Final Report Summary - FRAGILE X PATHWAYS (Molecular, cellular and metabolic neuronal pathways of Fragile X Syndrome)
Intellectual disability (ID) is a major cause of serious handicap and an important medical and social issue, affecting 1 to 1.5 % of the population. The fragile X syndrome (FXS) is the most frequent hereditary cause of ID affecting 1/4000 males and 1/7000 females, due to the inactivation of the X-linked fragile X mental retardation 1 gene (FMR1). In FXS patients and its mouse model - the Fmr1 knock-out mouse - the lack of the gene product, the RNA-binding protein FMRP, induces behavioural and cognitive abnormalities, coupled to alterations of synaptic plasticity and morphology. To better understand the role played by FMRP in this processes, I followed several research aims in this European Reintegration Grant (ERG) project. After the three years of ERG support, I have completed several aspects of the project while others are still under investigation:
Aim 1 - Characterisation of mRNA targets of FMRP specific to neuronal RNA granules
This aspect is still under investigation, in collaboration with Prof. Edouard Khandjian (Laval University, Québec, Canada). Meanwhile, I have participated to the characterisation of a novel neuronal mRNA target of FMRP, the mRNA encoding the Superoxide dismutase 1 (SOD1, Bechara et al., 2009, PloS Biology).
Aim 2 - Characterisation of the protein composition of neuronal RNA granules containing FMRP
I have developed and characterised new polyclonal antibodies directed against the C-terminus of FMRP which are able to immunoprecipitate the neuronal mRNP complexes containing FMRP. The mass-spec analysis of the immunoprecipitates is ongoing. Meanwhile, I have contributed to the characterisation of the involvment in cancer of an interactor of FMRP, MSP58 (Shi et al., 2009, Cancer Sci; Lin et al., 2009, J Cell Mol Med.), which I had previously characterised (Davidovic et al., 2006, Hum Mol Genet).
Aim 3 - Identification of neuronal pathways perturbed in the mouse model of FXS by proton nuclear magnetic resonance spectroscopy
This part of the project is completed. I have published as first and corresponding author (Davidovic et al., 2011, Genome research) the first comprehensive profiling of the metabolome of the brain of the mouse model of FXS, in close collaboration with Dr Marc-Emmanuel Dumas (Imperial College, London, United Kingdom (UK)). The term metabonomics or metabolic profiling refers to the large-scale analysis of the entire complement of low-weight molecules (or metabolome) produced by a biological system, just like the term proteome refers to the entire protein content of a given sample. Using 1H-NMR-based metabonomics in conjunction with multivariate pattern recognition, 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 on 25 brain metabolites, notably leading to alterations in levels of neurotransmitters and secondary messenger precursors.
Since FMRP modulates protein synthesis via direct regulation of the translation of multiple mRNAs, we hypothesised that Fmr1-deficiency could functionally affect protein interaction networks with direct consequences on signalling cascades and cellular metabolism. This novel 'integrated metabolome and interactome mapping' (iMIM) approach has highlighted that the FXS metabolic response is initiated by distinct mRNA targets and proteins interacting with FMRP, and then relayed by numerous regulatory proteins. This landmark study was recently acknowledged to 'add a new dimension to fragile X syndrome' (Heulens et al., 2011, Genome med) and now forms the conceptual basis for my future research. By integrating the molecular and metabolic data obtained following this trans-disciplinary approach, I expect to contribute significantly to the understanding of the neuronal alterations induced by the absence of FMRP in FXS patients, and to validate therapeutical molecules.
Aim 1 - Characterisation of mRNA targets of FMRP specific to neuronal RNA granules
This aspect is still under investigation, in collaboration with Prof. Edouard Khandjian (Laval University, Québec, Canada). Meanwhile, I have participated to the characterisation of a novel neuronal mRNA target of FMRP, the mRNA encoding the Superoxide dismutase 1 (SOD1, Bechara et al., 2009, PloS Biology).
Aim 2 - Characterisation of the protein composition of neuronal RNA granules containing FMRP
I have developed and characterised new polyclonal antibodies directed against the C-terminus of FMRP which are able to immunoprecipitate the neuronal mRNP complexes containing FMRP. The mass-spec analysis of the immunoprecipitates is ongoing. Meanwhile, I have contributed to the characterisation of the involvment in cancer of an interactor of FMRP, MSP58 (Shi et al., 2009, Cancer Sci; Lin et al., 2009, J Cell Mol Med.), which I had previously characterised (Davidovic et al., 2006, Hum Mol Genet).
Aim 3 - Identification of neuronal pathways perturbed in the mouse model of FXS by proton nuclear magnetic resonance spectroscopy
This part of the project is completed. I have published as first and corresponding author (Davidovic et al., 2011, Genome research) the first comprehensive profiling of the metabolome of the brain of the mouse model of FXS, in close collaboration with Dr Marc-Emmanuel Dumas (Imperial College, London, United Kingdom (UK)). The term metabonomics or metabolic profiling refers to the large-scale analysis of the entire complement of low-weight molecules (or metabolome) produced by a biological system, just like the term proteome refers to the entire protein content of a given sample. Using 1H-NMR-based metabonomics in conjunction with multivariate pattern recognition, 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 on 25 brain metabolites, notably leading to alterations in levels of neurotransmitters and secondary messenger precursors.
Since FMRP modulates protein synthesis via direct regulation of the translation of multiple mRNAs, we hypothesised that Fmr1-deficiency could functionally affect protein interaction networks with direct consequences on signalling cascades and cellular metabolism. This novel 'integrated metabolome and interactome mapping' (iMIM) approach has highlighted that the FXS metabolic response is initiated by distinct mRNA targets and proteins interacting with FMRP, and then relayed by numerous regulatory proteins. This landmark study was recently acknowledged to 'add a new dimension to fragile X syndrome' (Heulens et al., 2011, Genome med) and now forms the conceptual basis for my future research. By integrating the molecular and metabolic data obtained following this trans-disciplinary approach, I expect to contribute significantly to the understanding of the neuronal alterations induced by the absence of FMRP in FXS patients, and to validate therapeutical molecules.