Hong-Sheng Li

Activation of a TRPC3-dependent cation current through the neurotrophin BDNF.

Nonvoltage-gated cation currents, which are activated following stimulation of phospholipase C (PLC), appear to be major modes for Ca2+ and Na+ entry in mammalian cells. The TRPC channels may mediate some of these conductances since their expression in vitro leads to PLC-dependent cation influx. We found that the TRPC3 protein was highly enriched in neurons of the central nervous system (CNS). The temporal and spatial distribution of TRPC3 paralleled that of the neurotrophin receptor TrkB. Activation of TrkB by brain-derived nerve growth factor (BDNF) led to production of a PLC-dependent, nonselective cation conductance in pontine neurons. Evidence is provided that TRPC3 contributes to this current in vivo. Thus, activation of TrkB and PLC leads to a TRPC3-dependent cation influx in CNS neurons.

Coassembly of TRP and TRPL Produces a Distinct Store-Operated Conductance

The Drosophila retinal-specific protein, TRP (transient receptor potential), is the founding member of a family of store-operated channels (SOCs) conserved from C. elegans to humans. In vitro studies indicate that TRP is a SOC, but that the related retinal protein, TRPL, is constitutively active. In the current work, we report that coexpression of TRP and TRPL leads to a store-operated, outwardly rectifying current distinct from that owing to either TRP or TRPL alone. TRP and TRPL interact directly, indicating that the TRP-TRPL-dependent current is mediated by heteromultimeric association between the two subunits. We propose that the light-activated current in photoreceptor cells is produced by a combination of TRP homo- and TRP-TRPL heteromultimers.

Termination of phototransduction requires binding of the NINAC myosin III and the PDZ protein INAD.

Many of the proteins that are critical for Drosophila phototransduction assemble into a signaling complex, signalplex, through association with the PDZ-domain protein INAD. Some of these proteins depend on INAD for proper subcellular localization to the phototransducing organelle, the rhabdomere, making it difficult to assess any physiological function of this signaling complex independent of localization. Here we demonstrated that INAD bound directly to the NINAC myosin III, yet the subcellular localization of NINAC was normal in inaD mutants. Nevertheless, the INAD binding site was sufficient to target a heterologous protein to the rhabdomeres. Disruption of the NINAC/INAD interaction delayed termination of the photoreceptor response. Thus one role of this signaling complex is in rapid deactivation of the photoresponse.

RETINAL TARGETS FOR CALMODULIN INCLUDE PROTEINS IMPLICATED IN SYNAPTIC TRANSMISSION

Ca2+ influxes regulate multiple events in photoreceptor cells including phototransduction and synaptic transmission. An important Ca2+ sensor inDrosophila vision appears to be calmodulin since a reduction in levels of retinal calmodulin causes defects in adaptation and termination of the photoresponse. These functions of calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the TRP and TRPL ion channels, NINAC and INAD. To identify additional calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, we conducted a screen for retinal calmodulin-binding proteins. We found eight additional calmodulin-binding proteins that were expressed in the Drosophila retina. These included six targets that were related to proteins implicated in synaptic transmission. Among these six were a homolog of the diacylglycerol-binding protein, UNC13, and a protein, CRAG, related to Rab3 GTPase exchange proteins. Two other calmodulin-binding proteins included Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission.

A lysosomal tetraspanin associated with retinal degeneration identified via a genome-wide screen

The Drosophila visual system has provided a model to study phototransduction and retinal degeneration. To identify new candidate proteins that contribute to these processes, we conducted a genome‐wide screen for genes expressed predominately in the eye, using DNA microarrays. This screen appeared to be comprehensive as it led to the identification of all 22 eye‐enriched genes previously shown to function in phototransduction or implicated in retinal degeneration. In addition, we identified 93 eye‐enriched genes whose roles have not been previously defined. One of the eye‐enriched genes encoded a member of a large family of transmembrane proteins, referred to as tetraspanins. We created a null mutation in the eye‐enriched tetraspanin, Sunglasses (Sun), which resulted in light‐induced retinal degeneration. We found that the Sun protein was distributed primarily in lysosomes, and functioned in a long‐known but poorly understood phenomenon of light‐induced degradation of rhodopsin. We propose that lysosomal tetraspanins in mammalian cells may also function in the downregulation of rhodopsin and other G‐protein‐coupled receptors, in response to intense or prolonged agonist stimulation.

