freakoutcrazy

Blog des AK Psychiatriekritik der NFJ Berlin

Kategorie: Zoloft

Pfizer disputes claim against antidepressant

The maker of Zoloft is being sued in an unusual case alleging the popular antidepressant has no more benefit than a dummy pill and that patients who took it should be reimbursed for their costs.

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RxISK Stories: The Myth of the Magic Bullet – Flox Tox

by MadinAmerica

 

In July this year, Antimicrobrial Agents & Chemotherapy had an article – Fluoxetine is a Potent Inhibitor of Coxsackievirus Replication by Zuo J, Quinn KK and colleagues. “No antiviral drugs currently exist for the treatment of enterovirus infections, which are often severe and potentially life-threatening. Molecular screening of small molecule libraries identified fluoxetine, a selective serotonin reuptake inhibitor, as a potent inhibitor of coxsackievirus replication. Fluoxetine and its metabolite norfluoxetine markedly reduced the synthesis of viral RNA and protein”.

Another blog on the latest developments recently billed both Prozac and Zoloft as anti-microbials.

These come over as good news stories. That wonderful little Prozac, see what else it can do, isn’t it smart? Except the definition of a smart drug – a Magic Bullet – is that it should only do what it is told to do. Evidence that Prozac or Zoloft do more indicates they may be poisonous rather than medicinal. If we turn the story on its head, the problem may be a little clearer. If an antidepressant can be an antibiotic, what might an antibiotic do, and would we like it? If smart drugs do just what they are supposed to do, the fluoroquinolone antibiotics are in the running to be the dumbest drugs ever.

The Myth of Floxing by Billiam James, 2012

 

Joanne’s Story

In April 2011, I was prescribed Cipro for an uncomplicated, routine Urinary Tract Infection. After only 6 days of 250mg twice daily, I was suddenly hit with a host of symptoms. Within two hours I went from being a healthy, 49 year-old to someone mutilated from head to toe, fighting for my life. My life has changed irreversibly. I have medical documentation of partial paralysis, head to toe tendon damage, hearing loss, heart murmur, kidney and liver damage, erythema multiforme, extreme food allergies.

I was initially told that these symptoms, especially appearing collectively, was “rare”. While this did little to help me, at least I thought I was just unlucky. Imagine my horror when I found that, even using conservative numbers, hundreds of people are poisoned from fluoroquinolones each year with the same devastating result I endure. Not only are there countless scores of FaceBook, YouTube, and blogs on the internet from people with crippling stories almost exactly like mine, there are many people I have met within just my local area that are suffering in this same way.

There is ample documentation to show that the FDA knows that these effects appear syndromically. The FDA knows the devastation caused by fluoroquinolones. Yet, the FDA allows these “medications” to be used in a flippantly casual manner by unknowing doctors with only a black box warning of possible tendon damage. Possible tendon damage implies a bad case of tennis elbow or, at worst, a rupture of the Achilles tendon. Nothing can explain my terror at suddenly having every tendon in my body as fragile as wet tissue paper. Nothing can explain the heartache of having to be fed like a baby by my eight year-old child or being unable to use the bathroom without the assistance of others. Although I have made improvements in the last seven months my chance of complete recovery, based upon expert information, is almost non-existent.

MedWatch, as it currently stands, does not work as an accurate reporting system. It requires doctors to report on their own errors. If physicians recognized the error, it is doubtful they would have made it in the first place. My PCP is a conscientious physician but just did not have enough information on the effects of fluoroquinolones. He reported to MedWatch that I was “recovered” only about 10 weeks out from the onset of my initial symptoms because I was no longer using a wheelchair full-time. He simply could not believe that the plethora of symptoms I suddenly had could be caused by a drug. I have to agree that it is completely unbelievable that anything so dangerous would be out on the market.

The subsequent specialists I now see: cardiologist, nephrologist, endocrinologist, immunologist, orthopedist, GI specialist, neurologist, physical therapist, etc., feel they cannot report my symptoms to MedWatch because I was not under their care at the time of the poisoning and, so, cannot confirm the cause and effect.

There is no established first-aid protocol for those poisoned by fluoroquinolones. We called the Poison Control Center. They confirmed that the effects I was experiencing were caused by Cipro but when we asked what to do they said, “Well, if you rupture, go to the emergency room.” This was not helpful. My doctor also prescribed NSAIDS. Not only should physicians be informed that NSAIDS and steroids are contraindicated, there should be an intervention that includes the immediate administration of antacids or something to bind the remaining fluoroquinolone in an attempt to reduce damage.

The Myth of Floxing by Billiam James, 2012

 

Erin’s Story

May 5, 2004. I was having dinner at Le Cirque at the Belaggio Hotel in Las Vegas. I paid the price for 6 years. Eight hours later I was rushed by the hotel security to a cab because they didn’t think an ambulance would get there in time to save me. The description of what Ecoli is like is too gruesome to share. I was taken to the ER and shot up with Levaquin. The E-coli nightmare stopped. I was to take 500 mg twice a day for 7 days.

OK, I can do that, not a problem. I slept for 4 days straight in a hotel room and boarded a plane and went home where I was kept on Levaquin for a total of 14 days. I could not get out of bed for 2 months. No one recognized me because I looked so deathly. I was sure it was the E-Coli. I struggled with the diarrhea and cramping, the rashes, the memory loss, sore body and broken mind. Then the eight month came. It began with itching and my lips and eyes swelling shut. Well, I do have allergies so maybe that’s it. I began to have severe reactions to every food I ate so I became very selective. As my food choices narrowed my diet adjusted and I eliminated almost everything.

