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. 2023 Feb 4;4:100077. doi: 10.1016/j.crneur.2023.100077

The systemic inhibition of the terminal complement system reduces neuroinflammation but does not improve motor function in mouse models of CMT1A with overexpressed PMP22

Iliana Michailidou a, Jeroen Vreijling a, Matthijs Rumpf a, Maarten Loos b, Bastijn Koopmans b, Nina Vlek b, Nina Straat b, Cedrick Agaser c, Thomas B Kuipers c, Hailiang Mei c, Frank Baas a, Kees Fluiter a,∗
PMCID: PMC10011818  PMID: 36926597

Abstract

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most prevalent hereditary demyelinating neuropathy. This autosomal, dominantly inherited disease is caused by a duplication on chromosome 17p which includes the peripheral myelin protein 22 (PMP22) gene. There is clinical evidence that the disability in CMT1A is to a large extend due to axonal damage rather than demyelination. Over-expression of PMP22 is recently thought to impede cholesterol trafficking causing a total shutdown of local cholesterol and lipid synthesis in the Schwann cells, thus disturbing their ability to remyelinate. But there is a large variety in disease burden between CMT1A patients with the same genetic defect, indicating the presence of modifying factors that affect disease severity. One of these potential factors is the immune system. Several reports have described patients with co-occurrence of CMT1A with chronic inflammatory demyelinating disease or Guillain-Barré syndrome. We have previously shown in multiple animal models that the innate immune system and specifically the terminal complement system is a driver of inflammatory demyelination. To test the contribution of the terminal complement system to neuroinflammation and disease progression in CMT1A, we inhibited systemic complement C6 in two transgenic mouse models for CMT1A, the C3-PMP22 and C3-PMP22 c-JunP0Cre models. Both models over-express human PMP22, and one (C3-PMP22 c-JunP0Cre) also has a Schwann cell-specific knockout of c-Jun, a crucial regulator of myelination controlling autophagy. We found that systemic inhibition of C6 using antisense oligonucleotides affects the neuroinflammation, Rho GTPase and ERK/MAPK signalling pathways in the CMT1A mouse models. The cholesterol synthesis pathway remained unaffected. Analysis of motor function during treatment with C6 antisense oligonucleotides did not reveal any significant improvement in the CMT1A mouse models. This study shows that the contribution of the terminal complement system to progressive loss of motor function in the CMT1A mouse models tested is limited.

Graphical abstract

Image 1

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Highlights

  • •

    The role of the terminal complement system as a modifying factor for CMT1A was investigated.

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    Inhibition of complement factor C6 speeded up recovery after acute demyelination.

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    Inhibition of complement factor C6 reduced neuroinflammation in CMT1A mouse models.

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    Complement inhibition did not ameliorate the progressive loss of motor function.

1. Introduction

Charcot-Marie-Tooth disease type 1A (CMT1A) is a demyelinating peripheral neuropathy in which the Schwann cells progressively lose their ability to remyelinate the axons, causing slow nerve conduction velocities (Boerkoel Garcia, 2002). This autosomal, dominantly inherited disease is caused by a 1.4-Mb duplication on chromosome 17p that includes the peripheral myelin protein 22 (PMP22) gene (Lupski et al., 1991; Raeymaekers et al., 1991). Symptoms include distal loss of strength and sensation, more in the legs than in the arms, with onset in the first decades of life (Berciano J Garcia, 2000). Clinical disease progression might be caused by increasing axonal dysfunction (KrajewskiLewisFuerst, 2000; Verhamme and Koelman, 2004; ShyChen, 2008).

One of the main molecular pathways which is disturbed by PMP22 overexpression in the Schwann cells is the intracellular routing of cholesterol esters. A series of recent publications showed that both overexpression and mutations in PMP22 either trap cholesterol esters in the lysosomes or cause retention in the Golgi (Zhou et al., 2020). Therefore, there is a complete shut-down of cholesterol synthesis in the Schwann cells in the CMT1A animal models tested (Zhao et al., 2017; Giambonini-Brugnoli et al., 2005).

The clinical phenotype of CMT1A is very variable even within families, with some patients experiencing late disease onset and a relatively mild progression, while others carrying the same duplication of PMP22 show an early onset and severe progression (KrajewskiLewisFuerst, 2000; Verhamme and Koelman, 2004; ShyChen, 2008). This led to the hypothesis that factors other than the PMP22 gene act as modifiers of CMT1A course. One of these modifiers might be the complement system, a key arm of innate immunity, some components of which are highly expressed in the peripheral nervous system (PNS) (de Jonge et al., 2003a).

The complement system comprises a cascade of self-activating proteins which involves various components, including the C1q, mannan-binding lectin, C3 and C5 proteins. All pathways of complement activation lead to cleavage of the C5 component for generation of the anaphylatoxin C5a, and the opsonin C5b. C5b and the complement proteins C6 through C9, together form the membrane attack complex (MAC). Over-activation of the complement system is linked to neuroinflammation in several neurological diseases. In CMT1A, complement expression was found in human biopsies of peripheral nerves (de Jonge et al., 2003b). Whether activation of the complement system plays a beneficial or a detrimental role in demyelinating disease is still debated. Some studies showed that complement activation is needed for myelin clearance which is a prerequisite for efficient axon remyelination (Dailey et al., 1998; Brück and Friede, 1991). In contrast, other studies showed that inhibition of the complement system improved regeneration and remyelination efficiency in animal models (Ramaglia et al. 2007, 2008). Specifically, inhibition of the terminal complement system promoted regeneration and recovery in models of Wallerian degeneration of the peripheral nerve (Ramaglia et al., 2009) and other models of PNS or central nervous system (CNS) neuroinflammation (Michailidou et al., 2018: Fluiter et al., 2014; Bahia El Idrissi et al., 2015). Of note, CMT1A often co-occurs chronic inflammatory demyelinating disease or Guillain-Barré syndrome, two diseases which show complement activation (reviewed in Kokubun, 2020).

In this study we set out to investigate whether inhibition of complement C6, which is a critical component of MAC, the end product of terminal complement pathway activation, affects progression of CMT1A. For this purpose, we utilized two mouse models for CMT1A both of which overexpress the human PMP22 gene. The first CMT1A model is the C3-PMP22 model, which shows a mild disease phenotype that more resembles the human disease (Verhamme et al., 2011) as compared with the original C22-PMP22 mouse model (Huxley et al., 1996) of which the C3-PMP22 is a spontaneous revertant. C3-PMP22 (B6.Cg-Tg(PMP22)C3Fbas/J) transgenic mice carry 5 copies of the wild type human PMP22 gene. These C3-PMP22 mice show no overt clinical signs at 3 weeks and develop mild neuromuscular impairment in an age-dependent manner. Like CMT1A patients, C3-PMP22 mice show stable, low nerve conduction velocities (Verhamme et al., 2011). Myelination is delayed in these mice, and they contain reduced numbers of myelinated fibers at 3 weeks of age. The second CMT1A model is the C3-PMP22 model with an additional Schwann cell specific knockout of c-Jun (C3-PMP22 c-Jun P0Cre) (Hantke et al., 2014). c-Jun controls autophagy in Schwann cells (Gomez-Sanchez et al., 2015), a crucial step to clear dysfunctional Schwann cells and recycle molecular building blocks such as lipids and cholesterol, which are needed for remyelination. Thus, the C3-PMP22 c-Jun P0Cre model has a reduced capacity to recycle lipids by autophagy, one of the few rescue mechanisms which compensates for the loss of cholesterol in the Schwann cells over-expressing PMP22.

