Successful autologous hematopoietic stem cell transplantation in a refractory anti-Caspr1 antibody nodopathy.

AIM: Autoimmune nodopathies have specific clinicopathologic features, antibodies directed against nodal proteins (neurofascin 186) or paranodal proteins (neurofascin 155, contactin 1, contactin-associated protein 1 (Caspr1)), and usually have a poor response to first-line therapies for chronic inflammatory demyelinating polyradiculoneuropathy. Anti-Caspr1 nodopathy treated with autologous hematopoietic stem cell transplantation (AHSCT) has not been previously reported. METHODS: We report the first case of an anti-Caspr1 antibody-positive nodopathy refractory to high-intensity immunosuppressive treatment, including rituximab, that responded dramatically to AHSCT. RESULTS: A 53-year-old woman presented with a rapidly progressive generalized ataxic, painful motor, and inflammatory neuropathy supported by neurophysiologic and MRI studies. Initial tests for antibodies to nodal/paranodal proteins were negative. She was treated with multiple courses of intravenous immunoglobulin and methylprednisolone, plasma exchange, rituximab, and cyclophosphamide without significant clinical benefit. Repeated testing for antibodies to nodal/paranodal proteins yielded a positive result for anti-Caspr1/IgG4 isotype antibodies. Given the poor response to multiple high intensity treatments and the relatively young age of the patient, we decided to perform AHSCT at 30 months post-onset. Immediately after AHSCT, she stopped all immunomodulatory or immunosuppressive therapy. The Overall Neuropathy Limitation Score improved from 8/12 to 4/12 at 6 months post-AHSCT. At 3 months post-AHSCT, IgG4 against Caspr1 was negative and no reactivity against paranodes could be detected. CONCLUSION: We report a particularly severe anti-Caspr1 antibody autoimmune nodopathy that responded dramatically to AHSCT. Although the rarity of the disease limits the possibility of larger studies, AHSCT may be a valuable therapy in treatment-refractory cases.

Super-resolution imaging pinpoints the periodic ultrastructure at the human node of Ranvier and its disruption in patients with polyneuropathy.

The node of Ranvier is the key element in saltatory conduction along myelinated axons, but its specific protein organization remains elusive in the human species. To shed light on nanoscale anatomy of the human node of Ranvier in health and disease, we assessed human nerve biopsies of patients with polyneuropathy by super-resolution fluorescence microscopy. We applied direct stochastic optical reconstruction microscopy (dSTORM) and supported our data by high-content confocal imaging combined with deep learning-based analysis. As a result, we revealed a âˆ¼ 190 nm periodic protein arrangement of cytoskeletal proteins and axoglial cell adhesion molecules in human peripheral nerves. In patients with polyneuropathy, periodic distances increased at the paranodal region of the node of Ranvier, both at the axonal cytoskeleton and at the axoglial junction. In-depth image analysis revealed a partial loss of proteins of the axoglial complex (Caspr-1, neurofascin-155) in combination with detachment from the cytoskeletal anchor protein ß2-spectrin. High content analysis showed that such paranodal disorganization occurred especially in acute and severe axonal neuropathy with ongoing Wallerian degeneration and related cytoskeletal damage. We provide nanoscale and protein-specific evidence for the prominent, but vulnerable role of the node of Ranvier for axonal integrity. Furthermore, we show that super-resolution imaging can identify, quantify and map elongated periodic protein distances and protein interaction in histopathological tissue samples. We thus introduce a promising tool for further translational applications of super resolution microscopy.

Caspr1 antibodies autoimmune paranodopathy with severe tetraparesis: Potential relevance of antibody titers in monitoring treatment response.

