The neuroprotective agent, valproic acid, regulates the mitogen-activated protein kinase pathway through modulation of protein kinase A signalling in Dictyostelium discoideum

Activation of the mitogen-activated protein kinase (MAPK) cascade gives rise to a neuroprotective effect in a variety of cell types. The bipolar disorder treatment, valproic acid (VPA), increases the activity of this pathway by modulating extracellular signal-regulated kinase 2 (ERK2) phosphorylation through an unknown mechanism. To investigate the molecular basis of this effect, we have used the biomedical model system Dictyostelium discoideum to dissect this signalling pathway. We find that, similar to mammalian systems, VPA causes a transient increase in the activation of the MAPK signalling pathway, as shown by ERK2 phosphorylation. We show that the MAP kinase and phosphatase, protein kinase A (PKA) and glycogen synthase kinase signalling pathways all function in controlling the levels of phospho-ERK2 (pERK2). We find that VPA induces elevated pERK2 levels through attenuation of the PKA signalling pathway. Interestingly, pERK2 levels are also controlled by another bipolar disorder drug, lithium, providing a common effect of these two drugs. This work therefore suggests a conserved pathway in eukaryotes that is targeted by neuroprotective and bipolar disorder drugs and allows us to propose a model for this neuroprotective effect.     

The neuroprotective agent, valproic acid, regulates the mitogen-activated protein kinase pathway through modulation of protein kinase A signalling in Dictyostelium discoideum

Activation of the mitogen-activated protein kinase (MAPK) cascade gives rise to a neuroprotective effect in a variety of cell types. The bipolar disorder treatment, valproic acid (VPA), increases the activity of this pathway by modulating extracellular signal-regulated kinase 2 (ERK2) phosphorylation through an unknown mechanism. To investigate the molecular basis of this effect, we have used the biomedical model system Dictyostelium discoideum to dissect this signalling pathway. We find that, similar to mammalian systems, VPA causes a transient increase in the activation of the MAPK signalling pathway, as shown by ERK2 phosphorylation. We show that the MAP kinase and phosphatase, protein kinase A (PKA) and glycogen synthase kinase signalling pathways all function in controlling the levels of phospho-ERK2 (pERK2). We find that VPA induces elevated pERK2 levels through attenuation of the PKA signalling pathway. Interestingly, pERK2 levels are also controlled by another bipolar disorder drug, lithium, providing a common effect of these two drugs. This work therefore suggests a conserved pathway in eukaryotes that is targeted by neuroprotective and bipolar disorder drugs and allows us to propose a model for this neuroprotective effect.     

Towards a molecular understanding of human diseases using Dictyostelium discoideum

The social amoeba Dictyostelium discoideum is increasingly being used as a simple model for the investigation of problems that are relevant to human health. This article focuses on several recent examples of Dictyostelium-based biomedical research, including the analysis of immune-cell disease and chemotaxis, centrosomal abnormalities and lissencephaly, bacterial intracellular pathogenesis, and mechanisms of neuroprotective and anti-cancer drug action. The combination of cellular, genetic and molecular biology techniques that are available in Dictyostelium often makes the analysis of these problems more amenable to study in this system than in mammalian cell culture. Findings that have been made in these areas using Dictyostelium have driven research in mammalian systems and have established Dictyostelium as a powerful model for human-disease analysis.     

Dictyostelium as host model for pathogenesis

The haploid social soil amoeba Dictyostelium discoideum has been established as a host model for several pathogens including Pseudomonas aeruginosa, Cryptococcus neoformans, Mycobacterium spp. and Legionella pneumophila. The research areas presently pursued include (i) the use of Dictyostelium wild-type cells as screening system for virulence of extracellular and intracellular pathogens and their corresponding mutants, (ii) the use of Dictyostelium mutant cells to identify genetic host determinants of susceptibility and resistance to infection and (iii) the use of reporter systems in Dictyostelium cells which allow the dissection of the complex host-pathogen cross-talk. The body of information presented in this review demonstrates that the availability of host cell markers, the knowledge of cell signalling pathways, the completion of the genome sequencing project and the tractability for genetic studies qualifies Dictyostelium for the study of fundamental cellular processes of pathogenesis.     

