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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 8, 2004 as doi:10.1096/fj.03-0289fje. |
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* Department of Molecular Pathology,
Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany
2Correspondence: Department of Molecular Pathology, Im Neuenheimer Feld 220, University of Heidelberg, Heidelberg D-69120, Germany. E-mail: juergen_kopitz{at}med.uni-heidelberg.de
SPECIFIC AIMS
Lipofuscin accumulation in the retinal pigment epithelium (RPE) is associated with various blinding retinal diseases including age-related macular degeneration (AMD). The major lipofuscin fluorophore A2-E is thought to play an important pathogenetic role. In previous studies A2-E was shown to severely impair lysosomal function of RPE cells. However, the underlying molecular mechanism remained obscure. Therefore, we investigated whether A2-E exerts its inhibitory effects on lysosomal function by an inhibition of the ATP-driven lysosomal proton pump (V-type H+ATPase), which is in charge of maintaining an acidic milieu within the lysosomal compartment, a prerequisite for the activity of lysosomal hydrolases. Furthermore we examined the consequences of A2-E induced lysosomal dysfunction on phagocytosis of photoreceptor outer segments (POS) and on autophagic turnover of intracellular structures.
PRINCIPAL FINDINGS
1. A2-E inhibits the ATPase activity of the lysosomal proton pump in a dose-dependent manner
When the effect of synthetic A2-E on the ATPase activity of highly purified lysosomes from human RPE cells was measured by determination of the release of [33P]orthophosphate from [
-33P]ATP, a dose-dependent inhibition was observed. Lysosomal ATPase activity was reduced by 
50% at 1µM and by 90% at 2µM A2-E.
2. The ATP-dependent proton translocating activity is inhibited by A2-E
To measure the proton translocating activity in lysosomal membranes the ATP-dependent acidification of highly purified lysosomal vesicles was assayed by the determination of the incorporation of the lysosomotropic compound [methyl-3H]-methylamine into the vesicle. Methylamine accumulation was strikingly reduced by the presence of A2-E, indicating an inhibition of proton translocation to the intravesicular lumen (Fig. 1
).
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3. A2-E-loading of the lysosomal compartment in cultured RPE cells causes reduced capacity for degradation of POS with intracellular accumulation of undegraded POS components and subsequent congestion of the phagocytic process
Degradation of POS by cultured human RPE was assayed by adding iodinated POS to the culture medium and subsequent measurement of low-molecular weight radioactivity released by the cells. POS degradation was strongly reduced in RPE-cells that were loaded with A2-E (Fig. 2
A). Impaired degradation resulted in intracellular accumulation of undegraded POS (Fig. 2B
). Secondary to lysosomal dysfunction and intracellular accumulation of undegraded material, a striking reduction of phagocytosis of labeled POS occurred (Fig. 2C
).
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4. A2-E loaded RPE cells have significantly reduced autophagic sequestration rates
Turnover of intracellular proteins was measured in pulse-chase experiments using 3H-leucine as metabolic label for intracellular proteins. 3-methyladenine acts as a specific inhibitor of autophagic sequestration. Thus, by comparing protein degradation rates during the chase phase in the presence and absence of the specific inhibitor, the sequestration rates are reflected by the 3-methyladenine-sensitive part of total protein degradation. In these experiments A2-E loaded RPE cells had significantly reduced autophagic sequestration rates as compared with normal controls.
