Molecular and cellular mechanisms of mitochondria transfer in models of central nervous system disease

Abstract

In the central nervous system (CNS), neuronal function and dysfunction are critically dependent on mitochondrial integrity and activity. In damaged or diseased brains, mitochondrial dysfunction reduces adenosine triphosphate (ATP) levels and impairs ATP-dependent neural firing and neurotransmitter dynamics. Restoring mitochondrial capacity to generate ATP may be fundamental in restoring neuronal function. Recent studies in animals and humans have demonstrated that endogenous mitochondria may be released into the extracellular environment and transported or exchanged between cells in the CNS. Under pathological conditions in the CNS, intercellular mitochondria transfer contributes to new classes of signaling and multifunctional cellular activities, thereby triggering deleterious effects or promoting beneficial responses. Therefore, to take full advantage of the beneficial effects of mitochondria, it may be useful to transplant healthy and viable mitochondria into damaged tissues. In this review, we describe recent findings on the mechanisms of mitochondria transfer and provide an overview of experimental methodologies, including tissue sourcing, mitochondrial isolation, storage, and modification, aimed at optimizing mitochondria transplantation therapy for CNS disorders. Additionally, we examine the clinical relevance and potential strategies for the therapeutic application of mitochondria transplantation.

Introduction

Mitochondria are cell components that store energy and are essential for maintaining cellular function ¹ through the regulation of levels of adenosine triphosphate (ATP), fatty acids, and cellular calcium.^2–4 In central nervous system (CNS) diseases, the impairment of mitochondrial membrane permeability and the accumulation of mitochondrial reactive oxygen species (ROS) and inflammasomes may induce cell death and neuroinflammation. ⁵ Therefore, restoring intracellular mitochondrial function is a key therapeutic strategy for various CNS diseases, including ischemic stroke, ⁶ hemorrhagic stroke, ⁷ spinal cord injury (SCI), ⁸ Alzheimer’s disease, ⁹ and Parkinson’s disease (PD). ¹⁰

Recent research suggests that mitochondria can be released into the extracellular space and subsequently transferred between cells within the CNS.^11,12 This phenomenon has been demonstrated in both animal models and human studies involving human cells and clinical samples.^13–15 For example, Gao et al. showed that mitochondria are dynamically transferred between human astrocytes and neurons in cell culture models. ¹³ Additionally, Youn et al. and Caicedo et al. detected extracellular mitochondria in cerebrospinal fluid (CSF) and plasma from patients with subarachnoid hemorrhage, indicating their role in intercellular signaling.^14,15 The transfer of mitochondria between cells in the damaged or diseased brain may represent a novel form of intercellular communication, which can be either beneficial or harmful depending on the specific circumstances. In this context, transplanting healthy, functional mitochondria, including autologous mitochondria from non-lesioned tissue, into injured regions of the brain holds potential as a therapeutic approach to support the recovery of damaged cells and restore normal cellular function. Despite the tremendous potential that could be harnessed from mitochondria transplantation therapy, there are currently only a few clinical trials investigating mitochondria transplantation for CNS diseases. To advance the clinical application of this therapy to patients with CNS diseases, it is essential to further elucidate the mechanisms of mitochondria transfer and accumulate high-quality evidence on experimental techniques, including tissue sourcing, mitochondrial isolation, storage, and modification, to optimize transplantation therapy.

In this review, we present the latest findings on the mechanisms of intercellular mitochondria transfer and provide an overview of experimental methods aimed at optimizing mitochondria transplantation therapy for CNS diseases. Finally, we discuss the clinical relevance and strategies for the therapeutic use of mitochondria transplantation.

Mechanisms of mitochondria transfer in CNS disease

Intracellular mitochondria transfer and extracellular mitochondria uptake can occur through several mechanisms, including the formation of tunneling nanotubes (TNTs), gap junctions (GJs), extracellular vesicles (EVs), cell fusion, and endocytosis.^16–18 Functional mitochondria transferred from healthy to damaged cells may integrate with resident mitochondria, replacing the damaged ones and replenishing the mitochondrial pool to meet the cell’s energy demands. ¹⁹ As a result, intercellular mitochondria transfer plays a dynamic role in regulating cell signaling and is linked to the prevention, onset, and progression of various CNS diseases, such as PD, Alzheimer’s disease, ischemic stroke, and schizophrenia.^19,20 Currently, intercellular mitochondria are thought to be exchanged via TNTs, GJs, EVs, and cell fusion. Meanwhile, mitochondria released into the extracellular space and exogenous mitochondria introduced through transplantation therapy may be internalized into cells via endocytosis. Here, we describe various mechanisms of mitochondria transfer (Figure 1).

Figure 1.

Mechanisms of mitochondria transfer. Mitochondria are exchanged between cells via TNT, GJ, EV, endocytosis, and cell fusion. Normal cells transport healthy mitochondria to adjacent damaged cells via these mechanisms, and thus protect and repair the damaged cells. Moreover, mitochondria at different locations in a single cell repeatedly undergo fusion and fission with each other due to GTP hydrolases such as Drp1, Mfn, and OPA1. Mitochondrial fusion is controlled by Mfn and OPA1, while Drp1 controls mitochondrial fission. Mitochondria damaged by pathological conditions undergo fusion and fission with healthy mitochondria transported via these pathways and are finally degraded by autophagosomes (mitophagy). Subsequently, healthy mitochondria serve as new hosts to supply energy to the cells and protect and repair neuronal functions. Meanwhile, the mechanism by which transplanted exogenous mitochondria are incorporated into cells in pathological conditions has not been elucidated in detail, but it is believed that the above mechanisms of mitochondria transfer, including endocytosis, are involved. Drp1: dynamin-related protein 1; EV: extracellular vesicle; GJ: gap junction; Mfn: mitofusin; OPA1: optic atrophy 1; TNT: tunnel nanotube.

Tunneling nanotubes

TNTs are long, thin, membranous structures that facilitate direct intercellular communication through the transfer of various cellular components, such as proteins, lipids, and organelles, between neighboring cells. TNTs comprise cell membrane and cytoskeletal components, primarily filamentous actin (F-actin) and microtubules. ²¹ The length of TNTs varies from tens to hundreds of micrometers, and their diameters typically measure 50 to 200 nm. TNTs have been identified in various cell types, including neurons, astrocytes, ²² microglia, ²³ immune cells, ²⁴ and tumor cells. ²⁵ Moreover, TNTs are not solely an in vitro phenomenon but can also be observed in vivo. For example, TNTs have been identified between cardiomyocytes and fibroblasts in the mouse heart, as well as in some corneal dendritic tumor cells in humans.^26–28 A remarkable feature of TNTs is their ability to facilitate intercellular mitochondria transfer, providing a physical conduit for mitochondria to move from one cell to another. Intercellular mitochondria transfer can occur under diverse physiological and pathological conditions, such as cellular development, tissue regeneration, and cellular stress or injury. Importantly, the transfer of functional mitochondria from healthy to damaged cells via TNTs enhances the metabolic capacity of recipient cells, promoting cellular survival and preserving functionality under conditions of metabolic stress, oxidative injury, or disease.^29,30 The formation and regulation of TNTs, along with the facilitation of intercellular mitochondrial transfer, are regulated by intricate cellular and environmental cues, including cytoskeletal dynamics, calcium signaling, and local stress conditions. Movement via TNTs can occur through active and passive diffusion. Motor proteins, such as dynamin, myosin, and kinesin, can actively transport mitochondria along the cytoskeletal filaments within TNTs. ³¹ In neurons, motor proteins play a crucial role in the regulation of mitochondrial movement, with kinesin and dynein facilitating anterograde and retrograde transport, respectively.^32,33 Studies of neurons that have explored the interaction between mitochondria and various myosins (II, V, VI, and XIX) have shown that the downregulation of myosin V and VI notably increased the velocity of mitochondrial transport.^33,34 Furthermore, mitochondria can move passively within TNTs, driven by diffusion gradients or cytoplasmic flow. Factors such as calcium signaling, oxidative stress, cytoskeletal dynamics, and specific signaling pathways can influence TNT formation, stability, and function. Hayakawa et al. ¹¹ reported that intracellular cyclic ADP-ribose (cADPR) induces Ca²⁺ release and promotes events, such as F-actin polymerization, which are crucial for TNT-mediated intercellular mitochondria transfer. Active research endeavors investigating the molecular mechanisms that govern TNT formation and intercellular mitochondria transfer have implications for both molecular biology and therapeutic interventions. An improved understanding of the interplay between mitochondria and TNTs in health and disease may provide insight that could facilitate the development of novel therapeutic strategies that target these cellular processes.

