Toxicity of tunicamycin to cultured brain neurons: Ultrastructure of the degenerating neurons

Abstract

Our previous study has shown that tunicamycin irreversibly downregulates the expression of GABA A R and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mechanisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brains were incubated with 5 µg/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons treated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), nuclear envelope and components of Golgi apparatus, in addition to the degranulation of rER and disaggregation of ribosomal rosettes. In neurons subjected to the prolonged incubation, some ribosomes reattached 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. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons resemble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA-elicited current in the neurons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM 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.

Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein

“…2B, insets). The periphery of these structures contained both mutant PLPmh and calnexin, resembling the distended ER that has been observed under conditions of chronic ER stress (Alvarez et al, 1999;Dalal et al, 2004;Lass et al, 2008;Lin et al, 1999). The milddisease-associated mutants W162L and G245A were also present A twodimensional model of PLP topology illustrating the four TM domains, cytosolic N-and C-termini, palmitoylation sites (squiggles), and disulphide bonds (straight lines).…”

Distinct Roles for the AAA ATPases NSF and p97 in the Secretory Pathway

“…Such disorganization could result in changes in the ratio, size, and shape of ER cisternae and tubules. Morphologically similar swelling is seen in a few pathological conditions, including intoxication with the pore forming toxin aerolysin (Abrami et al, 1998), prolonged exposure of cells to BFA (Alvarez and Sztul, 1999), and treatment of neurons with tunicamycin (Lin et al, 1999). Further understanding of the events that lead to ER dilation will shed light on how p97 participates in ER homeostasis.…”

Effect of Bu-Zhong-Yi-Qi-Tang on deficiency of N-glycan/nitric oxide and islet damage induced by streptozotocin indiabetic rats

“…Alpha-mannosidase, a key enzyme converting precursor high-mannose-type N-glycan to matured complex-type structure, contributes to the establishment of an equitable glycoprotein quality control standard, by which the efficiency of Asn-linked glycoprotein conformational maturation results in dislocation of misfolded glycoproteins into the cytosol where proteins are degraded in the proteasome and maintain the homeostasis of ER [2] . Lin et al [24] cultured neurons derived from embryonic chicken brains with tunicamycin (TM), an inhibitor of N-linked glycosylation, and found that the light neurons resembled necrotic cells, but the dense neurons exhibited distinct morphological features of necrosis and apoptosis, indicating that TM has an irreversible toxicity to the neurons and a different mechanism underlying neuron death.…”