Alice Benham et al. (2007) International Worm Meeting "Regulation of GABA receptor levels at the neuromuscular junction requires lysosome function."

Bruce Bamber, Alice Benham, Aaron Rowland

[International Worm Meeting, 2007]

The UNC-49 GABA receptor is the inhibitory neurotransmitter receptor at the C. elegans neuromuscular junction. Its human homolog, the GABA_A receptor, is the primary inhibitory neurotransmitter receptor in the brain. The strength of inhibitory synaptic transmission in both worm muscles and the human brain depends on the abundance of GABA receptors. Receptor abundance in turn depends on the balance of receptor synthesis and degradation. What are the mechanisms of GABA receptor degradation? The lysosome and the proteasome have both been implicated in GABA_A receptor degradation in cultured cells, but convincing experiments in vivo have not been performed. Using quantitative immunofluorescence and electrophysiology, we have investigated the importance of the lysosome in GABA receptor regulation in C. elegans. We compared GABA receptor levels at synapses in wild-type worms and worms containing the cup-5(ar465) allele, which causes reduced lysosome function. cup-5(ar465) mutants had a slight but significant excess of synaptic GABA receptors, indicating a role for the lysosome. We then exposed wild-type and mutant worms to the GABA agonist muscimol. This treatment led to a strong reduction in synaptic GABA receptor levels in wild-type worms. cup-5(ar465) mutants were still able to reduce synaptic GABA receptor levels, but the reduction was slower and less complete. These results suggest that lysosomal degradation normally sets an upper limit to the levels of GABA receptors at the synapse, and that the lysosome degrades GABA receptors during homeostatic regulation. However, because some aspects of receptor regulation were maintained despite reduced lysosome function, it is likely that other mechanisms also come into play.

Bruce A Bamber et al. (2005) International Worm Meeting "Homeostatic regulation of the UNC-49 GABAA receptor"

Janet E Richmond, Aaron M Rowland, Alice Benham, Bruce A Bamber

[International Worm Meeting, 2005]

Inhibitory synaptic strength depends on the density of postsynaptic GABAA receptors. Plasticity of postsynaptic GABAA receptor density contributes to homeostatic regulation in the nervous system, and is associated with disease states such as epilepsy and anxiety. To understand the mechanisms that control GABAA receptor density at synapses, we are studying the inhibitory neuromuscular junction in C. elegans. When exposed to the GABA agonist muscimol, worms initially become paralyzed, but then adapt. Adapted worms have a shrinker Unc phenotype characteristic of worms that lack GABA neurotransmission. We hypothesized that adaptation is the result of downregulation of GABAA receptors at the neuromuscular junction, which are encoded by the unc-49 gene. We tested this hypothesis by comparing GABA currents in naïve and muscimol-adapted worms. GABA currents were six-fold reduced in amplitude in adapted animals. Using quantitative immunofluorescence, we demonstrated that UNC-49 abundance at the synapse was reduced nine-fold. A transcriptional mechanism does not appear to be involved: quantitative RT-PCR analysis demonstrated that unc-49 mRNA levels were reduced, but only transiently. unc-49 mRNA returned to its pre-exposure level before the majority of the protein was lost. Using the immunofluorescence assay, we are characterizing protein trafficking and cell signaling mutants to understand the mechanism of this homeostatic regulation of the UNC-49 GABAA receptor.