Ischemic stroke induces profound mitochondrial dysfunction across diverse central nervous system cell types, driving neuronal death, glial activation, and overall tissue injury. Collectively, these mitochondria-driven alterations across CNS cell types form an interconnected network of metabolic failure, oxidative stress, and cell death that amplifies ischemic brain damage, highlighting mitochondria as central therapeutic targets in stroke (Fig. 2).

The mitochondria are usually called the powerhouses of the cell because they produce adenosine triphosphate (ATP), and maintain energy metabolism and cellular homeostasis. An imbalance in mitochondrial bioenergetics can cause neurodegenerative disorders. Under pathological conditions like cerebral ischemia-reperfusion (CI/R) injury, ischemia can lead to mitochondrial dysfunction, resulting in ROS accumulation, dysfunction of mitochondrial permeability, impaired ATP production, neuroinflammation (Fig. 3) and increased release of pro-apoptotic factors for review [68]. The oxygen and glucose deprivation resulting from blockage of blood flow to the brain initiates a cascade of degenerative cellular events surrounding the mitochondria. Therefore, the proper functioning of mitochondrial enzymes is essential for maintaining neuronal survival and improving neurological outcomes after ischemic stroke.

The relationship between ischemic stroke and circadian rhythms is an emerging and important area in neuroscience and chronobiology. Stroke onset follows a circadian pattern; therefore, these rhythms affect the severity and outcome of stroke episodes. Disruptions of the circadian rhythm and reduced circulating levels of melatonin predispose the subject to ischemic stroke. The retinoic acid-related Orphan Receptor alpha (RORα) plays a critical role in regulating mitochondrial biogenesis, metabolism, and oxidative stress, and is also considered a circadian rhythm regulator and a mediator of certain melatonin effects. A CI/R injury in RORα-deficient mice was associated with greater cerebral infarct size, brain edema, and cerebral apoptosis compared with the wild-type model [80]. The relationship between ROR receptors and melatonin is complex. Melatonin and RORα are associated with the regulation of several molecular mechanisms, like modulation of RORα activity, antioxidant effects, and enhancement of circadian and metabolic gene expression [92], but the nuclear effects of melatonin might not be directly mediated via its interaction with the RORβ, despite the finding that melatonin increased both RORα and RORβ gene expressions [93]. Hence, melatonin-mediated RORα activation might protect neurons via mitochondrial support and circadian rhythm modulation.

Melatonin has been found to exert neuroprotective effects in ischemic episodes [94] by improving ischemia-induced disturbance in mitochondrial redox state, fusion and fission, biogenesis, mitophagy and mitochondrial transfer, calcium regulation, mitochondrial permeability transition pore (mPTP) modulation, and upregulation of mitochondrial antioxidants-superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, etc. Therefore, targeting mitochondrial repair and improving its function might proffer potential neuroprotective and restorative strategies. Melatonin is lipophilic in nature, and because of this property, it easily passes through the biological membranes and accumulates in organelles, such as mitochondria, a site of its active synthesis [95], which means that high levels of melatonin are required to protect the mitochondrial DNA from the continuous production of ROS [96]. Melatonin, synthesized in the mitochondria, functions indirectly to regulate the antioxidant enzymes as well as act as a direct free radical scavenger, while its secretion into the cytosol aids its binding to the receptors on the surface of the mitochondria [97], further helping to restore the expression and activity of complexes I and IV, which are downregulated in ischemic injury. In addition, increased calcium influx following ischemic brain injury is also responsible for mitochondrial dysfunction, severely affecting ATP production. In this context, [98] demonstrated that melatonin protects the homeostasis of mitochondria by controlling the calcium influx. Melatonin therapy preserved the brain architectural and functional integrity against ischemic stroke dependently through suppressing the inflammatory/oxidative stress downstream signaling pathways as well as modulating the mitochondrial-damaged markers like cytosolic cytochrome C/cyclophilin D/serpin-SRP1 [99]. Administration of melatonin after the stroke not only leads to a reduction in mitochondrial dysfunction but also alleviates endoplasmic reticulum stress and inflammation [100].

