Joint Transnational Call 2009 (JTC2009)


Moderate to severe mental retardation and developmental delay in Fragile X syndrome (FXS) is caused by the loss of the FX mental retardation protein (FMRP) encoded by the FX mental retardation 1 (FMR1) gene. FMRP is contained within ribonucleoprotein granules that traffic specific mRNAs to sites of synaptic transmission. Dendritic abnormalities that have been associated with human mental retardation are found in both FXS patients and Fmr1 knockout mice, but despite growing evidence suggesting a role for specific receptors and biochemical pathways in FXS pathogenesis, an effective therapy has not yet been developed. The central, integrating role of Rho GTPases in controlling actin dynamics and structural plasticity in FXS indicates that pharmacological intervention at the level of the GTPases themselves or their downstream effectors kinases may be an effective strategy to manipulate structural plasticity underlying memory in FXS. The project explored the possibility of cellular and behavioural rescue of FXS phenotypes using pharmacological Rho manipulation in a mouse model of FXS, the Fmr1 knockout mouse, and addressed the mechanistic link between effective Rho-signalling drugs, their target proteins, and the structural and functional alterations that occur at the synapse in association with memory effects.
Initial data indicated that the toxin Cytotoxic Necrotizing Factor 1 (CNF1), a bacterial protein endowed with activating effects on Rho GTPases, rescues hippocampus-dependent object recognition phenotypes; however, the project consortium also used new plasticity related therapeutic agents such as epigalocatechin gallate (EGCG) and rimonabant.

The Cure-FXS project aimed at identifying the Rho-signalling drugs that are most effective in FXS phenotypes, as well as their possible side-effects. In addition, the consortium wanted to generate an integrated understanding of how the different aspects involved in memory are correlated and mechanistically linked and thus might be able to use the knowledge obtained for studying this disease in a broader spectrum of mental disorders.

A major result is the analysis of the behavioral effects of Rho GTPase compounds and the proof of their effectiveness in wild type and Fmr1 knockout mice. One important aspect was to discard the possible toxicity of CNF-1. Primary and secondary SHIRPA screens were used to determine the primary toxicological evaluation suggests that the approach is non-toxic. Acute treatment with the Rho GTPase compounds showed no sign of toxicity as determined by monitoring the condition of the mice for several weeks after the treatment. Intracerebroventricular infusion of the compounds used did not affect the animal’s weight measured once a week for two weeks after injection, or the physical appearance of the animals by monitoring of various signs (coat appearance, appearance of the eyes , interaction with the experimenter, vocalizations, general behavior in the housing cage, etc).
Behavioral experiments have been conducted analyzing the effects of acute treatment with CNF1 on cognitive deficits present Fmr1 knockout (Fmr1−/y) mice, with very positive results. Specifically, we have shown that activation of the GTPase Rho signaling pathway partially reverses memory deficits in novel object recognition in mice Fmr1, without affecting their levels of exploration. Furthermore, the same treatment does not affect the decreased anxiety that these mice exhibit the elevated plus maze paradigm. As well as in general memory deficits, we were interested in observing the effects of drugs RhoGTPase in fear memories. This revealed a significant improvement of memory acquisition in Fmr1−/y mice. Besides studying the effects of CNF1, we also analyzed other possible drugs targeting neural plasticity. We have shown that a phenolic compound found naturally in green tea (EGCG), related to the promotion of synaptic plasticity improves memory consolidation in the same paradigm of novel object recognition in male Fmr1−/y mice. This effect may be mediated by Dyrk1A signalling, which phosphorilates synaptic plasticity related targets. Also, the endocannabinoid system (ECS) is a key modulator of synaptic plasticity, cognitive performance, and anxiety, all of which are affected in FXS. We found that cannabinoid 1 receptor (CB1R) blockade in male Fmr1−/y mice through pharmacological approaches normalized cognitive impairment, overactivated mTOR signaling and altered spine morphology. Some of these traits were also reversed by pharmacological inhibition of mTOR or mGluR5. Thus, blockade of ECS is a potential therapeutic approach to normalize specific plasticity alterations in FXS.
In addition, we have studied the effect of CNF1 on the total number of protrusions, spines and filopodia per 10 μm dendrite in cultured primary hippocampal neurons. The only significant difference between the untreated WT and Fmr1 KO neurons was the filopodia density. After exposure to CNF1 we noticed a significant reduction of the filopodia density in treated Fmr1 KO neurons compared with untreated Fmr1 KO neurons. In conclusion, increased neural plasticity through the modulation of Rho GTPase, mTOR and Dyrk1A signaling, is associated with improved learning ability and enhanced functional and structural CNS plasticity in FXS.

  • Dierssen, Mara (Coordinator)
    Center for Genomic Regulation Genes and <disease Program [SPAIN]
  • Bardoni, Barbara
  • Ramakers, Gerard
    University of Amsterdam department of Developmental Psychology [NETHERLANDS]
  • Diana, Giovanni
    Instituto Superiore de Sanita Department of Therapeutic Research and Medicines Evaluation [ITALY]
  • Stork, Oliver
    Otto von Guerick University Magdeburg Institute of Biology Department of Genetics & Molecular Neurobiology [GERMANY]