De novo non-synonymous DPYSL2 (CRMP2) variants in two patients with intellectual disabilities and documentation of functional relevance through zebrafish rescue and cellular transfection experiments

Abstract

Collapsin response mediator protein 2 (Crmp2) is an evolutionarily well-conserved tubulin-binding cytosolic protein that plays critical roles in the formation of neural circuitry in model organisms including zebrafish and rodents. No clinical evidence that CRMP2 variants are responsible for monogenic neurogenic disorders in humans presently exists. Here, we describe two patients with de novo non-synonymous variants (S14R and R565C) of CRMP2 and intellectual disability associated with hypoplasia of the corpus callosum. We further performed various functional assays of CRMP2 variants using zebrafish and zebrafish Crmp2 (abbreviated as z-CRMP2 hereafter) and an antisense morpholino oligonucleotide [AMO]-based experimental system in which crmp2-morphant zebrafish exhibit the ectopic positioning of caudal primary (CaP) motor neurons. Whereas the co-injection of wild-type z-CRMP2 mRNA suppressed the ectopic positioning of CaP motor neurons in Crmp2-morphant zebrafish, the co-injection of R566C or S15R, z-CRMP2, which corresponds to R565C and S14R of human CRMP2, failed to rescue the ectopic positioning. Transfection experiments of zebrafish or rat Crmp2 using plasmid vectors in HeLa cells, with or without a proteasome inhibitor, demonstrated that the expression levels of mutant Crmp2 protein encoded by R565C and S14R CRMP2 variants were decreased, presumably because of increased degradation by proteasomes. When we compared CRMP2-tubulin interactions using co-immunoprecipitation and cellular localization studies, the R565C and S14R mutations weakened the interactions. These results collectively suggest that the CRMP2 variants detected in the present study consistently led to the loss-of-function of CRMP2 protein and support the notion that pathogenic variants in CRMP2 can cause intellectual disabilities in humans.

Introduction

Collapsin response mediator protein 2 (Crmp2) is an evolutionarily well-conserved tubulin-binding cytosolic protein that plays critical roles in the formation of neural circuitry (1). Crmp2 was first identified in chick dorsal root ganglia as a protein responsible for growth cone retraction evoked by negative guidance signals in the Sem3A pathway of the developing central nervous system. During this process, Crmp2 binds to multiple proteins involved in microtubule dynamics, endocytosis and vesicle recycling, synaptic assembly, calcium channels and neurotransmitters. Crmp2 has been shown to be critical in the establishment of functional neuronal circuits in the developing central nervous system of zebrafish (2).

Reflecting the critical role of non-human Crmp2 in the development of neural circuitry in zebrafish and mice, the human CRMP2 gene has been implicated in a wide variety of neurological disorders including neurodegenerative disorders (e.g. Alzheimer (3)), sensory and motor neuron disorders (ALS (4), Batten disease (5), Huntington’s disease (6)) and central disorders (e.g. epilepsy, bipolar disorder, schizophrenia) (7–11). Several genome-wide association studies have mapped CRMP2 as a susceptibility locus for schizophrenia (12–14). Moreover, the loss-of-function intolerance scores calculated from a large human population without severe problems with a pediatric onset predicts that CRMP2 has a high score of 0.99 (15). Hence, haploinsufficiency for CRMP2 in humans reduces genetic fitness to zero from an epidemiologic standpoint. Nevertheless, no clinical evidence that CRMP2 variants are responsible for monogenic neurogenic disorders in humans presently exists. Here, we describe two patients with de novo non-synonymous variants (S14R and R565C) of CRMP2 who had intellectual disability associated with hypoplasia of the corpus callosum. Since brain-specific Crmp2 deletion in mice leads to neuronal development deficits and behavioral impairments associated with hypoplasia of the corpus callosum (16,17), these CRMP2 variants observed in the two patients may well have functional relevance.

