Christian G. Specht

Extracellular vesicles (EVs) play a critical role in the propagation of Tau pathology in Alzheimer’s disease (AD). The selective identification and characterization of Tau seed-carrying EVs would help developing new diagnostic tools and therapeutic strategies. While pathogenic forms of Tau can be detected biochemically in EV-enriched fractions, only super-resolution fluorescence imaging provides both morphological and compositional information at the level of single EVs on account of their small size and the low number of target molecules. This was the object of our study. Post-mortem human brain-derived EVs from AD patients and non-demented control individuals were characterized in depth using biochemistry, transmission electron microscopy (TEM) and single molecule localization microscopy (SMLM). Dual-colour SMLM of immobilized EVs, displaying size distributions that were consistent with TEM, enabled statistically meaningful measurements of thousands of EVs in parallel. Owing to the high spatial resolution and capability of SMLM to detect sparse epitopes, we identified pathological Tau proteins in individual EVs from AD samples. Reconstructed super-resolution images of permeabilized EVs demonstrate that pathological Tau forms are frequent cargo proteins of EVs from AD brains. Our data illustrate the power of SMLM to determine the ultrastructure, molecular composition and topology of single EVs and EV populations.

Gephyrin, the principal scaffolding protein of inhibitory postsynaptic densities, clusters glycine and GABA _A receptors via multivalent interactions. It features structured N and C terminal domains connected by an intrinsically disordered linker. Although the structural and functional properties of its terminal domains are well characterized, the mechanism by which full-length gephyrin organizes into higher-order complexes remains unresolved. Here, we combine biochemical reconstitution, cryo-electron microscopy, and mutational analyses to elucidate the structural logic of gephyrin oligomerization. We demonstrate that gephyrin adopts a stable dimeric assembly which constitutes the basic unit for both linear and oblique tetramers as well as linear hexameric arrangements. High resolution structures reveal a critical segment of the flexible linker that adopts two distinct conformations, one of which occludes the receptor-binding site. This segment harbors key phosphorylation sites, suggesting a regulatory control mechanism. Our findings redefine the architecture of inhibitory postsynaptic sites and reconcile gephyrin oligomerization models with published in-situ postsynaptic densities characterized by cryo-electron tomography.

Glycine receptors (GlyRs) are heteropentameric chloride channels that mediate fast inhibitory neurotransmission in the brainstem and spinal cord, where they regulate motor and sensory processes. GlyRs are clustered in the postsynaptic membrane by strong interactions of the β subunit with the scaffold protein gephyrin. Even though Glrb mRNA is highly expressed throughout the brain, the existence of synaptic GlyRs remains controversial as there is little conclusive evidence using conventional fluorescence microscopy and electrophysiological recordings. Here, we exploit the high sensitivity and spatial resolution of single molecule localisation microscopy (SMLM) to investigate the presence of GlyRs at inhibitory synapses in the brain, focusing on several areas of the telencephalon. Making use of a knock-in mouse model expressing endogenous mEos4b-tagged GlyRβ, we identified few GlyRs in sub-regions of the hippocampus. Dual-colour SMLM revealed that these sparse receptors are integrated within the postsynaptic gephyrin domain, pointing to a possible role in maintaining the structural integrity of inhibitory synapses. In contrast, we found functionally relevant numbers of synaptic GlyRs at inhibitory synapses in the ventral striatum. Our results highlight the strength of SMLM to detect few and sparsely distributed synaptic molecules in complex samples and to analyse their organisation with high spatial precision.

