tau deficiency induces a parkinsonism with dementia ... · 85 f 71 control multi-organ failure...

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Supplementary Information

Tau deficiency induces a parkinsonism with dementia

phenotype by obtunding APP-mediated iron export

Peng Lei, Scott Ayton, David I. Finkelstein, Loredana Spoerri, Giuseppe D. Ciccotosto,

David K. Wright, Bruce X.W. Wong, Paul A. Adlard, Robert A. Cherny, Linh Q. Lam,

Blaine R. Roberts, Irene Volitakis, Gary F. Egan, Catriona McLean, Roberto Cappai,

James A. Duce, and Ashley I. Bush

Inventory of Supplementary Information.

Supplementary Data

Supplementary Figure 1–11

Supplementary Table 1, 2

Supplementary Methods

Supplementary References

9 literature citations from within the Supplementary Information

Nature Medicine doi:10.1038/nm.2613

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Supplementary Figure 1 Decreased nigral tau levels in PD and effects of

L-DOPA treatment on nigral chemistry. (a) Representative soluble tau western

blots of post mortem SN samples from PD and age-matched control cases (HC).

(b) SN tau levels corrected by neuron-specific βIII tubulin, reduplicate the results

in Fig. 1a, indicating that the tau loss is independent of loss of neurons. (c)

Representative soluble tau western blots of SN tissues of L-DOPA-treated mice.

L-DOPA was administered in drinking water (20mg L–1 in 0.2% ascorbic acid) for

7 d, and mice were then analyzed. (d) No change of tau level (corrected by β-actin)

or iron content (corrected by tissue wet weight) in SN of L-DOPA-treated mice.

Data are means ± SEM, n as indicated.


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Supplementary Figure 2 No correlation between iron and soluble tau in PD or

insoluble tau in HC. (a) Lack of association between SN iron and soluble tau

levels in PD. (b) Lack of association between SN iron and insoluble tau levels in

HC.


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Supplementary Figure 3 Decreased nigral tau levels in MPTP-intoxicated mice.

(a) Representative tau western blots of SN tissues of MPTP-intoxicated mice.

After injection, mice were sacrificed at intervals indicated, and SN microdissected

and analyzed. (b) SN tau levels corrected by neuron-specific βIII tubulin,

reduplicate the results in Fig. 1c, suggesting the tau loss observed is independent

of loss of neurons. Data are means ± SEM, n = 5 per group.


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Supplementary Figure 4 Age-dependent motor deficits in tau KO mice.

(a–d)These data compare the performances of tau KO to WT mice at 6-,12- and

24-months of age. (a) Time-to-finish data for the Pole Test. (b) Distance of

locomotion data in the Open Field Test. (c) Average distance per movement in the

Open Field Test. (d) Distance of movement in Y maze test. Data are means ±

SEM. *, #P < 0.05; ***, ###P < 0.005, two-factor ANOVA, # represents comparison

against 6 month baseline means. n is indicated in a.


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Supplementary Figure 5 No parkinsonism in 6-month old tau KO mice. (a–c)

Representative TH-staining of 6-month old WT (a) and tau KO (b) mice.

Quantitative data (both Nissl and TH staining) are shown in c. Scale bar is 250

µm. (d) No CPu size difference between 6-month old WT and tau KO mice. (e) No

differences in striatal dopamine (DA) or DOPAC levels between 6-month old WT


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and tau KO mice. The levels are presented as a percentage of the mean of WT

controls. Data are means ± SEM, n as indicated in the figure.


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Supplementary Figure 6 No

cognitive dysfunction in

6-month old tau KO mice. (a)

No differences in body weight

between WT and tau KO mice

at 6 and 12 months of age. (b)

No differences in brain wet

weight between WT and tau KO

mice at 6 months of age. (c) No

differences of lateral ventricular

area (LV), cortical thickness

(Ctx), corpus callosum

thickness (cc), 4th ventricular

area (4V), or granular layer

thickness (GL) between WT

and tau KO mice at 6 months of

age. The levels are presented

as a percentage of the mean of

WT controls. (d) No difference in total entries in Y maze test between WT and tau

KO mice at 6- or 12- months of age. (e) No differences in Y maze performance

between WT and tau KO mice at 6 months of age. Data are means ± SEM, n as

indicated.


