Department of Cellular Neurophysiology (DCN) · Department of Cellular Neurophysiology (DCN) Report on the scientific activity of the team in 2010–2014 Ladislav Vyklický, MD, PhD.,

Department of Cellular Neurophysiology (DCN)

Report on the scientific activity of the team in 2010–2014

Ladislav Vyklický, MD, PhD., DSc. (Head of the Department)

http://www.fgu.cas.cz/en/departments/cellular-neurophysiology

NEUROPHYSIOLOGY CARDIOVASCULAR PHYSIOLOGY

METABOLISM

System level

- Circadian rhythms - Memory - Epilepsy - Alzheimer’s disease - Pain - Schizophrenia

- Central and peripheral blood pressure control

- Regulation of embryonic cardiac output

- Pathophysiology of heart failure

- Neurohumoral control - Energy expenditure

- Glucose homeostasis

- Nutritional interventions

- Metabolic syndrome - Biomarkers

Cellular level

- Synaptic transmission - Neuromodulation - Nociception - Ion channels:

NMDA, TRP, nicotinic,

purinergic - Metabotropic receptors - Secretion of pituitary

hormones

- Calcium influx and calcium sensitization in contractility of resistance arteries

- Calcium transients and ion channels

- Gap junctional coupling

- Isolated cardiac myocytes

- Cell proliferation in cardiac growth and regeneration

- Intracellular signalling - Mitochondrial

(dys)function - Membrane biophysics

Molecular level

- Receptor structure- function

- Gene and protein expression

- Adrenergic receptor number regulation

- Mitochondrial membrane potential

- RhoA/Rho kinase pathway in calcium sensitization

- Transport proteins

- Reactive oxygen species

- Structure of signalling proteins

Department of Cellular Neurophysiology (DCN)

Department of Cellular Neurophysiology – IP CAS 2

The goal of our research is to:

comprehend the relationship between the structure and function of ion channels, characterize the actions of pharmacologically active substances, contribute to the understanding of molecular processes involved in neurodegeneration, schizophrenia

and nociception where ionotropic glutamate receptors, TRP channels, nicotinic type of acetylcholine receptors are involved.

Genetic

Structural – functional

Trafficking

Quality of the results and share in their aquisition: Main research activities

Our research is :

Hypothesis based on model predictions Quantitative Methodologically advanced:

• Patch clamp technique: with fast application of

solutions and controlled temperature • PCR based methods • Biochemical methods • Confocal /immunofluorescence/FRET microscopy • Molecular modeling and structural bioinformatics • In vitro cellular models and recombinant receptors

Ligand-gated ion channels


Quality of the results: Structural – overall quality of publications - Phase I

Corresponding 63%

Non-corresponding 37%

Corresponding 55%

Non-corresponding 45%

Nu

mb

er o

f p

ub

lica

tio

ns

Nu

mb

er o

f p

ub

lica

tio

ns

Quality profile (evaluated outputs 16)

Quality of outputs by journal ranking (total number for bibliometry 42/58)


Determinants of voltage-dependent gating

S4 S5

Mechanism for camphor activation and desensitization of TRPV1

Domains involved in Ca2+ and PIP2 regulation (A1, V1)

Viktorie Vlachová

Quality of the results: Structural – functional studies on TRP channels. Molecular basis of multimodal regulation of nociceptive TRP channels. TRPV1,

TRPV3, TRPA1


Determinants of voltage-dependent gating

S4 S5

Mechanism for camphor activation and desensitization of TRPV1

Domains involved in Ca2+ and PIP2 regulation (A1, V1)

