Regulation of neurofilament interactions in vitro by natural and synthetic polypeptides sharing Lys-Ser-Pro sequences with the heavy neurofilament subunit NF-H: neurofilament crossbridging by antiparallel sidearm overlapping.

Neurofilaments are organised into parallel bundles in axons through crossbridges formed by lateral projections of neurofilament subunits. Pure neurofilaments form gels in vitro, consisting of interconnected parallel arrays of filaments regulated by the phosphorylation level of neurofilament subunits. Neurofilament-associated polypeptides sharing phosphorylated epitopes with the repetitive lysine-serine-proline (Lys-Ser-Pro) motifs of the neurofilament heavy subunit sidearm are characterised: they regulate in vitro the neurofilament gelation kinetics in a concentration- and phosphorylation-dependent manner. Studies with synthetic peptides show that interactions between neurofilaments involve both acid and base amino acid residues of neurofilament sidearms and demonstrate the opposite effects of peptides containing either one (inhibition) or two (activation) Lys-Ser-Pro motifs. Electron microscopy reveals an organised network of native neurofilament sidearms, regulated by the phosphorylation level of neurofilament subunits, suggesting a structural transition between intra- and inter-neurofilament sidearm interactions. These results favour the hypothesis of a mechanism of neurofilament crossbridging through the variable antiparallel overlapping of the phosphorylable Lys-Ser-Pro domains of neurofilament sidearms from adjacent filaments, following an equilibrium regulated by neurofilament-associated proteins, bivalent cations and the phosphorylation level of Lys-Ser-Pro motifs from both neurofilament sidearms and neurofilament-associated proteins.

Possible involvement of ubiquitination in neurofilament degradation.

Ubiquitinated proteins are components of intraneuronal inclusions found in several degenerative diseases. Immunohistochemical studies of neurofilament accumulations in Lewy bodies suggest their possible ubiquitination. We investigated in the present work the presence and the nature of ubiquitin epitopes in purified neurofilament preparations from spinal cord. Ubiquitin antibodies consistently label the medium molecular weight neurofilament subunit, and to a lower extent the two other subunits of the neurofilament triplet. Ubiquitinated neurofilament epitopes are removed in vitro by incubation of neurofilaments with a deubiquitinase purified from nervous tissues. Studies of neurofilament degradation in vitro revealed that addition of ATP and exogenous ubiquitin stimulates the proteolysis of neurofilament by crude soluble fractions from nervous tissues. These observations favor the hypothesis of a physiological function of ubiquitin-associated pathways in degradation of neurofilaments in situ.

Progressive hyperphosphorylation of neurofilament heavy subunits with aging: possible involvement in the mechanism of neurofilament accumulation.

Abnormal accumulations of phosphorylated neurofilaments occur both in normal senescence and in age-associated neurodegenerative diseases. In the present work, we study the physicochemical properties of neurofilaments isolated from rats of controlled ages. Aging induces in vivo hyperphosphorylation of the heavy neurofilament subunit without affecting in vitro neurofilament phosphorylation by the neurofilament-associated protein kinase. Interactions in vitro between neurofilaments from very old rats occur at higher rate and extent than that of neurofilaments from younger animals. These results support the hypothesis that the abnormal accumulation of neurofilaments observed in nervous tissues from aging mammals results from an altered equilibrium in situ between interconnected and independent neurofilaments.

Naftidrofuryl, a putative activator of neuron survival, stimulates the expression of neurofilament heavy subunit in cultivated spinal cord neurons from chicken.

The effect of naftidrofuryl, a drug used in ischemia for its vasodilator properties and its protective effect on neuronal survival, was investigated on the maturation of cultured chicken spinal cord neurons, focusing on the presence of proteins specific for the developing neuronal cytoskeleton. Although no influence of naftidrofuryl on the rate of growth of neurites was observed, the drug enhanced the relative amount of the high molecular weight neurofilament subunit without affecting the concentration of a microtubule-associated protein, MAP2. These findings suggest that the effect of naftidrofuryl on cultured spinal cord neurons might involve molecular events directly associated with the induction of a mature cytoskeleton architecture, instead of stimulating undifferentiated neurite growth.