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Supplementary Figure 7 Iron levels in 6M tau KO mice. Ctx= frontal cortex,

Hippo= hippocampus, Cb= cerebellum. All data were corrected by tissue wet

weight. Data are means ± SEM, n as indicated.


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Supplementary Figure 8 Effect of CQ treatment on copper and zinc levels in tau

KO mice. (a,b) CQ treatment for 5 months from 6.5 months of age did not change

Hippocampus (Hippo), SN or liver copper (a) or zinc levels (b) (normalized by

protein) in tau KO mice. Data are means (expressed as % of mean values for

sham-treated mice) ± SEM, n as indicated.


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Supplementary Figure 9

Effects of CQ on tau KO

mice and neurons. (a–d) CQ

in food was administered to

6.5-month old tau KO mice

for 5 months, and the

animals were studied for

performance during the trial

and for brain changes at

completion. (a) Rotarod

testing was performed

before and after 4 months of

CQ treatment, revealing that

CQ treatment prevented a

significant deterioration in

motor performance. (b) Y

maze test was performed

before and after 4 months of CQ treatment, revealing that CQ treatment

prevented a significant deterioration in cognitive performance. Data show

proportion of entries into the novel arm. (c) CQ treatment retarded age-dependent

loss of CPu size. (d) CQ treatment retarded age-dependent loss of CPu DA and

DOPAC. (e,f) In primary neuronal culture, CQ treatment (10 µM for 24 h) did not

reduce copper (e) or zinc levels (f). Data are means ± SEM, and n as indicated.

*P < 0.05; **P < 0.01; ***P < 0.005.


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Supplementary Figure 10 Tau deficiency impairs iron export selectively by

disrupting APP maturation. (a) No significant difference between copper levels in

tau KO and WT primary neuronal cultures prepared from cortices of embryonic

day 14 mice and incubated for 7 days in Neurobasal media, then harvested for

metal analysis. (b) No significant differences between zinc levels in primary

neuronal cultures prepared as described above. (c) 59Fe retention in tau KO and


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WT primary neurons following 1 h and 24 h incubations with 59Fe-loaded

transferrin. * P < 0.05. (d) Representative western blots of APP, Fpn and β-actin,

as well as enrichment by surface biotinylation. Tau KO neurons express higher

levels of immature APP (≈ 90 kDa). Tau KO neurons also express altered patterns

of Fpn fragments, which may reflect increased breakdown due to being

destabilized by lack of surface APP interaction, analogous to the effects of

ceruloplasmin ablation in cells that express ceruloplasmin1. The

immunoreactivities as illustrated were analyzed by densitometry. (e) Densitometry

values normalized to neuronal β-actin, quantified by Image J. Both cellular Fpn

and surface Fpn were unchanged in tau KO neurons. (f) Tau KO and WT primary

neuron homogenates were fractionated through an iodixanol step gradient.

Representative western blot shows the distribution of APP, tau, BiP (ER marker),

Syntaxin6 (Golgi marker) and EEA1 (early endosomes marker) from three

independent experiments. Mature APP (mAPP) sediments towards the top of the

gradient, compared to immature APP (imAPP) that sediments towards the bottom

of the gradient, with BiP, the ER marker. (g) Quantification of protein distribution

by Image J of f (fractions 1–20), showing a differentiated distribution of total APP

(both immature forms and mature forms) in WT and tau KO neurons. Note that

WT tau largely cofractionates with APP from WT neurons. Data are means

(expressed as percentage of the WT means) ± SEM, n as indicated.


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Supplementary Figure 11 Copper or zinc toxicity to neurons. (a, b) Primary

neurons were incubated with CuCl2 (a) or ZnCl2 (b) in the presence of ascorbic

acid (5 mM). Data are means ± SEM, n = 6. The differences between the WT and

tau KO survival curves were not significant.


