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Keywords:

  • Dpr1;
  • mitochondrial dynamics;
  • neurons;
  • oxygen–glucose deprivation;
  • PINK1

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Competing interest
  8. References
Thumbnail image of graphical abstract

Our previous study has shown that PTEN-induced novel kinase 1 (PINK1) knocking down significantly induced mitochondrial fragmentation. Although PINK1 is proved to be associated with autosomal recessive parkinsonism and its function in this chronic pathological process is widely studied, its role in acute energy crisis such as ischemic stroke is poorly known. In this study by employing an oxygen–glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. Human PINK1, two PINK1 mutants W437X and K219M, or Pink1 shRNA were transduced before OGD using lentiviral delivery. Our results showed that over-expression of wild-type PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential. PINK1 over-expression also reversed OGD increased mitochondrial fragmentation, and suppressed the translocation of the mitochondrial fission protein dynamin-related protein 1 (Drp1) from the cytosol to the mitochondria. Transduction of the mutant PINK1 failed to provide any protective effect, while knockdown of Pink1 significantly increased the severity of OGD-induced neuronal damage. Importantly, inhibition of Drp1 reversed the effects of knocking down Pink1 on neuronal death and ATP production in response to OGD. This study demonstrates that PINK1 prevents ischemic damage in neurons by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission. These novel findings implicate a pivotal role of PINK1 regulated mitochondrial dynamics in the pathology of ischemic stroke.

In this study by employing an oxygen–glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. We indicated that PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission.

Abbreviations used
DAPI

2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride

Drp1

dynamin-related protein 1

LDH

lactate dehydrogenase

MMP

mitochondrial membrane potential

MPP+

1-methyl-4-phenylpyridinium

OGD

oxygen–glucose deprivation

PD

Parkinson's disease

PINK1

PTEN-induced novel kinase 1

Mitochondria are ubiquitous intracellular organelles enclosed by a double membrane structure. The primary function of mitochondria is the production of cellular energy in the form of adenosine triphosphate (ATP). Mitochondrial shape is maintained by two opposing forces: fission and fusion (Chan 2006). In healthy neurons, fission and fusion balance equally; imbalances between these processes lead to abnormal mitochondrial function (Westermann 2002; Chan 2006). Fission and fusion are controlled by evolutionarily conserved, large GTPases belonging to the dynamin family.

Mitochondrial fission is controlled by dynamin-related protein 1 (Drp1) and the protein mitochondrial fission 1 (Fis1) (Knott et al. 2008). Although Drp1 mainly localizes within the cytoplasm, a small amount of Drp1 can translocate to the outer mitochondrial membrane where it promotes mitochondrial fragmentation (Smirnova et al. 2001). Fis1 is localized to the outer mitochondrial membrane (Yoon et al. 2003; Chang and Blackstone 2010). Drp1 and Fis1 are activated by increased free radical production in the mitochondria, which is critical for mitochondrial fission.

Mitochondrial fusion is also regulated by three other GTPase proteins: two outer-membrane-localized proteins, Mfn1 and Mfn2 (mitofusin 1 and 2), and the inner-membrane-localized protein Opa1 (optic atrophy 1) (Cerveny et al. 2007; Detmer and Chan 2007; Benard and Karbowski 2009). The C-terminus of Mfn2 mediates oligomerization between Mfn molecules from adjacent mitochondria and facilitates mitochondrial fusion (Ishihara et al. 2004; Zuchner et al. 2004).

Another important protein involved in mitochondrial dynamics is PTEN-induced novel kinase 1 (PINK1), which was originally reported to be associated with autosomal recessive parkinsonism (Hardy et al. 2006). Although debate still exists on the exact function of PINK1, recent studies have demonstrated the ability of PINK1 to alter the balance between mitochondrial fission and fusion (Exner et al. 2007; Yang et al. 2008; Dagda et al. 2009; Sandebring et al. 2009). Our previous research experience demonstrated that PINK1 exerts an overall fusion effect. Strategies impairing the function of Pink1, either by knocking down of endogenous Pink1 or over-expression of mutant forms of PINK1 induced severe mitochondrial fragmentation in N27 cells. By over-expressing dominant negative forms of Drp1 or mfn2 to inhibit fission or enhance fusion, we successfully reversed this morphological change (Cui et al. 2010).

The majority of literature on the influence of mitochondrial dynamics in pathologies of the central nervous system is associated with neuronal cell death in age-related neurodegenerative diseases. It is suggested that impaired regulation of mitochondrial dynamics towards fission would induce essential neuronal apoptosis (Otera and Mihara 2012). Although severe energy crisis occurs in the pathology of acute brain injury, such as ischemic stroke, support for the role of mitochondrial dynamics in these pathologies is strikingly rare. Only recently, evidence showed that mitochondrial fission may be associated with neuronal cell death induced by glutamate toxicity and oxygen–glucose deprivation (OGD), as inhibition of mitochondrial fission protected neurons from glutamate toxicity and ischemic damage (Grohm et al. 2012). Although, as mentioned above, the function of Pink1 in the regulation of mitochondrial dynamics is not fully characterized, Pink1 is mainly considered to be a neuroprotective protein. PINK1 over-expression confers resistance to staurosporine, MPP+, and rotenone toxicity (Petit et al. 2005; Haque et al. 2008; Sandebring et al. 2009). Based on our previous finding that Pink1 is a mitochondrial fusion protein, in this study we aimed to characterize the role of Pink1 in acute cerebral ischemia and its association with mitochondrial dynamics.

