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. 2024 Apr 4;21(4):e00355. doi: 10.1016/j.neurot.2024.e00355

Mitochondrial transplantation exhibits neuroprotective effects and improves behavioral deficits in an animal model of Parkinson's disease

Hyeyoon Eo a,1, Shin-Hye Yu b,1, Yujin Choi a, Yujin Kim b, Young Cheol Kang b, Hanbyeol Lee a, Jin Hee Kim a, Kyuboem Han b, Hong Kyu Lee b, Mi-Yoon Chang c,d, Myung Sook Oh a,e,⁎, Chun-Hyung Kim b,⁎
PMCID: PMC11067340  PMID: 38580511

Abstract

Mitochondria are essential organelles for cell survival that manage the cellular energy supply by producing ATP. Mitochondrial dysfunction is associated with various human diseases, including metabolic syndromes, aging, and neurodegenerative diseases. Among the diseases related to mitochondrial dysfunction, Parkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic neuronal loss and neuroinflammation. Recently, it was reported that mitochondrial transfer between cells occurred naturally and that exogenous mitochondrial transplantation was beneficial for treating mitochondrial dysfunction. The current study aimed to investigate the therapeutic effect of mitochondrial transfer on PD in vitro and in vivo. The results showed that PN-101 mitochondria isolated from human mesenchymal stem cells exhibited a neuroprotective effect against 1-methyl-4-phenylpyridinium, 6-hydroxydopamine and rotenone in dopaminergic cells and ameliorated dopaminergic neuronal loss in the brains of C57BL/6J mice injected 30 ​mg/kg of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally. In addition, PN-101 exhibited anti-inflammatory effects by reducing the expression of pro-inflammatory cytokines in microglial cells and suppressing microglial activation in the striatum. Furthermore, intravenous mitochondrial treatment was associated with behavioral improvements during the pole test and rotarod test in the MPTP-induced PD mice. These dual effects of neuroprotection and anti-neuroinflammation support the potential for mitochondrial transplantation as a novel therapeutic strategy for PD.

Keywords: Parkinson's disease, Mitochondria, Transplantation, Neuroprotective, Mitotherapy

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disorder and affects >2% of the population over age 65 [1]. The disease is pathologically characterized by the selective loss of dopaminergic neurons within the substantia nigra (SN) pars compacta, leading to dopamine deficiency in the basal ganglia and symptoms of resting tremor, rigidity, and bradykinesia [2]. Although the administration of the dopamine precursor, levodopa (L-3,4-dihydroxy-phenylalanine, l-DOPA), improves the quality of life for patients with PD [3], long-term exposure of l-DOPA can lead to the involuntary movements and dyskinesias [[3], [4], [5]]. Therefore, there is an urgent need for novel therapeutic approaches to stop or slow selective neuronal degeneration.

There are multiple pathologies involving in development and progression of PD. Among them, injured mitochondria is one of the crucial factor of PD progression as they can promote various pathogenic molecular mechanisms such as oxidative stress, inflammation and apoptosis which are necessary and sufficient condition in neurodegeneration [6]. Indeed, mitochondrial abnormalities have been observed not only in environmental toxin-induced models of the disease, but also in both sporadic and genetic forms of PD [2,7]. One of the toxins inducing PD-like symptoms is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is a complex I inhibitor of the electron transport chain in mitochondria [2,8,9]. In general, individuals with PD show impairments in mitochondrial membrane potential due to a decrease in complex I activity but an increase in reactive oxygen species (ROS) [10,11]. Impaired mitochondrial DNA homeostasis and alterations in mitochondrial morphology and transport are also features of patients with PD and PD-specific models [[12], [13], [14]]. Therefore, targeting mitochondrial abnormalities may lead to promising therapies for PD.

Recently, it was reported that mitochondria isolated from various cell sources can enter any cell type in vitro [[15], [16], [17], [18]] as well as be internalized into tissues via local or systemic injection in vivo [[19], [20], [21], [22]]. Furthermore, mitochondria intravenously injected preferentially travel to cells and tissues wherein mitochondria have been damaged [22]. With the accumulating evidence of mitochondrial transfer, transplantation of intact functional mitochondria has received attention as an attractive therapeutic strategy for treating various diseases. Mitochondrial transplantation therapy using autologous, allogeneic, and xenogeneic mitochondria has shown potential therapeutic effects in ischemia/reperfusion injury, neurodegenerative diseases, tissue injury, and inflammatory diseases by supplementing adenosine triphosphate, enhancing cell proliferation, alleviating excessive inflammation, and preventing oxidative damage [[23], [24], [25], [26], [27], [28]]. Together, these reports suggest that mitochondrial transplantation could be a potential therapeutic strategy for treating diseases caused by mitochondrial dysfunction.

Human umbilical cord mesenchymal stem cell (UC-MSCs)-derived mitochondria, named as a PN-101, showed a strong anti-inflammatory effect by mediating the blockade of the nuclear factor kappa B (NFκB) signaling pathway [27]. Recently, a phase 1/2a clinical trial of PN-101 in patients with refractory polymyositis (PM) and dermatomyositis (DM) was conducted to investigate safety and efficacy. The present study aim was to determine if PN-101 can be a potential treatment for PD. The results of in vitro and in vivo models of PD showed that PN-101 had neuroprotective and anti-inflammatory effects. Intravenous PN-101 treatment also led to behavioral improvements in MPTP-induced PD mice. Together, our data suggest that PN-101 can be developed as a novel therapeutic for PD.

