Li TM et al. (2015) G3 (Bethesda) "No Significant Increase in the 4- and 7-Dafachronic Acid Concentration in the Long-Lived glp-1 Mutant, nor in the Mutants Defective in Dauer Formation."

Zhang YP, Li X, Li TM, Liu W, Jia LM, Lei X, Lu S, Dong MQ, Chen J

[G3 (Bethesda), 2015]

The steroid hormone dafachronic acid (DA) regulates dauer formation and lifespan in Caenorhabditis elegans by binding to the nuclear receptor DAF-12. However, little is known about how DA concentrations change under various physiologic conditions and about how DA/DAF-12 signaling interacts with other signaling pathways that also regulate dauer formation and lifespan. Using a sensitive bioanalytical method, we quantified the endogenous DA concentrations in a long-lived germline-less glp-1 mutant and in the Dauer formation-defective (Daf-d) mutants daf-12, daf-16, daf-5, and daf-3. We found that the DA concentration in the glp-1 mutant was similar to that in the wild type (WT). This result is contrary to the long-held belief that germline loss-induced longevity involves increased DA production and suggests instead that this type of longevity involves an enhanced response to DA. We also found evidence suggesting that increased DA sensitivity underlies lifespan extension triggered by exogenous DA. At the L2/L3 stage, the DA concentration in a daf-12 null mutant decreased to 22% of the WT level. This finding is consistent with the previously proposed positive feedback regulation between DAF-12 and DA production. Surprisingly, the DA concentrations in the daf-16, daf-5, and daf-3 mutants were only 19-34% of the WT level at the L2/L3 stage, slightly greater than those in the Dauer formation-constitutive (Daf-c) mutants at the pre-dauer stage (4-15% of the WT L2 control). Our experimental evidence suggested that the positive feedback between DA and DAF-12 was partially induced in the three Daf-d mutants.

Mukherjee MM et al. (2017) Dev Biol "Dafachronic acid inhibits C. elegans germ cell proliferation in a DAF-12-dependent manner."

Vagasi AS, Mukherjee MM, Kipreos ET, Balachandran RS, Chaudhari SN

[Dev Biol, 2017]

Dafachronic acid (DA) is a bile acid-like steroid hormone that regulates dauer formation, heterochrony, and lifespan in C. elegans. Here, we describe that DA is an inhibitor of C. elegans germ stem cell proliferation in adult hermaphrodites. Using a C. elegans germ cell primary culture system, we show that DA inhibits the proliferation of germ cells in vitro. Exogenous DA reduces the frequency of large tumors in adult tumorous germline mutants and decreases the proliferation of wild-type germ stem cells in adult hermaphrodites. In contrast, DA has no appreciable effect on the proliferation of larval-stage germ cells in wild type. The inhibition of adult germ cell proliferation by DA requires its canonical receptor DAF-12. Blocking DA production by inactivating the cytochrome P450 DAF-9 increases germ cell proliferation in wild-type adult hermaphrodites and the frequency of large tumors in germline tumorous mutants, suggesting that DA inhibits the rate of germ cell proliferation under normal growth conditions.

Carvelli L et al. (2008) Proc Natl Acad Sci U S A "Dopamine transporter/syntaxin 1A interactions regulate transporter channel activity and dopaminergic synaptic transmission."

Blakely RD, Carvelli L, DeFelice LJ

[Proc Natl Acad Sci U S A, 2008]

The Caenorhabditis elegans (C. elegans) dopamine (DA) transporter (DAT-1) regulates DA signaling through efficient DA reuptake following synaptic release. In addition to its DA transport function, DAT-1 generates detectible DA-gated currents that may influence neuronal excitability. Previously, we provided evidence that single Cl-channel events underlie DAT-1 currents. In these studies, we identified a distinct population of altered DAT-1 currents arising from DAT-1 transgenic constructs bearing an N-terminal GFP fusion. The presence of these channels suggested disruption of an endogenous regulatory mechanism that modulates occupancy of DAT-1 channel states. A leading candidate for such a regulator is the SNARE protein syntaxin 1A (Syn1A), previously found to interact with homologous transporters through N-terminal interactions. Here we establish that UNC-64 (C. elegans Syn1A homologue) associates with DAT-1 and suppresses transporter channel properties. In contrast, GFP::DAT-1 is unable to form stable transporter/UNC-64 complexes that limit channel states. Although DAT-1 and GFP::DAT-1 expressing DA neurons exhibit comparable DA uptake, GFP::DAT-1 animals exhibit swimming-induced paralysis (SWIP), a phenotype associated with excess synaptic DA release and spillover. We propose that loss of UNC-64/DAT-1 interactions leads to enhanced synaptic DA release, providing a novel mechanism for DA neuron sensitization that may be relevant to mechanisms of DA-associated disorders.

