Elsevier

Neuroscience Research

Volume 139, February 2019, Pages 63-68
Neuroscience Research

Inhibition of collapsin response mediator protein-2 phosphorylation ameliorates motor phenotype of ALS model mice expressing SOD1G93A

https://doi.org/10.1016/j.neures.2018.08.016Get rights and content

Highlights

  • Inhibition of CRMP2 phosphorylation delays progression of the motor symptoms of SOD1G93A-Tg mice.

  • Inhibition of CRMP2 phosphorylation maintains the survival of motor axons in SOD1G93A-Tg mice.

  • Inhibition of CRMP2 phosphorylation maintains innervation of NMJs in SOD1G93A-Tg mice.

  • Phosphorylation of CRMP2 may promote dying back axonal degeneration in motor neurons in ALS.

Abstract

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disease characterized by the selective degeneration of motor neurons leading to paralysis and immobility. Missense mutations in the gene coding for the Cu2+/Zn2+ superoxide dismutase 1 (SOD1) accounts for 15–20% of familial ALS, and mice overexpressing ALS-linked SOD1 mutants have been frequently used as an animal model for ALS. Degeneration of motor neurons in ALS progresses in a manner called “dying back”, in which the degeneration of synapses and axons precedes the loss of cell bodies. Phosphorylation of collapsin response mediator protein 2 (CRMP2) is implicated in the progression of neuronal/axonal degeneration of different etiologies. To evaluate the role of CRMP2 phosphorylation in ALS pathogenesis, we utilized CRMP2 S522A knock-in (CRMP2ki/ki) mice, in which the serine residue 522 was homozygously replaced with alanine and thereby making CRMP2 no longer phosphorylatable by CDK5 or GSK3B. We found that the CRMP2ki/ki/SOD1G93A mice showed delay in the progression of the motor phenotype compared to their SOD1G93-Tg littermates. Histological analysis revealed that the CRMP2ki/ki/SOD1G93A mice retained more intact axons and NMJs than their SOD1G93A-Tg littermates. These results suggest that the phosphorylation of CRMP2 may contribute to the axonal degeneration of motor neurons in ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal, progressiveneurodegenerative disease affecting mostly motor neurons. The vast majority of ALS cases are of sporadic nature, while ∼10% are familial (FALS). Approximately 15%–20% of FALS patients have point mutations in cytosolic Cu2+/Zn2+ superoxide dismutase (SOD1) (Rosen et al., 1993; van Es et al., 2017). SOD1 is an antioxidant enzyme ubiquitously expressed in the cytosol, which converts superoxide anion radicals to hydrogen peroxide. More than 115 disease-causing mutations of SOD1 have been identified throughout the total 153 amino acid residues of SOD1 (Pasinelli and Brown, 2006). Previous studies have shown that the disease is not caused by the loss of its dismutase activity, but by the gain of abnormal properties caused by the mutation (Bruijn et al., 1998). It has also been shown that transgenic mice overexpressing human mutant SOD1 bearing a disease-causing point mutation identified in human ALS patients develop an ALS-like phenotype, including progressive degeneration of spinal motor neurons (Gurney et al., 1994). In the mice expressing human SOD1 bearing G93A mutation (SOD1G93A-Tg mice), degeneration of motor neurons proceeds in a “dying-back” degeneration pattern, in which motor synapses of neuromuscular junctions are lost initially, followed by axonal degeneration and subsequent loss of neuronal cell bodies (Dadon-Nachum et al., 2011; Fischer et al., 2004). “Dying back” degeneration can be explained by both cell autonomous and non-autonomous mechanisms. The cell autonomous mechanism includes impairment of axonal transport. Impaired axonal transport in mutant SOD1-Tg mice has been reported by different groups including ourselves (Tateno et al., 2009; Zhang et al., 1997). With regard to the non-cell-autonomous mechanism, previous reports have shown increased Semaphorin-3A (Sema-3A) expression in terminal Schwann cells of the SOD1G93A-Tg mice and in the motor cortex of ALS patients (De Winter et al., 2006). Sema-3A signals through Neuropilin 1 (NRP1) (Kolodkin and Ginty, 1997) and Plexin1 co-receptor (Takahashi et al., 1999) on neurons at neuromuscular junctions (NMJ) to cause repulsion of the synapse and subsequent degeneration of motor axons.

