Assembly properties of lamprey neurofilament subunits and their expression after spinal cord transection

Regenerating axon tips in transected sea lamprey (Petromyzon marinus) spinal cord contain dense accumulations of neurofilaments (NFs) (Lurie et al., 1994; Pijak et al., 1996; Hall et al., 1997) and NF expression by individual neurons after axotomy is correlated with their regenerative ability (Jacobs et al., 1997). This has suggested that NFs may play a role in the mechanism of axonal regeneration. NF proteins are intermediate filaments (IFs) found in the neuronal cytoskeleton. Mammalian NFs are composed of three polypeptide subunits: NFL, NFM, and NFH corresponding to light, medium, and heavy molecular mass. Rodent NFs are obligate heteropolymers, i.e., NFL must bind to either NFM or NFH to form filaments. By comparison, NFs in lampreys had been thought to contain only a single subunit, NF180 (Jacobs et al., 1995), which implied that NF180 self-assembles. However, in a previous study (Zhang et al., 2004) NF180 could not self-assemble in vitro, nor in cell cultures, including mammalian cell lines cultured at 37°C and fish cell lines cultured at lower temperatures. Furthermore, NF180 appeared to disrupt self-polymerization of rat NFL in vitro, and NF180 formed thick, rod-like filamentous structures when coexpressed with rat NFL in SW13c1.2Vim− cells, which lack endogenous IFs. More typical filament networks were observed when NF180 was coexpressed with NFL plus NFM. Transfection of NF180 into NB2a/d1 neuroblastoma cells, which contain endogenous NFs (Shea et al., 1985) resulted in the incorporation of NF180 into NFs (Hall et al., 2000). Thus, NF180 may require an NFL-like protein and possibly additional elements to form normal filaments in lamprey. Indeed, a 50 kDa neuronal IF (nlF50) was described based on sodium dodecyl sulfate (SDS) gels and immunohistochemistry (Jin et al., 2005). In attempting to clone this missing nIF we recently cloned and sequenced three new NF genes (Jin et al., 2011), two of them NFM-like peptides (NF132 and NF95) and the third a lamprey NFL homolog (L-NFL). None of the new sequences represented nlF50, the sequence of which is still unknown. In the present study the assembly properties of the four sequenced lamprey NFs and of truncated NF180 constructs were determined in SW13cl.2Vim−, a well-established system for assessing the assembly requirements of IFs, including NFs, due to its lack of any endogenous IF proteins (Sarria et al., 1994). The results suggest that L-NFL is essential to NF assembly and the full length of NF180 appears to be essential for the formation of thick NF bundles. In situ hybridization shows that like NF180, after spinal cord transection, expression of these newly discovered NFs first decreases, and then is recovered selectively in neurons whose axons have a high probability of regenerating.