Effects of 2,5-hexanedione on calpain-mediated degradation of human neurofilaments in vitro.
2,5-Hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, can structurally modify neurofilaments (NF) by pyrrole adduct formation and subsequent covalent cross-linking. 2,5-HD also induces accumulations of NF within the pre-terminal axon. We examined whether exposure of NF to 2,5-HD affected NF degradation. Two different models were used: (1) NF-enriched cytoskeletons isolated from human sciatic nerve were incubated with 2,5-HD in vitro and (2) differentiated human neuroblastoma cells (SK-N-SH) were exposed to 2, 5-HD in culture prior to isolation of cytoskeletal proteins. The cytoskeletal preparations were subsequently incubated with calpain II. The amount of NF-H and NF-L remaining after proteolysis was determined by SDS-PAGE and quantitative immunoblotting. NF-M proteolysis could not be quantified. Incubation of sciatic nerve cytoskeletal preparations with 2,5-HD resulted in cross-linking of all three NF proteins into high molecular weight (HMW) material with a range of molecular weights. Proteolysis of the NF-H and NF-L polypeptides was not affected by 2,5-HD-exposure. Degradation of the HMW material containing NF-H or NF-L was retarded when comparing with degradation of the NF-H and NF-L polypeptides, respectively, from control samples, but not as compared to the corresponding NF polypeptides from 2,5-HD-treated samples. Exposure of SK-N-SH cells to 2,5-HD also resulted in considerable cross-linking of NF. No differences were found between the proteolytic rates of NF-L and NF-H from exposed cells as compared with those subunits from control cells. Moreover, degradation of cross-linked NF-H was not different from monomeric NF-H. In conclusion, whether 2,5-HD affects calpain-mediated degradation of cross-linked NF proteins will depend on which model better reflects NF cross-linking as occurring in 2, 5-HD-induced axonopathy. However, with both models it was demonstrated that exposure of NF proteins to 2,5-HD without subsequent cross-linking is not adequate to inhibit NF proteolysis in vitro by added calpain.
Heijink
E
E
Coronel Institute for Occupational and Environmental Health, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE, Amsterdam, The Netherlands.
Scholten
S W
SW
Bolhuis
P A
PA
de Wolff
F A
FA
D000328:Adult; D002154:Calpain / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000032:analysis; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure*; D005260:Female; D006588:Hexanones / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008297:Male; D009447:Neuroblastoma; D009454:Neurofibrils / Q000187:drug effects / Q000648:ultrastructure; D016900:Neurofilament Proteins / Q000032:analysis; D009498:Neurotoxins / Q000494:pharmacology*; D012584:Sciatic Nerve / Q000187:drug effects / Q000648:ultrastructure*; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D006588:Hexanones; D016900:Neurofilament Proteins; D009498:Neurotoxins; C011269:2,5-hexanedione; D002154:Calpain