Materials and Methods

Cell culture and culture conditions

Human UC-MSCs were obtained by primary culture of the umbilical cord from a healthy pregnant woman who provided her informed consent. The study was approved by the Public Institutional Review Board (IRB) designated by the Ministry of Health and Welfare, Republic of Korea (IRB No. P01-202002-31-008). The UC-MSCs were cultured in Minimum Essential Medium Eagle Alpha Modification (α-MEM, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 10 ​ng/mL fibroblast growth factor-2 (FGF-2; CHA Meditech Co, Republic of Korea). Human LUHMES cells were grown in a plate pre-coated with 50 ​μg/mL poly-l-ornithine (Sigma, USA) and 1 ​μg/mL human plasma fibronectin (Gibco, USA). The cells were cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12; Gibco, USA) supplemented with 1x N-2 supplement (Invitrogen, USA) and 40 ​ng/mL FGF-2. For induction of differentiation, the cells were switched to DMEM/F-12 medium plus 1x N-2 supplement containing 1 ​μg/mL tetracycline hydrochloride (Sigma, USA), 0.1 ​mM dibutyryl cAMP (Sigma, USA), 10 ​μM Forskolin (Sigma, USA), and 10 ​ng/mL of human recombinant GDNF (PeproTech, USA). All experiments were conducted with LUHMES neuronal cells ≥5 days after induction of differentiation. Murine MN9D cells were grown in DMEM (Hyclone, USA) supplemented with 10% FBS. For induction of differentiation, the cells were switched to DMEM medium with 10% FBS, 1 ​mM n-butyrate (Sigma, USA), 1 ​mM dibutyryl cAMP, and 10 ​μM Forskolin. All experiments were performed with MN9D neuronal cells ≥6 days after induction of differentiation. Murine BV2 cells were cultured in DMEM supplemented with 10% FBS. HEK293T cells were cultured on poly-L-lysine-coated dishes in DMEM supplemented with 10% FBS.

Labeling of mitochondria

The mitochondrial targeting sequence (MTS) of succinate dehydrogenase complex subunit C was fused with green fluorescence protein (GFP) gene and red fluorescence protein (dsRED), resulting in MTS-GFP and MTS-dsRED, respectively. Lentiviral expression vectors were constructed by cloning MTS-dsRED and MTS-GFP genes into a lentiviral vector plasmid with EF1α promoter. For lentivirus production, HEK293T cells were seeded on 10-cm dishes at 70%–80% confluency. The cells were co-transfected with 4 ​μg lentiviral expression vector plasmid along with 4 ​μg pLP1, 4 ​μg pLP2, and 3 ​μg pLP/VCVG (Packaging plasmids, Invitrogen). Transfection mixtures were prepared in 1 ​mL of Opti-MEM containing the DNA and 30 ​μL of 293 Tran (Origene, USA) according to the manufacturer's protocol. After transfection, lentiviral supernatant samples were collected after 24, 48, and 72 ​h. The collected lentiviral supernatants were concentrated via ultracentrifugation, and the medium was exchanged with α-MEM medium. To label mitochondria with dsRED and GFP, UC-MSCs were seeded into 150-mm plates at 70%–80% density and then incubated with lentiviral supernatants and 4 ​μg/mL polybrene followed by further incubation for 72 ​h.

Mitochondria isolation

UC-MSCs were used at passage 12 for mitochondria preparation. The cells were harvested from culture flasks, depressurized in SHE buffers [0.25 ​M Sucrose, 20 ​mM HEPES (pH 7.4) and 2 ​mM EGTA] using an ultrasonic processor (VCX130, USA), and then centrifuged at 2000 ​× ​g for 10 ​min at 4 ​°C to remove cellular debris and nuclei. The supernatant was then centrifuged at 12,000 ​× ​g for 15 ​min at 4 ​°C to pellet the mitochondria. The pellet was washed twice by suspension in 1 ​mL of SHE buffer followed by centrifugation at 20,000 ​× ​g for 1 ​min at 4 ​°C. The final pellet was resuspended in 100 ​μL suspending buffer and kept on ice until use. Isolated mitochondria (PN-101) were quantified by determining protein concentrations using a bicinchoninic acid assay (BCA protein assay; Thermo, USA). The labeled mitochondria were treated in the differentiated LUHMES cells or MN9D cells for 24 ​h for in vitro experiment. For biodistribution of the labeled mitochondria, they were intravenously injected into ICR male mice (7-weeks-old), which were then sacrificed 24 ​h after the injection. For further assays, cells and brain tissues were fixed with pre-chilled 4% paraformaldehyde.

Cell cytotoxicity assay

For the cell cytotoxicity assay, we used three different neurotoxic substances. Differentiated LUHMES cells or MN9D cells were treated with 200 ​μM MPP+, 10 ​μM 6-hydroxydopamine (6-OHDA), or 0.2 ​μM rotenone for 6 ​h, followed by treatment with 10 ​μg PN-101. After 48 ​h, the cells were immunostained with an anti-TH antibody, and the supernatant was subjected to the lactate dehydrogenase activity assay (LDH assay; Promega, USA) according to the manufacturer's protocol. To evaluate the cytotoxicity of TH-positive cells, the fluorescence intensity of TH was quantified using ImageJ software and was normalized by that of DAPI. Data are mean ​± ​SD, n ​= ​5.

