Abstract

Diabetes-associated cognitive impairment (DACI) can increase the risk of major cardiovascular events and death. Neuronal functionality is highly dependent on mitochondria and emerging evidence has shown that mitochondrial transplantation is a potential and effective strategy that can reduce brain injury and associated disorders. Platelets are abundant in blood and can be considered a readily available source of small-size mitochondria. These cells can be easily acquired from the peripheral blood with minimal invasion via simple venipuncture. The present study aimed to investigate whether transplantation of platelet-derived mitochondria (Mito-Plt) could improve DACI. Cognitive behaviors were assessed using the Morris water maze test in db/db mice. The results demonstrated that Mito-Plt was internalized into hippocampal neurons 24 h following intracerebroventricular injection. Importantly, one month following Mito-Plt transplantation, DACI was alleviated in db/db mice and the effect was accompanied with increased mitochondrial number, restored mitochondrial function, attenuated oxidative stress and neuronal apoptosis, as well as decreased accumulation of Aβ and Tau in the hippocampus. Taken together, the data demonstrated that transplantation of Mito-Plt attenuated cognitive impairment and mitochondrial dysfunction in db/db mice. This method may be a potential therapeutic application for the treatment of DACI.

Introduction

The global convergence of lifestyle variation and population aging has resulted in the increased incidence of diabetes [1]. According to epidemiological analysis, the number of diabetic subjects has increased to 415 million worldwide by 2015 and is expected to increase to 642 million by 2040 [2]. Diabetics exhibit a 20–60% increased risk of cognitive impairment [3]. In a clinical research of 11,140 patients with Type 2 diabetes, severe cognitive dysfunction increased the risks of major cardiovascular events, cardiovascular death and all-cause death with hazard ratio of 1.42, 1.56 and 1.50, respectively [4]. Therefore, the development of effective methods that can prevent diabetes-associated cognitive impairment (DACI) is imperative.

Mitochondria are considered the hub of bioenergetic metabolism and are the main organelles that can maintain brain function [5]. Mitochondria facilitates intracellular physiological processes and can also initiate the development of pathological conditions. It has been shown that CNS neurons are extremely vulnerable to mitochondrial dysfunction in diabetic mice [6,7]. Under hyperglycemic conditions, impaired mitochondrial function promotes endless cellular energy disruption, oxidative stress and consequent neuronal loss in response to the energy replenishment required to load the self-sustaining demands of the neurons [8,9]. In addition, it has been shown that mitochondrial dysfunction is a central part of cerebrovascular alterations, insulin-resistance, oxidative stress, Tau hyperphosphorylation and Aβ deposition, and these processes are also involved in the process of DACI [10–14]. Therefore, mitochondrial defects may be the main reason for DACI.

Transplantation of exogenous mitochondria into damaged tissues has been proposed as a feasible therapeutic method for treating disorders with poor prognosis, such as myocardial ischemic injury, stroke, Parkinson’s disease and schizophrenia [15–18]. Our studies have also demonstrated that autologous mitochondria isolated from the pectoralis major were injected into the lateral ventricle of rats in order to exert a neuroprotective effect following stroke induction. This was achieved by reducing oxidative stress and apoptosis, by attenuating reactive astrogliosis and by promoting neurogenesis [16]. Skeletal muscles have been suggested as an appropriate source of mitochondria [19]. However, mitochondrial exaction from muscular tissue is invasive and limits clinical transplantation. Platelets are abundant in blood and are enucleated cells containing small-size mitochondria [20]. More significantly, platelets are the most metabolically active blood organelles and can be acquired from the peripheral blood via simple venipuncture [21]. Consequently, isolation of platelet-derived mitochondria (Mito-Plt) is minimally invasive and easy to perform.

In the present study, we hypothesized that the in vivo transplantation of Mito-Plt improves DACI by restoring mitochondrial function and alleviating neuron apoptosis in hippocampus of diabetes model. Therefore, we aimed to investigate the therapeutic effects of Mito-Plt transplantation on DACI in leptin knockout mouse (db/db mouse) as well as its underlying mechanisms.

Materials and methods

Animals

All experiments involving animals were granted approval from the Institutional Animal Care and Use Committees of Xi’an Jiaotong University (Xi'an, China; 2019-060) and conducted on the basis of the Regulation for Administration of Affairs Concerning Experimental Animals (China), the ARRIVE criteria established by The National Centre for the Replacement, Refinement and Reduction of Animals in Research (London, U.K.). Homozygous male db/db mice and littermate control animals were purchased from the Model Animal Research Center of the Nanjing University (Nanjing, China). Male Sprague–Dawley (SD, 8–10weeks) rats obtained from the Experimental Animal Center of the Xi’an Jiaotong University were sacrificed for Mito-Plt isolation. Following acclimatization for one week, db/db mice were allocated to the four following groups: (1) Control group including vehicle injection (Control, 17 weeks old); (2) Control group that contained Mito-Plt transplantation (Control+mito); (3) db/db mice including vehicle injection (db/db, 17 weeks old); (4) db/db mice that contained Mito-Plt transplantation (db/db+mito). The animals underwent intracerebroventricular (icv) injection (0.4 mm posterior, 1 mm lateral, and 2.5 mm ventral to the bregma) with 1 × 105 per 5 μl Mito-Plt suspension or isometric amount of vehicle for 10 min. One month after icv injection, the animals were scheduled for behavioral testing. All animal tries were performed at Center for Brain Science, The First Affiliated Hospital of Xi'an Jiaotong University.

