J Mol Neurosci DOI 10.1007/s12031-013-0198-y

Spatiotemporal Changes in NFATc4 Expression of Retinal Ganglion Cells After Light-Induced Damage Yue Xu & Lu Yang & Shanshan Yu & Qinmeng Shu & Cheng Yang & Jiawei Wang & Fan Xu & Aimin Sang & Xiaoling Liang

Received: 18 October 2013 / Accepted: 28 November 2013 # Springer Science+Business Media New York 2013

Abstract Nuclear factor of activated T cells, cytoplasmic 4 (NFATc4) is one of the four members of the NFAT family, which were described first as essential components of T cells activation and lately as important regulators for the initiation and coordination of the immune response, including B cells and natural killer cells. Accumulating evidence has demonstrated that NFATc4 exerted a pro-apoptotic effect in the pathogenesis of various experimental central nervous system diseases by upregulating Fas ligand (FasL) levels. However, the function of NFATc4 in the retina is still with limited acquaintance. To investigate whether NFATc4 is involved in retinal neuron apoptosis, we performed a light-induced retinal damage model in adult rats. A significant upregulation of NFATc4 was detected in the retina after light-induced damage by using Western blotting and reverse transcriptase PCR (RTPCR). Besides this, NFATc4 was observed to be localized mainly in the retinal ganglion cells (RGCs). In addition, the Yue Xu and Lu Yang contributed equally to this work. Y. Xu : L. Yang : S. Yu : J. Wang : F. Xu : X. Liang (*) State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, Guangdong Province, People’s Republic of China e-mail: [email protected] Q. Shu Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China C. Yang Department of Ophthalmology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong Province, People’s Republic of China A. Sang (*) Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province 226001, People’s Republic of China e-mail: [email protected]

expression patterns of active caspase-3, active caspase-8, and FasL were parallel with that of NFATc4. We also found the colocalization of NFATc4 with active caspase-3 and FasL in RGCs after light exposure. Collectively, we hypothesized that NFATc4 might participate in RGCs apoptosis by upregulating FasL levels. Keywords NFATc4 . Light-induced retinal damage . Retinal ganglion cells . Apoptosis . Rat

Introduction Age-related macular degeneration (AMD) is the principal cause of blindness among the elderly in the USA and in the Europe and Australia (Mitchell et al. 1995; van Leeuwen et al. 2003), but little is known about its etiology. Light-induced retinal damage, a well-established model of AMD, is often used to study the factors leading to photoreceptor death (Costa et al. 2008; Li et al. 2013b). However, the pathophysiologic mechanism underlying retinal ganglion cells (RGCs) death is poorly understood. Impressively, previous studies have demonstrated that RGCs death in this model is not secondary to photoreceptor degeneration, but rather to retinal remodeling after light exposure (Garcia-Ayuso et al. 2011; Marc et al. 2008). Thus, it seems crucial for the treatment of AMD to investigate the molecular mechanisms of RGCs apoptosis and by which they can be regulated suitably. The nuclear factors of activated T cells (NFATs) represent a family of at least four transcription factors (NFATc1, NFATc2, NFATc3, and NFATc4), which could regulate several processes in vertebrates, including the development and function of the cardiovascular system (Bushdid et al. 2003; Graef et al. 2001; Santana 2008), skeletal muscle (Hill-Eubanks et al. 2003), and nervous system (Graef et al. 1999; Nguyen and Di Giovanni 2008; Yoshida et al. 1998). In the nervous

J Mol Neurosci

system, NFAT regulates neurite outgrowth, synaptogenesis, neuronal development, and survival (Abdul et al. 2009; Benedito et al. 2005; Graef et al. 2003; Hudry et al. 2012; Nguyen et al. 2009; Schwartz et al. 2009; Vashishta et al. 2009). Despite the critical role of NFAT-dependent transcription in neurons, the respective functions and regulation in the retina is still unclear. Recently, numerous reports in the literature support that NFATc4 is involved in the regulation of neuronal apoptosis. For instance, NFATc4 can be activated by BDNF and NMDA in primary neurons where it promotes survival, synaptic plasticity, and axonal outgrowth (Vashishta et al. 2009). Furthermore, NFATc4 can induce upregulation of the Fas ligand (FasL)/Fas death pathway which is involved in methamphetamine-induced neuronal apoptosis (Jayanthi et al. 2005). Notably, in the brain of rats after intracerebral hemorrhage, the expression of NFATc4 gradually increased, which were parallel with that of active caspase-3, active caspase-8, and FasL in neurons. The result of transfecting NFATc4 RNA interference into PC12 cells further confirmed its role in neuronal apoptosis (Li et al. 2013a, b). Therefore, we hypothesized that NFATc4 might play a pivotal role in regulating RGCs apoptosis. In the present study, we reported the expression and distribution of NFATc4 in rat retina after light exposure for the first time. Our research is conducted to gain a better insight into the physiologic functions of NFATs family in the retina and its association with the cellular and molecular mechanisms after light-induced damage.

