Hilliard MA (2009) J Neurochem "Axonal degeneration and regeneration: a mechanistic tug-of-war."

Hilliard MA

[J Neurochem, 2009]

Axonal degeneration is a common hallmark of both nerve injury and many neurodegenerative conditions, including motor neuron disease, glaucoma, and Parkinson's, Alzheimer's, and Huntington's diseases. Degeneration of the axonal compartment is distinct from neuronal cell death, and often precedes or is associated with the appearance of the symptoms of the disease. A complementary process is the regeneration of the axon, which is commonly observed following nerve injury in many invertebrate neurons and in a number of vertebrate neurons of the PNS. Important discoveries, together with innovative imaging techniques, are now paving the way towards a better understanding of the dynamics and molecular mechanisms underlying these two processes. In this study, I will discuss these recent findings, focusing on the balance between axonal degeneration and regeneration.

Hilliard MA et al. (1998) European Worm Meeting "qui-1, a gene involved in quinine avoidance"

Bazzicalupo P, Hilliard MA

[European Worm Meeting, 1998]

We are studying chemoreception in C.elegans focusing our attention on the avoidance of water soluble substances. The mechanisms by which taste receptors function and how the signals are transduced is poorly understood compared with what is known about olfaction . Quinine is one of the substances found to act as repellents in C. elegans. 15 mutants that fail to avoid quinine when tested with the drop test have been previously isolated. Two of them gb404 and gb408 have been better characterised phenotypically studying the response to all the repellents discovered so far with the drop test. They show a diminished response to all the quinoline derivatives tested: amodiaquin, chloroquine, primaquine, quinacrine. They also fail to respond to etidium bromide, denatonium, acid pH, and SDS. However the mutants maintain unaltered the response to other repellents such as high osmotic strength (glycerol 2M), copper ions, garlic extract, levamisole. They also respond normally to light touch and when stained with FITC they are dyf+. The two mutations do not complement and map on the right arm of LG IV between dpy-4 and stP35 or immediately past stP35 but very close to it. We have started the molecular identification of the gene by transforming gb404 with a Yac of 520 Kb (Y57G11) which lies to the right of stP35. Neither the injection of Y57G11 nor the injection of PCR amplification products corresponding to three potential candidate genes present in the yac itself seem to be able to rescue the quinine non-avoider phenotype of gb404. We hope to be on the way of uncovering the identity of this gene in the near future.

Hilliard MA et al. (1997) International C. elegans Meeting "C.ELEGANS MUTANTS THAT FAIL TO AVOID QUININE"

Bazzicalupo P, Hilliard MA

[International C. elegans Meeting, 1997]

We are studying chemoreception in C.elegans focusing our attention on the avoidance of water soluble substances. We have used a new test, "drop test", which consists in delivering a small drop of the chosen solution behind a worm freely moving on the plate surface. By capillarity the solution reaches the anterior end of the animal where the amphids are located. If the solution contains a repellent, the worm stops immediately and moves backward for a few seconds; if no repellent is present the worm continues its forward movement. Using this test we have identified, including those already known, 16 chemicals acting as repellent at concentrations between 0.1 and 10 mM. We have chosen quinine, at a concentration of 10 mM, to screen the F2 progeny of EMS mutagenized worms. Out of 70.000 worms screened we have found 15 mutants which fail to avoid quinine. After preliminary behavioral characterization we have focused our attention on two, gb404 and gb408. These are allelic and identify a new gene, qui-1, which has been positioned on the right arm of the LG IV using STS mapping. The amphid neurons in both alleles stain normally with FITC. They respond normally to light touch and to some other repellent e.g. copper ions, zinc ions, veratridin, levamisole, garlic extract, high osmotic strenght. We hope we have identified either the receptor for quinine, if it exists, or some other component acting relatively early in the sensory transduction pathway used by C.elegans to avoid quinine.

Hilliard MA et al. (2006) Dev Cell "Wnt signals and frizzled activity orient anterior-posterior axon outgrowth in C. elegans."

Bargmann CI, Hilliard MA

[Dev Cell, 2006]

Secreted proteins of the Wnt family affect axon guidance, asymmetric cell division, and cell fate. We show here that C. elegans Wnts acting through Frizzled receptors can shape axon and dendrite trajectories by reversing the anterior-posterior polarity of neurons. In lin-44/Wnt and lin-17/Frizzled mutants, the polarity of the PLM mechanosensory neuron is reversed along the body axis: the long PLM process, PLM growth cone, and synapses are posterior to its cell body instead of anterior. Similarly, the polarity of the ALM mechanosensory neuron is reversed in cwn-1 egl-20 Wnt double mutants, suggesting that different Wnt signals regulate neuronal polarity at different anterior-posterior positions. LIN-17 protein is asymmetrically localized to the posterior process of PLM in a lin-44-dependent manner, indicating that Wnt signaling redistributes LIN-17 in PLM. In this context, Wnts appear to function not as instructive growth cone attractants or repellents, but as organizers of neuronal polarity.

