C698R mutation in Lrsam1 gene impairs nerve regeneration in a CMT2P mouse model

Missense mutation C694R in the RING domain of the LRSAM1 gene results in a dominantly inherited polyneuropathy, Charcot-Marie-Tooth disease type 2P (CMT2P). We have generated and characterized a Lrsam1C698R knock-in mouse model produced through CRISPR/Cas9 technology. Both heterozygous (Lrsam1+/C698R) and homozygous (Lrsam1C698/C698R) knock-in mice exhibited normal motor functions on behavioral tests as well as normal on nerve conduction studies. Axonal density and myelin thickness were not significantly different between mutants and wild-type mice by sciatic nerve morphometric analysis up to 17 months of age. In line with these normal findings, protein–protein interactions between mutant LRSAM1 and RNA-binding proteins (such as FUS and G3BP1) were still present in mouse cells, which differs from the disrupted interactions between these proteins in human CMT2P cells. However, after crush nerve injury, Lrsam1+/C698R mice had a mild, but statistically significant, reduced compound nerve action potential and conduction velocity during recovery. Therefore, C698R mutation results in a mild impaired nerve regeneration in mice. We speculate that repetitive nerve injuries may, at least partially, contribute to the slowly progressive axonal loss in CMT2P.

LRSAM1 is an E3 ubiquitin ligase with a RING domain at its c-terminal. The RING domain is crucial for the ubiquitination function of LRSAM1. CMT mutations have been shown to disrupt its function 13 . CMT2P was initially found in multiple European families with frame-shift mutations, which altered a large portion of the RING amino acid sequence 7,9,10 . Of the 12 CMT2P-causing mutations, 11 are dominantly inherited and located in the catalytic RING domain 13 ; which further supports the functional importance of the RING. Our group has described a missense mutation, Cys694Arg, that alters a highly conserved cysteine in the RING and is co-segregated with affected CMT2P members in an American family 6 . This conserved cysteine is required to maintain the structure of E3 ubiquitin ligase through its binding with two zinc ions 4,6,13 . We have further demonstrated that the C694R mutation altered the RNA-binding protein nuclear translocation likely by disrupting the protein-protein interaction between LRSAM1 and the RNA-binding proteins 6 , a potential mechanism in CMT2P pathogenesis.
To further explore this mechanism in vivo, we have generated a Lrsam1 C698R knock-in mouse model, the amino acid substitution equivalent to the human C694R mutation. Herein, we present our initial characterization of the Lrsam1 C698R mouse model. We did not observe axonal degeneration in the peripheral nerve in the naïve Lrsam1 C698R knock-in mice; instead, we found a mild impairment of nerve repair in the mouse model. www.nature.com/scientificreports/ substitution in the mouse Lrsam1 gene equivalent to the human C694R mutation (Fig. 1A). Heterozygous (Lrsam1 +/C698R ) mice were crossed to obtain homozygous (Lrsam1 C698R/C698R ) mice. DNA sequencing revealed that the mutant mice carry both C698R and a silent C to A mutation 7 bp upstream to C698R mutation (Fig. 1B). Mutant mice were further verified by restriction fragment length polymorphism analysis to determine whether Lrsam1 knock-in occurs in both alleles or one allele (Fig. 1C). These knock-in mice did not appear to differ from Lrsam1 +/+ mice in appearance and body weight (Fig. 1D,E).
Behavioral studies showed no abnormality in Lrsam1 C698R mice. Rotarod and hindlimb clasping test assess mouse motor coordination and limb weakness. At 12 months of age, Lrsam1 +/C698R and Lrsam1 C698R/C698R mice did not show any abnormalities in hindlimb clasping or paw extension during tail suspension ( Fig. 2A) when compared with Lrsam1 +/+ mice. The fall latency from the rotating bas of Rotarod was not different among Lrsam1 +/+ , Lrsam1 +/C698R and Lrsam1 C698R/C698R mice measured at 6-and 8-months of age (Fig. 2B).
