Loss of Glial Glutamate and Aspartate Transporter (Excitatory Amino Acid Transporter 1) Causes Locomotor Hyperactivity and Exaggerated Responses to Psychotomimetics: Rescue by Haloperidol and Metabotropic Glutamate 2/3 Agonist

Article · July 2008with145 Reads
DOI: 10.1016/j.biopsych.2008.05.001 · Source: PubMed
Abstract
Recent data suggest that excessive glutamatergic signaling in the prefrontal cortex may contribute to the pathophysiology of schizophrenia and that promoting presynaptic glutamate modulation via group II metabotropic glutamate 2/3 (mGlu2/3) receptor activation can exert antipsychotic efficacy. The glial glutamate and aspartate transporter (GLAST) (excitatory amino acid transporter 1 [EAAT1]) regulates extracellular glutamate levels via uptake into glia, but the consequences of GLAST dysfunction for schizophrenia are largely unknown. We examined GLAST knockout mice (KO) for behaviors thought to model positive symptoms in schizophrenia (locomotor hyperactivity to novelty, exaggerated locomotor response to N-methyl-d-aspartate receptor [NMDAR] antagonism) and the ability of haloperidol and the mGlu2/3 agonist LY379268 to normalize novelty-induced hyperactivity. Glial glutamate and aspartate transporter KO consistently showed locomotor hyperactivity to a novel but not familiar environment, relative to wild-type (WT) mice. The locomotor hyperactivity-inducing effects of the NMDAR antagonist MK-801 was exaggerated in GLAST KO relative to WT. Treatment with haloperidol or LY379268 normalized novelty-induced locomotor hyperactivity in GLAST KO. Schizophrenia-related abnormalities in GLAST KO raise the possibility that loss of GLAST-mediated glutamate clearance could be a pathophysiological risk factor for the disease. Our findings provide novel support for the hypothesis that glutamate dysregulation contributes to the pathophysiology of schizophrenia and for the antipsychotic potential of mGlu2/3 agonists.
3 Figures
RESEARCH REPORTS
Loss of Glial Glutamate and Aspartate Transporter
(Excitatory Amino Acid Transporter 1) Causes
Locomotor Hyperactivity and Exaggerated Responses
to Psychotomimetics: Rescue by Haloperidol and
Metabotropic Glutamate 2/3 Agonist
Rose-Marie Karlsson, Kohichi Tanaka, Markus Heilig, and Andrew Holmes
Background: Recent data suggest that excessive glutamatergic signaling in the prefrontal cortex may contribute to the pathophysiology
of schizophrenia and that promoting presynaptic glutamate modulation via group II metabotropic glutamate 2/3 (mGlu2/3) receptor
activation can exert antipsychotic efficacy. The glial glutamate and aspartate transporter (GLAST) (excitatory amino acid transporter 1
[EAAT1]) regulates extracellular glutamate levels via uptake into glia, but the consequences of GLAST dysfunction for schizophrenia are
largely unknown.
Methods: We examined GLAST knockout mice (KO) for behaviors thought to model positive symptoms in schizophrenia (locomotor
hyperactivity to novelty, exaggerated locomotor response to N-methyl-
D-aspartate receptor [NMDAR] antagonism) and the ability of
haloperidol and the mGlu2/3 agonist LY379268 to normalize novelty-induced hyperactivity.
Results: Glial glutamate and aspartate transporter KO consistently showed locomotor hyperactivity to a novel but not familiar environ-
ment, relative to wild-type (WT) mice. The locomotor hyperactivity-inducing effects of the NMDAR antagonist MK-801 was exaggerated in
GLAST KO relative to WT. Treatment with haloperidol or LY379268 normalized novelty-induced locomotor hyperactivity in GLAST KO.
Conclusions: Schizophrenia-related abnormalities in GLAST KO raise the possibility that loss of GLAST-mediated glutamate clearance could
be a pathophysiological risk factor for the disease. Our findings provide novel support for the hypothesis that glutamate dysregulation
contributes to the pathophysiology of schizophrenia and for the antipsychotic potential of mGlu2/3 agonists.
Key Words: GLAST, glutamate, mGlu2/3, NMDA, schizophrenia
G
lutamatergic dysfunction is increasingly implicated in the
pathophysiology of schizophrenia (1,2). Patients with
schizophrenia exhibit alterations in glutamate receptors
in brain regions functionally compromised in schizophrenia,
such as the hippocampus and prefrontal cortex (PFC) (3).
