Therapeutic and diagnostic potential of extracellular vesicle (EV)-mediated intercellular transfer of mitochondria and mitochondrial components

Abstract

Extracellular vesicles (EVs) facilitate the transfer of biological materials between cells throughout the body. Mitochondria, membrane-bound organelles present in the cytoplasm of nearly all eukaryotic cells, are vital for energy production and cellular homeostasis. Recent studies highlight the critical role of the transport of diverse mitochondrial content, such as mitochondrial DNA (mt-DNA), mitochondrial RNA (mt-RNA), mitochondrial proteins (mt-Prots), and intact mitochondria by small EVs (<200 nm) and large EVs (>200 nm) to recipient cells, where these cargos contribute to cellular and mitochondrial homeostasis. The interplay between EVs and mitochondrial components has significant implications for health, metabolic regulation, and potential as biomarkers. Despite advancements, the mechanisms governing EV-mitochondria crosstalk and the regulatory effect of mitochondrial EVs remain poorly understood. This review explores the roles of EVs and their mitochondrial cargos in health and disease, examines potential mechanisms underlying their interactions, and emphasizes the therapeutic potential of EVs for neurological and systemic conditions associated with mitochondrial dysfunction.

Introduction

Mitochondria play primary roles in energy production and cell function by regulating homeostatic cellular metabolism, particularly for high-energy-demanding neurons.^1–3 Dysfunctional mitochondria are hallmarks of various diseases, including neurodegenerative diseases.^2,4,5 Mitochondrial dysfunction often precedes and contributes to disease progression, largely via altered mitochondrial membrane potential, generation of reactive oxygen species (ROS), and decreased production of adenosine triphosphate (ATP).^5,6 Mitochondria can be transferred between cells under physiological and pathophysiological conditions.^1,7,8

Mitochondrial transfer refers to the transfer of intact mitochondria, mitochondrial components such as DNA, RNA, proteins, and fragmented mitochondria.^9–12 Transfer can occur via direct mechanisms, including tunneling nanotubes and gap junctions, or via EV-mediated pathways.^13,14 Extracellular vesicles (EVs) released by nearly all living cells mediate intercellular communication by transferring their cargo to recipient cells.^7,15,16 Emerging studies show that EV-mediated intercellular mitochondrial transfer by delivery of encapsulated mitochondria and its components to recipient cells has a profound impact on cellular function and disease progression.^7,17

EV-mediated mitochondrial transfer in the nervous system is a highly cell-type-specific process, serving as an important cellular communication mechanism both within the nervous system and between the nervous system and peripheral organs.^1,18,19 This transfer undergoes significant alterations under pathological conditions, reflecting its critical role in disease progression and intercellular signaling.

This review highlights recent advances in EV-mediated mitochondrial transfer and its underlying mechanisms in disease progression and repair, with a primary focus on neurological disorders. The ability of EVs to deliver mitochondrial cargo across cellular barriers and over long distances underscores their pivotal role in maintaining cellular homeostasis, modulating metabolic function, and orchestrating intercellular communication. These processes are particularly critical in the nervous system, where precise and efficient signaling is essential for both normal function and recovery from injury.

Search strategy

The literature search was predominantly conducted through the PubMed database and Google Scholar, encompassing articles published up to October 2024. To enhance search comprehensiveness and accuracy, various text combinations were employed, including terms such as “extracellular vesicles,” “exosomes,” “microvesicles,” and “mitochondria.” The search strategy involved filtering articles pertaining to extracellular vesicles and mitochondria based on their abstracts and results. No restrictions were placed on publication time or language. In this review, we included only studies that adhere to the latest EV research guidelines, the minimal information for studies of extracellular vesicles (MISEV) 2018 and 2023^17,20 to ensure accuracy and avoid the potential impact of ambiguous research.

Mitochondria and mitochondrial transfer

Mitochondria are double-membraned multifunctional organelles that are essential for cellular metabolism across nearly all eukaryotic cells. They are thought to have originated through an ancient endosymbiotic event in which proteobacteria were incorporated into host cells, giving rise to eukaryotic life forms ranging from yeast to plants to animals.^3,7,21 While mitochondria are typically inherited vertically during cell division, cells can also export mitochondria to other cells, facilitating intercellular mitochondrial transfer.^1,22 This transfer occurs among cells via contact-dependent mechanisms, such as tunneling nanotubes, connexin 43 (Cx43)-mediated gap junction channels, and contact-independent mechanisms, including free mitochondria transfer and EV-mediated transfer.^7,13,14 Consequently, mitochondrial transfer can be broadly categorized into direct and EV-mediated mechanisms (Figure 1).

Figure 1.

Mechanisms of mitochondrial transfer. (a) Tunnel-nanotubes transfer mitochondria mediated by microtubules and Myosin/Miro1 complex. (b) Mitochondrial Gap-junction-channel transfer mediated by Connexin 43. (c) Free mitochondria are released directly through the secretory autophagy pathway or an unclarified mechanism. (d) Mitochondrial fission promoted by PGC-1α signaling has been suggested to mediate extracellular mitochondrial secretion. (e) MDVs’ generation is mediated by the PINK1/Parkin, also regulated by Snx9 and Rab9. MDVs are delivered to endosomes, leading to the formation of MDV-containing MVBs (f). (g) Various pathological stimuli, such as hypoxia, induce mitochondria-containing apoptotic bodies releasing. (h) Kif5b and Myosin19 induce migrasome-mediated mitochondria transfer. (i) ARRDC1 mediates the transport of mitochondria and mitochondrial contents into microvesicles. (j–l) Mitochondria secreted extracellularly can be taken up by recipient cells through membrane fusion (j) or endocytosis (k). Extracellular free mitochondria may also interact with the recipient cell surface for uptake (l). Then these internalized mitochondria can elicit biological effects on recipient cells. (Created in BioRender.).

