Test-tube simulated lipofuscinogenesis. Effect of oxidative stress on autophagocytotic degradation

doi:10.1016/0047-6374(94)01580-F

Mechanisms of Ageing and Development

Volume 81, Issue 1, 30 June 1995, Pages 37-50

https://doi.org/10.1016/0047-6374(94)01580-F Get rights and content

Abstract

Cysteine-stimulated oxidation of a rat liver lysosomal-mitochondrial fraction (LMF) was studied. The process would simulate oxidative stress-related events during the degradation of autophagocytosed material within secondary lysosomes, which may contribute to the formation of lipofuscin or age pigment. Millimolar concentration of cysteine was needed to stimulate LMF lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS). The amount of endogenous LMF iron was 545 μg/l and was enough to initiate peroxidation, probably through the reduction of ferric to ferrous iron by cysteine with induction of Fenton chemistry. Peroxidation could be completely inhibited by the addition of the iron chelator desferal or the antioxidant BHT. A substantial amount of the formed TBARS was associated with trichloroacetic acid (TCA) precipitable proteins. Elevated protein carbonyls was observed 1–2 h after the increase of TBARS. The tryptophan-tyrosine related protein autofluorescence ( $280335 nm$ ) decreased sharply during the first few hours of incubation. In contrast, a lipofuscin-type autofluorescence ( $345430 nm$ ) appeared only after a few days, suggesting that the latter fluorophore is not an immediate product of protein oxidation. The sequential formation of TBARS, protein carbonyls and lipofuscin-type autofluorescence as well as their dependence on iron and a reducing agent add further support to the concept that lipofuscin forms in secondary lysosomes as a result of iron-catalyzed oxidative reactions involving autophagocytosed materials.

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The importance of proper lysosomal activity in cell and tissue homeostasis is underlined by “experiments of nature”, i.e. genetic defects in one of the at least 40 lysosomal enzymes/proteins present in the human cell. The complete lack of 1-4 α-glucosidase (glycogen storage disease type II (GSD II) or Pompe disease) is life-threatening. Patients suffering from GSD II commonly die before the age of 2 years because of cardiorespiratory insufficiency. Striated muscle cells appear to be particularly vulnerable in GSD II. The high cytoplasmic glycogen content in muscle cells most likely gives rise to a high rate of glycogen engulfment by the lysosomes. The polysaccharides become subsequently trapped in these organelles when 1-4 α-glucosidase activity is absent. During the course of the disease, muscle wasting occurs. It is hypothesised that the gradual loss of muscle mass is caused by a combination of disuse atrophy and lipofuscine-mediated apoptosis of myocytes. Moreover, we hypothesise that in the remaining skeletal muscle cells, longitudinal transmission of force is hampered by swollen lysosomes, clustering of non-contractile material and focal regions with degraded contractile proteins, which results in muscle weakness.
Seeing the wood through the trees: A review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence
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The accumulation of lipofuscin (LF)—a polymeric, electron-dense, autofluorescent substance—within postmitotic cells is a characteristic manifestation of aging. It is generally believed that LF is undegradable and formed due to peroxidative alterations of various macromolecules under intralysosomal autophagic degradation. We report here that a short-term exposure of cultured neonatal rat cardiac myocytes to the thiol protease-inhibitor leupeptin, causes an accumulation of numerous electron-dense autophagic lysosomes within the cells. Although very similar to LF by ultrastructure, these inclusions do not display LF-specific, yellow-orange autofluorescence when excited with blue light. Moreover, they rapidly disappear from the cells upon re-establishment of normal culture conditions. In contrast, prolonged leupeptin treatment results in an accumulation of dense lysosomes that also show LF-typical autofluorescence. This autofluorescent material remains in the cells after the end of leupeptin action. The results suggest that: (i) a certain amount of time is needed for autophagocytosed material to become peroxidized, autofluorescent and undegradable, i.e. to acquire properties typical of LF; (ii) protease-inhibition by itself does not lead to LF-formation but rather allows the prolonged time needed for oxidative modification of autophagocytosed material; (iii) mature LF is probably not subjected to either degradation or exocytosis.

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Test-tube simulated lipofuscinogenesis. Effect of oxidative stress on autophagocytotic degradation

Abstract

Free Radic. Biol. Med.

Mech. Ageing Dev.

Mutat. Res.

Free Radic. Biol. Med.

Arch. Biochem. Biophys.

J. Inorg. Biochem.

J. Biol. Chem.

Methods Enzymol.

Biochim. Biophys. Acta

Free Radic. Biol. Med.

Biochim. Biophys. Acta

J. Biol. Chem.

Arch. Biochem. Biophys.

Adv. Free Radic. Biol. Med.

Biochim. Biophys. Acta

J. Biol. Chem.

Mechanisms of lipofuscinogenesis: effect of the inhibition of lysosomal proteinases and lipases under varying concentrations of ambient oxygen in cultured rat neonatal myocardial cells

APMIS

Histochemical indications for lysosomal localization of heavy metals in normal rat brain and liver

J. Histochem. Cytochem.