We demonstrated that Pink1 deficiency sensitizes primary rat neurons to ischemic damage in vitro. Over-expression of human PINK1 could prevent mitochondrial fission, bioenergetic defects, loss of mitochondrial membrane potential (MMP), and cell death induced by OGD. PINK1 blocked mitochondrial fission induced by ischemic damage, by preventing translocation of Drp1 from the cytosol to the mitochondria. Our results indicate that a classic Parkinson's disease gene can also exert an important protective function during the response of the brain to stroke, which is likely to be mediated through the capacity of PINK1 to regulate mitochondrial dynamics.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Competing interest
  8. References

Cell culture

Sprague–Dawley rats were purchased from the Shanghai Institute of the Chinese Academy of Science. All experimental procedures were carried out in accordance with the experimental standards of Fudan University, as well as international guidelines on the ethical treatment of experimental animals. Neurons were isolated from embryonic day (E) 14.5 embryos (Rempe et al. 2007). Briefly, the embryos were removed and placed into ice-cold Dulbecco's minimal essential medium. Using a dissecting microscope, the cortex and hippocampus were dissected away from the brainstem, the meninges were removed and the telencephalon was placed into ice-cold HBSS. The tissue was digested in trypsin-EDTA (0.1%) for 20 min, washed three times in Dulbecco's minimal essential medium, and dispersed in neurobasal medium containing B27, l-glutamine and glutamic acid. The cells were plated on poly-l-lysine coated-glass coverslips, 96-well plates or 100 mm dishes at a density of 3–10 × 105 cells/mL and maintained at 37°C in a humidified 5% CO2 incubator. Under these conditions, neuronal purity was approximately 90%, as estimated by positive immunocytochemical staining for neurofilament proteins and the negative immunocytochemical staining for glial fibrillary acidic protein (data not shown). The cultured neurons were used for studies on in vitro days 8–10 (DIV 8–10).

The human neuroblastoma cell line SH-SY-5Y was used for the Drp1 translocation study. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 2 mM l-glutamine at 37°C with 5% CO2.

OGD

OGD was performed on mature cultures (DIV 8–10) as previously described, with minor modifications (Liu et al. 2011). Briefly, the original glucose-containing media were removed from cultured neurons and kept for future use. Neurons were washed three times with deoxygenated glucose-free ECF, and then incubated in glucose-free ECF in an anaerobic chamber (Model 1025; Forma Scientific, Vernon Hills, IL, USA) in an atmosphere (< 0.1% O2) containing 85% N2, 10% H2, and 5% CO2 at 37°C; control neurons were washed, incubated in ECF and placed in a normoxic incubator. OGD was terminated after 90 min by removing the cultures from the chamber, washing the cells with oxygenated glucose-containing ECF (pH 7.4), adding the original media and incubating the cultures at 37°C in the normoxic incubator for 8 h reoxygenation.

Lentivirus generation

Three plasmids were used for production of lentiviral vectors: (i) the packaging construct delta 8.91, in which the cytomegalovirus (CMV) early promoter drives the synthesis of all viral proteins besides the envelope (Zufferey et al. 1997); (ii) the plasmid Pvsvg producing the pseudotyping envelope vesicular stomatitis virus glycoprotein (Ronca et al. 1999); and (iii) the expression vector pGMLV-PA1, a CMV-MCS-IRES-GFP plasmid, in which the transgene is driven by the CMV promoter and GFP is expressed independently. For construction of the PINK1-expressing lentiviral vector, full length human PINK1 was subcloned from a pcDNA3.0-PINK1 vector (kindly provided by Dr. Kim Tieu, University of Rochester, USA). Transfection efficiency for pGMLV-PA1-PINK1 was > 80% in Human Embryonic Kidney 293 cells (HEK 293T cells). Viral particles pGMLV-PA1-PINK1 and the empty vector were produced by transiently co-transfecting the transfer plasmid pGMLV-PA1-PINK1 (12 μg) or pGMLV-PA1, the packaging plasmid delta 8.91 (9 μg) and Pvsvg (6 μg) into subconfluent HEK293T cells using the calcium phosphate method (Zennou et al. 2000). The medium was replaced after 16–18 h; the culture supernatant containing viral particles was collected 48 h post-transfection and stored at −80°C. Viral titer was determined in HEK 293T-transduced cells using serial dilutions of non-concentrated viral supernatant, and was found to be 3–4 × 109 TU/mL. Viral transduction was performed as follows: briefly, primary neurons were transduced with pGMLV-PA1-PINK1 or control pGMLV-PA1 vectors 48 h before OGD to generate non-concentrated viruses. The number of viral copies integrated in the genome of the transduced cells was estimated by western blotting.

