However, recent data indicate that serine racemase, the d-serine biosynthetic enzyme, is widely expressed in neurons of the brain, suggesting that d-serine also has a neuronal origin.. d
Trang 1D-Amino acids in the brain: D-serine in neurotransmission and neurodegeneration
Herman Wolosker, Elena Dumin, Livia Balan and Veronika N Foltyn
Department of Biochemistry, B Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel
N-methyl d-aspartate receptors (NMDARs) are key
excitatory neurotransmitter receptors in the brain and
are involved in many physiological processes, including
memory formation, synaptic plasticity and
develop-ment [1] The NMDARs are composed of multiple
subunits and their activity is regulated by numerous
mechanisms, including different ligands and interacting
proteins [2] The NMDARs display high permeability
to Ca2+, which is known to play a central role in
syn-aptic plasticity and many signal transduction
mecha-nisms [1] NMDAR overstimulation promotes
neurotoxicity and is implicated in several pathological
conditions, such as stroke and neurodegenerative
dis-eases [3]
The NMDARs are unique in their requirement for
more than one agonist to operate Glutamate, the main
NMDAR agonist, does not activate the receptors unless
a co-agonist binding site located at the NR1 subunit is occupied [4,5] d-Serine, an unusual d-amino acid pres-ent in mammalian brain, is now recognized as a physio-logical ligand of the NMDAR co-agonist site, mediating several NMDAR-dependent processes [6–15]
At first, the NMDAR co-agonist site was thought
to be occupied by glycine Hence, the co-agonist site
is also generally referred to as the ‘glycine site’ In addition, to be essential for NMDAR activity, the co-agonist site exerts neuromodulatory roles Thus, co-agonist binding increases the receptor’s affinity for glutamate [16], decreases its desensitization [17] and promotes NMDAR turnover by internalization [18] Since its discovery, the role of the co-agonist site in regulating the activity of the NMDAR has been
Keywords
D -serine; gliotransmitter; glutamate;
glycine; L -serine; neurodegeneration;
neurotransmission; neurotoxicity;
NMDA receptor; schizophrenia;
serine racemase
Correspondence
H Wolosker, Department of Biochemistry,
B Rappaport Faculty of Medicine,
Technion-Israel Institute of Technology, Haifa 31096,
Israel
Fax: +972 4 8295384
Tel: +972 4 8295386
E-mail: hwolosker@tx.technion.ac.il
(Received 30 January 2008, revised 14 April
2008, accepted 22 May 2008)
doi:10.1111/j.1742-4658.2008.06515.x
The mammalian brain contains unusually high levels of d-serine, a d-amino acid previously thought to be restricted to some bacteria and insects In the last few years, studies from several groups have demonstrated that d-serine
is a physiological co-agonist of the N-methyl d-aspartate (NMDA) type of glutamate receptor – a key excitatory neurotransmitter receptor in the brain d-Serine binds with high affinity to a co-agonist site at the NMDA receptors and, along with glutamate, mediates several important physio-logical and pathophysio-logical processes, including NMDA receptor transmission, synaptic plasticity and neurotoxicity In recent years, biosynthetic, degrada-tive and release pathways for d-serine have been identified, indicating that
d-serine may function as a transmitter At first, d-serine was described in astrocytes, a class of glial cells that ensheathes neurons and release several transmitters that modulate neurotransmission This led to the notion that
d-serine is a glia-derived transmitter (or gliotransmitter) However, recent data indicate that serine racemase, the d-serine biosynthetic enzyme, is widely expressed in neurons of the brain, suggesting that d-serine also has
a neuronal origin We now review these findings, focusing on recent ques-tions regarding the roles of glia versus neurons in d-serine signaling
Abbreviations
ALS, amyotrophic lateral sclerosis; AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; ISH, in situ hybridization; LTP, long-term potentiation; NMDA, N-methyl D -aspartate; NMDAR, N-methyl D -aspartate receptor.
