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

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

The effective CatB-enhancing agent PADK (chosen from Table 2) selectively enhances CatB activity in hippocampal slice cultures.

<p>The slice cultures were treated daily with vehicle or 3 μM PADK for 2 days before being collected into slice groups of 7–9 each. Panel a: Immunoblot assessments stained the 30- and 25-kDa CatB isoform (CatB-30 and CatB-25), GluR1, and synaptophysin (SNP). Panel b: Fluorogenic peptide assays assessed the harvested slice samples for cathepsin B and proteasome chymotrypsin-like activities. The two measures were normalized to their respective vehicle control groups (mean ± SEM). Cathepsin B activity exhibited a significant increase, whereas proteasome activity exhibited only a small increase. Panel c: Percent changes in CatB (white) and proteasome (black) activities compared to control are shown for 6-day Aβ₄₂ treatment (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182895#pone.0182895.g003" target="_blank">Fig 3a</a> data), for 4-day lactacystin treatment (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182895#pone.0182895.g004" target="_blank">Fig 4a</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182895#pone.0182895.t001" target="_blank">Table 1</a> data), and for 2-day PADK treatment (from Fig 7b data).</p

Proteasome activity is blocked by the inhibitor lactacystin in hippocampal slice cultures.

<p>The slices were treated daily with vehicle for 4 days (0-day control group) or with 5 μM lactacystin for 1–4 days, staggering the treatments in order for same-day preparation of slice groups of 7–9 each. Proteasome activity (mean Vmax/s ± SEM) was measured in control slices harvested at different times (dashed line) and in lactacystin-treated slice samples (a). The time-course data were analyzed by ANOVA (p<0.0001; post hoc tests compared to control: p<0.0001 at all 4 time points). A subset of the samples tested for proteasome activity was also assessed by immunoblot for the 20S proteasome α-1 subunit (20S) and actin (b), as well as for GluR1, synaptophysin (SNP), the 30-kDa CatB isoform (CatB-30), and again the actin load control (c).</p

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

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

Pre-aggregated Aβ₄₂ causes proteasomal dysfunction in correspondence with synaptic decline in rat hippocampal slice cultures.

<p>The brain tissue was maintained on culture inserts with media placed below the insert membrane—note the two Nissl-stained slices showing their insert positions and neuronal layers (a) (size bar = 3 mm). After several weeks in culture, a hippocampal slice was stained with anti-synaptophysin and DAPI to show the stable maintenance of CA1, CA3, and dentate gyrus subfields and their associated dense neuropil (b) (view-field width: 2.6 mm). Aliquots of pre-aggregated, human Aβ₄₂ peptide were diluted to 0.5–1.5 μM and applied daily to slice cultures alongside vehicle-treated slices. Proteasome chymotrypsin-like activity (mean Vmax/s ± SEM normalized to control; n = 6) was measured in hippocampal slices that were harvested after 4–6 days of treatment (c). For immunoblots, groups of 7–9 slices each were harvested after 6 days of treatment, sonicated, and equal protein aliquots assessed for the 20S proteasome α-1 subunit (d), synaptophysin (SNP; e), and actin. Mean immunoreactivity levels were normalized to their respective controls and percent ± SEM values are shown. Unpaired t-tests: ***p< 0.001.</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

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