For shRNA lentivirus production, the same system was used except that the expression vector was substituted with pGMLV-SC1, a CMV-GFP-U6-MCS-PGK plasmid. Based on our previous study (Cui et al. 2010), we selected the siRNA sequence GCGAAGCCAUCUUAAGCAAtt for knockdown of rat Pink1. This sequence was demonstrated to down-regulate endogenous Pink1 expression by > 80%. The corresponding DNA oligos were synthesized and subcloned into pGMLV-SC1; the packaging and transduction protocols were the same as for pGMLV-PA1-PINK1.

Quantitative PCR

The knockdown efficiency of Pink1-shRNA was confirmed by measuring Pink1 expression using quantitative real-time PCR, as previously described (Cui et al. 2010). Briefly, total RNA was purified using RNA easy columns (Qiagen, Valencia, CA, USA) and cDNA synthesis was performed using SuperScript III (Invitrogen, Carlsbad, CA, USA). The quantitative PCR reactions contained 1 μM sense and 1 μM antisense oligonucleotides with SYBR green I master mix (Bio-Rad, Hercules, CA, USA). Primers were designed to amplify a segment common to both rat Pink1 (accession number: NM_001106694) and human PINK1 (accession number: NM_032409) (Fwd 5′-CTGTCAGGAGATCCAGGCAATT-3′ and Rev 5′-GCATGGTGGCTTCATACACAGC-3′). Rat β-actin (accession number NM_031144) primers (Fwd 5′-ACCCTGTGCTGCTCACCGA-3′ and Rev 5′-CTGGATGGCTACGTACATGGCT-3′) were used in the same reactions to control for the amount of starting template. All samples were measured in duplicate and were compared with standard curves of known concentrations of different genes; all mRNA expression data are expressed relative to β-actin.

Cell viability assay

The viability of cells was examined by 3-(4, 5-dimethylthiazole-2-yl)- 2,5-dipenyltetrazolium bromide (MTT) assay. After followed by reoxygenation, MTT was added to a final concentration of 0.5 mg/mL for 4 h before the end of the experiment. The supernatant was removed and 150 μL dimethylsulfoxide was added for 20 min. The MTT optical density values were measured on a microplate reader at 570 nm and 630 nm wavelengths. Each experimental condition was repeated in triplicate, with each experiment containing eight readings.

Cell injury assays—lactate dehydrogenase (LDH) measurement

Cellular injury was determined by measuring concentration of LDH released into the medium (Koh and Choi 1987). Briefly, the media were removed and the cells were washed three times with ECF (pH 7.4), After OGD followed by reoxygenation, LDH in the medium and total cellular LDH were determined using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). Medium (50 μL) was transferred from the culture wells to 96-well plates, 50 μL reaction solution was added, incubated at 26°C in the dark for 30 min, then 50 μL of stop solution was added. Thirty minutes later, absorbance was read at 492 nm using a microplate reader. At the end of each experiment, the maximal LDH release was obtained in each well following repeated cycles of freezing and thawing. Each experimental condition was repeated in triplicate, with each experiment containing eight readings. Results were expressed as a percentage of maximal LDH release, after the subtraction of background levels was determined from the medium alone.

Measurement of ATP

ATP generation was detected after OGD followed by 8 h reoxygenation using HPLC. Briefly, culture medium was removed by aspiration followed by the immediate addition of liquid nitrogen. After evaporation on ice, 150 μL of ice-cold 0.4 M perchloric acid was added to 24-well plates. The cells were scraped off, centrifuged at 14 000 g for 15 min at 4°C and the pellets were used for protein determination. The supernatant was neutralized with 1 M K2CO3, kept at −80°C to promote precipitation of perchlorate, centrifuged and ATP content was analyzed using HPLC, as previously described (Cui et al. 2010). Briefly, samples were eluted on a reverse-phase column (Lichrospher-100; Merck, Philadephia, PA, USA) of 250 mm × 3 mm i.d. with a particle size of 5 μm, at a flow rate of 0.4 mL per min using a mobile phase containing 215 mM KH2PO4, 2.3 mM tetrabutylammonium hydrogen sulfate and 4% acetonitrile; the pH was adjusted to 6 with KOH. Signals were detected using a UV detector (D-UV 6000; Alltech, Deerfield, IL, USA) at 260 nm. Standard solutions were measured using the same process. All sample peak areas were within the linear range of the standard curve. The ATP concentrations of the samples were determined from the linear regression curve calculated from the standard solutions.

Measurement of MMP (Δψm)

MMP was measured using the cationic voltage-sensitive dye 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Invitrogen). The monomeric form of this dye has a green fluorescence, while at higher concentrations or potentials it forms red fluorescent J-aggregates. The ratio of red/green fluorescence is independent of mitochondrial shape, density or size, but depends on the membrane potential. Cortical neurons were incubated with JC-1 at 2 mg/mL for 20 min at 37°C in growth medium. Cells were then washed and scraped in cold phosphate-buffered saline. Ratio of red fluorescence (488 nm excitation, 590 nm emission) to green fluorescence (488 nm excitation and 525 nm emission) was quantified using a spectrofluorimeter (Perkin Elmer, Santa Clara, CA, USA).

