INVESTIGATIONS INTO NEURONAL CILIA UTILIZING MOUSE MODELS OF BARDET-BIEDL SYNDROME Dissertation Presented In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Nicolas F. Berbari, BS *****

The Ohio State University 2008

Dissertation Committee: Approved by: Kirk Mykytyn, PhD, Adviser Virginia Sanders, PhD ______Georgia Bishop, PhD Adviser Michael Robinson, PhD Integrated Biomedical Sciences Graduate Program

ABSTRACT

Cilia are hair-like microtubule based cellular appendages that extend 5-30 microns from the surface of most vertebrate cells. Since their initial discovery over a hundred years ago, cilia have been of interest to microbiologists and others studying the dynamics and physiological relevance of their motility. The more recent realization that immotile or primary cilia dysfunction is the basis of several human genetic disorders and diseases has brought the efforts of the biomedical research establishment to bear on this long overlooked and underappreciated organelle.

Several human genetic disorders caused by cilia defects have been identified, and include Bardet-Biedl syndrome, Joubert syndrome, Meckel-Gruber syndrome, Alstrom syndrome and orofaciodigital syndrome. One theme of these disorders is their multitude of clinical features such as blindness, cystic kidneys, cognitive deficits and obesity. The fact that many of these cilia disorders present with several features may be due to the ubiquitous nature of the primary cilium and their unrecognized roles in most tissues and cell types.

The lack of known function for most primary cilia is no more apparent than in the central nervous system. While it has been known for some time that neurons throughout the brain have primary cilia, their functions remain unknown. Research on neuronal cilia has suffered from a paucity of tools to study them. To this end, some of this work

ii describes the development of the first in vitro neuronal culture system where primary cilia are present (Chapter 2).

It is apparent that the functions of cilia, at least in part, are defined by the specific proteins that localize within and on the organelle. Indeed access of proteins and signaling modules into and out of the ciliary compartment appears to be tightly regulated.

In order to better understand the physiological roles of cilia throughout the body, an understanding of the signaling proteins that localize to the cilium and the mechanisms behind cilia localization is needed. To these ends, some of this work describes novel ciliary signaling proteins such as the localization of adenylyl cyclase III to primary cilia throughout the adult mouse brain, and the localization of the G protein-coupled receptor

(GPCR) melanin concentrating hormone receptor 1 to certain neuronal cilia (Chapter 3 and 4). We also have identified a specific GPCR sequence involved in the localization of

GPCRs to the ciliary compartment (Chapter 4). This sequence will aid in determining the mechanisms that target proteins to the cilium.

The development of these tools has allowed for the opportunity to test the hypothesis that certain clinical features of the ciliary disorder Bardet-Biedl Syndrome (BBS) are due to neuronal cilia dysfunction. Indeed, we have shown that mouse models of BBS fail to localize specific receptors to their neuronal cilia (Chapter 5). These data have set the ground work for more extensive investigations into neuronal cilia function. Further, this work suggests neuronal cilia dysfunction may contribute not only to the cognitive defects associated with ciliary disorders, but may also underlie the obesity observed in these disorders.

iii DEDICATION

Dedicated to

My Wife

iv ACKNOWLEDGMENTS

This work would not be possible without the help and guidance of my advisor and mentor, Dr. Kirk Mykytyn. I would like to thank you for your scientific integrity, optimism and endless patience in educating. This experience is one that I will remember forever.

I would like to thank each of my committee members, Dr. Michael Robinson, Dr.

Candice Askwith, and Dr. Georgia Bishop, who have been extremely generous with their time and have provided invaluable advice.

To my lab mates past and present, thank you for putting up with me. I would especially like to express my gratitude to Jackie Lewis for all of her help and patience both in and out of the lab. To Dr. David Cunningham, thank you for some of the most interesting conversations. I would like to thank Tom Sherwood and Dr. Brian Davy for reminding me that we do this because it is fun.

I am also grateful to the Raymond E. Mason Foundation for providing me a 2 year fellowship and The Ohio State University for the Presidential Fellowship this last year.

I would like to thank all of my friends and family for all of their support this year.

Finally, I would like to thank my wife who has been supportive in every possible way that one could support another.

v VITA

November 6, 1979 Born – Iowa City, Iowa, USA 2002 B.S. Biology, Indiana University

PUBLICATIONS

1. Lemon W.J., Swinton C.H., Wang M., Berbari N., Wang Y., You M. Single nucleotide polymorphism (SNP) analysis of mouse pulmonary adenoma susceptibility loci 1-4 for identification of candidate genes. Journal of Medical Genetics. 2003. 40(4):E36. 2. Kelly L.E., Davy B.E., Berbari N.F., Robinson M.L., El-Hodiri H.M. Recombineered Xenopus tropicalis BAC expresses a GFP reporter under the control of Arx transcriptional regulatory elements in transgenic Xenopus laevis embryos. Genesis. 2005. 41(4):185-91.

3. Doggett N.A., Xie G., Meincke L.J., Sutherland R.D., Mundt M.O., Berbari N.F., Davy B.E., Robinson M.L., Rudd M.K., Weber J.L., Stallings R.L., Han C. A 360-kb interchromosomal duplication of the human HYDIN locus. Genomics. 2006. Dec;88(6):762-71.

4. Berbari N.F., Bishop G.A., Askwith C.C., Lewis J.S., Mykytyn K. Hippocampal neurons develop primary cilia in culture. Journal of Neuroscience Research. 2007. April; 85(5):1095-100.

5. Bishop G.A., Berbari N.F., Lewis J.S., Mykytyn K. Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain. Journal of Comparative Neurology. 2007. Oct 9; 505(5): 562-71.

6. Berbari N.F., Askwith C.C., Lewis J.S., Mykytyn K. Identification of Ciliary Localization Sequences within the Third Intracellular Loop of G Protein-Coupled Receptors. Molecular Biology of the Cell [epub ahead of print Feb 2008].

7. Berbari N.F., Bishop G.A., Lewis J.S., Mykytyn K. Bardet-Biedl Syndrome Proteins are Required for G Protein-Coupled Receptor Localization to Neuronal Cilia. PNAS.

vi

FIELDS OF STUDY

Major Field: Integrated Biomedical Science

vii

TABLE OF CONTENTS

Page

ABSTRACT ...... ii

DEDICATION ...... iv

ACKNOWLEDGMENTS...... v

VITA ...... vi

LIST OF TABLES...... xi

LIST OF FIGURES...... xii

LIST OF ABBREVIATIONS...... xv

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

Summary ...... 1

Cilia Structure, Function, And Classification...... 2

Ciliogenesis And Intraflagellar Transport...... 3

Specialized Sensory Cilia and Disease ...... 4

Cilia Localization Sequences...... 4

Cilia Disorders and Clinical Features...... 6

viii The Cilia Disorder Bardet-Biedl Syndrome...... 7

Neuronal Cilia ...... 9

Hypothesis and Chapter 2-5 Overview ...... 10

CHAPTER 2: HIPPOCAMPAL NEURONS POSSESS CILIA IN CULTURE ...... 17

Summary ...... 17

Background...... 18

Results...... 19

Discussion ...... 23

Materials and Methods ...... 25

CHAPTER 3: TYPE III ADENYLYL CYCLASE LOCALIZES TO CILIA THROUGHOUT

THE ADULT MOUSE BRAIN ...... 33

Summary ...... 33

Background...... 34

Results...... 36

Discussion ...... 39

Materials and Methods ...... 44

CHAPTER 4: IDENTIFICATION OF CILIARY LOCLALIZATION SEQUENCES WITHIN

THE THIRD INTRACELLULAR LOOP OF G PROTEIN-COUPLED RECEPTORS ...... 55

Summary ...... 55

Background...... 56

ix Results...... 59

Discussion ...... 64

Materials and Methods ...... 68

CHAPTER 5: BARDET-BIEDL SYNDROME PROTEINS ARE REQUIRED FOR THE

LOCALIZATION OF G PROTEIN-COUPLED RECEPTORS TO PRIMARY CILIA ...... 83

Summary ...... 83

Background...... 84

Results...... 86

Discussion ...... 89

Materials and Methods ...... 92

CHAPTER 6: CONCLUSIONS AND DISCUSSION...... 104

Future Study of Neuronal Cilia In Vitro ...... 104

Future Study of Cilia Localization Sequences ...... 105

Discussion of the Role of BBS Proteins...... 105

Questions in The Field of Cilia Biology ...... 106

REFERENCES...... 108

x LIST OF TABLES

Table Page

Table 1.1 Common Clinical Features of Six Cilia Disorders ...... 15

Table 1.2 Analysis of BBS Genes ...... 16

Table 2.1 Composition of Day 7 Serum-Free Neuronal Cultures...... 32

Table 3.1 Distribution of ACIII-Positive Cilia in the Mouse Brain ...... 54

Table 4.1 G Protein-Coupled Receptors Identified by the Ciliary Localization

Consensus Sequence...... 82

Table 5.1 Neuronal and ACIII Cilia Frequency in Culture ...... 102

Table 5.2 Neuronal and Sstr3 Cilia Frequency In Culture ...... 102

Table 5.3 Quantification of Sstr3 Ciliary Rescue in Transfected Bbs2 Neurons...103

xi LIST OF FIGURES

Figure Page

Figure 1.1 Transmission Electron Micrograph of Motile and Primary Cilia...... 12

Figure 1.2 Immunolabeled Primary Cilia In the Hippocampus...... 13

Figure 1.3 Schematic Diagram of A Cilium...... 14

Figure 2.1 Cilia are detected in the developing mouse hippocampus...... 29

Figure 2.2 Cultured hippocampal neurons possess cilia...... 30

Figure 2.3 Hippocampal neurons possess different populations of cilia in vitro...31

Figure 3.1 Histochemical visualization of ACIII ciliary localization in the adult mouse hippocampus...... 47

Figure 3.2 Histochemical visualization of ACIII ciliary localization in regions of the adult mouse forebrain...... 48

Figure 3.3 Histochemical visualization of ACIII ciliary localization in medial and lateral regions of the adult mouse brain...... 49

Figure 3.4 Histochemical visualization of ACIII ciliary localization in regions of the adult mouse brainstem...... 50

xii Figure 3.5 Immunocytochemical visualization of cilia projecting from an astrocyte and choroid plexus cells in the adult mouse brain...... 51

Figure 4.1 Somatostatin receptor subtype 3 and serotonin receptor subtype 6 selectively localize to cilia when heterologously expressed in IMCD cells...... 73

Figure 4.2 Sequences between the fourth and sixth transmembrane domains of

Sstr3 are important for ciliary localization...... 74

Figure 4.3 The third intracellular (i3) loop of Sstr3 is sufficient to localize Sstr5 to cilia...... 75

Figure 4.4 The amino portion of the i3 loop of Htr6 is sufficient to localize Htr7 to cilia...... 76

Figure 4.5 Comparative genomics identifies unique residues in Sstr3 and Htr6 that are Potentially important for ciliary localization of GPCRs...... 77

Figure 4.6 Quantitation of Mutational Analysis Confirms Residues that are

Potentially Important for Ciliary Localization of GPCRS...... 78

Figure 4.7 Melanin concentrating hormone receptor 1 (Mchr1) localizes to cilia in vitro and in vivo...... 79

Figure 4.8 immunoblot of whole protein lysates from IMCD cells transiently transfected with Sstr5[TM4-5Sstr3]...... 80

Figure 4.9 Mutational analysis indicates that the A and Q are important for ciliary localization of chimeric receptors Sstr5[TM5-6Sstr3] and Htr7[TM5-V241Htr6]. ....81

Figure 5.1 BBS mice possess neuronal primary cilia in the brain but lack Sstr3- positive cilia...... 95

xiii Figure 5.2 Sstr3 ciliary localization can be restored in vitro...... 96

Figure 5.3 BBS mice lack Mchr1 ciliary labeling in the brain...... 97

Figure 5.4 Mchr1 ciliary localization can be restored in vitro...... 98

Figure 5.5 Western blot analysis From Adult Hippocampus for Sstr3 Levels ...... 99

Figure 5.6 Western Blot Analysis from Adult Hypothalmus and Accumbens and

Tubercle Regions for Mchr1 Levels ...... 100

Figure 5.7 Mchr1 localizes to cytoplasmic puncta in BBS neurons in vivo ...... 101

Figure 6.1 Model of Cilia Dysfunction In BBS ...... 107

xiv LIST OF ABBREVIATIONS

ACIII Adenylyl Cyclase III

Amy Amygdala

ARAC Arabinofuranoside

ATP Adenosine triphosphate

BBS Bardet-Biedl Syndrome

BSA Bovine Serum Albumin cAMP cyclic adenosine monophosphate

CBL VIII Cerebellum Lobule VIII

CBLIX Cerebellum Lobule IX

CNG Cyclic Nucleotide Gated Ion Channel

CP Choroid Plexus or Caudate Putamen

CSF Cerebral Spinal Fluid

DG Dentate Gyrus

DmHyp Dorsomedial hypothalamus

EcCx Ectorhinal cortex

GAD Glutamic Acid Decarboxylase

xv GFAP Glial Fibrillary Acid Protein

GL Granular Layer

Golf Olfactory G Protein

GPCR G Protein Coupled-Receptor

Hi Hilus

Htr6 Serotonin Receptor 6

Htr7 Serotonin Receptor 7

Hyp Hypothalamus

IC Inferior colliculi

IFT Intraflagellar Transport

IMCD Inner Medullary Collecting Duct Cell Line

JS Joubert Syndrome

Mchr1 Melanin Concentrating Hormone Receptor 1

MKS Meckel Syndrome

MoCx Motor Cortex

NA Nucleus Accumbens

Nf Neurofilament

OrCx Orbital Cortex

P Pontine

PBS Phosphate Buffered Saline

xvi PCL Olfactory Plexiform Cell Layer

PG Periaqueductal Gray

PiCx Piriform cortex

PL Pyramidal Layer

P0 Post natal day zero

P7 Post natal day seven

Rho Rhodopsin

RsCx Retrosplenial cortex

RsG Retrosplenial Granular

SC Superior colliculi

SEM Standard Error of the Mean

SN Substantia Nigra

SO Stratum Oriens

Sp5 Spinal Trigeminal Tract

SR Stratum Radiatum

Sstr3 Somatostatin Receptor 3

Sstr5 Somatostatin Receptor 5

Sub Subiculum

ViCx Visual Cortex

VmHyp Ventromedial hypothalmus

xvii VN Vestibular Nucleus

βTIII β-Tubulin III

xviii CHAPTER 1:

INTRODUCTION TO CILIA

SUMMARY

Currently, there is a rapidly expanding body of literature implicating cilia at the

center of a range of cellular processes from the control of cell division to the control of

diverse signaling pathways. Primary cilia have been observed on most vertebrate cell

types, and defects in ciliary structure and function have been linked to a spectrum of

phenotypes, including obesity, renal and hepatic cystic disease, alterations in cell

division, retinal degeneration, nervous system abnormalities, limb defects, and disrupted

left-right axis formation. Several human genetic disorders caused by ciliary defects have

also been identified, and include Bardet-Biedl syndrome, Joubert syndrome, Meckel-

Gruber syndrome, Alstrom syndrome, orofaciodigital syndrome, polycystic kidney

disease and nephronophthisis (1). Primary cilia have now been shown to play crucial

roles in receiving and communicating signals to the cell. Dysfunction of these ubiquitous

organelles may underlie the broad array of phenotypes observed in these disorders.

1 CILIA STRUCTURE, FUNCTION, AND CLASSIFICATION

Cilia and flagella have been highly conserved throughout evolution and are found on

most eukaryotic cells (2). In general, cilia are classified as either motile or primary. Motile

cilia, such as sperm flagella or respiratory cilia are involved in generating flow of

extracellular fluids or movement of individual cells. For example, the motile cilia found on

respiratory epithelia are responsible for mucociliary clearance in the airways

(Figure 1.1 A). Alternatively, non-motile primary cilia provide a sensory function. There are exceptions, as many unicellular protists and metazoan organisms possess flagella that play key roles in both motility and sensory reception (3). Additionally, vertebrates possess a subset of primary cilia that are motile and are involved in establishing the left- right body axis (4-6). Primary cilia are typically solitary organelles present on almost all mammalian cell types (7). They can be found extending from cells into fluid-filled compartments, such as in the kidney tubules (Figure 1.1 B), or can be found deep within tissue, such as in the brain on hippocampal neurons (Figure 1.2).

All cilia and flagella contain a central core structure called an axoneme that is comprised of microtubules arranged in a regular and specific pattern (Figure 1.3). The axoneme core of motile cilia and flagella is comprised of nine peripheral microtubule doublets surrounding two central microtubules that are important for the generation of movement. This is referred to as a 9+2 axoneme. In primary cilia, the axoneme lacks the central microtubules and thus has a 9+0 arrangement. The axoneme extends from the basal body, which is derived from the centriole and acts as a microtubule-organizing center just beneath the cell membrane (2). As the axoneme elongates from the basal body it becomes ensheathed by a ciliary membrane. Although the ciliary membrane is continuous with the plasma membrane, there is restricted access and only certain 2 proteins localize to the ciliary membrane (8, 9). Similarly, the cytosol of the cilium is

compartmentalized from the rest of the cell, apparently by the transition fibers that

extend from the distal portion of the basal body to the plasma membrane (10). These

fibers are thought to provide both a docking and sorting site for proteins bound for the

cilium (11). Little is known about the mechanisms of sorting and trafficking of proteins to

these discrete compartments.

CILIOGENESIS AND INTRAFLAGELLAR TRANSPORT

Proteins are not synthesized in the cilium, and a microtubule-based transport system called intraflagellar transport (IFT) has evolved to move macromolecular protein particles from the basal body to the tip of the axoneme and back again (12). Proper IFT is required for the assembly and maintenance of most eukaryotic cilia. IFT particles accumulate around the basal body and appear to associate with the transition fibers that function as a loading dock for IFT (10). Movement of particles from the base to the tip, antereograde transport, requires the action of the kinesin-II motors (12). Movement in the opposite direction, retrograde transport, requires the IFT-dynein motors (12).

Although it was once thought that only non-membrane bound proteins were transported by IFT, recent results also implicate IFT in the transport of membrane-associated proteins. Specifically, in C. elegans select cation channels undergo transport along the length of cilia on sensory neurons at rates comparable to IFT, and this motility is disrupted in IFT mutants (13). In the biflagellated algae Chlamydomonas, IFT is required for the movement of a membrane-associated kinase into the cilium (14). In mammalian cells, proper IFT is required for trafficking of the olfactory cyclic nucleotide-gated channel

1b into cilia (15). Further, IFT appears to play a role in the trafficking of ciliary membrane proteins from the Golgi complex to the cilium (16).There is also evidence from 3 Chlamydomonas that IFT plays a direct role in propagating signals from the cilium to the

cell (17). Thus, IFT has an evolutionarily conserved role in mediating ciliary signaling.

SPECIALIZED SENSORY CILIA AND DISEASE

Although primary cilia were once considered evolutionary holdovers, it is now clear

that they provide important and diverse sensory and signaling functions. In the eye,

photoreceptor cells, which are modified cilia, sense and respond to light. In the nose,

olfactory cilia detect odors and initiate signaling cascades in olfactory neurons. In the

kidney, bending of the cilium on kidney epithelial cells by fluid flow triggers an increase

in intracellular calcium mediated by a channel located on the cilium (18, 19). The

importance of primary cilia is highlighted by the fact that defects in the formation or

function of primary cilia have been implicated in the pathogenesis of many human

developmental disorders and diseases, including, obesity, renal cystic disease, retinal

degeneration, nervous system abnormalities, mental retardation, hepatic disease, limb

abnormalities, and laterality defects (1). It is likely that defects in primary cilia result in

such a broad range of phenotypes due to their presence on almost every human cell

type. In addition, recent studies have demonstrated that primary cilia play important roles

in morphogenetic signaling pathways, such as Hedgehog signaling (20-22). We are just

beginning to appreciate the importance that cilia play in normal development and cell

function. Remarkably, the functions of primary cilia on most cells are not known.

