IRE-1/XBP-1 pathway of the unfolded protein response is required for properly localizing neuronal UNC-6/Netrin for axon guidance in C. elegans.

During developing nervous system, neurons project axons to their targets precisely. In this process, axon guidance molecules provide positional information to the axons. Therefore, the spatially and temporally controlled localization of the axon guidance molecules is required for the proper structure formation of the complex nervous system. In C. elegans, UNC-6/Netrin is a secreted protein that elicits both attractive and repulsive response in axon guidance. UNC-6/Netrin secreted from ventral cells may establish a concentration gradient from the ventral to the dorsal side of the animal, thus providing dorso-ventral positional information. However, the mechanisms specifying positional information of UNC-6/Netrin are largely unknown. Here, we show that the ire-1/xbp-1 pathway of the unfolded protein response (UPR) is required for axonal distribution of UNC-6/Netrin in the ventral neurons. In addition, the ire-1/xbp-1 pathway is also required for dorso-ventral axon guidance mediated by UNC-6/Netrin. Our results suggest that the ire-1/xbp-1 pathway of the UPR is crucial for establishing positional information of UNC-6/Netrin. We propose that the proper secretion of UNC-6/Netrin from the ventral neurons requires the activity of IRE-1.

Amino- and carboxyl-terminal domains of Filamin-A interact with CRMP1 to mediate Sema3A signalling.

Reorganization of the actin cytoskeleton is an early cellular response to various extracellular signals. Sema3A, a repulsive axon guidance molecule, induces the reorganization of actin cytoskeleton in the growth cones. Collapsin response mediator protein 1 (CRMP1) mediates the intracellular Sema3A signalling through its Ser522 phosphorylation. Here we show that UNC-33, CRMP1 C. elegans homologue, interacts with FLN-1, an actin-binding Filamin-A orthologue. In nematodes, this interaction participates in the projection of DD/VD motor neurons. CRMP1 binds both the actin-binding domain and the last immunoglobulin-like repeat of Filamin-A. The alanine mutants of Filamin-A or CRMP1 in their interacting residues suppress the Sema3A repulsion in neurons. Conversely, a phosphor-mimicking mutant CRMP1(Ser522Asp) enhances the Sema3A response. Atomic-force microscopy analysis reveals that the V-shaped Filamin-A changes to a condensed form with CRMP1(Ser522Asp). CRMP1(Ser522Asp) weakens the F-actin gelation crosslinked by Filamin-A. Thus, phosphorylated CRMP1 may remove Filamin-A from the actin cytoskeleton to facilitate its remodelling.

Localization mechanisms of the axon guidance molecule UNC-6/Netrin and its receptors, UNC-5 and UNC-40, in Caenorhabditis elegans.

Netrin is an evolutionarily conserved, secretory axon guidance molecule. Netrin's receptors, UNC-5 and UNC-40/DCC, are single trans-membrane proteins with immunoglobulin domains at their extra-cellular regions. Netrin is thought to provide its positional information by establishing a concentration gradient. UNC-5 and UNC-40 act at growth cones, which are specialized axonal tip structures that are generally located at a long distance from the neural cell body. Thus, the proper localization of both Netrin and its receptors is critical for their function. This review addresses the localization mechanisms of UNC-6/Netrin and its receptors in Caenorhabditis elegans, focusing on our recent reports. These findings include novel insights on cytoplasmic proteins that function upstream of the receptors.

A seven-transmembrane receptor that mediates avoidance response to dihydrocaffeic acid, a water-soluble repellent in Caenorhabditis elegans.

The ability to detect harmful chemicals rapidly is essential for the survival of all animals. In Caenorhabditis elegans (C. elegans), repellents trigger an avoidance response, causing animals to move away from repellents. Dihydrocaffeic acid (DHCA) is a water-soluble repellent and nonflavonoid catecholic compound that can be found in plant products. Using a Xenopus laevis (X. laevis) oocyte expression system, we identified a candidate dihydrocaffeic acid receptor (DCAR), DCAR-1. DCAR-1 is a novel seven-transmembrane protein that is expressed in the ASH avoidance sensory neurons of C. elegans. dcar-1 mutant animals are defective in avoidance response to DHCA, and cell-specific expression of dcar-1 in the ASH neurons of dcar-1 mutant animals rescued the defect in avoidance response to DHCA. Our findings identify DCAR-1 as the first seven-transmembrane receptor required for avoidance of a water-soluble repellent, DHCA, in C. elegans.

