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Fragile X syndrome (FXS) is a common form of intellectual disability and autism caused by the lack of Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein involved in RNA transport and protein synthesis. Upon cellular stress, global protein synthesis is blocked and mRNAs are recruited into stress granules (SGs), together with RNA-binding proteins including FMRP. Activation of group-I metabotropic glutamate (mGlu) receptors stimulates FMRP-mediated mRNA transport and protein synthesis, but their role in SGs formation is unexplored. To this aim, we pre-treated wild type (WT) and Fmr1 knockout (KO) cultured astrocytes with the group-I-mGlu receptor agonist (S)-3,5-Dihydroxyphenylglycine (DHPG) and exposed them to sodium arsenite (NaAsO2), a widely used inducer of SGs formation. In WT cultures the activation of group-I mGlu receptors reduced SGs formation and recruitment of FMRP into SGs, and also attenuated phosphorylation of eIF2α, a key event crucially involved in SGs formation and inhibition of protein synthesis. In contrast, Fmr1 KO astrocytes, which exhibited a lower number of SGs than WT astrocytes, did not respond to agonist stimulation. Interestingly, the mGlu5 receptor negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl)pyridine (MPEP) antagonized DHPG-mediated SGs reduction in WT and reversed SGs formation in Fmr1 KO cultures. Our findings reveal a novel function of mGlu5 receptor as modulator of SGs formation and open new perspectives for understanding cellular response to stress in FXS pathophysiology.

Impaired energy metabolism may play a role in the pathogenesis of neurodevelopmental disorders including fragile X syndrome (FXS). We checked brain energy status and some aspects of cell bioenergetics, namely the activity of key glycolytic enzymes, glycerol-3-phosphate shuttle and mitochondrial respiratory chain (MRC) complexes, in the cerebral cortex of the Fmr1 knockout (KO) mouse model of FXS. We found that, despite a hyperactivation of MRC complexes, adenosine triphosphate (ATP) production via mitochondrial oxidative phosphorylation (OXPHOS) is compromised, resulting in brain energy impairment in juvenile and late-adult Fmr1 KO mice. Thus, an altered mitochondrial energy metabolism may contribute to neurological impairment in FXS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss of motor 29 neurons (MNs) and astrogliosis. Recent evidences suggest that factors secreted by activated astrocytes might 30 contribute to degeneration of MNs. We focused on endothelin-1 (ET-1), a peptide which is strongly up- 31 regulated in reactive astrocytes under different pathological conditions. We show that ET-1 is abundantly 32 expressed by reactive astrocytes in the spinal cord of the G93A mouse model and sporadic ALS patients. To test 33 if ET-1 might play a role in degeneration of MNs, we investigated its effect on MN survival in an in vitro model 34 of mixed rat spinal cord cultures (MSCs) enriched of astrocytes exhibiting a reactive phenotype. ET-1 exerted a 35 toxic effect on MNs in a time- and concentration-dependent manner, with an exposure to 100-200 nM ET-1 36 for 48 h resulting in 40-50% MN cell death. Importantly, ET-1 did not induce MN degeneration when adminis- 37 tered on cultures treated with AraC (5 ?M) or grown in a serum-freemediumthat did not favor astrocyte prolif- 38 eration and reactivity.We found that both ETA and ETB receptors are enriched in astrocytes in MSCs. The ET-1 39 toxic effect was mimicked by ET-3 (100 nM) and Sarafotoxin S6C (10 nM), two selective agonists of 40 endothelin-B receptors, andwas not additivewith that of ET-3 suggesting the involvement of ETB receptors. Sur- 41 prisingly, however, the ET-1 effect persisted in the presence of the ETB receptor antagonist BQ788 (200 nM- 42 2 ?M) andwas slightly reversed by the ETA receptor antagonist BQ123 (2 ?M), suggesting an atypical pharmaco- 43 logical profile of the astrocytic receptors responsible for ET-1 toxicity. The ET-1 effect was not undone by the 44 ionotropic glutamate receptor AMPA antagonist GYKI 52466 (20 ?M), indicating that it is not caused by an in- 45 creased glutamate release. Conversely, a 48-hour ET-1 treatment increased MN cell death induced by acute ex- 46 posure to AMPA (50 ?M), which is indicative of two distinct pathways leading to neuronal death. 47 Altogether these results indicate that ET-1 exerts a toxic effect on culturedMNs through mechanisms mediated 48 by reactive astrocytes and suggest that ET-1may contribute toMN degeneration in ALS. Thus, a treatment aimed 49 at lowering ET-1 levels or antagonizing its effect might be envisaged as a potential therapeutic strategy to slow 50 down MN degeneration in this devastating disease.

Fragile X syndrome (FXS) is the first cause of inherited intellectual disability, due to the silencing of the X-linked Fragile X Mental Retardation 1 gene encoding the RNA-binding protein FMRP. While extensive studies have focused on the cellular and molecular basis of FXS, neither human Fragile X patients nor the mouse model of FXS—the Fmr1-null mouse—have been profiled systematically at the metabolic and neurochemical level to provide a complementary perspective on the current, yet scattered, knowledge of FXS. Using proton high-resolution magic angle spinning nuclear magnetic resonance (1H HR-MAS NMR)-based metabolic profiling, we have identified a metabolic signature and biomarkers associated with FXS in various brain regions of Fmr1-deficient mice. Our study highlights for the first time that Fmr1 gene inactivation has profound, albeit coordinated consequences in brain metabolism leading to alterations in: (1) neurotransmitter levels, (2) osmoregulation, (3) energy metabolism, and (4) oxidative stress response. To functionally connect Fmr1-deficiency to its metabolic biomarkers, we derived a functional interaction network based on the existing knowledge (literature and databases) and show that the FXS metabolic response is initiated by distinct mRNA targets and proteins interacting with FMRP, and then relayed by numerous regulatory proteins. This novel “integrated metabolome and interactome mapping” (iMIM) approach advantageously unifies novel metabolic findings with previously unrelated knowledge and highlights the contribution of novel cellular pathways to the pathophysiology of FXS. These metabolomic and integrative systems biology strategies will contribute to the development of potential drug targets and novel therapeutic interventions, which will eventually benefit FXS patients.