Mechanisms and Implications of Mitochondrial Autophagy in Stroke

Abstract

Stroke is an acute cerebrovascular disease that is caused by disruptions in the cerebral blood supply and leads to brain tissue damage. Its pathological mechanisms remain to be fully elucidated. Stroke has high incidence, disability, and mortality rates, thus substantially affecting life and health. Against this backdrop, intracellular mitochondria, which are central to cellular energy metabolism and crucial for cell survival, have major roles in ischemic stroke. Ischemic stroke results in brain cell oxygen and nutrient deprivation, thereby triggering oxidative stress and inflammatory reactions, impairing mitochondrial function, and disrupting energy metabolism. Mitochondrial autophagy is a protective mechanism to improve the quality and quantity of mitrochondria, and exerts neuroprotective effects. In recent years, preserving mitochondrial function after ischemic stroke has emerged as a major research topic. Increasing evidence suggests a close relationship between abnormal mitochondrial autophagy and the occurrence, progression, and pathophysiology of ischemic stroke. However, current research has not adequately explained how ischemic stroke regulates the initiation and execution of mitochondrial autophagy. Elucidating this mechanism in detail will be critical for understanding the development of stroke injury, and identifying novel and effective intervention strategies. Additionally, the optimal treatment time window must be determined to enable effective interventions in mitochondrial autophagy and minimize brain damage. To deepen understanding of mitochondrial autophagy, this review summarizes mitochondrial autophagy’s signaling pathways and its major role in ischemic stroke pathophysiology. Further exploration in this field will provide a crucial theoretical foundation for developing novel therapeutic strategies and clinical applications.

Main article text

1. INTRODUCTION

Stroke, also known as cerebrovascular accident, is an acute vascular disease arising from sudden interruption of the blood supply to the brain [1,2]. The main causes of stroke are sudden blockages or damage to cerebral blood vessels, and are categorized as hemorrhagic and ischemic [3]. Stroke has diverse influencing factors and high rates of incidence, disability, and mortality, thus posing substantial economic and emotional burden on patients, their families, and society [4,5]. Ischemic stroke results in inadequate oxygen and glucose supply to brain tissues, thus triggering a cascade of events, including blood-brain barrier dysfunction, release of neuroinflammatory cytokines, excessive activation of oxidative stress, disrupted protein synthesis, mitochondrial metabolic abnormalities, increased cellular apoptosis, and other complex pathological mechanisms [6–10]. Moreover, stroke leads to various severe clinical symptoms, such as sudden loss of consciousness, facial distortion, slurred speech, or partial paralysis [11,12].

Stroke is marked by the onset of acute, chronic, and recovery phases. These three phases differ in their timeframes, mitophagy level, and function. The acute phase is characterized by elevated mitophagy due to rapid mitochondrial damage within minutes to hours after stroke onset. The chronic phase starts within days to weeks after stroke, and the level of mitophagy moderately increases. Thirdly, the recovery phase starts within weeks to months following a stroke. During this time, the levels begin to normalize or decrease as the population of mitochondria starts to regenerate. This regeneration plays a crucial role in restoring cellular function and promoting overall recovery in the affected areas.

Approximately 2 million new stroke cases occur annually in China, more than 80% of which are ischemic strokes. A total of 75% of patients with ischemic stroke face death or varying degrees of disability. Acute ischemic stroke treatments rely primarily on intravenous thrombolytic agents such as tissue plasminogen activator (tPA) and neuroprotective strategies [13]. However, owing to irreversible neuronal damage and the narrow treatment time window, current therapies have limited efficacy [14].

Mitochondria, crucial sites for aerobic respiration, are widely distributed across various cell types, and provide a continuous energy supply for cellular metabolic activities [15]. As dynamic organelles, mitochondria produce ATP through the electron transport chain, thus serving not only as the “powerhouse of the cell” but also as the primary site of reactive oxygen species (ROS) generation [16]. On the one hand, ROS serve as a signaling molecule in oxidative-reduction, by transmitting signals from the mitochondrial compartment to other cellular compartments. On the other hand, oxidative stress is a stimulus that can lead to excessive ROS production, thus further causing oxidative phosphorylation abnormalities, mitochondrial functional impairment, ATP depletion, intracellular calcium influx, and mitochondrial permeability transition pore opening, and eventually resulting in apoptosis, or cell death [17]. Because most ATP in the brain supports neuronal electrophysiological activities, the energy supplied by mitochondria is crucial for neuronal survival and excitatory functions. Given that neurons have high mitochondrial content to meet their high energy demand and weak antioxidant capacity, neuronal damage is frequently induced by ROS, owing to energy imbalances [18]. Several studies have convincingly demonstrated the pivotal role that oxidative stress, instigated by an excess of reactive oxygen species (ROS), plays in the pathological cascade leading to brain damage post-stroke. This oxidative stress is intimately linked to, and often precedes, neuronal cell damage or death in the context of ischemic stroke, highlighting its direct involvement in the deterioration of neuronal health [19,20].

Autophagy serves as an intracellular defense and stress-regulating mechanism, wherein cells clear, digest, and recycle damaged or aged organelles, denatured proteins, and nucleic acids, which provide essential materials for cellular repairing and reconstruction. Furthermore, multiple studies have indicated that various cells undergo autophagy after ischemia in the brain, at levels correlating with ischemia duration and severity [13]. Autophagy mitigates ischemic brain damage through various mechanisms, yet its detailed molecular mechanisms require further investigation [21]. According to the degradation substrate, autophagy can be categorized into mitophagy, reticulophagy, ribophagy, pexophagy, and many other types [22]. Mitophagy specifically refers to the selective clearance of damaged or dysfunctional mitochondria through autophagy to maintain normal physiological metabolic processes. This process can be induced by various factors, such as ROS, hypoxia, stroke, normal cellular metabolic processes, and viral infections [23,24].

