Male C57BL/6J mice and B6. Cg-Rptortm1.1Dmsa/J (The Jackson Laboratory, 013188) were housed in the Animal Center of the Tongji Medical College. All experimental animals were given free access to food and water in a 12-h light/12-h dark cycle environment. All animals were treated under protocols approved by ARVO Statement for the Use of Animals in Vision and Ophthalmic Research and the institutional IACUC committees of Huazhong University of Science and Technology. Animals of 2-month-old weighing 20-25g were used in all experiments.
Reagents and antibodies
Retinoic acid was obtained from Sigma-Aldrich China (Shanghai, China; R2625). Rapamycin was purchased from MedChemExpress (Shanghai, China; HY-10219). CTB-Alexa 488 and CTB-Alexa 555 were bought from BrainVTA (Wuhan, Hubei, China). The following antibodies were used in western blot: anti-CD82 (ab66400; Abcam; 1:1000); anti-synaptophysin (ab14692; Abcam; 1:1000); anti-p70S6k (2708T; Cell Signaling Technology; 1:1000); anti-p-p70S6k(9234T;Cell Signaling Technology; 1:1000); anti-β-actin (sc-47778; Santa Cruz; 1:1000); anti-TRAF2 (ab126758; Abcam; 1:1000); anti-Raptor (20984-1-AP; Proteintech; 1:1000).The following antibodies were used in immunofluorescence: anti-CD82 (ab66400; Abcam; 1:200); anti-Tuj1 (801201; Biolegend; 1:1000); anti-Iba1(ab48004;Abcam;1:100); anti-GAFP(ab4674; Abcam;1:100); anti-mCherry (ab167453; Abcam; 1:100); anti-synaptophysin (ab14692; Abcam; 1:200); anti-pS6(Ser 240/244)(5364T; Cell Signaling Technology; 1:1000); anti-TRAF2 (ab126758; Abcam; 1:400); anti-Raptor (20984-1-AP; Proteintech; 1:1000); anti-Lamp1 (15665, Cell Signaling Technology; 1:500).
Anti-β-Amyloid (2450; Cell Signaling Technology; 1:100) was used in immunohistochemistry.
Adeno-associated virus administration
rAAV2/9-hsyn-Cd82-2A-mCherry-WPRE-PA, rAAV2/9-hsyn-mCherry-WPRE-PA, rAAV2/9-hsyn-Cre-EGFP-WPRE-PA, rAAV2/9-hsyn-EGFP-WPRE-PA were obtained from BrainVTA (Wuhan, Hubei, China). Each virus preparation contained approximately 2.0x1012 genome copies/ml. One microliter of AAV was injected in one eye of each animal in the vitreous cavity using a 35-G needle with a 10-μl Hamilton microsyringe (Hamilton, Reno, NV, USA) at a constant rate over 30 s. The needle was held in place for 60 s to allow for intraocular pressure equilibration before removal. Animals were used for subsequent experiments three weeks after AAV injection.
Animal model of Acute Ocular Hypertension
Mouse model of acute glaucoma was performed as previously described(33). Animals undergoing surgery were anesthetized by intraperitoneal injections of 5% chloral hydrate (9 ml/kg). The corneas were topically anesthetized with 0.5% tetracaine hydrochloride, and the pupils were dilated with 1% tropicamide. A 30-gauge infusion needle connected to a standard saline reservoir was used to insert into the anterior chamber of 1 eye. The saline reservoir was elevated to a height of 1.2m for 1 hour. Whitening of the iris and loss of the red reflex suggested the retinal ischemia, and subsequent reperfusion was evident by the red reflex return. The other eye was served as a control with a sham procedure performed without elevating the pressure. After the process, eyes were covered with antibiotic ointment to prevent corneal desiccation and bacterial infection. The animals were allowed to recover for 8, 24, 48, 72 h or 7 days before sacrifice.
