Internalização e tráfego de PrPs

Abstract

Cellular prion protein (PrPsen ou PrPc) is expressed in a wide variety of cells, specialy neurons. This protein is rich in alpha helix structures, but it can assume an alternative conformational isoform known as PrPres (or PrPsc). PrPres presents mainly beta sheet structure and gains the ability to form aggregates as well as is protease resistant. PrPres is able to propagate by converting PrPsen molecules to the alternative conformation isoform PrPres in a new biologic mechanism, not yet fully understood. Then, PrPres molecules form aggregates that accumulates in the brain causing Prion Diseases, a group of neurodegenerative disorders very rare and fatal. PrPsen and PrPres internalization and subsequent intracellular trafficking may give important sights about the physiological role of PrPsen and the mechanisms of PrPres infection and propagation between cells. In order to pursue these mechanisms, we used a GFP-tagged version of the mouse cellular prion protein. This construction allowed us to check PrPsen distribution and follow its endocytosis and traffic in a living neuronal cell line, SN56. SN56 cells expressing GFP-PrPc showed fluorescence signal distributed at theplasma membrane and in intracellular compartments localized close to the nucleus, similarly to endogenous protein. In response to copper treatment, GFP-PrPc was internalized and directed to a perinuclear region, suggesting that Cu2+ may have an important role in PrPsen physiology. We also examined the steady state distribution of aN-terminal deletion mutant of PrPsen (Ä32 121) tagged with GFP. This mutant showed an accumulation at the plasma membrane and weak labeling of the perinuclear region suggesting an endocytosis deficiency. This mutant did not show internalization after copper treatment suggesting that N-terminal region may be involved with PrPsenendocytosis. Organelle specific markers suggest that the perinuclear region labeled with GFP-PrPc is composed of recycling endosomes and Golgi. We also evaluate the mechanism involved in GFP-PrPc endocytosis and our data suggests that dynamin I is important to both, constitutive and copper induced GFP-PrPc endocytosis. Cells expressing a C-terminal fragment of AP-180, a protein that recruits clathrin to the plasma membrane, showed 75% inhibition of clathrin dependentendocytosis. In these conditions, constitutive endocytosis of GFP-PrPc was not altered and the copper induced internalization showed a partial inhibition. These data suggest xvi that GFP-PrPc may present a complex endocytosis mechanism involving clathrin dependent and independent pathways. The clathrin independent mechanism does not involve caveolae, since we failed to detect caveolin, a caveolae marker, in SN56 cells. We also examined PrPres internalization and intracellular trafficking by labeling several PrPres strains with the fluorescent dye alexa568. SN56 cells were able to take up Chandler PrPres from aggregates and distribute this fluorescent protein in vesicles inside the cell body and neuritis. However, those cells were not able to internalize PrPres of other strains, like 263K and 87V, with the same efficiency seen for ChandlerPrPres. Moreover, cells treated with Chandler PrPres showed formation of new PrPres molecules, suggesting the establishment of a sustained infection, while cells treated with either 263K or 87V PrPres did not show PrPres formation. These observations suggest that uptake and intracellular trafficking of PrPres coincide with sustained infection inSN56 neuronal cells. Thus, the mechanisms and pathways taken by PrPres may be important to the establishment of a sustained infection.Chandler PrPres positive vesicles spread in the cell body and neuritis co-localized with late endosomes and lysosomes markers (dextran and lysotracker) and failed to colocalize with markers of clathrin dependent endocytosis (transferrin) or lipid raft endocytosis (cholera toxin), suggesting that those vesicles are directed to acidic organelles after internalization probably using a clathrin and raft independentmechanism. Chandler PrPres positive vesicles did not colocalize with GFP-VAChT, a marker for sinaptic vesicles, in neuritis or cell body.Overexpression of a constitutively active GTP-bound Rab7 fused to GFP (GFPRab7 Q67L) showed that Chandler PrPres localizes in late endosomes and lysosomes in the cell body and neuritis. Expression of Rab7 and mutants in another cell line persistently infected with PrPres (N2a) showed alteration of new PrPres formation. The results with infected SN56 and N2a expressing Rab7 mutants suggest that trafficking in late endosomes and lysosomes may play an important role in sustained PrPres formation.