hUCMSC mitochondrial EVs confer neuroprotection after ischemia by Tom1l2-mediated mitochondrial fusion and Crls1–cardiolipin axis reprogramming
Ziheng Li, Xingjia Zhu, Weiquan Liao, Rui Jiang, Enze Sang, Jue Zhu, Gaojia Sun, Zhichao Lu, Chenxing Wang, Yi Jiang, Jian Chen, Peipei Gong, Qianqian Liu
TL;DR
This study shows that mitochondrial extracellular vesicles from stem cells can protect neurons after stroke by repairing mitochondria and reducing damage.
Contribution
The study identifies the Tom1l2-Crls1 axis as a novel mechanism for mitochondrial repair in stroke therapy.
Findings
hUCMSC Mito-EVs transfer functional mitochondria to neurons via Tom1l2-mediated fusion.
Mito-EVs upregulate CRLS1, preserving mitochondrial integrity and function.
This therapy reduces ROS and pyroptosis, promoting neuronal recovery after stroke.
Abstract
Mitochondrial dysfunction is a central driver of irreversible neuronal injury following ischemic stroke (IS); yet effective strategies to restore mitochondrial function and promote long-term neurological recovery remain limited. In this study, we demonstrate that mitochondrial extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSC Mito-EVs) serve as a novel biotherapeutic vehicle capable of delivering functional mitochondria to damaged neurons. This process involves Target of Myb1-like 2 membrane trafficking protein (Tom1l2)-dependent membrane fusion between hUCMSC Mito-EVs and neuronal mitochondria, leading to the restoration of mitochondrial membrane potential and mitochondrial function. Mechanistically, Mito-EVs–mediated mitochondrial transfer upregulates cardiolipin synthase 1 (CRLS1), which preserves the inner mitochondrial membrane integrity and…
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Taxonomy
TopicsMitochondrial Function and Pathology · GDF15 and Related Biomarkers · Adipose Tissue and Metabolism
Introduction
1
Ischemic stroke (IS) is an acute cerebrovascular disease caused by acute occlusion of cerebral blood vessels, resulting in ischemic and hypoxic damage to brain tissue [1]. Although intravenous thrombolysis combined with mechanical thrombectomy constitutes the current standard clinical treatment, the secondary effects of ischemia–reperfusion injury (IRI) can significantly aggravate neurological deficits and result in poor long-term prognosis [2]. Therefore, antagonizing IRI and rescuing damaged neurons have become core scientific priorities for improving the prognosis of patients with IS [3].
As the core hub of cellular energy metabolism and signal transduction, mitochondrial dysfunction serves as a key pathological link in neuronal injury and apoptosis following IS [4]. However, current clinical interventions are difficult to precisely repair damaged mitochondria and cannot effectively halt the cascade of cell death [5]. The body inherently possesses an endogenous mitochondrial transfer mechanism, which mainly includes intercellular tunnel nanotubes (TNTs), mitochondrial extracellular vesicles (Mito-EVs), and transport mediated by free mitochondria [[6], [7], [8]]. The mitochondrial transplantation technology developed based on this mechanism provides a targeted therapeutic strategy for reconstructing the mitochondrial function in damaged cells [9,10]. A core advantage of Mito-EVs lies in their natural membrane structure, which can mimic cell membrane characteristics to maintain content homeostasis and ensure the structural integrity and activity of the functional mitochondrial fragments they carry [11]. Meanwhile, this membrane structure can also mediate crossing of biological barriers and participate in damage repair by regulating intercellular communication, demonstrating unique value in targeted therapy [12,13].
Target of Myb1-like 2 membrane trafficking protein (Tom1l2), a key regulatory molecule for vesicle sorting and transport, has an unclarified specific role and mechanism in Mito-EVs-mediated neuroprotection [14]. In particular, the molecular interaction network during the targeted fusion of Mito-EVs with damaged mitochondria remains to be analyzed. Meanwhile, the stability of the mitochondrial inner membrane is crucial for maintaining mitochondrial function [15]. As a characteristic phospholipid of the mitochondrial inner membrane, cardiolipin directly provides structural support for the assembly of respiratory chain complexes and regulates mitochondrial dynamics [16]. The expression level of cardiolipin synthase 1 (Crls1), a key enzyme in cardiolipin biosynthesis, plays a critical role in regulating cardiolipin production and maintaining its homeostasis. [17]. Whether mitochondrial function can be improved by regulating cardiolipin, and the overall therapeutic efficacy of this regulatory network in cerebral infarction, remain to be further explored.
This study confirms that Mito-EVs derived from human umbilical cord mesenchymal stem cells (hUCMSC) contain fully functional mitochondrial fragments [18]. The unique core regulatory molecule Tom1l2 is key to mediating mitochondrial fusion, as it can promote the fusion of vesicles with the target membrane of damaged mitochondria, laying a structural foundation for the repair of mitochondrial function. Following mitochondrial fusion, the vesicles upregulate the transcriptional level of Crls1 in damaged neurons to promote the synthesis of cardiolipin in the mitochondrial inner membrane [19]. Cardiolipin can provide structural support for the assembly of respiratory chain complexes and maintain inner membrane stability [20], directly facilitating mitochondrial structural repair and recovery of oxidative phosphorylation function [21]. This enables the reactivation of the damaged tricarboxylic acid (TCA) cycle and the restoration of cellular energy supply [22]. The dual recovery of energy metabolism and membrane function further inhibits the abnormal accumulation of reactive oxygen species and activation of pyroptosis pathways [23]. Ultimately, this effectively reduces neuronal death and achieves the repair of damaged cellular functions [24].
Materials and methods
2
Human cerebrospinal fluid (CSF) samples
2.1
All procedures involving human participants adhered to the Declaration of Helsinki and were approved by the Ethics Committee of the Affiliated Hospital of Nantong University (Approval No. 2024-L180). The CSF samples were collected from two groups: For the IS group, the inclusion criteria were: 1) acute IS confirmed by CT, CTP, or MRI; 2) CSF collection within 72 h after symptom onset; 3) age between 18 and 80 years; and 4) absence of severe systemic comorbidities (e.g., end-stage renal disease or advanced malignancy). The exclusion criteria included: 1) hemorrhagic stroke; 2) prior history of major neurological disorders; 3) contraindications to the sampling procedure; and 4) systemic inflammatory or infectious diseases that could potentially affect CNS biomarkers. The control group consisted of individuals aged 18–80 years without a history of stroke or major neurological disorders who underwent trigeminal or facial nerve decompression surgery. Individuals with systemic or neurological conditions that might influence CNS physiology were excluded.
All CSF samples were collected intraoperatively from the subarachnoid space immediately after dural opening. Patient characteristics are summarized in Table S1.
Animals
2.2
All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The experimental procedures were approved by the Nantong University Institutional Animal Care and Use Committee (Approval No. S20250522-003). Healthy Sprague–Dawley (SD) rats (220–250 g) were purchased from the Animal Center of Nantong University School of Medicine (Nantong, China). The rats were housed in an air-conditioned animal facility (22 °C to 25 °C) under a 12-h light/dark cycle with free access to food and water. The experimental animals were randomly assigned to treatment groups, and all experiments were performed in accordance with standard laboratory operating procedures for randomization and blinding. Euthanasia of the rats was performed using an overdose of isoflurane followed by cervical dislocation.
Cell culture
2.3
PC12 cells were obtained from Shangen Bio (SNL-124), and hUCMSCs were obtained from Shangen Bio (SNP–H198). DMEM/F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) was used for cell culture. All cells were seeded in 25 cm^2^ culture flasks (Corning) for proliferation and cultured in a constant-temperature and constant-humidity incubator with the conditions set at 37 °C, 95% air, and 5% CO_2_.
Middle cerebral artery occlusion (MCAO) model
2.4
SPF-grade SD rats (220–250 g) were selected for the experiment. Prior to surgery, the rats were fasted for 12 h with free access to water, followed by 4 h of water deprivation, and anesthetized using isoflurane inhalation. Following anesthesia, the rats were placed in a supine position and fixed. The cervical skin was disinfected, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery were exposed layer by layer. After occluding the proximal end of the CCA and distal end of the ICA, a 4-0 nylon monofilament with a silicone rubber-coated tip (Beijing Xinong Technology Co, 2634-A4) was inserted via the ECA stump into the ICA. The cerebral blood flow (CBF) was monitored in real time using a laser Doppler flowmeter (Perimed, Sweden) to ensure that occlusion resulted in a significant reduction in perfusion [25]. Animals with subarachnoid hemorrhage or unsuccessful occlusion were excluded. After 2 h of continuous occlusion, the nylon monofilament was gently withdrawn to restore perfusion. Postoperatively, the rats were monitored closely, and measures were taken to ensure their warmth and adequate food and water supply.
Oxygen-glucose deprivation/reperfusion (OGD/R) model
2.5
PC12 cells in the logarithmic growth phase were harvested and routinely cultured in DMEM/F12 (10% FBS). After washing the cells three times with phosphate-buffered saline (PBS), the medium was replaced with glucose-free DMEM, and the cells were subjected to hypoxia treatment at 37 °C under 1% O_2_, 5% CO_2_, and 94% N_2_ for 3 h. Subsequently, the medium was restored to DMEM/F12 (10% FBS), and the cells were reoxygenated in a conventional incubator (37 °C, 5% CO_2_) for 24 h [26]. This protocol established a reproducible OGD/R injury model.
Cellular fluorescence virus transfection
2.6
PC12 cells and hUCMSCs were seeded in 24-well plates at 3 × 10^4^ cells/well. Upon reaching 30% confluence, the cells were rinsed with PBS and prepared for viral transfection. An appropriate volume of viral suspension (based on prior experiments and respective viral titers, the optimal multiplicity of infection (MOI) for both cell types was determined to be 5) and 0.5 μL polybrene-plus sensitizing reagent were added to 500 μL antibiotic-free DMEM/F12 (10% FBS), which was then added to each well for transfection [27].
In the PC12 cell system, Mito-ZsGreen (Nanjing Kairis Biotech) and Mito-DsRed-WPRE (Heyuan Biotech, Cat. No. DH-1117) were employed for mitochondrial labeling and tracing, while Mito-SypHer3s (Heyuan Biotech, Cat. No. DH-1116) was used to assess the mitochondrial matrix pH. These three viral constructs were labeled separately. A co-transfection group was established using Mito-DsRed-WPRE and pcSLenti-CMV-Gsdmd-N-linker-EGFP-3 × FLAG-PGK (Heyuan Bio, Cat. No. HYKY-250730003-DLV) for tracing GSDMD-MT protein. In the hUCMSCs system, Mito-SypHer3s (Heyuan Bio, Cat. No. DH-1116) and Mito-DsRed-WPRE (Heyuan Bio, Cat. No. DH-1117) were added. These two markers were used to label the mitochondrial vesicles and monitor mitochondrial pH dynamics. The plates were incubated at 37 °C, 5% CO_2_ for 12 h. The viral supernatant was aspirated and replaced with fresh DMEM/F12 (10% FBS) containing puromycin for screening of successfully transfected cells; the optimal screening concentration of puromycin was 2 μg/mL for PC12 cells and 1 μg/mL for hUCMSCs. The cells were stably passaged three times. Transfection efficiency and fluorescent protein expression were verified using Zeiss laser scanning confocal microscopy (LSCM) prior to subsequent experiments.
