Guo, Xiaoyan et al. (2013) International Worm Meeting "SIR-2.1, an HDAC, is required to maintain male mating ability during aging of C. elegans."

Guo, Xiaoyan, Garcia, Luis Rene

[International Worm Meeting, 2013]

C. elegans males display a significant deterioration of mating behavior during 'early aging' prior to the structural dysfunction of neuromuscular circuitry. The mating deterioration is correlated with an increase of the excitability in the male mating circuitry(1). Here, we showed that the mating behavior of males with sir-2.1 null mutation declines even prematurely compared to that of wild-type males, and the mating circuitry of sir-2.1(ok434) is more excitable than that of wild type males. Through Ca2+ imaging in mating males, we demonstrated that the hyper excitation of sex muscles during mating blocks the process of sperm transferring in 2-day-old sir-2.1(ok434) males, hence leading to the failure of mating. Furthermore, we illustrated that sir-2.1(ok434) males generate more reactive oxygen species (ROS) possibly due to enhanced catabolism in the mating circuits and/or reduced ability to scavenge ROS because of reduced expression of ROS-scavenger genes such as superoxide dismutase sod-1and glutathione transferase gsto-1. Meanwhile, we found that artificially increasing ROS stress by feeding males with paraquat elevates the excitability of the mating circuitry and reduces the mating potency. In addition, reducing ROS by feeding males with N-Acetyl Cysteine (NAC), an antioxidant reagent, lowers the excitability of the mating circuitry and improves mating. Taken together, we conclude that SIR-2.1, a histone deacetylase (HDAC) regulates the physiological state of the mating circuitry possibly through regulation of the oxidative stress. Reference: 1.Guo X, Navetta A, Gualberto DG, Garcia LR. Behavioral decay in aging male C. elegans correlates with increased cell excitability. Neurobiol Aging. 2012.

LeBoeuf, Brigitte et al. (2021) International Worm Meeting "UPRmt required for anal depressor symmetry and male muscle remodeling"

Garcia, Luis Rene, LeBoeuf, Brigitte

[International Worm Meeting, 2021]

The mitochondrial unfolded protein response (UPRmt) is normally studied in the context of stress. When mitochondrial function is perturbed, the transcription factor ATFS-1 is trafficked to the nucleus, instead of the mitochondria, to promote gene expression involved in metabolic remodeling and repair of mitochondrial damage. However, the UPRmt is also necessary for normal development under normal conditions. One cell that uses UPRmt is the anal depressor (adp), which is required for defecation; when the adp contracts, waste is expelled from the anus. The H-shaped muscle has left and right sarcomeres that span from the dorsal hypodermis to the rectal epithelium. We used atfs-1 tissue-specific CRISPR to drive Cas9 expression in the enteric muscles, including the adp, and discovered that the muscle no longer makes the symmetrical H shape, but instead has hyper-expanded asymmetric sarcomeres. This suggests that the adp uses UPRmt to establish symmetry during early development. Furthermore, in L4 males, the adp remodels to change its form and function from a defecation muscle, with a dorsal/ventral sarcomere, to an anterior/posterior-oriented sex muscle. The remodeling process also requires atfs-1, since reduction in its function disrupts the disassembly of its dorsal/ventral sarcomere. Additionally, ATFS-1 is expressed in developing male adps, but is below detection in the hermaphrodite muscle, suggesting that ATFS-1 has sexually dimorphic functions during post-embryonic developmental. We asked if artificially inducing UPRmt via ethidium bromide (EtBr), while over-expressing ATFS-1 in the nucleus, could lead to adp defects. While EtBr treatment delays development in both sexes and impairs male tail development, it does not affect adp remodeling, indicating that while too little ATFS-1 prevents muscle remodeling, excess nuclear ATFS-1 is tolerated. Future work will focus on what impact ATFS-1 has on metabolic function in the adp and what role the previously identified WNT/Ca2+ pathway has on regulating UPRmt-controlled metabolism.

Correa, Paola et al. (2015) International Worm Meeting "Taurine is a cholinergic antagonist of the C. elegans male mating circuit."