Morris Water Maze (MWM)

One month after Mito-Plt transplantation, the MWM test was performed to evaluate spatial learning and long-term memory [22]. The apparatus was composed of a rotund plastic tank, filled with water, which was maintained at 23 ± 2°C. A 10 cm-diameter escape platform was concealed under water 1 cm below the surface. A camera connected with tracking system was used to record and analysis. The test was performed for six consecutive days by researchers blinded to the interventions. Initially, the mice performed 4 trials/day in the different quadrant for 5 days to search a subaqueous platform submerged in the fourth quadrant. The time expended by mice to seek the sightless platform was regarded as the escape latency (failed tries were treated as 90 s). At the end of the every trial in the first 5 days, either when the mouse had seeked out the platform or when 90 s had passed, mice were allowed to learn and rest on the platform for 60 s. On the last day, probe trials were conducted without the platform in the second quadrant for once. The escape latency, swimming speed, swimming distance and the times of platform location crossing was measured automatically by software (Taimeng, China). Since db/db mice were obese, slow swimming speed may lead to prolonged latency and influence the evaluation of spatial learning. Therefore, the path efficiency, which was calculated by the distance between the entry and end points over the total distance, was used to adjust the discrepancy in swimming velocity. Latency, swimming speed and path efficiency were analyzed by two-ANOVA. Crossing times were analyzed by one-ANOVA following by Tukey’s multiple comparisons test.

Mito-Plt isolation and number

The Mito-Plt was derived from SD rats and used for the transplantation experiments. The isolation of Mito-Plt was performed by a previously available method [23]. Briefly, total blood was obtained from healthy SD rats (about 10 ml) and stored in an aseptic anticoagulant tube followed by centrifugation for 15 min at 275 g at room temperature (RT). Following centrifugation for 2 min at 400 g to eliminate the erythrocytes, the pellet containing the platelets was obtained from centrifugation at 1300 g for 10 min. The pellet was subsequently resuspended in Isolation Buffer (IB) (sucrose 0.2 M, Tris 11 mM, EDTA 1 mM, pH 7.5). Following homogenization in the presence of proteinase K (Beyotime Biotechnology, China), the platelet suspension was added 0.2% BSA and centrifuged at 1300 g at 4°C for 10 min. Following centrifugation at 8000 g at 4°C for 10 min, the crude mitochondrial extract was processed for additional purification using germfree Percoll (Sigma, U.S.A.). The crude mitochondria were layered on a 15% Percoll stratum (15% Percoll, 10% sucrose 2.5 M, 75% IB) followed by centrifugation at 21,000 g for 8 min at 4°C. The purified mitochondria were resuspended in IB buffer followed by centrifugation at 13,000 g for 10 min at 4°C. Subsequently, the final pellet was resuspended in respiratory buffer (250 mM sucrose, 2 mM KH2PO4, 10 mM MgCl2, 20 mM K-HEPES Buffer (pH 7.2), 0.5 mM K-EGTA (pH 8.0)). About 0.98–1.22 × 106 purified Mito-Plt could be extracted from 10ml whole blood of SD rat. The total protein content of purified Mito-Plt were about 61.2–74.6 μg/ml proteins per 10 ml of the whole blood. Before each injection, the extracted Mito-Plt were counted and diluted to 1 × 105 with the respiratory buffer. The number of purified mitochondria was calculated using a Helber bacteria-count boards (Auvon, U.K.).

JC-1

Flow cytometry of isolated Mito-Plt was performed to determine the mitochondrial membrane potential (ΔΨm) using the JC-1 cationic dye (Beyotime Biotechnology, China). This assay is based on the theory that the cationic dye rapidly accumulates in energized and intact mitochondria owing to the high membrane potential, and forms aggregates that could be detect by fluorescence. When the mitochondrial membranes are damaged leading to low membrane potential of mitochondria, the cationic dye does not aggregate into the mitochondrion [23]. Isolated Mito-Plt suspension was incubated with 1.5 μM of the JC-1 dye and examined by flow cytometry. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, 20 μM) was used to induce the shift in the ΔΨm and was regarded as a positive control.

Direction of transplanted Mito-Plt

The direction of transplanted Mito-Plt was assessed as previously described [16]. Briefly, purified Mito-Plt were dyed with 0.1 μM MitoTracker Red CMXRos (Thermo Scientific, U.S.A.) for 10 min on ice. The brain tissues were fixed in 4% paraformaldehyde 24 h following icv injection with Mito-Plt. The transplantation of Mito-Plt was used to confirm the existence of exogenous mitochondria in neurons by confocal microscopy (Nikon, Japan).

Immunofluorescence staining (IF)

Following anesthesia through inhalation with isoflurane, animals were performed to transcardial perfusion with 4% paraformaldehyde. Subsequently extra fixation and sucrose gradient dehydration, the frozen section (16 μm in thickness) of brain tissue was used with a micro-slicer system. The following primary antibodies were used for immunostaining: anti-β-III tubulin (1:300, Abcam, U.K.), anti-NeuN (1:300, Millipore, U.S.A.), anti-Aβ (1:100, Abcam, U.K.), anti-COX IV (1:100, proteintech, U.S.A.), anti-GFAP (1:400, Genetex, U.S.A.) and anti-IBA-1 (1:300, Genetex, U.S.A.). The researchers were blinded to the interventions and animal genotypes. A fluorescence microscope (Olympus, Japan) or a confocal microscope (Nikon, Japan) was used for IF staining. TUNEL assay (TdT-mediated dUTP nick-end labeling) was performed using the ApopTag fluorescein in situ apoptosis detection kit (Abbkine, U.S.A.) in line with the manufacturer’s protocol.