Isolation, characterization of hUCMSC Mito-EVs, and co-culture with PC12 cells
2.7
hUCMSCs were cultured in a medium supplemented with 10% exosome-free FBS to produce hUCMSC Mito-EVs. The conditioned medium was collected and processed using a three-step centrifugation method. First, centrifugation was performed at 500×g for 10 min to remove cells. This was followed by centrifugation at 2,000×g for 10 min to eliminate cellular debris. Finally, centrifugation was conducted at 20,000×g for 90 min to precipitate hUCMSC Mito-EVs. The protein content was quantified using the bicinchoninic acid (BCA) assay, the mitochondrial components were verified by Western blot analysis, and their morphology and composition were characterized using transmission electron microscopy (TEM).
To generate mitochondria-dysfunctional Mito-EVs, isolated Mito-EVs were normalized by protein content (BCA assay). Mito-EVs (50 μg protein) were resuspended in 1.0 mL pre-warmed culture medium and incubated with 50 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at 37 °C for 90 min to dissipate the mitochondrial membrane potential (MMP). The loss of MMP was verified using JC-1 staining (quantified by the red/green fluorescence ratio) to assess the membrane potential collapse, and Mito-SypHer3s fluorescence labeling (measuring the F490/F405 ratio) to verify the disruption of mitochondrial matrix pH, confirming mitochondrial dysfunction. Subsequently, the suspension was centrifuged at 20,000×g for 90 min; the supernatant containing CCCP was discarded, and the pellet was resuspended in fresh medium to obtain CCCP-treated (mitochondria-dysfunctional) Mito-EVs.
For co-culture experiments, 1 × 10^5^ PC12 cells/well were seeded in 24-well plates and treated with Mito-EVs at 5 μg protein/well. After 24 h of co-culture with OGD/R-injured PC12 cells, the cells were collected for subsequent experiments.
Mitochondria isolation from cells and brain tissue
2.8
Mitochondria were isolated from treated PC12 cells and fresh rat brain tissue using the Cell Mitochondrial Isolation Kit (Beyotime, Cat. No. C3601) and Tissue Mitochondrial Isolation Kit (Beyotime, Cat. No. C3606), respectively, according to the manufacturers’ instructions: Adherent PC12 cells were washed with pre-cooled PBS, digested with a trypsin–EDTA solution (Beyotime, Cat. No. C0201), and centrifuged (200×g, RT, 10 min) to collect pellets, which were subsequently washed by centrifugation (600×g, 4 °C, 5 min). After pretreatment with mitochondrial isolation reagent (with PMSF) on ice for 15 min, the cells were homogenized and centrifuged at 600×g (4 °C, 10 min) to collect the supernatant (containing mitochondria). The supernatant was then centrifuged at 12,000×g (4 °C, 10 min) to obtain the mitochondrial pellet. The mitochondrial pellets were resuspended in storage buffer (Beyotime, Cat. No. C3609). For rat brain tissue, the minced samples (washed with pre-cooled PBS) were homogenized in a 10 × volume of isolation reagent on ice and subjected to the same centrifugation steps (600×g to collect supernatant and 12,000×g to collect pellet) to obtain mitochondria, which were resuspended in storage buffer.
MitoTracker staining
2.9
The culture medium of PC12 cells subjected to various interventions was aspirated. To evaluate the mitochondrial function and viability, the cells were co-stained with three fluorescent probes. MitoTracker Deep Red FM (MTDR; Beyotime, Cat. No. C1034) was used to monitor the mitochondrial membrane potential (MMP), while MitoTracker Green (MTG; Beyotime, Cat. No. C1048) was employed as an MMP-independent reference to normalize for mitochondrial mass. Propidium iodide (PI, Beyotime, Cat. No. C1008S) was included to assess the plasma membrane integrity. Preheated solutions of these dyes were added, gently mixed, and incubated at 37 °C in the dark for 15 min. Following incubation, the staining solution was aspirated, and the cells were washed three times with preheated PBS. Fresh culture medium was added to rewarm the cells for 5 min, after which they were observed using Zeiss LSCM [28].
Simultaneously, co-staining of hUCMSC Mito-EVs was performed using MTDR (Beyotime, Cat. No. C1034) and MTG (Beyotime, Cat. No. C1048). An appropriate volume of hUCMSC Mito-EVs suspension was taken, and preheated MTDR (Beyotime, Cat. No. C1034) and MTG (Beyotime, Cat. No. C1048) were added. This mixture was incubated at 37 °C in the dark for 15 min. After incubation, the suspension was washed three times with PBS and then placed on a slide. The images were observed and collected using Zeiss LSCM (LSM 990).
ATP measurement
2.10
The ATP levels of cells and tissues were detected using the ATP detection kit (Beyotime, Cat. No. S0026), and all procedures were performed at 4 °C. After discarding the culture medium for adherent cells, 200 μL of lysis buffer was added to each well of the 6-well plate, followed by pipetting for lysis. For brain tissue samples, 20 mg of tissue was homogenized with 200 μL of lysis buffer. All lysed samples were centrifuged at 12,000×g for 5 min at 4 °C, and the supernatant was collected for subsequent use. The ATP standard solution was serially diluted with lysis buffer and then mixed with the kit reagents at a ratio of 1:9 to prepare the working solution, which was maintained on ice. Overall, 100 μL of working solution was added to each well and incubated at room temperature for 3-5 min to eliminate background interference. Subsequently, 20 μL of sample or standard solution was added and mixed thoroughly. After standing for 2 s, the relative light unit (RLU) value was measured, and the ATP concentration was calculated based on the standard curve.
Pyruvate level detection
2.11
The CSF samples were immediately placed on ice and processed in accordance with the instructions of the Amplex Red Pyruvate Assay Kit (Beyotime, Cat. No. S0299S). Rat brain tissue was lysed with lysis buffer and homogenized at 4 °C (on ice). The homogenate was centrifuged at 12,000×g for 5 min at 4 °C, and the supernatant was retained on ice for subsequent analysis. The reaction working solution was prepared strictly according to the manufacturer's protocol. After incubating the samples with the working solution, the absorbance values were measured using a microplate reader.
L-lactic level detection
2.12
According to the L-lactic detection kit (WST-8 method) (Beyotime, Cat. No. S0208S), sample preparation was conducted at 4 °C. First, the PC12 cells were washed once with PBS, and any residual liquid was aspirated. For every 1 million cells, 100 μL of lysis buffer was added and mixed thoroughly. The mixture was incubated in an ice bath for 5–10 min, followed by centrifugation at 12,000×g for 5 min at 4 °C to collect the supernatant. For tissue samples, 100 μL of lysis buffer per 10 mg of tissue was added, followed by homogenization at low temperature. The samples were centrifuged under the same conditions, and the supernatants were collected. The specified buffer was dissolved and allowed to equilibrate to room temperature with thorough mixing. The WST-8 chromogenic working solution was prepared. After incubating the sample with the working solution, the absorbance values were measured using a microplate reader.
Cell viability assay
2.13
Cell viability was detected using the Cell Counting Kit-8 (Beyotime, Cat. No. C0037) following the manufacturer's instructions [29]. After incubation with CCK-8 reagent for 1 h at 37 °C, the absorbance value was measured at 450 nm.
qRT-PCR
2.14
Total RNA extraction was performed using Trizol reagent (Novozyme Biotechnology Co, China). Notably, 1 μg of the extracted total RNA was used as the template, and reverse transcription was conducted following the standard protocol provided with the cDNA Synthesis Kit (Novozyme Biotechnology Co, China) to synthesize the first strand of cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) amplification was performed on the ABI StepOnePlus Sequence Detection System using the synthesized cDNA as the template, along with gene-specific primers and SYBR Green reagent (Novozyme Biotechnology Co, China). The expression level of the target gene was normalized using GAPDH as the internal reference gene. The primers applied in the study are displayed in Table S6.
Western blot analysis
2.15
Cell samples and Mito-EVs were lysed using RIPA buffer (Beyotime, P0013) supplemented with protease and phosphatase inhibitors (Beyotime, P1045) followed by centrifugation at 13,000×g for 10 min at 4 °C. Protein concentrations were determined using a BCA kit (Beyotime, P0011). For cellular lysates, target protein expression was normalized to β-actin. For Mito-EVs samples, equal loading was performed based on the quantified total protein content to ensure reliable comparisons. The samples were mixed with loading buffer, denatured at 100 °C for 5 min, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skimmed milk and incubated with primary antibodies overnight at 4 °C. After incubation with secondary antibodies for 1 h at room temperature, signals were detected using the SuperPico ECL kit (Wozemay, 34578) and quantified with ImageJ software (NIH, USA). Complete, uncropped scans of representative blots are provided in the original files. The antibody details are listed in Table S2.
Seahorse mitochondrial stress test
2.16
PC12 cells subjected to different interventions were inoculated into Seahorse 96-well plates and cultured in DMEM/F12 (10% FBS). They were first subjected to hypoxia for 3 h, followed by reoxygenation for 24 h. The cells were divided into two groups: the experimental group was co-cultured with hUCMSC Mito-EVs, whereas the control group received no additional treatment. Both groups were further cultured for 24 h. Following incubation, the cells were washed twice with Seahorse detection medium. The culture medium was subsequently replaced with assay medium, and the cells were incubated for 1 h at 37 °C. The Agilent Seahorse XFe96 Extracellular Flux Analyzer was used to detect the oxygen consumption rate (OCR) under basal conditions and after the addition of a mixture containing 1.5 μM oligomycin, 1.5 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 0.5 μM rotenone, and 0.5 μM antimycin A [30].
JC-1 assay for MMP
2.17
In accordance with the JC-1 Assay Kit (Beyotime, Cat. No. C2006), PC12 cells subjected to various interventions were first washed with PBS to remove any residual culture medium. Subsequently, JC-1 staining solution was added to achieve a final concentration of 2 μM, and the cells were incubated for 20 min to ensure adequate dye incorporation. Following incubation, the cells were washed twice with the buffer provided in the kit to remove unbound dye. Finally, the samples were observed using Zeiss LSCM (LSM 990). MMP was quantified based on the red/green fluorescence intensity ratio, and the data were analyzed using ImageJ software (National Institutes of Health, USA).
Reactive oxygen species measurement
2.18
PC12 cells were seeded in 12-well plates and subjected to various experimental conditions after adherence. Each experimental group included wells designated for flow cytometry and confocal imaging. Following the interventions, all wells were rinsed twice with PBS in accordance with the manufacturer's protocol for the Reactive Oxygen Species Detection Kit (Beyotime, Cat. No. S0033S). Subsequently, 20 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was added, and the cells were incubated at 37 °C in a 5% CO_2_ environment for 20 min. After incubation, cells in the flow cytometry wells were detached with trypsin, resuspended in PBS, and analyzed for the fluorescence intensity of their oxidation product (2′,7′-dichlorofluorescein, DCF) using a BD FACS Calibur flow cytometer. In parallel, the adherent cells in the confocal imaging wells were directly visualized using Zeiss LSCM. The intracellular ROS levels in PC12 cells were quantitatively assessed using the two aforementioned detection methods.
