Overexpression of NAF-1 ameliorates Ischemic Stroke in rats by inhibiting PINK1/Parkin-mediated mitophagy

doi:10.21203/rs.3.rs-2003573/v1

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Overexpression of NAF-1 ameliorates Ischemic Stroke in rats by inhibiting PINK1/Parkin-mediated mitophagy

https://doi.org/10.21203/rs.3.rs-2003573/v1

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Ischemic stroke (IS) has a high mortality and disability rate worldwide. NAF-1 is involved in the occurrence and development of nervous system diseases, but its involvement in the pathophysiology of IS is unclear. In this study, we investigated the role of NAF-1 in IS. Middle cerebral artery occlusion and reperfusion in Sprague-Dawley rats was used as an in vivo model of IS. Moreover, oxygen-glucose deprivation and reoxygenation of PC12 cells was used for in vitro study. Intracellular NAF-1 protein expression was influenced by LV-NAF-1 or siRNA. Quantitative real-time PCR and western blot analysis showed that NAF-1 was significantly downregulated following IS. Overexpression of NAF-1 reduced mitophagy in PC12 cells, and it alleviated tissue damage in vivo in rats and cell injury in vitro. In contrast, knockdown of NAF-1 increased mitophagy and worsened cell damage. These findings suggest that NAF-1 may have potential as a therapeutic target for IS.

Ischemic stroke

NAF-1

Mitophagy

PINK1

Parkin

Ischemic stroke (IS) accounts for about 87% of strokes and is a significant cause of disability worldwide[1, 2]. Notably, it develops rapidly, has a high fatality rate, and is prone to relapse[3]. At present, the accepted treatment is thrombolysis or thrombus resection. However, a major limitation is the short time window, and only a small fraction of patients are consequently eligible for treatment[4]. Effective targets are therefore urgently needed to mitigate the progression of the disease.

In neurons, mitochondrial oxidative phosphorylation is the primary source of ATP[5]. During the reperfusion stage following stroke, reactive oxygen species are produced in large quantities, resulting in mitochondrial dysfunction[6]. Reducing mitochondrial damage is essential in IS[7, 8]. Mitophagy is a selective process of autophagy that removes dysfunctional or redundant mitochondria[9]. With moderate mitophagy, nerve cell damage can be reduced[10]. However, mitophagy is a double-edged sword. Excessive mitophagy can lead to the accumulation of toxic substances and extensive self-degradation. Studies have shown that cucurbitacin I can reduce oxygen-glucose deprivation and reoxygenation (OGD/R)-mediated damage to PC12 cells by inhibiting mitophagy[11]. The classical mitophagy pathway is the PTEN-induced putative kinase 1 (PINK1)/Parkin pathway. Inhibition of the PINK1/Parkin pathway has been shown to reduce mitophagy levels and injury induced by IS[11, 12].

NAF-1 (nutrient-deprivation autophagy factor-1) is localized on the endoplasmic reticulum and mitochondrial outer membrane, and is encoded by the CISD2 gene[13, 14]. An increasing number of studies show that NAF-1 is involved in the pathogenesis of many diseases. NAF-1 can ameliorate the damage caused by oxidative stress, reduce nutritional stress damage, and reduce mitochondrial damage. Furthermore, it can reduce damage to the endoplasmic reticulum, and regulate autophagy and iron accumulation[15–18]. Overexpression of CISD2 reduces mitochondrial damage and neuronal loss in a mouse model of Alzheimer’s disease[19]. CISD2 may target neurodegenerative dementia by affecting microglial inflammation[20]. Treatment with anti-CISD2 antibodies aggravates spinal cord and cerebral inflammation in mice with spinal cord injury[21]. However, it is unclear whether regulating NAF-1 ameliorates IS-mediated cell and tissue damage. In this study, we investigate whether NAF-1 attenuates IS injury by regulating mitophagy levels in vitro and in vivo.

Middle Cerebral Artery Occlusion and Reperfusion (MCAO/R)

A total of 72 male Sprague-Dawley rats (260–280 g) were housed in the Animal Center of Chongqing Medical University. The temperature was set at 25 °C, and the rats were allowed to drink water freely. The rats were maintained under a 12/12-h light/dark cycle. All animal procedures were conducted following the ethical regulations for animal experiments and were supervised by the Animal Center of Chongqing Medical University. (License Number: SYXK YU 2018-0003).

The IS model was carried out in accordance with previous studies[22, 23]. The rats were anesthetized by intraperitoneal injection with 3% pentobarbital sodium (1 ml/kg). Rats were placed in the supine position to expose the common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA). A silicone nylon suture was inserted from the CCA into the ICA to block the middle cerebral artery. After 90 min, the plug was slowly removed, and the CCA was ligated. The operation in the sham group was the same as in the model group, except the plugs were not inserted.

Experimental Groupings

To study the expression of NAF-1, the rats were randomly divided into the following five groups: Sham, MCAO/R 12 h, MCAO/R 24 h, MCAO/R 48 h, and MCAO/R 72 h. To study the role of NAF-1 in MCAO/R, the rats were grouped as follows: Sham, MCAO/R, MCAO/R+LV-NC, MCAO/R+LV-NAF-1.

Lateral ventricle injection: 14 days before MCAO surgery, either NAF-1-overexpressing lentivirus (5.5 µL of 1 × 10⁸ TU/mL, GENE, Shanghai, China) or control lentivirus (7.5 µL of 1 × 10⁸ TU/mL) was injected into the lateral ventricle. The rats were anesthetized with pentobarbital sodium by intraperitoneal injection, immobilized, and the bregma was exposed. The injection site was 1.5 mm to the right of the bregma and 0.8 mm posterior. The depth of the injection was 4.5 mm. A 10-µL volume of lentivirus stock was used for injection.

OGD/R

In this study, rat PC12 adrenal pheochromocytoma cells from the National Collection of Authenticated Cell Cultures (Shanghai, China) were used to simulate rat cortical neurons. Cells were cultured in DMEM containing 10% FBS (ExcellBio, Shanghai, China). For OGD, the medium was replaced with glucose-free DMEM (Gibco, Waltham, MA, USA) under a hypoxic environment (1% O₂,93.5% N₂,5% CO₂) for 2 h. Then, cells were cultured in a maintenance medium. To study the expression of NAF-1 in PC12 cells, the cells were divided into the following five groups: Control, OGD/R 6 h, OGD/R 12 h, OGD/R 24 h, and OGD/R 48 h.

Lentivirus Infection and siRNA Transfection

To study the function of NAF-1 in vitro, PC12 cells were divided into the following groups: Control, OGD/R, OGD/R+LV-NC, and OGD/R+LV-NAF-1. Cells were infected with LV-NAF-1 (MOI = 10) or LV-NC (MOI = 10) for 72 h. Then, stable lines were selected with puromycin (10 mg/mL) (BioFroxx, Einhausen, Germany) for 1 week. Next, to study the effect of NAF-1 downregulation, cells were divided into the following groups: Control, OGD/R, OGD/R+siNC, and OGD/R+siR-NAF-1. siR-NAF-1 sense (ACCUUGCAGUUCGUCCAUUCU-tt) and siNC sense (UUCUCCGAACGUGUCACGUdTdT) were synthesized by GeneBiogist (Shanghai, China). Cells were cultured to 50% confluency, and then, siRNA (60 nM) and riboFECT CP reagent (RIBBIO, Guangzhou, China) were added to the cells and maintained in culture until subsequent experiments.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

RNA from PC12 cells and rat cortical tissues were extracted with Trizol (Sangon Biotech, Shanghai, China), and then converted into cDNA using the Evo M-MLV RT Kit (Accurate Biotechnology, Hunan, China). cDNA and SYBR Green qPCR Master Mix (Bimake, Shanghai, China) were used for amplification with the following thermocycling parameters: 95 °C for 30 s, 38 cycles at 95 °C for 5 s and 62 °C for 30 s, 65 °C for 5 s, and 95 °C for 5 s. The primer sequences were as follows: rat NAF-1 (Fw: 5ʹ-GCTTACTGCCCTTCCTTGGT-3ʹ, Rev: 5ʹ-TCTGTTGCTTCTTCTTCGGGA-3ʹ) and rat β-actin (Fw: 5ʹ-GGAGATTACTGCCCTGGCTCCTA-3ʹ, Rev: 5ʹ-GACTCATCGTACTCCTGCTTGCTG-3ʹ).

