Biology subjects

Ladislav, M.

Publications and source records attributed to Ladislav, M..

6 recordsLinked to original sources

bioRxiv2025

The role of disulfide bonds in the GluN1 subunit in the early trafficking and functional properties of GluN1/GluN2 and GluN1/GluN3 NMDA receptors

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for excitatory neurotransmission. Previous studies proposed the existence of four disulfide bonds in the GluN1 subunit; however, their role in NMDAR trafficking remains unclear. Our study first confirmed the existence of four disulfide bonds in the GluN1 subunit using biochemistry in human embryonic kidney 293T (HEK293T) cells. Disrupting the individual disulfide bonds by serine replacements produced the following surface expression trend for GluN1/GluN2A, GluN1/GluN2B, and GluN1/GluN3A receptors: wild-type (WT) > GluN1-C744S-C798S > GluN1-C79S-C308S > GluN1-C420S-C454S > GluN1-C436S-C455S subunits. Electrophysiology revealed altered functional properties of NMDARs with disrupted disulfide bonds, specifically an increased probability of opening (Po) at the GluN1-C744S-C798S/GluN2 receptors. Synchronized release from the endoplasmic reticulum confirmed that disruption of disulfide bonds impaired early trafficking of NMDARs in HEK293T cells and primary hippocampal neurons prepared from Wistar rats of both sexes (embryonic day 18). The pathogenic GluN1-C744Y variant, associated with neurodevelopmental disorder and seizures, caused reduced surface expression and increased Po at GluN1/GluN2 receptors, consistent with findings for the GluN1-C744S-C798S subunit. The FDA-approved memantine inhibited GluN1-C744Y/GluN2 receptors more potently and with distinct kinetics compared to WT GluN1/GluN2 receptors. We also observed enhanced NMDA-induced excitotoxicity in hippocampal neurons expressing the GluN1-C744Y subunit, which memantine reduced more effectively compared to the WT GluN1 subunit. Lastly, we demonstrated that the presence of the hGluN1-1a-C744Y subunit counteracted the effect of the hGluN3A subunit on decreasing dendritic spine maturation, consistent with the reduced surface delivery of the NMDARs carrying this variant. Significance statementOur findings highlight the critical role of disulfide bonds in the GluN1 subunit in regulating trafficking and function of major conventional (GluN1/GluN2A, GluN1/GluN2B) and unconventional (GluN1/GluN3A) diheteromeric NMDAR subtypes in the postnatal forebrain. We further demonstrated that the pathogenic GluN1-C744Y variant reduces surface expression of all studied NMDARs, as well as increases the probability of opening (Po) of the GluN1/GluN2 receptors, leading to heightened NMDA-induced excitotoxicity in hippocampal neurons. Additionally, we introduced an ARIAD-based system for the synchronized release of NMDARs from the endoplasmic reticulum in hippocampal neurons. This system provides a powerful tool for studying pathogenic GRIN gene variants and addresses the current lack of molecular methods for analyzing the early trafficking of NMDARs.

neuroscience↗

bioRxiv2024

Dizocilpine derivatives with neuroprotective effect lacking the psychomimetic side effects

We aimed to prepare novel dibenzosuberane derivatives that act on N-methyl-D-aspartate (NMDA) receptors with potential neuroprotective effects. Our approach involved modifying the tropane moiety of MK-801, a potent open-channel blocker known for its psychomimetic side effects, by introducing a seven-membered ring with substituted base moieties specifically to alleviate these undesirable effects. Our in silico analyses showed that these derivatives should have high gastrointestinal absorption and cross the blood-brain barrier (BBB). Our pharmacokinetic studies in rats supported this conclusion and confirmed the ability of leading compounds 3l and 6f to penetrate the BBB. Electrophysiological experiments showed that all compounds exhibited different inhibitory activity towards the two major NMDA receptor subtypes, GluN1/GluN2A and GluN1/GluN2B. Of the selected compounds intentionally differing in the inhibitory efficacy, 6f showed high relative inhibition ([~]90% for GluN1/GluN2A), while 3l showed moderate inhibition ([~]50%). An in vivo toxicity study determined that compounds 3l and 6f were safe at 10 mg/kg doses with no adverse effects. Behavioral studies demonstrated that these compounds did not induce hyperlocomotion or impair prepulse inhibition of startle response in rats. Neuroprotective assays using a model of NMDA-induced hippocampal neurodegeneration showed that compound 3l at a concentration of 30 M significantly reduced hippocampal damage in rats. These results suggest that these novel dibenzosuberane derivatives are promising candidates for developing NMDA receptor-targeted therapies with minimal psychotomimetic side effects.