Gap junctions

GJs are specialized intercellular channels that facilitate direct connection with the cytoplasm of adjacent cells. These channels are formed by connexin proteins, which assemble into hexameric structures called connexons. ³⁵ Connexons on one cell align with those on a neighboring cell to create a continuous channel that facilitates the intercellular passage of ions, small molecules, and signaling molecules. GJ-mediated communication is essential for coordinating cellular activities within tissues, synchronizing physiological processes, such as neuronal signaling and tissue development, and maintaining cellular homeostasis. Recent evidence suggests the possibility of GJ-mediated intercellular mitochondria transfer, ³⁶ which involves the direct passage of mitochondria from the cytoplasm of one cell to another via interconnected channels that are formed by connexin proteins. More than 20 connexin subtypes have been identified, with Cx43 being the most widely expressed. Cx43 participates in various physiological processes, such as material exchange, vesicle transport, mitochondrial respiration, and ion transport. ³⁷ GJ-mediated intercellular mitochondria transfer plays a crucial role in maintaining cellular function and preserving tissue homeostasis. This process enables the direct exchange of mitochondria between adjacent cells, facilitating the restoration of metabolic balance and supporting cellular recovery in response to injury or stress. In situations involving cellular stress, injury, or disease, the transfer of healthy mitochondria from neighboring cells through GJs may promote cell survival, enhance bioenergetic capacity, and facilitate tissue repair and regeneration. ¹⁸ Conversely, the transfer of dysfunctional or damaged mitochondria by GJ could potentially propagate mitochondrial dysfunction and contribute to disease progression, such as in neurodegenerative disorders or metabolic diseases. ³⁸ GJ-mediated intercellular mitochondria transfer is regulated by multiple factors, including cellular signaling pathways, calcium dynamics, and the expression and assembly of connexin proteins.^18,37 For instance, fluctuations in intracellular calcium levels can modulate the opening and closing of GJ channels,^39,40 thereby influencing the intercellular transfer of mitochondria and other cellular components. Additionally, alterations in connexin expression or function, due to genetic mutations or environmental factors, may impair the efficiency of GJ-mediated mitochondrial transfer and contribute to the development of pathological conditions. ⁴¹ Thus, understanding the relationship between mitochondria transfer and GJs could have therapeutic implications for various CNS diseases.

Extracellular vesicles

EVs are membrane-bound vesicles that are released by cells into the extracellular environment. Based on their biogenesis and size, EVs are classified into different subtypes, including exosomes (30–150 nm), microvesicles (100–1,000 nm), and apoptotic bodies (1–5 µm).^42,43 EVs carry various biomolecules, including proteins, lipids, nucleic acids, and even organelles such as mitochondria.^44,45 EV-mediated intercellular mitochondria transfer primarily involves two mechanisms: the direct transfer of intact mitochondria enclosed within EVs, or the transfer of mitochondrial components, such as mitochondrial DNA, proteins, or RNA packaged inside EVs.^18,45 Intact mitochondria encapsulated within EVs and transferred to recipient cells can integrate with the cellular mitochondrial network and influence cellular metabolism and function.^17,46 Additionally, EVs can carry mitochondrial components, including mitochondrial DNA (mtDNA), mitochondrial proteins, and mitochondrial-derived vesicles, and modulate cellular processes in the recipient cells. ¹¹ EV-mediated intercellular mitochondria transfer contributes to a range of physiological functions and cellular processes and has been implicated in neuronal communication and synaptic plasticity, with significant potential implications for neurological function and the progression of neurological diseases. For example, after an ischemic stroke, astrocytes release mitochondria-containing vesicles for delivery to hypoxic neurons to support neuronal mitochondrial metabolism and survival. ¹¹ CD38 mediates this process,^11,47 and impairing it leads to more severe stroke pathology and worse outcomes in mice. Therefore, oxidative stress and nutrient deprivation can stimulate the release of mitochondria-containing EVs. However, the mechanisms underlying the mitochondrial packaging into EVs and their uptake by recipient cells remain unclear.

Cell fusion

Mitochondria can also be transferred via cell fusion, a biological process in which the plasma membranes of two or more cells merge to form a single multinucleated cell composed of similar or different cell types. ⁴⁸ Cell fusion can occur naturally in the body or be artificially induced by chemical agents. Naturally occurring cell fusion, typically involving stem cells and somatic cells, has been observed in several tissues and organs, including skeletal muscle, liver, lungs, heart, intestines, skin, brain, and retina. This process is thought to contribute to somatic cell reprogramming and tissue regeneration. ⁴⁹ During cell fusion, mitochondria from one cell are transferred to another, facilitating intercellular mitochondria transfer.

Endocytosis

Endocytosis is a fundamental process for the uptake of extracellular mitochondria, in which cells internalize external substances such as proteins, lipids, and other molecules through plasma membrane invagination to form vesicles. Studies have demonstrated the internalization of isolated mitochondria via simple co-incubation in vitro, without the need for transfection reagents, medium supplements, or additional interventions. ⁵⁰ This indicates that the cellular uptake of extracellular mitochondria likely occurs through endocytosis. Notably, the endocytosis of extracellular mitochondria into recipient cells is dependent on actin dynamics. ⁵¹ However, the regulation of actin-dependent endocytosis during the process of mitochondria transfer is not fully clear. Sun et al. ⁵¹ reported that, in cancer cells, the starvation signal induced via nicotinamide adenine dinucleotide NAD⁺-CD38-cADPR-Ca²⁺ pathway effectively involves actin-dependent endocytosis for intercellular communication. This signaling pathway, closely associated with TNTs, allows the exchange of cellular components and signal molecules, including vacuolar membrane components, calcium ions, and organelles such as mitochondria. Furthermore, they suggested that NAD⁺-CD38-cADPR-Ca²⁺-signaling-mediated endocytosis may be one of the mechanisms underlying mitochondria transfer via the entry of transplanted exogenous mitochondria into cells under pathological conditions. ⁵¹

Mitochondrial fusion

Functional mitochondria transferred from healthy to damaged cells may integrate with resident mitochondria, replacing the damaged ones and replenishing the mitochondrial pool to meet the cell’s energy demands. ¹⁹ Mitochondrial fusion is an important component of these reactions of replacement and restoration following intercellular mitochondria transfer. Mitochondria with different locations in a single cell repeatedly undergo fusion and fission.^16,52 This fusion involves the merging of the outer and inner mitochondrial membranes, facilitated by various proteins, such as mitofusins (Mfn) and optic atrophy 1 (OPA1). ⁵² Following mitochondrial fusion, the fused mitochondria undergo fission via dynamin-related protein 1 (Drp1). These proteins enable the fusion of pathologic or damaged mitochondria with healthy ones. Subsequently, damaged mitochondrial regions are fragmented through fission and eventually degraded by autophagosomes (mitophagy).^47,53 These processes rescue damaged cells from harmful stress, indicating that healthy mitochondria transfer plays an important role during pathological conditions.

