www.sciencetranslationalmedicine.org/cgi/content/full/4/163/163ra156/DC1

Supplementary Materials for

αααα-Synuclein–Induced Down-Regulation of Nurr1 Disrupts GDNF Signaling in Nigral Dopamine Neurons

Mickael Decressac,* Banafsheh Kadkhodaei, Bengt Mattsson, Ariadna Laguna, Thomas

Perlmann, Anders Björklund*

*To whom correspondence should be addressed. E-mail: Anders.Bjorklund@med.lu.se (A.B.); Mickael.Decressac@med.lu.se (M.D.)

Published 5 December 2012, Sci. Transl. Med. 4, 163ra156 (2012)

DOI: 10.1126/scitranslmed.3004676

The PDF file includes:

Materials and Methods Fig. S1. Trophic response induced by intrastriatal injection of human GDNF in nigral DA neurons. Fig. S2. Expression of GDNF and α-synuclein, GFP transgene expression, and survival of nigral DA neurons. Fig. S3. α-Synuclein overexpression represses transcriptional activity in DA neurons. Fig. S4. IGF-1 signaling is preserved in DA neurons overexpressing α-synuclein. Table S1. List of RT-qPCR primers.

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Supplementary Information

Supplemental Information includes Supplemental Experimental Procedures, Table S1,

Supplemental legends of Figures S1–S4 and Supplemental References.

Supplementary Experimental Procedures

Vector production

Production of AAV6-synapsin-α-synuclein, AAV6-synapsin-eGFP and AAV-6-CBA-Nurr1

was performed as described previously (16). Briefly, transfer plasmids carrying AAV2 ITRs

coding for either a wild-type human α-synuclein, enhanced GFP, or Nurr1 downstream to a

human synapsin-1 promoter were generated. Transfection into HEK 293 cells was carried out

using the calcium-phosphate method, and included the packaging plasmids pDP6 encoding

AAV6 capsid proteins (64-65). The cells were treated with a lysis buffer (50 mM Tris, 150

mM NaCl, pH 8.4) and freeze-thaw cycles in dry ice/ethanol bath. The crude lysates were

purified first by ultracentrifugation (1.5 hours at 350 000 x g at 18˚C) in a discontinuous

iodixanol gradient and the virus containing fractions were purified with ion-exchange

chromatography using FPLC. Genome copy titers were determined using real time

quantitative PCR. The genome copy titers for AAV6-α-synuclein, AAV6-GFP and AAV6-

Νurr1 were 2,5 x 1013, 2,5 x 1012 and 1,7 x 1012 genome copies/ml, respectively. Dilution of

the batches was performed so that an equivalent number of genome copies (2.0 x 107 gc/3µl)

were injected.

Tissue processing and histological procedure

Animals were deeply anesthetized with sodium pentobarbital i.p. (Apoteksbolaget, Sweden)

and then perfused through the ascending aorta with saline (0.9% w/v) at room temperature,

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followed by ice-cold paraformaldehyde (4% w/v in 0.1M phosphate buffered saline). The

brains were removed, post-fixed and cryoprotected overnight in sucrose (25% w/v in 0.1 M

phosphate buffered saline) before being sectioned on a freezing microtome (Leica). Coronal

sections were collected in 6 series at a thickness of 35 µm.

Immunohistochemical stainings were performed on free-floating sections as previously

described (16, 39) using antibodies raised against tyrosine hydroxylase (TH) (rabbit, 1:1500,

Chemicon or mouse, 1:5000, Incstar), VMAT-2 (guinea pig, 1:5000, Sigma), GDNF (goat,

1:1000, R&D System), GFRα1 (Rabbit, 1:200, R&D System), Ret (Rabbit, 1:100, Santa

Cruz), IGF-1R (Mouse, 1:500, Calbiochem), Nurr1 (rabbit, 1:1000, Santa Cruz), Nurr1

(Rabbit, 1:500, SantaCruz), phospho-S6 (Ser235/236) (rabbit, 1:300, Cell Signaling),

phospho-α-synuclein (Ser129) (rabbit, 1:3000, Wako) and α-synuclein 211 (mouse IgG,

1:3000; SantaCruz), MEF2D (rabbit, 1:1000, Abcam), phospho-CREB (Ser133) (rabbit,

1:500, Cell Signling), GFP (chicken, 1:1000, Abcam). For DAB staining, sections were

incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA),

followed with avidin-biotin-peroxidase complex (ABC Elite; Vector Laboratories,

Burlingame, CA), and visualized using 3,3-diaminobenzidine (DAB) as a chromogen,

mounted and coverslipped using the DPX mounting medium. For fluorescent stainings,

sections were incubated with appropriate Alexa 488, 568 or 642-conjugated antibodies

(Invitrogen) and slides were coverslipped using the FluorSave reagent (Millipore).

