Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.

Introduction to the Theme "Novel Therapeutics with the Potential to Advance Health Care".

The reviews in Volume 65 of the Annual Review of Pharmacology and Toxicology cover a wide variety of topics in pharmacology and toxicology focused upon the pathway from preclinical studies to clinical trials. Many of these reviews discuss the identification and validation of new therapeutic targets and/or novel therapeutic approaches. Examples include reviews that focus on the treatment of obesity, neuropsychiatric disorders, Parkinson's disease, substance use disorders, liver fibrosis, cardiac arrythmias, chronic intestinal inflammation, prostate cancer, immuno-oncology, sickle cell disease, and snakebite envenoming. Other topics include drug discovery of biologics, microphysiological systems, and human induced pluripotent stem cell-derived organoids and organ-on-chip technology integrated with artificial intelligence methodologies. Together, these and other reviews give new insights into the assessment of aspects of toxicology and provide readers a glimpse of advances in pharmacology and toxicology that we believe will advance health care and environmental safety.

Introduction to the Theme "Pharmacological Individuality: New Insights and Strategies for Personalized and Precise Drug Treatment".

The reviews in Volume 64 of the Annual Review of Pharmacology and Toxicology cover diverse topics. A common theme in many of the reviews is the interindividual variability in the clinical response to drugs. Highlighted areas include emerging developments in pharmacogenomics that can predict the personal risk for drug inefficacy and/or adverse drug reactions. Other reviews focus on the use of circulating biomarkers to define drug metabolism phenotypes and the effect of circadian regulation on drug response. Another emerging technology, digital twins that model individual patients, is used to generate computational simulations of drug effects and identify optimal personalized treatments. Another variable that may affect clinical outcomes, the nocebo response (an adverse reaction to a placebo), complicates clinical trials. These reviews further document that pharmacological individuality is an essential component of the concepts of personalized medicine and precision medicine and will likely have an important impact on patient care.

Introduction to the Theme "Development of New Drugs: Moving from the Bench to Bedside and Improved Patient Care".

Investigations in pharmacology and toxicology range from molecular studies to clinical care. Studies in basic and clinical pharmacology and in preclinical and clinical toxicology are all essential in bringing new knowledge and new drugs into clinical use. The 30 reviews in Volume 63 of the Annual Review of Pharmacology and Toxicology explore topics across this spectrum. Examples include "Zebrafish as a Mainstream Model for In Vivo Systems Pharmacology and Toxicology" and "Artificial Intelligence and Machine Learning for Lead-to-Candidate Decision-Making and Beyond." Other reviews discuss components important for drug discovery and development and the use of pharmaceuticals in a variety of diseases. Air pollution continues to increase globally; accordingly, "Air Pollution-Related Neurotoxicity Across the Life Span" is a timely and forward-thinking review. Volume 63 also explores the use of contemporary technologies such as electronic health records, pharmacogenetics, and new drug delivery systems that help enhance and improve the utility of new therapies.

Introduction to the Theme "New Insights, Strategies, and Therapeutics for Common Diseases".

The reviews in Volume 62 of the Annual Review of Pharmacology and Toxicology (ARPT) cover a diverse range of topics. A theme that encompasses many of these reviews is their relevance to common diseases and disorders, including type 2 diabetes, heart failure, cancer, tuberculosis, Alzheimer's disease, neurodegenerative disorders, and Down syndrome. Other reviews highlight important aspects of therapeutics, including placebos and patient-centric approaches to drug formulation. The reviews with this thematic focus, as well as other reviews in this volume, emphasize new mechanistic insights, experimental and therapeutic strategies, and novel insights regarding topics in the disciplines of pharmacology and toxicology. As the editors of ARPT, we believe that these reviews help advance those disciplines and, even more importantly, have the potential to improve the health care of the world's population.

Introduction to the Theme "Old and New Toxicology: Interfaces with Pharmacology".

The theme of Volume 61 is "Old and New Toxicology: Interfaces with Pharmacology." Old toxicology is exemplified by the authors of the autobiographical articles: B.M. Olivera's work on toxins and venoms from cone snails and P. Taylor's studies of acetylcholinesterase and the nicotinic cholinergic receptor, which serve as sites of action for numerous pesticides and venoms. Other articles in this volume focus on new understanding and new types of toxicology, including (a) arsenic toxicity, which is an ancient poison that, through evolution, has caused most multicellular organisms to express an active arsenic methyltransferase to methylate arsenite, which accelerates the excretion of arsenic from the body; (b) small molecules that react with lipid dicarbonyls, which are now considered the most toxic oxidative stress end products; (c) immune checkpoint inhibitors (ICIs), which have revolutionized cancer therapy but have numerous immune-related adverse events, including cardiovascular complications; (d) autoimmunity caused by the environment; (e) idiosyncratic drug-induced liver disease, which together with the toxicity of ICIs represents new toxicology interfacing with pharmacology; and (f) sex differences in the development of cardiovascular disease, with men more susceptible than women to vascular inflammation that initiates and perpetuates disease. These articles and others in Volume 61 reflect the interface and close integration of pharmacology and toxicology that began long ago but continues today.

