Prevalence of acute kidney injury among dengue cases: a systematic review and meta-analysis.

Numerous studies have shown a correlation between dengue virus (DENV) infection and kidney disease. However, there is no existing meta-analysis on the prevalence of kidney diseases in the dengue population. A thorough systematic review and meta-analysis were undertaken to determine the prevalence of renal problems in people with DENV infection in order to fill this knowledge gap. A rigorous electronic literature search was carried out up to 25 January 2023 in a number of databases, including ProQuest, EBSCOhost, Scopus, PubMed and Web of Science. The search aimed to find articles that reported on the prevalence of kidney diseases in patients with DENV infection. Using the modified Newcastle-Ottawa Scale, the quality of the included studies was assessed. The meta-analysis included a total of 37 studies with 21 764 participants reporting on the prevalence of acute kidney injury (AKI) in individuals with DENV infection. The pooled prevalence of AKI in dengue patients was found to be 8% (95% confidence interval 6 to 11), with high heterogeneity across studies. The studies included are of moderate quality. The study revealed a high AKI prevalence in dengue patients, underlining the need for regular renal examination to detect AKI early and reduce hospitalization risk. Further research is needed to understand the dengue-kidney relationship and develop effective management strategies.

Crosstalk between neurons and glia through G-protein coupled receptors: Insights from Caenorhabditis elegans.

The past decades have witnessed a dogmatic shift from glia as supporting cells in the nervous system to their active roles in neurocentric functions. Neurons and glia communicate and show bidirectional responses through tripartite synapses. Studies across species indicate that neurotransmitters released by neurons are perceived by glial receptors, which allow for gliotransmitter release. These gliotransmitters can result in activation of neurons via neuronal GPCR receptors. However, studies of these molecular interactions are in their infancy. Caenorhabditis elegans has a conserved neuron-glia architectural repertoire with molecular and functional resemblance to mammals. Further, glia in C. elegans can be manipulated through ablation and mutations allowing for deciphering of glial dependent processes in vivo at single glial resolutions. Here, we will review recent findings from vertebrate and invertebrate organisms with a focus on how C. elegans can be used to advance our understanding of neuron-glia interactions through GPCRs.

The cellular architecture and molecular determinants of the zebrafish fusogenic synapse.

Myoblast fusion is an indispensable process in skeletal muscle development and regeneration. Studies in Drosophila led to the discovery of the asymmetric fusogenic synapse, in which one cell invades its fusion partner with actin-propelled membrane protrusions to promote fusion. However, the timing and sites of vertebrate myoblast fusion remain elusive. Here, we show that fusion between zebrafish fast muscle cells is mediated by an F-actin-enriched invasive structure. Two cell adhesion molecules, Jam2a and Jam3b, are associated with the actin structure, with Jam2a being the major organizer. The Arp2/3 actin nucleation-promoting factors, WAVE and WASP-but not the bipartite fusogenic proteins, Myomaker or Myomixer-promote the formation of the invasive structure. Moreover, the convergence of fusogen-containing microdomains and the invasive protrusions is a prerequisite for cell membrane fusion. Thus, our study provides unprecedented insights into the cellular architecture and molecular determinants of the asymmetric fusogenic synapse in an intact vertebrate animal.

Stress Responses Elicited by Misfolded Proteins Targeted to Mitochondria.

The double-membrane-bound architecture of mitochondria, essential for ATP production, sub-divides the organelle into inter-membrane space (IMS) and matrix. IMS and matrix possess contrasting oxido-reductive environments and discrete protein quality control (PQC) machineries resulting inherent differences in their protein folding environments. To understand the nature of stress response elicited by equivalent proteotoxic stress to these sub-mitochondrial compartments, we took misfolding and aggregation-prone stressor proteins and fused it to well described signal sequences to specifically target and impart stress to yeast mitochondrial IMS or matrix. We show, mitochondrial proteotoxicity leads to growth arrest of yeast cells of varying degrees depending on nature of stressor proteins and the intra-mitochondrial location of stress. Next, by employing transcriptomics and proteomics, we report a comprehensive stress response elicited by stressor proteins specifically targeted to mitochondrial matrix or IMS. A general response to proteotoxic stress by mitochondria-targeted misfolded proteins is mitochondrial fragmentation, and an adaptive abrogation of mitochondrial respiration with concomitant upregulation of glycolysis. Beyond shared stress responses, specific signatures due to stress within mitochondrial sub-compartments are also revealed. We report that stress-imparted by bipartite signal sequence-fused stressor proteins to IMS, leads to specific upregulation of IMS-chaperones and TOM complex components. In contrast, matrix-targeted stressors lead to specific upregulation of matrix-chaperones and cytosolic PQC components. Finally, by systematic genetic interaction using deletion strains of differentially upregulated genes, we found prominent modulatory role of TOM complex components during IMS-stress response. In contrast, VMS1 markedly modulates the stress response originated from matrix.

