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Prof. Massimo Zollo

Professor of Genetics at the Department of Molecular Medicine and Medical Biotechnology (DMMBM), Federico II University, Naples, Italy


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The project has 3 objectives, defined in three phases:


The first goal

is to study the role that Prune-1 and Tubulin-alpha proteins in microtubule dynamics during mitotic division. Furthermore, we will evaluate whether some of the mutations most frequently identified in these two genes (PRUNE-1: p.D106N - / -, p.D106N + / - / L270P +/-, p.H292Qfs * 3 - / -; and TUBA1A: p.R2S - / -) are responsible for alterations both in the microtubule polymerization process and during mitosis. These studies will be performed using cells (fibroblasts) obtained from affected patients. In fact, our laboratory (located at CEINGE, Advanced Biotechnology, Naples) has collected primary fibroblasts obtained from skin biopsies of patients affected by neurodevelopmental diseases caused by different mutations in the PRUNE-1 genes (p.D106N - / -, p.D106N +/- / L270P +/-, p.H292Qfs * 3 - / -) and TUBA1A (p.R2S - / -). On these fibroblasts, immunofluorescence analysis will be performed to characterize mitotic defects and chromosomal segregation (eg presence of binucleate cells, cytokine defects, presence of micronuclei and/or chromosomes such as loss of chromosomes or "laggins", and alterations in the number of centrosomes), to study the polymerization of microtubules and defects in cell proliferation using a technological system (Cell Index) that records the rate of cell growth in real time (X-CELLigence, Roche). 

The second goal

of this project is to "produce in vitro" a neural system that mimics the pathology of the child to test the effectiveness of new molecules in "recovering" the mitotic and proliferation defects caused by mutations in the PRUNE-1 and TUBA1A genes. For this purpose, patients' fibroblasts (described above) will be induced to differentiate into neuronal cells to then develop "cerebral organoids", i.e. small human brains "grown in vitro" (in collaboration with the University of Cologne (Germany) in the laboratory of Prof. Gopalakrishnan). These organoids will allow us to validate the mitotic defects previously identified in patients' fibroblasts in a neuronal system allowing us to evaluate the presence of typical signs of neurodevelopmental diseases: microcephaly, hypoplasia of the corpus callosum, optic and cerebellar atrophy. In addition, we will test the efficacy of new small molecules synthesized in vitro derived from antagonists of the enzyme function of Prune-1 (in collaboration with Prof. Jemielity University of Warsaw of the "Center of New Technologies and the Division of Biophysics", Faculty of Physics and with Prof. Fattorusso: Department of Biological and Pharmaceutical Environmental Sciences and Technologies, Luigi Vanvitelli University of Campania). These molecules will be tested on the organoids obtained for the various mutations identified to date (PRUNE-1: p.D106N - / -, p.D106N + / - / L270P +/-, p.H292Qfs * 3 - / -; and TUBA1A: p.R2S - / -), and will also be used to characterize the site of interaction between the molecules and the recombinant PRUNE-1 and TUBA1A proteins to define the site of the interaction protein and to define, with structural and conformational studies (using Mass Spectrometry techniques), the site of binding to the proteins and their capacity of inhibition and/or their functional activation. This study will allow us to evaluate the ability of new-generation drugs to interact with recombinant Prune-1 and TUBA1A proteins and to demonstrate in vitro efficacy to recover the defect in fibroblasts of affected patients (see objective 1) during mitosis (in cell divisions) and/or in the generated organoids, showing an efficacy on the functionality of the neuronal system generated in vitro, through electrophysiological measurements.

The third goal

dof the project is to validate the efficacy of the best molecules tested in vitro on fibrobalasts and organoids in the murine knock-in model of PRUNE-1 p. D106N - / - of the latest generation in our laboratory. The murine model D106N - / - in homozygosity generates proliferation / differentiation / migration defects in the neuronal precursors of the mouse cortex. One of the objectives of this project is to evaluate the functionality of the newly synthesized compounds in "preventing" mitotic defects in early embryonic stages, with appropriate doses to be injected into uterus in mice during gestation. We expect these defects to be recovered to generate an embryo capable of completing brain development in the mouse. These preliminary studies will be helpful for translational studies in humans aimed at the future use of anti-Prune-1 drugs in the treatment of neurodevelopmental diseases and tubulinopathies in pregnancy where it is possible to observe, through a genetic investigation, the presence of the causative mutation of the disease in embryo, during gestation.

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