Date of Award

December 2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

James D. Foster

Abstract

The projects discussed in this dissertation outline the physiologic consequences of S-palmitoylation, a lipid-based post-translational modification, on the serotonin and norepinephrine transporters (SERT and NET, respectively). SERT and NET are trans-membrane proteins that belong to the SLC6 family of secondary active transporters. During neuro- and neuro-hormonal transmission, SERT and NET utilize the differential ionic concentrations of the extracellular and intracellular spaces to remove their corresponding substrates (serotonin [5HT] and norepinephrine [NE]) from the extracellular space. Each monoamine modulates distinct and overlapping physiologic functions, with dysregulation driving the pathogenesis of neuro-cognitive, neuro-humoral, sympathetic, cardiac, and vascular diseases. Because of this, these transporters are a target in the development of therapeutics to battle human disease associated with monoamine signaling. Prescription therapeutics that target SERT and NET include methylphenidate (Ritalin), bupropion (Wellbutrin), selective-serotonin (Lexapro, Prozac, Lustral), and serotonin-norepinephrine reuptake inhibitors (Pristiq, Cymbalta). SERT and NET are also targets for drugs of abuse like cocaine (COC), amphetamine (AMPH), methamphetamine (METH), and methylenedioxymethamphetamine (MDMA). As a medical student, I approached my graduate research training from the point-of-view that dysregulated physiology precedes pathophysiology, which can be observed through clinical phenotypes and rectified by intelligent chemo-therapeutic design and selection. More clearly, this is the pathogenesis-to-presentation model of medicine. The experimental design of this dissertation seeks to (1) outline the normal physiologic consequences of S-palmitoylation for SERT and NET, (2) describe how these processes become dysregulated and facilitate disease pathogenesis, and (3) use this knowledge to outline how therapeutics can potentially rectify these pathophysiology’s and re-justify monoamine homeostasis. The first project of this dissertation explores the regulation of SERT by S-palmitoylation and how this process is altered by therapeutic manipulation with escitalopram (Lexapro). Our studies revealed that when SERT-expressing cells are acutely challenged with the irreversible palmitoyl acyl-transferase (DHHC) inhibitor, 2-bromopalmitate (2BP), SERT palmitoylation was reduced in a time-wise fashion without changing surface or total SERT expression. Acute inhibition of SERT palmitoylation decreased SERT Vmax without altering surface expression or relative affinity of the transporter for 5HT (Km). This suggests that palmitoylation regulates SERT acutely by adjusting SERT kinetics without altering levels of SERT at the cellular surface. In longer time intervals with higher 2BP concentrations, inhibition of SERT palmitoylation promoted a loss in SERT surface density and total protein, suggesting that palmitoylation is involved in trafficking of SERT through cell surface recruitment or endocytosis, dependent on SERT’s state-of-palmitoylation. Additionally, palmitoylation may prevent a loss of total SERT protein by opposing lysosomal degradation or supporting biogenesis. When treated with escitalopram, SERT palmitoylation was reduced alongside 2BP inhibition. These results correlate with losses in surface SERT and 5HT uptake under the same conditions, suggesting that escitalopram may bind and configure SERT to a conformation that discourages palmitoylation, leading to internalization and downregulation of SERT activity. The second project explored the pathogenesis of autism and how escitalopram may be efficacious in its treatment. In previous research, functionally-rare SERT coding variants associated with autism and obsessive-compulsive disorders (ASD and OCD, respectively) exhibit increased surface expression and transport capacity. ASD is a disorder of developmental delay in cognitive processes characterized by difficulties in communication, interaction in social settings, and obsessive-compulsive patterns in thought and behavior. Here, we reveal that the ASD SERT coding variant, F465L, confers an increase in palmitoylation and confirm from previous studies an increase in F465L cell surface levels and Vmax when compared to WT hSERT. Promising studies from clinical and functional magnetic resonance imaging (fMRI) data describe therapeutic approaches for adults with severe forms of ASD/OCD that consist of SSRIs like escitalopram. Acyl-biotinyl exchange (ABE) and cell surface analysis of WT and F465L hSERTs treated with 2BP or escitalopram reveal reductions in both palmitoylation and SERT surface levels to basal WT conditions. These results suggest dysregulated palmitoylation is a step in the pathogenesis of autism and obsessive-compulsory based cognitive illnesses, and escitalopram may be effective in rectifying this process. The third project investigated the family of enzymes responsible for protein palmitoylation called palmitoyl-acyl transferases or DHHCs. We have previously published that 5 members of this family (DHHC2, 3, 8, 15, and 17) catalyze palmitoylation of the dopamine transporter (DAT). This information, alongside our data from project one, led us to hypothesize that these enzymes may play a role in regulating SERT trafficking, activity, and serotonergic tone. When SERT was co-expressed with 9 different DHHCs, we observed an increase in SERT palmitoylation from DHHC1, 8, 15, and 17. Increased SERT palmitoylation status was consistent with increased levels of SERT surface expression, transport capacity, and total cellular SERT protein. From this group, SERT saturation analysis was performed in the absence or presence of DHHC 1 and 8 with transport capacity normalized to total cellular protein (pmol/min/mg) and SERT surface density. We identified that DHHC 1 and 8 modulate SERT activity differently, with DHHC1 directing a trafficking-dependent increase in transport capacity, while DHHC 8 directly enhanced SERT Vmax independent of cell surface levels. These results outline the diversity of DHHC outcomes for SERT trafficking, activity, and expression. It is possible that DHHCs may operate independently to palmitoylate SERT on different intracellular cysteines, and at different points in SERTs life-cycle, to accomplish the cell’s current physiologic objectives. The fourth project examined palmitoylation of NET and how perturbance of this process controls NET processing, trafficking, and may be involved in the development of a vascular disorder termed orthostatic intolerance (OI). NET is a catecholamine transporter that facilitates the reuptake of epinephrine, norepinephrine, and/or dopamine from pre-synaptically stimulated neurons, controlling cognitive functions and sympathetic tone. In this study, we demonstrate that native rat and heterologous human NETs are palmitoylated. Treatment of heterologous cells expressing NET with 2BP resulted in acute time-dependent decreases in Net palmitoylation, surface density, transport capacity, and total NET protein levels. As inhibition of NET palmitoylation was increased by 2BP concentration and time of exposure, we observed losses of total NET protein without loss of beta-actin, suggesting no changes in cellular cyto-toxicity. Physiologically, OI is a syndrome characterized by hyperadrenergic symptoms involving postural tachycardia, syncope, and excessive plasma NE. We have demonstrated that the OI coding variant, A457P has reduced palmitoylation and total expression compared to WT. These processes were partially recovered upon co-expression of DHHC1, an ER bound DHHC, suggesting that co-translational palmitoylation may facilitate NET biogenesis and that its dysregulation may be a mechanism in the pathogenesis of orthostatic intolerance.

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