Mitochondrial transfer is closely linked to the metabolic state of donor cells, particularly their dependence on oxidative phosphorylation (OXPHOS) for energy production.^1,7,8 Cells that rely less on mitochondrial respiration, such as glycolytic tumor cells and stem cells, are more likely to engage in mitochondrial transfer.^23,24 This suggests that altered metabolic states in donor cells, often observed under pathological conditions, influence mitochondrial transfer patterns.

The concept of direct mitochondrial transfer was first demonstrated in 2006 when ρ0 cells, which lack mitochondrial DNA (mt-DNA), regained aerobic respiration by acquiring mitochondria from neighboring cells in co-culture. ²⁵ Subsequent in vivo studies in 2012 revealed that bone marrow-derived stromal cells could transfer intact mitochondria to damaged epithelial cells, alleviating acute lung injury (ALI). ²⁶ These pioneering studies established direct mitochondrial transfer as a critical mechanism for intercellular metabolic regulation and cellular repair.

EVs and mitochondrial transfer

EVs are membrane-bound particles released by cells that carry a diverse array of bioactive molecules, including proteins, lipids, and nucleic acids.^27,28 EVs naturally facilitate intercellular communication by delivering cargo biomaterials to nearby cells and transporting these materials via the circulatory system to distant organs.^1,27,28 Their role in mitochondrial transfer has garnered significant attention due to their potential in regulating cellular processes, promoting repair, and influencing disease progression.

The first evidence of EV-mediated mitochondrial transfer, specifically involving mt-DNA and proteins, was observed in skeletal muscle cells. ²⁹ In 2013, EVs (1–8 μm) released from cultured astrocytes were shown to transfer mitochondria and ATP, thereby activating neighboring cells. ²² Later, in vivo evidence demonstrated that astrocytes transfer mitochondria via EVs (>300 nm) to stroke-damaged neurons, thereby supporting neuronal survival after ischemic injury. ¹ Additionally, mitochondrial-associated RNAs were first identified in EVs released by monocytes in 2019, where they regulated inflammatory responses in endothelial cells. ³⁰

Mechanisms of mitochondrial sorting into EVs

While EVs are often classified based on their biogenesis and function—exosomes, ectosomes, apoptotic bodies, primary cilia protrusions, migrasomes, etc.—this review follows the MISEV 2018 and 2023 guidelines,^17,20 broadly classifying EVs into two main categories: small EVs (<200 nm), typically exosomes, and large EVs (>200 nm), primarily microvesicles. ¹⁷

The biogenesis of EVs is closely tied to their mitochondrial cargo.^31,32 Small EVs, mainly released by multivesicular bodies (MVBs), are enriched in mitochondrial fragments and smaller mitochondrial molecules, such as proteins, DNA, and RNA. In contrast, large EVs, which primarily bud from the plasma membrane, are more likely to carry structurally intact and functional mitochondria (Figure 1). These differences in biogenesis impart distinct roles to small and large EVs, influencing their specific contributions to disease pathogenesis and cellular repair mechanisms.^17,18,31

Importantly, various pathological stimuli, including oxidative stress, inflammation, and cell damage, can influence the selective sorting of mitochondria into EVs^19,33,34 (Figure 1). In placental cells exposed to antiphospholipid antibodies, mitochondrial disruption suggests that fragmented mitochondria may be incorporated into EVs. ³⁵ Recent studies propose that mitochondrial-derived vesicles (MDVs) or mitovesicles facilitate the packaging of mitochondria and their components into EVs, regulated by sorting nexin 9 (SNX9) and optic atrophy 1 (OPA1). ³⁶ Instead of undergoing lysosomal degradation, MDVs are released extracellularly under the influence of parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), ³⁷ ultimately forming exosomes that selectively carry mitochondrial content or small mitochondria.^38,39 Additionally, mitocytosis involves the transport of mitochondria to the plasma membrane via kinesin family member 5B (Kif5b), where Myosin19 anchors them to cortical actin before their encapsulation into migrasomes. ⁴⁰ These findings suggest a complex interplay between EV-mediated mitochondrial transfer and disease pathogenesis.

Targeted delivery of mitochondrial EVs

EVs carry surface markers from their cell-of-origin, enabling targeted delivery to recipient cells.^41,42 Macrophages (Mφs) transfer mitochondria via EVs to dorsal root ganglia (DRG) neurons, mitigating inflammatory pain responses. This process critically depends on the interaction between the CD200 receptor (CD200R) on Mφ-derived EVs and the intestinal secretory cell protein 1 (iSEC1) ligand on neurons, ⁴³ highlighting the importance of targeted delivery in EV-mediated mitochondrial transfer. Similarly, astrocyte-derived mitochondrial EVs have been shown to target hypoxic neurons following ischemic stroke through a calcium-dependent mechanism mediated by the CD38/circulating ADP ribose (cADPR) signaling pathway, supporting neuronal survival and mitochondrial function. ¹ Adipocyte-derived mitochondrial EVs circulate and target cardiomyocytes after ischemic injury organ-specifically, contributing to cardioprotection. ⁴⁴ These targeting properties underscore the therapeutic potential of EV-mediated mitochondrial transfer in various disease contexts.

