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Supporting Information
Animal procedures
DTMR labeling
After sectioning the ON a piece of spongostan with absorbed 0.1% DTMR (Dextrantetramethylrodamine, 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 m was regarded as RGCs
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. 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
L 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
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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
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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 PBSTween 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.
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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 AAVCre-GFP injected retina immnunostained with Brn-3a showing GFP-positive cells
stained for Brn-3a.
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List of abreviations
3-MA
AAV
AP
ATP
Brn-3a
cDNA
DAPI
3-Methyladenine
Adeno-associated virus
Autophagosome
Adenosine triphosphate
Brain 3a
complementary DNA
4',6-diamidino-2phenylindole
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 3phosphate 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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