The humanised CYP2C19 transgenic mouse exhibits cerebellar atrophy and movement impairment reminiscent of ataxia

CYP2C19 transgenic mouse expresses the human CYP2C19 gene in the liver and developing brain, and it exhibits altered neurodevelopment associated with impairments in emotionality and locomotion. Because the validation of new animal models is essential for the understanding of the aetiology and pathophysiology of movement disorders, the objective was to characterise motoric phenotype in CYP2C19 transgenic mice and to investigate its validity as a new animal model of ataxia.


INTRODUCTION
Cerebellar ataxia is an umbrella clinical term used for the group of severe illnesses caused by various hereditary and/or acquired factors [1]. The clinical phenotype is heterogeneous among different subtypes of cerebellar ataxia; the most common symptoms include unstable gait, lack of balance, blurred vision, slurred speech and loss of fine dexterity [1]. Despite the major negative impact on the quality of life in affected individuals, the aetiology and pathophysiology of cerebellar ataxia are not completely understood, and effective treatment strategies for many forms of ataxia are sparse [2,3]. Several animal models of cerebellar ataxia have been developed to clarify molecular and cellular mechanisms causing cerebellar dysfunction. Specifically, transgenic mice that carry gene variants found in human hereditary forms of ataxia and animals selectively bred for their ataxia-like phenotype are frequently used [4]. These animal models have previously been very useful for the investigation of cerebellar function and ataxic pathophysiology [2]; consequently, the characterisation of novel animal models of ataxia is expected to further expand this knowledge.
CYP2C19 humanised transgenic mouse is an animal model that can be useful in the research of cerebellar function and ataxia. This mouse carries human CYP2C19 and CYP2C18 genes, which do not possess orthologous mouse variants. Whereas the CYP2C18 gene is not translated into protein in this animal model, the CYP2C19 gene encodes the CYP2C19 human liver enzyme, which is also expressed in the human fetal brain, suggesting that the enzyme may affect brain development [5,6]. The exact mechanism behind the influence of the CYP2C19 enzyme on neural development has not been directly determined; however, it has been hypothesised that the CYP2C19 enzyme catalyses endogenous substances in the fetal brain, which subsequently affect brain development [5,7]. Known endogenous substrates of CYP2C19 include polyunsaturated fatty acids, sex hormones and endocannabinoids, all of which are important for the various aspects of neuronal development [5,7]. The CYP2C19 transgenic mouse is a very good tool to study the role of CYP2C19 in the developing brain in vivo, because this mutant exhibits almost identical CYP2C19 enzyme expression pattern to humans, including the expression in the brain-restricted development phase and abundant CYP2C19 enzyme concentrations in adult liver, comparable with human levels [8]. Previous research on the CYP2C19 mice revealed their complex emotional phenotype that includes increased susceptibility to stress [6], elevated depression-like behaviour [5], increased serotonergic turnover and decreased whole-brain and hippocampal volumes [6]; in addition, alterations in motoric functions have also been observed but not systematically investigated [6]. To our knowledge, the only study testing the association between the CYP2C19 genotype and any movement disorder is the study of Alonso-Navarro et al., [9] which showed a significantly higher frequency of CYP2C19 intermediate metabolizer status in the cohort of patients with essential tremor compared with healthy controls. Even though there is still no obvious link between CYP2C19 and ataxia, unique features of an animal model expressing the human CYP2C19 gene can be a useful addition to the preclinical research of cerebellar function and dysfunction.
The initial aim of this study was an in-depth characterisation of the motoric phenotype seen in CYP2C19 humanised transgenic mice, whereas the subsequent aim was to examine its potential validity as
• Altered gait is bilateral in young CYP2C19 mice but becomes unilateral in adulthood.
• Cerebellar volume is reduced by 12% in CYP2C19 mice compared with controls.
• CYP2C19 mice may be a useful model for the research of cerebellar ataxia. an animal model for cerebellar ataxia. The primary hypothesis was as follows: (1) Motoric phenotype in CYP2C19 transgenic mice resembles human ataxia and leads to impaired performance in locomotor tests. Secondary hypotheses were driven by preliminary data, in particular, (2) CYP2C19 mice exhibit structural abnormalities in the brain regions associated with locomotion; (3) oxidative stress and/or neuromelanin accumulation are involved in the structural brain alterations; (4) structural and functional alterations of dopaminergic system are involved in the observed motoric phenotype.

