Palmitoylation of Caveolin-1 and Its Importance for Structural and Functional Plasticity

Home , Caveolin

Palmitoylation of Caveolin-1 and its importance for structural

and functional plasticity

A DISSERTATION

SUBMITTED TO THE FACULTY OF

UNIVERSITY OF MINNESOTA

Katherine R. Tonn

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

Paul G. Mermelstein

November 2018

Acknowledgments

I would like to thank my adviser, Dr. Paul Mermelstein, for his mentorship, training, support, and scientific flexibility. Infinite thanks to past and present members of the Mermelstein and Meisel Labs, especially Dr. Brittni Peterson, Dr. Kelsey Moore, Dr.

Laura Been, Dr. Luis Martinez, Dr. Valerie Hedges, and Dr. John Meitzen, for being a source of scientific inspiration and friendship. I could not have asked for a better friend and lab colleague than Dr. Kellie Gross, whose input has kept me sane and made me a better scientist. Thanks also to stellar undergrads Kerry Trotter, Julia Dworsky, and Sam

Swanson for their help and energy. Thanks to the many people who collaborated or helped in some way on the experiments presented in this thesis, including Dr. Mark

Thomas, Dr. Lorene Lanier, Dr. Mark Dell’Acqua, Dr. Kevin Woolfrey, Dr. Brian Head,

Dr. Jing Tong, and Dr. John Meitzen. In particular, I am extremely grateful for the collaboration of Dr. Lorene Lanier, whose enthusiasm helped reinvigorate me and this project. Finally, thanks to the members of my thesis committee, Drs. Harry Orr, Timothy

Ebner, Anna Lee, and Robert Meisel, for their encouragement and guidance.

Dedication

For Mom, my first and most important example of what it is to be smart, versatile, and kind.

For Dad, who fostered my sense of curiosity, and taught me to be comfortable with the contents of a toolbox.

For Gina, whose intelligence, passion, ambition, and star power give me something to strive for.

For Bob, who gets it. And me.

Abstract

This dissertation examines the regulation and function of caveolin-1 (Cav1). Cav1 is an integral membrane protein that creates functional microdomains of neuronal proteins within lipid rafts. Cav1 regulates a variety of signaling pathways, including mGluR-activated G protein cascades, and is involved in membrane trafficking of proteins such as estrogen and dopamine receptors. The function of Cav1 is regulated by palmitoylation, a reversible post-translational addition of a 16-carbon lipid chain that is involved in trafficking and compartmentalizing target proteins. This regulatory mechanism is important not only for Cav1, but also for membrane association of estrogen receptors. Within the nervous system, palmitoylation of estrogen receptor alpha (ERα) is necessary for surface membrane localization and mediation of downstream signaling through the activation of metabotropic glutamate receptors (mGluRs). Mutation of the single palmitoylation site on ERα prevents its physical association with Cav1, which in turn is required for the formation of the estrogen receptor/mGluR signaling complex.

Interestingly, siRNA knockdown of either of two palmitoyl acyltransferases, DHHC7 or

DHHC21, also eliminates this signaling mechanism. As ERα has only one palmitoylation site, I hypothesized that one of these DHHCs palmitoylates another essential protein in this signaling complex, namely Cav1. I investigated this using an acyl-biotin exchange assay in HEK293 cells in conjunction with DHHC overexpression, and found that

DHHC7 increased Cav1 palmitoylation. Mutation of the palmitoylation sites on Cav1 eliminated this effect, but did not disrupt the ability of the DHHC enzyme to associate with the protein. In contrast, siRNA knockdown of DHHC7 alone was not sufficient to decrease Cav1 palmitoylation, but rather required simultaneous knockdown of DHHC21.

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These findings raise questions about the overall influence of palmitoylation on the membrane-initiated estrogen signaling pathway, and highlight the importance of considering the influence of palmitoylation on other Cav1-dependent processes.

Additionally, recent studies have shown that altering Cav1 expression influences neuronal plasticity and related behaviors in contexts ranging from learning and memory to chronic injury. Given this relationship between Cav1 and experience-dependent plasticity, I hypothesized that Cav1 expression would also be involved in drug-induced changes in neuronal signaling. I utilized a locomotor sensitization paradigm to test this hypothesis. Animals receiving repeated cocaine displayed behavioral sensitization and greater expression of Cav1 mRNA in the nucleus accumbens when compared to saline- treated controls. Overexpression of Cav1 in the nucleus accumbens enhanced cocaine- induced locomotor responses to cocaine, while Cav1 KO animals did not sensitize.

Cultured neurons from the nucleus accumbens, a brain region critical for the development of sensitization, had enhanced dendritic complexity in Cav1 KO mice and altered responses to cocaine. Finally, I report that Cav1 palmitoylation is required for its normal function. Together, these findings suggest that (1) Cav1 KO mice may be structurally saturated such that normal drug-induced plasticity is prevented, (2) Cav1 palmitoylation plays an important role in facilitating the proper activity of signaling molecules associated with Cav1, and (3) understanding Cav1 function will be necessary for fully understanding the development of addiction.

Table of Contents

Acknowledgments...... i

Dedication ...... ii

Abstract ...... iii

List of Tables ...... vii

List of Figures ...... viii

CHAPTER 1: Introduction and Literature Review ...... 1

Caveolin proteins ...... 2 Classical nuclear vs. membrane-associated estrogen receptors ...... 3 Estrogen-Sensitive Membrane Receptors ...... 5 mGluRs: A link to intracellular signaling pathways ...... 6 ER-mGluR Interactions ...... 8 Membrane-associated ER-mGluR Interactions in Medium Spiny Neurons ...... 12 Caveolin organizes functional signaling microdomains ...... 13 Palmitoylation: Regulating membrane interactions ...... 16 Purpose and Aims ...... 22

CHAPTER 2: Palmitoylation of Caveolin-1 is regulated by the same DHHC enzymes as steroid hormone receptors ...... 23

Introduction ...... 24 Methods ...... 25 Results ...... 33 Discussion ...... 45

CHAPTER 3: Caveolin-1 and cocaine-induced plasticity ...... 51

Introduction ...... 52 Methods ...... 53 Results ...... 59 Discussion ...... 70

CHAPTER 4: Overall discussion and conclusions ...... 75 v

Importance of Cav1 for ER/mGluR signaling ...... 76 ER/mGluR relationship ...... 76 Sex differences ...... 78 Cav1 and drug-induced plasticity ...... 81 Cav1 interacts with and/or influences many signaling molecules associated with drug-induced plasticity ...... 82 The nucleus accumbens and medium spiny neurons ...... 86 Conclusion ...... 88

References ...... 89

List of Tables

Table 1.1. ER – mGluR functional interactions ...... 11

Table 1.2. Summary of ER-Cav-mGluR interactions ...... 15

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List of Figures

Figure 1.1: Schematic of membrane-initiated 17β-Estradiol (17βE) signaling ...... 10

Figure 1.2. Schematic of estradiol/ER/mGluR signaling microdomains organized by caveolin and palmitoylation...... 14

Figure 1.3. Possible models by which the enzymes DHHC7 and DHHC21 work to facilitate membrane-initiated signaling through ERs...... 21

Figure 2.1. DHHC7 and DHHC21 are required for estradiol-induced CREB phosphorylation...... 35

Figure 2.2. Endogenous CAV1 exhibits palmitoylation permanence...... 37

Figure 2.3. Overexpression of DHHC7 or DHHC21 prevents cycloheximide-induced decreases in CAV1 palmitoylation ...... 39

Figure 2.4. DHHC7 increases HA-tagged CAV1 palmitoylation ...... 40

Figure 2.5. Palmitoylation-null CAV1 still associates with DHHC7 and the plasma membrane ...... 41

Figure 2.6. Simultaneous, but not single, knockdown of zDHHC7 and zDHHC21 decreases CAV1 palmitoylation...... 43

Figure 2.7. DHHC7-mediated CAV1 Palmitoylation is not affected by knockdown of zDHHC21 ...... 44

Figure 2.8. Differential subcellular localization of DHHC7 and DHHC21 ...... 45

Figure 3.1. Repeated cocaine exposure followed by withdrawal and cocaine challenge led to increased Cav1 mRNA expression in the nucleus accumbens ...... 59

Figure 3.2. Overexpression of caveolin-1 (Cav1) in the nucleus accumbens (NAc) facilitates cocaine-mediated behavioral sensitization in female rats...... 61

Figure 3.3. Cav1 KO animals do not display cocaine-induced behavioral sensitization .. 62

Figure 3.4. Cultured MSNs from Cav1 KO mice have enhanced dendritic arborization. 65

Figure 3.5. PLC inhibition eliminates enhancement of dendritic arborization in Cav1 KO MSNs ...... 66

Figure 3.6. Dopamine decreases dendritic arborization of MSNs from Cav1 KO mice .. 67

Figure 3.7. Effect of Cav1 KO on MSN arborization is MSN-intrinsic ...... 68

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Figure 3.8. Cav1 palmitoylation is required for normal dendritic arborization in MSNs. 70

Figure 4.1. Schematic of palmitoylated Cav1 interacting with signaling proteins at the plasma membrane...... 82

CHAPTER 1: Introduction and Literature Review

Caveolin proteins

Caveolins are small integral membrane proteins that physically organize signaling proteins, including mGluRs, into functional microdomains (Patel et al., 2008;

Francesconi et al., 2009; Takayasu et al., 2010). Both Cav terminals are located intracellularly, with a putative scaffolding domain on their N-terminus, and they are enriched in lipid rafts. In non-neural tissue, they form high molecular weight oligomers called caveolae which create invaginations in the plasma membrane. Interestingly, caveolae are not observed in the brain, and early reports of caveolin expression in the nervous system were limited to endothelial and glial cells (Cameron et al., 1997; Galbiati et al., 1998; Ikezu et al., 1998; Mikol et al., 1999). The first report of neuronal Cav came in 2000 from the laboratory of Daniel Madison, that demonstrated expression of Cav1 in hippocampal neurons (Braun and Madison, 2000). Around this time, our lab and others were searching for a structural link that could explain an association between membrane- localized estrogen receptors (ERs) and mGluRs in neurons. Although it was known that

Cav-ER association was important for membrane-associated ER signaling outside the brain (Razandi et al., 2002, 2003), Dominique Toran-Allerand proposed that flotillin, a protein with qualities similar to caveolin, might serve the same role in neurons (Toran-

Allerand et al., 2002; Toran-Allerand, 2004). Nevertheless, those early reports of neuronal Cav1 led to the finding that, in fact, all three Cav isoforms are expressed in neurons (Boulware et al., 2007). As a result, the foundations of our knowledge of neuronal caveolin stem from investigations into membrane-localized estrogen signaling.

Classical nuclear vs. membrane-associated estrogen receptors

The steroid sex hormone 17β-estradiol (estradiol) causes changes in neuronal properties that can be either temporary or permanent, and either rapid or slow. These changes affect molecular, structural, and physiological states, leading to system-level changes in circuit dynamics and, ultimately, behavior. The influence of estradiol can be seen in many contexts, including those expected to require sex hormone modulation like reproduction, sex-specific behaviors, and sexual differentiation, as well as less obvious arenas like sensorimotor control, learning and memory, reward, and pathological drug use. In order to influence these processes, estradiol must interact with receptors that can activate a variety of signaling mechanisms. Depending on which mechanisms are activated, estradiol action may or may not involve direct nuclear modulation of gene expression.

In the nucleus, estrogen receptors modulate gene expression when estradiol binds to ERα or ERβ. This causes the receptor to dimerize and interact with estrogen response elements (ERE) on DNA. Dimerized ERs may also influence both ERE- and non-ERE- expressing genes through associations with transcription factors and co-activators

(Charlier et al., 2010). Such nuclear activity is a critical component of estrogen action in the brain. However, after observing estradiol effects inconsistent with nuclear changes in gene expression, researchers began to suspect the existence of alternate estrogen pathways. As discussed below, these effects included rapid estradiol-mediated behavioral changes, estradiol signaling initiated outside of the nucleus, and fast estradiol-evoked changes in neuron electrophysiology.

Within the nervous system, rapid estradiol action was first demonstrated in preoptic/septal neurons, whose electrophysiological properties change within seconds of estradiol exposure (Kelly et al., 1976). Since those early experiments, a wealth of research has emerged in support of non-nuclear estrogen receptor action in neurons.

Compelling evidence includes rapid estradiol modulation of cell-autonomous and synaptic physiology in many brain regions, estradiol-mediated changes in neural structures such as dendritic spines, and estradiol-induced activation of intracellular signaling cascades that ultimately change neuronal properties and gene expression

(Mermelstein et al., 1996; Joëls, 1997; Chaban et al., 2004; Woolley, 2007). It is important to note that, although neurons are the focus here, many of the groundbreaking experiments investigating non-nuclear estrogen receptor signaling were not performed with nervous system tissue. For example, it was reported in 1967 that estradiol in uterine tissue increases cAMP accumulation within 15 seconds of exposure (Szego and Davis,

1967).

A common element of these findings is that the majority of rapid estradiol responses are initiated at the plasma membrane. This was illustrated in four main ways.

First, electron microscopy showed the presence of non-nuclear estrogen receptors

(Blaustein et al., 1992; Almey et al., 2012). Second, the observed estradiol actions sometimes occurred in a brain region that expresses little or no nuclear ER, as shown by immunocytochemistry or centrifugal fractionation (Becker et al., 1987; Remage-Healey et al., 2010). Third, rapid effects were reproduced using membrane-impermeable estrogen analogs, such as estradiol linked to BSA (Pappas et al., 1995; Mermelstein et al., 1996;

Boulware et al., 2005). Lastly, the effects of extracellular administration of estradiol were

not occluded by intracellular dialysis of the hormone into individual neurons

(Mermelstein et al., 1996). This literature collectively established that estradiol can induce not only direct nuclear signaling, but also surface membrane-initiated signaling in neurons.

Estrogen-Sensitive Membrane Receptors

Having demonstrated that rapid effects of estradiol are initiated at the plasma membrane, the next task was to identify the receptors responsible and the underlying mechanism of action. In the 1980s, it was discovered that steroid sex hormone receptors translocate to the membrane in Xenopus oocytes (Sadler and Maller, 1982; Sadler et al.,

1985). These findings were initially dismissed as a procedural artifact, but have since been validated in multiple brain regions and tissues using a variety of techniques

(Razandi et al., 1999; Micevych and Mermelstein, 2008; Pedram et al., 2009). Additional evidence of “nuclear” receptor involvement in rapid estradiol signaling came from experiments conducted by Allan Herbison and his colleagues. Using ER knockout mice, they showed that ERα and ERβ are involved in rapid activation of the MAPK/ERK signaling pathway (Ábrahám et al., 2004). Probing this pathway has become one of the major ways to study rapid estrogen effects, so I will take a moment to describe it below.

Estradiol activates the MAPK/ERK signaling pathway leading to the phosphorylation of serine 133 on the transcription factor CREB (Gu and Moss, 1996;

Zhou et al., 1996; Wade and Dorsa, 2003; Lee et al., 2004). Phosphorylated CREB

(pCREB) then modulates gene expression by interacting with CREB response elements on DNA (Lonze and Ginty, 2002; Carlezon Jr et al., 2005). Our pharmacological experiments found that the pan-specific ER inhibitor ICI 182,780 blocks such signaling,

while specific ERα and (depending on the specific signaling pathway examined) ERβ agonists mimic the effect of estradiol (Boulware et al., 2005). These findings, along with those from Herbison’s group, provided strong evidence for the involvement of membrane-associated ERα and ERβ in rapid signaling events, but they did not address how activation of these receptors could lead to activation of MAPK pathways. mGluRs: A link to intracellular signaling pathways

Given what was known about membrane-initiated estrogen signaling – that ERα and ERβ can somehow be trafficked to and present at the plasma membrane of neurons where they activate intracellular transduction cascades resulting in increased CREB phosphorylation – we wanted to investigate how exactly the ERs were operating at the membrane. CREB phosphorylation is typically dependent on G-protein signaling, but classical ERs are not G-protein coupled. Nevertheless, electrophysiological experiments revealed that rapid estradiol signaling is closely associated with G-protein activity

(Mermelstein et al., 1996; Qiu et al., 2003). These early studies suggested three possible mechanistic explanations:

Hypothesis 1: G-proteins directly associate with, and are activated by, membrane-

associated ERα and/or ERβ (Levin, 2005; Vasudevan and Pfaff, 2006). This

hypothesis is supported by at least one study showing that estradiol-induced

interaction of ERα and G-protein subunit αi is involved in estradiol-mediated

endothelial nitric oxide synthase activation (Wyckoff et al., 2001). However, these

data do not exclude the possibility that an intermediary protein (e.g., a GPCR) forms

the necessary association of ERα and the G-protein.

