Biology subjects

Russeau, M.

Publications and source records attributed to Russeau, M..

10 recordsLinked to original sources

bioRxiv2026

Sex-specific long-term alteration of hippocampal excitation/inhibition balance and behavior by transient caffeine exposure during synaptogenesis

Caffeine is the most widely consumed psychoactive substance worldwide, yet the long-term consequences of exposure during critical periods of brain development remain incompletely understood. Synaptogenesis represents a vulnerable window during which environmental factors can shape the maturation of neuronal circuits and influence lifelong brain function. Here, we investigated the impact of caffeine exposure during hippocampal synaptogenesis on synaptic development, neuronal function, behavior, and seizure susceptibility, with a particular focus on sex-dependent effects. Developmental caffeine exposure induced distinct, sex-specific trajectories of hippocampal synaptic remodeling. In the CA1 region, caffeine produced opposite patterns of glutamatergic synapse regulation, characterized by a delayed reduction in excitatory synapse density in males and an increase in females. In contrast, inhibitory synapse organization was selectively altered in males, with a transient increase in CA3 inhibitory synaptic density during development associated with enhanced inhibitory transmission, whereas females exhibited no significant changes. These findings reveal sex-specific and temporally divergent effects of developmental caffeine exposure on hippocampal synaptic maturation and function. At the behavioral level, developmental caffeine exposure produced distinct sex-dependent phenotypes : males exhibited increased anxiety-like behavior, whereas females developed a delayed impairment in recognition memory that became apparent only in adulthood. Furthermore, caffeine exposure selectively increased PTZ-induced seizure susceptibility in juvenile females, an effect that was no longer detected in adulthood. Together, these findings demonstrate that caffeine exposure during hippocampal synaptogenesis induces sex-specific and temporally dynamic alterations in circuit maturation, resulting in distinct behavioral and neuronal excitability outcomes. These results highlight the importance of considering both sex and developmental timing when assessing the neurodevelopmental consequences of caffeine exposure.

neuroscience↗

bioRxiv2026

Ganglioside GM1-enriched rafts regulate the neuronal chloride co-transporter 1 KCC2.

During brain development, dynamic remodeling of membrane lipid composition accompanies the maturation of inhibitory neurotransmission and the progressive establishment of low intracellular chloride levels. Central to this developmental transition is the neuronal K-Cl- cotransporter KCC2, whose stabilization at the plasma membrane enables the emergence of hyperpolarizing GABAergic signaling. Although KCC2 regulation by protein partners has been extensively characterized, whether lipid remodeling actively contributes to its membrane organization and chloride transport remains unclear. Here we identify the ganglioside GM1, a complex lipid abundant in plasma membrane of neurons, as a developmentally regulated lipid determinant of KCC2 membrane localization and function. We show that KCC2 interacts with GM1 within plasma membrane lipid rafts and that this interaction increases during postnatal brain maturation. Molecular modeling identified a conserved ganglioside-binding domain (GBD) in KCC2 centered on tryptophan 318 (W318). Biophysical analyses revealed a specific and saturable interaction between this domain and GM1 that is abolished by the epilepsy-associated W318S mutation. Disruption of KCC2-GM1 interactions, either by W318S mutation or by pharmacological depletion of GM1, excludes KCC2 from lipid rafts, alters its membrane diffusion and clustering, and reduces its surface stability. Functionally, these perturbations impair KCC2-mediated chloride extrusion and disrupt the somato-dendritic chloride gradient in hippocampal neurons. Consistent with these cellular effects, GM1-deficient (St3gal5-/-) mice exhibit selective reduced hippocampal KCC2 expression. Together, these findings reveal a lipid-protein mechanism that links developmental membrane remodeling to KCC2 stabilization and chloride homeostasis, highlighting membrane lipids as active regulators of transporter maturation and inhibitory circuit development.

neuroscience↗

bioRxiv2026

Differential isoform-specific control of KCC2 function in developing cortical neurons

The K-Cl cotransporter KCC2 is essential for fast synaptic inhibition in the mature brain. It is encoded by a single gene and expressed as two isoforms: KCC2a and KCC2b, which differ in their N-terminal domains. While KCC2b is predominant, the function of the weakly expressed KCC2a isoform remains unclear. Here, we reveal that KCC2a is a potent, bidirectional regulator of KCC2b membrane stability and function in cortical neurons. In immature neurons, where WNK-SPAK kinase activity is high, KCC2a promotes SPAK-dependent phosphorylation of KCC2b at Thr1007, which likely contributes to hindering its membrane expression and function. Conversely, in mature neurons with low basal WNK-SPAK activity, KCC2a promotes KCC2b expression, clustering, and function. At this stage, although accounting for less than 5% of total KCC2 mRNA, KCC2a is enriched in dendrites and within KCC2 clusters, where it prevents clathrin-mediated KCC2b endocytosis, as well as polyubiquitination and proteasomal degradation. Thus, KCC2a acts as a developmental switch that first inhibits KCC2b during early development and then ensures its membrane stability to support effective synaptic inhibition in the adult brain.

neuroscience↗

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.

