• Characterization of Neurotransmitter Transporter Gene Family in C. Elegans

      Jiang, Gouliang; Department of Biochemistry and Molecular Biology (2005-10)
      GABA functions as an inhibitory neurotransmitter in body muscles and as an excitatory neurotransmitter in enteric muscles in C. elegans. No transporter specific for this neurotransmitter has been identified to date in this organism. Here we report on the cloning and functional characterization of a GABA transporter from C. elegans (ceGAT- 1) and on the functional relevance of the transporter to the biology of body muscles and enteric muscles. ceGAT-1 is coded by snf-11 gene, a member of the sodium-dependent neurotransmitter symporter gene family in C. elegans. The cloned ceGAT-1 functions as a Na+/C f -coupled high-affinity transporter selective for GABA with aK t of~15 uM. The Na+:C1':GABA stoichiometry for ceGAT-1-mediated transport process is 2:1:1. The process is electrogenic as evidenced from GABA-induced inward currents in X laevis oocytes that express ceGAT-1 heterologously. The transporter is expressed exclusively in GABAergic neurons and in two other additional neurons. We also investigated the functional relevance of ceGAT-1 to the biology of body muscles and enteric muscles by ceGAT-1-specific RNAi in rrf-3 mutant, a strain of C. elegans in which neurons are not refractory to RNAi as in wild type strain. Downregulation of ceGAT-1 by RNAi leads to an interesting phenotype associated with altered function of body muscles and enteric muscles and also with altered sensitivity to aldicarb-induced paralysis. These findings provide unequivocal evidence for a modulatory role of GABA and ceGAT-1 in the biology of cholinergic neurons and in the function of body muscles and enteric muscles in this organism. We also cloned and functionally characterized for the first time a sodium-coupled transporter for amino acids in C. elegans. This transporter, designated ceNAT-1 (sodiumcoupled amino acid transporter-1), is identified in Worm database as snf-5, also a member of the sn f gene family (sodium/neurotransmitter symporter gene family). When expressed heterologously in mammalian cells, ceNAT-1 mediates the uptake of a broad spectrum of neutral amino acids in a Na+ dependent manner. The transport process exhibits a Na+: amino acid stoichiometry of 1:1. There is no involvement of C f in the transport process. When expressed heterologously in A! laevis oocytes, ceNAT-1 induces inward currents in response to neutral amino acids under voltage-clamp conditions, indicating that the transport process is electrogenic. Based on functional features, NAT-1 seems to be the C. elegans counterpart of the amino acid transporter B°AT in mammals. Mutations in the gene coding for B°AT cause Hartnup disease in humans. The clinical phenotype of Hartnup disease varies markedly depending on the environmental conditions. The present study shows that RNAi-mediated knockdown of NAT-1 or genetic deletion of NAT-1 in C. elegans is not associated with any detectable phenotype. This may be similar to the situation in humans where environmental conditions influence the clinical outcome of Hartnup disease. Further studies with altered experimental conditions are needed to determine if C. elegans with deletion of NAT-1 is a useful model system for investigations of Hartnup disease. Recently, a second isoform of B°AT has been identified in mammals. This transporter is expressed predominantly in the brain. Therefore, it is not clear at present whether the ceNAT-1 represents the worm counterpart of the Hartnup gene or the recently identified second isoform. We also report here on the cloning and functional characterization of a C. elegas betaine transporter which is encoded by snf-3, another member of the C. elegans sn f gene family. We named this transporter ceBGT-1. ceBGT-1 exhibits high specificity for betaine when expressed heterologouly in mammalian expression system and the uptake process mediated by ceBGT-1 is dependent on both sodium and chloride. The Na+: Cl': betaine stoichiometry for ceBGT-1-mediated transport process is 2:1:las confirmed y two-microelectrode voltage-clamp study. The Kt of ceBGT-1 for betaine is about 0.32 mM. Consistent with its role in osmoregulation, in vivo expression study using transgenic GFP fusion technique shows ceBGT-1 is expressed in the canal cells of C. elegans which represent the excretory represent the excretory organ in this organism. Investigation of the effects of hypertonicity on the expression of ceBGT-1 shows that hypertonicity increases its expression in C. elegans cultured with medium containing 350 mM NaCl compared to C. elegans cultured under normal conditions (50 mM NaCl).
    • The Role of Tumor Necrosis Factor Alpha (TNF-a) In the Eyes and Brain of HSV-1 Infected Euthymic BALB/C Mice

      Fields, Mark A; Department of Biochemistry and Molecular Biology (2007-12)
      After uniocular anterior chamber (AC) inoculation of HSV-1, virus and TNFalpha (TNF-a) are detected in the eyes and brain o f HSV-1 infected euthymic BALB/c mice. The overall goal o f this study was to investigate the role o f TN F-a in the eyes and brain o f HSV-1 infected BALB/c mice. Mice were treated with thalidomide for TN F-a inhibition or injected with clodronate liposomes to deplete macrophages, and the AC of one eye (ipsilateral) was injected with HSV-1 (KOS). In thalidomide-treated mice, both suprachiasmatic nuclei (SCN) were infected by day 5 p.i. and the titer of virus in the SCN contralateral to the side o f injection was increased. In macrophage depleted mice, both SCN were infected at day 6 p.i. and the titer o f virus in the SCN o f these mice was increased at day 6 and 7 p.i. compared with controls. The titer o f virus in the contralateral (uninoculated) eye o f macrophage depleted mice was increased at day 7 p.i. The results o f these studies suggest that TN F-a plays a role in limiting virus replication in the SCN o f euthymic BALB/c mice and that one source of TN F-a is macrophages. In order to further investigate the role of TNF-a, a recombinant o f HSV-1 (KOS77VF) was constructed that produces TNF-a constitutively. Euthymic BALB/c mice were injected in one anterior chamber with the TNF-a recombinant, with a recombinant containing the pCI plasmid, with a recombinant rescue virus, or with the parental virus. Mice from each group were sacrificed on day 1-9 p.i. and the uninjected eyes were removed. In the uninjected eye o f KOSTNF -infected mice, TN F-a expression was increased and there were more viral antigen positive cells and immune inflammatory cells. There was earlier microscopic evidence o f retinal infection and destruction in these mice. In addition, the titer o f virus in the uninjected eye was significantly increased in KOSTiVF-infected mice on day 7 p.i. compared with KOSpCI, KOS6{irescue, or with KOS6/? infected mice. These results suggest that overproduction o f TN F-a by HSV-1 (KOS7WF) facilitates spread o f virus infection in the eye through increased inflammation and TNF-a-mediated damage to retinal cells.