A NEW MECHANISM of SEROTONIN TRANSPORTER REGULATION by SIMVASTATIN and the ISOPRENYLATION PATHWAY Carmen Marie Deveau Submitted

A NEW MECHANISM OF SEROTONIN TRANSPORTER REGULATION BY

SIMVASTATIN AND THE ISOPRENYLATION PATHWAY

Carmen Marie Deveau

Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree Doctor of Philosophy in the Department of Pharmacology and Toxicology, Indiana University

July 2021

Accepted by the Graduate Faculty of Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Doctoral Committee

______Bryan K Yamamoto, PhD, Chair

______Patrick L. Sheets, PhD

May 13, 2021

______William J. Sullivan, PhD

______Brady K. Atwood, PhD

______Nickolay Brustovetsky, PhD

Carmen Marie Deveau

iii DEDICATION

I would like to dedicate this dissertation to my grandfather John Baljak who always taught me that trying my best was the only thing that mattered. His ambition, sacrifice, and faith continue to allow for opportunity in generations to come.

iv ACKNOWLEDGEMENT

The PhD path has been one of the most difficult processes I have had the privilege to experience. The resilience, grit, and perseverance displayed by those around me including my mentor, current and former lab colleagues, and the students and faculty in the department helped me to achieve an understanding and respect toward the difficult process of science. Through this understanding, I was able to develop the ability to ask new questions and consider the endless realm of possibilities when confronted with scientific and methodological obstacles. I would love to thank all of these individuals for the impact they had on my perspective during my PhD research.

I owe my development as a scientist to the guidance of my mentor Bryan. I thank

Bryan for his constant feedback toward new dissertation ideas, strategies, and approaches. Bryan has always put his students first and I have consequently received thorough mentoring on all aspects of being a good scientist. I will forever value his opinions and I feel lucky to have experienced scientific discovery under his care and guidance.

I would also like to thank the current and former lab colleagues from the

Yamamoto lab. In addition to my mentor’s guidance, my lab colleagues have helped me prepare for my science career through critical evaluation of the data and approaches used in this dissertation. They have also offered unwavering support during the challenging trials of research, and have provided me with lasting memories and lifelong friendships.

v Carmen Marie Deveau

A NEW MECHANISM OF SEROTONIN TRANSPORTER REGULATION BY

SIMVASTATIN AND THE ISOPRENYLATION PATHWAY

The serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on

SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function.

To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5-

HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by

vi statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase

(CAMKII).

Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs.

Bryan K Yamamoto, PhD, Chair

vii TABLE OF CONTENTS

List of Figures ...... xi List of Abbreviations ...... xii Chapter 1: Introduction to SERT in the Brain and Neuron ...... 1 The Serotonin System ...... 1 The Serotonin Transporter ...... 3 Pharmacological Regulation of SERT ...... 5 Trafficking Dependent Regulation of SERT ...... 6 Trafficking Independent Regulation of SERT ...... 8 Lipids and SERT ...... 12 Cholesterol Regulation in the Brain ...... 14 Cholesterol and Brain 5-HT ...... 15 Gaps in Knowledge ...... 16 Chapter 2: Serotonin Uptake and Statins ...... 18 Introduction ...... 18 Methods ...... 22 Preparation of RN46A-B14 cells with SERTMyc expression ...... 22 SERTMyc cell culture and reagents ...... 23 5-HT uptake ...... 23 5-HT measurement by high pressure liquid chromatography ...... 24 5-HT and myc measurement by immunocytochemical detection ...... 24 Biotinylation and separation of plasma membrane and intracellular SERT ...... 26 Western blot analysis ...... 27 In vivo simvastatin treatment and ex vivo synaptosome preparation ...... 27 Statistical analysis ...... 28 Results ...... 30 2.1 RN46-B14 cells exhibit myc-tagged SERT expression ...... 30 2.2 Statins enhance 5-HT uptake in a concentration-dependent manner ...... 32 2.3 Simvastatin-enhanced 5-HT uptake is consistent over time ...... 34 2.4 Simvastatin enhances 5-HT uptake in a SERT-dependent manner ...... 36 2.5 Simvastatin does not change endogenous 5-HT content ...... 38 2.6 Simvastatin decreases the Michaelis-Menten Km for 5-HT uptake without changing Vmax and decreases the Ki of fluoxetine ...... 40 2.7 Simvastatin does not alter total SERT or SERT localization ...... 42 2.8 Simvastatin increases 5-HT uptake in ex vivo synaptosomes from rats treated with simvastatin ...... 44 Discussion ...... 46 Chapter 3: Mediators of Simvastatin-Increased 5-HT Uptake ...... 50 Introduction ...... 50 Methods ...... 53 SERTMyc cell culture and reagents ...... 53 Cholesterol measurement in SERTMyc cells ...... 53 siRNA knockdown of Rac1 and RhoA mRNA ...... 54 Results ...... 55 3.1 Restoration of cholesterol does not block simvastatin-increased 5-HT

viii uptake ...... 55 3.2 Direct cholesterol depletion with mβCD decreased 5-HT uptake ...... 57 3.3 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis pathway intermediate mevalonate, but not squalene ...... 59 3.4 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis pathway intermediates FPP and GGPP ...... 61 3.5 Role of GGPP-targeted small GTPases in statin-increased 5-HT uptake ...... 63 3.5.1 Inhibition of GGPP-targeted small GTPase CDC42 does not block simvastatin-enhanced 5-HT uptake ...... 63 3.5.2 Inhibition of GGPP-targeted small GTPase RhoA does not block simvastatin-enhanced 5-HT uptake ...... 65 3.5.3 Inhibition of GGPP-targeted small GTPase Rac1 does not block simvastatin-enhanced 5-HT uptake ...... 67 3.6 Role of kinases and phosphatases in statin-increased 5-HT uptake ...... 69 3.6.1 Phospho-38MAPK is increased after simvastatin, but is not involved in simvastatin-enhanced 5-HT uptake ...... 79 3.6.2 Pharmacological inhibition of PP2A does not affect simvastatin- enhanced 5-HT uptake ...... 71 3.6.3 CAMKII is involved in simvastatin-enhanced 5-HT uptake ...... 73 Discussion ...... 75 Chapter 4: Mechanism of Increased 5-HT Uptake by Simvastatin and GGT Inhibition .. 79 Introduction ...... 79 Methods ...... 82 SERTMyc cell culture and reagents ...... 82 Results ...... 82 4.1 Inhibition of GGT enhances 5-HT uptake ...... 82 4.2 GGTI-298-enhanced 5-HT uptake is SERT-dependent ...... 85 4.3 GGT inhibition increases 5-HT uptake to a greater extent than simvastatin ... 87 4.4 CAMKII inhibition during Simvastatin and GGT Inhibition ...... 89 4.4.1 CAMKII peptide inhibitor Tat-CN21 does not block simvastatin or GGTI-298-increased 5-HT uptake ...... 89 4.4.2 CAMKII small molecule inhibitors 78 and 79 block simvastatin and GGTI-298-increased 5-HT uptake ...... 91 4.5 Whole cell and intracellular phosphoCAMKII were not increased after simvastatin and GGTI-298 ...... 93 Discussion ...... 95 Chapter 5: General Discussion ...... 98 Summary of Statin and GGT Inhibition on SERT Regulation ...... 98 Activity-Dependent Regulation of SERT by Isoprenylation ...... 100 Cholesterol-Independent Regulation of SERT by Isoprenylation ...... 100 Involvement of CAMKII in Statin and GGT-Regulated SERT ...... 102 Involvement of Other Kinases and Phosphatases ...... 103 Post-Translational Modifications Possibly Involved ...... 103 CAMKII Activation by Statins and GGT Inhibition ...... 105 Shared Mechanisms of Monoamine Transporter Regulation ...... 107 Dopamine Uptake by SERT ...... 108

ix Adverse Statin Effects in Humans ...... 108 New Indications for Statin Treatment in Overactive 5-HT Conditions ...... 109 Future Directions ...... 110 References ...... 113 Curriculum Vitae

x LIST OF FIGURES

Figure 1.0 SERT kinase phosphorylation sites ...... 11 Figure 1.1 Dissertation objectives ...... 17 Figure 2.0 Hypothetical model ...... 21 Figure 2.1 RN46-B14 cells exhibit myc-tagged SERT expression ...... 31 Figure 2.2 Statins enhance 5-HT uptake in a concentration-dependent manner ...... 33 Figure 2.3 Simvastatin-enhanced 5-HT uptake is consistent over time ...... 35 Figure 2.4 Simvastatin enhances 5-HT uptake in a SERT-dependent manner ...... 37 Figure 2.5 Simvastatin does not change endogenous 5-HT content ...... 39 Figure 2.6 Simvastatin decreases the Michaelis-Menten Km for 5-HT uptake without changing Vmax and decreases the Ki of fluoxetine ...... 41 Figure 2.7 Simvastatin does not alter total SERT or SERT localization ...... 43 Figure 2.8 Simvastatin increases 5-HT uptake in ex vivo synaptosomes from rats treated with simvastatin ...... 45 Figure 3.0 Hypothetical model ...... 52 Figure 3.1 Restoration of cholesterol does not block simvastatin-increased 5-HT uptake ...... 56 Figure 3.2 Direct cholesterol depletion with mβCD decreased 5-HT uptake ...... 58 Figure 3.3 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis pathway intermediate mevalonate, but not squalene ...... 60 Figure 3.4 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis pathway intermediates FPP and GGPP ...... 62 Figure 3.5.1 Inhibition of GGPP-targeted small GTPase CDC42 does not block simvastatin-enhanced 5-HT uptake ...... 64 Figure 3.5.2 Inhibition of GGPP-targeted small GTPase RhoA does not block simvastatin-enhanced 5-HT uptake ...... 66 Figure 3.5.3 Inhibition of GGPP-targeted small GTPase Rac1 does not block simvastatin-enhanced 5-HT uptake ...... 68 Figure 3.6.1 Phospho-38MAPK is increased after simvastatin, but is not involved in simvastatin-enhanced 5-HT uptake ...... 70 Figure 3.6.2 Pharmacological inhibition of PP2A does not affect simvastatin- enhanced 5-HT uptake ...... 72 Figure 3.6.3 CAMKII is involved in simvastatin-enhanced 5-HT uptake ...... 74 Figure 4.0 Hypothetical model ...... 81 Figure 4.1 Inhibition of GGT enhances 5-HT uptake ...... 84 Figure 4.2 GGTI-298-enhanced 5-HT uptake is SERT-dependent ...... 86 Figure 4.3 GGT inhibition increases 5-HT uptake to a greater extent than simvastatin ... 88 Figure 4.4.1 CAMKII peptide inhibitor Tat-CN21 does not block simvastatin or GGTI-298-increased 5-HT uptake ...... 90 Figure 4.4.2 CAMKII small molecule inhibitors 78 and 79 block simvastatin and GGTI-298-increased 5-HT uptake ...... 92 Figure 4.5 Whole cell and intracellular phosphoCAMKII were not increased after simvastatin and GGTI-298 ...... 94 Figure 5.0 Schematic representation of summarized results ...... 99

xi LIST OF ABBREVIATIONS

5-HT, Serotonin CAMKII, Ca2+/Calmodulin-Dependent Protein Kinase CaN, Ca2+-Activated Protein Phosphatase Calcineurin DA, Dopamine DAT, Dopamine Transporter ER, Endoplasmic Reticulum FPP, Farnesyl Pyrophosphate FT, Farnesyl Transferase GGPP, Geranylgeranyl Pyrophosphate GGT, Geranylgeranyl Transferase NET, Norepinephrine Transporter PFC, Prefrontal Cortex PKC, Protein Kinase C PKG, Protein Kinase G PP2A, Protein Phosphatase 2A OA, Okadaic Acid SERT, Serotonin Transporter SSRI, Selective Serotonin Reuptake Inhibitor

xii

Chapter 1: Introduction to SERT in the Brain and Neuron

The Serotonin System

The serotonergic system is comprised of a serotonin (5-HT, 5- hydroxytryptomine)-rich monoamine neurotransmitter connective network that spans the brain and spinal cord. 5-HT is synthesized in the midbrain raphe nucleus and is transported in dense projections from the raphe nucleus to brain regions involved in neurophysiological processes related to sleep, cognition, and mood regulation. Numerous studies support the role of raphe 5-HT and innervated brain regions in these neurophysiological processes. For instance, genetic ablation of 5-HT neurons in the raphe prevents sleep in zebrafish and mice, whereas stimulation of raphe 5-HT neurons promotes sleep (Iwasaki et al., 2018; Oikonomou et al., 2019). Selective loss of 5-HT neurons in brain regions such as the frontal cortex impairs learning and cognitive flexibility in monkeys (Clarke et al., 2004), while increases in hippocampal 5-HT in rats enhance performance in cognitive assessment tests (Haider et al., 2006). Deep brain stimulation of frontal cortex neurons in humans mitigates major depression disorder symptoms (Veerakumar et al., 2014) and reduces depression severity (Kennedy et al.,

2011). Similarly, direct stimulation of serotonergic neurons in the raphe of mice decreases immobility in depression-like behaviors (Nishitani et al., 2018). As a result, the

5-HTergic pathways in the brain have been targeted to treat diseases associated with cognition and mood and have resulted in further identification of 5-HT-related diseases including generalized anxiety and obsessive-compulsive disorder (OCD).

5-HT in the raphe and its projections is regulated by synthesis, degradation, and reuptake of 5-HT. 5-HT synthesis begins with the conversion of the essential amino acid

1 tryptophan into 5-hydroxytryptophan (5-HTP) by the rate-limiting enzyme tryptophan hydroxylase. 5-HTP is further converted to 5-hydroxytryptamine (5-HT) via amino acid decarboxylase (Rahman et al., 1982). Synthesized 5-HT is transported into presynaptic vesicles by a vesicular monoamine transporter (VMAT) by the uptake of 5-HT into the vesicle in exchange for two hydrogen ions out of the vesicle (Fon et al., 1997).

Propagation of an action potential to the presynaptic terminal results in the release of vesicular 5-HT from the presynaptic neuron through increases in gated ion channels that increase Ca2+ into the presynaptic neuron, and result in Ca2+-mediated release of 5-HT- containing vesicles (reviewed in Chanaday et al., 2018).

5-HT released into the synaptic cleft can subsequently interact with multiple subtypes of 5-HT receptors. There are seven main receptors (5-HT1, 5-HT2, 5-HT3, 5-

HT4, 5-HT5, 5-HT6) with multiple subtypes within each main receptor category, resulting in 14 different 5-HT receptors (reviewed in Pithadia and Jain 2009) and associated signaling mechanisms. For instance, 5-HT1 and 5-HT5 receptors are Gi/Go-coupled and result in reduced cAMP, and an inhibitory potential. Conversely, 5-HT4, 5-HT6, and 5-

HT7 are Gs-coupled, increase cAMP, and result in an excitatory potential. Lastly, 5-HT2 receptors are excitatory through increases in cellular IP3 and DAG, and 5-HT3 receptors are uniquely ligand-gated Na+ and K+ cation channels that depolarize the plasma membrane and result in an excitatory potential. Released 5-HT can also act on presynaptic autoreceptors, such as at 5-HT1A (reviewed in Hannon et al., 2008), to inhibit presynaptic 5-HT synthesis and release (Meller et al., 1990; Starkey and Skingle 1994) that in turn, prevents 5-HT from interacting with postsynaptic receptors.

2 The action of 5-HT is primarily terminated by uptake into the presynaptic neuron through the serotonin transporter (SERT) (Kuhar et al., 1972; Ross and Renyi 1967). 5-

HT is subsequently repackaged into presynaptic vesicles by VMAT or degraded by the mitochondrial-bound enzyme monoamine oxidase (MOA) by oxidative deamination.

The Serotonin Transporter

The first identification and characterization of SERT was on blood platelets

(Sneddon, 1969) and gut enterochromaffin cells (Kanner et al., 1979). SERT was later identified in the brain (Rehavi et al., 1983) and functions to transport extracellular 5-HT into neurons through the symport of sodium, protonated 5-HT, and chloride into the presynaptic terminal, followed by the transport of potassium out of the terminal. This transport sequence results in a net neutral transport of ions and one 5-HT (Na+, 5-HT+, Cl- in, K+ out). The theory of SERT transport mechanics is supported by the “rocking bundle” mechanism (Forrest and Rudnick 2009), which validate structural changes to

SERT that cause it to face in an outward, extracellular conformation when unbound. This conformational change permits the binding of extracellular 5-HT in a chloride and sodium-dependent manner (Zhang and Rudnick 2006). The transporter then moves as a unit to a cytoplasmic-open and extracellular-closed conformation, followed by release of

5-HT, sodium, and chloride into the cytoplasm. After 5-HT and ion release, the inward- facing transporter binds potassium and recycles back to an extracellular-facing conformation. Additionally, the sodium and potassium gradients across the cell membrane are maintained by the sodium/potassium ATPase antiporter.

3 Plasma membrane SERT at the presynaptic neuron is distributed along the active zone (defined as the location of vesicle release directly across from the postsynaptic neuron) and at extrasynaptic sites (adjacent to the active zone) (Pickel and Chan 1999).

This distribution of SERT allows for reuptake of 5-HT released at the synapse or that which diffuses to extrasynaptic sites (Bunin and Wightman 1998). Prior to plasma membrane distribution, SERT must undergo synthesis, post-translational modification, and transport to the plasma membrane to reuptake extracellular 5-HT. SERT is synthesized from the gene SLC6A4 (Solute Carrier family 6, member A4) and is under transcriptional control by pet1 (pheochromocytoma 12 ETS factor-1), lmx1b (LIM homeobox transcription factor 1 β), and Gata3 (Hendricks et al., 2003; Liu et al., 2010;

Zhao et al., 2006). Gata3 primarily regulates SERT expression during development, while pet1 and lmx1b are necessary for SERT expression in the adult brain (van

Doornink et al., 1999).