Mutation in a mitochondrial ribosomal protein causes increased sensitivity to oxygen with decreased longevity in the nematode Caenorhabditis elegans.

Oxygen is essential for animals, but high concentrations of oxygen are toxic to them probably because of an increase in reactive oxygen species (ROS). Many genes are involved in the reactions from which ROS are generated, but not much attention has been focused on them. To identify these genes, we screened for mutants with an altered sensitivity to oxidative stress in the nematode Caenorhabditis elegans and isolated a mutant, oxy-5(qa5002). oxy-5 showed an increased sensitivity to oxygen and decreased longevity. The decreased life span in oxy-5 was probably due to increased oxidative stress because it was recovered to a normal level when oxy-5 was cultured under hypoxic conditions. Our genetic analysis has revealed that the responsible gene for oxy-5 encodes a protein similar to mitochondrial ribosomal protein S36. The OXY-5 protein was highly expressed in the neurons, pharynx, and intestine, and expression of oxy-5 from the pan-neuronal H20 promoter efficiently suppressed the increased sensitivity to oxygen in oxy-5. These findings suggested that oxy-5 played an important role in the regulation of the sensitivity to oxygen in neuronal cells in C. elegans.

Genes required for cellular UNC-6/netrin localization in Caenorhabditis elegans.

UNC-6/Netrin is an evolutionarily conserved, secretory axon guidance molecule. In Caenorhabditis elegans, UNC-6 provides positional information to the axons of developing neurons, probably by establishing a concentration gradient from the ventral to the dorsal side of the animal. Although the proper localization of UNC-6 is important for accurate neuronal network formation, little is known about how its localization is regulated. Here, to examine the localization mechanism for UNC-6, we generated C. elegans expressing UNC-6 tagged with the fluorescent protein Venus and identified 13 genes, which are involved in the cellular localization of VenusUNC-6. For example, in unc-51, unc-14, and unc-104 mutants, the neurons showed an abnormal accumulation of VenusUNC-6 in the cell body and less than normal level of VenusUNC-6 in the axon. An aberrant accumulation of VenusUNC-6 in muscle cells was seen in unc-18 and unc-68 mutants. unc-51, unc-14, and unc-104 mutants also showed defects in the guidance of dorso-ventral axons, suggesting that the abnormal localization of UNC-6 disturbed the positional information it provides. We propose that these genes regulate the process of UNC-6 secretion: expression, maturation, sorting, transport, or exocytosis. Our findings provide novel insight into the localization mechanism of the axon guidance molecule UNC-6/Netrin.

Protein phosphatase 2A cooperates with the autophagy-related kinase UNC-51 to regulate axon guidance in Caenorhabditis elegans.

UNC-51 is a serine/threonine protein kinase conserved from yeast to humans. The yeast homolog Atg1 regulates autophagy (catabolic membrane trafficking) required for surviving starvation. In C. elegans, UNC-51 regulates the axon guidance of many neurons by a different mechanism than it and its homologs use for autophagy. UNC-51 regulates the subcellular localization (trafficking) of UNC-5, a receptor for the axon guidance molecule UNC-6/Netrin; however, the molecular details of the role for UNC-51 are largely unknown. Here, we report that UNC-51 physically interacts with LET-92, the catalytic subunit of serine/threonine protein phosphatase 2A (PP2A-C), which plays important roles in many cellular functions. A low allelic dose of LET-92 partially suppressed axon guidance defects of weak, but not severe, unc-51 mutants, and a low allelic dose of PP2A regulatory subunits A (PAA-1/PP2A-A) and B (SUR-6/PP2A-B) partially enhanced the weak unc-51 mutants. We also found that LET-92 can work cell-non-autonomously on axon guidance in neurons, and that LET-92 colocalized with UNC-51 in neurons. In addition, PP2A dephosphorylated phosphoproteins that had been phosphorylated by UNC-51. These results suggest that, by forming a complex, PP2A cooperates with UNC-51 to regulate axon guidance by regulating phosphorylation. This is the first report of a serine/threonine protein phosphatase functioning in axon guidance in vivo.

Roles of adenine anchoring and ion pairing at the coenzyme B12-binding site in diol dehydratase catalysis.

