Tortajada Perez*, Julia et al. (2021) International Worm Meeting "Downstream effectors of the synergic activation of AMPK by metformin and salicylate to reduce polyQ aggregation"

Gomez Escribano, Ana Pilar, Vazquez Manrique, Rafael, Tortajada Perez*, Julia, Trujillo del Rio*, Cristina

[International Worm Meeting, 2021]

Huntington disease (HD) is a rare neurodegenerative disorder of autosomal dominant inheritance, characterized by an expansion in the number of CAG triplets in the huntingtin gene's (HTT) first exon. No therapy has been proven effective for HD treatment thus-far, but some compounds, such as metformin, have been reported as potentially beneficial disease-modifiers of this pathology. Previous findings have shown that the synergistic activation of AMP-activated protein kinase (AMPK), with metformin and salicylate, reduces aggregation and neuronal damage in Caenorhabditis elegans models of toxicity induced by polyglutamines (polyQs). Furthermore, strong evidence suggests that activation of AMPK, with just metformin, is neuroprotective in mouse models of HD. AMPK is a master regulator of healthspan and lifespan modulating energy metabolism, stress resistance and cellular proteostasis; via pathways such as autophagy, oxidative stress, and lipid metabolism. For instance, AMPK activates Nrf2/SKN-1, FoxO/DAF-16, and SIRT1/SIR-2.1 signalling pathways; and inhibits the signalling of CRTC-1/CRTC-1, and mTOR/LET-363. An activation of autophagy has been detected in metformin/salicylate-treated worms promoting proteostasis in a poliQs-induced toxicity context, however, preliminary results in our laboratory indicate that this activation may be independent of AMPK. In this work, we have studied several targets downstream of AMPK, potentially responsible for the protective effect dependent on salicylate and metformin synergistic AMPK activation, and show their relationship with this signalling pathway.

HA Thieringer et al. (1999) International C. elegans Meeting "Analysis of a deletion mutation of del-1, a member of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily"

N Tavernarakis, M Driscoll, HA Thieringer

[International C. elegans Meeting, 1999]

del-1 ( de generin- l ike) encodes a protein that is a member of the DEG/ENaC superfamily. Reporter fusions to del-1 suggest that this protein is expressed in the V class of motor neurons (Neuron, 18: 107-119) and in SABVR and SABVL (David Miller pers. comm.). No degeneration-causing mutations in del-1 have been reported, and in fact there are no known mutations in this gene. del-1 is expressed in many of the same cells as another degenerin, unc-8 . Null alleles of unc-8 cause subtle defects in locomotion, most notably there is a decrease in both the wavelength and amplitude of the worm tracks. We have postulated that both UNC-8 and DEL-1 are components of a stretch-sensitive ion channel in the motor neurons that modulates the sinusoidal movement of the worm. We screened a mutagenized C. elegans library for deletions within the del-1 coding region, and isolated a 1380 base deletion, del-1(bz33) . This deletion begins at residue 256 just after the first cysteine-rich domain of the DEL-1 protein, and extends into the 12th intron. The mutant allele could at best produce a truncated protein with just one transmembrane domain. Our initial look at this mutant suggests del-1(bz33) does not have the same locomotion defect as unc-8(lf) . Crosses of this allele with both gain-of-function and loss-of-function unc-8 alleles may help to answer questions about the role of these degenerins in nematode locomotion and proprioception.

Miller DM et al. (2000) West Coast Worm Meeting "UNC-4 targets ACR-5 and DEL-1: Are they determinants of synaptic choice?"

