Sex Differences in Dopamine Receptor Signaling in Fmr1 Knockout Mice: A Pilot Study

Fragile X syndrome (FXS) is an X-chromosome-linked dominant genetic disorder that causes a variable degree of cognitive dysfunction and developmental disability. Current treatment is symptomatic and no existing medications target the specific cause of FXS. As with other X-linked disorders, FXS manifests differently in males and females, including abnormalities in the dopamine system that are also seen in Fmr1-knockout (KO) mice. We investigated sex differences in dopamine signaling in Fmr1-KO mice in response to L-stepholidine, a dopamine D1 receptor agonist and D2 receptor antagonist. We found significant sex differences in basal levels of phosphorylated protein kinase A (p-PKA) and glycogen synthase kinase (GSK)-3β in wild type mice that were absent in Fmr1-KO mice. In wild-type mice, L-stepholidine increased p-PKA in males but not female mice, decreased p-GSK-3 in female mice and increased p-GSK-3 in male mice. Conversely, in Fmr1-KO mice, L-stepholidine increased p-PKA and p-GSK-3β in females, and decreased p-PKA and p-GSK-3β in males.


Introduction
Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism, caused by mutation of the fragile X mental retardation (FMR1) gene that leads to insufficiency of the fragile X mental retardation protein (FMRP) [1,2]. FXS includes a spectrum of clinical manifestations, ranging from learning disabilities, attention deficits and hyperactivity to severe intellectual disability with autistic symptoms [3][4][5]. Current treatment for FXS consists of therapy for speech, physical, or behavioral problems, [6,7] and medications for FXS-associated seizures, mood dysregulation, hyperactivity, and attention deficits [8,9]. Better and more specific biological treatments targeting FXS disease mechanisms are needed [10,11].
FXS results from mutations in the fragile X mental retardation 1 (Fmr1) gene located at chromosome Xq27.3 that encodes FMRP. The most common Fmr1 mutation leading to FMRP deficiency is a trinucleotide repeat expansion, consisting of a CGG in the 5untranslated region . Normally there are 6-54 repeats [12] and >200 repeats is considered a mutation. The trinucleotide expansion triggers the methylation of CGG sequences and the FMR1 promoter along with deacetylation of associated histones and chromatin condensation [13][14][15][16] ultimately resulting in epigenetic transcriptional silencing and decreased FMRP. Low levels of FMRP are also associated with various other mental health diseases including schizophrenia, bipolar disorder and major depressive disorder [17].
The prevalence of FXS has been estimated as 1.4 per 10,000 males and 0.9 per 10,000 females [18,19], and this significant sex difference is consistent with an X-chromosome linked disorder [20]. FXS-associated attention deficits, hyperactivity, anxiety, and autism are less severe in females, [21] who consequently have better overall outcomes and quality of life [22]. Sex-specific behavioural abnormalities are also observed in Fmr1-KO mice, in motor coordination, social interaction, learning, memory, and anxiety-like and repetitive behaviours [23]. Male Fmr1-KO mice are hyperactive compared with females, consistent with the hyperactivity and attentional deficits seen in boys with FXS [24,25]. Male Fmr1-KO mice have more rearing behaviour and less ultrasonic vocalizations compared to females [26] while female Fmr1-KO mice have more repetitive behaviours, impaired response inhibition and better motor coordination.
One functional pathway that could explain the attention deficits and hyperactivity in FXS is the dopamine system. FMRP regulates how dopamine modulates AMPA glutamate receptor subtype 1 (GluR1) surface expression through the D1 receptor [27]. In Fmr1-KO mice, there are fewer D1 receptors [28] that are hyperphosphorylated and hyperactivity is rescued by D1 receptor agonists. Thus, we hypothesized that other aspects of dopamine signaling related to the dopamine system could be abnormal in the Fmr1-KO mouse. In addition to classical dopamine receptor signaling through G-proteins, the dopamine D2like receptors also regulate protein kinase B (Akt) through beta-arrestin 2 and glycogen synthase kinase 3β (GSK-3β) [29,30], which we examined in this paper.
We chose to modulate dopamine receptors with L-stepholidine, a natural compound derived from Stephania intermedia [31] that is both a D1 receptor agonist and D2 receptor inhibitor [32]. Stephania intermedia is a plant used in traditional Chinese medicine, and L-stepholidine has been studied as a potential antipsychotic agent. L-stepholidine can attenuate morphine-induced conditioned place preference [33] and has neuroprotective effects against memory deficits caused by chronic methamphetamine exposure [34]. In addition, L-stepholidine can improve memory and synaptic plasticity in Alzheimer's disease models through D1-mediated PKA signaling [35].

Animals
All procedures were approved by the local Animal Care Committee at the Centre for Addiction and Mental Health (CAMH), Toronto, Canada, following guidelines by the Canadian Committee for Animal Care. C57Bl/6 wild-type mice were purchased from Charles River Laboratories (Wilmington, MA, USA), and breeding pairs of Fmr1-KO mice (with C57Bl/6 background) were purchased from the Jackson Laboratory (B6.129P2-Fmr1 tm1Cgr /J, Stock No: 003025), and bred at the CAMH animal facility. Animals were acclimated to our facility for one week prior to the start of experiments.
Animals were housed at 20-23 • C with a 12-h day-night cycle (7 AM-7 PM). All animals were fed by standard Laboratory Rodent Diet 5001 (LabDiet, St. Louis, MO, USA), and the feed was available in the feeder above the cage on a free choice basis.

