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Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson's disease

Abstract

Aggregated forms of α-synuclein play a crucial role in the pathogenesis of synucleinopathies such as Parkinson's disease (PD). However, the molecular mechanisms underlying the pathogenic effects of α-synuclein are not completely understood. Here we show that asparagine endopeptidase (AEP) cleaves human α-synuclein, triggers its aggregation and escalates its neurotoxicity, thus leading to dopaminergic neuronal loss and motor impairments in a mouse model. AEP is activated and cleaves human α-synuclein at N103 in an age-dependent manner. AEP is highly activated in human brains with PD, and it fragments α-synuclein, which is found aggregated in Lewy bodies. Overexpression of the AEP-cleaved α-synuclein1–103 fragment in the substantia nigra induces both dopaminergic neuronal loss and movement defects in mice. In contrast, inhibition of AEP-mediated cleavage of α-synuclein (wild type and A53T mutant) diminishes α-synuclein's pathologic effects. Together, these findings support AEP's role as a key mediator of α-synuclein-related etiopathological effects in PD.

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Figure 1: α-synuclein is truncated after N103 in PD and LBD brains.
Figure 2: α-synuclein is a substrate of AEP.
Figure 3: AEP is activated and cleaves α-synuclein in an age-dependent manner.
Figure 4: AEP cleavage of α-synuclein promotes α-synuclein aggregation and toxic effects.
Figure 5: The α-synuclein1–103 fragment induces dopaminergic cell death.
Figure 6: Deletion of AEP attenuates α-synuclein aggregation and dopaminergic cell death in mice overexpressing human α-synuclein.
Figure 7: Preservation of dopaminergic neurons in mice expressing uncleavable α-synuclein A53TN103A.

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Acknowledgements

This work was supported by grants from the Michael J. Fox Foundation (grant ID 11137) to K.Y.; a grant from the National Natural Science Foundation (NSFC) of China (no. 81571249) to Zhentao Zhang; NSFC grant (no. 81528007) to K.Y. and J.-Z.W.; a National Key Basic Research Program of China grant (2010CB945202) to Y.E.S.; an NSFC grant (81330030) to Y.E.S.; and grants from the US Public Health Service (P30EY006360 and R01EY004864) to P.M.I. We thank the ADRC at Emory University for providing human PD, LBD and healthy-control samples, and C. Watts (University of Cambridge) for providing anti-AEP.

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K.Y. conceived the project, designed the experiments and wrote the manuscript. Zhentao Zhang designed and performed most of the experiments. S.S.K. and X.L. prepared primary neurons and assisted with animal experiments. M.J.B. and F.P.M. provided clones and packaged viral vectors. D.M.D. and N.T.S. performed the mass spectrometry analysis. L.H. and P.M.I. performed the HPLC experiments and critically read and edited the manuscript. Zhaohui Zhang, E.H.A., L.J., Y.E.S., F.P.M. and J.-Z.W. designed the experiments, assisted with data analysis and interpretation and critically read the manuscript.

Corresponding authors

Correspondence to Lingjing Jin, Jian-Zhi Wang or Keqiang Ye.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 α-synuclein is truncated in PD and LBD brains.

(a) Specificity of anti-N103 antibody. Anti-N103 antibody was coated on an ELISA plate and different concentrations of full-length α-synuclein or 1-103 fragment (0-16,000 ng/ml) was added to the the system. The signal was detected using the anti-α-synuclein N-terminal antibody. (b) Localization of α-synuclein N103 fragments in synaptic structures. LBD brain sections were immunostained with anti-α-synuclein N103 (red) and presynaptic marker synapsin I (green). Scale bar, 20 μm. Images are representative of 9 sections from three subjects. SNCA knockout mice brain was used as negative control. (c-d) Western blot showing the presence of α-synuclein N103 fragment in brain lysates from human LBD cortex and PD SN tissues (mean ± SEM; n = 6, *P < 0.05 compared with control, student’s t-test). The shown blots are the representative figures of three independent experiments. (e) The correlation between N103 fragment concentration and AEP activity.

Supplementary Figure 2 α-synuclein is cleaved at N103 by AEP.

(a) Recombinant mammalian glutathione transferase (mGST)-tagged α-synuclein was incubated with purified active AEP, and analyzed by immunoblotting. The AEP-derived α-synuclein fragment shows the same molecular weight as a.a. 1-103 fragment. The AEP-derived α-synuclein fragment was recognized by the anti-α-synuclein N103 antibody. (b) MS/MS spectrum showing that AEP cleaves α-synuclein after N103 in vitro. The purified mGST-α-synuclein was incubated with active AEP for 30 min. The fragment was subject to MS/MS spectrum assay. (c-d) Cleavage of mutant N-terminal GFP-tagged α-synuclein or C-terminal GFP-tagged α-synuclein by AEP. α-synuclein cleavage was analyzed by Western blot after recombinant α-synuclein wide-type, N65A, N103A, or N122A mutants were incubated with active mouse kidney lysates for 15 min. (e) Comparison of protein sequences of human and mouse α-synuclein. (f) Cleavage of human and mouse α-synuclein by recombinant AEP. The shown blots are the representative figures of three independent experiments.

Supplementary Figure 3 AEP interacts with α-synuclein.

