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Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro
Neuroscience Letters 579 (2014) 24–29
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro Tao Liu, Chen-chen Xue, Yu-long Shi, Xiang-jun Bai, Zhan-fei Li, Cheng-la Yi ∗ Department of Traumatic Surgery, Tong-ji Hospital, Tong-ji Medical College, Huazhong University of Science and Technology, Wuhan, China
h i g h l i g h t s • Scratch injury and starvation-serum stimulation induce reactive astrogliosis. • Mfn2 expression is down-regulated during the activation of astrocytes. • Overexpression of Mfn2 inhibits astrocytes proliferation by cell-cycle arrest.
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Article history: Received 6 May 2014 Received in revised form 14 June 2014 Accepted 1 July 2014 Available online 11 July 2014 Keywords: Mitofusin 2 Reactive astrogliosis Cell cycle arrest
a b s t r a c t Astrocytes become activated in response to central nervous system (CNS) injury, and excessive astrogliosis is considered an impediment to axonal regeneration by forming glial scar. Mitofusin 2 (Mfn2), a key protein in mitochondrial network, has been reported to negatively regulate cell proliferation. The present study aimed to explore whether reactive astrogliosis could be suppressed by Mfn2 overexpression. Scratch injury and starvation-serum stimulation models in cultured astrocytes were combined to address this issue. In scratch model, reactive proliferation status of damaged astrocytes was implicated by migration of high ratio of EdU(+) cells into lesion region and significantly increased expression of GFAP and PCNA. At meantime, Mfn2 expression was found to exert a down-regulated trend both in gen and protein levels. Pretreatment of cells with adenoviral vector encoding Mfn2 gene increased Mfn2 expression and subsequently attenuated injury-induced astrocytes hyperplasia, activation-relevant protein synthesis, cellular proliferation, eventually delayed wound healing process. Furthermore, Mfn2 overexpression markedly inhibited astrocytes proliferation induced by serum stimulation, by arresting the transition of cell cycle from G1 to S phase. Together, these in vitro results demonstrated that reactive astrogliosis can be effectively suppressed by up-regulation of Mfn2, which might contribute to a promising therapeutic intervention in CNS disease characterized by glia-related damage. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Reactive astrogliosis is an important cellular response to central nervous system (CNS) injury, which is characterized by hypertrophy of cellular processes, cell proliferation and increased expression of intermediate filament proteins, such as glia fibrillary acidic protein (GFAP) [1]. Reactive astrogliosis may play both a ben-
Abbreviations: CNS, central nervous system; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; Ad, adenovirus; GFP, green fluorescent protein; EdU, 5-Ethynyl-2 -deoxyuridine; GFAP, glial fibrillary acidic protein; PCNA, proliferating cell nuclear antigen; MAPK/ERK, Mitogen-activated protein kinases/extracellular-signal-regulated kinase. ∗ Corresponding author at: Jie Fang Road 1095, Wuhan, China. Tel.: +86 027 83665346; fax: +86 027 83665346. E-mail address: [email protected] (C.-l. Yi). http://dx.doi.org/10.1016/j.neulet.2014.07.002 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
eficial and a detrimental role in CNS repair depending on the timing of astrocyte activation and the local environment [2]. Initially, it may protect the intact tissue from exposure to toxic elements in the infarct area and secrete neurotrophic factors to provide a permissive substrate for neuron regeneration [3]. However, at later stages post injury, reactive astrocytes result in glial scar formation that impedes axonal regrowth by constituting a physical as well as a biochemical barrier [4]. Thus, early regulation of excessive reactive astrocyte proliferation may inhibit glial scar formation to create a favorable environment for neuronal regeneration and thereby enhance CNS injury recovery. Mitofusin 2 (Mfn2) is a protein that localizes to the mitochondrial outer membrane. Apart from its major function in mitochondrial network [5], Mfn2 also has a potential role in regulating cell proliferation [6]. Mfn2 has been reported to take part in various hyper-proliferative diseases such as atherosclerosis,
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cardiac hypertrophy, diabetic nephropathy and neoplasms [7–10]. In these studies, Mfn2 exerts its anti-proliferative effect by acting as an effector molecule of Ras and Raf-1, resulting in the inhibition of Mitogen-activated protein kinases/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathway. On one hand, several lines of evidence have revealed the close correlation between the MAPK/ERK pathway and astrocyte proliferation [11]. On the other hand, Mfn2 is abundantly expressed in brain [12]. Therefore, the present study aims to determine whether Mfn2 is associated with reactive astrogliosis induced by serum stimulation and mechanical injury. If Mfn2 is indeed involved, whether adenovirus-mediated Mfn2 overexpression is capable of inhibiting reactive astrocyte proliferation and the potential mechanisms will also be explored.
2. Materials and methods 2.1. Astrocyte culture and identification Cultures of astrocytes from neonatal SD rat cortex were prepared according to a standard procedure [13]. The cultures were maintained at 37 ◦ C and 95% O2 /5%CO2 in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 0.5 mg/ml penicillin/streptomycin. About 48–60 h in vitro maintenance, purification of astrocyte was undertaken by shaking. At the end of the two weeks culture, primary astrocytes were trypsinized and replated onto 35-mm dishes at 3 × 104 cell/cm2 density or into 96-well cell plates at the concentration of 1 × 104 cell/well for following experiments. The purity of astrocyte was identified by double-labeling with GFAP antibody (1:200, Sigma) and Hoechst 33342. The immunocytochemistry revealed that more than 95% of the cultured cells were GFAP-positive astrocytes.
2.2. Adenovirus infection and expression identification Replication-defective adenovirus encoding the complete rat Mfn2 open reading frame (Ad-Mfn2) and the control adenovirus encoding the green fluorescent protein open reading frame (Ad-GFP) were constructed by BGI Tech (Shenzhen, China). Astrocytes were incubated with adenovirus at the indicated multiplicity of infection (MOI) of 30 pfu/cell for 24 h, then the virus-containing medium was changed to fresh complete growth medium to continue incubation. Ad-Mfn2 expression was detected by western-blotting, which showed that Mfn2 was significantly overexpressed after 48 h infection.
