Introduction 10 35 26 5 6 24 5 7 24 3 3 16 , 15 20 8 27 32 2 30 9 36 Materials and methods Antibodies Primary antibodies Peroxiredoxin 6 (rabbit) was obtained from Antibody Technology Australia Pty, Ltd, Adelaide, Australia. Tau (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK. Tau (goat) was obtained from Santa Cruz Biotechnology Inc., California, USA. Amyloid beta (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK. Glial fibrillary acidic protein (GFAP) (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK. Human neuronal protein HuC/HuD (mouse) was obtained from Molecular Probes Inc., Eugene, USA. Human HLA (MHC2) (mouse) was obtained from Dakocytomation, Denmark A/S. Myelin basic protein (MBP) (mouse) was obtained from Novacastra Laboratories Ltd, Newcastle, UK. Secondary antibodies Donkey anti sheep Cy3 was obtained from Jackson Immunoresearch, West Grove, USA. Donkey antirabbit Cy3 and Cy5 were obtained from Jackson Immunoresearch, West Grove, USA. Goat antimouse Alexa 488 was obtained from Molecular Probes Inc., Eugene, USA. Goat antirabbit alkaline phosphatase was obtained from Sigma Chemical Company, St Louis, USA. Brain tissue 1 Table 1 List of cases used in cell counting Case number Sex Age (years) Diagnosis Region PMI (h) Braak stage NIA Reagan AD1 M 65 AD C, A, H, MC  3 5/6 High AD2 F 84 AD C, A, H, MC  16 5/6 High AD3 M 63 AD C, A, H, MC  33 5/6 High AD4 M 59 AD C, A, H, MC 20 5/6 High AD5 F 81 AD C, A, H, MC 19 5/6 High AD6 F 69 AD C, A, H, MC 38 5/6 High C1 F 79 ADL C, A, H, MC 4  Low C2 M 69 Unk C, A, H, MC 48  Low C3 F 61 CF C, A, H, MC 8  Low C4 F 84 SS C, A, H, MC 15  Low C5 F 86 DM C, A, H, MC 17  Low C6 F 86 HVD C, A, H, MC 6  Low PMI AD NIA Reagan , M F C A H MC C ADL Unk CF SS DM HVD Brain homogenate g 2 Table 2 List of cases used for 2D PAGE and Western blots Case number Sex Age (years) Diagnosis Region PMI (h) Braak stage NIA Reagan AD7 F 69 AD MC 22 5/6 High AD8 F 79 AD MC 18 5/6 High AD9 F 76 AD MC 23 5/6 High C6 F 86 HVD MC 6  Low C7 F 73 ABD MC 24  Low C8 M 63 SS MC 38  Low PMI AD F M MC C HVD ABD SS Sample preparation 2 2D polyacrylamide gel electrophoresis (PAGE) Isoelectric focusing Isoelectric focusing was performed on 13 cm immobilized pH gradient strips (pH 4–7NL, Amersham Pharmacia) using an IPGphor (Amersham Pharmacia) isoelectric focusing unit. Samples were applied in gel rehydration (50 V, overnight) using 7 M urea, 2 M thiourea, 4% CHAPS, 0.5% dithiothreitol, 0.5% pharmalyte pH 4–7 with a trace of bromophenol blue as the re-swell buffer with a total loading volume of 280 μl including sample. Stained gels received a 400 μg protein load and Western blotting gels received a 200 μg load. IEF was performed for ∼60 kV h (500 V 30 min, 1,000 V 30 min, 1,000–8,000 V 30 min, 8,000 V 5–7 h, 1,000 V hold to end). SDS PAGE SDS PAGE was performed using 12.5% acrylamide gels. Immobilized pH gradient strips were incubated in SDS equilibration buffer containing 1% w/v dithiothreitol for 15 min followed by SDS equilibration buffer containing 4% w/v iodoacetamide for 15 min. Strips were then placed above the second dimension gels and overlaid with 1% low melting point agarose in Tris glycine gel running buffer. Electrophoresis was performed at 350 V for 2.5–3 h at 10°C. Protein gels were visualized using SYPRO ruby fluorescent stain (incubated for 2 h) and scanned using a Typhoon 9400 Variable Mode Imager (Amersham Biosciences). Western blotting 2 Immunohistochemistry Localization of peroxiredoxin 6 30 Colocalization of peroxiredoxin 6, Aβ and Tau 30 Cell counting 2 2 3 Table 3 Interactions examined in cell counting data Interactions examined F F P Significance (>0.01) Are there differences in AD (C, A, H, MC) gray matter cell counts? 1.00 4.94 NS Are there differences in AD (C, A, H, MC) white matter cell counts? 1.23 4.94 NS Are there differences in control (C, A, H, MC) gray matter cell counts? 0.62 4.94 NS Are there differences in control (C, A, H, MC) white matter cell counts? 0.25 4.94 NS Are there differences between gray and white matter cell counts in AD tissue? 89.3 7.21 S Are there differences between gray and white matter cell counts in control tissue? 