Dictyostelium discoideum to human cells: pharmacogenetic studies demonstrate a role for sphingolipids in chemoresistance

Resistance to chemotherapy is a major obstacle for the treatment of cancer and a subject of extensive research. Numerous mechanisms of drug resistance have been proposed, and they differ for different drugs. Nevertheless, it is clear that our understanding of this important problem is still incomplete, and that new targets for modulating therapy still await discovery. The attractive biology and the availability of powerful molecular techniques have made the cellular slime mold Dictyostelium discoideum, a powerful non-mammalian model for drug target discovery, and the problem of drug resistance. To understand the molecular basis of chemoresistance to the widely used drug cisplatin, both genetic and pharmacological approaches have been applied to this versatile experimental system. These studies have resulted in the identification of novel molecular pathways which can be used to increase the efficacy of cisplatin, and brought attention to the role of sphingolipids in mediating the cellular response to chemotherapeutic drugs. In the following review, we will describe the history and utility of D. discoideum in pharmacogenetics, and discuss recent studies which focus attention on the role of sphingolipids in chemotherapy and chemoresistance.     

Mechanisms of genomic and non-genomic actions of carotenoids

Carotenoids are highly bioactive dietary compounds that have the potential to have significant effects on human health. It is becoming increasingly clear that the various biological effects that carotenoids exert could be driven via a number of different mechanisms. These include direct pro- and antioxidant effects, redox sensitive cell signalling, vitamin A signalling pathways and other as yet unidentified mechanisms. This article provides an overview of the known effects of carotenoids and discusses the use of model systems and functional genomic approaches further to elucidate their modes of action.     

Signalling pathways that direct prestalk and stalk cell differentiation in Dictyostelium

Prestalk cell differentiation in Dictyostelium is induced by DIF and two DIF-induced genes, ecmA and ecmB, have revealed the existence of multiple prestalk and stalk cell sub-types. These different sub-types are defined by the pattern of expression of subfragments derived from the ecmA and ecmB promoters. These markers have been utilised in three ways; for fate mapping in vivo, to investigate the molecular mechanisms underlying DIF signalling and to explore the relative requirement for DIF and other signalling molecules for prestalk and stalk cell differentiation in vitro. The heterogeneity of the prestalk and stalk populations seems to be reflected in differences in the cell signalling pathways that they utilise.     

Imaging of cell migration

Cell migration is an essential process during many phases of development and adult life. Cells can either migrate as individuals or move in the context of tissues. Movement is controlled by internal and external signals, which activate complex signal transduction cascades resulting in highly dynamic and localised remodelling of the cytoskeleton, cell-cell and cell-substrate interactions. To understand these processes, it will be necessary to identify the critical structural cytoskeletal components, their spatio-temporal dynamics as well as those of the signalling pathways that control them. Imaging plays an increasingly important and powerful role in the analysis of these spatio-temporal dynamics. We will highlight a variety of imaging techniques and their use in the investigation of various aspects of cell motility, and illustrate their role in the characterisation of chemotaxis in Dictyostelium and cell movement during gastrulation in chick embryos in more detail.     

Autophagy in Dictyostelium: Genes and pathways,cell death and infection

The use of simple organisms to understand the molecular and cellular function of complex processes is instrumental for the rapid development of biomedical research. A remarkable example has been the discovery in S. cerevisiae of a group of proteins involved in the pathways of autophagy. Orthologues of these proteins have been identified in humans and experimental model organisms. Interestingly, some mammalian autophagy proteins do not seem to have homologues in yeast but are present in Dictyostelium, a social amoeba with two distinctive life styles, a unicellular stage in nutrient-rich conditions that differentiates upon starvation into a multicellular stage that depends on autophagy. This review focuses on the identification and annotation of the putative Dictyostelium autophagy genes and on the role of autophagy in development, cell death and infection by bacterial pathogens.     