Viktorie Vlachová

Zíma et al. (2015) Neuropharmacology 93, 294-307. Witschas et al. (2015) BBA–Biomembranes 1848, 1147-1156. Boukalova et al. (2013) BBA–Mol. Cell Res. 1833(3), 520-528. Vetter et al. (2013) J. Neurosci. 33, 16627-16641. Marsakova et al. (2012) Anesthesiology 116, 903-917. Sura et al. (2012) J. Biol. Chem. 287, 18067-18077. Vetter et al. (2012) EMBO J. 31, 3795-3808. Samad et al. (2011) Biochemical J. 433, 197-204. Boukalova et al. (2010) J. Biol. Chem. 285, 41455-41462. Hot Topic Issue: Vlachova, Guest Editor, (2011) Curr. Pharm. Biotechnol. 12, 1: TRP Channels: From Understanding to Action

¨

Quality of the results: Structural – functional studies on TRP channels. Molecular basis of multimodal regulation of nociceptive TRP channels.

TRPV1, TRPV3, TRPA1


20

0 p

A

1 s

100 µM ACh

0.5 s 1 µM Lob

2 s 1 µM Lob

5 s 1 µM Lob

50

pA

1 s

5 s

50

0 p

A

3 µM ACh

0.1 µM Lob

0.3 µM Lob

1 µM Lob

3 µM Lob

10 µM Lob

30 µM Lob

Lobeline has been used as an emetic, respiratory stimulant and nicotine addiction cessation agent despite the mechanism(s) of its action being unclear.

Lobeline inhibits responses to high concentration of acetylcholine but potentiates

responses to low concentration of acetylcholine in α4β2 neuronal nicotinic receptors.

Muscle nicotinic receptors are inhibited by lobeline.

Jan Krůšek

Quality of the results: Structural – functional studies on AChR. Modulation of nicotinic receptors by plant alkaloid lobeline

.


20

0 p

A

1 s

100 µM ACh

0.5 s 1 µM Lob

2 s 1 µM Lob

5 s 1 µM Lob

50

pA

1 s

5 s

50

0 p

A

3 µM ACh

0.1 µM Lob

0.3 µM Lob

1 µM Lob

3 µM Lob

10 µM Lob

30 µM Lob

Lobeline inhibits responses to high concentration of acetylcholine but potentiates responses to low concentration of acetylcholine in a4b2 neuronal nicotinic receptors. Lobeline has been used for treatment of nicotine addiction.

H. Krůšek/F. Vyskočil - bilateral experimental cooperation with prof. E. E. Nikolsky and prof. E. A. Bukharaeva - Kazan branch of Russian Academy of Sciences and Kazan Medical University in in studying mechanisms of cholinergic transmission: Khuzakhmetova et al., Int J Dev Neurosci 34: 9, (2014) Lindovsky et al., Eur J Pharmacol 688: 22, (2012) Petrov et al., Br J Pharmacol 163: 732, (2011) Volkov et al., Comp Biochem Physiol A 158: 520, (2011)

Jan Krůšek

Kaniakova et al. Eur. J Pharmacol 658 (2011) 108-113 Kaniakova et al. Eur. J. Pharmacol 738 (2014) 352-359

Quality of the results: Structural – functional studies on AChR. Modulation of nicotinic receptors by plant alkaloid lobeline

.


Extracellular channel vestibule „THE SITE“

90°

Open conformation of the NMDA receptor channel

Extracellular vestibule

Steroids

Pregnanolone sulfate (PA-S)

Patch-clamp technique, Immunofluorescence, Mutagenesis, Molecular modeling and structural bioinformatics

Quality of the results: Structural – functional studies on NMDAR. Steroid inhibition of NMDAR channels.