AIM: Nodopathies and paranodopathies are autoimmune neuropathies associated with antibodies to nodal-paranodal antigens (neurofascin 140/186 and 155, contactin-1, contactin-associated protein 1 [Caspr1]) characterized by peculiar clinical features, poor response to standard immunotherapies (e.g., intravenous immunoglobulins, IVIg). Improvement after anti-CD20 monoclonal antibody therapy has been reported. Data on Caspr1 antibodies pathogenicity are still preliminary, and longitudinal titers have been poorly described. METHODS: We report on a young woman who developed a disabling neuropathy with antibodies to the Caspr1/contactin-1 complex showing a dramatic improvement after rituximab therapy, mirrored by the decrease of antibody titers. RESULTS: A 26-year-old woman presented with ataxic-stepping gait, severe motor weakness at four limbs, and low frequency postural tremor. For neurophysiological evidence of demyelinating neuropathy, she was diagnosed with chronic inflammatory demyelinating polyradiculoneuropathy and treated with IVIg without benefit. MRI showed symmetrical hypertrophy and marked signal hyperintensity of brachial and lumbosacral plexi. Cerebrospinal fluid showed 710 mg/dL protein. Despite intravenous methylprednisolone, the patient progressively worsened, and became wheelchair-bound. Antibodies to nodal-paranodal antigens were searched for by ELISA and cell-based assay. Anticontactin/Caspr1 IgG4 antibodies resulted positive. The patient underwent rituximab therapy with slow progressive improvement that mirrored the antibodies titer, measured throughout the disease course. CONCLUSIONS: Our patient had a severe progressive course with early disability and axonal damage, and slow recovery starting only a few months after antibody-depleting therapy. The close correlation between titer, disability, and treatment, supports the pathogenicity of Caspr1 antibodies, and suggest that their longitudinal evaluation might provide a potential biomarker to evaluate treatment response.

The vestibular calyceal junction is dismantled following subchronic streptomycin in rats and sensory epithelium stress in humans.

Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3-8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure.

A CASPR1-ATP1B3 protein interaction modulates plasma membrane localization of Na+/K+-ATPase in brain microvascular endothelial cells.

Contactin-associated protein 1 (CASPR1 or CNTNAP1) was recently reported to be expressed in brain microvascular endothelial cells (BMECs), the major component of the blood-brain barrier. To investigate CASPR1's physiological role in BMECs, here we used CASPR1 as a bait in a yeast two-hybrid screen to identify CASPR1-interacting proteins and identified the ß3 subunit of Na+/K+-ATPase (ATP1B3) as a CASPR1-binding protein. Using recombinant and purified CASPR1, RNAi, GST-pulldown, immunofluorescence, immunoprecipitation, and Na+/K+-ATPase activity assays, we found that ATP1B3's core proteins, but not its glycosylated forms, interact with CASPR1, which was primarily located in the endoplasmic reticulum of BMECs. CASPR1 knockdown reduced ATP1B3 glycosylation and prevented its plasma membrane localization, phenotypes that were reversed by expression of full-length CASPR1. We also found that the CASPR1 knockdown reduces the plasma membrane distribution of the α1 subunit of Na+/K+-ATPase, which is the major component assembled with ATP1B3 in the complete Na+/K+-ATPase complex. The binding of CASPR1 with ATP1B3, but not the α1 subunit, indicated that CASPR1 binds with ATP1B3 to facilitate the assembly of Na+/K+-ATPase. Furthermore, the activity of Na+/K+-ATPase was reduced in CASPR1-silenced BMECs. Interestingly, shRNA-mediated CASPR1 silencing reduced glutamate efflux through the BMECs. These results demonstrate that CASPR1 binds with ATP1B3 and thereby contributes to the regulation of Na+/K+-ATPase maturation and trafficking to the plasma membrane in BMECs. We conclude that CASPR1-mediated regulation of Na+/K+-ATPase activity is important for glutamate transport across the blood-brain barrier.