Western Blotting

Total proteins from rat cortical tissues and PC12 cells were extracted using RIPA buffer (Beyotime, Shanghai, China). A 12.5% PAGE gel fast preparation kit (Epizyme, Shanghai, China) was used to resolve the proteins, which were then transferred onto PVDF membranes (Millipore, Boston, MA, USA) and blocked with 5% BSA for 2 h. The membranes were then incubated overnight at 4 °C with the following primary antibodies: β-actin (1:6,000, Proteintech, #66009-1), LC3A/B (1:1,000, Cell Signaling Technology, #12741), TOMM20 (1:1,000, Abcam, #ab186735), NAF-1 (1:1,000, Cell Signaling Technology, #60758), Beclin1 (1:1,000, Affinity, #AF5128), p62 (1:1,000, Cell Signaling Technology, #5114), PINK1 (1:2,000, Affinity, #DF7742), Parkin (1:1,000, ABclonal, #A0968). The membranes were thereafter incubated with secondary antibody. Finally, an ECL FemtoLight Chemiluminescence Kit (Epizyme, Shanghai, China) was used for detection.

Tissue Mitochondrial Isolation

Mitochondria were isolated with a tissue mitochondrial isolation kit (Beyotime). Fresh cortical tissue was homogenized in mitochondrial isolation reagent containing PMSF, and then centrifuged at 600 × g at 4 °C for 5 min. The supernatant was then centrifuged at 11, 000 × g at 4 °C for 10 min. The pellet was mitochondria.

Cell Viability Assay

MTT (5 mg/mL) (BioFroxx) was used to measure cell viability. A 20-µL of MTT was added to cells and incubated for 2 h at 37 °C. Then, cells were incubated with 150 µL dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA). The absorbance was measured at 490 nm with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Flow Cytometry

An Annexin V-iFluor 488/propidium iodide (PI) Apoptosis Detection kit (CHAMOT, Shanghai, China) was used to detect apoptosis. The cells were reacted with Annexin V-iFluor 488 and PI for 15 min. Finally, the apoptosis level was measured using a BD Influx apparatus (BD Biosciences, San Diego, CA, USA).

ATP Content

ATP content was measured with an ATP assay kit (Beyotime). In short, cells and tissues were lysed and centrifuged. The chemiluminescence value of the supernatant was measured using a microplate reader (Thermo Fisher Scientific), and a standard curve was used to calculate the ATP content.

Mitochondrial Membrane Potential (MMP) Assay

MMP was measured using a JC-1 assay kit (Beyotime). Briefly, mitochondria were isolated from tissues with the tissue mitochondrial isolation kit. JC-1 staining solution was added to mitochondria, and the fluorescence intensity was detected at 485 nm excitation/590 nm emission on a microplate reader (Thermo Fisher Scientific). For cells, JC-1 staining solution was added to the cells and incubated for 20 min. The fluorescence intensity was detected on a fluorescence microscope (Nikon, Tokyo, Japan) after three washes.

Transmission Electron Microscopy (TEM)

The cells were digested and centrifuged at 1, 200 rpm for 10 min, and then gently fixed with glutaraldehyde. Mitochondria and autophagosomes were observed by TEM (Hitachi 7100, Tokyo, Japan).

Immunofluorescence Staining

Mitochondria were stained using Mito-Tracker Red CMXRos (Beyotime) for 30 min. Then, the cells were fixed with cold methanol for 5 min and permeabilized with 1% Triton X-100 (Beyotime) for 10 min. Thereafter, cells were blocked with 10% goat serum (Mengbio, Chongqing, China) for 1 h. Primary antibody was then added to the cells and incubated overnight at 4 °C. The secondary antibody (1:200, Abcam, #ab150077) was added to the cells and incubated at room temperature for 1 h. Finally, cells were incubated with DAPI (Servicebio, Wuhan, China) for 10 min, and the fluorescence intensity was observed on a fluorescence microscope (Nikon).

Neurological Deficits

To assess neurological deficits, 48 h after MCAO, a blinded researcher assessed each group of rats using the Zea-Longa[24] scoring system, as follows: 0 = no apparent damage, 1 = the contralateral forelimb cannot fully extend, 2 = flex to the opposite side and cannot extend, 3 = fall to the opposite side, 4 = unable to walk.

