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CFERV 2019 Yuan

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N-methyl-D-aspartate receptors (NMDARs) mediate a slow component of excitatory synaptic transmission that plays important roles in normal brain function and development. A large number of disease-associated variants in the GRIN gene family encoding NMDAR GluN subunits have been identified in patients with various neurological and neuropsychiatric disorders. Many of these variants reduce the function of NMDARs by a range of different mechanisms, including
reduced glutamate potency, reduced glycine potency, accelerated deactivation time course, reduced current response amplitude, decreased surface expression, and/or reduced open probability. This talk will focus on three positive allosteric modulators of NMDAR receptor function (24(S)-hydroxycholesterol, pregnenolone sulfate, tobramycin) and two co-agonists (D-serine and D-cycloserine) on their ability to mitigate the diminished function of NMDARs harboring GRIN variants. We examined the effects of these modulators on NMDARs that contained 24 different loss-of-function missense variants in GRIN1, GRIN2A, or GRIN2B. The clinical phenotypes of the patients hosting these variants include epilepsy/seizures and/or developmental delay/intellectual disability. Some patients have autistic behavior or show symptoms of ADHD. For all variants, some aspect of the
reduced function was partially restored. Moreover, some variants showed enhanced sensitivity to positive allosteric modulators compared to wild type receptors. These results raise the possibility that enhancement of NMDAR function by positive allosteric modulators may be a useful therapeutic strategy.

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CFERV 2019 Yuan

  1. 1. Neurosteroid Rescue of Functional Deficits in GRIN Loss-of-function Variants CFERV GRIN_2019_ATLANTA Hongjie Yuan Department of Pharmacology and Chemical Biology Center for Functional Evaluation of Rare Variants Emory University School of Medicine © Hongjie Yuan © Hongjie Yuan
  2. 2. To test whether positive allosteric modulators (neurosteroids and aminoglycosides) and co- agonists can partially rescue functional deficits for a range of GRIN LoF variants CFERV GRIN_2019_ATLANTA © Hongjie Yuan © Hongjie Yuan
  3. 3. Estimation of Overall Impact of Variants on NMDAR Function Amplitude is controlled by: Agonist EC50 Surface expression Open probability Mg2+ sensitivity Endogenous modulators Time course controlled by: Agonist unbinding rate Deactivation time course Desensitization time course Extent of desensitization Channel open time Synaptic-like activation ▼ Variant 1 mM glutamate for < 5 ms WT Amplitude and time course define the integral of the synaptic current (or charge transfer) Non-synaptic activation Amplitude and potency (EC50) can be used to calculate non-synaptic current at steady-state Steady-state current activated by 100 nM glutamate A B → 67% variants in agonist-binding domain (ABD) and 38% variants in transmembrane domain (TMD) reduce NMDAR-mediated current, defined as a loss-of-function (LoF) → variants reduce NMDAR function by ① decreased agonist potency, ② decreased channel opening, ③ decreased receptor expression, ④ enhanced proton/zinc inhibition, and/or ⑤ accelerated synaptic-like response time course CFERV GRIN_2019_ATLANTA XiangWei et al., 2018 © Hongjie Yuan © Hongjie Yuan