Extracellular channel vestibule „THE SITE“

90°

Open conformation of the NMDA receptor channel

Mem

bra

ne

Extracellular vestibule

Steroids

Pregnanolon sulfate (PA-S)

Patch clamp technique, Immunofluorescence, Mutagenesis, Molecular modeling and structural bioinformatics

Papers: Korinek et al., Neuroscience 165: 736, (2010) Borovska et al., British journal of pharmacology 166: 1069, (2012) Vidrna et al., Steroids 76: 1043, (2011) Stastna et al., Steroids 74: 256, (2009) Kapras et al., Steroids 77: 282, (2012) Cerny et al., Steroids 77: 1233, (2012) Rambousek et al., Neuropharmacology 61: 61, (2011) Korinek et al., Steroids 76: 1409, (2011) Vales et al., Behavioral brain research 235: 82, (2012) Kudova et. al Journal of medicinal chemistry 58:5950-5966 (2015) Vyklicky et al., Scientific reports 5:10935 (2015)

Book Chapter: Vyklicky et al., Analysis of whole-cell NMDAR currents, Springer Business + Science Media Series, editor: Wolfgang Walz; Ionotropic Glutamate Receptor Technologies, editor: Gabriela K Popescu

Patents: 1. Czech Patent (UPV) No.: 303037: Pregnanolone derivatives substituted in position 3alpha, process for their

preparation and use (Chodounska et al., CZ patent No. 303037, 2012). 2. Czech Patent (UPV) No.: 303443: Pregnanolone derivatives substituted in position 3alpha with cationic

group, process of their preparation, their use and composition containing them (Chodounska et al., CZ patent No. 303443, 2012);

3. US patent No.: US8575376: Steroid anionic compounds, method of their production, usage and pharmaceutical preparation involving them (Chodounska et al., US patent No. US8575376, 2014);

4. PCT Patent Application WO/2010/136000: Steroid anionic compounds, method of their production, usage and pharmaceutical preparation involving them.

5. PCT Patent Application 2015/ P1425PC00: Amphiphilic compounds with neuroprotective properties.


Plasma membrane cholesterol is essential for NMDA receptor function. Cholesterol is a strong endogenous potentiator of NMDA receptors. The mechanism of potentiation is through modulation of the open probability of NMDA receptors. Cholesterol depletion enhances the entry of NMDA receptors into desensitized state. Neurons contain ̴30% of sleeping/partially working receptors

cholesterol depetion by methyl-β-cyclodextrin or simvastatin cholesterol enrichment by cholesterol/cyclodextrin complex

Míla Kořínek

Korinek et al. Journal of Physiology (2015) 593: 2279 Perspectives article written by Lonnie Wollmuth - "Is cholesterol good or bad for your brain? - NMDARs have a say")



RNA editing alternative splicing in GluA2

G/R site

DG

Hippocampus

GluA2 ligand binding domain

R/G

flip/flop

mRNA processing (editing and alternative splicing) of AMPAR subunits determines functional properties of receptor and also plays a role in receptor assembly.

neuronal activity regulates mRNA processing and receptor biogenesis.

Aleš Balík

Balik et al. (2013) Nucleic Acids Res 41(2):1113-23. Penn et al. (2012) Neuron 76(3):503-10.

Quality of the results: Structural – functional studies on AMPAR. Main research activities – gene regulation

.


Confocal microscopy analysis of hippocampal neurons infected with lentiviruses overexpressing wild type (rGluN1) and deglycosylated NMDAR subunits

The M3 domains of both GluN1 and GluN2 subunits contain key amino acid residues that regulate the number of surface NMDARs

A single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit (L830) regulates the surface number of NMDARs.

Three distinct regions within the N-terminus, M3 transmembrane domain, and C-terminus of GluN2C subunits are required for proper intracellular processing and surface delivery of NMDARs.

GluN1 GluN2C

Martin Horák

Quality of the results: NMDA receptor trafficking. Structural determinants of NMDARs important for receptor trafficking


Confocal microscopy analysis of hippocampal neurons infected with lentiviruses overexpressing wild type (rGluN1) and deglycosylated NMDAR subunits

The M3 domains of both GluN1 and GluN2 subunits contain key amino acid residues that regulate the number of surface NMDARs

A single amino acid residue within the fourth membrane domain (M4) of GluN1 subunit (L830) regulates the surface number of NMDARs.

Three distinct regions within the N-terminus, M3 transmembrane domain, and C-terminus of GluN2C subunits are required for proper intracellular processing and surface delivery of NMDARs.

