Kazuo Emoto

Mitochondrial Phospholipid Hydroperoxide Glutathione Peroxidase Plays a Major Role in Preventing Oxidative Injury to Cells

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is synthesized as a long form (L-form; 23 kDa) and a short form (S-form; 20 kDa). The L-form contains a leader sequence that is required for transport to mitochondria, whereas the S-form lacks the leader sequence. A construct encoding the leader sequence of PHGPx tagged with green fluorescent protein was used to transfect RBL-2H3 cells, and the fusion protein was transported to mitochondria. The L-form of PHGPx was identified as the mitochondrial form of PHGPx and the S-form as the non-mitochondrial form of PHGPx since preferential enrichment of mitochondria for PHGPx was detected in M15 cells that overexpressed theL-form of PHGPx, whereas no similar enrichment was detected in L9 cells that overexpressed the S-form. Cell death caused by mitochondrial injury due to potassium cyanide (KCN) or rotenone (chemical hypoxia) was considerably suppressed in the M15 cells, whereas the L9 cells and control RBL-2H3 cells (S1 cells, transfected with the vector alone) succumbed to the cytotoxic effects of KCN. Flow cytometric analysis showed that mitochondrial PHGPx suppressed the generation of hydroperoxide, the loss of mitochondrial membrane potential, and the loss of plasma membrane integrity that are induced by KCN. Mitochondrial PHGPx might prevent changes in mitochondrial functions and cell death by reducing intracellular hydroperoxides. Mitochondrial PHGPx failed to protect M15 cells from mitochondrial injury by carbonyl cyanide m-chlorophenylhydrazone, which directly reduces membrane potential without the generation of hydroperoxides. M15 cells were more resistant than L9 cells to cell death caused by direct damage to mitochondria and to extracellular oxidative stress. L9 cells were more resistant totert-butylhydroperoxide than S1 cells, whereas resistance to t-butylhydroperoxide was even more pronounced in M15 cells than in L9 cells. These results suggest that mitochondria might be a target for intracellular and extracellular oxidative stress and that mitochondrial PHGPx, as distinct form non-mitochondrial PHGPx, might play a primary role in protecting cells from oxidative stress.

Control of Dendritic Branching and Tiling by the Tricornered-Kinase/Furry Signaling Pathway in Drosophila Sensory Neurons

To cover the receptive field completely but without redundancy, neurons of certain functional groups exhibit tiling of their dendrites via dendritic repulsion. Here we show that two evolutionarily conserved proteins, the Tricornered (Trc) kinase and Furry (Fry), are essential for tiling and branching control of Drosophila sensory neuron dendrites. Dendrites of fry and trc mutants display excessive terminal branching and fail to avoid homologous dendritic branches, resulting in significant overlap of the dendritic fields. Trc control of dendritic branching involves regulation of RacGTPase, a pathway distinct from the action of Trc in tiling. Timelapse analysis further reveals a specific loss of the ability of growing dendrites to turn away from nearby dendritic branches in fry mutants, suggestive of a defect in like-repels-like avoidance. Thus, the Trc/Fry signaling pathway plays a key role in patterning dendritic fields by promoting avoidance between homologous dendrites as well as by limiting dendritic branching.

Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization

A neurons dendrites typically do not cross one another. This intrinsic self-avoidance mechanism ensures unambiguous processing of sensory or synaptic inputs. Moreover, some neurons respect the territory of others of the same type, a phenomenon known as tiling. Different types of neurons, however, often have overlapping dendritic fields. We found that Downs syndrome Cell Adhesion Molecule (Dscam) is required for dendritic self-avoidance of all four classes of Drosophila dendritic arborization (da) neurons. However, neighboring mutant class IV da neurons still exhibited tiling, suggesting that self-avoidance and tiling differ in their recognition and repulsion mechanisms. Introducing 1 of the 38,016 Dscam isoforms to da neurons in Dscam mutants was sufficient to significantly restore self-avoidance. Remarkably, expression of a common Dscam isoform in da neurons of different classes prevented their dendrites from sharing the same territory, suggesting that coexistence of dendritic fields of different neuronal classes requires divergent expression of Dscam isoforms.

The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance.

Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a ‘like-repels-like’ behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. Here we report that the other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. We further provide biochemical and genetic evidence for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis.

