Journal: International Journal of Alzheimer's Disease

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

doi: 10.4061/2011/695413

Figure Lengend Snippet: SPTLC2 expression is reduced in the presence of functional AICD. (a) Mouse embryonic fibroblasts expressing an APP construct lacking the last 15 amino acids (aa) and therefore a functional AICD domain (MEF APP∆CT15) show increased SPT expression and activity compared to control fibroblasts (MEF WT). (b) MEF APP∆CT15 cells incubated with functional AICD peptide show compared to MEF APP∆CT15 cells incubated with solvent control decreased SPTLC2 expression. (c) MEF APP∆CT15 cells incubated with A β peptides and solvent control showed no difference in SPTLC2 expression. (d) SPTLC2 expression in Fe65 knock-down SH-SY5Y cells is increased.

Article Snippet: To determine the effect of A β 40 (10 ng/mL) and A β 42 (1 ng/mL) (B. Penke, Szeged, Hungary) or AICD (sequence in 1-letter code: KMQQNGYENPTYKFFEQMQN) (2 μ M) (Genscript Corporation, Piscatway, USA) synthetic peptides were incubated for 6 days in cell culture.

Techniques: Expressing, Functional Assay, Construct, Activity Assay, Control, Incubation, Solvent, Knockdown

Journal: Frontiers in Aging Neuroscience

Article Title: APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

doi: 10.3389/fnagi.2015.00077

Figure Lengend Snippet: NEP gene expression and activity is regulated by AICD. (A) Reduced NEP gene expression (23.4 ± 8.1%, p ≤ 0.001), protein level (68.0 ± 4.1%, p ≤ 0.001), activity (55.3 ± 3.4%, p ≤ 0.001) and total Aβ degradation (−Thiorphan: 54.7 ± 2.7%, p ≤ 0.001 compared to wt; +Thiorphan: 68.5 ± 4.1%, p ≤ 0.001 compared to wt; p = 0.02 for effects −Thiorphan vs. +Thiorphan) in MEF expressing an APP construct lacking the last 15 C-terminal amino acids (aa) and therefore a functional AICD domain (MEF APPΔCT15) compared to control fibroblasts. (B) Measurement of FITC-AICD uptake in lysates of MEF APPΔCT15 after short term (2.8 ± 0.1%, p ≤ 0.001, shown in x fold change of fluorescence) and long term incubation with FITC-AICD (18.0 ± 5.1%, p = 0.03, shown in x fold change of fluorescence) compared to cells treated with solvent control. (C) Enhanced NEP gene expression in MEF APPΔCT15 after lipofection based short term (12 h) (143.6 ± 29.8%, p = 0.193 compared to solvent control) and after long term (9 days) (168.0 ± 18.7%, p = 0.002 compared to solvent control) incubation with AICD peptides. Level of NEP gene expression in MEF wt cells (427.0 ± 74.0%, p ≤ 0.001 when compared to MEF APPΔCT15) indicates a partial rescue of NEP gene expression after AICD incubation. (D) Increased NEP activity in MEF APPΔCT15 after lipofection based short term (12 h) (115.8 ± 5.5%, p = 0.014 compared to solvent control) and after long term (9 days) (120.4 ± 4.9%, p = 0.008 compared to solvent control) incubation with AICD peptides. Level of NEP activity in MEF wt cells (180.0 ± 6.12%, p ≤ 0.001 when compared to MEF APPΔCT15) indicates a partial rescue of NEP activity after AICD incubation. (E) Unaltered viabiliy (99.98 ± 0.11%, p = 0.86) and total protein level (101.5 ± 0.87%, p = 0.33) in MEF APPΔCT15 incubated with AICD peptides. ( F) Increase in NEP gene expression (201.9 ± 21.4%, p = 0.009) in MEF APPΔCT15 retransfected with the last 50 C-terminal aa of APP (MEF APPΔCT15 + C50) compared to MEF APPΔCT15. Level of NEP gene expression in MEF wt cells (427.0 ± 74.0%, p ≤ 0.001 when compared to MEF APPΔCT15) indicates a partial rescue of NEP gene expression due to expression of C50. Statistical significance as described for Figure .

Article Snippet: To determine the effects of AICD peptide, MEF APPΔCT15 cells were treated with synthetic AICD peptide (sequence in 1-letter code: KMQQNGYENPTYKFFEQMQN) (Genscript Corporation, Piscatway, USA).

