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reperfusion injury
Neurobiology of Disease 93 (2016) 121–128
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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi
Calpain-1 and calpain-2 play opposite roles in retinal ganglion cell degeneration induced by retinal ischemia/reperfusion injury Yubin Wang a, Dulce Lopez a, Pinakin Gunvant Davey b, D. Joshua Cameron b, Katherine Nguyen a, Jennifer Tran a, Elizabeth Marquez a, Yan Liu a, Xiaoning Bi c, Michel Baudry a,⁎ a b c
Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA, United States College of Optometry, Western University of Health Sciences, Pomona, CA, United States College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA, United States
a r t i c l e
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Article history: Received 4 March 2016 Revised 4 May 2016 Accepted 12 May 2016 Available online 13 May 2016 Keywords: Glaucoma Retinal ganglion cell Calpain Neuroprotection Blindness
a b s t r a c t Calpain has been shown to be involved in neurodegeneration, and in particular in retinal ganglion cell (RGC) death resulting from increased intraocular pressure (IOP) and ischemia. However, the specific roles of the two major calpain isoforms, calpain-1 and calpain-2, in RGC death have not been investigated. Here, we show that calpain-1 and calpain-2 were sequentially activated in RGC dendrites after acute IOP elevation. By combining the use of a selective calpain-2 inhibitor (C2I) and calpain-1 KO mice, we demonstrated that calpain-1 activity supported survival, while calpain-2 activity promoted cell death of RGCs after IOP elevation. Calpain-1 activation cleaved PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and activated the Akt pro-survival pathway, while calpain-2 activation cleaved striatal-enriched protein tyrosine phosphatase (STEP) and activated STEP-mediated pro-death pathway in RGCs after IOP elevation. Systemic or intravitreal C2I injection to wild-type mice 2 h after IOP elevation promoted RGC survival and improved visual function. Our data indicate that calpain1 and calpain-2 play opposite roles in high IOP-induced ischemic injury and that a selective calpain-2 inhibitor could prevent acute glaucoma-induced RGC death and blindness. © 2016 Published by Elsevier Inc.
1. Introduction Calpains are a family of calcium-dependent proteases regulating many cellular functions through the truncation of a number of proteins, resulting in alterations of their properties and functions (Ono and Sorimachi, 2012). In particular, overactivation of calpains has been shown to participate in many neurological disorders associated with neurodegeneration, such as traumatic brain injury, stroke, neurodegenerative disorders and aging (Liu et al., 2008; Vosler et al., 2009; Xu et al., 2009). However, some studies have indicated that, under certain conditions, calpain activation could provide neuroprotection (Jourdi et al., 2009; Wu and Lynch, 2006). The main calpain isoforms in the brain are calpain-1 and calpain-2 (aka μ-calpain and m-calpain), which differ in their calcium requirement for activation, as calpain-1 requires micromolar calcium vs. millimolar for calpain-2. Most studies investigating the role of calpains in neurodegeneration have not addressed the respective roles of calpain-1 and calpain-2, and of the downstream cascades leading to neuronal death. ⁎ Corresponding author at: Western University of Health Sciences, Pomona, CA 91766, United States. E-mail address: [email protected] (M. Baudry). Available online on ScienceDirect (www.sciencedirect.com).
http://dx.doi.org/10.1016/j.nbd.2016.05.007 0969-9961/© 2016 Published by Elsevier Inc.
We recently reported that calpain-1 and calpain-2 play opposite roles in synaptic plasticity as well as in neuroprotection/neurodegeneration (Wang et al., 2013; Wang et al., 2014). Calpain-1 activation following stimulation of synaptic NMDA receptors is required for the induction of long-term potentiation (LTP), a widely recognized cellular mechanism for learning and memory, and is also neuroprotective against starvation- or hydrogen peroxide-induced neuronal death in cultured neurons and against NMDA-mediated neurotoxicity in mouse hippocampal slices through the activation of the Akt pathway. Conversely, calpain-2 activation following LTP induction restricts the extent of LTP, and its activation following stimulation of extrasynaptic NMDA receptors in cultured neurons results in neurodegeneration through the cleavage of the phosphatase STEP (Wang et al., 2013; Wang et al., 2014). Primary angle-closure glaucoma (PACG) is a major cause of irreversible blindness worldwide and is expected to become more prevalent as lifespan increases. Angle-closure produces a rapid rise in intraocular pressure (IOP), which results in retinal ischemia and retinal ganglion cell (RGC) death. Several hypotheses have been proposed to account for RGC death, including apoptosis, mitochondrial dysfunction, lack of neurotrophin supply, excitotoxicity, and dysfunctional neuron-glia interactions (Almasieh et al., 2012; Wang et al., 2002). In addition, caspase and inflammasome activation and production of toxic cytokines (Chi et
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al., 2014), and deregulation of the autophagy process through calpainmediated truncation of beclin-1 have been reported (Russo et al., 2011). NMDA receptor blockade, as well as several calpain inhibitors reduced RGC loss after retinal ischemia in various animal species, including primates (Nakajima et al., 2006; Nucci et al., 2005; Oka et al., 2006; Sakamoto et al., 2006; Sakamoto et al., 2000; Shimazawa et al., 2010). However, none of these studies have specifically addressed the roles of calpain-1 and calpain-2 in the retina following ischemia. In this study, we combined the use of a selective calpain-2 inhibitor and calpain-1 knock-out (KO) mice to determine the time-courses of calpain-1 and calpain-2 activation and their functions in retina after acute IOP elevation. We found that calpain-1 is rapidly activated in RGC and promotes RGC survival, while calpain-2 activation is delayed and results in RGC death. We also found that a single systemic or intraocular injection of a selective calpain-2 inhibitor following increased IOP prevented RGC death and loss of vision in mice. These results suggest the possibility of developing selective calpain-2 inhibitors for the treatment of acute glaucoma.
antibodies overnight at 4 °C and then with secondary antibodies for 2 h at room temperature. Protein bands were visualized with the Odyssey imaging system (LI-COR). The primary antibodies used were spectrin (1:1000, MAB1622, Millipore), calpain-1 (1:1000, 2556, CST), STEP (1:1000, 4396, CST), PHLPP1 (1:1000, 07-1341, Millipore), phospho-Akt Ser473 (1:3000, 4060, CST) and Akt (1:2000, 2920). The secondary antibodies were IRDye secondary antibodies (1:10,000, LI-COR).
2. Materials and methods
At the indicted times after acute IOP elevation, eyes were enucleated, fixed in 4% paraformaldehyde for at least 1 day and dehydrated in 30% sucrose for at least 1 day. Six frozen sections (10 μm-thick) cut through the optic disc of each eye were collected. In order to determine in situ calpain activation, we used immunohistochemistry to label two calpain substrates, the spectrin breakdown product (SBDP) generated by calpain truncation of spectrin, and the Phosphatase and tensin homolog (PTEN). While SBDP is generated by activation of both calpain-1 and calpain-2, PTEN is selectively cleaved by calpain-2 (Briz et al., 2013). Sections were blocked in 0.1 M PBS containing 10% goat or donkey serum and 0.3% Triton X-100 for 1 h, and then incubated in rabbit anti-SBDP (1:500, a gift from Dr. Saido, Riken, Japan), mouse antiPTEN (1:600, 9556, Cell Signaling) to assess local activation of calpain1 and calpain-2. Brain-specific homeobox/POU domain protein 3A (brn-3a) has been shown to label retinal ganglion cells (Nakazawa et al., 2006) and we used goat anti-brn3a (1:100, sc-31984, Santa Cruz) primary antibodies in 0.1 M PBS containing 5% goat or donkey serum and 0.3% Triton X-100 overnight at 4 °C to selectively label RGCs after increased IOP. Sections were washed 3 times in PBS (10 min each) and incubated in Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies), Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 555 donkey antigoat IgG (Life Technologies) for 2 h at room temperature. After three washes, sections were mounted with mounting medium containing DAPI (Vector Laboratories) and visualized using Nikon C1 confocal laser-scanning microscope. For quantification of SBDP and PTEN staining, 6 sections in each eye were analyzed. In each section, three 50 × 25 μm2 regions in IPL layer were selected and MFI (mean fluorescence intensities) were measured in ImageJ and averaged. Image acquisition and quantification were done by two persons in a blind fashion.
2.1. Animals Animal use in all experiments followed NIH guidelines and all protocols were approved by the Institution Animal Care and Use Committee of Western University of Health Sciences. Calpain-1 KO mice on a C57Bl/6 background were obtained from a breeding colony established from breeding pairs generously provided by Dr. Chishti (Tufts University). C57Bl/6 mice were purchased from Jackson Labs and were the corresponding WT. 2.2. Mouse retinal ischemia/reperfusion model Mouse retinal ischemia/reperfusion model was established following the protocol reported in previous publications (Buchi et al., 1991; Chi et al., 2014; Park et al., 2011). Mice were anesthetized with isoflurane and a gas anesthesia machine. Pupils were dilated with 1% tropicamide. The anterior chamber of the right eye was cannulated with a 33-gauge infusion needle connected to a normal saline reservoir, which was elevated to maintain an intraocular pressure of 110 mmHg for 60 min, which was confirmed using a Tonopen (Richert, Buffalo NY) (Reitsamer et al., 2004). Retinal ischemia was confirmed by whitening of the fundus. A sham procedure performed without elevating the pressure in the contralateral left eye was used as control. After 1 h, intraocular pressure was normalized during a 30-s period, and the needle was withdrawn. The animals were allowed to recover for various periods of time before sacrifice. 2.3. Spectral Domain Optical Coherence Tomography (SD-OCT) Spectral Domain optical coherence tomography (SD-OCT) was used to obtain images of the retina in anesthetized mice. Mice were anesthetized as above and SD-OCT images were obtained using Ivue (Optovue Inc. Fremont CA) with an anterior segment attachment (20 diopter lens). Images were quantitatively analyzed with the inbuilt software of the OCT, which allows for free-hand measurement of any structure imaged. 2.4. Western blot At various times after IOP elevation, mouse retinas were isolated and homogenized in RIPA buffer (89901, Thermo) with a protease inhibitor cocktail (78446, Thermo) and 5 mM EGTA. Protein concentrations were measured using the BCA protein assay kit (Thermo). Sample buffer was added, and samples were separated in SDS-PAGE and transferred onto PVDF membranes (IPFL00010, Millipore). After blocking with Odyssey® Blocking Buffer (LiCOR) for 1 h, membranes were incubated with primary
2.5. H&E staining in retinal vertical sections Mouse eyes were enucleated, fixed in 4% paraformaldehyde for at least 1 day and dehydrated in 30% sucrose for at least 1 day. Six frozen sections (10 μm-thick), cut through the optic disc of each eye, were collected and stained with Hematoxylin solution Harris modified (HHS32, Sigma) and Eosin Y solution aqueous (HT110232, Sigma). Retina images were photographed using light microscopy (Nikon). 2.6. Immunohistochemistry in retinal vertical sections
2.7. Cell counting in retinal vertical sections Cell counting was performed as previously reported (Shimazawa et al., 2010) with minor changes. Two 450 × 350 μm2 images were obtained at 500–1000 μm from the optic disc (both sides) in each retinal section stained with H&E or brn-3a antibody. Cell number in GCL of each image was counted. GCL length was measured using ImageJ (Freehand line). Cell density in GCL was calculated as the ratio of cell number to length (cell numbers/mm). Cell densities from 6 sections in each eye were averaged. Image acquisition and RGC counting were done by two persons in a blind fashion. 2.8. Immunohistochemistry and RGC counting in retinal whole-mounts Three days after glaucoma or sham surgery, mouse eyes were enucleated, fixed in 4% paraformaldehyde overnight. Retinas were then
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dissected as flattened whole-mounts and processed for brn-3a immunostaining as described above. Brn-3a-labeled RGCs were counted, as previously described (Nakazawa et al., 2006) with minor changes. Images of twelve 320 × 320 μm2 areas in each retina, including three areas per retinal quadrant at one-sixth (central retina), three-sixths (middle retina), and five-sixths (peripheral retina) of the retinal radius, were taken using a confocal microscope. The density of RGCs was defined as the average number of cells in the 12 areas. Cell counting was performed in a masked manner using the “analyze particles” function in ImageJ.
