Astrocytic mitochondrial transfer: a new horizon for metabolic rescue and precision therapy in ischemic stroke

Abstract

Ischemic stroke (IS) remains a leading cause of global mortality and neurological disability, with neuronal mitochondrial dysfunction as a central pathological mechanism. Astrocytes, the metabolic custodians of the central nervous system, exert neuroprotection by transferring functional mitochondria to compromised neurons via tunneling nanotubes (TNTs), extracellular vesicles (EVs), connexin 43 (Cx43) mediated gap junctions, and membrane fusion. These transfers replenish neuronal energy reserves, mitigate oxidative stress, and enhance synaptic plasticity. This review systematically delineates the molecular mechanisms of astrocyte-mediated mitochondrial transfer, its regulatory roles in oxidative stress, calcium dyshomeostasis, and ferroptosis, and its therapeutic potential in IS. Experimental models demonstrate that pharmacological enhancement of mitochondrial transfer or exogenous transplantation significantly reduces infarct volume and improves neuronal survival. However, clinical translation faces challenges including low mitochondrial viability, immune rejection, and inefficient delivery. Future research should integrate gene-editing tools, nanocarrier systems, and organoid models to optimize mitochondrial dynamics and develop precision therapies. By bridging mechanistic insights with translational innovations, astrocytic mitochondrial transfer emerges as a groundbreaking strategy for ischemic stroke treatment.

Ischemic stroke (IS) is a neurological disorder caused by acute occlusion of cerebral arteries, which leads to interrupted local blood flow and critical deprivation of oxygen and glucose in brain tissue [1, 2]. It has remained the second leading cause of age-standardized mortality globally since 1990 [3]. Over 60% of incident strokes were attributed to cerebral ischemia in 2019 alone [4]. The pathogenesis of IS involves an initial phase of ischemia, often followed by reperfusion—either spontaneous or therapeutically induced—which, despite being essential for tissue salvage, paradoxically exacerbates damage through oxidative stress and inflammatory responses. This secondary injury is collectively termed ischemia-reperfusion (I/R) injury.

This devastating condition triggers a cascade of pathological mechanisms, including energy depletion, oxidative stress, calcium overload, and ultimately neuronal death [5, 6]. The central role of mitochondria becomes evident here: as oxygen deprivation halts oxidative phosphorylation, adenosine triphosphate (ATP) production plummets, accelerating ionic imbalance and metabolic collapse. Subsequent reperfusion then fuels a burst of reactive oxygen species (ROS) from the compromised electron transport chains in mitochondria, overwhelming cellular antioxidant defenses and propagating oxidative damage (Figs. 1 and 2).

Fig. 1.

Schematic illustration of astrocyte-mediated mitochondrial transfer in IS. Astrocytes transfer functional mitochondria to compromised neurons via TNTs, EVs, Cx43-mediated gap junctions, and membrane fusion, replenishing neuronal energy, mitigating oxidative stress, calcium dyshomeostasis, and ferroptosis, while enhancing synaptic plasticity. Pharmacological enhancement or exogenous transplantation reduces infarct volume and improves neuronal survival, with future strategies incorporating gene editing, nanocarriers, and organoids to overcome translational challenges. Figure created by Nano Banana

Fig. 2.

The main pathways of mitochondrial transfer. Astrocytes support compromised neurons by transferring functional mitochondria via (1) TNTs for direct transport (2), EVs for cargo delivery (3), Cx43-mediated gap junctions for metabolic exchange, and (4) membrane fusion for content sharing. Figure created with Adobe Illustrator (version 24.1.)

As the most energy-demanding cells in the brain, neurons are acutely vulnerable to I/R injury, in which disrupted mitochondrial function rapidly compromises cellular energy metabolism—the cornerstone of neuronal survival. Mitochondria, the central hubs of cellular energy production and apoptosis regulation, play a dual role in IS. While transient mitochondrial preservation in brain vasculature has been observed post-injury [7], persistent dysfunction exacerbates oxidative stress, ROS overproduction, and inflammatory cascades; driving irreversible neuronal damage. A critical vulnerability of neuronal mitochondria is their weak self-repair capability [5, 6]. This is largely due to an impaired mitophagy, which hinders the autonomous clearance of damaged mitochondrial structures. This deficiency in self-repair mechanisms renders mitochondrial dysfunction a pivotal factor in ischemic cell death and establishes the disruption of mitochondrial homeostasis as a key driver of programmed neuronal death in the ischemic penumbra [8]. Consequently, exogenous mitochondrial transfer may serve as an important compensatory strategy to sustain neuronal function under ischemic conditions. The centrality of mitochondrial dysfunction in ischemic neuronal death cannot be overstated [9]. It manifests as a vicious cycle of energy depletion, calcium overload, and excessive ROS production, ultimately triggering apoptotic and necrotic pathways [10]. It is within this pathological context that the transfer of healthy mitochondria from astrocytes emerges not merely as a supportive phenomenon but as a crucial compensatory strategy, highlighting a fundamental shift from cell-autonomous survival to intercellular metabolic cooperation in the ischemic brain.

This vulnerability of neurons underscores the critical need for external support, a role increasingly attributed to astrocytes. As the most abundant glial cells in the brain, astrocytes exhibit a dual role in the progression of IS. During early injury phases, they protect neurons by maintaining ionic/neurotransmitter homeostasis, scavenging ROS, and delivering metabolic substrates [11, 12]. However, chronic activation promotes glial scar formation, impeding neural repair [11]. Notably, astrocytes serve as metabolic lifelines for neurons through indirect vascular coupling, a process by which they transport glucose, oxygen, and functional mitochondria to energy-deprived neurons [13]. Emerging evidence suggests that astrocyte-to-neuron mitochondrial transfer may mitigate neuronal energy crises, yet the precise mechanisms underlying this metabolic cooperation remain poorly understood [14–16].

This review addresses a critical gap in IS research: How do astrocytes leverage mitochondrial transfer or secretory mechanisms to sustain neuronal survival following ischemic injury? To answer this question, we analyze the multifaceted roles of astrocytic mitochondria in post-stroke pathology. Our review encompasses their functions in oxidative stress regulation, calcium homeostasis, and metabolic coupling, and extends to pathways of mitochondrial transfer, dual roles in neuronal injury and repair, key regulatory mechanisms, relevant experimental models, and translational therapeutic potential. Finally, we highlight unresolved questions to guide future research in this rapidly evolving field.

To provide a systematic delineation of the pathological role of astrocytic mitochondria in IS, we conducted a comprehensive literature search and review. The search was performed in the core databases of PubMed and Web of Science, covering the period from the inception of each database to May 2025. The search strategy utilized key terms such as “astrocyte mitochondria”, “ischemic stroke”, “neuronal death”, and “mitochondrial transfer”, among other relevant terms and combinations. The inclusion criteria for the literature were primarily as follows: [1] peer-reviewed original research articles; [2] studies directly investigating the association between astrocytic mitochondria and neuronal fate following ischemic stroke; and [3] publications in English. This approach aimed to ensure that the present review is based on currently available high-quality evidence and provides a critical synthesis of the field.

Pathways of mitochondrial transfer

The intercellular transfer of mitochondria, a crucial compensatory mechanism in pathological conditions such as IS [17, 18], is mediated through multiple highly coordinated pathways including [1] tunneling nanotubes (TNTs) [2], extracellular vesicles (EVs) [3], connexin 43 (Cx43)-mediated gap junctions, and [4] direct membrane fusion processes [19, 20].