Introduction to the Theme "New Therapeutic Targets".

"New Therapeutic Targets" is the theme of articles in the Annual Review of Pharmacology and Toxicology, Volume 59. Reviews in this volume discuss targets for a variety of conditions in need of new therapies, including type 2 diabetes, heart failure with preserved ejection fraction, obesity, thyroid-associated ophthalmopathy, tinnitus, multiple sclerosis, Parkinson's disease and other neurodegenerative diseases, pain, depression, post-traumatic stress disorder, muscle wasting diseases, cancer, and anemia associated with chronic renal disease. Numerous articles in this volume focus on the identification, validation, and utility of novel therapeutic targets, in particular, ones that involve new or unexpected molecular entities. This theme complements several previous themes, including "New Approaches for Studying Drug and Toxicant Action: Applications to Drug Discovery and Development," "Precision Medicine and Prediction in Pharmacology," and "New Methods and Novel Therapeutic Approaches in Pharmacology and Toxicology."

Amphetamines signal through intracellular TAAR1 receptors coupled to Gα13 and GαS in discrete subcellular domains.

The extensive use of amphetamines to treat attention deficit hyperactivity disorders in children provides a compelling rationale for understanding the mechanisms of action of amphetamines and amphetamine-related drugs. We have previously shown that acute amphetamine (AMPH) regulates the trafficking of both dopamine and glutamate transporters in dopamine neurons by increasing activation of the small GTPase RhoA and of protein kinase A. Here we demonstrate that these downstream signaling events depend upon the direct activation of a trace amine-associated receptor, TAAR1, an intracellular G-protein coupled receptor (GPCR) that can be activated by amphetamines, trace amines, and biogenic amine metabolites. Using cell lines and mouse lines in which TAAR1 expression has been disrupted, we demonstrate that TAAR1 mediates the effects of AMPH on both RhoA and cAMP signaling. Inhibition of different Gα signaling pathways in cell lines and in vivo using small cell-permeable peptides confirms that the endogenous intracellular TAAR1 couples to G13 and to GS α-subunits to increase RhoA and PKA activity, respectively. Results from experiments with RhoA- and PKA-FRET sensors targeted to different subcellular compartments indicate that AMPH-elicited PKA activation occurs throughout the cell, whereas G13-mediated RhoA activation is concentrated near the endoplasmic reticulum. These observations define TAAR1 as an obligate intracellular target for amphetamines in dopamine neurons and support a model in which distinct pools of TAAR1 mediate the activation of signaling pathways in different compartments to regulate excitatory and dopaminergic neurotransmission.

Introduction to the Theme "New Approaches for Studying Drug and Toxicant Action: Applications to Drug Discovery and Development".

The theme "New Approaches for Studying Drug and Toxicant Action: Applications to Drug Discovery and Development" links 13 articles in this volume of the Annual Review of Pharmacology and Toxicology (ARPT). The engaging prefatory articles by Arthur Cho and Robert Lefkowitz set the stage for this theme and for the reviews that insightfully describe new approaches that advance research and discovery in pharmacology and toxicology. Examples include the progress being made in developing Organs-on-Chips/microphysiological systems and human induced pluripotent stem cell-derived cells to aid in understanding cell and tissue pharmacokinetics, action, and toxicity; the recognition of the importance of circadian rhythm, the microbiome, and epigenetics in drug and toxicant responses; and the application of results from new types of patient-derived information to create personalized/precision medicine, including therapeutics for pain, which may perhaps provide help in dealing with the opioid epidemic in the United States. Such new information energizes discovery efforts in pharmacology and toxicology that seek to improve the efficacy and safety of drugs in patients and to minimize the consequences of exposure to toxins.

Introduction to the Theme "New Methods and Novel Therapeutic Approaches in Pharmacology and Toxicology".

Major advances in scientific discovery and insights can result from the development and use of new techniques, as exemplified by the work of Solomon Snyder, who writes a prefatory article in this volume. The Editors have chosen "New Methods and Novel Therapeutic Approaches in Pharmacology and Toxicology" as the Theme for a number of articles in this volume. These include ones that review the development and use of new experimental tools and approaches (e.g., nanobodies and techniques to explore protein-protein interactions), new types of therapeutics (e.g., aptamers and antisense oligonucleotides), and systems pharmacology, which assembles (big) data derived from omics studies together with information regarding drugs and patients. The application of these new methods and therapeutic approaches has the potential to have a major impact on basic and clinical research in pharmacology and toxicology as well as on patient care.

