Membrane-bound complement regulator expression in peripheral nerves

doi:10.21203/rs.3.rs-2738481/v1

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Membrane-bound complement regulator expression in peripheral nerves

https://doi.org/10.21203/rs.3.rs-2738481/v1

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published 24 Oct, 2023

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BACKGROUND: Homozygous CD59-deficient patients manifest with recurrent peripheral neuropathy resembling Guillain-Barré syndrome (GBS), hemolytic anemia and recurrent strokes. Variable mutations in CD59, leading to loss of function, have been described, and overall, 17/18 of patients with any mutation presented with recurrent GBS. Here we determine the expression and possible role of membrane-bound complement regulators including CD59, in the peripheral nervous system (PNS) of mice and humans.

MAIN: Methods. We examined the expression of membrane-bound complement regulators in the peripheral nerves of healthy humans and a CD59-deficient patient, as well as in wild-type (WT) and CD59a-deficient mice. Cross sections of teased sciatic nerves and myelinating dorsal root ganglia (DRG) neuron/Schwann cell cultures were examined by confocal and electron microscopy.

Results. We demonstrate that CD59-deficient mice display normal peripheral nerve morphology but develop myelin abnormalities in older age. They normally express P0, ankyrin G (AnkG), caspr, dystroglycan, and neurofascin. CD59 is colocalized with myelin basic protein (MBP) and P0 and is present in compact myelin and endothelium but absent from noncompact myelin. Furthermore, immunolabeling of WT nerves using antibodies to CD59 and AnkG revealed that CD59 was localized along the internode but was absent from the nodes of Ranvier. Finally, we show that the nodes of Ranvier lack other complement-membrane regulatory proteins, including CD46, CD55, CD35, and Crry, rendering this area highly exposed to complement attack.

CONCLUSION: Myelin in the human PNS is protected only by CD59 and CD55, and not by CD46 or CD35, rendering myelin in the PNS vulnerable to complement attack and demyelination in autoinflammatory Guillain-Barre syndrome, as seen in CD59 deficiency.

Guillain-Barré syndrome

CD59

Peripheral nervous system

Complement

Guillain-Barré syndrome (GBS), including its variants, is currently the most frequent cause of acute flaccid paralysis worldwide and constitutes a serious neurological emergency. The disease encompasses a group of peripheral nerve disorders, each distinguished by the distribution of weakness in the limbs or cranial nerve-innervated muscles and underlying pathophysiology. There is substantial evidence to support an autoimmune cause with autoantibodies for the classical syndrome¹ and an autoinflammatory pattern without autoantibodies in congenital mutated CD59.^2-4 In both conditions, complement activation was suggested to have a crucial role in pathogenesis, although it is not known why patients with GBS or those with CD59 deficiency are susceptible to complement attack.^1-4

Primary congenital CD59 deficiency manifested by recurrent GBS in humans is a germline mutation, described in 18 individuals and characterized by recurrent GBS (GBS) (17/18), hemolytic anemia (14/16), and recurrent strokes (8/18).^3-17

The Membrane attack complex (MAC) inhibitory function of CD59 is related to its capacity to bind C8 within C5b-8 and block recruitment of C9, which is essential for MAC formation. This accounts for the observed impact on lytic sensitivity.^{18, 19} We have recently shown that the four CD59 mutations described so far have similar clinical manifestations despite differences between the mutant proteins.² The frameshift mutants p.Asp24Valfs* and p.Ala16Alafs* generate truncated proteins with a distinct c-terminal sequence, common to both mutants, that are not present at the plasma membrane. In contrast, the missense mutants p.Cys64Tyr and p.Asp24Val do reach the cell surface. Despite this difference, all four mutants produce nonfunctional CD59 and are thus unable to protect the host from MAC attack. As judged by clinical manifestations, another two mutations, the Ser83Ter (Terminal) nonsense⁸ and Tyr4Asp missense mutations,⁹ produce nonfunctional CD59.

