Ubiquilin-1 protects cells from oxidative stress and ischemic stroke caused tissue injury in mice

Abstract

Ubiquilin-1 (Ubqln1 or Ubqln), a ubiquitin-like protein, mediates degradation of misfolded proteins and has been implicated in a number of pathological and physiological conditions. To better understand its function in vivo, we recently generated transgenic (Tg) mice that globally overexpress mouse Ubqln in a variety of tissues and ubqln conditional knock-out mice. The Tg mice were viable and did not show any developmental or behavioral abnormalities compared with their wild-type (WT) littermates. When subjected to oxidative stress or ischemia/reperfusion, however, ubqln Tg mice but not the WT littermates showed increased tolerance to these insults. Following ischemic stroke, ubqln Tg mice recovered motor function more rapidly than did the WT mice. In contrast, KO of ubqln exacerbated neuronal damage after stroke. In addition, KO of ubqln also caused accumulation of ubiquitinated proteins. When ubqln KO mice were crossed with a ubiquitin-proteasome system function reporter mouse, the accumulation of a proteasome surrogate substrate was observed. These results suggest that Ubqln protects mice from oxidative stress and ischemic stroke-caused neuronal injury through facilitating removal of damaged proteins. Thus, enhanced removal of unwanted proteins is a potential therapeutic strategy for treating stroke-caused neuronal injury.

Figure 1.

OE of Ubqln in the Tg mice reduces oxidative stress-induced cell death in the liver. A, B, Western blot analysis showing Ubqln OE in the indicated tissues in the Tg-2 (A) and Tg-4 line (B) mice at 2 months. Immunoblotting of β-tubulin was used as a loading control. C, Fluorescence microscopy analysis of intracellular ROS levels in hepatocytes. WT or Tg mice were intraperitoneally injected with 150 mg/kg menadione or vehicle (control). After 6 h following the injection, animals were killed and subjected to ROS analysis. D, Graph showing the measurement of ROS levels in WT and Tg livers in presence or absence of menadione treatment. Data are shown as mean ± SEM; n = 3; *p < 0.001, **p < 0.05. E, Representative liver sections were stained with H&E. Note that compared with the Tg mouse liver, menadione treatment in the WT mouse liver caused increased vacuolization, cell disruption, and karyolysis (pointed by arrows). F, Measurement of ATP levels of liver cells of Tg and WT mice at 2 months after 6 h of menadione treatment. Data are shown as mean ± SD; n = 3 for each group; *p < 0.01. G, Measurement of caspase-3 activity of liver cells of Tg and WT mice (at 2 months) following 6 h of menadione treatment. Data are shown as mean ± SD; n = 3 for each group; *p < 0.05. H, Confocal microscopy showing TUNEL positively stained cells (green) in the liver of the WT and Tg mice (at 2 months) after menadione treatment. Scale bar, 50 μm. I, Graph showing the percentage of TUNEL positively stained cells in livers. Data are shown as mean ± SD; n = 4 for each group; *p < 0.05.

Figure 2.

OE of Ubqln in the Tg mice reduces oxidative stress-induced cell death in the brain. A, Fluorescence microscopy analysis of intracellular ROS levels in the brain cortex. WT or Tg mice were intraperitoneally injected with 100 mg/kg menadione or vehicle (control) daily. After 3 d injections, animals were killed and subjected to ROS analysis. B, Graph showing the measurement of ROS levels in WT and Tg brain cortex in presence or absence of menadione treatment. Data are shown as mean ± SEM; n = 3; *p < 0.05. C, Caspase-3 activity of brain cortex of Tg and WT mice at 2 months after 3 d menadione treatment. Data are shown as mean ± SD; n = 3 for each group; *p < 0.01. D, Brain sections were stained with FJB to identify degeneration neurons. E, Graph showing the quantification of neurodegeneration in the WT and Tg mouse brain cortex after 3-d menadione injection. Data are shown as mean ± SEM; n = 3; *p < 0.05. F, Confocal microscopy showing apoptotic cells (green) in the brain cortex after 3 d menadione treatment in the mice at 2 months. Scale bar, 50 μm. G, Graph showing the percentage of TUNEL positively stained cells in brain cortex after 3 d menadione treatment. Data are shown as mean ± SD; n = 4 for each group; *p < 0.001. H, Confocal microscopy showing TUNEL positively stained cells (green) in the brain striatum after 3 d menadione treatment in the mice at 2 months. Scale bar, 50 μm. I, Graph showing the percentage of TUNEL positively stained cells in brain striatum after 3 d menadione treatment. Data are shown as mean ± SD; n = 4 for each group; *p < 0.001.