Immunofluorescence assay

The fixed cells and brain sections were washed, and then incubated with blocking buffer [phosphate-buffered saline (PBS), 10% goat serum (Invitrogen), 0.1% Triton X-100 (Sigma)] for 30 ​min. The cells were then incubated overnight at 4 ​°C with anti-TH antibody (1:1000, Merck Millipore, Burlington, MA, USA) in PBS containing 1% normal goat serum. For the biodistribution analysis, free floating brain sections from ICR mice were incubated with anti-TH antibody (1:1000, Merck Millipore), anti-glial fibrillary acidic protein (GFAP) antibody (1:2000, Invitrogen, Waltham, MA, USA) or anti-ionized calcium-binding adapter molecule-1 (Iba-1) antibody (1:1000, Fujifilm Wako, Chuo-Ku, Osaka, Japan) overnight at 4 ​°C. Following three washes with PBS, appropriate fluorescence-tagged secondary antibodies (Invitrogen) were used for visualization. Stained samples were mounted in VECTASHIELD with 4′,6-diamidino-2-phenylindole (DAPI) mounting solution (Vector Laboratories Inc., Burlingame, CA). The fluorescence images were evaluated under a confocal microscope (Fluoview FV3000, Olympus).

Enzyme-linked immunosorbent assay (ELISA) for cytokine

To measure the amount of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) released by the cells, 1.0 ​× ​105 BV2 cells were incubated with 1 ​μg/mL of lipopolysaccharide and 10 ​μg PN-101 for 6 ​h. The amount of TNF-α and IL-6 released in the culture medium was quantified using ELISA kits (Abcam, United Kingdom) according to the manufacturer's instructions. Each kit's standard curves (TNF-α and IL-6 respectively) were used to quantify the amount of TNF-α and IL-6 released by the cells.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR)

The mRNA expressions of TNF-α, IL-6, IL-1β, and inducible nitric oxide synthase (iNOS) were analyzed by quantitative RT-PCR. Then, 1.0 ​× ​105 BV2 cells were incubated with 1 ​μg/mL of LPS and 10 ​μg PN-101 for 6 ​h and harvested. Total RNAs from BV2 cells were isolated by use of a TRIzol reagent (Invitrogen, USA), according to the manufacturer's instructions. A 2-μg aliquot of RNA was reverse-transcribed into cDNA by used of an M-MLV cDNA synthesis kit (Enzynomics, Republic of Korea) according to the protocol provided. The specific primers used for the amplification of each gene were as follows: sense primer (5′ - GTC TCC TCT GAC TTC AAC AGC G - 3′) and antisense primer (5′- ACC ACC CTG TTG CTG TAG CCA A - 3′) for GAPDH; sense primer (5′ - TCT CAT CAG TTC TAT GGC CC - 3′) and antisense primer (5′ - GGG AGT AGA CAA GGT ACA AC - 3′) for TNF-α; sense primer (5′ - CCA AAC TGG ATA TAA TCA GGA AAT - 3′) and antisense primer (5′- CTA GGT TTG CCG AGT AGA TCT - 3′) for IL-6; sense primer (5′ - AAC CTG CTG GTG TGT GAC GTT C - 3′) and antisense primer (5′ - CAG CAC GAG GCT TTT TTG TTG T - 3′) for IL-1β; sense primer (5′ - CCT CCT CCA CCC TAC CAA GT - 3′) and antisense primer (5′ - CAC CCA AAG TGC TTC AGT CA - 3′) for iNOS. A QuantiSpeed SYBR Green Master Mix (Philekorea, Republic of Korea) was used to perform the quantitative real-time PCR. The housekeeping gene GAPDH was used as a control to normalize gene-expression levels.

Animals and experimental design

C57BL/6 male mice (7-weeks-old) were obtained from DBL (Eumseong, Republic of Korea). The mice were acclimatized under controlled conditions (temperature: 23 ​°C ​± ​1 ​°C, humidity: 60% ​± ​10%, 12-h light/dark cycle) for 1 week and were allowed free access to water and food. All of the animal studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals, 8th edition” (National Institutes of Health, 2011) and approved by the “Animal Care and Use Guidelines” of Kyung Hee University, Seoul, Republic of Korea (Approval number: KHSASP-23-204).

The mice were randomly divided into six groups to evaluate ameliorative effects of PN-101 on PD as follows [1]: control group: vehicle-injected plus vehicle-treated group, n ​= ​16) [2]; MPTP group: MPTP-injected plus vehicle-treated group, n ​= ​16 [3]; low-dose PN-101 group: MPTP-injected plus 0.5 ​μg of PN-101-treated group, n ​= ​8 [4]; medium-dose PN-101 group: MPTP-injected plus 2.5 ​μg of PN-101-treated group, n ​= ​8 [5]; high-dose PN-101 group: MPTP-injected plus 10 ​μg of PN-101-treated group, n ​= ​16); and [6] l-DOPA group: MPTP-injected plus 80 ​mg/kg of l-DOPA with 20 ​mg/kg benserazide-treated group, n ​= ​16). Specifically, 30 ​mg/kg of MPTP or an equal volume of the vehicle was injected intraperitoneally for 5 days, the PN-101 or equal volume of its vehicle was treated intravenously after MPTP induction, and 80 ​mg/kg of l-DOPA with benserazide was administered by oral gavage starting from day 6 daily until day 12. There was no dead mouse during any intervention process. All behavioral tests were performed after acclimation to the behavioral room environment for 1 ​h.