Optic nerve crush
Mice were anesthetized by intraperitoneal injections of 5% chloral hydrate (9 ml/kg), and eyes were locally anesthetized using 0.5% tetracaine hydrochloride. A small conjunctival incision was made in the superior posterior area using micro-scissors, and eye muscles were then carefully moved. The optic nerve was exposed intraorbitally and crushed by fine self-closing forceps for 5 s approximately 0.5mm behind the optic disc without damaging the underlying ophthalmic artery. Eyes were covered with antibiotic ointment to protect the cornea after surgery. Mice were euthanized at day 7 post-injury for RGC survival analysis and day 14 or day 28 for axonal regeneration evaluation.
Eyes for retina cross-sectional preparation were fixed in 4% paraformaldehyde (pH= 7.4) as whole globes at RT for 2 days. All specimens were embedded in paraffin to enable ONH to be cut in parallel with ON longitudinal axis. The paraffin-embedded retinal sections (5 μm) were gently washed three times with phosphate-buffered saline (PBS) pre-heated to 37℃ and then blocked in 5% donkey serum albumin for 1 hour to avoid nonspecific binding. Afterward, the sections were incubated in diluted primary antibodies at 4℃ overnight. Retinal paraffin sections were washed three times before incubated with the conjugated secondary antibody in 5% bovine serum albumin (RT, 90 min).
Eyes for retinal flat mount were dissected and fixed in 4% paraformaldehyde (pH= 7.4) for 2h at room temperature. The posterior segments of the eye were cut into a ‘petal’ shape with 4 to 5 radial incisions, and then the retinas could be carefully detached. Leave the retinas to cold methanol for at least 20 min to facilitate permeabilization. After rinsed in PBS, retinas were blocked in normal donkey serum for 1h at room temperature and then incubated for 48-72 h at 4℃ with primary antibodies. Afterward, retinas were washed thoroughly in PBS three times and incubated with secondary antibodies at room temperature for 90 min. Finally, retinas were transferred onto slides and mounted with glycerol.
The sections and flat mounts were examined with a laser scanning confocal microscope (Zeiss LSM 710; Zeiss, Oberkochen, Germany) under excitation wavelengths of 405 nm for DAPI, 488 nm for FITC, and 594 nm for cy3, respectively.
For colocalization analysis, Mander’s colocalization coefficient (MCC) was calculated using the ImageJ plugin ’JACoP’.
Evaluation of anterograde axon transport by CTB
Mice were anesthetized by 5% chloral hydrate and mydriasis with 1% tropicamide.1 μl CTB-Alexa 488 (BrainVTA, Wuhan, Hubei, China) was intravitreally injected in one eye using a 10-μl Hamilton microsyringe (Hamilton, Reno, NV, USA). 48h after the injection, animals were anesthetized and sacrificed via cardiac perfusion of normal saline and 4% PFA. Brains and eyes were post-fixed in 4% PFA for an additional 24 h, dehydrated with 30% sucrose in PBS overnight prior to embedding in OCT (Tissue-Tek, Sakura Finetek Inc, Tokyo, Japan). Brains were continuously sliced into 30 μm sections in area of superior colliculus. Eyes were sectioned into 10μm with ONH. Alexa 488 was visualized using a fluorescent microscope (Olympus, Tokyo, Japan).
Axon labeling for regeneration
CTB-Alexa 555 (BrainVTA, Wuhan, Hubei, China) was intravitreally injected 48 h before sacrifice to trace regenerating RGC axons 14- or 28-days post-injury. After 4% PFA perfusion, mice eyes and optic nerves were microdissected and post-fixed for 3 h in 4% PFA. The nerves were dehydrated with 30% sucrose in PBS overnight at 4℃ and embedded in OCT Compound (Tissue-Tek, Sakura Finetek Inc, Tokyo, Japan) for cryosection. Optic nerves were cut longitudinally at a thickness of 10 µm and mounted onto slides. Alexa 555 was imaged using a fluorescent microscope (Olympus, Tokyo, Japan). Regeneration axons were quantified by counting the number of CTB-labeled fibers extended past every 500 µm division from the crush site. The total number of regeneration axons in each optic nerve was estimated using the equation elaborated in the literature(34).
