The effects of 2,5-Hexanedione on the phosphorylation of proteins and phospholidpids in rat sciatic nerve

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2,5-Hexanedione (2,5-HD) is known to produce a central-peripheral distal axonopathy that is characterized by an accumulation of 10 nm neurofilaments (Spencer et al.,1980). The studies described herein test the hypothesis that 2,5-HD alters the phosphorylation of Cytoskeletal proteins resulting in a disruption of axonal cytoskeleton integrity. To test this proposal, temporal studies were conducted which included: 1. 7 day treatment; 2. 15 day treatment; 3. end-point (24 day treatment); and 4. recovery studies (17 day post injection). Male rats were given daily intraperitoneal (i.p.) injections of either 2,5-HD (400 mg/kg/day), or 1,6-hexanediol (414 mg/kg/day) or saline. Animals were sacrificed by decapitation and both sciatic nerves removed. Nerves were incubated for 2 hours in oxygenated Krebs buffer containing 32P orthophosphate. Nerves were then cut into proximal and distal halves and incorporation of radiolabel was assessed by liquid scintillation spectrometry. Immunoblotting was performed to identify proteins exhibiting changes in phosphorylation. Morphological analysis (axonal diameter) was conducted to correlate biochemical changes with nerve damage. Treated animals showed a significant increase in label incorporation in HNF (high molecular weight constituent of the neurofilament triplet) and 55K (tubulin) bands in proximal nerve segments when compared to controls at both 15 day and end-point. Recovery experiments showed no significant differences among control and treated animals in 32P incorporation into protein bands in proximal nerve segments. Morphological analysis showed treated animals to have a significant increase (68%,70%) in fiber diameter both proximally and distally when compared to controls respectively. Immunoblotting positively identified HNF, 68K (low molecular weight constituent of the neurofilament triplet) and 55K as components of the sample protein homogenates. These results suggest that alteration in phosphorylation of Cytoskeletal proteins might play a role in 2,5-HD induced nerve damage.

Neurotoxic agents--Physiological effect, Phospholipids