Title: Loss of kallikrein‐related peptidase 7 exacerbates amyloid pathology in Alzheimer's disease model mice
Abstract: Research Article8 January 2018Open Access Source DataTransparent process Loss of kallikrein-related peptidase 7 exacerbates amyloid pathology in Alzheimer's disease model mice Kiwami Kidana Kiwami Kidana Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Department of Internal Medicine, Komeikai Hospital, Tokyo, Japan Search for more papers by this author Takuya Tatebe Takuya Tatebe Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kaori Ito Kaori Ito Venture Science Laboratories, R&D Division, Daiichi-Sankyo Co. Ltd., Tokyo, Japan Search for more papers by this author Norikazu Hara Norikazu Hara Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Akiyoshi Kakita Akiyoshi Kakita Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Takashi Saito Takashi Saito Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Sho Takatori Sho Takatori Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yasuyoshi Ouchi Yasuyoshi Ouchi Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Federation of National Public Service Personnel Mutual Aid Associations, Toranomon Hospital, Tokyo, Japan Search for more papers by this author Takeshi Ikeuchi Takeshi Ikeuchi Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Mitsuhiro Makino Mitsuhiro Makino Venture Science Laboratories, R&D Division, Daiichi-Sankyo Co. Ltd., Tokyo, Japan Search for more papers by this author Takaomi C Saido Takaomi C Saido orcid.org/0000-0003-1970-6903 Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Masahiro Akishita Masahiro Akishita Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Takeshi Iwatsubo Takeshi Iwatsubo Department of Neuropathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yukiko Hori Yukiko Hori Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Taisuke Tomita Corresponding Author Taisuke Tomita [email protected] orcid.org/0000-0002-0075-5943 Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kiwami Kidana Kiwami Kidana Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Department of Internal Medicine, Komeikai Hospital, Tokyo, Japan Search for more papers by this author Takuya Tatebe Takuya Tatebe Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kaori Ito Kaori Ito Venture Science Laboratories, R&D Division, Daiichi-Sankyo Co. Ltd., Tokyo, Japan Search for more papers by this author Norikazu Hara Norikazu Hara Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Akiyoshi Kakita Akiyoshi Kakita Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Takashi Saito Takashi Saito Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Sho Takatori Sho Takatori Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yasuyoshi Ouchi Yasuyoshi Ouchi Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Federation of National Public Service Personnel Mutual Aid Associations, Toranomon Hospital, Tokyo, Japan Search for more papers by this author Takeshi Ikeuchi Takeshi Ikeuchi Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japan Search for more papers by this author Mitsuhiro Makino Mitsuhiro Makino Venture Science Laboratories, R&D Division, Daiichi-Sankyo Co. Ltd., Tokyo, Japan Search for more papers by this author Takaomi C Saido Takaomi C Saido orcid.org/0000-0003-1970-6903 Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan Search for more papers by this author Masahiro Akishita Masahiro Akishita Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Takeshi Iwatsubo Takeshi Iwatsubo Department of Neuropathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yukiko Hori Yukiko Hori Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Taisuke Tomita Corresponding Author Taisuke Tomita [email protected] orcid.org/0000-0002-0075-5943 Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Author Information Kiwami Kidana1,2,3,‡, Takuya Tatebe1,‡, Kaori Ito4, Norikazu Hara5, Akiyoshi Kakita6, Takashi Saito7, Sho Takatori1, Yasuyoshi Ouchi2,8, Takeshi Ikeuchi5, Mitsuhiro Makino4, Takaomi C Saido7, Masahiro Akishita2, Takeshi Iwatsubo9, Yukiko Hori1 and Taisuke Tomita *,1 1Laboratory of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan 2Department of