Infarct Area

The brain was frozen at −20 °C for 1 h, and then cut into coronal sections of 2 mm. The slices were placed into 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich) for 30 min at 37 °C, and then fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) overnight. The infarct area appeared white. Cerebral infarct area (%) = infarct area × 100/total area.

Hematoxylin-Eosin (HE) Staining

Forty-eight h after MCAO, rats were transcardially perfused with PBS and 4% paraformaldehyde (Servicebio). The whole brain was removed and fixed in 4% paraformaldehyde. The brain tissue was then paraffin-embedded, sectioned for HE staining, and photographed on a light microscope (Nikon). The damaged nuclei were pyknotic or vacuolated. Percentage of damaged cells (%) = number of damaged cells × 100/total number of cells[25].

Statistical Analysis

All data were analyzed with GraphPad Prism software (version 6.0) and expressed as mean ± SD. One-way ANOVA was used to compare the data. P < 0.05 indicated a significant difference.

NAF-1 Expression is Reduced In Vitro and In Vivo in IS

Compared with the sham group, mRNA and protein expression levels of NAF-1 were reduced at 12, 24, 48 and 72 h post-MCAO. The most significant decrease was observed at 48 h post-MCAO (Fig. 1a–c). Similarly, the expression of NAF-1 protein was decreased at 6, 12, 24 and 48 h post-OGD. The most significant decrease was observed at 12 h post-OGD (Fig. 1d, e). These results suggest that NAF-1 expression is decreased both in vitro and in vivo in IS. The 48-h time point was selected for further study in vivo, while the 12-h time point was selected for further study in vitro.

Analysis of Mitophagy Regulators by Metascape Online Analysis

Mitophagy is closely related to IS. We screened 33 mitophagy regulatory genes in the literature and analyzed them with Metascape (https://metascape.org/gp/index.html#/main/step1 (analyzed on 1 August, 2022)). The analysis showed that mitophagy is involved in regulating various biological processes, including growth and neurodegenerative diseases (Fig. 2a–c). The protein–protein interaction (PPI) analysis revealed an interaction between NAF-1 and Parkin (Fig. 2d). Previous studies found that Parkin is closely related to mitophagy[11,12], therefore, we hypothesized that NAF-1 might regulate mitophagy through the PINK1/Parkin pathway.

Overexpression of NAF-1 Reduces Injury Induced by OGD/R in PC12 Cells

Cells were infected with NAF-1 lentivirus, and mRNA and protein expression levels were assessed. Compared with the LV-NC group, NAF-1 mRNA and protein levels were significantly upregulated in the group given NAF-1 lentivirus (Fig. 3a–c). Cells were infected with NAF-1 lentivirus before OGD/R. Compared with the control group, NAF-1 was downregulated in the OGD/R group. There was no significant difference between the OGD/R+LV-NC and OGD/R groups. NAF-1 expression in the OGD/R+LV-NAF-1 group was significantly increased compared with the OGD/R+LV-NC group (Fig. 3d, e). To investigate the role of NAF-1 in PC12 cells after OGD/R, MTT was used to detect cell viability. Compared with the control group, cell viability was decreased after OGD/R. The OGD/R-mediated reduction in cell viability was countered by the NAF-1 lentivirus (Fig. 3f). Furthermore, the number of cells was increased, debris was reduced, and cellular atrophy was decreased in the OGD/R+LV-NAF-1 group compared with the OGD/R+LV-NC group (Fig. 3g).

Overexpression of NAF-1 Reduces OGD/R-induced Mitophagy by Suppressing the PINK1/Parkin Pathway in PC12 Cells

Next, we studied the expression of PINK1, Parkin and autophagy-related proteins after overexpression of NAF-1. PINK1, Parkin, beclin1 and LC3II/LC3I expression levels were decreased in the OGD/R+LV-NAF-1 group compared with the OGD/R+LV-NC group. In contrast, p62 and TOMM20 were upregulated (Fig. 4a–g). ATP content was significantly reduced after OGD/R, but was increased in the OGD/R+LV-NAF-1 group (Fig. 4h).

