Decrease of MtDNA copy number affects mitochondrial function and involves in the pathological consequences of ischaemic stroke

Abstract The mtDNA copy number can affect the function of mitochondria and play an important role in the development of diseases. However, there are few studies on the mechanism of mtDNA copy number variation and its effects in IS. The specific mechanism of mtDNA copy number variation is still unclear. In this study, mtDNA copy number of 101 IS patients and 101 normal controls were detected by qRT‐PCR, the effect of D‐loop variation on mtDNA copy number of IS patients was explored. Then, a TFAM gene KD‐OE PC12 cell model was constructed to explore the effect of mtDNA copy number variation on mitochondrial function. The results showed that the mtDNA copy number level of the IS group was significantly lower than that of the normal control group (p < 0.05). The relative expression of TFAM gene mRNA in the cells of the OGD/R treatment group was significantly lower than that of the control group (p < 0.05). In addition, after TFAM gene knockdown and over‐expression plasmids were transfected into HEK 293T cells, mtDNA copy number and ATP production level of Sh‐TFAM transfection group was significantly decreased (p < 0.05), while mtDNA copy number and ATP production level of OE‐TFAM transfected group were significantly higher than that of blank control group and OE‐ctrl negative control group (p < 0.01). Our study demonstrated that mitochondrial D‐loop mutation and TFAM gene dysfunction can cause the decrease of mtDNA copy number, thus affecting the mitochondrial metabolism and function of nerve cells, participating in the pathological damage mechanism of IS.


| INTRODUC TI ON
Ischaemic stroke (IS) refers to the disease of neurological dysfunction due to the stenosis or occlusion of cerebral feeding artery (carotid artery and vertebral artery) and insufficient cerebral blood supply. 1,2 The pathological mechanism of IS is extremely complex. After cerebral ischaemia, nerve cells will produce a series of cascade damage, such as oxidative stress, energy disorder, excitotoxicity, acidosis, inflammatory reaction, calcium homeostasis imbalance and apoptosis, which eventually lead to central nervous system dysfunction. 3,4 Mitochondria are highly dynamic double-membrane organelle that exist in almost all eukaryotic cells. 5 The main function of mitochondria is to produce adenosine 5′-triphosphate (ATP) through oxidative phosphorylation (OXPHOS) to meet most of the energy requirements of cells. 6,7 Human mitochondrial DNA (mtDNA) is a circular molecule composed of 16,568 bases, encoding ribosomal RNA (rRNA), transfer RNA (tRNA) and the important components of mitochondrial electron transport chain (ETC). 8 The number of mtDNA in the mitochondrial genome is called mtDNA copy number, which is specific in different types of tissue cells. 9 El-Hattab et al. have reported that the reduction of mtDNA copy number in cells can impair mitochondrial respiration and cause pathology as diverse as encephalopathy, neuropathy and ageing process. 10 Ed Reznik et al. found that the expression of mitochondrial metabolic genes and the occurrence of some mutations will change the copy number of mtDNA and affect the function of mitochondria. 11 The replacement loop region (D-loop) of mtDNA is the only noncoding region in the human mitochondrial genome, which contains about 1122bp, accounting for 6% of the total mtDNA molecular weight and involved in the regulation of mtDNA replication and transcription. 12,13 As the binding site of mtDNA and mitochondrial membrane, the D-loop region rich in A and T bases is sensitive to oxidative stress, which is a high-incidence region of mtDNA mutations.
The ratio of base substitution is 6-8 times higher than that in other regions of the mitochondrial genome. 14 A large number of replication and transcription factors are important to maintain the normal copy number of mtDNA and meet the energy requirements of cells. 15 Mitochondrial transcription factor A (TFAM) encoded by the TFAM gene is the first discovered mitochondrial transcription factor, which is involved in mtDNA replication, transcription and mtDNA repair. 16 Polymerase γ (Polγ) encoded by the POLG gene is the only DNA polymerase in human mitochondria, which participates in mtDNA replication and enhances the activity of enzymes by accelerating the polymerization rate and inhibiting the activity of exonucleases. 17 Moreover, POLG gene mutation can lead to various mtDNA mutations and deletions as well as mtDNA depletion syndrome. The TWINKLE helicase encoded by TWNK gene is a mitochondrial 5'-3' helicase, which can bind to double-stranded DNA (dsDNA), and dissociate it into single-stranded DNA by breaking the hydrogen bond between the annealed nucleotide bases. 18 Besides, TWINKLE helicase can cooperate with Polγ to participate in the replication of mtDNA. 19 Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a transcriptional coactivator with multiple functional activities, plays a major role in the regulation of cellular energy homeostasis and mitochondrial oxidative metabolism. 20 Studies have shown that PGC-1α regulates the transcription of the mitochondrial genome by binding with NRF-1 and NRF-2 and activating the expression of the downstream genes TFAM, TFB1 M and TFB2 M that regulate the replication and transcription of mitochondrial DNA. 21 However, there are few studies on the mechanism of variations in mtDNA copy number and its follow-up effects in patients with IS.
The specific mechanism of variations in mtDNA copy number caused by D-loop mutation is still unclear.
Therefore, in the present study, firstly, mtDNA copy number levels of IS patients and normal controls were detected by quantitative real-time PCR and the effect of mitochondrial genome D-loop variation on mtDNA copy number of IS patients was explored. Then, by simulating the pathological process of cerebral ischaemia/reperfusion in IS, an oxygen-glucose deprivation and reperfusion injury (OGD/R) model was constructed to seek genes that regulate mtDNA copy number changes in IS. Finally, the knockdown-over-expression cell model of TFAM gene was constructed, and the influence of variations in mtDNA copy number on mitochondrial function was explored by testing the enzyme activity of mitochondrial respiratory chain complex, mitochondrial membrane potential and ATP production.

