Introduction 1 11 32 15 14 31 7 22 23 5 3 9 30 19 Materials and methods Tissue samples from human SN and frontal cortex 6 19 20 http://www.ICDNS.org Quantitative real-time polymerase chain reaction ® 19 25 In situ hybridization (ISH) 18 29 http://www.premierbiosoft.com 35 Snap frozen, unfixed post-mortem tissue blocks of the frontal cortex and SN were cut on a cryostat (12 μm), collected onto Superfrost Plus slides (VWR) and stored at −80°C. Before use, tissue sections were fixed for 5 min in fresh 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Pre-hybridisation treatment included acetylation in 0.25 M acetic anhydride/0.1 M triethanolamine, dehydration in graded alcohol (70–100% ethanols) and delipidation in 100% chloroform. Hybridisation with the oligonucleotide probe was performed overnight at 37°C. Post-hybridisation sections were washed in standard saline citrate solutions with increasing stringencies. The sections were dehydrated rapidly through graded alcohols (70–100% ethanol), air-dried, dipped in autoradiographic emulsion (LM1, GE Healthcare, Amersham, UK) and exposed for 5–6 weeks. The autoradiographic emulsion was developed, sections were counterstained with toluidine blue (VWR) for Nissl substance and mounted for visualisation using bright field microscopy for the counterstain and epi-polarized illumination for the silver grains of the autoradiographic emulsion. Control slides run to check for specificity of hybridization included competition with an excess of oligonucleotide (250 times) and the use of two probes to different portions of the mRNA of interest which produced identical patterns of signal. Black and white photographs of emulsion-coated sections were taken using a microscope provided with a Hamamatsu C4742–95 digital camera (Herrsching, Germany) and HiPic software (Herrsching) to capture images. Digital colour images were captured with a Retiga 1300 monochrome 12-bit camera using a color option provided by a RGB-HM-S filter (QImaging, Burnaby, BC, Canada) and QCapture 1.1.6 software (QImaging). Adobe Photoshop was used to assemble images. Immunohistochemistry 24 For fluorescence microscopy, anti-SNCA (1:1,000) and anti-NPTX2 (1:25) were added to the sections sequentially (each was incubated overnight at 4°C). For the detection of the primary antibodies, sections were rinsed in 0.1 M Tris buffered saline (TBS) and then incubated with either the goat anti-rabbit IgG tagged with Alexa Fluor 546 (red) for 1 h at room temperature or the biotinylated anti-mouse antibody (Vector Laboratories 1:100) for 1 hour followed by incubation with streptavidin Alex Fluor 488 (green) for 1 h at room temperature. Finally, tissue sections were rinsed in TBS and incubated in sudan black for 10 min before mounting in 80% Tris-buffered glycerol. Western blotting Snap frozen midbrain tissue samples containing SN from PD and control cases were homogenized with RIPA buffer (pH 8), heated to 70°C for 10 min and subsequently centrifuged (13,000 rpm, 10 min). The protein supernatant was removed and the concentration of protein determined (BIO-RAD protein reagent; BIO-RAD, UK) using a spectrophotometer. Denaturing gels were run using 30 μg of protein from each sample in 19.5 μl RIPA buffer, 3 μl β-mercaptoethanol and 7.5 μl lithium dodecyl sulphate sample buffer (Invitrogen, UK). NuPAGE Bis–Tris 4–12% polyacrylamide gels (Invitrogen) were run in NuPage SDS running buffer (Invitrogen). Gels were blotted onto nitrocellulose membranes (Invitrogen), immersed in NuPage LDS transfer buffer (Invitrogen) in a cold room (4°C) overnight applying 30 V. Successful protein transfer was verified by staining the membrane in Ponceau S (VWR, UK) for 10 min. Prior to overnight incubation in polyclonal goat antibody to NPTX2 (N-20), membranes were washed in phosphate buffered saline (PBS) containing 0.1% Tween 20 and subsequently rinsed in PBS/0.1% Tween 20 containing 5% skimmed milk (blocking step) for 30 min. Incubation with the secondary antibody coupled to horseradish peroxidase (1:5,000; Vector Labs, UK) was carried out for 1 h at room temperature. Visualization of antibody binding using enhanced chemilluminescence (ECL; Amersham Biosciences, UK) was performed according to the manufacturer’s instructions. Semi-quantitative ratings The NPTX2 labelled SN sections were examined by two independent observers blinded to the identity of the respective cases. The occurrence of NPTX2 immunoreactive profiles in the SN was scored for the overall burden of NPTX2 immunoreactive deposits using a seven-point scale ranging from 0–0.5 to 3 (absent to severe). Correlation analysis (Spearman; Sigmatstat 2.03) was conducted within the PD cohort for NPTX2 mRNA expression (log 2 microarray data) and the corresponding semi-quantitative immunocytochemical results. Results NPTX2 is a novel component of Lewy bodies 19 P log 2 1 1 1 Fig. 1 a  b  c 25 d, e arrow d arrows e asterisks f asterisks arrow a f d a f d e  Localisation of NPTX2 and SNCA protein 1 1 2 2 2 3 3 3 3 r P 1  Fig. 2 a, b large arrow a arrows b arrow head b asterisks a  c, d, e red c green d arrows c yellow e a b c e  Fig. 3 a c Arrows longer arrows b  Arrows c long arrows small arrows a b c  Table 1 Correlation analysis for NPTX2 microarray expression data and the corresponding semi-quantitative immunocytochemical ratings for eight of the PD cases Case number Expression NPTX2 mRNA (log 2) Semi-quantitative ratings NPTX2 IR in SN 2 7.278105587 1 3 9.800657274 3 6 8.739679364 1.5 7 8.44508108 1.5 9 3.418203444 0.5 12 7.980139578 1 14 8.965264821 2 15 6.414495823 1 Discussion 1 8 31 1 10 26 17 4 7 N P log 2 P log 2   28 16 3 Fig. 4 red blue AMPA GRIA1 GRIA2 GRIA3 GRIA4 NPTX1 NPTX2 NPTXR Green line purple line dotted grey lines solid lines grey colour  2 30 9 30 30 27 21 4 12 13 4 Electronic supplementary material Below is the link to the electronic supplementary material.
 Supplemental Figure 1. Cluster analysis is a method to organize genes in a microarray dataset into groups (clusters) of similar expression values. We have used ArrayAssist 5.5 software (Stratagene) to produce this image which shows that NPTX2 is not the only gene that is significantly up-regulated in the Parkinsonian substantia nigra compared to control and whose expression varies within the cohort. The latter is important because unlike a number of other highly dysregulated genes which show greater similarity across cases and therefore higher p values, NPTX2 fits better with both the clinical and neuromorphological notion of different disease stages in different cases. There was a good correlation between expression values and immunocytochemical results (Table 1). Abbreviations: CON, control; LN, lateral nigra; MN medial nigra; PD, Parkinson’s disease. Codes CON10, CON2, CON3, CON9, PDC1, PD01- PD36 relate to study cases 19, 17, 18, 23, 16, 1-15, respectively (Supplemental Table). The colour range of expression values represents a logarithmic scale. The correlation of expression between different cases, or the distance between rows, i.e. their similarity measure, was determined as follows: a hierarchical clustering algorithm was applied with "Pearson centred" as the distance function, the "centroid" linkage rule was used, and clustering was performed on the whole nigral transcriptome. (JPG 3.35 mb) Supplement to Fig. 4 (PDF 85.7 kb) Supplemental Table 1. Cases used in the study (PDF 37.8 kb)