Enhanced ex vivo cosedimentation of high molecular weight neurofilament protein (NFH) with microtubules following in vivo aluminum chloride exposure: inhibition of dephosphorylation-dependent dissociation.

We have investigated the effect of acute in vivo aluminum exposure on the subsequent ex vivo cross-linking of the high molecular weight neurofilament protein (NFH) with polymerized microtubules. Young adult female New Zealand white rabbits were inoculated intracisternally with 1000 micrograms of AlCl3 in 0.9% NaCl or with 0.9% NaCl alone, and killed 48 hours later. Following isolation of a cytoskeletal-enriched protein fraction from the cervical spinal cord, NFH was purified by either electroelution or column chromatography. Tubulin was isolated from New Zealand white rabbit brains by repeated temperature-dependent polymerization and depolymerization, purified over phosphocellulose, and cosedimented with either phosphorylated or dephosphorylated NFH. Following incubation for 30 minutes at 32 degrees C with tubulin in the presence of 20 microM Taxol, 1.0 mM MgCl2 and 1.0 mM GTP, the insoluble pellet containing NFH/microtubules was isolated. Both the pellet and supernatent were fractionated by SDS.PAGE and the amount of NFH present quantified by transmission densitometry following silver-staining. Results were identical regardless of the technique utilized for the purification of NFH. Control NFH preferentially cosedimented with microtubules when in the fully phosphorylated isoform, but remained in the soluble fraction following dephosphorylation. Phosphorylated NFH derived from AlCl3-inoculated rabbits demonstrated similar binding characteristics to control NFH, but following exhaustive dephosphorylation, exhibited a 4.5 fold induction of NFH/microtubule binding (p = 0.0314). Incubating dephosphorylated control NFH with microtubules in the presence of increasing concentrations of AlCl3 failed to induce similar cosedimentation. These experiments suggest that phosphorylation promotes NFH cross-linking to microtubules. In addition, the phosphorylation/dephosphorylation dependent regulation of NFH cross-linking to microtubules is disrupted following in vivo AlCl3 exposure by a mechanism that s independent of NFH/Al3+ binding.