Assessment of CA1 injury after global ischemia using supervised 2D analyses of nuclear pyknosis.

Selective neuronal vulnerability is a common theme in both acute and chronic diseases affecting the nervous system. This phenomenon is particularly conspicuous after global cerebral ischemia wherein CA1 pyramidal neurons undergo delayed death while surrounding hippocampal regions are relatively spared. While injury in this model can be easily demonstrated using either histological or immunological stains, current methods used to assess the cellular injury present in these biological images lack the precision required to adequately compare treatment effects. To address this shortcoming, we devised a supervised work-flow that can be used to quantify ischemia-induced nuclear condensation using microscopic images. And while we demonstrate the utility of this technique using models of ischemic brain injury, the approach can be readily applied to other paradigms in which programmed cell death is a major component.

MKP-1 antagonizes C/EBPß activity and lowers the apoptotic threshold after ischemic injury.

The dual specificity phosphatase MAPK phosphatase-1 (MKP-1) feeds back on MAP kinase signaling to regulate metabolic, inflammatory and survival responses. MKP-1 is widely expressed in the central nervous system (CNS) and induced after ischemic stress, although its function in these contexts remains unclear. Here we report that MKP-1 activated several cell death factors, including BCL2 and adenovirus E1B 19 kDa interacting protein 3, and caspases 3 and 12 culminating in apoptotic cell death in vitro. MKP-1 also exerted inhibitory effects on the bZIP transcription factor CCAAT/enhancer-binding protein (C/EBPß), previously shown to have neuroprotective properties. These effects included reduced expression of the full-length C/EBPß variant and hypo-phosphorylation at the MEK-ERK1/2-sensitive Thr(188) site. Notably, enforced expression C/EBPß rescued cells from MKP-1-induced toxicity. Studies performed in knock-out mice indicate that the MKP-1 activity is required to exclude C/EBPß from the nucleus basally, and that MKP-1 antagonizes C/EBPß expression after global forebrain ischemia, particularly within the vulnerable CA1 sector of the hippocampus. Overall, MKP-1 appears to lower the cellular apoptotic threshold by inhibiting C/EBPß and enhancing both BH3 protein expression and cellular caspase activity. Thus, although manipulation of the MKP-1-C/EBPß axis could have therapeutic value in ischemic disorders, our observations using MKP-1 catalytic mutants suggest that approaches geared towards inhibiting MKP-1's phosphatase activity alone may be ineffective.