Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, Nos. 12-13, pp. 1972-1986 © Pleiades Publishing, Ltd., 2023.

Russian Text © The Author(s), 2023, published in Biokhimiya, 2023, Vol. 88, No. 12, pp. 2358-2374.

1972

Changes in the Glutamate/GABA System

in the Hippocampus of Rats with Age

and during Alzheimer’s Disease Signs Development

Alena O. Burnyasheva

, Natalia A. Stefanova

, Nataliya G. Kolosova

1,a

and Darya V. Telegina

Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia

e-mail: kolosova@bionet.nsc.ru

Received August 30, 2023

Revised October 12, 2023

Accepted October 17, 2023

Abstract— GABA and glutamate are the most abundant neurotransmitters in the CNS and play a pivotal part in synaptic

stability/plasticity. Glutamate and GABA homeostasis is important for healthy aging and reducing the risk of various neu-

rological diseases, while long-term imbalance can contribute to the development of neurodegenerative disorders, including

Alzheimer’s disease(AD). Normalization of the homeostasis has been discussed as a promising strategy for prevention

and/or treatment of AD, however, data on the changes in the GABAergic and glutamatergic systems with age, as well

ason the dynamics of AD development, are limited. It is not clear whether imbalance of the excitatory/inhibitory systems

is the cause or the consequence of the disease development. Here we analyzed the age-related alterations of the levels

of glutamate, GABA, as well as enzymes that synthesize them (glutaminase, glutamine synthetase, GABA-T, and GAD67),

transporters (GLAST, GLT-1, and GAT1), and relevant receptors (GluA1, NMDAR1, NMDA2B, and GABAAr1) in the

whole hippocampus of the Wistar rats and of the senescence-accelerated OXYS rats, a model of the most common (>95%)

sporadic AD. Our results suggest that there is a decline in glutamate and GABA signaling with age in hippocampus of

theboth rat strains. However, we have not identified significant changes or compensatory enhancements in this system in

thehippocampus of OXYS rats during the development of neurodegenerative processes that are characteristic of AD.

DOI: 10.1134/S0006297923120027

Keywords: aging, Alzheimer’s disease, glutamate, GABA, hippocampus, OXYS rats

Abbreviations: AD, Alzheimer’s disease; GABA,gamma aminobutyric acid; GABAAR1,GABA-A receptor subunit1; GABA-T,

GABA transaminase; GAD,glutamic acid decarboxylase (glutamate decarboxylase); GAD67,glutamic acid decarboxylase iso-

form; GAT1, type1 GABA transporter; GLAST,glial glutamate and aspartate transporter; GLT-1, glial glutamate transporter1;

GluA1, AMPA receptor subunit1; NMDAR1, NMDA receptor subunit1; NMDAR2B,NMDA receptor subunit2B.

* To whom correspondence should be addressed.

INTRODUCTION

According to the World Health Organization, Alz-

heimer’s disease(AD) becomes the major cause of se-

nile dementia, with its incidence increasing against the

background of longer life expectancy and aging of pop-

ulation in the developed and developing countries [1].

AD is manifested by the pronounced decline in cogni-

tive capacities against the background of accumulation

of toxic forms of amyloid-β peptide in the brain, for-

mation of amyloid plaques and neurofibrillary tangles,

synaptic failure, and neuronal death [2-4]. Molecular

mechanisms underlying AD development are unclear;

there are no efficient approaches that can slow down

or stop progression of this disease. It is supposed that

the age-related changes in the balance of neurotrans-

mitters, i.e., the excitatory glutamatergic and inhibitory

GABAergic systems in the brain, can be a prerequisite

for the development of AD and make a significant con-

tribution to its progression [5]. Glutamate and gamma-

aminobutyric acid (GABA) control many processes in

the CNS, including total excitation level in the brain.

CHANGES IN THE GLUTAMATE/GABA SYSTEM 1973

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

For example, excessive excitation of the inhibitory

GABAergic system suppresses and excessive activity of

the excitatory glutamatergic system causes excitotoxicity.

Balanced interaction between these two neurotransmit-

ters is necessary for physiological homeostasis, whereas

long-term imbalance can promote development of men-

tal and neurodegenerative disorders, including AD [6].

Elimination of the excitatory/inhibitory imbalance is

actively discussed as a promising strategy for AD pre-

vention and/or treatment [7]; at the same time, the data

on age-related changes in the GABAergic and gluta-

matergic systems, the more so at different stages of AD

development, are extremely limited. It remains unclear

whether imbalance of the excitatory/inhibitory systems

is a cause or a consequence of the AD development.

This is associated, first of all, with impossibility to study

early preclinical stages of this disease in humans and

with lack of its adequate models.

The present study was aimed at assessing contribu-

tion of the age-related changes in the balance between

the glutamatergic and GABAergic systems in the devel-

opment and progression of the most widespread (>95%

of cases) sporadic form of AD. The work was carried out

in the prematurely aging OXYS rats: a unique model of

the sporadic form of AD. In these animals, all of the key

signs of AD develop spontaneously in the absence of

mutations in the Psen1, App, and Psen2 genes typical of

hereditary AD [8-10]. Already at the age of 3-5months,

OXYS rats show behavioral disorders and decline in

cognitive functions, tau protein hyperphosphorylation,

impairment of long-term post-tetanic potentiation, syn-

aptic failure, and destructive changes in neurons, which

occur against the background of the increased level of

amyloid precursor protein(APP), enhanced accumula-

tion of β-amyloid, and formation of amyloid plaques in

the brain by the age of 12 months, and reach the clearly

marked stages of AD-like pathology by the age of 16-18

months [10, 11]. Previously we have studied age-related

changes in the glutamate/GABA system in the retina

of OXYS rats and assessed their potential contribution

to the development of retinopathy typical of rats [12].

The present work was aimed at comparing the age-

related changes in the glutamate/GABA system in the

hippocampus of Wistar rats (control) and OXYS rats

at different stages of the development of AD signs, in-

cluding preclinical stage. To this effect, we studied the

glutamate and GABA levels in the hippocampus, as well

as the levels of key enzymes regulating the glutamate/

GABA cycle: glutaminase catalyzing formation of glu-

tamate from glutamine; glutamine synthase catalyzing

synthesis of glutamine from glutamate; glutamate de-

carboxylase (GAD67) converting glutamate to GABA;

and the enzyme for GABA degradation: GABA trans-

aminase (GABA-T). In addition, we studied the lev-

els of glutamate and GABA receptors: subunit 1 of the

NMDA receptor (NMDAR1) and subunit 2B of the

receptor (NMDAR2B), subunit 1 of the AMPA recep-

tor (GluA1) and subunit α1 of the GABA-A receptor

(GABAAR1), as well as glutamate carriers: glial gluta-

mate and aspartate transporter (GLAST, also known as

EAAT1), glutamate 1 transporter (GLT-1, also known

as EAAT2), and GABA transporter (GAT1). Finally, we

compared age-related changes in the level of expression

of the genes associated with the glutamate and GABA

signaling pathways in the hippocampus of OXYS and

Wistar rats.

MATERIALS AND METHODS

Experimental animals. All experiments were carried

out using OXYS and Wistar (control) male rats. Animals

were kept under the standard conditions of vivarium

with light cycle 12-h light/12-h darkness; they received

granulated feed and water adlibitum.

Enzyme immunoassay (ELISA). Glutamate and

GABA levels in the hippocampus of OXYS and Wistar

rats aged 1.5, 3, 12, and 18 months (n = 5 for each group)

were estimated by enzyme immunoassay(EIA) using an

ELISA Kit for Glutamic Acid (Glu) CES122Ge and

an ELISA Kit for Gamma-Aminobutyric Acid (gABA)

CEA900Ge, according to the manufacturer’s protocol

(Cloud-Clone Corp., USA). ELISA kits were chosen

to detect only the levels of free glutamate and GABA.

Glutamate and GABA as protein components (bound

glutamate and GABA, respectively) were excluded from

the assay.

Total protein concentration was determined using a

ThermoFisher Pierce™ BCA Protein Assay kit (Thermo

Fisher Scientific, USA). Concentration was determined

by plotting a calibration curve using reference protein

provided by the manufacturer.

Western blot assay. Content of the key enzymes for

synthesis of GABA and glutamate [glutaminase, gluta-

mine synthase, glutamate decarboxylase (GAD67), and

GABA transaminase (GABA-T), transporter proteins

for glutamate (GLAST and GLT-1) and GABA (GAT1),

glutamate (NMDAR1, NMDAR2B, GluA1) and GABA

(GABAAR1) receptor subunits] were determined in the

hippocampus of OXYS and Wistar rats aged 1.5, 3, 12, and

18 months (n = 6 for each group) by Western blot analysis.

Rats were anesthetized by CO

inhalation and de-

capitated. Hippocampus was extracted on ice and frozen

in liquid nitrogen. The samples were stored at –70°C till

the moment of use. All stages of protein isolation were

carried out on ice at 4°C. Hippocampal samples were

homogenized using a RIPA lysis buffer (150 mM NaCl;

50 mM Tris-HCl (pH 7.4); 1% Triton X-100; 0.1% so-

dium dodecyl sulfate (SDS); 1% sodium deoxycholate

(Deoxycholic Acid, Sodium Salt), and 1 mM EDTA)

with protease and phosphatase inhibitors (P8340 and

P5726-5ML; Sigma-Aldrich, USA). After thorough

BURNYASHEVA et al.1974

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

homogenization, the protein solution was centrifuged

at 12,000g for 30 min at 4°C. Supernatant was stored

at –20°C. Total protein concentration was determined

using a ThermoFisher Pierce™ BCA Protein Assay kit

(Thermo Fisher; #23225). Calibration curve was used to

determine protein concentration using a reference pro-

tein from the kit.

Samples (50 μg of total protein) in a loading buf-

fer (10% SDS; 15% β-mercaptoethanol; 50% glycerol;

0.3 M Tris-HCl (pH 6.8); bromophenol blue) were ap-

plied onto the lanes of 8% polyacrylamide gel in

Tris-Glycine buffer (1.5 M, pH 8.8), separated by electro-

phoresis, and transferred onto a nitrocellulose membrane

(Bio-Rad, USA), which was then blocked with 5%BSA in

PBST(1 h). Next the membranes were incubated for 16 h

at 4°C with primary antibodies (anti-glutaminase, anti-

glutamate synthase, anti-NMDAR1, AMPA receptor an-

ti-subunit 1 (anti-GluA1), anti-GAD67, anti-GABA-T,

GABA-A receptor anti-α1, anti-GLT-1 and anti-GAT1

(ab93434, ab64613, ab109182, ab183797, ab26116,

ab152134, ab33299, ab41621 and ab426, respectively;

Abcam, USA; dilution of 1 : 1000), anti-GLAST and

anti-NMDA2B (PA519709 and 71-8600, Invitrogen,

USA; dilution of 1 : 1000). β-Actin (42 kDa; ab6276,

Abcam; 1 : 5000) and GAPDH (37 kDa; ab8245, Ab-

cam; 1 : 5000) were used as reference proteins. After

washing in PBST, the samples were incubated with anti-

mouse and anti-rabbit secondary antibodies (ab150115

and ab96886, respectively; Abcam; 1 : 5000) for 1 h

at room temperature. Fluorescence was detected with

a ChemiDoc MP Imaging System (Bio-Rad). Fluo-

rescence intensity of the bands was measured with the

ImageJ software (NIH,USA).

