Presynaptic terminals independently regulate synaptic clustering and autophagy of GABAA receptors in Caenorhabditis elegans

Abstract

Synaptic clustering of GABAA receptors is important for the function of inhibitory synapses, influencing synapse strength and, consequently, the balance of excitation and inhibition in the brain. Presynaptic terminals are known to induce GABAA receptor clustering during synaptogenesis, but the mechanisms of cluster formation and maintenance are not known. To study how presynaptic neurons direct the formation of GABAA receptor clusters, we have investigated GABAA receptor localization in postsynaptic cells that fail to receive presynaptic contacts in Caenorhabditis elegans. Postsynaptic muscles in C. elegans receive acetylcholine and GABA motor innervation, and GABAA receptors cluster opposite GABA terminals. Selective loss of GABA inputs caused GABAA receptors to be diffusely distributed at or near the muscle cell surface, confirming that GABA presynaptic terminals induce GABAA receptor clustering. In contrast, selective loss of acetylcholine innervation had no effect on GABAA receptor localization. However, loss of both GABA and acetylcholine inputs together caused GABAA receptors to traffic to intracellular autophagosomes. Autophagosomes normally transport bulk cytoplasm to the lysosome for degradation. However, we show that GABAA receptors traffic to autophagosomes after endocytic removal from the cell surface and that acetylcholine receptors in the same cells do not traffic to autophagosomes. Thus, autophagy can degrade cell-surface receptors and can do so selectively. Our results show that presynaptic terminals induce GABAA receptor clustering by independently controlling synaptic localization and surface stability of GABAA receptors. They also demonstrate a novel function for autophagy in GABAA receptor degradative trafficking.

Figure 1.

Normal neuromuscular anatomy in C. elegans. A, Illustration of normal C. elegans neuromuscular system. ACh (white) and GABA (black) motor neurons form synapses (triangles) on dorsal body-wall muscles (gray bodies). Cell bodies are ventral, so axons must grow dorsally (arrows) to reach dorsal muscles (neurons innervating ventral muscles not shown). B, C, Dorsal nerve cord contains GABA and ACh axons. Axons are visualized with GFP, expressed under the control of promoters specific for GABA (B) and ACh (C) neurons (see Materials and Methods). D, Normal synaptic localization of GABA_AR-GFP in dorsal muscles. Scale bar, 5.0 μm.

Figure 2.

Loss of innervation affects GABA_A receptor clustering and trafficking. A–C, Illustrations of dorsal muscles lacking motor inputs. D–F, GFP expressed in GABA presynaptic neurons. G–I, GFP expressed in ACh presynaptic neurons. J–L, Postsynaptic GABA_AR-GFP. A, D, G, J, GABA dorsal pathfinding is disrupted, and muscle cells lack GABA inputs. B, E, H, K, ACh dorsal pathfinding is disrupted, and muscle cells lack ACh inputs. C, F, I, L, Both ACh and GABA dorsal pathfinding disrupted, and dorsal muscles lack all synaptic inputs. Arrowheads in D, H, and I indicate axons that extend dorsally a short distance but do not reach the dorsal cord. Arrows in J and L indicate dorsal midline. Scale bars, 5.0 μm.

Figure 3.

Autophagy is increased in non-innervated muscle cells. A, B, Electron micrographs of autophagosomes in the non-innervated dorsal body-wall muscles of netrin-defective (unc-5) worms. Autophagosomes typically constitute large membrane-bound organelles containing complex contents including additional membrane layers inside. Morphologies typical of early (A) and late (B) autophagosomes or autophagolysosomes were observed. C, Autophagy is significantly enhanced in netrin receptor-deficient (unc-5) mutants, mainly in dorsal muscles. Sarcomere volume and cytoplasm volume are comparable between normal (D) and non-innervated (E) dorsal muscles, indicating that lack of innervation does not cause muscle cell death or degeneration. Arrows indicate the boundary between sarcomeres and cytoplasm. Scale bars, 0.1 μm. *p < 0.05 compared with wild type, by Mann–Whitney U test; n = 48 and 56 muscle quadrants in wild-type (wt) and unc-5 mutants, respectively.

