Biological

Psychiatry Archival Report

Rett-like Severe Encephalopathy Caused by a

De Novo GRIN2B Mutation Is Attenuated by
D-serine Dietary Supplement

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David Soto, Mireia Olivella, Cristina Grau, Judith Armstrong, Clara Alcon, Xavier Gasull,
Macarena Gómez de Salazar, Esther Gratacòs-Batlle, David Ramos-Vicente,
Víctor Fernández-Dueñas, Francisco Ciruela, Àlex Bayés, Carlos Sindreu, Anna López-Sala,
Àngels García-Cazorla, and Xavier Altafaj

ABSTRACT

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BACKGROUND: N-Methyl-D-aspartate receptors (NMDARs) play pivotal roles in synaptic development, plasticity,
neural survival, and cognition. Despite recent reports describing the genetic association between de novo mutations
of NMDAR subunits and severe psychiatric diseases, little is known about their pathogenic mechanisms and potential
therapeutic interventions. Here we report a case study of a 4-year-old Rett-like patient with severe encephalopathy
carrying a missense de novo mutation in GRIN2B(p.P553T) coding for the GluN2B subunit of NMDAR.
METHODS: We generated a dynamic molecular model of mutant GluN2B-containing NMDARs. We expressed the
mutation in cell lines and primary cultures, and we evaluated the putative morphological, electrophysiological, and
synaptic plasticity alterations. Finally, we evaluated D-serine administration as a therapeutic strategy and
translated it to the clinical practice.
RESULTS: Structural molecular modeling predicted a reduced pore size of mutant NMDARs. Electrophysiological
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recordings confirmed this prediction and also showed gating alterations, a reduced glutamate affinity associated with
a strong decrease of NMDA-evoked currents. Moreover, GluN2B(P553T)-expressing neurons showed decreased
spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of
GluA1 at stimulated synapses. Notably, the naturally occurring coagonist D-serine was able to attenuate
hypofunction of GluN2B(p.P553T)-containing NMDARs. Hence, D-serine dietary supplementation was initiated.
Importantly, the patient has shown remarkable motor, cognitive, and communication improvements after
17 months of D-serine dietary supplementation.
CONCLUSIONS: Our data suggest that hypofunctional NMDARs containing GluN2B(p.P553T) can contribute to Rett-
like encephalopathy and that their potentiation by D-serine treatment may underlie the associated clinical
improvement.
Keywords: De novo mutation, D-serine, Glutamatergic neurotransmission, Neuropsychiatric disorders, NMDA
T

receptor, Severe encephalopathy

http://dx.doi.org/10.1016/j.biopsych.2017.05.028
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Rett syndrome (RTT) (Online Mendelian Inheritance in Man
[OMIM]: 312750) is a neurodevelopmental disorder (NDD)
affecting 1 in 10,000 live female births (1,2). Girls with RTT
seem to normally develop until 6 to 18 months and regress
thereafter. Clinical manifestations include microcephaly, loss
of achieved psychomotor abilities, intellectual disability (ID),
and autistic behaviors (3). While most typical RTT cases harbor
loss-of-function mutations in X-linked gene encoding methyl-
CpG binding protein 2 (MECP2) (4), mutations in CDKL5 and
FOXG1 genes have been identified in RTT variants (5). Rett-like
syndrome mostly affects patients exhibiting symptoms that are
similar to those seen in RTT. However, the genetic and mo-
lecular etiology of this rare disease pointed out different
mutations of RTT-associated genes and/or different causative
genes.
A growing number of genetic and functional studies are
unraveling the complex scenario and molecular players
involved in NDD and, in particular, in Rett-like syndrome.
Notably, it has been shown that the dysregulation of synaptic
proteins can lead to NDD (6–8). Genes encoding for the
N-methyl-D-aspartate receptor (NMDAR) could play critical
roles in the dysfunction of glutamatergic transmission associ-
ated with RTT. NMDARs belong to the ionotropic group of
glutamate receptors (iGluRs). Functionally, NMDARs play
critical roles in neurogenesis, synaptogenesis, and synaptic
plasticity processes. Early in development, NMDAR subunit

160 ª 2017 Society of Biological Psychiatry.

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 Biological
D-serine Treatment of Pathogenic GRIN2B De Novo Mutation Psychiatry

GluN2B expression is particularly high (9). Accordingly, it has
been proposed that GRIN2B gene disturbance might dramat-
ically compromise critical steps of neuronal, synaptic, and
brain circuitry development. In agreement with this, animal
models with altered Grin2b expression exhibit neuronal
impairment of social interaction, and no interest in the envi-
ronment. Along with these alterations, the patient showed high
irritability together with sleeping disturbances. Considering
these symptoms, together with the presence of handwashing
stereotypies, the girl was tentatively diagnosed with RTT

dysfunction (10). Moreover, discrete de novo mutations of
GRIN2B gene have been recently associated with NDD
(11–13), including early infantile epileptic encephalopathy-27
(EIEE27) (OMIM: 616139) (14) and autosomal dominant mental
retardation (MRD6) (OMIM: 613970) (15–17). Therefore,
understanding the functional impact of discrete mutations
leading to NDD is fundamental to the development of precise
therapeutic strategies.
In the current work, we report the identification of a de novo
missense mutation in the GRIN2B gene in a Rett-like patient
with severe encephalopathy. Functional studies showed that
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phenotype, but further studies and disease progression lead to
the diagnosis of a Rett-like phenotype. Cytogenetic analysis,
brain magnetic resonance imaging, and neurometabolic anal-
ysis did not show alterations. At 2.5 years old, she was less
irritable and developed the capacity to hold up her head.
Behaviorally, she slightly improved in social interaction, and 1
year later her sleeping pattern was ameliorated. At that age, the
electroencephalogram indicated the presence of epileptiform
alterations of brain activity, and she was treated with valproic
acid; later, the treatment was changed to levetiracetam to
prevent increase in irritability. At 5 years old, the patient’s

CT
channel gating is altered in mutant NMDARs and that this
mutation drastically reduces synaptic NMDA-mediated cur-
rents. Importantly, dietary supplementation with D-serine—an
NMDAR coagonist (18,19)—during 17 months ameliorated
the intellectual, communication, and motor deficits of the
patient. These results support the pathogenicity of GRIN2B
mutation and suggest that enhancing NMDAR activity using
D-serine dietary supplement can have therapeutic benefits in
certain NDDs.
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METHODS AND MATERIALS
adaptive behavior was assessed by the Vineland test, with
scores indicative of a mental age below 1 year old (Table 1).
Genetic Studies
The patient was tentatively diagnosed with RTT-like pheno-
type. However, no mutations of RTT candidate genes (MECP2,
CDKL5, and FOXG1) were detected, and whole-exome
sequencing was performed from the trio (21). Following ge-
netic data filtering (Supplement), a missense de novo hetero-
zygous mutation of GRIN2B(p.P553T), coding for the GluN2B
subunit of NMDARs, was identified (Figure 1A).

