Neurofilaments Consist of Distinct Populations That Can Be Distinguished by C-Terminal Phosphorylation, Bundling, and Axonal Transport Rate in Growing Axonal Neurites

Abstract

We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

Munc18: A Presynaptic Transmitter Release Site N type (CaV2.2) Calcium Channel Interacting Protein

“…For electrophysiological recordings, adherent DRGs were cotransfected with cDNAs encoding the marker, enhanced green fluorescent protein (pEGFP; Clontech, Palo Alto, California) and wild-type Munc18-1 in pCMV or Munc18siRNA (vector pSupersiRNA-Munc18) constructs using Lipofectamine 2000 [25][26][27] (Supplemental data Fig. 2).…”

Neurofilament light chain serum levels correlate with 10‐year MRI outcomes in multiple sclerosis

“…Neurofilament light chain (NfL) is a major component of the neuronal cytoskeleton and is important for axonal growth, stability, and intracellular transport 2, 3. NfL are released upon axonal or neuronal damage or degeneration, and can be found as a consequence, in the CSF and blood.…”

Differential roles of kinesin and dynein in translocation of neurofilaments into axonal neurites

“…Introduction of haptenized exogenous NF fusion proteins into neurons has facilitated analyses of NF axonal transport. NF subunits translocate within axons in multiple forms, including as short NFs, non-filamentous punctate structures ('puncta') and diffuse material that might represent unassembled subunits or hetero-oligomeric subunit assemblies (Jung et al, 1998;Motil et al, 2006;Roy et al, 2000;Prahlad et al, 2000;Theiss et al, 2005;Wang et al, 2000;Yabe et al, 1999;Yabe et al, 2001a;Yabe et al, 2001b;Yuan et al, 2009). The interplay, if any, among these various forms remains unresolved.…”