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International audience; Bacillus Calmette-Guerin (BCG) remains the only FDA-approved first-line therapy in patients with high-risk non-muscle invasive bladder cancer. Recurrences, even after adequate BCG therapy, are common and the efficacy of second-line therapies remains modest. Therefore, early identification of patients likely to recur and treatment after recurrence remain critical unmet needs in the clinical care of bladder cancer patients. To address these deficits, a better understanding of the mechanisms of resistance to BCG-therapy is needed. The virtual update of the International Bladder Cancer Network (IBCN) on the biology of response to BCG focused on potential mechanisms and markers of resistance to intravesical BCG therapy. The insights from this meeting will be highlighted and put into context of previously reported mechanisms of resistance to BCG in this review.

Background and purpose Cannabidiol (CBD) has been shown to differentially regulate the mechanistic target of rapamycin complex 1 (mTORC1) in preclinical models of disease, where it reduces activity in models of epilepsies and cancer and increases it in models of multiple sclerosis (MS) and psychosis. Here we investigate the effects of phytocannabinoids on mTORC1 and define a molecular mechanism. Experimental approach A novel mechanism for phytocannabinoids was identified using the tractable model system, Dictyostelium discoideum. Using mouse embryonic fibroblasts, we further validate this new mechanism of action. We demonstrate clinical relevance using cells derived from healthy individuals and from people with MS (pwMS). Key results Both CBD and the more abundant cannabigerol (CBG) enhance mTORC1 activity in D. discoideum. We identify a mechanism for this effect involving inositol polyphosphate multikinase (IPMK), where elevated IPMK expression inverses the response to phytocannabinoids, decreasing mTORC1 activity upon treatment, providing new insight on phytocannabinoids' actions. We further validated this mechanism using mouse embryonic fibroblasts. Clinical relevance of this effect was shown in primary human peripheral blood mononuclear cells, where CBD and CBG treatment increased mTORC1 activity in cells derived from healthy individuals and decreased mTORC1 activity in cells derived from pwMS. Conclusion and implications Our findings suggest that both CBD and the abundant CBG differentially regulate mTORC1 signalling through a mechanism dependent on the activity of the upstream IPMK signalling pathway, with potential relevance to the treatment of mTOR-related disorders, including MS.

The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system.

Ketogenic diets have been utilized for many years to improve health, and as a dietary approach for the treatment of a range of diseases, where the mechanism of these low carbohydrate and high fat diets is widely considered to be through the production of metabolic products of fat breakdown, called ketones. One of these diets, the medium chain triglyceride ketogenic diet, involves high fat dietary intake in the form of medium chain fatty acids (MCFAs), decanoic and octanoic acid, and is commonly used in endurance and high intensity exercises but has also demonstrated beneficial effects in the treatment of numerous pathologies including drug resistant epilepsy, cancer, and diabetes. Recent advances, using Dictyostelium discoideum as a model, have controversially proposed several direct molecular mechanisms for decanoic acid in this diet, independent of ketone generation. Studies in this model have identified that decanoic acid reduces phosphoinositide turnover, diacylglycerol kinase (DGK) activity, and also inhibits the mechanistic target of rapamycin complex 1 (mTORC1). These discoveries could potentially impact the treatment of a range of disorders including epilepsy, cancer and bipolar disorder. In this review, we summarize the newly proposed mechanisms for decanoic acid, identified using D. discoideum, and highlight potential roles in health and disease treatment.

Mutations in the γ-secretase complex are strongly associated with familial Alzheimer disease. Both proteolytic and non-proteolytic functions for the γ-secretase complex have been previously...

Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O(2) (pO(2)) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO(2) for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O(2)-micro-sensor created by adsorbing O(2)-sensitive and O(2)-insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O(2), the microparticles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 micron) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO(2) and micro-gradients of pO(2) without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO(2) and intracellular events can be imaged simultaneously. In addition, the response-time (t(0.5) = 0.7 s, 0 - 160 mm Hg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO(2) while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨ(m)); and (2) OxySplots measuring pO(2) during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca(2+)](i) levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO(2) with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously.

This prospective open-label feasibility study aimed to evaluate acceptability, tolerability and compliance with dietary intervention with K.Vita, a medical food containing a unique ratio of decanoic acid to octanoic acid, in individuals with drug-resistant epilepsy. Adults and children aged 3–18 years with drug-resistant epilepsy took K.Vita daily whilst limiting high-refined sugar food and beverages. K.Vita was introduced incrementally with the aim of achieving ≤35% energy requirements for children or 240 ml for adults. Primary outcome measures were assessed by study completion, participant diary, acceptability questionnaire and K.Vita intake. Reduction in seizures or paroxysmal events was a secondary outcome. 23/35 (66%) children and 18/26 (69%) adults completed the study; completion rates were higher when K.Vita was introduced more gradually. Gastrointestinal disturbances were the primary reason for discontinuation, but symptoms were similar to those reported from ketogenic diets and incidence decreased over time. At least three-quarters of participants/caregivers reported favourably on sensory attributes of K.Vita, such as taste, texture and appearance, and ease of use. Adults achieved a median intake of 240 ml K.Vita, and children 120 ml (19% daily energy). Three children and one adult had ß-hydroxybutyrate >1 mmol/l. There was 50% (95% CI 39–61%) reduction in mean frequency of seizures/events. Reduction in seizures or paroxysmal events correlated significantly with blood concentrations of medium chain fatty acids (C10 and C8) but not ß-hydroxybutyrate. K.Vita was well accepted and tolerated. Side effects were mild and resolved with dietetic support. Individuals who completed the study complied with K.Vita and additional dietary modifications. Dietary intervention had a beneficial effect on frequency of seizures or paroxysmal events, despite absent or very low levels of ketosis. We suggest that K.Vita may be valuable to those with drug-resistant epilepsy, particularly those who cannot tolerate or do not have access to ketogenic diets, and may allow for more liberal dietary intake compared to ketogenic diets, with mechanisms of action perhaps unrelated to ketosis. Further studies of effectiveness of K.Vita are warranted., See Auvin (https://doi.org/10.1093/braincomms/fcab234) for a scientific commentary on this article. Schoeler et al. report that K.Vita, a decanoic acid-rich medical food, was well-tolerated when introduced gradually and flexibly to individuals with epilepsy. Seizures or paroxysmal events reduced by 50% (95% CI 39–61%). Only 4/61 (7%) patients had ß-hydroxybutyrate levels >1 mmol/l. K.Vita may be valuable for individuals with drug-resistant epilepsy., Graphical Abstract Graphical Abstract