Scope of this review

With their unique structural and functional advantages, EVs enable enhanced stability, long-distance transport, targeted delivery, and amplified activity in recipient cells. To provide an overview of the advancements of EV-based mitochondrial transfer, this review focuses on various mitochondrial cargos and their biological implications. We begin with the EV-mediated transfer of intact mitochondria, emphasizing its therapeutic potential (Table 1). Then, we review the transfer of mitochondrial RNA and proteins, highlighting their localized regulatory effects in recipient cells, such as in axonal regulation (Table 2). Finally, we discuss the EV-mediated transfer of mt-DNA, emphasizing its utility as a biomarker for disease progression (Table 2). For each mitochondrial cargo type, we explore its specific packaging mechanisms, functional significance in recipient cells, and implications in both disease and health contexts.

Table 1.

Mitochondria in EVs.

Key findings	EVs type (Size)	Pathogenic factors	Donor cells/organs	Recipient cells/organs	References
Promote metabolism/OXPHOS capacity	EVs	ALI	BMDSCs	Alveolar epithelial cells	26
	EVs (100–1000 nm)	Wound healing	MSCs	Articular chondrocytes	45
	EVs (100–300 nm)	Renal artery stenosis	IRSTCs	Tubular epithelial cells	119
	EVs	COPD	MSCs	ASMCs	120
	Microvesicles	Sepsis	MSCs	Intestinal epithelial cells	121
	EVs (100–1000 nm)	ARDS	MSCs	Airway epithelial and endothelial cell	122
	EVs (>220 nm)	Heart failure	Cardiomyocytes	Hypoxia-injured cardiomyocytes	123
	Extracellular mitochondrial particles (300–1100 nm)	Cerebral IS	Astrocytes	Neurons	1
	Mitochondria-containing vesicles	Inflammatory pain	M2-like Mφs	Sensory neurons	43
	MVs (>150 nm)	Cerebral IS	BECs	Recipient BECs and neurons	19,57
	EVs	GBM	TASCs	ρ0 GBM cells	13
	Microparticles	Breast Cancer	Platelet	Breast cancer cell	124
	EVs (∼442 nm)	IS	BECs	BECs	9,19
	EVs (100–1000 nm)	Mitochondrial diseases	Platelet	Neutrophils and monocytes	34
	MVs (>500 nm)	Acute pancreatitis	MSCs	Pancreatic acinar cells	125
	Large CD133+ EVs (2–6 μm)	Melanoma	Melanoma cells	TME	54
Mitochondrial quality‐control process	Axonal Protrusions/Evulsions	Neuronal dysfunction	RGC	Astrocytes	56
	Exophers (∼3.5 μm)	Heart biology	Cardiomyocytes	Cardiac-resident Mφs	126
	Large EVs (115–550 nm)	Danon disease	Heart	Mφs	50
Neutrophil immunoregulation	MVs (<1 μm)	ALI	Damaged alveolar epithelial cells	Neutrophils	127
T cell immunoregulation	EVs (>100 nm)	Asthma	MDRCs	T cell	39
	Small EVs	Asthma	MDRCs	CD4+ T cells	128
Macrophage immunoregulation	EVs (100–1,000 nm)	ARDS	MSCs	MDMs	129
	EVs (>300 nm)	Skin wound	MSCs	Mφs	130
	Exosomes	ALI	Adipocytes	Alveolar Mφs	131
	EVs	Corneal epithelial defects	Human BMSCs	Mφs	132
Proinflammatory response	Microparticles (100–1,000 nm)	RA	Platelet	Neutrophils/leukocyte	52,53
	MVs (∼206.6 nm)	Inflammation	Activated Monocytes	Endothelial Cells	30
Trigger a burst of ROS	Small EV (30–500 nm)	Cardiac ischemia/reperfusion injury	Adipocytes	Cardiomyocytes	44
Lead to target cell failure and apoptosis	EVs (50–150 nm)	Acute pancreatitis	M1 macrophages	Pancreatic beta cells	133
Induce mitochondrial dysfunction	MVs (100–1000 nm)	Sepsis	Macrophage	Neutrophils	33
Rescue the excessive accumulation of ROS	Microvesicles (∼141 nm)	Intervertebral disc degeneration	MSCs	Nucleus pulposus cells	51
	Microvesicles (0.1–1 μm)	ALI	Alveolar epithelial cells	Neutrophils	127
Induce cancer chemoresistant	EVs (100–1000 nm)	TNBS	Chemoresistant TNBC cells	Sensitive TNBC cells	55
Induce cancer growth	EVs	GBM	TASC	GBM cells	13
Augmented mitochondrial biogenesis and function	Large EVs (>200 nm)	Anthracycline-induced cardiomyopathy	iPSC	Cardiomyocytes	134
Optimizes the thermogenic program	MVs (∼350 nm)	Obesity	Brown adipocytes	Mφs	135

ALI: acute lung injury; COPD: chronic obstructive pulmonary disease; ARDS: acute respiratory distress syndrome; IS: ischemic stroke; GBM: glioblastoma; RA: rheumatoid arthritis; TNBC: triple-negative breast cancer; BMDSCs: bone marrow-derived stromal cells; MSCs: mesenchymal stem cells; IRSTCs: intrinsic renal scattered tubular cells; ASMCs: airway smooth muscle cells; Mφs: macrophages; BECs: brain endothelial cells; TASCs: tumor-activated stromal cells; TME: tumor microenvironment; RGC: retinal ganglion cell; MDMs: monocyte–derived macrophages; MDRCs: myeloid-derived regulatory cells; TASC: tumor-activated stromal cells.

Table 2.

Mitochondrial contents in EVs.