Furthermore, treatment with N-methyl-
D-aspartate receptor
(NMDAR) antagonists such as ketamine and phencyclidine (PCP)
mimic the symptoms of schizophrenia in healthy subjects and
provoke relapse in schizophrenic patients (4,5). In rodents,
NMDAR antagonists produce a range of schizophrenia-related
behavioral abnormalities including locomotor hyperactivity and
cognitive dysfunction (6,7). While the psychotomimetic effects of
NMDAR antagonists has fostered the notion of a hypoglutama-
tergic state in schizophrenia, recent data suggest that these
effects stem from PFC glutamate excess caused by disinhibition
of NMDAR-containing gamma-aminobutyric acid (GABA)ergic
interneurons (8–13). In support of this hypothesis is preliminary
evidence of antipsychotic efficacy of group II metabotropic
glutamate 2/3 (mGlu2/3) agonists (14) that negatively modulate
glutamate in PFC (15).
These developments raise the possibility that molecular ab-
normalities leading to PFC glutamate excess could be risk factors
for schizophrenia. Glial glutamate and aspartate transporter
(GLAST) (excitatory amino acid transporter 1 [EAAT1]), glial
glutamate transporter-1 (GLT-1) (excitatory amino acid trans-
porter 2 [EAAT2]), excitatory amino-acid carrier 1 (EAAC1)
(excitatory amino acid transporter 3 [EAAT3]), and excitatory
amino acid transporter 4 (EAAT4) belong to a family of sodium-
dependent glutamate transporters that tightly regulate extracel-
lular concentrations (16,17). Glial glutamate and aspartate trans-
porter is mainly expressed in astrocytes within the rodent
cerebellum but is also found, albeit in lesser concentrations, in
hippocampus, cerebral cortex, and thalamus (16,17). Antisense
knockdown of GLAST has been shown to cause increased levels
of extracellular glutamate concentrations in hippocampus and
striatum and increased vulnerability to glutamatergic toxicity
(18). Intriguingly, genetic mutation of human GLAST (SLC1A3)
was recently linked to schizophrenia (19). However, the conse-
quences of disrupted GLAST function for schizophrenia-related
behavioral phenotypes have not been studied, in part due to a
lack of GLAST-specific pharmacological tools.
Here, we tested a GLAST knockout (KO) model (20) for pheno-
types considered relevant to the positive symptoms of schizophre-
nia (locomotor hyperactivity to novelty, hypersensitivity to NMDAR
antagonists) (6,7). We then tested whether the novelty-induced
hyperlocomotor phenotype was reversible with a typical antipsy-
chotic (haloperidol) and an mGlu2/3 agonist (LY379268).
From the Laboratory for Clinical and Translational Studies (R-MK, MH), Na-
tional Institute on Alcoholism and Alcohol Abuse, National Institutes of
Health, Bethesda; Laboratory of Molecular Neuroscience (KT), School of
Biomedical Science and Medical Research Institute, Tokyo Medical and
Dental University Bunkyo-ku, Tokyo, Japan; and Section on Behavioral
Science and Genetics (AH), Laboratory for Integrative Neuroscience,
National Institute on Alcoholism and Alcohol Abuse, National Institutes
of Health, Rockville, Maryland.
Address reprint requests to Rose-Marie Karlsson, MSc, Laboratory for Clinical
and Translational Studies, National Institute on Alcohol Abuse and Alco-
holism, National Institutes of Health, 10 Center Drive, Building 10 Room
1-5330, Bethesda, MD 20892; E-mail: karlssonr@mail.nih.gov.
Received December 17, 2007; revised April 8, 2008; accepted May 6, 2008.
BIOL PSYCHIATRY 2008;64:810 8140006-3223/08/$34.00
doi:10.1016/j.biopsych.2008.05.001 Published by Society of Biological Psychiatry
Methods and Materials
Subjects
Glial glutamate and aspartate transporter KO were generated
as previously described (20) (Supplement 1). Experimental pro-
cedures were approved by the National Institute on Alcohol
Abuse and Alcoholism (NIAAA) Animal Care and Use Committee
and followed the National Institutes of Health (NIH) guidelines,
“Using Animals in Intramural Research.”
Locomotor Activity in Novel and Familiar Environment
Locomotor responses were tested in a novel square arena
(40 40 35 cm, 55 lux) constructed of white Plexiglas as
previously described (21). Distance traveled and time spent in
the center (20 20 cm) were measured over 60 min via
Ethovision (Noldus Information Technology Inc., Leesburg, Vir-
ginia).