Hilliard MA et al. (2005) EMBO J "In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents."

Suzuki H, Bazzicalupo P, Kerr R, Apicella AJ, Schafer WR, Hilliard MA

[EMBO J, 2005]

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca2+ responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper- and hypo- osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC- 1, a G- protein gamma-subunit expressed specifically in chemosensory neurons. We hypothesize that G- protein gamma- subunit heterogeneity provides a mechanism for repellent- specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.

Hilliard MA et al. (2005) EMBO J "In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents."

Kerr R, Suzuki H, Apicella AJ, Schafer WR, Bazzicalupo P, Hilliard MA

[EMBO J, 2005]

Due to a typesetting error, Figure 1 of the above article was published incorrectly.Correction to: The EMBO Journal (2005) 24, 63-72. doi:10.1038/sj.emboj.7600493.

Hilliard MA et al. (2004) EMBO J "Worms taste bitter: ASH neurons, QUI-1, GPA-3 and ODR-3 mediate quinine avoidance in Caenorhabditis elegans."

Hilliard MA, Plasterk RHA, Bazzicalupo P, Arbucci S, Bergamasco C

[EMBO J, 2004]

An animal's ability to detect and avoid toxic compounds in the environment is crucial for survival. We show that the nematode Caenorhabditis elegans avoids many water-soluble substances that are toxic and that taste bitter to humans. We have used laser ablation and a genetic cell rescue strategy to identify sensory neurons involved in the avoidance of the bitter substance quinine, and found that ASH, a polymodal nociceptive neuron that senses many aversive stimuli, is the principal player in this response. Two G protein alpha subunits GPA-3 and ODR-3, expressed in ASH and in different, nonoverlapping sets of sensory neurons, are necessary for the response to quinine, although the effect of odr-3 can only be appreciated in the absence of gpa-3. We identified and cloned a new gene, qui-1, necessary for quinine and SDS avoidance. qui-1 codes for a novel protein with WD-40 domains and which is expressed in the avoidance sensory neurons ASH and ADL.

MA Hilliard et al. (1999) International C. elegans Meeting "Avoidance of water soluble repellents"

MA Hilliard, P Bazzicalupo

[International C. elegans Meeting, 1999]

The mechanisms underlying water soluble sensitivity (taste) are poorly understood both in vertebrates and in invertebrates. We have developed a new assay to study, in C. elegans , the avoidance of water soluble repellents. This assay, the drop test, consists in delivering a small drop of the chosen solution behind a worm freely moving on the plate surface. By capillarity the solution reaches the anterior end of the animal where the amphids are located. If the solution contains a repellent, the worm immediately stops its forward movement and starts backward. If no repellent is present, the worm continues moving forward. With this test we have identified 16 repellents and the list of them will be presented. We also developed a variation, the dry drop test, in which the drop is delivered just in front of the worm but far enough that the animal reaches the drop only when it dries out. Quinine is one of the substances that induces in the worms a strong avoidance response. Humans perceive it as tremendously bitter; in the channel catfish it induces responses in neurons of the oropharyngeal taste system. In order to discover the genes involved in its sensitivity we used quinine, with the drop test, to screen for mutants. We isolated 15 mutants and two of them gb 404 and gb 408 , that do not complement, have been better characterized phenotypically. They show a partial specificity in that they continue to respond to a subset of substances; their amphids cilia appear normal when stained with DiO or FITC. Genetic data locate them on the right arm of the LGIV between dpy-4 and stP35 or immediately past stP35 but very close to it. So far initial rescue attempts have failed. We have tested the existing mutants of the osm series with some of the repellent substances and we found a partial overlap between the drop test and the ring test (barrier test). Finally, we are attempting to identify the cells responsible for the avoidance of different substances in the drop test by genetic manipulations. We are using different sensory neurons specific promoters to drive mec-4(d) as a dominant cell autonomous killer gene (M. Driscoll). We are also trying to rescue the function of specific cells in an osm-6 mutants background (thanks to C. Bargmann for the suggestion and to J.Shaw for osm-6 cDNA).

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

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

[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.

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.