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Akt isoforms differentially protect against stroke-induced neuronal injury by regulating mTOR activities - PubMed

Akt isoforms differentially protect against stroke-induced neuronal injury by regulating mTOR activities

Rong Xie et al. J Cereb Blood Flow Metab. 2013 Dec.

Abstract

Protein kinases Akt1 and Akt3 are considered to be more crucial to brain function than Akt2. We investigated the roles of Akt1 and Akt3 in stroke-induced brain injury and examined their interactions with the Akt/mTOR pathways. Focal ischemia was induced in rats. Lentiviral vectors expressing constitutively active Akt1 and Akt3 (cAkt1 and cAkt3) were injected into the ischemic cortex. Infarct sizes and gene and protein expressions in the Akt/mTOR pathways were evaluated. The results show that Akt1 and Akt3 proteins were degraded as early as 1 hour after stroke, whereas Akt2 proteins remained unchanged until 24 hours after stroke. Lentiviral-mediated overexpression of cAkt1 or cAkt3 reduced neuronal death after in vitro and in vivo ischemia. Interestingly, cAkt3 overexpression resulted in stronger protection than cAkt1 overexpression. Western blot analyses further showed that cAkt3 promoted significantly higher levels of phosphorylated Akt and phosphorylated mTOR than cAkt1. The mTOR inhibitor rapamycin blocked the protective effects of both cAkt1 and cAkt3. In conclusion, Akt isoforms are differentially regulated after stroke and Akt3 offers stronger protection than cAkt1 by maintaining Akt levels and promoting mTOR activity.

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Figures

Figure 1
Figure 1

Diagram of Akt/mTOR pathways, definition of ischemic penumbra and core, and expression of Akt isoforms after stroke. (A) Diagram shows that the PI3K/Akt pathway closely interacts with the mTOR pathway. (B) Diagram shows the definition of ischemic penumbra and core. Penumbra (P) refers to the ischemic region that can be rescued by various neuroprotectants. Core (C) refers to the region of permanent damage. (C) Representative DNA bands from RT-PCR for Akt1, Akt2, and Akt3 in both the ischemic penumbra and core. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control gene. Bar graphs represent relative optical densities of DNA bands of Akt isoforms in ischemic brains, normalized to densities in sham rats and expressed as percentages. (D) Representative western blot with protein bands for Akt1, Akt2, and Akt3. A β-actin control ensured equal protein loading. Bar graphs represent relative optical densities of Akt1, Akt2, and Akt3 protein bands, normalized to those in the sham group. N=6/group. *, **, ***, versus sham, P<0.05, 0.01, 0.001, respectively; Φ, ΦΦ P<0.05, 0.01, respectively, between the two indicated groups. n=5 to 6/group.

Figure 2
Figure 2

Construction of lentiviral vectors of constitutively active Akt1 (cAkt1), constitutively active Akt2 (cAkt2), and dominant-negative Akt (AktDN) and their expression in primary neuronal cultures. (A) Schematic backbone of lentiviral vector of pHR'tripCMV-IRES-eGFP. The genes were cloned into the lentiviral backbone plasmid, pHR'tripCMV-IRES-eGFP, which contains a CMV promoter and an IRES sequence between its multiple cloning sites (MCS) and eGFP. The IRES sequence enables independent expression of both the target gene and eGFP simultaneously. Akt1 and Akt3 genes lacking the pleckstrin homology (PH) domain are fused with a Gag polypeptide. A myristoylation signal enables cAkt1 and cAkt3 genes to attach to the cell membrane and be phosphorylated, thus activated. (B) Western blot confirmation of the effects of gene transfer in primary neuronal cultures on protein expression of pAkt, Akt, pPRAS40, PRAS40, pFKHR, FKHR, pmTOR, and mTOR in the Akt/mTOR pathways. Mixed neuron cells were grown in 6-well plates and transfected with lentiviral vectors. The cells were harvested at 48 hours after gene transfer, and homogenized in the cold cell extraction buffer containing 1 mmol/L phenylmethylsulfonyl fluoride and the protease inhibitor cocktail. The homogenate was centrifuged at 13,000 r.p.m. for 20 minutes at 4°C, and the supernatant was removed for protein detection. Protein concentrations were measured using the Bradford assay. The cAkt1 and cAkt3 plasmids lack the PH domain, so proteins translated by the cAkt1 and cAkt3 genes are truncated and have a smaller molecular weight (∼50 kDa). Phosphorylation of FKHR may change its molecular structure and shift its band from 78 to 82 kDa. Thus, pFKHR and FKHR are visible in the same gel as two separate bands. (C) Triple staining of GFP, HA, and 4′, 6-diamidino-2-phenylindole (DAPI) in a mixed primary neuronal culture transfected with cAkt3 vectors. The cAkt1, cAkt3, and AktDN vectors are fused with an HA tag; thus, HA expression is proportional to that of cAkt1. Scale bar, 50 μm. (D) Triple staining of GFP, MAP-2, and 4′, 6-diamidino-2-phenylindole (DAPI) in a mixed primary neuronal culture transfected with cAkt3 confirms the successful transfection of neurons. Triple-stained neurons are labeled by arrows.

