ASCT1 (Slc1a4) transporter is a physiologic regulator ofbrain D-serine and neurodevelopment
Eitan Kaplana, Salman Zubedatb, Inna Radzishevskya, Alec C. Valentac, Ohad Rechnitzd, Hagit Sasona, Clara Sajrawia,
Oded Bodnera, Kohtarou Konnoe, Kayoko Esakif, Dori Derdikmand, Takeo Yoshikawaf, Masahiko Watanabee,
Robert T. Kennedyc, Jean-Marie Billardg,h,1, Avi Avitalb,1, and Herman Woloskera,1aDepartment of Biochemistry, Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, 31096 Haifa, Israel; bDepartment of Physiology,
Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, 31096 Haifa, Israel; cDepartment of Chemistry, University of Michigan, Ann Arbor,
MI 48109; dDepartment of Neurosciene, Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, 31096 Haifa, Israel; eDepartment of
Anatomy, School of Medicine, Hokkaido University, 060-8638 Sapporo, Japan; fLaboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Wako,
351-0198 Saitama, Japan; gCenter of Psychiatry and Neuroscience, UMR 894, University Paris Descartes, 75014 Paris, France; and hU1075 INSERM, Universitéde Caen Normandie, 14032 Caen cedex 5, France

Edited by Robert H. Edwards, University of California, San Francisco, CA, and approved August 13, 2018 (received for review January 1, 2018)
D-serine is a physiologic coagonist of NMDA receptors, but little is

known about the regulation of its synthesis and synaptic turnover.
The amino acid exchangers ASCT1 (Slc1a4) and ASCT2 (Slc1a5) are
candidates for regulating D-serine levels. Using ASCT1 and ASCT2
KO mice, we report that ASCT1, rather than ASCT2, is a physiologic
regulator of D-serine metabolism. ASCT1 is a major D-serine uptake
system in astrocytes and can also export L-serine via heteroexchange, supplying neurons with the substrate for D-serine synthesis. ASCT1-KO mice display lower levels of brain D-serine alongwith higher levels of L-alanine, L-threonine, and glycine. Deletionof ASCT1 was associated with neurodevelopmental alterations including lower hippocampal and striatal volumes and changes inthe expression of neurodevelopmental-relevant genes. Furthermore, ASCT1-KO mice exhibited deficits in motor function, spatial
learning, and affective behavior, along with changes in the relative contributions of D-serine vs. glycine in mediating NMDA receptor activity. In vivo microdialysis demonstrated lower levels of
extracellular D-serine in ASCT1-KO mice, confirming altered D-serine metabolism. These alterations are reminiscent of some of theneurodevelopmental phenotypes exhibited by patients with ASCT1
mutations. ASCT1-KO mice provide a useful model for potential therapeutic interventions aimed at correcting the metabolic impairmentsin patients with ASCT1 mutations.
slc1a4

The alanine, serine, cysteine, and threonine exchangers
ASCT1 (SLC1a4) and ASCT2 (SLC1a5) transport D-serine intransfected HEK293 cells (11). ASCT2 has a broader substrateselectivity, acting on glutamine as well (12). However, their relevance in transporting D-serine or other brain amino acids in vivo
has not been evaluated.
To identify the L- and D-serine transporters in astrocytes andstudy their role in vivo, we investigated ASCT1-KO and ASCT2KO mice. We found that ASCT1, rather than ASCT2, is a component of the brain serine shuttle. ASCT1-KO mice display motor
and neurodevelopmental deficits reminiscent of ASCT1 missense
mutations in humans. The data indicate a role of ASCT1 inastroglia–neuron metabolic cross-talk and neurodevelopment.
Results
Role of ASCT1 and ASCT2 in Amino Acid Transport. We establishedASCT1-KO and ASCT2-KO mice colonies and studied their rolein brain amino acid transport. ASCT1-KO and ASCT2-KO

SignificanceD-serine regulates synaptic plasticity and behavior by binding

to the NMDA receptors. Astrocyte–neuron cross-talk provides
L-serine for the synthesis of D-serine in a process called the| D-serine | ASCT1 | Slc1a5 | glycine

serine shuttle. We show that the neutral amino acid antiporter
ASCT1 can release L-serine from astrocytes in exchange forD-serine and other amino acid substrates. Mice lacking ASCT1,
but not the paralogue ASCT2, display altered brain levels of
L-serine, D-serine, L-alanine, L-threonine, and glycine. Deletionof ASCT1 was associated with decreased volume of different
brain regions, altered gene expression, and motor and learning
deficits. Our study sheds light on the role of ASCT1 in brain
metabolism, with implications for the pathophysiology of theneurodevelopmental impairments observed in patients with
mutations in the ASCT1 gene.

