a. Synergy in the Trentino research system, international visibility.
The short term objective of the AXonomIX project is driven by the fact that this project is done in Trentino for Trentino, and incorporates collaborations aimed at bringing into the young, productive and growing biotechnology community the expertise necessary for the establishment of a solid scientific background in the study of neuronal degeneration processes. The intention is to make Trentino an European-level centre for studying mechanisms of MN degeneration .
b. A complete, unbiased and defined map of subcellular mRNA and protein changes underlying motor neuron degeneration.
The middle-term scientific objective we want to pursue with "AXonomIX" is to gather comprehensive and unbiased information about what happens to mRNAs and their translation machinery in MN diseases at the highest possible resolution at the sub-cellular level. "Comprehensive" information means that we will utilize many possible experimental models and technologies to obtain various levels of experimental data. "Unbiased" information means that we want to take a systemic approach to address the molecular basis of MNDs, without any preconceptions of what we will find. "At the highest possible resolution" means at the molecule resolution, since we are dealing with mRNAs under active translation. This resolution will be achieved by studying RNA Binding Proteins (RBPs) and mRNAs binding sites at almost nucleotide resolution with Cross Linking Immuno Precipitation (CLIPs) approaches. In addition, by highly innovative nanoimaging technologies (Atomic Force Microscopy and STED), we will obtained important complementary information about the organization of axonal and somatic polysomes and stress granules. Overall, we will obtain an unprecedented, quantitative appreciation of the molecular differences that exist between native and diseased subcellular compartments of MNs. This description will provide powerful new insights into the cellular and molecular basis of MNDs, by merging classical cell biology approaches with state-of-the-art transcriptome and proteome profiling technologies and bioinformatics.
c. Therapy of MNDs, technology transfer.
There is a strong focus internationally for the development of MND therapies, including both pharmacological and gene therapy approaches, but in every case the chosen targets are the proteins known to be mutated in the disease. The long-term scientific objective of the AXonomIX project is to gather molecular and structural information which would identify key mRNAs and proteins altered in MNDs that can be considered as targets for the identification of new drugs. We want to offer new candidates for the drug discovery process, either specific to a particular disease or relevant across a range of disease states. Once the genome-wide perspective on the alterations in translational control behind MN degeneration will be available, we will study potential mRNAs and proteins target starting the "translational" process of the project. A technology transfer spin-off project is currently in preparation at the CIBIO. The idea behind the project is to provide pharma companies with: i) a new cell-based screening technology to screen for small molecules able to influence translation in a sequence- specific way and ii) targets that could be further assessed for translational potential by the start-up company.
The main objective of AXonomIX is to provide an unsupervised molecular characterization of the messenger ribonucleoprotein (mRNPs and polysomes) complexes and of the translational machinery in the motor unit laying in such a way the groundworks for drug repositioning and/or drug discovery useful for MNDs. The goal is to earn a qualitative and quantitative description of the alterations occurring in healthy motor units when undergoing to MN pathologies by analyzing several mouse models of specific MN disease and MN degeneration. In particular, we expect to obtain easy and reproducible protocols for studying translation in MNs. The transcriptome and translatome of samples from various sources (tissues, primary cell cultures, sub-cellular compartments) will be analyzed by means of sequencing approaches (RNA-Seq, Pol-Seq, Ribo-Seq, CLIPs). In addition, this data will be integrated with mass spectrometry analysis, allowing an unprecedented in-depth molecular profiling of fundamental biological processes during motor unit degeneration. Taking advantage of this widespread profiling, a significant part of the project will be aimed at understanding the pathogenesis of MNDs at the molecular level. The molecules involved are expected to be mainly RBPs or proteins playing critical roles in various RNA pathways to cause motor neuron degeneration through translational deregulation of local protein synthesis. The analysis of RBPs and of transported and/or translated mRNAs will clarify the biological functions of individual components of axonal mRNPs. This information will be highly innovative, being the characterization of mRNPs and polysomes still largely unknown in the context of MNs. Alongside portraying mRNPs, the project will provide fundamental details of the native super-structural organization of the translation machinery in term of high resolution description of the polysomes and regulatory proteins that co-sediment with polysomes. This will give us the unprecedented opportunity to integrate structural and omics information to draw a comprehensive topological model of the translational machinery in MNs and providing a unique deliverable in term of know-where and know-who, that would offer unique opportunities to new drug design approaches. To this aim, an original procedure will quantitatively monitor the level of translation in MNs and a new method will be employed with the final purpose of screening molecular scaffolds able to modulate translation in MNs. These approaches will be used by the screening facility to probe a collection of compounds and provide the relevant result of a list of active compounds to set the stage for the ambitious goal of a treatment for MNs diseases under study. Another expectation for the project is to make possible an effective exchange of information and experiences between the partners of the project with the local, national and international communities on MND research. We will favor the dissemination of our results not only as scientific publications and patents, but also in term of congress participation, organization of summer schools, development of a website for accessing the database produced. Concluding, this project will create a new hub for MND research in the Trento Province, extensively connected internationally, and able also to promote awareness of the importance of these studies in the local population