Significance The mTORC1 complex provides a critical role in cell function, regulating a variety of processes including growth and autophagy. mTORC1 signaling is hyperactivated in a range of common diseases including cancer, epilepsy, and neurodegenerative disorders. Hence, mTORC1 signaling provides an important target for regulation in many contexts. Here, we show that decanoic acid, a key component of a widely used medicinal diet, reduces mTORC1 activity. We identify this in a tractable model system, and validate it in ex vivo rat brain tissue and in human iPSC-derived astrocytes from patients with a clinically relevant disease. Thus, we provide insight into an easily accessible therapeutic approach for a range of diseases., Low-glucose and -insulin conditions, associated with ketogenic diets, can reduce the activity of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, potentially leading to a range of positive medical and health-related effects. Here, we determined whether mTORC1 signaling is also a target for decanoic acid, a key component of the medium-chain triglyceride (MCT) ketogenic diet. Using a tractable model system, Dictyostelium, we show that decanoic acid can decrease mTORC1 activity, under conditions of constant glucose and in the absence of insulin, measured by phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). We determine that this effect of decanoic acid is dependent on a ubiquitin regulatory X domain-containing protein, mediating inhibition of a conserved Dictyostelium AAA ATPase, p97, a homolog of the human transitional endoplasmic reticulum ATPase (VCP/p97) protein. We then demonstrate that decanoic acid decreases mTORC1 activity in the absence of insulin and under high-glucose conditions in ex vivo rat hippocampus and in tuberous sclerosis complex (TSC) patient-derived astrocytes. Our data therefore indicate that dietary decanoic acid may provide a new therapeutic approach to down-regulate mTORC1 signaling.

AMPA receptors, which shape excitatory postsynaptic currents and are directly involved in overactivation of synaptic function during seizures, represent a well-accepted target for anti-epileptic drugs. Trans-4-butylcyclohexane carboxylic acid (4-BCCA) has emerged as a new promising anti-epileptic drug in several in vitro and in vivo seizure models, but the mechanism of its action remained unknown. The purpose of this study is to characterize structure and dynamics of 4-BCCA interaction with AMPA receptors.We studied the molecular mechanism of AMPA receptor inhibition by 4-BCCA using a combination of X-ray crystallography, mutagenesis, electrophysiological assays, and molecular dynamics simulations.We identified 4-BCCA binding sites in the transmembrane domain (TMD) of AMPA receptor, at the lateral portals formed by transmembrane segments M1-M4. At this binding site, 4-BCCA is very dynamic, assumes multiple poses, and can enter the ion channel pore.4-BCCA represents a low-affinity inhibitor of AMPA receptors that acts at the TMD sites distinct from non-competitive inhibitors, such as the anti-epileptic drug perampanel and the ion channel blockers. Further studies might examine the possibsility of synergistic use of these inhibitors in treatment of epilepsy and a wide range of neurological disorders and gliomas.This article is part of a themed issue on Structure Guided Pharmacology of Membrane Proteins (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.14/issuetoc.

The sympathetic nervous system is essential for maintenance of cardiac function via activation of post-junctional adrenergic receptors. Prolonged adrenergic receptor activation, however, has deleterious long-term effects leading to hypertrophy and the development of heart failure. Here we investigate the effect of chronic adrenergic receptors activation on excitation-contraction coupling (ECC) in ventricular cardiomyocytes from a previously characterized mouse model of chronic sympathetic hyperactivity, which are genetically deficient in the adrenoceptor α2A and α2C genes (ARDKO). When compared to wild-type (WT) cardiomyocytes, ARDKO displayed reduced fractional shortening (~33%) and slower relaxation (~20%). Furthermore, ARDKO cells exhibited several electrophysiological changes such as action potential (AP) prolongation (~50%), reduced L-type calcium channel (LCC) current (~33%), reduced outward potassium (K+) currents (~30%), and increased sodium/calcium exchanger (NCX) activity (~52%). Consistent with reduced contractility and calcium (Ca2+) currents, the cytosolic Ca2+ ([Ca2+]i) transient from ARDKO animals was smaller and decayed slower. Importantly, no changes were observed in membrane resting potential, AP amplitude, or the inward K+ current. Finally, we modified our existing cardiac ECC computational model to account for changes in the ARDKO heart. Simulations suggest that cellular changes in the ARDKO heart resulted in variable and dyssynchronous Ca2+-induced Ca2+ release therefore altering [Ca2+]i transient dynamics and reducing force generation. In conclusion, chronic sympathetic hyperactivity impairs ECC by changing the density of several ionic currents (and thus AP repolarization) causing altered Ca2+ dynamics and contractile activity. This demonstrates the important role of ECC remodeling in the cardiac dysfunction secondary to chronic sympathetic activity.