Pole test

The pole test was performed on the sixth (training session) and seventh (test session) day after the last MPTP injection. The mice were positioned head-up at the top of a 55-cm vertical pole with a diameter of 8 ​mm. The time to turn (T-turn) completely downward and time to land on the floor (T-LA) were recorded, respectively.

Rotarod

The rotarod test was performed on the sixth (training session) and seventh (test session) day after the last MPTP injection. The rotarod device contained a rotating spindle (3.0-cm diameter, Jeung Do Bio & Plant Co., Ltd.) and five separate compartments for the simultaneous examination of five mice. The rotarod tests were performed at a constant speed of 30 ​rpm for 3 ​min. The training was performed by placing the mouse on a spindle whenever it fell on the ground. During the test session, the test was performed at the same rotational speed as that of the training. The time when the mice first dropped from the spindle (latency time) and the number of total falls were recorded.

Preparation of brain tissues

On the last day of the animal experiment, the mice were anesthetized with tribromoethanol (312.5 ​mg/kg, i.p.), perfused transcardially with 0.05 ​M PBS, and then fixed with pre-chilled 4% paraformaldehyde in 0.1 ​M phosphate buffer. The whole brains were immersed in 0.05 ​M PBS containing 30% sucrose for cryoprotection. Serial 25-μm thick coronal sections were cut on a freezing microtome (Leica, Nussloch, Germany) and then stored in cryoprotectant (25% ethylene glycol, 25% glycerol, and 0.05 ​M phosphate buffer) at 4 ​°C before use.

Histological and immunohistochemical staining

Brain sections from C57BL/6 mice were rinsed in PBS and incubated with 1% H2O2 in 0.05 ​M PBS for 15 ​min. After washing with PBS again, the sections were incubated with anti-TH antibody (Merck Millipore, Burlington, MA, USA) or anti-ionized calcium-binding adapter molecule-1 (Iba-1) antibody (Fujifilm Wako, Chuo-Ku, Osaka, Japan) diluted in 0.3% Triton X-100 (1:1000 and 1:500, respectively) overnight at 4 ​°C. The sections were subsequently incubated with biotinylated anti-rabbit IgG antibody (1:500 dilution) and incubated in an avidin–biotin complex solution. To develop the color of each section, 3,3-diaminobenzidine was used. Nissl staining was performed to evaluate neuronal survival as previously described [29]. Briefly, brain sections were washed with PBS and then mounted onto a slide. After then the slide was incubated with 0.5% Cresyl violet (Sigma, USA) and then dehydrated by serial graded alcohol solution. Confocal microscopy (K1-Fluo; Nanoscope Systems, Daejeon, Republic of Korea) was used to capture images. Optical density of TH in striatum was measured by using the Image J software [National Institutes of Health (Bethesda, MD, USA)]. TH-immunoreactive cells in the substantia nigra pars compacta (SNpc) and Iba-1-immunoreactive cells with >3 branches in the striatum and cortex were quantified by stereological counting, and percentage of Nissl-positive neurons in SN were quantified by using Image J software [National Institutes of Health (Bethesda, MD, USA)]for analysis. The organizational area was determined by comparing the images with a stereotaxic atlas of the mouse brain [30].

Statistical analysis

Data are expressed as the mean ​± ​standard error of the mean (SEM). GraphPad Prism ver. 8.0.1 (GraphPad Software Inc., San Diego, CA, USA) was used to perform all statistical analyses. A one-way analysis of variance (ANOVA) with Dunnett's post-hoc test was performed for multiple comparisons, and differences between the MPTP group and another group were considered to be statistically significant for values of p ​< ​0.05 (shown in each figure).

Results

Migration of exogenous fluorescence-labeled mitochondria into dopaminergic cells

Exogenous mitochondria have been known to be internalized into cells via micropinocytosis [16]. We first tested whether or not fluorescence-labeled mitochondria could be transferred to dopaminergic cells. To prove the transfer efficacy, MTdsRED and MTGFP were isolated from UC-MSCs infected, lentivirus-mediated MTS-dsRED and MTS-GFP by differential centrifugation [22] and were co-incubated with dopaminergic cells differentiated from LUHMES and MN9D, respectively. As a result, the fluorescence-labeled mitochondria were successfully transferred into dopaminergic cells by simple coincubation (Fig. 1 and Supplementary Fig. 1.)

Fig. 1.

Fig. 1

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Transplantation of MTdsREDinto the LUHMES cell. The left panel indicates the schematic diagram of the treatment of the LUHMES cells with MTdsRED, and the right panel shows representative immunofluorescence images. MTdsRED was obtained from UC-MSCs, which were infected with lentivirus expressing MTS-dsRED. The cell nuclei were stained with DAPI. MTdsRED were successfully transferred into the LUHMES cells.

