engleski

Gamma-Secretase Substrates APP, Notch1 and CD44 Show Distinct Processing in NPC Cells

Background: It has been previously demonstrated that NPC1 dysfunction, that causes Niemann Pick type C disease (NPC), results in altered APP processing leading to increased levels of C99 and amyloid-beta peptide (Abeta). To elucidate whether the Abeta increase upon NPC1 loss of function is due to a common mechanism of altered gamma-secretase processing, we analyzed the levels of Abeta-like peptides and intracellular domains (ICDs) of APP, Notch1 and CD44 in wt (CHOwt) and NPC cells (CHO NPC1-null). Methods: The cells were transiently transfected with C99, Flag-Next or CD44ΔE-Flag constructs (all C-terminal myc tagged). In order to detect beta-peptides, media were collected, cleared and immunoprecipitated using appropriate antibodies. Immunoprecipitates were subjected to SDS-PAGE on Schägger gels and blotted onto nitrocellulose membranes. The membranes were probed with antibody FLAG-M2 (CD44-beta and Notch1-beta) or 6E10 (Abeta). For detection of ICDs, cell lysates were subjected to SDS-PAGE, blotted on PVDF membranes and immunoblotted with antibody 9E10. Results: In contrast to the markedly increased levels of Abeta, we did not observe an increase in Notch-beta and CD44-beta peptides in NPC compared to wt cells. Our observation that AICD levels were similar in wt and NPC cells, suggests that cholesterol accumulation upon NPC1 dysfunction may specifically affect gamma-secretase cleavage at Abeta40/42-site and not gamma-secretase cleavage at the epsilon-site. In addition, the levels of Notch1-ICD (NICD) and CD44-ICD were similar between wt and NPC cells, further supporting the hypothesis that gamma-secretase processing of Notch1 and CD44 is not altered in NPC cells. Conclusion: We found that NPC1 loss specifically affects Abeta levels and not the levels of other gamma-secretase generated Abeta-like peptides. These results suggest that there is not a common mechanism of gamma-secretase cleavage that would result in similar gamma-secretase processing of APP, Notch1 and CD44 in NPC cells. This finding supports a model of distinct processing pathways of gamma-secretase substrates, indicating that designing an Abeta-specific gamma-secretase inhibitor might be feasible. Alternatively, Abeta increase in NPC cells might be due to defective Abeta degradation. This work was funded by the grants: Bilateral project between Germany and Croatia (H.S. and S.H.) and Ministry of Science of the Republic of Croatia 098-0982522-2525 (S.H.).

gamma-secretase; APP; Notch1; CD44; intracellular domains; amyloid-beta like peptides; ICD; Abeta

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