Correa, Paola, Garcia, Luis Rene

[International Worm Meeting, 2015]

In the mammalian CNS, the non-essential amino acid taurine increases cognitive performance either as a neuroprotective agent or as a neuromodulator of cellular excitability. However, precise understanding how taurine regulates behaviors is scarce. Here, we use C. elegans copulation as a model to address how taurine signaling directs a goal-oriented behavior. A stereotyped behavioral output of C. elegans male mating is the intromission of the copulatory spicules into the hermaphrodite vulva. The intromission motor sub-routine is orchestrated by a neuro-muscular cholinergic circuit and is artificially induced by cholinergic agonists. The process from sex muscle contraction to spicule protraction is induced by nicotinic (nACh), muscarinic (mACh) and the non-specific acetylcholine agonist, arecoline (ARE). Since previous studies suggest that taurine hyperpolarizes neurons via GABA(A) receptor binding, we tested the inhibitory effect of taurine by exposing males to exogenous taurine and ARE. We assessed the percentage of males that protruded their spicules when exposed simultaneously or sequentially to these drugs. Our data suggests that taurine negatively and sequentially regulates ARE-induced protraction. Old males normally develop an endogenously enhanced cholinergic state, which ultimately decreases their reproductive capacity, to address why taurine would antagonize a hyper-excitable mating circuit, we exposed old males to taurine and tested their ability to sire progeny. We found that taurine treatment can improve potency of aged males, presumably via attenuating the heightened circuitry excitability. To identify cells that secrete taurine for post-synaptic target inhibition, we assessed the expression pattern of a putative taurine transporter (PTT). We find broad expression of PTT in several muscles and neurons. A YFP translational PTT fusion suggests juxtaposed localization with the vesicular marker synaptobrevin in cholinergic neurons. Interestingly, we find that PTT co-localizes with a mitochondrial marker in head muscles indicating a potential taurine interaction with the mitochondria. Taken together our data suggest that C. elegans neuro-muscular circuitry utilizes taurine to improve the performance of goal-oriented behaviors, as a down-modulator of neuronal activity and/or as a potential antioxidant agent.

Correa, Paola et al. (2015) International Worm Meeting "The UNC-7 gap junction mediates a D2-like signaling pathway in a sensory motor recurrent circuit during C. elegans copulation."

Correa, Paola, Garcia, Luis Rene

[International Worm Meeting, 2015]

Neuro-modulation of self-amplifying circuits directs context-dependent behavioral executions. Although, recurrent networks are found throughout the C. elegans connectome, few reports describe the mechanisms that regulate reciprocal neural activity during a complex behavior. We used C. elegans male copulation to dissect how a goal-oriented motor behavior is regulated by recurrently wired sensory-motor neurons. As the male tail presses against the hermaphrodite's vulva, cholinergic and glutamatergic reciprocal innervations of post-cloaca sensilla (PCS) neurons (PCA, PCB and PCC), hook neurons (HOA, HOB) and their post-synaptic sex muscles execute rhythmic copulatory spicule thrusts. These repetitive spicule movements continue until the male shifts off the vulva or genital penetration is accomplished. However, the signaling mechanism that temporally and spatially restricts repetitive intromission attempts to vulva chemical and/or mechanical cues was unclear. Here, we report that confinement of spicule insertion attempts to the vulva is facilitated by D2-like receptor modulation of gap-junctions between PCB and the hook sensillum. We isolated a missense mutation in an UNC-7L gap-junction isoform, which perturbs DOP-2 signaling in the PCB neuron and its electrical partner, HOA. Further pharmacogenetic analysis suggests that the AVR-14 glutamate-gated channel partially introduces chloride ions into HOA to mediate DA downmodulation of the spicule circuit. Consistently, our analysis of the unc-7(rg396) mutant allele indicates that when DOP-2 promotes UNC-7 electrical communication, AVR-14-mediated inhibitory signals pass from HOA to PCB. As a consequence, the cholinergic PCB neuron is less receptive to be stimulated by its recurrent glutamatergic synaptic partner, PCA. Behavioral observations suggest that DA neuromodulation of UNC-7 ensures attenuation of recursive intromission attempts when the male disengages or is dislodged from the hermaphrodite genitalia. .

