5-HT1A Receptor Agonist Treatment Partially Ameliorates Rett Syndrome Phenotypes in mecp2-Null Mice by Rescuing Impairment of Neuron Transmission and the CREB/BDNF Signaling Pathway

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene that encodes methyl CpG-binding protein 2 (MECP2) and is characterized by the loss of acquired motor and language skills, stereotypic movements, respiratory abnormalities and autistic features. There has been no effective treatment for this disorder until now. In this study, we used a Mecp2-null (KO) mouse model of RTT to investigate whether repeated intraperitoneal treatment with the 5-HT1A receptor agonist tandospirone could improve the RTT phenotype. The results showed that administration of tandospirone significantly extended the lifespan of Mecp2-KO mice and obviously ameliorated RTT phenotypes, including general condition, hindlimb clasping, gait, tremor and breathing in Mecp2-KO mice. Tandospirone treatment significantly improved the impairment in GABAergic, glutaminergic, dopaminergic and serotoninergic neurotransmission in the brainstem of Mecp2-KO mice. Decreased dopaminergic neurotransmission in the cerebellum of Mecp2-KO mice was also significantly increased by tandospirone treatment. Moreover, RNA-sequencing analysis found that tandospirone modulates the RTT phenotype, partially through the CREB1/BDNF signaling pathway in Mecp2-KO mice. These findings provide a new option for clinical treatment.


Introduction
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that affects approximately 1 in 10,000 girls. RTT patients are characterized by seemingly normal development up to 6-18 months of age and then display a sudden deceleration in growth and development, which are associated with the progressive loss of acquired motor and language skills, stereotypic hand movements, muscle hypotonia, autonomic dysfunctions and severe cognitive impairment [1]. Most RTT cases are caused by mutations in the gene coding for methyl CpG-binding protein 2 (MECP2) [2]. MeCP2 is most abundant in the central nervous system (CNS), associated with neuronal maturation and synaptogenesis [3][4][5]. MeCP2 deficiency in mice results in RTT-like symptoms consisting of hind limb clasping, tremors, breathing irregularities, loss of muscle tone and hypoactivity [6]. Defects in various neurotransmitter systems have been observed in patients and animal models of RTT syndrome [7,8]. Currently, there is no curative therapy for RTT, despite many clinical trials and basic research studies investigating RTT.
Serotonin (5-HT) is a modulatory neurotransmitter involved in a variety of physiological and behavioral functions, including anxiety, aggressive behavior, stress, blood pressure regulation, heart rate and the coagulation system. The cellular actions of serotonin are mediated by multiple receptors, which have been classified as 5-HT 1A-F, 5-HT 2A-C, 5-HT 3, 5-HT 4, 5-HT 5, 5-HT 6 and 5-HT 7, according to their structural and functional 2 of 11 characteristics [9]. Among all of the 5-HT receptors, the 5-HT 1A receptor (5-HT 1A ) is the most widespread, existing both pre-and postsynaptically [10]. In general, activation of these presynaptic 5-HT 1A autoreceptors hyperpolarizes the cell membrane and results in a reduction in the firing rate of serotonergic neurons in the raphe area, leading to suppressed serotonin synthesis, turnover and release. The activation of 5-HT 1A receptors on postsynaptic cells decreases the firing rate of postsynaptic cells [10,11]. In mammals, dysfunction of the serotonergic system has been linked to various neurological diseases, such as depression, schizophrenia, Alzheimer's disease, Parkinson's disease and autism spectrum disorder [12][13][14][15][16]. Additionally, disturbances in serotoninergic neurotransmission were also found in RTT patients and mouse models [7,17].
Tandospirone is a potent and selective 5-HT 1A partial agonist that also presents a high selectivity of two to three orders of magnitude over dopamine, adrenergic and other 5-HT receptors [18]. Tandospirone is an effective anxiolytic drug that is well tolerated and presents limited adverse effects and low abuse liability [19]. Improvement in motor dysfunction in Parkinson's disease and cognitive deficits in schizophrenia have been revealed in recent clinical and preclinical studies [20,21]. For an RTT patient, tandspirone administration was described to clinically recover respiratory condition and behavior [22]. However, the effect of tandospirone was only demonstrated in one care report. In this study, we verified whether repeated intraperitoneal treatment with tandospirone could improve the RTT phenotype in a mouse model. Here, we revealed that administration of tandospirone was able to improve RTT phenotypes, including general condition, hindlimb clasping, gait, tremor and breathing, which are related with the improvement in GABAergic, glutaminergic, dopaminergic and serotoninergic neurotransmission. Moreover, RNA-seq analysis found that tandospirone modulates the RTT phenotype partially through the CREB1/BDNF signaling pathway in Mecp2-KO mice.

