Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 6, pp. 1024-1030 © The Author(s) 2024. This article is an open access publication.

1024

The C886T Mutation in the Th Gene Reduces

the Activity of Tyrosine Hydroxylase in the Mouse Brain

Ismail Alsalloum

1,2

, Vitalii S. Moskaliuk

, Ilya A. Rakhov

Daria V. Bazovkina

, and Alexander V. Kulikov

1,2,a

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

Novosibirsk State University, 630090 Novosibirsk, Russia

e-mail: v_kulikov@bionet.nsc.ru

Received March 16, 2024

Revised April 15, 2024

Accepted April 25, 2024

Abstract— Tyrosine hydroxylase (TH) catalyzes hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine, the

initial and rate-limiting step in the synthesis of dopamine, noradrenaline, and adrenaline. Mutations in the human

TH gene are associated with hereditary motor disorders. The common C886T mutation identified in the mouse

Thgene results in the R278H substitution in the enzyme molecule. We investigated the impact of this mutation

on the TH activity in the mouse midbrain. The TH activity in the midbrain of Mus musculus castaneus (CAST)

mice homozygous for the 886C allele was higher compared to C57BL/6 and DBA/2 mice homozygous for the 886T

allele. Notably, this difference in the enzyme activity was not associated with changes in the Th gene mRNA lev-

els and TH protein content. Analysis of the TH activity in the midbrain in mice from the F2 population obtained

by crossbreeding of C57BL/6 and CAST mice revealed that the 886C allele is associated with a high TH activity.

Moreover, this allele showed complete dominance over the 886T allele. However, the C886T mutation did not affect

the levels of TH protein in the midbrain. These findings demonstrate that the C886T mutation is a major genetic

factor determining the activity of TH in the midbrain of common laboratory mouse strains. Moreover, it represents

the first common spontaneous mutation in the mouse Th gene whose influence on the enzyme activity has been

demonstrated. These results will help to understand the role of TH in the development of adaptive and patholog-

ical behavior, elucidate molecular mechanisms regulating the activity of TH, and explore pharmacological agents

for modulating its function.

DOI: 10.1134/S000629792406004X

Keywords: tyrosine hydroxylase, C886T mutation, activity, expression, brain, mice

Abbreviations: DA,dopamine; CAST,Mus musculus castaneus;

L-DOPA,L-3,4-dihydroxyphenylalanine; TH,tyrosine hydrox-

ylase.

* To whom correspondence should be addressed.

INTRODUCTION

The dopaminergic system of the brain plays a piv-

otal role in the regulation of the nervous system, endo-

crine glands, and adaptive and pathological behavior

[1-4]. The nigrostriatal dopaminergic system regulates

motor function; its overactivation has been linked to

hyperactivity [5-7], while a deficit of its activity is as-

sociated with dystonia [8-10] and catalepsy/catatonia

[11,12]. On the other hand, the mesolimbic dopami-

nergic system is involved in the assessment of signal

significance, learning [13-15], and substance addiction

[16-19].

In the brain, dopamine (DA) is synthesized from

the amino acid L-tyrosine in two steps. First, tyrosine

hydroxylase(TH) converts L-tyrosine to L-3,4-dihydrox-

yphenylalanine (L-DOPA), and then, aromatic amino

acid decarboxylase converts L-DOPA to DA. The hy-

droxylation of L-tyrosine is a key and rate-limiting

step determining the DA levels in the brain. Indeed,

the Thgene knockout reduced DA content in the mouse

brain [20,21]. Some mutations in the human TH gene

have been associated with childhood parkinsonism

[22], increased risk of Parkinson’s disease [22, 23],

dystonia [24-27], and bipolar disorders [28]. However,

C886T MUTATION IN Th GENE 1025

BIOCHEMISTRY (Moscow) Vol. 89 No. 6 2024

investigating molecular events linking THgene muta-

tions to the nervous system disorders, motor function

impairments, and human mental activity is restricted

due to social and ethical constraints. Therefore, mod-

eling disturbances caused by mutations in the Thgene

in laboratory rodents is an important task in modern

neuroscience.

The Ensembl genome database (https://www.

ensembl.org/index.html) contains information on 21

single-nucleotide polymorphisms (SNPs) in the mouse

Thgene that lead to the amino acid substitutions in the

enzyme molecule. Among those, only one SNP, the C886T

mutation resulting in the R278H substitution, has been

identified in the Th gene of commonly used laboratory

mouse strains. The 886T allele is prevalent in most of

these strains (C57BL/6, C3H, DBA/2, CBA), whereas the

886C allele was found in the Mus musculus castaneus

(CAST) subspecies. Previously, it was demonstrated that

the G1449A mutation in the mouse Tph2 gene resulting

in the R441H substitution in tryptophan hydroxylase2

(TPH2), significantly reduces enzyme activity in the

mouse brain [29,30]. Taking into account that TH and

TPH2 are related enzymes from the aromatic amino

acid hydroxylase family [31], it was reasonable to ex-

pect that the C886T mutation decreases the TH activity.