SERT is regulated at a steady state level by recurring synthesis and degradation.

The half-life of SERT is 3.4 days whereas SERT can be fully recovered after degradation within 14 days in the hippocampus and striatum brain regions (Vicentic et al., 1999).

Synthesized SERT is folded at the endoplasmic reticulum (ER) to a 12 transmembrane-spanning structure with 5 intracellular loops and 6 extracellular loops.

The N and C-terminal tails of SERT at the plasma membrane are located in the cytoplasm. SERT is also post-translationally modified in the ER by N-linked glycosylation, which has proven to be critical for SERT function (El-Kasaby et al., 2010;

Larsen et al., 2006; Tate et al., 1994). Mutation of N-linked glycosylation sites on extracellular loop 2 results in reduced Vmax and less SERT expression at the plasma

4 membrane. In contrast, there is a naturally occurring human SERT variant that has an additional N-linked glycosylation site that increases SERT expression by 30%. The mutation also increases 5-HT uptake, Vmax for 5-HT, binding of serotonin selective reuptake inhibitors (SSRI), and SERT trafficking to the plasma membrane (Rasmussen et al., 2009). Once trafficked to the membrane, SERT preferentially sorts to late Rab7 endosomes for lysosomal degradation (Rahbek-Clemmensen et al., 2014) but can be recycled to and from the plasma membrane to Rab4-rich early short loop recycling endosomes or Rab11 long-loop recycling endosomes.

Based on the findings that SERT is the primary regulator of 5-HT transmission at the synapse and can be regulated at multiple points involving its synthesis, folding, structural modification, and trafficking, SERT is a viable and strategic target for the treatment of 5-HT-related disorders.

Pharmacological Regulation of SERT

Drugs that target and inhibit SERT protein at the plasma membrane are used to treat depression, generalized anxiety, and OCD. SSRIs block SERT and prevent 5-HT uptake, thereby prolonging the availability of 5-HT in the synapse and sustaining 5-HT receptor interactions and post-synaptic signaling. SSRIs inhibit SERT at two primary sites designated S1 and S2. The S1 site of SERT is the central pathway for the permeation of 5-HT into the cell and the cavity where 5-HT and ions bind to SERT.

Alternatively, S2 is at the extracellular entry site of the permeation pathway, which is in close proximity but independent of where 5-HT and ions bind to SERT (Coleman et al.,

2016; Coleman et al., 2018). The majority of SSRIs (fluoxetine, citalopram, imipramine,

5 etc.) competitively bind S1 with high affinity (Andersen et al., 2009; Coleman et al.,

2018; Tavoulari et al., 2009). The S2 site of SERT is an allosteric binding site for the

SSRIs and is bound by SSRIs at a much lower affinity than S1 (Banala et al., 2013;

Coleman et al., 2016; Coleman et al., 2018). More specifically, binding to the S2 allosteric site results in a conformation change in the transporter so that 5-HT is unable to bind at the permeation pathway site. Therefore, SSRIs differ in their inhibitory properties based on their differential affinities for S1 compared to S2.

Although SSRI drugs have been a mainstay for the treatment of depression, they require chronic administration for at least 2-6 weeks to resolve depressive symptoms

(Katz et al., 2006; Stassen et al., 1993). This delayed action suggests long term changes in protein trafficking, membrane expression and/or synthesis are required. In fact, chronic

SSRI treatment changes SERT distribution. Specifically, chronic fluoxetine treatment in rats, but not acute treatment, causes the internalization of SERT into dorsal raphe neurons

(Benmansour et al., 1999; Descarries and Riad 2012). 5-HT1A autoreceptors also display delayed differences in downregulation with chronic compared to acute treatment with

SSRIs. The biochemical and consolidation mechanisms that contribute to the acute and chronic changes to SERT and 5-HT1A receptors may improve depressive symptoms, but the mechanism remains unclear and requires further investigation.

Trafficking Dependent Regulation of SERT

The amount and specific modifications of SERT at the membrane can modify 5-

HT uptake. SERT at the plasma membrane contains intracellular serine, threonine, and tyrosine amino acids on the intracellular N and C termini, and on intracellular loops. The

6 intracellular residues and loops can be phosphorylated by kinases like protein kinase C

(PKC) and subsequently affects SERT at the membrane. Constitutively active PKC phosphorylates SERT (Figure 1.0) (Sørenson et al., 2014), and pharmacological activation of PKC increases SERT internalization (Samuvel et al., 2005). In addition to

SERT phosphorylation, association of SERT with the focal adhesion protein Hic-5 is necessary for SERT internalization after PKC activation (Carneiro and Blakely, 2006).

PKC-dependent SERT internalization and Hic-5 association is also inhibited by addition of exogenous 5-HT. These findings support an additional role for 5-HT in the regulation of protein and kinase interactions with SERT to further modulate SERT.

Other protein kinases such as protein kinase G and A (PKG and PKA) can regulate the trafficking of SERT. Specifically, PKG can increase the function of SERT by increasing SERT Vmax and SERT at the membrane through activation of adenosine receptors (Miller and Hoffman, 1994; Zhu et al., 2004). This differs from the action of

PKC in that PKG does not directly phosphorylate SERT, and is suggested to require an intermediate (Sørenson et al., 2014). PKG-dependent regulation of SERT also appears to require amino acids threonine 4, glycine 56, glutamate 215, lysine 605, and proline 621, evidenced by the lack of a PKG-dependent increase in 5-HT uptake in individual SERT variants with mutations at each specific site (Prasad et al., 2005). Alternatively, PKG can also activate other SERT-modifying kinases, such as p38 mitogen-activated protein kinase (P38MAPK) (Zhu et al., 2004; Zhu et al., 2006), that in turn can directly phosphorylate SERT (Figure 1.0) (Sørenson et al., 2014) and increase SERT trafficking to the membrane (Lao et al., 2009). Similarly, protein kinase A (PKA) can increase SERT trafficking to the membrane, SERT phosphorylation (Figure 1.0), and 5-HT uptake

7 (Ramamoorthy et al., 1998). However, upstream activators and accessory proteins involved in PKA-mediated SERT trafficking have not been identified.

Trafficking Independent Regulation of SERT

Kinases also regulate SERT activity independent of trafficking and may rely on a combination with phosphatases. Increases or decreases in SERT catalytic activity may also be dependent on the type of kinase, the exact site of phosphorylation, and the time period of kinase activation or inhibition (reviewed in Bermingham and Blakely, 2016).

For example, P38MAPK can regulate SERT with and without changes to SERT trafficking according to acute or chronic activation of P38MAPK. Acute activation of

P38MAPK increases SERT affinity for 5-HT without changing Vmax or amount of

SERT at the plasma membrane whereas chronic P38MAPK activation increases the expression of SERT at the plasma membrane (Zhu et al., 2004). Moreover, increases in

SERT catalytic activity by P38MAPK is dependent on protein phosphatase 2 (PP2A).

Specifically, increases in SERT activity with P38MAPK stimulation are blocked with

PP2A inhibitors. Given that PP2A complexes and dephosphorylates SERT (Bauman et al., 2000; Ramamoorthy et al., 1998), P38MAPK and PP2A could modulate phosphorylation and dephosphorylation at different sites on SERT. Although sites of

PP2A-dependent dephosphorylation have yet to be defined, Zhu et al., (2005) demonstrated that P38MAPK activation increases SERT phosphorylation, which others have identified as occurring at the C-terminus threonine 616 (Figure 1.0) (Sørenson et al.,

2014). Therefore, P38MAPK and PP2A could by modifying different phosphorylation

8 sites on SERT that regulate trafficking-independent mechanisms of 5-HT uptake by

SERT.

PKC and PKG can also increase SERT catalytic activity without affecting SERT trafficking to the plasma membrane. Similar to the time-dependent effect of P38MAPK stimulation producing differential increases in catalytic SERT activity vs. trafficking, acute stimulation of PKC decreased affinity of SERT for 5-HT, without changing Vmax or plasma membrane distribution (Jayanthi et al., 2005). Contrary to PKC, acute stimulation of PKG increased 5-HT uptake and affinity of SERT for 5-HT without changing the distribution of SERT to the plasma membrane (Ramamoorthy et al., 2007) and appears to result in the phosphorylation of SERT (Figure 1.0) (Sørensen et al., 2014).

Ca2+/calmodulin-dependent protein kinase (CAMKII) is an additional kinase that regulates SERT. Similar to the actions of other kinases, CAMKII activation phosphorylates SERT at the N-terminus serine 13 (Figure 1.0) (Sørenson et al., 2014); however, the functional consequences of this regulation are more diverse than the traditional kinase-mediated change in SERT catalytic activity. CAMKII can uniquely enhance the electrogenic capability of SERT uptake. 5-HT uptake by SERT is canonically electroneutral, but during acute CAMKII inhibition, SERT assumes a channel-like state with a large sodium flux into the cell (Ciccone et al., 2008). This effect is dependent on the interaction between SERT and syntaxin 1, a vesicle fusion protein necessary for vesicle-mediated release of 5-HT. Others have demonstrated a direct interaction between SERT and CAMKII (Steinkellner et al., 2015) but the exact role of

CAMKII on the kinetics, plasma membrane distribution, and upstream regulation of

CAMKII-mediated electrogenic uptake of 5-HT remains to be determined.

9 Phosphatases also associate with and regulate SERT catalytic activity by affecting

SERT phosphorylation. While PP2A can act in concert with P38MAPK to increase 5-HT catalytic activity (Zhu et al., 2005), direct inhibition of PP2A with okadaic acid or calyculin A without stimulation of P38MAPK increased SERT phosphorylation, decreased plasma membrane SERT, and decreased SERT activity (Ramamoorthy et al.,

1998; Blakely and Bauman 2000). This indicates a direct role for phosphatases in SERT regulation but the effects of stimulation rather than inhibition of PP2A activity on SERT kinetics or trafficking is relatively unknown. Along these lines, overexpression of protein phosphatase Ca2+-activated protein phosphatase calcineurin (CaN), which complexes with SERT, increases 5-HT uptake (Seimandi et al., 2013) and supports a potential bimodal role for phosphatases in SERT regulation.

Figure 1.0 SERT kinase phosphorylation sites Schematic of sites on SERT identified by liquid chromatography-tandem mass spectrometry analysis of generated SERT peptides for the intracellular regions of SERT (Sørensen et al., 2014). In vitro phosphorylation assays were performed on SERT peptides to identify kinase-specific SERT phospho-sites.

11 Lipids and SERT

In contrast to the role of kinases and phosphatases in SERT regulation, the role of lipids in SERT regulation is less clear despite the fact that SERT primarily localizes in lipid-rich microdomains within the plasma membrane (Magnani et al., 2004; Scanlon et al., 2001). These lipid-rich segments of the membrane, known as lipid rafts, are distinctly enriched with cholesterol, sphingolipids, and saturated phospholipids (Brown and Rose

1992). The saturated phospholipids permit denser lipid and protein packing, and influences the fluidity of lipid rafts in comparison to the rest of the membrane.

Specifically, proteins within lipid rafts exhibit reduced fluidity and lateral movement

(Hjort et al., 1987; Lindblom et al., 1981) due to the tighter packing and higher density of proteins and lipids. Protein function as a consequence of movement in and out of lipid rafts is not entirely understood, but specific kinases may be involved. Both PKC and cholesterol can regulate SERT localization to lipid rafts. Acute PKC activation moves

SERT out of lipid raft fractions (Samuval et al., 2005) which may occur at the plasma membrane and at lipid rafts in intracellular organelle membranes. Given that PKC induces internalization of SERT (Carneiro and Blakely, 2006) and regulates SERT localization out of lipid rafts, it is possible that PKC primarily regulates SERT in lipid rafts within distinct subregions of the cell, such as at intracellular organelle membranes.

It has yet to be determined if other kinases and phosphatases also regulate SERT lipid raft localization.

SERT physically interacts with cholesterol at Cholesterol Recognition Amino

Acid Consensus (CRAC or CARC) motifs in transmembrane region 4 and 10 when localized in membrane (Ferraro et al., 2016). Consequently, SERT distribution can be

12 shifted by cholesterol depletion to detergent soluble non-raft fractions throughout the cell, including plasma membrane and intracellular organelle membrane non-raft fractions

(Magnani et al., 2004; Scanlon et al., 2001). Interestingly, the CRAC site is conserved between other monoamine transporters (Dopamine transporter (DAT), Norepinephrine

Transporter (NET)) (Kantcheva et al., 2013) and suggests a shared role for cholesterol in monoamine transporter regulation. Functional consequences of cholesterol elimination include reduced SERT affinity and transport of 5-HT (Laursen et al., 2018; Magnani et al., 2004; Scanlon et al., 2001) via increases in Km and decreases in Vmax without a change in SERT trafficking (Magnani et al., 2004; Scanlon et al., 2001). SERT also may require cholesterol for the conformational changes necessary for 5-HT transport. SERT transports 5-HT by changing from an extracellular-facing conformation that binds 5-HT and additional ions that facilitate transport, to an inward-facing conformation where 5-HT is released into the presynaptic neuron. SERT shifts between these conformations to determine the rate of 5-HT uptake. Mutation of a conserved cholesterol binding site on

SERT also results in reduced 5-HT transport kinetics and a preference of SERT toward an inward-facing conformation (Laursen et al., 2018). The reported reductions in 5-HT transport by SERT under cholesterol depletion conditions is thought to be due to a persistent intracellular-facing conformation of SERT.

Cholesterol within lipid rafts may also affect protein-protein interactions that regulate SERT activity (Sheng et al., 2012) but it remains unclear how cholesterol in lipid rafts regulate specific properties of SERT and its activity.

13 Cholesterol Regulation in the Brain

Cholesterol biosynthesis occurs primarily in the liver but hepatic derived cholesterol does not cross the blood brain barrier. Alternatively, cholesterol can be catabolized into brain-permeable oxysterols; however, oxysterols are unable to convert back into cholesterol once taken up by the brain. Therefore, the brain possesses a capacity for de novo cholesterol synthesis in the ER of neurons and glial cells (DeGrella and

Simoni 1982; Jeske and Dietschy 1980) through a multi-step biosynthetic mechanism that begins with the conversion of acetyl-CoA to mevalonate by the rate-limiting enzyme

HMG-CoA reductase. Through a series of enzymatic steps, mevalonate produces farnesyl pyrophosphate (FPP), followed by squalene, and an additional 19-step process to produce cholesterol (Berg, 2002). Cholesterol distributes to and is stored in the endocytic recycling compartment, trans golgi network, and in the plasma membrane (Lange et al.,

1989; Mukherjee et al., 1998). Cholesterol can also be esterified when in excess, providing a pool of stored cholesterol that localizes in the ER or in cytoplasmic lipid droplets. In addition to biosynthetic regulation of cholesterol through the aforementioned biosynthetic intermediates, cholesterol is regulated transcriptionally by sterol regulatory element binding proteins (SREBPs). These proteins act as sensors for membrane cholesterol and regulate the synthesis of HMG-CoA reductase and other enzymes in the cholesterol synthesis pathway when cholesterol levels are disrupted (reviewed in

Rawson, 2003).

14 Cholesterol and Brain 5-HT

Cholesterol synthesis and cell lipid requirements vary among brain regions (Quan et al., 2003). In fact, cholesterol and cholesterol precursors are highest in raphe where

SERT protein density is the highest (Jagust et al., 1996). Although a rigorous experimental analysis of cholesterol and SERT localization within the same study has not been performed, localization and correlations between cholesterol and 5-HT-ergic processes are evident. For instance, humans with major depression have lower stored cholesterol (Maes et al., 1994), and humans with low cholesterol are three times more likely to display depressive symptoms (Morgan et al., 1993). Patients with major depressive disorder also display low cholesterol that was correlated with depression severity and suicidal ideation, both 5-HT-related processes (Rab-Jablonska and

Poprawska 2000). Rodent models of depression-like behavior also display low brain cholesterol in the frontal cortex (Sun et al., 2015). Thus, there appears to be a close association of low cholesterol with the disruption of 5-HT-ergic processes.

The relationship between cholesterol and 5-HT is further supported by the neurophysiological effects associated with statins. Statins are a class of cholesterol- lowering drugs that block de novo cholesterol synthesis by inhibiting HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Statins drugs are one of the most widely prescribed therapeutics on the market (CDC 2017), with one in every four adults over 40 years old currently prescribed a statin (Gu et al., 2014). Despite their cardiovascular benefits, some statins are brain-permeable and can cause a number of central nervous system and 5-HT-ergic effects including disturbances of sleep, cognition, and mood. Moreover, statin treatment in hypercholesterolemic adults results in

15 decrements in attention and psychomotor speed (Muldoon et al., 2000; Muldoon et al.,

2004), and produces learning deficits in humans and animals (Kotti et al., 2006;

Suraweera et al., 2016). In fact, simvastatin is positively correlated with both depression and decrements in 5-HT neurotransmission (You et al., 2013) and is associated with cognition and learning deficits (Harmer et al., 2002).

Gaps in Knowledge

Although the brain is the most cholesterol-rich organ in the body (Sastry, 1985), it is unclear how SERT is regulated within cholesterol-rich microdomains of the neuron, and how cholesterol affects SERT trafficking and catalytic transport activity, as well as proteins that regulate SERT. The goal of this dissertation was to elucidate the effects of statins and cholesterol on SERT catalytic transport activity and trafficking in 5-HT neurons (Figure 1.1).

Figure 1.1 Dissertation objectives This dissertation will investigate the role and mechanism by which cholesterol and its biosynthetic intermediates regulate SERT.