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bioRxiv2025

Epileptic encephalopathy-related Kv2.1 mutants impair channel clustering and membrane distribution but not neuronal excitability

The voltage-gated potassium channel Kv2.1, which is encoded by the epileptic encephalopathy-associated gene KCNB1, is a primary driver of delayed-rectifier K+ currents in neurons. These currents contribute to high frequency firing by preventing depolarization block due to Na+ channel inactivation. Wild-type (WT) channels are localized at the soma, proximal dendrites and axons, forming large aggregates (clusters) via their C-terminal proximal restriction and clustering domain (PRC). This study investigates the biophysical and functional consequences of two C-terminal truncation mutations (Y529* and R579*), identified in patients with epileptic encephalopathy, which disrupt this critical clustering domain. Cluster formation was impaired, though not abolished, in neurons expressing mutated subunits together with endogenous WT subunits. Consistent with this clustering deficit, single-molecule imaging revealed altered channel dynamics, with WT channels remaining largely immobile, while mutated forms exhibited intermittent diffusion punctuated by transient immobilization events. This behavior was reproduced by diffusion-capture simulations considering channels with different number of PRC domains (i.e. with mutant subunits) and labile scaffolding interactions. Patch-clamp recordings revealed no significant difference in excitability between WT and mutant-expressing neurons. However, we observed increased firing in both conditions compared to non-transfected neurons. This suggests that Kv2.1 overexpression (for both WT and mutant channels) counterintuitively enhances neuronal excitability. Our data challenge a canonical role of this voltage-gated potassium channel in dampening neuronal firing, indicating that its overall expression level can be a critical and paradoxical driver of hyperexcitability.

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↗

bioRxiv2023

Enhancing KCC2 function reduces interictal activity and prevents seizures in mesial temporal lobe epilepsy

The neuronal K/Cl cotransporter KCC2 regulates the transmembrane chloride gradient, which controls the efficacy of GABAergic signaling. In mesial temporal lobe epilepsy (mTLE) and other neurological disorders, reduced KCC2 expression or function can result in depolarizing GABA signaling, which is thought to contribute to pathological activity and seizures. Therefore, restoring chloride homeostasis represents a promising therapeutic strategy. We investigated the mechanisms and antiseizure effects of two small molecules, prochlorperazine (PCPZ) and CLP-257, that have been identified as potential KCC2 enhancers. We found that both compounds enhance KCC2 function and clustering in cortical neurons while reducing its membrane diffusion, without altering canonical regulatory phosphorylation. CLP-257 also selectively increased extrasynaptic, but not synaptic, GABAA receptor-mediated currents. Using in vitro recordings from resected brain tissue of patients with drug-resistant mTLE and in vivo recordings from a mouse model, we show that PCPZ and CLP-257 (or its prodrug CLP-290) effectively suppressed spontaneous epileptiform activity in both models. These findings reveal that PCPZ and CLP-257 act as genuine KCC2 enhancers and provide experimental evidence of the therapeutic potential of such compounds for treating drug-resistant mTLE. Significance statementA major challenge in treating epilepsy is the high percentage of patients with drug-resistant forms, like mesial temporal lobe epilepsy (mTLE). This study investigates a therapeutic strategy by targeting the neuronal KCC2 transporter, which is often dysfunctional in epilepsy. Our findings identify two compounds, prochlorperazine and CLP-257, that enhance KCC2 function by promoting its clustering on the cell membrane, a previously uncharacterized mechanism. Importantly, these compounds effectively reduce spontaneous epileptiform activity in human brain tissue from mTLE patients and significantly reduce seizure frequency in a mouse model. This work provides a critical proof-of-concept for activating KCC2 as a viable therapeutic approach for drug-resistant epilepsy.

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bioRxiv2022

Lateral diffusion of NKCC1 contributes to neuronal chloride homeostasis and is rapidly regulated by the WNK signaling pathway

An upregulation of the Na+-K+-2Cl- co-transporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABAA receptors and thus to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cellular and molecular regulatory mechanisms of NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK1 kinase and the downstream SPAK kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl- concentration but it participates to the elevation of intraneuronal Cl- concentration in hyperactivity condition associated with an up-regulation of NKCC1. The fact that the chloride exporter KCC2 is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

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

5-HT1A and 5-HT2B receptor interaction and co-clustering regulates serotonergic neuron excitability

Many psychiatric diseases including depression, schizophrenia and anxiety have been associated with serotonin (5-HT) neuron dysfunction. Pacemaker-like firing of raphe 5-HT neurons was proposed to be under unique 5-HT1A receptor-mediated autoinhibition. We previously showed that 5-HT2B receptors were expressed by 5-HT neurons together with 5-HT1A receptors. However, functional consequences on 5-HT neurons of putative interaction between these receptors are unknown. Using co-immunoprecipitation, BRET, confocal and super-resolution microscopy in hippocampal and 5-HT neurons, we present converging evidence that 5-HT1A and 5-HT2B receptors can form heterodimers and co-cluster at the surface of dendrites. 5-HT2B receptor clusters were redistributed upon 5-HT1A receptor expression supporting functional interactions between the two receptors. Furthermore, 5-HT2B receptor expression prevented agonist-induced internalization of 5-HT1A receptors, whereas 5-HT1A receptors mimicked the clustering effect of 5-HT2B receptor stimulation on its surface expression. The functional impact of this interaction in-vivo was assessed by recording 5-HT neuron excitability from mice lacking 5-HT2B receptors in 5-HT neurons. Upon 5-HT1A receptor stimulation, the firing activity of 5-HT neurons was increased in the absence of 5-HT2B receptors and decreased in their presence through regulation of SK channels, thus demonstrating functional output of this interaction in controlling 5-HT neuron firing activity.

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