SUMO2/3 conjugation is an endogenous neuroprotective mechanism

Abstract

Small ubiquitin-like modifier (SUMO)2/3 but not SUMO1 conjugation is activated after transient cerebral ischemia. To investigate its function, we blocked neuronal SUMO2/3 translation through lentiviral microRNA delivery in primary cortical neurons. Viability was unaffected by SUMO2/3 silencing unless neurons were stressed by transient oxygen-glucose deprivation (OGD). Both 15 and 45 minutes of OGD were tolerated by control microRNA-expressing neurons but damaged >60% of neurons expressing SUMO2/3 microRNA. Damaging OGD (75 minutes) increased neuronal loss to 54% (control microRNA) and to 99% (SUMO2/3 microRNA). This suggests that activation of SUMO2/3 conjugation is an endogenous neuroprotective stress response.

Figure 1

Neuronal specificity and efficacy of RNA interference with SUMO2/3 and its effects on survival of mouse primary cortical neurons. At days in vitro (DIV) 3, primary cortical neurons were transduced with lentiviral particles expressing EGFP as a reporter and either SUMO2/3 or control microRNA driven by the neuron-specific synapsin promoter. (A) Verification of transduction efficacy and neuronal specificity of cultures transduced with lentiviral particles driven by the synapsin promoter. Immunohistochemistry was performed after paraformaldehyde (PFA) fixation on DIV 12 with antibodies against EGFP, microtubuli-associated protein 2 (MAP2), and nuclear counterstain with DAPI. The multiplicity of infection (MOI) was ∼50. Scale bar=100 μm. (B) Verification of knockdown efficiency of SUMO2/3 versus control microRNAs (1=LacZ, 2=non-targeting scrambled) with and without OGD (45 minutes and 3 hours reoxygenation) shown by a representative western blot analysis. Neuronal cultures were analyzed on DIV 12 and subjected to SDS-PAGE. Membranes were probed with antibodies against SUMO2/3, EGFP, and GAPDH. EGFP expression corresponded to an equal MOI of lentiviral particles and concomitant microRNA expression. GAPDH served as a housekeeping protein and equal loading control. (C) SUMO2/3 microRNA does not influence baseline survival over time up to DIV 12. In brief, microscopic pictures of EGFP fluorescence (indicative for microRNA delivery) were taken at DIV 6, 9, and 12 as described in the ‘Materials and methods' section. EGFP-expressing neurons were counted and ratios were calculated for DIV 9/6 (indicated in blue) and DIV 12/9 (red) to evaluate cell survival over time. We assumed an effect size >0.15 and performed a prospective power analysis with α=0.05 and β=0.20. There was no significant difference between groups in a two-way repeated-measures ANOVA followed by Tukey's post hoc analysis. ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MAP2, microtubule-associated protein 2; OGD, oxygen–glucose deprivation; SUMO2/3, small ubiquitin-like modifier-2/3.

Figure 2

Characterization of ATP depletion and recovery after different durations of OGD and effect of SUMO2/3 microRNA expression on neuronal survival after OGD. (A) ATP contents were analyzed after 15, 45, and 75 minutes of OGD at different reoxygenation time points. Cultures with 75 minutes of OGD were compared with lentivirally transduced cultures (control microRNA). Data are presented as ratios versus controls and mean±s.d.. Statistical analysis was performed for each length of OGD as one-way repeated-measures ANOVA (^*P<0.01 versus control). Interactions between duration of OGD and reoxygenation were analyzed by a two-way repeated-measures ANOVA with F_(1, 31)=39.5 (OGD duration), with F_(1, 31)=59.2 (reoxygenation) and F_(1, 31)=5.0 (duration × reoxygenation interaction), with P=0.018 for interaction. The power of performed tests (α=0.05) is 1 for the duration of OGD and reoxygenation and 0.7 for interaction. To isolate significant differences between groups, a Tukey post hoc analysis for multiple comparisons was performed. ATP recovery was significant for 45 and 75 minutes OGD length (⁺P<0.001; 180 minutes versus 0 minutes reoxygenation) and ^#P<0.05 indicates the degree of ATP depletion referred to the duration of OGD (75 versus 45 minutes and 45 versus 15 minutes, respectively) either at 0 minutes or after 180 minutes recovery time. ATP depletion at 0-minute reoxygenation and recovery at 180-minute reoxygenation after 75 minutes of OGD did not differ significantly from that in cultures transduced with lentiviral particles to untransduced cultures. (B and C) On DIV 3, primary cortical neurons were lentivirally transduced to achieve SUMO2/3 or control microRNA expression. Propidium iodide (PI) was added 24 hours after 75 minutes of OGD and phase-contrast images were merged to the red fluorescence channel. (Panel B) Representative pictures illustrate PI incorporation after OGD or in the control sister cultures. (Panel C) Quantification of viable, PI-negative (PI⁻) neurons as a ratio to all neurons counted in transmission images. Interactions between microRNA and OGD effects were analyzed by a two-way ANOVA with F_(1, 17)=211.7 with P<0.001 for interaction followed by Tukey's post hoc analysis. (D) Increase in LDH activity after 75 minutes of OGD or control cultures was analyzed after 24 hours but before PI staining. (E) On DIV3, primary neurons were lentivirally transduced with control microRNA. LDH activity was analyzed before and after total cell lysis with Triton-X in neuronal cultures subjected to the indicated durations of OGD after 24 hours. The survival of GFP⁺ neurons was assessed as shown in Figure 1C. The mean values of the ratios (before and after OGD) per well were plotted against LDH activity. There is an inverse correlation of the loss of viability to the increase in normalized LDH release with R²=0.79, F_(1, 7)=19.1, P=0.007. (F–I) On DIV 3, SUMO2/3 or control microRNA was expressed in primary cortical neurons using lentiviral transduction. On DIV 12, cultures were exposed to 15, 45, or 75 minutes of OGD resulting in no or significant damage of control microRNA-expressing cultures. Predefined regions of interest were recorded using a semi-automated software algorithm, and numbers of EGFP-expressing neurons were counted before and 24 hours after OGD. (Panel F) To illustrate the effects of microRNA expression on the extent of OGD-induced cell death, digital images of EGFP-expressing neurons were false colored before (green, left panel, DIV 12) and 24 hours after OGD (red, middle panel, DIV 13). Merged yellow neurons are indicative for survival, green cells are indicative for lost neurons (right panel, merged pictures from DIV 12 and 13). Scale bar=100 μm. (Panels G–I) Extent of OGD-induced cell death was evaluated by calculating the ratios between the numbers of EGFP-positive neurons on DIV 13/12 (directly before and 24 hours after OGD) and compared with baseline cell counts as a ratio (DIV 12/6). Interactions between microRNA and OGD effects were analyzed by a two-way repeated-measures ANOVA with F_(1, 67)=97.5 (15 minutes OGD, panel G), with F_(1, 57)=157.4 (45 minutes OGD, panel H) and F_(1, 41)=62.6 (75 minutes OGD, panel I) with P<0.001 for interaction. The power of performed tests (α=0.05) is 1. To isolate significant differences between groups, a Tukey post hoc analysis for multiple comparisons was performed and P-values are indicated in the figures. ANOVA, analysis of variance; ATP, adenosintriphosphate; EGFP, enhanced green fluorescent protein; LDH, lactate dehydrogenase activity; OGD, oxygen–glucose deprivation; SUMO2/3, small ubiquitin-like modifier-2/3.