WP1 - Isolation of MN components from mice models of MN degeneration
Task 1.1 - Culture of MN and separation of subcellular compartments
Task 1.2 - Isolation of MN/motor unit compartments from in vivo mouse models
Task 1.3 - Isolation of cytoplasm from in vivo Schwann cells
WP 2 - Comparative identification of alteration in mRNP networks in degenerating MN
Task 2.1 - Transcriptome and translatome analysis of subcellular domains of embryo MNs by RNAseq and POLseq in WT and mutant mice
Task 2.2 - Transcriptome and translatome analysis of parts of motor units in wild type and mutant mice
Task 2.3 - Transcriptome and translatome analysis of Schwann cells surrounding the neuromuscular junction
Task 2.4 - Identification of ribonucleic complexes and targets of RNA binding proteins implicated in MN degeneration and disease by immunoprecipitation and mass spectrometry and by PAR-CLIP analysis Task 2.5 - Machine learning inference of genetic features involved in MN degeneration and disease and implementation of Axobase, a data base resource of MNs
WP 3 - Alteration of single mRNP complexes and of the localization of single mRNAs in degenerating MNs
Task 3.1 - Prioritization of mRNAs and proteins qualitatively and/or qualitatively altered in MN diseases
Task 3.2 - Subcellular localization of the prioritized mRNAs, characterization of RNP complexes and RNAs involved in MN degeneration
Task 3.3 - Functional studies of prioritized proteins and imaging af axonal transport and potential impact on mRNA localization
WP 4 - Structural and functional alteration of the translational machinery in MNs
Task 4.1 - Polysome imaging in MNs
Task 4.2 - Structural characterization of MN polysomes by AFM and cryo-EM
Task 4.3 - Colocalization of proteins relevant for MN degeneration and disease within the polysome by AFM and Immonogold Staining
WP 5 - Cell-based assays for drug screening
Task 5.1 - "POLtrack", polysome tracing in MN cell lines
Task 5.2 - Metabolic labeling of newly synthesized proteins
Task 5.3 - High Content Assay of modulation of MN translation by small molecules
WP 6 - Management and dissemination
Task 6.1 - Scientific management
Task 6.2 - Collaboration with the Italian charity funding ALS research for dissemination of results and reesarch follow-up
Task 6.3 - Advanced Molecular Diagnostic at the Centre of Integrative Biology: focus on MN diseases
Task 6.4 - An European appointment for mRNA involvement in MND and other mRNA localization and translation in nervous system pathologies in Trentino: 2016 edition of the "mRNA fate" workshop in Riva del Garda