2.3. Starvation and serum stimulation assay Cells were starved with serum-free DMEM for 24 h to achieve cell synchronization and treated by 10% FBS mitotic stimulation for 24 h or 48 h [14]. Control cell samples included cells nontreated and only treated with starvation.
2.4. Scratch injury model Monolayer confluent astrocytes were scratched with sterile plastic pipette tips (100 L) longitudinally and latitudinally every 0.5 cm at right angles to each other in 35 mm dishes. This process has been described to establish a reproducible model of 30–40% damage [15]. Immediately, the detached cells and debris were washed out with fresh medium. Scratched cultures were maintained for 12 h, 24 h and 48 h for following experiments.
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2.5. Reverse transcription-polymerase chain reaction (RT-PCR) analysis The trend of Mfn2 mRNA expression during serum stimulation and scratch model was detected by RT-PCR. Total RNA were extracted from cultured astrocytes by using Trizol reagent (Invitrogen). RNA concentration and quantity were determined by ultraviolet spectrophotometry (absorbance at 260 nm/280 nm). RNA were reverse-transcribed with the Revertaid first strand cDNA synthesis Kit (Thermo Scientific), followed by cDNA amplification in a DNA thermal cycler. The primers were designed as follows: Mfn2 sense primer: 5 -CTCAGGAGCAGCGGGTTTATTGTCT-3 antisense primer: 5 -TGTCGAGGGACCAGCATGTCTATCT-3 , fragment size: 412 bp. -actin sense primer: 5 -GGAGATTACTGCCCTGGCTCCTA-3 antisense primer: 5 -GACTCATCGTACTCCTGCTTGCTG-3 , fragment size: 151 bp. The PCR products were separated by 1.0% agarose gel electrophoresis and visualized by ethidium bromide staining. 2.6. Western blotting and quantification Total proteins of each group were harvested and then centrifuged at 12,000 × g for 10 min at 4 ◦ C and protein concentrations in the supernatant were detected using the BCA protein assay kit (Pierce). Protein samples were separated by 10% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were blocked for 1 h in Tris-buffered saline (TBS) containing 5% nonfat milk at room temperature and respectively incubated with primary antibodies overnight at 4 ◦ C, which included Mfn2 (1:500, CST), GFAP (1:2000, Sigma), PCNA (1:1000, Abcam), PhosphoERK1/2 (1:500, CST) and -actin (1:1500, Santa Cruz). On the following day, the membranes were incubated with Odyssey secondary antibodies (780-conjugated goat anti-rabbit and antimouse IgG, 1:15,000) for 1 h and then visualized and quantitated by Odyssey IR imaging system (Li-COR Bioscience, USA), then expressed as the ratio of optical density (OD) from the tested proteins to that from actin. 2.7. Assessment of cell proliferation To assess the proliferation of astrocytes, EdU staining was used [16]. Briefly, cells were incubated with culture medium in presence of 10 M EdU (RiboBio) for 24 h. Cells were then fixed with 4% paraformaldehyde for 30 min. After naturalization with 2 mg/ml glycine and permeabilized with 0.2% triton X-100, cells were incubated with Apollo® staining solution for 30 min to chase the template of DNA. To label nuclei, cells were washed in 0.2% Triton X-100 for three times prior to incubation with Hoechst 33342 for 10 min. Finally cells were visualized and counted with a fluorescent microscope. 2.8. Wound healing assay At 0 h, 12 h, 24 h and 48 h after scratch, astrocytes were fixed for immunofluorescence staining. The membranes of fixed cells were permeabilized with Triton X-100 (15 min, 0.2% in PBS), followed by incubation with 5% bovine serum albumin in PBS at room temperature for 1 h to block nonspecific antibody binding and incubated overnight at 4 ◦ C with monoclonal mouse anti-GFAP (1:200, Sigma, USA). Photographs of cells were captured with a fluorescent microscope. Scratch areas before and after recovery were measured using Axiovision 4.1 software [17].
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2.9. Cell cycle analysis Astrocytes treated by 24 h starvation and 48 h serum stimulation in control, Adv-GFP and Adv-Mfn2 groups were collected, washed with PBS and fixed in 70% ice-cold ethanol. Then, cells were treated with 0.5 mg/ml RNase (Invitrogen) for 30 min and finally stained with propidium iodide (PI, Invitrogen) for additional 30 min at room temperature. Flow cytometry analysis (Becton-Dickinson) was performed to separate and quantify the cell-cycle distribution.
2.10. Statistical analysis All measurements are done in triplicate and denoted as mean ± SEM. Statistically significant differences (p < 0.05) between two groups and among more than two groups were evaluated by Student’s t-test and one-way ANOVA with Tukey’s post hoc test, respectively.
3. Results 3.1. Time-dependent changes in the mRNA and protein expression of Mfn2 during the activation of astrocytes induced by serum stimulation and scratch injury PCNA, an important factor required for DNA synthesis, is an index for cell proliferation. Western-blot showed that PCNA expression increased with time after scratch injury and reached a plateau at 24 h. GFAP, a basic and specific substance for astrocytes to participate in glial scar formation, increased persistently after scratching. Up-regulation of PCNA and GFAP level proved that scratch model in vitro successfully activated astrocytes and induced their proliferation in a time-dependent manner. During this period, we found that Mfn2 expression at both mRNA and protein levels decreased with time, negatively correlated with reactive astrocyte proliferation. Similar results were obtained in serum stimulation model, in which both PCNA and GFAP proteins were up-regulated in parallel after incubation with 10%FBS for 24 h and 48 h, while Mfn2 mRNA and protein expressions were reduced.
