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Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction.

Thellung S, Corsaro A, Villa V, Simi A, Vella S, Pagano A, Florio T - Cell Death Dis (2011)

Bottom Line: Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis.Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis.In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Department of Oncology, Biology and Genetics University of Genova, Genova, Italy.

ABSTRACT
To define the mechanisms by which hPrP90-231 induces cell death, we analyzed its interaction with living cells and monitored its intracellular fate. Treatment of SH-SY5Y cells with fluorescein-5-isothiocyanate (FITC)-conjugated hPrP90-231 caused the accumulation of cytosolic aggregates of the prion protein fragment that increased in number and size in a time-dependent manner. The formation of large intracellular hPrP90-231 aggregates correlated with the activation of apoptosis. hPrP90-231 aggregates occurred within lysotracker-positive vesicles and induced the formation of activated cathepsin D (CD), indicating that hPrP90-231 is partitioned into the endosomal-lysosomal system structures, activating the proteolytic machinery. Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis. Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis. By confocal microscopy analysis of lucifer yellow (LY) intracellular partition, we show that hPrP90-231 accumulation induces lysosome destabilization and loss of lysosomal membrane impermeability. In fact, although control cells evidenced a vesicular pattern of LY fluorescence (index of healthy lysosomes), hPrP90-231-treated cells showed diffuse cytosolic fluorescence, indicating LY diffusion through damaged lysosomes. In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.

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Intracellular β–hPrP90-231 activates endosomal–lysosomal system. (a) β–hPrP90-231-FITC accumulates into endosomal–lysosomal vesicles. Cells were treated with vehicle and β–hPrP90-231FITC (1 μM) for 24 h and counterstained with lysotracker red DND-99, before being observed in live cell confocal microscopy. Cells treated with β–hPrP90-231-FITC show fluorescent round clusters that almost completely colocalize with lysotracker. Vehicle-treated cells were stained with lysotracker red to provide images of lysosomes distribution and size in SH-SY5Y cells. (b) β–hPrP90-231 elicits the expression of active CD. Cells were treated for 2–48 h with hPrP90-231 (1 μM). The amount of active CD (MW 33 kDa) was detected by SDS-PAGE (10%), loading 50 μg of cell proteins/lane. A separate set of samples (10 μg/lane) was probed with the monoclonal anti-α-tubulin to normalize protein content. The expression of 33kDa CD, corresponding to the mature active form, is significantly increased after 2 h, reaches a sustained plateau after 6 h, and lasts up to 48 h of treatment. (c) β–hPrP90-231 treatment causes loss of lysosome membrane impermeability. Cells were treated for 3 days with vehicle (PBS) or β–hPrP90-231 (1 μM). In the last 15 h of treatment, cells were loaded with 100 μg/ml of LY. At 15 min before confocal microscope analysis, cells were loaded with lysotracker red DND-99 (50 nM). Control cells evidenced a vesicular distribution of LY that almost completely overlaps with lysotracker fluorescence, indicating an efficient loading of LY in endosomal–lysosomal vesicles (upper panels). β–hPrP90-231-treated cells exhibited a diffuse LY staining within the cytoplasm outside lysotracker-positive vesicles (lower panel), indicating that LY has been redistributed in the cytosol following the loss of lysosomal membrane impermeability. (Scale bars=10 μm)
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fig7: Intracellular β–hPrP90-231 activates endosomal–lysosomal system. (a) β–hPrP90-231-FITC accumulates into endosomal–lysosomal vesicles. Cells were treated with vehicle and β–hPrP90-231FITC (1 μM) for 24 h and counterstained with lysotracker red DND-99, before being observed in live cell confocal microscopy. Cells treated with β–hPrP90-231-FITC show fluorescent round clusters that almost completely colocalize with lysotracker. Vehicle-treated cells were stained with lysotracker red to provide images of lysosomes distribution and size in SH-SY5Y cells. (b) β–hPrP90-231 elicits the expression of active CD. Cells were treated for 2–48 h with hPrP90-231 (1 μM). The amount of active CD (MW 33 kDa) was detected by SDS-PAGE (10%), loading 50 μg of cell proteins/lane. A separate set of samples (10 μg/lane) was probed with the monoclonal anti-α-tubulin to normalize protein content. The expression of 33kDa CD, corresponding to the mature active form, is significantly increased after 2 h, reaches a sustained plateau after 6 h, and lasts up to 48 h of treatment. (c) β–hPrP90-231 treatment causes loss of lysosome membrane impermeability. Cells were treated for 3 days with vehicle (PBS) or β–hPrP90-231 (1 μM). In the last 15 h of treatment, cells were loaded with 100 μg/ml of LY. At 15 min before confocal microscope analysis, cells were loaded with lysotracker red DND-99 (50 nM). Control cells evidenced a vesicular distribution of LY that almost completely overlaps with lysotracker fluorescence, indicating an efficient loading of LY in endosomal–lysosomal vesicles (upper panels). β–hPrP90-231-treated cells exhibited a diffuse LY staining within the cytoplasm outside lysotracker-positive vesicles (lower panel), indicating that LY has been redistributed in the cytosol following the loss of lysosomal membrane impermeability. (Scale bars=10 μm)

