Supporting Information Salazar et al. 10.1073/pnas.0804373105 SI Materials and Methods and postfixed for 24 h (4% paraformaldehyde and 0.1 M PBS). Chemicals and Antibodies. MPTP hydrochloride, 6-hydroxydopa- Alternatively, for TH cell counting in hemimesencephali, brains mine hydrobromide, Chelex 100, and D-amphetamine sulfate were postfixed for 48 h. Next, fixed brains were cryopreserved in were purchased from Sigma. Peptide N-glycosidase (PNGase) F 30% sucrose and cut on a freezing Microtome. Free-floating was purchased from New England Biolabs. DMT1 antibodies, sections were permeabilized, blocked for nonspecific binding previously developed and characterized (1), were used to rec- sites, and incubated with primary antibodies. Immunolabeling ognize the ϩIRE C-terminal region, the ϪIRE C-terminal was visualized using Alexa 488- and Cy3-conjugated secondary region, the 4th extracellular domain (Alpha Diagnostics), and antibodies (Invitrogen and Jackson ImmunoResearch, respec- the 3rd extracellular domain (Biosonda). In addition, the fol- tively) or HRP-conjugated secondary antibodies and DAB rev- lowing antibodies were used: rabbit anti-TH (Pel-Freez Biologi- elation (Vector Laboratories). cals), mouse anti-TH (Diasorin), rat anti-CD11b/Mac1 (Sero- tec), and mouse anti-actin (Sigma). Animals. Male C57BL/6J mice 12 weeks old were obtained from Janvier Breeding Center. Microcytic mice (MK/ReJ-mk/ϩ) were Tissue Preparation for Iron Measurement and Western Blot Analysis. obtained from Funmei Yang, University of Texas Health Sci- Within2hafterautopsy, the brains were dissected and blocks of ences Center, San Antonio, TX, and Mark Fleming, Harvard hemibrainstem were frozen in dry ice and stored at Ϫ80 °C. University School of Medicine, Boston, MA, and subsequently Serial 20-␮m-thick sections were cut from the frozen blocks at maintained as an inbred stock by breeding heterozygotes in a Ϫ12 °C by using a cryostat, thaw-mounted onto gelatin/ 129sv background. Animals were maintained on a standard chromalum-coated glass slides, desiccated, and stored at Ϫ80 °C. rodent diet in the animal facility of the State University of New SN from PD patients and age-matched controls was collected York (Buffalo, NY). Rats from the Buffalo colony of Belgrade from frozen mesencephalic brain slices. The boundaries of rats that were crossed into a Fischer 344 background to the N16 subregions were delineated according to Hirsch et al. (2). Tissue generation and thus were Ϸ90% Fischer 344, were maintained (5–12 mg) was scraped with a cold plastic blade onto the glass on an iron-supplemented rat chow (4). As crosses were b/bϫϩ/b, slide, which was placed on dry ice. The collected material was littermate controls were used; ϩ/ϩ controls were age- and transferred to an Eppendorf tube and stored at Ϫ80 °C. sex-matched Fischer 344 rats. All studies were carried out in For iron and protein analyses, frozen specimens were sus- accordance with the Declaration of Helsinki and the Guide for pended and homogenized in chilled lysis buffer [50 mM Tris (pH the Care and Use of Laboratory Animals adopted and promul- 7.5), 150 mM NaCl, 10 mM NaF, 0.5% deoxycholate, 1% gated by the National Institutes of Health. The appropriate Nonidet P-40, 1 mM sodium orthovanadate, and 0.1% SDS], animal care committees approved the work. pretreated overnight with Chelex 100. From the resulting lysate -aliquot was taken for iron quantification, and the rest was then HPLC Measurement of Dopamine and MPP؉. Dopamine was mea 1 centrifuged at 3,500 ϫ g for 15 min at 4 °C. The supernatant sured by RP-HPLC with electrochemical detection (potential (soluble subcellular fraction) was removed, aliquotted, and 750 mV) under isocratic conditions with an Ag/AgCl reference stored at Ϫ80 °C. Protein quantification was performed using electrode. MPPϩ was determined by HPLC with UV detection the Bradford method. (wavelength 295 nm). Tissue Preparation for Immunohistochemistry and Perls Staining. For Amphetamine-Induced Rotation. Behavioral testing was performed immunohistochemistry, paraffin-embedded slices were irradi- visually in rotational bowls over 90 min after 10 min of latency. ated with a conventional neon tube (18 W) for 24 h in a cold One week after the striatal 6-OHDA lesion, right and left body chamber to decrease autofluorescence. Paraffin was removed, turnings were recorded after an i.p. injection of 2.5 mg/kg and the slices were incubated in pH 6.0 citrate buffer for 12 min D-amphetamine sulfate. The data are expressed as net full body in a microwave at 350 W for epitope retrieval. To confirm iron turns per minute, where rotation toward the side of the lesion accumulation in the mesencephalon of the individuals studied, was given a positive value. iron histochemistry was performed on adjacent sections using Perls Prussian blue reaction (nonheme iron), as described by Image Analysis