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Supplementary Table 1: Clinicopathological information for human cases.

Case No. Age Sex

Post- mortem interval

(h)

Diagnosis Cause of Death Used in Fig.

03/481 77 M 53.6 Control Acute myocardial

infarction STable2

03/833 79 M 57 Control Ischaemic heart

disease 1ab, S1b, S2b,

STable2

03/983 84 M 55 Control Ischemic heart

disease STable2

04/041 83 M 22 Control Myocardial infarction 1ab, S1b, S2b,

STable2

04/104 88 M 71 Control Acute myocardial

infarction 1ab, S1b, S2b,

STable2

04/107 82 F 57.5 Control Cardiac tapenade, ruptured aortic arch

aneurysm STable2

04/250 80 M 31.5 Control Metastatic malignant

pleural mesothelioma

1ab, S1b, S2b, STable2

04/556 76 F 44 Control Acute on chronic

COAD 1ab, S1b, S2b,

STable2 04/972 75 F 22.5 Control Lobar pneumonia STable2 05/103

7 85 M 43 Control

Ischaemic heart disease



disease STable2

05/318 71 F 25 Control Pulmonary embolis 1ab, S1b, S2b,

STable2

05/728 84 F 16 Control Bronchopneumonia 1ab, S1b, S2b,

STable2 05/854 63 F 30 Control Pulmonary embolism STable2 06/101

9 85 F 71 Control Multi-organ failure STable2

06/144 69 M 24 Control Pulmonary

thromboembolism 1ab, S1b, S2b,

STable2

06/542 85 M 39.5 Control Acute myocardial

Infarct 1ab, S1b, S2b,

STable2


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06/554 76 M 45.5 Control Sepsis in a man with

ischemic heart disease


06/972 75 M 46 Control Ruptured abdominal

aortic aneurysm STable2

06/993 80 F 59 Control Cardiac failure STable2

07/022 81 M 36.5 Control Ischemic heart


STable2

07/561 84 M 47.5 Control Multiorgan failure 1ab, S1b, S2b,

STable2



STable2

07/635 72 M 42.5 Control Ischemic heart

disease STable2



STable2

07/737 77 M 49 Control Coronary artery


STable2

07/808 77 F 15.5 Control Airway compression,

Thyroid gland hemorrhage


08/026 67 F 24 Control Pulmonary

thromboembolism 1ab, S1b, S2b,

STable2

03/819 63 F 56 PD Hypertensive heart

disease 1a,

S1b,S2a,STable2 05/101

5 84 M 50.5 PD

Bronchopneumonia, COAD.

1a, S1b,S2a,STable2

05/413 72 M 45 PD

Pulmonary thromboembolism,

Deep vein thrombosis

1a, S1b,S2a,STable2

05/738 78 F 20.5 PD Ischemic heart

disease 1a,

S1b,S2a,STable2

06/437 74 M 23 PD Ischemic heart

disease 1a,

S1b,S2a,STable2

07/294 82 F 26 PD Cardiac arrest 1a,

S1b,S2a,STable2

07/566 78 M 31 PD Parkinson’s disease

15 years 1a,

S1b,S2a,STable2 07/809 80 M 26 PD Hanging 1a,


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S1b,S2a,STable2

08/319 70 M 32.5 PD Coronary artery

disease 1a,

S1b,S2a,STable2

09/255 68 M 28 PD Lobar pneumonia 1a,

S1b,S2a,STable2

09/260 67 F 20 PD Ischemic heart

disease 1a,

S1b,S2a,STable2

09/292 80 M 59 PD Acute myeloid

leukemia Dementia

1a, S1b,S2a,STable2

10/148 82 M 67.5 PD Pneumonia Dementia

1a, S1b,S2a,STable2

10/271 72 F 48.5 PD Bronchopneumonia 1a,

S1b,S2a,STable2

A01-67 84 F 34 PD Cerebrovascular

infarcts 1a,

S1b,S2a,STable2

A94-42 71 M 56 PD Cardiac arrest 1a,

S1b,S2a,STable2

A97-35 76 F 12 PD Cardiac arrest 1a,

S1b,S2a,STable2

A97-43 74 M 24.5 PD Cerebrovascular

infarcts 1a,

S1b,S2a,STable2


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Supplementary Table 2: Levels of brain iron and tau forms in PD compared to HC

