Altered brain serotonin 5-HT1A receptor expression and function in juvenile Fmr1 knockout mice.

There are no approved pharmacotherapies for fragile X syndrome (FXS), a rare neurodevelopmental disorder caused by a mutation in the FMR1 promoter region that leads to various symptoms, including intellectual disability and auditory hypersensitivity. The gene that encodes inhibitory serotonin 1A receptors (5-HT1ARs) is differentially expressed in embryonic brain tissue from individuals with FXS, and 5-HT1ARs are highly expressed in neural systems that are disordered in FXS, providing a rationale to focus on 5-HT1ARs as targets to treat symptoms of FXS. We examined agonist-labeled 5-HT1AR densities in male and female Fmr1 knockout mice and found no differences in whole-brain 5-HT1AR expression in adult control compared to Fmr1 knockout mice. However, juvenile Fmr1 knockout mice had lower whole-brain 5-HT1AR expression than age-matched controls. Consistent with these results, juvenile Fmr1 knockout mice showed reduced behavioral responses elicited by the 5-HT1AR agonist (R)-8-OH-DPAT, effects blocked by the selective 5-HT1AR antagonist, WAY-100635. Also, treatment with the selective 5-HT1AR agonist, NLX-112, dose-dependently prevented audiogenic seizures (AGS) in juvenile Fmr1 knockout mice, an effect reversed by WAY-100635. Suggestive of a potential role for 5-HT1ARs in regulating AGS, compared to males, female Fmr1 knockout mice had a lower prevalence of AGS and higher expression of antagonist-labeled 5-HT1ARs in the inferior colliculus and auditory cortex. These results provide preclinical support that 5-HT1AR agonists may be therapeutic for young individuals with FXS hypersensitive to auditory stimuli.

Effects of Fmr1 Gene Mutations on Sex Differences in Autism-Like Behavior and Dendritic Spine Development in Mice and Transcriptomic Studies.

Fragile X syndrome (FXS) is the most common single gene disorder contributing to autism spectrum disorder (ASD). Although significant sex differences are observed in FXS, few studies have focused on the phenotypic characteristics as well as the differences in brain pathological changes and gene expression in FXS by sex. Therefore, we analyzed sex differences in autism-like behavior and dendritic spine development in two-month-old male and female Fmr1 KO and C57 mice and evaluated the mechanisms at transcriptome level. Results suggest that Fmr1 KO mice display sex differences in autism-like behavior and dendritic spine density. Compared to females, male had more severe effects on anxiety, repetitive stereotype-like behaviors, and socializing, with higher dendritic spine density. Furthermore, two male-biased and five female-biased expressed genes were screened based on KEGG pathway enrichment and protein-protein interaction (PPI) analyses. In conclusion, our findings show mutations in the Fmr1 gene lead to aberrant expression of related genes and affect the sex-differentiated behavioral phenotypes of Fmr1 KO mice by affecting brain development and functional architecture, and suggest future studies should focus on including female subjects to comprehensively reflect the differentiation of FXS in both sexes and develop more precise and effective therapeutic strategies.

Impaired GABAergic inhibition in the hippocampus of Fmr1 knockout mice.

Many clinical and molecular features of the fragile X syndrome, a common form of intellectual disability and autism, can be modeled by deletion of the Fmr1 protein (Fmrp) in mice. Previous studies showed a decreased expression of several components of the GABAergic system in Fmr1 knockout mice. Here, we used this mouse model to investigate the functional consequences of Fmrp deletion on hippocampal GABAergic inhibition in the CA1-region of the hippocampus. Whole-cell patch-clamp recordings demonstrated a significantly reduced amplitude of evoked inhibitory postsynaptic currents (eIPSCs) and a decrease in the amplitude and frequency of spontaneous IPSCs. In addition, miniature IPSCs were reduced in amplitude and frequency and decayed significantly slower than mIPSCs in controls. Quantitative real-time PCR revealed a significantly lower expression of α2, ß1 and δ GABAA receptor subunits in the hippocampus of the juvenile mice (P22) compared to wild-type littermates. Correspondingly, we found also at the protein level reduced amounts of α2, ß1 and δ subunits in Fmr1 knockout mice. Overall, these results demonstrate that the reduction in several components of the GABAergic system is already present at young age and that this reduction results in measurable abnormalities on GABAA receptor-mediated phasic inhibition. These abnormalities might contribute to the behavioral and cognitive deficits of this fragile X mouse model.

