Supporting Information

Animal procedures

DTMR labeling

After sectioning the ON a piece of spongostan with absorbed 0.1% DTMR (Dextran-tetramethylrodamine, Molecular Probes) was placed on the ON stump. The diameter of each DTMR labelled cell was measured and any cell with a rounded soma and diameter exceeding 8 mm was regarded as RGCs 36. Ganglion cells were easily differentiated from microglia using morphological criteria.

Rapamycin administration

Rapamycin (Euromedex) was dissolved at 10 mM in DMSO and diluted in 5% Tween 80, 5% polyethylene glycol 400, NaCl 0.9% before each administration. Rapamycin at 7.5 mg/kg or the vehicle (DMSO in 5% Tween 80, 5% polyethylene glycol 400, NaCl 0.9%) were given intraperitoneally two days before the ONT, and every day until the euthanasia.

Adenoviral infections

AAV2-GFP and AAV2-Cre-GFP were purchaesed from Vector Biolabs, and intraocular injected in Atg5flox/flox mice. Animals were anesthetized and the left eyes were injected with a needle of 35 gauge in the lateral zone 1mm behind sclero-corneal limbus, just behind the ora serrata. The trajectory of needle was visualized under microscopy and 1 mL of adenovirus were injected deliberately angled to avoid damage to the lens. The animals subjected to the optic nerve axotomy were kept during 10 days after the adenoviral injection prior to the surgery.

In vivo DHE labeling

Axotomyzed mice were injected with 25 mg/kg dihydroethidium (DHE, Invitrogen) dissolved in DMSO and diluted in 5% Tween 80, 5% polyethylene glycol 400, NaCl 0.9% 18 hours before euthanasia. Retinas were isolated, fixed and confocal images focusing in the GCL were taken from control and axotomyzed eyes. All images were acquired with a TCS SP2 confocal microscope (Leica Microsystems) using a 63X oil objective. The red channel settings were kept the same for all images so that the red channel signal intensities could be used to compare the levels of DHE probe in the uninjured and axotomized retinas.

Determination of autophagy induction in GFP-LC3 retinas using confocal microscopy

Autophagosomes were identified as green dots in the cytosol of RGCs using confocal microscopy with a 63X objective and a 2X digital zoom and analyzing the images in 0.5 μm confocal planes besides the maximal projections. Positive cells were identified according to the following criteria: (1) the presence of GFP-LC3-labeled puncta in the cytosol, (2) a clear increase in the endogenous levels of the green fluorescence (that could mask the punctuate staining of the APs), (3) and an evident exclusion of the green fluorescence from the nucleus. In figure 3B left panel displays several negative cells from a control retina, note the typical green homogeneous fluorescence distributed in the whole cell. The right panel displays an autophagosome-positive cell from a 6-day axotomized animal.

Western blot

Retinas were lysed in 200 μL of the extraction buffer containing 50 mM Tris-HCl pH 6.8, glycerol 10% (v/v), 2% SDS (w/v), sodium fluoride (1 μM), sodium orthovanadate (1 mM) and sodium pirofosfate (2 mM) and EDTA-free protease inhibitor cocktail using a 25 gauge needle for 15 times in ice. The samples were boiled at 100 ºC for 10 minutes and the insoluble fractions were excluded by centrifugation at 5000 rpm for 5 min. The protein concentrations of the samples were measured using a BCA Protein Assay Reagent kit following the protocol of the manufacturer. Right before loading the samples into the gels 10 mM DTT and 0.005% bromophenol blue were added to forty micrograms of proteins and resolved on a 15% SDS-PAGE gel. The proteins were then transferred to PVDF membranes (Bio-Rad, Hercules) that were blocked for 1 h in PBS-Tween 20 (0.05% (v/v)) containing 5% non-fat milk and probed with antibodies against β-III Tubulin (Covance), LC3 (MBL Internacional), Beclin-1 (Santa Cruz Biotechnology), P62 (Biomol), Ubiquitin (Santa Cruz Biotechnology), β-actin (Sigma) and GAPDH (Abcam). The antibodies were detected with the appropriate horseradish peroxidase-labelled secondary antibodies (Pierce, Rockford) and were visualised with the SuperSignal West Pico chemioluminiscent substrate (Pierce). Densitometric analysis was performed with Quantity One software (Bio-Rad).

