Caratterizzazione funzionale di geni codificanti metalloproteasi ADAM in Schmidtea mediterranea

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The ADAMs (A Disintegrin And Metalloproteinase) are a family of transmembrane and secreted proteins of approximately 750 amino acids in length, with functions in cell adhesion, migration and proteolytic processing of the ectodomains of various cell surface receptors and signaling molecules. These proteins have a modular conserved structure that includes from N-terminal to C-terminal an Pro-domain, an Zn2+-dependent metalloprotease domain, an disintegrin domain, an Cysteine-rich domain, an EGF-like domain an a transmembrane segment and a cytoplasmic tail. Their functions are conserved from the nematode C. elegans to Vertebrates. Several molecules are substrates of ADAM activity, including Growth factors as FGF, EGF, TGF-alpha and their receptors as FGFR, EGFR, TGFalphaR, cellular adhesion molecules, extracellular matrix (ECM) components, but also include molecules involved in cell fate decision, as Notch receptor and ligand Delta.\nCatalytically inactive ADAMs are involved in regulation of cell adhesion and migration through their disintegrin domain interacting with several members of integrins. ECM plays a pivotal role in regulating cell behavior through its effects on cell proliferation, growth, differentiation, adhesion and migration in tissue homeostasis, morphogenesis and regeneration. The model organism studied in this thesis work is the planarian Schmidtea mediterranea, well known for the extraordinary body plasticity and regenerative capacity. These abilities reside in the presence of a population of adult stem cells, called neoblasts that are widely distributed in the parenchyma. These pluripotent stem cells can give rise to all differentiated cell types, including neurons.\nThe aim of my thesis work is to characterize functionally two adam-related genes, Smed-Adam3 and Smed-kuzbanian3, formerly identified in the lab (Isolani ME, Ph.D thesis). These two genes are been selected for their specific expression in the central nervous system. Smed-Adam3 is expressed in the cephalic ganglia, visual area, ventral nerve cords and pharyngeal nerve plexus, and are up-regulated during anterior regeneration both ventrally, where cephalic ganglia are regenerating, both dorsally in a region compatible with the formation of eye spots. Smed-adam3 is also up-regulated during pharynx regeneration in the internal cell layer where the pharyngeal nerve plexus is regenerating. Phylogenetic analysis demonstrates that the putative Smed-ADAM3 is homologous to ADAM 11, 22 and 23 of Vertebrates. All these proteins share an expression that is restricted to the nervous system and lack of protease activity, thus exerting their function by the disintegrin domain. Smed-Adam3(RNAi) injected planarians show altered localization of photoreceptors and pigmented cells that form the optic cup, and alterations in optic chiasm formation, as observed by whole-mount immunostaining with anti-body against Arrestin (VC-1). These phenotypes are also functional, in fact Smed-Adam3 dsRNA-injected planarians lose their characteristic negative phototaxis behavior. Smed-Adam3(RNAi) also causes altered localization of Smed-cintillo expressing-cells, putative chemoreceptors, but not a significant reduction of their average number. Smed-Adam3(RNAi) planarians also lose ability to feed. Gene expression analysis performed by Real-Time PCR, showed that the relative expression level of a pan-neural marker does not vary between controls and Smed-Adam3(RNAi) animals, while Smed-GAD (marker of GABA-ergic neurons of the visual area) and Smed-otxA (marker of the visual area and photoreceptors) show an statistically significant increase of expression level. Smed-ADAM3 could then act in regulating the migration of specific nerve cells. An altered migration, inducing anomalous cell differentiation would explain the increase of the expression level of the marker of GABA-ergic neurons. Later on, Smed-ADAM3 could represent a molecule that regulates the growth of axons, presence of abnormal chiasma was in fact observed in Smed-Adam3(RNAi) planarians.\nThe second gene functionally characterized is Smed-kuzbanian3, expressed throughout the parenchyma and in the nervous system, and overexpressed during CNS regeneration. Smed-kuzbanian3(RNAi) causes alterations in the optic chiasm formation, probably due to lack in regulation of Netrin/Netrin receptors and Slit/ Robo pathways. In other animals ADAM10/KUZBANIAN is involved in ectodomain shedding of these receptors, during cell migration and axon guidance. Smed-kuzbanian3(RNAi) planarian also show an altered localization of cellular nuclei of the cephalic ganglia. Real-Time PCR, showed that the relative expression levels of a pan-neural marker increase in Smed-kuzbanian3(RNAi) animals. The increase of the neuron number is not due to increased cell proliferation, and does not occur at detriment of other differentiated cell types. It is therefore possible that in the absence of Smed-kuzbanian3 function, neoblasts do not receive signals for a correct differentiation, and create neurons as a default cell lineage.\n