J Korean Med Sci.  2010 Oct;25(10):1492-1498. 10.3346/jkms.2010.25.10.1492.

GD3 Accumulation in Cell Surface Lipid Rafts Prior to Mitochondrial Targeting Contributes to Amyloid-beta-induced Apoptosis

• Kim JK
• Kim SH
• Cho HY
• Shin HS
• Sung HR
• Jung JR
• Quan ML
• Jiang DH
• Bae HR

• KMID: 2157855
• DOI: http://doi.org/10.3346/jkms.2010.25.10.1492

Abstract

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.

Keyword

Amyloid beta-peptide; GD3; Apoptosis; Trafficking; Lipid Rafts; Methyl-beta-cyclodextrin

MeSH Terms

Amyloid beta-Peptides/*pharmacology
*Apoptosis
Cell Line
Gangliosides/*metabolism/physiology
Humans
Membrane Microdomains/*metabolism
Mitochondria/*metabolism
Sialyltransferases/genetics/metabolism
beta-Cyclodextrins/pharmacology

Figure

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  Fig. 1 Amyloid β-induced apoptosis. TE671 cells were treated with GD3 (100 µM), Aβ (40 µM) or H2O2 (0.5 mg/mL) for 24 hr. Apoptotic cell death was quantified by flow cytometry analysis using PI alone (A) or with annexin V-FITC (B). Results are given as the mean±S.D. of four individual experiments (C). *P<0.01.

• 

  Fig. 2 Mitochondrial involvement in amyloid β-induced apoptosis. (A) TE671 cells were exposed to GD3 (100 µM), Aβ (40 µM) or H2O2 (0.5 mg/mL) for 8 hr. Dissipation of mitochondrial membrane potential was assessed by shift of JC-1 aggregates from red to green fluorescence using flow cytometry. Numbers represent percent of cells in the lower right quadrant. (B) Mitochondrial integrity was evaluated using Mito Tracker Red after 24 hr of Aβ treatments. (C) Caspase 9 and PARP activation after Aβ treatment were assessed by western blotting using cells lysates.

• 

  Fig. 3 GD3 synthesis and trafficking during amyloid β-induced apoptosis. (A) TE671 cells were treated with either Aβ (40 µM) or H2O2 (0.5 mg/mL) for 16 hr. Intracellular GD3 levels were measured by thin layer chromatography immunostaining using the crude ganglioside cell extracts. (B) Expression of GD3 synthase after 4 hr of Aβ treatment was analyzed by real-time quantitative RT-PCR using GAPDH as an internal control. Results are given as the mean±S.D. of four individual experiments. *P<0.01. (C) Localization of intracellular GD3 after Aβ treatment was evaluated by double immunofluorescence staining technique with FITC-labeled anti-GD3 and Texas Red-labeled anti-caveolin-1 antibodies. (D) Cells were loaded with Mito Tracker Red for 30 min before fixation and then labeled with FITC-conjugated anti-GD3 antibody. (E) Cell surface GD3 levels after 4 hr of Aβ treatment were analyzed by flow cytometry using FITC-labeled anti-GD3 antibody.

• 

  Fig. 4 Blockage of amyloid β-induced apoptosis by preventing cell surface GD3 accumulation and trafficking. (A) TE671 cells were pretreated with methyl-β-cyclodextrin (0.5 mg/mL) for 1 hr and then exposed to Aβ (40 µM) for 4 hr. Localization of intracellular GD3 was evaluated by double immunofluorescence staining technique with FITC-labeled anti-GD3 and Texas Red-labeled anti-caveolin-1 antibodies. (B) Cell surface GD3 levels after 4 hr of Aβ treatment were analyzed by flow cytometry using FITC-labeled anti-GD3 antibody. (C) Apoptotic cell death was quantified after 24 hr of Aβ treatment by flow cytometry analysis using PI. Results are given as the mean±S.D. of four individual experiments. *P<0.05.

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