Modulation of Stress Granules formation: Role of mGlu5 receptor and FMRP and implications for pathophysiology of Fragile X Syndrome

Abstract

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The genetic defect in FXS is a CGG trinucleotide repeat expansion (>200) in the promoter region of the FMR1 (fragile X mental retardation 1) gene; this amplification causes the absence of the encoded protein FMRP (Fragile X Mental Retardation Protein). FMRP is an RNA-binding protein involved in mRNA transport and translation. Despite numerous studies, the available treatments are only symptomatic. There is no cure to replace FMRP expression, yet. FMRP can interact with RNA-binding proteins such as FXR1P, FXR2P, NUFIP and 82-FIP, and with proteins that do not bind RNA, like CYFIP1 and CYFIP2. The interaction with these different proteins may modulate FMRP functions and its RNA affinity. A new role of FMRP in mRNA metabolism as component of stress granules (SGs) has been identified. FMRP seems to lead mRNAs in SGs upon cellular stress, during which protein synthesis is blocked. SGs are ribonucleoproteic aggregates containing translation initiation components and RNA binding proteins, like eIF2a and FMRP. Several data also indicate that some of the FXS symptoms are a consequence of a defect in group-I metabotropic glutamate receptor, namely mGlu5; pharmacological blockade of mGlu5 receptors provide a therapeutic target in FXS. mGlu5 receptor, like FMRP, regulates protein synthesis but in a functionally opponent manner: mGlu5 receptor activates protein synthesis, FMRP suppresses it. In the absence of FMRP, mGlu5-dependent protein synthesis is unchecked, with consequent excessive translation. Activation of mGlu5 receptors stimulates FMRP-mediated mRNA transport and protein synthesis, but its role in SGs formation is unknown.The aim of this PhD thesis was to better investigate FMRP function studying the relationship of FMRP with its interacting proteins and the role of FMRP in stress response under activation of mGlu5 receptor. In Paper I, we analyzed the expression pattern of FMRP and its interacting proteins in different brain areas, at different ages in wild type (WT) mice to better define the interplay between FMRP and its interacting proteins during development. FMRP was strongly expressed at P3, peaked at P7-P14 and gradually decreases thereafter. The analysis of expression pattern of several proteins carried out, indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in early and late developmental stages. We found that the RNA binding and cytoskeleton remodeling functions of FMRP may be differently modulated during development. In Paper II we studied FMRP under stress condition using WT and Fmr1 knockout (KO) astrocytes. We have demonstrated that the lack of FMRP impairs SGs formation and furthermore that activation of mGlu5 receptor affects SGs formation through a FMRP-mediated mechanism in WT. Interestingly, the mGlu5 receptor blockade restores SGs formation in Fmr1 KO. Also, mGlu5 receptor activation before stress reduced FMRP recruitment in SGs and phosphorylation of eIF2a and FMRP. In contrast, mGlu5 receptor activation did not affect SGs formation in Fmr1 KO astrocytes. Since phosphorylation of eIF2a and FMRP are two crucial key events in SGs formation and modulation of protein synthesis, mGlu5 receptors may act by shifting the balance from inhibition to activation of protein synthesis during stress. These findings suggest a potential novel role for mGlu receptors in SGs formation. We suggest that FMRP may have a positive role in stress response, facilitating and enhancing SGs formation to prevent stress damages. This process is useful to understand what happens in FXS, in which can occur abnormal modulation of different proteins during development with consequent abnormal response during adversal conditions, like oxidative stress that represent a frequent component in FXS and neurodegenerative disorders.