The Higher Sensitivity of GABAergic Compared to Glutamatergic Neurons to Growth-Promoting C3bot Treatment Is Mediated by Vimentin

The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of β1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects

Maternal synapsin autoantibodies are associated with neurodevelopmental delay

Maternal autoantibodies can be transmitted diaplacentally, with potentially deleterious effects on neurodevelopment. Synapsin 1 (SYN1) is a neuronal protein that is important for synaptic communication and neuronal plasticity. While monoallelic loss of function (LoF) variants in the SYN1 gene result in X-linked intellectual disability (ID), learning disabilities, epilepsy, behavioral problems, and macrocephaly, the effect of SYN1 autoantibodies on neurodevelopment remains unclear. We recruited a clinical cohort of 208 mothers and their children with neurologic abnormalities and analyzed the role of maternal SYN1 autoantibodies. We identified seropositivity in 9.6% of mothers, and seropositivity was associated with an increased risk for ID and behavioral problems. Furthermore, children more frequently had epilepsy, macrocephaly, and developmental delay, in line with the SYN1 LoF phenotype. Whether SYN1 autoantibodies have a direct pathogenic effect on neurodevelopment or serve as biomarkers requires functional experiments

High prevalence of NMDA receptor IgA/IgM antibodies in different dementia types

OBJECTIVE: To retrospectively determine the frequency of N-Methyl-D-Aspartate (NMDA) receptor (NMDAR) autoantibodies in patients with different forms of dementia. METHODS: Clinical characterization of 660 patients with dementia, neurodegenerative disease without dementia, other neurological disorders and age-matched healthy controls combined with retrospective analysis of serum or cerebrospinal fluid (CSF) for the presence of NMDAR antibodies. Antibody binding to receptor mutants and the effect of immunotherapy were determined in a subgroup of patients. RESULTS: Serum NMDAR antibodies of IgM, IgA, or IgG subtypes were detected in 16.1% of 286 dementia patients (9.5% IgM, 4.9% IgA, and 1.7% IgG) and in 2.8% of 217 cognitively healthy controls (1.9% IgM and 0.9% IgA). Antibodies were rarely found in CSF. The highest prevalence of serum antibodies was detected in patients with “unclassified dementia” followed by progressive supranuclear palsy, corticobasal syndrome, Parkinson’s disease-related dementia, and primary progressive aphasia. Among the unclassified dementia group, 60% of 20 patients had NMDAR antibodies, accompanied by higher frequency of CSF abnormalities, and subacute or fluctuating disease progression. Immunotherapy in selected prospective cases resulted in clinical stabilization, loss of antibodies, and improvement of functional imaging parameters. Epitope mapping showed varied determinants in patients with NMDAR IgA-associated cognitive decline. INTERPRETATION: Serum IgA/IgM NMDAR antibodies occur in a significant number of patients with dementia. Whether these antibodies result from or contribute to the neurodegenerative disorder remains unknown, but our findings reveal a subgroup of patients with high antibody levels who can potentially benefit from immunotherapy