Electron microscopyand analysis
Eyes with optic nerves attached were carefully dissected from the orbit. A 1.5mm section of optic nerve proximal to the globe was isolated and fixed in ice-cold 2.5% glutaraldehyde in 0.1M cacodylate solution. Tissue embedding and ultrathin sectioning were processed as described(35). Sections were examined and photographed with HITACHI H-7000FA TEM.
Axons were classified into three categories according to the condition of the myelin sheath, representing the different degrees of axonal degenerative change. Five non-overlapping visual fields of each section were randomly selected, and the frequency of different degenerative axons was calculated. Observers conducting assessments were masked to the experimental conditions of the images.
Both eyes were injected with AAV-Cd82 or vehicle three weeks before binocular optic nerve crush. Visual function tests were performed on day 14 and day 28 post-injury. For the dark light preference test, apparatus consisting of a small dark chamber and a large illuminated chamber (550 lumens) with a door allowed mice to move freely for 10 min. The time spent in each compartment was recorded by a camera, and the time ratio was calculated automatically by SuperMaze Software (XinRuan Information Technology, Shanghai). For the optomotor response test, the visual acuity of mice was measured based on an innate visual-motor reflex using a testing chamber(36) and the software OptoTrack from XinRuan Information Technology (Shanghai, China). Mice moved freely on a platform located in the center of an area surrounded by four screens displaying a moving vertical sinusoidal grating pattern. The spatial frequency started from 0.01 to 0.06 cycles per degree with constant rotation speed (12°/s) and 100% contrast to determine the spatial frequency threshold at which the mice still tracked the moving grid. Different testing frequencies occurred randomly and repeated 10 times within one test to reduce the occasional error. Observers were blinded to the group of mice.
Cell culture and transfection
HEK-293T cells were purchased from Boster Biologic Technology. SH-SY5Y cells were given as a gift from the Department of Neurobiology, Tongji Medical College, Huazhong University of Science and Technology.
Cells were maintained in high-glucose DMEM (Hyclone Laboratories, Logan, UT, USA) supplemented with 10% FBS (Gibco, CA, USA) and penicillin (100 U/ml)/streptomycin (100 μg/ml) at 37°C in a humidified atmosphere with air containing 5% CO2. Confluent cell layers were split every three days.
HEK293 and SH-SY5Y cells were transfected with Flag-Cd82 plasmid/vehicle and siTraf2/siNC using Lipofectamine 3000 (Thermo Fisher Scientific) according to the transfection protocol. Cells were cultured for another 48-72 h for further biochemical analyses. Flag-Cd82 plasmid was constructed by cloning the corresponding cDNA into the pcDNA3-Flag vector. TRAF2 siRNA duplexes (5’ -CGACAUGAACAUCGCAAGC-3’) with 30 dTdT overhangs were synthesized at Tsingke biological technology.
Neurite outgrowth assay
RA (all-trans-retinoic acid, Sigma) was used to induce cell differentiation and neurite outgrowth as indicated previously(37). Briefly, SH-SY5Y cells with proper confluence were cultured in 1%FBS medium supplemented with 10μM RA for 7days prior to treatment. On day 4, the medium was replaced with fresh differentiation medium, and on day 7, cells were used for subsequent experiments.
Oxidative stress induced by hydrogen peroxide exposure was indicated to cause axonal degeneration in vitro(38). SH-SY5Y cells were exposed to 100μM hydrogen peroxide for 8h to inhibit neurite outgrowth and recovered in normal medium for another 12h before morphological analysis.
The formation of neurites was observed using an inverted IX71 microscope system (Olympus, Tokyo, Japan). The neurite length of each cell was measured by Image J software plugin ’AxonJ’. The average length of neurites and the ratio of cells with different neurite lengths were analyzed.