The nerve conduction study was not significantly different between mutants and wild-type mice. We measured compound muscle action potentials (CMAP) at the paw muscles and conduction velocities (CV) on the sciatic nerves. CMAPs and CVs were not significantly different between Lrsam1 +/+ , Lrsam1 +/C698R and Lrsam1 C698R/C698R mice up to 2.5 years of age (P > 0.05, Fig. 3A,B).
Myelin and axon were morphologically normal in Lrsam1 C698R mice. We further studied mouse nerves morphologically. Axons and myelin were examined and quantified at 17 months of age in the sciatic  www.nature.com/scientificreports/ nerves. Visual inspection on semithin sections did not find axonal degeneration or demyelination features, such as loss of axons, myelin debris, and clusters of regenerating axons (Fig. 4A). Myelinated nerve fibers on the crosssections of the sciatic nerves were quantified using deep-learning-based analysis 14 (Fig. 4B). There was no significant difference in axon diameter, fiber density, g-ratio, and myelin thickness in 17-month-old Lrsam1 +/C698R and Lrsam1 C698R/C698R sciatic nerves, compared with those in wild-type mice (Fig. 4C,F).
Protein-protein interaction between LRSAM1 and RNA-binding proteins was not disrupted by the C698R mutation. Our previous study in fibroblasts from patients with CMT2P showed that C694R mutation disrupts the protein-protein interactions between LRSAM1 and RNA-binding proteins (including FUS and G3BP1), leading to altered nuclear-cytoplasmic translocation of the RNA-binding proteins, a potential mechanism of neuronal degeneration in CMT2P 6 . To determine whether similar molecular abnormalities occur in mouse fibroblasts, we performed co-IP. We found that interactions were still present and not significantly different between FUS-mutant LRSAM1 and FUS-wild type LRSAM1 or between G3BP1-mutant LRSAM1 and G3BP1-wild type LRSAM1 (P > 0.05, Fig. 5A-D). There were no significant differences in nuclear / cytoplasmic ratios among LRSAM1, FUS and G3BP1 protein levels (P > 0.05, Fig. 5E-I). This finding differs from disrupted interactions between mutant LRSAM1 and RNA-binding proteins in human cells from patients with CMT2P.  www.nature.com/scientificreports/ Nerve regeneration was mildly impaired in Lrsam1 C698R nerves. Most patients with CMT2P are due to a heterozygous C694R mutation 6 . We thus examined nerve regeneration using crush-nerve injuries on Lrsam1 +/C698R and Lrsam1 +/+ mice that were age-and sex-matched. A baseline NCS found no difference in CMAP or CV between the two genotypic groups. After the nerve injury, NCS was done every 2-3 weeks. Sciatic nerves were dissected at 23 weeks after the second surgery or 43 weeks after the initial surgery respectively for morphometric analysis. Lrsam1 +/C698R mice had a statistically significant negative main effect on CMAP over the entire period (-0.516 mV), and during the second recovery period (− 0.606 mv) (Fig. 6A). The main effect of mutant on CV was statistically significant during the post-surgery periods: − 7.24 m/s over the whole time period, − 3.41 m/s during the first recovery, and − 7.25 m/s during the second recovery (Fig. 6B). These findings suggest an incomplete recovery of CMAP and CV after the injury 15 .
Time had a statistically significant positive effect on CMAP and CV in both genotypes, indicating an effect of recovery process. More importantly, CV recovery showed a statistically significant genotype x time interaction over the two recovery periods, with Lrsam1 +/+ recovering faster than Lrsam1 +/C698R mice (Fig. 6A,B). In addition, Lrsam1 +/C698R also showed a reduced rate of CV recovery as compared to Lrsam1 +/+ mice.
