Supplementary Material
Generation of Lamp2a stable cell lines and transfections
Lamp2a-HA was excised from the pcR 3.1 vector and cloned into the KpnI/ApaI sites
of the pcDNA3.1-zeo vector (Invitrogen). Naïve SH-SY5Y cells were transfected
overnight with Lamp2a-HA or empty vector using the Lipofectamine 2000 reagent
(Invitrogen), following the manufacturer’s recommendations. Selection was
performed with 500 μg/mL Zeocin (Invitrogen). Zeocin-resistant colonies were
isolated and tested for Lamp2a-HA expression by immunocytochemistry and western
blot analysis.
Intracellular protein degradation
Total protein degradation in cultured cells (SH-SY5Y cells, cortical neurons) was
measured by pulse-chase experiments. Briefly, confluent SH-SY5Y cells or cortical
neurons (day 7 in culture) were labeled with [3H] leucine (2 μCi/ml) (Leucine, L3,4,5, NEN-Perkin Elmer Life Sciences, Belgium) at 37oC for 24 h. The cultures were
then extensively washed with medium and returned in complete growth medium
containing 2 mM of unlabeled leucine for 6 h. This medium containing mainly shortlived proteins was removed and replaced with: fresh medium containing cold leucine
(control conditions), medium containing Bafilomycin (500 nM, SH-SY5Y cells) or
NH4Cl (20 mM, cortical neurons) (total lysosomal proteolysis) or medium containing
3MA (10 mM, macroautophagic degradation). Aliquots of the medium were taken at
14 h after labeling and proteins in the medium were precipitated with 20%
thrichloroacetic acid (TCA) for 20 min on ice and centrifuged (10.000 X g, 10 min,
4oC). Radioactivity in the supernatant (representing degraded proteins) and pellet
(representing undegraded proteins) was measured in a liquid scintillation counter
(Wallac T414, Perkin Elmer). At the last time point, cells were lysed with 0.1%
NaOH. Proteolysis was expressed as the percentage of the initial total acidprecipitable radioactivity (protein) in the cell lysates transformed to acid soluble
radioactivity (amino acids and small peptides) in the medium during the incubation
(Kaushik and Cuervo, 2009).
RNA extraction and real-time RT-PCR analysis
Total RNA was extracted from the EV5, 2.11 and 3.10 cell lines using Trizol
(Invitrogen) and cDNA was generated with the Reverse Transcription System
(Invitrogen), according to the manufacturer’s instructions. Quantitative RT-PCR
assays were done in triplicate using the LightCycler RNA amplification kit (Roche
Applied Science) on a LightCycler instrument in conjunction with gene-specific
unlabeled external forward and reverse primers and pairs of differentially labeled
forward internal primers (HybProbes FL and LC, emitting at 530 and 640 nm,
respectively; designed and produced by TIB MOLBIOL, Syntheselabor GmbH,
Berlin, Germany). Primers for human glyceraldehyde-3-phosphate dehydrogenase
mRNA (hGAPDH), which was used as an internal standard, and human alphasynuclein
(hAS)
mRNA
were
as
follows:
hGAPDH
forward,
5’-
GCACCACCAACTGCTTAG-3’, and reverse, 5’-GCCATCCACAGTCTTCTG-3’;
AS
forward,
5′-
CGCCTTGCCTTCAAGCCTTC-3’,
and
reverse,
5’-
CACCACACTGTCGTCGAATGG-3’. Quantification standard curves were obtained
using PCR products diluted in 10 μg/ml sonicated salmon sperm DNA. Normalization
of the AS expression was done against GAPDH.
Measurement of endogenous AS half-life
We cultured 85% confluent cell cultures in methionine/cysteine-deprived RPMI 1640
medium (Sigma) for 10 min and then labeled with a [35S] methionine/cysteine mixture
(0.2 mCi/mL) (Express Labeling Mix; PerkinElmer Life Sciences) for 2 h. After
extensive washing with medium, the cells were maintained in complete or low serum
medium (0.5% FBS) and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.6,
0.1% SDS, 1% Triton X-100, 2 mM EDTA, and 0.1% deoxycholate) with protease
inhibitors (complete mini, Roche Diagnostics) at the indicated times and subjected to
immunoprecipitation with the syn-1 antibody (1:1,000; BD Biosciences, 619787)
against AS. The protein G+ agarose beads were purchased from Santa Cruz
Biotechnology. Immunoprecipitates were resolved by SDS-PAGE (12%), and the gels
were dried and then exposed on a PhosphorImager Screen and quantified using Gel
Analyzer version 1.0 software (Biosure, Athens, Greece).
