GSK-3 mediates the okadaic acid-induced modification of collapsin response mediator protein-2 in human SK-N-SH neuroblastoma cells

Collapsin response mediator protein-2 (CRMP-2), a phosphoprotein involved in axonal outgrowth and microtubule dynamics, is aberrantly phosphorylated in Alzheimer's disease (AD) brain. Alteration of glycogen synthase kinase-3 (GSK-3) activity is associated with the pathogenesis of AD. Here, we show that CRMP-2 is one of the major substrates for GSK-3 in pig brain extracts. Both GSK-3alpha and 3beta phosphorylate purified pig brain CRMP-2 and significantly alter its mobility in SDS-gels, resembling the CRMP-2 modification observed in AD brain. Interestingly, this modification can be detected in SK-N-SH neuroblastoma cells treated with a phosphatase inhibitor, okadaic acid (OA), and GSK-3 inhibitors completely block this OA-induced event. Knockdown of both GSK-3alpha and 3beta, but not either kinase alone, impairs OA-induced modification of CRMP-2. Mutation of Ser-518 or Ser-522 of CRMP-2, which are highly phosphorylated in AD brain, to Ala blocks the OA-induced modification of CRMP-2 in SK-N-SH cells. Ser-522 prephosphorylated by Cdk5 is required for subsequent GSK-3alpha-mediated phosphorylation of CRMP-2 in vitro. Collectively, our results demonstrate for the first time that OA can induce phosphorylation of CRMP-2 in SK-N-SH cells at sites aberrantly phosphorylated in AD brain, and both GSK-3alpha and 3beta and Ser-522 kinase(s) are involved in this process.     

Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer's disease progression

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5-mediated) is increased in Alzheimer's disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin-1 (PS1)(M146V)KI; Thy1.2-amyloid precursor protein (APP)(swe); Thy1.2tau(P301L)] that develops AD-like plaques and tangles, as well as the double (PS1(M146V)KI; Thy1.2-APP(swe)) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid beta peptide levels (Tg2576 and APP/amyloid beta-binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over-expression and/or processing defect.     

Deregulated Cdk5 promotes oxidative stress and mitochondrial dysfunction

Oxidative stress is one of the earliest events in Alzheimer's disease (AD). A chemical genetic screen revealed that deregulated cyclin-dependent kinase 5 (Cdk5) may cause oxidative stress by compromising the cellular anti-oxidant defense system. Using novel Cdk5 modulators, we show the mechanism by which Cdk5 can induce oxidative stress in the disease's early stage and cell death in the late stage. Cdk5 dysregulation upon neurotoxic insults results in reactive oxygen species (ROS) accumulation in neuronal cells because of the inactivation of peroxiredoxin I and II. Sole temporal activation of Cdk5 also increases ROS, suggesting its major role in this process. Cdk5 inhibition rescues mitochondrial damage upon neurotoxic insults, thereby revealing Cdk5 as an upstream regulator of mitochondrial dysfunction. As mitochondrial damage results in elevated ROS and Ca(2+) levels, both of which activate Cdk5, we propose that a feedback loop occurs in late stage of AD and leads to cell death (active Cdk5 --> ROS --> excess ROS --> mitochondrial damage --> ROS --> hyperactive Cdk5 --> severe oxidative stress and cell injury --> cell death). Cdk5 inhibition upon neurotoxic insult prevents cell death significantly, supporting this hypothesis. As oxidative stress and mitochondrial dysfunction play pivotal roles in promoting neurodegeneration, Cdk5 could be a viable therapeutic target for AD.     

