Name: a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate
Uploaded: 2018-07-10T15:00:00
Description: Watch this Scientific Journal Video about A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate at JoVE.com

Forfatterne vil gerne takke studentereksamen lærde Program posthus og Novartis institutter for biomedicinsk forskning som helhed til finansiering af denne undersøgelse.

1. forberedelse af celler
Bemærk: Denne protokol, herunder data generation, bruger HEK293 celler transduced med en baculovirus kodning NR1 og NR2A celler.
<ol><li>Frø bør antallet af HEK293 celler og tilføje virussen NR1 og/eller NR2A på de relevante endelige koncentrationer (1,00 µL). For en 384-godt plade, bruge 10.000 celler/brønd i en endelige mængden af 30 µL.</li>
	<li>Alternativt, frø HEK293-NR1-NR2A celler på den relevante celle nummer og volumen (for en 384-godt plade, br...

<ol>
	<li>Farrall, A. J., Wardlaw, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier:+ageing+and+microvascular+disease--systematic+review+and+meta-analysis.">Blood-brain barrier: ageing and microvascular disease--systematic review and meta-analysis.</a> Neurobiology of Aging. 30 (3), 337-352 (2009).</li><li>Walhovd, K. B., Fjell, A. M., Espeseth, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+decline+and+brain+pathology+in+aging--need+for+a+dimensional,+lifespan+and+systems+vulnerability+view.">Cognitive decline and brain pathology in aging--need for a dimensional, lifespan and systems vulnerability view.</a> Scandinavian Journal of Psychology. 55 (3), 244-254 (2014).</li><li>Wittchen, H. 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A., Roche, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+in+NMDA+receptor+composition:+many+regulators,+many+consequences.">Diversity in NMDA receptor composition: many regulators, many consequences.</a> Neuroscientist. 19 (1), 62-75 (2013).</li><li>Neyton, J., Paoletti, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relating+NMDA+receptor+function+to+receptor+subunit+composition:+limitations+of+the+pharmacological+approach.">Relating NMDA receptor function to receptor subunit composition: limitations of the pharmacological approach.</a> Journal of Neuroscience. 26 (5), 1331-1333 (2006).</li><li>Hunt, D. L., Castillo, P. 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L., Westbrook, G. L., Guthrie, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage-dependent+block+by+Mg2++of+NMDA+responses+in+spinal+cord+neurones.">Voltage-dependent block by Mg2+ of NMDA responses in spinal cord neurones.</a> Nature. 309 (5965), 261-263 (1984).</li><li>Zhu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+NMDA+Receptor+Inhibition+and+Activation.">Mechanism of NMDA Receptor Inhibition and Activation.</a> Cell. 165 (3), 704-714 (2016).</li><li>Yildiz-Unal, A., Korulu, S., Karabay, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+strategies+against+calpain-mediated+neurodegeneration.">Neuroprotective strategies against calpain-mediated neurodegeneration.</a> Neuropsychiatric Disease and Treatment. 11, 297-310 (2015).</li><li>Gascon, S., Sobrado, M., Roda, J. M., Rodriguez-Pena, A., Diaz-Guerra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitotoxicity+and+focal+cerebral+ischemia+induce+truncation+of+the+NR2A+and+NR2B+subunits+of+the+NMDA+receptor+and+cleavage+of+the+scaffolding+protein+PSD-95.">Excitotoxicity and focal cerebral ischemia induce truncation of the NR2A and NR2B subunits of the NMDA receptor and cleavage of the scaffolding protein PSD-95.</a> Molecular Psychiatry. 13 (1), 99-114 (2008).</li><li>Uttara, B., Singh, A. V., Zamboni, P., Mahajan, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+and+neurodegenerative+diseases:+a+review+of+upstream+and+downstream+antioxidant+therapeutic+options.">Oxidative stress and neurodegenerative diseases: a review of upstream and downstream antioxidant therapeutic options.</a> Current Neuropharmacology. 7 (1), 65-74 (2009).