Hypothesis 2: Estradiol-sensitive GPCRs mediate membrane-initiated signaling.

GPER-1 supports this hypothesis (Roepke et al., 2011; Srivastava and Evans, 2013),

although it does not explain the rapid effects of estradiol discussed here. Also, there is

some debate as to whether these receptors are localized to the surface membrane or

are localized to the endoplasmic reticulum (Srivastava and Evans, 2013). The Gq-

coupled membrane estrogen receptor (Gq-mER) is another putative estradiol-sensitive

GPCR (Roepke et al., 2009), but data suggesting its existence are based solely on

pharmacological studies. The lack of genetic identity indicates that the Gq-mER

ligand, STX, may in fact activate other known GPCR receptors (Christensen and

Micevych, 2013).

Hypothesis 3: Membrane-associated ERα and ERβ transactivate surface

membrane proteins, such as GPCRs, which stimulate intracellular signaling pathways

(Cardona-Gómez et al., 2000; Kahlert et al., 2000; Razandi et al., 2003; Song et al.,

2007). This hypothesis is supported by experiments conducted in multiple brain

regions and model systems, including our own primary neuron preparations.

Furthermore, evidence that ERs transactivate tyrosine kinase receptors such as

epidermal growth factor receptors and insulin-like growth factor receptors (Kahlert et

al., 2000; Razandi et al., 2003) provides precedence for the possibility that ERs can

transactivate GPCRs.

While I acknowledge that all of these hypotheses could be correct in certain contexts, the remainder of the chapter will focus on data related to Hypothesis 3.

ER-mGluR Interactions

Studies of ER-mGluR signaling began with the observation that picomolar concentrations of estradiol produced rapid CREB phosphorylation in primary hippocampal neurons (Boulware et al., 2005). CREB phosphorylation peaked within two minutes of estradiol exposure. An ERα agonist and a membrane-impermeable estradiol analog reproduced the increase in pCREB, while an estrogen receptor inhibitor (ICI

182,780) blocked it. Moreover, ERβ and GPER-1 agonists had no effect on pCREB levels in these neurons. This effect was unique to female neurons, due to the lack of testosterone during the organizational period of development (Meitzen et al., 2012).

To solve the puzzle of how ERα could activate intracellular signaling cascades that lead to CREB phosphorylation, the fact that ERα itself is not a GPCR, and therefore must partner with one, needed to be addressed. Specifically, it was hypothesized that ERα stimulates mGluRs, 7-transmembrane domain receptors that stimulate G-protein signaling following glutamate binding. There are eight types of mGluR, which fall into three groups and link to specific signal cascades:

1. Group I (mGluR1a and 5): Gq

2. Group II (mGluR2 and 3): Gi/Go

3. Group III (mGluR4, 6, 7, 8): Gi/Go

Focus was placed on group I mGluRs because they activate Gq signaling, which leads to CREB phosphorylation through MAPK, and because they are present in hippocampal neurons (Masu et al., 1991; Shigemoto et al., 1992; Baude et al., 1993;

Gereau and Conn, 1995). mGluR1a agonists mimicked, whereas antagonists blocked, estradiol-induced CREB phosphorylation, confirming the hypothesis (Boulware et al.,

2005). Interestingly, mGluR5 regulation did not impact estrogen signaling in this cell type. Experiments that blocked components of the mGluR1a → CREB signaling cascade

(PKC, IP3, MEK; Choe and Wang, 2001; Warwick et al., 2005) were all found to prevent estradiol-induced CREB phosphorylation, further supporting the involvement of mGluR1a in these estrogen effects (Boulware et al., 2005). This work was an important advance in the field because it demonstrated for the first time that a membrane-associated

ER can harness mGluR signaling in neurons. However, all rapid estradiol actions, even within a single neuron type, cannot be fully explained by the activity of one mGluR isoform. For example, in addition to increasing pCREB, ERα or ERβ can act through a

GPCR to decrease L-type calcium channel currents (Mermelstein et al., 1996; Lee et al.,

2002; Chaban et al., 2003). This effect cannot be attributed to the Gq signaling activated by mGluR1a, so the next step was to investigate whether membrane-associated ERs could also stimulate other mGluRs. Subsequent experiments showed that the aforementioned decrease in hippocampal L-type calcium currents is mediated via activation of mGluR2 and, that in contrast to ERα-mGluR1a signaling, both ERα and

ERβ are able to stimulate this pathway (Boulware et al., 2005). Thus, membrane- associated ERs can transactivate different mGluRs within the same type of neuron to induce precise effects on neuronal properties (See Figure 1.1). Specifically, membrane- associated ERs can trigger different signaling pathways depending on which mGluR they

associate with.

Figure 1.1: Schematic of membrane-initiated 17β-Estradiol (17βE) signaling. 17βE binds to membrane-localized estrogen receptors (ER) to activate distinct signaling pathways via association with Group I or Group II mGluRs. Interaction with group I mGluRs activates Gq-mediated signaling through protein lipase C (PLC) and protein kinase C (PKC), leading to intracellular calcium release and activation of MAPK/ERK. Interaction with group II mGluRs activates Gi/o-mediated inhibition of adenylyl cyclase (AC), decreasing activity (indicated by gray dashed lines) of protein kinase A (PKA), resulting in reduced L-type calcium channel currents. CAV, caveolin. IP3, inositol triphosphate. IP3R, IP3 receptor. CaM, calmodulin. MEK, MAPK/ERK kinase. MAPK, mitogen-activated protein kinase. RSK, ribosomal S6 kinase. CaMKIV, calcium/calmodulin-dependent protein kinase type IV.

This phenomenon has now been described many times, and many of the key findings are summarized in Table 1.1. Additionally, in some cases, the same neuron can contain multiple types of mGluR-ER interactions, but in other cases these interactions can be consistent within, and different between, particular brain regions.

Table 1.1. ER – mGluR functional interactions

Tissue Findings

 Estradiol signals through group I and II mGluRs to have opposite effects on CREB phosphorylation in female neurons Hippocampal (Boulware et al., 2005, 2007). neurons  ERα activates mGluR1a leading to endocannabinoid signaling that subsequently decreases presynaptic GABA release in female neurons (Huang and Woolley, 2012).

 ERα activates mGluR5, stimulating MAPK-dependent CREB Dorsal Striatal phosphorylation; ERα and ERβ activate mGluR3 to inhibit L- neurons type calcium signaling (Grove-Strawser et al., 2010).

 mGluR5 is required for E2-enhancement of cocaine-induced locomotor sensitization in female rats (Martinez et al., 2014). Nucleus Accumbens Core  mGluR5 activation of endocannabinoid pathways is required for E2-induced effects on dendritic spine density in female rats (Peterson et al., 2014, 2016).

 ER – mGluR1a coupling affects NMDA- or amyloidβ- induced Cortex neurotoxicity in cortical cultures from fetal mice (Spampinato et al., 2012a, 2012b)

 ERα – mGluR1a signaling is involved in sexual receptivity in Arcuate Nucleus female rats (Dewing et al., 2007). and Medial Preoptic Area  ERα – mGluR1a activity differs depending on the level of estrogens present (Mahavongtrakul et al., 2013).

 ERα couples to mGluR2/3 to suppress L-type calcium Dorsal Root signaling in cultured DRGs from female rats (Chaban et al., Ganglion 2011).

Hypothalamic  ERα – mGluR1a signaling occurs in hypothalamic astrocytes Astrocytes from female rats (Kuo et al., 2009, 2010a, 2010b).

ER-mGluR signaling has been implicated in a variety of behaviors, including estradiol-induced modulation of lordosis (Dewing et al., 2007), memory consolidation

(Boulware et al., 2013), drug addiction (Martinez et al., 2014), nociception (Chaban et

al., 2011; Lu et al., 2013), and motivation (Seredynski et al., 2015). These findings underscore the functional importance of membrane-initiated estradiol signaling through mGluR interactions.

Membrane-associated ER-mGluR Interactions in Medium Spiny Neurons

Medium spiny neurons are the predominant output neurons of the striatum, and are capable of influencing motor and cognitive behaviors through projections to other brain regions (Smith et al., 2013; Yager et al., 2015). In addition to dopaminergic and

GABAergic inputs, they receive multiple glutamatergic inputs (Groenewegen et al.,

1999; Kelley, 2004). Excitatory synaptic input onto medium spiny neuron dendrites is essential for generating action potential output and regulating synaptic plasticity

(O’Donnell and Grace, 1995; Mulder et al., 1998; Sesack et al., 2003; Papp et al., 2011;

Stuber et al., 2011; Britt et al., 2012). Medium spiny neurons express GPER-1, membrane-associated ERα and ERβ, and aromatase, but express few (if any) nuclear ERs

(Foidart et al., 1995; Mermelstein et al., 1996; Küppers and Beyer, 1998, 1999; Küppers et al., 2008; Schultz et al., 2009; Grove-Strawser et al., 2010; Almey et al., 2012, 2015,

2016; Stanić et al., 2014). Moreover, as in hippocampal neurons, ERα activation elevates pCREB in cultured striatal neurons. Unlike in hippocampal neurons, however, these increases depend on mGluR5 rather than mGluR1a (Grove-Strawser et al., 2010).

The nucleus accumbens core (NAcC) region of the ventral striatum is known for its unique sex differences. In particular, NAcC medium spiny neurons receive increased numbers of excitatory glutamatergic synapses in females relative to males (Forlano and

Woolley, 2010; Wissman et al., 2011a, 2011b; Cao et al., 2016). This sex difference is either absent or not as robust in other striatal regions such as the NAc shell (Wissman et

al., 2011a; Dorris et al., 2015; Willett et al., 2016). Perinatal estradiol exposure in males causes the relative decrease (Cao et al., 2016). Interestingly, estradiol injections in adult female rats similarly decrease putative glutamatergic input to NAcC neurons, as indicated by decreased dendritic spine density (Staffend et al., 2010; Peterson et al., 2014). Pre- treatment with an mGluR5 antagonist, MPEP, eliminates the change in spine density caused by estradiol (Peterson et al., 2014), while administration of an mGluR5 positive allosteric modulator mimics the effects of estradiol in the core region (Gross et al., 2016), corroborating in vitro findings. Additionally, experiments using a drug abuse paradigm demonstrate how these effects are functionally relevant. Specifically, estradiol is known to facilitate cocaine-induced locomotor sensitization in female rats, and mGluR5 inhibition abolishes this effect (Martinez et al., 2014). mGluR5 seems to cooperate with endocannabinoid signaling to produce these changes, as cannabinoid-1 receptor inhibition also eliminated spine density decreases and behavioral effects (Peterson et al., 2016).

This example illustrates how a specific ER-mGluR interaction can facilitate molecular, structural, and behavioral changes.

Caveolin organizes functional signaling microdomains

Given the degree of fine spatial tuning of ER-mGluR interactions and their downstream effects, we wanted to identify a mechanism that could account for this specificity. Based on previous work conducted outside the nervous system, caveolin proteins were a candidate for an intermediary protein that would allow ERs and mGluRs to functionally interact in a spatially localized manner. Experiments exploring the relationship between neuronal caveolin and membrane-associated ER signaling demonstrated that modifying caveolin expression and/or activity interferes with the

specific actions of estradiol on CREB phosphorylation (Boulware et al., 2007; Grove-

Strawser et al., 2010; Stern and Mermelstein, 2010). Moreover, the particular caveolin isoform determines the nature of the ER-mGluR interaction: Cav1 is necessary for ERα- mGluR1a/mGluR5 mediated increases in pCREB, whereas Cav3 is necessary for

ERα/ERβ-mGluR2/mGluR3 mediated decreases in pCREB (Boulware et al., 2007). That is, Cav1 and Cav3 organize unique signaling microdomains that link membrane- associated ERs to specific mGluR partners in order to produce divergent effects. Figure

1.2 illustrates the interactions of these proteins at the plasma membrane. Additionally, links between ERα and/or ERβ and Cav1 have been reported in the brain tissues listed in

Table 1.2.

Figure 1.2. Schematic of estradiol/ER/mGluR signaling microdomains organized by caveolin and palmitoylation. The specific ER, mGluR, and caveolin isoforms differ by signaling pathway and brain region. Abbreviations: ER, estrogen receptor; mGluR, metabotropic glutamate receptor; G, G-protein.

Table 1.2. Summary of ER-Cav-mGluR interactions

Brain Tissue ER Cav mGluR References

Septal neurons ERα Cav1 ? Marin et al., 2009 ERα Cav1 mGluR1a Boulware et al., Hippocampus ERα/ 2007 Cav3 mGluR2 ERβ

Mesencephalic Volpicelli et al., ERα Cav1 ? cells 2014

N-38 NPY ERα Cav1 ? Titolo et al., 2008 neurons

(Ramírez et al., Cortex ERα Cav1 mGluR1a 2009); (Yang et al., 2015b)

CNS myelin and Arvanitis et al., ERβ Cav1 ? oligodendrocytes 2004

ERα Cav1 mGluR5 Striatum (Dorsal Grove-Strawser et and Nucleus ERα/ al., 2010 accumbens) Cav3 mGluR3 ERβ

Dominguez and Micevych, 2010; Hypothalamus ERα Cav1 mGluR1a Christensen and Micevych, 2012.

Dorsal Root mGluR2 Chaban et al., ERα ? Ganglion /mGluR3 2011

These data indicate that caveolin proteins play a major role in linking membrane- associated ER to specific mGluR signaling pathways.

In addition to creating the spatial organization needed to cluster ERs with mGluRs, caveolin proteins likely facilitate trafficking of ERs to the plasma membrane.

Caveolin has been implicated in subcellular trafficking and endocytosis of a number of proteins, including dopamine receptors (Kong et al., 2007), metabotropic glutamate receptors (Hong et al., 2009), and M1 muscarinic receptors (Shmuel et al., 2007) to name a few (see Stern and Mermelstein (2010) for review). Supporting this idea, Cav1 deficiency disrupts mGluR signaling in the hippocampus (Francesconi et al., 2009) and decreases membrane expression of ERα in the hypothalamus (Christensen and Micevych,

2012).

Palmitoylation: Regulating membrane interactions

The fact that the same ER isoforms responsible for nuclear signaling also act at the membrane suggested that an additional regulatory mechanism must determine whether ERα and ERβ are targeted to the plasma membrane instead of the nucleus. It had been shown in several non-neuronal cell types that ERα undergoes a post-translational modification called palmitoylation that allows trafficking to the plasma membrane, so we predicted that palmitoylation might regulate ER activity in neurons as well. There are several forms of palmitoylation, but ERs appear to undergo S-palmitoylation. S- palmitoylation is the only known reversible lipid modification, and has been shown to control transient membrane tethering of otherwise cytosolic proteins (Wedegaertner et al.,

1993; Topinka and Bredt, 1998; Linder and Deschenes, 2007). Proteins belonging to the palmitoyl acyltransferase (PAT) family of enzymes are responsible for adding the 16- carbon fatty acid palmitate to target proteins, usually via a thiol-ester bond at cysteine residues (Fukata and Fukata, 2010). Palmitate attachment increases the lipophilicity/hydrophobicity of the protein, facilitating association with lipid membranes

and lipophilic proteins. In addition to serving as a lipophilic anchor, palmitate may also signal to cellular trafficking mechanisms (Greaves and Chamberlain, 2007).

Twenty-three PATs have been identified to date, all members of the DHHC family of enzymes, named for their characteristic aspartate-histidine-histidine-cysteine motif that is responsible for transferring the palmitate group (Fukata et al., 2004; Hou et al., 2009). DHHCs are integral transmembrane proteins localized to different cellular compartments. In HEK293 cells, most DHHC proteins are localized to the endoplasmic reticulum and/or Golgi apparatus, with a few at the plasma membrane (Ohno et al.,

2006), but distribution likely depends on cellular context (Fukata et al., 2016).

