TY - JOUR A1 - Hecker, Katharina A1 - Grüner, Julia A1 - Hartmannsberger, Beate A1 - Appeltshauser, Luise A1 - Villmann, Carmen A1 - Sommer, Claudia A1 - Doppler, Kathrin T1 - Different binding and pathogenic effect of neurofascin and contactin–1 autoantibodies in autoimmune nodopathies JF - Frontiers in Immunology N2 - Introduction IgG4 autoantibodies against paranodal proteins are known to induce acute-onset and often severe sensorimotor autoimmune neuropathies. How autoantibodies reach their antigens at the paranode in spite of the myelin barrier is still unclear. Methods We performed in vitro incubation experiments with patient sera on unfixed and unpermeabilized nerve fibers and in vivo intraneural and intrathecal passive transfer of patient IgG to rats, to explore the access of IgG autoantibodies directed against neurofascin-155 and contactin-1 to the paranodes and their pathogenic effect. Results We found that in vitro incubation resulted in weak paranodal binding of anti-contactin-1 autoantibodies whereas anti-neurofascin-155 autoantibodies bound to the nodes more than to the paranodes. After short-term intraneural injection, no nodal or paranodal binding was detectable when using anti-neurofascin-155 antibodies. After repeated intrathecal injections, nodal more than paranodal binding could be detected in animals treated with anti-neurofascin-155, accompanied by sensorimotor neuropathy. In contrast, no paranodal binding was visible in rats intrathecally injected with anti-contactin-1 antibodies, and animals remained unaffected. Conclusion These data support the notion of different pathogenic mechanisms of anti-neurofascin-155 and anti-contactin-1 autoantibodies and different accessibility of paranodal and nodal structures. KW - autoimmune nodopathy KW - IgG4 KW - neurofascin KW - contactin KW - node of ranvier KW - inflammatory neuropathy KW - passive transfer Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320395 VL - 14 ER - TY - JOUR A1 - Breyer, Maximilian A1 - Grüner, Julia A1 - Klein, Alexandra A1 - Finke, Laura A1 - Klug, Katharina A1 - Sauer, Markus A1 - Üçeyler, Nurcan T1 - \(In\) \(vitro\) characterization of cells derived from a patient with the GLA variant c.376A>G (p.S126G) highlights a non-pathogenic role in Fabry disease JF - Molecular Genetics and Metabolism Reports N2 - Highlights • The GLA variant S126G is not associated with Fabry symptoms in the presented case • S126G has no effect on α-GAL A activity or Gb3 levels in this patient • S126G sensory neurons show no electrophysiological abnormalities Abstract Fabry disease (FD) is a life-limiting disorder characterized by intracellular globotriaosylceramide (Gb3) accumulations. The underlying α-galactosidase A (α-GAL A) deficiency is caused by variants in the gene GLA. Variants of unknown significance (VUS) are frequently found in GLA and challenge clinical management. Here, we investigated a 49-year old man with cryptogenic lacunar cerebral stroke and the chance finding of the VUS S126G, who was sent to our center for diagnosis and initiation of a costly and life-long FD-specific treatment. We combined clinical examination with in vitro investigations of dermal fibroblasts (HDF), induced pluripotent stem cells (iPSC), and iPSC-derived sensory neurons. We analyzed α-GAL A activity in iPSC, Gb3 accumulation in all three cell types, and action potential firing in sensory neurons. Neurological examination and small nerve fiber assessment was normal except for reduced distal skin innervation. S126G iPSC showed normal α-GAL A activity compared to controls and no Gb3 deposits were found in all three cell types. Baseline electrophysiological characteristics of S126G neurons showed no difference compared to healthy controls as investigated by patch-clamp recordings. We pioneer multi-level cellular characterization of the VUS S126G using three cell types derived from a patient and provide further evidence for the benign nature of S126G in GLA, which is of great importance in the management of such cases in clinical practice. KW - Fabry disease KW - variants of unknown significance KW - C.376A>G (p.S126G) KW - globotriaosylceramide KW - induced pluripotent stem cells KW - sensory neurons KW - disease model KW - α-Galactosidase A Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350295 SN - 22144269 VL - 38 ER - TY - THES A1 - Grüner, Julia T1 - Pathogenesis of anti-paranodal autoantibodies in peripheral neuropathies T1 - Pathogenese anti-paranodaler Autoantikörper bei peripheren Neuropathien N2 - Autoantibodies against proteins of the node of Ranvier have been identified in a subset of patients with chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). Main antigens targeted by autoantibodies are the paranodal proteins contactin 1 (CNTN1), neurofascin (NF) 155 or contactin associated protein (Caspr) as well as the nodal NF186. Several studies investigated the role of anti-paranodal autoantibodies in the pathophysiology of CIDP leading to the current knowledge that immunoglobulin G (IgG)4 deposition leads to detachment of myelin from the axon at the paranodes. However, many questions remain unsolved. Thus, autoantibodies against NF155 have been well studied and their pathogenicity has been proven in an animal model in vivo. However, in some patients, autoantibodies against all isoforms of NF are detectable. These anti-pan-NF autoantibodies occur more rarely and lead to a very severe clinical phenotype. As the pathogenesis of patient-derived autoantibodies against pan-NF has never been investigated in vivo before, we used an animal model to study the effect of acute exposure to anti-pan-NF IgG3 by intraneural injections to the rat sciatic nerve. In addition, we used anti-NF155 IgG4 from a seropositive patient. Behavioral testings as well as nerve conduction studies did not re- veal any deficits after injected neither for anti-NF155 nor for anti-pan-NF autoantibodies. This leads to the suspicion that the disease is more likely induced by a chronic process. A common symptom in patients with anti-CNTN1 associated neuropathy is sensory ataxia and therefore, an involvement of dorsal root ganglia (DRGs) is hypothesized. We show that sera from anti-CNTN1 positive patients specifically bind to DRG neurons in vitro and reduce surface expression of CNTN1. This is most probably due to internalization mediated by coexisting IgG3 although IgG4 is the predominant subclass of autoantibodies. As it is known that CNTN1 interacts with the β1 subunit of specific sodium channels we analyzed channel expression and sodium currents of DRG neurons after incubation with anti-CNTN1 positive patients’ sera. We identified reduced sodium currents after long-term treatment with patients’ material although surface channel expression remained stable. We therefore concluded that CNTN1 might influence channel properties indirectly through auxiliary β1 subunits. Moreover, we suggest an involvement of DRG neurons in the pathogenesis of anti-CNTN1 associated CIDP as medium-large size neurons are more affected than small neurons. However, the exact mechanism of how anti-CNTN1 autoantibodies influence sodium channels should be subject of further studies. Furthermore, preliminary results indicate that the epitope for anti-CNTN1 autoantibodies from seropositive patients might be associated with distinct clinical features. We could show that autoantibodies might be either directed against a conformational epitope as binding is prevented after deletion of the first immunoglobulin (Ig) domain of CNTN1 or against the fibronectin type III (FnIII) domains. Strikingly, both patients with FnIII do- main specificity had very high titers of anti-CNTN1 autoantibodies and a chronic disease progression, whereas patients binding to a conformational epitope or to the Ig domains are related to a relapsing-remitting or even monophasic disease course. However, these results need to be further confirmed before a clear statement can be made. In conclusion, the present study contributes to elucidate the pathogenesis of peripheral neuropathies associated with anti-paranodal autoantibodies. However, further studies are required including a higher number of patients as well as considering effects on structures like DRGs besides the node of Ranvier to fully understand the disease mechanisms. N2 - Autoantikörper gegen Proteine des Ranvierschen Schnürrings wurden bei einer Untergruppe von Patienten mit chronisch inflammatorischer demyelinisierender Polyradikuloneuropathie (CIDP) identifiziert. Antigene, gegen die sich die Autoantikörper