Caspase cleavage of tau: linking amyloid and neurofibrillary tangles in Alzheimer's disease

Abstract

The principal pathological features of Alzheimer's disease (AD) are extracellular amyloid plaques and intracellular neurofibrillary tangles, the latter composed of the microtubule-binding protein tau assembled into paired helical and straight filaments. Recent studies suggest that these pathological entities may be functionally linked, although the mechanisms by which amyloid deposition promotes pathological tau filament assembly are poorly understood. Here, we report that tau is proteolyzed by multiple caspases at a highly conserved aspartate residue (Asp421) in its C terminus in vitro and in neurons treated with amyloid-beta (Abeta) (1-42) peptide. Tau is rapidly cleaved at Asp421 in Abeta-treated neurons (within 2 h), and its proteolysis appears to precede the nuclear events of apoptosis. We also demonstrate that caspase cleavage of tau generates a truncated protein that lacks its C-terminal 20 amino acids and assembles more rapidly and more extensively into tau filaments in vitro than wild-type tau. Using a monoclonal antibody that specifically recognizes tau truncated at Asp421, we show that tau is proteolytically cleaved at this site in the fibrillar pathologies of AD brain. Taken together, our results suggest a novel mechanism linking amyloid deposition and neurofibrillary tangles in AD: Abeta peptides promote pathological tau filament assembly in neurons by triggering caspase cleavage of tau and generating a proteolytic product with enhanced polymerization kinetics.

Fig. 1.

Human tau is cleaved by multiple caspases at Asp⁴²¹ in vitro. (A) ³⁵S-labeled WT human tau (doublet) was incubated with buffer control (C) or 2.5 or 25 ng of caspases-1, -2, -3, -6, -7, or -8 (C1–C8) for 1 h. The major caspase cleavage product (doublet) is indicated by the arrow. (B) ³⁵S-labeled WT or mutant (MT) D421E tau was incubated with control buffer or 2.5 ng of caspases-3, -7, or -8 for 1 h. In both A and B, the cleavage products were resolved by SDS/PAGE and visualized by autoradiography. (C) TauC3 mAb specifically recognizes tau that is truncated at Asp⁴²¹. Recombinant human WT tau (containing four microtubule-binding repeats), three-repeat tau (produced by alternative splicing, labeled 3R), or truncated tau proteins encoding amino acids 1–391, 1–421, or 1–429 was immunoblotted with Tau5 (Left) or TauC3 (Right) mAbs. (D) Recombinant human WT tau was incubated with control buffer (C) or 25 ng of caspase-3 or -7 (C3 or C7) for 1 h, and the reaction products were immunoblotted with Tau1 (Left) or TauC3 (Right) mAbs. The molecular mass (kDa) of markers is shown at the left of A–C.

Fig. 2.

Treatment of cortical neurons with fibrillar Aβ induces tau proteolysis at Asp⁴²¹. (A) Primary cultures of cortical neurons prepared from embryonic day-18 rats were treated with 10 μM fibrillar Aβ (1–42) for 0–24 h, and lysates were immunoblotted with TauC3 (Upper) or PARP (Lower) mAbs. (B) Cortical neurons were preincubated in the absence or presence of 50 μM zVAD-fmk for 1 h and then treated for an additional 3 h with 10 μM fibrillar Aβ (1–42) prior to immunoblot analysis. (C) Laser-scanning confocal images of untreated control neurons (Upper) or a neuron treated with fibrillar Aβ (Lower) analyzed by indirect immunofluorescence with tubulin or TauC3 mAbs; the merged images are shown (Right). (Bar=10 μm.) (D) Tau proteolysis at Asp⁴²¹ is an early event in Aβ-induced neuronal apoptosis. Primary cortical neurons were treated with 20 μM Aβ for 0–12 h, and TauC3-negative (black bars) or TauC3-positive neurons (hatched bars) were scored for apoptosis by nuclear morphology (mean ± SEM).

Fig. 3.

Truncation of tau at Asp⁴²¹ increases the rate and extent of tau filament assembly in vitro.(A) Kinetic analysis of the assembly of recombinant WT or truncated (1–421) tau proteins into tau filaments in vitro. Tau polymerization was induced by the addition of 75 μM arachidonic acid to 4 μM recombinant WT or truncated tau proteins and measured by right-angle laser light scattering. Scattering intensities were measured on a scale of 0–256 and are proportional to the total mass of polymerized tau filaments (30). The data are presented as the mean ± SEM of three independent experiments. (B) Representative electron micrographs of tau filaments assembled in vitro by recombinant WT tau (Upper) or truncated tau (Lower) proteins. (Bar = 200 nm.)

Fig. 4.

The characteristic neurofibrillar pathologies of AD contain tau truncated at Asp⁴²¹. The CA1 hippocampal region from a patient with AD was analyzed by immunohistochemistry by using TauC3 (A) and Tau5 (B) mAbs. NFT and dystrophic neurites forming neuritic plaques (NP), two hallmark pathologic features of AD, are indicated. (Bars = 50 μm.) In C–E, laser-scanning confocal images of a single NFT from a patient with AD were stained with TauC3 (C) or Thiazin red (D); the latter is a marker for compact filaments (33). The merged image is shown in E. (Bar = 10 μm.)