Tofaris et al. 16 09 11 Supporting Information M 66 Nil documented Normal Incidental ... Digests...

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Supplementary Material Supplementary Figures: Figure S1 In situ hybridization studies in mouse brain sections showing the distribution of the WW HECT domain E3s and α-synuclein. Nedd4, Nedd4-2 and Smurf1 were the only three members of the family found to be expressed in brain. Only Nedd4 is expressed at high levels in the same brain regions as α- synuclein (note olfactory bulb and substantia nigra indicated with white arrows). All three are expressed in the hippocampus (data available on-line and were adapted from: www.mouse.brain-map.org ).

Transcript of Tofaris et al. 16 09 11 Supporting Information M 66 Nil documented Normal Incidental ... Digests...

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Supplementary Material

Supplementary Figures:

Figure S1 In situ hybridization studies in mouse brain sections showing the

distribution of the WW HECT domain E3s and α-synuclein. Nedd4, Nedd4-2 and

Smurf1 were the only three members of the family found to be expressed in

brain. Only Nedd4 is expressed at high levels in the same brain regions as α-

synuclein (note olfactory bulb and substantia nigra indicated with white arrows).

All three are expressed in the hippocampus (data available on-line and were

adapted from: www.mouse.brain-map.org).

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Figure S2 (A) Endogenous Nedd4 co-immunoprecipitates with Ser129

phosphorylated α-synuclein from brain homogenate. (B) Purified recombinant

proteins and Ser129 phosphorylated variants were stained with Coomassie. (C)

Nedd4 ubiquitinates Ser 129 phosphorylated and c-terminally truncated α-

synuclein variants (syn1-133 and syn1-135) in a fashion similar to wild-type

protein. Recombinant wild-type or phosphoserine 129 full-length or truncated α-

synuclein was incubated with purified Nedd4, UbcH5, ATP +/- ubiquitin.

Ubiquitination was assayed by the increase in the molecular weight of α-

synuclein (seen only in the presence of ubiquitin) using anti-α-synuclein

antibodies.

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Figure S3 In the presence of ubiquitin mutants containing only a single lysine

(upper panel), formation of long poly-ubiquitinated chains on -synuclein by

Nedd4 is seen only with ubiquitin lysine 63. In the presence of different ubiquitin

mutants in which a single lysine was mutated to arginine (K->R, lower panel),

only K63R caused the accumulation of multiple mono-ubiquitinated -synuclein

species, whereas with the other six mutant ubiquitins long poly-ubiquitin chains

were formed.

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Figure S4 -Synuclein can be precipitated from total brain lysate with both GST-

immobilized Nedd4 and Rsp5p, but not with GST alone or GST-immobilized

MuRF-1.

Figure S5 In SH-SY5Y cells transfected with Nedd4 RNAi, which partly reduced

Nedd4 content, the basal levels of endogenous -synuclein increased. Left

panel: A typical experiment. Right panel: The mean content of -synuclein was

measured as in Fig 4a, and the data averaged from 3 experiments are shown.

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Figure S6 A, Sections through the substantia nigra of healthy control brains were

stained with anti-Nedd4 polyclonal antibodies. Staining was primarily detected in

neuronal processes (arrows) of the pigmented neurons. B, In serial sections

where the primary antibody was omitted no specific staining was seen. The

punctuate staining in both sections is due to background fluorescence of brain

tissue. Nedd4 (panel C) and α-synuclein (panel D) staining in rat primary neurons

showed co-localization of the two proteins (panel E).

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Figure S7 In the presence of cycloheximide (CHX), the effect of Nedd4

overexpression on α-synuclein levels at 6h is blocked when the cells are treated

with chloroquine (CQ).