Assessment of mitochondrial morphology

Primary cultured neurons were grown on poly-d-lysine-coated glass coverslips and transfected with Mito-Dsred at the time of neuronal culture preparation and before plating (Halterman et al. 2009). The cortical neurons were then fixed with 4% paraformaldehyde, and coverslips were mounted using Prolong Gold Antifade Reagent with DAPI (Invitrogen). Images were captured using an inverted epifluorescence microscope (Olympus, Tokyo, Japan). The number of neurons with fragmented mitochondria was determined from 10 randomly selected 200× magnification fields. Measurements of mitochondrial size and shape were quantified in a blinded manner using Image J (NIH image). More than 200 clearly identifiable mitochondria from 10–15 randomly selected cells per experiment were measured in five independent experiments.

Transient transfection of PINK1 constructs and siRNA mediated Pink1 knockdown

Cultured human SH-SY-5Y cells were transfected with PINK1-WT plasmid (1 μg per 1 × 105 cells) to overexpress human PINK1. For PINK1 knockdown, SH-SY-5Y cells were transfected with siRNA against human PINK1 (5′-GAAAUCCGACAACAUCCUUUU-3′; Dharmacon, Lafayette, CO, USA) or a scrambled control siRNA with no significant homology to any known gene sequences (ID#4611; Ambion, Carlsbad, CA, USA) using Lipofectamine™ 2000 (Invitrogen), according to the manufacturer's instructions. After 5-h incubation, the medium was replaced with regular culture medium, and the cells were cultured for an additional 48 h. The efficiency of PINK1 knockdown was confirmed by real-time RT-PCR analysis.

Mitochondrial isolation

Mitochondria were isolated using a mitochondrial isolation kit (Pierce, Rockford, IL, USA), according to the manufacturer's instructions. Briefly, cultured SH-SY-5Y cells were homogenized in a dounce homogenizer, centrifuged at 750 g for 10 min at 4°C, the supernatant was further centrifuged at 12 000 g for 15 min at 4°C, then the pellet was then washed and kept as the mitochondrial fraction.

Western blotting

Mitochondrial proteins (20 μg) were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The blots were cut into two portions; the lower part was probed for hFis1 (1 : 3000), the upper part was probed for larger proteins in the following order: Drp1 (1 : 2000; BD Biosciences, San Jose, CA, USA), Mfn2 (1 : 10 000; Sigma, St. Louis, MO, USA) and then Hsp60 (1 : 2000; Santa Cruz, Santa Cruz, CA, USA) as a loading control. After each development, the antibodies on the blots were gently removed using stripping buffer (#21059; Thermo Scientific; Waltham, MA, USA). Secondary horseradish peroxidase-conjugated antibodies were used, and immunoreactivity was visualized using chemiluminescence (SuperSignal Ultra; Pierce). The bands of interest were analyzed and quantified using Scion Image (NIH image, Bethesda, MD, USA).

Drp1 translocation

SH-SY-5Y cells were cultured on poly-d-lysine coated glass coverslips and then transfected with 1 μg DsRed-Mito and 1 μg Drp1-myc, together with either 1 μg of PINK1-WT (the vectors were kindly provided by Dr. Kim Tieu, University of Rochester, Rochester, NY, USA) or 50 nM PINK1-siRNA. After OGD followed by 24 h reoxygenation, the cells were fixed in 4% paraformaldehyde prior to being immunostained using an anti-myc antibody, followed by an AlexaFluor 488 secondary antibody (Invitrogen). Images were captured and analyzed using confocal microscopy. About 50 cells were analyzed per group in a blinded manner in three independent experiments.

Statistical analysis

All values are expressed as mean ± SE. Differences were analyzed using either one-way or two-way anova followed by Newman-Keuls post hoc testing for pairwise comparisons using SigmaStat Version 3.5 (Systat Software, San Jose, CA, USA). The null hypothesis was rejected when the p value < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Competing interest
  8. References

PINK1 protects cortical neurons from OGD damage

We used a lentivirus-based gene transfer system to genetically manipulate the expression of wild-type PINK1 and its two mutant forms W437X mutant linked to familial Parkinson's disease (PD) or an engineered kinase-dead mutant K219M in primary rat neuronal cultures (Fig. 1a and c). Transduction efficiency was estimated by quantitative PCR; the exogenous constructs were expressed at approximately five-fold over endogenous Pink1 (Fig. 1d). To further corroborate the effects of mutant PINK1, endogenous rat Pink1 was knocked down using lentiviral-mediated delivery of siRNA. As no available antibodies screened to date are able to detect endogenous Pink1 without having additional protein bands (Sandebring et al. 2009), quantitative PCR was used to confirm knockdown of Pink1 after siRNA transfection; Pink1 expression decreased by more than 90% (Fig. 1b).

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Figure 1. Efficiency of lentivirus transduction and effects on the expression of Pink1/PINK1 in cultured rat cortical neurons. Rat cortical neurons were transduced with lentivirus expressing human PINK1-WT, PINK1-W437X, PINK1-K219M or an empty vector control for 48 h. (a) Over 80% of the cortical neurons transduced with empty vector lentivirus expressed GFP. (b) Q-PCR analysis demonstrated Pink1 shRNA lentivirus knocked down endogenous rat Pink1 mRNA expression by more than 90% (= 6 independent experiments, error bars indicate SEM). (c) Western blot of PINK1 protein expression 48 h after PINK1-WT, PINK1-W437X or PINK1-K219M lentivirus transduction, showing the effectiveness of lentiviral over-expression. (d) Q-PCR analysis demonstrating PINK1-WT, PINK1-W437X, and PINK1-K219M lentivirus increased PINK1 expression more than five-fold (= 6 independent experiments, error bars indicate SEM). *< 0.05 versus empty vector group, one-way anova followed by Newman–Keuls post hoc testing for pairwise comparisons.

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To evaluate the role of PINK1 in ischemic-related injury, we utilized a neuronal model of OGD. Primary cultured rat cortical neurons were transduced with PINK1-WT, the PINK1 recessive mutants or the respective control GFP vector 48 h before OGD. Over-expression of wild-type PINK1 prior to OGD elicited increased cell viability and decreased LDH release (Fig. 2a and c). However, expression of two PINK1 mutants associated with PD in humans (W437X and K219M) failed to provide any protection against OGD, compared with the vector control (Fig. 2a and c). We also examined cell viability after OGD in Pink1-deficient cells. Pink1-shRNA cells were significantly more sensitive to OGD damage (Fig. 2b and d). This evidence indicated a neuroprotective role for PINK1 during OGD damage.

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Figure 2. PTEN-induced novel kinase 1 (PINK1) inhibits oxygen–glucose deprivation (OGD)-induced cell death in cultured primary rat cortical neurons. Cortical neurons were transduced with human PINK1-WT, PINK1-W437X, PINK1- K219M or a control GFP vector (empty vector group) 48 h prior to OGD. (a,c) Over-expression of wild-type PINK1 inhibited lactate dehydrogenase (LDH) release and improved cell viability induced by OGD; OGD-induced but lack of efficacy in the PINK1-W437X or PINK1-K219M transduced cells; (b) Endogenous Pink1 was knocked down 48 h prior to OGD. OGD-induced LDH release significantly increased in Pink1-shRNA cells, compared with scramble-shRNA control cells. (d) The cell viability was also decreased in Pink1-shRNA cells, compared with scramble-shRNA control (= 6 independent experiments, error bars indicate the SEM); *< 0.05 versus empty vector or scramble-shRNA/OGD+ group, two-way anova followed by Newman–Keuls post hoc testing for pairwise comparison.

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PINK1 prevents bioenergetic defects induced by OGD damage

Studies have indicated that PINK1 is important for maintaining mitochondrial functional and structural integrity in mammalian cells, and also that PINK1 protects mammalian cells against mitochondrial dysfunction because of decreased energy supply. To test whether PINK1 exerts a neuroprotective function by modulating mitochondrial function during ischemic damage, we cultured primary cortical neurons and measured the levels of ATP using HPLC. As expected, oligomycin, an inhibitor of the electron transport chain complex V significantly reduced ATP levels by about 68% (Fig. 3a). OGD lead to an approximately 36% reduction in ATP levels (Fig. 3a); this metabolic deficit was partially abolished by over-expression of wild-type PINK1; however, the W437X and K219M mutants failed to provide this protective effect (Fig. 3a). Consistent with these observations, we observed an approximately 26% reduction in ATP production in cells transfected with Pink1-shRNA against endogenous rat Pink1, compared with OGD cells (Fig. 3b), suggesting that basal levels of endogenous Pink1 are required for proper ATP production after OGD damage.

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Figure 3. PTEN-induced novel kinase 1 (PINK1) prevents bioenergetic defects induced by oxygen–glucose deprivation (OGD) damage. (a) The positive control oligomycin (oligo, 10 μM, a complex V inhibitor) significantly reduced the levels of ATP. The results were normalized to oligomycin treated cells. OGD also led to a reduced ATP production; this effect was partially abolished when the cells were transduced with PINK1-WT 48 h prior to OGD. However, over-expression of PINK1 W437X or PINK1 K219M did not affect ATP production. (b) Reduced levels of ATP were detected in cells transfected with shRNA-Pink1. Compared to the scramble-shRNA group, a dramatic reduction in ATP production was observed in Pink1-shRNA cells after OGD. All data represent the mean ± SEM of six independent experiments; *< 0.05 versus empty vector or scramble-shRNA/OGD+ group #< 0.05 versus scramble-shRNA/OGD- group, two-way anova followed by Newman–Keuls post-hoc tests.