Laboratory animals
The CYP2C19 transgenic mice were kept on the C57Bl/6JOlaHsd genetic background, and they are hemizygous carriers of the insert, which contains 12 copies of human CYP2C19 and CYP2C18 genes [7].
Animals were housed in groups of 3-5 animals per cage. Housing conditions included a 12 h light/dark cycle, controlled room temperature (22 ± 1 C), humidity (40-70%) and illumination; and ad libitum access to water and pelleted food. Mice of both sexes were included, and they were divided into two test groups, based on their genotype: CYP2C19 transgenic mice and their wild-type littermates. The body weight of animals was measured every day in the postnatal weeks 3-9. Investigators conducting the experiments were blinded to the animals' genotypes whenever this was possible. General signs of animal well-being were checked at least twice a week, and if signs of severe distress were observed, such animals were euthanized to reduce their suffering. Animals showing abnormal behaviour such as repetitive movements or extreme agitation during handling were also excluded from the subsequent statistical analysis because this hinted at developmental disturbances caused by random factors in these animals.
Most of the experiments were performed in mice of standard adult age of 3-6 postnatal months. There were two exceptions: gait analysis in young animals and structural analysis of the midbrain dopaminergic nuclei in 15 months old mice. All experiments were performed according to the permit of the Ethical Committee on Animal Experimentation of the University of Belgrade -Faculty of Pharmacy, Serbia (permit number 23-07-00933/2019-05 to MMJ). The study was conducted and reported in accordance with ARRIVE 2.0 guidelines [10].

Motor tests
Mouse gait was analysed with a footprint test [11] in adult mice at one time point (CYP2C19 transgenic n = 15, wild-type n = 15) and in young animals at four time points (CYP2C19 transgenic n = 27, wildtype n = 26) once a week in the postnatal weeks 5-8. Young animals were included as it was observed during animals' handling that gait changes during animals' maturation. Measured parameters included stride lengths for every paw, the width of the front and hind base and the overlap of the steps [11]. Next, the maximal height of hind paw elevation while walking was derived from the video footage of the footprint test. The validity of this measurement was ensured by the fixed placement of the camera during every run, exactly 18.5 cm from the apparatus, at a fixed height from the ground and a fixed angle compared with the apparatus. Also, only steps captured at the centre of the frame were considered for the analysis, and all runs were analysed by the same observer to avoid bias. Change in any of the parameters in CYP2C19 transgenic animals compared with wild-types would indicate the presence of gait disturbance. Mice were also visually screened for a pathological clasping reflex, which is present in numerous motorically impaired rodent strains [12].
Motoric function was quantified by the rotarod (CYP2C19 transgenic n = 52, wild-type n = 45, 3-5 months old animals) and beamwalking (BW) tests (CYP2C19 transgenic n = 48, wild-type n = 37, 3-5 months old animals). An accelerating protocol was used in the rotarod test, and the latency to fall was measured [13]. An average of the three longest runs was used as a readout of motoric performance, because the motorically impaired mice tend to fall sooner than the non-impaired animals [13]. In the BW test, a rectangular 8 mm wide beam was used. Because trained animals tend to cross the beam as fast as they possibly can, beam-crossing time is prolonged mainly due to the presence of motoric disturbances [14]. Also, animals with poor motoric coordination tend to exhibit more paw slips during the beamcrossing [14]. Hence, the performance in the BW test was measured as the average of the three shortest beam-crossing times and as the average of the three smallest measured numbers of paw slips per run.