Since the term “mitophagy” was proposed by Lemasters in 2005, the role of mitophagy in neurological disorders, such as stroke, Parkinson’s disease, and traumatic brain injury, has garnered increasing attention [25]. During ischemic stroke, disruption of the dynamic balance maintained by mitochondria leads to activation of relevant signaling pathways and a cascade of neuronal damage [26]. During the ischemic phase of ischemic stroke, mitochondrial dysfunction occurs because of oxygen and energy substrate deprivation. In the reperfusion phase, lipid peroxidation of the mitochondrial membrane leads to oxidative stress damage; moreover, excessive ROS disrupt the calcium pumps located on mitochondrial membranes, thus causing calcium overload and inflammatory reactions. Beyond these pathological changes, cellular apoptosis, autophagy, and death in ischemic stroke are associated with a loss of mitochondrial function [27]. Therefore, studying the relationship between mitochondria and ischemic stroke not only deepens understanding of the underlying pathological mechanisms but also may guide innovative treatments for this condition (Fig 1).

Figure1 |

Mitochondrial autophagy associated with stroke.

When stroke occurs, oxidative stress caused by hypoxia and ischemia can damage intracellular mitochondria and cause dysfunction such as mitochondrial imbalance. Mitochondrial autophagy clears damaged mitochondria, thereby maintaining healthy mitochondrial function and aiding in repair of damaged nerve cells.

This article briefly summarizes the pathological factors triggering mitochondrial autophagy in stroke, and reviews the main pathological injury mechanisms in ischemic stroke and the signaling pathways associated with mitochondrial autophagy. Additionally, it analyzes the role of mitochondrial autophagy in the brain during ischemic stroke, to provide a theoretical basis for innovative treatments for ischemic stroke, autoimmune diseases, and other neurological metabolic disorders.

2. MITOCHONDRIAL AUTOPHAGY

Mitochondria play critical roles in eukaryotes by regulating energy metabolism, cellular apoptosis, autophagy, and necrosis. They serve as critical hubs for intracellular signaling, thus exerting multifaceted functions in neuronal development, growth, and remodeling. In recent years, preserving mitochondrial function after ischemic stroke has become a focus of mitochondrial research. Mitochondrial autophagy has a role in the ischemia core by triggering apoptotic pathways; mitophagy is insufficient to remove damaged mitochondria from the core area, whereas in the penumbra, it removes damaged mitochondria and prevents apoptosis.

Experimental evidence has indicated that ischemia, hypoxia, and reperfusion injury directly decrease mitochondrial ATP production and disrupt cell membrane ion channels, and consequently lead to a loss of the ability to maintain the normal electrochemical gradient across the membrane. Continuously open voltage-gated channels cause excessive calcium influx and result in neuroexcitotoxicity [28]. Mitochondrial calcium overload and oxidative stress induce lipid peroxidation or damage to the mitochondrial respiratory chain, thus causing massive opening of the mitochondrial permeability transition pore, altering mitochondrial structure, decreasing membrane potential, releasing cytochrome C, and ultimately triggering cellular apoptosis [29,30]. Furthermore, when the mitochondrial respiratory chain is impaired, excess ROS are generated. Accumulation of ROS induces mitochondrial depolarization and leads to selective engulfment of damaged mitochondria within autophagosomes, in a process known as mitophagy [31].

According to the physiological environment, mitochondrial autophagy can be classified into three types: basal mitophagy, programmed mitophagy, and stress-induced mitophagy [32]. Basal mitophagy refers to degradation of dysfunctional or aged mitochondria under normal physiological conditions. Programmed mitophagy occurs during developmental stages in various cell types, such as during retinal ganglion cell development, cellular reprogramming, cardiac maturation, and erythrocyte differentiation. Stress-induced mitophagy involves the rapid degradation of mitochondria due to external stress stimuli such as hypoxia, stroke, and excess ROS, thus affecting mitochondrial physiological functions [33]. Current research suggests that the fundamental process of mitochondrial autophagy involves multiple stages, whose associated characteristics and processes are summarized in Table 1.

Table1 |

Fundamental processes in mitochondrial autophagy.

Stages	Characteristics	Specific process
Initiation	Mitochondrial dysfunction	Mitochondrial depolarization and accumulation of surplus and damaged mitochondria [34].
Early stage	Formation of mitophagosomes	Formation of a fragmented tubular network, thus leading to the segregation of damaged mitochondria; aggregation and activation of mitophagy receptors or ubiquitin-autophagy adaptors; targeting of mitochondria by autophagy-related proteins, generation of isolation membranes/autophagosomes; closure and isolation of mitochondria within autophagosomes [35,36].
Middle stage	Fusion of mitophagosomes with lysosomes	Transport of autophagosomes to the degradation compartment and fusion with endosomes or lysosomes [37].
Late stage	Degradation of mitochondria within autolysosomes and content recycling	Entry of acidic hydrolytic enzymes from lysosomes or endosomes into autophagosomes, thus leading to the degradation of enclosed mitochondria and subsequent recycling of their contents [38].

3. REGULATORY MECHANISM OF MITOCHONDRIAL AUTOPHAGY

In studies of ischemic stroke, cellular exposure to oxygen-glucose deprivation (OGD) stimuli triggers mitochondrial damage mechanisms. Subsequently, mitochondrial autophagy is initiated to maintaining the quality and quantity of mitochondria within the cell. This process is an adaptive response of cells to ischemic stimuli, and is important for cell survival and functional recovery. Recent research has highlighted the critical role of mitochondrial autophagy in ischemic stroke pathophysiology, thus providing a comprehensive theoretical basis for the development of relevant therapeutic strategies. Several regulatory mechanisms associated with mitochondrial autophagy have been identified, including those shown in Fig 2.

Figure2 |

Regulatory mechanisms of mitochondrial autophagy.

The regulatory mechanisms of mitochondrial autophagy in ischemic stroke involve PINK1-dependent/Parkin-independent induction of mitophagy; LC3 adapter protein inducing mitophagy via a ubiquitin-independent mechanism; Fun14 domain-containing protein 1 (FUNDC1) receptor-mediated mitophagy; and Nix/BNIP3L receptor-mediated mitophagy.