Effect of Tandospirone Treatment on Lifespan, Body Weight and Pathological Phenotypes of Mecp2-KO Mice
Mecp2-KO mice manifest a progressive postnatal neurological phenotype. Mecp2-KO mice are apparently normal until 4 weeks of age and then develop RTT-like symptoms, including inertia, irregular breathing, abnormal gait and hindlimb clasping. The progression of symptoms in Mecp2-KO male mice leads to rapid weight loss and a shortened lifespan (death at~10 weeks of age) compared with WT mice [6,23]. Therefore, we started tandospirone treatment when the animals were 4 weeks of age. After 4 weeks of treatment, at 8 weeks of age, both lifespan and body weight were observed. The survival curve for the treatment of Mecp2-KO mice is shown in Supplementary Figure S1A. Statistical analysis indicated that tandospirone treatment significantly extended the lifespan of Mecp2-KO mice (50% longer, p < 0.001, Supplementary Figure S1A). We measured body weight at 8 weeks of age. The body weight of Mecp2-KO mice was significantly lower than that of WT littermates (Supplementary Figure S1B, p < 0.001), but there was no significant difference between vehicle and tandospirone treatment. These findings suggest that tandospirone treatment potentially extends the lifespan of Mecp2-KO mice but has no effect on the body weight.
Next, we analyzed the effect of tandospirone treatment in the Mecp2-KO mice on the pathological phenotype of the Rett syndrome animal model. The score test values were measured at 8 weeks of age according to previous reports [6,24], including general condition, hindlimb clasping, tremor, breathing, mobility and gait ( Figure 1A-F, respectively). The scores of Mecp2-KO mice (vehicle group) were significantly increased compared to WT littermates, but significantly decreased by tandospirone treatment in general condition ( Figure 1A), hindlimb clasping ( Figure 1B), gait ( Figure 1D), tremor ( Figure 1E) and breathing ( Figure 1F) were significantly decreased by tandspirone treatment, with no effect on mobility ( Figure 1C). These results indicate that tandospirone treatment could partially improve Rett syndrome phenotypes, including retraction of the legs, tremor, irregular breathing and difficulties with walking. condition ( Figure 1A), hindlimb clasping ( Figure 1B), gait ( Figure 1D), tremor (Figure and breathing ( Figure 1F) were significantly decreased by tandspirone treatment, with effect on mobility ( Figure 1C). These results indicate that tandospirone treatment co partially improve Rett syndrome phenotypes, including retraction of the legs, tremor regular breathing and difficulties with walking. Figure 1. Effect of tandospirone treatment on the Mecp2-KO phenotype. Both Mecp2 WT and Mec KO (Vehicle) mice were treated with saline. Score test (n = 12-15) related to general condition hindlimb clasping (B), mobility (C), gait (D), tremor (E) and breathing (F). Tandospirone treatm significantly improved the Mecp2-KO phenotypes of general condition, hindlimb clasping, g tremor and breathing but not in mobility. All values are expressed as the means ± SEMs. Statist analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple co parison post hoc test for intergroup comparisons. n = 12~15 in each test, * p < 0.05, *** p < 0.001, p < 0.0001, ns: not significant.