  4. 4. # Gene Genotype Protein Location Phenotype 1 GRIN1 c.1595C>A p.P532H ABD ID, myoclonus 2 GRIN1 c.1656C>A, or >G p.D552E S1-M1 link Epi, DD, ID 3 GRIN1 c.1858G>A, or >C p.G620R M2 ID 4 GRIN1 c.1984G>A p.E662K M3-S2 link ID 5 GRIN1 c.2443G>A, >C p.G815R M4 Epi, DD, ID 6 GRIN2A c.1306T>C p.C436R ABD Epi 7 GRIN2A c.1447G>A p.G483R ABD Epi, DD, ID 8 GRIN2A c.1580C>G p.P527R ABD Epi, language problems 9 GRIN2A c.1642G>C p.A548T pre-M1 Epi, ID 10 GRIN2A c.1928C>A p.A643D M3 ID, movement disorders 11 GRIN2A c.2054T>G p.V685G ABD Epi, DD, ID 12 GRIN2A c.2095C>T p.P699S ABD Epi 13 GRIN2A c.2113A>G p.M705V ABD Epi, DD 14 GRIN2A c.2146G>A p.A716T ABD Epi 15 GRIN2A c.2179G>A p.A727T ABD Epi, DD, ID 16 GRIN2A c.2191G>A p.D731N ABD Epi, DD, ID 17 GRIN2A c.2200G>C p.V734L ABD Epi, DD 18 GRIN2A c.2279G>T p.G760V ABD Epi 19 GRIN2A c.2314A>G p.K772E ABD Epi 20 GRIN2B c.1238A>G p.E413G ABD DD, ID, hypotonia 21 GRIN2B c.1306T>C p.C436R ABD Epi, ID 22 GRIN2B c.1367G>A p.C456Y ABD Autism Spectrum Disorder, ID 23 GRIN2B c.1382G>T p.C461F ABD Epi, ID 24 GRIN2B c.1623C>G p.S541R S1-M1 link Epi, ID, movement disorders TMD ABD ATD GluN1/GluN2 tetramer GluN2GluN1 Summary of Loss-of-Function GRIN1, GRIN2A, GRIN2B Variants DD: developmental delay, Epi: epilepsy/seizures, ID: intellectual disability© Hongjie Yuan
  5. 5. 0.1 1 10 100 1000 0 20 40 60 80 100 Loss-of-Function GRIN1-p.P532H Variant GRIN1-p.P532H → 20-yo female → profound developmental encephalopathy → no visual tracking, non-verbal, G-tube → striking stimulus-sensitive myoclonic jerks → marked brachycephaly and midface hypoplasia → MRI at 2-yo showed enlarged extra space, thinned corpus callosum Dr. Bozarth, Seattle Children’s Proline Histidine GluN1/GluN2B tetramer c.1595C>A (p.Pro532His) TMD ABD ATD P532H reduces glutamate potency 0.01 0.1 1 10 100 0 20 40 60 80 100 Glycine, mM 1-P532H/2A WT GluN1/2A MaximalResponse,% A Glutamate, mM 1-P532H/2A WT GluN1/2A 0.1 1 10 100 1000 0 20 40 60 80 100 Glutamate, mM 1-P532H/2B WT GluN1/2B MaximalResponse,% B GluN2GluN1 EC50: 1.7 mM Zhang, Traynelis, Yuan, unpublishedCFERV GRIN_2019_ATLANTA© Hongjie Yuan
  6. 6. 500 ms ▼ glutamate 1-P532H/2B tW: 28 ms WT GluN1/2B tW: 700 ms 0 30 60 90 120 * Receptor Expression(%WT) co-expressed with 2A surface/total total WT 100% Loss-of-Function GRIN1-p.P532H Variant Proline Histidine GluN1/GluN2B tetramer c.1595C>A (p.Pro532His) TMD ABD ATD GluN2GluN1 P532H reduces channel open probability, cell surface trafficking, and shortens synaptic-like response time course 0.0 0.1 0.2 0.3 * CalculatedPOPEN C D E 26% vs 11% Zhang, Traynelis, Yuan, unpublished 1-P532H CFERV GRIN_2019_ATLANTA GRIN1-p.P532H → 20-yo female → profound developmental encephalopathy → no visual tracking, non-verbal, G-tube → striking stimulus-sensitive myoclonic jerks → marked brachycephaly and midface hypoplasia → MRI at 2-yo showed enlarged extra space, thinned corpus callosum Dr. Bozarth, Seattle Children’s © Hongjie Yuan
