Charakterisierung von Autoantikörpern gegen das neuronale Protein Synapsin hinsichtlich ihrer Epitopspezifität und Klassenzugehörigkeit

In den letzten Jahren sind neben den klassischen paraneoplastischen Syndromen mit neuronaler Präsentation vermehrt die Autoantikörperenzephalitiden in den Fokus der Forschung gerückt. Diese Krankheitsbilder sind nicht unbedingt mit einer neoplastischen Erkrankung vergesellschaftet und durch immunsuppressive Strategien therapierbar. Der Entdeckung der häufigsten Form von Autoantikörperenzephalitis, der anti-NMDA-Rezeptor-Enzephalitis, folgen bis heute zahlreiche Entdeckungen weiterer antineuronaler Antikörper. So nahm unsere Arbeitsgruppe nach dem Nachweis von IgA-Antikörpern gegen das neuronale Protein Synapsin bei einem Patienten mit dem klinischen Bild einer limbischen Enzephalitis ohne maligne Grunderkrankung die Untersuchung einer Kohorte mit breit gefächerten neurologisch-psychiatrischen Diagnosen auf das Vorliegen von anti-Synapsin-Antikörpern vor. Bei einem Teil dieser Patienten konnten anti-Synapsin-Antikörper, allerdings vom IgG-Typ, nachgewiesen werden. Die Antikörper von achtzehn Patienten werden in der vorliegenden Arbeit in Zell-basierten Assays hinsichtlich ihrer Epitopspezifität charakterisiert. Das Protein Synapsin besteht aus abgegrenzten Untereinheiten mit verschiedenen Strukturen und Funktionen. HEK-Zellen wurden mit diesen Untereinheiten transfiziert und die in Seren der Patienten enthaltenen Antikörper auf Reaktivität mit den einzelnen Untereinheiten überprüft. Dabei zeigte sich kein einheitliches Bild, es scheinen aber präferentiell zwei Domänen, die A-Domäne und die D-Domäne, als antigene Epitope zu wirken. Die Antikörper gehören nicht alle der gleichen IgG-Subklasse an, sind aber in der Mehrheit der Fälle vom vergleichsweise selten vorkommenden IgG4 Typ. Darüberhinaus wurden Liquores von Patienten mit beginnenden Demenzen unterschiedlicher Ätiologie auf das Vorliegen von anti-Synapsin-Antikörpern überprüft. In 4% der Fälle konnten im Western-Blot-Verfahren mit Synapsin-Knock-Out Kontrolle IgA-Antikörper gegen Synapsin nachgewiesen werden. Mangels einer ausreichenden Anzahl an Kontrollen kann über die statistische Signifikanz dieser Entdeckung gegenwärtig noch keine Aussage gemacht werden. Es bleibt mittels funktioneller Studien zu untersuchen, in wie weit die Antikörper der untersuchten Patienten eine pathogenetische Relevanz haben bzw. in wie weit es sich lediglich um ein Epiphänomen handelt.The recently discovered autoantibody encephalitides have moved more and more into the focus of scientific research. In contrast to the long known paraneoplastic syndromes with neurological presentation, the autoantibody encephalitides are not necessarily associated with a neoplastic condition and respond to treatment with immune suppressive strategies. After the discovery of the most common form of autoantibody encephalitis, the NMDA receptor encephalitis, many other types of autoantibodies against neuronal antigens were found and the search for new entities in this field continues to the day. A few years ago, in serum and CSF of a patient with the clinical picture of limbic encephalitis in the absence of neoplastic disease, IgA-anitbodies against the neuronal protein Synapsin were found. This led to the examination of a group of patients with a broad spectrum of neurological and/or psychiatric diseases for the presence of antibodies against this protein in the serum. A part of this group tested positive for said antibodies, this time of the IgG-type. The protein synapsin consists of different subunits, each of them having their own structure and function. In the paper at hand, the anti-Synapsinautoantibodies of eighteen patients are characterized concerning their epitope specifity. This is done by transfecting HEK-cells with the different subunits and testing the sera of the patients for reactivity against the fractions. The results showed that, though different domains were antigenic for different patients, a preference for the A-domain and the domain exists. Likewise, the antibodies from different patients don’t belong to the same IgG-subtype, but antibodies of the IgG4 type were clearly most frequently found. Additionally, CSF samples from patients diagnosed with early forms of different kinds of dementia were examined for the presence of anti-Synapsin-antibodies. In 4% of cases, antibodies could be detected by Western blotting with synapsin-knock-out controls. Due to a limited number of control CSFs and therefore lacking statistical significance, a valid conclusion cannot yet be drawn. Concerning the pathological relevance of the here presented findings, further research, particularly regarding the functional effects of the found antibodies, remains to be done