Effect of PN-101 treatment on dopaminergic cells damaged by neurotoxins

Next, to investigate the neuroprotective effects of PN-101 in a therapeutic window context, dopaminergic cells differentiated from LUHMES and MN9D were exposed to various neurotoxins, including MPP+, 6-OHDA, or rotenone, followed by the treatment of PN-101 after 6 ​h. As shown in Fig. 2B and Supplementary Fig. 2A, neurotoxins prominently altered their cellular morphology into shortened and fragmented neurites but PN-101 improved the abnormalities of neurites. To quantitatively validate the effect of neurotoxins on TH expression, the intensity of TH immunoreactivities in each of neurotoxins was further evaluated (Fig. 2C). Similarly, MPP+, 6-OHDA and rotenone increased the cytotoxicity of the dopaminergic cells but the delayed treatment with the PN-101 significantly reduced neurotoxin-induced cell death.

Fig. 2.

Fig. 2

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Neuroprotective effects of PN-101 in the neurotoxin-induced LUHMES cells. (A) Schematic representation of the treatment of PN-101 to LUHMES. 1 ​× ​106 differentiated LUHMES cells treated with 200 ​μM MPP+, 10 ​μM 6-OHDA, and 0.2 ​μM rotenone, respectively. After 6 ​h, 10 ​μg PN-101 was administered for 48 ​h. (B) Immunocytochemistry of TH. TH-positive cells visualized by immunostaining against TH protein. The treatment of neurotoxins altered the cellular morphology into shortened and fragmented neurites in the TH-positive neurons, whereas PN-101 treatment restored the neurotoxin-induced neurite abnormalities of the TH-positive neurons. TH is in green and nucleus staining is in blue. Scale bar ​= ​100 ​μm. (C) Quantitative analysis of immunofluorescence intensity in TH-positive cells. Treatment of neurotoxins decreased the TH fluorescent signals, whereas PN-101 treatment restored the TH fluorescent signals in cells. (C) Quantitative analysis of immunofluorescence intensity in TH-positive cells. The fluorescence intensity of TH was quantified using ImageJ software and normalized with their DAPI intensity (n ​= ​5 images/group). The treatment of neurotoxins decreased the fluorescence intensity of TH, whereas PN-101 restored. One-way ANOVA with Dunnett's post-hoc test was used for statistical comparison. ∗p ​< ​0.05, ∗∗p ​< ​0.01, and ∗∗∗p ​< ​0.001 vs. MPP+, 6-OHDA, or rotenone-only treated group, respectively.

Effect of PN-101 treatment on neuroinflammation in LPS-treated BV2 cells

The effect of PN-101 on neuroinflammation was determined by measuring the mRNA expression of pro-inflammatory cytokines in murine microglia BV2 cells. As shown in Fig. 3A, the mRNA expressions of pro-inflammatory cytokines, such as TNF-α, iNOS, IL-6, and IL-1β were significantly higher in the LPS-treated BV2 cells than in the controls. However, the treatment of PN-101 in LPS-activated BV2 microglia attenuated the mRNA expressions of TNF-α (p ​< ​0.01, n ​= ​5 in each group) and iNOS (p ​< ​0.05, n ​= ​5 in each group) (Fig. 3A). Although statistically insignificant, PN-101 also tended to attenuate LPS-induced IL-6 and IL-1β expressions in BV2 cells.

Fig. 3.

Fig. 3

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Anti-inflammatory effects of PN-101 in LPS-induced BV2 cells. Murine microglia BV2 cells treated with 1 ​μg/mL LPS in the absence or presence of PN-101 for 6 ​h. (A) The mRNA expression of cytokine genes as measured by quantitative RT-PCR. Reduction of mRNA expression of TNF-α, IL-6, IL-1β, and iNOS by PN-101 in LPS-stimulated BV2 cells. (B) The amount of TNF-α and IL-6 proteins released to the cultured media measured by ELISA assays. PN-101 attenuated TNF-α and IL-6 production induced by LPS. One-way ANOVA was used for statistical comparison. Data are presented as the mean ​± ​SEM (n ​= ​5). ∗p ​< ​0.05, ∗∗p ​< ​0.01, and ∗∗∗p ​< ​0.001 vs. LPS-only treated group.

To further explore the effect of PN-101 on TNF-α and IL-6 release in BV2 microglia, TNF-α and IL-6 production in LPS-induced BV2 cells was measured by performing ELISA. The concentrations of TNF-α and IL-6 in the medium were significantly higher in the LPS-stimulated BV2 cells than in the controls, whereas the treatment of PN-101 significantly alleviated LPS-induced TNF-α and IL-6 release in a dose-dependent manner (Fig. 3B).

Biodistribution of intravenously injected fluorescence-labeled mitochondria in the midbrain

To examine the biodistribution of mitochondria in the midbrain, MTGFP was systemically administrated into mouse. Twenty-four hours after intravenous injection, the green fluorescence intensity was clearly observed in the midbrain, indicating that MTGFP migrate into the midbrain through the blood-brain barrier (BBB) (Fig. 4A–C). To further test whether MTGFP was transferred into which type of cells in the midbrain, the brain sections were labeled with antibodies against GFAP, TH, and Iba-1 to detect the astrocyte, dopaminergic neuron, and microglia, respectively. The fluorescence signal was remarkably detected in GFAP-positive cells (Fig. 4A), but rarely observed in TH and Iba1-positive cells (Fig. 4C), suggesting that the mitochondria were actively taken up by the astrocyte.