hauptsächlich richten, sind die paranodalen Proteine Contactin 1 (CNTN1), Neurofascin (NF) 155 oder das Contactin-assoziierte Protein (Caspr) sowie das nodal exprimierte NF186. Mehrere Studien untersuchten die Rolle von anti-paranodalen Autoantikörpern in der Pathophysiologie der CIDP, was zu der aktuellen Erkenntnis führte, dass Immunglobulin G (IgG) 4-Ablagerungen die Ablösung des Myelins vom Axon an den Paranodien zur Folge haben. Viele Fragen bleiben jedoch ungelöst. So sind Autoantikörper gegen NF155 gut untersucht worden und ihre Pathogenität wurde in einem Tiermodell in vivo nachgewiesen. Bei einigen Patienten sind jedoch Autoantikörper gegen alle Isoformen von NF nachweisbar. Diese anti-pan-NF-Autoantikörper treten seltener auf und führen zu einem sehr schweren klinischen Phänotyp. Da die Pathogenese von Autoantikörpern gegen pan-NF bisher nicht in vivo untersucht wurde, haben wir in einem Tiermodell die Wirkung der akuten Exposition von anti-pan-NF IgG3 durch intraneurale Injektionen in den Ischiasnerv der Ratte untersucht. Zusätzlich verwendeten wir anti-NF155 IgG4 von einem seropositiven Patienten. Verhaltenstests sowie Nervenleitfähigkeitsuntersuchungen zeigten weder nach Injektion von anti-NF155 noch von anti-pan-NF-Autoantikörpern Defizite. Dies lässt den Verdacht aufkommen, dass die Erkrankung eher durch einen chronischen Prozess ausgelöst wird. Ein häufiges Symptom bei Patienten mit anti-CNTN1-assoziierter Neuropathie ist die sensorische Ataxie, weshalb eine Beteiligung der Spinalganglien vermutet wird. Wir zeigen, dass Seren von anti-CNTN1-positiven Patienten in vitro spezifisch an Spinalganglion- Neurone binden und die Oberflächenexpression von CNTN1 reduzieren. Dies ist höchstwahrscheinlich auf eine Internalisierung zurückzuführen, die durch koexistierendes IgG3 vermittelt wird, obwohl IgG4 die vorherrschende Subklasse der Autoantikörper ist. Da bekannt ist, dass CNTN1 mit der β1-Untereinheit von spezifischen Natriumkanälen interagiert, haben wir die Kanalexpression und die Natriumströme von Spinalganglion-Neuronen nach Inkubation mit anti-CNTN1-positiven Patientenseren analysiert. Wir stellten reduzierte Natriumströme nach Langzeitbehandlung mit Patientenmaterial fest, obwohl die Oberflächenkanalexpression stabil blieb. Daraus schlossen wir, dass CNTN1 die Kanaleigenschaften möglicherweise indirekt über die β1-Untereinheiten beeinflusst. Darüber hinaus legen wir eine Beteiligung von Spinalganglion-Neuronen an der Pathogenese der anti-CNTN1-assoziierten CIDP nahe, da mittelgroße bis große Neurone stärker betroffen sind als kleine Neurone. Der genaue Mechanismus, wie anti-CNTN1-Autoantikörper Natriumkanäle beeinflussen, sollte jedoch Gegenstand weiterer Studien sein. Darüber hinaus deuten vorläufige Ergebnisse darauf hin, dass das Epitop für anti-CNTN1- Autoantikörper von seropositiven Patienten mit unterschiedlichen klinischen Merkmalen assoziiert sein könnte. Wir konnten zeigen, dass die Autoantikörper entweder gegen ein konformationelles Epitop gerichtet sein könnten, da die Bindung nach Deletion der ersten Immunglobulin (Ig)-Domäne von CNTN1 verhindert wird, oder gegen die Fibronektin Typ III (FnIII)-Domänen. Auffallend ist, dass beide Patienten mit FnIII-Domänen-Spezifität sehr hohe anti-CNTN1-Autoantikörper Titer und einen chronischen Krankheitsverlauf aufwiesen, während Patienten, die an ein konformationelles Epitop beziehungsweise die Ig- Domänen binden, mit einem schubförmigen oder sogar monophasischen Krankheitsverlauf assoziiert sind. Diese Ergebnisse müssen jedoch noch weiter bestätigt werden, bevor eine klare Aussage getroffen werden kann. Zusammenfassend trägt die vorliegende Studie dazu bei, die Pathogenese von peripheren Neuropathien, die mit anti-paranodalen Autoantikörpern assoziiert sind, aufzuklären. Es sind jedoch weitere Studien erforderlich, die eine größere Anzahl von Patienten einschließen und auch Auswirkungen auf Strukturen wie beispielsweise Spinalganglien neben dem Ranvierschen Schnürring berücksichtigen, um die Krankheitsmechanismen vollständig zu verstehen. KW - Autoantikörper KW - Polyneuropathie KW - Ranvier-Schnürring KW - Pathophysiologie KW - Contactin-1 KW - Neurofascin KW - CIDP KW - Pathogenesis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-248655 ER -