Geriatric Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 3Department of Internal Medicine, Komeikai Hospital, Tokyo, Japan 4Venture Science Laboratories, R&D Division, Daiichi-Sankyo Co. Ltd., Tokyo, Japan 5Department of Molecular Genetics, Brain Research Institute, Niigata University, Niigata, Japan 6Department of Pathology, Brain Research Institute, Niigata University, Niigata, Japan 7Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama, Japan 8Federation of National Public Service Personnel Mutual Aid Associations, Toranomon Hospital, Tokyo, Japan 9Department of Neuropathology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 3 5841 4868; E-mail: [email protected] EMBO Mol Med (2018)10:e8184https://doi.org/10.15252/emmm.201708184 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Deposition of amyloid-β (Aβ) as senile plaques is one of the pathological hallmarks in the brains of Alzheimer's disease (AD) patients. In addition, glial activation has been found in AD brains, although the precise pathological role of astrocytes remains unclear. Here, we identified kallikrein-related peptidase 7 (KLK7) as an astrocyte-derived Aβ degrading enzyme. Expression of KLK7 mRNA was significantly decreased in the brains of AD patients. Ablation of Klk7 exacerbated the thioflavin S-positive Aβ pathology in AD model mice. The expression of Klk7 was upregulated by Aβ treatment in the primary astrocyte, suggesting that Klk7 is homeostatically modulated by Aβ-induced responses. Finally, we found that the Food and Drug Administration-approved anti-dementia drug memantine can increase the expression of Klk7 and Aβ degradation activity specifically in the astrocytes. These data suggest that KLK7 is an important enzyme in the degradation and clearance of deposited Aβ species by astrocytes involved in the pathogenesis of AD. Synopsis Decreased clearance of Aβ from the brain is related to the pathogenesis of Alzheimer's disease (AD). Kallikrein-related peptidase 7 (KLK7) is an astrocyte-derived Aβ degrading enzyme that affects the amyloid pathology. Thus, astrocytes could be an effective cellular target for AD. KLK7-dependent Aβ degradation activity is associated with astrocytes. Expression of KLK7 mRNA is significantly reduced in AD brains. Genetic ablation of Klk7 accelerated the amyloid deposition and amyloid-related pathologies. Astrocytic Klk7 expression is selectively upregulated by the FDA-approved drug memantine. Introduction Alzheimer's disease (AD) is the most common type of dementia. Genetic and biochemical evidence suggests that the aggregation and deposition of amyloid-β (Aβ) are critical processes in the pathogenesis of AD (Holtzman et al, 2011). Aβ is produced upon proteolysis of amyloid precursor protein (APP). Several genetic mutations linked to familial AD increase the production or aggregation of Aβ. In contrast, sporadic AD patients have been reported to have a decreased clearance rate, rather than an increased production rate of brain Aβ (Mawuenyega et al, 2010). Thus, understanding the molecular mechanism of brain “Aβ economy” (Karran et al, 2011), which reflects the balance of the rates of production, clearance, and aggregation of Aβ, is crucial for the development of effective therapeutics for AD. However, the whole picture of the pathophysiological Aβ clearance/degradation pathway in the brain is still unclear (Saido & Leissring, 2012). To date, several enzymes, including neprilysin (membrane metallo-endopeptidase) and insulin-degrading enzyme, have been identified as Aβ-degrading enzymes (Nalivaeva et al, 2014). Among them, the knockout of neprilysin or insulin-degrading enzyme in mice showed an approximately twofold increase in the level of soluble brain Aβ, although the effect on Aβ deposition in vivo remains controversial (Iwata et al, 2000, 2001; Farris et al, 2003; Leissring et al, 2003). Importantly, in a therapeutic context, remaining neurons might not be a suitable target to remove Aβ deposits in the treatment for AD. Thus, we focused on astrocytes, which play important roles in physiological and pathological functions in the brain (De Strooper & Karran, 2016; Pekny et al, 2016). Of note, changes in the phenotypes of astrocytes, but in not