Telomerase activity in response to mild oxidative stress

We have analysed telomerase activity to determine whether it can be modified when BCL-2 is endogenously overexpressed in response to a mild oxidative stress treatment as part of a survival mechanism, in contrast with an exogenous bcl-2 overexpression due to a retroviral infection. Endogenous bcl-2 overexpression was induced after a low oxidative insult of H2O2 in mice primary lung fibroblasts and L929 cell, whereas bcl-2 exogenous overexpression was performed using a retroviral infection in L929 cells. Telomerase activity was quantified in Bcl-2 overexpressing cells by the TRAP assay. When the cells were treated with different H2O2 concentrations, only those exposed to 50 μM showed increased telomerase activity. This correlates with BCL-2 expression as part of the endogenous response to mild oxidative stress. Oxidative stress generated during the toxic mechanism of chemotherapeutic drugs might induce BCL-2 increment, enhancing telomerase activity and reactivating the oncogenic process. Clinical trials should take into consideration the possibility of telomerase activation following increased BCL-2 expression when treating patients with ROS (reactive oxygen species) generation by anti-cancer drugs.     

Proteomic identification of a novel isoform of collapsin response mediator protein-2 in spinal nerves peripheral to dorsal root ganglia

Primary afferent fibers are originated from pseudounipolar sensory cells in dorsal root ganglia (DRG) and transmit external stimuli received in the skin to the spinal cord. Here we undertook a proteomic approach to uncover the polarity of primary afferent fibers. Lumbar spinal nerve segments, peripheral and central to DRG, were dissected from 5-wk-old Wistar rats and the lysates were subjected to large-sized 2-DE at pH 5-6. Among approximately 800 protein spots detected in the central and peripheral fractions, one of the unique spots in the peripheral fraction with MW of 60 kDa and pI of 5.6 was identified as an isoform of collapsin response mediator protein-2 (CRMP-2) by MALDI-TOF MS and Western blots with anti-CRMP-2 antibodies that recognize 1-17 and 486-528 residues. Since this novel spot was detected only in the peripheral fraction, but not in the central fraction, DRG, and other regions of the brain, it was named periCRMP-2. The C-terminal fragment of CRMP-2 was not detected in periCRMP-2 by MS analyses. Expression of periCRMP-2 decreased following sciatic nerve injury. These results suggest that periCRMP-2 is a C-terminal truncated isoform polarized in the peripheral side of spinal nerves and may be involved in nerve degeneration and regeneration.     

Synaptic cell adhesion molecule-2 and collapsin response mediator protein-2 are novel members of the matrix metalloproteinase-9 degradome

The elucidation of entire sets of protease substrates ("proteodegradomes") is important for understanding proteolytic pathways, their networks, and their role in the regulation of cell function. Matrix metalloproteinase-9 (MMP-9) is an extracellularly operating protease that is expressed and released in the brain in response to enhanced neuronal activity. Under physiological conditions, MMP-9 is involved in neuronal plasticity, including long-term potentiation, learning, and memory. This function may be related to its activity at the synapse. Under pathological conditions (e.g., during excitotoxicity, stroke, and traumatic brain injury), when the concentration of glutamate is persistently increased, MMP-9 is detrimental to brain tissue. To assess the MMP-9 degradome, we used synaptoneurosomal fractions isolated from the hippocampus of wildtype and MMP-9 knockout mice. To induce MMP-9 activity, the synaptoneurosomal fractions were treated with 50 μM glutamate for 30 min at 37°C. To investigate MMP-9 targets, two-dimensional fluorescence difference gel electrophoresis was performed. This approach enabled the accurate analysis of differences in protein abundance between samples. The differential spots that contained potential MMP-9 substrates were excised from the gel, and proteins of interest were identified using mass spectrometry. Two novel MMP-9 targets were identified: synaptic cell adhesion molecule-2 and collapsin response mediator protein-2. The MMP-9-driven processing of the newly identified substrates was confirmed by western blot in primary hippocampal neurons after stimulation with either N-methyl-D-aspartate or glutamate or incubation with recombinant autoactivating MMP-9 and use of a specific inhibitor.     

Loss of mitofusin 2 links beta‐amyloid‐mediated mitochondrial fragmentation and Cdk5‐induced oxidative stress in neuron cells

Mitochondrial dysfunction is implicated in age‐related degenerative disorders such as Alzheimer's disease (AD). Maintenance of mitochondrial dynamics is essential for regulating mitochondrial function. Aβ oligomers (AβOs), the typical cause of AD, lead to mitochondrial dysfunction and neuronal loss. AβOs have been shown to induce mitochondrial fragmentation, and their inhibition suppresses mitochondrial dysfunction and neuronal cell death. Oxidative stress is one of the earliest hallmarks of AD. Cyclin‐dependent kinase 5 (Cdk5) may cause oxidative stress by disrupting the antioxidant system, including Prx2. Cdk5 is also regarded as a modulator of mitochondrial fission; however, a precise mechanistic link between Cdk5 and mitochondrial dynamics is lacking. We estimated mitochondrial morphology and alterations in mitochondrial morphology‐related proteins in Neuro‐2a (N2a) cells stably expressing the Swedish mutation of amyloid precursor protein (APP), which is known to increase AβO production. We demonstrated that mitochondrial fragmentation by AβOs accompanies reduced mitofusin 1 and 2 (Mfn1/2) levels. Interestingly, the Cdk5 pathway, including phosphorylation of the Prx2‐related oxidative stress, has been shown to regulate Mfn1 and Mfn2 levels. Furthermore, Mfn2, but not Mfn1, over‐expression significantly inhibits the AβO‐mediated cell death pathway. Therefore, these results indicate that AβO‐mediated oxidative stress triggers mitochondrial fragmentation via decreased Mfn2 expression by activating Cdk5‐induced Prx2 phosphorylation.

     

Mitochondrial oxidative stress index,activity of redox-sensitive aconitase and effects of endogenous anti- and pro-oxidants on its activity in control,Alzheimer's disease and Swedish Familial Alzheimer's disease brain

Efficient function of the mitochondrial respiratory chain and the citric acid cycle (CAC) enzymes is required for the maintenance of human brain function. A conception of oxidative stress (OxS) was recently advanced as a disruption of redox signalling and control. Mitochondrial OxS (MOxS) is implicated in the development of Alzheimer's disease (AD). Thus, both pro- and anti-oxidants of the human body and MOxS target primarily the redox-regulated CAC enzymes, like mitochondrial aconitase (MAc). We investigated the specific activity of the MAc and MOxS index (MOSI) in an age-matched control (Co), AD and Swedish Familial AD (SFAD) post-mortem autopsies collected from frontal cortex (FC) and occipital primary cortex (OC) regions of the brain. We also examined whether the mitochondrial neuroprotective signalling molecules glutathione, melatonin and 17-β-estradiol (17βE) and mitochondrially active pro-oxidant neurotoxic amyloid-β peptide can modulate the activity of the MAc isolated from FC and OC regions similarly or differently in the case of Co, AD and SFAD. The activity of redox-sensitive MAc may directly depend on the mitochondrial oxidant/antioxidant balance in age-matched Co, AD and SFAD brain regions.     

N-acetyl-L-cysteine protects SHSY5Y neuroblastoma cells from oxidative stress and cell cytotoxicity: effects on beta-amyloid secretion and tau phosphorylation

Redox changes within neurones are increasingly being implicated as an important causative agent in brain ageing and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). Cells have developed a number of defensive mechanisms to maintain intracellular redox homeostasis, including the glutathione (GSH) system and antioxidant enzymes. Here we examine the effects of N-acetyl-L-cysteine (NAC) on beta-amyloid (A beta) secretion and tau phosphorylation in SHSY5Y neuroblastoma cells after exposure to oxidative stress inducing/cytotoxic compounds (H(2)O(2), UV light and toxic A beta peptides). A beta and tau protein are hallmark molecules in the pathology of AD while the stress factors are implicated in the aetiology of AD. The results show that H(2)O(2), UV light, A beta 1-42 and toxic A beta 25-35, but not the inactive A beta 35-25, produce a significant induction of oxidative stress and cell cytotoxicity. The effects are reversed when cells are pre-treated with 30 mM NAC. Cells exposed to H(2)O(2), UV light and A beta 25-35, but not A beta 35-25, secrete significantly higher amounts of A beta 1-40 and A beta 1-42 into the culture medium. NAC pre-treatment increased the release of A beta 1-40 compared with controls and potentiated the release of both A beta 1-40 and A beta 1-42 in A beta 25-35-treated cells. Tau phosphorylation was markedly reduced by H(2)O(2) and UV light but increased by A beta 25-35. NAC strongly lowered phospho-tau levels in the presence or absence of stress treatment.     

Amyloid-beta peptide induces oligodendrocyte death by activating the neutral sphingomyelinase-ceramide pathway

Amyloid-beta peptide (Abeta) accumulation in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neuronal degeneration. We have recently demonstrated that Abeta induced oligodendrocyte (OLG) apoptosis, suggesting a role in white matter pathology in AD. Here, we explore the molecular mechanisms involved in Abeta-induced OLG death, examining the potential role of ceramide, a known apoptogenic mediator. Both Abeta and ceramide induced OLG death. In addition, Abeta activated neutral sphingomyelinase (nSMase), but not acidic sphingomyelinase, resulting in increased ceramide generation. Blocking ceramide degradation with N-oleoyl-ethanolamine exacerbated Abeta cytotoxicity; and addition of bacterial sphingomyelinase (mimicking cellular nSMase activity) induced OLG death. Furthermore, nSMase inhibition by 3-O-methyl-sphingomyelin or by gene knockdown using antisense oligonucleotides attenuated Abeta-induced OLG death. Glutathione (GSH) precursors inhibited Abeta activation of nSMase and prevented OLG death, whereas GSH depletors increased nSMase activity and Abeta-induced death. These results suggest that Abeta induces OLG death by activating the nSMase-ceramide cascade via an oxidative mechanism.     

Oxidative stress increases levels of endogenous amyloid-beta peptides secreted from primary chick brain neurons

Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.     

Evidence for a role of Collapsin response mediator protein-2 in signaling pathways that regulate the proliferation of non-neuronal cells

Collapsin response mediator protein-2 or Crmp-2 plays a critical role in the establishment of neuronal polarity. In this study, we present evidence that apart from its functions in neurodevelopment, Crmp-2 is also involved in pathways that regulate the proliferation of non-neuronal cells through its phosphorylation by regulatory proteins. We show that Crmp-2 undergoes dynamic phosphorylation changes in response to contact inhibition-induced quiescence and that hyperphosphorylation of Crmp-2 occurs in a tumor. We further suggest that de-regulation of Crmp-2 phosphorylation levels at certain amino acid residues may lead to aberrant cell proliferation and consequently, tumorigenesis.     

Identification of collapsin response mediator protein-2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes

The cancer cell secretome may contain many potentially useful biomarkers. We therefore sought to identify proteins in the conditioned media of colorectal carcinoma (CRC) cell lines but not in those from other cancer cell lines. The secretomes of 21 cancer cell lines derived from 12 cancer types were analyzed by SDS-PAGE combined with MALDI-TOF MS. Among the 325 proteins identified, collapsin response mediator protein-2 (CRMP-2) was chosen for evaluation as a potential CRC biomarker, since it was selectively detected in the CRC cell line secretome and has never been reported as a cancer biomarker. Immunohistochemical analysis of 169 CRC specimens showed that CRMP-2 was positively detected in 58.6% of the tumors, but weakly or not detected in >90% of the adjacent nontumor epithelial cells. Moreover, the CRMP-2-positive rate was significantly increased in earlier stage tumors and lymph node metastasis. Plasma CRMP-2 levels were significantly higher in CRC patients (N = 201) versus healthy controls (N = 201) (61.3 +/- 34.6 vs. 40.2 +/- 24.3 ng/mL, p = 0.001). Our results indicate that comparative analysis of cancer cell secretome is a feasible strategy for identifying potential cancer biomarkers, and that CRMP-2 may be a novel CRC biomarker.     

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimer's disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta-peptide (Abeta). This 42-mer peptide is derived from the beta-amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS-1 and APP genes, which increase production of the highly amyloidogenic amyloid beta-peptide (Abeta42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock-in mice expressing mutant human PS-1 and APP were compared with those from wild-type mice, in the presence or absence of various oxidizing agents, viz, Abeta(1-42), H2O2 and kainic acid (KA). APP/PS-1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3-nitrotyrosine when compared with the wild-type neurons (p < 0.0005). Elevated levels of human APP, PS-1 and Abeta(1-42) were found in APP/PS-1 cultures compared with wild-type neurons. APP/PS-1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.     

Towards Alzheimer's root cause: ECSIT as an integrating hub between oxidative stress, inflammation and mitochondrial dysfunction. Hypothetical role of the adapter protein ECSIT in familial and sporadic Alzheimer's disease pathogenesis

Here we postulate that the adapter protein evolutionarily conserved signalling intermediate in Toll pathway (ECSIT) might act as a molecular sensor in the pathogenesis of Alzheimer's disease (AD). Based on the analysis of our AD-associated protein interaction network, ECSIT emerges as an integrating signalling hub that ascertains cell homeostasis by the specific activation of protective molecular mechanisms in response to signals of amyloid-beta or oxidative damage. This converges into a complex cascade of patho-physiological processes. A failure to repair would generate severe mitochondrial damage and ultimately activate pro-apoptotic mechanisms, promoting synaptic dysfunction and neuronal death. Further support for our hypothesis is provided by increasing evidence of mitochondrial dysfunction in the disease etiology. Our model integrates seemingly controversial hypotheses for familial and sporadic forms of AD and envisions ECSIT as a biomarker to guide future therapies to halt or prevent AD.     

TpMRK regulates cell division of Tetrahymena in response to oxidative stress

TpMRK was identified as a stress‐responsive mitogen activated protein kinase (MAPK)‐related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade. Copyright © 2009 John Wiley & Sons, Ltd.     

Peripheral biomarkers of oxidative stress and their limited potential in evaluation of clinical features of Huntington's patients

Abstract

Context: Peripheral oxidative biomarkers could be useful for monitoring clinical features of Huntington's disease (HD).

Materials and methods: Cu/Zn-superoxide dismutase (Cu/Zn-SOD), neuron-specific enolase (NSE) and 8-hydroxy-2′-deoxyguanosine (8-oxoGua) serum levels were analysed in 18 HD patients and 10 controls. Clinical measures were recorded from each HD patients.

Results: Cu/Zn-SOD, NSE and 8-oxoGua values were higher in HD patients than in controls. Cu/Zn-SOD and NSE correlated positively. No correlation was observed between the biomarkers analysed and the clinical measures assessed.

Discussion and conclusion: Serum oxidative biomarkers could express the neuronal oxidative processes going on in HD patients but are inadequate to evaluate clinical features of the disease.     

Tolerance to oxidative stress is required for maximal xylem colonization by the xylem‐limited bacterial phytopathogen,Xylella fastidiosa

Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem‐limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H₂O₂) relative to the wild‐type. In addition, during early stages of grapevine infection, the survival rate was 1000‐fold lower for the oxyR mutant than for the wild‐type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell–cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization.     

Prolonged swimming promotes cellular oxidative stress and p66Shc phosphorylation,but does not induce oxidative stress in mitochondria in the rat heart

Abstract

Exercise-induced changes in p66Shc-dependent signaling pathway are still not fully understood. The p66Shc protein is one of the key players in cell signaling, particularly in response to oxidative stress. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the phosphorylation of p66Shc as well as the induction of mitochondrial and cellular oxidative stress in rat hearts.

Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their hearts were removed immediately for experiments.

The exercise protocol caused increased levels of the following oxidative stress parameters in cardiac cells: DNA damage, protein carbonyls, and lipid dienes. There was also increased phosphorylation of p66Shc without any alterations in Akt and extracellular signal-regulated kinases. Changes in the ferritin L levels and the L to H subunit ratio were also observed in the exercised hearts compared with the control hearts. Despite increased phosphorylation of p66Shc, no significant increase was observed in either mitochondrial H₂O₂ release or mitochondrial oxidative stress markers. Regardless of the changes in phosphorylation of p66Shc, the antioxidant enzyme activities (superoxide dismutase and catalase) and anti-apoptotic (Bcl2), and pro-apoptotic (Bax) protein levels were not affected by prolonged swimming. Further studies are required to investigate whether p66Shc phosphorylation is beneficial or detrimental to cardiac cells after exercise cessation.