Cell Mol Neurobiol DOI 10.1007/s10571-015-0198-2
ORIGINAL RESEARCH
Upregulation of Homer1a Promoted Retinal Ganglion Cell Survival After Retinal Ischemia and Reperfusion via Interacting with Erk Pathway Fei Fei1 • Juan Li2 • Wei Rao2 • Wenbo Liu2 • Xiaoyan Chen2 • Ning Su2 Yusheng Wang1 • Zhou Fei2
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Received: 13 January 2015 / Accepted: 16 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Retinal ischemia and reperfusion (I/R) is extensively involved in ocular diseases, causing retinal ganglion cell (RGCs) death resulting in visual impairment and blindness. Homer1a is considered as an endogenous neuroprotective protein in traumatic brain injury. However, the roles of Homer1a in RGCs I/R injury have not been elucidated. The present study investigated the changes in expression and effect of Homer1a in RGCs both in vitro and in vivo after I/R injury using Western blot, TUNEL assay, gene interference and overexpression, and gene knockout procedures. The levels of Homer1a and phosphorylated Erk (p-Erk) increased in RGCs and retinas after I/R injury. Upregulation of Homer1a in RGCs after I/R injury decreased the level of p-Erk, and mitigated RGCs apoptosis. Conversely, downregulation of Homer1a increased the level of p-Erk, and augmented RGCs apoptosis. Furthermore, inhibition of the p-ERK reduced RGCs apoptosis, and increased the expression of Homer 1a after I/R injury. Finally, the retinas of Homer1a KO mice treated with I/R injury had significantly less dendrites and RGCs, compared with Homer1a WT mice. These findings demonstrated that
Homer1a may contribute to RGCs survival after I/R injury by interacting with Erk pathway.
Keywords Homer1a Erk signaling Retinal ischemia and reperfusion Retinal ganglion cells Abbreviations I/R Ischemia and reperfusion RGC Retinal ganglion cell p-Erk Phosphorylated Erk IOP Intraocular pressure mGluRs Metabotropic glutamate receptors CC Coiled-coil WT Wild type KO Knockout PBS Phosphate-buffered saline shRNA Short hairpin RNA OGD Oxygen and Glucose Deprivation TBI Traumatic brain injury TUNEL TdT-mediated dUTP nick end-labeling
Fei Fei, Juan Li and Wei Rao have contributed equally to this work. & Yusheng Wang [email protected]
Introduction
& Zhou Fei [email protected]
Ischemia/reperfusion (I/R) causes irreversible RGCs apoptosis (Zhang et al. 2002) or necrosis (Joo et al. 1999) in the retina, which are linked to many retinal disorders including glaucoma, traumatic optic neuropathy, anterior ischemic optic neuropathy, and diabetic retinopathy. In animal models of retinal I/R induced by elevated intraocular pressure (IOP), an increased number of necrotic and apoptotic nuclei in the inner retina have been observed. I/R
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Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi’an 71032, People’s Republic of China
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Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, 15 Changle Xi Road, Xi’an 710032, People’s Republic of China
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injury due to dysfunctional autoregulation of retinal bloodflow, oxidative stress and aberrant immunity, is extensively associated with RGCs death and optic nerve damage (Dreyer et al. 1996; Liu and Neufeld 2001; Schwartz 2003; Tezel et al. 1999; Yan et al. 2000; Yoles et al. 2001). Therefore, it is necessary to elucidate the etiology of ocular diseases and find new neuroprotective agents that can protect RGCs from I/R injury (Hughes 1991). Homer1c is a key moiety related to neurodegeneration in a mild retinal ischemia rat model (Kaja et al. 2003). The Homer protein family consists of two major groups: longform proteins, including Homer1b/c, Homer2 and Homer3, which are constitutively expressed in neurons and myocytes (Duncan et al. 2005), and short variant Homer1a, which is induced by elevated synaptic activity due to alternate splicing or premature termination of transcription (Kato et al. 1998; Shiraishi et al. 1999; Sun et al. 1998; Xiao et al. 1998; Soloviev et al. 2000). Although all variants have a highly conserved EVH-1 domain within their N-terminal regions, differences in C-terminal regions regulate postsynaptic structure and signal transduction. Specifically, both Homer1b/c and Homer1a are important adaptors of group I metabotropic glutamate receptors (mGluRs). With a C-terminal coiled-coil (CC) domain, Homer1b/c assembles into multiprotein complexes with mGluRs accompanied by other molecules, and facilitates downstream signal transduction. Homer1a, which lacks the CC domain, competitively binds to target proteins and disrupts the formation of mGluRs complexes (Shiraishi-Yamaguchi and Furuichi 2007). Homer proteins are associated with several neuronal diseases, including neuropathic pain, fragile X syndrome (Ronesi et al. 2012), and stress-related cognitive deficits (Wagner et al. 2013; Miletic et al. 2005; Miletic et al. 2009; Miyabe et al. 2006; Obara et al. 2013; Yao et al. 2011). Our previous studies showed that Homer1a and Homer1b/c play an important role in the development of traumatic brain injury (TBI) via regulation of group I mGluRs (Luo et al. 2014; Fei et al. 2014). Homer1 proteins were also involved in the regulation of neuronal injury in a model of inflammation (Luo et al. 2012b) and oxidative stress (Luo et al. 2012a). In this study, we found that downregulation of Homer1a correlated with the loss of RGCs in I/R-injured retinas. Therefore, we investigated the role of Homer1a protein in the pathology of retinal I/R injury using primary cultured RGCs and a retina ischemia injury model.