Knockdown of NAF-1 Aggravates Damage to PC12 Cells after OGD/R

To examine the knockdown efficiency of the siRNA, the protein expression levels of NAF-1 were measured in normal cells after transfection with siRNA. NAF-1 protein levels in cell lines transfected with sequences 1 or 2 was significantly reduced compared with the control group (Fig. 5a, b). We then chose siRNA sequence 1 for further experiments. The cells were transfected with siRNA and treated with OGD/R. Compared with the OGD/R+siR-NC group, the expression of NAF-1 in the OGD/R+siR-NAF-1 group was significantly reduced (Fig. 5c, d). MTT was then used to assess cell viability. Compared with the OGD/R+siR-NC group, cell viability was reduced in the OGD/R+siR-NAF-1 group (Fig. 5e). Furthermore, cell morphological injury was aggravated in the OGD/R+siR-NAF-1 group (Fig. 5f). Next, we examined apoptosis by flow cytometry. Compared with the OGD/R+LV-NC group, the apoptosis rate was significantly decreased in the OGD/R+LV-NAF-1 group. In comparison, after NAF-1 knockdown, the apoptosis rate increased significantly (Fig. 5g).

Knockdown of NAF-1 Aggravates OGD/R-induced Mitophagy via the PINK1/Parkin Pathway in PC12 Cells

We investigated the expression of PINK1, Parkin and autophagy-related proteins after knockdown of NAF-1. Compared with the OGD/R+siNC group, PINK1, Parkin, beclin1, and LC3II/LC3I expression was increased, and p62 and TOMM20 expression levels were decreased in the OGD/R+siR-NAF-1 group (Fig. 6a–g). ATP content was significantly decreased after cells were transfected with siR-NAF-1 (Fig. 6h). MMP level is closely related to mitochondrial function, and was expressed as the ratio of red to green fluorescence. This ratio was decreased significantly in the OGD/R group compared with the control group. In contrast, the ratio was increased after infection with NAF-1 lentivirus, compared with the OGD/R+NC group. Furthermore, the ratio was reduced in the OGD/R+siR-NAF-1 group compared with the OGD/R+NC group (Fig. 6i). Additionally, to observe mitophagy level, LC3 and mitochondria labeled with mitotracker were observed by immunofluorescence staining. Compared with the control group, mitophagy level was increased in the OGD/R group. Furthermore, compared with the OGD/R group, mitophagy level was decreased in the OGD/R+LV-NAF-1 group. However, the mitophagy level was increased in the OGD/R+siR-NAF-1 group compared with the OGD/R group (Fig. 6j). Mitophagy was also examined by TEM. Compared with the control group, the number of autophagosomes and mitochondrial swelling were increased in the ODG/R group. Compared with the OGD/R group, the number of autophagosomes and mitochondrial swelling were reduced in the ODG/R+LV-NAF-1 group. In contrast, the number of autophagosomes and mitochondrial swelling were increased in the ODG/R+siR-NAF-1 group (Fig. 6k).

Overexpression of NAF-1 Alleviates MCAO/R-induced Injury in Rats

NAF-1 lentivirus was injected into the lateral ventricle of rats. Protein expression of NAF-1 was detected in each group. NAF-1 expression in the MCAO/R group was markedly downregulated compared with the sham group. Compared with the MCAO/R+LV-NC group, NAF-1 protein was increased significantly in the MCAO/R+LV-NAF-1 group (Fig. 7a, b). To study the role of NAF-1 in ischemia-reperfusion injury in rats, we assessed neurological deficit scores and examined cerebral infarction area and histopathological changes after MCAO/R. The neurological deficit score and infarct area were increased in the MCAO/R group compared with the sham group. Notably, they were reduced in the MCAO/R+LV-NAF-1 group compared with the MCAO/R+LV-NC group (Fig. 7 c–e). Compared with the sham group, nuclear pyknosis and vacuolation of neurons were increased in the MCAO/R group. These cytopathological changes were ameliorated in the MCAO/R+LV-NAF-1 group (Fig. 7f, g).

Overexpression of NAF-1 reduces MCAO/R-induced Mitophagy via Suppression of the PINK1/Parkin Pathway

Next, we studied the expression of PINK1, Parkin and autophagy-related proteins after overexpression of NAF-1. Western blotting showed that compared with the MCAO/R+LV-NC group, PINK1, Parkin, beclin1 and LC3II/LC3I expression were decreased, and p62 and TOMM20 expression levels were increased in the MCAO/R+LV-NAF-1 group (Fig. 8a–g). Tissue mitochondria were isolated to detect the protein expression levels of PINK1, Parkin and LC3II/LC3I. Their expression levels were increased in the MCAO/R group compared with the sham group. However, this upregulation was suppressed by infection with the NAF-1 lentivirus (Fig. 8h–k). ATP content was significantly decreased after MCAO/R, but was increased by infection with the NAF-1 lentivirus (Fig. 8l). The aggregate fluorescence was decreased in the MCAO/R group compared with the sham group. However, this was suppressed by infection with the NAF-1 lentivirus (Fig. 8m).

