Fig 1: Analysis of the influence of SHANK2 mutations on AKT serine/threonine kinase (AKT) and amyloid precursor protein (APP) expression (7 days of differentiation) (a) and Overview of the putative influence of SHANK2 mutations on tyrosine kinase receptor downstream signaling (b). (a) Western blot analysis was carried out to identify p-AKT levels, as an indicator for tyrosine kinase receptor signaling pathway activity, and APP expression levels. P-AKT and AKT levels were normalized against GAPDH and the ratio between the normalized p-AKT/AKT expression calculated. APP expression was normalized against GAPDH. The mean for both controls (WT and 1F11) was taken for comparison with the SHANK2 mutant lines. The cell line with compound heterozygous SHANK2 mutation (6A4) showed impaired p-AKT levels and APP expression levels. Cropped western blot membrane images are shown and full-length blots are presented in Supplementary Fig. 9. n = 5 experiments, one-way ANOVA (ANOVA results: p-AKT F = 16.65, P = 0.0003; APP F = 25.83, P < 0.0001). Each mutant line was compared against the mean of both controls. ***P ≤ 0.001, correction for multiple testing with Dunnett’s test. Data were presented as box-plots. (b) Scheme illustrating the potential regulation of AKT signaling in the differentiating SH-SY5Y cells downstream of BDNF/TRKB or insulin/insulin receptor signaling. Increased p-AKT levels probably led to reduced apoptosis, increased proliferation and reduced amounts of amyloid precursor protein (APP). Further investigation is needed regarding the ERK and mTOR signaling downstream of AKT, to elucidate an effect from SHANK2 mutations. These pathways have been already linked to ASD pathology61. The part of the signaling cascade which was analyzed in this study is illustrated in green. In this simplified scheme not all different factors which influence AKT and APP expression can be considered. BDNF brain derived neurotropic factor, TRKB tyrosine receptor kinase beta, ERK extracellular-signal regulated kinase, mTOR mammalian target of rapamycin.
Fig 2: Expression of neuronal disease-associated proteins in FtMt clones. (A) Relative levels of amyloid precursor protein (APP) in different FtMt clones. Results shown represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Relative levels of Tau in different FtMt clones. Results shown represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. (C). Relative levels of α-synuclein (α-syn) in different FtMt clones. Results shown represent mean ± SEM of triplicate experiments. Data analyzed by One way ANOVA with Tukey's post-hoc test of significance. Results indicate level of significance. *p < 0.05. (D) Composite Western blot showing pattern of expression for APP, tau, and α-synuclein. Representative western blots of one experiment showing patterns of expression in untransfected and different FtMt clones. Experiment represents the sequential probing of single membrane for APP, tau, and α-synuclein followed by normalization for β-actin.
Fig 3: PSMB5 overexpression increases degradation of APP machinery.(A and B) Immunoblot of 70-day-old Elav-GS-GAL4>UAS-hAPP;UAS-hBACE1 and Elav-GS-GAL4>UAS-hAPP;UAS-hBACE1;UAS-Prosβ5 flies fed with 400 μM RU-486. AD model, N = 12; AD + Prosβ5, N = 12. Values normalized by β-actin. (C and D) Immunoblot of APP levels in SK-N-SH cells transfected with either NSE vector (control) or NSE-PSMB5, N = 3. (E) Images of SK-N-SH cells transfected with APPGFP and either the NSE vector or NSE-PSMB5, N = 6. (F) NSE-PSMB5 mice crossed with hAPP(J20) AD model. (G) Anti-APP immunoblots in 7- to 8-month-old hAPP(J20) ± NSE-PSMB5 mouse brains. APP, N = 9; APP;PSMB5, N = 9. Values normalized by β-actin with quantification (left) and Aβ42 ELISA (right); APP, N = 9; APP;PSMB5, N = 9. (H) X34 staining on brain coronal sections from hAPP(J20) ± NSE-PSMB5 mice, N = 3 per genotype. Number of plaques per hemibrain were quantified using ImageJ software. (I) Postmortem human hippocampal tissue (patients with AD versus age-matched unaffected controls). (J) Postmortem human hippocampal tissue sample lysate, proteasome activity plotted against soluble Aβ ELISA or anti-APP immunoblot (N = 35 in total, 11 controls and 24 patients with AD). (K to O) Postmortem human hippocampal tissue sample lysate, proteasome activity plotted against phosphorylated tau/total tau ratio (L), BACE1 (M), K48-polyubiquitinated (K48-UB) proteins (N), and total polyubiquitinated (poly-UB) proteins (O). N = 23. *P < 0.05 and ***P < 0.001. Significance is based on Student’s t test in (B), (E), (G), and (H). Significance was based on linear regression in (J) to (O). N represents the number of animals, samples, or patients per group.
Fig 4: TAT1-8,9TOD reduces cognitive deficits, proteostatic dysfunction, and abundance of Aβ machinery in hAPP(J20) mice.(A) Treatment of MC65 cells with TAT1-8,9TOD reduces AD-related cell death, based on WST-1 viability assay, N = 24. (B) Proteasome activity in the brain of nontransgenic mice 24 hours after intraperitoneal injection with TAT1-8,9TOD, N = 5. (C) Treatment schematic; 6-month-old hAPP(J20) mice were treated every 2 days for 14 days with intraperitoneal injections of TAT1-8,9TOD (1.26 mg/kg) or vehicle (N = 5). (D) Novel object recognition assay in TAT1-8,9TOD–treated hAPP(J20) mice. (E) Representative immunoblot. (F and G) Immunoblots and ELISA of brain tissue from TAT1-8,9TOD–treated hAPP(J20) against (F) anti-APP, (G) anti-BACE1, (H) Aβ42 ELISA, (I) anti–K48-polyubiquitinated proteins, and (J) anti–K63-polyubiquitinated proteins. Values normalized by β-actin. *P < 0.05 and **P < 0.01. Significance was based on one- or two-way ANOVA in (A) and (B). Student’s t test was used in (D) to (J). N represents the number of animals or samples per group.
Fig 5: Effects of oxysterols on brain pathology and the expression of amyloid precursor protein in the brain. (A) HE staining of the whole brain (n = 3, scale bar: 100 and 20 μm), black arrows: Nuclei pyknosis; (B) number of neurons (/0.05976 mm2) (n = 3, mean ± SEM); (C) density of neurons (cell/mm2) (n = 3, mean ± SEM); (D) APP mRNA (n = 7, median with range); (E) SAA mRNA (n = 7, mean ± SEM); (F) Western blot results of APP and SAA; (G) APP protein (n = 7, mean ± SEM); (H) SAA protein (n = 7, mean ± SEM). *: p < 0.05, **: p < 0.01, ***: p < 0.001.
Supplier Page from Abcam for Anti-Amyloid Precursor Protein antibody [EPR5118-34]