Cortical neurons express postsynaptic Ca²⁺-permeable AMPAR that are functionally coupled to ERK phosphorylation and APP cleavage.

<p>A, Immunofluorescence staining of primary cortical neurons at 10 DIV. Left hand panels; single immunofluorescence staining for APP, GluA1 and GluA2. Middle panels; double immunofluorescence staining for APP, GluA1 and GluA2 (all green) with the nerve terminal marker synaptophysin (Syn, red). Right hand panels; double immunofluorescence staining for APP, GluA1 and GluA2 (all green) with the postsynaptic marker PSD95 (red). Nuclei are counterstained with DAPI (blue), scale bar 20 µM. B, Fura-2 AM microfluorimetry demonstrating AMPA-evoked increases in Ca²⁺ in primary cortical neurons. Pseudocoloured images illustrating [Ca²⁺]i at baseline (left hand panel) and maximum [Ca²⁺]i response evoked by 50 µM AMPA (right hand panel), scale bar 150 µM. C, Representative ratiometric 340 nm∶380 nm trace against time (s) for Fura-2 AM loaded cortical neurons microperfused with 50 µM AMPA. D, Average changes in [Ca²⁺]i (340 nm∶380 nm ratio) of 40 individual neurons in response to sequential application of 50 µM AMPA, 50 µM AMPA+2.5 µM MK801 and reapplication of 50 µM AMPA (AMPA Reapp) expressed as % of the initial AMPA response. E, Primary cultured cortical neurons at 10 DIV were treated with vehicle (Control), 50 µM AMPA or 50 µM NMDA for 1 h followed by immunoblotting of neuronal lysates with antibodies to APP CT20 to detect full length APP695 (APP) and APP CTFs, phosphorylated ERK2 (pERK2), and ERK2.</p

AMPAR-stimulation increases time-dependent APP processing that is not completely dependent on ERK activation.

<p>A, Primary cultured cortical neurons at 10 DIV were treated with vehicle (Control) or 50 µM AMPA for 0.3, 1, 3, 6 and 24 h followed by immunoblotting of neuronal lysates with antibodies to APP CT20 to detect full length APP695 (APP) and APP CTFs (α-CTF), phospho ERK1/ERK2 (pERK1/pERK2), ERK2 and synaptophysin (Syn). B, Full length APP695 levels (tAPP) and C, APP CTF (α-CTF) levels were analysed by ECL protein band densitometry using calibrated ImageJ software. Each column is the mean of +/−SEM of five independent experiments (n = 5; *p<0.05, **p<0.01, control (time 0, grey bar) vs treatment (black bars), one-way ANOVA with Dunnett's post hoc test). D, Primary cultured cortical neurons at 9 DIV were treated with vehicle (Control), 50 µM AMPA, 2 µM PD184352, 5 µM U0126 or AMPA in the presence of either PD184352 (AMPA+PD) or U0126 (AMPA+U0) for 1 h followed by immunoblotting of neuronal lysates with antibodies to APP CT20 to detect full length APP695 (APP), phospho ERK1/ERK2 (pERK1/pERK2) and ERK2. E, Full length APP695 levels (tAPP) were analysed by ECL protein band densitometry using calibrated ImageJ software. Each column is the mean of +/−SEM of five independent experiments (n = 4; *p<0.05, control (grey bar) vs AMPA (black bars), one-way ANOVA with Dunnett's post hoc test).</p

AMPAR-stimulation of APP processing is independent of NMDAR and L-type calcium channels is partially dependent on extracellular Ca²⁺ and is blocked by the ADAM inhibitor TAPI-1.

<p>A, Primary cultured cortical neurons at 10 DIV were treated for 1(Control), 50 µM AMPA, 2.5 µM MK801 (MK), 10 µM Nimodipine (Nim) or AMPA in the presence of either MK801 (AMPA+MK) or Nimodipine (AMPA+Nim) followed by immunoblotting of neuronal lysates with antibodies to APP CT20 to detect APP CTFs (α-CTF), phospho ERK1/ERK2 (pERK1/pERK2) and ERK2. B, APP CTF (α-CTF) levels were analysed by ECL protein band densitometry using calibrated ImageJ software. Each column is the mean of +/−SEM of six independent experiments (n = 6; *p<0.05, **p<0.01, control (white bar) vs AMPA, AMPA+MK801 and AMPA+Nimodipine (black bars), one-way ANOVA with Dunnett's post hoc test). C, Primary cultured cortical neurons at 10 DIV were treated for 1 h with vehicle (Control), 50 µM AMPA, 10 µM GYKI53655 (GYKI), 2 mM EGTA or AMPA in the presence of either GYKI53655 (AMPA+GYKI) or EGTA (AMPA+EGTA) followed by immunoblotting of neuronal lysates with antibodies to APP CT20 to detect APP CTFs (α-CTF), phospho ERK1/ERK2 (pERK1/pERK2) or ERK2. D, APP CTF (α-CTF) levels were analysed by ECL protein band densitometry using calibrated ImageJ software. Each column is the mean of +/−SEM of five independent experiments (n = 5; **p<0.01, control (white bar) vs AMPA (black bar) and AMPA vs AMPA+GYKI (black bars) one-way ANOVA with Bonferroni post hoc test). E, Primary cultured cortical neurons at 10 DIV were treated for 1 h with vehicle (Control), 50 µM AMPA or 50 µM NMDA, in the absence and presence of 50 µM TAPI-1 (AMPA+TAPI, NMDA+TAPI) followed by immunoblotting of neuronal lysates with antibodies to APP CT20 to detect APP CTFs (α-CTF), phospho ERK1/ERK2 (pERK1/pERK2) or ERK2. F, APP CTF (α-CTF) levels were analysed by ECL protein band densitometry using calibrated ImageJ software. Each column is the mean of +/−SEM of five independent experiments (n = 5; **p<0.01, AMPA vs and AMPA+TAPI (black bars) one-way ANOVA with Bonferroni post hoc test).</p