Among these, TNTs, which are F-actin-supported membranous nanotubes with diameters between 50 nm and 1 μm, represent a major pathway for astrocyte-mediated transfer of functional mitochondria to compromised neurons [21]. This transfer helps restore energy metabolism and suppress apoptosis under I/R injury [22, 23]. The process is regulated by several mechanisms, including ROS/mtROS and PI3K/AKT/mTOR signaling [24], DAMPs-CD38 activation [25], and Miro1-dependent transport [26]. Furthermore, post-transfer integration of mitochondria into recipient neurons is facilitated by fusion proteins such as MFN1/2 and OPA1 [27]. EVs are nanosized, lipid bilayer-enclosed particles that facilitate intercellular communication by transferring bioactive cargo [28], including functional mitochondria. Classified by size and origin into exosomes (30–150 nm), microvesicles(100–1000 nm), and apoptotic bodies(>1000 nm) [29], EVs play a key role in delivering intact mitochondria to neurons, supporting mitochondrial homeostasis and network integration [30–32]. Mitochondrial sorting into EVs involves active packaging mechanisms and may synergize with TNT-mediated transfer under certain conditions. Cx43-formed gap junctions provide another major route for direct mitochondrial transfer, often coordinating with TNTs and EVs to enhance delivery efficiency, as evidenced by the synergistic activation of these pathways in ischemic models. Furthermore, mitochondrial fusion proteins MFN1/2 play essential roles in membrane fusion-mediated transfer, with dysregulation exacerbating blood-brain barrier (BBB) disruption and impairing mitochondrial integration. Importantly, these pathways are not mutually exclusive but exhibit functional synergy: TNTs may cooperate with channels associated with F-actin-rich EVs for directional transport, while CD38-mediated endoplasmic reticulum-mitochondria crosstalk regulates mitochondrial extrusion and uptake via endocytosis/membrane fusion.

Currently, mitochondrial transfer is primarily understood to occur through four main pathways. However, most existing research has focused on TNTs and EVs, largely conducted at the cellular level. Further studies using in vivo models are needed to validate their functional roles and regulatory mechanisms. Moreover, it remains unclear how mitochondrial transfer via different mechanisms is precisely regulated following IS. For instance, the specific molecular mechanisms underlying TNT formation have not been fully elucidated. Regarding EVs, the processes of recipient cell recognition and internalization are still in the early stages of investigation and require further exploration (Table 1).

Table 1.

Comparison of major pathways of mitochondrial transfer

Pathways	Advantages	Limitations	References
TNTs	Direct intercellular connection, highly efficient transfer	Structurally unstable, susceptible to mechanical disruption	In vitro: [33, 34] In vivo: [35, 36]
	Capable of transferring large organelles (e.g., intact mitochondria)	Slow formation rate, dependent on cellular stress signals
EVs	Long-distance transport, overcomes spatial constraints	Low loading efficiency, difficulty in preserving mitochondrial integrity	In vitro: [37] In vivo: [38, 39]
	Native biocompatibility, low immunogenicity	Complex isolation and purification techniques
Gap junctions	Rapid exchange of ions and small molecules	Permits only small molecules (< 1 kDa); may require synergy with TNTs	In vitro: [40] In vivo: [40, 41]
	Stable connection, dynamically regulated by phosphorylation	Limited capacity for whole mitochondrial transfer
Membrane fusion	Avoids intracellular degradation, direct integration	Low efficiency, unclear mechanisms	In vitro: [42] In vivo: [43, 44]
	Suitable for large-scale material exchange	May cause membrane instability

Function of astrocytic mitochondria

Astrocytic mitochondria exert neuroprotective effects through multiple mechanisms following IS, with their functions extending beyond energy supply to encompass antioxidant and anti-apoptotic activities, as well as the promotion of synaptic plasticity and neural circuit reconstruction. These functions are interrelated and collectively form a synergistic defense system, with the core objective of maintaining neuronal metabolic homeostasis and facilitating post-injury repair. Subsequent sections will systematically elaborate on these functions and their intrinsic connections.

Energy supply and metabolic support

Astrocytic mitochondria sustain neuronal energy demands through diverse metabolic pathways, delivering substrates such as glucose, lactate, fatty acids, amino acids, and ketone bodies [45]. Their mitochondrial carbon metabolism transfers metabolites to neurons via anaplerotic inputs and carbon shuttle mechanisms, thereby supporting the high bioenergetic requirements of neurons [46]. Astrocytic mitochondria provide lactate to neurons through monocarboxylate transporters; this lactate is then converted to pyruvate by lactate dehydrogenase (LDH) in neuronal mitochondria, fueling the tricarboxylic acid (TCA) cycle and contributing 30–40% of neuronal ATP production. Notably, the astrocyte-neuron lactate shuttle underscores the central role of astrocytes in glycolytic lactate generation and its transfer to neurons as an energy substrate [47]. Astrocytic mitochondria dynamically adjust energy supply in response to neuronal activity: during heightened synaptic transmission, mitochondrial calcium homeostasis and ATP synthesis help meet localized energy demands [48]. Mitochondrial trafficking and anchoring mechanisms ensure precise energy delivery to sites of neuronal injury [49]. In ischemic or neurodegenerative conditions, direct mitochondrial transfer from astrocytes to compromised neurons exerts neuroprotection. Experimental evidence demonstrates that transplanted astrocytic mitochondria rescue neuronal degeneration by restoring redox balance, neurotransmitter recycling, and calcium signaling, indirectly bolstering neuronal metabolism and survival [50].

Antioxidant and anti-apoptosis

Astrocyte mitochondria maintain energy homeostasis through fatty acid metabolism and oxidative phosphorylation (OxPhos), degrading excess fatty acids (FAs) and thereby reducing the accumulation of ROS [51]. Their mitochondrial respiratory chain produces higher levels of ROS than of neurons but protects neurons from oxidative damage by regulating antioxidant systems through a physiologically elevated ROS environment [52]. Astrocytes can transfer functionally normal mitochondria to damaged neurons to directly complement their defective mitochondrial function, eliminate ROS-generating mitochondria during intrinsic metabolism of neurons, and enhance the antioxidant capacity of neurons through metabolic coupling mechanisms such as the supply of glutathione precursors [32].

Astrocyte mitochondria reduce neuronal apoptosis by upregulating the expression of anti-apoptotic proteins such as Bcl-2 and inhibiting mitochondria-dependent apoptotic pathways [53]. In addition, astrocyte mitochondrial dysfunction triggers a pro-inflammatory response, whereas normal mitochondria inhibit microglia activation and the release of pro-inflammatory factors, such as TNF-α and IL-6, indirectly protecting neurons from inflammation-related apoptosis [54]. Astrocyte mitochondria maintain neuronal energy requirements and avoid oxidative stress and apoptotic cascade through calcium buffering, ATP supply and glutamate metabolic homeostasis [32]. Studies have shown that targeting and enhancing astrocyte mitochondrial function by antioxidants or mitochondrial transplantation significantly reduces neuronal apoptosis and improves neurological function in models of cerebral ischemia and Parkinson’s disease [32, 48, 51].

Synaptic plasticity and neurocircuitry restoration

Astrocytic mitochondria regulate synaptic activity by maintaining homeostasis of glutamate, calcium ion (Ca²⁺), and ATP, thereby supporting energy demands for synaptic function [51, 55]. Evidence indicates that mitochondrial modulation of lactate metabolism plays a key role in regulating synaptic plasticity, with studies showing that restoration of astrocytic lactate supply can rescue synaptic plasticity impaired by cellular stress [56]. Notably, through ryanodine receptor-mediated Ca²⁺ signaling and metabolic intermediates such as lactate, astrocytes modulate long-term potentiation (LTP) in the hippocampus and cerebral cortex, influencing learning and memory [57]. Further evidence suggests that aquaporin-4-dependent volume transients induce morphological changes in astrocytes, directly coupling neuronal activity to synaptic strength regulation [58]. Mitochondrial dysfunction may disrupt the expression of synaptic proteins, impairing plasticity-associated pathways such as BDNF-ERK1/2 signaling [59, 60]. Following hypoxic-ischemic injury, astrocytic mitochondria promote neural circuit reconstruction by sustaining the dynamic balance of synaptic protein synthesis and degradation [61, 62]. Additionally, astrocytic mitochondria enhance computational efficiency in neural circuits by coupling energy metabolism with redox homeostasis, thereby repairing circuit dysfunction caused by mitochondrial deficits [45, 63] (Figs. 3 and 4).

