Jordan, Aurore et al. (2021) International Worm Meeting "Human pain gene ortholog screen in C. elegans"

Lia, Andrei-Stefan, Glauser, Dominique A., Jordan, Aurore

[International Worm Meeting, 2021]

Chronic pain affects up to 30% of the population and can be caused by different factors such as injuries or genetic mutations. Chronic pain can take various forms like migraines, different musculoskeletal conditions (low back pain, fibromyalgia), neuropathic pain disorders triggered by cancer or diabetes, as well as visceral pain disorders. Available drugs are not always effective and could trigger many undesirable side effects. In the recent years, genes responsible for those pathologies have been screened in the population with methods, such as linkage analysis and genome wide association studies. Those approaches have revealed a plethora of new candidates but, for the majority of them, we know nothing about their function in the pain pathway. Because studies on pain in human are very limited, we propose C. elegans as a complementary model organism to study conserved nociception genes. In the present study, we first determined a list of C. elegans orthologs for candidate human pain genes reported in the literature and recovered available mutants (109 mutants). We then screened these mutants for alterations in noxious heat-evoked reversals using as high-throughput, computer-assisted behavior analysis pipeline. Using different heat intensities and comparing naive animals with animals repeatedly stimulated with heat, we obtained a set of measures reflecting baseline sensitivity profiles, and the ability to adapt (desensitize) in response to repeated stimuli. Twenty-two mutants displayed significant alterations and could be clustered in different categories, including fast-adapting mutants, non-adapting mutants, and mutants with exacerbated naive sensitivity. As a whole, our study suggests that C. elegans represent a promising model to tackle the function of recently identified human pain-associated genes and set the bases for additional studies on specific candidates. Topics: neurobiology, behavior Keywords: noxious heat avoidance; sensory plasticity; pain modelling

Owens, Hannah et al. (2021) International Worm Meeting "Characterizing the Role of the Mechanosensitive Ion Channel TACAN in C. elegans Osmosensation"

Davidova, Albena, Hendricks, Michael, Owens, Hannah, Tala Tirera, Fouley, Sharif-Naeini, Reza

[International Worm Meeting, 2021]

Mechanotransduction occurs via ion channels whose gating is controlled by mechanical stimuli. Recently, the transmembrane protein TACAN was identified as a mechanosensitive ion channel crucial for sensing mechanical pain in mice, and TACAN homologs were shown to be highly conserved across other species such as humans and nematodes. The nematode C. elegans is an ideal model organism to study the molecular properties of TACAN, given its mapped connectome, simple behavior, and capacity for genetic manipulations. Our preliminary data suggest that the uncharacterized TACAN homolog in C. elegans is involved in worm mechanosensation, specifically contributing to the detection of osmotic stimuli.

Madhivanan, K. et al. (2017) International Worm Meeting "Modulating Transthyretin levels to ameliorate neuronal proteotoxicity in Caenorhabditis elegans models of Transthyretin Amyloidosis."

Madhivanan, K., Chapman, J., Encalada, S. E, Kelly, J. W, Dillin, A., Aguirre, C., Jiang, X., Rouzbeh, N., Greiner, E. R, Alves-Ferreira, M., Paulsson, J.

[International Worm Meeting, 2017]

Familial Amyloid Polyneuropathy (FAP) is a lethal autosomal dominant systemic amyloidosis caused by the aggregation of transthyretin (TTR), a tetrameric protein secreted primarily by the liver. The most common FAP mutation -V30M TTR- affects primarily peripheral nerves, resulting in loss of pain- and thermo-sensation, and sensory-motor function. In FAP, the V30M TTR tetramer dissociates post-secretion, leading to monomer misfolding, and aggregation around peripheral neurons. The non-native TTR species (i.e. oligomers, aggregates) have been hypothesized to result in peripheral neurodegeneration, but not tested due to the lack of models faithfully recapitulating the cell non-autonomous neuronal phenotypes. To investigate the mechanisms of neurodegeneration in FAP, we generated C. elegans models expressing human V30M TTR exclusively in the body-wall muscle. Using immunofluorescence and small molecule fluorogenic sensors, we confirmed secretion of theV30M TTR tetramer, which is then endocytosed into degradative macrophage-like coelomocytes. The V30M animals display impaired pain sensation and uncoordinated locomotion, phenotypes that we show depend on the expression of TTR, and that resemble FAP patient relevant neuronal symptoms. To test whether modulating TTR levels alters TTR proteotoxicity, we first genetically ablated coelomocytes in V30M TTR expressing animals, and observed that levels of TTR oligomers were significantly increased, followed by decreased pain sensation response. We also used RNAi to reduce TTR protein levels, and treated V30M TTR animals with tafamidis, a regulatory agency approved drug that binds strongly to the native TTR tetramer and stabilizes it and dramatically slows tetramer dissociation. Both approaches resulted in reduced TTR oligomer formation and in significant rescue of the uncoordinated phenotype in V30M TTR animals. Thus, we showed that in our FAP disease models that exhibit TTR-mediated patient relevant phenotypes, modulation of TTR levels can be used as a potential therapeutic opportunity.

