Figure 5Schematic representation of AAV9 constructs enabling the neuron-specific expression of i) Venus and ii) HA-tagged APPsβ (+ Venus). ITR: inverted terminal repeat, Synapsin: neuron specific promotor, T2A: Thoseaasigna virus 2A site, SP: signal peptide, HA: influenza hemagglutinin tag. APPsα differs from APPsβ in the last 16 C-terminal amino acids (CTα16, green). The epitope recognized by the M3.2 antibody is indicated. Overview of the hippocampus of a AAV-APPsβ (HA-tag, red) injected cDKO mouse. APPsβ is expressed in CA1, CA3 and DG. Scale bar: 500 μm. DG: dentate gyrus, CA: Cornus Ammonis. Western blot analysis of HA-APPsα and HA-APPsβ in hippocampi of injected mice (boxed upper panel), probed with an HA-tag specific antibody (lower band, arrowhead; *unspecific signal). Homogenates of AAV injected hippocampi were subjected to ultracentrifigation to detect soluble APPsα and APPsβ (UC supernatant; boxed lower panel). The APP C-terminal antibody Y188 was used to confirm the separation of soluble APPsα from membrane bound full-length APP that is detected in total lysates of AAV-Venus injected littermate controls. Hippocampi of APP-/- mice served as a negative control for antibody specificity. Quantification of HA-APPsα and HA-APPsβ expression in total hippocampal homogenates and of soluble HA-APPs after UC normalized to β-tubulin. Note the comparable expression in AAV-APPsα and AAV-APPsβ injected mice. Age: 5-6 months. N=Number of animals. Data represent mean ± SEM. Data were analyzed by unpaired two-tailed Student's t test. nsp>0.05. Activity dependent synaptic plasticity was investigated in cDKO after injection of AAV-Venus (red) or APPsβ (blue). F, G AAV-mediated overexpression of APPsß (n=23 slices, blue symbols) failed to rescue the LTP defect of cDKO mice (n=24 slices, red symbols). G TBS induced strengthening of fEPSPs resulted in similar potentiation levels for the last 5 minutes of recording (t75-80). The LTP induction rate is shown as percentage % of mean baseline slope. Data points were averaged over 6 time points. H PPF paradigm yielded no significant differences between viral vector injected cDKO mice. Age: 5-6 months. n=number of recorded slices. N=number of animals. Data represent mean ± SEM. Data were analyzed unpaired two-tailed Students t-test. Representative images of Golgi stained midapical and basal dendritic segments of LM control and cDKO animals injected with AAV-Venus or AAV-APPsβ. The significant spine density deficit of AAV-Venus injected cDKO mice (red bar) in basal (J) and apical dendrites (K) was comparable and not significantly different (nsp>0.05) from that of AAV-APPsβ injected cDKO mice (blue bar). White bar: AVV-Venus injected LM controls. Images are maximum projections of z-stacks. Scale bar: 5 µm. Spine density is normalized to LM control levels. Age: 5-6 months; n=number of neurons (from 5 animals per condition). Data represent mean ± SEM. Data were analyzed by one-way ANOVA followed by Bonferroni´s post-hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 . Acute hippocampal slices of cDKO were pre-incubated for 1 hour either with 10 nM CTα16 or CTα16scr before inducing LTP by TBS (arrowhead). Averaged potentiation levels at LTP induction (t20-25) revealed a significantly enhancement by CTα16 as compared to CTα16scr present over the whole recoding time. Potentiation levels at t75-80 are significantly increased in the presence of CTα16 (**p<0.01). The LTP induction rate is shown as percentage % of mean baseline slope. Data points were averaged over 6 time points. N CTα16 significantly enhances short-term synaptic plasticity at inter-stimulus intervals of 40 (**p<0.01), 80 (*p<0.05), and 160 (**p<0.01) ms compared to CTα16scr. Acute application of CTα16 and recAPPsα elevates LTP in cDKO slices to the same extent. O, P After 1 hour pre-incubation with either 10 nM CTα16 or 10 nM recAPPsα fEPSPs were recorded. The TBS induced LTP curve was closely overlapping for both conditions and resulted in similar, statistically not significantly different LTP values Q CTα16 and recAPPsα modulate presynaptic function of cDKO mice in the same manner. Age: 5-6 months. n=number of recorded slices. N=number of animals. Data represent mean ± SEM. Data were analyzed by unpaired two-tailed Students t-test.