Additionally, neuronal DHHCs can be dynamically relocated in response to synaptic changes (Fukata et al., 2013). Remarkably, mRNA for all 23 PAT enzymes has been detected in hippocampal neurons (Meitzen et al., 2013). Ellis Levin’s group identified two PATs necessary for ERα trafficking in MCF-7 cells: DHHC7 and DHHC21 (Pedram et al., 2012). Using this work as a foundation, we hypothesized that palmitoylation is required for membrane-initiated estrogen signaling in neurons, and that these same

DHHC proteins would be involved. To test these hypotheses, we first pharmacologically blocked palmitoylation in primary hippocampal neurons using the broad-spectrum palmitoylation inhibitor 2-bromopalmitate (2-Br). 2-Br exposure eliminated estradiol- induced changes in pCREB, indicating that palmitoylation is indeed necessary for membrane-associated ER signaling in neurons (Meitzen et al., 2013). As discussed below, subsequent experiments narrowed the investigation to determine whether ERα and

ERβ must be directly palmitoylated in order to carry out membrane-initiated signaling in neurons.

ERα and ERβ (as well as progesterone, androgen, and glucocorticoid (α, β, γ) receptors) contain a highly conserved motif designated as the palmitoylation sequence

(Pedram et al., 2007). In rats, the ERα palmitoylation site is at cysteine 452, which is part of the conserved amino acid motif (ERα 446-461: QGEEFVCLKSIILLNS) (Acconcia et al., 2004; Pedram et al., 2007). ERβ is palmitoylated at cysteine 354, also part of the palmitoylation motif (ERβ 348-363: QHKEYLCVKAMILLNS). Radiolabeled palmitate assays have demonstrated that mutation of the consensus palmitoylation sequence on

ERs, particularly the cysteine residue, prevents incorporation of palmitate, and also prevents ER – Cav interactions (Acconcia et al., 2004). Because Cav interaction is required for membrane signaling in neurons, it followed that ER palmitoylation would be required as well. To test this, we mutated the palmitoylation site on ERα and ERβ from cysteine to alanine in order to prevent palmitoylation (Meitzen et al., 2013). We first transfected neurons with the altered ERα (ERα C452A), which eliminated membrane- associated ERα-mediated changes in CREB phosphorylation, but did not affect membrane-associated ERβ signaling. We then transfected neurons with the altered ERβ

(ERβ C354A), and this eliminated membrane-associated ERβ-mediated changes in

CREB phosphorylation, but did not affect membrane-associated ERα signaling.

Importantly, overexpression of wild-type ER did not affect membrane-associated ER signaling. Nuclear signaling through these receptors remained functional in transfected neurons. These data demonstrated that the non-palmitoylatable mutants of ERα and ERβ act as dominant-negatives, eliminating membrane signaling of the respective endogenous

ERα or ERβ in neurons.

Having found that ER palmitoylation is required for membrane signaling through the receptor, we next tested whether DHHC7 and 21 are involved in this process in neurons (Meitzen et al., 2013). We transfected neurons with siRNA directed against

DHHC7 and DHHC21, along with appropriate controls – siRNA against no known target or against DHHC10, a DHHC with no known connection to hormone receptors (Fukata et al., 2004; Ohno et al., 2012; Pedram et al., 2012). We found that knockdown of either

DHHC7 or DHHC21 eliminates modulation of CREB phosphorylation through membrane-associated ERα and through membrane-associated ERβ. This result was surprising because redundancy exists for most palmitoyl substrates; that is, the different

DHHC PATs have overlapping targets (Hou et al., 2009). Importantly, glutamatergic mGluR signaling was unaffected in our experiments, consistent with previous work indicating that mGluRs are not palmitoylated (Alaluf et al., 1995; Pickering et al., 1995).

Because mGluR signaling remained intact, and because the siRNA did not affect expression levels of ERα, ERβ, Cav1, or Cav3, we attribute the observed impairments in membrane-initiated estradiol signaling to a direct loss of membrane-associated ER activity and/or to interruptions in the caveolin-dependent ER-mGluR interaction resulting from decreased DHHC7 and 21. It remains possible that other DHHC proteins also interact with membrane-associated ERs in neurons, but this would be a surprising departure from non-neural cells.

Because knocking down either DHHC7 or 21 interferes with membrane- associated ER signaling, each PAT alone must not be sufficient to palmitoylate and/or maintain palmitoylation of ER at the plasma membrane. There are at least three possible

models that can explain the relationship between DHHC7/21, and membrane-associated

ER signaling (Figure 1.3).

Simultaneous: The two DHHC proteins could form a functional heterodimer that palmitoylates ER. Though not well explored, there are some data indicating that such a process can occur in neurons (Fukata et al., 2004).

Serial: DHHC7 and 21 work in sequence to facilitate translocation and maintenance of ERs at the plasma membrane. For example, one DHHC could be responsible for the initial palmitoylation of ER while the second maintains the palmitoylation at the membrane. Consistent with this model, maintenance of the palmitoyl moiety on membrane-associated ERs would be necessary, since palmitoylation is a reversible process (Fukata et al., 2004). There are several findings that provide additional support for this model. First, membrane-associated ER can be cycled in and out of the membrane of non-neural cells (Dominguez and Micevych, 2010). Additionally, membrane-associated ERα is depleted after depalmitoylation caused by exposure to

Palmostatin B (Tabatadze et al., 2013). Finally, DHHC7 appears localized to the Golgi apparatus in hippocampal neurons (Thomas et al., 2012), which would be consistent with

DHHC7 providing a palmitate group as a signal for protein trafficking. Indeed, DHHC7 is known to regulate many proteins and is often involved in the first steps to target proteins to the membrane (Fukata et al., 2004; Fernández-Hernando et al., 2006; Greaves et al., 2008; Ponimaskin et al., 2008; Huang et al., 2009a; Tsutsumi et al., 2009). Much less is known about DHHC21, however.

Parallel: The two DHHCs palmitoylate separate but equally necessary proteins involved in membrane-associated ER signaling. For example, one DHHC could

palmitoylate ER, while the other could palmitoylate caveolin. Indeed, caveolin proteins have at least three putative palmitoylation sites located near the c-terminus. Although palmitoylation of caveolin is not necessary for its translocation to the cellular membrane, our preliminary data suggest that Cav1 palmitoylation supports the presence of ERα at the plasma membrane (Tonn and Mermelstein, 2014).

Figure 1.3. Possible models by which the enzymes DHHC7 and DHHC21 work to facilitate membrane-initiated signaling through ERs. Abbreviations: ER, estrogen receptor; 7, DHHC7; 21, DHHC21.

Regardless of the model, palmitoylation helps explain the dynamic relationship between estrogen receptors, caveolin proteins, and mGluRs at the membrane.

Palmitoylation is necessary for membrane trafficking and anchoring for originally soluble proteins (i.e., peripheral membrane proteins) but not for transmembrane proteins. It can, however, affect partitioning of transmembrane proteins into specialized domains, including lipid rafts (Fukata et al., 2016). The association of ERs with caveolin proteins facilitates trafficking to membrane lipid rafts, as caveolins are a key component of these

specialized domains. Therefore, palmitoylation of the ER is crucial not only for its interaction with Cav, but also for its existence within the membrane raft where it is available for transactivation of an mGluR (Levental et al., 2010). Importantly, the caveolin-lipid organizational system is critical for regulation of mGluRs as well

(Francesconi et al., 2009; Takayasu et al., 2010). Resolving the remaining questions of which DHHCs do what, and where, will further clarify our understanding of how membrane estrogen signaling is regulated.

Purpose and Aims

Because Cav1 facilitates ER/mGluR interaction, it is likely involved in determining when and how ERs and mGluRs associate. Moreover, its palmitoylation state is of particular interest, as this is another likely regulatory mechanism for

ER/mGluR signaling. Beyond its role in facilitating ER/mGluR signaling, the basic role of Cav1 in neurons remains poorly understood. Thus, the experiments described here were performed in order to fill a gap in our knowledge regarding Cav1 palmitoylation and the function of Cav1 in the context of drug-induced plasticity.

Aim 1: Determine whether DHHC7 and/or DHHC21 can palmitoylate Cav1.

Aim 2: Test the necessity and sufficiency of Cav1 for cocaine-induced behavioral sensitization. Identify cellular phenotype of Cav1 KO mice. Determine whether palmitoylation is required for normal Cav1 function.

CHAPTER 2: Palmitoylation of Caveolin-1 is regulated by the same DHHC

enzymes as steroid hormone receptors

Introduction

Palmitoylation is the posttranslational addition of a 16-carbon lipid chain that increases protein hydrophobicity. S-palmitoylation, occurring via thioester bonds at cysteine residues, is reversible, making it an important means of dynamic cellular regulation (Fukata et al., 2013). Palmitoylation has been implicated in many processes, including regulation of protein conformation, membrane association, protein-protein interactions, and compartmentalization and trafficking within cells (Greaves and

Chamberlain, 2007; Greaves et al., 2009; Fukata and Fukata, 2010; Blaskovic et al.,

2014). Through these mechanisms, palmitoylation can modulate a variety of signal transduction pathways. One such pathway is membrane-initiated estradiol signaling, which occurs in the nervous system via estrogen receptor (ER) interactions with metabotropic glutamate receptors (mGluRs). This interaction leads to estradiol-mediated mGluR signaling independent of glutamate (Boulware et al., 2005; Meitzen et al., 2013).

Neuronal ER/mGluR signaling in females underlies the effects of estradiol on learning and memory, nociception, motor control, sexual receptivity, and heightened responses to drugs of abuse (Tonn Eisinger et al., 2017). Recent findings also implicate ER/mGluR signaling in the male quail brain (Cornil et al., 2012; Seredynski et al., 2015), and the male and female rodent cerebellum (Hedges et al., 2018). The ERs that signal through mGluRs are the same proteins that regulate gene expression in the nuclei of cells, except they have undergone palmitoylation, thereby promoting their trafficking to the plasma membrane (Acconcia et al., 2004; Pedram et al., 2007). Two specific palmitoyl acyltransferases, DHHC7 and DHHC21, have been identified as crucial for ER palmitoylation; disrupting the expression of either enzyme will eliminate membrane ER

localization (Pedram et al., 2012) and neuronal ER/mGluR signaling (Meitzen et al.,

2013). These results are somewhat surprising. As there is only one palmitoylation site on

ERs, it has been unclear why knockdown of either enzyme would disrupt ER trafficking, and not require inhibition of both DHHC7 and DHHC21.

The fact that ER/mGluR interactions do not occur spontaneously, but rather require caveolin proteins to physically associate (Razandi et al., 2002; Boulware et al.,

2007; Christensen and Micevych, 2012) may provide clues to resolve this discrepancy.

Caveolins are integral membrane proteins enriched in lipid rafts, and are responsible for creating microdomains of signaling proteins, including mGluRs (Hansen and Nichols,

2010; Stern and Mermelstein, 2010). There are three caveolin isoforms with specific expression patterns and interaction partners. Caveolin proteins are also palmitoylated

(Dietzen et al., 1995), although the DHHC enzymes responsible are unknown. In this study, I examined the palmitoylation of caveolin-1 (CAV1), as it clusters group I mGluRs with estrogen receptor  (ER) (Boulware et al., 2007; Luoma et al., 2008). I find that the same enzymes responsible for ER palmitoylation can also affect CAV1 palmitoylation, supporting the hypothesis that the same DHHC enzymes cooperate to facilitate surface membrane signaling.

Methods

Estradiol-mediated CREB phosphorylation

Cell culture

Hippocampal neurons were cultured from female Sprague-Dawley rat pups at postnatal day 1 or 2 as described previously (Boulware et al., 2005), in accordance with the Institutional Animal Care and Use Committee at the University of Minnesota.

Hippocampi were isolated in ice-cold modified Hanks’ balanced salt solution containing

20% fetal bovine serum (FBS, Atlanta Biologicals) and 4.2 mM NAHCO3 and 1 mM

HEPES, pH 7.35, 300 mOsm. Tissue was then washed and subjected to a five minute digestion in a trypsin solution with 137 mM NaCl, 5 mM KCl, 7 mM Na2HPO4, 25 mM

HEPES, and 1500 U of DNase, pH 7.2, 300 mOsm. After additional washes, tissue was dissociated by trituration and pelleted twice by centrifugation. Cells were then plated on

Matrigel-treated coverslips and incubated for 20 minutes at room temperature prior to the addition of 2 mL MEM (Invitrogen, Carlsbad, CA) with 28 mM glucose, 2.4 mM

NaHCO3, 0.0013 mM transferrin (Calbiochem, San Diego, CA), 2 mM glutamine,

0.0042 mM insulin, 1% B-27 (Invitrogen), and 10% FBS (pH 7.35, 300 mOsm). 48 hours after plating, 1 mL of medium was replaced with a solution containing 4 μM cytosine 1-

β-D-arabinofuranoside to inhibit glial growth. Cells were fed four days later by replacement of 1 mL medium. Gentamicin (2 μg/mL; Invitrogen) was added to all media solutions to eliminate bacterial growth.

Immunocytochemistry

Cell stimulations and immunocytochemistry were performed as described previously (Boulware et al., 2005). Neurons (8-9 days in vitro) were pre-treated in a

Tyrode solution containing TTX (1 μM) and APV (25 μM) for two hours prior to a five- minute stimulation with 1 nM 17β-estradiol. Neurons were then fixed in ice-cold 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS containing 4 mM EGTA and subsequently washed in PBS, permeabilized with 0.1% Triton X-100, washed again, and then blocked at 37° C for 30 minutes in PBS plus 1% BSA and 2% goat serum (Jackson ImmunoResearch, West Grove, PA). The cells were stained with

primary antibodies directed against serine 133-phosphorylated cAMP response element binding protein (pCREB; monoclonal, 1:1000, Upstate Biotechnology) and against microtubule-associated protein 2 (MAP2; polyclonal, 1:1000, Calbiochem). Alexa Fluor

488 and 635 secondary antibodies (Invitrogen) were used for visualization of MAP2 and pCREB, respectively. Coverslips were washed and mounted using Citifluor (Ted Pella,

Redding, CA). Nuclear fluorescence intensities for pCREB were acquired using a Leica

DM5500Q confocal system and quantified with Leica LAS AF (version 1.9.0; Leica).

Neurons were selected randomly using MAP2 fluorescence, and images were captured through the approximate midline of each cell. For analysis, a region of interest was drawn around the nucleus of each neuron according to MAP2 staining, which allowed unbiased analysis of pCREB intensity. This was done by transferring the region of interest from the MAP2 image to the pCREB image and then measuring fluorescence intensities. Average pCREB fluorescence intensities within the nucleus were recorded (n

= approx. 25 neurons/group). The background from a region of the image not containing pCREB fluorescence was subtracted from the average pCREB fluorescence.

Measurement of protein palmitoylation

Acyl-biotin exchange (ABE)

HEK293 cells (ATCC, Manassas, VA) were maintained at 37 C, 5% CO2 in

Dulbecco’s Modified Eagle’s Medium supplemented with 1% penicillin/streptomycin and 5% fetal bovine serum. Acyl-biotin exchange was performed according to procedures outlined previously (Kang et al., 2008; Brigidi and Bamji, 2013; Woolfrey et al., 2015).

HEK293 cells were lysed in buffer containing 150 mM NaCl, 50 mM Tris pH 7.4, 5 mM

EDTA, 0.2% Triton X-100, 10 mM NEM, and protease inhibitor cocktail (Pierce) by

sonication. Additional Triton X-100 was then added for a total concentration of 1.7%.

Lysates were then mixed at 4 C for 1 hour, and then clarified at 12,000 x g for 10 min.

