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

Abstract

The dual hit hypothesis of neurodegeneration states that severe stress sensitizes vulnerable cells to subsequent challenges so that the two hits are synergistic in their toxic effects. Although the hippocampus is vulnerable to a number of neurodegenerative disorders, there are no models of synergistic cell death in hippocampal neurons in response to combined proteotoxic and oxidative stressors, the two major characteristics of these diseases. Therefore, we developed a relatively high-throughput dual hit model of stress synergy in primary hippocampal neurons. In order to increase the rigor of our study and strengthen our interpretations, we employed three independent, unbiased viability assays at multiple timepoints. Stress synergy was elicited when hippocampal neurons were treated with the proteasome inhibitor MG132 followed by exposure to the oxidative toxicant paraquat, but only after 48h. MG132 and paraquat only elicited additive effects 24h after the final hit and even loss of heat shock protein 70 activity and glutathione did not promote stress synergy at this early timepoint. Dual hits of MG132 elicited modest glutathione loss and slightly synergistic toxic effects 48h after the second hit, but only at some concentrations and only according to two viability assays (metabolic fitness and cytoskeletal integrity). The thiol N-acetyl cysteine protected hippocampal neurons against dual MG132/MG132 hits but not dual MG132/paraquat hits. Our findings support the view that proteotoxic and oxidative stress propel and propagate each other in hippocampal neurons, leading to synergistically toxic effects, but not as the default response and only after a delay. The neuronal stress synergy observed here lies in contrast to astrocytic responses to dual hits, because astrocytes that survive severe proteotoxic stress resist additional cell loss following second hits. In conclusion, we present a new model of hippocampal vulnerability in which to test therapies, because neuroprotective treatments that are effective against severe, synergistic stress are more likely to succeed in the clinic.

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

“…NAC has been shown to protect primary neurons and neuroblastoma cells against proteasome inhibition Jiang et al, 2013;Posimo et al, 2013;Heinemann et al, 2016). In contrast to our previous studies on neuroblastoma cells (Jiang et al, 2013), NAC did not preserve Hsp70 protein levels in primary astrocytes.…”

“…Total GSH levels were measured by In-Cell Western analyses and expressed as a fraction of Hoechst-stained nuclei in the same well to control for differences in cell density. This GSH In-Cell Western assay has been validated in previous studies, in which the signal for GSH was successfully decreased with buthionine sulfoximine and increased with NAC (Leak et al, 2008;Unnithan et al, 2012;Posimo et al, 2013;Titler et al, 2013;Gleixner et al, 2016;Heinemann et al, 2016). Reduced GSH levels were measured by the luminescent GSH-Glo assay according to the manufacturer's instructions (cat.…”

“…NAC Protects Astrocytes Independent of Glutathione Synthesis. Previous studies have shown that NAC can increase GSH levels in MG132-treated cells and primary neurons Cheng et al, 2016;Heinemann et al, 2016) as well as untreated astrocytes (Jurkowska and Wróbel, 2008). Therefore, we assessed GSH levels in MG132and NAC-treated astrocytes.…”

“…We recently showed that NAC protects neuroblastoma cells and primary cortical and hippocampal neurons against proteotoxic or oxidative stress, perhaps by raising the concentration of the antioxidant GSH (Unnithan et al, 2012Posimo et al, 2013;Heinemann et al, 2016). Indeed, most studies of NAC assume that it is effective because it provides a critical precursor, cysteine, for GSH biosynthesis (Martínez-Banaclocha, 2012;Pocernich and Butterfield, 2012).…”

Astrocytes Do Not Forfeit Their Neuroprotective Roles After Surviving Intense Oxidative Stress

“…Our previous report showed that neurons respond to dual hits of high concentrations of paraquat with additive cell loss (Heinemann et al, 2016), suggesting that neurons do not develop stress tolerance in response to severe oxidative toxicity. However, there are many other types of oxidative stimuli, including hydrogen peroxide.…”