Traumatic axonal injury induces proteolytic cleavage of the voltage-gated sodium channels modulated by tetrodotoxin and protease inhibitors

Abstract

We demonstrated previously that dynamic stretch injury of cultured axons induces structural changes and Ca2+ influx modulated by tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (NaChs). In the present study, we evaluated potential damage to the NaCh alpha-subunit, which can cause noninactivation of NaChs. In addition, we explored the effects of pre-injury and post-injury treatment with TTX and protease inhibition on proteolysis of the NaCh alpha-subunit and intra-axonal calcium levels ([Ca2+]i) over 60 min after trauma. After stretch injury, we found that [Ca2+]i continued to increase in untreated axons for at least 60 min. We also observed that the III-IV intra-axonal loop of the NaCh alpha-subunit was proteolyzed between 5 and 20 min after trauma. Pre-injury treatment of the axons with TTX completely abolished the posttraumatic increase in [Ca2+]i and proteolysis of the NaCh alpha-subunit. In addition, both pre-injury and post-injury inhibition of protease activity attenuated long-term increases in [Ca2+]i as well as mitigating degradation of the NaCh alpha-subunit. These results suggest a unique "feed-forward" deleterious process initiated by mechanical trauma of axons. Na+ influx through NaChs resulting from axonal deformation triggers initial increases in [Ca2+]i and subsequent proteolysis of the NaCh-subunit. In turn, degradation of the alpha-subunit promotes persistent elevations in [Ca2+]i, fueling additional pathologic changes. These observations may have important implications for developing therapeutic strategies for axonal trauma.

Figure 1.

A, B, Schematic illustration of axonal stretch injury with membrane pre-stretch (A) and post-stretch (B). Note the downward deflection of the membrane resulting in tensile elongation of axons crossing a cell-free gap. C, D, Neurites in the gap are entirely axons demonstrated in representative photomicrographs of immunocytochemical staining of MAP2 (C), a specific marker for the dendrites and neuronal somata, and NaCh protein (D) found on axons as well as the dendrites and neuronal somata. Scale bar, 50 μm.

Figure 2.

Proteolytic damage to the NaCh α-subunit on axonal fascicles after stretch injury (without treatment). A, Immunocytochemical staining for the III-IV loop region of the NaCh α-subunit was detected in sham injury and at 5 min after injury but disappeared by 20-60 min after injury. C, In contrast, staining for the I-II loop of NaCh α-subunit was found at all post-injury time points. Scale bars, 20 μm. B, Representative Western blot demonstrates 220 kDa bands corresponding to the NaCh α-subunit III-IV loop in sham injury and at 5 min after injury but not at 20 min after injury (left). Graphic representation of the staining intensities of these bands demonstrates relative changes over time (^**p < 0.01 vs sham) (B, right). D, In contrast, representative Western blot analysis of the I-II loop of the NaCh protein demonstrates that the 220 kDa band found at 5 min after injury almost disappeared by 20 min after injury, whereas a new 55 kDa band is found (^§§p < 0.01 vs sham). E, Similarly, the 220 kDa MW band found with Western blot analysis of the IV loop of the NaCh protein for sham axons also almost disappeared by 20 min after injury, whereas a new 55 kDa immunoreactive band was found (^§§p < 0.01 vs sham). All values are presented as means ± SE.

Figure 3.

Modulation of NaCh proteolytic damage with TTX and PIs after stretch injury of axon fascicles. A, Representative photomicrographs show that pre-injury treatment with TTX maintains immunoreactivity for the III-IV loop region of the NaCh α-subunit over 60 min after injury. B, However, delaying TTX treatment does not preserve immunoreactivity to the III-IV loop of the NaCh α-subunit. C, Representative Western blot demonstrates that pre-injury TTX treatment prevented the loss of the 220 kDa bands corresponding to the III-IV loop of the NaCh α-subunit after trauma. There was no significant difference of staining intensities between sham and 20 min groups. Scale bars: A, B, 20 μm. D, E, With both pre-injury and post-injury PI treatment, staining for the III-IV loop region of the NaCh α-subunit does not disappear over 60 min after injury. F, Representative Western blot demonstrates no loss of 220 kDa bands corresponding to the III-IV loop of the NaChα-subunit in sham injury and at 20 min after injury. There was no significant difference of staining intensities between sham and 20 min groups. Scale bars: D, E, 20 μm.

Figure 4.

Graphic representation of changes in intra-axonal Ca²⁺ fluorescence at 2, 20, and 60 min on axonal fascicles after stretch injury in CSS compared with pre-injury treatment of TTX (TTX-pre), 5 min post-injury treatment of TTX (TTX-5 min), 20 min post-injury treatment of TTX (TTX-20 min), pre-injury treatment of PI (PI-pre), and 5 min post-injury treatment of PI (PI-5 min). A, F/F₀ = change in Ca²⁺ fluorescence over initial fluorescence. Comparison with 2 min after injury in CSS: ^*p < 0.05; ^**p < 0.01. Comparison with 20 min after injury in CSS: #p < 0.05. Comparison with 60 min after injury in CSS: ^†p < 0.05; ^††p < 0.01. B, Representative photomicrographs demonstrate changes in Ca²⁺ fluorescence before (left) and 60 min after (right) axonal injury in CSS, with pre-injury treatment of TTX and with pre-injury treatment of PI.

Figure 5.

Proposed feed-forward pathway of Ca²⁺ entry and NaCh proteolysis resulting from traumatic mechanical deformation of axons. VGCCs, Voltage-gated Ca²⁺ channels.