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.

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. 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. 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. # 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. 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. 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. 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. 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. ▼
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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. -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