Fig. 4.

Fig. 4

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Biodistribution of MTGFP. Representative images of TH, Iba-1, and GFAP immunofluorescence staining in the midbrain of control and intravenously injected MTGFP mice. The SN tissues were immunostained with GFAP (A; red), TH (B; red), and Iba-1 (C; red). White box indicates area of enlargement. The cell nuclei were stained with DAPI. MTGFP were successfully taken up by the astrocytes in the midbrain. Scale bars represent 100 ​μm.

Effect of PN-101 administration on motor deficits in the MPTP-induced PD mice

To investigate the therapeutic effect of PN-101 in a mice PD model, an MPTP-induced PD mice model was used in which the mice were intravenously treated with PN-101 in a concentration-dependent manner after MPTP injection (Fig. 5A). The pole test and a rotarod were used to assess motor behavior in an assessor-blinded manner. Compared with the vehicle-treated mice, the MPTP-treated mice exhibited significant motor deficits in all behavioral tests. Significantly shorter T-turn and T-LA in the pole test were observed in the mice treated with 10 ​μg of PN-101 than in the mice treated only with MPTP (Fig. 5B and C). During the rotarod test, significantly shorter latency to fall but a greater number of falls were observed in the MPTP group than in the control group. However, after administration of 10 ​μg of PN-101 to the MPTP-treated mice, the number of falls significantly decreased relative to those in the MPTP group (Fig. 5D and E). As a positive control, shorter T-turn and T-LA during the pole test as well as shorter latency to fall and a smaller number of falls during the rotarod test were observed in the l-DOPA-treated group relative to those in the MPTP group.

Fig. 5.

Fig. 5

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Effects of PN-101 in the MPTP-induced PD mice model. (A) Schematic representation of the administration of PN-101 to MPTP-induced mice. The mice were administered PN-101 at different concentrations (0.5 ​μg, 2.5 ​μg, and 10 ​μg) or saline once intravenously on day 6 following MPTP treatment. 80 ​mg/kg of l-DOPA was treated for 7 consecutive days after MPTP injection. PN-101 improved the motor abnormalities in (B) time to turn completely downward, (C) time to land on the floor in the pole test, (D) latency to fall, and (E) number of falls in the rotarod test. One-way ANOVA with Dunnett's post-hoc test was used for statistical comparison. Data are presented as the mean ​± ​SEM (m ​= ​8). ∗p ​< ​0.05, ∗∗p ​< ​0.01, and ∗∗∗p ​< ​0.001 vs. the MPTP group. Scale bars represent 500 ​μm.

Effect of PN-101 treatment on dopaminergic neuronal loss and microglial activation in the MPTP-induced PD mice

To evaluate the ameliorative effect of PN-101 administration on dopaminergic cell loss, the optical density of striatal TH and number of TH+ cells in the SN region were measured by performing immunohistochemistry (Fig. 6A–C). First, the MPTP-treated mice showed less expression of striatal TH, but the high dose PN-101 group exhibited significantly higher striatal TH expression (approximately 136% increase, p ​< ​0.01, Fig. 6B). In addition, TH+ cells were counted in the region of SN. The MPTP-treated mice showed severe loss of TH+ cells, whereas post-treatment of PN-101 to MPTP-treated mice significantly restored the reduction in the number of TH+ cells in the SN. Quantification of the TH+ cells demonstrated that the number of TH+ cells in the SN of the MPTP-treated mice was approximately 72% of the number in the control group (p ​< ​0.001), whereas it was restored up to 53% in the MPTP-induced mice by administration of PN-101 (Fig. 6C). In addition, MPTP-treated mice showed lower percentage of Nissl+ neurons in SN compared to the control group, but PN-101 showed higher expression of Nissl+ neurons in SN than MPTP group (Fig. 6D).

Fig. 6.

Fig. 6

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Immunohistochemical analysis of MPTP-induced PD mice treated with 10 ​μg of PN-101. The mice were administered 10 ​μg of PN-101 or saline once intravenously on day 6 following MPTP treatment. l-DOPA group was treated with 80 ​mg/kg of l-DOPA for 7 days after MPTP injection. Images of anti-TH, Nissle, and anti-Iba-1 staining on the representative brain sections in the striatum and SN were shown in the upper, middle, and lower panels, respectively (A). Optical density of striatal TH expression quantified as shown in (B). TH-positive cell counting in the SN region (C). Percentage of Nissl-positive neurons were quantified as shown in (D). PN-101 treatment increased the number of TH+ cells in the ST and SN, and Nissle+ cells in the SN whereas l-DOPA did not show any significant change. Quantification of Iba-1 expression in the striatum (E) and cortex (F). Scale bar ​= ​500 ​μm. One-way ANOVA with Dunnett's post-hoc test was used for statistical comparison. Data are expressed as the mean ​± ​SEM (n ​= ​16). ∗∗p ​< ​0.01, and ∗∗∗p ​< ​0.001 vs. the MPTP group.