Effect of Tandospirone Treatment on Glutamate, GABA, Monoamines and Metabolites in Brains of Mecp2-KO Mice
It is known that Mecp2 deficit impairs neurotransmitter systems that correspo with neurological phenotypes observed in both Rett patients and Mecp2-KO mice [7,2 Therefore, in this study, we used an HPLC system to investigate the effect of tandospiro treatment on glutamate, GABA, monoamines and their metabolites in the brains of Mec KO mice by HPLC system. The data showed that both the GABA concentration (Figure and glutamate concentration ( Figure 3) were significantly decreased in all brain regio of Mecp2-KO mice compared with WT littermates. Tandospirone treatment significan improved the GABA concentration in the brainstem of Mecp2-KO mice compared to ve cle ( Figure 2E, p < 0.01), but the differences were not significant in the other brain regio The glutamate concentration was also significantly increased in the cerebellum and bra stem of Mecp2-KO mice when compared with vehicle ( Figure 3D,E, respectively; p < 0.0 These results suggest that tandospirone treatment may partially improve the deficits glutaminergic and GABAergic neurotransmission observed in Mecp2-KO mice. In ad tion, we also confirmed the effect of tandospirone treatment on monoaminergic neu transmission in the brains of Mecp2-KO mice, focusing on the dopaminergic and sero ninergic systems. The results indicated that Mecp2-KO mice showed a significantly low concentration of dopamine and its metabolites in the midbrain (Supplementary Fig  S3A,B, p < 0.05), cerebellum ( Figure 4A,B, p < 0.05) and brainstem ( Figure 5A,B, p < 0.0 than WT littermates, but the differences were not significant in the cortex (Supplement Figure S2A,B, p > 0.05) or hippocampus (Supplementary Figure S4A,B, p > 0.05). Tandos rone treatment significantly enhanced the dopamine concentrations of Mecp2-KO mice the cerebellum and brainstem compared to vehicle (Figures 4A and 5A, respectively; 0.05). The DOPAC concentration in the brainstem was also significantly increased by t dospirone treatment ( Figure 5B; p < 0.05). These results suggest that tandospirone tre ment significantly improves the dopaminergic neurotransmission deficits in the cereb lum and brainstem seen in Mecp2-KO mice. The serotonin concentration of Mecp2mice was significantly decreased in all brain regions when compared with W , mobility (C), gait (D), tremor (E) and breathing (F). Tandospirone treatment significantly improved the Mecp2-KO phenotypes of general condition, hindlimb clasping, gait, tremor and breathing but not in mobility. All values are expressed as the means ± SEMs. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison post hoc test for intergroup comparisons. n = 12~15 in each test, * p < 0.05, *** p < 0.001, **** p < 0.0001, ns: not significant.

Effect of Tandospirone Treatment on Glutamate, GABA, Monoamines and Metabolites in the Brains of Mecp2-KO Mice
It is known that Mecp2 deficit impairs neurotransmitter systems that correspond with neurological phenotypes observed in both Rett patients and Mecp2-KO mice [7,25]. Therefore, in this study, we used an HPLC system to investigate the effect of tandospirone treatment on glutamate, GABA, monoamines and their metabolites in the brains of Mecp2-KO mice by HPLC system. The data showed that both the GABA concentration ( Figure 2) and glutamate concentration ( Figure 3) were significantly decreased in all brain regions of Mecp2-KO mice compared with WT littermates. Tandospirone treatment significantly improved the GABA concentration in the brainstem of Mecp2-KO mice compared to vehicle ( Figure 2E, p < 0.01), but the differences were not significant in the other brain regions. The glutamate concentration was also significantly increased in the cerebellum and brainstem of Mecp2-KO mice when compared with vehicle ( Figure 3D,E, respectively; p < 0.05). These results suggest that tandospirone treatment may partially improve the deficits of glutaminergic and GABAergic neurotransmission observed in Mecp2-KO mice. In addition, we also confirmed the effect of tandospirone treatment on monoaminergic neurotransmission in the brains of Mecp2-KO mice, focusing on the dopaminergic and serotoninergic systems. The results indicated that Mecp2-KO mice showed a significantly lower concentration of dopamine and its metabolites in the midbrain (Supplementary Figure S3A,B, p < 0.05), cerebellum ( Figure 4A,B, p < 0.05) and brainstem ( Figure 5A,B, p < 0.001) than WT littermates, but the differences were not significant in the cortex (Supplementary Figure S2A Figure 5D,E, respectively; p < 0.05). Tandospirone treatment also had a tendency to increase the low contents of serotonin and its metabolites observed in Mecp2-KO mice in different brain regions, compared to vehicle but the increase was only significant in the brainstem ( Figure 5C,E, respectively; p < 0.05), suggesting that tandospirone treatment might ameliorate the impairment of serotoninergic neurotransmission in the brainstem observed in Mecp2-KO mice.
mentary Figures S2D,E and S3D, Figure 5D,E, respectively; p < 0.05). Tandospirone tre ment also had a tendency to increase the low contents of serotonin and its metaboli observed in Mecp2-KO mice in different brain regions, compared to vehicle but the crease was only significant in the brainstem ( Figure 5C,E, respectively; p < 0.05), sugge ing that tandospirone treatment might ameliorate the impairment of serotoninergic n rotransmission in the brainstem observed in Mecp2-KO mice.