17 Chapter 2: Serotonin Uptake and Statins

Introduction

Cholesterol-rich lipid rafts are present both intracellularly and at the plasma membrane and can directly interact with and regulate SERT activity (Laursen et al.,

2014; Magnani et al., 2004; Scanlon et al., 2001), however the specific and dynamic role of cholesterol on SERT protein trafficking, SERT sorting into lipid rafts, SERT modification, and SERT activity are not established (Fiedler et al., 1993; Hayashi et al.,

2003). On the other hand, evidence supports differential roles of cholesterol in these processes with other membrane-trafficked and raft-sorted proteins. For instance, surface localization of glycosylphosphatidylinositol (GPI)-anchored proteins is dependent on lipid raft cholesterol for sorting to the plasma membrane and into lipid rafts (Rothberg et al., 1990). Glycosylation of GPI-linked proteins can result in localization of GPI-linked proteins outside of lipid rafts once sorted to the plasma membrane (Benting et al., 1999;

Lipardi et al., 2000). In addition to GPI-linked proteins, rafts also contain protein- modifying enzymes that regulate SERT. For instance, lipid rafts contain and can activate kinases such as P38MAPK, PKC, and CAMKII (Cabrera-Poch et al., 2004; Fox et al.,

2007; Tsui et al., 2005) that in turn, could modify SERT activity through changes in

SERT phosphorylation (Annamalai et al., 2012; Jayanthi et al., 2005; Sørensen et al.,

2014). These dynamic interactions of cholesterol and subsequent effects on SERT activity have not been fully elucidated.

Previous models that investigate the role of cholesterol and monoamine transport primarily deplete or sequester plasma membrane cholesterol with methyl-β cyclodextrin

(mβCD) or nystatin, respectively. mβCD reduces SERT 5-HT uptake and affinity of

18 SERT for 5-HT and DAT dopamine uptake and affinity for DAT (Jones et al., 2012;

Magnani et al., 2004; Scanlon et al., 2001). However, manipulation of plasma membrane cholesterol with nystatin does not affect monoamine transport, suggesting distinct roles of cholesterol on monoamine transporter activity and function according to the compartment of cholesterol being manipulated. mβCD primarily depletes cell surface cholesterol at the

PM by up to 85% and can partially deplete intracellular cholesterol (Keller et al., 1998;

Scanlon et al., 2001). Comparatively, nystatin sequesters plasma membrane cholesterol and has no documented effects on intracellular cholesterol. The role of non-plasma membrane cholesterol throughout the cell on SERT function and activity has not been evaluated. Given the possibility of organelle-specific differences in cholesterol and SERT regulation, the goal was to selectively deplete cholesterol throughout the cell by disrupting cholesterol synthesis with the cholesterol-lowering statin class of drugs.

Statins block the rate-limiting enzyme in cholesterol synthesis HMG-CoA reductase, preventing conversion of acetyl-CoA to mevalonate, which will synthesize into cholesterol after a series of biosynthetic steps. Inhibition of HMG-CoA reductase occurs at the ER and will effectively reduce cholesterol throughout the cell given that the distribution of cholesterol throughout the cell is provided by the ER.

The serotonergic RN46-B14 cell line with expressed myc-tagged SERT (see

Methods) was selected to model the effects of statins on SERT activity. This cell line was advantageous due to the serotonergic signaling and release dynamics of this cell line, including 5-HT synthesis, vesicle-mediated 5-HT release (Bethea et al., 2003; Eaton et al., 1995a; Eaton et al., 1995b; White et al., 1994), and measurable levels of SERT- dependent uptake that are similar to previous uptake results in rat brain slices (see

19 Discussion). Furthermore, the cell line can be genetically manipulated to test the role of specific substrates involved in 5-HT uptake changes, and does not contain other neurotransmitters that SERT can promiscuously uptake under specific conditions

(Kannari et al., 2006). To test the impact of statins on SERT, statins were applied to cells for a minimum chronic period of time (24hr) previously demonstrated to reduce total cell cholesterol (Villanueva et al., 2013). Based on previous studies that have manipulated cholesterol and measured the impact on monoamine transporter function (Magnani et al.,

2004; Scanlon et al., 2001), we hypothesized that inhibition of cholesterol synthesis by statins would decrease SERT- dependent 5-HT uptake (Figure 2.0)

Figure 2.0 Hypothetical model Statin drugs inhibit the rate limiting enzyme in cholesterol synthesis HMG-CoA reductase, preventing synthesis of cholesterol. Reduced cholesterol is hypothesized to decrease SERT-dependent 5-HT uptake in RN46-B14 cells with myc-tagged SERT expression.

21 Methods

Preparation of RN46A-B14 cells with SERTMyc expression

Serotonergic RN46A-B14 cells with a temperature-sensitive SV40 large T- antigen (Eaton et al., 1995a; Eaton et al., 1995b) were provided by Scott Whittemore from University of Louisville. RN46A-B14 cells exhibit a neuronal phenotype, express vesicular monoamine transporter (VMAT), aromatic amino acid decarboxylase (AADC) and SERT, and constitutively release 5-HT via vesicular docking at the plasma membrane (Eaton et al., 1995b; Stansley and Yamamoto, 2013). Thus, these cells have the ability to synthesize 5-HT, express SERT, package 5-HT into vesicles, as well as release and reuptake 5-HT (Bethea et al., 2003; Eaton et al., 1995a; White et al., 1994).

The RN46A-B14 cell line has been authenticated by the European Collection of

Authenticated Cell Cultures (ECACC) under the Public Health England. Cells were stably transfected with c-myc-tagged SERT to provide ample SERT expression and to allow for identification of SERT using antibodies for c-myc. For stable SERTMyc expression, rat SERT cDNA was amplified in a pCMV6 vector (Origene, 0084884) with a c-myc tag. The insert was digested and cloned into the Xba1 and Kpn1 sites in the pcDNA3.1/Zeo (-) vector (Invitrogen, V86520) and contained the Zeocin resistance gene that permitted selection of stable cell lines with expression driven by the CMV promoter.

The pcDNA3.1-SERTMyc construct was transfected into the RN46A-B14 cells and colonies were selected with 200 μg/ml Zeocin. Colonies 3 and 12 out of 22 colonies were selected for the experiments in this study based on neuronal-like morphology, comparable SERT and myc expression between both colonies verified by western blot, and comparable SERT function verified by 5-HT uptake.

22 SERTMyc cell culture and reagents

RN46A-B14 cell stocks were incubated in T75 flasks at 33⁰C in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 250μg/ml G418,

100μg/ml Hygromycin and 200μg/ml Zeocin. G418 was required to select for the large T antigen and hygromycin was used to select for brain‐derived neurotrophic factor, both of which are important for the immortalized and serotonergic phenotype of the cells. The temperature at 33C⁰ is the permissive temperature which permits continued division in cell culture T75 stocks. Cells were switched to a 37C⁰ incubator when plated on 6 well plates prior to uptake. The switch to 37C⁰, combined with reducing serum to 2% helped to limit confluency during the 3-day pre-drug incubation prior to experimental assays.

Stocks were split onto 6 well plates (100,000 cells/well) for 72hr prior to addition of any reagents. After 72hr, cells were washed with serum- and antibiotic-free DMEM.

Simvastatin (Cayman Chemical, 10010345), atorvastatin (Cayman Chemical, 10493) and fluvastatin (Cayman Chemical, 10010337) were applied to cells in DMEM with 2% FBS for 24hr. Fluoxetine (Cayman Chemical, 14418) was applied 30min prior to the addition of 5-HT for uptake and was not included in the incubation period during the 24hr incubation with simvastatin.

5-HT uptake

Measurement of 5-HT uptake in SERTMyc cells was modified from that described by Zhu et al. (2007). Briefly, SERTMyc cells were incubated in 10μM MAO inhibitor pargyline for 30min to prevent the degradation of 5-HT (Pizzinat et al., 1999).

5-HT uptake was initiated by addition of 0.1μM 5-HT (or a range of 1nM-20μM 5-HT

23 for Michaelis Menten kinetics) to the media and incubated at 37⁰C for 10min. Uptake was terminated by the transfer of media and nonadherent cells to ice-cold HBSS followed by an ice cold HBSS wash over ice on attached cells. All cells were collected in ice cold

HBSS and centrifuged at 600xg. The supernatant was discarded, and pellets were stored at -20⁰C until assayed for 5-HT.

5-HT measurement by high pressure liquid chromatography

Cell pellets were sonicated in 0.25M perchloric acid and centrifuged at 14,000xg for 20min at 4⁰C to precipitate protein. Supernatant (20μl) was injected into a HPLC and analyzed with EZChrom software (Scientific Software Inc.). Samples were quantified by comparison of sample peak height to the peak height of a 400pg 5-HT standard. Cell pellets from initial spin down were dissolved in 1N NaOH and the cell protein pellet was quantified by a Bradford assay. 5-HT was normalized to total protein measured in the cell pellet (pg 5-HT/μg protein). Transformation of the fluoxetine inhibition kinetics (Figure

2.6) yielded the serotonin Ki for SERT in the presence of increasing fluoxetine concentrations. Calculations were based on the Cheng and Prusoff equation for a competitive inhibitor IC50= Ki (1+([5-HT]/Km) (Cheng and Prusoff 1973).

5-HT and myc measurement by immunocytochemical detection

Cells were plated on poly-D-lysine coated coverslips in 6 well plates (50,000 cells/well) and simvastatin treatment with fluoxetine was completed as described (see

SERTMyc cell culture and reagents). Following treatment with 5-HT for 10min, cells were washed twice with ice-cold phosphate-buffered saline (PBS), fixed with 4%

24 paraformaldehyde for 10min at room temperature, and washed twice with PBS for 5min.

Cells were permeabilized with 0.05% saponin in 0.5% PBS-Triton (PBS-T) solution for 2 min, washed twice with PBS for 5min, and then blocked with 10% normal goat serum

(NGS) in PBS-T for 1hr at room temperature. Cells were incubated with anti-SERT primary antibody (1:3000, Neuromics RA24330), anti-Myc primary antibody (1:3000,

Millipore 05-724), or anti-5-HT primary antibody (1:40000, Immunostar 20080) in 5%

NGS in PBS-T overnight at 4°C. Methods were not incorporated to distinctly measure endogenous SERT compared to expressed myc-tagged SERT. Subsequently, cells were washed three times with PBS-T and incubated with goat anti-mouse Alexa Fluor 488 secondary antibody and/or donkey anti-rabbit Alex Fluor 568 (1:1000, ThermoFisher

A11001) in 5% NGS in PBS-T for 1hr at room temperature. Cells were washed three times with PBS-T, then incubated with a solution containing 300nM DAPI (Sigma-

Aldrich D9542) and/or 165nM Rhodamine Phalloidin (ThermoFisher R415) for 5min at room temperature. Cells were washed again with PBS-T and mounted on slides with

Vectashield and kept at 4°C until evaluation. Images were collected at 6 x 2µm z axis steps using a Zeiss LSM800 laser scanning confocal microscope equipped with a 20x and

40x objective at excitation wavelengths of 405nm (DAPI, Em: 400-510nm), 488nm

(Alexa Fluor 488, Em: 510-575 nm) and 561nm (Alexa Fluor 568 or Rhodamine

Phalloidin, Em: 575-700nm). The microscope was equipped with Zeiss Zen 2.6 (blue edition) software (Carl Zeiss, Inc.). Maximum projection images of the exported Z-stack files were captured and visualized using ImageJ image software ver.1.51n (Abramoff et al., 2004). All treatment conditions within an experimental cohort were stained and imaged simultaneously, and cells within each experimental cohort were imaged using

25 identical microscope settings. No modifications of brightness or contrast were imposed by ImageJ.

Biotinylation and separation of plasma membrane and intracellular SERT

Separation of biotinylated plasma membrane SERT was modified from Loweth et al. (2014). In brief, cells were treated for 24hr with vehicle or 0.5μM simvastatin and a 5-

HT uptake assay was performed. Cell pellets were spun down at 600xg for 5min and pellets were resuspended and incubated for 30min in sodium-citrate buffer activated sulfo-NHS-S-S-Biotin (Thermo Scientific 21331) on a 360⁰ end-over-end rotator at 4⁰C.

The reaction was quenched with 100mM glycine for 20min, and samples were centrifuged at 20,000xg for 2min at 4⁰C. Biotinylated cell pellets were sonicated in lysis buffer (25mM HEPES, 500mMNaCl, 1mM PMSF, 20mM NaF, 0.1% IGEPAL, 2mM

EDTA, 1X HALT protease inhibitor), centrifuged at 20,000xg for 2min at 4⁰C, and supernatant was stored at -20⁰C until the avidin-biotin pull-down. Bradford protein analysis was performed on stored lysed biotinylated cell samples and 100μg of protein was incubated with equal amounts of PBS pre-washed NeutrAvidin agarose resin

(Thermo Scientific 29204), followed by overnight incubation. The following day, incubated sample was removed from the resin and the overnight incubation was repeated with freshly washed NeutrAvidin agarose resin. Sample-bound resin from the first incubation (membrane fraction) was stored in 2x LDS and 200mM DTT at -20⁰C. After the second overnight incubation in resin, non-biotinylated sample that did not bind to the resin was removed and placed in fresh eppendorf tubes (intracellular fraction) and stored in 2x LDS and 200mM DTT at -20⁰C. 2x LDS and 200mM DTT was also added to the

26 remaining second fraction of bound resin, and then the first and second bound fractions were pooled together, spun on a centrifugal filter unit with a 0.45μm filter (Millipore

UFC30HV00) at 11,000xg for 5min at 4⁰C. Samples were stored at -20⁰C until western blot analysis. Even volumes of protein in LDS were loaded for each sample.

Western blot analysis

Cell pellets were prepared in 2x LDS with 200mM DTT prior to loading onto a

NuPAGE4-12% bis tris polyacrylamide gel (Invitrogen NP0335BOX) for gel electrophoresis using the XCell SureLock™ Mini gel electrophoresis system in MOPS running buffer. Following SDS-PAGE, gels were transferred onto PVDF membranes and blocked for 1hr in 0.5% nonfat dry milk solubilized in 0.5% TBS-T. Membranes were then incubated overnight with primary antibody in 4⁰C on an orbital shaker. The following morning, membranes were removed from 4⁰C, washed 3x for 5min in 0.5%

TBS-T on a rocking shaker at room temperature, and incubated in secondary antibody at a 1:4500 dilution for 1hr, followed by three 5min washes in 0.5% TBS-T. Membranes were developed in chemiluminescent reagent (VWR PI34578) and imaged in a Fujifilm imager and analyzed with Multi Gauge V3.0 software. Anti-Myc primary antibody and anti-actin antibody (1:3000, Millipore MAB1501) were used in conjunction with anti- mouse secondary antibody (1:2500, Santa Cruz SC-516102).

In vivo simvastatin treatment and ex vivo synaptosome preparation

Male Sprague Dawley rats received 10mg/kg of simvastatin acid or vehicle, 1x a day for 7 days. The simvastatin dosing paradigm was selected after experimental

27 investigation of different treatments previously shown to reduce brain cholesterol (Eckert et al., 2005; Johnson-Anuna et al., 2005; Thelen et al., 2006). On the 7th day rats were sacrificed by rapid decapitation, and the prefrontal cortex (PFC) was dissected over wet ice. The PFC brain region was selected based on correlation to depression-like behaviors.

Synaptosomes were immediately prepared from PFC using a modified protocol from Zhu et al. (2006). PFC was homogenized in 1ml of ice-cold 0.32M sucrose with a glass homogenizer, and spun at 1,000g for 10min. Supernatant was spun again at 16,000g for

10min. Supernatant was removed and the synaptosome-containing pellet was resuspended in 1ml of ice-cold 0.32M sucrose. For each dissected PFC the prepared synaptosome fraction was divided into 5 different vials (200µl per vial) and incubated at

37⁰C with gentle agitation, with 10µM pargyline, with or without 1µM fluoxetine for

10min, prior to 5-HT uptake. 5-HT uptake was performed as described in Methods for

SERTMyc cells, but the uptake time was reduced from 10min to 5min and exogenously added 5-HT was reduced from 100nM to 20nM. The modified uptake was experimentally determined to allow for measurable increases in 5-HT uptake according to Michaelis-

Menten kinetic curves that were prepared and analyzed prior to the initiation of simvastatin treatment in rats.

Statistical analysis

Experiments were replicated with at least 3 independent experiments and each independent experiment had 2-3 technical replicates. Statistical analyses were performed on the mean of each independent experiment and data were represented as the mean ±

SEM. Under conditions in which samples were pooled from more than one well (Figure

28 2.7), the pooled data were treated as the mean of an independent experiment. Statistical analysis was performed using one- and two-way ANOVA where applicable, followed by the appropriate post-hoc test to determine statistical comparison between different treatments. p-values less than 0.05 were considered significant.

29 Results

2.1 RN46-B14 cells exhibit myc-tagged SERT expression

The RN46-B14 cell line was selected based on the serotonergic and neuron-like phenotype (see Methods). Due to low SERT expression in the RN46-B14 cell line and poor availability of strong SERT antibodies, cells were stably expressed with rat SERT containing a myc-tag on the C-terminus. Expression was verified by analyzing myc optical density by western blot. Myc optical density displayed at the molecular weight of

SERT (70.5kDa) in cells with the SERTMyc expression vector and was not evident in cells without the vector, although actin loading controls were present (Figure 2.1 A).

Expression was also verified with identification of SERTMyc immunoreactivity (Figure

2.1 B). Successful expression of SERTMyc allowed for the investigation into the role of cholesterol in SERT activity and function, and provided a methodological advantage for

SERTMyc optical density detection and immunoprecipitation procedures.

Figure 2.1 RN46-B14 cells exhibit myc-tagged SERT expression A.) Western blot optical density of Myc and actin antibody immunoreactivity in RN46-B14 cells with and without stable expression of a pcDNA3.1 vector with myc- tagged rat SERT. B.) Immunoreactivtiy images of RN46-B14 cells with stable SERTMyc expression. Cells were incubated in 5-HT for 10min, and fixed and stained with Myc antibody (green), SERT antibody (red) and DAPI (blue) for expressed SERTMyc and nuclear visualization. The MERGE image depicts Myc and SERT localization and the BRIGHTFIELD image displays the transmitted light microscopy from the fixed and permeabilized cells.