Mechanisms of intracellular transfer after mitochondria transplantation

The mechanism by which transplanted exogenous mitochondria are incorporated into cells under pathological conditions has not been elucidated. However, it is believed that the known mechanisms of mitochondria transfer, including endocytosis, are involved. Although transplanted exogenous mitochondria have the potential to act as an energy source, it is not clear whether they undergo fusion and fission with damaged mitochondria to promote mitophagy. Previous studies have demonstrated that transplanted healthy mitochondria play an important role in damaged cells. A recent in vivo study reported that after mitochondria transplantation into a mouse model of hindlimb ischemia, DsRed+ mitochondria immediately began to migrate to endothelial cells (ECs) and were observed in ECs lining the neovessels. ⁵⁴ However, once the vasculature stabilized, DsRed+ mitochondria were restricted to mesenchymal stromal cells around the blood vessels and were rarely seen in ECs, indicating that mitochondria transfer had stopped. These findings suggest that there is a dynamic relationship between mitochondria transfer and the physiological state of the vasculature (stress and activation vs. stability). Similarly, in nerves, the efficiency of mitochondrial uptake may change as injured nerves recover, warranting further investigation.

Experimental methods for mitochondria transplantation

Mitochondria transplantation constitutes one of the methods available to promote beneficial responses by mitochondria at injured and sites and is based on the delivery of isolated, viable mitochondria to the target region. Isolation of healthy mitochondria is necessary for mitochondria transplantation. However, conventional methods of mitochondrial isolation, such as multiple continuous homogenization and centrifugation, induce mitochondrial shortening and fragmentation, resulting in significantly decreased activity. Therefore, research into the optimization of methods for mitochondrial isolation is ongoing. Moreover, for mitochondria transplantation, it is important to note that naked mitochondria or mitochondrial surface lipids are injurious factors. Thus, mitochondrial modification may be essential to enable clinical translation. To optimize mitochondria transplantation, we reviewed experimental methods, including mitochondrial source, isolation, and modification. The experimental techniques used to detect intercellular mitochondria transfer have been previously described. ¹² Here, we summarize mitochondrial isolation methods, particularly the centrifugation conditions, and methods for checking the quality of the isolated mitochondria (Table 1). It should be noted that the criteria and methods for assessing the purity and quality of isolated mitochondria vary between papers, and not all papers include these data.

Table 1.

Summary of methods of isolation for mitochondria transplantation.

Animal	Source	Isolation methods/Quality check [Purity or quality]	Refs
Density gradient centrifugation
Rat	Soleus muscle	The source was homogenized in buffer and centrifuged at 1500 rcf for 5 min. The supernatant was removed and centrifuged at 13000 rcf for 10 min, after which the pellet was suspended in buffer and purified using Ficoll gradient (7.5%/10%) centrifugation at 32000 rpm for 30 min. The pellet was removed, resuspended in buffer, and centrifuged at 10000 rcf for 10 min. The resulting pellet contain purified mitochondria.	8
		Oxygen consumption rate [70–90%] a
Rat	Neonatal brain	The source was homogenized in buffer and purified using Percoll gradient (12%/40%) centrifugation at 30000 g for 5 min. Fraction containing the mitochondria was collected and centrifuged at 15000 g for 10 min. The pellet was resuspended in buffer and centrifuged at 15000 g for 5 min. The resulting pellet contain purified mitochondria.	64
		Respiratory control ratio [Similar levels to other methodologies]
Differential centrifugation
Mouse	Skeletal muscle	The source was homogenized in buffer and centrifuged at 800 g for 10 min. The supernatant was removed, and the pellet was resuspended in in buffer with the protease nagarse. The suspension was centrifuged at 5000 g for 5 min. The supernatant was collected and centrifuged at 800 g for 15 min. The supernatant was collected and centrifuged at 9000 g for 10 min. The resulting pellet contain purified mitochondria.	65
		Mitochondrial proteins [Increased expression of COX4 and VDAC] a
Mouse	Cerebral cortex	The source was homogenized using a glass grinder in buffer. Following removal of debris with repeated centrifugation at 1000 g for 5 minutes followed by 4000 g at 5 min. The supernatant was collected and centrifuged at 8000 g for 10 min. The resulting pellet contain purified mitochondria.	66
		MitoTracker dyes [81% positive marker cells]
Differential centrifugation and filtration
Mouse	Placenta	The source was homogenized in buffer and centrifuged at 1000 g for 5 min. The supernatant was collected and centrifuged again at 1000 g for 5 min. The supernatant was filtered through 1.2 μm mesh and then the filtrate was centrifuged at 4000 g for 10 min. The resulting pellet contain purified mitochondria.	57
		Membrane potential [87% positive markers]
Rat	Liver	The source was homogenized in buffer and centrifuged at 750 g for 4 min. The supernatant was filtered twice through a 40 μm mesh and then final filtration was performed through 5 μm syringe filter. The filtrate was centrifuged at 9000 g for 10 min. The resulting pellet contain purified mitochondria.	68
		MitoTracker dyes [93% positive marker cells]
Commercial cell mitochondria isolation kit and differential centrifugation
Human	Astrocyte cell line	The cells were incubated in mitochondrial isolation reagent with the Cell Mitochondria Isolation Kit (Beyotime) for 10 min and homogenized for 30 strokes using a tight pestle. The homogenate was centrifuged at 600 g for 10 min. The supernatant was collected and centrifuged at 12,000 g for 10 min. The resulting pellet contain purified mitochondria.	51
		Membrane potential (JC-1) [82% positive marker cells]
Mouse	Primary astrocyte	The cells were added to mitochondrial isolation reagent with the Cell Mitochondria Isolation Kit (Beyotime) and trypan blue, and centrifuged at 1000 g for 10 min. The supernatant was collected and centrifuged at 12000 g for 10 min. The resulting pellet contain purified mitochondria.	69
		MitoTracker dyes [95% positive marker cells]
Mouse	Hippocampus	The source was homogenized for 10 stroke using a tight pestle in mitochondrial isolation reagent with the Cell Mitochondria Isolation Kit (Beyotime) and centrifuged at 1000 g for 15 min. The supernatant was collected and centrifuged at 3500 g for 10 min. The resulting pellet contain purified mitochondria.	70
		Membrane potential (JC-1) [Similar levels to the positive control group]
Commercial tissue dissociator kit and differential filtration
Mouse	Liver	The source was homogenized using the gentle MACSTM Dissociator (Miltenyi Biotec) in buffer. The homogenate was filtered through 40 μm mesh and then the filtrate was centrifuged at 9000 g for 10 min. The resulting pellet contain purified mitochondria.	72,73
		Respiratory control ratio [Similar levels to other methodologies]
Pig	Psoas muscle	The source was homogenized using the gentle MACSTM Dissociator (Miltenyi Biotec) in buffer. Debris was removed centrifugation at 750 g and the cell suspension was added protease. The cell digest was filtered through a 40 µm polyethylenterephthalat mesh filter. After a second 40 µm filtration step and 2 passages through a 10 µm filter, mitochondria were isolated by centrifugation at 9000 g for 10 min. The resulting pellet contain purified mitochondria.	74
		MitoTracker dyes [93% positive marker cells]
Magnetic beads
Human	HEK293 cells	The cells were lysed in buffer by shearing through a needle approximately 20 times. For magnetic labeling, the cell lysate was incubated with microbeads for 15 to 60 minutes. The suspension was loaded onto a column, and then retained mitochondria were eluted with buffer after removing the column from the magnetic field. Following centrifugation at approximately 13,000 g for 1 min. The resulting pellet contain purified mitochondria.	75
		Respiratory control ratio [Similar levels to the preparations by differential centrifugation]
－	Yeast	The prepared mitochondria-enriched fraction was added to the magnetic beads and incubated with the beads for 10–60 minutes with gentle rotation. The mixture was placed in the separation rack for 1 min. The magnetic bead-bound mitochondria were then washed 3 times with imidazole in buffer. Mitochondria were eluted from the magnetic beads by incubation with imidazole in buffer for 5 min with rotation. The released mitochondria were collected and centrifuged at 12,000 g for 5 min.	76,77
		COX activity [Similar levels to the preparations by differential centrifugation]
Pump-controlled cell rupture system
Rat	Liver	The slightly minced source was pumped once using a pump-controlled cell rupture system and mitochondria were isolated.	78
		Respiratory control ratio [Similar levels to the preparations by density gradient centrifugation]

ATP; Adenosine triphosphate: COX; cytochrome c oxidase: HEK; human embryonic kidney: MSCs; mesenchymal stem cells: mNSC; mouse neural stem cells: VDAC; voltage-dependent anion channel.

