Figure 1.
Identification of biomarkers in the CSF of AD cases.
For the study, CSF was collected by lumbar puncture (n = 4 per group), centrifuged (1,300×g) at 4°C, aliquoted and stored at −80°C. 4 µg of CSF proteins from each case was analyzed by SELDI technology using the IMAC-Cu++ protein chip; only Cu++-binding proteins are analyzed using this chip. A, SELDI retention map; “peaks” represent individual detected proteins, and the area under the peak represents the signal intensity. Reference molecular sizes are indicated across the bottom of the panel. B, molecular weight frequency scatter graph indicating the quantitative distributions of individual Cu++-binding protein for each of the cases analyzed. Peak #3 shows the levels of expression of the 11.7-kDa species in the AD dementia and normal cognitive controls. C, western blot confirmation of elevated S100A7 contents in the CSF of AD cases. Bar graph represents mean±SEM and is shown as % of the control group; *P<0.05, by 2 way t-test, n = 4–7 per group. Inset: representative S100A7 western blot analysis. D. Immunoadsorption with the S100A7 antibody showed no visible bands in a western blot.
Figure 2.
Selective elevation of S100A7 protein in the CSF of AD cases relative to neurologically normal control cases or PD cases.
Monoclonal anti-human S100A7 antibody was used in a single antibody ELISA assay to quantify the content of S100A7 immunoreactivity in the CSF of non-medicated AD (n = 49), Parkinson's disease (n = 21), and neurological normal control (n = 10) cases. In this study, microtiter wells were coated with the antigen (0.5 µl CSF diluted 200-fold), dissolved in 100 µl of Buffer 42 (30 mM NaHCO3, 70 mM Na2CO3, 0.05% NaN3, pH 9.6), and incubated at 4°C overnight. Data are shown as a scatter plot. *P<0.05 vs. neurological controls or PD cases by one-way ANOVA.
Figure 3.
S100A7 mRNA expression in AD brain increases as a function AD dementia and amyloid neuropathology.
A, S100A7 content in BM 8 (quantified by real-time RT-PCR and normalized by cyclophilin) as a function of CDR representing cognitive normalcy (CDR 0), questionable dementia (CDR 0.5), mild dementia (CDR 1), and severe dementia (CDR 5). Data are expressed as mean±SEM and are shown as percent of CDR 0 group. B, C, S100A7 mRNA expression as a function of β-amyloid NP plaque and NFTs neuropathology, respectively, in accordance with the CERAD four-point scale for AD. Data represent mean±SEM and are shown as percent relative to CERAD 0 group, respectively; in A–C statistics were calculated by two-way ANOVA followed by Dunnett's t-test vs. control; *P<0.05. The number inside each bar indicates number of cases analyzed.
Figure 4.
S100A7 expression attenuates β-amyloid peptide generation in cultured CHO-APPswe cells.
Cells were infected with adeno human h-S100A7 virus or lacZ virus for 24 or 48 hours at 10 multiplicities of infection (MOI = 10). A, Amyloid-beta1–42 peptide content in the condition media was analyzed using a commercial ELISA assay. B, western blot analysis of soluble (s)APPα (6E10 antibody/total sAPP content (22C11 antibody) in the culture media. C, α-secretase activity of cell lysate assessed based on monitoring cleavage of a specific synthetic α-secretase peptide substrate using an α-secretase activity kit following manufacturer's instructions (R&D Systems). Inset, representative western blot analysis of h-S100A7 in culture media confirming the secretory feature of S100A7; Mature ADAM-10 (D) and mature ADAM-9 or ADAM-17 (TACE) (E) were assessed by western blot analysis. Insets, representative western blot analysis of precursor and mature ADAM-10 (D) and ADAM-9 or ADAM-17 (E). A–C, values represent mean±SEM and are shown as percent of LacZ control infected cells; n = 3 independent cultures per group from two independent studies; *P<0.05 by two-way t-test.
Figure 5.
Adenovirus-mediated overexpression of hr-S100A7 modulated β- amyloid generation in primary Tg2576 cortical neurons cultures.