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CASE

Age

Sex

Post-mortemDelay(hours) Symptoms

Clinicaldiagnosis

Pathologicaldiagnosis

OtherPathology

SNDegeneration

LBStaging

NFTstage

1

92 M 36

Unilateraltremor andhypophonia PD PD Nil

moderatepale LC Limbic Nil

2

77 F N/A Nil documented NormalIncidentalLB Nil Mild BS (SN) I/II

3

77 F 36

Progressivelanguage deficitseizures fromstrokes Stroke

IncidentalLB CAA Mild BS Nil

4

72 M 40

Gaitdisturbance,memorydifficulties PD PD

Lacunarstrokes Mild BS II

5

82 M 29 Nil documented NormalIncidentalLB SVD Mild BS Nil

6

71 M 88

Memorydecline , speechdisturbance DLB DLB Nil Severe Cortical Nil

7

76 M 70

Poor memory,ideationalapraxia DLB DLB Nil Severe Cortical I/II

8

77 M 90

Motorsymptoms withsubsequentmemorydecline PDD PDD SVD

Severe,Pale LC Limbic Nil

9

88 M 24 Nil documented NormalIncidentalLB

Small celllung cancer Mild BS Nil

10

82 M 55

Motorsymptomsmemorydecline PDD PDD Mild SVD

moderatepale LC

BSCingulateCortex Nil

11

92 M 24Motorsymptoms PD PD Nil Moderate

BSCingulate,EntorhinalCortex I/II

12

76 F 96

Hallucinations ,Mild motorfeatures DLB DLB Mild SVD Severe

DiffuseCortical I/II

13

86 M 66 Nil documented NormalIncidentalLB SAH Mild BS Nil

Table S1. Clinical and pathological data for cases selected for Nedd4 staining.

BS, brain stem; CAA, cerebral amyloid angiopathy; DLB, dementia with Lewy

bodies; PD, idiopathic Parkinson’s disease; LB, Lewy bodies; LC, locus

coeruleus; PDD, Parkinson’s disease dementia; SVD, small vessel disease;

SAH, sub-arachnoid haemorrhage

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Methods:

Yeast Strains and Genetic Procedures. Yeast strain FY56 (MATα his4-912ΔR5

lys2-128Δura3-52) and isogenic Rsp5-1 mutant strain FW1808 were obtained

from Fred Winston (HMS). At 30oC, the rsp5-1 mutant grows with a doubling time

only slightly longer than that of the isogenic wild-type, but its growth is severely

impaired at 37o. The rsp5-1 protein has a reduced ability to form a ubiquitin-

thioester intermediate and to catalyze substrate ubiquitination (S1). The α-

synuclein expressing yeast strain IntTox (α-synWT, MATα can1-100 his3-11, 15

leu2-3, 112 trp1-1 ura3-1 ade2-1 pRS303Gal-α-synWT-YFP pRS304Gal-

αSynWT-YFP) was used in the Rsp5p modifier study and the plasmids:

p426GPD-EGFP, p426Gal-aSynWT-GFP, p426Gal-aSynWT were kindly

provided by Dr Susan Lindquist (MIT). Centromere- and TRP1-based plasmids

pYES-HA-Rsp5 and pYES-HA-Rsp5C->A were a generous gift of Dr Jon

Huibregtse (Austin, Texas). Yeast manipulations were performed and media

were prepared using standard procedures (S2). Transformations of yeast were

carried out using a standard lithium acetate procedure (S3). Yeast strains

carrying the galactose-inducible α-synuclein constructs were pre-grown in

raffinose medium (which does not repress the galactose-inducible promoter) prior

to addition of galactose medium, to allow rapid, synchronous induction of

expression.

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Viability studies in yeast. Cells carrying either one or two copies of the α-

synuclein gene were grown overnight at 30 oC in liquid media containing

raffinose lacking uracil, for plasmid selection until they reached log or mid-log

phase. Cultures were then normalized for OD600, serially diluted and spotted onto

solid media containing either 2% glucose (α-synuclein off) or 2% galactose (α-

synuclein on), after which they were grown at 30 oC for 2-3 days.