Wheat inositol pyrophosphate kinase TaVIH2-3B modulates cell-wall composition and drought tolerance in Arabidopsis.

BACKGROUND: Inositol pyrophosphates (PP-InsPs) are high-energy derivatives of inositol, involved in different signalling and regulatory responses of eukaryotic cells. Distinct PP-InsPs species are characterized by the presence of phosphate at a variable number of the 6-carbon inositol ring backbone, and two distinct classes of inositol phosphate kinases responsible for their synthesis have been identified in Arabidopsis, namely ITPKinase (inositol 1,3,4 trisphosphate 5/6 kinase) and PP-IP5Kinase (diphosphoinositol pentakisphosphate kinases). Plant PP-IP5Ks are capable of synthesizing InsP8 and were previously shown to control defense against pathogens and phosphate response signals. However, other potential roles of plant PP-IP5Ks, especially towards abiotic stress, remain poorly understood. RESULTS: Here, we characterized the physiological functions of two Triticum aestivum L. (hexaploid wheat) PPIP5K homologs, TaVIH1 and TaVIH2. We demonstrate that wheat VIH proteins can utilize InsP7 as the substrate to produce InsP8, a process that requires the functional VIH-kinase domains. At the transcriptional level, both TaVIH1 and TaVIH2 are expressed in different wheat tissues, including developing grains, but show selective response to abiotic stresses during drought-mimic experiments. Ectopic overexpression of TaVIH2-3B in Arabidopsis confers tolerance to drought stress and rescues the sensitivity of Atvih2 mutants. RNAseq analysis of TaVIH2-3B-expressing transgenic lines of Arabidopsis shows genome-wide reprogramming with remarkable effects on genes involved in cell-wall biosynthesis, which is supported by the observation of enhanced accumulation of polysaccharides (arabinogalactan, cellulose, and arabinoxylan) in the transgenic plants. CONCLUSIONS: Overall, this work identifies a novel function of VIH proteins, implicating them in modulation of the expression of cell-wall homeostasis genes, and tolerance to water-deficit stress. This work suggests that plant VIH enzymes may be linked to drought tolerance and opens up the possibility of future research into using plant VIH-derived products to generate drought-resistant plants.

Infectivity of Human Olfactory Neurons to SARS-CoV-2: A Link to Anosmia.

OBJECTIVES: We sought to determine whether SARS-CoV-2 infections are associated with anosmia and if this virus infects other neuronal cells. We utilized male and female olfactory neuronal cell lines and other olfactory cell lines to determine the viral targets. METHODS: We used four undifferentiated and two partially differentiated human developing neuronal cell lines. Infectivity was confirmed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), immunofluorescence assay (IFA) probing with anti-SARS-CoV-2 antibody, evaluation of cytopathic effects, and neurite formation. We induced partial differentiation of all cell lines (since both olfactory cell lines were terminally differentiated) with retinoic acid (RA) to determine whether differentiation was a factor in viral permissiveness. The expression of serine protease, transmembrane serine protease 2 (TMPRSS2), and angiotensin-converting enzyme II (ACE2) receptors were examined by RT-qPCR and IFA to determine the mechanism of viral entry. RESULTS: Four to five days after exposure, both olfactory cell lines exhibited morphological evidence of infection; IFA analyses indicated that ~30% of the neurons were SARS-CoV-2 positive. At two weeks, 70-80% were positive for SARS-CoV-2 antigens. The partially differentiated (CRL-2266 and CRL-2267) and undifferentiated cell lines (CRL-2142, CRL-2149, CRL-127, and CDL-2271) were essentially non-permissive. After RA treatment, only CRL-127 exhibited slight permissiveness (RT-qPCR). The TMPRSS2 receptor showed high expression in olfactory neurons, but low expression in RA treated CRL-127. ACE2 exhibited high expression in olfactory neurons, whereas other cell lines showed low expression, including RA-treated cell lines. ACE2 expression slightly increased in CRL-127 post RA-treatment. CONCLUSIONS: Our studies confirm neurotropism of SARS-CoV-2 to olfactory neurons with viral entry likely mediated by TMPRSS2/ACE2. Other neuronal cell lines were non-permissive. Our results established that the nerve cells were infected regardless of male or female origin and strengthened the reported association of COVID-19 with loss of smell in infected individuals.