Chloroform-methanol precipitation was performed and then proteins incubated with 50 mM NEM overnight to block free cysteine residues. Protein was again precipitated the following day, and divided into two equal portions. One portion was incubated with 10 mM HPDP-biotin buffer containing hydroxylamine (1 hour, room temperature) to cleave thioesterase bonds and replace palmitoyl groups with biotin. The remaining half was incubated with 10 mM HPDP-biotin (Life Technologies) without hydroxylamine as a control. Chloroform-methanol precipitation was again performed, and then samples were incubated with streptavidin-agarose beads (GE Healthcare, Chicago, IL) in lysis buffer

(no NEM) overnight to capture biotin-labeled proteins. 10% of each sample portion was reserved prior to pull down for use as input control. The following morning, beads were washed four times with lysis buffer before proteins were eluted in Lamelli sample buffer

(Biorad, Hercules, CA) at 90 for 10 mins. Eluted proteins and input controls were then subjected to SDS-PAGE (4-20% Mini-PROTEAN TGX Precast gels, Biorad) and western blotting (nitrocellulose, Biorad). Blots were blocked with Odyssey Blocking

Buffer TBS (Licor, Lincoln, NE) and incubated overnight in primary antibody.

Antibodies: Rabbit anti CAV1 1:5000 (Abcam, Cambridge, MA, ab2910), Goat anti HA tag 1:5000 (Abcam, ab9134), Mouse anti HA tag 1:5000 (Abcam, ab18181), Mouse anti myc tag 1:5000 (Sigma, M4439). Blots were then washed in tris-buffered saline + tween and incubated with the appropriate fluorescent secondary antibodies (Licor). Blots were imaged and analyzed using Odyssey scanner and software (Licor). Palmitoylated proteins were normalized to their respective input control for data analysis.

Antibody validation

Tissue punches obtained from wild type (WT) or CAV1 knockout (KO) mice were homogenized with a bullet blender (Next Advance, Troy, NY) and lysed in RIPA buffer supplemented with protease and phosphatase inhibitors. Equal protein was run on

SDS-PAGE prior to Western blotting with 1:5000 dilution of rabbit anti Cav1 (Abcam, red) and 1:20,000 mouse anti GAPDH (Millipore, green). (Channels of two-color blot were merged and changed to gray scale using ImageJ.)

2-Bromopalmitate incubation

HEK293 cells were treated overnight with either 100 μM 2-Bromopalmitate (2-Br,

Sigma), prepared at 10 mM in DMSO and diluted to final concentration in culture media, or with vehicle alone. ABE was performed as described above. Following western blotting for CAV1 as described above, blots were stripped with 1X NewBlot IR Stripping

Buffer (LiCor) for 15 minutes, washed and blocked, and probed for Ras (rabbit anti pan-

Ras, Abcam 52939, 1:5000).

Immunoprecipitation

Cells were lysed in ice cold buffer containing 150 mM NaCl, 50 mM Tris pH 8, 1%

NP-40, and protease and phosphatase inhibitors (Pierce). Equal amounts of lysate per sample were incubated in anti myc antibody (Sigma M4439) in PBS for 1 hour at 4 C.

Dynabeads G (Novex, Invitrogen) were then added and the incubation continued overnight. Beads were washed the following morning before elution in 2X Lamelli

Buffer (Biorad). Eluted proteins were then subjected to SDS-PAGE and western blotting as described above.

Plasmid and siRNA transfection

CAV1 was inserted into a pCMV-HA vector (Addgene, 32530) using PCR-based cloning. mNeongreen-CAV1 was obtained from Allele Biotechnology (San Diego, CA).

DHHC plasmids were a gift from the Bamji Lab (University of British Columbia). Site- directed mutagenesis was performed using QuikChange Lightning Multi (Agilent, Santa

Clara, CA). Cells were transfected using Xtremegene HP (Roche, Indianapolis, IN).

siRNAs were purchased from GE Healthcare Dharmacon, specifically ON-

TARGETplus Non-targeting or SMARTpool. These contain a mixture of four distinct siRNAs against each target. Cells were transfected with 50nM siRNA using Dharmafect

(GE Healthcare). Knockdown was verified using qPCR as described previously (Meitzen et al., 2013). Briefly, RNA was extracted from cells using RNeasy kit (Qiagen, Hilden,

Germany), followed by cDNA synthesis from 1 μg of RNA using Roche Transcriptor

First Strand cDNA synthesis kit. qPCR was performed on Lightcycler 480 (Roche) using

Lightcycler 480 SYBR Green I Master. Primers: RPL13A, 5’-

TACTTCACTGTTTAGCCACGAT-3’ and 5’-CGAAGATGGCGGAGGTG-3’. zDHHC7, 5’-GTCCTGATGGCTGCATGA-3’ and 5’-

GACAGTATGCACCTTAAGATCCT-3’. zDHHC11, 5’GGATCACAGGGGCACCT-3’ and 5’-ATGGCACAGGAAGCAGATG-3’. zDHHC21, 5’-

CACTTGTTACATAATTCCCAGAACT-3’ and 5’-GGCCTCCATAACTGATCCAG-

3’. Results were analyzed using the ΔΔCT method (Schmittgen and Livak, 2008).

Total Internal Reflectance Fluorescence (TIRF) imaging

Immunocytochemistry

HEK293 cells were transiently-transfected with pMNG-CAV1-C10 or pMNG-

CAV1-MT with Lipofectamine 3000 according to the manufacturer’s suggestions

(Invitrogen). The next day, cells were scraped from flasks and seeded onto 35mm imaging dishes equipped with #1.5 coverglass (Eppendorf North America, Hauppauge,

NY). Two days post-transfection, the cell growth media was removed and the cells fixed using a solution of 2.0% formaldehyde, 7% picric acid in a tris and phosphate-buffered saline (a 1:1 mixture of TBS and PBS); pH 6.9 for 2 minutes at room temperature. The fixative solution was drawn off and replaced with a solution of 4% formaldehyde, 14% picric acid in PBS; pH 6.9 for 5-10 minutes at room temperature. The fixative was removed from the cells and washed several times with TBS. The cells were stained using a 1:1000 dilution of rabbit anti CAV1 (Abcam) in TBS containing 0.2% triton X-100

(Fisher Scientific) and 0.2% tween-20 (Sigma) for 4 hours to overnight at room temperature. The cells were washed for one hour with TBS and then stained with 1:1000 donkey anti rabbit Alexa 568 in the TBS/triton/tween diluent for two hours at room temperature. The cells were washed for one hour with TBS and the solution replaced with

DMEM containing antibiotics (Anti Anti, Invitrogen) to inhibit bacterial growth during storage before imaging at 4°C.

TIRF Microscopy

TIRF imaging was performed using a Zeiss Axio Observer Z1 inverted microscope (Carl Zeiss Microscopy, Thornwood, NY) equipped with a 100X/1.46na oil objective and Zen2 Acquisition and Analysis Software. Widefield images were obtained

by first locating endogenous caveolin-1 that was visualized using immunocytochemistry and Alexa 568 fluorescence. After a cell was identified and imaged using differential interference contrast (DIC) optics, the 561nm laser was engaged at 10% power and, focusing from the coverglass into the cell, at the first instance of in-focused fluorescence of caveolin 1 (Alexa568 stained structures), a widefield image was acquired. The 488nm laser was then engaged at 1% power and a widefield image of mNeongreen fluorescence was obtained. The microscope was then switched to TIRF mode, the optimal angle of incidence was determined (typically the angle was ~65°) and images obtained. The images were converted to TIFF using the Fiji build (version 2) of the freely-available

Image J software (ImageJ.net) and were minimally processed (adjusted contrast, brightness and unsharp mask) using Photoshop software (CS5.1, Adobe Systems Inc.).

For analysis, cells were traced in Fiji and mean TIRF fluorescence was normalized to the total wide-field fluorescence. Log transformation was performed to correct for unequal variances prior to quantification and use of the unpaired t-test.

Subcellular localization immunocytochemistry

HEK293 cells plated on poly-d-lysine coated coverslips in 6-well tissue culture plates were transfected with 0.5 µg plasmid DNA (DHHC7 or DHHC21) using

XtremeGENE HP (Roche). Twenty-four hours later, cells were fixed, permeabilized, and blocked using reagents from Image-iT Fixation/Permeabilization Kit (Thermo Fisher).

Briefly, cells were fixed for 15 minutes at room temperature in 4% formaldehyde, washed in PBS, and then permeabilized for 15 minutes at room temperature with 0.5%

Triton X-100. Cells were again washed and then blocked in 3% BSA for one hour at room temperature. Cells were then incubated for 24 hours at 4 C in primary antibodies

diluted in BlockAid (Thermo Fisher) at 1:200 (rabbit anti Myc-Tag 71D10, CST; mouse anti Golgin-97 CDF4, Thermo Fisher). Following PBS washes, cells were then incubated in secondary AlexaFluor+ antibodies diluted 1:1000 in Block Aid for one hour at room temperature (goat anti mouse 488, A32723; goat anti rabbit 555, A32732; Thermo

Fisher). Cells were again washed and then subjected to 5-minute incubation with 5 µM

DRAQ5 nuclear stain (CST). Coverslips were washed a final time and then inverted into

ProLong Diamond Antifade Mountant (Thermo Fisher) on slides for visualization.

Confocal images were taken using a Leica DM5500Q confocal system and Leica LAS X software. Z-stacks were taken through the cell using a 40X objective plus 3X digital zoom. Laser power, emission filters, and gain were set to optimize dynamic range. Fiji software was used to pseudocolor, minimally process, and create maximum projections of Z-stack images.

Statistical analysis

Data generated by Western blot and immunocytochemistry were analyzed by one- way ANOVA or unpaired t-test, as appropriate. All experiments were repeated at least three times, with exemplar blots shown in figures. Tukey’s tests were used for all post hoc analyses between treatment groups, with a determination of p < 0.05 for significance.

Graphs are presented as mean ± SEM.

Results

DHHC7 and DHHC21 are required for the rapid effects of estradiol on CREB phosphorylation

Estrogen receptors have a single palmitoylation site contained within a palmitoylation motif conserved across steroid hormone receptors. Of the 23 palmitoyl

acyltransferase enzymes, two have been found essential for palmitoylation of these receptors, DHHC7 and DHHC21. These two enzymes regulate estrogen receptor trafficking in MCF-7 cells (Pedram et al., 2012), and membrane estrogen receptor signaling in neurons (Meitzen et al., 2013). Here, we confirm that these enzymes are required for rapid membrane-initiated estradiol signaling. To do so, we transfected cultured hippocampal neurons derived from female rat pups with short-interfering

(si)RNAs targeting genes zDHHC7 and zDHHC21 and subjected the neurons to five- minute estradiol stimulation. Normally, this stimulation increases nuclear CREB phosphorylation, as was the case with a non-targeting control siRNA, or when targeting zDHHC11, which contains the transcript for DHHC10, an enzyme that does not palmitoylate steroid hormone receptors (Figure 2.1). In contrast, disrupting expression of either DHHC7 or DHHC21 abolished the estradiol-induced CREB phosphorylation. siRNA targeting DHHC7 produced a 92% decrease in mRNA (p<0.05, as measured via

RT-qPCR), without affecting DHHC21 (13%, n.s.). Reciprocally, siRNA targeting

DHHC21 produced a 72% decrease in its mRNA (p<0.05, as measured via RT-qPCR), without affecting DHHC7 (10%, n.s.).

Figure 2.1. DHHC7 and DHHC21 are required for estradiol-induced CREB phosphorylation. Primary cultures of female-derived rat hippocampal neurons were transfected with control siRNAs, or those targeting zDHHC7, zDHHC21, or zDHHC11 (DHHC10). Forty-eight hours later, cultures were stimulated for five minutes with 1nM 17β-estradiol (grey bars) or vehicle (white bar) prior to immunocytochemical processing for CREB phosphorylation (pCREB, red; microtubule associated protein 2, green). Estradiol-induced pCREB was eliminated when expression of either DHHC7 or DHHC21 was disrupted (*p<0.01 vs. no target siRNA estradiol-treated group).

CAV1 is endogenously palmitoylated

Given that estrogen receptors have only one palmitoylation site, but knockdown of either DHHC7 or DHHC21 eliminated estradiol-induced CREB phosphorylation, we

sought to ascertain whether these enzymes also play a role in CAV1 palmitoylation, since

CAV1 is essential for ER/mGluR coupling. To test our hypothesis that DHHC7 and/or

DHHC21 can affect CAV1 palmitoylation, I first validated the specificity of the CAV1 antibody used for quantification by western blotting samples from wild-type (WT) or

CAV1 knockout (KO) mice (Razani et al., 2001). I found that the antibody used in these studies detected a band of the correct size (approx. 22 kDa) in WT mice, but did not detect a similar band in KO mice (Figure 2.2A). I next determined whether CAV1 is palmitoylated under steady-state conditions in HEK293 cells using an acyl-biotin exchange assay (Figure 2.2B). I found that CAV1 is palmitoylated under unstimulated conditions (Figure 2.2B, first two lanes). I next overexpressed myc-tagged DHHC7,

DHHC21, and DHHC10 in these cells (Figure 2.2B, next 6 lanes), hypothesizing that

DHHC7 or DHHC21 would increase CAV1 palmitoylation, and that DHHC10 would have no effect. Counter to my prediction, none of the overexpressed enzymes increased palmitoylation of endogenous CAV1. As a control, I verified expression of the myc-

DHHC proteins via western blot (Figure 2.2C).

Figure 2.2. Endogenous CAV1 exhibits palmitoylation permanence. (A) Validation of the CAV1 antibody using striatal brain tissue from two wild-type versus two CAV1 knockout mice. GAPDH was used as a loading control. (B) Detection of CAV1 In the presence, but not the absence, of hydroxylamine (HAM) in the ABE assay indicates endogenous CAV1 palmitoylation in HEK293 cells. Endogenous CAV1 palmitoylation was unaffected by DHHC7, DHHC21, or DHHC10 overexpression. (C) Validation of overexpression of myc-tagged DHHC7, DHHC10, and DHHC21; two samples shown of each. (D and E) Overnight treatment with 100µM 2-Br decreased palmitoylation of Ras, but not of CAV1.

Although this finding was initially surprising, previous reports suggested a possible explanation. While CAV1 palmitoylation is technically reversible in that it occurs through S-palmitoylation (a bond that would be subject to depalmitoylation), Parat and Fox found that CAV1 palmitoylation is, in fact, an essentially permanent modification (Parat and Fox, 2001). It was hypothesized that once palmitoylated, these sites are immediately embedded in the membrane and become inaccessible to depalmitoylating enzymes. To determine whether this might explain the results, I treated cells with the pan-palmitoylation inhibitor, 2-Bromopalmitate (2-Br) overnight. I used small GTPase Ras proteins as a positive control because they are ubiquitously expressed and dynamically palmitoylated (Hancock et al., 1989; Rocks et al., 2005; Eisenberg et al.,

2013). Although 2-Br decreased palmitoylated Ras protein levels by approximately 80%

(Figure 2.2D, t(4) = 3.564, p < 0.05), 2-Br did not decrease CAV1 palmitoylation levels

(Figure 2.2E, t(4) = 0.7528, p = 0.49). Thus, it is likely that the timescale of these experiments was insufficient to detect any changes in endogenous CAV1 palmitoylation.

For further experiments, I needed to isolate a newly translated pool of CAV1. One strategy to do so would be to halt protein synthesis with cycloheximide (CHX) and then monitor changes in palmitoylation of newly translated protein following release from

CHX treatment. However, I found that overexpression of DHHC7 or DHHC21 eliminated the decrease in CAV1 palmitoylation following CHX treatment that was observed in non-transfected cells (Figure 2.3). Therefore, while this suggested a stabilization effect, it was not a viable approach for determining DHHC-substrate relationships.

Figure 2.3. Overexpression of DHHC7 or DHHC21 prevents cycloheximide-induced decreases in CAV1 palmitoylation. HEK293 cells were treated with 28 µg/mL cycloheximide (CHX) for one hour (a dose previously shown to reduce CAV1 palmitoylation) and harvested immediately or following a 45-minute washout period. Acyl-biotin exchange was conducted to detect palmitoylated CAV1. (A) CAV1 palmitoylation is reduced following CHX treatment, and restored by washout in non- transfected HEK293 cells (*p<0.05). (B) DHHC7 or (C) DHHC21 overexpression prevented the CHX-induced decrease in CAV1 palmitoylation and did not cause further increase following washout period.

This led us to overexpress CAV1 in order to make a pool of newly-synthesized

CAV1 available and overcome this limitation, yet still monitor the initial palmitoylation of the protein.

