Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than move extensively phosphorylated subunits in situ

Axonal transport of neurofilaments (NFs) has long been considered to be reg ulated by phosphorylation. We present evidence that in optic axons of norma l mice, the rate of NF axonal transport is inversely correlated with the NF phosphorylation state. In addition to 200 kDa NF-H and 145 kDa NF-M, axona l cytoskeletons from CNS contained a range of phospho-variants of NF-H migr ating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 k Da phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-varian t of NF-M since (I) pulse-chase metabolic labeling confirmed the 97 kDa spe cies to be a new synthesis product that was converted by phosphorylation ov er time into a form migratings at 145 kDa, (2) the 97 kDa protein reacted w ith multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autorad iographic analyses following metabolic radiolabeling demonstrated that hypo phosphorylated NF-H and NF-M isoforms underwent substantially more rapid tr ansport in situ than did extensively phosphorylated isoforms, while NF-H su bunits bearing a developmentally delayed C-terminal phospho-epitope transpo rted at a rate slower than that of total 200 kDa NF-H. Differential transpo rt of phospho-variants also highlights that these variants are not homogene ously distributed among NFs, but are segregated to some extent among distin ct, although probably overlapping, NF populations, indicating that axonal N Fs are not homogeneous with respect to phosphorylation state. (C) 2000 Wile y-Liss, Inc.