Neumann B et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Axonal Regeneration Proceeds Through Axonal Fusion in C. elegans Neurons"

Hall D H, Hilliard M A, Nguyen K CQ, Neumann B, Ben-Yakar A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Invertebrate axons and those of the mammalian peripheral nervous system are able to regenerate in the adult. Functional recovery takes place when a damaged axon regains connection with its target tissues. In C. elegans, following laser axotomy, the regrowing axon still attached to cell body (proximal) is able to reconnect with its separated distal segment through unknown mechanisms. Using the mechanosensory neurons ALM and PLM as a model system, we have found that during axonal regeneration reconnection between the proximal and distal axonal fragments occurs through a mechanism of axonal fusion, with reestablishment of cytoplasmic and membrane continuity. We found that when axonal fusion does not occur the distal fragment inevitably undergoes Wallerian degeneration and the original axonal tract cannot be restored. Through the use of dual colour labeling of adjacent axonal pairs, we found a high level of specific recognition occurring between a proximal re-growing axon and its own separated distal fragment, revealing possible cross talk between the two processes. Finally, from a candidate mutant approach, we have identified a molecule with homology to a human protein implicated in axonal degeneration, as being necessary for successful regeneration and specifically involved in the process of axonal fusion. We anticipate that a similar mechanism of axonal regeneration could be exploited to improve the outcome of axonal regeneration following injury in mammalian systems.

Vijayaraghavan, T. et al. (2019) International Worm Meeting "The dynamin GTPase is required for regenerative axonal fusion."

Vijayaraghavan, T., Neumann, B.

[International Worm Meeting, 2019]

Axonal fusion is an efficient regenerative process that restores neuronal function post-injury. It is a highly specific mechanism whereby a regrowing axon reconnects and fuses with its severed segment, restoring cytoplasmic and membrane integrity, and preventing the degeneration of the detached segment. While synaptic vesicle recycling has been linked to axonal regeneration, its role in axonal fusion remains largely unknown. Dynamin proteins are large GTPases that hydrolyse lipid binding membranes to carry out clathrin-mediated synaptic vesicle recycling. The role of dynamin in endocytosis was first discovered in Drosophila melanogaster, with disruption of the dynamin-like shibire gene causing blockage of synaptic vesicle transport in this species. In C. elegans, the dynamin-related protein DYN-1 functions in apoptotic cell clearance by mediating a signalling pathway involving the ABC transporter CED-7, as well as the transmembrane receptor CED-1/LRP1 and its intracellular adapter CED-6/GULP in the engulfing cell. CED-7 and CED-6 were previously reported to be important for axonal fusion, functioning to mediate the reconnection between separated axon segments. Thus, we hypothesized that DYN-1 may also be necessary for axonal fusion. To test this, we conducted UV-laser axotomy and analysed axonal regeneration in the posterior lateral microtubule neurons (PLMs) in animals carrying the temperature-sensitive dyn-1(ky51) allele. We show that disruption of DYN-1 function by raising animals at higher, restrictive temperatures (20oC and 25oC) leads to dramatically reduced levels of axonal fusion. Importantly, we demonstrate that these defects are reversible at lower, permissive temperatures, with the level of axonal fusion being restored to wild-type levels at 15oC. Quantification of the length of regrowth under these conditions has revealed no defect in dyn-1 mutants compared to wild-type animals at 15 deg C and 20 deg C but showed a significant defect at 25 deg C. Furthermore, we find that DYN-1 activity is only required from 16 hours post-axotomy for neuronal repair. Together, these results establish important roles for DYN-1 in regulating axonal regrowth and fusion.

Teoh, J-S. et al. (2019) International Worm Meeting "The alpha-tubulin acetyltransferase MEC-17/alphaTAT1 is essential for robust axonal regeneration."

Teoh, J-S., Neumann, B.

[International Worm Meeting, 2019]

Injury to the nervous system frequently results in devastating conditions for which very limited treatment options exist. Remodelling of the microtubule network is important for the repair of severed axons as it allows for the development of growth-promoting structures including the growth cone. Therefore, regulators of microtubule dynamics, including microtubule associated proteins and post-translational modifications, have been suggested to play an essential role in axon regeneration. Indeed, recent studies support the involvement of microtubule associated proteins in promoting stable microtubules for efficient repair. The acetylation of ?-tubulin increases the stability of microtubules and protects them from mechanical stresses by imparting structural elasticity. However, the role of this post-translational modification in promoting nerve regeneration remains poorly defined. To study the role of the ?-tubulin acetyltransferase MEC-17 in the repair of C. elegans neurons, we performed UV-laser surgery to transect the axons of individual sensory neurons and monitored their regenerative capacity at multiple time-points. Additionally, we studied the genetic pathways in which mec-17 acts using epistatic analyses. Our results suggest mec-17 to be important for efficient axonal regrowth and axonal fusion, for which it genetically interacts with mec-12/?-tubulin to promote axon repair. Additionally, we will present data on the impact of MEC-17 on microtubule structure, on the proteins with which it interacts, and how its misregulation impacts the expression of interacting genetic pathways. Collectively our data reveals the importance of MEC-17 for robust axonal regeneration after injury.