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1A). Some of these changes persisted over 3 days of observation. Eyes were collected at 0, 2, 4 and 6 h after IOP elevation and frozen retinal sections were prepared and processed for immunohistochemistry with an antibody against a spectrin breakdown product (SBDP), generated by calpain-1- or calpain-2-mediated spectrin cleavage. In WT mice, SBDP was clearly present in the inner plexiform layer (IPL), which
2.9. Intravitreal injection Mice were anesthetized with isoflurane and a gas anesthesia machine. Intravitreal injections were performed with a 33-gauge needle (0.5 in. long) attached to a 5 μl glass syringe (Hamilton). The needle was positioned 1 mm posterior to the limbus and 1 μl of the solution was slowly (3–5 s) injected into the vitreous chamber of the eye. A 20 s interval was kept before removing the needle. 2.10. OKR Visual Acuity measurement Visual acuity was measured by immobilizing the mouse head and restraining the mouse with a home-made OKR device, which was modified from a zebrafish device in order to accommodate a mouse, as previously published (Cahill and Nathans, 2008; Cameron et al., 2013; Sakatani and Isa, 2004). An infrared camera was used to monitor and record pupil movement and videos were analyzed with the Tracker video analysis and modeling tool. A 34.3 cm-diameter grating drum rotated around the mouse illuminated with a 200 lx-white light. The grating frequency was decreased using the staircase method until eye tracking ceased, identifying the smallest grating detected by the mouse's eye. The grating frequency was then converted into spatial acuity measured in cycles per degree (cpd) using the following formula: cpd = 1/ [2arctan (h/(2a))] where h is the grating cycle size in mm and a is the approximate distance from the center of the lens to the grating, also in mm. The range of spatial acuity we could test was 0.04–0.9 cpd. If the eye could not track the lowest spatial frequency (0.04 cpd), the acuity of this eye was recorded as 0.04 cpd. The clockwise rotation of gratings triggered OKR of the left eye (OS), while counterclockwise rotation triggered OKR of the right eye (OD) (Douglas et al., 2005). Thus, visual acuity of OS was measured when gratings rotated clockwise while OD was measured when gratings rotated counterclockwise. 2.11. Statistical analyses In all cases, error bars indicate standard error of the mean. N values represent numbers of eyes tested. To compute p values, unpaired student's t-test and one-way ANOVA followed by Bonferroni test were used, as indicated in figure legends. 3. Results 3.1. Sequential activation of calpain-1 and calpain-2 in retina after acute IOP elevation We implemented a widely used retinal ischemia/reperfusion model that produces features of acute angle closure glaucoma (Buchi et al., 1991; Chi et al., 2014; Osborne et al., 2004; Park et al., 2011). Intraocular pressure (IOP) was increased to 110 mmHg for 60 min by inserting a needle connected to an elevated reservoir of saline into the anterior chamber (see Materials and methods). This model reproduces several features of acute angle closure, including ischemia of retina and iris, as noted by the absence of red reflex and pupillary response to light. Anterior chamber synechae, resulting in a narrow angle and adhesion between the iris and the cornea, increased cells and flare in the anterior chamber and increased corneal thickness due to corneal edema (Fig.
Fig. 1. Time course of calpain-1 and calpain-2 activation in retina after acute IOP elevation. (A) Representative images of mouse eyes obtained with spectral domain optical coherence tomography (SD-OCT) following elevated IOP. Top left panel: image obtained prior to elevated IOP. The white arrow points to open angle with normal corneal anatomy and anterior chamber. Top right panel: 1 day after inducing IOP elevation for 1 h; the anterior chamber synechae is visible (white arrow) along with increased hyper reflectivity in anterior chamber (red arrow) indicating breakdown of blood aqueous barrier, which is caused by proteins and cells in the anterior chamber. Also note the increased corneal thickness (yellow arrow). Lower left panel: day two, corneal thickness and anterior camber reflectivity are decreased as compared to day 1, but anterior synechae is still present. Lower right Panel: day 3, showing marked decrease in cornea thickness and anterior chamber reflectivity; the synechae is now broken. (B) Immunostaining of SBDP (green) in ganglion cell layer (GCL) and inner plexiform layer (IPL) of retina in WT, calpain-1 KO and C2I-injected WT mice at 0, 2, 4 and 6 h after acute IOP elevation. Sections were counterstained with DAPI (blue). C2I (0.3 mg/kg) was injected i.p. to WT mice 2 h after IOP elevation. Scale bar = 20 μm. (C) Quantification of SBDP staining in IPL, n = 3–5 (eyes) at each time point. For each eye, 3 retinal sections were used for quantification. For each section, three 50 × 25 μm regions in the IPL were selected and MFI (mean fluorescence intensities) were measured and averaged. *p b 0.05, **p b 0.01, ***p b 0.001 versus control in the same group, One-way ANOVA followed by Bonferroni test.
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contains RGC dendrites, at 2, 4 and 6 h after IOP elevation. However in calpain-1 KO mice, SBDP was only evident in the IPL at 4 and 6 h but not at 2 h (Fig. 1B,C). These results suggest that calpain-1 is activated early and that calpain-2 activation is delayed in the IPL after IOP elevation. We then tested the effects of a calpain-2 selective inhibitor (C2I), Z-Leu-Abu-CONH-CH2-C6H3 (3, 5-(OMe)2) (Wang et al., 2013; Wang et al., 2014), injected intraperitoneally (i.p., 0.3 mg/kg) to WT mice 2 h after IOP elevation. C2I injection significantly reduced SBDP immunoreactivity in the IPL at 4 and 6 h after IOP elevation (Fig. 1B,C), confirming that calpain-2 was activated in the IPL following increased IOP. To further analyze the time course of calpain-2 activation in retina after increased IOP, retinal sections were immunostained with an antibody against full-length PTEN, a substrate of calpain-2 but not of calpain-1 (Briz et al., 2013). In both WT and calpain-1 KO mice, PTENimmunoreactivity in the IPL was unchanged at 2 h, but was significantly reduced at 4 and 6 h after IOP elevation (Fig. 2), confirming that calpain-2 was activated at 4 and 6 but not at 2 h after IOP elevation. When C2I was injected to WT mice 2 h after IOP elevation, PTEN degradation at 4 and 6 h was completely blocked (Fig. 2). Altogether, these results strongly suggest that calpain-1 is briefly activated in RGC dendrites after acute IOP elevation, while calpain-2 activation in the same dendrites is delayed and prolonged. 3.2. Calpain-1 and calpain-2 play opposite roles in RGC death induced by acute IOP elevation To evaluate elevated IOP-induced retinal damage, IOP of the right eye was elevated to 110 mmHg for 60 min, while a sham procedure was performed in the left eye. Retinal vertical sections were collected 3 days after surgery and H&E staining was performed to examine cell
numbers in the ganglion cell layer (GCL) (Fig. 3A,B). In WT mice injected with vehicle (i.p., 10% DMSO in PBS), cell counts in the right GCL were 62.1 ± 5.6 cells/mm, as compared to 113.4 ± 7.1 cells/mm in the left GCL (n = 7). We used three different protocols to examine the effect of C2I. First, C2I (0.3 mg/kg) was injected (i.p.) 30 min before and 2 h after acute IOP elevation (pre and post inj). Cell counts in GCL of sham eye and IOP-elevated eye were 125.1 ± 10.5 and 105.8 ± 4.5 cells/mm, respectively (n = 3, no significant difference (ns) sham vs. IOP). Second, C2I was injected (i.p.) 2 h after IOP elevation (one post inj). Cell counts in sham and IOP-elevated eye were 110.6 ± 3.6 and 86.4 ± 7.0 cells/mm (n = 10, ns). Third, C2I was injected (i.p.) 2 and 4 h after IOP elevation (two post inj). Cell counts in sham and IOP-elevated eye were 118.6 ± 3.7 and 96.1 ± 6.3 cells/mm (n = 6, ns). In all three C2I-injected groups, cell survival rate (ratio of cell count in IOP-elevated eye to that in sham eye) was significantly increased, as compared to vehicle-injected group (Fig. 3C). These results suggest that calpain-2 activation plays an important role in GCL cell death after IOP elevation and that C2I systemic injection has a protective effect against IOP-induced cell death. In calpain-1 KO mice, cell count in GCL of IOP-elevated eye was significantly lower than that of sham eye (37.7 ± 10.4 vs. 130.3 ± 7.0 cells/mm, n = 4). Importantly, cell survival rate in calpain-1 KO mice was significantly lower than that in WT mice (Fig. 3C), again suggesting that calpain-1 supports cell survival in GCL after IOP elevation. To specifically examine the effect of C2I on RGCs, which constitute approximately 40% of the cells in mouse GCL (Jeon et al., 1998), retinal sections from WT mice injected with vehicle or C2I 2 h after acute IOP elevation were immunostained with an antibody against brn-3a, a selective RGC marker (Nadal-Nicolas et al., 2009) (Fig. 3D). In WT mice injected with vehicle, RGC counts in sham eye and IOP-elevated eye were 40.9 ± 5.2 and 19.9 ± 3.4 cells/mm (p b 0.01 sham vs. IOP, n = 4). In WT mice injected with C2I, RGC counts in sham eye and IOP-elevated eye were 45.2 ± 5.0 and 37.1 ± 2.5 cells/mm (ns, sham vs. IOP, n = 5) (Fig. 3E). The survival rate of RGC with C2I injection was significantly improved, as compared to vehicle injection (Fig. 3F), suggesting that C2I systemic injection protects RGC against IOP-induced cell death. To explore the nature of the signaling pathways downstream of calpain-1 and calpain-2, retinas in WT, calpain-1 KO and C2I-injected WT mice were collected 3 h after IOP elevation or sham surgery, homogenized and aliquots processed for Western blots (Fig. 4A,B). In WT mice, levels of PH domain and Leucine-rich repeat Protein Phosphatase 1 (PHLPP1), a phosphatase downstream of calpain-1 (Wang et al., 2013), were significantly reduced, while levels of phospho-Akt Ser473 (pAkt), which can be dephosphorylated by PHLPP1, were significantly increased after IOP elevation. These changes in PHLPP1 and pAkt were absent in calpain-1 KO mice but present in C2I-injected WT mice, suggesting that calpain-1 but not calpain-2 mediates PHLPP1 degradation and Akt activation in retina after IOP elevation. STEP33, the product of calpain-2-mediated cleavage of striatal-enriched protein tyrosine phosphatase (STEP) (Wang et al., 2013), was present in WT and calpain-1 KO mice but not in C2I-injected WT mice following increased IOP, indicating that calpain-2 but not calpain-1 mediates STEP cleavage after IOP elevation. These results suggest that both calpain-1/PHLPP1/Akt prosurvival pathway and calpain-2/STEP pro-death pathway (Wang et al., 2013) are engaged in retina after IOP elevation, and that C2I selectively inhibits calpain-2/STEP pro-death pathway. 3.3. Intravitreal injection of C2I reduces cell death in GCL and prevents loss of vision caused by acute IOP elevation
Fig. 2. Time course of calpain-2 activation in retina after acute IOP elevation. (A) Immunostaining of full-length PTEN (red) in GCL and IPL of retina in WT, calpain-1 KO and C2I-injected WT mice at 0, 2, 4 and 6 h after acute IOP elevation. Sections were counterstained with DAPI (blue). C2I was injected to WT mice 2 h after IOP elevation. Scale bar = 20 μm. (B) Quantification of PTEN staining, n = 3–5 (eyes) at each time point. *p b 0.05, **p b 0.01 versus control in the same group, One-way ANOVA followed by Bonferroni test.
We used intravitreal C2I injection in order to locally deliver C2I to retina. First, we tested the delivery efficiency by injecting different doses of C2I intravitreally 2 h after IOP elevation in calpain-1 KO mice and analyzing SBDP levels in the IPL at 4 h (Fig. 5A,B). A clear dose-dependent inhibition of SBDP formation was observed, providing an apparent IC50 of 8 μM for C2I. In all subsequent experiments, we used 20 μM (1 μl) to examine the neuroprotective effects of intravitreal C2I
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injection. Eyes were collected 3 days after surgery for HbE staining. After vehicle injection (10% DMSO in PBS), GCL counts in sham eye and IOPelevated eye were 132.3 ± 4.5 and 62.0 ± 5.7 cells/mm (p b 0.001
Fig. 4. Calpain-1 activation cleaves PHLPP1 while calpain-2 activation cleaves STEP in retinal ganglion cells after acute IOP elevation. (A) Representative immunoblots of indicated proteins in retina of WT, calpain-1 KO and C2I-injected mice collected 3 h after sham surgery or acute IOP elevation. C2I (0.3 mg/kg) was injected systemically 2 h after sham surgery or IOP elevation. (B) Quantitative analysis of the levels of PHLPP1 and STEP33 and ratios of pAkt/Akt for each group. Results represent means ± S.E.M. of 4 experiments. *p b 0.05, **p b 0.01, ***p b 0.001, ns no significant difference, One-way ANOVA followed by Bonferroni test.
