Università degli Studi di Milano, 2017-03-07

Lysosomes are the principal site of the catabolism of sphingolipids, a class of bioactive lipids mainly associated with the external leaflet of cell plasma membrane. Several lines of evidence support a direct correlation between modifications in sphingolipid pattern and the activation of specific signaling pathways, including apoptosis and autophagy. Loss-of-function mutations in genes coding for lysosomal enzymes involved in sphingolipid catabolism result in severe clinical manifestations called sphingolipidoses. These pathologies belong to the group of Lysosomal Storage Diseases and are characterized by the accumulation of undegraded materials leading to lysosomal impairment and consequent cell damage. Until now, the molecular mechanisms by which the perturbation of lysosomal homeostasis affects cell functionality and viability are unknown. To investigate this issue, I used an artificial in vitro model of lysosomal impairment obtained by loading human fibroblasts with 88 mM sucrose for 14 days. In these experimental conditions, the absence of invertase induces sucrose accumulation into lysosomes. I found that sucrose loaded fibroblasts are characterized by a growth slowdown and by the activation of both apoptosis and autophagy. By RNA-sequencing, approximately a thousand of genes were found to be dysregulated after sucrose loading. In particular, 56 cell cycle-related genes are downregulated, whereas 37 lysosomal-related genes are upregulated. Using biochemical approaches, I found that sucrose loading activates lysosomal biogenesis although sucrose storage inhibits lysosomal functionality. In particular, in sucrose loaded cells lipid catabolism is blocked and complex lipids, such as phospholipids, cholesterol, glycosphingolipids, and gangliosides are accumulated. Moreover, I found that sucrose loading induces the nuclear translocation of the Transcription Factor EB (TFEB), a master-gene regulator of lysosomal function, which in turn promotes the increased fusion between lysosomes and the plasma membrane. This last event leads to higher levels of sphingolipid hydrolases at the cell surface resulting in the alteration of the plasma membrane sphingolipid composition and the consequent ectopic production of pro-apoptotic and pro-autophagic ceramide. Interestingly, in sucrose loaded fibroblasts the blocking of glycosphingolipid hydrolysis at the plasma membrane results in a reduction of autophagy and apoptosis. Similar results were also obtained in response to sphingomyelin accumulation in Niemann-Pick Type A disease (NPA). NPA is a sphingolipidosis caused by acid sphingomyelinase deficiency which leads to sphingomyelin storage. Interestingly, using NPA-derived human fibroblasts loaded with 50 µM exogenous sphingomyelin for 30 days, I found that the lysosomal impairment caused by sphingomyelin accumulation activates the same molecular pathways described in healthy fibroblasts subjected to sucrose loading. A pathogenic role of TFEB has also been suggested by biochemical analysis on brains from Acid Sphingomyelinase Knockout (ASMKO) mice. In fact, ASMKO mouse brains are characterized by TFEB nuclear translocation, increased lysosomal biogenesis, increased glycohydrolytic activities and onset of apoptosis and autophagy. Collectively, these data suggest the existence of a cross-talk among lysosomes and the cell plasma membrane. In this context, the lysosomal impairment caused by the accumulation of uncatabolized substrates leads to an altered composition of plasma membrane sphingolipids resulting in the ectopic production of ceramide which in turn is responsible for the onset of cell damage.

diritti: info:eu-repo/semantics/openAccess
In relazione con info:eu-repo/semantics/altIdentifier/hdl/2434/482301
tutor: Sandro Sonnino ; supervisore: Sandro Sonnino ; coordinatore: Sandro Sonnino
Settore BIO/10 - - Biochimica

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD18002726