We investigated the impact of the C886T muta-

tion in the Th gene on the TH activity in the mouse

brain and compared the activity of TH in the midbrain

(area containing the bodies of DA neurons) in C57BL/6

(886T), DBA/2 (886T), and CAST (886C) mouse strains,

as well as the studied the differences in the levels of

Th gene mRNA and TH protein between the studied

mouse strains. We also assessed the contribution of

the C886T mutation to the TH activity in the context

of other genes and investigated the linkage of the 886T

and 886C alleles with the TH activity and protein level

in the midbrain of F2 segregating intercross animals

obtained by crossbreeding of C57BL/6 and CAST mice.

MATERIALS AND METHODS

Animals. Adult male mice of C57BL/6 (n = 6),

DBA/2 (n = 6), and CAST (n = 5) strains, and F2 inter-

crosses between C57BL/6 and CAST mice (42 males and

females) were used in the experiments. The intercross-

es were obtained by crossing first-generation hybrids

F1(C57BL/6 × CAST) with each other. At the start of the

experiment, all mice were 12 weeks old and had the

specific pathogen-free (SPF) status that was maintained

during the study. The animals were housed under stan-

dard SPF conditions at a constant temperature of 23°C

and at a 14 h light/10 h dark cycle (lights switched on

at 01:00 and switched off at 15:00). The mice had an

adlibitum access to sterile dry food and water. At the

age of 3 weeks, young animals were separated from

their mothers and grouped into cages with 4-5 animals

of the same sex (Optimice, Animal Care Systems, Inc.).

The animals were marked with ear notches, and ear

samples obtained during marking were used for DNA

extraction and genotyping. Two days before the start of

the experiment, the animals were placed into individ-

ual cages to minimize the potential influence of group

effects on the TH activity. The animals were euthanized

using CO

asphyxia and decapitated; the midbrains

containing the bodies of TH-expressing DA neurons

were dissected, frozen in liquid nitrogen, and stored

at–80°C until assay.

Genotyping of 886T and 886C alleles. Genomic

DNA was isolated from ear punches obtained during

animal marking using precipitation with a saturat-

ed NaCl solution and then dissolved in sterile water.

The concentration of isolated genomic DNA was

measured with a NanoDrop 2000 (Thermo Fisher

Scientific, USA); genomic DNA was diluted to a con-

centration of 50 ng/μl. The 886T and 886C alleles

were identified by quantitative PCR (qPCR) using an

R-402 reagent kit (Syntol, Russia), a forward primer

(5′-GTAAGGGACCTCGCATCAGA-3′), and two allele-spe-

cific primers: T-allele (5′-CAGCTGGAGGATGTGTCACA-3′)

and C-allele (5′-CAGCTGGAGGATGTGTCACG-3′). To in-

crease the specificity of detection, we placed A instead

of T at position 18 of the allele-specific primers. For al-

lele identification, DNA samples (50ng) was amplified

with the forward primer and one of the allele-specific

primers using a CFX96 Touch Real-Time PCR Detection

System (Bio-Rad, USA) according to the protocol recom-

mended by the manufacturer (Syntol, Russia): 94°C for

5 min; 40 cycles of 94°C for 15 s, 60°C for 60 s, and

80°C for 2 s (fluorescence measurement). The amount

of formed PCR product was determined from the Sybr

Green fluorescence. If the allele presents in the DNA

sample matched the allele-specific primer, the number

of threshold cycle was 24-25; otherwise, it was greater

than 28. Each DNA sample was tested 3 times.

Preparation of samples for RT-qPCR and HPLC.

To determine the TH activity, TH content, and Th gene

mRNA level, the midbrain was homogenized in 400 μl

of cold 50 mM Tris-HCl (pH 6.0) containing 1mM dithio-

threitol, using a motor driven grinder (Z359971, Sigma-

Aldrich, Germany). An aliquot of 100μl of the homog-

enate was immediately mixed with 1ml of ExtractRNA

reagent (Eurogene, Russia) for total RNA extraction. The

RNA pellet was dissolved in 25μl of sterile water, treat-

ed with RNase-free DNase EM-100 (Biolabmix, Russia)

and the optical density was measured using a Nano-

Drop 2000 spectrophotometer (Thermo Scientific, USA).