Methods and Materials Animal Care Male Sprague–Dawley rats (10 weeks; Laboratory of Zhongshan Ophthalmic Center, Guangzhou, China) with an average body weight of 250 g (220–275 g) were used in this study. Rats were kept on a 12 h:12 h light–dark schedule and given food and water in a pathogen-free area. All procedures with animals in this study were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthamic Center. Light-Induced Retinal Damage Seventy-two rats were used for this experimental research. They were divided into six groups (n=12 at each group): the control (without light exposure), 6 h, 12 h, 1 day, 3 days, and 5 days after light exposure. In each group, three rats (six eyeballs) were used for Western blot analysis, three rats (six

eyeballs) were used for RNA isolation and reverse transcriptase PCR (RT-PCR) analysis, and six rats (12 eyeballs) were used for immunohistochemistry and immunofluorescence detection. Rat light-induced retinal damage was prepared following the procedure described in detail previously (Sang et al. 2011, 2013). Before light exposure, rats were allowed to adapt in the dark for 2 days. After pupil dilation with compound tropicamide (Santen Pharmaceutical, Osaka, Japan), 12 rats from each group were individually placed into the light box apparatus and exposed to 12, 000 lx of cool white fluorescent light for 3 h, beginning at 9 a.m. During light exposure, the room temperature was kept at 24 °C and the animals had free access to food and water. After light exposure, all rats were returned to the dark. Western Blot Analysis The extract samples (2.0 mg/ml, 10 μl) were loaded, subjected to 10 % SDS-PAGE, and electro-blotted onto polyvinylidene fluoride (PVDF) membranes using the Mini-PROTEAN 3 Electrophoresis System and the Mini Trans-Blot Electrophoretic Transfer System (Bio-Rad, Hercules, CA, USA). The membranes were blocked with skimmed milk at room temperature for 2 h, followed by incubation with primary antibody against NFATc4 (anti-rabbit, 1:500; Santa Cruz), FasL (anti-mouse, 1:500; Santa Cruz), active caspase-3 (antimouse, 1:1,000; Cell Signaling), active caspase-8 (antimouse, 1:500; Cell Signaling), and β-actin (anti-rabbit, 1:1,000, Santa Cruz) at 4 °C overnight. At last, the membrane was incubated with second antibody goat-anti-mouse or goatanti-rabbit conjugated horseradish peroxidase (1:2,000; Southern-Biotech) for 2 h and visualized using an enhanced chemiluminescence system (ECL; Pierce Company, USA). Values are responsible for at least three independent experiments. RT-PCR Analysis Total RNA was isolated from rat retina by Trizol (GibcoBRL). RNA concentration was determined by absorption at 260 nm. The reverse transcription step was carried using RevertAid First Strand cDNA Synthesis Kit (Fermentas). The upper and lower primer sequences for NFATc4 were 5′GAGGAGCCCCTACCAGACT-3′ and 5′-CCTCATTGTA GCCCTCAGGA-3′ (Seybold et al. 2006). Amplification was carried out with an initial denaturing step at 94 °C for 5 min, then 30 cycles at 94 °C for 45 s, 51 °C for 45 s, and 72 °C for 45 s, then a further extension at 72 °C for 10 min. The PCR product was sequenced. The cDNA of β-actin was adopted as an intrinsic standard during RT-PCR analysis. Values are responsible for at least three independent experiments.

J Mol Neurosci

Sections and Immunohistochemistry

Quantitative and Statistical Analysis

After adult rats were anesthetized and perfused, the superior conjunctiva was sutured with 8.0 vicryl as a reference that provided orientation (Sun et al. 2007). The eyes were enucleated and fixed in 4 % neutral-buffered formalin for 24 h at 4 °C. The tissue was dehydrated and made into frozen sections (8-μm thick). The superior hemisphere along the vertical meridian was chosen in this experiment. Sections were blocked with 10 % normal serum blocking solution and incubated overnight at 4 °C with NFATc4. Then, slides were incubated in biotinylated secondary antibody, followed by incubation in the complex avidin-biotin-peroxidase. The reaction product was revealed by 0.02 % diaminobenzidine tetrahydrochloride. Slides were examined at×20 or×40 magnifications on a Leica light microscope (Germany).

To avoid counting the same cell in more than one section, we counted every fifth section (50 μm apart). The number of NFATc4-positive cells in the ganglion cell layer (GCL) was counted at×400 magnification. For each section, three separate GCL regions were examined. All values were expressed as means±standard error of mean (SEM). The statistical significance of difference between groups was determined by one-way ANOVA followed by Tukey's post hoc multiple comparison tests. P

Spatiotemporal changes in NFATc4 expression of retinal ganglion cells after light-induced damage.

Nuclear factor of activated T cells, cytoplasmic 4 (NFATc4) is one of the four members of the NFAT family, which were described first as essential com...
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