Tipping the scales: Lessons from simple model systems on inositol imbalance in neurological disorders.

Inositol and inositol-containing compounds have signalling and regulatory roles in many cellular processes, suggesting that inositol imbalance may lead to wide-ranging changes in cellular functions. Indeed, changes in inositol-dependent signalling have been implicated in various diseases and cellular functions such as autophagy, and these changes have often been proposed as therapeutic targets. However, few studies have highlighted the links between inositol depletion and the downstream effects on inositol phosphates and phosphoinositides in disease states. For this research, many advances have employed simple model systems that include the social amoeba D. discoideum and the yeast S. cerevisiae, since these models enable a range of experimental approaches that are not possible in mammalian models. In this review, we discuss recent findings initiated in simple model systems and translated to higher model organisms where the effect of altered inositol, inositol phosphate and phosphoinositide levels impact on bipolar disorder, Alzheimer disease, epilepsy and autophagy.

Employing Dictyostelium as an Advantageous 3Rs Model for Pharmacogenetic Research.

Increasing concern regarding the use of animals in research has triggered a growing need for non-animal research models in a range of fields. The development of 3Rs (replacement, refinement, and reduction) approaches in research, to reduce the reliance on the use of animal tissue and whole-animal experiments, has recently included the use of Dictyostelium. In addition to not feeling pain and thus being relatively free of ethical constraints, Dictyostelium provides a range of distinct methodological advantages for researchers that has led to a number of breakthroughs. These methodologies include using cell behavior (cell movement and shape) as a rapid indicator of sensitivity to poorly characterized medicines, natural products, and other chemicals to help understand the molecular mechanism of action of compounds. Here, we outline a general approach to employing Dictyostelium as a 3Rs research model, using cell behavior as a readout to better understand how compounds, such as the active ingredient in chilli peppers, capsaicin, function at a cellular level. This chapter helps scientists unfamiliar with Dictyostelium to rapidly employ it as an advantageous model system for research, to reduce the use of animals in research, and to make paradigm shift advances in our understanding of biological chemistry.

Non-Catalytic Roles of Presenilin Throughout Evolution.

Research into Alzheimer's disease pathology and treatment has often focused on presenilin proteins. These proteins provide the key catalytic activity of the γ-secretase complex in the cleavage of amyloid-ß precursor protein and resultant amyloid tangle deposition. Over the last 25 years, screening novel drugs to control this aberrant proteolytic activity has yet to identify effective treatments for the disease. In the search for other mechanisms of presenilin pathology, several studies have demonstrated that mammalian presenilin proteins also act in a non-proteolytic role as a scaffold to co-localize key signaling proteins. This role is likely to represent an ancestral presenilin function, as it has been described in genetically distant species including non-mammalian animals, plants, and a simple eukaryotic amoeba Dictyostelium that diverged from the human lineage over a billion years ago. Here, we review the non-catalytic scaffold role of presenilin, from mammalian models to other biomedical models, and include recent insights using Dictyostelium, to suggest that this role may provide an early evolutionary function of presenilin proteins.

The Inositol-3-Phosphate Synthase Biosynthetic Enzyme Has Distinct Catalytic and Metabolic Roles.

Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders, including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium discoideum, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in an inositol auxotrophy that can be rescued only partially by exogenous inositol. Removal of inositol supplementation from the ino1(-) mutant resulted in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis, and substrate adhesion. Inositol depletion also caused a dramatic generalized decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 that is independent of inositol biosynthesis. To characterize this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) and having homology to mammalian macromolecular complex adaptor proteins was identified. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.

An ancestral non-proteolytic role for presenilin proteins in multicellular development of the social amoeba Dictyostelium discoideum.

Mutations in either of two presenilin genes can cause familial Alzheimer's disease. Presenilins have both proteolysis-dependent functions, as components of the γ-secretase complex, and proteolysis-independent functions in signalling. In this study, we investigate a conserved function of human presenilins in the development of the simple model organism Dictyostelium discoideum. We show that the block in Dictyostelium development caused by the ablation of both Dictyostelium presenilins is rescued by the expression of human presenilin 1, restoring the terminal differentiation of multiple cell types. This developmental role is independent of proteolytic activity, because the mutation of both catalytic aspartates does not affect presenilin ability to rescue development, and the ablation of nicastrin, a γ-secretase component that is crucial for proteolytic activity, does not block development. The role of presenilins during Dictyostelium development is therefore independent of their proteolytic activity. However, presenilin loss in Dictyostelium results in elevated cyclic AMP (cAMP) levels and enhanced stimulation-induced calcium release, suggesting that presenilins regulate these intracellular signalling pathways. Our data suggest that presenilin proteins perform an ancient non-proteolytic role in regulating intracellular signalling and development, and that Dictyostelium is a useful model for analysing human presenilin function.