Ethanol-induced Sedative Behavior: An Assay to Investigate Increased Dopamine Signaling in Caenorhabditis elegans.

Dopamine (DA) signaling affects locomotion, feeding, learning, and memory in C. elegans. Various assays have been developed to study the proteins involved in these behaviors; however, these assays show behavioral output only when there is a drastic change in DA levels. We designed an assay capable of observing behavioral output even with only slight alterations in DA levels. To achieve this, we designed a behavioral paradigm where we combined C. elegans movement with ethanol (EtOH) administration. The behavioral response to alcohol/EtOH and susceptibility to alcohol-use disorders (AUDs) have been linked to DA. Our assay correlates an increase in DA levels due to EtOH and movement obstruction due to a dry surface to a circular sedative behavior, which we designated as EtOH-induced sedative (EIS) behavior. We successfully utilized this assay to assign physiological and behavioral functions to a DA autoreceptor, DOP-2.

Differential expression of miRNAs in a human developing neuronal cell line chronically infected with Zika virus.

Zika virus (ZIKV) is a serious public health concern that may lead to neurological disorders in affected individuals. The virus can be transmitted from an infected mother to her fetus, via mosquitoes, or sexually. ZIKV infections are associated with increased risk for Guillain-Barré syndrome (GBS) and congenital microcephaly in newborns infected prenatally. Dysregulations of intracellular microRNAs (miRNAs) in infected neurons have been linked to different neurological diseases. To determine the potential role of miRNAs in ZIKV infection we developed a chronically infected neuroblastoma cell line and carried out differential expression analyses of miRNAs with reference to an uninfected neuroblastoma cell line. A total of 3192miRNAs were evaluated and 389 were found to be upregulated < 2-fold and 1291 were downregulated < 2-fold. In particular, we determined that hsa-mir-431-5p, hsa-mir-3687, hsa-mir-4655-5p, hsa-mir-6071, hsa-mir-762, hsa-mir-5787, and hsa-mir-6825-3p were significantly downregulated, ranging from -5711 to -660-fold whereas, has-mir-4315, hsa-mir-5681b, hsa-mir-6511a-3p, hsa-mir-1264, hsa-mir-4418, hsa-mir-4497, hsa-mir-4485-3p, hsa-mir-4715-3p, hsa-mir-4433-3p, hsa-mir-4708-3p, hsa-mir-1973 and hsa-mir-564 were upregulated, ranging from 20-0.8-fold. We carried out target gene alignment of these miRNAs with the ZIKV genome to predict the function of the differentially expressed miRNAs and their potential impact on ZIKV pathogenesis. These miRNAs might prove useful as novel diagnostic or therapeutic markers and targets for further research on ZIKV infection and neuronal injury resulting from ZIKV infectivity in developing fetal brain neurons.

Dauer Formation in C. elegans Is Modulated through AWC and ASI-Dependent Chemosensation.

