Tau Protein Disrupts Nucleocytoplasmic Transport in Alzheimer's Disease

Abstract

Tau is the major constituent of neurofibrillary tangles in Alzheimer's disease (AD), but the mechanism underlying tau-associated neural damage remains unclear. Here, we show that tau can directly interact with nucleoporins of the nuclear pore complex (NPC) and affect their structural and functional integrity. Pathological tau impairs nuclear import and export in tau-overexpressing transgenic mice and in human AD brain tissue. Furthermore, the nucleoporin Nup98 accumulates in the cell bodies of some tangle-bearing neurons and can facilitate tau aggregation in vitro. These data support the hypothesis that tau can directly interact with NPC components, leading to their mislocalization and consequent disruption of NPC function. This raises the possibility that NPC dysfunction contributes to tau-induced neurotoxicity in AD and tauopathies.

Keywords: Alzheimer’s disease; Nup98; nuclear pore complex; nucleocytoplasmic transport; tauopathies.

Figure 1.. In AD, tau is found at the nuclear membrane and NPCs mislocalize to the cytosol.

(A) Illustration describing how abnormal phospho-tau that mislocalizes into the soma during AD interfaces with the cytoplasmic side of nuclear pore complexes. At the nuclear pore, pathogenic tau can disrupt nuclear pore complex function. (B) Human AD (Braak VI) and age/sex matched control brains were immunolabeled for phospho-tau (green; p-tau mix of anti phospho-tau antibodies pT181, pS199, pT205, pT231, pS409), NPCs (red; using mab414), and Dapi (blue). In AD cortex, phospho-tau accumulates in the cytosol and NPCs mislocalize from the nuclear membrane into the cytosol (arrow heads). In neurons with diffuse phospho-tau staining in the cytosol, phospho-tau also localizes to the nuclear membrane (arrows).

Figure 2:. The FG-nucleoporin Nup98 mislocalizes to the cytoplasm in AD brains.

(A) Nup98 (green) is evenly distributed in the nuclear membrane (arrow heads) of hippocampal neurons in control brains, but colocalizes aberrantly with phospho-tau (phospho-tau mix; pink) in the neuronal cytosol of AD brains (arrows). The small nuclei (DAPI+) without obviuos Nup98 staining in the nuclear membrane are glia cell nuclei. Scale bars, 10 μm. (B+C) Pronounced cytosolic Nup98 (red in B, green in C, arrows) accumulation in cortical NFT neurons in AD brains labeled for different pathology associated phospho-tau epitopes (pink; phospho-tau mix in B, pT205 and pT231 in C). Scale bars, 10 μm. (D) Cytoplasmic mislocalization of Nup98 (DAB) in hippocampal AD neurons (arrows) compared to nuclear membrane localization (arrow head) in control brains. Scale bar, 50 m; scale bar in zoomed images, 10 μm. (E) Immunofluorescence labeling of hippocampal sections shows that neither FG-Nup Nup54 (green) nor non-FG structural Nup Pom121 (red) overtly mislocalize in the cytoplasm of phospho-tau (PHF1) containing neurons in AD, compared to control brains. Scale bars, 10 μm.

Figure 3:. The Nup98 C-terminal domain induces tau fibrilization in vitro.

(A) Recombinant human tau (2N4R; wild-type or P301L; 8 μM; 0.33 mg/ml) was incubated with the C-terminal half of Nup98 (aa645–911) at a final [Nup98:tau] ratio of [1:4], [1:8], or [1:27] in the absence of heparin. Tau aggregation was monitored by thioflavin-S fluorescence and EM (lower panel), Scale bar, 0.2 μm. (B) SDS-PAGE and Western blot analysis of supernatants (soluble fractions) and pellets (insoluble fractions containing aggregated and polymerized tau and Nup98) from in vitro Nup98-tau aggregations. Membranes probed for tau (tau5) and Nup98 show that for P301L tau a tau:Nup98 ratio of 1:8 is sufficient to initiate tau aggregation. Monomeric and HMW species of tau and Nup98 are indicated. HMW tau and Nup98 species in the insoluble fractions indicate that one protein induces the other one to shift to higher molecular weight species, presumably through co-aggregation.

Figure 4:. Phospho-tau iteracts with nucleoporins in nuclei from AD brain and in vitro.

(A) Principle of NPCs quantification and co-localization in whole nuclei extracted from brain tissue using high-resolution AiryScan imaging and 3D-rendering. (B+C) Images of nuclei extracted from control and AD hippocampal tissue after staining for phospho-tau (green) and either NPC (red, mab414 in B) or Nup98 (red in C). Displayed are maximum Z-projections from the midline to the dorsal Z-maximum of the nuclei. Note the accumulation of phospho-tau on the morphologically abnormal AD nuclei. Scale bar, 2 μm. (D-F) AD nuclei show higher levels of associates phospho-tau (D), colocalization of p-tau with NPC components (E), and a significantly reduced number of NPCs in the nuclear membrane (F). Graphs show quantification of punctae in 3D nuclei images using Imaris (see STAR methods); N=5 cases; N=20 nuclei per sample. All graphs are presented as mean + SEM. *p< 0.5, **p< 0.01 and ***p< 0.001 by one-way ANOVA with Tukey post-test. (G) Surface plasmon resonance (SPR) response curves show that recombinant human phsopho-tau (tau-p12; 2N4R) binds to immobilized Nup98-FG and to Nup98-S. In contrast, nonphosphoryliertes tau (tau-p0) does not bind Nup98-FG and Nup98-S. N=3 experiments. (H) Nup98 layers height changes corresponding to (G). N=3 experiments, data presented as mean + SD. (I) SPR of tau-p12 an tau-p0 applied to C-terminal half of Nup98 (C-Nup98) reveals no significant binding. N=3 experiments. (J) Tau protein was isolated from the HMW protein fraction (size exclusion chromatography) of AD brain homogenate using an anti-human tau affinity column (mab HT7 recognizing human tau aa159–163) contains phopho-tau (PHF-1) and Nup98. The identical sample was run simultaneously in parallel and Western blotted for PHF1 and Nup98.