ZUSAMMENFASSUNG: Die direkte Visualisierung postsynaptischer Gerüstproteine in lebenden Neuronen ist eine entscheidende Voraussetzung für die Untersuchung synaptischer Dynamik und Plastizität. Bisherige Methoden zur Live‐Synapsenbildgebung sind jedoch stark eingeschränkt: Sie erfordern genetische Manipulationen oder beruhen auf mehrstufigen Protokollen mit für Live‐Cell Imaging ungeeigneten Antikörpern bzw. Nanobodies. Zudem liefern verfügbare Affinitätssonden und Strategien zur intrazellulären Markierung oft nicht den erforderlichen Kontrast, etwa durch unvollständige zytosolische Abgabe oder überschüssige Sonde mit Hintergrundsignal. Hier stellen wir Sylives vor, eine Familie kompakter, synthetischer fluoreszenter Peptide zur kontrastreichen Live‐Bildgebung inhibitorischer (Gephyrin) und exzitatorischer (PSD‐95) postsynaptischer Gerüstproteine in primären Neuronen. Durch Vorreinigung des redoxspaltbaren Konjugats aus zellpenetrierendem Peptid und Sonde vermeiden wir Nebenprodukte der in‐situ‐Kopplung und erzielen eine reproduzierbare zytosolische Aufnahme mit nach intrazellulärer Reduktion vollständig wiederhergestellter Bindung an das Zielprotein. Die funktionelle Entkopplung von Zellaufnahme und Zielproteinadressierung ermöglicht saubere Markierung bei nanomolaren Sondenkonzentrationen, während die Aufnahme durch mikromolare Konzentrationen zellpenetrierender Peptide gewährleistet wird. Quantitative Experimente identifizieren die entscheidenden Assay‐Parameter und die optimalen Bedingungen für eine effiziente zytosolische Aufnahme der Sonden. Spezifität und nahezu rückstandsfreie Aufnahme belegen wir durch den direkten Vergleich mit transient exprimierten Proteinen sowie durch Immunfärbungen in fixierten Neuronen. Sylives ermöglichen damit eine kontrastreiche Markierung endogener postsynaptischer Strukturen ohne genetische Modifikation und bieten eine modulare Plattform zur Adressierung weiterer intrazellulärer Zielproteine in lebenden primären Neuronen.

Direct visualization of postsynaptic scaffolds in living neurons is essential for dissecting synaptic dynamics and plasticity. Existing methods for live synapse visualization have major constraints, relying on genetic engineering or multistep application of live‐cell incompatible antibodies or nanobodies. Available affinity probes and delivery strategies lack the required contrast due to incomplete or excess delivery. Here, we introduce Sylives, a set of compact, synthetic fluorescent peptides that enable high‐contrast live imaging of inhibitory (gephyrin) and excitatory (PSD‐95) postsynaptic scaffolds in native neurons. Critically, by pre‐purification of the redox‐cleavable CPP‐probe conjugate we overcome side‐product formation of in‐situ coupling strategies, achieving reliable cytosolic delivery and restored scaffold binding after intracellular reduction. The Sylive design addresses the need for nanomolar probe levels versus micromolar CPP for clean labelling and efficient delivery by decoupling targeting and uptake. Through quantitative evaluation of uptake and off‐target binding, we defined a transferrable parameter space for effective intracellular delivery. Near traceless Sylive uptake and target specificity are validated by direct comparison to transiently expressed proteins and immunolabeling in fixed neurons. The reduction‐sensitive Sylive conjugates enable high‐contrast, specificity‐restored labelling of endogenous postsynaptic sites without genetic modification and offer a modular platform for targeting alternative intracellular proteins in living primary neurons.

Glycine receptors (GlyR) and γ-aminobutyric acid type A receptors (GABA _A R) are ligand-gated chloride channels that mediate inhibitory neurotransmission throughout the central nervous system. The receptors colocalize widely at inhibitory synapses in the spinal cord and in the brainstem due to their interaction with an overlapping binding site of the synaptic scaffold protein gephyrin, pointing to a direct competition between the different receptor types. We have put this hypothesis to the test using single-molecule approaches to measure receptor–gephyrin interactions in cells and in vitro. We explored the effects of receptor competition at inhibitory synapses in living neurons by measuring the change in the accumulation and effective stabilization energy of glycine receptors in the presence of interfering GABA receptor complexes through single-molecule tracking and diffusion analysis. Second, using molecular tweezers, we quantified the thermodynamic properties of receptor–gephyrin binding, demonstrating direct and reversible competition through the addition of interacting peptides in solution. The relatively low affinity of GABA receptor subunits for gephyrin compared to the glycine receptor raises interesting questions about the role of this competition in synaptic plasticity. We hypothesize that GABA and glycine receptor competition constitutes a molecular system designed to reconcile synapse stability and plasticity at mixed inhibitory synapses.

Glycine receptors (GlyRs) are heteropentameric chloride channels that mediate fast inhibitory neurotransmission in the brainstem and spinal cord, where they regulate motor and sensory processes. GlyRs are clustered in the post-synaptic membrane by strong interactions of the β subunit with the scaffold protein gephyrin. Even though GlyRβ mRNA is highly expressed throughout the brain, the existence of synaptic GlyRs remains controversial as there is little conclusive evidence using conventional fluorescence microscopy and electrophysiological recordings. Here we exploit the high sensitivity and spatial resolution of single molecule localisation microscopy (SMLM) to investigate the presence of GlyRs at inhibitory synapses in the brain, focusing on several areas of the telencephalon. Making use of a knock-in mouse model expressing endogenous mEos4b-tagged GlyRβ, we identified few GlyRs in sub-regions of the hippocampus. Dual-colour SMLM revealed that these sparse receptors are integrated within the post-synaptic gephyrin domain, pointing to a possible role in maintaining the structural integrity of inhibitory synapses. In contrast, we found functionally relevant numbers of synaptic GlyRs at inhibitory synapses in the ventral striatum. Our results highlight the strength of SMLM to detect few and sparsely distributed synaptic molecules in complex samples and to analyse their organisation with high spatial precision.