Failure of the Expression of Phospholipid Hydroperoxide Glutathione Peroxidase in the Spermatozoa of Human Infertile Males

Abstract Phospholipid hydroperoxide glutathione peroxidase (PHGPx) was intensely expressed in mitochondria in the midpiece of human spermatozoa by immunostaining with anti-PHGPx monoclonal antibodies. The PHGPx not only reduced phospholipid hydroperoxide but also scavenged hydrogen peroxide in human spermatozoa. We found a dramatic decrease in the level of expression of PHGPx in the spermatozoa of some infertile males by immunoblotting with anti-PHGPx monoclonal antibodies. These individuals accounted for about 10% of the group of 73 infertile males that we examined. All seven patients with PHGPx-defective spermatozoa were classified as suffering from oligoasthenozoospermia, a defect in which both the number and the motility of spermatozoa are significantly below normal. Males with PHGPx-defective spermatozoa accounted for 26% of the 27 infertile males with oligoasthenozoospermia. No defects in expression of PHGPx in spermatozoa were observed in 31 fertile volunteers. After a 3-h incubation, the relative number of motile spermatozoa with low-level expression of PHGPx was significantly lower than that of spermatozoa with normal expression of PHGPx. The PHGPx-defective spermatozoa failed to incorporate rhodamine 123, revealing a loss of mitochondrial membrane potential. Ultrastructual analysis of mitochondria by electron microscopy demonstrated that the morphology of mitochondria in PHGPx-defective spermatozoa was abnormal. The results suggest that failure of the expression of mitochondrial PHGPx in spermatozoa might be one of the causes of oligoasthenozoospermia in infertile men.

Local Change in Phospholipid Composition at the Cleavage Furrow Is Essential for Completion of Cytokinesis

Cell division ends up with the membrane separation of two daughter cells, presumably by a membrane fusion that requires dynamic changes of the distribution and the composition of membrane lipids. We have previously shown that a membrane lipid phosphatidylethanolamine (PE) is exposed on the cell surface of the cleavage furrow during late cytokinesis and that this PE movement is involved in regulation of the contractile ring disassembly. Here we show that immobilization of cell surface PE by a PE-binding peptide blocks the RhoA inactivation in the late stage of cytokinesis. Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), but not other RhoA effectors, is co-localized with RhoA in the peptide-treated cells. Indeed, PIP5K and its product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are localized to the cleavage furrow of normally dividing cells. Both overexpression of a kinase-deficient PIP5K mutant and microinjection of anti-PI(4,5)P2 antibodies compromise cytokinesis by preventing local accumulation of PI(4,5)P2 in the cleavage furrow. These findings demonstrate that the localized production of PI(4,5)P2 is required for the proper completion of cytokinesis and that the possible formation of a unique lipid domain in the cleavage furrow membrane may play a crucial role in coordinating the contractile rearrangement with the membrane remodeling during late cytokinesis.

A Novel Membrane Protein, Ros3p, Is Required for Phospholipid Translocation across the Plasma Membrane in Saccharomyces cerevisiae

Ro09-0198 (Ro) is a tetracyclic peptide antibiotic that binds specifically to phosphatidylethanolamine (PE) and causes cytolysis. To investigate the molecular basis of transbilayer movement of PE in biological membranes, we have isolated a series of budding yeast mutants that are hypersensitive to the Ro peptide. One of the most sensitive mutants, designated ros3(Ro-sensitive 3), showed no significant change in the cellular phospholipid composition or in the sensitivity to amphotericin B, a sterol-binding polyene macrolide antibiotic. These results suggest that the mutation of ros3affects the PE organization on the plasma membrane, rather than PE synthesis or overall organization of the membrane structures. By functional complementation screening, we identified the geneROS3 affected in the mutant, and we showed that the hypersensitive phenotype was caused by the defective expression of theROS3 gene product, Ros3p, an evolutionarily conserved protein with two putative transmembrane domains. Disruption of theROS3 gene resulted in a marked decrease in the internalization of fluorescence-labeled analogs of PE and phosphatidylcholine, whereas the uptake of fluorescence-labeled phosphatidylserine and endocytic markers was not affected. Neither expression levels nor activities of ATP-binding cassette transporters of the ros3Δ cells differed from those of wild type cells, suggesting that Ros3p is not related to the multidrug resistance activities. Immunochemical analyses of the structure and subcellular localization showed that Ros3p was a glycosylated membrane protein localized in both the plasma membrane and the endoplasmic reticulum, and that a part of Ros3p was associated with the detergent-insoluble glycolipid-enriched complexes. These results indicate that Ros3p is a membrane glycoprotein that plays an important role in the phospholipid translocation across the plasma membrane.