Mitophagy is a dynamic process that removes damaged mitochondria by autophagy. The process of autophagy includes the formation of phagophores, the maturation of bilayer structures, and the fusion and degradation of autophago-lysosomes [5, 26]. The conversion of LC3I to LCII and the degradation of the latter are key events in the autophagy process[27]. p62 is a substrate of autophagy, and its expression reflects the level of autophagy[28]. As an autophagy regulator, beclin1 modulates the autophagy process by recruiting ATG[29]. TOMM20 is located in the outer membrane of mitochondria and reflects changes in mitochondrial number[30].

We found here that NAF-1 expression was downregulated in the IS model. Overexpression of NAF-1 reduced cellular apoptosis, LC3II/LC3I, beclin1, the neurological deficit score, the infarct area and neuronal vacuolation, and increased p62 levels and cell viability.

PINK1 and Parkin (E3-ubiquitin ligase)-mediated mitophagy affects many mitochondrial processes[31]. Normally, PINK1 is transferred from the outer membrane to the inner membrane of mitochondria, depending on membrane potential[32]. PINK1 is then cleaved and degraded to maintain it at a low level[33]. When the membrane potential dissipates, PINK1 accumulates in the outer membrane[34]. Next, Parkin is recruited by phosphorylation of accumulated PINK1. Finally, PINK1 produces poly-Ub chains to amplify the mitophagy signal[35, 36]. In our study, we found that PINK1 and Parkin protein levels increased and TOMM20 levels decreased after IS. After overexpression of NAF-1, the protein levels of PINK1 and Parkin decreased, and TOMM20 increased. The opposite trend was observed after knockdown of NAF-1. Similar trends were found for PINK1 and Parkin in mitochondria. After MCAO/R, PINK1 and Parkin protein levels in mitochondria increased, and these increases were inhibited by overexpression of NAF-1. Furthermore, MMP levels decreased after IS, but increased after overexpression of NAF-1. TEM revealed that mitochondrial damage was aggravated and the number of autophagosomes increased after OGD/R. After the overexpression of NAF-1, mitochondrial damage and the number of autophagosomes were reduced. These changes were inhibited by NAF-1 knockdown. The fluorescence analysis also showed that overexpression of NAF-1 reduces mitophagy. These results suggest that PINK1/Parkin-dependent mitophagy increases after IS. Overexpression of NAF-1 may lead to a decrease in PINK1 accumulation on the outer membrane and mitophagy levels by inhibiting its transfer from the outer to the inner mitochondrial membrane. However, further research is needed to verify this result.

Our findings show that mitophagy levels are increased after IS in vitro and in vivo, and that overexpression of NAF-1 inhibits mitophagy and excessive auto-degradation. The underlying mechanisms may involve inhibition of the recruitment of PINK1 and Parkin proteins in mitochondria. NAF-1 may be a target for the treatment of IS (Fig. 9).

Acknowledgments: This work was supported by the Chongqing Yuzhong District Science Technology Commission of China (Grant Number: 20210121), the Outstanding Researcher Fund of the College of Pharmacology of Chongqing Medical University (No. YXY2021BSH02), China Postdoctoral Science Foundation (NO:2022MD713710), and Chongqing Postdoctoral Special Grant (NO:2021XM3063). ZD conceived and supervised the experiment and wrote the manuscript. XF collected the data and wrote the manuscript. SC supervised the experiments and wrote the manuscript. YW and LD provided opinion. All authors read and approved the final manuscript. We thank Barry Patel, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn/), for editing the English text of a draft of this manuscript.

Compliance with ethical standards

Conflict of interest The authors declare that they have no financial conflicts of interest.

Ethical Approval Animal procedures were in accordance with animal ethical standards.

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Overexpression of NAF-1 ameliorates Ischemic Stroke in rats by inhibiting PINK1/Parkin-mediated mitophagy

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