We further performed various functional assays of CRMP2 amino acid substitutions using zebrafish and zebrafish Crmp2 (abbreviated as z-CRMP2 hereafter) and an antisense morpholino oligonucleotide [AMO]-based experimental system, which we developed previously: with this system, zebrafish spinal motor neurons can be used to study the cellular and molecular mechanisms that control neuronal specification and their positioning (2). Specifically, the injection of AMO of z-CRMP2 into fertilized zebrafish eggs at the one-to-two-cell stage causes the abnormal positioning of caudal primary (CaP) motor neurons outside the spinal cord in the injected embryos. These embryos subjected to the knock-down effect of z-CRMP2 AMO are collectively referred to as ‘z-CRMP2 morphants’. In the experimental system, the z-CRMP2 AMO was designed so that it would target the first codon of z-CRMP2.

Taking advantage of the high evolutionary conservation (i.e. 89.65%) of the human CRMP2 protein sequence and the z-CRMP2 protein, we tested whether the co-injection of z-CRMP2 AMO and in vitro-transcribed wild-type CRMP2 mRNA or mutant human CRMP2 mRNA encoding either the S14R or the R565C variant could rescue the abnormal positioning of CaP motor neurons in z-CRMP2 morphants. We also evaluated the expression levels of the mutant proteins by transfecting plasmid expression vectors into HeLa cells.

Clinical Reports

Patient 1

Patient 1 was a 12-year-old boy who was born to non-consanguineous Japanese parents with no significant family medical history. He was born at 37 weeks of gestation via a caesarean section. His birth weight was 2750 g (0 SD), his height was 47.4 cm (+0.03 SD) and his head circumference was 33.0 cm (+0.23 SD). Dysmorphic features at birth included a large mouth, broad forehead, high palate, short 5th finger and mild clinodactylies. He experienced respiratory distress requiring 3 days of ventilator management. He also required tube feeding until the age of 2 years and 10 months. He had a severe global developmental delay: he was able to walk with support at the age of 2 years and 8 months, he could walk independently at 5 years and he started speaking meaningful words at 5 years. At the age of 6 years, he developed hyperreflexia in his extremities. His intelligence quotient was 27. He was diagnosed as having premature puberty at the age of 9 years after the growth of pubic hair. At the age of 12 years, his most recent examination, his height was 153 cm (+0.51 SD) and his weight was 58 kg (+1.59 SD). When asked his name, he could respond correctly. All his responses were limited to a single word. His facial features are shown in Figure 1A–C. A physical examination revealed hyperactive tendon reflexes in his extremities, and the ankle clonus test was positive. Brain magnetic resonance imaging at the age of 9 years showed ventriculomegaly and a thin corpus callosum. An exome analysis revealed that the patient had a de novo heterozygous variant in the CRMP2 gene (NM_1386.6): c.1693C > T, Chr8 (GRCh37): g.26513196C > T, p.(Arg565Cys). CRMP2 is also known as DPYSL2. Rare variants (i.e. allele frequency less than 0.01 among 8300 Japanese) that were de novo heterozygous, homozygous, compound heterozygous or hemizygous were detected and have been summarized in Supplementary Material, Table S1.

Figure 1

Photographs of patients. (A–C) Patient 1. Note high anterior hairline, arched and thick eyebrows and thick lips. (D–G) Patient 2. Note high anterior hairline, arched and thick eyebrows, hypertelorism, malocclusion of teeth and micrognathia.

Patient 2

Patient 2 was a 9-year-old girl who was born to non-consanguineous Japanese parents. Her older brother had died at the age of 7 months from an unknown cause. At birth, her weight was 2401 g (−1.21 SD), her height was 48 cm (−0.05 SD) and her head circumference was 31 cm (−1.46 SD). Dysmorphic features included frontal bossing, large ears, large eyes, high arched eyebrows, hypertelorism and micrognathia (Fig. 1D–G). At 2 months of age, she developed upper airway stenosis arising from tracheomalacia. She experienced feeding difficulty because of muscular hypotonia, and she underwent a gastrostomy at the age of 1 year and 6 months. At the age of 5 years, she underwent glottic closure and a laryngeal tracheostomy because of recurrent dysphagic pneumonia and apnea. Brain magnetic resonance imaging showed a thin corpus callosum and cerebellar hypoplasia. An electroencephalogram did not detect any abnormal waves, but she had a history of myoclonic seizures and status epilepticus. At the age of 9 years, she had a profound global developmental delay. She was bedridden and could not roll over by herself. An exome analysis revealed that the patient had a de novo heterozygous variant in the CRMP2 gene (NM_1386.6): c.42C > A, Chr8 (GRCh37): g.26439487C > A, p.(Ser14Arg). Rare variants (i.e. allele frequency less than 0.01 among 8300 Japanese) that were de novo heterozygous, homozygous, compound heterozygous or hemizygous were detected and have been summarized in Supplementary Material, Table S1.