Amphetamine Stimulates Endocytosis of the Norepinephrine and Neuronal Glutamate Transporters in Cultured Locus Coeruleus Neurons.

Amphetamines and amphetamine-derivatives elevate neurotransmitter concentrations by competing with endogenous biogenic amines for reuptake. In addition, AMPHs have been shown to activate endocytosis of the dopamine transporter (DAT) which further elevates extracellular dopamine (DA). We previously found that the biochemical cascade leading to this cellular process involves entry of AMPH into the cell through the DAT, stimulation of an intracellular trace amine-associated receptor, TAAR1, and activation of the small GTPase, RhoA. We also showed that the neuronal glutamate transporter, EAAT3, undergoes endocytosis via the same cascade in DA neurons, leading to potentiation of glutamatergic inputs. Since AMPH is a transported inhibitor of both DAT and the norepinephrine transporter (NET), and EAAT3 is also expressed in norepinephrine (NE) neurons, we explored the possibility that this signaling cascade occurs in NE neurons. We found that AMPH can cause endocytosis of NET as well as EAAT3 in NE neurons. NET endocytosis is dependent on TAAR1, RhoA, intracellular calcium and CaMKII activation, similar to DAT. However, EAAT3 endocytosis is similar in all regards except its dependence upon CaMKII activation. RhoA activation is dependent on calcium, but not CaMKII, explaining a divergence in AMPH-mediated endocytosis of DAT and NET from that of EAAT3. These data indicate that AMPHs and other TAAR1 agonists can affect glutamate signaling through internalization of EAAT3 in NE as well as DA neurons.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.

A call for transparent reporting to optimize the predictive value of preclinical research.

The US National Institute of Neurological Disorders and Stroke convened major stakeholders in June 2012 to discuss how to improve the methodological reporting of animal studies in grant applications and publications. The main workshop recommendation is that at a minimum studies should report on sample-size estimation, whether and how animals were randomized, whether investigators were blind to the treatment, and the handling of data. We recognize that achieving a meaningful improvement in the quality of reporting will require a concerted effort by investigators, reviewers, funding agencies and journal editors. Requiring better reporting of animal studies will raise awareness of the importance of rigorous study design to accelerate scientific progress.

Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine.

Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH's effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action.

Differential regulation of two isoforms of the glial glutamate transporter EAAT2 by DLG1 and CaMKII.

The gene for EAAT2, the major astrocytic glutamate transporter, generates two carrier isoforms (EAAT2a and EAAT2b) that vary at their C termini as a consequence of alternative RNA splicing. The EAAT2b cytoplasmic C terminus contains a postsynaptic density-95/Discs large/zona occludens-1 (PDZ) ligand, which is absent in EAAT2a. To understand how the distinct C termini might affect transporter trafficking and surface localization, we generated Madin-Darby canine kidney (MDCK) cells that stably express EGFP-EAAT2a or EGFP-EAAT2b and found robust basolateral membrane expression of the EAAT2b isoform. In contrast, EAAT2a displayed a predominant distribution within intracellular vesicle compartments, constitutively cycling to and from the membrane. Addition of the PDZ ligand to EAAT2a as well as its deletion from EAAT2b confirmed the importance of the motif for cell-surface localization. Using EAAT2 constructs with an extracellular biotin acceptor tag to directly assess surface proteins, we observed significant PDZ ligand-dependent EAAT2b surface expression in cultured astrocytes, consistent with observations in cell lines. Discs large homolog 1 (DLG1; SAP97), a PDZ protein prominent in both astrocytes and MDCK cells, colocalized and coimmunoprecipitated with EAAT2b. shRNA knockdown of DLG1 expression decreased surface EAAT2b in both MDCK cells and cultured astrocytes, suggesting that the DLG scaffolding protein stabilizes EAAT2b at the surface. DLG1 can be phosphorylated by Ca(2+)/calmodulin-dependent protein kinase (CaMKII), resulting in disruption of its PDZ-mediated interaction. In murine astrocytes and acute brain slices, activation of CaMKII decreases EAAT2b surface expression but does not alter the distribution of EAAT2a. These data indicate that the surface expression and function of EAAT2b can be rapidly modulated through the disruption of its interaction with DLG1 by CaMKII activation.

A Mutation in Transmembrane Domain 7 (TM7) of Excitatory Amino Acid Transporters Disrupts the Substrate-dependent Gating of the Intrinsic Anion Conductance and Drives the Channel into a Constitutively Open State.