Fig. 3.

Astrocytic Mitochondrial function in neuronal. The diagram illustrates: (1) Energy supply via glycolysis, TCA cycle, and oxidative phosphorylation producing ATP; (2) Antioxidation and anti-apoptosis mechanisms counteracting ROS and regulating Bcl-2, cytochrome c, Apaf-1, and caspases; (3) Synaptic plasticity and neurocircuitry restoration driven by ATP, supporting synapse strength and protein expression for learning and memory. Figure created with Adobe Illustrator (version 24.1.)

Fig. 4.

Receptors Oxidative stress of found in IS related to astrocyte-neuron. The diagram illustrates the role of receptors related to oxidative stress in the astrocyte-neuron mitochondrial transfer, highlighting their contribution to neuronal dysfunction and neuroprotection in IS. Figure created with Adobe Illustrator (version 24.1.)

Pathological processes of astrocytic mitochondria in neuronal loss/repair

Oxidative stress

Oxidative stress is particularly prominent in the brain, a tissue that accounts for only 2–3% of body weight yet consumes 20% of total body oxygen, resulting in substantial basal free radical production [64]. This vulnerability is compounded following IS, as reperfusion—though necessary for salvage—exacerbates oxidative damage through a burst of ROS accumulation [65]. In this context, mitochondrial function becomes paramount. As cellular powerhouses, mitochondria primarily produce ATP, while astrocytes occupying about 50% of brain volume and serving as the sole glycogen reservoirs, provide metabolic support to neurons via intricate signaling [66, 67]. Their mitochondrial integrity is thus essential for maintaining cerebral energy balance and neuronal health [67]. Under anaerobic conditions, co-cultured astrocytes exhibit increased mortality when supporting damaged neurons, suggesting that energy transfer to neurons accelerates their demise [68, 69]. In isolation, astrocytes display greater ischemic tolerance than neurons [65]. Therefore, the mechanisms by which astrocytes provide metabolic support, particularly through the transfer of functional mitochondria to neurons, are crucial for modulating redox homeostasis and promoting survival under pathological conditions.

In IS, oxidative stress modulates astrocyte-neuron interactions through multiple molecular pathways that directly regulate the intercellular transfer of mitochondria, a process crucial for metabolic rescue and neuronal survival. However, the evidence supporting the relative importance of these pathways varies significantly in strength. The activation of the Nrf2-Keap1 pathway in astrocytes not only enhances cellular antioxidant defenses but also influences mitochondrial quality control and autophagic flux, thereby determining the quantity and functional capacity of mitochondria available for transfer to neurons [70]. Specifically, Nrf2 activation promotes the transcription of genes involved in mitochondrial biogenesis and quality control, potentially increasing the pool of functional mitochondria that can be mobilized for transfer via TNTs or EVs [71–73]. However, these mechanisms are mainly supported by in vitro studies of preclinical models, and their exact role in human pathophysiology still needs to be confirmed. Similarly, the multifunctional redox-sensitive protein DJ-1 integrates antioxidant defense, mitochondrial homeostasis, and RNA-binding activity to establish a cytoprotective network. Key mechanisms through which DJ-1 may influence mitochondrial transfer include: (1) scavenging ROS via conserved cysteine residues and suppressing MAO-B activity [74, 75]; to reduce dopaminergic neuron oxidative damage [76]; (2) maintaining mitochondrial membrane potential, ATP synthesis, and respiratory chain activity [77]; ensuring the functionality of mitochondria destined for transfer; and (3) protecting mitochondrial transcripts from oxidative damage through non-canonical RNA-binding mechanisms [78], which may support the synthesis of proteins essential for mitochondrial transport and integration. The astrocyte-specific DJ-1 enhances antioxidant capacity via the miR-155/SHP-1 axis [79] while its truncated isoform DJ-1∆C exerts paracrine protection in ischemic models. Although these findings are compelling, a direct causal relationship between DJ-1’s RNA-binding function and its putative role in mitochondrial transfer remains to be experimentally confirmed.

Conversely, some inflammatory pathways can negatively regulate mitochondrial transfer. The NOX-NF-κB pathway promotes lipocalin-2 secretion, driving neuronal apoptosis and axonal degeneration [80]. More importantly, this pro-inflammatory oxidative environment alters astrocytic physiological states, potentially impairing their ability to form TNTs or generate EVs, thereby directly inhibiting mitochondrial transfer mechanisms. Similarly, peroxiredoxin 6-mediated neuroinflammation via iPLA2-dependent ROS generation [81] and RIPK2-induced pro-inflammatory astrocyte polarization [82] further contribute to a microenvironment that disrupts intercellular mitochondrial transport. Within this context, molecules like Miro1 and TFAM serve as “help-me” signals from neurons to mediate mitochondrial transfer from astrocytes [80], highlighting how bidirectional communication coordinates this process. A key unanswered question is how the balance between pro-inflammatory and antioxidant pathways determines the net outcome of mitochondrial transfer. Collectively, these pathways regulate ROS generation, mitochondrial dynamics, and intercellular communication, forming an integrated regulatory network that determines the efficiency and directionality of mitochondrial transfer.

Calcium homeostasis

IS leads to an imbalance in calcium homeostasis within neurons and astrocytes, which is one of the major drivers of cell death and brain damage. The massive release of glutamate after stroke overactivates neuronal NMDA/AMPA receptors and metabotropic glutamate receptors, triggering a spike in intracellular Ca²⁺ concentration. Compounding this, neurons are unable to maintain normal calcium ion concentration under ischemic conditions, which leads to abnormally elevated intracellular calcium levels [83, 84]. Astrocytes regulate the uptake and release of calcium ions through their mitochondria, thereby affecting the calcium ion concentration in neurons [85]. Physiologically, mitochondria maintain Ca²⁺ homeostasis by actively utilizing Ca²⁺ to regulate ATP production, mitochondrial permeability, and signaling. However, in the presence of dysregulated calcium homeostasis, elevated Ca²⁺ levels cause mitochondria to swell and release their contents, ultimately leading to delayed neuronal death. This calcium overload triggers multiple cell death pathways, including excitotoxicity, mitochondrial dysfunction, and apoptosis [86].

