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Ageing-associated changes of lysosomal compartment: implications on cellular functions
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology. Linköping University, Faculty of Health Sciences.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The lysosomal compartment is a major site for intracellular degradation. Lysosomal degradation of the cell’s own constituents, so-called autophagy, not only provides a cell with nutrients, but also removes damaged and potentially dangerous endogenous structures, thus securing intracellular homeostasis. On the other hand, lysosomes have been shown to be involved in the initial stages of apoptosis, and the protective effect of autophagy has been suggested to switch to cell death when excessive.

Ageing-related changes of cellular structures result from damage caused by eactive oxygen species (ROS), which are an inevitable by-product of aerobic life. Intracellular turnover of compromised organelles and macromolecules, to which lysosomal degradation is a major contributor, does not function perfectly, even under favourable conditions. This inherent incompleteness of lysosomal degradation is responsible for the accumulation of a variety of nondegraded and functionally inefficient structures, which can be considered biological “garbage”. Biological “garbage” includes damaged non-degraded macromolecules and organelles, as well as intralysosomal non-degradable polymer-like structure called lipofuscin, or age pigment. Although accumulation of biological “garbage” has been suggested harmful, little is known about the mechanisms of its deleterious effects.

To gain a better understanding of ageing-related changes of the lysosomal compartment and their influence on cell functions, we focused on studying: (1) the role of macroautophagy in the turnover of organelles and lipofuscin formation; (2) the role of biological “garbage” accumulation in the development of ageing-related changes and eventual death of growth-arrested, postmitotic-like cells; (3) the possible cell-protective effect of mitosis; (4) the influence of lipofuscin on cell survival during complete starvation; and (5) the effects of lipofuscin on lysosomal stability.

As a model of induced biological “garbage” accumulation we used confluent human fibroblasts treated with the autophagy inhibitor 3-methyladenine (3MA). Alternatively, lysosomal degradation was suppressed by using the cysteine protease inhibitor leupeptin, or the cathepsin D inhibitor pepstatin A. As a cellular model of aged cells, we used lipofucsin-loaded human fibroblasts. Lipofuscin-loading was achieved by culturing confluent fibroblasts under hyperoxic conditions for 2-4 months. Using these in vitro models, the present study shows that: (1) inhibition of autophagy results in accumulation of lysosome-associated autofluorescent material and mitochondria with low membrane potential; (2) detrimental effect of biological “garbage” accumulation following inhibition of autophagy is prevented by continuous cell division; (3) lipofuscin-loaded cells are more resistant to starvation-induced cell death than control cells; (4) lysosomes of lipofuscinloaded fibroblasts are more resistant to the organelle-targeted stress then lysosomes of control cells.

Based on the results of the present study we conclude that properly operating autophagic machinery plays a crucial role in preventing age-related changes associated with accumulation of biological “garbage”. We also suggest that continual proliferation is the natural mechanism by which cells cope with the accumulation of non-degradable material, employing mechanical dilution during the cell division. Finally, we introduce an idea of lipofuscin being a hormetic agent, and possibly possessing some lysosome-stabilising properties. Better understanding of the influence of the age-related accumulation of biological “garbage” on cellular functions may be helpful for future development of anti-ageing therapy and management of age-associated pathologies.

Place, publisher, year, edition, pages
Institutionen för nervsystem och rörelseorgan , 2007. , 68 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 978
Keyword [en]
Ageing, Apoptosis, Biological gabarge, Hormesis, Lipofuscin, Lysosome
National Category
Pathobiology
Identifiers
URN: urn:nbn:se:liu:diva-8012ISBN: 91-85643-13-0 (print)OAI: oai:DiVA.org:liu-8012DiVA: diva2:22913
Public defence
2007-02-02, Berzeliussalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2007-01-09 Created: 2007-01-09 Last updated: 2017-08-30
List of papers
1. Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material
Open this publication in new window or tab >>Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material
2004 (English)In: European journal of cell biology, ISSN 0171-9335, Vol. 83, no 10, 583-590 p.Article in journal (Refereed) Published
Abstract [en]