How would nonfunctioning mutated CD59 contribute to the appearance of autoimmune (classical sporadic) and autoinflammatory (congenital, recurrent) GBS? In the current study, we use biopsies from healthy and CD59-deficient humans and from WT and CD59-deficient mice. We evaluated cross sections, teased sciatic nerve fibers, and myelinating DRG neuron/Schwann cell cultures for CD59 expression using a variety of antibodies to study the exact anatomical localization of CD59 and other membrane-bound complement with the aim of understanding their physiological and pathophysiological roles.

Ethical approval. Human (HMO0390-12-; HMO0379-18-) and animal (MD-17-14937-4; MD-12-13432-1) ethical approvals were issued by the committees of Hadassah Medical Center and Faculty of Medicine, respectively.

Immunofluorescence labeling of complement regulators in mice peripheral nerve. Mouse sciatic nerves were excised and fixed for 30 minutes in 4% paraformaldehyde. For cross-sections and longitudinal sections, all mouse sciatic nerves were cryoprotected overnight in 30% sucrose and then cut into 10μm-thick sections. Teased sciatic nerves, cross sections and longitudinal sections were immunostained as described previously.²⁰ Briefly, samples were permeabilized by methanol and blocked for 1 hour with 0.5% Triton X100 PBS and 5% normal goat serum, then incubated overnight at 4°C with different primary antibodies diluted in 0.1% Triton X100 PBS blocking solution, extensively washed, and incubated for 45 minutes at room temperature with secondary antibodies. Finally, slides were mounted with elvanol and analyzed.

Immunostaining was also performed with the tyramide signal amplification (TSA) method in teased fibers and longitudinal sections. Endogenous peroxidase was quenched by incubation for 30 minutes in 0.25% H₂O₂/methanol. Sections were washed in PBS and incubated for 30 minutes with blocking buffer (TSA Fluorescence System Kit, PerkinElmer, Waltham, MA, USA). They were then incubated for 30 minutes with 3µg/ml rat antibody against mouse DAF/CD55 (Cardiff University, 2C6) or with rabbit polyclonal antibody against mouse Crry (Complement receptor type-1 related gene Y) (Biorbyt, Cambridge, UK). The treated sections were subsequently washed with TNT buffer (0.1M tris, pH 7.5, 0.15M NaCl, 0.05%, tween-20) and incubated for 30 minutes with 3µg/ml HRP-conjugated mouse anti-rat/rabbit IgG (Jackson Laboratories, Bar Harbor, ME, USA). After a second wash with TNT buffer, the sections were stained for 6 minutes at 20°C with tyramide-red (PerkinElmer). Finally, slides were mounted with elvanol.

For murine sciatic nerve, teased, cross and longitudinal sections (including the TSA method), we used rabbit polyclonal antibody against mouse CD59 (Cardiff University), rat monoclonal antibody against mouse CD59 (Cardiff University), chicken polyclonal antibody against mouse P0 (AVES-2B Scientific, Oxfordshire, UK), mouse antibody against mouse ankyrin-G (NeuroMab, Davis, CA, USA), rat antibody against mouse myelin basic protein (MBP) (Millipore, Burlington, MA, USA), rabbit monoclonal antibody against mouse Caspr (Weizmann Institute²¹), rat antibody against mouse DAF/CD55 (Cardiff University, 2C6), rabbit polyclonal antibody against mouse CD46 (Abcam, Cambridge, UK), mouse monoclonal antibody against mouse beta dystroglycan (Novocastra, Newcastle on Tyre, UK), mouse antibody against mouse myelin-associated glycoprotein (MAG) (Chemicon International, Temecula, CA, USA), chicken polyclonal antibody against mouse neurofascin (R&D, Minneapolis, MN, USA), rabbit polyclonal antibody against mouse CD31 (Abcam, Cambridge, UK), and rat monoclonal antibody against mouse CD31 (Abcam, Cambridge, UK). Fluorophore-coupled secondary antibodies included Cy3-coupled anti-rabbit, anti-mouse and anti-rat IgG, 488-coupled anti-rabbit, anti-mouse and anti-rat IgG, Cy5-coupled anti-chicken IgG (Jackson Laboratories).