Ye P et al. (2023) Prog Neurobiol "TRPM2 as a conserved gatekeeper determines the vulnerability of DA neurons by mediating ROS sensing and calcium dyshomeostasis."

Kang L, Al-Sheikh U, Fang Q, Huang M, Cai X, Pan PY, Ke M, Su H, Ye P, Hua N, Xu S, Jiang P, Liu X, Yu P, Fang EF, Jiang LH, Zhang C, Luo J, Zou W, Li Y, Liu M, Yang W, Hu X

[Prog Neurobiol, 2023]

Different dopaminergic (DA) neuronal subgroups exhibit distinct vulnerability to stress, while the underlying mechanisms are elusive. Here we report that the transient receptor potential melastatin 2 (TRPM2) channel is preferentially expressed in vulnerable DA neuronal subgroups, which correlates positively with aging in Parkinson's Disease (PD) patients. Overexpression of human TRPM2 in the DA neurons of C. elegans resulted in selective death of ADE but not CEP neurons in aged worms. Mechanistically, TRPM2 activation mediates FZO-1/CED-9-dependent mitochondrial hyperfusion and mitochondrial permeability transition (MPT), leading to ADE death. In mice, TRPM2 knockout reduced vulnerable substantia nigra pars compacta (SNc) DA neuronal death induced by stress. Moreover, the TRPM2-mediated vulnerable DA neuronal death pathway is conserved from C. elegans to toxin-treated mice model and PD patient iPSC-derived DA neurons. The vulnerable SNc DA neuronal loss is the major symptom and cause of PD, and therefore the TRPM2-mediated pathway serves as a promising therapeutic target against PD.

Krause MW et al. (1997) Development "A C. elegans E/Daughterless bHLH protein marks neuronal but not striated muscle development."

Fire A, Zhang J-M, Yuan JY, Harfe BD, Park M, Xu SQ, Cole MD, Paterson BM, Greenwald IS, Krause MW

[Development, 1997]

The E proteins of mammals, and the related Daughterless (DA) protein of Drosophila, are ubiquitously expressed helix-loop-helix (HLH) transcription factors that play a role in many developmental processes. We report here the characterization of a related C. elegans protein, CeE/DA, which has a dynamic and restricted distribution during development. CeE/DA is present embryonically in neuronal precursors, some of which are marked by promoter activity of a newly described Achaete-scute-like gene hlh-3. In contrast, we have been unable to detect CeE/DA in CeMyoD-positive striated muscle cells. In vitro gel mobility shift analysis detects dimerization of CeE/DA with HLH-3 while efficient interaction of CeE/DA with CeMyoD is not seen. These studies suggest multiple roles for CeE/DA in C. elegans development and provide evidence that both common and alternative strategies have evolved for the use of related HLH proteins in controlling cell fates in different species.

Li TM et al. (2013) Anal Chem "Absolute quantification of a steroid hormone that regulates development in Caenorhabditis elegans."

Chen S, Lei X, Wu Y, Zhao LF, Dong MQ, Li TM, Chen J, Li X, Ding XJ

[Anal Chem, 2013]

Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 +/- 0.02 ng/mg protein in the L1 larval stage to 1.21 +/- 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.

Bernhard N et al. (2000) Learn Mem "A behavioral and genetic dissection of two forms of olfactory plasticity in Caenorhabditis elegans: adaptation and habituation."

Bernhard N, van der Kooy D

[Learn Mem, 2000]

Continuous presentation of an olfactory stimulus causes a decrement of the chemotaxis response in the nematode Caenorhabditis elegans. However, the differences between the learning process of habituation (a readily reversible decrease in behavioral response) and other types of olfactory plasticity such as adaptation (a decrement in response due to sensory fatigue, which cannot be dishabituated) have not been addressed. The volatile odorant diacetyl (DA) was used within a single paradigm to assess the distinct processes of olfactory adaptation and habituation. Preexposing and testing worms to 100% DA vapors caused a chemotaxis decrement that was not reversible despite the presentation of potentially dishabituating stimuli. This DA adaptation was abolished in worms with an odr-10 mutation (encoding a high-affinity DA receptor on the AWA neuron), even though naive chemotaxis remained unaffected. Conversely, DA adaptation remained intact in odr-1 mutants (defective in AWC neuron-mediated olfactory behavior), even though naive chemotaxis to DA decreased. Surprisingly, exposure to vapors of intermediate concentrations of DA (0.01% and 25%) did not cause worms to exhibit any response decrement. In contrast to preexposure to high DA concentrations, preexposure to low DA concentrations (0.001%) produced habituation of the chemotaxis response (a dishabituating stimulus could reverse the response decrement back to baseline levels). The distinct behavioral effects produced by DA preexposure highlight a concentration-dependent dissociation between two decremental olfactory processes: adaptation at high DA concentrations versus habituation at low DA concentrations.

