The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber, and decreases axonal microtubules in NB2a/d1 cells.
When cells were treated with dbcAMP for 3 days to induce the outgrowth of axonal neurites, the addition of the phosphatase inhibitor okadaic acid (OA; 5 nM) for the last 24 hr markedly increased neurofilament subunit immunoreactivity including phosphate-dependent NF-H epitopes in axonal neurites, increased axonal neurite caliber by approximately 30%, but did not increase neurite contour length. Ultrastructural analysis demonstrated a > 2-fold increase in neurofilaments and indicated that neurofilaments were phosphorylated to a similar extent in the presence and absence of OA. Vimentin immunoreactivity, which undergoes down-regulation during dbcAMP-mediated differentiation, was not increased by OA. OA did not induce the precocious appearance of delayed phosphate-dependent neurofilament epitopes suggesting that it did not induce the activation of additional neurofilament kinases. NF-H subunits from cytoskeletons of OA-treated cells were less susceptible to degradation by an endogenous calcium-dependent protease, providing a possible mechanism for neurofilament accumulation during OA treatment. By contrast, OA decreased axonal neurite microtubules, and eliminated stabilized (acetylated) axonal microtubules. OA treatment at earlier times prevented and reversed neurite outgrowth. Despite increased deposition of phosphorylated neurofilaments, OA did not hasten the development of colchicine resistance to neurites, suggesting that stabilization of the axonal cytoskeletal lattice requires neurofilament-microtubule interaction.
Laboratory for Molecular Neuroscience, Mailman Research Center, McLean Hospital, Belmont, MA 02178.
T B
TB
Shea
P A
PA
Paskevich
M L
ML
Beermann
D000818:Animals; D001369:Axons / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D003994:Bucladesine / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology*; D007382:Intermediate Filaments / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D016501:Neurites / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D009447:Neuroblastoma; D016900:Neurofilament Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D014407:Tumor Cells, Cultured
D004988:Ethers, Cyclic; D016900:Neurofilament Proteins; D019319:Okadaic Acid; D003994:Bucladesine; D010749:Phosphoprotein Phosphatases