Elevated post-ischemic ubiquitination results from suppression of deubiquitinase activity and not proteasome inhibition

Abstract

Cerebral ischemia-reperfusion increases intraneuronal levels of ubiquitinated proteins, but the factors driving ubiquitination and whether it results from altered proteostasis remain unclear. To address these questions, we used in vivo and in vitro models of cerebral ischemia-reperfusion, in which hippocampal slices were transiently deprived of oxygen and glucose to simulate ischemia followed by reperfusion, or the middle cerebral artery was temporarily occluded in mice. We found that post-ischemic ubiquitination results from two key steps: restoration of ATP at reperfusion, which allows initiation of protein ubiquitination, and free radical production, which, in the presence of sufficient ATP, increases ubiquitination above pre-ischemic levels. Surprisingly, free radicals did not augment ubiquitination through inhibition of the proteasome as previously believed. Although reduced proteasomal activity was detected after ischemia, this was neither caused by free radicals nor sufficient in magnitude to induce appreciable accumulation of proteasomal target proteins or ubiquitin-proteasome reporters. Instead, we found that ischemia-derived free radicals inhibit deubiquitinases, a class of proteases that cleaves ubiquitin chains from proteins, which was sufficient to elevate ubiquitination after ischemia. Our data provide evidence that free radical-dependent deubiquitinase inactivation rather than proteasomal inhibition drives ubiquitination following ischemia-reperfusion, and as such call for a reevaluation of the mechanisms of post-ischemic ubiquitination, previously attributed to altered proteostasis. Since deubiquitinase inhibition is considered an endogenous neuroprotective mechanism to shield proteins from oxidative damage, modulation of deubiquitinase activity may be of therapeutic value to maintain protein integrity after an ischemic insult.

Keywords: Cerebral ischemia–reperfusion; Deubiquitinase inhibition; Free radical production; Ubiquitin.

Fig. 1

Post-ischemic ubiquitination is enabled by reperfusion-dependent recovery of ATP production. a Hippocampal brain slices were exposed to control condition or OGD for 1 h followed by reperfusion of 0, 5, 20, 60, and 120 min. Tissue slices were harvested, and ubiquitinated proteins were isolated and detected by Western blotting with anti-ubiquitin antibody. β-Actin served as loading control. *P < 0.01 from co (60 min P = 0.0002; 120 min P = 0.0044); n = 5–7 slice pools/group. ATP concentrations were measured at the same timepoints. *P < 0.01 from co (0 min P < 0.0001; 5 min P = 0.0001; 20 min P = 0.007); n = 5–6 slice pools/group. b Hippocampal slices underwent OGD, followed by 60 min reperfusion in the presence of oxygen with and without glucose (left panel), or in the presence of glucose with and without oxygen (right panel). Ubiquitin levels were determined as in (a). Left panel: *P = 0.0014 from co; P = 0.052 from OGD (+ glu); n = 5 slice pools/group; right panel: *P = 0.0008 from co; ^#P  < 0.0001 from OGD (+ O₂); n = 3 slice pools/group. c ATP levels were determined in slices exposed to OGD/60 min reperfusion. *P < 0.001 from OGD/60 min reperfusion (–glu P < 0.0001; –O₂ P < 0.0001); n = 12 slice pools/group. d E1 ubiquitin-activating enzyme binding to ubiquitin was assessed in lysates obtained under non-reducing (upper panel) or reducing (lower panel) conditions from control and OGD samples with and without reperfusion. E1-ubiquitin (E1 + Ub) (upper band) and native E1 (lower band) were visualized by Western blotting with anti-E1 antibody. Blots are representative of three independent experiments. e Lysates from control and OGD-treated slices with and without reperfusion were incubated with ubiquitin/DHA in the absence and presence of ATP and the ATP-diphosphohydrolase apyrase. Labeling of E1 enzyme with ubiquitin/DHA was determined by Western Blotting with anti-E1 antibody. Ubiquitin levels were monitored with anti-ubiquitin antibody. The blot is representative of three independent experiments. f Ubiquitin accumulation after OGD/60 min reperfusion was examined in the absence and presence of the E1 activating enzyme inhibitor PYR41 by Western Blot and quantified. *P = 0.0016 from co, ^#P = 0.0145 from OGD-PYR41, n = 3 slice pools/group. ATP adenosine triphosphate, co control, glu glucose, min minutes, O₂ oxygen, OGD oxygen–glucose deprivation, rep reperfusion, Ub ubiquitin

