Institute of Physiology CAS, Vídeňská 1083, 142 20 Prague 4, Czech Republic Tel: +420 241 062 424, +420 241 062 815, +420 776 005 601 E-mail: fgu@fgu.cas.cz Website: www.fgu.cas.cz IČ: 67985823 DIČ: CZ67985823
Martin Balastik, Ph.D.
Dpt. of Molecular Neurobiology Institute of Physiology, CAS Videnska 1083, Room 306 142 20, Prague 4 Czech Republic
Report on the dissertation thesis
Functional and pharmacological properties of GluN1/GluN2 and GluN1/GluN3 subtypes of NMDA receptors
by Mgr. Marharyta Kolcheva
The Ph.D. thesis by Marharyta Kolcheva aimes to analyze in detail the roles of extracellular and transmembrane regions of different subtypes of NMDA receptors in the regulation of their function using electrophysiology and microscopy methods on HEK293 cells and cultured hippocampal neurons. The author focused primarily on the effect of altered N-glycosylation of GluN1/GluN3 receptors; analyzed several pathogenic mutations in the transmembrane domain of the GluN1 subunit; and characterized structural determinants in the glycine-binding sites of the GluN1 and GluN3A subunits in NMDAR trafficking.
The topic of the thesis is highly relevant as various NMDA receptor mutations have been associated with intellectual disability, schizophrenia, autism, movement disorders, epilepsy and other human disorders.
Consequently, analysis of NMDAR is important for our understanding of molecular and physiological causes of these diseases and can prove instrumental for development of new therapeutic approaches.
The thesis work was done at the Institute of Experimental Medicine of the Czech Academy of Sciences, in the Department of Neurochemistry under the supervision of Dr. Martin Horak, whose laboratory specializes on analysis of NMDA receptors and as such it provides an optimal environment to successfully address all the raised questions.
Due to high number of publications related to the topic of the doctoral thesis and which the author published during her PhD studies (4 publications, out of which one as first author and one as a co-first author and 2 publications unrelated to the thesis), she chose to writhe the PhD thesis in cumulative format.
The thesis is well written and easy to read. On the formal level, it is conventionally divided into Introduction, Literature review, Objectives, Methods, Discussion, Conclusion, References and the List of Publications:
Introduction opens the topics and the questions the author wanted to address.
Literature review nicely introduces the important aspects of NMDA receptors relevant for the thesis work and the questions later addressed in the results section. It covers both the basic information, as well as latest discoveries. The references throughout the introduction are current, and accurate.
The Objectives of the thesis are clearly stated and divided into 3 aims. In part they follow up on the previous work of the department, but develop it further on. The main objectives are:
1) To examine the impact of N-glycosylation and different lectins on the regulation of the functional properties of GluN1/GluN3 receptors.
2) To examine the effects of pathogenic mutations in the TMD of the GluN1 subunit on surface delivery and on the functional and pharmacological properties of conventional and non-conventional di- heteromeric NMDARs.
3) To examine how the integrity of the glycine-binding site of the GluN1 and GluN3A subunits influences trafficking and functional properties of NMDARs.
The aims are followed by Methods section, in which the methods used in the publications are summarized with references to the publication, where they are described in detail.
The results of each of the publication are summarized in the Results section and contribution of the author to each of the publication is well stated.
The obtained data are systematically discussed in relation to the previously published data in the Discussion section. The doctoral candidate clearly demonstrates a broad knowledge of the fundamental previous and current literature in the field of NMDA receptor regulation and her final conclusions are fully justified.
References are followed by the List of Publications both related and unrelated to the thesis.
In summary, this is a very well-written thesis and a successful PhD project. The experiments, performed by the doctoral candidate were well planned, executed with right controls, described in detail and properly discussed. In her multiple publications Marharyta Kolcheva successfully addressed some important questions related to NMDA receptor regulation in various pathological conditions. The obtained data will be useful not only in the specific field of NMDA receptor physiology, they will help our better understanding of the pathogenesis of various neurological disorders. The methods established on course of the thesis will be further used in the Department of Neurochemistry. Ms. Kolcheva demonstrated her ability to conduct independent research, evaluate the obtained data and use them to develop new hypothesis. Based on her successful dissertation work I fully recommend to award Ms. Kolcheva with a PhD degree.
I have following question related to the Ph.D. thesis:
1. While multiple experiments have been performed on both hippocamapl neurons, as well as on HEC293 cells with similar results, the excitotoxicity was analyzed only using the hippocampal neurons. Could HEC293 cells be also used for this purpose, and would you expect a different result than in neurons?
2. The sensitivity of the pathogenic GluN1 mutation A645S to memantine was significantly increased both in Mg2+-free, as well as in the physiological Mg2+ levels, while M641I mutation had un-changed IC50 levels in Mg2+-free, but significantly decreased IC50 value in physiological Mg2+ levels. Do you have any explanation, or hypothesis accounting for this altered memantine sensitivity of the two mutants and conditions? Do they reflect the different human pathology associated with these mutations?
3. Several of the experiments have been performed using HEC293 cells transfected with various combinations of NMDAR subunits. Contrary to the neurons, HEC293 cells undergo cell cycle with specific activation of
various (e.g. mitotic) kinases. As phosphorylation has been shown to regulate NMDAR activity, were the measurements done only at a specific phase of cell cycle or on a synchronized cells?
Prague, March 13, 2023
Martin Balastik, Ph.D.