Patient Neurodevelopmental Assessment/Adaptive Molecular Modeling of the Mutant (GluN1)2–

Behavior and Dietary D-serine Supplement (GluN2B(P553T))2 Receptor
The study and D-serine dietary supplement (500 mg/kg/day; To identify the structural changes induced by
three divided doses during the day) was approved by the GluN2B(p.P553T) mutation, the molecular models of wild-type
appropriate informed consent of the patient’s parents. The and mutant receptors were generated by homology modeling
Vineland Adaptive Behavior Scale-II (20) semistructured inter- and molecular dynamics simulation. The GluN2B(P553) res-
view, allowing the assessment of four domains of adaptive idue is located at the beginning of the transmembrane helix 1
behavior (communication, daily living skills, socialization, and (TMH1; Figure 1B). This model suggested a role for P553 in
motor skills), was conducted by a trained neuropsychologist breaking TMH1 and bending TMH1 toward TMH3 (Figure 2C,
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before (at 5 years 10 months old) and after (at 7 years 3 months upper panel), hence allowing P553 interaction with F653
old) 17 months of D-serine supplementation. (TMH3) of the GluN2B subunits. In addition, according to this
model, the residue P557 (TMH1) is also bending TMH1 to
In Silico, Biochemical, Cellular, and
Electrophysiological Studies
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In silico studies and experiments in cell lines and primary Table 1. Clinical Manifestations of the Patient Harboring De
Novo GRIN2B(p.P553T) Mutation Before and After 17
neuronal cultures were performed as fully detailed in the
Months of D-serine Dietary Supplement
Supplement, which also includes a description of the statistical
methods. Clinical Manifestations
After D-serine Dietary
Initial Assessment Supplement
RESULTS (5 Years 10 Months Old) (7 Years 3 Months Old)
Patient Clinical Information Behavior Irritability Reactive smiling
Poor visual contact Establishment of visual contact
The patient is a girl who was born after an uneventful preg- No gestural communication Some gestural communication
nancy. Prior to the patient’s birth, the familial history was Disconnected and lost look Interested in surroundings
negative for NDD, with nonconsanguineous parents and a Motor Inability to sit down Autonomy to sit down
healthy sister. She was referred to our clinic at 1 year old, and Clasping hands Walking with orthopedic
the primary clinical examination showed a psychomotor delay Severe hypotonia walker
with severe hypotonia (with the presence of osteotendinous Sleep Altered sleeping pattern Improved sleeping pattern
reflexes and devoid of pyramidal signs), inability to hold the EEG Epileptiform alterations Epileptiform alterations
head and to be seated with no support. Behaviorally, she had without seizures without seizures
an overall absent affect, as manifested by poor visual contact, EEG, electroencephalogram.

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alignment around residues P553 and F653. Representative sequences from a larger alignment containing 147 proteins from 12 metazoan species spanning
seven phyla are shown. Displayed protein sequences are from the following species: Homo sapiens GluN2B (Uniprot Ref. Q13224), Mus musculus GluN2B
(Uniprot Ref. Q01097), Danio rerio GluN2Bb (Ensembl Ref. ENSDARG00000030376), Branchiostoma belcheri 254360R.t1 (from the database B.belcher-
i_v18h27.r3_ref_protein included in LanceletDB Genome browser, Sun Yat-sen University), Strigamia maritima SmarNMDAR2b, Apis melifera GB48097,
Capitela teleta CapteT179505, and Lottia gigantea LotgiT137890 (all from Ensemble Metazoa).
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TMH3 through its proline kink, allowing the interaction with
F654 (TMH3) of the GluN1 subunit. Because the TMH3 of GluN
subunits contributes to the ion channel pore, our molecular
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model strongly suggests that the TMH3–TMH1 interaction
would bring TMH3 out of the center of the channel pore.
Importantly, this model predicts that GluN2B(P553T) mutation
would prevent TMH1 breaking and bending, Consequently,
TMH1–TMH3 interaction would be disrupted (Figure 2C, lower
panel), which in turn would bring TMH3 closer to the center of
the pore and potentially reduce its size or alter the gating
properties of mutant receptor.
Based on this structural model, we investigated the evolu-
tionary conservation of GluN2B proline 553 and its predicting
interacting residue GluN2B phenylalanine 653. Indeed, the
GluN2B(F653) and GluN1(F654) residues belong to the
SYTANLAAF motif, the most highly conserved motif among
mammalian iGluRs (22). Multiple sequence alignment of 147
metazoan iGluRs showed extremely high conservation of these
residues (Figure 1D). Indeed, the proline in position 553 was
observed in 144 of the 147 iGluR sequences investigated.
Similarly, phenylalanine 653 is conserved in 131 of all iGluRs.
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D-serine Treatment of Pathogenic GRIN2B De Novo Mutation
Figure 1. Identification of GRIN2B(p.P553T) mu-
tation associated with the case study and predicted
structural consequences. (A) (Left panel) Pedigree of
the family showing the absence of clinical manifes-
tations of the progenitors and the sister of the
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patient. (Right panels) Chromatograms of the com-
plementary DNA sequence of GRIN2B showing the
GRIN2B(c.1657C.A) mutated nucleotide (indicated
by an arrow) using forward and reverse oligonucle-
otides. (B) Structure of heterotetrameric (GluN1)2–
(GluN2B)2 N-methyl-D-aspartate receptor (Protein
Data Bank ID: 4PE5), according to Karakas and
Furukawa (41), showing the N-terminal domain
(NTD), the ligand binding domain (LBD), and the
transmembrane domain (TMD, containing the
mutated amino acid P553T). (Lower panel) Magnifi-
cation of the TMD, showing the topological position
of Pro553 residue at the beginning of trans-
membrane helix 1 (TMH1, in green) of the TMD,
facing TMH3 (in blue). (C) (Upper panel) Top view of
the TMD structural molecular model of wild-type
(GluN1)2–(GluN2B)2 receptor (from the extracellular
domain). The model predicts that P553 residue (in
orange) of TMH1–GluN2B (in green) is bending
TMH1 toward TMH3 (in dark blue), allowing the
interaction with F653 residue (in light blue) located in
TMH3. The interaction between TMH3 and TMH1
brings TMH3 out of the center of the pore channel.
(Lower panel) Top view of the TMD structural mo-
lecular model of mutant (GluN1)2–(GluN2B(P553T))2
receptor (from the extracellular domain). The model
predicts that T553 residue (in green) of TMH1–
GluN2B is not able to bend TMH1, abolishing the
interaction with F653 (located in TMH3, light blue).
According to this model, the breaking of the inter-
action between TMH3 and TMH1 brings TMH3
toward the center of the pore, inducing a reduction of
the channel pore. (D) GluN2B protein sequence
This high conservation strongly supports a critical role of
GluN2B(P553) in NMDAR channel activity. These data
prompted us to further assess the molecular and biophysical
consequences of the GluN2B(P553T) mutation.
Heteromerization and Trafficking of GluN1–
GluN2B(P553T) Receptors
To assess the effects of GluN2B(P553T) on NMDAR oligo-
merization and trafficking, we cotransfected HEK-293T cells
with GluN1 subunit and either green fluorescent protein (GFP)–
GluN2Bwt or GFP–GluN2B(P553T). Biochemical analysis
showed that GluN2B(P553T) protein levels were similar to
those of GluN2Bwt (Figure 2A). Furthermore, coimmunopreci-
pitation experiments showed the presence of GluN1 and
hemagglutinin–GluN2A subunits in anti-GFP pulled-down
complexes, indicating that GluN2B(P553T) subunit interacts
with GluN1 and/or GluN2A, similarly to GluN2Bwt (Figure 2B).
Surface expression of wild-type or mutant GFP–GluN2B was
also evaluated in COS-7 cells and in mouse primary cortical
neurons. Immunofluorescent analysis showed the ability of