CILIA LOCALIZATION SEQUENCES

Appropriate signaling through cilia depends on the localization of ciliary signaling

proteins (2, 21). The absence of cilia specific proteins can cause disease and altered

development (2). However, very little is known about the mechanisms of protein 4 trafficking to cilia. It is thought that ciliary proteins contain targeting signals that direct them to the ciliary compartment (11). Most of our understanding of the targeting of integral membrane proteins has been gained from studies looking at the trafficking of polycystin-2 (PC-2) and rhodopsin. PC-2 is a ciliary cation channel that is required for flow-mediated calcium signaling in renal epithelial cells. A conserved serine residue in the C-terminus of the C. elegans homolog of PC-2 is important for ciliary localization

(23). Additionally, in the NH2-terminus of PC-2 there is a conserved RVXP motif that is both necessary and sufficient for ciliary localization in mammalian cell lines (9). This motif is also found at the carboxy-terminus of olfactory cyclic nucleotide-gated channel

1b and is required for trafficking of the channel into cilia (15). It is presumed that the

RVXP motif is part of a protein interaction domain (9). This motif is not found in the ciliary GPCRs Sstr3 or Htr6, however, suggesting that a different mechanism mediates their ciliary localization. Studies in C. elegans sensory cilia, mammalian photoreceptors, and olfactory cilia indicate there are multiple pathways for trafficking ciliary membrane proteins, and each pathway may be specific for a particular class of proteins (13, 24-26).

Trafficking of the GPCR rhodopsin (rho) to the outer segment of photoreceptors provides potential insight into ciliary GPCR trafficking. Like other membrane proteins, rho is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi where it is further processed. Rho is then transported in post-Golgi vesicles that fuse to the inner segment plasma membrane surrounding the connecting cilium. Finally, rho is transported through the connecting cilium to the rod outer segments, possibly through an

IFT-mediated mechanism (24). Four highly conserved amino acids at the carboxy- terminus of rhodopsin (VXPX-COOH) have been implicated as the most important determinants in the sorting and transport of rhodopsin-containing membranes from the

5 trans-Golgi network to the base of the connecting cilium. Mutations in these residues

cause abnormal accumulation of rhodopsin in the Golgi and inner segment plasma

membrane (27). However, Sstr3 and Htr6 do not contain the VXPX-COOH motif,

suggesting the existence of additional GPCR ciliary localization sequences.

Investigation of the trafficking of olfactory GPCRs to cilia in C. elegans has

implicated adjacent hydrophobic and basic residues immediately C-terminal to the

seventh transmembrane segment as a ciliary localization motif (25). This motif is also

present and conserved in other GPCRs that localize to cilia, including Sstr3, Htr6, and

Smoothened, suggesting a role in cilia localization (21). However, this motif is present in

all of the somatostatin receptor subtypes, yet only Sstr3 localizes to cilia (28), indicating

this motif may be necessary but it cannot be sufficient to specify ciliary localization.

The signaling function of an individual cilium is determined by its signaling protein

complement. A better understanding of the underlying mechanisms of ciliary localization

will lend invaluable insights into the physiological role of this organelle. Once these

mechanisms are realized, the gap between the current lack of understanding in cilia

signaling and the clinical features observed in known cilary disorders can be bridged.

CILIA DISORDERS AND CLINICAL FEATURES

In recent years, several rare human genetic diseases have been linked to cilia

dysfunction. These disorders have heterogeneous genetics and are often pleitropic in

their clinical presentation. For example there are nine known nephronophthisis and

twelve known Bardet-Biedl Syndrome genes. Cilia disorders often present with a range

of clinical features including but not limited to: retinopathy, renal cystic disease, cognitive

deficits, polydactylyl, CNS anomalies, posterior encephalocele, hepatic disease, obesity,

6 anosmia, heart defects, hypogonadism, situs inversis, obesity, and deafness. It is

believed that the range in clinical features is due to the ubiquitous presence of the

primary cilia and their unknown functions in various cell types and tissues. Table 1.2

shows some clinical features of six known ciliary disorders: Bardet-Biedl Syndrome

(BBS), Orofaciodigital syndrome I (ORFD1), Senior-Loken syndrome, Meckel syndrome,

Joubert syndrome, and Alstrom syndrome. While each disorder has been attributed to

cilia dysfunction, clinical phenotypes vary, suggesting that unique aspects of cilia

function may be altered in each. It is clear that as more roles for primary cilia are

recognized the list of cilia disorders will expand.

THE CILIA DISORDER BARDET-BIEDL SYNDROME

Bardet-Biedl syndrome (BBS) is a rare human genetic disorder occurring in

approximately 1 in 100,000 live births (OMIM 29-9900) (29). BBS is characterized by

obesity, retinopathy, polydactyly, kidney and heart defects, hypogenitalism, and

cognitive deficits (30). To date, twelve causative genes have been identified that when

mutated in an autosomal recessive fashion present with the same spectrum of clinical

features (31-37) (Table 1.2). The BBS proteins are evolutionarily conserved with

homologs of nine of the genes identified in invertebrates. However, BBS3, 6, 10, 11, and

12 are the only BBS proteins that share similarity to any known proteins. BBS3 is ADP-

ribosylation factor (ARF)-like protein 6 (38, 39), which is a subgroup of the ARF family of

proteins that have diverse cellular functions, including regulation of intracellular transport

and membrane trafficking (40). Based on sequence similarity, BBS6, 10 and 12 are

putative chaperonins (41-43). BBS11 is an E3 ubiquitin ligase (44). The majority of the

BBS proteins do not display similarity to any proteins of known function. Additionally,

most of the BBS proteins do not contain any well-characterized motifs that would provide 7 insight into their functions. The exceptions are BBS4 and BBS8 that contain tetratricopeptide repeat (TPR) motifs, which are involved in mediating protein-protein interactions. The BBS proteins do not share significant similarity to each other (Table

1.2).

Numerous studies in diverse model systems have shown that the BBS proteins play important roles in cilia biology (29). The BBS proteins localize to basal bodies of ciliated cells and to ciliated structures in tissues (45, 46). Comparative genomics reveals that

BBS homologs are found in ciliated organisms and not in non-ciliated organisms (47,

48). Knockdown of the bbs5 gene in Chlamydomonas using RNA interference results in the loss of flagella (48). In C. elegans, the BBS orthologues are expressed exclusively in the ciliated neurons (39, 45, 48). It is believed that the BBS proteins in C. elegans act as linkers or adapters between the IFT particles and the molecular motors responsible for anterograde and retrograde transport (49). Further, mutations in the bbs7 and bbs8 genes result in structurally and functionally abnormal cilia due to the disruption of coordinated IFT in C. elegans (50, 51).

In ciliated mammalian cells, the functions of BBS proteins are less clear. Lacking a single BBS protein produces widely varying effects in different cell types. For example, knockout mouse models of BBS possess structurally normal motile tracheal cilia but sperm flagella are absent (47, 52, 53). With regard to primary cilia, BBS mice show loss of photoreceptors (47, 52, 53) and display defects in olfactory cilia structure and function

(54). Yet, primary cilia structure is not altered in other tissues that are associated with the phenotype. For example, cilia on renal epithelial cells appear structurally normal in

BBS mice (47, 55), despite the fact that some of the animals develop cystic kidney disease. This observation suggests that cilia function, rather than structure, is

8 compromised in these cells.

A recent seminal study provides insight into how the BBS proteins mediate cilia function in mammalian cells (56). Nachury et al. generated a retinal pigmented epithelial cell line expressing a tagged version of BBS4 and used this fusion protein to isolate a stable protein complex, called the BBSome. The BBSome is comprised of seven of the most evolutionarily conserved BBS proteins (BBS1, 2, 4, 5, 7, 8, and 9). This complex localizes to the centriolar satellites, travels to the basal body and appears to enter the cilium and associate with the ciliary membrane. Several additional proteins were shown to interact with the BBSome, including PCM1, tubulin and Rabin8, which is the Rab8 nucleotide exchange factor. This latter interaction is particularly interesting as Rab8 is involved in the docking and fusion of post-Golgi vesicles at the base of the cilium (57).

The interaction of the BBSome with Rabin8 is mediated by the BBS1 protein (56). BBS9 interacts with 5 of the other BBS proteins, suggesting that it is the structural core subunit of the BBSome. BBS5 binds to phosphoinositides and may mediate interactions between the BBSome and membrane. BBS4 and BBS8 bind to pericentriolar material 1 at the centriolar satellites, but not at the base of the cilium, suggesting that this interaction is not required for BBSome function at the cilium.

NEURONAL CILIA

It is not widely known that neurons throughout the brain possess primary cilia. In fact

many neuroscientists would be surprised to learn this fact. Early reports of neuronal cilia

were based upon the observation of cilia axoneme cross sections showing the unique

9+0 microtubule arrangement in electron microscopy neuro-anatomical studies (58).

Until relatively recently there were several hypotheses about the physiological relevance

9 of neuronal cilia. These ranged from a role for neuronal cilia as flow sensors to their

being a vestigial organelle serving no real purpose (59). It has recently been found that

specific GPCRs, somatostatin receptor 3 and serotonin receptor 6, preferentially localize

to the ciliary membrane in neurons (60, 61). This is the first evidence that implicates a

signaling role for neuronal cilia. However, the physiological relevance of the localization

of these receptors remains elusive. The role of primary cilia on neurons is particularly

interesting when one considers that neurons possess highly developed and tightly

regulated processes (axons and dendrites) specifically utilized for intracellular and

intercellular communication. Thus, in neurons cilia may function as unrecognized

signaling organelles with distinct roles. The further development of new tools to study

neuronal cilia will allow for extensive investigations into the roles of cilia in the central

nervous system.

HYPOTHESIS AND CHAPTER 2-5 OVERVIEW

This document presents a culmination of work that has focused on the hypothesis

that some of the clinical features observed in BBS are due to defects in neuronal primary

cilia. In testing this hypothesis we have also identified novel ciliary signaling proteins as

well as developed other tools to aid in investigating the roles of neuronal cilia. These

tools have allowed for the initial testing of the hypothesis.

The development of an important tool for future investigations into neuronal cilia is

described in Chapter 2. The first in vitro neuronal culture system in which primary

neuronal cilia have been observed is described, and has been published in The Journal

of Neuroscience Research. Chapter 3 also reports a new tool, and describes the

localization of type III adenylyl cyclase (ACIII) to neuronal cilia throughout the adult

10 mouse brain. These observations have been published in the Journal of Comparative

Neurology. Chapter 4 reports a primary protein sequence important for the localization of specific G protein-coupled receptors to cilia. This loose GPCR cilia localization sequence will aid future investigations into the localization of receptors to this sub- cellular compartment. Further, this sequence was used to predict novel ciliary GPCRs.

Chapter 4 has been published in the journal Molecular Biology of the Cell. Finally, in

Chapter 5 studies on the neuronal cilia of mouse models of BBS show that the ciliary

GPCRs Sstr3 and Mchr1 fail to localize properly to neuronal cilia. Further, the localization of these GPCRs appears to require the BBS proteins. The identification of a potential signaling pathway responsible for the obesity observed in ciliary disorders is also discussed. These studies show that BBS mice are models of defective neuronal ciliary signaling. The work in Chapter 5 has been published at the Proceedings of the

National Academy of the Sciences.

11

Figure 1.1 Transmission Electron Micrograph of Motile and Primary Cilia (A) Motile cilia of respiratory epithelial cells of the trachea which are responsible for mucociliary clearance. Note that multiple cilia per cell are present. (B) Arrows indicate primary cilia of renal epithelial cells which protrude into the lumen of tubules and are present as solitary appendages.

12

FIGURE 1.2 IMMUNOLABELED PRIMARY CILIA IN THE HIPPOCAMPUS Somatostatin Receptor 3 immunolabeled primary cilia, labeled in green, within the CA3 pyramidal layer of the mouse hippocampus. Draq5 nuclear labeling in blue.

13

FIGURE 1.3 SCHEMATIC DIAGRAM OF A CILIUM The internal microtubule structure of the axoneme is shown in cross section. The microtubule complement of motile and primary cilia is indicated. The cell membrane and cilia membrane as well as the transition fiber zone and basal body are labeled and indicated with arrows.

14

Disease BBS OFD1 Senior-Loken Meckel Joubert Alstrom

Retinitis Pigmentosa X X X X X

Cystic Kidney Disease X X X X X X

Polydactyly X X X X

Cognitive Deficits X X X X

CNS Anomalies X X X X X

Posterior X X encephalocele

Hepatic Disease X X X X X

Obesity X X

Anosmia X

Situs Inversis X X X X

Heart Anomalies X X

Hypogonadism X

TABLE 1.1 COMMON CLINICAL FEATURES OF SIX CILIA DISORDERS BBS OMIM (209900), OFD1 OMIM (311200), Senior-Loken OMIM (266900), Meckel OMIM (249000), Joubert OMIM (213300), Alstrom OMIM (203800).

15

Gene Location Protein Sequence Characterization Subcellular Localization BBS1 11q13 Novel Basal Body/Cilium BBS2 16q22 Novel Basal Body/Cilium BBS3 3p13 ADP-Ribosylation Factor-Like protein 6 Basal Body/Cilium BBS4 15q21 Novel, TPR Repeats Basal Body/Cilium BBS5 2q31 Novel Basal Body/Cilium BBS6 20p12 Chaperonin-like Basal Body BBS7 4q27 Novel Basal Body/Cilium BBS8 14q31 Novel, TPR Repeats Basal Body/Cilium BBS9 9q22 Novel Basal Body/Cilium BBS10 12q Chaperonin-like Unknown BBS11 9q31 E3 Ubiquitin Ligase Unknown BBS12 4q27 Chaperonin-like Unknown

TABLE 1.2 ANALYSIS OF BBS GENES Shows the BBS genes 1-12 and their chromosomal locations as well as analysis of their primary protein sequences for similarities to proteins of known function. The known sub-cellular localization of the BBS proteins is also noted in the last column.

16 CHAPTER 2:

HIPPOCAMPAL NEURONS POSSESS PRIMARY

CILIA IN CULTURE

SUMMARY

Primary cilia are cellular appendages that provide important sensory functions and defects in primary ciliary signaling have been implicated in the pathophysiology of human diseases and developmental abnormalities. Almost all human cell types possess a primary cilium. In fact, neurons throughout the brain possess primary cilia upon which certain receptors localize, suggesting that neurons possess cilia-mediated signaling.

However, the functional significance of neuronal cilia is unknown. Although there is a great deal of interest in understanding the functions of neuronal cilia, their study is hampered by the lack of an in vitro model system. Here we report that the majority of hippocampal neurons cultured from postnatal mice possess primary cilia in vitro. Further, we describe cilia proteins that can be labeled to readily visualize neuronal primary cilia in culture. These findings are the first characterization of neuronal primary cilia in vitro and should greatly facilitate further investigations into the function of these organelles.

17 BACKGROUND

Cilia are evolutionarily conserved cellular appendages that are classified as either

motile or primary. Motile cilia and flagella, such as airway epithelial cilia and sperm

flagella, are responsible for generating fluid flow or movement. Primary cilia are

generally immotile and are present as solitary organelles on almost all human cell types

(7) where they have been shown to provide important cellular sensory and signaling

functions (2, 62, 63). Defects in primary cilia have been implicated in renal cystic

diseases, retinal degeneration, liver fibrosis, anosmia, ataxia, cardiac defects, and situs

inversus (2, 63). Primary cilia are also important for normal patterning of tissues during

development (2) and primary cilia dysfunction is thought to underlie the etiology of

numerous human genetic disorders (2, 63).

The development of in vitro model systems has been instrumental in elucidating

the functions of primary cilia on certain cells. For example, the finding that bending of

primary cilia on a canine kidney cell line resulted in a cellular calcium signal was the first

evidence that renal primary cilia act as mechanosensors (18). Subsequent studies using

primary cultures of mouse kidney cells demonstrated that the proteins defective in

autosomal dominant polycystic kidney disease locate to the primary cilium and are

necessary for generating this signal (19). Together, these studies established that renal

primary cilia are mechanosensory signaling organelles and defects in this signaling are

associated with disease. However, the function of primary cilia on most cells remains

unknown.

Neurons throughout the adult rodent brain possess primary cilia upon which

certain G protein-coupled receptors localize, including somatostatin receptor 3 (28, 61)

and serotonin receptor 6 (60, 64), suggesting that there is cilia-mediated signaling on 18 neurons. However, there has been no direct evidence that signaling can be mediated

through neuronal cilia and the functions of these organelles remain obscure. Further, the

functions and significance of neuronal cilia during neural development are unknown. The

properties and potential functions of neuronal cilia have been discussed in several

recent reviews (58, 59, 65).

The study of neuronal primary cilia has been complicated by the fact that the marker

that is commonly used for visualizing primary cilia, acetylated α-tubulin (7), is not specific

for neuronal cilia and instead localizes throughout the cell body and processes of

neurons. Further, to our knowledge, the presence of neuronal cilia in primary cultures

has not been reported. Previous investigations have utilized electron microscopy or

immunocytochemistry with antibodies against ciliary receptors to visualize neuronal cilia

in tissue sections. Here, we describe a new marker for neuronal cilia and report that the

majority of cultured hippocampal neurons possess a primary cilium, thereby providing a

system that will facilitate investigations into the functions of neuronal cilia.

RESULTS

Primary Cilia were Present in the Hippocampus of Postnatal Mice

Somatostatin receptor 3 (Sstr3) has been shown to selectively localize to the plasma

membrane of neuronal primary cilia throughout the rodent brain, including the pyramidal

cell layer of the CA regions and the granule cell layer of the dentate gyrus in the

hippocampus (28, 61). However, these analyses were performed on adult animals and

the presence of Sstr3-immunoreactive cilia in the hippocampus during development has

not been reported. In order to investigate the potential that neurons cultured from early

postnatal brains would possess Sstr3-positive cilia, we labeled postnatal mouse brain 19 sections with an antibody to Sstr3. Although the presence of Sstr3-immunoreactive cilia

was rare in the hippocampus from of P0 mice and confined to the CA3 region (data not

shown), we observed an abundance of Sstr3-positive cilia in the CA3 region of P7

animals (Figure 2.1A). At this age, some cilia were also detected in the CA1 region

(data not shown). Thus, Sstr3-positive neuronal cilia are scarce in the mouse

hippocampus at birth and the distribution and abundance of these cilia increases during

early postnatal development.

Given that Sstr3 will only label cilia on neurons expressing Sstr3, rather than all

potentially ciliated neurons, we sought to identify an additional marker of neuronal cilia.

Type III adenylyl cyclase (ACIII) is enriched in olfactory neuronal cilia (66) and we

hypothesized that it would localize to neuronal cilia in the brain. In order to test whether

ACIII-positive cilia were present in the hippocampus of postnatal mice, we labeled

sections from P0 and P7 mice with anti-ACIII. Rod-shaped ACIII-immunoreactive

appendages were detected in a similar distribution and abundance to Sstr3-positive cilia

with rare detection at P0 but abundant labeled appendages in the CA3 region of sections

from P7 mice (Figure 2.1B). To confirm that these structures were cilia, we colabeled

sections with anti-Sstr3 and anti-ACIII. We found that Sstr3 and ACIII colocalized

(Figure 2.1 C-E), indicating that Sstr3 and ACIII are markers for neuronal cilia in the developing mouse hippocampus. Some cilia were detected that were positive for ACIII but not Sstr3, indicating that ACIII is an additional marker of neuronal cilia that colocalizes with Sstr3 in some, but not all, neuronal cilia in vivo.