Tissue Iron

(percentage of HC)

Total tau

(percentage of HC)

Ratio 3R/4R

(percentage of HC)

Ratio pTauS262/

total tau (percentage of HC)

Ratio pTauS396/

total tau (percentage of HC)

S 56.4±6.9***,# 82.1±6.1* 153.5±12.1** 201.7±24.4** SN 139.2±9.7***,#

P 112.7±11.4 78.2±12.1 104.9±18.6 105.8±14.1

S 87.9±11.2 39.8±6.5*** 130.4±8.8* 180.9±18.4** CTX 109.4±13.1

P 95.3±20.5 92.6±18.1 111.2±20.2 144.2±27.2

S 95.7±10.9 40.9±8.6* 145.1±13.5* 159.2±18.8* CB 91.5±6.2

P 102.2±11.2 79.6±14.7 105.5±14.7 173.6±19.6*

SN: substantia nigra; CTX: frontal cortex; CB: cerebellum; S: supernatant; P: pellet. 3R/4R: tau(3-repeat forms)/tau(4-repeat forms). Values are means ± SEM, n = 10 PD and n = 10 HC cases, except where indicated by #, which is n = 18 in each group. Iron levels were corrected by tissue wet weight, and the proteins are corrected by β-actin. pTauS262 and pTauS396 values were normalized against the total tau level of each individual sample, and then PD ratios (shown) expressed as a percentage of the mean HC ratio. *P < 0.05; **P < 0.01; ***P < 0.005, compared to HC mean values.


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Supplementary Methods

Reagents. Reagents were purchased from Sigma, Australia, unless specified. Mice. All mice were housed according to standard animal care protocols and fed standard laboratory chow and tap water ad libitum. All animal procedures were approved by the Mental Health Research Institute animal ethics committee and were performed in accordance with the National Health and Medical Research Council guidelines. Mouse tissue preparation. Mice were euthanized with an overdose of sodium pentobarbitone (Lethabard, 100mg kg–1) and perfused with ice-cold saline. Body weight and total brain wet weight were recorded. The right brain hemisphere was micro-dissected and stored at –80 °C until required. The left brain hemisphere was fixed in 4% paraformaldehyde for 24 h, and then either dehydrated in ascending ethanol and embedded in paraffin for Perl and Turnbull staining, or transferred to 30% Sucrose + PBS (pH 7.4) and kept at 4 °C overnight for TH immunohistochemistry and brain section analysis, respectively. L-DOPA treatment of C57/Bl6 mice. L-DOPA combined with the peripheral DOPA decarboxylase inhibitor, benserazide, was administered in drinking water (20 mg L–1 in 0.2% ascorbic acid) to mice (12 months of age) for 7 d. Mice were estimated to receive 20 mg kg–1 of L-DOPA and 5 mg kg–1 of benserazide per day based on daily water uptake. The drinking water was changed every second day to minimize the oxidation of L-DOPA. The SN was dissected for western blot and metal analysis. Animal performance studies. For the Pole Test, mice were placed vertically on a 30 cm vertical, 1cm diameter pole. On the day prior to testing (day 1), the animals were habituated to the pole and were allowed five consecutive trials. Animals were then recorded via digital video on the test day (day 2). The amount of time was recorded for the interval for the mouse to turn toward the ground (time to turn), and for the interval to reach the ground (time to finish). Each mouse underwent five trials and the average was used in analysis. For the Rotarod test, mice were assessed using a Panlab Rotarod apparatus in an accelerating model with triplicate measurements (maximum time of 2.5 min; speed increases every 8 s). The time on the rod was recorded and the triplicates averaged for analysis. For the Open Field Test, spontaneous motor activity of mice was measured using a photo-beam activity system (Truscan 2.0, Coulbourn Instruments). The test area was 25.4 cm wide by 25.4 cm deep by 40.64 cm high. The mouse was placed in the chamber for one hour to allow acclimatization to its surroundings. Parameters of movements were calculated from the interception of beams to provide an XY coordinate. For Y maze test, a Y-shaped grey-painted timber with arms 29.5 cm long × 7.5 cm wide × 15.5 cm high was used. All mice were subjected to a 2-trial Y-maze test separated by a 1 h intertrial interval to assess spatial recognition memory, with all testing performed during the light phase of the circadian cycle. The 3 identical arms were randomly designated start arm, novel arm, and other arm. Visual cues were placed on the walls of