RNA-Seq analysis. High-throughput RNA se-

quencing (RNA-Seq) of the hippocampal samples of

OXYS and Wistar rats aged 20 days, 5 and 18 months

(n = 3 for each group) was performed using an Illumina

Genome AnalyzerIIx instrument at the Genoanalitika,

Russia. For each sample, ~40 million reads of 50 nucle-

otides in length were obtained. The reads were mapped

to the reference genome of Rattus norvegicus (version

Rnor_5.0.76) using the TopHat software (v. 2.0.10).

Based on the RNA-Seq data at a significance level of

padj < 0.05, lists of the differentially expressed genes

were compiled [13,14].

The list of genes for the glutamatergic signaling

pathway was retrieved from the rat genome database

(RGD; 126 rat genes; https://rgd.mcw.edu/). The list

of genes for the GABAergic synapse was obtained from

the KEGG pathway database (89 human genes; https://

www.genome.jp/kegg/).

Statistical analysis. Statistical processing of the

results was performed with the STATISTICA soft-

ware package (version 10.0). The Kolmogorov–Smirnov

test was used to check normal distribution. The anal-

ysis included all values lying within the range of three

root mean square deviations from the sample mean.

The analysis of variance was used, followed by posthoc

comparison of intergroup average values using the

Newman–Keuls test. “Genotype” and “age” were con-

sidered as independent factors. The data are presented

as a mean ±standard deviation (M ± SD). Differences

were considered statistically significant at p<0.05.

RESULTS

Assessment of glutamate and GABA levels in the

hippocampus of OXYS and Wistar rats of different ages.

The first stage of the work included enzyme immuno-

assay (ELISA) of free glutamate and GABA levels in

the hippocampus of Wistar and OXYS rats of different

age. No any inter-lineage and age-related differences in

the levels of glutamate (Fig. 1a) and GABA (Fig. 1b) in

the hippocampus of rats of both lineages were revealed.

Two-factor analysis of variance (ANOVA) did not show

any effects of “genotype” and “age” factors. Thus, the

level of neurotransmitters under the study remained

stable in the hippocampus of OXYS and Wistar rats

throughout their life.

Age-related changes in the glutamatergic system in

the hippocampus of Wistar and OXYS rats. When assess-

ing age-related changes in the glutamatergic system in

the rat brain, we analyzed expression of the key enzymes

for glutamate synthesis and degradation: proteins gluta-

minase and glutamine synthase, as well as transporter

proteins GLAST, GLT-1, and NMDAR1, NMDAR2B,

and GluA1 glutamate receptor subunits (AMPA recep-

tor subunits). Glutamate cannot penetrate through the

blood–brain barrier; accordingly, glutamate is synthe-

sized in the brain de novo in astrocytes and neurons

from glutamine with involvement of glutaminase. In as-

trocytes, glutamate is converted into glutamine with in-

volvement of glutamine synthase [15].

According to the two-way analysis of variance, the

levels of glutaminase (Fig. 2a) and glutamine synthase

(Fig.2b) does not depend on the age or genotype of the

animals: we have not observed any differences between

the levels of these proteins in the hippocampus of OXYS

and Wistar rats.

An important factor determining availability of glu-

tamate for signaling processes is the system of its recap-

ture and recycling. Glutamate uptake from the synaptic

cleft is necessary for normal neurotransmission in glu-

tamatergic synapses, because high level of extracellular

glutamate may have a toxic effect on the neurons and

synapses. The GLAST and GLT-1 transporters remove

glutamate from the extracellular space, which is nec-

essary to maintain low nontoxic concentrations of this

neurotransmitter [16] As assessed with ANOVA, the lev-

els of GLAST and GLT-1 proteins in the hippocampus

depended on the “age” factor (F

3.37

= 10.2; p < 0.001

CHANGES IN THE GLUTAMATE/GABA SYSTEM 1975

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

Fig. 1. Levels of glutamate (a) and GABA (b) in the hippocampus of Wistar and OXYS rats of different age. The data are presented as

M±SD(n=5).

Fig. 2. Age-related changes in the protein levels of glutaminase(a), glutamine synthase(b), GLAST(c), GLT-1(d), NMDAR1(e), NMDAR2B(f),

GluA1 (g) in the hippocampus of Wistar and OXYS rats. h)Representative images of the Western blot analysis of the proteins: W, Wistar,

O,OXYS. The data are presented as M±SD(n=4-6).

and F

3.31

= 4.4; p < 0.01, respectively) but did not de-

pend on the “genotype” factor. Comparison of the av-

erage group values showed that the level of GLAST in the

rats of both lineages increased by the age of 12 months

and decreased to the level of 1.5-month-old animals by

the age of 18 months (Fig.2c). The level of GLT-1 in

the OXYS rats increased from 3 to 12 months and re-

mained at the same level at the age of 18 months, while

in the Wistar rats the level of GLT-1 did not significantly

change with age (Fig.2d). There were no inter-lineage dif-

ferences in the levels of GLAST and GLT-1 transporters.