neuroscience↗

bioRxiv2024

Potent and reversible open-channel blocker of NMDA receptor derived from dizocilpine with enhanced membrane-to-channel inhibition

N-methyl-D-aspartate receptors (NMDARs) play a significant role in developing several central nervous system (CNS) disorders. Currently, memantine, used for treating Alzheimers disease, and ketamine, known for its anesthetic and antidepressant properties, are two clinically used NMDAR open-channel blockers. However, despite extensive research into NMDAR modulators, many have shown either harmful side effects or inadequate effectiveness. For instance, dizocilpine (MK-801) is recognized for its powerful psychomimetic effects due to its high-affinity and nearly irreversible inhibition of the GluN1/GluN2 NMDAR subtypes. Unlike ketamine, memantine and MK-801 also act through a unique, low-affinity "membrane-to-channel inhibition" (MCI). We aimed to develop an open-channel blocker based on MK-801 with distinct inhibitory characteristics from memantine and MK-801. Our novel compound, K2060, demonstrated effective voltage-dependent inhibition in the micromolar range at key NMDAR subtypes, GluN1/GluN2A and GluN1/GluN2B, even in the presence of Mg2+. K2060 showed reversible inhibitory dynamics and a partially trapping open-channel blocking mechanism with a significantly stronger MCI than memantine. Using hippocampal slices, 30 {micro}M K2060 inhibited excitatory postsynaptic currents in CA1 hippocampal neurons by [~]51%, outperforming 30 {micro}M memantine ([~]21% inhibition). K2060 exhibited No Observed Adverse Effect Level (NOAEL) of 15 mg/kg upon intraperitoneal administration in mice. Administering K2060 at a 10 mg/kg dosage resulted in brain concentrations of approximately 2 {micro}M, with peak concentrations (Tmax) achieved within 15 minutes. Finally, applying K2060 with trimedoxime and atropine in mice exposed to tabun improved treatment outcomes. These results underscore K2060s potential as a therapeutic agent for CNS disorders linked to NMDAR dysfunction.

neuroscience↗

bioRxiv2024

Independent regulation of early trafficking of NMDA receptors by ligand-binding domains of the GluN1 and GluN2A subunits

The essential role of N-methyl-D-aspartate receptors (NMDARs) in excitatory neurotransmission is underscored by numerous pathogenic variants in the GluN subunits, including those identified in their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs; however, whether it controls the structure of LBDs or the functionality of NMDARs is unknown. Using alanine substitutions combined with microscopy and electrophysiology, we found that surface expression of GluN1/GluN2A receptors, the primary NMDAR subtype in the adult forebrain, strongly correlates with EC50 values for glycine and L-glutamate. Interestingly, co-expression of both GluN1 and GluN2A subunits with alanine substitutions led to an additive reduction in the surface number of GluN1/GluN2A receptors, as did co-expression of both GluN1 and GluN2A subunits containing closed cleft conformation of LBDs. The synchronized ER release confirmed the altered regulation of early trafficking of GluN1/GluN2A receptors bearing alanine substitutions in the LBDs. Furthermore, the human versions of GluN1/GluN2A receptors containing pathogenic GluN1-S688Y, GluN1-S688P, GluN1-D732E, GluN2A-S511L, and GluN2A-T690M variants exhibited distinct surface expression compared to the corresponding alanine substitutions. Mutant cycles of GluN1-S688, GluN1-D732, GluN2A-S511, and GluN2A-T690 residues revealed, in most cases, a weak correlation between surface expression of the mutant GluN1/GluN2A receptors and their EC50 values for glycine or L-glutamate. Consistent with our experimental data, molecular modeling and dynamics showed that the ER quality control machinery likely perceives structural changes of the LBDs but not the functionality of GluN1/GluN2A receptors. Significant statementOur study showed that structural changes in LBDs independently regulate the early trafficking of GluN1/GluN2A receptors and that the surface numbers of mutant GluN1/GluN2A receptors do not necessarily correlate with their agonist sensitivity. In addition, we validated a novel system of synchronized release of GluN1/GluN2A receptors from the ER. Together, our experimental and in silico findings support the urgency of further detailed research on the regulation of early trafficking of NMDARs, as it may open the avenue to targeted therapeutic intervention of CNS disorders associated with pathogenic variants in GluN subunits.