Postmortem human brain tissue samples were obtained from the UK Parkinson’s Disease

Society Tissue Bank. For immunohistochemical analyses, paraffin-embedded ventral

midbrain sections from 5 age-matched control subjects and 10 PD patients were used.

Following antigen retrieval (sections boiled in antigen retrieval solution (Dako) for 5 min),

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sections were incubated overnight at 4°C with antibodies against α-synuclein (1:2000, Santa-

Cruz), TH (1:2000, Incstar), or Ret (C-20, 1:50, Santa-Cruz). Staining was then revealed

using appropriate Alexa 488 or 568-conjugated secondary antibodies (Invitrogen) or the

Vector SG substrate as chromogen.

Analysis of protein expression by Western blot

The animals (n=5 per group) were sacrificed, brains were rapidly removed, and the ipsi- and

contra-lateral striatum and ventral midbrain were dissected and snap-frozen on dry ice.

Tissues were homogenised in RIPA buffer (Sigma) with phosphatase inhibitor and protease

inhibitor cocktail 2 (1:100, Sigma). Homogenates were centrifuged 10 min at 800 g or when

necessary were processed for differential centrifugation as described previously (66) and

stored at -80ºC until use. Protein concentration was determined using the DC protein assay kit

(Bradford method). 20-40 µg of protein were boiled at 90°C for 5 min in Laemmli buffer

(Biorad), separated on a 4-20% SDS-PAGE gel and then electrotransferred (100V, 1 hour) on

a PVDF membrane (Bio-Rad). After blocking for 1 h in Tris-buffered saline with 0.1%

Tween-20 (TBST) and 3% bovine serum albumin, membranes were incubated overnight at

4ºC with one of the following primary antibodies: rabbit anti-tyrosine hydroxylase TH (rabbit,

1:1500, Chemicon), DAT (rat, 1:500, Millipore) VMAT-2 (rabbit, 1:1000, Abcam), GFRα1

(rabbit, 1:200, R&D System), Ret (Rabbit, 1:100, Santa Cruz), phospho-Ret (Tyr1062)

(rabbit, 1:500, Santa Cruz), IGF-1R (Mouse, 1:500, Calbiochem), phospho-IGF-1R (Rabbit,

1:100, Abcam), Nurr1 (rabbit, 1:1000, Santa Cruz), Pitx-3 (1:500, rabbit, Zymed), phospho-

S6 (Ser235/236) (rabbit, 1:300, Cell signalling) S6 (mouse, 1:1000, Cell Signaling), alpha-

synuclein 211 (mouse IgG, 1:5000; Santa Cruz), Akt (rabbit, 1:1000, Cell Signaling),

phospho-Akt (Ser473) (rabbit, 1:2500, Cell Signaling), Erk1/2 (Rabbbit, 1:1000, Cell

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Signaling), phospho-Erk1/2 (Thr202/Tyr204) (rabbit, 1:1000, Cell Signaling), Bad (rabbit,

1:1000, Cell Signaling), phospho-Bad (Ser112) (rabbit, 1:800, Cell Signaling), Histone H3

(rabbit, 1:1000, Cell Signaling), acetyl-Histone H3 (Lys9) (rabbit, 1:1000, Cell Signaling), or

GFP (rabbit, 1:1000, Abcam). Membranes were also blotted with an anti-actin antibody

(mouse, 1:8000, Sigma) control protein loading. After washing for 30 min in TBST with

gentle agitation, membranes were incubated during 1 hour at room temperature with an Alexa

488, 568, or 642-conjugated secondary antibody (1:1000, Invitrogen). Protein expression was

also revealed using the Immun-Star kit (Biorad). Signal was detected using Gel VersaDoc

system (Biorad) and band intensities were quantified by densitometry using the ImageLab

software (Biorad).