Figure 4.

Evidence that GABA_AR-GFP traffics to autophagosomes. A, GABA_AR-GFP-containing organelles do not form in non-innervated muscles in an autophagy-defective mutant background (unc-5;unc-51 double mutant). GABA_AR-GFP-containing organelles also contain the autophagosome markers mRFP-LGG-1 (B) and beclin-1-mRFP (C). D, GABA_A receptor immunoreactivity is associated with beclin-1-GFP fluorescence in non-innervated muscle cells that do not overexpress GABA_AR-GFP, indicating that endogenous GABA_A receptors traffic to autophagosomes. E, In control worms lacking endogenous GABA_A receptors (unc-49 mutants), autophagosomes in non-innervated muscle cells do not contain GABA_A receptor immunoreactivity. Arrows in A indicate the position of the dorsal midline. Scale bars: A, 5.0 μm; B–E, 1.0 μm.

Figure 5.

GABA_A receptors traffic to autophagosomes in innervated muscles. GABA_A receptor immunoreactivity associates with beclin-1-GFP in correctly innervated muscle cells in worms with functional netrin receptors. Scale bar, 1.0 μm.

Figure 6.

GABA_A receptors are removed from the surface of non-innervated cells by trafficking to autophagosomes. A, Accumulation of GABA_AR-GFP in autophagosomes requires ongoing endocytosis. In worms with a temperature-sensitive endocytosis defect (open symbols, dashed line), the number of GABA_AR-GFP-containing autophagosomes decreases when animals are shifted from the permissive temperature (15°C) to the restrictive temperature (26°C). This decrease is not observed in control worms with normal endocytosis (filled symbols, solid line). Differences between endocytosis-defective and control strains are significant at 14 h and all subsequent time points (p < 0.05; n = 10 for each time point, except for t = 0, in which n = 6 for the control strain). B, GABA currents are reduced in non-innervated dorsal cells as a result of autophagy. Left trace and bar, Wild type; middle trace and bar, netrin deficient (unc-5 mutant); right trace and bar, netrin deficient and autophagy deficient (unc-5;unc-51 double mutant). *p < 0.05 indicates that currents are significantly reduced in non-innervated dorsal cells compared with either normally innervated dorsal cells or non-innervated autophagy-defective dorsal cells (one-way ANOVA). Error bars are SEM.

Figure 7.

Acetylcholine receptors do not traffic to autophagosomes in non-innervated muscle cells. A, B, Localization pattern of GFP-tagged ACh receptors expressed in body-wall muscles. A, In a worm with functional netrin receptors, and therefore normal neuromuscular innervation, AChR-GFP is visible along the ventral (left) and dorsal (right) nerve cords. Cellular fluorescence in the ventral cord (open arrowheads) is probably neuronal (White et al., 1986). B, In a worm lacking functional netrin receptors, and therefore lacking dorsal motor innervation, AChR-GFP is visible along the ventral cord (left) but is not observed dorsally in either synaptic puncta or intracellular organelles (right). C, Immunoreactivity for another ACh receptor subunit, UNC-29, does not localize to autophagosomes. In a netrin-defective worm, GABA_AR-GFP was visible along the ventral nerve cord and within an autophagosome in a dorsal muscle (left). UNC-29 immunoreactivity was also visible along the ventral nerve cord but not in the autophagosome (middle). Merged image shows localization of GABA_AR-GFP and UNC-29 localization to adjacent synapses but no colocalization in the autophagosome (right). D, Electrophysiological response to the ACh agonist is the same in innervated and non-innervated cells and is not increased when autophagy is blocked (p > 0.05, one-way ANOVA). Left trace and bar, Wild type; middle trace and bar, netrin deficient (unc-5 mutant); right trace and bar, netrin deficient and autophagy deficient (unc-5;unc-51 double mutant). Error bars are SEM. Scale bars, 5 μm.