Hippocampal Neurons Possessed Cilia in Culture

To determine whether hippocampal neurons possess cilia in vitro, we cultured

hippocampal neurons from newborn mice under serum-free conditions (67). These 20 conditions limit glial growth and produce cultures that are enriched for neurons. After 7 days in culture the cells were fixed and labeled with anti-Sstr3 and anti-ACIII. We detected the abundant presence of solitary cilia projecting from cells that appeared to be neurons based on morphology (Figure 2.2 A, B). In order to confirm that the ciliated cells were neurons, we colabeled cultures with anti-Sstr3 or anti-ACIII and an antibody against the neuronal-specific tubulin subunit, β-Tubulin III (β-TubIII). We found that the majority of cells displaying Sstr3- or ACIII-immunoreactive cilia were also positive for β-

TubIII (Figure 2.2 C, D). Quantification of the ciliated cells that were positive for β-TubIII and Sstr3 indicated that after 7 days in culture 22% of neurons on average possessed an Sstr3-positive cilium (Table 2.1). However, quantification of the ciliated cells that were positive for β-TubIII and ACIII indicated that on average 63% of cultured neurons possessed an ACIII-positive cilium after 7 days (Table 2.1). These results confirm that hippocampal neurons possess cilia in culture that can be visualized with antibodies to both Sstr3 and ACIII, with labeling for ACIII detecting almost three-fold more neuronal cilia than Sstr3.

As Sstr3 and ACIII ciliary localization is predominantly confined to the pyramidal layer of the CA3 and CA1 regions of the hippocampus in P7 mice, it is likely that the cilia primarily project from pyramidal neurons. To further characterize the ciliated neurons in culture, we colabeled cells with antibodies to Sstr3 or ACIII and glutamic acid decarboxylase (GAD), a marker of GABA containing neurons. Although the vast majority of ciliated neurons were negative for GAD, both Sstr3 and ACIII-positive cilia were detected on GAD positive neurons (Figure 2.2 E, F). Therefore, most ciliated neurons in culture are GAD-negative, but GABAergic neurons can possess cilia that are positive for

Sstr3 and/or ACIII.

21 Hippocampal Neurons Possessed Different Populations of Cilia In Vitro

The fact that the percentage of ACIII-positive neuronal cilia was three-times greater

than the percentage of Sstr3-positive cilia in vitro (Table 2.1) indicated that there was a

population of cilia that were positive for ACIII only. To test whether neurons also

possessed cilia to which both ACIII and Sstr3 localized, we colabeled cells with anti-

ACIII and anti-Sstr3. We found that ACIII and Sstr3 colocalized to neuronal cilia in vitro

(Figure 2.3 A, B) and most cilia were positive for only ACIII (Figure 2.3 C). Interestingly,

cilia that were positive for Sstr3 only were not detected. Thus, the ciliated neurons within

our cultures consisted of one population of cilia that were positive for ACIII only and a

second population of cilia that were positive for both markers.

Cultured Glial Cells Possessed Type 3 Adenylyl Cyclase Positive Cilia

Although our hippocampal neurons were cultured under serum-free conditions, only

79% of the cultured cells were positive for β-TubIII, indicating that the remaining cells

were non-neuronal. Further, ACIII-immunoreactive cilia were detected on a significant

number of β-TubIII-negative cells (Figure 2.3 D). Quantification of these cells revealed

that on average 50% of the non-neuronal cells possessed an ACIII-positive cilium at 7

days (Table 2.1). Interestingly, we did not detect Sstr3-immunoreactive cilia on β-TubIII- negative cells. To determine whether any of these ciliated β-TubIII-negative cells were glia, we colabeled the cultures with anti-ACIII and glial fibrillary acidic protein (GFAP), a marker of astrocytes. Of the cells that were positive for GFAP, approximately one-half also possessed an ACIII-immunoreactive cilium (Figure 2.3 E). These results indicate that glial cells also possess cilia in culture that can be detected by ACIII labeling.

22 DISCUSSION

In the present study, we report that Sstr3 and ACIII are markers of cilia on early

postnatal mouse hippocampal neurons in vivo. Localization of Sstr3 to neuronal cilia in

the hippocampus has previously been shown only in adult rodents. To our knowledge,

ACIII localization to neuronal cilia outside of the olfactory system has not been

previously reported. We show for the first time that hippocampal-derived neurons

possess cilia in vitro. Our results also show that both GAD-positive and -negative

neurons possess cilia in vitro. The ability to detect and characterize neuronal cilia in culture is critical for studying the functions of these organelles. Our initial characterization provides some insights into the properties of neuronal cilia.

In this study we show that Sstr3 ciliary localization in the hippocampus increases in abundance and distribution during early postnatal development. Sstr3 is one of six somatostatin receptor subtypes that are all members of the G protein-coupled receptor

(GPCR) superfamily. These receptors are activated by somatostatin, a regulatory peptide that primarily affects endocrine function and regulates neurotransmission (68,

69). Although a great deal is known about the pharmacologic and physiological properties of the individual somatostatin receptors, little is known about the significance of subcellular localization on receptor function. Therefore, the consequence of Sstr3 selectively localizing to neuronal cilia is unknown. It is of note that we consistently detected Sstr3 immunofluorescence within the nucleus of neurons. Whether this labeling is due to receptor localization in the nucleus or the result of non-specific staining has yet to be determined. Nevertheless, the availability of an in vitro system will allow for the characterization of Sstr3 signaling on neuronal cilia.

23 We report here that ACIII localizes to cilia on hippocampal neurons both in vivo and in vitro. Although ACIII is expressed in the brain (70), it has not been previously reported to localize to neuronal cilia in the brain. ACIII has been shown to localize to olfactory cilia

(66). It is required for olfaction and couples activation of odorant GPCRs to increased cAMP levels in olfactory cilia (71). As somatostatin receptors are functionally coupled to adenylyl cyclases (68, 69), the finding that ACIII and Sstr3 colocalize in a subset of neuronal cilia suggests that these proteins may functionally interact. Further studies are necessary to test this possibility. Interestingly, the majority of ciliated neurons were positive for ACIII but negative for Sstr3. If ACIII is coupled to GPCRs in neuronal cilia, this would suggest that Sstr3-negative neurons may express other receptors that mediate signaling through an interaction with ACIII. One possibility is serotonin receptor subtype 6, which has been shown to localize to neuronal cilia (60).

Importantly, our results indicate many similarities between cilia in vivo and in vitro.

For example, we observed colocalization of Sstr3 and ACIII to cilia on some neurons and localization of only ACIII to other neuronal cilia in both the brain and in culture.

Whether these similarities between neuronal cilia in the brain and neuronal cilia in vitro is the result of predetermined pathways or the result of environmental stimuli remains to be determined. Understanding whether certain neurons are fated to have cilia with specific types of receptors or whether receptor localization to cilia is a response to other cues will be an exciting avenue of investigation. This system should prove extremely useful for answering such questions.

Perhaps the most intriguing question raised by our findings is why neurons possess primary cilia in culture and what is the physiological significance of neuronal cilia?

Kidney primary cilia have been shown to act as mechanosensors of fluid-flow in renal

24 tubules (2, 63) and olfactory cilia are laced with odorant receptors and project into the

mucous in order to detect odors (66). Potential reasons for neurons to possess cilia-

mediated signaling include modulation of signaling by compartmentalization of the signal

and/or signaling molecules, association of the signal with specific cellular

organelles/processes such as the nucleus and gene regulation, and modulation of signal

transduction based on the high surface to volume ratio of the cilium (58, 59, 65). We

believe this in vitro model system of neuronal primary cilia provides the ability to

determine the roles of ciliary signaling for neuronal function.

MATERIALS AND METHODS

Experimental Animals The mice used in this study were on a 129:BL6 background.

All procedures were approved by the Institutional Animal Care and Use Committee at

The Ohio State University.

Neuronal Cell Culturing Hippocampal neurons were prepared as previously

described (67, 72, 73). Briefly, hippocampi were dissected from mouse pups on the day

of their birth (P0) and placed in a sterile solution of Leibovitz's L-15 medium (Invitrogen,

La Jolla, CA) containing 0.2 mg/ml bovine serum albumin (BSA). The meninges were

removed and the hippocampal tissue was torn into small pieces. The tissue was then

transferred into a solution of L-15/BSA containing 0.375 mg/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. After incubation, the tissue was washed three times with pre-warmed Neurobasal-A medium (Invitrogen) containing B-27 (Invitrogen) and triturated with a series of Pasteur pipettes of decreasing diameters. The diameters of the

Pasteur pipettes corresponded to approximately 2/3 and 1/3 the size of the starting

25 diameter, and were generated by briefly heating the pipette tips over a Bunsen burner.

Hippocampal neurons were plated onto poly-D-lysine coated coverslips (BD

Biosciences, Bedford, MA) in 24-well dishes in Neurobasal/B-27 with the addition of

insulin-transferrin-sodium selenite (Sigma-Aldrich). Each hippocampus was plated onto

3 coverslips. Glial replication was prevented by the addition of cytosine

arabinofuranoside (ARA-C; Sigma-Aldrich) to a final concentration of 10 µm after 2-3

days.

Immunostaining and Imaging Animals were anesthetized by a 0.1 ml/10 g

intraperitoneal injection of 2.5% tribromoethanol (Sigma-Aldrich), sacrificed by cardiac

puncture, and perfused with phosphate-buffered saline (PBS) followed by 4%

paraformaldehyde. The brains were cryoprotected in 20% sucrose, frozen, embedded in

Optimal Cutting Temperature compound (VWR, West Chester, PA), and coronally

sectioned in a cryostat at a thickness of 30 µm. Sections were permeabilized with 0.3%

Triton X-100 in PBS with 2% goat serum, 0.02% sodium azide and 10 mg/ml BSA. All

incubations and washes were carried out in PBS with 2% serum, 0.02% sodium azide

and 10 mg/ml BSA. All primary antibody incubations were carried out for 16-24 hours at

4°C and all secondary antibody incubations were carried out for 1 hour at room

temperature.

Cultured neurons were fixed with 4% paraformaldehyde, permeablized, and immuno-

labeled using the same procedure. Primary antibodies included; anti-adenylyl cyclase III

(sc-588; Santa Cruz, Santa Cruz, CA), anti-Sstr3 (ss-830; Gramsch, Schwabhausen,

Germany), anti-β-Tubulin III (T-8660; Sigma-Aldrich), anti-GFAP (G-3893; Sigma-

Aldrich) and anti-GAD (a gift from Dr. Richard Burry). Secondary antibodies included;

Alexa Fluor 488- and 546-conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 26 488- and 546-conjugated goat anti-rabbit IgG (Invitrogen). Nuclei were visualized by

either Sytox (Invitrogen) or DRAQ5 (Axxora, San Diego, CA).

For double labeling with two rabbit primary antibodies, samples were fixed,

permeabilized, and incubated with the first primary antibody and an Alexa Fluor-

conjugated goat anti-rabbit IgG secondary antibody as described above. The samples

were then incubated for 1 hour at room temperature in 5% normal rabbit serum (Jackson

ImmunoResearch, West Grove, PA) with 10mg/ml BSA in PBS, to block any remaining

IgG binding sites remaining on the first secondary. PBS washes were then followed by

incubation in goat anti-rabbit Fab (Jackson ImmunoResearch) for 1 hour at room

temperature to further block any remaining IgG binding sites on the first primary or

secondary antibodies. PBS washes were then followed by incubation with the second

primary antibody for 16-24 hours at 4°C. The second primary antibody was then labeled

with a different Alexa Fluor-conjugated goat anti-rabbit IgG secondary antibody, as

described above, followed by final PBS washes. Slips were then mounted onto slides

using Immu-Mount (Thermo Electron Corporation, Waltham, MA) for observation.

Samples were imaged on a Zeiss 510 META laser scanning confocal microscope in the

Ohio State University Central Microscopy facility.

Cellular Counting and Analysis Coverslips were centered on the microscope

objective and the number of nuclei and the number of fluorescent cells were counted

separately in 5-10 consecutive fields. The results were expressed as the percent positive

cells, calculated as the ratio of either fluorescent cells to nuclei (i.e. β-Tubulin III positive cells/total number of nuclei) or fluorescent marker to marker (i.e. β-Tubulin III positive cell and adenylyl cyclase III positive cilia/total number of β-Tubulin III positive cells, β-

Tubulin III positive cell and Sstr3 positive cilia/total number of β-Tubulin III positive cells). 27 The results for ciliated non-neuronal cells were expressed as the ratio of the cells that were β-Tubulin III negative and adenylyl cyclase III positive to total number of β-Tubulin

III negative cells. For colabeling of β-Tubulin III and ACIII, approximately 120 cells were counted from three distinct neuronal cultures (n = 3) for a total of 356 cells. For colabeling of β-Tubulin III and Sstr3, approximately 80 cells were counted from three distinct neuronal cultures (n = 3) for a total of 250 cells. Data are expressed as mean +

Standard Error of the Mean (SEM).

28

FIGURE 2.1 CILIA ARE DETECTED IN THE DEVELOPING MOUSE HIPPOCAMPUS. REPRESENTATIVE IMAGES OF THE CA3 REGION FROM P7 MICE (N=3) STAINED WITH ANTIBODIES TO SSTR3 (A) AND ACIII (B) REVEALS THE PRESENCE OF NEURONAL CILIA. NUCLEI WERE STAINED WITH SYTOX (BLUE). IMAGES OF THE DENTATE GYRUS FROM A P30 MOUSE COLABELED WITH ANTIBODIES TO SSTR3 (C) AND ACIII (D) AND MERGED (E) INDICATE THE PRESENCE OF SSTR3-POSITIVE CILIA (ARROWS; LEFT PANEL), ACIII- POSITIVE CILIA (ARROWS; MIDDLE PANEL) AND CILIA THAT ARE POSITIVE FOR BOTH (ARROWS; RIGHT PANEL). NUCLEI WERE LABELED WITH THE DNA STAIN DRAQ5 (BLUE).

29

FIGURE 2.2 CULTURED HIPPOCAMPAL NEURONS POSSESS CILIA. Immunostaining of day 7 hippocampal-derived cells with antibodies to Sstr3 (A) or ACIII (B) detect solitary cilia. Colabeling of day 7 hippocampal-derived cells with antibodies to β-Tubulin III and Sstr3 (C) or ACIII (D) confirms that the ciliated cells are neurons. Colabeling of day 7 hippocampal-derived cells with antibodies to GAD and Sstr3 (E) and ACIII (F) reveals ciliated GABAergic neurons. Cilia are indicated by arrows. Nuclei were labeled with the DNA stain DRAQ5 (blue).

30

FIGURE 2.3 HIPPOCAMPAL NEURONS POSSESS DIFFERENT POPULATIONS OF CILIA IN VITRO. IMAGES OF DAY 7 CULTURED HIPPOCAMPAL NEURONS COLABELED WITH ANTIBODIES TO ACIII (A) AND SSTR3 (B) AND MERGED (C) INDICATE THE PRESENCE OF ACIII-POSITIVE CILIA (ARROWS; LEFT PANEL), SSTR3-POSITIVE CILIA (ARROWS; MIDDLE PANEL) AND CILIA THAT ARE POSITIVE FOR BOTH (ARROWS; RIGHT PANEL). COLABELING OF DAY 7 CULTURED HIPPOCAMPAL NEURONS WITH ANTIBODIES TO ACIII AND β-TUBULIN III (D) OR GFAP (E) INDICATE THE PRESENCE OF CILIATED GLIAL CELLS. CILIA ARE INDICATED BY ARROWS. NUCLEI WERE LABELED WITH THE DNA STAIN DRAQ5 (BLUE).

31

Culture Characeteristic %

Neurons 78.3 ± 1.5

Neurons with an Sstr3 Positive Cilium 21.0 ± 2.1

Neurons with an ACIII Positive Cilium 62.6 ± 2.8

Non-Neuronal Cells with an ACIII positive Cilium 50.6 ± 1.8

TABLE 2.1 COMPOSITION OF DAY 7 SERUM-FREE NEURONAL CULTURES Percentage of cells after 7 days in culture that were positive for the neuronal marker β-Tubulin III (β-TubIII), somatostatin receptor 3 (Sstr3) and β-TubIII, Type 3 adenylyl cyclase (ACIII) and β- TubIII, and ACII but negative for β-TubIII. Values are expressed as the mean percentage ± SEM.

32

CHAPTER 3:

TYPE III ADENYLYL CYCLASE LOCALIZES TO

CILIA THROUGHOUT THE ADULT MOUSE BRAIN

SUMMARY

Solitary primary cilia project from nearly every cell type in the human body.

These organelles are considered to have important sensory and signaling

functions. Although primary cilia have been detected throughout the mammalian

brain, their functions are unknown. The study of primary cilia in the brain is constrained by the scarcity of specific markers for these organelles. We previously demonstrated that type III adenylyl cyclase is a marker for primary cilia on neonatal hippocampal neurons in vivo and in vitro. We further showed that

ACIII localizes to cilia on cultured glial cells. Here, we report that ACIII is a marker for primary cilia throughout many regions of the adult mouse brain.

Further, we report that ACIII localizes to primary cilia on choroid plexus cells and some astrocytes in the brain, which to our knowledge is the first report of a 33 marker for visualizing cilia on glia in vivo. Overall, our data indicate that ACIII is a prominent marker of primary cilia in the brain and will provide an important tool to facilitate further investigations into the functions of these organelles.

BACKGROUND

Cilia are microtubule-based organelles that project from the surface of cells in the

body (65). These organelles can be divided into two types; motile cilia which generate

movement or flow and non-motile primary cilia. Primary cilia are solitary appendages

that extend from the basal bodies of most human cells (7). These primary cilia provide

important sensory and signaling functions (2, 62, 63, 65, 74). The importance of primary

cilia throughout the body is highlighted by the fact that the etiology of several human

diseases and genetic syndromes is linked to cilia function (1, 2, 63). One type of primary

cilia that has been extensively studied is the cilia that project from the apical surface of

epithelial cells lining the kidney tubules. Renal primary cilia act as mechanosensors that

respond to flow-induced bending by initiating an intracellular calcium signal (75).

Disruptions in renal ciliary signaling are associated with renal cystic disease (19). The

recognition of an association between cilia function and human disease has sparked a

great deal of interest in these organelles. Yet, the functions of primary cilia on most cell

types remain unknown.

The potential functions of primary cilia in the brain have been discussed in

several reviews (58, 59, 65). It is suggested that they are involved in the transduction of

a multitude of stimuli, including concentrations of growth factors, hormones, and

developmental morphogens, as well as osmolarity, pH, and fluid flow. However, their 34 precise functions remain unclear.

A severe limitation in the study of primary cilia in the brain is the scarcity of

specific markers. The marker that is commonly used for visualizing primary cilia,

acetylated α-tubulin (7), strongly labels neuronal processes and cell bodies and is

therefore not appropriate for selectively detecting cilia within the brain. Traditionally,

primary cilia in the brain have been characterized by electron microscopy. Ultrastructural

studies revealed the presence of primary cilia on both neurons (76) and glial cells (77,

78). The utility of this approach, however, is limited by its labor intensiveness. More

recently, immunocytochemical studies showed that some neurons express G protein-

coupled receptors (GPCRs) that selectively localize to primary cilia. These GPCRs

include serotonin receptor 6 (Htr6) (60, 64) and somatostatin receptor 3 (Sstr3) (28, 61).

Therefore, neuronal cilia can be visualized by labeling with antibodies to Htr6 or Sstr3.

The limitation with this approach is that not all cells express these receptors. Further, these antibodies do not detect cilia on glial cells. Thus, populations of cilia remain undetectable and the abundance of cilia in the brain is likely underestimated. The identification and characterization of additional markers of primary cilia in the brain is necessary to facilitate the study of these organelles.