The roles of flotillin microdomains--endocytosis and beyond.

Flotillins are membrane proteins that form microdomains in the plasma membrane of all mammalian cell types studied to date. They span the evolutionary spectrum, with proteins related to flotillins present in bacteria, fungi, plants and metazoans, which suggests that they perform important, and probably conserved, functions. Flotillins have been implicated in myriad processes that include endocytosis, signal transduction and regulation of the cortical cytoskeleton, yet the molecular mechanisms that underlie flotillin function in these different cases are still poorly understood. In this Commentary, we will provide an introduction to these intriguing proteins, summarise their proposed functions and discuss in greater detail some recent insights into the role of flotillin microdomains in endocytosis that have been provided by several independent studies. Finally, we will focus on the questions that are raised by these new experiments and their implications for future studies.

Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment.

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.

A novel syntaxin 6-interacting protein, SHIP164, regulates syntaxin 6-dependent sorting from early endosomes.

Membrane fusion is dependent on the function of SNAREs and their alpha-helical SNARE motifs that form SNARE complexes. The Habc domains at the N-termini of some SNAREs can interact with their associated SNARE motif, Sec1/Munc18 (SM) proteins, tethering proteins or adaptor proteins, suggesting that they play an important regulatory function. We screened for proteins that interact with the Habc domain of Syntaxin 6, and isolated an uncharacterized 164-kDa protein that we named SHIP164. SHIP164 is part of a large (approximately 700 kDa) complex, and interacts with components of the Golgi-associated retrograde protein (GARP) tethering complex. Depletion of GARP subunits or overexpression of Syntaxin 6 results in a redistribution of soluble SHIP164 to endosomal structures. Co-overexpression of Syntaxin 6 and SHIP164 produced excessive tubulation of endosomes, and perturbed the transport of cation-independent mannose-6-phosphate receptor (CI-MPR) and transferrin receptor. Thus,we propose that SHIP164 functions in trafficking through the early/recycling endosomal system.

Function of the Dictyostelium discoideum Atg1 kinase during autophagy and development.

When starved, the amoebae of Dictyostelium discoideum initiate a developmental process that results in the formation of fruiting bodies in which stalks support balls of spores. The nutrients and energy necessary for development are provided by autophagy. Atg1 is a protein kinase that regulates the induction of autophagy in the budding yeast Saccharomyces cerevisiae. In addition to a conserved kinase domain, Dictyostelium Atg1 has a C-terminal region that has significant homology to the Caenorhabditis elegans and mammalian Atg1 homologues but not to the budding yeast Atg1. We investigated the function of the kinase and conserved C-terminal domains of D. discoideum Atg1 (DdAtg1) and showed that these domains are essential for autophagy and development. Kinase-negative DdAtg1 acts in a dominant-negative fashion, resulting in a mutant phenotype when expressed in the wild-type cells. Green fluorescent protein-tagged kinase-negative DdAtg1 colocalizes with red fluorescent protein (RFP)-tagged DdAtg8, a marker of preautophagosomal structures and autophagosomes. The conserved C-terminal region is essential for localization of kinase-negative DdAtg1 to autophagosomes labeled with RFP-tagged Dictyostelium Atg8. The dominant-negative effect of the kinase-defective mutant also depends on the C-terminal domain. In cells expressing dominant-negative DdAtg1, autophagosomes are formed and accumulate but seem not to be functional. By using a temperature-sensitive DdAtg1, we showed that DdAtg1 is required throughout development; development halts when the cells are shifted to the restrictive temperature, but resumes when cells are returned to the permissive temperature.

Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells.

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA-mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

Autophagy gene disruption reveals a non-vacuolar cell death pathway in Dictyostelium.