Flow cytometry
2.19
Relevant evaluations were conducted using a BD FACS Calibur flow cytometer. First, the endocytosis of hUCMSC Mito-EVs by OGD/R-injured PC12 cells was evaluated. After purifying Mito-EVs from hUCMSCs labeled with Mito-DsRed-WPRE, the Mito-EVs were co-cultured with OGD/R-treated PC12 cells labeled with Mito-ZsGreen for 24 h. Subsequently, the endocytosis rate was calculated by determining the proportion of Mito-DsRed and Mito-ZsGreen double-positive cells.
Next, the Annexin V-FITC/PI double-staining method (Beyotime, Cat. No. C1063) was used to evaluate the level of neuronal apoptosis. Neuronal samples from each experimental group were resuspended in binding buffer, and the concentration was adjusted accordingly. Subsequently, FITC-labeled Annexin V and PI staining solution were added, and the samples were incubated at room temperature in the dark for 20 min. The apoptosis rates were calculated by detecting and analyzing the positive signals.
Transmission electron microscopy
2.20
PC12 cells were scraped using a cell scraper, washed with PBS, centrifuged into pellets, and fixed with a 2.5% glutaraldehyde solution specifically formulated for TEM. Rat brain tissue samples were immediately immersed in the same glutaraldehyde solution, sectioned into 1 mm^3^ blocks, fixed at room temperature for 1 h, then overnight at 4 °C. All samples were subsequently washed with PBS, post-fixed in 1% osmium tetroxide (4 °C, 2 h), dehydrated using an ethanol/acetone gradient, impregnated, and embedded in epoxy resin. Semi-thin sections were stained with toluidine blue for light microscopic localization, followed by ultrathin sectioning. The ultrathin sections were transferred onto grids, stained with uranyl acetate and lead citrate, dried, and examined using TEM.
Immunofluorescence analysis
2.21
The rat brain tissue sections were fixed in 4% paraformaldehyde for 30 min and washed twice with PBS. After permeabilization and blocking, the sections were incubated overnight at 4 °C with an anti-NeuN primary antibody (Proteintech, Cat. No. 26975-1-AP). On the following day, the sections were washed with PBS and incubated with the corresponding fluorophore-conjugated secondary antibody at room temperature in the dark for 3 h. TUNEL staining was then performed using a detection solution prepared by mixing 5 μL of TdT enzyme with 45 μL of TRITC-dUTP labeling mix (final volume, 50 μL per section). The TUNEL reaction mixture was applied to the sections and incubated for 1 h, followed by three washes with PBS. The nuclei were subsequently counterstained with DAPI (Proteintech, Cat. No. 28718-90-3) for 5–10 min at room temperature in the dark, followed by PBS washes. Fluorescence images were acquired using a Leica fully automatic inverted fluorescence microscope (DMi3000), and a quantitative analysis was performed using ImageJ software.
Measurement of cytosolic mtDNA released from mitochondria
2.22
PC12 cells were subjected to several treatments. Mitochondria were isolated using a Cell Mitochondria Isolation Kit (Beyotime, Cat. No. C3601) in accordance with the manufacturer's instructions. Briefly, the cells were harvested, resuspended in cold isolation buffer, and gently homogenized. The homogenate was centrifuged at low speed (600×g for 10 min) to pellet nuclei and cellular debris (P1). The resulting supernatant (S1), containing mitochondria and cytosolic components, was further centrifuged at high speed (15,000×g for 20 min) to obtain the mitochondrial pellet (P2). For genomic DNA extraction from the remaining cellular components, the nuclear/cell debris pellet (P1) was combined with the post-mitochondrial supernatant (S2). Genomic DNA was subsequently extracted using the BeyoMag™ Genomic DNA Extraction Kit (Beyotime, D0088) following the standard magnetic bead-based protocol. The extracted DNA was subsequently diluted to 2.5–40 ng/μL. A 20 μL qPCR reaction mixture was prepared on ice: containing 10 μL BeyoFast™ SYBR Green qPCR Mix, 1 μL corresponding primers (DNA target gene: mtDNA ND1, internal control gene: nDNA 18S rRNA), 2 μL template, 0.4 μL ROX, and ultrapure water to a final volume of 20 μL. Each sample was analyzed in triplicate, with ultrapure water serving as the negative control (NTC). Following gentle mixing and brief centrifugation, the reaction plates were loaded into the QuantStudio™ 6 qPCR system. The qPCR program was set as follows: 50 °C UDG treatment for 5 min, 95 °C pre-denaturation for 2 min, followed by 95 °C denaturation for 15 s, 55 °C annealing/extend 15 s at 55 °C for 40 cycles, with the results analyzed using the instrument software (Beyotime, Cat. No. D8039). For NTC samples, the mt/nDNA primer detection CT values ≥ 38 were calculated using the formula ΔCT (mt) = CT (mt, experimental group) – CT (mt, control) and ΔCT(n) = CT (n, experimental group) – CT (n, control); ΔΔCT = ΔCT (mt) – ΔCT (n). Relative mtDNA copy number was calculated using the 2^−ΔΔCT^ method and expressed as fold change relative to the control group. The primers mt-ND1 as mitochondrial genes and 18S rRNA as nuclear DNA controls are presented in Table S3.
Enzyme-linked immunosorbent assay (ELISA)
2.23
After cell treatment, the supernatant was collected and centrifuged at 4 °C, 1,000×g for 10 min. The supernatant was then retained for subsequent use. The IL-1β ELISA Kit (Huamei, Cat. No. CSB-E08055r-IS) and IL-18 ELISA Kit (Huamei, Cat. No. CSB-E04610r-IS) were used for detection. The relevant reagents were prepared in accordance with the manufacturers’ instructions. The standards and supernatant samples were added sequentially, followed by incubation at 37 °C. The subsequent steps, including biotinylated antibody binding, enzyme conjugate incubation, washing, substrate chromogenic reaction, and reaction termination, were performed sequentially. The absorbance value was measured using a microplate reader, and the relative contents of IL-1β and IL-18 in the supernatant were calculated based on the standard curves.
The CSF was collected from patients with cerebral infarction and normal controls, centrifuged at 4 °C, 10,000×g for 10 min, and the supernatant was collected for subsequent use. The Human TOM1L2 (TOM1-like protein 2) ELISA Kit (Wuhan Weikesai Technology Co, Cat. No. ELI-17358h) and Human CRLS1 (Cardiolipin Synthase 1) ELISA Kit (Wuhan Weikesai Technology Co, Cat. No. ELA-E0245h) were selected. In accordance with the manufacturers’ instructions, the steps, including the sequential addition of standards and samples, incubation at 37 °C, biotinylated antibody binding, enzyme conjugate incubation, washing, substrate chromogenic reaction, and reaction termination were completed. The absorbance value was measured using a microplate reader, and the relative expression levels of TOM1L2 and CRLS1 in the CSF were calculated based on the standard curve.
Relative pyruvate dehydrogenase activity assay
2.24
PC12 cells (5 × 10^6^ per experimental replicate) exposed to distinct treatments were harvested, rinsed with pre-chilled PBS, and resuspended in extraction buffer supplemented with Extraction Buffer 1 and 2. The cell suspension was subjected to ice-cold sonication (20% power output/200 W; 3 s sonication pulses alternating with 7 s intervals, repeated 30 cycles), followed by centrifugation at 11,000×g for 10 min at 4 °C. The resulting supernatant was stored on ice pending subsequent analysis. The working solution was freshly prepared by combining Reagent 1, 2, and 3, followed by incubation at 37 °C for 10 min to equilibrate. For each assay reaction, 10 μL of the supernatant was mixed with 190 μL of the pre-equilibrated working solution. The absorbance at 605 nm was measured at 20 s (defined as A_1_) and 5 min 20 s (defined as A_2_) post-mixing; the absorbance change (ΔA) was calculated as ΔA = A_1_ – A_2_. The relative pyruvate dehydrogenase (PDH) activity across treatment groups was assessed by comparing the ΔA values of experimental groups against one another. All samples and reagents were maintained on ice throughout the procedure to mitigate enzyme denaturation and loss of activity.
Lactate dehydrogenase release assay
2.25
PC12 cells were seeded in 96-well plates and cultured until reaching the appropriate confluency. The culture medium was replaced, and the cells were subjected to designated treatments. After the treatment period, the plates were centrifuged at 400×g for 5 min. Subsequently, 120 μl of the supernatant from each well was transferred to a new 96-well plate. Next, 60 μL of lactate dehydrogenase (LDH) assay working solution was added to each well. The plates were mixed thoroughly and incubated at room temperature (approximately 25 °C) for 30 min in the dark. The absorbance level was measured at a wavelength of 490 nm, and LDH release levels were evaluated by comparing the absorbance values among the groups.
Transcriptome sequencing and bioinformatic analysis
2.26
The total RNA was extracted and evaluated for purity and integrity (NanoDrop and Agilent 2100). Poly(A) + mRNA was enriched using oligo(dT) magnetic beads, fragmented, and reverse-transcribed to generate cDNA libraries with adaptor ligation and PCR amplification. The libraries were quality-controlled (Qubit and Agilent) and sequenced on an Illumina NovaSeq (6),000 platform (PE150). Raw sequencing reads were subjected to quality filtering, followed by alignment to the reference genome, and quantification of gene expression levels. Differentially expressed genes were identified, followed by gene ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and GSEA, with principal component analysis (PCA) and clustering used for expression pattern visualization.
CSF sample preparation and untargeted metabolomics analysis
2.27
CSF 100 μL was mixed with 400 μL cold methanol/acetonitrile (1:1, v/v), sonicated for 1 h in an ice bath, incubated at −20 °C for 1 h, and centrifuged (14,000 g, 20 min, 4 °C). Supernatants were dried under vacuum for LC-MS analysis. QC samples were prepared by pooling aliquots from all samples and processed identically. Dried extracts were reconstituted in 50% acetonitrile, filtered (0.22 μm cellulose acetate), transferred to 2 mL HPLC vials, and stored at −80 °C. Raw MRM data were processed in MultiQuant (peak detection, alignment, extraction, and filtering). Differential metabolites were defined as FC > 1.5 and p < 0.05 (two-tailed Student's t-test; one-way ANOVA for multiple groups), with FC calculated as the logarithm of the mean peak-area ratio between groups. Significant metabolites were subjected to clustering in R and KEGG pathway enrichment (Fisher's exact test, FDR correction).