Ubiquitination Assays. Wild-type human α-synuclein protein (full-length and

fragments) was purified as previously described (S4). pRK172 plasmids

containing full-length α-synuclein or α-synuclein fragments (amino acids1-120 or

40-140) were a kind gift of Dr M. Goedert. Recombinant proteins were expressed

in E. coli (DH5α). Bacterial pellets were sonicated in 50 mM Tris, pH 8.0, 0.1 mM

EDTA, 0.1 mM dithiothreitol (DTT), 0.1 mM phenylmethylsulfonyl fluoride

(PMSF), and centrifuged for 15 min at 15 000 rpm. 30-50% ammonium sulfate

cuts of the supernatants were applied to an S-200 Sephacryl column equilibrated

in sonication buffer. For full-length wild-type and truncated α-synucleins, peak

protein fractions were applied to a Mono Q column equilibrated in sonication

buffer, and eluted using a 0-500 mM NaCl gradient. For truncated α-synuclein,

peak protein fractions from an S-200 Sephacryl column equilibrated in 50 mM

MES, pH 6.25, 0.1 mM DTT, 0.1 mM PMSF, were applied to a phosphocellulose

column equilibrated in the same buffer and eluted by salt step elution. GST-

tagged Nedd4, Rsp5, Nedd4-2, Smurf1&2 were expressed in E. coli (BL21) and

purified using sepharose beats. Ubiquitination of α-synuclein by the relevant E3s

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and auto-ubiquitination of E3s were assayed as described earlier (S5). Purified

α-synuclein was added to the ubiquitination mixture containing 6His-E1 (50 nM),

UbcH5 or UbcH7 (750 nM), 6His-E3 (500 nM) and ubiquitin (59 mM) in a buffer

composed of 20mM Tris–HCl (pH7.6), 20mM KCl, 5mM MgCl2, 2mM ATP, and

1mM DTT. Ubiquitination reaction was performed at 30 oC for 1 h. Ubiquitinated

α-synuclein was resolved on SDS–PAGE and detected by immunoblotting.

LC-MS analysis and database search: Coomassie blue stained gel regions,

containing α-synuclein and the ubiquitnated α-synuclein were sliced into sizes of

~ 1mm3 and transferred to a clean microcentrifuge tube. Gel slices were

sequentially digested using 12.5 ng/ul of sequencing grade trypsin (Promega, WI,

USA) and the tryptic digested peptides were secondly digested by 12.5 ng/ul of

Glu-C (Roche, Basel, Switzerland). The digested peptides were loaded onto a

fused silica microcapillary column (12 cm x 75 µm) which was packed with C18

resin (5 mm, 300 Å, Alltech, KY, USA) and separated by HPLC (Eksigent-Nano-

HPLC system; Eksigent Technologies, CA, USA) with a linear gradient of 5-50%

buffer B in 50 min (buffer A : 0.1% formic acid in H2O, buffer B : 0.1% formic acid

in ACN) at a flow rate of 250 nl/min. The column was connected directly to LTQ

XL ion-trap mass spectrometer (Finnigan, CA, USA) as described earlier (S6,

S7). For database search, all MS/MS spectra recorded were searched using the

small database, including α-synuclein and Ub protein by the SEQUEST algorithm

(ThermoFinnigan, CA, USA). The search parameters allowed for two missed

cleavages for trypsin. For database searching, TurboSEQUEST (Thermo

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Electron) was used with precursor and fragment ion mass tolerances of 1.5 and 1

Da, respectively. Oxidation on Met (+ 16 Da), and ubiquitination on Lys (+ 114.1

Da) were selected as variable modifications. For validation of peptide and protein

identification, Scaffold software (Proteome Software, OR, USA, version

3_00_02), which is based on peptide prophet and protein prophet algorithm was

used (S8). After validation with the combined peptide sequences, peptide

probability must be over than 95% and protein probability greater than 99%.

AQUA analysis of ubiquitin chains on ubiquitin- α-synuclein conjugates: In

order to quantify the polyubiquitinated chain, AQUA analysis was performed for

three ubiquitinated peptides on Ub (Table S2). Isotopically labeled synthetic

internal standard peptides were synthesized by Anygen (Gwangju, Korea) for

K11, K48 and K63 Ub branched chains. Quantification of the amount of each

internal standard peptide was carried out by amino acid analysis (Korea Basic

Science Institute, Daejon, Korea). Internal standard peptides were spiked for

quantification during the digestion step. Digests with added synthetic peptides

were then analyzed by reverse phase LC-MS/MS in selected reaction monitoring

(SRM) mode. SRM data were acquired within fragment ion mass ± 1.5 m/z, and

each SRM transition and respective retention time were validated for each

specific peptide (S9,S10). SRM transitions for each peptide sequence are

summarized in Table 1. Data were processed by integrating the appropriate

peaks for the light sample peptide and heavy internal standard peptide, followed

by calculation of the ratio of peak areas to estimate the abundance of the

peptide.