Mentions: SH-SY5Y cells plated into glass-bottom Petri dishes were treated with hPrP90-231-FITC, loaded with lysotracker red DND-99 that stains endosomal–lysosomal vesicles and analyzed by confocal microscopy in live cell imaging. Intracellular hPrP90-231-FITC was detected as fluorescent clusters colocalized with lysotracker (Figure 7a). Lysotracker-positive and hPrP90-231-FITC-loaded vesicles showed larger size than those that do not contain the peptide. To better analyze lysosomal involvement in hPrP90-231 effects, we tested, by immunoblotting, the expression of the active form of the lysosomal enzyme CD. The amount of active CD was greatly increased after 2 h, peaked at 6 h, and was still upregulated after 2 days of hPrP90-231 treatment (Figure 7b), with a significant temporal relationship with intracellular accumulation of hPrP90-231 (Figure 4b). These data provide important evidence that cell proteolytic machinery is activated in response to hPrP90-231 (i.e. accumulation within lysosomes and increase of CD activity), but suggest that it is not sufficient to remove hPrP90-231 from cells.


Human PrP90-231-induced cell death is associated with intracellular accumulation of insoluble and protease-resistant macroaggregates and lysosomal dysfunction.

Thellung S, Corsaro A, Villa V, Simi A, Vella S, Pagano A, Florio T - Cell Death Dis (2011)