Postsynaptic terminal recognizes glutamate using

glutamate receptors [6]. That is why we analyzed the

levels of subunits of the NMDA- and AMPA-type ion-

otropic glutamate receptors mediating fast neurotrans-

mission [AMPA receptor subunit 1 (also known as

GluA1) and NMDA receptor subunits NR1 and NR2B

(NMDAR2B and NMDAR1; also known as GluN2B

BURNYASHEVA et al.1976

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

Fig. 3. Age-related changes in the levels of proteins GAD67(a), GABA-T(b), GAT1(c), and GABAAR1(d) in the hippocampus of Wistar and

OXYS rats. e)Representative images of the Western blot analysis of the proteins: W,Wistar; O,OXYS. The data are presented as M±SD(n=4-6).

and GluN1, respectively)] in the hippocampus of Wistar

and OXYS rats of different age. Based on the two-way

analysis of variance, the level of NMDAR1 protein de-

pended only on the “age” factor (F

3.34

= 1.6; p < 0.05).

Comparison of the average group values showed that

the level of NMDAR1 decreased with age in the Wistar

rats: by the age of 12 months it became reliably low-

er (p < 0.05) compared to the 1.5-month-old animals

(Fig. 2e). In the OXYS rats no significant age-related

changes were detected; therefore, even at the age of

12 months, they demonstrated an increasing trend of

the level of NMDAR1 (p = 0.06) in comparison to the

Wistar rats; at the age of 18 months in the OXYS rats,

the level of NMDAR1 was reliably higher (p < 0.05;

Fig. 2e). The levels of NMDAR2B and GluA1 in the

hippocampus of OXYS and Wistar rats were not differ-

ent and did not change with age (Fig.2,fandg).

Age-related changes in the GABAergic system in

the hippocampus of Wistar and OXYS rats. Next, we as-

sessed expression of the key enzymes for GABA synthe-

sis (glutamate dexarboxylase, GAD67) and degradation

(GABA transaminase, GABA-T). As was shown by the

two-way analysis of variance, the level of GAD67 was

higher (F

1.38

= 4.2; p < 0.05) and the level of GABA-T

was lower (F

1.39

= 6.3; p < 0.02) in the OXYS rats com-

pared to the Wistar rats; at the same time, age had no

effect on the levels of these proteins (Fig. 3,a andb).

Age had no effect on the level of GAT1: the transporter

that removes GABA from the synaptic cleft. Differenc-

es in the GAT1 level in the hippocampus of OXYS and

Wistar rats were not detected either (Fig.3c).

The level of GABAAR1 depended on the age of an-

imals (F

3.40

= 39.7; p < 0.001) but was independent of

their genotype. The level of GABAAR1 significantly

increased by the age of 12 months and then decreased

by the age of 18 months in the rats of both lineages

(p < 0.05; Fig.3d). At the same time, comparison of the

average group values showed that the level of GABAAR1

at the age of 3 and 18 months in the OXYS rats was high-

er than in the Wistar rats (p<0.05).

Age-related changes in the expression of the genes as-

sociated with the glutamate and GABA signaling pathways

in the hippocampus of OXYS and Wistar rats. For assess-

ing the changes in expression of the genes associated with

glutamatergic and GABAergic synapses in the OXYS

rats, we analyzed the previous data obtained by RNA-Seq

of the hippocampus of 20-day-old, 5- and 18-month-

old OXYS and Wistar rats [13, 14]. In the OXYS rats,

expression of 29 out of 126 genes associated (according

to RGD) with glutamatergic synapse changed from the

age of 20 days to the age of 5 months, and expression

of 56 genes changed from 5 to 18 months. In the Wis-

tar rats, expression of 34 genes changed from the age of

20days to the age of 5 months and expression of 51 genes

changed from 5 to 18 months. Out of the 88 genes asso-

ciated (according to the KEGG pathway database) with

the GABAergic synapse, expression of 13 genes changed

from the age of 20 days to 5 months and expression of 41

genes changed from 5 to 18 months in the hippocampus

of OXYS rats; in the Wistar rats, expression of 21 genes

changed from the age of 20 days to 5 months and expres-

sion of 37 genes changed from the age of 5 to 18 months.

It should be noted that expression of the vast majority of

the genes associated with the glutamate/GABA signaling

pathways decreased at the age from 5 to 18 months in the

hippocampus of rats from both lineages (table).

CHANGES IN THE GLUTAMATE/GABA SYSTEM 1977

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

Age-related changes in expression of the genes associated with the glutamate/GABA signaling pathways in the

hippocampus of OXYS and Wistar rats

Gene name Gene symbol

20days-5months 5-18 months

OXYS Wistar OXYS Wistar

4-Aminobutyrate aminotransferase Abat

↓↓

Adenylate cyclase 1 Adcy1

↑↓↓

Adenylate cyclase 2 Adcy2

↑↓↓

Adenylate cyclase 4 Adcy4

↓↑

Adenylate cyclase 5 Adcy5

↓↓

Adenylate cyclase 6 Adcy6

↓↓

Adenylate cyclase 9 Adcy9

↑↑↓↓

Voltage-gated N-type calcium channel subunitalpha-1B Cacna1b

↓

Voltage-gated N-type calcium channel subunit alpha-1C Cacna1c

↓↓

Calcineurin-like EF-hand protein1 Chp1

↓↓

Discs large MAGUK scaffold protein4 Dlg4

↓↓

DLG associated protein 1 Dlgap1

↑↑↓↓

Type1 protein associated with type A GABA receptor Gabarapl1

↑

Type 2 protein associated with type A GABA receptor Gabarapl2

↑↑

Type B GABA receptor subunit1 Gabbr1

↑

Type B GABA receptor subunit2 Gabbr2

↓↓

Type A GABA receptor subunit alpha 1 Gabra1

↓↓

Type A GABA receptor subunit alpha2 Gabra2

↓↓

Type A GABA receptor subunit alpha 3 Gabra3

↓↓

Type A GABA receptor subunit alpha5 Gabra5

↓↓

Type A GABA receptor subunit beta 1 Gabrb1

↑↑↓↓

Type A GABA receptor subunit beta2 Gabrb2

↓↓

Type A GABA receptor subunit beta3 Gabrb3

↓↓

Type A GABA receptor subunit delta Gabrd

↑↑↑

Type A GABA receptor subunit gamma 2 Gabrg2

↑↓↓

Type A GABA receptor subunit gamma 3 Gabrg3

↓

Type A GABA receptor subunit theta Gabrq

↓

Glutamate decaboxylase 2 Gad2

↓↓

BURNYASHEVA et al.1978

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

Table (cont.)