Morphometric analysis showed that surgeries had main effects on axon density (an increase of 4758.8 fibers/ mm 2 ), a decrease of axon radius (− 0.74 µm), and a decrease of myelin thickness (− 0.30 µm), all statistically , which produces a segmentation of myelin (c) and inner axonal areas (d). The software's Region of Interest analysis tool is used to measure each fiber's inner and outer areas (e). The results are exported into a spreadsheet, where myelin radii, thickness, and g-ratio are calculated (f). (C-F) Morphometric analysis showed that the axon radius, axon density, myelin thickness, and g-ratio (determined by dividing axonal radius by the outer fiber diameter) were not significantly different between 17-month-old wild-type and Lrsam1 +/C698R or Lrsam1 C698R/C698R sciatic nerves (P > 0.05, t-test, n = 5 per group, 3 males and 2 females each genotype). www.nature.com/scientificreports/ Interactions of Genotype x Time were found to have a significant effect on CV and indicated a slowed rate of CV recovery in Lrsam1 +/C698R mice during the whole time period (Lrsam1 +/+ increased 0.641 mV (P < 0.001), Lrsam1 +/C698R increased 0.437 mV (P < 0.001), interaction P < 0.001), over the first postoperative period (Lrsam1 +/+ increase 2.078 mV (P < 0.001), Lrsam1 +/C698R increased 1.628 mV (P < 0.001), interaction P < 0.001), and over the second post-op period (Lrsam1 +/+ increased 1.906 mV (P < 0.001), Lrsam1 +/C698R increased 1.294 mV (P < 0.001), interaction P < 0.001). # = p < 0.001. CV was significantly different between 5-and 20-weeks post-surgery. * = P < 0.001. (C-E) Sciatic nerves were collected from the surgery and control hindlimbs, embedded in Epon resin, sliced and imaged under a brightfield microscope at 40X magnification. Automatic segmentation of myelin was performed and refined in Dragonfly software. Results were analyzed using a mixed linear model and showed that crash nerve surgery was associated with an increase of axon density (+ 4758.8 fibers/mm 2 ), a decrease of axon radius (− 0.74 µm), and a decrease of myelin thickness (− 0.30 µm), all statistically significant (P < 0.05). Genotype or interaction between genotype and surgery status were not found to be statistically significant. (F-G) Morphometric analysis of the nerve fibers revealed no significant difference in g-ratio and axon-diameter distribution in injured Lrsam1 +/C698R nerves as compared to injured Lrsam1 +/+ nerves (n = 6 per group, 4 males and 2 females each genotype). Ctrl = Control. Inj = crush injury. www.nature.com/scientificreports/ significant changes expected in regenerating nerves (P < 0.05). However, comparisons between Lrsam1 +/+ and Lrsam1 +/C698R nerve morphometrics, as well as the interaction of genotype and injury status were not significantly different (Fig. 6C-G). Additionally, in homozygous C698R mice, CV and CMAP were not significantly different from that of heterozygous C698R mice after injury; but were significantly lower than that of wild-type mice ( Supplementary  Fig. 1A-B). Again, the morphometric analysis did not find a significant change in homozygous injured nerves, compared with heterozygous and wild-type injured nerves ( Supplementary Fig. 1C-F).

Scientific Reports
Taken together, C698R Lrsam1 mutation only mildly impaired nerve function during regeneration.

Discussion
In this study, we have shown the production and initial characterization toward a new mouse model of CMT2P, where a knock-in of the C698R Lrsam1 mutation was introduced into the mouse genome. However, we observed no peripheral neuropathy in naïve Lrsam1 +/C698R and Lrsam1 C698R/C698R mice up to 2.5 years of age but did find a mildly impaired nerve function during regeneration after nerve injury. While the electrophysiological phenotype is subtle, it could contribute to the slowly progressive polyneuropathy in patients with CMT2P. As every person is exposed to many nerve injuries during their lifetime, we speculate that an accumulation of impaired nerve recoveries may eventually contribute to the polyneuropathy of CMT2P [16][17][18] . This finding also brings up a challenging issue. The nerve abnormality in the knock-in mice was disproportionally subtle, comparing with the polyneuropathy in persons with CMT2P 6 . This mild phenotype is in line with absent disruption of protein-protein interactions between mutant LRSAM1 and RNA-binding proteins (Fig. 5). The exact reason for the loss of this disruption of protein-protein interaction by the mutation in mice is unclear at this point. However, there have been many precedents where mouse models could not completely recapitulate phenotypes in its human counterpart 19 . Perhaps, CMT2P needs to be modeled in larger animals.