Moreover, the phosphorylated active forms of ERK1/2 (phospho-ERK1/2), a crucial signal protein for cellular proliferation did not show significant changes either in scratch injury or serum stimulation model. All above results can be found in Fig. 1. 3.2. Overexpression of Mfn2 regulated expression of relevant proteins associated with astrocyte activation The observation time chosen for western-blot detection was 24 h after scratch injury and 48 h after 10% FBS stimulation respectively due to the highest proliferation rate of reactive astrocyte in combination with lowest Mfn2 expression at these two timepoints. Fig. 2 demonstrated that infection of astrocytes with Mfn2 reduced the expression of activation related proteins PCNA and GFAP both in scratch injury (A) and serum stimulation models (B). However, p-ERK expression was not altered again.
3.3. Overexpressing Mfn2 inhibited the proliferation and cell cycle progression of astrocytes initiated by serum stimulation The percentage of EdU-positive cells accounting for the total cell number (counterstained with Hoechst 33342) indicated the cell proliferation situation. As Fig. 3A shows, after being deprived of serum for 24 h, cultured astrocytes were kept in a resting stage characterized by majority of cells were EdU-negative. The proportions of Edu(+) astrocyte in the control and Ad-GFP group increased gradually after being cultured with complete medium for 24 h and 48 h, which were remarkably higher than those in Adv-Mfn2 group. Flow cytometry analysis in Fig. 3B revealed that serumdeprived cells were mainly distributed in the G0/G1 phase (92.15 ± 1.37%). After 48 h serum stimulation, approximately 19% of astrocytes infected by uninfected astrocytes (19.4 ± 2.33%) or Adv-GFP (19.05 ± 3.16%) progressed into S phase, however astrocytes infected with Adv-Mfn2 remained mostly in the G0/G1 phases with only 6.92 ± 2.01% (p < 0.05) of cells entering S phase. The G0/G1 fraction of astrocytes in Adv-Mfn2 group was 86.83 ± 2.47% (p < 0.05), compared with 71.88 ± 2.42% for uninfected group and
Fig. 1. Detection of Mfn2 expression during the activation of astrocytes in scratch-injury and starve-serum stimulation models. (A) Expression of GFAP, PCNA, p-ERK1/2 and Mfn2 proteins were evaluated by Western blot with -actin as the control. (B) RT-PCR showed the decreased expression of Mfn2 mRNA in a time-dependent manner. Statistical graph describes the relative expression level compared to 0 h. (*p < 0.05, **p < 0.01).
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Fig. 2. Mfn2 overexpression suppressed expression of relevant proteins associated with reactive astrogliosis. Western blot analysis revealed that Mfn2 expression was increased by Ad-Mfn2 infection and up-regulation of Mfn2 reduced the levels of GFAP and PCNA in astrocytes after 24 h scratching injury (A) and 48 h serum stimulation (B), while p-ERK1/2 abundance was not altered. Relative level of target proteins in different groups were statistically analyzed (*p < 0.05, **p < 0.01, vs. the uninfected group).
Fig. 3. Mfn2-induced astrocytes proliferation inhibition and cell-cycle arrest. (A) EdU (red) and Hoechst 33342 (blue) were double stained to assess astrocytes proliferation in serum-stimulation model. Percentages of EdU-positive cells in each group for corresponding time points were statistically analyzed. (*p < 0.05, **p < 0.01, vs. the uninfected group). Scale bars = 200 m. (B) (I) Typical examples of cell cycle distribution in synchronized, or serum-stimulated astrocytes uninfected or infected with either Adv-GFP or Adv-Mfn2. (II) The average data of cell cycle distributions (*p < 0.05, vs. the uninfected group). (III) Mfn2-induced decrease in CyclinD1 expression. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Mfn2 transfection slowed down scratch wound healing by inhibiting reactive astrocytes proliferation. (A) GFAP immunostaining (green) images were taken from astrocyte cultures in uninfected, Adv-GFP and Adv-Mfn2 groups scratched for 0, 12, 24 and 48 h, respectively and astrocyte-absent areas were measured. (B) Proportions of proliferation cells in the scratch wounded regions were evaluated by Edu staining and statistically analyzed. (*p < 0.05, **p < 0.01, vs. the uninfected group). Scale bars = 200 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
72.61 ± 2.73% for Adv-GFP group. No significant difference in the G2-M fraction was observed between the Ad-Mfn2 (7.25 ± 0.86%), Ad-GFP (8.72 ± 1.38%), and uninfected (8.34 ± 1.12%) groups. CyclinD1, an important positive regulator for the transition of the cell cycle from the G1 to S phase, was found to decrease accordingly after Adv-Mfn2 infection.
3.4. Up-regulation of Mfn2 delayed wound healing by reducing reactive astrogliosis To acquire evidence for the hypothesis that cell cycle inhibition induced by up-regulation of Mfn2 could directly modulate astrogliosis, wound healing assay was used. Changes in cell morphology and proliferation in the wound area were evaluated by GFAP and EdU staining, respectively. As shown in Fig. 4, at 12 h after scratching, the number of proliferating astrocytes in the wound area increased and the cell bodies and cytoplasmic processes became hypertrophic and extended to the denuded area. Cells adjacent to the wound continued proliferating and migrating into the denuded area until cell density nearly approached confluence in the wound at 48 h after injury. In Ad-Mfn2 group, significant slower wound healing process was observed, as characterized by reduced astrocyte proliferation, increased inter-cellular space and shrunken cell volume. Cells in the area distant from the scratch did not show enhanced proportion of EdU(+) cells during the wound healing.
