Rodriguez, P. et al. (2019) International Worm Meeting "Glial-expressed SWIP-10 Regulates Mitochondrial Metabolism and Energetic Homeostasis Underlying Deficits in Dopamine Signaling and Neuronal Survival in Caenorhabditis elegans."

Blakely, R., Refai, O., Miller, G., Khalia, V., Rodriguez, P.

[International Worm Meeting, 2019]

Dopamine (DA) is a conserved neurotransmitter, well-known for its role in mammals in modulating neural activity vital to movement, attention and reward-driven motivation. Proper DA signaling relies heavily on dynamic regulation of extracellular DA imparted by the presynaptic DA transporter (DAT). Our lab has taken advantage of the simplicity of the C. elegansnervous system, containing only 8 DA neurons, and a robust phenotype discovered in loss-of-function dat-1mutants termed Swimming-induced paralysis (Swip) to pursue unbiased genetic screening to identify novel genetic contributors of DA signaling. In this screen, we identified the gene swip-10, which encodes an enzyme reliant on a conservedmetallo b-lactamase domain, whose mutation leads to DA- and glutamate (Glu)-dependent Swip as well as age-dependent DA neuron degeneration. Moreover, genetic rescue experiments indicate that the critical site of swip-10in support of normal swimming behavior and DA neuron viability is in glial cells that lie adjacent to and ensheath DA neurons. We find that swip-10 mutant animals exhibit a number of signs of mitochondrial and oxidative stress, globally, suggesting that SWIP-10 activity supports a broader contribution to C. elegansphysiology beyond that of DA neurons. Our recent studies suggest that SWIP-10activity is linked to fatty acid synthesis and where deficits lead to altered mitochondrial respiration and changes in cellular metabolites indicative of loss of mitochondrial function. Ongoing studies seek to link SWIP-10 function to the enzyme's endogenous substrate(s) and elucidate how deficits in enzyme activity lead to perturbations of DA homeostasis and neuronal vulnerability, possibly supported by a dual hit from excess Glu signaling and metabolic insufficiency.

Hardaway J A et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "A Forward Genetic Screen to Reveal Presynaptic Regulators of Dopamine Signaling"

Whitaker S M, Blakely R D, Hardaway J A, Hardie S L

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

The catecholamine neurotransmitter dopamine (DA) modulates multiple behavioral processes in man including learning,arousal, reward, cognition and motor control, faculties disrupted in neuropsychiatric and neurodegenerative disorders such as bipolar disorder, schizophrenia, attention deficit hyperactivity disorder (ADHD) and Parkinson's Disease. Overall DA signaling is regulated by modulation of the neurotransmitter's biosynthesis, packaging, release, metabolism and receptor activation, but the temporal and spatial duration of DA action is controlled by the presynaptic dopamine transporter (DAT). DATs clear synaptic DA via Na-dependent uptake, returning the catecholamine to the presynaptic terminal where it can be repackaged and re-released. In C. elegans, DAT-1 supports DA reuptake, is expressed solely within DA neurons, preferentially transports DA, and is blocked by psychoactive compounds and substrates such as cocaine and amphetamine as well as the tricyclic antidepressant imipramine. Our previous studies show that DAT-1 is an important modulator of endogenous DA levels because dat-1 worms exhibit a DA-dependent locomotory phenotype termed Swimming Induced Paralysis (SWIP). Briefly, whereas wild-type worms will swim continuously for 15-20 min when placed in a small volume of water, dat-1 worms will paralyze within 2-3 min. SWIP is mediated by an increase in extrasynaptic DA, as dat-1 animals are rescued by reserpine-induced depletion of DA from synaptic vesicles, by loss of DA biosynthesis (cat-2) or with loss of the postsynaptic receptor DOP-3. Although reverse genetic approaches have been successfully implemented to study DATs in worm and rodent models, unbiased forward genetic approaches are necessary to expand our knowledge of presynaptic factors regulating DA signaling. In the current study, we capitalized on the SWIP phenotype to pursue a forward genetic screen that identifies hyperdopaminergic strains. To date, we have screened ~ 10,000 haploid genomes and we have isolated 12 stable lines that maintain the SWIP phenotype after outcrossing. In addition, we have thoroughly characterized two of these lines, swip2 and swip3, demonstrating that they harbor loss-of-function mutations in DAT-1 coding sequences, providing vital proof-of-concept evidence that our screen targets genes involved in the presynaptic regulation of DA signaling. Supported by NIH award DA027739 to R.D.B. and S.L.H.