Key points Our group previously discovered and characterized the microtubule mechanotransduction pathway linking diastolic stretch to NADPH oxidase 2-derived reactive oxygen species signals that regulate calcium sparks and calcium influx pathways. Here we used focused experimental tests to constrain and expand our existing computational models of calcium signalling in heart. Mechanistic and quantitative modelling revealed new insights in disease including: changes in microtubule network density and properties, elevated NOX2 expression, altered calcium release dynamics, how NADPH oxidase 2 is activated by and responds to stretch, and finally the degree to which normalizing mechano-activated reactive oxygen species signals can prevent calcium-dependent arrhythmias. Abstract Microtubule (MT) mechanotransduction links diastolic stretch to generation of NADPH oxidase 2 (NOX2)-dependent reactive oxygen species (ROS), signals we term X-ROS. While stretch-elicited X-ROS primes intracellular calcium (Ca2+ ) channels for synchronized activation in the healthy heart, the dysregulated excess in this pathway underscores asynchronous Ca2+ release and arrhythmia. Here, we expanded our existing computational models of Ca2+ signalling in heart to include MT-dependent mechanotransduction through X-ROS. Informed by new focused experimental tests to properly constrain our model, we quantify the role of X-ROS on excitation-contraction coupling in healthy and pathological conditions. This approach allowed for a mechanistic investigation that revealed new insights into X-ROS signalling in disease including changes in MT network density and post-translational modifications (PTMs), elevated NOX2 expression, altered Ca2+ release dynamics (i.e. Ca2+ sparks and Ca2+ waves), how NOX2 is activated by and responds to stretch, and finally the degree to which normalizing X-ROS can prevent Ca2+ -dependent arrhythmias.

Visualizing mitochondria in living Dictyostelium discoideum cells using fluorescent dyes is often problematic due to variability in staining, metabolism of the dyes, and unknown potential effects of the dyes on mitochondrial function. We show that fluorescent labelling of mitochondria, using an N-terminal mitochondrial localization sequence derived from the D. discoideum protein GcvH1 (glycine cleavage system H1) attached to a red fluorescent protein enables clear mitochondrial imaging. We also show that this labelling has no effect upon mitochondria load or respiratory function., Highlights • Live-cell visualization of mitochondria dynamics is currently difficult in Dictyostelium discoideum. • We provide an easy and rapid procedure for visualizing mitochondria. • This technology employs a plasmid, REMIT-mRFPmars, expressing a mitochondrial targeting sequence linked to mRFPmars. • Expression of REMIT-mRFPmars does not alter mitochondrial load or function, providing a suitable tag for live-cell studies.

High-fat, low-carbohydrate diets, known as ketogenic diets, have been used as a non-pharmacological treatment for refractory epilepsy. A key mechanism of this treatment is thought to be the generation of ketones, which provide brain cells (neurons and astrocytes) with an energy source that is more efficient than glucose, resulting in beneficial downstream metabolic changes, such as increasing adenosine levels, which might have effects on seizure control. However, some studies have challenged the central role of ketones because medium-chain fatty acids, which are part of a commonly used variation of the diet (the medium-chain triglyceride ketogenic diet), have been shown to directly inhibit AMPA receptors (glutamate receptors), and to change cell energetics through mitochondrial biogenesis. Through these mechanisms, medium-chain fatty acids rather than ketones are likely to block seizure onset and raise seizure threshold. The mechanisms underlying the ketogenic diet might also have roles in other disorders, such as preventing neurodegeneration in Alzheimer's disease, the proliferation and spread of cancer, and insulin resistance in type 2 diabetes. Analysing medium-chain fatty acids in future ketogenic diet studies will provide further insights into their importance in modified forms of the diet. Moreover, the results of these studies could facilitate the development of new pharmacological and dietary therapies for epilepsy and other disorders.

Ketogenic diets, used in epilepsy treatment, are considered to work through reduced glucose and ketone generation to regulate a range of cellular process including autophagy induction. Recent studies into the medium-chain triglyceride (MCT) ketogenic diet have suggested that medium-chain fatty acids (MCFAs) provided in the diet, decanoic acid and octanoic acid, cause specific therapeutic effects independent of glucose reduction, although a role in autophagy has not been investigated. Both autophagy and MCFAs have been widely studied in Dictyostelium, with findings providing important advances in the study of autophagy-related pathologies such as neurodegenerative diseases. Here, we utilize this model to analyze a role for MCFAs in regulating autophagy. We show that treatment with decanoic acid but not octanoic acid induces autophagosome formation and modulates autophagic flux in high glucose conditions. To investigate this effect, decanoic acid, but not octanoic acid, was found to induce the expression of autophagy-inducing proteins (Atg1 and Atg8), providing a mechanism for this effect. Finally, we demonstrate a range of related fatty acid derivatives with seizure control activity, 4BCCA, 4EOA, and Epilim (valproic acid), also function to induce autophagosome formation in this model. Thus, our data suggest that decanoic acid and related compounds may provide a less-restrictive therapeutic approach to activate autophagy.

Alzheimer's disease is the most prevalent form of dementia in the elderly but the precise causal mechanisms are still not fully understood. Growing evidence supports a significant role for Aβ42 oligomers in the development and progression of Alzheimer's. For example, intracellular soluble Aβ oligomers are thought to contribute to the early synaptic dysfunction associated with Alzheimer's disease, but the molecular mechanisms underlying this effect are still unclear. Here, we identify a novel mechanism that contributes to our understanding of the reported synaptic dysfunction. Using primary rat hippocampal neurons exposed for a short period of time to Aβ42 oligomers, we show a disruption in the activity-dependent phosphorylation cycle of SynapsinI at Ser9. SynapsinI is a pre-synaptic protein that responds to neuronal activity and regulates the availability of synaptic vesicles to participate in neurotransmitter release. Phosphorylation of SynapsinI at Ser9, modulates its distribution and interaction with synaptic vesicles. Our results show that in neurons exposed to Aβ42 oligomers, the levels of phosphorylated Ser9 of SynapsinI remain elevated during the recovery period following neuronal activity. We then investigated if this effect could be targeted by a putative therapeutic regime using valproic acid (a short branch-chained fatty acid) that has been proposed as a treatment for Alzheimer's disease. Exposure of Aβ42 treated neurons to valproic acid, showed that it restores the physiological regulation of SynapsinI after depolarisation. Our data provide a new insight on Aβ42-mediated pathology in Alzheimer's disease and supports the use of Valproic acid as a possible pharmaceutical intervention for the treatment of Alzheimer's disease.