DHHC7 increases HA-CAV1 palmitoylation

In the remaining experiments, HA-tagged CAV1 was transfected into HEK293 cells to provide a newly expressed pool of CAV1 that was distinct from the endogenous protein. I hypothesized that this HA-tagged pool of CAV1 would be subject to palmitoylation by DHHC7 and/or DHHC21. To test this, cells were transfected with HA-

CAV1 alone, or co-transfected with HA-CAV1 and DHHC7, DHHC21, or DHHC10 and

subjected to acyl-biotin exchange. I found that DHHC7 overexpression increased HA-

CAV1 palmitoylation over that of the control (NT) but overexpression of DHHC21 or

DHHC10 did not (Figure 2.4, F(3,20) = 8.4, p < 0.001).

Figure 2.4. DHHC7 increases HA-tagged CAV1 palmitoylation. (A) Western Blot indicating HA-tagged CAV1 exhibits increased palmitoylation following overexpression of DHHC7, but not DHHC21 or DHHC10. (B) Average intensity of HA-CAV1 palmitoylation (*p<0.001 vs. NT group).

Mutant HA-CAV1 is not palmitoylated, but still associates with DHHC7 and plasma membrane

To establish that DHHC7-mediated HA-CAV1 palmitoylation occurred at the expected cysteine residues, I created a mutant version of the HA-CAV1 plasmid in which all three of the putative palmitoylation sites were changed from cysteine to alanine, rendering the protein palmitoylation-null (Figure 2.5A). No palmitoylation of this mutant

(MT) HA-CAV1 was detected, even when DHHC7 was overexpressed (Figure 2.5B), suggesting that DHHC7 increased CAV1 palmitoylation at the expected sites.

To further demonstrate that there were no gross abnormalities in the MT HA-

CAV1 that would interfere with palmitoylation, such as abnormal protein folding, etc., I performed immunoprecipitation experiments to see if it still associated with DHHC7 40

(Figure 2.5C). Co-immunoprecipitation of CAV1 and DHHC7 was not interrupted, suggesting that altering these residues did not interfere with DHHC-substrate binding, but only with palmitate attachment.

Figure 2.5. Palmitoylation-null CAV1 still associates with DHHC7 and the plasma membrane. (A) Amino acid sequence and protein schematic of wild-type (WT) and mutant palmitoylation-null (MT) HA-tagged CAV1. In the MT, all three cysteine residues found in WT CAV1 were changed to alanine. (B) DHHC7 increased WT HA- CAV1 palmitoylation, whereas palmitoylation of MT HA-CAV1 was completely absent. (C) Mutation of HA-CAV1 did not affect co-immunoprecipitation with DHHC7. (D) DIC (top) and TIRF (bottom) imaging of HEK293 cells transfected with WT (left) or MT (right) mNEONGreen (mNG)-tagged CAV1 (mNG-CAV1). Scale bar = 10 microns. (E) Membrane localization of CAV1 was unaffected by palmitoylation state. 41

Palmitoylation also often plays an important role in the trafficking of proteins to the plasma membrane. Thus, although biochemical fractionation experiments have suggested that CAV1 palmitoylation is not necessary for association with caveolae- and lipid raft-associated fractions (Dietzen et al., 1995), we wanted to know if imaging would reveal any palmitoylation-dependent changes in CAV1 trafficking. Consequently, we performed TIRF imaging of cells transfected with WT or MT CAV1 tagged with mNeonGreen to determine if membrane localization of CAV1 depends on its palmitoylation. This revealed both WT and MT CAV1 at or near the membrane (i.e., within 100nm) (Figure 2.5D). No difference in TIRF fluorescence was observed when normalized to the total wide-field fluorescence (Figure 2.5E, t=0.067, p = 0.95). These findings support the conclusion that palmitoylation is not essential for the membrane- targeting of CAV1.

Possible relationship between DHHC7 and DHHC21

Having demonstrated that overexpression of DHHC7 is sufficient to increase HA-

CAV1 palmitoylation, I sought to determine whether DHHC7 is also necessary for

CAV1 palmitoylation. To do so, I transfected HEK293 cells with siRNA targeting genes zDHHC7, zDHHC21, or a non-targeting siRNA, followed by overexpression of HA-

CAV1 and ABE analysis (Figure 2.6A, B). Surprisingly, knockdown of DHHC7 (or

DHHC21) did not cause a reduction in HA-CAV1 palmitoylation (Figure 2.6A, F(3,5) =

2.6, p = 0.17). siRNA effectiveness and specificity were verified by qPCR (Figure 2.6B,

*p<0.05 compared to NT condition).

Figure 2.6. Simultaneous, but not single, knockdown of zDHHC7 and zDHHC21 decreases CAV1 palmitoylation. (A) siRNAs targeting either zDHHC7 or zDHHC21 did not affect HA-CAV1 palmitoylation. (B) qPCR validation of siRNA knockdown (*p<0.05 vs NT). (C) Simultaneous knockdown of zDHHC7 and zDHHC21decreased HA-CAV1 palmitoylation (*p<0.01). (D) qPCR validation of double siRNA knockdown (*p<0.05 vs. NT).

Because of the requirement for both DHHC7 and DHHC21 for membrane- initiated estradiol signaling, I considered the possibility of a cooperative interaction between DHHC7 and DHHC21 in regards to CAV1 palmitoylation. Thus, I transfected

cells with siRNA targeting both DHHC7 and DHHC21 to see if simultaneous reduction would alter CAV1 palmitoylation. Interestingly, following disruption of both DHHC7 and DHHC21, I observed a decrease in HA-CAV1 palmitoylation (Figure 2.6C, t(6) =

4.11, p = 0.006), indicating a relationship or redundancy between these two enzymes in the context of CAV1 palmitoylation. Effectiveness of knockdown was again verified by qPCR (Figure 2.6D, *p<0.05 compared to NT condition).

One explanation why expression of both DHHC7 and DHHC21 must be compromised to decrease HA-CAV1 palmitoylation is that DHHC21 facilitates activity of DHHC7. To test this possibility, I examined whether siRNA knockdown of DHHC21 would eliminate the effect of DHHC7 overexpression (Figure 2.7A). As shown before,

DHHC7 overexpression increased HA-CAV1 palmitoylation, but this was not affected by

DHHC21 knockdown (Figure 2.7B, F=14.08, p < 0.01; Tukey p < 0.05), suggesting that

DHHC7 does not require DHHC21 to increase CAV1 palmitoylation.

Figure 2.7. DHHC7-mediated CAV1 Palmitoylation is not affected by knockdown of zDHHC21. (A) Palmitoylation of HA-CAV1 was increased by DHHC7, and was unaffected following disruption of DHHC21 expression. (B) Average palmitoylation of HA-CAV1 with overexpression of DHHC7 and/or DHHC21 knockdown (*p<0.05 in comparison to basal palmitoylation controls). 44

Given that DHHC7 activity does not appear to depend on the presence of

DHHC21, I wanted to know whether these two enzymes are localized to different cellular compartments in HEK293 cells. To address this, I transfected HEK293 cells with myc- tagged DHHC7 or DHHC21 and immunostained cells for myc tag expression and co- stained with markers for the Golgi apparatus and the nucleus. Confocal images revealed that DHHC7 expression was widespread, but DHHC21 expression appeared to be restricted to the Golgi apparatus (Figure 2.8).

Figure 2.8. Differential subcellular localization of DHHC7 and DHHC21. Confocal images of HEK293 cells transfected with myc-tagged DHHC7 or DHHC21. Cellular expression of DHHC7 was widespread, while DHHC21 expression was restricted to the Golgi. Color coding: green = myc tag, red = golgin-97, blue = nuclear counterstain. Scale bar = 10 microns.

Discussion

The current understanding of the regulatory role palmitoylation plays in ER membrane trafficking and ER/mGluR signaling has thus far been limited by focusing solely on the ER, to the exclusion of other regulatory proteins involved in this mechanism 45

of ER action. While effector proteins downstream of mGluR activity are likely subject to palmitoylation activity as well, it is critical to understand potential post-translational modifications to the proteins that facilitate the ER/mGluR interaction in the first place.

Our finding that the same DHHCs important for steroid receptor palmitoylation also affect CAV1 palmitoylation adds another layer of regulation to the ER/mGluR signaling model.

The finding that two DHHCs must be knocked down to decrease CAV1 palmitoylation suggests that there is some form of cooperation between DHHC7 and

DHHC21. Perhaps one palmitoylates the other, or perhaps they are localized to different subcellular compartments, as the immunostaining presented here and elsewhere (Pedram et al., 2012) suggest. Moreover, while it is clear that there is DHHC-substrate specificity, most protein substrates can be palmitoylated by more than one DHHC enzyme (Fukata et al., 2016). This PAT redundancy may be particularly apparent when the substrate is overexpressed, which would explain why siRNA knockdown of both DHHC7 and

DHHC21 was required here to decrease palmitoylation of overexpressed CAV1. An additional possibility is that these DHHCs can act as a functional heteromer. Lai and

Linder suggest that the oligomerization state of DHHC proteins affect their enzymatic activities (Lai and Linder, 2013). These alternative hypotheses await further study.

Our findings from TIRF imaging are also consistent with previous work indicating that palmitoylation does not affect CAV1 membrane trafficking (Dietzen et al.,

1995). Importantly, the presence of non-palmitoylated CAV1 at or near the membrane does not indicate CAV1 function remains intact. For example, CAV1 must be palmitoylated in order to interact with G-proteins (Galbiati et al., 1999). Moreover, as

CAV1 is an important component of lipid rafts, changes in CAV1 palmitoylation could alter lipid raft dynamics and therefore signal transduction.

Additionally, the present work did not distinguish between the three palmitoylation sites on CAV1. Previous reports suggest that while CAV1 is palmitoylated at all three cysteine residues, it is primarily palmitoylated at cysteine 133 (Dietzen et al.,

1995). It remains to be seen whether all three sites are palmitoylated by a single PAT, and if there is any order or sequence to possible site-specific palmitoylation. This could contribute to cell sorting, trafficking, and lipid raft association (Greaves and

Chamberlain, 2007; Levental et al., 2010).

Our ability to make conclusions about the role of DHHC7 and/or DHHC21 in

Cav1 regulation is limited by the current approach. The acyl-biotin exchange assay used here is binary: it differentiates between palmitoylated and non-palmitoylated proteins. It cannot tell us if a protein is more or less palmitoylated (i.e., how many palmitoylation sites are occupied). This is an issue because Cav1 has three palmitoylation sites. If, for example, most Cav1 proteins are palmitoylated at one site immediately after translation, we would not be able to detect further palmitoylation following manipulations such as overexpression of DHHC7 or DHHC21. Thus, the finding that DHHC7 overexpression increases Cav1 palmitoylation should be refined such that DHHC7 overexpression increases new or first-time palmitoylation of Cav1. This suggests that DHHC7 may be responsible for the initial palmitoylation of Cav1. The main concern is that our failure to detect enhancement of Cav1 palmitoylation following DHHC21 overexpression is inconclusive due to the fact that our approach cannot account for the possibility that

DHHC21 could palmitoylate additional sites on already-palmitoylated Cav1. Our siRNA

experiments suggest that this could be what is actually happening, as knockdown of both

DHHC7 and DHHC21 was required to cause a decrease in Cav1 palmitoylation. (Again, however, the ABE assay is only sensitive to complete loss of palmitoylation and is not capable of picking up intermediate palmitoylation states.) These findings could be greatly clarified by employing a technique that can measure how many palmitoyl groups are attached to a protein, such as the Acyl-PEGyl exchange gel shift (APEGS) assay (Yokoi et al., 2016). Additionally, many of the same techniques employed here could be repeated with versions of Cav1 having only one intact palmitoylation site in order to determine the site-specificity.

One goal of investigating the regulation of proteins involved in ER/mGluR signaling is to elucidate the mechanisms that confer sex specificity to this pathway.

Estradiol activation of mGluR G-protein activity through membrane localized estrogen receptors is often restricted to females (but see Seredynski et al., 2015 and Hedges et al.,

2018). Because there are no obvious differences in ER (or mGluR) expression between males and females in brain regions that exhibit a female only ER/mGluR signaling, we and others hypothesize that sex specificity may instead be explained by the regulatory processes that partner these signaling proteins together, such as palmitoylation and CAV1 association. Paradoxically, we have found that mRNA expression of both Cav1 and zDHHC7 is decreased in the hippocampus of adult female rats relative to males (Meitzen et al., 2017). No differences were observed in ER or zDHHC21 expression. In fact, no sex differences were observed in any of the remaining 22 palmitoyl acyltransferases.

Because estrous cycles were not monitored during these studies, the possibility of hormonal regulation of CAV1 and/or DHHC7 expression remains. In fact, this may be a

likely explanation based on findings from a variety of tissues that estradiol increases

CAV1 expression, including endothelial cells (Jayachandran et al., 2001), female mouse hypothalamus (Zschocke et al., 2002), female rat bladder (Zhu et al., 2004), rat adipose tissue to a greater degree in females (Mukherjee et al., 2014), and rat uterine tissue (Turi

Agnes et al., 2013). Moreover, this could be a locus of intersection between classical estrogen receptor regulation of gene expression and ER/mGluR signaling. It remains to be seen whether or not DHHC7 expression is subject to estradiol regulation.

S-palmitoylation is a powerful regulatory mechanism in large part because of its reversible nature. Indeed, many groups have reported activity-dependent fluctuations in palmitoylation that may contribute to signaling outcomes (Fukata et al., 2013; Brigidi et al., 2014; Woolfrey et al., 2015; Globa and Bamji, 2017). Dynamic ER palmitoylation has also been observed, with estradiol exposure decreasing ER palmitoylation in cancer cell lines (Acconcia et al., 2004) and hippocampal synaptosomes (Tabatadze et al., 2013).

However, our data support the previous findings from Parat and Fox that CAV1 palmitoylation is a relatively stable modification, at least in HEK293 cell lines (Parat and

Fox, 2001). Accordingly, in addition to elucidating the functional importance of CAV1 palmitoylation for ER/mGluR and other signaling mechanisms, future work must consider the relevance of its permanence.

Dysregulation of CAV1 has been implicated in a wide array of disease processes, including various cancers (Chatterjee et al., 2015), liver disease (Fernandez-Rojo and

Ramm, 2016), chronic pain (Yang et al., 2015a), neurodegeneration (Head et al., 2010), and schizophrenia (Kassan et al., 2016). CAV1 is likely to exert influence on these processes through its roles as both an organizer of signaling molecules and as a regulator

of membrane dynamics and lipid raft domains. Consequently, palmitoylation and palmitoyl acyltransferases not only provide a potential target for cellular regulation of

CAV1 with respect to estrogen receptor activity at the cell membrane, but may also provide insight into how this cell signaling regulatory protein affects distinct cellular processes both in and out of the nervous system.

CHAPTER 3: Caveolin-1 and cocaine-induced plasticity

Introduction

Caveolins are small integral membrane proteins that spatially organize signaling proteins, including mGluRs, into functional microdomains. In peripheral tissue, caveolins form large oligomers – caveolae – that create invaginations in the plasma membrane.

Caveolae are not observed in the brain, but caveolin proteins are still expressed, and serve many of the same functions (Zheng et al., 2011). Work in the hippocampus has suggested that overexpression of Cav1 can enhance structural and functional plasticity associated with learning and memory (Head et al., 2011), while Cav1 KO animals have impaired hippocampal mGluR-dependent LTD (Takayasu et al., 2010).

Most studies of neuronal Cav1 have focused on the hippocampus. I was interested in expanding this line of research to the striatum, specifically the nucleus accumbens.

Medium spiny neurons (MSNs) are the output neurons of the nucleus accumbens, and integrate multiple inputs (Groenewegen et al., 1999; Kelley, 2004), processing these signals in order to influence motor and cognitive behaviors through projections to other brain regions (Smith et al., 2013; Yager et al., 2015). Repeated exposure to psychostimulants enhances both glutamatergic and dopaminergic input to the nucleus accumbens (Nestler, 2001; D’Souza, 2015), causing physiological and structural plasticity in MSNs (Golden and Russo, 2012).

Given the important role of Cav1 for plasticity-related proteins, I asked whether repeated exposure to cocaine would affect its expression in the nucleus accumbens.