Content	Key findings	EVs type (Size)	Pathogenic factors	Donor cells/ organs	Recipient cells/ organs	References
mt-DNA	Biomarker	Exosomes	Epithelial ovarian cancer	Plasma	——	93
		Exosomes (50–400 nm)	Renal cell carcinoma	Plasma	——	94
		Exosomes	Non-small cell lung cancer	Plasma	——	91
		Exosomes (30–130 nm)	GBM	GBM cells, neural stem cells	——	98
	Proinflammatory response	Exosomes	Autism	Serum	Microglia	106
		Microparticles (50–1000 nm)	Excessive alcohol use	Liver	Neutrophil	103,107
		EVs	BS	Monocytes	Circulation	108
		EVs (∼100 nm)	On-Pump Heart Surgery	Plasma	Circulation	136
	Enhance mitochondrial respiration/tumor progression	EVs (<200 nm)	Colon cancer	Colon cancer cell	Colonic epithelial cells	11
	Promote metabolism/OXPHOS capacity	Exosomes	Breast cancer	CAF	HTR cancer cells	101
	Activate endothelial cells	Exosomes	Preeclampsia	Placental explants	Endothelial cells	35
	Induce hepatocyte damage and fibrosis	Exosomes	Liver fibrosis	Damaged-hepatocytes	Mouse primary hepatocytes	105
	Attenuate mtDNA damage and inflammation	EVs (∼160 nm)	ARI	MSCs	HK-2 cells	68
mt-Prots	Biomarker	Exosomes (40–300 nm)	Melanoma	Melanoma tissue	——	79
		EVs (70–118 nm)	AD	Neurons	Plasma	73
		Small EVs	PD	Serum	——	80
		Small EVs (<100 nm)	PF&S	Serum	——	78
		Exosomes (30–100 nm)	Metabolic disorders	Brown adipose tissue (BAT)	Plasma	76
	Promote metabolism/OXPHOS capacity	Engineered exosomes	Neurodegeneration	Tom40 overexpressing cells	Brain tissue	74
		EVs	IS	BECs	Brain tissue	19
	Enhance mitochondrial respiration/ promote tumor survive	EVs (50–400 nm)	Prostate cancer	PC3 cells	PC3 cells	83
	Mitochondrial RNA-binding protein	Small EVs (100–200 nm)	IS	Neurons	Neurons	64
		Exosomes (30–150 nm)	——	——	——	65
	Induce mitochondrial dysfunction	Mitovesicles	DS	Brain tissue	Hippocampal slices	12
	T cell immunoregulation	EVs (∼154.5 nm)	Autoimmune hepatitis	MSCs	CD4+ T-cells	82
mt-RNA	Biomarker	EVs (<600 nm)	AD	Plasma	——	10
		Microvesicles (30–200 nm)	CAD	Monocytes	Plasma	137
	Rescue the excessive accumulation of ROS	EVs	ARI	MSCs	Kidney	62
	Proinflammatory response	Exosomes	Alcoholic liver disease	Hepatocytes	Kupffer cells	66
	Promote OXPHOS capacity/promote the osteogenesis	Exosomes	Bone defect	MSCs	DPSCs	61

BS: Behçet’s syndrome; ARI: acute renal injury; CAD: coronary artery disease; AD: Alzheimer’s disease; PD: Parkinson’s disease; PF&S: physical frailty and sarcopenia; DS: Down syndrome; CAF: cancer-associated fibroblast; DPSCs: dental pulp stem cells.

EV-mediated transfer of intact mitochondria

Functional mitochondria are encapsulated in EVs derived from cells under physiological and pathophysiological conditions.^1,18,22,31 The transfer of intact mitochondria via EVs is a direct form of intercellular regulation of metabolic responses with emerging and important implications in disease pathology.^1,18 Notably, functional mitochondria can also be encapsulated in EVs released by healthy cells,^45–47 offering promising therapeutic potential for repairing mitochondrial dysfunction in recipient cells (Table 1).

A wide range of cells can produce mitochondria-containing EVs, though production levels and functionality vary with cell type, metabolic state, and external stimuli. ⁷ Cells with surplus mitochondria or low mitochondrial demands, such as brain endothelial cells (BECs), are more likely to release mitochondrial-containing EVs. For example, under cerebral ischemic conditions, BECs with high mitochondrial loads release mitochondria-enriched EVs to support recipient cells, such as neurons, thereby boosting ATP levels and improving neurological outcomes. ¹⁹ Endogenous factors like resveratrol-activated peroxisome proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α) can increase mitochondrial biogenesis in BECs, promoting the secretion of mitochondria-enriched EVs and supporting recipient cell metabolism under stress conditions. ⁴⁸

Environmental factors and pathological changes also influence mitochondrial EV production. Mφs under lipopolysaccharide (LPS)-induced necroptosis or pyroptosis can release large EVs containing mitochondria.^33,49 Molecules mediating intracellular transport, such as the actions of ras-related protein Rab7, Rab22a, and Kif5b, aid in transporting mitochondria to the plasma membrane for EV packaging.^50,51 These findings support the strong therapeutic potential of EV-mediated mitochondrial transfer, especially under conditions involving energy deficits and mitochondrial dysfunction.