Melatonin pre-treatment significantly increases mitochondrial transfer by influencing tunneling nanotubes (TNTs)-mediated transfer of functional mitochondria and antioxidants, and thereby inhibits apoptosis. Pre-treatment with melatonin not only reduced brain infarct area but also prevented oxidative stress, improved autophagic, mitochondrial/DNA-damaged biomarker indices with consequent improvement in neurological function [101]. Conjugation of mitochondrial targeting molecule triphenylphosphine (TPP) to melatonin (TPP-melatonin) increases the distribution of melatonin in intracellular mitochondria. mtPTP functions to regulate apoptotic pathways in the brain and is considered to be one of the major factors in causing tissue damage in ischemic and post-ischemic brain [102]. It is modulated by mK_ATP channels. Melatonin directly inhibits the mtPTP by significantly regulating the mK_ATP channels isolated in brain mitochondria [103] and diminishes NMDA-induced calcium upregulation with a subsequent decrement in cytochrome c release. A recent investigation revealed that melatonin can directly interact with the F1 domain of the F1·Fo-ATP synthase (F1FO-ATPase), a protein complex associated with the mPTP. This interaction can further desensitize the mPTP [104]. This study further demonstrated that melatonin prevented cell respiration impairment induced by hypoxia/reoxygenation in aortic endothelial cells and affected the mitochondrial bioenergetics, targeting the F₁F_o-ATPase. A study in a rat model of MCAO, melatonin decreased cytochrome c release, downregulated the activation of caspase-3, and apoptotic DNA fragmentation, essentially contributing to its anti-apoptotic effects in transient brain ischemia [75]. Yet another study in a rat MCAO model showed that melatonin inhibited inducible NOS activity, resulting in decreased tissue nitrite levels with significant reduction in COX activity and substantial recovery of mitochondrial enzyme activities [11].

Excessive activation of Notch 1 post-ischemic stroke is associated with increased expression of pro-apoptotic genes, neuronal apoptosis, and exacerbation of infarct size, where Notch1 interaction with hypoxia-inducible factor-1α (HIF-1α) and NF-κB promotes inflammation and cell death. In this context, it has been shown that melatonin significantly prevented the enhancement of the levels of cytokines (tumor necrosis factor alpha [TNF-α], IL-1β, IL-6) and NF-κB phosphorylation [105]. In the acute phase of an ischemic stroke, activation of Notch 1 promotes inflammation and cell death, and in the later recovery phase, its activation supports angiogenesis and neurogenesis. Therefore, selective Notch1 modulators like melatonin are required to fine-tune timing and cell-specific effects. Melatonin significantly prevented the Notch1 signaling activation in the hippocampus of hypoxic-ischemic neonatal rats, maintaining notch intracellular domain (NICD) and Hes Family BHLH Transcription Factor 1 (HES1) expression under regulated measures. Additionally, melatonin also prevented the sirtuin 3 (SIRT3) depletion. SIRT3 is a protein primarily located in mitochondria that accounts for managing mitochondrial homeostasis and is essential for cell survival during ischemic conditions [106]. Melatonin alleviated CI/R injury in diabetic mice by activating protein kinase B (Akt) and SIRT3/SOD2 signaling and subsequently improving mitochondrial damage [107] with substantial upregulation of mitochondrial biogenesis-related transcription factors.

Mitochondria contain a well-defined and imperviously controlled antioxidant defense system (Fig. 4), which works synergistically to interdict ROS in order to minimize oxidative damage. Melatonin can play a role in protecting the brain from oxidative damage by boosting its antioxidant defense mechanisms. Although several studies have previously shown that melatonin enhances the expressions of catalase, SOD, and GPx in various diseased models where mitochondrial activity is compromised, leading to elevated levels of oxidative stress [108]. A recent investigation in a rat model of MCAO showed that melatonin could modulate SOD, catalase, GPx, and Nrf2, expression [77]. Nrf2 is an oxidation-sensitive transcription factor that is the upstream regulator of antioxidant genes such as SOD, catalase, and GPx [109]. Inferences from various studies reveal that cerebral ischemia decreases antioxidant levels, and melatonin can induce their upregulation (SOD, CAT, GPx, and Nrf2) [110,111].

A proteomic analysis revealed decreased expression of the endogenously produced antioxidant enzyme thioredoxin (Trx) in the ischemic striatum of a MCAO rat model, and melatonin treatment prevented this decrease [112] and modulated the thioredoxin pathway in a similar kind of MCAO model of rats [113]. These findings are supported by a recent investigation where melatonin upregulates the levels of Trx1 and Thioredoxin reductase 1, thus abolishing oxidative stress-induced apoptosis in retinal ganglion cells via the activation of the thioredoxin-1 pathway [114]. Although it is evident that melatonin modulates the status and function of peroxiredoxins [115], which are involved in redox-sensitive death signaling during ischemic neuronal injury [116], more investigations are needed to understand the direct interaction between melatonin and the mitochondrial peroxiredoxins. Interestingly, peroxiredoxins are also regulated by the thioredoxin system; therefore, it could be envisioned that melatonin might regulate peroxiredoxins via the Trx pathway.