Fig. 3. Calpain-2 inhibition reduces, while calpain-1 knockout exacerbates cell death in ganglion cell layer induced by acute IOP elevation. (A) H&E staining of retinal sections from the right eye of vehicle- or C2I-injected wild-type and calpain-1 KO mice and the left eye where sham surgery was performed. Vehicle, 10% DMSO in PBS, was injected i.p. 30 min before and 2 h after acute IOP elevation. Pre- and post-injection C2I (0.3 mg/kg) was done i.p. 30 min before and 2 h after acute IOP elevation. For the One post injection group, C2I was injected 2 h after IOP elevation. For the Two post inj group, C2I was injected 2 and 4 h after IOP elevation. H&E staining was performed 3 days after surgery. Scale bar = 30 μm. (B) Quantification of H&E staining. Data represent means ± S.E.M. n = 7 (eyes) for vehicle, n = 3 for pre and post inj, n = 10 for one post inj, n = 6 for two post inj, n = 4 for KO. ns, no significant difference. ***p b 0.001, Two-tailed t-test. (C) Survival rate for each mouse was calculated as the ratio of cell density in GCL of IOPelevated eye to cell density in GCL of sham eye. *p b 0.05, **p b 0.01 versus vehicle, Oneway ANOVA followed by Bonferroni test. (D) Brn-3a immunostaining in the retina of vehicle- or C2I-injected WT mice 3 days after surgery. Vehicle, 10% DMSO, or C2I, C2I (0.3 mg/kg) was injected i.p. 2 h after acute IOP elevation. Scale bar = 60 μm. (E) Brn3apositive cells in GCL were counted. n = 4 for vehicle, n = 5 for C2I. Ns, no significant difference, **p b 0.01 sham versus IOP elevation, two-tailed t-test. (F) Comparison of survival rates. n = 4 for vehicle, n = 5 for C2I. **p b 0.01 vehicle versus C2I, two-tailed t-test.
sham vs. IOP, n = 4). In C2I-treated eyes, GCL counts in sham eye and IOP-elevated eyes were 128.1 ± 7.2 and 101.3 ± 9.2 cells/mm (no significant difference, sham vs. IOP, n = 5) (Fig. 5C,D). Survival rate with C2I injection was significantly improved, as compared to vehicle injection (80.8 ± 8.4% vs. 47.2 ± 5.4%, p b 0.01) (Fig. 5E). To further confirm that RGCs were protected against IOP-induced degeneration by C2I injection, retinal whole-mounts were prepared and immunostained with brn-3a antibody to label RGCs 3 days after treatment, and average RGC densities in retina whole-mounts were quantified. After intravitreal vehicle injection, RGC densities in sham eye and IOP elevated eye were 2.3 ± 0.2 and 0.6 ± 0.3 103 cells/mm2 (p b 0.001, n = 4). A large proportion of RGCs degenerated leaving cell debris in retina after IOP elevation (Fig. 5F,G). After intravitreal C2I injection, RGC densities in sham eye and IOP elevated eyes were 2.4 ± 0.2 and 1.6 ± 0.6 103 cells/mm2 (no significant difference, n = 4). The difference in RGC densities between eyes treated with vehicle or C2I following increased IOP was highly significant (p b 0.01, n = 4) (Fig. 5G). The above results indicate that intravitreal C2I injection 2 h after IOP elevation is neuroprotective against IOP-induced RGC death. To examine vision of mice after glaucoma, we tested their optokinetic reflex (OKR). OKR is the saccadic eye movement triggered by the movement of gratings in front of the mouse eye. Changing the frequency of gratings and determining the lowest frequency triggering OKR, allows analyzing visual acuity of each eye. Acute IOP elevation or sham surgery was performed in the right eye (OD). Left eye (OS) served as a naive control. Intravitreal C2I or vehicle injection was performed 2 h after surgery. Three and 21 days after surgery, OKR was determined in both eyes (Fig. 6A,B). Visual acuity of sham eye with vehicle injection was 0.47 ± 0.11 cpd (mean ± SEM, n = 7) at day 3 and 0.41 ± 0.16 (n = 7) at day 21, in good agreement with published results (Douglas et al., 2005; Prusky et al., 2004). Visual acuity was dramatically reduced after increased IOP at both time points, which was significantly improved by C2I injection. C2I injection in the sham eye did not affect visual acuity, as compared to vehicle injection. Mice were sacrificed after OKR test at day 21 and RGC densities were analyzed with brn-3a
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4. Discussion
Fig. 5. Intravitreal injection of C2I reduces cell death in ganglion cell layer induced by acute IOP elevation. (A) SBDP immunostaining in retina of calpain-1 KO mice after acute IOP elevation. Vehicle (10% DMSO in PBS, 1 μl) or different doses of C2I (2–80 μM, 1 μl) were injected intravitreally 2 h after IOP elevation. Eyes were collected 4 h after IOP elevation for SBDP staining. Scale bar = 20 μm. (B) Quantification of SBDP staining in IPL. N = 3 (eyes) at each concentration. The inhibition of SBDP signal was calculated by (MFIVehicleMFIC2I)/MFIVehicle %. Data represent means ± S.E.M. (C) H&E staining in retina of WT mice after IOP elevation. Vehicle or C2I (20 μM, 1 μl) was injected 2 h after IOP elevation or sham surgery. Eyes were collected 3 days after surgery for H&E staining. Scale bar = 30 μm. (D) Quantification of H&E staining. n = 7 for naive, n = 4 for vehicle, n = 6 for C2I. *p b 0.05, ***p b 0.001, two-tailed t-test. (E) Comparison of survival rates. *p b 0.05, **p b 0.01, One-way ANOVA followed by Bonferroni test. (F) Brn-3a immunostaining in retina whole-mounts of WT mice after IOP elevation or sham surgery. Vehicle or C2I (20 μM, 1 μl) was injected 2 h after surgery. Eyes were collected 3 days after the surgery. Representative images show area of middle retina. Scale bar = 100 μm. (G) Quantification of brn-3a positive cell numbers in retina whole-mounts under different treatments. The cell density in each retina was calculated as the average cell density of twelve 320 × 320 μm2 areas at central, middle and peripheral retina. N = 4. **p b 0.01, ***p b 0.001. Ns, no significant difference. Two-way ANOVA followed by Sidak's multiple comparisons test.
immunostaining in retinal sections (Fig. 6C). As expected, RGC densities were significantly reduced in eyes treated with vehicle after increased IOP, but recovered in eyes treated with C2I after increased IOP. Moreover, visual acuity was highly correlated with the number of surviving RGCs (Fig. 6D), further supporting the prominent role of calpain-2 in triggering RGC death after increased IOP.
While several mechanisms have been involved in RGC death in acute glaucoma (Almasieh et al., 2012; Chi et al., 2014; Wang et al., 2002), including calpain activation (Russo et al., 2011), our results clearly indicate that calpain-1 and calpain-2 play opposite functions in increased IOP-induced retinal damage. Thus, calpain-1 is neuroprotective, since retinal damage is exacerbated in calpain-1 KO mice, as compared to WT mice. This effect is due to the activation by calpain-1 of a survival pathway previously identified in a different system (Wang et al., 2013), calpain-1 mediated cleavage of PHLPP1 leading to activation of the prosurvival Akt pathway. On the other hand, calpain-2 is neurodegenerative, as evidenced by the significant protection against retinal damage provided by a selective calpain-2 inhibitor. The differential functions of the two calpain isoforms are due in part to their differential time-course of activation, and their different signaling pathways. Thus, calpain-1 is rapidly and briefly activated following increased IOP, and stimulates a pro-survival pathway, probably due to the rapid and transient activation of synaptic NMDA receptors, composed of GluN2A subunits, as we previously reported (Wang et al., 2014). In contrast, calpain-2 activation is delayed and more prolonged. It is activated between 2 and 4 h after increased IOP, which could be due to the stimulation of extrasynaptic NMDA receptors, composed of GluN2B subunits, as a result of glutamate spill-over or inhibition of glutamate transport known to take place following ischemia (Brassai et al., 2015; Parsons and Raymond, 2014). It also triggers the degradation and inhibition of the phosphatase STEP, which activates STEP substrate p38 and results in neurodegeneration (Wang et al., 2013; Xu et al., 2009). This interpretation is consistent with the differential roles of GluN2A- and GluN2Bcontaining NMDA receptors in NMDA-induced neurotoxicity in retina (Bai et al., 2013). We exploited the differential temporal activation of calpain-1 and calpain-2 to show that systemic or intravitreal injection of a selective calpain-2 inhibitor 2 h after IOP elevation completely inhibited calpain-2 activation and provided significant protection against retinal damage. Furthermore, this treatment also restored vision following increased IOP to a level not significantly different from that of shamoperated eyes, as assessed with OKR sensitivity. Interestingly, the neuroprotection provided by the single injection of the calpain-2 inhibitor was still present 3 weeks following increased IOP, indicating that calpain-2 activation is the trigger of the neurodegenerative events. Our results thus indicate that calpain-2 activation is a critical step in RGC death following acute IOP elevation, possibly the converging node downstream of other pathways previously implicated in RGC neurodegeneration (Almasieh et al., 2012). Calpain has previously been implicated in retinal damage following ischemia or other types of insults (Nakajima et al., 2006; Oka et al., 2006; Sakamoto et al., 2000; Shimazawa et al., 2010). However, the lack of understanding of the different roles of the two major calpain isoforms in retina and in other regions of central nervous system is probably the reason why non-selective calpain inhibitors have never reached clinical trials, although they have shown some protection against retinal damage in certain animal models of eye disorders (Das et al., 2013; Sakamoto et al., 2000; Shimazawa et al., 2010; Smith et al., 2011). In addition to reducing optic nerve damage and RGC apoptosis, calpain inhibition reduced astrogliosis and microgliosis in acute optic neuritis(Das et al., 2013; Smith et al., 2011). Whether C2I has a similar effect in the acute glaucoma model remains to be determined. In any event, our results underscore the need to use a selective calpain-2 inhibitor to reduce retinal damage after a variety of insults. Any type of ocular drug delivery methods will inevitably cause systemic absorption of the drug (Urtti, 2006). Ocular delivery of non-selective calpain inhibitors may inhibit the physiological function of calpain-1 such as neuroprotection in retina and learning and memory. In contrast, local delivery of a selective calpain-2 inhibitor may avoid those negative side effects.
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Fig. 6. Intravitreal injection of calpain-2 inhibitor protects vision assessed with the optokinetic reflex. (A) OKR spatial frequency thresholds of eyes measured 3 days after IOP elevation or sham surgery. Vehicle or C2I (20 μM, 1 μl) was injected intravitreally 2 h after surgery. Surgery and injection were always performed in the right eye (OD). OKR of the Left eye (OS) was measured as control. n = 7. ****p b 0.0001, **p b 0.01. One-way ANOVA followed by Bonferroni test. (B) OKR was re-measured 21 days after surgery. n = 7. ****p b 0.0001, *p b 0.05. (C) RGC density in the retina of the eyes at 21 days after surgery. Eyes were collected after the OKR test. Brn-3a immunostaining was performed. Brn-3a-positive cells in GCL were counted. n = 7 for IOP elevation + C2I. n = 4 for other groups. ***p b 0.001, **p b 0.01. (D) Linear regression analysis of OKR spatial frequency thresholds and RGC densities measured 21 days after surgery. Black symbol, data from Sham plus Vehicle group; Blue, Sham plus C2I. Red, IOP elevation plus Vehicle. Green, IOP elevation plus C2I. N = 19. R2 = 0.86.
While the ischemia/reperfusion model we used reproduces several features of PACG, it also exhibits some limitations, and the relationship between the actual events taking place in human PACG and in our model remains to be completely validated. In human PACG, IOP does not rise and drop as quickly as in the model used in this study. Nevertheless, there is consensus regarding the major events leading to RCG death in PACG, including ischemia, overactivation of glutamate receptors and activation of neurodegenerative cascades, which also take place in our models. The identification of the neuroprotective role of calpain-1 and the neurodegenerative function of calpain-2 combined with the neuroprotective effects of a selective calpain-2 inhibitor administered 2 h after the increase in IOP in our model strengthen the notion that it might be possible to develop new treatments for a number of neurodegenerative eye disorders previously linked to calpain activation. Thus, selective targeting of calpain-2 may provide a potential therapeutic approach for these different eye disorders. Of particular interest will be chronic open angle and angle closure glaucoma. Although the disease process varies in different types of glaucoma, the consequences of elevated IOP and the pathway to RGC death are extremely similar. Thus, the next steps will be to evaluate the neuroprotective efficacy of selective calpain-2 inhibitors, along with other standard IOP-lowering therapy, on vision preservation in models of chronic glaucoma. To our knowledge, a calpain inhibitor has never been tested in clinical trials for the treatment of eye disorders. On the other hand, Abbvie is currently carrying a Phase I clinical trial with an orally active non-selective calpain inhibitor for the treatment of Alzheimer's disease (https:// clinicaltrials.gov/ct2/show/NCT02220738?term=Abbvie+and+ABT957&rank=1). As our results indicate that a single intraocular administration of a selective calpain-2 inhibitor is neuroprotective when delivered 2 h after the increased IOP, the path to testing this or similar compounds in PACG could be relatively straightforward and lead to the validation of the critical role of calpain-2 in PACG.
5. Conclusion Calpain-1 and calpain-2 play opposite functions following ischemic injury in the retina. While calpain-1 is rapidly activated its activation is neuroprotective through the stimulation of the Akt pathway, as a result of cleavage of PHLPP1. Thus, lack of calpain-1 exacerbates ischemic injury in the retina. On the other hand, calpain-2 activation is delayed and prolonged and is neurodegenerative, as a result of STEP truncation. A selective calpain-2 inhibitor injected either systemically or intraocularly 2 h after the ischemic episode protects retinal ganglion cells and maintains vision to normal levels. These findings suggest that selective targeting of calpain-2 might produce better neuroprotection and less side-effects than non-selective calpain inhibitors not only for the treatment of eye disorders but also for other neurodegenerative conditions.
Authors contributions YW performed the majority of experiments and wrote the manuscript; DL participated in many experiments, PGD performed the OCT studies, DJC helped with the OKR experiments, KN, JT, EM and YL helped with many experiments, XB designed experiments and wrote the manuscript, MB designed the project and wrote the manuscript.
Conflict of interest The authors have declared that no conflict of interest exists. A patent entitled “Isoform-Selective Calpain Inhibitors, Methods of Identification, and Uses Thereof” has been filed by Western University of Health Sciences to the USPTO.
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Acknowledgements This work was supported by grant P01NS045260 from NINDS (PI: Dr. C.M. Gall). The authors want to thank Western University of Health Sciences for the financial support to MB. XB is also supported by funds from the Daljit and Elaine Sarkaria Chair. The authors want to acknowledge the generous gift of the calpain-1 KO mice from Dr. A. Chishti (Tufts University), and the technical assistance of Mr. Joseph Gray and Mr. Farouk Bruce from the College of Optometry. References Almasieh, M., Wilson, A.M., Morquette, B., Cueva Vargas, J.L., Di Polo, A., 2012. The molecular basis of retinal ganglion cell death in glaucoma. Prog. Retin. Eye Res. 31, 152–181. Bai, N., Aida, T., Yanagisawa, M., Katou, S., Sakimura, K., Mishina, M., Tanaka, K., 2013. NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina. Mol. Brain 6, 1. Brassai, A., Suvanjeiev, R.-G., Bán, E.-G., Lakatos, M., 2015. Role of synaptic and nonsynaptic glutamate receptors in ischaemia induced neurotoxicity. Brain Res. Bull. 112, 1–6. Briz, V., Hsu, Y.T., Li, Y., Lee, E., Bi, X., Baudry, M., 2013. Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis. J. Neurosci. 33, 4317–4328. Buchi, E.R., Suivaizdis, I., Fu, J., 1991. Pressure-induced retinal ischemia in rats: an experimental model for quantitative study. Ophthalmologica 203, 138–147. Cahill, H., Nathans, J., 2008. The optokinetic reflex as a tool for quantitative analyses of nervous system function in mice: application to genetic and drug-induced variation. PLoS ONE 3, e2055. Cameron, D.J., Rassamdana, F., Tam, P., Dang, K., Yanez, C., Ghaemmaghami, S., Dehkordi, M.I., 2013. The optokinetic response as a quantitative measure of visual acuity in zebrafish. J. Vis. Exp. Chi, W., Li, F., Chen, H., Wang, Y., Zhu, Y., Yang, X., Zhu, J., Wu, F., Ouyang, H., Ge, J., Weinreb, R.N., Zhang, K., Zhuo, Y., 2014. Caspase-8 promotes NLRP1/NLRP3 inflammasome activation and IL-1beta production in acute glaucoma. Proc. Natl. Acad. Sci. U. S. A. 111, 11181–11186. Das, A., Guyton, M.K., Smith, A., Wallace, G., McDowell, M.L., Matzelle, D.D., Ray, S.K., Banik, N.L., 2013. Calpain inhibitor attenuated optic nerve damage in acute optic neuritis in rats. J. Neurochem. 124, 133–146. Douglas, R.M., Alam, N.M., Silver, B.D., McGill, T.J., Tschetter, W.W., Prusky, G.T., 2005. Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system. Vis. Neurosci. 22, 677–684. Jeon, C.J., Strettoi, E., Masland, R.H., 1998. The major cell populations of the mouse retina. J. Neurosci. 18, 8936–8946. Jourdi, H., Hsu, Y.T., Zhou, M., Qin, Q., Bi, X., Baudry, M., 2009. Positive AMPA receptor modulation rapidly stimulates BDNF release and increases dendritic mRNA translation. J. Neurosci. 29, 8688–8697. Liu, J., Liu, M.C., Wang, K.K., 2008. Calpain in the CNS: from synaptic function to neurotoxicity. Sci. Signal. 1, re1. Nadal-Nicolas, F.M., Jimenez-Lopez, M., Sobrado-Calvo, P., Nieto-Lopez, L., CanovasMartinez, I., Salinas-Navarro, M., Vidal-Sanz, M., Agudo, M., 2009. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest. Ophthalmol. Vis. Sci. 50, 3860–3868. Nakajima, E., David, L.L., Bystrom, C., Shearer, T.R., Azuma, M., 2006. Calpain-specific proteolysis in primate retina: contribution of calpains in cell death. Invest. Ophthalmol. Vis. Sci. 47, 5469–5475. Nakazawa, T., Nakazawa, C., Matsubara, A., Noda, K., Hisatomi, T., She, H., Michaud, N., Hafezi-Moghadam, A., Miller, J.W., Benowitz, L.I., 2006. Tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma. J. Neurosci. 26, 12633–12641.
Nucci, C., Tartaglione, R., Rombola, L., Morrone, L.A., Fazzi, E., Bagetta, G., 2005. Neurochemical evidence to implicate elevated glutamate in the mechanisms of high intraocular pressure (IOP)-induced retinal ganglion cell death in rat. Neurotoxicology 26, 935–941. Oka, T., Tamada, Y., Nakajima, E., Shearer, T.R., Azuma, M., 2006. Presence of calpain-induced proteolysis in retinal degeneration and dysfunction in a rat model of acute ocular hypertension. J. Neurosci. Res. 83, 1342–1351. Ono, Y., Sorimachi, H., 2012. Calpains: an elaborate proteolytic system. Biochim. Biophys. Acta 1824, 224–236. Osborne, N.N., Casson, R.J., Wood, J.P., Chidlow, G., Graham, M., Melena, J., 2004. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog. Retin. Eye Res. 23, 91–147. Park, S.W., Kim, K.Y., Lindsey, J.D., Dai, Y., Heo, H., Nguyen, D.H., Ellisman, M.H., Weinreb, R.N., Ju, W.K., 2011. A selective inhibitor of drp1, mdivi-1, increases retinal ganglion cell survival in acute ischemic mouse retina. Invest. Ophthalmol. Vis. Sci. 52, 2837–2843. Parsons, M.P., Raymond, L.A., 2014. Extrasynaptic NMDA receptor involvement in central nervous system disorders. Neuron 82, 279–293. Prusky, G.T., Alam, N.M., Beekman, S., Douglas, R.M., 2004. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest. Ophthalmol. Vis. Sci. 45, 4611–4616. Reitsamer, H.A., Kiel, J.W., Harrison, J.M., Ransom, N.L., McKinnon, S.J., 2004. Tonopen measurement of intraocular pressure in mice. Exp. Eye Res. 78, 799–804. Russo, R., Berliocchi, L., Adornetto, A., Varano, G.P., Cavaliere, F., Nucci, C., Rotiroti, D., Morrone, L.A., Bagetta, G., Corasaniti, M.T., 2011. Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. Cell Death Dis. 2, e144. Sakamoto, Y.R., Nakajima, T.R., Fukiage, C.R., Sakai, O.R., Yoshida, Y.R., Azuma, M.R., Shearer, T.R., 2000. Involvement of calpain isoforms in ischemia-reperfusion injury in rat retina. Curr. Eye Res. 21, 571–580. Sakamoto, K., Yonoki, Y., Kubota, Y., Kuwagata, M., Saito, M., Nakahara, T., Ishii, K., 2006. Inducible nitric oxide synthase inhibitors abolished histological protection by late ischemic preconditioning in rat retina. Exp. Eye Res. 82, 512–518. Sakatani, T., Isa, T., 2004. PC-based high-speed video-oculography for measuring rapid eye movements in mice. Neurosci. Res. 49, 123–131. Shimazawa, M., Suemori, S., Inokuchi, Y., Matsunaga, N., Nakajima, Y., Oka, T., Yamamoto, T., Hara, H., 2010. A novel calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-me thylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), reduces murine retinal cell death in vitro and in vivo. J. Pharmacol. Exp. Ther. 332, 380–387. Smith, A.W., Das, A., Guyton, M.K., Ray, S.K., Rohrer, B., Banik, N.L., 2011. Calpain inhibition attenuates apoptosis of retinal ganglion cells in acute optic neuritis. Invest. Ophthalmol. Vis. Sci. 52, 4935–4941. Urtti, A., 2006. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv. Drug Deliv. Rev. 58, 1131–1135. Vosler, P.S., Sun, D., Wang, S., Gao, Y., Kintner, D.B., Signore, A.P., Cao, G., Chen, J., 2009. Calcium dysregulation induces apoptosis-inducing factor release: cross-talk between PARP-1- and calpain-signaling pathways. Exp. Neurol. 218, 213–220. Wang, Y., Briz, V., Chishti, A., Bi, X., Baudry, M., 2013. Distinct roles for mu-calpain and mcalpain in synaptic NMDAR-mediated neuroprotection and extrasynaptic NMDARmediated neurodegeneration. J. Neurosci. 33, 18880–18892. Wang, L., Cioffi, G.A., Cull, G., Dong, J., Fortune, B., 2002. Immunohistologic evidence for retinal glial cell changes in human glaucoma. Invest. Ophthalmol. Vis. Sci. 43, 1088–1094. Wang, Y., Zhu, G., Briz, V., Hsu, Y.T., Bi, X., Baudry, M., 2014. A molecular brake controls the magnitude of long-term potentiation. Nat. Commun. 5, 3051. Wu, H.Y., Lynch, D.R., 2006. Calpain and synaptic function. Mol. Neurobiol. 33, 215–236. Xu, J., Kurup, P., Zhang, Y., Goebel-Goody, S.M., Wu, P.H., Hawasli, A.H., Baum, M.L., Bibb, J.A., Lombroso, P.J., 2009. Extrasynaptic NMDA receptors couple preferentially to excitotoxicity via calpain-mediated cleavage of STEP. J. Neurosci. 29, 9330–9343.
