Effect of Different Phospholipids on α-Secretase Activity in the Non-Amyloidogenic Pathway of Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by extracellular accumulation of amyloid-β peptide (Aβ), generated by proteolytic processing of the amyloid precursor protein (APP) by β- and γ-secretase. Aβ generation is inhibited when the initial ectodomain shedding is caused by α-secretase, cleaving APP within the Aβ domain. Therefore, an increase in α-secretase activity is an attractive therapeutic target for AD treatment. APP and the APP-cleaving secretases are all transmembrane proteins, thus local membrane lipid composition is proposed to influence APP processing. Although several studies have focused on γ-secretase, the effect of the membrane lipid microenvironment on α-secretase is poorly understood. In the present study, we systematically investigated the effect of fatty acid (FA) acyl chain length (10:0, 12:0, 14:0, 16:0, 18:0, 20:0, 22:0, 24:0), membrane polar lipid headgroup (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine), saturation grade and the FA double-bond position on α-secretase activity. We found that α-secretase activity is significantly elevated in the presence of FAs with short chain length and in the presence of polyunsaturated FAs, whereas variations in the phospholipid headgroups, as well as the double-bond position, have little or no effect on α-secretase activity. Overall, our study shows that local lipid membrane composition can influence α-secretase activity and might have beneficial effects for AD

Plasmalogens Inhibit APP Processing by Directly Affecting γ-Secretase Activity in Alzheimer's Disease

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD). Previously it has been shown that plasmalogens, the major brain phospholipids, are altered in AD. However, it remained unclear whether plasmalogens themselves are able to modulate amyloid precursor protein (APP) processing or if the reduced plasmalogen level is a consequence of AD. Here we identify the plasmalogens which are altered in human AD postmortem brains and investigate their impact on APP processing resulting in Aβ production. All tested plasmalogen species showed a reduction in γ-secretase activity whereas β- and α-secretase activity mainly remained unchanged. Plasmalogens directly affected γ-secretase activity, protein and RNA level of the secretases were unaffected, pointing towards a direct influence of plasmalogens on γ-secretase activity. Plasmalogens were also able to decrease γ-secretase activity in human postmortem AD brains emphasizing the impact of plasmalogens in AD. In summary our findings show that decreased plasmalogen levels are not only a consequence of AD but that plasmalogens also decrease APP processing by directly affecting γ-secretase activity, resulting in a vicious cycle: Aβ reduces plasmalogen levels and reduced plasmalogen levels directly increase γ-secretase activity leading to an even stronger production of Aβ peptides

Intracellular APP Domain Regulates Serine-Palmitoyl-CoA Transferase Expression and Is Affected in Alzheimer's Disease

Lipids play an important role as risk or protective factors in Alzheimer's disease (AD), a disease biochemically characterized by the accumulation of amyloid beta peptides (Aβ), released by proteolytic processing of the amyloid precursor protein (APP). Changes in sphingolipid metabolism have been associated to the development of AD. The key enzyme in sphingolipid de novo synthesis is serine-palmitoyl-CoA transferase (SPT). In the present study we identified a new physiological function of APP in sphingolipid synthesis. The APP intracellular domain (AICD) was found to decrease the expression of the SPT subunit SPTLC2, the catalytic subunit of the SPT heterodimer, resulting in that decreased SPT activity. AICD function was dependent on Fe65 and SPTLC2 levels are increased in APP knock-in mice missing a functional AICD domain. SPTLC2 levels are also increased in familial and sporadic AD postmortem brains, suggesting that SPT is involved in AD pathology

Evaluation of a health promotion program in children: Study protocol and design of the cluster-randomized Baden-Württemberg primary school study [DRKS-ID: DRKS00000494]

<p>Abstract</p> <p>Background</p> <p>Increasing prevalences of overweight and obesity in children are known problems in industrialized countries. Early prevention is important as overweight and obesity persist over time and are related with health problems later in adulthood. "Komm mit in das gesunde Boot - Grundschule" is a school-based program to promote a healthier lifestyle. Main goals of the intervention are to increase physical activity, decrease the consumption of sugar-sweetened beverages, and to decrease time spent sedentary by promoting active choices for healthy lifestyle. The program to date is distributed by 34 project delivery consultants in the state of Baden-Württemberg and is currently implemented in 427 primary schools. The efficacy of this large scale intervention is examined via the Baden-Württemberg Study.</p> <p>Methods/Design</p> <p>The Baden-Württemberg Study is a prospective, stratified, cluster-randomized, and longitudinal study with two groups (intervention group and control group). Measurements were taken at the beginning of the academic years 2010/2011 and 2011/2012. Efficacy of the intervention is being assessed using three main outcomes: changes in waist circumference, skinfold thickness and 6 minutes run. Stratified cluster-randomization (according to class grade level) was performed for primary schools; pupils, teachers/principals, and parents were investigated. An approximately balanced number of classes in intervention group and control group could be reached by stratified randomization and was maintained at follow-up.</p> <p>Discussion</p> <p>At present, "Komm mit in das Gesunde Boot - Grundschule" is the largest school-based health promotion program in Germany. Comparative objective main outcomes are used for the evaluation of efficacy. Simulations showed sufficient power with the existing sample size. Therefore, the results will show whether the promotion of a healthier lifestyle in primary school children is possible using a relatively low effort within a school-based program involving children, teachers and parents. The research team anticipates that not only efficacy will be proven in this study but also expects many other positive effects of the program.</p> <p>Trial registration</p> <p>German Clinical Trials Register (DRKS), DRKS-ID: DRKS00000494</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

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

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