Nitration of tau protein is linked to neurodegeneration in tauopathies

Abstract

Oxidative and nitrative injury is implicated in the pathogenesis of Alzheimer's disease (AD) and Down syndrome (DS), but no direct evidence links this type of injury to the formation of neurofibrillary tau lesions. To address this, we generated a monoclonal antibody (mAb), n847, which recognizes nitrated tau and alpha-synuclein. n847 detected nitrated tau in the insoluble fraction of AD, corticobasal degeneration (CBD), and Pick's disease (PiD) brains by Western blots. Immunohistochemistry (IHC) showed that n847 labeled neurons in the hippocampus and neocortex of AD and DS brains. Double-label immunofluorescence with n847 and an anti-tau antibody revealed partial co-localization of tau and n847 positive tangles, while n847 immunofluorescence and Thioflavin-S double-staining showed that a subset of n847-labeled neurons were Thioflavin-S-positive. In addition, immuno-electron microscopy revealed that tau-positive filaments in tangle-bearing neurons were also labeled by n847 and IHC of other tauopathies showed that some of glial and neuronal tau pathologies in CBD, progressive supranuclear palsy, PiD, and frontotemporal dementia with parkinsonism linked to chromosome 17 also were n847-positive. Finally, nitrated and Thioflavin-S-positive tau aggregates were generated in a oligodendrocytic cell line after treatment with peroxynitrite. Taken together, these findings imply that nitrative injury is directly linked to the formation of filamentous tau inclusions.

Figure 1.

Characterization of the n847 mAb. A: The specificity of n847 for nitrated tau. Fifty ng of each protein shown were loaded in corresponding lanes of 7.5% (left) or 12% (right) SDS-polyacrylamide gels, separated by electrophoresis, and transferred to nitrocellulose membranes that were probed with n847 and developed by enhanced chemiluminescence (ECL). B: The n847 mAb recognized sarkosyl-insoluble nitrated tau protein in AD (AD1-AD3), CBD, and PiD but not in normal (N1-N3) brains. PHF-tau fractions from frontal cortical gray matter of each brain were isolated biochemically and analyzed by Western blot using n847, T14/T46, and PHF-1 antibodies as described in the text. C: Similar PHF-tau bands are recognized by n847 and the 3-NT antibodies, but 3-NT also recognizes other nitrated proteins in AD brains. Shown in (C) is a PHF-tau fraction isolated from AD brain (as in B) analyzed by Western blot with n847, PHF-1, and anti-3-NT. D: The n847 mAb specifically recognizes nitrated tau proteins. Shown in (D) are native (control lanes) and denitrated PHF-tau proteins from AD brains examined by Western blot illustrating the reduced staining with n847 in the denitrated AD PHF-tau sample compared to the control sample. Denitration did not affect PHF-tau staining by the nitration independent anti-tau mAbs T14/T46. E: Western blots demonstrate the specificity of mAb n847 for 3-NT modified hα-Syn proteins. Wild-type and Tyr (Y) to Phe (F) point mutants of recombinant hα-Syn were exposed to peroxynitrite as described in the text, and 25 ng of each protein were loaded in separate lanes of 12% SDS polyacrylamide gels for Western blot analysis with mAb LB509 to native hα-Syn and the n847 mAb. Note that LB509 shows that equal amounts of hα-Syn proteins were loaded in each lane, but n847 preferentially recognizes peroxynitrite-treated α-Syn containing at least one Tyr residue, but not the Tyr-deficient hα-Syn protein (4Y→F). The hα-Syn trimers and dimers (identified on the right) probably reflect dityrosine cross-linking.

Figure 2.

IHC analyses of the AD hippocampus with mAb n847. A: N847 IR in the CA1 region of AD patients (Braak and Braak stage III-IV, CERAD criteria - probable AD 44 ). Low-power views of CA1 (a corresponds to the boxed region of f) showing diverse n847 IR in pyramidal neurons. Four types of n847 IR in neuron are shown (ie, types 1, 2, 3, and 4 in b, c, d, and e, respectively, as described in the text). As the Braak stage advanced, the number of type 3 and 4 neurons in the CA1 region increased but only type 1 neurons (b) were observed in young normal control brains (g, low-power view), while n847 IR was very weak in cerebellum of AD and normal brain (h, Braak stage V-VI in AD). *, indicates Purkinje cells. Bars, 10 μm. B and C: Semi-quantitative analysis shows a shift of n847 IR with progression of AD. The signature of early stage (I-II) of AD was predominance of type 2 n847 IR, but this type of staining diminished as the Braak stage advanced. On the other hand, types 3 and 4 n847 IR were insignificant in stage I-II, but increased significantly at later Braak stages while type 4 n847 IR predominated at Braak stage V-VI.

