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. 2004 Nov;24(11):1219-25.
doi: 10.1097/01.WCB.0000136706.77918.21.

Protein ubiquitination in postsynaptic densities after transient cerebral ischemia

Chen Li Liu  1 , Maryann E Martone, Bingren R Hu
Affiliations

Protein ubiquitination in postsynaptic densities after transient cerebral ischemia

Chen Li Liu et al. J Cereb Blood Flow Metab. 2004 Nov.

Abstract

The mechanisms underlying neurologic deficits and delayed neuronal death after ischemia are not fully understood. In the present study, we report that transient cerebral ischemia induces accumulation of ubiquitinated proteins (ubi-proteins) in postsynaptic densities (PSDs). By immunoelectron microscopy, we demonstrated that ubi-proteins were highly accumulated in PSD structures after ischemia. On Western blots, ubi-proteins were markedly increased in purified PSDs at 30 minutes of reperfusion, and the increase persisted until cell death in the CA1 region after ischemia. In the resistant DG area, however, the changes were transient and significantly less pronounced. Deposition of ubi-proteins in PSDs after ischemia correlates well with PSD structural damage in the CA1 region as viewed by electron microscopy. These results suggest that the ubiquitin-proteasome system fails to repair and remove damaged proteins in PSDs. The changes may demolish synaptic neurotransmission, contribute to neurologic deficits, and eventually lead to delayed neuronal death after transient cerebral ischemia.

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Figures

FIG. 1
FIG. 1
(A) Electron micrographs of PSDs isolated from hippocampal tissue. Isolated PSDs from control rats (Ctr) are small and often curved, whereas PSDs at 24 hours of reperfusion are larger in size (arrows). (B) Immunoblots of ubi-proteins in PSDs. PSDs were prepared from sham-control rats (Ctr) and rats subjected to 15 minutes of ischemia followed by 4 and 24 hours of reperfusion, respectively. The blots were labeled with the anti-ubi-protein antibody and visualized with an ECL system. Molecular sizes are indicated on the left. (C) Quantification of ubi-proteins on PSD Western blots with Kodak 1D image software. Mean optical intensities of immunoblot bands are expressed as mean ± SD (n = 4). *P < 0.01 between control and experimental conditions. One-way ANOVA followed by Fischer’s PLSD post hoc test was used to assess statistical significance. PSD, postsynaptic density; ubi-proteins, ubiquitinated proteins.
FIG. 2
FIG. 2
Immuno-electron micrographs of ubiquitin immunolabeling in the dendritic region of CA1 and DG sections from a sham-operated control rat and a rat subjected to 15 minutes of ischemia followed by 24 hours of reperfusion. The immunolabeling is distributed throughout dendrites in the CA1 and DG control (Ctr, arrowheads), as well as in the DG region after ischemia (DG, 24 hours, arrowheads) but is more concentrated in CA1 PSD structures after ischemia (CA1, 24 hours, arrows). Scale bar = 0.5 μm.
FIG. 3
FIG. 3
Double-staining confocal microscopic images of CA1 (upper) and DG (lower) regions. Brain sections were double-labeled with mouse anti-MAP-2 and rabbit anti-free ubiquitin antibodies. Sections are from sham-control (Ctr) rat and rats subjected to 15 minutes of ichemia followed by 30 minutes, 4, 24, and 72 hours of reperfusion, respectively. Free ubiquitin immunostaining (green color) is thoroughly depleted persistently in CA1 neurons after ischemia. MAP-2 immunlabeling is lost after 72 hours of reperfusion, indicative of delayed CA1 neuronal death after ischemia.
FIG. 4
FIG. 4
A) Immunoblots of ubiquitin in Triton-insoluble pellets (upper) and cytosol (S3, lower). Samples of hippocampal CA1 and DG were from sham-control rats (Ctr) and rats subjected to 15 minutes of ischemia followed by 30 minutes, 4, and 24 hours of reperfusion, respectively. Two separate samples derived from two rats in each experimental group were subjected to the SDS-PAGE. The blots were labeled with the anti-ubiquitin antibody and visualized with the ECL system. Molecular sizes are indicated on the left. (B) Changes in ubi-proteins in the synaptic pellets and cytosol were evaluated with Kodak 1D image software. Mean optical intensities of immunoblot bands are expressed as mean ± SD (n = 4). *P < 0.01 between control and experimental conditions. One-way ANOVA followed by Fischer’s PLSD post hoc test was used to assess statistical significance.
FIG. 5
FIG. 5
A) Immunoblots of ubi-proteins in postsynaptic densities (PSDs), cytosol (S3), intracellular membranes (P3) and light membranes (LM). Samples were prepared from sham-control rats (Ctr) and rats subjected to 15 minutes of ischemia followed by 4 h of reperfusion. The blots were labeled with the anti-ubi-protein antibody and visualized with the ECL system. Molecular sizes are indicated on the left. (B) Changes in ubi-proteins among subcellular fractions were evaluated with Kodak 1D image software. Mean optical intensities of immunoblot bands are expressed as mean ± SD (n = 4). *P < 0.01 between control and experimental conditions. One-way ANOVA followed by Fischer’s PLSD post hoc test was used to assess statistical significance.
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