Many antidepressants are cationic amphipaths, which spontaneously accumulate in natural or reconstituted membranes in the absence of their specific protein targets. However, the clinical relevance of cellular membrane accumulation by antidepressants in the human brain is unknown and hotly debated. Here we take a novel, evolutionarily informed approach to studying the effects of the selective-serotonin reuptake inhibitor sertraline/Zoloft® on cell physiology in the model eukaryote Saccharomyces cerevisiae (budding yeast), which lacks a serotonin transporter entirely. We biochemically and pharmacologically characterized cellular uptake and subcellular distribution of radiolabeled sertraline, and in parallel performed a quantitative ultrastructural analysis of organellar membrane homeostasis in untreated vs. sertraline-treated cells. These experiments have revealed that sertraline enters yeast cells and then reshapes vesiculogenic membranes by a complex process. Internalization of the neutral species proceeds by simple diffusion, is accelerated by proton motive forces generated by the vacuolar H + -ATPase, but is counteracted by energy-dependent xenobiotic efflux pumps. At equilibrium, a small fraction (10–15%) of reprotonated sertraline is soluble while the bulk (90–85%) partitions into organellar membranes by adsorption to interfacial anionic sites or by intercalation into the hydrophobic phase of the bilayer. Asymmetric accumulation of sertraline in vesiculogenic membranes leads to local membrane curvature stresses that trigger an adaptive autophagic response. In mutants with altered clathrin function, this adaptive response is associated with increased lipid droplet formation. Our data not only support the notion of a serotonin transporter-independent component of antidepressant function, but also enable a conceptual framework for characterizing the physiological states associated with chronic but not acute antidepressant administration in a model eukaryote.

Copyright: © 2012 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here we build on an effort begun in our previous study of sertraline-induced “overdose” [12] , in which we demonstrated that the model eukaryote Saccharomyces cerevisiae (budding yeast) is an ideal experimental system in which to combine the biophysical insights of the bilayer couple model with the physiological insights of lysosomotropism. In that study, we reported the isolation and genetic characterization of sertraline overdose-resistant mutants (sert R ) with altered clathrin function or reduced vacuolar H+-ATPase complex activity. Others have also shown that yeast is amenable to studying cellular membrane accumulation by CAD [13] – [15] . However, a caveat of our previous study is that selection for (sert R ) mutants required supra-therapeutic (∼10 −5 M) drug concentrations. Here we applied techniques of classical pharmacology to yeast, which enabled us to measure membrane accumulation by radiolabeled sertraline – hereafter [ 3 H]sertraline – at clinically relevant (∼10 −9 M) concentrations. We conclude the present study by proposing an evolutionarily informed model of antidepressant function that may provide a molecular basis for neurotrophism induced by chronic treatment with antidepressants in rodent models of human depression, and by extension the therapeutic lag observed in patients taking antidepressants.

The goal of the present study is to begin developing and validating a comprehensive model of complex antidepressant function in humans. The first step in this arduous process is to reconcile two pharmacological perspectives that have historically dominated conventional thinking about CAD activity in cells lacking specific integral membrane protein targets. On the one hand, a molecular view of drug-membrane interactions derives from the seminal work of Singer and Sheetz on amphipath-induced morphological transformations of freshly isolated human erythrocytes, a cell-based model system superior to reconstituted liposomes but still lacking endomembranes. Singer and Sheetz proposed the bilayer couple/balance model, which states that a charged amphipath preferentially accumulates at equilibrium in the leaflet (monolayer) exhibiting the opposite net charge [10] . A disparity in inter-leaflet surface area of less than 1% resulting from asymmetric partitioning by charged amphipaths can be readily observed as dramatic macroscopic changes in the topology of the erythrocyte plasma membrane. On the other hand, a physiological view was developed around the same time by Christian de Duve and colleagues, and is called lysosomotropism, or “ion trapping.” Lysosomotropism is defined as the concentrative capacity of acidic organelles to trap protonated weak bases within, and cannot be modeled by red blood cells [11] . Lysosomotropism has been documented in various mammalian cell lines and in whole organisms treated with CAD.