^aChanges compared to the control.

Mitochondrial source

Mitochondria with normal respiratory activity are essential for mitochondria transfer and functional integration. Therefore, virtually any healthy tissue or cell located far from the lesion site can be used as a source for mitochondrial isolation. Several studies have been conducted on mitochondria isolated from various tissues, including muscle, liver, heart, placenta, and brain.^55–59 In a clinical study, McCully et al. proposed that selecting tissue sources for mitochondrial isolation based on the incision site required for surgical access could have therapeutic applications without the need for secondary intervention. ⁶⁰

For clinical applications, using autologous tissue for mitochondrial isolation, skeletal muscles such as rectus abdominis, pectoralis major, and gastrocnemius may offer high clinical utility and carry a low risk of immunogenic complications. However, the isolation of mitochondria from muscles requires fresh tissue. For instance, frozen-thawed mitochondria have reduced membrane potential and were ineffective in experimental rat models of disease. ⁶¹ This reduction in effectiveness is attributed to mitochondrial membrane disruption, which impairs the uptake of mitochondria by recipient cells. ⁶⁰ Therefore, mitochondria derived from these tissues cannot be stored or prepared in advance.

In our previous study, ⁵⁹ we obtained mitochondrial-enriched fractions from various tissues, including snap-frozen placenta, and evaluated mitochondrial activity. Our findings revealed that the placenta-derived mitochondrial fraction exhibited ATP levels comparable to those from skeletal muscle and brown fat tissue. Moreover, western blot analysis demonstrated high expression of key mitochondrial proteins, including glutathione reductase, manganese superoxide dismutase (MnSOD), and heat shock protein (HSP) 70 in the placenta-derived fractions. Our results indicated that viable membrane potentials could be isolated from cryopreserved placenta with preserved ATP levels and high expression of mitochondrial proteins. Given that placental tissues have already been used for cell-based therapies and cryopreservation does not appear to negatively affect this specific compartment,^62,63 the cryopreserved placenta could be a promising source for viable organelle isolation in a non-invasive manner. However, since placental tissue is non-autologous, future studies are necessary to assess potential immune-related complications.

Mitochondrial isolation

Differential or density gradient centrifugation is widely used in mitochondrial isolation methods,^8,64–66 with differential centrifugation yielding higher quantities and lower purity, ⁶⁷ and density gradient centrifugation yielding lower quantities and higher purity. These isolation methods require repeated centrifugation-related steps and are time-consuming. For therapeutic applications, rapid mitochondrial isolation is required. Researchers have reported a rapid and simple isolation procedure using filtration^57,68 or commercial kits^51,69,70; however, it has been noted that even using these methods, mitochondrial isolation could can be time-consuming. ⁷¹

In our previous study, ⁵⁷ mitochondria-enriched fractions were obtained from tissues and suspended in a mitochondrial isolation buffer (10 mM HEPES [pH 7.5], 250 mM sucrose, 1 mM ATP, 0.1 mM ADP, 5 mM sodium succinate, and 2 mM K₂HPO₄). Each tissue was homogenized in glass tissue grinders in the mitochondrial isolation buffer at 4°C. Cellular debris was removed with repeated centrifugation at 1,000 g at 4°C for 5 minutes, followed by 4,000 g at 4°C for 5 min. The supernatants obtained were strained through 1.2-µm filters, and the pellets (mitochondria-enriched fractions) were obtained by centrifugation at 4,000 g at 4°C for 10 min. This process was completed in 30 to 60 min. ⁵⁷ Cardiac and cerebrovascular surgery, as well as many other surgical procedures, have limited intervention time windows; therefore, mitochondrial isolation times of 60 min may be too long for clinical application. The methods proposed by Preble et al. ⁷² and McCully et al. ⁷³ may be better suited for clinical settings where an intervention time of less than 60 min is required. These methods employ the gentleMACS^TM Dissociator (Miltenyi Biotec Inc., San Diego, CA, USA), a tissue dissociator kit used to homogenize freshly isolated tissue. This benchtop instrument facilitates the semi-automated dissociation of tissues into single-cell suspensions or homogenates. Semi-automated and programmable protocols ensure uniform and consistent tissue homogenization, which is difficult to achieve with manual methods. Following homogenization, mitochondria-enriched fractions are obtained by digesting the tissue with subtilisin A, a protease derived from Bacillus licheniformis type VIII, and filtering it through a mesh before centrifugation. This process is considered simple and rapid, typically completed in less than 30 min.^72,73 Rossi et al. also used the gentleMACS^TM Dissociator kit to efficiently isolate high-quality mitochondria from large animal organs. ⁷⁴

In addition to these methods, the use of magnetic beads or a pump-controlled cell rupture system (PCC) has also been reported as techniques to isolate mitochondria.^75–78 Mitochondria isolated using magnetic beads exhibit fewer contaminants and retain similar ultrastructure, protein import, and cytochrome c oxidase complex activity compared to mitochondria isolated by differential centrifugation. This method offers a valuable option for obtaining higher quality and purer mitochondria. However, a typical separation procedure requires at least 1 to 2 h. ⁷⁷ On the other hand, PCC has the potential to rapidly isolate mitochondria through a streamlined process. Schmitt et al. described PCC as a semi-automatic device, composed of a high-precision pump, gas-tight syringes, a Balch homogenizer, and tungsten carbide balls of varying diameters. ⁷⁸ The combination of the adjustable Balch homogenizer with a precisely controlled high-precision pump ensures reproducibility, operator independence, and comparability across experiments. As these devices become more widely available and capable of processing large sample volumes, the practicality of these methods will increase.

Overall, it is important that isolated mitochondria remain intact and viable. The use of dead, nonviable mitochondria, mitochondrial proteins or complexes, mitochondrial DNA/RNA, or high-energy phosphates, either alone or in combination, will not provide sufficient therapeutic benefit. After isolation, mitochondria should be used immediately for transplantation. Isolated mitochondria can be stored on ice for approximately 1 h, and storage beyond this time point greatly reduces efficacy. ⁷³ Therefore, the development of clinical applications involving mitochondria transplantation also requires the identification of ways to store the isolated mitochondria.

