Stroke is currently the third leading cause of death and severe long-term disability worldwide, and the number of new and recurrent stroke patients continues to increase each year [1]. Ischemic stroke, a major subtype of stroke, is caused by a lack of blood supply to the brain due to a cerebral infarction or thrombus in the brain, leading to loss of brain function. Ischemic strokes account for approximately 70–80% of all strokes and have a recurrence rate of up to 41% within 5 years in survivors [1]. Current therapeutic options for ischemic stroke focus on improving cerebral circulation and ischemic prognosis [2,3]. Intravenous thrombolysis is the most prominent measure to restore blood flow [4]. However, severe ischemia-reperfusion (I/R) injury often occurs after blood flow reestablishment [5]. The mechanisms of cerebral I/R injury mainly involve oxidative stress, calcium overload, neuroexcitotoxicity, and inflammation [6,7]. Neurons are particularly sensitive to oxidative stress due to their high energy requirements. Neurons are more prone to ferroptosis under conditions of oxidative stress than under normal conditions; indeed, oxidative stress is the fundamental mechanism underlying ferroptosis. Ferroptosis results from the accumulation of cellular reactive oxygen species (ROS) that exceed the redox content maintained by glutathione (GSH) and phospholipid hydroperoxidase, which uses GSH as a substrate, ultimately resulting in the accumulation of excess peroxides [8,9]. Importantly, lipid ROS/peroxides, rather than cytosolic ROS, trigger ferroptosis [10,11]. Therefore exploring endogenous antioxidants as inhibitors of ferroptosis may lead to a breakthrough in ischemic stroke treatment.

Disruptions in mitochondrial energy metabolism, lipid metabolism, iron homeostasis, glutamine metabolism, and other key regulatory signalling pathways all have an impact on ferroptosis susceptibility. Recent studies of changes in mitochondrial metabolism during I/R injury have unlocked new therapy possibilities. In the ischaemic heart, succinate, a metabolite of the tricarboxylic acid (TCA) cycle, accumulates during ischemia due to reversal of succinate dehydrogenase (SDH) [12]. After reperfusion, the accumulated succinate is rapidly oxidized by SDH to maintain the reduction of the Q pool in order to maintain a large proton potential through the traditional electron transfer of complexes III and IV. At the same time, the SDH oxidation also drives reverse electron transfer (RET) at complex I to produce a large amount of ROS and result in oxidative stress injury. Many studies have proven that inhibition of SDH by malonic acid can reduce reperfusion injury [13,14]. However, recent articles have also reported that changes in HIF-1α and IL-1β production are related to the efficiency of succinate oxidation by SDH and that succinate processing is more important than accumulation for inflammatory reorganization in macrophages [[14], [15], [16]] This suggests that the signalling effect of succinate may not depend on the accumulation of succinate but may also be related to the efficiency and directionality of the electron transport chain (ETC). The efficiency of succinate oxidation by SDH is the key driver of signal transduction. It has been shown that cellular ferroptosis can be inhibited by blocking lipid peroxidation by reducing the amount of fuel for mitochondrial oxidative phosphorylation [17]. Inhibition of the mitochondrial TCA cycle or ETC mitigates mitochondrial membrane potential (MMP) hyperpolarization, lipid peroxide accumulation, and ferroptosis [18].

TP53-induced regulator of glycolysis and apoptosis (TIGAR) produces neuroprotective effects by inhibiting cytoplasmic glycolysis shifting glucose metabolism to the pentose phosphate pathway (PPP) increasing NADPH and GSH levels and decreasing intracellular ROS levels and oxidative stress. However, prolonged ischemia disrupts PPP in the brain, causing the glucose metabolic pathway to be reshifted to glycolysis, which in turn impairs glucose availability and NADPH production. Adequate glucose supply is necessary for the production of NADPH via the PPP. Moreover, glucose is metabolised during reperfusion mainly by glycolysis rather than PPP [19]. Thus, in long-term ischemic brains whatever the level of TIGAR fails to inhibit neuronal ferroptosis through the production of NADPH and GSH. Contradictionally, TIGAR shows a fourfold upregulation after 2 h of ischemia [20], and in mice, TIGAR was consistently expressed at a high level within 24 h of reperfusion [21]. These studies all suggest the existence of a PPP-independent pathway for TIGAR to mitigate oxidative stress injury [22]. Our previous study found that TIGAR concentrates ectopically in mitochondria under hypoxic conditions [23,24]. However, it is not fully understood the purpose of TIGAR translocation to mitochondria and whether and how mitochondria-localised TIGAR exerts antioxidant activity and neuroprotection in the brain in the presence of prolonged ischemia.

In the present study, we describe the mechanisms by which mitochondria-localised TIGAR promotes antioxidant activity and inhibits neuronal ferroptosis during prolonged ischemia. We identified and demonstrated that TIGAR can reduce neuronal ferroptosis during prolonged ischemia in a PPP-independent manner. Specifically, TIGAR maintains redox homeostasis during prolonged ischemia by translocating to mitochondria and interacting with SDH to inhibit ischemia-induced enhancement of SDH activity, which in turn reduces ROS, MitoROS and lipid peroxidation levels to attenuate neuronal ferroptosis.