ARTHUR ASBURY LECTURE: Chronic inflammatory demyelinating polyradiculoneuropathy: clinical aspects and new animal models of auto-immunity to nodal components.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune disorder of the peripheral nerves with clinical and immunological heterogeneity. Both cellular and humoral immune mechanisms against peripheral nerve antigens are considered to contribute to the pathogenesis of the disorder. Currently, the diagnosis of CIDP is based on clinical, laboratory and electrophysiological criteria. The field of CIDP recently underwent a major change with the identification of autoantibodies directed against paranodal (CNTN1, CASPR1 and NF155) and nodal (NF186/140) proteins. Over the last 5 years, correlations have been found between these autoantibodies and CIDP clinical subtypes including the likelihood of response to specific immunotherapies. Additionally, during this time a series of experimental studies have unraveled the underlying immunopathogenesis for CNTN1 and NF155 antibody associated CIDP. Although paranodal and nodal autoantibodies are only found in a small subset of patients with CIDP, the detection of these immune biomarkers should be incorporated in the evaluation of patients, considering the implications of their presence on prognosis, follow-up, and treatment decisions.

Actions and interactions of alcohol and transforming growth factor ß1 on prepubertal hypothalamic gonadotropin-releasing hormone.

BACKGROUND: Alcohol (ALC) diminishes gonadotropin-releasing hormone (GnRH) secretion and delays puberty. Glial transforming growth factor ß1 (TGFß1) plays a role in glial-neuronal communications facilitating prepubertal GnRH secretion. We assessed the effects of acute ALC administration on TGFß1-induced GnRH gene expression in the brain preoptic area (POA) and release of the peptide from the medial basal hypothalamus (MBH). Furthermore, we assessed actions and interactions of TGFß1 and ALC on an adhesion/signaling gene family involved in glial-neuronal communications. METHODS: Prepubertal female rats were administered ALC or water via gastric gavage at 7:30 am. At 9:00 am, saline or TGFß1 (100 ng/3 µl) was administered into the third ventricle. At 3:00 pm, the POA was removed and frozen for gene expression analysis and repeated for protein assessments. In another experiment, the MBH was removed from ALC-free rats. After equilibration, tissues were incubated in Locke's medium only or medium containing TGFß1 with or without 50 mM ALC for measurement of GnRH peptide released in vitro. RESULTS: TGFß1 induced GnRH gene expression in the POA, and this effect was blocked by ALC. We also described the presence and responsiveness of the TGFß1 receptor in the POA and showed that acute ALC exposure not only altered the TGFß1-induced increase in TGFß-R1 protein expression but also the activation of receptor-associated proteins, Smad2 and Smad3, key downstream components of the TGFß1 signaling pathway. Assessment of an adhesion/signaling family consisting of glial receptor protein tyrosine phosphatase beta and neuronal contactin-associated protein-1 (Caspr1) and contactin showed that the neuronal components were induced by TGFß1 and that ALC blocked these effects. Finally, TGFß1 was shown to induce release of the GnRH peptide in vitro, an action that was blocked by ALC. CONCLUSIONS: We have demonstrated glial-derived TGFß1 induces GnRH gene expression in the POA and stimulates release of the peptide from the MBH, actions necessary for driving the pubertal process. Importantly, ALC acted at both brain regions to block stimulatory effects of TGFß1. Furthermore, ALC altered neuronal components of an adhesion/signaling family previously shown to be expressed on GnRH neurons and implicated in glial-GnRH neuronal communications. These results further demonstrate detrimental effects of ALC at puberty.

Maturation of type I and type II rat vestibular hair cells in vivo and in vitro.

Vestibular sensory epithelia contain type I and type II sensory hair cells (HCI and HCII). Recent studies have revealed molecular markers for the identification of these cells, but the precise composition of each vestibular epithelium (saccule, utricle, lateral crista, anterior crista, posterior crista) and their postnatal maturation have not been described in detail. Moreover, in vitro methods to study this maturation are not well developed. We obtained total HCI and HCII counts in adult rats and studied the maturation of the epithelia from birth (P0) to postnatal day 28 (P28). Adult vestibular epithelia hair cells were found to comprise ∼65% HCI expressing osteopontin and PMCA2, ∼30% HCII expressing calretinin, and ∼4% HCII expressing SOX2 but neither osteopontin nor calretinin. At birth, immature HCs express both osteopontin and calretinin. P28 epithelia showed an almost adult-like composition but still contained 1.3% of immature HCs. In addition, we obtained free-floating 3D cultures of the epithelia at P1, which formed a fluid-filled cyst, and studied their survival and maturation in vitro up to day 28 (28 DIV). These cultures showed good HC resiliency and maturation. Using an enriched medium for the initial 4 days, a HCI/calretinin+-HCII ratio close to the in vivo ratio was obtained. These cultures are suitable to study HC maturation and mature HCs in pharmacological, toxicological and molecular research.

