Napoli, Barbara (2019) Endoplasmic reticulum homeostasis, lipid droplets biogenesis and autophagy in Drosophila models of Hereditary Spastic Paraplegia. [Ph.D. thesis]
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Abstract (italian or english)
Mutations in the SPG4 gene (Spastin), SPG31 gene (REEP1) and SPG3A gene (Atlastin) are the most common causes of autosomal dominant Hereditary Spastic Paraplegia (HSP), a complex genetic disorder characterized by the axonal degeneration of corticospinal tracts. Interactions between REEP1, Atlastin and Spastin, have a crucial role in modifying ER architecture and lipid metabolism, two important emerging cellular aspects potentially underlying HSP pathological mechanism. The role of lipid droplets (LDs) in HSP has been highlighted by recent evidence that proteins such as Seipin/SPG17, Erlin2/SPG18, Atlastin/SPG3A, Spartin/SPG20, REEP1/SPG31 and Spastin/SPG4 affect cellular LD turnover. Moreover, the authophagy/lysosomes degradative pathway is another important process that crosses LD and ER homeostasis. Different studies have shown that Spastin modulates the endosomal tubule fission and has a crucial role in the tethering of cellular organelles such as LDs, endosomes and peroxisomes. The depletion of spastin alters the ER-endosome contacts, impairs endosomal tubule fission and induces lysosome abnormalities. Furthermore, spastin reduces the LD-peroxisome contacts and affects the fatty acid (FA) trafficking from LDs to peroxisomes. In spite of these findings, the relationship between ER homeostasis and the lipid pathway, with the related implications for neuronal dysfunction in HSP, still remains unknown.
In this work we used the common fruit fly, Drosophila melanogaster, as model organism to perform in vivo studies aimed at describing ReepA function in endoplasmic reticulum homeostasis and morphology and, establishing the role of ReepA and Dspastin in LD biogenesis and autophagy. In order to investigate these pathways, we manipulated the expression of Drosophila REEPA and Spastin by using loss of function alleles and RNA interference approaches. Since immunostaining experiments have shown that loss of ReepA function modifies ER morphology. We investigated the role of ReepA in ER homeostasis, quantifying the mRNA levels of the main genes involved in unfolded protein response (UPR). The results indicate that absence of ReepA triggers a selective activation of the Ire1 and Atf6 branches of UPR. Drosophila lacking ReepA exhibit locomotor dysfunction and shortened lifespan and display a decrease in LD number and size in nerves and muscles phenotypes reminiscent of those caused by Dspastin-RNAi. In order to understand the link between ER homeostasis and LD turnover, we quantified the relative mRNA expression of genes (Mino and Mdy) involved in lipid metabolism. Both HSP models displayed a reduction of Mino and Mdy mRNA levels suggesting a role for Spastin and ReepA in LD biogenesis. Moreover, we investigated the formation of early and late autophagosomes, lysosomes and autolysosomes using fluorescent monomeric tandem constructs that allow the visualization of autophagosomes and lysosomes in vivo. Loss of ReepA and Spastin function impaired the autophagic flux, increasing the number of early/immature and late autophagosomes. Moreover, we showed that loss of DSpastin and ReepA function produces larger lysosomes, consistent with the studies in mammalian models. We also found that naringenin, a flavonoid that possesses strong antioxidant activity and is considered a neuroprotective phytochemical, is able to rescue the cellular phenotypes, the lifespan and locomotor disability associated with loss of ReepA and Dspastin-RNAi. Our data highlight the importance of ER homeostasis in nervous system functionality and in HSP neurodegenerative mechanisms opening new avenues for HSP treatment.
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|EPrint type:||Ph.D. thesis|
|Ph.D. course:||Ciclo 32 > Corsi 32 > SCIENZE FARMACOLOGICHE > FARMACOLOGIA, TOSSICOLOGIA E TERAPIA|
|Data di deposito della tesi:||02 December 2019|
|Anno di Pubblicazione:||02 December 2019|
|Key Words:||Drosophila melanogaster, Endoplasmic Reticulum, Hereditary Spastic Paraplegia, Naringenin, REEP1, ReepA, Spastin, UPR, LDs, Autophagy|
|Settori scientifico-disciplinari MIUR:||Area 05 - Scienze biologiche > BIO/14 Farmacologia|
|Struttura di riferimento:||Dipartimenti > Dipartimento di Scienze del Farmaco|
|Depositato il:||25 Jan 2021 15:16|
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