Pathological roles of EV-mediated mitochondrial transfer in disease

EV-mediated mitochondrial transfer can contribute to disease progression. For example, activated platelets release EVs containing mitochondria, which can lead to inflammatory responses, contributing to neuroinflammation in Alzheimer’s disease (AD) and Parkinson’s disease (PD).^52,53 In cancer, mitochondrial transfer via EVs provides energetic support to tumor cells, meeting the demands of rapid cell division and enabling tumor growth. Glioblastoma multiforme (GBM) cells receive mitochondria-containing EVs from astrocytes, enhancing their tumorigenicity and resistance to therapies such as radiotherapy and chemotherapy. ¹³ In breast cancer, EV-mediated mitochondrial transfer from stromal cells contributes to chemoresistance and metastasis.^54,55 Similarly, stress-induced EVs from adipocytes carry oxidatively damaged but respiration-competent mitochondria to cardiomyocytes, affecting ROS production and heart tissue resilience to oxidative stress. ⁴⁴

Endogenous repair and protective mechanisms via EV-mediated mitochondrial transfer

EV-mediated mitochondrial transfer maintains tissue homeostasis and promotes cell survival and recovery. For example, retinal ganglion cells (RGCs) package damaged axonal mitochondria into large EVs for transfer to adjacent astrocytes, facilitating the clearance of dysfunctional organelles. ⁵⁶ This mechanism also plays a key role in tissue repair and protection. Following stroke, astrocytes release large EVs containing functional mitochondria that can be taken up by hypoxic neurons, supporting neuronal mitochondrial function and survival. ¹ BEC-derived EVs containing mitochondrial proteins, such as ATP synthase F1 subunit alpha (ATP5A), contribute to ATP production in recipient cells, enhancing metabolic recovery in ischemic models. ¹⁹ In mouse models of ischemic stroke, intravenous administration of mitochondria-containing EVs from BECs significantly reduce infarct volume and improve neurological function, demonstrating therapeutic promise.^9,57

Neural stem cells release EVs with intact mitochondria that modulate recipient immune cells, such as Mφs, by restoring OXPHOS and maintaining mitochondrial function, which helps regulate inflammatory responses.^24,39 In pain modulation, Mφ-derived EVs transfer mitochondria to DRG neurons, aiding in the resolution of inflammation and reducing inflammatory pain transmission to the brain. ⁴³

In neurodegenerative disease models, mesenchymal stem cell-derived EVs (MSC-EVs) carrying intact mitochondria can attenuate oxidative stress, inhibit apoptosis, and improve mitochondrial function.^45,58 In AD models, MSC-EVs enhance cellular metabolism and reduce cell death by delivering functional mitochondria to recipient cells. ⁵⁸ These findings underscore the therapeutic potential of EV-mediated mitochondrial transfer for neuroprotection and metabolic support.

Given the role of EV-mediated mitochondrial transfer in energy homeostasis, encapsulating functional mitochondria in EVs has become an area of interest for therapeutic applications in neurological and systemic diseases.^1,48,51 Elucidating the mechanisms of EV-mediated mitochondrial transfer may, therefore, lead to the development of targeted therapies in conditions marked by mitochondrial dysfunction.

EV-mediated transfer of mitochondrial RNA (mt-RNA)

EVs surpass synthetic delivery systems in RNA transport efficiency and can encapsulate and deliver diverse types of mt-RNA, including messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA).^59,60 mt-RNA, sourced from both the mitochondrial and nuclear genomes, is abundant in small and large EVs,^34,61 enabling targeted delivery to recipient cells. ⁶² This EV-mediated mt-RNA transfer plays multiple roles in disease progression; it can act as a pathogenic factor, contributing to cellular dysfunction and inflammation, and support cellular repair mechanisms by stabilizing mitochondrial function and protecting cells from damage (Table 2).

During the transport of coding and noncoding RNAs, RNA-binding proteins (RBPs) assist in packaging RNA into small EVs and facilitate RNA transport via RNA-ribonucleoprotein (RNP) complexes.^63,64 They also contribute to mRNA localization at synapses, enabling neurons to respond swiftly to stimuli by promoting rapid protein synthesis.^63,64 Key RBPs, e.g., fused in sarcoma (FUS), selectively bind specific mitochondrial mRNAs, packaging them into small EVs and delivering them to recipient cells. For example, hypoxic neuron-derived EVs (HypEVs) containing FUS transport mitochondrial mRNA to dendrites in recipient neurons, where they promote translation on endosomes, compensating for the loss of mitochondrial proteins. In stroke models, the knockdown of FUS significantly reduces the neuroprotective effect of HypEVs. ⁶⁴ Another RBP, Y-box binding protein 1 (YBX1), selectively binds mitochondrial microRNA miR-223 through a specific sequence motif (UCAGU), promoting its sorting into small EVs. ⁶⁵

EV-mt-RNA transfer as a pathogenic factor in disease

mt-RNA transported via EVs can promote disease pathology by triggering inflammation, immune activation, and cellular dysfunction in recipient cells. Under conditions of mitochondrial dysfunction, excessive ROS generation can damage mt-RNA, leading to the release of fragmented mt-RNAs through EVs.^66,67 These damaged mitochondrial RNAs act as damage-associated molecular patterns (DAMPs), activating inflammatory responses and contributing to autoimmune reactions. For example, EVs containing mitochondrial mRNAs such as mt-Nd5 and mt-Nd6 can activate toll-like receptor 3 (TLR3) in recipient cells, initiating self-RNA-mediated autoimmunity. ⁶⁶ Additionally, conditions such as metabolic syndrome can alter the miRNA cargo of EVs, disrupting the regulation of mitochondrial genes in recipient cells. This can impair the therapeutic efficacy of stem cell-derived EVs and exacerbate disease progression. ⁶⁷ In ischemic and injured cells, mt-RNA and other mitochondrial components, like electron transport chain (ETC) proteins, are released through EVs, inducing pro-inflammatory cytokines, such as interleukin (IL)-8 and type I interferons (IFNs). ³⁰ The robust inflammatory response triggered by mt-RNA-carrying EVs underscores the role of these vesicles as potential pathogenic factors that can intensify disease progression in inflammatory and autoimmune disorders. ⁶⁶

