Dopey-dependent regulation of extracellular vesicles maintains neuronal morphology.

Mature neurons maintain their distinctive morphology for extended periods in adult life. Compared to developmental neurite outgrowth, axon guidance, and target selection, relatively little is known of mechanisms that maintain mature neuron morphology. Loss of function in C. elegans DIP-2, a member of the conserved lipid metabolic regulator Dip2 family, results in progressive overgrowth of neurites in adults. We find that dip-2 mutants display specific genetic interactions with sax-2, the C. elegans ortholog of Drosophila Furry and mammalian FRY. Combined loss of DIP-2 and SAX-2 results in severe disruption of neuronal morphology maintenance accompanied by increased release of neuronal extracellular vesicles (EVs). By screening for suppressors of dip-2 sax-2 double mutant defects we identified gain-of-function (gf) mutations in the conserved Dopey family protein PAD-1 and its associated phospholipid flippase TAT-5/ATP9A. In dip-2 sax-2 double mutants carrying either pad-1(gf) or tat-5(gf) mutation, EV release is reduced and neuronal morphology across multiple neuron types is restored to largely normal. PAD-1(gf) acts cell autonomously in neurons. The domain containing pad-1(gf) is essential for PAD-1 function, and PAD-1(gf) protein displays increased association with the plasma membrane and inhibits EV release. Our findings uncover a novel functional network of DIP-2, SAX-2, PAD-1, and TAT-5 that maintains morphology of neurons and other types of cells, shedding light on the mechanistic basis of neurological disorders involving human orthologs of these genes.

DIP-2 suppresses ectopic neurite sprouting and axonal regeneration in mature neurons.

Neuronal morphology and circuitry established during early development must often be maintained over the entirety of animal lifespans. Compared with neuronal development, the mechanisms that maintain mature neuronal structures and architecture are little understood. The conserved disco-interacting protein 2 (DIP2) consists of a DMAP1-binding domain and two adenylate-forming domains (AFDs). We show that the Caenorhabditis elegans DIP-2 maintains morphology of mature neurons. dip-2 loss-of-function mutants display a progressive increase in ectopic neurite sprouting and branching during late larval and adult life. In adults, dip-2 also inhibits initial stages of axon regeneration cell autonomously and acts in parallel to DLK-1 MAP kinase and EFA-6 pathways. The function of DIP-2 in maintenance of neuron morphology and in axon regrowth requires its AFD domains and is independent of its DMAP1-binding domain. Our findings reveal a new conserved regulator of neuronal morphology maintenance and axon regrowth after injury.

Mitigating Motor Neuronal Loss in C. elegans Model of ALS8.

ALS8 is a late-onset familial autosomal dominant form of Amyotrophic Lateral Sclerosis (ALS) caused by a point mutation (P56S) in the VAPB gene (VAMP associated protein isoform B). Here, we generated two C. elegans models of the disease: a transgenic model where human VAPB wild-type (WT) or P56S mutant was expressed in a subset of motor neurons, and a second model that targeted inducible knockdown of the worm's orthologue, vpr-1. Overexpression of human VAPB in DA neurons caused a backward locomotion defect, axonal misguidance, and premature neuronal death. Knockdown of vpr-1 recapitulated the reduction in VAPB expression associated with sporadic cases of human ALS. It also caused backward locomotion defects as well as an uncoordinated phenotype, and age-dependent, progressive motor neuronal death. Furthermore, inhibiting phosphatidylinositol-4 (PtdIns 4)-kinase activity with PIK-93 reduced the incidence of DA motor neuron loss and improved backward locomotion. This supports the loss of VAPB function in ALS8 pathogenesis and suggests that reducing intracellular PtdIns4P might be an effective therapeutic strategy in delaying progressive loss of motor neurons.

A novel small RNA is important for biofilm formation and pathogenicity in Pseudomonas aeruginosa.

The regulation of biofilm development requires multiple mechanisms and pathways, but it is not fully understood how these are integrated. Small RNA post-transcriptional regulators are a strong candidate as a regulatory mechanism of biofilm formation. More than 200 small RNAs in the P. aeruginosa genome have been characterized in the literature to date; however, little is known about their biological roles in the cell. Here we describe the identification of the novel regulatory small RNA, SrbA. This locus was up-regulated 45-fold in P. aeruginosa strain PA14 biofilm cultures. Loss of SrbA expression in a deletion strain resulted in a 66% reduction in biofilm mass. Furthermore, the mortality rate over 72 hours in C. elegans infections was reduced to 39% when infected with the srbA deletion strain compared to 78% mortality when infected with the parental wild-type P. aeruginosa strain. There was no significant effect on culture growth or adherence to surfaces with loss of SrbA expression. Also loss of SrbA expression had no effect on antibiotic resistance to ciprofloxacin, gentamicin, and tobramycin. We conclude that SrbA is important for biofilm formation and full pathogenicity of P. aeruginosa.

