STABLE INTERACTION BETWEEN G-ACTIN AND NEUROFILAMENT LIGHT SUBUNIT INDOPAMINERGIC-NEURONS

Excessive accumulation of neurofilaments in the cell bodies and proxim al axons of motor neurons is a major pathological hallmark of motor ne uron diseases. In this communication we provide evidence that the neur ofilament light subunit (68 kDa) and G-actin are capable of forming a stable interaction. Cytochalasin B, a cytoskeleton disrupting agent th at interrupts actin-based microfilaments, caused aggregation of neurof ilaments in cultured mesencephalic dopaminergic neurons, suggesting a possible interaction between neurofilaments and actin; which was teste d further by using crosslinking reaction and affinity chromatography t echniques. In the cross-linking experiment, G-actin interacted with in dividual neurofilament subunits and covalently cross-linked with disuc cinimidyl suberate, a homobifunctional crosslinking reagent. Furthermo re, G-actin was extensively cross-linked to the light neurofilament su bunit with this reagent. The other two neurofilament subunits showed n o cross-linking to G-actin. Moreover, neurofilament subunits were reta ined on a G-actin coupled affinity column and were eluted from this co lumn by increasing salt concentration. All three neurofilament subunit s became bound to the G-actin affinity column. However, a portion of t he 160 and 200 kDa neurofilament subunits did not bind to the column, and the remainder of these two subunits eluted prior to the 68 kDa sub unit, suggesting that the light subunit exhibited the highest affinity for G-actin. Moreover, neurofilaments demonstrated little or no bindi ng to F-actin coupled affinity columns. The phosphorylation of neurofi lament proteins with protein kinase C reduced its cross-linking to G-a ctin. The results of these studies are interpreted to suggest that the interaction between neurofilaments and actin, regulated by neurofilam ent phosphorylation, may play a role in maintaining the structure and hence the function of dopaminergic neurons in culture. (C) 1997 Elsevi er Science Ltd. All rights reserved.