Coakley, S. et al. (2017) International Worm Meeting "The epidermis protects sensory axons from degeneration."

Hilliard, M.A., Ritchie, F., Coakley, S.

[International Worm Meeting, 2017]

Maintenance of neuronal integrity is essential for the preservation of correct neuronal function. Sensory neurons and their axons are subject to continuous mechanical stress due to their location within the skin, muscles, and moveable joints. Despite the strong forces experienced, these neurons are able to maintain their structure and functional circuitry. The ability to resist strain has been shown to be a combination of intrinsic and extrinsic protection mechanisms, but the precise interplay between cell autonomous and cell-non-autonomous stress resistance is not known. Mutations in C. elegans beta -spectrin/unc-70 cause spontaneous axonal breakage due to mechanical strain. Through an unbiased forward genetic screen using unc-70 mutants as a sensitised background, we have identified a novel mutant allele of the conserved gene tbc-10, which results in enhanced axonal damage to the PLM mechanosensory neuron. TBC-10 is a Rab-GTPase-activating protein that we demonstrate localises to the membrane of the hypodermis surrounding the PLM axon and functions non-cell-autonomously within this tissue to exert an axonal protective effect via inactivation of the conserved small GTP-ase RAB-35. Inactivation of RAB-35 within the hypodermis, by either expression of a GDP-locked RAB-35 or a loss of function mutation, is sufficient to rescue the enhanced axonal breakage phenotype in tbc-10 mutants. We show that in C. elegans the epidermis acts to protect the axons of mechanosensory neurons from spontaneous degeneration induced by disruption of the spectrin network, demonstrating a crucial role for non-neuronal support cells in maintaining an intact and functional nervous system.

Coakley S et al. (2020) Nat Commun "Epidermal control of axonal attachment via -spectrin and the GTPase-activating protein TBC-10 prevents axonal degeneration."

Hilliard MA, Ritchie FK, Coakley S, Galbraith KM

[Nat Commun, 2020]

Neurons are subjected to strain due to body movement and their location within organs and tissues. However, how they withstand these forces over the lifetime of an organism is still poorly understood. Here, focusing on touch receptor neuron-epidermis interactions using Caenorhabditis elegans as a model system, we show that UNC-70/-spectrin and TBC-10, a conserved GTPase-activating protein, function non-cell-autonomously within the epidermis to dynamically maintain attachment of the axon. We reveal that, in response to strain, UNC-70/-spectrin and TBC-10 stabilize trans-epidermal hemidesmosome attachment structures which otherwise become lost, causing axonal breakage and degeneration. Furthermore, we show that TBC-10 regulates axonal attachment and maintenance by inactivating RAB-35, and reveal functional conservation of these molecules with their vertebrate orthologs. Finally, we demonstrate that -spectrin functions in this context non-cell-autonomously. We propose a model in which mechanically resistant epidermal attachment structures are maintained by UNC-70/-spectrin and TBC-10 during movement, preventing axonal detachment and degeneration.

Coakley S et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "NFI-1 (nuclear factor one) transcription factor regulates development of sensory rays in C. elegans male tail"

Coakley S, Richards L J, Lazakovitch E, Gronostajski R M, Hilliard M A

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

The NFI family of transcription factors contains four members in all vertebrates (Nfia, Nfib, Nfic and Nfix). Three of these family members (Nfia, Nfib and Nfix) are expressed within the mammalian CNS and function to regulate different aspects of neural development, including axonal guidance, gliogenesis and neurogenesis. A single NFI gene, nfi-1, is present in the C. elegans genome. Loss of nfi-1 results in slower pharyngeal pumping rates, egg laying defects, impaired motility and a decreased life span (Lazakovitch et al., 2005). We investigated the function of nfi-1 in different classes of C. elegans neurons including the DD and VD GABAergic motor neurons, the mechanosensory neurons and the oxygen sensory neurons AQR, PQR and URX without finding obvious morphological or functional defects. Analysis of the amphid and phasmid glia also appeared functionally intact in the nfi-1 mutant animals. On the contrary, analysis of the male tail revealed that loss of nfi-1 resulted in abnormal development of the sensory rays derived from the V cells (rays 1 to 6). The defects included missing, anteriorly displaced or fused rays, or rays with gross abnormal morphologies. Interestingly, we also observed that overexpression of nfi-1 from the endogenous promoter, as well as from a pan-neuronal promoter (F25B3.3), caused defects in V-ray formation. We propose a previously unknown role for NFI-1 in regulating V-ray development in the C. elegans male tail, a novel paradigm to investigate in detail the function and targets of this transcription factor.

Ho, X.Y. et al. (2019) International Worm Meeting "The metalloprotease ADM-4 promotes regenerative axonal fusion."

Ho, X.Y., Coakley, S., Hilliard, M.A.