Figure 2.
Effect of tandospirone treatment on GABA concentration in the cortex (A), midbrain hippocampus (C), cerebellum (D) and brainstem (E). Mecp2-KO mice showed a significantly creased GABA concentration in all brain regions, but administration of tandospirone only sign cantly increased GABA concentration in the brainstem. All values are expressed as the mean SEMs. Statistical analysis was performed using one-way analysis of variance (ANOVA) w Tukey's multiple comparison post hoc test for intergroup comparisons. n = 6, * p < 0.05, ** p < 0 *** p < 0.001, **** p < 0.0001, ns: not significant. . Mecp2-KO mice showed a significan decreased glutamate concentration in the cortex, hippocampus, cerebellum and brainstem w compared with WT mice. Administration of tandospirone significantly increased the lower glu mate concentration in the cerebellum and brainstem but no significant changes in the cortex, m brain or hippocampus were observed. All values are expressed as the means ± SEMs. Statist analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple co parison post hoc test for intergroup comparisons. n = 6, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: significant. Mecp2-KO mice showed a significantly decreased GABA concentration in all brain regions, but administration of tandospirone only significantly increased GABA concentration in the brainstem. All values are expressed as the means ± SEMs. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison post hoc test for intergroup comparisons. n = 6, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. ment also had a tendency to increase the low contents of serotonin and its metaboli observed in Mecp2-KO mice in different brain regions, compared to vehicle but the crease was only significant in the brainstem ( Figure 5C,E, respectively; p < 0.05), sugge ing that tandospirone treatment might ameliorate the impairment of serotoninergic ne rotransmission in the brainstem observed in Mecp2-KO mice.

Figure 2.
Effect of tandospirone treatment on GABA concentration in the cortex (A), midbrain hippocampus (C), cerebellum (D) and brainstem (E). Mecp2-KO mice showed a significantly creased GABA concentration in all brain regions, but administration of tandospirone only sign cantly increased GABA concentration in the brainstem. All values are expressed as the mean SEMs. Statistical analysis was performed using one-way analysis of variance (ANOVA) w Tukey's multiple comparison post hoc test for intergroup comparisons. n = 6, * p < 0.05, ** p < 0 *** p < 0.001, **** p < 0.0001, ns: not significant. . Mecp2-KO mice showed a significan decreased glutamate concentration in the cortex, hippocampus, cerebellum and brainstem wh compared with WT mice. Administration of tandospirone significantly increased the lower glu mate concentration in the cerebellum and brainstem but no significant changes in the cortex, m brain or hippocampus were observed. All values are expressed as the means ± SEMs. Statist analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple co parison post hoc test for intergroup comparisons. n = 6, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns: significant.