RNA samples were diluted with sterile water to a con-

centration of 125 ng/μl and stored at –80°C. The quality

of isolated total RNA was assessed by electrophoresis in

1% agarose gel; only samples with clearly visible two

ribosomal RNA bands were included in further analysis.

ALSALLOUM et al.1026

BIOCHEMISTRY (Moscow) Vol. 89 No. 6 2024

The remaining 300μl of the homogenate was cen-

trifuged for 15 min at 12,700 rpm (4°C). The superna-

tant was transferred to clean tubes, and protein con-

centration was determined by the Bradford method

using the Bio-Rad Protein Assay kit (Bio-Rad). Thesu-

pernatant was stored at –80°C and used for the TH ac-

tivity and protein assays.

TH activity was assessed using the HPLC method

developed by us for determining the activity of TH in

brain tissues based on the rate of L-DOPA synthesis*.

A 15-μl aliquot of the TH-containing supernatant was

incubated for 15min at 37°C with 0.3 mM L-tyrosine

(Sigma-Aldrich), 0.3mM artificial cofactor 6,7-dimethyl-

5,6,7,8-tetrahydropterin (DMPH4, Sigma- Aldrich)**,

0.3 mM m- hydroxybenzylhydrazine decarboxylase in-

hibitor (Sigma-Aldrich), and 5 units of catalase (Sigma-

Aldrich) in a final volume of 25 μl. Theincubation was

stopped by adding 75 μl of 0.6 M HClO

. Protein was pre-

cipitated by centrifugation for 15 min at 14,000 rpm.

The clear supernatant was diluted two-fold with ultra-

pure water to reduce the acid concentration to a de-

tector-safe level of 0.3 M. Synthesized L-DOPA was de-

termined by HPLC on a Luna C18(2) column (length,

100 mm; diameter, 4.6 mm; 5 μm particle size, Phe-

nomenex, USA) equipped with a Zorbax SB-C8 guard

column (length, 12.5 mm; diameter, 4.6 mm; 5 μm par-

ticle size, Agilent, USA) using an LC-20AD chromato-

graph (Shimadzu Corporation, Japan). The mobile

phase (pH 3.2) contained 13.06 g of KH

, 200 μl of

0.5 M Na

EDTA, 300 mg of sodium 1-octanesulfonate,

940 μl of concentrated H

, and 130 ml of metha-

nol (13%) in 1 liter. The mobile phase flow rate was

0.6 ml/min. The concentration of synthesized L-DOPA

was determined using a DECADEII™ electrochemical

detector and a VT-03 glassy carbon electrode (3 mm;

Antec, Netherlands). The temperature of the column

and the detector was 40°C. Under these conditions, the

retention time of L-DOPA on the column was 4 min.

Thearea of the L-DOPA peak was determined with the

LabSolution LG/GC v. 5.54 software (Shimadzu Cor-

poration, Japan) using L-DOPA standards (25, 50, and

100 pmol). The activity of TH was expressed in pmol of

L-DOPA synthesized per minute per mg protein (mea-

sured by the Bradford method).

Assessment of Th gene mRNA level was carried

out by RT-qPCR. cDNA was synthesized on the isolated

RNA using random hexanucleotide primers and R01 kit

(Biolabmix, Russia). The content of Th gene cDNA was

determined by qPCR using R-402 kit (Syntol, Russia) and

forward (5′-CCGTACACCCTGGCCATTGATG-3′) and reverse

(5′-ATGAAGGCCAGGAGGAATGCAGG-3′) primers specific

to the nucleotide sequence of the mouse Th gene exon

in the following regime: 94°C for 5 min; 40 cycles of

94°C for 15 s, 64°C for 60 s, 80°C for 2 s (fluorescence

measurement) [31]. The Polr2a housekeeping gene was

used for normalization after amplification with the for-

ward (5′-GTTGTCGGGCAGCAGAATGTAG-3′) and reverse

(5′-TCAATGAGACCTTCTCGTCCTCC-3′) primers specific

to the nucleotide sequence of the mouse Polr2a gene

exon in the following regime: 94°C for 5 min, 40 cycles

of 94°C for 15 s, 63°C for 60 s, 80°C for 2 s (fluorescence

measurement) [32]. Mouse genomic DNA standards (25,

50, 100, 200, 400, 800, 1600, and 3200 copies of mouse

genomic DNA per 1 µl) were used for the threshold cy-

cle calibration. The level of the Th gene expression was

expressed as the number of copies of cDNA of this gene

per 100 copies of Polr2a gene cDNA [33].