Introduction In all types of muscle cells, contraction is initiated by an increase in intracellular Ca2+. Another similarity among skeletal, cardiac, and smooth muscle cells is that much of the Ca2+ responsible for contraction is released from the SR. Beyond these common features, however

Background and Purpose: Epidiolex, a form of highly purified cannabidiol (CBD) derived from Cannabis plants has demonstrated seizure control activity in patients with Dravet syndrome, without a fully elucidated mechanism of action. We have employed an unbiased approach to investigate this mechanism at a cellular level.\ud Experimental Approach: We use a tractable biomedical model organism, Dictyostelium, to identify protein controlling the effect of CBD and characterize this mechanism. We then translate these results to a Dravet Syndrome mouse model and an acute in vitro seizure model.\ud Key Results: CBD activity is partially dependent upon the mitochondrial glycine cleavage system component, GcvH1 in Dictyostelium, orthologous to the human GCSH protein, which is functionally linked to folate one-carbon metabolism (FOCM). Analysis of FOCM components identified a mechanism for CBD in directly inhibiting methionine synthesis. Analysis of brain tissue from a Dravet syndrome mouse model also showed drastically altered levels of one-carbon components including methionine, and an in vitro rat seizure model showed an elevated level of methionine that is attenuated following CBD treatment. \ud Conclusions and Implications: Our results suggest a novel mechanism for CBD in the regulating methionine levels, and identify altered one-carbon metabolism in Dravet syndrome and seizure activity.\ud voltage-dependent anion selective channel proteins, VDAC1.

Developing novel compounds for the treatment of diseases remains one of the highest priorities in biomedical research, where it is critical to identify their targets and how they work at a cellular level. Most studies in this area employ mammalian models, since rodents or non-human primates are seen as a good approximation for humans. However, using mammalian models can be problematic for a range of reasons, including high genetic redundancy and the essential role for many proteins in development. More importantly, it is very difficult to identify how compounds function at a cellular or molecular level in these models without a previously suggested mechanism or target. So how can we identify targets of medicinal compounds? In this review we outline the use of an innovative and tractable model system, Dictyostelium discoideum, to provide useful insight to the cellular and molecular functions of both therapeutic drugs and pharmacologically active natural products. We outline the advantages of using this model, and then provide a range of exemplar studies using D. discoideum in pharmacological research to demonstrate breakthroughs in understanding the action and effects of compounds, and the subsequent translational of these advances to mammalian models leading to potential improvements in societal health.

The workshop 'Nutrition for the Ageing Brain: Towards Evidence of an Optimal Diet' was organized with funds from the ILSI Europe Nutrition and Mental Performance Task Force. Industry members of this task force are listed on the ILSI Europe website at www.ilsi.eu. For further information about ILSI Europe, please email or call +32 2 771 00 14. This review was prepared taken into account the presentations at the workshop mentioned in the abstract and was conducted by an expert group of ILSI Europe. This publication was coordinated by Mr Jeroen Schuermans, Scientific Project Manager at ILSI Europe during the proceedings process. The authors would like to acknowledge Ms Catherine Collins, Ms Lilou van Lieshout and Dr Lucie Geurts for the finalisation and submission of this manuscript; As people age they become increasingly susceptible to chronic and extremely debilitating brain diseases. The precise cause of the neuronal degeneration underlying these disorders, and indeed normal brain ageing remains however elusive. Considering the limits of existing preventive methods, there is a desire to develop effective and safe strategies. Growing preclinical and clinical research in healthy individuals or at the early stage of cognitive decline has demonstrated the beneficial impact of nutrition on cognitive functions. The present review is the most recent in a series produced by the Nutrition and Mental Performance Task Force under the auspice of the International Life Sciences Institute Europe (ILSI Europe). The latest scientific advances specific to how dietary nutrients and non-nutrient may affect cognitive ageing are presented. Furthermore, several key points related to mechanisms contributing to brain ageing, pathological conditions affecting brain function, and brain biomarkers are also discussed. Overall, findings are inconsistent and fragmented and more research is warranted to determine the underlying mechanisms and to establish dose-response relationships for optimal brain maintenance in different population subgroups. Such approaches are likely to provide the necessary evidence to develop research portfolios that will inform about new dietary recommendations on how to prevent cognitive decline

Much of Alzheimer disease (AD) research has been traditionally based on the use of animals, which have been extensively applied in an effort to both improve our understanding of the pathophysiological mechanisms of the disease and to test novel therapeutic approaches. However, decades of such research have not effectively translated into substantial therapeutic success for human patients. Here we critically discuss these issues in order to determine how existing human-based methods can be applied to study AD pathology and develop novel therapeutics. These methods, which include patient-derived cells, computational analysis and models, together with large-scale epidemiological studies represent novel and exciting tools to enhance and forward AD research. In particular, these methods are helping advance AD research by contributing multifactorial and multidimensional perspectives, especially considering the crucial role played by lifestyle risk factors in the determination of AD risk. In addition to research techniques, we also consider related pitfalls and flaws in the current research funding system. Conversely, we identify encouraging new trends in research and government policy. In light of these new research directions, we provide recommendations regarding prioritization of research funding. The goal of this document is to stimulate scientific and public discussion on the need to explore new avenues in AD research, considering outcome and ethics as core principles to reliably judge traditional research efforts and eventually undertake new research strategies.