Effects on Structure and Function of Neuronal and Glial Cells

In den vorgestellten Studien wurde der Einfluss von C3 Proteinen auf die Morphologie und Funktion von Neuronen und glialen Zellen des ZNS untersucht. Im Zentrum der Untersuchungen standen dabei enzymvermittelte sowie enzymunabhängige Effekte. Es konnte zum ersten Mal gezeigt werden, dass Clostridium botulinum C3 Protein (C3bot) neben der bekannten ADP- Ribosyltransferase Aktivität eine zusätzliche neurotrophe Aktivität aufweist, die nicht enzymatisch vermittelt ist. An murinen hippocampalen Primärkulturen konnte durch enzymkompetentes C3 Protein die Länge und die Anzahl von axonalen und dendritischen Verzweigungen gesteigert werden. Durch die Herstellung verschiedener überlappender Peptidfragmente aus der Gesamtsequenz von C3bot wurde ein Bereich innerhalb der Aminosäuren 154-182 identifiziert, der vergleichbare neurotrophe Effekte aufwies, die jedoch nicht auf einer enzymatischen Inaktivierung von Rho beruhten. Weiterhin konnte gezeigt werden, dass die erhöhte strukturelle Komplexität hippocampaler Neurone auch von einer vermehrt ausgebildeten Anzahl synaptischer Kontakte begleitet wurde. An organotypischen Kultursystemen konnten die an dissoziierten Zellen beobachteten Effekte bestätigt und ergänzt werden. An Hirnschnitten des entorhinalen Cortex der Maus wurde sowohl durch C3bot als auch durch enzymdefizientes C3bot154-182 das Wiederauswachsen von Axonen gesteigert. An einem hippocampal-entorhinalen Läsionsmodell konnte zudem eine verbesserte Reinnervierung von hippocampalen Zielgebieten durch Axone des entorhinalen Tractus perforans dargestellt werden. An Astrozytenkulturen konnten ausschließlich durch enzymkompetentes C3bot bzw. durch Hemmung von Rho nachgeschalteten Signalwegen fördernde Effekte auf die zelluläre Fortsatzbildung und die Migrationsgeschwindigkeit beim Wundverschluss als Modell für gliale Narbenbildung festgestellt werden. Weiterhin wiesen mit C3bot behandelte Astrozyten eine erhöhte Glutamataufnahme auf, die auf einer Rho/NFkB-abhängig induzierten Expression des glialen Glutamat-Transporters GLT-1 beruhte. Gleichzeitig wurde die Fähigkeit von Astrozyten gesteigert, vermehrt vesikulär gespeichertes Glutamat exozytotisch freizusetzen. An kultivierter Mikroglia konnte durch C3bot, ebenfalls ausschließlich enzymgebunden, eine proinflammatorische Antwort hervorgerufen werden. Eine deutliche morphologische Aktivierung wurde begleitet von der vermehrten Freisetzung von charakteristischen Mediatoren wie NO, TNF-alpha und weiteren proinflammatorischen Zytokinen und Chemokinen. Insgesamt liefern die Ergebnisse einen wesentlichen Beitrag zum Verständnis der zentralen Bedeutung von Rho GTPasen für morphogenetische und funktionelle Mechanismen in Neuronen und glialen Zellen. Weiterhin konnte ein Peptid aus der Sequenz von C3bot identifiziert werden, das ein hohes Potential zur Förderung neuronaler Regeneration aufweisen dürfte.The present studies investigated the influence of C3 proteins on morphological and functional parameters of neurons and glial cells of the CNS. Both enzyme- dependent as well as enzyme-independent effects were looked at. We could show for the first time that Clostridium botulinum C3 protein (C3bot) in addition to its well known ADP-ribosyltransferase activity harbors a neurotrophic activity not executed by its enzymatical mode of action. Enzyme-competent C3bot increased axonal and dendritic outgrowth of cultured hippocampal neurons. By generating overlapping peptidic fragments from C3bot we were able to identify a region spanning the amino acids 154-182 (C3bot154-182) that exhibited comparable neurotrophic effects to full length C3bot. However, these effects did not rely on enzymatic inactivation of Rho proteins. Furthermore, we demonstrated that the observed improved morphological complexity was accompanied by an increased number of synaptic contacts. Using organotypical brain slice cultures we could show that incubation with C3bot154-182 resulted in a promoted axonal outgrowth and reinnervation of hippocampal target regions by entorhinal cortex fibers after lesion. Using murine astrocyte cultures we showed that increased process outgrowth and in vitro scar formation strictly relied on enzymatical inactivation of Rho proteins by C3bot or inhibition of Rho-downstream targets. Moreover, astrocytes incubated with C3bot exhibited a higher glutamate uptake capacity than control cells, mediated by an Rho/NFkB- dependent upregulation of the glutamate transporter GLT-1. At the same time exocytotic release of glutamate was promoted in astrocytes treated with C3bot. Using primary cultures of murine microglia we were able to show that incubation with C3bot resulted in microglia activation characterized by morphological alterations and an increased release of pro-inflammatory mediators such as nitric oxide, TNF-a and other cyto- and chemokines. In conclusion, these data represent an essential contribution towards a better understanding of the central role of Rho proteins in the regulation of morphological and functional processes. In addition to that we were able to identify a peptidic fragment derived from C3bot that exhibits a high potential to foster neuronal regeneration

Regulation der Aktivität der vesikulären Monoamintransporter VMAT1 und VMAT2 in neuroendokrinen Zellen und Neuronen