The perception of our surrounding environment is an amalgamation of stimuli detected by sensory neurons. In Caenorhabditis elegans, olfaction is an essential behavior that determines various behavioral functions such as locomotion, feeding and development. Sensory olfactory cues also initiate downstream neuroendocrine signaling that controls aging, learning, development and reproduction. Innate sensory preferences toward odors (food, pathogens) and reproductive pheromones are modulated by 11 pairs of amphid chemosensory neurons in the head region of C. elegans Amongst these sensory neurons, the ASI neuron has neuroendocrine functions and secretes neuropeptides, insulin-like peptide (DAF-28) and the TGF-ß protein, DAF-7. Its expression levels are modulated by the presence of food (increased levels) and population density (decreased levels). A recent study has shown that EXP-1, an excitatory GABA receptor regulates DAF-7/TGF-ß levels and participates in DAF-7/TGF-ß-mediated behaviors such as aggregation and bordering. Here, we show that exp-1 mutants show defective responses toward AWC-sensed attractive odors in a non-autonomous manner through ASI neurons. Our dauer experiments reveal that in daf-7 mutants, ASI expressed EXP-1 and STR-2 (a G-protein-coupled receptor; GPCR) that partially maintained reproductive growth of animals. Further, studies suggest that neuronal connections between ASI and AWC neurons are allowed at least partially through ASI secreted DAF-7 or through alternate TGF- ß pathway/s regulated by EXP-1 and STR-2. Together, our behavioral, genetic and imaging experiments propose that EXP-1 and STR-2 integrate food cues and allow the animals to display DAF-7/TGF-ß neuroendocrine dependent or independent behavioral responses contributing to chemosensensory and developmental plasticity.

Increased dopaminergic neurotransmission results in ethanol dependent sedative behaviors in Caenorhabditis elegans.

Ethanol is a widely used drug, excessive consumption of which could lead to medical conditions with diverse symptoms. Ethanol abuse causes dysfunction of memory, attention, speech and locomotion across species. Dopamine signaling plays an essential role in ethanol dependent behaviors in animals ranging from C. elegans to humans. We devised an ethanol dependent assay in which mutants in the dopamine autoreceptor, dop-2, displayed a unique sedative locomotory behavior causing the animals to move in circles while dragging the posterior half of their body. Here, we identify the posterior dopaminergic sensory neuron as being essential to modulate this behavior. We further demonstrate that in dop-2 mutants, ethanol exposure increases dopamine secretion and functions in a DVA interneuron dependent manner. DVA releases the neuropeptide NLP-12 that is known to function through cholinergic motor neurons and affect movement. Thus, DOP-2 modulates dopamine levels at the synapse and regulates alcohol induced movement through NLP-12.

Drug Resistance Reversal Potential of Nanoparticles/Nanocomposites via Antibiotic's Potentiation in Multi Drug Resistant P. aeruginosa.

Bacteria employ numerous resistance mechanisms against structurally distinct drugs by the process of multidrug resistance. A study was planned to discover the antibacterial potential of a graphene oxide nanosheet (GO), a graphene oxide-zinc oxide nanocomposite (GO/ZnO), a graphene oxide-chitosan nanocomposite (GO-CS), a zinc oxide decorated graphene oxide-chitosan nanocomposite (GO-CS/ZnO), and zinc oxide nanoparticles (ZnO) alone and in a blend with antibiotics against a PS-2 isolate of Pseudomonas aeruginosa. These nanocomposites reduced the MIC of tetracycline (TET) from 16 folds to 64 folds against a multidrug-resistant clinical isolate. Efflux pumps were interfered, as evident by an ethidium bromide synergy study with nanocomposites, as well as inhibiting biofilm synthesis. These nanoparticles/nanocomposites also decreased the mutant prevention concentration (MPC) of TET. To the best of our knowledge, this is the first report on nanomaterials as a synergistic agent via inhibition of efflux and biofilm synthesis.

Dynamin regulates the dynamics and mechanical strength of the actin cytoskeleton as a multifilament actin-bundling protein.

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.

Control of Locomotory Behavior of Caenorhabditis elegans by the Immunoglobulin Superfamily Protein RIG-3.