N

MDA receptors (NMDARs) play key roles in neurodevelopment, synaptic plasticity, and learning and memory(1). Unlike other neurotransmitter receptors, the NMDARs require the binding of a coagonist (D-serine or glycine) in addition to
glutamate (1). D-serine is synthesized from L-serine by the serine
racemase (SR) (2). SR-KO mice display a 90% decrease in brain Dserine and exhibit impairments in NMDAR-dependent synaptic
plasticity (3–5). However, the mechanisms regulating D-serine
metabolism in the forebrain remain unknown. Early studies
reported exclusive astrocytic localizations of D-serine (6), leading to
the suggestion that D-serine is a gliotransmitter. However, recent
studies using more selective antibodies and SR-KO as controls
indicate that SR is predominantly expressed by neurons (7–9). In
agreement, selective deletion of SR from neurons decreases thesynaptic plasticity at the Schaffer collateral-CA1 hippocampal
synapses both in vitro and in vivo (5, 9).
Brain L-serine is synthesized from glucose by a series of enzymes
that include 3-phosphoglycerate dehydrogenase (3PGDH), whichis exclusively expressed in astroglia. Astrocyte-selective deletion of
3PGDH in mice strongly decreases the levels of neuronal D-serine(8). These data led us to propose an astrocyte–neuron metabolic
cross-talk called the serine shuttle, whereby L-serine synthesized
in astrocytes shuttles to neurons and fuels the synthesis of Dserine (10).
9628–9633 | PNAS | September 18, 2018 | vol. 115 | no. 38

Author contributions: H.W. conceived the project; D.D., J.-M.B., A.A., and H.W. designed
research; E.K., S.Z., I.R., A.C.V., O.R., H.S., C.S., K.K., K.E., M.W., J.-M.B., A.A., and H.W.
performed research; O.B., K.E., T.Y., and R.T.K. contributed new reagents/analytic tools;
E.K., S.Z., I.R., A.C.V., O.R., H.S., C.S., D.D., T.Y., M.W., R.T.K., J.-M.B., A.A., and H.W.
analyzed data; and H.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no.
GSE108675).
1

To whom correspondence may be addressed. Email: jean-marie.billard@inserm.fr,
avital@technion.ac.il, or hwolosker@tx.technion.ac.il.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1722677115/-/DCSupplemental.
Published online September 5, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1722677115

homozygotes are viable and fertile. ASCT1-KO mice had nodetectable ASCT1 protein in the brain (SI Appendix, Fig. S1).
Since the expression of ASCT2 in the brain is below the affinityof our antibody, we confirmed the lack of ASCT2 critical exonsin the brain of ASCT2-KO mice by PCR, along with lack ofprotein expression in the kidneys, where ASCT2 is highly
expressed (SI Appendix, Fig. S1).
We observed an 80–90% decrease in the transport of D-serineand L-serine in cultured astrocytes from ASCT1-KO mice (Fig. 1
A and B). Transport of typical ASCT1 substrates (e.g., L- alanineand L- threonine) was also significantly decreased (Fig. 1 C and
D). In addition, glycine transport by ASCT1-KO astrocytes was
40% lower than WT (Fig. 1E), while glutamate uptake was unaffected (Fig. 1F).
Under physiological conditions, other extracellular amino acidsubstrates may bind with high affinity to ASCT1 and prevent Dserine transport. Therefore, we tested the uptake of D-[3H]serinein the presence of typical extracellular concentrations of ASCT1
substrates [14 μM L-alanine, 36 μM L-serine, 2 μM L-cysteine,and 18 μM L-threonine (13)]. Under these conditions, ASCT1
still transported D-serine (Fig. 1G). In contrast, no ASCT1dependent uptake of glycine was detectable in the presence ofthe other ASCT1 substrates (Fig. 1H), suggesting glycine is apoor ASCT1 substrate.
However, the uptake of D-serine, L-serine, L-glutamine, andL-glutamate was unaltered in primary astrocyte cultures of
ASCT2-KO mice (Fig. 1 I–L).

Fig. 1. Amino acid transport by astroglia of ASCT1-KO and ASCT2-KO mice.
(A–H) Amino acid uptake in primary astrocyte cultures from WT and ASCT1KO mice carried out in Hepes-buffered saline solution (HBSS) supplemented
with 10 μM D-[3H]serine (A), L-[3H]serine (B), L-[14C]alanine (C), L-[3H]threonine (D), [3H]glycine, (E) or L-[3H]glutamate (F). (G and H) Uptake of either
10 μM D-[3H]serine (G) or [3H]glycine (F) was measured in HBSS supplemented with 14 μM alanine, 36 μM L-serine, 2 μM cysteine, and 18 μM
threonine (A,S,C,T). (I–L) Amino acid uptake in primary astrocyte culturesfrom WT and ASCT2-KO mice in HBSS supplemented with 10 μM D-[3H]serine(I), L-[3H]serine (J), L-[3H]glutamine (K), or L-[3H]glutamate (L). The results
represent the average ± SEM of four to seven experiments with different
astroglia preparations. Different from control at ***P < 0.001, **0.01, and*0.05 (paired two-tailed Student’s t test). ns, not a significant difference.

