Molecular mechanisms involved in the death of motor neurons in patients with sporadic ALS have not been yet elucidated. To date many candidate genes has been detected as involved in a variety of more or less complex pathways. Recently, the post-transcriptional molecular phenomenon of RNA editing has been investigated to demonstrate the motor neuron degeneration through cell excitotoxicity mediated by the AMPA glutamate receptor. In particular, the product of GRIA2 gene plays a basic role in the calcium influx through the AMPA receptor. Indeed, a specific A-to-I RNA editing of GRIA2 mRNA is necessary for the normal neuronal function, whereas the product of the unedited mRNA increases the calcium influx activating the neuronal death pathway. These findings indicate that RNA editing could be relevant in ALS pathogenesis. For this reason, the present pilot project aims to investigate the RNA editing landscape of motor neurons using newly developed techniques for massive RNA sequencing referred to as RNA-Seq. In this way, millions of reads will be produced per biological sample. Each read will be mapped onto the complete human genome in order to detect potential A-to-I editing sites corresponding to A-to-G (inosine is read as guanosine by sequencing enzymes) variations. We will carry out the deep sequencing of the poly-A RNA fraction extracted from motor neurons of three patients with sporadic ALS using the Illumina/Solexa sequencing technology, performing at least three complete runs per biological sample. At the same time we will sequence total RNA from non-ALS patients as controls. All produced reads will be analyzed by using specific bioinformatics tools, to be developed in the present project, suitable to handle huge amount of data. Editing sites, detected through the comparative analysis against the reference human genome, potentially correlated to ALS for their differential representation with respect to controls, will be experimentally confirmed by direct sequencing of genomic DNA extracted from the same samples. The main objective of the project is to find editing events potentially involved in motor neuron degeneration providing also a collection of candidate biomarkers. Additional novelty issues of this project concern the use of technologically advanced methods for massive sequencing and data analysis.

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