Drug handling and administration
Retinoic acid (R2625, Sigma) powder was prepared in DMSO at 3 mg/ml (0.01M) as stock solution and stored in light-protected vials at -80℃. Tissue culture medium was used to dilute the stock solution at a final concentration of 10 µM to induce differentiation of SH-SY5Y cells.
For animal experiments, rapamycin (HY-10219, MedChemExpress) was dissolved in DMSO at 20mg/ml for temporary storage in -20℃. Before each administration, rapamycin stock solution was diluted in sterile saline solution and given intraperitoneally at 6 mg/kg every two days. For cell experiments, rapamycin was dissolved in DMSO at 20mM. Subsequent dilutions were made in growth medium with a final concentration of 100nM and maintained for 2 hours before cells were harvested.
PPI Network visualization
To identify new interaction partners and the corresponding interaction networks between CD82 and mTORC1, we used the online database Molecular Interaction Search Tool (MIST; http://fgrtools.hms.harvard.edu/MIST/)(39)and visualized the protein-protein interaction networks by Cytoscape software 3.8.0(40).
Protein extraction, immunoblots and immunoprecipitation
Cells and tissue from retina, ONH and ON were lysed in RIPA buffer (Applygen Technologies, Beijing China) respectively at designed time-points. BCA Protein Assay Reagent (Boster Biologic Technology) was used to quantify protein concentration. For immunoprecipitation, the same amounts of whole-cell lysate were incubated with the primary antibodies (0.5-2μg) overnight at 4℃. Protein A/G sepharose beads (P2012, Beyotime Biotechnology) were added into the incubation tubes, and the mixture was incubated at 4℃ with gentle shaking for 3 hours. The precipitated complexes were washed five times with RIPA buffer and then mixed with loading buffer (Boster Biologic Technology) and boiled for 5 min. For western blot analysis, equivalent amounts of total protein or immunoprecipitate were fractionated by SDS-PAGE and then transferred to PVDF membrane (MilliporeSigma). Membranes were blocked at room temperature using 5% nonfat milk in TBST buffer for 1 h and treated overnight at 4°C with diluted primary antibodies. The following day membranes were washed three times with TBST before incubated with horseradish peroxidase-coupled secondary antibodies for 60 min at room temperature. Immunoreactive bands were detected with a chemiluminescence substrate kit (ECLPlus; PerkinElmer Inc, Covina, CA, USA) prior to exposure using either film or digital detection equipment (BLT GelView 6000 pro). Target protein expression levels were quantified using ImageJ software normalized to β-actin or GAPDH level.
RNA isolation and Real-time quantitative PCR
Total RNA was extracted with RNAiso plus (Takara Biomedical Technology, Beijing, China). RNA concentration and quality were assessed on NanoDrop 2000 (Thermo Fisher Scientific). The eligible RNA samples were reverse-transcribed with PrimeScript™RT reagent Kit (RR047A; Takara Biomedical Technology) according to the manufacturer’s instructions. The amplified cDNA templates were diluted and used for quantitative PCR with TB GreenPremix Ex Taq (RR420A; Takara Biomedical Technology) on the Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific). Primer sequences were designed as follows:
Cd82: forward 5′-TGTCCTGCAAACCTCCTCCS-3’, reverse5′-CCATGAGCATAGTGACTGCCC-3′;
Traf2: forward 5′-GCTCATGCTGACCGAATGTC-3’,
reverse 5’- GCCGTCACAAGTTAAGGGGAA-3’;
Gapdh: forward 5′-GGAGTCCACTGGCGTCTTCA-3’,
reverse 5’- GTCATGAGTCCTTCCACGATACC-3’.
All samples were run in triplicate with blank controls. The relative expression of target genes was calculated by the 2-ΔΔCT method with normalization against Gapdh level.
The results are expressed by mean ± S.E.M from at least three independent experiments, specific sample size was indicated in the context. Graphing and statistical analysis were performed in statistical software Prism (v.7.03; GraphPad Software, La Jolla, CA, USA). Differences within the experimental groups were assessed by Student’s t-test or one-way analysis of variance (ANOVA). P values were considered significant for P < 0.05.