Autoantibodies Against the Node of Ranvier in Seropositive Chronic inflammatory Demyelinating Polyneuropathy: Diagnostic, Pathogenic, and Therapeutic Relevance

Discovery of disease-associated autoantibodies has transformed the clinical management of a variety of neurological disorders. Detection of autoantibodies aids diagnosis and allows patient stratification resulting in treatment optimization. In the last years, a set of autoantibodies against proteins located at the node of Ranvier has been identified in patients with chronic inflammatory demyelinating polyneuropathy (CIDP). These antibodies target neurofascin, contactin1, or contactin-associated protein 1, and we propose to name CIDP patients with these antibodies collectively as seropositive. They have unique clinical characteristics that differ from seronegative CIDP. Moreover, there is compelling evidence that autoantibodies are relevant for the pathogenesis. In this article, we review the current knowledge on the characteristics of autoantibodies against the node of Ranvier proteins and their clinical relevance in CIDP. We start with a description of the structure of the node of Ranvier followed by a summary of assays used to identify seropositive patients;and then, we describe clinical features and characteristics linked to seropositivity. We review knowledge on the role of these autoantibodies for the pathogenesis with relevance for the emerging concept of nodopathy/paranodopathy and summarize the treatment implications

Neurofascin-155 IgM autoantibodies in patients with inflammatory neuropathies

Objectives Recently, IgG autoantibodies against different paranodal proteins have been detected and this has led to important advances in the management of inflammatory neuropathies. In contrast, not much is known on IgM autoantibodies against paranodal proteins. Methods In the present study, we screened a large cohort of patients (n=140) with inflammatory neuropathies for IgM autoantibodies against neurofascin-155, neurofascin-186 or contactin-1. Results IgM autoantibodies against neurofascin-155 were detected by ELISA in five patients, four with inflammatory demyelinating polyradiculoneuropathy (CIDP) and one with Guillain-Barre syndrome (GBS), and were confirmed by ELISA-based preabsorption experiments and Western blot. Titres ranged from 1:100 to 1:400. We did not detect IgM anti-neurofascin-186 or anti-contactin-1 antibodies in this cohort. All patients presented with distally accentuated tetraparesis and hypesthesia. Remarkably, tremor was present in three of the patients with CIDP and occurred in the patients with GBS after the acute phase of disease. Nerve conduction studies revealed prolonged distal motor latencies and F wave latencies. Nerve biopsies showed signs of secondary axonal damage in three of the patients, demyelinating features in one patient. Teased fibre preparations did not demonstrate paranodal damage. Conclusion In summary, IgM neurofascin-155 autoantibodies may be worth testing in patients with inflammatory neuropathies. Their pathogenic role needs to be determined in future experiments

Dermal Phospho-Alpha-Synuclein Deposition in Patients With Parkinson's Disease and Mutation of the Glucocerebrosidase Gene

Heterozygous mutations in the glucocerebrosidase gene (GBA1) represent the most common genetic risk factor for Parkinson's disease (PD) and are histopathologically associated with a widespread load of alpha-synuclein in the brain. Therefore, PD patients with GBA1 mutations are a cohort of high interest for clinical trials on disease-modifying therapies targeting alpha-synuclein. There is evidence that detection of phospho-alpha-synuclein (p-syn) in dermal nerve fibers might be a biomarker for the histopathological identification of PD patients even at premotor or very early stages of disease. It is so far unknown whether dermal p-syn deposition can also be found in PD patients with GBA1 mutations and may serve as a biomarker for PD in these patients. Skin biopsies of 10 PD patients with different GBA1 mutations (six N370S, three E326K, one L444P) were analyzed by double-immunofluorescence labeling with anti-p-syn and anti-protein gene product 9.5 (PGP9.5, axonal marker) to detect intraaxonal p-syn deposition. Four biopsy sites (distal, proximal leg, paravertebral Th10, and C7) per patient were studied. P-syn was found in six patients (three N370S, three E326K). P-syn deposition was mainly detected in autonomic nerve fibers, but also in somatosensory fibers and was not restricted to a certain GBA1 mutation. In summary, dermal p-syn in PD patients with GBA1 mutations seems to offer a similar distribution and frequency as observed in patients without a known mutation. Skin biopsy may be suitable to study p-syn deposition in these patients or even to identify premotor patients with GBA1 mutations