Gd-enhanced neuroimaging
To assess the structural integrity of various brain regions associated with motoric function, brains of 6 months old wild-types (n = 30) and CYP2C19 transgenic mice (n = 29) were investigated with gadolinium (Gd) enhanced post-mortem neuroimaging. Additionally, volumes of non-motoric regions were analysed either to replicate the previous finding of hippocampal atrophy [6] or for exploratory purposes. The samples were prepared for imaging through transcardial formaldehyde perfusion, supplemented with the magnetic resonance imaging (MRI) contrast agent gadoteridol (ProHance, Bracco Diagnostic Inc., NJ, USA) [15]. Scanning was performed using a 9.4 T horizontal bore MRI scanner (Varian, Yarnton, UK) equipped with a millipede coil with an inner diameter of 30 mm. The samples were scanned using a multigradient echo 3D with the following parameters: matrix size 1024 Â 512 Â 512, field-of-view 51.2 Â 25.6 Â 25.6 mm 3 , recovery time 30 ms, time to echo 2.82 ms, flip angle 35 . Two echoes separated by 4.96 ms were acquired, and 512 dummy excitations to establish a steady state were used. Obtained data were segmented automatically based on the 3D Waxholm Space 2012 mouse brain atlas [16,17] into 39 brain regions, but 19 regions such as ventricles, inner ear, medulla oblongata, various nerves and white matter structures were not analysed due to proneness of substantial variability induced by tissue processing. Volumes of each brain region were quantified by multiplying the number of voxels the region comprises with the volume of individual voxel (50 μm voxel edge, 12.5 Â 10 À5 mm 3 ), whereas the total brain volume was calculated as the sum of volumes of all 39 brain regions.
Subsequently, between-group differences in local grey matter (GM) volume were further investigated by performing voxel-based morphometry (VBM). We employed FMRIB Software Library voxelbased morphometry (FSL-VBM) [18], an optimised VBM protocol [19] carried out with FSL tools [20]. Of note, the protocol was slightly modified to be adapted to the present mouse brain (see Supporting Information) images and to compensate for the difference between human and mouse brains. Local differences between groups were investigated by calculating voxel-wise F statistics for genotype, with genotype and sex as factors.

Antioxidative enzyme concentration and activity
Antioxidative enzyme status was determined in the brain tissues of 6 months old wild-types (n = 32) and CYP2C19 transgenic mice

Dopamine level determination and dopamine receptor antagonist treatment
The concentration of dopamine and its two major metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), was determined with High performance liquid chromatography -tandem mass spectrometry (HPLC-MS/MS) method in the brain hemispheres, hippocampi and cerebella of adult 3 months old mice (CYP2C19 transgenic n = 11, wild-type n = 13) harvested after the decapitation as previously described [21]. Of note, this was the only experiment that included only males as the females had to be spared for the breading and colony expanding purpose at the time this experiment was conducted. Also, dopamine, DOPAC and HVA levels were measured in the whole brain samples of embryos at E18.5 day (CYP2C19 transgenic n = 13, wild-type n = 7).
Because the determination of dopamine concentration revealed hyperdopaminergism in adult CYP2C19 mice, their dopaminergic system was characterised to assess its involvement in their motoric phenotype. First, the BW test (CYP2C19 transgenic n = 47, wild-type n = 42, 3-5 months old animals) was repeated after the treatment with the 0.1 mg/kg selective D1 antagonist ecopipam hydrobromide (SCH-39166, Tocris Bioscience, UK) or the 0.25 mg/kg D2 antagonist raclopride (Tocris Bioscience, UK). If ecopipam administration was to reverse motoric impairment in CYP2C19 mice observed in the BW test, this would indicate that excessive dopamine in their brains overactivated the D1 receptor in the striatum causing the impairment this way, and the same would be true for raclopride administration and D2 receptor involvement. Drugs were dissolved in a saline solution and administered in a single intraperitoneal injection 30 min before the BW test. Doses were selected to be sufficient to impact the performance of animals with hyperactivated dopaminergic systems but not to cause general sedation of the animals, as previously described in the literature [22].  Figure 5C) and compared in nine transgenic/wild-type littermate pairs; four pairs were adults (6 months old), and five pairs were at the old age (15 months old). Nine representative coronal slides were analysed and the neuron was considered TH + if the cytoplasm was stained and if the nucleus was visible.

