scholarly journals Flavin Monooxygenases Regulate C. elegans Axon Guidance and Growth Cone Protrusion with UNC-6/Netrin signaling and Rac GTPases

2017 ◽  
Author(s):  
Mahekta Gujar ◽  
Aubrie M. Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Molecular Mechanisms ◽  
Direct Oxidation ◽  
Rac Gtpase ◽  
C Elegans ◽  
Growth Cone Collapse ◽  
Rac Gtpases ◽  
Inhibition Of Growth

AbstractThe guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5/UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5/UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL.Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies, we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.Author SummaryMolecular mechanisms of axon repulsion mediated by UNC-6/Netrin are not well understood. Inhibition of growth cone lamellipodial and filopodial protrusion is critical to repulsive axon guidance. Previous work identified a novel pathway involving Rac GTPases and the cytoskeletal interacting molecule UNC-33/CRMP required for UNC-6/Netrin-mediated inhibition of growth cone protrusion. In other systems, CRMP mediates growth cone collapse in response to semaphorin. Here we demonstrate a novel role of flavoprotein monooxygenases (FMOs) in repulsive axon guidance and inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling and Rac GTPases. In Drosophila and vertebrates, the multidomain MICAL FMO mediates semaphorin-dependent growth cone collapse by direct oxidation and depolymerization of F-actin. The C. elegans genome does not encode a multidomain MICAL-like molecule, and we speculate that the C. elegans FMOs might have an equivalent role downstream of UNC-6/Netrin signaling. Indeed, we show that EHBP-1, similar to the non-FMO portion of MICAL, also controls repulsive axon guidance and growth cone inhibition, suggesting that in C. elegans, the functions of the multidomain MICAL molecule might be distributed across different molecules. In sum, we show conservation of function of molecules involved in semaphorin growth cone collapse with inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling.

scholarly journals RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in C. elegans

2019 ◽  
Author(s):  
Mahekta R. Gujar ◽  
Aubrie M. Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Neural Circuit ◽  
Growth Cones ◽  
Negative Regulator ◽  
Microtubule Organization ◽  
C Elegans ◽  
Guidance Cue ◽  
Rho Gef

AbstractUNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions suggest that RHO-1 and RHGF-1 act with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.Author SummaryNeural circuits are formed by precise connections between axons. During axon formation, the growth cone leads the axon to its proper target in a process called axon guidance. Growth cone outgrowth involves asymmetric protrusion driven by extracellular cues that stimulate and inhibit protrusion. How guidance cues regulate growth cone protrusion in neural circuit formation is incompletely understood. This work shows that the signaling molecule RHO-1 acts downstream of the UNC-6/Netrin guidance cue to inhibit growth cone protrusion in part by excluding microtubules from the growth cone, which are structural elements that drive protrusion.

scholarly journals UNC-6/Netrin and its Receptors UNC-5 and UNC-40/DCC Control Growth Cone Polarity, Microtubule Accumulation, and Protrusion

2018 ◽  
Author(s):  
Mahekta R. Gujar ◽  
Lakshmi Sundararajan ◽  
Aubrie Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Growth Cones ◽  
Microtubule Organization ◽  
C Elegans ◽  
Control Growth ◽  
Receptor Interactions ◽  
Independent Mechanism ◽  
Actin Localization

AbstractMany axon guidance ligands and their receptors have been identified, but it is still unclear how these ligand-receptor interactions regulate events in the growth cone, such as protrusion and cytoskeletal arrangement, during directed outgrowth in vivo. In this work, we dissect the multiple and complex effects of UNC-6/Netrin on the growth cone. Previous studies showed that in C. elegans, the UNC-6/Netrin receptor UNC-5 regulates growth cone polarity, as evidenced by loss of asymmetric dorsal F-actin localization and protrusion in unc-5 mutants. UNC-5 and another UNC-6/Netrin receptor UNC-40/DCC also regulate the extent of protrusion, with UNC-40/DCC driving protrusion and UNC-5 inhibiting protrusion. In this work we analyze the roles of UNC-6/Netrin, UNC-40/DCC, and UNC-5 in coordinating growth cone F-actin localization, microtubule organization, and protrusion that results in directed outgrowth away from UNC-6/Netrin. We find that a previously-described pathway involving the UNC-73/Trio Rac GEF and UNC-33/CRMP that acts downstream of UNC-5, regulates growth cone dorsal asymmetric F-actin accumulation and protrusion. unc-5 and unc-33 mutants also display excess EBP-2::GFP puncta, suggesting that MT + end accumulation is important in growth cone polarity and/or protrusion. unc-73 Rac GEF mutants did not display excess EBP-2::GFP puncta despite larger and more protrusive growth cones, indicating a MT-independent mechanism to polarize the growth cone and to inhibit protrusion, possibly via actin. Finally, we show that UNC-6/Netrin and UNC-40/DCC are required for excess protrusion in unc-5 mutants, but not for loss of F-actin asymmetry or MT + end accumulation, indicating that UNC-6/Netrin and UNC-40/DCC are required for protrusion downstream of F-actin asymmetry and MT + end entry. Our data suggest a model in which UNC-6/Netrin polarizes the growth cone via UNC-5, and then regulates a balance of pro- and anti-protrusive forces driven by UNC-40 and UNC-5, respectively, that result in directed protrusion and outgrowth.