their number, were associated with the clinical pathology of AD (Serrano-Pozo et al, 2013). However, the precise mechanism of astrocyte-mediated Aβ clearance remains unclear. Fifteen kallikrein-related peptidase (KLK) family proteins have been identified and act in a complex network as a cascade reaction. Among them, KLK6 (neurosin) and KLK8 (neuropsin) are major kallikrein-related peptidase proteins in the central nervous system (Sotiropoulou et al, 2009; Prassas et al, 2015). KLK6 is widely expressed in several cells, and KLK8 is expressed in neurons. Also, KLK8 has been implicated in the pathogenesis of AD (Herring et al, 2016). KLK7 was originally identified as an inflammation-induced proteolytic enzyme in the skin. However, the expression level of KLK7 was decreased in the cerebrospinal fluid and brain of AD patients (Diamandis et al, 2004; Bossers et al, 2010). Moreover, it was reported that KLK7 is capable to cleave the hydrophobic core motif of Aβ fibrils, thereby attenuating neurotoxicity in vitro (Shropshire et al, 2014). In this manuscript, we have investigated the pathophysiological impact of KLK7 in brain Aβ economy and identified KLK7 as the astrocyte-derived Aβ-degrading enzyme that regulates amyloid pathology in vivo. Results Identification of KLK7 as the Aβ-degrading enzyme To identify the proteolytic enzyme that is capable to degrade secreted Aβ species, we utilized the conditioned medium from 7PA2 cells as a substrate source, as 7PA2 cells secretes toxic human Aβ oligomer species (Podlisny et al, 1995). We took the conditioned medium from several cell lines and mixed with that from 7PA2 cells. The mixture was separated by Urea-containing SDS–PAGE gel, which enables us to discriminate different C-terminal length of Aβ immunoblot (Klafki et al, 1996; Qi-Takahara et al, 2005). We found that the media from astrocytoma CCF-STTG1, neuroglioma H4, neuroblastoma SH-SY5Y, and glioblastoma U87 cells showed robust Aβ-degrading activity in this condition (Fig 1A, and Appendix Fig S1A and B). In addition, the conditioned medium of CCF-STTG1 cells also showed the degrading activity on Aβ derived from human neuroblastoma BE(2)-C cells (Appendix Fig S1C). We then further characterized the degradation activity using CCF-STTG1 cells that showed strongest activity. This activity was specifically inhibited by diisopropyl fluorophosphates as well as tosyl phenylalanyl chloromethyl ketone (Fig 1B and C), indicating that a secreted chymotrypsin-type serine protease is required for Aβ degradation. Neither phosphoramidon, ethylenediaminetetraacetic acid nor GM6001 inhibited this activity, indicating that metalloprotease was not involved in this catabolic pathway (Fig 1B and D) (Iwata et al, 2000; Yin et al, 2006). However, inhibitors against known Aβ-degrading serine proteases [i.e., anti-plasmin for plasmin (Van Nostrand & Porter, 1999) and acetylmethionine for acyl-peptide hydrolase (Yamin et al, 2007)] failed to inhibit this activity (Fig 1E). In contrast, the activity was reduced by zinc ions (Fig 1F). This unusual character led us to investigate the possible function of KLK7, one of the secreted chymotrypsin-type serine proteases with zinc sensitivity (Debela et al, 2007). Figure 1. Pharmacological analysis of Aβ degradation activity in the conditioned medium of astrocytoma cell lines Aβ degradation activity in the conditioned media from CCF-STTG1 and U87 cells. The media were incubated for 24 h with the conditioned medium of 7PA2 cells. Remaining Aβ in the mixture was visualized by immunoblotting. Aβ secreted from 7PA2 cells completely disappeared after 24 h, and this was inhibited by the addition of a complete protease inhibitor cocktail. Aβ remained in the mixed medium by the addition of diisopropyl fluorophosphates or complete protease inhibitor cocktail. DIFP, diisopropyl fluorophosphates; PR, phosphoramidon; PepA, pepstatin A. Effect of tosyl-L-lysyl-chloromethane hydrochloride or tosyl phenylalanyl chloromethyl ketone on Aβ degradation activity in the conditioned medium of CCF-STTG1 cells. TLCK, tosyl-L-lysyl-chloromethane hydrochloride; TPCK, tosyl phenylalanyl chloromethyl ketone. Effect of matrix metalloprotease inhibitor, GM6001, on Aβ degradation