3.1. PINK1/Parkin-dependent mitochondrial autophagy

The PINK1/Parkin signaling pathway, the classical mitochondrial autophagy pathway, has been extensively studied in mammals. Moreover, PINK1/Parkin-dependent mitochondrial autophagy plays a major role in the repair of ischemic stroke-induced injuries [13,39]. PINK1, a serine/threonine kinase located on the outer mitochondrial membrane, bears an N-terminal mitochondrial targeting sequence. Under normal physiological conditions, PINK1 is a precursor that is synthesized in the cytoplasm and translocated to the mitochondria through the concerted action of translocase of the outer mitochondrial membrane (TOM) and translocase of the inner mitochondrial membrane (TIM) [40]. Stroke-induced mitochondrial depolarization triggers the PINK1/Parkin signaling pathway [39]. Loss of membrane potential prevents the precursor of PINK1 from entering the inner mitochondrial membrane via TIM23, thereby stabilizing it on the outer mitochondrial membrane via the mitochondrial outer membrane protein TOMM7. At this point, PINK1, located on the outer mitochondrial membrane, phosphorylates ubiquitin chains that are attached to mitochondrial proteins, facilitating the recruitment of Parkin, which primarily resides in the cytoplasm as an E3 ubiquitin ligase [41–43]. This phosphorylation event enhances Parkin’s affinity for ubiquitinated substrates on the mitochondrial surface, enabling its translocation from the cytoplasm to the outer mitochondrial membrane and subsequent activation [44–46]. Activated Parkin polyubiquitinates various mitochondrial outer membrane proteins, thus forming ubiquitin chains that are recognized by autophagy adaptors, which in turn bind LC3, facilitate autophagosome formation, and subsequently induce mitochondrial autophagy [41,47–49].

Under physiological conditions, PINK1 and Parkin form a tight mitochondrial regulatory mechanism maintaining a balance that prevents autophagic damage through excessive mitochondrial autophagy [50]. During mitochondrial dysfunction induced by ischemic stroke, PINK1 rapidly senses the abnormal membrane potential in damaged mitochondria and recruits Parkin, which in turn aggregates at damaged mitochondria. The PINK1/Parkin pathway induces mitochondrial autophagy by mediating polyubiquitination of functional or structural proteins within damaged mitochondria [50,51]. Furthermore, within 24–48 hours, Parkin translocation results in the complete clearance of damaged mitochondria. After silencing of the Parkin gene, decreasing expression of LC3-II, PINK1, and Parkin have been shown to significantly decrease the loss of mitochondrial membrane potential, suppress ROS levels, and decrease apoptosis rates [52]. Studies have also indicated that OGD/reperfusion activate PINK1/Parkin-mediated mitochondrial autophagy, thus demonstrating the involvement of PINK1 and Parkin in the pathological process of brain ischemia-reperfusion injury [53].

3.2. PINK1-dependent/Parkin-independent induction of mitochondrial autophagy

Beyond the classical signaling pathway mediated by PINK1/Parkin in mitochondrial autophagy, PINK1 promotes mitochondrial aggregation and activation of the serine kinase TBK1 through ubiquitination. Mitochondrial autophagy is subsequently induced in a Parkin-independent manner through autophagy adaptor proteins such as OPTN and NDP52, which contain LIR motifs (a characteristic amino acid sequence) [49]. In the context of ischemic stroke, both PINK1-dependent and Parkin-independent mechanisms have been shown to induce mitochondrial autophagy.

In ischemic stroke, PINK1 might participate in regulating mitochondrial autophagy in a manner independent of Parkin. This mechanism involves the activation of other signaling pathways on the mitochondrial membrane, thus initiating mitochondrial clearance. A complex interplay exists between ischemic stroke and the induction of mitochondrial autophagy through PINK1-dependent and Parkin-independent pathways. This interplay is likely to involve multilayered regulatory networks encompassing mitochondrial membrane potential, PINK1 activity, Parkin function, and other potential regulatory factors. Comprehensive understanding of these mechanisms will be important in revealing the pathophysiology of ischemic stroke and identifying potential therapeutic targets. These findings might provide a new biological foundation for future therapeutic strategies targeting stroke.

3.3. LC3 adaptor protein induces mitochondrial autophagy through a ubiquitin-independent mechanism

The LC3 adaptor protein, whether through the PINK1/Parkin-dependent mitochondrial autophagy pathway or the PINK1-dependent/Parkin-independent induction of mitochondrial autophagy pathways, identifies ubiquitinated mitochondrial proteins through a ubiquitin-dependent mechanism and induces mitochondrial autophagy. Under normal physiological conditions, Choline dehydrogenase (CHDH) localizes within both the inner and outer mitochondrial membranes. However, when the mitochondrial membrane potential is disrupted, CHDH accumulates on the outer mitochondrial membrane and interacts with p62/SQSTM1 through the Phox and Bem1 (PB1) domains, thus forming the CHDH-p62/SQSTM1-LC3 complex and inducing mitochondrial autophagy [54].

During ischemic stroke, disruption of the mitochondrial membrane potential may trigger LC3 adaptor protein and lead to mitochondrial autophagy. Furthermore, LC3, via a specific structural domain, binds and fuses with the mitochondrial membrane. This mechanism differs from the conventional ubiquitin-dependent process; LC3 induces mitochondrial autophagy by directly interacting with the mitochondrial membrane. This ubiquitin-independent mechanism provides a more rapid and flexible pathway for autophagy, which is particularly suitable for acute conditions such as ischemic stroke. In summary, the LC3 adaptor protein induces mitochondrial autophagy in ischemic stroke through a ubiquitin-independent process. This mechanism provides a new perspective on autophagy regulation under conditions of brain ischemia, including crucial insights into stroke pathophysiology and potential therapeutic targets. These findings may lay a biological foundation for future stroke treatment strategies.