Intracranial self-stimulation (ICSS) in rodents to study the neurobiology of motivation

It has become increasingly important to assess mood states in laboratory animals. Tests that reflect reward, reduced ability to experience reward (anhedonia) and aversion (dysphoria) are in high demand because many psychiatric conditions that are currently intractable in humans (e.g., major depression, bipolar disorder, addiction) are characterized by dysregulated motivation. Intracranial self-stimulation (ICSS) can be utilized in rodents (rats, mice) to understand how pharmacological or molecular manipulations affect the function of brain reward systems. Although many different methodologies are possible, we will describe in this protocol the use of medial forebrain bundle (MFB) stimulation together with the 'curve-shift' variant of analysis. This combination is particularly powerful because it produces a highly reliable behavioral output that enables clear distinctions between the treatment effects on motivation and the treatment effects on the capability to perform the task.     

Comparison of molecular mechanisms mediating cell contact phenomena in model developmental systems: an exploration of universality

Are there universal molecular mechanisms associated with cell contact phenomena during metazoan ontogenesis? Comparison of adhesion systems in disparate model systems indicates the existence of unifying principles. Requirements for multicellularity are (a) the construction of three‐dimensional structures involving a crucial balance between adhesiveness and motility; and (b) the establishment of integration at molecular, cellular, tissue, and organismal levels of organization. Mechanisms for (i) cell–cell and cell–substrate adhesion, (if) cell movement, (Hi) cell‐cell communication, (iv) cellular responses, (v) regulation of these processes, and (vi) their integration with patterning, growth, and other developmental processes are all crucial to metazoan development, and must have been present for the emergence and radiation of Metazoa. The principal unifying themes of this review are the dynamics and regulation of cell contact phenomena. Our knowledge of the dynamic molecular mechanisms underlying cell contact phenomena remains fragmentary. Here we examine the molecular bases of cell contact phenomena using extant model developmental systems (representing a wide range of phyla) including the simplest i.e. sponges, and the eukaryotic protist Dictyostelium discoideum, the more complex Drosophila melanogaster, and vertebrate systems. We discuss cell contact phenomena in a broad developmental context. The molecular language of cell contact phenomena is complex; it involves a plethora of structurally and functionally diverse molecules, and diverse modes of intermolecular interactions mediated by protein and/or carbohydrate moieties. Reasons for this are presumably the necessity for a high degree of specificity of inter‐molecular interactions, the requirement for a multitude of different signals, and the apparent requirement for an increasingly large repertoire of cell contact molecules in more complex developmental systems, such as the developing vertebrate nervous system. However, comparison of molecular models for dynamic adhesion in sponges and in vertebrates indicates that, in spite of significant differences in the details of the way specific cell–cell adhesion is mediated, similar principles are involved in the mechanisms employed by members of disparate phyla. Universal requirements are likely to include (a) rapidly reversible intermolecular interactions; (b) low‐affinity intermolecular interactions with fast on–off rates; (c) the compounding of multiple intermolecular interactions; (d) associated regulatory signalling systems. The apparent widespread employment of molecular mechanisms involving cadherin‐like cell adhesion molecules suggests the fundamental importance of cadherin function during development, particularly in epithelial morphogenesis, cell sorting, and segregation of cells.     