The determination of protein quantity via by

Western blotting was conducted as described in [34].

Proteins were separated by SDS-PAGE in 10% PAG

(10µg of total protein per lane) and transferred onto

a membrane. The membrane was stained with rabbit

polyclonal anti-TH antibodies (1 : 500; ab112, Abcam,

UK). Rabbit polyclonal antibodies to GAPDH (1 : 2000,

ab9485, Abcam) and mouse monoclonal antibodies to

GAPDH (1 : 10,000, HC301, TransGen, China) served as

internal controls. The content of TH protein (60 kDa)

was normalized to the content of GAPDH (37 kDa) and

expressed in relative units.

Statistical analysis of the results was performed

using Statistica9.0 (StatSoft, Inc.). The levels of the Th

gene mRNA, TH protein content, and TH activity in the

midbrain were expressed as mean ±SEM and analyzed

using one-way (interstrain differences) and two-way

(F2) ANOVA followed by the intergroup comparison us-

ing the Fisher’s least significant difference (LSD) meth-

od. The segregation ratio of the TT, TC, and CC geno-

types among the F2 intercrosses was tested against the

1 : 2 : 1 ratio using the Pearson χ

-test. The significance

level was set at 0.05.

RESULTS

Comparison of the Th gene expression levels, TH

protein content, and TH activity in the midbrain of

C57BL/6 (886T), DBA/2 (886T), and CAST (886C) male

mice. Mice from the three strains exhibited differenc-

es in the TH activity in the midbrain [F(2,14) = 21.68,

p < 0.001]. Thus, the TH activity in the midbrain of

CAST mice was significantly higher than in C57BL/6

(p < 0.001) and DBA/2 mice (p < 0.001) (Fig. 1). However,

no differences between the strains were found in the

levels of Th gene transcripts [F(2,13) = 2.67, p = 0.11]

* Since no traces of endogenous L-DOPA were detected in the supernatant, no tissue control samples were used.

** Because the retention time of L-DOPA (4min) coincided with the retention times of the 5,6,7,8-tetrahydrobiopterin and

6-methyl-5,6,7,8-tetrahydropterin cofactors, only DMPH4 was used as a cofactor.

C886T MUTATION IN Th GENE 1027

BIOCHEMISTRY (Moscow) Vol. 89 No. 6 2024

Fig. 1. TH activity (a), Th genemRNA level (b), and TH protein content(c) in the midbrain of C57BL/6 (TT), DBA/2 (TT), and (CAST)

(CC) mouse strains. Individual values are presented along with means ±SEM. Expression of the Th gene was normalized to the

Polr2a gene expression; TH protein levels were normalized to the GAPDH levels; ***p<0.001 vs. CAST mice.

Fig. 2. TH activity (a) and protein levels(b) in the midbrain of F2 intercrosses with the TT, TC, and CC genotypes. The data for

males and females of the same genotype are combined. Individual values are presented along with means ±SEM. The TH protein

level was normalized to the GAPDH protein level. *p<0.05, ***p<0.001 vs. TT.

and TH protein content [F(2,14) = 1.23, p = 0.32] in the

midbrain (Fig.1).

TH protein level and TH activity in the mid-

brain of F2 intercrosses. The number of males and

females with the TT, TC, and CC genotypes among the

F2 intercrosses (42 animals) is presented in the table.

For statistical analysis, the data for males and females

with the same genotype were combined. The obtained

values closely correspond to the expected distribution

of 1 : 2 : 1 (χ

(1) = 0.805, p > 0.05).

Two-way ANOVA indicated a substantial contribu-

tion of the “genotype” factor to the TH activity in the

midbrain of F2 animals [F(2,34) = 9.47, p < 0.001]. How-

ever, no influence of the “gender”› factor [F(1,34) < 1]

and interaction between the factors [F(2,34) = 1.74,

p = 0.19] on the TH activity in the midbrain of F2 mice

was detected. This allowed us to combine the values

obtained for males and females of the same genotype

in order to increase the sample size for each genotype.

The activity of TH in the midbrain was lower in indi-

viduals with the TT genotype compared to those with

the TC (p < 0.001) and CC (p = 0.02) genotypes (Fig. 2).

No differences between the genotypes were observed

in the TH protein levels [F(2,38) = 2.22, p = 0.12] (Fig. 2).