Many neurodegenerative diseases present the loss of synapses as a common pathological feature. Here we have employed an in vitro model for synaptic loss to investigate the molecular mechanism of a therapeutic treatment, valproic acid (VPA). We show that amyloid-β (Aβ), isolated from patient tissue and thought to be the causative agent of Alzheimer's disease, caused the loss of synaptic proteins including synaptophysin, synapsin-1 and cysteine-string protein from cultured mouse neurons. Aβ-induced synapse damage was reduced by pre-treatment with physiologically relevant concentrations of VPA (10 μM) and a structural variant propylisopropylacetic acid (PIA). These drugs also reduced synaptic damage induced by other neurodegenerative-associated proteins α-synuclein, linked to Lewy body dementia and Parkinson's disease, and the prion-derived peptide PrP82-146. Consistent with these effects, synaptic vesicle recycling was also inhibited by these proteins and protected by VPA and PIA. We show a mechanism for this damage through aberrant activation of cytoplasmic phospholipase A2 (cPLA2) that is reduced by both drugs. Furthermore, Aβ-dependent cPLA2 activation correlates with its accumulation in lipid rafts, and is likely to be caused by elevated cholesterol (stabilising rafts) and decreased cholesterol ester levels, and this mechanism is reduced by VPA and PIA. Such observations suggest that VPA and PIA may provide protection against synaptic damage that occurs during Alzheimer's and Parkinson's and prion diseases.

Valproic acid (VPA) provides a common treatment for both epilepsy and bipolar disorder; however, common cellular mechanisms relating to both disorders have yet to be proposed. Here, we explore the possibility of a diacylglycerol kinase (DGK) playing a role in regulating the effect of VPA relating to the treatment of both disorders, using the biomedical model Dictyostelium discoideum. DGK enzymes provide the first step in the phosphoinositide recycling pathway, implicated in seizure activity. They also regulate levels of diacylglycerol (DAG), thereby regulating the protein kinase C (PKC) activity that is linked to bipolar disorder-related signalling. Here, we show that ablation of the single Dictyostelium dgkA gene results in reduced sensitivity to the acute effects of VPA on cell behaviour. Loss of dgkA also provides reduced sensitivity to VPA in extended exposure during development. To differentiate a potential role for this DGKA-dependent mechanism in epilepsy and bipolar disorder treatment, we further show that the dgkA null mutant is resistant to the developmental effects of a range of structurally distinct branched medium-chain fatty acids with seizure control activity and to the bipolar disorder treatment lithium. Finally, we show that VPA, lithium and novel epilepsy treatments function through DAG regulation, and the presence of DGKA is necessary for compound-specific increases in DAG levels following treatment. Thus, these experiments suggest that, in Dictyostelium, loss of DGKA attenuates a common cellular effect of VPA relating to both epilepsy and bipolar disorder treatments, and that a range of new compounds with this effect should be investigated as alternative therapeutic agents. This article has an associated First Person interview with the first author of the paper.

Perampanel is an adjunctive treatment for epilepsy that works through the direct inhibition of AMPA receptors. The same molecular mechanism has recently been shown for a fatty acid, decanoic acid, prescribed in the medium chain triglyceride ketogenic diet for the treatment of patients with drug-resistant epilepsy. Because each compound has been proposed to act through a distinct AMPA receptor binding site, we predicted that perampanel and decanoic acid would act synergistically against AMPA receptors and, consequently, seizures. Here, we show a synergistic interaction between perampanel and decanoic acid in direct AMPA receptor inhibition, in an ex vivo model of seizure activity, and against seizure-induced activity in human brain slices. These data support a potential role for combination treatment using perampanel and dietary decanoic acid to provide enhanced seizure control.

Since the original report of seizure control through starvation in the 1920s, the ketogenic diet has been considered an energy-related therapy. The diet was assumed to be functioning through the effect of reduced carbohydrate intake regulating cellular energy state, thus giving rise to seizure control. From this assumption, the generation of ketones during starvation provided an attractive mechanism for this altered energy state; however, many years of research has sought and largely failed to correlate seizure control and ketone levels. Due to this focus on ketones, few studies have examined a role for free fatty acids, as metabolic intermediates between the triglycerides provided in the diet and ketones, in seizure control. Recent discoveries have now suggested that the medium-chain fats, delivered through the medium-chain triglyceride (MCT) ketogenic diet, may provide a key therapeutic mechanism of the diet in seizure control. Here we describe an unusual pathway leading to this discovery, beginning with the use of a tractable non-animal model—Dictyostelium, through to the demonstration that medium-chain fats play a direct role in seizure control, and finally the identification of a mechanism of action of these fats and related congeners leading to reduced neural excitability and seizure control.

Background and Purpose Valproic acid (VPA), a widely used epilepsy and bipolar disorder treatment, provides acute protection against haemorrhagic shock-induced mortality in a range of in vivo models through an unknown mechanism. In the liver, this effect occurs with a concomitant protection against a decrease in GSK3β-Ser9 phosphorylation. Here, we developed an in vitro model to investigate this protective effect of VPA and define a molecular mechanism. Experimental Approach The human hepatocarcinoma cell line (Huh7) was exposed to conditions occurring during haemorrhagic shock (hypoxia, hypercapnia and hypothermia) to investigate the changes in GSK3β-Ser9 phosphorylation for a 4 h period following treatment with VPA, related congeners, PPAR agonists, antagonists and siRNA. Key Results Huh7 cells undergoing combined hypoxia, hypercapnia, and hypothermia reproduced the reduced GSK3β-Ser9 phosphorylation shown in vivo during haemorrhagic shock, and this change was blocked by VPA. The protective effect occurred through upstream PTEN and Akt signalling, and prevented downstream β-catenin degradation while increasing histone 2/3 acetylation. This effect was reproduced by several VPA-related compounds with known PPARγ agonist activity, independent of histone deacetylase (HDAC) inhibitory activity. Specific pharmacological inhibition (by T0070907) or knockdown of PPARγ blocked the protective effect of VPA against these signalling changes and apoptosis. In addition, specific activation of PPARγ using ciglitazone reproduced the changes induced by VPA in haemorrhagic shock-like conditions. Conclusion and Implications Changes in GSK3β-Ser9 phosphorylation in in vivo haemorrhagic shock models can be modelled in vitro, and this has identified a role for PPARγ activation in the protective role of VPA.

