Neurofilament (NF) Assembly; Divergent Characteristics of Human and Rodent NF-L Subunits

Abstract

Previous studies have shown that rodent neurofilaments (NF) are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation. We have assessed the competence of human NF-L and NF-M to assemble and find that unlike rat NF-L, human NF-L is capable of self-assembly. However, human NF-M cannot form homopolymers and requires the presence of NF-L for incorporation into filaments. To investigate the stage at which filament formation is blocked, the rod domains or the full-length subunits of human NF-L, human NF-M, and rodent NF-L were analyzed in the yeast "interaction trap" system. These studies demonstrated that the fundamental block to filament formation in those neurofilaments that do not form homopolymers is at the level of dimer formation. Based on theoretical biophysical considerations of the requirements for the formation of coiled-coil structures, we predicted which amino acid differences were likely to be responsible for the differing dimerization potentials of the rat and human NF-L rod domains. We tested these predictions using site-specific mutagenesis. Interestingly, single amino acid changes in the rod domains designed to restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and human NF-L into a protein that is no longer able to self-assemble. Our results additionally suggest that the functional properties of the L12 linker region of human NF-L, generally thought to assume an extended ␤-sheet conformation, are consonant with an ␣-helix that positions the heptad repeats before and after it in an orientation that allows coiled-coil dimerization. These studies reveal an important difference between the assembly properties of the human and rodent NF-L subunits possibly suggesting that the initiating events in neurofilament assembly may differ in the two species.Intermediate filaments (IFs) 1 are a heterogeneous family of proteins sharing common structural features that can be subdivided into six types (I-VI) based on sequence homology (1). IF genes are expressed in a cell type-specific and developmentally regulated manner with cells frequently containing only a single IF type at a particular stage of differentiation. Neurofilaments (NFs) are the predominant IF in mature neurons but are preceded during neuronal differentiation by a succession of other IFs including vimentin (2) nestin (49), ␣-internexin (3, 4), and peripherin (5). Neurofilaments are assemblies of three subunits, the NF-L (molecular mass, 68 kDa), NF-M (150 kDa), and NF-H (200 kDa) (6). These three components form heteropolymeric 10-nm filaments that run parallel along the length of the axon with frequent cross-bridges between neighboring filaments. Axonal neurofilaments are thought to serve a primarily structural function. Evidence from a Japanese quail (quiverer) with a spontaneous mutation in NF-L (7) and a line of transgenic mice expressing an NF-H-␤ galactosidase fusion protein (8) suggest that a loss of axonal neurofilaments re...

Effects of Charcot-Marie-Tooth-linked mutations of the neurofilament light subunit on intermediate filament formation

“…Previous studies using the Vim -cells have shown that rat and mouse neurofilament triplet proteins are obligate heteropolymers in vivo (Ching and Liem, 1993;Lee et al, 1993). In contrast, human NFL was shown to be able to selfassemble into filaments (Carter et al, 1998). This unique selfassembly property of hNFL was thought to be the result of the differences in the primary sequences between human and rodent NFL in the rod domain.…”

“…Effects of CMT2 mutations on hNFL self-assembly Human NFL has been reported to form homopolymers in vivo, as opposed to rat or mouse NFL (Carter et al, 1998). The authors noted that there were eight differences between rNFL and hNFL in the rod domain.…”

“…Rat and mouse NFs are obligate heteropolymers in vivo, requiring the NFL subunit and either the NFM or NFH subunit for the formation of extensive filamentous networks in the absence of other cytoplasmic IFs (Ching and Liem, 1993;Lee et al, 1993). In contrast, human NFL has been reported to self-assemble into a filamentous network (Carter et al, 1998). Mice null for NFL are viable but display a 15-20% reduction in the number of myelinated axons at 2 months of age.…”

Neurofilaments at a glance

“…Neurons may also express other intermediate filament proteins, including nestin, synemin, syncoilin and vimentin (Perrin et al, 2005). Mouse NFH, NFM and NFL subunits are unable to selfassemble into homopolymer filaments, although -at least in vitro -human NFL can do so (Carter et al, 1998). Among the notable properties of NFs are their exceptionally long half-lives (Millecamps et al, 2007;Nixon and Logvinenko, 1986;Yuan et al, 2009) and their elastic fibrous properties that enable them to maintain the markedly asymmetrical shape of neurons (Wagner et al, 2007).…”

Multicenter Immunoassay Validation of Cerebrospinal Fluid Neurofilament Light: A Biomarker for Neurodegeneration

“…Alternatively, failure to observe parallelism, and indeed some of the other validation parameters, could be in part due to some of the less practiced operators experiencing difficulty with interpretation or complexity of the plate plans and SOPs. It has been argued that as NfL has the tendency to re-aggregate following suspension [39][40][41][42] that delays in sample and standard incubation could account for the failure to satisfactorily measure NfL in many experiments 26 . The data show that between 15 and 60 minutes were taken, from the time standards were reconstituted, to incubate the standards and samples on the ELISA plates.…”