D-serine Treatment of Pathogenic GRIN2B De Novo Mutation
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neurons (n = 25–62 cells per condition, from three or four independent experiments; *p = .026; ns, nonsignificant; Student’s t test). Scale bar = 40 mm
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(low magnification) or 3 mm (dendrites).
mutant GFP–GluN2B(P553T) to reach COS-7 plasma
membrane (100 6 10.3 vs. 146.6 6 13.1 in GFP–GluN2Bwt/
P553T-expressing cells; Figure 2C). In a neuronal context,
GluN2B(P553T) dendritic surface/intracellular ratio expression
was normal at day in vitro 7 (DIV7) and DIV11, with a slight
decrease at DIV16 (100 6 2.6 vs. 92.0 6 1.6 GFP–GluN2Bwt/
P553T-transfected primary cortical neurons; Figure 2D).
Electrophysiological Changes in GluN2B(P553T)
Subunit-Containing NMDARs
Patch-clamp experiments were performed to evaluate the
biophysical properties of GluN2B(P553T)-containing NMDARs.
Following fast application of glutamate (1 mM) and glycine (50
mM), whole-cell NMDA-mediated currents were evoked in
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Figure 2. Protein interactions and cellular
trafficking of GluN2Bwt- and GluN2B(P553T)-
containing N-methyl-D-aspartate receptors. (A)
Western blot analysis of heterologous expression
of wild-type (first lane) and (P553T) mutant green
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fluorescent protein (GFP)–GluN2B (second lane) in
equal amounts of proteins (actin loads) extracted
from transiently transfected HEK-293T cells. (B)
Representative Western blot analysis of coimmu-
noprecipitation (co-IP) experiments from HEK-293T
cells transiently cotransfected with hemagglutinin
(HA)–GluN2A, GluN1, and either GFP–GluN2Bwt or
GFP–GluN2B(P553T). The coimmunoprecipitation
was performed either with anti-GFP (pulling
down GFP–GluN2B construct products and their
interacting proteins) or immunoglobulin G (IgG)
(negative control). Interaction studies showed the
ability of GFP-tagged GluN2B(P553T) subunit to
interact with HA–GluN2A and GluN1 subunits,
similarly to GluN2Bwt. Bar graph represents the
densitometric analysis of immunoprecipitated pro-
teins (relative to immunoprecipitated GFP–GluN2B
for GluN2A and GluN1 subunits; relative to GFP–
GluN2B load for GluN2B subunit) [mean 6 SEM
densitometric of three independent experiments;
white bars: GluN2Bwt condition; red bars:
GluN2B(P553T) condition]. (C) Left panel: Immu-
nofluorescent analysis of cell surface expression
(red channel) and total expression (green channel)
of wild-type (left panels) and mutant GFP–GluN2B
(right panels) in COS-7 cells. Right panel: Bar
graph representing the relative surface to total
expression of GFP–GluN2B constructs in COS-7
cells (n = 35–38 cells per condition, from three
independent experiments; **p = .007, Student’s
t test). (D) Time course analysis of GFP–GluN2B
cell surface expression (green channel) and intra-
cellular expression (red channel) of wild-type
(left panels) and mutant GFP–GluN2B (right
panels) in primary cortical neuronal cultures
transiently transfected. Primary cultures were
transfected at day in vitro 4 (DIV4) or DIV7, and
immunofluorescence analysis of surface (green
channel) and intracellular (red channel) expression
was performed at DIV6, DIV11, and DIV16.
Bottom: Bar graph showing the ability of mutant
GluN2B(P553T)-containing N-methyl-D-aspartate
receptors to reach the plasma membrane of the
dendritic processes (insets) of transfected cortical
transfected HEK-293T cells. Current amplitudes were drasti-
cally reduced in cells expressing GluN1–GluN2B(P553T)
receptors compared with GluN1–GluN2B-expressing cells
[280.56 6 14.25 pA/pF for GluN2Bwt vs. 215.49 6 3.16
pA/pF for GluN2B(P553T); Figure 3A–C]. The GluN2B(p.P553T)
mutation spared the voltage-dependent channel block by
extracellular Mg21 (Figure 3D–F). Because GluN2B(P553T)
mutation is located in the vicinity of the agonist binding site
and the channel pore, we investigated possible effects of the
mutation on channel kinetics. Electrophysiological recordings
showed a significantly faster deactivation rate in mutant
receptors [0.60 6 0.06 seconds for GluN2Bwt vs. 0.26 6 0.03
seconds for GluN2B(P553T); Figure 4A, B] and faster desen-
sitization quantified on 5-second-duration jumps [1.87 6 0.33
seconds for GluN2Bwt vs. 0.79 6 0.22 seconds for
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Figure 3. GluN2B(P553T) mutation reduces N-methyl-D-aspartate receptor (NMDAR)-mediated whole-cell currents without affecting magnesium block.
(A) Representative whole-cell currents evoked by rapid application of 1 mM glutamate plus 50 mM glycine (0.5 seconds duration; 260 mV) for HEK-293T cells
expressing GluN1–GluN2B (black trace) or GluN1–GluN2B(P553T) NMDARs (red trace). Note that currents for GluN2B(P553T)-containing receptors were notably
smaller even when both cells displayed a similar membrane capacitance (5.5 and 7.0 pF). (B) Average of raw peak currents for GluN2B- and GluN2B(P553T)-
expressing cells with single experiment values superimposed (open circles; p = .0003, Mann-Whitney U test, n = 19 and 21, respectively). (C) Normalized peak
currents (2pA/pF) for GluN2B- and GluN2B(P553T)-expressing cells with single experiment values superimposed (open circles; p , .0001, Mann-Whitney U test,
n = 19 and 21, respectively). (D) Traces recorded at 260 mV for a GluN2B(P553T)-expressing cell showing the characteristic magnesium block of NMDARs. (E)
Percentage of current block at 260 mV by Mg21 (1mM) for GluN2Bwt- and GluN2B(P553T)-containing receptors. Single-value experiments are denoted as open
circles for each condition [p = .0732, Mann-Whitney U test, n = 5 and 7 for GluN2B and GluN2B(P553T) respectively]. (F) Current–voltage relationship for GluN2B-
and GluN2B(P553T)-containing receptors where it can be observed that Mg21 blocks similarly both NMDARs at the whole range of negative potentials. ***p , .001;
****p , .0001; n.s., nonsignificant.
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GluN2B(P553T); Figure 4C, D]. Moreover, in agreement
with aforementioned modeling predictions, nonstationary
fluctuation analysis (23) showed a reduction of the single-
channel conductance in mutant receptors [49.73 6 2.61 pS
for GluN2Bwt vs. 39.15 6 2.60 pS for GluN2B(P553T);
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GluN1–GluN2B(P553T)-expressing cells (14.9 6 3.25% at 10
mM, 43.4 6 11.08% at 100 mM, and 32.3 6 7.12% at 300 mM,
ps = .006, .004, and .001, respectively; Figure 5) (p = .022 at
100 mM D-serine between mutant and wild-type receptors;