Whitaker S M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Behavioral, Pharmacological, and Molecular Characterization of swip Mutants"

Blakely R D, Whitaker S M, Hardaway J A, Hardie S

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

Through an EMS-based forward genetic screen, we isolated twelve stable mutant lines that demonstrate the dopamine (DA)–dependent, reserpine-sensitive phenotype termed Swimming Induced Paralysis (SWIP). Since SWIP is mediated by an increase in extrasynaptic DA as observed, for example, with the loss of presynaptic DA transporters (DAT-1), we hypothesize that these swip strains may harbor mutations that result in a loss of dopamine transport or enhanced DA release. In order to determine the number of complementation groups present in this screen, we have crossed these mutants to N2, dat-1, dop-3 and amongst themselves. To date, these experiments demonstrate the presence of at least five complementation groups. Two lines harbor loss of function coding mutations in DAT-1, with their SWIP phenotype rescued by reserpine treatment to eliminate DA release or by loss of DOP-3 to inhibit DA signaling. To demonstrate the effect of these mutations in vivo, we have characterized several locomotory behaviors of these lines including the use of automated thrashing analysis, movement on solid substrate with exogenous DA, and the induction of SWIP by the DAT-1 targeted agents amphetamine and imipramine. These experiments demonstrate that several of the swip mutants demonstrate locomotory behaviors that mimic those of dat-1, whereas some lines display behavioral profiles distinct from dat-1. For swip mutants lacking a fully penetrant SWIP phenotype and which do not bear DAT-1 coding mutations, the induction of SWIP by amphetamine and imipramine is consistent with the presence of residual DAT-1 function. To identify the molecular lesions of fully penetrant lines, we sequenced the genomic DAT-1 locus and identified two strains, swip2 and swip3, that harbor dat-1 mutations. For those swip lines lacking DAT-1 coding mutations, we are using SNP mapping to map the sites of mutation, an approach that has been used to map the mutation associated with swip10 to a 4 megabase interval on LGI, consistent with a locus distinct from DAT-1. For swip10 and other mutant lines, we are employing whole-genome sequencing to identify candidate mutation sites. Supported by NIH award DA027739 to R.D.B. and S.L.H.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Refai, O.M. et al. (2017) International Worm Meeting "A Screen for Contributors to Dopamine Signaling Perturbations in Swimming-Induced Paralysis (Swip)."

Freeman, P., Hardie, S. L., Hardaway, J. A., Snarrenberg, C.L., Refai, O.M., Blakely, R. B.

[International Worm Meeting, 2017]

The monoamine neurotransmitter dopamine (DA) regulates a wide variety of complex behaviors across phylogeny. The presynaptic DA transporter (DAT) is critical for restricting DA actions in space and time by limiting DA availability at synaptic and extra-synaptic DA receptors. Mutations impacting DAT protein expression or structure have been identified in subjects with multiple brain disorders in humans, including attention-deficit hyperactivity disorder (ADHD), juvenile Parkinsonism/dystonia, bipolar disorder and autism. In the nematode C. elegans, loss of function mutations in dat-1 gene result in a hyperdopaminergic state that causes animals to paralyze in water, a phenotype termed Swimming-induced paralysis (Swip). This phenotype can be rescued by either treatment of animals with the vesicular monoamine transporter (CAT-1) inhibitor reserpine or through mutation of the TH ortholog CAT-2, or the D2-type DA receptor DOP-3. We have implemented a forward genetic screen to identify novel dat-1 alleles as well as novel determinants of DA signaling. Here we describe our efforts to characterize two mutant lines that express DA-dependent Swip, vt39 and vt44, using genetic and pharmacological approaches. Evaluation of sensitivity to osmotic tone and the DAT-1 antagonist nisoxetine, as well as genetic reversal experiments, are consistent with the genes mutated in these lines as DA signaling dependent. SNP mapping and genome sequencing analyses have revealed multiple candidates as the site of the molecular lesion responsible for the Swip phenotype. Further studies related to the identity of the genes encoding these mutations and their functional analyses will be presented. Supported by NIH Award MH095044 (RDB, PF) and MH093102 (JAH).

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.

Birsoy B et al. (2010) Biology of the C. elegans Male, Madison, WI "Novel Left-Right Asymmetries of Male C.elegans"

Rothman J H, Choi S, Birsoy B, Downes J C, Bloss T A

[Biology of the C. elegans Male, Madison, WI, 2010]

While the mechanism that breaks left-right (L-R) anatomical asymmetry has been described in C. elegans, we have found that at least one additional mechanism must exist to generate L-R differences in the deployment of alternative cell death pathways in the male tail, male mating behavior and patterning of the male gonad. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite's body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites' body (away from the agar surface) and >55% of turns when made under the hermaphrodites' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays, we examined the turning bias in egl-1(n1084n3082) mutants. Wild-type males lack rays more frequently on the right and egl-1 mutant males have no ray loss but still display the right-hand turning bias. To assess whether this L-R behavioral bias is dependent on anatomical asymmetry, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that an asymmetry-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. During the characterization of the gonad reversals of males, we also observed a previously unreported temperature sensitive reversal of L-R asymmetry of the male gonads in N2 (Bristol) and a Hawaiian wild isolate. We will describe our recent findings on these novel L-R asymmetries of the C.elegans male anatomy and behavior.