Distribution of the TT, TC, and CC genotypes among the

males and females from the F2 intercross offspring

obtained by crossing F1 hybrids (C57BL/6×CAST)

Genotype Males Females Males + Females

TT 5 3 8

TC 13 10 23

CC 5 6 11

ALSALLOUM et al.1028

BIOCHEMISTRY (Moscow) Vol. 89 No. 6 2024

DISCUSSION

This study provides the first experimental evi-

dence of the influence of the prevalent C886T mutation

in the Th gene on the TH activity in the midbrain of

laboratory mice. The midbrain was chosen for exam-

ination because TH is expressed in the bodies of do-

paminergic and noradrenergic neurons located in the

midbrain and then distributed via axonal transport to

other brain structures receiving projections from these

neurons. First, we compared the activity of TH in the

midbrain of mice with the TT (C57BL/6 and DBA/2) and

CC (CAST) genotypes. CAST mice demonstrated an in-

creased activity of this enzyme compared to TT mice.

Although the C886T mutation is located in the TH cat-

alytic domain and theoretically cannot influence ex-

pression of the Th gene, we could not rule out that

the increased enzyme activity observed in CAST mice

may be due to some unknown genetic factors upreg-

ulating expression of the Th gene and/or TH protein

level. To test this assumption, we measured expression

of the Th gene and TH protein levels in the midbrain

of C57BL/6, DBA/2, and CAST mice and found no dif-

ferences in these parameters. It can be hypothesized

that the differences in the enzyme activity among these

three strains are not associated with the regulation of

the Thgene expression and/or TH protein stability.

To study a linkage between the 886C and 886T al-

leles with a high and low TH activity, we produced F2

intercrosses with different genotypes (TT, TC, and CC)

and investigated the relationship between the TH ac-

tivity and genotype of these mice. The distribution of

the TT, TC, and CC genotypes among F2 corresponded

well to the expected Mendelian segregation of 1 : 2 : 1,

which, in turn, may serve as an evidence of the ab-

sence of the effect of the C886T mutation on mouse sur-

vival. The TH activity in the brains of F2 mice with the

TC and CC genotypes was higher than in the mice with

the TT genotype. This result not only demonstrated the

linkage of high enzyme activity with the 886C allele

but also indicated that the C886T polymorphism in the

Th gene is a major genetic factor determining the TH

activity in mouse brain. Indeed, the influence of this

factor on the TH activity is so significant that segrega-

tion by a large number of mutations, which distinguish

C57BL/6 and CAST mice but theoretically may affect the

TH activity, could not mask the effect of the C886T mu-

tation. At the same time, no linkage of this mutation

with the TH protein level was found. This result can be

considered as an additional experimental evidence that

the C886T polymorphism does not affect expression of

the TH protein and/or its stability.

A question arises regarding possible molecular

mechanisms underlying reduction in the TH activity

caused by the R278H substitution. The most obvious

mechanism involves a decrease in the level of active

enzyme due to the reduced stability and lifetime of the

mutant protein. In this case, the TH activity in hetero-

zygotes should be equal to the arithmetic mean of TH

activities in the two homozygotes. Recently, it has been

demonstrated that the C1473G mutation in the mouse

Tph2 gene, resulting in the P447R substitution in TPH2,

reduces the stability and the lifetime of the enzyme

and, therefore, decreases the number of active enzyme

molecules [35]. It has been shown that the TPH2 activ-

ity in the brain of 1473CG heterozygotes is equal to the

arithmetic mean of the enzyme activities in homozy-

gous 1473CC and 1473GG mice [36]. We found no dif-

ferences in the TH activity in the mice with the 886TC

and 886CC genotypes, indicating complete dominance

of the 886C allele. This inheritance pattern contradicts

the hypothesis of the influence of the C886T mutation

on the level of TH protein. Since TH, like all aromatic

amino acid hydroxylases, is a tetramer consisting of

four subunits, it can be assumed that the presence of a

“normal” subunit (allele 886C) in heterozygous animals

somehow corrects the negative effect of the “defective”

subunit (allele 886T) on the activity of the entire te-

tramer. However, elucidating the exact mechanism

underlying the effect of the R278H substitution on the

THactivity requires special investigation using recom-

binant TH molecules.

Since TH is a key enzyme in the synthesis of DA,

a neurotransmitter that regulates motor activity, it can

be assumed that the 886C allele is associated with the

increased levels/metabolism of DA and motor activity.

However, at present, it is impossible to verify these

assumptions due to a significant uncontrolled genetic

variability of these traits. To study the association of

the C886T mutation with DA levels and motor activity,

it is necessary to significantly reduce the proportion

of uncontrolled variations in these traits by conducting

atleast 10 consecutive backcrosses of heterozygous in-

dividuals (886CT) onto C57BL/6 (886TT) mice.

In this pilot study, we have undoubtedly demon-

strated that the C886T polymorphism is a key factor

determining the activity of TH in the mouse brain.