Glycine receptors (GlyRs) are heteropentameric chloride channels that mediate fast inhibitory neurotransmission in the brainstem and spinal cord, where they regulate motor and sensory processes. GlyRs are clustered in the post-synaptic membrane by strong interactions of the β subunit with the scaffold protein gephyrin. Even though GlyRβ mRNA is highly expressed throughout the brain, the existence of synaptic GlyRs remains controversial as there is little conclusive evidence using conventional fluorescence microscopy and electrophysiological recordings. Here we exploit the high sensitivity and spatial resolution of single molecule localisation microscopy (SMLM) to investigate the presence of GlyRs at inhibitory synapses in the brain, focusing on several areas of the telencephalon. Making use of a knock-in mouse model expressing endogenous mEos4b-tagged GlyRβ, we identified few GlyRs in sub-regions of the hippocampus. Dual-colour SMLM revealed that these sparse receptors are integrated within the post-synaptic gephyrin domain, pointing to a possible role in maintaining the structural integrity of inhibitory synapses. In contrast, we found functionally relevant numbers of synaptic GlyRs at inhibitory synapses in the ventral striatum. Our results highlight the strength of SMLM to detect few and sparsely distributed synaptic molecules in complex samples and to analyse their organisation with high spatial precision.

Glycine and GABA receptors are ligand-gated chloride channels that mediate inhibitory neurotransmission throughout the central nervous system. The receptors co-localise widely at inhibitory synapses in the spinal cord and in the brainstem due to their interaction with an overlapping binding site of the synaptic scaffold protein gephyrin, pointing to a direct competition between the different receptor types. We have put this hypothesis to the test using single molecule approaches to measure receptor-gephyrin interactions in cells and in vitro . We explored the effects of receptor competition at inhibitory synapses in living neurons by measuring the change in the accumulation and effective stabilisation energy of glycine receptors in the presence of interfering GABA receptor complexes through single molecule tracking and diffusion analysis. Secondly, using molecular tweezers, we quantified the thermodynamic properties of receptor-gephyrin binding, demonstrating direct and reversible competition through the addition of interacting peptides in solution. The relatively low affinity of GABA receptor subunits for gephyrin compared to the glycine receptor raises interesting questions about the role of this competition in synaptic plasticity. We hypothesize that GABA and glycine receptor competition constitutes a molecular system designed to reconcile synapse stability and plasticity at mixed inhibitory synapses.

Startle disease is due to the disruption of recurrent inhibition in the spinal cord. Most common causes are genetic variants in genes ( GLRA1 , GLRB ) encoding inhibitory glycine receptor (GlyR) subunits. The adult GlyR is a heteropentameric complex composed of α1 and β subunits that localizes at postsynaptic sites and replaces embryonically expressed GlyRα2 homomers. The human GlyR variants of GLRA1 and GLRB , dominant and recessive, have been intensively studied in vitro. However, the role of unaffected GlyRβ, essential for synaptic GlyR localization, in the presence of mutated GlyRα1 in vivo is not fully understood. Here, we used knock-in mice expressing endogenous mEos4b-tagged GlyRβ that were crossed with mouse Glra1 startle disease mutants. We explored the role of GlyRβ under disease conditions in mice carrying a missense mutation ( shaky ) or resulting from the loss of GlyRα1 ( oscillator ). Interestingly, synaptic targeting of GlyRβ was largely unaffected in both mouse mutants. While synaptic morphology appears unaltered in shaky animals, synapses were notably smaller in homozygous oscillator animals. Hence, GlyRβ enables transport of functionally impaired GlyRα1 missense variants to synaptic sites in shaky animals, which has an impact on the efficacy of possible compensatory mechanisms. The observed enhanced GlyRα2 expression in oscillator animals points to a compensation by other GlyRα subunits. However, trafficking of GlyRα2β complexes to synaptic sites remains functionally insufficient, and homozygous oscillator mice still die at 3 weeks after birth. Thus, both functional and structural deficits can affect glycinergic neurotransmission in severe startle disease, eliciting different compensatory mechanisms in vivo.