The amount of background signal with APP-KO tissue varies widely between different ELISAs.

<p><b>A</b>) The Covance Colorimetric BetaMark™ Beta-Amyloid x-40 ELISA Kit (left) and x-42 Kit (right). Both kits give a signal with APP-KO tissue that is significantly above baseline. The x-40 kit gives a signal with APP-KO tissue that is not significantly different from WT (the difference between APP-KO and WT is significant for the x-42 kit). <b>B</b>) The Invitrogen Aβ 40 Mouse ELISA Kit (left) and Aβ 42 Mouse ELISA Kit (right). Both kits give a signal with APP-KO tissue that is significantly above baseline, but also significantly different from WT tissue. <b>C</b>) The Wako β-Amyloid (40) ELISA Kit (left) and β-Amyloid (42) ELISA High-Sensitive Kit (right). Both kits give a signal with APP-KO tissue that is not statistically significant from baseline. <b>D</b>) Both WT and APP-KO give a similar level of background signal with an ELISA for human β-amyloid (6E10 capture antibody – left), but show a clear difference when a rodent-specific capture antibody is used (M3.2 antibody – right). <b>E</b>) “Signal to noise” ratio of the WT signal divided by the APP-KO signal for each ELISA. Error bars in A–D are standard error.</p

Several β-amyloid antibodies show high non-specificity for β-amyloid by western blot.

<p><b>A</b>) Western blot with the antibody M3.2 (specific for rodent β-amyloid) using hippocampal tissue from APP-KO, WT, and APP transgenic mice expressing human APP (Hu<i>APP</i>695SWE). All three genotypes show a similar band pattern. <b>B</b>) Western blot with the antibody 6E10 (specific for human β-amyloid). All three genotypes show a similar band pattern. However, with 6E10, the APP transgenic mouse tissue shows an additional 8 kD band (presumably a human β-amyloid dimer) as well as a band near 87 kD (presumably full-length human APP). In addition, a 4.5 kDa band (β-amyloid) is detected when older mouse brain is used that has high levels of β-amyloid protein. <b>C</b>) Western blot with 4G8. All three genotypes also showed a similar band pattern with this antibody as well. In addition, a 4.5 kDa band (β-amyloid) is detected when older mouse brain is used that has high levels of β-amyloid protein.</p

A Thr⁶⁶⁸Ala mutation on APP prevents the synaptic deficits of FDD_KI mice.

<p>Normal LTP in FDD_KI/<i>APP^TA/TA</i> and <i>APP^TA/TA</i> compared with WT mice by two-way ANOVA (FDD_KI/<i>APP^TA/TA</i> versus WT mice: F(1,12) = 1.936; P = 0.187; <i>APP^TA/TA</i> versus WT F(1,12) = 0.989; P = 0.338). Two-way ANOVA shows impaired LTP in FDD_KI mice when compared with WT (F(1,13) = 15.125; P = 0.002), to FDD_KI/<i>APP^TA/TA</i> (F(1,13) = 12.759; P = 0.004) or to <i>APP^TA/TA</i> mice littermates (F(1,13) = 22.396; P<0.0001).</p

Dynamin inhibition by dynasore affects LTP, a type of synaptic plasticity due to sustained activity in hippocampus.

<p><b>A</b>, Dynasore (80 μM, 20 minute perfusion, open triangles) decreases LTP induced by theta-burst stimulation in CA₃–CA₁ synapses compared to vehicle-treated slices (black circles)(F_1,10 = 9.081, <i>p</i> = 0.013). The horizontal bar indicates the period of perfusion with dynasore before tetanic stimulation. <b>B</b>, Post-tetanus dynamin inhibition by dynasore (80 μm, 20 minute perfusion <i>after</i> the tetanus delivery, open triangle) induced by theta-burst stimulation in CA₃–CA₁ synapses compared to vehicle-treated slices (black circles; F_1,7 = 0.209, <i>p</i> = 0.662). The horizontal bar indicates the period of perfusion with dynasore <i>after</i> tetanic stimulation. <b>C</b>, Basal synaptic transmission is unmodified by dynamin inhibition with dynasore. Averaged evoked field potential slopes as a function of stimulation intensity measured in volts (V) at CA₃–CA₁ synapses in slices do not show significant differences between vehicle-treated (black circles) and dynasore (80 μM, open triangles) treated slices (F_1,11 = 40.081, <i>p</i> = 0.7013). <b>D</b>, Dynamin inhibition by dynasore (open triangles; 80 μm, 20 minute perfusion before the tetanus) does not produce changes in solely post-synaptic LTP induced by three tetani at 50 Hz for 1 second, each tetanus separated by 20 seconds, at the CA₃–CA₁ synapse compared to vehicle-treated slices (black circles; F1,8 = 1.538, p = 0.250). The horizontal bar indicates the period of perfusion with dynasore before tetanic stimulation. Error bars indicate SEM.</p

A Thr⁶⁶⁸Ala mutation on APP prevents the short-term memory deficit of FDD_KI mice.