Reply to Witthöft et al. Comment on “Wardzinski et al. Mobile Phone Radiation Deflects Brain Energy Homeostasis and Prompts Human Food Ingestion. Nutrients 2022, 14, 339”

We are somewhat surprised about the extent of the feedback that we received upon our publication [1], in terms of the not entirely new connection between mobile phone radiation, brain activity, and food intake, being previously explored by EEG, association studies, and animal experiments (as outlined in the introduction of our paper). Ten years ago, scientists found “alarming” evidence of a long-term association between mobile phone radiation and obesity in humans [2]. Specifically, we are perplexed by the partly emotional character of the discussion among our readers. However, back to the facts: We thank our scientific colleagues for their detailed analyses and considerations [3] regarding our study and are pleased to explain the open points for more clarity

Deep RNA sequencing of muscle tissue reveals absence of viral signatures in dermatomyositis

Objective: To explore a possible connection between active viral infections and manifestation of Dermatomyositis (DM). Methods: Skeletal muscle biopsies were analyzed from patients diagnosed with juvenile (n=10) and adult (n=12) DM. Adult DM patients harbored autoantibodies against either TIF-1γ (n=7) or MDA5 (n=5). Additionally, we investigated skeletal muscle biopsies from non-diseased controls (NDC, n=5). We used an unbiased high-throughput sequencing (HTS) approach to detect viral sequences. To further increase sequencing depth, a host depletion approach was applied. Results: In this observational study, no relevant viral sequences were detected either by native sequencing or after host depletion. The absence of detectable viral sequences makes an active viral infection of the muscle tissue unlikely to be the cause of DM in our cohorts. Discussion: Type I interferons (IFN) play a major role in the pathogenesis of both juvenile and adult dermatomyositis (DM). The IFN response is remarkably conserved between DM subtypes classified by specific autoantibodies. Certain acute viral infections are accompanied by a prominent type I IFN response involving similar downstream mechanisms as in DM. Aiming to elucidate the pathogenesis of DM in skeletal muscle tissue, we used an untargeted high-throughput sequencing and a host depletion approach to detect possible causative viruses

Effect of Mycorrhizae on Artemisia Annua

This project explored the symbiotic relationship between the plant Artemisia annua, which produces the antimalarial drug artemisinin, and the arbuscular mycorrhizal root fungus Glomus intraradices. A high-producing cultivar, SAM from the Weathers lab, was grown in two soil types, one seeded with fungal spores, to determine whether an increase in biomass, flavonoids, and artemisinin could occur. Differences in metabolites between plants grown from rooted cuttings and plants derived from tissue culture suggest a possible signal or mechanism for further study

Mobile Phone Radiation Deflects Brain Energy Homeostasis and Prompts Human Food Ingestion

Obesity and mobile phone usage have simultaneously spread worldwide. Radio frequency-modulated electromagnetic fields (RF-EMFs) emitted by mobile phones are largely absorbed by the head of the user, influence cerebral glucose metabolism, and modulate neuronal excitability. Body weight adjustment, in turn, is one of the main brain functions as food intake behavior and appetite perception underlie hypothalamic regulation. Against this background, we questioned if mobile phone radiation and food intake may be related. In a single-blind, sham-controlled, randomized crossover comparison, 15 normal-weight young men (23.47 ± 0.68 years) were exposed to 25 min of RF-EMFs emitted by two different mobile phone types vs. sham radiation under fasting conditions. Spontaneous food intake was assessed by an ad libitum standard buffet test and cerebral energy homeostasis was monitored by 31phosphorus-magnetic resonance spectroscopy measurements. Exposure to both mobile phones strikingly increased overall caloric intake by 22–27% compared with the sham condition. Differential analyses of macronutrient ingestion revealed that higher calorie consumption was mainly due to enhanced carbohydrate intake. Measurements of the cerebral energy content, i.e., adenosine triphosphate and phosphocreatine ratios to inorganic phosphate, displayed an increase upon mobile phone radiation. Our results identify RF-EMFs as a potential contributing factor to overeating, which underlies the obesity epidemic. Beyond that, the observed RF-EMFs-induced alterations of the brain energy homeostasis may put our data into a broader context because a balanced brain energy homeostasis is of fundamental importance for all brain functions. Potential disturbances by electromagnetic fields may therefore exert some generalized neurobiological effects, which are not yet foreseeable

Epitope specificity of anti-synapsin autoantibodies: Differential targeting of synapsin I domains.

