The N-glycosylation inhibitors interfere the functional expression and subcellular distribution of GABAA receptor and induce cell death in cultured neurons

• Main Authors: Tzu-Yung Lin, 林自勇
• Other Authors: Hsiang-Shu Yin
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/47946920193611707767

博士 === 國立臺灣大學 === 解剖學研究所 === 87 === gamma-Aminobutyric acid type A receptor (GABAAR) is the major inhibitory neurotransmitter receptor on the post-synaptic membrane of the neurons in the CNS. It is known as a multi-subunit membrane glycoprotein containing an integral ligand-gated Cl- ion channel. Through combining with the receptor, GABA increases the Cl- ion conductance to modulate neuronal excitability. Using inhibitors to N-glycosylation and oligosaccharide processing, the significance of N-linked glycosylation and oligosaccharide processing was examined for the expression of GABAAR in cultured neurons derived from chick embryo brains. Tunicamycin, a specific inhibitor to N-glycosylation, blocks the first step in the synthesis of N-linked oligosaccharides, resulting in the absence of N-glycans on glycoproteins. Incubation of cultures with 5 ug/ml of tunicamycin for 24 h decreased the binding of 3H-flunitrazepam and 3H-muscimol, probes for the benzodiazepine and GABA sites on the receptor, by about 20% and 28%. The loss of ligand binding was due to a reduction in the number of binding sites with no significant changes in receptor affinity. Light microscopic immunocytochemistry also revealed that the treatment reduced approximately 13% of the intensity of GABAAR immunoreactivity in the neuronal somata. Furthermore, the fraction of intracellular receptors was decreased from 34% to 24% in the presence of tunicamycin, as revealed by trypsinization of the cells in situ followed by 3H-flunitrazepam binding. Immuno-electron microscopy also revealed that the GABAAR appeared down-regulated on the cell membrane and the membranes of rough endoplasmic reticulum (rER) and Golgi apparatus following the tunicamycin treatment. In addition, electrophysiological recordings disclosed that the tunicamycin treatment reduced GABA-elicited current amplitude by 40% in the neurons. The molecular weight of the receptor subunit protein was lowered 0.5 kDa by tunicamycin, in accordance with that following N-glycosidase F digestion, indicating the blockade of N-linked glycosylation of GABAAR by tunicamycin. Moreover, intense inhibitions of 91% and 44% to the general galactosylation and mannosylation were also detected in the tunicamycin-treated cells, whereas protein synthesis was blocked by 13%, by assaying the incorporation of 3H-sugars and 3H-leucine. Nevertheless, treatment with castanospermine or swainsonine (10 ug/ml, 24 h), inhibitors to maturation of oligosaccharides, failed to produce significant changes in 3H-flunitrazepam binding. In situ hybridization analysis showed that these three inhibitors did not perturb the expression of mRNA of GABAAR a1 subunit. Although the cells were surviving after 24 h treatment of 5 ug/ml tunicamycin by Trypan blue exclusion experiment, after 3 h removal of the agent the cells started to die, and the level of 3H-flunitrazepam binding continued to decline. Neurons of the 24 h tunicamycin treatment showed dilated rER, nuclear envelope and components of Golgi apparatus, in addition to degranulation of rER and disaggregation of ribosomal rosettes. In neurons of the 4 prolonged incubation groups, some ribosomes were seen to reattach to the membranes of rER, the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle-like structure appeared in the perikarya; this structure is implicated to involve the neuronal degeneration. Extracellular cell debris was increased with time of incubation. The ratio of "light" neurons, containing lower density of cytoplasmic matrix than control, decreased from 28% to 3% at 3 h and 24 h after removal of tunicamycin, and 45% to 6% for the further 3 h and 24 h tunicamycin incubation, whereas "dense" neurons only appeared in these two 24 h groups, as 44% and 34%. The "light" neurons resemble necrotic cells, but the "dense" neurons exhibit distinct morphological features. Nevertheless, gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. The data suggest that tunicamycin cause the down-regulation and subcellular redistribution of GABAAR by producing unglycosylated receptors and modifying their localization. Both galactosylation and mannosylation during the process of N-linked glycosylation of GABAAR proteins may be important for its functional expression and intracellular transport. The results also indicate irreversible toxicity of chronic tunicamycin treatment to the neurons and suggest differential mechanisms for the neuronal death among various populations of cells. It is evident that the N-glycosylation plays a critical role for neuronal survival.