Locomotor activity in a familiar environment was assessed
over 24 hours (after 48 hours acclimation) in individual home
cages under normal vivarium conditions via the photocell-based
Opto M3 activity monitor (Columbus Instruments, Columbus,
Ohio) (21).
Locomotor Response to MK-801
To circumvent basal hyperactivity to novelty in GLAST KO
confounding assessment of the hyperactivity-inducing effects of
MK-801, mice tested for the response to novel stimulus (above)
were re-exposed to the open field and acclimated for a further 60
min. Mice were then injected with .2 mg/kg MK-801 and returned
to the open field for 60 min (further details in Supplement 1).
Locomotor Effects of Haloperidol and mGlu2/3 Agonist
LY379268
Open field naïve mice were injected intraperitoneal (IP) with
vehicle or .3 mg/kg haloperidol and tested in the open field for
30 min as above.
Before testing the effects of LY379268 in GLAST KO, we first
established doses sufficient to normalize phencyclidine-induced
locomotor activity in nonmutant male C57BL/6J mice. Mice were
injected with saline or .3, 1.0, or 3.0 mg/kg LY379268 and 30 min
later, were placed in the open field. After 60 min of acclimation,
mice were injected with 5.0 mg/kg phencyclidine and tested in
the open field for 60 min as above.
On the basis of the C57BL/6J dose-response data (Supplement
2), open field naïve GLAST KO were injected with vehicle or 1.0
mg/kg LY379268 and 30 min later tested in the open field for 30
min as above.
Statistical Analysis
Data were analyzed using Statistica (Statsoft, Tulsa, Okla-
homa). Effects of genotype, sex, drug treatment, and time were
analyzed using analysis of variance (ANOVA) (repeated mea-
sures for time) followed by Tukey post hoc tests.
Results
Locomotor Activity in Novel and Familiar Environment
In the novel open field, there was a significant main effect of
genotype [F (2,45) 10.58, p .01] and time [F(11,495) 2.00,
p .05] but no genotype time interaction for distance traveled
(Figure 1A). Post hoc analysis revealed that KO were significantly
more active than wild-type (WT) during novel open field exposure.
We also noted a significant main effect of sex [F(1,45) 4.70, p
.05] and significant genotype sex interaction [F (2,45) 3.39, p
.05] for distance traveled and therefore further analyzed
locomotor activity separately within each sex. This confirmed a
robust and significant effect of genotype in both male [F(2,22)
6.70, p .01] and female mice [F (2,23) 7.18, p .01], with a
more pronounced difference between female KO and WT than
between male KO and WT underlying the interaction effect in the
global analysis. Time spent in the center of open field, a putative
measure of anxiety-like behavior, was unaffected by genotype or
sex (Figure 1B). There was no significant effect of genotype, sex,
or genotype sex interaction for locomotor activity in the
familiar home cage (Figure 1C). Two mice were removed from
the home cage test due to scores higher than two standard
deviations (SD) above mean (SD 209695).
Locomotor Response to MK-801
Analyzing predrug time block, there was a main effect of time
[F(11,242) 19.85, p .01], reflecting some residual habituation
in all genotype groups, but no main effect of genotype or
genotype time interaction. The lack of genotype effect,
Figure 1. Locomotor hyperactivity in response to environmental novelty in
GLAST KO. (A) GLAST KO showed elevated locomotor activity than WT
during exposure to a novel open field (n 14 –20). (B) Time spent in the
center of the open field did not differ between genotypes. (C) Home cage
locomotor activity was no different between genotypes (n 13–16). **p
.01. Data are mean SEM. GLAST, glial glutamate and aspartate transporter;
HET, heterozygous; KO, knockout; WT, wild-type.
R.-M. Karlsson et al. BIOL PSYCHIATRY 2008;64:810 814 811
www.sobp.org/journal
together with the lower overall activity levels compared with the
novel open field, shows that hyperactivity in GLAST KOs is
selectively found under conditions of novelty.
The response to MK-801 challenge was subsequently evalu-
ated using mixed-model ANOVA for postinjection time points
and controlling for individual differences in baseline as covari-
ates using the last 15 min of predrug baseline. There was a
significant effect of genotype [F (2,21) 4.21, p .05], time
[F(5,105) 2.57, p .05], and a genotype time interaction
[F(22,231) 1.87, p .05] for total distance traveled. Post hoc
analysis showed that MK-801 increased activity in KO to a greater
extent than WT (Figure 2). There was also significant effect of sex
[F(2,21) 6.88, p .05] and a sex time interaction [F(11,231)
1.99, p .05] due to female mice responding to MK-801
challenge more profoundly than male mice. Sex did not signifi-
cantly influence the genotypic differences in responsivity to
MK-801, as shown by a lack of genotype sex or genotype
sex time interactions.
