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CFERV 2019 Roche_kwr
- 1. NMDAR subunit rare variants: Receptor trafficking and synaptic deficits Katherine Roche, NINDS © Katherine Roche, NINDS
- 2. Neurons actively communicate in the brain via synapses © Katherine Roche, NINDS
- 3. Neurotransmitters are released and bind to receptors © Katherine Roche, NINDS
- 4. Imaging of hippocampal neurons Eric Bloss, PhD and Nelson Spruston, PhD., HHMI, Janelia Research Campus © Katherine Roche, NINDS
- 5. Excitatory synapses on dendritic spines Ferreira, et al., J.Neurosci., 2015 © Katherine Roche, NINDS
- 6. Spacek & Harris J. Neuroscience 17, 190. © Katherine Roche, NINDS
- 7. Kim, E. and Sheng M., (2004) Excitatory Synapses © Katherine Roche, NINDS
- 8. GluN1 GluN2A GluN2B GluN2C GluN2D GluN3A GluN3B 1 2 3 NTD P Ligand glycineglutamate Ca2+ GluN2 GluN1 Mg2+ Structure of NMDA Receptors © Katherine Roche, NINDS
- 9. PDZ PDZ GK PSD-95 CortactinActindynamin clathrin adaptor clathrin coated-pit endosome Shank SH3PDZ FNGSSNGHVYEKLSSIESDV 1482 LNSCSNRRVYKKMPSIESDV 1464GluN2A GluN2B * PSD-95 (MAGUK) AP-2 clathrin adaptor complex © Katherine Roche, NINDS
- 10. GluN 2B ESDV MAGUK P CK2 P GluN 2B GluN 2A GluN2B: …G H V Y E K L S S I E S D V 14801472 © Katherine Roche, NINDS
- 11. GluN2B PDZ ligand mutants - Surface expression - GluN2B: …G H V Y E K L S S I E S D V 14801472 Sanz-Clemente et al., 2010 © Katherine Roche, NINDS
- 12. Molecular replacement of NMDARs using double-floxed GluN2A/2B mice John Gray and Roger Nicoll UCSF © Katherine Roche, NINDS
- 13. GluN 2B ESDV MAGUK P CK2 P GluN 2B GluN 2A 14801472 FNGSSNGHVYEKLSSIESDV 1482 LNSCSNRRVYKKMPSIESDV 1464GluN2A GluN2B © Katherine Roche, NINDS
- 14. How best to probe structure function relationships within the large C- terminal domains of GluN2 subunits? © Katherine Roche, NINDS
- 15. Petrovski et al., 2013 GRIN genes are intolerant to variation The GRIN genes encoding NMDARs are some of the most intolerant in the genome RVIS is a score for lack of genetic variation in the healthy population (EXaC server). There are fewer SNPs than expected for intolerant genes HGNC gene Residual Variation Intolerance Score (RVIS, Percentile) GRIA1 2.67 GRIA2 10.8 GRIA3 6.36 GRIA4 3.06 GRID1 2.05 GRID2 6.95 GRIK1 10.7 GRIK2 5.81 GRIK3 2.30 GRIK4 10.1 GRIK5 9.04 GRIN1 4.67 GRIN2A 1.17 GRIN2B 1.09 GRIN2C 81.7 GRIN2D 4.56 GRIN3A 67.5 * * * * * * * * © Katherine Roche, NINDS
- 16. QSNPCVPNNKN-----PRAFNGSSNGHVYEKLSSIESDV 1484 ...||| ||..|..||..||:|:.|||||| -----YAANKNNMYSTPRVLNSCSNRRVYKKMPSIESDV 1464 GluN2B 1451 GluN2A 1431 ********** * ********** * *** * ** 141K samples - gnomAD Intolerance analysis across functional domains GluN2A GluN2B Ogden et al, 2017 © Katherine Roche, NINDS
- 17. GluN2B S1413L shows reduced surface expression, spine density, and synaptic currents GFP + GluN2B vehicle S1413L L1422F S1450F WT ΔSTOP Liu et al., 2017 WT S1415L L1422F S1452F 0.0 0.5 1.0 1.5 Surface/IntracellularRatio ** Surface IntracellularGluN2B WT S1413L © Katherine Roche, NINDS
- 18. Cortex WT KI PSD GluN1 Actin GluN2A GluN2B PSD-95 Hippocampus WT KI PSD GluN1 Actin GluN2A GluN2B PSD-95 GluN2B S1413L knock-in mouse Marta Vieira, unpublished © Katherine Roche, NINDS
- 19. • Rare variants in GRIN2B C-terminal domain • We have generated a mouse with an ASD variant in C-terminal domain and it has synaptic deficits in the hippocampus • Analyzing other variants in the C- terminal domain Summary Part 1 © Katherine Roche, NINDS
- 20. Novel GluN2A phosphorylation site: Ser1459 Proteomic studies that identified GluN2A phospho-Ser1459 Lundby et al. 2012 Rat brain Nature Communications Trinidad et al. 2012 Adult mouse synaptic membranes Molecular & Cellular Proteomics Goswami et al. 2012 Proteomics Huttlin et al. 2011 3 w.o. mouse brain Cell Wisnlewski et al. 2010 Mouse brain Journal of Proteome Research Tweedie-Cullen et al. 2009 6-12 m.o. mouse synaptosomes Journal of Proteome Research Munton et al. 2007 2-5 m.o. mouse cortical synaptosomes Molecular & Cellular Proteomics Trinidad et al. 2006 2-16 m.o. mouse PSDs Molecular & Cellular ProteomicsClairfeuille et al. (2016) Nat Struct Mol Biol, 23(10):921-32 © Katherine Roche, NINDS