Importantly, C6 is not expressed within the nervous system by resident cells but is derived from the blood circulation after production in the liver (Hobart et al., 1977) and is therefore an accessible target for antisense oligonucleotide intervention strategies. For this reason, we inhibited C6 systemically in the two mouse models for CMT1A using C6 antisense oligonucleotides, targeting liver C6 production to limit the capacity of MAC formation. As proof-of-concept of this oligonucleotide intervention strategy we included a crush nerve injury model for Wallerian degeneration of the peripheral nerve. In this model inhibition of MAC formation is known to speed up recovery after damage (Ramaglia et al. 2008, 2009).

We show that complement proteins are deposited on Schwann cells over the natural disease course in the two CMT1A models tested. RNAseq analysis of sciatic nerves showed an upregulation of pathways related to neuroinflammation and a strong downregulation of pathways of cholesterol production in the C3-PMP22 mice. Systemic complement C6 inhibition with an antisense oligonucleotide improved clinical recovery in the nerve crush model of Wallerian degeneration. In the CMT1A models, C6 treatment affected the neuroinflammation, Rho GTPase and ERK/MAPK signalling pathways in the sciatic nerve. However, comprehensive analyses of the motor function of these mice showed no differences between treated and untreated mice. Treatment with the C6 antisense oligonucleotide also did not restore the disease-related de-activation of the cholesterol synthesis pathway.

2. Materials and methods

2.1. Animals

All experiments were performed after ethical approval by the institutional ethical committee and the central (national) commission for animal experiments according to the EU directive 2010/63/EU. Experiments were overseen by the local animal welfare bodies at the LUMC (Leiden, The Netherlands) and VU University (Amsterdam, The Netherlands). The ARRIVE guidelines essential 10 (Percie du Sert et al., 2020) were followed. C3-PMP22 mouse model (B6.Cg-Tg(PMP22)C3Fbas/J) is a spontaneous revertant of the C22-PMP22 model after backcrossing into C57Bl/6 mice (Verhamme et al., 2011). The generation of C3-PMP22 mice with a Schwann cell-specific knockout of c-Jun (C3-PMP22 c-JunP0Cre) is previously described (Hantke et al., 2014). Both mouse lines were bred and kept Specific Pathogen Free, according to FELASA 2014 recommendations) at Janvier-labs (Le Genest Saint Isle, France). Breeding was performed for all PMP22 transgenes by mating heterozygous PMP22 transgene-positive females with transgene-negative males. Wild type littermates derived from this breeding were used as controls. Upon weaning mice received an RFID chip (Biolog id, Bernay cedex, France) which was used throughout the experiment to identify individual mice during procedures. Before experiments, animals were allowed to acclimatize for at least one week. All animals were housed socially, initially with at least 3–4 mice per cage, but at least 2 mice per cage in case cage-mates were taken out of the experiment earlier. Mice were housed in conventional cages (type 2). The animal room was maintained at a temperature of 21 ± 2 °C and a relative humidity between 45% and 65%. Lighting was artificial by fluorescent tubes, time switch controlled at a sequence of 12 h light, 12 h dark (lights on from 7.00 a.m. to 7.00 p.m.). All cages were provided with sawdust as bedding material, and nesting material. Food and drinking water were provided ad libitum from the arrival of the mice until the end of the study. The mice were fed a commercial rodent diet (Harlan Teklad, 2018). Each cage was supplied with domestic mains tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.

2.2. Antisense oligonucleotides

The antisense oligonucleotide used in this study was fully described before (Michailidou et al., 2018). The sequence of the C6 antisense oligonucleotide is 5′-AACttgctgggAAT-3′ [Locked Nucleic Acid (LNA) in capital letters, DNA in lowercase letters]. This C6 LNA oligonucleotide (RGS1104) referred to as C6 antisense, targeting the mRNA of the complement component C6, was synthesized with phosphorothioate backbones and methylated DNA-C (medC) by Ribotask (Odense, Denmark), on a Mermade 12, using 2 g NittoPhase (BioAutomation). A control oligonucleotide (5′-ATCttcgcgtgaaTAA-3′) was also synthesized by Ribotask using the same method. Throughout the process, the oligonucleotide constitution was confirmed by MALDI-TOF mass spectrometry analysis on a Bruker Autoflex using 3-hydroxypicolinic acid as matrix. The antisense oligonucleotides (5 mg/kg) were dissolved in phosphate buffered saline (PBS, pH 7.4, Life technologies, Bleiswijk, The Netherlands) when administered to the mice. The antisense oligonucleotides were administered subcutaneously (s.c.) either by injection or through osmotic minipumps (Alzet micro-osmotic pumps model 1007D, Durect Corporation, Cupertino, CA, USA) as indicated for each experiment.

2.3. Verification of C6 antisense oligonucleotides target knockdown efficacy

The efficacy of the C6 oligonucleotide treatment was determined by measuring the level of C6 mRNA knockdown using qPCR in the liver and the sciatic nerve. The full qPCR method was published before (Fluiter et al., 2014). In short, a LightCycler 480 (Roche) with the Universal probe system (Roche) was used. Hypoxanthine phosphoribosyl transferase was used as a reference gene. The following primers were used (Sigma-Aldrich): C6 mouse F 5′-cagagaaaaatgaacattcccatta-3′, C6 mouse R 5′-ttcttgtgggaagctttaatgac-3′; for HPRT, HPRT rat/mouse F 5′-ggtccattcctatgactgtagatttt-3′, HPRT rat/mouse R 5′-caatcaagacgttctttccagtt-3′; Universal Probe #13 (Roche reference no. 04685121001) for C6; Universal Probe #22 (Roche reference no. 04686969001) for HPRT. To test the effect of C6 knockdown on the systemic activity of the terminal complement system in the serum of mice we used a classical pathway hemolysis assay using antibody-sensitized sheep erythrocytes ready-to-use (Virion\Serion GMBH, Wurzburg, Germany) in CFT buffer (Virion\Serion GMBH, Wurzburg, Germany). Mouse serum was diluted 1:10 in this assay. Plates were incubated at 37° for 30 min, centrifuged, and haemoglobin in the supernatant was measured by absorbance at 405 nm. Percentage lysis was calculated according to: %Lysis = (Absorbance [Abs] sample − Abs background)/(Abs max − Abs background) × 100%. Measurements were done in triplicate.