2.2 Statins enhance 5-HT uptake in a concentration-dependent manner

The objective of this study was to investigate the role of cholesterol in SERT activity and function. Three FDA-approved cholesterol synthesis inhibitors were selected based on brain permeability and cholesterol reducing properties, and 5-HT uptake was subsequently measured. Atorvastatin, fluvastatin, and simvastatin were separately applied to SERTMyc cells for 24hr, which has previously been shown to produce measurable decreases in cell cholesterol (Villanueva et al., 2013). Atorvastatin, fluvastatin, and simvastatin treatment significantly enhanced 5-HT uptake at concentrations of 1μM and

10μM but did not significantly affect uptake at 0.1μM (Figure 2.2 A, B, C).

Figure 2.2 Statins enhance 5-HT uptake in a concentration-dependent manner. A.) Atorvastatin, B.) Fluvastatin, and C.) Simvastatin were applied to SERTMyc cells for 24hr at 0.1, 1, and 10μM concentrations. Each statin significantly enhanced 5-HT uptake at 1 and 10μM (n=3/grp). 5-HT uptake is expressed as pg 5-HT normalized to µg protein per sample. (One-Way ANOVA significant drug effect (p<0.0001), *p<0.01 Tukey Post-Hoc comparison to Vehicle). DMSO vehicle was used for all three statins.

2.3 Simvastatin-enhanced 5-HT uptake is consistent over time.

Of the three statins tested, simvastatin is the most commonly prescribed and was selected for all future investigations based on this statistic (Gu et al., 2014). To determine the persistence of SERT regulation by simvastatin, simvastatin incubation was extended to 48, 72, and 96hr. Simvastatin (1µM) significantly enhanced 5-HT uptake at each timepoint compared to vehicle, and simvastatin significantly increased uptake after 96hr compared to 48hr incubation (Figure 2.3).

Figure 2.3 Simvastatin-enhanced 5-HT uptake is consistent over time. 1μM simvastatin treatment for 48, 72, or 96hr increased 5-HT uptake compared to Vehicle (Veh) (n=3/grp). 5-HT uptake is expressed as pg 5-HT normalized to µg protein per sample, and compared across timepoints by standardizing to vehicle at 100%. (One-Way ANOVA significant simvastatin effect (*p<0.001), Tukey Post-Hoc comparisons to Vehicle were significant at all time points represented. Tukey Post- Hoc comparisons between 48hr and 96hr was significantly increased (#p<0.05).

35 2.4 Simvastatin enhances 5-HT uptake in a SERT-dependent manner.

Additional and lower concentrations of simvastatin ranging between 0.1-1μM were tested to reproduce concentrations measured in the brain after oral simvastatin dosing in mice (Johnson-Anuna et al., 2005). Simvastatin also enhanced 5-HT uptake at

0.1μM and 0.5μM (Figure 2.4 A). To assess that simvastatin-enhanced uptake was mediated by SERT, the SERT inhibitor fluoxetine was used in the presence of exogenous

5-HT application (Zhu et al., 2006). Fluoxetine (1μM) blocked 5-HT uptake in both vehicle and simvastatin-treated cells, indicating a SERT-specific mechanism of 5-HT uptake and its enhancement by simvastatin (Figure 2.4, B, C).

Figure 2.4 Simvastatin enhances 5-HT uptake in a SERT-dependent manner. A) Simvastatin (0.1, 0.5, and 1μM) enhanced 5-HT uptake (n=4-5/grp) (One-Way ANOVA simvastatin effect (p<0.001), *p<0.01 Tukey Post-Hoc comparison to Vehicle). B) Enhanced 5-HT uptake by 0.5μM simvastatin was blocked by 1μM of the SERT inhibitor fluoxetine, (n=4/grp, except Veh/Fluox n=2) (Two-way ANOVA Fluoxetine effect (p<0.01), #p<0.05 Tukey Post-Hoc comparison to Veh/Veh, *p<0.001 comparison to 0.5μM Sim/Veh). C) SERTMyc cells post-fixed after 5-HT uptake and stained for 5-HT after 0.5μM simvastatin or vehicle, followed by 1μM fluoxetine.

2.5 Simvastatin does not change endogenous 5-HT content.

The fluoxetine-sensitive uptake by SERT reveal a primary effect of simvastatin on SERT. However, to further verify that 5-HT changes were a result of SERT and not due to a change in 5-HT synthesis within the cell, endogenous intracellular 5-HT was measured after simvastatin. 0.5μM simvastatin was applied to cells for 24hr and cells were collected without the addition of exogenous 5-HT. Endogenous 5-HT content did not change with simvastatin treatment (Figure 2.5).

Figure 2.5 Simvastatin does not change endogenous 5-HT content SERTMyc cells treated with 0.5μM simvastatin for 24hr does not change endogenous basal 5-HT content (no added 5-HT) within SERTMyc cells (n=3/grp). (t-test between Veh and Sim not significant (p=0.745)).

39 2.6 Simvastatin decreases the Michaelis-Menten Km for 5-HT uptake without

changing Vmax and decreases the Ki of fluoxetine.

SERT catalytic activity after simvastatin was assessed by Michaelis-Menten kinetics after 0.5μM simvastatin or vehicle for 24hr and uptake was measured in the presence of increasing concentrations of 5-HT. The 5-HT concentration range selected was based on the maximal plateau in 5-HT uptake for Vmax calculation in the RN46-B14 expression system. Simvastatin reduced Km values from a Km of 2.213μM in vehicle- treated cells to 0.679μM (Figure 2.6 A, C). The Vmax for 5-HT uptake was not significantly different between simvastatin (232.2μM) compared to vehicle-treated

(246.1μM) cells. Values for vehicle-treated cells correspond with previously published

Km and Vmax values from raphe rat brain slices from which these cells were derived and brain synaptosomal preparations (Bunin et al., 1998a; Bunin et al., 1998b; Butler et al.,

1988; Montañez et al., 2003; Shaskan and Snyder, 1970). The Ki was determined at a fixed concentration of 100nM 5-HT and increasing concentrations of the SERT inhibitor, fluoxetine. The fluoxetine concentration range (0.1nM-10µM) was selected based on a plateau of inhibition at higher concentrations that represented maximal SERT saturation by fluoxetine. Simvastatin reduced the Ki for fluoxetine from 5.98nM to 1.18nM at a 5-

HT concentration of 100nM (Figure 2.6 B, C). The transformed Ki values (see Cheng-

Prusoff equation in Methods) in vehicle-treated samples corresponded with previously published Ki values for SERT (Barker et al., 1998).

Figure 2.6 Simvastatin decreases the Michaelis-Menten Km for 5-HT uptake without changing Vmax and decreases the Ki of fluoxetine. A.) SERTMyc cells treated with 0.5μM simvastatin for 24hr significantly decreased the Michaelis-Menten Km, but did not change SERT uptake Vmax (n=5/grp) (*p<0.0001 t-test comparing vehicle vs. simvastatin Km, t-test comparing vehicle vs simvastatin Vmax not significant). B.) SERTMyc cells treated with 0.5μM simvastatin for 24hr reduced the SERT Ki at 100nM 5-HT, with increasing concentrations of SERT inhibitor fluoxetine (n=4/grp) (*p<0.05 t-test comparing vehicle vs. simvastatin Ki). C.) Mean values and SEM for SERT uptake Km and Vmax (from graph A) and IC50 and Ki (from graph B) in vehicle and simvastatin-treated cells. Data are represented as mean ± SEM.

2.7 Simvastatin does not alter total SERT or SERT localization.

Total, intracellular, and plasma membrane SERT were measured by biotinylation of SERT at the membrane and subsequent streptavidin separation of membrane and intracellular SERT. Densitometry of SERT optical density by western blot revealed no changes in SERT in total cell homogenate, or the corresponding membrane and intracellular fractions of these samples (Figure 2.7). Intracellular optical density was faint in the selected western blot from which the optical density was quantified, but values were quantifiable compared to background and representative after overexposure of the western blot. Representative optical density images of pan-cadherin and SERT on the same western blot indicate membrane enrichment in the total and membrane fractions.

Optical density westerns for lamin B1 on the same western blot revealed intracellular protein within the lanes loaded with intracellular fraction samples.

Figure 2.7 Simvastatin does not alter total SERT or SERT localization Simvastatin does not change densitometry measures of SERT in total, membrane separated, and intracellular fractions after 0.5μM, 24hr simvastatin treatment. Representative blots demonstrate enrichment of membrane localized pan-cadherin in membrane and total fractions that is not present in the intracellular fraction, and intracellular fractions with lamin B1. T=Total SERT, M and Mem.=Membrane SERT, I and Intra.=Intracellular SERT, V=Vehicle, S=Simvastatin. Data are represented as mean ± SEM.

2.8 Simvastatin increases 5-HT uptake in ex vivo synaptosomes from rats

treated with simvastatin

The limitations of the SERTMyc cell model prompted exploration of the statin effect into in vivo and alternative cell models. To achieve an additional comparison of simvastatin’s effects on SERT uptake in an alternative model, 5-HT uptake was performed in ex vivo synaptosomes prepared from rats treated with simvastatin.

Specifically, 5-HT uptake was measured in rat PFC synaptosomes prepared from rats treated daily with 10mg/kg simvastatin i.p. for 7 days. 5-HT uptake was significantly increased in synaptosomes from simvastatin-treated rats compared to vehicle (Figure 2.8

A). In both vehicle and simvastatin-treated rat synaptosomes, 1μM fluoxetine SSRI attenuated 5-HT uptake. However, the specific synaptosome uptake (Veh/Veh -

Veh/SSRI or Sim/Veh - Sim/SSRI) was not significantly different between vehicle and simvastatin-treated rats (Figure 2.8 B).

Figure 2.8 Simvastatin increases 5-HT uptake in ex vivo synaptosomes from rats treated with simvastatin 10mg/kg simvastatin was administered intraperitoneal (i.p.) to rats daily for 7 days. Synaptosomes were exposed to 20nM 5-HT for 5min with and without 1µM fluoxetine. A.) Total 5-HT uptake in ex vivo synaptosomes prepared from the prefrontal cortex (PFC) of simvastatin-treated rats was significantly increased compared to Veh/Veh. (n=7-8/grp) (Two-way ANOVA simvastatin effect (p<0.01), *p<0.01 Tukey Post-Hoc comparison Veh/Veh and Veh/SSRI, *p<0.01 comparison Sim/Veh and Sim/SSRI, *p<0.001 comparison Veh/Veh and Sim/Veh). B.) Specific 5-HT uptake (defined as Veh/Veh-Veh/SSRI and Sim,Veh-Sim/SSRI) was not significantly (n.s.) different (p=0.575, t-test comparison between vehicle and simvastatin).

Discussion

The results demonstrate an unexpected increase in SERT-dependent, 5-HT catalytic uptake with statin treatment. The results suggest an alternative or independent effect of cholesterol in SERT regulation that differs from previously published findings on SERT regulation by cholesterol (Jones et al., 2012; Magnani et al., 2004; Scanlon et al., 2001).

The concentration-dependent increase in 5-HT uptake by lipid-lowering statin drugs (Figure 2.2, 2.4) suggests a broad range of concentrations at which statins enhance

SERT uptake, and a sustained time course by which statins increase 5-HT uptake (Figure

2.3). The statins tested (atorvastatin, fluvastatin, simvastatin) differ according to their affinity for HMG-CoA reductase and their chemical structure. However, all three statins tested are capable of crossing the blood brain barrier (Guillot et al., 1993; Kandiah and

Feldman 2009; Saheki et al., 1994) and the concentrations of simvastatin and atorvastatin that increase 5-HT uptake are within the range of concentrations in the brain reported after acute systemic treatment (Eckert et al., 2005; Johnson-Anuna et al., 2005; Thelen et al., 2006). Simvastatin treatment produced the most consistent increases in 5-HT uptake at concentrations of 0.5µM and higher. Concentrations below 0.5µM, such as at 0.1µM, displayed variability in 5-HT uptake increases (Figure 2.2 C compared to Figure 2.4 A), and provided rationale for the 0.5µM concentration of simvastatin selected for the remainder of dissertation research.

To test the role of SERT in the statin-enhanced 5-HT uptake, the SSRI fluoxetine was used to block SERT during 5-HT uptake. Fluoxetine is an established SERT inhibitor known to block uptake of 5-HT by RN46A-B14 cells (Zhu et al., 2006). Our findings

illustrate that fluoxetine also blocks the effects of simvastatin on 5-HT uptake measured by biochemical and immunocytochemical Methods (Figure 2.4) and supports the role of

SERT in mediating 5-HT uptake by these cells. Further kinetic analyses with fluoxetine show decreases in the Ki of fluoxetine for SERT after simvastatin (Figure 2.6 B, C), and suggest changes in fluoxetine affinity for SERT after simvastatin treatment.

In addition to the quantification of 5-HT by HPLC Methods, immunocytochemistry techniques were employed to visualize the 5-HT uptake by SERT.

The immunocytochemical evidence of enhanced 5-HT uptake provides robust visualization of the enhanced intracellular accumulation of 5-HT (Figure 2.4) that is blocked with fluoxetine. Furthermore, the distribution of 5-HT throughout the cell and perinuclear space are likely due to the broad distribution of total SERT throughout the cell processes and soma (Figure 2.1 B).

These findings are also suggestive of changes in SERT activity without changes in the localization or amount of SERT with simvastatin treatment. Michaelis-Menten kinetics revealed significant decreases in the Km of SERT for 5-HT without changes in

Vmax (Figure 2.6 A, C). The kinetic results using SERTMyc cells are similar to literature reports of Km values ranging from 1-4μM using brain slices or synaptosomes (Butler et al., 1988; Codd and Walker 1987; Daws et al., 2005) and transformed Ki values of 6.6nM

(Barker et al., 1998). The kinetic changes demonstrate that alterations in 5-HT uptake by simvastatin are due to activity-dependent effects of SERT since simvastatin also did not change the amount of plasma membrane, intracellular, or total SERT (myc-tagged and non myc-tagged SERT) (Figure 2.7) and did not alter endogenous 5-HT in the absence of added 5-HT (Figure 2.5).

Although simvastatin does not alter SERT trafficking to the membrane, it may alter SERT distribution within the plasma membrane. For instance, SERT may localize outside of lipid-raft fractions of the plasma membrane after simvastatin treatment. Lipid raft distribution of SERT was investigated in SERTMyc cells after simvastatin. However, the distribution of SERT appeared to change within segments of the plasma membrane enriched in the lipid raft marker flotillin (data not shown). Although this could indicate differential SERT distribution at the plasma membrane after simvastatin treatment, it is unclear how to interpret movement to different flotillin-rich regions of the lipid rafts.

In addition to the increased 5-HT uptake in vitro observed in SERTMyc cells, the results also demonstrate that simvastatin affects the serotonergic system after in vivo administration (Figure 2.8). However, it remains to be determined whether the increase in synaptosomal 5-HT with simvastatin is dependent on SERT specifically. The simvastatin treatment paradigm utilized did not change specific 5-HT uptake of exogenously added 5-

HT during a 5min uptake assay (Figure 2.8 B). However, total synaptosomal 5-HT uptake was increased in simvastatin-treated rats (Figure 2.8 A) and could indicate changes in 5-

HT uptake or synthesis throughout the 7-day simvastatin treatment paradigm and prior to the 5min uptake assay. Additional simvastatin treatment paradigms or a Michaelis-

Menten kinetic analysis could reveal changes in specific 5-HT uptake by SERT not captured under the exact conditions tested.

The content of cholesterol in the SERTMyc cells after simvastatin was not determined in these experiments but will be explored in subsequent Chapters. The low but pharmacologically relevant concentrations of simvastatin used over 24hr may have been insufficient to significantly deplete plasma membrane cholesterol and disrupt SERT

(Ferraro et al., 2016; Laursen et al., 2018; Ikonen, 2008). Regardless, simvastatin at the concentrations used in these experiments was sufficient to increase 5-HT uptake. In addition, it is possible that statins preferentially lower intracellular vs plasma membrane cholesterol and affect modifications of trafficked SERT to the plasma membrane.

Cholesterol is synthesized in the ER and transports throughout the cell. Thus, intracellular cholesterol sources could be contributing to SERT activity changes by statins. There is no evidence of a direct interaction between statins and SERT, but statins could regulate

SERT through cholesterol-independent biosynthetic intermediates or nonspecific targets to enhance 5-HT uptake by SERT. The cholesterol-independent nature of these data will be explored in subsequent Chapters to further define the mechanism of statin-enhanced 5-

HT uptake.

Chapter 3: Mediators of Simvastatin-Increased 5-HT Uptake

Introduction

The enhanced 5-HT uptake by statins is a novel and previously undiscovered effect on SERT activity (Chapter 2, Deveau et al., 2020). While the cholesterol-lowering effect of statins is presumed to be a mechanism by which statins increase 5-HT uptake, the biosynthetic intermediates related to cholesterol synthesis may be the critical mediators. These intermediates include mevalonate, farnesyl pyrophosphate (FPP), and squalene (Figure 3.0). FPP can also be converted to geranylgeranyl pyrophosphate

(GGPP). Both FPP and GGPP are substrates for lipidation enzymes and together comprise the cholesterol-independent isoprenylation pathway. Both FPP and GGPP also are upstream effectors of small GTPases like Rho and Rac. Therefore, inhibition of these biosynthetic intermediates or small GTPases may contribute to a cholesterol-independent mechanism by which 5-HT uptake is enhanced after statin treatment.

Regardless of the role of cholesterol, kinases or phosphatases may also be involved in the observed statin-enhanced 5-HT uptake. Kinases and phosphatases are the primary mechanism by which SERT is regulated. Although numerous kinases (PKC,

PKG, CAMKII, P38MAPK,) and phosphatases (PP2A, CaN) can regulate SERT activity

(see Chapter 1), changes in P38MAPK, PP2A, and CAMKII can produce robust increases in SERT-dependent 5-HT uptake similar to the robust increases in SERT activity after statins (Bauman et al., 2000; Ramamoorthy et al., 1998; Zhu et al., 2005).