Cationic amphiphilic/amphipathic drugs (CAD) represent a subset of Food and Drug Administration (FDA) approved compounds that promiscuously interact with both proteinaceous and non-proteinaceous targets, the latter being cellular membranes [1] , [2] . CAD association with cellular membranes depends on an ionizable amine that is positively charged at physiological pH and a lipophilic polycyclic scaffold, but does not depend on stereochemistry, as in the peculiar case of the antidepressant sertraline/Zoloft® moonlighting as a fungicide [3] . The primary protein target of sertraline is thought to be the human serotonin transporter (hSERT), which localizes to synaptic clefts and recycles the monoamine neurotransmitter serotonin after each burst of neurotransmission. According to the monoamine hypothesis of depression, antidepressants like sertraline bind hSERT and acutely block reuptake of serotonin in the brain [4] . However, a latency period whose molecular basis is unknown precedes the emergence of the actual antidepressant effect in humans, and in rodent behavorial models of depression, suggesting that antidepressants exert additional effects at targets besides hSERT. Given the well known and wide-ranging effects of CAD on cellular membrane homeostasis in the absence of specific proteins targets [5] , [6] , the clinical relevance of antidepressant accumulation in neuronal cell membranes has been vigorously debated. For example, there is evidence that supports the existence of serotonin transporter-independent components of antidepressant function in vertebrate cellular models [7] , some of which appears to involve membrane accumulation by antidepressants [8] , [9] . Yet a comprehensive model of antidepressant function that accounts for all drug-target interactions in the human brain has so far been elusive.

Results

Two thermodynamic drivers of sertraline entry into yeast cells We treated wildtype BY4716 cells (hereafter “wildtype) with [3H]sertraline, which we obtained by custom synthesis (see Materials and Methods). We report a total [3H]sertraline cellular accumulation (B max ) equal to 0.019 picomoles (pmol) per 107 cells (+/−0.0014 SEM), and a half-maximal [3H]sertraline cellular uptake rate equal to 3.1 minutes (+/−0.97 SEM) (Fig. 1A). These data are consistent with the lysosomotropic mechanism originally described de Duve and colleagues [11]. Briefly, as the pH of the extracellular medium increases, the deprotonation of sertraline is favored; the ratio of neutral to cationic species reaches unity at the pKa of sertraline. Neutral sertraline is membrane-permeable while charged sertraline is not. Therefore, more [3H]sertraline is internalized by cells growing in alkaline media compared to acidic media. Several classical studies showed that cellular uptake and accumulation of radiolabeled tricyclic antidepressants by primary neurons and fibroblast cell lines is lysosomotropic and Na+-independent [16], [17]. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 1. [3H]sertraline cellular uptake and accumulation is rapid, partially lysosomotropic and varies from cell to cell. (A) Wildtype BY4716 cells accumulate [3H]sertraline (picomoles of radioligand/107cells) in a time-dependent manner as a function of ambient pH: pH 6.0 (diamonds); pH 6.5 (triangles); pH 7.0 (squares). Notice the break in the x-axis between 10 and 20. (B) Bar graph showing fraction of [3H]sertraline accumulated with a 30-minute 2 µM bafilomycin A pre-treatment (“before”) compared to no bafilomycin A pre-treatment; and fraction of [3H]sertraline accumulated with a 30-minute 2 µM bafilomycin A-post-treatment (“after”) compared to no bafilomycin A post-treatment. Wildtype BY4716 (black, unfilled) is contrasted with a vma9 mutant (black, filled). Before and after treatments of wildtype BY4716 treated with 1 µM FCCP (blue, unfilled), 1 µM FCCP+2 µM bafilomycin A (blue, filled), or 10 µg/mL oligomycin (red, unfilled) are also shown. Statistical significance determined by two-tailed t-test. *** P = 0.0004; * P = 0.0118. (C) [3H]sertraline uptake and accumulation time course with wildtype BY4742 cells (black circles, filled) a dnf1Δ dnf2Δ dnf3Δ triple mutant cells (red circles, filled), and a vma9 mutant (red circles, unfilled). Notice the break in the y-axis between 0.02 and 0.05. (D) Single-cell dose response of sertraline-induced cytotoxicity (“overdose”) for wildtype BY4716 (black), vma9 (red), and cup5 (cyan) cells. Error bars indicate SEM. Means were generated from three independent biological replicates. https://doi.org/10.1371/journal.pone.0034024.g001 However, [3H]sertraline cellular uptake is only partially