14.5 7.21 S Are there differences in gray matter cell counts between control and AD tissue? 299 7.21 S Are there differences in white matter cell counts between control and AD tissue? 28.0 7.21 S AD NS S C A H MC Results Molecular forms of peroxiredoxin 6 in human brain 30 31 1 2 Fig. 1 a b c d pI 4–6 Y box spots  Fig. 2 a b 1–8  2 2 Cellular distribution of peroxiredoxin 6 in control and AD brain tissue 3 3 3 3 Fig. 3 a green b red c bar d green e red f bar g green h red i bar j green k red l bar  4 5 Fig. 4 a b  Fig. 5 gray a white b gray c white d  Cell counting 30 6 3 Fig. 6 bar chart WM GM Cing Amy MFC Hip N  Colocalization of peroxiredoxin 6 and AD pathology The major defining pathology in AD is the presence of extracellular plaques and intracellular hyperphosphorylated tau in the form of tangles. Plaques are present in brain tissue at different stages of development, ranging from diffuse plaques to fibrillar and neuritic plaques. It is generally considered that diffuse plaques do not contain tau, but neuritic plaques contain both Aβ and tau. Colocalization of peroxiredoxin 6 and the Aβ peptide, a major component of plaques, and tau was carried out to determine if peroxiredoxin 6 positive astrocytes were associated with AD pathology. Tau pathology 7 Fig. 7 a–c a green b red c bar d–f d green e f bar g–j green light blue bar k Arrows  Plaques 7 7 Peroxiredoxin 6 staining of blood vessels 7 Discussion 13 23 29 9 30 11 33 34 11 Tissue selection is also a variable that needs to be considered. While the AD tissue had all the hallmarks of AD pathology and was easy to define, age-matched control tissues are somewhat more variable. All the control tissue was from people without clinically defined AD, but most brains in this age group usually contain some neurodegenerative pathology. The age span of the two groups was similar, 59–84 years for the AD group and 61–86 years for the control group, but the mean age for the control group was 7 years older. We feel confident that, although some control tissue may have had some elements of subclinical AD pathology, the reduced level of astrocyte activation and the clear differences in the cell counting would indicate that all the control tissues were not experiencing high levels of oxidative stress as compared to the AD tissue. 30 F 25 14 22 12 18 12 4 19 17 1 Neurofibrillary tangles comprising hyperphosphorylated tau were observed in many regions in AD tissue. Two antibodies were used to localize tau, a monoclonal antibody (Novacastra) and one specific for paired helical filaments (Santa Cruz) and both produced a similar staining pattern. Neurons containing tau were not surrounded by activated astrocytes as were the diffuse plaques and to a lesser extent the neuritic plaques, suggesting that these cells are not secreting toxic or activating products that are stimulating to astrocytes. In PD, Lewy bodies contained a dense core of peroxiredoxin 6 staining suggesting that it was trying to detoxify oxidative stress produced by Lewy bodies, but we did not observe any interaction between tau and peroxiredoxin 6 in AD tissue. Although there is considerable evidence to implicate oxidative stress in the hyperphosphorylation of tau, this does not appear to upregulate peroxiredoxin 6 in tau positive neurons. 28 21 In conclusion, we have shown for the first time the range of peroxiredoxin 6 variants, which are either isoforms or post-translational modifications in human brain tissue. We have also shown that peroxiredoxin 6 is primarily an astrocytic enzyme with very low levels in neurons and is not detectable in microglia or oligodendrocytes. This enzyme is markedly elevated in astrocytes in both white and gray matter in AD. Strongly staining peroxiredoxin 6 astrocytes appear to be involved in the detoxification of diffuse plaques and to a lesser extent in neuritic plaques, but is not associated with tau aggregations within neurons. From this work and previous work on PD and dementia with Lewy bodies, we suggest that peroxiredoxin 6 is a major antioxidant enzyme in human neural tissue.