Aβ42-mediated proteasome inhibition and associated tau pathology in hippocampus are governed by a lysosomal response involving cathepsin B: Evidence for protective crosstalk between protein clearance pathways.

Impaired protein clearance likely increases the risk of protein accumulation disorders including Alzheimer's disease (AD). Protein degradation through the proteasome pathway decreases with age and in AD brains, and the Aβ42 peptide has been shown to impair proteasome function in cultured cells and in a cell-free model. Here, Aβ42 was studied in brain tissue to measure changes in protein clearance pathways and related secondary pathology. Oligomerized Aβ42 (0.5-1.5 μM) reduced proteasome activity by 62% in hippocampal slice cultures over a 4-6-day period, corresponding with increased tau phosphorylation and reduced synaptophysin levels. Interestingly, the decrease in proteasome activity was associated with a delayed inverse effect, >2-fold increase, regarding lysosomal cathepsin B (CatB) activity. The CatB enhancement did not correspond with the Aβ42-mediated phospho-tau alterations since the latter occurred prior to the CatB response. Hippocampal slices treated with the proteasome inhibitor lactacystin also exhibited an inverse effect on CatB activity with respect to diminished proteasome function. Lactacystin caused earlier CatB enhancement than Aβ42, and no correspondence was evident between up-regulated CatB levels and the delayed synaptic pathology indicated by the loss of pre- and postsynaptic markers. Contrasting the inverse effects on the proteasomal and lysosomal pathways by Aβ42 and lactacystin, such were not found when CatB activity was up-regulated two-fold with Z-Phe-Ala-diazomethylketone (PADK). Instead of an inverse decline, proteasome function was increased marginally in PADK-treated hippocampal slices. Unexpectedly, the proteasomal augmentation was significantly pronounced in Aβ42-compromised slices, while absent in lactacystin-treated tissue, resulting in >2-fold improvement for nearly complete recovery of proteasome function by the CatB-enhancing compound. The PADK treatment also reduced Aβ42-mediated tau phosphorylation and synaptic marker declines, corresponding with the positive modulation of both proteasome activity and the lysosomal CatB enzyme. These findings indicate that proteasomal stress contributes to AD-type pathogenesis and that governing such pathology occurs through crosstalk between the two protein clearance pathways

Aβ₄₂–induced increase in phosphorylated tau is attenuated by PADK.

<p>Hippocampal slice cultures were treated daily with vehicle for 6 days (0-day control) or with 1.5 μM pre-aggregated Aβ₄₂ for 4–6 days in the absence of presence of 3 μM PADK. The treatment schedule was staggered in order for same-day harvesting of 7–9 slices per group. Equal protein aliquots of the slice samples were assessed by immunoblot with antibodies against phospho-tau-Ser^199/202 and against actin (a). Positions of molecular weight standards of 49–76 kDa are shown on the right. Integrated optical densities were measured and within-sample ratios between the two antigens were plotted for the 6-day treatments (b); note that the ratios were normalized to vehicle-treated samples (mean ± SEM). Unpaired t tests compared to vehicle control: **p<0.01; compared to Aβ₄₂ alone: #p<0.05. Additional immunblot samples were stained for phospho-tau-Ser^199/202, the 30-kDa CatB isoform (CatB-30), synaptophysin (SNP), GluR1, and actin (c).</p

Among screened compounds, PADK was chosen as an effective CatB-enhancing agent for further testing in the hippocampal slice model.

<p>Among screened compounds, PADK was chosen as an effective CatB-enhancing agent for further testing in the hippocampal slice model.</p

The proteasome inhibitor lactacystin causes delayed synaptic marker loss in hippocampal slice cultures.

<p>The hippocampal slices were treated daily with vehicle for 4 days (0-day control group) or with 5 μM lactacystin for 1–4 days, and treatments were staggered for same-day harvesting of 7–9 slices per group. The samples were assessed by immunoblot (a), staining the proteins synaptophysin (SNP), synapsin IIa (syn IIa) and IIb (syn IIb), GluR1, and actin. Mean synapsin levels were normalized to their respective controls and percent ± SEM values are shown for the time points with distinct effects between the two isoforms (b). Across the set of immunoblot samples, the levels of synaptophysin were plotted against the within-sample measures of synapsin IIb (c) and synapsin IIa (d). Linear regression analysis was conducted (c: R = 0.913, p<0.001; d: R = -0.430, N.S.).</p

Model of the putative crosstalk between the two major protein clearance pathways.

<p>The autophagic-lysosomal system consists of autophagosomes, endosomes, primary lysosomes, and secondary lysosomes. The proteasomal system is composed of multimeric complexes with a catalytic core and regulatory subunits. Oligomerized Aβ₄₂ and lactacystin elicit proteasomal inhibition which is linked to positive crosstalk with a component of the lysosomal pathway. Enhancing such compensatory lysosomal responses with a CatB-enhancing agent also leads to positive crosstalk back to the proteasome system.</p

Treatment with pre-aggregated Aβ₄₂ peptide leads to increased phosphorylation of tau residues Ser199 and Ser202.

<p>Hippocampal slice cultures were treated daily with Aβ₄₂ alongside vehicle-treated slices. After 6 days the tissue was gently harvested into groups of 7–9 slices each and equal protein aliquots assessed by immunoblot with antibodies against phospho-tau-Ser^199/202 and against actin (a). Positions of molecular weight standards are shown. The 55–70-kDa phospho-tau immunoreactivity levels were normalized to within-sample actin measures and plotted as mean percent of control ± SEM (b). Unpaired t-test: ***p< 0.001.</p

Aβ₄₂–induced decline in proteasome activity is diminished by delayed PADK treatment.

<p>Panel a: Hippocampal slice cultures were treated daily with vehicle (veh) or 1.5 μM Aβ₄₂ for 6 days, or they were subjected to the 6 daily Aβ₄₂ treatments but 3 μM PADK was included during the last 3 days of Aβ₄₂ incubations. Proteasomal activity used a fluorogenic peptide assay in harvested slice samples and Vmax/s measures were normalized to vehicle-treated control samples (mean ± SEM). Panel b: The harvested samples were also assessed by immunoblot for the 30-kDa active CatB isoform (CatB-30) and the actin load control. Mean CatB-30 levels were normalized to control values and percent ± SEM values are shown. Unpaired t tests compared to Aβ₄₂ alone: #p<0.05.</p

Lactacystin-induced proteasome inhibition leads to the enhancement of CatB activity.

<p>Lactacystin-induced proteasome inhibition leads to the enhancement of CatB activity.</p

Aβ₄₂-induced proteasome inhibition is associated with a delayed, inverse effect on CatB activity.

<p>Hippocampal slice cultures were treated daily with vehicle for 6 days (0-day control group) or with pre-aggregated Aβ₄₂ for 4–6 days, staggering the treatments in order for same-day harvesting of slice groups of 7–9 each. Proteasome activity (gray plot of mean Vmax/s ± SEM; ANOVA: p<0.01) and cathepsin B activity (black plot of mean fluorometric units ± SEM; ANOVA: p<0.01) were measured in equal protein aliquots from the same samples (a). The time-course samples were also assessed for 55–70-kDa pTau-Ser^199/202 and actin in order to plot the within-sample ratios between the immunoreactivity levels (b; mean ± SEM). Tukey post hoc tests compared to vehicle-treated slices: **p<0.01, ***p<0.001.</p