Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in

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1 Supplementary information Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer s disease Zhentao Zhang, Mingke Song, Xia Liu, Seong Su Kang, Il-Sun Kwon, Duc M. Duong, Nicholas T. Seyfried, William T. Hu, Zhixue Liu, Jian-zhi Wang, Liming Cheng, Yi E. Sun, Shan Ping Yu, Allan I. Levey and Keqiang Ye 1

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3 Supplementary Figure 1. The cleavage of tau by AEP is independent of caspases, calpain, or other proteinases. (a) Cleavage of tau by AEP was not blocked by caspase, calpain, thrombin, cathepsin or PSA inhibitors. HEK293 cells were transfected with GST-tau. The cell lysates were incubated with kidney lysates at 37 C for 30 min in the presence of pan-caspase inhibitor ZVAD-fmk, calpain inhibitor ALLN, thrombin inhibitor PMSF, cathepsin inhibitor E64, PSA inhibitor puromycin, or AEP inhibitor AENK. Tau cleavage was analyzed by Western blot. Only AEP inhibitory peptide AENK but not any other small molecular inhibitors antagonized tau cleavage by AEP. (b) Caspase, calpain and other proteinases cleavage site mutation dose not interfere with the cleavage of tau by AEP. HEK293 cells were transfected with GST-tau WT, or thrombin cleavage site (R155A and K257A), cathepisn cleavage site (M419A), caspase cleavage site (D421E), calpain cleavage site (L43A/V229A), or AEP cleavage site (N255A/N368A) mutated tau and incubated in mouse kidney lysates at ph 7.4 or ph 6.0. Tau cleavage was analyzed by Western blot. (c) Tau cleavage by AEP does not interfere with its cleavage by calpain. HEK293 cells transfected with GST-tau wild-type, N255A/N368A or GST-tau were incubated with recombinant calpain-1 for 0, 30 or 60 min, the generation of the apparent relative molecular weigh 17 kda tau fragment was monitored by immunoblotting with anti tau-1 antibody. (d) Tau cleavage by AEP does not affect its cleavage by caspase-3. HEK293 cells transfected with GST-tau wild-type or N255A/N368A were incubated with recombinant caspase-3 for 0, 30 or 60 min, the cleavage of tau by caspase-3 was monitored. (e) Protein phosphatase inhibitor Okadaic acid (OA) induces tau hyperphosphorylation. HEK 293 cells transfected with GST-tau was treated with okadaic acid (OA, 0.2 μm) for 1 h before harvest. Cell extracts was analyzed by 3

4 immunoblotting with AT8 and AT100 antibodies to demonstrate the phosphorylation of tau. (f) Tau phosphorylation status does not affect its proteolytic degradation. Cell extracts was incubated with AEP for 0, 5, 10, and 30 min, and analyzed by immunoblotting. The phosphorylated and unphosphorylated tau was cleaved at the similar rate. 4

5 Supplementary Figure 2. Development of anti-tau N368 specific antibody and stain with human AD brain senctions. (a) Specificity of anti-tau N368 antibody. mgst-tau was incubated with active AEP, and the cleavage of tau was detected by immunoblotting using anti-tau-1 and anti-tau N368 antibodies, respectively. Anti-tau N368 antibody recognized the AEP-generated tau fragment at N368, but not the full-length tau or tau (b) The cleavage of tau in mouse brain lysate. Two-month-old mouse brain tissues were incubated at ph 6.0 or ph 7.4, and the cleavage of tau was analyzed by Western blot using anti-tau-5 antibody and anti-tau N368 antibody. (c) Immunoprecipitation of tau fragments with anti-tau N368 antibody from human AD brain lysates. The immunoprecipitated bands were detected 5

6 by the pan tau antibody (tau-5) and non-phosphorylated tau antibody (tau-1). (d) Confirmation of the specificity of anti-tau N368 antibody. The anti-tau N368 antibody was preincubated with a peptide (tau ) or nonspecific peptide before immunohistochemistry. The signal was blocked by tau peptide but not by nonspecific peptide. Scale bar, 20 μm. (e) Immunostaining showing the colocalization of AEP-derived tau fragments and PHFs in human AD brain slides with mouse anti-tau N368 antibody (red) and rabbit anti-phosphorylated tau antibody (green). Scale bar, 20 μm. 6