</li><li>Hansen, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementation+of+a+fluorescence-based+screening+assay+identifies+histamine+H3+receptor+antagonists+clobenpropit+and+iodophenpropit+as+subunit-selective+N-methyl-D-aspartate+receptor+antagonists.">Implementation of a fluorescence-based screening assay identifies histamine H3 receptor antagonists clobenpropit and iodophenpropit as subunit-selective N-methyl-D-aspartate receptor antagonists.</a> Journal of Pharmacology and Experimental Therapeutics. 333 (3), 650-662 (2010).</li><li>Bettini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+novel+NMDA+receptor+antagonists+selective+for+NR2A-+over+NR2B-containing+receptors.">Identification and characterization of novel NMDA receptor antagonists selective for NR2A- over NR2B-containing receptors.</a> Journal of Pharmacology and Experimental Therapeutics. 335 (3), 636-644 (2010).</li><li>Feuerbach, D., Loetscher, E., Neurdin, S., Koller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pharmacology+of+the+human+NMDA-receptor+subtypes+R1-2A,+R1-2B,+R1-2C+and+R1-2D+using+an+inducible+expression+system.">Comparative pharmacology of the human NMDA-receptor subtypes R1-2A, R1-2B, R1-2C and R1-2D using an inducible expression system.</a> European Journal of Pharmacology. 637 (1-3), 46-54 (2010).</li><li>Hansen, K. B., Brauner-Osborne, H., Egebjerg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+characterization+of+ligands+at+recombinant+NMDA+receptor+subtypes+by+electrophysiological+recordings+and+intracellular+calcium+measurements.">Pharmacological characterization of ligands at recombinant NMDA receptor subtypes by electrophysiological recordings and intracellular calcium measurements.</a> Combinatorial Chemistry and High Throughput Screening. 11 (4), 304-315 (2008).</li><li>Guo, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+NMDA-receptor+calcium+influx+assay+sensitive+to+stimulation+by+glutamate+and+glycine/D-serine.">A NMDA-receptor calcium influx assay sensitive to stimulation by glutamate and glycine/D-serine.</a> Scientific Reports. 7 (1), 11608 (2017).</li><li>Hackos, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+Allosteric+Modulators+of+GluN2A-Containing+NMDARs+with+Distinct+Modes+of+Action+and+Impacts+on+Circuit+Function.">Positive Allosteric Modulators of GluN2A-Containing NMDARs with Distinct Modes of Action and Impacts on Circuit Function.</a> Neuron. 89 (5), 983-999 (2016).</li><li>Romero-Hernandez, A., Furukawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Mode+of+Antagonist+Binding+in+NMDA+Receptors+Revealed+by+the+Crystal+Structure+of+the+GluN1-GluN2A+Ligand-Binding+Domain+Complexed+to+NVP-AAM077.">Novel Mode of Antagonist Binding in NMDA Receptors Revealed by the Crystal Structure of the GluN1-GluN2A Ligand-Binding Domain Complexed to NVP-AAM077.</a> Molecular Pharmacology. 92 (1), 22-29 (2017).</li><li>Auberson, Y. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Phosphonomethylquinoxalinediones+as+competitive+NMDA+receptor+antagonists+with+a+preference+for+the+human+1A/2A,+rather+than+1A/2B+receptor+composition.">5-Phosphonomethylquinoxalinediones as competitive NMDA receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition.</a> Bioorganic and Medicinal Chemistry Letters. 12 (7), 1099-1102 (2002).</li><li>Danysz, W., Parsons, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycine+and+N-methyl-D-aspartate+receptors:+physiological+significance+and+possible+therapeutic+applications.">Glycine and N-methyl-D-aspartate receptors: physiological significance and possible therapeutic applications.</a> Pharmacological Reviews. 50 (4), 597-664 (1998).</li><li>Liu, M. 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Dette assay succes afhænger i høj grad sundhed HEK celler anvendes. Celler gennemgår eksponentiel vækst og med lav passage nummer bør anvendes. Denne analyse indebærer mange overførsler og tilføjelser af løsninger, så ved hjælp af forsigtighed vil sikre højere præcision i sine resultater. Koncentrationer af forbindelser og alle andre reagenser skal krydscheckes også at minimere fejl. Når du udskifter celle medier med analysebuffer til calcium flux assay, skal pladen vippes forsigtigt så at cellerne ikke f...

