Calpain Inhibition Restores Basal Autophagy and Suppresses MNU-induced Photoreceptor Cell Death in Mice

Materials and Methods

Animals. Six-week-old, female BALB/c mice were purchased from Charles River Japan (Atsugi, Japan). Animals were housed in groups of 8 in plastic cages with paper bedding (Paper Clean, SLC, Hamamatsu, Japan), in a room maintained at 22±2°C and 60±10% relative humidity with a 12-h light/dark cycle (with lights on from 8:00 in the morning to 8:00 at night and an illumination intensity less than 60 lux at the cage level). Animals were maintained on a commercial pellet diet (CMF 30 kGy, Oriental Yeast, Chiba, Japan) and had free access to water. After a 1-week acclimatization period, experiments were begun when mice were 7 weeks old. Throughout the experiments, animals were cared for in accordance with the Guidelines for Animal Experimentation of Kansai Medical University.

Chemicals. MNU in a powder form was obtained from Sigma-Aldrich (St. Louis, MO, USA), stored at 4°C in the dark, and dissolved in physiologic saline containing 0.05% acetic acid (5 mg/ml) immediately before use. A powder form of calpain inhibitor (1S-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl) amino) carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945) was a gift from Senju Pharmaceutical Co. Ltd. (Kobe, Japan). SNJ-1945 was suspended at 0.8% (w/v) in distilled water containing 0.5% carboxymethyl cellulose.

Experimental procedure. Female mice received a single intraperitoneal (IP) injection of 60 mg/kg MNU at 7 weeks of age (75 mg/kg MNU was given in sequential apoptosis and/or autophagy detection experiments); the dose of MNU was determined from published data (4). Animals were sacrificed by cervical dislocation. The time at sacrifice and the number of mice examined at each time point are described below under each experiment. A dose of 80 mg/kg SNJ-1945 was administered IP 3 h prior to MNU and thereafter once daily until sacrifice. Control mice received the same volume of vehicle solution (distilled water containing 0.5% carboxymethyl cellulose) at the same time points.

Western blotting. In all experiments, protein extracts were prepared from 4 retinas (one from each of 4 mice sacrificed at each time point) by homogenization in lysis buffer solution (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% deoxycholate-Na, 0.1% sodium dodecylsulfate, and 1% protease inhibitor (Halt protease inhibitor cocktail; Thermo-Pierce, Rockford, IL, USA). The homogenates were sonicated and centrifuged at 18,000 × g. The supernatants were collected, and the pellets were discarded. Protein concentrations were determined by the DC protein assay method (Bio-Rad, Hercules, CA, USA). Aliquots of lysates equivalent to 15 μg of protein were subjected to electrophoresis through 15% polyacrylamide gels for the detection of microtubule-associated protein light chain 3 (LC3) and caspase-3, 10% polyacrylamide gels for the detection of autophagy-related protein 12 (Atg12)-Atg5 and full-length Atg5, and 4-15% gels (Mini-Protean TGX precast gel; Bio-Rad) for the detection of α-spectrin and cleaved Atg5. Then, the separated proteins were transferred to Hybond-P PVDF membranes (Amersham Biosciences, Buckinghamshire, UK). The membranes were blocked with 5% non-fat powdered milk prior to treatment with each primary antibody. Subsequently, the blots were washed and treated with an appropriate secondary antibody. The antigen-antibody complexes were detected by ECL system (Amersham Biosciences). The following antibodies were used: anti-cleaved caspase-3 antibody (clone 5A1E; Cell Signaling Technology, Danvers, MA, USA); anti-LC3 antibody (polyclonal; Abgent, San Diego, CA, USA); anti-α-spectrin antibody (nonerythroid) (clone AA6; Enzo, Farmingdale, NY, USA); two polyclonal anti-Atg5 antibodies, one from Cell Signaling Technology (Danvers, MA, USA) for Atg12-Atg5 conjugate detection and the other from Santa Cruz Biotechnology (Santa Cruz, CA, USA) for full-length and truncated Atg5 detection; and HRP-conjugated anti-actin antibody (polyclonal; Santa Cruz Biotechnology). To evaluate caspase-3 and LC3 expression over time, retinal samples of MNU-exposed mice were obtained at 6, 12, 24, and 72 h after MNU treatment and compared with age-matched, MNU-unexposed, control retinas. Retinal samples for Atg5 evaluation were obtained 24 h after MNU injection and compared with age-matched control retinas. For α-spectrin and LC3 evaluation, respectively, age-matched control retinas and MNU-exposed mice treated with SNJ-1945 or vehicle were obtained 24 hrs after MNU injection.

Tissue fixation and processing. Four eyes (one from each of the 4 MNU-exposed mice and age-matched untreated controls) were obtained at each time point and were fixed in 10% neutral buffered formalin overnight to examine the morphologic progression of the disease and to detect cell death. Six eyes (one from each of 6 MNU-exposed mice treated with SNJ-1945 or vehicle) were obtained 7 days after MNU treatment, together with age-matched controls. The eyes were fixed in methacarn (60% methanol, 30% chloroform and 10% acetic acid) overnight and used for morphometrical analysis. The formalin-fixed and methacarn-fixed samples were embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (HE). Sections including the ora serrata and optic nerve were used for the evaluation.

Morphometric analysis of photoreceptor cell ratio and retinal damage ratio. As described previously (5, 12), NDP.view was used to measure the total retinal thickness (from the internal limiting membrane to the pigment epithelial cell) and photoreceptor thickness (the outer nuclear layer and the inner and outer segments) in HE preparations. The measurements were collected at the peripheral retina (approximately 400 μm from both sides of the ciliary body) and central retina (approximately 400 μm from the optic nerve). The photoreceptor cell ratio was calculated as [(photoreceptor thickness/total retinal thickness)×100]. To determine the area of retinal damage, the entire length of retina and the length of the damaged area were measured. A damaged retina was defined as the presence of less than four rows of photoreceptor cell nuclei in the outer nuclear layer, and the retinal damage ratio was calculated as [(length of damaged retina/whole retinal length) ×100].

In situ detection of apoptosis. Formalin-fixed, paraffin-embedded sections obtained 72 h after MNU treatment and the age-matched controls were used for apoptosis detection. Apoptosis was observed by TUNEL, with an in situ apoptosis detection kit (Apop-Tag; Millipore, Bellerica, MA, USA). The reaction products were visualized with 3,3’-diaminobenzidine as the chromogen.

Electron microscopy. Retinas collected 72 h after MNU exposure, as well as age-matched control retinas, were fixed in 5% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 2% OsO₄, and embedded in Luveac-812. Semi-thin sections stained with toluidine blue were used to locate areas, and ultrathin sections stained with uranyl acetate and lead citrate were examined with a JEOL JEM-1400A electron microscope (JEOL, Tokyo, Japan).

Statistics. All in vitro experiments were performed at least in triplicate. All data are presented as mean±SEM. Caspase-3 and LC3-II expression was analyzed by one-way ANOVA followed by Dunnett's test, Atg12-Atg5 and Atg5 expression was analyzed by the Student's t-test, and the rest of the data were compared by one-way ANOVA followed by Tukey's test. All statistical analyses were performed with GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). P-values less than 0.05 were considered statistically significant.