activity in the conditioned medium of CCF-STTG1 cells. Effect of known Aβ degrading serine protease inhibitors on Aβ degradation activity in the conditioned medium from CCF-STTG1 cells. AcMet, acetylmethionine; αplasmin, α2-anti-plasmin. Effect of zinc ions on Aβ degradation activity in the conditioned medium of CCF-STTG1 cells. Source data are available online for this figure. Source Data for Figure 1 [emmm201708184-sup-0002-SDataFig1.zip] Download figure Download PowerPoint It was reported that KLK7 is capable to cleave the hydrophobic core motif of Aβ fibrils in vitro (Shropshire et al, 2014). Moreover, the expression level of KLK7 was decreased in the cerebrospinal fluid and brain of AD patients (Diamandis et al, 2004; Bossers et al, 2010). However, it remains unclear whether KLK7 is involved in the brain amyloid pathology in vivo. We confirmed the ~50% reduction of KLK7 mRNA expression in the brains of Japanese AD patients (Fig 2) (Miyashita et al, 2014) in good correlation with Braak NFT stage (Appendix Fig S2). In addition, we analyzed the levels of human KLK7 mRNA expression in two public RNAseq datasets deposited at the AMP-AD knowledge portal: the Mayo RNAseq (MayoRNAseq) (Allen et al, 2016) and Mount Sinai Brain Bank (MSBB) AD cohorts. In the Mayo sample set, KLK7 repression was significantly decreased in the temporal cortex of AD patients (false discovery rate (FDR) < 0.05, β = −0.623). Similarly, KLK7 expression was significantly reduced in relation to increased amyloid plaque burden in the MSBB sample set (FDR < 0.01 for Brodmann area (BM) 22, FDR < 0.05 for BM36). Figure 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. Download figure Download PowerPoint To test whether KLK7 is involved in Aβ degradation, we examined a specific neutralizing antibody against KLK7 (MAB2624) (Bin et al, 2011). Addition of MAB2624 significantly reduced Aβ degradation activity in CCF-STTG1 cells (Fig 3A and B, and Appendix Fig S3A). Moreover, the conditioned medium from COS-1 cells overexpressing KLK7 showed the degradation activity against naturally secreted Aβ (Fig 3C and Appendix Fig S3B), which was abolished by the addition of MAB2624. Finally, coincubation of recombinant purified human KLK7, but not KLK6, derived from either mammalian cells or bacteria with 7PA2-derived Aβ or the synthetic Aβ resulted in a significant Aβ degradation in vitro (Fig 3D and Appendix Fig S3C–E), indicating that KLK7 is directly involved in the Aβ degradation. Consistent with previous result (Shropshire et al, 2014), KLK7 degraded synthetic Aβ fibrils (Fig 3E). To further elucidate the role of KLK7 in murine astrocyte, we analyzed the primary glial cells obtained from rat pups. This culture is mainly comprised of primary astrocytes, but still contained the primary microglia. Notably, Klk7-positive puncta were detected only in Aldh1L1-positive primary astrocytes, but not in Iba-1-positive microglia (Appendix Fig S4A). Conditioned medium from the primary glial cell culture also showed chymotrypsin-type serine protease-dependent Aβ degradation activity inhibited by the neutralizing antibody against KLK7 (Fig 4A and B, and Appendix Fig S4B). Other proteases might be also involved in the Aβ degradation because the MAB2624 showed partial inhibition in this assay. However, these results implicated that Klk7-dependent Aβ degradation activity was associated with the astrocytes. Figure 3. KLK7 is involved in the Aβ degradation activity Inhibition of Aβ degradation activity of CCF-STTG1 cells with MAB2624, a KLK7-neutralizing antibody. Quantification of the relative remaining Aβ40 and Aβ42 in a mixture of normal culture medium and conditioned medium is shown below the blot (n = 3, mean ± s.e.m., *P < 0.05 by Student's t-test). Dose-dependent inhibition of Aβ degradation activity of CCF-STTG1 cells with MAB2624. Quantification of relative remaining Aβ40 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 Tukey's test). Aβ degradation activity in the conditioned medium of COS-1 cells expressing human KLK7. Quantification of relative remaining Aβ40 in the mixture of normal culture medium and conditioned medium is shown below the blot (n = 3, mean ± s.e.m., ***P < 0.001 by Tukey's