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the maze. The first trial (training) was for 10 min, and the mice were allowed to explore only 2 arms (starting arm and other arm). For the second trial (retention) mice were placed back in the maze in the same starting arm, and allowed to explore for 5 min with free access to all 3 arms. Behaviours were recorded on video during a 5 min trial and the Ethovision video-tracking system (Noldus, Netherlands) was used for analysis. Data are expressed as the percentage of frequency and duration for novel arm entries made during the 5-min second trial. Tyrosine hydroxylase immunohistochemistry. Brains were cryoprotected in 30% sucrose/PBS until frozen sectioned on a calibrated Leica Cryostat in 30 µm sections for SN and VTA. Sections (1:3 series) were collected through the SN pars compacta (SNpc) (anteroposterior –2.92 to –3.64 mm from bregma, Mouse Atlas Figure 55 to Figure 612), generating 8 sections per mouse (the second of the three sections was analyzed). For TH-immunoreactive terminal counting, 20 µm sections (1:15 series) were prepared through the CPu (anteroposterior 1.54 to –0.22 mm from bregma, Mouse Atlas Figure 18 to Figure 332), generating 6 sections per mouse (the second of the 15 sections was analyzed). After brief fixation (4% paraformaldehyde for 30 s), the sections were blocked in 3% normal goat serum (Millipore) and incubated with rabbit polyclonal primary antibody to TH (1:3000, Millipore) overnight. The sections were then incubated with secondary HRP-conjugated goat antibody to rabbit antibodies for 3 h (Millipore), followed by diaminobenzidine solution (1% in PBS + 1% CoCl2, 1% NiSO4) + 3% hydrogen peroxide (1:3000). TH immunostained slides were counter-stained with Neutral Red to visualize the Nissl substance in all neurons, and then were mounted on Superfrost-Plus slides. Stereological estimation of nigral and VTA neurons. The numbers of neurons within the SNpc were estimated using a stereological fractionator design as previously described3,4,5. The neurons in the SNpc were distinguished primarily by anatomical location, but adjunctive features of orientation, presence of nucleolus and cell density were used to distinguish them from the smaller, sparsely packed, rostral-medial orientated neurons of the VTA5. The total number of neurons was obtained by counting TH-positive and large TH-negative, Nissl-positive neurons (which were of comparable size, ~16 µm, to TH-positive neurons). Both TH-positive and TH-negative, Nissl-positive neurons were scored according to the optical fractionator rules, where the principle involves the selection of a series of random sampled sections at a random starting point. The counts were taken using an unbiased counting frame of x = 35 µm, y = 45µm (1575 µm2) at regular intervals on a sampling grid of x = 140µm, y = 140µm (19600 µm2), viewed with a 60 × 1.3 N.A. oil objective (DMLB Leica Microscope) by the morphometry and design-based stereology software package (Stereo Investigator 10.04, Microbrightfield, Colchester, VT). The actual section thickness after processing was measured by focusing through the section with the same software package. The coefficients of error (CE) and coefficients of variance (CV) were calculated as estimates


