De Rosa, Laura (2024) Study of the functional cross-talk between the endolysosomal pathway and Synaptojanin 1 (Synj1): impact on neurophysiopathology. [Tesi di dottorato]

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Endosomal trafficking is essential for cellular homeostasis. At the crossroads of exocytic and endocytic fluxes, the endolysosomal system is a critical hub to meet the demands of metabolism, survival and growth by sorting proteins throughout the cell, by modulating cellular signaling and by controlling the cellular proteostasis. Neurons seem to be more sensitive to endosomal perturbations, as evidenced by the increasing number of neurological disorders associated to alterations of the molecular machinery regulating this pathway. Among the molecular components the ubiquitous inositol phosphatase Synaptojanin 1 (Synj1), whose gene maps to chromosome 21 (21q22.11), has a critical role for this cellular hub. Interestingly, loss-of-function mutations of Synj1 are causative of the hereditary form of Parkinson’s disease PARK20, while the excessive expression of Synj1 was observed in the post-mortem brains of individuals with Down syndrome (DS), the leading cause of intellectual disability. Main goal of my PhD project was to study the functional cross-talk among endolysosomal trafficking, Synj1 and neuronal homeostasis. Alterations of endosomal compartments were observed in cells from adult individuals with DS, suggesting that the dysfunction of the endosomal pathway may contribute to DS pathogenesis. However, the nature and the degree of impairment, the timing of onset as well as the link with neuronal dysfunction observed in DS remain elusive. By applying imaging and biochemical approaches, we have shown that the structure and dynamics of early endosomes (EEs), as well as the recycling trafficking, are altered in human fetal trisomic fibroblasts, supporting the involvement of endosomal pathway in DS pathogenesis and indicating that these alterations are present early in development. Moreover, by taking the advantage of novel and efficient techniques to differentiate induced-pluripotent stem cells (iPSCs) into neural precursors (NPCs) we have highlighted that i) the homeostasis and dynamics of EEs is already perturbed at the undifferentiated state and these alterations are exacerbated during neural differentiation with a compromising of EE functions, suggesting that the dysfunction of the endosomal hub may be an early neuropathological mechanism of disease. Remarkably, we have also shown that i) the overexpression of Synj1 in SH-SY5Y cells, a widely used human neuronal cell line, recapitulates the alterations observed in DS cells and ii) the knockdown of Synj1 expression restores the homeostasis of early endosomes in trisomic fibroblasts, altogether demonstrating that such endosomal perturbations are the results of Synj1 upregulation. On the mechanistic view, we determined that the Synj1 overexpression affects the dynamics of early endosomal compartments as evidenced by double immunofluorescence assays and colocalization analyses. Interestingly, comparable results were obtained when the Synj1 expression was knockdown in neural precursors, thus strengthen the role of Synj1 as key player in controlling early endosomal compartments. Altogether these data highlight the detrimental link between Synj1 and endosomal pathway in DS from one side, to the other they also point out a critical role of Synj1 for neuronal cells. To support this, we found that Synj1 expression increases along neural differentiation. In addition, transcriptomic analysis of wild-type and Synj1-overexpresisng SH-SY5Y cells revealed that the major part of deregulated genes are implicated in transmembrane transport and synaptic activity, in neurogenesis, and axon guidance. On the other side, it is worth noting that genes involved in cell-cell adhesion, cell-matrix interaction, all processes critical during neuronal development, were found deregulated upon Synj1 knockdown. Overall, our data highlight that the early alteration of the endosomal pathway may concur to neuropathogenesis of DS and point out that proper levels of Synj1 are critical for neuronal physiology.

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