PADK selectively enhances cathepsin B levels in two transgenic mouse models.

<p>APP_SwInd and APP-PS1 mice were injected i.p. daily with PADK (20 mg/kg; n = 11−13) or vehicle (n = 10) for 9–11 days. Hippocampal homogenates were analyzed by immunoblot and mean immunoreactivities are shown for active cathepsin B (CB), neprilysin (nep), insulin-degrading enzyme (IDE), α-secretase (α-sec), and LAMP1.</p><p>***<i>P</i><0.0001, unpaired t-test.</p

PADK reduces behavioral deficits in APP_SwInd and APP-PS1 mice.

<p>In the first model, vehicle-treated wildtype mice (n = 11) were tested with groups of vehicle- (n = 10) and PADK-treated APP_SwInd mice (n = 13) across trials on the suspended rod test (A), and time maintained on the rod during the third trial was plotted (means±SEM). The animal groups were also tested across consecutive days in the same novel field, and the percent change±SEM in exploratory distance on the second day compared to the first was determined (B). In the second model, age-matched vehicle-treated wildtypes were tested with groups of vehicle- (n = 10) and PADK-treated APP-PS1 mice (n = 11) for spontaneous alternation behavior in a T-maze (C); data are plotted as percent of maximum alternations possible (mean±SEM). Open field mobility assessment confirmed no change in mean grid crossings±SEM across the three groups of mice (D). Post hoc tests compared to vehicle-treated transgenics: *<i>P</i>≤0.01, **<i>P</i><0.001.</p

PADK decreases intra- and extracellular 6E10 staining in APPswe/PS1ΔE9 (APP-PS1) mice of 20–22 months.

<p>The APP-PS1 mice received 11 daily injections of PADK (i.p., 20 mg/kg; n = 11) or vehicle (veh; n = 10), and non-transgenic control mice (wt) received vehicle injections. Fixed brain sections from the different groups were hematoxylin-eosin stained (A; arrows denote typical deposits) and 6E10 immunolabeled (B), indicating that PADK treatment reduces intra- and extracellular deposition in hippocampus. Equal protein samples from vehicle- (–) and PADK-treated (+) APP-PS1 mouse brains were immunoblotted with 6E10 antibody to assess the 4-kDa Aβ peptide and the parent hAPP, and with selective antibodies to label sAPPα and sAPPβ (C). Mean integrated optical densities±SEM for the different species were normalized to 100% as shown. The same brain samples were also tested by Aβ_x-42 sandwich ELISA to determine femtomoles of peptide per milligram protein (D). ANOVA: <i>P</i><0.0001; post hoc test compared to APP-PS1+vehicle: **<i>P</i><0.001. Unpaired t-test: *<i>P</i><0.01. Size bar: 400 µm, A; 50 µm, B. DG, dentate gyrus; sp, stratum pyramidale; sr, stratum radiatum.</p

PADK-modulated cathepsin B is localized to lysosomes.

<p>From PADK-treated mice (20 mg/kg/day×9 days) exhibiting increased levels of active cathepsin B, fixed hemi-brains were sectioned and double-stained for cathepsin B (green) and the lysosomal marker LAMP1 (red). Individual antigen labeling and the merged image from hippocampal CA1 show that PADK-modulated cathepsin B highly co-localizes with LAMP1-positive organelles in pyramidal neurons. View-field width: 35 µm. To localize cathepsin B activity, APP_SwInd mice were injected daily with vehicle (–) or 18 mg/kg PADK (+) for 10 days, and cortical and hippocampal regions were subsequently dissected to isolate lysosomes. Lysosomal fractions (Lys) and non-lysosomal fractions (non) were separated using Percoll gradients, and the two types of fractions were separately pooled and assessed for protein content and hydrolase activity with Z-Arg-Arg AMC (mean specific activity plotted±SEM). Unpaired Mann-Whitney U-test compared to lysosomal fractions from vehicle-treated mice: ***<i>P</i><0.0001.</p

Reduced intracellular Aβ_1–42 staining corresponds with enhanced cathepsin B.

<p>Fixed brain sections from vehicle-treated wildtype mice (wt) and from the APP-PS1 mice treated with vehicle (veh) or PADK were double-stained for Aβ_1–42 (green) and cathepsin B (red). Immunofluorescence images of CA1 pyramidal neurons (arrows) are shown, with view-field widths of 56 µm.</p

PADK has no inhibitory effect on β-secretase activity.