Gene name Gene symbol

20days-5months 5-18 months

OXYS Wistar OXYS Wistar

Glutaminase Gls

↓↓

Glutaminase2 Gls2

↑

Subunit G protein subunit alphai1 Gnai1

↓↓

Gprotein subunitalphai3 Gnai3

↓↓

G protein subunit alphao1 Gnao1

↓↓

G protein subunit alphaq Gnaq

↓↓

Complex locus GNAS Gnas

↑↑

G protein subunit beta 1 Gnb1

↓↓

G protein subunit beta 2 Gnb2

↑

G protein subunit beta 4 Gnb4

↓↓↓↓

G protein subunit beta 5 Gnb5

↑

G protein subunit gamma 12 Gng12

↓↓↓↓

G protein subunit gamma 2 Gng2

↓↓↓

G protein subunit gamma 3 Gng3

↓↑

G protein subunit gamma 4 Gng4

↓↓

G protein subunit gamma 5 Gng5

↑↑

G protein subunit gamma 8 Gng8

↑

Glutamate ionotropic receptor AMPA type subunit 1 Gria1

↑↓↓

Glutamate ionotropic receptor AMPA type subunit3 Gria3

↓

Glutamate ionotropic receptor kainite type subunit 2 Grik2

↓

Glutamate ionotropic receptor kainite type subunit 3 Grik3

↓↓

Glutamate ionotropic receptor kainite type subunit 4 Grik4

↑

Glutamate ionotropic receptor NMDA type subunit 2A Grin2a

↑↑↓↓

Glutamate ionotropic receptor NMDA type subunit2B Grin2b

↑↑ ↓

Glutamate ionotropic receptorNMDA type subunit2D Grin2d

↓

Glutamate ionotropic receptorNMDA type subunit3A Grin3a

↓↓

Glutamate ionotropic receptorNMDA type subunit3B Grin3b

↑

Glutamate metabotropic receptor 1 Grm1

↑↑↓↓

CHANGES IN THE GLUTAMATE/GABA SYSTEM 1979

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

Gene name Gene symbol

20days-5months 5-18 months

OXYS Wistar OXYS Wistar

Glutamate metabotropic receptor 2 Grm2

↓

Glutamate metabotropic receptor 3 Grm3

↑↑↓↓

Glutamate metabotropic receptor 4 Grm4

↓↓

Glutamate5 Grm5

↓↓

Glutamate metabotropic receptor 7 Grm7

↓↓

Huntingtin-associated protein 1 Hap1

↑↑

Homer scaffold protein 1 Homer1

↓↓

Homer scaffold protein 2 Homer2

↓↓

Homer scaffold protein 3 Homer3

↑↑

Inositol-1,4,5-triphosphate receptor type 1 Itpr1

↑↑↓↓

Inositol-1,4,5-triphosphate receptor type 2 Itpr2

↓↓

Member 3 of potassium inwardly rectifying channel subfamilyJ Kcnj3

↓↓

Member 6 of potassium inwardly rectifying channel subfamilyJ Kcnj6

↓

Mitogen-activated protein kinase1 Mapk1

↑↑↓↓

N-ethylmaleimide-sensitive factor, vesicle-fusing ATPase Nsf

↑

Phospholipase A2, group IIC Pla2g2c

↑

Phospholipase A2, group III Pla2g3

↓↓↑↑

Phospholipase A2, group IVE Pla2g4e

↓

Phospholipase A2, group V Pla2g5

↓

Phospholipase A2, group VI Pla2g6

↓

Phospholipase C beta 1 Plcb1

↑↓↓

Phospholipase C beta 4 Plcb4

↓↓

Phospholipase C like 1 (inactive) Plcl1

↓

Phospholipase D1 Pld1

↓↓↓↓

Phospholipase D2 Pld2

↓↓

Protein phosphatase3, catalytic subunit alpha Ppp3ca

↑↓

Protein phosphatase3, catalytic subunit beta Ppp3cb

↓

Protein phosphatase3, catalytic subunit gamma Ppp3cc

↑

BURNYASHEVA et al.1980

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

Table (cont.)

Gene name Gene symbol

20days-5months 5-18 months

OXYS Wistar OXYS Wistar

Protein phosphatase3, regulatory subunitB, alpha Ppp3r1

↑↓

cAMP-dependent protein kinase, catalytic subunit beta Prkacb

↓↓

Protein kinase C alpha Prkca

↓↓

Protein kinase C beta Prkcb

↓↓

Protein kinase C gamma Prkcg

↑↓

cAMP-dependent protein kinase X-linked catalytic subunit Prkx

↓

SH3 and multiple ankyrin repeat domains1 Shank1

↑

Solute carrier family 12 member 5 Slc12a5

↑↑

Solute carrier family 17 member 6 Slc17a6

↓↓↓↓

Solute carrier family 17 member 8 Slc17a8

↓↓

Solute carrier family 1 member 1 Slc1a1

↓↓

Solute carrier family 1 member 2 Slc1a2

↑↑↓↓

Solute carrier family 1 member 3 Slc1a3

↓

Solute carrier family 1 member 7 Slc1a7

↓↓

Solute carrier family 38 member 1 Slc38a1

↓↓

Solute carrier family 1 member 2 Slc38a2

↓

Solute carrier family 38 member 3 Slc38a3

↓↓

Solute carrier family 6 member 1 Slc6a1

↓

Solute carrier family 6 member 11 Slc6a11

↑↓↓

Solute carrier family 6 member 1 Slc6a12

↓

Solute carrier family 6 member 13 Slc6a13

↓

Motor protein kinesin 2 Trak2

↓↓

Member 1 of subfamily C of transient receptor cation

potential channels

Trpc1

↓↓

Note. Upward pointing arrow,genes with the age-related increase in expression; downward pointing arrow,genes with the age-re-

lated decrease in expression.