Contents lists available at ScienceDirect
Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi
Calpain-1 and calpain-2 play opposite roles in retinal ganglion cell degeneration induced by retinal ischemia/reperfusion injury Yubin Wang a, Dulce Lopez a, Pinakin Gunvant Davey b, D. Joshua Cameron b, Katherine Nguyen a, Jennifer Tran a, Elizabeth Marquez a, Yan Liu a, Xiaoning Bi c, Michel Baudry a,⁎ a b c
Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA, United States College of Optometry, Western University of Health Sciences, Pomona, CA, United States College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA, United States
a r t i c l e
i n f o
Article history: Received 4 March 2016 Revised 4 May 2016 Accepted 12 May 2016 Available online 13 May 2016 Keywords: Glaucoma Retinal ganglion cell Calpain Neuroprotection Blindness
a b s t r a c t Calpain has been shown to be involved in neurodegeneration, and in particular in retinal ganglion cell (RGC) death resulting from increased intraocular pressure (IOP) and ischemia. However, the specific roles of the two major calpain isoforms, calpain-1 and calpain-2, in RGC death have not been investigated. Here, we show that calpain-1 and calpain-2 were sequentially activated in RGC dendrites after acute IOP elevation. By combining the use of a selective calpain-2 inhibitor (C2I) and calpain-1 KO mice, we demonstrated that calpain-1 activity supported survival, while calpain-2 activity promoted cell death of RGCs after IOP elevation. Calpain-1 activation cleaved PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and activated the Akt pro-survival pathway, while calpain-2 activation cleaved striatal-enriched protein tyrosine phosphatase (STEP) and activated STEP-mediated pro-death pathway in RGCs after IOP elevation. Systemic or intravitreal C2I injection to wild-type mice 2 h after IOP elevation promoted RGC survival and improved visual function. Our data indicate that calpain1 and calpain-2 play opposite roles in high IOP-induced ischemic injury and that a selective calpain-2 inhibitor could prevent acute glaucoma-induced RGC death and blindness. © 2016 Published by Elsevier Inc.
1. Introduction Calpains are a family of calcium-dependent proteases regulating many cellular functions through the truncation of a number of proteins, resulting in alterations of their properties and functions (Ono and Sorimachi, 2012). In particular, overactivation of calpains has been shown to participate in many neurological disorders associated with neurodegeneration, such as traumatic brain injury, stroke, neurodegenerative disorders and aging (Liu et al., 2008; Vosler et al., 2009; Xu et al., 2009). However, some studies have indicated that, under certain conditions, calpain activation could provide neuroprotection (Jourdi et al., 2009; Wu and Lynch, 2006). The main calpain isoforms in the brain are calpain-1 and calpain-2 (aka μ-calpain and m-calpain), which differ in their calcium requirement for activation, as calpain-1 requires micromolar calcium vs. millimolar for calpain-2. Most studies investigating the role of calpains in neurodegeneration have not addressed the respective roles of calpain-1 and calpain-2, and of the downstream cascades leading to neuronal death. ⁎ Corresponding author at: Western University of Health Sciences, Pomona, CA 91766, United States. E-mail address: [email protected] (M. Baudry). Available online on ScienceDirect (www.sciencedirect.com).
http://dx.doi.org/10.1016/j.nbd.2016.05.007 0969-9961/© 2016 Published by Elsevier Inc.
We recently reported that calpain-1 and calpain-2 play opposite roles in synaptic plasticity as well as in neuroprotection/neurodegeneration (Wang et al., 2013; Wang et al., 2014). Calpain-1 activation following stimulation of synaptic NMDA receptors is required for the induction of long-term potentiation (LTP), a widely recognized cellular mechanism for learning and memory, and is also neuroprotective against starvation- or hydrogen peroxide-induced neuronal death in cultured neurons and against NMDA-mediated neurotoxicity in mouse hippocampal slices through the activation of the Akt pathway. Conversely, calpain-2 activation following LTP induction restricts the extent of LTP, and its activation following stimulation of extrasynaptic NMDA receptors in cultured neurons results in neurodegeneration through the cleavage of the phosphatase STEP (Wang et al., 2013; Wang et al., 2014). Primary angle-closure glaucoma (PACG) is a major cause of irreversible blindness worldwide and is expected to become more prevalent as lifespan increases. Angle-closure produces a rapid rise in intraocular pressure (IOP), which results in retinal ischemia and retinal ganglion cell (RGC) death. Several hypotheses have been proposed to account for RGC death, including apoptosis, mitochondrial dysfunction, lack of neurotrophin supply, excitotoxicity, and dysfunctional neuron-glia interactions (Almasieh et al., 2012; Wang et al., 2002). In addition, caspase and inflammasome activation and production of toxic cytokines (Chi et
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al., 2014), and deregulation of the autophagy process through calpainmediated truncation of beclin-1 have been reported (Russo et al., 2011). NMDA receptor blockade, as well as several calpain inhibitors reduced RGC loss after retinal ischemia in various animal species, including primates (Nakajima et al., 2006; Nucci et al., 2005; Oka et al., 2006; Sakamoto et al., 2006; Sakamoto et al., 2000; Shimazawa et al., 2010). However, none of these studies have specifically addressed the roles of calpain-1 and calpain-2 in the retina following ischemia. In this study, we combined the use of a selective calpain-2 inhibitor and calpain-1 knock-out (KO) mice to determine the time-courses of calpain-1 and calpain-2 activation and their functions in retina after acute IOP elevation. We found that calpain-1 is rapidly activated in RGC and promotes RGC survival, while calpain-2 activation is delayed and results in RGC death. We also found that a single systemic or intraocular injection of a selective calpain-2 inhibitor following increased IOP prevented RGC death and loss of vision in mice. These results suggest the possibility of developing selective calpain-2 inhibitors for the treatment of acute glaucoma.
antibodies overnight at 4 °C and then with secondary antibodies for 2 h at room temperature. Protein bands were visualized with the Odyssey imaging system (LI-COR). The primary antibodies used were spectrin (1:1000, MAB1622, Millipore), calpain-1 (1:1000, 2556, CST), STEP (1:1000, 4396, CST), PHLPP1 (1:1000, 07-1341, Millipore), phospho-Akt Ser473 (1:3000, 4060, CST) and Akt (1:2000, 2920). The secondary antibodies were IRDye secondary antibodies (1:10,000, LI-COR).
2. Materials and methods
At the indicted times after acute IOP elevation, eyes were enucleated, fixed in 4% paraformaldehyde for at least 1 day and dehydrated in 30% sucrose for at least 1 day. Six frozen sections (10 μm-thick) cut through the optic disc of each eye were collected. In order to determine in situ calpain activation, we used immunohistochemistry to label two calpain substrates, the spectrin breakdown product (SBDP) generated by calpain truncation of spectrin, and the Phosphatase and tensin homolog (PTEN). While SBDP is generated by activation of both calpain-1 and calpain-2, PTEN is selectively cleaved by calpain-2 (Briz et al., 2013). Sections were blocked in 0.1 M PBS containing 10% goat or donkey serum and 0.3% Triton X-100 for 1 h, and then incubated in rabbit anti-SBDP (1:500, a gift from Dr. Saido, Riken, Japan), mouse antiPTEN (1:600, 9556, Cell Signaling) to assess local activation of calpain1 and calpain-2. Brain-specific homeobox/POU domain protein 3A (brn-3a) has been shown to label retinal ganglion cells (Nakazawa et al., 2006) and we used goat anti-brn3a (1:100, sc-31984, Santa Cruz) primary antibodies in 0.1 M PBS containing 5% goat or donkey serum and 0.3% Triton X-100 overnight at 4 °C to selectively label RGCs after increased IOP. Sections were washed 3 times in PBS (10 min each) and incubated in Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies), Alexa Fluor 594 goat anti-mouse IgG or Alexa Fluor 555 donkey antigoat IgG (Life Technologies) for 2 h at room temperature. After three washes, sections were mounted with mounting medium containing DAPI (Vector Laboratories) and visualized using Nikon C1 confocal laser-scanning microscope. For quantification of SBDP and PTEN staining, 6 sections in each eye were analyzed. In each section, three 50 × 25 μm2 regions in IPL layer were selected and MFI (mean fluorescence intensities) were measured in ImageJ and averaged. Image acquisition and quantification were done by two persons in a blind fashion.
2.1. Animals Animal use in all experiments followed NIH guidelines and all protocols were approved by the Institution Animal Care and Use Committee of Western University of Health Sciences. Calpain-1 KO mice on a C57Bl/6 background were obtained from a breeding colony established from breeding pairs generously provided by Dr. Chishti (Tufts University). C57Bl/6 mice were purchased from Jackson Labs and were the corresponding WT. 2.2. Mouse retinal ischemia/reperfusion model Mouse retinal ischemia/reperfusion model was established following the protocol reported in previous publications (Buchi et al., 1991; Chi et al., 2014; Park et al., 2011). Mice were anesthetized with isoflurane and a gas anesthesia machine. Pupils were dilated with 1% tropicamide. The anterior chamber of the right eye was cannulated with a 33-gauge infusion needle connected to a normal saline reservoir, which was elevated to maintain an intraocular pressure of 110 mmHg for 60 min, which was confirmed using a Tonopen (Richert, Buffalo NY) (Reitsamer et al., 2004). Retinal ischemia was confirmed by whitening of the fundus. A sham procedure performed without elevating the pressure in the contralateral left eye was used as control. After 1 h, intraocular pressure was normalized during a 30-s period, and the needle was withdrawn. The animals were allowed to recover for various periods of time before sacrifice. 2.3. Spectral Domain Optical Coherence Tomography (SD-OCT) Spectral Domain optical coherence tomography (SD-OCT) was used to obtain images of the retina in anesthetized mice. Mice were anesthetized as above and SD-OCT images were obtained using Ivue (Optovue Inc. Fremont CA) with an anterior segment attachment (20 diopter lens). Images were quantitatively analyzed with the inbuilt software of the OCT, which allows for free-hand measurement of any structure imaged. 2.4. Western blot At various times after IOP elevation, mouse retinas were isolated and homogenized in RIPA buffer (89901, Thermo) with a protease inhibitor cocktail (78446, Thermo) and 5 mM EGTA. Protein concentrations were measured using the BCA protein assay kit (Thermo). Sample buffer was added, and samples were separated in SDS-PAGE and transferred onto PVDF membranes (IPFL00010, Millipore). After blocking with Odyssey® Blocking Buffer (LiCOR) for 1 h, membranes were incubated with primary
2.5. H&E staining in retinal vertical sections Mouse eyes were enucleated, fixed in 4% paraformaldehyde for at least 1 day and dehydrated in 30% sucrose for at least 1 day. Six frozen sections (10 μm-thick), cut through the optic disc of each eye, were collected and stained with Hematoxylin solution Harris modified (HHS32, Sigma) and Eosin Y solution aqueous (HT110232, Sigma). Retina images were photographed using light microscopy (Nikon). 2.6. Immunohistochemistry in retinal vertical sections
2.7. Cell counting in retinal vertical sections Cell counting was performed as previously reported (Shimazawa et al., 2010) with minor changes. Two 450 × 350 μm2 images were obtained at 500–1000 μm from the optic disc (both sides) in each retinal section stained with H&E or brn-3a antibody. Cell number in GCL of each image was counted. GCL length was measured using ImageJ (Freehand line). Cell density in GCL was calculated as the ratio of cell number to length (cell numbers/mm). Cell densities from 6 sections in each eye were averaged. Image acquisition and RGC counting were done by two persons in a blind fashion. 2.8. Immunohistochemistry and RGC counting in retinal whole-mounts Three days after glaucoma or sham surgery, mouse eyes were enucleated, fixed in 4% paraformaldehyde overnight. Retinas were then
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dissected as flattened whole-mounts and processed for brn-3a immunostaining as described above. Brn-3a-labeled RGCs were counted, as previously described (Nakazawa et al., 2006) with minor changes. Images of twelve 320 × 320 μm2 areas in each retina, including three areas per retinal quadrant at one-sixth (central retina), three-sixths (middle retina), and five-sixths (peripheral retina) of the retinal radius, were taken using a confocal microscope. The density of RGCs was defined as the average number of cells in the 12 areas. Cell counting was performed in a masked manner using the “analyze particles” function in ImageJ.
