α-Internexin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament (NF) triplet proteins (NF-L, NF-M, and NF-H) but has an unknown function. The earlier peak expression of α-internexin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that α-internexin and neurofilament triplet form separate filament systems. Here, we demonstrate, however, that despite a postnatal decline in expression, α-internexin is as abundant as the triplet in the adult CNS and exists in a relatively fixed stoichiometry with these subunits. α-Internexin exhibits transport and turnover rates identical to those of triplet proteins in optic axons and colocalizes with NF-M on single neurofilaments by immunogold electron microscopy. α-Internexin also coassembles with all three neurofilament proteins into a single network of filaments in quadruple-transfected SW13vim(−) cells. Genetically deleting NF-M alone or together with NF-H in mice dramatically reduces α-internexin transport and content in axons throughout the CNS. Moreover, deleting α-internexin potentiates the effects of NF-M deletion on NF-H and NF-L transport. Finally, overexpressing a NF-H–LacZ fusion protein in mice induces α-internexin and neurofilament triplet to aggregate in neuronal perikarya and greatly reduces their transport and content selectively in axons. Our data show that α-internexin and the neurofilament proteins are functionally interdependent. The results strongly support the view that α-internexin is a fourth subunit of neurofilaments in the adult CNS, providing a basis for its close relationship with neurofilaments in CNS diseases associated with neurofilament accumulation.

Neurofilaments assemble from three intermediate-filament proteins, contribute to the radial growth of axons, and are exceptionally stable. Microtubules are dynamic structures that assemble from tubulin dimers to support intracellular transport of molecules and organelles. We show here that neurofilaments, and other intermediate-filament proteins, contain motifs in their N-terminal domains that bind unassembled tubulin. Peptides containing such motifs inhibit thein vitropolymerization of microtubules and can be taken up by cultured cells in which they disrupt microtubules leading to altered cell shapes and an arrest of division. In transgenic mice in which neurofilaments are withheld from the axonal compartment, axonal tubulin accumulation is normal but microtubules assemble in excessive numbers. These observations suggest a model in which axonal neurofilaments modulate local microtubule assembly. This capacity also suggests novel mechanisms through which inherited or acquired disruptions in intermediate filaments might contribute to pathogenesis in multiple conditions.