Materials and Methods Animal Models Male C57BL/6 J mice (10–12 weeks) and post-natal 4- to 6-day pups were obtained from the Laboratory Animal
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Center of the Fourth Military Medical University. The mice had continuous access to food and water, and were caged at a temperature of 20–22 °C with a 12:12 h light–dark cycle. Homer1a KO mice were kindly provided by Paul Worley (Hu et al. 2010; Mahan et al. 2012). We crossed heterozygous Homer1a mice (Het, Homer1a?/-) to generate wild type (WT, Homer1a?/?) and knockout (KO, Homer1a-/-) littermates. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Briefly, mice were anesthetized by an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine (SigmaAldrich, St. Louis, MO, USA). The corneas were topically anesthetized with 0.5 % tetracaine hydrochloride, and the pupils were dilated with 1 % tropicamide (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The right eyeball of each mouse was cannulated with a 30-gauge needle connected to a saline reservoir to produce and maintain an IOP of 110 mmHg for 60 min. Successful ischemia was confirmed by the ophthalmoscopic observation of whitened iris and loss of the red reflex under microscopy. With subsequent reperfusion the return of the red reflex was confirmed. The contralateral eye was cannulated and maintained at a normal level of IOP to serve as a normotensive control. All the mice were allowed to recover for 21 days after I/R. Tissue Procurement and Retinal Flat-Mounts After treatment for 21 days, all the mice were humanely sacrificed by transcardial perfusion with 4 % paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (Gibco, Gaithersburg, MD, USA) under general anesthesia with an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. Retinas from both ipsilateral and contralateral eyeballs were dissected, post-fixed in 2 % paraformaldehyde (pH 7.4) for 1 h and washed with 0.1 M PBS (pH 7.4). RGCs density was quantified in the retinal flat-mounts as previously described (Dvoriantchikova et al. 2010; Zhang et al. 2011). Briefly, retinas were incubated at 4 °C overnight with an anti-Tuj1 antibody (Sigma-Aldrich, St. Louis, MO, USA), which was specifically bound to a RGCs-specific marker, b-III-tubulin, and then incubated with an Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). The retinas were mounted flat on pre-coated glass slides. b-IIItubulin-positive RGCs were observed in flat-mounted retinas using a 960 epifluorescent microscope containing a motorized X–Y–Z stage (Provis AX70; Olympus, Tokyo, Japan). A total of 16 images from four quadrants in each
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retina were obtained at four distances from the optic disk. After adjusting the image threshold levels and manually excluding artifacts, the loss of RGCs in the ischemic retinas was calculated as the mean cell density (RGCs number/corresponding areas) in normotensive control eyes. Primary Culture of RGCs RGCs were isolated according to the two-step immunopanning method, as previously described (Ivanov et al. 2006; Park et al. 2015; Dvoriantchikova et al. 2014). Briefly, the C57BL/6J pups were euthanized and the retinas were removed, mechanically dissected, minced, and incubated in papain solution (15 U/ml, Worthington, Lakewood, NJ, USA) for 30 min. The macrophages were removed from the cell suspension by panning with the antimacrophage antiserum (Accurate Chemical, Westbury, NY, USA). RGCs were specifically bound to the panning plates containing anti-Thy1.2 antibody. After washing with PBS, adherent cells were dissociated by incubation with trypsin (Gibco, Gaithersburg, MD, USA) and mechanically aspirating with a pipette. Finally, RGCs were seeded onto 35, 60-mm petri dishes, and 6-well plates treated with a poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) Neurobasal/2 % B27 supplement medium (Gibco, Gaithersburg, MD, USA) and cultured for the following experiments. Lentiviral Design for Homer1a RNAi and Overexpression Short hairpin RNA (shRNA) lentiviruses were designed by subcloning an oligo siRNA targeting the coding region of 1190–1208 bp into a lentiviral expression vector, pFUGW-RNAi (GeneChem Company, Shanghai, China). Following digestion and ligation, clones were selected and verified for the inserted sequence. The lentiviral constructco-expressed EGFP was driven by the ubiquitin C promoter, and shRNA was driven by the U6 promoter. To control for the off-target and non-specific effects of shRNA, a negative control shRNA (50 -TTCTCCG AACGTGTCACGT-30 ) was used. The lentiviral overexpression system was developed by removing the EGFP open reading frame from the pGC-FU-EGFP-3FLAG construct (GeneChem Company, Shanghai, China) using an AgeI/NheI digestion and replacing it with the cDNA of Homer1a (NM_011982.3). Oxygen and Glucose Deprivation (OGD) of RGCs OGD was induced by replacing the normal RGCs media with glucose- and sodium pyruvate-free Neurobasal media (Gibco, Gaithersburg, MD, USA). Primary cultured RGCs
were deprived of oxygen (0–0.1 % O2, 5 % CO2, and 95 % N2) using a ProOx model C21 hypoxia chamber (BioSpherix, Redfield, NY, USA) for 6 h at 37 °C. After OGD, the culture medium was substituted with fresh Neurobasal/B27 media, and the RGCs were incubated for 6, 12, and 24 h in a 5 % CO2 atmosphere. Parallel cultures were exposed to oxygenated Neurobasal/B27 media in an incubator under normoxic atmosphere (37 °C; 5 % CO2) as controls. Fluorometric TUNEL (TdT-mediated dUTP Nick End-Labeling) Analysis Apoptosis was analyzed using the DeadEndTM Fluorometirc TUNEL system (Promega, WI, USA) according to the manufacturer’s protocol (Fei et al. 2014). Hoechst (SigmaAldrich, MO, USA) staining was performed to assess the total number of cells per well. RGCs were observed immediately under a fluorescence microscope (Leica, Germany) using a standard fluorescein filter set. Green nuclei were positive for apoptotic cells and blue nuclei were an indicator of total non-apoptotic RGCs. An apoptotic index (AI = positive apoptotic cells/total cells 9 100 %) was used to quantify the level of apoptosis. At least 10 highpower fields (40X)/well were observed. Western Blot After various treatments, the RGCs or mice retinas were lysed with the radioimmunoprecipitation assay buffer (Pierce, Rockford, IL, USA) supplied with protease inhibitors (Roche Applied Bioscience, Indianapolis, IN, USA). Protein concentration was quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were loaded onto a 10 % SDS-PAGE gel (Beyotime, Shanghai, China). After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5 % skim milk and incubated at 4 °C overnight with the appropriate primary antibodies: anti-Homer1a (1:100; Santa Cruz Biotech, Santa Cruz, CA, USA), anti-Caspase3, and anti-cleaved-caspase3, and antip-Erk (1:1000; CST, Danvers, MA, USA). After washing, the immunoreactivity of the membrane was determined by incubated it with the corresponding horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA) followed by enhanced chemical luminescence development (Pierce, Rockford, IL, USA). The expression of b-actin was used to standardize protein loading. Western blotting analysis was performed in triplicate for each protein and the optical densities of the bands were calculated using Image Analysis Software (Bio-Rad, Los Angeles, CA, USA).
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Statistical Analysis Data are expressed as the mean ± standard error of the mean. Statistical significance was tested using ANOVA followed by a Bonferroni’s test using SPSS 22.0 (IBM, Armonk, New York, USA). Data were defined as significantly different when P \ 0.05. The use of additional statistics is explained in the text as needed.
Results Increased Expression of Homer1a and p-Erk in OGD-Induced RGCs Apoptosis As shown in Fig. 1a and b, compared with that of control group (no OGD treatment), the expression of Homer1a significantly increased 6, 12, or 24 h after OGD (P \ 0.05). We also observed a similar change in the expression of p-Erk (Fig. 1a, c). To evaluate OGD-induced RGCs apoptosis, a TUNEL assay was performed and the level of cleaved-caspase3 was measured. We found that the expression of cleaved-caspase3 was significantly higher (P \ 0.05) at periods of 6, 12, and 24 h after OGD treatment and peaked at 24 h after OGD, compared with that of the control group (Fig. 1a, d). Similarly, the TUNEL assay also showed that the number of apoptotic cells significantly increased after OGD treatment for 12 and 24 h (P \ 0.05) but not after 6 h (Fig. 1e, f). Ischemia for 6 h and reperfusion for 24 h, induced a higher level of Homer1a expression and apoptosis, and therefore, these time points were used in the following experiments. Upregulated Homer1a and p-Erk were Associated with the Loss of RGCs in I/R-Injured Retinas The expression of Homer1a was measured in vivo in I/Rinjured retinas. The expression of Homer1a and p-Erk in ischemic retinas was detected as early as 1 h after I/R. The expression of Homer1a significantly (P \ 0.05) increased at 12 h, peaked at 48 h, and then declined after the 7th day following I/R (Fig. 2a, b). The expression of p-Erk also significantly increased at 12 h (P \ 0.05) and declined to the physiological level at 72 h after I/R (Fig. 2a, c). These findings suggested that Homer1a might also be involved in the loss of RGCs in vivo. Inhibiting Erk Activation Increased the Expression of Homer1a and Mitigated RGCs Apoptosis After OGD To determine whether Erk activation affected RGCs apoptosis and the expression of Homer1a, RGCs were
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pretreated with 40 lM PD98059 (Sigma-Aldrich, St. Louis, MO, USA), a widely used Erk inhibitor, for 1 h followed by OGD. OGD-induced Erk activation was inhibited by PD98059 after OGD treatment for 24 h (Fig. 3a, c). Furthermore, the inhibition of Erk activation correlated with the increased expression of Homer1a (Fig. 3a, b), suggesting that Erk pathway might regulate the expression of Homer1a. Additionally, compared with vehicle groups, inhibition of Erk activation significantly (P \ 0.05) decreased the cleavage level of caspase3 (Fig. 3a, d) and TUNEL-positive cells (Fig. 3e), demonstrating that Erk activation mediated OGD-induced RGCs apoptosis and that inhibition of Erk promoted RGCs survival.
