Marion Bergmann

Inhibition of the ATP-driven proton pump in RPE lysosomes by the major lipofuscin fluorophore A2-E may contribute to the pathogenesis of age-related macular degeneration

Lipofuscin accumulation in the retinal pigment epithelium (RPE) is associated with various blinding retinal diseases, including age‐related macular degeneration (AMD). The major lipofuscin fluorophor 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. Using purified lysosomes from RPE cells we now demonstrate that A2‐E is a potent inhibitor of the ATP‐driven proton pump located in the lysosomal membrane. Such inhibition of proton transport to the lysosomal lumen results in an increase of the lysosomal pH with subsequent inhibition of lysosomal hydrolases. An essential task of the lysosomal apparatus of postmitotic RPE for normal photoreceptor function is phagocytosis and degradation of membranous discs shed from photoreceptor outer segments (POS) and of biomolecules from autophagy. When the lysosomes of cultured RPE cells were experimentally loaded with A2‐E, we observed intracellular accumulation of exogenously added POS with subsequent congestion of the phagocytic process. Moreover, the autophagic sequestration of cytoplasmic material was also markedly reduced after A2‐E loading. These data support the hypothesis that A2‐E‐induced lysosomal dysfunction contributes to the pathogenesis of AMD and other retinal diseases associated with excessive lipofuscin accumulation.

How adhesion/growth‐regulatory galectins‐1 and ‐3 attain cell specificity: Case study defining their target on neuroblastoma cells (SK‐N‐MC) and marked affinity regulation by affecting microdomain organization of the membrane

Galectins are potent effectors with conspicuous cell‐type‐specific activity profile. Its occurrence poses the question on the nature of the underlying biochemical determinants, in human SK‐N‐MC neuroblastoma cells involved in negative growth regulation. Since increase of surface presentation of ganglioside GM1 and homodimeric galectin‐1 precedes growth inhibition, a direct interaction is suggested. We thus examined cell binding depending on glucosylceramide synthesis. It was drastically reduced by N‐butyldeoxynojirimycin and threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol, adding decisive evidence for the assumed galectin/ganglioside binding. Glycoproteins do not compensate ganglioside depletion which was verified by measuring lipid‐bound sialic acid. Binding affinity is significantly lowered by disrupting microdomain integrity, also effective for the competitive inhibitor galectin‐3. This was caused by cell treatment with either 2‐hydroxypropyl‐β‐cyclodextrin or filipin III. In this cell system, target specificity and topology of ligand presentation act together to enable high‐affinity binding.

Chromosomal gains and losses in human papillomavirus-associated neoplasia of the lower genital tract – A systematic review and meta-analysis

BACKGROUND Overexpression of the human papillomavirus (HPV) oncogenes E6 and E7 is necessary for the development of distinct lower genital tract cancers. However, secondary cellular genomic alterations are mandatory to promote progression of HPV-induced premalignant stages. We aimed at identifying the chromosomal regions most frequently gained and lost and the disease stage at which the latter occurs. These regions might be relevant for carcinogenesis and could serve as diagnostic markers to identify premalignant lesions with high progression risk towards invasive cancer. METHODS We performed a systematic literature review and meta-analysis of studies listed in PubMed that analysed chromosomal copy number alterations by comparative genomic hybridisation (CGH) in HPV-positive and -negative cancers or premalignant lesions of the anogenital tract (cervix, anus, vagina, penis and vulva). FINDINGS Data were extracted and analysed from 32 studies. The most common alterations in cervical squamous cell carcinoma (SCC) (12 studies, 293 samples) were gains at 3q with a rate of 0.55 (95% confidence interval (CI) 0.43-0.70), losses at 3p (0.36, 95%CI 0.27-0.48) and losses at 11q (0.33, 95%CI 0.26-0.43). Gains at 3q were particularly frequent in HPV16-positive cervical SCC (0.84, 95%CI 0.78-0.90). Also more than one quarter of high grade cervical intraepithelial neoplasia (CIN) harboured gains of 3q (0.27, 95%CI 0.20-0.36), but the rate in low grade CIN was low (0.02, 95%CI 0.00-0.09). For HPV-associated vulvar SCC (four studies, 30 samples) the same common alterations as in cervical SCC were reported. Studies on non-cervical and non-vulvar SCC and premalignant lesions of the lower genital tract are scarce. INTERPRETATION 3q gains were most frequently found in HPV16-positive cervical SCC. The results suggest the selection of HPV-transformed cell clones harbouring 3q gains in high grade premalignant lesions, while alterations in low grade lesions are rare.

