Tau-dependent microtubule disassembly initiated by prefibrillar beta-amyloid

Abstract

Alzheimer's Disease (AD) is defined histopathologically by extracellular beta-amyloid (Abeta) fibrils plus intraneuronal tau filaments. Studies of transgenic mice and cultured cells indicate that AD is caused by a pathological cascade in which Abeta lies upstream of tau, but the steps that connect Abeta to tau have remained undefined. We demonstrate that tau confers acute hypersensitivity of microtubules to prefibrillar, extracellular Abeta in nonneuronal cells that express transfected tau and in cultured neurons that express endogenous tau. Prefibrillar Abeta42 was active at submicromolar concentrations, several-fold below those required for equivalent effects of prefibrillar Abeta40, and microtubules were insensitive to fibrillar Abeta. The active region of tau was localized to an N-terminal domain that does not bind microtubules and is not part of the region of tau that assembles into filaments. These results suggest that a seminal cell biological event in AD pathogenesis is acute, tau-dependent loss of microtubule integrity caused by exposure of neurons to readily diffusible Abeta.

Figure 1.

Tau-dependent hypersensitivity of CV-1 cell microtubules to prefibrillar Aβ42. CV-1 cells transfected with tau-CFP and YFP-tubulin were treated with prefibrillar Aβ42 as indicated and imaged by time-lapse fluorescence microscopy or analyzed by a biochemical assay for unassembled and polymerized tubulin (Black et al., 1996). (A) 1 μM prefibrillar Aβ42 caused tau to dissociate from microtubules and the microtubules to disassemble soon thereafter. (B) This effect required tau expression, because microtubules remained intact in cells that expressed only YFP-tubulin and were treated with 3 μM prefibrillar Aβ42. (C) Microtubules were unaffected in tau-expressing cells exposed to 3 μM fibrillar Aβ42. (D and E) Time-dependent microtubule loss induced by prefibrillar Aβ42 documented in D by fractionation of tubulin into soluble (S) and polymerized (P) pools (Black et al., 1996), and quantitation of fluorescence micrographs of fixed cells expressing Tau-CFP and counterstained with anti–α-tubulin (E). Error bars indicate the SD, and transfected and nontransfected refer to cells that did and did not express Tau-CFP, respectively.

Figure 2.

Tau-dependent hypersensitivity of neuronal microtubules to prefibrillar Aβ42. Primary rat cortical neurons were cultured for at least 8 d before treatment with prefibrillar Aβ42. (A) Cells were stained by immunofluorescence for tubulin (DM1A) and tau (R1tau) at the time points indicated after the addition of 1 μM prefibrillar Aβ42. Note the swollen varicosities induced by the Aβ42 (arrows). (B) Neurites in neurons treated for 2 h with 1 μM prefibrillar Aβ42 were found by electron microscopy to contain numerous varicosities that were filled with membrane-bound organelles (asterisks) and lacked microtubules, and additional regions with sparse, poorly organized microtubules (arrowheads). (C) Primary hippocampal neurons were extracted with Triton X-100 to separate the soluble (S) from polymerized (P) tubulin (Black et al., 1996). Note that ∼90% of the tubulin was polymerized in control cultures, that only ∼45% was polymerized after 2 h of cellular exposure to 1 μM prefibrillar Aβ42, and that only a modest loss of polymerized tubulin (65% polymerized) was caused by a 2-h exposure to 3 μM fibrillar Aβ42. (D) Primary neurons were transfected with a tau-specific siRNA and treated with 2 μM prefibrillar Aβ42 for 2 h before extraction. The siRNA-treated cells expressed between 1/16 and 1/32 the normal level of tau (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200605187/DC1) and showed no change in tubulin levels in response to the Aβ42 treatment when compared with the tau-expressing cells.

Figure 3.

Prefibrillar Aβ42 does not induce AD-like tau phosphorylation. Primary rat hippocampal neurons were treated with 1 μM Aβ42 for 2 h before Western blotting with the indicated anti-tau antibodies: PHF-1 (phosphoserine 396 and 404), AT180 (phosphoserine 231), and tau-1 (dephosphoserine 199 and 202). The phosphorylation of tau in treated neurons was compared with whole brain extract from an AD brain, as well as to paired helical filaments purified from an AD brain. Note that AD-like tau phosphorylation was not increased by prefibrillar Aβ42.

Figure 4.

The active portion of tau resides within an N-terminal fragment that does not target to microtubules. CV-1 cells transfected with the indicated fluorescently tagged proteins were treated with 1 μM prefibrillar Aβ42 and imaged by time-lapse fluorescence microscopy. (A) Microtubules remained intact in cells expressing GFP-MAP2c or GFP-MAP2c chimera after >2 h of exposure to Aβ42. In contrast, microtubules depolymerized in cells expressing GFP-tau chimera after <1 h of exposure to Aβ42. (B) Microtubules also depolymerized in cells that were exposed to Aβ42 and expressed the N-terminal arm of tau coupled to CFP.

Figure 5.

Quantitation of fluorescence micrographs for Aβ42-induced microtubule loss. CV-1 cells expressing the indicated proteins with 40–50% transfection efficiency were exposed to the form of Aβ42 specified on the figure. The cells were then fixed and stained with anti-tubulin and scored for microtubules as described in Materials and methods. Transfected and nontransfected refer to cells that did and did not express the indicated transgenes, respectively. Error bars indicate the SD, and asterisks mark statistically significant differences at α = 0.02 between the indicated pairs of transfected and nontransfected cells after 3 h of Aβ exposure. Statistically significant differences were not found for any pair of transfected versus nontransfected cells at 0 h, nor for any nontransfected pair at 0 versus 3 h. The collective results shown here confirm the qualitative results shown in Figs. 1 and 4 and Videos 1 and 4–8 (available at http://www.jcb.org/cgi/content/full/jcb.200605187/DC1).