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The role of cathepsin D in apoptosis induced by oxidative stress
Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The lysosomal protease cathepsin D is translocated from lysosomes to the cytosol during apoptosis induced by oxidative stress. In the present studies, the redox-cycling, xenobiotic compound naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) was used to create oxidative stress in rat cardiomyocytes and human foreskin fibroblasts. In naphthazarin exposed cells, lysosomal release of cathepsin D preceded liberation of cytochrome c from mitochondria and a decrease in the mitochondrial transmembrane potential (ΔΨm).

A pre-embedding immunocytochemical method was used for ultrastructural examination of cathepsin D and cytochrome c in cultured cells. Electron microscopic morphometry confirmed that a statistically significant amount of cathepsin D was transferred from lysosome-like structures to the cytosol before any biochemical or morphological signs of apoptosis were detected. Pretreatment of the cells with atocopherol succinate largely prevented translocation of cathepsin D and also significantly decreased apoptosis. Electron microscopy also revealed that, during exposure to naphthazarin, a minor release of cytochrome c has occured after one hour and a more extensive release after two hours, and these results were verified by Western blotting. After the translocation of cathepsin D and cytochrome c, a decrease in ΔΨm was detected using the ΔΨm-sensitive probe JC-1 and confocal microscopy or measured by flow cytornetry. Pretreatment with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria, maintained the ΔΨm and inhibited apoptosis.

In conclusion, these findings show that translocation of cathepsin D precedes important incidents in mitochondria, such as release of cytochrome c and loss of ΔΨm during apoptosis induced by oxidative stress. Moreover, inhibition of cathepsin D prevented the apoptosis and the mitochondrial changes, which indicates that cathepsin D is an inducer of apoptosis upstream of cytochrome c release.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 87 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 688
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28070Local ID: 12834ISBN: 91-7219-979-2 (print)OAI: oai:DiVA.org:liu-28070DiVA: diva2:248621
Public defence
2001-09-28, Berzeliussalen, Hälsouniversitetet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2013-07-08Bibliographically approved
List of papers
1. A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress
Open this publication in new window or tab >>A Pre-embedding Technique for Immunocytochemical Visualization of Cathepsin D in Cultured Cells Subjected to Oxidative Stress
1998 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 46, no 3, 411-418 p.Article in journal (Refereed) Published
Abstract [en]

We describe a pre-embedding immunocytochemical method for visualization of the lysosomal enzyme cathepsin D in cultured cells. The protein was demonstrated at both light and electron microscopic levels by neutral-pH silver enhancement of ultrasmall (0.8-nm) gold particles conjugated to the antibodies. The best morphological preservation and the highest labeling density were achieved by initial fixation for 20 min at 4C in 4% paraformaldehyde (PFA) and 0.05% glutaraldehyde (GA) in 0.15 M sodium cacodylate buffer, followed by permeabilization in sodium borohydride. Three cell types were used: human foreskin fibroblasts, histocytic lymphoma (J-774) cells, and primary rat heart myocytes. In all three, cathepsin D was demonstrated in lysosome-like structures. The rat heart myocytes were also exposed to the redox cycling substance naphthazarin (5,8-dihydroxy-1,4-naph-thoquinone) to induce oxidative stress. This was done for such a short period of time that the cells initially did not show any signs of morphological damage and retained normal plasma membrane stability, although an early and clear redistribution of cathepsin D from membrane-bound structures to the cytosol was apparent. This redistribution was followed by cell degeneration and, eventually, by cell death.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80993 (URN)10.1177/002215549804600316 (DOI)
Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2017-12-07Bibliographically approved
2. Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes
Open this publication in new window or tab >>Oxidative stress causes relocation of the lysosomal enzyme cathepsin D with ensuing apoptosis in neonatal rat cardiomyocytes
1998 (English)In: American Journal of Pathology, ISSN 0002-9440, E-ISSN 1525-2191, Vol. 152, no 5, 1151-1156 p.Article in journal (Refereed) Published
Abstract [en]