Danger Signaling Protein HMGB1 Induces a Distinct Form of Cell Death Accompanied by Formation of Giant Mitochondria

Cells dying by necrosis release the high-mobility group box 1 (HMGB1) protein, which has immunostimulatory effects. However, little is known about the direct actions of extracellular HMGB1 protein on cancer cells. Here, we show that recombinant human HMGB1 (rhHMGB1) exerts strong cytotoxic effects on malignant tumor cells. The rhHMGB1-induced cytotoxicity depends on the presence of mitochondria and leads to fast depletion of mitochondrial DNA, severe damage of the mitochondrial proteome by toxic malondialdehyde adducts, and formation of giant mitochondria. The formation of giant mitochondria is independent of direct nuclear signaling events, because giant mitochondria are also observed in cytoplasts lacking nuclei. Further, the reactive oxygen species scavenger N-acetylcysteine as well as c-Jun NH(2)-terminal kinase blockade inhibited the cytotoxic effect of rhHMGB1. Importantly, glioblastoma cells, but not normal astrocytes, were highly susceptible to rhHMGB1-induced cell death. Systemic treatment with rhHMGB1 results in significant growth inhibition of xenografted tumors in vivo. In summary, rhHMGB1 induces a distinct form of cell death in cancer cells, which differs from the known forms of apoptosis, autophagy, and senescence, possibly representing an important novel mechanism of specialized necrosis. Further, our findings suggest that rhHMGB1 may offer therapeutic applications in treatment of patients with malignant brain tumors.

In-depth mass spectrometric mapping of the human vitreous proteome

Mapping of proteins involved in normal eye functions is a prerequisite to identify pathological changes during eye disease processes. We therefore analysed the proteome of human vitreous by applying in-depth proteomic screening technologies. For ethical reasons human vitreous samples were obtained by vitrectomy from “surrogate normal patients” with epiretinal gliosis that is considered to constitute only negligible pathological vitreoretinal changes. We applied different protein prefractionation strategies including liquid phase isoelectric focussing, 1D SDS gel electrophoresis and a combination of both and compared the number of identified proteins obtained by the respective method. Liquid phase isoelectric focussing followed by SDS gel electrophoresis increased the number of identified proteins by a factor of five compared to the analysis of crude unseparated human vitreous. Depending on the prefractionation method proteins were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a UPLC system coupled online to an LTQ Orbitrap XL mass spectrometer. The obtained mass spectra were searched against the SwissProt database using the Mascot search engine. Bioinformatics tools were used to annotate known biological functions to the detected proteins. Following this strategy we examined the vitreous proteomes of three individuals and identified 1111 unique proteins. Besides structural, transport and binding proteins, we detected 261 proteins with known enzymatic activity, 51 proteases, 35 protease inhibitors, 35 members of complement and coagulation cascades, 15 peptide hormones, 5 growth factors, 11 cytokines, 47 receptors, 30 proteins of visual perception, 91 proteins involved in apoptosis regulation and 265 proteins with signalling activity. This highly complex mixture strikingly differs from the human plasma proteome. Thus human vitreous fluid seems to be a unique body fluid. 262 unique proteins were detected which are present in all three patient samples indicating that these might represent the constitutive protein pattern of human vitreous. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies on pathological vitreous proteome changes.

