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
1
, Ilya A. Rakhov
2
,
Daria V. Bazovkina
1
, and Alexander V. Kulikov
1,2,a
*
1
Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, Russia
2
Novosibirsk State University, 630090 Novosibirsk, Russia
a
e-mail: v_kulikov@bionet.nsc.ru
Received March 16, 2024
Revised April 15, 2024
Accepted April 25, 2024
AbstractTyrosine 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
Thgene 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 Thgene 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 THgene 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 Thgene
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
Thgene 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
2
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
4
. 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
2
PO
4
, 200 μl of
0.5 M Na
2
EDTA, 300 mg of sodium 1-octanesulfonate,
940 μl of concentrated H
3
PO
4
, 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 Fishers 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
2
-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
2
(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
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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 Thgene 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 Thgene
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.
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