Morphological and physiological characterization of filamentous Lentzea aerocolonigenes: Comparison of biopellets by microscopy and flow cytometry

Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process conditions. Hence, the characterization of cell morphology is of major importance to improve the understanding of industrial processes with filamentous microorganisms. For this purpose, reliable and robust methods are necessary. In this study, pellet morphology and physiology of the rebeccamycin producing filamentous actinomycete Lentzea aerocolonigenes were investigated by microscopy and flow cytometry. Both methods were compared regarding their applicability. To achieve different morphologies, a cultivation with glass bead addition (Ø = 969 μm, 100 g L-1) was compared to an unsupplemented cultivation. This led to two different macro-morphologies. Furthermore, glass bead addition increased rebeccamycin titers after 10 days of cultivation (95 mg L-1 with glass beads, 38 mg L-1 without glass beads). Macro-morphology and viability were investigated through microscopy and flow cytometry. For viability assessment fluorescent staining was used additionally. Smaller, more regular pellets were found for glass bead addition. Pellet diameters resulting from microscopy followed by image analysis were 172 μm without and 106 μm with glass beads, diameters from flow cytometry were 170 and 100 μm, respectively. These results show excellent agreement of both methods, each considering several thousand pellets. Furthermore, the pellet viability obtained from both methods suggested an enhanced metabolic activity in glass bead treated pellets during the exponential production phase. However, total viability values differ for flow cytometry (0.32 without and 0.41 with glass beads) and confocal laser scanning microscopy of single stained pellet slices (life ratio in production phase of 0.10 without and 0.22 with glass beads), which is probably caused by the different numbers of investigated pellets. In confocal laser scanning microscopy only one pellet per sample could be investigated while flow cytometry considered at least 50 pellets per sample, resulting in an increased statistical reliability

Glycine receptor autoantibody binding to the extracellular domain is independent from receptor glycosylation

Glycine receptor (GlyR) autoantibodies are associated with stiff-person syndrome and the life-threatening progressive encephalomyelitis with rigidity and myoclonus in children and adults. Patient histories show variability in symptoms and responses to therapeutic treatments. A better understanding of the autoantibody pathology is required to develop improved therapeutic strategies. So far, the underlying molecular pathomechanisms include enhanced receptor internalization and direct receptor blocking altering GlyR function. A common epitope of autoantibodies against the GlyRα1 has been previously defined to residues 1 A- 33 G at the N-terminus of the mature GlyR extracellular domain. However, if other autoantibody binding sites exist or additional GlyR residues are involved in autoantibody binding is yet unknown. The present study investigates the importance of receptor glycosylation for binding of anti-GlyR autoantibodies. The glycine receptor α1 harbors only one glycosylation site at the amino acid residue asparagine 38 localized in close vicinity to the identified common autoantibody epitope. First, non-glycosylated GlyRs were characterized using protein biochemical approaches as well as electrophysiological recordings and molecular modeling. Molecular modeling of non - glycosylated GlyRα1 did not show major structural alterations. Moreover, non-glycosylation of the GlyRα1 N38Q did not prevent the receptor from surface expression. At the functional level, the non-glycosylated GlyR demonstrated reduced glycine potency, but patient GlyR autoantibodies still bound to the surface-expressed non-glycosylated receptor protein in living cells. Efficient adsorption of GlyR autoantibodies from patient samples was possible by binding to native glycosylated and non-glycosylated GlyRα1 expressed in living not fixed transfected HEK293 cells. Binding of patient-derived GlyR autoantibodies to the non-glycosylated GlyRα1 offered the possibility to use purified non-glycosylated GlyR extracellular domain constructs coated on ELISA plates and use them as a fast screening readout for the presence of GlyR autoantibodies in patient serum samples. Following successful adsorption of patient autoantibodies by GlyR ECDs, binding to primary motoneurons and transfected cells was absent. Our results indicate that the glycine receptor autoantibody binding is independent of the receptor’s glycosylation state. Purified non-glycosylated receptor domains harbouring the autoantibody epitope thus provide, an additional reliable experimental tool besides binding to native receptors in cell-based assays for detection of autoantibody presence in patient sera