<p>(<b>a</b> and <b>b</b>) In RAWM testing, FDD_KI/<i>APP^TA/TA</i>, FDD_KI/<i>APP^TA/WT</i>, <i>APP^TA/TA</i>, <i>APP^TA/WT</i> mice made the same number of errors as WT mice at both 5.5 months and 9 months of age. At 5.5 months of age, FDD_KI mice made significantly more errors at A4 (versus FDD_KI/APP^TA/TA<i>P</i> = 0.0007; versus FDD_KI/APP^TA/WT P = 0.0028; versus WT <i>P = </i>0.0005) and R (versus FDD_KI/APP^TA/TA<i>P</i> = 0.0017; versus FDD_KI/APP^TA/WT P = 0.0005; versus WT <i>P</i> = 0.0005) (<b>a</b>). Similar results are found at 9 months of age; FDD_KI mice made significantly more errors at A4 (versus FDD_KI/APP^TA/TA<i>P</i> = 0.0004; versus FDD_KI/APP^TA/WT P = 0.019; versus WT <i>P</i> = 0.0003) and R (versus FDD_KI/APP^TA/TA<i>P</i> = 0.0006; versus FDD_KI/APP^TA/WT P = 0.004; versus WT <i>P</i><0.0001). Thus, the APP^TA/TA and APP^TA/WT point mutations prevent the development of working memory deficits in FDD_KI mice (<b>b</b>). (<b>c</b> and <b>d</b>) WT, FDD_KI, FDD_KI/<i>APP^TA/TA</i>, FDD_KI/<i>APP^TA/WT</i>, <i>APP^TA/TA</i> and <i>APP^TA/WT</i> mice have similar speed (c) and need similar time (d) to reach a visible platform.</p

A Thr⁶⁶⁸Ala mutation on APP prevents the object recognition memory deficit of FDD_KI mice.

<p>(<b>a</b>) Western blot analysis of hippocampal synaptosomal preparations shown that the Thr to Ala mutation abolishes phosphorylation of Thr⁶⁶⁸ (APP^pThr⁶⁶⁸). Interestingly, only the mature form of APP (mAPP) and not the immature (imAPP), is found phosphorylated on this Thr in hippocampal synaptic fractions of WT mice. (<b>b</b> and <b>c</b>) Open field is a sensorimotor test for habituation, exploratory, emotional behavior, and anxiety-like behavior, in novel environments. The percent of time in the center (b) and the number of entries into the center (c) are indicators of anxiety levels. The more the mouse enters the center and explores it, the lower the level of anxiety-like behavior. Since the FDD_KI, FDD_KI/<i>APP^TA/TA</i>, FDD_KI/<i>APP^TA/WT</i>, <i>APP^TA/TA</i>, <i>APP^TA/WT</i> mice are similar to the WT animals there is no deficit or excess of anxiety. (<b>d</b>) All six genotypes (WT, FDD_KI, FDD_KI/<i>APP^TA/TA</i>, FDD_KI/<i>APP^TA/WT</i>, <i>APP^TA/TA</i>, <i>APP^TA/WT</i>) mice spent similar amounts of time exploring the two identical objects on day 1. (<b>e</b>) FDD_KI/APP^TA/TA and FDD_KI/APP^TA/WT mice behaved similarly to WT mice and prevented the deficit in the NOR tests found in FDD_KI mice at 6 months of age (FDD_KI versus FDD_KI/APP^TA/TA P = 0.011; FDD_KI versus FDD_KI/APP^TA/WT P = 0.0083; FDD_KI versus WT P<0.001), (<b>f)</b> 9 months of age (FDD_KI versus FDD_KI/APP^TA/TA P = 0.01; FDD_KI versus FDD_KI/APP^TA/WT P = 0.347; FDD_KI versus WT P = 0.000995), and (<b>g</b>) 12 months of age (FDD_KI versus FDD_KI/APP^TA/TA P = 0.0003; FDD_KI versus FDD_KI/APP^TA/WT P = 0.0002; FDD_KI versus WT P<0.0001). Thus the APP^TA point mutation prevented the novel object recognition deficit of FDD_KI mice.</p

Dynamin inhibition affects presynaptic mechanisms underlying synaptic plasticity evoked by sustained activity.