Furthermore, P38MAPK, PP2A, and CAMKII can localize to lipid rafts in the plasma membrane (Sui et al., 2006; Young et al., 2003) and P38MAPK and CAMKII can be

activated through inhibition of the cholesterol biosynthetic pathway (Borahay et al.,

2014; Dunford et al., 2006; Khan et al., 2011).

To test the cholesterol dependency of the enhanced 5-HT uptake after statin treatment, simvastatin was applied to RN46-B14 neuron-like cells with expressed myc- tagged SERT for 24hr as previously performed, but cholesterol or cholesterol biosynthetic intermediates (mevalonate, FPP, GGPP, and squalene) were restored during the 24hr simvastatin treatment (Figure 3.0). In addition, the roles of selected small

GTPases (CDC42, RhoA, and Rac1) and kinases and phosphatases (P38MAPK, PP2A, and CAMKII) in simvastatin-induced SERT function were explored.

Figure 3.0 Hypothetical Model Statin drugs inhibit the rate limiting enzyme in cholesterol synthesis HMG-CoA reductase, preventing synthesis of cholesterol, but also preventing the synthesis of cholesterol biosynthetic intermediates mevalonate, FPP, GGPP, and squalene. Reduced cholesterol is hypothesized to increase SERT-dependent 5-HT uptake in RN46-B14 cells with expressed SERT.

Methods SERTMyc cell culture and reagents

RN46-B14 cells expressing myc-tagged SERT were prepared as described in

Chapter 2 Methods. The following reagents were used independently and applied for a

20min incubation in DMEM with 2% FBS followed by simvastatin (Cayman Chemical

10010345) for 24hr: mevalonate (Sigma Aldrich, 41288), squalene (Sigma Aldrich

S3626), synthechol (Sigma Aldrich S5442), farnesyl pyrophosphate (Cayman Chemical,

63250), geranylgeranyl pyrophosphate (Cayman Chemical, 63330), CDC42 inhibitor

ZCL 278 (Cayman Chemical 14849), RhoA inhibitor Rhosin HCl (Tocris 5003), Rac1 inhibitor NSC 23766 (Tocris 2161), P38MAPK inhibitor BIRB 796 (Selleckchem

S1574), PP2A inhibitor Okadaic Acid (Santa Cruz sc-202269A), and CAMKII inhibitor

TATCN21 (provided by Andy Hudmon, Purdue University). Methyl-β-Cyclodextrin

(mβCD) was applied 30min prior to 5-HT uptake, independent of treatment with other reagents. 5-HT uptake or immunofluorescent analysis was performed as previously described in Chapter 2 Methods, after 24hr incubation in reagents.

Cholesterol measurement in SERTMyc cells

The Amplex Red Assay (ThermoFisher A12216) was used to measure total cholesterol in SERTMyc cells, treated with and without simvastatin and synthechol. Cells were collected over ice and washed with HBSS, centrifuged at 600xg for 5min, and the pellets were washed in ice cold HBSS. Pellets were then sonicated in 300μl of the provided reaction buffer and plated onto a 96 well plate for a 30min incubation with amplex red working solution containing HRP, cholesterol oxidase, cholesterol esterase, and reaction buffer. Total cell cholesterol content was quantified using a cholesterol

standard curve based on increasing concentrations of cholesterol incubated in a 96 well plate with working solution prior to measurement of the fluorescent signal. A fluorescent plate reader was used to excite (520nM) and measure emitted fluorescence (590nM) from the samples.

siRNA knockdown of Rac1 and RhoA mRNA

Rac1 and RhoA mRNA knockdowns were performed in 6-well plates prepared as described in Chapter 2 Methods. After 3 days in 6-well plates, cells were washed with

2ml of OptiMEM (ThermoFisher 31985088), and 1ml OptiMEM was added to each well.

Meanwhile, the siRNA duplexes were prepared. Pre-designed stealth siRNA for Rac1

(1330001) and RhoA (RSS350533) or the negative control siRNA duplex (12935112) were purchased from ThermoFisher. siRNA duplexes were diluted in OptiMEM and mixed with diluted lipofectamine 2000 (ThermoFisher 13778500) that was previously incubated in OptiMEM for 20min. The duplex and lipofectamine solutions were brought up to 1ml for each well, and the prepared solutions were added to each well in the 6-well plate to a final volume of 2ml and final concentration of 20nM siRNA for RhoA and

100nM siRNA for Rac1. Cells were incubated in the siRNA solution for 24hr, and solution was removed and replaced with 2% FBS and DMEM with and without 0.5μM simvastatin. 5-HT uptake and cell collections for protein knockdown measurements were performed 24hr after incubation in simvastatin.

Results

3.1 Restoration of cholesterol does not block simvastatin-increased 5-HT uptake

To test the contribution and role of cholesterol in simvastatin-enhanced uptake, total cell cholesterol content was measured (see Methods) to determine the effect of simvastatin after the 24hr timeframe of exposure. Simvastatin decreased total cell cholesterol by 20% (Figure 3.1 A). The cholesterol delivery reagent synthechol restored total cell cholesterol. The consequence of cholesterol restoration on increased 5-HT uptake by simvastatin was also investigated. Although synthechol restored total cholesterol in the cells, it did not block the enhanced 5-HT uptake by simvastatin (Figure

3.1 B.).

Figure 3.1 Restoration of cholesterol does not block simvastatin-increased 5-HT uptake A.) Restoration of total cell cholesterol with synthechol during treatment with simvastatin for 24hr blocked the simvastatin-induced cholesterol depletion (n=8- 12/grp) (2-Way ANOVA interaction (p<0.001), *p<0.01 Tukey Post-Hoc comparison Veh and Sim, #p<0.01 comparison Sim and Sim+Chol). B.) However, synthechol did not block enhanced 5-HT uptake by simvastatin treatment (n=6-7/grp) (Two-way ANOVA simvastatin effect (p<0.001), no significant Tukey Post-Hoc comparison Sim, and Sim+Chol). Data are represented as mean ± SEM. Veh=DMSO vehicle, Sim=simvastatin, Chol=synthechol.

3.2 Direct cholesterol depletion with mβCD decreased 5-HT uptake

To provide further evidence that cholesterol was not involved in increased 5-HT uptake by SERT, 5-HT uptake was measured after direct depletion of cholesterol without simvastatin. To achieve direct cholesterol depletion without disrupting cholesterol biosynthetic intermediates, mβCD was utilized to deplete cell cholesterol at 10 and

20mg/ml, for 30min in SERTMyc cells. mβCD is a cyclic ring structure made up of 7 glucose subunits and forms a toroid structure with a hydrophobic inner compartment that can extract membrane cholesterol. Due to the numerous studies demonstrating reduced cell cholesterol after mβCD (Magnani et al., 2004; Scanlon et al., 2001), total cell cholesterol was not measured. Similar to observations in the literature, application of mβCD produced >60% reductions in 5-HT uptake at both 10 and 20mg/ml mβCD concentrations (Figure 3.2). Since this result was opposite to the increased uptake induced by statin treatment, investigation into the cholesterol-independent mechanism by which statins increased 5-HT uptake was warranted.

Figure 3.2 Direct cholesterol depletion with mβCD decreased 5-HT uptake SERTMyc cells treated with 10 and 20mg/ml mβCD for 30min significantly decreased 5-HT uptake (n=4/grp) (One-Way ANOVA mβCD effect (p<0.001), *p<0.01 Tukey Post-Hoc comparison 10 or 20mg/ml and 0mg/ml). Data are represented as mean ± SEM. mβCD=methyl-β-Cyclodextrin.

3.3 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis

pathway intermediate mevalonate, but not squalene.

Simvastatin inhibits the synthesis of mevalonate, squalene, FPP, GPP, and cholesterol (Figure 3.0). To test the role of cholesterol biosynthetic intermediates in simvastatin-enhanced uptake, intermediates mevalonate and squalene were included in the media during simvastatin treatment. The addition of mevalonate blocked simvastatin- enhanced 5-HT uptake in a concentration-dependent manner (Figure 3.3 A). In contrast, the addition of the more immediate cholesterol precursor, squalene, did not block simvastatin-enhanced 5-HT uptake (Figure 3.3 B). Simvastatin increased uptake was variable in Figure 3.3A compared to 3.3B, but displayed consistent 6-fold increases in the uptake ratio of simvastatin to vehicle.

Figure 3.3 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis pathway intermediate mevalonate, but not squalene. Mevalonate or squalene was applied to SERTMyc cells during treatment with 1μM simvastatin for 24hr. Restoration of mevalonate blocked simvastatin-enhanced 5-HT uptake at 10μM and 50μM (n=3/grp) (One-Way ANOVA significant simvastatin effect within simvastatin groups (p<0.0001), *p<0.01 Tukey Post-Hoc comparison to simvastatin/0μM mevalonate) (Two-way ANOVA simvastatin/mevalonate interaction within veh, veh+50μM mevalonate, simvastatin, simvastatin+50μM mevalonate (p<0.05), #p<0.01 Tukey Post-Hoc comparison veh and simvastatin/0μM mevalonate). Squalene restoration did not block enhanced 5-HT uptake by simvastatin (n=3/grp) (One-Way ANOVA not significant within simvastatin-treated groups) (#p<0.01 t-test comparing veh and simvastatin). Data are represented as mean ± SEM.

3.4 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis

pathway intermediates FPP and GGPP.

In addition to the inhibition of cholesterol synthesis, simvastatin inhibits the synthesis of the isoprenylation pathway intermediates FPP and GGPP prior to the production of cholesterol (Figure 3.0). Lower concentrations of simvastatin (0.5µM) were utilized to better approximate conditions in the brain. The addition of FPP and GGPP blocked simvastatin-enhanced uptake in a concentration-dependent manner (Figure 3.4

A, B). Inhibition of simvastatin-enhanced uptake was also visualized with immunofluorescent images depicting qualitative inhibition of intracellular 5-HT (green) with GGPP treatment (Figure 3.4 C).

C.) 6 2

Figure 3.4 Simvastatin-enhanced 5-HT uptake is dependent on cholesterol synthesis pathway intermediates FPP and GGPP A.) FPP was applied to SERTMyc cells during treatment with 0.5μM simvastatin for 24hr. FPP restoration blocked simvastatin-enhanced 5-HT uptake concentration dependently at 2μM and 4μM FPP, but not at 0.5μM FPP (n=4- 6/grp). B.) GGPP restoration also blocked enhanced 5-HT uptake concentration dependently at 4μM GGPP, but not at 2 and 0.5μM GGPP (n=3-5/grp). (One-Way ANOVA simvastatin effect within simvastatin-treated groups (p<0.0001), *p<0.05 Tukey Post-Hoc comparison simvastatin/0μM FPP or GGPP) (Two-way ANOVA simvastatin FPP or GGPP interaction within veh,veh+4μM FPP or GGPP,simvastatin, simvastatin+4μM FPP or GGPP (p<0.05 or p<0.01), #p<0.01 Tukey Post-Hoc comparison veh and simvastatin/0μM FPP or GGPP). Data are represented as mean ± SEM. Veh=DMSO vehicle. C.) SERTMyc cells post-fixed after 5-HT uptake assay and stained for 5-HT. Prior to 5-HT uptake assay and fixation, SERTMyc cells were treated with or without 0.5μM simvastatin and 4μM GGPP.

3.5 Role of GGPP-targeted small GTPases in statin-increased 5-HT uptake

A role for GGPP was established in statin-increased 5-HT uptake (Figure 3.4 B,

C), however the downstream target of GGPP necessary for statin-enhanced 5-HT uptake by SERT is unclear. We measured and manipulated downstream small GTPases CDC42,

RhoA, and Rac1 that are lipidated by GGPP and subsequently transported to the plasma membrane for GTPase activity.

3.5.1 Inhibition of GGPP-targeted small GTPase CDC42 does not block

simvastatin-enhanced 5-HT uptake

The possible role of GGPP suggests that GGPP-targeted small GTPases may be involved in simvastatin-enhanced 5-HT uptake. To test the canonical small GTPases targeted by GGPP, small GTPases were pharmacologically inhibited and/or the mRNA for the respective small GTPase protein was silenced with siRNA. CDC42 was pharmacologically inhibited with ZCL 278 at concentrations surrounding the Kd of the drug (11.4μM). ZCL 278 did not block simvastatin-enhanced uptake at 1, 10, or 50μM

(Figure 3.5.1). Alternatively, inhibition of CDC42 at 50µM ZCL-278 further increased statin-increased 5-HT uptake.

Figure 3.5.1 Inhibition of GGPP-targeted small GTPase CDC42 does not block simvastatin-enhanced 5-HT uptake CDC42 was inhibited with ZCL 278 during treatment with 0.5μM simvastatin for 24hr. Inhibition of CDC42 did not block simvastatin enhanced 5-HT uptake at 1, 10, or 50μM ZCL 278 (n=3/grp) (Two-way ANOVA simvastatin interaction (p<0.001), (*p<0.05) Tukey Post-Hoc comparison simvastatin/50μM ZCL 278 and vehicle/50µM ZCL 278. Data are represented as mean ± SEM.

3.5.2 Inhibition of GGPP-targeted small GTPase RhoA does not block simvastatin-enhanced 5-HT uptake RhoA was pharmacologically inhibited with Rhosin HCl at concentrations around the Kd of the drug (0.4μM). Pharmacological inhibition of RhoA decreased vehicle 5-HT uptake and blocked simvastatin-enhanced uptake in a concentration-dependent manner

(Figure 3.5.2 A). To determine if the effects of rhosin HCl on simvastatin-enhanced uptake were specific, siRNA was utilized to degrade RhoA mRNA and prevent translation of new RhoA protein. The negative control siRNA duplex did not affect simvastatin-enhanced uptake or RhoA protein levels. siRNA for RhoA mRNA reduced

RhoA protein levels, but did not affect simvastatin-enhanced uptake or basal uptake in vehicle treated groups (Figure 3.5.2 B). The effects of RhoA protein knock-down on

RhoA activity was not assessed.

Figure 3.5.2 Inhibition of GGPP-targeted small GTPase RhoA does not block simvastatin-enhanced 5-HT uptake A.) RhoA was inhibited with Rhosin HCl during treatment with 0.5μM simvastatin for 24hr. Inhibition of RhoA with Rhosin HCl blocked simvastatin-enhanced uptake (n=3/grp) (Two-wayTwo-way ANOVA, simvastatin and rhosin HCl interaction (p<0.01), *p<0.01 Tukey Post-Hoc comparison simvastatin and 0μM rhosin HCl, no significant comparison vehicle to 0μM rhosin HCl, #p<0.01 comparison within 0μM rhosin HCl). B.) Knockdown (KD) of RhoA protein was verified with western blot. RhoA KD did not block simvastatin-enhanced uptake or uptake in vehicle-treated cells (n=4/grp) (Two-wayTwo-way ANOVA, Sim effect (p<0.001), no significant siRNA effect (p=0.824), no significant siRNA/Sim interaction (p=0.691). Data are represented as mean ± SEM. V,Veh=DMSO vehicle, S,Sim=Simvastatin, Neg Ctrl=Negative Control siRNA, KD=knock-down.

3.5.3 Inhibition of GGPP-targeted small GTPase Rac1 does not block

simvastatin-enhanced 5-HT uptake

Rac1 was pharmacologically inhibited with NSC 23766 at concentrations around the Kd of the drug (0.4μM). Pharmacological inhibition of Rac1 with NSC 23766 blocked simvastatin-enhanced uptake in a concentration-dependent manner and uptake within vehicle-treated cells (Figure 3.5.3 A). To determine if the effects of NSC 23766 on simvastatin-enhanced uptake were specific, siRNA was utilized to degrade Rac1 mRNA and prevent translation of new Rac1 protein. siRNA for Rac1 reduced Rac1 protein levels, but did not affect simvastatin-enhanced uptake or basal uptake in vehicle treated groups (Figure 3.5.3 B). The effect of Rac1 protein knock-down on Rac1 activity was not assessed.

Figure 3.5.3 Inhibition of GGPP-targeted small GTPase Rac1 does not block simvastatin-enhanced 5-HT uptake A.) Rac1 was inhibited with NSC 23766 during treatment with 0.5μM simvastatin for 24hr. Inhibition of Rac1 blocked simvastatin-enhanced uptake and uptake in vehicle- treated cells (n=3-4/grp) (Two-way ANOVA, interaction (p<0.001), *p<0.001 Tukey Post-Hoc comparison within simvastatin to 0μM NSC 23766, ^p<0.01 comparison within vehicle to 0μM NSC 23766, #p<0.01 comparison within 0μM NSC 23766 between vehicle and simvastatin). B.) Knockdown (KD) of Rac1 protein was verified with western blot. Rac1 KD did not block simvastatin-enhanced uptake or basal uptake in vehicle-treated cells (n=3/grp) (Two-way ANOVA, Sim effect p<001, no significant siRNA effect (p=0.918), no significant siRNA/Sim interaction (p=0.174). Data are represented as mean ± SEM. V,Veh=DMSO vehicle, S,Sim=Simvastatin, Neg Ctrl=Negative Control siRNA, KD=knock-down.

3.6 Role of kinases and phosphatases in statin-increased 5-HT uptake

Given the role of kinases in the regulation of SERT activity, we sought to measure and manipulate kinases or phosphatases that may regulate an increase in 5-HT uptake by SERT. The kinases and phosphatases tested include P38MAPK, PP2A, and

CAMKII.

3.6.1 Phospho-38MAPK is increased after simvastatin, but is not involved in

simvastatin-enhanced 5-HT uptake

P38MAPK specifically regulates increases in SERT activity and can be regulated downstream of GGPP. Phospho-P38MAPK was increased after 24hr simvastatin treatment. Inhibition of P38MAPK with 5nM of the pharmacological inhibitor BIRB 796 inhibited increases in phospho-P38MAPK from simvastatin (Figure 3.6.1 A). However,

BIRB 796 did not block simvastatin-enhanced 5-HT uptake (Figure 3.6.1 B).