[International Worm Meeting, 2019]

Axonal damage, such as that observed in nerve and spinal cord injuries, interrupts the communication between a neuron and its target tissue. Functional recovery is achieved when the regenerating axon re-innervates its original target tissue. However, despite progress in medicine, intervention to repair such damage is still not achievable. C. elegans and other invertebrate species have evolved a specific repair mechanism, whereby the proximal axonal fragment (still attached to the cell body) first regrows, reaches its own separated distal axonal fragment, and then through a membrane fusion event, known as axonal fusion, re-establishes the original axonal tract and neuronal function. Axonal fusion therefore represents an innovative and potentially highly-effective repair strategy for axonal injuries. Using a candidate gene approach, and the PLM mechanosensory neurons as a model system, we have identified ADM-4 as a key regulator of axonal fusion. ADM-4 is a member of the ADAM (A Disintegrin and Metalloprotease) family, and ortholog of the human ADAM17/TACE (Tumor necrosis factor Alpha-Converting Enzyme). adm-4 loss of function leads to severe reduction of axonal fusion in PLM neurons without affecting axonal regrowth. We demonstrate that ADM-4 regulates axonal fusion by functioning cell-autonomously in PLM neurons. Furthermore, overexpression of this molecule selectively in the PLM neurons is sufficient to enhance axonal fusion in wild-type animals. We reveal dynamic changes in the subcellular localisation of ADM-4 after axotomy, and identify the metalloprotease domain as essential to promote axonal fusion. We have previously demonstrated that phosphatidylserine (PS) exposure on the damaged axon functions as a "save-me" signal, which allows for successful axonal fusion. We show that putative PS-binding sites in ADM-4 are required for axonal fusion, and propose that PS exposure induced by injury, activates ADM-4 metalloproteinase and promotes fusion. Thus, our results reveal an essential function for ADM-4 in promoting axonal repair by fusion, and provide a strong foundation for designing novel therapeutics for injuries to the nervous system.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.

Bonacossa-Pereira I et al. (2022) Cell Rep "Neuron-epidermal attachment protects hyper-fragile axons from mechanical strain."

Hilliard MA, Coakley S, Bonacossa-Pereira I

[Cell Rep, 2022]

Axons experience significant strain caused by organismal development and movement. A combination of intrinsic mechanical resistance and external shielding by surrounding tissues prevents axonal damage, although the precise mechanisms are unknown. Here, we reveal a neuroprotective function of neuron-epidermal attachment in Caenorhabditis elegans. We show that a gain-of-function mutation in the epidermal hemidesmosome component LET-805/myotactin, in combination with a loss-of-function mutation in UNC-70/β-spectrin, disrupts the uniform attachment and subsequent embedment of sensory axons within the epidermis during development. This generates regions of high tension within axons, leading to spontaneous axonal breaks and degeneration. Completely preventing attachment, by disrupting HIM-4/hemicentin or MEC-5/collagen, eliminates tension and alleviates damage. Finally, we demonstrate that progressive neuron-epidermal attachment via LET-805/myotactin is induced by the axon during development, as well as during regeneration after injury. Together, these results reveal that establishment of uniform neuron-epidermal attachment is critical to protect axons from mechanical strain during development.

Vandecandelaere I et al. (2017) PLoS One "Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus."

Deforce D, Coenye T, Van Nieuwerburgh F, Vandecandelaere I

[PLoS One, 2017]

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.

Kirszenblat L et al. (2013) Mol Biol Cell "A dominant mutation in mec-7/-tubulin affects axon development and regeneration in Caenorhabditis elegans neurons."

Kirszenblat L, Neumann B, Coakley S, Hilliard MA

[Mol Biol Cell, 2013]

Microtubules have been known for decades to be basic elements of the cytoskeleton. They form long, dynamic, rope-like structures within the cell that are essential for mitosis, maintenance of cell shape, and intracellular transport. More recently, in vitro studies have implicated microtubules as signaling molecules that, through changes in their stability, have the potential to trigger growth of axons and dendrites in developing neurons. In this study, we show that specific mutations in the Caenorhabditis elegans mec-7/-tubulin gene cause ectopic axon formation in mechanosensory neurons in vivo. In mec-7 mutants, the ALM mechanosensory neuron forms a long ectopic neurite that extends posteriorly, a phenotype that can be mimicked in wild-type worms with a microtubule-stabilizing drug (paclitaxel), and suppressed by mutations in unc-33/CRMP2 and the kinesin-related gene, vab-8. Our results also reveal that these ectopic neurites contain RAB-3, a marker for presynaptic loci, suggesting that they have axon-like properties. Interestingly, in contrast with the excessive axonal growth observed during development, mec-7 mutants are inhibited in axonal regrowth and remodeling following axonal injury. Together our results suggest that MEC-7/-tubulin integrity is necessary for the correct number of neurites a neuron generates in vivo and for the capacity of an axon to regenerate.

Vandecandelaere I et al. (2014) Pathog Dis "Protease production by Staphylococcus epidermidis and its effect on Staphylococcus aureus biofilms."

Coenye T, Vandecandelaere I, Depuydt P, Nelis HJ

[Pathog Dis, 2014]

Due to the resistance of Staphylococcus aureus to several antibiotics, treatment of S. aureus infections is often difficult. As an alternative to conventional antibiotics, the field of bacterial interference is investigated. Staphylococcus epidermidis produces a serine protease (Esp) which inhibits S. aureus biofilm formation and which degrades S. aureus biofilms. In this study, we investigated the protease production of 114 S. epidermidis isolates, obtained from biofilms on endotracheal tubes (ET). Most of the S. epidermidis isolates secreted a mixture of serine, cysteine and metalloproteases. We found a link between high protease production by S. epidermidis and the absence of S. aureus in ET biofilms obtained from the same patient. Treating S. aureus biofilms with the supernatant (SN) of the most active protease producing S. epidermidis isolates resulted in a significant biomass decrease compared to untreated controls, while the number of metabolically active cells was not affected. The effect on the biofilm biomass was mainly due to serine proteases. Staphylococcus aureus biofilms treated with the SN of protease producing S. epidermidis were thinner with almost no extracellular matrix. An increased survival of Caenorhabditis elegans, infected with S. aureus Mu50, was observed when the SN of protease positive S. epidermidis was added.