Results

Evolutionary conservation of amino acid residues Ser14 and Arg565 and flanking sequences between human CRMP2 and zebrafish Crmp2 proteins

We first compared the amino acid sequences of CRMP2 among humans, mice, rats and zebrafish. As shown in Supplementary Material, Figure S1, the homology between the human CRMP2 protein and its rodent homolog is high: 98.78% for human and mouse proteins and 98.78% for human and rat proteins. A comparison between human and zebrafish showed an 89.65% homology for all regions (Table 1). Importantly, the amino acids at Ser14 and Arg565 in human CRMP2 protein are conserved among these species (Supplementary Material, Fig. S1). The human CRMP2 codon Ser14 and Arg565 (abbreviated as h-S14 and h-R565 hereafter) correspond to codon Ser15 and Arg566 of zebrafish Crmp2 (abbreviated as z-S15 and z-R566 hereafter), respectively. Since the h-S14 and h-R565 are located at the N-terminus and C-terminus, respectively, we compared the sequences of the first and last 30 amino acids of CRMP2. These 30 amino acid sequences showed a high homology between humans and zebrafish: 93.33% for the N-terminal 30 amino acids and 93.33% for the C-terminal amino acids. Therefore, we considered that substitution at S15R and R566C in the z-CRMP2 protein would result in effects comparable to those arising from S14R and R565C substitutions in the human CRMP2 protein.

Table 1

Amino acid homology of CRMP2 among species

	All	N-terminal   30 AA	C-terminal   30 AA
human versus mouse	97.64%	96.67%	100%
human versus rat	97.37%	63.33%	100%
human versus zebrafish	88.11%	66.67%	93.33%

Lack of rescue of z-CRMP2 AMO morphant phenotype by R566C or S15R z-CRMP2 mRNA co-injection

As we reported previously (2), Crmp2-morphant zebrafish embryos display the ectopic positioning of CaP motor neurons outside the spinal cord (Supplementary Material, Fig. S2). As a control for the morpholino-mediated oligonucleotide knockdown of Crmp2, we injected a 5-base-pair mismatched oligonucleotide (MO-Crmp2 MM) to evaluate the specificity of the morpholino-oligonucleotide knockdown. The injection of the mismatched morpholino oligonucleotide (MO-CRMP2 MM) did not cause any abnormal positioning of CaP motor neurons in the injected zebrafish embryos, confirming the specificity of the MO-oligonucleotide-mediated knockdown of crmp2 (Supplementary Material, Fig. S2). As we previously reported, wild-type z-CRMP2 mRNA could normalize the ectopic positioning of CaP motor neurons in zDypls2-morphants (n = 50 embryos in total) (2).

We next tested whether mutant z-CRMP2 mRNA with z-R566C or z-S15R substitutions could also normalize the ectopic positioning of motor neurons in z-CRMP2-morphants. In contrast to wild-type Crmp2 mRNA, neither the mutated Crmp2 z-R566C mRNA (n = 50 embryos in total) nor the mutated Crmp2 z-S15R mRNA (n = 18 embryos in total) were capable of suppressing the ectopic positioning of CaP motor neurons in Crmp2–morphant zebrafish embryos (Fig. 2).