GluN1 GluN2C

Martin Horák

Kaniakova et al., Journal of Biological Chemistry 287: 26423-34, (2012) Kaniakova et al., Journal of Neurochemistry 123: 385-95, (2012). Lichnerova et al., Front Cell Neurosci 8: 375, (2014). Lichnerova et al., Journal of Biological Chemistry 290(30):18379-90, (2015)

Quality of the results: NMDA receptor trafficking. Structural determinants of NMDARs important for receptor trafficking


Involvement of PhD students in research Quantity and quality

PhD students:

1. Mgr. Jiřina Borovská 2. Ing. Pavla Hubálková 3. Mgr. Anna Hynková 4. Mgr. Barbora Krausová 5. Mgr. Marek Ladislav 6. Mgr. Katarína Lichnerová (Slovakia) 7. Mgr. Filip Touška (Bi-nationally supervised doctoral

thesis Friedrich-Alexander-Universitat – Nurnberg and Charles University in Prague)

8. Mgr. Kristýna Skřenková

Undergraduate students:

1. Bc. Viktor Synytsya (Ukraine) 2. Denisa Fraňková 3. Šárka Danačíková 4. David Novák 5. Dominika Chudá (Slovakia)

Magister: 9 – defended Bachelor: 8 – defended PhD: 13 - defended


Postdoctoral fellows (defended PhD 2010 -1014 ) gained postdoctoral positions at:

1. Lucie Svobodová PhD: Instituto de Neurociencias Universidad Miguel Hernández-CSIC, Alicante, Spain 2. Ondřej Cais, PhD: MRC Laboratory of Molecular Biology, Cambridge Biomedical Campus, UK 3. Miloslav Sedláček, PhD: NIH/NIDCD, 31 Center Drive, MSC 2320, Bethesda, MD USA 4. Miloš Petrovic, MD, PhD: SPBS, University of Central Lancashire, Lancashire, UK 5. Jan Benedikt, PhD: Universidad Central del Caribe; Puerto Rico, USA 6. Jiří Lindovský, PhD: Institut de Neurobiologie Alfred Fessard CNRS, Gif-sur-Yvette, France 7. Martina Kaniaková, Ph.D. - Inserm Research Centre, Bordeaux, France (2015) 8. Lenka Maršáková, Ph.D. UT Health Science Center, School of Dentistry, San Antonio, TX, USA 9. Tereza Smejkalová, Ph.D. Washington University in St. Louis, MO, USA 10. Štepána Boukalová Ph.D. BIOCEV 11. Abdul Samad PhD (Pakistan) – Institute of Microbiology CAS Prague 12. Vojtěch Vyklický, Ph.D. (defended 2015) Expected to leave for postdoc 2016 13. Lucie Surá-Zímová, Ph.D. (defended 2015) Expected to leave for postdoc 2016

MODELS

In vitro cellular models preparation of primary hippocampal neurons and cerebellar granule cells neuronal micro-island cultures acute brain slices organotypic brain slices

Recombinant receptors heterologous expression of receptors in cell lines transfection of neurons virus-assisted transfection/infection

TECHNIQUES

Patch-clamp technique: with fast application of solutions and controlled temperature

PCR-based methods: Quantitative and qualitative PCR, mutagenesis and cloning

Biochemical methods: SDS/PAGE electrophoresis with Western blotting

Confocal microscopy

Immunofluorescence microscopy

Ca2+- imaging

Fluorescence Resonance Energy Transfer (FRET) Microscopy

Molecular modeling and structural bioinformatics protein/protein and protein/small molecule docking, homology modeling, molecular dynamics, quantum chemical calculations, genome/RNA expression data analysis

Involvement of PhD students in research Methods students get familiarized with


Awards Best Diploma Thesis 2013, Department of Physiology, Faculty of Science, Charles University in Prague (2013). Student: Mgr.