Guo, Xiaoyan et al. (2015) International Worm Meeting "Regulation of gluconeogenesis and its role in attenuating excitability of the male mating circuitry."

Garcia, Luis Rene, Guo, Xiaoyan

[International Worm Meeting, 2015]

Our previous study showed that a key enzyme involved in gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) plays a compensatory role to delay the copulation behavior deterioration during male aging. Behavioral decay is correlated with increased excitability in multiple cellular components of the male's insemination circuitry. In aged male, the expression of pck-2 (ortholog of pepck in C. elegans) genes is up regulated, resulting in the accumulation of fat and glycogen. However, the mechanisms used to regulate the expression of pck-2 are yet to be determined. To study the regulation of pck-2, we used the CRISPR/CAS-9 technique to first generate a worm strain containing the pck-2 gene fused with yfp. Then we conducted an EMS mutagenesis to screen for mutants displaying aberrant expression of pck-2. Currently, we are characterizing two mutants; one mutation that causes increased pck-2 expression, and another mutation that causes PCK-2 proteins to accumulate into functional puncta.Mutation of pck-2 increases the prc (spontaneous spicules protraction) phenotype induced by unc-103(n1213), suggesting that gluconeogenesis is important to reduce cell excitability, at least in the mating circuitry. To dissect which tissue gluconeogenesis is important to maintain the male mating circuitry excitability, we are generating a worm strain with two loxP sites in the pck-2 genes. Through expression of cre in a tissue specific manner, we can knock out pck-2 genes in different tissues, and ask how the tissue specific pck-2 mutation affects the excitability of the mating circuitry, via either pharmacological drug test or genetic interaction with unc-103.Taken together, through these studies we are hoping to manipulate the activity of pepck gene in a tissue specific manner to either extend the mating ability during aging or alleviate muscle spontaneous contraction induced by mutations in the potassium channels or other genes. .

Chen, Xin et al. (2015) International Worm Meeting "Wnt canonical and calcium pathways regulate cytoskeletal changes in the C. elegans male anal depressor ."

Chen, Xin, Garcia, Luis Rene

[International Worm Meeting, 2015]

We studied the C. elegans anal depressor development in males and hermaphrodites to address how a differentiated muscle cell sex-specifically reprograms developmental paths to achieve sarcomere rearrangement and functional alterations. In both larval males and hermaphrodites, the anal depressor muscle possesses a dorsal-ventrally oriented sarcomere. The sarcomere is dorsally attached to the hypodermis and is ventrally attached to the rectum. In larval males and hermaphrodites, its contraction facilitates defecation behavior; however in adult males, the anal depressor reorganizes its sarcomere on top of the dorsal protractors and becomes a copulation muscle. To identify the cytoskeletal alteration events, as well as the sex-determination mechanism that contribute to the sarcomere rearrangement, we used YFP:actin to monitor, and mutant analysis, laser-ablation and transgenic feminization to perturb the cell's morphological dynamics. In young larva, the muscle of both sexes has similar sarcomere morphology. However later in L4, the male extrinsic sex mechanism promotes slit formation at the anal depressor's ventral region, demarcating the sarcomere into anterior and posterior half. The male intrinsic sex determination mechanism then functions to promote the disassembly of both the anterior and posterior sarcomere. The anterior sarcomere first disassembles, detaching the myofilaments from the dorsal and ventral attachments. The anterior edge of the muscle then extends filopodia anteriorly towards the developing protractors. Subsequently, the posterior sarcomere then disassembles, but retains a vestigial attachment to ventral body wall. Finally, the anterior domain establishes a novel ventral attachment to the sex muscles, and reassembles an anterior-posteriorly oriented sarcomere. To identify the signaling pathways that are sex-differentially activated to promote sarcomere disassembly, we conducted EMS mutagenesis and RNAi screens. We discovered a series of Wnt canonical and non-canonical mutants that possess sarcomere disassembly defects in the male anal depressor. egl-20 and lin-44 (Wnt ligands), and the lin-17 (Frizzled) mutants possess posterior sarcomere disassembly defects. Meanwhile, we found that the Wnt calcium pathway components, EGL-8/PLCbeta, GOA-1/Galpha, ITR-1/IP3R, as well as the canonical pathway component, BAR-1/beta-catenin, are involved in the posterior disassembly process. We are further identifying the functional relationship between these two pathways to investigate how the Wnt canonical and non-canonical pathways interact to regulate cytoskeletal rearrangement.