Effect of Tandospirone on Cellular Mechanisms in Mecp2-KO Mice
To study the therapeutic effect and molecular mechanisms of tandospirone in Mecp2-KO mice, the cortex, midbrain, cerebellum and brainstem were collected for RNA-seq analysis after 4 weeks of tandospirone treatment. Bioinformatic and statistical approaches were used to identify the differentially expressed genes (DEGs) from the WT, vehicle and tandospirone-treated Mecp2-KO groups. In detail, the dysregulated genes in Mecp2-KO mice were first identified by comparing WT and vehicle groups. We defined gene upregulation as vehicle/WT ≥ 2 and downregulation as vehicle/WT ≤ 0.5. Next, the expression of these dysregulated genes observed in brains of Mecp2-KO mice was further compared with that in tandospirone-treated Mecp2-KO mice. The results showed a total of 129 genes with the potential for recovery of dysregulation by tandospirone treatment in four brain regions, of which 111 were upregulated and 18 were downregulated. Finally, these 129 genes were used for functional pathway analysis using the KEGG pathway database. The

Effect of Tandospirone on Cellular Mechanisms in Mecp2-KO Mice
To study the therapeutic effect and molecular mechanisms of tandospirone in Mecp2-KO mice, the cortex, midbrain, cerebellum and brainstem were collected for RNA-seq analysis after 4 weeks of tandospirone treatment. Bioinformatic and statistical approaches were used to identify the differentially expressed genes (DEGs) from the WT, vehicle and tandospirone-treated Mecp2-KO groups. In detail, the dysregulated genes in Mecp2-KO mice were first identified by comparing WT and vehicle groups. We defined gene upregulation as vehicle/WT ≥ 2 and downregulation as vehicle/WT ≤ 0.5. Next, the expression of these dysregulated genes observed in brains of Mecp2-KO mice was further compared with that in tandospirone-treated Mecp2-KO mice. The results showed a total of 129 genes with the potential for recovery of dysregulation by tandospirone treatment in four brain regions, of which 111 were upregulated and 18 were downregulated. Finally, these 129 genes were used for functional pathway analysis using the KEGG pathway database. The Interestingly, tandospirone also recovered the concentrations of noradrenalin and its metabolite (MHPG; 3-methoxy-4-hydroxy-phenylglycol) in the brainstem of Mecp2-KO mice (Supplementary Figure S5).