test). Aβ degradation activity of the recombinant KLK7 protein. Immunoblot analysis of the remaining Aβ in the mixture of normal cultured medium and conditioned medium of 7PA2 cells, and recombinant human KLK7 and KLK6 protein is shown. In vitro degradation of preformed Aβ fibril by purified MBP-tagged hKLK7 protein. Amounts of Aβ fibrils were measured by thioflavin T fluorescence, and relative fluorescence levels at each time point were shown (n = 3, mean ± s.e.m., *P < 0.05, **P < 0.01 by Tukey's test). Source data are available online for this figure. Source Data for Figure 3 [emmm201708184-sup-0003-SDataFig3.zip] Download figure Download PowerPoint Figure 4. Klk7 is involved in the Aβ degradation activity detected in the conditioned medium of primary astrocytes Aβ 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 Aβ degradation activity of primary astrocytes. Quantification of relative remaining Aβ40 and Aβ42 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. Source Data for Figure 4 [emmm201708184-sup-0004-SDataFig4.zip] Download figure Download PowerPoint Importance of KLK7 in the brain Aβ pathology We then generated homozygous Klk7 knockout mice (Klk7−/−) using ES cells carrying Klk7tm1(KOMP)Vlcg VelociGene deletion allele. Klk7−/− mice appeared normal and were fertile, and their expression of Klk7 mRNA was completely abolished (Appendix Fig S5A–C). Moreover, MAB2624 failed to inhibit the Aβ degradation in the conditioned medium from the primary glial culture obtained from Klk7−/− mice, indicating that MAB2624 specifically inhibited the proteolytic activity of KLK7 protein in the degradation assay (Appendix Fig S5D). However, no commercial antibody detected the endogenous brain KLK7 protein on the immunoblot. Then, we analyzed the levels of endogenous murine brain Aβ and found 1.4-fold to twofold increase in both male and female Klk7−/− mouse brains (Appendix Fig S5E). No change was observed in the expression levels of APP, proteolytic fragments of APP, ADAM10, BACE1, γ-secretase, ApoE, neprilysin, insulin-degrading enzyme or matrix metalloprotease-9 (Appendix Fig S5F). Injection of recombinant KLK7 protein into hippocampi of wild-type mouse significantly reduced the murine brain Aβ levels, supporting the notion that Klk7 is physiologically involved in the catabolism of brain Aβ (Appendix Fig S5G). Then, we examined the amyloid pathology of App knockin mice (Saito et al, 2014, 2016) under a Klk7-null background. Homozygous AppNL-G-F/NL-G-F mice showed cortical amyloid deposition at 1–2 months of age, and thioflavin S-positive plaques that were surrounded by glial fibrillary acidic protein (GFAP)-positive astrocytes appeared at 6–7 months of age. As the Arctic mutation in the knockin allele promotes aggregation (Nilsberth et al, 2001) and decreases the immunoreactivity in the sandwich enzyme-linked immunosorbent assay (Saito et al, 2014), we compared the brain Aβ levels by immunoblot (Fig 5A–C and Appendix Fig S6A–C). We observed a significant increase in the levels of Tris buffer-soluble human Aβ in the brains of AppNL-G-F/NL-G-F mice at 3 months of age by genetic Klk7 ablation without affecting APP and related proteins (Appendix Fig S7A). Moreover, amounts of insoluble Aβ (i.e., SDS-soluble and formic acid-soluble) were also increased. Consistent with these biochemical analyses, brain amyloid deposition was drastically increased (5.6-fold) in the brains of AppNL-G-F/NL-G-F; Klk7−/− mice (Fig 5D and E). These data implicated a significant impact of loss of Klk7 not only on the biochemical Aβ economy, but on the deposition pattern of the Aβ plaques. Figure 5. Klk7 gene deficiency increases the brain Aβ levels and amyloid pathology in AppNL-G-F/NL-G-F mice A–C. Biochemical analyses of human Aβ in Tris buffer-soluble (A), SDS-soluble (B), and formic acid-soluble (C) fractions of the brains of 3-month-old male AppNL-G-F/NL-G-F; Klk7−/− mice. Arrowheads indicate total Aβ. Quantification of relative levels of Aβ is shown in Appendix Fig S6. D. Immunohistochemical analysis of the brains of 3-month-old male AppNL-G-F/NL-G-F; Klk7−/− mice using the 82E1 antibody (red). Magnified images were shown below. Scale bar, 100 μm. E. Quantification results