Contactin-1 and Neurofascin-155/-186 Are Not Targets of Auto-Antibodies in Multifocal Motor Neuropathy

Multifocal motor neuropathy is an immune mediated disease presenting with multifocal muscle weakness and conduction block. IgM auto-antibodies against the ganglioside GM1 are detectable in about 50% of the patients. Auto-antibodies against the paranodal proteins contactin-1 and neurofascin-155 and the nodal protein neurofascin-186 have been detected in subgroups of patients with chronic inflammatory demyelinating polyneuropathy. Recently, auto-antibodies against neurofascin-186 and gliomedin were described in more than 60% of patients with multifocal motor neuropathy. In the current study, we aimed to validate this finding, using a combination of different assays for auto-antibody detection. In addition we intended to detect further auto-antibodies against paranodal proteins, specifically contactin-1 and neurofascin-155 in multifocal motor neuropathy patients’ sera. We analyzed sera of 33 patients with well-characterized multifocal motor neuropathy for IgM or IgG anti-contactin-1, anti-neurofascin-155 or -186 antibodies using enzyme-linked immunosorbent assay, binding assays with transfected human embryonic kidney 293 cells and murine teased fibers. We did not detect any IgM or IgG auto-antibodies against contactin-1, neurofascin-155 or -186 in any of our multifocal motor neuropathy patients. We conclude that auto-antibodies against contactin-1, neurofascin-155 and -186 do not play a relevant role in the pathogenesis in this cohort with multifocal motor neuropathy

Biomarkers of conversion to alpha-synucleinopathy in isolated rapid-eye-movement sleep behaviour disorder

Patients with isolated rapid-eye-movement sleep behaviour disorder (RBD) are commonly regarded as being in the early stages of a progressive neurodegenerative disease involving \u3b1-synuclein pathology, such as Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy. Abnormal \u3b1-synuclein deposition occurs early in the neurodegenerative process across the central and peripheral nervous systems and might precede the appearance of motor symptoms and cognitive decline by several decades. These findings provide the rationale to develop reliable biomarkers that can better predict conversion to clinically manifest \u3b1-synucleinopathies. In addition, biomarkers of disease progression will be essential to monitor treatment response once disease-modifying therapies become available, and biomarkers of disease subtype will be essential to enable prediction of which subtype of \u3b1-synucleinopathy patients with isolated RBD might develop