PCP and SAX-3/Robo Pathways Cooperate to Regulate Convergent Extension-Based Nerve Cord Assembly in C. elegans.

Formation and resolution of multicellular rosettes can drive convergent extension (CE) type cell rearrangements during tissue morphogenesis. Rosette dynamics are regulated by both planar cell polarity (PCP)-dependent and -independent pathways. Here we show that CE is involved in ventral nerve cord (VNC) assembly in Caenorhabditis elegans. We show that a VANG-1/Van Gogh and PRKL-1/Prickle containing PCP pathway and a Slit-independent SAX-3/Robo pathway cooperate to regulate, via rosette intermediaries, the intercalation of post-mitotic neuronal cell bodies during VNC formation. We show that VANG-1 and SAX-3 are localized to contracting edges and rosette foci and act to specify edge contraction during rosette formation and to mediate timely rosette resolution. Simultaneous loss of both pathways severely curtails CE resulting in a shortened, anteriorly displaced distribution of VNC neurons at hatching. Our results establish rosette-based CE as an evolutionarily conserved mechanism of nerve cord morphogenesis and reveal a role for SAX-3/Robo in this process.

A Farnesyltransferase Acts to Inhibit Ectopic Neurite Formation in C. elegans.

Genetic pathways that regulate nascent neurite formation play a critical role in neuronal morphogenesis. The core planar cell polarity components VANG-1/Van Gogh and PRKL-1/Prickle are involved in blocking inappropriate neurite formation in a subset of motor neurons in C. elegans. A genetic screen for mutants that display supernumerary neurites was performed to identify additional factors involved in this process. This screen identified mutations in fntb-1, the ß subunit of farnesyltransferase. We show that fntb-1 is expressed in neurons and acts cell-autonomously to regulate neurite formation. Prickle proteins are known to be post-translationally modified by farnesylation at their C-terminal CAAX motifs. We show that PRKL-1 can be recruited to the plasma membrane in both a CAAX-dependent and CAAX-independent manner but that PRKL-1 can only inhibit neurite formation in a CAAX-dependent manner.

Identification of genes involved in Pseudomonas aeruginosa biofilm-specific resistance to antibiotics.

Pseudomonas aeruginosa is a key opportunistic pathogen characterized by its biofilm formation ability and high-level multiple antibiotic resistance. By screening a library of random transposon insertion mutants with an increased biofilm-specifc antibiotic susceptibility, we previously identified 3 genes or operons of P. aeruginosa UCBPP-PA14 (ndvB, PA1875-1877 and tssC1) that do not affect biofilm formation but are involved in biofilm-specific antibiotic resistance. In this study, we demonstrate that PA0756-0757 (encoding a putative two-component regulatory system), PA2070 and PA5033 (encoding hypothetical proteins of unknown function) display increased expression in biofilm cells and also have a role in biofilm-specific antibiotic resistance. Furthermore, deletion of each of PA0756, PA2070 and PA5033 resulted in a significant reduction of lethality in Caenorhabditis elegans, indicating a role for these genes in both biofilm-specific antibiotic resistance and persistence in vivo. Together, these data suggest that these genes are potential targets for antimicrobial agents.

Planar polarity genes and inhibition of supernumerary neurites.

Planar cell polarity (PCP) genes have recently emerged as important players in sculpting neuronal connections. The bipolar VC neurons display stereotypical differences in axon extension along the anterior-posterior (AP) body axis: VC1-3 and VC6 polarize along the AP axis while VC4 and VC5 polarize along the orthogonal left-right (LR) axis generated by the developing vulva. vang-1 and prkl-1, the worm orthologs of Van Gogh and Prickle, are required to restrict the polarity of neurite emergence to a specific tissue axis. vang-1 and prkl-1 loss results in ectopic VC4 and VC5 neurites extending inappropriately along the AP axis. Conversely, prkl-1 overexpression in VC neurons suppresses neurite formation. These findings suggest that a PCP-like pathway acts to silence or antagonize neuronal responses to polarity cues that would otherwise be permissive for neurite growth.

VANG-1 and PRKL-1 cooperate to negatively regulate neurite formation in Caenorhabditis elegans.