In der vorliegenden Arbeit wurde die Regulation der Aktivität der vesikulären Monoamintransporter VMAT1 und VMAT2 durch heterotrimere G-Proteine untersucht. In der humanen neuroendokrinen Zellinie BON werden VMAT1 und VMAT2 exprimiert. Sie colokalisieren in diesen Zellen mit der a-Untereinheit des heterotrimeren G-Proteins Go2 vorwiegend auf großen elektronendichten Vesikeln, den LDCVs. Die Aktivität beider Transporter unterliegt einer Regulation durch Gao2. Nach Aktivierung des G-Proteins kommt es zu einer Hemmung der vesikulären Monoaminaufnahme. Die Aktivität von VMAT2 wird dabei empfindlicher reguliert als die Aktivität von VMAT1. In Primärkulturen von Rapheneuronen der Ratte wird VMAT2 als neuronale Variante des Transporters exprimiert. VMAT2 lokalisiert in diesen Neuronen überwiegend auf kleinen synaptischen Vesikeln, den SSVs. Hier kommt es zu einer Colokalisation mit Gao2 auf diesem Vesikeltyp. Auch in Rapheneuronen wird die Aktivität von VMAT2 durch diese G-Protein Untereinheit gehemmt. Elektronenmikroskopische Befunde belegen die Lokalisation von VMAT2 und Gao2 auf SSVs von serotonergen Axonterminalen im präfrontalen Cortex der Ratte. An einer Präparation synaptischer Vesikel aus diesem Gehirnbereich konnte ebenfalls eine Hemmung der Transportaktivität von VMAT2 durch Gao2 nachgewiesen werden. Auch in Thrombozyten der Maus unterliegt die vesikuläre Serotoninaufnahme einer Hemmung durch ein heterotrimeres G-Protein. In chronisch entleerten Vesikeln aus Mäusen, in denen das Gen für die periphere Tryptophanhydroxylase deletionsmutiert vorlag, konnte zunächst keine Hemmung der Serotoninaufnahme durch heterotrimere G-Proteine beobachtet werden. Nach Vorbeladung der Vesikel mit Serotonin war dies jedoch der Fall. Die Aktivierung des G-Proteins wird somit sehr wahrscheinlich über den Füllungszustand der Vesikel gesteuert.In this study we investigated the regulation of the activity of the vesicular monoamine transporters VMAT1 and VMAT2 by heterotrimeric G-proteins. In the human neuroendocrine cell line BON both transporters are expressed. They colocalize in these cells with the a-subunit of the heterotrimeric G-protein Go2 predominantely on Large Dense Core Vesicles (LDCVs). The activity of both VMAT1 and VMAT2 is regulated by Gao2. G-protein activation results in a down-regulation of vesicular monoamine uptake. VMAT2 appears to be more sensitive towards the observed G-protein regulation than VMAT1. Serotonergic raphe neurons in primary culture express VMAT2 as the neuronal form of the transporter. In these neurons VMAT2 predominantely localizes to Small Synaptic Vesicles (SSVs). Here, VMAT2 colocalizes with Gao2 on SSVs. In these neurons Gao2-dependent down-regulation of VMAT2 activity was observed, too. Immunoelectron microscopic analysis confirmed a localization of VMAT2 and Gao2 on SSVs from serotonergic terminals in the rat prefrontal cortex. In addition, Gao2-dependent regulation of VMAT2 activity could also be demonstrated when using a crude synaptic vesicle preparation of this brain area. Even in platelets obtained from mice the vesicular serotonin uptake is down-regulated by heterotrimeric G-proteins. In serotonin-depleted platelets from peripheral tryptophane-hydroxylase knockout mice no G-protein-dependent down-regulation of monoamine uptake was observed. After preincubation of the platelets with serotonin, the G-protein regulation was restored. Therefore, the vesicular transmitter content appears to be a likely factor of G-protein activation in platelets

Transfer of an interprofessional emergency caesarean section training program: using questionnaire combined with outcome data of newborn

Puprose!#!An emergency caesarean section is a potentially life-threatening situation both for the mother and the newborn. Non-technical skills can be improved by simulation training and are necessary to manage this urgent situation successfully. The objective of this study was to investigate, if training of emergency caesarean section can be transferred into daily work to improve the outcome parameters pH an APGAR of the newborn.!##!Methods!#!In this pre-post study, 141 professionals took part in a training for emergency caesarean section. Participants received a questionnaire, based on the tools 'Training Evaluation Inventory' and 'Transfer Climate Questionnaire' 1 year after training. Outcome data of the newborn were collected from the hospitals information system.!##!Results!#!Except the scale 'extinction', Cronbach's alpha was higher than 0.62. All scales were rated lower than 2.02 on a 5-point Likert Scale (1 = fullest approval; 5 = complete rejection). 'Negative reinforcement' was rated with 2.87 (SD 0.73). There were no significant differences in outcome data prior. The questionnaire fulfils criteria for application except the scale 'extinction'.!##!Conclusion!#!The presented training course was perceived as useful by the professionals and attitudes toward training were positive; the content was positively reinforced in practice 1 year after training. Parameters of the newborn did not change. It is conceivable that other outcome parameters (e.g. posttraumatic stress disorder) are addressed by the training. The development of relevant outcome parameters for the quality of emergency sections needs further investigation