3.4. FUNDC1 receptor-mediated mitochondrial autophagy

FUNDC1, a receptor protein located on the outer mitochondrial membrane, is involved in mitochondrial autophagy, through interacting with LC3 or Atg8 under low oxygen conditions. This kinase shows decreased activity during hypoxic conditions, thus leading to FUNDC1 dephosphorylation, promoting interaction between FUNDC1 and LC3 or yeast homolog of Atg8, and consequently inducing mitochondrial autophagy [55]. This process provides a regulatory mechanism for mitochondrial clearance, and is associated with the removal of damaged mitochondria in ischemic stroke. Additionally, FUNDC1 activity closely correlates with changes in mitochondrial membrane potential. Under ischemic conditions, the decrease in mitochondrial membrane potential activates FUNDC1 and facilitates its binding to LC3, thereby directing mitochondria toward the autophagic pathway. This process is a crucial step in regulating mitochondrial autophagy during ischemic stroke.

Research has highlighted the major role of tPA in the treatment of acute ischemic brain injury. tPA is a thrombolytic agent in blood vessels and is an essential neuroprotective agent in hyperacute ischemic stroke [56,57]. Cai et al. have discovered that when brain ischemic injury impairs mitochondrial function, tPA increases FUNDC1 expression levels through AMP-activated protein kinase (AMPK) phosphorylation, thus restoring mitochondrial function, decreasing neuronal apoptosis, exerting neuroprotection, and ultimately limiting the extent of ischemic injury [58]. Moreover, tPA exerts neuroprotective effects by increasing AMPK phosphorylation and FUNDC1 expression, thus inhibiting cell apoptosis and improving mitochondrial function, whereas deletion of the tPA gene significantly worsens brain injury, increases neuronal apoptosis, and exacerbates mitochondrial damage [59–61]. On the basis of these studies, the expression level of FUNDC1 might be influenced by ischemic stroke. Under ischemic conditions, significant upregulation of FUNDC1 expression is observed, in agreement with this protein’s crucial role in regulating mitochondrial autophagy [58]. Therefore, modulating the expression level of FUNDC1 through external genetic editing to regulate mitochondrial autophagy for maintaining energy balance and cell survival could be considered a cellular stress response to the hypoxic-ischemic environment in ischemic stroke. In conclusion, receptor-mediated mitochondrial autophagy through FUNDC1 may play a critical role in ischemic stroke by modulating mechanisms such as binding to LC3, sensing changes in mitochondrial membrane potential, and altering expression levels. This discovery offers new insights into the pathophysiology of ischemic stroke and may provide a potential target for future stroke therapies.

3.5. Nix/BNIP3L receptor-mediated mitochondrial autophagy

Bcl-2/adenovirus E1B 19kDa interacting protein 3-like (BNIP3L), also known as NIP3-like protein X (Nix), is a mammalian mitochondrial autophagy receptor and a member of the Bcl-2 family. Unlike other apoptosis-inducing proteins in the Bcl-2 family, Nix facilitates mitochondrial autophagy through its distinctive autophagic receptor structure, and was initially reported to be involved in mitochondrial autophagy during erythrocyte maturation [62,63]. Normally, BNIP3/NIX expression remains low in most organs; however, under hypoxic-ischemic conditions, its transcriptional levels are directly regulated and activated by HIF-1α [64]. During cerebral ischemia, excitotoxic damage to neurons can lead to mitochondrial autophagy. This process may be triggered by the upregulation of p53, a key protein involved in stress responses, along with the autophagy regulator DRAM. Together, these factors help promote the degradation of damaged mitochondria, supporting cellular health and mitigating neuronal injury during ischemic events. In contrast, modulation of mitochondrial autophagy can be achieved by influencing Beclin-1 and LC3 expression [65].

In mouse models of ischemic brain injury, Nix induces mitochondrial autophagy through a Parkin-independent mechanism and consequently protects mice against ischemic brain damage. The Nix protein is widely believed to directly bind LC3 through its BH3 domain and subsequently induce mitochondrial autophagy [66]. Additionally, Nix has been suggested to participate in Parkin-dependent mitochondrial autophagy, thus serving as a substrate for Parkin ubiquitination, recruitment of the LC3 adapter protein NBR1 to target mitochondria to the autophagosome, and induction of mitochondrial autophagy [67]. In summary, BNIP3L functions as a mitochondrial autophagy receptor through its association with LC3; mediates mitochondrial autophagy; participates in mitochondrial clearance during erythrocyte development; and plays major roles in various diseases.

3.6. Temporal and spatial differences in mitophagy in acute ischemic stroke

In the onset of ischemic stroke, a heterogeneous decrease in blood flow is observed, including a marked decline in the ischemic core [68]. This diminished blood flow in the core region falls far below the metabolic requirements of the brain tissue, and results in severe, often irreversible cellular damage culminating in extensive necrosis [69]. Concurrently, mitochondria within this compromised area sustain substantial harm, thereby triggering robust mitochondrial autophagy. Whereas the elimination of damaged mitochondria halts further release of detrimental substances, this active autophagy also facilitates the release of pro-apoptotic molecules such as cytochrome C from mitochondria, and consequently initiates the downstream caspase-mediated apoptotic cascade, and ultimately leads to apoptosis and the elimination of irreparable cells to accommodate healthy cellular regeneration [70]. The infarcted core is surrounded by the ischemic penumbra, a region where blood flow is diminished, yet tissues retain a degree of viability. Within this penumbra, mitochondrial autophagy has a crucial role in sequestering damaged mitochondria, thereby mitigating the production of ROS and other noxious byproducts, preserving cellular homeostasis, and potentially delaying or averting apoptosis [71].

The ischemic penumbra is a crucial therapeutic target in stroke management. Despite functional impairment, the brain tissue within this zone remains non-necrotic and consequently presents an opportunity for recovery. Prompt restoration of blood flow and enhancement of tissue metabolism may rejuvenate the function of this brain region, thereby mitigating the progression of infarction and preserving neurological function [72].

