Sumoylation of LYS590 of NCX3 f-Loop by SUMO1 Participates in Brain Neuroprotection Induced by Ischemic Preconditioning

Abstract

Background and Purpose— The small ubiquitin-like modifier (SUMO), a ubiquitin-like protein involved in posttranslational protein modifications, is activated by several conditions, such as heat stress, hypoxia, and hibernation and confers neuroprotection. Sumoylation enzymes and substrates are expressed also at the plasma membrane level. Among the numerous plasma membrane proteins controlling ionic homeostasis during cerebral ischemia, 1 of the 3 brain sodium/calcium exchangers (NCX3), exerts a protective role during ischemic preconditioning. In this study, we evaluated whether NCX3 is a target for sumoylation and whether this posttranslational modification participates in ischemic preconditioning-induced neuroprotection. To test these hypotheses, we analyzed (1) SUMO1 conjugation pattern after ischemic preconditioning; (2) the effect of SUMO1 knockdown on the ischemic damage after transient middle cerebral artery occlusion and ischemic preconditioning, (3) the possible interaction between SUMO1 and NCX3 and (4) the molecular determinants of NCX3 sequence responsible for sumoylation. Methods— Focal brain ischemia and ischemic preconditioning were induced in rats by middle cerebral artery occlusion. SUMOylation was evaluated by western blot and immunohistochemistry. SUMO1 and NCX3 interaction was analyzed by site-directed mutagenesis and immunoprecipitation assay. Results— We found that (1) SUMO1 knockdown worsened ischemic damage and reduced the protective effect of preconditioning; (2) SUMO1 bound to NCX3 at lysine residue 590, and its silencing increased NCX3 degradation; and (3) NCX3 sumoylation participates in SUMO1 protective role during ischemic preconditioning. Thus, our results demonstrate that NCX3 sumoylation confers additional neuroprotection in ischemic preconditioning. Conclusions— Finally, this study suggests that NCX3 sumoylation might be a new target to enhance ischemic preconditioning-induced neuroprotection.

SUMOylation in brain ischemia: Patterns, targets, and translational implications

“…Resistance to OGD in immortal cells and primary neurons with increased SUMOylation (overexpressing Ubc9 or SUMO1, or silencing SENP3) 1,36,40 # Resistance to OGD in immortal cells and primary neurons with decreased SUMOylation (overexpressing dominant negative Ubc9, silencing SUMO1 or SUMO2/3, or overexpressing SENP1) 1,36,37,41 In vivo (rodents) " SUMO-conjugation in the brain after transient forebrain ischemia 4,5 # SUMO-conjugation in the brain during transient forebrain ischemia 5 " SUMO-conjugation in the brain after transient ischemic stroke 3,23,39 " Stroke outcomes in transgenic mice overexpressing Ubc9 37 # Functional outcomes post-forebrain ischemia in transgenic mice with silenced neuronal SUMO1-3 expression…”

“…In vitro and in vivo experimental approaches have been used to determine whether post-ischemic SUMOylation is indeed a neuroprotective stress response that shields neurons from the damage induced by ischemia, or is simply an epiphenomenon that is not directly related to ischemiainduced pathobiology (summarized in Table 1). 1,22,[36][37][38][39][40] Cell-based studies have been conducted by exposing primary neuronal cells or neuroblastoma cells to transient oxygen/glucose deprivation (OGD), which is considered to be an acceptable in vitro model of ischemia. The effect of SUMOylation on outcomes after OGD has been assessed by genetically manipulating the SUMO pathway in the cells, such as by increasing the levels of SUMOylated proteins via the overexpression of SUMO isoforms or the conjugating enzyme Ubc9, 1,36 or suppressing SUMOylation by microRNA-induced silencing of SUMO isoforms or through overexpressing SENP1.…”

The Role of Ubiquitin-Proteasome Pathway and Autophagy-Lysosome Pathway in Cerebral Ischemia

“…SUMOylation of NaV1.2 channels contributes to the early response to acute hypoxia in central neurons [53]. Ischemic preconditioning confers neuroprotection in ischemic stroke, which is mediated by SUMO1-induced SUMOylation of LYS590 of NCX3 f-Loop [54] (Table 3). E2-25K is an E2-conjugating enzyme and is SUMOylated during oxidative stress.…”

Knocking-out the Siah2 E3 ubiquitin ligase prevents mitochondrial NCX3 degradation, regulates mitochondrial fission and fusion, and restores mitochondrial function in hypoxic neurons

“…Transient focal ischemia was induced, as previously described [35], by suture occlusion of the right middle cerebral artery in male mice anesthetized with 1.5% sevoflurane, 70% N 2 O, and 28.5% O 2 . Achievement of ischemia was confirmed by monitoring regional cerebral blood flow in the area of the right middle cerebral artery through a laser Doppler (PF5001; Perimed, Sweden).…”