Neuroprotective action of cycloheximide involves induction of bcl-2 and antioxidant pathways

Abstract

The ability of the protein synthesis inhibitor cycloheximide (CHX) to prevent neuronal death in different paradigms has been interpreted to indicate that the cell death process requires synthesis of "killer" proteins. On the other hand, data indicate that neurotrophic factors protect neurons in the same death paradigms by inducing expression of neuroprotective gene products. We now provide evidence that in embryonic rat hippocampal cell cultures, CHX protects neurons against oxidative insults by a mechanism involving induction of neuroprotective gene products including the antiapoptotic gene bcl-2 and antioxidant enzymes. Neuronal survival after exposure to glutamate, FeSO4, and amyloid beta-peptide was increased in cultures pretreated with CHX at concentrations of 50-500 nM; higher and lower concentrations were ineffective. Neuroprotective concentrations of CHX caused only a moderate (20-40%) reduction in overall protein synthesis, and induced an increase in c-fos, c-jun, and bcl-2 mRNAs and protein levels as determined by reverse transcription-PCR analysis and immunocytochemistry, respectively. At neuroprotective CHX concentrations, levels of c-fos heteronuclear RNA increased in parallel with c-fos mRNA, indicating that CHX acts by inducing transcription. Neuroprotective concentrations of CHX suppressed accumulation of H2O2 induced by FeSO4, suggesting activation of antioxidant pathways. Treatment of cultures with an antisense oligodeoxynucleotide directed against bcl-2 mRNA decreased Bcl-2 protein levels and significantly reduced the neuroprotective action of CHX, suggesting that induction of Bcl-2 expression was mechanistically involved in the neuroprotective actions of CHX. In addition, activity levels of the antioxidant enzymes Cu/Zn-superoxide dismutase, Mn-superoxide dismutase, and catalase were significantly increased in cultures exposed to neuroprotective levels of CHX. Our data suggest that low concentrations of CHX can promote neuron survival by inducing increased levels of gene products that function in antioxidant pathways, a neuroprotective mechanism similar to that used by neurotrophic factors.

Figure 1

CHX protects cultured hippocampal neurons against excitotoxic and oxidative insults in a concentration-dependent manner. Cultures were preincubated for 16 h with vehicle or the indicated concentrations of CHX, and then were exposed to 10 μM glutamate (A), 5 μM FeSO₄ (B), 5 μM Aβ (C), or vehicle control (D). Neuronal survival was quantified 20 h later. Values represent the mean and SEM (n = 4 separate cultures). *P < 0.05, **P < 0.01 compared with 0 CHX value. ANOVA with Scheffe's post-hoc tests.

Figure 2

(A) Time course of effects of neuroprotective (100 nM) and high (10 μM) concentrations of CHX on protein synthesis. Cultures were exposed to 100 nM or 10 μM CHX for 1, 4, 12, or 24 h, and incorporation of [³⁵S]methionine into TCA-precipitable protein was quantified. Values are the mean and SEM of determinations made in three separate cultures. For 100 nM CHX, the 1 h value was significantly less than the 0 h value (P < 0.05). For 10 μM CHX, the 1, 4, 12, and 24 h values were each significantly less than the 0 h value and each value for cultures exposed to 100 nM CHX (P < 0.001). (B) Effect of increasing concentrations of CHX on protein synthesis. Cultures were exposed for 1 h to the indicated concentrations of CHX, and incorporation of [³⁵S]methionine into TCA-precipitable protein was quantified. Values are the mean and SEM of determinations made in three separate cultures. The values for cultures exposed to either 100 or 300 nM CHX were different than the values for cultures exposed to 0 (P < 0.01), 10 (P < 0.05), 1,000 (P < 0.001), and 10,000 (P < 0.001) nM CHX. Statistical comparisons used ANOVA with Scheffe's post-hoc tests.

Figure 3

Neuroprotective concentrations of CHX induce expression of c-fos, bcl-2, and c-jun mRNAs. Hippocampal cultures were exposed for 1 h to the indicated concentrations of CHX (A and B), or were exposed to 100 nM CHX for the indicated time periods (C). RNA was isolated, and c-fos, c-jun, bcl-2, and neurofilament (NFM) mRNAs were amplified by RT-PCR. Data in B and C are representative of at least three independent experiments.

Figure 3

Neuroprotective concentrations of CHX induce expression of c-fos, bcl-2, and c-jun mRNAs. Hippocampal cultures were exposed for 1 h to the indicated concentrations of CHX (A and B), or were exposed to 100 nM CHX for the indicated time periods (C). RNA was isolated, and c-fos, c-jun, bcl-2, and neurofilament (NFM) mRNAs were amplified by RT-PCR. Data in B and C are representative of at least three independent experiments.

Figure 3

Neuroprotective concentrations of CHX induce expression of c-fos, bcl-2, and c-jun mRNAs. Hippocampal cultures were exposed for 1 h to the indicated concentrations of CHX (A and B), or were exposed to 100 nM CHX for the indicated time periods (C). RNA was isolated, and c-fos, c-jun, bcl-2, and neurofilament (NFM) mRNAs were amplified by RT-PCR. Data in B and C are representative of at least three independent experiments.