Trang 2controversial The high extracellular concentration of
glycine was assumed to saturate the co-agonist site
in vivo, with some supporting studies demonstrating
that this site is indeed saturated in tissue slices [19,20]
However, most studies now agree that the co-agonist
site is not saturated under resting conditions,
indicat-ing that co-agonist bindindicat-ing exerts a dynamic regulation
of the NMDARs [1,21]
As the dispute about the degree of saturation of the
co-agonist site became abated, a new controversy
emerged regarding the identity of the physiological
NMDAR co-agonist Like glycine, d-serine is a
high-affinity ligand of the co-agonist site, displaying up to
threefold higher affinity than glycine [22,23] At first,
d-amino acids like d-serine were not thought to exist
at significant quantities in eukaryotes Therefore, in
contrast to glycine, d-serine was not viewed as a
physi-ological ligand of NMDARs
The serendipitous discovery of large amounts of
endogenous d-serine in the brain, by Hashimoto et al.,
quickly changed this view [24] Following this
discov-ery, studies from several laboratories have shown that
endogenous d-serine is a physiological regulator of
NMDARs through binding to the co-agonist site
Endogenous d-serine has been implicated in several
physiological and pathological NMDAR-dependent
processes, including normal NMDAR transmission
and synaptic plasticity [6,7,10–12,14,15], cell migration
[9] and neurotoxicity [8,25–29]
A structural explanation for the selective effects of
d-serine on NMDARs comes from inspection of the
crystal structure of the binding core of the NR1
sub-unit of NMDARs d-Serine binds more tightly to the
receptor in comparison with glycine because it makes
three additional hydrogen bonds and displaces a water
molecule from the binding pocket [22] There is also
unique selectivity for the d-isomer of serine, as the
hydroxyl group of l-serine interacts unfavorably in the
binding pocket [22]
D-Serine – a physiological co-agonist
of NMDARs
d-Serine is present at very high levels in the
mamma-lian brain and at a much lower concentration in the
peripheral tissues (Fig 1) Brain d-serine accounts for
one-third of the l-serine and its levels are higher than
most essential amino acids [24,30] In contrast to
l-amino acids, d-serine is not incorporated into
pro-teins or peptides, thus constituting a free amino acid
pool Experiments of brain microdialysis show that
the extracellular concentration of endogenous d-serine
is twice that of glycine in the striatum and
compara-ble to the concentration of glycine in the cerebral cortex [31]
Hashimoto et al initially observed that d-serine was enriched in rat forebrain areas, where NMDARs are abundant [32] Subsequently, immunohistochemical studies carried out by the Snyder group (Fig 1) dem-onstrated that the regional distribution of d-serine in rat brain co-localized almost perfectly with that
of NMDARs [33,34] The density of d-serine is much lower in the caudal part of the brain, including the adult cerebellum and brainstem (Fig 1) This is because of the emergence of d-amino acid oxidase
in adult animals, which degrades endogenous d-serine almost completely in these regions [34,35]
In contrast with d-serine, glycine immunoreactivity
is higher in the caudal areas of the brain, where the density of NMDARs is lower [36] The inverse local-izations of d-serine and glycine led Schell et al to propose that endogenous d-serine was physically closer
to NMDARs than glycine
d-Serine is enriched in protoplasmic astrocytes, rais-ing the possibility that it is released from astrocytes ensheathing the synapse to activate neuronal NMDARs [33,34] d-Serine was subsequently shown to be present also in neurons, where the d-serine biosynthetic enzyme, serine racemase, is robustly expressed, indicat-ing that d-serine also has a neuronal origin [25,37–41]
A more direct demonstration that d-serine is a phys-iological NMDAR co-agonist arose from experiments that employed d-serine metabolic enzymes to remove,
in a selective manner, endogenous d-serine from brain slices and cultures, leaving the levels of glycine unchanged Using this strategy, endogenous d-serine was shown to mediate a variety of physiological NMDAR-dependent events
In a pioneer study, Snyder, Mothet et al depleted endogenous d-serine from neural cultures by applying
d-amino acid oxidase, which specifically degrades
H
T
S C
AON
OT
MOL
D -Serine
Fig 1 Localization of D -serine in the rat brain The highest D -serine densities (white areas) are observed in the forebrain AON, anterior olfactory nuclei; C, cerebral cortex; H, hippocampus; MOL, mole-cular layer of the cerebellum; OT, olfactory tubercule; S, striatum;
T, thalamus Reproduced from Schell et al [34].