Subtle Phenotype Differences in Psychiatric Patients With and Without Serum Immunoglobulin G Antibodies to Synapsin

The discovery that antibodies targeting neuronal antigens can induce severe psychiatric symptoms has been a significant progress in the understanding of psychiatric disorders. Antibodies targeting synapsin I in serum and cerebrospinal fluid (CSF) were first reported in 2015 in a patient with limbic encephalitis. Because of its regulatory function for neurotransmitter release, synapsin I has been suggested to play a role in psychiatric disorders. It is, however, unknown whether or not synapsin antibodies are of clinical significance in patients with psychiatric disorders. In the present study, we aimed to investigate if synapsin I immunoglobulin (Ig)G serum antibody positive patients admitted to acute psychiatric care have a different psychiatric phenotype than synapsin IgG antibody negative patients. A total of 13 anti-synapsin positive patients were matched for age, sex, and psychiatric diagnosis with 39 anti-synapsin negative patients from the same cohort. The groups were compared regarding 11 clinical features frequently seen in anti-neuronal antibody associated disorders. Anti-synapsin positive patients had higher agitation scores as measured with the Positive and Negative Syndrome Scale Excited Component [median (interquartile range) 11 (8) versus 7 (7), p = 0.04] compared to controls. However, the absolute scores were low in both groups, and the difference may not be clinically significant. Other clinical features assessed (e.g. hallucinations, delusions) did not differ between groups. We conclude that synapsin serum IgG antibodies lack syndrome specificity in patients admitted to acute psychiatric inpatient care. However, further studies addressing functional effects of synapsin antibodies are needed to conclude whether or not they have a pathophysiological relevance

Immunoreactivity to astrocytes in different forms of dementia: High prevalence of autoantibodies to GFAP

Objective: To study the prevalence of autoantibodies to glial and neuronal antigens with a focus on glial acidic fibrillary protein (GFAP) in patients with dementia. Methods: Sera of 127 patients with different forms of dementia and sera of 82 age-matched patients with various neurological diseases except for dementia, as well as sera from 15 age-matched healthy controls were analyzed for anti-glial or anti-neuronal IgG using 1) primary murine embryonic hippocampus cell cultures, 2) murine brain sections, 3) immunoblotting on mouse brain homogenates and 4) astrocyte cultures. Sera reacting with astrocytes in hippocampus cell cultures were further analyzed using HEK293 cells transfected with human GFAP. Results: IgG in serum from 45 of 127 (35.5%) patients with dementia but only 8 of 97 (8.2%, p ≤ 0.001) controls bound to either glial or neuronal structures in cultured murine hippocampus cells. In these cultures antibodies to astrocytes were detected in 35 of 127 (27.5%) of the dementia patients, whereas in controls antibodies to astrocytes were detected in 4 sera only (4.1%, p ≤ 0.001). Among the sera exhibiting reactivity to astrocytes, 14 of 35 (40%) showed immunoreaction to HEK293 cells transfected with GFAP in dementia patients, representing 11% of all sera. Within the 4 immunoreactive control sera reacting with astrocytes one reacted with GFAP (1.0% of total immunoreactivity, p = 0.003). Conclusions: Autoantibodies to glial epitopes in general and to GFAP in particular are more frequent in patients with dementia than in age-matched controls without dementia, thus indicating the need for further investigations regarding the potential pathophysiological relevance of these antibodies

The neuronal monoamine transporter VMAT2 is regulated by the trimeric GTPase Go(2

Monoamines such as noradrenaline and serotonin are stored in secretory vesicles and released by exocytosis. Two related monoamine transporters, VMAT1 and VMAT2, mediate vesicular transmitter uptake. Previously we have reported that in the rat pheochromocytoma cell line PC 12 VMAT1, localized to peptide-containing secretory granules, is controlled by the heterotrimeric G-protein Go 2. We now show that in BON cells, a human serotonergic neuroendocrine cell line derived from a pancreatic tumor expressing both transporters on large, densecore vesicles, VMAT2 is even more sensitive to G-protein regulation than VMAT1. The activity of both transporters is only downregulated by G�o 2, whereas comparable concentrations of G�o 1 are without effect. In serotonergic raphe neurons in primary culture VMAT2 is also downregulated by pertussis toxin-sensitive Go 2. By electron microscopic analysis from prefronta