We recently reported that type III adenylyl cyclase (ACIII) localizes to primary cilia on neonatal hippocampal neurons in vivo and in vitro (79). We further showed that ACIII

localizes to cilia on cultured glial cells. Here, we demonstrate that ACIII is a prominent

marker of primary cilia throughout the adult mouse brain. Further, we show that ACIII

localizes to cilia on neurons, choroid plexus cells, and astrocytes in the brain. To our

knowledge, this is the first report of a marker for visualizing astrocytic cilia in vivo.

35 RESULTS

ACIII Localizes to Cilia in the Adult Hippocampus

We previously reported that ACIII ciliary localization can be detected on cells in the pyramidal and granular cell layers of the developing hippocampus (79). To determine whether ACIII also localizes to cilia in the adult hippocampus, we performed immunohistochemistry with an antibody to ACIII on sagittal sections from 6 month old wild-type mouse brains. Figure 3.1 shows a sagittal section through the adult mouse hippocampus approximately 1 mm from midline. The letters indicate regions that are shown at higher magnification. We detected abundant labeling of rod-shaped structures associated with cell bodies throughout the pyramidal cell layer of the CA regions and the granule cell layer of the dentate gyrus (Figure 3.1 A-C). Labeling hippocampal sections with an antibody to Sstr3, which is a known marker of primary cilia on neurons, produced an identical pattern of immunoreactivity (data not shown). Thus, we conclude that these structures correspond to primary cilia. Although the vast majority of detected cilia were located within the pyramidal and granule cell layers, occasional ACIII-positive cilia were seen in other layers of the hippocampus, including the stratum oriens, stratum radiatum, and hilus (Figure 3.1 D-F). Thus, ACIII-positive cilia are present in the juvenile and adult mouse hippocampus.

ACIII Localizes to Cilia in the Adult Cerebral Cortex and Forebrain

We then examined the distribution of ACIII ciliary localization in additional regions of

the brain (summarized in Table 3.1). Figure 3.2 shows a sagittal section through the

adult mouse brain approximately 1 mm from midline. The letters indicate regions that are 36 shown at higher magnification. ACIII-positive cilia were detected throughout the cortex

and forebrain, including the subiculum (Figure 3.2 A), as well as the retrosplenial

granular (Figure 3.2 B), visual (Figure 3.2 C), motor (Figure 3.2 D), and orbital cortices

(Figure 3.2 E). These cilia appeared evenly distributed in the cortical cell layers, but were absent from the most superficial layer (Figure 3.2 B-E). ACIII-positive cilia were

present on cells throughout the olfactory bulb, including cells within the plexiform and

granular cell layers (Figure 3.2 F-G). Labeled cilia were especially abundant in the

olfactory tubercle (Figure 3.2 H) and appeared longer than cilia in the other regions.

ACIII-positive cilia were also observed throughout nuclei of the deep forebrain, including the nucleus accumbens (Figure 3.2 I), caudate/putamen (Figure 3.2 J), thalamus (Figure 3.2 K), and hypothalamus (Figure 3.2 L). The density of these cilia was highest in the nucleus accumbens and caudate/putamen. The density of ACIII- positive cilia in the thalamus was noticeably lower than in other regions and was nuclear specific (Table 3.1). The dorsolateral thalamus contained the largest number of cells with ACIII-immunopositive cilia and only a few cilia were detected in the ventral posterior medial or posterior group. Few, if any, were present in the ventral posterior lateral or reticular nuclei. Other regions of the forebrain that contained ACIII-positive cilia included the subthalamus, zona incerta, and substantia inominata (Table 3.1).

To evaluate more medial and lateral regions, we analyzed data from transverse sections from 6 month old wild-type mouse brains. Figure 3.3 shows a transverse section through the adult mouse brain approximately 1.5 mm caudal to Bregma. The letters indicate regions that are shown at higher magnification. ACIII-positive cilia were observed throughout the cortex, including the retrosplenial (Figure 3.3 A), ectorhinal

(Figure 3.3 B), and piriform cortices (Figure 3.3 C). These cilia were particularly

37 abundant in the piriform cortex. As mentioned above, ACIII-positive cilia were absent

from the superficial layers of the cortex. Abundant ACIII-positive cilia were also observed

in the amygdala (Figure 3-3 D) and medial regions of the hypothalamus (Figure 3.3 E,

F), with an especially high density of detected cilia in the ventromedial hypothalamus

(Figure 3.3 F).

ACIII Localizes to Cilia throughout the Adult Mouse Brainstem

In general, ACIII-positive cilia were less abundant in the brainstem than in the

forebrain (Table 3.1). Figure 3.4 is the same section as shown in Figure 3.2. The boxes indicate areas of the brainstem that are shown at higher magnification. ACIII-positive cilia were detected throughout the brainstem, including the substantia nigra (Figure 3.4

A), basilar pons (Figure 3.4 B), superior and inferior colliculi (Figure 3.4 C, D), lateral periaqueductal grey (Figure 3.4 E), vestibular nucleus (Figure 3.4 F), and spinal trigeminal tract (Figure3.4 G). ACIII-positive cilia were sparse in the cerebellum and their laminar and lobular distribution varied (Table 3.1). In lobules I - VIII of the vermis,

ACIII-positive cilia were located primarily on cells in the Purkinje cell layer (Figure 3.4

H). In lobules IX and X, ACIII-positive cilia were found on cells in discrete regions of the granule cell layer (Figure 3.4 I) and virtually no immunolabeling was present in the

Purkinje cell layer. Other regions of the brainstem that contained ACIII-positive cilia included the peripeduncular nucleus, retrorubral nucleus, and nucleus of the lateral leminiscus (Table 3.1).

ACIII Localizes to Cilia on Astrocytes and Choroid Plexus Cells in the Brain

We previously showed that some cultured astrocytes possess cilia that are positive

for ACIII. To test whether ACIII also localizes to cilia on astrocytes in vivo, we colabeled 38 transverse sections from P30 wild-type mouse brains with antibodies to ACIII and glial

fibrillary acidic protein (GFAP), a marker of astrocytes. The vast majority of ACIII-positive

cilia were not associated with GFAP-labeled cells. However, we did detect rare ACIII-

positive cilia projecting from GFAP-positive cells (Figure 3.5 A). We did not detect any

ACIII-positive cilia on cells colabeled with oligodendrocyte or microglial markers (data

not shown), suggesting that ACIII ciliary expression is limited to astrocytic glia.

It has recently been reported that epithelial cells of the choroid plexus (CP) possess

primary cilia (80). We examined CP tissue in the ventricles of the brain and detected the

presence of ACIII-immunoreactive cilia projecting from CP cells (Figure 3.5 B). We did

not detect ACIII labeling of motile cilia on choroid plexus or ependymal cells lining the

ventricles, suggesting that ACIII is specifically expressed in primary cilia in the brain.

DISCUSSION

The goal of this study was to determine whether ACIII-immunoreactive primary cilia

are present in the adult mouse brain. We have demonstrated that ACIII-positive cilia are

prevalent throughout the adult mouse brain. Our data suggest that many cells in the

forebrain as well as select areas of the brainstem possess ACIII-positive cilia. We have

further shown that ACIII localizes to primary cilia on CP cells and select populations of

astrocytes in the brain. Overall, ACIII is an important new tool for studies of primary cilia

in the central nervous system.

Abundance and Distribution of ACIII-Positive Cilia

We detected ACIII-positive cilia throughout most regions of the brain (Table 3.1).

Other ciliary markers have a more limited distribution. Ciliary localization of Htr6 is

confined to the striatum, nucleus accumbens, olfactory tubercle and islands of Calleja 39 (60, 64). Sstr3 localizes to neuronal cilia throughout the brain (61), but does not localize to cilia in regions where ACIII-positive cilia are abundant, such as the nucleus accumbens and caudate/putamen. In the hippocampus, there is a population of cilia that is positive for both ACIII and Sstr3, and another population that is positive for ACIII only

(79). Finally, neither 5-Htr6 nor Sstr3 is a marker for cilia on glial cells. Thus, ACIII is an important supplement to the repertoire of ciliary markers as it detects cilia in the brain that are not positive for Htr6 or Sstr3. Additionally, the ACIII antibody described in this work recognizes the rat protein and we detect a similar pattern of ciliary localization in rat brain sections (G.B. unpublished data).

ACIII-positive cilia were most commonly found in nuclei related to sensory processing of information, such as the olfactory bulb and associated structures, including the olfactory tubercle and the piriform cortex. In the brainstem, the areas with the most

ACIII-positive cilia were in cranial nerve nuclei related to sensory processing, such as the sensory trigeminal nucleus, the cochlear nucleus, and the vestibular nuclei. Regions of the brain involved in visceral or limbic functions also contained numerous ACIII- positive cilia.

Several areas of the brain contained few cells expressing ACIII-positive cilia. Two areas of the forebrain that showed a clear paucity were the thalamus and the globus pallidus. Further, nuclei and structures related to the motor system showed only sparse immunolabeling. These findings do not mean cilia are not present on cells in these regions, only that they are not positive for ACIII. As the complement of signaling proteins varies between cilia in the brain (79), it is possible that there are populations of cilia that do not express ACIII. Further, based on the distribution of these cilia and our findings that ACIII-positive cilia are rare on astrocytes and absent from oligodendrocytes and

40 microglia, it appears that the vast majority of ACIII-positive cilia extend from neurons in

the brain.

Functional Significance of ACIII Expression in Cilia

There are ten known mammalian isoforms of adenylyl cyclase that function to

convert ATP to cyclic AMP (cAMP) in response to activation by a variety of hormones,

neurotransmitters, and other regulatory molecules (81). Cyclic AMP, in turn, activates

several other target molecules to control a broad range of intracellular processes. ACIII

was first described in the olfactory epithelium where it is enriched in cilia on the olfactory

sensory neurons (66). It is also expressed in male germ cells and is required for

spermatid or spermatozoa function and male fertility (82). However, the functions of

ACIII outside the olfactory system are less well understood. In olfactory sensory neurons

it plays a vital role in the olfactory signal transduction cascade that transforms

chemosensory information into electrical signals (71). This cascade involves sequential

activation of odorant GPCRs, an olfactory-specific stimulatory G protein (Golf), ACIII, and cyclic nucleotide-gated (CNG) ion channels. In olfactory sensory neurons, activation of

CNGs by increased concentrations of cAMP leads to increased permeability to Ca2+ ions

and alters membrane potential. It is possible that ACIII plays a similar role in cilia in the

brain to affect membrane potential and alter neuronal firing rate. An important step in

evaluating this hypothesis will be to look for the presence of other signaling components,

such as G proteins and CNGs, in cilia in the central nervous system.

Potential Functions of Primary Cilia in the Brain

Despite the widespread distribution and abundance of cilia in the brain, their

functions are unknown. It is likely that their functions are defined by a number of factors,

41 including the signaling components present in the cilium, the cell type that it projects from, and the location of the cilium within the brain. The functional importance of these cilia is suggested by the fact that several human genetic disorders associated with cilia dysfunction, including Bardet-Biedl syndrome (BBS), Joubert syndrome (JS), and

Meckel syndrome (MKS), have prominent functional and structural CNS phenotypes (1).

Specifically, each syndrome is characterized by mental retardation/developmental delay and structural malformations. It appears the functions of primary cilia in the brain are important for proper neural development and function.

The presence of somatostatin and serotonin receptors suggests that cilia may sense levels of neuromodulators in the local environment of neurons. The consequences of such signaling on neuronal function and the particular importance of ciliary localization of specific receptors can only be speculated. It has been suggested that the physical structure of cilia may confer particular functional traits (58, 65). For example, the small size of the cilium could allow second messenger levels to be sustained for a prolonged period of time, in contrast to the relatively large size of the soma and dendrites where these second messengers would be quickly diluted. The prolonged presence of these second messengers could produce sustained changes in ionic conductances and other cellular effects. Additional studies are required to test these theories and determine the functions of this organelle in the CNS.

The functions of primary cilia on astrocytes are also unknown. Astrocytes, a sub- type of glia in the central nervous system, serve critical roles in the regulation of synaptic activity in the central nervous system. They integrate neuronal inputs, exhibit calcium excitability, and in turn release chemical transmitters that regulate neuronal activity and strength (83). Astrocytes in the subventricular zone extend a cilium into the ventricle

42 (78), suggesting they sense factors in the cerebrospinal fluid. Thus, it could be postulated that cilia serve as an additional mechanism by which these non-neuronal cells sense the chemical makeup of their immediate environment. We found that the minority of astrocytes in the brain possessed an ACIII-positive cilium. Currently, we do not know whether the remaining astrocytes are not ciliated or whether they posses cilia but lack ACIII expression. Additionally, we did not detect cilia on oligodendrocytes or microglia. It is possible there are different populations of glial cilia in the brain that, similarly to neuronal cilia, are comprised of different signaling proteins. The fact that some astrocytes express ACIII-positive cilia suggests that they use cAMP as a second messenger to affect cellular processes. This finding provides an additional mechanism that may affect the role of astrocytes in regulating synaptic transmission in the nervous system.

Finally, we detected ACIII-immunoreactive cilia on CP cells. The choroid plexuses are structures found in the lateral, third, and forth ventricles of the brain, whose primary functions are the production and homeostasis of cerebrospinal fluid (CSF). It has been reported that some CP cells have small tufts of motile cilia and other cells have a single primary cilium (80). Although the functions of CP primary cilia are unknown, disruption of these cilia is associated with elevated chloride levels in the CSF combined with hydrocephalus (80). Intriguingly, the increased chloride level in the CSF is coupled with elevated intracellular cAMP concentration in the CP epithelium. Thus, dysregulation of ACIII-mediated cAMP signaling in CP epithelial cells may be involved in the onset of hydrocephalus.

43 MATERIALS AND METHODS

Experimental Animals

The mice used in this study were on a 129:BL6 background. All procedures were approved by the Institutional Animal Care and Use Committee at The Ohio State

University.

Primary Antibodies

The adenylyl cyclase III (ACIII) rabbit polyclonal antibody (Santa Cruz, Santa Cruz,

CA catalog No. sc-588) was prepared against a synthetic peptide representing the C- terminal 20 amino acids of mouse ACIII (PAAFPNGSSVTLPHQVVDNP). This antibody has been shown to stain a broad band of approximately 200 kDa on Western blot analysis of mouse olfactory cilia and does not label tissue from ACIII knockout mice (71).

Further, preadsorption of diluted ACIII antibody with the peptide (Santa Cruz; catalog

No. sc-588P) prevented ciliary staining. The GFAP antibody (Sigma-Aldrich, St. Louis,

MO; clone G-A-5, catalog No. G-3893) is a mouse monoclonal antibody in ascites fluid.

It was derived from the hybridoma produced by the fusion of mouse myeloma cells and splenocytes from a mouse immunized with purified GFAP from pig spinal cord. The

GFAP antibody labeled only cells that were consistent with fibrillary astrocytes, based on morphology and distribution (Figure 3-5; see (84, 85)).

Perfusion/Fixation/Tissue Processing

Animals were anesthetized by a 0.1 ml/10 g intraperitoneal injection of 2.5% tribromoethanol (Sigma-Aldrich), sacrificed by cardiac puncture, and perfused with phosphate-buffered saline (PBS, pH 7.6) followed by 4% paraformaldehyde (PFA). The brains were then further fixed in 4% PFA for 16-24 hours at 4°C followed by 44 cryoprotection in 30% sucrose in PBS for 16-24 hours. A freezing microtome was used to generate 60 µm sections in both sagittal and transverse planes for peroxidase anti- peroxidase immunohistochemistry. For immunofluorescence procedures, cryoprotected brains were embedded in Optimal Cutting Temperature compound (VWR, West Chester,

PA), and sectioned in a cryostat at a thickness of 30 µm. All results were verified in at least three different animals.

Immunohistochemical Procedures

Floating sections were permeabilized with 0.3% Triton X-100 in PBS with 2% goat serum, and 10 mg/ml BSA (PBT). All incubations and washes were carried out in PBT.

Sections were incubated in anti-ACIII at 1:500 in PBT for 16-24 hours at 4°C with constant agitation. The sections were then rinsed in PBT and sequentially incubated in rabbit IgG (1:500 in PBT) and rabbit peroxidase anti-peroxidase (1:500 in PBT) for 1 hour each at room temperature with constant agitation. Following PBS rinses the sections were then processed using the glucose oxidase procedure (86). Sections were mounted on glass slides and coverslipped with Permount (Fisher Scientific, Pittsburgh,

PA).

Immunofluorescence Procedures

The ACIII antibody, made in a rabbit, was combined with the mouse monoclonal

GFAP antibody. Sections were permeabilized with 0.3% Triton X-100 in PBS with 2% goat serum, 0.02% sodium azide and 10 mg/ml BSA. All incubations and washes were carried out in PBS with 2% serum, 0.02% sodium azide and 10 mg/ml BSA. Anti-ACIII, used at 1:500, and anti-GFAP, used at 1:1000, were applied simultaneously to the tissue sections. Primary antibody incubations were carried out for 16-24 hours at 4°C and

45 secondary antibody incubations were carried out for 1 hour at room temperature.

Secondary antibodies included; Alexa Fluor 488-conjugated goat anti-rabbit IgG

(Invitrogen) and Alexa Fluor 546-conjugated goat anti-mouse IgG (Invitrogen). Nuclei were visualized by DRAQ5 (Axxora, San Diego, CA). Slides were mounted using Immu-

Mount (Fisher Scientific).

Data Analysis

PAP stained tissue sections were analyzed with a light microscope (Zeiss Axioskop), and images were captured with a digital camera (Axiocam, Zeiss) using Axiovision software (Zeiss). Either single or multiple consecutive focal planes, spaced at 0.5-1 µm intervals, (Z-stack) were captured. The Z-stacked images of tissue processed for the

PAP technique were further processed using algorithms included in a deconvolution program in the Axiovision® software. Overlapping low power images were assembled in

Photoshop. The contrast and brightness of all images was adjusted using either

Photoshop or the Axiovision software. Immunofluorescent samples were imaged on a

Zeiss 510 META laser scanning confocal microscope in the Ohio State University

Campus Microscopy and Imaging Facility. For all collected images, the brightness and contrast of each channel was adjusted using the Zeiss LSM Image Browser program.

46

FIGURE 3.1 HISTOCHEMICAL VISUALIZATION OF ACIII CILIARY LOCALIZATION IN THE ADULT MOUSE HIPPOCAMPUS. (A) REPRESENTATIVE IMAGE OF ACIII-IMMUNOREACTIVE CILIA IN THE PYRAMIDAL LAYER (PL) IN THE CA1 REGION. STRATUM ORIENS (SO). STRATUM RADIATUM (SR). (B) REPRESENTATIVE IMAGE OF ACIII-IMMUNOREACTIVE CILIA IN THE CA3 REGION. (C) REPRESENTATIVE IMAGE OF ACIII-IMMUNOREACTIVE CILIA IN THE GRANULAR LAYER (GL) IN THE DENTATE GYRUS (DG). HILUS (HI). (D) REPRESENTATIVE IMAGE OF ACIII- IMMUNOREACTIVE CILIA IN THE STRATUM ORIENS. (E) REPRESENTATIVE IMAGE OF ACIII- IMMUNOREACTIVE CILIA IN THE STRATUM RADIATUM. (F) REPRESENTATIVE IMAGE OF ACIII- IMMUNOREACTIVE CILIA IN THE HILUS. SCALE BARS ARE 20 µM.