Kaplan et al.

In cortical synaptosomes, consisting of a mixture of neuronaland glial particles, we detected 30% lower uptake of L-serine andD-serine in preparations from ASCT1-KO mice (Fig. 2 A and B),
a percentage that represents the estimated glial contamination in
the synaptosomal preparation (14). We also monitored the effectof trans-4-hydroxy-L-proline (OH-Pro), a selective ASCT1 substrate that competes with other substrates (15). In agreement,
OH-Pro inhibited D-serine transport in HEK293 transfected with
ASCT1 to the same extent as excess L-alanine (SI Appendix, Fig.
S2A). OH-Pro was without effect on ASCT2 or the D-serine
transporter Asc-1 (SI Appendix, Fig. S2 B and C). OH-Pro
inhibited the uptake of D-serine (Fig. 2B) and L-serine (Fig.
2A) in WT synaptosomes but was without effect on preparations
from ASCT1-KO. In contrast with ASCT1, the uptake of typical
ASCT2 substrates (e.g., glutamine, L-serine, and D-serine) was
unaffected in synaptosomes prepared from ASCT2-KO mice
(Fig. 2D).
Brain Amino Acids in ASCT1-KO Mice. We measured the levels of
ASCT1 and ASCT2 substrates in the brains of their respective
KO mice and WT littermates at different ages. Brains from 4-dold (P4) ASCT1-KO mice exhibited a 20 and 25% decrease in Land D-serine levels, respectively (Fig. 2E). The levels of glycine,L-threonine, and L-alanine were 60–40% higher in ASCT1-KO
than in WT mice (Fig. 2E). A small 7% decrease in brain
L-glutamate was also detected in ASCT1-KO mice. It is noteworthy
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Fig. 2. Amino acid uptake in synaptosomes of ASCT1-KO and ASCT2-KO
mice and changes in brain amino acids. (A–C) Amino acid uptake in synaptosomes from WT and ASCT1-KO mice monitored in HBSS supplemented
with 1 μM D-[3H]serine (A), L-[3H]serine (B), or L-[3H]glutamate (C) either in
the absence or in the presence of 1 mM OH-Pro. (D) Amino acid uptake insynaptosomes of WT and ASCT2-KO mice. (E) Amino acid levels in wholebrains of P4 WT (n = 10) and ASCT1-KO (n = 11) mice. The results represent
the average ± SEM of four (A and B), five (D), and seven (C) experimentswith different preparations. Different from control at ***P < 0.001, **0.01
and *0.05. n.s., not a significant difference. Repeated measures one-way
ANOVA followed by Tukey’s post hoc test (A–C) or two-tailed Student’s
t test (D and E).

cerebral cortex, hippocampal CA1 region, and dentate gyrus(Fig. 3A). Immunofluorescence was abolished in ASCT1-KO mice
(Fig. 3B). Triple immunofluorescence experiments showed lackof colocalization of ASCT1 with cells expressing the neuronalmarker MAP2 (Fig. 3C). At high magnification, punctated
ASCT1 immunoreactivity is observed at the cell bodies andastroglial processes, where it colocalizes with 3PHGDH (Fig.
3C). The data indicate ASCT1 is mainly an astrocytic transporterin adult mice.

