J Mol Neurosci DOI 10.1007/s12031-015-0534-5
Upregulation of SYF2 Relates to Retinal Ganglion Cell Apoptosis and Retinal Glia Cell Proliferation After Light-Induced Retinal Damage Aimin Sang & Xiaowei Yang & Hui Chen & Bai Qin & Manhui Zhu & Ming Dai & Rongrong Zhu & Xiaojuan Liu
Received: 28 December 2014 / Accepted: 19 February 2015 # Springer Science+Business Media New York 2015
Abstract SYF2 (SYF2 homologue, RNA splicing factor), also known as CCNDBP1-interactor or p29, belongs to the SYF2 family, which are involved in pre-mRNA splicing and cell cycle progression. Accumulating evidences demonstrate that SYF2 exerted multiple effects including pro-apoptosis, cell differentiation, and glial activation in the pathogenesis of various experimental central nervous system (CNS) diseases. However, SYF2 expression and functions in the retina are still with limited acquaintance. To investigate whether SYF2 was involved in retinal degeneration, we performed a light-induced retinal damage model in adult rats. The SYF2 protein expression was dramatically upregulated after retinal damage. Besides that, SYF2 localized in the retinal ganglion cell (RGC) layer (GCL), inner unclear layer (INL), and outer nuclear layer (ONL) after light exposure. In addition, the expression of cyclin D1, CDK4, and active caspase-3 was parallel with SYF2. We also found the co-localization of SYF2 with active caspase-3, PCNA, and CD11b. Collectively, SYF2 might participate in RGC apoptosis and retinal glia cell proliferation after light-induced retinal damage. Aimin Sang, Xiaowei Yang, Rongrong Zhu and Xiaojuan Liu contributed equally to this work. A. Sang : X. Yang : H. Chen : B. Qin : M. Zhu : M. Dai : R. Zhu Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China X. Liu Department of Pathogen Biology, Medical College, Nantong University, Nantong, Jiangsu 226001, China A. Sang : X. Yang : H. Chen : B. Qin : M. Zhu : M. Dai : R. Zhu (*) : X. Liu (*) Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China e-mail: [email protected] e-mail: [email protected]
Keywords SYF2 . Light-induced retinal damage . Apoptosis . Retinal ganglion cells . Cell cycle . Retinal glia cells
Introduction Age-related macular degeneration (AMD) is a common and painless eye condition as well as a leading cause of vision loss for people older than 50 years, but the molecule mechanism of AMD is ambiguous (Randolph 2014). Light-induced retinal damage is a model covering the essential characteristics of human AMD (Marc et al. 2008). After light exposure, apoptosis occurs in the retinal cells including photoreceptors and RGCs (Marc et al. 2008; Wenzel et al. 2005). But the molecular mechanism of light-induced photoreceptors and RGC damage remains unclear. In addition, Müller glia cells (MGCs) and microglia cells which are the major retinal glia cells, are activated during retinal degeneration (Bringmann et al. 2006; Langmann. 2007; Levine et al. 2014). However, the pathophysiologic mechanism underlying retinal degeneration is poorly understood. Thus, it is crucial to investigate the molecular mechanisms of RGC apoptosis and retinal homeostasis. SYF2 is regarded to be part of splicing complex, which is involved in both pre-mRNA splicing and cell cycle progression (Ben-Yehuda et al. 2000). Previous researches showed that SYF2 interacts with Grap2 and cyclinD-interacting protein (GCIP). GCIP interacting with cyclin D1 may regulate proteins involved in the G1/S cell cycle progression (Chang et al. 2000). In addition, SYF2 is also considered to be involved in cell cycle progression by modulating transcriptional and post-transcriptional regulation of α-tubulin (Chen et al. 2012; Dahan and Kupiec 2002). Depletion of SYF2 using small interfering RNA duplexes induced disordered cell cycle
J Mol Neurosci
progression and DNA damage checkpoint responses (Chu et al. 2006). SYF2 is functionally related with cyclin D1 in LPS-induced neuroinflammation and required for astrocyte activation and neuronal apoptosis (Xu et al. 2014). However, the biological function of SYF2 after light-induced retinal damage is unknown. The elevation of markers for cell cycle re-entry was observed in the injured RGCs (Galan et al. 2014; Shu et al. 2014). Microglia cells show co-localization with PCNA in rd-1 mouse (a kind of photoreceptor degeneration model mouse) (Zeiss and Johnson 2004). In addition, there is a proliferative response in MGCs in C57BL/6 mouse after photoreceptor death (Suga et al. 2014). In