Notably, the regulation of calcium homeostasis is intrinsically linked to intercellular mitochondrial transfer. Mitochondrial calcium overload is a central driver of neuronal injury [87, 88], and crucially, the transfer of functional mitochondria from astrocytes to neurons directly counteracts this pathology. Through an LRP1 receptor mediated mechanism [49], astrocytes provide healthy mitochondria to damaged neurons, which restore calcium homeostasis by buffering excess Ca²⁺, replenishing ATP, and supporting the clearance of cytosolic calcium loads [83]. This transfer is particularly critical for recovering synaptic function, as astrocyte-derived mitochondria help maintain calcium homeostasis at synaptic sites and inhibit excitotoxicity by supporting glutamate metabolism and ATP-dependent neurotransmission [55, 89]. The process of mitochondrial transfer is further coordinated by specialized signaling platforms and metabolic coupling. Mitochondria-associated endoplasmic reticulum membranes (MAMs) serve as key microdomains for calcium crosstalk between astrocytes and neurons [90]. Through MAMs, astrocytes coordinate ER-mitochondrial calcium exchange, indirectly priming mitochondria for transfer and enhancing the capacity of recipient neurons to handle calcium loads. The endoplasmic reticulum regulates mitochondrial calcium uptake through ryanodine receptors (RyR), a process independent of the mitochondrial calcium uniporter (MCU) [91, 92]. Additionally, astrocytes contribute to the restoration of calcium homeostasis after ischemia via STIM1/ORAI-mediated store-operated calcium entry (SOCE). Meanwhile, the sodium/calcium/lithium exchanger (NCLX) and the MCU complex jointly regulate the uptake and extrusion of mitochondrial calcium [91, 93]. Dysfunction in these mechanisms can exacerbate ischemic injury. Metabolic and organellar quality control mechanisms also support this transfer process. Enhanced astrocytic glycolysis supplies reduced glutathione, mitigating oxidative stress and indirectly promoting mitochondrial transfer by preserving donor organellar function [94]. Lactate, a key astrocytic metabolite, facilitates mitochondrial translocation via ARF1 protein modification, and higher lactate levels correlate with improved transfer efficiency [49]. Furthermore, astrocyte autophagy maintains mitochondrial fitness and calcium buffering capacity by removing damaged organelles, thereby ensuring that transferred mitochondria are functionally competent to alleviate neuronal calcium overload [95]. Disruption of these processes impairs mitochondrial dynamics and calcium handling, directly contributing to post-ischemic neuronal death [96].

Although these mechanisms are well-supported by in vitro studies, assessment of their relevance in vivo remains limited. For instance, the quantitative contribution of LRP1-mediated transfer has not been definitively established in living models of ischemic stroke. A significant knowledge gap exists regarding the relative importance of different calcium-handling mechanisms—such as SOCE, MCU, and NCLX—within the specific context of mitochondrial transfer. In summary, calcium dyshomeostasis and mitochondrial transfer are deeply intertwined in ischemic stroke. Astrocytes not only modulate calcium dynamics through their own mitochondrial networks but also directly supply functional mitochondria to neurons via specific molecular mechanisms such as LRP1. These integrated mitochondria then support neuronal recovery through three key mechanisms: calcium buffering, restoration of bioenergetics, and synaptic repair. Consequently, facilitating the transfer of mitochondria emerges as a critical therapeutic strategy, directly targeting the calcium-mediated pathways that underlie neurodegeneration (Figs. 5 and 6).

Fig. 5.

Astrocyte-neuron metabolic coupling and calcium signaling in neuronal health. The diagram illustrates glycolysis in neurons producing ATP and pyruvate, astrocyte-derived lactate as an energy source, and calcium regulation via MCU, NCLX, and RyR. Dysregulated calcium signaling and MPTP opening contribute to mitochondrial dysfunction and neuronal damage, while GSH and lactate support neuroprotection. Figure created with Adobe Illustrator (version 24.1.)

Fig. 6.

Mitochondrial Autophagy on Astrocyte-neuron. The diagram illustrates the clearance of damaged mitochondria in neurons triggered by ROS and AMPK activation, involving PGAM5, FUNDC1, PINK1, Parkin, and LC3. Key processes include DNM1, ubiquitination, and autophagosome formation, with contributions from tPA, Bnip3, and HIF-1, supported by astrocytes. Figure created with Adobe Illustrator (version 24.1.)

Lipid metabolism

Lipids play a foundational role in astrocyte function, a role that encompasses energy production, membrane fluidity, and intercellular signaling. Lipids and their metabolic intermediates constitute 50% of the brain’s dry weight, critically maintaining its normal structure and function. Studies demonstrate that astrocytes store lipids in the form of lipid droplets and utilize them to execute essential physiological and protective roles in the central nervous system [97, 98]. The maintenance of lipid homeostasis heavily relies on mitochondrial function [98], as mitochondria dynamically balance intracellular lipid levels by regulating fatty acid metabolic enzyme activity and lipid droplet formation.

Astrocytic lipid metabolism is intrinsically linked to mitochondrial transfer, which is a key neuroprotective mechanism. By storing excess fatty acids as lipid droplets, astrocytes prevent lipotoxicity—a condition that compromises mitochondrial function and impedes mitochondrial transfer to neurons [99]. Beyond energy provision, astrocytes supply neurons with essential lipids, including cholesterol and phospholipids that are critical for mitochondrial membrane integrity and fusion following transfer [98]. This lipid support stabilizes neuronal homeostasis, enhancing the receptivity and functional integration of transferred mitochondria. Thus, astrocytic lipid handling not only sustains neuronal metabolism but also establishes the necessary conditions for successful mitochondrial donation, ultimately promoting neuronal survival and recovery after injury. In the pathological context of IS, the connection between lipid metabolism dysregulation and mitochondrial transfer becomes particularly evident. Long-chain acylcarnitines (LCACs) have been identified as novel diagnostic and prognostic biomarkers for acute ischemic stroke. Elevated LCACs levels exacerbate stroke injury, while their reduction mitigates damage [100]. Astrocytes generate LCACs by releasing free fatty acids from lipid droplets and transporting them to mitochondria. Impairment in this process may induce mitochondrial damage in astrocytes, thereby weakening their capacity to transfer healthy mitochondria to neurons. More importantly, mitochondrial damage and lipid droplet metabolic dysfunction form a vicious cycle: impaired mitochondria fail to efficiently process fatty acid intermediates, further promoting pathological LCAC accumulation, ultimately disrupting both lipid homeostasis and mitochondrial transfer potential [100]. Additionally, post-stroke dynamic changes in cerebral lipid profiles are closely associated with microglial function. Lipid droplet accumulation and lipidomic alterations may modulate post-stroke inflammatory responses [101], and the inflammatory environment significantly regulates astrocyte-mediated mitochondrial transfer.

While these associations are compelling, the causal relationship between lipid metabolism and mitochondrial transfer remains partially unverified by direct evidence. For instance, it remains controversial whether astrocytic lipid droplet formation serves as a prerequisite for mitochondrial transfer or as merely an epiphenomenon. Although the value of LCACs as biomarkers has been established, their capacity to directly impair mitochondrial transfer within astrocytes requires functional validation. A critical knowledge gap lies in understanding how specific lipid species influence the fusion and functional integration of transferred mitochondria within neurons. Collectively, lipid metabolism should not be regarded as an isolated process; rather, it directly modulates the efficiency and functional outcomes of astrocyte-to-neuron mitochondrial transfer through multiple mechanisms including energy provision, membrane property regulation, and signaling molecule transmission.

Mitophagy

Mitophagy is a cellular process that degrades or removes damaged or superfluous mitochondria through phagocytosis. Under ischemic conditions, dysfunctional mitochondria can promote cell death by increasing the release of pro-apoptotic factors, while mitophagy primarily functions to eliminate damaged mitochondria to maintain intracellular mitochondrial homeostasis [102]. Studies indicate that mitophagy may play distinct roles during the early ischemic phase and late reperfusion phase [103]. In IS, mitophagy exhibits dual effects: moderate mitophagy is widely regarded as neuroprotective. During the early ischemic phase, mitophagy protects neurons by clearing damaged mitochondria, thereby reducing ROS production and pro-apoptotic factor release [104, 105] However, excessive mitophagy activation not only fails to protect cells but also triggers nuclear degradation and cell death [106]. During reperfusion, hyperactivation of mitophagy may lead to excessive mitochondrial degradation, resulting in cellular energy crisis and death [107]. This duality positions mitophagy as a critical regulatory mechanism in ischemic stroke pathology.