Western blotting

After animals were anesthetized through inhalation with isoflurane, their hippocampus were immediately removed followed by mechanical homogenization with lysis buffer encompassing proteinase inhibitor. After sonication and centrifugation at 12,000 g for 20 min, the concentrations of separated protein were measured by BCA method. Equal amount of protein (40 μg) was loaded on 10% or 12% TGX Stain-Free Fast Cast Acrylam (Bio-Rad, U.S.A.). The proteins were immunoblotted with primary antibodies. The detection of the bands was conducted by the Image Lab v5.2.1 software (Bio-Rad, U.S.A.). The list of primary antibodies involved in the Western blotting experiments is displayed in Supplementary Table S1.

Table 1
Effect of Mito-Plt transplantation on body weight and blood glucose levels
GroupsControlControl+mitodb/dbdb/db+mito
Body weight (g) 25.01 ± 1.379 25.78 ± 1.917 49.82 ± 6.15**** 51.03 ± 6.385#### 
Blood glucose (mmol/l) 6.322 ± 1.166 6.878 ± 0.7678 30.98 ± 1.701**** 29.42 ± 2.241#### 
GroupsControlControl+mitodb/dbdb/db+mito
Body weight (g) 25.01 ± 1.379 25.78 ± 1.917 49.82 ± 6.15**** 51.03 ± 6.385#### 
Blood glucose (mmol/l) 6.322 ± 1.166 6.878 ± 0.7678 30.98 ± 1.701**** 29.42 ± 2.241#### 

N=9 mice/group. Control = control group including vehicle injection; Control+mito = control group that contained Mito-Plt transplantation; db/db = db/db mice including vehicle injection; db/db+mito = db/db mice that contained Mito-Plt transplantation.*P<0.05 versus Control; #P<0.05 versus db/db (****P or ####P≤0.0001).

Measurement of oxidative stress and ATP levels

The proteins derived from the hippocampal tissues were obtained by the homogenate samples as soon as the animals were sacrificed. The levels of MDA, H2O2, ROS, 8-OHdG, NT, TAOC and ATP in the hippocampal tissues were measured by specific kits in accordance with the instructions provided by the manufacturer.

Transmission electron microscopy examination

Transmission electron microscopy was performed as described previously [24]. Briefly, isolated Mito-Plt and hippocampal tissues were fixed in 2.5% glutaraldehyde in PBS and post-fixed in 1% OsO4 at 4°C. Following dehydration in a graded series of alcohol, the samples were embedded upon Poly/Bed 812 resin followed by ultrathin (80-nm) sections with an ultramicrotome. Imaging was performed by a Hitachi H-7650 transmission electron microscope (Hitachi, Japan) at 80 kV. Quantitative analysis of mitochondrial damage was performed independently by two investigators blinded to groups, who reviewed each enlarged electron microscopy image for the presence of structurally abnormal mitochondria. The number of damaged mitochondria (loss of cristae) and total mitochondria per image in neurons was quantified, and analyzed by using one-way ANOVA [25].

Statistical analysis

Statistical analysis was performed with the GraphPad Prism software 7.0 (Version7.00, U.S.A.). The data were analyzed by the Student’s t-test for two group comparisons and one-way ANOVA followed by Tukey’s multiple comparisons test for four group comparisons of independent samples. Two-way ANOVA was used to analyze the MWM (group × day). A P value lower than 0.05 (P<0.05) was considered for significant differences (*P or #P<0.05; **P or ##P≤0.01; ***P or ###P≤0.001; ****P or ####P≤0.0001). All the measurement data were presented as mean ± SEM.

Results

Isolation and properties of viable Mito-Plt

Prior to Mito-Plt transplantation, the viability, integrity and purity of Mito-Plt were evaluated. MitoTracker Red CMXRos (Mito Red) staining demonstrated that the isolated Mito-Plt were functional (Figure 1A). TEM indicated that the morphology of isolated Mito-Plt was intact (Figure 1B). The membrane potential (ΔΨm) of Mito-Plt was detected by flow cytometry in order to assess integrity. Red fluorescence of JC-1 was noted in isolated Mito-Plt, and was reduced following pretreatment with CCCP (Figure 1C). The ratio of aggregate/monomer (red/green) was also significantly decreased in the CCCP group (Figure 1D). Thus, above data detected by flow cytometry showed the isolated Mito-Plt were electron-dense with no damage or disintegration. The total platelet were regarded as positive control and the high expression of COX IV (mitochondrial maker) was observed in the lysate of the purified Mito-Plt except β-tubulin (cytoplasmic maker) (Figure 1E). These results demonstrated that the isolated Mito-Plt were intact, functional and purified.

Isolation and properties of viable Mito-Plt

Figure 1
Isolation and properties of viable Mito-Plt

(A) Mito-Plt are shown under bright field (BF) and following staining with Mitotracker red CMXRos; scale bar: 20 μm. (B) TEM of Mito-Plt; scale bar: 200 nm. (C and D) Mitochondrial membrane potential (ΔΨm) of Mito-Plt labeled with JC-1 was measured by flow cytometry (n=3 for each). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is regarded as a positive control group. (E) Immunoblotting analysis of the purity of isolated Mito-Plt. The protein extracts from purified mitochondria and total platelets, the latter were considered as a positive control. The data are shown as mean ± SD and were analyzed with the Student’s t-test. *P<0.05 versus mitochondria .

Figure 1
Isolation and properties of viable Mito-Plt

(A) Mito-Plt are shown under bright field (BF) and following staining with Mitotracker red CMXRos; scale bar: 20 μm. (B) TEM of Mito-Plt; scale bar: 200 nm. (C and D) Mitochondrial membrane potential (ΔΨm) of Mito-Plt labeled with JC-1 was measured by flow cytometry (n=3 for each). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is regarded as a positive control group. (E) Immunoblotting analysis of the purity of isolated Mito-Plt. The protein extracts from purified mitochondria and total platelets, the latter were considered as a positive control. The data are shown as mean ± SD and were analyzed with the Student’s t-test. *P<0.05 versus mitochondria .

