REGULATORY ELEMENTS AND BIOMIMETIC SIGNALS IN NEURITOGENESIS, NEURAL DEVELOPMENT AND REGENERATIVE MEDICINE

Homodimerization between Cell Adhesion Molecules (CAMs) of the immunoglobulin family play a central role in cell-cell and cell-extracellular matrix interactions. Specific Ig-like repeats from the extracellular domains of neuronal CAMs share a highly conserved Neurite Outgrowth and Guidance (NOG) motif, which is crucial in mediating such interactions during neural development and repair. Synthetic peptides derived from the NOG motif of such proteins can promote neurite sprouting and elongation in cell cultures at levels comparable to whole recombinant proteins by binding to a cleft between the second and third Ig-like domain of L1CAM. This protein is known to be crucial to neuronal differentiation in both mature and developing nervous systems. Several studies suggest that many of its functional interactions are mediated by homodimerization via Ig2 domains. X-linked mutations in the human L1CAM gene cause the L1 syndrome, a severe neurodevelopmental disorder that, in most cases, abrogates L1CAM homodimerization and neurite outgrowth in cell models. In silico simulations provided a molecular rationale for L1 syndrome phenotypes resulting from mutations I179S and R184Q in the NOG region of L1CAM. Simulations allowed the identification of a structural rationale for the severity of the phenotype. Notably, even if the dimerization interface was disrupted upon mutation, simulations proved that the peptide binding pocket on the L1CAM surface is preserved even upon R184Q mutation. Notably, while treatment with recombinant L1CAM ectodomain proved ineffective, NOG peptides could rescue neuritogenesis in a cellular model of the CRASH syndrome. Evidence presented in this thesis describes the role of the NOG motif in neuronal CAMs structure and function to using NOG-derived peptides as biotechnological and therapeutic tools. L1CAM is transported towards the plasma membrane in a process depending on VAMP7, a member of the SNARE and longin families involved in vesicular trafficking. Splicing of VAMP7 results in a total of 7 different protein-coding isoforms. Among the minor isoforms identified so far, VAMP7j is the most expressed in the human brain. Functional and structural evidence suggests VAMP7j lacks a functional SNARE motif but retains both the longin and transmembrane domains. Evidence presented here suggests VAMP7j modulates L1CAM-dependent neurite growth by mediating the transport of L1CAM toward the plasma membrane in a mechanism requiring LRRK1 and dependent on the phosphorylation of the longin domain. The importance of VAMP7 splicing in neuronal maturation was also considered. The Methyl CpG binding protein 2 (MeCP2) has been previously identified to bind the VAMP7 gene, suggesting MeCP2 could be relevant in modulating VAMP7 splicing. A human iPSCs line carrying the MeCP2T158M mutation and its isogenic control was differentiated into mature cortical neurons and harvested for RT-qPCR. qRT-PCR analysis of isogenic controls indicated all but one VAMP7 isoform exhibits a strong upregulation upon cortical differentiation of hiPSCs. When isogenic controls were compared with mutant cell lines, all isoforms stopped showing any modulation over time. Strikingly, VAMP7b and VAMP7j, missing exon 6 and exon 5 and 6, respectively, showed a steep decrease (up to 400%) in expression level upon mutation of MeCP2. Overall, this thesis work identified in the structural features of the NOG region and in the regulation of L1CAM trafficking mediated by VAMP7j, two elements contributing to the regulation of neurite outgrowth. Moreover, an application of NOG-derived peptides was also investigated, and a proof of concept for their use to rescue an L1-syndrome phenotype in a cell model is presented.