mchr1 and Sstr3 colocalize in a subset of piriform cortical neuronal cilia. (A-F) Representative image of the piriform cortex from an adult wild type (WT) mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the piriform cortex. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D-F) reveals a subset of neuronal cilia that are positive for both Mchr1 and Sstr3 (arrowhead). (G) Quantification of day 7 WT piriform cortical neurons colabeled for Mchr1 and ACIII or Sstr3 and ACIII reveals that Mchr1 localizes to 55.62 ± 3.63% (n = 186) of ACIII-positive cilia and Sstr3 localizes to 14.03 ± 1.71% (n = 159) of ACIII-positive cilia. (H) Graphical representation of the quantification of day 7 WT piriform cortical neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 23.36 ± 5.11% (n = 86) of Mchr1-positive cilia. Scale bars represent 10µm.

Figure 4.4: Mchr1 and Sstr3 colocalize in a subset of hypothalamic neuronal cilia. (A-F) Representative image of the hypothalamus from an adult wild type (WT) mouse colabeled for Mchr1 (green) and Sstr3 (red). Nuclei are stained with DRAQ5 (blue). Labeling for Mchr1 (A) and Sstr3 (B) reveals the presence of Mchr1- and Sstr3-positive cilia in the hypothalamus. Merged image (C) shows colocalization of Mchr1 and Sstr3 on a subset of neuronal cilia. Zoomed in image (D-F) reveals a subset of neuronal cilia that are positive for both Mchr1 and Sstr3 (arrowhead). (G) Quantification of day 7 WT hypothalamic neurons colabeled for Mchr1 and ACIII or Sstr3 and ACIII reveals that Mchr1localizes to 63.42 ± 4.59% (n = 108) of ACIII-positive cilia and Sstr3 localizes to 33.34 ± 5.24% (n = 129) of ACIII-positive cilia. (H) Graphical representation of the quantification of day 7 WT piriform cortex neurons colabeled for Mchr1 and Sstr3 shows Sstr3 colocalizes to 20.36 ± 4.39% (n = 85) of Mchr1-positive cilia. Scale bars represent 10µm.

A Co-IP Input Cotransfected Gapdh- Mchr1- Cotransfected Gapdh- Mchr1- with Sstr3 myc myc kDa with Sstr3 myc myc kDa 200 200 150 150 100 100 IP: Myc 75 IB: Sstr3 75 IP IB: Sstr3 50 50 37 37 Input 200 25 150 100 75 IB: Myc 50 37

B Co-IP Input Cotransfected Gapdh- Mchr1- Cotransfected Gapdh- Mchr1- with Sstr3 myc myc kDa with Sstr3 myc myc kDa 200 200 150 150 100 IP: Sstr3 100 IP 75 IB: Myc 75 IB: Myc 50 50 37 37 25 Input 25 200 150 100 IB: Sstr3 75 50 37 18

Figure 4.5: Mchr1 and Sstr3 proteins interact. Sstr3 was co-expressed with myc-tagged glyceraldehyde-3-phosphate dehydrogenase (Gapdh-myc) or myc-tagged Mchr1 (Mchr1) in HEK293T cells. (A) Cell extracts were immunoprecipitated (IP) with an anti-myc antibody. Immunoprecipitates were analyzed by western blotting (IB) with an anti-Sstr3 antibody (left). Note that Sstr3 is immunoprecipitated with Mchr1, as indicated by the 45kDa band, but not Gapdh. (B) In the reverse experiment, cell extracts were immunoprecipitated (IP) with an anti-Sstr3 antibody and immunoprecipitates were analyzed by western blotting (IB) with an anti- myc antibody (left). Note that Mchr1 is immunoprecipitated with Sstr3, as indicated by the 37kDa band, but not Gapdh. The input, confirming expression of each protein, is also shown (right). Sstr3 appears as a 45kDa band, which agrees with its predicted size, and 75kDa and 150kDa bands, which may represent higher order oligomers. Gapdh and Mchr1 are observed as 39kDa and 37kDa bands, respectively. The expression pattern of GPCRs often results in a multi-band pattern due to various oligomeric combinations and post translational modifications.

A B C

Mchr1-CFP Sstr3-YFP Merge D E

FRETcorrected 19

Figure 4.6: Mchr1 and Sstr3 interact in live cells. HEK293T cells were transiently co-transfected with constructs encoding CFP-tagged Mchr1 and YFP-tagged Sstr3. Mchr1-CFP and Sstr3-YFP colocalize at the plasma membrane and in intracellular compartments (A-C). Robust FRET signals are observed between Mchr1-CFP and Sstr3-YFP localizing in intracellular compartments and moderate FRET signals are observed between Mchr1-CFP and Sstr3-YFP localizing in the cell membrane (D). The fluorescence intensity profile along the red-dotted line within the FRETcorrected image is shown (E). The intensity scale for CFP (blue) and FRETcorrected (red) are on the left. The intensity scale for YFP (green) is on the right. AU = Arbitrary Unit

IP kDa Input  Mchr1 IgG kDa Input 150 100 75 100 75 50 37 50 IB: G  Mchr1 IB: R  Sstr3 20