Production of recombinant AVs
Viral vector stocks were amplified from plaque isolates in order to guarantee
homogeneity. The final vector stocks were purified and concentrated using double
discontinuous and continuous CsCl gradients. Viral titers of purified vector stocks
were determined using an Adeno-X Rapid Titer Kit (Clontech) and OD260
measurements. The following titers were obtained, expressed as viral particles
(vp)/μL: 2.8 × 108 vp/μL for rAd-WTAS, 2.4 × 108 vp/μL for rAd-Lamp2a-HA, and
1.43 × 109 vp/μL for rAd-GFP.
Production of rAAVs
The transgenes were cloned into a rAAV backbone plasmid containing the synapse-1
promoter, the woodchuck hepatitis post-transcriptional regulatory element, and a
bovine growth hormone polyA site. The expression cassette is flanked by AAV2
inverted terminal repeats (ITR). HEK293 cells grown to approximately 70–80%
confluence were double-transfected using the calcium-phosphate method including
the rAAV plasmid and helper plasmids encoding for essential AV packaging and
AAV6 capsid genes. After 3 days of incubation, the cells were harvested and lysed by
performing 3 freeze-thaw cycles in a dry ice/ethanol bath. After treatment with
Benzonase nuclease (Sigma, Stockholm, Sweden), the lysate was purified using a
discontinuous iodixanol gradient followed by Sepharose Q column chromatography
and finally concentrated with a 100-kD cut-off column (Millipore Amicon Ultra;
Millipore, Solna, Sweden). To determine the titer of the viral stock solutions
quantitative PCR with primers and probes targeting the ITR sequence was performed.
Primary antibodies for Western blot analysis
Primary antibodies included antibodies to HA (1:1,000; Covance, MMS-101P),
human Lamp2a (1:1,000, Abcam, 18528), TH (1:2,000; Calbiochem, 657012), AS (C20, 1:1,000, Santa Cruz, 7011-R and syn-1 [42/α-Synuclein], 1:1,000, BD
Biosciences, 619787), human-specific AS (LB509) (1:1,000; Covance, SIG-39725),
phospho S129 AS (EP1536Y) (1:1,000, Abcam, 51253), ERK 2 (1:5,000; Santa Cruz,
sc-154), and β-actin (ACTBD11B7) (1:1,000, Santa Cruz, 81178). Detection and
quantification were performed using the Gel Analyzer Imaging System.
HPLC
The mobile phase consisted of an acetonitrile (Merck KgaA, Darmstadt, Germany)-50
mM phosphate buffer (10.5:89.5) pH 3.0, containing 300 mg/L 5-octylsulfonic acid
sodium salt (Merck KgaA, Darmstadt, Germany) as the ion-pair reagent and 20 mg/L
Na2EDTA (Riedel-de Haën AG, Seelze, Germany). The working electrode was glassy
carbon; the columns were Thermo Hypersil-Keystone, 150 x 2.1 mm 5 μ Hypersil,
Elite C18 (Thermo Electron, Cheshire, UK). The HPLC system was connected to a
computer, which was used to quantify all compounds by comparison of the area under
the peaks with the area of reference standards with specific HPLC software
(Chromatography Station for Windows).
Immunohistochemistry
The primary antibodies used were against TH (1:2000; Calbiochem, 657012), HA-tag
(6E2) (1:400; Cell Signaling, 2367), Lamp2a (lgp96) (1:400; Zymed Laboratories, 512200), AS (clone Syn211) (1:150,000; Millipore, 36-008), Lamp1 (LY1C6) (1:400;
Santa Cruz, 65236), and GFP (1:1,000; Santa Cruz Biotechnology). Antibodies were
solubilized in 2% NGS in PBS and blocking of nonspecific binding sites was done
with 2% NGS and 0.1% Triton-X-100 in PBS. In the case of Lamp1/2 staining,
antigen retrieval was performed in citric acid (pH 4.0) for 30 min at 80°C. For
double-fluorescence labelling, secondary antibodies conjugated to Cy2 and Cy3
(Jackson ImmunoResearch, Suffolk, UK) were used. For visualization by the 3,3'diaminobenzidine (DAB) chromogen (DAB+, Dako A/S, Denmark) with bright-field
microscopy, the same primary antibodies were used in conjunction with biotinylated
universal horse antibody (Vecta Stain ABC Kit; Vectorlabs, Southfield, MI, USA),
followed by an avidin-biotin-peroxidase complex (Vectorlabs). After processing, all
sections were mounted on chrome-alum-coated glass slides and coverslipped.