Elsevier

Neuroscience Letters

Volume 519, Issue 1, 21 June 2012, Pages 56-61
Neuroscience Letters

CRMP4 mediates MAG-induced inhibition of axonal outgrowth and protection against Vincristine-induced axonal degeneration

https://doi.org/10.1016/j.neulet.2012.05.020Get rights and content

Abstract

Suppression of inhibition of axonal outgrowth and promotion of axonal protection from progressive axonal degeneration are both therapeutic strategies for the treatment of neuronal diseases characterized by axonal loss. Myelin-associated inhibitors (MAIs) have been shown to suppress axonal outgrowth, but a specific MAI, myelin-associated glycoprotein (MAG), has also been shown to protect neurons from axonal degeneration through activation of the small GTPase protein RhoA. Recent in vitro studies have shown that collapsin response mediator protein 4 (CRMP4) interacts with RhoA and that the CRMP4b/RhoA complex mediates MAG-induced inhibitory signaling against axonal outgrowth. However, whether CRMP4 is involved in MAG-mediated axon protection signaling remains unclear. Here, we show involvement of CRMP4 in MAG-induced inhibition of axonal outgrowth and axonal protection using the CRMP4−/− mouse model. In dorsal root ganglion (DRG) neurons, loss of CRMP4 prevents MAG-induced inhibition of axonal outgrowth and growth cone collapse and increases sensitivity to microtubule destabilizing factor Vincristine (VNC)-induced axonal degeneration. MAG-mediated axon protection against VNC is suppressed in CRMP4−/− DRG neurons. Understanding the molecular mechanism of MAG-mediated inhibition and protection via CRMP4 may provide novel opportunities to control axonal degeneration and regeneration.

Highlights

► We examine the role of CRMP4 in MAG-induced axonal responses using CRMP4−/− mice. ► We show involvement of CRMP4 in MAG-induced inhibition of axonal outgrowth. ► MAG-induced axonal protection against acute toxic insult also requires CRMP4. ► CRMP4−/− DRG neurons have elevated sensitivity to Vincristine-induced axonal degeneration.

Introduction

Axonal degeneration and limited regeneration are common features of many neurodegenerative diseases, such as Alzheimer disease and Guillain–Barré syndrome, as well as, in axonal injuries caused by toxic, ischemic, or traumatic insults [6]. To treat such neurological diseases and axonal injuries, strategies have focused on suppression of axonal degeneration and promotion of neuronal regeneration. Axonal regeneration is limited by the activation of an intracellular signaling pathway within the injured neurons. Specifically, myelin-associated inhibitors (MAIs) lead to failure of spontaneous regeneration in injured axons by binding the Nogo receptor, expressed on axonal membranes, and activating the small GTPase protein RhoA to mediate inhibition of neurite outgrowth [11]. Disruption of Rho-GTPase-dependent cytoskeleton dynamics promotes axonal regeneration both in vitro and in vivo [3], [7], [8]. In addition, one of the MAIs, myelin-associated glycoprotein (MAG), may function in the inhibition of neurite outgrowth [12], [15], [19]. MAG also gained the attention of researchers because of its protective effect against acute toxic insults on axons. MAG−/− mice undergo progressive axon degeneration in the central and peripheral nervous system that results in increased vulnerability to axonal degeneration from additional stresses [16], [18], [26]. These results suggest that MAG maintains axonal stability. In vitro studies have shown that MAG binds gangliosides, another MAG receptor on axonal membranes, and protects degenerating axons from acute toxic insult also via Rho-GTPase [14], [16].

Recent evidence has linked the collapsin response mediator protein (CRMP) family, which consists of 5 cytosolic phosphoproteins that are expressed redundantly in the developing nervous system [4], [22], [23], with MAI-induced inhibition of axonal outgrowth. CRMP4 mechanically regulates F-actin bundling and binds tubulin [9], [20]. The CRMP4 allele produces 2 transcripts that differ in their N-terminus length and have been referred to as “a” (short isoform) and “b” (long isoform) [27]. In vitro studies have shown that siRNA-mediated knockdown of CRMP4, which suppressed the expression of both CRMP4a and CRMP4b attenuates MAIs-mediated inhibition of neurite outgrowth and that CRMP4b interacts with RhoA physically and functionally in a MAI-dependent manner [1]. However, the involvement of CRMP4 in MAG-mediated axon protection signaling remains unknown.

Here, we analyzed the involvement of CRMP4 in these 2 MAG-mediated functions using our recently established CRMP4−/− mouse model [17]. Dorsal root ganglion (DRG) neurons derived from postnatal CRMP4−/− mice exhibit a marked reduction in MAG-induced inhibition of axonal outgrowth and growth cone collapse, indicating the requirement for CRMP4 in this process. We also demonstrate that CRMP4−/− DRG neurons have enhanced sensitivity to microtubule destabilizing factor Vincristine (VNC)-induced axonal degeneration, mimicking axonal degeneration and suggesting that CRMP4 is also necessary for microtubule stabilization. It is interesting that MAG-mediated axonal protection from VCN-induced axonal degeneration is remarkably suppressed in CRMP4−/− DRG neurons. These findings implicate CRMP4 is involved in both MAG-mediated axonal outgrowth inhibition and axonal protection.

Section snippets

Mice

The mice used in the experiments were housed in accordance with the protocols approved by the Institutional Animal Care and Use Committee of Waseda University. CRMP4 mutant mice were generated and genotyped as described previously [17]. CRMP4+/+ and CRMP4−/− mice were obtained from the mating of intercrossing of offspring of CRMP4+/− × CRMP4+/− mating for experiments.

Axonal outgrowth assay

DRG neurons derived from postnatal day (P) 3–8 mice were dissociated with collagenase type III (Worthington) for 2 h and 0.25%

MAG-induced inhibition of axonal outgrowth is attenuated in CRMP4-null DRG neurons

A previous study has shown that siRNA-mediated knockdown of CRMP4 expression attenuates inhibition of neurite outgrowth with myelin [1]. Although CRMP4 gene function is greatly attenuated by siRNA, it is not lost completely. To confirm whether CRMP4 function is necessary for MAG-dependent neurite outgrowth inhibition, axonal outgrowth inhibition by MAG-Fc was assessed in dissociated DRG neurons using the recently generated CRMP4−/− mice [17]. Axonal outgrowth of CRMP4+/+ DRG neurons was

Discussion

A previous study has shown that siRNA-mediated knockdown of CRMP4 expression attenuates inhibition of neurite outgrowth with myelin. Although siRNA robustly inhibits CRMP4 expression, it is not lost completely [1]. We confirmed that knockout of CRMP4 attenuates MAG-induced inhibition of axonal outgrowth using DRG neurons derived from CRMP4−/− mice in which complete loss of CRMP4 has been demonstrated [17]. RhoA and CRMP4b co-localize within the growth cone after myelin stimulation, suggesting

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