Fig. 2

ROS are necessary and sufficient to promote post-ischemic ubiquitination. a Slices were exposed to control conditions or OGD/60 min reperfusion in the absence and presence of the free radical scavengers 4-hydroxy-Tempo or MnTBAP. Free radical production was assessed in CA regions of slices by DHE fluorescence. Representative images are shown. Scale bar = 400 µm. b Slices were treated as in a and harvested for determination of ubiquitin accumulation. Tem: *P < 0.0001 from co; ^#P = 0.0013 from OGD (–Tem); n = 6/group; MnT: *P = 0.0032 from co; ^#P = 0.0027 from OGD (–MnT); n = 3 slice pools/group. c ROS production was induced in hippocampal slices by incubation with H₂O₂ for 1 h. ROS levels were assessed by DHE fluorescence. *P < 0.0001, n = 12 slices/group. Scale bar = 400 µm. d Free radical production was initiated by treatment with X/XO for 1 hr and ROS levels were measured by DHE fluorescence. *P < 0.0001, n = 6 slices/group. Scale bar = 400 µm. e ROS production was induced as in c. Slices were harvested and ubiquitin accumulation in the absence and presence of H₂O₂ was tested by Western Blotting with anti-ubiquitin antibody. β-Actin served as loading control. *P = 0.0043, n = 13–14 slice pools/group. f ROS production was induced as in d and the level of ubiquitin accumulation was determined. *P = 0.003, n = 6–7 slice pools/group. co control, DHE dihydroethidium, H₂O₂ hydrogen peroxide, MnT MnTBAP, O₂ oxygen, OGD oxygen–glucose deprivation, rep reperfusion, Tem 4-hydroxy-Tempo, Ub ubiquitin, X/XO xanthine/xanthine oxidase

Fig. 3

ROS induce Lys⁴⁸ and Lys⁶³ ubiquitination. a Hippocampal slices exposed to H₂O₂ were harvested and lysates were probed for the presence of Lys⁶³- and Lys⁴⁸-linked ubiquitin chains. β-Actin served as loading control. Representative blots and quantifications are shown. Lys⁶³: *P < 0.0001; Lys⁴⁸: *P = 0.0035, n = 8 slice pools/group. b Same as in a, but treated with X/XO. Lys⁶³: *P < 0.0001; Lys⁴⁸: *P < 0.0001, n = 7 slice pools/group. H₂O₂ hydrogen peroxide, Lys⁴⁸ lysine 48, Lys⁶³ lysine 63, Ub ubiquitin, X/XO xanthine/xanthine oxidase