Non-targeted metabolic flux
2.28
Non-targeted metabolic flux analysis was performed using LipidALL technology based on a previously reported method. Polar metabolites were extracted using ice-cold methanol containing phenylhydrazine. The mixture was incubated at 4 °C with agitation at 1,500 r/min for 30 min, followed by storage at −20 °C for 1 h to complete α-keto acid derivatization. Subsequently, the mixture was centrifuged at 12,000 r/min for 15 min at 4 °C, and the supernatant was dried in water bath mode using a vacuum centrifugal concentrator. The total precipitated protein was quantified using the BCA Protein Assay Kit, following the manufacturer's instructions. The dried extract was reconstituted in 5% acetonitrile aqueous solution and analyzed using an Agilent 1290 II ultra-performance liquid chromatography system coupled with a Sciex 5600+ quadrupole-time-of-flight mass spectrometer. Hydrophilic interaction chromatography (HILIC) separation was performed using a Waters ACQUITY BEH Amide column. The mass spectrometer was operated in electrospray ionization negative ion mode, with the voltage set at approximately 4.5 kV, atomization temperature at 500 °C, and drying gas, nebulizing gas, and curtain gas all using nitrogen at pressures of 50, 50, and 35 pounds per square inch (psi), respectively. The reversed-phase chromatography scanning range was m/z 60–700, and the HILIC scanning range was m/z 70–850. MS/MS employed information-dependent acquisition, with collision energy set at −35 ± 15 eV. The data were collected using Analyst TF 1.7.1 software. MarkerView 1.3 was used to extract ion information, which was then imported into Excel. PeakView 2.2 was used to extract MS/MS data, and ion identification was performed by integrating metabolite databases, the Human Metabolome Database, along with reference standards. The ionization differences between samples were corrected using l-leucine-D10 as the internal standard, followed by peak area normalization [31].
Protocol for intracerebroventricular siRNA injection in rats
2.29
SPF-grade male SD rats aged 6-8 weeks were selected and randomly divided into the negative control group and the Crls1 siRNA experimental group. All procedures were approved by the institutional Animal Care and Use Committee. After isoflurane anesthesia, the rats were fixed on a stereotactic frame, the anterior fontanelle was exposed, and the lateral ventricles were localized (0.8 mm posterior to the anterior fontanelle, 1.2 mm lateral to the midline, and 2.5 mm deep) by referring to a brain atlas. The working solution (final concentration: 0.5 μg/μL), prepared by incubating Crls1 siRNA with an in vivo transfection reagent, was microinjected into the lateral ventricles at a rate of 0.5 μL/min for a total volume of 2 μL (MedChemExpress, Cat. No. HY-RS27412). The needle was retained for 5 min to prevent reflux, followed by sealing the burr hole and suturing the incision. 72 h after injection, the rats were anesthetized, and the target brain region was dissected for subsequent verification of silencing efficiency and functional analysis.
Intravenous injection of mitochondrial vesicles into the orbital vein
2.30
SPF-grade male SD rats aged 6-8 weeks, all of which had been successfully subjected to the MCAO model, were selected for the experiment. They were first anesthetized with isoflurane, followed by topical anesthesia with 0.5% procaine hydrochloride eye drops. Mild pressure was applied to evert the eyes. The prepared hUCMSC Mito-EVs suspension (concentration adjusted to 50 μg/mL and calibrated via the BCA protein quantification method) was taken. A 29G insulin syringe was inserted into the inner canthus of the rat's eye, with the bevel facing outward, and then advanced at a 45° angle to a depth of approximately 5 mm into the center of the retro-orbital venous sinus. A total of 200 μL of the suspension was injected slowly. Control animals received an equal volume of vehicle solution (PBS). After completion of the injection, the syringe was withdrawn gradually, and the rats were placed on a warm pad for resuscitation prior to subsequent experiments.
Morris water maze
2.31
Fourteen days after MCAO injury, the spatial learning and memory abilities of rats were evaluated using the Morris Water Maze (MWM). The experiment was conducted in a circular opaque water pool maintained at 25 ± 2 °C. The pool was divided into four quadrants, and a transparent platform (10 cm in diameter, 1 cm below the water surface) was placed in one of the quadrants. Visual cues were affixed to the external walls of the pool. The training phase lasted for 5 days, with four trials per day. Rats were randomly placed into the water from four different starting points and allowed 60 s to locate the hidden platform. 24 h after the final training session, the platform was removed for the probe trial. The procedure was consistent with the training phase, and the time spent in the target quadrant was used as the primary evaluation index.
Quantification and statistical analysis
2.32
Statistical analysis was performed with GraphPad Prism 8. Independent experiments were repeated ≥3 times, and data are shown as mean ± standard error of the mean. Two-group comparisons were performed using unpaired t-tests. Comparisons involving multiple groups were analyzed by one-way or two-way ANOVA, followed by appropriate post-hoc tests. Statistical significance was defined as follows: ns, not significant; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001.
Results
3
Elevated l-lactate and mitochondrial dysfunction drive energy metabolic disturbance in ischemic stroke
3.1
CSF reflects dynamic alterations in brain energy metabolism [32]; therefore, we collected CSF from patients with IS after obtaining ethical approval and informed consent for non-targeted metabolomics analysis (Fig. 1A). The metabolomic profile was altered following cerebral ischemia, with bidirectional changes observed in metabolite levels. (Fig. 1B–D). A quantitative analysis of CSF metabolites revealed substantially higher concentrations of l-lactate and pyruvate in patients with IS. Among these metabolites, l-lactate exhibited the most significant increase and clearest discriminatory pattern (SFig. 1A–I, Fig. 1E–H) KEGG pathway analysis indicated that, compared with the normal control group, the TCA cycle, pyruvate metabolism, and related pathways were altered in the CSF of patients with IS (Fig. 1I). The level of l-lactate in CSF was positively correlated with infarct volume and National Institutes of Health Stroke Scale (NIHSS) scores, suggesting that higher l-lactate levels are linked to greater stroke severity. Analysis of modified Rankin Scale (mRS) scores also indicated that elevated l-lactate is associated with a poorer prognosis in IS patients. Further examination of serological indicators revealed a correlation between elevated average blood glucose levels during the acute phase of IS (within 3 days) and increased l-lactate [33]. (Fig. 1J–O). These findings highlight that elevated l-lactate not only reflects stroke severity but also serves as a potential marker for both poor functional outcomes and early metabolic disturbances in IS patients.Fig. 1l-lactate-centered energy metabolic reprogramming is associated with ischemic stroke severity(A) Cerebrospinal fluid (CSF) samples were collected from both healthy controls and patients with cerebral infarction. (B) Principal component analysis (PCA) of CSF energy metabolism profiles comparing healthy controls and patients with cerebral infarction (n = 4 per group; representative subset from the full cohort, used for preliminary metabolic profile screening). (C) Heatmap showing differentially abundant metabolites in CSF between the two groups. (D) l-lactate identified as the top contributor to cerebral metabolic alterations. (E) Relative abundance of pyruvate in CSF. (F) Relative abundance of l-lactate in CSF. (G) Absolute content of pyruvate in CSF (n = 10). (H) Absolute content of l-lactate in CSF (n = 10). (I) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed metabolites. (J–K) Representative magnetic resonance imaging (T1 sequence) of patients with stroke, used for infarct focus visualization and volume measurement. (L–O) Pearson correlation analyses of CSF l-lactate levels with clinical parameters in 30 patients, including NIHSS scores, infarct volume, mRS scores, and blood glucose levels.Data are presented as mean ± standard error of the mean. Statistical analysis for panels (E, F, G, H) was performed using an unpaired t-test. ns. P ≥ 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Fig. 1
To further explore the pathological relevance and underlying mechanisms suggested by the clinical findings, we established a rat MCAO model using the intraluminal filament method. Successful model construction was confirmed by 2,3,5-triphenyltetrazolium chloride (TTC) staining (SFig. 1J). On postoperative day 3, we collected brain tissue from the infarct region of MCAO model rats and from normal control rats for transcriptome sequencing. PCA and differential expression gene (DEG) analysis both showed significant differences in global gene expression between the two groups (SFig. 1K–L). GO analysis demonstrated that energy metabolism-related pathways, particularly ATP generation from ADP and glycolytic process, were markedly dysregulated in the brain tissues derived from MCAO model rats (SFig. 1M). KEGG pathway analysis showed marked activation of the hypoxia-inducible factor 1 (HIF-1) signaling pathway and pyruvate metabolism in the brain tissue of MCAO model rats (SFig. 1N). These pathway alterations were consistent with dysregulation of the TCA cycle, glycolysis/gluconeogenesis, oxidative phosphorylation, and pyruvate metabolism observed in the untargeted metabolomic analysis of CSF from patients with IS. To directly confirm changes in energy metabolism, we measured metabolite levels in brain tissue. The results showed a significant reduction in the ATP levels in the brain tissue adjacent to the infarct (SFig. 1O), while elevated l-lactate and pyruvate levels indicated that cerebral infarction directly disrupts energy metabolism (SFig. 1P–Q). In the MWM test, MCAO model rats exhibited significantly impaired memory and cognitive function compared with the normal control group (SFig. 1R–S). Nissl staining showed a marked decrease in the neuronal density around the infarct in the MCAO model group (SFig. 1T–U). This finding indicates that energy metabolism dysfunction following cerebral infarction is a major contributor to neuronal loss and resulting cognitive impairment [34].
Mitochondria, as the central organelles for cellular energy metabolism, play a critical role in ischemic brain injury through dysfunction-induced metabolic disturbances [35]. To elucidate the underlying molecular mechanisms, this study used PC12 cells to establish an in vitro OGD/R model of 3-h oxygen-glucose deprivation followed by 24-h reoxygenation. We labeled mitochondria using a Mito-ZsGreen-expressing viral vector. After OGD/R treatment, the mitochondria displayed pronounced fragmentation and shortening (SFig. 2A–B). Western blot analysis showed that OGD/R treatment markedly decreased the expression of the mitochondrial markers TOM20 (translocase of the outer mitochondrial membrane 20), VDAC (voltage-dependent anion channel), and COX IV (cytochrome c oxidase subunit IV) [36] (SFig. 2C–D). JC-1 staining showed that MMP decreased significantly after OGD/R treatment (SFig. 2E–F). Concurrently, cellular ATP levels, a key product of energy metabolism, were also markedly reduced (SFig. 2G). The results indicate that OGD/R injury causes marked abnormalities in mitochondrial morphology and function. To define the temporal relationship between mitochondrial dysfunction and cellular damage, we used triple fluorescence staining—MTG, MTDR, and PI—to dynamically track MMP and cell membrane integrity. LSCM showed that after 100 min of oxygen-glucose deprivation followed by 24 h of reoxygenation, the cellular MMP was depolarized. After 160 min of oxygen-glucose deprivation followed by 24 h of reoxygenation, the cell membrane integrity was compromised. After 300 min of oxygen-glucose deprivation followed by 24 h of reoxygenation, the MMP was completely lost, and the cell membranes exhibited irreversible damage. These results indicate that OGD/R-induced mitochondrial dysfunction precedes global cellular damage and constitutes an early event in OGD/R-mediated cellular injury (SFig. 2H–I). The quantitative measurements of intracellular metabolites showed that l-lactate and pyruvate levels increased significantly after OGD/R treatment (SFig. 2J–K). Collectively, these results indicate that disrupted l-lactate metabolism is tightly linked to the IS outcomes, and that mitochondrial structural and functional dysfunction is a key trigger of this pathological process [37].
Mito-EVs retain essential mitochondrial properties and can be internalized by PC12 cells
3.2
Previous studies have demonstrated that astrocytes can repair OGD/R-damaged neurons via mitochondrial transfer [38]. However, the dysfunction of donor cells induced by the ischemic microenvironment limits this endogenous repair mechanism [39]. Mito-EVs, as carriers of functional mitochondrial components, offer a novel strategy for this purpose [40]. Given the advantages of hUCMSCs including high mitochondrial content, strong proliferative capacity, and ease of in vitro expansion [41], we hypothesized that hUCMSC Mito-EVs may promote mitochondrial repair in ischemically damaged neurons by delivering functional mitochondrial fragments.
