Peptides
Peptain-1 and peptain-3a were obtained from Peptide 2.0 (>95% pure, Chantilly, VA). All peptides used in this study were confirmed by mass spectrometry to have the expected molecular weights. For in vivo studies, peptains were dissolved in a small amount of DMSO and then diluted in PBS to the desired concentration. The final concentration of DMSO was <0.1%. For in vitro tests, peptains were dissolved in PBS.
Chaperone activity
The chaperone activity of peptain-1 and peptain-3a was assessed using insulin (Cat# I5500, Sigma–Aldrich, St. Louis, MO, USA) and citrate synthase (Cat# C3260, Sigma–Aldrich) as client proteins, as previously described (38). Insulin (0.3 mg/mL) and dithiothreitol (20 mM, Cat# D9163, Sigma–Aldrich) were incubated in 50 mM phosphate buffer, pH 7.4, at 25 °C for 1 h in the presence or absence of peptain-1 (20-80 μM) or peptain-3a (0.07-8.7 μM). Citrate synthase (0.167 mg/mL) was incubated in 10 mM HEPES buffer, pH 7.4, at 43 °C for 1 h in the presence or absence of peptain-1 or peptain-3a. The assay volume in each case was 200 μl. The kinetic profile of aggregation was monitored by measuring light scattering at 360 nm in a 96-well microplate reader (SpectraMax 190, Molecular Devices, Sunnyvale, CA).
Primary RGC apoptosis assay
The isolation of rat primary RGCs from postnatal day 4-6 Sprague–Dawley pups was performed according to a previously published protocol (69). The purity of the culture was found to be between 90%-95%. RGCs were seeded onto glass coverslips, and after 5 days of culture, cells were incubated with vehicle (DPBS), peptain-1 (12.5 μg/μl), or peptain-3a (1.25 μg/μl) in either DMEM with high glucose (no trophic factors) or in full RGC (Sato) media (with trophic factors). After 48 h of incubation at 37 °C with 10% CO2, the Image-iT LIVE Green Caspase-3 and -7 Detection Kit (Cat# I35106, Invitrogen) was used to stain cells with FLICA (labeling caspase-3 and -7 in apoptotic cells) and with Hoechst, labeling all cells, and propidium iodide (PI, labeling dead cells). Briefly, RGCs were incubated with 1x FLICA for 60 min followed by Hoechst/PI for 5 min. The cells were then washed and imaged using a Cytation5 Cell Imaging Multimode Reader (Agilent, Santa Clara, CA). Manual cell counting was performed using ImageJ software for Hoechst-positive cells with 0.3-1.0 circularity, which passed the threshold (determined by the level of background to ensure that only true positives were counted). Cells costained with PI and FLICA were categorized into cells undergoing apoptosis. Cells labelled only with PI were marked as dead. The experiment was repeated three times. The data are presented as the percentages of apoptotic and dead cells compared to controls (vehicle-treated group).
Animals
All animal experiments were reviewed and approved by the University of Colorado and North Texas Eye Research Institute, UNT Health Science Center’s Institutional Animal Care and Use Committee and performed under adherence to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Both male and female wild-type mice were obtained from Jackson Laboratories (C57BL/6J, Stock No: 000664, Bar Harbor, ME, USA) or bred in-house with a 12:12-h light:dark cycle with ad libitum food and water.
Retinal ischemia–reperfusion (I/R) injury
I/R injury was performed as previously described (70). Briefly, mice were anesthetized with an intraperitoneal injection of ketamine/xylazine, eyes were anesthetized with topical 0.5% proparacaine hydrochloride ophthalmic solution (Akorn, Somerset, New Jersey), and anesthesia was confirmed by the toe-pinch pain test. The animals were then placed on a heating pad to maintain their body temperature throughout the procedure. The anterior chamber of the right eye of each mouse was cannulated with a 33-gauge needle connected to an elevated saline reservoir. The height of the reservoir was adjusted to achieve an IOP of 120 mm Hg. After 60 min, the needle was rapidly removed. Peptain-1 or peptain-3a (1 μl from 1 mg/mL stock) was intravitreally injected immediately after I/R injury and after 2 days. The control group was injected with 1 μl PBS containing 0.1% DMSO. The animals were euthanized on day 14 post- I/R injury.
