Elsevier

Neuroscience Letters

Volume 328, Issue 2, 9 August 2002, Pages 150-154
Neuroscience Letters

Calpain-dependent neurofilament breakdown in anoxic and ischemic rat central axons

https://doi.org/10.1016/S0304-3940(02)00469-XGet rights and content

Abstract

Neurofilaments are key structural components of white matter axons. The effect of in vitro anoxia or oxygen-glucose deprivation (OGD) on the integrity of the 160 and 200 kDa neurofilament isoforms was studied by immunoblot, and correlated with physiological function. Adult rat optic nerves were exposed to 60 min of either anoxia or OGD. Compound action potential area recovered to 22±6% of control after 60 min of anoxia, and to 4±1% after 60 min of OGD. Ca2+-free (+EGTA) perfusate allowed complete recovery after OGD (108±42%). Tetrodotoxin (TTX, 1 μM) was less protective (45±6%). Both anoxia and OGD induced breakdown of neurofilament 160 (NF160) and NF200 revealed by the appearance of multiple lower molecular weight bands mainly in the 75–100 kDa range. Zero-Ca2+/EGTA completely prevented NF breakdown. TTX only partially reduced NF160 degradation. Non-phosphorylated NF200 appeared after reperfusion post-anoxia or OGD, and was also greatly reduced by zero-Ca2+ or TTX. Calpain inhibitors (10 μM calpain inhibitor I or 50 μM MDL 28,170) significantly reduced NF160 and NF200 breakdown/dephosphorylation, but did not improve electrophysiological recovery. Significant calpain-mediated breakdown of NF160 and NF200 indicates structural damage to the axonal cytoskeleton, which was completely Ca2+-dependent. While pharmacological inhibition of calpain alone greatly reduced NF proteolysis, there was no concomitant improvement in function. These results imply that calpain inhibition is necessary but not sufficient for white matter protection, and emphasize the existence of multiple Ca2+-dependent degradative pathways activated in injured white matter.

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Acknowledgements

Supported by operating grant # B-3743; PKS is supported by a Career Investigator Award, both from the Heart and Stroke Foundation of Ontario.

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