Purpose: Various neurological diseases baring defects in neurofilaments (NF's) are known including Parkinson's disease, Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth disease etc. Though a critical role of NF's has been ascertained little is known about how these diseases develop on the molecular level, critical to facilitate future drug design. Nematodes have been increasingly used to dissect these underlying molecular mechanisms. However, whether an NF homolog exists in the nematode still remains unclear. Therefore, the major goal of this study was to identify and characterise a putative NF homolog in C.elegans. Using bioinformatics tools, we identified TAG-63 with numerous sequence homologies to NEFH, three coiled coils as well as various phosphorylation sites. Methods: We then employed a broad range of techniques like Western blotting, Immunohistochemistry, Worm imaging, motility analysis etc. to delineate the role of
tag-63 in C.elegans. Result: To identify NF homologs, we employed WormBase and received hits from C.elegans gene
tag-63. Using KEGG database, we received hits for NEFH orthologs. Using KEGG, we also note a cluster of NEFH orthologs in a rooted phylogenetic tree emerging from a common TAG-63 ancestor. Though we cannot detect KSP repeats in TAG-63, we identified nine potential phosphorylation sites using Scansite tool and coiled coil prediction tool identified three potential coiled coils in TAG-63. Using worm lysates from N2 wild-type animals, we clearly identified a band around 68kDa in Western blots by a monoclonal mouse anti-NEFH antibody, but this band cannot be detected in lysates from
tag-63 knockout worms VC275 (
ok471). To understand if
tag-63 is expressed in neuronal tissues, we microinjected transcriptional fusion into existing worms. We mostly see expression in a broad range of body, head and tail neurons as well as in sheath cells. Anti-NF antibodies also stain cultured primary nematode neurons and these neurons grow shorter axons when incubated with acrylamide and TCP known to disintegrate the NF network. As it has been reported that NF's affect axonal transport, we investigated the effect of
tag-63 knockout on synaptic vesicle transport. We found that anterograde transport of UNC-104 is significantly reduced in
tag-63 mutant animals pointing to a motor-activating role of C.elegans NF TAG-63. Though we do not reveal similar effects on UNC-104's cargo SNB-1, we do measure increased retrograde movements of this cargo. Further, velocity and flux of kinesin-3 UNC-104(KIF1A) are largely diminished in
tag-63 knockout worms leading to increased accumulations of motors along axons. In summary, we identified and characterised an NEFH ortholog in C.elegans, and demonstrate that lack of this protein limits axonal transport efficiencies suggesting that this model organism may be used for studying neurofilament-based neurological diseases.