Storage

Mitochondrial storage is an important aspect in clinical practice. The function of isolated mitochondria can be affected by the cooling rate, thawing rate, and cryopreservation temperature. ¹² Regarding mitochondrial function, freshly isolated mitochondria are the best option for mitochondria transplantation. However, if mitochondria could be stored, their usefulness would be greater. Nukala et al. ⁷⁹ reported that brain-derived mitochondria were suspended in 10% (v/v) dimethyl sulfoxide as a cryoprotectant and cooled at a uniform rate of 1°C/min for storage at −80°C. Compared to freshly isolated mitochondria, mitochondria cryopreserved for 1 week retained approximately 50% of their normal respiratory function. Another study found that liver-derived mitochondria, suspended in trehalose buffer and stored at −80°C for 2 weeks, maintained mitochondrial ultrastructure and retained the ability to produce ATP and import proteins. ⁸⁰

Additionally, in our previous study, we demonstrated that the function of mitochondria was preserved after the whole placenta was rapidly cooled in liquid nitrogen and stored at −80°C for 2 weeks, even without the use of a specialized buffer. ⁵⁷ The conditions for directly preserving mitochondria versus preserving tissue sources for later mitochondrial isolation may differ. If isolated mitochondria can be frozen and thawed without causing functional disruption, mitochondria transplantation therapy could potentially be applied at the bedside. Further studies are warranted to investigate this hypothesis and evaluate the feasibility of mitochondrial storage until transplantation.

Modification

Extracellular mitochondria and their components act as damage-associated molecular patterns (DAMPs) that induce inflammation. Furthermore, oxidized cardiolipin on the mitochondrial surface may activate proinflammatory processes in leukocytes. ⁸¹ In the extracellular environment, mitochondria may be susceptible to glycation and consequently produce danger signals. ⁸² Therefore, artificially modifying isolated mitochondria may reduce their toxicity and minimize the adverse effects of mitochondria transplantation. The feasibility and utility of mitochondrial modifications have recently been reported. For instance, peptide-labeled mitochondria enhanced delivery into dopaminergic neurons and ameliorated oxidative DNA damage, improving locomotor activity in a rat model of PD. ⁸³ Similarly, dextran triphenylphosphonium-based polymer functionalization of mitochondria was shown to have a threefold increase in cellular internalization of cardiac cells compared to uncoated mitochondria. ⁸⁴ This modification technique is worth considering for neurons. Notably, isolated mitochondria are susceptible to advanced glycation end product (AGE) modification, and these glycated mitochondria, when transferred into neurons, induce receptors for advanced glycation end product (RAGE)-mediated autophagy and oxidative stress. ⁸² However, modifying mitochondria with O-GlcNAcylation counteracts glycation, diminishes RAGE-mediated effects, and improves the viability of mitochondria recipient neurons. These findings suggest that AGE modification in extracellular mitochondria may induce danger signals, whereas O-GlcNAcylation can prevent glycation and improve the therapeutic efficacy of transplanted mitochondria in the CNS. Furthermore, we focused on mitochondrial coating to enhance the efficiency of mitochondria transfer and coated mitochondria with cationic and fusogenic lipids, specifically 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). ⁶⁶ Endocytosis is one of the common mechanisms by which extracellular mitochondria are transferred into cells. However, mitochondria entering the cytoplasm via endocytosis are often recognized as foreign substance, with some being degraded by lysosomes. ⁸⁵ In contrast, mitochondria transferred by fusogenic lipids may bypass this degradation process. Fusogenic lipids fuse with the cell membrane to create a channel that allows direct transfer of mitochondria into the cytoplasm. ⁸⁵ Therefore, coating mitochondria with fusogenic lipids may enhance the efficiency of cellular uptake and retention. In fact, DOTAP/DOPE-coated mitochondria, demonstrated increased neuronal uptake and neuroprotective effects against ischemic damage. ⁷⁰ Additionally, western blot and fluorescence-activated cell sorting (FACS) analyses demonstrated that DOTAP/DOPE coating did not affect key mitochondrial proteins, including TOM40, ATP5A, ACADM, HSP60, and COX IV. Furthermore, the surface modification did not significantly alter ATP content or mitochondrial structure, and FACS analysis revealed improved mitochondrial purity and stabilized mitochondrial membrane potentials.

Coating the mitochondrial surface may also prevent the exposure of components that may induce unwanted detrimental effects on cells. As such, encapsulating isolated mitochondria could improve mitochondrial delivery and reduce toxicity. However, this coating process requires over an hour, highlighting the need for further optimization for clinical application, particularly in treating acute CNS disease symptoms. Additionally, fusion-promoting lipids such as DOPE are strong destabilizers for the lipid bilayer and can cause membrane rupture during the fusion process. ⁸⁶ Although this side effect was not observed in our study, further investigation into the potential side effects of fusogenic lipids is warranted.

Mechanism of action of extracellular mitochondria in therapeutic interventions for CNS disease

Various techniques of exogenous mitochondria transplantation have been evaluated in CNS disease models. The experimental conditions, including the dosages of mitochondria, sources, and administration routes, have been summarized in Table 2.

Table 2.

Summary of methods or techniques for in vivo studies of mitochondria transplantation in CNS disease or injury.

Animal	Source	Dose (numbers of µg protein)	Modification/modulation	Functional outcomes	Refs
		Route of transplantation
SCI model
Rat	PC12 cell/rat skeletal muscle (allograft)	50–150 µg	None	Oxygen consumption rate was improved, but long-term motor and sensory functions were not improved.	8
		DI into spinal cord
Rat	Rat skeletal muscle (allograft)	50 μg	None	Mitochondrial fragmentation, neuroapoptosis, neuroinflammation, oxidative stress were reduced, and functional recovery was improved.	87
		DI into spinal cord
Rat	Rat platelet (allograft)	10 μg (3 × 105 mitochondria)	Photobiomodulation	ATP production was increased, oxidative stress and neuronal apoptosis levels were reduced, and motor function recovery were promoted.	88
		DI into spinal cord
Mouse	Mouse bone marrow-derived macrophages (allograft)	0.1–0.25 × 106 mitochondria	Engineered mitochondria	Engineered mitochondria enhanced macrophage.	89
		IV
TBI model
Rat	hUC-MSCs (xenograft）	harvested from 3 × 107 hUC-MSCs	None	Neuronal apoptosis levels were reduced, astrogliosis and microglia activation were alleviated, normal brain morphology and cytoarchitecture were retained, and sensorimotor functions were improved.	90
		ICVI
SZ model
Rat	Human lymphocyte/rat brain (heterograft/allograft)	100 µg	None	Prevented dissipation in mitochondrial membrane potential and attentional deficit	91
		ICDI
PD model
Rat	PC12 (Allogeneic) or human osteosarcoma cybrids (xenogeneic)	1.05 µg (Pep-1–labeled)	None	locomotive activity was improved and deterioration of dopaminergic neurons was attenuated.	83
		ICDI
Mouse	HepG2	0.5 mg/kg	None	Improved behavior test, ATP, increased ETC activity, decreased ROS formation, apoptosis and necrosis.	92
		IV
ICH model
Mouse	Mouse or rat primary astrocyte culture medium (allograft or heterograft)	200 μL medium	None	Mn-SOD levels were restored and neurological deficits was improved.	93
		IV
IS model
Mouse	Mouse cryopreserved placenta (allograft)	100 μg	None	Infarction volume was reduced, but functional outcome was not evaluated.	57
		IV
Mouse	Mouse cerebral cortex (allograft)	6 μg	O-GlcNAcylation	Neuronal injury was reduced and neurological deficits were improved.	82
		ICVI
Rat	BHK cells	75 µg, 750 µg	None	Improve motor function, decrease infarct area and cell death.	94
		ICDI
Rat	Rat pectoralis major muscle (autologous)	5 × 106	None	Neuronal apoptosis levels were reduced, reactive astrogliosis was alleviated, neurogenesis was promoted, infarct volume was reduced, and neurological deficits were improved.	95
		ICVI
Rat	Rat MSCs (allograft)	5 × 105 MSCs	None	Mitochondrial activity of injured microvasculature was improved, angiogenesis was enhanced, infarct volume was reduced, and functional recovery was improved.	96
		ICDI
Mouse	Mouse gastrocnemius muscle (allograft)	NA	Focused ultrasound activation of microbubbles	Cell viability was improved and infarct volume was reduced, but functional outcome was not evaluated.	97
		IA or ICVI
Mouse	hUC-MSCs (heterograft)	Extracted functional mitochondria from 5 × 106 hUC-MSCs	None	Internalized transplanted mitochondria decreases host cell ROS levels and rescues survival, and functional outcome was not evaluated.	98
		ICDI
Rat	hUC-MSCs (heterograft)	Extracted mitochondria from 3 × 107 hUC-MSCs	None	Blood creatine phosphokinase, apoptosis, glial activation, and infarct volume were decreased, and motor function was improved.	99
		ICVI
Rat	Rat primary astrocyte culture (allograft)	NA	None	Infarction volume was reduced, but functional outcome was not evaluated.	100
		ICDI
Mouse	Mouse liver (allograft)	100 μg	None	Oxidative stress and neuroinflammation were attenuated, infarct volume and edema were reduced, and neurological deficits were improved.	101
		IND
Mouse	Mouse bone marrow MSCs (allograft)	85 μg, 170 μg, and 340 μg	None	Memory impairment was improved, ROS was reduced, ATP generation was restored, and mitochondrial function in brain was improved.	102
		IND
Mouse	Mouse liver (allograft)	2 × 107 mitochondria	None	Mitochondrial function was restored, and antidegenerating and myelination-promoting effects were observed.	103
		ICDI