Von Stetina SE, Miller DM

[West Coast Worm Meeting, 2000]

Mutations in the UNC-4 homeodomain transcription factor disrupt backward locomotion. EM reconstruction of an unc-4 mutant revealed that the VA motor neurons fail to receive synapses from their usual interneuron partners and instead accept inputs normally reserved for their sisters, the VB motor neurons. Work done in this laboratory has shown that UNC-4 and the Groucho homolog UNC-37 function together in the VA motor neurons to repress VB-specific genes. Two genes, del-1 (DEG/ENaC sodium channel subunit) and acr-5 (alpha-like nictonic acetylcholine receptor subunit), are ectopically expressed in the VA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins and ion channel components, ACR-5 and DEL-1 are attractive candidates for mediators of synaptic specificity. We have now performed genetic experiments, however, that rule out a necessary role for either ACR-5 or DEL-1 in the specification of VB-type inputs. Deletion mutants of del-1 and acr-5, as well as the double mutant (acr-5;del-1), do not perturb forward locomotion as would be expected for a mutation which disrupts normal inputs to the VBs. Furthermore, assays designed to quantitate locomotion have detected no differences between wild type and these mutants. Placement of acr-5 and del-1 mutants in an unc-4 background does not suppress the Unc-4 phenotype, suggesting that ACR-5 and DEL-1 are also not required to promote VB-type inputs in VA motor neurons. A future goal is to ectopically express ACR-5 and DEL-1 in VA motor neurons to determine if these proteins are sufficient to cause miswiring. Also, we will define the UNC-4 mediated repression domains in the acr-5 and del-1 promoters in an effort to identify additional UNC-4 targets.

Petratou, Dionysia et al. (2021) International Worm Meeting "The DEG/ΕNaC ion channel DEL-4 maintains neuronal ionstasis and promotes neuronal survival under stress"

Kaulich, Eva, Petratou, Dionysia, Gjikolaj, Martha, Schafer, William, Tavernarakis, Nektarios

[International Worm Meeting, 2021]

Physiological stress is sensed and processed by neurons, which initiate systemic stress responses. At the cellular level, ion homeostasis is of utmost importance for neuronal function, both for the maintenance of resting potential, and for the creation and propagation of action potentials. Indeed, imbalance in neuronal sodium homeostasis has been linked with many pathologies of the nervous system. However, the effects of stress on neuronal sodium homeostasis, excitability and survival are not understood. We find that DEL-4, an ENaC/DEG family member, which forms proton-inactivated homomeric sodium channels, is differentially regulated under stress to trigger appropriate cellular stress responses and motor adaptation. DEL-4 exhibits neuronal, non-synaptic localization and modulates the characteristics of Caenorhabditis elegans locomotory behavior. Heat stress and starvation alter DEL-4 expression, which in turn modulates the expression and activity of key stress response transcription factors, leading to increased autophagy and triggering of the ER stress response. Notably, similar to heat stress and starvation, DEL-4 deficiency causes hyperpolarization of dopaminergic neurons and impacts neurotransmission in dopaminergic and motor neurons. Utilizing two humanized models of Parkinson's and Alzheimer's disease in C. elegans, we show that DEL-4 promotes neuronal survival in the context of these proteinopathies. Our findings provide insight on the molecular mechanisms via which sodium channels uphold neuronal function and promote adaptation upon stress.

Stephen E Von Stetina et al. (2001) International C. elegans Meeting "In search of UNC-4 target genes, mediators of synaptic specificity."

David M Miller III, Stephen E Von Stetina

[International C. elegans Meeting, 2001]

