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. 2014 Nov;20(11):1254-62.
doi: 10.1038/nm.3700. Epub 2014 Oct 19.

Cleavage of Tau by Asparagine Endopeptidase Mediates the Neurofibrillary Pathology in Alzheimer's Disease

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Free PMC article

Cleavage of Tau by Asparagine Endopeptidase Mediates the Neurofibrillary Pathology in Alzheimer's Disease

Zhentao Zhang et al. Nat Med. .
Free PMC article

Abstract

Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation and triggers neurodegeneration. AEP is upregulated and active during aging and is activated in human AD brain and tau P301S-transgenic mice with synaptic pathology and behavioral impairments, leading to tau truncation in NFTs. Tau P301S-transgenic mice with deletion of the gene encoding AEP show substantially reduced tau hyperphosphorylation, less synapse loss and rescue of impaired hippocampal synaptic function and cognitive deficits. Mice infected with adeno-associated virus encoding an uncleavable tau mutant showed attenuated pathological and behavioral defects compared to mice injected with adeno-associated virus encoding tau P301S. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.

Conflict of interest statement

COMPETING FINANCIAL INTERESTRS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. AEP cleaves tau in vitro
(a) Tau cleavage assay. GST-tau was incubated with kidney lysates from Lgmn+/+ or Lgmn−/− mice at pH 7.4 or pH 6.0 at 37°C for 30 min. Western blot shows that tau was cleaved at pH 6.0 by Lgmn+/+ kidney lysates (left panel) when AEP was activated (right panel) (mean ± SEM; n =3). (b) Tau cleavage by wild-type and mutant AEP. HEK293 cells cotransfected with GST-tau and myc-AEP WT, myc-AEP C189S, myc-AEP N323A or control plasmid were incubated in buffer (pH 6.0) at 37°C for 30 min. Tau was selectively cleaved in cells expressing wild-type but not mutant AEP. (c) The proteolysis of tau is blocked by AENK peptide, but not AEQK (upper panel). AEP activity was inhibited by AENK (bottom panel) (mean ± SEM; n =3). (d) Antibody titration assay. AEP specific antibody inhibited the cleavage of tau. Mouse IgG was used as negative control. (e) Purified active recombinant AEP potently cleaves purified GST-tau recombinant protein. (f) Endogenous tau cleavage by AEP. Lgmn+/+, Lgmn+/−, and Lgmn−/− mouse brain tissues were lysed in buffer at pH 6.0 or 7.4 and subjected to Western blot. (g) Validation of AEP enzymatic activities by fluorescent substrate cleavage assay (mean ± SEM; n =3).
Figure 2
Figure 2. AEP cleaves tau at N255 and N368 residues
(a) Cleavage of purified GST-tau analyzed by immunoblotting (upper panel) or Coomassie blue staining (bottom panel). (b) Mass spectrometry analysis of recombinant tau fragmented by AEP. The detected MS/MS peptide spectra are listed. (c) Cleavage of mutant tau by AEP. Tau cleavage was analyzed by Western blot after GST-tau wide-type, N255A, N368A, or N255A/N368A mutant were incubated with active mouse kidney lysates. (d) MS/MS spectrum showing the cleavage of tau after N368 in brain samples from subjects with AD. (e) Representative extracted ion chromatograms for tau N255 and N368 peptide from wild-type and Lgmn−/− mouse brain samples. Signal intensities were then normalized to wild-type samples, setting the maximum signal intensity to 100%. Values are represented as raw peptide extracted ion intensity.
Figure 3
Figure 3. AEP is upregulated and cleaves tau during aging and in Alzheimer’s disease
(a) Western blot analysis of tau and AEP in mouse brain during aging process. (b) AEP activity assay (mean ± SEM; n = 6; *P < 0.05 compared with 1- and 2-months old mouse brain, **P < 0.01 compared with 1-, 2-, and 4-months old mouse brain, one-way ANOVA). (c) Western blot detection of tau fragments in the Lgmn−/− and Lgmn+/+ mice brain. (d) Immunostaining of tau N368 fragments in brain sections of subjects with AD (mean ± SEM, *P < 0.01, Student’s t-test). Scale bar, 50 μm. (e) Western blot detection of tau fragments in human brain samples from subjects with AD and age-matched controls. (f) Immunostaining showing colocalization of tau N368 fragment with PHFs. Brain sections from subjects with AD were immunostained with anti-tau N368 antibody, and then stained with Thioflavin S, which labels both the senile plaque (arrow head) and PHFs (arrow). Scale bar, 50 μm. (g) AEP activity assay in brain samples from subjects with AD and age-matched controls (mean ± SEM; *P < 0.05 compared with control group, one-way ANOVA). (h) AEP activity assay in 6-month-old tau P301S mice and non-transgenic controls (mean ± SEM; n = 6; *P < 0.01, Student’s t-test). (i) pH in the brain cortex and hippocampus of control and tau P301S transgenic mice (n = 6, *P < 0.01, Student’s t-test).
Figure 4
Figure 4. Tau cleavage by AEP disrupts its microtubule assembly activity and is toxic to neurons