Homer1a Mitigated OGD-Induced RGCs Apoptosis and Interacted with Erk To evaluate the effects of Homer1a on RGCs survival, lentivirus-mediated genetic interventions were used to regulate the expression of Homer1a in RGCs. Endogenous expression of Homer1a was significantly downregulated by a lentiviral shRNA targeting Homer1a (Homer1a-RNAi) 24 h after OGD (P \ 0.05, Fig. 4a, b). Exogenous expression of Homer1a was significantly upregulated by lentiviral transduction and induced expression of 3-Flagtagged Homer1a (Flag-Homer1a) (P \ 0.05), compared with the expression of Homer1a in the vector group (ScrRNAi). Downregulation of Homer1a significantly increased the level of p-Erk (Fig. 4a, c), cleaved caspase3 (Fig. 4a, d) and stimulated apoptosis of RGCs (Fig. 4e) after OGD for 24 h (P \ 0.05). Further, upregulation of Homer1a decreased the level of p-Erk (Fig. 4a, c) and protected RGCs from OGD injury by significantly mitigating apoptosis (Fig. 4a, d, e) 24 h after OGD treatment (P \ 0.05). These results suggested that Homer1a might be an important endogenous protective protein in vitro. We investigated the potential interaction between Homer1a and Erk using Erk inhibitor 48 h after Homer1alentivirus infection. Inhibition of Erk increased OGD-induced expression of Homer1a in Scr-RNAi ? PD98059 group compared with that of the Scr-RNAi ? Vehicle group (Fig. 4a, b) 24 h after OGD injury, which was consistent with the results above. No significant difference of expression of Homer1a was observed between Homer1a-RNAi ? Vehicle group and the Homer1aRNAi ? PD98059 group, or between the Flag-Homer1a ? Vehicle group and Flag-Homer1a ? PD98059 group. Compared with that of the Homer1a-RNAi ? Vehicle group, inhibition of Erk significantly decreased the level of cleaved-capase3 (Fig. 4a, d) and reduced RGCs apoptosis (Fig. 4e) induced by Homer1a downregulation
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Fig. 1 OGD-induced the expression of Homer 1a, p-Erk and apoptosis in RGCs. Primary cultured RGCs were treated with glucose-free Neurobasal and deprived of oxygen (0–0.1 % O2, 5 % CO2, and 95 % N2) using a hypoxia chamber for 6 h at 37 °C. After OGD, the culture medium was exchanged for fresh Neurobasal/B27 media, and the RGCs were incubated for 6, 12, and 24 h in a 5 % CO2 atmosphere. Protein expression was measured by Western blot
analysis (a). Quantitative analysis of Homer 1a (b), p-Erk (c), and cleaved-capsase3 (d). TUNEL assay of apoptosis (e) scored as apoptotic index (AI positive apoptotic cells/total cells 9 100 %) (f). Scale bars 50 lm. The data are presented as mean ± SEM. One-way ANOVA followed by a Bonferroni’s test, n = 4 for each group, * P \ 0.05 versus control
Fig. 2 Upregulated Homer1a and p-Erk might be involved in the loss of RGCs in I/R-injured retinas. C57BL/6J mice were subjected to retinal ischemia injury for 60 min followed with reperfusion at different times (1, 6, 12, 24, 48, 72 h, and 7 days). The protein
expression of Homer1a (a, b) and p-Erk (a, c) was measured by Western blot analysis. The data are presented as mean ± SEM. Oneway ANOVA followed by a Bonferroni’s test, n = 8 for each group, * P \ 0.05 versus control
followed by OGD injury, demonstrating that Homer1a might be involved in regulating Erk-mediated RGCs apoptosis. However, no significant difference of RGCs apoptosis between Flag-Homer1a ? Vehicle and Flag-Homer1a ? PD98059 group after OGD injury (Fig. 4d, e).
Knockout of Homer1a Gene Increased the Loss of RGCs in I/R-Injured Retinas The potential role of Homer1a in vivo was tested using Homer1a gene knockout mice. Genotype analysis using agarose gel was performed to determine the genotype of
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Fig. 3 Inhibition of Erk increased the expression of Homer1a and mitigated RGCs apoptosis after OGD injury. After pretreatment with Erk inhibitor (PD98059, 40 lM) for 1 h, the RGCs were exposed to OGD for 6 h and reperfused for 24 h. Protein expression was measured by Western blot analysis (a). Quantitative analysis of Homer 1a (b), p-Erk (c), and cleaved-capsase3 (d); TUNEL assay of
apoptosis scored as apoptotic index. AI positive apoptotic cells/total cells 9 100 % (e). The data are presented as mean ± SEM. One-way ANOVA followed by a Bonferroni’s test, n = 4 for each group, *P \ 0.05 versus control, #P \ 0.05 versus vehicle ? 24 h postOGD group
Fig. 4 Homer1a mitigated OGD-induced RGCs apoptosis and interacted with Erk. RGCs were transfected with lentiviral vector (ScrRNAi), lentivirus-mediated Homer1a shRNA (Homer1a-RANi) or 3Flag-tagged Homer1a (Flag-Homer1a) separately. After 48 h of transfection, RGCs were pretreated with vehicle (0.5 % DMSO) or Erk inhibitor (PD98059) for 1 h, and subjected to OGD injury. Protein expression was measured by Western blot analysis (a). Quantitative analysis of Homer 1a (b) (29 KD, endogenous protein
band; 32 KD, exogenous protein band, Homer 1a-3-Flag expression cassette vector), p-Erk (c), and cleaved-capsase3 (d). Apoptosis was measured using TUNEL assay (e). The data are presented as mean ± SEM. Two-way ANOVA followed by a Tukey’s test, n = 4 for each group, *P \ 0.05 versus Scr-RNAi ? vehicle ? 24 h post-OGD group, #P \ 0.05 versus Homer1a-RNAi ? vehicle ? 24 h post-OGD group
the mice (Fig. 5a) (Hu et al. 2010). Retinal flat-mounts and b-III-tubulin staining were used to evaluate the loss of RGCs in the retinas of Homer1a WT and KO mice, which
were both subjected to retinal I/R injury. The densities of dendrites and RGCs in the retinas ipsilateral to injury were significantly lower in both WT and KO mice on 21th day
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after retinal I/R injury (P \ 0.05, Fig. 5b, c), compared with that of contralateral retinas. Furthermore, a significantly increased loss of dendrites and RGCs was observed in retinas ipsilateral to injury in Homer1a KO mice (P \ 0.05) compared with Homer1a WT mice. However, there was no significant difference in contralateral retinas between the Homer1a WT and KO groups. Therefore, Homer1a might protect RGCs from retinal I/R injury.