Genetic approaches to molecular mechanisms of programmed cell death in Dictyostelium

Caspase-independent cell deaths have been observed in many species including the human. However, the molecular mechanisms which govern them are largely unknown. Our present work makes use of a model organism, the protist Dictyostelium discoideum, which displays a caspase-independent cell death during its development. In rich medium, Dictyostelium multiplies vegetatively as a unicellular organism, but in starvation conditions, Dictyostelium cells aggregate, differentiate and morphogenize into a multicellular structure, called sorocarp, containing a mass of spores supported by a stalk. Cells in the stalk are considered dead on the basis of non-regrowth in a rich medium and are vacuolized. This programmed cell death is therefore developmental and vacuolar, and in addition, caspase-independent since the Dictyostelium genome does not contain caspases genes. In order to study in detail this cell death without induction of development, an in vitro experimental protocol has been adopted, which enabled us to describe the cascade of morphological events during this cell death. An insertional mutagenesis approach, followed by appropriate selection or screening of mutants potentially resistant to death, attempted at establishing the cascade of molecular events leading to vacuolar death of Dictyostelium cells. A better understanding of alternative death pathways may allow to control different types of cell deaths in the cases of cancers or neurodegenerative diseases. In this short review, we will discuss briefly some generalities about the development of Dictyostelium in starvation conditions, and we will focus on the course of programmed cell death in Dictyostelium and on the genetic tools used to elucidate the corresponding molecular mechanisms.     

Lymphokine signal transduction

Lymphokines are a group of signalling molecules involved in communication between cells, mainly those of the immune system. The lymphokines are multi-functional and most of them have mitogenic or co-mitogenic activity. An understanding of lymphokine biology is essential to understand how the immune system develops and functions and to provide a rationale for their use in immunotherapy.The potential to understand the cell biology of the lymphokines has recently become more apparent as molecular biological techniques have first of all produced recombinant factors and secondly have provided clues to the signal transduction pathways by cloning receptors, applying site-directed mutational analysis and also probing for specific promoters and enhancers that are activated along the signal pathway.This review discusses the information that has come from these recent analyses which blends with the biochemical analysis of the second messenger systems in an effort to understand the signalling pathways of the lymphokines.     

Dopamine and glutamate in schizophrenia: biology,symptoms and treatment

Glutamate and dopamine systems play distinct roles in terms of neuronal signalling, yet both have been proposed to contribute significantly to the pathophysiology of schizophrenia. In this paper we assess research that has implicated both systems in the aetiology of this disorder. We examine evidence from post‐mortem, preclinical, pharmacological and in vivo neuroimaging studies. Pharmacological and preclinical studies implicate both systems, and in vivo imaging of the dopamine system has consistently identified elevated striatal dopamine synthesis and release capacity in schizophrenia. Imaging of the glutamate system and other aspects of research on the dopamine system have produced less consistent findings, potentially due to methodological limitations and the heterogeneity of the disorder. Converging evidence indicates that genetic and environmental risk factors for schizophrenia underlie disruption of glutamatergic and dopaminergic function. However, while genetic influences may directly underlie glutamatergic dysfunction, few genetic risk variants directly implicate the dopamine system, indicating that aberrant dopamine signalling is likely to be predominantly due to other factors. We discuss the neural circuits through which the two systems interact, and how their disruption may cause psychotic symptoms. We also discuss mechanisms through which existing treatments operate, and how recent research has highlighted opportunities for the development of novel pharmacological therapies. Finally, we consider outstanding questions for the field, including what remains unknown regarding the nature of glutamate and dopamine function in schizophrenia, and what needs to be achieved to make progress in developing new treatments.     

A Functional Genome-Wide In Vivo Screen Identifies New Regulators of Signalling Pathways during Early Xenopus Embryogenesis

Embryonic development requires exquisite regulation of several essential processes, such as patterning of tissues and organs, cell fate decisions, and morphogenesis. Intriguingly, these diverse processes are controlled by only a handful of signalling pathways, and mis-regulation in one or more of these pathways may result in a variety of congenital defects and diseases. Consequently, investigating how these signalling pathways are regulated at the molecular level is essential to understanding the mechanisms underlying vertebrate embryogenesis, as well as developing treatments for human diseases. Here, we designed and performed a large-scale gain-of-function screen in Xenopus embryos aimed at identifying new regulators of MAPK/Erk, PI3K/Akt, BMP, and TGF-β/Nodal signalling pathways. Our gain-of-function screen is based on the identification of gene products that alter the phosphorylation state of key signalling molecules, which report the activation state of the pathways. In total, we have identified 20 new molecules that regulate the activity of one or more signalling pathways during early Xenopus development. This is the first time that such a functional screen has been performed, and the findings pave the way toward a more comprehensive understanding of the molecular mechanisms regulating the activity of important signalling pathways under normal and pathological conditions.     