Figure 3.6.1 Phospho-38MAPK is increased after simvastatin, but is not involved in simvastatin-enhanced 5-HT uptake A.) Western blot and corresponding densitometry of phospho-P38MAPK /total P38MAPK protein after 0.5μM simvastatin for 24hr, with and without 5nM P38MAPK inhibitor BIRB 796 (n=3/grp) (Two-way ANOVA, ^p<0.05 Tukey Post-Hoc comparison Veh/Veh and Sim/Veh, *p<0.01 comparison Sim/Veh and Sim/BIRB, #p<0.05 comparison Veh/Veh and Veh/BIRB). B.) Inhibition of P38MAPK with BIRB 796 did not block simvastatin-enhanced uptake or uptake in vehicle- treated cells (n=3-4/grp) (Two-way ANOVA significant interaction (p<0.001), not significant via Tukey-Post-Hoc comparison between Sim/Veh and Sim/BIRB or Veh/Veh and Veh/BIRB). Data are represented as mean ± SEM. Veh=DMSO vehicle, Sim=Simvastatin.

3.6.2 Pharmacological inhibition of PP2A does not affect simvastatin-enhanced

5-HT uptake

PP2A specifically regulates increases in SERT activity and can be activated downstream of GGPP. Okadaic Acid (OA) was selected to inhibit PP2A at concentrations that are specific to PP2A and do not affect PP1A ranging from 0.1nM-10nM. Inhibition of PP2A during 24hr treatment with 0.5μM simvastatin did not block simvastatin- enhanced 5-HT uptake (Figure 3.6.2). The trending increases in statin-increased uptake with OA were not significant.

Figure 3.6.2 Pharmacological inhibition of PP2A does not affect simvastatin- enhanced 5-HT uptake Inhibition of PP2A with 0.1-10nM okadaic acid did not block simvastatin-enhanced uptake or uptake in vehicle-treated cells (n=3-6/grp) (Two-way ANOVA significant simvastatin interaction (*p<0.001), (One-Way ANOVA not significant within simvastatin-treated groups (p=0.623)). Data are represented as mean ± SEM. Veh=DMSO vehicle, Sim=Simvastatin.

3.6.3 CAMKII is involved in simvastatin-enhanced 5-HT uptake

CAMKII specifically regulates SERT activity. CAMKII was inhibited with the peptide inhibitor TATCN21 at previously published concentrations that inhibit CAMKII activity (Ashpole and Hudmon 2011, Zybura et al., 2020). The inhibitor contains a TAT sequence that allows for translocation of the applied inhibitor into the cell. Inhibition of

CAMKII during 24hr treatment with 0.5μM simvastatin blocked simvastatin-enhanced 5-

HT uptake, but did not block uptake in vehicle-treated SERTMyc cells (Figure 3.6.3).

Figure 3.6.3 CAMKII is involved in simvastatin-enhanced 5-HT uptake Inhibition of CAMKII with 0.1-40μM peptide inhibitor TATCN21 blocked simvastatin-enhanced uptake (n=3-7/grp) (Two-way ANOVA significant interaction (p<0.05), #p<0.01 Tukey Post-Hoc comparison between Sim and Veh, *p<0.05 comparison between Sim/0μM and Sim/40μM or Sim/20μM) (not significant Tukey Post-Hoc comparison between Veh/0uM and Veh/100nM, 10,20,40μM). Data are represented as mean ± SEM. Veh=DMSO vehicle, Sim=Simvastatin.

Discussion

Experiments on the mechanism of simvastatin-enhanced 5-HT uptake by SERT revealed that simvastatin enhances uptake through isoprenylation intermediates FPP and

GGPP, and not cholesterol. Results also demonstrate a possible CAMKII-mediated effect on statin-enhanced 5-HT uptake.

The findings show that statin-enhanced 5-HT uptake is independent of cholesterol based on the results illustrating that cholesterol restoration with synthechol or the introduction of the immediate cholesterol precursor squalene did not alter the effect of simvastatin on enhanced 5-HT uptake (Figure 3.1 B, Figure 3.3 B). Moreover, the direct depletion of cholesterol with mβCD did not increase 5-HT uptake (Figure 3.2) as observed with simvastatin, but rather decreased 5-HT uptake. The current findings with mβCD on 5-HT uptake are consistent with previous findings from others (Magnani et al.,

2004; Scanlon et al., 2001). Although cell death or apoptosis was not measured after mβCD treatment, there was no observationally distinct change in the cells after 30min mβCD treatment. Furthermore, literature suggests a time course of a few hours before mβCD initiates apoptotic mechanisms (Fortalezas et al., 2018). The apparent difference between the findings with mβCD and simvastatin can be explained by the mechanistic differences between the drugs and the difference in the magnitude of cholesterol depletion. The magnitude of cholesterol depletion produced by statins is modest (about

20%) in the SERTMyc cells (Figure 3.1 A) compared to treatment with mβCD, which typically depletes free cholesterol by 40-50% in HEK cells (Magnani et al., 2004) and up to 85% in the plasma membrane (Scanlon et al., 2001). The low concentration of simvastatin used over 24hr is likely insufficient to significantly deplete plasma membrane

cholesterol where SERT and cholesterol interact, yet is sufficient to alter 5-HT uptake through inhibition of isoprenylation intermediates that diverge from the cholesterol biosynthetic pathway. Thus, the interactions of cholesterol with SERT at the plasma membrane likely remain intact with statin treatment (Ikonen, 2008; Ferraro et al., 2016;

Laursen et al., 2018) and support an effect independent of a SERT-cholesterol interaction.

The addition of the isoprenylation intermediates FPP and GGPP also blocked simvastatin-enhanced uptake and further illustrates that simvastatin enhances 5-HT uptake through inhibition of isoprenylation intermediates. It remains unknown whether

FPP is converted into GGPP and whether GGPP is ultimately necessary for statin- enhanced uptake, or if FPP and GGPP share a common target that modifies SERT- dependent 5-HT uptake. Direct inhibition of GGPP will be explored in Chapter 4 to further elucidate whether geranylgeranylation exclusively regulates SERT. GGPP geranylgeranylates small GTPases, such as CDC42, RhoA, and Rac1 (Hoffman et al.,

2000; Joyce and Cox, 2003; Park et al., 2002). Prolonged inhibition of GGPP (24hr) can increase the activity of GTPases such as Rac1 (Dunford et al., 2006; Khan et al., 2011) that in turn, can activate kinase signaling pathways that regulate SERT (reviewed in

Bermingham and Blakely 2016). Therefore, each of the canonical small GTPases were evaluated (Figure 3.5.1, 3.5.2, 3.5.3) and results support a CDC42, RhoA, and Rac- independent effect of simvastatin-increased 5-HT uptake. However, a discrepancy is evident between the pharmacological inhibition of RhoA and Rac1 during simvastatin- increased uptake compared to the mRNA knockdown during simvastatin-increased uptake (Figure 3.5.2, 3.5.3). A possible explanation could be that the pharmacological

reagents utilized (Rhosin HCl and NSC 23766) have nonspecific effects on SERT or

SERT-regulating proteins. This conclusion is supported by the inhibition of vehicle- treated 5-HT uptake. Although it is widely accepted that Rhosin HCl and NSC 23766 are specific for their respective small GTPases without affecting other small GTPases, evidence in the literature demonstrating cellular effects in Rac-1-deficient cells during

Rac-1 inhibition reveal that there may be off-target effects of NSC 23766 at higher concentrations (Dütting et al., 2015). Alternatively, decreased 5-HT uptake after 24hr inhibition of RhoA and Rac1 could describe a mechanism by which basal activity SERT is regulated directly by small GTPases. A possible explanation as to why siRNA experiments did not reveal this effect could be due to the required time frame of the siRNA protocol for protein knockdown (48hr) compared to the shorter exposure of pharmacological inhibitors (24hr). It is also possible that the siRNA experiments did not block the activity of RhoA and Rac1 compared to the pharmacological inhibitors. Since activity was not measured after protein knockdown, this remains a possible outcome.

Given the role of P38MAPK in regulating SERT and the notable increases in

P38MAPK phosphorylation after simvastatin or isoprenylation inhibition (Figure 3.6.1,

Dunford et al., 2006; Goncalves et al., 2020; Khan et al., 2011), a clear rationale for

P38MAPK in statin-increased 5-HT uptake was evident. However, investigation into

P38MAPK in statin-increased 5-HT uptake revealed no effect (Figure 3.6.1), irrespective of increases in active phospho-P38MAPK induced by statin treatment in SERTMyc cells.

Statins can also increase PP2A in neurons (Zhu et al., 2012) which is known to regulate

SERT activity (Annamalai et al., 2012). However, manipulation of PP2A during simvastatin treatment also did not affect statin-enhanced 5-HT uptake (3.6.2). This is

likely due to the difference in “inhibitory” and “stimulatory” phosphorylation sites on

SERT. Statins may increase PP2A and thus dephosphorylation of SERT, but the

“inhibitory” sites regulated by PP2A are not involved with “stimulatory” phosphorylation sites regulated by statins.

Inhibition of CAMKII blocked simvastatin-enhanced 5-HT uptake. Contrary to the other SERT-regulating kinases, CAMKII plays a unique role in establishing electrogenic, channel-like uptake by SERT (Ciccone et al., 2008) and can also regulate 5-

HT uptake by SERT (Annamalai et al., 2020; Ragu Varman et al., 2021). However, previous findings have not established how CAMKII can regulate SERT. How CAMKII regulates statin-increased uptake is explored in Chapter 4.

It is unknown how statins and isoprenylation can regulate CAMKII signaling and activation that subsequently affects 5-HT uptake by SERT. It is also unclear if direct manipulation of isoprenylation independent of statins can regulate SERT. The next chapter addresses the role of geranylgeranylation on 5-HT uptake and the role of

CAMKII in statin-increased 5-HT uptake.

Chapter 4: Mechanism of Increased 5-HT Uptake by Simvastatin and GGT

Inhibition

Introduction

SERT activity was increased by simvastatin (Chapter 2-3, Deveau et al., 2020) in a GGPP-dependent manner, independent of cholesterol. The role of isoprenylation and

GGPP in SERT regulation has not been investigated and indicates a potentially novel mechanism of SERT regulation. Along these lines, the CAMKII inhibitor TATCN21 blocked statin-increased 5-HT uptake (Figure 3.6.3) and indicates a role for CAMKII in the regulation of statin and GGPP-regulated SERT, and a role for GGPP in CAMKII regulation.

GGPP is necessary for the lipidation of proteins through the transfer of a geranylgeranyl hydrocarbon group by geranylgeranyl transferase I enzyme (GGT).

Statin-increased uptake that is CAMKII-dependent suggests a novel mechanism by which

CAMKII regulates GGPP. Given that CAMKII can directly interact with, phosphorylate, and regulate SERT activity (Sørensen et al., 2014; Steinkeller et al., 2015), CAMKII is a plausible mediator in statin- increased 5-HT uptake. It remains unclear if CAMKII is directly activated through inhibition of geranylgeranylation by simvastatin, and if direct inhibition of geranylgeranylation through inhibition of GGT produces a similar CAMKII- dependent increase in 5-HT uptake by SERT.

To further understand the suggested activation of CAMKII by simvastatin (Figure

3.6.3), the time-dependent phosphorylation and inhibition of CAMKII was further explored after simvastatin and after direct inhibition of geranylgeranylation with GGT inhibition. Previous data suggest the hypothetical mechanism illustrated below in which

simvastatin and GGT inhibition increases CAMKII phosphorylation and CAMKII- dependent increases in 5-HT uptake by SERT (Figure 4.0).

Figure 4.0 Hypothetical Model Statin drugs inhibit the rate limiting enzyme in cholesterol synthesis HMG-CoA reductase, preventing synthesis of cholesterol and biosynthetic intermediates mevalonate, FPP, GGPP, and squalene. GGTI-298 inhibits GGTase and prevents protein geranylgeranylation by GGPP. Reduced GGPP and inhibition of protein geranylgeranylation is hypothesized to increase SERT-dependent 5-HT uptake in RN46-B14 cells with expressed SERT, by a CAMKII activity-dependent mechanism.

Methods

SERTMyc cell culture and reagents

RN46-B14 cells with myc-tagged SERT expression were prepared as described in

Chapter 2 Methods. The following reagents were used independently and applied for a

20min incubation in DMEM with 2% FBS followed by 0.5µM simvastatin (Cayman

Chemical 10010345) or 1-10µM GGTI 298 (Santa Cruz sc-361184) for 24hr: CAMKII peptide inhibitor TATCN21, or CAMKII small molecule inhibitors 78 or 79 (synthesized and provided by Andy Hudmon, Purdue University).

5-HT uptake, western blot, biotinylation, and immunofluorescence analysis were performed as previously described in Chapter 2 Methods, after 24hr incubation in reagents. For western blot application, the following antibodies were utilized: phosphoCAMKII (1:1000, Novus Biologicals NB110-96869), Actin (1:2500, Millipore

Sigma MAB1501).

Results

4.1 Inhibition of GGT enhances 5-HT uptake

Similar to inhibition of isoprenylation with simvastatin treatment, GGTI-298 was utilized to inhibit geranylgeranylation specifically, independent of simvastatin. The cell permeable FTase Inhibitor I was utilized to inhibit farnesylation specifically and independent of simvastatin. Concentrations of 1-500nM were selected based on selectivity of FT Inhibitor I for FT and not GGT, and based on the IC50 of the compound for FT at 21nM (Garcia et al., 1993). Inhibition of FT at the selected concentrations did not increase 5-HT uptake after 24hr treatment (Figure 4.1 A). Concentrations of 1-10µM

were selected based on selectivity of GGTI-298 for GGT and not FT, and based on a 3-

4µM IC50 of the compound for GGT (McGuire et al., 1996; Miquel et al., 1997).

Inhibition of GGT concentration-dependently increased 5-HT uptake after 24hr treatment at 10µM, but not at 1,2, or 4µM (Figure 4.1 B). Concentration-dependent changes in 5-

HT uptake by GGTI-298 treatment increased intracellular 5-HT (green) visualized with immunofluorescent images (Figure 4.1 C).

C.)

Figure 4.1 Inhibition of GGT enhances 5-HT uptake A.) Farnesyl Transferase inhibitor I up to IC70 inhibitor concentrations (500nM Farnesyl Transferase Inhibitor I) did not enhance 5-HT uptake (n=3/grp) (One-Way ANOVA not significant (p=0.714)). B.) Geranylgeranyl transferase Type 1 (GGT-1) inhibitor GGTI-298 concentration-dependently increased 5-HT uptake at concentrations up to IC70 inhibitor concentrations (10μM GGTI-298) (n=4/grp) (One- Way ANOVA GGTI-298 effect (p<0.001), *p<0.05 Tukey Post-Hoc comparison 10µM GGTI-298 and vehicle). C.) SERTMyc cells were post-fixed after 5-HT uptake and stained for 5-HT (green). Prior to 5-HT uptake assay and fixation, SERTMyc cells were treated with vehicle, 1, 2, 4, and 10μM GGTI-298. GGTI-298 concentration- dependently increased cellular 5-HT accumulation. Data are represented as mean ± SEM. Veh=DMSO vehicle.

4.2 GGTI-298-enhanced 5-HT uptake is SERT-dependent Similar to the SERT-dependency measurements after simvastatin treatment, the

SERT-dependency of GGTI-298-increased 5-HT uptake was also elucidated. Enhanced

5-HT uptake by GGTI-298 (10μM) applied for 24hr was blocked by the SERT inhibitor fluoxetine (1μM), applied 30min prior to 5-HT uptake (Figure 4.2 A). GGTI-298 treatment during inhibition of SERT with fluoxetine was also visualized with immunofluorescent images depicting qualitative inhibition of intracellular 5-HT (green) with GGTI-298 and fluoxetine treatment (Figure 4.2 B).

A.)

B.)

Figure 4.2 GGTI-298-enhanced 5-HT uptake is SERT-dependent A.) SERT inhibitor fluoxetine blocked vehicle and GGTI-increased 5-HT uptake (n=3/grp) (Two-way ANOVA significant fluoxetine effect (p<0.01), #p<0.05 Tukey Post-Hoc comparison to Veh,Veh, *p<0.001 Tukey Post-Hoc comparison to 10μM GGTI-298). B.) SERTMyc cells were post-fixed after 5-HT uptake and stained for 5- HT. Prior to 5-HT uptake and fixation, SERTMyc cells were treated with 10μM GGTI-298 +/- 1µM fluoxetine 30min prior to uptake. Data are represented as mean ± SEM. Veh=DMSO vehicle, Fluox=Fluoxetine, GGTI=GGTI-298. 86

4.3 GGT inhibition increases 5-HT uptake to a greater extent than simvastatin

Changes in 5-HT uptake after GGT inhibition vs after simvastatin treatment were compared with and without addition of exogenous 5-HT for 10min. GGTI-298 increased

5-HT uptake more than simvastatin, without changing basal 5-HT (Figure 4.3).

Figure 4.3 GGT inhibition increases 5-HT uptake to a greater extent than simvastatin 0.5µM simvastatin and 10µM GGTI-298 increased 5-HT uptake after 24hr (n=4/grp) (Two-way ANOVA with Tukey’s Post-Hoc comparison to Veh *p<0.05, and comparison Sim and GGTI #p<0.001). Data are represented as mean ± SEM. Veh=DMSO vehicle, Sim=Simvastatin, GGTI=GGTI-298.

4.4 CAMKII inhibition during simvastatin and GGT inhibition

Previous findings demonstrated that high concentrations of CAMKII peptide inhibitor (40µM) blocked simvastatin-increased 5-HT uptake (Figure 3.6.3). This was further investigated using a panel of CAMKII inhibitors including peptide and small molecule inhibitors with respective appropriate controls.