Retinal I/R causes irreversible pathological damage to RGCs, especially affecting the inner retinal neurons (Hughes 1991; Dvoriantchikova et al. 2014). In the present study, using both OGD in vitro and I/R injury in vivo, we studied the changes and effects of Homer1a after retinal I/R injury, demonstrating that Homer1a may be involved in the pathogenesis of retinal I/R injury and that it protects RGCs from I/R injury via interaction with the Erk pathway. Homer signaling plays important roles in the pathogenesis of ocular diseases and other neurological disorders, such as TBI (Luo et al. 2012b; Menard and Quirion 2012; Kaja et al. 2014). In this study, we observed that the expression of Homer1a protein was significantly elevated in primary cultured RGCs after OGD injury and in the retinas subjected to I/R injury, consistent with our previous study involving TBI (Luo et al. 2014). Our results were partially inconsistent with those of Kaja et al., who found no
significant difference between Homer1a expression in the retinas from 9-month-old DBA/2J mice and 6-week old DBA/2J mice, but Homer 1a was moderately correlated with IOP (Kaja et al. 2014). The inconsistency of Homer1a expression might partially be attributed to different models and observation times. Kaja et al.’s study focused on chronic changes, while our study observed a significant increase of Homer1a expression after retinal I/R injury for a maximum of 7 days. Additionally, the different stages of the disease may also have contributed to the inconsistencies. For instance, different levels of Homer1a expression may occur in the acute and chronic stages of the disease (Kaja et al. 2014). Furthermore, related studies suggest that Homer1a was highly expressed in a premature stage and gradually declined with aging, suggesting that the expression of Homer1a was age-specific in the development of the nervous system (Bek et al. 2009; Colucciello 2004; Kammermeier and Worley 2007; Kaja et al. 2003). The age-specific expression may partially explain the non-significant difference in the increase of Homer1a expression between 9 months and 6 weeks in glaucomatous DBA/2J mice (Kaja et al. 2014). Given the key role of Homer1a in several neurological conditions (Ronesi et al. 2012; Luo et al. 2014; Luo et al. 2012a), it is imperative to investigate its role in promoting RGCs survival. Herein, we found that upregulation of Homer1a in RGCs decreased OGD-induced RGCs apoptosis. Conversely, downregulation of Homer1a increased apoptosis after OGD treatment. These findings were
Fig. 5 Knockout of Homer1a gene aggravated the loss of dendrites and RGCs in I/R-injured retinas. Agarose gels were used for genotype analysis of Homer1a mutant mice. The top gel was amplified with the Homer1a WT primer and the bottom gel was amplified with Homer1a KO primer (a). After 21 days of treatment, the retinas of Homer1a
WT and KO mice were stained for b-III-tubulin (Tuj1, green) and nuclei (Hochest, blue) (b). Quantitative analysis of RGCs density (c); scale bars 50 lm. The data are presented as mean ± SEM. Two-way ANOVA followed by a Tukey’s test, WT, n = 7; KO, n = 8, *P \ 0.05 (Color figure online)
Discussion
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consistent with our previous work on TBI and oxidative injury (Luo et al. 2014; Luo et al. 2012a). Furthermore, in a retinal I/R injury model, a significant increase in the loss of dendrites and RGCs was found in Homer1a KO mice, compared with those of Homer1a WT mice. Taken together, these results indicate that Homer1a might promote RGCs survival both in vitro and in vivo and be a potential endogenous anti-apoptotic agent in RGCs. Classic Erk signaling in the nervous system was strongly associated with retinal injury and cell survival. Previous studies have suggested that p-Erk was upregulated in injured retinas and inhibition of Erk activation provided significant protection of RGCs against ischemic damage and axotomy (Roth et al. 2003; Gao et al. 2014; Galan et al. 2014; Luo et al. 2007). Consistent with previous studies, we also observed a significant increase of p-Erk expression after OGD injury, accompanied with increased expression of Homer1a, both in vivo and in vitro. Inhibition of Erk activation mitigated OGD-induced RGCs apoptosis. Homer1a, as an endogenic neuroprotective protein, was upregulated in neurons by some insults, like glutamate, trauma, and oxidative stress (Luo et al. 2012a; Luo et al. 2014; Luo et al. 2012b). Similar changes of Homer1a and p-Erk were also evident in our previous study in PC12 cells (Luo et al. 2012b). Additionally, inverse correlation between Homer1a and Erk was also observed in OGD-induced RGCs injury in vitro. Inhibition of Erk increased the expression of Homer1a and reduced the injured effect of Homer1a downregulation in OGD-induced RGCs apoptosis, similar to a previous brain study (Luo et al. 2012b). Furthermore, downregulation of Homer1a was associated with an increase in the level of p-Erk and reduced the protective effect of Erk inhibitor against OGD-induced RGCs apoptosis. Upregulation of Homer1a was associated with a decrease in the level of p-Erk but had no effect on the protective effect of Erk inhibitor against OGD-induced RGCs apoptosis. The association between Homer1a and Erk may be causative and not coincidental. Previous studies have demonstrated that Homer1 proteins coordinated mGluRs-Erk signaling under various conditions (Tappe-Theodor et al. 2011; Ronesi et al. 2012; Mao et al. 2005). Homer1a has been thought to downregulate the increase in the intracellular Ca2? response and negatively regulate the level of p-Erk (Curran and Orman 2009; Chen et al. 2012; Obara et al. 2013), both of which are associated with the regulation of mGluRs function (Kammermeier and Worley 2007; Tu et al. 1998; Tappe-Theodor et al. 2011; Ronesi et al. 2012). Our previous study suggested that intracellular Ca2? overload contributed to the mGluR1-induced activation of Erk after TBI. Our study also suggested that the upregulation of Homer1a disrupted mGluR1-Erk signaling and inhibited the level of p-Erk by inhibiting the release of intracellular Ca2? in response to neuronal injury
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(Chen et al. 2012; Luo et al. 2012a; Luo et al. 2014). Thus, the role of Homer 1a and intracellular Ca2? mobilization may contribute to its interaction with the Erk pathway. Nevertheless, the underlying mechanisms involving Homer1a and Erk interactions need to be elucidated to better understand the pathophysiology of I/R injury in RGCs. We conclude that Homer1a is an endogenous protective gene that could potentially be used therapeutically in RGCs I/R injury and related diseases. However, the underlying mechanisms of protection including the interaction of Homer1a with Erk, and additional detailed mechanisms mediating RGCs I/R injury need further elucidation (Duncan et al. 2005; Hwang et al. 2003; Tanaka et al. 2006; Westhoff et al. 2003; Kaja et al. 2014). Acknowledgments We express our thanks to Professors Bo Xiao and Paul Worley for providing the Homer1a KO mice. This study was supported by the National Natural Science Foundation of China (Nos. 81430043 and 30930093), the National Science and Technology Major Project of China (2013ZX 09J13109-02C), the National Science and Technology Pillar Program of China (No. 2012BAI11B02), and the Science and Technology Project of Shaanxi (No. 2013KTCQ03-01). Conflicts of interest of interest.