the model of lightinduced zebrafish photoreceptor damage, MGCs can re-enter the cell cycle to produce undifferentiated neuronal progenitors that continue to proliferate, migrate to the outer nuclear layer, and differentiate into photoreceptors (Nelson et al. 2013). Inhibition of cell cycle progression reduced reactive astrocytosis and neuronal apoptosis in mouse cerebral ischemia model (Zhu et al. 2007). These studies indicate that cell cycle re-entry may participate in neurodegeneration diseases. SYF2 is relevant to the cell cycle progression, and cell cycle re-entry is responsible for RGC apoptosis and retinal glia cell activation; thus, we hypothesized that SYF2 may be associated with light-induced retinal damage. In this study, we constructed adult rat light-induced retina damage model and explored the expression, distribution, and functions of SYF2 in the rat retina. Our work may provide a useful clue to the pathogenesis of retinal degeneration and may serve as a potential new therapeutic target for neurodegenerative diseases.
Materials and Methods Animals and the Light-Induced Retinal Damage Model Adult male Sprague Dawley rats weighing 180–200 g (Department of Animal Center, Nantong University) were used in our experiments. All experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Before light exposure, rats were dark adapted for 1 day. After pupil dilation with compound tropicamide (Santen Pharmaceutical, Osaka, Japan), dark-adapted rats were placed in cages and exposed to white light (20,000 lx) for 3 h, beginning at 9 AM. During light exposure, the room temperature (RT) was kept at 24 °C. The animals had free access to food and water. After light exposure, all rats were returned to darkness and randomly divided into six groups (6 h, 12 h, 1 day, 3 days, 5 days, 7 days).
Western Blot To obtain samples for Western blot, all rats were given an overdose of chloral hydrate (400 mg/kg, i.p.) and sacrificed at different time points after light exposure (total 18 rats at each time point). Then, the cornea, lens, and vitreous were removed; retina was extruded gently from the eye cup. Twelve retinas were put on one pool for further protein extraction and analysis. The extract samples (2.0 mg/ml, 10 μl) were loaded, subjected to 10 % SDS-PAGE, and electro-blotted onto PVDF membranes using the MiniPROTEAN 3 Electrophoresis System and the Mini TransBlot Electrophoretic Transfer System (BioRad, Hercules, CA, USA). The membranes were blocked with 5 % nonfat milk at RT for 2 h, followed by incubation with primary antibody against SYF2 (rabbit, 1:1000; Santa Cruz), PCNA (mouse, 1:500; Santa Cruz), active caspase-3 (mouse, 1:1000; Cell Signaling), cyclin D1 (mouse, 1:500; Santa Cruz), CDK4 (rabbit, 1:500; Santa Cruz), and GAPDH (mouse,1:3000; Santa Cruz) at 4 °C overnight. Next, immunoreactive bands were detected by chemiluminescence using corresponding HRP-conjugated secondary antibodies (1:2000; GE Healthcare, Piscataway, NJ, USA), enhanced chemiluminescence detection reagents (GE Healthcare), and LAS 3000 image analyzer (FUJIFILM, Japan). Quantitative changes in band intensities were evaluated with Image Quant 5.2 software (GE Healthcare). Values are responsible for at least three independent experiments. Quantitative Real-Time PCR Quantitative real-time PCR was used to determine the SYF2 mRNA level in different survival time after light exposure. Total RNA was isolated from retina of rats using RISO reagent (Biomics, Nantong, China) and treated with DNase I. Complementary DNA (cDNA) was synthesized by reverse transcriptase from total RNA with oligo-d (T) primers. Quantitative real-time PCR analysis was performed with the Bio-Rad IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using the SYBR Master mixture (Biomics, Nantong, China). The PCR reactions were performed in triplicate on each cDNA template along with triplicate reactions of a house-keeping gene, GAPDH. We used the following primers: for SYF2, forward (5′-ATGGCGGCTG TGACTGAAG-3′) and reverse (5′-TCACACAGCTGTGC C-3′); for GAPDH, forward (5′-GAAGGTGAAGGTCGGA GTC-3′) and reverse (5-GAAGATGGTGATGGGATTTC-3′). The specific amplification was verified by melting curve analysis. The data was normalized against GAPDH. The expression levels were determined using the ΔΔCT method (IQ5 sotware version 2.0, Bio-Rad) and presented as fold changes. Experiments were performed in triplicate with three biological samples (two rats) from each group (six rats).