Figure 4.7: Mchr1 and Sstr3 interact in mouse hippocampal lysate. Membrane protein enriched cell lysate from the hippocampus of 5 week old adult wild type mice was immunoprecipitated (IP) with a goat anti-Mchr1 antibody or goat IgG, as a negative control. Immunoprecipitates were analyzed by western blotting (IB) with a rabbit anti-Sstr3 antibody. Note that Sstr3 is immunoprecipitated with Mchr1 but not with the IgG negative control. The input probed with anti-Sstr3 (left) or anti-Mchr1 (right), confirms the expression of Sstr3 and Mchr1. The ~55kDa bands in the IP lanes may be IgG heavy chain that is detected due to cross-reactivity of the secondary antibody.

TABLE 4.1: DISTRIBUTION OF MCHR1 POSITIVE CILIA IN THE MOUSE BRAIN

REGION OF THE BRAIN RELATIVE IMMUNOLABELING

Amygdala ++

Cortex-Motor +

Cortex-Somatosensory +

Cortex-Visual +

Cortex-Auditory +

Cortex-Piriform +++

Fasciolar Gyrus +++

Hippocampus-CA1 +++

Hippocampus-CA2 ++

Hippocampus-CA3 +

Hypothalamus ++

Nucleus Accumbens ++++

Olfactory Tubercle +++

Striatum ++ 4 Table 4.1: Distribution of Mchr1 immunoreactive cilia in the central nervous system. The relative number of Mchr1-positive cilia in each brain region, normalized to ACIII-positive cilia, is designated by: +, sparse distribution of Mchr1- positive cilia; ++, moderate distribution of Mchr1-positive cilia; +++, extensive distribution of Mchr1-positive cilia; and ++++, highest detection of Mchr1-positive cilia.

CHAPTER 5: CONCLUSIONS AND DISCUSSIONS

Implications and future studies elucidating the function of BBS proteins

Previous studies from our laboratory have shown that BBS proteins are required for proper localization of ciliary GPCRs on central neurons. Specifically, we have shown in our lab the ciliary GPCRs, Sstr3 and Mchr1 require BBS proteins to be trafficked properly to cilia [55], and the ciliary GPCR, D1, requires BBS proteins to be properly trafficked out of cilia [56]. Based on these findings we hypothesized that one function of the BBS proteins is to traffic ciliary GPCRs into and out of cilia upon appropriate cell signals. Utilizing yeast two-hybrid analysis and co-immunoprecipitation we have shown that Bbs5, a BBSome subunit, directly interacts with multiple ciliary GPCRs providing evidence that indeed one function of BBS proteins is to mediate the localization of ciliary

GPCRs through direct binding. However, from these studies many questions still remained. Do other neuronal ciliary membrane proteins utilize the same trafficking pathway as ciliary GPCRs? Could the BBS proteins interact with other novel ciliary signaling machinery and mediate their trafficking into and out of neuronal cilia?

To begin to address these questions, we developed and optimized a new proteomics tool to further investigate the function of BBS proteins. Data obtained from these experiments have provided exciting new avenues to pursue for future studies.

Through proteomic analysis we have identified putative Bbs4/BBSome-interacting proteins which include various phospholipid phosphatases and kinases. Although, additional experiments are needed to validate these interactions, these findings suggest that BBS proteins may be involved in cellular processes other than ciliary GPCR

85 trafficking. It would be interesting to determine if these new putative Bbs4/BBSome- interacting proteins contain a novel consensus sequence that mediates interaction with the BBSome and/or mediates ciliary localization. This knowledge could provide a bioinformatics tool to allow for the discovery of novel ciliary proteins which may implicate new signaling cascades which utilize the primary cilium. Moreover, determining the role

BBS proteins play in various cellular processes may help reveal new connections between ciliary dysfunction and BBS pathology.

Implications and future studies investigating dynamic localization of neuronal ciliary signaling machinery

Although it was discovered over ten years ago that the GPCR, Sstr3, selectively localizes to neuronal cilia throughout the brain [52], it has yet to be determined whether

Sstr3 is active and can signal on the ciliary membrane. The results presented in

Chapter 3 provide strong evidence that Sstr3 is functional within the ciliary membrane.

Our results show that upon SST treatment, the natural ligand for Sstr3, there is a time- dependent reduction in Sstr3 ciliary localization combined with an accumulation of Sstr3 in punctate vesicles within the cell body. It is tempting to speculate that upon Sstr3 activation the receptor is trafficked out of the cilium and is endocytosed to be recycled back to the ciliary membrane or degraded. Future co-labeling immunocytochemistry studies could help determine the fate of activated ciliary Sstr3.

Arrestin proteins play a critical role in the canonical pathway of activated GPCR desensitization and internalization [84]. We have discovered that upon Sstr3 activation within the ciliary membrane, βarr2 is trafficked into the cilium and colocalizes with the receptor. Furthermore, this dynamic localization of βarr2 into the ciliary compartment is prevented by expressing a phosphorylation Sstr3 mutant. Although four residues in the

C-terminal tail of Sstr3 have been shown to play a critical role in desensitization and

86 internalization of the receptor [88], results presented in Chapter 3 show that only phosphorylation at threonine 357 is necessary for βarr2 translocation into the cilium.