In IS, the PINK1/Parkin pathway and mitophagy receptors including NIX, BNIP3, FUNDC1, and BCL2L13 synergistically regulate mitochondrial quality control through distinct mechanisms to eliminate damaged mitochondria and maintain cellular homeostasis. The mechanism, function and roles of mitophagy in IS of mitophagy are summarized in Table 2. In general, the PINK1/Parkin pathway and mitophagy receptors collectively establish a dual-layered quality control network: a ubiquitin-dependent system and a LIR-ATG8 direct-binding system. Furthermore, key executors of mitochondrial fission such as DNM1 (Drp1) and regulators of metabolism like PGAMS are also integral to this process, facilitating the segregation and removal of damaged organelles. Beyond preserving mitochondrial integrity within cells, mitophagy also plays a critical role in intercellular mitochondrial transfer. In astrocytes, it clears dysfunctional mitochondria, ensuring that a healthy pool is available for donation. In recipient neurons, mitophagy facilitates the integration of newly transferred mitochondria by removing pre-existing damaged ones. This coordinated clearance underscores the functional importance of mitophagy in both donor and recipient cells, highlighting its potential as a therapeutic target in IS.

Table 2.

Functional characteristics of major mitophagy pathways and receptors in IS

classification	Pathway/Receptors	Primary Mechanism	Primary Function	Roles in IS
Pathway	PINK1/Parkin [107–110]	1.Stabilizes on the outer mitochondrial membrane, phosphorylates ubiquitin (e.g., at Ser65), recruits and activates Parkin; 2.Mediates ubiquitination of mitochondrial proteins, forming signals for autophagy adapter recognition	1.Initiates ubiquitin-dependent mitophagy; 2.Promotes clearance of damaged mitochondria	1.Clears mitochondria overproducing ROS; loss of function exacerbates neurodegenerative pathology; 2.Reduces neuronal oxidative damage and apoptosis; interacts with mitochondrial fission/fusion dynamics
Mitophagy receptors	NIX/BNIP3L [111, 112]	Binds ATG8/LC3 via LIR domain; MER domain facilitates autophagosome formation	Mediates mitochondrial clearance during development and hypoxia	Deficiency aggravates mitochondrial accumulation and oxidative stress; overexpression enhances neuroprotective mitophagy
	BNIP3 [113]	Initiates mitophagy via LIR-LC3 interaction	Regulates pluripotency and mitochondrial clearance	Loss leads to abnormal mitochondrial accumulation and increased oxidative stress
	FUNDC1 [114]	Mitochondrial outer membrane protein; binds LC3 via LIR, with phosphorylation status dynamically controlling initiation	Hypoxia-specific mitophagy receptor	Src inhibition rescues its mediated neuronal mitophagy
	BCL2L13 [115]	Promotes clearance of damaged mitochondria via LIR-LC3 binding	Essential for maintaining mitochondrial homeostasis	Essential for maintaining mitochondrial homeostasis

Ferroptosis

Ferroptosis, a form of cell death distint from apoptosis, necrosis, and autophagy, has been implicated in multiple neurological pathologies, including neurodegenerative diseases [116], IS [117], and traumatic brain injury [118]. Its mechanisms primarily involve glutathione (GSH) depletion, inactivation of glutathione peroxidase 4 (GPX4), accumulation of lipid peroxides, and high levels of free iron that bind to polyunsaturated fatty acids, which trigger the Fenton reaction to release excessive ROS [119]. The production of oxidized phospholipids (oxPLs) is a key execution step in ferroptosis [120]. Dysregulated cysteine metabolism promotes tripeptide biosynthesis, leading to tripeptide-GSH conjugation, intracellular GSH exhaustion, and ultimately the cell’s inability to repair oxPLs. Morphological features of ferroptosis, such as mitochondrial shrinkage, increased membrane density, outer membrane rupture, and reduced cristae, can be detected using transmission electron microscopy [121, 122].

As iron-rich and ROS-generating organelles, mitochondria are proposed to act as central hubs in ferroptosis. Post-ischemia, neuronal iron overload, GSH depletion, GPX4 activity suppression, and lipid peroxidation accumulation are hallmark features of ferroptosis. These changes form a vicious cycle with mitochondrial dysfunction, exacerbating neuronal damage. Emerging evidence indicates that exogenous mitochondrial transplantation or astrocyte-derived mitochondria can integrate into the neuronal mitochondrial network via fusion, restoring oxidative phosphorylation and reducing ROS accumulation [121, 123]. Studies demonstrate that mesenchymal stem cell (MSC)-mediated mitochondrial transfer significantly inhibits neuronal ferroptosis [123], though further validation in ischemia models is needed.

Mitochondrial transfer has been hypothesized to modulate ferroptosis through multi-pathway coordination. Proposed mechanisms include the following. First, the restoration of the GSH/GPX4 pathway through supplemental functional mitochondria rejuvenates GPX4 enzymatic activity [124], clearing lipid peroxides by maintaining reduced GSH levels, thereby blocking the core execution of ferroptosis. Second, mitochondrial transfer activates the Nrf2 signaling pathway, upregulating antioxidant genes such as heme oxygenase-1 (HO-1) to enhance cellular defense against oxidative stress and negatively regulate ferroptosis [125]. Third, healthy mitochondria stabilize intracellular iron levels by participating in iron metabolism, reducing free iron-driven Fenton reactions and subsequent lipid radical chain damage, thereby suppressing ferroptosis at its origin. Although these mechanisms are supported by in vitro or preliminary in vivo studies, their efficacy and temporal dynamics in IS remain areas of active investigation. Astrocyte-to-neuron mitochondrial transfer represents a promising therapeutic avenue, potentially restoring energy metabolism, reducing ROS, and regulating iron flux, but more research is required to establish causal evidence in ischemic contexts (Figs. 7 and 8).

Fig. 7.

Ferroptosis receptors found in IS related to Astrocyte-neuron. The diagram illustrates cystine uptake via SLC3A2/SLC7A11, GSH synthesis by GPX4, and iron metabolism dysregulation. Increased Fe²⁺ triggers the Fenton reaction, leading to ROS accumulation, lipid peroxidation by ACSL4, and ferritin degradation via FtMt/NCOA4, culminating in ferroptosis, marked by HMGB1 release. Figure created with Adobe Illustrator (version 24.1.)

Fig. 8.

Schematic illustration of key molecular pathways and phenotypic heterogeneity governing astrocyte-mediated mitochondrial transfer. Molecular pathways (left) and astrocyte phenotypic heterogeneity (middle and right) collectively regulate mitochondrial transfer. Neuroprotective signals (via AMPK, BDNF) and A2-type astrocytes enhance transfer, while inflammatory stress and A1-type astrocytes impair it. Regional differences (cortex > cerebellum) further modulate this process, ultimately promoting neuronal recovery by restoring energy, reducing oxidative stress, and modulating Ca²⁺. Figure created with Microsoft Powerpoint (version 16.78)

Key regulatory factors of astrocytic mitochondrial transfer

The regulation of astrocytic mitochondrial transfer involves a multifactorial synergistic network, with its efficiency and functional state governed by molecular signaling pathways and cellular phenotypic heterogeneity.

Molecular signaling pathways

Indirect interplay and dynamic balance of inflammatory cytokines

Inflammatory cytokines dynamically modulate astrocytic mitochondrial transfer through a dual mechanism involving pro- and anti-inflammatory factors. In the context of neuroinflammation, elevated levels of pro-inflammatory cytokines often coexist with neuronal damage and mitochondrial dysfunction. Meanwhile, the transfer of mitochondria from astrocytes to neurons is activated as an endogenous protective mechanism, likely in indirect response to inflammatory conditions. In models of ischemia, lipopolysaccharide (LPS) exposure, or intracerebral hemorrhage, an inflammatory environment is accompanied by enhanced mitochondrial transfer. This process may involve mechanisms such as the formation of TNTs and molecular mediators including CD38 and Rho GTPase. However, cytokines themselves have not been established as direct regulatory factors. On the contrary, mitochondrial transfer may suppress neuroinflammation through feedback mechanisms by restoring neuronal energy metabolism, reducing oxidative stress, and modulating immune responses, such as promoting the production of the anti-inflammatory cytokine IL‑10, thereby forming a protective cycle. Therefore, inflammatory cytokines are more likely to constitute a background condition rather than direct regulators of mitochondrial transfer. Their specific signaling pathways and causal relationships still require further investigation.