The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway

To cover the receptive field completely and non‐redundantly, neurons of certain functional groups arrange tiling of their dendrites. In Drosophila class IV dendrite arborization (da) neurons, the NDR family kinase Tricornered (Trc) is required for homotypic repulsion of dendrites that facilitates dendritic tiling. We here report that Sin1, Rictor, and target of rapamycin (TOR), components of the TOR complex 2 (TORC2), are required for dendritic tiling of class IV da neurons. Similar to trc mutants, dendrites of sin1 and rictor mutants show inappropriate overlap of the dendritic fields. TORC2 components physically and genetically interact with Trc, consistent with a shared role in regulating dendritic tiling. Moreover, TORC2 is essential for Trc phosphorylation on a residue that is critical for Trc activity in vivo and in vitro. Remarkably, neuronal expression of a dominant active form of Trc rescues the tiling defects in sin1 and rictor mutants. These findings suggest that TORC2 likely acts together with the Trc signalling pathway to regulate the dendritic tiling of class IV da neurons, and thus uncover the first neuronal function of TORC2 in vivo.

Compartmentalized Calcium Transients Trigger Dendrite Pruning in Drosophila Sensory Neurons

Dendritic Pruning During metamorphosis, Drosophila sensory neurons eliminate their dendritic trees, but axons and soma remain intact. Kanamori et al. (p. 1475, published online 30 May) demonstrate that compartmentalized calcium transients in dendrites function as the spatiotemporal cue for pruning of unwanted branches. Such a localized calcium signal, induced by a local elevation of branch excitability, activates calcium-dependent proteinases and eventually causes branch death. During fruit fly metamorphosis, dendritic calcium signaling defines the branches to be eliminated in sensory neurons. Dendrite pruning is critical for sculpting the final connectivity of neural circuits as it removes inappropriate projections, yet how neurons can selectively eliminate unnecessary dendritic branches remains elusive. Here, we show that calcium transients that are compartmentalized in specific dendritic branches act as temporal and spatial cues to trigger pruning in Drosophila sensory neurons. Calcium transients occurred in local dendrites at ~3 hours before branch elimination. In dendritic branches, intrinsic excitability increased locally to activate calcium influx via the voltage-gated calcium channels (VGCCs), and blockade of the VGCC activities impaired pruning. Further genetic analyses suggest that the calcium-activated protease calpain functions downstream of the calcium transients. Our findings reveal the importance of the compartmentalized subdendritic calcium signaling in spatiotemporally selective elimination of dendritic branches.

The Drosophila caspase DRONC is required for metamorphosis and cell death in response to irradiation and developmental signals

Cell death is essential for eliminating excess cells during development as well as removing damaged cells. While multiple conserved apoptosis pathways involving different cascades of caspases, which are cysteine proteases, have been identified, their regulation in the context of a developing organism is not very well understood. Expression of the Drosophila caspase-9 homolog, DRONC, can be induced by ecdysone, a steroid hormone, which induces metamorphosis. To elucidate the functional role of DRONC during metamorphosis and for cell death during development we have generated and analyzed two loss-of-function alleles of DRONC. We report that DRONC is required for developmentally induced neuroblast cell death and apoptosis in response to X irradiation. DRONC mutants show reduced pupariation even in the presence of high levels of ecdysone and impaired cell death of larval midgut. The levels of ecdysone-inducible transcripts such as E75A and Reaper (Rpr) are normal in the absence of DRONC, suggesting that DRONC acts downstream of these genes. In addition, Reaper and Grim, but not Hid induced apoptosis is sensitive to a reduction of DRONC levels. Our study places DRONC at a central point of convergence for multiple cell death pathways and for the ecdysone pathway regulating metamorphosis.