At the age of 20 days, expression of only four genes

associated with the glutamatergic synapse in the hip-

pocampus of the OXYS rats was different from that in

the Wistar rats: the mRNA levels of Grin3b, Grm6, and

Slc1a2 were increased and that of Pla2g5 was decreased

(padj < 0.05). In the OXYS rats aged 5months, expres-

sion of the Grin3b and Pla2g2d was changed (increased);

at the age of 18 months, expression of the Pla2g6 gene

was increased and that of the Gng12, Grm6, Ppp3r1,

and Slc1a1 genes was decreased. Among the genes

CHANGES IN THE GLUTAMATE/GABA SYSTEM 1981

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

associated with the GABAergic synapse, only at the age

of 18 months we identified differentially expressed genes

with reduced expression in the OXYS rats: Gad2, Gng12,

Plcl1, and Trak2.

Thus, we have failed to detect any significant inter-

lineage differences in the level of expression of the genes

associated with glutamatergic and GABAergic synaps-

es. With age, the hippocampus of rats of both lineages

showed, in general, similar changes in the expression of

the genes associated with glutamate and GABA signal-

ing pathways; at the same time, the mRNA level of the

vast majority of these genes showed a regular decrease

inboth Wistar and OXYS rats.

DISCUSSION

Cox et al. [5] consider age-related decline in the ef-

ficiency of glutamatergic signal transduction, which has

been found in different animal species, as one of the con-

servative manifestations of aging, similar to sarcopenia

or osteoporosis. Naturally, decline and impairment of

synaptic plasticity is accelerated in the case of prema-

ture aging, including AD. The main goal of the pres-

ent study was to assess contribution of the age-related

changes in the glutamatergic and GABAergic systems

to the emergence and progression of the signs of AD in

the brain of OXYS rats, the model of its sporadic form.

Unexpectedly, we have not revealed significant differ-

ences between functioning of these systems in the hip-

pocampus of OXYS rats and control Wistar rats. One of

the possible causes was the decrease in total protein lev-

el used to normalize the levels of glutamate and GABA

in the present work. In another work, where we stud-

ied hippocampal metabolome by NMR spectroscopy,

all parameters were normalized to the sample weight

and showed increase in the GABA level from the age of

20days to the age of 5 months and its decrease from the

age of 5 months to the age of 18 months in the rats of

both lineages, as well as analogous age-related changes

in the glutamate level in the OXYS rats [17]. Such results

indicate that the age-related changes in the balance of

the glutamate/GABA system occur in the hippocampus

of rats of both lineages. It should be noted that the pub-

lished data on changes in the level of glutamate and the

enzymes for its synthesis and degradation with age and

during AD development are contradictory. Some works

report age-related decrease in the level of glutamate in

the anterior cingulate gyrus, hippocampus, and other

brain regions [18-22]. Other authors state that there are

no age-related changes in the glutamate and glutamine

synthesis in the rat brain and in the glutamate cycle, as

confirmed by the data on the absence of changes in the

glutamate synthase activity [23,24].

The results of the current study have shown that

the levels of the key enzymes for glutamate synthesis

(neuronal glutaminase) and degradation (glial glutamate

synthase) do not change with age and are not different

in the OXYS and Wistar rats, indicating stability of its

synthesis in the hippocampus of rats throughout their

entire life. At the same time, it should be noted that

the absence of changes in the glutamate level cannot be

unambiguously considered as an indicator of stability

of the glutamatergic system in aging and AD develop-

ment. Glutamate excitotoxicity is mediated mainly by

the impairment of its recapture system, which leads to

the high level of glutamate in synaptic clefts and, as a

consequence, hyperactivation of NMDA receptors [25].

Thus, an important factor that determines avail-

ability of glutamate for the signal transduction pathway

is the system of its recapture and recycling. Glutamate

cannot penetrate through the blood–brain barrier and

is produced mainly by neurons and astrocytes. Never-

theless, neurons per se are unable to synthesize gluta-

mate from glucose via the tricarboxylic acid cycle due

to the absence of pyruvate carboxylase [15]. In view of

the above, glutamate formation in astrocytes plays an

important role; it occurs through the two pathways:

synthesis de novo in the tricarboxylic acid cycle (it ac-

counts for ~15% of glutamate) or glutamine “recycling”

from GABA and glutamate through recapture of neu-

rotransmitters [6]. Under physiological conditions, as-

trocytes remove ~90% of all glutamate released by the

CNS using excitatory amino acid transporters GLAST

and GLT-1 required for maintaining low, nontoxic con-

centrations of this neurotransmitter [26]. As our study

has shown, the level of GLAST in the hippocampus

of both Wistar and OXYS rats increased by the age

of 12months and decreased by the age of 18 months.

Wewere unable to find any age-related changes in the

level of GLT-1 in the Wistar rats, while in the OXYS rats

it increased considerably from the age of 3 to 12 months,

which may indicate the change in glutamate recapture.