J Mol Neurosci
Sections and Immunohistochemistry
Quantitative Analysis
At defined survival time, normal and light-exposed rats were terminally anesthetized and perfused through the ascending aorta with saline, followed by 4 % paraformaldehyde. After perfusion, the superior conjunctiva was sutured with 8.0 vicryl. The eyes were fixed in 4 % paraformaldehyde solution, followed by immersion in sucrose solution for cryoprotection. Then, the tissues were embedded in OCT compound, and 7-μm frozen sections were prepared. The superior hemisphere along the vertical meridian was chosen in this experiment. After the sections had been prepared, they were kept in an oven at 37 °C for 60 min and rinsed twice with 0.01 M PBS. The sections were blocked and incubated with SYF2 antibody (rabbit, 1:200; Santa Cruz) at RT for 2 h, followed by incubation with the primary and the secondary antibodies for 30 min at 37 °C, then color reaction with a liquid mixture (0.02 % DAB, 3 % H2O2, and 0.1 % PBS). Finally, the sections were dehydrated, cleared, coverslipped, and examined under a light microscope (Leica, Germany). All the acquired images were used for quantification. The cell counting and assessment were performed in a double-blind manner (Xu et al. 2014).
We counted the cell by using the software Image J. To avoid counting the same cell in more than one section, we counted every fifth section (50 μm apart). The number of SYF2 positive cells in the retina was counted at ×400 magnification. For each section, three separate regions were examined. The cell counts in the three sections were then used to determine the total number of SYF2 positive cells and whole retinal cells per square millimeter.
Double Immunostaining The cryosections were blocked and then incubated with antiSYF2 antibody (rabbit, 1:200; Santa Cruz). The co-incubated antibodies were NeuN (mouse, 1:200; Cell Signaling), CD11b (rabbit, 1:400; Chemicon, Temecula, CA), active caspase-3 (mouse, 1:200; Cell Signaling), PCNA (rabbit, 1:100; Santa Cruz), and cyclin D1 (mouse, 1:500; Santa Cruz) separately for 10 h at 4 °C. On the following day, a mixture of FITC-, CY3-, and AMCA-conjugated secondary antibodies and Hoechst (Pierce Biotechnology, USA) were added in dark and incubated for 2 h at 4 °C. The stained sections were examined with a Leica fluorescence microscope (Germany). Intravitreal Treatment After the rats were anesthetized, their corneas were anesthetized with a drop of 0.5 % proparacaine hydrochloride (Alcaine; Alcon-Couvreur, Puurs, Belgium), pupils were dilated with 1 % tropicamide, and then the eyes were gently protruded with a rubber sleeve. Intravitreal injection of different remedies was performed 1 mm behind the limbus with a 33-gauge blunt-tip needle (Hamilton, Reno, NV, USA) and leaving the needle for 1 min to reduce the reflux. The rats intravitreal injection of liposome encapsulated 5-fluorouracil (1.5 mg/ml) (TCI, shanghai, China) were divided into seven subgroups: control; 6 h; 12 h; 1 day; 3 days; 5 days; and 7 days. The animals were sacrificed after the start of light exposure, and the eyes were then enucleated for Western blot analyses.
