Protective Effects of Positive Lysosomal Modulation in Alzheimer's Disease Transgenic Mouse Models

Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ1–42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APPSwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβx-42 sandwich ELISA measures in APPSwInd mice of 10–11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ1–38 occurs as Aβ1–42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ1–42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof-of-principle for small molecule therapeutic development for AD and possibly other protein accumulation disorders

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-mediated enhancement across brain regions of transgenic mouse models.

<p>APP_SwInd (10–11 months of age) and APPswe/PS1ΔE9 mice (APP-PS1; 20–22 months) were injected i.p. daily with PADK (20 mg/kg; n = 11−13) or vehicle (n = 10) for 9–11 days. Active cathepsin B in tissue homogenates was measured by immunoblot, and the mean levels were compared to the respective mean immunoreactivity in vehicle-injected transgenic samples to determine the fold increase across brain regions (±SEM).</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

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

PADK-mediated reductions of intraneuronal accumulation in the APP_SwInd mice.

<p>Non-transgenic control (wt) and APP_SwInd mice were treated with 9 daily injections of PADK or vehicle. Brain sections were stained with the 6E10 antibody, and photomicrographs indicate PADK-mediated reductions of the intracellular labeling in hippocampal subfields and piriform cortex (A). Image analysis for densitometric quantification was conducted across view-fields of four different neuronal layers (B), and mean integrated optical densities were plotted (±SEM). Individual ANOVAs: <i>P</i><0.0001; Tukey's post hoc tests compared to APP_SwInd+vehicle: **<i>P</i><0.001. Size bar: 40 µm, CA1 and DG; 65 µm, piriform cortex. DG, dentate gyrus; sg, stratum granulosum; sp, stratum pyramidale.</p

Mature forms of cathepsin B exhibit pronounced modulation by PADK.

<p>APP_SwInd mice subjected to 9 daily injections of PADK (20 mg/kg; n = 10) or vehicle (n = 9) were assessed for the different cathepsin B species in hippocampal samples. Homogenates from the transgenic mice and from vehicle-treated wildtype mice were analyzed by immunoblot to label the approximately 40-kDa procathepsin B species (proCB) and the 25- and 30-kDa active mature forms (arrows) within the same samples (A). Immunoreactivity levels of the proenzyme and mature forms (means±SEM) in vehicle- (–) and PADK-treated transgenic samples (+) were determined by image analysis (B). Unpaired Mann-Whitney U-test: *<i>P</i> = 0.0296, **<i>P</i><0.001.</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