(a) Co-immunoprecipitation of α-synuclein and AEP in PD brain samples. α-synuclein was immunoprecipitated with anti-α-synuclein N-terminal antibody, and analyzed by immunoblotting with anti-AEP antibody. (b) Anti-AEP antibody abolishes the cleavage of α-synuclein by AEP. Anti-AEP antibody or control IgG was added into human brain lysates and incubated at pH 6.0 for 15 min. The proteolytic processing of α-synuclein was analyzed using Western blot. The shown blots are the representative figures of three independent experiments. Data represent mean ± SEM of three experiments. n = 3, *P < 0.05, one-way ANOVA. (c) Immunostaining with lysosomal marker LAMP1 and AEP showing strict lysosomal localization of AEP in the control brain slides and diffuse staining of AEP in PD brain slides. Scale bar, 20 μm. (d) AEP activity in lysosomal and cytoplasmic fractions of control and PD brain samples (mean ± SEM; n = 3, *P < 0.05, one-way ANOVA). (e) Cleavage of recombinant α-synuclein by lysosomal and cytoplasmic fraction. Recombinant α-synuclein was incubated in lysosomal and cytoplasmic fraction of human brain tissue at pH 6.0 for 0, 5 and 10 min. The production of α-synuclein N103 fragment was analyzed using Western blot.

Supplementary Figure 4 The cleavage of α-synuclein by AEP is independent of its phosphorylation and mutations.

(a) Cleavage rate of α-synuclein S129A and S129D mutations by AEP. HEK293 cells were transfected with GFP-α-synuclein wild-type, S129A, and S129D mutations of α-synuclein, and incubated with active kidney lysates for 0, 5, 10, or 20 min at pH 6.0, and analyzed by immunoblotting. (b) Cleavage rate of α-synuclein Y125F mutation by AEP. (c) Overexpression of constitutively active Fyn induced the phosphorylation of α-synuclein at Y125. (d) Cleavage assay indicates that constitutively active (CA) Fyn or kinase dead (KD) Fyn does not affect the cleavage rate of α-synuclein. (e) A30P and A53T mutant α-synuclein were cleaved by AEP at a similar rate as wild-type (WT) α-synuclein. (f) Cleavage of α-synuclein by AEP was not blocked by calpain, cathepsin or protease inhibitor cocktail. HEK293 cells were transfected with GFP-α-synuclein. The cell lysates were incubated with active kidney lysates at 37°C for 10 min in the presence of calpain inhibitor ALLN, cathepsin inhibitor E64, protease inhibitor cocktail, or AEP inhibitor AENK. α-synuclein cleavage was analyzed by Western blot. Only AEP inhibitory peptide AENK but not any other small molecular inhibitors antagonized α-synuclein cleavage by AEP. The shown blots are the representative figures of three independent experiments. The shown blots are the representative figures of three independent experiments. Data represent mean ± SEM of three experiments. *P < 0.05, one-way ANOVA.

Supplementary Figure 5 AEP activity assay in wild-type mice.

AEP activity is escalated in the cortex and SN tissues of wild-type mice in an age-dependent style (mean ± SEM; n = 6, *P < 0.05, one-way ANOVA). AFU, arbitrary fluorescence units.

Supplementary Figure 6 Aggregation of full-length α-synuclein and 1–103 fragments in vitro.

(a) Electron microscopy visualizing the aggregated full-length α-synuclein and 1-103 fragment. Scale bar, 100 nm. (b) Immunostaining showing the presence of 1-103 fragment in neurons. Scale bar, 20 μm. (c) Western blots showing the localization of α-synuclein in the cytosol and nuclear fraction. Most of full-length α-synuclein and its 1-103 fragment localizes in the cytosol fraction.

Supplementary Figure 7 Expression of α-synuclein full-length or 1–103 fragments induces intraneuronal inclusions containing ubiquitin and 14-3-3.

(a) Immunohistochemistry using anti-human α-synuclein antibody (LB509) showing the expressing of FL α-synuclein. Scale bar, 200 μm. (b) Immunostaining using anti-α-synuclein N103 antibody showing the expression of α-synuclein 1-103 fragment. Scale bar, 20 μm. (c-f) Brain slides injected with AAVs encoding α-synuclein full-length (c, d) or 1-103 (e, f) were immunostained with α-synuclein antibody (c, d), anti-α-synuclein N103 antibody (e, f), 14-3-3 antibody (c, e) and ubiquitin antibody (d, f). (g) Relative AEP activity in lysosomal and cytosolic fractions of SN tissue from control mice and mice injected with AAV-α-synuclein FL and AAV-α-synuclein A53T (mean ± SEM; n = 3, *P < 0.05, one-way ANOVA).

Supplementary Figure 8 Expression level of N103 fragments.

Western blot shows the expression level of α-synuclein 1-103 fragments in SN tissues from PD patients, mice SN tissue injected with human α-synuclein virus, and mice SN tissue injected with α-synuclein 1-103 fragment virus (mean ± SEM; n = 3, *P < 0.05, student’s t-test).

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Zhang, Z., Kang, S., Liu, X. et al. Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson's disease. Nat Struct Mol Biol 24, 632–642 (2017). https://doi.org/10.1038/nsmb.3433

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