Figure 5B). This differential potency of D-serine might result

Figure 4E, F]. Together with biochemical data indicating normal
expression and oligomerization, we conclude that GluN1–
GluN2B(P553T) receptors are intrinsically hypofunctional.
D-serine Administration Enhances NMDA Currents
in GluN1–GluN2B(P553T) Receptors
Next, we sought to enhance activity of mutant NMDARs by
using compounds with unlikely side effects or impaired as a
result of the GluN2B(P553T) mutation. D-serine, a naturally
occurring coagonist of the NMDAR, may fulfill both criteria.
In agreement with previous reports (19), D-serine administra-
tion dose-dependently increased GluN1–GluN2Bwt-mediated
current density in HEK-293T cells (13.3 6 4.61% increase at
100 mM and 16.9 6 3.97% increase at 300 mM, ps = .018 and
.002, respectively; Figure 5). By comparison, NMDA-evoked
currents were more strongly potentiated by D-serine in
from a reduced affinity for GluN1–GluN2B(P553T), leading to
the enhanced potentiation and faster deactivation/desensiti-
zation rates at higher concentrations. Alternatively, because
glutamate binding increases the dissociation rate of glycine/
D-serine coagonist with NMDARs (24), seeming changes in
D-serine potency might be explained by an altered glutamate
affinity. Concentration–response experiments showed no
changes in D-serine EC50 [0.51 mM for GluN2Bwt vs. 0.66 mM
for GluN2B(P553T); Figure 5C], while the EC50 for glutamate
increased by sevenfold in GluN1-GluN2B(P553T) receptors
compared with wild-type receptors [3.09 mM for GluN2Bwt vs.
20.94 mM for GluN2B(P553T); Figure 5D].
Biophysical Alterations in Triheteromeric GluN1–
GluN2A–GluN2B(P553T) Receptors
GluN2B subunit interacts with different GluN subunits, forming
both (GluN1)2–(GluN2B)2 heterodimers (prominent NMDARs at

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analysis for these recording cells were 53.9 pS (GluN2Bwt) and 36.8 pS [GluN2B(P553T)]. (F) Bar graph showing that single-channel conductance values are
decreased in GluN2B(P553T)-containing N-methyl-D-aspartate receptors [49.73 6 2.61 pS for GluN1–GluN2B vs. 39.15 6 2.60 pS for GluN1–GluN2B(P553T),
p = .0209, Mann-Whitney U test; n = 12 and 9, respectively]. Single cells are shown as open circles superimposed to bar graph. *p , .05; **p , .01;
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****p , .0001.
initial developmental stages) and (GluN1)2–GluN2A–GluN2B
triheteromers. Both (GluN1)2–(GluN2B)2 and (GluN1)2–
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GluN2A–GluN2B receptor complexes exist in adult hippo-
campus and cortex (25). Thus, we explored whether the
GluN2B(P553T) mutation may also impair the subpopulation
of triheteromeric receptors or, instead, its deleterious ef-
fects might be mitigated when coassembled with GluN2A
subunit. To record triheteromeric-mediated NMDA currents,
HEK-293T cells were transfected with GluN constructs
harboring ectopic retention signals (26). NMDA current
amplitudes were not significantly reduced in
GluN2B(P553T)-containing triheteromers (Figure 6A, B).
However, administration of 100 mM D-serine potentiated
GluN2B(P553T)-containing triheteromers more strongly than
wild-type ones (30.59 6 4.20% for GluN2A–GluN2Bwt vs.
54.76 6 10.10% for GluN2A–GluN2B(P553T); Figure 6A–C).
Likewise, the desensitization and deactivation kinetics of
triheteromeric mutants were increased compared with
control subjects (Figure 6D–H), recapitulating some of the
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Figure 4. Faster deactivation and lower single-
channel conductance contribute to GluN2B(P553T)
loss of function. (A) Peak-scaled representative re-
sponses to 1 mM glutamate plus 50 mM glycine (0.5
second agonist application; 260 mV) for GluN2B
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(black trace) and GluN2B(P553T) (red trace), showing
a faster inactivation of GluN2B(P553T)-containing N-
methyl-D-aspartate receptors following coagonist
removal. (B) Average deactivation time constant
(fitted to a double exponential) fitted from tail cur-
rents for GluN2B and GluN2B(P553T). P553T muta-
tion accelerates channel closure after coagonists
unbind [0.60 6 0.06 seconds for GluN2B vs. 0.26 6
0.03 seconds for GluN2B(P553T), p , .0001; Mann-
Whitney U test; n = 16 and 17, respectively]. Single-
experiment values are shown as open circles for
each condition. (C) Peak-scaled representative re-
sponses to 1 mM glutamate plus 50 mM glycine (long
jumps of 5 seconds duration; 260 mV) for GluN2B
(black trace) and GluN2B(P553T) (red trace), where it
can be observed the faster desensitization of the
current mediated by GluN2B(P553T) in the presence
of coagonists. (D) Desensitization weighted time
constant for GluN2Bwt and GluN2B(P553T), showing
enhanced desensitization in GluN2B(P553T)-
containing N-methyl-D-aspartate receptors. Single-
experiment values are shown as open circles for
each condition [1.87 6 0.33 seconds for GluN2B vs.
0.79 6 0.22 seconds for GluN2B(P553T), p = .0054,
Mann-Whitney U test, n = 14 for both). (E) Whole-cell
current activated by rapid application of 1 mM
glutamate plus 50 mM glycine (0.5 second; 260 mV)
from a cell expressing GluN2Bwt (left panel) or
GluN2B(P553T) (right panel). Gray traces represent
single responses, and black lines are the averages of
69 and 33 responses for wild-type and mutant
GluN2B, respectively. Insets denote the current
variance versus mean current plot calculated from
the deactivating tail current for this cell. The
weighted mean single-channel conductance (g)
values estimated from nonstationary fluctuation
effects observed in the mutant (GluN1)2–(GluN2B(P553T))2
diheterodimers.
GluN2B(P553T) Overexpression Decreases Spine
Density and Reduces Excitatory Postsynaptic
Currents
To evaluate the effect(s) of the GluN2B(P553T) mutation in
central neurons, we overexpressed GluN2B(P553T) in primary
hippocampal neurons and measured both morphological
parameters and NMDAR-mediated synaptic currents. Sholl
analysis of dendrites labeled with GFP–GluN2B(P553T) or
GFP–GluN2Bwt indicated similar distributions of the subunit
across the dendritic arbor (F = 0.71, p = .884; Figure 7A).
Interestingly, however, the spine density was significantly
reduced in neurons expressing GluN2B(P553T) [21.1 6 1.6
vs. 13.1 6 1.0 spines/10 mm dendrite for GluN2Bwt and
GluN2B(P553T), respectively, p = .0002; Figure 7A]. The
reduction in spine density mainly resulted from a decrease in
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Figure 5. N-Methyl-D-aspartate receptor coactivation by D-serine is enhanced in GluN2B(P553T)-containing receptors. (A) Representative traces evoked by
physiological concentrations of 1 mM glutamate plus 1 mM glycine (0.5 second; 260mV) from GluN2Bwt- or GluN2B(P553T)-expressing HEK-293T cells, either in the
absence (black traces) or in the presence (red traces) of D-serine. The coagonist D-serine was applied at different concentrations (1 mM: upper panel; 10 mM: second
panel from top; 100 mM: second panel from bottom; 300 mM: lower panel). Note that GluN2B(P553T)-mediated currents are smaller and have faster deactivation
kinetics than GluN2B-mediated currents. (B) Average of normalized peak currents evoked by 1 mM glutamate 1 1 mM glycine at different D-serine concentrations
(gray scale colored bars) compared with absence of D-serine (white bar: 100% of the response). Although D-serine was able to enhance evoked currents for both
GluN2B and GluN2B(P553T) at high concentrations, the modulatory effect was greater for GluN2B(P553T)-containing receptors, whose currents were significantly
increased at 10 mM D-serine compared with GluN2B. (*p , .05 and **p , .01 for comparisons between different D-serine concentrations within same receptor type;
#
p , .05 for comparisons between GluN2B and GluN2B(P553T) at the same D-serine concentration). Numbers inside the bars denote the number of recordings for
each condition. (C) D-serine concentration–response curves constructed from responses to 1 mM glutamate plus the specified D-serine concentration in the absence
of glycine. GluN2Bwt- and GluN2B(P553T)-containing N-methyl-D-aspartate receptors expressed in HEK-293T cells showed similar EC50 values [0.51 mM for
GluN2B vs. 0.66 mM for GluN2B(P553T); n = 5 for each construct]. (D) Glutamate concentration–response curves constructed from responses to 1 mM D-serine plus
the specified glutamate concentration in the absence of glycine of GluN2Bwt- and GluN2B(P553T)-containing N-methyl-D-aspartate receptors. GluN2B(P553T)
mutation produces an approximately sevenfold increase in EC50 value for glutamate [3.09 mM for GluN2B vs. 20.94 mM for GluN2B(P553T); n = 5 and 7, respectively].

