Selective vulnerability of neurons to acute toxicity after proteasome inhibitor treatment: implications for oxidative stress and insolubility of newly synthesized proteins - PubMed (original) (raw)

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

Kalavathi Dasuri et al. Free Radic Biol Med. 2010.

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.

Copyright © 2010. Published by Elsevier Inc.

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Figures

Figure 1

Figure 1. Proteasome inhibitors induce impairment of 20S and 26S proteasomes in both neurons and astrocytes

Primary rat astrocytes and neurons analyzed for sensitivity to proteasome inhibition following exposure to MG132. Activities of 20S and 26S were determined 1 hr following exposure to proteasome inhibitor MG132 for 1 hour, with 20S and 26S proteasome activity measured as described in methods. Data is representative of results from 2 separate experiments.

Figure 2

Figure 2. Proteasome inhibition induces more cell death in primary rat neurons as compared to astrocytes

Cells were treated with increasing concentrations of the proteasome inhibitor MG132 and analyzed for cell viability 24 hours post treatment. Neurons were observed to have significantly higher levels of cell death in response to proteasome inhibitor administration (A) as compared to astrocyte cultures (B). Results using morphological criteria as well as nuclear condensation/fragmentation gave nearly identical results. Data are presented as the mean and S.E.M. of results from 3 different sets of independent experiments (5 dishes per experiment).

Figure 3

Figure 3. The levels of oxidized proteins, but not ubiquitinated proteins, are increased to larger extent in neurons as compared to astrocytes following proteasome inhibition

Rat primary cortical neurons and astrocyte cultures were analyzed for the levels of ubiquitinated (A) and oxidized proteins (B) following treatment with proteasome inhibitors. Cells were treated with increasing concentrations of the proteasome inhibitor 10 μM MG132 and analyzed for ubiquitinated and oxidized protein levels following proteasome inhibitor treatment for 15 hours. Neurons were observed to have more severe increases in oxidized proteins following proteasome inhibitor treatment. Data are representative of results from three separate experiments.

Figure 4

Figure 4. The levels of short-lived protein synthesis and degradation are similar in neurons and astrocytes

Rat primary cortical neurons and astrocyte cultures were analyzed for protein synthesis following a 5 minute pulse of 35S-methionine. The levels of short lived protein degradation were analyzed in neuron and astrocyte cultures following 5 minute pulse of 35S-methionine and corresponding increasing lengths of chase period. Results indicate that neurons and astrocytes have similar levels of short lived protein synthesis (A) and short lived protein degradation (B). Data are presented as the mean and S.E.M. of results from 3 independent set of experiments with 4 dishes for each time point in an experiment.

Figure 5

Figure 5. Heat shock proteins in neurons and astrocytes following proteasome inhibition

Lysates from rat primary cortical neurons and astrocyte cultures were analyzed by Western blotting for the levels of heat shock protein induction following proteasome inhibition for 15 hours. Antibodies against the Hsp40, Hsp70 or Hsp90 were used in the analysis. Beta actin was used to show the equal loading of protein lysates. Data represent the 3 independent set of experiments done under similar conditions.

Figure 6

Figure 6. Proteasome inhibition resulted in increased accumulation of recently synthesized proteins in Triton x-100 insoluble fraction of neurons, as compared with astrocytes

Rat primary neurons and astrocytes were pulsed for one hour with 35S-methionine and chased for indicated time points in the presence or absence of proteasome inhibitor, MG132. Whole cell lysates were separated in to triton X-100 soluble and insoluble fractions and TCA insoluble radioactivity of these fractions was measured as described in methods. Proteasome inhibition resulted in the accumulation of higher levels of recently synthesized short lived proteins in triton X-100 insoluble fractions of primary neurons (A) when compared with the astrocytes (B). Data are presented as the mean and S.E.M. of results from 3 different sets of independent experiments.

Figure 7

Figure 7. Proteasome inhibition resulted in increased accumulation of recently synthesized proteins, that are ubiquitinated, in triton x-100 insoluble pool of neurons as compared with astrocytes

Rat primary Neurons and astrocytes were collected after treatment with cyclohexamide for indicated time points in the presence or absence of proteasome inhibitor, MG132, as described in methods. Whole cell lysates were fractionated by Triton X-100 and the amounts of ubiquitinated proteins in Triton X-100 soluble and insoluble fractions were analyzed using western blot analysis. Results indicate the increased accumulation of recently synthesized proteins, that are higher molecular weight ubiquitinated proteins, in triton x-100 insoluble pool of neurons (A), as compared with astrocytes (B), following proteasome inhibition.

Figure 8

Figure 8. Primary Neuron and astrocytes showed no differences in the levels of oxidized proteins in triton x-100 soluble and insoluble fractions following proteasome inhibition

Rat primary neurons and astrocytes were collected after treatment with cyclohexamide for indicated time points in the presence or absence of proteasome inhibitor, MG132, as described in methods. Whole cell lysates were fractionated by triton X-100 and the amounts of oxidized proteins in triton X-100 soluble and insoluble fractions were analyzed as described in methods. Results did not show differences in the levels of oxidized proteins in triton x-100 insoluble and soluble fractions between neurons (A) and astrocytes (B) following proteasome inhibition.

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