The temporal progression of ischemic stroke can be delineated into three distinct phases—the acute, chronic, and recovery stages—each characterized by varying degrees and roles of mitochondrial autophagy. In the acute stage, cerebral ischemia and hypoxia induce severe cellular injury concomitant with mitochondrial disruption. During this period, a rapid increase in mitochondrial autophagy serves as a primary mechanism for the elimination of compromised mitochondria. This surge in autophagy triggers apoptosis in severely damaged cells—a crucial process that eliminates irreparable cells to preserve bodily homeostasis [73,74]. A delicate dynamic equilibrium exists between mitochondrial autophagy and apoptosis, wherein autophagy mitigates excessive apoptosis by sequestering damaged mitochondria, whereas apoptosis curbs the expansion of tissue damage by eliminating non-viable cells. As neurons sustain substantial harm, the apoptotic pathway is activated and synergistically functions alongside mitochondrial autophagy in maintaining homeostasis [75]. In the transition into the chronic phase, the state of ischemia and hypoxia is ameliorated, apoptosis rates decline, and cells undergo repair and regeneration. In this stage, mitochondria remain instrumental, because mitochondrial autophagy not only removes dysfunctional mitochondria but also contributes to the biogenesis of new mitochondria, thereby safeguarding the neuronal energy supply and metabolic equilibrium. This dual role of autophagy fosters neuronal repair, regeneration, and eventual recovery [76].

Ultimately, in the recovery period, most damaged neurons undergo either restoration or replacement by nascent neurons, as evidenced by the progressive improvement in neuronal function and a further reduction in apoptosis rates. Mitochondrial autophagy returns to normal levels, primarily serving its crucial role in mitochondrial quality control, thereby ensuring the sustained health and vitality of the neuronal network [58].

4. MITOCHONDRIAL AUTOPHAGY UNDER METABOLIC IMBALANCE IN THE BRAIN AFTER STROKE

Stroke triggers a cascade of pathophysiological events, wherein ischemia causes interruption of blood flow to the brain, and subsequent halting of the oxygen and glucose supply to brain tissues. This process induces neuroexcitotoxicity, mitochondrial dysfunction, cellular apoptosis, intracellular Ca²⁺ accumulation, opening of voltage-gated ion channels, neuroinflammation, oxidative stress, and other physiological events [77–80] (Fig 3). These events substantially influence the homeostasis of neurons, microglial cells, and other neural cells. In this scenario, cells initiate mitochondrial autophagy to regulate the quantity and quality of mitochondria, thereby protecting neurons and decreasing the likelihood of cell death [81].

Figure3 |

The onset of mitochondrial autophagy in stroke is driven by factors such as calcium overload, excitotoxicity, and oxidative stress. Ischemia and hypoxia trigger electron leakage in the electron transport chain, thus producing substantial reactive oxygen species (ROS). Meanwhile, decreased mitochondrial energy production results in an inability of intracellular antioxidant systems to maintain homeostasis, thereby leading to oxidative stress in cells. Additionally, the decrease in mitochondrial energy production causes neuronal depolarization, which in turn triggers influx of calcium ions mediated by sodium-calcium exchanger proteins (NCX) on the presynaptic membrane. This influx leads to substantial release of glutamate, which binds the NMDAR and AMPA receptors on the postsynaptic membrane. The resultant calcium overload and surge in intracellular calcium ions substantially affect mitochondrial function and worsen oxidative stress.

4.1. Excitotoxicity

Ischemic stroke results in an inadequate supply of oxygen and glucose in brain tissues, thus impeding oxidative phosphorylation, affecting sodium-potassium pumps, and disrupting ion concentration gradients [82]. Neurons release excessive glutamate, thereby overactivating N-methyl-D-aspartate receptors (NMDARs) and other glutamate receptors, inducing calcium influx, causing neuronal calcium overload, and resulting in neuroexcitotoxicity [83]. Only several minutes of hypoxia can lead to ATP depletion, Na⁺/K⁺ pump failure, neuronal depolarization, and excessive release of L-glutaminetamine. Ion channels are activated in this context and subsequently induce mitochondrial damage and promote mitochondrial autophagy. Excessive extracellular L-glutaminetamine, which cannot be rapidly absorbed neurons or astrocytes, causes Ca²⁺ entry into cells. Subsequently, Ca²⁺ overload triggers the activation of intracellular proteases, esterases, and endonucleases, and eventually leads to neuronal death [80,84]. Elevated intracellular Ca²⁺ concentrations activate the regulatory mechanisms of mitochondrial autophagy [80,84]. Brain tissues experience severe damage when cerebral blood flow decreases by 20%. Excitotoxic damage typically occurs rapidly and is considered a primary cause of acute cerebral ischemic injury [85]. Moreover, L-glutaminetamine excitotoxicity has been reported to be a fundamental indicator of ischemia-induced neurodegeneration [86]. Dou et al. have discovered that L-glutaminetamine-induced cell damage decreases mitochondrial membrane potential, enhances activation of the PINK1/Parkin signaling pathway, and consequently triggers mitochondrial autophagy [87]. Importantly, the precise mechanism of excitotoxicity may vary by context and disease state. Further investigation of the interactions and regulatory mechanisms of these factors is necessary to provide a more comprehensive understanding of how excitotoxicity affects mitochondrial autophagy.

4.2. Excessive activation of oxidative stress

Oxidative stress is a critical factor in autophagy activation. After cerebral ischemia-reperfusion, succinate substantially accumulates and is rapidly oxidized by succinate dehydrogenase, thus leading to extensive electron flow through mitochondrial complex, resulting in massive accumulation of mitochondrial-derived ROS, and inducing oxidative stress [77]. However, because damaged neuronal cells cannot eliminate excess ROS, disruption of the lipid-protein redox balance and widespread cellular damage result, and further exacerbate mitochondrial injury [88]. Excessive activation of oxidative stress and ROS overproduction activate hypoxia-inducible factor 1 (HIF-1), which subsequently induces the transcription of BNIP3 and NIX. These protein products competitively bind Bcl-2, thereby releasing Beclin-1; consequently, autophagy is induced, and ischemic hypoxic damage is prevented [89,90]. Moreover, oxidative stress initiates nuclear factor-erythroid2-associated factor 2 (Nrf2), thus inducing expression of multiple antioxidant enzymes. Nrf2 is believed to bind the autophagy protein P62 and consequently regulate autophagy [91]. Although oxidative stress-induced mitochondrial autophagy has been extensively researched, future studies must explore the metabolic pathways associated with oxidative stress in ischemic stroke and their potential effects on mitochondrial autophagy. Additionally, investigating the regulatory role of neuro-immune interactions in mitochondrial autophagy under oxidative stress conditions in stroke may be of interest.