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1A). Some of these changes persisted over 3 days of observation. Eyes were collected at 0, 2, 4 and 6 h after IOP elevation and frozen retinal sections were prepared and processed for immunohistochemistry with an antibody against a spectrin breakdown product (SBDP), generated by calpain-1- or calpain-2-mediated spectrin cleavage. In WT mice, SBDP was clearly present in the inner plexiform layer (IPL), which
2.9. Intravitreal injection Mice were anesthetized with isoflurane and a gas anesthesia machine. Intravitreal injections were performed with a 33-gauge needle (0.5 in. long) attached to a 5 μl glass syringe (Hamilton). The needle was positioned 1 mm posterior to the limbus and 1 μl of the solution was slowly (3–5 s) injected into the vitreous chamber of the eye. A 20 s interval was kept before removing the needle. 2.10. OKR Visual Acuity measurement Visual acuity was measured by immobilizing the mouse head and restraining the mouse with a home-made OKR device, which was modified from a zebrafish device in order to accommodate a mouse, as previously published (Cahill and Nathans, 2008; Cameron et al., 2013; Sakatani and Isa, 2004). An infrared camera was used to monitor and record pupil movement and videos were analyzed with the Tracker video analysis and modeling tool. A 34.3 cm-diameter grating drum rotated around the mouse illuminated with a 200 lx-white light. The grating frequency was decreased using the staircase method until eye tracking ceased, identifying the smallest grating detected by the mouse's eye. The grating frequency was then converted into spatial acuity measured in cycles per degree (cpd) using the following formula: cpd = 1/ [2arctan (h/(2a))] where h is the grating cycle size in mm and a is the approximate distance from the center of the lens to the grating, also in mm. The range of spatial acuity we could test was 0.04–0.9 cpd. If the eye could not track the lowest spatial frequency (0.04 cpd), the acuity of this eye was recorded as 0.04 cpd. The clockwise rotation of gratings triggered OKR of the left eye (OS), while counterclockwise rotation triggered OKR of the right eye (OD) (Douglas et al., 2005). Thus, visual acuity of OS was measured when gratings rotated clockwise while OD was measured when gratings rotated counterclockwise. 2.11. Statistical analyses In all cases, error bars indicate standard error of the mean. N values represent numbers of eyes tested. To compute p values, unpaired student's t-test and one-way ANOVA followed by Bonferroni test were used, as indicated in figure legends. 3. Results 3.1. Sequential activation of calpain-1 and calpain-2 in retina after acute IOP elevation We implemented a widely used retinal ischemia/reperfusion model that produces features of acute angle closure glaucoma (Buchi et al., 1991; Chi et al., 2014; Osborne et al., 2004; Park et al., 2011). Intraocular pressure (IOP) was increased to 110 mmHg for 60 min by inserting a needle connected to an elevated reservoir of saline into the anterior chamber (see Materials and methods). This model reproduces several features of acute angle closure, including ischemia of retina and iris, as noted by the absence of red reflex and pupillary response to light. Anterior chamber synechae, resulting in a narrow angle and adhesion between the iris and the cornea, increased cells and flare in the anterior chamber and increased corneal thickness due to corneal edema (Fig.
Fig. 1. Time course of calpain-1 and calpain-2 activation in retina after acute IOP elevation. (A) Representative images of mouse eyes obtained with spectral domain optical coherence tomography (SD-OCT) following elevated IOP. Top left panel: image obtained prior to elevated IOP. The white arrow points to open angle with normal corneal anatomy and anterior chamber. Top right panel: 1 day after inducing IOP elevation for 1 h; the anterior chamber synechae is visible (white arrow) along with increased hyper reflectivity in anterior chamber (red arrow) indicating breakdown of blood aqueous barrier, which is caused by proteins and cells in the anterior chamber. Also note the increased corneal thickness (yellow arrow). Lower left panel: day two, corneal thickness and anterior camber reflectivity are decreased as compared to day 1, but anterior synechae is still present. Lower right Panel: day 3, showing marked decrease in cornea thickness and anterior chamber reflectivity; the synechae is now broken. (B) Immunostaining of SBDP (green) in ganglion cell layer (GCL) and inner plexiform layer (IPL) of retina in WT, calpain-1 KO and C2I-injected WT mice at 0, 2, 4 and 6 h after acute IOP elevation. Sections were counterstained with DAPI (blue). C2I (0.3 mg/kg) was injected i.p. to WT mice 2 h after IOP elevation. Scale bar = 20 μm. (C) Quantification of SBDP staining in IPL, n = 3–5 (eyes) at each time point. For each eye, 3 retinal sections were used for quantification. For each section, three 50 × 25 μm regions in the IPL were selected and MFI (mean fluorescence intensities) were measured and averaged. *p b 0.05, **p b 0.01, ***p b 0.001 versus control in the same group, One-way ANOVA followed by Bonferroni test.
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contains RGC dendrites, at 2, 4 and 6 h after IOP elevation. However in calpain-1 KO mice, SBDP was only evident in the IPL at 4 and 6 h but not at 2 h (Fig. 1B,C). These results suggest that calpain-1 is activated early and that calpain-2 activation is delayed in the IPL after IOP elevation. We then tested the effects of a calpain-2 selective inhibitor (C2I), Z-Leu-Abu-CONH-CH2-C6H3 (3, 5-(OMe)2) (Wang et al., 2013; Wang et al., 2014), injected intraperitoneally (i.p., 0.3 mg/kg) to WT mice 2 h after IOP elevation. C2I injection significantly reduced SBDP immunoreactivity in the IPL at 4 and 6 h after IOP elevation (Fig. 1B,C), confirming that calpain-2 was activated in the IPL following increased IOP. To further analyze the time course of calpain-2 activation in retina after increased IOP, retinal sections were immunostained with an antibody against full-length PTEN, a substrate of calpain-2 but not of calpain-1 (Briz et al., 2013). In both WT and calpain-1 KO mice, PTENimmunoreactivity in the IPL was unchanged at 2 h, but was significantly reduced at 4 and 6 h after IOP elevation (Fig. 2), confirming that calpain-2 was activated at 4 and 6 but not at 2 h after IOP elevation. When C2I was injected to WT mice 2 h after IOP elevation, PTEN degradation at 4 and 6 h was completely blocked (Fig. 2). Altogether, these results strongly suggest that calpain-1 is briefly activated in RGC dendrites after acute IOP elevation, while calpain-2 activation in the same dendrites is delayed and prolonged. 3.2. Calpain-1 and calpain-2 play opposite roles in RGC death induced by acute IOP elevation To evaluate elevated IOP-induced retinal damage, IOP of the right eye was elevated to 110 mmHg for 60 min, while a sham procedure was performed in the left eye. Retinal vertical sections were collected 3 days after surgery and H&E staining was performed to examine cell
numbers in the ganglion cell layer (GCL) (Fig. 3A,B). In WT mice injected with vehicle (i.p., 10% DMSO in PBS), cell counts in the right GCL were 62.1 ± 5.6 cells/mm, as compared to 113.4 ± 7.1 cells/mm in the left GCL (n = 7). We used three different protocols to examine the effect of C2I. First, C2I (0.3 mg/kg) was injected (i.p.) 30 min before and 2 h after acute IOP elevation (pre and post inj). Cell counts in GCL of sham eye and IOP-elevated eye were 125.1 ± 10.5 and 105.8 ± 4.5 cells/mm, respectively (n = 3, no significant difference (ns) sham vs. IOP). Second, C2I was injected (i.p.) 2 h after IOP elevation (one post inj). Cell counts in sham and IOP-elevated eye were 110.6 ± 3.6 and 86.4 ± 7.0 cells/mm (n = 10, ns). Third, C2I was injected (i.p.) 2 and 4 h after IOP elevation (two post inj). Cell counts in sham and IOP-elevated eye were 118.6 ± 3.7 and 96.1 ± 6.3 cells/mm (n = 6, ns). In all three C2I-injected groups, cell survival rate (ratio of cell count in IOP-elevated eye to that in sham eye) was significantly increased, as compared to vehicle-injected group (Fig. 3C). These results suggest that calpain-2 activation plays an important role in GCL cell death after IOP elevation and that C2I systemic injection has a protective effect against IOP-induced cell death. In calpain-1 KO mice, cell count in GCL of IOP-elevated eye was significantly lower than that of sham eye (37.7 ± 10.4 vs. 130.3 ± 7.0 cells/mm, n = 4). Importantly, cell survival rate in calpain-1 KO mice was significantly lower than that in WT mice (Fig. 3C), again suggesting that calpain-1 supports cell survival in GCL after IOP elevation. To specifically examine the effect of C2I on RGCs, which constitute approximately 40% of the cells in mouse GCL (Jeon et al., 1998), retinal sections from WT mice injected with vehicle or C2I 2 h after acute IOP elevation were immunostained with an antibody against brn-3a, a selective RGC marker (Nadal-Nicolas et al., 2009) (Fig. 3D). In WT mice injected with vehicle, RGC counts in sham eye and IOP-elevated eye were 40.9 ± 5.2 and 19.9 ± 3.4 cells/mm (p b 0.01 sham vs. IOP, n = 4). In WT mice injected with C2I, RGC counts in sham eye and IOP-elevated eye were 45.2 ± 5.0 and 37.1 ± 2.5 cells/mm (ns, sham vs. IOP, n = 5) (Fig. 3E). The survival rate of RGC with C2I injection was significantly improved, as compared to vehicle injection (Fig. 3F), suggesting that C2I systemic injection protects RGC against IOP-induced cell death. To explore the nature of the signaling pathways downstream of calpain-1 and calpain-2, retinas in WT, calpain-1 KO and C2I-injected WT mice were collected 3 h after IOP elevation or sham surgery, homogenized and aliquots processed for Western blots (Fig. 4A,B). In WT mice, levels of PH domain and Leucine-rich repeat Protein Phosphatase 1 (PHLPP1), a phosphatase downstream of calpain-1 (Wang et al., 2013), were significantly reduced, while levels of phospho-Akt Ser473 (pAkt), which can be dephosphorylated by PHLPP1, were significantly increased after IOP elevation. These changes in PHLPP1 and pAkt were absent in calpain-1 KO mice but present in C2I-injected WT mice, suggesting that calpain-1 but not calpain-2 mediates PHLPP1 degradation and Akt activation in retina after IOP elevation. STEP33, the product of calpain-2-mediated cleavage of striatal-enriched protein tyrosine phosphatase (STEP) (Wang et al., 2013), was present in WT and calpain-1 KO mice but not in C2I-injected WT mice following increased IOP, indicating that calpain-2 but not calpain-1 mediates STEP cleavage after IOP elevation. These results suggest that both calpain-1/PHLPP1/Akt prosurvival pathway and calpain-2/STEP pro-death pathway (Wang et al., 2013) are engaged in retina after IOP elevation, and that C2I selectively inhibits calpain-2/STEP pro-death pathway. 3.3. Intravitreal injection of C2I reduces cell death in GCL and prevents loss of vision caused by acute IOP elevation
Fig. 2. Time course of calpain-2 activation in retina after acute IOP elevation. (A) Immunostaining of full-length PTEN (red) in GCL and IPL of retina in WT, calpain-1 KO and C2I-injected WT mice at 0, 2, 4 and 6 h after acute IOP elevation. Sections were counterstained with DAPI (blue). C2I was injected to WT mice 2 h after IOP elevation. Scale bar = 20 μm. (B) Quantification of PTEN staining, n = 3–5 (eyes) at each time point. *p b 0.05, **p b 0.01 versus control in the same group, One-way ANOVA followed by Bonferroni test.
We used intravitreal C2I injection in order to locally deliver C2I to retina. First, we tested the delivery efficiency by injecting different doses of C2I intravitreally 2 h after IOP elevation in calpain-1 KO mice and analyzing SBDP levels in the IPL at 4 h (Fig. 5A,B). A clear dose-dependent inhibition of SBDP formation was observed, providing an apparent IC50 of 8 μM for C2I. In all subsequent experiments, we used 20 μM (1 μl) to examine the neuroprotective effects of intravitreal C2I
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injection. Eyes were collected 3 days after surgery for HbE staining. After vehicle injection (10% DMSO in PBS), GCL counts in sham eye and IOPelevated eye were 132.3 ± 4.5 and 62.0 ± 5.7 cells/mm (p b 0.001
Fig. 4. Calpain-1 activation cleaves PHLPP1 while calpain-2 activation cleaves STEP in retinal ganglion cells after acute IOP elevation. (A) Representative immunoblots of indicated proteins in retina of WT, calpain-1 KO and C2I-injected mice collected 3 h after sham surgery or acute IOP elevation. C2I (0.3 mg/kg) was injected systemically 2 h after sham surgery or IOP elevation. (B) Quantitative analysis of the levels of PHLPP1 and STEP33 and ratios of pAkt/Akt for each group. Results represent means ± S.E.M. of 4 experiments. *p b 0.05, **p b 0.01, ***p b 0.001, ns no significant difference, One-way ANOVA followed by Bonferroni test.