Figure 5:. Tau pathology causes nuclear leakiness and Ran mislocalization in AD.

(A) Dextran size exclusion assay of nuclei extracted from hippocampus (left) and cerebellum (right) of Braak I (control), Braak III (mild AD), and Braak VI (severe AD) brains. Nuclei were isolated with a sucrose gradient and incubated with 70-kDa (green) and 500-kDa (red) fluorescent dextrans. Intranuclear 70-kDa or 500-kDa dextran indicated leakiness of the nuclear membrane. N = 3 brains per Braak stage. Scale bars, 15 μm. (B) The percent of leaky nuclei (70-kDa dextran intra-nuclear fluorescent signal >20% of the extra-nuclear signal; (D’Angelo et al., 2009)) in hippocampi of AD brains correlates with the AD Braak staging. Nuclei from cerebellum, a region that remains unaffected by tau pathology in AD, show no leakiness. N = 3 patients per Braak stage, N = 100 nuclei/Braak stage. (C) Immunolabeling of phospho-tau (p-tau mix) and Ran in AD (Braak VI; N = 10) and control (Braak I and II; N = 10) hippocampal CA1 tissue. Controls show diffuse nuclear and cytoplasmic Ran, whereas Ran is cleared from the nucleus in phospho-tau-positive AD neurons. Scale bars, 10 μm. (D) Quantification of nuclear Ran intensity in hippocampal control and AD neurons. N = 10 control and AD cases; 5 fields per case; 100 neuronal nuclei per case. Data are presented as mean + SEM. **p< 0.01, ***p< 0.001 by one-way ANOVA with Tukey post-test.

Figure 6.. Cytosolic HMW tau disrupts nuclear import and export of proteins.

(A) The fluorescent reporter NES:tdTomato:NLS was used to monitor the nuclear import capability of neurons treated with fluorescently labeled HMW protein from AD brain. Representative images of untreated and HMW AD protein treated (for 24 h) neurons before (pre-bleach *) and after (bleached **) photo-bleaching and after recovery (post-bleached). (B) Florescence recovery after photobleaching (FRAP) of nuclei after 24, 48, and 72 h incubation with HMW AD protein. Most FRAP is lost after 24 h, and no further decrease is detected after longer times of HMW AD protein treatment. Data is presented as area under the average curve. N = 15 nuclei for untreated and treated neurons. (C) The fluorescenct reporter 2xGFP:NES-IRES-2xRFP:NLS is used to monitor nuclear import and export of proteins in neurons. Schematic illustration of nuclear transport disruption after treatment with HMW AD protein. (D) Confocal images of primary neurons expressing 2xGFP:NES-IRES-2xRFP:NLS and treated with HMW AD protein. Untreated neurons show clear separation of nuclear RFP and cytosolic GFP. GFP:NES occurs the nucleus after 24 h, RFP:NLS diffuses into the cytoplasm at 48 h upon treatment with HMW AD protein fraction containing tau. Scale bars, 10 μm.

Figure 7:. Severe Ran mislocalization in transgenic mice with tau aggregation.

(A) 14-month-old WT, rTg21221, rTg4510, and APP/PS1 brains stained for phospho-tau (green) and Ran (pink) reveal Ran mislocalization into the cytoplasm in hippocampal neurons positive for phospho-tau. rTg21221 and APP/PS1 mice have Ran intensities similar to WT, whereas phospho-tau-positive neurons in rTg4510 mice show a substantial depletion of Ran in the nucleus. Scale bars, 40 μm; scale bars in zoomed images, 10 μm. (B) Quantification of nuclear (white outline) Ran protein intensity in CA1 neuron. N = 3 per group, 100 nuclei per group. Data are presented as mean + SEM. ***p< 0.001.

Figure 8:. Reducing tau mitigates Nup98 aggregation and rescues Ran gradient In mice.

(A) Transgenic human tau expression in rTg4510 mice can be repressed by feeding doxicylin (DOX). Comparing hippocampal (CA1) brain sections from age-matched WT and rTg4510 mice with or without DOX treatment from 6 to 12 months of age, we found that Ran (pink) was depleted from most neuronal nuclei (white outlined) in rTg4510. Ran was present in the nucleus in DOX-treated rTg4510 even in presence of remaining aggregated phospho-tau (green) in the soma. Nup98 (red) mislocalized to the somata in rTg4510 and rTg4510+DOX neurons with cytosolic phospho-tau (green). Scale bars in merged images, 40 μm; scale bars in zoomed images, 10 μm. (B) Quantification shows that the nuclear Ran intensity was fully restored in DOX-treated animals, which have decreased soluble phospho-tau. N = 3 per group, 100 nuclei per group. Data are presented as mean + SEM. **p< 0.01, ***p< 0.001, ****p< 0.0001 by one-way ANOVA with Tukey post-test. (C) The colocalization of Nup98 with phospho-tau significantly decreased in DOX-treated animals. N = 3 per group. Data are presented as mean + SEM. **p< 0.01, ***p< 0.001, ****p< 0.0001 by one-way ANOVA with Tukey post-test.