Ubiquitin proteasome-mediated synaptic reorganization: a novel mechanism underlying rapid ischemic tolerance

Abstract

Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin-beta-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.

Figure 1.

Rapid ischemic tolerance is blocked by proteasome inhibition. A, Schematic of the rapid ischemic tolerance paradigm. PC (30 min OGD) is administered 1 h before harmful ischemia (120 min OGD). Cell death is assessed 24 h later by propidium iodide from exposure to 120 min OGD. B, Addition of the proteasome inhibitors MG132 (0.1 μm), MG115 (0.1 μm), or clasto-lactacystin-β-lactone (1 μm) after preconditioning blocks neuroprotection (data shown are mean ± SEM; n = 9, 5, 4, and 3 independent experiments, respectively; *p < 0.001 vs PC + 120 min OGD). C, Representative 40× images of DAPI- (blue) and propidium iodide- (red) stained cultures. Control, MG132, MG115, or clasto-lactacystin-β-lactone-treated cells were subject to no ischemia, 120 min OGD, or PC + 120 min OGD and recovered for 24 h. Scale bar, 25 μm. All experiments were performed on cortical neurons in culture at 10–14 DIV.

Figure 2.

Degradation of MARCKS and fascin after preconditioning ischemia. A, Immunoblot images of MARCKS, fascin, and actin protein levels in control (C) and 1 h after PC (30 min OGD) showing a reduction in fascin and MARCKS, but not actin protein levels after PC that is reversed by MG132 (0.1 μm). Blots are representative images of four and six independent experiments, respectively. Data are quantified in supplemental Figure 3A (available at www.jneurosci.org as supplemental material). B, Immunoblot of MARCKS and fascin after immunoprecipitation of actin in cells subject to preconditioning, showing reduced interaction of actin with MARCKS and fascin after PC that is reversed by MG132. Blots are representative images of three and four independent experiments, respectively. Data are quantified in supplemental Figure 3B (available at www.jneurosci.org as supplemental material). C, D, MARCKS immunostaining in control neuronal cultures (C) and 1 h after preconditioning ischemia (D). MARCKS staining was reduced after preconditioning ischemia. MARCKS (red) and DAPI (blue) staining were visualized using a 100× oil-immersion objective. Scale bar, 20 μm. E, F, Fascin immunostaining in neuronal cultures. E, Note process-like staining pattern in control untreated cells. F, Fascin staining after preconditioning ischemia and 1 h recovery. Note translocation of fascin to cell soma. Fascin staining (green) was visualized using a 40× objective. Scale bar, 20 μm. Images shown are representative images of at least two experiments. All experiments were performed on cortical neurons in culture at 10–14 DIV.

Figure 3.

Reorganization of actin after preconditioning ischemia. A, Actin filaments are revealed by staining with phalloidin (conjugated with Oregon green 488). B, One hour after PC (30 min OGD), the staining pattern changes from punctate to filamentous, with staining also observed around the cell body. Phalloidin (green) and DAPI (blue) staining were visualized using a 40× objective. Scale bar, 20 μm. Arrows denote cells in control (yellow) and preconditioned cells (white). C, D, Higher-magnification images (100×) of phalloidin staining in control (C) and preconditioned cells recovered for 1 h (D). Images were acquired using a Zeiss Apotome. Scale bar, 10 μm. E, Quantification of puncta in control and preconditioned cells. Data shown are mean ± SEM (n = 6). F, Solubilization of filamentous F-actin [in pellet (P)] into globular G-actin [in supernatant (S)] after preconditioning ischemia. After separation, actin was visualized by immunoblotting. Data shown are mean ± SEM (n = 3). The ratio of F-actin to G-actin was determined using a kit (Cytoskeleton, Denver, CO). G, Quantitation of G-actin/F-actin ratios. Data shown are mean ± SEM (n = 3). H, Rapid ischemic tolerance is blocked by the actin-stabilizing compound jasplakinolide. Addition of jasplakinolide (1.0 μm) to cells after PC blocks the preconditioning-induced neuroprotection. Data shown are mean ± SEM (n = 5). *p < 0.05 versus 120 min OGD; **p < 0.05 versus PC + 120 min OGD. All experiments were performed on cortical neurons in culture at 10–14 DIV.

Figure 4.