<p><b>A</b>, Tetanic efficiency, expressed as percent in evoked potential area across a train of 10 bursts at 5 Hz, each consisting of 4 pulses at 100 Hz, is reduced by dynasore (80 μM, open bars) with respect to percent response area in vehicle-treated slices (black bars) (F_1,9 = 12.071, <i>p</i> = 0.007). The reduction reaches statistical significance at the third burst (Mann-Whitney U_{51, 15} = 0.00001, <i>p</i> = 0.0043); <b>B</b>, Dynasore (80 μM, open bars) already produces a partial reduction of the area within the first group of 4 pulses at 100 Hz compared to vehicle-treated slices (black bars); <b>C</b>, Raw <i>f</i>EPSP signals recorded during tetanic stimulation in vehicle-treated and dynasore (80 μM, 20 minutes before tetanus)-treated hippocampal slices. Calibration: 0.5 mV, 2 ms;. <b>D</b>, Perfusion of hippocampal slices with dynasore (80 μM) in the presence of D-APV (100 μM) for 20 minutes prior to theta-burst stimulation diminishes PTP to about 50% of the values obtained with vehicle perfusion (F_1,12 = 6.924, <i>p</i> = 0.022). The effect was reversed by washout with vehicle. Error bars indicate SEM.</p

Dynamin inhibition with dynasore does not affect paired-pulse facilitation, a type of synaptic plasticity that is not linked with sustained activity.

<p>Paired-pulse-induced change is calculated as the ratio of the slope of the second evoked field potential to the first one at different interpulse intervals (1–1000 ms). Paired-pulse ratio is not modified in dynasore (80 μM, open triangles) treated slices with respect to vehicle-treated slices (black circles)(F1,12 = 0.339, p = 0.914). No effect is observed by subsequent washout with ACSF (open squares). Error bars indicate SEM.</p

Dynamin inhibition by dynasore impairs associative memory.

<p><b>A</b>, Schematic representation of the hippocampi bilaterally implanted with cannulas. <b>B</b>, Bilateral injections of dynasore (80 μM in a final volume of 1.5 μl over 1 minute) into dorsal hippocampi, 20 minutes before training, dramatically impairs contextual fear memory (open bars) compared to vehicle treated mice (black bars) (Mann-Whitney U_{137, 73} = 18.00, <i>p</i> = 0.00147). <b>C</b>, Mice do not show changes in cued fear conditioning following dynasore infusions (open bars) compared to vehicle-treated animals (black bars) (Mann-Whitney U_{121, 89} = 34.00, <i>p</i> = 0.2412). Each bar represents the average percent of time spent in freezing posture. Error bars indicate SEM. <b>D</b>, Sensory threshold was not affected regardless of treatment (n = 10).</p

Selective dynamin 1 inhibition through siRNA impairs both synaptic plasticity and associative memory.

<p><b>A</b>, siRNA specific for murine dynamin 1 reduces protein expression. An example of western blot showing that Penetratin 1- conjugated dynamin 1 siRNA reduces protein expression. Cells are lysed 48 hours after the treatment with siRNA. Dynamin 1 is detected using a rabbit polyclonal anti-dynamin1 antibody. Penetratin 1- conjugated Control siRNA, that does not affect dynamin 1 expression, does not change protein levels. n = 3 for each group. <b>B</b>, Penetratin 1- conjugated dynamin 1 siRNA (open bars) (80 nM in a final volume of 1.5 μl over 1 minute, bilateral injections twice a day for 3 days) impairs contextual fear memory compared to control siRNA infused mice (grey bars) (Mann-Whitney U_{150, 126} = 21.00, <i>p</i> = 0.0089). Moreover, control siRNA infused mice show similar amount of freezing as vehicle-infused animals (black bars). <b>C</b>, Penetratin 1- conjugated dynamin 1 siRNA (open bars) does not modify cued fear memory compared to Penetratin 1- conjugated Control siRNA (grey bars) (Mann-Whitney U_{99, 90} = 44.50, <i>p</i> = 1.00) in mice previously tested for contextual fear conditioning at 24 hours after the shock. <b>D</b>, Bilateral infusions of Penetratin 1- conjugated dynamin 1 siRNA (open triangles) (80 nM in a final volume of 1.5 μl over 1 minute, repeated 2 times a day for three days) into dorsal hippocampi decrease LTP compared to Penetratin 1- conjugated Control siRNA treatment (grey squares) (F_1,9 = 5.578, <i>p</i> = 0.001). As an internal control, slices from vehicle-infused animals (black circles) show similar amounts of potentiation as those from Penetratin 1- conjugated Control siRNA treated animals. Error bars indicate SEM.</p