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First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02473

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Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates

¹ Department of Biochemical Sciences
² Department of Anatomy, Histology and Forensic Medicine
³ Center of Excellence for Molecular and Clinical Studies in Chronic, Inflammatory, Degenerative and Tumoural Diseases for the Development of New Therapies
⁴ Interuniversity Centre for the Study of the Molecular Basis of Neurodegenerative Diseases, University of Florence, Florence, 50134, Italy

View larger version (17K): [in a new window]   Fig. 1. Various cell lines are differently affected by pre-fibrillar aggregates. Cell viability was checked by the MTT test, after supplementing cell culture media with 2.0 µM HypF-N pre-fibrillar aggregates. Values are relative to control cells treated with the soluble HypF-N (means ±s.d.). The inset shows the percentage of cell death in the most susceptible cell type (Hend) exposed to varying amounts of fibrillar (F) or pre-fibrillar (G) aggregates. Hend cells were pre-treated to enrich the cholesterol content of the plasma membrane before exposure to pre-fibrillar aggregates (G+cholesterol). For technical details see under Materials and Methods. The reported values are representative of six independent experiments, each performed in duplicate. *P0.05, significant difference vs cells exposed to the soluble HypF-N. **P0.05, significant difference vs not pre-treated cells exposed to the HypF-N pre-fibrillar aggregates.

View larger version (10K): [in a new window]   Fig. 2. Time-course of HypF-N binding to HeLa and Hend cells (either normal or enriched in membrane cholesterol). Both cell lines were exposed for 5, 10, 15, 20, 30, 60 minutes to 2.0 µM HypF-N pre-fibrillar aggregates and then washed twice with PBS. The residual aggregate-cell complex was stained with 2.0 µM Congo Red for 20 minutes. Under these conditions, Congo Red-staining is a measure of the amount of prefibrillar aggregates adsorbed to cell membrane surface. Binding of HypF-N aggregates to Hend cell membranes was highly reduced following supplementation of the culture media with 0.5 mM water-soluble cholesterol.

View larger version (16K): [in a new window]   Fig. 3. Correlation between susceptibility and total antioxidant capacity. Basal TAC was correlated with cell viability (as determined by the MTT test) in cells exposed to 2.0 µM aggregated HypF-N for 24 hours. TAC values are expressed as pmol of ABTS+ produced per mg of protein. For technical details see under Materials and Methods. TAC values are the means of three experiments carried out in duplicate.

View larger version (11K): [in a new window]   Fig. 4. Time course of ROS accumulation. Confocal microscopy analysis of the redox status of two different cell lines exposed for 15, 30, 60, 180 minutes to 2.0 µM HypF-N pre-fibrillar aggregates. ROS production was checked by incubating the exposed cells, for 10 minutes, in the presence of the redox fluorescent probe CM-H2 DCFA. For technical details see under Materials and Methods. The values shown are means of three experiments carried out in duplicate.

View larger version (8K): [in a new window]   Fig. 5. Changes of intracellular ROS and free Ca2+ levels in Hend cells checked by confocal analysis. Cells were exposed for 15, 30, 60, 180 minutes and 24 hours to 2.0 µM HypF-N pre-fibrillar aggregates and then fixed with 2.0% paraformaldehyde. ROS were obtained by incubating for 10 minutes the exposed cells in the presence of the redox fluorescent probe and by measuring the fluorescence of CM-H2 DCFA. Intracellular free Ca2+ levels were revealed by incubating the exposed cells for 15 minutes in the presence of the fluorescent dye Fluo-3AM. The data are reported as percentages of the values determined at time 0 and are expressed as means ±s.d. of four experiments each carried out in duplicate. For technical details see under Materials and Methods.

View larger version (48K): [in a new window]   Fig. 6. Continuous confocal microscopy analysis of intracellular free Ca2+ levels in unfixed Hend and HeLa cells. Intracellular free Ca2+ levels were imaged by confocal microscopy using the fluorescent dye Fluo-3AM as a probe as described under Materials and Methods. Cells were exposed for 20 minutes to 2.0 µM HypF-N aggregates. Fluorescence is expressed as fractional change above the resting baseline, F/F, where F is the average baseline fluorescence before aggregate exposure and F is the fluorescence change over the baseline. The values shown are means ±s.d. of three independent experiments each carried out in triplicate. Bar, 30 µm.