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of precision and values of < 0.1 were accepted. < 5% variation between-operators and < 2% for individual experimenters were obtained. TH-ir terminal estimation in CPu. TH-ir terminals in the dorsal 400 µm of the CPu that predominantly receives the SNpc projection6 were counted, as previously described3. Briefly, counts of TH-ir terminals were made using an unbiased counting frame of x = 5 µm, y = 4 µm (20 µm2) at regular intervals on a sampling grid of x = 140 µm, y = 140 µm (19600 µm2), viewed with a 60 × 1.3 N.A. oil objective (DMLB Leica Microscope) by a stereological program (Stereo Investigator 10.04, Microbrightfield, Colchester, VT). TH-ir terminals were identified as round swellings in association with axonal processes. Total terminal numbers (TH number) and density were estimated using a fractionator design. T2-weighted magnetic resonance imaging. Animals (n = 2 in each group) overdosed with anesthetic were positioned in a stereotactic animal cradle with an anatomically shaped surface coil positioned on top of the head. The cradle was then inserted into a decoupled transmit coil fixed inside a BGA12S gradient set for imaging with a Bruker Biospec 47/30 scanner. A 3-plane localizer sequence and axial, coronal and sagittal scout images were acquired to ascertain the orientation of the brain and set the slice position. A T2-weighted image was acquired using a rapid acquisition, relaxation enhanced (RARE) sequence with the following imaging parameters: recovery time (TR) = 4159 ms, RARE factor = 8, effective echo time (TEeff) = 60 ms, field of view (FOV) = 2.5 cm2, in-plane resolution = 78 µm2, number of slices = 30, slice thickness = 0.5 mm and averages (NEX) = 16. Quantification from brain section. CPu and cerebellum sections from mice were sectioned using a Leica Cryostat set at 50 µm thickness and areas of interest were measured using Image-J (v1.45i, NIH). Briefly, landmarks (anterior commissure for CPu and flocculus for cerebellum) were used to identify the level of coronal sectioning (bregma 0.26± 0.01 mm and bregma –6.12± 0.01 mm) and two sections per mouse per area were analyzed. Lateral ventricles and 4th ventricular size were measured as marked in Fig. 2c. CPu area was defined by the boundaries of corpus callosum, lateral ventricle, and anterior commissure as marked in Fig. 2c. Corpus callosum, neocortical and cerebellar cortical thicknesses were averaged from 5 measurements. All quantifications were blinded. Fe2+ and Fe3+ histological staining. Paraffin-embedded sections were used to detect non-heme iron species by Perls’ and Turnbull staining methods, as previously described7. Briefly, deparaffinized and rehydrated tissue sections (7 µm) were incubated at 37 °C for 2 h in 7% potassium ferrocyanide or potassium ferricyanide in 3% HCl and subsequently incubated in DAB for 10 min. Hematoxylin and eosin was used for nuclei staining. Computer-assisted staining analysis was as described previously8. The SN area was identified as above, and by TH-staining. The area of iron-positive structures present in each section was quantitated using colour threshold (brown) to separate the stain from