The C886T mutation is the first naturally occurring

common mutation in the mouse Th gene for which the

effect on the enzyme activity has been shown. This

opens up new possibilities for experimental modeling

of the influence of functional mutations in the Thgene

on the physiological functions under normal and

pathological conditions.

Contributions. K.A.V. developed the concept and

supervised the study; A.I., R.I.A., K.A.V., and M.V.S. con-

ducted the experiments; B.D.V. and K.A.V. discussed the

results; K.A.V. wrote the manuscript; B.D.V. and K.A.V.

edited the manuscript.

Funding. This work was supported by Budget Proj-

ect FWNR-2022-0023.

C886T MUTATION IN Th GENE 1029

BIOCHEMISTRY (Moscow) Vol. 89 No. 6 2024

Ethics declarations. All procedures were con-

ducted following international guidelines for the care

and use of laboratory animals (National Institute of

Health Guide for the Care and Use of Laboratory An-

imals, NIH Publications No.80023, 1996) and the de-

cree of the Ministry of Health of the Russian Federa-

tion dated 01.04.2016 No. 119n “On the Approval of

the Rules of Good Laboratory Practice” (registered on

15.08.2016 No. 43232). Animal housing conditions and

experimental procedures were approved by the Ethics

Committee of the Institute of Cytology and Genetics,

Siberian Branch of the Russian Academy of Sciences.

Theauthors of this work declare that they have no con-

flicts of interest.

Open access. This article is licensed under a Cre-

ative Commons Attribution4.0 International License,

which permits use, sharing, adaptation, distribution,

and reproduction in any medium or format, as long

as you give appropriate credit to the original author(s)

and the source, provide a link to the Creative Commons

license, and indicate if changes were made. Theimages

or other third-party material in this article are includ-

ed in the article’s Creative Commons license, unless

indicated otherwise in a credit line to the material.

Ifmaterial is not included in the article’s Creative Com-

mons license and your intended use is not permitted

by statutory regulation or exceeds the permitted use,

you will need to obtain permission directly from the

https://creativecommons.org/licenses/by/4.0/.

REFERENCES

1. Chinta, S. J., and Andersen, J. K. (2005) Dopaminer-

gic neurons, Int. J. Biochem. Cell Biol., 37, 942-946,

doi:10.1016/j.biocel.2004.09.009.

2. Björklund, A., and Dunnett, S. B. (2007) Dopamine

neuron systems in the brain: an update, Trends Neu-

rosci., 30, 194-202, doi:10.1016/j.tins.2007.03.006.

3. Klein, M. O., Battagello, D.S., Cardoso, A. R., Hauser,

D. N., Bittencourt, J. C., and Correa, R. G. (2019) Do-

pamine: functions, signaling, and association with

neurological diseases, Cell. Mol. Neurobiol., 39, 31-59,

doi:10.1007/s10571-018-0632-3.

4. Channer, B., Matt, S. M., Nickoloff-Bybel, E. A., Pap-

pa,V., Agarwal,Y., Wickman,J., and Gaskill, P.J. (2023)

Dopamine, immunity, and disease, Pharmacol. Rev.,

75, 62-158, doi:10.1124/pharmrev.122.000618.

5. Tripp, G., and Wickens, J. R. (2009) Neurobiology of

ADHD, Neuropharmacology, 57, 579-589, doi:10.1016/

j.neuropharm.2009.07.026.

6. Del Campo, N., Chamberlain, S. R., Sahakian, B. J.,

and Robbins, T. W. (2011) The roles of dopamine

and noradrenaline in the pathophysiology and treat-

ment of attention-deficit/hyperactivity disorder, Biol.

Psychiatry, 69, e145-e157, doi: 10.1016/j.biopsych.

2011.02.036.

7. Faraone, S. V. (2018) The pharmacology of amphet-

amine and methylphenidate: Relevance to the neu-

robiology of attention-deficit/hyperactivity disor-

der and other psychiatric comorbidities, Neurosci.

Biobehav. Rev., 87, 255-270, doi: 10.1016/j.neubiorev.

2018.02.001.

8. Ribot,B., Aupy,J., Vidailhet,M., Mazère,J., Pisani,A.,

Bezard,E., Guehl,D., and Burbaud,P. (2019) Dystonia

and dopamine: from phenomenology to pathophys-

iology, Prog. Neurobiol., 182, 101678, doi: 10.1016/

j.pneurobio.2019.101678.

9. Cherian, A., Paramasivan, N. K., and Divya, K. P.

(2021) Dopa-responsive dystonia, DRD-plus and DRD

look-alike: a pragmatic review, Acta Neurol. Belg., 121,

613-623, doi:10.1007/s13760-020-01574-1.