Katarína Lichnerová. Supervisor: Martin Horák

Best Original Research Study by a Young Investigator 2012, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Martin Horák

Otto Wichterle Award, Academy of Sciences of the Czech Republic (2011-2013). Martin Horak

Project evaluator and rapporteur, “Marie Curie Grants“ in the FP7 and Horizon 2020 programme, Brussels, Belgium M. Horak

Award for the best presentation at the Advancement report seminar at Institute of Physiology CAS; 13 November 2014 Barbora Krausová; Supervisor: Ladislav Vyklicky

Award for the best presentation at the Advancement report seminar at Institute of Physiology CAS; 13 November 2014 Anna Hynková; Supervisor: Viktorie Vlachová

Award from the vice-dean for science & research for presentation at the Scientific Conference at the Second Faculty of Medicine; 22 and 23 April 2015 Barbora Krausová Supervisor: Ladislav Vyklicky

Czech Neuroscience Society award for an excellent scientific publication published in 2012: Marsakova, Touska, Krusek, Vlachova

Grants for PhD students (Charles University Grant Agency - GAUK) 1. Mechanisms of activation and modulation of TRP receptors: structure-function relationship 25409; Provider: PřF UK; Main

contractor: Mgr. Filip Touška; Research years: 2009 – 2011

2. Structural determinants of polymodal activation of vanilloid receptors 26110; Provider: PřF UK; Main contractor: Mgr. Štěpána Boukalová; Research years: 2010 – 2011

3. Mechanisms of proton-induced inhibition of thermosensitive TRP receptors 500512; Provider: PřF UK; Main contractor: Mgr. Štěpána Boukalová; Research years: 2012 - 2013

4. Role of cholesterol in modulation of functional and pharmacological properties of NMDA receptor 800313; Provider: 2.LF UK; Main contractor: Mgr. Barbora Krausová; Research years: 2013 – 2014

5. Elucidating the role of SALMs in the regulation of synapses 688213; Provider: PřF UK; Main contractor: Mgr. Katarína Lichnerová; Research years: 2013 – 2015

Involvement of PhD students in research


Postdoctoral stays at the department :

1. Katja Witschas, Ph.D. (Dr. rer. nat.), (Germany) Postdoctoral Research Assistant – currently at: Department of Pharmacology, University of Oxford, UK

2. Tereza Smejkalová, Ph.D., (Slovakia/USA) Postdoctoral Research Assistant – currently still at the department

3. Inma Gonzales Ph.D. (Spain) Postdoctoral Research Assistant – currently at: Centre for applied Medical Research (CIMA), University of Navarra

Avda, Pamplona, Spain.

4. Miloš Petrovic, M.D., Ph.D. (Serbia) Postdoctoral Research Assistant – currently at: School of Pharmacy and Biomedical Sciences, Maudland Building;

University of Central Lancashire, Preston, Lancashire, PR1 2HE, UK

Involvement of postdocs in research


Member of the Board for Ph.D. studies in the field of Animal Physiology

Senior scientists: • Ladislav Vyklický, MD, PhD., DSc. (Undergraduate and PhD student mentor) • Viktorie Vlachová, PhD., DSc. (Undergraduate and PhD student mentor) • Jan Krůšek, Ph.D. (Undergraduate and PhD student mentor) • Martin Horák, Ph.D. (Undergraduate and PhD student mentor) • Aleš Balík, Ph.D (Undregraduate student mentor)

• Prof. František Vyskočil

Junior scientists: • Tereza Smejkalová, Ph.D., postdoc (Undergraduate student mentor)

Member of the Board for Ph.D. studies in the field of Neuroscience

Coordinator of the PhD progrm at the IP CAS

Lecturer at the Progress in Neuroscience –certified course for PhD students

Quality profile (evaluated outputs 16)

Member of the Board for Ph.D. studies in the program Biophysics, chemical and macromolecular physics.