Transmission electron microscopy

Retinas were fixed for 4 h at 4 ºC in Karnowsky buffer (4% paraformaldehyde (w/v) and 2.5% glutaraldehyde (v/v) in cacodylate buffer (Sigma) pH 7.4), washed and fixed again in aqueous 5% (w/v) osmium tetroxide, 1% potassium ferricianide, dehydrated and immersed in propylene oxide 10 minutes for three times and embedded in Epon resin (EMS). The resin was polymerized in a 50°C oven overnight. Sectioning for electron microscopic examination followed was accomplished with an ultramicrotome (Vitracut E, Reichert-Jung) and electron microscopy was performed with a Zeiss EM 902 transmission electron microscope (Germany), at 90 kV, on ultra-thin sections (50 nm) stained with uranyl acetate and lead citrate.


Supplementary Figure 1: (A) Immunoblot analysis of Brn-3a and the neuronal marker β-III-tubulin at different time points after ONT, C-control eye, A-axotomied eye. (B) Quantification of the total number of cells in the GCL stained with DAPI in control and injured retinas. (C) Intranucleosomal DNA fragmentation determined by ELISA in control and axotomized retinas at different time points after the injury.

Supplementary Figure 2: (A) Confocal representative image of Brn-3a immunostaining in a 6 day-axotomized retina to show that the GFP-LC3 fluorescence, belongs to RGCs stained with the specific marker Brn-3a. (B) Representative western blot of the levels of LC3-I, LC3-II and Beclin-1 in control and axotomized retinas 4, 6 and 14 days after the lesion.

Supplementary Figure 3: (A) Biparametric FACS diagrams of RGC-5 cells stained with DiOC(6)(3) to measure mitochondrial membrane potential and propidium iodide (PI) to determine cell viability 24h after treatments. (B) Quantification of PI positive cells 24h after treatments.

Supplementary Figure 4: (A) Quantification of the LC3-II/LC3-I ratio by immunoblot in retinas from uninjured mice treated with vehicle or rapamycin during 10 days. (B) Quantification of the LC3-II/LC3-I ratio by immunoblot and (C) p62 levels in retinas from uninjured WT and Atg4B-/- mice. (D) Representative confocal image taken at 10X objective of retina from an Atg5flox/flox mouse injected with the AAV-Cre-GFP adenovirus showing GFP expression both in the somas and in the axons of RGCs. White asterisk represents the optic nerve. (E) Representative magnification of an AAV-Cre-GFP injected retina immnunostained with Brn-3a showing GFP-positive cells stained for Brn-3a.

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List of abreviations

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3-MA 3-Methyladenine

AAV Adeno-associated virus

AP Autophagosome

ATP Adenosine triphosphate

Brn-3a Brain 3a

cDNA complementary DNA

DAPI 4',6-diamidino-2-phenylindole

DCF Dicloro-fluorescein

DHE di-hydroethidium

DiOC6(3) 3,3´-dihexyloxacarbocyanine iodide

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic acid

DTMR Dextran tetramethylrhodamine

ELISA Enzyme-Linked ImmunoSorbent Assay

EM Electron microscope

ER Endoplasmic Reticulum

FACS Fluorescence Activated Cell Sorting

FBS Fetal bovine serum

Fig. Figure

GAPDH Glyceraldehyde 3-phosphate dehydrogenase

GCL Ganglion Cell Layer

GFP Green Fluorescent Protein

i.p. intraperitoneal

LC3 microtubule-associated protein 1 light chain 3

mRNA messenger RiboNucleic Acid

NaCl Sodium Chloride

ONT Optic Nerve Transection

PCR Polymerase Chain Reaction

PD Parkinson's disease

PQ paraquat

RGC Retinal Ganglion Cell

RGC-5 Retinal ganglion cell clon 5, cell line

ROS Reactive oxygen species

RNA RiboNucleic Acid

SEM Standard error

Sup. Supplementary

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