The authors declare that they have no conflicts
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ORIGINAL RESEARCH
Upregulation of Homer1a Promoted Retinal Ganglion Cell Survival After Retinal Ischemia and Reperfusion via Interacting with Erk Pathway Fei Fei1 • Juan Li2 • Wei Rao2 • Wenbo Liu2 • Xiaoyan Chen2 • Ning Su2 Yusheng Wang1 • Zhou Fei2
•
Received: 13 January 2015 / Accepted: 16 April 2015 Ó Springer Science+Business Media New York 2015
Abstract Retinal ischemia and reperfusion (I/R) is extensively involved in ocular diseases, causing retinal ganglion cell (RGCs) death resulting in visual impairment and blindness. Homer1a is considered as an endogenous neuroprotective protein in traumatic brain injury. However, the roles of Homer1a in RGCs I/R injury have not been elucidated. The present study investigated the changes in expression and effect of Homer1a in RGCs both in vitro and in vivo after I/R injury using Western blot, TUNEL assay, gene interference and overexpression, and gene knockout procedures. The levels of Homer1a and phosphorylated Erk (p-Erk) increased in RGCs and retinas after I/R injury. Upregulation of Homer1a in RGCs after I/R injury decreased the level of p-Erk, and mitigated RGCs apoptosis. Conversely, downregulation of Homer1a increased the level of p-Erk, and augmented RGCs apoptosis. Furthermore, inhibition of the p-ERK reduced RGCs apoptosis, and increased the expression of Homer 1a after I/R injury. Finally, the retinas of Homer1a KO mice treated with I/R injury had significantly less dendrites and RGCs, compared with Homer1a WT mice. These findings demonstrated that
Homer1a may contribute to RGCs survival after I/R injury by interacting with Erk pathway.
Keywords Homer1a Erk signaling Retinal ischemia and reperfusion Retinal ganglion cells Abbreviations I/R Ischemia and reperfusion RGC Retinal ganglion cell p-Erk Phosphorylated Erk IOP Intraocular pressure mGluRs Metabotropic glutamate receptors CC Coiled-coil WT Wild type KO Knockout PBS Phosphate-buffered saline shRNA Short hairpin RNA OGD Oxygen and Glucose Deprivation TBI Traumatic brain injury TUNEL TdT-mediated dUTP nick end-labeling
Fei Fei, Juan Li and Wei Rao have contributed equally to this work. & Yusheng Wang [email protected]
Introduction
& Zhou Fei [email protected]
Ischemia/reperfusion (I/R) causes irreversible RGCs apoptosis (Zhang et al. 2002) or necrosis (Joo et al. 1999) in the retina, which are linked to many retinal disorders including glaucoma, traumatic optic neuropathy, anterior ischemic optic neuropathy, and diabetic retinopathy. In animal models of retinal I/R induced by elevated intraocular pressure (IOP), an increased number of necrotic and apoptotic nuclei in the inner retina have been observed. I/R
1
Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi’an 71032, People’s Republic of China
2
Department of Neurosurgery, Xijing Hospital, Fourth Military Medical University, 15 Changle Xi Road, Xi’an 710032, People’s Republic of China
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injury due to dysfunctional autoregulation of retinal bloodflow, oxidative stress and aberrant immunity, is extensively associated with RGCs death and optic nerve damage (Dreyer et al. 1996; Liu and Neufeld 2001; Schwartz 2003; Tezel et al. 1999; Yan et al. 2000; Yoles et al. 2001). Therefore, it is necessary to elucidate the etiology of ocular diseases and find new neuroprotective agents that can protect RGCs from I/R injury (Hughes 1991). Homer1c is a key moiety related to neurodegeneration in a mild retinal ischemia rat model (Kaja et al. 2003). The Homer protein family consists of two major groups: longform proteins, including Homer1b/c, Homer2 and Homer3, which are constitutively expressed in neurons and myocytes (Duncan et al. 2005), and short variant Homer1a, which is induced by elevated synaptic activity due to alternate splicing or premature termination of transcription (Kato et al. 1998; Shiraishi et al. 1999; Sun et al. 1998; Xiao et al. 1998; Soloviev et al. 2000). Although all variants have a highly conserved EVH-1 domain within their N-terminal regions, differences in C-terminal regions regulate postsynaptic structure and signal transduction. Specifically, both Homer1b/c and Homer1a are important adaptors of group I metabotropic glutamate receptors (mGluRs). With a C-terminal coiled-coil (CC) domain, Homer1b/c assembles into multiprotein complexes with mGluRs accompanied by other molecules, and facilitates downstream signal transduction. Homer1a, which lacks the CC domain, competitively binds to target proteins and disrupts the formation of mGluRs complexes (Shiraishi-Yamaguchi and Furuichi 2007). Homer proteins are associated with several neuronal diseases, including neuropathic pain, fragile X syndrome (Ronesi et al. 2012), and stress-related cognitive deficits (Wagner et al. 2013; Miletic et al. 2005; Miletic et al. 2009; Miyabe et al. 2006; Obara et al. 2013; Yao et al. 2011). Our previous studies showed that Homer1a and Homer1b/c play an important role in the development of traumatic brain injury (TBI) via regulation of group I mGluRs (Luo et al. 2014; Fei et al. 2014). Homer1 proteins were also involved in the regulation of neuronal injury in a model of inflammation (Luo et al. 2012b) and oxidative stress (Luo et al. 2012a). In this study, we found that downregulation of Homer1a correlated with the loss of RGCs in I/R-injured retinas. Therefore, we investigated the role of Homer1a protein in the pathology of retinal I/R injury using primary cultured RGCs and a retina ischemia injury model.