Cell surface immunoglobulin superfamily (IgSF) proteins play important roles in the development and function of the nervous system . Here we define the role of a Caenorhabditis elegans IgSF protein, RIG-3, in the function of the AVA command interneuron. This study reveals that RIG-3 regulates the abundance of the glutamate receptor subunit, GLR-1, in the AVA command interneuron and also regulates reversal behavior in C. elegans The mutant strain lacking rig-3 (rig-3 (ok2156)) shows increased reversal frequency during local search behaviors. Genetic and behavioral experiments suggest that RIG-3 functions through GLR-1 to regulate reversal behavior. We also show that the increased reversal frequency seen in rig-3 mutants is dependent on the increase in GLR-1 abundance at synaptic inputs to AVA, suggesting that RIG-3 alters the synaptic strength of incoming synapses through GLR-1 Consistent with the imaging experiments, altered synaptic strength was also reflected in increased calcium transients in rig-3 mutants when compared to wild-type control animals. Our results further suggest that animals lacking rig-3 show increased AVA activity, allowing the release of FLP-18 neuropeptide from AVA, which is an activity-dependent signaling molecule. Finally, we show that FLP-18 functions through the neuropeptide receptor, NPR-5, to modulate reversal behavior in C. elegans.

Cellular Targets and Receptor of Sexual Transmission of Zika Virus.

STUDY QUESTION: What is the mechanism of sexual transmission of Zika virus (ZIKV)? SUMMARY ANSWER: By utilizing exquisite reverse transcriptase-initiated in situ polymerase chain reaction (RT-in situ PCR), which enables an improved visualization of spermatozoa's subcellular compartment, we precisely localized the mid-piece of sperm that carry receptors for ZIKV. WHAT IS ALREADY KNOWN: ZIKV is transmitted sexually and recent studies have verified ZIKV presence in semen of previously Zika-infected patients for >6-month postinfection when ZIKV had disappeared from blood, saliva, and urine. Strong serial analyses of various body fluids suggest that ZIKV can be transmitted between sexual partners. Currently, there is limited information on the association of the virus with human semen cell types that may carry the virus. STUDY DESIGN, SIZE, DURATION: Analyses were carried out to localize ZIKV for subcellular localization of ZIKV on cell types. The Tyro3 receptor for ZIKV was colocalized by dual immunocytochemistry with specific monoclonal antibodies. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three semen specimens were purchased from a commercial sperm bank. Motile sperm was separated from nonmotile cells by the "swim-up" technique. Each of the semen fractions was infected with ZIKV at the multiplicity of infection of 0.1.0 and 1.0 and evaluated for the primary targets of ZIKV in the semen cells by RT-in situ PCR and confirmed by real-time RT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE: ZIKV was present primarily at the mid-piece of mature spermatozoa in about 30% of the sperm. In addition, we determined that Tyro3 receptors, primarily expressed on mid-piece of human spermatozoa, play a role in ZIKV-binding and entry into spermatozoa. Our data strongly suggest a potential sexual/horizontal route of transmission for ZIKV primarily via infected sperms; most likely ZIKV enters the sperm via the Tyro3 receptor found at the mid-piece of the mature spermatozoa. LIMITATIONS, REASONS FOR CAUTION: We are uncertain as to what phase of spermatogenesis, that in human takes about 120 days, sperms are permissive to ZIKV. If permissiveness was very early during spermatogenesis males could be infectious for ∼120 days after the disappearance of viremia in an infected man. WIDER IMPLICATIONS OF THE FINDINGS: Our findings bring a new focus on the current affords to develop ZIKV vaccine. Why in the presence of anti-ZIKV antibodies infected men are still able to transmit the virus sexually? We suggest that only certain subclass of immunoglobulin (Ig)G (ie, IgG4) can cross the blood-Sertoli barrier therefore, a successful vaccine must provoke a subclass of IgG can quell ZIKV inside the seminiferous tubules.

Regulation of WNT Signaling at the Neuromuscular Junction by the Immunoglobulin Superfamily Protein RIG-3 in Caenorhabditis elegans.