Doctors had no idea what this was and kept giving me cortisone shots and telling me to take Advil for pain. They knew as much about Levaquin as I did. I had developed full blown angioedema, a life threatening condition that makes everything from the neck up swell so badly that the windpipe can swell shut. I couldn’t go anywhere. I needed to be within 15 minutes of a hospital at all times. I was down to being able to eat rice, milk products, apples and green vegatables. I did this for 6 years. No meat, a very hard task for a carnivore like me. I was lucky enough to have a good marriage. My husband watched me go through all of this in disbelief but gave me complete support.

Then after 6 years without any sign it was going to happen, it did pass. The constant burning of my neck and face and the looming threat of the angioedema quietly came to an end.

Three months later I ended up in the ER with a nasty case of cellulitis. I have run out of standard antibiotics due to severe allergic reactions and so Levaquin, once again was the drug of last resort. After my very verbal protest as to how horrible my last round of Levaquin was the ER doctor actually told me that it was this or nothing at all. It was actually the only drug that could get me out of there with a pill rather than an IV administered drug that had to be done once a day for 4 days as an out patient. Cost is everything here. With the thought of losing my leg, I said OK, maybe this time it will be better.

I could not have been more wrong. I took one pill and immediately checked out mentally. I have very few recollections of the 7 days of 500 mg once a day. All I knew was that I was determined to finish this pill and go on with my life. My symptoms on the medication ranged from extreme nightmares, depersonalization, inability to sleep, anxiety attacks, missing time, delirium, fever for 2 hours after I took the pill, then dropped to 96.2 degrees and I was unable to get my body temp up, frozen to the core, inability to empty my bladder, itching, tinnitus, hair falling out, burning skin, broken teeth both times, irregular heartbeats, shaking so bad that I could barely feed myself, semi-hysteria, depression, suicidal thoughts, my skin literally hung from my bones due to collagen degeneration. I lost 15 pounds.

I now have to wear glasses because my vision was damaged. My bones hurt. My tendons snapped. My body ached. My joints swelled. A week after I stopped the meds, I suffered a severe tendon pull from opening a bottle of water. The same arm had tendonnitis of the shoulder and felt like it would fall out of the socket. I could not extend my arm for 3 months. It burned like fire from the pull and my elbow filled with blood. My thyroid seemed to stop working as all of my hypothyroid symptoms returned on top of the Levaquin induced symptoms. The first two month after the discontinuation of the drug was hell, really hell. My blood pressure was so high it terrified my doctor. I was a stroke just waiting to happen.

It took me four months to regain my memory. I still remember very little of the floxing week. I do remember freaking out over the periods of missing time. I did tell everyone around me that I was having dark thoughts and voices telling me to kill myself. Fortunately, no matter how out of my mind I am, I know in my soul that God determines my check out time, not me. I now know that I probably had a small stroke as I could barely talk for almost a month.

It has been  almost 2 years. Here’s the good news. The first time I was poisoned I knew nothing. I did everything wrong and allowed doctors to do everything wrong to me. The first time I was given 3 cortisone shots for the rashes and told to take Advil for the pain. A more appropriate recommendation would have been to dowse myself in gasoline and have a cigarette.

References:

1. Fluoroquinolone-induced suicidal ideation
https://www.deepdyve.com/lp/elsevier/fluoroquinolone-induced-suicidal-ideation-LfMBv3r2mB
A case study about man who became suicidal after being given fluoroquinolone. The introduction notes that adverse CNS reactions to FQs affect up to 1in 25 patients, with severe problems affecting 1 in 200.

2. Fluoroquinolone therapy and idiosyncratic acute liver injury: a population-based study
http://www.cmaj.ca/content/early/2012/08/13/cmaj.111823, published Aug 13, 2012: “Fluoroquinolones are among the most widely prescribed antibiotic agents in North America, and the use of broad spectrum fluoroquinolones such as levofloxacin and moxifloxacin is increasing. Despite their popularity, safety concerns have led to the restriction and, in some cases, withdrawal of several members of this class of drugs… The varied and unpredictable nature of these adverse reactions has led to the ongoing scrutiny of the entire class of drugs.”

3. Based on number direct reports to FDA in 2011, Levaquin is FDA’s 3rd most dangerous drug.
QuarterWatch: http://www.ismp.org/quarterwatch/pdfs/2011Q4.pdf

Illustrations: The Myth of Flox and The Truth of Flox,  2012 © Billiam James

Zoloft, Prozac, Paxil and Other Antidepressants – Anencephaly Birth Defect

by drugwatch

Even before a woman realizes she is pregnant, she could be harming her baby with the chemical substances that pass through her body and into the baby. Doctors counsel their patients about what is considered safe to take during pregnancy, but if drug manufacturers don’t disclose risks of certain antidepressants, how can they — or anyone — offer an accurate assessment?

A mother’s use of antidepressants during pregnancy has been linked to several severe neural tube defects — defects of the brain and spinal cord. Every year, 1 in 4,859 babies in the United States is born with anencephaly.

Researchers have found anencephaly — a fatal birth defect characterized by the absence of a large portion of the brain, skull and scalp — in more babies whose mothers had taken selective serotonin reuptake inhibitors (SSRIs) one month prior to conception or while pregnant. Prozac, Zoloft and Paxil are all part of the SSRI family of drugs.

Antidepressant use during pregnancy has also been linked to other serious birth defects, including respiratory distress, Persistent Pulmonary Hypertension (PPHN) and heart problems. Many women have filed lawsuits against antidepressant manufacturers after giving birth to a baby with birth defects or who later developed problems related to SSRI use during pregnancy.