dependent on proton motive forces generated by vacuolar H+-ATPase complexes (V-ATPases) [18], which can be specifically inhibited by the macrolide antibiotic bafilomycin A (BAF). Pre-treatment of wildtype cells with BAF for 30 minutes resulted in a 65% reduction in [3H]sertraline cellular accumulation compared to the control condition, while treatment of wildtype cells with BAF 30 minutes after exposure to [3H]sertraline resulted in reduced [3H]sertraline cellular accumulation that was 57% of the control amount (Fig. 1B). We performed four controls in order to demonstrate the specificity of V-ATPase-dependent proton motive forces. First, a dnf1,2,3Δ triple mutant, which exhibits constitutive vacuolar hyper-acidification [19], significantly hyper-accumulates [3H]sertraline while the vma9 mutant, which exhibits constitutive vacuolar alkalinization, hypo-accumulates [3H]sertraline (Fig. 1C). Second, the effects of BAF on [3H]sertraline accumulation are completely abolished in a vma9 (YCL005W-A) mutant, which normally encodes subunit e of the V0 subunit of the V-ATPase complex (Fig. 1B). Third, before and after treatments of wildtype cells with oligomycin, a specific chemical inhibitor of the F1-F0 mitochondrial ATPase, actually resulted in slightly increased [3H]sertraline accumulation (Fig. 1B). Fourth, FCCP, a non-specific proton ionophore, phenocopies the effects BAF but co-administration of these two agents does not exhibit additivity (Fig. 1B). Interestingly, single cells overdose in the presence of sertraline in a stochastic manner (Fig. 1D). Thus, at the population level and at the level of single cells, the cellular uptake of [3H]sertraline appears to be non-uniform; a fraction of internalized sertraline is “ion trapped,” while the remainder is associated with cellular membrane sites. Next we measured [3H]sertraline cellular uptake and accumulation in response to several environmental perturbations that affect cellular membrane function globally. First, we examined the effect of low temperature, as low temperature promotes the liquid crystalline-gel transition of membranes, i.e., decreases membrane fluidity. Membrane fluidity has been shown to be a determinant of local anesthetic partitioning into reconstituted liposomes [20]. The initial rate of [3H]sertraline cellular uptake by wildtype cells is four times slower at 0°C versus 25°C; after 60 minutes, cells incubated at 0°C accumulate 45% of the total [3H]sertraline taken up by isogenic cells incubated at 25°C (Fig. 2A). To rule out that low temperature mediates this dampening effect through cessation of vesicle-mediated transport, we also measured [3H]sertraline cellular accumulation by a sec18ts mutant, which is conditionally unable to perform membrane-membrane fusion reactions after temperature up-shift [21]. We observed no significant differences between sec18ts and its wildtype reference after a short (25 minute) or long (60 minute) incubation at the non-permissive temperature (Fig. 2B). Next, we tested whether [3H]sertraline cellular uptake and accumulation depends on energy. We pretreated wildtype cells with a cellular ATP depleting cocktail containing 10 mM sodium azide and 10 mM 2-deoxy-D-glucose. Total [3H]sertraline cellular accumulation was increased 3.4-fold in the presence of energy poisons (Fig. 2C). We interpret this result to mean that energy-dependent xenobiotic efflux pumps constitutively extrude [3H]sertraline from the cell. Thus, the association of [3H]sertraline with cellular membranes appears to depend on bulk physical properties of the bilayer. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 2. Solubility properties of cellular membranes affect the rate and extent of [3H]sertraline accumulation. (A) One-hour time course of [3H]sertraline accumulation in BY4716 cells incubated at 25°C (black circles, filled), wildtype incubated at 0°C (black circles, unfilled). Low temperature values were normalized by room temperatures values for ease of comparison. (B) [3H]sertraline accumulation was measured in wildtype BY4742 cells (unfilled) and sec18ts mutant cells (hatches). Times indicate duration of radioligand exposure at 37°C. (C) Total [3H]sertraline accumulation in the presence and absence of energy poisons. Statistical significance determined by two-tailed t-test. * P = 0.0113. Error bars indicate SEM. Means were generated from three independent biological replicates (except for (B), which was generated from two independent biological replicates). https://doi.org/10.1371/journal.pone.0034024.g002