88 % publications were those with PhD students from the DCN

Involvement of PhD students in research


Social relevance

FOUR members are University teachers (Faculty of Natural Sciences; Faculty of Medicine) There were 78 presentations - Radio and TV lectures, Public lectures, newspaper articles We participate in ONE project oriented on Development of Original Drugs (joint project of academia and business) We have filed 6 national and international patents We have prepared FOUR – Courses for undergraduate students: o Ion channels in the excitable tissue - Advanced course for PhD students „Progress in the field of

neuroscience“

o Basics of Receptor Neurophysiology – organized anually - In the frame of The Center of Biomedical Research project, supported by the Ministry of Education, Youth and Sports of the Czech Republic with support from the EU funds

o Electrical Measurements in Physiology – organized anually - 1 week theoretical and practical course of applied electronics for Master's degree students of the Faculty of Science; taught at the Department of Cellular Neurophysiology

o Molecular Basis of Nerve Excitability– organized anually - 1 week theoretical and practical course of molecular and cellular electrophysiology for Master's degree students of the Faculty of Science; taught at the Department of Cellular Neurophysiology

Seven articles were published in Czech medical journanls


Intramural cooperation (within the institute):

Dpt. Neurophysiology of Memory

Dpt. Protein Structure

Department of Cellular Neurophysiology

List of cooperation (Common projects (papers), short term visits (1-6 months), common grants):

Extramural cooperation (within the country):

Institute of Organic Chemistry and Biochemistry CAS - Prague

National Institute of Mental Health – Prague

Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague

International cooperation:

Seoul National University College of Medicine, South Korea

Ingo Greger PhD - MRC-LMB, Cambridge, UK

David Ogden PhD. - ENS Ecole Normale Supérieure - Paris

Prof. E. E. Nikolsky and prof. E. A. Bukharaeva - Kazan branch of Russian Academy of Sciences and Kazan Medical University

Steven Mennerick, PhD; Washington University in St. Louis; St. Louis, MO, USA

Christian Rosenmund, PhD; Charité Universitaetsmedizin; Berlin, Germany

Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Germany

CBMN-UMR, CNRS, University of Bordeaux, France

Declaration on international and national context


Junior scientists and Postdoctoral fellows:

•Martina Kaniaková, Ph.D. •Lenka Maršáková, Ph.D. •Vojtěch Vyklický, Ph.D. •Lucie Surá-Zímová, Ph.D. •Milos Petrovič MD, Ph.D. (Serbia/UK) •Inma Gonzales Ph.D. (Spain) •Tereza Smejkalová, Ph.D. (Slovakia/USA) •Katja Witschas, Ph.D. (Germany)

PhD students:

•Mgr. Jiřina Borovská •Mgr. Anna Hynková •Mgr. Barbora Krausová •Mgr. Marek Ladislav •Mgr. Katarína Lichnerová (Slovakia) •Mgr. Filip Touška •Mgr. Kristýna Skřenková •Abdul Samad PhD (Pakistan)

Undergraduate students

•Bc. Štěpán Chvojka •Bc. Pavel Švehla •Bc. Viktor Synytsya (Ukraine) •Denisa Fraňková •Šárka Danačíková •David Novák •Dominika Chudá (Slovakia)

Czech citizen / foreigner

Declaration on international and national context


50%

29%

25%

Senior scientists:

Ladislav Vyklický, MD, PhD., DrSc. (age 60) postdoctoral experience: 1987 - 1988 and 1990 - 1992 NICHHD/NIH, Bethesda, MD, USA1; 1995 Max Planck Institut für Zellphysiologie, Heidelberg, NIH Bethesda and Max Planck Heidelberg

Viktorie Vlachová, PhD., DrSc. (age 58) Jan Krůšek, Ph.D. (age 54) 1992 Dept. of Pharmacology, University College London (prof. S.G.Cull-

Candy). 1996 Institute of Neurobiology, University of Puerto Rico Medical Campus, San Juan (prof. R.K.Orkand)

Martin Horák, Ph.D. (age 37) 2005 – 2010 Postdoctoral fellow - National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA; (Robert J. Wenthold, Ph.D.)