Denervation and reinnervation in human muscle biopsies with neurogenic atrophy

Denervation von Muskulatur führt zu neurogener Atrophie. Diese geht mit morphologischen Veränderungen, wie dem Auftreten von angulär-atrophen Fasern, einher und führt zur Expression von Denervationsmarkern, wie NCAM, MHCn, Myogenin und Tenascin. In humanen Muskelbiopsien liegt ein heterogenes Muster an Fasern vor. Im Gegensatz zur Denervation im Tierexperiment kann nicht direkt auf den Innervationzustand geschlossen werden. Stattdessen liegen in humanen Muskelbiopsien mit neurogener Atrophie denervierte, reinnervierte und normale Fasern nebeneinander vor. In der Literatur wurden zwar sowohl morphologische Veränderungen beschrieben, die auf Denervation einer Faser hinweisen, als auch einige Denervationsmarker, die nach Denervation aufreguliert werden. Doch wurden bisher weder mehrere Denervationsmarker im Zusammenhang noch der Zusammenhang zwischen der Expression von Denervationsmarkern und dem Vorliegen von morphologischen Veränderungen, wie Atrophie untersucht, so dass es momentan noch nicht möglich ist, genaue Aussagen über den Innervationszustand von Muskelfasern in humanen Muskelbiopsien zu treffen. Ziel dieser Arbeit war es daher, durch vergleichende Untersuchung mehrerer Denervationsmarker (NCAM, MHCn, Myogenin und Tenascin) und durch Vergleich der Expression dieser Marker in Fasern mit verschiedenem Ausmaß an Atrophie eine genauere Aussage über Denervation und Reinnervation in einer humanen Muskelbiopsie zu treffen, als dies anhand der bisherigen Erkenntnisse möglich war. Es wurden dabei 66 humane Muskelbiopsien mit neurogener Atrophie immunhistochemisch auf ihre Reaktivität mit Anti-NCAM, Anti-MHCn, Anti-Myogenin und Anti-Tenascin überprüft. Um speziell die Expression dieser Marker nach Denervation zu untersuchen, wurden Biopsien die für Vimentin, einem regenerationsspezifischen Marker, positiv waren, ausgeschlossen. Zur Untersuchung des Zusammenhangs zur Morphologie der Fasern wurden die Muskelfasern in die Kategorien „normal groß“, „angulär-atroph“ und „klein“ eingeteilt. Die Menge der positiven Fasern wurde getrennt für diese drei Kategorien ermittelt. Überraschend war das Vorliegen einer großen Menge an kleinen Fasern, was für humane Muskelbiopsien so bisher nie beschrieben worden war. Diese kleinen Muskelfasern sind signifikant häufiger MHCn-positiv als NCAM- oder Myogenin-positiv (p<0,0001). Entstanden sind diese Muskelfasern vermutlich im Rahmen von durch Denervation ausgelösten Myogenesevorgängen mittels Aktivierung und Fusion von Satellitenzellen. Myogenese spielt demnach in denervierter humaner Muskulatur eine weitaus größere Rolle als bisher angenommen. Angulär-atrophe Fasern waren insgesamt ähnlich häufig für NCAM und MHCn positiv (p=0,28). Erstaunlicherweise unterschied sich die Menge der NCAM- und MHCn-positiven Fasern in den einzelnen Biopsien jedoch häufig. Dies deutet darauf hin, dass NCAM und MHCn zeitlich nicht exakt dem gleichen Expressionsmuster folgen. Einige angulär-atrophe Fasern sind MHCn-positiv, jedoch NCAM-negativ. Dies liegt vermutlich daran, dass die Expresssion von NCAM, wie in der Literatur beschrieben, nach Reinnervation sehr rasch zurückgeht. Normal große Fasern waren häufiger NCAM-positiv als MHCn- oder Myogenin-positiv. Dies entspricht den Erwartungen, da NCAM auch im Tierexperiment früher als MHCn oder Myogenin exprimiert wird. Auffällig war jedoch das Vorliegen von erstaunlich vielen NCAM- und MHCn-negativen angulär-atrophen Fasern. Dies kann nur durch Reinnervation erklärt werden. Reinnervation liegt demnach in humanen Muskelbiopsien mit neurogener Atrophie überraschend häufig vor. Myogenin-positive Fasern waren erstaunlicherweise nur in wenigen Biopsien zu finden. Wenn Myogenin-positive Fasern vorlagen, dann handelte es sich vor allem um angulär-atrophe und kleine Fasern. Ebenfalls erstaunlich war das Expressionsmuster von Tenascin: Tenascin unterschied sich von den anderen Denervationsmarkern dahingehend, dass es eher Felder von positiven Fasern bildete, anstatt einzelne Fasern anzufärben und von allen Faserformen ähnlich häufig exprimiert wurde. Tenascin korreliert dabei eher mit dem Ausmaß an Atrophie insgesamt ohne speziell von denervierten Fasern exprimiert zu werden und kann daher nicht als spezifischer Denervationsmarker angesehen werden. NCAM und MHCn stellen demnach brauchbare Denervationsmarker dar und lassen bei kombinierter Anwendung gute Schlüsse auf das Vorliegen von Denervation und Reinnervation in humanen Muskelbiopsien zu. Myogenin und Tenascin sind als Denervationsmarker nicht empfehlenswert. Myogenin wird nur von einem geringen Teil der denervierten Fasern exprimiert, während Tenascin überhaupt nicht dem Muster der anderen Denervationsmarker