Raj V et al. (2022) J Neurosci Res "Dopamine plays a critical role in the olfactory adaptive learning pathway in Caenorhabditis elegans."

Raj V, Thekkuveettil A

[J Neurosci Res, 2022]

Encoding and consolidating information through learning and memory is vital in adaptation and survival. Dopamine (DA) is a critical neurotransmitter that modulates behavior. However, the role of DA in learning and memory processes is not well defined. Herein, we used the olfactory adaptive learning paradigm in Caenorhabditis elegans to elucidate the role of DA in the memory pathway. Cat-2 mutant worms with low DA synthesis showed a significant reduction in chemotaxis index (CI) compared to the wild type (WT) after short-term conditioning. In dat-1::ICE worms, having degeneration of DA neurons, there was a significant reduction in adaptive learning and memory. When the worms were trained in the presence of exogenous DA (10 mM) instead of food, a substantial increase in CI value was observed. Furthermore, our results suggest that both dop-1 and dop-3 DA receptors are involved in memory retention. The release of DA during conditioning is essential to initiate the learning pathway. We also noted an enhanced cholinergic receptor activity in the absence of dopaminergic neurons. The strains expressing GCaMP6 in DA neurons (pdat-1::GCaMP-6::mCherry) showed a rise in intracellular calcium influx in the presence of the conditional stimulus after training, suggesting DA neurons are activated during memory recall. These results reveal the critical role of DA in adaptive learning and memory, indicating that DA neurons play a crucial role in the effective processing of cognitive function.

Bermingham DP et al. (2016) Neurochem Int "Acute Blockade of the C. elegans Dopamine Transporter DAT-1 by the Mammalian Norepinephrine Transporter Inhibitor Nisoxetine Reveals the Influence of Genetic Modifications of Dopamine Signaling In Vivo."

Salimando GJ, Bermingham DP, Snarrenberg CL, Blakely RD, Folkes OM, Jinnah H, Hardaway JA, Robinson SB

[Neurochem Int, 2016]

Modulation of neurotransmission by the catecholamine dopamine (DA) is conserved across phylogeny. In the nematode C. elegans, excess DA signaling triggers Swimming-Induced Paralysis (Swip), a phenotype first described in animals with loss of function mutations in the presynaptic DA transporter (dat-1). Swip has proven to be a phenotype suitable for the identification of novel dat-1 mutations as well as the identification of novel genes that impact DA signaling. Pharmacological manipulations can also induce Swip, though the reagents employed to date lack specificity and potency, limiting their use in evaluation of dat-1 expression and function. Our lab previously established the mammalian norepinephrine transporter (NET) inhibitor nisoxetine to be a potent antagonist of DA uptake conferred by DAT-1 following heterologous expression. Here we demonstrate the ability of low (M) concentrations of nisoxetine to trigger Swip within minutes of incubation, with paralysis dependent on DA release and signaling, and non-additive with Swip triggered by dat-1 deletion. Using nisoxetine in combination with genetic mutations that impact DA release, we further demonstrate the utility of the drug for demonstrating contributions of presynaptic DA receptors and ion channels to Swip. Together, these findings reveal nisoxetine as a powerful reagent for monitoring multiple dimensions of DA signaling in vivo, thus providing a new resource that can be used to evaluate contributions of DAT-1 and other genes linked to DA signaling without the potential for compensations that attend constitutive genetic mutations.

Hossain M et al. (2014) Neurochem Int "-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons."

Hossain M, Wickramasekara RN, Carvelli L

[Neurochem Int, 2014]

-Phenylethylamine (PEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by PEA, however the mechanism through which PEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the PEA-induced increase of extracellular DA required DAT-1. Our data show that PEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the PEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by PEA. These results suggest that PEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that PEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of PEA to increase extracellular DA. Finally, our electrophysiology data show that PEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, PEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA.