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1.  Phosphorylated MAPK/ERK1/2 may not always represent its kinase activity in a rat model of focal cerebral ischemia with or without ischemic preconditioning 
Neuroscience  2012;209:155-160.
The ERK 1/2 protein require a dual phosphorylation at conserved threonine and tyrosine residues to be fully activated under normal physiological conditions. Thus, ERK1/2 kinase activity is often defined by the quantity of phosphorylated kinase. However, this may not accurately represent its true activity under certain pathological conditions. We investigated whether ERK1/2 kinase activity is proportional to its phosphorylation state in a rat focal ischemia model with and without rapid ischemic preconditioning. We showed that phosphorylated-ERK1/2 protein levels were increased 2.6±0.07 fold, and ERK1/2 kinase activity was increased 10.6±1.9 fold in animals receiving ischemic preconditioning alone without test ischemia compared with sham group (P<0.05, n=6/group), suggesting that phosphorylated-ERK1/2 protein levels represent its kinase activity under these conditions. However, preconditioning plus test ischemia robustly blocked ERK1/2 kinase activity, while it increased phosphorylated-ERK1/2 protein levels beyond those receiving test ischemia alone, suggesting that phosphorylated-ERK1/2 protein levels were not representative of actual kinase activity in this pathological condition. In conclusion, protein phosphorylation levels of ERK1/2 do not always correspond to kinase activity, thus, measuring the true kinase activity is essential.
PMCID: PMC3322316  PMID: 22366512
ischemic preconditioning; kinase activity; MAPK; ERK1/2; focal ischemia; stroke
2.  Lithium Treatment Reduces Brain Injury Induced by Focal Ischemia with Partial Reperfusion and the Protective Mechanisms Dispute the Importance of Akt Activity 
Aging and Disease  2012;3(3):226-233.
Lithium is a mood stabilizer shown to have neuroprotective effects against several chronic and acute neuronal injuries, including stroke. However, it is unknown whether lithium treatment protects against brain injury post-stroke in a rat model of permanent distal middle cerebral artery occlusion (MCAo) combined with transient bilateral common carotid artery occlusion (CCAo), a model that mimics human stroke with partial reperfusion. In addition, whether lithium treatment alters Akt activity as measured by the kinase activity assay has not been reported, although it is known to inhibit GSK3β activity. After stroke, Akt activity contributes to neuronal survival while GSK3β activity causes neuronal death. We report that a bolus of lithium injection at stroke onset robustly reduced infarct size measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining at 48 h post-stroke and inhibited cell death in the ischemic penumbra, but not in the ischemic core, as shown by TUNEL staining performed 24 h post-stroke. However, lithium treatment did not alter the reduction in Akt activity as measured by Akt kinase assay. We further showed that lithium did not alter phosphorylated GSK3β protein levels, or the degradation of β-catenin, a substrate of GSK3β, which is consistent with previous findings that long-term treatment is required for lithium to alter GSK3β phosphorylation. In summary, we show innovative data that lithium protects against stroke in a focal ischemia model with partial reperfusion, however, our results dispute the importance of Akt activity in the protective effects of lithium.
PMCID: PMC3375079  PMID: 22724081
Lithium; Akt; Cerebral focal ischemia; GSK3β; β-catenin
3.  The Akt signaling pathway contributes to postconditioning’s protection against stroke; the protection is associated with the MAPK and PKC pathways 
Journal of neurochemistry  2008;105(3):943-955.
We previously reported that ischemic postconditioning with a series of mechanical interruptions of reperfusion reduced infarct volume 2 days after focal ischemia in rats. Here, we extend this data by examining long-term protection and exploring underlying mechanisms involving the Akt, mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways. Post-conditioning reduced infarct and improved behavioral function assessed 30 days after stroke. Additionally, postconditioning increased levels of phosphorylated Akt (Ser473) as measured by western blot and Akt activity as measured by an in vitro kinase assay. Inhibiting Akt activity by a phosphoinositide 3-kinase inhibitor, LY294002, enlarged infarct in postconditioned rats. Postconditioning did not affect protein levels of phosphorylated-phosphatase and tensin homologue deleted on chromosome 10 or -phosphoinositide-dependent protein kinase-1 (molecules upstream of Akt) but did inhibit an increase in phosphorylated-glycogen synthase kinase 3β, an Akt effector. In addition, postconditioning blocked β-catenin phosphorylation subsequent to glycogen synthase kinase, but had no effect on total or non-phosphorylated active β-catenin protein levels. Furthermore, postconditioning inhibited increases in the amount of phosphorylated-c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in the MAPK pathway. Finally, postconditioning blocked death-promoting δPKC cleavage and attenuated reduction in phosphorylation of survival-promoting εPKC. In conclusion, our data suggest that postconditioning provides long-term protection against stroke in rats. Additionally, we found that Akt activity contributes to postconditioning’s protection; furthermore, increases in εPKC activity, a survival-promoting pathway, and reductions in MAPK and δPKC activity; two putative death-promoting pathways correlate with postconditioning’s protection.
PMCID: PMC2746404  PMID: 18182053
Akt; cerebral ischemia; mitogen-activated protein kinase; postconditioning; protein kinase C; β-catenin
4.  Hypothermia Blocks β-catenin Degradation after Focal Ischemia in Rats 
Brain research  2008;1198:182-187.
Dephosphorylated and activated glycogen synthase kinase (GSK) 3β hyperphophorylates β-catenin, leading to its ubiquitin-proteosome-mediated degradation. β-catenin-knockdown increases while β-catenin overexpression prevents neuronal death in vitro; in addition, protein levels of β-catenin are reduced in the brain of Alzheimer’s patients. However, whether β-catenin degradation is involved in stroke-induced brain injury is unknown. Here we studied activities of GSK3 β and β-catenin, and the protective effect of moderate hypothermia (30 °C) on these activities after focal ischemia in rats. The results of Western blot showed that GSK3 β was dephosphorylated at 5 and 24 hours after stroke in the normothermic (37 °C) brain; hypothermia augmented GSK3β dephosphorylation. Because hypothermia reduces infarction, these results contradict with previous studies showing that GSK3β dephosphorylation worsens neuronal death. Nevertheless, hypothermia blocked degradation of total GSK3β protein. Corresponding to GSK3β activity in normothermic rats, β-catenin phosphorylation transiently increased at 5 hours in both the ischemic penumbra and core, and the total protein level of β-catenin degraded after normothermic stroke. Hypothermia did not inhibit β-catenin phosphorylation, but it blocked β-catenin degradation in the ischemic penumbra. In conclusion, moderate hypothermia can stabilize β-catenin, which may contribute to the protective effect of moderate hypothermia.
PMCID: PMC2350209  PMID: 18241848
Focal ischemia; hypothermia; GSK-3β; β-catenin

Results 1-4 (4)