4.4.1 CAMKII peptide inhibitor Tat-CN21 does not block simvastatin or GGTI-

298-increased 5-HT uptake

A fixed concentration of Tat-CN21 (10µM) was chosen based on selective inhibition of CAMKII demonstrated in the literature (Vest et al., 2007). The corresponding inactive peptide inhibitor Tat-Ala was utilized as a Tat-CN21 control reagent that could serve as an additional control for nonspecific effects of the Tat-CN21 compound not measured in Figure 3.6.1. Results demonstrated that simvastatin and

GGTI-298-increased uptake were not blocked by 10µM Tat-CN21

A.) B.)

Figure 4.4.1 CAMKII peptide inhibitor Tat-CN21 does not block simvastatin or GGTI- 298-increased 5-HT uptake A.) Tat-CN21 did not block simvastatin-enhanced 5-HT uptake (n=3/grp) (Two-way ANOVA, significant Sim effect (*p<0.001), no significant Tat-CN21 effect (p=0.599) no significant Tat- CN21/Sim interaction (p=0.351)) or B.) GGTI-enhanced 5-HT uptake (n=4/grp) (Two-way ANOVA, significant GGTI-298 effect (*p<0.05), no significant Tat-CN21 effect (p=0.487), no significant Tat-CN21/GGTI-298 interaction (p=0.633)). Data are represented as mean ± SEM. Veh=DMSO vehicle, Sim=Simvastatin, Tat-Ala=Inactive peptide control, Tat-CN21= Active CAMKII peptide inhibitor.

4.4.2 CAMKII small molecule inhibitors 78 and 79 block simvastatin and

GGTI-298-increased 5-HT uptake

Inhibitors 78 and 79 are pyrimidine-based ATP-competitive CAMKII inhibitors synthesized by Allosteros Therapeutics and based on compounds 15b (inhibitor 78) and

15e (inhibitor 79) from computational structure-activity relationship analyses by Scios

(Mavunkel et al., 2008). Concentrations of inhibitor 78 and 79 were selected based on the cell based IC50s at 0.11-0.32µM in cardiac HL-2 cells and selective inhibition of

CAMKII. For full inhibition of CAMKII, the concentration at which half of the maximal inhibitory effect is observed (IC50) is about 10-fold higher (Swinney, 2011). Therefore, concentrations 1-5µM were sufficient for the predicted 90% inhibition of CAMKII based on the assumption of a single binding site. CAMKII inhibitors 78 and 79 were applied to

SERTMyc cells during 0.5µM or 10µM GGTI-298 treatment. Results demonstrated that

1µM of inhibitor 78 attenuated GGTI-298-increased 5-HT uptake, and 5µM blocked

GGTI-298-increased 5-HT uptake (Figure 4.4.2 A). Simvastatin-increased 5-HT uptake was blocked at both 1µM and 5µM inhibitor 78. Similarly, inhibitor 79 also blocked

GGTI-298-increased 5-HT uptake at 1µM and 5µM (Figure 4.4.2 B). However, only

5µM inhibitor 79 blocked simvastatin-increased 5-HT uptake.

Figure 4.4.2 CAMKII small molecule inhibitors 78 and 79 block simvastatin and GGTI-298-increased 5-HT uptake A.) Inhibition of CAMKII with small molecule inhibitors 78 and B.) 79 blocked simvastatin and GGTI-enhanced 5-HT uptake at 5µM, but not consistently with 1µM (78: n=3-4/grp, Two-way ANOVA interaction (p=0.005) #p<0.001 Tukey Post-Hoc comparison to Veh, *p<0.03 comparison to GGT,Veh or Sim,Veh) (79: n=3-4/grp, Two-way ANOVA interaction (p=0.003) #p<0.001 Tukey Post-Hoc comparison to Veh, *p<0.03 comparison to GGT,Veh or Sim,Veh). Data are represented as mean ± SEM. Veh=DMSO vehicle, GGT=GGTI-298, Sim=Simvastatin.

4.5 Whole cell and intracellular phosphoCAMKII were not increased after

simvastatin and GGTI-298

CAMKII is activated by phosphorylation of Thr286/287 and remains persistently active. To determine if CAMKII Thr286/287 phosphorylation increased early, throughout, or at the 24hr timepoint of 5-HT uptake, CAMKII Thr286/287 phosphorylation was measured at early (30min) and mid (6hr) timepoints, and at the time of uptake (24hr). Preservation of CAMKII phosphorylation was maintained with phosphatase inhibitors (see Chapter 4 Methods) and increases in phospho-CAMKII were verified with calcium ionomycin-stimulated positive controls on separate western blots

(data not shown). Previous concentrations of simvastatin (0.5µM) and GGTI-298 (10µM) known to increase 5-HT uptake (Deveau et al., 2020) did not increase CAMKII phosphorylation at 30min, 6hr, or 24hr. Total pan-CAMKII was not measurable with available antibodies, thus comparison to total pan-CAMKII was not possible.

Intracellular phosphoCAMKII was measured utilizing a biotinylation tag of plasma membrane proteins and subsequent streptavidin separation of plasma membrane from intracellular proteins. 24hr treatment with simvastatin or GGTI-298 did not significantly increase phosphoCAMKII under basal conditions without 5-HT stimulation or with 5-HT stimulation.

Figure 4.5 Whole cell and intracellular phosphoCAMKII were not increased after simvastatin and GGTI-298 A.) Simvastatin (0.5µM) and GGTI-298 (10µM) did not change phosphoCAMKII (normalized to actin and expressed as % vehicle 30min) at 30min, 6hr, or 24hr after treatment (n=3/grp) (One-Way ANOVA repeated measures, not significant). Cells were not exposed to exogenous 5-HT when collected at specified timepoint. B.) Simvastatin (0.5µM) and GGTI-298 (10µM) did not increase intracellular phosphoCAMKII (normalized to actin and expressed as % veh/-5HT or +5HT) regardless of 10min 5-HT stimulation (n=5/grp) (Two-way ANOVA not significant treatment/5HT interaction (p=0.293), treatment effect (p=0.086), or 5HT effect (p=0.454)). Veh=DMSO vehicle, S=Simvastatin, G=GGTI-298, PM=Plasma membrane, Intra=Intracelular.

Discussion

These results further support a mechanism by which SERT is regulated by statins and isoprenylation. Direct inhibition of GGT produced similar SERT-dependent increases in 5-HT uptake as simvastatin. Increased 5-HT uptake by simvastatin and GGTI-298 were blocked with several CAMKII inhibitors, although CAMKII phosphorylation increases were not evident.

Inhibition of geranylgeranyl transferase with GGTI-298 reproduced the effects of simvastatin and further supports the role of decreased isoprenylation and the GGPP modification of proteins in mediating increased 5-HT uptake through SERT (Figure 4.1).

However, inhibition of farnesyl transferase with farnesyl transferase inhibitor I did not reproduce the effects of simvastatin and indicates more specifically that inhibition of geranylgeranylation and not farnesylation may increase 5-HT uptake by SERT. The lack of effect on 5-HT uptake by FTase inhibition could also be a consequence of ineffective

FTase inhibition. Attempts were made to demonstrate cytoplasmic localization of FTase- targeted Ras as a measure of FTase inhibition, but we were unable to confirm cytoplasmic localization of Ras during FTase treatment up to 100µM or with alternative

FTase inhibitors up to 100µM (data not shown). While previous studies have demonstrated cytoplasmic localization of Ras with FTase inhibitors with similar incubation periods (Novotny et al., 2017), these data were not reproducible in the

SERTMyc cell model. More evidence such as the investigation of FTase with siRNA approaches as opposed to the pharmacological approach shown is required to fully support the conclusion that FTase does not regulate SERT.

The 10µM concentration of GGTI-298 produced a greater effect on 5-HT uptake by SERT compared to simvastatin (Figure 4.3). Although GGT inhibition is more proximal to changes in SERT compared to HMG-CoA reductase inhibition that could produce a greater increase in 5-HT uptake, it is also likely that the concentrations of simvastatin compared to GGTI-298 differ in magnitude of inhibition on isoprenylation and thus differences in 5-HT uptake by SERT.

Inhibition of CAMKII during 24hr simvastatin and GGT inhibition blocked enhanced 5-HT uptake. CAMKII small molecule inhibitors 78 and 79 (5µM) maximally blocked increases in 5-HT uptake after statin and GGTI-298. Lower concentrations

(1µM) of CAMKII inhibitors 78 and 79 reduced the enhanced 5-HT uptake by GGTI-

298. Interestingly, 10µM of the peptide inhibitor Tat-CN21 was ineffective at reducing 5-

HT uptake by both simvastatin and GGTI-298 (Figure 4.4.1) but blocked statin-increased uptake at 40µM (Figure 3.6.3). The 40µM concentration is well beyond concentrations required to block CAMKII (Vest et al., 2007) and may be non-specific for CAMKII at this concentration. However, Tat-CN21 could be required at higher concentrations to sustain inhibition of CAMKII throughout the chronic 24hr simvastatin and GGTI-298 incubation. Importantly, CAMKII activity inhibition or reductions in CAMKII phosphorylation, were not verified after use of any of the CAMKII inhibitors (78. 79,

Tat-CN21). Thus, it is possible that the CAMKII inhibitors Tat-CN21 (40µM), 78, and 79 nonspecifically inhibit other kinases or proteins involved in statin and GGTI-increased 5-

HT uptake. However, the effectiveness of two separate small molecule inhibitors (Figure

4.4.2) do suggest that statin and GGT regulated SERT is CAMKII-dependent.

CAMKII can be persistently phosphorylated and active for over 2hr and past 24hr

(Chen et al., 2018; Deng et al., 2017; Otmakhov et al., 2004; Tscheschner et al., 2019), and suggests the timepoints selected (30min, 6hr, and 24hr) were feasible for measurements of long-term and persistent changes in CAMKII phosphorylation after statin and GGTI-298. However, no increases were evident in phosphorylation of

CAMKII at either of the time points, or with more selective isolation of cytoplasmic localized CAMKII (Figure 4.5). Further investigation into CAMKII activity and time- dependent changes in intracellular phosphoCAMKII would further elucidate the statin and GGT-mediated regulation of CAMKII, and thus SERT.

Chapter 5: General Discussion

Summary of Statin and GGT Inhibition on SERT Regulation

Evidence presented in the preceding chapters demonstrated that statins increased

5-HT uptake by SERT in an activity-dependent and trafficking-independent manner. The increase in 5-HT uptake by statins is dependent on isoprenylation intermediates FPP and

GGPP, and not dependent on cholesterol per se. Furthermore, inhibition of GGT and protein geranylgeranylation recapitulated the increased 5-HT uptake through SERT.

Finally, both statins and GGT inhibition increased 5-HT uptake in a manner dependent on

CAMKII inhibitors. This body of research provides evidence for the lipid signaling isoprenylation pathway in SERT regulation and is summarized in Figure 5.0.

Figure 5.0 Schematic representation of summarized results Increases in 5-HT uptake by statins are SERT and activity-dependent, and dependent on mevalonate, FPP, and GGPP cholesterol biosynthetic intermediates. Inhibition of geranylgeranyl transferase also increased 5-HT uptake in a SERT- dependent manner, independent of statins. Both statins and GGTI-298 increased 5-HT uptake through CAMKII.

Activity-Dependent Regulation of SERT by Isoprenylation

Evidence was provided on SERT kinetics and the cellular distribution of SERT after 24hr simvastatin treatment. Simvastatin increased SERT affinity for 5-HT displayed by a reduced Michaelis-Menten constant Km and Ki (Figure 2.6), and occurred in a manner sensitive to the SSRI fluoxetine (Figure 2.4). However, simvastatin did not significantly change Vmax for 5-HT uptake and simvastatin did not change the distribution of SERT at the membrane compared to the intracellular compartment (Figure

2.7). These data suggest that increased 5-HT uptake is a result of catalytic changes in

SERT, rather than an increase in trafficking of SERT to the plasma membrane.

Reductions in Km and Ki indicate increased affinity for 5-HT and a higher capacity for 5-

HT uptake at lower 5-HT concentrations. A lack of change in Vmax is interpretable as a lack of change in the maximal rate of the reaction, thus supporting a lack of change in

SERT trafficking and are consistent with the protein measurements at the membrane and intracellular compartments.

Cholesterol-Independent Regulation of SERT by Isoprenylation

The initial results illustrated increases in 5-HT uptake by 3 different statins

(Figure 2.2). The findings that increases in 5-HT uptake were blocked by the SERT inhibitor fluoxetine demonstrate that the changes in 5-HT uptake are mediated by SERT

(Figure 2.4) and are not limited to a particular statin. Based on the ability of statins to reduce cholesterol via inhibition of the rate-limiting enzyme HMG-CoA reductase, cholesterol was the suspected mediator of the increased SERT-dependent 5-HT uptake.

However, concentrations that restored the statin-induced decreases in cholesterol or the

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administration of cholesterol precursor squalene, did not block statin-increased 5-HT uptake (Figure 3.1 B, Figure 3.3 B). Moreover, the direct depletion of cholesterol decreased rather than increased 5-HT uptake (Figure 3.2).

We next examined the role of the isoprenylation pathway in mediating the effects of simvastatin. The isoprenylation pathway diverges from the cholesterol biosynthetic pathway prior to the actual production of cholesterol. Therefore, it was hypothesized that inhibition of HMG-CoA reductase may also decrease intermediates of the isoprenylation pathway independent of a reduction in cholesterol. Although specific measures of decreases in isoprenylation intermediates were not examined, the addition of GGPP and

FPP after statin treatment blocked statin-dependent increases in 5-HT uptake.

Furthermore, the inhibition of GGT mirrored the increases in 5-HT uptake in a manner that was blocked by fluoxetine. Therefore, the increases in 5-HT uptake by GGT inhibition is likely independent of cholesterol and dependent on SERT.

Evidence that both FPP and GGPP restoration block statin-increased 5-HT uptake

(Figure 3.4) does not differentiate distinct roles for FPP or GGPP since FPP can be immediately converted into GGPP. Therefore, the selective role of FPP independent of

GGP was examined by the inhibition the farnesylation enzyme FTase. Attempts to verify inhibition of farnesylation by FT inhibition did not reduce cytoplasmic localization of small GTPase Ras, and indicates farnesylation of Ras was not blocked (data not shown).

However, direct inhibition of GGT, which does not affect farnesylation, mirrors the effect of statins. Therefore, it can be concluded that SERT regulation in general can be geranylgeranylation-dependent, but it remains to be determined if increased 5-HT uptake by statins is also dependent on farnesylation. Regardless, the findings support a

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cholesterol-independent effect by which statins and isoprenylation can regulate SERT through inhibition of isoprenylation and geranylgeranylation, and not through inhibition of cholesterol.

Involvement of CAMKII in Statin and GGT-Regulated SERT

The inhibition of CAMKII during either statin exposure or GGT inhibition effectively blocked the increased 5-HT uptake (Figure 4.4.2). Two different small molecule CAMKII inhibitors effectively antagonized the enhanced 5-HT uptake by statin and GGT inhibition (Figure 4.4.2). These findings support the role for CAMKII in mediating the effects of statins on 5-HT uptake by GGT. Attempts to assess CAMKII activity by changes in CAMKII phosphorylation did not indicate increases in whole cell or intracellular measurement of phospho-CAMKII (Figure 4.5). However, intracellular phospho-CAMKII was unexpectedly decreased in 5-HT-stimulated conditions. Since total CAMKII measurements were not performed, there may be a change in the ratio of phospho-CAMKII to total CAMKII. On the other hand, a lack of increase in CAMKII phosphorylation, or the reductions observed, may indicate a kinase-independent function of CAMKII in SERT regulation. For instance, CAMKII can function as a scaffold, irrespective of phosphorylation changes or kinase activity (Hojjati et al., 2007). As phosphorylation changes are dynamic and change rapidly, it would be important to determine the best time point to detect CAMKII phosphorylation changes throughout the

24hr treatment incubation. It is also possible that CAMKII is not involved in statin and

GGT-increased 5-HT uptake, suggesting that the inhibitors for CAMKII nonspecifically- target a different kinase or protein in the cells. However, given the strong specificity of

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the small molecule inhibitors used, statin-enhanced 5-HT uptake is altered by specific

CAMKII inhibitors and suggests a CAMKII-dependent effect that requires further elucidation.

Involvement of Other Kinases and Phosphatases

P38MAPK, PP2A, and CAMKII were selected as possible statin and GGT- regulated kinase signaling pathways. The basis for this selection was determined from literature supporting that these kinases and phosphatase regulate robust increases after chronic activation/inhibition, and/or evidence that statins can regulate these kinases and phosphatase. For example, chronic inhibition of isoprenylation can increase phospho-

P38MAPK (Dunford et al.,2006; Khan et al., 2011), and stimulation of P38MAPK activation increases 5-HT uptake by 20-40% in RN46-B14 cells (Zhu et al., 2005).

Although the effects of PP2A and CAMKII activation on SERT activity has not been investigated, chronic statin treatment (12-24hr) can increase CAMKII phosphorylation and CAMKII activity (Borahay et al., 2014), and decrease PP2A phosphorylation known to increase PP2A activity (Raina et al., 2013). Numerous other kinases could still be involved in statin and GGT-regulated SERT. For instance, reductions in FPP and GGPP increase recruitment of novel PKC isoforms (δ,ε,η,θ) as opposed to conventional isoforms (α,β,γ) (Chen et al., 2002) but have not been specifically investigated in SERT regulation.

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Post-Translational Modifications Possibly Involved

The role of post-translational phosphorylation in the regulation of SERT activity is well-established and a plausible mechanism by which statins and GGT regulate SERT.