CNTNAP1-encephalopathy: Six novel patients surviving the neonatal period.

CNTNAP1 encodes CASPR1, involved in the paranodal junction. Thirty-three patients, with CNTNAP1 biallelic mutations have been described previously. Most of them had a very severe neurological impairment and passed away in the first months of life. We identified four patients, from two unrelated families, who survived over the neonatal period. Exome sequencing showed compound heterozygous or homozygous variants. Severe hypotonia was a constant feature. When compared to previous reports, the most important clinical differences observed in our patients were the absence of antenatal problems and, in two of them, the lack of respiratory distress. Less commonly reported characteristics such as epileptic seizures, dystonia, and impaired communication skills were also observed. MRIs revealed hypomyelination or abnormal white matter signal, cerebral or cerebellar atrophy. The present observations support a wider than initially reported clinical spectrum, including survival after the neonatal period and additional neurological features. They contribute to better delineate the phenotype-genotype correlations for CNTNAP1. In addition, we report one more family with two sibs who carry a missense variant of uncertain significance which we propose could be associated with a milder phenotype.

Case report: Immunoadsorption therapy for anti-caspr1 antibody-associated nodopathy.

Background and objectives: Several autoantibodies against proteins located at the node of Ranvier has been identified in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) in the last few years. Then a new concept, autoimmune nodo-paranodopathies was proposed. Cases of Caspr1 autoantibodies are the most rare. Here we describe an anti-Caspr1 nodopathy patient, summarized his clinical, physiological and pathological features. Case presentation: We present the case of a 56-year-old male patient with proprioceptive loss, ataxia, coarse tremor and distal limb weakness without any painess and cranial involvement. Electrophysiological studies showed prolonged distal motor latencies, conduction slowing and reduced amplitude distal compound muscle action potential (CMAP) amplitude. Antibodies against the nodes of Ranvier in serum samples revealed a positive finding for the anti-Caspr1 antibody (1:10).Myelinated fiber loss could be revealed in nerve biopsy. Longitudinal ultrathin sections of the nodal region was discovered in electron microscope, the paranodal/nodal architecture was destructed. It was lack of transverse bands and enlargement of the space between the axon and the paranodal loops was seen. The patient improved obviously after three times immunoadsorption(IA) therapy. Conclusion: Anti-Caspr1 nodopathy patient may present atypical symptoms without any neuropathic pain and cranial palsy. The destruction of paranodal/nodal architecture could be observed in nerve biopsy, which may be caused by the lost of axoglial complex formed by NF155, CNTN1 and Caspr1. Antibodies detection is important for the diagnosis, while IA therapy could be regarded as an option for the patients allergic to rituximab (RTX).

Caspr1 Facilitates sAPPα Production by Regulating α-Secretase ADAM9 in Brain Endothelial Cells.