Figure 3
Figure 3

Effects of gene transfer on cell death in primary neuronal cultures. Effects of gene transfer of constitutively active Akt1 (cAkt1), constitutively active Akt3 (cAkt3), and dominant-negative Akt (AktDN) on neuronal death as measured by lactate dehydrogenase (LDH) release. Cultured mixed neurons were transfected with vectors containing cAkt1, cAkt3, and AktDN; cultures transfected with GFP vectors or treated with vehicle solution served as controls. Cultures were subjected to 6 hours of oxygen glucose deprivation (OGD) 2 days after transfection. LHD release was measured at 16 hours after OGD. All data after OGD were normalized to the values of control, nonOGD samples transfected with the corresponding vectors. n=18 to 24/group. *, **, P<0.05, 0.001, respectively, versus GFP control vector receiving OGD treatment. Φ, ΦΦ, P<0.05, 0.01, respectively, between the two indicated groups.

Figure 4
Figure 4

Gene transfer of constitutively active Akt1 and Akt3 (cAkt1 and cAkt3), but not dominant-negative Akt (AktDN), reduced brain infarction after focal ischemia. (A) Representative coronal sections of brains show infarction areas and injection sites. Injection sites are marked by squares in which needle tracks are visible. (B) Average infarct size is determined from brain sections with needle tracts. Infarct area was measured and normalized to the nonischemic contralateral cortex, and expressed as a percentage. **, ***, versus control vector of GFP, P<0.01, 0.001, respectively. ΦΦ, cAkt1 versus cAkt3, P<0.01. (C) Representative immunostaining of GFP in brains transfected with various lentiviral vectors. (D) GFP levels measured by western blot indicate the effects of gene transfer on brain injury. All protein bands presented are derived from the same gel (see Supplementary Figure 5), but were cut and rearranged for convenient comparison. Transfected brain tissues at the needle tracks were dissected for western blotting. The average protein levels of GFP are presented in the bar graphs. N=6/group. ** versus control vector of GFP, P<0.01, respectively. ΦΦ, P<0.05, 0.01, respectively, between the two indicated groups.

Figure 5
Figure 5

Effects of constitutively active Akt1 and Akt3 (cAkt1 and cAkt3) gene transfer on expression of endogenous and exogenous Akt1, 2, and 3 after stroke. (A) Representative protein bands of Akt1, Akt2, and Akt3 as shown by western blot. β-Actin was used to show even protein loading. (B) Quantified Akt1 protein levels in ischemic brains transfected with GFP, cAkt1, and cAkt3 are presented in bar graphs. Left and right bar graphs show endogenous and exogenous Akt1 protein levels, respectively. (C) Effects of gene transfer on Akt2 protein expression. (D) Effects of gene transfer on both endogenous and exogenous Akt3 expression. N=6/group. *, ** versus GFP, 5 hours, P<0.05, 0.01, respectively; #, ## versus GFP, 24 hours, P<0.05, 0.01, respectively; Φ, ΦΦ show significance difference between the two indicated groups, P<0.05, 0.01, respectively.

Figure 6
Figure 6

The effects of constitutively active Akt1 (cAkt1), constitutively active Akt3 (cAkt3), and dominant-negative Akt (AktDN) gene transfer on protein phosphorylation in the Akt/mTOR pathways 5 and 24 hours after stroke. (A) Representative protein bands of critical molecules in the Akt/mTOR pathways, including phosphorylated and total protein expression as measured by western blot. All protein bands presented are derived from the same gel, but were cut and rearranged for convenient comparison (see Supplementary Figure 6). (B) The bar graphs show quantified protein levels of pPTEN, pAkt, pFKHR, pGSK3β, pPRAS40, and pmTOR. N=6/group. & and &&, versus the nonischemic group treated with control GFP vectors, P<0.05, 0.001, respectively; *, ** versus ischemic brain transfected with control GFP vectors 5 hours after stroke, P<0.05, 0.01, respectively; #, ## versus ischemic brain transfected with control GFP vectors 24 hours after stroke, P<0.05, 0.01, respectively; Φ, ΦΦ show significance difference between the two indicated groups, P<0.05, 0.01, respectively.

Figure 7
Figure 7

The mTOR inhibitor rapamycin blocked the protective effects of gene transfer of cAkt1 and cAkt3. (A) Infarct sizes in animals injected with control GFP, cAkt1, and cAkt3, with and without rapamycin. (B) Western blot was used to detect the effect of rapamycin on protein expression in the Akt/mTOR pathways. Representative protein bands of pAkt, pmTOR, and pS6K in ischemic brain transfected with control GFP vectors, cAkt1, and cAkt3, with and without rapamycin treatment are shown. (C) Bar graphs show the relative average optical densities of protein bands of endogenous pAkt, exogenous pAkt, pmTOR, and pS6K, respectively. N=6 to 8/group. &, *, #, versus corresponding control GFP group, P<0.05; Φ, ΦΦ, compare between the two indicated groups, P<0.05, 0.01, respectively.

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