The X-ray structure of the diol dehydratase-adeninylpentylcobalamin complex revealed that the adenine moiety of adenosylcobalamin is anchored in the adenine-binding pocket of the enzyme by hydrogen bonding of N3 with the side chain OH group of Seralpha224, and of 6-NH(2), N1 and N7 with main chain amide groups of other residues. A salt bridge is formed between the epsilon-NH(2) group of Lysbeta135 and the phosphate group of cobalamin. To assess the importance of adenine anchoring and ion pairing, Seralpha224 and Lysbeta135 mutants of diol dehydratase were prepared, and their catalytic properties investigated. The Salpha224A, Salpha224N and Kbeta135E mutants were 19-2% as active as the wild-type enzyme, whereas the Kbeta135A, Kbeta135Q and Kbeta135R mutants retained 58-76% of the wild-type activity. The presence of a positive charge at the beta135 residue increased the affinity for cobalamins but was not essential for catalysis, and the introduction of a negative charge there prevented the enzyme-cobalamin interaction. The Salpha224A and Salpha224N mutants showed a k(cat)/k(inact) value that was less than 2% that of the wild-type, whereas for Lysbeta135 mutants this value was in the range 25-75%, except for the Kbeta135E mutant (7%). Unlike the wild-type holoenzyme, the Salpha224N and Salpha224A holoenzymes showed very low susceptibility to oxygen in the absence of substrate. These findings suggest that Seralpha224 is important for cobalt-carbon bond activation and for preventing the enzyme from being inactivated. Upon inactivation of the Salpha224A holoenzyme during catalysis, cob(II)alamin accumulated, and a trace of doublet signal due to an organic radical disappeared in EPR. 5'-Deoxyadenosine was formed from the adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus considered to be a mechanism-based one.

Histidine-alpha143 assists 1,2-hydroxyl group migration and protects radical intermediates in coenzyme B12-dependent diol dehydratase.

Diol dehydratase of Klebsiella oxytoca contains an essential histidine residue. Its X-ray structure revealed that the migrating hydroxyl group on C2 of substrate is hydrogen-bonded to Hisalpha143. Mutant enzymes in which Hisalpha143 was mutated to another amino acid residue were expressed in Escherichia coli, purified, and examined for enzymatic activity. The Halpha143Q mutant was 34% as active as the wild-type enzyme. Halpha143A and Halpha143L showed only a trace of activity. Kinetic analyses indicated that the hydrogen bonding interaction between the hydroxyl group on C2 of substrate and the side chain of residue alpha143 is important not only for catalysis but also for protecting radical intermediates. Halpha143E and Halpha143K that did not exist as (alphabetagamma) 2 complexes were inactive. The deuterium kinetic isotope effect on the overall reaction suggested that a hydrogen abstraction step is fully rate-determining for the wild type and Halpha143Q and partially rate-determining for Halpha143A. The preference for substrate enantiomers was reversed by the Halpha143Q mutation in both substrate binding and catalysis. Upon the inactivation of the Halpha143A holoenzyme by 1,2-propanediol, cob(II)alamin without an organic radical coupling partner accumulated, 5'-deoxyadenosine was quantitatively formed from the coenzyme adenosyl group, and the apoenzyme itself was not damaged. This inactivation was thus concluded to be a mechanism-based inactivation. The holoenzyme of Halpha143Q underwent irreversible inactivation by O 2 in the absence of substrate at a much lower rate than the wild type.

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons.

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.

C. elegans VAB-8 and UNC-73 regulate the SAX-3 receptor to direct cell and growth-cone migrations.

During nervous system development, a small number of conserved guidance cues and receptors regulate many axon trajectories. How could a limited number of cues and receptors regulate such complex projection patterns? One way is to modulate receptor function. Here we show that the Caenorhabditis elegans kinesin-related protein VAB-8L, which is necessary and sufficient for posterior cell and growth-cone migrations, directs these migrations by regulating the levels of the guidance receptor SAX-3 (also known as robo). Genetic experiments indicate that VAB-8L and the Rac guanine nucleotide exchange factor activity of UNC-73 (trio) increase the ability of the SLT-1 (slit) and UNC-6 (netrin) guidance pathways to promote posterior guidance. The observations of higher SAX-3 receptor abundance in animals with increasing amounts of VAB-8L, and of physical interactions between UNC-73 and both VAB-8L and the intracellular domain of the SAX-3, support a model whereby VAB-8L directs cell and growth-cone migrations by promoting localization of guidance receptors to the cell surface.

The autophagy-related kinase UNC-51 and its binding partner UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5 in Caenorhabditis elegans.