The metalloproteinase ADAM10 is required for retinal ganglion cell axon guidance in the developing visual systems

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Y. Y. Chen ◽  
C. L. Hehr ◽  
K. Atkinson-Leadbeater ◽  
J. C. Hocking ◽  
S. McFarlane
Keyword(s):  
Ganglion Cell ◽  
Axon Guidance ◽  
Retinal Ganglion Cell ◽  
Growth Cone ◽  
Expression Patterns ◽  
Optic Tract ◽  
Axon Growth ◽  
Proteolytic Processing ◽  
Retinal Ganglion

Background: The growth cone interprets cues in its environment in order to reach its target. We want to identify molecules that regulate growth cone behaviour in the developing embryo. We investigated the role of A disintegrin and metalloproteinase 10 (ADAM10) in axon guidance in the developing visual system of African frog, Xenopus laevis. Methods: We first examined the expression patterns of adam10 mRNA by in situ hybridization. We then exposed the developing optic tract to an ADAM10 inhibitor, GI254023X, in vivo. Lastly, we inhibited ADAM10 function in diencephalic neuroepithelial cells (through which retinal ganglion cell (RGC) axons extend) or RGCs by electroporating or transfecting an ADAM10 dominant negative (dn-adam10). Results: We show that adam10 mRNA is expressed in the dorsal neuroepithelium over the time RGC axons extend towards their target, the optic tectum. Second, pharmacological inhibition of ADAM10 in an in vivo exposed brain preparation causes the failure of RGC axons to recognize their target at low concentrations (0.5, 1 μM), and the failure of the axons to make a caudal turn in the mid-diencephalon at higher concentration (5 μM). Thus, ADAM10 function is required for RGC axon guidance at two key guidance decisions. Finally, molecular inhibition of ADAM10 function by electroporating dn-adam10 in the brain neuroepithelium causes defects in RGC axon target recognition (57%) and/or defects in caudal turn (12%), as seen with the pharmacological inhibitor. In contrast, molecular inhibition of ADAM10 within the RGC axons has no effect. Conclusions: These data argue strongly that ADAM10 acts cell non-autonomously within the neuroepithelium to regulate the guidance of RGC axons. This study shows for the first time that a metalloproteinase acts in a cell non-autonomous fashion to direct vertebrate axon growth. It will provide important insights into candidate molecules that could be used to reform nerve connections if destroyed because of injury or disease. References Hattori M, Osterfield M, Flanagan JG. Regulated cleavage of a contact-mediated axon repellent. Science 2000; 289(5483):1360-5. Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123(2):291-304. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997; 90(2):271-80.

scholarly journals Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

2021 ◽  
pp. 1-9
Author(s):  
Dayana Torres Valladares ◽  
Sirisha Kudumala ◽  
Murad Hossain ◽  
Lucia Carvelli
Keyword(s):  
Caenorhabditis Elegans ◽  
Molecular Mechanisms ◽  
Drugs Of Abuse ◽  
Genetic Model ◽  
In Vivo Model ◽  
C Elegans ◽  
Hyperactivity Disorder ◽  
In Vitro Data

Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in <i>Caenorhabditis elegans</i> to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that <i>C. elegans</i> is a suitable system to study tolerance to drugs of abuse such as amphetamines.

scholarly journals Regulation of axon repulsion by MAX-1 SUMOylation and AP-3

2018 ◽  
Vol 115 (35) ◽  
pp. E8236-E8245
Author(s):  
Shih-Yu Chen ◽  
Chun-Ta Ho ◽  
Wei-Wen Liu ◽  
Mark Lucanic ◽  
Hsiu-Ming Shih ◽  
...  
Keyword(s):  
Axon Guidance ◽  
Neural Development ◽  
Biochemical Analysis ◽  
Genetic Interaction ◽  
Motor Axons ◽  
Surface Receptors ◽  
C Elegans ◽  
Guidance Receptors ◽  
Axon Repulsion