Drug Treatment
L-stepholidine was dissolved in sterile dimethyl sulfoxide (DMSO, Sigma-Aldrich, Burlington, MA, USA) at 1 mg/mL and stored at −20 • C. Prior to injection, stock solutions were diluted in filtered phosphate-buffered saline (PBS). Ten-week old Fmr1-KO and wildtype mice were injected intraperitoneally (i.p.) with L-stepholidine at a dosage of 10 mg/kg for seven consecutive days; control animals received PBS only. On the day following the last injection, mice were sacrificed by cervical dislocation, and the whole brain was removed for further analysis.

Statistical Analysis
Results are presented as the mean ± standard error of the mean (SEM). The statistical tests were performed using GraphPad Prism 9 (La Jolla, CA, USA). Before conducting statistical analyses, the normality of data was confirmed using the Shapiro-Wilk test. The student's t-test (unpaired, two-sided) was used to compare two groups and one-way ANOVA followed by Tukey's multiple comparisons test was used to analyze multiple groups.

Discussion
We sought to expand knowledge on dopamine signaling and function in FXS by examining sex differences in the phosphorylated PKA and GSK-3 response to L-stepholidine in wild-type and Fmr1-KO mice. We found sex differences in p-PKA and p-GSK-3β in wild type mice but not Fmr1-KO mice. L-stepholidine had opposite effects in wild-type mice compared to Fmr1-KO mice on these dopamine signaling components. L-stepholidine increased p-PKA and p-GSK-3β in male wild-type mice and decreased p-GSK-3 in female wild-type mice. In contrast, L-stepholidine increased p-PKA in female Fmr1-KO mice, but decreased p-PKA in male Fmr1-KO mice. This drug also increased p-GSK-3β in female Fmr1-KO mice while decreasing p-GSK-3β in male Fmr1-KO mice. Overall, we observed many opposing effects of the Fmr1-KO and stepholidine on intracellular dopamine signals in male and female animals.
Our results confirm and extend the literature on sex differences associated with FXS in humans and in Fmr1-KO mice. The complex pattern of changes in dopamine systemrelated phenotypes is consistent with the combined effects of a sex-linked mutation (Fmr1 gene deletion) combined with more general sex differences in the dopamine system. For example, PET studies revealed greater D2-like receptor levels in the frontal cortex of women vs. men [36], with further sex differences in the regulation of dopamine release in the striatum [37]. The D1-D2 receptor complex is also observed at higher density in female vs. male rodents and non-human primates [38].
The sex differences we observed in Fmr1-KO mice are consistent with the many sex differences reported in human FXS patients. For example, adolescent male FXS patients have a greater cerebral volume than female patients [39], consistent with a larger volume of grey matter, cortical grey matter and the caudate nucleus in boys with FXS children [39]. Boys with FXS also tend to perseverate in speech more than girls [40]. Male FXS patients are more likely than females to have epilepsy [41]. Two antipsychotics, aripiprazole and Risperdal are also commonly used for ameliorating severe behavioural abnormalities patients such as irritability associated with aggression in male adolescents FXS [5,[42][43][44].

Discussion
We sought to expand knowledge on dopamine signaling and function in FXS by examining sex differences in the phosphorylated PKA and GSK-3 response to L-stepholidine in wild-type and Fmr1-KO mice. We found sex differences in p-PKA and p-GSK-3β in wild type mice but not Fmr1-KO mice. L-stepholidine had opposite effects in wild-type mice compared to Fmr1-KO mice on these dopamine signaling components. L-stepholidine increased p-PKA and p-GSK-3β in male wild-type mice and decreased p-GSK-3 in female wild-type mice. In contrast, L-stepholidine increased p-PKA in female Fmr1-KO mice, but decreased p-PKA in male Fmr1-KO mice. This drug also increased p-GSK-3β in female Fmr1-KO mice while decreasing p-GSK-3β in male Fmr1-KO mice. Overall, we observed many opposing effects of the Fmr1-KO and stepholidine on intracellular dopamine signals in male and female animals.
Our results confirm and extend the literature on sex differences associated with FXS in humans and in Fmr1-KO mice. The complex pattern of changes in dopamine systemrelated phenotypes is consistent with the combined effects of a sex-linked mutation (Fmr1 gene deletion) combined with more general sex differences in the dopamine system. For example, PET studies revealed greater D2-like receptor levels in the frontal cortex of women vs. men [36], with further sex differences in the regulation of dopamine release in the striatum [37]. The D1-D2 receptor complex is also observed at higher density in female vs. male rodents and non-human primates [38].
The sex differences we observed in Fmr1-KO mice are consistent with the many sex differences reported in human FXS patients. For example, adolescent male FXS patients have a greater cerebral volume than female patients [39], consistent with a larger volume of grey matter, cortical grey matter and the caudate nucleus in boys with FXS children [39]. Boys with FXS also tend to perseverate in speech more than girls [40]. Male FXS patients are more likely than females to have epilepsy [41]. Two antipsychotics, aripiprazole and Risperdal are also commonly used for ameliorating severe behavioural abnormalities patients such as irritability associated with aggression in male adolescents FXS [5,[42][43][44].
These examples are not a comprehensive list, but serve to illustrate the variety of sex differences in brain structure and function caused by mutations in the Fmr1 gene.

Conclusions
Overall, our results produced a complex picture of sex differences in dopamine signaling components and response to medications that are associated with Fmr1 gene knock-out. While we did not investigate the mechanisms underlying these observations, they suggest that further research into these results could help to understand the clinical differences between male and female FXS patients. Our findings also suggest that there could be sex differences in responses to medication used in the management of patients with FXS, and this certainly warrants additional investigation [45]. Our experiments also reinforce that the Fmr1 gene has a broad range of functions in the brain, and that disparate aspects of neurotransmitter signaling can be affected by the insufficiency of the FMR protein. These pleiotropic effects could be relevant to the wide range of clinical severity and manifestations of FXS, which complicates management of this complex disorder.