Bag S et al. (2010) Acta Trop "In vitro biological evaluation of biguanides and dihydrotriazines against Brugia malayi and folate reversal studies."

Goswami K, Degani MS, Bag S, Sharma R, Reddy MV, Tawari NR

[Acta Trop, 2010]

Dihydrofolate reductase (DHFR) is a well-known target for antibacterial and anticancer therapy. DHFR inhibitors are useful for protozoan parasites, but are yet to be explored against metazoan species; hence the present work was designed to evaluate the efficacy of DHFR inhibitors against filariasis, one of the major neglected tropical diseases. Molecules from our in-house library of synthetic antifolate agents (biguanide and dihydrotriazine derivatives) were evaluated along with the antimalarial drug pyrimethamine and the antibacterial drug trimethoprim in an in vitro model against Brugia malayi microfilariae (Mf). Three biguanides and two dihydrotriazines were more potent than trimethoprim and pyrimethamine against B. malayi Mf. Trimethoprim, pyrimethamine and four of the five compounds active against Mf were also active against adult worms. To probe the mechanism of action of the compounds, reversal of activity of active compounds by folic acid and folinic acid was studied. In conclusion, DHFR inhibitors could be used as leads for new antifilarial drugs.

Takayama S et al. (1999) J Biol Chem "An evolutionarily conserved family of Hsp70/Hsc70 molecular chaperone regulators."

Takayama S, Reed JC, Xie ZH

[J Biol Chem, 1999]

Heat Shock Protein 70 kDa (Hsp70) family molecular chaperones play critical roles in protein folding and trafficking in all eukaryotic cells. The mechanisms by which Hsp70 family chaperones are regulated, however, are only partly understood. BAG-1 binds the ATPase domains of Hsp70 and Hsc70, modulating their chaperone activity and functioning as a competitive antagonist of the co-chaperone Hip. We describe the identification of a family of BAG-1-related proteins from humans (BAG-2, BAG-3, BAG-4, BAG-5), the invertebrate Caenorhabditis elegans (BAG-1, BAG-2), and the fission yeast Schizosaccharomyces pombe (BAG-1A, BAG-1B). These proteins all contain a conserved approximately 45-amino acid region near their C termini (the BAG domain) that binds Hsc70/Hsp70, but they differ widely in their N-terminal domains. The human BAG-1, BAG-2, and BAG-3 proteins bind with high affinity (KD congruent with 1-10 nM) to the ATPase domain of Hsc70 and inhibit its chaperone activity in a Hip-repressible manner. The findings suggest opportunities for specification and diversification of Hsp70/Hsc70 chaperone functions through interactions with various BAG-family proteins.

Juozaityte, Vaida et al. (2011) International Worm Meeting "Searching for factors required for BAG cell fate determination."

Juozaityte, Vaida, Pocock, Roger

[International Worm Meeting, 2011]

The BAG neurons enable C. elegans to generate rapid behavioral responses to oxygen and carbon dioxide. However, the molecular factors that determine BAG cell fate are not known. The aim of this project is to identify the molecular factors required for the differentiation of BAG neurons. First, we aim to identify elements in the promoters of genes expressed in the BAG neurons that are required for BAG expression. We are systematically dissecting the promoters of genes expressed in the BAG neurons - flp-11, flp-13, flp-17, flp-19, gcy-31 and gcy-33. Such deletion analysis will enable the identification of minimal elements within these promoters that are required for BAG expression. Second, we will search for putative transcription factor binding sites within these minimal elements that are required for BAG expression. We will subsequently use BAG::GFP reporter strains to examine BAG cell fate in relevant transcription factor mutant strains. The results of the project will enable us to better understand how the specification of BAG neurons is regulated in C. elegans and potentially identify crucial factors required for oxygen and carbon dioxide sensing in higher organisms.

Ringstad, Niels et al. (2009) International Worm Meeting "The BAG Sensory Neurons are Activated by Environmental Carbon Dioxide."

Hallem, Elissa, Sternberg, Paul, Ringstad, Niels, Horvitz, Bob

[International Worm Meeting, 2009]

The BAG neurons are ciliated sensory neurons that recently have been shown to mediate avoidance behavior to carbon dioxide (CO2) and to inhibit egg-laying behavior. To test whether the BAG neurons are CO2 sensors, we generated transgenic animals expressing the high-affinity genetically encoded calcium sensor cameleon YC3.60 in the BAG neurons. We observed increases in BAG neuron calcium in response to application of as little as 0.1% CO2 with half maximal increases evoked by application of 0.9% CO2. The BAG neurons require the TAX-2 / TAX-4 cyclic nucleotide-gated cation channel to respond to environmental CO2. The BAG neurons of rgs-3 mutants, which lack a negative regulator of heterotrimeric G proteins, are defective for CO2-evoked calcium responses, indicating that the BAG neurons are negatively regulated by a G protein signaling pathway. Using in vivo calcium imaging, we are seeking neurons that function downstream of the BAG sensory neurons in neuronal circuits that mediate CO2 avoidance and the regulation of egg laying.

