Targeting the Erk1/2 and autophagy signaling easily improved the neurobalst differentiation and cognitive function after young transient forebrain ischemia compared to old gerbils

Abstract

The hippocampal neurogenesis occurs constitutively throughout adulthood in mammalian species, but declines with age. In this study, we overtly found that the neuroblast proliferation and differentiation in the subgranular zone and the maturation into fully functional and integrated neurons in the granule-cell layer in young gerbils following cerebral ischemia/reperfusion was much more than those in old gerbils. The neurological function and cognitive and memory-function rehabilitation in the young gerbils improved faster than those in the old one. These results demonstrated that, during long term after cerebral ischemia/reperfusion, the ability of neurogenesis and recovery of nerve function in young animals were significantly higher than that in the old animals. We found that, after 14- and 28-day cerebral ischemia/reperfusion, the phosphorylation of MEK1/2, ERK1/2, p90RSK, and MSK1/2 protein levels in the hippocampus of young gerbils was significantly much higher than that of old gerbils. The levels of autophagy-related proteins, including Beclin-1, Atg3, Atg5, and LC3 in the hippocampus were effectively maintained and elevated at 28 days after cerebral ischemia/reperfusion in the young gerbils compared with those in the old gerbils. These results indicated that an increase or maintenance of the phosphorylation of ERK1/2 signal pathway and autophagy-related proteins was closely associated with the neuroblast proliferation and differentiation and the process of maturation into neurons. Further, we proved that neuroblast proliferation and differentiation in the dentate gyrus and cognitive function were significantly reversed in young cerebral ischemic gerbils by administering the ERK inhibitor (U0126) and autophagy inhibitor (3MA). In brief, following experimental young ischemic stroke, the long-term promotion of the neurogenesis in the young gerbil's hippocampal dentate gyrus by upregulating the phosphorylation of ERK signaling pathway and maintaining autophagy-related protein levels, it overtly improved the neurological function and cognitive and memory function.

Fig. 1. Observation of neurological function and recovery.

Neurological-function score of young and old gerbils after I/R (A). The maps of computer printouts of the swimming trajectories on the fifth day of each group (B). The time spent in the removed escape-platform quadrant (C). Number of crossings over the original platform position by gerbils (D) (n = 7 per group; *p < 0.05, significantly different from the corresponding young group, #p < 0.05, significantly different from the young I/R group). Bars indicate mean ± SD.

Fig. 2. Observation of cell proliferation and neuroblast differentiation.

Immunohistochemistry for BrdU in the DG region of the young and old of the sham and ischemia groups (A). Immunohistochemistry for DCX in the DG region of the young and old of the sham and ischemia groups (B). Double-immunofluorescence staining for BrdU, NeuN, and merged images in the DG 28 days after I/R in the young and old groups (C). The number of BrdU cells after I/R in the young and old groups. Data are represented by the mean number of BrdU cells per animal (D). Relative optical density as % of DCX immunoreactive structures in the young and old groups (E). Quantification of BrdU/NeuN cells at 28 days post ischemia. Data are represented by the mean percentage per animal (F). Scale bar = 100 μm (n = 7 per group; *p < 0.05, significantly different from the sham young group at the same reperfusion time point, #p < 0.05, significantly different from the corresponding young I/R group at the same reperfusion time point). Bars indicate mean ± SD.

Fig. 3. Observation of Erk1/2 signaling and autophagy-related protein levels.

Western blot analysis of p-MEK1/2, MEK1/2, p-Erk1/2, Erk1/2, p-p90RSK and p-MSK1/2 in the hippocampus of the young and old groups after I/R (A). Western blot analysis of Beclin-1, LC3-I, LC3-II, Atg3, and Atg5 in the hippocampus of the young and old groups after I/R (B) (n = 7 per group; *p < 0.05, significantly different from the old group at the same reperfusion time point, #p < 0.05, significantly different from the sham group of the same age). Bars indicate mean ± SD.

Fig. 4. Change of neurological function and recovery by U0126 or 3MA treatment.

Neurological-function score of young gerbils treated with U0126 or 3MA after I/R (A). The maps of computer printouts of the swimming trajectories on the fifth day of each group (B). The escape latency on the 5th day of each group (C). The time spent in target quadrant on the last day of each group (D). Number of crossings over the original platform position by gerbils (E) (n = 7 per group; *p < 0.05, significantly different from the young group at the same reperfusion time). Bars indicate mean ± SD.

Fig. 5. Change of Cell proliferation and neuroblast differentiation by U0126 or 3MA treatment.

Immunohistochemistry for BrdU in the DG region of the young gerbils treated with U0126 or 3MA after I/R (A). Immunohistochemistry for DCX in the DG region of the young gerbils treated with U0126 or 3MA after I/R (B). Double-immunofluorescence staining for BrdU, NeuN, and merged images in the DG 28 days after I/R in the young gerbils treated with U0126 or 3MA (C). The number of BrdU cells after I/R in the young gerbils treated with U0126 or 3MA. Data are represented by the mean number of BrdU cells per animal (D). Relative optical density as % of DCX immunoreactive structures in the young gerbils treated with U0126 or 3MA (E). Quantification of BrdU/NeuN cells at 28 days post ischemia. Data are represented by the mean percentage per animal (F). Scale bar = 100 μm (n = 7 per group; *p < 0.05, significantly different from the young group at the same reperfusion time). Bars indicate mean ± SD.

Fig. 6. Change of Erk1/2 signaling and autophagy-related protein levels by U0126 treatment.

Western blot analysis of p-MEK1/2, MEK1/2, p-Erk1/2, Erk1/2, p-p90RSK and p-MSK1/2 in the hippocampus of the young gerbils treated with U0126 (A). Western blot analysis of Beclin-1, LC3-I and LC3-II in the hippocampus of the young gerbils treated with U0126 (B) (n = 7 per group; *p < 0.05, significantly different from the 14 days young group, #p < 0.05, significantly different from the young group at the same reperfusion time). Bars indicate mean ± SD.

Fig. 7. Change of Erk1/2 signaling and autophagy-related protein levels by 3MA treatment.

Western blot analysis of Beclin-1, LC3-I, LC3-II, and Atg3 in the hippocampus of the young gerbils treated with 3MA (A). Western blot analysis of p-MEK1/2, MEK1/2, p-Erk1/2, Erk1/2, and p-p90RSK in the hippocampus of the young gerbils treated with 3MA (B) (n = 7 per group; *p < 0.05, significantly different from the 14-d young group, #p < 0.05, significantly different from the young group at the same reperfusion time). Bars indicate mean ± SD.