Autophagy (which includes macro-, micro-, and chaperone-mediated autophagy) is an important biological mechanism for degradation of damaged/obsolete macromolecules and organelles. Ageing non-dividing cells, however, progressively accumulate oxidised proteins, defective organelles and intralysosomal lipofuscin inclusions, suggesting inherent insufficiency of autophagy. To learn more about the role of macroautophagy in the turnover of organelles and lipofuscin formation, we inhibited autophagic sequestration with 3-methyladenine (3 MA) in growth-arrested human fibroblasts, a classical model of cellular ageing. Such treatment resulted in a dramatic accumulation of altered lysosomes, displaying lipofuscin-like autofluorescence, as well as in a moderate increase of mitochondria with lowered membrane potential. The size of the late endosomal compartment appeared not to be significantly altered following 3 MA exposure. The accumulation of lipofuscin-like material was enhanced when 3 MA administration was combined with hyperoxia. The findings suggest that macroautophagy is essential for normal turnover of lysosomes. This notion is supported by reports in the literature of lysosomal membrane proteins inside lysosomes and/or late endosomes, as well as lysosomes with active hydrolases within autophagosomes following vinblastine-induced block of fusion between lysosomes and autophagosomes. The data also suggest that specific components of lysosomes, such as membranes and proteins, may be direct sources of lipofuscin.

Keyword
Ageing, Autophagocytosis, Fibroblasts, Lysosomes, 3-Methyladenine
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14211 (URN)10.1078/0171-9335-00433 (DOI)
Available from: 2007-01-09 Created: 2007-01-09 Last updated: 2009-06-04
2. Testing the “garbage” accumulation theory of ageing: mitotic activity protects cells from death induced by inhibition of autophagy
Open this publication in new window or tab >>Testing the “garbage” accumulation theory of ageing: mitotic activity protects cells from death induced by inhibition of autophagy
2005 (English)In: Biogerontology, ISSN 1389-5729, Vol. 6, no 1, 39-47 p.Article in journal (Refereed) Published
Abstract [en]

Imperfect autophagic degradation of oxidatively damaged macromolecules and organelles (so-called biological garbage) is considered an important contributor to ageing and consequent death of postmitotic (non-dividing) cells, such as neurons and cardiac myocytes. In contrast, proliferating cells apparently escape senescence by a continuous dilution and repair of damaged structures during division. Postmitotic ageing can be mimicked and studied in cultures of potentially dividing cells if their mitotic activity is inhibited. To test the garbage accumulation theory of ageing, we compared survival of density-dependent growth-arrested (confluent) and proliferating human fibroblasts and astrocytes following inhibition of autophagic sequestration with 3-methyladenine (3MA). Exposure of confluent fibroblast cultures to 3MA for two weeks resulted in a significantly increased proportion of dying cells compared to both untreated confluent cultures and dividing cells with 3MA-inhibited autophagy. Similar results were obtained when autophagic degradation was suppressed by the protease inhibitor leupeptin. In 3MA- or leupeptin-exposed cultures, dying cells were overloaded with undegraded autofluorescent material. The results support a key role of biological lysosomal garbage accumulation in the triggering of ageing and death of postmitotic cells, as well as the anti-ageing role of cell division.

Keyword
ageing, apoptosis, astrocytes, autophagocytosis, fibroblasts, 3-methyladenine, leupeptin, mitosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14212 (URN)10.1007/s10522-004-7382-y (DOI)
Available from: 2007-01-09 Created: 2007-01-09 Last updated: 2009-06-04
3. Increased resistance of lipofuscin-loaded prematurely senescent fibroblasts to starvation-induced programmed cell death
Open this publication in new window or tab >>Increased resistance of lipofuscin-loaded prematurely senescent fibroblasts to starvation-induced programmed cell death
2007 (English)In: Biogerontology (Dordrecht), ISSN 1389-5729, E-ISSN 1573-6768, Vol. 8, no 1, 43-53 p.Article in journal (Refereed) Published
Abstract [en]

