Naslov
Subcellular Distribution Of APP/C99 And Amyloid-beta Peptide Is Altered In CHO NPC1-null Cells Compared To CHOwt

Autori
Posavec, Melanija ; Omerbašić, Damir ; Goate, Alison ; Hećimović, Silva

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Alzheimer's & Dementia 4 (Suppl 2) / - New York (NY) : Elsevier, 2008, T353-T353

Skup
International Conference on Alzheimer's Disease - ICAD 2008

Mjesto i datum
Chicago (IL), Sjedinjene Američke Države, 26.07.2008. - 31.07.2008

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
Alzheimer's disease; cholesterol; APP protein; Abeta peptide; neurodegeneration

Sažetak
Background: A sphingolipid storage disease (SLSD) Niemann Pick type C (NPC) is caused by dysfunction of NPC1 protein which leads to accumulation of free cholesterol and glycosphingolipids in endosomal/lysosomal compartments. It has been recently shown that this defect leads to increased formation of amyloid-beta (Abeta) peptide, and is accompanied by altered localization of presenilin 1 to early/late endosomes. We hypothesized that cholesterol accumulation upon NPC1 loss of function leads to increased APP/C99 localization in endosomes and increased formation of Abeta in these compartments. To test this we monitored subcellular localization of APP/C99 and Abeta between CHO NPC1-null (M12) and CHOwt cells. Methods: The cells were stably transfected with APPsw-6myc construct. Subcellular distribution of APP processing products and organelle markers was analyzed by subcellular fractionation in an Iodixanol gradient. After centrifugation (20h and 100, 000xg), fractions were collected from the top and protein (DC Protein Assay, BioRad) and cholesterol concentrations (AmplexRed cholesterol assay, Molecular Probes) were determined in each fraction. After acetone precipitation, APP processing products and organelle markers were detected by western blotting using specific antibodies, while Abeta levels were determined by ELISA assay (BioSource International, Inc.). Results: We observed that the majority of subcellular markers tested (BIP/GRP78, EEA1 and Rab7) showed an altered subcellular distribution in M12 cells compared to CHOwt, indicating that loss of NPC1 leads to altered membrane/protein trafficking. In addition, markedly increased levels of BIP/GRP78 were detected in M12 vs. CHOwt cells, which has been previously reported in another SLSD - GM1 gangliosidosis. Although APP was found in similar fractions of M12 and CHOwt cells, the proportion of APP in early/late endosomes was higher in M12 cells. In these cells we also observed increased levels of Abeta40 in endosomal fractions. Conclusion: Our results suggest that increased formation of Abeta in CHO NPC1-null cells is due to redistribution of APP/C99 to early/late endosomes. We propose that NPC1 dysfunction leads to increased coupling of presenilin 1 and its substrate C99 in endosome compartments generating increased Abeta. This work was funded by the grants: NIH-FIRCA 1R03TW007335-01 (to A.G.) and Ministry of Science of the Republic of Croatia 098-0982522-2525 (to S.H.).

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA