Elsevier

Free Radical Biology and Medicine

Volume 49, Issue 8, 1 November 2010, Pages 1290-1297
Free Radical Biology and Medicine

Original Contribution
Selective vulnerability of neurons to acute toxicity after proteasome inhibitor treatment: Implications for oxidative stress and insolubility of newly synthesized proteins

https://doi.org/10.1016/j.freeradbiomed.2010.07.014Get rights and content

Abstract

Maintaining protein homeostasis is vital to cell viability, with numerous studies demonstrating a role for proteasome inhibition occurring during the aging of a variety of tissues and, presumably, contributing to the disruption of cellular homeostasis during aging. In this study we sought to elucidate the differences between neurons and astrocytes in regard to basal levels of protein synthesis, proteasome-mediated protein degradation, and sensitivity to cytotoxicity after proteasome inhibitor treatment. In these studies we demonstrate that neurons have an increased vulnerability, compared to astrocyte cultures, to proteasome-inhibitor-induced cytotoxicity. No significant difference was observed between these two cell types in regard to the basal rates of protein synthesis, or basal rates of protein degradation, in the pool of short-lived proteins. After proteasome inhibitor treatment neuronal crude lysates were observed to undergo greater increases in the levels of ubiquitinated and oxidized proteins and selectively exhibited increased levels of newly synthesized proteins accumulating within the insoluble protein pool, compared to astrocytes. Together, these data suggest a role for increased oxidized proteins and sequestration of newly synthesized proteins in the insoluble protein pool, as potential mediators of the selective neurotoxicity after proteasome inhibitor treatment. The implications for neurons exhibiting increased sensitivity to acute proteasome inhibitor exposure, and the corresponding changes in protein homeostasis observed after proteasome inhibition, are discussed in the context of both aging and age-related disorders of the nervous system.

Section snippets

Materials

The antibodies to β-actin (SC-47778) and ubiquitin (SC - 8017) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MG132 was purchased from EMD Chemicals (Gibbstown, NJ, USA). The BCA reagent was purchased from Thermo Scientific (Waltham, IL, USA). The OxyBlot kit was purchased from Millipore (Billerica, MA, USA). [35S]Methionine was purchased from PerkinElmer (Cat. No. NEG009A500UC; Shelton, CT, USA). All the chemicals, including Hoechst, H33342 (bisbenzamide trihydrochloride)

Neuronal cells exhibit increased vulnerability to the toxicity of proteasome inhibitors

To begin to elucidate the relative susceptibilities of primary rat neuronal and astrocyte cultures to the toxicity of proteasome inhibitors, we conducted studies analyzing the severity of proteasome inhibition and induction of cell death after treatment with the proteasome inhibitor MG132. In these studies we observed that proteasome inhibitor treatment resulted in a robust dose-dependent impairment of 26S and 20S chymotrypsin-like activity in both neurons and astrocytes (Fig. 1).

Discussion

Our studies identify for the first time that primary rat neuron cultures undergo a greater degree of cytotoxicity after proteasome inhibitor treatment compared to primary rat astrocyte cultures. These data suggest that neurons may be preferentially affected by the proteasome inhibition reported to occur in the brain during aging and age-related diseases of the nervous system [30]. In this model the negative effects of proteasome inhibition in the brain during aging and age-related diseases are

Acknowledgments

This work was supported by grants from the NIA (AG029885, AG025771) and the Hibernia National Bank/Edward G. Schlieder Chair (J.N.K.).

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