Statistical Analysis All data were analyzed with IBM SPSS Statistics 19 statistical software. All values were expressed as means ± SEM and were analyzed by one-way variance (ANOVA) followed by Tukey’s post hoc multiple comparisons tests. P
Upregulation of SYF2 Relates to Retinal Ganglion Cell Apoptosis and Retinal Glia Cell Proliferation After Light-Induced Retinal Damage Aimin Sang & Xiaowei Yang & Hui Chen & Bai Qin & Manhui Zhu & Ming Dai & Rongrong Zhu & Xiaojuan Liu
Received: 28 December 2014 / Accepted: 19 February 2015 # Springer Science+Business Media New York 2015
Abstract SYF2 (SYF2 homologue, RNA splicing factor), also known as CCNDBP1-interactor or p29, belongs to the SYF2 family, which are involved in pre-mRNA splicing and cell cycle progression. Accumulating evidences demonstrate that SYF2 exerted multiple effects including pro-apoptosis, cell differentiation, and glial activation in the pathogenesis of various experimental central nervous system (CNS) diseases. However, SYF2 expression and functions in the retina are still with limited acquaintance. To investigate whether SYF2 was involved in retinal degeneration, we performed a light-induced retinal damage model in adult rats. The SYF2 protein expression was dramatically upregulated after retinal damage. Besides that, SYF2 localized in the retinal ganglion cell (RGC) layer (GCL), inner unclear layer (INL), and outer nuclear layer (ONL) after light exposure. In addition, the expression of cyclin D1, CDK4, and active caspase-3 was parallel with SYF2. We also found the co-localization of SYF2 with active caspase-3, PCNA, and CD11b. Collectively, SYF2 might participate in RGC apoptosis and retinal glia cell proliferation after light-induced retinal damage. Aimin Sang, Xiaowei Yang, Rongrong Zhu and Xiaojuan Liu contributed equally to this work. A. Sang : X. Yang : H. Chen : B. Qin : M. Zhu : M. Dai : R. Zhu Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China X. Liu Department of Pathogen Biology, Medical College, Nantong University, Nantong, Jiangsu 226001, China A. Sang : X. Yang : H. Chen : B. Qin : M. Zhu : M. Dai : R. Zhu (*) : X. Liu (*) Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu 226001, People’s Republic of China e-mail: [email protected] e-mail: [email protected]
Keywords SYF2 . Light-induced retinal damage . Apoptosis . Retinal ganglion cells . Cell cycle . Retinal glia cells
Introduction Age-related macular degeneration (AMD) is a common and painless eye condition as well as a leading cause of vision loss for people older than 50 years, but the molecule mechanism of AMD is ambiguous (Randolph 2014). Light-induced retinal damage is a model covering the essential characteristics of human AMD (Marc et al. 2008). After light exposure, apoptosis occurs in the retinal cells including photoreceptors and RGCs (Marc et al. 2008; Wenzel et al. 2005). But the molecular mechanism of light-induced photoreceptors and RGC damage remains unclear. In addition, Müller glia cells (MGCs) and microglia cells which are the major retinal glia cells, are activated during retinal degeneration (Bringmann et al. 2006; Langmann. 2007; Levine et al. 2014). However, the pathophysiologic mechanism underlying retinal degeneration is poorly understood. Thus, it is crucial to investigate the molecular mechanisms of RGC apoptosis and retinal homeostasis. SYF2 is regarded to be part of splicing complex, which is involved in both pre-mRNA splicing and cell cycle progression (Ben-Yehuda et al. 2000). Previous researches showed that SYF2 interacts with Grap2 and cyclinD-interacting protein (GCIP). GCIP interacting with cyclin D1 may regulate proteins involved in the G1/S cell cycle progression (Chang et al. 2000). In addition, SYF2 is also considered to be involved in cell cycle progression by modulating transcriptional and post-transcriptional regulation of α-tubulin (Chen et al. 2012; Dahan and Kupiec 2002). Depletion of SYF2 using small interfering RNA duplexes induced disordered cell cycle
J Mol Neurosci