A major site for CAMKII-dependent SERT phosphorylation occurs at Serine 13 on the

N-terminal (Sørensen et al., 2014). It is also possible that CAMKII interacts with an additional SERT-modifying kinase to produce changes at a different kinase-specific phosphorylation site on SERT. The SERT C-terminal is also critical for CAMKII interaction (Steinkeller et al., 2015) and as mentioned above, CAMKII can also act as a scaffold in a kinase-independent manner (Hojjati et al., 2007) that may involve interaction with SERT. However, the function of CAMKII scaffolding in relation to monoamine transporter regulation and phosphorylation has not been investigated.

Phosphorylation is also regulated by phosphatases and inhibition of protein phosphatase 2 A (PP2A) increases SERT phosphorylation but decreases 5-HT uptake by

SERT (Ramamoorthy et al., 1998). These apparent opposing roles of phosphorylation on the activity of SERT might be explained by the specific site of PP2A dephosphorylation that could include glycine 56, since glycine 56 mutations are refractory to the effects of inhibition of PP2A (Prasad et al., 2009). Many kinases, including P38MAPK, that regulate SERT at “stimulatory” phosphorylation sites like threonine 616, also rely on

PP2A for dephosphorylation at hypothesized “inhibitory” phosphorylation sites

(Sørensen et al., 2014; Zhu et al., 2005). Therefore, changes in SERT phosphorylation may be a result of opposing phosphorylation changes rather than a general increase or decrease in SERT phosphorylation.

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Measurement of CAMKII-dependent SERT phosphorylation after statins or GGT inhibition was attempted after simvastatin or GGT-treatment. Immunoprecipitation of

SERT or the myc-tag on expressed SERT were investigated with western blot approaches and mass spectrometry, but did not yield measurable post-translational site-specific or general phosphorylation changes. To date, few studies have been able to successfully detect measurable and site-specific SERT phosphorylation differences in vitro and this experimental approach remains a technical challenge. Regardless, successful mass spectrometry approaches have used generated SERT peptides (Sørensen et al., 2014) and could be a plausible method for identifying CAMKII-dependent SERT phosphorylation changes by statins and GGT inhibition.

In addition to phosphorylation, other involved post-translational modifications could include glycosylation or palmitoylation. For example, FPP can react with isopentenyl diphosphate to produce dolichol, which contributes to the N-glycosylation of proteins like SERT. While N-glycosylation has been shown to occur prior to the availability of SERT at the plasma membrane (Nobukuni et al, 2009), it is unknown if N- glycosylation can result in rapid modulation of membrane SERT. It is also unclear if

GGPP can regulate dolichol synthesis independent of FPP. Palmitoylation can also be regulated by isoprenylation. Both statins and isoprenylation protein knockdown can increase palmitate precursors (Murthy et al, 2005) and palmitoylation substrates

(Fiorentino et al, 2008), and palmitoylation of DAT can increase transporter activity

(Moritz et al, 2015).

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CAMKII Activation by Statins and GGT Inhibition

Simvastatin has been shown to increase CAMKII phosphorylation at Thr286

(Chen et al., 2018). We were unable to measure increases in CAMKII phosphorylation, which could be due to several aforementioned reasons, such as the wrong timepoints, different CAMKII functions in our cells, or even based on our phospho-antibody only measuring α and β isoforms and not γ and δ. However, given the impact of CAMKII specific inhibitors, CAMKII could still be activated after statins and GGT inhibition through a number of CAMKII-specific mechanisms. For instance, CAMKII is a Ca2+- dependent kinase and can be activated by Ca2+. Simvastatin can increase basal free Ca2+

(Heinke et al., 2004), that in turn, could bind and consequently activate CAMKII. Along these lines, statins and GGT could impact intracellular Ca2+ through GPCR signaling

(reviewed in Dhyani et al., 2020). Of the three GPCR subunits (α, β, and γ), the γ subunit is geranylgeranylated (de Loof and Schoofs, 2019) and could alter intracellular Ca2+ and

GPCR signaling. Consequently, the prevention of geranylgeranylation could contribute to intracellular Ca2+ and CAMKII changes after simvastatin and GGT inhibition.

Regardless, the consequence of geranylgeranylation inhibition on GPCR-mediated Ca2+ changes in neuronal cells remains to be investigated.

The role of RACK (Receptor for Activated Kinase C) may also be involved in

CAMKII regulation by statins and GGT inhibition. RACK can recruit PKC to the membrane of neurons and in the presence of low FPP and GGPP, PKC recruitment by

RACK is increased (Chen et al., 2002). RACK also directly interacts with DAT (Torres

2006) and was identified in numerous SERT immunoprecipitation preparations analyzed by mass spectrometry in our studies (data not shown). Thus, numerous kinases that

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interact with DAT and SERT (reviewed in Bermingham and Blakely, 2016) including kinase-kinase interactions between CAMKII and PKC may also be mechanisms by which

CAMKII is regulated by statins and GGT.

RhoA, Rac1, and CDC42 were examined based on their regulation by geranylgeranylation, but the current evidence demonstrated a lack of involvement by these small GTPases (Chapter 3). Other small GTPases that are geranylgeranylated and could regulate 5-HT uptake by SERT include RhoC, RhoG, RhoH, Rac2, Rac3, Wrch1, and Rif. Of these small GTPases, Rac3 is highly expressed in the brain and may interact with CAMKII in the neuron, similar to the highly homologous Rac1 (de Curtis, 2019).

The role of Rac3 and other GGT-targeted small GTPases in CAMKII activation/phosphorylation or monoamine transporter regulation remains to be explored in the neuron.

Shared Mechanisms of Monoamine Transporter Regulation

The findings of the current study related to regulation by isoprenylation may not be limited to SERT. The 630 amino acid structure of SERT is 50-55% homologous to the

Dopamine Transporter (DAT) and the Norepinephrine Transporter (NET) (reviewed in

Aggarwal and Mortensen 2017). The overlapping amino acids occur primarily in transmembrane and intracellular loops to participate in both DAT and SERT folding at the plasma membrane. Citalopram or paroxetine-bound SERT (Coleman et al., 2016;

Coleman and Gouaux, 2018; Coleman et al., 2020) and nortriptyline-bound DAT observed by X-ray crystallography show clear similarities in structural folding between the transporters. In addition, similarities in the regulation of the activities of DAT and

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SERT exist. CAMKII interacts with and phosphorylates DAT and stimulates dopamine efflux during amphetamine reversal of DAT (Fog et al., 2006). CAMKII also interacts with and phosphorylates SERT, and regulates 5-HT efflux during amphetamine reversal of SERT uptake (Sørensen et al., 2014; Steinkeller et al., 2015). Overall, similarities in

CAMKII-dependent mechanisms between SERT and DAT suggest that regulation of

SERT by statins and GGT could also regulate DAT.

Statins may regulate DAT within the context of drug abuse models. Since extracellular dopamine is reinforcing in drug abuse, statin-increased dopamine uptake by

DAT would result in reduced reinforcement of abused drugs by increase dopamine uptake. Along these lines, brain-permeable statins reduce cocaine and nicotine self- administration in rats (Chauvet et al., 2016) and supports future investigation into the role of statins and GGT in DAT regulation.

Dopamine Uptake by SERT

SERT also can uptake dopamine under certain conditions. Specifically, SERT uptakes dopamine in a SSRI-sensitive manner in both human and rat SERT variants

(Larsen et al. 2011). However, dopamine uptake by SERT occurs at a different affinity compared to 5-HT uptake and requires a higher sodium and chloride demand (Kannari et al., 2006). Since increased SERT affinity for 5-HT after statins (Figure 2.6) are similar to the affinity changes required for DA uptake by SERT, statin-induced enhancement of DA uptake by SERT is possible and could be explored.

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Adverse Statin Effects in Humans

Statins produce a variety of adverse neurological effects, such as sleep abnormalities, suicidal tendencies, aggression, cognitive abnormalities, and mood changes. The adverse neurological effects of statins remain controversial as some studies suggest no adverse effects with statin therapy (Molero et al, 2020). However, recent studies indicate age, sex, and time-dependent neurological effects from statin therapy that could be masked in large uncategorized datasets. Aggression has been documented to persist throughout the length of statin treatment, resolves immediately upon stopping use, and selectively impacts women more than men (Golomb et al., 2004; Golomb et al.,

2015). Recent studies on the adverse cognitive effects of statins reveal a negative impact on working memory in subjects under 65 years old, but not older than 65 years old

(Alselhi et al., 2020). Although the benefits of statins drugs, including reductions in cholesterol and decreased cardiovascular and stroke risk, outweigh the adverse neurological risks, additional consideration should be given to statins that do not cross the blood brain barrier. Regardless, the current findings suggest a potential mechanism for the adverse neurological effects from statin therapy.

New Indications for Statin Treatment in Overactive 5-HT Conditions

Conditions that are produced by an excess of 5-HT may benefit from statin or

GGTI-targeted treatment. For instance, movement disorders are caused or exacerbated by

SSRI treatment in some individuals (Gerber and Lynd et al., 1998). This consequence presumably occurs due to an increase in extracellular 5-HT produced by SSRI treatment, and are manifested as akathisia, dystonia, dyskinesia, tardive dyskinesia, Parkinsonism,

109

and bruxism. These movement disorders are similar to the extrapyramidal side effects associated with antipsychotic medications and is a primary concern with patient compliance (Kane 2001). In fact, SSRI treatment further exacerbates extrapyramidal side effects when given concomitantly with antipsychotic medication and can be reduced with statin treatment (Nomura et al., 2017; Sommer et al., 2021). Therefore, consideration could be given to statins as an adjuvant therapy with antipsychotics to prevent heightened increases in synaptic 5-HT, unwanted extrapyramidal side effects and thereby improve patient compliance and comfort.

SERT regulation by statins could also benefit brain cancers exacerbated by 5-HT.

Metastasis of brain-derived gliomas are impacted by 5-HT and 5-HT receptors (Merzak et al., 1996). Increases in extracellular 5-HT facilitates brain tumor progression and metastasis in brain-derived cancers (reviewed in Caragher et al., 2018). Furthermore, gliomas in the densely 5-HT innervated pons brain region are more aggressively graded and associated with poor prognoses (Hargrave et al., 2006). Statin treatment has been demonstrated to reduce glioma migration and invasion (Xiao et al., 2019). While this could be due to any of the pleiotropic effects of statins (including anti-inflammatory effects), the effects of statins on monoamines could be further explored in glioma models and could be an adjuvant treatment for brain-derived gliomas.

Future Directions

Several critical aspects of SERT regulation by statins and GGT inhibition remain unclear. Investigation into site-specific post-translational modifications on SERT could reveal specific residues regulated by lipid signaling and provide more information on

110

kinases, phosphatases, and accessory protein interactions involved in SERT regulation by isoprenylation.

CAMKII activation is also an important feature of this study. Identification of the timepoint at which CAMKII is activated, and what protein(s) are necessary for CAMKII activation remain(s) unknown. The role of Rac3 or other GGT-targeted small GTPases or

GPCR signaling-dependency could be further elucidated. It is also unclear if CAMKII changes occur as a result of altered intracellular Ca2+. The proteins involved and whether isoprenylation regulates SERT by a Ca2+-dependent mechanism should reveal insight into the mechanism by which SERT is regulated by lipid signaling.

It will also be important to determine whether statins and GGT inhibition also increase DA uptake by SERT, or DA uptake by DAT. This will provide new information on the generic role of isoprenylation on monoamine transporters and the promiscuity of neurotransmitter regulation by isoprenylation.

The consequences of increased SERT activity were not investigated but could reveal important insights into serotonergic signaling in general. For example, measurement of 5-HT vesicle release in cells treated with statins could reveal consequences of the increased 5-HT uptake. Proteins can also be modified by serotonylation, and could be a downstream consequence of increased 5-HT uptake.

The impact of the current findings could be strengthened by in vivo studies. The involvement of statins and GGT inhibition in rodent models and the role of CAMKII activation in vivo will strengthen the translatability of SERT regulation by isoprenylation.

Further elucidation of the impact of statin and GGT-regulated SERT on serotonergic signaling could be further explored in vivo. For instance, the consequences of long-term

111

reductions in synaptic 5-HT through increased SERT activity could be assessed. This could reveal long-term synaptic plasticity changes, altered serotonergic receptor expression, and may impact long term potentiation/depression (LTP, LTD).

The identification of human disease states in which isoprenylation is decreased could help to elucidate the role of 5-HT in these diseases. For instance, geranyleranyl transferase is decreased in post-mortem brain tissue from humans with schizophrenia

(Pinner et al., 2020), and patients with Alzheimer’s disease exhibit reduced protein isoprenylation (Hoof et al., 2010). Finally, the impact of prenylation-mediated SERT regulation could reveal key mechanistic insights into the contributions of 5-HT and SERT toward disease progression and symptom development.

112

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Curriculum Vitae

Carmen Marie Deveau

Education

PhD in Pharmacology & Toxicology

July 2015- July 2021

Indiana University School of Medicine, Indianapolis, IN

BSPS in Medicinal & Biological Chemistry

August 2009- May 2013

The University of Toledo, Toledo, OH

Research Experience

PhD Dissertation Research

Department of Pharmacology, Indiana University School of Medicine

Major Advisor: Bryan Yamamoto, PhD

• Nonspecific effects of cholesterol-lowering drugs on serotonin transporter

regulation in the neuron.

• Benefits of anti-inflammatory pulmonary drugs on methamphetamine drug-

dependence and relapse in the rat.

Undergraduate Honor Thesis Research

Department of Neurosciences, University of Toledo College of Medicine

• Axonal plasticity in striatal terminals after methamphetamine neurotoxicity in

the rat.

Undergraduate Fellowship Research

Department of Neurosciences, University of Toledo College of Medicine

• Glutamate sequestration in the thalamostriatal pathway after methamphetamine

neurotoxicity in the rat.

Publications

Baek, J.B., Deveau, C.M., Kline, H., & Yamamoto, B.K. (2021). Roflumilast treatment during forced abstinence reduces relapse to methamphetamine seeking and taking.

Addiction Biology, submitted.

Deveau, C.M., Rodriguez, E., Shroering, A., & Yamamoto, B.K. (2020). Serotonin transporter regulation by cholesterol-independent lipid signaling. Biochemical

Pharmacology, 183, 114349.

Mitchell, C. M., El Jordi, O., & Yamamoto, B. K. (2019). Inflammatory mechanisms of abused drugs. In Role of Inflammation in Environmental Neurotoxicity (pp. 133–168).

Elsevier.

Natarajan, R., Mitchell, C., Harless, N., & Yamamoto, B.K. (2018). Cerebrovascular injury after serial exposure to chronic stress and abstinence from methamphetamine self- administration. Scientific Reports, 8(1) 10558.

Blaker ,A.L., Mitchell, C., & Semple, E. (2015). Identifying the role of novel protein kinase C isoforms in mediating paclitaxel-induced peripheral neuropathy. Journal of

Neuroscience, 35(28) 10101-10102.

Abstracts

Deveau, C.M., Hudmon, A., & Yamamoto, B.K. (2021, April 27-30). Regulation of serotonin transport by geranylgeranyl transferase and Ca2+/calmodulin-dependent protein kinase II [Conference poster abstract]. Experimental Biology, Remote.

Mitchell, C.M. & Yamamoto, B.K. (2019, October 19-23). A mechanism of serotonin transporter regulation by cholesterol biosynthetic intermediates [Conference poster abstract]. Society for Neuroscience, Chicago, IL.

Baek, J., Mitchell, C., Natarajan, R., & Yamamoto, B.K. (2018, November 3-7).

Phosphodiesterase inhibitor roflumilast attenuates relapse to methamphetamine self- administration after forced abstinence [Conference poster abstract]. Society for

Neuroscience, San Diego, CA.

Mitchell, C.M., Schroering, A., & Yamamoto, B.K. (2018, November 3-7). Cholesterol- independent effects on serotonin transporter regulation: role of simvastatin [Conference poster abstract]. Society for Neuroscience, San Diego, CA.

Mitchell, C.M., Schroering, A., & Yamamoto, B.K. (2017, November 11-15). Roles for cholesterol and the isoprenylation pathway in serotonin transporter regulation

[Conference poster abstract]. Society for Neuroscience, Washington, D.C.

Chiu, V.M., Mitchell C, & Yamamoto, B.K. (2014, November 15-19). Acute stress to adolescent rats reduces p-CREB in adulthood [Conference poster abstract]. Society for

Neuroscience, Washington D.C.

Messer WM, Yang T, Mitchell C, Zheng G. (2014, November 15-19). Development of

M5 muscarinic antagonists: Interaction of GZ-002-05 with muscarinic receptor subtypes.

[Conference presentation abstract]. Society for Neuroscience, Washington, D.C.

Presentations

Deveau, C.M. (2020, November 20). Challenges of measuring phosphorylated proteins:

Attempts measuring serotonin transporter phosphorylation [Department Seminar].

Department of Pharmacology and Toxicology, Remote.

Deveau, C.M. (2020, November 19). Serotonin in the brain: A new role for cholesterol precursors [Conference presentation]. Preparing Future Faculty and Professional

Program, Remote.

Mitchell, C.M. (2018, February 2). Serotonin transporter regulation by isoprenylation:

An off-target effect of statins [Department Seminar]. Department of Pharmacology and

Toxicology, Indianapolis, IN.

Mitchell, C.M. (2017, May 5). Roles for cholesterol and isoprenylation in the regulation of the serotonin transporter [Department Seminar]. Department of Pharmacology and

Toxicology, Indianapolis, IN.

Involvement

University Involvement

NetworkIN Committee Chair, 2020-2021, Indiana University

Department of Pharmacology and Toxicology Student Representative, 2018-2019,

Indiana University

College of Biomedical Graduate Students (CBGS) Treasurer, 2014-2015, University of

Toledo

Volunteer

School on Wheels Tutor, 2018-present

Drug Information Association (DIA) Module Beta Tester, 2018-2019

Awards

Cagiantas Scholar, 2019-2020, Indiana University

Predoctoral Fellowship, 2018-2020, PhRMA

Paradise Travel Award, 2018, Indiana University

Summer Undergraduate Research Fellowship, 2012, University of Toledo