  7. 7. 1E-9 1E-8 1E-7 1E-6 0 20 40 60 80 100 Loss-of-Function GRIN2A-p.D731N Variant GRIN2A-p.D731N → three patients, onset at 2,4,5 yo, atypical Rolandic epilepsy, partial seizures, generalized tonic-clonic seizures → refractory to valproate and oxcarbazepine, levetiracetam and clonazepam partially controlled the seizures → global developmental delay, intellectual, motor and cognitive regression, psychomotor and language problem (verbal dyspraxia) Lesca 2013, Dyment 2015, Gao 2017 D731N reduces agonist potency and enhances endogenous proton and zinc inhibition GluN2GluN1 GluN1/GluN2A tetramer Ligand (glutamate) 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 10 7 0 20 40 60 80 100 0.1 1 10 100 1000 20 40 60 80 100 Glutamate, mM WT 2A 2A-D731N MaximalResponse,% A H+, M WT 2A 2A-D731N WT 2A 2A-D731N Zinc, nM MaximalResponse,% B MaximalResponse,% Cphysiological pH Swanger et al., 2016; Gao et al., 2017CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  8. 8. 0.0 0.1 0.2 CalculatedPOPEN * 0 25 50 75 100 * WT 100% Loss-of-Function GRIN2A-p.D731N Variant GRIN2A-p.D731N → three patients, onset at 2,4,5 yo, atypical Rolandic epilepsy, partial seizures, generalized tonic-clonic seizures → refractory to valproate and oxcarbazepine, levetiracetam and clonazepam partially controlled the seizures → global developmental delay, intellectual, motor and cognitive regression, psychomotor and language problem (verbal dyspraxia) Lesca 2013, Dyment 2015, Gao 2017 D731N reduces channel opening, cell surface trafficking, and shortens synaptic-like response time course GluN2GluN1 GluN1/GluN2A tetramer Ligand (glutamate) ▼ glutamate ▼ glutamate D731N WT 2A Swanger et al., 2016; Gao et al., 2017 800 pA 100 ms 100 ms * Receptor Expression(%WT) D E F 22% vs 4% 2A-D731N CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  9. 9. ▼ glutamate 500 ms ▼ glutamate 150 pA 500 ms WT 2B E413G Loss-of-Function GRIN2B-p.E413G Variant GRIN2B-p.E413G → an 8-year old girl → profound developmental delay → rolled by 6 months → sat by 19 months → walked by 4 years → spoke only a single word at 5 years → brain MRI: NO any abnormalities Adam et al., 2014 GluN1 GluN2B GluN1 GluN2B E413 ATD ABD TMD Adam et al., 2014; Swanger et al., 2016; Wells et al., 2018 Ligand (glutamate) E413G reduces agonist potency, shortens synaptic-like response time course, and reduces receptor trafficking in cultured hippocampal neurons 0.1 1 10 100 1000 0 20 40 60 80 100 Glutamate, mM MaximalResponse,% WT 2B E413G A B C CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  10. 10. Loss-of-Function GRIN2B-p.E413G Variant Swanger et al., 2016; Wells et al., 2018 E413G increases ligand mobility and water access, promoting unbinding and ABD opening C A B WT 2B E413G water glutamate hydrogen bonding E413G WT E413G CFERV GRIN_2019_ATLANTA Glutamate partially detached from binding pocket in E413G by breaking many of the interactions © Hongjie Yuan
  11. 11. Positive Allosteric Modulators, PAMs, for NMDARs GluN1 (grey) – GluN2B (blue) PYD-106 Spermine Aminoglycosides GNE-6901 GNE-0723 CIQ 1180-55 Pregnenolone Sulfate 24(S)-hydroxycholesterol SGE-201, SGE-301 GNE-9278 Extracellular Intercellular ABD TMD ATD Modified from Dr. Riley Perszyk’s PhD Dissertation ● 24(S)-hydroxycholesterol, 24(S)-HC ● Pregnenolone sulfate, PS ● Tobramycin ● D-serine and D-cycloserine → the major cholesterol metabolite in brain → Alzheimer’s and Parkinson’s, exhibit altered levels of 24(S)-HC compared to age-matched controls (Lund 1999; Linsenbardt 2014) → PS can potentiate neuronal 2A- and 2B-containing NMDAR function (Wu 1991; Ceccon 2001) → aminoglycoside antibiotics are PAMs for 2B- containing NMDARs → it’s ototoxicity involves the excitotoxic activation of cochlear NMDARs (Basile 1996) → beneficial effects in treatment refractory schizophrenia, depression, and anxiety disorders (Durrant 2014)CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  12. 