Mito-Plt is internalized and the mitochondrial density is augmented in the hippocampal neurons of the db/db mice following transplantation

A total of 1 × 105 isolated Mito-Plt or vehicle were pre-labeled with Mito Red and were injected into the left icv (5 μl). Following 24 h of the injection, the biodistribution and internalization efficacy of dyed Mito-Plt in the hippocampus were observed by confocal microscopy. Mito Red-only was regarded as an internal control. Mito Red was detected in neurons (colocalization with β-III tubulin) and the ratio of the Mito-Plt uptake was 5.3 ± 0.75% and 9.4 ± 0.33% in the control+mito and db/db+mito groups, respectively (Figure 2A,B).

Biodistribution of Mito-Plt and measurement of mitochnodrial density in the hippocampal neurons of db/db mice following transplantation

Figure 2
Biodistribution of Mito-Plt and measurement of mitochnodrial density in the hippocampal neurons of db/db mice following transplantation

(A) The biodistribution of transplanted Mito-Plt in the hippocampal neurons 24 h following intracerebroventricular (icv) injection was determined using immunofluorescent analysis. The neuronal specific marker β-III tubulin (green) and the mitochondria prelabeled MitoTracker Red (red) were performed by immunohistochemical staining analysis in the brain tissues of mice; scale bar: 20 μm. (B) Quantification of mitochondrial uptake ratio of the control+mito and db/db+mito groups. (C) Representative images for β-III tubulin (green) and COXIV (red) staining in hippocampal pyramidal neurons derived from brain tissues of db/db mice one month after mitochondrial transplantation. The nuclei (blue) were stained by DAPI; scale bar: 20 μm. (D) The immunofluorescence intensity of COXIV was quantified in the hippocampal neurons of control and db/db mice. (E) Representative images of the mitochondria in hippocampal neurons by TEM; scale bar: 1 μm. (F–H) Quantification of mitochondrial number, mitochondrial area and ratio of damaged mitochondria in the hippocampal neurons. The analyses of (B), (D) and (F–H) were performed by one-way ANOVA followed by Tukey’s multiple comparisons test. The data are shown as mean ± SD. N=3 for each group. *P<0.05 versus Control; #P<0.05 versus db/db(#P<0.05; **P or ##P≤0.01; ***P≤0.001).

Figure 2
Biodistribution of Mito-Plt and measurement of mitochnodrial density in the hippocampal neurons of db/db mice following transplantation

(A) The biodistribution of transplanted Mito-Plt in the hippocampal neurons 24 h following intracerebroventricular (icv) injection was determined using immunofluorescent analysis. The neuronal specific marker β-III tubulin (green) and the mitochondria prelabeled MitoTracker Red (red) were performed by immunohistochemical staining analysis in the brain tissues of mice; scale bar: 20 μm. (B) Quantification of mitochondrial uptake ratio of the control+mito and db/db+mito groups. (C) Representative images for β-III tubulin (green) and COXIV (red) staining in hippocampal pyramidal neurons derived from brain tissues of db/db mice one month after mitochondrial transplantation. The nuclei (blue) were stained by DAPI; scale bar: 20 μm. (D) The immunofluorescence intensity of COXIV was quantified in the hippocampal neurons of control and db/db mice. (E) Representative images of the mitochondria in hippocampal neurons by TEM; scale bar: 1 μm. (F–H) Quantification of mitochondrial number, mitochondrial area and ratio of damaged mitochondria in the hippocampal neurons. The analyses of (B), (D) and (F–H) were performed by one-way ANOVA followed by Tukey’s multiple comparisons test. The data are shown as mean ± SD. N=3 for each group. *P<0.05 versus Control; #P<0.05 versus db/db(#P<0.05; **P or ##P≤0.01; ***P≤0.001).

Immunostaining and TEM were performed one month later in order to examine the extent of mitochondrial density alterations in the hippocampal neurons. As shown in Figure 2C,D, the fluorescence density of COX IV was apparently elevated in the db/db+mito group compared with db/db group (Figure 2C,D). Furthermore, the mitochondrial number and area proportion in the hippocampal neurons were also significantly reversed by treatment with Mito-Plt in comparison with db/db mice. (Figure 2E–G). In addition, the number of damaged mitochondria was decreased effectively in the db/db+mito group compared with that of the db/db group (Figure 2H). This evidence suggested that the transplantation of Mito-Plt via icv injection could cause effective internalization of mitochondria into the hippocampal neurons and consequently augment mitochondrial density.

Mito-Plt transplantation attenuates mitochondrial dysfunction in the hippocampal tissues of db/db mice

It has been previously shown that the reduction in energetic metabolism and mitochondrial dysfunction is major contributors to the development of DACI [6,26]. The expression levels of the mitochondrial respiration complex protein were examined to evaluate the capacity of oxidative phosphorylation (OXPHOS) in the hippocampus of db/db mice. The present study indicated that the downregulation of the expression of Complex I, III, IV and V was recovered following injection of Mito-Plt in the hippocampus of db/db mice compared with control group (Figure 3A,C–E). It is important to note that the expression of Complex II was unaffected following Mito-Plt transplantation (Figure 3B).

Effect of Mito-Plt transplantation on mitochondrial function in the hippocampal tissues of db/db mice

Figure 3
Effect of Mito-Plt transplantation on mitochondrial function in the hippocampal tissues of db/db mice

(A–E) Mitochondrial function in the hippocampus one month following Mito-Plt transplantation revealed by the expression levels of the mitochondrial respiratory protein complexes I–V. (F) The ATP content in the hippocampus was determined using the ATP determination kit in accordance with the instructions of the manufacturer. One-way ANOVA followed by Tukey’s multiple comparisons test was used in (A–F). The data are presented as mean ± SD. N=3 for each group. *P<0.05 versus Control; #P<0.05 versus db/db (*P or #P<0.05; **P or ##P≤0.01; ***P≤0.001).