This is probably due to accumulation of the toxic forms

of β-amyloid in the hippocampus of OXYS rats by the

age of 12 months[9]. It should be noted that insignifi-

cant changes in the amount of GLT-1 were also detected

in the hippocampus of AD patients: it has been reported

that the spatial pattern of expression of this transporter

ischanged and the increased immunostaining of GLT-1

is observed in the astrocyte processes and in neuropile,

especially in the CA1 and CA3 areas of the hippocam-

pus, as well as in the dentate gyrus [27].

The NMDA and AMPA receptors are present in

approximately 70% of synapses of the mammalian brain,

mainly in the cerebral cortex, amygdaloid body, striated

body, and hippocampus. Specific localization of these

receptors is of great significance, because glutamatergic

system plays an important role not only in neuroplasti-

city but also in excitotoxicity [28]. It has been shown that

the glutamatergic system mediated by NMDA recep-

tors becomes hypofunctional with age, and this deficit

BURNYASHEVA et al.1982

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

can lead to cognitive dysfunction under both normal

and pathological conditions [5]. In addition, there is

evidence that the number of NMDA receptors on the

postsynaptic terminals of neurons in the hippocampus

decreases with age [29, 30], thereby considerably re-

ducing glutamate bioavalability [31]. It is known that

NMDA receptors are heterotetramers consisting of two

obligatory subunits NMDAR1 and two regulatory sub-

units GluN2(A–D) or GluN3(A or B) localized mainly

in the dendrites of neurons. Since the hippocampus is

a cerebral area regulating cognitive functions, regula-

tory subunits are mainly represented by NMDA2A or

NMDA2B (GluN2A and GluN2B, respectively) [32].

In the present work, we have assessed the age-relat-

ed changes in the level of obligate subunit NMDAR1 and

subunit NMDA2B mediating excitotoxic effects of gluta-

mate in the rat hippocampus [33]. The NMDAR2B pro-

tein level did not change significantly with age and was

not different in the Wistar and OXYS rats, while the level

of subunit NMDAR1 decreased with age in the hippo-

campus of Wistar rats but did not change significantly in

the OXYS rats. As a result, the level of NMDAR1 became

much higher by the age of 18 months in the OXYS rats

compared to the Wistar rats. It should be noted that sim-

ilar changes in the level of NMDAR1 have been found in

the AD patients [34]. It is likely that the increased level

of NMDAR1 in the hippocampus in the case of AD is

a compensatory mechanism, because it has been report-

ed that the increased number of subunits NMDAR1 and

NMDA2A, but not of NMDAR2B, is associated with

spatial memory consolidation and formation [34].

In the hippocampus, AMPA receptors localized

mainly in neurons are in composition of the majori-

ty of excitatory synapses, especially in the CA1 region

(~80% of all receptors). The best studied AMPA recep-

tor subunit is GluA1[35]. Moreover, the GluA1-related

impairment of synaptic plasticity is considered by many

authors as one of the key events at the early stages of

AD development [36]. Our analysis of the GluA1 lev-

el did not reveal any significant changes with aging and

development of the signs of AD in the OXYS rats. Prob-

ably, this is due to the fact that we have assessed it in the

whole hippocampus, while the changes in the GluA1

expression may be differently regulated in the different

areas of this brain structure [34,35].

Previously it has been considered that the GAB-

Aergic neurons are more resistant to the pathological

effects of β-amyloid compared to the cholinergic or glu-

tamatergic neurons [36]. The hypothesis that has been

put forward in recent years suggests that the excitato-

ry/inhibitory imbalance could cause GABAergic dys-

function, which increases susceptibility of the neurons

to unfavorable external factors and pathological stress,

contributing to the impairment of functional connec-

tions in the brain during the development of AD [37]. In

the present study, we have not revealed any differences

in the GABA levels in the hippocampus of OXYS and

Wistar rats. The only direct precursor of GABA in the

CNS is glutamate, which is converted into GABA by

glutamic acid decarboxylase, or glutamate decarboxylase

(GAD). In the mammalian brain, GAD has two iso-

forms: GAD65 and GAD67[38]. GAD65 is mainly lo-

calized on the presynaptic nerve endings, while GAD67

is distributed all around a cell. It should be noted that

more than 90% of GABA in the brain is synthesized by

GAD67 [39, 40]. Mice with the GAD67 gene knock-

out die within a week after birth; however, mice with

the GAD67 expression deficiency are viable, though

exhibit abnormal behavior [41]. On the contrary, mice

with the GAD65 gene knockout survive but are prone

to convulsions [42]. Dysfunction of GAD67 is related to

the brain disorders such as schizophrenia [43], bipolar

disorder [44] and Parkinson’s disease [45]. It has been

reported that the expression of GAD67 is unchanged in

the post mortem samples of the brain tissues from AD

patients; however, at the same time, it is still unclear

whether GAD67 is involved in progression of the dis-

ease [46]. In addition, it has been shown that age and

sex have no effect on the GAD67 expression in the hu-

man hippocampus and cerebral cortex [47]. According

to our data, in the hippocampus of OXYS rats, the level

of GABA-T (enzyme responsible for GABA degradation

in the brain and localized mainly in astrocytes) is con-

siderably lower, while the level of GAD67, which cata-

lyzes GABA formation in neurons, is higher than in the

Wistar rats. These results indicate the enhanced demand

for GABA formation in the hippocampus of OXYS rats.

At the same time, we have shown no reliable differences

in the content of GABA transporter GAT1, which re-

moves GABA from the synaptic cleft.