Lysosomal stress and lipid peroxidation products induce VEGF-121 and VEGF-165 expression in ARPE-19 cells

BackgroundPathologically increased VEGF-A expression is considered a major pathogenic factor in exudative AMD. Since VEGF-A can exist in isoforms with different individual functions, for a detailed understanding of the role of VEGF-A in normal and disease associated processes, in particular in wet AMD, the expression pattern of VEGF-A isoforms has to be taken into account. Therefore in the present study, adressing the effects of lysosomal dysfunction on VEGF expression and secretion by RPE cells induced by lipid peroxidation products and an inhibitor of lysosomal acidification, we applied quantitative methods discriminating the major VEGF-A isoforms.MethodsARPE-19 cells were treated with the primary lipid peroxidation products 4-hydroxynonenal (HNE), malondialdehyde (MDA) or the lysosomal inhibitor bafilomycin A. VEGF-A isoforms were determined by splice-variant-specific RT-PCR. For detection of protein levels, a protein prefractionation strategy based on the strikingly different isoelectric points of VEGF isoforms was used prior to quantification of VEGF-A 121, −165, −189 and −206 expression by ELISA.ResultsOn mRNA level, VEGF-A 165 represents the major isoform (60%), VEGF-A 121 accounts for about one-third, and VEGF-A 189 for about 10% of total mRNA detected in untreated cells. No VEGF-A 206 mRNA was detected. Treatment with bafilomycin A increased VEGF-A 121 and VEGF-A 165 mRNA levels. VEGF-A 189 expression remained unaffected, and no induction of VEGF-A 206 mRNA was detectable. Similar effects were observed when cells were stressed with HNE or MDA. On protein level, bafilomycin A as well as the lipid peroxidation products caused an increase of total VEGF-A protein secretion into the culture medium. In analysis of VEGF-A for different splice variants, only VEGF-A 121 and VEGF-A 165 were detected, the latter representing the major secreted isoform, with the ratio of both isoforms being slightly changed in favour of VEGF-A 165 secretion.ConclusionLysosomal dysfunction and lipid peroxidation damage might be an inducer of VEGF-A 121 and VEGF-A 165 expression in the retina. Furthermore, the novel technique used to analyze the protein expression pattern of VEGF- A isoforms in biological samples may represent a valuable tool in future analyses of specific VEGF-A isoforms in normal and pathogenic functions.

Mechanismen der Hemmung lysosomaler Funktionen im retinalen Pigmentepithel durch den Lipofuszin-Retinoidbestandteil A2-E