| Study population
The case group consisted of 101 patients with IS (68 males and 33 females) with the average age of 53.02 ± 9.15 years who were diagnosed in the First Affiliated Hospital of Henan University of Traditional Chinese Medicine from April to August in 2018. The diagnosis of all IS patients was based on the IS diagnostic criteria revised by the fourth National Conference on Cerebrovascular Diseases. All IS patients were first onset and confirmed by clinical examination (physical signs, history, biochemical tests, CT/MRI and other ancillary diagnostic investigations), without a family history of cardiovascular disease, diabetes and hypertension. The control group consisted of 101 healthy individuals (61 males and 40 females) with an average age of 52.05 ± 5.87 years whose gender and age matched that of IS group from the same hospital during the same period, excluding those with a family history of cardiovascular disease, diabetes and hypertension. All the subjects were from the Henan Han population without blood relationship between them.

The study was permitted by the Ethics Committee on Human
Research of Zhengzhou University. Written informed consent was obtained from all subjects. All experiments were performed in accordance with relevant guidelines and regulations.

| Extraction and purification of peripheral blood DNA
2-5mL of fasting peripheral venous blood was collected from subjects into tubes containing 2% EDTA-K 2 and stored in a refrigerator at −20℃ for subsequent assays. High-purity genomic DNA was isolated from peripheral blood cells of subjects using the Blood DNA Extraction Kit (TIANGEN). Concentration and quality of DNA were detected by NanoDrop2000 (Thermo Fisher).

| Detection of mtDNA copy number in population by Real-time PCR
Real-time quantitative PCR was performed on the mtDNA copy number of IS patients and normal subjects using the SYBR ® Premix Ex Taq TM kit (Takara) and a QS5 quantitative PCR Systems (Thermo

| Cell culture and oxygen-glucose deprivation/ re-oxygenation (OGD/R) treatments
Rat adrenal medulla pheochromocytoma cell line PC12 was purchased from Shanghai Cell Bank of Chinese Academy of Sciences.
PC12 cells were cultured in the prepared high-glucose complete medium and maintained in an incubator with 5% CO 2 , 37°C, and 95% relative humidity. CCK8 Kit (Meilunbio) was used to detect the viability of cells by testing the absorbance at 450 nm by ELIAS (Thermo Fisher). An oxygen-glucose deprivation/re-oxygenation (OGD/R) treatment was performed on cultured PC12 cells. In short, when in the logarithmic growth phase, PC12 cells were cultured in serumfree low-glucose DMEM medium after washing with 1 × PBS and placed in an anaerobic incubator with 95% N 2 , 5% CO 2 and 1% O 2

| Plasmids transfection in HEK293T cells
Human embryonic kidney cells HEK 293T was purchased from Shanghai Cell Bank of Chinese Academy of Sciences.  Goat Anti-Mouse IgG Secondary Antibody (Sino Biological). Protein bands were detected using an ECL analysis system.