Mutations in the UNC-4 homeodomain transcription factor disrupt backward locomotion. In unc-4 mutants, VA motor neurons fail to receive synapses from their usual interneuron partners and instead accept inputs normally reserved for their lineal sisters, the VB motor neurons. Work done in this laboratory has shown that UNC-4 and the Groucho homolog UNC-37 function together in VA motor neurons to repress VB-specific genes. GFP reporters constructed from two genes, del-1 (DEG/ENaC sodium channel subunit) and acr-5 ( a -like nicotinic acetylcholine receptor subunit), are ectopically expressed in the VA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins and ion channel components, ACR-5 and DEL-1 are attractive candidates for mediators of neuron-specific synapses. We have now performed genetic experiments, however, that rule out a necessary role for either ACR-5 or DEL-1 in the specification of VB-type inputs. Deletion mutants of del-1 and acr-5 , as well as the double mutant ( acr-5;del-1 ), do not perturb forward locomotion as would be expected for a mutation which disrupts normal inputs to the VBs. Assays designed to quantitate locomotion have detected no differences between wildtype worms and these mutants. In addition, placement of acr-5 and del-1 mutations in an unc-4 background does not suppress the Unc-4 phenotype, suggesting that ACR-5 and DEL-1 are also not required to promote VB-type inputs in VA motor neurons. Although acr-5 and del-1 are not determinants of synaptic specificity, they are regulated by UNC-4 and are therefore likely to contain UNC-4 Response Elements (U4REs) in their promoter regions that are also located in other UNC-4 target genes. A 1.8 kb upstream region of the C. elegans (Ce) del-1 gene contains four subregions that are conserved in the promoter of the C. briggsae (Cb) del-1 gene. These motifs will be targeted for deletion and their effects on del-1 expression will be determined; removal of an U4RE should result in ectopic expression in VA motor neurons in wildtype animals. A comparison of the Ce and Cb acr-5 upstream regions has not identified conserved sequences. However, deletion analysis of the Ce acr-5 promoter has revealed a modular structure that segregates the U4RE region from sites that are likely to respond to other classes of transcription factors. The 4.2 kb acr-5 promoter is expressed in head and tail neurons as well as in DB/VB motor neurons in the ventral nerve cord (VNC). We have found that a 1 kb segment of this region is sufficient to drive expression in only the VNC and is regulated by unc-4 . Further deletion analysis of these regions should reveal specific sequence subdomains that can then be tested for direct UNC-4 binding. We plan to use the C. elegans microarray to detect genes that are de-repressed in unc-4 and unc-37 mutants. These candidate UNC-4 target genes will be scanned for U4RE sequences and then tested for expression and unc-4 regulation in vivo .

Ross JM et al. (1997) International C. elegans Meeting "UNC-4/UNC-37 REPRESSION OF VB-SPECIFIC GENES DEFINES THE VA PATTERN OF SYNAPTIC INPUT"

Tavernarakis NN, Miller DM, Ishihara T, Ross JM, Driscoll MA, Katsura I

[International C. elegans Meeting, 1997]

The homeoprotein UNC-4 specifies synaptic input to the VA motor neurons; in unc-4 mutants, the VAs receive synaptic input normally reserved for their VB sisters. We have proposed that UNC-4 and UNC-37 (Groucho) function together to repress genes which specify VB-type inputs, thereby ensuring VA-type inputs. We have identified two genes, del-1 (degenerin) and acr-4 (a2 acetylcholine receptor subunit) that are negatively regulated by UNC-4/UNC-37 in the VAs. del-1::gfp and acr-4::gfp are normally expressed in the VBs during the period in which motor neuron inputs are established (late L1). However, in unc-4(lf) and unc-37(e262) mutants, del-1::gfp and acr-4::gfp are expressed in both the VAs and VBs. We speculate that either of these cell surface proteins could mediate synaptic specificity. To test whether UNC-4/UNC-37 repression of VB-specific genes is sufficient to specify VA-type synaptic input, we expressed UNC-4 in the VBs (UNC-37 is ubiquitous) using the del-1 promoter. Animals harboring integrated del-1::unc-4 [wdIs2] display a movement defect indicative of VBs receiving synaptic input characteristic of their VA sisters. During forward movement, the body of wdIs2 animals remains inert and is pulled along by the independent motor neuron circuit in the head. When backing, wdIs2 animals coil ventrally, suggesting excitation by both the VAs and respecified VBs on the ventral side.(del-1::unc-4 escapes autoinhibition perhaps due to high levels of UNC-4 expression.) The apparent respecification of VB inputs by ectopic UNC-4 is consistent with a model in which UNC-4/UNC-37 repress VB specific genes to distinguish VA from VB motor neurons during synapse formation.

van der Burght, Servaas N. et al. (2017) International Worm Meeting "Contribution of protein localization to variability in salt chemotaxis behavior."

