Age-dependent induction of congophilic neurofibrillary tau inclusions in tau transgenic mice

Abstract

Intraneuronal filamentous tau inclusions such as neurofibrillary tangles (NFTs) are neuropathological hallmarks of Alzheimer's disease (AD) and related sporadic and familial tauopathies. NFTs identical to those found in AD brains have also been detected in the hippocampus and entorhinal cortex of cognitively normal individuals as they age. To recapitulate age-induced NFT formation in a mouse model, we examined 12- to 24-month-old transgenic (Tg) mice overexpressing the smallest human brain tau isoform. These Tg mice develop congophilic tau inclusions in several brain regions including the hippocampus, amygdala, and entorhinal cortex. NFT-like inclusions were first detected in Tg mice at 18 to 20 months of age and they were detected by histochemical dyes that bind specifically to crossed beta-pleated sheet structures (eg, Congo red, Thioflavin S). Moreover, ultrastructurally these lesions contained straight tau filaments comprised of both mouse and human tau proteins but not other cytoskeletal proteins (eg, neurofilaments, microtubules). Isolated tau filaments were also recovered from detergent-insoluble tau fractions and insoluble tau proteins accumulated in brain in an age-dependent manner. Thus, overexpression of the smallest human brain tau isoform resulted in late onset and age-dependent formation of congophilic tau inclusions with properties similar to those in the tangles of human tauopathies, thereby implicating aging in the pathogenesis of fibrous tau inclusions.

Figure 1.

Filamentous tau lesions in the brains of 24-month-old Tg mice. A: NFT-like intraneuronal tau lesions in entorhinal cortex stained with anti-tau antibody 17026. B: NFT-like intraneuronal tau lesions associated with neuropil thread-like structures in the amygdala stained with the PHF1 mAb. C: Congo red detected flame-shaped intraneuronal tangles in the hippocampus. D: Silver-positive NFT-like intraneuronal inclusion in neocortex stained with the Gallyas method. E and F: Double-labeled NFT-like intraneuronal inclusion in the entorhinal cortex stained with the 17026 anti-tau antibody (E) and Thioflavin-S (F). All photomicrographs are at the same magnification. Scale bar, 10 μm.

Figure 2.

Double immunofluorescence staining of filamentous tau lesions in CNS neurons of 24-month-old Tg mice. A–D: Double label immunofluorescence of a spinal cord spheroid (A, B) and NFT-like intraneuronal tau lesion in entorhinal cortex (C, D) of a 24-month-old Tg mouse using the T14 mAb (A, C) and the exon 2-specific antibody (B, D). Only the NFT-like lesions in entorhinal cortex show exon 2 immunoreactivity. E–H: Double immunofluo-rescence staining of NFT-like intraneuronal tau lesions using the T14 mAb (E, G), the anti-NFL antibody (F), and the anti-β-tubulin antibody (H) showing that the tau immunoreactivity does not colocalize with NFL or β-tubulin. (I, J) Double label immunofluorescence staining of a NFT-like tau lesion with the 17026 anti-tau antibody (I) and the anti-ubiquitin mAb (J) showing colocalization of tau and ubiquitin. All photomicrographs are at the same magnification. Scale bar, 10 μm.

Figure 3.

Intraneuronal fibrillary tau lesions contain tau immunoreactive straight filaments. Pre-embedding immuno-EM shows tau immunoreactive straight filaments using the 17026 (A, C, and E) and PHF1 (B, D, and F) antibodies. The tau immunoreactive filaments labeled with 17206 anti-tau antibody in A are shown at higher magnification (see arrows in C) (bottom box) and E (top box) from areas marked by the squares in A. Arrows and arrowheads in B point to PHF1-labeled filaments around the nucleus and in neurite, respectively. Irregularly arranged PHF1-labeled straight filaments in neuronal perikarya and processes (see arrows in F) from the area in the square in D. Scale bars, 2 μm (A, B, and D) and 500 nm (C, E, and F).

Figure 4.

Accumulation of insoluble tau proteins in the CNS of old tau Tg mice. Brain cortex and spinal cord of 3-, 12-, and 24-month WT and 3-, 12-, 18-, 21-, and 24-month Tg mice were sequentially extracted with RAB high salt buffer, RIPA buffer, and 70% FA, and immunoblotted with antibodies T49 (A) or 17026 (B) and quantified using the appropriate ¹²⁵I-labeled secondary antibodies. In the Tg mice, high salt insoluble tau recovered from RIPA and FA fractions progressively accumulated in brain and spinal cord. The accumulated tau in RIPA and FA extracted fractions was mainly Tg tau and there was no obvious difference between the levels of endogenous mouse tau in RIPA fractions in WT and Tg mice spinal cord, whereas in the cortex, the levels of mouse tau in RIPA fractions of Tg mice were significantly higher than those of WT mice.

Figure 5.

Immunogold labeling of tau filaments isolated from the old Tg mouse CNS. A: Negative staining alone shows a bundle of straight 10- to 20-nm diameter filaments in the insoluble fraction from the Tg mouse CNS. Using immuno-EM, intense immunogold labeling of these filaments is seen using the 17026 anti-tau antibody (B–D), but no immunogold labeling of the filaments was seen using the antibodies to NFL (E) and peripherin (F). All electron photomicrographs are at the same magnification. Scale bar, 200 nm.