| Detection of mitochondrial respiratory chain complex activity, mitochondrial membrane potential, and mitochondrial ATP level
The activity of the mitochondrial respiratory chain complex was, respectively, measured using the respiratory chain complex activity detection kit (Solarbio). In brief, mitochondria were first extracted from HEK 293T cells transfected with plasmids for subsequent determination of complex activity and concentration, then the pre- The level of ATP was calculated according to the standard curve.

| Statistical analysis
All statistical analyses were performed using SPSS21.0 software.
The experimental data was evaluated for normality and homogeneity of variance. The quantitative data was analysed by Independent Sample t-test or one-way ANOVA, which were expressed as Mean ±SD. The Student's t-test or rank sum test was used to analyse differences of the biochemical indexes and the D-loop region mutation between IS group and control group. The copy numbers of mtDNA for IS group and control group also accepted the test by the Student's t-test. The correlation between oxidative stress index and mtDNA copy number was analysed by Pearson correlation analysis or Spearman correlation analysis. The value of p < 0.05 indicated that the difference was statistically significant.

| Comparison of variations in mtDNA copy number in peripheral blood between IS group and control group
The clinical information of the study populations and the comparison results of the clinical data were summarized in Table S1. The level of mtDNA copy number in the IS group (1.11±0.8) was significantly lower than that in control group (1.52 ± 1.37; p < 0.05; Figure 1). Moreover, the results of gender stratification showed that the mtDNA copy number of IS patients was significantly lower than that of controls (p < 0.05) in the male group, but there was no statistical difference between the IS cases and the controls in the female group (p < 0.05), as shown in Table S2 and Figure S1; while age stratification results indicated that the mtDNA copy number of IS patients was significantly lower than that of the controls (p < 0.05) in the over-50-year-old group, but there was no statistical difference between the IS group and the control group under the age of 50 years (p < 0.05), as shown in Table S3 and Figure S2.

| The effect of mitochondrial genome D-loop mutation on mtDNA copy number
In our previous study, 7 mutation sites located in the D-loop region of the mitochondrial genome were detected in both the IS group and the control group, of which there was a significant difference in the mutation proportion of the m.T195C site between the IS group and the control group (p < 0.01, Table S4). In this study, the mtDNA copy number of IS patients with and without the above 7 D-loop mutation sites was statistically analysed. The results showed that the mtDNA copy F I G U R E 1 MtDNA copy number level in peripheral blood of IS group and control group (*p < 0.05) number (1.08 ± 0.82) of IS patients with the above D-loop mutation site was significantly lower than that of IS patients without the above D-loop mutation site (1.55 ± 1.23; p < 0.05; Figure 2A). Furthermore, a single analysis of the 7 mutation sites showed that the mtDNA copy number of IS patients with m.16215A > G and m.16355C > A mutations (1.07 ± 0.82, 0.98 ± 0.74 respectively) was significantly lower than that of IS patients without D-loop mutations (1.55 ± 1.24, 1.59 ± 1.23 respectively; p < 0.05). The results were shown in Figure 2B-C.

| Detection of mtDNA copy number in OGD/R cells model
The results of cell viability test at different time points after OGD/R treatment showed that the cell viability decreased to 60% after 4 h of oxygen-glucose deprivation, and the cell viability continued to decrease with a longer treat time, but it was easy to cause irreversible damage to the cell. When 4 h for oxygen-glucose deprivation was chosen, the cell viability was reduced to the lowest after 4 h of reperfusion; therefore, OGD 4h/R 4 h is the optimal modelling time for oxygen-glucose deprivation/reperfusion model (OGD/R). The results were shown in Figure 3A-B. The mtDNA copy number of cells in the OGD/R group (0.49±0.14) was significantly lower than that in the control group (1.03 ± 0.29; p < 0.05), which was consistent with the results of the population study ( Figure 3C).
In addition, heat shock protein 60 (HSP60) is a chaperone protein necessary for the folding of mitochondrial proteins and the formation of multimeric complexes, which is an important indicator for changes in mitochondrial expression. The results showed there was no difference in HSP60 protein expression between the OGD/R group and the control group (p > 0.05), which indicated that the mitochondria expression in the cells did not change after OGD/R injury. The results are shown in Figure S3.

| Research on genes regulating mtDNA copy number in OGD/R cells model
To explore genes that regulate mtDNA copy number of cells in the OGD/R group, real-time fluorescence quantitative PCR was  Table 1 and    Figure 5E.