Immunohistochemistry and neuromelanin staining
Neuromelanin is a side-product of catecholamine synthesis and is potentially involved in the pathogenesis of dopamine-induced cell death in humans [23], and because CYP2C19 mice are hyperdopaminergic, it is possible that neuromelanin aggregates in their midbrain dopaminergic neurons due to the higher-than-normal dopamine production. The presence of neuromelanin was assessed in the brain sections of 15 months old mice, stained with Fontana-Masson melanin stain (ab150669, Abcam, UK) according to the manufacturer-supplied protocol [24]. A histological sample of the human skin was used as a positive control.

Statistical data interpretation
Data were analysed with SPSS statistics 20 software (IBM, USA).
Genotype was the principle independent variable in all experiments.
Determination of sample sizes and power analysis are presented in detail in the Supporting Information. In short, the rotarod, BW test (both preliminary and after pharmacological treatments), neuroimaging and oxidative stress analyses involved the same n = 99 cohort of animals. Originally, power analysis [25] estimated that n = 126 animals are needed to detect mild motoric disturbance (10% change) with 95% confidence, 80% power and 20% standard deviation known from the pilot test [25], but this number was reduced to n = 99 due to ethical concerns. Other tests utilised the smallest number of animals needed to yield meaningful results.
The normality of data distributions was assessed with the Shapiro-Wilk test; if the distribution was normal, all outliers were excluded based on the 2.2 interquartile range (IQR) rule [26]. If the experiment included covariates such as age or sex, one-way analysis of covariance (ANCOVA) was performed; otherwise, Student's t test for independent samples was used. Exceptions were experiments that included drug treatments or multiple time points where two-way mixed ANCOVA was performed and non-normally distributed data where Mann Witney and Kruskal Wallis tests were used. All tests were two-tailed with a p value < 0.05 as a measure of significance.
False discovery rate (FDR) correction of p values for multiple comparisons was applied when multiple brain regions or multiple antioxidant enzyme activities were analysed. Differences between test groups were textually presented as the ratio-of-means with a 95% confidence interval (95% CI) for normally distributed data or as median and IQR for each group for non-Gaussian data. For the VBM analysis, the threshold-free cluster enhancement method was used to obtain cor-  (Table S5, Video S1). The number of adult CYP2C19 transgenic mice with affected left vs right hind paws was similar. In addition, the pathological clasping reflex, which occurred after several seconds of struggle upon elevation from the firm surface, was observed in all tested CYP2C19 transgenic animals ( Figure 1C). Next, gait analysis did not show significant genotype-specific changes in young and adult animals ( Figure 1D). There was a trend towards the 7% narrower hind base in CYP2C19 transgenic mice at the 5th postnatal week, but the effect was no longer significant after the correction for animals' sex.
Several animals were excluded from the motoric tests because they were unmotivated to complete the tasks or the training; a similar number of exclusions occurred in transgenic and wild-type mice. Animals excluded for this reason lost motivation gradually, indicating habituation to the test conditions.
To investigate the potential functional implications of the observed motoric phenotype in CYP2C19 transgenic mice, the latency to fall was measured on the rotarod test, whereas the beam-crossing time and number of paw slips were measured on the BW test. On the rotarod test, no significant motoric impairment was observed, as CYP2C19 transgenic and wild-type mice did not exhibit different latencies to fall ( Figure 1E). However, impairment of motor function was observed on the BW test, as CYP2C19 transgenic mice exhibited slightly, 1.14-fold longer beam crossing times and profoundly 5.6-fold increased numbers of paw slips, compared with wild-types ( Figure 1F).
In summary, CYP2C19 transgenic mice exhibit a visually pronounced motoric phenotype, which is in its true nature subtle and impacts motoric function only in challenging tasks.
Next, potential neuroanatomical changes in CYP2C19 transgenic mice were investigated throughout the brain with Gd-enhanced ex vivo structural MRI neuroimaging. Significant, structural GM alterations identified through VBM are graphically represented in 3D ( Figure 2A) and as a collection of representative coronal neuroimaging slides ( Figure 2B). Because the total brain volume in the CYP2C19  .59], F 1,57 = 11.9, p = 0.0011, q = 0.023). Graphic representations of the brain were adopted from the reference atlas [27]. Data presented as means with whiskers as 95% CI or as Tukey box-plot. Bars and whiskers represent means and 95% CI. *Statistical significance p < 0.05. unrelated to the age and genotype. In summary, CYP2C19 transgenic mice exhibit mild hyperdopaminergism and increased cerebellar dopamine turnover, which is unlikely the cause behind the observed motor phenotype and which is not accompanied by any pronounced structural changes within dopaminergic nuclei.