Figure 2

Rescue experiments of wild-type and mutant Crmp2 mRNAs in Crmp2-morphants. (A) Representative images of tubulin-staining of Crmp2-morphants injected with wild-type, S15R-mutant or R566C-mutant Crmp2 mRNAs. Lateral view, anterior right. Scale bar: 50 μm. (B) Quantitative analysis of the percentage of ectopic CaP motor neurons (n = 40, 25, 25, 18 and 50, respectively). Data are shown as the mean ± SEM. ns, not significant. ^****, P < 0.0001. Two-tailed paired Student t-test.

Protein levels of wild-type and mutant z-CRMP2 in zebrafish embryos

Proteins were extracted from 30 to 50 embryos and subjected to a western blot analysis. We found a significant increase in z-CRMP2 protein expression in embryos injected with 100 ng/μL of wild-type z-CRMP2 (Supplementary Material, Fig. S3). On the other hand, we found no increase in z-CRMP2 protein levels when we injected Crmp2 R566C mRNA (Supplementary Material, Fig. S3).

Protein levels of wild-type and mutant Crmp2 in cultured cells

Transfection studies using appropriate expression plasmids and HeLa cells were performed to evaluate the intracellular levels of proteins encoded by zebrafish Crmp2 mRNA containing S15R or R566C substitutions and rat Crmp2 mRNA containing S14R or R565C substitutions (correspondence to S14R and R565C in human CRMP2, abbreviated as r-S14R and r-R565C hereafter). The HeLa cells were transfected with the pCS2-zCrmp2 wild type or z-R566C mutant expression vectors that were used in the zebrafish experiments. β-Tubulin was used as the internal reference, and repeated experiments were conducted. Western blotting showed that the Crmp2 protein size did not change as a result of the amino acid substitutions, but the protein expression levels decreased when vectors containing the z-R566C substitution were used (Fig. 3A). The application of the proteasome inhibitor MG-132 induced the recovery of the z-CRMP2 protein level, as shown in Figure 3A.

Figure 3

Western blot of HeLa cells transfected with zebrafish Crmp2 (z-CRMP2) or rat CRMP2 (r-CRMP2) wild-type or mutant plasmid DNAs. (A–C) Analysis of CRMP2 protein levels in HeLa cells transfected with wild-type or mutant plasmid DNAs of zebrafish (A), rat R565C (B) or rat S14R (C). Zebrafish CRMP2 protein was detected using anti-CRMP2 antibody, and tubulin was used as a loading control. The CRMP2 protein levels in MG132-treated cells were also analyzed (A). Rat CRMP2 protein was detected using anti-myc because the r-CRMP2 plasmid constructs contained a myc-tag in their C-terminus; actin was used as a loading control. The CRMP2 protein levels in MG132-treated cells were also analyzed (B, C). Quantitative analysis of CRMP2 protein levels per loading controls. Data are shown as the mean ± SEM. (n = 4) ns, not significant. ^*, P < 0.05, ^**, P < 0.01, ^***, P < 0.001. ^****, P < 0.0001. One-way ANOVA post-hoc Tukey test.

We further conducted comparable experiments using rat CRMP2 expression vectors in which we had introduced either the S14R or R565C substitution. The use of rat CRMP2 vectors carrying either an S14R or R565C substitution resulted in decreased protein expression from the vectors. To exclude the possibility that a poor transfection efficiency contributed to the low protein levels, we co-transfected an EGFP-expression vector and detected both CRMP2 protein and EGFP proteins. As shown in Supplementary Material, Figure S4, cells transfected with r-S14R or r-R565C CRMP2 had lower CRMP2 protein levels, although the EGFP protein levels were comparable to those observed in wild-type CRMP2-transfected cells. As observed for the zebrafish CRMP2 R566C mutant, the reduction in the CRMP2 protein levels was restored by treatment with MG132, a potent cell-permeable proteasome and calpain inhibitor (Fig. 3).

CRMP2 mutation affects its association with tubulin

CRMP2 protein binds to its binding partners, including tubulin proteins (18). We conducted co-immunoprecipitation (co-IP) experiments using anti-myc antibody and detected bound tubulin protein. As shown in Figure 4, we observed that binding to tubulin was weakened for both mutant r-S14R (Fig. 4B) and r-R565C (Fig. 4A) CRMP2 proteins, compared with that for wild-type CRMP2.

