Cittelly Diana M, Nesic-Taylor Olivera, Perez-Polo J Regino
Neuroscience and Cell Biology Department, University of Texas Medical Branch, Galveston, Texas 77555-1072, USA.
J Neurosci Res. 2007 Jul;85(9):1894-911. doi: 10.1002/jnr.21313.
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-ser(62)-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-ser(62)-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-ser(62)-Bcl-x(L) levels in organelles. However, P-ser(62)-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-ser(62)-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.
脊髓损伤（SCI）所致的功能障碍是由继发性凋亡引起的，而这种凋亡部分受SCI诱导的抗凋亡蛋白Bcl-x(L)减少的调控。我们评估了Bcl-x(L)亚细胞重排和翻译后磷酸化在大鼠SCI挫伤模型中Bcl-x(L)减少过程中所起的作用。免疫组织化学分析显示，Bcl-x(L)存在于神经元和少突胶质细胞中，但不存在于星形胶质细胞和小胶质细胞中，而磷酸化的Bcl-x(L)（P-ser(62)-Bcl-x(L)）仅存在于神经元中。蛋白质印迹分析表明，Bcl-x(L)存在于线粒体、内质网、细胞核和胞质提取物中，而P-ser(62)-Bcl-x(L)则局限于细胞器。在SCI后的最初24小时内，所有组分中的Bcl-x(L)水平均下降，但时间进程不同，这表明SCI后Bcl-x(L)从胞质穿梭至各组分的过程受到独立调控。SCI并未影响细胞器中P-ser(62)-Bcl-x(L)的水平。然而，在未损伤脊髓的胞质组分中未检测到的P-ser(62)-Bcl-x(L)，在挫伤后15分钟就最早出现在胞质中，这表明磷酸化在SCI诱导的Bcl-x(L)减少中发挥了作用。使用体外模型，我们观察到胞质磷酸化Bcl-x(L)水平与神经元凋亡之间存在相关性，支持了Bcl-x(L)磷酸化具有促凋亡作用的假说。SCI后7天，活化的小胶质细胞/巨噬细胞强烈表达Bcl-x(L)，并且显示核浓缩的亚群也表达P-ser(62)-Bcl-x(L)。因此，Bcl-x(L)的磷酸化在损伤脊髓中可能具有相反的作用：1）它可能降低神经元中抗凋亡Bcl-x(L)的水平，导致神经元死亡；2）它可能促进活化的小胶质细胞/巨噬细胞凋亡，从而减少与SCI相关的炎症级联反应。