4. Discussion Mfn2 is highly expressed in cells with high-energy requirement, such as smooth muscle cells, cardiomyocytes, cancer cells and nerve cells. Proliferation inhibitory capacity of Mfn2 on these cells has recently been reported in succession, except for nerve cells. The role of Mfn2 in CNS is little discovered and limited to its conventional function in mitochondrial fusion [18]. It is known that various forms of injuries such as trauma, hypoxia, ischemia and infection in CNS can activate astrocytes and cause reactive gliosis, which result in scar formation and eventually inhibit CNS reconstruction. Therefore, astrocyte activation after CNS injury is also some kind of hyper-proliferative disease. In present study, we intend to explore the pathological change of Mfn2 expression during astrocyte activation and whether overexpressing Mfn2 can inhibit reactive astrogliosis. To investigate the process of astrocyte activation, a set of vitro models have been proposed [19]. A simple but well-described “scratching injury model” was established in this study, in which confluent monolayers of cultured astrocytes were scratched by pipette tips to simulate mechanical injury of CNS in vivo. Astroglia cells in the scratch area have been reported to exhibit some typical signs of activation, including process extension, nuclei hypertrophy, increased proliferation and up-regulation of GFAP [20,21]. Based on proliferating cell markers such as PCNA and EdU in combination with astrocytes hyperplasia indicators such as GFAP, how astrocytes responded to scratching was monitored in the present
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study. Initially, most of astrocytes both adjacent to and far away the wounds were in stationary growth phase. Cells far from the wounds remained a resting state during the wound healing period. However, as time went on, astrocytes next to the wound site started extending flat, cytoplasmic processes to the denuded area and proportion of proliferating cells in cells filling the wound was increased. Astrocytic migration and enhanced proliferation lasted until cell density within the wound approached confluence again. Relevant changes in the key protein expression were observed for PCNA and GFAP postlesion. These results revealed that astrocytes could become reactive and proliferate in response to mechanical injury in vitro. Meanwhile, Mfn2 expression both in gen and protein level showed an opposite trend. This finding implied that down-regulation of Mfn2 might contribute to the initiation and development of astrocytes activation. This implication was supported by the observation that Mfn2 overexpression alleviated astrocyte proliferation and decelerated wound healing process. To further confirm the involvement of Mfn2 in the regulation of astrocyte proliferation, starvation and serum stimulation model was conducted. Serum contains an astroglial mitogen, thus it is also an optimal system to test relevant proliferating proteins [22]. As respected, increased GFAP and PCNA expression was consistent with significant cellular proliferation, while Mfn2 level declined. Together, these results suggested that over-expressing Mfn2 suppressed reactive astrogliosis in vitro through cessation of proliferation. Following the finding that Mfn2 negatively regulated reactive astrocytes proliferation, we further explored the underlying mechanism. Since cell cycle retardation is an important way of inhibiting reactive astrogliosis, the cell cycle distribution after Adv-Mfn2 infection was chosen to investigate. Here, we found the suppressive effect of Mfn2 on astrocyte proliferation was mediated by G0/G1 cell-cycle arrest, which was also manifested by Mfn2-mediated alterations in key components of cell-cycle regulatory machinery. It was consistent with the anti-proliferation mechanism of Mfn2 reported in some other cell types [6,23]. To further dissect the possible pathway linking Mfn2 with cell cycle arrest, we sought to determine ERK1/2, well recognized as the downstream substrate of Mfn2 in controlling cell proliferation. Unexpectedly, marked activation of ERK1/2 was not observed. In fact, there were strong evidences to implicate the ERK/MAPK pathway as an obligatory step in either the acute trigger or chronic maintenance of reactive astroglia [24]. It was proved by previous studies based on scratching model that focal mechanical injury could induce a rapid activation and spreading of ERK1/2. However, it was a transient manner that p-ERK1/2 began to increase as early as 10 min postlesion, which peaked at 30 min and lasted for as long as 8 h, then returned to baseline at 24 h after scratching [25,26]. In addition, it was found that in astrocytes, ERK activation is more prominent and long-lasting after ischemia injury [27] but not after mechanical injury. Taken together, cell-type and modeltype dependence of ERK activation might contribute to the failure to observe the change of p-ERK1/2 expression and to correlate Mfn2 with ERK pathway in present study. It is likely that multiple parallel and interconnected pathways regulate the complex activating reaction of astroglia. 5. Conclusions Overall, the findings presented here reveals that the activation and proliferation of astrocytes occurred in response to stimuli such as scratch injury and serum in vitro, which is paralleled by pathological down-regulation of Mfn2 expression. Mfn2 over-expression can alleviate the responsive activation of astrogliosis and delay wound healing process by inhibiting cell cycle progression.
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Anti-proliferative effect of Mfn2 may serve as a promising therapeutic intervention in CNS diseases with excessive astrogliosis. Acknowledgements This study was funded by the National Nature Science Foundation of China (Grant NO. 81271348) and the National key Technology R&D Program (Grant NO. 2012BAI11B00). References [1] M.V. Sofroniew, Molecular dissection of reactive astrogliosis and glial scar formation, Trends Neurosci. 32 (2009) 638–647. [2] S. Karimi-Abdolrezaee, R. Billakanti, Reactive astrogliosis after spinal cord injury-beneficial and detrimental effects, Mol. Neurobiol. 46 (2012) 251–264. [3] R.E. White, L.B. Jakeman, Don’t fence me in: harnessing the beneficial roles of astrocytes for spinal cord repair, Restor. Neurol. Neurosci. 26 (2008) 197–214. [4] Z. Chen, M. Negra, A. Levine, Y. Ughrin, J.M. Levine, Oligodendrocyte precursor cells: reactive cells that inhibit axon growth and regeneration, J. Neurocytol. 31 (2002) 481–495. [5] O.M. de Brito, L. Scorrano, Mitofusin 2 tethers endoplasmic reticulum to mitochondria, Nature 456 (2008) 605–610. [6] K. Chen, X. Guo, D. Ma, Y. Guo, Q. Li, D. Yang, Dysregulation of HSG triggers vascular proliferative disorders, Nat. Cell Biol. 6 (2004) 872–883. [7] Y. Guo, K. Chen, W. Gao, Q. Li, L. Chen, G.S. Wang, Overexpression of mitofusin 2 inhibited oxidized low-density lipoprotein induced vascular smooth muscle cell proliferation and reduced atherosclerotic lesion formation in rabbit, Biochem. Biophys. Res. Commun. 363 (2007) 411–417. [8] H. Yu, Y. Guo, L. Mi, X. Wang, L. Li, W. Gao, Mitofusin 2 inhibits angiotensin II-induced myocardial hypertrophy, J. Cardiovasc. Pharmacol. Ther. 16 (2011) 205–211. [9] W. Tang, T. Cui, B. Wang, W. Wu, Effect of mitofusin 2 overexpression on the proliferation and apoptosis of high-glucose-induced rat glomerular mesangial cells, J. Nephrol. 25 (2012) 1023–1030. [10] K.H. Chen, A. Dasgupta, J. Ding, F.E. Indig, P. Ghosh, D.L. 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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Overexpression of mitofusin 2 inhibits reactive astrogliosis proliferation in vitro Tao Liu, Chen-chen Xue, Yu-long Shi, Xiang-jun Bai, Zhan-fei Li, Cheng-la Yi ∗ Department of Traumatic Surgery, Tong-ji Hospital, Tong-ji Medical College, Huazhong University of Science and Technology, Wuhan, China
h i g h l i g h t s • Scratch injury and starvation-serum stimulation induce reactive astrogliosis. • Mfn2 expression is down-regulated during the activation of astrocytes. • Overexpression of Mfn2 inhibits astrocytes proliferation by cell-cycle arrest.
a r t i c l e
i n f o
Article history: Received 6 May 2014 Received in revised form 14 June 2014 Accepted 1 July 2014 Available online 11 July 2014 Keywords: Mitofusin 2 Reactive astrogliosis Cell cycle arrest
a b s t r a c t Astrocytes become activated in response to central nervous system (CNS) injury, and excessive astrogliosis is considered an impediment to axonal regeneration by forming glial scar. Mitofusin 2 (Mfn2), a key protein in mitochondrial network, has been reported to negatively regulate cell proliferation. The present study aimed to explore whether reactive astrogliosis could be suppressed by Mfn2 overexpression. Scratch injury and starvation-serum stimulation models in cultured astrocytes were combined to address this issue. In scratch model, reactive proliferation status of damaged astrocytes was implicated by migration of high ratio of EdU(+) cells into lesion region and significantly increased expression of GFAP and PCNA. At meantime, Mfn2 expression was found to exert a down-regulated trend both in gen and protein levels. Pretreatment of cells with adenoviral vector encoding Mfn2 gene increased Mfn2 expression and subsequently attenuated injury-induced astrocytes hyperplasia, activation-relevant protein synthesis, cellular proliferation, eventually delayed wound healing process. Furthermore, Mfn2 overexpression markedly inhibited astrocytes proliferation induced by serum stimulation, by arresting the transition of cell cycle from G1 to S phase. Together, these in vitro results demonstrated that reactive astrogliosis can be effectively suppressed by up-regulation of Mfn2, which might contribute to a promising therapeutic intervention in CNS disease characterized by glia-related damage. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Reactive astrogliosis is an important cellular response to central nervous system (CNS) injury, which is characterized by hypertrophy of cellular processes, cell proliferation and increased expression of intermediate filament proteins, such as glia fibrillary acidic protein (GFAP) [1]. Reactive astrogliosis may play both a ben-
Abbreviations: CNS, central nervous system; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; Ad, adenovirus; GFP, green fluorescent protein; EdU, 5-Ethynyl-2 -deoxyuridine; GFAP, glial fibrillary acidic protein; PCNA, proliferating cell nuclear antigen; MAPK/ERK, Mitogen-activated protein kinases/extracellular-signal-regulated kinase. ∗ Corresponding author at: Jie Fang Road 1095, Wuhan, China. Tel.: +86 027 83665346; fax: +86 027 83665346. E-mail address: [email protected] (C.-l. Yi). http://dx.doi.org/10.1016/j.neulet.2014.07.002 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
eficial and a detrimental role in CNS repair depending on the timing of astrocyte activation and the local environment [2]. Initially, it may protect the intact tissue from exposure to toxic elements in the infarct area and secrete neurotrophic factors to provide a permissive substrate for neuron regeneration [3]. However, at later stages post injury, reactive astrocytes result in glial scar formation that impedes axonal regrowth by constituting a physical as well as a biochemical barrier [4]. Thus, early regulation of excessive reactive astrocyte proliferation may inhibit glial scar formation to create a favorable environment for neuronal regeneration and thereby enhance CNS injury recovery. Mitofusin 2 (Mfn2) is a protein that localizes to the mitochondrial outer membrane. Apart from its major function in mitochondrial network [5], Mfn2 also has a potential role in regulating cell proliferation [6]. Mfn2 has been reported to take part in various hyper-proliferative diseases such as atherosclerosis,
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cardiac hypertrophy, diabetic nephropathy and neoplasms [7–10]. In these studies, Mfn2 exerts its anti-proliferative effect by acting as an effector molecule of Ras and Raf-1, resulting in the inhibition of Mitogen-activated protein kinases/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathway. On one hand, several lines of evidence have revealed the close correlation between the MAPK/ERK pathway and astrocyte proliferation [11]. On the other hand, Mfn2 is abundantly expressed in brain [12]. Therefore, the present study aims to determine whether Mfn2 is associated with reactive astrogliosis induced by serum stimulation and mechanical injury. If Mfn2 is indeed involved, whether adenovirus-mediated Mfn2 overexpression is capable of inhibiting reactive astrocyte proliferation and the potential mechanisms will also be explored.
2. Materials and methods 2.1. Astrocyte culture and identification Cultures of astrocytes from neonatal SD rat cortex were prepared according to a standard procedure [13]. The cultures were maintained at 37 ◦ C and 95% O2 /5%CO2 in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 0.5 mg/ml penicillin/streptomycin. About 48–60 h in vitro maintenance, purification of astrocyte was undertaken by shaking. At the end of the two weeks culture, primary astrocytes were trypsinized and replated onto 35-mm dishes at 3 × 104 cell/cm2 density or into 96-well cell plates at the concentration of 1 × 104 cell/well for following experiments. The purity of astrocyte was identified by double-labeling with GFAP antibody (1:200, Sigma) and Hoechst 33342. The immunocytochemistry revealed that more than 95% of the cultured cells were GFAP-positive astrocytes.