Fig. 3. Astroglial localization of ASCT1 in adult mice. (A) ASCT1 protein(red) colocalizes with the astrocytic marker 3PGDH (green) in WT mice but
not with neuronal marker microtubule-associated protein 2 (MAP2). (B) Lackof ASCT1immunoreactivity in ASCT1-KO mice. (C) High magnification of a
cortical astrocyte showing punctated immunoreactivity for ASCT1 thatcolocalizes with 3PGDH but is absent from MAP2-expressing cells. CA1, CA1
subfield of the hippocampus; Cx, neocortex; DG, dentate gyrus; gr, granulecells; py, pyramidal cell. (Scale bars, 10 μm.)that the ratio between the two NMDAR coagonists (glycine/D-serine) in ASCT1-KO mice brains was almost twice that in WT mice
(1.9-fold increase, P < 0.001, unpaired Student’s t test), indicating asignificant increase in total brain glycine relative to D-serine.
Serum levels of L-threonine in P4 ASCT1-KO mice pups weretwice the values of WT (SI Appendix, Fig. S3D). However, except
for this essential amino acid, serum levels of the other aminoacids in P4 mice were not correlated with their brain levels(compare Fig. 2E and SI Appendix, Fig. S3D). The decrease in Dserine levels along with the increase in glycine, L-alanine, andthreonine persisted in some brain regions of adult ASCT1-KO
mice (SI Appendix, Fig. S3 A–C), whereas their serum aminoacids were unchanged (SI Appendix, Fig. S3E). Therefore, thedevelopmental changes in brain amino acids observed in ASCT1KO mice are due to intrinsic changes in brain metabolism unrelated to the levels of serum amino acids.
The levels of different L-serine–derived sphingolipids in the
cerebral cortex and striatum were unaltered in ASCT1-KO mice
(SI Appendix, Fig. S3 F and G), suggesting that the supply of Lserine to lipid synthesis was largely unaffected in adult mice.
However, ASCT2-KO mice had normal levels of brain aminoacids (SI Appendix, Fig. S4), further indicating this transporter
does not affect mouse brain amino acid metabolism under normal conditions. Therefore, in the subsequent experiments, we
focused on investigating the role of its paralog ASCT1.
Astroglial Localizations of ASCT1. ASCT1 expression has been
demonstrated either in astrocytes (16) or predominantly inneurons (17). To determine the localizations of ASCT1, we
carried out immunohistochemistry using ASCT1-KO mice as
negative controls to ensure antibody specificity. We found that
ASCT1 colocalizes with the astrocytic marker 3PGDH in the9630 | www.pnas.org/cgi/doi/10.1073/pnas.1722677115

Neurodevelopmental Role of ASCT1. Mutations in ASCT1 cause
microcephaly in humans (18, 19). Using MRI we found a significant decrease in the volumes of the hippocampus and striatum of ASCT1-KO mice, indicating that they recapitulate someof the alterations seen in humans (Fig. 4). The results of thesemiautomated analysis were confirmed by manual analysis of
the hippocampus (SI Appendix, Fig. S5). No differences in volumes were observed at the neocortex, cerebellar cortex, andwhole brain (Fig. 4B).
We next carried out RNA sequencing of the striatum, which
was altered in MRI analysis, and compared it with the neocortex,
which was unaffected. We found significant changes in 375 transcripts in the striatum of adult mice at adjusted P values lower
than 0.05 (Fig. 4C and Dataset S1). No changes in gene expression
were observed in the neocortex (Fig. 4D). In the striatum, enrichment analysis using nonredundant reciprocal linkage of genes
identified 11 metagroups, with overrepresentation of genes linked
to axonal guidance and neurodevelopment (Fig. 4E and Dataset
S2). All metagroups had silhouette width >0.5 (Dataset S2), indicating a high degree of the internal tightness and distancefrom other metagroups (20).The most significant changes wereseen in axon guidance genes (Fig. 4E), suggesting changes inneurodevelopment-related gene transcripts in ASCT1-KO mice.

Fig. 4. Brain region volumes and gene expression in ASCT1-KO mice. (A)Hippocampus masking (red) on an average of WT population MRI scans. (B)Total brain and subregions volumes of 9- to 11-mo age-matched WT (n = 12)
and ASCT1-KO (n = 12) mice. (C and D) Plots of the delta difference (Log2) ingene expression between WT and ASCT1-KO mice (y axis) and mean normalized counts (x axis) generated in the striatum and neocortex. Differentially expressed genes are depicted in red symbols. (E) Enrichment analysis inmetagroups of differentially expressed genes in the striatum of WT and
ASCT1-KO mice according to Gene Ontology biological processes analyzed
by nonredundant reciprocal linkage of genes and biological terms. y axis
depicts adjusted P values. Different from control at ***P < 0.001 and **0.01.
n.s., not a significant difference. Unpaired two-tailed Student’s t test (B).

Kaplan et al.

Fig. 5. Behavioral characterization of ASCT1-KO mice. (A) Ambulation
scores of P8 WT (n = 42) and ASCT1-KO (n = 19) mice pups. (B) Righting timeshowing longer time latency in ASCT1-KO (n = 19) compared with WT (n =42) P8 mice (**P < 0.01). (C) ASCT1-KO mice (n = 19) display weaker grip
than WT (n = 23) P8 mice (**P < 0.01). (D) Hindlimb hanging scores of ASCT1KO (n = 19) and WT (n = 42) P8 mice. (E) ASCT1-KO mice (n = 19) display
shorter latency to fall in the hindlimb suspension test compared with WT(n = 42) P8 mice (***P < 0.001). (F) Reduced open-field activity of 4-mo-old
ASCT1-KO mice (**P < 0.01). (G) Rotor-rod performance of WT and ASCT1KO mice (*P < 0.05). (H) In the Morris water maze task, ASCT1-KO micereached the platform after longer periods of time on days 2 and 3 (**P <0.01) but attained the same performance as WT at day 4. Memory retrieval
by returning the platform and exposing the mice to the same context at days
50 and 51 show identical performance between WT and ASCT1-KO mice.
Spatial reversal carried out at days 52–54 revealed significant longer latency
in the ASCT1-KO mice (*P < 0.05, **P < 0.01). (I) ASCT1-KO mice displayed
slower swimming in the first 3 d of the spatial acquisition phase, but notduring spatial reversal (*P < 0.05, **P < 0.01, ***P < 0.001). Experimentalgroups in F–I consisted of 4- to 5-mo age-matched male mice (12 mice in eachgroup). (A–E) Two-tailed Mann–Whitney U test; (F–I) unpaired two-tailed
Student’s t test.