Antidepressant Use by a Mother Can Be Fatal to Her Baby

Anencephaly happens during the first month after conception, when the top part of the neural tube fails to close and form the brain and spinal cord. Usually, babies with anencephaly do not have the forebrain, the front part of the brain, or the cerebrum, where complex thinking takes place. Furthermore, the parts of the brain that do form are not protected by the normal skin or bone.

An ultrasound or a blood test of the mother often can detect anencephaly during pregnancy.

The lack of an organ to direct the body’s higher-level functions means that the baby cannot survive. Typically, babies with anencephaly are stillborn or die within a few hours of being born. If a baby with anencephaly is born alive, he or she is likely to be unconscious for its short life. The baby is usually blind, deaf and unable to feel pain.

Babies born with the birth defect are kept comfortable with hydration and nutrition, but heroic measures, such as ventilation and surgery, are not employed.

It is thought that a folic acid supplement can reduce the incidence of neural tube defects (NTDs) such as anencephaly.

Studies Linking Antidepressants to Anencephaly

A study that was published in 2007 in The New England Journal of Medicine found that maternal SSRI use is associated with anencephaly. The study, which was associated with the Centers for Disease Control and Prevention (CDC), found that anencephaly “showed a 2.4 times greater occurrence in women who had taken SSRIs in the first trimester.”

Yet, the authors maintain that “the absolute risks associated with SSRIs appear small. …”

Families who have suffered the loss of a baby after antidepressant use during pregnancy have a different point of view. They say they were not warned about the dangers associated with antidepressant use. Many are choosing to hold the drug manufacturers responsible for their loss.

Teenagers on SSRIs

by MadinAmerica

Last week, the Wall Street Journal has an article titled The Medication Generation by Katherine Sharpe which questioned the fact that a large number of teenagers are currently taking antidepressants. In several respects the article was a bit of a refreshing change from how the topic has often been presented  as it raises important questions about what it means for a generation of teenagers to grow up on medications.  It also has some refreshing quotes from experts who point that there are no easy answers. Of course, all this is nothing new to the readers of this website. But at least some of the major newspapers are beginning to publish skeptical articles. (The article never brings up the efficay problem with the SSRIs but that is another story).

However, one aspect that stands out in the current paper is the author’s discussion about the chemical imbalance theory of depression.  Sharpe is on the fence about the theory and can’t quite decide if it is a good thing or bad thing that people have been told that depression is caused by low serotonin. On one hand she points out a potential problem with biological theories: “there are the consequences of teaching young people to think about their problems in biomedical terms.” But then on the other hand she sees a silver lining because the chemical imbalance theory has supposedly removed any negative stigma: “the acceptance of depression as a biological illness has been hailed for removing shame and stigma from the condition.”

But rather than discuss the theory’s utility shouldn’t the questions focus on the accuracy of the theory? If it is not scientifically accurate, isn’t it misleading to portray it as a scientific fact? Several years ago we monitored the media for mention of the chemical imbalance theory. Whenever an article in the main stream media mentioned the theory as a well-established proven fact an email was sent to the author asking if they could supply a reference in support of their statement. None of the reporters provided any citations that could be considered sound evidence to support the theory. Many of the responses seemed to equate this request with asking for a citation that the world was really round. Some of the organizations supplied citations which were little more than similar statements from another organization.

As just one example, consider the following. In the Philadelphia Inquirer, a reporter wrote that “mental illnesses are simply chemical imbalances.”  For her reference she supplied  a statement made by the President of the Society of Neuroscience, which was part of a request made to a congressional panel for more funding. He said: “mental illnesses were due to a chemical imbalance.” When it was pointed out to the reporter that this was not a scientific reference, she replied, “I did not conduct an extensive literature search, as I assumed that if an individual such as the president of the Society of Neuroscience, among others, stated that a mental illness represents a chemical imbalance, there must be some evidence to that fact.”  In the published paper there are many more examples like this.

What would probably come as a surprise to many of these reporters that we talked to is that some leaders in the field have even stated that the psychiatry profession never accepted the theory in the first place and that the chemical imbalance theory is really nothing more than a straw man argument developed by scientologists.

Any discussion about too many teenagers taking SSRIs needs to examine the role of a now largely discredited theory about depression. The presentation of a false scientific theory cannot be excused by some sort of utilitarian argument that ultimitely the public is well served by a falsehood.

Zoloft Causes Cells to Eat Themselves

by PLoS One

Researchers at Princeton found that Zoloft accumulates in yeast cells, leading to curvature of the cellular membrane that triggers “autophagy,” wherein cells eat themselves as a protective measure. “So these SSRIs were supposed to be safe, and yet we’re finding that these yeast are overdosing quite strongly in response to Zoloft,” said one author. The finding suggests that Zoloft has unexpected side effects, given that yeast cells lack serotonin transporters. Results are in PLoS One.

Jingqiu Chen#, Daniel Korostyshevsky#, Sean Lee, Ethan O. Perlstein*

Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, United States of America

Abstract Top

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.

Citation: Chen J, Korostyshevsky D, Lee S, Perlstein EO (2012) Accumulation of an Antidepressant in Vesiculogenic Membranes of Yeast Cells Triggers Autophagy. PLoS ONE 7(4): e34024. doi:10.1371/journal.pone.0034024

Editor: Kenji Hashimoto, Chiba University Center for Forensic Mental Health, Japan

Received: October 17, 2011; Accepted: February 20, 2012; Published: April 18, 2012

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.

Funding: The Lewis-Sigler Institute provided all financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

* E-mail: eperlste@princeton.edu

# These authors contributed equally to this work.

Introduction Top

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.

