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-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

Changes in Rab proteins in cultured hippocampal slices treated with PADK.

<p>Stable slice cultures prepared from rat hippocampus were treated daily with PADK (10 µM; n = 12 groups of 8 slices each) or with the corresponding vehicle (final condition of 0.02% DMSO; n = 14 groups of slices) for 4 days. Slice groups were homogenized and equal protein aliquots analyzed by immunoblot for Rab5a, Rab7, active form of cathepsin B (act CB), LAMP1, and actin (A). Rab protein levels (means±SEM) in slices treated without (–) or with PADK (+) were determined by image analysis (B). Unpaired Mann-Whitney U-test: *<i>P</i> = 0.0208, ***<i>P</i><0.0001.</p

PADK modulates cathepsin B more than cathepsin D in APP_SwInd mice.

<p>The APP_SwInd transgenic mice (tg) were subjected to 9 daily injections of PADK (20 mg/kg; n = 13) or vehicle (n = 10), and wildtype mice (wt; n = 13) were injected with vehicle. Hippocampal homogenate samples were analyzed by immunoblot for the active isoform of cathepsin B (CB), the 33-kDa mature cathepsin D (CD), the lysosomal marker LAMP1, and actin (A). Mean immunoreactivities±SEM for CB and CD were determined and the respective data normalized with vehicle-treated transgenic groups (–) set at 100% (B). ANOVA across the 8 normalized groups: <i>P</i><0.0001; Tukey post hoc tests: **<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

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

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

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 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