Essential role of the redox-sensitive kinase p66shc in determining energetic and oxidative status and cell fate in neuronal preconditioning

Abstract

Ischemic preconditioning is a phenomenon in which low-level stressful stimuli upregulate endogenous defensive programs, resulting in subsequent resistance to otherwise lethal injuries. We previously observed that signal transduction systems typically associated with neurodegeneration such as caspase activation are requisite events for the expression of tolerance and induction of HSP70. In this work, we sought to determine the extent and duration of oxidative and energetic dysfunction as well as the role of effector kinases on metabolic function in preconditioned cells. Using an in vitro neuronal culture model, we observed a robust increase in Raf and p66(Shc) activation within 1 h of preconditioning. Total ATP content decreased by 25% 3 h after preconditioning but returned to baseline by 24 h. Use of a free radical spin trap or p66(shc) inhibitor increased ATP content whereas a Raf inhibitor had no effect. Phosphorylated p66(shc) rapidly relocalized to the mitochondria and in the absence of activated p66(shc), autophagic processing increased. The constitutively expressed chaperone HSC70 relocalized to autophagosomes. Preconditioned cells experience significant total oxidative stress measured by F(2)-isoprostanes and neuronal stress evaluated by F(4)-neuroprostane measurement. Neuroprostane levels were enhanced in the presence of Shc inhibitors. Finally, we found that inhibiting either p66(shc) or Raf blocked neuroprotection afforded by preconditioning as well as upregulation of HSP70, suggesting both kinases are critical for preconditioning but function in fundamentally different ways. This is the first work to demonstrate the essential role of p66(shc) in mediating requisite mitochondrial and energetic compensation after preconditioning and suggests a mechanism by which protein and organelle damage mediated by ROS can increase HSP70.

Figure 1.

Mild mitochondrial stress protects primary neuronal cultures against excitotoxicity. Cortical cultures were chemically preconditioned for 90 min before exposure to an otherwise lethal dose of NMDA. A–F, Representative photos and LDH measurement of viability were taken 24 h after preconditioning alone (A–C) or 24 h after NMDA exposure (D–F). A, Naive cultures were washed but not exposed to preconditioning or 100 μm NMDA and exhibit phase bright neuronal somas with little or no cell death. B, Cells that were preconditioned with 3 mm KCN and oxygen glucose deprivation but not exposed to NMDA are also intact and healthy, demonstrating that preconditioning is not toxic to cells. C, Lack of cell death induced by preconditioning is demonstrated by LDH release in naive versus preconditioning alone. D, Cells, which were not preconditioned but were exposed to 100 μm NMDA for 1 h experienced near total neuronal death after 24 h. E, After 24 h, many of the preconditioned cells remained phase bright and morphologically intact after 1 h exposure to an otherwise lethal NMDA dose. F, LDH data comparing the effects of NMDA toxicity in naive and preconditioned cells demonstrate that prior preconditioning significantly decreases cell death. Pooled data are expressed as the ratio of the LDH released in NMDA-treated cultures to that released in glycine-only-treated sister cultures. LDH data are pooled from >12 experiments and were analyzed using a two-way ANOVA. *p < 0.01.

Figure 2.

p66^Shc and Raf are rapidly and independently activated in preconditioned cells. Whole-cell extracts of neuronal cultures were harvested at various time points after initiation of preconditioning. Proteins were separated on SDS-PAGE gels and probed with antibodies specific to the phosphorylated forms of p42/44 ERK, p38, S338 Raf, and p66^Shc or HSC70 as a loading control. A, Lysates from 5 min to 24 h after preconditioning reveal a rapid as well as a delayed increase in Raf activation and modest increases in ERK and p38. B, Phosphorylation of p66^Shc S36 was increased within 1 h of initiation of preconditioning and remained high for several hours. C, Cells harvested 3 h after the initiation of preconditioning revealed no effect of Raf inhibition on phospho-p66^Shc activation, whereas the free radical spin trap N-tert-butyl-α-phenylnitrone (PBN) decreased p66^Shc phosphorylation. D, Raf phosphorylation was evaluated at 30 min (first 4 lanes) or 24 h (last lane). Quantification of these data is presented in supplemental Figure 1 (available at www.jneurosci.org as supplemental material). Similar results were obtained in three to five additional independent experiments.

Figure 3.

Acute impairment of energetic status of preconditioned cultures is dependent on redox stress and Shc activation. A, Primary cultures were exposed to preconditioning and harvested 3 or 24 h later, and total ATP content was measured. Data represent the raw luminescent units ± SEM. B, To test the efficacy of various agents on ATP content at the 3 h time point, the K_ATP channel blocker glibenclamide (1 μm), the free radical spin trap (PBN; 500 μm), the Raf kinase inhibitor 1 (Raf_l 5 nm), or the Shc inhibitor PP2 (Shci 50 nm) were added as described in Materials and Methods. Preconditioning alone decreased total ATP by 20%, an effect that was blocked only by the free radical spin trap or Shc inhibitor. Data represent the mean ± SEM of five to eight independent experiments performed in duplicate. Asterisks are used to demonstrate a significant effect compared with naive cells. Statistical significance between groups compared with preconditioning is denoted with a bracket and Δ. Data were analyzed using Tukey test after two-way ANOVA by comparing groups to PC alone. Both asterisks and Δ represent p < 0.05.