Exposing neonatal rat heart myocytes to the redox cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) for 15 to 45 minutes led to a time-dependent release of cathepsin D from many secondary lysosomes to the cytosol, as analyzed by morphometry. Cathepsin D was detected electron microscopically using a pre-embedding immunostaining technique that utilizes antibodies conjugated to ultra-small (0.8-nm) gold particles and subsequent silver enhancement. The exposure to naphthazarin also caused a decrease in both the pH and the ATP level of the cells within the same time frame. Lipid peroxidation was, however, not detected. Pretreatment of the cultures with alpha-tocopherol succinate prevented cathepsin D relocation, as shown by immunofluorescence. After exposure to naphthazarin, cells were washed, and normal culture conditions were re-established for 18 hours. Many cells then showed apoptotic morphology (ie, cellular shrinkage and chromatin condensation) as analyzed by Giemsa staining. Also, 41% of the cells stained positive with the TUNEL technique, and DNA fragmentation was detected by separation of intact and fragmented DNA. Apoptosis was significantly decreased in cultures pretreated with alpha-tocopherol succinate.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80994 (URN)9588882 (PubMedID)
Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2017-12-07Bibliographically approved
3. Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress
Open this publication in new window or tab >>Lysosomal release of Cathepsin D precedes relocation of Cytochrome C and loss of mitochondrial transmembrane potential during apoptosis induced by oxidative stress
1999 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 27, no 11-12, 1228-1237 p.Article in journal (Refereed) Published
Abstract [en]

Apoptosis was induced in human foreskin fibroblasts by the redox-cycling quinone naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Most of the cells displayed ultrastructure typical of apoptosis after 8 h of exposure to naphthazarin. Apoptosis was inhibited in fibroblasts pretreated with the cathepsin D inhibitor pepstatin A. Immunofluorescence analysis of the intracellular distribution of cathepsin D revealed a distinct granular pattern in control cells, whereas cells treated with naphthazarin for 30 min exhibited more diffuse staining that corresponded to release of the enzyme from lysosomes to the cytosol. After 2 h, release of cytochrome c from mitochondria to the cytosol was indicated by immunofluorescence. The membrane-potential–sensitive probe JC-1 and flow cytometry did not detect a permanent decrease in mitochondrial transmembrane potential (ΔΨm) until after 5 h of naphthazarin treatment. Our findings show that, during naphthazarin-induced apoptosis, lysosomal destabilization (measured as release of cathepsin D) precedes release of cytochrome c, loss of ΔΨm, and morphologic alterations. Moreover, apoptosis could be inhibited by pretreatment with pepstatin A.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80995 (URN)10.1016/S0891-5849(99)00146-X (DOI)
Available from: 2012-09-05 Created: 2012-09-05 Last updated: 2017-12-07Bibliographically approved
4. Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy
Open this publication in new window or tab >>Relocalization of Cathepsin D and Cytochrome c Early in Apoptosis Revealed by Immunoelectron Microscopy
2001 (English)In: Laboratory Investigation, ISSN 0023-6837, E-ISSN 1530-0307, Vol. 81, no 2, 149-158 p.Article in journal (Refereed) Published
Abstract [en]

Cathepsin D was translocated from lysosomal structures to the cytosol in primary cultures of neonatal rat cardiomyocytes exposed to oxidative stress, and these cells underwent apoptotic death during subsequent incubation. Temporal aspects of cathepsin D relocalization, cytochrome c release, and decrease in mitochondrial transmembrane potential (Δψm) were studied in myocytes exposed to the redox-cycling xenobiotic naphthazarin (5,8-dihydroxy-1,4-naphthoquinone). Immunofluorescence labeling revealed that cathepsin D was translocated to the cytosol after 30 minutes of naphthazarin treatment, and cytochrome c was released from mitochondria to the cytosol after 2 hours. Western blotting and immunoelectron microscopy indicated a minor release of cytochrome c after only 30 minutes and 1 hour, respectively. Thereafter, a decrease in Δψm was detected using the Δψm-sensitive dye JC-1 and confocal microscopy, and ultrastructural analysis indicated apoptotic morphology. Pretreatment of the cultures with the cathepsin D inhibitor pepstatin A prevented release of cytochrome c from mitochondria and maintained the Δψm. Moreover, ultrastructural examination showed no apoptotic morphology. These findings suggest that lysosomal destabilization (detected as the release of cathepsin D) and release of cytochrome c from mitochondria take place early in apoptosis. Also, the former event probably occurs before the latter during apoptosis induced by oxidative stress because pretreatment with pepstatin A prevented release of cytochrome c and loss of Δψm in cardiomyocytes exposed to naphthazarin.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-27927 (URN)12688 (Local ID)12688 (Archive number)12688 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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