- 21. CaMKIIa phosphorylates GluN2A S1459 HEK293T co-expressing GluN1, GluN2A and CaMKIIa Adult WT mouse brain GluN2A -Tubulin GluN2A- pS1459 kDa 250 150 250 150 50 GST CaMKIIa S1459A GluN2A W T GST 32 P GluA1 50 37 25 kDa 50 37 GST- GluN2A S1459A GluN2A W T GST GluA1 GFP- GluN2A CaMKIIa b-Tubulin GluN2A- pS1459 S1459A +CaM KIIa NT GluN2A W T +CaM KIIa GluN2A W T +CaM KIIa K42R kDa 250 150 250 150 50 50 GFP- GluN2A CaMKIIa b-Tubulin GluN2A- pS1459 S1459A +CaM KIIa NT GluN2A W T +CaM KIIa GluN2A W T +CaM KIIa K42R kDa 250 150 250 150 50 50 GluN2A C-terminus phosphorylated by CaMKII © Katherine Roche, NINDS
- 22. GluN2A-S1459 phosphorylation determines SNX27 vs. PSD-95 binding GFP-GluN2A Myc-PSD-95 Inputs2% GFP-GluN2A Myc-PSD-95 IPGFPGFP-GluN2A Myc-SNX27 Inputs2% GFP-GluN2A Myc-SNX27 IPGFP © Katherine Roche, NINDS
- 23. GluN2A S1459 phosphorylation modulates receptor trafficking S1459D – Phospho-mimetic S1459A – Phospho-dead G luN 2A W T S1459D S1459A 0 5 10 15 spinenumber/20mm ** **** WT S1459G S1459D S1459A 0 5 10 15 spinenumber/20µm n=4 * GluN2A WT S1459G S1459D S1459A WT S1459G S1459D S1459A 0 5 10 15 spinenumber/20µm n=4 * GluN2A WT S1459G S1459D S1459A WT S1459G S1459D S1459A 0 5 10 15 n=4 * GluN2A WT S1459G S1459D S1459A © Katherine Roche, NINDS
- 24. • S1459 is a novel phosphorylation site in GluN2A, mediated by CaMKIIa • Phosphorylation of GluN2A-S1459 modulates the binding to SNX27 and PSD-95 • Phosphorylation affects subunit trafficking and spine density What can we learn from human genetics? © Katherine Roche, NINDS
- 25. Tool Score Prediction Poly-Phen 0.992 Probably damaging MutationTaster 0.9999994002 Disease causing subRVIS -2.0798/1.6141% Highly intolerant Intellectual disability (moderate); Facial dysmorphism; Speech delay; Relative microcephaly; MRI Brain: increased signal white matter at level of external capsules bilaterally and periphery of centrum semiovale Identified using WES GluN2A S1459G rare variant Not found in the healthy population (gnomAD database – 150K samples) Bowling et al. (2017) Genome Med, 9(1):43 Karczewski et al. (2019) BioRXiv © Katherine Roche, NINDS
- 26. The GluN2A S1459G variant impairs GluN2A interactions GST-GluN2A S1459G – Epilepsy variant GFP-GluN2A GFP-GluN2A Myc-PSD-95 Myc-PSD-95 IPGFPInputs2% GFP-GluN2A GFP-GluN2A Myc-SNX27 Myc-SNX27 IPGFPInputs2% GST-GluN2A GFP-GluN2A GFP-GluN2A Myc-SNX27 Myc-SNX27 IPGFPInputs2% GFP-GluN2A GFP-GluN2A Myc-PSD-95 Myc-PSD-95 IPGFPInputs2% © Katherine Roche, NINDS
- 27. S1459G impairs GluN2A surface expression and spine density Merge Spine density G luN2A W T S1459G 0.0 2.5 5.0 7.5 10.0 Spinenumber/20mm * WT S1459G S1459D S1459A 0 5 10 15 spinenumber/20µm n=4 * GluN2A WT S1459G S1459D S1459A © Katherine Roche, NINDS
- 28. • The epilepsy variant S1459G affects GluN2A protein interactions and trafficking • GluN2A-S1459G dysfunction offers insight into disease mechanism In summary… The epilepsy variant GluN2A S1459G affects GluN2A protein interactions, protein trafficking, and spine density © Katherine Roche, NINDS
- 29. Summary •The C-termini of GluN2A and GluN2B are important for receptor trafficking and synaptic targeting. •Rare variants in the C-terminus can be analyzed by using imaging and biochemistry to evaluate synaptic localization and spine density. •GluN2B variants in the extreme C-terminus are very rare. •Mouse models can be made by making knock-in mice with a particular mutation. © Katherine Roche, NINDS
- 30. John Badger Kai Chang Jaehoon Jeong Richa Lomash Elena Bagatelas Thien Nguyen Jeremiah Paskus Marta Vieira Sehoon Won Erin Fingleton NINDS Wei Lu Liang Zhou Gene Dx Shuxi Liu Emory Steve Traynelis Hongjie Yuan Roche Lab © Katherine Roche, NINDS