p66^shc's role as an essential mitophaghic molecule in controlling neuronal redox and energetic tone

Abstract

Stroke is the leading cause of adult disability in the U.S. and is now recognized as a global epidemic. There are currently no FDA-approved drugs to block the cell death that results from oxygen and glucose deprivation. This void in clinical medicine has sparked an intense interest in understanding endogenous cellular protective pathways that might be exploited for therapeutic development. The work highlighted here describes the critical role between redox tone and energetic stress signaling in mediating mitophagy and determining neuronal cell fate following acute oxygen glucose deprivation.

Preconditioning (PC) is a phenomenon in which a sublethal insult induces neuroprotection against subsequent injury. This type of protection is highly conserved across species and is effective in eliciting defensive responses in several tissues including heart and brain. We previously reported that activation of the chaperone and neuroprotective protein, heat shock protein 70 (HSP70), is an essential mediator of cell fate in our model of neuronal PC. Loss of HSP70 or its transcription factor, heat shock factor 1, is associated with increased vulnerability to a host of stressors including hypoxia and ischemia. The goal of our recently published work in Journal of Neuroscience was to determine the early events evoked by mitochondrial stress that trigger HSP70 activation and protection.

Neuronal cultures were preconditioned via exposure to an inhibitor of oxidative phosphorylation for 90 min in glucose-free media. The following day, cells were treated with the glutamate receptor agonist NMDA. Not only is PC nontoxic, but preconditioned cells exhibit 50% less death compared to naïve cells following a potentially lethal insult. We initially assessed the role of mitogen and redox-activated protein kinases in mediating PC protection as these proteins are poised to integrate changes in cellular redox status, mitochondrial calcium buffering and ionic dysfunction. We observed that both the redox-sensitive kinase p66^shc and the mitogen-activated protein kinase (MAPK) target Raf are rapidly and intensely activated in response to PC. Raf1 is phosphorylated within minutes of PC, decreases within an hour and increases again at 24 h. Similarly, p66^shc phosphorylation increases early, but unlike Raf remains elevated for 4 h, only returning to baseline 24 h after PC.

In order to determine if these kinases were essential to the neuroprotective pathways elicited by preconditioning, p66^shc or Raf inhibitors were added during incubation in PC media. The following day NMDA was applied, then viability was assessed 24 h later. Inhibition of either kinase results in a significant increase in neuronal cell death compared to naïve cells, suggesting that activation of these kinases can be neuroprotective, an observation supported by the fact that inhibition of these kinases prevents HSP70 upregulation. These effects are not additive, suggesting that both kinases are required if PC is to be protective, albeit at different times and in response to different signals.

We were surprised to find that although preconditioning is nontoxic, it does involve far more intense oxidative stress than we had anticipated. Total oxidative stress in both neurons and glia was assessed via F₂-isoprostane (IsoPs) formation, while neuron-specific oxidative stress was measured by F₄-neuroprostane (NeuroPs) formation. Whereas preconditioning causes no death, it does evoke profound increases in total lipid injury and neuron-specific lipid peroxidation. In spite of this stress, the energetic status of preconditioned cells remains remarkably well preserved with only a 20% decrease in total ATP 3 h after PC onset, which rebounded to control levels by the time of secondary stress.

The implication that p66^shc activation is associated with increased survival is counter to the prevailing literature. Activation of p66^shc by redox stress has been associated with shortened life span and mitochondrial dysfunction. So how does activated p66^shc provide neuroprotection against secondary stress? In tracking p66^shc following preconditioning, we find that p66^shc relocates to the nucleus and mitochondria. p66^shc also plays an essential role in determining both energetic and redox tone; decreasing ATP stores immediately after PC and dampening cumulative reactive oxygen species production. These data helped us develop a model in which p66^shc acts as an essential gatekeeping molecule rapidly sensing changes in oxidative stress and redistributing to evoke alterations in mitochondrial function and transcription of proteins including HSP70 (Fig. 1).

Figure 1. The role of mitogen and redox-activated kinases in mediating mitophagy in response to PC. Inhibition of oxidative phosphorylation combined with glucose deprivation leads to a block of the electron transport chain (ETC), decreased ATP and significantly increased reactive oxygen species (ROS). This stress results in the relocalization of p66^shc to the mitochondria where it works in concert with chaperones, stress-activated kinases and channel proteins to dampen ROS. Additional upstream kinases including Raf increase expression of HSP70 and other neuroprotective genes which include other small molecule chaperones and hypoxia inducible factor genes. The sequestration of damaged mitochondria and mild metabolic dysfunction induced by altering aerobic respiration at the level of the Kreb’s cycle and electron transport chain ensure secondary insults are less devastating to cell survival.

In prior work, we established that pro-apoptotic events, such as caspase-3 activation, were essential to elicit the neuroprotective pathway evoked by PC. Indeed, temporally and spatially constrained caspase-3 activation depletes constitutively expressed chaperones, triggering subsequent inducible HSP70 upregulation at the time of secondary stress. In the current study, we observe extensive LC3 cleavage and signs of mitophagy in preconditioned cells. By inhibiting p66^shc, LC3 cleavage is significantly enhanced, suggesting that without p66^shc, oxidative and mitochondrial stress can no longer be harnessed into adaptive and pro-survival measures. Without p66^shc to dampen the signaling and upregulate HSP70, oxidative and energetic dysfunction go unchecked, priming cells to die upon subsequent injury.

These data present the first evidence of the relationship between extensive neuronal oxidative stress, mitophagy signaling and p66^shc in evoking preconditioning protection, and offer new and exciting avenues to be investigated. We have long appreciated that aerobic failure leads to compromised mitochondrial membrane potential, morphological changes and molecular events that can include combinations of the following: Cytochrome c release, caspase activation, aberrant mitochondrial fission and fusion reactions and mitophagy. We see our data as a part of a growing body of literature in which we can begin to think of mitophaghic signaling as not just a series of protein release events that trigger organelle containment and cellular adaptation. Rather, during mitophagy, p66^shc along with chaperone proteins and their associated protein complex machinery can actively be recruited to stressed mitochondria. These molecules, which include chaperone proteins, E3 ligases and kinases, combine to determine the fate of mitochondria and in turn the entire cell.

Brown JE, Zeiger SLH, Hettinger JC, Brooks JD, Holt B, Morrow JD, et al. Essential role of the redox-sensitive kinase p66shc in determining energetic and oxidative status and cell fate in neuronal preconditioning. J Neurosci. 2010;30:5242–52. doi: 10.1523/JNEUROSCI.6366-09.2010.