Trang 3d-amino acids, but not l-amino acids Depletion of
d-serine led to a 60% decrease in the spontaneous
activity attributed to the postsynaptic NMDAR,
whereas
a-amino-3-hydroxy-5-methylisoxazole-4-propi-onic acid (AMPA) responses were unaffected [10]
Sub-sequent studies demonstrated that endogenous d-serine
is required for the NMDAR-mediated light-evoked
responses in the retina [6,12] Likewise, along with
glu-tamate, endogenous d-serine mediates the long-term
potentiation of synaptic activity in the hippocampus, a
region associated with learning and memory
[7,14,15,42]
One concern with the experiments employing
d-amino acid oxidase to deplete d-serine is that the
enzyme displays a very low affinity for d-serine –
about 50 mm at physiological conditions [43] As the
affinity of NMDARs to d-serine is at least five orders
of magnitude higher than that of d-amino acid
oxi-dase, it is conceivable that a significant fraction of
endogenous d-serine remains bound to the receptors
Furthermore, some commercial preparations of
d-amino acid oxidase contain many impurities that
may negatively affect the tissue preparation, including
large quantities of d-aspartate oxidase, which quickly
degrades N-methyl d-aspartate (NMDA) itself [28]
To overcome some of the limitations of the d-amino
acid oxidase treatment, new enzyme preparations were
developed to allow direct comparison between the
effects of d-serine and glycine in stimulating
NMDARs One of these new enzyme preparations is
the recombinant bacterial d-serine deaminase enzyme
This enzyme displays both high affinity and high
speci-ficity to d-serine, and efficiently degrades it in
organo-typic hippocampal slices [28], neuronal cultures [25] and retina preparations [6] We found that depletion
of endogenous d-serine by d-serine deaminase virtually abolished NMDA-elicited neurotoxicity in organotypic hippocampal slices (Fig 2) This indicates that d-ser-ine, and not glycd-ser-ine, is the dominant co-agonist required for the neuronal cell death elicited by NMDAR stimulation in the hippocampus (Fig 2) Likewise, the essential role of d-serine for NMDAR neurotoxicity was also observed in cortical slices subjected to ischemic cell death induced by oxygen⁄ glucose deprivation [8,26]
The dominant role of d-serine for NMDAR activity observed in neurotoxicity experiments is also sup-ported by electrophysiological experiments d-Serine is essential for the NMDAR-mediated light-evoked responses in the rat retina, shown by Miller et al to contain endogenous d-serine [6,12,44] A similar domi-nant role for endogenous d-serine in NMDAR trans-mission was observed in the supra-optic nucleus of the hypothalamus [11] In this study, Panatier et al dem-onstrated that a more efficient recombinant d-amino acid oxidase preparation destroyed d-serine from hypothalamic slices and blocked the NMDAR responses By contrast, the degradation of endogenous glycine by a glycine oxidase enzyme had no effect, suggesting that d-serine, rather than glycine, is the dominant NMDAR co-agonist in the supra-optic nucleus [11]
The role of endogenous d-serine as the foremost co-agonist of NMDARs, as suggested by some studies,
is at odds with the very high levels of extracellular glycine [31] In hippocampal organotypic slice cultures,
A B
C D
E F
NMDA + DsdA
(No D-serine) (No D-serine)
NMDA + MK-801 NMDA +DNQ
DsdA
Fig 2 The role of endogenous D -serine in NMDAR-elicited neurotoxicity The removal
of D -serine by D -serine deaminase enzyme (DsdA) completely prevented NMDA-elicited cell death (A) Control (B) NMDA (500 l M ) elicited robust cell death in all hippocampal areas, as measured by propidium iodide (PI) uptake (C) Control treated with DsdA (10 lgÆmL)1for 90 min) (D) Destruction of
D -serine by DsdA protected against NMDA-elicited cell death (E) The NMDA effect was prevented by addition of the antagonist MK-801 (F) The NMDA effect was not pre-vented by addition of the AMPA receptor antagonist, DNQX Densitometric analysis of
PI uptake revealed almost complete neuro-protection by the removal of endogenous
D -serine Reproduced with slight modifications from Shleper et al [28].