Neuritogenesis is a critical early step in the development and maturation of neurons and neuronal circuits. While extracellular directional cues are known to specify the site and orientation of nascent neurite formation in vivo, little is known about the genetic pathways that block inappropriate neurite emergence in order to maintain proper neuronal polarity. Here we report that the Caenorhabditis elegans orthologues of Van Gogh (vang-1), Prickle (prkl-1), and Dishevelled (dsh-1), core components of planar cell polarity (PCP) signaling, are required in a subset of peripheral motor neurons to restrict neurite emergence to a specific organ axis. In loss-of-function mutants, neurons display supernumerary neurites that extend inappropriately along the orthogonal anteroposterior (A/P) body axis. We show that autonomous and non-autonomous gene activities are required early and persistently to inhibit the formation or consolidation of growth cone protrusions directed away from organ precursor cells. Furthermore, prkl-1 overexpression is sufficient to suppress neurite formation and reorient neuronal polarity in a vang-1- and dsh-1-dependent manner. Our findings suggest a novel role for a PCP-like pathway in maintaining polarized neuronal morphology by inhibiting neuronal responses to extrinsic or intrinsic cues that would otherwise promote extraneous neurite formation.

The N-glycanase png-1 acts to limit axon branching during organ formation in Caenorhabditis elegans.

Peptide:N-glycanases (PNGases) are cytoplasmic de-N-glycosylation enzymes that have been shown in cultured cells to facilitate the degradation of misfolded glycoproteins during endoplasmic reticulum-associated degradation and in the processing of major histocompatibility complex class I antigens for proper cell-surface presentation. The gene encoding PNGase activity was initially described in budding yeast (Png1p) and shown to be highly conserved from yeast to humans, but physiological roles in higher organisms have not been elucidated. Here we describe peripheral nervous system defects associated with the first loss-of-function mutations in an animal PNGase. Mutations in png-1, the Caenorhabditis elegans PNGase ortholog, result in an increase in axon branching during morphogenesis of the vulval egg-laying organ and egg-laying behavior changes. Neuronal defects include an increase in the branched morphology of the VC4 and VC5 egg-laying neurons as well as inappropriate branches from axons that run adjacent to the vulva but would normally remain unbranched. We show that png-1 is widely expressed and can act from both neurons and epithelial cells to restrict axon branching. A deletion allele of the DNA repair gene rad-23, orthologs of which are known to physically interact with PNGases in yeast and mammals, displays similar axon branching defects and genetic interactions with png-1. In summary, our analysis reveals a novel developmental role for a PNGase and Rad-23 in the regulation of neuronal branching during organ innervation.

C. elegans seu-1 encodes novel nuclear proteins that regulate responses to UNC-6/netrin guidance cues.

In C. elegans, ectopic expression of the UNC-5 netrin receptor is sufficient to cause repulsion of growth cones and cells away from ventral sources of the UNC-6/netrin guidance cue. A genetic suppressor screen identified the seu-1 gene as required for repulsion of touch neuron growth cones ectopically expressing unc-5. We report here that seu-1 mutations also enhance the frequency of distal tip cell migrations of unc-5 or unc-40 mutants. The seu-1 gene encodes two novel proteins (SEU-1A and SEU-1B) containing a charged central domain and several regions of low amino acid complexity. Transgenic rescue experiments indicate that seu-1 can act cell autonomously in the touch neurons and distal tip cells and that SEU-1 function requires both the SEU-1A and SEU-1B isoforms. A GFP fusion construct was expressed in a dynamic pattern throughout development and localized in the nuclei of neuronal and non-neuronal cells, including gonadal leader cells. These results implicate nuclear SEU-1 in the interpretation of UNC-6/netrin directional information by migrating growth cones and cells.

Dual enzymatic properties of the cytoplasmic peptide: N-glycanase in C. elegans.

The endoplasmic reticulum-associated degradation (ERAD) of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the ER and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, some of them are subjected to deglycosylation by the cytoplasmic peptide:N-glycanase (PNGase). The cytosolic PNGase is widely distributed throughout eukaryotes. Here we show that the nematode Caenorhabditis elegans PNG-1, the cytoplasmic PNGase orthologue in this organism, exhibits dual enzyme functions, not only as PNGase but also as an oxidoreductase (thioredoxin). Using an in vitro assay as well as an in vivo assay system in budding yeast, the N-terminal thioredoxin domain and the central transglutaminase domain were found to be essential for oxidoreductase activity and PNGase activity, respectively. Occurrence of a C. elegans mutation affecting a catalytic residue in the PNGase domain strongly suggests the functional importance of this protein in higher eukaryotes.

A Neurexin-related protein, BAM-2, terminates axonal branches in C. elegans.

Neuronal axons connect to multiple target cells through the formation of collateral branches, but the mechanisms that regulate this process are largely unknown. We show that BAM-2, a neurexin-related transmembrane protein, is required for development of VC motoneuron branches in the worm Caenorhabditis elegans. Expression analysis and ectopic expression experiments suggest that BAM-2 functions as a branch termination cue and reveal a mechanism for selective control of branches that sprout off a primary axon.