Effect of Tandospirone on Cellular Mechanisms in Mecp2-KO Mice
To study the therapeutic effect and molecular mechanisms of tandospirone in Mecp2-KO mice, the cortex, midbrain, cerebellum and brainstem were collected for RNA-seq analysis after 4 weeks of tandospirone treatment. Bioinformatic and statistical approaches were used to identify the differentially expressed genes (DEGs) from the WT, vehicle and tandospirone-treated Mecp2-KO groups. In detail, the dysregulated genes in Mecp2-KO mice were first identified by comparing WT and vehicle groups. We defined gene upregulation as vehicle/WT ≥ 2 and downregulation as vehicle/WT ≤ 0.5. Next, the expression of these dysregulated genes observed in brains of Mecp2-KO mice was further compared with that in tandospirone-treated Mecp2-KO mice. The results showed a total of 129 genes with the potential for recovery of dysregulation by tandospirone treatment in four brain regions, of which 111 were upregulated and 18 were downregulated. Finally, these 129 genes were used for functional pathway analysis using the KEGG pathway database. The results indicated that several candidate genes in the midbrain were associated with the adenylyl cyclase-cyclic adenosine monophosphate (cAMP)-cAMP response element-binding (CREB) signaling pathway, AMP-activated protein kinase (AMPK)-CREB signaling pathway or mitogen-activated protein kinase (MAPK) signaling pathway, including CAMK2D (calcium/calmodulin-dependent protein kinase type II subunit delta), PRKAG2 (protein kinase AMP-activated noncatalytic subunit gamma), CREB1 (cyclic AMPresponsive element-binding protein 1), BDNF (brain-derived neurotrophic factor) and MAPKAPK2 (MAP kinase-activated protein kinase 2). The GABRA4 (gamma-aminobutyric acid receptor subunit alpha-4) gene is associated with GABAergic synapse transmission. No pathway was found in the cortex, cerebellum or brainstem. Quantitative PCR (qPCR) indicated that the expression of CAMK2D, PRKAG2, CREB1, BDNF and GABRA4 was significantly decreased in Mecp2-KO mice in comparison with WT mice but significantly increased in Mecp2-KO tandospirone-treated mice when compared with vehicle Mecp2-KO mice ( Figure 6A-D,F, respectively; p < 0.05). There was no significant change in the expression of MAPKAPK2 ( Figure 6E). These results suggested that tandospirone treatment might activate CREB1 by the cAMP and AMPK signaling pathways in the midbrains of Mecp2-KO mice, resulting in increased BDNF expression. adenylyl cyclase-cyclic adenosine monophosphate (cAMP)-cAMP response elementbinding (CREB) signaling pathway, AMP-activated protein kinase (AMPK)-CREB signaling pathway or mitogen-activated protein kinase (MAPK) signaling pathway, including CAMK2D (calcium/calmodulin-dependent protein kinase type II subunit delta), PRKAG2 (protein kinase AMP-activated noncatalytic subunit gamma), CREB1 (cyclic AMP-responsive element-binding protein 1), BDNF (brain-derived neurotrophic factor) and MAP-KAPK2 (MAP kinase-activated protein kinase 2). The GABRA4 (gamma-aminobutyric acid receptor subunit alpha-4) gene is associated with GABAergic synapse transmission. No pathway was found in the cortex, cerebellum or brainstem. Quantitative PCR (qPCR) indicated that the expression of CAMK2D, PRKAG2, CREB1, BDNF and GABRA4 was significantly decreased in Mecp2-KO mice in comparison with WT mice but significantly increased in Mecp2-KO tandospirone-treated mice when compared with vehicle Mecp2-KO mice ( Figure 6A, 6B, 6C, 6D and 6F, respectively; p < 0.05). There was no significant change in the expression of MAPKAPK2 ( Figure 6E). These results suggested that tandospirone treatment might activate CREB1 by the cAMP and AMPK signaling pathways in the midbrains of Mecp2-KO mice, resulting in increased BDNF expression.  All values are expressed as the means ± SEMs. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison post hoc test for intergroup comparisons. n = 4, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant.

