Deficits in ERK and CREB activation in the hippocampus after traumatic brain injury

Abstract

Traumatic brain injury (TBI) activates several protein kinase signaling pathways in the hippocampus that are critical for hippocampal-dependent memory formation. In particular, extracellular signal-regulated kinase (ERK), a protein kinase activated during and necessary for hippocampal-dependent learning, is transiently activated after TBI. However, TBI patients experience hippocampal-dependent cognitive deficits that occur for several months to years after the initial injury. Although basal activation levels of ERK return to sham levels within hours after TBI, we hypothesized that activation of ERK may be impaired after TBI. Adult male Sprague-Dawley rats received either sham surgery or moderate parasagittal fluid-percussion brain injury. At 2, 8, or 12 weeks after surgery, the ipsilateral hippocampi of sham surgery and TBI animals were sectioned into transverse slices. After 2h of recovery in oxygenated artificial cerebrospinal fluid, the hippocampal slices were stimulated with glutamate or KCl depolarization, then analyzed by western blotting for phosphorylated, activated ERK and one of its downstream effectors, the transcription factor cAMP response element-binding protein (CREB). We found that activation of ERK (p<0.05) and CREB (p<0.05) after 30s of glutamate stimulation or KCl depolarization was decreased in hippocampal slices from animals at 2, 8, or 12 weeks after TBI as compared to sham animals. Basal levels of phosphorylated or total ERK were not significantly altered at 2, 8, or 12 weeks after TBI, although basal levels of phosphorylated CREB were decreased 12 weeks post-trauma. These results suggest that TBI results in chronic signaling deficits through the ERK-CREB pathway in the hippocampus.

Fig. 1

TBI results in deficits in ERK activation. Hippocampal slices at 2, 8 or 12 weeks after sham surgery (n=4 animals/time point) or TBI surgery (n=6 animals/time point) were stimulated with KCl (90 mM, n=6–10 slices/time point), glutamate (Glu, 200 μM, or vehicle (Ctl, n=6–8 slices/time point), then analyzed by western blotting for changes in phospho-ERK2 (pERK2). Phosphorylated ERK2 significantly increased in hippocampal slices stimulated with high KCl or glutamate from sham animals, but not in hippocampal slices from TBI animals. Values are mean ± SEM. **p<0.01, ***p<0.001, stimulated slices compared to non-stimulated slices, #p<0.05 stimulated slices from sham animals compared to stimulated slices from TBI animals

Fig. 2

Impairments in CREB activation after TBI. Hippocampal slices at 2, 8 or 12 weeks after sham surgery (n=4 animals/time point) or TBI (n=6 animals/time point) were stimulated with high KCl (90 mM, 30 s, n=6–10 slices/time point), glutamate (Glu, 200 μM, 30 s, n=7–9 slices/time point), or vehicle (Ctl, n=6–8 slices/time point), then analyzed by western blotting for changes in phospho-CREB (pCREB). Levels of phospho-CREB were significantly increased in stimulated hippocampal slices from sham animals, but not from TBI animals. Values are mean ± SEM. **p<0.01, ***p<0.001, non-stimulated slices compared to stimulated slices; #p<0.05 stimulated slices from sham animals compared to stimulated slices from TBI animals

Fig. 3

Basal levels of phosphorylated ERK at chronic time points after TBI. Basal levels of phospho-ERK2 and total ERK2 levels were analyzed in non-stimulated hippocampal slices by western blotting at 2 weeks (n=8 sham slices, n=4 TBI slices), 8 weeks (n=7 sham slices, n=7 TBI slices) and 12 weeks (n=8 sham slices, n=5 TBI slices) after surgery. Basal levels of phospho- and total ERK2 were not significantly different between TBI and sham surgery animals. Values are mean ± SEM.

Fig. 4

Reduced levels of phospho-CREB after TBI. Basal levels of phospho-CREB and total CREB were analyzed by western blotting at 2 weeks (n=8 sham slices, n=4 TBI slices), 8 weeks (n=7 sham slices, n=5 TBI slices) and 12 weeks (n=8 sham slices, n=5 TBI slices) after surgery. Basal phospho-CREB levels significantly decreased at 12 weeks after TBI in non-stimulated hippocampal slices as compared to non-stimulated levels in sham hippocampal slices. Values are mean ± SEM. **p<0.01, sham animals compared to TBI animals