Evidence of Phosphorylation of Akt and Neuronal Survival after Transient Focal Cerebral Ischemia in Mice

Abstract

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways

“…Moreover, no protection was observed by suppression of caspase-8 or -9, the upstream regulatory caspases, which control caspase-3 activity and modulate the extrinsic or intrinsic pathways of neuronal apoptosis, respectively. [51][52][53][54] In addition, anandamide-induced cytotoxicity appeared prior to changes in caspase activity. These findings were further confirmed using neuroblastoma cell lines that expressed either caspase-9 or -8 dominant-negative mutants; again, anandamide-induced cell death was not modified in cells transfected with the dominantnegative constructs versus the empty vector.…”

Activation of the Akt/GSK3β Signaling Pathway Mediates Survival of Vulnerable Hippocampal Neurons after Transient Global Cerebral Ischemia in Rats

“…We have reported that activation of Akt signaling has neuroprotective effects against ischemic neuronal injury (Noshita et al, 2001(Noshita et al, , 2003. Several potential substrates for Akt that are related to cell survival after cerebral Figure 5 (A) Apoptotic-related DNA fragmentation after ischemia was analyzed with a commercial cell death detection kit (n = 4 each).…”

“…Sublethal global ischemia induced persistent Akt phosphorylation in the hippocampal CA1 subregion, and Yano et al (2001) concluded that this persistent Akt activation resulted in neuronal survival against lethal global ischemia. In the tFCI model, the Akt phosphorylation observed in the ischemic penumbra after ischemia was prevented by LY294002, a PI3-K inhibitor, which facilitated ischemic injury (Noshita et al, 2001). This Akt phosphorylation was enhanced and persistent after tFCI in SOD1 Tg mice, Quantitative analysis showed that phospho-GSK3b (Ser9) expression significantly increased in the SOD1 Tg rats 8 h and 1 day after tGCI compared with the Wt rats (n = 4, *P < 0.05).…”

PH domain leucine-rich repeat protein phosphatase 1 contributes to maintain the activation of the PI3K/Akt pro-survival pathway in Huntington's disease striatum

“…6 In fact, we detected increased levels of pGSK3b and pFoxO1, indicating that pAkt inactivates these proteins in the striatum of R6/1 mice. Notably, GSK3b or FoxO1 inactivation by Akt has been shown to be neuroprotective in several types of brain injury, such as ischemia 9,10 or traumatic lesion, 11 thereby suggesting a neuroprotective function for Akt mediated by inactivation of GSK3b and FoxO1 also in the R6/ 1 model of HD. Supporting this idea, the resistance of striatal cells of R6/1 mice to excitotoxic-induced cell death can in part be related to the presence of increased levels of pAkt (Ser473), and inactivation of GSK3b and FoxO1.…”

“…6 This kinase is present at low levels in the adult brain, 7 but its expression and activation increase dramatically in neurons during cellular stress or injury. 8 For instance, activated Akt has been proposed as an important neuroprotective pathway in distinct acute 7,9,10,11 and chronic 12,13 models of neurodegeneration. In HD, Akt has been proposed as a crucial neuroprotective pathway because it is one of the serine/threonine kinases that phosphorylate the Ser421 of mutant htt (mhtt) attenuating its toxicity.…”