Interference with kinesin‐based anterograde neurofilament axonal transport increases neurofilament‐neurofilament bundling

Abstract

Neurofilaments (NFs) associate with each other and with other cytoskeletal elements to form a lattice that supports the mature axon. Phosphorylation contributes to formation of this stationary population of NFs by fostering cation-dependent interactions among NF sidearms. Association of NFs with the stationary phase indirectly competes with NF axonal transport by withdrawing NFs from kinesin-dependent motility along microtubules. We therefore hypothesized that inhibition of anterograde NF transport may increase incorporation into the stationary phase. To test this hypothesis, we treated differentiated NB2a/d1 cells expressing GFP-tagged NF subunits with monastrol, a specific inhibitor of kinesin-5. Monastrol significantly inhibited anterograde axonal transport of NF-H but not NF-M, and increased the incorporation of newly-transported NF subunits into axonal NF bundles. These findings support the notion that NF transport and bundling exert opposing forces on axonal NF dynamics, and that inhibition of anterograde transport of NFs can increase their incorporation into the stationary phase.

Neurofilaments and Neurofilament Proteins in Health and Disease

“…The principal motor protein superfamilies (Miki et al 2001; Brown and Bridgman 2004)—MT-based kinesins and dynein, and actin filament-based myosins—have often been considered candidate motors for the translocation of NFs at slow rates (Prahlad et al 2000; Alami et al 2009; Sunil et al 2012). Kinesin-I has been proposed as an anterograde motor for NF based on its reported interactions with NF-H or NF-M subunits (Yabe et al 2000; Jung et al 2005) and on the inhibitory effect of antibodies against kinesin-I on NF transport (Yabe et al 1999).…”

Neurofilaments at a glance

“…The molecular motors that regulate NF transport are believed to be the fast microtubule-based motors kinesin and dynein (Prahlad et al, 2000;Sunil et al, 2012), and microfilament-based motor myosin Va ). Kinesin-I has been proposed to be an anterograde motor for NF because it interacts with the NFH or NFM subunits (Jung et al, 2005;Yabe et al, 2000) and because antibody against kinesin-I blocks NF transport (Yabe et al, 1999).…”

Early expression of the high molecular weight neurofilament subunit attenuates axonal neurite outgrowth

“…Axonal outgrowth and maturation is also critically dependent upon microtubules (MTs) and their associated proteins. Axonal neurites develop a population of long, stationary MTs [30][31][32] that both mediate NF axonal transport [17,19,26] and participate in axonal cytoskeletal stabilization by crosslinking with phospho-NFs via their associated proteins MAP1B and tau [2,10,33]. Of interest would be to manipulate expression of these MAP1B and tau in these cells and in cultured primary neurons, along with manipulation of NF-H, and monitor the impact on axonal neurite elongation and stabilization.…”

“…We previously demonstrated that a GFP-tagged form of NF-H lacking this region (GFP-H∆187) undergoes axonal transport, and incorporates into filamentous structures [9,19]. Herein, cells expressing GFP-H∆187 immediately after differentiation elaborated neurites within 48hr that were statistically identical in length and caliber to those expressing GFP alone, and were statistically longer and thinner than those expressing GFP-H ( Fig.…”