Types of cell death include apoptosis, necrosis, and autophagic cell death. The latter can be defined as death of cells containing autophagosomes, autophagic bodies, and/or vacuoles. Are autophagy and vacuolization causes, consequences, or side effects in cell death with autophagy? Would control of autophagy suffice to control this type of cell death? We disrupted the atg1 autophagy gene in Dictyostelium discoideum, a genetically tractable model for developmental autophagic vacuolar cell death. The procedure that induced autophagy, vacuolization, and death in wild-type cells led in atg1 mutant cells to impaired autophagy and to no vacuolization, demonstrating that atg1 is required for vacuolization. Unexpectedly, however, cell death still took place, with a non-vacuolar and centrally condensed morphology. Thus, a cell death mechanism that does not require vacuolization can operate in this cell death model showing conspicuous vacuolization. The revelation of non-vacuolar cell death in this protist by autophagy gene disruption is reminiscent of caspase inhibition revealing necrotic cell death in animal cells. Thus, hidden alternative cell death pathways may be found across kingdoms and for diverse types of cell death.

Dictyostelium macroautophagy mutants vary in the severity of their developmental defects.

Macroautophagy is the major mechanism that eukaryotes use to recycle cellular components during stressful conditions. We have shown previously that the Atg12-Atg5 conjugation system, required for autophagosome formation in yeast, is necessary for Dictyostelium development. A second conjugation reaction, Aut7/Atg8 lipidation with phosphatidylethanolamine, as well as a protein kinase complex and a phosphatidylinositol 3-kinase complex are also required for macroautophagy in yeast. In this study, we characterize mutations in the putative Dictyostelium discoideum orthologues of budding yeast genes that are involved in one of each of these functions, ATG1, ATG6, and ATG8. All three genes are required for macroautophagy in Dictyostelium. Mutant amoebae display reduced survival during nitrogen starvation and reduced protein degradation during development. Mutations in the three genes produce aberrant development with defects of varying severity. As with other Dictyostelium macroautophagy mutants, development of atg1-1, atg6(-), and atg8(-) is more aberrant in plaques on bacterial lawns than on nitrocellulose filters. The most severe defect is observed in the atg1-1 mutant, which does not aggregate on bacterial lawns and arrests as loose mounds on nitrocellulose filters. The atg6(-) and atg8(-) mutants display almost normal development on nitrocellulose filters, producing multi-tipped aggregates that mature into small fruiting bodies. The distribution of a green fluorescent protein fusion of the autophagosome marker, Atg8, is aberrant in both atg1-1 and atg6(-) mutants.

Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum.

The Gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free-living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D. discoideum. Furthermore, a green fluorescent protein (GFP)-tagged marker of autophagosomes, Apg8, does not systematically co-localize with DsRed-labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum.

Macroautophagy is required for multicellular development of the social amoeba Dictyostelium discoideum.

Macroautophagy is a mechanism employed by eukaryotic cells to recycle non-essential cellular components during starvation, differentiation, and development. Two conjugation reactions related to ubiquitination are essential for autophagy: Apg12p conjugation to Apg5p, and Apg8p conjugation to the lipid phosphatidylethanolamine. These reactions require the action of the E1-like enzyme, Apg7p, and the E2-like enzymes, Apg3p and Apg10p. In Dictyostelium, development is induced by starvation, conditions under which autophagy is required for survival in yeast and plants. We have identified Dictyostelium homologues of 10 budding yeast autophagy genes. We have generated mutations in apg5 and apg7 that produce defects typically associated with an abrogation of autophagy. Mutants are not grossly affected in growth, but survival during nitrogen starvation is severely reduced. Starved mutant cells show little turnover of cellular constituents by electron microscopy, whereas wild-type cells show significant cytoplasmic degradation and reduced organelle number. Bulk protein degradation during starvation-induced development is reduced in the autophagy mutants. Development is aberrant; the autophagy mutants do not aggregate in plaques on bacterial lawns, but they do proceed further in development on nitrocellulose filters, forming defective fruiting bodies. The autophagy mutations are cell autonomous, because wild-type cells in a chimaera do not rescue development of the autophagy mutants. We have complemented the mutant phenotypes by expression of the cognate gene fused to green fluorescent protein. A green fluorescent protein fusion of the autophagosome marker Apg8 mislocalizes in the two autophagy mutants. We show that the Apg5-Apg12 conjugation system is conserved in Dictyostelium.