Small sensory neurons in the rat dorsal root ganglia express functional NK-1 tachykinin receptor

The tachykinins substance P (SP) and neurokinin A, released by the C‐type primary afferent fibre terminals of the small dorsal root ganglion (DRG) neurons, play important roles in spinal nociception. By means of non‐radioactive in situ hybridization and whole‐cell recording, we showed that the small rat DRG neurons also express the NK‐1 tachykinin receptor. In situ hybridization demonstrated that the positive neurons in rat DRG sections were mainly small cells (85.9%) with diameters less than 25 μm. The remaining positive neurons (14.1%) were cells with medium diameters between 26 and 40 μm. No positive large neurons (diameters > 40 μm) were observed. Expression in small DRG neurons (diameter < 21 μm) was confirmed by in situ hybridization of isolated cells, which were demonstrated to express NK‐1 receptor mRNA at a very high frequency (> 90% of small DRG neurons) and therefore were subjected to whole‐cell recording. In 57 of 61 cells recorded, SP or the selective NK‐1 receptor agonist [Sar9, Met(O2)11]SP (Sar‐SP, 1 or 2 μm) produced a delayed vibrating inward current (50–300 nA) with a long duration of 0.5–2 h. These currents were blocked by co‐application of the NK‐1 receptor antagonist L‐668, 169 (1 μm), but were not affected by the NK‐2 antagonist

The Fly CAMTA Transcription Factor Potentiates Deactivation of Rhodopsin, a G Protein-Coupled Light Receptor

Control of membrane-receptor activity is required not only for the accuracy of sensory responses, but also to protect cells from excitotoxicity. Here we report the isolation of two noncomplementary fly mutants with slow termination of photoresponses. Genetic and electrophysiological analyses of the mutants revealed a defect in the deactivation of rhodopsin, a visual G protein-coupled receptor (GPCR). The mutant gene was identified as the calmodulin-binding transcription activator (dCAMTA). The known rhodopsin regulator Arr2 does not mediate this visual function of dCAMTA. A genome-wide screen identified five dCAMTA target genes. Of these, overexpression of the F box gene dFbxl4 rescued the mutant phenotypes. We further showed that dCAMTA is stimulated in vivo through interaction with the Ca(2+) sensor calmodulin. Our data suggest that calmodulin/CAMTA/Fbxl4 may mediate a long-term feedback regulation of the activity of Ca(2+)-stimulating GPCRs, which could prevent cell damage due to extra Ca(2+) influx.

RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance

To facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.

Prolonged Gq activity triggers fly rhodopsin endocytosis and degradation, and reduces photoreceptor sensitivity

Rapid deactivation of the Drosophila light receptor rhodopsin, through a visual arrestin Arr2 and a pathway that involves a transcription factor dCAMTA, is required for timely termination of light responses in the photoreceptor neuron. Here we report that this process is also critical for maintenance of the photoreceptor sensitivity. In both dCAMTA‐ and arr2‐mutant flies, the endocytosis of the major rhodopsin Rh1 was dramatically increased, which was mediated by a Gq protein that signals downstream of rhodopsin in the visual transduction pathway. Consequently, the Rh1 level was downregulated and the photoreceptor became less sensitive to light. Remarkably, the Gq‐stimulated Rh1 endocytosis does not require phospholipase C, a known effector of Gq, but depends on a tetraspanin protein. Our work has identified an arrestin‐independent endocytic pathway of G protein‐coupled receptor in the fly. This pathway may also function in mammals and mediate an early feedback regulation of receptor signaling.

Long-distance mechanism of neurotransmitter recycling mediated by glial network facilitates visual function in Drosophila

Significance Neurons communicate at synapses through neurotransmitters. For sustained neuronal signaling, neurotransmitters are recycled after release from neuronal terminals. During this process, perisynaptic glial cells take in and convert neurotransmitters such as glutamate, GABA, and histamine into inactive metabolites for transport. It has been assumed that inactive metabolites are delivered directly into neighboring neuronal terminals for neurotransmitter regeneration. Our work in the fruit fly, however, demonstrates that a gap junction-dependent multicellular glial network transports metabolites of histamine between perisynaptic glia and cell bodies of photoreceptor neurons, and this is required for visual signal transmission and alert behavior. Thus, by mediating this novel, long-distance recycling of neurotransmitters, intercellular glial networks play an important role in the maintenance of neuronal functions. Neurons rely on glia to recycle neurotransmitters such as glutamate and histamine for sustained signaling. Both mammalian and insect glia form intercellular gap-junction networks, but their functional significance underlying neurotransmitter recycling is unknown. Using the Drosophila visual system as a genetic model, here we show that a multicellular glial network transports neurotransmitter metabolites between perisynaptic glia and neuronal cell bodies to mediate long-distance recycling of neurotransmitter. In the first visual neuropil (lamina), which contains a multilayer glial network, photoreceptor axons release histamine to hyperpolarize secondary sensory neurons. Subsequently, the released histamine is taken up by perisynaptic epithelial glia and converted into inactive carcinine through conjugation with β-alanine for transport. In contrast to a previous assumption that epithelial glia deliver carcinine directly back to photoreceptor axons for histamine regeneration within the lamina, we detected both carcinine and β-alanine in the fly retina, where they are found in photoreceptor cell bodies and surrounding pigment glial cells. Downregulating Inx2 gap junctions within the laminar glial network causes β-alanine accumulation in retinal pigment cells and impairs carcinine synthesis, leading to reduced histamine levels and photoreceptor synaptic vesicles. Consequently, visual transmission is impaired and the fly is less responsive in a visual alert analysis compared with wild type. Our results suggest that a gap junction-dependent laminar and retinal glial network transports histamine metabolites between perisynaptic glia and photoreceptor cell bodies to mediate a novel, long-distance mechanism of neurotransmitter recycling, highlighting the importance of glial networks in the regulation of neuronal functions.