ZusammenfassungHintergrund. Die exzessive Akkumulation von Lipofuszin in retinalen Pigmentepithelzellen ist sowohl bei der altersabhängigen Makuladegeneration (AMD) als auch bei verschiedenen erblichen Makuladystrophien von pathogenetischer Bedeutung. Wir haben gezeigt, dass der Lipofuszinfluorophor A2-E (N-Retinyliden-N-Retinyl-Ethanolamin) speziell den lysosomalen Stoffwechsel humaner retinaler RPE-Zellen hemmt und phototoxische Eigenschaften besitzt. Hier wurden neben der vorher gezeigten intralysosomalen pH-Wert-Erhöhung weitere Mechanismen der Hemmung lysosomaler Enzyme untersucht. Methoden. RPE-Zellen wurden durch Potter-Elvejem-Homogenisation aufgeschlossen und in den resultierenden RPE-Zellhomogenaten mithilfe spezieller Enzymtests die Aktivitäten 24 lysosomaler Enzyme vor und nach Inkubation mit A2-E gemessen. Ergebnisse. In humanen RPE-Zellen wurde erstmals ein breites Spektrum lysosomaler Hydrolasen, das Proteasen, Lipasen, Glykosidasen, Nukleasen, Sulfatasen und Phosphatasen umfasst, gemessen. Lysosomale Enzymaktivitäten können hierbei in RPE-Zellen exakt und reproduzierbar bestimmt werden. Selbst hohe Konzentrationen von A2-E (10 μM) zeigten keine hemmende Wirkung auf lysosomale Hydrolasen im RPE. Schlussfolgerung. Mit den hier verwendeten Methoden lassen sich reproduzierbar lysosomale Enzymaktivitäten in humanen RPE-Zellen bestimmen. Die A2-E bedingte Hemmung des lysosomalen Stoffwechsels wird offensichtlich nicht durch eine direkte Enzymhemmung vermittelt. Ursächlich scheint ein intralysosomaler pH-Anstieg mit konsekutivem Verlust der lysosomalen Enzymaktivität zu sein. Für diese pH-Verschiebung könnte eine inhibitorische Wirkung auf die membranständige Protonenpumpe verantwortlich sein. Das genaue Verständnis der Hemmungsmechanismen von A2-E auf die Funktion von RPE-Zellen kann neue Wege der gezielten pharmakologischen Intervention bei Erkrankungen mit exzessiver Lipofuszinakkumulation aufzeigen.AbstractBackground. Excessive accumulation of lipofuscin in the retinal pigment epithelium with age and in various hereditary and degenerative retinal diseases, is of pathogenetic significance. We have shown that the major lipofuscin fluorophor A2-E (N-retinylidene-N-retinylethanolamine) affects the lysosomal degradation of human RPE cells and damages the cellular metabolism by phototoxic properties. Herein we sought to determine mechanisms for the inhibitory effect on lysosomal function apart from pH elevation. Methods. Potter-Elvejem homogenates of RPE cells were used to measure the activity of 24 lysosomal enzymes before and after incubation with A2-E. Results. This is the first time that RPE cells have been screened for a large spectrum of lysosomal hydrolases including proteases, lipidases, glycosidases, nucleases, sulfatases and phosphatases. The activities of these hydrolases were readily detectable in cultured RPE cells. Incubation of RPE cell homogenates even with high A2-E concentrations (up to 10 μM) did not affect the activity of isolated lysosomal enzymes. Conclusions. The results suggest that a direct inhibition of lysosomal enzyme activity would not explain the inhibitory effect on lysosomal degradation. A2-E increases the acidic intralysosomal pH thereby probably hindering pH-dependent lysosomal enzymatic activities. The understanding of the inhibitory effects of A2-E on RPE cell metabolism may contribute to new approaches for treatment of retinal diseases with excessive lipofuscin accumulation such as ARMD or M. Stargardt.

Detergenzienwirkung des Lipofuszin-Retinoidbestandteils A2-E in retinalen Pigmentepithelzellen