DISCUSSION
CYP2C19 transgenic mice exhibit altered gait and slightly worse performance in more demanding motoric tasks, including impaired balance. This motoric phenotype shares several attributes of ataxia arguing for the face validity of the model; because abnormal walking, diminished balance and loss of fine dexterity are indeed some of the most commonly described symptoms of cerebellar ataxia [1]. Of note, the absence of a wider hind base on the footprint test, nonprogressive nature, unilateral manifestation and mild intensity does not resemble commonly observed symptoms of cerebellar ataxia in humans, but our model may still be of relevance for certain disease subtypes.
Another important ataxia-like characteristic of CYP2C19 mice is the presence of cerebellar atrophy in adulthood, which is in concordance with neuroimaging observations in humans with ataxia [1]. Therefore, it is quite likely that cerebellar atrophy is one of the causes of the observed motoric phenotype in CYP2C19 transgenic mice.  [29,30], which is most likely the case with the CYP2C19 mouse model as well.
Profoundly increased levels of HVA in the cerebella of CYP2C19 mice are likely associated with altered emotionality rather than locomotion in this model as a recent in vivo rodent study revealed association with dopaminergic innervation in a cerebellum and social behaviour alterations but not motoric alterations [31]. Also, hyperdopaminergism was not associated with dopaminergic cell loss in the midbrain in both F I G U R E 4 CYP2C19 mice exhibit hyperdopaminergism in adulthood but not during development. There was an initial statistical trend towards 21% (t 20 = À1.95, p = 0.065) increased dopamine concentrations in the cerebella of CYP2C19 mice, which was no longer present after p-value correction (q = 0.45). Also, homovanillic acid (HVA) level was 46% ([95% CI: 21%, 71%], t 21 = À3.52, p = 0.0020, q = 0.016) increased in CYP2C19 mice compared with controls (A). Hippocampal levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and HVA were not different between test groups (B). CYP2C19 transgenic mice were hyperdopaminergic with slightly increased (1.15-fold [95% CI: 1.08, 1.23]; t 22 = À4.26, p = 0.0003, q = 0.0029) dopamine concentrations in the hemisphere compared to wild-types (C). Graphic representations of the brain [27] and embryo [28] were adopted from reference atlases. Bars and whiskers represent means and 95% CI. *Statistical significance p < 0.05.
adult and older CYP2C19 transgenic mice, despite hypothetically increased vulnerability of dopaminergic neurons to cell death due to chronic hyper-activation [32]. Even though neuromelanin aggregates are not normally detectable in rodents due to their relatively short lifespans compared with the long process of neuromelanin production [33], a few animal models with altered dopaminergic nigro-striatal pathways have been described to exhibit detectable neuromelanin levels [33,34]. However, neuromelanin was not detected in 15 months old CYP2C19 transgenic mice in this study, arguing that the increase in dopamine production was not sufficient to cause quantifiable neuromelanin aggregation in this particular transgenic mouse.
Several animal models exhibit a motoric phenotype similar to the one observed in CYP2C19 transgenic mice [35][36][37]. For example, Atcay ji-hes mouse exhibits more extreme gait disturbances and involuntary hind-paw movements while walking, which were not observed in the CYP2C19 transgenic mice (Movie 2 from Luna-Cancalon et al. [35]). Next, the Reeler mouse can be considered as the closest phenocopy of the CYP2C19 transgenic mouse, as it has a very similar gait (Video S1 in Machado et al. [36]); this behaviour is also accompanied by impaired performance in rotarod and BW tests. Reeler mice also exhibit an aberrant formation of the hippocampus and profoundly reduced cerebellar size [36]. Finally, Sgce m+/pGt mice, besides similar gait to CYP2C19 transgenic mice, also exhibit slight tremors, more slips in