Figure 3
Effect of Mito-Plt transplantation on mitochondrial function in the hippocampal tissues of db/db mice

(A–E) Mitochondrial function in the hippocampus one month following Mito-Plt transplantation revealed by the expression levels of the mitochondrial respiratory protein complexes I–V. (F) The ATP content in the hippocampus was determined using the ATP determination kit in accordance with the instructions of the manufacturer. One-way ANOVA followed by Tukey’s multiple comparisons test was used in (A–F). The data are presented as mean ± SD. N=3 for each group. *P<0.05 versus Control; #P<0.05 versus db/db (*P or #P<0.05; **P or ##P≤0.01; ***P≤0.001).

In addition, the content of ATP was depleted in the hippocampal tissues of db/db mice. The effect was significantly improved in the db/db+mito group (Figure 3F). These results implied that the Mito-Plt transplantation could promote mitochondrial function in the hippocampal tissues of db/db mice.

Mito-Plt transplantation alleviates oxidative stress in the hippocampal tissues of db/db mice

The by-products of oxidative stress can directly cause disorders in the metabolism of intracellular lipids, proteins and nucleic acids leading to irreversible damage of the hippocampus. Therefore, the contents of ROS and H2O2 were significantly diminished in the db/db+mito group (Figure 4A,B). Furthermore, the levels of malondialdehyde (MDA) and nitrotyrosine (NT), which are products of endocellular lipid oxidation and oxidation of peroxynitrite with tyrosine respectively, were increased in the db/db group but did not change significantly in the db/db+mito group compared with those in the control group (Figure 4C,D). 8-Hydroxydeoxyguanosine (8-OHdG), which is a biomarker of DNA oxidative lesions, was also decreased following Mito-Plt replenishment (Figure 4E). Moreover, oxidation resistance was an effective defense technique against oxidative stress and as anticipated, the total antioxidant capacity (TAOC) was significantly higher in the db/db group than that of the db/db+mito group (Figure 4F). In summary, these results demonstrated that exogenous Mito-Plt could inhibit oxidative stress injury in the brain tissues of T2DM mice.

Effect of Mito-Plt transplantation against oxidative stress in the hippocampal tissues of db/db mice

Figure 4
Effect of Mito-Plt transplantation against oxidative stress in the hippocampal tissues of db/db mice

(A–F) Right hippocampus samples were harvested one month following mitochondrial transplantation for further measurement. The total levels of ROS, H2O2, MDA, NT, 8-OHdG and TAOC in the homogenates of the mouse hippocampal tissues were determined using specific assay kits, respectively. One-way ANOVA followed by Tukey’s multiple comparisons test was used in (A–F). The data are shown as mean ± SD. N = 9 for each group. *P<0.05 versus Control; #P<0.05 versus db/db (*P or #P<0.05; **P or ##P≤0.01; ***P ≤0.001).

Figure 4
Effect of Mito-Plt transplantation against oxidative stress in the hippocampal tissues of db/db mice

(A–F) Right hippocampus samples were harvested one month following mitochondrial transplantation for further measurement. The total levels of ROS, H2O2, MDA, NT, 8-OHdG and TAOC in the homogenates of the mouse hippocampal tissues were determined using specific assay kits, respectively. One-way ANOVA followed by Tukey’s multiple comparisons test was used in (A–F). The data are shown as mean ± SD. N = 9 for each group. *P<0.05 versus Control; #P<0.05 versus db/db (*P or #P<0.05; **P or ##P≤0.01; ***P ≤0.001).

Mito-Plt transplantation inhibits cell apoptosis and caspase activation in the hippocampal tissues of db/db mice

Immunofluorescence analysis and western blotting were performed one month after Mito-Plt injection to detect the TUNEL positive cells and the expression levels of cleaved caspase 3. These markers were assessed to confirm the induction of cell and mitochondrial apoptosis. The number of TUNEL positive cells was significantly decreased in the db/db+mito group compared with that of the db/db group (Figure 5A,B). The expression levels of cleaved caspase 3 proteins declined in the db/db+mito group (Figure 5C,D).

Effects of Mito-Plt transplantation on cellular apoptosis and caspase activation in the hippocampus of db/db mice

Figure 5
Effects of Mito-Plt transplantation on cellular apoptosis and caspase activation in the hippocampus of db/db mice

(A) Cell apoptosis in the hippocampus was assessed using TUNEL staining following one month of vehicle or Mito-Plt treatment. Scale bar: 20 μm. (B) The TUNEL positive cells were counted in all groups. (C) The expression levels of full length caspase 3 and cleaved caspase 3 in the lysates of the hippocampal tissues were analyzed with immunoblotting. (D) Representative examples of NeuN immunofluorescence staining in the hippocampal tissues. Scale bar: 20 μm. (E) The number of positive cells of NeuN was quantified following transplantation with Mito-Plt or vehicle. One-way ANOVA followed by Tukey's multiple comparisons test was used in B, C and E. The data are shown as mean ± SD. N = 3 for each group in A-D; N = 4 for each group in D-E. *P<0.05 versus Control; #P<0.05 versus db/db ( #P<0.05; **P ≤0.01; ***P or ###P≤0.001).

