Biology subjects

Schwabe, T.

Publications and source records attributed to Schwabe, T..

5 recordsLinked to original sources

bioRxiv2024

The Alzheimers Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

Genetic variations in MS4A4A and MS4A6A are linked to the regulation of cerebrospinal fluid soluble TREM2 (sTREM2) levels and are associated with Alzheimers disease (AD) risk and progression. Using CRISPR knockout and MS4A4A-degrading antibodies in primary human microglia, non-human primates (NHP), and a xenotransplantation model of amyloid pathology, we provide evidence that MS4A4A and MS4A6A are negative regulators of both the transmembrane and soluble TREM2 proteins. They also negatively regulate microglia proliferation, survival, metabolism, lysosomal function, energetics, phagocytosis, and disease-fighting states. Mechanistically, we find that MS4A4A exerts negative regulation by interacting with MS4A6A and protecting it from degradation. MS4A6A in turn forms a complex with and blocks the co-receptor DAP12, which is required for the stability, cell surface localization, and signaling of TREM2 and other receptors. Taken together, the data indicate that MS4A4A and MS4A6A are cooperating, post-transcriptional negative regulators of TREM2 and microglial function, and potential drug targets for AD.

neuroscience↗

bioRxiv2023

cAMP binding to closed pacemaker ion channels is cooperative

Most of receptor proteins in biomembranes are oligomers of either homologue or identical subunits, that govern a cooperative activation process. HCN ion channels, generating electric rhythmicity in specialized brain neurons and cardiomyocytes1, are tetramers activated by hyperpolarizing voltage and modulated by the intracellular binding of the second messenger cAMP2,3. The molecular processes underlying the cooperative action of the subunits4-6 by the binding are not fully understood, in particular if they are necessarily associated with channel activation7 or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides8. Here we show positive cooperativity in ligand binding to resting HCN2 channels in native cell membranes by analyzing the binding of fluorescence-labelled single cAMP derivatives to the channels. Kinetic modelling reveals, that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in resting HCN2 channels is already cooperative prior to channel activation. This shows that single-molecule binding studies can quantify cooperativity in ligand binding to receptors in native membranes based on equilibrium measurements.

biophysics↗

bioRxiv2021

The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation

The blood-brain barrier (BBB) of Drosophila is comprised of a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity, by precisely regulating the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKAs molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments and not their total length is crucial for generating a proper paracellular seal. Based on these findings, we propose a model that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical for maintain tissue size and brain homeostasis during organogenesis.

developmental biology↗

bioRxiv2020

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics simulations to show that residue K464 of the C-linker is essential for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that both cAMP and K464E induce a rotation of the intracellular domain relative to the channel pore, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. The adopted poses are in excellent agreement with structural results.

biophysics↗

bioRxiv2020

Pathways of inter- and intrasubunit allosteric signaling in the C-linker disk and cyclic nucleotide-binding domain of HCN2 channels

Opening of hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels is controlled by membrane hyperpolarization and binding of cyclic nucleotides to the tetrameric cyclic nucleotide-binding domain (CNBD), attached to the C-linker disk (CL). Confocal patch-clamp fluorometry revealed a pronounced cooperativity of ligand binding among protomers. However, by which pathways allosteric signal transmission occurs remained elusive. Here, we investigate how changes in the structural dynamics of the CL- CNBD of mouse HCN2 upon cAMP binding relate to inter- and intrasubunit signal transmission. Applying a rigidity theory-based approach, we identify two intersubunit and one intrasubunit pathways that differ in allosteric coupling strength between cAMP binding sites or towards the CL. These predictions agree with results from electrophysiological and patch-clamp fluorometry experiments. Our results map out distinct routes within the CL-CNBD that modulate different cAMP binding responses in HCN2 channels. They signify that functionally relevant submodules may exist within and across structurally discernable subunits in HCN channels.

biophysics↗