To clarify the therapeutic potential of hUCMSC Mito-EVs for mitochondrial dysfunction in IS-injured neurons, this study first used the Mito-DsRed-WPRE virus to specifically trace the mitochondria derived from hUCMSCs, followed by stepwise isolation and purification of Mito-EVs from their culture supernatant (Fig. 2A) [42]. TEM revealed that purified Mito-EVs exhibited a roughly spherical morphology, with diameters ranging from 30 to 1000 nm (Fig. 2B) [43]. LSCM further confirmed the presence of fragmented mitochondrial structures enclosed within these vesicles (Fig. 2C). Western blot analysis showed that purified Mito-EVs not only expressed the vesicle-associated protein Annexin A, but were also enriched with mitochondrial core proteins such as COX IV, HSP60, and VDAC. These findings confirmed that they possess both vesicle properties and mitochondrial component characteristics (Fig. 2D). To systematically verify the functional preservation of mitochondrial fragments within hUCMSC Mito-EVs, hUCMSCs were first labeled with the mitochondrial matrix pH-specific probe Mito-SypHer virus. This probe dynamically reflects changes in mitochondrial matrix pH through the fluorescence intensity ratio of F490/F405 [44]. Following Mito-EVs isolation and quantitative LSCM analysis, the fluorescence ratio showed no statistically significant difference compared with mitochondria in normal hUCMSCs, suggesting that the mitochondrial fragments within hUCMSC Mito-EVs maintain physiological pH homeostasis (Fig. 2E and F). Subsequently, we assessed MMP using simultaneous staining with MTG and MTDR. The MTDR/MTG fluorescence ratio of Mito-EVs showed no statistically significant difference compared with mitochondria within normal hUCMSCs, indicating preserved MMP (Fig. 2G and H). Western blot assays revealed the presence of key oxidative phosphorylation system (OXPHOS) components in the isolated vesicles, indicating that the purified Mito-EVs retain key components of the OXPHOS machinery (Fig. 2I). Collectively, these results indicate that isolated hUCMSC Mito-EVs preserve key mitochondrial properties, including matrix pH homeostasis, membrane potential, and core components of the oxidative phosphorylation machinery.Fig. 2. Mitochondrial extracellular vesicles (Mito-EVs) retain essential mitochondrial properties and can be internalized by PC12 cells(A) Isolation workflow of mitochondria-derived extracellular vesicles. (B) Morphological characterization of Mito-EVs via transmission electron microscopy (TEM). (C) Morphological characterization of Mito-EVs via laser scanning confocal microscopy (LSCM). (D) Western blot of vesicle marker Annexin A and mitochondrial markers (COX IV, VDAC, HSP60) in Mito-EVs lysates. (E) Visualization of Mito-EVs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) transfected with Mito-SypHer virus (pH-sensitive), where the F490/F405 fluorescence ratio increases with the elevation of mitochondrial matrix pH. (F) Quantification of F490/F405 ratio for mitochondrial pH (n = 10). (G) Co-staining of MitoTracker Green (MTG; labels mitochondria) and MitoTracker Deep Red (MTDR; reflects MMP) in Mito-EVs and hUCMSCs. (H) Quantification of MTDR/MTG ratio for mitochondrial function (n = 10). (I) Western blot of oxidative phosphorylation (OXPHOS proteins) in Mito-EVs lysates. (J) Co-culture of Mito-EVs with PC12 cells. (K) Flow cytometry of Mito-EVs uptake by PC12 cells (L) Quantification of uptake rate (positive cell percentage; n = 10). (M) Fluorescence images of Mito-EVs uptake by healthy PC12 cells. (N) Immunofluorescence quantification confirming co-localization and fusion in healthy PC12 cells. (O) Fluorescence images of Mito-EVs uptake by oxygen-glucose deprivation/reperfusion (OGD/R)-injured PC12 cells. (P) Immunofluorescence quantification confirming co-localization and fusion in OGD/R-injured PC12 cells. (Q) Comparison of the mitochondrial fusion efficiency between groups (Pearson's correlation; n = 10).Data are presented as mean ± standard error of the mean. Statistical analysis: unpaired t-test for two-group comparisons (F, H, L, Q), ns. P ≥ 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Fig. 2
To assess PC12 cell uptake of Mito-EVs, we labeled endogenous mitochondria in PC12 cells with Mito-ZsGreen and co-cultured them for 24 h with hUCMSC Mito-EVs labeled with Mito-DsRed-WPRE (Fig. 2J). To clarify the uptake of hUCMSC Mito-EVs and their interaction with endogenous mitochondria, flow cytometry showed comparable uptake in normal and OGD/R-modeled PC12 cells, indicating that OGD/R does not impair Mito-EVs internalization (Fig. 2K and L). LSCM analysis confirmed the co-localization of internalized Mito-EVs with endogenous mitochondria and fusion of exogenous mitochondria into the endogenous mitochondrial pool. Although the fusion efficiency was unchanged, OGD/R-induced polarized mitochondrial redistribution, with both endogenous and exogenous mitochondria biased toward one side of the cell, leading to a corresponding shift in exogenous mitochondrial distribution (Fig. 2M–Q). Collectively, these data indicate that OGD/R does not measurably impair Mito-EVs uptake or mitochondrial fusion under the experimental conditions used, although it induces a redistribution of mitochondrial localization within injured cells.
Mito-EVs ameliorate mitochondrial dysfunction in OGD/R-injured PC12 cells
3.3
To further evaluate the therapeutic effect of hUCMSC Mito-EVs on injured neurons, we co-cultured the isolated Mito-EVs with PC12 cells subjected to OGD/R for 24 h. LSCM revealed that mitochondria in the hUCMSC Mito-EVs treatment group showed markedly less fragmentation than those in the OGD/R injury group, with a greater average mitochondrial length (Fig. 3A and B). JC-1 fluorescence probing further confirmed that the hUCMSC Mito-EVs treatment group showed a clear recovery of MMP compared with the OGD/R injury group (Fig. 3C and D). Further assessment using co-staining with MTG, MTDR, and PI showed that Mito-EVs treatment partially restored MMP and plasma membrane integrity (Fig. 3E–G). Western blot analysis showed that the core mitochondrial functional proteins and OXPHOS chain proteins were significantly upregulated in the hUCMSC Mito-EVs treatment group compared with those in the OGD/R injury group, suggesting that mitochondrial vesicle treatment supports the recovery of mitochondrial protein expression (SFig. 3A–B and Fig. 3H and I). The Seahorse Mitochondrial Stress Test showed that basal respiration, maximal respiration, and spare respiratory capacity were significantly increased in the hUCMSC Mito-EVs-treated group compared with the OGD/R group (Fig. 3J and K). Following treatment with hUCMSC Mito-EVs, cytosolic free mtDNA levels in OGD/R-injured PC12 cells were significantly lower than in the injury-only group (Fig. 3L). This finding suggests improved mitochondrial membrane integrity and reduced mitochondrial damage, potentially associated with decreased mitochondrial permeability transition pore (mPTP) opening. At the whole-cell metabolic level, hUCMSC Mito-EVs treatment restored energy metabolism: the intracellular ATP levels were significantly higher, l-lactate content was reduced, and LDH release decreased compared with the OGD/R injury group, indicating a recovery of cellular energy metabolism (Fig. 3M − O). Untargeted metabolomic analysis revealed significant differences in the metabolite profiles between the two groups (SFig. 3C). Compared with the OGD/R injury group, the hUCMSC Mito-EVs-treated group showed increased levels of TCA cycle intermediates and a marked reduction in l-lactate (SFig. 3D), suggesting reduced reliance on glycolytic metabolism. KEGG pathway enrichment analysis showed significant activation of the TCA cycle and pyruvate metabolism pathways in the hUCMSC Mito-EVs treatment group, confirming reconstruction of the cellular energy metabolism network (SFig. 3E). Flow cytometry confirmed that treatment with hUCMSC Mito-EVs significantly reduced apoptosis in PC12 cells after OGD/R injury (Fig. 3P and Q). These results indicate that Mito-EVs can be taken up by PC12 cells injured by hypoxia, partially restore mitochondrial function, and attenuate OGD/R-induced injury.Fig. 3. Mitochondrial extracellular vesicles (Mito-EVs) can salvage the mitochondrial function of PC12 cells damaged by oxygen-glucose deprivation/reperfusion (OGD/R) (A) Mitochondrial morphology of OGD/R-injured PC12 cells before and after treatment with Mito-EVs (B) Quantitative analysis of the mitochondrial length (n = 10). (C) Mitochondrial membrane potential (MMP) assessed by JC-1 staining (red fluorescence: healthy mitochondria with high MMP; green fluorescence: depolarized mitochondria with low MMP; the red/green ratio reflects the MMP status). (D) Relative quantification of JC-1 red/green fluorescence ratio (n = 10). (E) Co-staining of PC12 cells with MitoTracker Green (MTG), MitoTracker Deep Red (MTDR), and propidium iodide (PI) before and after Mito-EVs treatment. (F–G) Quantitative analysis of MTDR/MTG and PI/MTG fluorescence intensity ratios (MTDR/MTG reflects MMP; PI/MTG reflect cell membrane permeability; n = 10). (H) Western blot analysis of oxidative phosphorylation (OXPHOS) chain proteins in PC12 cells before and after treatment with Mito-EVs. (I) Quantitative analysis of Western blot gray values for OXPHOS chain proteins (n = 3). (J) Oxygen consumption rate (OCR) of PC12 cells measured via Seahorse Mitochondrial Stress Test (n = 5). (K) Quantitative statistics of the mitochondrial respiratory rate (n = 5). (L) Relative quantification of the cytoplasmic mtDNA content in PC12 cells before and after Mito-EVs treatment (n = 10). (M) Quantitative analysis of ATP levels in PC12 cells before and after Mito-EVs treatment (n = 10). (N) Quantitative analysis of l-lactate levels in PC12 cells before and after Mito-EVs treatment (n = 10). (O) Relative quantification of lactate dehydrogenase (LDH) release in the culture medium of PC12 cells before and after Mito-EVs treatment (n = 10). (P–Q) Annexin V/PI double-staining flow cytometry scatter plots (P) and quantitative analysis (Q) of PC12 cells before and after Mito-EVs treatment (n = 3).Data are presented as mean ± standard error of the mean. Statistical analysis: unpaired t-test was used for two-group comparisons (B, D, F, G, L, M, N, O); two-way ANOVA followed by Tukey's post hoc test was used for multiple-group comparisons (I, J, K, Q). ns. P ≥ 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Fig. 3
To further define the role of functional mitochondria in Mito-EVs, we treated them with CCCP to deplete the MMP and impair their function (SFig. 3F). CCCP-treated Mito-EVs (hUCMSC Mito-EVs-CCCP) showed significantly reduced expression of OXPHOS chain proteins (SFig. 3G–H), a drastic loss of MMP, and a decrease in mitochondrial matrix pH (SFig. 3I–L), confirming functional impairment. Subsequently, we treated OGD/R-injured PC12 cells with either hUCMSC Mito-EVs or hUCMSC Mito-EVs-CCCP. The results indicated that hUCMSC Mito-EVs-CCCP failed to restore the MMP, matrix homeostasis, or OXPHOS chain protein expression, with mitochondrial function-related proteins downregulated (SFig. 3M–T). These findings confirm that functional mitochondria are crucial for Mito-EVs’ therapeutic efficacy, and loss of mitochondrial function abolishes their reparative effects.