Jansen, Gert, van der Burght, Servaas N.

[International Worm Meeting, 2017]

Many behavioral responses of the nematode C. elegans show a degree of variation that suggests stochasticity in the underlying molecular pathways. We use chemotaxis to NaCl to investigate this process. C. elegans shows attraction to NaCl concentrations between 0.1-200 mM and avoidance of higher concentrations. This response can be analyzed behaviorally with quadrant assays in which worms are presented with a choice between buffered agar with or without NaCl. When animals are given a choice between 0 and 100 mM NaCl, WT worms move to the salt containing quadrants. However, a small fraction of (genetically identical) animals will end up on the quadrants without salt. We aim to explain the variability in C. elegans behavior by examining stochasticity at a molecular and cellular level. Perception of NaCl probably occurs by receptors and channels in the sensory cilia of the ASE neurons, including the guanylate cyclase subunits gcy-22 and gcy-14 and the epithelial sodium channel (ENaC) subunits del-2, del-4 and del-6. Stochasticity could be caused by variability in the localization of these proteins, their in- and export into and from the cilia, and by variability in intraflagellar transport (IFT). We use CRISPR/Cas9 technology to tag proteins of interest with full length or split GFP (spGFP) to visualize their transport dynamics and localization, and correlate this with the cellular or behavioral response to NaCl. Thus far, we have generated del-6::spGFP, del-2::spGFP and gcy-22::spGFP worm strains. We found that GCY-22::spGFP localizes at the tip of the cilium and at the base of the cilium in the peri ciliary membrane compartment (PCMC) in ASE(R), similar to the published localization pattern of a GCY-22::GFP fusion construct and the previously reported localization of GCY-14::GFP in ASE(L). Thus far, we were unable to show active transport of GCY-22::spGFP between the PCMC and the ciliary tip using live imaging and kymography. FRAP experiments suggest that GCY-22::spGFP fluorescence at the tip or PCMC recovers very slowly. We are currently performing FRAP experiments to determine the motility of the protein within these regions. DEL-6::spGFP localizes to the body wall muscles and hypodermis, but neuronal expression remains unclear. DEL-2::spGFP, on the other hand, shows clear localization at the base of the cilium in several head neurons, including ASE. In future experiments, we will examine the variability in transport and localization of NaCl-receptor/channel proteins in ASE neurons and its cilia and test if this variability correlates with variation in gene expression, intracellular transport and/or IFT. Finally, we will analyze if this variability correlates with stochasticity of the response to NaCl.

Stephen E Von Stetina et al. (2002) West Coast Worm Meeting "In search of UNC-4 response elements (U4REs)"

Nina Xu, Penelope J Brockie, David M Miller III, Stephen E Von Stetina, Andres V Maricq

[West Coast Worm Meeting, 2002]

Mutations in the UNC-4 homeodomain transcription factor disrupt backward locomotion. In unc-4 mutants, VA motor neurons fail to receive synapses from their usual interneuron partners and instead accept inputs normally reserved for their lineal sisters, the VB motor neurons. Work done in this laboratory has shown that UNC-4 and the Groucho homolog UNC-37 function together in VA motor neurons to repress VB-specific genes. De-repression of these VB genes in unc-4 mutant VA motor neurons presumptively imposes VB-type inputs. GFP reporters constructed from two genes, del-1 (DEG/ENaC sodium channel subunit) and acr-5 (nicotinic acetylcholine receptor subunit), are ectopically expressed in the VA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins and ion channel components, ACR-5 and DEL-1 are attractive candidates for mediators of neuron-specific synapses.