For mouse DRG culture we used rabbit polyclonal antibody against mouse CD59 (Cardiff University), rat antibody against MBP (Millipore, Burlington, MA, USA), and chicken polyclonal antibody against mouse neurofascin (R&D, Minneapolis, MN, USA). Fluorophore-coupled secondary antibodies included Cy3-coupled anti-rat IgG, Cy5-coupled anti-chicken IgG, and 488-coupled anti-rabbit IgG (Jackson Laboratories).

Fluorescence images were obtained using a confocal microscope (LSM700, Carl Zeiss, Oberkochen, Germany) fitted with an ORCA-ER CCD camera (Hamamatsu, Hamamatsu, Japan). Images were acquired and processed using the Zen2012 (Carl Zeiss) and PhotoShop software (Adobe, San Jose, CA, USA).

Immunohistochemistry labeling of complement regulators in human peripheral nerve. Paraffin-embedded sections from human sural nerve biopsies were stained with hematoxylin and eosin (H&E), Luxol fast blue-periodic acid Schiff (LFB-PAS), Bielschowsky, Gomori-trichrome (GTC), and Congo-red according to the standard procedures. Paraffin-embedded sections of human sural biopsies were stained using immunohistochemistry methods for mouse monoclonal antibody against human CD59 (BIO-RAD, Hercules, CA, USA) at 1:1000 and 1:400 dilutions, mouse monoclonal antibody against human CD55 antibody (Abcam, CB, UK) at 1:400 dilution, mouse monoclonal antibody against human CD46 (Abcam, CB, UK) at 1:1000 dilution, mouse monoclonal antibody against human CD35 (Abcam, CB, UK) at 1:100 dilution, rabbit polyclonal antibody against human MBP (Zymed, Waltham, MA, USA), mouse monoclonal antibody against human neurofilament (NF) (Dako, Glostrup South, Denmark), and rabbit polyclonal antibody against human S-100 (Dako, Glostrup South, Denmark). Universal secondary Horseradish peroxidase (HRP) antibody (UltraView universal HRP Multimer) was used for all staining (Ventana, Tucson, AZ, USA). All immunohistochemistry was performed using an automated stainer (Ventana Benchmark-Ultra, Roche Diagnostics, Indianapolis, IN, USA) according to standard procedures and protocols.

Staining peripheral nerve for autoantibodies was done using patients’ sera at a dilution of 1:100 or IVIg control at 1:10,000. βIV spectrin 1:600 and neurofascin 1:300 also used for this set of stains.

Images were obtained using a Nikon Ti Automated Inverted Microscope fitted with a Nikon DS-Fi1 color CCD camera. Images were acquired and processed using NIS-Elements image acquisition software (Nikon).

Myelinating dorsal root ganglia (DRG) cultures. DRG cultures were prepared from mouse embryos at day 13.5 of gestation. DRGs were dissociated and plated at a density of 4×10⁴ per 13 mm slide and coated with matrigel and poly-l-lysine. Cultures were grown for 2 days in neurobasal medium (Gibco) supplemented with B-27 (Gibco), glutamax (Gibco), penicillin /streptomycin, and 50 ng/ml NGF (Alomone Labs). Cultures were then grown for 4–5 additional days in BN medium containing Basal Medium Eagle (Sigma), ITS supplement (Sigma), glutamax (Gibco), 0.2% BSA, 4 mg/ml D-glucose, 50 ng/ml NGF, and antibiotics. To induce myelination, cultures were grown in BNC, namely a BN medium supplemented with 15% heat-inactivated fetal calf serum (Gibco, replacing the BSA) and 50 μg/ml L ascorbic acid. Cultures were fixed after 10 additional days.

Electron microscopy. Adult WT and CD59 knock-out (KO) mice were anesthetized using a lethal dose of ketamine/xylazine (1:10) injected intraperitoneally. Sciatic nerves were fixed for 30 min in situ with a freshly prepared fixative containing 4% paraformaldehyde, 2.5% glutaraldehyde, 0.13 M NaH4PO4, and 0.11 M NaOH pH 7.4. Nerves were subsequently dissected out and cleaned from surrounded tissue, and further fixed with the above fixative overnight at room temperature while shaking. Fixed sciatic nerves were analyzed using a transmission electron microscope (CM-12, Eagle 2K×2K; FEI, Philips, Amsterdam, The Netherlands). Semi-thin sections were stained with toluidine blue for further analysis.