2.4. Nerve crush

The nerve crush model was described in full before (de Koning et al., 1986). In this study, we used female mice to perform the nerve crush experiments. One hind paw was always crushed and the other hind paw was used as control. The animals were either treated with the C6 antisense oligonucleotide RGS1104 (5 mg/kg, 5 subcutaneous injections (s.c.), once daily, starting three days before the nerve crush) or with the anti-C5 monoclonal antibody BB5.1 (200 μg/mouse, total three intra-peritoneal (i.p.) injections, first dose 24 h before the nerve crush, kind gift from Paul Morgan of the University of Cardiff]. The BB5.1 antibody (Frei et al., 1987) was administrated to 10 to 12-week-old mice, housed 6 per cage. A control group was administered with PBS for comparison with the BB5.1-treated group. A second control group was administered with a control oligonucleotide (5 mg/kg) dissolved in PBS for comparison with the C6 antisense oligonucleotide. The recovery of sensory function as measured using an electric stimulation of the footpad (de Koning et al., 1986) was used to monitor restoration of function. The sciatic nerves were isolated after the experiment and either snap-frozen in liquid nitrogen or fixed in buffered formalin.

2.5. Functional testing of C3-PMP22 and C3-PMP22 c-JunP0Cre mice

The primary personnel performing injections, behavioural tests and data analyses were blind to treatment and genotype. On the day of the first injection with antisense oligonucleotide, body weight was measured. Mice were assigned to the treatment groups, with the factors body weight and sex stratified (randomized block design).

Several behavioural observations or tests were used to evaluate the effect of genotype. Tremor score as well as hanging wire score were performed using the modified SHIRPA test (Rogers et al., 2001). Strength of the combination of front and hind paws was assessed by sensing the peak amount of force (N) applied in mice grasping a pull bar connected to a force meter (1027DSM Grip Strength Meter, Columbus instruments, Columbus, OH, USA). Mice were allowed to grasp the pull bar 5 times with front paws only, followed by grasping 5 times with front and hind paws. The median of each 5 repetitions was taken as grip strength. The balance beam test was used to score the ability of mice to traverse a stationary horizontal rod and measures sensorimotor coordination as assessed by the latency to cross the beam and number of foot slips. Mice were placed at a platform at the start of a wide training beam (100 cm long, 5 cm wide) and allowed to walk along the beam into an enclosed box. All mice had three training runs on a wide beam, and on the subsequent day were given three runs on a narrow beam (1 cm wide). Gait measurements were recorded and analysed using the CatWalk XT system from Noldus (Noldus IT, Wageningen, the Netherlands). Mice were introduced to the CatWalk system on day 1 for habituation and got tested on day 2 (next day).

2.6. Statistics

Primary outcome measures were tested using ANOVA with factor ‘genotype’, ‘treatment’ and ‘sex’. When p < 0.05, differences were considered statistically significant. In case of an overall treatment group effect or an interaction effect, Tukey post-hoc testing with Bonferroni correction was performed. Error bars indicate the standard error of the mean (SEM) unless stated otherwise. Since the data are measured over different time-points, repeated measures ANOVA were used for data analysis, except for nonparametric data acquired in the hanging wire test and the tremor score in the SHIRPA test, where a modified repeated measures ANOVA for non-parametric data was used as described in (Noguchi et al., 2012) and in (Brunner and Puri, 2001). As this method does not allow for testing more than 2 groups plus a time factor, the Genotype and sex factors were grouped resulting in 4 groups denoted as ‘Sex + Genotype’ in the results section. A Mann-Whitney U (MWU) test was performed, comparing the results between all groups at all time points.

2.7. Immunohistochemistry

Sciatic nerves were fixed in formalin for 1 week and subsequently embedded in paraffin blocks. Briefly, sections were deparaffinized in xylene and rehydrated through a series of ethanol. Endogenous peroxidase activity was blocked by incubation in methanol (Merck, Darmstadt, Germany) with 0.3% H2O2 (Merck) for 25 min at room temperature. Sections were pretreated with heat-induced antigen retrieval (96 °C) in 10 mM citric acid buffer pH 6.0 for 1 h and incubated overnight at 4 °C in the appropriate primary antibody (mouse anti-C1q clone JL-1, Biotin, 1:100, ab72355; rat anti-C3 clone 11H9, 1:200, NB200-540; rat anti-C9 clone C9-6-25-5, 1:100, HM1134) diluted in a 10% fetal bovine serum (FBS, Sigma-Aldrich)/DAKO washing buffer. The next day, the rat anti-mouse C3 and C9 antibodies were visualized with biotinylated anti-rat secondary antibodies (1:1500; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) after incubation with avidin-peroxidase (1:100; Sigma-Aldrich, Steinheim, Germany) and development with 3-amino-9-ethyl-carabzole (AEC, Sigma-Aldrich). The biotinylated C1q antibody was immediately incubated with avidin-peroxidase and developed with the EnVision detection kit, containing a high sensitivity DAB chromogenic substrate system (Dako, Glostrup, Denmark). DAB-stained sections were counterstained with hematoxylin (Sigma-Aldrich). Iba-1 (Aif1) detection was done using anti-Iba-1 rabbit polyclonal (FUJIFILM Wako Pure Chemical Corporation, Japan). The Iba-1 antibody was visualized with Brightvision poly-AP anti-rabbit IgG (Immunologic, The Netherlands) and Vector® Blue Substrate Kit, Alkaline Phosphatase (AP) (Vector laboratories) and nuclear fast red (Sigma) as counter stain. All sections were mounted with permanent or aqueous VectaMount (Vector Laboratories).

2.8. RNA-seq

Tissue was lysed using a Mikro-Dismembrator S (B. Braun Biotech International GmbH, Sartorius group) in TRIzol (Thermo Fischer Scientific). After phenol/chloroform extraction and precipitation, RNA pellet was dissolved in 100 μl buffer RA1 (Machery-Nagel, Duren, Germany). RNA was purified using NucleoSpin RNA XS Kit (Machery-Nagel, Duren, Germany). RNA concentration was measured by using Qubit, RNA-BR Kit (Thermo Fischer Scientific) and quality check was done with a BioAnalyzer RNA Nano Chip (Agilent).

cDNA synthesis was performed using NuGEN Ovation RNA-Seq System v2 (7102-A01; NuGEN, San Carlos, CA, USA) followed by purification with the Qiagen MinElute Kit. DNA was sheared to 200 to 400-bp fragments. The DNA was end polished and dA tailed, and adaptors with Bioo barcodes were ligated (Life Technologies). The fragments were amplified (eight cycles) and quantified with a QuBit (Thermo Fisher Scientific). Sequencing was done by Genomescan (Leiden, the Netherlands; https://www.genomescan.nl/) using a Novaseq 6000 PE150 (Illumina).

2.9. Pathway analysis

RNA-Seq files were processed using the open source BIOWDL RNAseq pipeline v3.0.0 developed at the LUMC. This pipeline performs FASTQ preprocessing (including quality control, quality trimming, and adapter clipping), RNA-Seq alignment and read quantification. FastQC (v0.11.7) was used for checking raw read QC. Adapter clipping was performed using Cutadapt (v2.4) with default settings. RNA-Seq reads were aligned using STAR (v2.7.3a) on the GRCm38 reference genome. The gene read quantification was performed using HTSeq-count (v0.11.2) with setting “–stranded yes”. The gene annotation used for quantification was Ensembl version 99. The raw counts were converted to CPM counts using R package EdgeR (v3.28.1) and from this, fold changes were calculated for every comparison. Data were analysed using the Ingenuity Pathway Analysis (IPA) software (Qiagen) (Krämer et al., 2014).

2.10. Graphs and drawings

The graphical abstract and schematical drawings were created by BioRender.com. Graphs of data were created using GraphPad Prism version 9.3.1 for Windows (GraphPad Software, San Diego, California USA, www.graphpad.com).