47

FIGURE 3.2 HISTOCHEMICAL VISUALIZATION OF ACIII CILIARY LOCALIZATION IN REGIONS OF THE ADULT MOUSE FOREBRAIN. (A) SUBICULUM (SUB). (B) RETROSPLENIAL GRANULAR (RSG). (C) VISUAL CORTEX (VICX). (D) MOTOR CORTEX (MOCX). (E) ORBITAL CORTEX (ORCX). (F) OLFACTORY PLEXIFORM CELL LAYER (PCL). (G) OLFACTORY GRANULAR CELL LAYER (GCL). (H) OLFACTORY TUBERCLE (OT). (I) NUCLEUS ACCUMBENS (NA). J: CAUDATE/PUTAMEN (CP). (K) THALAMUS (TH). (L) HYPOTHALAMUS (HYP). SCALE BARS ARE 20 µM. 48

FIGURE 3.3 HISTOCHEMICAL VISUALIZATION OF ACIII CILIARY LOCALIZATION IN MEDIAL AND LATERAL REGIONS OF THE ADULT MOUSE BRAIN. (A) RETROSPLENIAL CORTEX (RSCX). (B) ECTORHINAL CORTEX (ECCX). (C) PIRIFORM CORTEX (PICX). (D) AMYGDALA (AMY). (E) DORSOMEDIAL HYPOTHALAMUS (DMHYP). (F) VENTROMEDIAL HYPOTHALAMUS (VMHYP). SCALE BARS ARE 20 µM.

49

FIGURE 3.4 HISTOCHEMICAL VISUALIZATION OF ACIII CILIARY LOCALIZATION IN REGIONS OF THE ADULT MOUSE BRAINSTEM. (A) SUBSTANTIA NIGRA (SN). (B) PONTINE (P). (C) SUPERIOR COLLICULI (SC). (D) INFERIOR COLLICULI (IC). (E) PERIAQUEDUCTAL GRAY (PG). (F) VESTIBULAR NUCLEUS (VN). (G) SPINAL TRIGEMINAL TRACT (SP5). (H) CEREBELLUM LOBULE VIII (CBLVIII). (I) CEREBELLUM LOBULE IX (CBLIX). SCALE BARS ARE 20 µM.

50

FIGURE 3.5 IMMUNOCYTOCHEMICAL VISUALIZATION OF CILIA PROJECTING FROM AN ASTROCYTE AND CHOROID PLEXUS CELLS IN THE ADULT MOUSE BRAIN. (A) IMAGE OF A CELL IN THE CEREBRAL CORTEX COLABELED WITH ANTIBODIES TO ACIII (GREEN) AND GFAP (RED). NUCLEI WERE LABELED WITH THE DNA STAIN DRAQ5 (BLUE). THE CILIUM IS INDICATED BY AN ARROW. SCALE BAR IS 5 µM. (B) IMAGE OF CHOROID PLEXUS IN THE LATERAL VENTRICLE LABELED WITH AN ANTIBODY TO ACIII (GREEN) AND STAINED WITH DRAQ5 (BLUE). CILIA ARE INDICATED BY ARROWS. SCALE BAR IS 10 µM.

51

Region of the Brain Relative Abundance of Cilia Cerebral Cortex, Forebrain & Diencephalon Motor cortex ++ Visual cortex ++ Auditory cortex ++ Entorhinal (Piriform) cortex +++ Retrosplenial granular cortex ++ Thalamus Medial geniculate + Lateral geniculate + Ventral posterior lateral - Ventral posterior medial +/- Posterior +/- Dorsomedial +/- Dorsolateral + Subthalamus + Hypothalamus (all regions) +++ Mammilary nucleus + Habenula ++ Hippocampus CA1 (pyramidal cell layer) +++ CA3 (pyramidal cell layer) +++ Dentate gyrus (granule cell layer) +++ Amygdala (all subnuclei) +++ Nucleus accumbens +++ Caudate/Putamen +++ Globus pallidus - Zona incerta + Substantia inominata ++

Continued Table 3.1 Distribution of ACIII Positive Cilia in the Mouse Brain

52 Region of the Brain Relative Abundance of Cilia Midbrain & Pons Substantia nigra ++ Ventral tegmental area + Periaqueductal gray (all regions) ++ Oculomotor (III) nucleus +/- Edinger-Westphal nucleus + Interstitial nucleus of Cajal + Nucleus of Darkschewitsch + Nucleus of posterior commissure + Retrorubral nucleus ++ Nucleus of lateral leminiscus +/- Peripeduncular nucleus ++ Pretectal nucleus +/- Red nucleus +/- Superior colliculus ++ Inferior colliculus ++ Lateral leminiscus +/- Dorsal raphe nucleus ++ Dorsomedial raphe +/- Facial nucleus +/- Pontine nuclei (basilar pons) ++ Peripeduncular nucleus + Locus coeruleus +/- Medulla & Cerebellum Cochlear nucleus + Vestibular nuclei (all regions) ++ Dorsal vagal nucleus +/- Spinal trigeminal nucleus ++ Inferior olive Medial accessory olive +/-

Continued Table 3.1 Distribution of ACIII Positive Cilia in the Mouse Brain

53 Region of the Brain Relative Abundance of Cilia Principal olivary nucleus - Reticular formation Paragigantocellular nucleus +/- Gigantocellular - Raphe nucleus - Cerebellar cortex Hemisphere +/- Flocculus/Paraflocculus + Vermis +/- Cerebellar nuclei +/- Table 3.1 Distribution of ACIII-Positive Cilia in the Mouse Brain Distribution of ACIII-immunoreactive cilia in the central nervous system. The relative number of ciliated cells in each region is designated by – (no ACIII-positive cilia are present in the region or nucleus), +/- (sparse distribution of ACIII-positive cilia), + (ACIII-positive cilia are observed on some cells), ++ (relatively more cells express ACIII-positive cilia), or +++ (most cells express ACIII-positive cilia).

54

CHAPTER 4:

IDENTIFICATION OF CILIARY LOCALIZATION

SEQUENCES WITHIN THE THIRD

INTRACELLULAR LOOP OF G PROTEIN-COUPLED

RECEPTORS

SUMMARY

Primary cilia are sensory organelles present on most mammalian cells. The functions of cilia are defined by the signaling proteins localized to the ciliary membrane.

Certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) and serotonin receptor 6 (Htr6), localize to cilia. As Sstr3 and Htr6 are the only somatostatin and serotonin receptor subtypes that localize to cilia, we hypothesized they contain ciliary localization sequences. To test this hypothesis we expressed chimeric 55 receptors containing fragments of Sstr3 and Htr6 in the non-ciliary receptors Sstr5 and

Htr7, respectively, in ciliated cells. We found the third intracellular loop of Sstr3 or Htr6 is

sufficient for ciliary localization. Comparison of these loops revealed a loose consensus

sequence. To determine whether this consensus sequence predicts ciliary localization of

other GPCRs, we compared it against the third intracellular loop of all human GPCRs.

We identified the consensus sequence in melanin-concentrating hormone receptor 1

(Mchr1) and confirmed Mchr1 localizes to primary cilia in vitro and in vivo. Thus, we

have identified a putative GPCR ciliary localization sequence and used this sequence to

identify a novel ciliary GPCR. As Mchr1 mediates feeding behavior and metabolism, our

results implicate ciliary signaling in the regulation of body weight.

BACKGROUND

Primary cilia are appendages that project from almost all human cell types (7). It

is generally accepted that primary cilia serve important specialized signaling functions

(62, 65, 74). In the eye, photoreceptors, which are modified primary cilia, sense and

respond to light. In the nose, specialized olfactory cilia detect odors and initiate signaling

cascades in olfactory neurons. In the kidney, it is proposed that bending of cilia on

epithelial cells by fluid flow triggers an increase in intracellular calcium mediated by an

ion channel located on the cilium (18, 19). In each case, the function of the cilium is

defined by the signaling proteins that are enriched in the ciliary membrane (i.e. light

receptors, odorant receptors, and mechanoreceptors). Importantly, disruption of the

signaling mediated by these receptors can cause disease and altered development (2,

63, 87, 88). Yet, the specific signaling proteins that localize to the vast majority of cilia in

the mammalian body are unknown. Thus, the functions of primary cilia on most cell

types in the body are unknown. 56 Neuronal primary cilia are abundant throughout the rodent brain (89). The functional importance of these cilia is suggested by the fact that several human ciliary disorders, including Bardet-Biedl syndrome (BBS), Joubert syndrome (JS), and Meckel syndrome (MKS), have prominent functional and structural CNS phenotypes (1).

Although the specific functions of neuronal cilia are unknown, certain G protein-coupled receptors (GPCRs), including somatostatin receptor 3 (Sstr3) (28, 61) and serotonin receptor 6 (Htr6) (60, 64), localize to the ciliary membrane on neurons. This observation suggests that cilia may sense levels of neuromodulators in the local environment of neurons. Given that the minority of the total ciliated neurons are positive for Sstr3 (79) and the distribution of Htr6 ciliary localization is restricted to a few regions of the brain

(60, 64), it is likely that additional GPCRs localize to neuronal cilia.

Although the ciliary membrane is continuous with the plasma membrane, only

certain GPCRs localize to cilia. This is clearly demonstrated by the somatostatin

receptors. There are five somatostatin receptor genes present in humans and rodents

(Sstr1-5) (69). In addition, the carboxy (C)-tail of Sstr2 can undergo alternative splicing

to yield Sstr2a and Sstr2b. They are all members of the GPCR superfamily of cell-

surface receptors that couple to heterotrimeric G-proteins and regulate numerous

downstream effectors. There are approximately 950 GPCRs in the human genome, with

500 of those coding for odorant or taste receptors and the remaining 450 coding for

receptors with endogenous ligands (90). All GPCRs share a common molecular topology

of seven transmembrane-spanning domains, three intracellular loops, three extracellular

loops, an amino (N)-terminus outside the cell, and a C-terminus inside the cell (91). The

six somatostatin receptor subtypes are expressed in the rat central nervous system and

show overlapping regional distributions (28). Yet, Sstr3 is selectively targeted to the

57 ciliary membrane (28, 61). Similarly, of the 13 mammalian G protein-coupled serotonin

receptor subtypes only Htr6 has been shown to localize to cilia (60, 64). These

observations suggest that Sstr3 and Htr6 contain unique sequences that mediate their

localization to cilia.

Very little is known about the underlying mechanisms of sorting and trafficking of

GPCRs to the cilium. One GPCR ciliary localization sequence that has been described is

an adjacent hydrophobic and basic residue motif immediately C-terminal to the seventh

transmembrane segment. In C. elegans, this sequence is required for ciliary localization

of olfactory GPCRs (25). In mammalian cells, this motif is required for the ciliary

localization of the GPCR Smoothened (21). Although this motif is present in Sstr3 and

Htr6, it is also present and conserved in all of the somatostatin and serotonin receptor

subtypes. Yet, only Sstr3 and Htr6 localize to cilia. Thus, this motif may be necessary

but it is not sufficient to specify ciliary localization.

We hypothesized that ciliary GPCRs contain unique sequences that mediate

localization to cilia. Here, we report that the third intracellular loop of Sstr3 and Htr6 is

sufficient to localize non-ciliary GPCRs to cilia, suggesting that Sstr3 and Htr6 are

targeted to cilia through similar mechanisms. Comparison of the sequences within these

loops reveals a putative consensus sequence. This consensus sequence is present in

the third intracellular loop of numerous known ciliary GPCRs and also identifies a

number of candidate ciliary GPCRs. We subsequently show that one of these

candidates, melanin-concentrating hormone receptor 1 (Mchr1), localizes to cilia in vitro

and in vivo. These findings identify a new role for cilia in MCH signaling and, as Mchr1 is involved in the regulation of energy homeostasis, implicate ciliary signaling in the regulation of body weight.

58 RESULTS

To determine whether heterologous expression could be used to identify GPCR

ciliary localization sequences, we generated constructs encoding mouse somatostatin

receptors one through five (Sstr1-5) fused at the C-terminus to enhanced green

fluorescent protein (EGFP). These constructs were then expressed in inner medullary

collecting duct (IMCD) cells, which are derived from mouse kidney and develop cilia in

culture. Visualization of the subcellular localization of the fluorescently labeled receptors

24-48 hours post transfection showed that of the somatostatin receptor subtypes, only

Sstr3 selectively localized to cilia (Figure 4.1 A-E). The remaining somatostatin receptor

subtypes failed to localize to cilia, as indicated by a lack of colocalization with the ciliary

marker acetylated α-tubulin, and instead localized to the plasma membrane or within

intracellular compartments. Similarly, transfection of IMCD cells with serotonin receptor

six (Htr6) and the closely related Htr7 fused at the C-terminus to EGFP revealed that

Htr6 preferentially localized to cilia while Htr7 localized primarily to the plasma

membrane (Figure 4.1 F and G). Interestingly, cilia on cells expressing Sstr3 or Htr6

consistently appeared longer than cilia on untransfected cells or on cells expressing non-

ciliary GPCRs (Figure 4.1 A-G). The underlying mechanism for this difference is

unknown. Overall, these results suggest that Sstr3 and Htr6 contain sequences that

specify ciliary localization and IMCD cells possess the necessary machinery to traffic

specific receptors to cilia.

To identify the region of Sstr3 containing ciliary localization sequences, we utilized a

fusion PCR approach to construct EGFP-fused chimeric receptors. Chimeras were

generated containing segments of Sstr3 and the structurally and pharmacologically

similar Sstr5. In order to minimize the likelihood of protein misfolding, the chimeric 59 receptor fusion sites were engineered at conserved residues located within the

transmembrane domains. Initially, we generated chimeric receptors of Sstr5 in which the

sequence from the N-terminus to the second, fourth, or sixth transmembrane (TM)

domains was substituted with the corresponding sequence from Sstr3 (Figure 4.2 A).

Expression of these chimeric receptors in IMCD cells revealed that only chimeric

receptor Sstr5[N-TM6Sstr3] selectively localized to cilia (Figure 4.2 B-D), suggesting that sequences between the TM4 and TM6 domains in Sstr3 mediate ciliary localization.

To test this hypothesis we generated a chimeric receptor of Sstr5 in which the sequence between the TM4 and TM6 domains was replaced with the corresponding sequence from Sstr3 (Figure 4.3 A). This chimera also localized to cilia (Figure 4.3 B), suggesting

that ciliary localization sequences are located within the third extracellular (e3) loop or

the third intracellular (i3) loop of Sstr3. To distinguish between these two possibilities, we

generated chimeric receptors of Sstr5 containing only the e3 or i3 loop of Sstr3 (Figure

4.3 A). Notably, chimeric receptor Sstr5[TM4-5Sstr3] did not localize to cilia (Figure 4.3

C) but chimeric receptor Sstr5[TM5-6Sstr3] did localize to cilia (Figure 4.3 D), indicating

that sequences within the i3 loop of Sstr3 are sufficient to localize Sstr5 to cilia.

Immunoblotting of proteins isolated from cells transiently transfected with chimeric

receptors Sstr5[TM4-5Sstr3] and Sstr5[TM5-6Sstr3] revealed similar expression patterns

(Figure 4.8), indicating that absence of cilia localization was not due to the lack of

receptor expression or stability.

To determine whether the i3 loop of Htr6 also mediates ciliary localization, we

generated a chimeric receptor in which the i3 loop of Htr7 had been substituted with the

i3 loop of Htr6 (Figure 4.4 A). Notably, this chimeric receptor selectively localized to cilia

(Figure 4.4 B). This result indicates that Sstr3 and Htr6 contain ciliary localization

60 sequences within the same domain and suggest that they may be targeted to cilia

through similar mechanisms. As the predicted i3 loop of Htr6 (63 residues) is

significantly larger than the i3 loop of Sstr3 (36 residues), we further narrowed the region

of Htr6 sufficient to traffic Htr7 to the cilium by generating chimeric receptors containing

only the N-portion or C-portion of the i3 loop (Figure 4.4 A). V241 in Htr6, which

corresponds to V295 in Htr7, was used as the fusion site. Chimeric receptor Htr7[TM5-

V241Htr6] selectively localized to cilia (Figure 4.4 C) but chimeric receptor Htr7[V241-

TM6Htr6] did not (Figure 4.4 D), indicating that sequences within the N-portion of the i3

loop of Htr6 are sufficient to localize Htr7 to cilia.

We hypothesized that ciliary localization sequences would be unique to the receptor

subtype that localizes to cilia, would be conserved in species in which the receptor is

ciliary, and would be similar between Sstr3 and Htr6. Comparison of the i3 loop

sequences of the mouse somatostatin receptors reveals similarity between Sstr3 and the

other subtypes, except for several residues in the N-portion and an insertion of eleven

amino acids that are

unique to Sstr3 (Figure 4.5 A). To further narrow the sequences of interest we compared the i3 loop sequences of Sstr3 across species. Comparison of the mouse and human sequences reveals that the unique residues in the N-portion of the loop are not conserved, but five of the eleven amino acids inserted in mouse Sstr3 (APSCQ) are completely conserved in human SSTR3 (Figure 4.5 B). Given that human SSTR3 also localizes to cilia when expressed in IMCD cells (data not shown), this suggested that residues within “APSCQ” might confer ciliary localization.

We then examined the N-portion of the i3 loop of Htr6 and identified a sequence

(ATAGQ) with modest similarity that is not present in Htr7 (Figure 4.5 C). As the A and 61 Q at the ends of the sequence are identical between Sstr3 and Htr6, we performed site- directed mutagenesis on these residues to verify they are important for ciliary localization. The A and Q residues in chimeric receptor Sstr5[TM5-6Sstr3] were mutated to phenylalanine and ciliary localization was then quantified in IMCD cells expressing

Sstr3, Sstr5, Sstr5[TM5-6Sstr3], or Sstr5[TM5-6Sstr3mut]. Sstr3 localized to cilia in approximately 91% of transfected cells, while Sstr5 never localized to cilia (Figure 4.5

D). Chimeric receptor Sstr5[TM5-6Sstr3] showed a similar percentage of cilia localization

(~93%) as Sstr3 (Figure 4.5 D and Figure 4.9 A). The mutated version of this chimera

(Sstr5[TM5-6Sstr3mut1]) showed a dramatic reduction in the percentage of ciliary localization but still localized to cilia in ~50% of cells (Figure 4.5 D). However, the i3 loop of mouse Sstr3 contains a second ciliary localization consensus sequence (APACQ;

Figure 4.5 B). To test whether the second site was contributing to ciliary localization we mutated the A and Q residues to F in Sstr5[TM5-6Sstr3mut1] and quantified ciliary localization. Notably, when all four residues were mutated in this chimera (Sstr5[TM5-

6Sstr3mut2]) ciliary localization was reduced to ~6% of cells (Figure 4.5 D and Figure

4.9 B). Immunoblotting of proteins isolated from transiently transfected cells expressing chimera Sstr5[TM5-6Sstr3mut2] confirmed that the mutant chimeric receptor was expressed (Figure 4.8).

Similarly, the A and Q residues in chimeric receptor Htr7[TM5-V241Htr6] were mutated to phenylalanine and ciliary localization was then quantified in IMCD cells expressing Htr6, Htr7, Htr7[TM5-V241Htr6], or Htr7[TM5-V241Htr6mut]. Htr6 localized to cilia in approximately 93% of transfected cells, while Htr7 localized to cilia in approximately 20% of transfected cells (Figure 4.5 E). Of note, ciliary localization of Htr7 was almost exclusively seen in cells that were expressing the receptor at a very high

62 level. Chimeric receptor Htr7[TM5-V241Htr6] localized to cilia in approximately 70% of

transfected cells (Figure 5 E and Figure 4.9 C). Remarkably, the mutated version of this

chimera (Htr7[TM5-V241Htr6mut]) almost never localized to cilia (~4%) (Figure 4.5 E

and Figure 4.9 D). Together, these results indicate that the A and Q residues in the i3

loop of Sstr3 and Htr6 are important for ciliary localization.