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PINK1 protects against the collapse of MMP (Δψm) induced by OGD damage

Ischemia/reperfusion is believed to provoke the collapse of MMP (Δψm) (Anderson and Sims 1999), while PINK1 is important for maintaining MMP (Δψm) in mammalian cells under oxidative stress (Yang et al. 2006; Haque et al. 2008). To evaluate whether maintenance of Δψm is one of the mechanisms by which PINK1 exerts a protective effect after ischemic damage, we used a fluorescent cationic dye to measure Δψm in cortical neurons (Fig. 4a and b). As expected, the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone led to a significant collapse of Δψm (data not shown). Consistent with the hypothesis that PINK1 maintains Δψm, over-expression of PINK1-WT conferred protection against Δψm reduction induced by OGD damage (Fig. 4a). However, the W437X or K219M mutant failed to provide this protective effect (Fig. 4a). Consistent with this result, knockdown of endogenous Pink1 significantly reduced Δψm after OGD (Fig. 4b).

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Figure 4. PTEN-induced novel kinase 1 (PINK1) prevents against the collapse of mitochondrial membrane potential (MMP) induced by oxygen–glucose deprivation (OGD) damage. The mitochondrial membrane potential (Δψm) of cultured primary rat cortical neurons was measured using the fluorescent cationic dye JC-1. And the background fluorescence contributed by JC-1 staining (the ratio of FCCP treated cells) was substracted from the total JC-1 transmembrane potential. (a) After OGD/reoxygenation, over-expression of wild-type PINK1, but not PINK1-W437X or PINK1-219M restored Δψm; (b) Cells transduced with Pink1-shRNA displayed reduced Δψm after OGD. The data are presented as values of the 590/525 nm emission ratio that were normalized to the starting ratio. All data represent the mean ± SEM of six independent experiments; *< 0.05 versus empty vector or scramble-shRNA/OGD+ group #< 0.05 versus scramble-shRNA/OGD- group, two-way anova followed by Newman–Keuls post-hoc tests.

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PINK1 influences mitochondrial morphology in response to OGD damage

We examined mitochondrial morphology in primary cultured neurons using an approach to classify mitochondrial morphology as intact or fragmented. By using MitoDs-red to label mitochondria, it has been shown that OGD leading to dramatic mitochondrial fragmentation. Increased expression of wild-type PINK1 limited the number of rat neurons with fragmented mitochondria after exposure to OGD (Fig. 5e). Knocking down of Pink1 increased the number of cells containing fragmented mitochondria (Fig. 5b and e). To compare mitochondrial morphology more objectively, the shapes of individual mitochondria were measured (Fig. 5c, d, f and g). This quantitative data confirmed a higher proportion of smaller, rounder mitochondria in the Pink1 knockdown cells after OGD. Consistent with this observation, cells over-expressing wild-type PINK1 displayed increased numbers of tubular, long mitochondria (Fig. 5f and g). These results suggested that OGD may cause excessive mitochondrial fission, and that PINK1 induced morphological changes in the mitochondria through enhanced mitochondrial fusion.

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Figure 5. PTEN-induced novel kinase 1 (PINK1) influences mitochondrial morphology in response to oxygen–glucose deprivation (OGD) damage. (a) Primary cultured rat neurons were labeled with MitoDs-red to visualize mitochondrial morphology. Control cells showed tubular, long mitochondria, in contrast to the fragmented mitochondria observed after OGD/reoxygenation. Scale bars = 5 μm. (b. e) Percentage of cultured neurons with truncated or fragmented mitochondria in cells over-expressing different forms of PINK1 or lacking Pink1 (determined in a blinded manner from more than 200 cells in 10 randomly chosen 200× microscopic fields in three independent experiments). (c–g) Quantification of mitochondrial shape (from 10–15 randomly selected cells in three independent experiments with > 200 mitochondria per experiment). Roundness was calculated as perimeter2/4π area. Aspect ratio is a measurement of major/minor axes. Both of these values approach 1 as the particle becomes more circular. All data represent the mean ± SEM; *< 0.05 versus empty vector or scramble-shRNA/OGD+ group #< 0.05 versus scramble-shRNA/OGD- group, two-way anova followed by Newman-Keuls post-hoc tests.

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PINK1 prevents Drp1 mitochondrial translocation induced by OGD

The balance between mitochondrial fission and fusion proteins is critical for the maintenance of proper mitochondrial morphology. Therefore, we used human SH-SY-5Y cells to further investigate the molecular mechanism behind mitochondrial fragmentation after ischemic damage. Mitochondria were isolated from different groups of cells for the assessment of fission and fusion protein levels (Fig. 6a–c). As illustrated in the immunoblots, no changes were detectable in the mitochondrial fission protein hFis and fusion protein Mfn2. However, the levels of the fission protein Drp1 level increased about 1.88-fold after OGD, compared with the control group (Fig. 6b).

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Figure 6. Alterations of the mitochondrial fission and fusion proteins induced by oxygen–glucose deprivation (OGD) damage. (a–b) Mitochondria were isolated from human SH-SY-5Y cells transduced with either Pink1-shRNA or wild-type PINK1. Alterations in the levels of the mitochondrial fission proteins dynamin-related protein 1 (Drp1) and hFis1 and the mitochondrial fusion protein Mfn2 were assessed by western blotting, (c) The protein expression levels were quantified from individual band intensities and expressed as a percentage of the respective control groups. Data represent the mean ± SEM of six independent experiments, *< 0.05 versus empty vector group, #< 0.05 versus scramble-shRNA group, two-way anova followed by Newman–Keuls post-hoc tests.