Tom1l2 promotes the fusion of mitochondria in Mito-EVs with those in damaged cells
3.4
To elucidate the molecular mechanism by which hUCMSC Mito-EVs repair OGD/R injury in PC12 cells, we performed transcriptome sequencing on OGD/R-exposed PC12 cells treated with hUCMSC Mito-EVs and on untreated control cells. PCA and DEG analyses indicated significant differences in the gene expression profiles between the two groups of samples (SFig. 4A–B). KEGG enrichment analysis showed that hUCMSC Mito-EVs treatment was closely related to the HIF-1 signaling pathway and glycolysis/gluconeogenesis (SFig. 4C). Given the central role of vesicle trafficking in injury repair, we focused on differentially expressed genes related to transport. After treatment with hUCMSC Mito-EVs, the expression of the vesicle-transport protein-encoding gene Tom1l2 was markedly upregulated in PC12 cells (Fig. 4A and B). Western blot analysis further confirmed that Tom1l2 protein expression was significantly upregulated in OGD/R-injured PC12 cells treated with hUCMSC Mito-EVs (Fig. 4C and D). To clarify how Tom1l2 was upregulated and whether it was functionally required, we generated hUCMSCs with stable Tom1l2 knockout and isolated mitochondria-containing extracellular vesicles derived from them (Tom1l2-KO Mito-EVs). qRT-PCR and Western blot analyses confirmed that the Tom1l2 mRNA and protein levels were markedly lower in Tom1l2-KO Mito-EVs than in hUCMSC Mito-EVs (SFig. 4D–F), and that these Tom1l2-KO Mito-EVs failed to upregulate the Tom1l2 expression in OGD/R-injured PC12 cells (SFig. 4G–I). Notably, Tom1l2 expression in the CSF of patients with IS was significantly reduced compared with that in normal controls (Fig. 4E). Collectively, this finding further suggests that the downregulation of Tom1l2 may be associated with the pathological processes underlying IS-induced brain injury. These results indicate that the therapeutic effect of hUCMSC Mito-EVs on OGD/R-injured PC12 cells is dependent on Tom1l2, and that the increase in Tom1l2 expression in recipient PC12 cells relies on Tom1l2 delivered by Mito-EVs.Fig. 4. Tom1l2 promotes the fusion of mitochondria in mitochondrial extracellular vesicles (Mito-EVs) with those in damaged cells(A) Heatmap of differential genes in oxygen-glucose deprivation/reperfusion (OGD/R)-injured PC12 cells before and after treatment with Mito-EVs (n = 3) (B) Quantitative qRT-PCR detection of differential genes in OGD/R-injured PC12 cells before and after Mito-EVs treatment (n = 10). (C–D) Western blot detection of TOM1L2 protein expression and its quantitative analysis in OGD/R-injured PC12 cells before and after treatment with Mito-EVs (n = 3). (E) Enzyme-linked immunosorbent assay (ELISA) detection of TOM1L2 expression level in CSF (n = 10). (F–G) Observation of co-culture of OGD/R-injured PC12 cells with human umbilical cord mesenchymal stem cell (hUCMSC) Mito-EVs and Tom1l2-KO Mito-EVs via fluorescence microscopy (hUCMSC Mito-EVs and Tom1l2-KO Mito-EVs were labeled with red fluorescence, PC12 cells' mitochondria with green fluorescence). (H) Comparison of the exogenous mitochondrial uptake between groups (n = 10) (I) Quantitative comparison of Pearson correlation coefficient for colocalization degree between red fluorescent-labeled Mito-EVs and green fluorescent-labeled mitochondria in PC12 cells (n = 10). (J–K) Observation (via transmission electron microscopy) and quantitative analysis of the mitochondrial aspect ratio (length/width ratio) in PC12 cells (n = 10). (L–M) Observation and quantification of PC12 cell mitochondria labeled with Mito-SypHer (pH-sensitive, detects mitochondrial matrix pH via F490/F405 ratio; n = 10). (N) Flow cytometry histogram of the ROS levels in PC12 cells. (O–P) Western blot detection of oxidative phosphorylation (OXPHOS) chain proteins and their quantitative analysis in PC12 cells (n = 3). (Q–R) Annexin V/PI double-staining flow cytometry scatter plots and quantitative analysis of PC12 cells (n = 3).Data are presented as mean ± standard error of the mean. Statistical analysis: unpaired t-test was used for two-group comparisons (D, E, H, I); one-way ANOVA followed by Tukey's post hoc test was used for multiple-group comparisons (K, M); two-way ANOVA followed by Tukey's post hoc test was used for comparisons involving two variables (B, P, R). ns. P ≥ 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Fig. 4
To determine whether Tom1l2 deletion in hUCMSCs altered the function of mitochondrial fragments within their derived Mito-EVs, we performed a Western blot analysis. No significant differences were observed in the expression levels of OXPHOS chain proteins between Tom1l2-KO Mito-EVs and hUCMSC Mito-EVs (SFig. 4J–K). Additionally, the mitochondrial matrix pH and membrane potential of Tom1l2-KO Mito-EVs did not differ significantly from those in hUCMSC Mito-EVs (SFig. 4L–O), indicating that Tom1l2 deletion did not impair mitochondrial function in the derived Mito-EVs. Subsequently, we co-cultured each set of Mito-EVs with PC12 cells subjected to OGD/R. Quantitative LSCM analysis revealed no significant difference in the fluorescence intensity of exogenous mitochondria between the hUCMSC Mito-EVs and Tom1l2-KO Mito-EVs groups, confirming that the mitochondrial delivery efficiency of Tom1l2-KO Mito-EVs was comparable to that of hUCMSC Mito-EVs (Fig. 4F–H). Notably, Pearson correlation analysis revealed that hUCMSC Mito-EVs effectively fused with endogenous mitochondria in OGD/R-injured PC12 cells, whereas Tom1l2-KO Mito-EVs showed markedly reduced fusion with recipient cell mitochondria (Fig. 4I). These results indicate that Tom1l2 knockout in Mito-EVs does not affect vesicle uptake, but specifically impairs the fusion between exogenous and endogenous mitochondria, underscoring the critical role of Tom1l2 in mediating mitochondrial fusion. TEM showed that mitochondria in the hUCMSC Mito-EVs treatment group exhibited restored cristae structure and reduced swelling, whereas mitochondria in the Tom1l2-KO Mito-EVs group exhibited severe cristae disruption and swelling. These findings confirm that Tom1l2 deficiency impairs the structural repair function of hUCMSC Mito-EVs (Fig. 4J and K). Further analysis showed that the Tom1l2-KO Mito-EVs group had a lower Mito-SypHer fluorescence ratio (F490/F405), indicating that, relative to the hUCMSC Mito-EVs group, mitochondrial matrix pH dysregulation and functional impairment in OGD/R-injured PC12 cells were not improved (Fig. 4L and M). Additionally, neither MMP nor cell membrane integrity was restored (SFig. 5A–C). ROS-specific staining and quantitative mitochondrial fluorescence imaging showed that, compared with the hUCMSC Mito-EVs treatment group, the cells treated with Tom1l2-KO Mito-EVs exhibited persistent and significantly increased intracellular ROS accumulation (Fig. 4N and SFig. 5D–E). Moreover, the mitochondrial mean length remained shortened, indicating no signs of restoration (SFig. 5F–G). Western blot analysis confirmed that treatment with hUCMSC Mito-EVs significantly increased mitochondrial functional proteins TOM20, VDAC, and COX IV (SFig. 5H–I) and upregulated OXPHOS proteins (Fig. 4O and P). Concurrently, the treatment reduced cytosolic mtDNA leakage, restored ATP production (SFig. 5J–K), and decreased apoptosis (Fig. 4Q and R). In contrast, Tom1l2-KO Mito-EVs treatment failed to induce the upregulation of mitochondrial functional proteins and respiratory chain-related proteins, leaving mitochondrial function and membrane integrity persistently impaired.
In summary, these results demonstrate that although Tom1l2 deletion in hUCMSCs does not impair the mitochondrial function within their derived Mito-EVs, it abolishes their ability to fuse with mitochondria in OGD/R-injured PC12 cells. This fusion defect prevents the restoration of mitochondrial morphology and function, disrupts cellular energy metabolism and membrane integrity, and ultimately eliminates the neuroprotective effects of hUCMSC Mito-EVs.
