KyeongJin Kang

An internal thermal sensor controlling temperature preference in Drosophila

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly’s preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly’s preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.

PDF Cells Are a GABA-Responsive Wake-Promoting Component of the Drosophila Sleep Circuit

Daily sleep cycles in humans are driven by a complex circuit within which GABAergic sleep-promoting neurons oppose arousal. Drosophila sleep has recently been shown to be controlled by GABA, which acts on unknown cells expressing the Rdl GABAA receptor. We identify here the relevant Rdl-containing cells as PDF-expressing small and large ventral lateral neurons (LNvs) of the circadian clock. LNv activity regulates total sleep as well as the rate of sleep onset; both large and small LNvs are part of the sleep circuit. Flies mutant for pdf or its receptor are hypersomnolent, and PDF acts on the LNvs themselves to control sleep. These features of the Drosophila sleep circuit, GABAergic control of onset and maintenance as well as peptidergic control of arousal, support the idea that features of sleep-circuit architecture as well as the mechanisms governing the behavioral transitions between sleep and wake are conserved between mammals and insects.

Analysis of Drosophila TRPA1 reveals an ancient origin for human chemical nociception

Chemical nociception, the detection of tissue-damaging chemicals, is important for animal survival and causes human pain and inflammation, but its evolutionary origins are largely unknown. Reactive electrophiles are a class of noxious compounds humans find pungent and irritating, such as allyl isothiocyanate (in wasabi) and acrolein (in cigarette smoke). Diverse animals, from insects to humans, find reactive electrophiles aversive, but whether this reflects conservation of an ancient sensory modality has been unclear. Here we identify the molecular basis of reactive electrophile detection in flies. We demonstrate that Drosophila TRPA1 (Transient receptor potential A1), the Drosophila melanogaster orthologue of the human irritant sensor, acts in gustatory chemosensors to inhibit reactive electrophile ingestion. We show that fly and mosquito TRPA1 orthologues are molecular sensors of electrophiles, using a mechanism conserved with vertebrate TRPA1s. Phylogenetic analyses indicate that invertebrate and vertebrate TRPA1s share a common ancestor that possessed critical characteristics required for electrophile detection. These findings support emergence of TRPA1-based electrophile detection in a common bilaterian ancestor, with widespread conservation throughout vertebrate and invertebrate evolution. Such conservation contrasts with the evolutionary divergence of canonical olfactory and gustatory receptors and may relate to electrophile toxicity. We propose that human pain perception relies on an ancient chemical sensor conserved across ∼500 million years of animal evolution.

Modulation of TRPA1 thermal sensitivity enables sensory discrimination in Drosophila

Discriminating among sensory stimuli is critical for animal survival. This discrimination is particularly essential when evaluating whether a stimulus is noxious or innocuous. From insects to humans, transient receptor potential (TRP) channels are key transducers of thermal, chemical and other sensory cues. Many TRPs are multimodal receptors that respond to diverse stimuli, but how animals distinguish sensory inputs activating the same TRP is largely unknown. Here we determine how stimuli activating Drosophila TRPA1 are discriminated. Although Drosophila TRPA1 responds to both noxious chemicals and innocuous warming, we find that TRPA1-expressing chemosensory neurons respond to chemicals but not warmth, a specificity conferred by a chemosensory-specific TRPA1 isoform with reduced thermosensitivity compared to the previously described isoform. At the molecular level, this reduction results from a unique region that robustly reduces the channel’s thermosensitivity. Cell-type segregation of TRPA1 activity is critical: when the thermosensory isoform is expressed in chemosensors, flies respond to innocuous warming with regurgitation, a nocifensive response. TRPA1 isoform diversity is conserved in malaria mosquitoes, indicating that similar mechanisms may allow discrimination of host-derived warmth—an attractant—from chemical repellents. These findings indicate that reducing thermosensitivity can be critical for TRP channel functional diversification, facilitating their use in contexts in which thermal sensitivity can be maladaptive.

Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster.

The ability to sense and respond to subtle variations in environmental temperature is critical for animal survival. Animals avoid temperatures that are too cold or too warm and seek out temperatures favorable for their survival. At the molecular level, members of the transient receptor potential (TRP) family of cation channels contribute to thermosensory behaviors in animals from flies to humans. In Drosophila melanogaster larvae, avoidance of excessively warm temperatures is known to require the TRP protein dTRPA1. Whether larval avoidance of excessively cool temperatures also requires TRP channel function, and whether warm and cool avoidance use the same or distinct TRP channels has been unknown. Here we identify two TRP channels required for cool avoidance, TRPL and TRP. Although TRPL and TRP have previously characterized roles in phototransduction, their function in cool avoidance appears to be distinct, as neither photoreceptor neurons nor the phototransduction regulators NORPA and INAF are required for cool avoidance. TRPL and TRP are required for cool avoidance; however they are dispensable for warm avoidance. Furthermore, cold-activated neurons in the larvae are required for cool but not warm avoidance. Conversely, dTRPA1 is essential for warm avoidance, but not cool avoidance. Taken together, these data demonstrate that warm and cool avoidance in the Drosophila larva involves distinct TRP channels and circuits.

