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

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 hours, 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 co-cultures 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.

Synergistic stress exacerbation in hippocampal neurons: Evidence favoring the dual‐hit hypothesis of neurodegeneration

“…Considering our previous findings on stress resistance in astrocytes versus the stress synergy we reported in neuroblastoma cells (Unnithan et al, ; Titler et al, ; Unnithan et al, ; Gleixner et al, ), the present findings lend further support to the notion that cell type influences the direction of the stress response. Even more specifically, brain subregion and neuronal subtype may also determine whether the stress response takes a toxic turn.…”

“…The present findings support the view that hippocampal neurons are even more vulnerable to injury if loss of protein homeostasis is followed by loss of redox homeostasis and that NAC supplementation can protect hippocampal neurons against some but not all types of injuries. This phenomenon of stress exacerbation in neurons lies in contrast to the stress tolerance exhibited by primary astrocytes when exposed to dual proteotoxic hits, supporting the view that neurons are more vulnerable to stress exacerbation (Titler et al, ; Gleixner et al, ). The new model presented here also serves as a robust method to test potential therapies, because treatments that can protect against unremitting, severe stress are more likely to succeed in the clinic, where exposures to stress in real life almost never occur in isolation or as singular events.…”

“…Our previous work showed that glutathione loss exacerbates the response to dual MG132 hits in primary cortical astrocytes (Titler et al, ; Gleixner et al, ) and that dual MG132 hits elicit synergistic loss of glutathione in neuroblastoma cells (Unnithan et al, ). As we observed no evidence of stress synergy thus far, we challenged the cells further by inhibiting glutathione synthesis with buthionine sulfoximine.…”

“…In contrast to synergistic stress exacerbation, if two hits are only additive in their toxic effects, then the first hit is not hypothesized to have any impact on the response to the second hit (Leak, ). Alternatively, if the first hit blocks the toxic response to the second exposure, the cells have developed tolerance and are preconditioned against further challenges, as reported in primary astrocytes and dopaminergic cells (Leak et al, ; Titler et al, ; Gleixner et al, ). In the primary astrocyte studies, the cells that managed to survive an initial proteotoxic hit tolerated subsequent insults better than stress‐naïve astrocytes and continued to protect neighboring primary neurons in neuron/glia co‐cultures, suggesting that some cells can be preconditioned even by high‐dose toxic stimuli and continue to fulfill their evolutionary roles.…”

N-Acetyl-l-Cysteine Protects Astrocytes against Proteotoxicity without Recourse to Glutathione

“…All images were collected from the 3 and 9 o'clock positions in each well. Cell viability was then determined by blinded counts of Hoechst 1 cells using the Image J software (NIH Image, Bethesda, MD), as described previously (Gleixner et al, 2016).…”