Changes in dendritic morphology after preconditioning ischemia. A, B, Dendritic morphology was visualized using DiO incorporation into membranes of cortical cells. Cells were incubated with DiO overnight before ischemia. DiO staining was visualized using a confocal microscope and a 63× water-immersion lens. C, D, One hour after PC (30 min OGD), there is a reduction in the number of visible spines. Scale bar, A, C, 10 μm. B, D, Enlarged images of the regions marked by the yellow dashed box. E, Quantification of dendritic spines 1 and 4 h after PC (30 min OGD). Data shown are mean ± SEM (n = 4). **p < 0.01 versus control. F, Analysis of spine number by dendrite location 1 h after preconditioning ischemia. Dendrites were determined to be primary (touching the cell soma), secondary (contacting a primary), or tertiary (contacting a secondary). Ischemic preconditioning reduced spine density on all dendrites. Data shown are mean ± SEM (n = 8). **p < 0.01 versus region control. G, Some cultures were incubated with the proteasome inhibitor MG132 (0.1 μm) or the actin stabilizer jasplakinolide (Jas) (1.0 μm) after the preconditioning ischemia. Spine density was determined using DiO staining 1 h after preconditioning ischemia. A further breakdown of the compound effects by region is in supplemental Figure 2A (available at www.jneurosci.org as supplemental material). Data shown are mean ± SEM (n = 4). *p < 0.001 versus control; **p < 0.01 versus PC. All experiments were performed on cortical neurons in culture at 10–14 DIV.

Figure 5.

Preconditioning ischemia induces formation of varicosities on dendrites. A, Quantification of number of varicosities 1 and 4 h after PC (30 min OGD). Data shown are mean ± SEM (n = 4). *p < 0.05 versus control. B, Analysis of varicosity density by dendrite location. Dendrites were determined to be primary (touching the cell soma), secondary (contacting a primary), or tertiary (contacting a secondary). Data shown are mean ± SEM (n = 4). **p < 0.01 versus control. C, D, MAP 2 immunocytochemistry (green) in control (C) and preconditioned cells (D). Scale bar, 20 μm. E, Some cultures received the proteasome inhibitor MG132 (0.1 μm) or the actin stabilizer jasplakinolide (1.0 μm) (Jas) after the PC. Varicosity density was determined 1 h after preconditioning ischemia, as above using DiO staining. A further breakdown of the compound effects on varicosities by region is in supplemental Figure 2B (available at www.jneurosci.org as supplemental material). Data shown are mean ± SEM (n = 4). ^†p < 0.001 versus control; *p < 0.05, **p < 0.01 versus PC. All experiments were performed on cortical neurons in culture at 10–14 DIV.

Figure 6.

Uncoupling of NMDA receptors from the actin cytoskeleton and reduced excitotoxicity in rapid ischemic tolerance. A, Immunoblot images showing the expression of NR1, NR2A, NR2B, and PSD-95 protein expression in controls (C) and 1 h after PC (30 min OGD). Data shown are representative images of three independent experiments, respectively. Data are quantified in supplemental Figure 3C (available at www.jneurosci.org as supplemental material). B, Immunoblot images showing expression of NR2B and PSD-95 after actin immunoprecipitation. Note the loss of NR2B and PSD-95 association with actin after preconditioning ischemia. Data shown are representative images of four experiments. Data are quantified in supplemental Figure 3D (available at www.jneurosci.org as supplemental material). C, Whole-cell patch-clamp recording show a smaller peak current in response to NMDA in cells preconditioned with 30 min OGD (PC). Currents are recorded by voltage clamp and exposing the cells to 200 μm NMDA and 5 μm glycine 1 h after the preconditioning stimulus. D, Quantification of electrophysiological recordings. Data shown are mean ± SEM (n = 18 and 17). *p < 0.05 versus control cells. E, Preconditioning induces tolerance to NMDA excitotoxicity. After PC (30 min OGD) and 1 h recovery, cells were incubated with 200 μm NMDA for 1 h, and then cell death was assessed 24 h later. Preconditioning the cells reduced the toxic effects of NMDA. The protection against NMDA was reduced in cells incubated with either MG132 (0.1 μm) or jasplakinolide (1.0 μm) for 1 h after the preconditioning ischemia. Data shown are mean ± SEM (n = 7). ^†p < 0.05 versus NMDA; *p < 0.05 versus PC + NMDA; **p < 0.01 vs PC + NMDA. All experiments were performed on cortical neurons in culture at 10–14 DIV.