12. 24(S)-HC enhances the current response of variant receptors WT 2B 50 sec 200 nA 2B-E413G Glu/GlyGlu/Gly 24(S)-HC 400 nA 50 sec Glu/GlyGlu/Gly 24(S)-HC WT 2A Glu/GlyGlu/Gly 24(S)-HC C Glu/GlyGlu/Gly 24(S)-HC 50 nA 50 sec 2A-D731N D A B 50 sec 200 nA I24(S)HC/IGG,% WT 2A 1-P532H/2A 1-D552E/2A 1-G620R/2A 1-E662K/2A 1-G815R/2A 2A-C436R 2A-G483R 2A-P527R 2A-A643D 2A-V685G 2A-P699S 2A-M705V 2A-A716T 2A-A727T 2A-D731N 2A-V734L 2A-G760V 2A-K772E 100 120 140 160 180 200 WT 2B 1-P532H/2B 1-D552E/2B 1-G620R/2B 1-E662K/2B 1-G815R/2B 2B-E413G 2B-C436R 2B-C456Y 2B-C461F 2B-S541R 100 140 180 220 I24(S)HC/IGG,% Tang, Traynelis, Yuan, unpublished Error bar: 95% confidence interval CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  13. 13. 24(S)-HC prolongs response time course without changing agonist potency 10 100 1000 0 80 160 240 Glutamate, mM MaximalResponse% Control + 24(S)-HC 2B-E413G 10 100 1000 0 25 50 75 100 Glutamate, mM Control + 24(S)-HC 2B-E413G A Fast perfusion system to mimic synaptic events B glutamate Control 24(S)-HC 500 ms 80 pA 2B-E413G C 0 150 300 1000 1200 0 300 600 900 1200D Currentamplitude,pA Deactivationtw,ms Chargetransfer, pAxms×103/pF 0 2000 4000 60006.0 4.0 2.0 0 Tang, Traynelis, Yuan, unpublishedCFERV GRIN_2019_ATLANTA© Hongjie Yuan
  14. 14. PS enhances the current response of variant receptors 30 sec WT 2A 200 nA Glu/Gly PS WT 2B Glu/Gly PS 30 sec 200 nA 2B-E413G Glu/Gly PS 200 nA 30 sec Glu/Gly PS 2A-D731N 200 nA 30 sec C D A B IPS/IGG,% W T 2A 1-P532H/2A 1-D552E/2A 1-G620R/2A 1-E662K/2A 1-G815R/2A 2A-C436R 2A-G483R 2A-P527R 2A-A643D 2A-V685G 2A-P699S 2A-M 705V 2A-A716T 2A-A727T 2A-D731N 2A-V734L 2A-G760V 2A-K772E 100 300 500 700 900 1100 W T 2B 1-P532H/2B 1-D552E/2B 1-G620R/2B 1-E662K/2B 1-G815R/2B 2B-E413G 2B-C436R 2B-C456Y 2B-C461F 2B-S541R 300 600 900 1200 IPS/IGG,% Tang, Traynelis, Yuan, unpublishedCFERV GRIN_2019_ATLANTA© Hongjie Yuan
  15. 15. 20 pA 1 sec PS enhances the current response of variant receptors Pregnenolone sulfate, mM MaximalResponse% WT 2A 2A-D731N 2A-V685G Pregnenolone sulfate, mM WT 2B 2B-C461F 2B-E413G MaximalResponse% A 500 pA 1 sec 40 pA 1 sec glutamate WT 2B 2B-E413G glutamate Control + PS Control + PS Control + PS glutamate 2B-C461FB Swanger et al., 2016; Tang, Traynelis, Yuan, unpublishedCFERV GRIN_2019_ATLANTA© Hongjie Yuan
  16. 16. 100 pA 1 sec glutamate WT 2B Control + Tobramycin Tobramycin enhances the current response of variant receptors 80 nA Tobramycin GG WT 2B 20 sec 2B-E413G 100 nA 20 sec GG Tobramycin W T 2B 1-P532H /2B 1-D 552E/2B 1-G 620R /2B 1-E662K/2B 1-G 815R /2B 2B-E413G 2B-C 436R 2B-C 456Y 2B-C 461F 2B-S541R 100 200 300 400 500 ITobramycin/IGG,%A C B 0 20 pA 1 sec Control + Tobramycin glutamate 2B-E413G Tobramycin potential current response without changing deactivation rate CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  17. 17. 1 10 100 1000 100 150 200 250 300 MaximalResponse% A Tobramycin, mM WT 2B 2B-C461F 2B-E413G Other aminoglycosides enhance the current response of variant receptors WT 2B 2B-E413G 2B-C461F Gentamicin 172  11% 169  25% 182  12% Amikacin 212  20% 234  8.3% n.a. Tobramycin 178  9.5% 198  15% 252  15% Paromomycin 222  24% 236  27% n.a. Kanamycin 162  9.9% 183  12% 193  4.8% Summary of effects of aminoglycosides Data are mean  SEM%, for 300 mM aminoglycosides on current amplitude evoked by 1000 mM glutamate and 100 mM glycine (VHOLD -40 mV) 1 10 100 1000 100 125 150 175 200 Gentamicin, mM WT 2B 2B-C461F 2B-E413G B Tang, Traynelis, Yuan, unpublishedCFERV GRIN_2019_ATLANTAMaximalResponse%© Hongjie Yuan