Figure 4

Change in interactions between tubulin and CRMP2 because of R565C and S14R mutations. (A, B) Western blot analysis of co-immunoprecipitates (IP) of tubulin protein with CRMP2 with a C-terminal myc-tag. The input shows the protein levels of CRMP2 and tubulin in cell lysates. CRMP2 protein (anti-myc) and tubulin protein were detected after co-immunoprecipitation. Quantitative analysis of tubulin proteins that were co-immunoprecipitated with wild-type CRMP2 (WT), R565C (A) or S14R CRMP2 (C). Data are shown as the mean ± SEM. ^*, P < 0.05, ^**, P < 0.01, ^***, P < 0.001. One-way ANOVA post-hoc Tukey test.

We then visualized the co-localization of CRMP and tubulin in cultured HeLa cells that had been transfected with wild-type or mutant CRMP2 plasmid DNAs. The transfected HeLa cells were stained with anti-tubulin and anti-myc-tag. Imaging of the wild-type CRMP2-transfected cells showed a broad area in which tubulin and CRMP2 were co-localized (white area in merged images, Fig. 5A and C). When we quantitatively analyzed the results of immunofluorescence staining in transfected cells, the co-localization levels of tubulin and CRMP2 were significantly lower for the mutant z-S15R (Fig. 5D) and z-R566C (Fig. 5B) CRMP2 proteins than for the wild-type CRMP2 (^*P < 0.05). These results showed that CRMP2 proteins carrying z-S15R or z-R566C substitutions had lower levels of tubulin binding.

Figure 5

Change in colocalization between tubulin and CRMP2 because of R565C and S14R mutations. (A, C) Images of double staining of CRMP2 with a myc-tag and tubulin in HeLa cells transfected with wild-type CRMP2 (WT), R565C-mutant CRMP2 (A) or S14R-mutant CRMP2 (C). The white color shows the colocalization of CRMP2 and tubulin in merged images of HeLa cells. Quantitative analysis shows less colocalization of CRMP2 and tubulin in R565C-mutant (B) and S14R-mutant (D) CRMP2, compared with wild-type CRMP2. Data are shown as the mean ± SEM. (n = 4) ^**, P < 0.01. ^****, P < 0.0001. Two-tailed paired Student t-test.

Discussion

Two patients with de novo CRMP2 non-synonymous variants (h-S14R and h-R565C) and intellectual disability with hypoplasia of the corpus callosum are described in the present report. The de novo occurrence of these variants in two unrelated patients exhibiting a similar phenotype (i.e. intellectual disability) gives credence to the concept that these non-synonymous variants are clinically relevant.

The functional relevance of the de novo variants was evaluated by the injection of AMO into fertilized zebrafish eggs. The validity of this experimental system was established by our group in previous studies (2). The knock-down of z-CRMP2 by AMO injection leads to the ectopic positioning of CaP motor neurons in zebrafish ‘morphants’. Using this ectopic positioning of CaP motor neurons as the read-out, we tested the significance of the amino acid substitutions found in the presently reported patients using rescue experiments. When wild-type z-CRMP2 mRNA was expressed in z-CRMP2-knockdown zebrafish embryos (morphants), we observed the normalization of the ectopic CaP motor neuron phenotype. However, when we expressed z-CRMP2 S15R- or R566C-mutant mRNAs in Crmp2-morphants, the ectopic CaP motor neuron phenotype was not reversed. Therefore, we concluded that the z-S15R and z-R566C substitutions were functionally significant. Furthermore, the mode of action of these substitutions was likely to involve a loss of function.

The mechanistic basis of this loss-of-function effect of the two amino acid substitutions was evaluated at the protein expression level using a cell transfection assay.

The Crmp2 protein expression levels were lower when we transfected HeLa cells with z-CRMP2 expression vector carrying z-R566C substitutions or rat Crmp2 expression vector carrying r-S14R or r-R565C compared with the level in the wild-type construct. Incubation with MG-132, which inhibits ubiquitin-proteasome degradation, partially ameliorated the reduction in protein expression (Fig. 3). Based on these results, we inferred that CRMP2 proteins with z-S15R or z-R566C substitutions may be prone to proteasome degradation.