Abay, Z.C. et al. (2017) International Worm Meeting "Phosphatidylserine 'save-me' signals drive functional recovery of severed axons."

Hilliard, M.A., Abay, Z.C., Neumann, B.

[International Worm Meeting, 2017]

Functional regeneration after axonal injury requires transected axons to regrow and re-establish connection with their original target tissue. The spontaneous regenerative mechanism known as axonal fusion provides a highly efficient means of achieving targeted reconnection, as a regrowing axon is able to recognize and fuse with its own detached axon segment, thereby rapidly re-establishing the original axonal tract. Here we use behavioral assays and fluorescent reporters to demonstrate that axonal fusion enables full recovery of function following axotomy of Caenorhabditis elegans mechanosensory neurons. Furthermore, we reveal that the phospholipid phosphatidylserine, which becomes exposed on the damaged axon to function as a 'save-me' signal, defines the rate of axonal fusion. We also show that successful axonal fusion correlates with the regrowth potential and branching of the proximal fragment, and with the retraction length and degeneration of the separated segment. Finally, we identify discrete axonal domains that vary in their propensity to regrow through fusion, and demonstrate that the rate of axonal fusion can be genetically modulated. Taken together, our results reveal that axonal fusion restores full function to injured neurons, is dependent on exposure of phospholipid signals, and is achieved through the balance between regenerative potential and rate of degeneration.

Kirszenblat L et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Wnt signals and Frizzled receptors regulate dendrite formation in C. elegans"

Kirszenblat L, Hilliard M A, Neumann B, Pattabiraman D

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Neurons exhibit distinct morphological domains, axons and dendrites, which are essential for functional wiring of the nervous system. While many molecules involved in axon development have been discovered, there is little known about the ligands and receptors that regulate dendrite development. To understand how dendrites develop in C. elegans we focused on the PQR oxygen sensory neuron. PQR has its cell body positioned in the left lumbar ganglion on the posterior-lateral side of the body. A single dendrite extends posterior with sensory cilia at its tip, while the axon extends anterior along the ventral nerve cord. PQR is born post-embryonically allowing easy visualization of dendrite development using the gcy-36::GFP transgene. In a genetic screen for dendrite defective mutants we isolated a previously uncharacterized mutation in lin-17, a C. elegans Frizzled receptor gene. We found that in lin-17(vd002), the PQR dendrite was absent, shortened or misrouted anterior. Similar dendrite defects were also observed in other known alleles of lin-17. Cell-specific expression of wild-type LIN-17 in PQR indicated a non-cell-autonomous role of this molecule in regulating dendrite development. LIN-44 is a Wnt ligand known to bind the Frizzled receptor LIN-17 and is expressed by four hypodermal cells in the tip of the tail. We found that lin-44 mutants presented PQR dendrite defects similar to those observed in lin-17 mutants. We expressed LIN-44 ectopically from more anterior regions of the body and found that it rescued the PQR dendrite defects of lin-44 mutants, indicating LIN-44 functions as a permissive cue. Using a heat-shock promoter to drive LIN-44 we determined that the presence of this ligand at the time of PQR dendrite formation was sufficient to rescue the dendrite defects of the lin-44 mutant. Analysis of the lin-17 lin-44 double mutant indicated a genetic interaction between these molecules. Our studies provide the first direct evidence that specific Wnt signals and Frizzled receptors regulate dendrite formation in vivo. We propose a model in which PQR dendrite formation is achieved by the interaction of LIN-44 and LIN-17 acting on PQR through its neighbouring cells.

