Expression and activation of ephexin is altered after spinal cord injury

Abstract

Failure of axon regeneration after traumatic spinal cord injury (SCI) is attributable in part to the presence of inhibitory molecular interactions. Recent evidence demonstrates that activation of Eph signaling pathways leads to modulation of growth cone dynamics and repulsion through the activation of ephexin, a novel guanine nucleotide exchange factor (GEF). However, little is known about the expression and modulation of Eph molecular targets in the injured spinal cord. In this study, we determined the expression profile of ephexin after a moderate spinal cord contusion at thoracic level (T10) in young adult rats. Western Blot studies showed increased protein expression in injured rats at 4, and 7 days post-injury (DPI) when compared to control animals. The protein levels returned to normal at 14 DPI and remained steady until 28 DPI. However, immunoprecipitation studies of the phosphorylated ephexin demonstrated that this protein is activated by day 2 until 14 DPI. Expression of ephexin was noticeable in neurons, axons, microglia/macrophages and reactive astrocytes, and co-localized with EphA3, A4 and A7. These results demonstrate the presence of ephexin in the adult spinal cord, and its activation after SCI. Therefore, we show, for the first time, the spatio-temporal pattern of ephexin expression and activation after contusive spinal cord injury. Collectively, our data supports our previous findings on the putative non-permissive roles of Eph receptors after spinal cord injury, and the possible involvement of ephexin in the intracellular cascade of events.

Expression Profile and Role of EphrinA1 Ligand After Spinal Cord Injury

“…Then the sections were incubated with mouse anti-GFAP (1:100, Chemicon International Inc, Temecula, CA, USA), mouse anti-NeuN (1:200, Chemicon International Inc), mouse anti-ED1 (1:500, Serotec, Raleigh, NC, USA), mouse anti-NF-H (1:1000, Chemicon International Inc.), mouse anti-MAG (1:250, Chemicon International Inc, Temecula, CA, USA), and rabbit anti-ephrinA1 antibody (1:200 [sc-911], Santa Cruz Biotechnology, Santa Cruz, CA, USA) to identify reactive astrocytes, motorneurons, macrophages, axons or myelin, respectively. For the double-labeling assay related to Eph receptors, anti m-EphA4 (3 μg/μl), and anti m-EphA7 (5 μg/μl) (R&D Systems, Minneapolis, MN, USA) were used as standardized by Rosas et al (2010). After a 24 h incubation at 4°C with the primary antibodies and three washes with PBS, donkey anti-rabbit Rhodamine (1:200, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), donkey anti-mouse Alexa (1:250), and donkey anti-goat Alexa (1:200, Invitrogen Detection Technologies, Eugene, OR, USA) were applied to the sections for 2 h at room temperature.…”

Dietary Omega-3 Polyunsaturated Fatty Acids Improve the Neurolipidome and Restore the DHA Status while Promoting Functional Recovery after Experimental Spinal Cord Injury

“…Eight weeks after the operations, the spinal cord segments were processed for Western blot analysis as described previously. 32 Briefly, 5 mm spinal cord segments were dissected and immediately frozen. The tissue containing injured epicenter-to-caudal regions was then homogenized manually with a Dounce tissue grinder in 200 lL of 1% SDS in Tris-EDTA buffer with proteinase inhibitors (10 lg/mL aprotinin, 1 lg/mL leupeptin, and 1 mM PMSF) and then sonicated.…”

Tamoxifen Administration Immediately or 24 Hours after Spinal Cord Injury Improves Locomotor Recovery and Reduces Secondary Damage in Female Rats

“…All surgical procedures were performed as described previously. 23,24 Vaginal smears were performed on at least five animals from each group to determine the estrus cycle stage of the animals. Cytology evaluation was performed blinded to treatment at the moment of surgery and gave us a representation of the cycle of the complete population of animals used for this study.…”