Another possible mechanism leading to a loss of function in CRMP2 proteins with z-S15R or z-R566C substitutions is a decrease in binding to tubulins, which is a critical process in CRMP2’s regulation of the formation of neural circuits (18). Immunoprecipitation experiments showed that both amino acid substitutions r-R565C and r-S14R decreased the binding of mutant CRMP2 proteins to tubulin (Fig. 4).

Severity in the two patients differed significantly. It is possible that variants of genes other than CRMP2 may have modified the phenotype. Evaluation of de novo heterozygous, homozygous, compound heterozygous or hemizygous variants did not support such a notion (Supplementary Material, Table S1). We cannot rule out the possibility that a de novo heterozygous non-synonymous variant in the COBLL1 gene, which has never been shown to be disease-causative in humans, may have contributed to the severer phenotype of Patient 2, since the Combined Annotation Dependent Depletion score of 25.8 for the COBLL1 variant was relatively high (19).

In the present study, we have shown the functional relevance of the two amino acid substitutions using a zebrafish model system that allows us to detect the ectopic positioning of CaP motor neurons. This observation in a fish model did substantiate the functional relevance of the h-R565C and h-S14R variants. However, the exact mechanisms by which intellectual disability or hypoplasia of the corpus callosum occurred in the presently reported patients remains unknown. However, given the fundamental role of CRMP2 protein in the development of the central nervous system documented in rodents (20), it is reasonable to infer that the abnormal patterning of the nervous system observed in the zebrafish experimental system can be extrapolated to mammals, including humans. Indeed, it is critical to note that one of the two patients reported herein had hypoplasia of the corpus callosum, a condition that has been documented in neuron-specific CRMP2 conditional knockout (cKO) mice (17). The phenotypic spectrum of CRMP2-deficiency in humans should be explored in future studies.

Materials and Methods

Patients

The two patients were recruited through the ‘Initiative on Rare and Undiagnosed Disease’ project (21). The study was approved by the central institutional review board at Tohoku University in Japan (approval number: 20851). After written consent was obtained, peripheral blood samples were collected from each patient and his or her parents.

Exome analysis

Genomic DNA was extracted from the peripheral blood leukocytes of the patients and their parents. Whole-exome sequencing was performed as described previously (22). Briefly, all the exons were captured using the SureSelect XT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA); then, exome analyses were performed using the HiSeq 2500 platform (Illumina, San Diego, CA) for patient 1 and the NovaSeq 6000 platform (Illumina) for patient 2.

Experimental animals and feeding conditions

Male and female zebrafish (Danio rerio) of the RIKEN Wako (RW) wild-type strain were obtained from the Zebrafish National BioResource Center of Japan (http://www.shigen.nig.ac.jp/zebra/) and used as the experimental animals. Adult zebrafish were kept in a constant temperature circulation system, where the water temperature was 28 ± 0.5°C and the lighting pattern was 14 h light/10 h dark (23). Fertilized eggs were collected via natural spawning. To prevent pigmentation within the embryos, the embryos were raised in fish water containing 0.003% 1-phenyl-2-thiourea (PTU; Nacalai Tesque, Kyoto, Japan) at 12 h post-fertilization (hpf). All experiments involving live zebrafish (D. rerio) were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University.

Antisense morpholino oligonucleotides

The design of the AMOs (Gene Tools, Philomath, OR) for zDpysl2 and their effectiveness and specificity in knocking down z-CRMP2 have been previously documented elsewhere (2,23). The Crmp2 AMO targets the first start codon of Crmp2 mRNA: Crmp2 AMO 5′–CTTCTTGCCCTGATAGCCAGACATC-3′ (the translation initiation codon is underlined). The control experiment was performed using 5-base-pair-mismatched AMO: Crmp2 5mis AMO 5′- CTTgTTcCCCTcATAGCgAGAgATC-3′ (lowercase indicates the mis-paired residue).