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Over-expression of wild-type PINK1 reduced the level of Drp1 by approximately 21% after OGD, and knocking down of endogenous PINK1 increased the level of Drp1 by approximately 25% after OGD, compared to the empty vector group (Fig. 6b). Given that only about 3% of Drp1 is associated with mitochondria and the rest is cytosolic (Smirnova et al. 2001), these observations indicated that OGD induced translocation of Drp1 from the cytosol to the mitochondria. To test this hypothesis, we co-transfected cells with Drp1-myc and DsRed-Mito. As shown in Fig. 7a and b, Drp1 aggregated and colocalized with the mitochondria after OGD. When assessed in a blinded manner using confocal microscopy, over-expression of wild-type PINK1 significantly reduced Drp1 mitochondrial translation and knocking down of endogenous PINK1 had the opposite effect (Fig. 7a and b); these changes were significant compared to the empty vector group.

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Figure 7. PTEN-induced novel kinase 1 (PINK1) prevents dynamin-related protein 1 (Drp1) mitochondrial translocation induced by oxygen–glucose deprivation (OGD) damage. (a–b) Human SH-SY-5Y cells were co-transfected with DsRed-Mito and Drp1-myc for 24 h, subjected to OGD, then the cells were then fixed and immunostained for myc. In control cells, Drp1 appeared diffused. After OGD/reoxygenation, the distribution of Drp1 was punctuate and co-localized with mitochondria. This altered distribution of Drp1 could be prevented by over-expression of wild-type PINK1. However, Pink1-siRNA increased both the number and size of Drp1 aggregates that colocalized with mitochondria after OGD. Data represent the mean ± SEM, *< 0.05 versus empty vector/OGD+ group, two-way anova followed by Newman–Keuls post-hoc test (= 3 experiments). Scale bar = 50 μm.

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The deleterious effects of Pink1 down-regulation on OGD-induced neuronal death are reversed by Drp1 inhibitor

Although several critical proteins including hFis, Mfn2, and Drp1 have been proven to be involved in the regulation of mitochondrial morphology, our results showed that PINK1 only affected the translocation of Drp1 after OGD. This suggested that the deleterious effects of PINK1 down-regulation on OGD-induced cell death may be an outcome of Drp1 translocation. To test this hypothesis, we employed a specific Drp1 inhibitor, Mdivi1, together with Pink1 shRNA, during the process of OGD in primary rat neurons (Fig. 8c and d). As expected, down-regulation of Pink1 significantly increased cell death and decreased the levels of ATP after OGD. Importantly, inhibition of Drp1 obviously reversed the deleterious effects of OGD together with knocking down of Pink1 on both cell death and energy production (Fig. 8c and d). Interestingly, we noticed a dose-dependent effect of Mdivi1 on cell death in the LDH release experiment, although this trend was not statistically significant (Fig. 8c).

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Figure 8. The deleterious effects of Pink1 down-regulation on oxygen–glucose deprivation (OGD)-induced neuronal death are reversed by dynamin-related protein 1 (Drp1) inhibitor. Primary rat cortical neurons were transduced with lentivirus expressing Pink1 shRNA 48 h prior to OGD; the Drp1 inhibitor Mdivi was added right before OGD. (a and b) Drp1 inhibitor Mdivi-1 blocked the cell death and bioenergetic defects induced by OGD in a dose-dependent manner. (c) Cellular lactate dehydrogenase (LDH) release. As expected, knockdown of Pink1 significantly increased cell death induced by OGD, while inhibition of Drp1 obviously reversed this effect. A dose-dependent relationship was observed for Mdivi-1; however, this trend was not statistically significant. (d) Knockdown of Pink1 significantly decreased cellular ATP production after OGD, while inhibition of Drp1 reversed this effect. Data represent the mean ± SEM, *< 0.05 versus OGD group. Two-way anova followed by Newman–Keuls post-hoc tests (= 6).

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Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Competing interest
  8. References

Mitochondria are dynamic organelles that continually undergo the opposing processes of fusion and fission to maintain their distinct morphology. The balance between fission and fusion events regulates mitochondrial morphology. Mitochondrial shape corresponds to metabolic status (Rossignol et al. 2004) and the health of the cell (Youle and Karbowski 2005). Recently, several neurodegenerative diseases, including PD, have been linked to perturbations of mitochondrial dynamics (Han et al. 2011). Disruption of the PD-associated genes PINK1, Parkin, DJ-1 or LRRK2 results in mitochondrial defects and aberrant mitochondrial morphology (Greene et al. 2003; Clark et al. 2006; Park et al. 2006; Irrcher et al. 2010; Krebiehl et al. 2010; Wang et al. 2012). Although the importance of mitochondrial dynamics in neurodegenerative diseases has been widely addressed, the importance of mitochondrial dynamics in acute brain injuries, such as stroke, remains poorly understood. A recent study showed that inhibition of the mitochondrial fission protein Drp1 using small molecules (Mdivi compounds) prevented cell death after glutamate toxicity and OGD (Grohm et al. 2012), indicating that perturbations of mitochondrial dynamics may participate in OGD-mediated cell damage.