3. Results

3.1. Complement deposition on sciatic nerves of C3-PMP22 and C3-PMP22 c-JunP0Cre mice

To test whether complement is involved in the natural disease course of the C3-PMP22 and the C3-PMP22 c-JunP0Cre mice we performed immunohistochemistry for the markers C1q, C3 and C9 on paraffin embedded tissue from sciatic nerves. We found deposition of all three proteins on the sciatic nerve of both models (Fig. 1A–F). None of the proteins was present in the wt mice.

Fig. 1.

Fig. 1

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Deposition of complement components in the sciatic nerve of C3-PMP22 and C3-PMP22 c-jun P0Cre mice. Immunohistochemical detection of C1q, C3 and C9 on sciatic nerves obtained from C3-PMP22 c-Jun P0Cre (A–C), and C3-PMP22 mice (D–F). Inserts show magnification of the selected region.

3.2. Systemic C6 inhibition by an antisense oligonucleotide speeds up recovery of sciatic nerve function after nerve crush

C6 is primarily produced in the liver (Hobart et al., 1977). In rodents, most antisense oligonucleotides with a phosphorothioate backbone accumulate in the liver (Rifai et al., 1996). Therefore, we selected this chemistry to knockdown liver C6 expression in the two CMT1A mouse models tested. Systemic subcutaneous dosing (5 mg/kg) of a LNA wing-mer antisense oligonucleotide against C6 lowered expression in the liver in both the C3-PMP22 and C3-PMP22 c-JunP0Cre models (Supplemental Fig. 1). In both sexes, the C6 antisense reduced C6 mRNA levels (Supplemental Fig. 1). The duration of C6 mRNA knockdown in the liver and sciatic nerve and its effect on systemic MAC was further tested and found to be stable up to 30 days after the end of treatment depending on the dosing regimen (Supplemental Fig. 2). These data are in line with our earlier studies showing a link between inhibition of liver C6 mRNA expression and C6 protein levels in the circulation (Michailidou et al., 2018; Fluiter et al., 2014; Bahia El Idrissi et al., 2015). We first studied the effect of the achieved systemic inhibition of C6 in the nerve crush model for Wallerian degeneration that was previously shown to be dependent on complement C6 in the rat (Ramaglia V et al., 2007). For this experiment sciatic nerves were crushed in one hind paw while the other paw only received a sham operation as control. Nerve crush was followed by Wallerian degeneration and loss of sensory function. Recovery of sensory function was faster in animals treated with the C6 antisense compared to the non-treated controls (Fig. 2B). To demonstrate that this effect was due to MAC depletion caused by the C6 knockdown we tested the effect of antibody (BB5.1)-mediated C5 inhibition on sensory function recovery of the crushed nerve. Similar to the C6 antisense oligonucleotide treatment, the C5 antibody promoted recovery of sensory function to the injured nerve (Fig. 2C). This shows that inhibition of the terminal complement pathway increases the speed of functional recovery of the mouse sciatic nerve after crush, similar to what was previously seen in the rat (Ramaglia et al., 2008, 2009).

Fig. 2.

Fig. 2

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Inhibition of MAC improves recovery of sensory function after nerve crush. A: Schematic drawing of the experimental setup. B, Graphical depiction of the terminal complement pathway and the two points of intervention. C: Effect of C6 inhibition using a C6 locked nucleic acid (LNA) modified antisense oligonucleotide on sensory recovery post-acute crush of the sciatic nerve. D: Effect of treatment with an anti-C5 monoclonal antibody (BB5.1) on sensory recovery after acute crush of the sciatic nerve (for all experiments: n = 7 per group, SEM).

3.3. Characterization of motor function and sciatic nerve transcriptome of PMP22 over-expressing CMT1A models

Motor function of the C3-PMP22 and C3-PMP22 c-JunP0Cre mice was analysed during the first 9 weeks after birth (Fig. 3). Functional motor impairment developed between week 4 and 12, resulting in a CMT1A phenotype in the C3 mice. The c-Jun deficient animals had a more severe phenotype as compared to the c-Jun sufficient C3-PMP22 animals, confirming earlier reports (Hantke et al., 2014; Gomez-Sanchez et al., 2015). Notably, aging affected the balance of the c-Jun deficient but not of the c-Jun sufficient mice based on data obtained from the balance beam slips test (Fig. 3C). In addition, tremor increased in the c-Jun deficient animals until week 7 post-birth but returned to baseline levels on week 9, showing the same phenotype as the c-Jun sufficient mice (Fig. 3A). With respect to sex, differences in performance were noted, which might be directly related to differences in the body weight or the body size between male and female mice. For tests that produced parametric data, and for which parametric RM-ANOVA was performed, there were no significant sex*genotype interaction effects, suggesting that the C3 transgene had similar effects in males and females (see Supplemental Table 1). RNA sequencing was performed on sciatic nerves obtained from adult (>14 weeks old) C3-PMP22, C3-PMP22 c-JunP0Cre, and wild type littermates. Pathway analysis of RNA expression data showed that similar pathway activity pattern changes were seen in the two tested CMT1A models when compared with wild type controls. In these analyses the cholesterol synthesis pathways were the most severely affected pathways (Fig. 4), being completely shut down.

Fig. 3.

Fig. 3

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Functional tests (tremor, balance beam slips, hanging wire and paw strength) showing differences in motor function between the C3-PMP22 (n = 18 per group) and the C3-PMP22 c-JunP0Cre mice (n = 13 per group), including sex-related differences.

Fig. 4.

Fig. 4

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RNA expression data of sciatic nerves from C3-PMP22 and C3-PMP22 c-JunP0Cre mice compared with wildtype mice reveals that pathway activity patterns are similar in the two models. Cholesterol synthesis is completely shut down in sciatic nerves of both strains. A (left): Heatmap depicting the z scores of each pathway activity. A negative z score (blue) means a predicted down regulation of a pathway. B: Expression fold changes of gene expression in the cholesterol synthesis pathway. For both graphs dataset A = C3-PMP22 and dataset B Created by potrace 1.16, written by Peter Selinger 2001-2019 C3-PMP22 c-JunP0Cre. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.4. Systemic C6 inhibition in PMP22 over-expressing mice results in changes in the expression of genes involved in the neuroinflammation, rho GTPase and ERK/MAPK signalling pathways in the sciatic nerve

To study the effect of systemic C6 inhibition on the two models of CMT1A, we administered the C6 antisense oligonucleotide at a dose of 5 mg/kg by subcutaneous injections twice a week, from week 4 -when disease is not yet started (Verhamme et al., 2011)- until week 9 of age (10 injections in total). RNA-Seq was performed on sciatic nerves obtained from treated C3-PMP22, treated C3-PMP22 c-JunP0Cre, control-treated C3-PMP22, and control-treated C3-PMP22 c-JunP0Cre mice. Pathway analysis revealed that only a limited set of biological pathways including the neuroinflammation, Rho GTPase and ERK/MAPK pathways, were affected by the C6 antisense treatment in the C3-PMP22 and C3-PMP22 cJunP0Cre mouse models as compared to control treated animals (Fig. 5). These effects are not caused by loss of macrophages in the sciatic nerve. The number of macrophages did not change in the sciatic nerve after C6 antisense oligonucleotide treatment as determined by quantification of Iba-1 positive cells and by analysis of gene expression data of the sciatic nerves for markers that reflect macrophage presence (Supplemental Fig. 3). The downregulation of the cholesterol synthesis pathway did not change during the treatment with the C6 antisense oligonucleotide.