<p>Recombinant human β-secretase (10 ng/ml) was incubated with different concentrations of PADK (open triangles), CA074me (circles), and β-secretase inhibitor IV (closed triangles), and the enzyme activity was determined with the SensiZyme assay kit that uses the procaspase-3 variant containing the β-secretase cleavage sequence Gly-Ser-Ser-Glu-Ile-Ser-Tyr-Glu-Val-Glu-Phe-Arg-Glu-Phe (A). Activity was expressed in absorbance units (mean±SEM), and only β-secretase inhibitor IV elicited inhibition with an IC₅₀ of 19.8±2.4 nM. The three compounds were also tested against cathepsin B activity using the fluorogenic substrate Z-Arg-Arg AMC (mean fluorescence units±SEM plotted). β-secretase inhibitor IV had no effect on the cathepsin B activity, and PADK and CA074me resulted in IC₅₀ values of 9,200±1,030 and 120±13 nM, respectively (B).</p

The lysosomal modulator PADK selectively enhances cathepsin B levels in APP_SwInd mice.

<p>The 10–11-month transgenic mice (tg) were injected i.p. daily with either PADK (20 mg/kg; n = 13) or vehicle (veh; n = 10) for 9 days. Brain homogenates from the transgenic mice and from vehicle-treated wildtypes (wt; n = 13) were analyzed by immunoblot for the active form of cathepsin B (CB), neprilysin (nep), insulin-degrading enzyme (IDE), α-secretase (α-sec), and actin (A). Mean immunoreactivities±SEM were determined by image analysis and plotted. Hippocampal photomicrographs from vehicle- (B) and PADK-treated mice (C) show cathepsin B immunostaining (green) in pyramidal neurons counterstained with anti-NeuN (red); view-field width is 75 µm. The CA1 zone was assessed for cathepsin B immunoreactivity (mean intensity±SEM) in the pyramidal layer (D) and for the number of cathepsin B-positive puncta per neuron (E). Tukey post hoc test: **<i>P</i><0.001; unpaired t-test: ***<i>P</i><0.0001.</p

Lysosomal modulator treatment promotes truncation of the Aβ_1-42 peptide.

<p>APP_SwInd and APP-PS1 mice treated with vehicle or PADK were assessed for Aβ_x-42 and truncated Aβ_x-38 species in brain samples (n = 9−11 per group), using selective sandwich ELISAs. The PADK-mediated changes in Aβ_x-42 were determined from data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020501#pone-0020501-g006" target="_blank">Figures 6E</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020501#pone-0020501-g009" target="_blank">9D</a>. In the APP_SwInd and APP-PS1 samples, PADK increased Aβ_x-38 species from 60.6±7.7 to 92.2±5.7 fmol/mg and from 104±7.0 to 137±18 fmol/mg, respectively. The plotted PADK effects are expressed as mean percent change±SEM. Post hoc tests compared to the corresponding vehicle-treated transgenic data: *<i>P</i><0.01.</p

PADK decreases 6E10 immunostaining in APP-PS1 mouse brain.

<p>APP-PS1 mice were injected i.p. daily with PADK (20 mg/kg; n = 11) or vehicle (n = 10) for 11 days. Fixed tissue was sectioned and stained with the 6E10 antibody. Image analysis for densitometric quantification of the immunostaining (mean integrated optical density±SEM) was conducted across view-fields of the hippocampal CA1 stratum pyramidale (sp). Area of deposit labeling above background was also measured for view-fields of the hippocampal stratum radiatum (sr) and piriform cortex (mean percent of total measured area±SEM). ANOVAs: <i>P</i><0.0001; Tukey's post hoc tests compared to APP−PS1+vehicle.</p><p>**<i>P</i><0.001.</p

Lysosomal modulation is associated with preservation of synaptic markers in APP_SwInd and APP-PS1 mice.

<p>Transgenic and wildtype (wt) mice were injected daily with PADK (+) or vehicle (–) for 9–11 days. Equal protein aliquots of hippocampal homogenates were analyzed by immunoblot for synaptic markers and actin, showing PADK-improved levels of GluA1 and synapsin II (syn II) in transgenic mice (A). The mean GluA1 immunoreactivities±SEM are shown for vehicle-treated wildtypes and for the vehicle- and PADK-treated transgenics (B). Post hoc tests compared to vehicle-treated transgenics: **<i>P</i><0.001.</p