To test whether these sequences could be used to predict novel ciliary GPCRs, we formulated a consensus sequence based on Sstr3 and Htr6. As serine and alanine belong to a "strong" Gonnet Pam250 matrix conservation group (92), we formulated the following loose consensus sequence (AX[S/A]XQ), where position 1 is an A, position 5 is a Q, position 3 is an S or A, and positions 2 and 4 are any amino acid. As the ciliary localization sequences were identified within the i3 loop of both Sstr3 and Htr6, we searched for the consensus sequence in a database containing the sequences of the predicted i3 loop of all human GPCRs. The consensus sequence is present in the i3 loop of 11 additional GPCRs (Table 4.1). Remarkably, these 11 GPCRs include 3 odorant receptors, 3 cone opsins, and rhodopsin, all of which are ciliary proteins. The remaining four GPCRs (alpha-2A adrenergic receptor, chemokine orphan receptor 1, melanin- concentrating hormone receptor 1, and muscarinic acetylcholine receptor M5) have not been shown to localize to cilia, and were classified as candidate ciliary GPCRs. As melanin-concentrating hormone receptor 1 (Mchr1) is involved in the regulation of feeding behavior and energy balance (93) and multiple human ciliary disorders are associated with obesity (1), Mchr1 was considered a particularly good candidate ciliary

GPCR.

To test whether Mchr1 localizes to cilia, we cloned the coding sequence of Mchr1 from mouse cDNA, fused it to EGFP, expressed it in IMCD cells, and assessed

63 subcellular localization. Notably, Mchr1 localized to cilia when expressed in IMCD cells

(Figure 4.6 A). We then tested whether Mchr1 localizes to cilia in tissue by labeling

mouse brain sections with an antibody to Mchr1. We detected ciliary localization of

Mchr1 in several brain regions (Figure 4.6 B), including the hippocampus, nucleus accumbens, olfactory bulb, and hypothalamus. Ciliary localization was confirmed by colabeling with adenylyl cyclase III, which is a marker of neuronal cilia (79, 89). These results indicate, for the first time, that Mchr1 localizes to cilia in vitro and in vivo.

DISCUSSION

It is presumed that ciliary membrane proteins are synthesized in the endoplasmic

reticulum, trafficked through the Golgi, and then transported in post-Golgi vesicles to the

base of the cilium (94). As access to the cilium is restricted and only certain proteins

localize to the ciliary membrane (94), it is thought that ciliary proteins contain targeting

signals that direct them to the cilial compartment (11). Few mammalian ciliary

localization sequences have been described (9, 15, 21, 23, 95) and only one sequence

has been shown to be sufficient for ciliary targeting in mammalian cells. The N-terminal

15 amino acids of the ciliary cation channel polycystin-2 are sufficient to localize

heterologous proteins to cilia (9). Within this domain is a conserved motif, RVXP, that is

required for ciliary localization (9). This motif is also found at the C-terminus of olfactory

cyclic nucleotide-gated channel 1b and is required for trafficking of the channel into cilia

(15). This motif is not found in Sstr3 or Htr6, but is found in the i3 loop of Htr7, which

does not localize to cilia, suggesting this motif is not functional in Htr7 or is not involved

in the targeting of GPCRs to cilia.

64 In this work, we show that sequences within the i3 loop of Sstr3 and Htr6 are sufficient to localize non-ciliary GPCRs to cilia on IMCD cells. Notably, we see the same subcellular localization results when we express the constructs in cultured primary hippocampal neurons (data not shown). This suggests that the trafficking machinery for ciliary receptors is conserved between neurons and IMCD cells and supports the utility of this system for identifying the mechanisms of ciliary protein sorting and trafficking.

Interestingly, replacing the i3 loop in Sstr3 and Htr6 with the i3 loop from Sstr5 and Htr7, respectively, does not prevent ciliary localization (data not shown). Thus, although the i3 loops are sufficient to localize non-ciliary GPCRs to cilia, they are not necessary for ciliary localization of Sstr3 and Htr6. This is an intriguing result that suggests there are additional ciliary localization sequences within Sstr3 and Htr6.

The i3 loops of GPCRs, including somatostatin and serotonin receptors, are normally associated with G protein coupling and desensitization (96-98). This is the first time the i3 loop has been implicated in GPCR ciliary localization. Most known GPCR trafficking motifs are located within the C-terminus (91). However, the i3 loop of mouse vasopressin V2 receptor contains two RXR ER retention motifs that are thought to be masked under normal conditions but can be unmasked in mutant receptors and block trafficking to the plasma membrane (99). An RXR motif is present in the i3 loop of Sstr3 and Sstr5 but neither Htr6 nor Htr7 contain an RXR motif in the i3 loop. Thus, it is unlikely that ER retention is a mechanism for affecting ciliary localization in our system.

The i3 loop of many GPCRs also contains a loose consensus sequence (BBXXB), where B is a basic residue and X is a non-basic residue, at the junction of the i3 loop and the TM6 domain that is important for GPCR structure and activity (100, 101). This consensus sequence is present in the C-terminal end of the i3 loop of Sstr3, Sstr5, Htr6,

65 and Htr7, and is required for Htr6 activity (102). However, it is likely unrelated to ciliary localization given the ciliary localization domain in Htr6 maps to the N-portion of the i3 loop.

Comparison of the sequences within the i3 loops of Sstr3 and Htr6 reveals a consensus sequence (AX[S/A]XQ) that may comprise a ciliary localization sequence. In support of this idea, the consensus sequence is also present in the i3 loop of Mchr1, which we have shown localizes to cilia. Further, mutating the A and Q residues in chimeric receptor Htr7[TM5-V241Htr6] decreases ciliary localization from ~70% of cells to ~4%. Mutating the A and Q residues in both consensus sequences in chimeric receptor Sstr5[TM5-6Sstr3] decreases ciliary localization from ~93% of cells to ~6%. It is interesting that Htr7 localizes to cilia in ~20% of cells, whereas Sstr5 never localizes to cilia. This may be due to the fact that Sstr5 appears to be associated primarily with intracellular structures (Figure 4.1 E), as opposed to Htr7 that appears to localize mainly to the plasma membrane (Figure 4.1 G). It is likely that high levels of heterologously expressed proteins can overcome restrictions on ciliary localization when they are on the plasma membrane. Indeed, cotransfecting Htr7 with an empty expression vector

(pcDNA3.1) at a ratio of 1:4, to lower the level of Htr7 expression, reduces ciliary localization of Htr7 to 6% of cells (data not shown).

Comparison of our consensus ciliary localization sequence against the i3 loop sequence of all human GPCRs identified known ciliary GPCRs and four candidate ciliary

GPCRs. We have confirmed that one of these candidates, Mchr1, localizes to cilia. The consensus sequences of the remaining three candidates are present in the human protein but are not completely conserved in lower organisms. An exciting possibility is ciliary localization of some GPCRs may have evolved only in higher organisms. The

66 development of ciliary localization would potentially be a mechanism for organisms to create additional complexity and more specialized functions in existing signaling pathways. Another possibility is our consensus sequence is too stringent and the sequences in lower organisms correspond to ciliary localization sequences. In support of this possibility, the ciliary localization sequence in human HTR6 contains a G rather than an A at position 1, suggesting there is flexibility to the consensus sequence. We also hypothesized that localization of the sequence within the i3 loop was a requirement for ciliary localization and limited our search to the i3 loop. The amount of flexibility in the consensus sequence and its location within receptors could potentially increase the number of candidate ciliary GPCRs dramatically. It will be necessary to experimentally determine what residues are required and permissive and whether the consensus sequence can be present in other domains in order to accurately predict all ciliary

GPCRs.

Although the ciliary localization sequence we identified is common to Sstr3, Htr6, and

Mchr1, it is not present in Smo, despite the fact that Smo localizes to cilia in vitro and in vivo (21). One possibility is that Smo is targeted to the cilium through a different mechanism. Indeed, substitution of the hydrophobic-basic residues in the C-terminal tail of Smo with alanines prevents ciliary localization (21) but mutation of this motif in Sstr3 or Htr6 does not prevent ciliary localization (N. F. Berbari and K. M. Mykytyn, unpublished results). Perhaps there are divergent mechanisms of ciliary trafficking between the different GPCR families. Sstr3, Htr6, and Mchr1 are members of the

Rhodopsin family of GPCRs while Smo is a member of the Frizzled/Smoothened family of GPCRs. Several studies have found that defects in ciliary protein transport can be

67 associated with mislocalization of some membrane proteins but not others (13, 24-26),

suggesting there are multiple pathways for targeting membrane proteins to the cilium.

An important outcome from these studies is the demonstration that Mchr1

localizes to neuronal cilia in regions of the mouse brain, including the hypothalamus. A

role for melanin-concentrating hormone (MCH) and its receptor in the regulation of

feeding and metabolism is well established. Specifically, injection of MCH into the brains

of mice induces a rapid increase in feeding behavior (103) while injection of Mchr1

antagonists reduces feeding behavior (104). Further, transgenic mice overexpressing

MCH (105) are obese and mice lacking expression of either MCH (106) or Mchr1 (107)

are lean. The fact that Mchr1 localizes to cilia in regions of the brain known to regulate

feeding behavior suggests that localization of Mchr1 to cilia may be important for

signaling through the receptor and proper regulation of these processes. Interestingly,

mice lacking cilia in the brain or specifically on pro-opiomelanocortin-expressing cells in

the hypothalamus are hyperphagic and become obese (108). Further, obesity is a

hallmark of some human ciliary disorders. The role of cilia in this phenotype in particular

has been a long standing mystery. The identification of Mchr1 as a ciliary GPCR

provides, for the first time, a potential molecular mechanism to link defects in cilia with

obesity.

MATERIALS AND METHODS

The mice used in this study were on a 129:BL6 background. All procedures were

approved by the Institutional Animal Care and Use Committee at The Ohio State

University. Animals were anesthetized by a 0.1 ml/10 g intraperitoneal injection of 2.5%

tribromoethanol (Sigma-Aldrich, St. Louis, MO), sacrificed by cardiac puncture, and

68 perfused with phosphate-buffered saline (PBS) followed by a mixture of 2% paraformaldehyde and HistoChoice (Amresco, Solon, OH). The brains were then further fixed in 2% PFA/HistoChoice for 16-24 hours at 4°C followed by cryoprotection in 30% sucrose in PBS for 16-24 hours. Cryoprotected brains were embedded in Optimal

Cutting Temperature compound (VWR, West Chester, PA), and sectioned in a cryostat at a thickness of 30 µm. Labelings were performed in three different animals.

Plasmid Construction

The coding sequences of the somatostatin receptors were amplified from mouse genomic DNA. The unspliced version of Sstr2 that was amplified corresponds to Sstr2a.

The coding sequences of serotonin receptors 6 and 7 and melanin-concentrating hormone receptor 1 were amplified from reverse-transcribed mouse whole brain RNA using the Superscript First-Strand Synthesis RT-PCR Kit (Invitrogen, Carlsbad, CA). All coding sequences were cloned into a TA cloning vector (pSTBlue-1; Novagen, San

Diego, CA). Receptor open reading frames were utilized as templates for overlap extension PCR to generate chimeric receptors, as previously described (109). Briefly, oligonucleotide primers (36mers; IDT, Coralville, IA) corresponding to the nucleotide sequence of the junction regions were generated with 5’ nucleotide sequence from one receptor and 3’ sequence from the other. The junctions were designed at conserved residues, the coding frames were maintained, and no sequences were inserted or deleted. Primers corresponding to the N-terminal and C-terminal regions of both receptors were designed with 5’ restriction sites for directional cloning. The initial PCR generated fragments of the two receptors to be combined with complementary overhanging sequence. The products from the first reaction were gel purified and combined as template for the fusion PCR. All amplifications were performed with HiFi

69 Platinum Taq Polymerase (Invitrogen). The final PCR products were cloned into the

pEGFP-N vector (Clontech, Mountain View, CA) using the added restriction enzyme

sites. All DNA constructs were sequence verified. Primer sequences are available upon

request. Chimeric receptor sequences were mutated using the QuikChange Site

Directed Mutagenesis Kit (Stratagene, La Jolla, CA).

Cell Culture and Transient Transfections

IMCD-3 cells (ATCC, Manassas, VA) were maintained in DMEM:F12 media

supplemented with 10% FBS, 1.2 g/L of sodium bicarbonate, and 0.5 mM sodium

pyruvate (Invitrogen). 5×106 cells were electroporated with 10 µg of DNA and plated at

high density on glass coverslips. Cells were harvested 24 hours post-transfection for

immunocytochemistry.

Immunofluorescence Procedures

Cells were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100

in PBS with 2% goat serum, 0.02% sodium azide and 10 mg/ml bovine serum albumin

(BSA). The IMCD cells were then labeled with anti-acetylated α-tubulin (T-6793; Sigma-

Aldrich). Brain sections were permeabilized with 0.3% Triton X-100 in PBS with 2% donkey serum, 0.02% sodium azide and 10 mg/ml BSA and simultaneously labeled with anti-adenylyl cyclase III (ACIII) rabbit polyclonal antibody (sc-588; Santa Cruz, Santa

Cruz, CA), used at 1:500, and anti-melanin-concentrating hormone receptor 1 (sc-5534;

Santa Cruz), used at 1:250. All incubations and washes were carried out in PBS with 2% serum, 0.02% sodium azide and 10 mg/ml BSA. 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. Secondary antibodies included; Alexa Fluor-546

70 conjugated goat anti-mouse IgG (Invitrogen), Alexa Fluor 488-conjugated donkey anti-

goat IgG (Invitrogen), or Cy3-conjugated donkey anti-rabbit IgG (Jackson

ImmunoResearch, West Grove, PA). Nuclei were visualized by DRAQ5 (Axxora, San

Diego, CA). Slides were mounted using Immu-Mount (Fisher Scientific, Pittsburgh, PA).

Data Analysis

All samples were imaged on a Zeiss 510 META laser scanning confocal microscope at the Ohio State University Central Microscopy Imaging Facility. Multiple consecutive focal planes (Z-stack), spaced at 0.5-1 µm intervals, were captured. For all collected images, the brightness and contrast of each channel was adjusted using the Zeiss LSM

Image Browser program. For quantitative analysis, coverslips were centered on the microscope objective and 3-5 consecutive fields were imaged. The number of ciliated transfected cells and the number of transfected cells expressing cilia localization in each image were counted by individuals blinded to the experimental conditions. The results are expressed as the percent of transfected cells showing ciliary localization. A total of approximately 100 cells were counted from three distinct transfections (n = 3) for each construct. Data are expressed as mean + Standard Error of the Mean (SEM).

Prediction of Human GPCR Third Intracellular Loop Sequences

Human GPCR protein sequences were downloaded from the GPCRDB information

system (http://www.gpcr.org/7tm/) (110). The protein sequences were input into the transmembrane hidden Markov model (TMHMM) program (version 2.0) to generate predictions of transmembrane, intracellular, and extracellular sequences (111). A Perl program was then applied to the TMHMM output to extract the predicted third intracellular loop sequence for all human GPCRDB proteins. The resulting database was

71 searched using the ambiguous regular expression “AX[S/A]XQ” to identify GPCRs predicted to contain this motif within their third intracellular loop (Table 4.1).

72

FIGURE 4.1 SOMATOSTATIN RECEPTOR SUBTYPE 3 AND SEROTONIN RECEPTOR SUBTYPE 6 SELECTIVELY LOCALIZE TO CILIA WHEN HETEROLOGOUSLY EXPRESSED IN IMCD CELLS. (A-G) REPRESENTATIVE CONFOCAL MICROSCOPY IMAGES OF TRANSIENTLY TRANSFECTED IMCD CELLS EXPRESSING SOMATOSTATIN RECEPTORS 1 THROUGH 5 (SSTR1-5) AND SEROTONIN RECEPTORS 6 (HTR6) AND 7 (HTR7) FUSED AT THE C- TERMINUS TO EGFP. (LEFT PANELS) EGFP FLUORESCENCE (GREEN) SHOWS EXPRESSION OF THE RECEPTORS; (MIDDLE PANELS) ACETYLATED Α-TUBULIN (RED) MARKS THE CILIA; (RIGHT PANELS) MERGED IMAGES. BELOW EACH PANEL IS A CONFOCAL IMAGE IN THE XZ PLANE TO SHOW CILIA PROJECTING VERTICALLY FROM THE APICAL SURFACE OF CELLS. NOTE THAT ONLY SSTR3-EGFP (C) AND HTR6-EGFP (F) SHOW LOCALIZATION THAT OVERLAPS WITH ACETYLATED-Α TUBULIN CILIARY LABELING. SSTR2 CORRESPONDS TO SSTR2A. NUCLEI ARE LABELED WITH THE DNA STAIN DRAQ5 (BLUE). ALL SCALE BARS, 10 µM.

73

FIGURE 4.2 SEQUENCES BETWEEN THE FOURTH AND SIXTH TRANSMEMBRANE DOMAINS OF SSTR3 ARE IMPORTANT FOR CILIARY LOCALIZATION. (A) SCHEMATIC OF CHIMERIC RECEPTORS CONTAINING PORTIONS OF SSTR3 (INDICATED BY BLACK LINES) AND SSTR5 (INDICATED BY WHITE LINES) FUSED AT THE C-TERMINUS TO EGFP. TRANSMEMBRANE DOMAINS (TM) ARE DEPICTED AS BOXES. (B-D) REPRESENTATIVE IMAGES OF TRANSIENTLY TRANSFECTED IMCD CELLS EXPRESSING THE INDICATED CHIMERIC RECEPTORS. (LEFT PANELS) EGFP FLUORESCENCE (GREEN); (MIDDLE PANELS) ACETYLATED Α-TUBULIN (RED); (RIGHT PANELS) MERGED IMAGES. CHIMERIC RECEPTORS SSTR5[N-TM2SSTR3] (B) AND SSTR5[N-TM4SSTR3] (C) DO NOT LOCALIZE TO CILIA. CHIMERIC RECEPTOR SSTR5[N-TM6SSTR3] DOES LOCALIZE TO CILIA, SUGGESTING THAT CILIARY LOCALIZATION OF SSTR3 IS MEDIATED BY SEQUENCES BETWEEN TM4 AND TM6. NUCLEI ARE LABELED WITH DRAQ5 (BLUE). ALL SCALE BARS, 10 µM. 74

FIGURE 4.3 THE THIRD INTRACELLULAR (I3) LOOP OF SSTR3 IS SUFFICIENT TO LOCALIZE SSTR5 TO CILIA. (A) SCHEMATIC OF CHIMERIC RECEPTORS CONTAINING PORTIONS OF SSTR3 (BLACK LINES) AND SSTR5 (WHITE LINES) FUSED AT THE C-TERMINUS TO EGFP. TM DOMAINS ARE DEPICTED AS BOXES. (B-D) REPRESENTATIVE IMAGES OF TRANSIENTLY TRANSFECTED IMCD CELLS EXPRESSING THE INDICATED CHIMERIC RECEPTORS. (LEFT PANELS) EGFP FLUORESCENCE (GREEN); (MIDDLE PANELS) ACETYLATED Α-TUBULIN (RED); (RIGHT PANELS) MERGED IMAGES. CHIMERIC RECEPTORS SSTR5[TM4-6SSTR3] (B) AND SSTR5[TM5-6SSTR3] (D) LOCALIZE TO CILIA, WHILE CHIMERIC RECEPTOR SSTR5[TM4- 5SSTR3] (C) DOES NOT LOCALIZE TO CILIA, SUGGESTING THAT THE I3 LOOP OF SSTR3 CONTAINS CILIARY LOCALIZATION SEQUENCES. NUCLEI ARE LABELED WITH DRAQ5 (BLUE). ALL SCALE BARS, 10 µM.

