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CFERV 2019 Roche_kwr

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Synaptic dysfunction is implicated in a variety of neurological disorders. De novo mutations in certain synaptic proteins are pathogenic. In particular, rare variants of two NMDA receptor
subunits, GluN2A and GluN2B, underlie some cases of epilepsy and neurodevelopmental disorders.
For many years, my laboratory has worked on mechanisms regulating NMDA receptor trafficking to excitatory synapses and how synaptic localization is dynamically regulated during development and in response to experience. In particular, we have studied how the long C-terminal domains of GluN2A and GluN2B regulate trafficking through protein-protein interactions and posttranslational modifications such as phosphorylation. In particular we have evaluated a missense mutation within
the C-terminal domain of GluN2B identified in a patient with ASD and described deficits in receptor surface expression and dendritic spine density. We are moving forward to make a mouse model of this mutation. More recently we are studying a missense mutation located within the C-domain of GluN2A identified in a patient with epilepsy and intellectual disability. Thus residue is a phosphorylation site and affects receptor trafficking. Our studies validate a ‘bedside to bench’
approach of studying rare variants associated with disease to better understand the function and signaling properties of NMDA receptors. We anticipate our results will allow us to better understand the structure, function and signaling regulated by the C-termini. Importantly, by characterizing these variants, we hope to gain insight into the pathogenesis of various neurodevelopmental disorders.

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CFERV 2019 Roche_kwr

  1. 1. NMDAR subunit rare variants: Receptor trafficking and synaptic deficits Katherine Roche, NINDS © Katherine Roche, NINDS
  2. 2. Neurons actively communicate in the brain via synapses © Katherine Roche, NINDS
  3. 3. Neurotransmitters are released and bind to receptors © Katherine Roche, NINDS
  4. 4. Imaging of hippocampal neurons Eric Bloss, PhD and Nelson Spruston, PhD., HHMI, Janelia Research Campus © Katherine Roche, NINDS
  5. 5. Excitatory synapses on dendritic spines Ferreira, et al., J.Neurosci., 2015 © Katherine Roche, NINDS
  6. 6. Spacek & Harris J. Neuroscience 17, 190. © Katherine Roche, NINDS
  7. 7. Kim, E. and Sheng M., (2004) Excitatory Synapses © Katherine Roche, NINDS
  8. 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. 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. 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. 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. 12. Molecular replacement of NMDARs using double-floxed GluN2A/2B mice John Gray and Roger Nicoll UCSF © Katherine Roche, NINDS
  13. 13. GluN 2B ESDV MAGUK P CK2 P GluN 2B GluN 2A 14801472 FNGSSNGHVYEKLSSIESDV 1482 LNSCSNRRVYKKMPSIESDV 1464GluN2A GluN2B © Katherine Roche, NINDS
  14. 14. How best to probe structure function relationships within the large C- terminal domains of GluN2 subunits? © Katherine Roche, NINDS
  15. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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