Future studies investigating the phosphorylation mechanism might reveal which kinase is responsible for “shutting off” the GPCR signaling cascade and allowing for βarr2 to further desensitize the receptor. Using an online predictor tool, it is likely that a GRK mediates the phosphorylation of Thr357 in the C-terminus tail of Sstr3. Heparin has been shown to be an inhibitor of all GRKs. Future studies pretreating cells with heparin, might reveal that indeed a GRK is mediating the phosphorylation of activated Sstr3 by blocking the recruitment of βarr2 into the cilium. Furthermore, dominant negative GRK constructs have been developed which might further reveal the precise kinase responsible.

Taken together, we have provided evidence that Sstr3, localized on the ciliary membrane, can receive stimuli from the extracellular environment, become activated, and transmit these signals back to the cell. As primary cilia are often defined by the complement of proteins that localize to them, these studies help support our model that neuronal cilia are specialized non-synaptic organelles. Improper trafficking of the receptor or any part of the signaling cascade could result in altered ciliary signaling.

Aberrant ciliary signaling could explain many phenotypes commonly observed in ciliopathy patients.

Implications and future studies exploring ciliary GPCR heteromerization

Although it is known that neurons can signal through intricate networks of axons and dendrites, understanding how neuronal cilia contribute to overall neuronal function is an exciting new field in cell biology. Data presented in Chapter 4 represents an unrecognized layer of complexity in ciliary signaling. We show for the first time that two

87 ciliary GPCRs can colocalize and form heteromers in the mouse brain. Specifically we show that Mchr1 and Sstr3 colocalize within distinct populations of neuronal cilia and can form heteromers. GPCR heteromerization can alter ligand binding properties, G protein coupling, and/or receptor desensitization and internalization rates [115], therefore ciliary GPCR heteromerization could substantially influence the signal generated by a neuronal cilium. Future studies aimed at determining the functional relevance of Mchr1 and Sstr3 heteromerization will provide insight into the roles neuronal cilia play in non- synaptic signaling. Interestingly, Mchr1 and Sstr3 have both been shown to couple to

Gαi. Perhaps ligand binding to either partner in the heteromer causes a synergistic inhibitory signal. In contrast, it is possible that the Mchr1-Sstr3 heteromer generates a unique signal by coupling to a different G protein, such as Gαq. Future studies utilizing signaling assays will help elucidate the functional relevance of ciliary GPCR heteromerization. As more and more ciliary GPCRs are discovered, it will be interesting to determine if various GPCRs can form heteromers. Furthermore, because various

GPCR heteromers have been shown to be upregulated in disease states, such as depression [130], determining the clinical relevance of Mchr1-Sstr3 heteromers could provide exciting new therapeutic avenues for ciliopathies.

Implications of understanding the role neuronal cilia play in cell biology

Experiments presented in this work tested various aspects of neuronal cilia biology. Specifically we tested the hypothesis that like other cilia types, neuronal cilia are specialized sensory and signaling organelles. When this investigation began, little was known about the function of neuronal cilia. To test our hypothesis we examined which proteins dynamically localized to neuronal cilia and the proteins that mediated this specificity. Determining the proteins that localize to cilia and the trafficking mechanisms

88 involved will not only shed light on cell-type specific cilia functions but also provide potential therapeutic targets. This strategy is illustrated in the development of drugs to treat PKD. Although there is currently no treatment or cure for PKD, the discoveries made over the past decade in understanding the disease pathophysiology and specifically the role of the primary cilium have led to many potential therapeutics. The dominant form of PKD is caused by mutations in either PKD1 or PKD2. As mentioned in

Chapter 1 the gene products polycystin-1 and polycystin-2 form a mechanosensor complex on renal epithelial cilia and regulate Ca2+ signaling. It has also been discovered, that the GPCR vasopressin receptor 2 (V2R) localizes to renal cilia. V2R couples to Gαs and stimulates cAMP/PKA signaling. PKA activity affects the fluid transport within the kidney epithelia by targeting various receptors including the aquaporin-2 (AQP2) and cystic fibrosis transmembrane conductance regulator (CFTR). In concurrence with these findings, Ca2+ and cAMP levels are altered in PKD cells, potentially leading to cyst formation and PKD pathogenesis. By dissecting these ciliary-mediated signaling pathways researchers have been able to unveil many therapeutic targets and potential treatments for individuals affected by this ciliopathy. Although, the findings presented in this dissertation are in their infancy they provide a good foundation for exploring the various signaling cascades neuronal cilia are involved in. Understanding how neuronal cilia can affect overall neuronal function in cellular homeostasis and more importantly in disease will provide many therapeutic targets to pursue in future studies. It is important to remember due to the abundant presence of neuronal cilia in the brain, ciliary signaling and putative therapeutics could be pursued in not just ciliopathy treatment, but treatment for more common neurodegenerative diseases such as Alzheimer’s disease or

Parkinson’s disease.

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