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.

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 (sertR) 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 (sertR) 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 [3H]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.

Results Top

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 (Bmax) 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].

thumbnailFigure 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.

doi: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.

thumbnailFigure 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).

doi: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 P10,000 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 P100,000 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 P10,000-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 P10,000 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.

thumbnailFigure 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 P10,000-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 (P100,000) 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.

doi:10.1371/journal.pone.0034024.g003

A P10,000 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 P100,000 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.

Pharmacological characterization of mutants with altered sertraline sensitivity

We reasoned that comparison of measurements of [3H]sertraline cellular uptake and accumulation in a panel of mutant strains with altered sertraline cellular response would allow us to develop a cell biological model of cellular membrane accumulation by sertraline. The wildtype strain serves as a reference for three previously described de novo sertR mutants: vma9 (described above), swa2 (YDR320C) and chc1 (YGL206C) [12]. ACY769 is a wildtype prototrophic strain derived from S288c (hereafter “prototroph”), which serves as a reference for four sertraline-hypersensitive (sertHS) homozygous gene deletion mutants involved in clathrin coat formation: arf1Δ, cdc50Δ, drs2Δ and sac1Δ. ARF1 (YDL192W) encodes a small GTPase that is thought to be a master regulator of vesiculogenesis at internal membranes. DRS2 (YAL026C) encodes an aminophospholipid flippase that localizes to Golgi and endosomal membranes, and CDC50 (YCR094W) encodes its regulatory subunit. SAC1 (YKL212W) encodes a lipid phosphatase with specific activity against phosphatidylinositol 4-phosphate (PIP), and localizes to the endoplasmic reticulum (ER) and Golgi. Together these sertR and sertHS mutants comprise a “vesiculogenesis” module centered around ARF1 and PIP, as deletion of ARF1 is synthetically lethal with loss of SWA2, DRS2 or CDC50 [22], [23]. Total [3H]sertraline cellular accumulations (Bmax) for each of the mutants appear in Table 1.

thumbnailTable 1. Total [3H]sertraline cellular accumulation (Bmax) as a function of pH. Bmax is defined as pmol/107 cells/hour.

doi:10.1371/journal.pone.0034024.t001

Unlike the vma9 mutant or BAF-treated wildtype cells, the chc1 mutant and to a lesser extent the swa2 mutant, unexpectedly hyper-accumulate [3H]sertraline. Therefore it may be more appropriate to classify chc1 and swa2 mutants as “sertraline-tolerant.” This hyper-accumulation is distinct from that exhibited by dnf1,2,3Δ, as both chc1 and swa2 have a constitutive vacuolar acidification defect that phenocopies V-ATPase deficiency [12]. Controlling for cell number and cell size, we observed that two other mutants hyper-accumulate [3H]sertraline: sac1Δ and arf1Δ. drs2Δ and cdc50Δ mutants are indistinguishable from the control condition. These results indicate that there are at least two qualitatively distinct phases of the cellular response to sertraline: the first phase affects acute sertraline uptake, while the second phase, which is typified by drs2Δ, has no effect on acute sertraline uptake, and so instead must be involved in the adaptation to chronic sertraline exposure.

To explore this phenomenon more rigorously, we measured the kinetics of [3H]sertraline cellular uptake as a function of substrate concentration. Kinetic experiments with both wildtype references and mutants with altered sertraline cellular response indicate a single non-saturable component of [3H]sertraline cellular uptake, i.e., internalization by simple diffusion (Fig. 4A–B). We estimate Vmax to be 0.62 pmol [3H]sertraline/107 cells/min, which translates to ~1000 sertraline molecules per cell per minute subject, of course, to individual differences between clones. We estimate the Km of sertraline to be 71.5 nM, which represents high-affinity binding but less than the affinity exhibited by it for the human serotonin transporter [24]. The panel of mutants exhibited a range of acute [3H]sertraline cellular uptake rates. Neither vma9 nor drs2Δ mutants show a measurable difference in the rate of [3H]sertraline cellular uptake compared to wildtype. However, as expected chc1, swa2, arf1Δ and sac1Δ take up more [3H]sertraline per unit time, in the order sac1Δ>chc1 = arf1Δ>swa2. Acute sertraline hyper-accumulation appears to have two different molecular bases, as indicated by the results of screening the mutants on a secondary phenotype of BAF-induced release of internalized [3H]sertraline (Fig. 4C–D).

thumbnailFigure 4. Altered kinetics of [3H]sertraline cellular uptake in sertraline-resistant (sertR) and sertraline-hypersensitive (sertHS) mutants.

(A) Kinetic analysis of [3H]sertraline cellular uptake after two minutes for sertR mutants (chc1, swa2 and vma9). (B) Kinetic analysis of [3H]sertraline cellular uptake after two minutes for sertHS mutants (arf1Δ, drs2Δ and sac1Δ). The concentrations of [3H]sertraline tested were 0.3125 nM, 0.625 nM, 1.563 nM, 3.125 nM, 6.25 nM, 12.5 nM, and 25 nM. Wildtype reference strains are plotted as black unfilled circles. Strains are identified by a fixed color scheme. Before and after treatments with bafilomycin A are shown for sertR (C) and sertHS (D) strains. Multi-hour growth curve for wildtype and thre sertraline-hypersensitive mutants. Growth in the absence (E) and in 20 µM sertraline (F) are shown. Statistical significance determined by two-tailed t-test: * P<0.05; ** P<0.01. Fits were generated by nonlinear regression. Error bars indicate SEM. Means were generated from three independent biological replicates.

