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Astrocytes Surviving Severe Stress Can Still Protect Neighboring Neurons from Proteotoxic Injury

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Abstract

Astrocytes are one of the major cell types to combat cellular stress and protect neighboring neurons from injury. In order to fulfill this important role, astrocytes must sense and respond to toxic stimuli, perhaps including stimuli that are severely stressful and kill some of the astrocytes. The present study demonstrates that primary astrocytes that managed to survive severe proteotoxic stress were protected against subsequent challenges. These findings suggest that the phenomenon of preconditioning or tolerance can be extended from mild to severe stress for this cell type. Astrocytic stress adaptation lasted at least 96 h, the longest interval tested. Heat shock protein 70 (Hsp70) was raised in stressed astrocytes, but inhibition of neither Hsp70 nor Hsp32 activity abolished their resistance against a second proteotoxic challenge. Only inhibition of glutathione synthesis abolished astrocytic stress adaptation, consistent with our previous report. Primary neurons were plated upon previously stressed astrocytes, and the cocultures were then exposed to another proteotoxic challenge. Severely stressed astrocytes were still able to protect neighboring neurons against this injury, and the protection was unexpectedly independent of glutathione synthesis. Stressed astrocytes were even able to protect neurons after simultaneous application of proteasome and Hsp70 inhibitors, which otherwise elicited synergistic, severe loss of neurons when applied together. Astrocyte-induced neuroprotection against proteotoxicity was not elicited with astrocyte-conditioned media, suggesting that physical cell-to-cell contacts may be essential. These findings suggest that astrocytes may adapt to severe stress so that they can continue to protect neighboring cell types from profound injury.

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Acknowledgments

Wrote the paper: RKL and AMG. Designed the experiments: RKL. Conducted the experiments and analyzed the data: AMG, JMP, DP, MPH. Generated the figures: AMG. We are grateful to Mary Caruso, Deborah Willson, and Jackie Farrer for excellent administrative support and to Denise Butler-Bucilli and Christine Close for outstanding animal care. These studies were supported by a Hillman Foundation award (109033) and an R15 award from NIH (1R15NS093539) to RKL. The authors have no conflicts to disclose.

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  1. Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, 407 Mellon Hall, 600 Forbes Avenue, Pittsburgh, PA, 15282, USA

    Amanda M. Gleixner, Jessica M. Posimo, Deepti B. Pant, Matthew P. Henderson & Rehana K. Leak

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  1. Amanda M. Gleixner
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Supplementary Fig 1

Lactacystin-mediated severe proteotoxic stress preconditions astrocytes against subsequent insults. A) Five days after plating, astrocytes were treated with various concentrations of lactacystin or an equivalent v/v of vehicle (phosphate buffered saline) as the first hit. On the following day, the astrocytes were treated with vehicle or a second hit of lactacystin at the indicated concentrations. Viability was assayed 24 h after the second hit by counting Hoechst-stained nuclei. B) Representative images of Hoechst-stained nuclei for experiments shown in panel A. * p ≤ 0.05 vs 0 μM 2nd lactacystin hit; + p ≤ 0.05, ++ p ≤ 0.01, +++ p ≤ 0.001 vs 0 μM 1st lactacystin hit, two-way ANOVA followed by Bonferroni post hoc correction (GIF 109 kb)

High Resolution Image (TIFF 1989 kb)

Supplementary Fig 2

MG132 treatment is more lethal to small, bright nuclei than large nuclei. A) Hoechst-stained nuclei were categorized into two groups: those exhibiting small, brightly stained nuclei (50–150 μm2 in size and staining intensity greater than 20 arbitrary units for the mean gray value measurements) or large nuclei (greater than 150 μm2; all staining intensities). Raw counts of the numbers of nuclei per microscopic field of view (200× magnification) are shown here to supplement the transformed data in Figure 3P. * p ≤ 0.05, ** p ≤ 0.01 vs 0 μM 2nd MG132 hit; ++ p ≤ 0.01 vs vs 0 μM 1st MG132 hit; ^^ p ≤ 0.01, ^^^ p ≤ 0.001 vs small, bright nuclei, three-way ANOVA followed by Bonferroni post hoc correction (GIF 17 kb)

High Resolution Image (TIFF 298 kb)

Supplementary Fig 3

Stressed astrocytes can resist dual hits despite inhibition of heme oxygenase 1 (HO1, also known as Hsp32). A) Astrocytes were treated with the first and second MG132 hits or vehicle 24 h apart in the absence or presence of the HO1 inhibitor SnPPx. Viability was assayed 24 h after the second hit. B) Representative images of Hoechst stained nuclei. * p ≤ 0.05, *** p ≤ 0.001 vs 0 μM 2nd MG132 hit; + p ≤ 0.05, +++ p ≤ 0.001 vs 0 μM 1st MG132 hit, two-way ANOVA followed by Bonferroni post hoc correction (GIF 52 kb)

High Resolution Image (TIFF 1109 kb)

Supplementary Fig 4

Impact of first hit on glutathione-related enzymes. Astrocytes were treated with the first MG132 hit or vehicle and the following glutathione-related proteins were measured in lysates collected 24 h later: A) glutamate cysteine ligase catalytic subunit (GCLC), B) glutathione S-transferase π (GST-π). No significant difference was determined with the two-tailed paired Student’s t test. (GIF 23 kb)

High Resolution Image (TIFF 355 kb)

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Gleixner, A.M., Posimo, J.M., Pant, D.B. et al. Astrocytes Surviving Severe Stress Can Still Protect Neighboring Neurons from Proteotoxic Injury. Mol Neurobiol 53, 4939–4960 (2016). https://doi.org/10.1007/s12035-015-9427-4

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