The effect of PN-101 treatment on microglial activation also was investigated by immunohistochemical analysis of the microglial marker Iba-1in the microglia population. Microglial activation was prominently observed in the striatum (Fig. 6E), but not in the cortex regions in the MPTP group. In contrast, the post-treatment of PN-101 significantly reduced the numbers of MPTP-increased Iba-1+ microglia to the levels observed in the striatum of the controls (p ​< ​0.001). On the other hand, as a positive control, l-DOPA-treated group did not show significant change in TH+ cells in the ST and SN, Nissl+ cells in the SN or Iba-1+ microglia in the ST compared to the MPTP group.

Discussion

In this study, we demonstrated that mitochondrial transplantation ameliorated dopaminergic cell damage and neuroinflammation in vitro and in vivo. We demonstrated that exogenous fluorescence-labeled mitochondria were successfully transferred into dopaminergic neurons in vitro and astrocytes in vivo. Moreover, the results showed that isolated PN-101 mitochondria were successfully transferred into dopaminergic cells and reversed neurotoxins-induced cytotoxicity. In addition, PN-101 reduced mRNA expression and secretion of pro-inflammatory cytokines in microglial cells. Furthermore, intravenous PN-101 administration improved MPTP-induced motor declines observed in the mice model. Lastly, PN-101 treatment ameliorated dopaminergic neuronal loss and suppressed microglial activation in the brain. These results suggest that PN-101 can be a potential therapeutic treatment against PD by mediating both the neuroprotective and anti-inflammatory effects.

PD is one of the most prevalent neurodegenerative diseases and is caused by specific and progressive damage of mid-brain dopaminergic neurons. Although the cause and origin of PD remain largely elusive, the prevailing hypothesis is that PD-related neurodegeneration is associated with the mitochondrial dysfunction. This hypothesis was particularly strengthened by the pharmacological finding that the risk of developing PD was increased by exposure of neurotoxins, such as MPTP, rotenone, and paraquat, which impair the complex I activity of the electron transport chain located in mitochondria [9,31,32]. Those reports are consistent with the finding of a clinical study that complex I activity is remarkably compromised in patients with PD [10]. Interestingly, mitochondrial injury and dysfunction of mitochondrial complex I is closely related to various pathogenic molecular mechanisms including oxidative stress, endoplasmic reticulum stress, neuroinflammation and alpha-synuclein aggregation [33]. Hence, the best PD treatment would be one that can cure or replace certain damaged mitochondria directly. In this context, transplantation of healthy mitochondria to replace damaged ones is an emerging therapy in PD [22]. Interestingly, there are numerous pre-clinical studies on mitochondrial transplantation in various disease models, including models of ischemia and mental illness, in which direct or systemic injection of functional mitochondria enhanced tissue function, lowered oxidative stress, reduced inflammation and cell death, and rescued mitochondrial dysfunction [[34], [35], [36], [37], [38], [39], [40], [41], [42], [43]]. In addition, beneficial effects of mitochondrial transplantation have been observed in neurodegenerative diseases, such as Alzheimer's disease [40] and PD [20,44,45]. A research group previously demonstrated that intravenous mitochondrial administration ameliorated striatal oxidative stress induced by MPTP injection. However, the data from the previous study do not provide any evidence of neuroprotective and anti-inflammatory effects of mitochondrial transplantation at the organism level [20]. Thus, there is a need to explore the question of whether or not mitochondrial transplantation can directly recover dopaminergic neuronal loss and suppress neuroinflammation, which is a key therapeutic strategy in PD.

Recently, we developed clinical good manufacturing practices (GMP)-grade mitochondria (PN-101), which are isolated from human UC-MSCs. In a human phase 1/2a clinical trial, PN-101 demonstrated a good safety profile and encouraging efficacy in patients with PM/DM. Based on these promising results, the current study investigated the question of whether or not PN-101 could be a potential therapy for the treatment of PD, especially from the perspectives of neuronal damage and inflammation in vitro and in vivo.

First, we determined if PN-101 could be successfully transferred into dopaminergic neurons. Exogenous mitochondria have been shown to be successfully transferred into various cells by simple co-incubation [46,47], centrifugation [18], MitoCeption [48], and Mitopunch [49]. We also previously reported that GFP-labeled mitochondria isolated from HEK293 could be efficiently transferred to fibroblast cells by simple co-incubation [22]. Similarly, the current study demonstrated that fluorescence-labeled PN-101 mitochondria were directly transferred into differentiated LUHMES and MN9D cells.

Interestingly, exogenous mitochondria tagged with GFP were detected in the SN region and they were mostly colocalized with GFAP-positive cells which is a biomarker of astrocyte. This data is in line with the previous report that astrocytes can transfer the mitochondria into contiguous neurons intercellularly after injury in vitro [[50], [51], [52], [53]]. Taken together, our results imply that the exogenous mitochondria were first taken by astrocytes and might be further transferred into damaged neurons nearby.