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background labelling, analysed using Image J (v1.45i, NIH). An average was taken from 3–5 sections from each animal, and from results of three independent, blinded, assessors. Dopamine and DOPAC Measurement. CPu tissues dissected from each experimental condition were homogenized in HPLC sample buffer (0.4 M perchloric acid, 0.15% sodium metabisulfite and 0.05% EDTA) before centrifugation at 10,000 g at 4 ˚C for 10 min. Supernatants were used for dopamine measurement by a HPLC system (ESA Biosciences; model 584) coupled to an electrochemical detector (ESA Biosciences; Coulochem III detector) (E1: –150 mV, E2:+220 mV, and guard cell: +250 mV). 50 µL was injected onto a MD–150 reverse phase C18 column (ESA Biosciences) and elution was performed at a flow rate of 0.6 ml min–1 in the mobile phase (75 mM sodium dihydrogen phosphate, 1.7 mM 1-octanesulfonic acid sodium salt, 100 mL L–1 triethylamine, 25 mM EDTA, 10% acetonitrile, pH 3.0). Peaks were identified by retention times set to known standards. Data were normalized to wet weight tissue. Preparation of primary neuron cultures. Primary neurons from the cortices of wild type and tau knockout embryos (day 14) were prepared as previously described9. Cortices were dissected and dissociated in 0.025% trypsin. The neurons were plated (Nunc) at a density of 600,000 cells cm2 –1 in plating medium (DMEM with 10% FCS, 5% HS, and 10 mg L–1 gentamycin sulfate, from Invitrogen or Sigma). After 2 h incubation, the neurons were changed to Neurobasal supplemented medium (with B27, 500 µM glutaMAX and 10µg ml–1 gentamycin sulphate). The experiments were performed 7 d later. For CQ treatment, neurons were cultured in 6-well plates for 7 d, and 10µM CQ was incubated with neurons for a further 24 h before harvesting for metal analysis. Metal Toxicity Assay. Toxicity was assessed by cell viability assays (CCK assay, Dojindo Molecular Technologies). Neurons were cultured in a 48-well plate and treated with a series concentrations of Fe ((NH4)2Fe(SO4)2•6H2O), Cu (CuCl2), and Zn (ZnCl2) for 48 h in the presence of 5 mM L-Ascorbic acid. The pH of the metal-Neurobasal medium solution was adjusted to 7.0. Fresh medium containing 10% v/v CCK-8 was then added for further 2 h incubation before reading the absorbance at 460 nm. Cell surface biotinylation assay. Cell surface proteins were isolated using a cell surface protein isolation kit (Pierce) following manufacturer’s instructions. Briefly, primary neurons (wild type or tau knockout) were cultured in 75 cm2 flasks for 7 d, and labelled with Sulfo-NHS-SS-Biotin in PBS for 30 min. After quenching the unreacted biotin, cells were harvested by scraping and lysed using the kit lysis solution + Phosphatase Inhibitor I and II (1:1000). To precipitate biotinylated surface proteins, equal amounts of protein from cell lysates were incubated with NeutrAvidin Agarose, eluted with NuPAGE LDS sample buffer (Invitrogen) containing 50 mM DTT, and analyzed by western blotting. Total proteins were normalized to β-actin, and the ratio of surface protein to normalized total protein was expressed as a percentage of the untreated control. Iodixonal step gradient. Primary neurons (wild type or tau knockout) were cultured in 175 cm2 flasks for 7days. The day of the fractionation cells were washed with PBS and homogenized in 1 ml ice-cold homogenization buffer (20 mM Hepes/NaOH pH 7.4, 1


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mM EDTA, 1 mM EGTA, 0.25 M sucrose) containing protease inhibitors by 15 passages through a 26-gauge needle and 30 strokes with a Dounce homogenizer in ice. The cell homogenate was centrifuged at 1,000 × g for 10 min at 4 °C, the resulting post-nuclear-supernatant collected and its iodixanol concentration adjusted to 25% with ice cold 50% iodixanol (5 volumes of 60% iodixanol (Optiprep) diluted with one volume of dilution buffer (120 mM Hepes/NaOH pH 7.4, 6 mM EDTA, 6 mM EGTA, 0.25 M sucrose)) containing protease inhibitors. The sample was placed on the bottom of an ultracentrifugation tube and fractions of 20, 18.5, 17, 15.5, 14, 12.5, 11, 9.5, 8 and 6% ice cold iodixanol (50% iodixanol diluted with homogenization buffer) containing protease inhibitor were successively layered above it. After 20 h of centrifugation at 90,000 g at 4 °C (SW 41Ti rotor, Beckman), fractions of 0.5 ml were collected from the top of the tube, precipitated using acetone, resuspended in 2× sample buffer, incubated 10 min at 90 °C and equal volume of each fractions used for electrophoresis followed by immunoblot with 22C11 (APP), Tau, BiP (ER marker), Syntaxin6 (Golgi marker) and EEA1 (early endosome marker) specific antibodies. Sample preparation and western blot. Samples from each experiments were homogenized in PBS (pH=7.4) with EDTA-free protease inhibitor cocktail (1:50, Roche) + phosphatase Inhibitor I and II (1:1000) and centrifuged at 40,000 g for 30 minutes. Both supernatant and pellet were collected. Total protein concentration was determined by BCA protein assay (Pierce) or Nanodrop 1000 (Thermo). Aliquots of homogenate with equal protein concentrations were separated in 4–12% bis-Tris gels with NuPAGE MES running buffer (Invitrogen), and transferred to nitrocellulose membranes by iBlot (Invitrogen). The membranes were blocked with milk (10% v/v) and probed with appropriate primary and secondary IgG-HRP conjugated antibodies (Dako). Enhanced chemiluminiscence detection system (GE Healthcare) was used for developing and Fujifilm LAS-3000 was used for visualization. Densitometry quantification of immunoreactive signals was performed by ImageJ (1.45i, NIH) and normalized to the relative amount of β-actin (and in some cases βIII tubulin) and expressed as percentage of the mean of the control group. The following antibodies were used in this study: antibody to APP (in house, 22C11); antibody to β-actin (Sigma); antibody to BDNF (Abcam); antibody to Bip (Cell Signaling); antibody to EEA1 (Cell Signaling); antibody to Ferroportin (MAP23, from T.A. Rouault); antibody to neuron-specific βIII tubulin (Abcam); antibody to ProBDNF (Biosensis); antibody to Syntaxin6 (Cell Signaling); antibody to Synaptophysin (Millipore); antibody to tau (Dako); antibody to tau (3-repeat isoform, Millipore); antibody to tau (4-repeat isoform, Millipore); antibody to phosphorylated tau262 (Invitrogen); antibody to phosphorylated tau396 (Invitrogen); and antibody to TrkB (Cell Signaling). Statistics. Linear regression was used for the correlation between iron and tau levels in human samples (Fig. 1b, Supplementary Fig. 2a,b). One-way ANOVA with post-test was used for MPTP-treated mice studies (Fig. 1c, Supplementary Fig. 3b). Two-way ANOVA with post-test was used for pole test results of CQ-treated mice (Fig. 3f).


