Phosphorylation of Bcl-x_L after spinal cord injury

Spinal cord injury (SCI)-induced functional impairment results from secondary apoptosis regulated in part by SCI-induced decreases in the antiapoptotic protein Bcl-x_L. We assessed the role that Bcl-x _L subcellular rerouting and posttranslational phosphorylation play in Bcl-x_L decreases in a contusion model of rat SCI. Immunohistochemical analysis showed the presence of Bcl-x_L in neurons and oligodendrocytes, but not in astrocytes and microglia, whereas phosphorylated Bcl-x_L (P-ser⁶²-Bcl-x_L) was present only in neurons. Western blot analyses showed Bcl-x_L present in mitochondria, endoplasmic reticulum, nuclei, and cytosolic extracts, whereas P-ser⁶²-Bcl-x_L was restricted to organelles. During the first 24 hr after SCI, Bcl-x_L levels decreased in all fractions but with a different time course, suggesting an independent regulation of Bcl-x _L shuttling from the cytosol to each compartment after SCI. SCI did not affect P-ser⁶²-Bcl-x_L levels in organelles. However, P-ser⁶²-Bcl-x_L, which was not detected in the cytosolic fraction of uninjured spinal cord, appeared in the cytosol as early as 15 min postcontusion, suggesting a role for phosphorylation in SCI-induced Bcl-x _L-decreases. Using an in vitro model, we observed a correlation between levels of cytosolic phosphorylated Bcl-x_L and neuronal apoptosis, supporting the hypothesis that Bcl-x_L phosphorylation is proapoptotic. Activated microglia/macrophages robustly expressed Bcl-x _L 7 days after SCI, and a subpopulation showing nuclear condensation also expressed P-ser⁶²-Bcl-x_L. Therefore, phosphorylation of Bcl-x_L may have opposite effects in injured spinal cords: 1) it may decrease levels of the antiapoptotic Bcl-x_L in neurons contributing to neuronal death, and 2) it may promote apoptosis in activated microglia/macrophages, thus curtailing the inflammatory cascades associated with SCI.