Integration of metabolic stress signals

IS leads to hypoxia and insufficient glucose supply in brain tissue, triggering cellular metabolic stress. Key manifestations of metabolic stress include mitochondrial dysfunction, reduced ATP production, accumulation of ROS, and activation of apoptotic signaling [126]. Under metabolic stress, astrocytes alter their metabolic state by enhancing glycolysis and increasing lactate production to sustain neuronal energy supply [94]; however, excessive activation may exacerbate oxidative stress. Additionally, metabolic stress induces inflammatory responses in astrocytes, prompting the release of pro-inflammatory factors and neurotrophic factors, thereby influencing neuronal survival and function [127]. Metabolic stress signals regulate mitochondrial biogenesis and transfer through activation of the AMPK and NAD+/SIRT1 pathways. AMPK, an energy sensor, is activated under cellular energy depletion to promote mitochondrial biogenesis and functional recovery, thereby enhancing mitochondrial transfer capacity. Upon activation of the AMPK/SIRT1 pathway, it suppresses NF-κB acetylation-dependent activation to reduce inflammation while promoting mitochondrial biogenesis. Meanwhile, the NAD+/SIRT1 pathway maintains mitochondrial dynamic balance by regulating mitochondrial fusion and fission. Studies have shown that supplementation with NAD + precursors enhances SIRT1 activity, improves expression of the mitochondrial fusion protein MFN2, and boosts mitochondrial transfer efficiency. Furthermore, metabolic stress modulates mitochondrial transfer efficiency by regulating Ca²⁺ homeostasis and mitochondrial membrane potential [128].

Neuroprotective factor-driven mechanisms

Following IS, neuroprotective factors drive mitochondrial transfer from astrocytes to damaged neurons through multidimensional mechanisms, contributing to neural repair. These regulatory processes involve phenotypic transformation, metabolic reprogramming, and inflammation modulation. Brain-derived neurotrophic factor (BDNF) enhances mitochondrial transfer efficiency and functional integrity by activating TrkB receptors to promote TNTs formation and upregulate mitochondrial fusion protein MFN2 [129]. Mesencephalic astrocyte-derived neurotrophic factor (MANF) suppresses endoplasmic reticulum stress and TLR4/NF-κB-mediated inflammatory responses, reducing transfer barriers and improving mitochondrial delivery [130]. Glial cell line-derived neurotrophic factor (GDNF) drives astrocyte metabolic reprogramming via the PI3K-AKT-mTOR pathway, enhancing oxidative phosphorylation to increase the supply of functional mitochondria [131].

Phenotypic heterogeneity impacts

Functional divergence of A1/A2 subtypes

Reactive astrocytes are broadly categorized into two functional phenotypes: the harmful A1 type and the protective A2 type. Notably, A2 astrocytes demonstrate enhanced glycogen metabolism and greater mitochondrial biogenesis [132]. In middle cerebral artery occlusion (MCAO) models, A2 cells activate glycogen phosphorylase to promote glycogenolysis, generating ATP to fuel mitochondrial transfer [132]. Their mitochondria display more stable membrane potential (ΔΨm) and higher respiratory chain complex activity, closely associated with elevated PGC-1α expression. In contrast, A1 astrocytes under sustained pro-inflammatory stimuli exhibit fragmented mitochondria due to excessive Drp1-mediated fission and ROS accumulation [133]. This metabolic dysfunction leads to mitochondrial calcium overload via MCUb channels, significantly impairing transfer efficiency to neurons.

Region-specific regulation

Astrocytes in different brain regions show marked heterogeneity in mitochondrial transfer capacity, driven by variations in metabolic demands, mitochondrial function, injury responses, and molecular mechanisms. Cortical astrocytes, with higher mitochondrial respiration rates, glycolytic activity, and ATP production [134], demonstrate superior mitochondrial transfer potential under ischemia or oxidative stress. MCAO experiments reveal that cortical astrocyte-derived mitochondria effectively rescue neuronal damage, while cerebellar astrocytes show weaker repair efficacy. Mitochondrial functional differences include higher ΔΨm in hippocampal and brainstem astrocytes [135], along with region-specific Ca²⁺ buffering capacity and morphological plasticity. At the molecular level, CD38/cADPR signaling and MIRO GTPase expression vary across regions [136, 137]: cortical MFN2 upregulation enhances mitochondrial transfer to vascular endothelial cells, whereas age-related MFN2 decline in the cortex disrupts BBB integrity and impairs transfer. Notably, brainstem astrocyte transfer capacity remains relatively preserved during aging [138]. These findings highlight that astrocytic mitochondrial transfer is regulated by local microenvironments, energy metabolism features, and region-specific molecular signatures. Future research should dissect regional mechanisms to advance precision therapies for different diseases.

As discussed above, despite the diversity of regulatory signals and cellular contexts, the factors governing astrocytic mitochondrial transfer can be ultimately categorized into two major types: those promoting transfer and those inhibiting it. Critically, the net effect of these regulatory factors is further modulated by key biological variables including age, sex, and cellular phenotype. Promotive interventions enhance mitochondrial transfer by facilitating the formation of transfer pathways such as TNTs, improving transfer efficiency, or optimizing the transfer microenvironment; inhibitory interventions, on the other hand, primarily impede the process by disrupting cytoskeletal architecture, interfering with intercellular connections, or reducing mitochondrial quality. Future research and therapeutic strategies must therefore account for these subgroup influences, such as the decline in transfer efficiency with age, sex-dependent differences in inflammatory responses, and the opposing roles of A1 versus A2 astrocytic phenotypes, to maximize therapeutic efficacy across diverse patient populations. These strategies aim to amplify endogenous neuroprotective mechanisms or remove barriers to mitochondrial transfer, thereby offering novel therapeutic avenues for neurological disorders (Table 3).

Table 3.

Regulatory factors and molecular mechanisms of Astrocyte-to-Neuron mitochondrial transfer

Effect	Factor/Pathway	Mechanism	Outcome and Function	参考文献
Promote	Miro1	A key adaptor protein on the mitochondrial outer membrane, responsible for anchoring mitochondria to motor proteins for transport.	Knockdown of Miro1 reduces the ability of mitochondria to transfer to damaged neurons, impairing the restoration of calcium homeostasis.	[136, 137]
	MFN2	A key regulator of mitochondrial fusion that modulates the efficiency of mitochondrial transfer.	Knockdown of the MFN2 gene reduces mitochondrial transfer efficiency and leads to BBB leakage.	[138]
	CD38/cADPR	Mediates Ca²⁺ signaling and activates Rho GTPases to promote the formation of TNTs.	A key initiator of mitochondrial transfer, enhancing connectivity and transfer efficiency between astrocytes and neurons.	[92, 139]
	PSPH	PSPH-derived serine metabolism generates α-ketoglutarate, which activates the neuronal OXGR1 receptor to promote TNTs formation between astrocytes and neurons.	PSPH regulates serine metabolism, influencing mitochondrial function and quality, thereby indirectly promoting astrocyte-to-neuron mitochondrial transfer.	[140]
	ERK1/2	Mild hypothermia upregulates ERK1/2 to activate the CD38-cADPR-Ca²⁺ signaling pathway, promoting the transfer of mitochondria from astrocytes into the extracellular space.	Improves neuronal survival and alleviates ischemic injury.	[141]
Inhabit	S100a4	Influences TNTs formation via the S100a4-IL-10 axis.	Elevated S100a4 levels impair mitochondrial transfer between neurons and astrocytes, accompanied by increased mitophagy regulators and elevated ROS levels.	[142]
	GFAP Mutation	Disrupts astrocytic cytoskeletal dynamics, impairing the intracellular transport capacity of mitochondria.	Leads to impaired mitochondrial transfer function, which is associated with the pathogenesis of neurodegenerative disease.	[137]

Experimental evidence and model systems

In vitro studies

In vitro studies primarily employ astrocyte-neuron co-culture systems combined with oxygen-glucose deprivation/reperfusion (OGD/R) models [139] to simulate intercellular interactions using transwell systems or microfluidic chips [30]. Research confirms that astrocytes actively deliver functional mitochondria to ischemic neurons via TNTs or EVs. This process can be dynamically tracked using MitoTracker or mitochondrial-targeted fluorescent proteins [143]. Transferred mitochondria significantly alleviate neuronal mitochondrial dysfunction, characterized by reduced ROS accumulation and downregulated apoptosis-related protein expression [32]. For example, ginsenoside Rb1 enhances astrocyte-mediated neuroprotection by suppressing CD38-dependent complex I dysfunction [139].