Figure 4

Neuroprotective concentrations of CHX induce c-fos transcription. Cultures were exposed for 1 h to the indicated concentrations of CHX and RNA was isolated. (A) RT-PCR analysis of RNA amplified with c-fos, c-jun, and NFM primers. Locations of c-fos heteronuclear RNA (hnRNA) and mRNA are indicated at the left. Levels of c-fos hnRNA were increased in cells exposed to 100 and 300 nM CHX, but not in cells exposed to 1 μM CHX. (B) Relative levels of c-fos mRNA and c-fos hnRNA in cultures exposed for 1 h to the indicated concentrations of CHX. Similar results were obtained in three independent experiments.

Figure 4

Neuroprotective concentrations of CHX induce c-fos transcription. Cultures were exposed for 1 h to the indicated concentrations of CHX and RNA was isolated. (A) RT-PCR analysis of RNA amplified with c-fos, c-jun, and NFM primers. Locations of c-fos heteronuclear RNA (hnRNA) and mRNA are indicated at the left. Levels of c-fos hnRNA were increased in cells exposed to 100 and 300 nM CHX, but not in cells exposed to 1 μM CHX. (B) Relative levels of c-fos mRNA and c-fos hnRNA in cultures exposed for 1 h to the indicated concentrations of CHX. Similar results were obtained in three independent experiments.

Figure 5

CHX induces a concentration-dependent increase in levels of Bcl-2, c-Fos, and c-Jun protein in cultured hippocampal neurons. (A) Cultures were exposed for 5 h to vehicle or 100 nM CHX. Cultures were then immunostained with antibodies to c-Fos or Bcl-2. Note increased nuclear c-Fos and Bcl-2 immunoreactivities in neurons exposed to CHX compared with neurons in control cultures. (B) Relative levels of immunoreactivity of neurons with antibodies to c-Fos, Bcl-2, or c-Jun were determined in cultures exposed for 5 h to the indicated concentrations of CHX. Staining intensity was rated on a scale from 0 to 4 (0, no staining; 1, weak; 2, moderate; 3, strong; 4, very strong). Values are the mean and SEM of determinations made in three separate cultures (100 neurons scored per culture). *P < 0.01 compared with corresponding value for cultures not exposed to CHX; ANOVA with Scheffe's post-hoc tests.

Figure 5

CHX induces a concentration-dependent increase in levels of Bcl-2, c-Fos, and c-Jun protein in cultured hippocampal neurons. (A) Cultures were exposed for 5 h to vehicle or 100 nM CHX. Cultures were then immunostained with antibodies to c-Fos or Bcl-2. Note increased nuclear c-Fos and Bcl-2 immunoreactivities in neurons exposed to CHX compared with neurons in control cultures. (B) Relative levels of immunoreactivity of neurons with antibodies to c-Fos, Bcl-2, or c-Jun were determined in cultures exposed for 5 h to the indicated concentrations of CHX. Staining intensity was rated on a scale from 0 to 4 (0, no staining; 1, weak; 2, moderate; 3, strong; 4, very strong). Values are the mean and SEM of determinations made in three separate cultures (100 neurons scored per culture). *P < 0.01 compared with corresponding value for cultures not exposed to CHX; ANOVA with Scheffe's post-hoc tests.

Figure 6

Neuroprotective concentrations of CHX suppress H₂O₂ accumulation induced by FeSO₄. (A) Representative confocal laser scanning microscope images of DCF fluorescence in neurons in a vehicletreated control culture (left), a culture exposed to 10 μM FeSO₄ for 20 min (middle), and a culture that had been pretreated with 100 nM CHX for 16 h and then exposed to 10 μM FeSO₄ for 20 min (right). Fluorescence intensity is represented by the color scale at the lower left. (B) Cultures were preincubated for 16 h with 100 nM CHX or vehicle (Control and FeSO₄), and then were loaded with 2,7-dichlorofluorescin diacetate and exposed to vehicle or 10 μM FeSO₄ for 20 min. Average fluorescence in neuronal cell bodies was quantified (see Materials and Methods). Values are the mean and SEM of determinations made in a total of 30–48 neurons in three separate cultures. *P < 0.02; ANOVA with Scheffe's post-hoc test. (C) Hippocampal cultures were pretreated with vehicle or 100 nM CHX for 16 h, and then were exposed for 20 h to glutamate alone or in combination with BSO as indicated. Values are the mean and SEM of determinations made in four separate cultures. *P < 0.05; ANOVA with Scheffe's post-hoc test.

