Non-receptor tyrosine kinase Src is required for ischemia-stimulated neuronal cell proliferation via Raf/ERK/CREB activation in the dentate gyrus

Abstract

Background: Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. However, Molecular mechanisms that regulate neuronal cell proliferation in the dentate gyrus (DG) following ischemic stroke insult are poorly understood. This study was designed to investigate the potential regulatory capacity of non-receptor tyrosine kinase Src on ischemia-stimulated cell proliferation in the adult DG and its underlying mechanism.

Results: Src kinase activated continuously in the DG 24 h and 72 h after transient global ischemia, while SU6656, the Src kinase inhibitor significantly decreased the number of bromodeoxyuridine (BrdU) labeling-positive cells of rats 7 days after cerebral ischemia in the DG, as well as down-regulated Raf phosphorylation at Tyr(340/341) site, and its down-stream signaling molecules ERK and CREB expression followed by 24 h and 72 h of reperfusion, suggesting a role of Src kinase as an enhancer on neuronal cell proliferation in the DG via modifying the Raf/ERK/CREB cascade. This hypothesis is supported by further findings that U0126, the ERK inhibitor, induced a reduction of adult hippocampal progenitor cells in DG after cerebral ischemia and down-regulated phospho-ERK and phospho-CREB expression, but no effect was detected on the activities of Src and Raf.

Conclusion: Src kinase increase numbers of newborn neuronal cells in the DG via the activation of Raf/ERK/CREB signaling cascade after cerebral ischemia.

Figure 1

Src kinase sustainedly activated in the DG field of hippocampus after ischemia, while its inhibitor SU6656 blocked cell proliferation induced by ischemia. SU6656 was intracerebroventricularly (i.c.v) administratered 20 min before ischemia. (A) Western blot for Src kinase detected by anti-p-Src antibody (Tyr527). (B) DAB staining for Brdu-positive cells in the SGZ of dentate gyrus. (a) Sham rats (n = 4), (b) Rats with 7 days of reperfusion postischemia (n = 5), (c) SU6656-treated ischemic rats (n = 5), and (d) vehicle-treated ischemic rats (n = 4). *P < 0.05 vs. sham group; ^#P < 0.05 or ^aP < 0.05 vs. the respective reperfusion group. Bar: 200 μm.

Figure 2

Inhibiting the activation of Src kinase by SU6656 down-regulate Raf (Y340)/ERK/CREB cascade after ischemia- reperfusion. Samples were used as in Fig. 1. (A) Raf phosphorylation at Tyr340/341. (B) ERK phosphorylation at Thr202/Tyr204. (C) CREB phosphorylation at Ser133. Data are expressed as mean ± S.D. (n = 4/group) *P < 0.05 vs. sham group, ^#P < 0.05 vs. respective reperfusion group.

Figure 3

Inhibition on activated ERK blocked ischemia-stimulated cell proliferation in the dentate gyrus and CREB activation, but it did not affect expression of p-Src and p-Raf. U0126 was intracerebroventricularly (i.c.v) administratered 20 min before ischemia. (A) ERK phosphorylation after reperfusion 24 h post ischemia.(B) DAB staining for BrdU-positive cells in the SGZ at the 7^th post-ischemic day. (a) Rats with 7 days of reperfusion after ischemia (n = 5), (b) U0126-treated ischemic rats (n = 4), (c) vehicle-treated ischemic rats (n = 4), and (d) SU6656-treated ischemic rats (n = 5). (C) CREB phosphorylation, (D) Src phosphorylation or (E) Raf phosphorylation. Data are expressed as mean ± S.D. *P < 0.05 vs. sham, ^#P < 0.05 or ^aP < 0.05 vs. respective reperfusion. Bar: 200 μm

Figure 4

The region of DG is resistant to ischemic damage while CA1 is vulnerable. Blockage activation of Src instead of ERK significantly decreased neuronal loss in hippocampal CA1 after ischemia. NISSL staining was performed to examine the survival of hippocampal neurons following 5 days reperfusion. (A) sham group, (B) 5 days of reperfusion postischemia (R5d), (C) SU6656 administration, (D) U0126 administration, and (E) vehicle administration (n = 5). Data are expressed as means ± S.D. *P < 0.05 versus sham; ^#P < 0.05 versus I/R 5 days groups (n = 5). Scale bars in A-E = 200 μm; bars in CA1 and DG = 20 μm.

Figure 5

Suggested pathways through which Src kinase regulates cell proliferation in the DG after ischemia. Transient global ischemia triggered sustained activation of Src kinase by intracellular Ca²⁺ influx via NMDA and L-VGCC or released from ER and mitochondria. Subsequently, activated Src induced continuous phosphorylation of ERK through direct phosphorylation of Raf-1 at its Tyr 340/341, and in turn, was translocated from the cytosol to the nucleus where it regulated transcription via CREB and some genes relating to cell proliferation expression.