Trang 4the removal of endogenous d-serine completely
blocked the NMDAR-elicited neurotoxicity, even
though extracellular glycine was present at a level was
10-fold higher than d-serine [28] Glycine alone was
very inefficient in promoting NMDAR neurotoxicity
When d-serine was completely removed from the slice
cultures, the amount of glycine needed to cause
maxi-mal NMDAR neurotoxicity was two orders of
magni-tude higher than its dissociation constant from purified
NMDARs [28] Likewise, in hypoglossal neurons,
exogenously applied d-serine was almost two orders of
magnitude more effective than glycine in stimulating
NMDAR responses [45]
Why is glycine much less efficient than d-serine in
slice preparations? Glycine and d-serine display
com-parable affinities to NMDARs, and therefore the
dif-ference in their functional efficiency may be related to
the availability of the two co-agonists at synaptic or
extra-synaptic sites The synaptic glycine concentration
is efficiently regulated by a high-affinity glycine
trans-port that limits glycine access to NMDAR sites
[45,46] Accordingly, the addition of a selective
inhibi-tor of the GlyT1 transporter potentiates NMDAR
responses [47] and elicits NMDAR neurotoxicity after
the endogenous d-serine has been enzymatically
destroyed [28]
In contrast to glycine, d-serine behaves as a poorly
transported analogue Specific d-serine transporters
have not yet been identified, and neutral amino acid
uptake systems capable of transporting d-serine
dis-play only low-to-moderate affinity [48–50] It is
there-fore conceivable that d-serine can more easily reach
synaptic or extrasynaptic NMDARs by evading the
neutral amino acid re-uptake systems Nevertheless,
the relative contributions of d-serine versus glycine in
mediating NMDAR transmission are still largely
unknown Further studies will be required to map the
relative contributions of d-serine and glycine for
NMDAR transmission in different brain regions
The levels of d-serine are high in the cerebellum of
neonatal rats, decreasing to very low levels in the third
week of life as a result of the emergence of the
d-amino acid oxidase enzyme, which destroys
endoge-nous d-serine [34,35] The transient presence of
d-ser-ine in the cerebellum coincides with the postnatal
cerebellar development, in which granule cells migrate
from the external to the internal granule cell layer in
an NMDAR-dependent manner [51] Blockage of the
NMDAR at granule cells decreases the rate of
migra-tion [51] Bergman glia cells, which contain high levels
of endogenous d-serine, serve as a scaffold for granule
cell migration Endogenous d-serine, presumably
released by the Bergman glia, mediates the
NMDAR-dependent neuronal migration in the cerebellum [9]
As migrating granule cells do not make conventional synaptic connections, the modulatory action of glial-released d-serine reflects a novel mechanism for neuromodulation [9]
Origin of brain D-serine
The role of d-serine as a possible regulator of NMDARs was initially viewed with scepticism, or even ignored, for d-amino acids were not thought to be syn-thesized in mammals The discovery of the biosynthetic enzyme for d-serine paved the way for additional advances in the field We found that endogenous d-ser-ine is synthesized from l-serd-ser-ine by serd-ser-ine racemase, a brain-enriched enzyme [52–54] Serine racemase requires pyridoxal 5¢-phosphate as a cofactor and, in addition to racemization, it de-aminates l-serine into pyruvate and ammonia [52,55] The enzyme is unique among the pyridoxal 5¢-phosphate enzymes as a result
of its requirement for divalent cations and the Mg.ATP complex for its activity [52,56–58]
The regional localization of serine racemase matches those of endogenous d-serine, indicating a physiologi-cal role in d-serine synthesis [53] Preliminary reports indicate that serine racemase knockout mice display an 80–90% decrease in brain d-serine levels, confirming the role of serine racemase as the biosynthetic enzyme for d-serine [59–62] Serine racemase knockout animals exhibit decreased NMDAR transmission, impaired long-term potentiation of synaptic activity in the hippocampus, and are more resistant to stroke damage upon middle-cerebral artery occlusion [60,61] These preliminary reports support previous biochemical and electrophysiological data, indicating that d-serine is indeed a physiologically relevant endogenous co-agonist of NMDARs
Is D-serine a transmitter?