Further, I hypothesized that alterations in Cav1 expression would affect the development of cocaine-induced behavioral sensitization. I also investigated the possibility of a

cellular phenotype in MSNs that could contribute to behavioral changes. in Cav1 KO mice.

Methods

Animals

Animal procedures were performed at the University of Minnesota in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal

Care (AAALAC) and in accordance with protocols approved by the University of

Minnesota Institutional Animal Care and Use Committee (IACUC), as well as the principles outlined in the National Institutes of Health Guide for the Care and Use of

Laboratory Animals. qPCR Experiment

Male C57BL/6J mice, 10 weeks of age, were housed 4 to a cage with food and water available ad libitum. Prior to experiment onset, animals were handled and placed in the testing chamber for one day of habituation. On the subsequent five days, animals were placed into the chamber for a 20 minute pre-injection session, followed by intraperitoneal injection of cocaine (15 mg/kg, 3 mg/ml) or saline vehicle and a 50 minute post-injection session. A week of withdrawal was followed by a challenge day in which animals again received a 20 minute pre-injection session and then a challenge dose of cocaine or saline. Locomotor activity was video recorded and analyzed using ANY- maze tracking software (Stoelting). Animals were sacrificed 24 hours post challenge, and bilateral tissue punches from nucleus accumbens and caudate were submerged in

RNAlater (Qiagen) for subsequent RNA extraction using RNeasy (Qiagen). cDNA was generated from 500 ng mRNA using First Strand cDNA Synthesis Kit (Roche). Real-time

PCR was performed on a Roche LightCycler 480 using KAPA SYBR Fast. Results were analyzed using the ΔΔCT method (Schmittgen and Livak, 2008).

Stereotaxic injections

Eight-week old ovariectomized Sprague-Dawley rats received bilateral intracranial infusions (1 μl/side) into the NAc of AAV9-SynCav1 or AAV9-SynRFP.

Specifically, animals were anesthetized with a 2.5-4% isoflurane (Piramal Critical Care,

Bethlehem, PA)/oxygen mixture and placed in a stereotaxic apparatus. Drug or vehicle was injected bilaterally via a Hamilton microinjection syringe at the following coordinates targeting the core-shell border: AP: +1.80 mm from bregma, ML: ±1.50 mm from bregma, DV: -6.20 mm from dura. Infusions of 1 μl were given manually over the course of 5 minutes. The injection needle was then left in place for an additional 5 minutes to allow for diffusion of the drug away from the needle tip. Animals were given a subcutaneous injection of 2.5 mg/kg ketoprofen 20 minutes prior to surgery and every

12 hours for the 3 days following surgery to induce analgesia and a subcutaneous injection of 10 mg/kg Baytril at the time of surgery to prevent infection. Immediately following the surgery, animals were monitored until they recovered ambulatory posture.

Afterwards, animals were observed for eating, drinking, and or the display of any discomfort. None of the animals exhibited any symptoms of problems. Location of the injection site was verified following euthanasia.

Habituation to behavioral chambers began two weeks following stereotaxic surgery. Two habituation days were followed by seven test days consisting of a 30 minute preinjection session in the behavioral chamber, i.p. injection of cocaine (15 mg/kg/ml, a dose not expected to produce sensitization (Martinez et al., 2014)) or saline

vehicle, and a 60 minute post-injection session. Locomotor activity was measured using photobeam sensing and Motor Monitor Software (Kinder Scientific).

To assess viral expression, animals were anesthetized with Beuthanasia-D (0.3 ml i.p., Schering, Union, NJ) and transcardially perfused with 25 mM phosphate buffered saline (PBS, pH = 7.2) for 3 minutes followed by 4% paraformaldehyde in 25 mM PBS for 20 mins. Brains were post-fixed for one hour in 4% PFA and then submerged in 30% sucrose until saturated. 40 μM slices were obtained using a freezing microtome and stored at -80C in cryoprotectant (25 mM PBS supplemented with 1% w/v polyvinylpyrrolidone, 30% v/v ethylene glycol, 30% w/v sucrose). For immunohistochemistry, slices were washed in 25 mM PBS and incubated overnight with rabbit anti Cav1 antibody (CST, 1:100) followed by washes and incubation with

AlexaFluor488 secondary antibody (Invitrogen) for 1 hour at room temperature. Slices were then washed again and mounted on slides with ProLong Diamond mounting media

(Invitrogen).

Additionally, a subset of animals was euthanized and fresh tissue was collected for western blotting analysis. Bilateral tissue punches were homogenized in RIPA buffer supplemented with HALT Phosphatase and Protease inhibitors (Pierce) using Bullet

Blender (Next Advance). Homogenates were lysed at 4C for 45 minutes with rotation and then spun at 12,000 rpm at 4C for 20 minutes to pellet cellular debris. Supernatants were analyzed using the DC Protein Assay (Biorad). 20 μg of each sample was subjected to SDS-PAGE on 4-20% gradient Mini-PROTEAN TGX precast gels (Biorad) at 120V for 90 minutes, transferred to nitrocellulose (Mini Trans-Blot Cell from Biorad, 100 V for

40 minutes), blocked for 1 hour at room temperature in Odyssey Blocking Buffer (TBS

formulation, Licor) and probed with rabbit anti-Cav1 (Abcam, 1:5000) and mouse anti-

GAPDH (Millipore, 1:10000) diluted in 1:1 TBST + blocking buffer overnight at 4° C with gentle shaking. Blots were then washed and stained with Licor secondary antibodies for 1 hour at room temperature prior to imaging on a Licor Odyssey infrared scanner.

Cav1 KO animals

10-week old male wild-type (WT) and CAV1 knockout (KO) littermates (Razani et al., 2001) were handled and habituated to locomotor chambers for two days prior to behavioral testing. On test and challenge days, animals were placed in the chamber for a

20-minute preinjection session, followed by an intraperitoneal injection of cocaine (15 mg/kg, 3 mg/ml) or saline vehicle, and returned to the chamber for a 50 minute postinjection session. Video of test sessions was analyzed using ANYmaze tracking software. Five days of cocaine or saline injection were followed by one week of withdrawal and a subsequent challenge day.

Cortico-striatal co-culture

Primary striatal-cortical cultures were prepared as previously described (Penrod et al, 2011). Briefly, the ganglionic eminence (presumptive striatum) and prefrontal cortex were removed from day 16 mouse embryos. Tissue from these brain regions were maintained separately until plating. Tissue was digested at 37C for 15-30 min in 0.25% trypsin-EDTA (Sigma), rinsed in calcium/magnesium-free Hanks balanced salts (HBSS), and resuspended in neuronal plating media (10 mM HEPES, 10 mM sodium pyruvate,

0.5 mM glutamine, 12.5 μM glutamate, 10% Newborn Calf Serum, 0.6% glucose in minimal essential media plus Earl’s salt (EMEM)). Tissue was dissociated by trituration with a fire-polished pipette. Cells were then counted using trypan blue and a

hemocytometer. Cells were plated on 12 mm acid-washed glass coverslips coated with

100 μg/ml poly-d-lysine/4 μg/ml laminin contained five to a 35 mm dish. Cells were plated in a 3:2 cortex to striatum ratio at a final cell density of 200 cells/mm2. Dishes were maintained at 37C, 5% C02. Following cell adhesion 1-3 hours after plating, plating media was replaced with growth media (Neurobasal, 1 x B27 (Invitrogen), 0.5 mM glutamine) pre-conditioned on glia. After four days in vitro (DIV), this glia- conditioned media was replaced with unconditioned growth media. Subsequently, on

DIV7 and DIV14, half of the media was replaced with fresh, unconditioned growth media. For experiments with DA (Sigma), AAV (UMN Viral Core), or PLC inhibitor

U73122 (Tocris), 1 μm DA, 1 μl AAV, and/or 100 nM U73122 was added during the media change at DIV4. All drugs were administered chronically, and media was supplemented with fresh drug every 3-4 days.

Glia cultures were prepared as previously described (Penrod et al 2011). Briefly, cortices of postnatal P1-2 mice were dissected into small pieces and incubated with

0.25% trypsin-EDTA and 1 μl/ml Benzonase (Novagen) or 3 mg/ml DNAse1 (Sigma) for

30 min at 37C. After digestion, an equal volume of glia plating media (10 mM HEPES,

1 mM sodium pyruvate, 2 mM glutamine, 10% Newborn Calf Serum, 0.6% glucose, 1 x

Penicillin-Streptomycin) was added and cells were collected by centrifugation (1000xg for 2 min). Cells were resuspended in glia plating media, triturated, and filtered through a

0.7 um cell strainer. Cells were plated on uncoated 10 cm tissue culture dishes. Glia media was replaced one day after plating and then once per week subsequently. Glia- conditioned media was prepared by incubating 10 ml of neuron growth media on

confluent glial plates for 48 hours. Following conditioning, the conditioned media was removed and replaced with fresh glia plating media to maintain the glial cultures.

On DIV19, cells were fixed at 37C with 4% PFA/PHEM (60 mM PIPES pH 7.0,

25 mM HEPES pH 7.0, 10 mM EGTA, 2 mM MgCl2)/0.12 M sucrose-buffered fixative for 15 minutes. Cells were then rinsed in PBS and blocked in 3% bovine serum albumin

(BSA)/PBS for 30 minutes at room temperature. Cells were permeabilized using 0.2% triton in PBS for 10 minutes at room temperature, rinsed in PBS, and blocked for another

30 minutes in 3% BSA/PBS. All antibodies were prepared in 1% BSA/PBS and coverslips were incubated overnight at 4C with primary antibody mixture. To identify

MSNs, cells were stained with polyclonal rabbit anti Dopamine and cAMP regulated phosphor-protein of 32 KDa (DARPP-32; CST2302, 1:250). For verification of Cav1

KO, cells were instead stained with polyclonal rabbit anti Caveolin-1 primary antibody

(Abcam, ab2910, 1:2000). Following overnight primary incubation, coverslips were rinsed in PBS and incubated secondary antibody (Alexafluor goat anti rabbit 488, 1:100) for 1 hour at room temperature. Coverslips were then rinsed in PBS and mounted on glass slides with 2.5% 1,4-Diazabicyclo-[2.2.2]Octane (DABCO), 150 mM Tris pH 8.0, and

80% glycerol mountant to reduce photobleaching.

For neurite tracing and Sholl analysis (Sholl, 1953), MSNs were identified by their high level of DARPP-32 expression and imaged with a 20X objective on a Zeiss

Axiovert 200M microscope controlled by MicroManager software. Image analysis was performed using FIJI/ImageJ (NIH) using the Simple Neurite Tracer plugin. Statistical analyses were performed using GraphPad Prism. For Sholl analyses, crossings were compared at each ring (10 µm intervals centered on the soma) and between experimental

treatments using two-way ANOVA and post hoc tests using Bonferroni’s correction for multiple comparisons.

Results

Repeated cocaine exposure increases Cav1 expression in the nucleus accumbens

To test whether repeated exposure to cocaine would affect expression of Cav1, I administered cocaine (15 mg/kg) or saline vehicle to male C57BL/6J mice on five consecutive days. Mice then underwent a week of withdrawal prior to a challenge dose of cocaine or saline. Mice receiving five days of repeated cocaine followed by a cocaine challenge exhibited greater locomotor activity on the fifth and challenge day than their first exposure (i.e., sensitized; data not shown). This group also had increased Cav1 mRNA in the nucleus accumbens (Figure 3.1A; F(2,9) = 5.27, p = 0.031), and decreased

Cav2 mRNA in the caudate/putamen (CPu) (Figure 3.1B; F(2,9) = 5.98, p = 0.022).

A B

Figure 3.1. Repeated cocaine exposure followed by withdrawal and cocaine challenge led to increased Cav1 mRNA expression in the nucleus accumbens (A, Coc-Coc) but not in the dorsal striatum (B). *Indicates Tukey’s multiple comparisons yielded p <0.05.

There was no difference in Cav3 expression in either brain region (data not shown). Importantly, the increase in Cav1 expression was not seen in mice receiving their first dose of cocaine on the challenge day, but rather required previous repeated exposure.

This suggests that Cav1 could be involved in maintaining the long-term plasticity caused by cocaine and withdrawal.

Overexpression of CAV1 in the nucleus accumbens increases cocaine-induced locomotor behavior

Given that behavioral sensitization following repeated cocaine exposure was accompanied by elevated NAc Cav1, we hypothesized that increasing Cav1 expression in this brain region would enhance cocaine-induced behavioral plasticity. To test this, we overexpressed Cav1 in neurons of the nucleus accumbens in ovariectomized rats (Figure

3.2A) and measured differences in locomotor responses to cocaine (Figure 3.2B). we used a dose of cocaine previously shown to not produce behavioral sensitization in ovariectomized rats without estradiol supplementation, and hypothesized that Cav1 overexpression would mimic the enhancement normally seen with estradiol (Martinez et al., 2014; Tonn Eisinger et al., 2018). Indeed, Cav1 animals increased their locomotor responses from the first to last day of cocaine exposure, while control animals did not

(Figure 3.2B). This indicated that Cav1 overexpression facilitated cocaine-induced plasticity.

Figure 3.2. Overexpression of caveolin-1 (Cav1) in the nucleus accumbens (NAc) facilitates cocaine-mediated behavioral sensitization in female rats. (A) Immunohistochemistry confirmation of Cav1 overexpression (regulated under the synapsin (Syn) promoter) two weeks following AAV9-mediated delivery (left panel). Control animals overexpressed red fluorescent protein (RFP) (middle panels). Western blot confirmation of Cav1 overexpression in the NAc compared to caudate putamen (CPu) control region. GAPDH was used as a loading control (lower panel). (B) Only animals overexpressing Cav1 exhibited behavioral sensitization between the first and seventh day of cocaine treatment (n = 9-10; *p = 0.01, paired-sample t-test between groups within day following main effect of two-way ANOVA). Dashed line indicates baseline ambulatory behavior one-day prior to cocaine administration. No differences were observed on these habituation days or in animals receiving repeated saline (data not shown).

Cav1 knockout mice do not display cocaine-induced behavioral sensitization

To determine the importance of Cav1 for cocaine-induced locomotor activity,

Cav1 knockout (KO) and wildtype (WT) littermates were given repeated cocaine (15 mg/kg) or saline vehicle i.p. for five days followed by withdrawal and challenge (Figure

3.3B). Baseline activity did not differ between WT and KO animals (Figure 3.3C).

However, while WT cocaine-treated animals sensitized as expected, displaying higher levels of locomotor behavior on the challenge day relative to the first day of cocaine, KO animals did not (Figure 3.3D). Although KO animals had intact acute responses to cocaine, they did not increase in locomotor activity after subsequent exposures or withdrawal and challenge.

Figure 3.3. Cav1 KO animals do not display cocaine-induced behavioral sensitization. (A) Western blot confirmation of Cav1 knockout in NAc of KO animals (right) compared to WT (left), two animals each. GAPDH was included as a loading 62

control (lower panel). (B) Behavioral scheme, each box indicating a separate day of testing. (C) Locomotor activity recorded on second day of habituation shows no baseline differences between WT and Cav1 KO animals. (D) WT animals receiving repeated cocaine (15 mg/kg i.p.) (black circle, n=8) increased locomotor activity over the course of the experiment, while KO animals (open black square, n=8) did not. Animals receiving repeated saline (gray, n=6-8) served as a control, and received an acute dose of cocaine (15 mg/kg) on the challenge day. (Two-way RM ANOVA yielded significant variation from interaction, day, and genotype. *Bonferroni post-test yielded p<0.01.)

MSNs from Cav1 KO mice have altered dendritic arborization

Given that Cav1 KO mice have impaired long-term behavioral plasticity following cocaine exposure, I expected that these mice would have an altered cellular phenotype that could potentially explain this effect. In particular, I hypothesized that there would be alterations in the nucleus accumbens, the brain region largely responsible for long-term drug-induced plasticity (Kauer and Malenka, 2007; Russo et al., 2010). To investigate this, we cultured medium spiny neurons (MSNs) from E16 WT or KO mice

(Figure 3.4A). We utilized a striatal-cortical co-culture method that has been shown to yield robust MSN morphology similar to that seen in vivo (Segal et al., 2003; Penrod et al., 2011), and examined dendritic arbors at DIV19, when dendritic complexity is normally stable and maximal (Penrod et al., 2015). Sholl analysis revealed that MSNs from Cav1 KO animals had significantly enhanced dendritic arborization compared to those from WT animals (Figure 3.4B, C). Neurite tracing revealed that the increased number of Sholl crossings in KO MSNs could be attributed to increased number and length of primary, secondary (not shown), and total dendrites per neuron (Figure 3.4D,

E).