2.2. Adenovirus infection and expression identification Replication-defective adenovirus encoding the complete rat Mfn2 open reading frame (Ad-Mfn2) and the control adenovirus encoding the green fluorescent protein open reading frame (Ad-GFP) were constructed by BGI Tech (Shenzhen, China). Astrocytes were incubated with adenovirus at the indicated multiplicity of infection (MOI) of 30 pfu/cell for 24 h, then the virus-containing medium was changed to fresh complete growth medium to continue incubation. Ad-Mfn2 expression was detected by western-blotting, which showed that Mfn2 was significantly overexpressed after 48 h infection.
2.3. Starvation and serum stimulation assay Cells were starved with serum-free DMEM for 24 h to achieve cell synchronization and treated by 10% FBS mitotic stimulation for 24 h or 48 h [14]. Control cell samples included cells nontreated and only treated with starvation.
2.4. Scratch injury model Monolayer confluent astrocytes were scratched with sterile plastic pipette tips (100 L) longitudinally and latitudinally every 0.5 cm at right angles to each other in 35 mm dishes. This process has been described to establish a reproducible model of 30–40% damage [15]. Immediately, the detached cells and debris were washed out with fresh medium. Scratched cultures were maintained for 12 h, 24 h and 48 h for following experiments.
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2.5. Reverse transcription-polymerase chain reaction (RT-PCR) analysis The trend of Mfn2 mRNA expression during serum stimulation and scratch model was detected by RT-PCR. Total RNA were extracted from cultured astrocytes by using Trizol reagent (Invitrogen). RNA concentration and quantity were determined by ultraviolet spectrophotometry (absorbance at 260 nm/280 nm). RNA were reverse-transcribed with the Revertaid first strand cDNA synthesis Kit (Thermo Scientific), followed by cDNA amplification in a DNA thermal cycler. The primers were designed as follows: Mfn2 sense primer: 5 -CTCAGGAGCAGCGGGTTTATTGTCT-3 antisense primer: 5 -TGTCGAGGGACCAGCATGTCTATCT-3 , fragment size: 412 bp. -actin sense primer: 5 -GGAGATTACTGCCCTGGCTCCTA-3 antisense primer: 5 -GACTCATCGTACTCCTGCTTGCTG-3 , fragment size: 151 bp. The PCR products were separated by 1.0% agarose gel electrophoresis and visualized by ethidium bromide staining. 2.6. Western blotting and quantification Total proteins of each group were harvested and then centrifuged at 12,000 × g for 10 min at 4 ◦ C and protein concentrations in the supernatant were detected using the BCA protein assay kit (Pierce). Protein samples were separated by 10% SDS-PAGE and then transferred onto nitrocellulose membranes. The membranes were blocked for 1 h in Tris-buffered saline (TBS) containing 5% nonfat milk at room temperature and respectively incubated with primary antibodies overnight at 4 ◦ C, which included Mfn2 (1:500, CST), GFAP (1:2000, Sigma), PCNA (1:1000, Abcam), PhosphoERK1/2 (1:500, CST) and -actin (1:1500, Santa Cruz). On the following day, the membranes were incubated with Odyssey secondary antibodies (780-conjugated goat anti-rabbit and antimouse IgG, 1:15,000) for 1 h and then visualized and quantitated by Odyssey IR imaging system (Li-COR Bioscience, USA), then expressed as the ratio of optical density (OD) from the tested proteins to that from actin. 2.7. Assessment of cell proliferation To assess the proliferation of astrocytes, EdU staining was used [16]. Briefly, cells were incubated with culture medium in presence of 10 M EdU (RiboBio) for 24 h. Cells were then fixed with 4% paraformaldehyde for 30 min. After naturalization with 2 mg/ml glycine and permeabilized with 0.2% triton X-100, cells were incubated with Apollo® staining solution for 30 min to chase the template of DNA. To label nuclei, cells were washed in 0.2% Triton X-100 for three times prior to incubation with Hoechst 33342 for 10 min. Finally cells were visualized and counted with a fluorescent microscope. 2.8. Wound healing assay At 0 h, 12 h, 24 h and 48 h after scratch, astrocytes were fixed for immunofluorescence staining. The membranes of fixed cells were permeabilized with Triton X-100 (15 min, 0.2% in PBS), followed by incubation with 5% bovine serum albumin in PBS at room temperature for 1 h to block nonspecific antibody binding and incubated overnight at 4 ◦ C with monoclonal mouse anti-GFAP (1:200, Sigma, USA). Photographs of cells were captured with a fluorescent microscope. Scratch areas before and after recovery were measured using Axiovision 4.1 software [17].
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2.9. Cell cycle analysis Astrocytes treated by 24 h starvation and 48 h serum stimulation in control, Adv-GFP and Adv-Mfn2 groups were collected, washed with PBS and fixed in 70% ice-cold ethanol. Then, cells were treated with 0.5 mg/ml RNase (Invitrogen) for 30 min and finally stained with propidium iodide (PI, Invitrogen) for additional 30 min at room temperature. Flow cytometry analysis (Becton-Dickinson) was performed to separate and quantify the cell-cycle distribution.
2.10. Statistical analysis All measurements are done in triplicate and denoted as mean ± SEM. Statistically significant differences (p < 0.05) between two groups and among more than two groups were evaluated by Student’s t-test and one-way ANOVA with Tukey’s post hoc test, respectively.