Choi, Jung-Hwan et al. (2013) International Worm Meeting "Redundant mechanisms for modulation of the serotonergic HSNs by an environmental cue."

Choi, Jung-Hwan, Ringstad, Niels

[International Worm Meeting, 2013]

Egg-laying in worms is regulated by hermaphrodite-specific neurons (HSNs), which stimulate the vulval muscles through serotonin. Previous studies have shown that cholinergic and peptidergic input to HSNs inhibits egg-laying. However, it has been shown that removal of only one of these inhibitory inputs to HSNs cannot induce egg-laying, but removal of both inputs induces precocious egg-laying. BAG neurons have been shown to the primary source of HSN inhibition through the neuropeptide, FLP-17, and its receptor, EGL-6, a G protein-coupled receptor expressed in HSNs. BAG-ablated wildtype animals show normal egg-laying behavior, whereas BAG-ablated unc-17 mutants, which lack the vesicular acetylcholine transporter, have a precocious egg-laying phenotype. This suggests there are redundant inhibitory pathways, one that requires BAG neurons and FLP-17 and another that requires cholinergic transmission. To better understand how the BAG-dependent and BAG-independent pathways contribute to HSN inhibition in vivo, we propose to perform calcium imaging of HSNs in response to BAG neuron stimulation in wildtype and mutant animals which lack cholinergic or peptidergic input.

Guillermin ML et al. (2011) Genetics "Differentiation of carbon dioxide-sensing neurons in Caenorhabditis elegans requires the ETS-5 transcription factor."

Castelletto ML, Guillermin ML, Hallem EA

[Genetics, 2011]

Many animals sense environmental gases such as carbon dioxide and oxygen using specialized populations of gas-sensing neurons. The proper development and function of these neurons is critical for survival, as the inability to respond to changes in ambient carbon dioxide and oxygen levels can result in reduced neural activity and ultimately death. Despite the importance of gas-sensing neurons for survival, little is known about the developmental programs that underlie their formation. Here we identify the ETS-family transcription factor ETS-5 as critical for the normal differentiation of the carbon dioxide-sensing BAG neurons in Caenorhabditis elegans. Whereas wild-type animals show acute behavioral avoidance of carbon dioxide, ets-5 mutant animals do not respond to carbon dioxide. The ets-5 gene is expressed in BAG neurons and is required for the normal expression of the BAG neuron gene battery. ets-5 may also autoregulate its expression in BAG neurons. ets-5 is not required for BAG neuron formation, indicating that it is specifically involved in BAG neuron differentiation and the maintenance of BAG neuron cell fate. Our results demonstrate a novel role for ETS genes in the development and function of gas-detecting sensory neurons.

Cornils, Astrid et al. (2011) International Worm Meeting "Mechanisms of BAG sensory cilia specification."

Cornils, Astrid, Sengupta, Piali

[International Worm Meeting, 2011]

The nervous system relies on specialized sensory neurons to sense environmental conditions and initiate appropriate physiological and behavioral responses. Functions of many sensory neurons are dependent on the presence of morphologically specialized cilia which are microtubule-based organelles acting as cellular 'antennae'. A mechanism called intraflagellar transport (IFT) is required to build cilia and transport cargo and ciliary precursors into the ciliary compartment. However, little is known about how diverse, specialized sensory cilia are shaped. Caenorhabditis elegans is an established model for the study of cilia generation and maintenance. Worms possess 60 ciliated neurons, each of which is specialized to sense specific cues. The BAGL/R sensory neurons, so-called due to the bag-like appearance of their cilia, sense carbon dioxide and decreases in oxygen levels. Little is known about the mechanisms by which BAG cilia are formed, although interestingly, the essential IFT component osm-6/IFT52 has been reported not to be expressed in the BAG neurons. To investigate the formation of BAG cilia, I am first analyzing BAG cilia structure in known ciliary mutant backgrounds to determine the extent to which BAG ciliogenesis depends on known ciliary genes. I am analyzing in vivo IFT as well as the ultrastructure of BAG cilia in wild-type animals, and plan to carry out a forward genetic screen to identify new genes required for BAG ciliogenesis. Comparing the pathways for BAG cilia specialization to those of other specialized C. elegans neuronal cilia will allow me to define new mechanisms for the generation of ciliary structure diversity.