Fig. 3. Calpain-2 inhibition reduces, while calpain-1 knockout exacerbates cell death in ganglion cell layer induced by acute IOP elevation. (A) H&E staining of retinal sections from the right eye of vehicle- or C2I-injected wild-type and calpain-1 KO mice and the left eye where sham surgery was performed. Vehicle, 10% DMSO in PBS, was injected i.p. 30 min before and 2 h after acute IOP elevation. Pre- and post-injection C2I (0.3 mg/kg) was done i.p. 30 min before and 2 h after acute IOP elevation. For the One post injection group, C2I was injected 2 h after IOP elevation. For the Two post inj group, C2I was injected 2 and 4 h after IOP elevation. H&E staining was performed 3 days after surgery. Scale bar = 30 μm. (B) Quantification of H&E staining. Data represent means ± S.E.M. n = 7 (eyes) for vehicle, n = 3 for pre and post inj, n = 10 for one post inj, n = 6 for two post inj, n = 4 for KO. ns, no significant difference. ***p b 0.001, Two-tailed t-test. (C) Survival rate for each mouse was calculated as the ratio of cell density in GCL of IOPelevated eye to cell density in GCL of sham eye. *p b 0.05, **p b 0.01 versus vehicle, Oneway ANOVA followed by Bonferroni test. (D) Brn-3a immunostaining in the retina of vehicle- or C2I-injected WT mice 3 days after surgery. Vehicle, 10% DMSO, or C2I, C2I (0.3 mg/kg) was injected i.p. 2 h after acute IOP elevation. Scale bar = 60 μm. (E) Brn3apositive cells in GCL were counted. n = 4 for vehicle, n = 5 for C2I. Ns, no significant difference, **p b 0.01 sham versus IOP elevation, two-tailed t-test. (F) Comparison of survival rates. n = 4 for vehicle, n = 5 for C2I. **p b 0.01 vehicle versus C2I, two-tailed t-test.
sham vs. IOP, n = 4). In C2I-treated eyes, GCL counts in sham eye and IOP-elevated eyes were 128.1 ± 7.2 and 101.3 ± 9.2 cells/mm (no significant difference, sham vs. IOP, n = 5) (Fig. 5C,D). Survival rate with C2I injection was significantly improved, as compared to vehicle injection (80.8 ± 8.4% vs. 47.2 ± 5.4%, p b 0.01) (Fig. 5E). To further confirm that RGCs were protected against IOP-induced degeneration by C2I injection, retinal whole-mounts were prepared and immunostained with brn-3a antibody to label RGCs 3 days after treatment, and average RGC densities in retina whole-mounts were quantified. After intravitreal vehicle injection, RGC densities in sham eye and IOP elevated eye were 2.3 ± 0.2 and 0.6 ± 0.3 103 cells/mm2 (p b 0.001, n = 4). A large proportion of RGCs degenerated leaving cell debris in retina after IOP elevation (Fig. 5F,G). After intravitreal C2I injection, RGC densities in sham eye and IOP elevated eyes were 2.4 ± 0.2 and 1.6 ± 0.6 103 cells/mm2 (no significant difference, n = 4). The difference in RGC densities between eyes treated with vehicle or C2I following increased IOP was highly significant (p b 0.01, n = 4) (Fig. 5G). The above results indicate that intravitreal C2I injection 2 h after IOP elevation is neuroprotective against IOP-induced RGC death. To examine vision of mice after glaucoma, we tested their optokinetic reflex (OKR). OKR is the saccadic eye movement triggered by the movement of gratings in front of the mouse eye. Changing the frequency of gratings and determining the lowest frequency triggering OKR, allows analyzing visual acuity of each eye. Acute IOP elevation or sham surgery was performed in the right eye (OD). Left eye (OS) served as a naive control. Intravitreal C2I or vehicle injection was performed 2 h after surgery. Three and 21 days after surgery, OKR was determined in both eyes (Fig. 6A,B). Visual acuity of sham eye with vehicle injection was 0.47 ± 0.11 cpd (mean ± SEM, n = 7) at day 3 and 0.41 ± 0.16 (n = 7) at day 21, in good agreement with published results (Douglas et al., 2005; Prusky et al., 2004). Visual acuity was dramatically reduced after increased IOP at both time points, which was significantly improved by C2I injection. C2I injection in the sham eye did not affect visual acuity, as compared to vehicle injection. Mice were sacrificed after OKR test at day 21 and RGC densities were analyzed with brn-3a
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4. Discussion
Fig. 5. Intravitreal injection of C2I reduces cell death in ganglion cell layer induced by acute IOP elevation. (A) SBDP immunostaining in retina of calpain-1 KO mice after acute IOP elevation. Vehicle (10% DMSO in PBS, 1 μl) or different doses of C2I (2–80 μM, 1 μl) were injected intravitreally 2 h after IOP elevation. Eyes were collected 4 h after IOP elevation for SBDP staining. Scale bar = 20 μm. (B) Quantification of SBDP staining in IPL. N = 3 (eyes) at each concentration. The inhibition of SBDP signal was calculated by (MFIVehicleMFIC2I)/MFIVehicle %. Data represent means ± S.E.M. (C) H&E staining in retina of WT mice after IOP elevation. Vehicle or C2I (20 μM, 1 μl) was injected 2 h after IOP elevation or sham surgery. Eyes were collected 3 days after surgery for H&E staining. Scale bar = 30 μm. (D) Quantification of H&E staining. n = 7 for naive, n = 4 for vehicle, n = 6 for C2I. *p b 0.05, ***p b 0.001, two-tailed t-test. (E) Comparison of survival rates. *p b 0.05, **p b 0.01, One-way ANOVA followed by Bonferroni test. (F) Brn-3a immunostaining in retina whole-mounts of WT mice after IOP elevation or sham surgery. Vehicle or C2I (20 μM, 1 μl) was injected 2 h after surgery. Eyes were collected 3 days after the surgery. Representative images show area of middle retina. Scale bar = 100 μm. (G) Quantification of brn-3a positive cell numbers in retina whole-mounts under different treatments. The cell density in each retina was calculated as the average cell density of twelve 320 × 320 μm2 areas at central, middle and peripheral retina. N = 4. **p b 0.01, ***p b 0.001. Ns, no significant difference. Two-way ANOVA followed by Sidak's multiple comparisons test.
immunostaining in retinal sections (Fig. 6C). As expected, RGC densities were significantly reduced in eyes treated with vehicle after increased IOP, but recovered in eyes treated with C2I after increased IOP. Moreover, visual acuity was highly correlated with the number of surviving RGCs (Fig. 6D), further supporting the prominent role of calpain-2 in triggering RGC death after increased IOP.
While several mechanisms have been involved in RGC death in acute glaucoma (Almasieh et al., 2012; Chi et al., 2014; Wang et al., 2002), including calpain activation (Russo et al., 2011), our results clearly indicate that calpain-1 and calpain-2 play opposite functions in increased IOP-induced retinal damage. Thus, calpain-1 is neuroprotective, since retinal damage is exacerbated in calpain-1 KO mice, as compared to WT mice. This effect is due to the activation by calpain-1 of a survival pathway previously identified in a different system (Wang et al., 2013), calpain-1 mediated cleavage of PHLPP1 leading to activation of the prosurvival Akt pathway. On the other hand, calpain-2 is neurodegenerative, as evidenced by the significant protection against retinal damage provided by a selective calpain-2 inhibitor. The differential functions of the two calpain isoforms are due in part to their differential time-course of activation, and their different signaling pathways. Thus, calpain-1 is rapidly and briefly activated following increased IOP, and stimulates a pro-survival pathway, probably due to the rapid and transient activation of synaptic NMDA receptors, composed of GluN2A subunits, as we previously reported (Wang et al., 2014). In contrast, calpain-2 activation is delayed and more prolonged. It is activated between 2 and 4 h after increased IOP, which could be due to the stimulation of extrasynaptic NMDA receptors, composed of GluN2B subunits, as a result of glutamate spill-over or inhibition of glutamate transport known to take place following ischemia (Brassai et al., 2015; Parsons and Raymond, 2014). It also triggers the degradation and inhibition of the phosphatase STEP, which activates STEP substrate p38 and results in neurodegeneration (Wang et al., 2013; Xu et al., 2009). This interpretation is consistent with the differential roles of GluN2A- and GluN2Bcontaining NMDA receptors in NMDA-induced neurotoxicity in retina (Bai et al., 2013). We exploited the differential temporal activation of calpain-1 and calpain-2 to show that systemic or intravitreal injection of a selective calpain-2 inhibitor 2 h after IOP elevation completely inhibited calpain-2 activation and provided significant protection against retinal damage. Furthermore, this treatment also restored vision following increased IOP to a level not significantly different from that of shamoperated eyes, as assessed with OKR sensitivity. Interestingly, the neuroprotection provided by the single injection of the calpain-2 inhibitor was still present 3 weeks following increased IOP, indicating that calpain-2 activation is the trigger of the neurodegenerative events. Our results thus indicate that calpain-2 activation is a critical step in RGC death following acute IOP elevation, possibly the converging node downstream of other pathways previously implicated in RGC neurodegeneration (Almasieh et al., 2012). Calpain has previously been implicated in retinal damage following ischemia or other types of insults (Nakajima et al., 2006; Oka et al., 2006; Sakamoto et al., 2000; Shimazawa et al., 2010). However, the lack of understanding of the different roles of the two major calpain isoforms in retina and in other regions of central nervous system is probably the reason why non-selective calpain inhibitors have never reached clinical trials, although they have shown some protection against retinal damage in certain animal models of eye disorders (Das et al., 2013; Sakamoto et al., 2000; Shimazawa et al., 2010; Smith et al., 2011). In addition to reducing optic nerve damage and RGC apoptosis, calpain inhibition reduced astrogliosis and microgliosis in acute optic neuritis(Das et al., 2013; Smith et al., 2011). Whether C2I has a similar effect in the acute glaucoma model remains to be determined. In any event, our results underscore the need to use a selective calpain-2 inhibitor to reduce retinal damage after a variety of insults. Any type of ocular drug delivery methods will inevitably cause systemic absorption of the drug (Urtti, 2006). Ocular delivery of non-selective calpain inhibitors may inhibit the physiological function of calpain-1 such as neuroprotection in retina and learning and memory. In contrast, local delivery of a selective calpain-2 inhibitor may avoid those negative side effects.
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Fig. 6. Intravitreal injection of calpain-2 inhibitor protects vision assessed with the optokinetic reflex. (A) OKR spatial frequency thresholds of eyes measured 3 days after IOP elevation or sham surgery. Vehicle or C2I (20 μM, 1 μl) was injected intravitreally 2 h after surgery. Surgery and injection were always performed in the right eye (OD). OKR of the Left eye (OS) was measured as control. n = 7. ****p b 0.0001, **p b 0.01. One-way ANOVA followed by Bonferroni test. (B) OKR was re-measured 21 days after surgery. n = 7. ****p b 0.0001, *p b 0.05. (C) RGC density in the retina of the eyes at 21 days after surgery. Eyes were collected after the OKR test. Brn-3a immunostaining was performed. Brn-3a-positive cells in GCL were counted. n = 7 for IOP elevation + C2I. n = 4 for other groups. ***p b 0.001, **p b 0.01. (D) Linear regression analysis of OKR spatial frequency thresholds and RGC densities measured 21 days after surgery. Black symbol, data from Sham plus Vehicle group; Blue, Sham plus C2I. Red, IOP elevation plus Vehicle. Green, IOP elevation plus C2I. N = 19. R2 = 0.86.
