Molecular mechanisms of APP cleavage products in the regulation of GD3S enzyme activity.

<p>(<i>A</i>) In absence of Aβ peptides <i>a</i>-series ganglioside GM3 binds to GD3S and is converted to the <i>b</i>-series ganglioside GD3. In presence of Aβ, Aβ binds ganglioside GM3, forming an Aβ-GM3 complex. This complex still binds to GD3S, but cannot be converted to GD3. (<i>B</i>) Dual function of Aβ and AICD in GD3S regulation. Aβ reduces enzyme activity of GD3S by forming an Aβ-GM3 complex, resulting in reduced turnover of GM3 to GD3. AICD binds the adaptor protein Fe65 and reduces GD3S gene transcription, which also results in reduced turnover of GM3 to GD3.</p

Effect of GM3 and GD3 on APP processing.

<p>(<i>A</i>) Dose-dependent effect of GM3 on Aβ production: COS7 cells stably transfected with SPC99, representing the β-secretase cleaved C-terminal fragment of APP, show a dose-dependent decrease in Aβ production in presence of GM3. Aβ levels were determined by IP and Western blot (WB) analysis. (<i>B</i>) Dose-dependent effect of GD3 on Aβ generation in SPC99 expressing COS7 cells. GD3 enhances Aβ generation dose-dependently. Aβ levels were determined by IP and WB analysis.</p

Effect of the APP intracellular domain (AICD) on GD3S.

<p>(<i>A</i>) MEF, deficient in full-length APP expressing a truncated APP construct lacking 15 aa from the C-terminus (APPΔCT15) show increased GD3S expression and activity. The level of the GD3S activity of the APP/APLP2 knock-out cells (MEF APP/APLP2-/-) are indicated (horizontal dotted black line). Interestingly the MEF APP/APLP2 knock-out level showed a slight less effect strength compared to the MEF APPΔCT15 cells. However this difference did not reach a significant level and might be due to clonal heterogeneity. (<i>B</i>) Increased GD3S expression in APPΔCT15 mouse brains. (<i>C</i>) GD3S gene expression: AICD peptide partially rescues elevated GD3S gene expression in MEF cells expressing the C-terminal truncated APP. An AICD peptide consisting of the last 20 aa from the APP C-terminus (AICD) is able to decrease GD3S expression. (<i>D</i>) shRNA generated Fe65 knock-down cells show increased GD3S gene transcription.</p

Hypothetic model of the physiological functions of Aβ and AICD in the regulation of GD3-synthase (GD3S) – the enzyme controlling major brain ganglioside composition.

<p>(<i>A</i>) Amyloidogenic proteolytic processing of the Alzheimer's amyloid precursor protein (APP) releases amyloid-beta peptides (Aβ) and the intracellular domain of APP (AICD). Aβ and AICD inhibit GD3S, resulting in reduced conversion of <i>a</i>- to <i>b</i>-series gangliosides. As a consequence of reduced conversion of <i>a</i>- to <i>b</i>-series gangliosides, GM3 increases whereas GD3 decreases. In return, both gangliosides, GM3 and GD3, themselves regulate the proteolytic cleavage of APP. The <i>b</i>-series ganglioside GD3 increases, whereas the <i>a</i>-series gangliosides GM3 decreases amyloidogenic proteolytic processing of APP.</p

Influence of Aβ on GD3S activity.

<p>(<i>A</i>) Effect of Aβ on GD3S activity. MEF lacking Aβ because of PS- or APP-deficiency (MEF PS1/2-/- and MEF APP/APLP2-/-, respectively) were incubated with physiological Aβ40 or Aβ42 concentrations or the solvent used for the Aβ peptides (control). The level of the GD3S activity of the corresponding wildtype cells are indicated (horizontal dotted black line); corresponding wildtype/control cells for MEF PS1/2-/-: PS1/2 deficient MEF retransfected with PS1 (MEF PS1r); corresponding wildtype/control cells for MEF APP/APLP2: mouse embryonic fibroblasts of wildtype mice (MEF wt). Both Aβ species partially rescued the increased GD3S activity in PS- or APP-deficient MEF. No significant differences were observed between Aβ40 and Aβ42 peptides. (<i>B</i>) Direct effect of Aβ40 and Aβ42 on GD3S activity. Homogenates of PS1/2-/- cells incubated with Aβ40 and Aβ42 show decreased GD3S activity. Similar results are obtained with Aβ40 and Aβ42 in a cell-free assay containing only purified GD3S and the substrate GM3. (<i>C</i>) Influence of aggregated Aβ and inverted Aβ on GD3S activity. Inverted Aβ peptides and aggregated Aβ showed no influence on GD3S activity. (<i>D</i>) GM3, the substrate for GD3S, binds to Aβ: Physiological concentrations of cellular derived Aβ bind GM3 analyzed by co-immunoprecipitation (co-IP) of Aβ in presence of GD3 or GM3. GM3, but not GD3, binds to Aβ. After IP, Aβ bound gangliosides were detected via TLC. GM3 binds to synthetic Aβ40 and Aβ42 (shown for equimolar concentrations), and cellular derived Aβ40 and Aβ42 (shown for physiological concentrations, approx. 10∶1 ratio). Thin black vertical lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034095#pone-0034095-g002" target="_blank">Fig. 2D</a> left) indicate that the TLC plates were scraped to separate lines. (<i>E</i>) Co-IP of Aβ and GD3S in dependence of GM3. Aβ only binds to GD3S in presence of the substrate GM3. Data are represented as mean +/− SEM.</p