7 Supplementary Figure 3. AEP cleaves tau in primary neurons. (a c) Aβ oligomers induce the activation of AEP and cleavage of tau in primary neurons. (a) Western blots of oligomeric Aβ separated by SDS-PAGE and probed with anti-aβ antibody. (b) Active AEP fragment was detected after the neurons were exposed to Aβ oligomers for 24 h. The AEP generated tau fragment was detected using anti-tau N368 antibody. (c) AEP enzymatic activities were 7

8 validated by fluorescent substrate cleavage assay (mean ± SEM; n = 4; *P < 0.01, one-way ANOVA). (d, e) The effect of Aβ oligomers on caspase and cathepsin activity. Aβ treatment induced the activation of caspase (d), but not cathepsin (e), in primary neurons (mean ± SEM; n = 4; *P < 0.01, one-way ANOVA). (f) Overexpressed AEP cleaves tau in primary neurons. Primary neurons at DIV 7 was transfected with myc-aep. The cleavage of tau was detected by immunoflurescence staining using anti-tau N368 antibody (red) and anti-myc antibody (green). The proposition of cells positive for tau N368 was much higher in AEP-expressing cells than in control cell (mean ± SEM; n = 3; *P < 0.05, Student's t-test). Neurons transfected with control plasmid were used as control. Scale bar, 20 μm. 8

9 Supplementary Figure 4. Neurotoxic effect of tau Primary neurons (DIV 7) were infected with AAV-tau full-length or AAV-tau (a) Equal levels of tau full-length and tau were detected by Western blot using HT-7, a monoclonal antibody specific for human tau. (b) Cell apoptosis was detected by TUNEL staining. The apoptotic rate is higher in neurons expressing tau than in neurons expressing full-length tau (mean ± SEM; n = 4; *P < 0.05, Student's t-test). Scale bar, 20 μm. 9

10 Supplementary Figure 5. Cleavage of tau by AEP promotes the formation of neurofibrillary tangles. (a) Thioflavin S assay showing the kinetics of aggregation of tau fragments (mean ± SEM; n = 3). (b) Electron micrographs of tau assembly reactions. Filaments were formed by full-length tau, tau 1 368, and , and a mixture of 10

11 tau fragments, but not by tau and Scale bar, 0.2 μm. (c) Ultracentrifugation assay. The soluble and aggregated tau was analyzed with Western blotting (S, supernatant; P, pellet). 11

12 Supplementary Figure 6. Cleavage of tau by AEP promotes tau phosphorylation in primary neurons. Primary neurons were transfected with HA-tagged full-length tau or tau fragments (1 255 and 1 368). The phosphorylation of tau was detected by AT8 antibody. The percentage AT8-positive cells were significantly higher in neurons expressing tau or tau fragments than in neurons expressing full-length tau (mean ± SEM; n = 3; *P < 0.05, one-way ANOVA). Scale bar, 20 μm. 12

13 Supplementary Figure 7. Genotyping of wild-type, Lgmn /, tau P301S and tau P301S/Lgmn / mice. Tau P301S mice was crossed with Lgmn / mice. The genotype of the mice was examined by PCR. 13

14 Supplementary Figure 8. AEP gene deletion attenuates tau phosphorylation in tau P301S mice. The brain sections of WT, Lgmn /, tau P301S, tau P301S/ Lgmn / mice were immunostained with AT100 antibody (mean ± SEM; *P < 0.05, Student's t-test). Less AT100-positive cells were found in tau P301S/ Lgmn / mice than in tau P301S mice. Scale bar, 50 μm. 14

15 Supplementary Figure 9. AEP deletion attenuates synaptic loss in tau P301S mice. (a) Electron microscopy of the synapses. Tau P301S mice show decreased synaptic density, which was attenuated by AEP gene deletion. Arrows indicate the synapses. Scale bar, 1 μm. (b) AEP deletion increases the density of dendritic spines in tau P301S mice. Golgi staining revealed the dendritic spines from apical dendritic layer of the CA1 region. Scale bar, 5 μm. (c) Quantification of spine density (mean ± SEM; n = 6; *P < 0.01, one-way ANOVA). 15

16 Supplementary Figure 10. AEP deletion ameliorates basal synaptic transmission deficits in the tau P301S mice. (a) Construction of input/output (I/O) relation between stimuli intensity ( V) and fepsp slopes in the hippocampus of WT, tau P301S and tau P301S/ Lgmn / mice. (b) Averaged slopes of I/O curves are significantly greater in WT and tau P301S/ Lgmn / mice compared with tau P301S mice (mean ± SD; n = 6; *P < 0.05, one-way ANOVA). 16