Small GTPases in Dictyostelium: lessons from a social amoeba

Although the process of sequencing the Dictyostelium genome is not complete, it is already producing surprises, including an unexpectedly large number of Ras- and Rho-subfamily GTPases. Members of these families control a wide variety of cellular processes in eukaryotes, including proliferation, differentiation, cell motility and cell polarity. Comparison of small GTPases from Dictyostelium with those from higher eukaryotes provides an intriguing view of their cellular and evolutionary roles. In particular, although mammalian Ras proteins interact with several signalling pathways, the Dictyostelium pathways appear more linear, with each Ras apparently performing a specific cellular function.     

Regulation of phagocytosis and endo-phagosomal trafficking pathways in Dictyostelium discoideum

Phagocytosis, a critically important process employed by leukocytes against invading pathogens, is an actin-dependent clathrin-independent process that results in the internalization of particles >0.5 microm in diameter. Phagocytosis consists of a number of stages, including the binding of particles to the cell surface via interaction with a receptor, engulfment of the particle by pseudopod extension, and fission and fusion reactions to form phago-lysosomes. Much remains to be learned concerning the molecular mechanisms that regulate particle internalization and phagosome maturation. Dictyostelium is a genetically tractable professional phagocyte that has proven useful in determining the molecular steps involved in these processes. We will summarize, in this chapter, what we currently understand concerning the molecular mechanisms that regulate the process of phagocytosis in Dictyostelium, and we will compare and contrast this body of information with that available describing phagocytosis in higher organisms. We will also present current information that suggests that macropinocytosis, a process morphologically similar to phagocytosis, utilizes a different signaling pathway than phagocytosis. Finally, we will discuss the process of maturation of phagosomes, which requires membrane trafficking events, and we will summarize data that support the use of Dictyostelium as a model to determine how intracellular pathogens survive.     

Lead genetic studies in Dictyostelium discoideum and translational studies in human cells demonstrate that sphingolipids are key regulators of sensitivity to cisplatin and other anticancer drugs

A Dictyostelium discoideum mutant with a disruption in the sphingosine-1-phosphate (S-1-P) lyase gene was obtained in an unbiased genetic analysis, using random insertional mutagenesis, for mutants with increased resistance to the widely used cancer chemotherapeutic drug cisplatin. This finding opened the way to extensive studies in both D. discoideum and human cells on the role and mechanism of action of the bioactive sphingolipids S-1-P and ceramide in regulating the response to chemotherapeutic drugs. These studies showed that the levels of activities of the sphingolipid metabolizing enzymes S-1-P lyase, sphingosine kinase and ceramide synthase, affect whether a cell dies or lives in the presence of specific drugs. The demonstration that multiple enzymes of this biochemical pathway were involved in regulating drug sensitivity provided new opportunities to test whether pharmacological intervention might increase sensitivity. Thus it is of considerable clinical significance that pharmacological inhibition of sphingosine kinase synergistically sensitizes cells to cisplatin, both in D. discoideum and human cells. Linkage to the p38 MAP kinase and protein kinase C (PKC) signaling pathways has been demonstrated. This work demonstrates the utility of D. discoideum as a lead genetic system to interrogate molecular mechanisms controlling the sensitivity of tumor cells to chemotherapeutic agents and for determining novel ways of increasing efficacy. The D. discoideum system could be easily adapted to a high throughput screen for novel chemotherapeutic agents.