Sertraline permeates vesiculogenic membranes We characterized the subcellular distribution of [3H]sertraline in wildtype cells by biochemical fractionation experiments. As shown in Figure 3A, over 80% of intracellular [3H]sertraline sediments in the P 10,000 fraction following osmotic lysis; that percentage climbs to 90% after mechanical lysis (Fig. 3B). After accounting for the trace amount of [3H]sertraline present in the P 100,000 microsomal fraction, only 10–15% of intracellular [3H]sertraline appears to be truly soluble, demonstrating that at equilibrium the vast majority of [3H]sertraline stably partitions into cellular membranes, presumably as a neutral species. To verify that [3H]sertraline partitioned into cellular membranes we performed the same experiment but in the presence of the nonionic detergent Triton X-100. Triton X-100 completely solubilizes P 10,000 -associated [3H]sertraline (P<0.0001, ANOVA; Fig. 3A). However, detergent-sensitive cellular membrane binding sites may not be chemically uniform. We reasoned that we could distinguish between at least two types of membrane association – adsorption (topical) versus intercalation (deep) – on the basis of chemical extractability of [3H]sertraline from the P 10,000 fraction. The results of this analysis are presented in Figure 3B. Chemical agents that disrupt electrostatic interactions (e.g., 0.5 M Tris) either have no or little solubilizing effect on membrane-associated [3H]sertraline, while chemical agents that disrupt hydrophobic interactions, including the mild detergent digitonin, solubilize membrane-associated [3H]sertraline to varying degrees. Proteolytic digestion of surface-exposed membrane proteins has a modest solubilizing effect, ruling out membrane proteins as essential for amphipath-membrane association. Interestingly, excess (100 µM) “cold” sertraline only has a modest solubilizing effect, which is consistent with the notion that sertraline is buried in the hydrophobic phase of the bilayer at equilibrium. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Figure 3. Subcellular fractionation demonstrates [3H]sertraline accumulation in vesiculogenic membranes. (A) The percent [3H]sertraline detected in soluble (“S”) versus pellet (“P”) fractions following sequential centrifugations (10,000×g and 100,000×g) in the absence (white columns) or presence of 0.5% Triton X-100 detergent (hatches). (B) The ratio of soluble (yellow) to P 10,000 -associated (blue) [3H]sertraline following extraction with a diverse panel of chemical agents. * P<0.05, ANOVA; ** P<0.0001, ANOVA. (C) Distribution of [3H]sertraline across ten equal volume fractions following Optiprep gradient separation of total organellar membranes (P 100,000 ) from wildtype BY4716 cells treated with [3H]sertraline for one hour at 25°C. The refractive index is plotted (blue slashed line) on the left y-axis. (D) Densitometry plots of six organellar markers distributed across the ten gradient fractions. Error bars indicate SEM. Means were generated from three independent biological replicates. https://doi.org/10.1371/journal.pone.0034024.g003 A P 10,000 fraction is thought to be enriched in large organelles like vacuoles and mitochondria at the expense of small organelles like Golgi and ER. As an initial attempt to biochemically purify sertraline-associated cellular membranes, we performed analytical density-gradient centrifugation on a P 100,000 fraction after a single-step centrifugation of lysates from wildtype cells. A single peak spanning two fractions of intermediate density (refractive index in the range 1.37–1.38) contains on average ∼70% of membrane-associated [3H]sertraline, with ∼60% concentrated in a single fraction (fraction 6) (Fig. 3C). We screened these gradient fractions against a panel of antibodies specific for markers residing in different subcellular compartments, and densitometry plots are shown for each marker in Figure 3D. Although our gradient had limited resolving power at fractions 6–7, we observed the strongest co-localization of markers specific for ER and vacuolar membrane markers, as well as the vacuolar resident enzyme carboxypeptidase, with the [3H]sertraline peak in fraction 6, but we did not observe co-localization with a plasma membrane marker (data not shown). We observed less albeit still significant co-localization of Golgi and endosomal membrane markers with [3H]sertraline, though these markers themselves peak in the slightly denser fraction 7. Although the mitochondrial marker porin is also present in fractions 6–7, we showed above that disrupting proton motive forces with oligomycin actually increased [3H]sertraline accumulation (Fig. 1B), so while association with mitochondrial cannot be ruled out it appears coincidental.