ATP; adenosine triphosphate: BHK; baby hamster kidney fibroblasts: DI; direct injection: ETC; electron transfer chain: HepG2; hepatocellular carcinoma cell line: hUC-MSCs; human umbilical cord-derived mesenchymal stem cells: IA; intra-arterial: ICDI; intracerebral direct injection: ICH; intracranial hemorrhage: ICVI; intracerebroventricular injection: IND; intranasal delivery: IS; ischemic stroke: IV; intravenous: MSCs; mesenchymal stem cells: NA; not applicable: PC12; pheochromocytoma cell line: PD; Parkinson's disease: ROS; reactive oxygen species: SCI; spinal cord injury: SZ; schizophrenia: TBI; traumatic brain injury.

Spinal cord injury

Some studies have reported that mitochondria transplantation may have a therapeutic potential in SCI. Gollihue et al. ⁸ reported that when mitochondria (50, 100, or 150 µg) isolated from pheochromocytoma cell line (PC12) cells or rat skeletal muscle were transplanted into the site of injury within 30 minutes after induction of SCI, mitochondrial incorporation was observed in macrophages, ECs, and astrocytes, but not in neurons. Mitochondria transplantation enhanced acute mitochondrial bioenergetics in injured tissue, as evidenced by the oxygen consumption ratio analysis conducted using a metabolic analyzer. However, mechanical hypersensitivity was assessed 6 weeks post-injury, and the Basso, Beattie, Bresnahan locomotor rating scale was used on multiple days following the injury, extending up to 42 days. Mitochondrial transplantation did not result in improvements in either metric. Additionally, tissue sparing morphometric analyses, conducted 6 weeks after injury, showed no significant differences among the groups in total lesion volume or in gray and white matter sparing at various spinal cord levels relative to the injury epicenter. Collectively, the proof-of-concept study advances the burgeoning field of mitochondria transplantation and indicates that an increase in incorporation efficiency and cell-type targeting may be key factors for improving long-term functional neuroprotection in models of SCI.

Lin et al. ⁸⁷ reported that 100 µg of soleus-derived allogenic-labeled mitochondria injected directly into the injured spinal cord in rat SCI models were detectable 28 days post-treatment. The transplanted group showed better recovery of locomotor and sensory functions and also exhibited reduced apoptosis and inflammatory response.

Zhu et al. ⁸⁸ demonstrated that photobiomodulation (PBM) combined with platelet-derived mitochondrial allograft transplantation increased ATP production and reduced oxidative stress and neuronal apoptosis, promoting tissue repair and motor function recovery. In vitro experiments showed that CX36 mediates the transfer of mitochondria to neurons and, both in vivo and in vitro, is facilitated by PBM via CX36. Thus, PBM can be used as a novel therapeutic adjuvant in mitochondria transplantation.

Xu et al. ⁸⁹ developed an engineered mitochondrial compound targeting macrophages in the SCI region. Mitochondria sourced from interleukin-10-induced mer tyrosine kinase high expression (Mertkhi) bone marrow-derived macrophages (BMDM) were conjugated with a peptide sequence of cations (cysteine-alanine-glutamine-lysine), to optimize its affinity for the injury site. These mitochondria isolated from mouse BMDMs were engineered and injected intravenously into a mouse model of SCI. Mitochondria transplantation significantly enhanced macrophagic phagocytosis of myelin debris, curtailed lipid buildup, ameliorated mitochondrial dysfunction, and attenuated proinflammatory profiles in macrophages, both in vitro and in vivo.

Traumatic brain injury

Mitochondria isolated from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were transplanted into rat models of traumatic brain injury by intracerebroventricular (ICV) injection. The transplanted mitochondria were successfully internalized into the neuronal cells. This significantly reduced the number of brain cells undergoing apoptosis, alleviated astrogliosis and microglia activation, and led to the retention of normal brain morphology and cytoarchitecture, resulting in improved sensorimotor functions. ⁹⁰

Schizophrenia

Robicsek et al. ⁹¹ reported the effectiveness of mitochondria transplantation in a schizophrenia model. The experimental schizophrenia model was prepared by maternal immune activation, which was achieved by intravenously injecting 4 mg/kg/ml of poly-I:C in pregnant rats. On postnatal day 21, the offspring were separated from their parents and housed. Mitochondria isolated from rat brains were bilaterally injected into the intra-prefrontal cortex of the rat model of schizophrenia. Mitochondria transplantation was observed to prevent attention-deficit-characterized cognitive impairment in schizophrenia and concurrently improve the mitochondrial membrane potential.

Parkinson’s disease

Chang et al. ⁸³ investigated the efficacy of mitochondria transplantation in a rat model of PD induced by 6-hydroxydopamine (6-OHDA) injection. The researchers utilized allogeneic PC12 or xenogeneic (human osteosarcoma cybrids) mitochondria, with or without Pep-1 conjugation. Rats received mitochondria injections (1.05 µg/5 µL) locally 3 weeks after the 6-OHDA injection. Analysis of behavioral tests revealed that Pep-1–labeled allogeneic (PMD) and xenogeneic mitochondria (peptide-mediated delivery of xenogeneic mitochondria [xPMD]) significantly improved locomotion, travel distance, movement speed, and number of crossed zone entries as compared to the vehicle or allogeneic mitochondria transplantation (without Pep-1 label) groups. Additionally, both PMD and xPMD mitigated dopaminergic neuronal deterioration compared to the vehicle-treated group. Allogeneic transplantation was more effective than xenogeneic transplantation in some locomotor activity tests, indicating that mitochondria transplantation efficacy may be influenced by the host species (i.e., allogeneic or xenogeneic).