Aleš Balík, Ph.D (age 38) 2008 - 2011 Postdoctoral fellow - MRC-LMB, Cambridge,UK (Ingo Greger, Ph.D.)

Jiří Černý, Ph.D. (age 37) 2006 - 2007 The University of Manchester, UK; research associate

Junior scientists:

Miloslav Kořínek, Ph.D., (age 41) -Postdoctoral fellow - Laboratory of Biomolecular Dynamics, Department of Biochemsitry, Katholieke Universiteit Leuven, Belgium

Tereza Smejkalová, Ph.D., (age 36) – 2003-2010 Ph.D. student at Northwestern University, Evanston, IL, USA (Catherine Woolley, Ph.D.); 2014-2015 Postdoctoral stay at Washington University in St. Louis, MO, USA (Steven Mennerick, Ph.D.)

Vitality and sustainability - postdoctoral experience


INTERNATIONAL GRANTS

1. SIXTH FRAMEWORK PROGRAMME - PRIORITY 1 – LifeSciHealth - LSHM-CT-2007-037765; Life sciences, genomics and biotechnology for health; PHOTOLYSIS - Development of flash photolysis for deep uncaging in vivo and high throughput characterization of neurotransmitter gated ion channels in drug discovery; L. Vyklický, (2008-2011)

2. Elucidating mechanisms that regulate early trafficking of NMDA receptors (PIMarie Curie IRG (FP7) - PIRG-GA-2010-27682; EU; M. Horák, (2011-2015)

MAJOR CZECH GRANTS „CENTERS“ (MULTILATERAL WITH SEVERAL INSTITUTES INVOLVED )

1. Project of excellence in the field of neuroscience - GBP304/12/G069; Czech Science Foundation; coordinator L. Vyklický, (2012-2018) - (four institutes, 8 laboratories in the IP CAS)

2. Neuroscience Centre - LC554; Ministry of Education, Youth and Sport; co-investigator L. Vyklický, (2005 -2011) - Institute of Experimental medicine CAS (two institutes, 8 laboratories in the IPCAS)

3. Center of Neuropsychiatric Studies (Neurobiology in Clinical Application) - 1M0517; Ministry of Education, Youth and Sports; co-investigator L. Vyklický, (2005-2011) – National Institute of Mental Health (two institutes, 8 laboratories in the IP CAS)

4. Center of Biomedical Research - CZ.1.07/2.3.00/30.0025; Ministry of Education, Youth and Sport; L. Vyklický, V. Vlachová, (2012-2015)

5. Center for Development of Original Drugs - TE01020028; Technology Agency of the Czech Republic; co-investigator L. Vyklický, (2012 – 2019) - Institute of Organic Chemistry and Biochemistry CAS

Vitality and sustainability - grants


Czech Science Foundation (CSF)

1. Study of the effects of neurosteroids and temperature on NMDA receptors. A309/07/0271; Vyklický 2007-2011.

2. Synthesis and activity evaluation of NMDA receptor antagonists. GA203/08/1498; Vyklický 2008-2010. 3. Acetylcholine release and nicotinic receptor IAA500110905; Vyskočil 2009-2013. 4. Activation and regulation mechanisms of nociceptor-specific transient receptor potential channels:

physiological role in acute and chronic pain. GA305/09/0081; Vlachová 2009-2013. 5. Cooperativity of nicotinic receptors. GA202/09/0806; Krůšek 2009-2012. 6. Role of membrane domains of NR1 and NR2 subunits in the assembly and trafficking of the NMDA receptor to

the cell surface. GAP303/11/0075; Horák 2011-2013. 7. The effect of cell membrane fluidity on the functional properties of NMDA receptors. GPP303/11/P391;