Primary cortical neuronal cultures generated from hemizygote E14 Tg2576 mouse embryos were maintained in a serum-free Neurobasal media in the presence of B27-supplement (GIBCO) essentially as previously described [42]. Seven-day-old Tg2576 neuronal cultures were infected with adeno-S100A7 virus (or adeno-LacZ virus as control) with a dose of virus at 10 MOI. (A) Aβ1–40 and Aβ1–42 (B) peptide contents in the condition media 48 hrs after viral infection was analyzed using commercial ELISA assays. C, α-secretase activity of neuronal cell lysate using the α-secretase activity kit (R&D Systems).
Figure 6.
h-S100A7 adenoviral infection in primary cortico-hippocampal neuron cultures derived from Tg 2576 embryos coincides with activation of p42/44MAPK and PKC signaling.
A,B, cortico-hippocampal neuron cultures derived from Tg2576 embryos were infected with LacZ or h-S100A7 adenovirus for 24 hours at 10 MOI. The phosphorylation of p42/44 MAPK and PKC were analyzed in cell lysate and detected by pTpY185/187-p42/44 MAPK antibody or total p42/44 MAPK antibody (A) and phosphor-PKC (pan) antibody or PKCα antibody (B). In this study, tissue-culture dishes were placed on ice (4°C) and, following the removal of conditioned media, cells were immediately collected in ice-cold 1× SDS-sample buffer containing protease inhibitors. Data represent mean±SEM of determinations made in three separate cultures; n = 3 to 4 per culture; two-tailed Dunnett's t-test vs. LacZ infection: *P<0.05, two-way t-test.
Figure 7.
h-S100A7-mediated promotion of α-secretase activity and sAPPα secretion and decreased amyloid-beta1–42 level are prevented by p42/44MAPK and PKC signal-transduction inhibitors.
In this study cortico-hippocampal neuron cultures derived from Tg2576 embryos were pre-incubated for 30 min with vehicle alone or with PD98059 (10 µM) (A–C) or with GF109203X (5 µM) (D–F). Following the preincubation time, the cells were infected with LacZ or h-S100A7 adenovirus for 24 h at 10 MOI. A, resulting cell lysates were analyzed for α-secretase activity. B, C, proteins released into the conditioned media were collected and analyzed for β-amyloid by ELISA or sAPP α and total sAPP by western blotting. Data represent mean±SEM of determinations made in two separate cultures; n = 3 to per culture; two-tailed Dunnett's t-test vs. control (vehicle treated): *P<0.05.
Figure 8.
In situ S100A7 receptor binding activities in primary embryonic cortical neurons derived from Tg2576 mice, CHO-APPswe cells, and mouse brain.
Seven-day-old Tg2576 neuronal cultures (A), CHO-APPswe cells (B) and 10 µm frozen tissue sections from mouse brain (C) were incubated with AP-S100A7 fusion protein or control AP protein. Blue (A) and purple (B, C) staining detects interaction between AP-S100A7 with a putative S100A7 receptor. Length bar = 10 µm. n = 3 independent cultures per group from two independent experiments.
Figure 9.
S100A7 receptor-binding activity in human platelets and modulation of α-secretase activity and ADAM-10 expression in platelet cells by S100A7 exposure.
In this study, human platelet was prepared from blood collected from a cognitively normal control case. Cultured platelets were used for AP-S100A7 ligand-binding assay (bottom); parallel study using AP served as control staining (A). B, C, cultured platelets were treated with 100 nM h-S100A7 for 24 hours; parallel platelet cultures treated with vehicle served as controls. B, α -secretase activity in platelet lysates assessed using the α-secretase activity kit (R&D Systems). C, precursor and mature ADAM-10 content assessed by western blot analysis. Inset, representative western blot analysis of precursor and mature ADAM-10 in platelet lysates. B, C, values represent mean±SEM and are shown as percent of control cells; n = 3 independent cultures per group. *P<0.05, two-tailed Student t-test, hr-S100A7 vs. control group.
Figure 10.
Elevated α-secretase activity in the brain of h-S100A7 transgenic mice.
In this study, h-S100A7 transgenic mice were identified by dot blot hybridization of tail skin DNA samples with labeled h-S100A7 cDNA (for the h-S100A7 transgene). Expression of serum S100A7 (A) content in 1-month old h-S100A7 and age-, gender-, and stain-matched WT control mice was assessed by western blot analysis. The brains of h-S100A7 transgenic mice and WT control mice were assessed for α-secretase activity (B) using an activity assay kit (R&D Systems). Values represent mean±SEM and are expressed as percent of WT control mice group. *P<0.05, two-tailed Student t-test; n = 4 per group.