THE STUDY OF SNAP29 IN MITOSIS AND IN CEDNIK PATHOGENESIS.

Intracellular trafficking includes a series of regulated events that allow the transport of proteins and macromolecules. A key step of intracellular trafficking is the fusion between a containing-cargo vesicle and a target membrane, mediated by Soluble N-ethylmaleimide-sensitive fusion Attachment protein REceptor (SNARE) proteins. Snap29 is a cytosolic SNARE protein containing two SNARE domains required for fusion, whose specificity and activity is unclear. During the last few years, we and others have discovered that Snap29 is a key regulator of autophagy required for fusion of autophagosomes with lysosomes, the last trafficking step before cargo degradation. During the first part of my PhD, I contributed to uncover a novel function of Snap29 using Drosophila melanogaster, as a model system. We demonstrated that during mitosis Snap29 is repurposed as an outer kinetochore component, and that its localization depends on known kinetochore proteins, but does not require membranes or the autophagy process. Depletion of Snap29 in Drosophila S2 cells leads to cell division defects, such as failure to form a proper metaphase plate and segregate chromosomes correctly, or formation of aberrant mitotic spindles, ultimately leading to generation of micronuclei, aneuploidy and cell death. In addition, we observed that Snap29 is fundamental to determine correct tissue development and homeostasis in Drosophila, since its depletion or mutation determines disorganization and multilayering in the follicular epithelium, and tumor-like tissue alterations in eye imaginal discs. Since mutations affecting autophagy genes are not sufficient per se to induce such disruptions in the epithelial architecture, we hypothesize that these defects might be due to loss of Snap29 activity during mitosis. Mutations of SNAP29 human gene cause a rare neurocutaneous syndrome called CEDNIK (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma), which causes severe neurological and dermatological congenital manifestations associated with short life expectancy. So far, the most investigated aspects of this syndrome are dermatological alterations, likely caused by the impairment of SNAP29 activity during membrane trafficking. Other symptoms such as neonatal feeding impairment, muscle hypotonia, and neurological defects were never investigated neither in human patients nor in CEDNIK animal models. To study uncharacterized CEDNIK traits, in the second part of my PhD, we took advantage of an uncharacterized snap29 mutant in zebrafish. The presence of CEDNIK traits in homozygous mutant fish, such as keratoderma and microcephaly, indicated that snap29 zebrafish mutant could be a valid CEDNIK disease model. Importantly, by studying the homozygous fish, we found that they display trigeminal nerve formation and axon branching defects, suggesting the requirement of Snap29 for correct nervous system development. Such alterations correlate with mouth opening problems and swimming difficulties, as well as feeding impairment. In addition, we are currently characterizing defects in muscle fibers organization and angiogenesis and we are assessing whether Snap29 plays a role in autophagy and cell division in vivo. Overall, our findings demonstrate that Snap29 is a key regulator of cell division and shed light on uncharacterized aspects of CEDNIK syndrome, highlighting a pivotal role of Snap29 in nervous system development.