Discussion
In our present study, we investigated the effect of chronic tandospirone treatment on RTT using a mouse model. By comparing the phenotypes of Mecp2-KO tandospironetreated mice with vehicle, we found that tandospirone treatment significantly extended the lifespan of Mecp2-KO mice and obviously ameliorated RTT phenotypes, including general condition, hindlimb clasping, gait, tremor and breathing, in Mecp2-KO mice; however, there was no effect of tandospirone treatment on body weight or mobility in these mice. Tandospirone treatment also significantly improved the impairment in GABAergic, glutaminergic, dopaminergic and serotoninergic neurotransmission in the brainstem of Mecp2-KO mice. Decreased dopaminergic neurotransmission in the cerebellum of Mecp2-KO mice was also significantly increased by tandospirone treatment. Moreover, RNA-seq analysis found that tandospirone modulated the RTT phenotype partially through the CREB1/BDNF signaling pathway in our mouse model.
Respiratory disorders are prominent and one of the most disturbing features of RTT. This phenotype was faithfully mimicked in our mouse model of RTT, suggesting that breathing dysregulation in RTT results from progressive neurochemical dysfunction in the respiratory network, including GABAergic, glutamatergic and monoaminergic neurotransmission [26]. Recently, several studies have reported that administration of a 5-HT 1A receptor agonist reduced apneas, improved breathing irregularity and extended survival in a mouse model of RTT [27][28][29]. Our present results were consistent with these previous studies, proving that treatment with a 5-HT 1A receptor agonist could significantly improve RTT brainstem dysfunctions, particularly in the GABAergic, glutamatergic and serotoninergic neurotransmissions, responsible for breathing and lifespan. Moreover, tandospirone is not only a selective 5-HT 1A receptor agonist but also binds to the dopamine D2 receptor and affects the dopaminergic pathway [30,31]. It has been reported that dopaminergic stimulation by treatment with levodopa and a dopa-decarboxylase inhibitor improved motor activities, such as tremor, hindlimb clasping, gait, mobility and breathing, in a Mecp2 mouse model [24]. Thus, we consider improvement in the RTT phenotype upon treatment with tandospirone to be partially modulated by the dopaminergic pathway in the cerebellum and brainstem. In addition, tandospirone treatment may recover noradrenergic neuronal function.
In addition, disruption in the expression of the brain-derived neurotrophic factor (BDNF) gene has been proposed to contribute to the molecular pathogenesis of RTT [32]. Enhancement of brain BDNF levels improved symptoms of a mouse model of RTT, including locomotor activity, motor coordination and lifespan [33]. BNDF expression is regulated by the CREB family, a nuclear transcription factor expressed in all cells in the brain [34]. In stress-maladaptive mice, chronic administration of 5-HT 1A receptor agonists may enhance CREB expression, which, in turn, facilitates transcription factor binding to the BDNF promoter domain to stimulate BDNF synthesis [35]. Therefore, in our present study, we consider that tandospirone treatment increased BDNF expression in Mecp2-KO mice due to the activation of CREB1 via CAMK2D and PRKAG2 signaling. Increased BDNF expression partially ameliorates the phenotypes of Mecp2-KO mice. However, there are some limitations to completely understanding the molecular mechanism from the results.
In conclusion, our data indicate that chronic administration of tandospirone may partially ameliorate Rett syndrome phenotypes in Mecp2-KO mice by rescuing the impairment of neurotransmission and the CREB/BDNF signaling pathway. These findings provide a new option for clinical treatment.

Mecp2 Knockout (KO) Mice and Preparation
Mecp2 knockout (KO) mice have been reported previously and genotyping was performed as previously described [36]. Littermate male mice were used in the study and divided into three groups: Mecp2 wild-type saline-treated (WT), Mecp2-KO saline-treated (Vehicle) and Mecp2-KO tandospirone-treated (Tandospirone). Mice were intraperitoneally (i.p.) injected with tandospirone (T6704; Merck & Co., Kenilworth, NJ, USA) at a dose of 15 mg/kg/day every day from 4 to 8 weeks of age, according to a previous report [37]. Both the WT and vehicle groups were injected with saline as a control. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Experimental protocols were approved by the Ethical Committee for Animal Experiments at our institute (Approved protocol number; 2020010).

Score Test of Clinical Symptoms
To analyze the effect of tandospirone treatment on the pathological phenotype of Rett syndrome, the "Score test" was evaluated in Mecp2-KO mice according to a previous report adopted in the study of the Mecp2 null mouse model [24,38]. In detail, the pharmacological treatment started at 4 weeks of age. After 4 weeks of treatment, neurological defects in Mecp2-KO mice were scored (blind to the experimenter and treatment), focusing on general condition, hindlimb clasping, tremor, breathing, mobility and gait. Each symptom was scored from 0 to 2; 0 corresponds to the symptom being absent or the same as in the WT mouse, 1 corresponds to the symptom being present and 2 corresponds to the symptom being severe. (D) Breathing: movement of the flanks observed while the animal is standing still. 0 = Normal breathing. 1 = Periods of regular breathing interspersed with short periods of more rapid breathing or with pauses in breathing. 2 = Very irregular breathing-gasping or panting.
(E) Mobility: the mouse is observed when placed on a bench and then handled gently. 0 = WT. 1 = Reduced movement when compared with WT: extended freezing period when first placed on bench and longer periods spent immobile. 2 = No spontaneous movement when placed on the bench; mouse can move in response to a gentle prod or a food pellet placed nearby.
(F) Gait: 0 = WT. 1 = Hind legs are spread wider than WT when walking or running with reduced pelvic elevation, resulting in a 'waddling' gait. 2 = More severe abnormalities: tremor when feet are lifted, walks backward or 'bunny hops' by lifting both rear feet at once.