Intracellular β–hPrP90-231 activates endosomal–lysosomal system. (a) β–hPrP90-231-FITC accumulates into endosomal–lysosomal vesicles. Cells were treated with vehicle and β–hPrP90-231FITC (1 μM) for 24 h and counterstained with lysotracker red DND-99, before being observed in live cell confocal microscopy. Cells treated with β–hPrP90-231-FITC show fluorescent round clusters that almost completely colocalize with lysotracker. Vehicle-treated cells were stained with lysotracker red to provide images of lysosomes distribution and size in SH-SY5Y cells. (b) β–hPrP90-231 elicits the expression of active CD. Cells were treated for 2–48 h with hPrP90-231 (1 μM). The amount of active CD (MW 33 kDa) was detected by SDS-PAGE (10%), loading 50 μg of cell proteins/lane. A separate set of samples (10 μg/lane) was probed with the monoclonal anti-α-tubulin to normalize protein content. The expression of 33kDa CD, corresponding to the mature active form, is significantly increased after 2 h, reaches a sustained plateau after 6 h, and lasts up to 48 h of treatment. (c) β–hPrP90-231 treatment causes loss of lysosome membrane impermeability. Cells were treated for 3 days with vehicle (PBS) or β–hPrP90-231 (1 μM). In the last 15 h of treatment, cells were loaded with 100 μg/ml of LY. At 15 min before confocal microscope analysis, cells were loaded with lysotracker red DND-99 (50 nM). Control cells evidenced a vesicular distribution of LY that almost completely overlaps with lysotracker fluorescence, indicating an efficient loading of LY in endosomal–lysosomal vesicles (upper panels). β–hPrP90-231-treated cells exhibited a diffuse LY staining within the cytoplasm outside lysotracker-positive vesicles (lower panel), indicating that LY has been redistributed in the cytosol following the loss of lysosomal membrane impermeability. (Scale bars=10 μm)
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fig7: Intracellular β–hPrP90-231 activates endosomal–lysosomal system. (a) β–hPrP90-231-FITC accumulates into endosomal–lysosomal vesicles. Cells were treated with vehicle and β–hPrP90-231FITC (1 μM) for 24 h and counterstained with lysotracker red DND-99, before being observed in live cell confocal microscopy. Cells treated with β–hPrP90-231-FITC show fluorescent round clusters that almost completely colocalize with lysotracker. Vehicle-treated cells were stained with lysotracker red to provide images of lysosomes distribution and size in SH-SY5Y cells. (b) β–hPrP90-231 elicits the expression of active CD. Cells were treated for 2–48 h with hPrP90-231 (1 μM). The amount of active CD (MW 33 kDa) was detected by SDS-PAGE (10%), loading 50 μg of cell proteins/lane. A separate set of samples (10 μg/lane) was probed with the monoclonal anti-α-tubulin to normalize protein content. The expression of 33kDa CD, corresponding to the mature active form, is significantly increased after 2 h, reaches a sustained plateau after 6 h, and lasts up to 48 h of treatment. (c) β–hPrP90-231 treatment causes loss of lysosome membrane impermeability. Cells were treated for 3 days with vehicle (PBS) or β–hPrP90-231 (1 μM). In the last 15 h of treatment, cells were loaded with 100 μg/ml of LY. At 15 min before confocal microscope analysis, cells were loaded with lysotracker red DND-99 (50 nM). Control cells evidenced a vesicular distribution of LY that almost completely overlaps with lysotracker fluorescence, indicating an efficient loading of LY in endosomal–lysosomal vesicles (upper panels). β–hPrP90-231-treated cells exhibited a diffuse LY staining within the cytoplasm outside lysotracker-positive vesicles (lower panel), indicating that LY has been redistributed in the cytosol following the loss of lysosomal membrane impermeability. (Scale bars=10 μm)
Mentions: SH-SY5Y cells plated into glass-bottom Petri dishes were treated with hPrP90-231-FITC, loaded with lysotracker red DND-99 that stains endosomal–lysosomal vesicles and analyzed by confocal microscopy in live cell imaging. Intracellular hPrP90-231-FITC was detected as fluorescent clusters colocalized with lysotracker (Figure 7a). Lysotracker-positive and hPrP90-231-FITC-loaded vesicles showed larger size than those that do not contain the peptide. To better analyze lysosomal involvement in hPrP90-231 effects, we tested, by immunoblotting, the expression of the active form of the lysosomal enzyme CD. The amount of active CD was greatly increased after 2 h, peaked at 6 h, and was still upregulated after 2 days of hPrP90-231 treatment (Figure 7b), with a significant temporal relationship with intracellular accumulation of hPrP90-231 (Figure 4b). These data provide important evidence that cell proteolytic machinery is activated in response to hPrP90-231 (i.e. accumulation within lysosomes and increase of CD activity), but suggest that it is not sufficient to remove hPrP90-231 from cells.

Bottom Line: Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis.Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis.In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmacology, Department of Oncology, Biology and Genetics University of Genova, Genova, Italy.

ABSTRACT
To define the mechanisms by which hPrP90-231 induces cell death, we analyzed its interaction with living cells and monitored its intracellular fate. Treatment of SH-SY5Y cells with fluorescein-5-isothiocyanate (FITC)-conjugated hPrP90-231 caused the accumulation of cytosolic aggregates of the prion protein fragment that increased in number and size in a time-dependent manner. The formation of large intracellular hPrP90-231 aggregates correlated with the activation of apoptosis. hPrP90-231 aggregates occurred within lysotracker-positive vesicles and induced the formation of activated cathepsin D (CD), indicating that hPrP90-231 is partitioned into the endosomal-lysosomal system structures, activating the proteolytic machinery. Remarkably, the inhibition of CD activity significantly reduced hPrP-90-231-dependent apoptosis. Internalized hPrP90-231 forms detergent-insoluble and SDS-stable aggregates, displaying partial resistance to proteolysis. By confocal microscopy analysis of lucifer yellow (LY) intracellular partition, we show that hPrP90-231 accumulation induces lysosome destabilization and loss of lysosomal membrane impermeability. In fact, although control cells evidenced a vesicular pattern of LY fluorescence (index of healthy lysosomes), hPrP90-231-treated cells showed diffuse cytosolic fluorescence, indicating LY diffusion through damaged lysosomes. In conclusion, these data indicate that exogenously added hPrP90-231 forms intralysosomal deposits having features of insoluble, protease-resistant aggregates and could trigger a lysosome-mediated apoptosis by inducing lysosome membrane permeabilization, followed by the release of hydrolytic enzymes.

Show MeSH
Related in: MedlinePlus