MB-induced ocular hypertensive mouse model
Mice were anesthetized as described above. Ocular hypertension was induced unilaterally by injection of polystyrene MB (10-μm diameter, FluoSpheres, Invitrogen, Carlsbad, CA) into the anterior chamber of the right eye of each animal, as previously described (71). Briefly, MB was reformulated at a concentration of 5x106 beads/mL in PBS. The cornea was gently punctured near the center using a 33 G needle, and a small air bubble was injected to deepen the anterior chamber. MB (2 μl) was injected into the anterior chamber under the bubble via a blunt 33 G needle connected to a 10 μl Hamilton syringe. Antibiotic ophthalmic ointment (Akorn, Inc. Cat# AP704009, Lake Forest, IL) was applied topically to the injected eye to prevent infection. IOP was monitored once weekly for 6 weeks using the TonoLab tonometer (Colonial Medical Supply, Espoo, Finland). Three weeks after MB injection, peptain-1 or peptain-3a was injected intravitreally (1 µg in 1 µL PBS), and then subsequently once a week for 3 weeks.
Induction of IOP elevation by injection of SO
Mice were anesthetized as described above. SO-induced reversible ocular hypertension was induced as previously described (52). Briefly, a 33 G needle was inserted superotemporally into the anterior chamber without injuring the lens or iris. Through this puncture, approximately 2 μl SO (1,000 mPa.s, Silikon, Alcon Laboratories, Fort Worth, Texas) was gradually injected intracamerally until the oil droplet expanded to cover most of the iris, and the needle was then slowly withdrawn. After the injection, ophthalmic antibiotic ointment was applied to the surface of the injected eye. After 2 weeks, the SO droplet was removed from the anterior chamber by aspiration using a 33 G needle. PBS was then injected into the anterior chamber using a 33 G needle attached to a 3 mL Luer lock syringe to allow SO outflow through an inferior tunnel incision. Antibiotic ophthalmic ointment was applied topically to the eye after SO removal. The IOP of the eye was monitored once weekly for 4 weeks after SO injection using the TonoLab tonometer under anesthesia (5% isoflurane at 2 L/min mixed with oxygen). Peptains were injected intravitreally 2 days after SO removal.
Whole mount immunostaining of mouse retina
The animals were euthanized at the indicated times, and the eyes were enucleated and fixed with 4% PFA overnight at 4 °C. The next day, the retinas were dissected out and washed extensively in PBS before blocking (5% normal donkey serum and 1% Triton X-100 in PBS) overnight. The whole-mounted retinas were immunostained with Brn3a (1∶400 dilution, Cat# MAB1585, EMD Millipore, Bedford, MA) and βIII-tubulin antibodies (1:400 dilution, Cat# T8578, Sigma–Aldrich, St. Louis, MO) as markers for RGCs or Iba1 (1:400 dilution, Cat# ab178846, Abcam, Cambridge, MA) or GFAP (1:400 dilution, Cat# 12389, Cell Signaling Technology) antibodies as markers for activated microglia and activated astrocytes, respectively. After 3 days, Alexa Fluor 488-conjugated donkey anti-mouse (1:250 dilution, Cat# A-11001, Invitrogen), Alexa Fluor 488-conjugated donkey anti-rabbit (1:250 dilution, Cat# A-21206, Invitrogen), or Texas red-conjugated goat anti-rabbit IgG (1:250 dilution, Cat# T-2767, Invitrogen) antibody was incubated overnight at 4 °C. Four fields from mid-peripheral regions of the retina were imaged using a confocal microscope (Nikon ECLIPSE Ti, Japan).
Axonal transportation
CT-B, a neuronal tracer, was used to assess anterograde axonal transport as previously described (29). Mice were anesthetized and intravitreally injected with 1 μl of 0.2% CT-B (Alexa Fluor 555-conjugate, Thermo Fisher, Cat# C22843) a day before euthanasia. After 24 h, the mice were sacrificed, and the optic nerves were dissected, fixed in 4% PFA overnight, and dehydrated in methanol for 5 min. The optic nerves were cleared by incubation with Visikol® HISTO-1™ for 1 week, then transferred to Visikol® HISTO-2™ and incubated for an additional 1 week. The optic nerves were imaged using confocal microscopy along the entire length to the optic chiasm and stitching with 15% overlap. The mean fluorescence intensities were calculated using ImageJ software (NIH).
Statistical analysis
GraphPad Prism software version 9.2.0 (GraphPad Prism Software, Inc., San Diego, CA) was used for statistical analyses. We used one-way ANOVA and multicomparison tests to determine the significance of the differences among the animal groups. A p value of <0.05 was considered statistically significant.