Notice that we did not find an age-effect in the NCS of 30-month-old mice, while CV is known to decline over time 20 . This is not unexpected. Age-effect in CV is typically detectable in consecutive recordings in same animals. This was not possible in this study as tissues had to be collected at different ages; thus, our CV data in the 30-month-old mice were cross-sectional; thus may and may not show the expected age-effect. However, it would not affect the overall conclusion of this study. This was not likely a technical issue as the same NCS technique was used in our studies before and has detected age-effect 21 .
Another potential limitation relates to small n-numbers of some experimental groups. While our data do not provide evidence of obvious progressive neuropathy in mutant mice without the challenge of nerve injury, subtle alterations could be undetected due to the small n-numbers. However, impaired nerve regeneration after injury was reproduced in repeated experiments and also in a different experimental group (Fig S1), supporting the key conclusion of this study.
In summary, we have produced a knock-in mouse with C698R mutation in order to model CMT2P. While the phenotype is not robust, mild abnormality in nerve repair provides a helpful clue toward the slowly progressing polyneuropathy in CMT2P.

Methods
Animal generation. The Lrsam1 C698R knock-in mice were generated on a C57Bl/6J background using the CRISPR/Cas9 technique. Briefly, single-cell zygotes from C57BL/6J mice were microinjected with mRNA encoding Cas9 and a guide sequence (5′-ACA GCA GCA GAC GTG GCC AC-3′ at 20 ng/µl) to target the exon 26 of Lrsam1. A single stranded DNA oligo carrying the C698R mutation was co-injected at 20 ng/µl to promote homology-directed repair. The sequence of the oligo was (…ACGTC CGCTG…). The underlined "C" changes the first base of codon 698, creating the C to R substitution. Following implantation of the embryos into surrogate females, the microinjection resulted in 49 live-born mice. Fourteen of them carried idles consistent with non-homologous end joining (NHEJ) events. Two female offspring carried the C698R, as well as an upstream silent mutation. These mice were bred with wild-type C57BL/6 J males to establish the germline transmission of the mutation. The offspring were confirmed to carry both the C698R and the silent C to A mutation 7 bp upstream to C698R mutation. The silent mutation was likely a not-targeted change accidentally introduced by the genetic manipulation as we did not find this silent mutation in the progeny of Lrsam1 +/+ mice. After germline transmission of the targeted variant allele, we used the congenic Lrsam1 +/C698R mice derived from two founders to backcross with C57BL/6J mouse for at least 10 generations. A mixed mouse line from both founders were used for experiments. The experimental mice were obtained by intercrossing heterozygous mice (Lrsam1 +/C698R ) and were confirmed by both restriction fragment length polymorphism analysis and sequencing. The mice used in this study were age-matched and sex-mixed littermates. Mice were maintained on a standard 12 h light/12 h dark cycle, ad libitum access to food and water, and housed in plastic cages with standard rodent bedding. We did not observe any difference in mortality between genotypes.

Animal ethics statements.
All animal experiments and procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Wayne State University and were performed in accordance with federal and university guidelines and regulations for the care and use of experimental vertebrate animals. Authors have complied with the ARRIVE guidelines for reporting.
Mouse PCR genotyping. DNA was extracted from ear clips by incubation in 100 μl DirectPCR Lysis buffer (Cat # 102T, Viagen) with 2 μl of 10 mg/ml proteinase K (Cat #P4850, Sigma) at 55 °C overnight. After deactivating the mixture at 85 °C for 45 min, the supernatant was used for genotyping by PCR. DNA segments around the C698 were amplified by the following primers: forward primer, 5′-CCA GGT AAG CAG TAC ACG CCT Rotarod test. Wild-type Lrsam1 +/+ and Lrsam1 C698R knock-in mice were placed on an accelerating (0.1 rpm/ second) rotarod (Columbus Instruments, Columbus, OH) that progressed from 16 to 28 rpm over 2 min. If mice jumped off, turned around, or hung to the rod, they were given an additional attempt for the trial (up to a maximum of 3 attempts for any given trial). Once mice were in position, facing the correct direction, the device was turned on, and they walked until they lost balance and fell off or until the 2-min run-time ended. All mice were given 2 days of training prior to the test. Each mouse underwent 3 trials a day for 3 days following the training, with each trial separated by 30-min rest. The results were averaged for each animal. The cohorts of mice at 6-and 8-months of age were two separate groups; and were tested concurrently on the Rotarod. This was not a longitudinal test on the same group of mice at two different time points.