Wood, Jordan et al. (2015) International Worm Meeting "Developmental specification of a polymodal nociceptor in C. elegans."

Ferkey, Denise, Wood, Jordan

[International Worm Meeting, 2015]

All animals rely on their ability to sense and respond to the environment to survive. Nociception (the perception of noxious/painful stimuli) serves an important protective function, eliciting withdrawal and avoidance behaviors in response to potentially damaging stimuli. The primary nociceptors in C. elegans are the two ASH head sensory neurons, which detect multiple forms of aversive stimuli. However, while the sensation of pain is particularly valuable in helping an organism to avoid potentially harmful stimuli, there are still large gaps in our understanding of the mechanisms that govern nociceptor development across species. Based upon previously published experiments from other groups, we hypothesized that the paired-like homeodomain transcription factor UNC-42 functions as a terminal selector of ASH identity. However, our data indicate that although UNC-42 regulates a significant portion of the functionally mature ASH state, it does not function as the sole driver of the terminal ASH identity. In addition to UNC-42, a diverse array of transcription factors are expressed in the ASH neurons, which in combination likely comprise the genetic program underlying ASH specification and thus drive expression of the sensory signaling apparatus that endows the adult ASHs with polymodal nociceptive sensitivity. Using molecular, genetic and behavioral approaches, we have identified and are currently characterizing the function of several transcription factors in ASH specification.

Wood, Jordan et al. (2013) International Worm Meeting "Developmental Specification of a Polymodal Nociceptor in C. elegans."

Ferkey, Denise, Wood, Jordan

[International Worm Meeting, 2013]

All animals rely on their ability to sense and respond to the environment to survive. Nociception serves an important protective function, eliciting withdrawal and avoidance behaviors in response to potentially damaging stimuli. However, while pain sensation is particularly valuable in helping an organism to avoid potentially harmful stimuli, there are still large gaps in our understanding of the mechanisms that govern nociceptor development across species. In C. elegans, the pair of polymodal nociceptive ASH head sensory neurons responds to a broad range of aversive stimuli, including soluble chemicals, odorants, ions, osmotic stress and mechanosensory stimulation, with ASH detection activating backward locomotion and aversive responses. Importantly, the ASHs are believed to be analogous to nociceptors that detect multiple pain modalities in systems ranging from other invertebrates such as Drosophila to vertebrates. To understand the continuum that connects embryonic neuronal developmental specification with neuronal physiological function in adult animals, we aim to characterize the developmental transcriptional hierarchy that bestows the C. elegans ASH nociceptors with polymodal sensitivity and to determine the extent to which identified modality-specific gene batteries contribute to associated adult animal sensory behaviors. Our central hypothesis is that the paired-like homeodomain transcription factor UNC-42 functions as a "terminal selector" in the developmental specification of ASH cell identity. We will present behavioral and ASH-reporter data examining the role for UNC-42 in coordinating multiple cellular characteristics during development in the specification of the functionally distinct ASH neuronal identity.