Materials and Methods Animal Models Male C57BL/6 J mice (10–12 weeks) and post-natal 4- to 6-day pups were obtained from the Laboratory Animal
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Center of the Fourth Military Medical University. The mice had continuous access to food and water, and were caged at a temperature of 20–22 °C with a 12:12 h light–dark cycle. Homer1a KO mice were kindly provided by Paul Worley (Hu et al. 2010; Mahan et al. 2012). We crossed heterozygous Homer1a mice (Het, Homer1a?/-) to generate wild type (WT, Homer1a?/?) and knockout (KO, Homer1a-/-) littermates. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Briefly, mice were anesthetized by an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine (SigmaAldrich, St. Louis, MO, USA). The corneas were topically anesthetized with 0.5 % tetracaine hydrochloride, and the pupils were dilated with 1 % tropicamide (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The right eyeball of each mouse was cannulated with a 30-gauge needle connected to a saline reservoir to produce and maintain an IOP of 110 mmHg for 60 min. Successful ischemia was confirmed by the ophthalmoscopic observation of whitened iris and loss of the red reflex under microscopy. With subsequent reperfusion the return of the red reflex was confirmed. The contralateral eye was cannulated and maintained at a normal level of IOP to serve as a normotensive control. All the mice were allowed to recover for 21 days after I/R. Tissue Procurement and Retinal Flat-Mounts After treatment for 21 days, all the mice were humanely sacrificed by transcardial perfusion with 4 % paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (Gibco, Gaithersburg, MD, USA) under general anesthesia with an intraperitoneal injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. Retinas from both ipsilateral and contralateral eyeballs were dissected, post-fixed in 2 % paraformaldehyde (pH 7.4) for 1 h and washed with 0.1 M PBS (pH 7.4). RGCs density was quantified in the retinal flat-mounts as previously described (Dvoriantchikova et al. 2010; Zhang et al. 2011). Briefly, retinas were incubated at 4 °C overnight with an anti-Tuj1 antibody (Sigma-Aldrich, St. Louis, MO, USA), which was specifically bound to a RGCs-specific marker, b-III-tubulin, and then incubated with an Alexa Fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA). The retinas were mounted flat on pre-coated glass slides. b-IIItubulin-positive RGCs were observed in flat-mounted retinas using a 960 epifluorescent microscope containing a motorized X–Y–Z stage (Provis AX70; Olympus, Tokyo, Japan). A total of 16 images from four quadrants in each
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retina were obtained at four distances from the optic disk. After adjusting the image threshold levels and manually excluding artifacts, the loss of RGCs in the ischemic retinas was calculated as the mean cell density (RGCs number/corresponding areas) in normotensive control eyes. Primary Culture of RGCs RGCs were isolated according to the two-step immunopanning method, as previously described (Ivanov et al. 2006; Park et al. 2015; Dvoriantchikova et al. 2014). Briefly, the C57BL/6J pups were euthanized and the retinas were removed, mechanically dissected, minced, and incubated in papain solution (15 U/ml, Worthington, Lakewood, NJ, USA) for 30 min. The macrophages were removed from the cell suspension by panning with the antimacrophage antiserum (Accurate Chemical, Westbury, NY, USA). RGCs were specifically bound to the panning plates containing anti-Thy1.2 antibody. After washing with PBS, adherent cells were dissociated by incubation with trypsin (Gibco, Gaithersburg, MD, USA) and mechanically aspirating with a pipette. Finally, RGCs were seeded onto 35, 60-mm petri dishes, and 6-well plates treated with a poly-D-lysine (Sigma-Aldrich, St. Louis, MO, USA) Neurobasal/2 % B27 supplement medium (Gibco, Gaithersburg, MD, USA) and cultured for the following experiments. Lentiviral Design for Homer1a RNAi and Overexpression Short hairpin RNA (shRNA) lentiviruses were designed by subcloning an oligo siRNA targeting the coding region of 1190–1208 bp into a lentiviral expression vector, pFUGW-RNAi (GeneChem Company, Shanghai, China). Following digestion and ligation, clones were selected and verified for the inserted sequence. The lentiviral constructco-expressed EGFP was driven by the ubiquitin C promoter, and shRNA was driven by the U6 promoter. To control for the off-target and non-specific effects of shRNA, a negative control shRNA (50 -TTCTCCG AACGTGTCACGT-30 ) was used. The lentiviral overexpression system was developed by removing the EGFP open reading frame from the pGC-FU-EGFP-3FLAG construct (GeneChem Company, Shanghai, China) using an AgeI/NheI digestion and replacing it with the cDNA of Homer1a (NM_011982.3). Oxygen and Glucose Deprivation (OGD) of RGCs OGD was induced by replacing the normal RGCs media with glucose- and sodium pyruvate-free Neurobasal media (Gibco, Gaithersburg, MD, USA). Primary cultured RGCs
were deprived of oxygen (0–0.1 % O2, 5 % CO2, and 95 % N2) using a ProOx model C21 hypoxia chamber (BioSpherix, Redfield, NY, USA) for 6 h at 37 °C. After OGD, the culture medium was substituted with fresh Neurobasal/B27 media, and the RGCs were incubated for 6, 12, and 24 h in a 5 % CO2 atmosphere. Parallel cultures were exposed to oxygenated Neurobasal/B27 media in an incubator under normoxic atmosphere (37 °C; 5 % CO2) as controls. Fluorometric TUNEL (TdT-mediated dUTP Nick End-Labeling) Analysis Apoptosis was analyzed using the DeadEndTM Fluorometirc TUNEL system (Promega, WI, USA) according to the manufacturer’s protocol (Fei et al. 2014). Hoechst (SigmaAldrich, MO, USA) staining was performed to assess the total number of cells per well. RGCs were observed immediately under a fluorescence microscope (Leica, Germany) using a standard fluorescein filter set. Green nuclei were positive for apoptotic cells and blue nuclei were an indicator of total non-apoptotic RGCs. An apoptotic index (AI = positive apoptotic cells/total cells 9 100 %) was used to quantify the level of apoptosis. At least 10 highpower fields (40X)/well were observed. Western Blot After various treatments, the RGCs or mice retinas were lysed with the radioimmunoprecipitation assay buffer (Pierce, Rockford, IL, USA) supplied with protease inhibitors (Roche Applied Bioscience, Indianapolis, IN, USA). Protein concentration was quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein were loaded onto a 10 % SDS-PAGE gel (Beyotime, Shanghai, China). After electrophoresis, the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5 % skim milk and incubated at 4 °C overnight with the appropriate primary antibodies: anti-Homer1a (1:100; Santa Cruz Biotech, Santa Cruz, CA, USA), anti-Caspase3, and anti-cleaved-caspase3, and antip-Erk (1:1000; CST, Danvers, MA, USA). After washing, the immunoreactivity of the membrane was determined by incubated it with the corresponding horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA) followed by enhanced chemical luminescence development (Pierce, Rockford, IL, USA). The expression of b-actin was used to standardize protein loading. Western blotting analysis was performed in triplicate for each protein and the optical densities of the bands were calculated using Image Analysis Software (Bio-Rad, Los Angeles, CA, USA).
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Statistical Analysis Data are expressed as the mean ± standard error of the mean. Statistical significance was tested using ANOVA followed by a Bonferroni’s test using SPSS 22.0 (IBM, Armonk, New York, USA). Data were defined as significantly different when P \ 0.05. The use of additional statistics is explained in the text as needed.
Results Increased Expression of Homer1a and p-Erk in OGD-Induced RGCs Apoptosis As shown in Fig. 1a and b, compared with that of control group (no OGD treatment), the expression of Homer1a significantly increased 6, 12, or 24 h after OGD (P \ 0.05). We also observed a similar change in the expression of p-Erk (Fig. 1a, c). To evaluate OGD-induced RGCs apoptosis, a TUNEL assay was performed and the level of cleaved-caspase3 was measured. We found that the expression of cleaved-caspase3 was significantly higher (P \ 0.05) at periods of 6, 12, and 24 h after OGD treatment and peaked at 24 h after OGD, compared with that of the control group (Fig. 1a, d). Similarly, the TUNEL assay also showed that the number of apoptotic cells significantly increased after OGD treatment for 12 and 24 h (P \ 0.05) but not after 6 h (Fig. 1e, f). Ischemia for 6 h and reperfusion for 24 h, induced a higher level of Homer1a expression and apoptosis, and therefore, these time points were used in the following experiments. Upregulated Homer1a and p-Erk were Associated with the Loss of RGCs in I/R-Injured Retinas The expression of Homer1a was measured in vivo in I/Rinjured retinas. The expression of Homer1a and p-Erk in ischemic retinas was detected as early as 1 h after I/R. The expression of Homer1a significantly (P \ 0.05) increased at 12 h, peaked at 48 h, and then declined after the 7th day following I/R (Fig. 2a, b). The expression of p-Erk also significantly increased at 12 h (P \ 0.05) and declined to the physiological level at 72 h after I/R (Fig. 2a, c). These findings suggested that Homer1a might also be involved in the loss of RGCs in vivo. Inhibiting Erk Activation Increased the Expression of Homer1a and Mitigated RGCs Apoptosis After OGD To determine whether Erk activation affected RGCs apoptosis and the expression of Homer1a, RGCs were
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pretreated with 40 lM PD98059 (Sigma-Aldrich, St. Louis, MO, USA), a widely used Erk inhibitor, for 1 h followed by OGD. OGD-induced Erk activation was inhibited by PD98059 after OGD treatment for 24 h (Fig. 3a, c). Furthermore, the inhibition of Erk activation correlated with the increased expression of Homer1a (Fig. 3a, b), suggesting that Erk pathway might regulate the expression of Homer1a. Additionally, compared with vehicle groups, inhibition of Erk activation significantly (P \ 0.05) decreased the cleavage level of caspase3 (Fig. 3a, d) and TUNEL-positive cells (Fig. 3e), demonstrating that Erk activation mediated OGD-induced RGCs apoptosis and that inhibition of Erk promoted RGCs survival.