Figure 3.4. Cultured MSNs from Cav1 KO mice have enhanced dendritic arborization. (A) Verification of Cav1 KO by immunostaining for Cav1 in cultures from WT (upper panel) and Cav1 KO (lower panel) mice. (B) DARPP32 staining of exemplar neurons representing the average Sholl profile for each group (upper panels) and example Sholl overlay with rings every 10 µm (lower panel). Scale bar = 50 µm. (C) Sholl analysis of dendritic complexity of MSNs from WT (blue) or KO (orange) mice. (D) Quantification of number and length of primary dendrites. (E) Quantification of the number of dendrites and total dendrite length per neuron. For (C), (D) and (E) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data displayed as Mean ± SEM. N = 90-107 neurons per group.

Enhancement of dendritic arborization in Cav1 KO MSNs is eliminated by PLC inhibition

Because previous reports have suggested that Cav1 KO animals have altered basal

Gq signaling (Francesconi et al., 2009), I hypothesized that alterations in arborization may be a result of changes to this pathway. To test this, we chronically blocked phospholipase C (PLC), a signaling molecule downstream of Gq whose activity results in increased calcium release from intracellular stores. Addition of PLC inhibitor U73122

(100 nM) decreased KO arborization to WT levels, but did not affect arborization of WT

MSNs (Figure 3.5; F(103, 4725) = 1.804). These data suggest that enhanced arborization in Cav1 KO MSNs may be a result of increased Gq activity.

Figure 3.5. PLC inhibition eliminates enhancement of dendritic arborization in Cav1 KO MSNs. Chronic application of PLC inhibitor U73122 (U7, 100 nM) returned dendritic complexity of Cav1 KO MSNs to WT levels, but did not further decrease WT complexity. Orange asterisks indicate rings at which Bonferroni post hoc tests showed that KO MSNs had more crossings than all other groups. (*p<0.05, **p<0.01, ***p<0.001)

Dopamine has opposite effects on WT and Cav1 KO MSNs

Dopamine (DA) has previously been shown to have a modulatory effect on development of MSN dendritic arborization (Penrod 2015). I was particularly interested in this effect given the behavioral phenotype seen in Cav1 KO mice following repeated exposure to cocaine, which increases dopamine availability in the nucleus accumbens.

DA normally enhances MSN arborization, so I hypothesized that it would have no further effect on Cav1 KO MSNs that already have increased arborization. To test this, we chronically treated MSNs with 1 µM DA beginning at DIV4 as previously described

(Penrod et al., 2015). DA increased the arborization of WT neurons as expected, but, surprisingly, decreased arborization of KO neurons (Figure 3.6; F(102, 14384) = 8.235, p<0.0001).

Figure 3.6. Dopamine decreases dendritic arborization of MSNs from Cav1 KO mice, as revealed by Sholl analysis. Blue asterisks = significant difference between WT and WT + DA groups; Orange asterisks = significant difference between KO and KO + DA groups (Bonferroni post hoc comparisons; *p<0.05, **p<0.01, ***p<0.001).

Decreased arborization in Cav1 KO cultures is MSN-intrinsic

Because these co-cultures include both striatal MSNs and cortical neurons, I wanted to determine whether the cortical neurons were contributing to the KO phenotype.

To do so, we created mixed cultures, comprised of cortical tissue from WT mice and striatal tissue from Cav1 KOs (Figure 3.7A or cortical tissue from Cav1 KO mice and striatal tissue from WT (Figure 3.7B). Mixed cultures containing KO striatal tissue recapitulated the KO phenotype (Figure 3.7A, F(102, 8294) = 2.45, p<0.0001) while 67

mixed cultures containing KO cortex did not (Figure 3.7B, F(102, 8644) = 3.18, p<0.0001). These data suggest that the KO MSN phenotype is MSN-intrinsic. It is likely that cortical cells may have their own phenotype, but it appears to have little effect on dendritic arborization of MSNs at baseline. However, the attenuated response to dopamine in cultures with WT cortical tissue (KOstr + DA, Figure 3.7A, does not have as dramatic of a reduction in arborization as KO + DA, Figure 3.6) suggests that KO cortical neurons in these cultures may be contributing to the reversal of dopamine- induced arborization seen in KO cultures.

Figure 3.7. Effect of Cav1 KO on MSN arborization is MSN-intrinsic. (A) Sholl analysis of cultures in which all have WT cortical cells, accompanied by either WT (blue) or KO (KOstr, orange) striatal tissue. KOstr cultures have enhanced dendritic arborization relative to WT, and this is reversed by dopamine. (B) Sholl analysis of cultures in which all have WT striatal cells, accompanied by either WT (blue) or KO (KOctx, orange) cortical neurons. KOctx cultures are largely indistinguishable from WT, and dopamine enhances dendritic arborization of KOctx cultures as in WT. (Bonferroni post hoc comparisons; *p<0.05, **p<0.01, ***p<0.001.) 68

Normal development of MSN dendritic arbor requires Cav1 palmitoylation

Cav1 is an adaptor protein that functions at the membrane to facilitate interactions between receptors and their effector molecules, often within specialized membrane compartments known as lipid rafts. Palmitoylation, the posttranslational addition of a 16- carbon lipid group, is an important regulator of proteins that associate with lipid rafts, including Cav1. Moreover, a previous report suggested that Cav1 palmitoylation is required for normal function of G-protein subunits at the membrane (Galbiati et al.,

1999). For these reasons, I am interested in the possibility that Cav1 regulation by palmitoylation is important for Cav1 function, especially in the context of the nucleus accumbens and MSNs. To test whether Cav1 palmitoylation is required for normal Cav1 function in vitro, we transduced Cav1 KO cultures with AAV9 expressing Cav1 under the control of the Synapsin promoter to ensure neuron-specific expression. The Cav1 construct was either wildtype (WT SynCav1) or palmitoylation-null, in which all three palmitoylation sites had been mutated from cysteine to alanine (mt SynCav1). AAV9-

SynRFP was used to confirm viral expression (Figure 3.8A). Expression of WT SynCav1 in KO cultures restored dendritic arborization to levels seen in WT MSNs, but palmitoylation-null mt SynCav1 did not (Figure 3.8B, F(102, 9590) = 5.68, p<0.0001).

This suggested that Cav1 palmitoylation plays a critical role in normal development of

MSN dendritic arborization.

Figure 3.8. Cav1 palmitoylation is required for normal dendritic arborization in MSNs. (A) Verification of viral expression by immunostaining for DARPP32 (left panel, to identify MSNs) and imaging red fluorescent protein (RFP, middle panel, control virus AAV9-SynRFP) confirmed strong expression in MSNs (merged image, right panel). (B) Sholl analysis reveals that restoration of WT Cav1 to KO neurons (dark orange) returns arborization to WT levels, but expression of palmitoylation-null Cav1 does not (red). (Bonferroni post hoc comparisons; *p<0.05, **p<0.01, ***p<0.001.)

Discussion

In this study, I combined in vivo behavioral approaches with in vitro morphological studies to examine the role of Cav1 in plasticity. The main findings are (1) 70

that Cav1 expression is both altered by, and critical for, exposure to cocaine and subsequent behavioral plasticity, (2) cultured MSNs from Cav1 KO mice display a phenotype consisting of altered dendritic arborization and response to dopamine, and (3) palmitoylation of Cav1 is required for rescue of this cellular phenotype by re-expression of Cav1.

The effect of repeated cocaine exposure on MSN dendritic morphology is clear and consistent: it causes increases in both arborization and spine density (Robinson et al.,

2001; Robinson and Kolb, 2004). However, the relationship between dendritic structure,

MSN function, and animal behavior remains unresolved. For example, the physiological role of increased spine density has been the cause for some debate. In one case, blocking cocaine-induced increases in spine density heightened cocaine sensitivity, suggesting that

MSN spine density increases may actually be a compensatory mechanism reducing sensitivity to the rewarding effects of cocaine (Pulipparacharuvil et al., 2008). Another experiment paradoxically found the opposite (Russo et al., 2009). Of course, interpretations are limited because these studies utilized different methods of blocking cocaine-induced spine increases and also had different measures of cocaine sensitivity

(locomotor sensitization vs. conditioned place preference). Expanding the present experimental direction could help clarify the mechanistic relevance of MSN dendritic structure for cell function and behavior. For example, I report both that Cav1 KO mice have impaired cocaine-induced behavioral plasticity and also that MSNs cultured from these mice have enhanced baseline dendritic arborization. Adding spine analyses and complementary morphological studies from brain slices to determine if the in vitro findings match the in vivo state of NAc MSNs could support the idea that increased

dendritic complexity may actually dilute the effects of cocaine. Moreover, comparison of in vivo MSN morphology before and after cocaine exposure in the Cav1 KO mice would reveal whether the dopamine-induced decrease in arborization seen in vitro is related to the altered behavioral response in these animals.

Follow-up of this co-culture approach with in vivo studies will be critical. The neurons examined here were cultured from embryonic mice, and the fact that dendrites are pruned postnatally (Paolicelli et al., 2011; Lefebvre et al., 2015) should not be overlooked. It is possible that pruning mechanisms would compensate for the enhanced arbor seen in MSNs cultured from Cav1 KO mice, thus eliminating in vivo detection of this phenotype.

An additional caveat to consider is the likelihood that the expression pattern of neurotransmitter receptors differs across developmental time points. In particular, while we typically think of striatal MSNs being segregated into D1-expressing or D2- expressing subtypes, there are reports of MSNs expressing both D1- and D2-type dopamine receptors, possibly as a heteromer. Although the existence of such a heterodimer in neostriatal cells in vitro is fairly accepted, the idea of D1/D2 receptor heterodimers in adults in vivo remains controversial (Frederick et al., 2015; Asher et al.,

2017; Gagnon et al., 2017). It is probably the case that D1/D2 heterodimer expression is limited to a small subset of NAc MSNs in adult rodents in vivo, but is ubiquitous in embryonic culture (Aizman et al., 2000; Biezonski et al., 2015; Hasbi et al., 2018).

The D1/D2 heterodimer is proposed to signal through a Gq-coupled signaling pathway. Preliminary results suggest that the enhancement of MSN dendritic complexity resulting from chronic exposure to dopamine in culture occurs via D1/D2 signaling to Gq

(Pelkey, 2018; Lorene Lanier, unpublished data). Thus, the findings that dopamine- induced dendritic enhancement is reversed in Cav1 KO neurons suggests that the formation of the heterodimer, or something downstream of it, is altered in these cells.

One possibility is that in the case of Cav1 KO, dopamine signaling is restricted to the D1 receptor, leading to the observed arborization decrease. There is presently no selective agonist for the D1/D2 receptor heteromer, but future experiments could utilize the recently-developed D1/D2 heteromer antagonist (TAT-D1 peptide, Hasbi et al., 2014).

Inconsistent with this possibility, however, are recent reports that interruption of D1/D2 heteromer signaling actually enhances locomotor sensitization to cocaine (Hasbi et al.,

2018).

A potentially more plausible explanation of our behavioral data is that Cav1 KO impairs mGluR signaling in the nucleus accumbens, preventing cocaine-induced mGluR- mediated plasticity. Supporting this, experiments using mGluR antagonists (Huang et al.,

2015) and in mGluR knockout animals (Chiamulera et al., 2001) show impaired responses to cocaine and elimination of sensitization. This explanation is especially compelling given reports that mGluR signaling is altered in the hippocampus of Cav1 KO mice (Takayasu et al., 2010). In contrast, however, the fact that the Cav1 KO mice retain the acute response to cocaine suggests that these animals do not have a complete loss of mGluR function. Rather, perhaps Cav1 KO has disrupted the connection between mGluR activity and insertion of calcium-permeable AMPA receptors necessary for the expression of long-term cocaine-induced plasticity. Future studies investigating basal and cocaine-induced electrophysiological properties of Cav1 KO MSNs will test the hypothesis that impaired mGluR signaling contributes to the observed phenotype.

Together, these findings further our understanding of the role of Cav1 in cellular and behavioral plasticity, and suggest that understanding Cav1 function may help identify therapeutic targets for ameliorating addiction.

CHAPTER 4: Overall discussion and conclusions

Importance of Cav1 for ER/mGluR signaling

First, we demonstrated that Cav1 palmitoylation is regulated by the same palmitoylation enzymes that modify steroid hormone receptors. This is particularly important because Cav1 facilitates the interaction between estrogen receptors and mGluRs at the plasma membrane of neurons, whereby estradiol can activate G-protein signaling pathways in a glutamate-independent fashion. This ER/mGluR signaling provides an avenue through which estrogens can alter the plasticity state of a variety of neuronal systems to affect diverse behaviors. While this is adaptive for reproduction- related processes, it is detrimental when plastic motivational systems are usurped by drugs of abuse (Yoest et al., 2014; Tonn Eisinger et al., 2017). Because of the vast implications of ER/mGluR association, it is important to understand what regulates their interaction. Such regulatory mechanisms are likely to be dynamic, allowing the coupling of these receptors in not only a sex-specific manner, but also a cell-specific manner. The contributions made by this dissertation to our understanding of both Cav1 and palmitoylation bring us one step closer to understanding these mechanisms.

ER/mGluR relationship

A commonality between the behaviors affected by ER/mGluR signaling is their reliance on plasticity over time. ER/mGluR enhancement of cocaine-induced locomotor activity does not affect acute psychomotor responses but rather is restricted to effects following repeated drug exposure that lead to sensitization. This is important because this type of long-term plasticity is thought to drive the chronic, relapsing nature of addiction.

For example, the development of behavioral sensitization requires plasticity within the same neural circuits that underlie incubation of craving in humans – a hallmark of

addiction (Robinson and Berridge, 1993; Vanderschuren and Kalivas, 2000; Thomas et al., 2001). Unsurprisingly, ER/mGluR signaling similarly influences other aspects of addiction mediated by long-term plasticity. This can be seen in estradiol enhancement of cocaine self-administration (Martinez et al., 2016) and estradiol enhancement of cocaine- conditioned place preference reinstatement (Tonn Eisinger et al., 2017). It is interesting that the effects of Cav1 KO in male mice reported here were similarly limited to long- term changes, and had no effect on acute responses.

It is becoming increasingly clear that the same mGluRs can pair with distinct downstream signaling partners to have differential effects both within and across brain regions (Mannaioni et al., 2001; Valenti et al., 2002; Poisik et al., 2003; Gross et al.,

2016). In other cases, mGluR signaling may result in the same outcome, but through distinct pathways (Benquet et al., 2002; Thandi et al., 2002). These nuances in mGluR signaling are an important consideration, as they likely contribute to the varied effects of estradiol on structural plasticity. The flexibility and diversity of ER/mGluR signaling outcomes are thus conferred at multiple levels. Learning the precise mechanism that determines which mGluR an ER pairs with, and the nature of downstream effects of that mGluR, will clarify our understanding of how estradiol modulates neural systems in specific and complex ways.

Additionally, although membrane-localized ER signaling has generally been studied in isolation from nuclear signaling, it is apparent that integration of these mechanisms must be considered (Frick, 2015). Perhaps the distinct estradiol signaling mechanisms serve as a sort of coincidence detector in various systems, whereby the convergence of rapid and slow effects promotes a certain outcome. This can be seen in

the case of sexual receptivity, as both the slower and more rapid effects of estradiol are required in the hypothalamus for the normal expression of sexual receptivity in females

(Kow and Pfaff, 2004). Specifically, ERα/mGluR1a signaling leads to rapid internalization of µ-opioid receptors in the medial preoptic area (Dewing et al., 2007), followed by a slower, enduring increase in dendritic spine density in the arcuate nucleus

(Christensen et al., 2011). Similarly, estradiol signaling through ERα/mGluR5 affects neuronal excitability in striatal cells on the order of seconds or minutes (Grove-Strawser et al., 2010), followed by slower effects on dendritic spine plasticity (Peterson et al.,

2014). These differing time courses converge to enhance motivated behaviors in females, as seen in the findings from our lab and others on estradiol facilitation of cocaine-induced plasticity. Although nuclear estrogen receptors are not found in abundance in the NAc, nuclear ERs in other reward circuitry brain regions could contribute to the effects of estrogens on the development and maintenance of addiction. Thus, it stands to reason that nuclear estradiol signaling adds another layer to the processes discussed here, and careful work must be undertaken to dissect these signaling mechanisms. Doing so will help explain the special nature of the ER/mGluR relationship that allows estradiol to powerfully influence neuronal physiology, structure, and behavior.