4.3. Apoptosis

From the subacute stage after ischemia to a more prolonged period, neuronal cells may undergo apoptosis via mitochondrial-dependent and death receptor-dependent pathways. Stroke results in oxygen and nutrient deprivation, and triggers oxidative stress. Mitochondrial dysfunction, ROS release, mitochondrial membrane rupture, and loss of membrane potential may result. These events in turn trigger mitochondrial autophagy and cellular apoptosis. Damaged mitochondria might release cellular factors such as cytochrome C, thereby activating the cellular apoptosis pathway. Mitochondrial autophagy aids in clearing these damaged mitochondria, and hence limits the release of cellular factors and slows the occurrence of cellular apoptosis. According to experimental evidence, autophagy, by clearing damaged mitochondria, decreases neuronal apoptosis [6,92]. Huang et al. have found that mitochondrial autophagy exerts neuroprotective effects by inhibiting cellular apoptosis in a transient ischemia-hypoxia model. Increased expression of Beclin-1 protein and the LC3-II/I ratio, and decreased expression of p62, TOM20, and HSP60, attenuates stroke progression [93]. As observed in models of long-term ischemia or permanent middle cerebral artery occlusion (MCAO), alterations in the expression patterns of mitochondrial autophagy-related proteins—specifically, the downregulation of Beclin-1 and LC3 along with the upregulation of p62, TOM20, and HSP60—collectively signify an inhibition of mitochondrial autophagy processes. This inhibition is accompanied by a marked augmentation in the brain infarction volume, highlighting the detrimental consequences of impaired mitochondrial clearance mechanisms under these pathological conditions. Changes in the levels of mitochondrial autophagy-related proteins—such as Beclin-1, LC3, p62, TOM20, and HSP60—can indicate a suppression of mitochondrial autophagy. Beclin-1 is essential for initiating autophagy, so a decrease suggests reduced autophagic activity. LC3 is involved in the formation of autophagosomes. A lower level of LC3-II, the lipidated form associated with autophagosomes, indicates impaired autophagosome formation. p62 serves as a cargo receptor for autophagic degradation and typically accumulates when autophagy is inhibited; high levels of p62 can signal a backlog of damaged proteins and organelles. TOM20 is a receptor on the outer mitochondrial membrane that helps in recognizing damaged mitochondria for autophagy. Increased TOM20 levels can imply that damaged mitochondria are not being adequately cleared. HSP60 is a chaperone protein that helps maintain mitochondrial protein integrity. Elevated levels may suggest mitochondrial stress and reduced autophagic clearance. So, alterations in these proteins reflect a disruption in mitochondrial autophagy, leading to the accumulation of damaged mitochondria and contributing to cellular dysfunction. Beclin-1 protein engages with ULK1 to initiate mitochondrial autophagy, a specialized form of autophagy targeting mitochondria. However, excessive mitochondrial autophagy can, paradoxically, trigger cell apoptosis [77]. Conversely, when the interaction between the anti-apoptotic proteins Bcl-2/Bcl-xL and Beclin-1 increases, it serves to inhibit mitochondrial autophagy, thus potentially modulating the balance between cell survival and death.

Neurological dysfunction resulting from mitochondrial impairment is significantly associated with stroke, through mechanisms primarily involving elevated adenosine triphosphate (ATP), increased levels of ROS, and an overload of oxidative stress. During mild ischemia or manageable hypoxic stress, mitochondrial autophagy may contribute to cellular homeostasis maintenance and support cell survival. In the context of persistent ischemia or reperfusion, vulnerable brain cells in the ischemic core area begin to undergo excessive long-term autophagy hyperactivity resulting in cell damage or death [31], and therefore may provide a potential approach to protecting neurons and averting cell death. During a stroke, the regulation of both the quantity and quality of mitochondria is achieved through the process of mitochondrial autophagy, as shown in the Fig 2.

5. MITOCHONDRIAL AUTOPHAGY STATUS OF VARIOUS CELL TYPES IN THE BRAIN AFTER STROKE

Ischemia in the brain may result in irreversible damage to neural networks and lead to functional impairments [94,95]. Targeted repair of various types of neurons post-stroke enhance the formation of new neural connections, and may potentially provide benefits in stroke treatment (Fig 4). Neurons, the fundamental units of the brain and nervous system, are responsible for transmitting electrical signals and information. They play crucial roles in cognition, motor control, and emotional regulation, among many other aspects [96]. After stroke, neurons face severe oxidative stress and disrupted energy metabolism, which may compromise mitochondrial function [97]. Mitochondrial autophagy in neurons is likely to have a protective role by eliminating damaged mitochondria, and maintaining cellular energy metabolism and homeostasis [98,99]. Therefore, promoting mitochondrial autophagy in neurons might potentially alleviate stroke-induced neuronal damage. Astrocytes are supportive cells in the nervous system that are closely associated with neurons [100]. They participate in maintaining the health and stability of neurons, including providing nutrients, clearing metabolic byproducts, and regulating synapse formation and function [101]. Microglia, another type of glial cell in the nervous system distinct from astrocytes, play major roles in immune regulation and inflammatory responses. Both astrocytes and microglia contribute to inflammation modulation and neural protection [102]. Hence, mitochondrial autophagy within these cells might aid in preserving their function, decreasing inflammatory responses, and consequently shielding surrounding neurons from additional damage. Endothelial cells form the vascular wall and are crucial for maintaining cerebral blood flow and the blood-brain barrier. Post-stroke, endothelial cells are also affected, thus triggering vascular damage and inflammatory responses [103]. Mitochondrial autophagy might assist in repairing and protecting endothelial cells, and promoting vascular regeneration and functional recovery. The enhancement of mitochondrial quality in astrocytes and microglia, achieved through the process of mitochondrial autophagy, serves to modulate their immune status, thereby mitigating the detrimental effects on neurons and ultimately facilitating the therapeutic management of stroke. The selective elimination of dysfunctional mitochondria and promotion of healthy mitochondrial turnover serves to restore cellular homeostasis in glial cells, thereby reducing neuroinflammation and neuronal damage. This mechanism represents a promising therapeutic strategy for stroke.