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Figure 6. Altered biophysical properties of triheteromeric GluN1–GluN2A–GluN2B(P553T) N-methyl-D-aspartate receptors (NMDARs). (A) Representative
traces evoked by 1 mM glutamate plus 1 mM glycine (0.5 second; 260 mV) in HEK-293T cells expressing triheteromeric GluN1–GluN2A–GluN2Bwt (2A-2B, left
traces) or GluN1–GluN2A-2B(P553T) [2A-2B(P553T), right traces] in the absence (black traces) or presence (red traces) of 100 mM D-serine. (B) Normalized
peak currents (2pA/pF) for GluN2A-2B- and GluN2A-2B(P553T)-expressing cells with single-experiment values superimposed (open circles; p . .05, Mann-
Whitney U test, n = 18 and 13, respectively). (C) Percentage of current increment in the presence of 100 mM D-serine for GluN2A-2B- and GluN2A-2B(P553T)-
expressing cells with single-experiment values superimposed (open circles; p , .05, Mann-Whitney U test, n = 14 and 13, respectively). (D) Representative
whole-cell currents evoked by 1 mM glutamate plus 1 mM glycine from HEK-293T cells expressing GluN2A-2B (black trace) or GluN2A-2B(P553T) (red trace),
showing the faster kinetics of triheteromeric NMDARs containing GluN2B(P553T) subunit. Responses have been peak scaled for comparison purposes.
(E) Magnification of the tail-deactivating currents (shown in panel D) after glutamate and glycine removal. Traces (aligned at the coagonist removal time point)
showed a faster deactivating kinetics of mutant triheteromeric NMDARs (0.14 second for depicted cell, red trace) compared with GluN2Bwt-containing tri-
heteromers (0.29 second for depicted cell, black trace). Fits are shown overlapped in green. (F) Magnification of triheteromeric NMDAR desensitization (shown
in panel D). Both currents have been aligned at the peak current. GluN2B(P553T)-containing receptors show a faster desensitization time constant
(0.12 second for depicted cell, red trace) compared with GluN2Bwt-containing triheteromers (0.38 second for depicted cell, black trace). Fits are shown in
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green. (G) Bar graph representing the deactivation time constant fitted from tail currents for triheteromeric GluN2A-2B and GluN2A-2B(P553T). GluN2B(P553T)
mutation accelerates channel closure after coagonist removal [0.30 6 0.02 second for GluN2A-2B vs. 0.21 6 0.02 second for GluN2A-2B(P553T), p , .01,
Mann-Whitney U test, n = 17 and 14, respectively]. Single-experiment values are shown as open circles for each condition. (H) Bar graph representing the
average desensitization time constant for GluN2A-2B- and GluN2A-2B(P553T)-expressing cells, measured in the presence of the agonists (as shown in panel
F). P553T mutation accelerates desensitization of triheteromeric NMDARs [0.30 6 0.02 second for GluN2A-2B vs. 0.15 6 0.02 second for GluN2A-2B(P553T),
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p , .0001, Mann-Whitney U test, n = 17 and 14, respectively]. Single-experiment values are shown as open circles for each condition. *p , .05; **p , .01;
****p , .0001; n.s., nonsignificant.