and Stereological Cell Counting. For confocal studies Smith et al. (3). of DMT1 distribution, a Leica microscope was used. Total numbers of TH-positive cells were calculated by stereological Immunohistochemistry. Prepared human mesencephalic slices 5 methods by using Explora Nova software. ␮m thick were blocked for nonspecific binding sites and incu- bated with primary antibodies. Immunolabeling was visualized Atomic Absorbance Spectroscopy. Total iron was determined by using Cy5-conjugated secondary antibodies, biotinylated sec- atomic absorption spectroscopy. Ventral hemimesencephalon of ondary antibodies/Cy3-conjugated streptavidin, or HRP- control and MPTP mice was homogenized in chilled lysis buffer. conjugated secondary antibodies/nickel-enhanced DAB revela- One hundred microliters (20–50 ␮g of protein) of sample was tion (Vector Laboratories). During the analysis of human mixed with 100 ␮L of ultrapure 5% nitric acid and incubated at mesencephalic sections, we considered a neuron to be strongly 60 °C for 12 h. The digest was cooled and centrifuged at 12,000 ϫ labeled when staining intensity was in the same range as the g for 2 min, and the supernatant was diluted to 1 mL with 0.2% intensity of neuromelanin color and when nerve processes were ultrapure nitric acid. The final sample was colorless and trans- clearly labeled. In contrast, in lightly labeled neurons staining parent. Fe content was determined in an atomic absorption intensity was less intense than the endogenous color of neu- spectrometer with a graphite furnace (SIMAA 6100; Perkin– romelanin, and no processes were stained. DMT1 antibody Elmer). MR-CCHEN-002 (Venus antiqua) preparation from the specificity was confirmed by preabsorption control experiments. Comisio´n Chilena de Energía Nuclear was used as the reference Rodent brains were fixed by transcardiac perfusion, dissected, material to validate the mineral analysis. Salazar et al. www.pnas.org/cgi/content/short/0804373105 1of3 Perfusion–Perls. Mice were perfused with a solution containing protein extracts were denatured at 65 °C in buffer containing 1% HCl, 1% potassium ferrocyanide, and 10% formalin (pH 0.5% SDS and 1% 2-mercaptoethanol, then incubated at 37 °C 1.0). Brains were postfixed, cryopreserved, and cut into 20-␮m for 1 h in buffer supplemented with Nonidet P-40 (1% final) and coronal slices. Sections were pretreated with 0.3% H2O2 and 0.01 PNGase F (25 units/␮g of particulate protein). M NaN3 in methanol for 30 min to block endogenous peroxidase activity, and enhanced in 0.025% DAB and 0.005% H2O2 in 0.1 Western Blots and Quantification. The immunoblots were visual- M phosphate buffer (pH 7.4). ized using HRP-conjugated secondary antibodies against IgG and a chemiluminescent substrate (SuperSignal Ultra; Pierce). Protein Deglycosylation. Protein extracts were deglycosylated by Images were quantified using National Institutes of Health using PNGase F according to the supplier’s instructions. Briefly, ImageJ software. 1. Roth JA, et al. (2000) Differential localization of divalent metal transporter 1 with and 3. Smith MA, Harris PL, Sayre LM, Perry G (1997) Iron accumulation in Alzheimer disease without iron response element in rat PC12 and sympathetic neuronal cells. J Neurosci is a source of redox-generated free radicals. Proc Natl Acad Sci USA 94:9866–9868. 20:7595–7601. 4. Garrick M, et al. (1997) Iron supplementation moderates but does not cure the 2. Hirsch E, Graybiel AM, Agid YA (1988) Melanized dopaminergic neurons are differen- Belgrade anemia. BioMetals 10:65–76. tially susceptible to degeneration in Parkinson’s disease. Nature 334:345–348. Salazar et al. www.pnas.org/cgi/content/short/0804373105 2of3 Fig. S1. Changes in iron content and distribution in the ventral mesencephalon of acutely MPTP-intoxicated mice. (A) Time course of iron content changes in the ventral mesencephalon of controls and MPTP-intoxicated mice, as measured by AAS. **, P Ͻ 0.01. (B and C) Photomontage of perfusion–Perls iron histochemistry (black) and TH immunohistochemistry (cyan) in the ventral mesencephalon of control and MPTP-intoxicated mice. (B) In the SNpc (delineated) of control mice, there was only mild iron staining. Moreover, iron staining was present in small cells (Ͻ10 ␮m; black arrowhead), whereas it was totally absent from TH-positive cells (Inset and detail; Right). (C) Two days after MPTP intoxication there was stronger iron staining in the ventral aspects of the SNpc (black arrowheads; Upper Left). Iron accumulation is observed in some DNs with weak TH expression (white arrowhead, Inset and detail; Right). (D) Immunohisto- chemistry against 4-hydroxynonenal adduct in the coronal slices of control and MPTP-intoxicated mice. (Scale bars: in central images in C and D, 200 ␮m; in Right Insets and detail widths in C and D,20␮m.) Salazar et al. www.pnas.org/cgi/content/short/0804373105 3of3.