Locomotor Effects of Haloperidol in GLAST KO Mice
For the haloperidol rescue test, there was a significant main
effect of genotype [F (2,62) 5.00, p .01] and drug [F(1,62)
30.93, p .01] and a significant genotype drug interaction
[F(2,62) 5.97, p .01] for total distance traveled. Post hoc tests
showed that KO were more active than WT following vehicle
treatment but not following haloperidol treatment (Figure 3A).
Haloperidol significantly reduced locomotor activity in heterozy-
gous (HET) and KO but not WT. There was an overall effect of
sex [F (1,62) 4.29, p . 05] due to higher scores in female mice
than male mice (post hoc test: p . 01) but sex did not interact
with either drug or genotype.
Reversal of PCP-Induced Hyperlocomotion by the mGlu2/3
Agonist LY379268 in C57BL/6J Mice
There was an overall significant effect of treatment [F(3,32)
11.08, p .01], time [F(3,32) 27.88, p .01], and a time drug
interaction for total distance traveled [F(69,736) 3.76, p .01].
Analyzing pre-PCP, there was a significant effect of treatment
[F(3,32) 4.58, p .01] and time [F(11,352) 32.94, p .01] but
no treatment time interaction. Post hoc tests showed that prior to
the PCP challenge, 3.0 but not .3 or 1.0 mg/kg LY379268 signifi-
cantly decreased activity relative to vehicle (Supplement 2).
The response to PCP challenge was evaluated using mixed-
model ANOVA for postinjection time points and controlling for
individual differences in baseline as covariates using the last 15
min of predrug baseline. There was a significant effect of
treatment [F(3,31) 11.64, p .01] but no time or treatment
time interaction effect. Post hoc test showed that 1.0 and 3.0
mg/kg LY379268 prevented PCP-induced hyperactivity relative
to vehicle (Supplement 2).
Locomotor Effects of LY379268 in GLAST KO Mice
There was a significant effect of genotype [F (2,68) 20.29,
p .01], drug [F (1,68) 29.33, p .01], and genotype drug
interaction [F(2,68) 12.31, p .01] for total distance traveled.
Post hoc tests showed that KO were more active than WT
following vehicle treatment but not following LY379268 treat-
ment (Figure 3B). LY379268 significantly reduced locomotor
activity in KO but not HET or WT. Because we observed a
significant effect of sex [F (1,68) 12.77, p .01] and significant
genotype sex interaction [F (2,68) 13.27, p .01], we also
analyzed genotype and LY379268 effects separately within each
sex and found a rescue of GLAST KO hyperlocomotion in both
male and female mice. Thus, in male mice, there was a significant
effect of genotype [F (2,32) 4.76, p .05], LY379268 [F(1,32)
6.18, p .05], and their interaction [F(2,32) 4.63, p .05]. Post
hoc analysis showed that KO were more active than WT after
vehicle but not LY379268 treatment. Similarly, in female mice,
Figure 3. Locomotor hyperactivity in GLAST KO is rescued by haloperidol
and the mGlu2/3 receptor agonist LY379268. (A) Treatment with .3 mg/kg
haloperidol normalized novel open field locomotor hyperactivity in GLAST
KO (n 10 –14/genotype/treatment). (B) Treatment with 1 mg/kg
LY379268 normalized novel open field locomotor hyperactivity in GLAST KO
(n 10 –15/genotype/treatment). *p .05, **p .01 versus WT/same
treatment; #p .05, ##p .01 versus vehicle/same genotype. Data are
mean SEM. GLAST, glial glutamate and aspartate transporter; HET, het-
erozygous; KO, knockout; mGlu2/3, metabotropic glutamate 2/3; WT, wild-
type.
Figure 2. Increased sensitivity to the locomotor hyperactivity-inducing ef-
fects of MK-801 in GLAST KO. Treatment with .2 mg/kg MK-801 produced a
significantly greater increase in open field locomotor activity in GLAST KO
than WT (n 9 –10). *p .05 versus WT. Data are mean SEM. GLAST, glial
glutamate and aspartate transporter; HET, heterozygous; KO, knockout; WT,
wild-type.
812 BIOL PSYCHIATRY 2008;64:810814 R.-M. Karlsson et al.
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there was a significant effect of genotype [F (2,36) 18.51, p
.01], LY379268 [F (1,36) 26.47, p .01], and their interaction
[F(2,36) 9.77, p .01]. Again, post hoc tests demonstrated that
KO were more active than WT after vehicle but not after
LY379268 treatment.