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Non-linear regression with extra sum-of-squares F test was used for Fe59 release and iron toxicity experiments (Fig. 4b,f, Supplementary Fig. 11a,b). For the rest, two-tailed t-test was used for comparing between two groups; two-factor ANOVA was used for comparison of multiple groups.


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Supplementary References 1. De Domenico, I. et al. Ferroxidase activity is required for the stability of cell

surface ferroportin in cells expressing GPI-ceruloplasmin. EMBO J. 26, 2823-2831 (2007).

2. Paxinos, G. & Franklin, K. B. J. The Mouse Brain in Stereotaxic Coordinates. 2 edn, (Academic Press, 2001).

3. Parish, C. L., Finkelstein, D. I., Drago, J., Borrelli, E. & Horne, M. K. The role of dopamine receptors in regulating the size of axonal arbors. J. Neurosci. 21, 5147-5157 (2001).

4. Finkelstein, D. I. et al. Axonal sprouting following lesions of the rat substantia nigra. Neuroscience 97, 99-112 (2000).

5. Parish, C. L. et al. Haloperidol treatment reverses behavioural and anatomical changes in cocaine-dependent mice. Neurobiol. Dis. 19, 301-311 (2005).

6. Gerfen, C. R., Herkenham, M. & Thibault, J. The neostriatal mosaic: II. Patch- and matrix-directed mesostriatal dopaminergic and non-dopaminergic systems. J. Neurosci. 7, 3915-3934 (1987).

7. Meguro, R., Asano, Y., Odagiri, S., Li, C. & Shoumura, K. Cellular and subcellular localizations of nonheme ferric and ferrous iron in the rat brain: a light and electron microscopic study by the perfusion-Perls and -Turnbull methods. Arch. Histol. Cytol. 71, 205-222 (2008).

8. Duce, J. A. et al. Iron-export ferroxidase activity of β-amyloid precursor protein is inhibited by zinc in Alzheimer's disease. Cell 142, 857-867 (2010).

9. Ciccotosto, G. D. et al. Enhanced toxicity and cellular binding of a modified amyloid beta peptide with a methionine to valine substitution. J. Biol. Chem. 279, 42528-42534 (2004).


tau deficiency induces a parkinsonism with dementia ... · 85 f 71 control multi-organ failure...

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