While the ischemia/reperfusion model we used reproduces several features of PACG, it also exhibits some limitations, and the relationship between the actual events taking place in human PACG and in our model remains to be completely validated. In human PACG, IOP does not rise and drop as quickly as in the model used in this study. Nevertheless, there is consensus regarding the major events leading to RCG death in PACG, including ischemia, overactivation of glutamate receptors and activation of neurodegenerative cascades, which also take place in our models. The identification of the neuroprotective role of calpain-1 and the neurodegenerative function of calpain-2 combined with the neuroprotective effects of a selective calpain-2 inhibitor administered 2 h after the increase in IOP in our model strengthen the notion that it might be possible to develop new treatments for a number of neurodegenerative eye disorders previously linked to calpain activation. Thus, selective targeting of calpain-2 may provide a potential therapeutic approach for these different eye disorders. Of particular interest will be chronic open angle and angle closure glaucoma. Although the disease process varies in different types of glaucoma, the consequences of elevated IOP and the pathway to RGC death are extremely similar. Thus, the next steps will be to evaluate the neuroprotective efficacy of selective calpain-2 inhibitors, along with other standard IOP-lowering therapy, on vision preservation in models of chronic glaucoma. To our knowledge, a calpain inhibitor has never been tested in clinical trials for the treatment of eye disorders. On the other hand, Abbvie is currently carrying a Phase I clinical trial with an orally active non-selective calpain inhibitor for the treatment of Alzheimer's disease (https:// clinicaltrials.gov/ct2/show/NCT02220738?term=Abbvie+and+ABT957&rank=1). As our results indicate that a single intraocular administration of a selective calpain-2 inhibitor is neuroprotective when delivered 2 h after the increased IOP, the path to testing this or similar compounds in PACG could be relatively straightforward and lead to the validation of the critical role of calpain-2 in PACG.
5. Conclusion Calpain-1 and calpain-2 play opposite functions following ischemic injury in the retina. While calpain-1 is rapidly activated its activation is neuroprotective through the stimulation of the Akt pathway, as a result of cleavage of PHLPP1. Thus, lack of calpain-1 exacerbates ischemic injury in the retina. On the other hand, calpain-2 activation is delayed and prolonged and is neurodegenerative, as a result of STEP truncation. A selective calpain-2 inhibitor injected either systemically or intraocularly 2 h after the ischemic episode protects retinal ganglion cells and maintains vision to normal levels. These findings suggest that selective targeting of calpain-2 might produce better neuroprotection and less side-effects than non-selective calpain inhibitors not only for the treatment of eye disorders but also for other neurodegenerative conditions.
Authors contributions YW performed the majority of experiments and wrote the manuscript; DL participated in many experiments, PGD performed the OCT studies, DJC helped with the OKR experiments, KN, JT, EM and YL helped with many experiments, XB designed experiments and wrote the manuscript, MB designed the project and wrote the manuscript.
Conflict of interest The authors have declared that no conflict of interest exists. A patent entitled “Isoform-Selective Calpain Inhibitors, Methods of Identification, and Uses Thereof” has been filed by Western University of Health Sciences to the USPTO.
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Acknowledgements This work was supported by grant P01NS045260 from NINDS (PI: Dr. C.M. Gall). The authors want to thank Western University of Health Sciences for the financial support to MB. XB is also supported by funds from the Daljit and Elaine Sarkaria Chair. The authors want to acknowledge the generous gift of the calpain-1 KO mice from Dr. A. Chishti (Tufts University), and the technical assistance of Mr. Joseph Gray and Mr. Farouk Bruce from the College of Optometry. References Almasieh, M., Wilson, A.M., Morquette, B., Cueva Vargas, J.L., Di Polo, A., 2012. The molecular basis of retinal ganglion cell death in glaucoma. Prog. Retin. Eye Res. 31, 152–181. Bai, N., Aida, T., Yanagisawa, M., Katou, S., Sakimura, K., Mishina, M., Tanaka, K., 2013. NMDA receptor subunits have different roles in NMDA-induced neurotoxicity in the retina. Mol. Brain 6, 1. Brassai, A., Suvanjeiev, R.-G., Bán, E.-G., Lakatos, M., 2015. Role of synaptic and nonsynaptic glutamate receptors in ischaemia induced neurotoxicity. Brain Res. Bull. 112, 1–6. Briz, V., Hsu, Y.T., Li, Y., Lee, E., Bi, X., Baudry, M., 2013. Calpain-2-mediated PTEN degradation contributes to BDNF-induced stimulation of dendritic protein synthesis. J. Neurosci. 33, 4317–4328. Buchi, E.R., Suivaizdis, I., Fu, J., 1991. Pressure-induced retinal ischemia in rats: an experimental model for quantitative study. Ophthalmologica 203, 138–147. Cahill, H., Nathans, J., 2008. The optokinetic reflex as a tool for quantitative analyses of nervous system function in mice: application to genetic and drug-induced variation. PLoS ONE 3, e2055. Cameron, D.J., Rassamdana, F., Tam, P., Dang, K., Yanez, C., Ghaemmaghami, S., Dehkordi, M.I., 2013. The optokinetic response as a quantitative measure of visual acuity in zebrafish. J. Vis. Exp. Chi, W., Li, F., Chen, H., Wang, Y., Zhu, Y., Yang, X., Zhu, J., Wu, F., Ouyang, H., Ge, J., Weinreb, R.N., Zhang, K., Zhuo, Y., 2014. Caspase-8 promotes NLRP1/NLRP3 inflammasome activation and IL-1beta production in acute glaucoma. Proc. Natl. Acad. Sci. U. S. A. 111, 11181–11186. Das, A., Guyton, M.K., Smith, A., Wallace, G., McDowell, M.L., Matzelle, D.D., Ray, S.K., Banik, N.L., 2013. Calpain inhibitor attenuated optic nerve damage in acute optic neuritis in rats. J. Neurochem. 124, 133–146. Douglas, R.M., Alam, N.M., Silver, B.D., McGill, T.J., Tschetter, W.W., Prusky, G.T., 2005. Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system. Vis. Neurosci. 22, 677–684. Jeon, C.J., Strettoi, E., Masland, R.H., 1998. The major cell populations of the mouse retina. J. Neurosci. 18, 8936–8946. Jourdi, H., Hsu, Y.T., Zhou, M., Qin, Q., Bi, X., Baudry, M., 2009. Positive AMPA receptor modulation rapidly stimulates BDNF release and increases dendritic mRNA translation. J. Neurosci. 29, 8688–8697. Liu, J., Liu, M.C., Wang, K.K., 2008. Calpain in the CNS: from synaptic function to neurotoxicity. Sci. Signal. 1, re1. Nadal-Nicolas, F.M., Jimenez-Lopez, M., Sobrado-Calvo, P., Nieto-Lopez, L., CanovasMartinez, I., Salinas-Navarro, M., Vidal-Sanz, M., Agudo, M., 2009. Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas. Invest. Ophthalmol. Vis. Sci. 50, 3860–3868. Nakajima, E., David, L.L., Bystrom, C., Shearer, T.R., Azuma, M., 2006. Calpain-specific proteolysis in primate retina: contribution of calpains in cell death. Invest. Ophthalmol. Vis. Sci. 47, 5469–5475. Nakazawa, T., Nakazawa, C., Matsubara, A., Noda, K., Hisatomi, T., She, H., Michaud, N., Hafezi-Moghadam, A., Miller, J.W., Benowitz, L.I., 2006. Tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma. J. Neurosci. 26, 12633–12641.
Nucci, C., Tartaglione, R., Rombola, L., Morrone, L.A., Fazzi, E., Bagetta, G., 2005. Neurochemical evidence to implicate elevated glutamate in the mechanisms of high intraocular pressure (IOP)-induced retinal ganglion cell death in rat. Neurotoxicology 26, 935–941. Oka, T., Tamada, Y., Nakajima, E., Shearer, T.R., Azuma, M., 2006. Presence of calpain-induced proteolysis in retinal degeneration and dysfunction in a rat model of acute ocular hypertension. J. Neurosci. Res. 83, 1342–1351. Ono, Y., Sorimachi, H., 2012. Calpains: an elaborate proteolytic system. Biochim. Biophys. Acta 1824, 224–236. Osborne, N.N., Casson, R.J., Wood, J.P., Chidlow, G., Graham, M., Melena, J., 2004. Retinal ischemia: mechanisms of damage and potential therapeutic strategies. Prog. Retin. Eye Res. 23, 91–147. Park, S.W., Kim, K.Y., Lindsey, J.D., Dai, Y., Heo, H., Nguyen, D.H., Ellisman, M.H., Weinreb, R.N., Ju, W.K., 2011. A selective inhibitor of drp1, mdivi-1, increases retinal ganglion cell survival in acute ischemic mouse retina. Invest. Ophthalmol. Vis. Sci. 52, 2837–2843. Parsons, M.P., Raymond, L.A., 2014. Extrasynaptic NMDA receptor involvement in central nervous system disorders. Neuron 82, 279–293. Prusky, G.T., Alam, N.M., Beekman, S., Douglas, R.M., 2004. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest. Ophthalmol. Vis. Sci. 45, 4611–4616. Reitsamer, H.A., Kiel, J.W., Harrison, J.M., Ransom, N.L., McKinnon, S.J., 2004. Tonopen measurement of intraocular pressure in mice. Exp. Eye Res. 78, 799–804. Russo, R., Berliocchi, L., Adornetto, A., Varano, G.P., Cavaliere, F., Nucci, C., Rotiroti, D., Morrone, L.A., Bagetta, G., Corasaniti, M.T., 2011. Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. Cell Death Dis. 2, e144. Sakamoto, Y.R., Nakajima, T.R., Fukiage, C.R., Sakai, O.R., Yoshida, Y.R., Azuma, M.R., Shearer, T.R., 2000. Involvement of calpain isoforms in ischemia-reperfusion injury in rat retina. Curr. Eye Res. 21, 571–580. Sakamoto, K., Yonoki, Y., Kubota, Y., Kuwagata, M., Saito, M., Nakahara, T., Ishii, K., 2006. Inducible nitric oxide synthase inhibitors abolished histological protection by late ischemic preconditioning in rat retina. Exp. Eye Res. 82, 512–518. Sakatani, T., Isa, T., 2004. PC-based high-speed video-oculography for measuring rapid eye movements in mice. Neurosci. Res. 49, 123–131. Shimazawa, M., Suemori, S., Inokuchi, Y., Matsunaga, N., Nakajima, Y., Oka, T., Yamamoto, T., Hara, H., 2010. A novel calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-me thylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), reduces murine retinal cell death in vitro and in vivo. J. Pharmacol. Exp. Ther. 332, 380–387. Smith, A.W., Das, A., Guyton, M.K., Ray, S.K., Rohrer, B., Banik, N.L., 2011. Calpain inhibition attenuates apoptosis of retinal ganglion cells in acute optic neuritis. Invest. Ophthalmol. Vis. Sci. 52, 4935–4941. Urtti, A., 2006. Challenges and obstacles of ocular pharmacokinetics and drug delivery. Adv. Drug Deliv. Rev. 58, 1131–1135. Vosler, P.S., Sun, D., Wang, S., Gao, Y., Kintner, D.B., Signore, A.P., Cao, G., Chen, J., 2009. Calcium dysregulation induces apoptosis-inducing factor release: cross-talk between PARP-1- and calpain-signaling pathways. Exp. Neurol. 218, 213–220. Wang, Y., Briz, V., Chishti, A., Bi, X., Baudry, M., 2013. Distinct roles for mu-calpain and mcalpain in synaptic NMDAR-mediated neuroprotection and extrasynaptic NMDARmediated neurodegeneration. J. Neurosci. 33, 18880–18892. Wang, L., Cioffi, G.A., Cull, G., Dong, J., Fortune, B., 2002. Immunohistologic evidence for retinal glial cell changes in human glaucoma. Invest. Ophthalmol. Vis. Sci. 43, 1088–1094. Wang, Y., Zhu, G., Briz, V., Hsu, Y.T., Bi, X., Baudry, M., 2014. A molecular brake controls the magnitude of long-term potentiation. Nat. Commun. 5, 3051. Wu, H.Y., Lynch, D.R., 2006. Calpain and synaptic function. Mol. Neurobiol. 33, 215–236. Xu, J., Kurup, P., Zhang, Y., Goebel-Goody, S.M., Wu, P.H., Hawasli, A.H., Baum, M.L., Bibb, J.A., Lombroso, P.J., 2009. Extrasynaptic NMDA receptors couple preferentially to excitotoxicity via calpain-mediated cleavage of STEP. J. Neurosci. 29, 9330–9343.