the BW test and abnormal expression of several genes in the cerebellum that is potentially associated with aberrant cerebellar development [37]. Because none of the mentioned models gives detailed enough reports of the circuitry involved in their phenotype, it is hard to extrapolate which motoric pathway is affected in CYP2C19 transgenic mice. Still, all phenotypically similar models exhibit abnormal cerebellar development and/or reduced output from Purkinje cells suggesting that the same might be true in the CYP2C19 transgenic mouse model, but further research is needed to investigate this. Additionally, CYP2C19 transgenic mice exhibit a clasping reflex that, besides being a non-specific symptom, can be narrowed down to disturbances in either cerebello-cortico-reticular or cortico-striato-pallido-reticular pathway [12] and that can be triggered by alterations in either noradrenergic or serotonergic transmission. Because the CYP2C19 model has cerebellar atrophy and abnormal serotonergic system activity [5], it can be speculated that the clasping reflex in CYP2C19 transgenic mice is caused by cerebello-cortico-reticular pathway disturbance combined with abnormal serotonergic system function, but further research on this topic is needed for firmer claims.
Importantly, CYP2C19 transgenic mice possess a few unique properties, not previously observed in any other animal model of F I G U R E 5 The dopaminergic system is not associated with motoric impairment in CYP2C19 transgenic mice. Dopaminergic antagonists failed to ameliorate motoric impairment in CYP2C19 transgenic mice in the beam-walking test, and they in fact slightly prolonged beam walking time (Ecopipam: 1.2-fold [95% CI: 1.17, 1.30], F 1,76 = 13.9, p < 0.0001; raclopride: 1.2-fold [95% CI: 1.12, 1.25], F 1,76 = 13.9, p < 0.0001) in them (A, B). Anti-tyrosine hydroxylase (TH) immunohistochemistry (C) revealed no significant changes in the numbers of midbrain TH+ neurons (D) after false discovery rate (FDR) correction. Data presented as means with whiskers as 95% CI or as Tukey box-plot. *Statistical significance p < 0.05. ataxia: (1) CYP2C19 transgenic mice either stagnate or spontaneously improve phenotypic characteristics, whereas in most other ataxia models, progressive worsening of motoric function is observed [4]; (2) Although most animal models of ataxia exhibit altered movements in both sides of the body [4], only one side of the body is affected with motoric impairment in the CYP2C19 transgenic mice in their later adulthood; (3) Although many animal models of ataxia exhibit wider hind base on a footprint test, CYP2C19 transgenic mice nearly do not exhibit abnormalities in footprint test parameters. In conclusion,

Limitations
Most importantly, the time course for several observed effects is still virtually unknown; measurements of brain morphology at different time points would be necessary to determine if the changes occurred during development or after birth and if the latter is the case, when exactly. Next, CYP2C19 mice are more susceptible to stress, and it cannot be excluded that the usage of a light source in the BW and footprint test to motivate animals to complete the tasks could affect two test groups asymmetrically; however, the animals were exposed to several training sessions, and they were habituated to the light, and it is, therefore, unlikely that the light as a stressor affected the result in a meaningful way. Next, antioxidative enzyme activity and expression only provide information related to the general state in the observed regions; detection of ROS or oxidative damage is still needed to confirm the presence of disrupted oxidative/antioxidative balance in brain regions of interest.

CONFLICTS OF INTEREST
All authors declare no conflict of interest.

PEER REVIEW
The peer review history for this article is available at https://publons. com/publon/10.1111/nan.12867.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available in the Supporting Information of this article.