(a) Schematic diagram of tau isoform 4 domains and its cleavage by AEP. I, Inserts; P, proline rich; R, repeats; R′, pseudo-repeat; C, C-terminal tail. (b) Western blot analysis of purified His-tagged tau fragments. (c) Microtubule assembly assay. (d)Axon elongation in primary neurons transfected with control plasmid, HA-tau, or HA-tau fragments (mean ± SEM; n = 5; *P < 0.05, one-way ANOVA). (e, f) TUNEL assay showing the neurotoxicity of AEP-derived tau fragments. Mixed tau fragments (e), tau 1–368 and tau 256–368 (f) induced significant neuronal apoptosis (mean ± SEM; n = 3; *P < 0.01, one-way ANOV). Scale bar, 10 μm. (g) Effect of kinase inhibitors on the neurotoxic effect of tau 1–368. Primary neurons infected with AAV-tau 1–368 were treated with 10 μM roscovitine (CDK5 inhibitor), SB216763 (GSK3 inhibitor), or H89 (PKA inhibitor) for 12 h. Cell apoptosis was detected by TUNEL staining (n = 4). Scale bar, 20 μm.
Figure 5
Figure 5. AEP gene deficiency prevents tau phosphorylation, synaptic dysfunction, and memory deficits in the tau P301S mouse
(a) The processing of tau in WT, Lgmn−/−, tau P301S, and tau P301S/Lgmn−/− mice. (b) Immunostaining of AEP-derived tau fragments in brain sections of tau P301S and tau P301S/Lgmn−/− mice. Scale bar, 20 μm. (c) AT8 immunostaining of hippocampal and cortex neurons (mean ± SEM; *P < 0.05, **P < 0.01, Student’s t-test). Scale bar, 50 μm. (d) Synaptic density analysis (mean ± SEM; n = 6; **P < 0.01, one-way ANOVA). (e) The ratio of paired pulses (mean ± SD; n = 6 in each group; *P < 0.05, one-way ANOVA). (f) LTP of fEPSPs (mean ± SD; n = 6 in each group; *P < 0.05, one-way ANOVA). Shown traces are representative fEPSPs recorded at the time point 1 and 2 (tau P301S/Lgmn−/−), 3 and 4 (tau P301S). (g, h) Morris water maze analysis as distance traveled (millimeters) and integrated distance (AUC) for WT, Lgmn−/−, tau P301S and tau P301S/Lgmn−/− mice (mean ± SEM; n = 8; *P < 0.05, one-way ANOVA). (i) Probe trail of Morris water maze test (left panel, mean ± SEM; n = 8; *P < 0.05, **P < 0.01, one-way ANOVA). AEP gene deletion did not interfere with the swim speed (right panel, mean ± SEM; n = 8; P = 0.786, one-way ANOVA). (j) Fear conditioning test. Tau P301S/Lgmn−/− mice show more freezing time than tau P301S littermates both in cued fear conditioning test (left panel), and contextual fear conditioning test (right panel) (mean ± SEM; n = 8; *P < 0.05, **P < 0.01, one-way ANOVA).
Figure 6
Figure 6. Blocking of tau cleavage by AEP prevents tau P301S-induced synaptic dysfunction and cognitive impairment
(a, b) Immunostaining and Western blot showing the expression of tau P301S and tau P301SN255AN368A in mice brain. Scale bar, 50 μm. (c) Electron microscopy of the synapses. Arrows indicate the synapses. Scale bar, 1 μm. (d) Quantification of synaptic density (mean ± SEM; n = 6; *P < 0.05, ** P < 0.01, one-way ANOVA). (e) The ratio of paired pulses is greater in tau P301SN255AN368A mice than in tau P301S mice (mean ± SD; n = 6 in each group; *P < 0.05, one-way ANOVA). (f) LTP of fEPSPs (mean ± SD; n = 6 in each group; *P < 0.05, one-way ANOVA). Shown traces are representative fEPSPs recorded at the time point 1 and 2 (tau P301S), 3 and 4 (tau P301SN255AN368A). (g) I/O curves obtained in hippocampal slices prepared from mice injected with AAV-GFP control, AAV-tau P301S, and AAV-tau P301SN255AN368A. (h) Averaged slope of I/O curves (mean ± SD; n = 6; *P < 0.05, one-way ANOVA). (i) Morris water maze analysis as time to platform (sec) and integrated latency (AUC) for mice injected with AAV-GFP control, AAV-tau P301S, or AAV-tau P301SN255AN368A (mean ± SEM; n = 10; **P < 0.01, one-way ANOVA). (j) Probe trail result (left panel, mean ± SEM; n = 10; *P < 0.05, **P < 0.05, one-way ANOVA). The swim speed of the mice was not affect by the expression of tau P301S or tau P301SN255N368 (right panel, mean ± SEM; n = 10; P = 0.512, one-way ANOVA).

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References

    1. Ballatore C, Lee VM, Trojanowski JQ. Tau-mediated neurodegeneration in Alzheimer’s disease and related disorders. Nat Rev Neurosci. 2007;8:663–672. - PubMed
    1. Binder LI, Frankfurter A, Rebhun LI. The distribution of tau in the mammalian central nervous system. J Cell Biol. 2565;101:1371–1378. - PMC - PubMed
    1. Harada A, et al. Altered microtubule organization in small-calibre axons of mice lacking tau protein. Nature. 1994;369:488–491. - PubMed
    1. Esmaeli-Azad B, McCarty JH, Feinstein SC. Sense and antisense transfection analysis of tau function: tau influences net microtubule assembly, neurite outgrowth and neuritic stability. J Cell Sci. 1994;107 (Pt 4):869–879. - PubMed
    1. Caceres A, Kosik KS. Inhibition of neurite polarity by tau antisense oligonucleotides in primary cerebellar neurons. Nature. 1990;343:461–463. - PubMed

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