Biology subjects

Levi, S.

Publications and source records attributed to Levi, S..

8 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↗

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

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↗

bioRxiv2025

USP15: the fourth Proteasome-associated DUB

The human genome encodes approximately 100 deubiquitinating enzymes (DUBs), but only three are considered proteasome-associated DUBs (pDUBs): PSMD14/Rpn11, USP14, and UCHL5. Among these, only PSMD14 is an integral 19S subunit, whereas USP14 and UCHL5 bind transiently to specific proteasomal subunits. Given the dynamic nature of proteasome composition, we searched for additional pDUBs. USP15 was found to be associated with 26S proteasomes purified from cultured cells. In proteasome preparations from erythrocytes, USP15 was identified as the most abundant transient pDUB. It was even feasible to separate proteasomes containing USP15 from those containing USP14. Although USP15 utilizes an internal ubiquitin-like (UBL) domain for positioning itself at the proteasome, it did not compete with the UBL-containing USP14. USP15 facilitated substrate selection at the proteasome by efficiently disassembling short polyubiquitin (polyUb) chains, while sparing K48-linked tetra-ubiquitin conjugates from deubiquitination. This feature may aid the proteasome in differentiating between substrates to be rescued from those committed to proteolysis. Identification of a fourth pDUB encourages the continued search for additional proteasome-interacting proteins that modulate its substrate specificity in a context-specific manner.

biochemistry↗

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.

neuroscience↗

bioRxiv2022

ECPAS/Ecm29-Mediated 26S Proteasome Disassembly Is an Adaptive Response to Glucose Starvation

The 26S proteasome consists of loosely associated 20S catalytic and 19S regulatory complexes. Approximately half of the proteasomes in eukaryotic cells exist as free 20S complexes; however, our mechanistic and physiological understanding of what determines the ratio of 26S to 20S species remains incomplete. Here, we show that glucose starvation in mammalian cells results in the uncoupling of 26S holoenzymes into intact 20S and 19S subcomplexes. Subcomplex affinity-purification and quantitative mass spectrometry revealed that Ecm29 proteasome adaptor and scaffold (ECPAS) is a crucial mediator of this structural remodeling. The loss of ECPAS abrogated 26S dissociation, leading to decreased degradation of 20S proteasome substrates such as puromycylated polypeptides and lysine-less cyclin B. In silico modeling analysis suggested that the conformational changes of ECPAS may commence the disassembly process. ECPAS was also essential for proper endoplasmic reticulum stress response and cell survival during glucose starvation. In addition, we evaluated the role of ECPAS in vivo using the mouse xenograft model and observed that glucose-deprived tumor tissues had significantly elevated 20S proteasome levels. Collectively, our results indicate that the 20S-19S disassembly mediated by ECPAS is a novel mechanism adapting global proteolysis to physiological needs and an effective cellular strategy against proteotoxic stress.

biochemistry↗