Discussion
The first major novel finding of the present study was that
GLAST KO showed a significant and robust open field locomotor
hyperactivity relative to WT. This was restricted to conditions of
novelty and not seen after prior habituation to the open field (see
MK-801-challenge experiment) or in the familiar environment of
the home cage. Together, these data demonstrate an exaggerated
GLAST KO locomotor response under conditions of sufficient
environmental challenge.
The basal locomotor hyperactivity in GLAST KO phenocopies
the effects of NMDAR antagonists in nonmutant mice (6,7). These
drugs also aggravate symptoms in schizophrenic patients and
simulate psychosis in normal subjects (4,5). Therefore, providing
further support for a schizophrenia-related abnormality in GLAST
KO, these mice exhibited an exaggerated locomotor hyperactiv-
ity response to the noncompetitive NMDAR antagonist MK-801.
A final important observation was that basal GLAST KO locomo-
tor hyperactivity was rescued by the prototypical antipsychotic
haloperidol and the mGlu2/3 agonist LY379268 recently shown
to have therapeutic efficacy in schizophrenia (14). These findings
provide a pharmacological validation that reversal of GLAST KO
induced hyperactivity may have predictive activity for clinical
efficacy in schizophrenia and therefore provide a useful tool for
screening novel antipsychotics.
Mechanistically, the ability of NMDAR antagonists to provoke
human psychosis and produce schizophrenia-related behaviors
in rodents has been linked to a loss of NMDAR-mediated
GABAergic inhibition, leading to excessive glutamate release and
neuronal hyperexcitability in PFC (8 –13). Loss of GLAST is
predicted to cause glutamate excess and extrasynaptic spillover
under conditions that provoke glutamate release, including
novelty and NMDAR-mediated GABA inhibition. Although vol-
tametry or microdialysis measures of PFC extracellular glutamate
in behaving GLAST KO would provide direct evidence of this,
indirect support for this notion is given by the ability of LY379268
to rescue GLAST KO phenotype and is entirely consistent with
the notion that extrasynaptic mGlu2/3 receptors facilitate inhib-
itory control of glutamate release and mitigate the effects of
impaired clearance (22). In this context, our data offer novel
support for the hypothesis that excessive glutamate contributes
to the pathophysiology of schizophrenia and lend further cre-
dence to the antipsychotic potential of mGlu2/3 agonists (14).
A number of issues await clarification. First, the relative
contribution of GLAST to forebrain glutamate signaling remains
to be established. Glial glutamate and aspartate transporter
messenger RNA and protein expression is most heavily concen-
trated in the cerebellum rather than the forebrain regions impli-
cated in schizophrenia, such as the PFC and hippocampus, and
neurons in these areas are not surrounded by high numbers of
astrocytes where the majority of GLAST-mediated glutamate
reuptake is thought to occur (16,17). Second, although acute loss
of glutamate clearance provides a plausible mechanism for the
schizophrenia-related abnormalities in GLAST KO, this does not
exclude the potential for constitutive loss of GLAST to cause more
permanent neural changes resulting from excitotoxicity (18,20) or
developmental abnormalities. The neurodevelopment issue is par-
ticularly salient given that high transient GLAST gene promoter
activity is present during early postnatal mouse development in
both the cortex and hippocampus (23) and the posited neurode-
velopmental ontogeny of schizophrenia. Third, possible GLAST KO
abnormalities on behaviors pertinent to the negative and cognitive
symptoms of schizophrenia await further study.
In summary, present data demonstrate that GLAST KO exhibit
abnormalities on behavioral measures thought to model positive
symptoms of schizophrenia. These disturbances were normal-
ized by the prototypical antipsychotic haloperidol and the
mGlu2/3 receptor agonist LY379268. Present findings suggest
that loss of GLAST-mediated glutamate clearance could be a
pathophysiological risk factor for schizophrenia and are partic-
ularly intriguing given recent evidence linking genetic mutation
of human GLAST (SLC1A3) with schizophrenia (19). More gen-
erally, our data provide novel support for the hypothesis that
excessive glutamate neurotransmission contributes to psychosis.
This work was supported by the National Institute on Alcohol
Abuse and Alcoholism (NIAAA)-Intramural Research Program
(IRP). Tanaka was supported by The Novartis Foundation (Japan)
for the Promotion of Science, Takeda Science Foundation, The
Tokyo Biochemical Research Foundation, Research Foundation of
Opto-Science and Technology, and Grants-in-Aids for Scientific
Research on Priority Area (20022013 and 18053006) provided by
the Ministry of Education, Culture, Sports, Science, and Technology
of Japan.