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Table S2. Detailed information of ubiquitinated peptides for quantification by

AQUA analysis.

Fragment ions (m/z)UbiqitinatedPeptide

Type SequenceParent ion

(m/z) First Second

K11 Isotope TLTGK@TI*LE 599.4. 983.4 882.3

Wild type TLTGK@TILE 595.9 976.4 875.3

K48 Isotope LIFAGK@QL*E 570.4 913.3 695.3

Wild type LIFAGK@QLE 566.9 906.3 688.3

K63 Isotope TLSDYNI*QK@E 666.5 1117.3 942.3

Wild type TLSDYNIQK@E 663.0 1110.3 935.3

K@ : Ubiquitinated site K

X* : Isotope labeled X

Quantitative Immunoblotting: Protein content was determined using the

Bradford assay (BioRad). Equal amounts of protein were resolved by SDS-PAGE

(Invitrogen), transferred to PVDF membranes and analysed by immunoblotting

with anti-syn-1 antibody (BD Transduction), LB509 antibodies (Zymed

Laboratories). The quantity of each band was calculated by Quantity One

software (BioRad).

Degradation Assays in yeast. Cells were grown overnight in raffinose until lag-

phase. Equal amount of cells were subsequently diluted to OD 0.3 and grown to

O.D 0.6-0.7 prior to addition of 2% (w/v) galactose for 2h to induce α-synuclein

expression. Cells were washed in 2% (w/v) glucose twice (to switch off α-

synuclein expression) and incubated in medium containing 2% (w/v) glucose for

the indicated time-points at 37 oC. Equal amounts of cells were collected,

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centrifuged and placed on dry ice until further use. Once all samples were

collected, cells were lysed on ice using glass beats (Sigma) and lysis buffer

(50mM Tris pH 7.4, 0.1mM PMSF) with Complete, (Roche) and centrifuged for

5min at 1500g at 4 oC.

Pull-down Assays. Whole mouse brains or HEK cells were homogenised in

lysis buffer (50mM Tris pH 7.4, 100mM KCl, 1mM EDTA, 0.1 mM PMSF, 1mM

NEM, 1% Triton X100 and Complete, Roche) on ice and centrifuged at 20

000rpm. Brain extract was incubated with GST-tagged E3s, or GST alone pre-

bound to sepharose beats overnight. Cell extract was incubated with anti-T7

antibodies (Sigma) overnight and subsequently with sepharose beats for 3h. All

steps were performed at 4 oC. Beats were centrifuged and washed three times in

buffer containing 200mM NaCl prior to addition of loading buffer and

immunoblotting.

Staining of human sections. Human sections through the substantia nigra and

locus coeruleus from ILDD, PD, DLB and control brains (Table S1) were kindly

provided by the Thomas Willis Brain Bank (Oxford University). Sections were

dewaxed, autoclaved in 10mM citrate buffer (pH6) for 10 minutes at 121oC then

incubated with rabbit anti-Nedd4 antibodies (Abcam) in TBS overnight at 4oC.

Detection of the primary antibody was performed using a biotin free indirect

labeling kit (Histar 3000c). Sections were then rinsed and counterstained in

haematoxylin before mounting with DPX. Primary antibody was omitted for

negative controls. Images were taken with an Olympus DP20 camera. For

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immunofluorescence, sections were permeabilised in 0.5% triton X100 for 20min

in room temperature and blocked in 3% serum and incubated with anti-Nedd4

polyclonal (Abcam, MA) and syn-1 monoclonal (BD Transduction) antibodies

overnight at 4 oC. Sections were washed three times in TBS-T and incubated for

1h at room temperature with appropriate Alexa-fluor secondary antibodies

(Molecular Probes) and imaged using fluorescence Nikon 80i Upright

Microscope.

Fluorescence Microscopy. For analysis of α-Syn-GFP distribution, strains

carrying either one or two copies of α-Syn-GFP were grown overnight in raffinose

medium. After normalizing the culture density, α-synuclein expression was

induced in medium containing 2% galactose and the cells were imaged at the

indicated times with Nikon Ti Inverted Fluorescence microscope. Experiments

were replicated in triplicates for quantitative analysis. Localization to the

membrane preceded the formation of inclusions as previously seen (S11).