With this context, the current study also evaluated the therapeutic effect of PN-101 treatment on dopaminergic cells, which were damaged with various types of common neurotoxins, including MPP+, 6-OHDA, and rotenone, at the cellular level. As a result, post-treatment of PN-101 significantly reduced neurotoxins-induced cytotoxicity regardless of the types of neurotoxins. In addition, abnormal morphology found in neurotoxin-treated dopaminergic cells were repaired when the cells were treated with PN-101. This data is consistent with our result showing that PN-101 administration improved dopaminergic neuronal loss in the striatum and SN induced by the MPTP injection. Thus, the results imply that mitochondrial transplantation has therapeutic potential for treatment of dopaminergic neuronal damage mainly found in the PD brain.

In addition to neuronal damage, we used in vitro and in vivo models of PD to evaluate the therapeutic effect of PN-101 on neuroinflammation. Neuroinflammation is one of the key features found in individuals with PD [54]. Generally, mitochondrial dysfunction generates excessive ROS, which can promote inflammatory response through activation of the NFκB pathway and NLR family pyrin domain-containing 3 inflammasomes [55]. Especially in neurological disorders, activated glia amplify ROS production and pro-inflammatory cytokine secretion, resulting in further mitochondrial dysfunction [55]. Previously, mitochondrial transplantation demonstrated potent anti-inflammatory effects in human macrophages by suppressing the NFκB signaling pathway [27]. Hence, we hypothesized that mitochondrial transplantation suppresses neuroinflammation by halting a vicious cycle of microglial activation and mitochondrial dysfunction. In the current study, PN-101 reduced mRNA expression of TNF-α and iNOS as well as secretion of pro-inflammatory cytokines, including TNF-α and IL-6 in the BV2 microglia activated by LPS. Likewise, PN-101 post-treatment significantly reduced expression of Iba-1+ cells in the striatum of the MPTP-injected mice. All of these results suggest that mitochondrial transplantation can alleviate striatal neuroinflammation by suppressing hyperactive microglia cells. Lastly, our study determined if PN-101 treatment can improve declines in movement function in MPTP-induced PD mice models. Since patients with PD have lower basal ganglia activity [56] and impaired motor coordination [57], the pole test to evaluate basal ganglia-related motor function and the rotarod test for motor control were performed. The results showed that PN-101 reduced the T-turn and L-LA during the pole test and improved latency to falls and number of falls during the rotarod test, suggesting that PN-101 post-administration significantly modified motor deficits observed in the MPTP-induced PD mice. Notably, all behavioral outcomes were consistent with our biochemical analysis results in the current study. Although several drugs have shown a neuroprotective effect against nigral cell death in preclinical studies, none of the agents with claimed neuroprotective activity have been approved yet for treatment. The nigral cell death process in PD involves complex etiological factors, including oxidative stress, inflammation, apoptosis, and mitochondrial complex I impairment, all of which have been directly or indirectly linked to mitochondrial dysfunction [58,59]. Given that PN-101 has both neuroprotective and anti-inflammatory effects, PN-101 transplantation could be a fundamental therapy for PD by restoring mitochondrial dysfunction and halting further PD progression.

Taken together, the study findings provide evidence that mitochondria transplantation can be developed as a novel therapeutic for PD. Especially, exogenous mitochondria were shown to be directly transferable into dopaminergic cells and to reduce dopaminergic neuronal damage and neuroinflammation. Thus, mitochondrial transplantation has great potential as a candidate treatment for neurodegenerative disorders, including PD.

Author Contributions

Hyeyoon Eo and Shin-Hye Yu: Data curation, Formal Analysis, Investigation, Visualization, Hyeyoon Eo and Shin-Hye Yu: Data curation, Formal Analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft, Writing – review & editing. Yujin Choi and Yujin Kim: Formal Analysis, Validation, Methodology. Young Cheol Kang: Resources, Validation, Methodology. Hanbyeol Lee and Jin Hee Kim: Investigation, Validation. Kyuboem Han: Funding acquisition, Project administration. Hong Kyu Lee: Project administration. Mi-Yoon Chang: Visualization, Writing – review & editing, Myung Sook Oh: Data curation, Supervision, Project Administration, Writing – original draft, Writing – review & editing. Chun-Hyung Kim: Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Project Administration, Writing – original draft, Writing – review & editing.

Data Availability

Data will be available on request.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Shin-Hye Yu reports a relationship with Paean Biotechnology that includes: employment. Yujin Kim reports a relationship with Paean Biotechnology that includes: employment. Young Cheol Kang reports a relationship with Paean Biotechnology that includes: equity or stocks. Kyuboem Han reports a relationship with Paean Biotechnology that includes: employment. Hong Kyu Lee reports a relationship with Paean Biotechnology that includes: employment. Chun-Hyung Kim reports a relationship with Paean Biotechnology that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

We thank the various members of Paean Biotechnology Inc. who participated in the project. This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2022R1A6A3A01087061 to H Eo and NRF-2022R1A2C2006061 to C.H. Kim).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.neurot.2024.e00355.

Contributor Information

Myung Sook Oh, Email: msohok@khu.ac.kr.

Chun-Hyung Kim, Email: chkim@paeanbio.com.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Multimedia component 1
mmc1.docx (557.1KB, docx)

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Supplementary Materials

Multimedia component 1
mmc1.docx (557.1KB, docx)

Data Availability Statement

Data will be available on request.

Articles from Neurotherapeutics are provided here courtesy of Elsevier

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