Sex differences

Importantly, the relationship between estrogen receptors and mGluRs appears unique to females across many brain regions (but see Hedges et al., 2018), and appears to exist even in the absence of ER activation. Co-immunoprecipitation studies have indicated a physical association between estrogen receptors and mGluRs (Dewing et al.,

2007). The transactivation hypothesis is further supported by the fact in cultured neurons

generated from female rat pups, the estrogen receptor antagonist, ICI 182,780, will attenuate DHPG-induced group I mGluR activation, which leads to increased CREB phosphorylation (Tonn Eisinger et al., 2017). Furthermore, disrupting ERα interacting with group I mGluRs via use of a dominant-negative CAV1 construct eliminates the effect. Additional potential off-target effects of ICI 182,780 were ruled out using male- derived cultures, which lack ER/mGluR coupling (Boulware et al., 2005). In this preparation, ICI 182,780 had no effect on DHPG-induced CREB phosphorylation.

Interactions between hormones and responses to drugs of abuse are revealed in part by the sex differences observed between men and women in addiction. For example,

16-17% of drug users progress to an addicted state (SAMHSA, 2014), but women appear more likely to reach this addicted state than men (Wagner and Anthony, 2007; Becker et al., 2012). Additionally, women begin psychostimulant use at younger ages, have increased acute responses, more rapidly escalate use, and progress to addiction faster

(Etten et al., 1999; Justice and Wit, 1999; Van Etten and Anthony, 2001). This is especially evident with psychostimulants, but is also seen with opioids, nicotine, alcohol, and marijuana (Carroll et al., 2004; Becker and Hu, 2008). Women also report that psychostimulants produce greater subjective effects when estradiol levels are elevated, due either to natural hormonal cycles or to exogenous administration (Justice and Wit,

1999; Evans et al., 2002; Terner and Wit, 2006; Maria et al., 2014). These effects have been reproduced in rodent studies, where ovariectomy eliminated sex differences in models of addiction, and estradiol replacement restored those differences (Sircar and

Kim, 1999; Becker and Hu, 2008; Anker and Carroll, 2010; Segarra et al., 2010). One of the consequences of neuronal plasticity in the motivational system is that sex behavior

and exposure to drugs of abuse cross-sensitize. This also solidifies the notion that the motivational pathways for reproduction and drugs of abuse are interrelated (Hedges et al.,

2010). For example, prior sexual experience produces increased dopamine release in the nucleus accumbens during subsequent mating (Kohlert and Meisel, 1999) and increases drug-induced locomotor responsiveness (Bradley and Meisel, 2001). The reverse has also been demonstrated; that is, previous exposure to drugs of abuse enhances motivation to engage in sex behavior (Afonso et al., 2009).

Sex differences in drug abuse extend to the nucleus accumbens, where drugs of abuse can increase MSN spine density to a greater degree in females than males

(Wissman et al., 2011a; Strong et al., 2017). Catherine Woolley’s group has shown that structural differences are associated with physiological differences, in that the frequency of miniature excitatory postsynaptic currents in NAc MSNs increased with cocaine in females more than males (Wissman et al., 2011b). Their work demonstrated that differences exist in excitatory synapse number per neuron rather than in presynaptic release probability, further supporting the importance of dendritic spine changes.

Modulation of neurotransmission systems in the nucleus accumbens by drugs of abuse produces long-lasting changes in MSN excitability and structure; these changes are seen as the neurobiological basis for drug addiction (Russo et al., 2010; Golden and Russo,

2012; Kourrich et al., 2015). Thus, it follows that estradiol-induced plasticity within the female nucleus accumbens could impact the effects of drugs of abuse.

Current work focusing on the role ER/mGluR signaling plays in regulating various aspects of drug addiction builds on a growing body of evidence linking group I mGluRs (especially mGluR5) to responses to drugs of abuse (Pomierny-Chamioło et al.,

2014). We find that estradiol replacement facilitates cocaine-induced locomotor sensitization in ovariectomized female rats in an mGluR5-dependent manner (Martinez et al., 2014). This ERα/mGluR5 mediated effect also relies on endocannabinoid signaling and CB1 receptor activation (Peterson et al., 2016). It will be critical to extend the present research to female Cav1 KO mice in order to investigate sex differences and better understand its role in ER/mGluR signaling.

Cav1 and drug-induced plasticity

Addiction is a disease of aberrant plasticity in the brain, in which drugs of abuse modify the neural environment to create long-lasting adaptations in synaptic and circuit connectivity. The molecular changes underlying these adaptations are varied and vast, while at the same time highly specific. They involve upregulation and downregulation of both ionotropic and metabotropic receptors, altered by changes in transcription, translation, localization, structure, and function, and in turn cause further changes in these same processes. In the case of psychostimulants, dopaminergic and glutamatergic signaling converge in the nucleus accumbens, where group I mGluR signaling leads to insertion of calcium-permeable AMPA receptors that contributes to enduring cocaine craving, a hallmark of addiction that leads to relapse. While it is clear that mGluRs play a key role in this process, the mechanistic link between the effects of cocaine and altered mGluR function remains unclear. Investigating the relationship between cocaine and proteins that can regulate mGluR function, like Cav1, is an important step towards fully understanding how drugs of abuse cause indelible changes to brain physiology.

Cav1 interacts with and/or influences many signaling molecules associated with drug- induced plasticity

Figure 4.1. Schematic of palmitoylated Cav1 interacting with signaling proteins at the plasma membrane.

Lipid rafts

Cav1 is a prevalent component of lipid rafts, often serving as a marker for these specialized membrane compartments. Cav1 preferentially associates with inactive forms of lipid-modified signaling molecules, including G, H-ras, Src (Li et al., 1995, 1996;

Oka et al., 1997). Along with Cav1, many of these associating proteins are palmitoylated, which increases their hydrophobicity and ability to interact with lipids (Levental et al.,

2010). Cav1 may function to negatively regulate activation state of these proteins by anchoring otherwise cytosolic signaling molecules at the membrane in their inactive form. This dual function could be seen as both facilitative, in that it localizes effector proteins conveniently near their signaling partners, and inhibitory, in that Cav1 association seems incompatible with activation.

Interestingly, repeated cocaine exposure alters the “lipidome” in brain regions associated with drug-induced plasticity, including the NAc, PFC, hippocampus, and

dorsal striatum (Lin et al., 2017). Some of these lipids (i.e., sphingolipids, cholesterol, and gangliosides) are enriched in the eponymous lipid rafts, and alterations to their expression could be indicative of impactful changes to membrane structure and cellular function, particularly related to Cav1 and associating proteins.

Extracellular signal-related kinase

Cav1 deficiency is associated with increased basal extracellular signal-related kinase (ERK) phosphorylation (Francesconi et al., 2009; Takayasu et al., 2010). ERK activation via phosphorylation plays an important role in carrying out the long-term effects of drugs of abuse. It serves as a coincidence detector in the nucleus accumbens, where simultaneous glutamate and dopamine signals are required for ERK activation, which then facilitates plasticity (Girault et al., 2007). For Cav1 KO mice, it could be that elevated basal ERK means that drug-induced changes cannot occur; by starting at a higher level, the dynamic range has been dampened and gain control available for drugs of abuse to exert changes is lost.

The induction and expression of cocaine-induced behavioral sensitization require an intricate combination of various signaling processes, including dopamine, glutamate, neuropeptides, and trophic factors. These signals may converge on the ERK pathway, which integrates these varied extracellular events and translates them into long-term effects (Lu et al., 2006). Systemic inhibition of ERK phosphorylation blocks the development of sensitization without greatly altering acute responses (Valjent et al.,

2006). Moreover, psychostimulant exposure increases phosphorylation of ERK in a subset of striatal MSNs (Valjent et al., 2006; Kim and Kim, 2008). This increase requires both D1 dopamine and glutamatergic signaling. Increased ERK activity in the NAc could

contribute to secondary neuroplastic events that permit input from the VTA to have a larger output, as seen in sensitization. Losing the ability to upregulate pERK as a result of psychostimulant exposure could occlude cellular plasticity, preventing the development and expression of behavioral plasticity. Thus, disrupted ERK signaling could explain the lack of behavioral sensitization seen in Cav1 KO mice.

NMDA Receptors

Cav1 may influence additional processes involved in drug-induced plasticity as well. For example, repeated cocaine exposure increases so-called “silent synapses” which contain NMDA receptors but no AMPA receptors (Huang et al., 2009b; Scofield et al.,

2016). Withdrawal from cocaine leads to synapse maturation through AMPAR insertion.

Overexpression of Cav1 in the hippocampus increases NMDAR subunit localization to membrane/lipid rafts in the hippocampus and increases synapse number (Egawa et al.,

2017), and so it follows that Cav1 could somehow be involved in the development of silent synapses. Perhaps Cav1 KO animals have a higher percentage of silent synapses at baseline, occluding the normal increase in such synapses, thereby preventing drug- induced plasticity. Interestingly, however, NMDAR-LTD at Schaffer-CA1 synapses is normal in slices from Cav1 KO mice (Takayasu et al., 2010). Together with other findings, this suggests that the role of Cav1 at CA1 synapses may be selective for mGluR-dependent plasticity.

mGluR-dependent plasticity is crucial for drug-induced changes. Blocking group I mGluRs inhibits cocaine sensitization (Russo et al., 2010). Group I mGluRs (mGluR1a and mGluR5) are primarily post-synaptic, located at the edge of the postsynaptic density, where they interact with Homer, Shank, and other scaffolding proteins to form a

signaling complex with ionotropic receptors and downstream effectors (Verpelli et al.,

2011; Xiao et al., 2000). This organization is critical for their proper function (Ronesi et al., 2012; Ronesi and Huber, 2008), which involves modulation of excitatory neurotransmission (Saugstad and Ingram, 2008). Group I mGluRs are important for synaptic plasticity (Fitzjohn and Bashir, 2008), and their activation can be involved in both LTD and LTP (Anwyl, 2009; Lüscher and Huber, 2010). Group I mGluR activity is highly associated with structural changes, particularly in dendritic spines (Vanderklish and Edelman, 2002). Many of these changes are associated with synaptic weakening or spine loss, and this modulation of spine structure may be relevant as a mechanism for synaptic refinement (Wilkerson et al., 2014).

The two group I mGluRs, mGluR1a and mGluR5, are often co-expressed, but not always to the same degree (Shigemoto et al., 1992, 1993). They are also often thought of as interchangeable, but increasing evidence suggests they have distinct and sometimes cooperative functions. For example, in the striatum and globus pallidus, mGluR5 activity regulates the outcome of mGluR1 on neuronal excitability (Kramer and Williams, 2015;

Poisik et al., 2003). Cocaine exposure alters the influence of mGluR1 versus mGluR5 on excitatory neurotransmission (McCutcheon et al., 2011). In the context of drug abuse, mGluR5 is more thoroughly studied than mGluR1. Blockade of mGluR5 eliminates responses to acute and long-term cocaine exposure (Chiamulera et al., 2001).

Cav1 KO mice show impaired mGluR-dependent (DHPG-induced) LTD in the hippocampus (Takayasu et al., 2010). This results from impaired rapid activation of ERK by mGluRs, which is possibly occluded by elevated basal ERK phosphorylation. This suggests that group I mGluRs are uncoupled to their downstream signaling effectors in

Cav1 KO mice. LTD is not completely abolished, however, and residual LTD appears to be mediated by the same signaling mechanism as in WT animals. Moreover, a greater degree of depression was achieved following repeated application of DHPG, bringing

Cav1 KO levels in line with WT. This suggests that the mechanisms for mGluR-LTD are in place, only less sensitive and therefore resistant to activation. Perhaps something about the increased dendritic complexity of the Cav1 KO neurons requires more effort to achieve the same degree of LTD.

The nucleus accumbens and medium spiny neurons

The nucleus accumbens (NAc) region of the striatum carries out complicated processing required for motivated behaviors. Current models posit that glutamatergic afferents to the NAc from the hippocampus, prefrontal cortex, and amygdala provide signals needed for prediction, dopaminergic inputs from the ventral tegmental area provide reinforcement information, and GABAergic inputs engage in action selection and subsequent motor output (Nestler, 2001; Sesack and Grace, 2010; Lüscher and Malenka,

2011). The structure of the NAc is specialized in order to perform this integration. The nucleus accumbens is divided into two sub regions: the core, which is predominately interconnected with motor circuitry, and the shell, which is connected to other limbic structures. Together, these two regions control the execution of conditioned behaviors

(NAc core) and their reinforcement through interaction with reward circuitry (NAc shell)

(Meredith et al., 2008; Haber, 2011). Dendritic spines on medium spiny neurons (MSNs) integrate dopamine and glutamate inputs, with dopamine modulating the signal of incoming glutamatergic input (Surmeier et al., 2007). MSNs are the predominant output neurons of the striatum, and are capable of influencing motor and cognitive behaviors

through projections to other brain regions (Smith et al., 2013; Yager et al., 2015).

Excitatory synaptic input on to MSN dendrites is essential for generating action potential output and regulating synaptic plasticity (O’Donnell and Grace, 1995; Mulder et al.,

1998; Sesack et al., 2003; Papp et al., 2011; Stuber et al., 2011; Britt et al., 2012).

Although our behavioral data showing impaired sensitization in Cav1 KO mice is from a global knockout, there are several reasons we believe that Cav1 KO exerts changes in the nucleus accumbens. First, we showed that repeated cocaine enhanced

Cav1 mRNA in the nucleus accumbens but not the dorsal striatum (caudate/putamen).

Secondly, long-lasting plasticity in the nucleus accumbens underlies the development of cocaine sensitization (Thomas et al., 2001). Finally, cultured MSNs from Cav1 KO mice have altered dendritic arborization and responses to dopamine.

Dendritic arbors are highly dynamic, and the extent of arbor formation depends on both intrinsic factors and external cues, including neuronal activity (Dailey and Smith,

1996; Cline, 2001; Niell et al., 2004; Parrish et al., 2007). Alterations in neuronal activity caused by drug-induced plasticity can in turn lead to alterations in dendritic morphology

(Kauer and Malenka, 2007). Culturing MSNs for dendrite analysis as performed here allows experimental control of the myriad variables that affect the dendritic arborization, allowing conclusions to be made about the direct effects of pharmacological and genetic manipulations. The cellular phenotype described here, wherein Cav1 KO MSNs have enhanced dendritic complexity which is reversed by dopamine, raises many new questions about the role of Cav1 in neuronal plasticity. Future work will need to determine the signaling pathways involved and the implications for physiology and behavior. Nevertheless, this phenotype could have relevance for drug-related behaviors

like those reported here, as well as Cav1-related disorders such as schizophrenia (Kassan et al., 2016).

Conclusion

This dissertation began with the goal of determining whether Cav1 palmitoylation was related to membrane-initiated ER/mGluR signaling, and found that the same enzymes that palmitoylate the estrogen receptor are implicated in Cav1 palmitoylation.

This work went on to demonstrate the importance of Cav1 for cocaine-induced plasticity, and offered insight into a potentially associated cellular phenotype. Moreover, it highlighted the importance of Cav1 palmitoylation for normal function.

There is much left to learn about the role of neuronal Cav1, and our understanding of dynamic palmitoylation is in its infancy. It can be difficult to make meaningful conclusions about how the mechanisms of disease/addiction/sex difference are affected by Cav1 and palmitoylation while the basic science is still so patchy. So although we started with the framework of ER/mGluR signaling and its facilitation by caveolin and palmitoylation, it became necessary to strip away that context and focus on Cav1 in isolation. Consequently, the knowledge gained here has extremely broad implications for many critical neuronal signaling mechanisms.

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