Starting with the established antiepileptic drug, valproic acid, we have taken a novel approach to develop new antiseizure drugs that may be effective in status epilepticus. We first identified that valproic acid has a potent effect on a biochemical pathway, the phosphoinositide pathway, in Dictyostelium discoideum, and we demonstrated that this may relate to its mechanism of action against seizures in mammalian systems. Through screening in this pathway, we have identified a large array of fatty acids and fatty acid derivatives with antiseizure potential. These were then evaluated in an in vitro mammalian system. One compound that we identified through this process is a major constituent of the ketogenic diet, strongly arguing that it may be the fatty acids that are mediating the antiseizure effect of this diet. We further tested two of the more potent compounds in an in vivo model of status epilepticus and demonstrated that they were more effective than valproic acid in treating the status epilepticus. This article is part of a Special Issue entitled "Status Epilepticus".

Natural compounds often have complex molecular structures and unknown\ud molecular targets. These characteristics make them difficult to analyse using a classical\ud pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex\ud molecule possessing wide-ranging biological activities, cellular mechanisms and roles in\ud potential therapeutic treatment including Alzheimer’s disease and cancer. Here, we\ud investigate the physiological effects and molecular targets of curcumin in Dictyostelium\ud discoideum. We show curcumin causes acute effects on cell behaviour, reduces cell growth,\ud and slows multicellular development. We then employ a range of structurally related\ud compounds to show the distinct role of different structural groups cell behaviour, growth, and\ud development, highlighting active moieties in cell function, and showing that these cellular\ud effects are unrelated to the well-known antioxidant activity of curcumin. Molecular\ud mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then\ud investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A\ud regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue\ud (PsenB). Using in-silico docking analysis, we then show that curcumin may function through\ud direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and\ud molecular mechanisms for the function of curcumin and related compounds.

The amyloid hypothesis of Alzheimer's disease suggests that synaptic degeneration and pathology is caused by the accumulation of amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP). Subsequently, soluble Aβ oligomers cause the loss of synaptic proteins from neurons, a histopathological feature of Alzheimer's disease that correlates with the degree of dementia. In this study, the production of toxic forms of Aβ was examined in vitro using 7PA2 cells stably transfected with human APP. We show that conditioned media from 7PA2 cells containing Aβ oligomers caused synapse degeneration as measured by the loss of synaptic proteins, including synaptophysin and cysteine-string protein, from cultured neurons. Critically, conditioned media from 7PA2 cells treated with valproic acid (2-propylpentanoic acid (VPA)) or propylisopropylacetic acid (PIA) did not cause synapse damage. Treatment with VPA or PIA did not significantly affect total Aβ42 concentrations; rather these drugs selectively reduced the concentrations of Aβ42 oligomers in conditioned media. In contrast, treatment significantly increased the concentrations of Aβ42 monomers in conditioned media. VPA or PIA treatment reduced the concentrations of APP within lipid rafts, membrane compartments associated with Aβ production. These effects of VPA and PIA were reversed by the addition of platelet-activating factor, a bioactive phospholipid produced following activation of phospholipase A2, an enzyme sensitive to VPA and PIA. Collectively these data suggest that VPA and PIA reduce Aβ oligomers through inhibition of phospholipase A2 and suggest a novel therapeutic approach to Alzheimer's treatment.

Getting older usually means becoming increasingly forgetful, but for some of us it will mean much more than simple absent-mindedness. For millions of older people worldwide, advancing age can also mean the beginning of a steady decline in mental faculties that is characteristic of the devastating neurodegenerative deterioration in Alzheimer's disease. This form of senile dementia is a burden not only for the patient, because of the debilitation that the condition causes, but also for the families of sufferers, who must dedicate increasing time and resources to caring for their affected loved ones. The cost to society is also staggering, currently about £23 billion in Britain annually, and projected to rise to £50 billion by 20381. Thus understanding what causes the neurodegeneration and how to treat it is a major priority for biomedical researchers. This research is dominated by the use of animals to study changes in the brain during development of the disease.

Drug-resistant epilepsy has remained a problem since the inception of antiepileptic drug development, despite the large variety of antiepileptic drugs available today. Moreover, the mechanism-of-action of these drugs is often unknown. This is due to the widespread screening of compounds through animal models. We have taken a different approach to antiepileptic drug discovery and have identified a biochemical pathway in Dictyostelium discoideum (a ‘slime mould’) that may relate to the mechanism-of-action of valproate, one of the most commonly used and effective antiepileptic drugs. Through screening in this pathway, we have been able to identify a whole host of fatty acids and fatty acid derivatives with potential antiepileptic activity; this was then confirmed in in vitro and in vivo mammalian seizure models. Some of these compounds are more potent than valproate and potentially lack many of the major side effects of valproate (including birth defects and liver toxicity). In addition, one of the compounds that we have identified is a major constituent of the ketogenic diet, strongly arguing that it may be the fatty acids and not the ketogenesis that are mediating the effect of this diet.

