The polypeptide composition of moving and stationary neurofilaments in cultured sympathetic neurons

Abstract

Studies on the axonal transport of neurofilament proteins in cultured neurons have shown they move at fast rates, but their overall rate of movement is slow because they spend most of their time not moving. Using correlative light and electron microscopy, we have shown that these proteins move in the form of assembled neurofilament polymers. However, the polypeptide composition of these moving polymers is not known. To address this, we visualized neurofilaments in cultured neonatal mouse sympathetic neurons using GFP-tagged neurofilament protein M and performed time-lapse fluorescence microscopy of naturally occurring gaps in the axonal neurofilament array. When neurofilaments entered the gaps, we stopped them in their tracks using a rapid perfusion and permeabilization technique and then processed them for immunofluorescence microscopy. To compare moving neurofilaments to the total neurofilament population, most of which are stationary at any point in time, we also performed immunofluorescence microscopy on neurofilaments in detergent-splayed axonal cytoskeletons. All neurofilaments, both moving and stationary, contained NFL, NFM, peripherin and α-internexin along >85% of their length. NFH was absent due to low expression levels in these neonatal neurons. These data indicate that peripherin and α-internexin are integral and abundant components of neurofilament polymers in these neurons and that both moving and stationary neurofilaments in these neurons are complex heteropolymers of at least four different neuronal intermediate filament proteins.

Review of the Multiple Aspects of Neurofilament Functions, and their Possible Contribution to Neurodegeneration

“…In these studies, intact filaments (1-15 μm of length) represent 95% of the moving structures observed, strongly supporting the polymer model. The transport of NF polymers was recently confirmed by Yan and Brown [326] who showed that NF move in the form of assembled polymers in axons of cultured neonatal mouse sympathetic neurons and, more interestingly, that moving and stationary NF are complex heteropolymers also containing peripherin and α-internexin along >85% of their length [327]. Yuan et al [328] also demonstrated that α-internexin is a key determinant for the axonal transport of NF in CNS.…”

Cerebrospinal fluid biomarkers in Guillain-Barré syndrome – Where do we stand?

“…Neurofilaments are particularly abundant in large myelinated axons [52]. They are obligate heteropolymers that are composed of four subunits: a light (NfL), a medium (NfM) and a heavy (NfH) chain and alpha-internexin [52,53,79]. Elevated levels of CSF neurofilaments have been observed in several neurodegenerative diseases including MS [9,50], amyotrophic lateral sclerosis [8,60] or different subtypes of dementia [7].…”

Neurofilaments at a glance

“…Neurofilaments from the central nervous system (CNS) are heteropolymers that are composed of four subunits, namely neurofilament heavy, medium and light (See poster insert) polypeptides (NFH, NFM and NFL, respectively; also known as NEFH, NEFM and NEFL), as well as a-internexin (Int), whereas in the peripheral nervous system, neurofilaments are made up of NFH, NFM, NFL and peripherin (Beaulieu et al, 1999;Yan et al, 2007;Yuan et al, 2006b). Neurons may also express other intermediate filament proteins, including nestin, synemin, syncoilin and vimentin (Perrin et al, 2005).…”