Med de nuværende fremskridt i medicin, er levealder steget betydeligt; men så har forekomsten af aldersbetingede sygdomme. Sygdomme i centralnervesystemet (CNS) som skizofreni, amyotrofisk lateral sklerose (ALS), Alzheimers sygdom og Parkinsons sygdom, blandt andre, er ingen undtagelse og har blevet fremskrevet til at stige i det næste årti1, 2 , 3. fejlfunktion af ionotropic glutamat receptorer kendt som N-methyl-D-aspartat receptorer (NMDAR) har været forbundet med Alzheimers sygdom, skizofreni, traumatisk hjerneskade, slagtilfælde, diabetes og glaukom blandt andre, som garanterer de nødt til at undersøge deres biologi for udviklingen af effektive, Sygdomsmodificerende behandlinger4,5,6,7.
NMDARs er sammensat af fire monomerer eller underenheder4,8,9. Den strukturelle sammensætning af NMDAR viser udviklingsmæssige og regional variation inden for hjernen7,10. NMDARs er involveret i synaptisk plasticitet, kognition og generation af rytmer for vejrtrækning og bevægelse11,12,13. Som en spænding-gated kanal, det er stort set ikke-ledende på hvile membran potentiale (-70 mV) og blokeres af magnesium til at forhindre yderligere gennemtrængning af ioner. Kanalen er aktiveret ved at binde to ligander, glutamat og glycin/D-Serin, og en samtidig depolarisering på synaptic membranen medieret af AMPA-receptorer, et andet underklasse af ionotropic glutamat receptorer. Depolarisering fjerner magnesium blokering af NMDAR, at tilstrømningen af kationer, især calcium14,15,16. Selvom aktivering af NMDAR er afgørende for celle overlevelse, kan overdreven aktivering føre til celle død17,18,19 gennem excitotoxicity. Dette, ud over den komplekse karakter af receptoren, gør det udfordrende for at udføre undersøgelser nødvendige for at udvikle effektive behandlinger.
Forskellige metoder er blevet udviklet for at studere NMDAR. Hver enkelt har dog ledsager forbehold. For eksempel, er en meget udbredt teknik et fluorescens-baseret analyse, der måler NMDAR-medieret ændringer i intracellulære calcium i en stabil cellelinie under kontrol af en tetracyklin-inducerbar promotor (Tet-On)20. Men i dette system, supramaximal koncentrationer af ligander, der er nødvendige, og kravet om at NMDAR hæmmere er til stede under målingen gør det næsten umuligt at opdage aktivitet af konkurrencedygtige antagonist. I andre tilsvarende systemer forårsager udtryk for den funktionelle receptor toksicitet, der kræver kanal blokkere såsom ketamin21,22 at bevare cellekulturer. Disse kanal blokkere sidde kernen i receptoren og er svære at vaske ud, især i en plade-baseret format, så de forstyrrer funktionelle studier af receptor. I elektrofysiologiske målinger såsom patch fastspænding, der er begrænset overførselshastighed og store undersøgelser er meget dyre23. Trods er de ovennævnte systemer ufølsom over for glycin stimulation; dermed, at studere glycin-afhængig aktivitet af NMDAR bliver en udfordring.
Her, beskrive vi en ny tilgang til at studere NMDAR, der overvinder de omtalte begrænsninger. Vores teknik udnytter baculovirus ekspressionssystem udtrykke receptor på funktionelle niveauer med et optimalt forhold for underenheder i så lidt som 16 timer. Desuden brug af baculovirus giver mulighed for en simpel og kombinatorisk tilgang, som giver en bred karakterisering af de særskilte rekombinante NMDAR undertyper. I modsætning til andre assays kræver denne protokol ikke kanal blokkere på grund af brugen af svage antagonister. Metodens stærkeste fordel er, at efter udvaskning af de svage antagonist, receptoren er følsomme over for graduering af de enkelte glycin - og glutamat-bindingssteder ud over dobbelt graduering af både glycin/D-Serin og glutamat ligand-bindende websteder. Analysen indeholder kendte Farmakologi af NMDAR receptoren og virkningerne af dens kendte positive og negative modulatorer. Endelig, generation af denne i vitro cellulære assay overvinder den cellulære toksicitet forårsaget af overdreven calcium tilstrømning og giver mulighed for funktionelle studier af receptor i en høj overførselshastighed mode, som kan fremskynde opdagelser af NMDAR modulatorer i sygdomstilstande.

<table>
	<tbody>
		<tr>
			<td>HEK-293</td>
			<td>ATCC</td>
			<td>CRL-1573</td>
			<td></td>
		</tr>
		<tr>
			<td>Human NMDA (NR1/NR2A) Receptor Cell Line</td>
			<td>ChanTest Corporation</td>
			<td>CT6120</td>
			<td></td>
		</tr>
		<tr>
			<td>pFastBac Dual Expression Vector</td>
			<td>ThermoFisher Scientific</td>
			<td>10712-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 384-well Clear Flat Bottom Microplate</td>
			<td>Corning Life Sciences</td>
			<td>3844</td>
			<td></td>
		</tr>
		<tr>
			<td>FLIPR Calcium 6-QF Assay Kit</td>
			<td>Molecular Devices</td>
			<td>R8192</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>49621</td>
			<td></td>
		</tr>
		<tr>
			<td>D-serine</td>
			<td>Sigma-Aldrich</td>
			<td>S4250</td>
			<td></td>
		</tr>
		<tr>
			<td>L701,324</td>
			<td>Tocris</td>
			<td>907</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Boston Bio Product</td>
			<td>BB-103</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride Solution</td>
			<td>Sigma-Aldrich</td>
			<td>63069</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>E506</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher Scientific</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Probenecid</td>
			<td>ThermoFisher Scientific</td>
			<td>P36400</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, GlutaMAX media</td>
			<td>ThermoFisher Scientific</td>
			<td>10565018</td>
			<td></td>
		</tr>
		<tr>
			<td>MDL105,519</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>NVP-AAM077</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
		<tr>
			<td>CGP070667</td>
			<td>NIBR</td>
			<td>Synthesized in house</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput calcium-flux assay to study nmda-receptors with sensitivity to glycine/d-serine and glutamate

N-methyl-D-aspartat (NMDA) receptorer (NMDAR) er klassificeret som ionotropic glutamat receptorer og spiller en kritisk rolle i indlæring og hukommelse. NMDAR fejl, udtrykt som enten over - eller under - activity forårsaget af mutationer, ændrede udtrykket, menneskehandel eller lokalisering, kan bidrage til mange sygdomme, især i det centrale nervesystem. Derfor er forstå den receptor biologi samt lette opdagelsen af forbindelser og små molekyler afgørende i igangværende bestræbelser på at bekæmpe neurologiske sygdomme. Nuværende metoder til at studere receptoren har begrænsninger herunder lav overførselshastighed, høje omkostninger og manglende evne til at studere dets funktionelle evner på grund af den nødvendige tilstedeværelse af kanal blokkere at forhindre NMDAR-medieret excitotoxicity. Derudover de eksisterende assay systemer er følsomme over for stimulation af glutamat kun og manglende følsomhed over for stimulation af glycin, de andre Co ligand på NMDAR. Her præsenterer vi den første plade-baseret analyse med høj overførselshastighed magt til at studere en NMDA-receptor med følsomhed over for både Co ligander, glutamat og D-Serin/glycin. Denne tilgang giver mulighed for undersøgelse af forskellige NMDAR subunit kompositioner og tillader funktionelle studier af receptor i glycin - og/eller glutamat-følsomme tilstande. Metoden kræver desuden ikke tilstedeværelsen af hæmmere under målingerne. Effekten af positive og negative allosteriske modulatorer kan registreres med denne analyse og den kendte Farmakologi af NMDAR er blevet replikeret i vores system. Denne teknik overvinder begrænsningerne af eksisterende metoder og er omkostningseffektivt. Vi mener, at denne nye teknik vil fremskynde opdagelsen af terapier for NMDAR-medieret patologier.

Før testning virkningerne af små molekyler, fastlægger en optimal udtryk niveauer af NMDARs samt optimal ligand koncentrationer. Som beskrevet, HEK293 celler var seedet på 10.000 celler pr. brønd i en 384-godt plade, i overværelse af 5 µM transduced CGP060667, derefter med varierende mængder af NR1 og NR2A virus. Efter inkubering natten, ligand-induceret calcium flux blev målt (figur 1). Resultaterne af disse eksperimenter afslører bedste mængden o...

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A High-throughput Calcium-flux Assay to Study NMDA-receptors with Sensitivity to Glycine/D-serine and Glutamate

En høj overførselshastighed Calcium-flux Assay at studere NMDA-receptorer med følsomhed over for glycin/D-Serin og glutamat

Målet med denne protokol er at lette undersøgelsen af NMDA-receptorer (NMDAR) på en større skala og giver mulighed for undersøgelse af modulerende virkningerne af små molekyler og deres terapeutiske anvendelsesmuligheder.

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are classified as ionotropic glutamate receptors and have critical roles in learning and memory. NMDAR ...

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