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Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy.

Gao C, Cai Y, Zhang X, Huang H, Wang J, Wang Y, Tong X, Wang J, Wu J - PLoS ONE (2015)

Bottom Line: Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group.The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA.In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tianjin Huanhu Hospital, Tianjin Key Laboratory of Cerebrovascular and Neurodegenerative Diseases, Tianjin, China.

ABSTRACT
The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm2 in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm2 in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.

No MeSH data available.


Related in: MedlinePlus

IPC-induces autophagy conferred neuroprotection independent of Akt phosphorylation.(A) Representative image of western blot analysis for Akt phosphorylated at serine 473 (pAkt), total Akt (tAkt), and β-actin in lysates from hippocampal neurons 6 h after exposure to normoxic conditions, 15 min of OGD alone, 55 min of OGD alone, or preconditioning. (B) Mean survival in hippocampal neurons exposed to 55 min of OGD; pretreated with IPC alone, 10 nM rapamycin alone, a combination of IPC and 10 nM GDC-0068, or a combination of 10 nM rapamycin and 10 nM GDC-0068 24 h before 55 min of OGD; or pretreated with 10 nM GDC-0068 alone under normoxic conditions. (C) Representative images of Nissl staining of the hippocampus. Mice were exposed to 1 min × 3 sublethal BCCAO or with a combination of IPC and intraventricular injection of wortmannin followed by 20 min of lethal BCCAO 24 h later. Cell survival was quantified by Nissl staining of the hippocampal CA1 and CA3 layers 24 h later. Values are given as the mean number of neurons. Error bars denote SDs. *P < 0.05 compared with other groups. **P < 0.05 compared with cells under normoxic conditions. #P < 0.05 compared with neurons maintained under 55 min of OGD. ☆P > 0.05 compared with the control. ☆☆P < 0.05 compared with the control.
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pone.0137146.g006: IPC-induces autophagy conferred neuroprotection independent of Akt phosphorylation.(A) Representative image of western blot analysis for Akt phosphorylated at serine 473 (pAkt), total Akt (tAkt), and β-actin in lysates from hippocampal neurons 6 h after exposure to normoxic conditions, 15 min of OGD alone, 55 min of OGD alone, or preconditioning. (B) Mean survival in hippocampal neurons exposed to 55 min of OGD; pretreated with IPC alone, 10 nM rapamycin alone, a combination of IPC and 10 nM GDC-0068, or a combination of 10 nM rapamycin and 10 nM GDC-0068 24 h before 55 min of OGD; or pretreated with 10 nM GDC-0068 alone under normoxic conditions. (C) Representative images of Nissl staining of the hippocampus. Mice were exposed to 1 min × 3 sublethal BCCAO or with a combination of IPC and intraventricular injection of wortmannin followed by 20 min of lethal BCCAO 24 h later. Cell survival was quantified by Nissl staining of the hippocampal CA1 and CA3 layers 24 h later. Values are given as the mean number of neurons. Error bars denote SDs. *P < 0.05 compared with other groups. **P < 0.05 compared with cells under normoxic conditions. #P < 0.05 compared with neurons maintained under 55 min of OGD. ☆P > 0.05 compared with the control. ☆☆P < 0.05 compared with the control.

Mentions: Although IPC can protect neurons through induction of Akt phosphorylation [7], Akt phosphorylation has been shown to be a negative regulator of autophagy. Therefore, we examined whether IPC induced the activation of autophagy in an Akt-dependent manner in our IPC model. We performed western blot analysis to measure Akt phosphorylation at serine 473 (phospho-Akt) in the preconditioning group, in which cell lysates prepared from hippocampal neurons were collected at different times (0, 1, 6, or 24 h) after exposure to lethal OGD. We found that Akt phosphorylation began to rise at 1 h, peaked at 6 h (p < 0.05), and returned to baseline levels at 24 h (Fig 6A). Next, we used GDC-0068, a highly selective ATP-competitive Akt inhibitor, to evaluate the role of Akt phosphorylation on autophagy-mediated hippocampal neuronal survival in the OGD model in vitro. The survival of hippocampal neurons after 55 min of OGD increased from 50.7% ± 5.8% in nonpreconditioned cells to 74.6% ± 6.9% in preconditioned neurons and to 76.8% ± 8.2% in neurons pretreated with 10 nM rapamycin. Moreover, pretreatment with a combination of IPC and 10 nM GDC-0068 or 10 nM rapamycin and 10 nM GDC-0068 did not block the protective effects of IPC or autophagy (70.1% ± 6.8% and 72.2% ± 6.9%, respectively). Neuronal survival following incubation with 10 nM GDC-0068 under normoxic conditions remained unchanged compared with the control (92.2% ± 5.8%; Fig 6B). In the preconditioning group, pretreatment with intraventricular injection of wortmannin did not alter LC3 expression or improve neuronal survival in the hippocampal CA1 layer. Moreover, in hippocampal CA1 and CA3 neurons treated with a combination of IPC and intraventricular injection of wortmannin before lethal BCCAO, cell numbers (260 ± 27 and 148 ± 22 cells/ mm2, respectively) did not differ significantly compared with those of the preconditioning group (p > 0.05; Fig 6C).


Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy.

Gao C, Cai Y, Zhang X, Huang H, Wang J, Wang Y, Tong X, Wang J, Wu J - PLoS ONE (2015)

IPC-induces autophagy conferred neuroprotection independent of Akt phosphorylation.(A) Representative image of western blot analysis for Akt phosphorylated at serine 473 (pAkt), total Akt (tAkt), and β-actin in lysates from hippocampal neurons 6 h after exposure to normoxic conditions, 15 min of OGD alone, 55 min of OGD alone, or preconditioning. (B) Mean survival in hippocampal neurons exposed to 55 min of OGD; pretreated with IPC alone, 10 nM rapamycin alone, a combination of IPC and 10 nM GDC-0068, or a combination of 10 nM rapamycin and 10 nM GDC-0068 24 h before 55 min of OGD; or pretreated with 10 nM GDC-0068 alone under normoxic conditions. (C) Representative images of Nissl staining of the hippocampus. Mice were exposed to 1 min × 3 sublethal BCCAO or with a combination of IPC and intraventricular injection of wortmannin followed by 20 min of lethal BCCAO 24 h later. Cell survival was quantified by Nissl staining of the hippocampal CA1 and CA3 layers 24 h later. Values are given as the mean number of neurons. Error bars denote SDs. *P < 0.05 compared with other groups. **P < 0.05 compared with cells under normoxic conditions. #P < 0.05 compared with neurons maintained under 55 min of OGD. ☆P > 0.05 compared with the control. ☆☆P < 0.05 compared with the control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0137146.g006: IPC-induces autophagy conferred neuroprotection independent of Akt phosphorylation.(A) Representative image of western blot analysis for Akt phosphorylated at serine 473 (pAkt), total Akt (tAkt), and β-actin in lysates from hippocampal neurons 6 h after exposure to normoxic conditions, 15 min of OGD alone, 55 min of OGD alone, or preconditioning. (B) Mean survival in hippocampal neurons exposed to 55 min of OGD; pretreated with IPC alone, 10 nM rapamycin alone, a combination of IPC and 10 nM GDC-0068, or a combination of 10 nM rapamycin and 10 nM GDC-0068 24 h before 55 min of OGD; or pretreated with 10 nM GDC-0068 alone under normoxic conditions. (C) Representative images of Nissl staining of the hippocampus. Mice were exposed to 1 min × 3 sublethal BCCAO or with a combination of IPC and intraventricular injection of wortmannin followed by 20 min of lethal BCCAO 24 h later. Cell survival was quantified by Nissl staining of the hippocampal CA1 and CA3 layers 24 h later. Values are given as the mean number of neurons. Error bars denote SDs. *P < 0.05 compared with other groups. **P < 0.05 compared with cells under normoxic conditions. #P < 0.05 compared with neurons maintained under 55 min of OGD. ☆P > 0.05 compared with the control. ☆☆P < 0.05 compared with the control.
Mentions: Although IPC can protect neurons through induction of Akt phosphorylation [7], Akt phosphorylation has been shown to be a negative regulator of autophagy. Therefore, we examined whether IPC induced the activation of autophagy in an Akt-dependent manner in our IPC model. We performed western blot analysis to measure Akt phosphorylation at serine 473 (phospho-Akt) in the preconditioning group, in which cell lysates prepared from hippocampal neurons were collected at different times (0, 1, 6, or 24 h) after exposure to lethal OGD. We found that Akt phosphorylation began to rise at 1 h, peaked at 6 h (p < 0.05), and returned to baseline levels at 24 h (Fig 6A). Next, we used GDC-0068, a highly selective ATP-competitive Akt inhibitor, to evaluate the role of Akt phosphorylation on autophagy-mediated hippocampal neuronal survival in the OGD model in vitro. The survival of hippocampal neurons after 55 min of OGD increased from 50.7% ± 5.8% in nonpreconditioned cells to 74.6% ± 6.9% in preconditioned neurons and to 76.8% ± 8.2% in neurons pretreated with 10 nM rapamycin. Moreover, pretreatment with a combination of IPC and 10 nM GDC-0068 or 10 nM rapamycin and 10 nM GDC-0068 did not block the protective effects of IPC or autophagy (70.1% ± 6.8% and 72.2% ± 6.9%, respectively). Neuronal survival following incubation with 10 nM GDC-0068 under normoxic conditions remained unchanged compared with the control (92.2% ± 5.8%; Fig 6B). In the preconditioning group, pretreatment with intraventricular injection of wortmannin did not alter LC3 expression or improve neuronal survival in the hippocampal CA1 layer. Moreover, in hippocampal CA1 and CA3 neurons treated with a combination of IPC and intraventricular injection of wortmannin before lethal BCCAO, cell numbers (260 ± 27 and 148 ± 22 cells/ mm2, respectively) did not differ significantly compared with those of the preconditioning group (p > 0.05; Fig 6C).

Bottom Line: Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group.The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA.In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Tianjin Huanhu Hospital, Tianjin Key Laboratory of Cerebrovascular and Neurodegenerative Diseases, Tianjin, China.

ABSTRACT
The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm2 in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm2 in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.

No MeSH data available.


Related in: MedlinePlus