The expression of contactin-associated protein 1 (Caspr1) in brain microvascular endothelial cells (BMECs), one of the major cellular components of the neurovascular unit (NVU), has been revealed recently. However, the physiological role of Caspr1 in BMECs remains unclear. We previously reported the nonamyloidogenic processing of amyloid protein precursor (APP) pathway in the human BMECs (HBMECs). In this study, we found Caspr1 depletion reduced the levels of soluble amyloid protein precursor α (sAPPα) in the supernatant of HBMECs, which could be rescued by expression of full-length Caspr1. Our further results showed that ADAM9, the α-secretase essential for processing of APP to generate sAPPα, was decreased in Caspr1-depleted HBMECs. The reduced sAPPα secretion in Caspr1-depleted HBMECs was recovered by expression of exogenous ADAM9. Then, we identified that Caspr1 specifically regulates the expression of ADAM9, but not ADAM10 and ADAM17, at transcriptional level by nuclear factor-κB (NF-κB) signaling pathway. Caspr1 knockout attenuated the activation of NF-κB and prevented the nuclear translocation of p65 in brain endothelial cells, which was reversed by expression of full-length Caspr1. The reduced sAPPα production and ADAM9 expression upon Caspr1 depletion were effectively recovered by NF-κB agonist. The results of luciferase assays indicated that the NF-κB binding sites are located at -859 bp to -571 bp of ADAM9 promoter. Taken together, our results demonstrated that Caspr1 facilitates sAPPα production by transcriptional regulation of α-secretase ADAM9 in brain endothelial cells.

Antibodies against the node of Ranvier: a real-life evaluation of incidence, clinical features and response to treatment based on a prospective analysis of 1500 sera.

INTRODUCTION: IgG4 antibodies against neurofascin (Nfasc155 and Nfasc140/186), contactin (CNTN1) and contactin-associated protein (Caspr1) are described in specific subtypes of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Our objective was to assess, in a real-life practice, the incidence, the clinical features and the response to treatment of these forms of CIDP. METHODS: 1500 sera of patients suspected of having CIDP from France, Belgium and Switzerland were prospectively tested using a flow cytometry technique. The characteristics of patients with antibodies against the node of Ranvier were compared to 100 seronegative CIDP from our department. RESULTS: IgG4 antibodies against Nfasc155, CNTN1, and Caspr1 were, respectively, detected in 15 (prevalence 1%), 10 (0.7%) and 2 (0.2%) sera. Antibodies specific of the Nfasc140/186 were not detected. All subjects with antibodies against the node of Ranvier fulfilled diagnostic criteria for CIDP. CIDP with anti-Nfasc155 were younger, had more sensory ataxia and postural tremor than seronegative CIDP. CIDP with anti-CNTN1 had more frequent subacute onset and facial paralysis, commoner renal involvement with membranous glomerulonephritis and greater disability, than seronegative CIDP. CIDP with anti-Caspr1 had more frequent respiratory failure and cranial nerve involvement but not more neuropathic pain than seronegative CIDP. Intravenous immunoglobulins were ineffective in most seropositive patients. Rituximab produced dramatic improvement in disability and decreased antibodies titres in 13 seropositive patients (8 with anti-Nfasc155 and 5 with anti-CNTN1 antibodies). CONCLUSIONS: Although rare, anti-paranodal antibodies are clinically valuable, because they are associated with specific phenotypes and therapeutic response.

Early auto-immune targeting of photoreceptor ribbon synapses in mouse models of multiple sclerosis.

Optic neuritis is one of the first manifestations of multiple sclerosis. Its pathogenesis is incompletely understood, but considered to be initiated by an auto-immune response directed against myelin sheaths of the optic nerve. Here, we demonstrate in two frequently used and well-validated mouse models of optic neuritis that ribbon synapses in the myelin-free retina are targeted by an auto-reactive immune system even before alterations in the optic nerve have developed. The auto-immune response is directed against two adhesion proteins (CASPR1/CNTN1) that are present both in the paranodal region of myelinated nerves as well as at retinal ribbon synapses. This occurs in parallel with altered synaptic vesicle cycling in retinal ribbon synapses and altered visual behavior before the onset of optic nerve demyelination. These findings indicate that early synaptic dysfunctions in the retina contribute to the pathology of optic neuritis in multiple sclerosis.

Escherichia coli hijack Caspr1 receptor to invade cerebral vascular and neuronal hosts.