Perturbations in synaptic function could affect the normal behavior of an animal, making it important to understand the regulatory mechanisms of synaptic signaling. Previous work has shown that in Caenorhabditis elegans an immunoglobulin superfamily protein, RIG-3, functions in presynaptic neurons to maintain normal acetylcholine receptor levels at the neuromuscular junction (NMJ). In this study, we elucidate the molecular and functional mechanism of RIG-3. We demonstrate by genetic and BiFC (Bi-molecular Fluorescence Complementation) assays that presynaptic RIG-3 functions by directly interacting with the immunoglobulin domain of the nonconventional Wnt receptor, ROR receptor tyrosine kinase (RTK), CAM-1, which functions in postsynaptic body-wall muscles. This interaction in turn inhibits Wnt/LIN-44 signaling through the ROR/CAM-1 receptor, and allows for maintenance of normal acetylcholine receptor, AChR/ACR-16, levels at the neuromuscular synapse. Further, this work reveals that RIG-3 and ROR/CAM-1 function through the ß-catenin/HMP-2 at the NMJ. Taken together, our results demonstrate that RIG-3 functions as an inhibitory molecule of the Wnt/LIN-44 signaling pathway through the RTK, CAM-1.

The tumor promoter-activated protein kinase Cs are a system for regulating filopodia.

Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, ß, γ) and novel (δ, ɛ, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter-stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC ɛ hydrophobic sequence, but also by a myristoylated PKC α/ß pseudosubstrate sequence, and an inhibitor of T-cell protein tyrosine phosphatase (TC-PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC ɛ predominated over other isoforms. PKC ɛ could decrease filopodia significantly in promoter-treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC ɛ-mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady-state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC ɛ. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC ɛ can create a long-term "memory" of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal.

A synergistic approach towards understanding the functional significance of dopamine receptor interactions.

The importance of the neurotransmitter dopamine (DA) in the nervous system is underscored by its role in a wide variety of physiological and neural functions in both vertebrates and invertebrates. Binding of dopamine to its membrane receptors initiates precise signaling cascades that result in specific cellular responses. Dopamine receptors belong to a super-family of G-protein coupled receptors (GPCRs) that are characterized by seven trans-membrane domains. In mammals, five dopamine receptors have been identified which are grouped into two different categories D1- and D2-like receptors. The interactions of DA receptors with other proteins including specific Gα subunits are critical in deciding the fate of downstream molecular events carried out by effector proteins. In this mini-review we provide a synopsis of known protein-protein interactions of DA receptors and a perspective on the potential synergistic utility of Caenorhabditis elegans as a model eukaryote with a comparatively simpler nervous system to gain insight on the neuronal and behavioral consequences of the receptor interactions.

GPA-14, a Gα(i) subunit mediates dopaminergic behavioral plasticity in C. elegans.

BACKGROUND: Precise levels of specific neurotransmitters are required for appropriate neuronal functioning. The neurotransmitter dopamine is implicated in modulating behaviors, such as cognition, reward and memory. In the nematode Caenorhabditis elegans, the release of dopamine during behavioral plasticity is in part modulated through an acid-sensing ion channel expressed in its eight dopaminergic neurons. A D2-like C. elegans dopamine receptor DOP-2 co-expresses along with a Gα(i) subunit (GPA-14) in the anterior deirid (ADE) pair of dopaminergic neurons. FINDINGS: In follow-up experiments to our recently reported in vitro physical interaction between DOP-2 and GPA-14, we have behaviorally characterized worms carrying deletion mutations in gpa-14 and/or dop-2. We found both mutants to display behavioral abnormalities in habituation as well as associative learning, and exogenous supply of dopamine was able to revert the observed behavioral deficits. The behavioral phenotypes of dop-2 and gpa-14 loss-of-function mutants were found to be remarkably similar, and we did not observe any cumulative defects in their double mutants. CONCLUSION: Our results provide genetic and phenotypic support to our earlier in vitro results where we had shown that the DOP-2 dopamine receptor and the GPA-14 Gα(i) subunit physically interact with each other. Results from behavioral experiments presented here together with our previous in-vitro work suggests that the DOP-2 functions as a dopamine auto-receptor to modulate two types of learning, anterior touch habituation and chemosensory associative conditioning, through a G-protein complex that comprises GPA-14 as its Gα subunit.

The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit.

Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a Gα coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.