Fig 2: Expression analyses of KLK7 mRNA in the brains of AD patientsRelative mRNA expression levels of KLK7 in human autopsied brain samples from control (Ctrl) subjects (n = 24) and AD patients (n = 29) are shown by box-and-whisker plots. The box plots indicate median (solid line in the middle) ± 25th percentile. The whiskers indicate the smallest or highest values that are within 1.5 times the interquartile range below the 25th or above the 75th percentile, respectively. Three different TaqMan probes for KLK7 mRNA (Hs01012730_g1, Hs00192503_m1, Hs01012731_m1) were used for the qRT–PCR analysis. Relative expression of KLK7 mRNA was standardized by GUSB mRNA levels (TaqMan probe Hs99999908_m1), which exhibited comparable expression between AD patients and control subjects. Statistical analysis was performed by Mann–Whitney U-test between control and AD. ***P < 0.001.

Fig 3: Klk7 gene deficiency increases the brain Aß levels and amyloid pathology in AppNL -G-F/ NL -G-F mice A–CBiochemical analyses of human Aß in Tris buffer-soluble (A), SDS-soluble (B), and formic acid-soluble (C) fractions of the brains of 3-month-old male App NL-G-F/NL-G-F ; Klk7 -/- mice. Arrowheads indicate total Aß. Quantification of relative levels of Aß is shown in Appendix Fig S6.DImmunohistochemical analysis of the brains of 3-month-old male App NL-G-F/NL-G-F ; Klk7 -/- mice using the 82E1 antibody (red). Magnified images were shown below. Scale bar, 100 µm.EQuantification results of cortical 82E1-positive total Aß plaques (left) and the cortical thioflavin S-positive Aß plaques (right) are shown (n = 3 or 4, mean ± s.e.m., ***P < 0.001 by Student's t-test). Source data are available online for this figure.

Fig 4: Effect of memantine on Klk7-dependent Aß degradation activity A, BEffect of memantine in human Aß spike experiment. Primary neuron culture or neuron and glia coculture were treated with or without 30 µM of memantine (Mem) at DIV8. 30 nM of synthetic human Aß42 peptide was spiked to these cultures. Levels of remaining human Aß42 at days in vitro measured by human Aß-specific sandwich enzyme immunosorbent assay are shown in (B) (n = 6, mean ± s.e.m., ***P < 0.001 by Student's t-test).CRelative levels of Klk5, Klk6, Klk7, and Spink5 mRNA in the brains of memantine (Mem)-treated female Tg2576 mice (n = 5 or 4, mean ± s.e.m., *P < 0.05 by Student's t-test). Veh, vehicle.DEffect of 30 µM memantine or 10 µM MK-801 on Klk7 mRNA expression in primary astrocytes obtained from wild-type mice (n = 5 or 6, mean ± s.e.m., **P < 0.01 by Tukey's test).EEffect of 20 µM l-glutamate or 50 µM N-methyl-d-aspartate on Klk7 mRNA expression in primary astrocytes obtained from wild-type mice (n = 5 or 6, mean ± s.e.m., **P < 0.01 and ***P < 0.001 by Tukey's test).FEffect of 30 µM memantine on Aß degradation activity in the conditioned medium of primary astrocytes obtained from wild-type or Klk7 -/- mice. Remaining Aß40 and Aß42 are quantified (n = 4, mean ± s.e.m., *P < 0.05, **P < 0.01 by Student's t-test).

Fig 5: Klk7 is involved in the Aß degradation activity detected in the conditioned medium of primary astrocytes Aß degradation activity in the conditioned medium of primary astrocytes. Note that diisopropyl fluorophosphates and tosyl phenylalanyl chloromethyl ketone inhibited this activity in a similar manner to those observed in the conditioned medium of CCF-STTG1 cells. DIFP, diisopropyl fluorophosphates; TLCK, tosyl-L-lysyl-chloromethane hydrochloride; TPCK, tosyl phenylalanyl chloromethyl ketone.Effect of the KLK7-neutralizing antibody MAB2624 on the Aß degradation activity of primary astrocytes. Quantification of relative remaining Aß40 and Aß42 in the mixture of normal culture medium and conditioned medium is shown below the blot (n = 3, mean ± s.e.m., **P < 0.01 by Student's t-test). Source data are available online for this figure.