Fig. 5.

Fig. 5

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Effect of the C6 antisense oligonucleotide on gene expression in sciatic nerves obtained from C3-PMP22 and C3-PMP22 c-JjunP0Cre mice. Panel A: z scores depicting activation status of differentially regulated canonical biological pathways as calculated by IPA. A positive z score indicates a predicted activation of a pathway; a negative z score indicates a predicted inhibition of a pathway. The differential gene expression analysis is done by comparison with WT littermates for the C3-PMP22 (column A) and C3-PMP22 c-JunP0Cre (column B) mice and, with control-treated mice for the C6 antisense-treated mice (columns C, D). Panel B: Color-coded depiction of gene expression (expression fold changes) of three selected pathways that are differentially regulated. The differential gene expression analysis is done by comparison with WT littermates for the C3-PMP22 mice (column A) and with control treated mice for the C6 antisense treated mice (columns B, C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.5. Systemic C6 inhibition in PMP22 over-expressing mice does not improve motor function

To study whether treatment with the C6 antisense affects motor performance, we analysed the tested parameters hanging wire, paw strength and balance beam test, in the two CMT1A mouse models (Fig. 6A–F). No significant differences were detected between treated and non-treated mice in any of the two models. Analysis also showed no differences between males and females. Both models were also tested on a gait analysis system (catwalk). We focussed on the parameters stride length and support diagonal, as these were considered the most informative based on data from pilot studies aiming to characterize the C3-PMP22 mice (data not shown). Our data show that there was no effect of C6 inhibition on stride length and support diagonal in any of the two models (Fig. 7, see statistics in Supplemental Table 2).

Fig. 6.

Fig. 6

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Treatment of C3-PMP22 (n = 18 per group) or C3-PMP22 c-JunP0Cre mice (n = 14 per group) with C6 antisense oligonucleotides did not result in a significant difference in disease progression compared to controls as measured with multiple motor function tests. A. Schematic drawing of the experimental setup. B-G, Motor function tests: Hanging Wire test (B and C). Strength of front and Hind paw (D and E), Balance beam slips (F and G).

Fig. 7.

Fig. 7

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Catwalk analysis of stride length (A) and support diagonal (B) of C3-PMP22 (n = 18 per group) and C3-PMP22 c-JunP0Cre mice (n = 14 per group) with or without the C6 antisense oligonucleotide treatment.

4. Discussion

Inhibition of the complement system improved recovery in rodent models of Wallerian degeneration (Ramaglia et al., 2008). Specifically, it was demonstrated that C6 deficiency improved recovery of sensory function after a nerve crush (Ramaglia et al., 2009). In addition, it was shown that systemic C6 inhibition using antisense oligonucleotides is beneficial in models of peripheral nerve injury and CNS neuroinflammation (Michailidou et al., 2018; Fluiter et al., 2014; Bahia El Idrissi et al., 2015). In this study we tested whether systemic inhibition of the terminal complement system by blocking synthesis of C6 in the liver using antisense oligonucleotides affects disease progression in two transgenic mouse models overexpressing the human PMP22 gene, resembling human CMT1A disease. We present data showing that C6 inhibition does not ameliorate disease progression in any of the two transgenic models examined but it does affect the activation status of important immune pathways and it improves sensory recovery after acute nerve crush.

We set out to investigate the role of complement in two transgenic models of CMT1A each having up to 5 copies of the PMP22 gene reproducing a disease which mimics human CMT1A. We found substantial deposition of complement C1q, C3 and C9 within the sciatic nerve parenchyma in both the C3-PMP22 and C3-PMP22 c-Jun P0Cre mice, suggestive of complement activation until the terminal point. To get insights into the molecular contribution of complement to these models we tested the effect of systemic C6 inhibition on the C3-PMP22 and C3-PMP22 c-JunP0Cre models. It was earlier shown that the lack of c-Jun in Schwann cells hampers autophagy (Gomez-Sanchez et al., 2015), a mechanism which might compensate for the apparent shutdown of cholesterol synthesis caused by the overexpression of PMP22 (Zhao et al. 2018; Zhou et al., 2019; Zhou et al., 2020). Pathway analysis of RNAseq data of sciatic nerves obtained from C3-PMP22 and C3-PMP22 c-JunP0Cre mice showed that both lines are very similar in differentially regulated pathways as compared to wild type littermates. Notably, a strong downregulation of cholesterol synthesis pathways was found in the sciatic nerves of both mouse lines. Why cholesterol synthesis is inhibited in PMP22 overexpressing mice is still unclear. Previous research in the C22 PMP22 over-expressing murine model (with 10 copies of human PMP22) showed deregulation of cholesterol synthesis and amelioration of this effect when PMP22 overexpression was lowered by antisense oligonucleotides (Zhao et al., 2017), suggesting a link between cholesterol synthesis and the PMP22 protein. PMP22 contains a highly conserved cholesterol recognition CRAC motif in the fourth transmembrane domain (Sedzik et al., 2013) and a conserved palmitoylation site at C85 (Zoltewicz et al., 2012) both suggesting a direct interaction with lipids. Indeed, a recent study demonstrated that Schwann cells from nerves of PMP22 knockout mice show a different cholesterol distribution and aberrant lipid raft morphology, indicating that PMP22 plays a role in cellular lipid metabolism. In addition, PMP22 was involved in the regulation of cholesterol trafficking (Zhou et al., 2019) and PMP22 overexpression resulted in lysosomal sequestering of cholesterol esters (Zhou et al., 2020).

We show that inhibition of C6 protein synthesis using antisense oligonucleotides in the liver resulted in reduced liver C6 mRNA levels in both males and females; the drop of liver C6 mRNA results in reduced systemic C6 protein levels, and reduced amounts of MAC formation on nervous tissues (Michailidou et al., 2018; Fluiter et al., 2014; Bahia El Idrissi et al., 2015). Here we show that overexpression of PMP22 resulted in upregulation of pathways involved in neuroinflammation, whereas the Rho signalling and ERK/MAPK pathways were downregulated. Treatment with a C6 antisense oligonucleotide ameliorated these effects (Fig. 5). However, the C6 antisense treatment did not affect the number of macrophages within the sciatic nerve endoneurium and the cholesterol synthesis pathways remained shut down.

The ERK/MAPK pathway is known to regulate myelination and control the thickness of the myelin layer (Ishii et al., 2012). Peripheral nerve injury induces the ERK signalling to promote remyelination following Wallerian degeneration (Napoli et al., 2012). However, strong induction of the ERK/MAPK signalling in the adult peripheral nerve induces demyelination (Cervellini et al., 2018) leading to the hypothesis that the ERK/MAPK pathway is a double-edged sword.