Mito-EVs promote the functional recovery of damaged mitochondria through Crls1-mediated mitochondrial inner membrane homeostasis
3.5
To elucidate how Tom1l2 mediates the therapeutic effects during the fusion of hUCMSC Mito-EVs with damaged mitochondria, we treated OGD/R-injured PC12 cells with either fusion-competent hUCMSC Mito-EVs or fusion-incompetent Tom1l2-KO Mito-EVs, then performed transcriptome sequencing on the PC12 cells. The results showed significant differences in PCA clustering and DEG profiles between the two groups (SFig. 6A and Fig. 5A–B). The expression of genes, including Crls1, Tfam, and Hk2, was significantly reduced in the Tom1l2-KO Mito-EVs treatment group (SFig. 6B). GO and KEGG enrichment analyses revealed that pathways enriched in the hUCMSC Mito-EVs treatment group primarily involved activation of mitochondrial-related pathways, exemplified by cardiolipin biosynthesis, and alterations in the HIF-1 signal pathway and glycolysis/gluconeogenesis (Fig. 5C and D). Moreover, a subsequent analysis showed that OGD/R injury downregulated the expression of Crls1 in PC12 cells, which encodes a key enzyme involved in cardiolipin synthesis. This downregulation was accompanied by a concurrent reduction in cardiolipin expression, further confirming the impact of Crls1 on cardiolipin production [45] (SFig. 6C–F). In line with these in vitro findings, Crls1 levels were also significantly reduced in the CSF of patients with IS compared with healthy controls (Fig. 5E). Consistently, treatment with hUCMSC Mito-EVs upregulated Crls1 expression in damaged PC12 cells, whereas Tom1l2-KO Mito-EVs reversed this effect. Similarly, the changes in Crls1 expression were mirrored by corresponding alterations in cardiolipin levels, further supporting the role of Tom1l2 in both Crls1 regulation and cardiolipin production (SFig. 6G–J). To directly assess the role of Crls1 in the protective effects of hUCMSC Mito-EVs, injured PC12 cells were co-treated with Mito-EVs and Crls1 siRNA. As expected, this siCrls1-mediated knockdown effectively abolished the ability of Mito-EVs to restore Crls1 expression in injured PC12 cells, and, consequently, cardiolipin levels were also not restored, thereby further linking the changes in Crls1 expression to alterations in cardiolipin production (Fig. 5F–I).Fig. 5. Mitochondrial extracellular vesicles (Mito-EVs) promote the functional recovery of damaged mitochondria through Crls1-mediated mitochondrial inner membrane homeostasis(A) Heatmap of differential genes in oxygen-glucose deprivation/reperfusion (OGD/R)-injured PC12 cells treated with human umbilical cord mesenchymal stem cell (hUCMSC) Mito-EVs and Tom1l2-KO Mito-EVs (n = 3). (B) Volcano plot of differentially expressed genes (DEGs). (C) Gene ontology (GO) functional enrichment analysis of DEGs. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs. (E) Enzyme-linked immunosorbent assay (ELISA) detection of the CRLS1 expression level in cerebrospinal fluid (CSF) (n = 10). (F–H) PCR (n = 10) and Western blot analysis of the CRLS1 expression in PC12 cells and corresponding quantitative statistics (n = 3). (I) Expression level of cardiolipin (n = 10). (J–L) Observation of co-culture of OGD/R-injured PC12 cells and Mito-EVs via fluorescence microscopy (hUCMSC Mito-EVs were labeled with red fluorescence, PC12 cells' mitochondria with green fluorescence). (M) Statistical analysis of relative mitochondrial length in PC12 cells (n = 10). (N) Quantitative analysis of the ATP levels in PC12 cells (n = 10). (O–P) Western blot detection of mitochondrial function-related proteins in PC12 cells and quantitative analysis of the corresponding blots (n = 3). (Q) Viability of PC12 cells after different treatments (n = 3).Data are presented as mean ± standard error of the mean. Statistical analysis: unpaired t-test was used for two-group comparisons (E); one-way ANOVA followed by Tukey's post hoc test was used for multiple-group comparisons (F, H, I, M, N); two-way ANOVA followed by Tukey's post hoc test was used for comparisons involving two variables (P, Q). ns. P ≥ 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Fig. 5
LSCM showed that hUCMSC Mito-EVs promoted fusion between exogenous and damaged mitochondria and restored mitochondrial morphology. However, in the combined treatment group with siCrls1, although the fusion of Mito-EVs with damaged mitochondria was not affected, the recovery of the average mitochondrial length was markedly less than that in the hUCMSC Mito-EVs-only group, indicating an impaired restoration of the mitochondrial morphology (Fig. 5J–M and SFig. 6K). Energy metabolism assays showed that siCrls1 prevented hUCMSC Mito-EVs from restoring the ATP levels (Fig. 5N). This impairment was accompanied by abnormal mitochondrial matrix pH and accumulation of ROS, indicating defective functional recovery (SFig. 6L–O). Western blot confirmed that Crls1 deletion prevented hUCMSC Mito-EVs from upregulating mitochondrial functional proteins such as TOM20, VDAC, and COX IV (Fig. 5O and P), thereby inhibiting the recovery of cellular viability (Fig. 5Q).
In conclusion, Crls1 is a key molecule that mediates the therapeutic effects following the fusion of Mito-EVs with damaged mitochondria. hUCMSC Mito-EVs achieve mitochondrial repair, restoration of energy metabolism, and relief of oxidative stress by upregulating the Crls1 expression in recipient cells and regulating pathways such as cardiolipin biosynthesis [46]. Crls1 deficiency does not abolish the fusion process itself but markedly impairs the subsequent restoration of mitochondrial morphology and function.
Crls1 can reshape the glucose metabolism of hypoxia-damaged neurons and inhibit pyroptosis
3.6
To define how hUCMSC Mito-EVs modulate energy metabolism via Crls1, we cultured the cells with ^13^C-labeled glucose and applied high-resolution metabolic flux analysis (Fig. 6A). This approach showed that hUCMSC Mito-EVs substantially restored the glucose metabolism in OGD/R-injured PC12 cells. Compared with the OGD/R injury group, the hUCMSC Mito-EVs-treated group showed a significant reduction in the proportion of ^13^C-labeled l-lactate among cellular metabolites, indicating reversal of excessive glycolytic activation and reduced l-lactate accumulation [47]. Concurrently, the fractional ^13^C labeling of key TCA cycle metabolites—citrate, α-ketoglutarate, and malate—increased significantly, while the fraction of unlabeled metabolites markedly decreased, confirming TCA cycle reactivation, enhanced oxidative phosphorylation, and restoration of ATP-generating capacity. It is noteworthy that the metabolic recovery in the Tom1l2-KO Mito-EVs group and hUCMSC Mito-EVs + siCrls1 group was significantly weaker than that in the hUCMSC Mito-EVs group alone. There were no significant differences in the proportion of ^13^C-labeled l-lactate and TCA cycle metabolites compared to the OGD/R injury group (Fig. 6B–E). Further detection revealed that hUCMSC Mito-EVs could effectively increase the intracellular PDH levels, further confirming TCA cycle reactivation. However, in both the Tom1l2-KO Mito-EVs treatment group and hUCMSC Mito-EVs combined with siCrls1 treatment group, PDH activity recovery was suppressed to varying degrees (SFig. 7A). Thus, these findings indicate that hUCMSC Mito-EVs mediate the shift of glucose metabolism in hypoxia-injured neurons from anaerobic glycolysis toward the TCA cycle through the Tom1l2-Crls1 axis.Fig. 6. Crls1 can reshape the glucose metabolism of hypoxia-damaged neurons and inhibit pyroptosis(A) Schematic of ^13^C-glucose metabolic tracing. (B) Quantitative analysis of ^13^C-labeled metabolite ratio in l-lactate (n = 6). (C) Quantitative analysis of ^13^C-labeled metabolite ratio in Citrate (n = 6). (D) Quantitative analysis of ^13^C-labeled metabolite ratio in α-Ketoglutarate (n = 6). (E) Quantitative analysis of ^13^C-labeled metabolite ratio in Malate (n = 6). (F–H) Western blot detection and quantification of GSDMD-FL and GSDMD-NT protein in PC12 cells mitochondria and cytoplasm (n = 3). (I–M) Confocal observation and quantification of the fluorescence intensity of green fluorescent–labeled GSDMD-NT in PC12 cells (n = 10), with mitochondria labeled in red. (N) Relative quantification of lactate dehydrogenase (LDH) release in the culture medium of PC12 cells (n = 10).Data are presented as mean ± standard error of the mean. Statistical analysis: one-way ANOVA followed by Tukey's post hoc test was used for multiple-group comparisons (B, C, D, E, M, N); two-way ANOVA followed by Tukey's post hoc test was used for comparisons involving two variables (G, H). ns. P ≥ 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Fig. 6
Cardiolipin is synthesized by Crls1 and plays a key role in maintaining inner mitochondrial membrane (IMM) homeostasis [48]. Previous studies have shown that under cellular stress, cardiolipin translocates to the outer mitochondrial membrane and serves as the lipid ligand for GSDMD-NT, mediating GSDMD-NT-dependent mitochondrial damage and thereby modulating the course of pyroptosis [49]. Western blot analysis showed that hUCMSC Mito-EVs reduced the levels of activated pyroptosis protein GSDMD-NT in the cytoplasm and mitochondria of OGD/R-injured PC12 cells, thereby alleviating mitochondrial inflammatory pyroptosis. The pyroptosis-suppressive effect of hUCMSC Mito-EVs was abolished in both the Tom1l2-KO Mito-EVs group and injured PC12 cells co-treated with hUCMSC Mito-EVs and siCRLS1. Meanwhile, the GSDMD-NT expression was increased in both the cytoplasm and mitochondria of these cells. This confirmed that Crls1 is a key molecule for hUCMSC Mito-EVs to regulate the pyroptosis pathway (Fig. 6F–H). LSCM observations revealed that GSDMD-NT fluorescence intensity in PC12 cells increased after OGD/R injury and decreased after hUCMSC Mito-EVs treatment, suggesting that hUCMSC Mito-EVs can alleviate pyroptosis. However, the GSDMD-NT fluorescence intensity rebounded in both the Tom1l2-KO Mito-EVs group and the hUCMSC Mito-EVs-treated group with Crls1 inhibition, indicating that pyroptosis in hypoxia-injured PC12 cells were not alleviated (Fig. 6I–M). Further experiments indicated that Crls1 inhibition in both the Tom1l2-KO Mito-EVs group and hUCMSC Mito-EVs treatment group weakened the regulatory effect on inflammatory pyroptosis, as manifested by a significant increase in the extracellular LDH release and secretion levels of IL-1β and IL-18. These findings suggest that the absence of Tom1l2 and Crls1 leads to the loss of Mito-EVs’ regulatory function on inflammatory pyroptosis, thereby abrogating their neuroprotective effect (Fig. 6N and SFig. 7B–C). To further determine whether mitochondrial functional recovery is required for suppressing ROS accumulation and pyroptotic progression after OGD/R injury, we subsequently examined CCCP-inactivated Mito-EVs. Consistent with their inability to restore mitochondrial function, hUCMSC Mito-EVs-CCCP also failed to reduce the ROS levels or inhibit pyroptosis. These results support that restoration of mitochondrial function is a prerequisite for the anti-pyroptotic and neuroprotective effects of Mito-EVs (SFig. 7D–J).
In summary, following the fusion of hUCMSC Mito-EVs with damaged mitochondria in OGD/R-injured neurons, Crls1 expression is upregulated in recipient cells. First, this upregulation reshapes glucose-centered energy metabolism, reverses abnormal glycolysis, and reactivates the TCA cycle to restore oxidative phosphorylation. Second, it enhances cardiolipin synthesis, thereby inhibiting the pyroptosis pathway and reducing GSDMD-NT-mediated damage and the secretion of inflammatory factors.