EV-mt-RNA transfer as an endogenous repair mechanism

Conversely, mt-RNA transferred by EVs can support cellular repair and enhance mitochondrial function in recipient cells, particularly under conditions of stress or injury.^61,62,64 EVs from healthy cells, including stem cells and neurons, transport mt-RNA to assist damaged cells in restoring mitochondrial function. For example, stem cell-derived small EVs deliver mitochondrial transcription factor A (TFAM) mRNA, which upregulates TFAM expression and regulates OXPHOS by enhancing glutamate dehydrogenase (GDH) activity, restoring mitochondrial function, and exerting anti-inflammatory effects. ⁶¹ Notably, the EV-mediated TFAM mRNA transfer to recipient cells is unaffected by transcriptional inhibition, highlighting the robust mechanism of EV-mediated mRNA delivery. ⁶⁸ Neuronal small EVs generated under hypoxic conditions deliver mitochondrial mRNA (e.g., Cox7a1, mt-Nd6, mt-Co1) to neurons, promoting cell survival by preserving neuronal morphology and function after ischemic injury. ⁶⁴ Similarly, in ischemia-reperfusion-induced acute kidney injury, MSC-EVs enriched with superoxide dismutase 2 (SOD2) mRNA reduce oxidative stress in recipient cells, protecting against ROS damage. ⁶²

In neurodegenerative diseases, mt-RNA delivered to axonal termini supports mitochondrial function, essential for axon elongation, regeneration, and synapse stability. ⁶⁹ We and others have demonstrated that EVs can deliver cargo miRNA to distal axons and facilitate localized protein regulation.^15,16,70,71 The localized RNA regulatory pathogenesis plays a pivotal role in several neurodegenerative diseases, including amyotrophic lateral sclerosis/frontotemporal dementia, spinal muscular atrophy, AD, and fragile X syndrome (FXS). ⁷² This suggests that the mt-RNA EV transfer contributes to the rapid and steady supply of mitochondrial proteins to damaged or regenerating axons, potentially aiding recovery, and therefore, holds promise as an intrinsic mechanism for mitochondrial maintenance and neuroprotection in various disease states.

EV-mediated transfer of mitochondrial proteins (mt-prots)

The encapsulation and transfer of mitochondrial component proteins via EVs play significant roles in cellular communication, which can serve as biomarkers, metabolism regulators, and mt-RNA carriers^63,64,73,74 (Table 2). Certain pathways may contribute to the sorting of mt-Prots into EVs. The deletion of arrestin domain-containing protein 1 (ARRDC1) in wild-type mouse embryonic fibroblasts led to an enrichment of mt-Prots in EVs, suggesting that ARRDC1 plays a role in regulating the incorporation of mt-Prots into microvesicles. ⁷⁵

EV-mt-Prots transfer as a pathogenic factor in disease

The presence and altered levels of mitochondrial proteins in EVs have been associated with various diseases, particularly in neurodegenerative disorders, cancers, and metabolic diseases.^12,76,77 Pathological conditions affect mitochondrial protein sorting mechanisms, leading to the selective packaging of disease-related mitochondrial proteins into EVs. ⁷⁷

Mitochondrial proteins carried by small EVs are largely related to mitochondrial respiratory function, particularly in the nervous system.^9,73 Neuronal-derived EVs (NDEVs) from the plasma of AD patients contain mitochondrial proteins, including electron transport chain (ETC) components, ATP synthase, and superoxide dismutase 1 (SOD1). ⁷³ These proteins are present in differing levels between AD patients and controls, with reduced catalytic activity of ATP synthase and OXPHOS complex IV observed in NDEVs from AD patients. Similarly, Down Syndrome (DS) and AD models secrete EVs with elevated levels of monoamine oxidase B (MAO-B), a mitochondrial protein linked to impaired long-term potentiation (LTP) and cognitive decline, highlighting a common pathway in neurodegenerative disease progression. ¹² In addition to neurological disease, small EVs from patients with Sarcopenia show reduced levels of key mitochondrial proteins, specifically ATP5A complex V, ubiquinone oxidoreductase subunit S3 (NDUFS3) complex I, and succinate dehydrogenase complex iron sulfur subunit B (SDHB) complex II, compared to controls. ⁷⁸ This altered mitochondrial protein profile may contribute to reduced cellular energy and increased vulnerability to muscle degeneration in Sarcopenia. When brown adipose tissue (BAT) is activated, a mitochondrial tetrahydrofuran synthesis enzyme, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like (MTHFD1L), is enriched in EVs, suggesting its potential as a plasma biomarker for BAT activity and metabolic health. ⁷⁶

In addition to mitochondrial respiratory proteins, cancer-derived EVs have been shown to contain mitochondrial membrane proteins such as mitochondrially encoded cytochrome c oxidase II (MT-CO2) and cytochrome c oxidase subunit VIc (COX6c), which are associated with cancer-related mitochondrial functions and can be detected in blood, making them as potential biomarkers for cancer. ⁷⁹ Additionally, mitochondrial proteins like ATP5A are present in EVs from murine cancer-associated fibroblasts (CAFs) and in serum from adults with PD, suggesting that these proteins may serve as indicators of metabolic dysregulation in various pathological states. ⁸⁰

EV-mt-Prots transfer as an endogenous repair mechanism

EV-mediated transfer of mitochondrial proteins also plays protective and reparative roles in injured or metabolically stressed cells, particularly in the nervous system and immune systems, where mitochondrial protein transfer supports cellular recovery, metabolic reprogramming, and survival.^9,81–83