Fig. 4

Proteasome inhibition does not contribute to post-ischemic ubiquitination. a Hippocampal slice cultures were exposed to control conditions or 1 h OGD followed by 0, 5, 20, 60, and 120 min reperfusion. Chymotryptic, tryptic, and caspase-like proteasomal activities were determined in lysates by incubation with AMC-labeled fluorogenic peptides carrying respective cleavage sites. The graph shows relative proteasomal activity levels in percent at each time point. Maximal decrease was detected after 20 min reperfusion to 47.2 ± 1.7% (chymotryptic activity), 47.7 ± 1.6% (tryptic activity), and 50.2 ± 2.8% (caspase-like activity) of control. *P < 0.01 from co (chymotryptic: 0 min P = 0.0025, 5 min P = 0.0006, 20 min P < 0.0001, 60 min P = 0.0002, 120 min P = 0.0002; tryptic: 0 min P = 0.0002, 5 min P < 0.0001, 20 min P < 0.0001, 60 min P < 0.0001, 120 min P = 0.0005; caspase-like: 0 min P = 0.0006, 5 min P = 0.0004, 20 min P = 0.0001, 60 min P = 0.0001, 120 min P = 0.0004); n = 3 slice pools/group. b Slices were treated with the proteasomal inhibitor epoxomicin at 0.2 and 10 µM for 2 h, and proteasomal activities were measured as in (A). Chymotryptic: ^#P < 0.0001; tryptic: ^#P < 0.0001; caspase-like: ^#P = 0.0006 from untreated; chymotryptic: *P = 0.0021; tryptic: *P < 0.0001; caspase-like *P < 0.0001 from 0.2 µM; n = 4 slice pools/group. c Ubiquitin accumulation was assessed in slices treated with epoxomicin for 2h and compared to OGD/60 min reperfusion by Western Blotting. Optical densities of ubiquitin-stained bands were measured and changes in ubiquitin levels were expressed relative to untreated controls. β-Actin served as normalization and loading control. ^#P = 0.018 and *P < 0.0001 from untreated; n = 4 slice pools/group. d Protein levels of neuronal proteasomal targets GluN1 and Shank were determined before and after OGD with and without addition of 10 µM epoxomicin, and quantified relative to β-actin. GluN1: *P = 0.0016; Shank: *P = 0.018 from OGD/rep –epox; n = 7 slice pools/group. e Hippocampal slices were transduced with AAV2/1 expressing the UPS reporter Ub-R-GFP and a control reporter mCherry, both under the neuronal SYN1 promoter. Green and red fluorescence was monitored in control and OGD-treated slices after 6 h reperfusion. As positive control, after OGD the proteasome was inhibited with epoxomicin (10 µM). Ub-R-GFP levels relative to mCherry were quantified in the CA region. *P < 0.0001; n = 24 slices/group. Scale bar = 400 µm. f Mice were intracortically injected with neuronal AAV2/1 expressing Ub-R-GFP or GFPu together with mCherry. The targeted expression area is shown in a representative DAPI-stained coronal brain section, with a scale bar of 1 mm. Mice underwent sham or MCAO surgery followed by 16 h reperfusion. Intracerebroventricular injection with 50 µg Bortezomib was performed to show maximal possible accumulation of UPS reporters. Fluorescence levels of reporters were monitored and representative images are shown. Scale bar = 100 µm. Ub-R-GFP n = 3 mice/group. GFPu n = 2–3 mice/group. co control, epox epoxomicin, h hours, min minutes, ns not significant, OGD oxygen–glucose deprivation, rep reperfusion, Ub ubiquitin

Fig. 5

ROS promote ubiquitination without causing proteasomal inhibition. a Proteasomal chymotryptic, tryptic, and caspase-like activities were measured using fluorogenic substrate peptides in slices exposed to control or OGD with or without reperfusion. *P < 0.001 from co (chymotryptic: OGD + rep P < 0.0001; OGD − rep P = 0.001; tryptic: OGD + rep P < 0.0001; OGD-rep P = 0.0001; caspase-like: OGD + rep P < 0.0001; OGD-rep P = 0.0003); n = 3 slice pools/group. b Proteasomal activities were measured and quantified as in a in slices maintained under control conditions or OGD followed by reperfusion in presence and absence of oxygen. *P < 0.05 from co (chymotryptic: OGD/re P + O₂ P = 0.0021; OGD/rep − O₂ P < 0.0001; tryptic: OGD/re P + O₂ P = 0.0006; OGD/rep − O₂ P < 0.0001; caspase-like: OGD/re P + O₂ P = 0.042; OGD/rep − O₂ P = 0.0009); n = 6 slice pools/group. c ROS production was assessed by DHE fluorescence in CA regions of hippocampal slices treated as in a. Representative images as well as quantification of n = 24–30 slices/group are shown. *P < 0.0001 from OGD-rep. Scale bar = 400 µm. d Free radical production was determined by DHE fluorescence in slices treated as in b. *P < 0.0001 from OGD/re P + O₂; n = 24 slices/group. Representative images and quantification are shown. Scale bar = 400 µm. e ROS production was induced in hippocampal slices by incubation with H₂O₂ for 1 h, after which proteasomal activities were measured. n = 9–10 slice pools/group. f Free radical production was initiated by treatment with X/XO and proteasomal activities were measured. n = 6–7 slice pools/group. co control, DHE dihydroethidium, H₂O₂ hydrogen peroxide, O₂ oxygen, OGD oxygen–glucose deprivation, rep reperfusion, X/XO xanthine/xanthine oxidase