Mito-EVs can rescue damaged neurons in MCAO rats and improve prognosis through mitochondrial transplantation
3.7
To clarify the in vivo therapeutic efficacy of hUCMSC Mito-EVs, this study adopted a rat MCAO model and administered the vesicles via the orbital vein [50] (Fig. 7A). First, rat brain tissues were sectioned. Co-labeling with NeuN and Mito-EVs markers confirmed that hUCMSC Mito-EVs could cross the blood-brain barrier and be taken up by neurons in the infarct region (Fig. 7B).Fig. 7. Mitochondrial extracellular vesicles (Mito-EVs) can rescue damaged neurons in middle cerebral artery occlusion (MCAO) rats and improve prognosis through mitochondrial transplantation(A) Schematic diagram of MCAO model establishment and Mito-EVs administration via orbital venous injection. (B) Neuronal uptake of Mito-EVs in vivo (Mito-EVs labeled with red fluorescence; neurons labeled with green fluorescence via NeuN immunostaining). (C–D) Transmission electron microscopy (TEM) observation and quantitative analysis of mitochondrial morphology (length/width ratio) in peri-infarct brain tissue of rats (n = 10). (E) Relative level of l-lactate in the peri-infarct area of rat brains (n = 6). (F–G) Representative 2,3,5-Triphenyltetrazolium chloride (TTC) staining images of MCAO rat brain tissue (red: normal brain tissue; white: infarcted tissue) and quantitative analysis of cerebral infarct volume n = 6). (H) TUNEL-NeuN double immunofluorescence staining and quantitative analysis of apoptotic neurons in the peri-infarct area (n = 3). (I–J) Representative trajectory heatmaps and quantitative analysis of target zone residence time of rats in Morris water maze (MWM) test (n = 6). (K) Modified neurological severity score (mNSS) results of MCAO rats after Mito-EVs treatment (n = 6). (L) Rotarod test results of MCAO rats after Mito-EVs treatment (n = 6).Data are presented as mean ± standard error of the mean. Statistical analysis: one-way ANOVA followed by Tukey's post hoc test was used for multiple-group comparisons (D, E, G, H, J); two-way ANOVA followed by Tukey's post hoc test was used for comparisons involving two variables (K, L). ns. P ≥ 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.Fig. 7
Brain tissue samples were collected from the peri-infarct region. TEM revealed that mitochondrial swelling was significantly alleviated and cristae architecture was restored in the hUCMSC Mito-EVs treatment group. However, both Tom1l2-KO Mito-EVs and hUCMSC Mito-EVs combined with siCRLS1-mediated Crls1 knockdown markedly impaired mitochondrial structural repair (Fig. 7C and D). We further examined the ATP and l-lactate levels, as well as the expression of key OXPHOS proteins and mitochondrial function-related proteins in the brain tissue surrounding the infarct. The results demonstrated that, compared with the MCAO model group, the hUCMSC Mito-EVs treatment group effectively restored the ATP content and reduced the l-lactate levels. Additionally, the key OXPHOS proteins, along with Tom1l2 and Crls1 expression, were significantly upregulated, and cardiolipin expression was also restored, thereby restoring the phosphatidylserine levels, stabilizing mitochondrial inner membrane homeostasis, and enhancing mitochondrial function. Conversely, both the Tom1l2-KO Mito-EVs treatment group and siCrls1-mediated knockdown group exhibited diminished therapeutic effects (Fig. 7E and SFig. 8A–H). We further isolated the mitochondrial and cytoplasmic fractions from peri-infarct brain tissue for Western blot analysis. The results showed that hUCMSC Mito-EVs treatment significantly reduced the expression of the pyroptosis-related molecule GSDMD-NT in both the mitochondria and cytoplasm, indicating an effective inhibition of mitochondrial pyroptosis [51]. Conversely, either Tom1l2-KO Mito-EVs or siCrls1-mediated knockdown in MCAO model rats reversed the inhibitory effect of hUCMSC Mito-EVs on mitochondrial pyroptosis and abrogated the recovery of damaged mitochondrial function (SFig. 8I–K).
TTC staining further confirmed that hUCMSC Mito-EVs treatment significantly reduced the cerebral infarction area in MCAO model rats (Fig. 7F and G). Simultaneous verification via TUNEL staining and Nissl staining showed that hUCMSC Mito-EVs moderately increased the number of surviving neurons around the infarction area (Fig. 7H and SFig. 8L–N). Behavioral experiments further revealed that compared with the MCAO model group, MCAO rats treated with hUCMSC Mito-EVs had a prolonged stay in the water maze target quadrant, a significantly lower modified neurological severity score (mNSS) score, and an extended rotarod retention time (Fig. 7I–L), indicating improvements in the neuromotor function and spatial learning and memory. These results confirmed that hUCMSC Mito-EVs alleviated pathological damage to brain tissue by promoting mitochondrial repair and ultimately restore neurological function. In contrast, compared with the hUCMSC Mito-EVs treatment group, both the Tom1l2-KO Mito-EVs treatment group and the group of MCAO rats co-treated with hUCMSC Mito-EVs and siCrls1 exhibited significantly inferior outcomes in the mitochondrial morphology and function repair, infarction area pathological improvement, and neurological function scores. This further confirms that Tom1l2 and Crls1 play a core regulatory role in the neuroprotective effect mediated by hUCMSC Mito-EVs.
Finally, we assessed the biosafety profile of hUCMSC Mito-EVs in vivo. Paraffin-embedded sections of major organs, including the heart, liver, spleen, lungs, and kidneys, were subjected to hematoxylin and eosin (HE) staining for histological analysis using a light microscopy (SFig. 9A). Consistent with previous reports, HE staining revealed normal tissue architecture without any evidence of pathological alterations or tumorigenic potential (unrestrained cell growth) associated with the administered Mito-EVs.
In conclusion, hUCMSC Mito-EVs transplantation in MCAO rats can effectively repair the structure of damaged mitochondria, improve the function of the mitochondrial oxidative respiratory chain, thereby rescuing damaged neurons around the infarct area, and ultimately improving neurological outcomes [52].
Discussion
4
IS remains the leading cause of irreversible neurological deficits worldwide, with ischemia–reperfusion injury (IRI)–induced mitochondrial dysfunction representing a central pathological driver of neuronal death [53]. Current clinical interventions largely fail to address mitochondrial damage itself, thereby limiting sustained neurological recovery [54]. In this study, we found that hUCMSC Mito-EVs, acting as a mitochondrial repair system, integrate into the damaged neuronal mitochondria via a Tom1l2-dependent mechanism, providing a structural foundation for restoring their bioenergetic homeostasis. The restoration of mitochondrial structural integration promotes elevated Crls1-mediated repair of the inner mitochondrial membrane and reestablishes oxidative phosphorylation (OXPHOS), thereby shifting the metabolism of damaged neurons from glycolysis to aerobic respiration. hUCMSC Mito-EVs synergistically promote both structural repair and functional improvement of the mitochondria, ultimately alleviating ischemic neuronal injury and offering a novel therapeutic approach for IS [55].
Our study demonstrates that Tom1l2 is an enrichment factor of Mito-EVs, and that its depletion in donor hUCMSCs markedly reduces the integration of transferred mitochondria with damaged neuronal mitochondria, thereby attenuating downstream bioenergetic recovery. Notably, Tom1l2 is significantly reduced under ischemic injury conditions and in the CSF of patients; however, the underlying mechanisms responsible for this reduction remain unclear. Multiple non-mutually exclusive mechanisms may contribute. Ischemia–reperfusion and its associated inflammatory milieu may reprogram the transcriptional states of neurons and glial cells, potentially suppressing Tom1l2 expression [56,57]. Oxidative stress and proteotoxic stress may accelerate Tom1l2 turnover through the ubiquitin–proteasome or lysosomal/autophagy pathways [58]. In parallel, energy metabolic dysfunction accompanied by impaired membrane trafficking may disrupt secretory processes and extracellular vesicle biogenesis, thereby reducing the release of Tom1l2-containing vesicles into the CSF. Moreover, injury-induced alterations in the cellular composition of CSF-contacting sources, such as neuronal loss and glial activation, may further modulate the extracellular release of Tom1l2. Although the current data do not allow discrimination among these possibilities, the observed pathological decline suggests that Tom1l2 availability is limited under ischemic stress, providing a theoretical rationale for delivering Mito-EVs endowed with Tom1l2 function to restore efficient mitochondrial integration and subsequent bioenergetic recovery.
Mitochondrial transfer has been reported in several scenarios, including via tunnel nanotubes (TNTs), cell fusion, or vesicle-mediated delivery [59]. Mitochondrial interactions via TNTs rely on direct intercellular contact [60]; however, such contact is often disrupted after cerebral ischemia, which may partially impede the intercellular mitochondrial transfer [61]. Conventional mitochondrial transplantation typically relies on the direct transfer of intact mitochondria, but suffers from poor stability, limited blood-brain barrier permeability, and high immunogenicity [62]. Cell- or vesicle-based mitochondrial transplantation, encapsulating functional mitochondria within native lipids, has laid the foundation for engineered applications [63]. However, such approaches face numerous challenges including safety concerns. Our study identifies a cell-free alternative that preserves mitochondrial health while avoiding tumorigenicity concerns associated with direct delivery by tool cells. hUCMSC Mito-EVs bind to dysfunctional mitochondria via Tom1l2-mediated interaction and restore the inner mitochondrial membrane (IMM) function through the Tom1l2–Crls1 axis, thereby salvaging the cellular function.
Despite these advantages, this study has several limitations that warrant attention. First, although our experiments demonstrated a significant reduction in the mitochondrial fusion between Tom1l2-deficient mitochondrial vesicles and neuronal mitochondria, whether TOM1L2 directly mediates mitochondrial fusion and the specific mechanism involved remain to be further clarified. Second, the inherent heterogeneity of Mito-EVs, including variations in the mitochondrial content, cargo composition, and potential off-target effects, is an unavoidable yet critical issue. It is important to note that vesicles extracted under different centrifugation parameters exhibit significant differences [64]. To mitigate this, we standardized the extraction conditions for each batch of vesicles and minimized the variability through BCA protein quantification. The carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment results indicate that mitochondria within the vesicles primarily mediate the therapeutic effects in this study, while nucleic acids, lipids, or other small proteins in the vesicles play a relatively limited role. In this study, the mitochondrial vesicles do not specifically target damaged neurons, as they may also be taken up by cells such as microglia and astrocytes [65]. Nevertheless, we primarily focused on the direct effects of mitochondrial vesicles on neurons and confirmed these effects through in vitro experiments. Future work could incorporate engineering modifications to enhance the targeting specificity of mitochondrial vesicles. Finally, although the Mito-EVs used in this study were derived from hUCMSCs, the in vivo experiments were limited to the rat middle cerebral artery occlusion (MCAO) model, making clinical translation a formidable challenge. Future work requires further validation in non-human primates, along with detailed assessments of dosage, immunogenicity, and long-term safety [66,67].
In conclusion, this study mechanistically elucidates a novel therapeutic strategy by which hUCMSC Mito-EVs rescue ischemic neurons via the Tom1l2–Crls1 axis. Tom1l2 mediates the targeted fusion of healthy mitochondrial components carried by Mito-EVs with damaged neuronal mitochondria, while Crls1 restores cardiolipin synthesis, repairs the inner mitochondrial membrane, reconstructs OXPHOS function, and inhibits mitochondrial pyroptosis [68]. By simultaneously addressing metabolic dysfunction and inflammatory cell death, this work provides critical mechanistic insight into mitochondrial replacement therapy and offers a promising foundation for the development of targeted translational interventions aimed at improving the prognosis of patients with IS.
Fundings
National Natural Science Foundation of China (Grant No. 82301561 and 82471422), Postgraduate Research &Practice Innovation Program of Jiangsu Province (SJCX24_2054 and KYCX25_3813).
CRediT authorship contribution statement
Ziheng Li: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Xingjia Zhu: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Weiquan Liao: Data curation, Formal analysis, Investigation, Methodology, Software, Validation. Rui Jiang: Data curation, Investigation, Software, Validation. Enze Sang: Data curation, Investigation, Software, Validation. Jue Zhu: Formal analysis, Investigation, Methodology. Gaojia Sun: Formal analysis, Writing – review & editing. Zhichao Lu: Investigation, Methodology, Writing – review & editing. Chenxing Wang: Formal analysis, Investigation. Yi Jiang: Data curation, Methodology, Writing – review & editing. Jian Chen: Data curation, Methodology, Writing – review & editing. Peipei Gong: Data curation, Methodology, Supervision, Writing – review & editing. Qianqian Liu: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Specifically, none of the authors have any financial or non-financial associations (including but not limited to employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties) with any organization or entity that has a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.
All authors confirm that the content of this manuscript is free from any conflicts of interest, and the research was conducted in accordance with the principles of academic integrity.
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