CD59 expression in murine sciatic nerve and myelinated cultures. To evaluate CD59 expression in the murine peripheral nervous system, we labeled cross sections of sciatic nerves from both WT and CD59 KO mice with antibodies to CD59 together with antibodies to dystroglycan and P0 (myelin protein zero (P0), a type I transmembrane protein is the most abundant protein in PNS myelin and is localized in compact myelin). This immunolabeling revealed that CD59 is present in compact myelin and merges with P0 in WT nerves (Fig. 1 A-D). As expected, CD59 was not detected in KO sciatic nerves (Fig. 1 A-D, small squares), confirming the antibody’s specificity. Immunolabeling of anti-CD59 together with anti-MAG (present in noncompact myelin and here as cross section mark for the abaxonal and adaxonals of Schwann cell membrane) showed nonoverlapping signals, confirming that CD59 was indeed localized in compact myelin (Fig. 1 E-G). This was supported by immunolabeling of WT nerves using antibodies to CD59 and AnkG, which revealed that CD59 was localized along the internodal areas but was absent from the nodes of Ranvier (Fig. 2 A). Additional immunolabeling of WT nerves using antibodies to CD59, caspr, and neurofascin (Caspr is present in the paranodal loops domain and neurofascin is present in paranodal loops and Schmidt Lanterman Incisure (SLI) domains. These domains are in noncompact myelin areas), showed that CD59 was also present in the paranodal loops and SLI (Fig. 2 B-C).

To further establish the localization of CD59 in murine PNS compact myelin and its absence in the nodes of Ranvier, we immunolabeled myelinated Schwann cells/DRG neuron cultures using antibodies to MBP and P0 together with CD59. As shown, CD59 was absent from the nodes of Ranvier and colocalized with MBP (Fig. 2 D) and P0 (Additional file 1: Fig. S1) in WT cultures.

Expression of CD59 in human sural nerve. To test whether the observations made in mouse nerves apply to humans, we first tested the expression of CD59 in paraffin-embedded cross sections of healthy human sural nerve. As seen in the results obtained in mice, immunohistochemical staining revealed CD59 expression in the area of myelinated nerve fibers (Fig. 3 C, vertical black arrow heads). CD59 was also detected in the endothelial blood vessels of the epineurium and endoneurium (Fig. 3 A-C, vertical black arrows for epineurium, and horizontal black arrow heads for endoneurium), and in the perineurium (Fig. 3 A-B, horizontal black arrows). In human sural nerve longitudinal sections, CD59 was expressed in areas that correspond to exon fibers and compact myelin (Fig. 3 D).

CD59 is not required for murine PNS myelination, and its absence leads to abnormal myelination only at older age. To examine whether genetic deletion of CD59 affected myelin morphology, we compared cross sections of sciatic nerves obtained from WT and KO animals by Electron microscopy (EM) and toluene blue (TB) staining. We found no significant morphological differences between the WT and KO nerves (Fig. 4 A, E). Similar results were obtained from 1.5 and 6-month-old animals (Fig.4 B-C, F-G). EM analysis of 18-month-old animals revealed abnormalities in both genotypes (Fig. 4 D, H-L), including the appearance conspicuous axoplasmatic organelles that were often present at the paranodal region (Fig. 4 I-J). These axoplasmatic organelles were more frequently detected in KO compared to WT mice (Fig. 4 L). In addition, in the KO nerves we detected areas that contained mitochondria and dense bodies (Fig. 4 K) and with some neurofilaments that had lost their normal orientation and formed disorganized bundles (Additional file 2: Fig. S2). These results may indicate that CD59 KO nerves are more sensitive to nerve damage and degeneration, suggesting a protective role for CD59 in the PNS.