Behavioral Changes in ASCT1-KO Mice. We monitored the motor
function of mice pups at P8 to evaluate early dysfunction. The
ambulation pattern of ASCT1-KO mice was the same as that of
WT mice, both exhibiting slow crawling and some asymmetriclimb movements (Fig. 5A). However, ASCT1-KO mice exhibiteddelayed righting response when placed on their backs (Fig. 5B).
When placed on an iron mesh that was gradually inverted,
ASCT1-KO mice fell at lower angles, suggesting weaker grip
strength in the four limbs (Fig. 5C). When suspended by theirhind legs at the rim of a conical tube, ASCT1-KO mice pups
displayed normal posture (Fig. 5D) but exhibited a shorter latency to fall, suggesting weaker hindlimb strength (Fig. 5E).
Adult ASCT1-KO mice display some degree of motor dysfunction along with impairments in learning and affective domain. Adult ASCT1-KO mice exhibited lower activity in the
open field but no significant changes in freezing time or center
entries (Fig. 5F). ASCT1-KO mice had a 25% lower latency tofall off the rotor rod on the first day, indicating slightly impaired
sensorimotor coordination, although they reached the same latency of the WT in subsequent tests (Fig. 5G). In spatial learning
using a Morris water maze, it took ASCT1-KO mice longer tolearn the platform location (Fig. 5H, spatial acquisition).
Kaplan et al.

Synaptic Activity in ASCT1-KO Mice. The long-term potentiation(LTP) at the Schaffer collateral-CA1 synapses of the hippocampus was normal in ASCT1-KO mice (SI Appendix, Fig. S7A).
LTP in WT animals was unchanged by the addition of the
ASCT1 substrate OH-Pro (SI Appendix, Fig. S7B). The longterm depression induced by low-frequency stimulation was normal in ASCT1-KO mice (SI Appendix, Fig.S7C). No changes
were detected in basal neurotransmission or isolated NMDAR
potentials (SI Appendix, Fig. S7 D and E).
In light of their normal NMDAR activity, we wondered if thehigher levels of brain glycine in ASCT1-KO mice might compensate for the decrease in D-serine (Fig. 2E and SI Appendix,
Fig. S3 A–C). To investigate this possibility, we treated hippocampal slices of WT and ASCT1-KO mice with glycine oxidase
enzyme (GO) to remove glycine (6). To increase the sensitivity ofthe method, we employed six- to sevenfold lower GO (in activity
units) than in previous reports (6, 21). The use of a limiting
amount of GO allows only partial removal of endogenous glycine. Under this condition, GO treatment will likely be less effective in samples displaying higher glycine levels or increased
glycine release. We found that treatment with low GO titer decreased the expression of the NMDAR-dependent LTP from
WT mice by almost 50% (SI Appendix, Fig. S7F). LTP in slices
from ASCT1-KO mice were unaffected by the treatment withlow GO titer (SI Appendix, Fig. S7G), suggesting their higher
glycine levels might surpass the GO enzymatic capacity. In addition, we also monitored the effect of the glycine transporter 1
inhibitor ALX 5407, known to increase synaptic glycine (1). ALX
5407 augmented the isolated NMDAR potentials of both WT
and ASCT1-KO mice (SI Appendix, Fig. S7H). However, theextent of stimulation by ALX 5407 was slightly lower in slices
from ASCT1-KO mice compared with WT controls (SI Appendix, Fig. S7H), indicating that NMDARs from ASCT1-KO may
be slightly more saturated with glycine. We also examined place
cells in the CA1 region of mice by in vivo electrophysiology,
which showed their activity is preserved in ASCT1-KO mice,excluding significant changes (SI Appendix, Fig. S8).
Amino Acid Exchange by ASCT1 in Vitro and in Vivo. To investigate
the ability of ASCT1 in releasing endogenous amino acids, weinvestigated the effects of OH-Pro in acute cortical slices. As a
selective ASCT1 substrate, it is expected that OH-Pro will exchange with intracellular ASCT1 substrates. Perfusion with OHPro elicited release of L-serine in slices of WT mice but did notaffect the release of L-serine in slices of ASCT1-KO mice (Fig.
6A). Endogenous L-alanine and L-threonine were also released
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However, once formed, the retention of the spatial memory in
ASCT1-KO was the same as in WT mice as observed in theprobe trial (SI Appendix, Fig. S6 A and B) and memory retrieval
test (Fig. 5H). However, ASCT1-KO mice were significantly
impaired in spatial learning reversal when the platform location
was changed (Fig. 5H, spatial reversal), indicating impairments
in learning flexibility. The swimming speed of adult ASCT1-KO
mice was 25% slower than WT, indicating possible motor impairment (Fig. 5I). However, the lower swimming velocity per sedoes not explain the learning deficits. On day 1, despite thedifference in swimming speeds, WT and ASCT1-KO micereached the escape platform after similar delay (Fig. 5 H and I).
Furthermore, the swimming performance of ASCT1-KO mice
improved with subsequent trials and was identical to that of WT
during the spatial reversal test (Fig. 5I).
Amygdala-dependent fear learning monitored by the active
two-way avoidance test was normal in ASCT1-KO mice (SIAppendix, Fig. S6C). ASCT1-KO mice displayed a 60% increasein startle response, known to be enhanced in anxiety (SI Appendix, Fig. S6D). However, the ASCT1-KO mice did not exhibitchanges in prepulse inhibition (SI Appendix, Fig. S6E), indicating
an intact sensorimotor gating. A sucrose preference test showeddecreased preference at 72 h (SI Appendix, Fig. S6F) but normal
fluid intake (SI Appendix, Fig. S6G).