Figure 3.

ubqln Tg mice showed reduced neuronal damage after stroke. A, TTC staining of coronal sections from ischemic mouse brains. The white indicates infarction. B, Measurement of infarct volume. Data are shown as mean ± SD; n = 7 for the WT mice; n = 5 for the Tg mice; *p < 0.05.

Figure 4.

ubqln Tg mice show enhanced motor function recovery after transient focal cerebral ischemia. A, Body weight of Tg and WT mice measured each d after surgery. n = 9 for the WT mice; n = 11 for the Tg mice. B, Rotarod test results following the surgery. The cutoff time for mice staying on the rotarod was set at 250 s. A two-way ANOVA (time by genotype) revealed a significant effect of genotype (F_(10,241) = 53.83, p < 0.0001), and a significant effect of time (F_(10,241) = 5.79, p < 0.001). Data are shown as mean ± SD; n = 9 for the wild-type mice; n = 11 for the Tg mice.

Figure 5.

Generation of ubqln cKO mice. A, An illustration of ubqln WT allele, targeting vector, and recombinant ubqln alleles. Red bars, probes for Southern blot. B, C, Genotyping of ubqln-floxed mice by Southern blot analysis of BamHI digested genomic DNA with a 5′ probe (B) or with a 3′ probe (C) from the WT mice (lanes 1, 2), targeted embryonic stem (ES) cells (lanes 3, 4), and heterozygous floxed F2 generation mice (lanes 5–7). D, PCR analysis of the floxed F2 generation mice following chimera breeding. WT: lanes 2 and 9; targeted ES cells: lanes 3 and 4; buffer control: lane 5; heterozygous floxed mice: lanes 6–8. Lane 1, DNA ladder. E, Examination of Ubqln protein levels in the brain in the synapsin-I promoter-regulated Cre recombinase mediated neuron-specific KO and WT control mice by Western blotting.

Figure 6.

Synapsin-I neuron-specific KO of ubqln exacerbates ischemia/reperfusion-caused brain injury and delays functional recovery. A, KO of ubqln in neurons exacerbates neuronal injury as indicated by TTC staining 24 h after ischemic stroke. B, Measurement of infarct volume. Data are shown as mean ± SD; n = 7 for each group; *p < 0.05. C, Rotarod test results suggesting that KO of ubqln in neurons delays functional recovery after stroke. The cutoff time for mice staying on the rotarod was set at 250 s. A two-way ANOVA (time by genotype) revealed a significant effect of genotype (F_(10,248) = 6.30, p = 0.0127), and a significant effect of time (F_(10,248) = 7.04, p < 0.001). Data are shown as mean ± SD; n = 6 for the WT mice; n = 7 for the KO mice.

Figure 7.

Accumulation of ubiquitinated proteins and a UPS function reporter protein, GFPu, in the brain cortex of the synapsin-I neuron-specific ubqln cKO mice. A, Western blot analysis of ubiquitinated proteins in WT and ubqln KO brain cortex at 2 months. B, Quantification of ubiquitinated protein levels. Data are shown as mean ± SD; n = 3; *p < 0.05. C, Western blot analysis of GFPu expression in brain cortex derived from GFPu, WT control, and ubqln-KO/ubqln mice at 2 months. D, GFP expression was quantified and normalized against tubulin levels. Data are shown as mean ± SD; n = 3; *p < 0.05.