Abnormal neural precursor cell regulation in the early postnatal Fragile X mouse hippocampus.

The regulation of neural precursor cells (NPCs) is indispensable for a properly functioning brain. Abnormalities in NPC proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome. Yet, no studies have examined NPCs from the early postnatal Fragile X mouse hippocampus despite the importance of this developmental time point, which marks the highest expression level of FMRP, the protein missing in Fragile X, in the rodent hippocampus and is when hippocampal NPCs have migrated to the dentate gyrus (DG) to give rise to lifelong neurogenesis. In this study, we examined NPCs from the early postnatal hippocampus and DG of Fragile X mice (Fmr1-KO). Immunocytochemistry on neurospheres showed increased Nestin expression and decreased Ki67 expression, which collectively indicated aberrant NPC biology. Intriguingly, flow cytometric analysis of the expression of the antigens CD15, CD24, CD133, GLAST, and PSA-NCAM showed a decreased proportion of neural stem cells (GLAST+CD15+CD133+) and an increased proportion of neuroblasts (PSA-NCAM+CD15+) in the DG of P7 Fmr1-KO mice. This was mirrored by lower expression levels of Nestin and the mitotic marker phospho-histone H3 in vivo in the P9 hippocampus, as well as a decreased proportion of cells in the G2/M phases of the P7 DG. Thus, the absence of FMRP leads to fewer actively cycling NPCs, coinciding with a decrease in neural stem cells and an increase in neuroblasts. Together, these results show the importance of FMRP in the developing hippocampal formation and suggest abnormalities in cell cycle regulation in Fragile X.

Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse.

UNLABELLED: An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain. HIGHLIGHTS: • We studied the proliferation of neural stem and progenitor cells in Fragile X.• We examined the role of astrocyte-secreted factors in neural precursor cell biology.• Astrocyte-secreted factors with differential expression in Fragile X identified.

GABAAergic stimulation modulates intracellular protein arginine methylation.

Changes in cytoplasmic pH are known to regulate diverse cellular processes and influence neuronal activities. In neurons, the intracellular alkalization is shown to occur after stimulating several channels and receptors. For example, it has previously demonstrated in P19 neurons that a sustained intracellular alkalinization can be mediated by the Na(+)/H(+) antiporter. In addition, the benzodiazepine binding subtypes of the γ-amino butyric acid type A (GABAA) receptor mediate a transient intracellular alkalinization when they are stimulated. Because the activities of many enzymes are sensitive to pH shift, here we investigate the effects of intracellular pH modulation resulted from stimulating GABAA receptor on the protein arginine methyltransferases (PRMT) activities. We show that the major benzodiazepine subtype (2α1, 2ß2, 1γ2) is constitutively expressed in both undifferentiated P19 cells and retinoic acid (RA) differentiated P19 neurons. Furthermore stimulation with diazepam and, diazepam plus muscimol produce an intracellular alkalinization that can be detected ex vivo with the fluorescence dye. The alkalinization results in significant perturbation in protein arginine methylation activity as measured in methylation assays with specific protein substrates. Altered protein arginine methylation is also observed when cells are treated with the GABAA agonist muscimol but not an antagonist, bicuculline. These data suggest that pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation, which we verified using hippocampal slice preparations from a mouse model of fragile X syndrome.

Behavioral analysis of male and female Fmr1 knockout mice on C57BL/6 background.

Fragile X syndrome (FXS) is a monogenic disease caused by mutations in the FMR1 gene. The Fmr1 knockout (KO) mice show many aspects of FXS-related phenotypes, and have been used as a major pre-clinical model for FXS. Although FXS occurs in both male and female patients, most studies on the mouse model use male animals. Few studies test whether gender affects the face validity of the mouse model. Here, we examined multiple behavioral phenotypes with male hemizygous and female homozygous Fmr1 KO mice on C57BL/6 background. For each behavioral paradigm, we examined multiple cohorts from different litters. We found that both male and female Fmr1 KO mice displayed significant audiogenic seizures, hyperactivity in the open field test, deficits in passive avoidance and contextual fear memory, and significant enhancement of PPI at low stimulus intensity. Male and female Fmr1 KO mice also showed more transitional movement between the lit and dark chambers in the light-dark tests. The lack of gender effects suggests that the Fmr1 KO mouse is a reasonable tool to test the efficacy of potential FXS therapies.