by OH-Pro in acute slices (Fig. 6 B and C). Although D-serine isa bona fide ASCT1 substrate for uptake, addition of OH-Pro did
not elicit the release of endogenous D-serine, indicating D-serine
is preferentially taken up via ASCT1 (Fig. 6D). Likewise, OHPro did not elicit detectable release of glycine (Fig. 6E).
We subsequently monitored the extracellular concentrationsof typical ASCT1 substrates by in vivo microdialysis of thestriatum. Values were corrected by the in vivo recovery throughinfusion of stable-isotope-labeled amino acids (22). ASCT1-KO
mice displayed a 25% decrease in the steady-state extracellularlevels of D-serine, with no change in the extracellular L-serine
levels (Fig. 6 F and G). In addition, ASCT1-KO mice exhibiteda decrease in the ratio of extracellular D-serine/total serine(Fig. 6H). We found no change in the extracellular concentrations of alanine, threonine, and glycine in adult ASCT1-KO mice
(Fig. 6 I–K).
Discussion
We identified ASCT1 as an important D- and L-serine transporter in astrocytes and a potential player in the serine shuttle
between glia and neurons (Fig. 6L). ASCT1 is an obligatoryexchanger that displays faster kinetics than unidirectional aminoacid transporters (12). Its exchange activity will likely facilitate
reaching a steady-state concentration gradient across the membrane by increasing the rates of transport of its substrates. This isin agreement with our observations in primary astrocyte cultures,
synaptosomes, and acute brain slices. Our in vivo microdialysis
data, however, indicate that ASCT1 does not alter the absolute
extracellular amino acid concentrations at steady state, except
for D-serine. Extracellular levels of D-serine were 25% lower inthe striatum of ASCT1-KO mice (Fig. 6G). The decrease in
extracellular D-serine is in agreement with the lower levels of
total brain D-serine in young and adult ASCT1-KO mice (Fig. 2
and SI Appendix, Fig. S3 A–C).
We propose that ASCT1 carries out bidirectional transport of all
its neutral amino acid substrates but D-serine (Fig. 6L). Although
ASCT1 catalyzes the release of previously loaded D-[3H]serine from
cells (11), activation of ASCT1 heteroexchange by OH-Pro in acutecortical slices did not release endogenous D-serine. This result isin agreement with previous studies demonstrating much lower
levels of endogenous D-serine in astrocytes compared with neurons (5, 7–10). Conceivably, the low amounts of D-serine in thecytosol of astrocytes fall below the affinity of the ASCT1.
The decrease in both the total and the extracellular levels of Dserine observed in ASCT1-KO mice suggests a partial impairment in brain D-serine production. L-serine made in astrocytes
appears to shuttle to neurons to fuel the synthesis of D-serine in aprocess called the serine shuttle (Fig. 6L) (10). Even thoughextracellular L-serine was unchanged in adult ASCT1-KO mice,
our observations that ASCT1-KO mice are deficient in L-serine
transport imply that the equilibrative nature of the ASCT1
transporter is required to provide enough L-serine for D-serine
synthesis in the local contacts between astrocytes and neurons(Fig. 6L). Further evidence supporting a role of ASCT1 in theserine shuttle comes from our findings demonstrating a significant decrease in both L-serine and D-serine in brains from
ASCT1-KO P4 mice (Fig. 2E). However, in vivo microdialysis
was impractical in P4 mice pups, where changes in L-serine andother ASCT1 substrates were more prominent than in adult mice(compare Fig. 2E and SI Appendix, Fig. S3). Our data, however,
do not exclude that other L-serine transporters may partiallycompensate for the lack of ASCT1 in the KO mice. Other
components of the serine shuttle, such as the neuronal uptake
system for L-serine, still remain to be identified.
In contrast to D-serine, levels of L-serine–derived sphingolipids
were normal in ASCT1-KO mice. This might be due to the highaffinity exhibited by the serine palmitoyltransferase (SPT), the key
enzyme for sphingolipid biosynthesis. SPT has a 30-fold higher
affinity for L-serine (Km ∼ 0.3 mM) (23) compared with the Dserine biosynthetic enzyme, SR (Km ∼ 10 mM) (10). Therefore,
9632 | www.pnas.org/cgi/doi/10.1073/pnas.1722677115