Escherichia coli (E. coli) penetration of the blood-brain barrier (BBB) is the key step essential for the development of meningitis. In a recent paper (Nat Commun 9:2296), we identify Caspr1 as a host receptor for E. coli virulence factor IbeA to pave the way the penetration of bacteria through the BBB. Bacterial IbeA interacts with endothelial Caspr1 to trigger intracellular focal adhesion kinase activation, leading to E. coli internalization into the brain endothelial cells. Importantly, endothelial knockout of Caspr1 in mice significantly reduced E. coli crossing through the BBB. Based on the results that extracellular aa 203-355 of Caspr1 bind with IbeA, we tested the blocking effect of recombinant Caspr1(203-355) peptides in neonatal rat model of meningitis. The results showed that Caspr1(203-355) peptides effectively attenuated E. coli penetration into the brain during meningitis, indicating that Caspr1(203-355) peptides could be used to neutralize the virulent IbeA to prevent meningitis. We further found that E. coli can directly invade into hippocampal neurons causing apoptosis which required the interaction between bacterial IbeA and neuronal Caspr1. These findings demonstrate that E. coli hijack Caspr1 as a host receptor for penetration of BBB and invasion of hippocampal neurons, resulting in progression of meningitis.

Absence of Axoglial Paranodal Junctions in a Child With CNTNAP1 Mutations, Hypomyelination, and Arthrogryposis.

Leukodystrophies and genetic leukoencephalopathies are a heterogeneous group of heritable disorders that affect the glial-axonal unit. As more patients with unsolved leukodystrophies and genetic leukoencephalopathies undergo next generation sequencing, causative mutations in genes leading to central hypomyelination are being identified. Two such individuals presented with arthrogryposis multiplex congenita, congenital hypomyelinating neuropathy, and central hypomyelination with early respiratory failure. Whole exome sequencing identified biallelic mutations in the CNTNAP1 gene: homozygous c.1163G>C (p.Arg388Pro) and compound heterozygous c.967T>C (p.Cys323Arg) and c.319C>T (p.Arg107*). Sural nerve and quadriceps muscle biopsies demonstrated progressive, severe onion bulb and axonal pathology. By ultrastructural evaluation, septate axoglial paranodal junctions were absent from nodes of Ranvier. Serial brain magnetic resonance images revealed hypomyelination, progressive atrophy, and reduced diffusion in the globus pallidus in both patients. These 2 families illustrate severe progressive peripheral demyelinating neuropathy due to the absence of septate paranodal junctions and central hypomyelination with neurodegeneration in CNTNAP1-associated arthrogryposis multiplex congenita.

Transient alteration of the vestibular calyceal junction and synapse in response to chronic ototoxic insult in rats.

Ototoxicity is known to cause permanent loss of vestibule function through degeneration of sensory hair cells (HCs). However, functional recovery has been reported during washout after chronic ototoxicity, although the mechanisms underlying this reversible dysfunction are unknown. Here, we study this question in rats chronically exposed to the ototoxic compound 3,3'-iminodipropionitrile (IDPN). Pronounced alterations in vestibular function appeared before significant loss of HCs or stereociliary coalescence became evident by ultrastructural analyses. This early dysfunction was fully reversible if the exposure was terminated promptly. In cristae and utricles, the distinct junctions formed between type I HCs (HCI) and calyx endings were completely dismantled at these early stages of reversible dysfunction, and completely rebuilt during washout. Immunohistochemical observations revealed loss and recovery of the junction proteins CASPR1 and tenascin-C and RT-PCR indicated that their loss was not due to decreased gene expression. KCNQ4 was mislocalized during intoxication and recovered control-like localization after washout. At early stages of the intoxication, the calyces could be classified as showing intact or lost junctions, indicating that calyceal junction dismantlement is triggered on a calyx-by-calyx basis. Chronic toxicity also altered the presence of ribeye, PSD-95 and GluA2 puncta in the calyces. These synaptic alterations varied between the two types of calyx endings (formed by calyx-only or dimorphic afferents) and some persisted at the end of the washout period. The present data reveal new forms of plasticity of the calyx endings in adult mammals, including a robust capacity for rebuilding the calyceal junction. These findings contribute to a better understanding of the phenomena involved in progressive vestibular dysfunction and its potential recovery during and after ototoxic exposure.