Sema-3A -NRP1/Plexin1 signaling in motor neurons triggers axonal retraction by destabilizing microtubules and microfilament networks via a mechanism involving the collapsin response mediator protein (CRMP) (Uchida et al., 2005). CRMP is a family of tubulin-binding proteins, and among them CRMP2 serves as a molecule to stabilize microtubule structure in axons (Goshima et al., 1995). Among a variety of signaling pathways elicited by Sema-3A-NRP1/Plexin1, sequential CRMP2 phosphorylation by CDK5 at Ser522, followed by GSK3B at Ser518, Thr514, and Thr509 has been reported as a signal for regulation of microtubule integrity (Brown et al., 2004; Uchida et al., 2005; Yoshimura et al., 2005). Phosphorylated CRMP2 decreases affinity to tubulin and promotes regression of axons (Chae et al., 2009). CRMP2 phosphorylation is implicated in neurodegeneration of various etiologies (Cole et al., 2007; Hensley and Kursula, 2016), but the pathophysiological significance of phosphorylation of CRMP2 has not been established in ALS.

We hypothesized that CRMP2 phosphorylation is involved in ALS pathogenesis, and inhibition of CRMP2 phosphorylation might improve the pathology of ALS. To investigate the role of CRMP2 phosphorylation in ALS, we employed CRMP2 S522 A knock in (CRMP2ki/ki) mice, in which the 522nd serine residue was replaced with alanine, thereby changing CRMP2 in a way that it can no longer be phosphorylated by CDK5 or GSK3B (Yamashita et al., 2012). We crossed CRMP2ki/ki mice with the SOD1G93A-Tg and examined the progression of their ALS-like phenotype under inhibition of CRMP2 phosphorylation. We found that the CRMP2ki/ki/SOD1G93A mice showed delay in the progression of the motor phenotype compared to their SOD1G93A-Tg littermates. Histological analysis revealed that the CRMP2ki/ki/SOD1G93A mice retained more axons and NMJs than their SOD1G93A-Tg littermates. These results suggest that the phosphorylation of CRMP2 may contribute to degeneration of motor neurons in ALS.

Section snippets

Mice

We obtained transgenic mice harboring the G93A-mutated human SOD1 gene (B6SJL-TgN(SOD1-G93A)1Gur) from Jackson Laboratories. CRMP2ki/ki mice in which the 522nd serine residue of CRMP2 was replaced with alanine were established as described previously (Yamashita et al., 2012). Both transgenic mouse lines were backcrossed with C57BL/6J for more than 15 generations.

Transgenic progeny for SOD1 G93A were identified by a polymerase chain reaction (PCR) on genomic tail DNA using a primer set as

Results

To analyze how the ALS-like phenotype of SOD1G93A-Tg mice is affected by the inhibition of CRMP2 phosphorylation, we crossed SOD1G93A-Tg mice with CRMP2ki/ki mice to obtain CRMP2ki/ki/SOD1G93A-Tg mice. To compare general conditions of the CRMP2ki/ki/SOD1G93A mice with SOD1G93A mice, we monitored the body weight of the both lines of mice until reaching humane endpoints. We found that the body weight profile of the CRMP2ki/ki/SOD1G93A-Tg mice was not significantly different from that of SOD1G93A

Discussion

In this study, we reported that the inhibition of the phosphorylation of CRMP2 at Ser522 is sufficient to ameliorate motor abnormality of SOD1G93A-Tg mice. We found progression of axonal degeneration from spinal motor neurons in CRMP2ki/ki/SOD1G93A-Tg mice was delayed at the levels of both ventral root and NMJ compared with that in SOD1G93A-Tg mice. The axons of CRMP2ki/ki/SOD1G93A-Tg mouse motor neurons at the ventral root level seemed more preserved than those at the NMJ level. While the

Conclusions

CRMP2ki/ki/SOD1G93A-Tg mice showed delay in the progression of the motor phenotype compared to their SOD1G93A-Tg littermates. Histological analysis revealed that the CRMP2ki/ki/SOD1G93A-Tg mice retained more axons and NMJs than their SOD1 G93A-Tg littermates. These results suggest that the phosphorylation of CRMP2 may contribute to the degeneration of motor neurons in ALS.

Acknowledgements

This work was supported in part by Intramural Research Grant for Neurological and Psychiatric Disorders of NCNP (T.A.); grants from Takeda Science Foundation (S.W., and T.A.), and Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program in the Project for Developing Innovation System from the MEXT (no. 42890001 to YG).

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