Fig. 6. ASCT1-mediated amino acid exchange in slices and in vivo microdialysis. (A–E) Release of endogenous L-serine (A), L-alanine (B), L-threonine(C), D-serine (D), and glycine (E) was elicited by perfusion of slices with 1 mM
OH-pro at the indicated times. The data are average ± SEM of sevenexperiments. Different from ASCT1-KO mice at *P < 0.05 and ***0.001 (twoway repeated measures ANOVA and Bonferroni’s post hoc test). (F–K) Extracellular amino acid concentrations in the striatum of 6- to 7-mo-old miceby in vivo microdialysis (12 mice per group). The data represent the mean ±SEM of the extracellular amino acid concentrations calibrated by therecovery in vivo. **Different from WT at P < 0.01, unpaired two-tailed
Student’s t test. (L) Proposed model of ASCT1 function in the serine shuttle. Astrocytic ASCT1 releases L-serine, which is subsequently taken up by
neurons to fuel the synthesis of D-serine by the SR. D-serine may be releasedby neurons via the Asc-1 (Slc7a10) transporter (26) and subsequently taken
up by ASCT1 in astrocytes. L-alanine, L-threonine, and D-serine may serve as
extracellular substrates that stimulate L-serine export via ASCT1 by heteroexchange. Impairment in L-serine transport in ASCT1-KO mice may lead toincreased conversion of L-serine into glycine by the serine hydroxymethyltransferase (SHMT) enzyme. When the serine shuttle is impaired, glycine
taken up by neurons (dashed arrow) may provide a compensatory pathway
for supplying L-serine to neurons via neuronal SHMT. Adapted with permission from Servier Medical Art, https://smart.servier.com/.

the low affinity of SR for L-serine makes the D-serine synthesis
particularly sensitive to impairments in the serine shuttle.
Our study also uncovers the role of ASCT1 in alanine, threonine, and glycine metabolism, as their total brain levels increased in ASCT1-KO mice. Astrocytes are known to carry out
the metabolism of alanine into lactate (24), whereas threonine isan essential amino acid that reaches the brain via the blood–
brain barrier. Conceivably, deficient alanine and threonine
transport in ASCT1-KO mice will partially impair their metabolism in astrocytes and lead to their accumulation. However,glycine is a poor ASCT1 substrate (Fig. 1H). Higher levels ofglycine in ASCT1-KO mice may be a consequence of L-serine
transport impairment. Deficient exchange of L-serine in ASCT1KO astrocytes may favor L-serine conversion into glycine by theserine hydroxymethyl transferase enzyme (Fig. 6L).
Kaplan et al.

developing therapeutic interventions to prevent or reduce theneurodevelopmental deficits in patients with mutations in ASCT1.