We thank Dr. Jeffrey Rothstein at the John Hopkins University
for providing the mutant mice for breeding and Dr. Judith Davis
and Monique Melige for assistance with breeding.
The authors reported no biomedical financial interests or
potential conflicts of interest.
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    • Astroglial plasmalemmal glutamate transporters EAAT1/2 were decreased in the prefrontal cortex and hippocampus (Ohnuma et al. 2000 ; Bauer et al. 2008 Bauer et al. , 2010 Shan et al. 2013 ). At the same time, animals in which EAAT1 was genetically deleted demonstrated aberrant social behaviour and locomotor hyperactivity , resembling certain symptoms of schizophrenia (Karlsson et al. 2008Karlsson et al. , 2009). Expression of the plasmalemmal cystine-glutamate exchanger (Sxc-), which controls extrasynaptic concentration of glutamate in the CNS (Bridges et al. 2012 ) has been found to be up-regulated in the rodent phencyclidine model of schizophrenia (Baker et al. 2008 ).
    [Show abstract] [Hide abstract] ABSTRACT: Astrocytes are primary homeostatic cells of the central nervous system. They regulate glutamatergic transmission through the removal of glutamate from the extracellular space and by supplying neurons with glutamine. Glutamatergic transmission is generally believed to be significantly impaired in the contexts of all major neuropsychiatric diseases. In most of these neuropsychiatric diseases, astrocytes show signs of degeneration and atrophy, which is likely to be translated into reduced homeostatic capabilities. Astroglial glutamate uptake/release and glutamate homeostasis are affected in all forms of major psychiatric disorders and represent a common mechanism underlying neurotransmission disbalance, aberrant connectome and overall failure on information processing by neuronal networks, which underlie pathogenesis of neuropsychiatric diseases.
    Full-text · Chapter · Jan 2016 · Moleculer Cells
    • Thus, downregulation of excitatory glutamate transporters is expected to enhance neuroexcitability. Along those lines, consequences of EAAT1 deficiency include locomotor hyperactivity, abnormal behavior, reduced acoustic startle response, and impaired memory consolidation (Karlsson et al. 2008Karlsson et al. , 2009). EAAT2 deficiency may contribute to the pathophysiology of Alzheimer disease (Li et al. 1997; Tian et al. 2007), schizophrenia (Lang et al. 2007 ), HIV associated dementia (Rumbaugh et al. 2007), multiple sclerosis (Pampliega et al. 2008; Vercellino et al. 2007 ), leukomalacia (Desilva et al. 2008), epilepsy (Rakhade and Loeb 2008; Rakhade et al. 2007), brain trauma (van Landeghem et al. 2006), hypoxia, and stroke (Boycott et al. 2007; Hurtado et al. 2008; Munch et al. 2008; Sheldon and Robinson 2007), reward dependence (Matsumoto et al. 2007), and amyotrophic lateral sclerosis (ALS) (Gibb et al. 2007; Rothstein et al. 1992).
    [Show abstract] [Hide abstract] ABSTRACT: Excitatory amino acid transporters EAAT1 (SLC1A3), EAAT2 (SLC1A2), EAAT3 (SLC1A1), and EAAT4 (SLC1A6) serve to clear l-glutamate from the synaptic cleft and are thus important for the limitation of neuronal excitation. EAAT3 has previously been shown to form complexes with caveolin-1, a major component of caveolae, which participate in the regulation of transport proteins. The present study explored the impact of caveolin-1 on electrogenic transport by excitatory amino acid transporter isoforms EAAT1-4. To this end cRNA encoding EAAT1, EAAT2, EAAT3, or EAAT4 was injected into Xenopus oocytes without or with additional injection of cRNA encoding caveolin-1. The l-glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1-, EAAT2-, EAAT3-, or EAAT4-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of caveolin-1. Caveolin-1 decreased significantly the maximal transport rate. Treatment of EAATs-expressing oocytes with brefeldin A (5 µM) was followed by a decrease in conductance, which was similar in oocytes expressing EAAT together with caveolin-1 as in oocytes expressing EAAT1-4 alone. Thus, caveolin-1 apparently does not accelerate transporter protein retrieval from the cell membrane. In conclusion, caveolin-1 is a powerful negative regulator of the excitatory glutamate transporters EAAT1, EAAT2, EAAT3, and EAAT4.