the number of thin and mushroom spines [8.6 6 1.0 vs.
3.5 6 0.5 for thin spines/10 mm dendrite and 5.8 6 0.4 vs.
3.5 6 0.3 for mushroom spines/10 mm dendrite for GluN2Bwt
and GluN2B(P553T), ps = .0001 and .0002, respectively;
Figure 7A], while the number of stubby spines remained un-
affected [6.7 6 0.5 vs. 6.1 6 0.5/10 mm dendrite for
GluN2Bwt and GluN2B(P553T), respectively; Figure 7A].
Together with the reduction in spine density, immunofluo-
rescence analysis of GluA1 (alpha-amino-3-hydroxy-5-
methyl-4-isoxazole propionic acid receptor [AMPAR]
subunit) expression [overexpressed in RTT murine models
(27)] revealed a significant increase of GluA1 at DIV11 in
neurons overexpressing GluN2B(P553T) mutation (10.5 6
1.7 vs. 23.3 6 2.6 arbitrary units (A.U.) for GluN2Bwt and
GluN2B(P553T) respectively, p = .0002; Supplemental
Figure S1). Overall, these morphological and molecular
changes indicate deficient spine development in hippocampal
neurons expressing GluN2B(P553T).
Patch-clamp recordings revealed a decrease in the ampli-
tude of spontaneous excitatory postsynaptic currents (EPSCs)
mediated by NMDARs in neurons overexpressing mutant
GluN2B(P553T) compared with GluN2Bwt [2593.62 6 59.58
pA for GluN2Bwt vs. 2212.98 6 48.27 pA for GluN2B(P553T);
Figure 7B, top traces, and Figure 7C, left bar graph], directly
demonstrating an effect of this mutation in the synaptic pop-
ulation of NMDARs. Because D-serine administration
enhanced the activity of GluN2B(P553T)-containing NMDARs
in heterologous cells (see above), we assessed its effect

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in primary neurons. The addition of 100 mM D-serine
similarly increased EPSC frequency in GluN2Bwt- and
GluN2B(P553T)-overexpressing neurons (measured as a
shortening of the inter-event interval; Figure 7B, bottom traces,
and Figure 7C, right bar graph). Furthermore, the charge
transfer (pA*ms) selectively increased in GluN2B(P553T)-
overexpressing neurons (1687 pA*ms before D-serine vs.
2210 pA*ms after D-serine, i.e., a 34% change) but not in
neurons overexpressing GluN2Bwt (4089 pA*ms before D-
serine vs. 3979 pA*ms after D-serine, a 20.1% change) (data
not shown). In contrast to heterologous cell line data, the
addition of 100 mM D-serine did not increase the EPSC am-
plitudes in either group (Figure 7C, left bar graph), perhaps due
to the recruitment of new synapses or to increased
168 Biological Psychiatry January 15, 2018; 83:160–172 www.sobp.org/journal
D-serine Treatment of Pathogenic GRIN2B De Novo Mutation
Figure 7. GluN2B(P553T) mutation alters spine
morphology and decreases synaptic N-methyl-D-
aspartate receptor (NMDAR)-mediated currents in
hippocampal neurons. (A) Representative images of
primary hippocampal neurons transfected at day
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in vitro 7 with green fluorescence protein (GFP)–
GluN2Bwt (top image) or GFP–GluN2B(P553T) (bot-
tom image). Immunofluorescent detection of GFP–
GluN2B allowed the analysis of the dendritic arbori-
zation of GFP–GluN2Bwt- or GFP–GluN2B(P553T)-
expressing hippocampal neurons (upper and lower
panels, respectively). GFP–GluN2B immunode-
tection allowed the quantification of dendritic
complexity by Sholl analysis (upper left panel), which
is similar between neurons expressing GFP–
GluN2Bwt and GFP–GluN2B(P553T) (F = 0.71, p =
0.884, repeated-measures two-way analysis of vari-
ance and Bonferroni posttest, n = 16–18 transfected
neurons per condition, from three independent
experiments). Immunolabeling of GFP–GluN2B
transfected neurons was used to visualize spines
(insets, indicated by yellow arrows) and to quantify
spine density and morphology (bar graph, bottom
right). Compared with GFP–GluN2Bwt, the expres-
sion of GFP–GluN2B(P553T) reduced spine density,
resulting from a significant reduction of mushroom-
and thin-type spines, while stubby spines were un-
affected (n = 11–14 dendrites per condition, from two
independent experiments; ***p , .001; ns, nonsig-
nificant; Student’s t test). (B) Representative traces
from spontaneous activity-dependent NMDAR-
mediated excitatory postsynaptic current (EPSC)
recordings from hippocampal neuronal cultures.
EPSCs from neurons transfected with either wild-
type GluN2B (left traces) or mutant GluN2B(P553T)
(right traces) were recorded at 270 mV in the pres-
ence of NBQX (50 mM) and picrotoxin (10 mM) and in
the absence of TTX. The addition of 100 mM D-serine
(bottom traces) produced an increase in the fre-
quency of EPSCs (decrease in the interevent inter-
val). (C) Left: Mean amplitudes of NMDAR-mediated
EPSCs for GluN2Bwt and GluN2B(P553T) before
(basal) and after D-serine addition (n = 8 and 6
neurons, respectively). Right: Mean time of inter-
event intervals of NMDA-mediated EPSCs in primary
hippocampal neurons transfected with GFP–
GluN2Bwt or GFP–GluN2B(P553T), before and after
addition of D-serine (**p , .01 GluN2Bwt vs.
GluN2B(P553T), #p , .05 baseline vs. D-serine
administration).
desensitization at higher frequencies, thereby masking a pu-
tative effect on EPSC amplitudes. Indeed, the rate of recovery
from desensitization for NMDARs is quite slow, spanning
several seconds (28).
D-serine Rescues Altered Activity-Induced
Recruitment of AMPARs in GluN2B(P553T)-
Expressing Neurons
Sustained activation of functional NMDARs can lead to the
insertion of surface AMPARs at synapses and consequently
induce long-term potentiation (LTP) (29). Therefore, we
assessed the ability of neurons expressing GluN2B(P553T)-
containing NMDARs to support this form of synaptic