Substitution of a Single Residue, Asp575, Renders the NCKX2 K+-dependent Na+/Ca2+ Exchanger Independent of K+

The Na+/Ca2+-K+ exchanger (NCKX) is a polytopic membrane protein that uses both the inward Na+ gradient and the outward K+ gradient to drive Ca2+ extrusion across the plasma membrane. NCKX1 is found in retinal rod photoreceptors, while NCKX2 is found in retinal cone photoreceptors and is also widely expressed in the brain. Here, we have identified a single residue (out of >100 tested) for which substitution removed the K+ dependence of NCKX-mediated Ca2+ transport. Charge-removing replacement of Asp575 by either asparagine or cysteine rendered the mutant NCKX2 proteins independent of K+, whereas the charge-conservative substitution of Asp575 to glutamate resulted in a nonfunctional mutant NCKX2 protein, accentuating the critical nature of this residue. Asp575 is conserved in the NCKX1–5 genes, while an asparagine is found in this position in the three NCX genes, coding for the K+-independent Na+/Ca2+ exchanger.

Residues Contributing to the Ca2+ and K+ Binding Pocket of the NCKX2 Na+/Ca2+-K+ Exchanger

The Na+/Ca2+-K+ exchanger (NCKX) extrudes Ca2+ from cells utilizing both the inward Na+ gradient and the outward K+ gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca2+ and K+ and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K+ and Ca2+ dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K+ and Ca2+ dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu188 and Asp548, whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro187 and Pro547), three additional acidic residues (Asp258, Glu265, Glu533), and two hydroxyl-containing residues (Ser185 and Ser545) showed smaller shifts, but shifts in Ca2+ dependence were invariably accompanied by shifts in K+ dependence. We conclude that Glu188 and Asp548 are the central residues of a single cation binding pocket that can accommodate both K+ and Ca2+. Furthermore, a single set of residues lines a transport pathway for both K+ and Ca2+.

Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation

Hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H2O2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H2O2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H2O2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H2O2 was reversed by treatment with dithiothreitol. H2O2-elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca2+-activated K+ channels and inward rectifier K+ channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H2O2 increased Kv currents in a concentration-dependent manner. H2O2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H2O2-mediated channel activation was prevented by glutathione reductase. Consistent with S-glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H2O2. Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H2O2 to stimulate Kv channels. Not only was the H2O2 stimulatory effect much weaker, but the inhibitory effect of H2O2 was unmasked. These data suggest that H2O2 activates 4-AP-sensitive Kv channels, possibly through S-glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H2O2.

Natural killer (NK) cells inhibit systemic metastasis of glioblastoma cells and have therapeutic effects against glioblastomas in the brain

BackgroundGlioblastoma multiforme (GBM) is characterized by extensive local invasion, which is in contrast with extremely rare systemic metastasis of GBM. Molecular mechanisms inhibiting systemic metastasis of GBM would be a novel therapeutic candidate for GBM in the brain.MethodsPatient-derived GBM cells were primarily cultured from surgical samples of GBM patients and were inoculated into the brains of immune deficient BALB/c-nude or NOD-SCID IL2Rgammanull (NSG) mice. Human NK cells were isolated from peripheral blood mononucleated cells and expanded in vitro.ResultsPatient-derived GBM cells in the brains of NSG mice unexpectedly induced spontaneous lung metastasis although no metastasis was detected in BALB/c-nude mice. Based on the difference of the innate immunity between two mouse strains, NK cell activities of orthotopic GBM xenograft models based on BALB/c-nude mice were inhibited. NK cell inactivation induced spontaneous lung metastasis of GBM cells, which indicated that NK cells inhibit the systemic metastasis. In vitro cytotoxic activities of human NK cells against GBM cells indicated that cytotoxic activity of NK cells against GBM cells prevents systemic metastasis of GBM and that NK cells could be effective cell therapeutics against GBM. Accordingly, NK cells transplanted into orthotopic GBM xenograft models intravenously or intratumorally induced apoptosis of GBM cells in the brain and showed significant therapeutic effects.ConclusionsOur results suggest that innate NK immunity is responsible for rare systemic metastasis of GBM and that sufficient supplementation of NK cells could be a promising immunotherapeutic strategy for GBM in the brain.

Isoform-specific expression of the neuropeptide orcokinin in Drosophila melanogaster

Orcokinins are neuropeptides that have been identified in diverse arthropods. In some species, an orcokinin gene encodes two isoforms of mature orcokinin peptide through alternative mRNA splicing. The existence of two orcokinin isoforms was predicted in Drosophila melanogaster as well, but the expression pattern of both isoforms has not been characterized. Here, we use in situ hybridization, antibody staining, and enhancer fusion GAL4 transgenic flies to examine the expression patterns of the A and B forms of orcokinin, and provide evidence that they are expressed differentially in the central nervous system (CNS) and the intestinal enteroendocrine system. The orcokinin A isoform is mainly expressed in the CNS of both larvae and adults. The A form is expressed in 5 pairs of neurons in abdominal neuromeres 1-5 of the larval CNS. In the adult brain, the A form is expressed in one pair of neurons in the posteriorlateral protocerebrum, and an additional four pairs of neurons located near the basement of the accessory medulla. Orcokinin A expression is also observed in two pairs of neurons in the ventral nerve cord (VNC). The orcokinin B form is mainly expressed in intestinal enteroendocrine cells in the larva and adult, with additional expression in one unpaired neuron in the adult abdominal ganglion. Together, our results provide elucidation of the existence and differential expression of the two orcokinin isoforms in the Drosophila brain and gut, setting the stage for future functional studies of orcokinins utilizing the genetically amenable fly model.