Figure 5
Effects of Mito-Plt transplantation on cellular apoptosis and caspase activation in the hippocampus of db/db mice

(A) Cell apoptosis in the hippocampus was assessed using TUNEL staining following one month of vehicle or Mito-Plt treatment. Scale bar: 20 μm. (B) The TUNEL positive cells were counted in all groups. (C) The expression levels of full length caspase 3 and cleaved caspase 3 in the lysates of the hippocampal tissues were analyzed with immunoblotting. (D) Representative examples of NeuN immunofluorescence staining in the hippocampal tissues. Scale bar: 20 μm. (E) The number of positive cells of NeuN was quantified following transplantation with Mito-Plt or vehicle. One-way ANOVA followed by Tukey's multiple comparisons test was used in B, C and E. The data are shown as mean ± SD. N = 3 for each group in A-D; N = 4 for each group in D-E. *P<0.05 versus Control; #P<0.05 versus db/db ( #P<0.05; **P ≤0.01; ***P or ###P≤0.001).

The present study also demonstrated that the number of positive NeuN cells was significantly increased following treatment with Mito-Plt in db/db mice (Figure 5E,F). HE staining was used to assess the hippocampus injury in db/db mice. In db/db group, neuronal loss and pyknotic nuclei were showed in the hippocampus compared with control group. After Mito-Plt treatment, neuronal loss and pyknotic nuclei were improved in the hippocampus in db/db+mito group (Supplementary Fig 1A). We also explored the changing of astrocyte and microglia in the hippocampus after Mito-Plt infusion. The indensity of IBA-1 (marker of microglia) significantly elevated in the hippocampus of db/db group compared with control group, which was significantly restored by Mito-Plt infusion (Supplementary Fig 1B-C). However, No significant differences of GFAP (marker of astrocyte) were noted among the various groups (Supplementary Fig 1D-E). Moreover, the change of microglia after Mito-Plt transplantation in diabetic mice needs further experimental investigation. The data indicated that Mito-Plt could considerably diminish cell and mitochondrial apoptotic signals and improve the reduction noted in the number of neurons in the hippocampus.

Mito-Plt transplantation mitigates the accumulation of Aβ and Tau in the hippocampal tissues of db/db mice

Several investigations have demonstrated that hyperglycaemia can promote the accumulation of Aβ and total-Tau (t-Tau) and increase the hyperphosphorylation of Tau (p-Tau) [27,28]. Furthermore, upregulation of Aβ and tau levels is considered an important step in the pathogenesis of DACI [13,29]. The present study demonstrated that the Aβ burden in the hippocampus (percentage area overlay by Aβ immunoreactivity) was higher in the db/db group than that in the db/db+mito group as shown by immunofluorescence analysis (Figure 6A,B). These data were in agreement with the expression levels detected by immunoblotting (Figure 6C).

Effects of Mito-Plt transplantation on the accumulation of Aβ and Tau in the hippocampal tissues of db/db mice

Figure 6
Effects of Mito-Plt transplantation on the accumulation of Aβ and Tau in the hippocampal tissues of db/db mice

(A) Immunofluorescence staining of mouse Aβ (red) in the hippocampus. Scale bar: 10μm. (B) The Aβ burden was quantified following transplantation with Mito-Plt or vehicle. (C) The expression levels of oligomer Aβ in the lysate of hippocampus were analyzed with immunoblotting. (D,E) Immunoblotting analysis of p-Tau serine 202 and t-Tau in the hippocampus. (F) Quantification of the ratios of p-Tau and t-Tau. One-way ANOVA followed by Tukey's multiple comparisons test was used in B-F. The data are shown as mean ± SD. N = 4 for each group in A-B; N = 3 for each group in C-F. *P<0.05 versus Control; #P<0.05 versus db/db(*P or #P<0.05; **P ≤0.01; ****P ≤0.0001).

Figure 6
Effects of Mito-Plt transplantation on the accumulation of Aβ and Tau in the hippocampal tissues of db/db mice

(A) Immunofluorescence staining of mouse Aβ (red) in the hippocampus. Scale bar: 10μm. (B) The Aβ burden was quantified following transplantation with Mito-Plt or vehicle. (C) The expression levels of oligomer Aβ in the lysate of hippocampus were analyzed with immunoblotting. (D,E) Immunoblotting analysis of p-Tau serine 202 and t-Tau in the hippocampus. (F) Quantification of the ratios of p-Tau and t-Tau. One-way ANOVA followed by Tukey's multiple comparisons test was used in B-F. The data are shown as mean ± SD. N = 4 for each group in A-B; N = 3 for each group in C-F. *P<0.05 versus Control; #P<0.05 versus db/db(*P or #P<0.05; **P ≤0.01; ****P ≤0.0001).

Subsequently, we explored whether replenishing mitochondria induces alterations in the levels of total Tau (t-Tau) and phosphorylated Tau (p-Tau at Ser202). The results demonstrated a significant increase in t-Tau and p-Tau levels. This change was significantly restored by treatment with Mito-Plt (Figure 6D,E). However, the ratio of p-Tau/ t-Tau was not significantly different among the various groups (Figure 6F). These results suggested that the effects noted in t-Tau could be rescued by transplantation of Mito-Plt. However, this effect was not noted for p-Tau. The data implied that treatment of db/db mice with Mito-Plt could decrease the levels of oligomeric Aβ and t-Tau.

Mito-Plt transplantation improves cognition of db/db mice

Db/db mice exhibited a significant increase in weight and in the levels of random blood glucose at 22 weeks compared to age-matched WT mice (Table 1). No significant differences were noted between the db/db and db/db+mito groups. These results demonstrated that Mito-Plt transplantation did not affect weight and blood glucose levels in db/db mice.