ZusammenfassungHintergrund. Lipofuszin akkumuliert in postmitotischen retinalen Pigmentepithelzellen (RPE) mit dem Alter und in Assoziation mit verschiedenen Makula-/Netzhautdegenerationen. Wir konnten zeigen, dass der Lipofuszinretinoid-Bestandteil A2-E (N-Retinyliden-N-Retinyl-Ethanolamin) lysosomale Funktionen hemmt. Um weitere Wirkungsmechanismen für toxische Effekte von A2-E zu evaluieren, untersuchten wir nach Etablierung einer Methode zur Isolation intakter Lysosomen aus RPE-Zellkulturen Detergenzieneffekte auf lysosomale und andere zelluläre Membranen mittels Latenzmessungen. Methoden. Zur Isolation intakter Lysosomen wurde der postnukleäre Überstand humaner RPE-Zellen mittels sequenzieller Dichtegradientenzentrifugation fraktioniert. Die Ruptur der lysosomalen Membran wurde durch Latenzmessungen des lysosomalen Leitenzyms β-Hexosaminidase an mit A2-E inkubierten isolierten Lysosomen erfasst. Latenzen der Markerenzyme Succinatdehydrogenase and UDP-Galactosyltransferase zeigen Effekte des A2-E auf Membranen isolierter Mitochondrien und Mikrosomen. Nach Zugabe von A2-E in das Kulturmedium konnten durch Messung der Laktatdehydrogenase Schäden der Plasmamembran erfasst werden. Ergebnisse. Bis auf eine geringe Restaktivität mitochondrialer Leitenzyme konnte eine 100fache Anreicherung der lysosomalen Fraktion erzielt werden. Eine Kontamination mit anderen Zellfraktionen wurde nicht beobachtet. In geeigneten isotonischen Puffern besitzen die Lysosomen eine ausreichende Stabilität, um mögliche Detergenzieneffekte des A2-E auf lysosomale Membranen nachzuweisen. Bei Konzentrationen über 2 μM A2-E kam es zur Leckage des lysosomalen Leitenzyms. Leckage des mitochondrialen Leitenzyms erfolgte bereits ab 1 μM A2-E, während ER/Golgi-Membranen und Plasmamembranen relativ resistent gegenüber Detergenzieneffekten von A2-E waren. Schlussfolgerung. Die beschriebene Methode zur Isolation intakter Lysosomen aus RPE-Zellen ist auch für zukünftige Untersuchungen für lysosomale Effekte verschiedener Biomoleküle nutzbar. A2-E induziert bereits in niedriger Konzentration eine Desintegration der lysosomalen Membran. Detergenzieneffekte an der mitochondrialen Membran stellen offensichtlich einen weiteren Pathomechanismus dar, wodurch Reaktionskaskaden, die letztlich zum Zelltod führen können, induziert werden. Unsere Beobachtungen sprechen für einen Detergenzieneffekt von A2-E an verschiedenen zellulären Membranen, der bei allen retinalen Erkrankungen mit exzessiver Lipofuszinakkumulation, wie der AMD, von Bedeutung sein könnte. Ein besseres Verständnis dieser molekularen Mechanismen eröffnet neue therapeutische Perspektiven.AbstractPurpose. Several lines of evidence suggest that excessive accumulation of lipofuscin in postmitotic retinal pigment epithelial (RPE) cells with age and in various hereditary retinal diseases, plays a pathogenetic role. The lipofuscin retinoid component A2-E (N-retinylidene-N-retinylethanolamine) inhibits lysosomal degradation. Here we sought to evaluate additional toxic mechanisms of A2-E, whereby possible detergent-like effects on various membranes in human RPE cells were investigated by latency measurements. Methods. A postnuclear supernatant prepared from cultured human RPE cells was used to isolate intact lysosomes by fractionation of cellular organelles in two sequential gradients. Destabilization of the lysosomal membrane was tested by incubating the purified lysosomal fraction in the presence of A2-E and subsequent measurement of the latency of the lysosomal luminal marker β-hexosaminidase. In order to compare the effect of A2-E on other cellular membranes, latencies of the specific markers succinate dehydrogenase and UDP-galactosyltransferase were assessed using partially purified mitochondria and microsomes. Intactness of the plasma membrane was tested by including A2-E in the culture medium before leakage of lactate dehydrogenase into the medium was determined. Results. A more than 100-fold purification of the lysosomal fraction was achieved. Except for a minor activity of the mitochondrial marker, no contamination with other cell fractions was observed. Intactness of the purified lysosomes was well preserved during incubation in isotonic media and provided the basis for investigations on a possible detergent-like action of A2-E on lysosomal integrity. At concentrations above 2 μM A2-E, progressive leakage of the lysosomal marker was observed. In comparison leakage of the mitochondrial marker was induced at significantly lower concentrations (1 μM), whereas ER/Golgi membranes and the plasma membrane were relatively insensitive to a detergent effect of the retinoid. Conclusions. The described practical and fast methodology to obtain highly purified and intact lysosomes from RPE cells, provides a very suitable tool for investigations on compounds affecting the lysosomal structure. The results suggest that A2-E causes disintegration of the lysosomal membrane at relatively low concentrations which may implicate an involvement of such a mechanism in triggering lipofuscin-induced dysfunction of aged RPE in vivo. Secondary to disintegration of the lysosomal membrane, damage to mitochondria might be an additional pathogenic mechanism. Our data provide evidence for surfactant-like properties of A2-E on biomembranes which might be operative in retinal diseases associated with excessive lipofuscin accumulation including age-related macular degeneration.