Shiqiang Gao

Transfer of autoantibodies targeting ionotropic N ‐methyl‐ d ‐aspartate (NMDA) receptors from patients with autoimmune encephalitis to mice induces characteristic disease signs. The long‐term effects of pathogenic antibodies include immunoglobulin G (IgG)‐induced cross‐linking and receptor internalization. We investigated the direct and immediate impact of two specific pathogenic patient‐derived monoclonal autoantibodies (IgG #003‐102 and #007‐168) on NMDA receptor (NMDAR) function. We performed cell‐attached recordings from cells transfected with the GluN1‐3a and GluN2A subunits of NMDARs. IgG #003‐102 binds to the R2 lobe of the amino‐terminal domain (ATD) of the glycine‐binding GluN1 subunit. It significantly reduced the number of simultaneous NMDAR openings compared to controls at both low and high concentrations of glutamate and glycine. Closer examination revealed that IgG #003‐102 rapidly reduced the number of open receptors and shortened receptor‐opening bursts. However, antigen‐binding fragments of IgG #003‐102 did not reduce receptor openings. By contrast, IgG #007‐168, which binds to the top of the R1 lobe of the GluN1‐ATD, potentiated the NMDAR response. In conclusion, patient‐derived IgG #003‐102 inhibits NMDARs within seconds, before receptor internalization, and the full IgG is required for this acute inhibitory effect. This suggests that monovalent IgG #003‐102 constructs could be a new treatment strategy to shield a pathogenic epitope on NMDARs. image Key points: N ‐methyl‐ d ‐aspartate (NMDA) receptors (NMDARs) are ionotropic glutamate receptors central to brain development and function. Anti‐NMDAR encephalitis is the most common cause of non‐viral autoimmune brain inflammation and is characterized by psychosis, dyskinesias, autonomic dysfunction and seizures. Symptoms are considered to result from autoantibodies that internalize surface NMDARs on neurons. Using patch clamp recordings, we tested the acute effects of two monoclonal anti‐GluN1 autoantibodies and the Fab fragment of one of them on NMDARs. The autoantibody (IgG #003‐102), which binds to the R2 lobe of the GluN1‐amino‐terminal domain (ATD), inhibits receptors within seconds. The entire IgG is required for this inhibition. By contrast, IgG #007‐168, which binds to the top of the R1 lobe of the GluN1‐ATD, potentiates receptor responses. By capitalizing on the null phenotype of a Fab from IgG #003‐102, we propose monovalent constructs as a potential new treatment strategy to prevent pathogenic antibodies from causing receptor dysfunction.

Aims: Large animal models for atrial fibrillation (AF) research are ethically challenging and costly. Suitable humanized in vitro models could circumvent these problems. Here, we aimed to combine tissue engineering, human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCM), and optogenetic pacing to trigger an in vitro fibrillation-like state, which can be analysed by simple video recording. Methods and results: Atrial engineered heart tissue (aEHT) was created from hiPSC-aCM and transduced with adeno-associated virus vectors to express the channelrhodopsins CheRiff2.0, Chronos, or PsCatCh2.0f, respectively. We developed novel optogenetic hardware, based on microcontroller programmable light-emitting diodes. The interplay between aEHTs and hardware was optimized, and different pacing patterns were evaluated. Electrical burst pacing was evaluated for arrhythmia induction. Tissue constructs were analysed with regard to action potential, gene and protein expression, and structure. We found our optimized CheRiff2.0 best suitable for optogenetic pacing of aEHT at up to 5 Hz. Atrial EHT could be faithfully tachypaced at 4 Hz for 2.5 weeks. After 5 weeks, electrical burst pacing (20 Hz, 0.5 s) successfully induced self-sustained episodes of a state of fast beating, lower force, and incomplete relaxation. The propensity of these burst-evoked episodes of fibrillation was 3.2-fold higher in long-term paced aEHT than in non-paced controls. Action potential shape and gene expression recapitulated typical features of AF in the tachypaced aEHTs. Conclusion: We successfully created a first-of-its-kind in vitro model of AF, recapitulating the self-sustainability of atrial arrhythmia (‘AF begets AF’). Fibrillation-like episodes can be verified by visual inspection, rendering analysis possible without an electrophysiological setup.