The free Ca(2+) concentration within the mitochondrial matrix ([Ca(2+)]m) regulates the rate of ATP production and other [Ca(2+)]m sensitive processes. It is set by the balance between total Ca(2+) influx (through the mitochondrial Ca(2+) uniporter (MCU) and any other influx pathways) and the total Ca(2+) efflux (by the mitochondrial Na(+)/Ca(2+) exchanger and any other efflux pathways). Here we review and analyze the experimental evidence reported over the past 40years which suggest that in the heart and many other mammalian tissues a putative Na(+)/Ca(2+) exchanger is the major pathway for Ca(2+) efflux from the mitochondrial matrix. We discuss those reports with respect to a recent discovery that the protein product of the human FLJ22233 gene mediates such Na(+)/Ca(2+) exchange across the mitochondrial inner membrane. Among its many functional similarities to other Na(+)/Ca(2+) exchanger proteins is a unique feature: it efficiently mediates Li(+)/Ca(2+) exchange (as well as Na(+)/Ca(2+) exchange) and was therefore named NCLX. The discovery of NCLX provides both the identity of a novel protein and new molecular means of studying various unresolved quantitative aspects of mitochondrial Ca(2+) movement out of the matrix. Quantitative and qualitative features of NCLX are discussed as is the controversy regarding the stoichiometry of the NCLX Na(+)/Ca(2+) exchange, the electrogenicity of NCLX, the [Na(+)]i dependency of NCLX and the magnitude of NCLX Ca(2+) efflux. Metabolic features attributable to NCLX and the physiological implication of the Ca(2+) efflux rate via NCLX during systole and diastole are also briefly discussed.

The medium chain triglyceride (MCT) ketogenic diet is used extensively for treating refractory childhood epilepsy. This diet increases the plasma levels of medium straight chain fatty acids. A role for these and related fatty acids in seizure control has not been established. We compared the potency of an established epilepsy treatment, Valproate (VPA), with a range of MCT diet-associated fatty acids (and related branched compounds), using in vitro seizure and in vivo epilepsy models, and assessed side effect potential in vitro for one aspect of teratogenicity, for liver toxicology and in vivo for sedation, and for a neuroprotective effect. We identify specific medium chain fatty acids (both prescribed in the MCT diet, and related compounds branched on the fourth carbon) that provide significantly enhanced in vitro seizure control compared to VPA. The activity of these compounds on seizure control is independent of histone deacetylase inhibitory activity (associated with the teratogenicity of VPA), and does not correlate with liver cell toxicity. In vivo, these compounds were more potent in epilepsy control (perforant pathway stimulation induced status epilepticus), showed less sedation and enhanced neuroprotection compared to VPA. Our data therefore implicates medium chain fatty acids in the mechanism of the MCT ketogenic diet, and highlights a related new family of compounds that are more potent than VPA in seizure control with a reduced potential for side effects. This article is part of the Special Issue entitled ‘New Targets and Approaches to the Treatment of Epilepsy’., Highlights ► Medium chain straight and branched fatty acids were examined for seizure control in vitro. ► Structural specificity was shown for these compounds. ► Side effects were examined for active seizure-control compounds. ► In vivo seizure control, sedation and neuroprotection were shown for these compounds. ► Medium chain fatty acids relating to the MCT ketogenic diet may be active in seizure control.

Zizimin proteins belong to the Dock (Dedicator of Cytokinesis) superfamily of Guanine nucleotide Exchange Factor (GEF) proteins. This family of proteins plays a role in the regulation of Rho family small GTPases. Together the Rho family of small GTPases and the Dock/Zizimin proteins play a vital role in a number of cell processes including cell migration, apoptosis, cell division and cell adhesion. Our recent studies of Zizimin proteins, using a simple biomedical model, the eukaryotic social amoeba Dictyostelium discoideum, have helped to elucidate the cellular role of these proteins. In this article, we discuss the domain structure of Zizimin proteins from an evolutionary viewpoint. We also compare what is currently known about the mammalian Zizimin proteins to that of related Dock proteins. Understanding the cellular functions of these proteins will provide a better insight into their role in cell signaling, and may help in treating disease pathology associated with mutations in Dock/Zizimin proteins.

Calcium (Ca 2+ ) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca 2+ levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca 2+ ([Ca 2+ ] i ) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca 2+ uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca 2+ uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca 2+ uniporter (MCU) candidate. This quantitative analysis yields four clear results: ( i ) under physiological conditions, Ca 2+ influx into the mitochondria via the MCU is small relative to other cytosolic Ca 2+ extrusion pathways; ( ii ) single MCU conductance is ∼6–7 pS (105 mM [Ca 2+ ]), and MCU flux appears to be modulated by [Ca 2+ ] i , suggesting Ca 2+ regulation of MCU open probability ( P O ); ( iii ) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and ( iv ) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca 2+ critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca 2+ under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca 2+ ] i , mitochondrial Ca 2+ uptake can increase 10- to 1,000-fold and begin to shape [Ca 2+ ] i dynamics.

The medium chain triglyceride (MCT) ketogenic diet provides a highly effective and commonly used approach for treating drug-resistant epilepsy. It is associated with elevated levels of two MCT-derived fatty acids, decanoioc and octanoic acids. Researchers have identified a role for decanoic acid and a range of novel related chemicals in seizure control in multiple acute in vitro and in vivo models. A principal mechanism of decanoic acid is direct inhibition of AMPA receptors, key excitatory neurotransmitter receptors widely recognized as a target for seizure control. These data suggest a therapeutic mechanism of the MCT ketogenic diet through a direct fatty acid–dependent mechanism, independent of ketosis. This discovery will enable the development of an improved and, potentially, better-tolerated diet and the generation of a corresponding pharmaceutical approach. The diet should be termed the MCT diet, as the consequent ketosis may not be necessary for seizure control.