Hindlimb clasping test. Mice were held by the tail 30 cm above a tabletop, and video-recorded for 20 s.
Abnormal postures such as hindlimb clasping and sustained straining of paws were recorded. Videos were analyzed by a researcher blinded to the genotype. The time span for clasping and paw straining was also calculated.
Nerve conduction studies (NCS). NCS data were acquired as described 21 . Mice were anesthetized with isoflurane. CMAP was recorded from the intrinsic foot muscles using needle electrodes. Stimulation electrodes were placed in the mouse sciatic notch and at the ankle and recording was made on the paw. Supramaximal stimulations at each stimulation point evoked CMAP; and conduction velocity was calculated between the two stimulation points.
Sciatic nerve semithin section. This technique has been described previously 22 . Sciatic nerves were dissected and fixed in 4% paraformaldehyde and 2.5% glutaraldehyde mixture. Nerves embedded in Epon were sectioned to 1 µm thickness. The entire field of transverse sections of each nerve was imaged for morphometric analysis.
Nerve morphometrics. 40X  Co-immunoprecipitation (co-IP) and immunoblotting. This technique has been described 6 . Wholecell proteins were extracted using RIPA buffer (Cat# R0278, Sigma). Nuclear or cytoplasmic fractionation of mouse fibroblasts was isolated using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Cat#78833, Thermo Fisher Scientific). Cell lysates were incubated with primary antibodies overnight at 4 °C. Protein G agarose beads (Cat# 15920-010, Life technologies) were added for another 2 h incubation at 4 °C. Samples were eluted with Laemmli sample buffer, loaded into SDS-PAGE gels, and transferred to a PVDF membrane. The membranes were blotted with 5% milk and incubated overnight at 4 °C with primary antibodies and followed by secondary antibodies. The immune complexes were detected by the enhanced chemoilluminescence (Cat# NEL103001, Perkin Elmer). In some cases, the blots were stripped and re-probed with additional antibodies. Quantification of band intensity was performed using the ImageJ (http:// rsbweb. nih. gov/ ij/).
Sciatic nerve crush injury. While the mouse was under anesthesia with avertin (250 mg/kg, Cat #T48402, Sigma-Aldrich), a 1-2 cm incision just below the sciatic notch was made to expose the sciatic nerve. Forceps (Cat #11254-20, Fine Science Tools) were used to squeeze the nerve for 30 s; and turned 90 degrees to squeeze for another 30 s, which ensured that the whole nerve was crushed. Once the wound was clipped, Carprofen was administered over 3 days. NCS were performed immediately prior to the surgery and after the procedure to track the recovery of nerve function. The contralateral sciatic nerve was used as a sham control. The veterinarians and experimenters are always required to closely monitor the mouse's body weight after surgeries per IACUC proto- www.nature.com/scientificreports/ col and found no significant weight loss. Mice among different experimental groups were evaluated simultaneously for NCS and tissue collections in nerve injury experiments.
Statistical analysis. The data were represented as the mean ± SD. P-values were obtained from the Student's two-tailed t-test or repeated-measures ANOVA. For NCS data, we used a linear mixed model where time and genotype were coded as fixed effects on CMAP and CV. A linear mixed model was also chosen to analyze the crush nerve experiments as we were interested in determining effects on continuous outcome variables and dealing with clustered data (thousands of nerve fibers in each animal for morphometric analysis, as well as repeated measurements as part of NCS). Direct comparisons between genotypes were accomplished using a two-tailed t-test. We compared continuous variables between genotype/surgery status groups for morphometric analysis using another linear mixed model with genotype and surgical status as fixed effects. All statistical analysis was performed using SPSS version 27. P-value < 0.05 was used to determine significance.

Data availability
All data generated or analyzed in this study are provided in the Supplementary Information/Source data file. Additional details on protocols used in this study are available from the corresponding author on request. ORS Dragonfly myelin segmentation model is also available upon request.