doi:10.1371/journal.pone.0034024.g004

Among the sertR mutants, swa2 is more resistant to the effects of BAF than wildtype, while chc1 is unchanged compared to wildtype, indicating that one component of sertraline’s cellular membrane association may be complexly regulated by the rate of clathrin assembly and disassembly. The sac1Δ mutant appears to accumulate sertraline by a completely distinct mechanism. sac1Δ cells exhibit a near 20% reduction in the fraction of [3H]sertraline retained after inhibition of V-ATPase complexes compared to wildtype (Fig. 4D). This result suggests that another component of sertraline’s cellular membrane association may be regulated by the levels of the phosphoinositide PIP. Phosphoinositides have been shown to constitute anionic binding sites for CAD at the interfacial region of the bilayer in addition to the phosphate groups forming the backbone of glycerophospholipids [25]. We also observed that cell growth rate in the presence of overdose concentrations of sertraline is not correlated with the rate of nanomolar [3H]sertraline cellular uptake, as clearly demonstrated by a comparison between the sertHS mutants sac1Δ and drs2Δ (Fig. 4E–F). This result demonstrates that phospholipid asymmetry, as opposed to membrane anionicity per se, is a key component of the adaptive physiological response to chronic cellular membrane accumulation by sertraline, as was originally proposed by Huestis and colleagues in experiments on erythrocytes [26].

Ultrastructural analysis of autophagy induction and membrane quality control

The pharmacological and biochemical experiments described above localize sertraline to vesiculogenic membranes. We therefore performed an ultrastructural examination of organellar membrane homeostasis on yeast cells exposed to 60 µM sertraline for 45 minutes, which constitutes a sub-lethal chronic treatment (Fig. S1). If the bilayer couple model holds, then exogenous cationic amphipaths will favor the more negatively charged leaflet of organellar membranes. In the specific case of sertraline, we expected to find examples of damaging local curvature stress in membranes comprising the vesicular transport pathway. Initial support for this hypothesis was found in the observation that micromolar sertraline treatment has three noteworthy effects on vacuole homeostasis, which serves as a quantitative cell biological read out. First, we observed a decrease in the number vacuoles per cell in wildtype cells treated with 60 µM sertraline (Fig. 5A). The percentage of vacuole-less cells increases (36% vs. 12%) as does the percentage of cells containing one large consolidated vacuole (49% vs. 29%). In fact, loss of vacuoles was observed in all strains treated with 60 µM sertraline (Fig. 5B–H). Second, the steady-state distributions of vacuoles per cell are increased relative to wildtype in three mutants: chc1, swa2 and arf1Δ. Interestingly, the vacuoles of the sertHS mutant arf1Δ exhibits polygonal and tubulated morphologies (Fig. 5H), while the vacuoles of the sertR mutants chc1 and swa2 by and large exhibit wildtype vacuolar morphology. However, the vacuoles of chc1 and swa2 cells are more likely to have regions of thickened bilayer (Fig. 5D, inset). The seminal study by Lieber et al documented membrane thickening in erythrocytes bathed in the phenothiazine chlorpromazine [27]. Third, we observed a sharp increase in the percentage of electron-lucent wildtype vacuoles (24% vs 97%) (Fig. 5A, inset), and comparable increases were observed across all the strains (Fig. 5, red insets).

thumbnailFigure 5. Vacuolar number, morphology and contents assessed from transmission electron micrographs of untreated and sertraline-treated cells.

(A) BY4716; (B) vma9; (C) swa2; (D) chc1; (E) prototroph; (F) sac1Δ; (G) drs2Δ; (H) arf1Δ. For each strain, the distribution of vacuoles per cell is shown for a population of untreated cells (black columns) and 60 µM sertraline-treated cells (red columns). Insets contain a representative vacuole from the untreated (black outline) and 60 µM sertraline-treated (red outline) populations.

doi:10.1371/journal.pone.0034024.g005

We reasoned that the distribution of vacuoles per cell would allow us to test the hypothesis that sertraline hyper-accumulation is caused by an increase in the surface-area-to-volume ratio of vacuoles or other V-ATPase-acidified organelles of the vesicular transport pathway. The steady-state number of vacuoles per cell separates the four sertraline hyper-accumulating mutants into two unequal groups. Group One is arf1Δ, chc1, and swa2, three genes that interact physically and genetically [22]. Group Two is sac1Δ; sac1Δ cells accumulate the phosphoinositide PIP, which recruits and activates ARF1 and presumably stimulates vesiculogenesis. Changes in the number or surface-area-to-volume ratio of organelles may explain the hyper-accumulation phenotype of Group One mutants, but does not appear to explain the hyper-accumulation phenotype of sac1Δ cells, which have fewer vacuoles per cell at steady state. Also, the vacuolar membranes of sac1Δ cells appear to have an aberrant crenated morphology (Fig. 5F, inset).