To further establish that genetic deletion of CD59 did not affect myelin morphology, we compared immunolabeling of sciatic nerve stains obtained from 4-month-old WT and KO animals using antibodies to CD59 and AnkG (Fig. 4 M), caspr (Fig.4 N), neurofascin (Fig.4 O), and MAG (Fig.4 P). There was no significant difference in the intensity or localization of these molecular markers between WT and KO nerves, indicating that the molecular organization of the myelin unit is preserved in the absence of CD59 (Fig. 4 M-P).

Expression of CD59 in peripheral nerve of CD59-deficient patients. To test whether the observations made in mouse nerves apply to humans, we also examined paraffin-embedded sections of sural nerve biopsied from a CD59-deficient patient between GBS episodes. In immunohistochemical staining for CD59, there was no detection of CD59 protein in patient cross-sections compared to healthy control subjects (Fig. 5 A-B). Paraffin-embedded sections from a CD59-deficient patient were stained with H&E (Fig. 5 C-D), LFB-PAS, Bielschowsky, Gomori TC, and Congo-red (data not shown). Epoxy resin-embedded semi-thin sections stained with toluidine-blue (Fig. 5 E-F), immunohistochemical stains for MBP (Fig. 5 G), NF (Fig. 5 H), and S-100 protein (data not shown) displayed normal nerve fiber density. Nerve fibers appeared normal, without evidence of active axonal or myelin damage. There was no apparent excess of endoneurial cells or collagen. Neither inflammatory infiltrate nor amyloid were identified. EM (Fig. 5 I) showed occasional thin myelin sheaths relative to axonal diameter with a relatively high g-ratio,^{22, 23} suggestive of remyelination. The myelin sheath lamellar structure appeared to be normal. Macrophages or other signs of active axonal or myelin damage were not identified. Axons and Schwann cells were unremarkable. These findings suggest that in CD59-deficient patients there is a process of demyelination followed by remyelination, leading to a relatively thinner myelin layer.

No autoantibodies to peripheral nerve are found in the sera of children with CD59 deficiency. We examined autoantibodies that classically express in GBS in the serum of four patients with CD59 deficiency. No anti-GD1b or anti-GM1 were found in four patients’ sera during 1–7 years following diagnosis (data not shown). We also exposed teased mouse peripheral nerve fibers to the four patients’ sera, collected up to 5 years after the diagnosis, with no detection of autoantibodies (data not shown).

Differentially expressed complement membrane regulatory proteins in peripheral nerve and endothelial blood vessels. Complement regulatory proteins include CD59, CD55, CD46, and CD35 (CR1) in humans, and CD59, CD55, CD46, and Crry in mice.^24-26

As shown in Figures 1–2, CD59 was absent from the nodes of Ranvier and co-localized with MBP and P0 in WT mice. To determine whether CD59 is expressed in endothelial blood vessels, we labeled longitudinal sections of murine sciatic nerve with antibodies to CD59, neurofascin, and CD31 as an endothelial blood vessel marker. Immune-labeling revealed CD59 expression in internodes corresponding to compact myelin but there was no CD59 expression in blood vessels (Additional file 3: Fig. S3A, B). In humans, however, and as discussed earlier, CD59 was also detected in endothelial blood vessels of the epineurium, endoneurium, and perineurium (Fig. 3 A-C).

Interestingly, CD55 was not expressed in compact myelin in mouse peripheral nerve teased fibers and was expressed in SLI but not in compact myelin (Fig. 6A and Additional file 4: Fig. S4A). This observation was confirmed in murine longitudinal sections (Additional file 4: Fig. S4B). No CD55 expression was seen in the endothelial blood vessels, and no colocalization with antibodies to CD31 was seen (Additional file 4: Fig. S4B). However, normal human immunohistochemical staining cross sections revealed CD55 expression similar to that of CD59 in compact myelin and in the endothelial blood vessels of the epineurium, endoneurium, and perineurium (Fig. 7 A-B). In longitudinal human sural nerve sections, CD55 was expressed in areas that correspond to exon fibers and compact myelin (Fig. 7 C).

Taken together, this suggests that CD55 is expressed in myelin and blood vessels in human peripheral nerves; however, in mice, CD59 expression is seen in compact myelin but CD55 is expressed in SLI of the noncompact myelin compartment.