JM Ross et al. (1999) International C. elegans Meeting "UNC-4 and UNC-37 repress VB-specifying genes to define VA traits"

JM Ross, E German, DM Miller, DH Hall

[International C. elegans Meeting, 1999]

VA and VB motor neurons arise as sister cells, but adopt different morphologies and patterns of synaptic input. VA-type synaptic inputs define a circuit for backward movement, while VB-type synaptic inputs define a circuit for forward movement. The UNC-4 homeoprotein is required to specify the VA pattern of synaptic input; in unc-4 mutants VAs receive the VB pattern of synaptic input. We have proposed a model in which UNC-4 acts in the VAs to repress genes that normally specify VB-type inputs (Winnier et al., this meeting). Two observations support the hypothesis that UNC-4 acts as a repressor: 1) UNC-4 requires the activity of UNC-37, a homolog of the Drosophila co-repressor Groucho. 2) The UNC-4 C-terminus contains an Engrailed repressor domain (eh1). We have identified two targets of UNC-4/UNC-37 repression. del-1::gfp is normally expressed in VB motor neurons, but is ectopically expressed in VA motor neurons in unc-4 and unc-37 mutants. acr-5::gfp is normally expressed in VB and DB motor neurons, but is ectopically expressed in VA and DA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins, either DEL-1 (degenerin-like Na channel subunit) or ACR-5 (nicotinic acetylcholine receptor alpha subunit), could specify VB synaptic inputs or define other VB traits. If our model of negative regulation by UNC-4 and UNC-37 is correct, then ectopic expression of UNC-4 in the VBs should repress VB-specific gene expression, and may be sufficient to respecify the VBs with VA-type inputs. To test this hypothesis, we expressed UNC-4 in the VBs using the del-1 promoter. Ectopic expression of UNC-4 in the VBs represses acr-5::gfp , while del-1::gfp expression remains unchanged. Worms harboring an integrated del-1::unc-4 array ( wdIs2 ) display a phenotype which is consistent with the VBs receiving VA-type synaptic inputs and functioning in backward movement: wdIs2 worms are impaired in forward movement and coil ventrally when backing. EM reconstruction of the wdIs2 ventral nerve cord will determine whether this wdIs2 Unc phenotype is a result of miswiring of the VBs with VA-type inputs.

Kaulich, Eva et al. (2021) International Worm Meeting "Characterization of C. elegans acid-sensing DEG/ENaCs and their role in rhythmic behavior"

Ackley, Brian D., Schafer, William R., Kaulich, Eva, Tang, Yi-Quan, Walker, Denise S.

[International Worm Meeting, 2021]

While the roles of classical neurotransmitters and neuromodulators in neurotransmission are well-established, less attention has been paid to the effect of unconventional signaling molecules such as protons on neuronal signaling. In C. elegans, protons secreted from the intestine are able to generate motor contractions of the defecation motor program (DMP) independent of the nervous system (Beg et al., 2008). Proton-receptors from the DEG/ENaC family; called acid-sensing ion channels (ASICs) have been described in vertebrates, but their roles in behavior are mostly uncharacterized. We have been investigating C. elegans acid-sensing DEG/ENaCs and their role in modulating behavior via neuronal and non-neuronal tissues. In an initial screen using heterologous expression in Xenopus oocytes and two-electrode voltage recording, we identified two groups of C. elegans pH-sensitive DEG/ENaCs based on their response to neutral and low pH. One group, including ACD-5, DEL-4 and the previously characterized ACD-1, is open at neutral pH and blocked at low pH, while the other group, including ASIC-1, ACD-2 and DEL-9, becomes activated at low pH. The proton-inactivated channel ACD-5 is highly expressed at the apical lumen in the intestine where it senses proton-fluctuation and maintains the intestinal pH. Gain-of-function mutations show a more neutral intestinal pH and prolonged DMP intervals demonstrating the importance of protons in the generation of behavior. In contrast, the proton-activated DEL-9 is expressed in neurons and muscles. In the motoneuron AVL, DEL-9 drives the execution of the DMP enteric muscle contraction, while in the vulva muscles it is responsible for the timing of egg-laying. Mutants show prolonged inactive states during the rhythmic behavior of egg-laying and an egg-retention phenotype that can be rescued. This demonstrates that proton signaling is important for diverse physiological processes and behaviors.