Mitochondria are essential to maintain cellular energy homeostasis, where mitophagy regulation accounts for cell survival, determining the course of neurological disorders like stroke. It is a specialized form of autophagy that selectively degrades damaged or dysfunctional mitochondria and plays an important role in ischemic stroke. Several signaling pathways regulate mitophagy during ischemia, like; PTEN-induced kinase 1 (PINK1)/Parkin pathway, BCL2 and adenovirus E1B 19 kDa-interacting protein 3 (BNIP3)/BNIP3-like BNIP3L (NIX)-mediated pathway, and the FUN14 Domain Containing 1(FUNDC1) pathway.

The receptor-mediated clearance of damaged mitochondria by autophagy, known as mitophagy, plays a key role in controlling mitochondrial homeostasis [117]. Recent studies have highlighted that the mechanisms governing mitophagy can vary significantly between different cell types, such as neurons and astrocytes, reflecting their unique physiological roles and stress responses. In neurons, the PINK1/Parkin pathway is the predominant mechanism for mitophagy. Under normal conditions, healthy mitochondria maintain a membrane potential that allows the import of PINK1 into the inner mitochondrial membrane, where it is cleaved and degraded. However, upon mitochondrial depolarization, PINK1 accumulates on the outer mitochondrial membrane. This accumulation leads to the recruitment of Parkin, an E3 ubiquitin ligase, which ubiquitinates outer mitochondrial membrane proteins, marking them for degradation via autophagy [118,119]. Whereas, astrocytes utilize a different mitophagy mechanism. In these cells, BNIP3 and NIX are the primary receptors that mediate mitophagy. These receptors are localized to the outer mitochondrial membrane and facilitate the recruitment of autophagic machinery to damaged mitochondria. Unlike the PINK1/Parkin pathway, BNIP3/NIX-mediated mitophagy does not require TBK1 (TANK-binding kinase 1) activation but instead relies on the ULK1 (Unc-51-like kinase 1) complex for autophagosome formation 9 [120].

The PINK1/Parkin pathway is activated in response to stress induced by ischemic stroke. Accumulation of damaged mitochondria occurs due to loss of membrane potential and oxidative stress. During this process, PINK1 stabilizes on the outer mitochondrial membrane and recruits Parkin, which helps to clear damaged mitochondria, thereby reducing ROS production, inflammation, and apoptotic signaling. Melatonin exerts these effects by activating the PINK1/Parkin pathway, which promotes mitophagy by removing dysfunctional mitochondria after ischemic insult. Melatonin has been shown to inhibit excessive mitophagy both in vivo and in vitro by modulating the levels of mitophagy proteins PINK1 and Parkin and reversing post-ischemia changes in dynamin-related protein 1 (DRP1), p62, and translocase of the outer membrane (TOM 20) [81].

The BNIP3/NIX pathway helps recruit autophagosomes to damaged mitochondria independently of the PINK1/Parkin pathway and inhibits mitophagy, thus providing neuroprotection. The basal level of mitophagy essentially controls mitochondrial physiological homeostasis, whereas activation of excessive mitophagy leads to cellular death in cases of ischemic stroke [121]. An in vitro/in vivo study demonstrated that melatonin attenuated sodium iodate (NaIO3)-induced mitophagy by reducing ROS-mediated HIF-1α targeted BNIP3/microtubule-associated protein 1 light chain 3 beta (LC3B) signaling in ARPE-19 cells [122]. Additionally, in the mouse model of age-related macular degeneration, melatonin treatment reduced both BNIP3 and HIF-1α levels. This potential mechanism aids in treating age-related macular degeneration. Melatonin’s effect on BNIP3 is dependent on the kind of tissue and/or stress, based on which it can inhibit or activate BNIP3-mediated mitophagy. However, in the scope of ischemic injury, there is no direct evidence of melatonin action. NIX, otherwise known as BCL-2/adenovirus E1B 19kda protein-interacting protein 3-like (BNIP3L) signaling, supports basal mitophagy and is neuroprotective. BNIP3L/NIX-mediated mitophagy protects against ischemic brain injury independently of Parkin in a mouse model [123].

Considering the FUNDC1 pathway in ischemic conditions, FUNDC1 interacts with LC3 (an autophagosome marker) to recruit autophagosomes to damaged mitochondria. In diabetic cardiomyocytes, melatonin has been shown to suppress hyperglycemia-induced mitophagy by reducing FUNDC1 expression (FUNDC1-DRP1 axis), mitochondrial fragmentation, and oxidative stress [124]. Melatonin-mediated mitophagy protects against early brain injury after subarachnoid hemorrhage through inhibition of NLRP3 inflammasome activation.