As shown in Figure 6, CD46 was expressed in the paranodal and juxtaparanodal domains and not in the nodes or compact myelin in murine peripheral nerve teased fibers (Fig. 6 B). Immune-labeling to mouse CD46, neurofascin, and CD31 as an endothelial blood vessel marker in murine longitudinal sections, confirmed that CD46 was expressed in the paranodal-juxtaparanodal area, and was also weakly expressed in the endothelium of blood vessels (Additional file 5: Fig. S5A, B)).

In human sural nerve cross sections, CD46 was expressed in endothelial blood vessels of the epineurium and endoneurium, and to a lesser extent in the perineurium (Fig. 7 D-F). These results suggest that CD46 is expressed in blood vessels of the peripheral nerve in both humans and mice, and in the paranodal region of murine peripheral nerves.

CD35 (CR1) was not expressed in the human peripheral nervous system (Fig. 7 G).

An additional complement regulator, Crry, an inhibitor of complement activation, is expressed on rodent but not human cell membranes, and is a functional and structural analog of human CD55/DAF and CF46/MCP.²⁷ We therefore stained for Crry, and found that it is expressed in the paranodal areas and in the internodes, corresponding to compact myelin (Fig. 6 C and Additional file 6: Fig. S6A, B). Thus, as shown in Figures 1-3 and 6-7, complement membrane-regulatory proteins are differentially expressed in the PNS in mice and humans. In compact myelin, CD59 and CD55 are expressed in humans, whereas CD59 and Crry are expressed in mice. In endothelial blood vessels, CD59, CD55, and CD46 are expressed in humans, whereas only CD46 is expressed in mice. These observations may explain human clinical observations associated with CD55 and CD59 deficiencies. In patients with CD59 deficiency, CD59 is not present in the nodes of Ranvier (classical sporadic GBS) but as it is normally abundant in compact myelin (congenital recurrent GBS), its absence leads to GBS,^{2, 3} whereas CD55 deficiency does not.²⁸^, ²⁹ In mice, however, lack of CD59 does not lead to GBS following viral infection (Karbian and Mevorach, unpublished data) as Crry may prevent complement activation.

Healthy host tissues counteract complement-mediated damage by relying on efficient regulators that are either membrane-anchored or circulate as soluble proteins. Constant tick-over activation within the complement cascade marks a potential hazard to any surface; therefore, membrane-bound complement regulators on self-cells ensure that host surfaces are not affected, even after initial opsonization. Membrane-bound complement regulators are embedded in the cell membrane and their regulatory functions are restricted to the host surfaces they protect. Of the four members that are tethered to the plasma membrane, CD35 (CR1) and CD46 (MCP) are embedded via transmembrane domains, whereas CD55 (DAF) and CD59 (protectin) are fixed to the outside membrane leaflet via a GPI anchor.³⁰

CD35 is widely expressed on human cells, especially B cells and RBCs, and acts as a cofactor for the inactivation of both C3b and C4b via Factor I (FI).^{30, 31} As shown here, It is not expressed in the peripheral nervous system. CD46 is widely expressed on most cell surfaces, with the notable exception of erythrocytes, and protects cells by acting as a cofactor for the FI-mediated degradation of both C3b and C4b.³² Here we were able to show that CD46 is not expressed in compact myelin and therefore has no role in myelin protection from complement attack. It is expressed in the paranodal area, which is in line with its role in cell signaling,^{33, 34} metabolism, and development of the neuronal retina, Retinal pigment epithelium (RPE), and choroid in C57BL/6 mice, and has an important role in the development of retinal macular degeneration.^{34, 35}

Decay-accelerating factor (DAF, CD55) is expressed on almost all peripheral blood cells as well as endothelial and epithelial cells and has wide expression in multiple tissues. It prevents the formation of new C3 and C5 convertases and accelerates their decay.^{36, 37} Recently we have described patients that carry nonfunctional CD55, which led to protein-losing enteropathy.^{28, 38} Here we were able to show that CD59, which inhibits the final and most important step of MAC formation, is present in the myelin membrane of both human and murine PNS, together with CD55 in humans and Crry in mice. However, only CD59-deficient patients, and not CD55-deficient patients, are under frequent PNS attack leading to recurrent PNS demyelination episodes and conduction block,^{3, 4} indicating the crucial role of CD59 in protecting myelin from complement attack.