Morphants

Morphants are defined as organisms that have been treated with an AMO to knock down the expression of a targeted gene. Morphants of Crmp2 were generated by the injection of AMOs (Gene Tools, Philomath, OR) to block the translation of both maternal and zygotic mRNAs. Stock solutions were diluted as required, and the final concentration of the injection buffer was as indicated; approximately 1 nL of AMO (1 μg/μL) was injected into fertilized embryos at the 1-to-2-cell stage, as previously described (24).

Generation of AMO-resistant wild-type and mutant z-CRMP2 mRNA

We planned to rescue z-CRMP2 morphants through the co-injection of z-CRMP2 AMO and in vitro transcribed wild-type or mutant z-CRMP2 mRNA into fertilized zebrafish eggs.

To avoid translation inhibition by the z-CRMP2 AMOs in the rescue experiments of z-CRMP2 morphants, in vitro-transcribed z-CRMP2 mRNAs were designed for co-injection with z-CRMP2 AMO so that the third nucleotides of the codons within the targeted sequences of the AMOs were substituted without amino acid substitution: the 5′ end sequence z-CRMP2 was designed as 5′-GATGTCcGGgTATCAaGGgAAaAAG-3′ (the translation initiation codon is underlined) (24) in the wild-type AMO-resistant z-CRMP2 mRNA or the mutant z-CRMP2 mRNA encoding proteins with Cys- (R566C) or Arg- (S15R). Corresponding nucleotide substitutions were induced using the Quick Change Lightning Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). All mRNAs for the rescue experiments were transcribed in vitro using the mMessage mMachine Kit (Ambion, Austin, TX).

Rescue experiment for z-CRMP2 morphants

For the rescue experiment using z-CRMP2 morphants, 10 ng/μL of the above-described AMO-resistant z-CRMP2 mRNA were co-injected with z-CRMP2 AMO as described previously (2).

Whole mount immunohistochemistry using anti-acetylated alpha-tubulin

Immunohistochemistry was performed using whole-mount embryos at 28 hpf in 4% PFA. Each sample was washed twice in 0.1% PBTw (PBS containing 0.1% Tween 20) and then blocked in 0.1% PBT with 10% Blocking Reagent (Roche, Basel, Switzerland) for 1 h at room temperature or overnight at 4°C before application of the primary antibody. For the primary antibody, mouse anti-acetylated α-tubulin (1:2000; Sigma-Aldrich, St. Louis, MO) was used. The antibody was visualized using Histofine Simple Stain MAXPO (M) (Nichirei, Tokyo, Japan) for conventional microscopy. Incubation with the primary antibody and staining with Histofine were performed at 4°C overnight. Prior to visualization, the samples were developed using 3,3′-diaminobenzidine (DAB) at room temperature in a shaker for up to 1 h.

Imaging and quantification of ectopic CaP motor neurons

For conventional microscopy, a BX50 microscope (Olympus) with a UPlanApo 20x (NA = 0.70) and UPlanApo 40x (NA = 0.75) objective was used. The obtained images were processed using Adobe Photoshop and Adobe Illustrator. To quantify the percentage of CaP motor neurons with abnormal cell bodies located outside the spinal cord, images of ectopic CaP motor neurons at the 10–15 somite level were captured from a lateral view using a BX50 compound microscope with UPlanSApo 20X and 40X (NA = 0.95) objective lenses and a DP70 digital camera (Olympus, Tokyo, Tokyo). To evaluate the results in a statistical manner, we examined 20–50 embryos. In the captured images, we scored the number of CaP motor neurons with abnormal cell body locations outside the spinal cord (2).