By adopting a more liberal conceptualization of a neu-rotransmitter, Snyder and Ferris proposed that d-ser-ine belongs to a new class of transmitters that only partially fulfill the criteria previously used to define classic neurotransmitters [63] The existence of a bio-synthetic pathway, a target receptor, an uptake system and a degradative enzyme for d-serine favors the notion that d-serine is indeed a neurotransmitter (Table 1) Unlike classical chemical transmitters, how-ever, d-serine was originally thought to be specifically produced and released from astrocytes, suggesting that
d-serine is a glial-transmitter (also known as a glio-transmitter) [33,34,64] A boost to the notion that
Trang 5d-serine is a gliotransmitter was recently provided by
Mothet et al who demonstrated that cultured
astro-cytes are capable of the vesicular release of d-serine
[65] In this study, AMPA receptor stimulation was
shown to promote the release of d-serine by
exocyto-sis, an effect blocked by cocanamycin A, an inhibitor
of the vesicular filling of neurotransmitters that blocks
the generation of the electrochemical proton gradient
across the vesicles [65] In support for a possible
ular localization of d-serine, Pow et al observed
vesic-ular-like structures containing d-serine in astrocytes in
situ, which may correspond to synaptic-like vesicles
[66] The question remains, however, whether the
vesic-ular pathway in cultured astrocytes surpasses
nonvesic-ular forms of d-serine release and whether the
vesicular release of d-serine occurs in more
physiologi-cal preparations or in vivo
In order to function as a gliotransmitter, d-serine
actions should depend on an intimate relationship
between astrocytes and neurons In an elegant study,
Oliet et al found that NMDAR transmission in the
supra-optic nucleus depends on the degree of astrocytic
coverage of neurons [11] The neuronal centers in the
supra-optic nucleus undergo an extensive reduction of
astrocytic ensheathing of its neurons and synapses under conditions such as lactation Using this model, the authors showed that lactating rats display reduced NMDAR activity compared with virgin rats as a result
of reduced levels of d-serine release The data indicate that variations in the astrocytic environment of neu-rons and synapses play a prominent role in the post-synaptic control of excitatory neurotransmission by releasing d-serine [11]
Key to the hypothesis that d-serine is a gliotransmit-ter is the notion that the electrophysiological effects of
d-serine should be attributable to astrocytic rather than neuronal release of d-serine Most studies demon-strating a role for d-serine in mediating NMDAR activity attributed its effects solely to glial d-serine and overlooked a possible neuronal origin Although glial
d-serine is prominent, a number of recent studies have reported the presence of d-serine also in neurons Thus, purified neuronal cultures were recently shown
to synthesize large amounts of d-serine [25] d-Serine was also identified in situ by immunohistochemistry in neurons of the nervous system (Fig 3), including the cerebral cortex [25,39], some nuclei of the hindbrain [38,39,66] and in ganglion cells of the retina [67]
Table 1 Some transmitter-like properties of brain D -serine ASCT, alanine, serine, cysteine and thrionine transporter.
Actions Modulates NMDAR transmission, long-term potentiation of synaptic activity,
NMDAR-elicited neurotoxicity and NMDAR-dependent cell migration
[6–12,14,15,25–29]
Metabolism D -amino acid oxidase enzyme and b-elimination catalyzed by serine racemase [34,55,96]
Transport Neutral amino acid transporters; Asc-1 in neurons and ASCT-like in astrocytes [49,50,97,98] Release Vesicular and nonvesicular release modes described in neurons and astrocytes [25,50,65,99,100]
D-ser
Fig 3 D -Serine localizes to neurons and astrocytes in the brain Staining for D -serine was performed in pyramidal neurons of layer V of the cerebral cortex and in astro-cytes in the corpus callosum of a P9 rat The lower panels depict double-labeling immunofluorescence for D -serine (labeled for SR in the original publication) and a neuronal nucleus marker (NeuN) in layer VI
of the cerebral cortex of a P9 rat Repro-duced with slight modifications from Kartvelishvily et al [25].
Trang 6Recently, Puyal et al showed that d-serine displays a
developmental glia-to-neuron switch In the vestibular
nuclei of young rats, d-serine is predominantly glial,
whereas in adult rats, d-serine is exclusively present in
neurons in these regions [38]
NeuronalD-serine in NMDAR regulation
Is there a role for neurons in synthesizing and
releas-ing d-serine? Although present at a lower level than in
astrocytes, d-serine is detectable in pyramidal neurons
of the cerebral cortex in situ (Fig 3) [25,39] Originally
regarded as an elusive or nonimportant source of
d-serine, the extent of the neuronal pool of d-serine
became apparent when we re-investigated the
expres-sion of serine racemase using new antibodies [25] We
observed widespread and prominent neuronal serine
racemase in situ, especially in the cerebral cortex and hippocampal formation, in which neuronal serine race-mase predominates (Fig 4A–C) Furthermore, recent studies indicate that cultured neurons contain both ser-ine racemase mRNA and protein, and catalyze the synthesis of d-serine to levels comparable to that observed with astrocytes [25,40,41] Neuronal staining for serine racemase was also recently observed in ganglion cells of the retina [67]
The neuronal expression of serine racemase was con-firmed by in situ hybridization (ISH), which revealed prominent serine racemase mRNA in neurons of the brain [41] and in neuronal ganglion cells of the retina [67] Like the immunohistochemistry for serine race-mase, the ISH of rat brain shows striking neuronal predominance [41] The neuronal-like distribution of serine racemase mRNA in the hippocampus is evident,
Fig 4 Localizations of serine racemase protein and mRNA in the brain (A) Staining for serine racemase in the cerebral cortex (Ctx) of a P7 rat (layers IV–VI) (B) Staining of neurons in the stratum pyramidale (Pyr) of the CA1 region of the hippocampus (C) Staining for serine race-mase in the pyramidal cell layers and dentate gyrus of the hippocampus (D) In situ hybridization (ISH) for serine racerace-mase in the hippocam-pus of adult mice, showing the highest serine racemase mRNA levels in pyramidal cell and dentate gyrus layers (saggital image series
392945, Srr_110, Allen Brain Atlas) (E) ISH of adult mouse brain (saggital image series 392945, Srr_110, Allen Brain Atlas) (F) Dark-field ISH for serine racemase in the hippocampus of adult mice using a 33 P-labelled RNA probe and silver grain emulsion (saggital image 38687, Gen-sat project) (G) Dark-field ISH for serine racemase in the hippocampus of adult mice (coronal image 36854, GenGen-sat project) (A–C) Repro-duced with slight modifications from Kartvelishvily et al [25] (D–E) ISH images from the Allen Institute of Brain Science [101,102], and the Gensat project [103] bs, brainstem; cb, cerebellum; cc, corpus callosum; ctx, cerebral cortex; DG, dentate gyrus; H or Hipp, hippocampus;
ob, olfactory bulb; st, striatum.