CONCLUSIONS AND SIGNIFICANCE
Excessive lipofuscin accumulation in the lysosomal compartment of the retinal pigment epithelium is a characteristic feature of various blinding diseases including AMD, and has been implicated as a common downstream pathogenetic pathway. However, the underlying molecular mechanism of how lipofuscin compounds may interfere with normal RPE cell function has been obscure, mainly due to the lack of characterization of lipofuscin compounds. The identification and molecular characterization of A2-E as the major fluorophore of ocular RPE-lipofuscin represents an important milestone toward a detailed understanding of lipofuscin toxicity, since for the first time a single characteristic compound of ocular lipofuscin has become available for research purposes. Since lipofuscin and its compound A2-E form and accumulate in the lysosomes, this cellular compartment is considered a primary target of the compounds’ potential adverse effects. In the present study we provide evidence that A2-E acts as a potent inhibitor of the lysosomal proton pump (V-type H+ATPase) that physiologically maintains the acidic intralysosomal pH. This result explains former observations of an increase of intralysosomal pH after experimentally induced A2-E accumulation in the lysosomal compartment of cultured RPE cells. The major cellular function of lysosomes is the digestion of intra- and extracellular materials after autophagy, phagocytosis, and endocytosis with the help of some 40 lytic enzymes. These hydrolases require an acidic environment for activity and become inactivated a neutral pH. Therefore, maintenance of an intralysosomal pH around 4.5 is an absolute prerequisite for proper lysosomal function. Consequently, the inhibitory effect of A2-E on the lysosomal proton pump may be considered a key event in its RPE-damaging action.
The severely impaired cellular dysfunction caused by the inhibitory effect of A2-E on the lysosomal proton transporter is demonstrated in the experiments on phagocytosis and degradation of POS, which represents a major function of the RPE to preserve vision. We applied a previously developed test system in which lysosomes of cultured RPE cells were specifically loaded with A2-E. Our results clearly indicate the cells’ capacity to phagocytose and degrade POS is strongly reduced by A2-E-induced lysosomal dysfunction. We suggest that these effects are not the result of a direct interference of A2-E with the phagocytic process, but rather represent a consequence of vast intracellular storage and/or of increasing intralysosomal pH.
Intracellular accumulation of undigested phagocytosed material is known to cause severe cell damage and to promote lipofuscinogenesis. Furthermore, extracellular deposits of undegraded material resulting from reduced phagocytic capacity may trigger further damage, as exemplified by the formation of drusen (i.e., extracellular deposits posterior to the RPE cell monolayer which represent a hallmark of of early AMD).
Inhibition of lysosomal function is also known to affect autophagy, a process for bulk degradation of cytoplasm. In postmitotic cells, including the RPE, this process is a prerequisite for ongoing renewal of aged or damaged organelles or macromolecules. Therefore, defects in autophagy are thought to have deleterious effects, resulting in age-dependent degeneration of postmitotic tissues. In the present study an inhibitory effect of A2-E on autophagy in human RPE cells was demonstrated, suggesting that such pathomechanism may also contribute to development of AMD and related diseases. Autophagy was already shown to contribute to lipofuscinogenesis in RPE.
Our present results in combination with previous studies suggest the intralysosomal accumulation of the retinoid A2-E and subsequent inhibition of the lysosomal V-type H+ATPase as an initial step leading to RPE dysfunction and degeneration. The first consequence of impaired lysosomal acidification would be inactivation of lysosomal hydrolases with subsequent intralysosomal storage of undigested material and promotion of lipofuscin formation. Cellular obstipation, extracellular deposition of undegraded material, reduced autophagic capacity and phototoxicity of lipofuscin cause cell damage by various mechanisms (Fig. 3
). Recently described clinical findings, obtained by confocal scanning laser ophthalmoscopy, are in accordance with such a sequence of events in that excessive lipofuscin accumulations have been shown to precede atrophy of outer retinal layers including the RPE with concurrent severe functional loss. Considering the vital tasks RPE has to serve in order to maintain survival and function of the retinal cell layer in vivo, such RPE damage is likely to be an initial event in the pathogenesis of degenerative retinal diseases, including AMD.
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Further characterization of ocular lipofuscin is necessary to reveal whether the induction of lysosomal dysfunction by the inhibition of the V-type H+ATPase is a unique property of A2-E or, whether it is shared by other lipofuscin compounds.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0289fje; 
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) were compared with untreated controls in the absence (•) or presence (
) of 20 mM ammonium chloride. Results are the means ± SD of 3 independent determinations.