progression and DNA damage checkpoint responses (Chu et al. 2006). SYF2 is functionally related with cyclin D1 in LPS-induced neuroinflammation and required for astrocyte activation and neuronal apoptosis (Xu et al. 2014). However, the biological function of SYF2 after light-induced retinal damage is unknown. The elevation of markers for cell cycle re-entry was observed in the injured RGCs (Galan et al. 2014; Shu et al. 2014). Microglia cells show co-localization with PCNA in rd-1 mouse (a kind of photoreceptor degeneration model mouse) (Zeiss and Johnson 2004). In addition, there is a proliferative response in MGCs in C57BL/6 mouse after photoreceptor death (Suga et al. 2014). In the model of lightinduced zebrafish photoreceptor damage, MGCs can re-enter the cell cycle to produce undifferentiated neuronal progenitors that continue to proliferate, migrate to the outer nuclear layer, and differentiate into photoreceptors (Nelson et al. 2013). Inhibition of cell cycle progression reduced reactive astrocytosis and neuronal apoptosis in mouse cerebral ischemia model (Zhu et al. 2007). These studies indicate that cell cycle re-entry may participate in neurodegeneration diseases. SYF2 is relevant to the cell cycle progression, and cell cycle re-entry is responsible for RGC apoptosis and retinal glia cell activation; thus, we hypothesized that SYF2 may be associated with light-induced retinal damage. In this study, we constructed adult rat light-induced retina damage model and explored the expression, distribution, and functions of SYF2 in the rat retina. Our work may provide a useful clue to the pathogenesis of retinal degeneration and may serve as a potential new therapeutic target for neurodegenerative diseases.
Materials and Methods Animals and the Light-Induced Retinal Damage Model Adult male Sprague Dawley rats weighing 180–200 g (Department of Animal Center, Nantong University) were used in our experiments. All experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Before light exposure, rats were dark adapted for 1 day. After pupil dilation with compound tropicamide (Santen Pharmaceutical, Osaka, Japan), dark-adapted rats were placed in cages and exposed to white light (20,000 lx) for 3 h, beginning at 9 AM. During light exposure, the room temperature (RT) was kept at 24 °C. The animals had free access to food and water. After light exposure, all rats were returned to darkness and randomly divided into six groups (6 h, 12 h, 1 day, 3 days, 5 days, 7 days).
Western Blot To obtain samples for Western blot, all rats were given an overdose of chloral hydrate (400 mg/kg, i.p.) and sacrificed at different time points after light exposure (total 18 rats at each time point). Then, the cornea, lens, and vitreous were removed; retina was extruded gently from the eye cup. Twelve retinas were put on one pool for further protein extraction and analysis. The extract samples (2.0 mg/ml, 10 μl) were loaded, subjected to 10 % SDS-PAGE, and electro-blotted onto PVDF membranes using the MiniPROTEAN 3 Electrophoresis System and the Mini TransBlot Electrophoretic Transfer System (BioRad, Hercules, CA, USA). The membranes were blocked with 5 % nonfat milk at RT for 2 h, followed by incubation with primary antibody against SYF2 (rabbit, 1:1000; Santa Cruz), PCNA (mouse, 1:500; Santa Cruz), active caspase-3 (mouse, 1:1000; Cell Signaling), cyclin D1 (mouse, 1:500; Santa Cruz), CDK4 (rabbit, 1:500; Santa Cruz), and GAPDH (mouse,1:3000; Santa Cruz) at 4 °C overnight. Next, immunoreactive bands were detected by chemiluminescence using corresponding HRP-conjugated secondary antibodies (1:2000; GE Healthcare, Piscataway, NJ, USA), enhanced chemiluminescence detection reagents (GE Healthcare), and LAS 3000 image analyzer (FUJIFILM, Japan). Quantitative changes in band intensities were evaluated with Image Quant 5.2 software (GE Healthcare). Values are responsible for at least three independent experiments. Quantitative Real-Time PCR Quantitative real-time PCR was used to determine the SYF2 mRNA level in different survival time after light exposure. Total RNA was isolated from retina of rats using RISO reagent (Biomics, Nantong, China) and treated with DNase I. Complementary DNA (cDNA) was synthesized by reverse transcriptase from total RNA with oligo-d (T) primers. Quantitative real-time PCR analysis was performed with the Bio-Rad IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA, USA) using the SYBR Master mixture (Biomics, Nantong, China). The PCR reactions were performed in triplicate on each cDNA template along with triplicate reactions of a house-keeping gene, GAPDH. We used the following primers: for SYF2, forward (5′-ATGGCGGCTG TGACTGAAG-3′) and reverse (5′-TCACACAGCTGTGC C-3′); for GAPDH, forward (5′-GAAGGTGAAGGTCGGA GTC-3′) and reverse (5-GAAGATGGTGATGGGATTTC-3′). The specific amplification was verified by melting curve analysis. The data was normalized against GAPDH. The expression levels were determined using the ΔΔCT method (IQ5 sotware version 2.0, Bio-Rad) and presented as fold changes. Experiments were performed in triplicate with three biological samples (two rats) from each group (six rats).
