Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ.
Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation. We present evidence that in optic axons of normal 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, axonal cytoskeletons from CNS contained a range of phospho-variants of NF-H migrating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 kDa phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-variant of NF-M since (1) pulse-chase metabolic labeling confirmed the 97 kDa species to be a new synthesis product that was converted by phosphorylation over time into a form migrating at 145 kDa, (2) the 97 kDa protein reacted with multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autoradiographic analyses following metabolic radiolabeling demonstrated that hypophosphorylated NF-H and NF-M isoforms underwent substantially more rapid transport in situ than did extensively phosphorylated isoforms, while NF-H subunits bearing a developmentally delayed C-terminal phospho-epitope transported at a rate slower than that of total 200 kDa NF-H. Differential transport of phospho-variants also highlights that these variants are not homogeneously distributed among NFs, but are segregated to some extent among distinct, although probably overlapping, NF populations, indicating that axonal NFs are not homogeneous with respect to phosphorylation state.
Jung
C
C
Center for Cellular Neurobiology and Neurodegeneration Research, Department of Biological Sciences, University of Massachusetts-Lowell, 01854, USA.
Yabe
J T
JT
Lee
S
S
Shea
T B
TB
D000818:Animals; D001370:Axonal Transport; D001369:Axons / Q000502:physiology*; D015153:Blotting, Western; D051379:Mice; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000276:immunology / Q000378:metabolism*; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000276:immunology / Q000378:metabolism; D021122:Protein Subunits; D012165:Retinal Ganglion Cells / Q000276:immunology / Q000502:physiology*
D016900:Neurofilament Proteins; D010455:Peptides; D020033:Protein Isoforms; D021122:Protein Subunits; D010744:Phosphoric Monoester Hydrolases