BDNF translational control as a therapeutic target in Rett syndorme

Rett syndrome (RTT, MIM 312750) is a debilitating neurodevelopmental disorder that manifests in early childhood and affects almost exclusively girls. It 's a genetic disorder and is present worldwide with an estimated average incidence of 1:10,000 / 15,000 newborn girls. RTT is the second leading cause of mental retardation in female often misdiagnosed as autism or an unspecified developmental delay. In its classic version, this disease is caused by mutations in the transcription factor Methyl-CpG binding protein 2 (MeCP2) located on the female X chromosome (Amir et al., 1999). MeCP2 is a transcriptional regulator of many genes, including the neurotrophic factor Brain Derived Neurotrophic Factor (BDNF). BDNF is a member of the neurotrophins family and represents a key molecule for neuronal survival and development, and it is involved in learning and memory processes. BDNF levels significantly increase during the first period of postnatal life till it reaches the threshold necessary for the maturations of neurons with the development of dendrites, axons, dendritic spines and synapses. Recent studies have shown that in transgenic mice, in which MECP2 gene has been inactivated to mimic the symptoms of RTT, the mRNA levels of BDNF are reduced and, in these animals at 6-8 weeks of age, the total content of BDNF protein is reduced by 70%. Furthermore, the overexpression of BDNF in MeCP2-/Y mice leads to an increase in lifespan, improved locomotor deficits and electrophysiological defects, while the deletion of the gene coding for BDNF in the same animals leads to an early onset of the disease symptoms. The main goal of this thesis is to identify a possible drug treatment for the treatment of RTT. The idea is based on the fact that in animal models of RTT there are residual levels of mRNA coding for BDNF and that many drugs, mostly used for the treatment of depression, are able to increase the synthesis of BDNF. In this study we analyzed the residual expression of different BDNF transcripts in human brain and in two models of RTT (the mouse and the cellular model). Using quantitative PCR, we determined the residual expression of BDNF transcripts in post mortem brain samples of RTT patients, using tissues from somatosensory and motor cortex (Broadmann areas 1-5), which are the most affected by the disease. In addition, we determined the expression of BDNF transcripts in the cortex and hippocampus of MeCP2-/Y at different of post-natal ages. Finally, in order to establish a cellular model of disease, we disrupted the expression of MeCP2 gene in human neuroblastoma cell line SHSY-5Y (via RNA interference), and then we measured the expression levels of BDNF transcripts. Using the same cell line we obtained information on the translatability of the different BDNF transcripts through a luminescence assay, based on firefly luciferase as a reporter gene. We observed that in the absence of stimuli, each exon contained the 5'UTR of the BDNF leads to a different level of translation of the reporter gene. We therefore conducted a systematic pharmacological translation of each BDNF transcript to determine what compounds may be used to stimulate the synthesis of BDNF starting from its own specific transcripts. The treatment with serotonin and norepinephrine separately, or with two antidepressants such as desipramine and mirtazapine were found to be most effective in stimulating the BDNF synthesis. These results encourage the planning of future experiments to test the efficacy of existing antidepressant drugs in restoring the compromised BDNF levels in RTT.