OBJECTIVE:To identify the specific domains of the presynaptic protein synapsin targeted by recently described autoantibodies to synapsin. METHODS:Sera of 20 and CSF of two patients with different psychiatric and neurological disorders previously tested positive for immunoglobulin (Ig)G antibodies to full-length synapsin were screened for IgG against synapsin I domains using HEK293 cells transfected with constructs encoding different domains of rat synapsin Ia. Additionally, IgG subclasses were determined using full-length synapsin Ia. Serum and CSF from one patient were also screened for IgA autoantibodies to synapsin I domains. Sera from nine and CSF from two healthy subjects were analyzed as controls. RESULTS:IgG in serum from 12 of 20 IgG synapsin full-length positive patients, but from none of the healthy controls, bound to synapsin domains. Of these 12 sera, six bound to the A domain, five to the D domain, and one to the B- (and possibly A-), D-, and E-domains of synapsin I. IgG antibodies to the D-domain were also detected in one of the CSF samples. Determination of IgG subclasses detected IgG1 in two sera and one CSF, IgG2 in none of the samples, IgG3 in two sera, and IgG4 in eight sera. One patient known to be positive for IgA antibodies to full-length synapsin had IgA antibodies to the D-domain in serum and CSF. CONCLUSIONS:Anti-synapsin autoantibodies preferentially bind to either the A- or the D-domain of synapsin I

Multi-level profiling unravels mitochondrial dysfunction in myotonic dystrophy type 2.

Acknowledgements: This study was supported by a research grant from Deutsche Gesellschaft für Muskelkranke e.V. (grant number KI 3/2).Funder: Charité - Universitätsmedizin Berlin (3093)Myotonic dystrophy type 2 (DM2) is an autosomal-dominant multisystemic disease with a core manifestation of proximal muscle weakness, muscle atrophy, myotonia, and myalgia. The disease-causing CCTG tetranucleotide expansion within the CNBP gene on chromosome 3 leads to an RNA-dominated spliceopathy, which is currently untreatable. Research exploring the pathophysiological mechanisms in myotonic dystrophy type 1 has resulted in new insights into disease mechanisms and identified mitochondrial dysfunction as a promising therapeutic target. It remains unclear whether similar mechanisms underlie DM2 and, if so, whether these might also serve as potential therapeutic targets. In this cross-sectional study, we studied DM2 skeletal muscle biopsy specimens on proteomic, molecular, and morphological, including ultrastructural levels in two separate patient cohorts consisting of 8 (explorative cohort) and 40 (confirmatory cohort) patients. Seven muscle biopsy specimens from four female and three male DM2 patients underwent proteomic analysis and respiratory chain enzymology. We performed bulk RNA sequencing, immunoblotting of respiratory chain complexes, mitochondrial DNA copy number determination, and long-range PCR (LR-PCR) to study mitochondrial DNA deletions on six biopsies. Proteomic and transcriptomic analyses revealed a downregulation of essential mitochondrial proteins and their respective RNA transcripts, namely of subunits of respiratory chain complexes I, III, and IV (e.g., mt-CO1, mt-ND1, mt-CYB, NDUFB6) and associated translation factors (TACO1). Light microscopy showed mitochondrial abnormalities (e.g., an age-inappropriate amount of COX-deficient fibers, subsarcolemmal accumulation) in most biopsy specimens. Electron microscopy revealed widespread ultrastructural mitochondrial abnormalities, including dysmorphic mitochondria with paracrystalline inclusions. Immunofluorescence studies with co-localization of autophagy (p62, LC-3) and mitochondrial marker proteins (TOM20, COX-IV), as well as immunohistochemistry for mitophagy marker BNIP3 indicated impaired mitophagic flux. Immunoblotting and LR-PCR did not reveal significant differences between patients and controls. In contrast, mtDNA copy number measurement showed a reduction of mtDNA copy numbers in the patient group compared to controls. This first multi-level study of DM2 unravels thus far undescribed functional and structural mitochondrial abnormalities. However, the molecular link between the tetranucleotide expansion and mitochondrial dysfunction needs to be further elucidated