Cell culture. SH-SY5Y (ATCC) human dopaminergic neuroblastoma or HEK cell

lines were used. Cells were maintained at 37 °C, 5% CO2 in DMEM/F12

(Dulbecco's modified Eagle's medium / Ham’s F-12 medium 50/50 mix) for SH-

SY5Y cells or DMEM for HEK cells with L-glutamine, supplemented with 10%

(v/v) fetal bovine serum (Sigma) and 1% (v/v) penicillin/streptomycin (Cellgro,

Mediatech). For transfection, cells were grown to 60-80% confluency on 6-well

plates, washed once with PBS and exposed to 400µl Optimem (Invitrogen)

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containing 20 µl of ExGen500 (Fermentas) and 4 µg of plasmid DNA for 2-3 h at

37 °C, 5% CO2, after which time fresh culture medium was added. Twenty four to

48h after transfection, cells were treated for 6h with 1 µM velcade (gift from

Millenium), 10mM 3-methyladenine (3MA, Sigma) or 50µM chloroquine (Sigma).

A stock solution of Velcade (1mM in H2O) was kept at -20°C. To inhibit protein

synthesis, cells were treated with 20 µg/ml cycloheximide (Sigma) for the

indicated times. The plasmid used to over-express Nedd4 (S12) consisted of full-

length rat Nedd4 epitope-tagged with T7 (MASMTGGQQMG) placed at the N

terminus of the molecule and subcloned into the pRC-CMV vector (Invitrogen),

which was a kind gift from Dr Daniela Rotin (The Hospital for Sick Children,

Departments of Biochemistry and Molecular Genetics, University of Toronto,

Ontario, Canada).

Primary neuronal cultures: Primary cultures of hippocampal neurons were

prepared from P1 rat pups. Rat pups were killed by decapitation. Hippocampi

were isolated from the brain and collected in chilled HBSS (Hanks’ Balanced Salt

Solution), with antibiotic-antimytotic, and incubated in1% trypsin-EDTA solution

at 37°C for 30 minutes. The solution was aspirated and the hippocampi triturated

using a 1 ml and 200 μL pipette in 2-3 ml of complete MEM (Minimal Essential

Medium) with 10% foetal calf serum and penicillin/streptomycin. After low speed

centrifugation (1000 rpm, 4 minutes) the supernatant was discarded and the cells

were resuspended in complete MEM and plated onto 13mm diameter glass

coverslips coated with poly-L-lysine in 6-well plates. Cultures were grown at 37°C

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in a humidified 95% O2 5% CO2 atmosphere. Twenty four hours after plating, and

then twice weekly, half of the medium was replaced with neurobasal culture

medium with added glutamine, antibiotic-antimytotic and B27 (Invitrogen). For

double immunofluorescence, cells fixed in 3% PFA for 5min, washed and then

permeabilised with 0.3% PBST (0.3% Triton X-100 in PBS) for 15 min at RT and

incubated with mouse monoclonal syn1 (BD Transduction) diluted 1:500 and/or

polyclonal anti-Nedd4 antibodies (Abcam) diluted 1:1000 in 1% BSA 5% normal

goat serum for 1 h at RT, followed by incubation with Alexafluor conjugated

antibodies (Invitrogen, UK) for 45 minutes. Cells were mounted and visualized

using fluorescence Nikon 80i Upright Microscope.

RNAi Experiments. Plasmids expressing microRNA targeting the expression of

VPS24 were created by annealing self-complementary oligonucleotides and

cloning into the pcDNA6.2-GW/EmGFP-miR vector (BLOCK-iT Pol II miR RNAi

expression vector, Invitrogen). pcDNA6.2-GW/EmGFP-miR-neg control plasmid

(Invitrogen) containing an insert that can form an hairpin predicted not to target

any known vertebrate gene, was used as control. siRNA targeting nedd4, tsg101

or scrambled siRNA (Santa Cruz) was used in HEK cells.

Statistical Analysis: Statistical comparisons were done by unpaired t-test.

Means in all figures are shown+/-SEM.

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