Biology subjects

Pol, E.

Publications and source records attributed to Pol, E..

4 recordsLinked to original sources

bioRxiv2025

NKCC1 as a signaling hub regulating KCC2 stability, chloride homeostasis, and seizure susceptibility

Chloride homeostasis relies on the dynamic balance between the neuronal co-transporters NKCC1 and KCC2. We reveal an unexpected mechanism by which NKCC1 governs KCC2 membrane stability. NKCC1 clusters recruits SPAK and PP1 to dynamically trap KCC2, compensating for its lack of a direct SPAK-binding site. Single-particle tracking shows that these NKCC1-rich assemblies operate as signaling hubs, enabling either SPAK-driven KCC2 phosphorylation and its membrane destabilization or PP1-mediated dephosphorylation of SPAK and KCC2 membrane stabilization. Peptides that activate SPAK by engaging NKCC1s PP1-binding motif lower KCC2 surface levels and reduce chloride extrusion, whereas a SPAK-inhibiting peptide prevents SPAK recruitment to NKCC1, stabilizes KCC2 in membrane clusters, and enhances chloride extrusion. An optimized peptide analog preserves KCC2 clustering under hyperexcitable conditions, reduces seizure frequency and severity in PTZ-induced epilepsy, and suppresses ictal activity in human epileptic tissue. These findings identify NKCC1-KCC2 coupling as a central regulatory axis for inhibitory signaling, and position our peptides as promising therapeutic candidates to restore chloride homeostasis in epilepsy and other disorders marked by impaired KCC2 membrane stability.

neuroscience↗

bioRxiv2025

WNK-Dependent Phosphorylation of Gephyrin Tunes GABAA Receptors at Inhibitory Synapses and Modulates Anxiety Behavior

The role of the chloride-sensitive kinase WNK1 and its effector SPAK in the brain remains poorly understood. Here, we identify a regulatory mechanism involving WNK signaling that directly controls the synaptic diffusion and clustering, as well as the membrane stability and endocytosis of inhibitory GABAA receptors (GABAARs). We show that activation of WNK signaling stabilizes GABAARs at inhibitory synapses, while inhibition enhances receptor internalization. This regulation depends on the phosphorylation state of two previously uncharacterized residues in the central linker region of the gephyrin scaffold protein. Modulating WNK activity alters neuronal activity and the kinetics of GABAergic currents. In vivo, expression of a phospho-mimetic form of gephyrin at WNK-targeted sites produces anxiolytic effects. By orchestrating the recruitment of GABAARs at inhibitory synapses, the WNK pathway emerges as a master regulator of GABAergic transmission and establishes chloride as a bona fide second messenger in inhibitory synaptic signaling. SignificanceSynaptic transmission relies on signaling pathways that control how neurotransmitter receptors move and stabilize at synapses. Many of these pathways use calcium as a second messenger. In contrast, how chloride might regulate synapses has remained unclear. In this study, we identify a chloride-sensitive pathway involving the WNK1 kinase and its partner SPAK, which controls the movement and stability of inhibitory GABAA receptors at synapses. This occurs through the phosphorylation of gephyrin, a key scaffolding protein, at two newly identified sites. Our results show that intracellular chloride can act as a second messenger, reshaping inhibitory synapses and altering behavior in mice, including reducing anxiety-like responses.

neuroscience↗

bioRxiv2022

The fast diffusion of NKCC1 along the axon is driven by glutamatergic activity

NKCC1 and KCC2 transporters regulate neuronal chloride homeostasis and thus synaptic inhibition. KCC2 activity is tuned by diffusion-capture in an activity-dependent manner. The mechanisms controlling NKCC1 in neurons are unknown. We found using super-resolution imaging that NKCC1 like KCC2 form nanodomains in the somato-dendritic membrane at extrasynaptic sites and at the periphery of excitatory and inhibitory synapses. NKCC1 nanoclusters are half the size and density in molecules than KCC2 clusters. This is accompanied by a higher mobility of NKCC1 compared to KCC2 in the dendritic membrane, suggesting a weaker NKCC1 anchoring to the cytoskeleton. In contrast, NKCC1, but not KCC2, is confined to endocytic zones, which would explain its controlled surface expression and the fact that endocytic zones would provide a reservoir from which NKCC1 could be released into the membrane. Finally, we show an increased confinement of NKCC1 in axons but not in dendrites upon glutamatergic activity blockade, indicating a selective mechanism of regulation in the axon. We propose that a rapid regulation of NKCC1 by lateral diffusion in the axon would control presynaptic glutamate release and the firing of action potentials.

neuroscience↗

bioRxiv2022

AN IMAGING FLOW CYTOMETRY-BASED METHODOLOGY FOR THE ANALYSIS OF SINGLE EXTRACELLULAR VESICLES IN UNPROCESSED HUMAN PLASMA

Extracellular vesicles (EVs) are tissue-specific particles released by cells containing valuable diagnostic information in the form of various biomolecules. To rule out selection bias or introduction of artefacts caused by EV isolation techniques, we present a clinically feasible, imaging flow cytometry (IFCM)-based methodology to phenotype and determine the concentration of EVs with a diameter [≤]400 nm in human platelet-poor plasma (PPP) without prior isolation of EVs. Instrument calibration (both size and fluorescence) were performed with commercial polystyrene beads. Detergent treatment of EVs was performed to discriminate true vesicular events from artefacts. Using a combination of markers (CFSE & Tetraspanins, or CD9 & CD31) we found that >90% of double-positive fluorescent events represented single EVs. Through this work, we provide a framework that will allow the application of IFCM for EV analysis in peripheral blood plasma in a plethora of experimental and potentially diagnostic settings. Additionally, this direct approach for EV analysis will enable researchers to explore new corners of EV as cellular messengers in healthy and pathological conditions.

cell biology↗