In this study, we provide evidence that the OGD/reoxygenation procedure induced excessive fission in primary cultured neurons. After OGD, the cells displayed severe mitochondrial fragmentation, with a dramatic collapse in MMP and reduced neuronal ATP levels. Moreover, we demonstrated that OGD dramatically increased the mitochondrial Drp1 level by recruiting Drp1 from the cytosol to the mitochondria; OGD had no significant influence on the expression of other fission/fusion proteins. Overall, these changes tipped the balance of the mitochondrial fission/fusion machinery towards excessive fission.

As a major energetic crisis in the central nervous system, stroke undoubtedly has tremendous impact on mitochondrial function. Although the precise role of mitochondrial dynamics in the pathology of stroke is not clear, there are clues from the DJ-1 study that mitochondrial fission may deteriorate stroke damage. Loss of DJ-1 increased the sensitivity to excitotoxicity and ischemia (Aleyasin et al. 2007). Recently, DJ-1 is implicated to be involved in the regulation of mitochondrial dynamics (Irrcher et al. 2010; Krebiehl et al. 2010; Thomas et al. 2011). Over-expression of various pathogenic DJ-1 mutants induces mitochondrial fragmentation in neurons. It has been demonstrated that PINK1 acts upstream of or in parallel to DJ-1 to maintain proper mitochondrial morphology and function (Thomas et al. 2011). Although controversy remains regarding the exact function of PINK1, several recent studies suggest that PINK1 may alter the balance between fusion and fission, according to the intimate interaction between Pink1 and DJ-1 (Cookson 2010; Thomas et al. 2011). Therefore, it is of great interest to explore the role of PINK1 in cerebral ischemia.

Using lentiviral mediated siRNA to knockdown endogenous Pink1, we demonstrated that loss of function in Pink1 increased the sensitivity of primary neurons to ischemic damage. In contrast, over-expression of wild-type PINK1 remarkably rescued neurons from OGD injury by improving mitochondrial morphology and bioenergetic production. These findings support the suggestion that PINK1 plays a neuroprotective role in ischemic stroke.

The exact mechanisms by which PINK1 protects neurons from ischemic damage remained unclear. To this end, we over-expressed human wild-type PINK1 and two recessive mutants (PINK1-W437X and PINK1-K219M) in primary cultured cortical neurons. Mutations that impair kinase activity (the PD-associated mutation W437X and the artificial kinase-dead construct K219M) failed to protect the neurons from ischemic damage, indicating that kinase activity might be required for the neuroprotective function of Pink1.

Mitochondrial morphology is determined by the balance between mitochondrial fission and fusion and it was linked to the maintenance of proper mitochondrial functions. There has been controversy over whether the PINK1 modulates mitochondrial fusion or fission. In Drosophila, PINK1 loss of function results in swelling or enlargement of mitochondria, and these defective phenotypes of the dPINK1-null flies are strongly suppressed by the over-expression of Drp1 (Deng et al. 2008; Poole et al. 2008; Yang et al. 2008; Park et al. 2009). However, PINK1 deficient mammalian cells have a fragmented and truncated mitochondrial morphology, which would suggest an imbalance towards fission (Exner et al. 2007; Cui et al. 2010). In our study, we found that the influence of PINK1 on mitochondrial morphology was consistent with changes in mitochondrial fission/fusion proteins after OGD. OGD led to the recruitment of a large amount of Drp1 from the cytosol to the mitochondria, and induced mitochondrial fragmentation and dysfunction. Over-expression of PINK1 inhibited the translocation of Drp1 and shifted mitochondrial fission back towards the normal balance between fusion and fission, thereby preventing neuronal dysfunction and death in response to OGD.

To further corroborate the mechanisms by which PINK1 exerts a neuroprotective effect under ischemic conditions, endogenous rat Pink1 was knocked down using siRNA. Not surprisingly, the opposite effects were detected. Thus, these results suggested that PINK1 may prevent neurons from ischemic damage through maintaining proper mitochondrial functions by attenuating Drp1 translocation and reversing the mitochondrial fission induced by ischemia. To confirm this hypothesis, we manipulated the fission/fusion process by inhibiting Drp1 activity using the small molecule Mdivi-1. Inhibition of the fission process attenuated the effects of Pink1 knockdown on neuronal cell death and ATP generation after OGD. These observations demonstrated the protective effects of PINK1 against OGD injury are mediated, at least in part, through the mitochondrial fission/fusion pathway. Because of the fact that PINK1 is normally considered an autosomal recessive gene in PD, this is a novel and significant finding as the involvement of PINK1 in the pathology of stroke has rarely been explored.

Taken together, our findings provide evidence to support a neuroprotective role of PINK1 in mammalian neuronal models of ischemic injury. Given the pathological changes which occur in the mitochondria after stroke, PINK1 may be a promising new target for the prevention of stroke damage.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Competing interest
  8. References

This study was supported by National Natural Science Foundation of China 81000487(to M.C) 81171023(to Y.Z) and Shanghai Rising-Star Program (11QA1400900) and Shanghai Pujiang Program (12PJ1407200)

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Competing interest
  8. References