    Full-text · Article · Dec 2015
    • Decreased expression of EAAT1/2 plasmalemmal glutamate transporters is documented for the prefrontal cortex (Bauer et al., 2008Bauer et al., , 2010) and hippocampus (Ohnuma et al., 2000; Shan et al., 2013). Of note, genetic deletion of EAAT1 resulted in endophenotypes reflective of schizophrenia, including locomotor hyperactivity and abnormal social behavior (Karlsson et al., 2008Karlsson et al., , 2009). In addition, expression of hexokinase 1 (which contributes to regulation of glutamate-glutamine shuttle) was decreased in the post mortem tissue from schizophrenia sufferers (Shan et al., 2014a).
    [Show abstract] [Hide abstract] ABSTRACT: Fundamentally, all brain disorders can be broadly defined as the homeostatic failure of this organ. As the brain is composed of many different cells types, including but not limited to neurons and glia, it is only logical that all the cell types/constituents could play a role in health and disease. Yet, for a long time the sole conceptualization of brain pathology was focused on the well-being of neurons. Here, we challenge this neuron-centric view and present neuroglia as a key element in neuropathology, a process that has a toll on astrocytes, which undergo complex morpho-functional changes that can in turn affect the course of the disorder. Such changes can be grossly identified as reactivity, atrophy with loss of function and pathological remodeling. We outline the pathogenic potential of astrocytes in variety of disorders, ranging from neurotrauma, infection, toxic damage, stroke, epilepsy, neurodevelopmental, neurodegenerative and psychiatric disorders, Alexander disease to neoplastic changes seen in gliomas. We hope that in near future we would witness glial-based translational medicine with generation of deliverables for the containment and cure of disorders. We point out that such as a task will require a holistic and multi-disciplinary approach that will take in consideration the concerted operation of all the cell types in the brain.
    Full-text · Article · Sep 2015
    • ***(p \ 0.001) indicates statistically significant difference from the absence of OSR1 A. Abousaab et al.: Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2… 1113 several clinical conditions associated with neuronal hyperactivity , such as epilepsy, spasticity, neuropathic pain, schizophrenia, and autism (Alessi et al. 2014; Yang et al. 2013). As shown in mice, EAAT1 deficiency may lead to locomotor hyperactivity, abnormal behaviour with reduced preference for a novel social stimulus, reduced acoustic startle response, and impaired memory consolidation (Karlsson et al. 2008Karlsson et al. , 2009). Defective EAAT2 may contribute to several neurological disorders including Alzheimer disease (Li et al. 1997; Tian et al. 2007), schizophrenia (Lang et al. 2007), HIV-associated dementia (Rumbaugh et al. 2007), multiple sclerosis (Pampliega et al. 2008; Vercellino et al. 2007), leukomalacia (Desilva et al. 2008), epilepsy (Rakhade and Loeb 2008; Rakhade et al. 2007), brain trauma (van Landeghem et al. 2006), hypoxia and stroke (Boycott et al. 2007; Hurtado et al. 2008; Munch et al. 2008 ; Sheldon and Robinson 2007), reward dependence (Matsumoto et al. 2007), as well as amyotrophic lateral sclerosis (ALS) (Gibb et al. 2007; Rothstein et al. 1992 Rothstein et al. , 1995).
    [Show abstract] [Hide abstract] ABSTRACT: SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are cell volume-sensitive kinases regulated by WNK (with-no-K[Lys]) kinases. SPAK/OSR1 regulate several channels and carriers. SPAK/OSR1 sensitive functions include neuronal excitability. Orchestration of neuronal excitation involves the excitatory glutamate transporters EAAT1 and EAAT2. Sensitivity of those carriers to SPAK/OSR1 has never been shown. The present study thus explored whether SPAK and/or OSR1 contribute to the regulation of EAAT1 and/or EAAT2. To this end, cRNA encoding EAAT1 or EAAT2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type SPAK or wild-type OSR1, constitutively active T233ESPAK, WNK insensitive T233ASPAK, catalytically inactive D212ASPAK, constitutively active T185EOSR1, WNK insensitive T185AOSR1 or catalytically inactive D164AOSR1. The glutamate (2 mM)-induced inward current (I Glu) was taken as a measure of glutamate transport. As a result, I Glu was observed in EAAT1- and in EAAT2-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of SPAK and OSR1. As shown for EAAT2, SPAK, and OSR1 decreased significantly the maximal transport rate but significantly enhanced the affinity of the carrier. The effect of wild-type SPAK/OSR1 on EAAT1 and EAAT2 was mimicked by T233ESPAK and T185EOSR1, but not by T233ASPAK, D212ASPAK, T185AOSR1, or D164AOSR1. Coexpression of either SPAK or OSR1 decreased the EAAT2 protein abundance in the cell membrane of EAAT2-expressing oocytes. In conclusion, SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2.