Correa, Paola et al. (2013) International Worm Meeting "Dopamine down-modulates the activity of a re-current circuit via D2-like signaling during male mating."

Correa, Paola, Garcia, Luis Rene

[International Worm Meeting, 2013]

Neuro-modulation of self-amplifying circuits is required to drive the execution of behaviors to their appropriate context. Even though recurrent neuronal networks are found throughout the C. elegans connectome, the mechanism that fine-tunes the activity of these reciprocal synapses is unknown. In our work we dissect the cellular and molecular components involved in male copulation, a goal oriented behavior that encompasses multiple sub-steps that entail initiation and termination under appropriate circumstances. The C. elegans male mating circuit integrates sensory-motor cues that result in successful insertion of a pair of copulatory spicules into the hermaphrodite vulva. During mating, the post-cloaca-sensila (p.c.s.) and their post-synaptic sex muscles maintain the male's position over the vulva. Simultaneously, the cholinergic p.c.s. neurons and the reciprocally synapsed glutamatergic PCA neuron trigger rhythmic spicule thrusts. However, distinct signaling mechanisms that restrict repetitive spicule movements to vulva cues are unclear. We found that dopamine (DA) signaling directs spicule insertion attempts to the hermaphrodite vulva by dampening stimulus-independent activity of the spicule circuit. Our pharmacogenetic analyses indicate that DA antagonizes stimulatory ACh signaling via the D2-like receptors, DOP2 and DOP3 and Gao/i proteins, GOA-1and GPA-7. Live calcium imaging results suggest that DA ray neurons are coincidentally active with the cholinergic and glutamatergic cloacal ganglia when the male thrusts his spicules against the vulva. Via optogenetics and targeted illumination we find that activity in a pair of DA neurons can be triggered by stimulation of the p.c.s., and that in a hypodopaminergic state spurious spicule thrusts last even after artificial PCA stimulation is removed. Furthermore, D2-like receptor signaling attenuates the excitability of additional mating circuits to reduce the duration of unproductive mating attempts. Therefore, during a looped behavioral routine, such as spicule insertion attempts, DA down-modulates the activity threshold of a recurrent circuit, confining the behavior to a proper context.

Dengg M. et al. (2006) European Worm Meeting "Using Caenorhabditis elegans to Study Cellular Responses to Endogenous DNA Damage"

Dengg M., Nilsen H., Boulton S., Garcia-Muse T.

[European Worm Meeting, 2006]

Dengg, M. 1, Garcia-Muse, T.2, Boulton, S.2, and Nilsen, H1 DNA repair pathways and intracellular signalling cascades contribute to DNA damage response (DDR) mechanisms that determine cellular responses to DNA damage. C.elegans has been used successfully to study DDR in response to DNA damage induced by physical or chemical agents. Whether DDR is activated in response to the most abundant forms of DNA damage, arising spontaneously through the reaction of water and reactive oxygen species with DNA, is poorly understood. Compared with treatment with physical or chemical DNA damaging agents, the study of DDR in response to endogenous lesions must overcome technical problems with respect to inducing sufficiently high levels of DNA lesions to allow studies of downstream responses. We have exploited RNA interference (RNAi) technology in C.elegans to modulate dUTP pools in order to achieve incorporation of uracil above background levels. Here, we present data describing the phenotypical consequences of increased incorporation of uracil into DNA. The role of DNA repair and DNA damage checkpoints in activating DNA damage response pathways in response to uracil will be presented.