The spike in electron lucency combined with a reduction in the total number of vacuoles per cell is accompanied by the appearance of double membrane-bound autophagosomes, a tell-tale sign of autophagy. A typical sertraline-treated wildtype cell after five minutes of exposure to 60 µM sertraline is illustrative (Fig. 6A). Changes in gross vacuolar morphology, electron lucency, and fine vacuolar membrane structure are typical of sertraline-treated cells at all time points, and a magnified view of a representative vacuole highlights the salient changes (Fig. 6B). The aberrant multilamellar membranous structure marked by an asterisk in Figure 6A is clearly encased in an autophagosome (Fig. 6C). Quantification of autophagosomes like those appearing in Figure 6C–D in wide-field images corroborates the single-cell snapshots: untreated wildtype cells contain on average 0.11 autophagosomes, while sertraline-treated cells contain 0.48, an increase of 4.2-fold. Multilamellar structures encapsulated by autophagosomes were observed in wildtype cells at all time points. Interestingly, similar membranous structures have been observed in brain tissues of rodents treated with the illicit psychoactive CAD 3,4-Methylenedioxymethamphetamine (MDMA) [28]. Two particularly striking examples are highlighted. In Figure 6D, black arrows mark the sites where two unilamellar vesicles, one of which contains a densely packed membranous whorl, are clearly discerned from the outer membrane of the autophagosome. And in Figure 6E, lamellar plumes of membrane form a Medusa-like structure trapped inside a large electron-lucent vacuole.

thumbnailFigure 6. A time course of sertraline treatment reveals early and persistent induction of autophagy and the appearance of aberrant multilamellar structures.

(A) Representative 60 µM sertraline-treated wildtype BY4716 cell after 5 minutes of sertraline treatment. Magnification is 6,300× (scale bar = 0.5 micron). The lone vacuole of the cell in (A) is shown at higher magnification (16,000×; scale bar = 0.1 micron) in panel (B). The black asterisk denotes an autophagosome that has encapsulated a membranous whorl, which is shown at higher magnification (25,000×; scale bar = 0.1 micron) in (C). The black arrows in (D) mark the telltale double membrane of an autophagosome at higher magnification (25,000×; scale bar = 0.1 micron). An elaborated multilamellar structure from a BY4716 cell treated with 60 µM sertraline for 20 minutes is shown at higher magnification (16,000×; scale bar = 0.1 micron) in (E).

doi:10.1371/journal.pone.0034024.g006

If autophagy is triggered by sertraline accumulating in organellar membranes, one would expect to observe ultrastructural evidence of membrane curvature stress throughout the vesicular transport pathway, not just in vacuolar membranes. In this instance, a representative wildtype cell treated with 60 µM sertraline for 45 minutes is illustrative (Fig. 7A). The structure marked by an asterisk is a shown at higher magnification to be a dilated cisterna, possibly a Golgi stack or an autophagosomal precursor, with circular or crescent morphology (Fig. 7B). Similar structures were observed in other sertraline-treated cells at this time point, including an example of a dilated cisterna with clearly thickened membranes on the convex surface. (Fig. 7C). Comparison of an untreated arf1Δ cell to a sertraline-treated arf1Δ cell reveals several localized exaggerated regions of membrane expansion in comparable organellar structures (Fig. 7D–E).

thumbnailFigure 7. Ultrastructural evidence of local membrane curvature stress in sertraline-treated cells.

(A) Representative 60 µM sertraline-treated wildtype BY4716 cell at 8,000× (scale bar = 0.5 micron). Asterisk denotes circularized Golgi-like structure, which is shown at higher magnification (31,500×; scale bar = 0.1 micron) in (B). An enlarged Golgi-like cisterna with visible expansion of the convex membrane from a 60 µM sertraline-treated wildtype prototroph cell at high magnification (40,000×; scale bar = 0.1 micron) is shown in (C). An irregular organelle with normal membrane thickness from a typical untreated arf1Δ cell at 8,000× (scale bar = 0.5 micron) is shown in (D). A comparable structure exhibiting localized membrane expansion in a 60 µM sertraline-treated arf1Δ cell at 8,000× (scale bar = 0.5 micron) is shown in (E).

doi:10.1371/journal.pone.0034024.g007

Finally, two mutants, chc1 and swa2, exhibited a unique ultrastructural phenotype that may be involved in adaptive cellular pathways that degrade and regenerate cellular membranes infiltrated by exogenous cationic amphipaths. At steady state, chc1 and swa2 mutants exhibit a vacuolar expansion/fragmentation phenotype that is “normalized” after 60 µM sertraline treatment for 45 minutes insofar as the number of vacuoles per cell decreases (Fig. 5C–D). However, these vacuoles exhibit a non-random distribution of osmiophilic lumenal contents and appear to contain undigested vesicular compartments (Fig. 8B). Interesting, the number of lipid droplets, which are storage depots for neutral lipids (e.g., triglycerides) and marked by red asterisks (Fig. 8B), increased after sertraline treatment in wildtype and sertR and sertHS mutant strains by an unknown mechanism (Fig. 8C). This increase is most pronounced in sertraline-treated chc1 and swa2 cells; untreated chc1 cells have a mean lipid droplet count equal to 0.32, while sertraline-treated chc1 cells have a mean lipid droplet count equal to 2.2. By contrast, untreated wildtype cells have a mean lipid droplet count equal to 0.31, while sertraline-treated wildtype cells have a lipid droplet count equal to 0.55. One explanation for this result is that lipid droplets form during adaptation of yeast cells to secretory pathway stress, as has been suggested by others [29].

thumbnailFigure 8. The chc1 mutant exhibits unique adaptation to chronic sertraline exposure.