neuroscience↗

bioRxiv2022

Ventral posterolateral and ventral posteromedial thalamus neurons have distinct synaptic and cellular physiology

Somatosensory information is propagated from the periphery to the cerebral cortex by two parallel pathways through the ventral posterolateral (VPL) and ventral posteromedial (VPM) thalamus. VPL and VPM neurons receive somatosensory signals from the body and head, respectively. VPL and VPM neurons also receive cell-type-specific GABAergic input from the reticular nucleus of the thalamus (nRT). Although VPL and VPM neurons have distinct connectivity and physiological roles, differences in the functional properties of VPL and VPM neurons remain unclear as they are often studied as one ventrobasal (VB) thalamus neuron population. Here, we directly compared synaptic and intrinsic properties of VPL and VPM neurons in C57Bl/6J mice of both sexes aged P25-P32. Recordings of spontaneous synaptic transmission suggested that VPL neurons receive excitatory synaptic input with higher frequency and strength than VPM neurons, while VPL neurons exhibited weaker inhibitory synapse strength than VPM neurons. Furthermore, VPL neurons showed enhanced depolarization-induced spike firing and greater spike frequency adaptation than VPM neurons. VPL and VPM neurons fired similar numbers of spikes during hyperpolarization rebound bursts, but VPM neurons exhibited shorter burst latency compared to VPL neurons, which correlated with increased sag potential during hyperpolarization. This work indicates that VPL and VPM thalamocortical neurons are functionally distinct populations. The observed functional differences could have important implications for their specific physiological and pathophysiological roles within the somatosensory thalamocortical network.

neuroscience↗

bioRxiv2021

Synaptic and intrinsic mechanisms impair reticular thalamus and thalamocortical neuron function in a Dravet syndrome mouse model

Thalamocortical network dysfunction contributes to seizures and sleep deficits in Dravet syndrome (DS), an infantile epileptic encephalopathy, but the underlying molecular and cellular mechanisms remain elusive. DS is primarily caused by mutations in the SCN1A gene encoding the voltage-gated sodium channel NaV1.1, which is highly expressed in GABAergic reticular thalamus (nRT) neurons as well as glutamatergic thalamocortical neurons. We hypothesized that NaV1.1 haploinsufficiency alters somatosensory corticothalamic circuit function through both intrinsic and synaptic mechanisms in nRT and thalamocortical neurons. Using Scn1a heterozygous mice of both sexes aged P25-P30, we discovered reduced intrinsic excitability in nRT neurons and thalamocortical neurons in the ventral posterolateral (VPL) thalamus, while thalamocortical ventral posteromedial (VPM) neurons exhibited enhanced excitability. NaV1.1 haploinsufficiency enhanced GABAergic synaptic input and reduced ascending glutamatergic sensory input to VPL neurons, but not VPM neurons. In addition, glutamatergic cortical input to nRT neurons was reduced in Scn1a heterozygous mice, whereas cortical input to VPL and VPM neurons remained unchanged. These findings introduce input-specific alterations in glutamatergic synapse function and aberrant glutamatergic neuron excitability in the thalamus as disease mechanisms in Dravet syndrome, which has been widely considered a disease of GABAergic neurons. This work reveals additional complexity that expands current models of thalamic dysfunction in Dravet syndrome and identifies new components of corticothalamic circuitry as potential therapeutic targets. HIGHLIGHTSO_LIGABAergic reticular thalamus neurons have impaired tonic and burst firing properties in a NaV1.1 haploinsufficiency mouse model of Dravet syndrome. C_LIO_LINaV1.1 haploinsufficiency has opposing effects on spike firing in two distinct glutamatergic thalamocortical neuron populations. C_LIO_LINaV1.1 haploinsufficiency alters glutamatergic synaptic connectivity in an input-specific manner in the thalamus. C_LIO_LIDysregulation of both intrinsic and synaptic mechanisms contribute to imbalanced thalamic excitation and inhibition in this Dravet syndrome mouse model. C_LI

neuroscience↗