Fig 1.
Pre-aggregated Aβ42 causes proteasomal dysfunction in correspondence with synaptic decline in rat hippocampal slice cultures.
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.
Fig 2.
Treatment with pre-aggregated Aβ42 peptide leads to increased phosphorylation of tau residues Ser199 and Ser202.
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-Ser199/202 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.
Fig 3.
Aβ42-induced proteasome inhibition is associated with a delayed, inverse effect on CatB activity.
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-Ser199/202 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.
Fig 4.
Proteasome activity is blocked by the inhibitor lactacystin in hippocampal slice cultures.
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).
Fig 5.
Hippocampal slice cultures exhibit stable proteasomal activity.
Proteasome activity was measured with a fluorogenic peptide assay in control hippocampal slices harvested across a wide range of culture days. The mean Vmax/s ± SEM data were plotted across days in culture for the different slice samples in order to verify stable maintenance of proteasome function in the tissue model.
Table 1.
Lactacystin-induced proteasome inhibition leads to the enhancement of CatB activity.
Fig 6.
The proteasome inhibitor lactacystin causes delayed synaptic marker loss in hippocampal slice cultures.
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.).
Table 2.
Among screened compounds, PADK was chosen as an effective CatB-enhancing agent for further testing in the hippocampal slice model.
Fig 7.
The effective CatB-enhancing agent PADK (chosen from Table 2) selectively enhances CatB activity in hippocampal slice cultures.
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 Fig 3a data), for 4-day lactacystin treatment (from Fig 4a and Table 1 data), and for 2-day PADK treatment (from Fig 7b data).
Fig 8.
The CatB-enhancing compound PADK positively modulates the proteasome pathway in Aβ42–treated hippocampal slice cultures.
Panel a: Slice cultures were treated daily with vehicle (veh) or 1.5 μM pre-aggregated Aβ42 for 6 days, or they were pre-treated with 3 μM PADK for 1 day before starting the 6 daily Aβ42 treatments in combination with 3 μM PADK. Proteasome activity was measured with a fluorogenic peptide assay and Vmax/s measures were normalized to vehicle-treated samples (mean ± SEM). Unpaired t test compared to control: *p = 0.0196; compared to Aβ42 alone: ##p = 0.027. Panel b: Slice cultures were treated with vehicle (veh), 10 μM lactacystin (LAC), or with 10 μM LAC in the presence of PADK for 2 days. Proteasome activity was assessed in harvested slices and measures were normalized to vehicle-treated samples (mean ± SEM). Unpaired t test compared to vehicle control: ***p<0.0001.
Fig 9.
Aβ42–induced decline in proteasome activity is diminished by delayed PADK treatment.
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.
Fig 10.
Aβ42–induced increase in phosphorylated tau is attenuated by PADK.
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-Ser199/202 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-Ser199/202, the 30-kDa CatB isoform (CatB-30), synaptophysin (SNP), GluR1, and actin (c).
Fig 11.
Model of the putative crosstalk between the two major protein clearance pathways.
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.