The canonical atrial myocyte (AM) is characterized by sparse transverse tubule (TT) invaginations and slow intracellular Ca2+ propagation but exhibits rapid contractile activation that is susceptible to loss of function during hypertrophic remodeling. Here, we have identified a membrane structure and Ca2+-signaling complex that may enhance the speed of atrial contraction independently of phospholamban regulation. This axial couplon was observed in human and mouse atria and is composed of voluminous axial tubules (ATs) with extensive junctions to the sarcoplasmic reticulum (SR) that include ryanodine receptor 2 (RyR2) clusters. In mouse AM, AT structures triggered Ca2+ release from the SR approximately 2 times faster at the AM center than at the surface. Rapid Ca2+ release correlated with colocalization of highly phosphorylated RyR2 clusters at AT-SR junctions and earlier, more rapid shortening of central sarcomeres. In contrast, mice expressing phosphorylation-incompetent RyR2 displayed depressed AM sarcomere shortening and reduced in vivo atrial contractile function. Moreover, left atrial hypertrophy led to AT proliferation, with a marked increase in the highly phosphorylated RyR2-pS2808 cluster fraction, thereby maintaining cytosolic Ca2+ signaling despite decreases in RyR2 cluster density and RyR2 protein expression. AT couplon "super-hubs" thus underlie faster excitation-contraction coupling in health as well as hypertrophic compensatory adaptation and represent a structural and metabolic mechanism that may contribute to contractile dysfunction and arrhythmias.

Endocytic membrane structures called transverse tubules (TTs) are thought to represent the main mechanism for contacts with the junctional sarcoplasmic reticulum (jSR) and intracellular Ca2+ release in ventricular cardiomyocytes (VCM). In contrast, in atrial cardiomyocytes (ACM) we identify previously not described large axial membrane tubules (ATs) at the cell core. Using optimized ACM isolation and live cell superresolution microscopy, we show that AT membrane structures represent both the major (ie. >90% of endocytic tubule structures) and largest tubule component (mean AT circumference 921±24 nm). Due to the unusually large AT membrane surface with a positive curvature exposed to the cytoplasm, we hypothesized that the endocytic AT membrane surface functions as central-axial Ca2+ signaling hub during excitation-contraction (EC)-coupling. To test this hypothesis, we combined imaging of AT membranes with fast intracellular Ca2+ imaging. Indeed, high-amplitude Ca2+ transients originated in a highly localized fashion rapidly from the AT-hubs. Importantly, Ca2+ transients originating from AT-hubs occured significantly earlier compared to cellular surface locations (p

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca 2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca 2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca 2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

The importance of [Ca2+] in the mitochondrial matrix, [Ca2+]mito, had been proposed by early work of Carafoli and others [1], [2] and [3]. The key suggestion in the 1970s [4] was that regulatory [Ca2+]mito played a role in controlling the rate of activation of tricarboxylic acid cycle dehydrogenases, important in the regulation of ATP production by the electron transport chain (ETC) during oxidative phosphorylation. This view is now established [5] and [6] and the key questions currently debated are to what extent do the mitochondria acquire and release Ca2+, and what impact do mitochondria have on the dynamic Ca2+ signal in the cardiac ventricular myocyte [7]. Although investigations of Ca2+ dynamics in mitochondria have been problematic, disparate and inconclusive, they have also been both provocative and exciting. A recent special issue of this journal presented contrasting perspectives on the speed, extent and mechanisms of changes in [Ca2+]mito, and how these changes may influence cellular spatio-temporal [Ca2+]i dynamics [8]. An audio discussion is also available online [9]. The uncertain nature of the signaling pathways is noted in Table 1 (see below) which shows mitochondrial proteins and processes that are of current focus and which remain contentious. Each of the “items” listed is largely unsettled, or is a “work in progress”. There may be advocates for opposing positions noted or recent discoveries that must still be tested at multiple levels by diverse laboratories. Currently, the first item, the mitochondrial sodium/calcium exchanger (NCLX) [10], appears the most solid with respect to the molecular identification and physiological function, whereas, the recently described candidates of the mitochondrial Ca2+ uniporter (MCU) [11] and [12] still need to be verified and broadly examined by the scientific community.

Bio-electrospraying (BES) and aerodynamically assisted bio-jetting (AABJ) have recently been established as important novel biospray technologies for directly manipulating living cells. To elucidate their potential in medical and clinical sciences, these bio-aerosol techniques have been subjected to increasingly rigorous investigations. In parallel to these studies, we wish to introduce these unique biotechnologies for use in the basic biological sciences, for handling a wide range of cell types and systems, thus increasing the range and the scope of these techniques for modern research. Here, the authors present the analysis of the new use of these biospray techniques for the direct handling of the simple eukaryotic biomedical model organism Dictyostelium discoideum . These cells are widely used as a model for immune cell chemotaxis and as a simple model for development. We demonstrate that AABJ of these cells did not cause cell stress, as defined by the stress-gene induction, nor affect cell development. Furthermore, although BES induced the increased expression of one stress-related gene ( gapA ), this was not a generalized stress response nor did it affect cell development. These data suggest that these biospray techniques can be used to directly manipulate single cells of this biomedical model without inducing a generalized stress response or perturbing later development.

Understanding the mechanisms of drug action has been the primary focus for pharmacological researchers, traditionally using rodent models. However, non-sentient model systems are now increasingly being used as an alternative approach to better understand drug action or targets. One of these model systems, the social amoeba Dictyostelium, enables the rapid ablation or over-expression of genes, and the subsequent use of isogenic cell culture for the analysis of cell signalling pathways in pharmacological research. The model also supports an increasingly important ethical view of research, involving the reduction, replacement and refinement of animals in biomedical research. This review outlines the use of Dictyostelium in understanding the pharmacological action of two commonly used bipolar disorder treatments (valproic acid and lithium). Both of these compounds regulate mitogen activated protein (MAP) kinase and inositol phospholipid-based signalling by unknown means. Analysis of the molecular pathways targeted by these drugs in Dictyostelium and translation of discoveries to animal systems has helped to further understand the molecular mechanisms of these bipolar disorder treatments.