In another study by Shi et al., ⁹² a mouse model of PD was prepared by intraperitoneal injection of 10 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered once daily for 5 days. Subsequently, HepG2-derived mitochondria (500 µg/kg body weight) were intravenously injected on day 5 with the final MPTP dose. The mitochondria-treated group showed significantly improved behavioral performance (pole test and rotarod test), complex I activity, ATP content, cell apoptosis and necrosis, glutathione levels, and ROS levels in the striatum compared to the vehicle group. ⁹²

Hemorrhagic stroke

Tashiro et al. ⁹³ reported that intravenous transplantation of primary mouse or rat astrocyte culture cell medium containing mitochondria restored manganese superoxide dismutase (Mn-SOD) levels and reduced neurological deficits in models of intracerebral hemorrhage (ICH) mice upon entering the brain neurons. Furthermore, using an in vitro ICH-like injury model in cultured neurons, neuronal entry of astrocytic mitochondria prevented ROS-induced oxidative stress and neuronal death by restoring neuronal Mn-SOD levels while simultaneously promoting neurite extension and upregulating synaptogenesis-related gene expression. The study demonstrated that transplantation of astrocytic mitochondria modulates neuronal antioxidant defense and neuroplasticity and also promotes post-ICH functional recovery.

Ischemic stroke

In recent years, an increasing number of studies have demonstrated the efficacy of mitochondria transplantation therapy for ischemic brain injury. We reported the utility of mitochondria transplantation using cryopreserved placenta for ischemic stroke therapy. ⁵⁷ Mitochondria were isolated from cryopreserved mouse placentas and evaluated mitochondrial purity and function using flow cytometry. Approximately 87% of the placental mitochondria were viable and maintained JC1 membrane potentials after isolation. Furthermore, the placental mitochondrial fraction contained ATP equivalent to the mitochondrial fractions isolated from skeletal muscle and brown fat tissue. Normalized mitochondrial antioxidant enzymes (glutathione reductase and MnSOD) and HSP70 were highly preserved in the placental mitochondrial fractions. When intravenously injected after reperfusion in transient MCAO mouse models, mitochondria transplantation significantly reduced infarct volume. These results indicate that cryopreserved placentas are a feasible source for the isolation of viable mitochondria.

Park et al. ⁸² showed that mitochondrial O-GlcNAc-modification was amplified by recombinant O-GlcNAc transferase and UDP-GlcNAc. Mitochondria isolated from the mouse cerebral cortex and modified with O-GlcNAc were injected into the striatum of a mouse model of transient focal cerebral ischemia/reperfusion. Treatment with extracellular mitochondria modified by O-GlcNAcylation reduced neuronal injury and improved neurological deficits.

Huang et al. ⁹⁴ performed exogenous mitochondria transplantation in a rat model of focal ischemia. Mitochondria were isolated from the hamster kidney fibroblast cell line and injected directly into the ischemic striatum (75 µg) or administered as an intra-arterial infusion (750 µg) following transient focal ischemia. The results indicated that, compared to animals in the vehicle-treated group, mitochondria transplantation significantly improved motor function, reduced infarct area, and resulted in fewer TUNEL-positive cells. ⁹⁴

Zhang et al. ⁹⁵ allografted skeletal muscle-derived mitochondria in a rat model of focal ischemia. Mitochondria (5 × 10⁶/10 µL) were infused into the lateral ventricle after immediate reperfusion in transient focal ischemia. Consequently, mitochondrial allografts significantly decreased the infarct volume and alleviated neurological deficits, cellular oxidative stress, and apoptosis at 28 days after the ischemic stroke. Furthermore, mitochondrial treatment mitigated reactive astrogliosis and promoted neurogenesis post-stroke, suggesting that it may be effective not only for acute neuroprotection but also for improving long-term outcomes.

Stem cell-mediated mitochondria transfer may be a pivotal mechanism for neuroprotection and neurorepair. Liu et al. carried out transplantation with 5 × 10⁵ mesenchymal stem cells (MSCs) administered via the common carotid artery 24 h after transient focal ischemia in rats and found that MSCs improved microvasculature and mitochondrial function (as evaluated by the oxygen consumption rate in the peri-infarct area), infarct volume reduction, and functional recovery. ⁹⁶

Norat et al. ⁹⁷ reported that gastrocnemius-derived allogenic mitochondria were administered by intra-arterial infusion into a transient proximal middle cerebral artery occlusion (MCAO) mouse model. The researchers evaluated the effects of concurrent focused ultrasound (FUS) activation of microbubbles, which open the blood–brain barrier, on mitochondrial delivery efficacy. Following intra-arterial delivery, mitochondria are distributed throughout the affected hemisphere and integrated into neural and glial cells in the brain parenchyma. Consistent with functional integration in the ischemic tissue, transplanted mitochondria elevated the concentration of ATP in the affected hemisphere, reduced infarct volume, and increased cell viability. Therefore, FUS may be used as an adjuvant therapy for mitochondria transplantation.

Li et al. ⁹⁸ presented that functional mitochondria (F-Mito) isolated from hUC-MSCs were labeled with a lentivirus of the HBLV-mito-dsred-Null-PURO vector. The ability of stressed cells to internalize F-Mito was analyzed in a mouse MCAO and an oxygen-glucose deprivation/reoxygenation cell model. F-Mito was transplanted via intracerebral direct injection in vivo. Neurons and endothelial cells were more effective at internalizing mitochondria than astrocytes, both in vitro and in vivo. Moreover, internalized F-Mito decreased host cell ROS levels and improved survival. The ROS response in stressed cells after ischemia is a crucial determinant of the internalization of F-Mito by host cells, and inhibiting ROS generation in host cells may decrease F-Mito internalization. These results offer insights into how exogenous mitochondria rescue neural cells via the ROS response in an ischemic stroke model.

Pourmohammadi-Bejarpasi et al. ⁹⁹ reported that isolated healthy mitochondria from hUC-MSCs transplanted into a rat model of MCAO by ICV injection ameliorated reperfusion/ischemia-induced damage as evidenced by decreased blood creatine phosphokinase levels, reduced apoptosis, decreased astrogliosis and microglial activation, reduced infarct size, and improved motor function.

Lee et al. ¹⁰⁰ reported that treatment with mitochondria isolated from rat primary astrocytes significantly reduced infarct volume in rat MCAO models. The mitochondria were injected into the striatum at 60 min and 24 h after MCAO, and labeled mitochondria were confirmed in the neurons by immunostaining.

Salmn et al. ¹⁰¹ conducted an experiment by administering isolated mice liver mitochondria (100 µg protein) intranasally at 30 min, 24 h, and 48 h following photothrombotic ischemic stroke. After 72 h post-stroke, mice were tested for neurobehavioral outcomes and euthanized to assess infarct volume and brain edema and conduct molecular analyses. The mitochondria-treated group showed significantly decreased infarct volume and brain edema, as well as improvement in neurological dysfunction. Furthermore, ischemic stroke-induced oxidative stress and neuroinflammation were attenuated. In addition, mitochondrial treatment modulated AMPK and SIRT1/PGC-1α signaling pathway, and inhibited NLRP3 inflammasome activation. The researchers concluded that mitochondria therapy exerts neuroprotective effects by inhibiting oxidative damage and inflammation, mainly dependent on the heightening activation of the AMPK and SIRT1/PGC-1α signaling pathway.