Co-immunoprecipitation

Ventral midbrain tissue was isolated and homogenization was performed as described in the

western blot protocol (n=4 per group). Separation of the nuclear and cytoplasmic fraction was

performed using the NE-PER extraction kit (Thermo Scientific). Validation of extraction was

confirmed by SDS-PAGE, followed by blotting of the membrane with an antibody

recognizing the nuclear marker NeuN (Mouse, 1:1000, Chemicon) or the cytoplasmic marker

GAPDH (rabbit, 1:2000, Cell Signaling). Dynabeads (Invitrogen) were coated with an anti-

histone H3 antibody (Abcam) following manufacturer’s recommendations. The nuclear lysate

was then incubated with beads for immunoprecipation, complexes were then extracted and

applied to 4-20% SDS-PAGE. Membranes were blotted with an anti-histone H3 or anti-α-

synuclein antibody and revelation was performed as described in the Western blot section.

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Supplemental Figures

Figure S1: Trophic response induced by intrastriatal injection of human GDNF in nigral DA

neurons.

Double immunofluorescence staining against TH (red) and phospho-S6 (green) confirming

that the trophic response seen on the GDNF-injected side (top panel, right) was confined to

the TH-positive DA neurons. This response was absent in the α-synuclein-overexpressing

cells (bottom panel, left), but retained in the 6-OHDA intoxicated cells (bottom panel, right)

Scale bar: 200 µm.

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Figure S2: Expression of GDNF and α-synuclein, GFP transgene expression, and survival of

nigral DA neurons.

(A): Staining of human GDNF showing distribution of the protein 48 hours after unilateral

(right) injection of 1µg of human recombinant GDNF in the rat striatum (top) or substantia

nigra (bottom). Scale bar: 2.0 mm.

(B-C): Expression of GFP and human α-synuclein in midbrain and forebrain 2 weeks after

intra-nigral injection of AAV-GFP (B) or AAV-α-synuclein (C). Double

immunofluorescence show that the vast majority of nigral TH-positive neurons (red) express

the two transgenes (green). Scale bar: 2.0 mm.

(D-E): Two weeks after AAV vector injection, stereological analysis showed that GFP or α-

synuclein overexpression did not cause significant loss of TH-positive DA neurons, while 6-

OHDA injection in the medial forebrain bundle induced a significant death of this population

as seen 2 days post toxin injection. Data are expressed as mean ± SEM (n=20/group). *:

P<0.05; one-way ANOVA, compared to control group. Scale bar: 1.2 mm.

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Figure S3: α-Synuclein overexpression represses transcriptional activity in DA neurons.

(A-B): Immunofluorescence from an AAV-α-synuclein injected rat (2 weeks survival)

showing levels of phosphorylated α-synuclein (green) in the nuclear compartment (stained

with MEFD2 in blue) of VMAT-2-positive nigral DA neurons (red). The confocal images

illustrate that nigral DA neurons showing high expression of phospho-α-synuclein in the

nucleus have reduced levels of VMAT-2 and MEF2D (arrowheads in A), which is in contrast

to transduced DA cells with low nuclear expression of phospho-α-synuclein (arrowheads in

B). Scale bar: 12 µm.

(C): Western blot analysis of acetyl histone H3, total histone H3, cytoplasmic and nuclear α-

synuclein levels in the control, GFP and α-synuclein group 2 weeks after intra-nigral injection

of AAV-GFP or AAV-α-synuclein vectors. (D): Co-immunoprecipitation (IP) of histone H3

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with α-synuclein in α-synuclein overexpressing midbrain tissue. Lysates were subjected to IP

for histone H3 or control IgG, and immunoblotted for α-synuclein and histone H3 (Inp: input;

Ab: H3 antibody; IgG: control antibody).(E): Western blot analysis of acetyl-α-tubulin, α-

tubulin, total histone H3, Sirtuin-2 and p300 levels in midbrains from control, AAV-GFP and

AAV- α-synuclein animals 2 weeks after intra-nigral injection of AAV vectors.

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Figure S4: IGF-1 signaling is preserved in DA neurons overexpressing α-synuclein.

(A): qPCR analysis of IGF-1 receptor level in the midbrain of rats injected with AAV-GFP or

AAV- α-synuclein. Data are expressed as mean ± SEM (n=5/group).

(B): Phospho-S6 immunoreactivity in control, 6-OHDA-injected, GFP or α-synuclein

overexpressing rats 6 hours after unilateral intra-striatal injection of IGF-1 (right side). Scale

bar: 500 µm.

(C): Western blot analysis of the activation of IGF-1R, Akt and S6 in the same conditions.

Data show no significant difference in the activation of these pathways between groups. Data

are expressed as mean ± SEM (n=5/group).