Figure4 |

Effects of mitochondrial autophagy on various cells after stroke.

Ischemic stroke-induced energy depletion and hypoxia lead to neuronal death, thereby activating resident glial cells that can elicit diverse, even conflicting, immune-mediated effects. This activation might potentially facilitate neuronal repair, differentiation, and regeneration, and provide insights into novel potential therapeutic strategies.

5.1. Neurons

Neurons are the fundamental structural and functional units of the nervous system. Irreversible neuronal damage during ischemic stroke directly leads to neurological impairments. According to recent evidence, autophagy plays a major role in suppressing neuronal death during cerebral ischemia [98,99]. However, some studies have proposed that autophagy may promote neuronal death after cerebral ischemia. In PC12 neuronal cells under serum deprivation, the autophagy inhibitor 3-MA significantly decreases apoptosis rates [104]. Atg7 inhibits autophagy and decreases cell death in multiple brain regions [105]. Recent comprehensive investigations have conclusively established that, under conditions of OGD, mitochondrial constituents within neuronal axons embark on a well-orchestrated retrograde trafficking pathway toward the soma, where they subsequently undergo a highly regulated process of autophagy [106]. Targeted and precise intervention strategies aimed at modulating axonal mitochondrial autophagy might substantially mitigate injury to distal neurons, and provide a promising avenue for neuroprotective therapeutics, thereby advancing the field of neuronal resilience and protection research [107].

5.2. Astrocytes

Astrocytes play crucial roles in controlling neural circuits, ion homeostasis, inflammation, and the stability of neurotrophic factors. Activation of autophagy has been observed in primary cultured astrocytes under in vitro hypoxic conditions and in animal models of focal cerebral ischemia [108]. Knockdown of the Atg5 gene in primary cultured astrocytes reverses the increased expression of LC3-II induced by OGD, whereas knockdown of the Beclin1 gene in astrocytes reverses the upregulation of autophagy induced by OGD through miR-30d inhibition. Accumulation of autophagic vacuoles has been observed in astrocytes in ischemic brain tissue [109,110]. Administration of the autophagy inhibitors 3-MA and bafilomycin A1 significantly alleviates ischemic injury-induced astrocytic death. In astrocytes subjected to OGD, decreased autophagy after 3-MA treatment correlates with diminished apoptosis and necrosis levels [110,111]. Together these findings indicate that, during ischemia, autophagy in astrocytes is activated and exacerbates damage.

5.3. Microglia

Microglial cells, intrinsic immune-effector cells in the central nervous system, play crucial roles in physiological processes. During cerebral ischemia, microglia phagocytose dead neurons and promote repair of ischemic injury [112,113]. Activation of autophagy has been observed in murine models of cerebral ischemia and in primary cultured microglia treated with OGD [114,115]. Moreover, hypoxia-reoxygenation activates microglial autophagy through the ROS-regulated Akt/mTOR signaling pathway, and consequently induces microglial apoptosis. The decreased autophagy levels after 3-MA treatment are accompanied by decreased brain infarct volume and improved neurological scores; therefore, autophagy in microglia may exacerbate ischemic brain injury [116].

5.4. Endothelial cells

Ischemic stroke leads to blood-brain barrier disruption and increased vascular permeability, thereby causing cerebral edema. Brain microvascular endothelial cells (BMVECs) are specialized endothelial cells crucial for maintaining the integrity of the vascular barrier [117]. Inhibition of autophagy with 3-MA has been shown to increase apoptosis levels in BMVECs after ischemia; therefore, autophagy activation in BMVECs during the middle phase of ischemic stroke is beneficial for blood-brain barrier integrity. Another study has suggested that chloroquine inhibition of autophagy in BMVECs increases blood-brain barrier permeability and the degree of cerebral edema. These findings suggest that autophagy activation in BMVECs might have a beneficial role during cerebral ischemia [118,119]. However, 3-MA has been shown to reverse OGD/R-induced morphological changes in cerebral endothelial cells, increase cell survival rates, and inhibit apoptosis. Knockdown of Atg7 in cerebral endothelial cells to inhibit autophagy has been reported to significantly decrease OGD/R-induced pro-inflammatory cytokine expression, thus exerting neuroprotective effects [120]. Therefore, in cerebral ischemia, the role of mitochondrial autophagy in brain microvascular endothelial cells requires further clarification.

6. POTENTIAL THERAPEUTIC APPROACHES TO AMELIORATE STROKE PATHOLOGY BY TARGETING MITOCHONDRIAL AUTOPHAGY

Under normal conditions, cells use a cleaning process called autophagy that helps eliminate damaged proteins and worn-out cell components. However, excessive autophagy can lead to cell death. Another cleanup process, mitophagy, specifically targets damaged or dysfunctional mitochondria, thus helping to prevent the buildup of harmful substances and protecting against cell death [121]. Many clinical and experimental investigations have shown that mitophagy serves as a protective mechanism for brain cells in ischemic stroke. However, its role shifts when the brain experiences reperfusion injury [122].

In the ischemic phase of ischemic stroke, carnosine has shown potential in attenuating ischemic injury by safeguarding against mitophagy. Furthermore, acidic postconditioning in early stages of ischemic stroke (within 6 hours) has been observed to enhance mitophagy and thereby increase the resilience of brain tissue to ischemia in mice [122].