of cortical 82E1-positive total Aβ plaques (left) and the cortical thioflavin S-positive Aβ 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. Source Data for Figure 5 [emmm201708184-sup-0005-SDataFig5.zip] Download figure Download PowerPoint We further analyzed the pathological changes related to the Aβ deposition. We observed the accelerated phosphorylation of murine endogenous tau (Appendix Fig S8A and B) as well as formation of BACE1-accumulated dystrophic neurites (Appendix Fig S8C) (Kandalepas et al, 2013) in the brains of AppNL-G-F/NL-G-F; Klk7−/− mice. In addition, increased thioflavin S-positive amyloid plaques (Fig 5E) as well as GFAP-positive gliosis around the plaques were detected in the congenic mice (Appendix Fig S8D), supporting our notion that amyloid-induced neuritic/astrocytic changes were augmented in the congenic mice. Notably, although the expression of GFAP was significantly increased, Aldh1L1 levels were not affected by Klk7 gene ablation, suggesting that there was an activation without a change in astrocyte number (Appendix Fig S7B). Collectively, these results indicate that Klk7 is a crucial component of brain Aβ economy and attenuates brain amyloid pathology in AD model mice. Activation of Klk7 expression by Aβ treatment and memantine KLK7 is a terminal protease in the kallikrein-related peptidase cascade and is directly activated by KLK5-mediated prodomain removal (Sotiropoulou et al, 2009). Importantly, the upregulation of KLK5 and KLK7 activity by loss of the endogenous KLK inhibitor serine peptidase inhibitor Kazal type 5 (SPINK5) causes atopic dermatitis-associated diseases, including Netherton syndrome (Furio & Hovnanian, 2014). However, Klk7 mRNA expression is selectively regulated by cytokines in the skin (Morizane et al, 2012), suggesting that transcription of Klk7 mRNA is controlled by a specific mechanism. In fact, Klk7 mRNA expression was significantly and selectively increased in AppNL-G-F/NL-G-F mice (Fig 6A). Moreover, mRNA expression of Klk7, but not other Aβ-degrading enzymes (i.e., matrix metalloproteases, neprilysin, and insulin-degrading enzyme), was further specifically augmented in an age-dependent manner (Fig 6B and Appendix Fig S9). Intriguingly, treatment of primary astrocytes with Aβ42, but not with lipopolysaccharide, significantly increased the expression of Klk7 (Fig 6C and D). Although we are unable to exclude the possibility that lipopolysaccharide has some regulatory role in the Klk7, these results suggest that Klk7 transcription was selectively modulated by Aβ in the astrocytes and that selective augmentation of Klk7 expression is possible. Figure 6. Upregulation of Klk7 expression by Aβ in the astrocytes Relative levels of Klk5, Klk6, Klk7, and Spink5 mRNA in the brains of 5-month-old male AppNL-G-F/NL-G-F mice (n = 5 or 6, mean ± s.e.m., **P < 0.01 by Student's t-test). Levels of Klk7 mRNA in 5-month-old and 13-month-old male AppNL-G-F/NL-G-F mice (n = 5 or 6, mean ± s.e.m., **P < 0.01 by Student's t-test). Effect of the Aβ peptide on Klk7 mRNA expression in primary astrocytes obtained from wild-type mice (n = 6, mean ± s.e.m., *P < 0.05 by Tukey's t-test). Effect of 1 μg/ml lipopolysaccharide on Klk7 mRNA expression in primary astrocytes obtained from wild-type mice (n = 6, mean ± s.e.m., *P < 0.05 by Student's t-test). Download figure Download PowerPoint We recently investigated the effect of the memantine, which is the Food and Drug Administration-approved anti-dementia drug and an N-methyl-d-aspartate receptor antagonist, on the Aβ metabolism in the primary cultures (Ito et al, 2017). We noticed that the memantine treatment significantly reduced the spiked human Aβ only in the conditioned medium of primary neuron and glia coculture, but not in that of primary neuronal culture (Fig 7A and B). In addition, we as well as others have found that the chronic treatment of Tg2576 APP transgenic mice with memantine reduced Aβ levels (Dong et al, 2008; Ito et al, 2017). Thus, we hypothesized that memantine would affect Klk7 mRNA expression in the astrocytes and Aβ deposition in the brain. Supporting this notion, Klk7 mRNA level was specifically upregulated in the brains of memantine-treated Tg25