Kořínek 2011-2013. 8. Neurosteroid modulation of NMDA receptors. GAP303/12/1464; Vyklický 2012-2016. 9. Structural and functional determinants of NMDA receptor trafficking. 14-09220P; Kaniaková 2014-2016. 10. Regulation of NMDA receptor trafficking and function by N-glycosylation. 14-02219S ; Horák 2014-2016. 11. Regulatory mechanisms of nociceptive transient receptor potential ion channels. 15-15839S ; Vlachová 2015-

2017. Ministry of Health

1. Genetic and functional studies of NMDA receptors targeted on the prospective diagnosis and treatment of schizophrenia. 15-29370A Vyklický 2015 – 2020

Vitality and sustainability


Reduced NMDAR expression induced by genetically altered promoters (SNP, deletion, insertion)? PCR, sequencing, activity assay

Increased genetic variations disrupting the net of > 200 postsynaptic genes (Glutamate Receptor Complex)? whole gene sequencing, GWAS

promoter

PCR amplification

SCH association study

SNP identification

National Institute of Mental Health - Prague

The strategy and plans for the future. „We aim to determine how gene variants of the glutamate receptor complex contribute to the emergence and development of schizophrenia. „


~100 known mutated enzymes causing also neurological abnormalities in humans suffering from congenital disorders of glycosylation

Future approaches

Molecular replacement strategy (overexpression of mutated enzymes in hippocampal neurons).

Mouse models of the human congenital disorders of glycosylation

Human skin fibroblasts.

Lectin association with native glutamate receptors

Preliminary results suggest extensive glycan processing of glutamate receptors in brain tissue.

Co

ntr

ol

GSL

I

PSA

MA

L I

LC

A

PH

A-L

PH

A-E

SJA

GluN2B 180

GluN1 82

115

The strategy and plans for the future. „The wide diversity of glycan structures present in mammalian cells (>7,000 structures have been identified to date) provides an additional level of information content that can add another dimension to the mechanisms that regulate NMDA receptors“


Identify mechanisms for Ca2+ regulation in TRPA1 and TRPM3 (sensitization and inactivation)

Characterize as-yet-unrecognized putative regulatory sites in TRPV3 (sensitization and synergism)

Agonist

The strategy and plans for the future. „What is the molecular basis of multimodal regulation of nociceptive TRP channels: TRPV1, TRPV3, TRPM3, TRPA1“


75% inhibition of tonically activated receptors

Phasic activation (NMDAR EPSC) synaptic extra synaptic

The strategy and plans for the future. „Some steroids are potent inhibitors of tonically activated NMDAR with no effect at phasically activated (synaptic) receptors“


Glu PES

CTD

NTD Pregnenolone sulphate PE-S

~60 pregnenolone derivatives prepared and tested for their activity at NMDAR

The strategy and plans for the future. „What is the molecular mechanism and site of action of steroids with potentaiting action at NMDAR“


Preliminary results: NMDA EPSCs are more sensitive to cholesterol depletion than whole cell responses

Cholesterol depletion

Control Single Particle tracking (Quantum Dot) Diffusion coefficient (synaptic/extrasyn)

The strategy and plans for the future. „How does cholesterol manipulation affect glutamatergic synaptic transmission?“


SWOT


Strengths advanced techniques talented students experienced researchers with

excellent postdoctoral training - spanning from young to senior category

internationally recognized

Opportunities participation in international

projects including grant applications

use of advanced models of human diseases

Weaknesses small number of students that

return from postdoctoral training in foreign laboratories

slow process of publishing the results in prestigious international journals

Threats excessive administrative load inadequate recruitment of the

most talented students to the PhD program

… we have made a small step forward, but we have a long way ahead.

Thank you for your attention.

The presentation can be found on the Department web pages (Study section)

http://www.fgu.cas.cz/en/departments/cellular-neurophysiology

Documents

Department of Cellular Neurophysiology (DCN) · Department of Cellular Neurophysiology (DCN) Report on the scientific activity of the team in 2010–2014 Ladislav Vyklický, MD, PhD.,