(A) A representative untreated chc1 cell contains five electron-dense vacuoles (10,000×; scale bar = 0.5 micron). (B) A representative sertraline-treated chc1 cell contains a single vacuole and four lipid droplets marked by red asterisks (10,000×; scale bar = 0.5 micron). (C) Quantification of lipid droplet formation in untreated and sertraline-treated cells. The “-“ column correspond to lipid droplet counts performed on untreated cells. The “+” column correspond to lipid droplet counts performed on cells treated with 60 µM sertraline for 45 minutes. The strain identities are: BY4716 (black); chc1 (red); swa2 (green); vma9 (magenta); arf1Δ (orange); drs2Δ (yellow); sac1Δ (blue).

doi:10.1371/journal.pone.0034024.g008

Discussion Top

We presented evidence that supports an evolutionarily informed, cell biological explanatory model of cellular membrane accumulation by sertraline in a simple eukaryote with an intact secretory pathway. Explication of our model begins with the passive internalization of neutral sertraline molecules at the plasma membrane. Lysosomotropism acts as an amplifier of simple diffusion, and suggests a mechanism whereby sertraline may distribute non-uniformly throughout vertebrate tissues as a result of tissue-specific activity or regulation of V-ATPase-dependent acidification [30]. The internalization of sertraline appears to follow an entry route that is orthogonal to ATP-dependent, SEC gene-requiring internalization of larger aryl cationic amphipaths (e.g., lysophospholipids), i.e., does not depend on the endocytic pathway [31]. However, passively internalized sertraline appears to be a potent substrate for ATP-dependent efflux pumps. In fact, efflux may explain the paradoxical observation that at physiological pH, sertraline is predicted to be over 99% ionized yet only a minority fraction (10–15%) is observed to be soluble at equilibrium. Our interpretation is that the ionized pool of sertraline is actively depleted both by ATP-dependent efflux and by sequestration in cellular membranes as a neutral species.

Despite our best efforts to separate vesiculogenic membranes into discrete organelles by density-gradient centrifugation, parsimony dictates that sertraline asymmetrically accumulates in the membranes of all V-ATPase-acidified organelles, presumably in proportion to local lysosomotropic driving forces. Specifically, sertraline associates with organellar membranes by two mechanisms: adsorption to solvent-exposed anionic sites, and intercalation into the bulk hydrophobic phase of the bilayer. A two-state, weakly binding and strongly binding model has been proposed for local anesthetic association with reconstituted liposomes [32], and we argue that adsorptive binding by sertraline in yeast cells may be mediated by electrostatic interactions, while intercalative binding by sertraline in yeast cells may be mediated by lipophilic interactions. However, several uncertainties remain in part because in vivo experiments on CAD in living cells are unlike experiments on CAD in reconstituted liposomes, in which the ionization state of CAD can be experimentally manipulated. Therefore, we conclude that sertraline-membrane association is a composite of more than one binding interaction, a conclusion supported by molecular dynamic simulations [33], [34].

The central finding of our study is that at micromolar doses, cellular membrane accumulation by sertraline induced curvature stresses throughout the organelles of the vesicular transport pathway. This membrane curvature stress triggers an autophagy-dependent membrane quality control response that appears to be enhanced in mutants with altered clathrin function. Although we have not provided biochemical evidence of autophagy induction, our ultrastructural approach revealed unambiguous induction of autophagy. We argue that autophagy mitigates cationic amphipath accumulation in cellular membranes. This interpretation is supported by a recent study that documented induction of autophagy by antidepressants in mammalian neuronal cell lines [35]. Autophagy may be one of several buffering systems that evolved to preserve cellular membrane homeostasis in the face of endogenous (or exogenous) charged amphipath accumulation, but when sertraline-induced membrane curvature stresses become too punishing at high or sustained doses, cytotoxicity ensues [36]. However, the cell-physiological effects of sub-lethal doses of sertraline may not be deleterious. We previously showed that low micromolar sertraline partially rescues the constitutive growth defect of yeast mutants with altered clathrin function [12]. If these buffering systems are defective due to genetic (e.g., clathrin dysregulation) and/or environmental stressors – resulting in a maladaptive homeostatic set point – sub-lethal accumulation of amphipath may normalize this set point. A “trophic” or cytoprotective effect might ensue given the ancient coupling between membrane transport and cell growth [37]. A similar argument was proffered by researchers explaining the cytotoxic effects of the phenothiazine antipsychotic drug chlorpromazine in yeast cells [13]. In conclusion, our model supports the notion of a serotonin transporter-independent component of antidepressant pharmacology in humans, and appears to buttress the neurotrophic hypothesis of depression [38]. It is tempting to speculate that amphipath accumulation may, in specific contexts, provide a trophic signal through direct modulation of the physical properties of cellular membranes, perhaps resulting in vesicle formation, thereby exploiting (or “short-circuiting”) the aforementioned coupling between membrane transport and cell growth. Such a mechanism would be expected to unfold over long time scales given the vigilance of those buffering systems, and the large effective volume of cellular membranes of the mammalian brain.

Materials and Methods Top

Yeast strains and culture conditions

Standard growth conditions were YPD media (1% yeast extract, 2% peptone and 2% dextrose) buffered with 10 mM HEPES to the desired pH. Some experiments with prototrophic strains were carried out in HEPES-buffered minimal media (yeast nitrogen base containing ammonium sulfate, 2% dextrose). BY4716, BY4742 or ACY769 were employed as wildtype reference strains as appropriate, and are all derived from S288c. sertR mutant strains were previously described [12]. sertHS deletion strains were derived from ACY769 and belong to a prototrophic homozygous deletion collection generated by D. Hess (Santa Clara University) and A. Caudy (University of Toronto). The dnf1Δ dnf2Δ dnf3Δ mutant was kindly provided by T. Graham (Vanderbilt). The sec18ts mutant was kindly provided by W. Prinz (NIH).

Chemical compounds

Sertraline hydrochloride (Sigma-Aldrich) was resuspended in dimethyl sulfoxide (DMSO) to a final concentration of 25 mg/mL (~73 mM), and 100 µL aliquots were stored in glass vials at −20°C until use and subjected to a maximum of one freeze/thaw cycle. Sertraline [N-methyl-3H] hydrochloride (American Radiolabled Chemicals, Inc.) is 99% pure by HPLC, has a specific activity of 80 Ci/mmol, and was kept at a stock concentration of 1 mCi/mL in ethanol.