Debnath, Arunima et al. (2021) International Worm Meeting "Neuromodulation and the relationship between Ca2+ transients and neuronal states"

Bamber, Bruce, Debnath, Arunima

[International Worm Meeting, 2021]

Neuromodulators (monoamines and neuropeptides) shape nervous system function by regulating neuronal depolarization and synaptic strengths. We are studying neuromodulation in the context of nociception, to better understand pain perception and pain treatment strategies. The ASH neuron is a major nociceptor in C. elegans. ASH senses 1-octanol and drives an aversive response, modulated by the monoamines serotonin (5-HT, potentiating) and octopamine (OA, inhibitory). To better understand neuromodulation, we are focusing on 1-octanol stimulated Ca2+ dynamics in ASH, and the quantitative relationship between Ca2+ signals and depolarization amplitudes. We showed that 5-HT potentiates ASH depolarization, but surprisingly, inhibits ASH Ca2+ transient amplitudes. These effects, like the 5-HT stimulation of behavior, depend on the SER-5 receptor in ASH. This paradoxical finding is explained by existence of a Ca2+-dependent inhibitory feedback loop: Ca2+ inhibits ASH depolarization through SLO-1 IKCa channels, and 5-HT inhibits EGL-19 L-type Ca2+ channels in ASH (via SER-5), thus disinhibiting the neuron. We are currently investigating modulation of 1-octanol responses by OA. OA inhibits 1-octanol behavioral responses, and antagonizes 5-HT potentiation, dependent on the OCTR-1 receptor in ASH. OA also inhibits ASH Ca2+ transients, representing another paradox: how can OA and 5-HT have opposite effects on ASH-dependent aversive behaviors, but the same effect on ASH Ca2+ transients? Our results show that 5-HT and OA utilize distinct signaling pathways in ASH (Galphaq for 5-HT and Galphao for OA), and that OA does not inhibit EGL-19. We are currently testing the hypothesis that OA hyperpolarizes ASH through Galphao-dependent activation of IRK K+ channels. These results further emphasize that neuronal Ca2+ transients, as key reporters of neuronal depolarization, are also critical signaling intermediates in and of themselves, with multiple upstream inputs and downstream consequences.

Li, Wei et al. (2011) International Worm Meeting "The neural circuits underlying harsh touch sensation in C. elegans."

Feng, Zhaoyang, Xu, Shawn, Li, Wei, Kang, Lijun, Piggott, Beverly

[International Worm Meeting, 2011]

Most animals can distinguish two distinct types of touch stimuli: gentle (innocuous) and harsh (noxious/painful) touch, but the underlying mechanisms are not well understood. C. elegans is a highly successful model for the study of gentle touch sensation, and work in C. elegans has derived a thorough understanding of the mechanisms of gentle touch sensation. However, little is known about harsh touch sensation. Here we characterized harsh touch behavior in C. elegans. C. elegans exhibits differential behavioral responses to harsh touch and gentle touch. Laser ablations identified distinct sets of sensory neurons and interneurons required for harsh touch sensation at different body segments. Optogenetic stimulation of the circuitry can drive behavioral responses. Both TRP family and amiloride-sensitive Na+ channels regulate harsh touch sensation. Our results identify the neural circuits required for harsh touch sensation in C. elegans and establish C. elegans as a valuable model for studying this sensory modality.

Husson, Steven J. et al. (2011) International Worm Meeting "Optogenetics dissection of the nociceptive PVD network: RNAi of PVD-specific genes reveals TRP channels as signal amplifiers."

Miller III, David M., Gottschalk, Alexander, Spencer, W. Clay, Treinin, Millet, Stirman, Jeffrey N., Watson, Joseph D., Steuer Costa, Wagner, Husson, Steven J., Lu, Hang

[International Worm Meeting, 2011]

Higher animals display myriads of neurons contributing to pain sensation, making it extremely difficult to study the neuronal circuits underlying nociception. In contrast, only a handful of neurons mediate nociception in C. elegans. Nociceptive neurons contain highly branched dendrites and trigger the sensation of pain in response to noxious insults. The FLP and PVD neurons adopt similar complex dendritic arbors and have been implicated in harsh mechanical touch responses, while other touch receptor neurons (TRNs) detect gentle mechanical stimuli. Studying the behavioral output of the mechanosensory modality of PVD or FLP cells has so far been complicated, because a harsh touch inevitably agitates the whole body of the animal, thus making it impossible to prevent contributions of other cells. In contrast, optogenetic tools like the blue light-activated depolarizing channelrhodopsin-2 (ChR2), allow non-invasive stimulation of the sensory neuron of interest. Expression of ChR2 in the PVD neurons, FLP neurons or downstream command interneurons and subsequent photoactivation, allows us to mimic the harsh touch response, without interfering signaling from the TRN neurons. We show that behavioral responses to harsh touch are shaped by both the FLP and PVD sensors to jointly determine the output response as forward or reverse escape. Molecular players required for PVD function were identified by microarray profiling of mRNAs specifically isolated from PVD. Candidate nociceptive genes were knocked down by RNAi and effects on photo-triggered, ChR2-mediated escape responses were quantified. We found the VGCC a- and b-subunits UNC-2 and CCB-1 to be required for chemical synaptic output from PVD. Targeting synaptobrevin by the Tetanus toxin in PVD to impair synaptic output abolished PVD-dependent behavior. Knockdown of regulators of PVD differentiation severely impaired PVD function, while a DEG/ENaC channel and a TRP channel act cell-autonomously in PVD to shape its dynamic range and amplify the output signal, uncovering a novel role of TRP channels downstream of primary sensor molecules.