UNC-51 and UNC-14 are required for the axon guidance of many neurons in Caenorhabditis elegans. UNC-51 is a serine/threonine kinase homologous to yeast Atg1, which is required for autophagy. The binding partner of UNC-51, UNC-14, contains a RUN domain that is predicted to play an important role in multiple Ras-like GTPase signaling pathways. How these molecules function in axon guidance is largely unknown. Here we observed that, in unc-51 and unc-14 mutants, UNC-5, the receptor for axon-guidance protein Netrin/UNC-6, abnormally localized in neuronal cell bodies. By contrast, the localization of many other proteins required for axon guidance was undisturbed. Moreover, UNC-5 localization was normal in animals with mutations in the genes for axon guidance proteins, several motor proteins, vesicle components and autophagy-related proteins. We also found that unc-5 and unc-6 interacted genetically with unc-51 and unc-14 to affect axon guidance, and that UNC-5 co-localized with UNC-51 and UNC-14 in neurons. These results suggest that UNC-51 and UNC-14 regulate the subcellular localization of the Netrin receptor UNC-5, and that UNC-5 uses a unique mechanism for its localization; the functionality of UNC-5 is probably regulated by this localization.

Survey of catalytic residues and essential roles of glutamate-alpha170 and aspartate-alpha335 in coenzyme B12-dependent diol dehydratase.

The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K(+) was mutated to Ala or another amino acid residue. The Ealpha170A and Dalpha335A mutants were totally inactive, and the Halpha143A mutant showed only a trace of activity, indicating that Glu-alpha170, Asp-alpha335, and His-alpha143 are catalytic residues. The Qalpha141A, Qalpha296A, and Salpha362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-alpha296 is important for substrate binding and Gln-alpha296 and Gln-alpha141 for preventing the enzyme from mechanism-based inactivation. The Ealpha221A, Ealpha170H, and Dalpha335A did not form the (alphabetagamma)(2) complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-alpha170 and Asp-alpha335 mutants, Ealpha170D and Ealpha170Q were 2.2 +/- 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dalpha335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue alpha170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-alpha170 and Asp-alpha335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-alpha170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.

Translational control of maternal glp-1 mRNA by POS-1 and its interacting protein SPN-4 in Caenorhabditis elegans.

The translation of maternal glp-1 mRNAs is regulated temporally and spatially in C. elegans embryos. The 3' UTR (untranslated region) of the maternal glp-1 mRNA is important for both kinds of regulation. The spatial control region is required to suppress translation in the posterior blastomeres. The temporal one is required to suppress translation in oocytes and one-cell stage embryos. We show that a CCCH zinc-finger protein, POS-1, represses glp-1 mRNA translation by binding to the spatial control region. We identified an RNP-type RNA-binding protein, SPN-4, as a POS-1-interacting protein. SPN-4 is present developmentally from the oocyte to the early embryo and its distribution overlaps with that of POS-1 in the cytoplasm and P granules of the posterior blastomeres. SPN-4 binds to a subregion of the temporal control region in the 3' UTR and is required for the translation of glp-1 mRNA in the anterior blastomeres. We propose that the balance between POS-1 and SPN-4 controls the translation of maternal glp-1 mRNA.

Inclusions in novel perivascular macrophages (Mato's fluorescent granular perithelial cells) and neurons in the cerebral cortex of Hex A- and Hex B-deficient mice.

Beta-hexosaminidases are important enzymes for lipid and saccharide metabolism in the brain. In mice deficient in these enzymes, indigestible metabolic intermediates deposit in neurons. Inclusions such as membranous cytoplasmic bodies (MCB) and zebra bodies were seen in neurons of Tay-Sachs (TS) model mice, Sandhoff's disease (SD) model mice, and double knockout (DKO) mice. However, the cerebral perivascular macrophages discovered by Mato are active in the uptake of waste products and regarded as scavenger cells under steady-state conditions. We observed that indigestible components derived from neurons were taken up by the perivascular macrophages of TS mice by pinocytosis, but those of SD and DKO mice contained only pale inclusions and had marked vacuolations, and pinocytosis was rarely observed. Histochemically, the inclusions in the perivascular macrophages of TS mice were positive for the PAS stain, but those of SD and DKO mice were negative. In addition, the perivascular cells of TS mice expressed clear positive immunoreactivity against BM-8 and F4/80, but those of DKO mice had very weak BM-8 and F4/80 immunoreactivity. These differences between TS, SD, and DKO mice are based on their metabolism of oligosaccharides and glycosaminoglycans (GAG). Thus, hexosaminidase B is more important for keeping normal morphology and function of perivascular macrophages than hexosaminidase A. The foamy cells that appeared along the cerebral microvessels in lipidosis and saccharidosis were identified as perivascular macrophages (Mato's fluorescent granular perithelial cells: FGP cells).