In the mammalian central nervous system, excitatory amino acid transporters (EAATs) are responsible for the clearance of glutamate after synaptic release. This energetically demanding activity is crucial for precise neuronal communication and for maintaining extracellular glutamate concentrations below neurotoxic levels. In addition to their ability to recapture glutamate from the extracellular space, EAATs exhibit a sodium- and glutamate-gated anion conductance. Here we show that substitution of a conserved positively charged residue (Arg-388, hEAAT1) in transmembrane domain 7 with a negatively charged amino acid eliminates the ability of glutamate to further activate the anion conductance. When expressed in oocytes, R388D or R388E mutants show large anion currents that display no further increase in amplitude after application of saturating concentrations of Na(+) and glutamate. They also show a substantially reduced transport activity. The mutant transporters appear to exist preferentially in a sodium- and glutamate-independent constitutive open channel state that rarely transitions to complete the transport cycle. In addition, the accessibility of cytoplasmic residues to membrane-permeant modifying reagents supports the idea that this substrate-independent open state correlates with an intermediate outward facing conformation of the transporter. Our data provide additional insights into the mechanism by which substrates gate the anion conductance in EAATs and suggest that in EAAT1, Arg-388 is a critical element for the structural coupling between the substrate translocation and the gating mechanisms of the EAAT-associated anion channel.

Neuroscience in recession?

As the global financial downturn continues, its impact on neuroscientists - both on an individual level and at the level of their research institute - becomes increasingly apparent. How is the economic crisis affecting neuroscience funding, career prospects, international collaborations and scientists' morale in different parts of the world? Nature Reviews Neuroscience gauged the opinions of a number of leading neuroscientists: the President of the Society for Neuroscience, the President Elect of the British Neuroscience Association, the former President of the Japan Neuroscience Society, the President of the Federation of European Neuroscience Societies and the Director of the US National Institute of Mental Health. Their responses provide interesting and important insights into the regional impact of the global financial downturn, with some causes for optimism for the future of neuroscience research.

Differential targeting of the dopamine transporter to recycling or degradative pathways during amphetamine- or PKC-regulated endocytosis in dopamine neurons.

The dopamine transporter (DAT) clears the extracellular dopamine released during neurotransmission and is a major target for both therapeutic and addictive psychostimulant amphetamines. Amphetamine exposure or activation of protein kinase C (PKC) by the phorbol ester PMA has been shown to down-regulate cell surface DAT. However, in dopamine neurons, the trafficking itinerary and fate of internalized DAT has not been elucidated. By monitoring surface-labeled DAT in transfected dopamine neurons from embryonic rat mesencephalic cultures, we find distinct sorting and fates of internalized DAT after amphetamine or PMA treatment. Although both drugs promote DAT internalization above constitutive endocytosis in dopamine neurons, PMA induces ubiquitination of DAT and leads to accumulation of DAT on LAMP1-positive endosomes. In contrast, after amphetamine exposure DAT is sorted to recycling endosomes positive for Rab11 and the transferrin receptor. Furthermore, quantitative assessment of DAT recycling using an antibody-feeding assay reveals that significantly less DAT returns to the surface of dopamine neurons after internalization by PMA, compared with vehicle or amphetamine treatment. These results demonstrate that, in neurons, the DAT is sorted differentially to recycling and degradative pathways after psychostimulant exposure or PKC activation, which may allow for either the transient or sustained inhibition of DAT during dopamine neurotransmission.

Large collective motions regulate the functional properties of glutamate transporter trimers.

Glutamate transporters clear synaptically released glutamate to maintain precise communication between neurons and limit glutamate neurotoxicity. Although much progress has been made on the topology, structure, and function of these carriers, few studies have addressed large-scale structural motions collectively associated with substrate transport. Here we show that a series of single cysteine substitutions in the helical hairpin HP2 of excitatory amino acid transporter 1 form intersubunit disulfide cross-links within the trimer. After cross-linking, substrate uptake, but not substrate-activated anion conductance, is completely inhibited in these mutants. These disulfide bridges link residue pairs > 40 Å apart in the outward-facing crystal structure, and can be explained by concerted subunit movements predicted by the anisotropic network model (ANM). The existence of these global motions is further supported by the observation that single cysteine substitutions at the extracellular part of the transmembrane domain 8 can also be cross-linked by copper phenanthroline as predicted by the ANM. Interestingly, the transport domain in the un-cross-linked subunit of the trimer assumes an inward-facing orientation, suggesting that individual subunits potentially undergo separate transitions between outward- and inward-facing forms, rather than an all-or-none transition of the three subunits, a mechanism also supported by ANM-predicted intrinsic dynamics. These results shed light on how large collective motions contribute to the functional dynamics of glutamate transporters.