Opsins are known as proteins that bind retinal to form light-sensing rhodopsins, which mediate diverse forms of photoreception and are widely used for optogenetic control of cellular activity. Yet many opsins lack canonical retinal-binding features or, in their native biological contexts, are expressed where chromophore availability is limited, raising the question whether retinal-free opsins are silent. Here we show that retinal-free ion-transporting microbial opsins are not inert but adopt intrinsic, light-independent functional states alongside their canonical light-driven activity. Using developmental phenotyping in transgenic Drosophila melanogaster, electrophysiology in Xenopus oocytes and mammalian cells, together with structural and computational analyses, we demonstrate robust light-independent ion conductance in multiple channel-type opsins and an ion-pumping opsin. Structural modeling and molecular dynamics simulations reveal conformational rearrangements in transmembrane helices and internal water networks that are consistent with stabilization of apo-conductive states. Together, these findings establish apo-opsin activity as a defined functional state of microbial opsins, expand the current framework of rhodopsin biology, and highlight the need to consider chromophore-independent activity in both basic research and optogenetic applications.

Reactive oxygen species (ROS) have been implicated in multiple signaling processes in plants, but the underlying mechanisms and roles remain enigmatic. In this study, we developed a method of live imaging of apoplastic ROS at the root surface. Distinct signals, including auxin, extracellular adenosine triphosphate, and rapid alkalinization factor 1 peptide, induce cytosolic calcium transients and apoplastic ROS bursts. Genetic and optogenetic manipulations of Arabidopsis identified calcium transients as necessary and sufficient for ROS bursts through activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases RBOHC and RBOHF. Apoplastic ROS bursts are not required, but they do limit gravity-induced root bending. Root bending is sensed by the stretch-activated calcium channel MCA1, leading to NADPH oxidase activation. The resulting ROS production stiffens cell walls to facilitate soil penetration. Apoplastic ROS thus provides a means to balance tissue flexibility and stiffness to navigate soil.

Effective optogenetic inhibition of neuronal activity requires tools that reliably silence neurons across cell types and conditions. K ⁺ ‐selective channelrhodopsins (KCRs) have emerged as attractive alternatives to chloride‐conducting channels for optogenetic inhibition of cellular excitability, but many KCR variants exhibit an ion selectivity shift toward Na ⁺ under prolonged illumination. Through behavioral and electrophysiological analyses in Drosophila , and Caenorhabditis elegans , it is found that both the absolute K ⁺ to Na ⁺ permeability ratio and its stability over time determine the inhibition to activation transition, which limits their utility for silencing neural circuits. Among tested variants, the KCR1‐C29D mutant shows a relatively high and the most stable K ⁺ to Na ⁺ permeability ratio during illumination. While other KCR variants often evoke excitatory responses, KCR1‐C29D consistently provides robust in vivo inhibition across cell types, illumination conditions, and species. This work addresses a key limitation of KCR optogenetics and establishes KCR1‐C29D as a superior and reliable inhibitory tool. These findings highlight the stability of ion selectivity as a design criterion and provide guidance for the design of next generation optogenetic tools.

Calcium signaling is crucial across various cell types, but its spatiotemporal dynamics remain difficult to study due to limited methods. Optogenetics, with its high precision, can address this challenge. In this study, we introduced the channelrhodopsin variant ChR2 XXM2.0, which exhibits high light sensitivity and enhanced Ca²⁺ conductance in Xenopus oocytes, into bone marrow-derived megakaryocytes through viral transduction, aiming to clarify the poorly understood role of Ca²⁺ dynamics in these cells. ChR2 XXM2.0 expression was confirmed in megakaryocyte membranes, and its functionality validated through whole-cell patch-clamp and calcium imaging. Localized activation of ChR2 XXM2.0 at the cell periphery induced cell polarization, dependent on localized calcium influx, myosin IIA, and integrin αIIbβ3-fibrinogen interaction. Furthermore, we generated a transgenic mouse line with Pf4-Cre-dependent expression of ChR2 XXM2.0, enabling optogenetic manipulation of anucleate blood platelets via light-triggered calcium signaling. Illumination induced phosphatidylserine and P-selectin exposure in spread platelets. Our results highlight the importance of asymmetric subcellular calcium events in megakaryocyte polarity and demonstrate the feasibility of manipulating platelet function using optogenetics. Taken together, our study introduces the ChR2 XXM2.0 construct and its corresponding Cre-dependent transgenic mouse line as powerful tools for manipulating subcellular Ca²⁺ signaling, with potential applications for different cell types.