Fig. 6

ROS production induced by H₂O₂ or OGD reduces intracellular DUB activity. a Neuro2a cells were treated for 60 min with 500 µM H₂O₂ and harvested for measuring ubiquitination levels. A representative blot and quantification are shown. *P = 0.0043, n = 4–6/group. b Cells were treated as in a and DUB activity was measured by incubating lysates with a fluorogenic ubiquitin molecule coupled to AMC. Addition of the DUB inhibitor NEM showed maximal achievable inhibition. *P < 0.0001, ^#P < 0.0001 from control; n = 8/group. c Experiment was performed as in b. Lysates were incubated with the DUB activity probe ubiquitin-PA coupled to Cy5. Binding of DUBs to the probe was examined after SDS-PAGE by direct detection of Cy5 fluorescence on PVDF membranes. Band intensities, which are directly proportional to the activities of nine unidentified DUBs (#1–9), were quantified. DUB1 *P < 0.0001, DUB2 *P = 0.0002, DUB3 *P < 0.0001, DUB4 *P = 0.0014, DUB5 *P = 0.0007, DUB6 *P = 0.0005, DUB7 *P < 0.0001, DUB8 *P = 0.0002; n = 8/group. d Neuro2a cells were exposed to OGD/30 min reperfusion and harvested to determine ubiquitination levels. A representative blot and quantification are shown. *P < 0.0001, n = 11–12/group. e Cells were treated as in d to measure DUB activity using ubiquitin-AMC as substrate. *P < 0.0001, ^#P < 0.0001 from control; n = 5–10/group. f DUB activity was analyzed as in c using ubiquitin-PA coupled to Cy5. DUB1 *P = 0.012, DUB4 *P = 0.011, DUB5 *P < 0.0001, DUB6 *P = 0.0014, DUB7 *P = 0.012, DUB8 *P = 0.002, DUB9 *P = 0.042; n = 4/group. g Neuro2a cells were exposed to OGD/reperfusion in the absence and presence of the free radical scavenger MnTBAP, and DUB activity was measured with ubiquitin-AMC. *P = 0.0002 from control, ^#P = 0.0004 from OGD (–MnT); n = 12/group. h Hippocampal slices exposed to H₂O₂ were harvested and lysates were probed for DUB activity with ubiquitin-AMC. *P < 0.0001, n = 10–11 slice pools/group. i Hippocampal slices exposed to OGD/reperfusion were harvested and lysates were probed for DUB activity with ubiquitin-AMC. *P < 0.0001, n = 10–12 slice pools/group. co control, DUB deubiquitinase, H₂O₂ hydrogen peroxide, MnT MnTBAP, NEM N-ethylmaleimide, OGD oxygen glucose deprivation, Ub ubiquitin

Fig. 7

The OGD-mediated decrease in DUB activity is sufficient to induce ubiquitination. a Neuro2a cells were treated for 60 min with 0, 20, and 30 µM broad-spectrum DUB inhibitor PR619, and DUB activity was measured by incubating lysates with a fluorogenic ubiquitin molecule coupled to AMC. *P = 0.0022, ^#P < 0.0001 from 0 µM PR619; n = 8/group. b Cells were treated as in a and harvested for measuring ubiquitination levels. A representative blot and quantification are shown. *P < 0.0001, n = 6/group. c Hippocampal brain slices were exposed to 3 mM PR619 for 2 h. DUB activity was determined as in a. *P = 0.0062 from 0 µM PR619; n = 5 slice pools/group. d PR619-treated brain slices were harvested and analyzed for induction of ubiquitination by Western Blotting. *P < 0.0001, n = 5 slice pools/group. DUB deubiquitinase, Ub ubiquitin