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Neurochemistry

Research Department

Group photo of the Department of Neurochemistry team

The Department of Neurochemistry was established in 2018 to conduct cutting-edge research in regulating N-methyl-D-aspartate (NMDA) receptors in mammalian neurons under normal and pathological conditions. Specifically, we study the molecular mechanisms that regulate the surface numbers of NMDA receptors, including their maturation in the endoplasmic reticulum and transport across the Golgi apparatus, and the surface mobility and localization of NMDA receptors in synaptic and extrasynaptic regions of mammalian neurons. We are also studying the functional impact of selected pathogenic variants in genes encoding GluN subunits of NMDA receptors associated with developing epilepsy and other neurological diseases and developing pharmacological modulators of NMDA receptors with unique mechanisms of action. Our results contribute to the understanding and future therapy of disorders associated with abnormal regulation of NMDA receptors, including, for example, epilepsy and Alzheimer's disease.

Martin Horák

Head of the Department
Martin Horák, Ph.D.

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People

Deputy Head

Petra Zahumenská, M.Sc.

Researchers

Katarína Hemelíková, Ph.D.

Martin Horák, Ph.D.

Marharyta Kolcheva, Ph.D.

Štěpán Kortus, Ph.D.

Marek Ladislav, Ph.D.

PhD Students

Michaela Hrochová, M.Sc.

Emily Langore, M.Sc.

Anna Misiachna, M.Sc.

Jakub Netolický, M.Sc.

Petra Zahumenská, M.Sc.

Laboratory Technicians

Anežka Pelikánová, M.Sc.

  • The department has a total of ~140 m2, including laboratory, office, and storage space. Our culture room contains a laminar box, four CO2 incubators, a dissecting magnifying glass, and a microscope; we also have equipment for molecular biology (UV DNA/RNA decontamination box, incubators, shakers, PCR cyclers, centrifuges), biochemistry (homogenizers, sonicators, gel electrophoresis, Western blotting apparatus) and electrophysiology (micropipette puller, three fully equipped electrophysiology apparatus with rapid application systems, one newly built electrophysiology apparatus for recording from acute brain tissue sections). Our experimental research is conducted using a combination of advanced molecular biology methods (preparation of DNA expression vectors, targeted mutagenesis, lentiviral particle production, genotyping of mouse models), microscopy (classical confocal microscopy, live quantum dot microscopy, uPAINT, dSTORM), biochemistry (SDS-PAGE, "Western blotting", colorimetric essays) and electrophysiology (patch-clamp method in the whole-cell or single-channel configuration in cell cultures or brain sections). We maintain breeding colonies of mice carrying genes for NMDA receptor subunits with conditional "knock-out" (cKO-Grin2A, cKO-Grin2B, cKO-Grin2A/Grin2B, cKO-Grin1) or "knock-in" (globally expressing the Grin2aN615S gene; a model of sound-induced epileptic seizures).


Important Results


The pathogenic N650K variant in the GluN1 subunit regulates open probability of NMDA receptors and reduces NMDA-induced excitotoxicity

First, we reveal that pathogenic variant N650K in GluN1 subunit reduces open probability (Po) of GluN1-N650K/GluN2A receptors compared to wild-type. Next, using NMDA-induced excitotoxicity method – treating hippocampal neurons expressing either GluN1 or GluN1-N650K subunit for 1 h with various concentrations of NMDA – we found that neurons expressing the GluN1-N650K variant have increased resistance to NMDA impulse.

Reprezentativní snímky fluorescence YFP a Hoechst 33342 v hipokampálních neuronech s výsledkovými grafy. Detailní popis naleznete pod obrázkem.

The pathogenic N650K variant in the GluN1 subunit regulates open probability of NMDA receptors and reduces NMDA-induced excitotoxicity. (A) Structural model of the M1, M2, and M3 domains in the hGluN1/hGluN2A heterotetramer; the residue studied here is indicated in red. (B) Single channel activity recordings in HEK293 cells expressing either GluN1/GluN2A (left) or GluN1-N650K/GluN2A (right) receptors (C) Representative images of YFP and Hoechst 33342 fluorescence in hippocampal neurons expressing YFP-tagged GluN1 or GluN1-N650K subunits. (D) Summary of the percentage of dead (i.e., pyknotic) neurons expressing the indicated hGluN1-1a subunits and treated with the indicated concentrations of NMDA.

 

Publication:

Kolcheva, M., Ladislav, M., Netolicky, J., Kortus, S., Rehakova, K., Krausova, B.H., Hemelikova, K., Misiachna, A., Kadkova, A., Klima, M., Chalupska, D., Horak, M.: (2023) The pathogenic N650K variant in the GluN1 subunit regulates the trafficking, conductance, and pharmacological properties of NMDA receptors. Neuropharmacology, 1; 222:109297.


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