Fluet A et al. (1998) West Coast Worm Meeting "AGGREGATION AND TOXICITY OF HUMAN b-AMYLOID PEPTIDE VARIANTS EXPRESSED IN C. ELEGANS"

Fluet A, Fay DS, Link CD, Johnson CJ

[West Coast Worm Meeting, 1998]

We have engineered transgenic C. elegans that express the human b-amyloid peptide, which appears to play a central role in the pathology of Alzheimer's disease. As described previously, these worms, which express the peptide under the control of the unc-54 promoter, are unhealthy, have larval vacuoles, and exhibit a progressive paralysis. To better understand the molecular mechanism of b peptide toxicity, we would like to determine which, if any, of these phenotypes are specific to the expression of b peptide, and which are non-specific effects resulting from overexpression of a foreign peptide. To address this question we have sought to develop non-toxic b peptide variants, which should 'unmask' the non-specific phenotypes. We have specifically attempted to design b peptide variants that are incapable of aggregating into amyloid fibers, which have previously been reported to be directly correlated with b peptide toxicity. Single amino acid changes in the b(1-42) peptide have provided us with two interesting variants that both fail to produce amyloid aggregates: L17P and M35C. Intriguingly, multiple L17P transgenic lines all show the progressive paralysis observed in strains expressing wild-type b peptide. All M35C lines, however, are significantly healthier than the strains expressing wild-type b peptide. While the L17P variant may allow us to differentiate between b peptide's ability to form aggregates and its toxicity, the M35C variant is currently being used to differentiate between specific and non-specific toxicity resulting from b peptide expression. We have also isolated a second non-aggregating version of the b peptide, the single-chain b dimer. Like the M35C variant, this version of the b peptide does not aggregate and animals that express it are healthier than those expressing the wild-type b peptide. Quantitative immunoblot analyses on these strains demonstrate that the differences we see in aggregation of the variants are due to qualitative differences in the peptides and not quantitative differences in their levels of expression. These results also suggest that the progressive paralysis phenotype is specifically due to b peptide toxicity. We are currently using the non- or less-toxic b peptide variants in conjunction with the wild-type b peptide to look for molecular correlates of b peptide toxicity. We have preliminary results from immunoblots of Hsp16 expression that suggest that this heat shock protein is upregulated in response to b amyloid expression, and that this upregulation is significantly reduced in lines expressing the non-aggregating b dimer.

Soh, M.S. et al. (2017) International Worm Meeting "Characterising behavioural defects of the C. elegans model of CMT2A."

Byrne, J.J., Yapa, N.M., Chandhok, G.K., Neumann, B., Soh, M.S., Whitbread-Phee, G.

[International Worm Meeting, 2017]

Charcot-Marie-Tooth disease (CMT) is characterised by progressive motor and sensory neuropathy, resulting in muscle weakness and mobility impairments. The most common axonal form of CMT, CMT2A, is caused by mutations in the Mitofusin 2 (Mfn2) protein, which is a large GTPase critical for optimal mitochondrial functioning. Despite mutations of Mfn2 first being described as causative for CMT2A more than a decade ago, we still lack a precise understanding of how Mfn2 mutations cause the disease, and thus have a complete lack of effective therapeutics. We aim to model CMT2A in C. elegans by targeting the ortholog of Mfn2 in this species (fzo-1) in order to better understand the disease pathophysiology and to discover novel drugs that can modulate the disease. Using CRISPR-Cas9 methods, we have generated a novel allele of fzo-1 with the entire genomic region deleted (cjn020). We have used this allele, as well as the fzo-1(tm1133) allele, to assess the consequences of fzo-1 loss-of-function on animal behaviour, with a focus on locomotion and pharyngeal pumping. Notably, the absence of FZO-1 resulted in a significant reduction in the rate of locomotion compared to wild-type (WT) across all age groups tested, as demonstrated in both thrash and body bending assays. These observations mirror the mobility impairments endured by CMT2A patients. Furthermore, the two fzo-1 mutant strains displayed progressive defects in pharyngeal pumping compared to WT, with irregular and slower pumping rates observed. As the rate of pharyngeal pumping depends on the cycle of contraction and relaxation of the pharyngeal muscles, which in turn is determined by the pharyngeal muscle action potential, a deficit in pharyngeal pumping rate implies a corresponding defect in the neuronal control of the pharyngeal muscle contraction/relaxation. Finally, we have exploited the locomotor dysfunction of fzo-1 mutants as a platform for high-throughput screening of small compounds, in order to discover novel modulators that can reverse the movement defects in fzo-1 mutant animals. Our results help to define the normal biological role of Mfn-2/FZO-1, how this relates to CMT2A, and will also identify effective modulators of the disease phenotypes.

Xu, T. et al. (2019) International Worm Meeting "How does an animal move? An integrative model of C. elegans forward locomotion."

Lu, Y., Huo, J., Kawano, T., Po, M., Wen, Q., Xu, T., Wu, M., Shao, S., Zhen, M.