Recent studies have suggested that the specific inhibition of mTOR protein activity by rapamycin is known to trigger the initiation of autophagy, a process that has been demonstrated to significantly promote mitophagy. Consequently, in a rat model of transient middle cerebral artery occlusion (MCAO), rapamycin has been demonstrated to effectively alleviate mitochondrial dysfunction, thereby providing protection against cerebral ischemia-reperfusion (I/R) injury. It is important to note, however, that the introduction of 3-methyladenine, an inhibitor of phosphatidylinositol 3-kinase (PI3K), blocks the crucial step of membrane curvature of the initial autophagic vesicles mediated by the Beclin1-VPS34 complex. This effectively suppresses the initiation of autophagy, leading to a significant attenuation or neutralisation of the neuroprotective effects originally mediated by rapamycin [123]. An in vitro experiment has demonstrated that H2 exhibits neuroprotective effects on neurons damaged by OGD/R, through enhancing mitophagy via the PINK1/Parkin signaling pathway [124]. Parkin-dependent mitophagy has been found to decrease excessive inflammation caused by blood reperfusion by slowing NLRP3 inflammasome activation.

Several studies have confirmed the protective role of mitophagy in attenuating brain injury during the reperfusion phase [125,126]. Recent studies have uncovered potentially harmful effects of mitophagy. For instance, an in vitro study has revealed that the upregulation of small nucleolar RNA host gene 14 in OGD/R-damaged mouse hippocampal neurons contributes to mitophagy and leads to substantial cell apoptosis. In the context of ischemic stroke and the MCAO model, an increase in mitophagy-related proteins in brain tissues has been demonstrated. Additionally, inhibiting mitophagy may offer protective benefits against cerebral I/R injury in MCAO rats, potentially through mitigating excessive mitochondrial autophagy [127]. In addition, rehmapicroside inhibits mitophagy by preventing the accumulation of mitophagy-associated proteins in mitochondria and consequently improves neurological deficit scores.

7. DISCUSSION

Because ischemic stroke is a primary cause of death and disability worldwide, a deep understanding of its pathogenesis is crucial [128–131]. Neurons have high demand for energy, and mitochondria, the main energy producers, are particularly important in the nervous system [19,20]. Mitochondrial autophagy contributes to the elimination of dysfunctional mitochondria, prevention of the release of harmful substances, and maintenance of normal neuronal function [31]. Dysregulated mitochondrial autophagy might lead to the accumulation of damaged mitochondria in neurons, thus triggering oxidative stress and inflammatory reactions, affecting neuronal health, and exacerbating stroke progression [89,90]. Therefore, understanding and regulating mitochondrial autophagy in the nervous system is important for preventing and treating stroke.

In recent years, the relationship between stroke and mitochondrial autophagy has been extensively researched, and remarkable progress has been achieved. Although current studies indicate close correlations between neuronal damage in the ischemic brain and physiological events such as excitotoxicity, mitochondrial dysfunction, apoptosis, intracellular Ca²⁺ accumulation, voltage-gated ion channel opening, neuroinflammation, and oxidative stress [80,84], the precise regulation of these processes, and effective prevention or mitigation of pathological damage after interruption of the oxygen and glucose supply to brain tissues, remain important areas for exploration. Moreover, the molecular mechanisms and signaling pathways involved in mitochondrial autophagy may differ according to variations in ischemic stages, duration, stroke models, and animal ages. Additionally, mitochondrial autophagy’s role within ischemic brains might even exhibit contradictory effects. Hence, understanding the dynamic changes and effects of mitochondrial autophagy in different ages, stages, and stroke models is essential, and further exploration and clarification remain necessary. Furthermore, given the complexity of mitochondrial autophagy pathways and regulatory mechanisms, understanding how to precisely control the physiological state of mitochondria and maintain their balance remains to be addressed. Maximizing the benefits of mitochondrial autophagy is a direction for future investigations.

This article discussed current research hotspots and anticipated major breakthroughs in this field. With technological advancements, understanding of cellular mitochondrial autophagy regulation after stroke continues to deepen. However, many critical questions remain unanswered, including how stroke activates the initiation and execution of mitochondrial autophagy, and what role this mechanism might play in stroke injury development. Future research should focus on understanding the differences in various types of intracellular mitochondrial autophagy in stroke, and the role of inflammation and the immune system in this process. In coming years, this field will contribute to a comprehensive understanding of the interrelationships among stages of stroke. Additionally, exploring the optimal treatment window for intervention in mitochondrial autophagy to significantly decrease brain damage is a current research focus. Understanding the precise timeframe for interventions targeting mitochondrial autophagy to maximize the alleviation of brain damage will be crucial for translating basic research outcomes into clinical practice.

Furthermore, although some studies have investigated therapeutic approaches targeting mitochondrial autophagy for treating cerebral ischemia, research on the safety of specific treatments, pharmacokinetics of specific drugs, and routes of administration is limited. Moreover, whereas the effects of mitochondrial autophagy in various stroke models have been extensively studied in preclinical animal research in clinical trials, no clinical evidence has been reported to date. Hence, identifying and refining the clinical therapeutic effects of targeting mitochondrial autophagy for stroke treatment requires further assessment.

With the maturation of single-cell technology, proteomics, and gene editing techniques, a more comprehensive and in-depth understanding of the relationship between stroke and mitochondrial autophagy is expected. This understanding would provide a solid theoretical basis for developing new treatment strategies, innovative drugs, and personalized medicine. Interdisciplinary collaboration is expected to usher in a new era in stroke treatment. In summary, mitochondrial autophagy helps maintain mitochondrial integrity and intracellular homeostasis, thereby playing a neuroprotective role in cerebral ischemia. Investigating the molecular mechanisms and regulatory pathways of intracellular mitochondrial autophagy after ischemic stroke might offer new perspectives and breakthroughs for clinical stroke treatment.

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