In this study we show that CD59 is found in compact myelin and in endothelial cells in human peripheral nerve cells. Furthermore, we clearly show the absence of CD59 expression in regions devoid of compact myelin such as the node of Ranvier, the site at which nerve impulses propagate.³⁹ Erne et al.⁴⁰ demonstrated that rafts found in adult human and murine peripheral compact myelin membranes contain myelin and lymphocyte protein (MAL), the GPI-anchored protein CD59, and substantial amounts of the PMP22 and P0. Colocalization studies show that CD59 and MAL have an almost identical expression pattern within compact myelin where detergent-insoluble glycolipid-enriched complexes are also found. Our findings agree with Erne et al.,⁴⁰ who localized CD59 in compact myelin, as shown by us in cross sections, teased fibers, and DRG cultures. In addition, we were able to clearly demonstrate here that neither CD59, CD55, or CD46 are expressed in the nodes of Ranvier, as verified in teased fibers and DRGs. In blood vessels, on the other hand, all three membrane-bound regulators, CD59, CD55, and CD46, were found to be expressed in the endothelium of the PNS.

The association of CD59 deficiency with demyelination was previously suggested in CD59 KO mice, where induction of acute experimental allergic encephalomyelitis resulted in increased demyelination compared to WT mice.⁴¹ In humans, GBS has been specifically linked to hyperactivity of the complement system by the observed upregulation of terminal complement complex C5b-9 in Schwann cells of the demyelinating fibers. Moreover, deposition of complement components and MAC formation are found in the acute motor neuropathy form of GBS.^{1, 42-44} In addition, in a murine model of antiganglioside antibody-mediated neuropathy, the passive transfer of anti-GM1 or antiGD1a antibodies produced a GBS-like syndrome only in the presence of human complement components,^{45, 46} and eculizumab (which inhibits terminal complement activation), was shown to prevent dysfunction and structural nerve damage.^3,⁴⁷ While there are no known autoantibodies in patients with primary CD59 deficiency, nonfunctional CD59 allows spontaneous uncontrolled complement amplification that results in severe demyelination and conduction block in the PNS following viral infection³ and possibly additional triggers.

Along with a reversible conduction block in the hands in these patients, electrophysiological studies of irreversible damage in the foot showed marked delay of distal latencies, low compound muscle action potential (CMAP) amplitudes, and slow conduction velocities (right peroneal and left tibial). The latency of F responses of the left tibial nerve were prolonged and sympathetic skin responses showed no response. Improvement of the conduction block in distal or intermediate nerve segments was detected following eculizumab treatment,³ suggesting a rapid mechanism of action in the area of the nodes of Ranvier. Thus, some patients with both autoimmune (post-infectious antibody-dependent) and autoinflammatory (CD59-mutated) GBS have reversible conduction failure attributed to blockade at the nodes of Ranvier without tissue destruction, at least in the early stages of disease progression.

In summary, we were able to show that the nodes of Ranvier have no complement-membrane regulatory proteinsCD59, CD46, CD35, or CD55 (or Crry in mice), rendering this area exposed to complement attack. This provides an explanation for observations of complement attack in the nodes of Ranvier in classical autoantibody-dependent GBS.¹ Furthermore, we showed that myelin in the PNS is protected only by CD59 and CD55, but not by CD46 or CD35, rendering it susceptible to complement attack, conduction block, and demyelination in autoinflammatory (non-autoantibody-dependent) situations like CD59 deficiency.³