Previously, experiments in vitro have shown that

β-amyloid neurotoxicity reduces activity of the GAB-

Aergic neurons and attenuates inhibitory postsynaptic

potentials by suppressing postsynaptic GABA receptors

[48, 49]. However, in the hippocampus of OXYS rats,

the level of postsynaptic GABA receptor GABAAR1 at

the age of 3 months (in the period of manifestation of

the signs of AD) and in the period of their active pro-

gression (12months) was higher than in the Wistar rats.

In the hippocampus, the highest level of GABAAR1 ex-

pression is observed in the CA1 area and, according to

some studies, its expression does not change with age

[50,51]. We believe that the enhanced GABAAR1 ex-

pression in the hippocampus of the one-year-old OXYS

rats that we have observed may be due to neurodegener-

ative changes, which are noted as early as at the age of

3-5 months and progress with aging [52]. There is con-

siderable accumulation of β-amyloid in the brain struc-

tures of the OXYS rats by the age of 12 months [9], and

we consider it as a possible cause of increase in GAB-

AAR1; however, this assumption needs further experi-

mental verification.

CHANGES IN THE GLUTAMATE/GABA SYSTEM 1983

BIOCHEMISTRY (Moscow) Vol. 88 Nos. 12-13 2023

In order to assess age-related changes in the glu-

tamatergic and GABAergic systems and their poten-

tial contributions to the development of the signs of

AD, we have analyzed gene expression in the hippo-

campus of OXYS and Wistar rats of different ages us-

ing the RNA-Seq data. Our analysis has shown their

significant age-related changes in the hippocampus

of the rats in both lineages; however, we have failed to

find any inter-lineage differences. Thus, the mRNA

level of the genes encoding components of the gluta-

mate and GABA signaling pathways are not different in

the Wistar and OXYS rats at all stages of development

of the signs of AD. The exception that we have noted

is the genes encoding glutamate receptors (Grin3b and

Grm6), glutamate decarboxylase 2 (Gad2), G protein

subunit (Gng12), the family of solute carriers (Slc1a1

and Slc1a2), phospholipase A2 (Pla2g2d, Pla2g5, and

Pla2g6), phospholipaseC (Plcl1), protein phosphatase3

(Ppp3r1), and transport protein kinesin2 (Trak2). Nev-

ertheless, the changes in expression of these genes at

different stages of neurodegeneration in OXYS rats did

not allow us to formulate any hypothesis about contri-

bution of these genes to the pathogenesis of AD. On the

contrary, we have revealed a clear age-related decrease

in the expression of the genes associated with glutamate/

GABA signal transduction in the rats of both lineages.

Generally, our results indicate absence of the

changes or compensatory activation in the glutamate-

and GABAergic systems with aging and development of

the signs of AD in the OXYS rats, which seem to be a

consequence of the development of neurodegenerative

processes.

CONCLUSIONS

According to Cox et al. [5], the decrease in glu-

tamatergic transmission can be used as a biomarker of

transition from physiological to pathological aging.

Based on the results of our study, we can conclude that

there is a considerable age-related decrease in the glu-

tamate and GABA signal transduction in the hippo-

campus of Wistar rats; however, in the hippocampus of

OXYS rats there are no substantial changes or compen-

satory increase in this system during the development of

neurodegenerative processes typical of AD. The study

of AD pathogenesis is complicated by its heterogeneity,

various pathophysiological scenarios, and existence of

several molecular subtypes of this disease [53]. Further

studies are required to detect the changes in the balance

of neurotransmitter systems. This knowledge will be an

important step on the pathway to personalized medicine

for the patients with this neurodegenerative disease.

Contributions. A.O.B. and N.A.S. experimental

work; A.O.B., N.A.S., D.V.T., and N.G.K. discussion

of research results; A.O.B. and D.V.T. writing the man-

uscript; N.G.K., N.A.S., and D.V.T. editing the manu-

script.

Funding. The work was financially supported by the

Russian Science Foundation (project no.19-15-00044).

Ethics declarations. The authors declare no conflict

of interest in financial or any other sphere. All applica-

ble international, national, and/or institutional guide-

lines for the care and use of animals were followed. All

studies were carried out with OXYS and Wistar (control)

male rats on the basis of the Center for Collective Use

“Gene Pools of Laboratory Animals”, Institute of Cy-

tology and Genetics, Siberian Branch of the Russian

Academy of Sciences, in accordance with the ethical

standards of European Union Directive 2010/63/EU

of September 22, 2010. All experiments were approved

and performed in accordance with the guidelines of the

Ethics Committee on Animal Trials at the Institute of

Cytology and Genetics, Siberian Branch of the Russian

Academy of Sciences, Novosibirsk, Russia (Resolution

no.12000-496 of April2, 1980).

REFERENCES

1. Wang, R., and Reddy, P. H. (2017) Role of glutamate and

NMDA receptors in Alzheimer’s disease, J. Alzheimers

Dis., 57, 1041-1048, doi:10.3233/JAD-160763.

2. Selkoe, D. J., and Hardy, J. (2016) Theamyloid hypothe-

sis of Alzheimer’s disease at 25 years, EMBO Mol. Med., 8,

595-608, doi:10.15252/emmm.201606210.

3. Wyss-Coray, T. (2016) Ageing, neurodegeneration and

brain rejuvenation, Nature, 539, 180-186, doi:10.1038/na-

ture20411.

4. Polanco, J. C., Li, C., Bodea, L. G., Martinez-Mar-

mol,R., Meunier, F.A., and Götz, J. (2017) Amyloid-β and

tau complexity– towards improved biomarkers and target-

ed therapies, Nat. Rev. Neurol., 14, 22-39, doi:10.1038/

nrneurol.2017.162.

5. Cox, M. F., Hascup, E. R., Bartke, A., and Hascup, K.N.

(2022) Friend or foe? Defining the role of glutamate in

aging and Alzheimer’s disease, Front. Aging