  18. 18. Activation of LoF variants by glycine-site co-agonists D-serine and D-cycloserine 0.1 1 10 100 0 20 40 60 80 100 D-serine, mM MaximalResponse% WT 2A 2A-D731N 0.1 1 10 0 20 40 60 80 100 D-serine, mM WT 2B 2B-E413G 1 10 100 1000 0 20 40 60 80 100 WT 2B D-cycloserine, mM 2B-E413G A 1 10 100 1000 0 20 40 60 80 100 D-cycloserine, mM 2A-D731N WT 2A MaximalResponse% B Tang, Traynelis, Yuan, unpublished D-serine and D-cycloserine can replace glycine to enhance NMDAR function, raising the possibility that therapeutic administration of these agents might allow full occupancy of the glycine site and thereby enhance NMDAR-mediated currents. CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  19. 19. SUMMARY 1. The effects of three PAMs and two co-agonists were evaluated on 24 LoF GRIN variants associated with developmental delay/intellectual disability, epileptic encephalopathy, autism, and/or movement disorders 2. The neurosteroids potentiated current response for almost all LoF GRIN variants in all subunits evaluated (GluN1, GluN2A, GluN2B), raising the possibility that they may provide broad utility against LoF variants 3. The aminoglycoside tobramycin potentiates the function of GRIN2B and GRIN1 variants 4. Co-agonists D-serine and D-cycloserine can activate almost all LoF GRIN variants with a similar potency compared to the wild type receptors 5. PAMs and co-agonists can rectify certain aspect of the loss of function, raising the possibility that enhancement of NMDAR function may be a useful therapeutic strategy CFERV GRIN_2019_ATLANTA© Hongjie Yuan
  20. 20. DISCLOSURE HY is PI on a research grant from SAGE Therapeutics to Emory University School of Medicine. The grant is about the effects of neurosteroids on NMDAR function. Part of data related to the grant is included in today’s talk. © Hongjie Yuan
  21. 21. Collaborators (Mutations) Slave Petrovski (Melbourne) David Goldstein (Columbia) William Gahl, David Adams (NIH) Marni Falk, Eric Marsh, David Lynch, Ingo Helbig (Penn) Tim Benke (Colorado) Ann Poduri (Harvard) John Millichap (Northwestern) Katherine Roche (NIH) Johannes Lemke (Germany) Andrew Fry (Cardiff) Yuwu Jiang (Beijing) Sooky Koh (Emory) Xiuhua Bozarth (Seattle) Amy Ramsey (Toronto) Plus over 30 academic clinical centers Laboratory members Wendy Chen Jin Zhang Weiting Tang Wenshu XiangWei Jia Li Gil Shaulsky Jan Zhu Yuchen Xu Zhaoshi Zheng Ding Liu Nana Liu Rui Song Collaborators (Function) Adam Cohen (Harvard) Elias Aizenman (Pittsburgh) Wayne Frankel (Columbia) Collaborators (Modelling) Pieter B Burger Gordon Wells Funding NIH-NICHD (PI: YUAN) NIH-UDN (PI: YUAN) URC-ACTSI (PI: YUAN) Emory+Children’s Pediatric Center Seed Grant Program (PI: KIM and YUAN) SAGE Therapeutics (PI: YUAN) Stephen Traynelis, Hongjie Yuan, Scott Myers, Sukhan Kim, Dan Tenscher, James Allen, Wei Han Center for Functional Evaluation of Rare Variants (CFERV, Emory University) Disclosure Research support from SAGE © Hongjie Yuan
  22. 22. -90-75 -60-45 -30-15 10203040 -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2B-E413G Glu/Gly + 24(S)-HC -90-75-60-45-30-15 10203040 -3 -2 -1 1 2 2B-E413G Glu/Gly + PS -90 -75 -60 -45 -30 -15 10203040 -1.5 -1.0 -0.5 0.5 1.0 1.5 Glu/Gly + Tobramycin 2B-E413G © Hongjie Yuan

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