75

FIGURE 4.4 THE AMINO PORTION OF THE I3 LOOP OF HTR6 IS SUFFICIENT TO LOCALIZE HTR7 TO CILIA. (A) SCHEMATIC OF CHIMERIC RECEPTORS CONTAINING PORTIONS OF HTR6 (BLACK LINES) AND HTR7 (WHITE LINES) FUSED AT THE C-TERMINUS TO EGFP. TM DOMAINS ARE DEPICTED AS BOXES. (B-D) REPRESENTATIVE IMAGES OF TRANSIENTLY TRANSFECTED IMCD CELLS EXPRESSING THE INDICATED CHIMERIC RECEPTORS. (LEFT PANELS) EGFP FLUORESCENCE (GREEN); (MIDDLE PANELS) ACETYLATED Α-TUBULIN (RED); (RIGHT PANELS) MERGED IMAGES. CHIMERIC RECEPTORS HTR7[TM5-6HTR6] (B) AND HTR7[TM5-V241HTR6] (C) SELECTIVELY LOCALIZE TO CILIA, WHILE CHIMERIC RECEPTOR HTR7[V241-TM6HTR6] (D) DOES NOT, SUGGESTING THAT THE N-PORTION OF THE I3 LOOP OF HTR6 CONTAINS CILIARY LOCALIZATION SEQUENCES. NUCLEI ARE LABELED WITH DRAQ5 (BLUE). ALL SCALE BARS, 10 µM.

76

FIGURE 4.5 COMPARATIVE GENOMICS IDENTIFIES UNIQUE RESIDUES IN SSTR3 AND HTR6 THAT ARE POTENTIALLY IMPORTANT FOR CILIARY LOCALIZATION OF GPCRS. (A) ALIGNMENT OF THE PREDICTED I3 LOOP SEQUENCES OF THE MOUSE SOMATOSTATIN RECEPTORS REVEALS AN INSERTION OF ELEVEN UNIQUE RESIDUES IN SSTR3. THE CORRESPONDING POSITIONS OF THE RESIDUES ARE INDICATED. RED SIGNIFIES IDENTICAL RESIDUES AND BLUE SIGNIFIES CONSERVED RESIDUES. (B) ALIGNMENT OF THE PREDICTED I3 LOOP SEQUENCE OF SOMATOSTATIN RECEPTOR SUBTYPE 3 FROM MOUSE (SSTR3) AND HUMAN (SSTR3). FIVE OF THE UNIQUE ELEVEN RESIDUES (APSCQ; BOXED) ARE COMPLETELY CONSERVED BETWEEN MOUSE AND HUMAN. (C) ALIGNMENT OF THE PREDICTED I3 LOOP SEQUENCES OF MOUSE SEROTONIN RECEPTORS 6 AND 7 SHOWS THE PRESENCE OF A UNIQUE SEQUENCE IN HTR6 (ATAGQ; BOXED) WITH MODEST SIMILARITY TO THE SSTR3 SEQUENCE.

77

FIGURE 4.6 QUANTITATION OF MUTATIONAL ANALYSIS CONFIRMS RESIDUES THAT ARE POTENTIALLY IMPORTANT FOR CILIARY LOCALIZATION OF GPCRS. (A)Percentage of transiently transfected IMCD cells that show ciliary localization when expressing Sstr3, Sstr5, Sstr5[TM5-6Sstr3], Sstr5[TM5-6Sstr3mut1], or Sstr5[TM5- 6Sstr3mut2]. Sstr3 localizes to cilia in ~91% of transfected cells. Sstr5 never localizes to cilia. Chimeric receptor Sstr5[TM5-6Sstr3] localizes to cilia in ~93% of transfected cells. Chimeric receptor Sstr5[TM5-6Sstr3mut1], in which the A and Q in the conserved consensus sequence have been mutated to F, localizes to cilia in ~50% of transfected cells. Chimeric receptor Sstr5[TM5-6Sstr3mut2], in which the A and Q in the second consensus sequence have also been mutated to F, localizes to cilia in ~6% of transfected cells. Values are expressed as mean ± SEM. *Significantly different from Sstr3 and Sstr5[TM5-6Sstr3] percentages. (B) Percentage of transiently transfected IMCD cells that show ciliary localization when expressing Htr6, Htr7, Htr7[TM5-V241Htr6], or Htr7[TM5-V241Htr6mut]. Htr6 localizes to cilia in ~93% of transfected cells. Htr7 localizes to cilia in ~20% of transfected cells. Chimeric receptor Htr7[TM5-V241Htr6] localizes to cilia in ~70% of transfected cells. Chimeric receptor Htr7[TM5-V241Htr6mut], in which the A and Q have been mutated to F, localizes to cilia in ~4% of transfected cells. Values are expressed as mean ± SEM. *Significantly different from Htr6 and Htr7[TM5-V241Htr6] percentages.

78

FIGURE 4.7 MELANIN CONCENTRATING HORMONE RECEPTOR 1 (MCHR1) LOCALIZES TO CILIA IN VITRO AND IN VIVO. (A) Representative image of transiently transfected IMCD cells expressing Mchr1 fused at the C-terminus to EGFP. (Left panels) EGFP fluorescence (green); (middle panels) acetylated α-tubulin (red); (right panels) merged images. (B) Representative image of the nucleus accumbens from an adult mouse colabeled with antibodies to Mchr1 (green; left panel), ACIII (red; middle panel), and merged (right panel). The majority of cilia are positive for both Mchr1 and ACIII. Cilia that are positive for ACIII but negative for Mchr1 are indicated with arrows. Nuclei are labeled with DRAQ5 (blue). All scale bars, 10 µm.

79

FIGURE 4.8 IMMUNOBLOT OF WHOLE PROTEIN LYSATES FROM IMCD CELLS TRANSIENTLY TRANSFECTED WITH SSTR5[TM4-5SSTR3] (A), SSTR5[TM5-6SSTR3] (B), AND SSTR5[TM5-6SSTR3MUT2] (C) REVEALS SIMILAR EXPRESSION OF EACH CHIMERIC RECEPTOR. PROTEINS WERE PROBED WITH ANTIBODIES TO GFP (UPPER PANEL) AND ACTIN (LOWER PANEL), AS A LOADING CONTROL.

80

FIGURE 4.9 MUTATIONAL ANALYSIS INDICATES THAT THE A AND Q ARE IMPORTANT FOR CILIARY LOCALIZATION OF CHIMERIC RECEPTORS SSTR5[TM5-6SSTR3] AND HTR7[TM5- V241HTR6]. Representative images of transiently transfected IMCD cells expressing Sstr5[TM5- 6Sstr3] (A), or Sstr5[TM5-6Sstr3mut2] (B), in which the A and Q in both consensus sequences have been mutated to F, fused at the C-terminus to EGFP. (Left panels) EGFP fluorescence (green); (middle panels) acetylated α-tubulin (red); (right panels) merged images. Ciliary localization is rarely seen in cells expressing Sstr5[TM5- 6Sstr3mut2] (B). Representative images of transiently transfected IMCD cells expressing Htr7[TM5-V241Htr6] (C), or Htr7[TM5-V241Htr6mut] (D), in which the A and Q have been mutated to F, fused at the C-terminus to EGFP. (Left panels) EGFP fluorescence (green); (middle panels) acetylated α-tubulin (red); (right panels) merged images. Ciliary localization is rarely seen in cells expressing Htr7[TM5- V241Htr6mut] (D). Nuclei are labeled with DRAQ5 (blue). All scale bars, 10 µm.

81

G Protein-Coupled Receptor I3 Domain Consensus Localization Sequence Olfactory receptor 52N1 ADARQ Ciliary Olfactory receptor 52N4 ADARQ Ciliary Olfactory receptor 6V1 ASSCQ Ciliary Opsin 1 (short-wave-sensitive 2) AVAAQ Ciliary Opsin 1 (medium-wave-sensitive 2) AVAKQ Ciliary Opsin 1 (long-wave-sensitive) AVAKQ Ciliary Rhodopsin AAAQQ Ciliary Alpha-2A adrenergic receptor ARASQ ? Melanin-concentrating hormone APAEQ ? receptor 1 Muscarinic acetylcholine receptor AKAEQ ? M5 Chemokine orphan receptor 1 ASSDQ ?

TABLE 4.1 G PROTEIN-COUPLED RECEPTORS IDENTIFIED BY THE CILIARY LOCALIZATION CONSENSUS SEQUENCE Human G protein-coupled receptors that contain the putative ciliary localization sequence within the predicted i3 domain. The consensus sequences are shown with the obligatory residues indicated in bold. Known ciliary GPCRs are indicated.

82

CHAPTER 5:

BARDET-BIEDL SYNDROME PROTEINS ARE

REQUIRED FOR THE LOCALIZATION OF G

PROTEIN-COUPLED RECEPTORS TO PRIMARY

CILIA

SUMMARY

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet-Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors (GPCRs) to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the

83 Bbs2 or Bbs4 gene. As Mchr1 is involved in the regulation of feeding behavior, and BBS

is associated with hyperphagia-induced obesity, our results suggest that altered

signaling due to mislocalization of ciliary signaling proteins underlies the BBS

phenotypes. Our results also provide, for the first time, a potential molecular mechanism

to link cilia defects with obesity.

BACKGROUND

Cilia are microtubule-based appendages that extend from the basal bodies of cells

and are classified as either motile or primary. Motile cilia and flagella are responsible for

generating flow or movement. Primary cilia are generally immotile solitary organelles that

are present on almost all human cell types (7). It is generally accepted that primary cilia

serve important specialized signaling functions (22, 65, 74, 87). Photoreceptors, which

are modified primary cilia, sense and respond to light. Specialized olfactory cilia detect

odors and initiate signaling cascades in olfactory neurons. Primary cilia on epithelial cells

in the kidney act as mechanosensors to detect and respond to fluid flow (18, 19). The

significance of primary cilia is exemplified by the fact that defects in cilia formation or

function cause diseases, including renal cystic disease, retinal degeneration, liver

fibrosis, anosmia, ataxia, cardiac defects, and situs inversus (2, 63). Primary cilia also

serve important roles in the patterning of tissues during development (2, 22), and

primary cilia dysfunction is thought to underlie the etiology of numerous human genetic

disorders (112). Yet, the specific role of primary cilia on the vast majority of cells is

unknown.

Bardet-Biedl syndrome (BBS) is a rare human genetic disorder characterized by

obesity, retinal dystrophy, renal anomalies, hypogenitalism, polydactyly, and cognitive

84 deficits (113). BBS is a heterogeneous disorder and twelve causative genes (BBS1-12) have been identified (113). Although the precise functions of the BBS proteins are still unresolved, numerous studies in diverse model systems have implicated the BBS proteins in cilia function (113, 114). A recent study provides important insight into how the BBS proteins mediate cilia function in mammalian cells. Seven of the most evolutionarily conserved BBS proteins (BBS1, 2, 4, 5, 7, 8, and 9) form a stable complex, called the BBSome, that may mediate vesicular transport to the cilium (56).

Defects in neuronal signaling are likely components of many of the BBS phenotypes, including obesity, hypogenitalism, and cognitive deficits. It has been known for more than forty years that neurons in the brain possess primary cilia (76), but the specific functions of these organelles remain unknown. The GPCRs somatostatin receptor 3

(Sstr3) (61) and serotonin receptor 6 (60, 64), specifically localize to neuronal cilia, suggesting a role for cilia in signaling on neurons. The functional importance of these cilia is suggested by the fact that several human ciliary disorders, including BBS, Joubert syndrome, and Meckel syndrome, have prominent functional and structural CNS phenotypes (1).

Here, we investigate the hypothesis that BBS proteins are required for assembly or function of primary cilia on central neurons. We show that neurons both in vivo and in vitro from mice lacking either the Bbs2 or Bbs4 protein possess apparently normal primary cilia but lack ciliary localization of Sstr3 or Mchr1. Remarkably, the lack of ciliary localization can be corrected in BBS neurons by heterologous expression of the missing

BBS protein. Our studies indicate that the BBS proteins are required for the localization of GPCRs to cilia on central neurons and suggest that some BBS phenotypes are the result of altered signaling due to ciliary GPCR mislocalization.

85 RESULTS

BBS Mutant Mice Lack Sstr3-Positive Cilia.

To investigate whether the BBS proteins are required for assembly of primary cilia on

central neurons we colabeled brain sections from adult WT, Bbs2-null (Bbs2-/-) and

Bbs4-null (Bbs4-/-) mice with antibodies to Sstr3 and type III adenylyl cyclase (ACIII). We

recently reported that ACIII is a prominent marker of primary cilia throughout the mouse

brain (89) and in some brain regions, such as the hippocampus, a subset of these cilia

are also positive for Sstr3 (79). Examination of the distribution and abundance of ACIII-

immunoreactive cilia throughout the brains of WT, Bbs2-/- and Bbs4-/- mice revealed no obvious differences (Figure 5.1 A-C), indicating that Bbs2 and Bbs4 are not required for ciliogenesis in the brain. In the hippocampus, a significant proportion of the ACIII- positive cilia in the WT sections were also positive for Sstr3 (Figure 5.1 A, D, and G).

Interestingly, we failed to detect any Sstr3-immunoreactive cilia in the hippocampus of

Bbs2-/- or Bbs4-/- sections (Figure 5.1 E, F, H, and I). We reasoned that the absence of

Sstr3-positive cilia in BBS mice could be the result of one or more of the following; a loss of Sstr3 expression, a loss of neurons that normally localize Sstr3 to the cilium, or a failure of Sstr3 to localize to cilia. To test whether Sstr3 is expressed in BBS animals, we analyzed Sstr3 protein levels in the hippocampus of adult WT, Bbs2-/- and Bbs4-/- mice

by immunoblotting (Figure 5.5). We found similar Sstr3 protein levels in all genotypes,

indicating that the lack of Sstr3-positive cilia in BBS mice is not due a deficiency in Sstr3

expression.

86 Bbs2 and Bbs4 are required for Sstr3 Ciliary Localization.

To further investigate the role of the BBS proteins in neuronal cilia, we cultured

hippocampal neurons from newborn WT, Bbs2-/- and Bbs4-/- mice. We previously

reported that hippocampal neurons cultured from postnatal mice possess primary cilia

that can be visualized with antibodies to ACIII and Sstr3 (79). Colabeling of cells after 7

days in culture with antibodies to ACIII and β-tubulin type III (βTIII), which is a marker of

neurons, revealed the presence of neuronal cilia in all genotypes (Figure 5.2 A-C). No

obvious differences in cilia length or structure were detected and quantification of the

percentage of neurons and the percentage of neurons possessing an ACIII-positive

cilium indicated no differences between WT, Bbs2-/- or Bbs4-/- cultures (Table 5.1).

These results confirm that Bbs2 and Bbs4 are not required for neuronal ciliogenesis. In

WT cultures, some cilia were also positive for Sstr3 (Figure 5.2 D and Table 5.2).

However, we never detected Sstr3-immunolabeled cilia in Bbs2-/-or Bbs4-/- cultures

(Table 5.2). Rather, we consistently observed apparent cell membrane labeling in a subset of the Bbs2-/- and Bbs4-/- neurons (Figure 5.2 E, F), suggesting that the receptor

was expressed in BBS neurons but failed to localize to cilia. The fact that there were no

differences in the percentages of neurons or neurons possessing an ACIII-positive cilium

between WT, Bbs2-/- and Bbs4-/- cultures indicates that the lack of Sstr3-positive cilia in

BBS cultures is not due to the loss of a subpopulation of neurons. To test whether Sstr3

ciliary localization could be restored, we heterologously expressed Bbs2 and Bbs4 in

cultured Bbs2-/- and Bbs4-/- hippocampal neurons, respectively. Notably, Sstr3 ciliary

localization was observed on transfected Bbs2-/- and Bbs4-/- neurons (Figure 5.2 G, H).

Quantification of these neurons revealed that Sstr3 ciliary localization was restored to an

87 equivalent frequency as WT neurons (Table 5.3). Overall, these results indicate that the

BBS proteins are required for the localization of Sstr3 to cilia on hippocampal neurons.

Bbs2 and Bbs4 are required for Ciliary Localization of

Melanin-Concentrating Hormone Receptor 1.

Defects in the localization of ciliary signaling proteins could be the basis for the BBS

phenotypes. Therefore, we asked whether other GPCRs, and specifically GPCRs whose

functions are consistent with the BBS phenotypes, fail to localize to cilia in BBS animals.

We recently found that Mchr1, which is involved in the regulation of feeding and energy

balance (93), localizes to cilia in regions of the brain involved in feeding and reward

pathways, including the olfactory bulb, olfactory tubercle, nucleus accumbens, and

hypothalamus (Berbari et al., in press). To test whether the BBS proteins are required for

Mchr1 ciliary localization, we colabeled WT, Bbs2-/- and Bbs4-/- brain sections with anti-

ACIII and anti-Mchr1. In WT brains, we observed cilia that were positive for both ACIII and Mchr1 in the olfactory tubercle, hypothalamus, and nucleus accumbens (Figure 5.3

A, D, G). Remarkably, we did not detect any Mchr1-positive cilia in these regions in

Bbs2-/-or Bbs4-/- mice (Figure 5.3 E, F, H, I), but instead observed obvious punctate labeling. Immunoblotting of proteins from the nucleus accumbens/olfactory tubercle and hypothalamus of WT, Bbs2-/- and Bbs4-/- brains confirmed that Mchr1 is expressed in

BBS brains at levels comparable to WT brains (Figure 5.6). To further investigate the localization of the punctate Mchr1 labeling in BBS mice, we colabeled Bbs2-/- and Bbs4-/- brain sections with anti-Mchr1 and anti-γ-aminobutyric acid (GABA), a marker of

GABAergic neurons that primarily labels the cell body. In Bbs2-/- and Bbs4-/- brain

sections, we consistently observed colocalization of Mchr1-positive puncta with GABA

88 labeling (Figure 5.7), suggesting that in the absence of ciliary localization Mchr1

accumulates in cytoplasmic puncta in BBS neurons.

We then generated primary cultures enriched for neurons from the nucleus

accumbens/olfactory tubercle from newborn WT, Bbs2-/- and Bbs4-/- mice. Colabeling the cells after 7 days in culture with anti-ACIII and anti-βTIII revealed the presence of ACIII- positive neuronal cilia in all genotypes (Figure 5.4 A-C). However, colabeling the cells with anti-Mchr1 and anti-βTIII revealed the presence of Mchr1-positive cilia exclusively in the WT cultures (Figure 5.4 D). Similar to our results for Sstr3 in hippocampal neurons, a subset of Bbs2-/- and Bbs4-/- nucleus accumbens/olfactory tubercle enriched neurons

displayed punctate Mchr1 labeling (Figure 5.4 E, F) and we were able to restore Mchr1 ciliary localization in BBS hypothalamic neurons by heterologous expression of BBS protein (Figure 5.4 G, H). Thus, BBS proteins are required for proper localization of both

Sstr3 and Mchr1 to neuronal cilia.

DISCUSSION

Our results suggest a novel function for the BBS proteins in the localization of

GPCRs to cilia on central neurons. This is consistent with the previous findings

suggesting the BBSome mediates vesicular transport to the cilium (56) and further

implicates the BBS proteins in the transport of specific signaling proteins to cilia. The fact

that another membrane-bound ciliary protein, ACIII, localizes normally in Bbs2-/- and

Bbs4-/- neurons indicates that the BBS proteins mediate ciliary localization of distinct

signaling proteins. Several studies have found that defects in ciliary protein transport can

be associated with mislocalization of some signaling proteins but not others, suggesting

there are multiple mechanisms for trafficking proteins to the cilium (13, 24-26).

89 Interestingly, hypomorphic mutations in CEP290/NPHP6, which are known to cause the

early onset retinopathy Leber congenital amaurosis (115), are associated with defective

ciliary localization of olfactory G proteins but not other olfactory signaling proteins,

including odorant GPCRs and ACIII (26). It is possible that CEP290/NPHP6 and the

BBS proteins are part of separate ciliary protein transport mechanisms in neurons, with

the BBS proteins specifically mediating GPCR transport to cilia.

