Enhanced SUMOylation and SENP-1 protein levels following oxygen and glucose deprivation in neurones

Abstract

Here, we show that oxygen and glucose deprivation (OGD) causes increased small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 conjugation to substrate proteins in cultured hippocampal neurones. Surprisingly, the SUMO protease SENP-1, which removes SUMO from conjugated proteins, was also increased by OGD, suggesting that the neuronal response to OGD involves a complex interplay between SUMOylation and deSUMOylation. Importantly, decreasing global SUMOylation in cultured hippocampal neurones by overexpression of the catalytic domain of SENP-1 increased neuronal vulnerability to OGD-induced cell death. Taken together, these results suggest a neuroprotective role for neuronal SUMOylation after OGD.

Figure 1

Effects of oxygen and glucose deprivation (OGD) on global protein SUMOylation, UBC9, and SENP-1 levels in hippocampal neurons. (A, B) Representative patterns of SUMO-1 and SUMO-2/3 immunoreactive bands in total cell lysates from control and 75 minutes OGD-treated hippocampal neurons. (C, D) Representative UBC9 and SENP-1 blots. In all cases, the data were quantified from separate immunoblots using cells from three to five different cultures. The results are presented as percentage of control±s.e.m.; ^* indicates significant difference compared with control P<0.05. (E) The OGD-induced increase in SUMO-1 and SUMO-2/3 conjugation and SENP-1 protein levels is not due to increased transcription. quantitative polymerase chain reaction (qPCR) of RNA extracted from control and OGD-treated hippocampal neurons immediately after OGD (0 hours) revealed no changes in SUMO-1, SUMO-2, or SENP-1 mRNA levels. Graphs showing quantified data from duplicates of five experiments. The results are presented as mean±s.e.m.; no significant difference compared with control P>0.05. SUMO, small ubiquitin-like modifiers.

Figure 2

Decreasing global protein SUMOylation increases vulnerability of hippocampal neurons to oxygen and glucose deprivation (OGD). (A) Overexpression of the GFP-tagged catalytic domain of SENP-1 wild type (WT), but not the catalytically inactive SENP-1 C603S, globally downregulates SUMO-1 (left panel) and SUMO-2/3 (middle panel) conjugation to target proteins. GFP blots show effective viral expression (right panel) (B) Representative images showing the effects of overexpressing SENP-1 on OGD-induced cell death. Hippocampal neurones were infected 15 hours before OGD with Sindbis virus expressing either SENP-1 WT or SENP-1 C603S. After 75 minutes of OGD, cells were washed, returned to normal conditioned growth medium, and incubated for a further 24 hours. They were then stained with Hoechst and propidium iodide (PI), and the proportion of nuclei stained with PI was counted. Graph showing quantified data from four separate experiments. Overexpression of SENP-1 WT significantly increases cellular death in the cultures exposed to OGD. ^* and # indicate significant differences compared with all groups, P<0.05. SUMO, small ubiquitin-like modifiers.