During neural development, growing axons express specific surface receptors in response to various environmental guidance cues. These axon guidance receptors are regulated through intracellular trafficking and degradation to enable navigating axons to reach their targets. In Caenorhabditis elegans, the UNC-5 receptor is necessary for dorsal migration of developing motor axons. We previously found that MAX-1 is required for UNC-5–mediated axon repulsion, but its mechanism of action remained unclear. Here, we demonstrate that UNC-5–mediated axon repulsion in C. elegans motor axons requires both max-1 SUMOylation and the AP-3 complex β subunit gene, apb-3. Genetic interaction studies show that max-1 is SUMOylated by gei-17/PIAS1 and acts upstream of apb-3. Biochemical analysis suggests that constitutive interaction of MAX-1 and UNC-5 receptor is weakened by MAX-1 SUMOylation and by the presence of APB-3, a competitive interactor with UNC-5. Overexpression of APB-3 reroutes the trafficking of UNC-5 receptor into the lysosome for protein degradation. In vivo fluorescence recovery after photobleaching experiments shows that MAX-1 SUMOylation and APB-3 are required for proper trafficking of UNC-5 receptor in the axon. Our results demonstrate that SUMOylation of MAX-1 plays an important role in regulating AP-3–mediated trafficking and degradation of UNC-5 receptors during axon guidance.

The expression, lamin-dependent localization and RNAi depletion phenotype for emerin inC. elegans

2002 ◽  
Vol 115 (5) ◽  
pp. 923-929 ◽  
Author(s):  
Yosef Gruenbaum ◽  
Kenneth K. Lee ◽  
Jun Liu ◽  
Merav Cohen ◽  
Katherine L. Wilson
Keyword(s):  
Nuclear Envelope ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Growth Conditions ◽  
Rnai Phenotype ◽  
Lamin B ◽  
C Elegans ◽  
Nuclear Lamins ◽  
First Time

Emerin belongs to the LEM-domain family of nuclear membrane proteins, which are conserved in metazoans from C. elegans to humans. Loss of emerin in humans causes the X-linked form of Emery-Dreifuss muscular dystrophy(EDMD), but the disease mechanism is not understood. We have begun to address the function of emerin in C. elegans, a genetically tractable nematode. The emerin gene (emr-1) is conserved in C. elegans. We detect Ce-emerin protein in the nuclear envelopes of all cell types except sperm, and find that Ce-emerin co-immunoprecipitates with Ce-lamin from embryo lysates. We show for the first time in any organism that nuclear lamins are essential for the nuclear envelope localization of emerin during early development. We further show that four other types of nuclear envelope proteins, including fellow LEM-domain protein Ce-MAN1, as well as Ce-lamin, UNC-84 and nucleoporins do not depend on Ce-emerin for their localization. This result suggests that emerin is not essential to organize or localize the only lamin (B-type) expressed in C. elegans. We also analyzed the RNAi phenotype resulting from the loss of emerin function in C. elegans under laboratory growth conditions, and found no detectable phenotype throughout development. We propose that C. elegans is an appropriate system in which to study the molecular mechanisms of emerin function in vivo.

scholarly journals Plexin-B1/RhoGEF–mediated RhoA activation involves the receptor tyrosine kinase ErbB-2

2004 ◽  
Vol 165 (6) ◽  
pp. 869-880 ◽  
Author(s):  
Jakub M. Swiercz ◽  
Rohini Kuner ◽  
Stefan Offermanns
Keyword(s):  
Tyrosine Kinase ◽  
Growth Cone ◽  
Receptor Tyrosine Kinase ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Axonal Growth ◽  
Dominant Negative ◽  
Growth Cone Collapse ◽  
Rhoa Activation ◽  
Axonal Growth Cone

Plexins are widely expressed transmembrane proteins that mediate the effects of semaphorins. The molecular mechanisms of plexin-mediated signal transduction are still rather unclear. Plexin-B1 has recently been shown to mediate activation of RhoA through a stable interaction with the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG. However, it is unclear how the activity of plexin-B1 and its downstream effectors is regulated by its ligand Sema4D. Here, we show that plexin-B family members stably associate with the receptor tyrosine kinase ErbB-2. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. A dominant-negative form of ErbB-2 blocks Sema4D-induced RhoA activation as well as axonal growth cone collapse in primary hippocampal neurons. Our data indicate that ErbB-2 is an important component of the plexin-B receptor system and that ErbB-2–mediated phosphorylation of plexin-B1 is critically involved in Sema4D-induced RhoA activation, which underlies cellular phenomena downstream of plexin-B1, including axonal growth cone collapse.

scholarly journals C. elegans PVD Neurons: A Platform for Functionally Validating and Characterizing Neuropsychiatric Risk Genes