Loss of BBS proteins has previously been associated with defective protein transport

in specialized sensory cilia. Rhodopsin (a GPCR) accumulates in the cell bodies of

photoreceptors in Bbs2-/-, Bbs4-/-, and Bbs6-/- mice (52, 53, 116). Normally, rhodopsin is synthesized in the cell body and transported across the connecting cilium to the outer segment of the photoreceptor. However, in BBS mice rhodopsin can be detected in the cell body of some photoreceptors before the onset of apoptosis. It is interesting that in

BBS photoreceptors rhodopsin transport is not completely abrogated and significant amounts of protein are properly localized in the outer segments, whereas we did not detect any Sstr3 or Mchr1 in cilia on BBS neurons. This may reflect differences in the mechanisms of ciliary protein trafficking between connecting cilia and cilia on central neurons or indicate that the precise functions of the BBS proteins vary between cell types.

Loss of olfactory neuronal cilia and mislocalization of ciliary signaling proteins, including ACIII, has been reported in Bbs1-/-, Bbs4-/-, and Bbs6-/- mice (54, 116). In this case, protein mislocalization was accompanied by disorganization of microtubules within the olfactory bulb, raising the possibility that the trafficking defects were the result of microtubule disruption. The BBS proteins have been previously implicated in microtubule stability (46), which could be an underlying mechanism in the mislocalization of ciliary

90 signaling proteins. However, it is doubtful that alterations in microtubule structure are

involved in the lack of Sstr3 or Mchr1 ciliary localization in BBS neurons. We found that

ACIII localized to neuronal cilia on BBS neurons and the cilia were indistinguishable from

cilia on WT neurons. Further, β-Tubulin III labeling did not reveal any alterations in

microtubule structure in Bbs2-/- or Bbs4-/- neurons. This suggests that in some mammalian cells the function of the BBS proteins is to localize specific signaling proteins to cilia. A recent study showed altered distribution of the thermosensory channel TRPV1 and the mechanosensory channel STOML3 within the soma of peripheral neurons in

BBS animals (117), suggesting the BBS proteins may also contribute to trafficking of proteins within the cell body in some cell types.

We hypothesize that the fundamental mechanism underlying the pathophysiology of the pleiotropic BBS phenotypes involves mislocalization of cell-type specific ciliary signaling proteins and disruption of cellular signaling. Somatostatin regulates neurotransmission (69) and aberrant neuronal somatostatin levels are associated with

CNS diseases and cognitive impairment (118). Thus, it is possible that lack of Sstr3 ciliary localization could disrupt neuronal signaling and contribute to cognitive deficits in

BBS patients. Melanin concentrating hormone (MCH) and its receptor, Mchr1, are important regulators of feeding and energy balance (93). Injection of MCH induces a rapid increase in feeding behavior (103) while injection of Mchr1 antagonists reduces feeding behavior (104). Further, transgenic mice overexpressing MCH are obese (105) and mice lacking expression of either MCH or Mchr1 are lean (106, 107). Our results showing Mchr1 fails to localize to cilia in mouse models of obesity suggest that ciliary localization of Mchr1 is important for proper signaling. Interestingly, lack of cilia in the brain, and specifically on the pro-opiomelanocortin expressing neurons of the

91 hypothalamus results in hyperphagia-induced obesity (108). Our findings now provide a

potential molecular mechanism to link loss of cilia or cilia dysfunction with obesity.

Overall, these results provide important insights into the pathophysiology of BBS and

other ciliary disorders.

MATERIALS AND METHODS

Mice and Tissue Preparation

The generation and characterization of Bbs2- and Bbs4-null mice has been

previously described (47, 52). All procedures were approved by the Institutional Animal

Care and Use Committee at The Ohio State University. WT, Bbs2-/- and Bbs4-/- littermates were generated by intercrossing heterozygous animals. Animals were anesthetized by a 0.1 ml/10 g intraperitoneal injection of 2.5% tribromoethanol (Sigma-

Aldrich), sacrificed by cardiac puncture, and perfused with phosphate-buffered saline

(PBS) followed by a 1:1 mixture of 4% paraformaldehyde:HistoChoice (Amresco). The brains were further fixed in 4% paraformaldehyde:HistoChoice for 16-24 hours at 4°C followed by cryoprotection in 30% sucrose in PBS for 16-24 hours. For immunofluorescence procedures, cryoprotected brains were embedded in Optimal

Cutting Temperature compound (VWR), and sectioned in a cryostat at a thickness of 30

µm.

Neuronal Cell Culturing and Transfections

Primary hippocampal neurons from postnatal day 0-1 mouse pups were cultured as

previously described (79). Primary neurons were transfected after 5 days in culture using

Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instructions. The 92 expression vectors used in these studies included pcDNA3.1 (Invitrogen) and pEGFP-

N3 (Clontech). Bbs2-/- or Bbs4-/- neurons were cotransfected with pcDNA3.1 expressing either Bbs2 or Bbs4, respectively, and a vector expressing EGFP alone as a transfection marker. Neuronal cultures were fixed 48 hours post transfection.

Immunofluorescence

Day 7 neurons were fixed with a 4% paraformaldehyde/10% sucrose solution for 10 minutes at room temperature, followed by a 5 minute PBS wash. Neurons were then post fixed with cold MeOH at -20 °C for 15 minutes and permeabilized with 0.1% Triton

X-100 in PBS for 7 minutes. Following permeabilization, the cells were put in a blocking solution of PBS with 2% serum, 0.02% sodium azide and 10 mg/ml bovine serum albumin (BSA) for approximately 1 hour at room temperature. All antibody incubations and washes were carried out in PBS with 2% serum, 0.02% sodium azide and 10 mg/ml

BSA. All primary antibody incubations were carried out for 16-24 hours at 4°C. Primary antibodies included; anti-adenylyl cyclase III (sc-588; Santa Cruz), anti-Sstr3 (ss-830;

Gramsch), anti-Sstr3 (sc-11617; Santa Cruz), anti-Mchr1 (sc-5534; Santa Cruz), anti-β- tubulin III (T-8660; Sigma-Aldrich), and anti-γ-aminobutyric acid (A-0310; Sigma-Aldrich).

Secondary antibodies included; Alexa Fluor 488- and 546-conjugated goat anti-mouse

IgG, Alexa Fluor 488- and 546-conjugated goat anti-rabbit IgG, Alexa Fluor 488- conjugated donkey anti-goat IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG,

Alexa Fluor 488-conjugated donkey anti-mouse IgG (Invitrogen), Cy3-conjugated donkey anti-goat IgG and Cy3 conjugated donkey anti-rabbit IgG (Jackson Immuno Research).

Nucleic acids were stained with DRAQ5 (Axxora). All samples were imaged on a Zeiss

510 META laser scanning confocal microscope at the Ohio State University Central

Microscopy Imaging Facility. Multiple consecutive focal planes (Z-stack), spaced at 0.5 93 µm intervals, were captured. For all collected images, the brightness and contrast of each channel was adjusted using the Zeiss LSM Image Browser program.

94 FIGURE 5.1 BBS MICE POSSESS NEURONAL PRIMARY CILIA IN THE BRAIN BUT LACK SSTR3-POSITIVE CILIA. (A-C) REPRESENTATIVE IMAGES OF THE CA3 REGION OF THE HIPPOCAMPUS IN ADULT WT (A) BBS2-/- (B) AND BBS4-/- (C) MICE (N=9) SHOWING LABELING FOR ACIII (RED). NUCLEI ARE STAINED WITH DRAQ5 (BLUE). THE APPEARANCE AND DISTRIBUTION OF ACIII- POSITIVE CILIA IS SIMILAR BETWEEN ALL GENOTYPES. (D-F) THE IDENTICAL FIELDS SHOWING LABELING FOR SSTR3 (GREEN) REVEALS SSTR3-POSITIVE CILIA IN THE WT (D) SECTION BUT A COMPLETE LACK OF SSTR3-POSITIVE CILIA IN THE BBS2-/- (E) OR BBS4-/- (F) SECTIONS. (G-I) THE MERGED IMAGES SHOWING COLOCALIZATION OF ACIII AND SSTR3 TO CILIA IN THE WT (G) SECTION AND NO SSTR3 LABELING OF CILIA IN THE BBS2-/- (H) OR BBS4-/- (I) SECTIONS. ALL SCALE BARS = 10µM.

95

FIGURE 5.2 SSTR3 CILIARY LOCALIZATION CAN BE RESTORED IN VITRO. CO-IMMUNOLABELING OF DAY 7 HIPPOCAMPAL NEURONS FROM WT (A) BBS2-/- (B) AND BBS4-/- (C) MICE WITH ANTIBODIES TO ACIII (GREEN) AND TIII (RED) SHOWS THE PRESENCE OF CILIA (ARROWS) IN ALL THREE GENOTYPES. CO-IMMUNOLABELING OF DAY 7 HIPPOCAMPAL NEURONS FROM WT (D) BBS2-/- (E) AND BBS4-/- (F) MICE WITH ANTIBODIES TO SSTR3 (GREEN) AND TIII (RED) SHOWS THE PRESENCE OF CILIA (ARROW) ONLY ON THE WT NEURON. NOTE THE APPARENT PUNCTATE SSTR3 LABELING IN THE BBS2-/- (E) AND BBS4-/- (F) NEURONS. IMMUNOLABELING OF DAY 7 HIPPOCAMPAL NEURONS FROM BBS2-/- (G) AND BBS4-/- (H) MICE FOR SSTR3 (RED) TWO DAYS POST- TRANSFECTION WITH AN EXPRESSION VECTOR ENCODING BBS2 AND BBS4, RESPECTIVELY, AND A VECTOR EXPRESSING ENHANCED GREEN FLUORESCENCE PROTEIN (GFP; GREEN) AS A TRANSFECTION MARKER. NOTE THAT HETEROLOGOUS EXPRESSION OF BBS PROTEINS RESTORES SSTR3 CILIARY LABELING (ARROWS). NUCLEI ARE STAINED WITH DRAQ5 (BLUE). ALL SCALE BARS = 5µM.

96

FIGURE 5.3 BBS MICE LACK MCHR1 CILIARY LABELING IN THE BRAIN. (A-C) Representative images of the nucleus accumbens in adult WT (A) Bbs2-/- (B) and Bbs4- /- (C) mice (n=9) showing labeling for ACIII (red). The appearance and distribution of ACIII- positive cilia is similar between all genotypes. (D-F) The identical fields showing labeling for Mchr1 (green) reveals Mchr1-positive cilia in the WT (D) section but a complete lack of Mchr1- positive cilia in the Bbs2-/- (E) or Bbs4-/- (F) sections. Note the presence of increased punctate labeling in the Bbs2-/- (E) and Bbs4-/- (F) sections. (G-I) The merged images showing colocalization of ACIII and Mchr1 to cilia in the WT (G) section and no Mchr1 labeling of cilia in the Bbs2-/- (H) or Bbs4-/- (I) sections. Nuclei are stained with DRAQ5 (blue). All scale bars = 10µm.

97

FIGURE 5.4 MCHR1 CILIARY LOCALIZATION CAN BE RESTORED IN VITRO. CO-IMMUNOLABELING OF DAY 7 NUCLEUS ACCUMBENS/OLFACTORY TUBERCLE ENRICHED NEURONS FROM WT (A) BBS2-/- (B) AND BBS4-/- (C) MICE WITH ANTIBODIES TO ACIII (GREEN) AND TIII (RED). CILIA (ARROWS) ARE PRESENT IN ALL THREE GENOTYPES. CO- IMMUNOLABELING OF DAY 7 NUCLEUS ACCUMBENS/OLFACTORY TUBERCLE ENRICHED NEURONS FROM WT (D) BBS2-/- (E) AND BBS4-/- (F) MICE WITH ANTIBODIES TO MCHR1 (GREEN) AND TIII (RED) SHOWS THE PRESENCE OF CILIA (ARROW) ONLY ON THE WT NEURON. NOTE THE PUNCTATE STAINING IN (E) AND (F). IMMUNOLABELING OF DAY 7 HYPOTHALAMIC NEURONS FROM BBS2-/- (G) AND BBS4-/- (H) MICE FOR MCHR1 (RED) TWO DAYS POST-TRANSFECTION WITH AN EXPRESSION VECTOR ENCODING BBS2 AND BBS4, RESPECTIVELY, AND A VECTOR EXPRESSING ENHANCED GREEN FLUORESCENCE PROTEIN (GFP; GREEN) AS A TRANSFECTION MARKER. NOTE THAT HETEROLOGOUS EXPRESSION OF BBS PROTEINS RESTORES MCHR1 CILIARY LABELING (ARROWS). NUCLEI ARE STAINED WITH DRAQ5 (BLUE). ALL SCALE BARS = 5µM.

98

FIGURE 5.5 WESTERN BLOT ANALYSIS FROM ADULT HIPPOCAMPUS FOR SSTR3 LEVELS Whole protein lysates from the hippocampus of adult WT, Bbs2-/- and Bbs4-/- mice probed with antibodies to Sstr3 and actin, as a loading control. Sstr3 expression is shown in the upper panel and actin expression is shown in the lower panel. Note that Sstr3 expression is similar in all genotypes.

99

FIGURE 5.6 WESTERN BLOT ANALYSIS FROM ADULT HYPOTHALMUS AND ACCUMBENS/TUBERCLE REGIONS FOR MCHR1 LEVELS Immunoblot of whole protein lysates from the hypothalamus and nucleus accumbens/olfactory tubercle of adult WT, Bbs2-/- and Bbs4-/- mice probed with antibodies to Mchr1 and actin, as a loading control. Mchr1 expression is shown in the upper panels and actin expression is shown in the lower panels. Note that Mchr1 expression is similar in all genotypes.

100

Figure 5.7 Mchr1 localizes to cytoplasmic puncta in BBS neurons in vivo. Representative images of hypothalamic neurons in adult Bbs2-/- (A) and Bbs4-/- (B) brains showing labeling for GABA (green). (C, D) The identical fields showing labeling for Mchr1 (red). (E, F) The merged images showing colocalization of GABA and Mchr1 punctate labeling in the cytoplasm of neurons. Nuclei are stained with DRAQ5 (blue). All scale bars = 10µm.

101

WT Bbs2-/- WT Bbs4-/-

Neuron (%) 75±4.3 74±3.6 78±4.3 77±2

ACIII Positive Neuronal Cilia (%) 56±1.3 52±4.5 71±11.9 57±8

TABLE 5.1 NEURONAL AND ACIII CILIA FREQUENCY IN CULTURE Percentage of cells (n=1148) after 7 days in culture that were positive for the neuronal marker Β- tubulin type III and percentage of neurons possessing a type III adenylyl cyclase (ACIII) positive cilium. Values are expressed as the mean percentage ± SEM. For each genotype n=3 mice.

WT Bbs2-/- WT Bbs4-/-

Neuron (%) 76±0.6 73±1.4 75±0.7 78±1.4

Sstr3 Positive Neuronal Cilia (%) 14±0.3 0 18±3.5 0

TABLE 5.2 NEURONAL AND SSTR3 CILIA FREQUENCY IN CULTURE Percentage of cells (n=1311) after 7 days in culture that were positive for the neuronal marker neurofilament and percentage of neurons possessing a somatostatin receptor 3 (Sstr3) positive cilium. Values are expressed as the mean percentage ± SEM. For each genotype n=3 mice.

102 Bbs2-/- Neurons Bbs2-/- Neurons

Transfected with an Transfected with a Bbs2

Empty Vector Expression Vector

Sstr3 Positve Cilia (%) 0 15±2.7

Sstr3 Positive Neuronal 0 0

Cilia (%)

TABLE 5.3 QUANTIFICATION OF SSTR3 CILIARY RESCUE IN TRANSFECTED BBS2 NEURONS Percentage of transfected day 7 Bbs2-/- neurons (n=212) possessing an Sstr3 positive cilium 2 days post-transfection with empty vector or a Bbs2 expression vector. Values are expressed as the mean percentage ± SEM. Neurons were isolated from two Bbs2-/- mice.

103 CHAPTER 6:

CONCLUSIONS AND DISCUSSION

FUTURE STUDY OF NEURONAL CILIA IN VITRO

The use of in vitro systems to study neuronal cilia will allow investigators to dissect

the physiological roles of cilia in the central nervous system. The system described in

Chapter 2 will serve as a good foundation for delving into specific signaling pathways

where a role for neuronal cilia has been implicated. The system’s versatility will allow for

monitoring of potential downstream effects of cilia mediated signaling events. It will be

interesting to investigate the role that neuronal cilia play with regards to second

messengers and subsequent cellular responses. Further, the system may be utilized for

live cell imaging to test whether pathways involve dynamic cilia localization. Dynamic

cilia localization has been described for components of the hedgehog pathway (21, 119).

Whether this will emerge as a general theme for other primary cilia signaling pathways

remains to be determined.

104 FUTURE STUDY OF CILIA LOCALIZATION SEQUENCES

The identification of a loose cilia localization sequence in the third intracellular loop of

GPCRs will serve as a launching point for research into the mechanisms of cilia

localization. The most immediate question from this finding is: how do cilia localization

sequences impart localization? These sequences could directly interact with the

localization machinery, potentially even components of the BBSome. Identifying the

sequences themselves will facilitate experiments aimed at determining the mechanisms

and events required for entry into the cilium.

It will be interesting to determine if these underlying mechanisms are specific to cilia

GPCRs or are shared with other cilia membrane proteins. For example, do cilia specific

receptor tyrosine kinases, such as platelet derived growth factor receptor alpha, utilize

the same pathway as GPCRs. Work described here and by others suggests that multiple

pathways are involved in localization to the primary cilium (13, 24-26). For example the

BBS proteins are not required for the localization of ACIII to the primary cilium but are

required for GPCR ciliary localization (Chapter 5). Research pursuing the determinants

of cilia localization will provide valuable clues to the physiological functions of this

organelle throughout the body by Identifying novel ciliary signaling proteins and

pathways.

DISCUSSION OF THE ROLE OF BBS PROTEINS

Data presented in Chapter 5 show that the BBS proteins are required for the

localization of certain cilia GPCRs (Figure 6.1). However, the precise roles for the

BBSome, and how it mediates cilia localization, remain unknown. Future genetic and biochemical analysis of the BBS proteins and the BBSome will likely reveal other

105 proteins that both directly and transiently interact with the complex. The underlying

mechanisms and the sequence of events involved in receptor trafficking to the cilium will

provide insight into the functions of this organelle and its role in signaling.

QUESTIONS IN THE FIELD OF CILIA BIOLOGY

The realization that several human genetic disorders and disease states are the

result of cilia dysfunction has led to a fast growing body of literature aimed at

understanding the functions of this organelle. It is now accepted that primary cilia serve

very important and diverse roles as sensory organelles. There are numerous fascinating

questions in cilia biology. What precise roles in signaling do they play and what

pathways utilize the cilium? Does the cilium quantitatively modulate known signaling

pathways or is it utilized to add complexity to signaling pathways by qualitatively

modulating pathways? What other developmental pathways and processes require

cilia? Is the embryonic node the only site of motile primary cilia throughout the body?

Are primary cilia capable of sending signals to neighboring cells or just receiving

signals? What roles do primary cilia play in regulating the cell cycle? These are just a

few of the exciting questions in this rapidly growing field. It is becoming clear that the

primary cilium is a complex organelle, and that there will be complex answers to each of

these questions. Research on these questions will ultimately provide insights into basic

biological processes and human disease.

106

FIGURE 6.1 MODEL OF CILIA DYSFUNCTION IN BBS In a WT cell (left panel) GPCRs containing ciliary localization sequences within the third intracellular (i3) loop are trafficked into the cilium by the BBSome. This ciliary localization then plays a role in the signaling of these GPCRs. In a BBS cell (Bbs4-/- right panel) the BBSome is unable to mediate ciliary localization of GPCRs, resulting in a loss or gain of signaling. This model has been simplified and does not indicate the presence of the BBSome in the cilium or likely interactions with IFT components. (The structure of the BBSome is adapted from Nachury et. al. Cell 2007).

107

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