AMPAR-activity stimulates sAPP release, inhibits β-secretase processing and reduces Aβ secretion from primary cultured cortical neurons.

<p>A, Primary cultured cortical neurons at 10 DIV were treated with vehicle (Control) or 50 µM AMPA for 0.3, 1 or 3 h, followed by immunoblotting of the growth media with an N-terminal APP antibody APP13M to detect secreted APP (sAPP) and immunoblotting of the corresponding neuronal lysates with APP CT20 and ERK2. B, sAPP levels in the media from vehicle (Control) and following treatment with AMPA for 3 h were analysed by ECL protein band densitometry using calibrated ImageJ software. Each column is the mean +/−SEM of four independent experiments (n = 4; **p<0.05, Control (white bar) vs AMPA (black bar) unpaired two-tailed Student's t-test). C, Primary cultured cortical neurons at 8 DIV were cotransfected with APP695-GAL4, pFR-Luc Firefly luciferase reporter gene and phRL-TK plasmids and then treated with vehicle (Control), 10 µM DAPT, 10 µM β-secretase inhibitor (BSI) or 50 µM AMPA. Dual-Glo luciferase activity assays were performed 24 h after transfection for quantification of Firefly and Renilla luciferase expression. Firefly luciferase reporter activity was normalized using the constitutive Renilla activity. Each column is the mean +/−SEM of 12 separate transfections prepared from 3 independent cultures (n = 12; *p<0.05; **p<0.01; ***p<0,001; control vs DAPT, control vs BSI, control vs AMPA one-way ANOVA with Dunnett's post hoc test). D, Primary cultured cortical neurons at 10 DIV had a media change and were then treated with vehicle (Control) or 50 µM AMPA for 1, 3 or 6 h. The neuronal culture medium was removed and Aβ_1–40 levels were measured by ELISA. Each column represents the mean +/−SEM of three independent experiments (n = 3; *p<0.05; ***p<0.001; control (white bars) vs AMPA (black bars).</p

DNA-vaccine groups and plasmid combinations.

<p>DNA-vaccine groups and plasmid combinations.</p

Characterization and evaluation of the effect of co-delivering chIFNγ with a DNA vaccination system.

<p>Plasmids expressing NDV F and chIFNγ genes were developed and characterized for their use as DNA vaccines and adjuvant, respectively. DF-1 cells were transfected with pTriEX, pTriEX-ZJ1-F and pTriEX- IFNγ. Cell culture supernatants were tested by western blotting for the presence of F protein <b>(A)</b> and chIFNγ <b>(B)</b>, respectively. Eighteen-day-old SPF ECEs were inoculated with TE buffer, pTriEX, pTriEX-ZJ1-F, or pTriEX-ZJ1-F plus pTriEX- IFNγ and boosted 2 weeks after hatched. Two weeks after booster vaccination, birds were challenged with vZJ1. Oropharyngeal <b>(C)</b> and cloacal <b>(D)</b> swab samples were collected 3 days after challenge to measure the amount of challenge virus shed into the environment. Viral titers were determined by quantitative real time reverse transcription polymerase chain reaction (qRRT-PCR). A standard was prepared with a vZJ1 virus stock of know concentration, this was included in every plate and was used to obtain viral titers expressed as EID₅₀/mL. Morbidity <b>(E)</b> and mortality <b>(F)</b> were also evaluated. Viral shedding results were analyzed with One-way ANOVA followed by a multiple comparisons Tukey's test. Differences in morbidity among groups were evaluated using a two-tailed Z test for comparison of sample proportions. Survival curves were analyzed using the Long-Rank test. Statistical difference was considered with a <i>P<0</i>.<i>05</i>. Significant differences are denoted by different letters.</p

Effect of chIFNγ on hatchability and survival after <i>in ovo</i> vaccination with rZJ1*L/IFNγ and challenge with vZJ1.

<p>Effect of chIFNγ on hatchability and survival after <i>in ovo</i> vaccination with rZJ1*L/IFNγ and challenge with vZJ1.</p