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Inhibition of autophagy with 3-methyladenine results in impaired turnover of lysosomes and accumulation of lipofuscin-like material
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Experimental Pathology . Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Geriatric . Linköping University, Faculty of Health Sciences.
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.

Place, publisher, year, edition, pages
2004. Vol. 83, no 10, 583-590 p.
Keyword [en]
Ageing, Autophagocytosis, Fibroblasts, Lysosomes, 3-Methyladenine
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-14211DOI: 10.1078/0171-9335-00433OAI: diva2:22909
Available from: 2007-01-09 Created: 2007-01-09 Last updated: 2009-06-04
In thesis
1. Ageing-associated changes of lysosomal compartment: implications on cellular functions
Open this publication in new window or tab >>Ageing-associated changes of lysosomal compartment: implications on cellular functions
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.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 978
Ageing, Apoptosis, Biological gabarge, Hormesis, Lipofuscin, Lysosome
National Category
urn:nbn:se:liu:diva-8012 (URN)91-85643-13-0 (ISBN)
Public defence
2007-02-02, Berzeliussalen, Campus US, Linköpings Universitet, Linköping, 09:00 (English)
Available from: 2007-01-09 Created: 2007-01-09 Last updated: 2009-08-22

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