folgt und eher ein Marker für pathologische Veränderungen im Allgemeinen als speziell für Denervation zu sein scheint. Die Bezeichnung als Denervationsmarker ist bei Tenascin daher in Frage zu stellen. Das Vorkommen einer großen Menge an kleinen Fasern zeigt, dass Myogenesevorgänge in humanen Muskelbiopsien mit neurogener Atrophie in überraschend großem Maße ablaufen und erweitert die ohnehin schon hohe Komplexität von neurogener Atrophie um einen weiteren wichtigen Aspekt.Denervation of muscle tissue leads to neurogenic atrophy characterized by certain morphologic changes like angular-atrophic myofibers and leads to the expression of markers of denervation like Neural Cell Adhesion Molecule (NCAM), Myosin Heavy Chain neonatal (MHCn), myogenin and tenascin. In contrast to experimentally denervated muscles in animals, human muscle biopsies with neurogenic atrophy consist of myofibers in different states of innervation. A mixture of denervated, reinnervated and healthy myofibers can be found in each biopsy. In literature morphologic changes in denervated muscle biopsies as well as markers that are upregulated after denervation have been described. At present, little is known about the co-expression of different markers of denervation or about the co-existence of markers of denervation and morphologic changes what makes it difficult to infer the state of innervation of myofibers in human muscle biopsies. The objective of the present study is to give better evidence about denervation and reinnervation in human muscle biopsies by comparison of different markers of denervation (NCAM, MHCn, myogenin and tenascin) and by comparing their expression with the degree of atrophy of the myofibers. Skeletal muscle biopsies of 66 patients diagnosed with neurogenic atrophy were immunolabeled with anti-NCAM, anti-MHCn, anti-myogenin and anti-tenascin. Immunolabeling with anti-vimentin was performed to exclude regeneration. To compare the expression of markers of denervation with the morphology of the fibers, all myofibers were divided into the categories “normal”, “angular-atrophic” and “small”. The amount of immune-positive fibers was detected for each category. Both anti-NCAM and anti-MHCn studies indicated small myofibers that have not been described in denervated human muscle so far. The percentage of NCAM-positive small myofibers was lower in every biopsy compared with the percentage of MHCn-positive fibers (p<0.0001). These small fibers are most likely the result of myogenesis by activation and fusion of satellite cells. The percentage of MHCn-positive angular atrophic fibers was not significantly different from that of NCAM-positive fibers (p=0.28), but often differed within one biopsy. This indicates a difference in the time of upregulation of the two markers. Some angular-atrophic fibers are MHCn-positive, but NCAM-negative. This can be explained by the rapid downregulation of NCAM following reinnervation. A large number of NCAM- and MHCn-negative angular atrophic fibers could be explained by reinnervation, suggesting a larger degree of reinnervation in human muscle biopsies with neurogenic atrophy than suspected so far. The percentage of MHCn-positive or myogenin-positive morphologically normal myofibers was lower in every biopsy compared with the percentage of NCAM-positive normal myofibers. This can be explained by the earlier expression of NCAM after denervation, known from animal experiments. Myogenin-positive fibers could only be found in a few biopsies, they mainly belonged to the categories “angular-atrophic” or “small”. Tenascin showed a completely different expression: In contrast to the other markers of denervation, it formed areas of positivity instead of being expressed by single denervated fibers and it was expressed by angular-atrophic, small and normal fibers in the same amount. Tenascin seemed to correlate with the degree of atrophy in general without being predominantly expressed by denervated fibers. As a conclusion, tenascin cannot be regarded as a specific marker of denervation. NCAM and MHCn can be regarded as good markers of denervation, allowing conclusions about the existence of denervation and reinnervation within human muscle biopsies, especially if used in combination. Myogenin and tenascin cannot be recommended as markers of denervation. Myogenin is only expressed by a small part of denervated fibers, tenascin shows a completely different pattern of expression than all other markers and seems to be a marker of pathologic changes in general more than of denervation in specifity. The occurance of a large amount of small fibers suggests that myogenesis can frequently be found in human denervated muscles and is likely to play a more important role than so far suspected