Trang 7with little or no serine racemase message in astrocytes
at the corpus callosum, as revealed by ISH from both
the Allen Institute for Brain Science and the Gensat
project (Fig 4D–G)
Does neuronal d-serine activate NMDARs? We
found that endogenous d-serine released by neuronal
cultures lacking significant levels of astrocytes mediates
a considerable fraction of NMDAR-elicited
neurotox-icity [25] Like astrocytes, cultured neurons release
d-serine in a regulated manner, involving ionotropic
glutamate receptor stimulation and depolarization by
KCl [25] In contrast to that previously reported with
cultured astrocytes [65], however, the neuronal d-serine
was not released through exocytosis of synaptic
vesi-cles under our experimental conditions [25] It remains
to be established whether neuron-derived d-serine
affects normal NMDAR transmission and if neurons
indeed release d-serine in more physiological
prep-arations
In light of the widespread expression of serine
race-mase in forebrain neurons, which lack significant levels
of d-amino acid oxidase, one would predict that
d-ser-ine should be present in all neurons A few studies
detected the presence of d-serine in some neuronal
populations in situ, including the pyramidal neurons of
the cerebral cortex [25,38,39] Neuronal d-serine,
how-ever, is scarcely seen in most studies We speculate that
this may be attributed to technical difficulties or to low sensitivity of the immunohistochemical methods to detect d-serine Being a small amino acid, d-serine may be poorly fixed by the commonly used fixatives,
or even released from cells during the perfusion of the brain In this framework, it is conceivable that the antibodies against d-serine miss many neuronal popu-lations that contain significant levels of d-serine Many questions remain to be solved regarding the relative roles of glia versus neurons in the synthesis and release of d-serine In the original model of d-ser-ine signaling, d-serd-ser-ine was thought to be exclusively released from astrocytes The predominance of serine racemase expression in neurons led us to propose an alternative model of d-serine signaling, in which d-ser-ine may be released from both neurons and astrocytes (Fig 5) This model assumes that the neuronal serine racemase enzyme is active towards d-serine synthesis and, like astrocytes, neurons release d-serine in a regu-lated manner (Fig 5)
The notion that neurons play a role in d-serine sig-naling does not exclude a role of glia in releasing d-serine, as d-serine is clearly enriched in protoplasmic astrocytes in the forebrain [33,34] One possibility is that the higher level of d-serine in astrocytes reflects the glial uptake of d-serine synthesized and released by neurons In this case, one would expect that d-serine
Fig 5 Proposed roles of glia and neurons in D -serine signaling The scheme depicts two modes of D -serine release A glia to neuron D -ser-ine flux would be achieved through activation of glial AMPA receptors by glutamate (reaction 1) [34] This leads to the release of astrocytic
D -serine, possibly from a vesicular pool [65], to activate neuronal NMDARs (reaction 2) Because serine racemase (SR) occurs predominantly
in neurons [25,41], astrocytes may obtain D -serine by re-uptake from the extracellular medium Alternatively, the higher ability of astrocytes
to synthesize L -serine from glucose [69] might also allow the synthesis of D -serine by some astrocytes containing serine racemase; the rela-tive importance of each pathway leading to astrocytic accumulation of D -serine is unknown A neuron to glia D -serine flux would be achieved
by the release of D -serine from neurons, presumably by membrane depolarization (reaction 3) [25] Released D -serine will activate NMDARs
or be taken up by astrocytes (reaction 4) It is not clear whether neuronal D -serine synthesis and release occur at presynaptic or postsynaptic sites Because neurons are mostly devoid of the ability to synthesize L -serine from glucose [69], they should rely on the export of L -serine from astrocytes (reaction 5) [70].