Nucleotide-binding oligomerization domain and leucine-rich repeat-containing protein X1 (NLRX1) has emerged as a critical regulator of mitochondrial dynamics and neuronal death that participates in the pathology of diverse diseases. Melatonin protected neonates from hypoxic-ischemic brain damage (HIBD) through NLRX1-mediated mitophagy in vitro and in vivo, where the neuroprotective effects of melatonin were abolished by silencing NLRX1 after oxygen-glucose deprivation [125]. Concerning hemorrhagic strokes, melatonin treatment in a SAH model increased the Bcl-2 levels and decreased the levels of Bax and cleaved caspase-3. Additionally, melatonin increased the expression of Nrf2 thus regulating mitophagy and providing protection against the hemorrhagic insult [58].

Of interest is that melatonin has been shown to regulate and modulate mitophagy in various other diseased conditions. For instance, Sun et al., 2020, showed that long-term oral administration of melatonin restrained damage of mitochondrial structure and decreased the number of mitophagy vesicles along with the levels of mitophagy factors (PINK1, Parkin, LC3-II/LC3-I) in APP/PS1 transgenic mice model of AD. Another study in the 5XFAD mice model of AD revealed that oral melatonin treatment restored mitophagy by improving mitophagosome-lysosome fusion via Mcoln1, a ROS sensor localized on the lysosomal membrane, and is involved in autophagy and oxidative stress [126].

Melatonin formulated into nanoparticles enhances its bioavailability and therapeutic potential. A study by Sakar et al. [127] demonstrated that application of nanocapsulated melatonin rescued the neuronal cells and mitochondria during cerebral ischemia-reperfusion insult observed in the aged brain. The use of nanocapsulated melatonin not only restored the activities of antioxidative enzymes and matrix metalloproteinases, but it also exerted protection with a much higher potential and at a comparatively lower dosage regime. Furthermore, in a rotenone-induced in vitro PD model, administration of nanomelatonin improved mitophagy by enhancing the expression of BMI1 (a member of the PRC1 complex) and enhanced mitochondrial biogenesis, showing excelling antioxidative and neuroprotective properties [128]. Investigation of glutamate cytotoxicity in mouse HT22 hippocampal neurons showed that melatonin significantly reduced the fluorescence intensity of mitophagy via the Beclin-1/Bcl-2 pathway, thereby reducing oxidative stress and maintaining mitochondrial homeostasis [129].

Melatonin acts as a broad-spectrum therapeutic agent in ischemic stroke pathophysiology about mitochondria function and homeostasis (Fig. 5), where it accounts for redox balance, mitochondrial structural stability, survival signaling, selective clearance of organelle, and intercellular mitochondrial support. Moreover, melatonin can both suppress and stimulate autophagy/mitophagy depending upon the cellular profile and state of injury in order to sensitively optimize neuronal survival.

Ferroptosis is regulated by a complex network of molecules and signaling pathways. Ischemic conditions dismember iron metabolism, resulting in enhanced iron accumulation in neurons. Based on some studies, ferroptosis is also considered a form of autophagic cell death where nuclear receptor coactivator 4(NCOA4) mediated ferritinophagy was found to regulate ferroptosis in cerebral ischemia-reperfusion injury by interfering with iron metabolism [136]. Moreover, ischemia-induced inflammation produces proinflammatory signals resulting in excessive intracellular iron that triggers the Fenton reaction, causing excessive free radical production, lipid peroxidation, damage to cellular membranes, and oxidative stress, consequently activating ferroptosis [137]. Accumulation of glutamate plays a crucial role in neuroexcitotoxicity and ferroptosis. Over-stimulation of glutamate receptors (NMDAR) results in glutamate excitotoxicity, while in the non-receptor-mediated pathway, high glutamate concentrations lead to intracellular glutathione depletion, further resulting in ROS accumulation, leading to enhanced lipid peroxidation and ferroptosis [138]. Cerebral ischemia reduces levels of GSH and GPX4, an antioxidant enzyme that protects against lipid peroxidation by maintaining redox balance by reducing lipid hydroperoxides. Lack of GPX4 causes ROS accumulation, leading to ferroptosis [139]. Interestingly, genetic knockout models of GPX4 present debilitating stroke damage.

Ischemic damage induces oxidation of polyunsaturated fatty acids (PUFAs) in neuronal membranes, leading to membrane damage and ferroptotic cell death. PUFAs have shown potential benefits in both preventing and treating ischemic stroke, mainly due to their anti-inflammatory and neuroprotective properties [140]. Some studies using membranes rich in PUFAs (e.g., retinal rod outer segment membranes) demonstrated that melatonin protects PUFAs, especially DHA and arachidonic acid, from oxidative degradation during lipid peroxidation [141,142]. Our brain is high in DHA and susceptible to oxidative stress, and melatonin’s antioxidative protection targeting PUFAs in brain-specific studies about ischemic stroke needs prompt validation. Interestingly, PUFAs support melatonin production and melatonin protects PUFAs against oxidative damage, depicting a reciprocal relationship an area requiring further research.