Alterations of cellular structures often found in ageing cells is mainly the result of production of reactive oxygen species and a consequence of aerobic life. Both oxidative stress and decreased degradative capacity of lysosomal system cause accumulation of intralysosomal age-related pigment called lipofuscin. To investigate the influence of lipofuscin on cell function, we compared survival of lipofuscin-loaded and control human fibroblasts following complete starvation induced by exposure to phosphate-buffered saline (PBS). Starving of control fibroblasts resulted in lysosomal alkalinisation, relocation of cathepsin D to the cytosol, caspase-3 activation and, finally, cell death, which became evident 72 h after the start of exposure to PBS. Increase of lysosomal pH was significantly less prominent in lipofuscin-loaded cells than in controls and was accompanied neither by leakage of cathepsin D nor by caspase-3 activation even 96 h after the initiation of starvation. Suppression of autophagy by 3-methyladenine (3-MA) accelerated cell death, while inhibition of cathepsin D delayed it, implying an important role of autophagy in cell survival during starvation and showing the involvement of lysosomes in starvation-induced cell death. Disturbed apoptotic response found in lipofuscin-loaded cells can be interpreted as an example of hormesis—an adaptation to low doses of otherwise harmful agents, in this case of lipofuscin, which has a protective effect at moderate amounts but becomes toxic at large quantities.

Keyword
Ageing, Apoptosis, Autophagy, Cathepsin D, Hormesis, Lysosomal pH, 3-Methyladenine, Pepstatin A
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-14213 (URN)10.1007/s10522-006-9029-7 (DOI)
Available from: 2007-01-09 Created: 2007-01-09 Last updated: 2017-12-13
4. Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes
Open this publication in new window or tab >>Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes
Show others...
2008 (English)In: Age (Omaha), ISSN 0161-9152, E-ISSN 1574-4647, Vol. 30, no 1, 31-42 p.Article in journal (Refereed) Published
Abstract [en]

Cellular ageing is associated with accumulation of undegradable intralysosomal material, called lipofuscin. In order to accelerate the lipofuscin-accumulation, confluent, growth arrested human fibroblasts were cultured under hyperoxic conditions. To provide a better insight into the effects of lipofuscin on cellular functions, we compared lysosomal stability in control and lipofuscin-loaded human fibroblasts under conditions of lysosome-targeted stress induced by exposure to either the lysosomotropic detergent MSDH or the redox-cycling quinone naphthazarin. We show that lysosomal damage, assessed by acridine-orange relocation, translocation of cathepsin D to the cytosol, and alkalinization of lysosomes is more pronounced in control than in lipofuscin-loaded fibroblasts. Finding that lysosomal integrity was less affected or even preserved in case of lipofuscin-loaded cells enables us to suggest that lipofuscin exerts lysosome-stabilizing properties.

Place, publisher, year, edition, pages
Springer, 2008
Keyword
alkalinization, autophagolysosomes, bafilomycin A1, cathepsin D, MSDH, naphthazarin quinone
National Category
Physiology Pathobiology Pharmacology and Toxicology
Identifiers
urn:nbn:se:liu:diva-11051 (URN)10.1007/s11357-007-9045-9 (DOI)
Note

Note: Because of an embargo period with Springer journals, the full text will become available at LiU E-Press, January 2009. Original publication: Yuri Stroikin, Hanna Mild, Uno Johansson, Karin Roberg and Karin Öllinger, Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes, 2008, AGE http://dx.doi.org/10.1007/S11357-007-9045-9. Copyright: The original publication is available at http://www.springerlink.com

The previous status of this article was Manuscript and the working title was Lipofuscin preserves lysosome integrity under conditions of organelle-targeted stress.

Available from: 2009-01-12 Created: 2008-04-15 Last updated: 2017-12-13

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