Teixeira, J. et al. (2019) International Worm Meeting "Identification of the ubiquinone pathway as a suppressor of Myotonic Dystrophy-associated toxicity."

Brandao, A., Harju, A., Teixeira, J., Garcia, S.

[International Worm Meeting, 2019]

Myotonic dystrophy type 1 (DM1) is the most common type of dystrophy in adulthood. It is caused by the accumulation of mutant RNA in the nucleus due to the expression of expanded CUG repeats in the 3' UTR of the myotonic dystrophy protein kinase (DMPK) gene. These RNA-bearing CUG repeats form hairpin structures that interact inappropriately to RNA binding proteins such as splicing factors, namely muscleblind-like (Mbnl) and CUG-binding protein (CUGBP) families, thereby causing aberrant alternative splicing leading to a multisystemic disease. Notably, due to the multisystemic nature of DM1, the full extent of cellular processes affected by these toxic RNAs is still unknown. Our aim is to identify new modulators and mechanisms of RNA toxicity. To address this aim, we performed an RNAi screen by using a previously characterized C. elegans DM1 model[1]. Since this C. elegans model mimics DM1 phenotypes, changes in its motility defect were used as readout for toxicity. We identified the ubiquinone (CoQ) pathway as a suppressor of DM1, as downregulation of this pathway increases DM1 toxicity, whereas CoQ supplementation partially rescues the DM1 motility defect. Furthermore, our data also suggest that complex II of mitochondrial electron transport chain is implicated in DM1 dysfunction. The role of mitochondria in DM1 pathogenesis is further underlined by our preliminary results showing that DM1 animals have an altered mitochondrial morphology. Taken together, we established a genetic connection between DM1, ubiquinone pathway and mitochondrial function and are currently examining the mechanisms of DM1 mitochondrial dysfunction regulation. 1. Garcia, S.M., et al., Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans. Nat Struct Mol Biol, 2014. 21(8): p. 712-20.

Jolanta Polanowska et al. (2006) European Worm Meeting "Regulation of BRC-1 - Dependent Ubiquitylation at DNA Damage Sites"

Jolanta Polanowska, Simon Boulton, Tatiana Garcia-Muse, Julie Martin

[European Worm Meeting, 2006]

Jolanta Polanowska1,2, Julie Martin1, Tatiana Garcia-Muse1 and Simon Boulton1. The BRCA1 tumour suppressor and its heterodimeric partner BARD1 constitute an E3-ubiquitin (Ub) ligase and function in DNA repair by unknown mechanisms. We have previously described C. elegans BRCA1 and BARD1 orthologues (brc-1 and brd-1, respectively) that possess many of the functional domains present in their human counterparts, including RING, ankyrin, and BRCT domains (Boulton et al., 2004). Consistent with conserved roles in DNA repair, BRC-1 and BRD-1 interact to form a heterodimer via their respective RING domains. To explore the mechanistic role of BRC-1 and BRD-1 in DNA repair processes we have characterized a C. elegans BRC-1/BRD-1 complex (CeBCD) purified by tandem immunoaffinity before and at different time points after IR-treatment. This approach is a first for C. elegans and demonstrates that protein complexes purified in this manner are amenable to biochemical analysis and can be used in combination with genetics and cell biology to accelerate functional discoveries. We present evidence that the CeBCD complex possesses an E3-Ub ligase that is activated on chromatin in response to IR-treatment and further demonstrate that the DNA damage checkpoint promotes association of the CeBCD complex with E2-Ub conjugating enzyme, Ubc5(LET-70), to form an active E3-Ub ligase in response to DNA damage. We also show that ubiquitylation events at DNA damage sites require brc-1, brd-1, ubc5(let-70), mre-11 and atl-1, thus providing in vivo evidence to support our biochemical analysis.. Boulton, S.J., Martin, J.S., Polanowska, J., Hill, D.E., Gartner, A. and Vidal, M. (2004) Curr Biol, 14, 33-39.