Selective tau tyrosine nitration in non-AD tauopathies

Abstract

Previously, we reported the characterization of two novel antibodies that react with tau nitrated at tyrosine 197 (Tau-nY197) and tyrosine 394 (Tau-nY394) in Alzheimer's disease (AD). In this report, we examined whether tau nitration at these sites also occurs in corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and Pick's disease (PiD), three neurodegenerative tauopathies that contain abundant tau deposits within glial and neuronal cell types but lack amyloid deposition. The reactivity of these antibodies was also compared to two previously characterized antibodies Tau-nY18 and Tau-nY29, specific for tau nitrated at tyrosine 18 and tyrosine 29, respectively. In the present experiments, Tau-nY18 did not label the classical pathological lesions of CBD or PSP but did label the neuronal lesions associated with PiD to a limited extent. In contrast, Tau-nY29 revealed some, but not all classes of tau inclusions associated with both CBD and PSP but did label numerous Pick body inclusions in PiD. Tau-nY197 was restricted to the neuropil threads in both CBD and PSP; however, similar to Tau-nY29, extensive Pick body pathology was clearly labeled. Tau-nY394 did not detect any of the lesions associated with these disorders. In contrast, extensive neuronal and glial tau pathology within these diseases was labeled by Tau-Y197, a monoclonal antibody that reacts within the Y-197-containing proline-rich region of the molecule. Based on our Western and IHC experiments, it appears that nitration of tau at tyrosine 29 is a pathological modification that might be associated with neurodegeneration. Collectively, our data suggest that site-specific tau tyrosine nitration events occur in a disease and lesion-specific manner, indicating that nitration appears to be a highly controlled modification in AD and non-AD tauopathies.

Fig. 1

Determination of relative antibody affinities to non-nitrated and nitrated tau proteins by ELISA. a Tau-nY18, Tau-nY29, Tau-nY197, and Tau-nY394 were incubated with wild type tau (ht40), wild type nitrated tau (nht40) and nitrated mutant tau proteins containing a single tyrosine residue at position Y18, Y29, Y197 or Y394. Note that each antibody selectively binds to wild type nitrated tau and mutant proteins that correspond to its epitope. b The selectivity of these antibodies is due to both the nitro-tyrosine group and the amino acids surrounding it. c Total tau within these samples was labeled using Tau-Y197 and Tau-5, antibodies that bind to both 3R and 4R tau. Tau-Y197 did not react with mutant proteins nitrated at Y18, Y29 and Y394 as these proteins have a Y → F substitution at position 197. Note that all peptides contain an additional cysteine residue at the carboxy end of the sequence to facilitate binding to maleimide-activated KLH for immunizations

Fig. 2

Limited tau tyrosine nitration in CBD. a In most cases pathologically diagnosed with CBD, Tau-nY18 did not stain the characteristic lesions of the disease, although one case b did show some neuropil thread staining following citric acid treatment. c Tau-nY29 reacted with the perinuclear inclusions as well as some f globose tangles. d Tau-nY197 only labeled the neuropil treads following CIP treatment. g Tau-nY394 did not label any inclusions. e Numerous globose tangles, coiled bodies (arrow), h, i as well as the astrocytic plaques were labeled using Tau-Y197. j Western blot analysis of soluble and insoluble tau extracts demonstrates that Tau-nY18 did not label soluble tau but did label the insoluble fractions. Conversely, Tau-nY29 labeled soluble but not insoluble tau. Tau-nY197 labeled both, the soluble and insoluble fractions, while Tau-nY394 did not label either of the fractions analyzed. k Abundant soluble tau was detected with RD3, RD4 and Tau-12. The insoluble fractions, however, were only labeled with RD4 and Tau-12 antibodies. In all panels, calibration bars represent 20 μm

Fig. 3

Selective tau tyrosine nitration in PSP. a Tau-nY18 did not label the pathological inclusions associated with PSP. b Tau-nY29 reacted with the globose tangles and with c the perinuclear inclusions but d, e Tau-nY197 localized to the neuropil threads following CIP treatment. f Tau-nY394 did not label any of the pathological lesions associated with PSP. g Tau-Y197 reacted with numerous neuropil threads and h globose tangles (asterisk) as well as i the thorny (asterisk) and tufted astrocytes associated with PSP. j By Western blot analysis Tau-nY18 did not react with the soluble tau but did label the insoluble fractions whereas Tau-nY29 labeled soluble tau but not insoluble fractions. Tau-nY197 labeled both the soluble and insoluble fractions, while Tau-nY394 did not react with any fractions analyzed. k Abundant soluble tau was detected with RD3, RD4 and Tau-12. The insoluble fractions, however, were only labeled with the Tau-12 antibody. In all panels, calibration bars represent 20 μm

Fig. 4

Tau nitration in PiD. a Only a limited number of Pick body aggregates were labeled with Tau-nY18. In contrast, numerous Pick body inclusions reacted with Tau-nY29 (b, c arrowheads) and Tau-nY197 (d, e arrowhead). f Tau-nY394 did not react with any of the lesions of PiD, g, h but were clearly labeled using Tau-Y197. The coiled bodies (i arrowheads), and the ramified glial pathology were also labeled using Tau-Y197 (j arrowheads). k Western blot analysis indicated that Tau-nY18 did not label soluble tau but did label the insoluble fractions. No reactivity for soluble or insoluble tau was observed using Tau-nY29. Tau-nY197 sparsely labeled tau within the soluble and insoluble fractions. Tau-nY394 did not label either of the fractions analyzed. l However, the soluble fractions were clearly labeled using RD3, and to a limited extent using the Tau-12 antibody. The insoluble fractions were only labeled with RD3 and Tau-12 but did not react with the RD4 antibody. In all panels, calibration bars represent 20 μm

Fig. 5

Nitration of tau at Y18, Y29 and Y197 co-localizes with the Alz-50 antibody in PiD. Double-label immunofluorescence was performed on tissue sections from areas of the frontal cortex using Tau-nY18, Tau-nY29, Tau-nY197, and the Alz-50 antibody. a–c Pick body inclusions positive for Tau-nY18 clearly co-localized with the Alz-50 antibody. d–i (arrowheads) Only a fraction of Tau-nY29-positive inclusions co-localized with Alz-50 epitope. Limited co-localization was also observed when Tau-nY197 was paired with Alz-50, both Pick body inclusions (j–o), and some ramified glial pathology (j–l asterisk) showed some co-localization however, most Tau-nY197 reactive Pick bodies were not labeled by the Alz-50 antibody (m–o asterisk). In all panels, calibration bars represent 20 μm