Combining super-resolution microcopy with neuronal network recording using magnesium fluoride thin films as cover layer for multi-electrode array technology

We present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates. These show great advantages for use in super-resolution microscopy as well as for multi-electrode-array fabrication and are especially suited for combination of these techniques. The transparency of the coated substrates with the low refractive index material is adjustable by the layer thickness and can be increased above 92%. Due to the hydrophobic and lipophilic properties of the thin crystalline MgF2 layers, the temporal stable adhesion needed for fixation of thin tissue, e.g. cryogenic brain slices is given. This has been tested using localization-based super-resolution microscopy with currently highest spatial resolution in light microscopy. We demonstrated that direct stochastic optical reconstruction microscopy revealed in reliable imaging of structures of central synapses by use of double immunostaining of post- (homer1 and GluA2) and presynaptic (bassoon) marker structure in a 10 µm brain slice without additional fixing of the slices. Due to the proven additional electrical insulating effect of MgF2 layers, surfaces of multi-electrode-arrays were coated with this material and tested by voltage-current-measurements. MgF2 coated multi-electrode-arrays can be used as a functionalized microscope cover slip for combination with live-cell super-resolution microscopy

Human NMDAR autoantibodies disrupt excitatory-inhibitory balance, leading to hippocampal network hypersynchrony

Anti-NMDA receptor autoantibodies (NMDAR-Abs) in patients with NMDAR encephalitis cause severe dis-ease symptoms resembling psychosis and cause cognitive dysfunction. After passive transfer of patients' cerebrospinal fluid or human monoclonal anti-GluN1-autoantibodies in mice, we find a disrupted excit-atory-inhibitory balance resulting from CA1 neuronal hypoexcitability, reduced AMPA receptor (AMPAR) signaling, and faster synaptic inhibition in acute hippocampal slices. Functional alterations are also reflected in widespread remodeling of the hippocampal proteome, including changes in glutamatergic and GABAergic neurotransmission. NMDAR-Abs amplify network g oscillations and disrupt q -g coupling. A data-informed network model reveals that lower AMPAR strength and faster GABAA receptor current kinetics chiefly ac-count for these abnormal oscillations. As predicted in silico and evidenced ex vivo, positive allosteric mod-ulation of AMPARs alleviates aberrant g activity, reinforcing the causative effects of the excitatory-inhibitory imbalance. Collectively, NMDAR-Ab-induced aberrant synaptic, cellular, and network dynamics provide con-ceptual insights into NMDAR-Ab-mediated pathomechanisms and reveal promising therapeutic targets that merit future in vivo validation

Human Autoantibodies against the AMPA Receptor Subunit GluA2 Induce Receptor Reorganization and Memory Dysfunction

AMPA receptors are essential for fast excitatorytransmission in the CNS. Autoantibodies to AMPAreceptors have been identified in humans withautoimmune encephalitis and severe defects ofhippocampal function. Here, combining electrophysiologyand high-resolution imaging withneuronal culture preparations and passive-transfermodels in wild-type and GluA1-knockout mice,we analyze how specific human autoantibodiesagainst the AMPA receptor subunit GluA2 affect receptorfunction and composition, synaptic transmission,and plasticity. Anti-GluA2 antibodiesinduce receptor internalization and a reduction ofsynaptic GluA2-containing AMPARs followed bycompensatory ryanodine receptor-dependent incorporationof synaptic non-GluA2 AMPARs. Furthermore,application of human pathogenic anti-GluA2antibodies to mice impairs long-term synaptic plasticityin vitro and affects learning and memoryin vivo. Our results identify a specific immuneneuronalrearrangement of AMPA receptor subunits,providing a framework to explain diseasesymptoms

Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation.

Double membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses

Where Do Firms Issue Debt? An Empirical Analysis of Issuer Location and Regulatory Competition in Europe

In this article, we study the choice of issuer location and regulatory competition in the European corporate debt market. We find that, in absolute terms, Germany has by far the highest outflow of debt issues, while the Netherlands, the UK, Luxembourg and Ireland see the most inflows (in that order). We use a panel gravity model to investigate country specific factors attracting foreign subsidiaries as issuer. The data clearly support the prediction that the locational choice is positively influenced by a low withholding tax rate. There is also some evidence that corporate tax rates play a role. We do not find support for creditor protection rules in bankruptcy as a driver of cross-border debt securities issues. Hence, countries who wish to attract issuers are well-advised to reduce their withholding tax rates – creditor rights seem not to matter