Spatial learning and long-term memory were markedly reduced in 5-6 month old db/db mice as demonstrated previously [22,30]. In the MWM test, the escape latency of the db/db group was significantly prolonged compared with that of the control group (Figure 7A). The db/db+mito group displayed superior presentation from the second day of training (Figure 7A). Since db/db mice were obese, their swimming velocity was significantly decreased compared with that of the control group animals (Figure 7B), which may lead to prolonged latency and influence the evaluation of spatial learning. Therefore, the path efficiency, which was calculated by the distance between the entry and end points over the total distance, was used to adjust the discrepancy in swimming velocity [31]. The db/db group indicated significantly decreased path efficiency from the second day compared with that of the control group. This effect was significantly improved by Mito-Plt treatment (Figure 7C). During the probe trial, the number of crossing times of original platform location was dramatically recovered by Mito-Plt treatment of db/db mice (Figure 7D). However, no significant difference was noted in the swimming velocity between the db/db and db/db+mito groups, indicating that the cognitive function was recovered and that this effect was not due to mobility variation. Therefore, the data indicated that exogenous transplantation of Mito-Plt could significantly improve DACI (Figure 7E).

Effects of Mito-Plt transplantation on cognition of db/db mice

Figure 7
Effects of Mito-Plt transplantation on cognition of db/db mice

The MWM analysis was quantified to obtain the (A) latency, (B) swimming speed, (C) path efficiency and (D) crossing times. (E) Representative traces from Morris water maze (MWM) test. Two-way ANOVA was used in A-C, and one-way ANOVA followed by Tukey's multiple comparisons test was used in D. The results represented as mean ± SD. N = 9 for per group. *P<0.05 versus Control; #P<0.05 versus db/db (*P or #P<0.05; **P or ##P≤0.01; ***P ≤0.001;****P or ####P ≤0.0001).

Figure 7
Effects of Mito-Plt transplantation on cognition of db/db mice

The MWM analysis was quantified to obtain the (A) latency, (B) swimming speed, (C) path efficiency and (D) crossing times. (E) Representative traces from Morris water maze (MWM) test. Two-way ANOVA was used in A-C, and one-way ANOVA followed by Tukey's multiple comparisons test was used in D. The results represented as mean ± SD. N = 9 for per group. *P<0.05 versus Control; #P<0.05 versus db/db (*P or #P<0.05; **P or ##P≤0.01; ***P ≤0.001;****P or ####P ≤0.0001).

Discussion

The present study demonstrated the efficacy of Mito-Plt transplantation in treating DACI in db/db mice. Intracerebroventricular (icv) injection of Mito-Plt was able to improve DACI and this effect was accompanied with restored mitochondrial function, attenuated oxidative stress, reduced neuronal apoptosis and decreased accumulation of Aβ and Tau in the hippocampal tissues of db/db mice. In the present study, we present a potential therapeutic strategy for DACI.

Autologous transplantation of mitochondria has demonstrated successful therapeutic effects in several diseases [16,32]. However, polysystemic mitochondrial dysfunction has been noted in certain patients and autologous transplantation of mitochondria is not practicable. Previous studies have shown that xenogeneic mitochondrial transplantation provides operative prevention against mitochondrial disruption, such as Parkinson`s disease and stroke [15,33]. In addition, a previous study has shown lack of immune response following injection of autogenetic and allogeneic mitochondria [34]. The present study indicated neuroprotective effects of xenogeneic Mito-Plt. Therefore, it is feasible to consider that xenogeneic Mito-Plt transplantation can be used in clinical practice.

In the present study, Mito-Plt pre-labeling with Mito Red was performed prior to icv to verify the internalization of exogenous mitochondria. Several studies have shown mitochondria stained with Mito Red were viable and visible after injection and could be dispersed at a distance >2-3 mm from the point of injection [19,35]. Their successful entrance into the neurons also has been reported in vitro and in vivo [15]. Successful internalization of mitochondria in primary neurons at a rate of 18-22% has been previously shown in vitro following 24 h of co-culture [15,33]. The co-localization rate of exogenous mitochondria and neurons reached 6.6±1.9% by in vivo intercerebral injection following MACO and the ratio was increased to 36±4.1% following administration of peptide-labeled mitochondria [15,33]. The present study further indicated that Mito-Plt could successfully enter into the hippocampal neurons following 24 h of transplantation. In addition, the proportion of internalization into the hippocampal neurons was 9.4±0.33% in db/db mice and 5.3±0.75% in the control mice, suggesting that damaged neurons may require higher number of healthy mitochondria. These data revealed that internalization of exogenous mitochondria may be different in diverse pathological processes. Following successful mitochondrial transplantation, the intensity of mitochondria in the hippocampal neurons was significantly increased in db/db mice 1 month later, suggesting that exogenous mitochondrial supplementation are more than a powerplant and their metabolites could act as substrates and regulators of the epigenome [36]. This hypothesis requires further exploration.

As an organ with high metabolic function, the brain requires consistent energy support from healthy mitochondria. The deficiency of ATP due to the dysfunction of the respiratory complex is tightly connected with neuronal injury [37]. Our data indicated significantly reduced expression levels of the mitochondrial respiration complexes I and III-V resulting in lowered ATP content. However, the expression levels of complex II were not downregulated, which may contribute to an extreme electron flow in the mitochondria as a result of oxidative stress induction under severe hyperglycemia [38]. In addition, the neurons were severely affected by oxidative stress under hyperglycemic conditions. The present study indicated that the indices of oxidative stress (ROS, H2O2, MDA, NT and 8-OHdG) were augmented in the hippocampal tissues of db/db mice, which is consistent with previous studies in T2DM patients [39]. It was also proposed that the Mito-Plt supplement might expedite ROS scavenging via augmenting the ATP addition and decreasing the consequent activation of apoptosis signaling pathway [16]. In addition, the antioxidant capacity was also recovered following transplantation in accordance with a previous study [16]. Therefore, transplantation of healthy Mito-Plt into diabetic mouse brain tissues was conducive to augment energy production, diminish oxidative stress and reserve mitochondrial function. As a consequence, the ameliorated mitochondrial function increased the number of neurons in the hippocampus by reducing neuronal apoptosis.