J Mol Neurosci
Sections and Immunohistochemistry
Quantitative Analysis
At defined survival time, normal and light-exposed rats were terminally anesthetized and perfused through the ascending aorta with saline, followed by 4 % paraformaldehyde. After perfusion, the superior conjunctiva was sutured with 8.0 vicryl. The eyes were fixed in 4 % paraformaldehyde solution, followed by immersion in sucrose solution for cryoprotection. Then, the tissues were embedded in OCT compound, and 7-μm frozen sections were prepared. The superior hemisphere along the vertical meridian was chosen in this experiment. After the sections had been prepared, they were kept in an oven at 37 °C for 60 min and rinsed twice with 0.01 M PBS. The sections were blocked and incubated with SYF2 antibody (rabbit, 1:200; Santa Cruz) at RT for 2 h, followed by incubation with the primary and the secondary antibodies for 30 min at 37 °C, then color reaction with a liquid mixture (0.02 % DAB, 3 % H2O2, and 0.1 % PBS). Finally, the sections were dehydrated, cleared, coverslipped, and examined under a light microscope (Leica, Germany). All the acquired images were used for quantification. The cell counting and assessment were performed in a double-blind manner (Xu et al. 2014).
We counted the cell by using the software Image J. To avoid counting the same cell in more than one section, we counted every fifth section (50 μm apart). The number of SYF2 positive cells in the retina was counted at ×400 magnification. For each section, three separate regions were examined. The cell counts in the three sections were then used to determine the total number of SYF2 positive cells and whole retinal cells per square millimeter.
Double Immunostaining The cryosections were blocked and then incubated with antiSYF2 antibody (rabbit, 1:200; Santa Cruz). The co-incubated antibodies were NeuN (mouse, 1:200; Cell Signaling), CD11b (rabbit, 1:400; Chemicon, Temecula, CA), active caspase-3 (mouse, 1:200; Cell Signaling), PCNA (rabbit, 1:100; Santa Cruz), and cyclin D1 (mouse, 1:500; Santa Cruz) separately for 10 h at 4 °C. On the following day, a mixture of FITC-, CY3-, and AMCA-conjugated secondary antibodies and Hoechst (Pierce Biotechnology, USA) were added in dark and incubated for 2 h at 4 °C. The stained sections were examined with a Leica fluorescence microscope (Germany). Intravitreal Treatment After the rats were anesthetized, their corneas were anesthetized with a drop of 0.5 % proparacaine hydrochloride (Alcaine; Alcon-Couvreur, Puurs, Belgium), pupils were dilated with 1 % tropicamide, and then the eyes were gently protruded with a rubber sleeve. Intravitreal injection of different remedies was performed 1 mm behind the limbus with a 33-gauge blunt-tip needle (Hamilton, Reno, NV, USA) and leaving the needle for 1 min to reduce the reflux. The rats intravitreal injection of liposome encapsulated 5-fluorouracil (1.5 mg/ml) (TCI, shanghai, China) were divided into seven subgroups: control; 6 h; 12 h; 1 day; 3 days; 5 days; and 7 days. The animals were sacrificed after the start of light exposure, and the eyes were then enucleated for Western blot analyses.
Statistical Analysis All data were analyzed with IBM SPSS Statistics 19 statistical software. All values were expressed as means ± SEM and were analyzed by one-way variance (ANOVA) followed by Tukey’s post hoc multiple comparisons tests. P
