Role of Astrocytic Glutamate Transporter GLT1 in Fragile X Syndrome

Mutations in MECP2, encoding methyl CpG-binding protein 2, cause Rett syndrome, the most severe autism spectrum disorder. Re-expressing Mecp2 in symptomatic Mecp2-null mice markedly improves function and longevity, providing hope that therapeutic intervention is possible in humans. To identify pathways in disease pathology for therapeutic intervention, we carried out a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis suppressor screen in Mecp2-null mice and isolated five suppressors that ameliorate the symptoms of Mecp2 loss. We show that a stop codon mutation in Sqle, encoding squalene epoxidase, a rate-limiting enzyme in cholesterol biosynthesis, underlies suppression in one line. Subsequently, we also show that lipid metabolism is perturbed in the brains and livers of Mecp2-null male mice. Consistently, statin drugs improve systemic perturbations of lipid metabolism, alleviate motor symptoms and confer increased longevity in Mecp2 mutant mice. Our genetic screen therefore points to cholesterol homeostasis as a potential target for the treatment of patients with Rett syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the loss-of-function of fragile X mental retardation protein (FMRP). The loss of FMRP function in neurons abolishes its suppression on mGluR1/5-dependent dendritic protein translation, enhancing mGluR1/5-dependent synaptic plasticity and other disease phenotypes in FXS. In this study, we describe a new activation function of FMRP in regulating protein expression in astroglial cells. We found that astroglial glutamate transporter subtype glutamate transporter 1 (GLT1) and glutamate uptake is significantly reduced in the cortex of fmr1(-/-) mice. Correspondingly, neuronal excitability is also enhanced in acute fmr1(-/-) (but not in fmr1(+/+) control) cortical slices treated with low doses (10 μm) of the GLT1-specific inhibitor dihydrokainate (DHK). Using mismatched astrocyte and neuron co-cultures, we demonstrate that the loss of astroglial (but not neuronal) FMRP particularly reduces neuron-dependent GLT1 expression and glutamate uptake in co-cultures. Interestingly, protein (but not mRNA) expression and the (S)-3,5-dihydroxyphenylglycine-dependent Ca(2+) responses of astroglial mGluR5 receptor are also selectively reduced in fmr1(-/-) astrocytes and brain slices, attenuating neuron-dependent GLT1 expression. Subsequent FMRP immunoprecipitation and QRT-PCR analysis showed that astroglial mGluR5 (but not GLT1) mRNA is associated with FMRP. In summary, our results provide evidence that FMRP positively regulates translational expression of mGluR5 in astroglial cells, and FMRP-dependent down-regulation of mGluR5 underlies GLT1 dysregulation in fmr1(-/-) astrocytes. The dysregulation of GLT1 and reduced glutamate uptake may potentially contribute to enhanced neuronal excitability observed in the mouse model of FXS.

The Shank3 gene encodes a scaffolding protein that anchors multiple elements of the postsynaptic density at the synapse. Previous attempts to delete the Shank3 gene have not resulted in a complete loss of the predominant naturally occurring Shank3 isoforms. We have now characterized a homozygous Shank3 mutation in mice that deletes exon 21, including the Homer binding domain. In the homozygous state, deletion of exon 21 results in loss of the major naturally occurring Shank3 protein bands detected by C-terminal and N-terminal antibodies, allowing us to more definitively examine the role of Shank3 in synaptic function and behavior. This loss of Shank3 leads to an increased localization of mGluR5 to both synaptosome and postsynaptic density-enriched fractions in the hippocampus. These mice exhibit a decrease in NMDA/AMPA excitatory postsynaptic current ratio in area CA1 of the hippocampus, reduced long-term potentiation in area CA1, and deficits in hippocampus-dependent spatial learning and memory. In addition, these mice also exhibit motor-coordination deficits, hypersensitivity to heat, novelty avoidance, altered locomotor response to novelty, and minimal social abnormalities. These data suggest that Shank3 isoforms are required for normal synaptic transmission/plasticity in the hippocampus, as well as hippocampus-dependent spatial learning and memory.

Phelan-McDermid syndrome (PMDS) is a complex neurodevelopmental disorder characterized by global developmental delay, severely impaired speech, intellectual disability, and an increased risk of autism spectrum disorders (ASDs). PMDS is caused by heterozygous deletions of chromosome 22q13.3. Among the genes in the deleted region is SHANK3, which encodes a protein in the postsynaptic density (PSD). Rare mutations in SHANK3 have been associated with idiopathic ASDs, non-syndromic intellectual disability, and schizophrenia. Although SHANK3 is considered to be the most likely candidate gene for the neurological abnormalities in PMDS patients, the cellular and molecular phenotypes associated with this syndrome in human neurons are unknown. We generated induced pluripotent stem (iPS) cells from individuals with PMDS and autism and used them to produce functional neurons. We show that PMDS neurons have reduced SHANK3 expression and major defects in excitatory, but not inhibitory, synaptic transmission. Excitatory synaptic transmission in PMDS neurons can be corrected by restoring SHANK3 expression or by treating neurons with insulin-like growth factor 1 (IGF1). IGF1 treatment promotes formation of mature excitatory synapses that lack SHANK3 but contain PSD95 and N-methyl-D-aspartate (NMDA) receptors with fast deactivation kinetics. Our findings provide direct evidence for a disruption in the ratio of cellular excitation and inhibition in PMDS neurons, and point to a molecular pathway that can be recruited to restore it.