(D): Phospho-S6 immunoreactivity detected 6 hours after striatal injection of IGF-1 (right

side) in Nurr1WT/WT and Nurr1cKO/cKO mice treated 8-10 weeks before with tamoxifen. Scale

bar: 700 µm.

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(E): qPCR analysis of IGF-1 receptor expression 8-10 weeks after tamoxifen injection in

control (Nurr1WT/WT/ DATCreERT-2/+) and Nurr-1 floxed mice. Data are expressed as mean ±

SEM (n=5/group).

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Supplementary Table

Table S1: List of RT-qPCR primers.

Species Target gene Primer sequence (5' -> 3')

Mouse

Nurr1 (nr4a2) Fwd: GAGCTGGGCTGCTCGACCACT Rev: CCGTCAGATCTCCCTGCCCAGC

Pitx3 Fwd: GGGACGCACTAGACCTCCCTCC Rev: CGATAACGACAGCGCAGGGCTT

Th Fwd: AAGGTTCATTGGACGGCGGCAG Rev: CCAATGGGTTCCCAGGTTCCGC

Vmat-2 (slc18a2) Fwd: CAGCGCTCACAGCCTCCACTTC Rev: CGTGGCATTCCCCGTGAACACA

Dat (slc6a3) Fwd: TGCAGCTGACCAACTCCACCCT Rev: GCTCCAGGTCTCCCGCTCTTGA

Aadc (ddc) Fwd: TCGCAGAGCTGGACAATCCCGA Rev: ACCATCTCCTTGCCTCTCCTCCG

Igf1r Fwd: ATGGAGAGCGTCCCACTGGACC Rev: CCGGGCCATTCTCAGCCTTGTG

Gapdh Fwd: TCCATGACAACTTTGGCATTG Rev: CAGTCTTCTGGGTGGCAGTGA

Actin Fwd: CTAAGGCCAACCGTGAAAAGAT Rev: CACAGCCTGGATGGCTACGT

Ret Fwd: TCCCTTCCACATGGATTGA Rev: ATCGGCTCTCGTGAGTGGTA

Gfra1 Fwd: TTCCCACACACGTTTTACCA Rev: GCCCGATACATTGGATTTCA

Rat

Snca Fwd: GCCCAGAGCCTTTCACCCCTCT Rev: GCTCCACACGGCTCTCTAGGCT

Nurr1 (nr4a2) Fwd: GCCTCTGCACGTCCCCATGAAC Rev: AATGGGATTGGGCACGGCGAAG

Pitx3 Fwd: CTCGGTCAACGTGGGGCCTCTA Rev: CATGGAGGCAGCGATGGAGCTG

Th Fwd: GAAGGGCCGTCTCAGAGCAGGA Rev: TCTGTCGCCGTCCGATGAACCT

Vmat-2 (slc18a2) Fwd: GCCACTCGCGCAAACTGATCC Rev: AACCACGACGGTGAGCAGCAT

Dat (slc6a3) Fwd: CGGGGTCCTTCCGGGAGAAAC Rev: GTGAATTGGCGCACCTCCCCT

Aadc (ddc) Fwd: ACGGCATTGAGGGACGTCCAGT Rev: GATCAGGGCCCGAAGGTAGCCA

Igf1r Fwd: GCTGTGGGGGCTCGTGTTTCTC Rev: GATGTCAATGCCGGGCCCACAA

Ret Fwd: AACGCAGTCCCCTCTGCACCAT Rev: GGAGGCTTTGGTTTCGGTGGGA

Gfra1 Fwd: CTGAACCCCTGAAAGCGGGTCC Rev: CCAGGGTGAGAGCAGACTCCCC

Dj-1 (park7) Fwd: CGTGCTTCACAGTGGCGGCTAA Rev: AGGAGGTCAACCCTGCCAGACG

Bdnf Fwd:GTCTTCCCGCCCTAGCCTGACA Rev: TCCAATTTGCACGCCGCTCCTT

Actin Fwd: CTAAGGCCAACCGTGAAAAGA Rev: CACAGCCTGGATGGCTACGT

Gapdh Fwd: GGGGCTCTCTGCTCCTCCCTG

Rev: GACGAGGCTGGCACTGCACAAG

Girk-2 Fwd: AAGCCATGGGTGTGGGGACAGT

Rev: AGACAGCTCCGTGGAGAGCCTG

Mef2d Fwd: AGGAGTTGCCTTTGGAGCCCGA Rev: AGTGCCAGAGAGGAAGGGGCTG