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, 22 (1), 105-13

Molecular Pharmacology in a Simple Model System: Implicating MAP Kinase and Phosphoinositide Signalling in Bipolar Disorder


Molecular Pharmacology in a Simple Model System: Implicating MAP Kinase and Phosphoinositide Signalling in Bipolar Disorder

Marthe H R Ludtmann et al. Semin Cell Dev Biol.


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.


Fig. 1
Fig. 1
Dictyostelium as a simple model for molecular pharmacology research. Under control growth conditions; (1) Dictyostelium cells multiply by binary fission either in liquid media or in association with bacteria as a food source, until resources are depleted. (2) Induction of starvation causes cells to stop diving and enter a developmental phase, whereby expression of a range of developmental genes enables cells to move together in a process called chemotaxis (where cells move towards cyclic AMP). (3) Following aggregation of cells, Dictyostelium undergoes developmental differentiation to form a multi-cellular fruiting body, of around 1 mm in height, consisting primarily of spore and stalk cells. The development of molecular cell biology techniques for Dictyostelium research has enabled this model to be used in molecular pharmacology studies using either: (4a) Cell lines containing single or multiple ablated (or over-expressed) genes (green); or (4b) Pools of random insertional mutants (prepared using REMI; yellow) to provide mutant libraries for drug resistance screens. (5) The cellular role of pharmacological treatments (such as VPA and lithium (Li+)) can be analysed using either wild type cells, or cell lines containing ablated or over-expressed genes. (6) Analysis of resistance to the developmental block by VPA/Li+ can also help to understand and characterise the role of specific genes or identify novel genes involved in the action of bipolar disorder treatments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Fig. 2
Fig. 2
MAPK regulation by bipolar disorder treatments in the simple biomedical model Dictyostelium. In both Dictyostelium and mammalian systems, valproic acid (VPA) and lithium (Li+) treatment leads to an increase in activated MAPK, illustrated here for the phosphorylation of the ERK2 (extracellular regulated kinase 2 [ERK2] to form pERK2). (A) Regulation of pERK2 levels is complex, provided by a balance between kinase-dependent phosphorylation (catalysed by MEKK [mitogen-activated protein kinase kinase kinse], and MEK [MAPK kinase kinase]) and by de-phosphorylation (catalysed by MKP [MAP kinase phosphatase] in the PKA [protein kinase A] and GSK3/A [glycogen synthase kinase 3/A] signalling pathway). (B) Treatment of Dictyostelium cells with a single pulse of cyclic AMP (cAMP) gives rise to a transient increase in pERK2 detected by using phosphorylation-specific antibodies. Pre-incubation of Dictyostelium cells with VPA (1 mM, 60 min) increases and elongates the pathway activation by an unknown mechanism. (C) Research in Dictyostelium enables the use of isogenic cell lines containing either ablated or over-expressed genes, providing valuable tools for pharmacological research. For example, we can examine the role of GSKA (homologous to the human GSK3 proteins) in this effect by comparing pERK2 levels in wild-type cells and those lacking GSKA, under control conditions (no added drug) or following Li+ (10 mM, 60 min, dark grey) OR VPA (1 mM, 60 min, light grey) treatment. GSKA ablation increases pERK2 levels, suggesting that it functions as a negative regulator of this pathway, as does pharmacological inhibition with Li+. The lithium-dependent increase in pERK2 is also significantly reduced in the GSKA null, in agreement with a lithium/GSKA-dependent mechanism of action. In contrast, VPA causes a hypersensitive pERK2 increase in this mutant, suggesting VPA functions through a GSKA-independent mechanism. This approach shows an unambiguous role of GSKA in the transient formation of pERK2. * P < 0.05 compared to control. (D) Novel compounds related to VPA can also be analysed in structure–activity relationship studies (SARs), where for example, the amide derivative of VPA (valpromide), enantiomeric compounds (R- and S-2-pentyl-4-pentynoic acid), and altered branch derivatives (2-ethyl-4-methylpentanoic acid) show altered pathway activation. **P < 0.01, *P < 0.05 compared to control.
Fig. 3
Fig. 3
Inositol phosphate and phosphoinositide signalling regulation by bipolar disorder treatments in the simple biomedical model Dictyostelium. (A) Inositol signalling initiates from the phospholipase C (PLC)-generated cleavage of membrane-bound phosphatidylinositol 4,5-biphosphate (PIP2) to produce diacylglycerol (DAG) and cytosolic inositol 1,4,5-triphosphate (InsP3). Prolyl oligopeptidase (PO) negatively regulates multiple inositol polyphosphate phosphatase (MIPP) which in turn catalyses higher order inositol (InsP4, InsP5, InsP6) breakdown to form InsP3. InsP3 is hydrolysed to InsP2 and further broken down by Inositol polyphosphate phosphatase (IPPase) and inositol monophophatase (IMPase) to produce inositol. This is then incorporated into phosphoinositol signalling to form phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and then PIP2. DAG is recycled via phosphatidic acid (PA). Lithium plays a well-documented role in inhibiting IMPase and IPPase to reduce inositol-based signalling, and VPA has been suggested to reduce de novo inositol biosynthesis through indirect inhibition of inositol synthase 1 (INO1), and the turnover of phosphoinositides (all indicated in green). (B) The inositol head group of the family of phosphoinositide compounds act as a substrate for a range of kinases, providing a dynamic and complex network of products and substrates. Addition of radio-labelled phosphate (red) to these kinase reactions gives rise to incorporation of radiolabel into defined places on the inositol ring (shown here for phosphatidylinositol 3-kinase (PI3K)). (C) Multiple substrates (shown here for PI3K) produce multiple products. Inositol ring (hexagon), P (black) phosphorylation site, P (Bold red) identified through Dictyostelium radio-labelling turnover experiments, enzymes in blue (italics); ↓ decreased. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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