Role of BCL-XL in cell death after spinal cord injury


Long term functional impairment after rat spinal cord injury (SCI) results from secondary apoptosis regulated in part, by SCI-induced decreases in protein levels of the anti-apoptotic protein Bcl-xL. In this dissertation, I assessed the role that Bcl-xL subcellular re-routing and post-translational phosphorylation play in SCI-induced Bcl-xL decreases, and evaluated the therapeutic potential of Bcl-xL-administration after SCI. Immunohistochemical analysis showed non-phosphorylated Bcl-xL in neurons and oligodendrocytes, but not in astrocytes and microglia. Bcl-xL levels decreased in mitochondria, endoplasmic reticulum, nuclei and cytosolic extracts during the first 24h after SCI, but with a different time course for each organelle; suggesting an independent regulation of Bcl-xL shuttling from the cytosol to each compartment in the injured spinal cords. A membrane-bound phosphorylated form of Bcl-xL (P-ser62-Bcl-xL) was found in neurons in the uninjured SC. SCI did not affect P-ser62-Bcl-xL levels in organelles; however, P-ser62-Bcl-xL appeared in the cytosol early after SCI, suggesting a role for phosphorylation in SCI-induced decreases of Bcl-xL levels. Vinblastine-induced apoptosis of neuronal PC12 cells, showed that cytosolic phosphorylated Bcl-xL correlated with apoptotic cell death of neurons, suggestive of Bcl-xL-phosphorylation as a pro-apoptotic event. I found that activated microglia/macrophages robustly expressed Bcl-xL, 7 days after SCI, and a fraction of this population undergoing apoptosis, expressed P-ser62-Bcl-xL. Therefore, phosphorylation of Bcl-xL may have two opposite effects in injured spinal cords: (a) it may decrease levels of the anti-apoptotic Bcl-xL in neurons and therefore contribute to their death and, (b) it may regulate apoptosis in activated microglia/macrophages, thus curtailing the inflammatory cascades associated with SCI.\r\nTo counteract SCI-induced decreases in Bcl-xL and resulting apoptosis, I used a fusion protein made up of the TAT protein transduction domain and the Bcl-xL protein (Tat-Bcl-xL), or to its anti-apoptotic domain BH4 (Tat-BH4). Intrathecal delivery of Tat-Bcl-xL, or Tat-BH4 for 24h or 7 days after SCI, resulted in a significant decrease in apoptosis at the site of injury. However, the 7 day delivery of Tat-Bcl-xL or Tat-BH4 impaired locomotor recovery beyond the drug delivery time. Here I show that the 7 day application of Tat-Bcl-xL or Tat-BH4 increased microglia/macrophage activation and/or survival associated with an increase in neuronal losses. These results suggest that the anti-apoptotic treatment may shift neuronal apoptosis to necrosis, and initiate an inflammatory response (microglial activation) in SCI rats. As a result, Tat-Bcl-xL/Tat-BH4-induced increases in proinflammatory reactions may amplify SCI-induced neuronal cell death and additionally impair functional recovery. Given that microglial activation and inflammation are main players in shaping pathological outcomes after SCI, these results suggest that the therapeutic potential of Tat-Bcl-xL or Tat-BH4 in injured spinal cords may be limited. Moreover, chronic treatment of SCI with Tat-Bcl-xL or other anti-apoptotic treatments targeting Bcl-xL could be detrimental.\r\n



Tat-BH4, Tat-Bcl-xL, subcellular localization, spinal cord trauma, phosphorylation, neuronal death, microglial activation, Apoptosis, anti-apoptotic therapy