Rho GTPases regulate the changes of the cellular cytoskeleton which are related to cell differentiation the regeneration of peripheral nerves (Kalpachidou et al., 2019; Stankiewicz Trisha and Linseman Daniel, 2014). They regulate processes which are essential for neuronal survival and re-innervation of the de-nervated target following injury. Notably, CMT disease type 4H is linked to dysfunction of the Rho GTPase pathways (Stendel et al., 2007; Delague et al., 2007). In Schwann cells, Rho GTPases get activated during Wallerian degeneration (Kalpachidou et al., 2019) and are important for Schwann cell proliferation, migration and differentiation mediated by the downstream c-Jun N-terminal kinase (JNK) pathway and p38 mitogen-activated protein kinase (MAPK) (Ishii et al., 2012). It is therefore interesting that C6 inhibition in the C3-PMP22 and C3-PMP22 c-JunP0Cre mice downregulates genes involved in the canonical Rho GTPase pathway. More research is necessary to determine the effect on the Schwann cell's capacity to proliferate and migrate.

The effect of C6 inhibition on the pathways involved in neuroinflammation is in line with the known effect of (sub)lytic MAC levels on NLRP3 inflammasome activation (Michailidou et al., 2018; Triantafilou et al., 2013). Systemic C6 inhibition was shown to confer protection in a variety of neuroinflammation models (Michailidou et al., 2018; Fluiter et al., 2014; Bahia El Idrissi et al., 2015). However, we could not detect any significant effect on motor function in the two tested CMT1A mouse models. Thus, the complement system is linked to neuroinflammation, but its contribution to disease development in these models is minor, if any. This contrasts with our previous studies (Ramaglia et al. 2007, 2008, 2009) which show that the inhibition of the complement system is involved in Wallerian degeneration. At first sight this seems contradictory. However, in the studies by Ramaglia et al. a nerve crush model was used which showed acute neuroinflammation and Wallerian degeneration. In this acute model the contribution of the complement system, which is key in the immune response to tissue damage, drove important downstream effects such as macrophage activation and demyelination. Here we show, that in the C3-PMP22 model of chronic disease, the complement system is less activated and apparently not as important as in acute tissue damage and autoimmune responses. In the CMT1A disease models demyelination occurs slowly but is progressively propagating. The other rodent models with significant effects of complement inhibitors on disease progression were all models of acute neuroinflammatory disease and showed high levels of complement activation (Michailidou et al., 2018; Fluiter et al., 2014; Bahia El Idrissi et al., 2015; Gytz Olesen et al., 2022). Our work presented here suggests that terminal complement activation and neuroinflammation is not a major force in disease progression in the CMT1A mouse models.

5. Conclusions

Our data show that MAC formation is a driver for changes in the expression levels of genes involved in the neuroinflammation, EKR/MAPK and Rho GTPase pathways. However, despite the potentially beneficial knockdown of systemic C6 on these pathways, a C6 antisense oligonucleotide treatment does not lead to a clinically relevant improvement of the motor function in mice overexpressing PMP22. In addition, the effect of PMP22 overexpression on cholesterol synthesis is not dependent on MAC formation.

CRediT authorship contribution statement

Iliana Michailidou: Methodology, and . Jeroen Vreijling: Methodology, and . Matthijs Rumpf: Methodology, and . Maarten Loos: Formal analysis, Writing – review & editing. Bastijn Koopmans: Formal analysis. Nina Vlek: Methodology, and . Nina Straat: Methodology, and . Cedrick Agaser: Formal analysis. Thomas B. Kuipers: Formal analysis. Hailiang Mei: Formal analysis, Writing – review & editing. Frank Baas: Funding acquisition, Conceptualization, Writing – review & editing, Supervision. Kees Fluiter: Conceptualization, Methodology, and .

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Frank Baas reports a relationship with Complement Pharma BV that includes: board membership. Kees Fluiter reports a relationship with Complement Pharma BV that includes: consulting or advisory. Maarten Loos reports a relationship with Synaptologics BV that includes: employment. Bastijn Koopmans reports a relationship with Synaptologics BV that includes: employment. Nina Vlek reports a relationship with Synaptologics BV that includes: employment. Nina Straat reports a relationship with Synaptologics BV that includes: employment. Frank Baas has patent Complement inhibition for improved nerve regeneration issued to Regenesance BV. Frank Baas has patent COMPLEMENT ANTAGONISTS AND USES THEREOF issued to Regenesance BV. Kees Fluiter has patent COMPLEMENT ANTAGONISTS AND USES THEREOF issued to Regenesance BV. Frank Baas has patent #ANTIBODIES THAT BIND HUMAN C6 AND USES THEREOF issued to Regenesance BV.

Acknowledgements Funding

The work was supported financially by a grant from the “Prinses Beatrix Spierfonds “grant no. W.OR14-09 and AFM Telethon (France) grant 16523.

The sponsors did not have any role in the design of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crneur.2023.100077.

Appendix A. Supplementary data

The following are the Supplementary data to this article.

Multimedia component 1
mmc1.pdf (182.6KB, pdf)
Multimedia component 2
mmc2.docx (1.1MB, docx)

Data availability

Data will be made available on request.

