2,5-Hexanedione (HD) intoxication is associated with axon
atrophy that might be responsible for the characteristic gait abnormalities, hindlimb skeletal muscle weakness and other neurological deficits that accompany neurotoxicity. Although previous mechanistic research focused on
neurofilament triplet proteins (NFL, NFM, NFH), other cytoskeletal targets are possible. Therefore, to identify potential non-NF
protein targets, we characterized the effects of HD on
protein-
protein interactions in cosedimentation assays using microtubules and NFs prepared from spinal cord of rats intoxicated at different daily dose rates (175 and 400 mg/kg/day). Results indicate that HD did not alter the presence of alpha- or beta-tubulins in these preparations, nor were changes noted in the distribution of either anterograde (KIF1A, KIF3, KIF5) or retrograde (
dynein) molecular motors. The cosedimentation of
dynactin, a
dynein-associated
protein, also was not affected. Immunoblot analysis of
microtubule-associated proteins (MAPs) in microtubule preparations revealed substantial reductions (45-80%) in MAP1A,
MAP1B heavy chain, MAP2, and tau regardless of HD dose rate.
MAP1B light chain content was not altered. Finally, HD intoxication did not influence native NF
protein content in either preparation. As per previous research, microtubule and NF preparations were enriched in high-molecular weight NF species. However, these NF derivatives were common to both HD and control samples, suggesting a lack of pathognomonic relevance. These data indicate that, although motor
proteins were not affected, HD selectively impaired MAP-microtubule binding, presumably through adduction of
lysine residues that mediate such interactions. Given their critical role in cytoskeletal physiology, MAPs could represent a relevant target for the induction of gamma-diketone axonopathy.