Figure 6

Neuroprotective concentrations of CHX suppress H₂O₂ accumulation induced by FeSO₄. (A) Representative confocal laser scanning microscope images of DCF fluorescence in neurons in a vehicletreated control culture (left), a culture exposed to 10 μM FeSO₄ for 20 min (middle), and a culture that had been pretreated with 100 nM CHX for 16 h and then exposed to 10 μM FeSO₄ for 20 min (right). Fluorescence intensity is represented by the color scale at the lower left. (B) Cultures were preincubated for 16 h with 100 nM CHX or vehicle (Control and FeSO₄), and then were loaded with 2,7-dichlorofluorescin diacetate and exposed to vehicle or 10 μM FeSO₄ for 20 min. Average fluorescence in neuronal cell bodies was quantified (see Materials and Methods). Values are the mean and SEM of determinations made in a total of 30–48 neurons in three separate cultures. *P < 0.02; ANOVA with Scheffe's post-hoc test. (C) Hippocampal cultures were pretreated with vehicle or 100 nM CHX for 16 h, and then were exposed for 20 h to glutamate alone or in combination with BSO as indicated. Values are the mean and SEM of determinations made in four separate cultures. *P < 0.05; ANOVA with Scheffe's post-hoc test.

Figure 6

Neuroprotective concentrations of CHX suppress H₂O₂ accumulation induced by FeSO₄. (A) Representative confocal laser scanning microscope images of DCF fluorescence in neurons in a vehicletreated control culture (left), a culture exposed to 10 μM FeSO₄ for 20 min (middle), and a culture that had been pretreated with 100 nM CHX for 16 h and then exposed to 10 μM FeSO₄ for 20 min (right). Fluorescence intensity is represented by the color scale at the lower left. (B) Cultures were preincubated for 16 h with 100 nM CHX or vehicle (Control and FeSO₄), and then were loaded with 2,7-dichlorofluorescin diacetate and exposed to vehicle or 10 μM FeSO₄ for 20 min. Average fluorescence in neuronal cell bodies was quantified (see Materials and Methods). Values are the mean and SEM of determinations made in a total of 30–48 neurons in three separate cultures. *P < 0.02; ANOVA with Scheffe's post-hoc test. (C) Hippocampal cultures were pretreated with vehicle or 100 nM CHX for 16 h, and then were exposed for 20 h to glutamate alone or in combination with BSO as indicated. Values are the mean and SEM of determinations made in four separate cultures. *P < 0.05; ANOVA with Scheffe's post-hoc test.

Figure 7

Exposure of hippocampal cell cultures to Bcl-2 antisense oligodeoxynucleotide decreases levels of Bcl-2. (A) Parallel cultures were exposed for 16 h to 50 μM control (sense) ODN or 50 μM Bcl-2 antisense ODN. Cultures were then fixed and immunostained in parallel using an anti–Bcl-2 primary antibody. Shown are phase-contrast (left) and bright-field (right) micrographs. Note that levels of Bcl-2 immunoreactivity are greater in neurons in the control culture than in the culture exposed to Bcl-2 antisense ODN (arrowheads point to neuron cell bodies). (B) Cultures were exposed for 16 h to vehicle (water), 50 μM Bcl-2 antisense ODN, or 50 μM sense ODN. Solubilized proteins were electrophoretically separated, transferred to a nitrocellulose sheet, and immunoreacted with a Bcl-2 antibody. Levels of Bcl-2 were markedly decreased in the culture exposed to Bcl-2 antisense ODN compared with the control cultures.

Figure 7

Exposure of hippocampal cell cultures to Bcl-2 antisense oligodeoxynucleotide decreases levels of Bcl-2. (A) Parallel cultures were exposed for 16 h to 50 μM control (sense) ODN or 50 μM Bcl-2 antisense ODN. Cultures were then fixed and immunostained in parallel using an anti–Bcl-2 primary antibody. Shown are phase-contrast (left) and bright-field (right) micrographs. Note that levels of Bcl-2 immunoreactivity are greater in neurons in the control culture than in the culture exposed to Bcl-2 antisense ODN (arrowheads point to neuron cell bodies). (B) Cultures were exposed for 16 h to vehicle (water), 50 μM Bcl-2 antisense ODN, or 50 μM sense ODN. Solubilized proteins were electrophoretically separated, transferred to a nitrocellulose sheet, and immunoreacted with a Bcl-2 antibody. Levels of Bcl-2 were markedly decreased in the culture exposed to Bcl-2 antisense ODN compared with the control cultures.

Figure 8

Blockade of the neuroprotective actions of CHX in cultures treated with bcl-2 antisense oligodeoxynucleotide. Cultures were pretreated for 12 h with 10 or 50 μM bcl-2 antisense ODN (bcl-2 AS) or control missense ODN (MS). Cultures were then exposed to vehicle (saline) or 100 nM CHX for 16 h, followed by a 20-h exposure to vehicle (saline) or 10 μM (A) or 50 μM (B) glutamate (Glut) in the continued presence of ODN. Neuronal survival was quantified and values (mean and SEM; n = 3 cultures) are expressed as a percentage of the initial number of neurons. *P < 0.05; *P < 0.01.

Figure 9

CHX induces increases in antioxidant enzyme activities in cultured hippocampal cells. Cultures were exposed to the indicated concentrations of CHX for 24 h. Activity levels of catalase, Cu/Zn-SOD, and Mn-SOD in cell homogenates were then quantified. Values are expressed in units of enzyme activity per mg protein and represent the mean and SEM of determinations made in three separate cultures. *P < 0.01 compared with corresponding cultures not exposed to CHX.