Peroxiredoxin 6 in human brain: molecular forms, cellular distribution and association with Alzheimer's disease pathology

Abstract

Peroxiredoxin 6 is an antioxidant enzyme and is the 1-cys member of the peroxiredoxin family. Using two-dimensional electrophoresis and Western blotting, we have shown for the first time that, in human control and brain tissue of patient's with Alzheimer's disease (AD), this enzyme exists as three major and five minor forms with pIs from 5.3 to 6.1. Using specific cellular markers, we have shown that peroxiredoxin 6 is present in astrocytes with very low levels in neurons, but not detectable in microglia or oligodendrocytes. In control brains, there was a very low level of peroxiredoxin 6 staining in astrocytes that was confined to a "halo" around the nucleus. In AD, there were marked increases in the number and staining intensity of peroxiredoxin 6 positive astrocytes in both gray and white matter in the midfrontal cortex, cingulate, hippocampus and amygdala. Confocal microscopy using antibodies to A beta peptide, tau and peroxiredoxin 6 showed that peroxiredoxin 6 positive astrocytes are closely involved with diffuse plaques and to a lesser extent with neuritic plaques, suggesting that plaques are producing reactive oxygen species. There appeared to be little astrocytic response to tau containing neurons. Although peroxiredoxin 6 positive astrocytes were seen to make multiple contacts with tau positive neurons, there was no intraneuronal colocalization. In brain tissue of patients with AD, many blood vessels exhibited peroxiredoxin 6 staining that appeared to be due to the astrocytic foot processes. These results suggest that oxidative stress conditions exist in AD and that peroxiredoxin 6 is an important antioxidant enzyme in human brain defenses.

Fig. 1

a, b A two-dimensional PAGE gel and blot of AD brain tissue (Case AD7) and a corresponding control brain gel and blot panels (c, d) (Case C7). pI 4–6 is indicated between the gel and blots and the molecular weight of the standards (Std) in kilo Daltons is shown on the Y-axis. The gels were stained with SYPRO ruby fluorescent stain. The box indicates the location of the molecular forms of peroxiredoxin 6. Some spots are shown on the blots to indicate the range of peroxiredoxin 6 pIs

Fig. 2

A composite figure at higher magnification showing the range of molecular forms in three cases of AD tissue (a AD7, AD8 and AD9) and three cases of control tissue (b C7, C6 and C8). The range of molecular forms 1–8 is shown, but not all forms are present in all cases. Although there are slight variations between blots, the respective pIs are as follows: 1 (5.3), 2 (5.4), 3 (5.6), 4 (5.75), 5 (5.85), 6 (5.9), 7 (5.95), 8 (6.1)

Fig. 3

Confocal localization of peroxiredoxin 6 (P6) with GFAP (astrocyte marker) (a P6, Cy2-green; b GFAP, Cy3-red; c merged image; bar 25 μM), MHC2 (microglia marker) (d P6, Cy2-green; e MHC2, Cy3-red; f merged image; bar 25 μM), MBP (oligodendrocyte marker) (g P6, Cy2-green; h MBP, Cy3-red; i merged image; bar 10 μM), Hu (neuronal marker) (j P6, Cy2-green; k Hu, Cy3-red; l merged image; bar 25 μM). Cell localization was performed using AD tissue

Fig. 4

Oil immersion image (×1,000) of typical astrocyte seen in AD cortex (a) and in control cortex (b)

Fig. 5

Light immunohistochemistry (DAB substrate) of peroxiredoxin 6 staining of astrocytes in AD gray matter (a), AD white matter (b), control tissue gray matter (c) and control tissue white matter (d). All images at ×200 magnification

Fig. 6

A bar chart showing the range of cell counts in different brain regions in gray and white matter in control tissue and in AD tissue. WM white matter, GM gray matter, Cing cingulate, Amy amygdala, MFC midfrontal cortex, Hip hippocampus. Counts are shown as mean ± SE, N = 6 in all cases

Fig. 7

a–c Confocal localization of tau within neurons (a Cy2-green) and peroxiredoxin 6 positive astrocytes (b Cy3-red) and (c) the merged image in AD brain tissue (bar = 10 μM). d–f Confocal localization of Aβ peptide (d Alexa 488, green) and peroxiredoxin 6 positive astrocytes (e Cy3-red) and (f) the merged image in a diffuse plaque in AD brain tissue (bar = 50 μM). g–j Confocal localization of Aβ peptide (Alexa 488, green) and peroxiredoxin 6 positive astrocytes (Cy5-light blue) in four different plaques in AD brain tissue (bar = 50 μM). k Immunohistochemical staining (DAB substrate) showing peroxiredoxin 6 staining of astrocytes and astrocyte foot processes in the walls of blood vessels in the midfrontal cortex in Alzheimer’s disease (×400). Arrows indicate peroxiredoxin 6 positive astrocytic processes