References

  1. Bahia El Idrissi N., Das P.K., Fluiter K., Rosa P.S., Vreijling J., Troost D., Morgan B.P., Baas F., Ramaglia V. M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator. Acta Neuropathol. 2015;129(5):653–667. doi: 10.1007/s00401-015-1404-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berciano J., Garcia A., Calleja J. Clinico-electrophysiological correlation of extensor digitorum brevis muscle atrophy in children with Charcot-Marie-Tooth disease 1A duplication. Neuromuscul. Disord. 2000;10:419–424. doi: 10.1016/s0960-8966(99)00114-5. [DOI] [PubMed] [Google Scholar]
  3. Boerkoel C.F., Takashima H., Garcia C.A. Charcot-Marie-Tooth disease and related neuropathies: mutation distribution and genotype-phenotype correlation. Ann. Neurol. 2002;51:190–201. doi: 10.1002/ana.10089. [DOI] [PubMed] [Google Scholar]
  4. Brück W., Friede R.L. The role of complement in myelin phagocytosis during PNS wallerian degeneration. J. Neurol. Sci. 1991;103(2):182–187. doi: 10.1016/0022-510x(91)90162-z. PMID: 1880536. [DOI] [PubMed] [Google Scholar]
  5. Brunner E., Puri M.L. Nonparametric methods in factorial designs. Stat. Pap. 2001;42:1–52. doi: 10.1007/s003620000039. [DOI] [Google Scholar]
  6. Cervellini I., Galino J., Zhu N., Allen S., Birchmeier C., Bennett D.L. Sustained MAPK/ERK activation in adult Schwann cells impairs nerve repair. J. Neurosci. 2018;(3):679–690. doi: 10.1523/JNEUROSCI.2255-17.2017. 17;38. PMID: 29217688; PMCID: PMC5777114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dailey A.T., Avellino A.M., Benthem L., Silver J., Kliot M. Complement depletion reduces macrophage infiltration and activation during Wallerian degeneration and axonal regeneration. J. Neurosci. 1998;18(17):6713–6722. doi: 10.1523/JNEUROSCI.18-17-06713.1998. PMID: 9712643; PMCID: PMC6792968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. de Jonge R.R., Vreijling J.P., Meintjes A., Kwa M.S., van Kampen A.H., van Schaik I.N., Baas F. Transcriptional profile of the human peripheral nervous system by serial analysis of gene expression. Genomics. 2003;82(2):97–108. doi: 10.1016/s0888-7543(03)00124-1. PMID: 12837261. [DOI] [PubMed] [Google Scholar]
  9. de Koning P., Brakkee J.H., Gispen W.H. Methods for producing a reproducible crush in the sciatic and tibial nerve of the rat and rapid and precise testing of return of sensory function. Beneficial effects of melanocortins. J. Neurol. Sci. 1986;74(2–3):237–246. doi: 10.1016/0022-510x(86)90109-7. 1986 Jul. PMID: 3016200. [DOI] [PubMed] [Google Scholar]
  10. Delague V., Jacquier A., Hamadouche T., Poitelon Y., Baudot C., Boccaccio I., Chouery E., Chaouch M., Kassouri N., Jabbour R., Grid D., Mégarbané A., Haase G., Lévy N. Mutations in FGD4 encoding the Rho GDP/GTP exchange factor FRABIN cause autosomal recessive Charcot-Marie-Tooth type 4H. Am. J. Hum. Genet. 2007;81(1):1–16. doi: 10.1086/518428. 2007 Jul. PMID: 17564959; PMCID: PMC1950914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fluiter K., Opperhuizen A.L., Morgan B.P., Baas F., Ramaglia V. Inhibition of the membrane attack complex of the complement system reduces secondary neuroaxonal loss and promotes neurologic recovery after traumatic brain injury in mice. J. Immunol. 2014;192(5):2339–2348. doi: 10.4049/jimmunol.1302793. PMID: 24489093. [DOI] [PubMed] [Google Scholar]
  12. Frei Y., Lambris J.D., Stockinger B. Generation of a monoclonal antibody to mouse C5 application in an ELISA assay for detection of anti-C5 antibodies. Mol. Cell. Probes. 1987;(2):141–149. doi: 10.1016/0890-8508(87)90022-3. PMID: 3453897. [DOI] [PubMed] [Google Scholar]
  13. Giambonini-Brugnoli G., Buchstaller J., Sommer L., Suter U., Mantei N. Distinct disease mechanisms in peripheral neuropathies due to altered peripheral myelin protein 22 gene dosage or a PMP22 point mutation. Neurobiol. Dis. 2005;18(3):656–668. doi: 10.1016/j.nbd.2004.10.023. PMID: 15755691. [DOI] [PubMed] [Google Scholar]
  14. Gomez-Sanchez J.A., Carty L., Iruarrizaga-Lejarreta M., Palomo-Irigoyen M., Varela-Rey M., Griffith M., Hantke J., Macias-Camara N., Azkargorta M., Aurrekoetxea I., De Juan V.G., Jefferies H.B., Aspichueta P., Elortza F., Aransay A.M., Martínez-Chantar M.L., Baas F., Mato J.M., Mirsky R., Woodhoo A., Jessen K.R. Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves. J. Cell Biol. 2015;210(1):153–168. doi: 10.1083/jcb.201503019. PMID: 26150392; PMCID: PMC4494002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gytz Olesen H., Michailidou I., Zelek W.M., Vreijling J., Ruizendaal P., de Klein F., Marquart J.A., Kuipers T.B., Mei H., Zhang Y., Ahasan M., Johnson K.K., Wang Y., Morgan B.P., van Dijk M., Fluiter K., Andersen G.R., Baas F. Development, characterization, and in vivo validation of a humanized C6 monoclonal antibody that inhibits the membrane attack complex. J Innate Immun. 2022;12:1–21. doi: 10.1159/000524587. PMID: 35551129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hantke J., Carty L., Wagstaff L.J., Turmaine M., Wilton D.K., Quintes S., Koltzenburg M., Baas F., Mirsky R., Jessen K.R. c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy. Brain. 2014;137(Pt 11):2922–2937. doi: 10.1093/brain/awu257. PMID: 25216747; PMCID: PMC4208468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hobart M.J., Lachmann P.J., Calne R.Y. C6: synthesis by the liver in vivo. J. Exp. Med. 1977;146(2):629–630. doi: 10.1084/jem.146.2.629. PMID: 327019; PMCID: PMC2180766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huxley C., Passage E., Manson A. Construction of a mouse model of Charcot-Marie-Tooth disease type 1A by pronuclear injection of human YAC DNA. Hum. Mol. Genet. 1996;5:563–569. doi: 10.1093/hmg/5.5.563. [DOI] [PubMed] [Google Scholar]
  19. Ishii A., Fyffe-Maricich S.L., Furusho M., Miller R.H., Bansal R. ERK1/ERK2 MAPK signalling is required to increase myelin thickness independent of oligodendrocyte differentiation and initiation of myelination. J. Neurosci. 2012;32(26):8855–8864. doi: 10.1523/JNEUROSCI.0137-12.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kalpachidou T., Spiecker L., Kress M., Quarta S. Rho GTPases in the physiology and pathophysiology of peripheral sensory neurons. Cells. 2019;8(6):591. doi: 10.3390/cells8060591. . Published 2019 Jun 15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kokubun N. Charcot–marie–tooth disease and neuroinflammation. Clinical & experimental neuroimmunology. 2020;11(2):109–116. doi: 10.1111/cen3.12566. ISSN: 1759-1961 , 1759-1961. [DOI] [Google Scholar]
  22. Krajewski K.M., Lewis R.A., Fuerst D.R. Neurological dysfunction and axonal degeneration in Charcot-Marie-Tooth disease type 1A. Brain. 2000:1516–1527. doi: 10.1093/brain/123.7.1516. PMID: 10869062. [DOI] [PubMed] [Google Scholar]
