Protein aggregation and proteasome dysfunction after brain ischemia

Abstract

Background and purpose: Protein unfolding and aggregation are dominant early pathogenic events in neurons after brain ischemia. This study used a transient cerebral ischemia model to investigate whether overproduction of unfolded proteins after brain ischemia is a consequence of proteasome dysfunction.

Methods: Proteasome peptidase activity and proteasome subcellular redistribution and assembly were studied by peptidase activity assay, Western blot analysis, and size-exclusion chromatography.

Results: Proteasome peptidase activity, as determined with the peptide substrate succinyl-LLVY-7-amino-4-methylcoumarin, was moderately decreased, and the 26S proteasome was disassembled during the early period of reperfusion after transient brain ischemia. Furthermore, the proteasome subunits, particularly the 19S components, were deposited into the protein aggregate-containing fraction after an episode of transient cerebral ischemia.

Conclusions: These results clearly demonstrate that after an episode of brain ischemia, proteasomes are disassembled and aggregated and thus fail to function normally. Deposition of proteasomes into protein aggregates may also indicate that proteasomes attempt to degrade ubiquitin-conjugated proteins (ubiproteins) overproduced after brain ischemia. However, ubiproteins are too numerous to be degraded and trap some of the proteasomes into their aggregates after brain ischemia.

Figure 1

A, Electron photomicrographs of neocortical neurons from a sham-operated control rat and rats subjected to 20 minutes of ischemia followed by 24 and 72 hours of reperfusion. Sham control neurons contained normal polyribosomes (arrow) and cellular organelles. Large quantities of abnormal aggregates (arrows) and intracellular vacuoles (arrowheads) had accumulated in neurons at 24 hours of reperfusion. Ischemic dead neurons at 72 hours of reperfusion contained no viable organelles (small arrow) and clumped chromatin (large arrows). N indicates nucleus; ER, rough endoplasmic reticulum; M, mitochondria. B: Acid fuchsin– and celestine blue–stained neocortical neurons in brain sections adjacent to the brain sections used for the EM study (see Methods). Neuronal nuclei from controls and after 24 hours of reperfusion are round (arrowheads). Ischemic dead neurons at 72 hours of reperfusion have acidophilic cytoplasm, as well as dark and shrunken or polygonal nuclei (arrowheads).

Figure 2

A and B, Immunoblots of ubi-proteins in TX-insoluble aggregates. Brain samples were obtained from neo-cortical tissues of sham-operated control rats and rats subjected to 20 minutes of cerebral ischemia followed by 0.5, 4, 24, and 72 hours of reperfusion. TX-insoluble fractions were immunoblotted with antibody to ubiproteins. A, Two separate samples derived from 2 different rats in each experimental group are shown. B, Changes in optical density of ubiproteins from 4 different individual rat samples were evaluated. Data are mean±SD (n=4). *P<0.05 between control and experimental conditions, 1-way ANOVA followed by Dunnett’s test.

Figure 3

Proteasome peptidase activity in supernatant and pellet fractions after brain ischemia. Brain samples were obtained from the same sets of samples as those described in Figure 2. Four different individual rat samples in each group were assayed with the succinyl-LLVY-AMC substrate in the presence or absence of 30 μmol/L lactacystin. Proteasome activity was calculated by subtracting the lactacystin-insensitive (or nonproteasome) peptidase activity from total peptidase activity. White bars indicate the supernatant. Black bars indicate the pellet. Data are mean±SD (n=4). *P<0.05 between control and experimental conditions, 1-way ANOVA followed by Dunnett’s test.

Figure 4

Immunoblots (A and C) and quantitative analysis (B and D) of preoteasome subunits and actin in TX-insoluble and cytosolic fractions after brain ischemia. Brain samples were obtained from the same sets of rats as those described in Figure 2. Proteasome subunits were immunoblotted with antibodies against 20Sα-1 (25 kDa) and 19S-S10B (40/42 kDa). Actin-β was used as an endogenous protein loading control. Molecular sizes are indicated by arrows. Data are mean±SD (n=4). *P<0.05 between control and experimental conditions, 1-way ANOVA followed by Dunnett’s test.

Figure 5

Assessment of 26S proteasomes and 20S and 19S proteasomal subcomplexes by size-exclusion chromatography. Tissue samples from the same rats as those described in Figure 2 were used. A, Succinyl-LLVY-AMC–hydrolyzing proteasome activity was assayed as described in Methods. B, The chromatographic fractions 1 to 48 were subjected to SDS-PAGE and immunoblotted with antibodies to 20Sα-1 and 19S-S10B. The chromatographic fractions 17 to 40 containing 20Sα-1 and 19S-S10B protein bands are shown. C: Quantitative analysis of immunoblot intensity ratio between the 26S proteasomal peak and either the 20S or the 19S proteasomal peaks. The sums of immunoblot intensities of the 26S chromatographic peak (fractions 18 to 22), 20S peak (fractions 23 to 28), and 19S peak (fractions 29 to 35) were measured with Kodak 1D image software. Data are mean±SD (n=3). *P<0.05 between control and experimental conditions, 1-way ANOVA followed by Dunnett’s test.