| Effect of variations in mtDNA copy number on mitochondrial membrane potential
Compared with the control group, the mitochondrial membrane potential of HEK 293T cells significantly decreased in the sh-TFAM group (p < 0.05); while the mitochondrial membrane potential of HEK 293T cells in the OE-TFAM group was dramatically higher than that in the OE-Ctrl group (p < 0.05; Figure 5F-G).

| Effect of variations in mtDNA copy number on mitochondrial ATP production
The ATP generation level of HEK 293T cells transfected in the sh-TFAM group was significantly lower than that in the control group and sh-Ctrl group (p < 0.05), and compared with the control group and the OE-Ctrl group, the ATP production level of the OE-TFAM group was markedly increased (p < 0.01; Figure 5H).

| DISCUSS ION
MtDNA copy number is the number of mtDNA in the mitochondrial genome. Each contains 2-10 mtDNA copies. The copy number of mtDNA in each somatic cell of normal people is about 10 3 -10 4 , which is specific in different types of tissue cells. 22 The mtDNA copy number of tissues and organs with high energy dependence is relatively higher than that of tissues and organs with low energy dependence. 23 The change of mtDNA copy number can affect the function of mitochondria and play an important role in the occurrence and development of diseases.
Our results showed that the mtDNA copy number level of the IS group was significantly lower than that of the normal control group (p < 0.05). In the gender stratification analysis, it was found that the mtDNA copy number of the male IS group was significantly lower than that of the male control group (p < 0.05), but there was no significant difference between the female IS group and the female control group (p > 0.05). In the age stratification analysis, the mtDNA copy number in the IS patients over 50 years old was significantly lower than that in the normal control group (p < 0.05), but there was no significant difference between the IS group and the control group under 50 years old (p > 0.05). Lien et al. found that the mtDNA copy number of IS patients was significantly lower than that of the normal population. 24 Ashar et al.
also reported that the copy number of mtDNA in stroke patients was significantly reduced in large samples of cardiovascular disease, and the copy number of mtDNA was negatively correlated with the incidence of stroke. 25  The energy released by the mitochondrial respiratory chain during electron transfer is used to drive protons from the mitochondrial matrix to the membrane space. 37 Due to the high impermeability of the inner membrane to H + , a potential gradient across the mitochondrial inner membrane is created, resulting in a negative internal membrane potential. 38 Normal membrane potential is necessary to maintain mitochondrial oxidative phosphorylation function.
We used JC-1 fluorescent probe to detect the changes in mitochon- The respiratory chain of mitochondria is coupled with the phosphorylation of ADP in the process of electron transfer. Under the action of ATP synthase, ADP and 1-molecule phosphate are combined to form ATP, providing energy for life activities. 40 Our results showed that after sh-TFAM transfection, mtDNA copy number decreased, ATP production level was significantly decreased compared with blank control group and Sh-Ctrl negative control group (p < 0.05). The ATP production level of the OE-TFAM transfected group was significantly higher than that of blank control group and OE-ctrl negative control group (p < 0.01), indicating that the increase of mtDNA copy number can promote intracellular ATP production.
Hori A et al. found that mtDNA encodes proteins that are essential for cellular ATP production. 41 The present study includes several limitations. Firstly, further research of specific mechanism of variations in mtDNA copy number caused by mutations in the D-loop region is needed. Secondly, the mechanism of TFAM gene function damage after OGD/R injury is still unclear. Thirdly, the mechanism of mitochondrial copy number regulation of mitochondrial function needs further study.
In brief, the study demonstrated that mitochondrial D-loop mutation and TFAM gene dysfunction can cause the decrease of mtDNA copy number, resulting in the decrease of mitochondrial respiratory chain complex activity, membrane potential and ATP production, F I G U R E 6 Influence of mtDNA copy number mutation on mitochondrial function thus affecting the mitochondrial metabolism and function of nerve cells and participating in the pathological damage mechanism of IS.
In addition, two new mutations, m.16215 G > A and m.16355 C > A, which affect variations in mtDNA copy number, were found in the D-loop region and provide a new theoretical basis for variations in mtDNA copy number ( Figure 6).

ACK N OWLED G EM ENTS
We acknowledge the technical assistance of staff members of the department of the first affiliated hospital of Henan University of CM and the First People Hospital of Zhengzhou. We also thank all patients and controls for providing blood samples.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest with the contents of this article. Supervision (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.