EVs from BECs contain mitochondrial structural and functional proteins, such as translocase of outer mitochondrial membrane 20 (TOMM20) and ATP5A, which help maintain ATP production in ischemic stroke models. ¹⁹ In addition, combining mitochondrial protein transfer with mitochondria-containing EV complexes can further increase intracellular ATP levels and enhance cell viability in damaged endothelial cells, supporting neuroprotection and brain repair in ischemic conditions. ⁵⁷ Treating BECs with heat shock protein 27 (HSP27), in conjunction with mitochondria-containing EVs, further elevates ATP levels and mitochondrial respiration in ischemic BECs, potentially enhancing recovery post-stroke. ⁹

EV-mediated mitochondrial protein transfer has also been shown to regulate immune cell function, notably in CD4+ T cells, by inducing metabolic reprogramming. ⁸² The transfer of mitochondrial antioxidant proteins such as peroxiredoxin 3 (PRDX3) and glutathione peroxidase 4 (GPX4) via EVs can increase the survival of recipient cells, as demonstrated in PC3 cell models, by reducing oxidative stress and protecting against apoptosis. ⁸³

EV-mediated transfer of mt-DNA

EVs have been shown to carry DNA fragments, with the first report in 1990 of DNA within EVs emerging from human epithelial prostate cells. ⁸⁴ More recently, DNA fragments representing entire genomes, including mutations, have been identified in EVs derived from cancer cell lines and plasma from patients with pancreatic cancer.^85,86 Mitochondria contain their own genome encoding 13 essential polypeptides involved in the ETC and ATP synthase, which are critical for OXPHOS. ⁸⁷ Genetic mutations and changes in mt-DNA copy number are associated with cell survival, aging, and developing severe diseases, such as cancer. ⁸⁸ The first evidence that EVs from astrocytes and GBM cells carry mt-DNA was reported in 2010. ⁸⁹ Subsequent studies have shown that small EVs secreted by C2C12 myoblasts contain both mt-DNA and mt-DNA-associated proteins. Notably, proteins such as insulin-like growth factor binding protein 5 (IGFBP-5) in micro-vesicles, associated with mt-DNA, enhance stability and facilitate long-range transfer of mt-DNA. ²⁹

EV-mt-DNA transfer as a pathogenic factor in disease

Due to the absence of histone protection and direct exposure to ROS, mt-DNA is more prone to mutations than nuclear DNA.^90,91 EVs carrying mutated or aberrant mt-DNA serve as potential biomarkers, as mt-DNA abnormalities often emerge in early disease stages and persist throughout disease progression.^11,27,92 Bronchoalveolar lavage (BAL) fluid from asthmatic patients contains significantly higher mt-DNA levels than healthy controls. ³⁹ In ovarian cancer patients, mt-DNA copy numbers in plasma EVs indicate cancer progression. ⁹³ Similarly, high mt-DNA content in small EVs has been suggested as a promising biomarker for renal cell carcinoma. ⁹⁴ In non-small cell lung cancer (NSCLC), EV-encapsulated mt-DNA strongly correlates with aggressive tumor features, such as larger tumor size, advanced tumor stage, lymph node involvement, and metastasis, offering improved sensitivity and specificity for diagnosis. ⁹¹

mt-DNA abnormalities and mutations have also been associated with neurodegenerative diseases and cancers, including GBM.^95,96 In GBM patients, higher mt-DNA copy numbers in plasma EVs correlate with disease state. ⁹⁷ In addition, aberrations in mt-DNA are detected in small EVs from GBM cells but not from neural stem cells. ⁹⁸ Since brain tissue is difficult to access, EV analysis provides a noninvasive approach to detect disease and cancer in the CNS. EVs also carry surface markers that reflect their cellular origins.^42,99 Combined detection of EV surface proteins and mt-DNA anomalies may enhance diagnostic accuracy, potentially transforming mt-DNA-containing EVs into powerful biomarkers for disease prediction and monitoring.

Moreover, EV-encapsulated mt-DNA contains the entire mitochondrial genome, allowing it to exert functional effects in recipient cells.^11,100 EVs derived from CAFs transfer the complete mitochondrial genome to breast cancer stem-like cells, enhancing their metabolism and promoting tumor growth through OXPHOS upregulation. ¹⁰¹ Similarly, small EVs carrying the full mitochondrial genome facilitated metabolic crosstalk between colorectal cancer (CRC) cells and adjacent stromal cells (ASCs), impacting cellular respiration and ROS production to support tumor progression. ¹¹

Interestingly, research has shown that mt-DNA levels of plasma EVs decrease with age, potentially affecting mitochondrial energetics in an age-dependent manner. ¹⁰² Although there is no current evidence of EV-mediated mt-DNA transfer altering the metabolic state in CNS-related diseases, these findings suggest that mt-DNA transfer could be therapeutically relevant for CNS disorders such as stroke.

EV-mt-DNA transfer and the inflammatory response

In addition to its biomarker role, mt-DNA functions as DAMPs when released from mitochondria, initiating inflammatory signaling by activating pathways such as TLR9, cyclic GMP-AMP synthase- stimulator of interferon genes (cGAS-STING), and ataxia-telangiectasia mutated- checkpoint kinase 2 (ATM-Chk2). ^{35 , 103 , 104} EV-encapsulated mt-DNA may trigger a stronger immune response than free mt-DNA due to its protection within the EV lipid bilayer. For example, ethanol-induced endoplasmic reticulum (ER) stress prompts hepatocytes to release mt-DNA-rich EVs, promoting neutrophil infiltration and liver damage. ¹⁰³ Inhibition of mt-DNA transfer via tetramethylpyrazine (TMP) has shown promise in reducing EV-mediated inflammation in liver injury. ¹⁰⁵ Similarly, breast cancer cells package mt-DNA into EVs through a PINK1-dependent mechanism, triggering invasive responses by activating TLR9 in recipient cells. ³⁷