2016 ◽  
Author(s):  
Cristina Aguirre-Chen ◽  
Nuri Kim ◽  
Olivia Mendivil Ramos ◽  
Melissa Kramer ◽  
W. Richard McCombie ◽  
...  
Keyword(s):  
Protein Function ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
De Novo ◽  
Genetic Interaction ◽  
Cost Effective ◽  
Chromatin Remodelers ◽  
C Elegans ◽  
Effective Manner

AbstractOne of the primary challenges in the field of psychiatric genetics is the lack of an in vivo model system in which to functionally validate candidate neuropsychiatric risk genes (NRGs) in a rapid and cost-effective manner1−3. To overcome this obstacle, we performed a candidate-based RNAi screen in which C. elegans orthologs of human NRGs were assayed for dendritic arborization and cell specification defects using C. elegans PVD neurons. Of 66 NRGs, identified via exome sequencing of autism (ASD)4 or schizophrenia (SCZ)5−9 probands and whose mutations are de novo and predicted to result in a complete or partial loss of protein function, the C. elegans orthologs of 7 NRGs were found to be required for proper neuronal development and represent a variety of functional classes, including transcriptional regulators and chromatin remodelers, molecular chaperones, and cytoskeleton-related proteins. Notably, the positive hit rate, when selectively assaying C. elegans orthologs of ASD and SCZ NRGs, is enriched >14-fold as compared to unbiased RNAi screening10. Furthermore, we find that RNAi phenotypes associated with the depletion of NRG orthologs is recapitulated in genetic mutant animals, and, via genetic interaction studies, we show that the NRG ortholog of ANK2, unc-44, is required for SAX-7/MNR-1/DMA-1 signaling. Collectively, our studies demonstrate that C. elegans PVD neurons are a tractable model in which to discover and dissect the fundamental molecular mechanisms underlying neuropsychiatric disease pathogenesis.

scholarly journals APP-induced patterned neurodegeneration exacerbated by APOE4 in C. elegans

2018 ◽  
Author(s):  
Wisath Sae-Lee ◽  
Luisa L. Scott ◽  
Aliyah J. Encarnacion ◽  
Pragati Kore ◽  
Lashaun O. Oyibo ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Late Onset ◽  
Epidemiological Studies ◽  
Extra Copy ◽  
C Elegans ◽  
Age Related ◽  
Progressive Neurodegeneration ◽  
Ε4 Allele ◽  
Worm Model

AbstractGenetic and epidemiological studies have found that variations in the amyloid precursor protein (APP) and the apoliopoprotein E (APOE) genes represent major modifiers of the progressive neurodegeneration in Alzheimer’s disease (AD). An extra copy or gain-of-function mutations in APP lower age of AD onset. Compared to the other isoforms (APOE3 and APOE2), the ε4 allele of APOE (APOE4) hastens and exacerbates early and late onset forms of AD. Convenient in vivo models to study how APP and APOE4 interact at the cellular and molecular level to influence neurodegeneration are lacking. Here, we show that the nematode C. elegans can model important aspects of AD including age-related, patterned neurodegeneration that is exacerbated by APOE4. Specifically, we found that APOE4, but not APOE3, acts with APP to hasten and expand the pattern of cholinergic neurodegeneration caused by APP. Molecular mechanisms underlying how APP and APOE4 synergize to kill some neurons while leaving others unaffected may be uncovered using this convenient worm model of neurodegeneration.

scholarly journals Caenorhabditis elegans, a Host to Investigate the Probiotic Properties of Beneficial Microorganisms

2020 ◽  
Vol 7 ◽  
Author(s):  
Cyril Poupet ◽  
Christophe Chassard ◽  
Adrien Nivoliez ◽  
Stéphanie Bornes
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput Screening ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Probiotic Properties ◽  
C Elegans ◽  
Probiotic Potential ◽  
Challenges And Opportunities ◽  
Future Challenges

Caenorhabditis elegans, a non-parasitic nematode emerges as a relevant and powerful candidate as an in vivo model for microorganisms-microorganisms and microorganisms-host interactions studies. Experiments have demonstrated the probiotic potential of bacteria since they can provide to the worm a longer lifespan, an increased resistance to pathogens and to oxidative or heat stresses. Probiotics are used to prevent or treat microbiota dysbiosis and associated pathologies but the molecular mechanisms underlying their capacities are still unknown. Beyond safety and healthy aspects of probiotics, C. elegans represents a powerful way to design large-scale studies to explore transkingdom interactions and to solve questioning about the molecular aspect of these interactions. Future challenges and opportunities would be to validate C. elegans as an in vivo tool for high-throughput screening of microorganisms for their potential probiotic use on human health and to enlarge the panels of microorganisms studied as well as the human diseases investigated.

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