Figure 3.

Double-labeling of AD NFTs at various stages with n847 and 17024/Thio-S. A: The n847 IR co-localizes with 17024 IR NFTs. Shown in a, b, and c of A are representative images of types 2, 3 and 4 n847 IR cells, respectively. Co-localization of staining is seen in the merged images and by confocal microscopy. Bars, 10 μm. B: The n847 IR is evident in pre-tangles. To identify neurons containing nitrated NFTs, adjacent sections were labeled with combination of n847 and 17024 (section 1) or Thio-S and 17024 (section 2) as described in the text. Thio-S-positive and -negative neurons are indicated by arrowhead and arrow, respectively. C: Double-label studies of NFTs by n847 and Thio-S. Four representative labeling patterns of Thio-S are shown: (a) none; (b) uniformly moderate; (c) mixed intense and moderate; (d) uniformly intense. Note that the n847 IR decreases as Thio-S labeling increases. Bars, 10 μm.

Figure 4.

IHC n847 analyses of diverse tauopathy lesions and immuno-EM. A: Immunostaining with n847. Globose NFTs in the pons of PSP (a), substantia nigra of CBD (b), degenerating midfrontal cortical neurons in CBD (c), a ballooned neuron (inset, top) compared to a normal one (inset, bottom) in CBD, a Pick body in entorhinal cortex of PiD (d) and in hippocampus of FTDP-17 (e), a glial tangle (coiled body) in hippocampus of FTDP-17 (f). Bars, 10 μm. B: Double-labeling of various tauopathy lesions with n847 (red) and 17024 (green). (a) a globose NFT in substantia nigra of PSP, (b) a Pick body in the PiD hippocampus, (c) a glial tangle (coiled body) in the CBD basal ganglia, (d) a ballooned neuron in CBD midfrontal cortex. e and f are confocal microscopic images of a and b, respectively. Bars, 10 μm. C: Filaments in NFTs show n847 IR by immuno-EM as described in the text. a: Individual filaments are tau and n847 IR by double-label immune-EM using 17024 (5-nm gold particles, arrows) and n847 (10-nm gold particles, arrowheads) (b). Bars, 100 nm.

Figure 5.

IHC and Western blot studies of cultured oligodendroglia. OLN-Tau40 cells were peroxynitrite treated (0.5 mmol/L) and analyzed 24 hours after treatment. A: Control (Co) or peroxynitrite (PN)-treated cells were studied by FIHC with the 17024 polyclonal antibody (a, d, g, j), followed by Thio-S (b and e) or n847 staining (h and k) and nuclei of cells (c, f, i, l) were counterstained by DAPI. Images are shown in single (a, b, d, e, g, h, j, k) or merged (c, f, i, l) views. Bar, 10 μm. B: Tau is nitrated in cultured OLN-Tau40 cells treated with peroxynitrite. Shown are Western blots of cell lysates from untreated control (Co) or peroxynitrite-treated OLN-Tau40 cells at 1, 3, and 7 hours post-nitration as described in the text. Note that n847 IR bands co-migrate with tau IR bands labeled by 17024, but in addition, higher molecular weight n847 IR bands are seen that are tau negative which may represent nitratively cross-linked species of tau that may have lost tau IR due to nitrative damage.

Figure 6.

Schematic model of the possible role of nitrative/oxidative modifications in the formation of PHF. Under normal conditions, tau (ovals) is in equilibrium between a free and a microtubule-bound (rectangular bars) pool, where tau stabilizes microtubules. Nitrative damage of tau may reduce its affinity for microtubules resulting in unbound nitrated (triangles) tau, which may serve as a nidus for oligomer formation, but nitration also may enhance tau fibrillization. This polymerization process can lead to the formation of complex aggregates of PHFs. o-o’ Dityrosine cross-linking (brackets) that occurs concomitantly with nitration could also contribute to the stabilization of polymers.