AnkG: Ankyrin G; BSA: Bovine serum albumin; CMAP: Compound muscle action potential; CR1: Complement receptor 1; Crry: Complement receptor type-1 related gene Y (Crry/p65); DAB: 3, 3'-diaminobenzidine; DAF: Decay accelerating factor; DG: Dystroglycan; DRG: Dorsal root ganglia; EM: Electron microscopy; FI: Factor I; GBS: Guillain-Barré syndrome; GTC: Gomori-trichrome; GPI: Glycosyl phosphatidylinositol; H&E: Hematoxylin and eosin; HRP: Horseradish peroxidase; KO: knock-out; LFB-PAS: Luxol fast blue, Periodic Acid Schiff; MAC: Membrane attack complex; MAG: Myelin-associated glycoprotein; MAL: Myelin and lymphocyte protein; MBP: Myelin basic protein; MCP: Membrane cofactor protein; NF: Neurofilament; Nfasc: Pan Neurofascin; NGF: Nerve growth factor; PMP22: Peripheral myelin protein-22; RPE: Retinal pigment epithelium; SLI: Schmidt Lanterman incisures; WT: Wild-type; TB: Toluidine blue; TSA: Tyramide signal amplification.

Ethics approval and consent to participate

Consent for publication

Was received via ethical approval: HMO0390-12-; HMO0379-18-

Availability of data and materials

The datasets used during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Funding

This research was supported by the Legacy Heritage Biomedical Program of the Israel Science Foundation (grant No. 1192/18 to D.M.).

Authors' contributions

This work was part of PhD thesis of N.K. in the Faculty of Medicine, Hebrew University and Hadassah Medical Center. N.K. helped in study design made most of the actual work and helped writing the manuscript; Y.E.E. helped in study design, instructed practical work in animals, and helped writing the manuscript, M.Z., A.T., N.S., and A.V., participated in the actual work in animals, B.P.M. provided reagents and helped writing the manuscript, Y.F., instructed practical work in human tissue, and helped writing the manuscript; E.P. was a co-instructor in PhD thesis of N.K., helped in study design and helped writing the manuscript , D.M. was the instructor of PhD thesis of N.K., was responsible of study design and responsible of writing the manuscript.

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No competing interests reported.

Additionalfile1FigureS1.tif
Additional file 1: Figure S1. Expression of CD59 in murine myelinated cultures. CD59 (green), P0 (red), and neurofascin (blue). CD59 is co-localized with P0 and expressed in the internodes and paranodes. There is no expression in the nodes of Ranvier. Scale bars = 50 μm.
Additionalfile2FigureS2.tif
Additional file 2: Figure S2. Electron microscopy (EM) of murine sciatic nerve longitudinal sections. EM pictures of CD59-deficient murine sciatic nerves. Abnormality in neurofilament orientation is seen. Scale bar = 500 nm.
Additionalfile3FigureS3.tif
Additional file 3: Figure S3. Expression of complement membrane regulatory CD59 in murine longitudinal sections (A) and a cross-section (B) of sciatic nerve. Staining of WT murine sciatic nerve by CD59 (red), CD31 (green), and neurofascin (Nfacs, blue). Section from a 4.5-month-old mouse. Immunolabeling procedure performed with methanol and 0.1% Triton. Scale bars = 50 μm.
Additionalfile5FigureS5.tif
Additional file 5: Figure S5. Expression of complement membrane regulatory CD46 in murine longitudinal sections (A) and cross section (B) of sciatic nerve. Staining of WT murine sciatic nerve by CD55(red), CD31 (green), and neurofascin (Nfacs, blue). Section from a 4.5-month-old mouse. Immunolabeling procedure performed with methanol and 0.1% triton. Scale bars = 20 μm.
Additionalfile6FigureS6.tif
Additional file 6: Figure S6. Expression of complement membrane regulatory Crry in murine teased sections (A) and cross section (B) of sciatic nerve. A. Left panels - staining of WT teased fibers. Crry (red), neurofascin (Nfacs, blue), and CD59 (green). Right panels, control usingonly secondary antibody. B. Staining of WT cross section. Crry (red) and CD59 (green). Sections taken from a 4-month-old mouse. Crry staining was expressed in the paranodes and the internodes corresponding to compact myelin. Immunolabeling procedure performed with the TSA method. Scale bars = 20 μm.

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Membrane-bound complement regulator expression in peripheral nerves

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