Mitochondria isolated from mouse bone marrow MSCs were transplanted into the medial prefrontal cortex of a model of photothrombotic-induced ischemic stroke affecting the medial prefrontal cortex (mPFC). Mitochondria were administered intranasally on alternate days (3 days per week) for 1 week. Subjected mice were divided into groups based on different dosing regimens of mitochondria (85 µg, 170 µg, and 340 µg). Behavioral results revealed that mitotherapy alleviated ischemia-induced memory impairment. In addition, transplantation of exogenous mitochondria lowered ROS levels, restored ATP generation, and improved mitochondrial membrane potential. Induction of ischemia decreased the levels of synaptic markers in mPFC, while exogenous mitochondria (170 and 340 mg) significantly upregulated the expression of GAP-43 and PSD-95 after ischemic stroke. ¹⁰²

Chen et al. ¹⁰³ reported that isolated allogeneic mouse liver mitochondria were transplanted into the bilateral cortex of photochemical ischemic mice models. This procedure efficiently restored the overall mitochondrial functions, such as ATP levels and activity of mitochondrial complexes I to V. Furthermore, mitochondria transplantation was effectively internalized by oligodendrocyte progenitor cells (OPCs) in the ischemic cortex. In comparison with the control cortex, the mitochondria-treated cortex exhibited significantly less apoptotic and more proliferative OPCs. Higher levels of myelin basic protein and morphologically normal myelin-wrapped axons were observed using light and electron microscopy in the mitochondria-treated cortex at 21 days post-injury. Behavioral assays also showed better locomotion recovery in mitochondria-treated mice. Further analysis demonstrated that olig2 and lipid synthesis signaling were significantly increased in the mitochondria-treated cortex. These results reflect the antidegenerative and myelination-promoting effects of exogenous mitochondria, making mitochondria transplantation a potentially valuable treatment for ischemic stroke.

Multiple sclerosis

There are few studies that have applied exogenous mitochondria transplantation to animal models of multiple sclerosis. However, Peruzzotti-Jametti et al. reported that ICV injection of neural stem cells (NSCs) into mice with myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, actively transferred functional mitochondria via EVs to mononuclear phagocytes and resulted in significant amelioration of clinical deficits. ⁴⁶ Analysis revealed that mitochondria were predominantly transferred to F4/80⁺ mononuclear phagocytes (52.5%) and, to a lesser extent, GFAP⁺ astrocytes (38.25%) after a single ICV injection of NSCs. A lower proportion of transfer was observed in neurons, oligodendrocytes, and T cells. This study demonstrates that mitochondria transfer from NSCs occurs in vivo and that the process is modulated under neuroinflammatory conditions to target mononuclear phagocytes and host astrocytes. The findings suggest that mitochondria transfer may play a significant role in ameliorating multiple sclerosis.

Recent clinical trials, clinical relevance, and strategy for therapeutic use of mitochondria

The first clinical trial on mitochondria transplantation was reported in 2017 by Emani et al. ¹⁰⁴ In this trial, mitochondria harvested from a patient’s healthy muscle tissue were used autologous transplantation. The subjects were five pediatric patients with myocardial ischemia-reperfusion injury requiring extracorporeal membrane oxygenation (ECMO) support, and mitochondria were injected via epicardial injection. Cardiac function improved, and patients were able to withdraw from ECMO support with no observed severe adverse events. Since then, several clinical studies of mitochondria transplantation therapy have been conducted. However, only one clinical trial to date specifically targets CNS disorders, focusing on cerebral ischemia (NCT04998357).

Ischemic stroke is a leading cause of disability and death worldwide, and its neurological impact is both permanent and devastating. Therefore, the development of new therapies is urgently required. Patients with acute cerebral infarction may require thrombolytic agents, such as tissue plasminogen activator and endovascular therapy, as recommended treatments; however, these treatments are time-sensitive, ¹⁰⁵ and bleeding complications may occur. Prompt transport, diagnosis, and medical treatment are required in patients with ischemic stroke. Furthermore, the extraction of mitochondria from a patient’s tissue is complicated in emergency settings. Therefore, in emergency medicine, it may be necessary to extract and administer functionally preserved mitochondria from allogeneic and heterogeneous tissues, as well as cryopreserved tissues. Recent animal studies of CNS disease have reported mitochondrial isolation from not only autologous but also xenograft and allograft mitochondria (Table 2), as well as frozen allograft tissue. ⁵⁷ These reports are promising for clinical application. Another consideration is that mitochondrial components induce immune responses as molecular pattern molecules (DAMPs such as mtDNA, cardiolipin, and ATP).^106,107 Therefore, a high degree of purity may be required for candidate mitochondria used in transplantation.

Numerous clinical trials are evaluating mitochondria transplantation for various conditions, such as extracorporeal membrane oxygenation complications (NCT02851758), oocyte quality and developmental potential (NCT03639506), microcatheter infusion of autologous mitochondria from muscle tissues into ischemic brain area, and the safety and efficacy of autologous mitochondria transplantation for cerebral ischemia (NCT04998357), and co-transplantation of autologous mitochondria and MSC-derived exosomes for surgical candidates of coronary artery bypass grafting (NCT05669144). Additionally, allogeneic mitochondria transplantation is being investigated for mitochondrial diseases such as Pearson’s marrow/pancreas syndrome (NCT03384420), while mitochondria from hUC-MSCs intravenously injected into patients is being investigated for dermatomyositis or polymyositis (NCT04976140). These pilot trials may contribute significantly to the future of mitochondria transplantation in clinical settings. Ongoing or completed clinical trials are summarized in Table 3, which includes details on the intervention methods, trial status, phase, and available reports.

Table 3.

Summary of ongoing or completed clinical trials.

Clinical Trial No or Report	Patient or condition	Phase	Intervention	Status or outcome
Emani et al. 104	Pediatric patients with myocardial ischemia-reperfusion injury	Phase 1	Patient’s muscle tissues for autologous transplantation	Cardiac function improved, and no observed severe adverse events.
NCT04998357	Cerebral ischemia	Not applicable	Microcatheter infusion of autologous mitochondria from muscle tissues into ischemic brain area	Recruiting (2021–2024)
NCT02851758	Myocardial ischemia-reperfusion injury in pediatric patients requiring extracorporeal membrane oxygenation	Not applicable	Direct injection of autogenic mitochondria into the ischemic myocardium.	Unknown status.
NCT03639506	Repetition failure	Not applicable	Autologous mitochondria from bone marrow mesenchymal stem cells into oocyte as well as intracytoplasmic sperm injection.	Completed (2018–2021)
NCT05669144	Myocardial infarction	Phase I/II	Intracoronary and intra-myocardial injection of mitochondria from mesenchymal stem cells from the human umbilical cord (US-MSCs) or autologous muscle.	Recruiting (2022–2024)
NCT03384420	Pearson’s marrow/pancreas syndrome	Phase I/II	Transplantation of autologous stem cell enriched with MNV-BLD (blood-derived mitochondria).	Completed (2019–2021)
NCT04976140	Refractory polymyositis or dermatomyositis	Phase I/IIa	Intravenously administration using mitochondria isolated from Allogeneic Umbilical Cord-derived Mesenchymal Stem Cells.	Completed (2021–2023)

Conclusion and future perspectives

The specific mechanisms of endogenous mitochondria transfer in various CNS diseases remain to be elucidated, and further studies are needed to develop therapeutic intervention methods aimed at modulating mitochondria transfer and function. A full understanding of these mechanisms will provide insights into novel therapeutic strategies for mitigating mitochondrial dysfunction, promoting neuronal survival, and improving functional outcomes. Although unresolved issues regarding mitochondria transfer and mitochondrial dynamics in pathological conditions, the therapeutic use of mitochondria transplantation may be a promising approach for the treatment of various CNS diseases. Furthermore, its efficacy has been shown not only in autologous mitochondria but also in allogeneic and exogenous mitochondria. Ongoing clinical trials may contribute to the clinical application of mitochondria transplantation. Additionally, technological advancements, such as the modification and modulation of isolated mitochondria, will optimize mitochondria transplantation. Overall, research on the application of mitochondria transplantation in CNS diseases is still in its infancy, and further preclinical and clinical studies are required to fully elucidate its therapeutic potential, safety, and efficacy. Challenges, such as optimizing mitochondrial isolation and delivery methods, addressing immunological considerations, and ensuring the long-term quality and function of transplanted or isolated mitochondria, must be addressed. Nonetheless, mitochondria transplantation has the potential to be a novel and promising approach to target mitochondrial dysfunction and neurodegeneration in various CNS diseases.