Homer1a Mitigated OGD-Induced RGCs Apoptosis and Interacted with Erk To evaluate the effects of Homer1a on RGCs survival, lentivirus-mediated genetic interventions were used to regulate the expression of Homer1a in RGCs. Endogenous expression of Homer1a was significantly downregulated by a lentiviral shRNA targeting Homer1a (Homer1a-RNAi) 24 h after OGD (P \ 0.05, Fig. 4a, b). Exogenous expression of Homer1a was significantly upregulated by lentiviral transduction and induced expression of 3-Flagtagged Homer1a (Flag-Homer1a) (P \ 0.05), compared with the expression of Homer1a in the vector group (ScrRNAi). Downregulation of Homer1a significantly increased the level of p-Erk (Fig. 4a, c), cleaved caspase3 (Fig. 4a, d) and stimulated apoptosis of RGCs (Fig. 4e) after OGD for 24 h (P \ 0.05). Further, upregulation of Homer1a decreased the level of p-Erk (Fig. 4a, c) and protected RGCs from OGD injury by significantly mitigating apoptosis (Fig. 4a, d, e) 24 h after OGD treatment (P \ 0.05). These results suggested that Homer1a might be an important endogenous protective protein in vitro. We investigated the potential interaction between Homer1a and Erk using Erk inhibitor 48 h after Homer1alentivirus infection. Inhibition of Erk increased OGD-induced expression of Homer1a in Scr-RNAi ? PD98059 group compared with that of the Scr-RNAi ? Vehicle group (Fig. 4a, b) 24 h after OGD injury, which was consistent with the results above. No significant difference of expression of Homer1a was observed between Homer1a-RNAi ? Vehicle group and the Homer1aRNAi ? PD98059 group, or between the Flag-Homer1a ? Vehicle group and Flag-Homer1a ? PD98059 group. Compared with that of the Homer1a-RNAi ? Vehicle group, inhibition of Erk significantly decreased the level of cleaved-capase3 (Fig. 4a, d) and reduced RGCs apoptosis (Fig. 4e) induced by Homer1a downregulation
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Fig. 1 OGD-induced the expression of Homer 1a, p-Erk and apoptosis in RGCs. Primary cultured RGCs were treated with glucose-free Neurobasal and deprived of oxygen (0–0.1 % O2, 5 % CO2, and 95 % N2) using a hypoxia chamber for 6 h at 37 °C. After OGD, the culture medium was exchanged for fresh Neurobasal/B27 media, and the RGCs were incubated for 6, 12, and 24 h in a 5 % CO2 atmosphere. Protein expression was measured by Western blot
analysis (a). Quantitative analysis of Homer 1a (b), p-Erk (c), and cleaved-capsase3 (d). TUNEL assay of apoptosis (e) scored as apoptotic index (AI positive apoptotic cells/total cells 9 100 %) (f). Scale bars 50 lm. The data are presented as mean ± SEM. One-way ANOVA followed by a Bonferroni’s test, n = 4 for each group, * P \ 0.05 versus control
Fig. 2 Upregulated Homer1a and p-Erk might be involved in the loss of RGCs in I/R-injured retinas. C57BL/6J mice were subjected to retinal ischemia injury for 60 min followed with reperfusion at different times (1, 6, 12, 24, 48, 72 h, and 7 days). The protein
expression of Homer1a (a, b) and p-Erk (a, c) was measured by Western blot analysis. The data are presented as mean ± SEM. Oneway ANOVA followed by a Bonferroni’s test, n = 8 for each group, * P \ 0.05 versus control
followed by OGD injury, demonstrating that Homer1a might be involved in regulating Erk-mediated RGCs apoptosis. However, no significant difference of RGCs apoptosis between Flag-Homer1a ? Vehicle and Flag-Homer1a ? PD98059 group after OGD injury (Fig. 4d, e).
Knockout of Homer1a Gene Increased the Loss of RGCs in I/R-Injured Retinas The potential role of Homer1a in vivo was tested using Homer1a gene knockout mice. Genotype analysis using agarose gel was performed to determine the genotype of
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Fig. 3 Inhibition of Erk increased the expression of Homer1a and mitigated RGCs apoptosis after OGD injury. After pretreatment with Erk inhibitor (PD98059, 40 lM) for 1 h, the RGCs were exposed to OGD for 6 h and reperfused for 24 h. Protein expression was measured by Western blot analysis (a). Quantitative analysis of Homer 1a (b), p-Erk (c), and cleaved-capsase3 (d); TUNEL assay of
apoptosis scored as apoptotic index. AI positive apoptotic cells/total cells 9 100 % (e). The data are presented as mean ± SEM. One-way ANOVA followed by a Bonferroni’s test, n = 4 for each group, *P \ 0.05 versus control, #P \ 0.05 versus vehicle ? 24 h postOGD group
Fig. 4 Homer1a mitigated OGD-induced RGCs apoptosis and interacted with Erk. RGCs were transfected with lentiviral vector (ScrRNAi), lentivirus-mediated Homer1a shRNA (Homer1a-RANi) or 3Flag-tagged Homer1a (Flag-Homer1a) separately. After 48 h of transfection, RGCs were pretreated with vehicle (0.5 % DMSO) or Erk inhibitor (PD98059) for 1 h, and subjected to OGD injury. Protein expression was measured by Western blot analysis (a). Quantitative analysis of Homer 1a (b) (29 KD, endogenous protein
band; 32 KD, exogenous protein band, Homer 1a-3-Flag expression cassette vector), p-Erk (c), and cleaved-capsase3 (d). Apoptosis was measured using TUNEL assay (e). The data are presented as mean ± SEM. Two-way ANOVA followed by a Tukey’s test, n = 4 for each group, *P \ 0.05 versus Scr-RNAi ? vehicle ? 24 h post-OGD group, #P \ 0.05 versus Homer1a-RNAi ? vehicle ? 24 h post-OGD group
the mice (Fig. 5a) (Hu et al. 2010). Retinal flat-mounts and b-III-tubulin staining were used to evaluate the loss of RGCs in the retinas of Homer1a WT and KO mice, which
were both subjected to retinal I/R injury. The densities of dendrites and RGCs in the retinas ipsilateral to injury were significantly lower in both WT and KO mice on 21th day
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after retinal I/R injury (P \ 0.05, Fig. 5b, c), compared with that of contralateral retinas. Furthermore, a significantly increased loss of dendrites and RGCs was observed in retinas ipsilateral to injury in Homer1a KO mice (P \ 0.05) compared with Homer1a WT mice. However, there was no significant difference in contralateral retinas between the Homer1a WT and KO groups. Therefore, Homer1a might protect RGCs from retinal I/R injury.