Rudgalvyte, Martina et al. (2021) International Worm Meeting "Identification of calcium/calmodulin-dependent protein kinase I (CMK-1) phospho-targets relevant for nociceptive plasticity in C. elegans"

Glauser, Dominique, Hu, Zehan, Dengjel, Joern, Stumpe, Michael, Rudgalvyte, Martina, Jordan, Aurore, Kressler, Dieter

[International Worm Meeting, 2021]

Nociception is a conserved process serving as a self-protection system alerting animals of potential damage and underpinning different forms of pain in humans. Some chronic pain conditions may arise from maladaptive modulation in the nociceptive pathway, including within nociceptors, the primary nociceptive sensory neurons. We use Caenorhabditis elegans as a model due to its ability to detect noxious stimuli, perform avoidance behaviors in the form of stimulus-evoked reversals and adapt to repeated stimuli causing a desensitized, analgesia-like state. The worm ortholog of mammalian CaMKI/IV, CMK-1 (calcium/calmodulin-dependent kinase-1) mediates cellular responses to increased calcium levels and is crucial in nociceptors for this avoidance behavior plasticity. However, the downstream elements of the CMK-1 pathway remain unclear. Here, we performed in vitro CMK-1 kinase assays on both peptide and protein from total worm isolates in order to identify direct kinase target candidates via shotgun phosphoproteomics. For in vivo direct/indirect target determination, we carried out stable isotope labeling by amino acids (SILAC) using high-throughput quantitative phosphoproteomics. We used duplex SILAC, where ''light'' 12-carbon and ''heavy'' 13-carbon amino acids were incorporated into nematode proteins for measuring the amounts of phosphorylated proteins in wild type and cmk-1 null animals. By combining results obtained from these different studies, we were able to ascertain CMK-1 phosphorylation consensus and develop a list of potential CMK-1 targets. Mutants for these candidates were then tested for heat avoidance behavior to determine changes in naive sensitivity to noxious heat and/or adaptation. We used a computer-assisted high-throughput analysis pipeline to quantify heat-evoked reversals in naive animals and animals exposed to repeated stimuli. While wild type animal sensitivity decreased in response to the repeated heat stimuli, some mutant animals failed to adapt. In conclusion, our study reveals several potential CMK-1 targets that may have an important role in behavioral plasticity.

Alexander, Courtney et al. (2021) International Worm Meeting "Pseudognaphalium obtusifolium Extract Improves Lifespan and Thermotolerance in C. elegans"

Mosely, Dontae, Deberry, Kazhmiri, Edwards, Ashley, Mariani, Hannah, Alexander, Courtney, Sandefur, Conner

[International Worm Meeting, 2021]

For centuries, Lumbee Indians of North Carolina have used indigenous plants like Pseudognaphalium obtusifolium, rabbit tobacco, to make medicinal teas. Other tribes, like Cherokees and Creeks, used rabbit tobacco teas to combat a host of illnesses and maladies from muscle pain to colds. These teas were believed to have anti-inflammatory properties, though the biology has not been explored. We harvested and dried local rabbit tobacco and prepared aqueous extracts. The extract improved the lifespan, thermotolerance and motility of young adult nematodes, compared to vehicle control. Animals were age-synchronized and exposed to either the aqueous extract or control media. Then, worms were tested for thermotolerance or motility. A lifespan analysis was run in parallel. Nematodes that were treated with rabbit tobacco extract lived longer and had improved thermotolerance and motility. Work is ongoing to determine the molecular mechanism for this phenotype, as well as testing other Lumbee plants. This work helps pave the way for a biological basis for indigenous medicine.