Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport

doi:10.1016/0169-328X(89)90001-6

Molecular Brain Research

Volume 5, Issue 2, March 1989, Pages 93-108

https://doi.org/10.1016/0169-328X(89)90001-6 Get rights and content

Abstract

We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [³⁵S]methionine or [³²P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [³⁵S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [³²P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6–18 h for NF-M, 18–24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [³²P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195–210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non-phosphorylated neurofilaments to predominantly phosphorylated ones within a region of the axon between 200 and 1000 μm downstream from the cell body. These experiments demonstrate that the addition of most phosphate groups to NF-M and NF-H takes place within a proximal region of the axon. The rapid appearance of modified forms of NF-L after synthesis may imply that processing of this subunit occurs at least partly in the cell body. The presence of a substantial pool of Triton-insoluble, unmodified subunits early after synthesis indicates that the heaviest incorporation of phosphate occurs after neurofilament proteins are polymerized. Since neurofilaments composed of relatively unmodified NF-M and NF-H may enter axons, completion of phosphorylation is not a requirement for the axoplasmic transport of neurofilaments.

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Research reportEarly posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport

Abstract

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta.

FEBS Lett.

Brain Res.

Brain Res.

J. Biol. Chem.

J. Biol. Chem.

Neurosci. Lett.

J. Biol. Chem.

FEBS Lett.

Monoclonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants

Nature (Lond.)

Light dependent antibody labelling of photoreceptors

Nature (Lond.)

Slow posttranslational modification of a neurofilament protein

J. Cell Biol.

Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons

J. Neurosci.

Slow components of axonal transport: two cytoskeletal networks

J. Cell Biol.

Characterization and comparison of neurofilament proteins from rat and mouse CNS

J. Neurochem.

Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: Dye-binding characteristics and amino acid compositions

J. Neurochem.

Preparation of antisera to neurofilament protein from chicken brain and human sciatic nerve

J. Comp. Neurol.

Masking of epitopes in tissue sections

Antibodies to heavy neurofilament subunit detect a subpopulation of damaged ganglion cells in retina

Nature (Lond.)

Research report
Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport