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Genetic variant in SLC1A2 is associated with elevated anterior cingulate cortex glutamate and lifetime history of rapid cycling

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Glutamatergic dysregulation is implicated in the neurobiology of mood disorders. This study investigated the relationship between the anterior cingulate cortex (AC) glutamate, as measured by proton magnetic resonance spectroscopy (1H-MRS), and single-nucleotide polymorphisms (SNPs) from four genes (GLUL, SLC1A3, SLC1A2, and SLC1A7) that regulate the extracellular glutamate in 26 depressed patients with major depressive disorder (MDD; n = 15) and bipolar disorder (BD; n = 11). Two SNPs (rs3812778 and rs3829280), in perfect linkage disequilibrium, in the 3′ untranslated region of the EAAT2 gene SLC1A2, were associated with AC glutamate, with minor allele carriers having significantly higher glutamate levels (p < 0.001) in comparison with common allele homozygotes. In silico analysis revealed an association of minor allele carriers of rs3812778/rs382920 with an upregulation of the astrocytic marker CD44 localized downstream of SLC1A2 on chromosome 11. Finally, we tested the disease relevance of these SNPs in a large group of depressed patients [MDD (n = 458); BD (n = 1473)] and found that minor allele carriers had a significantly higher risk for rapid cycling (p = 0.006). Further work is encouraged to delineate the functional impact of excitatory amino acid transporter genetic variation on CD44 associated physiology and glutamatergic neurotransmission, specifically glutamate–glutamine cycling, and its contribution to subphenotypes of mood disorders.
Anterior cingulate cortex glutamate levels in common homozygotes and minor allele carriers for SLC1A2 single-nucleotide polymorphisms (SNPs) rs3812778/rs3829280 a MRI location for the pregenual anterior cingulate cortex ¹H-MRS voxel acquisition. The reference image of an 8-cm³ voxel (2 × 2 × 2 cm) of predominantly (prefrontal) gray matter was centered on the frontal interhemispheric fissure. The posterior margin of the voxel was placed immediately anterior to the genu of the corpus callosum in an area corresponding to the pregenual anterior cingulate cortex (Brodmann area 24a, 24b, and 32). b Glutamate–glutamine cycle and glutamate neurotransmission in the anterior cingulate cortex. Glutamate exerts its action on a variety of ionotropic (AMPA, NMDA, Kainate) and metabotropic (mGLUR 1–8) glutamate receptors. Glutamate is transported from the synaptic cleft into astrocytes by excitatory amino acid transporters. In astrocytes, glutamate is converted to glutamine by the astrocyte-specific enzyme glutamine synthetase and shuttled to the presynaptic neuron by sodium-coupled neutral amino acid transporters. In presynaptic neurons, phosphate-activated glutaminase converts glutamine back to glutamate. Glu glutamate, Gln glutamine, EAAT excitatory amino acid transporter, SNAT sodium-coupled neutral amino acid transporter, mGLUR metabotropic glutamate receptors, AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, NMDA N-methyl-D-aspartate receptor, Kainate kainate receptor, GS glutamine synthetase, GA glutaminase, Pre-SN presynaptic neuron Post-SN post-synaptic neuron. c Boxplot representations (median, 25th and 75th percentile) of glutamate levels measured by two-dimensional J-resolved averaged PRESS sequence in a combined group of unipolar and bipolar depressed common homozygotes and minor allele carriers for SLC1A2 SNPs rs3812778 (G/A) and rs3829280 (A/T). ***Homozygotes versus minor allele carriers, p = 0.00078 for both SNPs
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Veldic et al. Translational Psychiatry (2019) 9:149
https://doi.org/10.1038/s41398-019-0483-9
T
ranslational Psychiatry
ARTICLE Open Access
Genetic variant in SLC1A2 is associated with
elevated anterior cingulate cortex
glutamate and lifetime history of rapid
cycling
Marin Veldic
1
, Vincent Millischer
2,3
, John D. Port
4
, Ada Man-Choi Ho
5
,Yun-FangJia
5
,JenniferR.Geske
6
,
Joanna M. Biernacka
1,6
,LenaBacklund
2,3
, Susan L. McElroy
7
, David J. Bond
8
, J. Carlos Villaescusa
2,3
,
Michelle Skime
1
, Doo-Sup Choi
1,5
, Catharina Lavebratt
2,3
, Martin Schalling
2,3
and Mark A. Frye
1
Abstract
Glutamatergic dysregulation is implicated in the neurobiology of mood disorders. This study investigated the
relationship between the anterior cingulate cortex (AC) glutamate, as measured by proton magnetic resonance
spectroscopy (
1
H-MRS), and single-nucleotide polymorphisms (SNPs) from four genes (GLUL,SLC1A3,SLC1A2, and
SLC1A7) that regulate the extracellular glutamate in 26 depressed patients with major depressive disorder (MDD;
n=15) and bipolar disorder (BD; n=11). Two SNPs (rs3812778 and rs3829280), in perfect linkage disequilibrium, in the
3untranslated region of the EAAT2 gene SLC1A2, were associated with AC glutamate, with minor allele carriers having
signicantly higher glutamate levels (p< 0.001) in comparison with common allele homozygotes. In silico analysis
revealed an association of minor allele carriers of rs3812778/rs382920 with an upregulation of the astrocytic marker
CD44 localized downstream of SLC1A2 on chromosome 11. Finally, we tested the disease relevance of these SNPs in a
large group of depressed patients [MDD (n=458); BD (n=1473)] and found that minor allele carriers had a
signicantly higher risk for rapid cycling (p=0.006). Further work is encouraged to delineate the functional impact of
excitatory amino acid transporter genetic variation on CD44 associated physiology and glutamatergic
neurotransmission, specically glutamateglutamine cycling, and its contribution to subphenotypes of mood
disorders.
Introduction
There is increasing recognition that glutamatergic dys-
regulation is implicated in the neurobiology of mood dis-
orders. The evidence base spans animal studies,
1
postmortem
2
, imaging
35
, and pharmacological studies
35
,
as well as the latest genome-wide association studies in
major depressive disorder (MDD)
6
and bipolar disorder
(BD)
7
.
Most of the glutamate functional neuroimaging work in
mood disorders has focused on the prefrontal and cingu-
late cortices, recognizing the anterior cingulate cortex (AC)
as a regulator of emotional and cognitive behavior
8
.Mag-
netic resonance spectroscopy (MRS) is a functional brain
imaging method uniquely positioned to investigate gluta-
matergic biochemical mechanism of action
9,10
. Previous
work indicates that glutamate, glutamine, or the composite
glutamate/glutamine levels in depression may differ by
diagnostic subtype
3,11,12
. While brain regions, magnet
strength, glutamate MRS sequence, and post-processing
© The Author(s) 2019
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Correspondence: Marin Veldic (Veldic.Marin@mayo.edu)
1
Department of Psychiatry and Psychology, Mayo Clinic, Rochester, MN, USA
2
Department of Molecular Medicine and Surgery (MMK), Karolinska Institutet,
Stockholm, Sweden
Full list of author information is available at the end of the article.
These authors contributed equally: Marin Veldic, Vincent Millischer
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methods differ, available MR spectroscopic evidence to
date suggests that glutamate levels are increased in BD and
reduced in MDD
1113
.
In this study, in a mixed population of patients with
MDD and BD, we evaluated the relationship between
1
H-
MRS glutamate in the AC and single-nucleotide poly-
morphisms (SNPs) of astrocyte-specic genes, GLUL,
SLC1A3, and SLC1A2, encoding for glutamine synthetase
(GS), excitatory amino acid transporter (EAAT) 1 and
EAAT2, respectively, which are known to regulate
synaptic or extracellular glutamate levels in the astrocyte.
We also included SLC1A7, which encodes for EAAT5,
and is co-expressed with SLC1A2
14
(Fig. 1b). Positive hits
were followed up in silico. Finally, based on our previous
work on rapid cycling (RC)
15,16
, including genetic ndings
linking RC to glutamate physiology, we investigated the
association between positive hits and this phenotype in a
large group of unipolar and bipolar depressed patients.
Materials and methods
Participants for the MRS study
The MRS study was approved by the Mayo Clinic
Institutional Review Board (IRB# 06-006659). Potential
subjects were identied and referred to the study by Mayo
Clinic psychiatrists and psychologists from inpatient and
outpatient services, as well as a general intra campus
newsletter. After obtaining written informed consent, 51
individuals, ages 1865 were diagnosed using the Struc-
tured Clinical Interview for DSM-IV (SCID)
17
; this diag-
nostic interview was administered by trained raters
directly supervised by the principal investigator (MAF).
The inclusion criteria for this study were a current DSM-
IV diagnosis of a major depressive episode associated with
MDD, BD I, or BD II, based on SCID, and a negative
toxicology screen and pregnancy test. Exclusion criteria
included: inability to speak English or provide informed
consent, current treatment with an antidepressant, history
of active substance abuse within the last 6 months,
abnormal thyroid-stimulating hormone, unstable medical
illness, Young Mania Rating Scale (YMRS)
18
> 12 con-
sistent with hypomania, active suicidal ideation with plan,
current psychosis, and antipsychotic treatment within
4 weeks.
Depressive and manic symptom severity was assessed
with the Hamilton Depression Rating Scale-28 Item
Version (HAM-D28)
19,20
, to assess for atypical neurove-
getative symptoms, and the YMRS respectively. All ratings
were conducted by the principal investigator (MAF) or
inter-rater-reliable assistants.
Participants for the genotyping cohort
The cohort consisted of patients with BD and MDD.
The BD cohort consisted of patients from the Mayo Clinic
Individualized Medicine Biobank for Bipolar Disorder
(IRB# 08-008794)
21
and patients recruited from the Unit
of Affective Disorders, Psychiatry Southwest, Karolinska
University Hospital, Huddinge, Stockholm, Sweden
16
. The
assessment was based on interviews, medical records, and
Fig. 1 Anterior cingulate cortex glutamate levels in common
homozygotes and minor allele carriers for SLC1A2 single-
nucleotide polymorphisms (SNPs) rs3812778/rs3829280. a MRI
location for the pregenual anterior cingulate cortex
1
H-MRS voxel
acquisition. The reference image of an 8-cm
3
voxel (2 × 2 × 2 cm) of
predominantly (prefrontal) gray matter was centered on the frontal
interhemispheric ssure. The posterior margin of the voxel was placed
immediately anterior to the genu of the corpus callosum in an area
corresponding to the pregenual anterior cingulate cortex (Brodmann
area 24a, 24b, and 32). bGlutamateglutamine cycle and glutamate
neurotransmission in the anterior cingulate cortex. Glutamate exerts
its action on a variety of ionotropic (AMPA, NMDA, Kainate) and
metabotropic (mGLUR 18) glutamate receptors. Glutamate is
transported from the synaptic cleft into astrocytes by excitatory amino
acid transporters. In astrocytes, glutamate is converted to glutamine
by the astrocyte-specic enzyme glutamine synthetase and shuttled
to the presynaptic neuron by sodium-coupled neutral amino acid
transporters. In presynaptic neurons, phosphate-activated glutaminase
converts glutamine back to glutamate. Glu glutamate, Gln glutamine,
EAAT excitatory amino acid transporter, SNAT sodium-coupled neutral
amino acid transporter, mGLUR metabotropic glutamate receptors,
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor, NMDA N-methyl-D-aspartate receptor, Kainate kainate
receptor, GS glutamine synthetase, GA glutaminase, Pre-SN
presynaptic neuron Post-SN post-synaptic neuron. cBoxplot
representations (median, 25th and 75th percentile) of glutamate levels
measured by two-dimensional J-resolved averaged PRESS sequence in
a combined group of unipolar and bipolar depressed common
homozygotes and minor allele carriers for SLC1A2 SNPs rs3812778 (G/
A) and rs3829280 (A/T). ***Homozygotes versus minor allele carriers,
p=0.00078 for both SNPs
Veldic et al. Translational Psychiatry (2019) 9:149 Page 2 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
questionnaires and performed by specialized psychiatrists
or by trained psychiatric nurses. Patients with MDD were
selected from the PART study
22
, a longitudinal
population-based study in Stockholm County, Sweden,
utilizing the Major Depression Inventory (MDI)
23
.
Rapid cycling has been identied by the biobank as a
clinical phenotype to further investigate the underlying
genetics and neurobiology
15
. Lifetime history of rapid
cycling (RC) was dened as a self-reported history of
having four or more distinct bipolar mood episodes in a
12-month period, with each episode separated by a return
to baseline mood state for at least 2 months, or a switch to
the opposite mood pole. Manic and hypomanic episodes
were counted as being on the same mood pole.
Further description of cohort including clinical variables
quantied can be found in the Supplementary Information.
MR imaging (MRI) and 1H-MRS acquisition
Imaging and acquisition was completed with a GE 3T
Discovery 750 MRI scanner with 22.1 software and an 8-
channel head coil by a neuroradiologist blinded to the
group allocation throughout the entire study who did not
participate in assessing the outcome. The axial plane was
landmarked in all subjects at the center of the forehead,
1 cm above the eyebrows to standardize head position
from scan to scan. A neuroradiologist reviewed baseline
and posttreatment structural MRI data for potential
exclusionary head and brain pathology.
A FAST 3D SPGR sequence was used to acquire volu-
metric data for cerebrospinal uid (CSF) correction (axial
acquisition; repetition time [TR] =12.6 ms, echo time
[TE] =5.6 ms, ip angle =15°, voxel dimensions =0.49 ×
0.49 × 1.5 mm). Voxel positioning for the midline anterior
cingulate cortex (MACC) and for the left dorsolateral
prefrontal cortex (LDLPFC) voxels followed a systematic
approach during all scans (Fig. 1a; Supplementary
Information).
Based on the prior literature
24,25
, we chose two different
1H-MRS sequences for our glutamate and glutamine
measurements, each with its own strengths. A TE-
optimized PRESS sequence was used to measure both
glutamate and glutamine (PROBE-P PRESS; TE =80 ms,
TR =2000 ms, no. of excitations =8, no. of acquisitions =
128)
25
. A two-dimensional J-resolved averaged PRESS
sequence was used with the goal of collecting an optimized
measure of glutamate (2DJ PRESS; TE =35195 ms in
16 steps, TR =2000 ms, excitations =8)
26,27
.
Reconstruction and quantication of spectra
Spectroscopic imaging data were transferred to a Sun
workstation running SAGE-IDL (GE Medical Systems).
The data integrity was veried visually; scans with artifact
were excluded from the study. A quantitative analysis of
brain metabolites was performed using the LC Model
software. Basis sets for both the 3T-PRESS and 3T-2DJ
were provided by the vendor. The lower bound of mea-
surement error for glutamate quantication was a
CramerRao lower bound of 20 or less. For glutamine
quantication, the lower bound measurement error was
relaxed to 30 or less to optimize both limited data and
goodness of t
28,29
.
The SPGR anatomical data were segmented into gray
matter, white matter, and CSF using a technique modied
from a previous study
30
revised to use the FSL package
from FMRIB Oxford
31
. Briey, SPGR data were converted
into NIFTI format using mri_convert. The T1 volume was
skull-stripped using BET, then segmented into gray
matter, white matter, and CSF using FAST with default
parameters. The segmented data were then overlaid with
the voxel location using in-house software, and the
number of pixels of each tissue type within the voxel was
counted. These counts were then normalized to the total
number of pixels within the voxel to arrive at the fraction
of each tissue within the 1H-MRS voxel. The tissue
volume-corrected metabolite concentrations, [M]TVC,
were then calculated by taking the measured metabolite
concentration, [M]M, and applying a correction factor as
follows: [M]TVC =[M]M x (1/[1FCSF]) where FCSF is
fraction of CSF. This generated absolute(vs relative to
creatine) metabolite concentrations in institutional units
specic to our scanner and technique. These CSF-
corrected metabolite concentrations were used for all
statistical analyses.
AC and DLPFC MRS data acquisition of both TE80 and
2DJ Press, spectra reconstruction and quantication were
successfully completed in 39 individuals (BD: N=18,
UD: N=21); remaining subjects were either screening
failures or MRS was of a poor quality (i.e., inadequate
CramerRao bound, head movements during data
acquisition).
Genetic analysis of the MRS cohort
Of the 39 MRS-examined individuals, 26 subjects con-
sented to a blood draw for genetic analysis. Prior to study
initiation, we designated 16 SNPs located in essential
regulatory elements and coding sequences of GLUL (2
SNPs), SLC1A3 (1 SNP), SLC1A2 (12 SNPs), and SLC1A7
(1 SNP). We amplied genomic DNA regions containing
targeted SNPs and sequenced amplicons using an ABI
3730xl automated sequencer (Applied Biosystems, Foster
City, CA, USA). Sequence variants were then analyzed by
Mutation Surveyor version 2.2 (Softgenetics, PA). One
SLC1A2 SNP, rs12360706, was excluded from the analysis
due to poor sequencing quality (Supplementary Table 1).
Three groups in perfect LD (R
2
=1) could be determined:
(1) rs1043101, rs10768121, rs11033046, rs12361171, and
rs3088168; (2) rs3812778 and rs3829280; (3) rs10742338
and rs2229894, leaving nine independent (R
2
< 0.6) loci.
Veldic et al. Translational Psychiatry (2019) 9:149 Page 3 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
In silico analyses
LDlink (https://analysistools.nci.nih.gov/LDlink/) was
used to perform proxy search for SNPs in LD with
rs3812778, using populations of European descent.
Expression quantitative trait loci (eQTL) were identied
in the DLPFC using the gene expression database Brain-
Cloud (http://braincloud.jhmi.edu/)
32
, based on RNA
sequencing and genotype data of 412 subjects. The
modeling tested for additive genetic effects on expression,
adjusted for sex, ancestry, and expression heterogeneity. A
SNP-feature pair was considered signicant with a false
discovery rate less than 1%. Raw data for the signicant
pair were obtained from the website. Furthermore, data
were obtained for CD44 from the UK Brain Expression
Consortium (UKBEC) (http://www.braineac.org/), which
includes microarray data and genetic markers from dif-
ferent brain regions from 134 subjects. Genomic anno-
tations were used from UCSC genome for histone
modications and DNAseI-sensitive regions
33
. The
development transcriptome dataset summarized to genes
from the BrainSpan project (http://www.brainspan.org/)
34
was used to assess correlations between expression of
CD44 and several genes of interest. This data set contains
RNA-sequencing data from up to sixteen brain regions
from 42 donors across the full course of human brain
development. SNPs were functionally annotated using the
genome-wide annotation of variants (GWAVA) tool,
which supports prioritization of noncoding variants by
integrating various genomic and epigenomic annotations
(https://www.sanger.ac.uk/science/tools/gwava)
35
.
Genetic analyses of the genotyping cohort
DNA samples from peripheral blood collected in Swe-
den and at the Mayo Clinic were genotyped for the SNPs
rs3812778 and rs3829280 in SLC1A2 using TaqMan SNP
genotyping assays on QuantStudio 7 Flex instrument
(Applied Biosystems, Foster City, CA, USA). The geno-
typing was performed by an investigator blinded to the
disease status of the patients. The genotyping efciency
was 98%.
Statistical analysis
Normality was assessed with quantilequantile plots,
homogeneity of variance was tested using the
Levenes test.
Demographic and clinical measures are presented using
descriptive statistics. Comparisons between MDD and BD
groups were made using ttests for continuous measures
and a chi-square test for sex.
Linear regression models were used to test the additive
effect of the minor allele (coded as 0, 1, 2) on midline AC
and LDLPFC glutamate concentration for each SNP, fol-
lowed by a two-sided ttest in a dominant model when the
number of minor allele homozygotes was low (i.e.,
grouping A/G and A/A for rs3812778, and A/T and T/T
for rs3829280). A Bonferroni correction was applied for
36 (nine loci, two regions, two methods) independent
tests (p
cor
). Two-sided ttests were used to test for dif-
ferences in glutamate levels between BD and MDD.
The association between CD44 expression and the
genetic data was tested by two-sided ttest using a
dominant model. In the UKBEC data set, q-values were
used to estimate false discovery rates (FDR). Correlations
between the logarithm of CD44 expression and the
logarithm of the expression of the genes of the
glutamateglutamine cycle were assessed using Spearman
correlation coefcient.
Differences in genotype between diagnoses, as well as
between RC BD and non-rapid cycling (NRC) were tested
using chi-square, as well as logistic regression to correct
for sex and age. A Bonferroni correction for two inde-
pendent tests was applied (p
cor
).
Statistical analyses were conducted using SAS (version
9.4; Cary, NC) and R programming language.
Results
rs3812778/rs3829280 are associated with AC glutamate
levels
As presented in Table 1, there was no statistically sig-
nicant difference for age (p=0.075), sex (p=1.0), or
mood symptom severity, as measured by HAM-D28 (p=
0.073) between mood disorder subtypes.
The minor alleles of the two SNPs rs3812778 and
rs3829280 (in perfect linkage disequilibrium (LD, r
2
=1)
in the 3UTR region of SLC1A2 gene) were associated
with elevated 2D JPRESS mean AC glutamate levels
(common allele homozygotes: 105 ± 21 units, minor allele
carriers 135 ± 15 units; p=0.00078, p
cor
=0.028) (Fig. 1c).
No association between glutamate levels and diagnosis
(p=0.68), or depression symptom severity (p=0.75) was
found. There was no association between glutamate levels
and any other SNP. There was also no association
between any SNPs, including rs3812778/rs3829280, when
combined glutamate/glutamine levels were analyzed using
the TE80 method. No association was found in the
LDLPFC (Supplementary Table 1).
rs3812778/rs3829280 are associated with CD44 levels
In silico analyses, using the BrainCloud eQTL-browser,
we found an association between the minor allele of
rs3829280 and higher levels of the SLC1A2 neighboring
gene CD44 mRNA (chr11:35240935-35243200(*)) (Fig.
2a, p=0.00010). These ndings were strengthened with
data from the UK Brain Expression Consortium, where
signicant associations were identied between the minor
allele of rs3812778/rs3829280 and higher levels of the
full-length transcript of CD44 in the cerebellar cortex,
putamen, and substantia nigra, as well as in the average of
Veldic et al. Translational Psychiatry (2019) 9:149 Page 4 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
all measured brain regions (Fig. 2b). Signicant associa-
tions were also found for other CD44 transcripts (Sup-
plementary Table 2). When searching for potential
surrounding functional SNPs, we found six SNPs in per-
fect LD (R
2
=1) with rs3812778/rs3829280: rs10836358,
rs67384276, rs56193087, rs1570216, rs4508184,
rs12360706. Analysis with GWAVA, a tool for functional
annotation of noncoding sequence, revealed high values
across all prediction scores (> 0.6) for rs1570216, indi-
cating high probability for functionality for this SNP lying
in the 3-UTR of SLC1A2 in a genomic area sensitive for
DNaseI also rich in H3K27 acetylation and H3K4
monomethylation (Supplementary Table 3).
CD44 strongly correlates with astrocytic markers
Given the important role CD44 plays in brain devel-
opment
36
, the correlation between CD44 expression and
the expression of genes of the glutamateglutamine cycle
were assessed across several brain regions pre- and
postnatally using data from the BrainSpan project
34
(Fig.
3a). Strong positive correlations between CD44 and genes
typically expressed in astrocytes (GLUL, SLC1A3,
SLC1A2, and SLC38A3) were seen both pre- and post-
natally, while no or negative correlations were observed
for genes typically expressed in neurons (GLS, SLC1A1,
SLC1A6, SLC38A1, SLC17A7, SLC17A6, SLC17A8, and
SLC1A7). Furthermore, CD44 also very strongly corre-
lated with typical astrocytic markers like AQP4, S100b,
and GFAP (Fig. 3b).
rs3812778/rs3829280 and rapid cycling (RC) prevalence
We then performed an exploratory investigation to
check for disease relevance. Similar to the genetic
spectroscopic study, the follow-up cohort was com-
posed of both MDD and BD individuals of Swedish and
Caucasian American origin. Demographic character-
istics can be found in Table 1. rs3812778/rs3829280
were in HardyWeinberg equilibrium, and the minor
allele frequencies (MAF) of both SNPs in the whole
cohort were 13%, corresponding to those in European
populations
37
.
There was no signicant difference in the percentage of
minor allele carriers of rs3812778/rs3829280 in BD vs
MDD participants (odds ratio (OR): 1.05 [95% condence
Fig. 2 Expression of CD44 in different brain regions. Boxplot representations (median, 25th and 75th percentile) of CD44 expression stratied by
rs3812778/rs3829280 aCD44 (chr11:35240935-35243200(*)) in the dorsolateral prefrontal cortex as measured by RNA sequencing (data from
BrainCloud), (b)CD44 (Afmetrix transcript t3326635) in ten different brain regions, measured by microarray (data from the UK Brain Expression
Consortium). ***p=0.00010, *p< 0.05
Table 1 Subject demographics
MR spectroscopygenotyping Genotyping
BD MDD Rapid cycling BD Non-rapid cycling BD MDD
Number of participants
1
11 15 638 835 458
Age (mean ± SD) 33 (11.3) 35.2 (13.3) 41.2 (14.2) 47.2 (15.4) 51.8 (11.9)
Sex (female/male) 8/3 11/4 403/235 463/372 330/128
HAM-D (mean, SD) 36.3 (9.1) 29.9 (7.7) NA NA NA
BD bipolar depression, MDD major depressive disorder, NA not applicable
1
All participants were of Swedish or Caucasian American origin
Veldic et al. Translational Psychiatry (2019) 9:149 Page 5 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
interval (CI): 0.841.32], p=0.647). While the percentage
of minor allele carriers was comparable between MDD
(21.9% [95% CI: 19.124.9]) and non-RC (NRC) BD
(21.8% [95% CI: 17.925.8]), RC BD participants had a
signicantly higher percentage of minor allele carriers in
comparison with the MDD+NRC BD group (26.9% [95%
CI: 23.530.5]) (age- and sex-adjusted OR: 1.38 [95% CI:
1.091.73], p=0.006, p
cor
=0.012; unadjusted OR: 1.32
[95% CI: 1.051.64], p=0.015, p
cor
=0.03).
Focusing only on patients with BD, a similar effect could
be observed between RC BD and NRC BD (age- and sex-
adjusted OR: 1.37 [95% CI: 1.071.76], p=0.012, p
cor
=
0.024; unadjusted OR: 1.31 [95% CI: 1.031.67], p=0.028,
p
cor
=0.056; Fig. 4). The lifetime history of rapid cycling
was higher (58%) in the American sites (tertiary referral
clinic) than at the Swedish site (a primary referral site,
28%). The model was therefore corrected for site, without
signicantly affecting the outcome (age, sex, and site-
adjusted OR: 1.40 [95% CI: 1.081.82], p=0.011, p
cor
=
0.22).
Discussion
Here, we report a signicant association between the
minor alleles of rs3812778/rs3829280, two SNPs in per-
fect linkage disequilibrium in the 3UTR of the EAAT2
gene SLC1A2, and 2DJ glutamate levels in the AC. After
being released from presynaptic nerve terminals, the
extracellular glutamate is cleared by a family of excitatory
amino acid transporters (EAAT1-5)
38
(Fig. 1b). Astrocytes
play a major role in glutamate homeostasis in the
Fig. 3 Correlations between CD44 expression and genes of the glutamate/glutamine cycle across several brain regions pre- and
postnatally. a Correlations between CD44 expression (log) and the genes of the glutamate/glutamine cycle (log) across several brain regions in
prenatal brains (N
donors
=20, N
datapoints
=237) and postnatal brains (N
donors
=22, N
datapoints
=287), reported as Spearmans correlation coefcients
(error bars 95% CI). bSpearman correlation between the expression of CD44 and the astrocytic marker GFAP in pre- and postnatal brains
Veldic et al. Translational Psychiatry (2019) 9:149 Page 6 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
neocortex, with EAAT2, the most abundant glutamate
transporter in the forebrain, responsible for up to 95% of
glutamate clearance in the mammalian brain, mainly
being expressed on astrocytic plasma membranes.
39,40
Glutamate levels in the AC have been associated with
mood disorders. There has been previous speculation that
glutamate levels may distinguish BD (i.e., increased glu-
tamate) from MDD (i.e., decreased glutamate)
12,13,41
.
However, glutamate levels in the brain are also regulated
by genetic variations in molecules, such as EAAT and
enzymes responsible for glutamateglutamine conversion
and glutamineglutamate conversion which have not
been studied comparatively in different types of mood
disorders. For instance, Ongur et al.
42
showed that a
specic haplotype of four SNPs within GLS1, the gene
encoding for the enzyme glutaminase generating gluta-
mate from glutamine, was signicantly associated with
glutamine/glutamate in the parietooccipital cortex and
rs956572 in a mixed group of healthy controls and
patients with bipolar disorder and schizophrenia
42
.A
second example is the work that identied a SNP in B-cell
lymphoma 2 (Bcl-2) shown to be associated with
increased anterior cingulate cortical glutamate solely in
euthymic bipolar I disorder
43
.
In silico analysis showed that the minor allele of
rs3812778/rs3829280 was associated with increased levels
of CD44 mRNA. CD44 is situated downstream of SLC1A2
on chromosome 11 (Supplementary Fig. 1a) and codes for
a transmembrane glycoprotein acting as a receptor for
hyaluronan, a key component of the extracellular matrix
in the brain. It is implicated in cell-matrix binding, sig-
naling, and cell migration
44
, as well as in the activation
and the resolution of inammatory processes
45
and plays
important roles in physiology (e.g., organogenesis) and
pathology (e.g., cancer and metastasis)
44
. In the CNS,
CD44 is mainly expressed on glial cells, in particular
astrocytes, but expression has also been shown on neu-
rons
36
, in neural stem cells, astrocyte, and oligoden-
drocyte precursor cells at early postnatal stages
46
.CD44
has been implicated in many physiological CNS functions,
such as neural development, axon guidance, and astrocyte
differentiation
36
. In humans, CD44 has been suggested as
a candidate gene associated with BD using convergent
functional genomics
47,48
. Furthermore, CD44 has been
identied in a brain GWAS study as a possible risk gene
for suicidal behavior
49
and the CD44 ligand hyaluronic
acid was reported to be elevated in the CSF of suicide
attempters, correlating with blood-brain barrier perme-
ability, a hallmark of neuroinammation
50
. Higher levels
of CD44 were also reported in the white matter of patients
with multiple sclerosis
51
and astrocytes of patients with
Alzheimers disease
52
.CD44 has also been associated with
disorders of the CNS in animal models: while CD44
deciency is protective against cerebral ischemia injury in
mice
53
,CD44 levels have also been shown to be changed
by omega-3 fatty acid treatment in female, but not male
mice a stress-reactive knockout animal model of bipolar
disorder and co-morbid alcoholism
54
. Finally, an invol-
vement of CD44 in synaptic transmission has been sug-
gested with Matzke et al. showing that CD44-decient
mice had markedly reduced glutamatergic synaptic exci-
tation
55
. Taken together, this evidence points toward a
central role of CD44 in CNS functions, and it can there-
fore be hypothesized that a disturbance in CD44 signaling
can lead to a change in glutamate turnover.
The strong positive correlations between CD44 and
astrocytic markers, as well as with the genes of the
glutamineglutamate cycle expressed in astrocytes could
be indicative of an effect of rs3812778/rs3829280 on
astrocyte numbers. The changes in glutamate metabolism
could therefore also be explained by changed levels of
astrocytic glutamate transporters, including SLC1A2.
Therefore, we cannot exclude that rs3812778/rs3829280
also affect the expression of SLC1A2, but that this effect
cannot be detected in brain homogenates. Indeed,
SLC1A2 is highly regulated with various transcription
factor-binding sites, as well as regulatory elements in the
UTRs
1,56
. EAAT2 has been implicated in the pathophy-
siology of several disorders of the CNS, including Par-
kinsons disease, epilepsy, amyotrophic lateral sclerosis,
Alzheimers disease, addiction, schizophrenia, as well as
MDD and BD.
1
On the molecular level, there is strong
evidence of downregulation of EAAT2 in diverse brain
regions in MDD
2,57
. Early stress impact on the gray matter
has been shown to be inuenced by a functional poly-
morphism in EAAT2 in BD in the hippocampus, a brain
region with greater atrophy in BD versus MDD
58
; this
Fig. 4 Percentage of rs3812778 and rs3829280 minor allele
carriers in patients with rapid cycling BD, non-rapid cycling BD
and major depressive disorder. Bar graph representation of the
percentage (error bars: 95% CI) of minor allele carriers (rs3812778: A/G
and G/G; rs3829289: A/T and T/T) in the different diagnostic groups.
*Patients with rapid cycling BD versus patients with non-rapid cycling
BD, chi-square test, p=0.028;
#
patients with rapid-cycling BD versus
combined patients with non-rapid cycling BD and MDD, chi-square
test, p=0.015. RC BD rapid-cycling BD, NRC BD non-rapid cycling BD,
MDD major depressive disorder
Veldic et al. Translational Psychiatry (2019) 9:149 Page 7 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
gene-by-environment interaction in the hippocampus has
not been described in MDD
59
. Also, epigenetically
mediated effects of early-life stress and addiction on
EAAT2 expression regulation may play an important role
in determining glutamate clearance rates and subsequent
in vivo glutamate measurements
60
. Finally, EAAT2 also
affects synaptic transmission, as blocking it with dihy-
drokainate, a specic inhibitor for EAAT2, leads to
extended N-methyl-D-aspartate (NMDA)-receptor-
mediated excitatory post-synaptic currents
61
.
The regulatory potential of rs3812778/rs3829280 is
supported by the fact that these SNPs are in perfect LD
with rs1570216, also situated in the 3UTR of SLC1A2,in
a region sensitive for DNaseI, with high H3K27 acetyla-
tion and H3K4 monomethylation, pointing toward an
active regulatory area. The results from GWAVA, an
annotation tool for noncoding variants that integrates
various genomic and epigenomic variables, also point
toward the functionality of rs1570216
35
.
Our group has previously shown associations between
RC and rs2230912, a genetic variation in P2RX7
16
,
encoding for P2X purinoreceptor 7, a ligand-gated non-
selective cation channel, which has also been implicated
in modulating glutamatergic signaling
62
. Reporting a
novel association between the minor alleles of rs3812778/
rs3829280 and an increased risk for RC, we decided to test
whether we could nd an interaction of the two genetic
variants in our cohort and included rs2230912 in our
model. However, we did not see an interactive effect
between both SNPs, and the effect of rs3812778/
rs3829280 on RC was not changed by this additional
variable (Supplementary Table 4).
Summarizing our ndings, we hypothesize that the
minor alleles of rs3812778/rs3829280 are associated with
an upregulation of CD44, possibly indicative of an
increase in astrocyte numbers in the brain which in
combination with excitatory amino acid transporter
modulation, is associated with an increased glutamate
recycling resulting in dysregulated glutamatergic neuro-
transmission, associated with an increased risk of RC
(Supplementary Fig. 1b). This hypothesis is supported by
ndings from Michael et al. who has shown that elevated
glutamate/glutamine in the DLPFC of BD II patients is
associated with RC
63
. To what extent anti-glutamatergic
mood-stabilizing anticonvulsants such as lamotrigine,
which has an evidence base in treating rapid cycling
bipolar II disorder, could impact this interaction remains
to be investigated
64
. One can therefore question whether
the currently reported differences seen in functional
imaging between BD and MDD, are being driven by
currently established diagnostic criteria (i.e., presence of
absence of a history of hypo/mania) or rather by clinical
subphenotypes like the presence of RC
63
, psychosis
65
,or
melancholic vs non-melancholic depression subtypes
3
.
Limitations
An important limitation of our study is the small
sample size of the MRS study, and replication in a larger
sample is warranted. Furthermore, we only analyzed a
small number of genes. Examining additional genes
known to be implicated in depression and involved in
either the glutamate/glutamine cycle (e.g., GLS1), reg-
ulation of neuronal plasticity and cellular resilience (e.g.,
BCL2), or purinergic signaling (e.g., P2RX7) may provide
a better understanding of the underlying neurobiology of
these glutamate-level alterations
6668
. In addition, no
experimental evidence proves that the glutamine/gluta-
mate ratio directly reects synaptic neurotransmission of
glutamate. However, J-resolved MRS sequence is opti-
mized for glutamate detection. This sequence attempts to
address some major challenges, including resolving
glutamine and glutamate signal from underlying macro-
molecule resonances as well as those from glutamate-
conjugate compounds, such as glutathione. Moreover,
studying the glutamine/glutamate ratio has been fruitful,
and several lines of evidence reviewed above indicate that
changes in glutamine/glutamate correlate with and thus
are a measure of changes in glutamatergic activity
42
.
Another limitation is heterogeneity of diagnostic assess-
ment between BD and MDD. BD assessment was based
on interviews, medical records review, and ques-
tionnaires in a clinical sample while MDD cases in the
genotyping cohort were selected in a random population
cohort and dened by MDI, However, validation studies
for the use of MDI in making DSM-IV-based diagnosis of
depression have been performed in population-based
settings
23
, clinical settings
69
, and outpatient settings
70
.In
addition, a population-based sample may reduce the
effect of confounders, such as propensity toward help
seeking. Also, given that the nature of our cohorts, the
sample size of our genotype cohorts was xed. However,
power calculations showed that, given our sample size
and the allelic frequencies, we would be able to detect,
with a power of 80%, an effect size corresponding to an
OR of 1.36 for RC versus NRC and 1.4 for the compar-
ison of MDD versus BD. Finally, inter-rater reliability
assessment was not conducted between Swedish and
American sites.
Conclusion
This study is the rst to associate spectroscopic ndings
with gene variants in molecules central to glutamate
processing in mood disorders. Future studies combining
neuroimaging, genotyping, epigenetic, and possibly other
quantiable diagnostic measurements, with deep clinical
phenotyping may provide enough elements to construct
nosological categories, and invest in developing biological
psychiatric phenotypes that can contribute to diagnostic
classication and treatment intervention
3,11,71
.
Veldic et al. Translational Psychiatry (2019) 9:149 Page 8 of 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Acknowledgements
We thank Inger Romer Ek, MSc, for the phenotyping of the bipolar patients. as
well as all the patients who participated in this study.
Author details
1
Department of Psychiatry and Psychology, Mayo Clinic, Rochester, MN, USA.
2
Department of Molecular Medicine and Surgery (MMK), Karolinska Institutet,
Stockholm, Sweden.
3
Neurogenetics Unit, Center for Molecular Medicine,
Karolinska University Hospital, Stockholm, Sweden.
4
Department of Radiology,
Mayo Clinic, Rochester, MN, USA.
5
Department of Molecular Pharmacology &
Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA.
6
Department of
Health Sciences Research, Mayo Clinic, Rochester, MN, USA.
7
Lindner Center of
Hope, University of Cincinnati, Cincinnati, OH, USA.
8
Department of Psychiatry,
University of Minnesota, Minneapolis, MN, USA
Authors' contributions
Project conception and design: M.V., V.M., J.D.P., M.S., D.S.C., C.L., and M.A.F.;
patient recruitment: L.B., C.L., M.S., M.A.F., and S.L.M.; MRS experiments: J.D.P.;
genotyping of patients: V.M., C.L., M.C.H., and Y.F.J.; in silico analyses: V.M. andJ.
C.V.; the data analysis: M.V., V.M., J.R.G., J.M.B., and C.L.; paper writing: M.V., V.M.,
J.D.P., D.S.C., C.L., M.S., and M.A.F.; revision of the manuscript: all authors.
Competing interests
This work was supported by the National Institute of Mental Health
RO1MH079261, National Alliance for Research in Depression and
Schizophrenia (NARSAD) Independent Investigator Award, the Marriott
Foundation and Mayo Clinic Genomics of Addition to Dr. Frye, and by the
Mayo Foundation for Medical Education and Research as well as the J. Willard
and Alice S. Marriott Foundation grant to Dr. Veldic. The project was supported
by grants from the Karolinska Institutet, the KI-Mayo Collaboration (MV, CL, VM),
the Swedish Research Council (2016-02653 (MS); 2014-10171 (CL)), the Swedish
Brain Foundation (FO2017-0129 (CL); FO2018-0141 (CL)) and grants from the
regional agreement on medical training and clinical research (ALF) between
the Stockholm County Council and the Karolinska Institutet (SLL20170292 (CL)).
Dr. Choi is a scientic advisory board member to Peptron Inc. Dr. Frye is a
consultant (for Mayo Clinic) to Janssen, Mitsubishi Tanabe Pharma Corporation,
Myriad, Sunovion, and Teva Pharmaceuticals. None of this funding contributed
to work carried out in this study.
Publishers note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional afliations.
Supplementary information accompanies this paper at (https://doi.org/
10.1038/s41398-019-0483-9).
Received: 7 December 2018 Revised: 7 March 2019 Accepted: 10 April 2019
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... To elucidate the topography of gene expression profiles of known neurotransmitter receptors and transporters in OT (Table S2), we employed a module-scoring approach using our spatial data. [36][37][38][39][40][41][42][43] This approach involved scoring the expression levels of key categories of nerve transport and receptor genes, including GABA, glutamate, monoamine, and acetylcholine ( Figures S3A and S3B). As anticipated, GABA and glutamate were the main neurotransmitters in the visual processing of OT, as the superficial layers of OT undertook the most signal transmission tasks. ...
Article
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The avian optic tectum (OT) has been studied for its diverse functions, yet a comprehensive molecular landscape at the cellular level has been lacking. In this study, we applied spatial transcriptome sequencing and single-nucleus RNA sequencing (snRNA-seq) to explore the cellular organization and molecular characteristics of the avian OT from two species: Columba livia and Taeniopygia guttata. We identified precise layer structures and provided comprehensive layer-specific signatures of avian OT. Furthermore, we elucidated diverse functions in different layers, with the stratum griseum periventriculare (SGP) potentially playing a key role in advanced functions of OT, like fear response and associative learning. We characterized detailed neuronal subtypes and identified a population of FOXG1+ excitatory neurons, resembling those found in the mouse neocortex, potentially involved in neocortex-related functions and expansion of avian OT. These findings could contribute to our understanding of the architecture of OT, shedding light on visual perception and multifunctional association.
... To date, minimal knowledge exists regarding the potential utilization of ESK-NS in the context of RBCD and whether, similarly to other antidepressant compounds, it may result in an exacerbation of affective switches. Based on the evidence of glutamate dysregulation in RBCD pathophysiology (Michael et al., 2009;Veldic et al., 2019), we hypothesized an atypical, mood-stabilizing, antidepressant action of ESK-NS in these clinical conditions. ...
Article
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Rapid Cyclic Bipolar Disorder (RCBD) is a debilitating condition that manifests as four or more episodes of depression, mania, or hypomania in a span of one year, with depression being the predominant event. RCBD is prevalent among individuals diagnosed with bipolar disorder and is associated with elevated suicide risk, prolonged disease duration, unfavorable clinical outcomes, and cognitive decline. The treatment of RCBD is challenging, owing to the poor response to lithium and other conventional treatments for bipolar disorder and the possibility of exacerbating rapid-cycling oscillations during depressive phases with the use of antidepressant therapies. Esketamine Nasal Spray (ESK-NS) has been approved for the treatment of Treatment-Resistant Depression but its application in the context of RCBD is limited and unknown. This study presents the case of a 56-year-old male diagnosed with RCBD who received ESK-NS treatment during a depressive episode and was followed up for 18 months. During the observation period, the subject exhibited a significant mood stabilization, with only a transient depressive episode observed during a three-month period of cessation of ESK-NS treatment, which promptly resolved upon reinstating ESK-NS therapy. Mild dissociative symptoms were observed during the initial doses of ESK-NS administration, but no other significant adverse events were reported. This case report provides initial evidence for the potential utilization of ESK-NS in the treatment of RCBD, supporting the hypothesis of an additional mood-stabilizing effect.
... However, the association between SLC1A2 gene polymorphism and drug dependence with drug abuse remains unknown. Recently, Veldic et al. (2019) discovered that the SLC1A2 gene is associated with a higher risk of pathological conditions related to changes in the extracellular glutamate levels [16]. Indeed, the SLC1A2 gene is considered a strong candidate for drug dependence and associated psychosis since its variants are hypothesized to be associated with schizophrenia, and schizophrenia's symptoms, including paranoid hallucinations and delusions, resemble drug-induced psychosis [17,18]. ...
Article
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SLC1A2 is a gene encoded for the excitatory amino acid transporter 2 which is responsible for glutamate reuptake from the synaptic cleft in the central nervous system. Recent studies have suggested that polymorphisms on glutamate transporters can affect drug dependence, leading to the development of neurological diseases and psychiatric disorders. Our study investigated the association of rs4755404 single nucleotide polymorphism (SNP) of the SLC1A2 gene with methamphetamine (METH) dependence and METH-induced psychosis and mania in a Malaysian population. The rs4755404 gene polymorphism was genotyped in METH-dependent male subjects (n = 285) and male control subjects (n = 251). The subjects consisted of the four ethnic groups in Malaysia (Malay, Chinese, Kadazan-Dusun, and Bajau). Interestingly, there was a significant association between rs4755404 polymorphism and METH-induced psychosis in the pooled METH-dependent subjects in terms of genotype frequency (p = 0.041). However, there was no significant association between rs4755404 polymorphism and METH dependence. Also, the rs455404 polymorphism was not significantly associated with METH-induced mania for both genotype frequencies and allele frequencies in the METH-dependent subjects, regardless of stratification into the different ethnicities. Our study suggests that the SLC1A2 rs4755404 gene polymorphism confers some susceptibility to METH-induced psychosis, especially for those who carry the GG homozygous genotype.
... Another meta-analysis of data from 41,917 patients identified 64 loci associated with bipolar disorder. The genes implicated in bipolar disorder included CACNB2 and KCNB1, encoding proteins that regulate calcium ion-channels through modulation of calcium currents across neuronal membranes [26]; HTR6, encoding a serotonin receptor which mediates neurotransmission [27]; and SLC1A2, encoding a sodium dependent amino acid transporter with a role in the regulation and neurotransmission of glutamate [28]. These studies demonstrated that both schizophrenia and bipolar disorder are polygenic heritable disorders. ...
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Background Psychiatric disorders are characterized by alteration in emotions, mood and behavior. Genetics is known to play a significant role in the development of psychiatric disorders. Genome-wide association studies have identified several loci associated with psychiatric illnesses. We hypothesize the existence of rare variants following Mendelian recessive mode of inheritance. These variants can be identified in families with multiple affected individuals born to unaffected consanguineous parents. Methods We visited psychiatric outpatient departments of multiple hospitals in Lahore, Pakistan. We focused on psychosis, as it can occur in several DSM disorders such as schizophrenia, dementia and bipolar disorder. After clinical diagnosis by an American trained psychiatrist, detailed clinical assessments using Diagnostic Interview for Genetic Studies (DIGS), Diagnostic Interview for Psychosis and Affective Disorders (DI-PAD), Positive and Negative Syndrome Scale (PANSS), Hamilton Depression and Anxiety Rating Scale (HAM-D; HAM-A) were administered to all willing affected and unaffected participants. Results We identified eight pedigrees with two or more psychotic individuals in each family. Clinical diagnoses determined by their psychiatrists included ten individuals with schizophrenia; four individuals with psychosis and bipolar disorder; and two patients with “unspecified psychosis.” The rating instruments rigorously confirmed the diagnosis of psychosis in the affected patients from the six families as well as the absence of psychotic disorders in unaffected individuals from the six families. We obtained DNA samples from willing members of all eight families for future genetic analyses. Conclusion Our research highlights an alternative approach to discovery of rare recessively inherited genetic variants causing psychiatric disorders that have remained unidentified to date. These findings could illuminate underlying biological mechanisms leading toward development of targeted therapies in future.
... As far as we know, only a few studies on the genetic polymorphism of SLC1A2 in MDD were conducted. The majority of studies reported an association of SLC1A2 SNPs with bipolar disorder [54][55][56][57] and schizophrenia [58,59]. Polymorphism rs4354668 evaluated in this paper was also analyzed in a Thai sample [60]. ...
Article
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The membrane excitatory amino acid transporter 2 (EAAT2), encoded by SLC1A2, is responsible for the uptake and redistribution of synaptic glutamate. Glycine modulates excitatory neurotransmission. The clearance of synaptic glycine is performed by glycine transporters encoded by SLC6A9 and SLC6A5. Higher synaptic glycine and glutamate levels could enhance the activation of NMDA receptors and counteract the hypofunction of glutamate neurotransmission described in major depressive disorder (MDD). The aim of the study was to assess whether polymorphisms of SCL1A2 (rs4354668), SCL6A5 (rs2000959), and SCL6A9 (rs2486001) play a role in the development of MDD and its clinical picture in the Polish population. The study group consisted of 161 unrelated Caucasian patients with MDD and 462 healthy unrelated individuals for control. Polymorphisms were genotyped with PCR-RLFP assay. We observed that the frequency of genotype CC and allele C of the SLC1A2 polymorphism rs4354668 was twice as high in the MDD group as in control. Such differences were not detected in SLC6A5 and SLC6A9 polymorphisms. No statistically significant association of the studied SNPs (Single Nucleotide Polymorphisms) on clinical variables of the MDD was observed. The current study indicates an association of polymorphism rs4354668 in SCL1A2 with depression occurrence in the Polish population; however, further studies with larger samples should be performed to clarify these findings.
... Of note, GSK-3β acts as a powerful regulator of EAAT3/4 activity and thus of the regulation of NMDARs and glutamatergic currents (Abousaab and Lang, 2016). EAATs 1-5 superfamily's specific alleles have been linked to BD and particularly with the risk to develop a rapid cycle phenotype (Veldic et al., 2019). Lithium treatment has been found to affect GSK-3 protein levels and phosphorylation status in an animal model of mania (Cechinel-Recco et al., 2012) and to inhibit GSK-3, either via competition with magnesium for the active site of the enzyme (Ryves and Harwood, 2001) or via increased phosphorylation at the GSK-3β Ser-9 site and GSK-3α Ser-21 site (De Sarno et al., 2002), putatively via PI-3K/Akt dependent mechanisms (Pan et al., 2011). ...
Article
The monoamine hypothesis has dominated research on the pathophysiology of mood disorders as well as the development of therapeutic drugs by over half a century. Nowadays a change of perspective is taking place. The glutamate system is increasingly implicated in the pathophysiology of mood disorders. The evidence spans from animal, post-mortem, imaging, pharmacological and genome-wide association studies. Bipolar disorder has been recently re-conceptualized as a synaptic plasticity-related disorder rather than simply as a result of deficits or excesses in individual neurotransmitters. A paradigm shift from a monoamine hypothesis to a neuroplasticity hypothesis focused on glutamate may represent a substantial advancement in the research for new drugs and therapies. In this review we summarize data from clinical and pre-clinical studies that have addressed glutamatergic alterations in bipolar disorder. Along with an in-depth discussion of glutamatergic alterations in bipolar disorder, we also report available data on the neuroprotective and neuroplastic potential of the classic mood stabilizers, ketamine, and psychedelics. The glutamatergic mechanisms underlying the efficacy of these drugs are described and discussed.
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Addressing the formidable challenge posed by the development of effective and personalized interventions for major depressive disorder (MDD) necessitates a comprehensive comprehension of the intricate role that plasma amino acids play and their implications in MDD pathology and pharmacology. Amino acids, owing to their indispensable functions in neurotransmission, metabolism, and immune regulation, emerge as pivotal entities in this intricate disorder. Our primary objective entails unraveling the underlying mechanisms and unveiling tailored treatments through a meticulous investigation into the interplay between plasma amino acids, MDD, and pharmacological strategies. By conducting a thorough and exhaustive review of the existing literature, we have identified pertinent studies on plasma amino acids in MDD, thereby uncovering noteworthy disturbances in the profiles of amino acids among individuals afflicted by MDD when compared to their healthy counterparts. Specifically, disruptions in the metabolism of tryptophan, phenylalanine, and tyrosine, which serve as precursors to essential neurotransmitters, have emerged as prospective biomarkers and critical contributors to the pathophysiology of depression. Amnio acids play an essential role in MDD and could represent an attractive pharmacological target, more studies are further required to fully reveal their underlying mechanisms.
Article
Background Rapid cycling (RC) at ≥4 recurrent episodes per year in bipolar disorder (BD) has been recognized since the 1970s. We now comment on our recent review of the topic and extensive RC analysis in a large clinical cohort, emphasizing therapeutics research. Comments Prevalence of RC-BD averages 36% for any year versus 22% in the preceding year. Rapid cycling is not a consistent feature over many years, although average long-term, annual recurrence rates are greater in RC-BD patients. Risk of RC may be somewhat greater among women and with older ages. It is also associated with cyclothymic temperament, prominent depression, and mood-switching with antidepressant treatment and is associated with increased suicidal risk. Treatment of individual episodes in RC-BD and effective long-term prevention remain inadequately studied, although antidepressant treatment can worsen RC. Some research supports treatment with aripiprazole, lamotrigine, and lithium, and interest in second-generation antipsychotics is emerging. All such options are used in various inadequately evaluated combinations. Conclusions Rapid cycling is prevalent among BD patients but seems to vary in risk over time without evidence of progressive worsening. Treatment of acute episodes in RC-BD patients and effective long-term preventive management require much more intensive investigation.
Article
Rapid-cycling in bipolar disorder (RC-BD) is associated with greater illness morbidity and inferior treatment response but many aspects remain unclear, prompting this systematic review of its definitions, prevalence, and clinical characteristics. We searched multiple literature databases through April 2022 for systematic reviews or meta-analyses on RC-BD and extracted associated definitions, prevalence, risk-factors, and clinical outcomes. We assessed study quality (NIH Quality Assessment Tool) and levels of evidence (Oxford criteria). Of 146 identified reviews, 22 fulfilling selection criteria were included, yielding 30 studies involving 13,698 BD patients, of whom 3777 (27.6% [CI: 26.8-28.3]) were considered RC-BD, as defined in 14 reports by ≥4 recurrences/year within the past 12 months or in any year, without considering responsiveness to treatment. Random-effects meta-analytically pooled one-year prevalence was 22.3% [CI: 14.4-32.9] in 12 reports and lifetime prevalence was 35.5% [27.6-44.3] in 18 heterogenous reports. Meta-regression indicated greater lifetime prevalence of RC-BD among women than men (p=0.003). Association of RC-BD with suicide attempts, and unsatisfactory response to mood-stabilizers was supported by strong evidence (Level 1); associations with childhood maltreatment, mixed-features, female sex, and type-II BD had moderate evidence (Level 2). Other factors: genetic predisposition, metabolic disturbances or hypothyroidism, antidepressant exposure, predominant depressive polarity (Level 3), along with greater illness duration and immune-inflammatory dysfunction (Level 4) require further study. RC-BD was consistently recognized as having high prevalence (22.3%-35.5% of BD cases) and inferior treatment response. Identified associated factors can inform clinical practice. Long-term illness-course, metabolic factors, and optimal treatment require further investigation.
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Bipolar disorder is a highly heritable psychiatric disorder. We performed a genome-wide association study (GWAS) including 20,352 cases and 31,358 controls of European descent, with follow-up analysis of 822 variants with P < 1 × 10−4 in an additional 9,412 cases and 137,760 controls. Eight of the 19 variants that were genome-wide significant (P < 5 × 10−8) in the discovery GWAS were not genome-wide significant in the combined analysis, consistent with small effect sizes and limited power but also with genetic heterogeneity. In the combined analysis, 30 loci were genome-wide significant, including 20 newly identified loci. The significant loci contain genes encoding ion channels, neurotransmitter transporters and synaptic components. Pathway analysis revealed nine significantly enriched gene sets, including regulation of insulin secretion and endocannabinoid signaling. Bipolar I disorder is strongly genetically correlated with schizophrenia, driven by psychosis, whereas bipolar II disorder is more strongly correlated with major depressive disorder. These findings address key clinical questions and provide potential biological mechanisms for bipolar disorder. Genome-wide analysis identifies 30 loci associated with bipolar disorder, allowing for comparisons of shared genes and pathways with other psychiatric disorders, including schizophrenia and depression.
Article
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Major depressive disorder (MDD) is a common illness accompanied by considerable morbidity, mortality, costs, and heightened risk of suicide. We conducted a genome-wide association meta-analysis based in 135,458 cases and 344,901 controls and identified 44 independent and significant loci. The genetic findings were associated with clinical features of major depression and implicated brain regions exhibiting anatomical differences in cases. Targets of antidepressant medications and genes involved in gene splicing were enriched for smaller association signal. We found important relationships of genetic risk for major depression with educational attainment, body mass, and schizophrenia: lower educational attainment and higher body mass were putatively causal, whereas major depression and schizophrenia reflected a partly shared biological etiology. All humans carry lesser or greater numbers of genetic risk factors for major depression. These findings help refine the basis of major depression and imply that a continuous measure of risk underlies the clinical phenotype.
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Astrocyte glutamate release can modulate synaptic activity and participate in brain intercellular signaling. P2X7 receptors form large ion channels when activated by ATP or other ligands. Here we show that P2X7 receptors provide a route for excitatory amino acid release from astrocytes. Studies were performed using murine cortical astrocyte cultures. ATP produced an inward current in patch-clamped astrocytes with properties characteristic of P2X7 receptor activation: the current was amplified in low divalent cation medium, blocked by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and more potently activated by 3'-O-(4-benzoyl)benzoyl ATP (BzATP) than by ATP itself. Measurement of current reversal potentials showed the relative BzATP-induced permeabilities to different substrates to be Na+, 1 > Cl-, 0.34 > N-methyl-D-glucamine, 0.27 > L-glutamate, 0.15 approximately D-aspartate, 0.16. Astrocytes exposed to BzATP also became permeable to Lucifer yellow, indicating a large channel opening. Release of L-glutamate and D-aspartate through P2X7 channels was confirmed using radiolabeled tracers. As with the inward current, release of glutamate and D-aspartate was induced by BzATP more potently than ATP, amplified in Ca2+/Mg2+-free medium, and blocked by PPADS or oxidized ATP. Efflux through P2X7 channels is a previously unrecognized route of ligand-stimulated, nonvesicular astrocyte glutamate release.