Figure 4.

Phosphorylated Shc is rapidly activated and relocalizes after preconditioning. Primary cultures were incubated with the potentiometric dye Mitotracker (red) and then fixed 3–4 h after the initiation of preconditioning for immunofluorescent detection of phosphorylated p66^Shc. Mitochondria (red) are well dispersed in both naive and preconditioned neurons (green, MAP2 staining). Processes are intact in both conditions and mitochondria are present in both soma and processes. Phosphorylated p66^Shc (blue) is markedly increased after preconditioning with staining evident in both the nucleus and adjacent to mitochondria.

Figure 5.

Shc activation regulates the extent of oxidative stress in preconditioning. Formation of F₂-Isoprostanes (F₂-IsoP) and F₄-Neuroprostanes (NeuroP) is dependent upon lipid environment and initiated by oxidative stress. A, The pathway by which each class of compounds is formed is outlined. B, Primary cultures were exposed to preconditioning (PC) and harvested 24 h later to evaluate total F₂-IsoP content to test the effects of the Shc inhibitor PP2 (50 nm) on preconditioning-induced oxidative stress. Control cultures incubated in 100 μm NMDA for 1 h, which causes 100% neuronal death, were used as an index of oxidative injury. Data represent the mean ± SEM for three to six independent experiments and were analyzed using one-way ANOVA. *p < 0.05 versus naive. C, Neuroprostane levels were measured at 24 h using incubation conditions outlined in the prior panel. Data represent the mean ± SEM of five to eight independent experiments performed in duplicate. Asterisks are used to demonstrate a significant effect compared with naive cells; statistical analysis between groups is denoted by Δ where post hoc analysis of data using Tukey test was performed comparing groups to PC alone. Both asterisks and Δ represent p < 0.05.

Figure 6.

Preconditioning increases neuronal autophagic signaling. Cultures were fixed 24 h after the initiation of preconditioning and stained with the nuclear dye DAPI (blue), the neuronal cytoskeletal marker MAP2 (green), and LC3 (red), a hallmark of autophagic activation. A, Control cultures had well defined processes, and light dispersed LC3 activation. B, Autophagosome punctae containing LC3 increased in response to preconditioning (bottom left, in red). C, Exposure to the Shc_i PP2 (50 nm) exacerbated this effect. D, Cell counts by two independent investigators of >400 neurons from three independent experiments revealed a 20% increase in intensely labeled LC3II-positive cells in preconditioned cells, and a further increase of 15% when Shc_i was present.

Figure 7.

Raf activation is required for preconditioning protection and upregulation of HSP70. Inhibitors of Raf, p42/44 ERK, or p38 were added during preconditioning as outlined in Materials and Methods. Twenty-four hours after preconditioning, cultures were washed and exposed to 100 μm NMDA with 10 μm glycine for 1 h. A, Cell death was assessed 24 h later by LDH release from dead and dying neurons. Data represent the mean ± SEM for five independent experiments and were analyzed using one-way ANOVA. Post hoc analysis was done using Tukey test comparing groups to PC alone. Δ is used to denote statistical significance compared with preconditioning alone. Both asterisks and Δ represent p < 0.05. B, The upper blot demonstrates that HSP70 expression is increased at 24 h and that Raf inhibition blocks HSP70 upregulation. HSC70 is constitutively expressed and is used as a loading control.

Figure 8.

Shc activation is required for preconditioning protection and upregulation of HSP70. The Shc inhibitor PP2 was added during preconditioning as outlined in Materials and Methods. Twenty-four hours after preconditioning, cultures were washed and exposed to 100 μm NMDA with 10 μm glycine for 1 h. A, Cell death was assessed 24 h later by LDH release from dead and dying neurons. B, A redox-insensitive dominant-negative form of p66shc (S36A) was also used to verify that loss of function of this kinase blocked neuroprotection. Cells were transfected with vectors, which included a firefly luciferase reported 24 h prior to preconditioning and then were treated with preconditioning stress or washed. The following day, NMDA exposure was performed, and 24 h later cell survival was assessed by measuring luciferase signal from surviving cells. Data from A represent the mean ± SEM for five independent experiments and from B represent three experiments performed in duplicate. All averages were analyzed using one-way ANOVA. Post hoc analysis was done using Tukey test comparing groups to PC alone. Δ is used to denote statistical significance compared with preconditioning alone. Both asterisks and Δ represent p < 0.05. C, The upper blot demonstrates that HSP70 expression is increased at 24 h and that Shc inhibition blocks HSP70 upregulation. HSC70 is constitutively expressed and is used as a loading control.

Figure 9.

Model of Raf and p66^Shc contribution to oxidative and energetic dysfunction in preconditioning. Preconditioning oxygen glucose deprivation induces an early but modest decrease in total ATP content with a larger increase in total oxidative stress assessed by formation of F₂-IsoPs and F₄-NeuroPs. Raf and p66^Shc kinases are rapidly activated, and p66^Shc relocates to subcellular organelles including the mitochondria. In the absence of p66^Shc, cellular ATP, oxidative and protein stress persist, resulting in increased formation of F₄-NeuroPs and formation of autophagosomes. We hypothesize that the limited autophagy experienced by preconditioning functions to sequester damaged organelles and proteins via HSC70 and other pathways which ultimately enhance the induction of HSP70 and other protective proteins upregulated at the time of secondary stressors.