Techniques: Gene Expression, Activity Assay, Expressing, Construct, Functional Assay, Control, Fluorescence, Incubation, Solvent

Journal: Frontiers in Aging Neuroscience

Article Title: APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

doi: 10.3389/fnagi.2015.00077

Figure Lengend Snippet: NEP gene expression and activity is influenced by amyloidogenic, but not by non-amyloidogenic APP processing due to degradation of α-/γ-APP-cleavage derived AICD by IDE. (A) Increased NEP gene expression (139.6 ± 52.3%, p = 0.49) and activity (127.5 ± 3.3%, p ≤ 0.001) in SH-SY5Y cells overexpressing APP carrying the swedish mutation (APPswe) compared to SH-SY5Y APP cells. Level of NEP gene expression in SH-SY5Y wt cells is indicated by a dotted line (p = 0.01 for NEP gene expression in SH-SY5Y APPswe compared to SH-SY5Ywt). (B) Reduction of NEP gene expression (64.0 ± 4.4%, p = 0.0012) and activity (50.0 ± 5.6%, p ≤ 0.001) in MEF PS1/2 deficient cells retransfected with PS1 carrying the T354I mutation (MEF PS1-T354I) compared to MEF PS1/2 deficient cells retransfected with PS1 wt (MEF PS1rescue). Level of NEP gene expression in MEF wt cells is indicated by a dotted line. (C) NEP gene expression and activity in SH-SY5Y overexpressing the APP α-cleaved C-terminal fragment (α-CFT) (RNA-level: 86.9 ± 9.5%, p = 0.241; activity: 100.3 ± 6.0%, p = 0.967) and the APP β-cleaved C-terminal fragment (β-CFT) (RNA-level: 223.5 ± 36.3%, p = 0.027; activity: 192.5 ± 3.9%, p ≤ 0.001). (D) Increased NEP gene expression (157.2 ± 12.2%, p ≤ 0.001) in SH-SY5Y α-CFT cells with reduced IDE expression (SH-SY5Y α-CTF + IDE-KD). (E) Enhanced NEP expression (195.2 ± 6.9%, p ≤ 0.001) in SH-SY5Y α-CTF cells after pharmacological IDE inhibition (SH-SY5Y α-CTF + IDE inhibitor). (F) NEP gene expression in SH-SY5Y wt cells treated with α- (98.3 ± 5.4%, p = 0.764), β - (80.6 ± 3.5%, p = 0.005) or γ- (80.7 ± 0.6%, p ≤ 0.001) secretase inhibitor. (G) Reduction in NEP gene expression (63.0 ± 5.1%, p = 0.002) and activity (71.5 ± 4.0%, p ≤ 0.001) in SH-SY5Y Fe65 knock-down (SH-SY5Y Fe65-KD) compared to mock-transfected control cells. Statistical significance as described for Figure .

Article Snippet: To determine the effects of AICD peptide, MEF APPΔCT15 cells were treated with synthetic AICD peptide (sequence in 1-letter code: KMQQNGYENPTYKFFEQMQN) (Genscript Corporation, Piscatway, USA).

Techniques: Gene Expression, Activity Assay, Derivative Assay, Mutagenesis, Expressing, Inhibition, Knockdown, Transfection, Control

Journal: Frontiers in Aging Neuroscience

Article Title: APP intracellular domain derived from amyloidogenic β- and γ-secretase cleavage regulates neprilysin expression

doi: 10.3389/fnagi.2015.00077

Figure Lengend Snippet: NEP gene expression in brain tissue of transgenic mice. (A) NEP gene expression in brain tissue of APP knock-out (APP −/− ) (81.3 ± 8.5%, p = 0.049), APLP2 knock-out (APLP2 −/− ) (106.2 ± 6.1%, p = 0.317) and in APP/APLP2-double knockout mice (APP/APLP2 −/− ) (83.8 ± 6.1%, p = 0.015). (B) NEP gene expression in mice devoid of the last 15 APP C-terminal aa and hence AICD (APPΔCT15) (75.8 ± 5.7%, p ≤ 0.001). (C) NEP gene expression in APPswe (149.5 ± 19.4%, p = 0.016) and APPswe/PS1ΔE9 (78.6 ± 10.5%, p = 0.058) mouse brains. Statistical significance as described for Figure .

Article Snippet: To determine the effects of AICD peptide, MEF APPΔCT15 cells were treated with synthetic AICD peptide (sequence in 1-letter code: KMQQNGYENPTYKFFEQMQN) (Genscript Corporation, Piscatway, USA).

Techniques: Gene Expression, Transgenic Assay, Knock-Out, Double Knockout