In addition to direct co-culture, conditioned medium transfer models are widely utilized. By collecting astrocyte-conditioned medium (ACM) post-OGD treatment [144], researchers analyze its mitochondrial content and regulatory effects on neuronal energy metabolism. Experiments demonstrate that in Parkinson’s disease in vitro models, ACM significantly mitigates dopaminergic neuron degeneration through intercellular mitochondrial transfer [144]. In cerebral ischemia models [139], ACM co-culture enhances neuronal mitochondrial membrane potential and oxygen consumption rate, suggesting astrocyte-mediated neuroprotection via paracrine pathways. However, current in vitro models fail to fully replicate in vivo microenvironment features such as BBB disruption and immune cell infiltration. Future studies should integrate 3D organoid models and single-cell spatial transcriptomics to elucidate the spatiotemporal specificity of mitochondrial transfer and its interaction with the neurovascular unit.

In vivo studies

In vivo experiments utilize MCAO models to validate mitochondrial transfer phenomena through fluorescent labeling and live imaging, loss-of-function/gain-of-function studies, and metabolomic/proteomic analyses. Transgenic mouse models or viral vector-based labeling combined with two-photon microscopy enable real-time observation of mitochondrial transfer [145, 146]. For example, one study shows that astrocytes in the ischemic penumbra of MCAO model rats transfer mitochondria to damaged neurons via TNTs, a process that significantly reduces neuronal apoptosis. In other approaches, mitochondrial transfer-related genes in astrocytes are specifically knocked down using the Cre-LoxP system. It was found that Miro1-deficient mice had lower mitochondrial transfer efficiency and significantly impaired neurological function recovery [26].In addition, Miro1 defects affect mitochondrial interactions with the cytoskeleton and impede their targeted transport in TNTs [147]. Knockdown of the connexin 43 gene in astrocytes disrupts the formation and stabilization of TNTs, reduces mitochondrial translocation, and decreases mitochondrial respiratory function and neuronal survival [148]. Cx43 also affects the communication efficiency of TNTs through the modulation of intercellular junctions [149]. Metabolomics [150, 151] revealed that transferred mitochondria improved neuronal metabolic homeostasis by replenishing tricarboxylic acid cycle intermediates and reducing ROS accumulation. Proteomic analysis [123, 152] revealed that mitochondrial transfer upregulated the expression of antioxidant enzymes in neurons, activated mitochondrial fusion proteins and inhibited proteins associated with excessive fission, thereby maintaining mitochondrial network integrity (Table 4).

Table 4.

Summary of experimental models for studying Astrocyte-Neuron mitochondrial transfer

Model type	Specific method	Labeling/Tracking method	Key findings	Limitations
In Vitro Models
Co-culture Systems	Astrocyte-neuron co-culture combined with OGD/R treatment	MitoTracker, mitochondria-targeted fluorescent proteins (e.g., mito-GFP)	Astrocytes actively transfer functional mitochondria to ischemic neurons via TNTs or EVs; reduces neuronal ROS accumulation and apoptosis	Fails to fully replicate the in vivo microenvironment (e.g., lacks BBB and immune cell infiltration)
Conditioned Medium Transfer	Culture neurons with astrocyte-conditioned medium (ACM) collected post-OGD	Measure mitochondrial content in ACM; analyze neuronal ΔΨm and oxygen consumption rate (OCR)	ACM mediates neuroprotection and enhances neuronal mitochondrial function, suggesting the presence of paracrine pathways	Difficult to distinguish between direct mitochondrial transfer and effects of paracrine factors
In Vivo Models
MCAO Model	Middle cerebral artery occlusion in rats/mice	Transgenic mice, viral vector labeling (e.g., AAV-GFAP-mito-DsRed), two-photon in vivo imaging	Real-time observation of astrocyte-to-neuron mitochondrial transfer via TNTs in the ischemic penumbra; reduces neuronal apoptosis	1.In vivo imaging is technically complex and requires advanced equipment 2. Significant heterogeneity in occlusion time, animal strain, and age limits generalizability of findings
Genetic Functional Studies	Conditional knockout of specific genes in astrocytes (e.g., Miro1, Cx43) using Cre-LoxP system	Immunohistochemistry, behavioral functional scoring	Miro1 deficiency reduces transfer efficiency and impairs neurological recovery; Cx43 knockout disrupts TNT formation/stability and reduces mitochondrial transfer and neuronal survival	Potential for genetic compensation effects or developmental adaptation
Omics Analysis	Metabolomic/proteomic analysis of brain tissue or neurons that received mitochondrial transfer	Mass spectrometry	Transferred mitochondria replenish TCA cycle intermediates and improve neuronal metabolic homeostasis; upregulate neuronal antioxidant enzymes and suppress excessive fission-associated proteins	Results are largely correlative; requires functional validation experiments to establish causality
Common Limitations	1.Significant Heterogeneity: Marked variations in experimental models, methodologies, and outcome measurements hinder direct comparison and meta-analysis. 2. Lack of Quantitative Synthesis: The field lacks standardized metrics and comprehensive quantitative analyses to definitively establish the absolute efficiency and functional contribution of mitochondrial transfer. 3. Potential Publication Bias: The literature likely overrepresents positive findings demonstrating successful transfer and neuroprotection, while underreporting negative or neutral results.

Translational medicine and therapeutic potential

In the pathological progression of IS, mitochondrial dysfunction is a core mechanism underlying neuronal death and has emerged as a promising therapeutic target and source of biomarkers [88, 105]. Circulating mitochondrial DNA is being explored as potential peripheral indicators of cellular damage and treatment response [153]. Recent advancements in enhancing mitochondrial transfer through multi-target interventions and technological innovations demonstrate significant neuroprotective potential. By transferring or transplanting exogenous healthy mitochondria to replace damaged ones, cellular energy metabolism can be restored, neuronal survival improved, and neurological function recovered, offering a multidimensional therapeutic strategy for IS.

Strategies to enhance mitochondrial transfer

Research in mitochondrial transfer strategies for IS has evolved from single-mechanism exploration to innovative multi-dimensional synergistic interventions. Recent studies indicate that targeting mitochondrial dynamics and intercellular communication networks can markedly improve neuronal energy metabolism and reduce oxidative stress damage. At the molecular pathway level, pharmacological intervention with Chrysophanol significantly increased Miro1 expression in the brain (P < 0.01), promoted astrocyte-neuron interneuron mitochondrial transfer, and resulted in a reduction in cerebral infarct volume in the MCAO model (P < 0.05) [146]. This highlights Miro1 not only as a therapeutic target but also as a potential companion biomarker for verifying target engagement in future clinical trials. AMPK activators suppress Drp1-mediated excessive mitochondrial fission and activate PINK1/Parkin pathway-dependent autophagy, lowering neuronal ROS levels (p < 0.01) [154]. Notably, rapamycin inhibits the mTORC1 pathway, reducing TDP-43 protein aggregation (p < 0.05) while restoring mitochondrial function and alleviating gliosis, effectively rebalancing mitochondrial dynamics [155]. The blood-brain barrier permeability of such compounds, often assessed via LC-MS/MS in preclinical models, is a critical translational consideration. Concurrently, breakthroughs in enhancing intercellular communication include activating the PI3K/AKT signaling pathway to increase TNT density and boost TNT-mediated mitochondrial transfer [156]. Additionally, accelerating neuronal release of damaged mitochondria and promoting astrocytic phagocytosis and clearance of these mitochondria help maintain cellular homeostasis [92].

The integration of gene editing technologies provides precise tools for mitochondrial quality control. The application of CRISPR/dCas9 to modulate mitochondrial calcium homeostasis demonstrates therapeutic promise, including strategies to activate UCP2 for elevating anti-inflammatory factor levels (IL-13, IL-10 and IL-4, p < 0.05) [157]. For translational application, the development of efficient and safe in vivo delivery vectors for the central nervous system, such as engineered AAVs, is a paramount next step. In autophagy regulation, selective activation of the PINK1/Parkin pathway enhances ubiquitination and clearance of damaged mitochondria, increasing levels of the autophagosome marker LC3-II [102], which could serve as a pharmacodynamic biomarker in preclinical models.

Multimodal synergistic strategies amplify therapeutic efficacy. For example, combined drug-gene interventions enhance neuronal survival rates. Pretreatment of recipient cells with rapamycin increases exogenous mitochondrial coverage in the peri-infarct region. Functional analyses reveal that transplanted healthy mitochondria restore electron transport chain activity to boost ATP production while rebalancing MFN2/Drp1 phosphorylation to remodel mitochondrial network integrity. However, clinical translation faces several challenges, including the need for standardized mitochondrial preparation protocols, the development of smart-responsive nanocarriers to overcome glial scar barriers, and the systematic evaluation of mitochondrial transplant immunogenicity in non-human primate models. Future studies should integrate single-cell omics and organoid models to decode patient-specific mitochondrial dysfunction subtypes, enabling precise design of personalized therapies to bridge the gap from bench to bedside.

Exogenous mitochondrial transplantation

In addition to enhancing endogenous mitochondrial transfer, exogenous mitochondrial transplantation is another promising therapeutic strategy. Exogenous mitochondrial transplantation exerts neuroprotection through energy metabolism remodeling and signaling pathway modulation. Innovations in engineered delivery systems, such as lipid-based or polymeric nanoparticles, magnetic nanoparticles, and other nanocarriers, overcome biological barriers to enhance delivery efficiency and targeting precision. For instance, artificial mitochondrial transplantation (AMT/T) technology combined with nanocarriers addresses challenges such as mitochondrial heterogeneity and poor in vivo operability [158, 159]. Magnetic Fe3O4 nanoparticles coupled with near-infrared-controlled release technology enable high-precision mitochondrial localization, offering targeted therapy for ischemic or degenerative diseases [160, 161]. Mitochondrial transplantation must overcome limitations such as cellular uptake, enzymatic degradation, and mitochondrial membrane barriers. Optimization of nanoparticle size, surface charge, and functionalization can improve mitochondrial delivery efficiency [162]. Exosomes or engineered EVs, which naturally cross the BBB, are emerging as promising mitochondrial delivery vehicles [162, 163]. From a clinical application perspective, autologous mitochondrial transplantation could circumvent immune rejection issues and has demonstrated feasibility in pediatric cardiac surgery models [164], providing a valuable reference for neurological applications.

Conclusion

IS remains a devastating neurological condition with significant mortality and long-term disability, primarily due to neuronal mitochondrial dysfunction. This review has synthesized current evidence regarding the role of astrocytic mitochondrial transfer as an emerging therapeutic strategy for IS.

There is compelling evidence from in vitro and in vivo models that astrocytes transfer functional mitochondria to compromised neurons via TNTs, EVs, and connexin 43-mediated gap junctions. This transfer supports neuronal survival by restoring energy metabolism, reducing oxidative stress, and improving calcium homeostasis. Pharmacological or genetic enhancement of mitochondrial transfer has been consistently shown to reduce infarct volume and improve functional outcomes in animal models of IS.

Several critical challenges hinder the clinical translation of astrocyte-mediated mitochondrial transfer for ischemic stroke treatment. These include the limited viability and integration efficiency of transferred mitochondria, potential immune responses, and the absence of precise delivery systems. Additionally, the dynamics of mitochondrial transfer in humans—encompassing its spatiotemporal profile, dose-response relationship, and influence of comorbidities—remain poorly understood. Moreover, the dual role of certain pathways, such as inflammatory signaling, under varying temporal or metabolic conditions requires further investigation to fully elucidate their therapeutic implications.

Targeting astrocytic mitochondrial transfer represents a promising therapeutic strategy for IS. Future efforts should focus on overcoming delivery challenges using nanoparticle-based carriers, improving mitochondrial engraftment via mitochondrial fitness enhancement, and tailoring interventions using patient-derived cells or organoids. Combining mitochondrial transfer with neuroprotective agents or rehabilitation strategies may offer synergistic benefits. Translational studies should prioritize the validation of these approaches in clinically relevant models and identify biomarkers to select patients most likely to benefit from these interventions.

In summary, while astrocytic mitochondrial transfer holds groundbreaking potential for the treatment of ischemic stroke, addressing these unresolved questions through collaborative and multidisciplinary research will be essential to translate this promise into tangible clinical benefits.

Abbreviations

ACM

Astrocyte-conditioned medium

AMT/T

Artificial mitochondrial transplantation

ATP

Adenosine triphosphate

BBB

Blood-brain barrier

BDNF

Brain-derived neurotrophic factor

Cx43

Connexin 43

EVs

Extracellular vesicles

FAs

Fatty acids

GDNF

Glial cell line-derived neurotrophic factor

GPX4

Glutathione peroxidase 4

GSH

Glutathione

HO-1

Heme oxygenase-1

I/R injury

Ischemia-reperfusion injury

IS

Ischemic stroke

LCACs

Long-chain acylcarnitines

LDH

Lactate dehydrogenase

LPS

Lipopolysaccharide

LTP

Long-term potentiation

MAMs

Mitochondria-associated endoplasmic reticulum membranes

MANF

Mesencephalic astrocyte-derived neurotrophic factor

MCAO

Mddle cerebral artery occlusion

MCU

Mitochondrial calcium uniporter

MSC

Sodium/calcium/lithium exchanger

OGD/R

Oxygen-glucose deprivation/reperfusion

OxPhos

Oxidative phosphorylation

oxPLs

Oxidized phospholipids

ROS

Reactive oxygen species

RyR

Ryanodine receptor

SOCE

Store-operated calcium entry

TCA

Tricarboxylic acid

TNTs

Tunneling nanotubes

ΔΨm

Membrane potential

Author contributions

Xin Lan: Writing – original draft. Chuxin Zhang: Writing – review & editing, Data curation: Writing – review & editing, Jialin Cheng: Visualization. Congai Chen: Data curation. Yuxiao Zheng: Data curation. Jinhua Han: Data curation. Yang Zhao: Resources. Jiaming Li: Software. Fafeng Cheng: Project administration. Xueqian Wang: Project administration, Supervision. Qingguo Wang: Project administration, Supervision. Changxiang Li: Supervision, Funding acquisition.

Funding

This study was supported by supported by the Fundamental Research Funds for the Central Universities(No. 2024-JYB-JBZD-043, 2025-JYB-XJSJJ-002))and the National Natural Science Foundation of China (No. U21A20400).

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

No conflicts of interest, financial or otherwise, are declared by the authors.