Mitochondrial uncoupling protein-2 (UCP2) is a member of the UCP family, which regulates the production of mitochondrial superoxide anion. In a microglial study, melatonin was shown to decrease M1 polarization via attenuating mitochondrial oxidative damage. Additionally, melatonin decreased the redox ratio nicotinamide adenine dinucleotide phosphate. (NADP+/NADP(H-hydrogen)), and the p47phox and gp91phox subunits of NADPH oxidase expression with subsequent reduction in ROS levels via upregulating UCP2 [143]. UCP2 modulates neuroinflammation and regulates neuronal ferroptosis following ischemic stroke in vitro and in vivo. A recent study in a MCAO mouse model and cellular model (BV2, HT-22), UCP2 expression was found to be markedly reduced in both in vitro and in vivo ischemic stroke models. UCP2 overexpression protects the brain from ischemia-induced ferroptosis by activating adenosine monophosphate-activated protein kinase (AMPK)α/Nrf1 signaling [144]. Inference from the above-mentioned investigations clearly indicates another positive mechanism of melatonin action on downregulating ferroptosis in ischemic stroke incidences.

Activation of transcriptional pathways of genes responsible for antioxidant defense (GSH, CoQ10, and Nrf2) and membrane repair endosomal sorting complex required for transport-III (ESCRT-III) limits membrane damage during ferroptosis [138,145]. A study in rat hippocampus demonstrated that melatonin prevented oxygen-glucose-deprivation (OGD) and glutamate excitotoxicity-induced neuronal injury by restoring the reduction of GSH content in the CA1 and CA3 hippocampal regions [146]. Regarding Nrf2, melatonin is a potent modulator of Nrf2 signaling, and a recent investigation showed that melatonin administration mitigated brain edema through upregulating the phosphatidylinositol 3-kinase (PI3K)/AKT/Nrf2 signaling pathway in a cerebral ischemia-reperfusion injury-induced rat model [147]. NCOA4 is a cargo receptor for ferritinophagy and is a confirmed contributor to neuronal ferroptosis in ischemic stroke, and disrupting the NCOA4-ferritin heavy chain 1 (FTH1) interaction is yet another mechanism to inhibit ferroptosis [148]. Ferroptosis and ferritinophagy activation have pathogenic implications in cases of diabetic brain injury, where miR-214-3p (negative regulator of NCOA4-mediated ferritinophagy) is involved in p53-mediated ferroptosis. A study in the brains of diabetes mellitus mice, melatonin administration inhibited p53-mediated ferroptosis and NCOA4-mediated ferritinophagy via regulating miR-214-3p [149]. Given that melatonin inhibits ferroptosis via other pathways like ACSL4, and NCOA4 is a key driver of ferroptosis via ferritin degradation, the possibility that melatonin could modulate NCOA4 in stroke remains unexplored to date.

In summary, melatonin modulates ferroptosis and prevents its detrimental effects in ischemic stroke episodes by 1. Enhancing the antioxidant defense system by upregulating GPX4, which is considered the key enzyme in protecting cells from ferroptosis and boosts GSH synthesis, which is essential for GPX4 activity, 2. decreases lipid peroxidation by reducing ROS and malondialdehyde (MDA) levels. Moreover, 3. melatonin chelates free iron and reduces iron overload, limiting Fenton reaction-driven ROS and influences ferritin and transferrin receptor expression to modulate cellular iron levels. Additionally, 4. it activates Nrf2, a transcription factor that upregulates antioxidant genes (e.g., HO-1, SLC7A11, GPX4). Nrf2 also controls genes involved in iron metabolism and lipid detoxification. 5. Melatonin also decreases the expression of ACSL4, which is a pro-ferroptotic enzyme promoting PUFA incorporation. Apart from the above-mentioned mechanisms, there are several other molecular targets of melatonin in regulating ferroptosis in ischemia that need substantial exploration. Nonetheless, melatonin-induced suppression of ferroptosis in ischemic incidences is multitarget and involves various mechanisms, which are well-illustrated in Table 5. Collectively, these actions position melatonin as a multifunctional neuroprotective agent that integrates mitochondrial maintenance, ferroptosis inhibition, and survival signaling to limit ischemic brain injury (Fig. 6).