Trang 8uptake by astrocytes in vivo will be more efficient than
in neurons or, alternatively, that the d-serine half-life
would be longer in astrocytes Nevertheless,
experi-mental data demonstrating specific vesicular or
non-vesicular release of d-serine from astrocytes in brain
slices or in vivo are still lacking
The levels of l-serine relative to serine racemase
expression will also influence the distribution of
d-ser-ine l-Serine can be synthesized from either glycine or
glucose or be obtained by uptake from the extracellular
medium [68,69] It is known that, along with many
growth factors, astrocytes release l-serine to neurons
[70,71] Astrocytes have a higher l-serine content and
can synthesize it from the glycolytic intermediate
3-phosphoglycerate, an ability that neurons apparently
lack [72] The importance of the 3-phosphoglycerate
pathway for the synthesis of d-serine arose from the
observation that children exhibiting 3-phosphoglycerate
dehydrogenase deficiency display severe
neurodevelop-mental problems associated with lower levels of d-serine
in the cerebrospinal fluid [73] Thus, the ability of
astro-cytes to synthesize l-serine by the 3-phosphoglycerate
pathway might allow higher synthesis of d-serine, even
if the expression of serine racemase in astrocytes is lower
than in neurons On the other hand, neuronal synthesis
of d-serine will require conversion of glycine into
l-ser-ine by the serl-ser-ine hydroxymethyltransferase enzyme [74]
or uptake of l-serine from the extracellular medium
Serine racemase knockout mice will be a valuable tool
to ascertain definitively the cellular origin of d-serine
In light of the new data indicating a neuronal source of
d-serine, serine racemase knockout mice will be useful
in defining the specificity of serine racemase and
d-ser-ine antibodies previously employed, hopefully providing
a more definitive answer as to whether d-serine
origi-nates from neurons or astrocytes, or from both Indeed,
a preliminary study by Mori et al demonstrated, using
serine racemase knockout mice as controls, that serine
racemase is present mainly in neurons [62]
D-Serine in disease
As well as being important for normal NMDAR
trans-mission, NMDAR-dependent plasticity and
develop-mental processes (Fig 6), d-serine signaling
dysregulation might also be involved in the NMDAR
dysfunction that occurs in several pathologies,
includ-ing neuro-psychiatric and neurodegenerative diseases
(Fig 6)
An important pathological aspect of d-serine
signal-ing relates to NMDAR hypofunction thought to occur
in schizophrenia [75] NMDA antagonists, such as
phencyclidine, induce schizophrenic-like symptoms in
healthy volunteers, and precipitate thought disorder and delusions in schizophrenia patients [75,76] In mice, d-serine antagonizes the stereotypical behavior and ataxia caused by NMDAR antagonists [77] Mice expressing lower levels of the NMDAR1 (NR1) sub-unit display behavioral abnormalities, including increased motor activity and stereotypy, and deficits in social and sexual interactions, which are ameliorated
by conventional antipsychotic treatment [78]
Based on the NMDA hypofunction hypothesis, sev-eral clinical trials were carried out to evaluate the effi-cacy of stimulation of NMDAR in schizophrenia The administration of d-serine greatly ameliorated the posi-tive, negative and cognitive symptoms of schizophrenia when associated with conventional neuroleptics [79–81] Currently, five additional clinical trials are evaluating the effects of d-serine administration in schizophrenia in larger patient groups, which include both phase II and phase III studies
In addition to being a promising pharmacological treatment for schizophrenia, a number of recent stud-ies indicate that the level of endogenous d-serine may also be altered in the disease Schizophrenic patients display a higher ratio of l-serine to d-serine in the blood and cerebrospinal fluid [82–84] The possible involvement of d-serine in schizophrenia was also highlighted by genetic studies showing polymorphisms
in the genes of serine racemase [85] and of the d-serine metabolic enzyme, d-amino acid oxidase [86] Confir-mation of the above studies in larger populations will
be important to ascertain the role of endogenous d-ser-ine in the pathophysiology of schizophrenia
Fig 6 Multitude of D -serine functions D -Serine has been impli-cated in several physiological NMDAR-dependent processes, includ-ing normal transmission, synaptic plasticity and cell migration in the developing cerebellum D -Serine dysregulation may also play patho-logical roles in schizophrenia, ageing and acute and chronic neurodegeneration (see the text for references).