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While downregulation of excitatory amino acid transporter 2 (EAAT2), the main transporter removing glutamate from the synapse, has been recognized in bipolar disorder (BD), the underlying mechanisms of downregulation have not been elucidated. BD is influenced by environmental factors, which may, via epigenetic modulation of gene expression, differentially affect illness presentation. This study thus focused on epigenetic DNA methylation regulation of SLC1A2, encoding for EAAT2, in BD with variable environmental influences of addiction. High resolution melting PCR (HRM-PCR) and thymine–adenine (TA) cloning with sequence analysis were conducted to examine methylation of the promoter region of the SLC1A2. DNA was isolated from blood samples drawn from BD patients (N = 150) with or without addiction to alcohol, nicotine, or food, defined as binge eating, and matched controls (N = 32). In comparison to controls, the SLC1A2 promoter region was hypermethylated in BD without addiction but was hypomethylated in BD with addiction. After adjusting for age and sex, the association of methylation levels with nicotine addiction (p = 0.0009) and binge eating (p = 0.0002) remained significant. Consistent with HRM-PCR, direct sequencing revealed increased methylation in CpG site 6 in BD, but decreased methylation in three CpG sites (6, 48, 156) in BD with alcohol and nicotine addictions. These results suggest that individual point methylation within the SLC1A2 promoter region may be modified by exogenous addiction and may have a potential for developing clinically valuable epigenetic biomarkers for BD diagnosis and monitoring.
Article
Introduction The activation of the ATP-gated P2RX7 (purinergic receptor P2X, ligand-gated ion channel, 7) produces microglial activation, a process which has been demonstrated in depression, bipolar disorder, and schizophrenia. Emerging data over the last years highlighted the importance of P2X7 cation channel as a potential drug target for these central nervous system disorders. The Gln460Arg (rs2230912) polymorphism of the P2RX7 gene has been widely studied in mood disorders, however the results are still controversial. Therefore, we aimed to investigate the C-terminal region of this gene in major depressive and bipolar disorders (MDD and BD) by studying possibly functional, non-synonymous polymorphisms within a 7 kb long region around the Gln460Arg, including Ala348Thr (rs1718119), Thr357Ser (rs2230911), and Glu496Ala (rs3751143) variants. Since Gln460Arg is located at the 3′ end of the P2RX7 gene, we included additional, potentially functional single nucleotide polymorphisms (SNPs) from the 3′ untranslated region (UTR), which can be in linkage with Gln460Arg. Based on in silico search, we chose two SNPs in putative microRNA target sites which are located in consecutive positions: rs1653625 and rs1718106. Methods P2RX7 SNPs from the C-terminal region were selected based on previous functional assays, 3′ UTR variants were chosen using PolymiRTS and Patrocles databases. The genotyping of the non-synonymous SNPs was carried out by pre-designed TaqMan® kits, while the 3′ UTR variants were analyzed by PCR-RFLP method. Case-control analyses were carried out between 315 inpatients with acute major depressive episode (195 MDD, 120 BD) and 406 healthy control subjects. The two subscales of the Hospital Anxiety and Depression Scale (HADS) self-report questionnaire were used for quantitative analyses, including an additional, “at-risk” population of 218 patients with diabetes mellitus. The in vitro reporter gene assays were carried out on HEK and SK-N-FI cells transiently transfected with pMIR vector constructs containing the P2RX7 3′ UTR downstream of the luciferase gene. Results Haplotype analysis indicated a relatively high linkage between the analyzed P2RX7 SNPs. Our case-control study did not yield any association between P2RX7 gene variants and depression. However, dimensional analyses showed significant associations of the HADS depression severity scores with Gln460Arg (rs2230912) and Ala348Thr (rs1718119) in the depressed and diabetic patient groups. In the in vitro experiments, the P2RX7 3′ UTR constructs with the lowest predicted binding efficiency to their miRNAs showed the highest expression of the gene. The combination of the depression-associated P2RX7 C-terminal and 3′ UTR SNPs contributed to the highest depression severity score in the haplotype analysis. Conclusion Based on our findings, we propose that a P2RX7 haplotype combination of the Gln460Arg and neighboring SNPs contribute to the observed genetic association with depressive symptoms.
Article
Significant alterations in glutamatergic neurotransmission have been reported in major depressive disorder (MDD) that could underlie psychiatric traits. Studies were mainly interested in synaptic dysfunction in the prefrontal cortex, a key structure involved in depressive-like behavior, however hippocampus has been shown to be important in MDD. As cognitive deficits such as hippocampus-memory process were observed in MDD, we investigated in a mild hypoglutamatergic model behaviors related to depression and memory, synaptic transmission parameters and glutamatergic state specifically in the hippocampus. We thus characterized these phenotypes in adult male mice partially depleted in glutaminase type 1 or GLS1 (GLS1 HET), the enzyme responsible for glutamate synthesis in neurons, that we previously characterized as displaying moderate lower levels of glutamate in brain. We showed that GLS1 mutant mice display AMPA-R-mediated response deficits after prolonged repetitive stimulation with electrophysiological recording and inability to sustain glutamate release by microdialysis experiments with no consequences on behavioral spatial learning performances. However, their ability to escape from unpleasant but repeated escapable condition was attenuated whereas they were more immobile in the unescapable situation in the FST during re-test. These results show that GLS1 mutant mice display moderate impairments of hippocampal glutamatergic neurotransmission and moderate changes in adaptive behaviors that have been shown to participate to the development of depressive-like state.
Article
Background: Accumulating evidence suggests the involvement of abnormal glutamateric neurotransmission and N-methyl-D-aspartate receptor hypofunction in the pathophysiology of psychotic disorders. The purpose of this study was to quantify in vivo glutamate (Glu) and glycine (Gly) levels in patients with first-episode psychosis as well as age-matched healthy control subjects with magnetic resonance spectroscopy (MRS). Methods: The subjects were 46 patients with first-episode psychosis (20 with a schizophrenia spectrum disorder, 26 with bipolar disorder) and 50 age-matched healthy control subjects. Glu and Gly levels were measured in vivo in the anterior cingulate cortex and posterior cingulate cortex of the subjects by using the echo time-averaged proton MRS technique at 4T (i.e., modified point resolved spectroscopy sequence: 24 echo time steps with 20-ms increments). Metabolite levels were quantified using LCModel with simulated basis sets. Results: Significantly higher Glu and Gly levels were found in both the anterior cingulate cortex and posterior cingulate cortex of patients with first-episode psychosis as compared with healthy control subjects. Glu and Gly levels were positively correlated in patients. Patients with a schizophrenia spectrum disorder and bipolar disorder showed similar abnormalities. Conclusions: Our findings demonstrate abnormally elevated brain Glu and Gly levels in patients with first-episode psychosis by means of echo time-averaged proton MRS at 4T. The findings implicate dysfunction of N-methyl-D-aspartate receptor and glutamatergic neurotransmission in the pathophysiology of the acute early phase of psychotic illnesses.
Article
Although the precise drug mechanism of action of acamprosate remains unclear, its antidipsotropic effect is mediated in part through glutamatergic neurotransmission. We evaluated the effect of 4 weeks of acamprosate treatment in a cohort of 13 subjects with alcohol dependence (confirmed by a structured interview, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision) on proton magnetic resonance spectroscopy glutamate levels in the midline anterior cingulate cortex (MACC). We compared levels of metabolites with a group of 16 healthy controls. The Pennsylvania Alcohol Craving Scale was used to assess craving intensity. At baseline, before treatment, the mean cerebrospinal fluid-corrected MACC glutamate (Glu) level was significantly elevated in subjects with alcohol dependence compared with controls (P = 0.004). Four weeks of acamprosate treatment reduced glutamate levels (P = 0.025), an effect that was not observed in subjects who did not take acamprosate. At baseline, there was a significant positive correlation between cravings, measured by the Pennsylvania Alcohol Craving Scale, and MACC (Glu) levels (P = 0.019). Overall, these data would suggest a normalizing effect of acamprosate on a hyperglutamatergic state observed in recently withdrawn patients with alcohol dependence and a positive association between MACC glutamate levels and craving intensity in early abstinence. Further research is needed to evaluate the use of these findings for clinical practice, including monitoring of craving intensity and individualized selection of treatment with antidipsotropic medications in subjects with alcohol dependence.
Article
We outline an ambitious project to characterize the genetic and epigenetic regulation of multiple facets of transcription in distinct brain regions across the human lifespan in samples of major neuropsychiatric disorders and controls. Initially focused on schizophrenia and mood disorders, the goal of this consortium is to elucidate the underlying molecular mechanisms of genetic associations with the goal of identifying novel therapeutic targets. The consortium currently consists of seven pharmaceutical companies and a not-for-profit medical research institution working as a precompetitive team to generate and analyze publicly available archival brain genomic data related to neuropsychiatric illness.