3. Results 3.1. Time-dependent changes in the mRNA and protein expression of Mfn2 during the activation of astrocytes induced by serum stimulation and scratch injury PCNA, an important factor required for DNA synthesis, is an index for cell proliferation. Western-blot showed that PCNA expression increased with time after scratch injury and reached a plateau at 24 h. GFAP, a basic and specific substance for astrocytes to participate in glial scar formation, increased persistently after scratching. Up-regulation of PCNA and GFAP level proved that scratch model in vitro successfully activated astrocytes and induced their proliferation in a time-dependent manner. During this period, we found that Mfn2 expression at both mRNA and protein levels decreased with time, negatively correlated with reactive astrocyte proliferation. Similar results were obtained in serum stimulation model, in which both PCNA and GFAP proteins were up-regulated in parallel after incubation with 10%FBS for 24 h and 48 h, while Mfn2 mRNA and protein expressions were reduced.
Moreover, the phosphorylated active forms of ERK1/2 (phospho-ERK1/2), a crucial signal protein for cellular proliferation did not show significant changes either in scratch injury or serum stimulation model. All above results can be found in Fig. 1. 3.2. Overexpression of Mfn2 regulated expression of relevant proteins associated with astrocyte activation The observation time chosen for western-blot detection was 24 h after scratch injury and 48 h after 10% FBS stimulation respectively due to the highest proliferation rate of reactive astrocyte in combination with lowest Mfn2 expression at these two timepoints. Fig. 2 demonstrated that infection of astrocytes with Mfn2 reduced the expression of activation related proteins PCNA and GFAP both in scratch injury (A) and serum stimulation models (B). However, p-ERK expression was not altered again.
3.3. Overexpressing Mfn2 inhibited the proliferation and cell cycle progression of astrocytes initiated by serum stimulation The percentage of EdU-positive cells accounting for the total cell number (counterstained with Hoechst 33342) indicated the cell proliferation situation. As Fig. 3A shows, after being deprived of serum for 24 h, cultured astrocytes were kept in a resting stage characterized by majority of cells were EdU-negative. The proportions of Edu(+) astrocyte in the control and Ad-GFP group increased gradually after being cultured with complete medium for 24 h and 48 h, which were remarkably higher than those in Adv-Mfn2 group. Flow cytometry analysis in Fig. 3B revealed that serumdeprived cells were mainly distributed in the G0/G1 phase (92.15 ± 1.37%). After 48 h serum stimulation, approximately 19% of astrocytes infected by uninfected astrocytes (19.4 ± 2.33%) or Adv-GFP (19.05 ± 3.16%) progressed into S phase, however astrocytes infected with Adv-Mfn2 remained mostly in the G0/G1 phases with only 6.92 ± 2.01% (p < 0.05) of cells entering S phase. The G0/G1 fraction of astrocytes in Adv-Mfn2 group was 86.83 ± 2.47% (p < 0.05), compared with 71.88 ± 2.42% for uninfected group and
Fig. 1. Detection of Mfn2 expression during the activation of astrocytes in scratch-injury and starve-serum stimulation models. (A) Expression of GFAP, PCNA, p-ERK1/2 and Mfn2 proteins were evaluated by Western blot with -actin as the control. (B) RT-PCR showed the decreased expression of Mfn2 mRNA in a time-dependent manner. Statistical graph describes the relative expression level compared to 0 h. (*p < 0.05, **p < 0.01).
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Fig. 2. Mfn2 overexpression suppressed expression of relevant proteins associated with reactive astrogliosis. Western blot analysis revealed that Mfn2 expression was increased by Ad-Mfn2 infection and up-regulation of Mfn2 reduced the levels of GFAP and PCNA in astrocytes after 24 h scratching injury (A) and 48 h serum stimulation (B), while p-ERK1/2 abundance was not altered. Relative level of target proteins in different groups were statistically analyzed (*p < 0.05, **p < 0.01, vs. the uninfected group).
Fig. 3. Mfn2-induced astrocytes proliferation inhibition and cell-cycle arrest. (A) EdU (red) and Hoechst 33342 (blue) were double stained to assess astrocytes proliferation in serum-stimulation model. Percentages of EdU-positive cells in each group for corresponding time points were statistically analyzed. (*p < 0.05, **p < 0.01, vs. the uninfected group). Scale bars = 200 m. (B) (I) Typical examples of cell cycle distribution in synchronized, or serum-stimulated astrocytes uninfected or infected with either Adv-GFP or Adv-Mfn2. (II) The average data of cell cycle distributions (*p < 0.05, vs. the uninfected group). (III) Mfn2-induced decrease in CyclinD1 expression. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Mfn2 transfection slowed down scratch wound healing by inhibiting reactive astrocytes proliferation. (A) GFAP immunostaining (green) images were taken from astrocyte cultures in uninfected, Adv-GFP and Adv-Mfn2 groups scratched for 0, 12, 24 and 48 h, respectively and astrocyte-absent areas were measured. (B) Proportions of proliferation cells in the scratch wounded regions were evaluated by Edu staining and statistically analyzed. (*p < 0.05, **p < 0.01, vs. the uninfected group). Scale bars = 200 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
72.61 ± 2.73% for Adv-GFP group. No significant difference in the G2-M fraction was observed between the Ad-Mfn2 (7.25 ± 0.86%), Ad-GFP (8.72 ± 1.38%), and uninfected (8.34 ± 1.12%) groups. CyclinD1, an important positive regulator for the transition of the cell cycle from the G1 to S phase, was found to decrease accordingly after Adv-Mfn2 infection.
3.4. Up-regulation of Mfn2 delayed wound healing by reducing reactive astrogliosis To acquire evidence for the hypothesis that cell cycle inhibition induced by up-regulation of Mfn2 could directly modulate astrogliosis, wound healing assay was used. Changes in cell morphology and proliferation in the wound area were evaluated by GFAP and EdU staining, respectively. As shown in Fig. 4, at 12 h after scratching, the number of proliferating astrocytes in the wound area increased and the cell bodies and cytoplasmic processes became hypertrophic and extended to the denuded area. Cells adjacent to the wound continued proliferating and migrating into the denuded area until cell density nearly approached confluence in the wound at 48 h after injury. In Ad-Mfn2 group, significant slower wound healing process was observed, as characterized by reduced astrocyte proliferation, increased inter-cellular space and shrunken cell volume. Cells in the area distant from the scratch did not show enhanced proportion of EdU(+) cells during the wound healing.