  23. Krämer A., Green J., Pollard J., Jr., Tugendreich S. Causal analysis approaches in ingenuity pathway analysis. Bioinformatics. 2014;30(4):523–530. doi: 10.1093/bioinformatics/btt703. PMID: 24336805; PMCID: PMC3928520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Michailidou I., Jongejan A., Vreijling J.P., Georgakopoulou T., de Wissel M.B., Wolterman R.A., Ruizendaal P., Klar-Mohamad N., Grootemaat A.E., Picavet D.I., Kumar V., van Kooten C., Woodruff T.M., Morgan B.P., van der Wel N.N., Ramaglia V., Fluiter K., Baas F. Systemic inhibition of the membrane attack complex impedes neuroinflammation in chronic relapsing experimental autoimmune encephalomyelitis. Acta Neuropathol Commun. 2018;6(1):36. doi: 10.1186/s40478-018-0536-y. PMID: 29724241; PMCID: PMC5932802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Napoli I., Noon L.A., Ribeiro S., Kerai A.P., Parrinello S., Rosenberg L.H., Collins M.J., Harrisingh M.C., White I.J., Woodhoo A., Lloyd A.C. A central role for the ERK-signalling pathway in controlling Schwann cell plasticity and peripheral nerve regeneration in vivo. Neuron. 2012;73:729–742. doi: 10.1016/j.neuron.2011.11.031. pmid:22365547. [DOI] [PubMed] [Google Scholar]
  26. Noguchi K., Gel Y.R., Brunner E., Konietschke F. nparLD: an R software package for the nonparametric analysis of longitudinal data in factorial experiments. J. Stat. Software. 2012;50(12):1–23. doi: 10.18637/jss.v050.i12. [DOI] [Google Scholar]
  27. Percie du Sert N., Hurst V., Ahluwalia A., Alam S., Avey M.T., Baker M., et al. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. PLoS Biol. 2020;18(7) doi: 10.1371/journal.pbio.3000410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Raeymaekers P., Timmerman V., Nelis E. Duplication in chromosome 17p11.2 in charcot-marie-tooth neuropathy type 1a (CMT1a). The HMSN collaborative research group. Neuromuscul. Disord. 1991;1:93–97. doi: 10.1016/0960-8966(91)90055-w. PMID: 1822787. [DOI] [PubMed] [Google Scholar]
  29. Ramaglia V., King R.H., Nourallah M., Wolterman R., de Jonge R., Ramkema M., Vigar M.A., van der Wetering S., Morgan B.P., Troost D., Baas F. The membrane attack complex of the complement system is essential for rapid Wallerian degeneration. J. Neurosci. 2007;27(29):7663–7672. doi: 10.1523/JNEUROSCI.5623-06.2007. PMID: 17634361; PMCID: PMC6672891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ramaglia V., Wolterman R., de Kok M., Vigar M.A., Wagenaar-Bos I., King R.H., Morgan B.P., Baas F. Soluble complement receptor 1 protects the peripheral nerve from early axon loss after injury. Am. J. Pathol. 2008;172(4):1043–1052. doi: 10.2353/ajpath.2008.070660. PMID: 18349134; PMCID: PMC2276415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ramaglia V., Tannemaat M.R., de Kok M., Wolterman R., Vigar M.A., King R.H., Morgan B.P., Baas F. Complement inhibition accelerates regeneration in a model of peripheral nerve injury. Mol. Immunol. 2009;47(2–3):302–309. doi: 10.1016/j.molimm.2009.09.019. PMID: 19833392. [DOI] [PubMed] [Google Scholar]
  32. Rifai A., Brysch W., Fadden K., Clark J., Schlingensiepen K.H. Clearance kinetics, biodistribution, and organ saturability of phosphorothioate oligodeoxynucleotides in mice. Am. J. Pathol. 1996;149(2):717–725. PMID: 8702009; PMCID: PMC1865299. [PMC free article] [PubMed] [Google Scholar]
  33. Rogers D.C., Peters J., Martin J.E., Ball S., Nicholson S.J., Witherden A.S., Hafezparast M., Latcham J., Robinson T.L., Quilter C.A., Fisher E.M. SHIRPA, a protocol for behavioral assessment: validation for longitudinal study of neurological dysfunction in mice. Neurosci. Lett. 2001;306(1–2):89–92. doi: 10.1016/s0304-3940(01)01885-7. PMID: 11403965. [DOI] [PubMed] [Google Scholar]
  34. Sedzik J., Jastrzebski J.P., Ikenaka K. Sequence motifs of myelin membrane proteins: towards the molecular basis of diseases. J. Neurosci. Res. 2013;91:479–493. doi: 10.1002/jnr.23177. [DOI] [PubMed] [Google Scholar]
  35. Shy M.E., Chen L., Swan E.R. Neuropathy progression in Charcot-Marie-Tooth disease type 1A. Neurology. 2008;70:378–383. doi: 10.1212/01.wnl.0000297553.36441.ce. PMID: 18227419. [DOI] [PubMed] [Google Scholar]
  36. Stankiewicz Trisha R., Linseman Daniel A. Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration. Front. Cell. Neurosci. 2014;7(8):314. doi: 10.3389/fncel.2014.00314. PMID: 25339865; PMCID: PMC4187614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stendel C., Roos A., Deconinck T., Pereira J., Castagner F., Niemann A., Kirschner J., Korinthenberg R., Ketelsen U.P., Battaloglu E., Parman Y., Nicholson G., Ouvrier R., Seeger J., De Jonghe P., Weis J., Krüttgen A., Rudnik-Schöneborn S., Bergmann C., Suter U., Zerres K., Timmerman V., Relvas J.B., Senderek J. Peripheral nerve demyelination caused by a mutant Rho GTPase guanine nucleotide exchange factor, frabin/FGD4. Am. J. Hum. Genet. 2007;81(1):158–164. doi: 10.1086/518770. PMID: 17564972; PMCID: PMC1950925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Triantafilou K., Hughes T.R., Triantafilou M. Morgan BP. The complement membrane attack complex triggers intracellular Ca2+ fluxes leading to NLRP3 inflammasome activation. J. Cell Sci. 2013;126(Pt 13):2903–2913. doi: 10.1242/jcs.124388. PMID: 23613465. [DOI] [PubMed] [Google Scholar]
  39. Verhamme C., van Schaik I.N., Koelman J.H.T.M. Clinical disease severity and axonal dysfunction in hereditary motor and sensory neuropathy Ia. J. Neurol. 2004;251:1491–1497. doi: 10.1007/s00415-004-0578-x. PMID: 15645349. [DOI] [PubMed] [Google Scholar]
  40. Verhamme C., King R.H.M., ten Asbroek A.L.M.A., Muddle J.R., Nourallah M., Wolterman R., Baas F., MD. van Schaik I.N. Myelin and axon pathology in a long-term study of PMP22-overexpressing mice. J. Neuropathol. Exp. Neurol. 2011;70(5):386–398. doi: 10.1097/NEN.0b013e318217eba0. PMID: 21487305. [DOI] [PubMed] [Google Scholar]
  41. Zhao H.T., Damle S., Ikeda-Lee K., Kuntz S., Li J., Mohan A., Kim A., Hung G., Scheideler M.A., Scherer S.S., Svaren J., Swayze E.E., Kordasiewicz H.B. PMP22 antisense oligonucleotides reverse Charcot-Marie-Tooth disease type 1A features in rodent models. J. Clin. Invest. 2017;128(1):359–368. doi: 10.1172/JCI96499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhou Y., Miles J.R., Tavori H., Lin M., Khoshbouei H., Borchelt D.R., Bazick H., Landreth G.E., Lee S., Fazio S., Notterpek L. PMP22 regulates cholesterol trafficking and ABCA1-mediated cholesterol efflux. J. Neurosci. 2019;39(27):5404–5418. doi: 10.1523/JNEUROSCI.2942-18.2019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhou Y., Borchelt D., Bauson J.C., Fazio S., Miles J.R., Tavori H., Notterpek L. Subcellular diversion of cholesterol by gain- and loss-of-function mutations in PMP22. Glia. 2020;68:2300–2315. doi: 10.1002/glia.23840. [DOI] [PubMed] [Google Scholar]
  44. Zoltewicz S.J., Lee S., Chittoor V.G., Freeland S.M., Rangaraju S., Zacharias D.A., Notterpek L. The palmitoylation state of PMP22 modulates epithelial cell morphology and migration. ASN Neuro. 2012;4(6):409–421. doi: 10.1042/AN20120045. PMID: 23127255. PMCID: PMC3563111. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Multimedia component 1
mmc1.pdf (182.6KB, pdf)
Multimedia component 2
mmc2.docx (1.1MB, docx)

Data Availability Statement

Data will be made available on request.

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