Several pathophysiological features of T2D are similar to AD, such as insulin resistance, oxidative stress and amyloid deposition [14]. Postmortem studies have demonstrated the accumulation of Aβ and tau in the brain of diabetic patients compared with those of age-matched controls [28,40]. Oligomeric Aβ can induce a marked rise of oxidative stress in neurons, which was similar to the increased levels of glucose [41]. Accumulated evidence has indicated that the excessive phosphorylation of tau is attributed to several factors, including chronic oxidative stress, Aβ-oligomer deposition, mitochondrial dysfunction and caspase activation [12]. Mitochondria-targeted nanozymes and pharmacological compounds effectively improve mitochondrial function, inhibit Aβ deposits and decrease tau hyperphosphorylation [42–44]. In the present study, Mito-Plt treatment could reduce total tau levels but not influence phosphorylated tau (Ser202) expression, indicating that lessening of total tau levels was accompanied with decreased phosphorylated tau levels. In fact, several studies have pointed out that damaged mitochondria can lead to abnormal expression and modification of tau protein, and loss of axonal mitochondria also could promote tau-mediated neurodegeneration [44–47]. Another study showed that mitochondrial transplantation could increase the number of axons and improve oxidative metabolism after optic nerve crush in the retina [48]. Thus, it is a reasonable hypothesis that Mito-Plt transplantation could reduce tau protein by improving mitochondrial function in axons. The data of the current study also were consistent with those reported by a previous study showing that tau deletion caused a significant improvement in memory [49]. Therefore, the improvement of the mitochondrial function could be an effective strategy to recover cognitive changes in T2DM and AD.

Collectively, our study demonstrated that the icv transplantation of Mito-Plt alleviated DACI in db/db mice. Platelets are abundant in the peripheral blood and can be acquired easily. More importantly, they are enucleated cells containing small-size mitochondria. Therefore, transplantation of Mito-Plt, obtained from the patient body with viable, structurally intact, respiration competent mitochondria can replace native mitochondria that are damaged by diabetes or other diseases. This strategy can be developed as a novel method to offset cognitive damage in T2DM. Further investigations should be focused on the pathway of exogenous mitochondria responsible for their internalization into the neurons and the specific intracellular mechanisms of mitochondrial treatment. It is reasonable to presume that injected mitochondria via peripheral veins may be a feasible strategy. However, their reduced ability to cross the blood–brain barrier is still a major challenge.

Consequent translation of these findings to the clinical setting may provide novel ways for mitochondrial transplantation in order to protect and repair damaged tissues from CNS-associated diseases.

Clinical perspectives

  • Diabetics exhibit increased risk of cognitive impairment which is associated with mitochondrial dysfunction. Our studies have demonstrated that transplantation of mitochondria exert neuroprotective effect following stroke induction. Hence, mitochondrial transplantation may be a potential strategy to reduce diabetics associated cognitive impairment (DACI).

  • Our results demonstrated that platelet-derived mitochondria (Mito-Plt) was internalized into hippocampal neurons 24 h following icv injection. Importantly, one month following Mito-Plt transplantation, DACI was alleviated in db/db mice and the effect was accompanied with increased mitochondrial number, restored mitochondrial function, attenuated oxidative stress and neuronal apoptosis, as well as decreased accumulation of Aβ and Tau in the hippocampus.

  • Platelets are abundant in the peripheral blood and can be acquired easily. More importantly, they are enucleated cells containing small-size mitochondria and isolation of Mito-Plt is minimally invasive and easy-to perform. Transplantation of Mito-Plt, obtained from the patient body with viable, structurally intact, respiration competent mitochondria can replace native mitochondria that are damaged by diabetes or other diseases. This strategy can be developed as a novel method to offset cognitive damage in T2DM.

Competing Interests

The authors declare that they have no conflict of interest.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 81774113, 81974540, 81971290 and 81801899] and Natural Science Basic Research Program of Shaanxi [grant number 2017JZ029].

Author Contribution

Q.W., Y.L., W.G., G.Z., H.M. designed the study. H.M., T.J., W.T., K.P., performed experiments. Z.M., F.X., H.C., H.M. analyzed data. Q.W., Y.L., H.M. wrote the manuscript. Q.W. is the guarantor of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Abbreviations

     
  • ATP

    Adenosine triphosphate

  •  
  • Amyloid β-protein

  •  
  • CCCP

    Carbonyl cyanide m-chlorophenylhydrazone

  •  
  • CNS

    Central Nervous System

  •  
  • DACI

    Diabetes-associated cognitive impairment

  •  
  • EDTA

    Ethylene Diamine Tetraacetic Acid

  •  
  • EGTA

    Ethylenebis (oxyethylenenitrilo) Tetraacetic Acid

  •  
  • GFAP

    Glial fibrillary acidic protein

  •  
  • HEPES

    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

  •  
  • IBA-1

    Ionized calcium-binding adapter molecule 1

  •  
  • JC-1

    5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine

  •  
  • MDA

    Malonaldehyde

  •  
  • Mito-Plt

    Platelet-derived mitochondria

  •  
  • MWM

    Morris Water Maze

  •  
  • NT

    Nitrotyrosine

  •  
  • OXPHOS

    Oxidative phosphorylation

  •  
  • ROS

    Reactive Oxygen Species

  •  
  • TAOC

    Total Antioxidant Capacity

  •  
  • TUNEL

    TdT-mediated dUTP nick-end labeling

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Supplementary data