10. Scarduzio, M., Hess, E. J., Standaert, D. G., and Es-

kow Jaunarajs, K. L. (2022) Striatal synaptic dys-

function in dystonia and levodopa-induced dyskine-

sia, Neurobiol. Dis., 166, 105650, doi: 10.1016/j.nbd.

2022.105650.

11. Lander, M., Bastiampillai, T., and Sareen, J. (2018)

Review of withdrawal catatonia: what does this re-

veal about clozapine? Transl. Psychiatry, 8, 139,

doi:10.1038/s41398-018-0192-9.

12. Slavnic, B., Barnett, B. S., McIntire, S., Becker, R.,

Saba, S., Vellanki, K. D., Honaker, L., Weleff, J., and

Carroll, B. T. (2023) Methamphetamine-associated

catatonia: case series and systematic review of the

literature from 1943-2020, Ann. Clin. Psychiatry, 35,

167-177, doi:10.12788/acp.0116.

13. Puig, M.V., Antzoulatos, E.G., and Miller, E.K. (2014)

Prefrontal dopamine in associative learning and

memory, Neuroscience, 282, 217-229, doi: 10.1016/

j.neuroscience.2014.09.026.

14. Zafiri, D., and Duvarci, S. (2022) Dopaminergic cir-

cuits underlying associative aversive learning,

Front. Behav. Neurosci., 16, 1041929, doi: 10.3389/

fnbeh.2022.1041929.

15. Kourosh-Arami,M., Komaki,A., and Zarrindast, M.R.

(2023) Dopamine as a potential target for learning

and memory: contributing to related neurological dis-

orders, CNS Neurol. Disord. Drug Targets, 22, 558-576,

doi:10.2174/1871527321666220418115503.

16. Baik, J. H. (2013) Dopamine signaling in reward-re-

lated behaviors, Front. Neural Circuits, 7, 152,

doi:10.3389/fncir.2013.00152.

17. Hou, H., Wang, C., Jia, S., Hu, S., and Tian, M. (2014)

Brain dopaminergic system changes in drug ad-

diction: a review of positron emission tomography

findings, Neurosci. Bull., 30, 765-776, doi: 10.1007/

s12264-014-1469-5.

18. Solinas,M., Belujon,P., Fernagut, P.O., Jaber,M., and

Thiriet, N. (2019) Dopamine and addiction: what

have we learned from 40 years of research, J.Neural.

ALSALLOUM et al.1030

BIOCHEMISTRY (Moscow) Vol. 89 No. 6 2024

Transm. (Vienna), 126, 481-516, doi: 10.1007/s00702-

018-1957-2.

19. Poisson, C.L., Engel,L., and Saunders, B.T. (2021) Do-

pamine circuit mechanisms of addiction-like behav-

iors, Front. Neural Circuits, 15, 752420, doi: 10.3389/

fncir.2021.752420.

20. Kobayashi, K., Morita, S., Sawada, H., Mizuguchi, T.,

Yamada, K., Nagatsu, I., Hata, T., Watanabe, Y., Fu-

jita, K., and Nagatsu, T. (1995) Targeted disruption

of the tyrosine hydroxylase locus results in severe

catecholamine depletion and perinatal lethality in

mice, J. Biol. Chem., 270, 27235-27243, doi: 10.1074/

jbc.270.45.27235.

21. Kobayashi, K., Noda, Y., Matsushita, N., Nishii, K.,

Sawada, H., Nagatsu, T., Nakahara, D., Fukabori, R.,

Yasoshima, Y., Yamamoto, T., Miura, M., Kano, M.,

Mamiya, T., Miyamoto, Y., and Nabeshima, T. (2000)

Modest neuropsychological deficits caused by re-

duced noradrenaline metabolism in mice hetero-

zygous for a mutated tyrosine hydroxylase gene,

J. Neurosci., 20, 2418-2426, doi: 10.1523/JNEUROSCI.

20-06-02418.2000.

22. Bademci, G., Vance, J. M., and Wang, L. (2012) Ty-

rosine hydroxylase gene: another piece of the

genetic puzzle of Parkinson’s disease, CNS Neu-

rol Disord Drug Targets, 11, 469-481, doi: 10.2174/

187152712800792866.

23. Nagatsu, T., Nakashima, A., Ichinose, H., and Ko-

bayashi, K. (2019) Human tyrosine hydroxylase in

Parkinson’s disease and in related disorders, J. Neu-

ral Transm. (Vienna), 126, 397-409, doi: 10.1007/

s00702-018-1903-3.

24. Kobayashi,K., and Nagatsu,T. (2005) Molecular genet-

ics of tyrosine 3-monooxygenase and inherited dis-

eases, Biochem. Biophys. Res. Commun., 338, 267-270,

doi:10.1016/j.bbrc.2005.07.186.

25. Asmus, F., and Gasser, T. (2010) Dystonia-plus syn-

dromes, Eur. J. Neurol., 17, 37-45, doi: 10.1111/

j.1468-1331.2010.03049.x.

26. Lee, W. W., and Jeon, B. S. (2014) Clinical spectrum

of dopa-responsive dystonia and related disorders,

Curr. Neurol Neurosci. Rep., 14, 461, doi: 10.1007/

s11910-014-0461-9.

27. Dong, H. Y., Feng, J. Y., Yue, X. J., Shan, L., and Jia,

F.Y. (2020) Dopa-responsive dystonia caused by tyro-

sine hydroxylase deficiency: three cases report and

literature review, Medicine (Baltimore), 99, e21753,

doi:10.1097/MD.0000000000021753.

28. Craddock, N., Davé, S., and Greening, J. (2001) Asso-

ciation studies of bipolar disorder, Bipolar Disord.,

3, 284-298, doi:10.1034/j.1399-5618.2001.30604.x.

29. Beaulieu, J.M., Zhang,X., Rodriguiz, R.M., Sotnikova,

T.D., Cools, M.J., Wetsel, W.C., Gainetdinov, R.R., and

Caron, M. G. (2008) Role of GSK3 beta in behavioral

abnormalities induced by serotonin deficiency, Proc.

Natl. Acad. Sci. USA, 105, 1333-1338, doi: 10.1073/

pnas.0711496105.

30. Kulikova, E.A., and Kulikov, A.V. (2019) Tryptophan

hydroxylase 2 as a therapeutic target for psychiat-

ric disorders: focus on animal models, Expert Opin.

Ther. Targets, 23, 655-667, doi:10.1080/14728222.2019.

1634691.

31. Fitzpatrick, P. F. (2023) The aromatic amino acid hy-

droxylases: structures, catalysis, and regulation of

phenylalanine hydroxylase, tyrosine hydroxylase,

and tryptophan hydroxylase, Arch. Biochem. Biophys.,

735, 109518, doi:10.1016/j.abb.2023.109518.

32. Popova, N.K., Kulikov, A.V., Kondaurova, E.M., Tsyb-

ko, A. S., Kulikova, E. A., Krasnov, I. B., Shenkman,

B. S., Bazhenova, E. Y., Sinyakova, N. A., and Nau-

menko, V. S. (2015) Risk neurogenes for long-term

spaceflight: dopamine and serotonin brain system,

Mol. Neurobiol., 51, 1443-1451, doi: 10.1007/s12035-

014-8821-7.

33. Naumenko, V.S., Osipova, D.V., Kostina, E.V., and Ku-

likov, A.V. (2008) Utilization of a two-standard system

in real-time PCR for quantification of gene expres-

sion in the brain, J. Neurosci. Methods, 170, 197-203,

doi:10.1016/j.jneumeth.2008.01.008.

34. Moskaliuk, V. S., Kozhemyakina, R. V., Bazovkina,

D. V., Terenina, E., Khomenko, T. M., Volcho, K. P.,

Salakhutdinov, N. F., Kulikov, A. V., Naumenko, V. S.,

and Kulikova, E. (2022) On an association between

fear- induced aggression and striatal-enriched protein

tyrosine phosphatase (STEP) in the brain of Norway

rats, Biomed. Pharmacother., 147, 112667, doi:10.1016/

j.biopha.2022.112667.

35. Arefieva, A. B., Komleva, P. D., Naumenko, V. S.,

Khotskin, N.V., and Kulikov, A.V. (2023) Invitro and

invivo chaperone effect of (R)-2-amino-6-(1R, 2S)-1,2-

dihydroxypropyl)-5,6,7,8-tetrahydropterin-4(3H)-one

on the C1473G mutant tryptophan hydroxylase 2,

Biomolecules, 13, 1458, doi:10.3390/biom13101458.

36. Kulikov, A.V., Osipova, D.V., and Popova, N.K. (2007)

The C1473G polymorphism in gene tph2 is the main

factor mediating the genetically defined variability of

tryptophan hydroxylase-2 activity in the mouse brain

[in Russian], Genetika, 43, 1676-1681.

Publisher’s Note. Pleiades Publishing remains

neutral with regard to jurisdictional claims in pub-

lished maps and institutional affiliations.