4. Discussion Mfn2 is highly expressed in cells with high-energy requirement, such as smooth muscle cells, cardiomyocytes, cancer cells and nerve cells. Proliferation inhibitory capacity of Mfn2 on these cells has recently been reported in succession, except for nerve cells. The role of Mfn2 in CNS is little discovered and limited to its conventional function in mitochondrial fusion [18]. It is known that various forms of injuries such as trauma, hypoxia, ischemia and infection in CNS can activate astrocytes and cause reactive gliosis, which result in scar formation and eventually inhibit CNS reconstruction. Therefore, astrocyte activation after CNS injury is also some kind of hyper-proliferative disease. In present study, we intend to explore the pathological change of Mfn2 expression during astrocyte activation and whether overexpressing Mfn2 can inhibit reactive astrogliosis. To investigate the process of astrocyte activation, a set of vitro models have been proposed [19]. A simple but well-described “scratching injury model” was established in this study, in which confluent monolayers of cultured astrocytes were scratched by pipette tips to simulate mechanical injury of CNS in vivo. Astroglia cells in the scratch area have been reported to exhibit some typical signs of activation, including process extension, nuclei hypertrophy, increased proliferation and up-regulation of GFAP [20,21]. Based on proliferating cell markers such as PCNA and EdU in combination with astrocytes hyperplasia indicators such as GFAP, how astrocytes responded to scratching was monitored in the present
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study. Initially, most of astrocytes both adjacent to and far away the wounds were in stationary growth phase. Cells far from the wounds remained a resting state during the wound healing period. However, as time went on, astrocytes next to the wound site started extending flat, cytoplasmic processes to the denuded area and proportion of proliferating cells in cells filling the wound was increased. Astrocytic migration and enhanced proliferation lasted until cell density within the wound approached confluence again. Relevant changes in the key protein expression were observed for PCNA and GFAP postlesion. These results revealed that astrocytes could become reactive and proliferate in response to mechanical injury in vitro. Meanwhile, Mfn2 expression both in gen and protein level showed an opposite trend. This finding implied that down-regulation of Mfn2 might contribute to the initiation and development of astrocytes activation. This implication was supported by the observation that Mfn2 overexpression alleviated astrocyte proliferation and decelerated wound healing process. To further confirm the involvement of Mfn2 in the regulation of astrocyte proliferation, starvation and serum stimulation model was conducted. Serum contains an astroglial mitogen, thus it is also an optimal system to test relevant proliferating proteins [22]. As respected, increased GFAP and PCNA expression was consistent with significant cellular proliferation, while Mfn2 level declined. Together, these results suggested that over-expressing Mfn2 suppressed reactive astrogliosis in vitro through cessation of proliferation. Following the finding that Mfn2 negatively regulated reactive astrocytes proliferation, we further explored the underlying mechanism. Since cell cycle retardation is an important way of inhibiting reactive astrogliosis, the cell cycle distribution after Adv-Mfn2 infection was chosen to investigate. Here, we found the suppressive effect of Mfn2 on astrocyte proliferation was mediated by G0/G1 cell-cycle arrest, which was also manifested by Mfn2-mediated alterations in key components of cell-cycle regulatory machinery. It was consistent with the anti-proliferation mechanism of Mfn2 reported in some other cell types [6,23]. To further dissect the possible pathway linking Mfn2 with cell cycle arrest, we sought to determine ERK1/2, well recognized as the downstream substrate of Mfn2 in controlling cell proliferation. Unexpectedly, marked activation of ERK1/2 was not observed. In fact, there were strong evidences to implicate the ERK/MAPK pathway as an obligatory step in either the acute trigger or chronic maintenance of reactive astroglia [24]. It was proved by previous studies based on scratching model that focal mechanical injury could induce a rapid activation and spreading of ERK1/2. However, it was a transient manner that p-ERK1/2 began to increase as early as 10 min postlesion, which peaked at 30 min and lasted for as long as 8 h, then returned to baseline at 24 h after scratching [25,26]. In addition, it was found that in astrocytes, ERK activation is more prominent and long-lasting after ischemia injury [27] but not after mechanical injury. Taken together, cell-type and modeltype dependence of ERK activation might contribute to the failure to observe the change of p-ERK1/2 expression and to correlate Mfn2 with ERK pathway in present study. It is likely that multiple parallel and interconnected pathways regulate the complex activating reaction of astroglia. 5. Conclusions Overall, the findings presented here reveals that the activation and proliferation of astrocytes occurred in response to stimuli such as scratch injury and serum in vitro, which is paralleled by pathological down-regulation of Mfn2 expression. Mfn2 over-expression can alleviate the responsive activation of astrogliosis and delay wound healing process by inhibiting cell cycle progression.
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Anti-proliferative effect of Mfn2 may serve as a promising therapeutic intervention in CNS diseases with excessive astrogliosis. Acknowledgements This study was funded by the National Nature Science Foundation of China (Grant NO. 81271348) and the National key Technology R&D Program (Grant NO. 2012BAI11B00). References [1] M.V. Sofroniew, Molecular dissection of reactive astrogliosis and glial scar formation, Trends Neurosci. 32 (2009) 638–647. [2] S. Karimi-Abdolrezaee, R. Billakanti, Reactive astrogliosis after spinal cord injury-beneficial and detrimental effects, Mol. Neurobiol. 46 (2012) 251–264. [3] R.E. White, L.B. Jakeman, Don’t fence me in: harnessing the beneficial roles of astrocytes for spinal cord repair, Restor. Neurol. Neurosci. 26 (2008) 197–214. [4] Z. Chen, M. Negra, A. Levine, Y. Ughrin, J.M. Levine, Oligodendrocyte precursor cells: reactive cells that inhibit axon growth and regeneration, J. Neurocytol. 31 (2002) 481–495. [5] O.M. de Brito, L. 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