Pharmacological/biochemical assays and analysis

Overnight cultures were diluted in fresh pH-buffered YPD medium and incubated at 30°C till log phase. Cell number and cell size were determined by the Coulter counter method. For uptake and accumulation experiments, 0.25 µCi 3H-sertraline (American Radiolabeled Chemicals) was added to 2 mL yeast culture aliquot (all 0-minute time point aliquots contained 1 µM sodium azide and sodium fluoride). Cells were collected on Durapore* PVDF filters (Millipore) by passing through a filter unit, and washed with 18 ml ice cold pH-buffered YPD. Filters were incubated with 300 µL cold lysis buffer (acetonitrile:methanol:water, 40:40:20) at −20°C for 10 minutes. Cell pellets were collected by pipetting and transferred to scintillation vials containing 4 mL Cytoscint fluid (Fisher Scientific, Inc.). Counts were obtained using a Perkin Elmer Tri-Carb 2800TR liquid scintillation analyzer. For kinetic experiments, cells were incubated with specified concentrations of [3H]sertraline for 2 minutes, then collected on filters and washed with ice cold YPD containing 10 µM unlabeled sertraline [39]. Subcellular fractionation was carried out as follows. Cells were sphereplasted following Zymolyase (Zymo Research) digestion at 30°C for 1 hour according to manufacturers protocol. Spheroplasts were osmotically lysed in lysis buffer (50 mM Tris-HCl, 0.8 M sorbitol). The resulting lysate was centrifuged at 400 rcf for 10 minutes, yielding a post-nuclear lysate. An initial spin at 10,000 rcf for 10 minutes yielded P10,000 (enriched in large organellar membranes) and S10,000 fractions; S10,000 was spun at 100,000 rcf for 50 minutes, yielding a microsomal (P100,000) and a true soluble fraction (S100,000). All pellets were resuspended in equal volumes of the same buffer as the corresponding supernatant fractions. For experiments involving density-gradient centrifugation, the 10,000 rcf pellet obtained during fractionation was resuspended in 1 mL 35% Optiprep solution (Sigma Aldrich), on top of which 1 mL 30% Optiprep solution and 1 mL base solution (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 8% sucrose) were layered consecutively. The gradient was centrifuged at 61,000 rpm for 17 hours and 45 minutes using a TLA100.3 rotor (Beckman). Ten equal fractions were collected from each gradient. Optiprep gradient fractions were pelleted with TCA, washed twice with ice-cold acetone, and resuspended in 30 µL Nu-PAGE SDS sample buffer (Invitrogen). Primary antibodies were used at the following dilutions: the late-Golgi marker Vsp10p 1:250 (A-21274, Invitrogen); the vacuolar membrane marker ALP 1:500 (A-6458, Invitrogen); the endoplasmic reticulum marker Dpm1p 1:2000 (A-6429, Invitrogen); the endosomal marker Pep12p 1:2000 (A-21273, Invitrogen); the mitochondrial marker Porin 1:2000 (A-6449, Invitrogen); carboxypeptidase Y (ab113685, Abcam); and the plasma membrane marker Pma1p 1:10,000 (ab4645, Abcam). For immunoblotting, total protein was transferred to PVDF membrane after electrophoresis using the iBlot system (Invitrogen). PVDF membranes were washed with SNAP I.D. Protein Detection System (Millipore), and chemiluminescence was detected using ECL plus Western Blotting Substrate (Pierce). Analysis of pharmacological data and figure generation were performed using Prism 5 (GraphPad Software, Inc).

Transmission electron microscopy and analysis

We based our protocol on a membrane-preserving procedure previously described [40]. Our step-by-step protocol is available as Methods S1. 10 mL of fresh YPD media were inoculated with cells from an overnight culture and allowed to double several times to a maximum OD600 of ~0.5. For drug treatments, cells were treated with 60 µM sertraline for 5, 10, 20 and 45 minutes. Multi-cell and single-cell fields were collected. Quantification of ultrastructural phenotypes was performed on a dataset compiled from wide fields as previously described [12]. Wide-field cell counts for the 45-minute dataset were as follows: BY4716 (untreated n = 216, sertraline-treated n = 193); prototroph (untreated n = 247, sertraline-treated n = 184); chc1 (untreated n = 234, sertraline-treated n = 202); swa2 (untreated n = 165, sertraline-treated n = 168); vma9 (untreated n = 212, sertraline-treated n = 166); sac1Δ (untreated n = 219, sertraline-treated n = 151); drs2Δ (untreated n = 220, sertraline-treated n = 158); arf1Δ (untreated n = 194, sertraline-treated n = 133).

Supporting Information Top

Figure S1.

Growth rate of BY4716 cells as a function of sertraline concentration. Cells at optical density (OD600) equal to 1.0 were used as the initial inoculum.

(TIFF)

Methods S1.

Detailed protocol describing “ROTO” (Reduced Osmium tetroxide Thiocarbohydrazide – reduced Osmium) transmission electron microscopy.

(DOCX)

Acknowledgments Top

We thank Ben de Bivort and David Spiegel for general comments on the manuscript. We thank Christina DeCoste for technical assistance with cell sorting experiments. We thank Woo Jung Cho for technical assistance with electron microscopy experiments.

Author Contributions Top

Conceived and designed the experiments: EOP. Performed the experiments: JC DK. Analyzed the data: EP JC DK. Contributed reagents/materials/analysis tools: SL. Wrote the paper: EOP.

References Top

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