Regulation of neurofilament axonal transport by phosphorylation in optic axons in situ

Axonal transport of neurofilament (NFs) is considered to be regulated by ph osphorylation. While existing evidence for this hypothesis is compelling, s upportive studies have been largely restricted to correlative evidence and/ or experimental systems involving mutants. We tested this hypothesis in ret inal ganglion cells of normal mice in situ by comparing subunit transport w ith regional phosphorylation state coupled with inhibition of phosphatases. NF subunits were radiolabeled by intravitreal injection of S-35-methionine . NF axonal transport was monitored by following the location of the peak o f radiolabeled subunits immunoprecipitated from 9 x 1.1 mm segments of opti c axons. An abrupt decline transport rate was observed between days 1 and 6 , which corresponded to translocation of the peak of radiolabeled subunits from axonal segment 2 into segment 3. Notably, this is far downstream from the only caliber increase of optic axons at 150 mu from the retina. Immunob lot analysis demonstrated a unique threefold increase between segments 3 an d 3 in levels of a "late-appearing" C-terminal NF-H phospho-epitope (RT97). Intravitreal injection of the phosphatase inhibitor okadaic acid increased RT97 immunoreactivity within retinas and proximal axons. and markedly decr eased NF transport rate out of retinas and proximal axons. These findings p rovide in situ experimental evidence for regulation of NF transport by site -specific phosphorylation, Cell Motil. Cytoskeleton 42:230-240, 1999. (C) 1 999 Wiley-Liss, Inc.