D-serine Treatment of Pathogenic GRIN2B De Novo Mutation
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plasticity. Primary hippocampal cultures transfected with either
GFP–GluN2Bwt or GFP–GluN2B(P553T) were stimulated with
200 mM glycine in the absence of Mg21 (glycine-mediated
chemical LTP [Gly-cLTP]) or simultaneously with 200 mM
glycine plus 100 mM D-serine. Immunofluorescent analysis
showed that Gly-cLTP significantly increased surface GluA1
levels at spine-like structures in GluN2Bwt-transfected neu-
rons (100 6 5.6 A.U. baseline vs. 121.8 6 8.4 A.U. Gly-cLTP;
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Figure 8A, left panels, and Figure 8B). In contrast, the sur-
face levels of GluA1 were not significantly increased in neurons
expressing GluN2B(P553T) (100 6 10.6 A.U. baseline vs.
112 6 9.5 A.U. Gly-cLTP; Figure 8A, right panels, and
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Figure 8B). Importantly, the defects observed in AMPAR
recruitment in GluN2B(P553T)-expressing neurons could be
rescued by simultaneous administration of glycine and
D-serine (100.0 6 4.3 A.U. baseline vs. 127.0 6 6.0 A.U.
glycine 1 D-serine cLTP; Figure 8A, right panels, and
Figure 8B). These results indicate that D-serine can also
facilitate a major mechanism of NMDAR-dependent plasticity
in the context of GluN2B(P553T) mutation.
Dietary Supplement of D-serine Improves Motor
and Communication Skills of the Patient Carrying
GluN2B(P553T) Mutation
The partial restoration of mutant NMDAR currents by D-serine,
together with the lack of side effects of D-serine treatment (30),
prompted us to start a dietary supplement treatment of the
patient. At 5 years 10 months of age, the patient was admin-
istered D-serine (500 mg/kg/day) and continued to date (i.e., 17
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Figure 8. Glycine-induced chemical long-term
potentiation (Gly-cLTP) deficits of primary hippo-
campal neurons expressing GluN2B(P553T) muta-
tion are rescued by D-serine administration.
(A) Immunofluorescent analysis of surface expres-
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sion of the alpha-amino-3-hydroxy-5-methyl-4-
isoxazole propionic acid receptor GluA1 subunit in
murine primary hippocampal neurons at day in vitro
16, transiently transfected at day in vitro 11 with
green fluorescent protein (GFP)–GluN2Bwt (left
panels) and GFP–GluN2B(P553T) (right panels).
Transfected neurons (green channel) were analyzed
for the detection of surface GluA1 in dendritic pro-
cesses (red channel), under basal conditions (left),
after Gly-cLTP (middle), or following Gly-cLTP in the
presence of 100 mM D-serine (right). (B) Bar graph
representing GluA1 surface fluorescence intensity
(arbitrary units, compared with basal conditions) in
murine primary hippocampal neurons transfected
with GFP–GluN2Bwt (light boxes) and GFP–
GluN2B(P553T) (dark boxes). Histograms represent
the average 6 SEM. Measurements were performed
with 14 to 40 dendrites per condition (30–40 spines
per dendrite; 3–7 neurons per condition; **p , .05,
***p , .001, Student’s t test).
months). Before treatment, the patient had few communication
skills; had poor eye contact; did not pay attention to the
activities of parents, teachers, and schoolmates; did not have
verbal language (any word/sounds) but was able to wave
bye-bye (although with no pointing); did not engage in sym-
bolic play; could sit without support but could not move from
prone lying to the sitting position. The Vineland test scores
(where standard score [SS] is mean = 100, SD = 15) before
treatment were as follows: communication, 42 SS; daily living
skills, 36 SS; socialization, 49 SS; motor skills, 31 SS.
After 17 months of treatment, the patient remarkably
improved communication, social, and motor skills (Table 1).
Behaviorally, she is interested in faces and has persistent eye
contact, follows the surrounding human activities with interest,
is able to stretch the arms in order to be held and turn her head
when called, laughs at funny situations, and is in general
happier. She can also imitate toys and animal sounds, creep
on the floor and move from prone lying to the sitting position,
and move from sitting to standing position with support. She
has also improved her sleeping patterns, is able to sit down
without help, and (importantly) can walk with the assistance of
an orthopedic walker. The electroencephalogram shows
epileptiform alterations, but clinical seizures were not present.
Despite pharmacological interventions (valproate first and
levetiracetam later), the epileptiform alterations detected in the
electroencephalogram are still present; thus, these treatments
have been stopped. In summary, the Vineland test (devoid of
motor score that cannot be performed beyond 7 years old)
after D-serine treatment resulted in the following test scores:
communication, 48 SS; daily living skills, 48 SS; socialization,
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50 SS. Overall, the clinical assessment indicates attenuation of
both cognitive and motor impairments.
DISCUSSION
NMDARs are critical players of glutamatergic neurotransmis-
sion and are fundamental actors in neuritogenesis, synapto-
genesis, and synaptic plasticity processes. Currently, on the
development of next-generation sequencing, there is a growing
body of data implicating de novo mutations of ionotropic
glutamate receptors (7,8) in mental and behavioral disorders.
Indeed, several mutations in subunits of the NMDAR have been
related to neurodevelopmental diseases (11–13). However,
these data require functional validation to unveil whether these
mutations are really pathogenic. In the current study, we iden-
tified a GRIN2B(p.P553T) missense de novo mutation of the
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GluN2B subunit of the NMDAR in a Rett-like patient with severe
encephalopathy. This mutation resulted in the exchange of
highly evolutionary conserved proline into threonine. Impor-
tantly, a mutation affecting the same amino acid position
GluN2B(p.P553L) was previously described in a patient from a
cohort of individuals with ID (16). This patient exhibited
phenotypic alterations similar to those of the case in the current
study, including severe ID, hypotonia, and no speech. The
phenotypic similarity between these two cases provides strong
evidence for a pathogenic role of GluN2B(P553) mutations in
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these neurodevelopmental conditions.
Electrophysiological studies confirmed the in silico struc-
tural studies predicting the hypofunctionality of mutant
NMDARs. In addition to a reduction of the channel conduc-
tance, we found a significant increase in both the desensiti-
zation and deactivation rates of GluN1–GluN2B(P553T)
receptors. Whether the P553T mutation located far from the
binding site is altering deactivation kinetics, an intrinsic prop-
erty of the receptor, is unknown, but electrophysiological
concentration–response experiments indicate decreased
glutamate binding affinity of mutant NMDARs, triggering
decreased receptor efficacy that seems to be physiologically
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relevant. NMDAR function is largely determined by the high
amount of Ca21 influx, which is mostly dependent on channel
kinetics, in particular the rates of desensitization and deacti-
vation. Thus, we can speculate that kinetics changes detected
on GluN2B(P553T) mutant receptors might be limiting Ca21
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influx, which in turn would alter Ca21-mediated signaling
pathways and synaptic plasticity.
Overall, these changes dramatically reduce NMDAR-
mediated currents and might be underlying the severe
phenotype of the patient. Indeed, GluN2B subunits are highly
expressed at embryonic and initial postnatal stages, playing a
critical role in neurodevelopmental processes (31). Conse-
quently, the hypofunctionality of this major subtype of
NMDARs might certainly affect neuritogenesis and synapto-
genesis, leading to altered synaptic transmission. This hy-
pothesis is supported by the morphological and biochemical
findings, showing a significant decrease of spine density
together with increased levels of GluA1 subunit of AMPARs.
Interestingly, Pozzo-Miller’s group has reported similar syn-
aptic outcomes in patients with RTT and in primary murine
neuronal cultures deficient in or harboring mutations of MeCP2
(27,32). Therefore, our data suggest that NMDAR-induced
170 Biological Psychiatry January 15, 2018; 83:160–172 www.sobp.org/journal
alterations of glutamatergic synapses might be involved in the
pathophysiology of the classical RTT condition.
In agreement with the proposed functional impact of de
novo mutations of GluN2B subunit, previous studies have
associated de novo mutations of GRIN2B with severe pheno-
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typic alterations (6,14,15,33). Lemke et al. elegantly described
the functional consequences of GRIN2B mutations in patients
with West syndrome as well as in individuals with ID with focal
epilepsy (14). In the latter study, the patient with ID and focal
epilepsy had a missense mutation in the extracellular
glutamate-binding domain. Interestingly, de novo GRIN2B
mutations lead to a gain of function, either significantly
reducing Mg21 block and increasing Ca21 permeability (N615I
and V618G mutations affecting the M2 domain in the pore of
the channel) or increasing the apparent glutamate binding
affinity (R540H mutation within the extracellular S1 domain).
These gain-of-function mutations point out the important role
of facilitated NMDAR signaling in epileptogenesis, with further
therapeutic strategies involving the selective blockade of
mutant leak/hyperactive channels. On the contrary, loss-of-
function mutations might be causing a hypoglutamatergic
function that could be potentially rescued by increasing
NMDAR activity with a therapeutic purpose.
The glutamatergic synapse is an extremely sophisticated
system where a plethora of molecular actors reside and
interact to finely tune neurotransmission. However, under
pathological conditions, the dysregulation of critical players
might compromise glutamatergic neurotransmission, resulting
in an enhancement or a reduction of glutamate signaling (8). In
the current work, in silico and in vitro experiments concluded
that GluN2B(P553T)-containing NMDARs are hypofunctional.
Thus, we envisioned that enhancing NMDAR activity might
recover normal glutamatergic neurotransmission and attenuate
clinical manifestations. To this end, we evaluated the effect of
D-serine, a coagonist of the GluN1 subunit of the NMDAR,
coapplied with physiological glycine concentrations (34).
Interestingly, our findings indicate an enhancement of mutant
GluN1–GluN2B receptor activity, suggesting that the structural
changes induced by the mutation are not transduced to GluN1
ligand-binding domain. Noteworthy, because D-serine acti-
vates all NMDAR subtypes (35,36), it should have a general
effect on glutamatergic function. Recently, the beneficial effect
of D-serine in healthy individuals was described in a clinical
trial (37). In their work, Heresco-Levy et al. showed the pro-
cognitive effects of D-serine throughout NMDAR function and,
as we proposed in the current study, intended the develop-
ment of NMDAR–glycine site strategies for treating synapt-
opathy conditions. In agreement with this, D-serine deficits
have been associated with aging in rats, with a functional
rescue observed following exogenous D-serine administration
(38,39). In addition, serine deficiency disorders also provoke
neurological phenotypes (psychomotor retardation, micro-
cephaly, and seizures in newborns and children) that can be
safely treated by serine oral replenishment (40). Taken
together, these works and ours indicate that, independent of
the molecular etiology (serine racemase deficit, NMDAR
hypofunctionality), D-serine-potentiated NMDAR activity can
partially rescue hypoglutamatergic function. Noteworthy, in
addition to the beneficial effect of D-serine dietary supple-
mentation, the improved condition of the patient might also be