In the CNS, mt-DNA-containing small EVs from children with autism spectrum disorder have been shown to stimulate microglial IL-1β secretion, contributing to neuroinflammation. ¹⁰⁶ Additionally, long-term alcohol exposure induces the release of mt-DNA-enriched EVs, promoting liver inflammation via apoptosis signal-regulating kinase 1 (ASK1) and p38 signaling. ¹⁰⁷ Caspase-1 activation in hepatocytes can also promote mt-DNA packaging into EVs, resulting in sterile inflammation and exacerbating inflammatory diseases. ¹⁰⁸

Although there is no direct evidence yet, increased secretion of mt-DNA-containing EVs following traumatic brain injury (TBI) or stroke could potentially lead to sterile neuroinflammation. Suppressing mt-DNA release or blocking its downstream effectors may thus provide a therapeutic approach for mitigating inflammation following trauma or surgery.

Discussion and prospective in neurological disorders

Emerging studies on EV-mediated mitochondria and their component transfer have revealed new aspects of intercellular communication, with intact mitochondria and mitochondrial components (mt-RNA, mt-Prot, and mt-DNA) playing distinct yet complementary roles in cellular maintenance and disease progression. As EV-based research advances, the therapeutic potential of mitochondrial transfer holds promise to combat various diseases, particularly within the CNS where localized, and for cell-specific regulation and communication between the CNS and peripheral organs ^109–111 (Figure 2).

Figure 2.

The EV-mediated cell-type-specific mitochondrial transfer and its potential role in neurological disease. EVs, carrying cell-specific surface markers, transfer mitochondrial components such as DNA, RNA, proteins, and intact mitochondria between neurons, astrocytes, and microglia (color indicated), supporting axonal maintenance, localized metabolic regulation in EV-targeting cells, and brain remodeling. These EVs can also cross the BBB and deliver their mitochondrial cargo to distant organs, contributing to the pathological biomarker and systemic metabolic homeostasis factor. (Created in BioRender.).

The transfer of functional mitochondria via EVs could support neural regeneration, enhance cellular resilience, and stabilize metabolic processes across neural networks and distal organs by promoting ATP production, balancing ROS, and maintaining overall metabolic function.^1,18 Given the scalability and ability to cross the blood-brain barrier (BBB),^24,48,112 EV-mediated mitochondrial transfer provides an opportunity to develop therapies for neurological dysfunction.

The transfer of mt-RNA and mt-Prots by EVs facilitates rapid, localized regulation, particularly at axonal terminals, where such interventions could preserve or restore neural integrity (Figure 2). This feature enables treating neurological disorders with compromised axonal health, such as neurodegenerative diseases and CNS injuries. Mitochondria-specific miRNAs have been identified as key regulators of mitochondrial function, ¹¹³ and their EV-mediated transfer is suggested by miRNA motifs that facilitate sorting into EVs. ¹¹⁴ Furthermore, during the transport of coding and noncoding mt-RNA, RBPs in mitochondria play a crucial role in packaging mt-RNA into EVs and aiding mt-RNA transport via RNP complexes.^64,65 EV-encapsulated mt-DNA offers both a biomarker for disease progression and a means to modulate mitochondrial function in recipient cells. Future research is warranted to investigate the therapeutic potential of controlled mt-DNA delivery to manage cellular energetics and mitigate metabolic dysregulation. Additionally, further studies could clarify the diagnostic potential of mt-DNA in EVs, providing non-invasive, cell-type-specific biomarkers for neurodegenerative conditions and other diseases.

Small and large EVs transport distinct mitochondrial components, but their interactions in mitochondrial transfer remain unclear. For instance, treating BECs with HSP27 and mitochondria-containing large EVs significantly enhances ATP production in ischemic cells. ⁹ Similarly, ATP5A-enriched large EVs correlate with improved ATP levels in BECs. ⁵⁷ As small EVs primarily deliver RNAs and proteins, it is worth exploring whether their localized transfer primes cells for the uptake of intact mitochondria from large EVs, resulting in a synergistic metabolic response.

While pathways mediating mitochondrial encapsulation into EVs are well-documented,^31,32 the molecular mechanisms governing their regulation remain poorly understood and warrant further investigation. For example, oxidative stress, inflammation, and cellular damage can influence mitochondrial sorting into EVs,^33,34 but whether mitochondria enter different EV subtypes through shared or distinct mechanisms remains an open question.

Additionally, the intracellular destination of EV components within recipient cells plays a pivotal role in their functional impact.^15,32,115 For example, mitochondrial RNAs delivered via EVs may facilitate communication between the nucleus and mitochondria. ⁵⁰ EV-mediated transfer of mitochondrial components can increase cellular bioenergetics, survival, mt-DNA copy number, and modulate anti-inflammatory responses of recipient cells.^18,32,97 However, further research is needed to clarify how EVs carrying intact mitochondria or specific mitochondrial components are routed within recipient cells to affect recipient cell metabolic function. The ER, through its direct interaction with mitochondria via mitochondria-associated membranes (MAMs), may act as a critical hub for coordinating these regulatory functions.^116–118 Elucidating these pathways could advance the therapeutic potential of EV-mediated mitochondrial transfer.

The future of EV-based therapies, in-part, lies in improving delivery methods, increasing target specificity, and combining diverse mitochondrial components to treat multifactorial diseases. With ongoing research, EV-mediated mitochondrial transfer has the potential to become a powerful platform for diagnosing and treating conditions where mitochondrial health is critical for cellular resilience and function.