Western blot analysis using rabbit anti-Crmp2 antibody

Homogenized morphant embryos, rescued morphant embryos and control embryos were subjected to a western blot analysis. Zebrafish embryos were collected at 28 hpf; some of the embryos were injected with 100 ng of z-CRMP2 mRNA with the R566C substitution; others were injected with 100 ng of z-CRMP2 wild-type mRNA and some were not injected with mRNA. Proteins were extracted from 30 to 50 embryos by homogenization in a lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM Na₃VO₄, 1% Nonidet P-40, 50 mM p-APMSF and 10 μg/mL aprotinin; pH 8.0). Equal amounts of protein were then separated on a 12.5% SDS-PAGE gel, and the proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA). The membranes were blocked in 5% skim milk in TBS buffer containing 0.05% Tween20 (TBST) and then incubated with rabbit anti-CRMP2 antibody (Ab62661, Abcam, Cambridge, UK) at 4°C overnight. After washing with TBST, the membranes were incubated with HRP-conjugated anti-rabbit IgG secondary antibodies. After reaction with ECL Plus reagent (GE Healthcare, Chicago, IL), the signals were detected using ImageQuant (GE Healthcare).

Cell culture and transfection

Mutant Crmp2 proteins were transfected into HeLa cells to evaluate the expression levels of mutant proteins in the cells

HeLa cells were cultured in DMEM containing 10% (v/v) of fetal bovine serum, 100 U/mL penicillin and 0.1 mg/mL streptomycin. Plasmids expressing the rat CRMP2 protein tagged with myc protein were used. These plasmids were constructed previously and have been described elsewhere. The rat Crmp2 protein sequence and human CRMP2 protein sequence are almost identical (Supplementary Material, Fig. S1). The Myc-tag allows the detection of CRMP2 protein transcribed from the vector in the presence of intrinsic h-CRMP2 protein. The original rat CRMP2 vector pcDNA3-CRMP2WT-myc was modified so that the vector would expresses CRMP2S14R-myc (pcDNA3-CRMP2WT-myc) or CRMP2R565C-myc (pcDNA3-CRMP2R565C-myc) proteins.

Transfection of the plasmid DNA was conducted using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s manual. After 24 h, the cells were washed with PBS twice and fixed with 4% (v/v) PFA for 20 min at 37°C. After washing with PBS twice, the cells were subjected to immunocytochemistry. In some experiments, 10 uM MG132 (Sigma-Aldrich, St Louis, MO) was added to the media and incubated for 8 h to inhibit protein degradation by proteasomes.

Immunocytochemistry of transfected HeLa cells

Cells were treated with 0.1% (v/v) Triton X-100 and 3% (v/v) skim milk in PBS for permeabilization and blocking. Then, mouse anti-myc antibody (M192-3, MBL) and rabbit anti-α-tubulin antibody (Proteintech, cat NO:11224-1-AP) were added and incubated overnight at 4°C. After washing with PBS thrice, secondary antibodies were added and incubated for 1 h at RT. After washing with PBS twice, the cells were mounted in Fluoromount Aqueous Mounting Medium (Sigma-Aldrich, St Louis, MO). The immunofluorescence staining results were quantitatively analyzed by comparing the ratio of white area to the tubulin area after merging. After quantification, the wild type was used as the standard to standardize the protein expression position.

Co-immunoprecipitation experiments

For the immunoprecipitation experiments, cell lysates from transfected HeLa cells were disrupted in 20 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% (v/v) Nonidet P-40, phos STOP EASY Pack and cOmplete Protease Inhibitor Cocktail (Roche), EDTA-free on ice for 10 min. After centrifugation at 14 000 rpm for 20 min at 4°C, the supernatants were incubated with anti-myc antibody for 1 h on ice and then with Protein G Sepharose beads (Pierce, Rockford, IL). After centrifugation at 5000 xg for 1 min, the pellets were washed in homogenate buffer thrice. After removing the supernatants, the pellets were subjected to a western blot analysis.

Statistical analysis

Data were presented as the mean ± SEM. A one-way ANOVA post-hoc Tukey was mainly used. Comparisons between two independent experimental groups were performed using a t-test (two-tailed) for parametric samples. A P-value <0.05 was considered statistically significant.

Conflict of Interest statement. The authors have no conflict of interests.

Funding

Japan Agency for Medical Research and Development JP18ek0109288 to T.O. Japan Agency for Medical Research and Development JP21ek0109549 to K.K.

Author notes

The first two authors (H.S. and S.L.) contributed equally to this work.