Symersky J et al. (2004) Acta Crystallogr D Biol Crystallogr "Structural genomics of Caenorhabditis elegans: structure of the BAG domain."

Schormann N, Luo M, Luan CH, Symersky J, Bunzel R, Zhang Y, Li S, Pruett P

[Acta Crystallogr D Biol Crystallogr, 2004]

Binding of the BAG domain to the eukaryotic chaperone heat-shock protein (Hsp70) promotes ATP-dependent release of the protein substrate from Hsp70. Although the murine and human BAG domains have been shown to form an antiparallel three-helix bundle, the Caenorhabditis elegans BAG domain is formed by two antiparallel helices, while the third helix is extended away and stabilized by crystal-packing interactions. A small beta-sheet between helices 2 and 3 interferes with formation of the intramolecular three-helix bundle. However, intermolecular three-helix bundles are observed throughout the crystal packing and suggest that stable functional dimers and tetramers can be formed in solution. The structure may represent a new folding type of the BAG domain.

Garrett, Destane et al. (2017) International Worm Meeting "The transcription factor fkh-8 is involved in sustaining CO2 function in C. elegans."

Garrett, Destane, Cawthon, Bryan, Nelms, Brian

[International Worm Meeting, 2017]

Sensing and responding to changing levels of oxygen and carbon dioxide is an important function across the animal kingdom. In the model organism Caenorhabditis elegans, the inability to sense fluctuations in CO2 levels can potentially lead to death. The BAG neuron is one cell type that can function as a mediator of high CO2 avoidance and can sense decreases in O2 levels. Some transcription factors, such as ETS-5 and EGL-13, are already known to be required for BAG neuron CO2 and O2 sensing. We have observed through genetics and behavioral studies that the transcription factor FKH-8 likely also plays a role in CO2 sensing. We are more specifically studying the role of FKH-8 in regulating BAG neuron gene expression by examining expression decreases or increases in specific BAG neuron reporter genes in fkh-8 mutants. Through these experiments, we hope to identify which important signaling components in BAG neuron CO2-sensing are controlled by FKH-8.

Beverly, Matthew H. et al. (2009) International Worm Meeting "The BAG neurons respond to temperature, and are necessary for cryophilic behavior."

Sengupta, Piali, Beverly, Matthew H.

[International Worm Meeting, 2009]

C. elegans exhibits complex behavioral responses to thermal stimuli. When placed on a spatial thermal gradient at temperatures above its cultivation temperature (Tc), worms will move down the gradient towards colder temperatures (cryophilic behavior), whereas at temperatures below the Tc, worms are atactic. At temperatures around the Tc, worms track isotherms. The underlying circuit required for these behaviors includes the AFD sensory neurons, as well as the AIY and AIZ interneurons. A current model of circuit function in thermosensory navigation behavior suggests that there is an underlying default cryophilic drive regulated by the AIZ interneurons and as yet unknown sensory neurons. Exhibition of the cryophilic drive is restricted to temperatures above the Tc via activity of the AFD and AIY neurons, which are also essential for isothermal tracking behavior. The goal of my project is to identify additional components of the thermosensory circuit, and in particular, to identify the sensory neuron(s) mediating cryophilic behavior. We found that animals with genetically ablated BAG neurons exhibit strong defects in cryophilic behavior. Although the BAG neurons are not directly presynaptic to the AIZ interneurons required for cryophilic behavior, this neuron type is presynaptic to the RIR and RIG interneurons which in turn synapse directly onto AIZ. The BAG neurons have previously been implicated in oxygen and carbon dioxide sensation; however, our experiments suggest that temperature responses are independent of gas sensation in this neuron type. BAG expresses known genes involved in sensory pathways including the tax-2/4 cyclic nucleotide gated ion channels as well as the soluble guanylyl cyclases gcy-31 and -33, gcy-31 and -33 mutants also show defects in cryophilic behavior on a thermal gradient. Preliminary calcium imaging data indicate that BAG responds to both small increases and decreases in temperature (0.2 degC) above and below the animal''s cultivation temperature. I am further characterizing the responses of BAG to temperature by examining changes in intracellular calcium and cGMP levels using genetically encoded sensors. I will also determine whether communication between the AFD and BAG neurons play a role in mediating temperature responses, and identify additional BAG-expressed genes required for thermosensory signal transduction.