Interestingly, our results using both enzymatic removal ofendogenous glycine and a glycine transport inhibitor suggest
higher occupancy of NMDARs by glycine in ASCT1-KO mice.
Since glycine is also a regulator of synaptic NMDARs (4, 11, 21),higher glycine occupancy might help to preserve NMDAR, as wefound in ASCT1-KO mice. Furthermore, higher brain glycine
may provide a compensatory mechanism to overcome impairments in the serine shuttle, as glycine released from astrocytes
may be taken up by neurons and subsequently converted into Lserine (Fig. 6L, dotted line). Future studies will be required toclarify the exact role of ASCT1 in alanine, threonine, and glycine
metabolism. Our study also does not provide information on therole of ASCT1 in cysteine transport. Although L-cysteine is a
substrate for ASCT1 in vitro, almost all extracellular cysteine ispresent in the form of cystine, which is imported into the cells bya dedicated cystine/glutamate transporter (25).
Our finding of lower striatal and hippocampal volumes in
ASCT1-KO mice associated with changes in the expression ofneurodevelopment-relevant genes partially recapitulates some of
the neurodevelopmental alterations in patients with mutations in
ASCT1 transporter (18, 19). We found that ASCT1-KO micealso display motor impairments and behavioral deficits inlearning and affective tests. ASCT1-KO mice exhibited impaired
spatial learning in the Morris water maze, which cannot be solely
explained by their lower swimming speed. Although the ASCT1KO mice displayed smaller hippocampal volumes, extensive exvivo and in vivo electrophysiology did not unveil hippocampal
dysfunction under our recording conditions. It is likely that the
combined impairments of motor function (e.g., lower swimming
velocity, open field activity, and latency in the rotor rod) alongwith changes in the affective behavior (lower sucrose preference
and possible anxiety as revealed by higher startle response) may
have contributed to the learning impairment in the Morris
water maze.
The phenotypic abnormalities of ASCT1-KO mice differ fromthose exhibited by SR-KO mice, which mostly lack D-serine (3).
SR-KO mice have normal motor function, unaltered startle response, and no evidence of impairment in learning acquisition in
the Morris water maze. Therefore, the motor and learning abnormalities observed in ASCT1-KO mice likely result from the
combination of D-serine deficits with a broader metabolic change
in amino acids we described here, including alanine, threonine,and glycine. In this framework, ASCT1-KO mice will be useful for

ACKNOWLEDGMENTS. We thank O. Schwartz and Dr. E. Suss-Toby (BCF
Bioimaging Center, Faculty of Medicine, Technion) for the MRI; Dr. Itamar
Khan for guidance and supervision in MRI analysis; and Drs. Liat Linde,
Dr.Ronit Modai-Hod, Elizabeta Ginzburg, and Doron Fogel (BCF Genomics
Center, Faculty of Medicine, Technion) for RNA sequencing. This work was
funded by the Israel Science Foundation (H.W.) and the Vivian and Oscar
Lasko fund for Parkinson's and Alzheimer Research (H.W.), the Allen andJewell Prince Center for Neurodegenerative Disorders of the Brain (H.W.,
D.D., and A.A.), and Michigan-Israel Partnership for Research and Education
funds (H.W. and R.T.K.). J.-M.B. was supported by the Institut National de laSanté et de la Recherche Scientifique. T.Y.’s research was supported by Japan
Agency for Medical Research and Development (AMED) under Grant
JP18dm0107083, AMED-Core Research for Evolutional Science and Technology under Grant JP18gm0910004, and Ministry of Education, Culture, Sports,
Science and Technology under Grant JP18H05435.

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Kaplan et al.

Materials and Methods
Animals. Slc1a4tm1e(KOMP)Wtsi mice (ASCT1-KO) were obtained from Sanger Institute on a C57BL/6N genetic background. Slc1a5 KO mice (ASCT2-KO) wereobtained from Taconic and back-crossed in our facility with C57BL/6J mice for 10
generations. All animal procedures were in accordance with the Committee for
the Supervision of Animal Experiments (Technion – Israel Institute of Technology).
Western Blots. The brains were homogenized in buffer containing 20 mM Tris·
HCl (pH 7.4), 100 mM NaCl, 0.4 mM PMSF, and 1 mM EDTA. The homogenate
was centrifuged at 1,500 × g for 10 min and the supernatant was collected
and then centrifuged at 200,000 × g for 30 min. The pellet, containing themembrane fraction, was subjected to SDS/PAGE followed by Western blot.
For ASCT2 western blot, the samples were first deglycosylated by adding
1,000 units of PNGase F under nondenaturing conditions according to the
manufacturer’s instructions (New England Biolabs). To minimize the aggregation of membrane proteins, the samples were not boiled after addition ofsample buffer containing SDS.

PNAS | September 18, 2018 | vol. 115 | no. 38 | 9633

NEUROSCIENCE

Amino Acid Uptake in Cell Cultures. Primary astrocyte cultures were obtained
from the cortices of P0–P2 mice as previously described (11) and used
12–14 d after plating. For uptake experiments, cultures were incubated in
96-well plates for 5–7 min with 1 μM [3H]amino acids in Hepes-bufferedsaline (HBS, in millimolar: 137 NaCl, 5.4 KCl, 0.18 Na2HPO4, 0.44 KH2PO4,
0.41 MgSO4, 0.49 MgCl2, 1.07 CaCl2, 5.6 D-glucose, 4.2 NaHCO3, 1 Na-pyruvate,
and 10 Hepes, pH 7.4) at 25 °C. The [3H]amino acid transport was terminatedby washing the cells four times with ice-cold HBS and processed as previously
described (11).
Additional methods are described in SI Appendix, Supplemental Methods.