Measurements of Glutamate, GABA, Monoamines and Their Metabolites in the Brain
Mice were anesthetized and the brain regions being studied, the cortex, hippocampus, midbrain, cerebellum and brainstem, were dissected, immediately frozen in liquid nitrogen and stored at −80 • C until assay. A high-performance liquid chromatography (HPLC) system was used to measure the contents of glutamate, GABA, monoamine and their metabolites in each brain region. To measure the contents of glutamate and GABA, the brain samples were weighed and homogenized in 50 vols 0.3 N perchloric acid containing 1 µM 3,4-dihydroxybenzylamine hydrobromide (DHBA) as an internal standard. Then, the homogenate was centrifuged at 12,000 rpm for 10 min at 4 • C. After filtration (0.45 µm), a 1/10 dilution (20 µL) was injected into the HPLC system. Both glutamate and GABA were separated using an HPLC system at 50 • C on a reverse-phase analytical column (Acclaim 120 C18, 3 mm × 150 mm, 3 µm particle diameter, Thermo Fisher Scientific, Waltham, MA, USA) and detected by an electrochemical detector (CoulArray 8-electrode detector, ESA, MA, USA). The column was eluted with 0.1 M phosphate buffer (pH 6.75) containing 20% methanol. All separations were performed at a flow rate of 1.0 mL/min. The measurement of monoamines and metabolites was also carried out as described in our previous study [39].
The following monoamines and their metabolites were measured: dopamine (DA), 3,4dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (serotonin, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). Mouse brains regions, including the cortex, midbrain, cerebellum and brainstem, in the WT, vehicle and tandspirone groups (n = 4 for each group) were dissected. Total RNA for each brain region was extracted using an RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA) including gDNA eliminator treatment according to the manufacturer's protocol. RNA-seq was performed as follows. In brief, the library was prepared using a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech laboratories Inc., Mountain View, CA, USA), Nextera XT DNA Library Prep Kit and Nextera XT Index Kit v2 SetA/B/C/D (Illumina Inc., San Diego, CA, USA) and then sequenced on a NovaSeq 6000 system (Illumina). Data analysis was processed by the following software: Genedata Profiler Genome v13.0.11 (Gendata KK, Tokyo, Japan), STAR v2.6.0c [40] and CLC Genomics Workbench v12 (Qiagen). An absolute value of log2 (fold change) ≥ 1 and adjusted p < 0.05 were taken as the screening criteria. After filtering, functional pathways were obtained by using the Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/kegg_ja.html) pathway database, accessed on 13 June 2021.

Quantitative PCR (qPCR)
To validate the genes that were differentially expressed in RNA-Seq, we performed real-time qPCR. Total RNA from the cortex, midbrain, cerebellum and brainstem (n = 4) was extracted using an RNeasy Mini Kit (Qiagen). The cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) according to the manufacturer's instructions. qPCR was measured by a LightCycler 480 Instrument (Roche Applied Science; Upper Bavaria, Germany). The primers used for the amplification of candidate genes are presented in Supplementary Table S1. Amplifications were performed in a 384-well optical plate and the thermocycling conditions were 30 s at 96 • C, 30 s at 60 • C and 30 s at 72 • C for 45 cycles. A quantitative analysis was performed using the ∆∆CT method with normalization to GAPDH expression. Data were obtained from three independent qPCR experiments.

Statistical Analysis
Data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's multiple comparison post hoc test for intergroup comparisons. All results were analyzed using GraphPad 9.0 Prism software. Lifespan data were plotted as Kaplan-Meier survival curves. The results were considered significant for values of * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Institutional Review Board Statement: The study was approved by the Institutional Animal Ethics Committee of the National Center of Neurology and Psychiatry (protocol code; 2020001 and date of approval; 29 January 2020).

Informed Consent Statement: Not applicable.
Data Availability Statement: Data are available from the authors upon reasonable request and with permission from the Ethics Committee of the National Center of Neurology and Psychiatry.