Phosphorylation of neurofilament subunit NF‐M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape

Abstract

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.

Neurofilament localization and phosphorylation in the developing inner ear of the rat

“…Because regulation of phosphorylation may account for variations in size, morphology and dynamics of the neurofilament network (Shea et al, 2003;Millecamps et al, 2006;Fiumelli et al, 2008;Yates et al, 2009), data on NF phosphorylation in the inner ear during development are essential. It is conceivable that NF phosphorylation in the auditory nerve plays a critical role in the sensitivity of hearing, since growth of the axon diameter is a function of NF phosphorylation (Martin et al, 1999) and axonal caliber determines the conduction velocity of the nerve (Braissant, 2007).…”

Specialized roles of neurofilament proteins in synapses: Relevance to neuropsychiatric disorders

“…On the one hand, D1R is a primary target of stimulant drugs and chronic exposure to drugs of abuse lower levels of NF proteins and alter their phosphorylation state [16]. On the other hand, NMDA receptor inhibition increases the phosphorylation state and the solubility of NFM protein [64]. Glutamate has been shown to inhibit NF transport, which can be reversed by NMDA receptor blocker MK-801 [1].…”

Calcineurin regulation of cytoskeleton organization: a new paradigm to analyse the effects of calcineurin inhibitors on the kidney

“…Today, CaN is known to directly control microtubules assembly, through dephosphorylation of tubulin and microtubules associated proteins such as Tau and MAP-2 [10, 14–16], and neurofilaments organization in cultured-neuronal cells [8, 17, 18]. These effects were confirmed in mice lacking neuronal CaN Aα subunit, which displayed abnormal phosphorylation of microtubules, diminution of neurofilament content and abnormal cytoskeleton organization [19].…”