Retinal I/R causes irreversible pathological damage to RGCs, especially affecting the inner retinal neurons (Hughes 1991; Dvoriantchikova et al. 2014). In the present study, using both OGD in vitro and I/R injury in vivo, we studied the changes and effects of Homer1a after retinal I/R injury, demonstrating that Homer1a may be involved in the pathogenesis of retinal I/R injury and that it protects RGCs from I/R injury via interaction with the Erk pathway. Homer signaling plays important roles in the pathogenesis of ocular diseases and other neurological disorders, such as TBI (Luo et al. 2012b; Menard and Quirion 2012; Kaja et al. 2014). In this study, we observed that the expression of Homer1a protein was significantly elevated in primary cultured RGCs after OGD injury and in the retinas subjected to I/R injury, consistent with our previous study involving TBI (Luo et al. 2014). Our results were partially inconsistent with those of Kaja et al., who found no
significant difference between Homer1a expression in the retinas from 9-month-old DBA/2J mice and 6-week old DBA/2J mice, but Homer 1a was moderately correlated with IOP (Kaja et al. 2014). The inconsistency of Homer1a expression might partially be attributed to different models and observation times. Kaja et al.’s study focused on chronic changes, while our study observed a significant increase of Homer1a expression after retinal I/R injury for a maximum of 7 days. Additionally, the different stages of the disease may also have contributed to the inconsistencies. For instance, different levels of Homer1a expression may occur in the acute and chronic stages of the disease (Kaja et al. 2014). Furthermore, related studies suggest that Homer1a was highly expressed in a premature stage and gradually declined with aging, suggesting that the expression of Homer1a was age-specific in the development of the nervous system (Bek et al. 2009; Colucciello 2004; Kammermeier and Worley 2007; Kaja et al. 2003). The age-specific expression may partially explain the non-significant difference in the increase of Homer1a expression between 9 months and 6 weeks in glaucomatous DBA/2J mice (Kaja et al. 2014). Given the key role of Homer1a in several neurological conditions (Ronesi et al. 2012; Luo et al. 2014; Luo et al. 2012a), it is imperative to investigate its role in promoting RGCs survival. Herein, we found that upregulation of Homer1a in RGCs decreased OGD-induced RGCs apoptosis. Conversely, downregulation of Homer1a increased apoptosis after OGD treatment. These findings were
Fig. 5 Knockout of Homer1a gene aggravated the loss of dendrites and RGCs in I/R-injured retinas. Agarose gels were used for genotype analysis of Homer1a mutant mice. The top gel was amplified with the Homer1a WT primer and the bottom gel was amplified with Homer1a KO primer (a). After 21 days of treatment, the retinas of Homer1a
WT and KO mice were stained for b-III-tubulin (Tuj1, green) and nuclei (Hochest, blue) (b). Quantitative analysis of RGCs density (c); scale bars 50 lm. The data are presented as mean ± SEM. Two-way ANOVA followed by a Tukey’s test, WT, n = 7; KO, n = 8, *P \ 0.05 (Color figure online)
Discussion
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consistent with our previous work on TBI and oxidative injury (Luo et al. 2014; Luo et al. 2012a). Furthermore, in a retinal I/R injury model, a significant increase in the loss of dendrites and RGCs was found in Homer1a KO mice, compared with those of Homer1a WT mice. Taken together, these results indicate that Homer1a might promote RGCs survival both in vitro and in vivo and be a potential endogenous anti-apoptotic agent in RGCs. Classic Erk signaling in the nervous system was strongly associated with retinal injury and cell survival. Previous studies have suggested that p-Erk was upregulated in injured retinas and inhibition of Erk activation provided significant protection of RGCs against ischemic damage and axotomy (Roth et al. 2003; Gao et al. 2014; Galan et al. 2014; Luo et al. 2007). Consistent with previous studies, we also observed a significant increase of p-Erk expression after OGD injury, accompanied with increased expression of Homer1a, both in vivo and in vitro. Inhibition of Erk activation mitigated OGD-induced RGCs apoptosis. Homer1a, as an endogenic neuroprotective protein, was upregulated in neurons by some insults, like glutamate, trauma, and oxidative stress (Luo et al. 2012a; Luo et al. 2014; Luo et al. 2012b). Similar changes of Homer1a and p-Erk were also evident in our previous study in PC12 cells (Luo et al. 2012b). Additionally, inverse correlation between Homer1a and Erk was also observed in OGD-induced RGCs injury in vitro. Inhibition of Erk increased the expression of Homer1a and reduced the injured effect of Homer1a downregulation in OGD-induced RGCs apoptosis, similar to a previous brain study (Luo et al. 2012b). Furthermore, downregulation of Homer1a was associated with an increase in the level of p-Erk and reduced the protective effect of Erk inhibitor against OGD-induced RGCs apoptosis. Upregulation of Homer1a was associated with a decrease in the level of p-Erk but had no effect on the protective effect of Erk inhibitor against OGD-induced RGCs apoptosis. The association between Homer1a and Erk may be causative and not coincidental. Previous studies have demonstrated that Homer1 proteins coordinated mGluRs-Erk signaling under various conditions (Tappe-Theodor et al. 2011; Ronesi et al. 2012; Mao et al. 2005). Homer1a has been thought to downregulate the increase in the intracellular Ca2? response and negatively regulate the level of p-Erk (Curran and Orman 2009; Chen et al. 2012; Obara et al. 2013), both of which are associated with the regulation of mGluRs function (Kammermeier and Worley 2007; Tu et al. 1998; Tappe-Theodor et al. 2011; Ronesi et al. 2012). Our previous study suggested that intracellular Ca2? overload contributed to the mGluR1-induced activation of Erk after TBI. Our study also suggested that the upregulation of Homer1a disrupted mGluR1-Erk signaling and inhibited the level of p-Erk by inhibiting the release of intracellular Ca2? in response to neuronal injury
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(Chen et al. 2012; Luo et al. 2012a; Luo et al. 2014). Thus, the role of Homer 1a and intracellular Ca2? mobilization may contribute to its interaction with the Erk pathway. Nevertheless, the underlying mechanisms involving Homer1a and Erk interactions need to be elucidated to better understand the pathophysiology of I/R injury in RGCs. We conclude that Homer1a is an endogenous protective gene that could potentially be used therapeutically in RGCs I/R injury and related diseases. However, the underlying mechanisms of protection including the interaction of Homer1a with Erk, and additional detailed mechanisms mediating RGCs I/R injury need further elucidation (Duncan et al. 2005; Hwang et al. 2003; Tanaka et al. 2006; Westhoff et al. 2003; Kaja et al. 2014). Acknowledgments We express our thanks to Professors Bo Xiao and Paul Worley for providing the Homer1a KO mice. This study was supported by the National Natural Science Foundation of China (Nos. 81430043 and 30930093), the National Science and Technology Major Project of China (2013ZX 09J13109-02C), the National Science and Technology Pillar Program of China (No. 2012BAI11B02), and the Science and Technology Project of Shaanxi (No. 2013KTCQ03-01). Conflicts of interest of interest.
The authors declare that they have no conflicts
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