[International Worm Meeting, 2019]

Descending signals from the brain play critical roles in controlling and modulating locomotion kinematics. In the Caenorhabditis elegans nervous system, descending AVB premotor interneurons exclusively form gap junctions with the B-type motor neurons that execute forward locomotion. We combined genetic analysis, optogenetic manipulation, calcium imaging, and computational modeling to elucidate the function of AVB-B gap junctions during forward locomotion. First, we found that some B-type motor neurons generate rhythmic activity, constituting distributed oscillators. Second, AVB premotor interneurons use their electric inputs to drive bifurcation of B-type motor neuron dynamics, triggering their transition from stationary to oscillatory activity. Third, proprioceptive couplings between neighboring B-type motor neurons entrain the frequency of body oscillators, forcing coherent bending wave propagation. Despite substantial anatomical differences between the motor circuits of C. elegans and higher model organisms, converging principles govern coordinated locomotion.

Melissa Harrison et al. (2001) International C. elegans Meeting "FUNCTIONAL STUDIES OF THE CLASS B SYNMUVS REQUIRED FOR THE NEGATIVE REGULATION OF VULVAL INDUCTION AND CHARACTERIZATION OF A NOVEL CLASS B SYNMUV GENE"

Bob Horvitz, Melissa Harrison

[International C. elegans Meeting, 2001]

A receptor tyrosine kinase/Ras pathway necessary for vulval induction in C. elegans has been shown to be negatively regulated by two redundant pathways, defined by the synthetic Multivulva (synMuv) class A and B genes. Mutations in members of either class alone do not result in a Multivulva phenotype. However, animals containing loss-of-function mutations in both a class A and a class B gene display a synMuv phenotype. While most of the identified class A genes encode novel proteins, many of the class B synMuv genes, including lin-35 Rb, dpl-1 DP, efl-1 E2F, lin-53 RbAp48, hda-1 HDAC, and let-418 Mi-2, have homologs in other organisms known to be involved in chromatin modification complexes. Three mutations that define a new class B synMuv were identified in a recent screen for additional class B genes performed with Craig Ceol and Frank Stegmeier in our laboratory. We mapped these mutations to the far left of the X chromosome and are currently attempting to clone the gene by cosmid rescue. Several mammalian homologs of the class B synMuv genes have been shown to be components of the NuRD chromatin remodelling complex. Additionally, some of the proteins in the synMuv B pathway have been shown to physically interact either in vitro (1,2) or in yeast two-hybrid assays (3). We are attempting to use co-immunoprecipitation experiments to expand the current knowledge of the presumptive class B protein complex in C. elegans . Analysis of the effects of various mutations on co-immunoprecipitation may allow us to assign functionality to some novel class B genes. Currently, there is little understanding of how the class A and B synMuvs redundantly regulate vulval induction. Class A and B synMuvs might play redundant roles in the formation of a chromatin modifying complex, and such physical interactions could also be demonstrated through co-immunoprecipitaion experiments. (1) Ceol, C. and H.R. Horvitz (in press). (2) Lu and Horvitz. (1998) Cell 95 : 981. (3) Walhout et al. (2000) Science 287 :116.

Sun, Simo et al. (2017) International Worm Meeting "Neural and molecular basis involved in low preference to Bifidobacterium infantis in Caenorhabditis elegans."

Kage-Nakadai, Eriko, Nishikawa, Yoshikazu, Sun, Simo

[International Worm Meeting, 2017]

Neural and molecular mechanisms underlying food preference have been poorly understood. We previously showed that Bifidobacterium infantis, one of the well-known probiotic bacteria, extend the lifespan of Caenorhabditis elegans (Ikeda et al., Appl. Environ. Microbiol., 2007). In this study, we found that C. elegans exhibited low preference to B. infantis when compared with a standard food E. coli. Genetic screens identified tol-1 (a sole homolog of mammalian Toll-like receptors) and daf-16/FoxO as positive regulators of low preference to B. infantis. Worms with mutations in canonical TLR signaling molecules (pmk-3, mom-4 and ikb-1) that are employed in avoidance behavior to a pathogenic bacterium Serratia marcescens (Brand et al.,Curr. Biol., 2015) showed a normal behavior to B. infantis, suggesting alternative mechanisms. Genetic analyses revealed that the function of daf-16 isoform b in AIY neurons is responsible for the behavior to B. infantis. Because daf-16b regulates the AIY neurons morphology during development (Christensen et al., Development, 2011), daf-16b may play roles in AIY development to organize low preference to B. infantis.