D-serine Treatment of Pathogenic GRIN2B De Novo Mutation
influenced by the developmental changes in expression of
GluN2 subunits, increasing the GluN2A/GluN2B subunit ratio
along development (9). Indeed, our findings indicate that
mutant triheteromeric NMDARs are less affected than the
diheteromeric mutant receptors. Therefore, in addition to the
NMDAR-potentiating effect of D-serine, the developmental
switch of GluN2 subunits might also contribute to improve the
clinical symptoms of the patient to some extent.
In summary, our data represent a proof-of-concept study to
identify the pathogenicity of de novo mutations of NMDARs
and the development of precision therapeutic strategies.
Indeed, the methodological pipeline developed in this study
might be further implemented to functionally stratificate de
novo iGluR mutations associated with synaptic dysfunctions
and to define therapeutic strategies. Moreover, it supports the
use of D-serine as a dietary supplement for the enhancement
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of glutamatergic neurotransmission and/or excitatory/inhibi-
tory neurotransmitter imbalance affected in a large spectrum of
neurological disorders.
ACKNOWLEDGMENTS AND DISCLOSURES
This work was supported by the Grant No. PI16/00851 (National Institute
of Health Carlos III [ISCIII]), La Marató (Project No. 20140210), PCIN-2014-
105 (Ministry of Economy and Competitiveness [MINECO]) and Miguel
Servet Program (Grant No. CPII16/00021, ISCIII) (all to XA); Grant No.
BFU2014-57562-P (Ministerio de Ciencia e Innovacion, to DS); Grant No.
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PI15/01082 (ISCIII) to AG-C); Grant No. SAF2016-77830-R (to MO); Grant
Nos. BFU2012-34398 and BFU2015-69717-P (MINECO), a Career Inte-
gration Grant (Ref. 304111), a Marie Curie Intra-European Fellowship
(Ref. 221540), and a Ramón y Cajal Fellowship (Ref. RYC-2011-08391p)
(all to AB); Grant No. SAF2012-40102 (MINECO/European Regional
Development Fund [FEDER]), an FP7 Marie Curie CIG Grant (Ref. 631035),
and Ramón y Cajal (Grant No. RYC-2011-08026) (all to CS); and Grant
Nos. PI14/00141 and RETIC RD16/0008/0014 (to XG). CA received an FPI
contract (MINECO). XA, CS, and AB also benefit from the financial support
of Agency for Management of University and Research Grants (Ref. No.
SGR14-297).
We thank Sandra Jurado for guidance in chemical long-term potentiation
experiments and the Medicinal Computational Laboratory from Universitat
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Autònoma de Barcelona for providing computing facilities. We also
acknowledge Pierre Paoletti (Ecole Normale Supérieure, Paris) for kindly
providing the plasmids allowing the studies with triheteromeric N-methyl-
D-aspartate receptors.
The authors report no biomedical financial interests or potential conflicts
of interest.
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ARTICLE INFORMATION
From the Institute of Neurosciences (DS, CA, XG, EG-B, CS, FC, VF-D),
Department of Biomedicine (DS, XG, EG-B), Department of Pathology and
Experimental Therapeutics (FC, VF-D), Bellvitge Biomedical Research
Institute–Unit of Neuropharmacology and Pain Group (CG, MGdS, VF-D, FC,
XA), and Department of Clinical Foundations (CA, CS), University of Bar-
celona, August Pi i Sunyer Biomedical Research Institute (DS, XG, EG-B),
Bioinfomatics and Medical Statistics Group (MO), University of Vic–Central
University of Catalonia, Genetics and Molecular Medicine Service (JA, AG-C)
and Department of Neurology (AL-S, AG-C), Neurometabolic Unit, Hospital
Sant Joan de Déu and Centro de Investigación Biomédica en Red de
Enfermedades Raras, Molecular Physiology of the Synapse Laboratory
(DR-V, AB), Biomedical Research Institute Sant Pau, and Autonomous
University of Barcelona, Bellaterra (DR-V, AB), Barcelona, Spain.
DS, MO, and CG contributed equally to this work.
AG-C and XA contributed equally to this work.
Address correspondence to Xavier Altafaj, Ph.D., Neuropharmacology
and Pain Unit–IDIBELL, Hosp. Duran Reynals, Av. Gran Via 199-203, 3rd
Biological Psychiatry January 15, 2018; 83:160–172 www.sobp.org/journal
Biological
Psychiatry
Floor, 08908 L’Hospitalet de Llobregat, Barcelona, Catalonia, Spain; E-mail:
xaltafaj@idibell.cat.
Received Sep 20, 2016; revised Apr 26, 2017; accepted May 17, 2017.
Supplementary material cited in this article is available online at http://dx.
doi.org/10.1016/j.biopsych.2017.05.028.
ED
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