Trang 9Is d-serine dysregulation linked to cognitive deficits?
The long-term potentiation (LTP) of the synaptic
activity in the hippocampus has been thought to play
a role in memory formation [87] The role of
endo-genous d-serine in LTP [7,14,15,42] raises the possibility
that d-serine dysfunction might cause cognitive deficits
Although this possibility has not been directly
investi-gated, aged rats display a sharp decrease in
hippocam-pal d-serine and serine racemase expression [42] This
is associated with impaired LTP, which is reversed by
the addition of exogenous d-serine in aged rats [7]
By contrast, in young rats, LTP is not enhanced by
exogenous d-serine Thus, it is possible that the LTP
impairment observed in aged rats is caused by specific
deficits in local d-serine synthesis
The overproduction or excessive release of glutamate
has been widely implicated in a large number of acute
and chronic degenerative diseases The harmful effects
of excessive glutamate occur mainly through activation
of the NMDARs and consequently by massive calcium
influx into the cell [1] NMDAR over-activation is the
main culprit in the cell death that occurs following
stroke and in neurodegenerative diseases [3]
Blockers of NMDARs are neuroprotective in animal
models of stroke, but they were not well tolerated in
clinical trials because of the side effects caused by
NMDAR blockage, such as hallucinations [88,89]
Recently, low-affinity NMDAR inhibitors, like
memantine, have been proposed as an alternative to
high-affinity NMDAR blockers, and are indeed well
tolerated by patients [88,89] Similarly to low-affinity
NMDAR antagonists, serine racemase inhibitors offer
a more gentle approach to decrease NMDAR
activa-tion, and are likely to be better tolerated than
high-affinity antagonists In this framework, selective serine
racemase inhibitors provide a new strategy to prevent
stroke damage and cell death in neurodegenerative
dis-eases
Excessive production or release of d-serine may also
be involved in chronic neurodegeneration The levels
of d-serine and its biosynthetic enzyme, serine
race-mase, are greatly increased in the spinal cord of
patients with familiar and sporadic forms of
amyo-trophic lateral sclerosis (ALS) [27] Although the
motoneuronal cell death in ALS is widely attributed to
excessive AMPA receptor stimulation [90], a recent
study indicates that endogenous d-serine mediates
motoneuron cell death by excessive stimulation of
NMDAR in the spinal cord of ALS mice [27] In ALS
transgenic mice harboring the G93A mutation in
superoxide dismutase 1, activated microglia seem to be
the main source of spinal d-serine, constituting a
potential therapeutic target for ALS [27] Activation of
microglia by inflammatory stimuli induces overexpres-sion of serine racemase, an effect mediated by the c-Jun terminal kinase [27,91] Additionally, overexpres-sion of the G39A mutant, superoxide dismutase 1, pro-motes the upregulation of serine racemase in a c-Jun terminal kinase-independent manner [27] Removal of endogenous d-serine from spinal cord cultures of ALS transgenic mice protects the motoneurons against NMDAR-mediated cell death, linking d-serine to motoneuron degeneration [27] The overproduction of
d-serine by glia in ALS fits the notion that glial activa-tion⁄ dysfunction plays a role in the disease [92] In this context, inhibitors of serine racemase may provide a new neuroprotective strategy against ALS
Conclusion
d-Serine is now widely recognized as an important player in NMDAR transmission and in pathologies linked to NMDAR dysfunction Whether or not d-ser-ine satisfies all the criteria for a transmitter, its role in regulating NMDARs indicates an important physio-logical role There is still much to be learned regarding the regulation of d-serine signaling, including its bio-synthesis regulation and mechanisms of release While the experimental data so far favor a role of d-serine as
a transmitter, many of the effects previously attributed
to astrocytic d-serine release may also be caused by neuronal d-serine Furthermore, it is unclear whether
d-serine is physiologically released in a tonic manner
or in a fast and activity-dependent manner Further studies will be required to define the release pathways for d-serine from both neurons and astrocytes, and to clarify their relative contributions in d-serine-mediated NMDAR signaling
Acknowledgements
HW is supported by a grant from Israel Science Foun-dation
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