    Full-text · Article · Aug 2015
    • It is important to address how astrocytosis might affect behavioral manifestations in Git1 −/− mice. Several lines of evidence suggest links between hyperactivity and astrocytes: 1) the perturbation of the glutamate metabolism by the deletion of the glial glutamate transporter, GLAST, causes schizophrenia-like noveltyinduced hyperactivity (Karlsson et al., 2008); 2) astrocytosis is detected in a repetitive mild traumatic brain injury (rmTBI) animal model that exhibits hyperactivity (Mannix et al., 2014); and 3) the ablation of D1 dopamine receptor expressing cells causes astrocytosis in the striatum, which has been connected to hyperactivity (Gantois et al., 2007). However, the astrocytosis reported in these studies is hypothesized to result from neuronal cell death, which initiates reactive astrocytes.
    [Show abstract] [Hide abstract] ABSTRACT: Attention deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorders, affecting approximately 5% of children. However, the neural mechanisms underlying its development and treatment are yet to be elucidated. In this study, we report that an ADHD mouse model, which harbors a deletion in the Git1 locus, exhibits severe astrocytosis in the globus pallidus (GP) and thalamic reticular nucleus (TRN), which send modulatory GABAergic inputs to the thalamus. A moderate level of astrocytosis was displayed in other regions of the basal ganglia pathway, including the ventrobasal thalamus and cortex, but not in other brain regions, such as the caudate putamen, basolateral amygdala, and hippocampal CA1. This basal ganglia circuit-selective astrocytosis was detected in both in adult (2-3 months old) and juvenile (4 weeks old) Git1(-/-) mice, suggesting a developmental origin. Astrocytes play an active role in the developing synaptic circuit; therefore, we performed an immunohistochemical analysis of synaptic markers. We detected increased and decreased levels of GABA and parvalbumin (PV), respectively, in the GP. This suggests that astrocytosis may alter synaptic transmission in the basal ganglia. Intriguingly, increased GABA expression colocalized with the astrocyte marker, GFAP, indicative of an astrocytic origin. Collectively, these results suggest that defects in basal ganglia circuitry, leading to impaired inhibitory modulation of the thalamus, are neural correlates for the ADHD-associated behavioral manifestations in Git1(-/-) mice.
    Full-text · Article · May 2015
    • In mammalian forebrain, three glutamate transporters including astroglial GLT1 and GLAST, and neuronal EAAC1, are expressed. Although there are many studies about behavioral phenotypes of GLAST-and EAACdeficient mice (Karlsson et al, 2008Karlsson et al, , 2009Karlsson et al, , 2012 Peghini et al, 1997; Watase et al, 1998), early postnatal lethality in GLT1 KO mice hindered their comprehensive behavioral analysis. To overcome the premature lethality of GLT1 KO mice, we generated GLT1 iKO mice that exhibit a 60–70% decrease in GLT1 protein levels.
    [Show abstract] [Hide abstract] ABSTRACT: An increase in the ratio of cellular excitation to inhibition (E/I ratio) has been proposed to underlie the pathogenesis of neuropsychiatric disorders, such as autism spectrum disorders (ASD), obsessive-compulsive disorder (OCD) and Tourette's syndrome (TS). A proper E/I ratio is achieved via factors expressed in neuron and glia. In astrocytes, the glutamate transporter GLT1 is critical for regulating an E/I ratio. However, the role of GLT1 dysfunction in the pathogenesis of neuropsychiatric disorders remains unknown because mice with a complete deficiency of GLT1 exhibited seizures and premature death. Here, we show that astrocyte-specific GLT1 inducible knockout (GLAST(CreERT2/+)/GLT1(flox/flox), iKO) mice exhibit pathological repetitive behaviors including excessive and injurious levels of self-grooming and tic-like head shakes. Electrophysiological studies reveal that excitatory transmission at corticostriatal synapse is normal in a basal state but is increased after repetitive stimulation. Furthermore, treatment with an N-methyl-D-aspartate (NMDA) receptor antagonist memantine ameliorated the pathological repetitive behaviors in iKO mice. These results suggest that astroglial GLT1 plays a critical role in controlling the synaptic efficacy at cortico-striatal synapses and its dysfunction causes pathological repetitive behaviors.Neuropsychopharmacology accepted article preview online, 09 February 2015. doi:10.1038/npp.2015.26.
    Full-text · Article · Feb 2015
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