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  1. Article ; Online: A Dual-Function "TRE-Lox" System for Genetic Deletion or Reversible, Titratable, and Near-Complete Downregulation of Cathepsin D.

    Terron, Heather M / Maranan, Derek S / Burgard, Luke A / LaFerla, Frank M / Lane, Shelley / Leissring, Malcolm A

    International journal of molecular sciences

    2023  Volume 24, Issue 7

    Abstract: Commonly employed methods for reversibly disrupting gene expression, such as those based on RNAi or CRISPRi, are rarely capable of achieving >80-90% downregulation, making them unsuitable for targeting genes that require more complete disruption to ... ...

    Abstract Commonly employed methods for reversibly disrupting gene expression, such as those based on RNAi or CRISPRi, are rarely capable of achieving >80-90% downregulation, making them unsuitable for targeting genes that require more complete disruption to elicit a phenotype. Genetic deletion, on the other hand, while enabling complete disruption of target genes, often produces undesirable irreversible consequences such as cytotoxicity or cell death. Here we describe the design, development, and detailed characterization of a dual-function "TRE-Lox" system for effecting either (a) doxycycline (Dox)-mediated downregulation or (b) genetic deletion of a target gene-the lysosomal aspartyl protease cathepsin D (CatD)-based on targeted insertion of a tetracycline-response element (TRE) and two LoxP sites into the 5' end of the endogenous CatD gene (
    MeSH term(s) Animals ; Mice ; Cathepsin D/genetics ; Cathepsin D/metabolism ; Down-Regulation/genetics ; Fibroblasts/metabolism ; Tetracycline ; Doxycycline/pharmacology ; Response Elements
    Chemical Substances Cathepsin D (EC 3.4.23.5) ; Tetracycline (F8VB5M810T) ; Doxycycline (N12000U13O)
    Language English
    Publishing date 2023-04-04
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2019364-6
    ISSN 1422-0067 ; 1422-0067 ; 1661-6596
    ISSN (online) 1422-0067
    ISSN 1422-0067 ; 1661-6596
    DOI 10.3390/ijms24076745
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article ; Online: Prominent tauopathy and intracellular β-amyloid accumulation triggered by genetic deletion of cathepsin D: implications for Alzheimer disease pathogenesis.

    Terron, Heather M / Parikh, Sagar J / Abdul-Hay, Samer O / Sahara, Tomoko / Kang, Dongcheul / Dickson, Dennis W / Saftig, Paul / LaFerla, Frank M / Lane, Shelley / Leissring, Malcolm A

    Alzheimer's research & therapy

    2024  Volume 16, Issue 1, Page(s) 70

    Abstract: Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-β protein (Aβ) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of ... ...

    Abstract Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-β protein (Aβ) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aβ pathology and tauopathy in vivo.
    Methods: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model of a disease (Krabbe A) characterized by pronounced lysosomal dysfunction. Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau.
    Results: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aβ, manifesting as intense, exclusively intracellular aggregates; extracellular Aβ deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatD-KO mice were found to develop prominent tauopathy by just ∼ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology present in aged JNPL3 mice. CatD-KO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (∼ 1250%) are present in CatD-KO mice but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aβ42.
    Conclusions: Our findings support a major role for CatD in the proteostasis of both Aβ and tau in vivo. To our knowledge, the CatD-KO mouse line is the only model to develop detectable Aβ accumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aβ42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.
    MeSH term(s) Aged ; Animals ; Humans ; Mice ; Alzheimer Disease/pathology ; Amyloid beta-Peptides/metabolism ; Amyloid beta-Protein Precursor/genetics ; Amyloid beta-Protein Precursor/metabolism ; Cathepsin D ; Disease Models, Animal ; Mice, Knockout ; Mice, Transgenic ; tau Proteins/genetics ; tau Proteins/metabolism ; Tauopathies/genetics ; Tauopathies/metabolism
    Chemical Substances Amyloid beta-Peptides ; Amyloid beta-Protein Precursor ; Cathepsin D (EC 3.4.23.5) ; tau Proteins ; Ctsd protein, mouse (EC 3.4.23.5)
    Language English
    Publishing date 2024-04-04
    Publishing country England
    Document type Journal Article
    ZDB-ID 2506521-X
    ISSN 1758-9193 ; 1758-9193
    ISSN (online) 1758-9193
    ISSN 1758-9193
    DOI 10.1186/s13195-024-01443-6
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  3. Article: Prominent tauopathy and intracellular β-amyloid accumulation triggered by genetic deletion of cathepsin D: Implications for Alzheimer disease pathogenesis.

    Terron, Heather M / Parikh, Sagar J / Abdul-Hay, Samer O / Sahara, Tomoko / Kang, Dongcheul / Dickson, Dennis W / Saftig, Paul / LaFerla, Frank M / Lane, Shelley / Leissring, Malcolm A

    Research square

    2023  

    Abstract: Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-β protein (Aβ) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of ... ...

    Abstract Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid-β protein (Aβ) and the microtubule-associated protein, tau, which accumulate pathognomonically in Alzheimer disease (AD), but few studies have examined the role of CatD in the development of Aβ pathology and tauopathy in vivo.
    Methods: CatD knockout (KO) mice were crossed to human amyloid precursor protein (hAPP) transgenic mice, and amyloid burden was quantified by ELISA and immunohistochemistry (IHC). Tauopathy in CatD-KO mice, as initially suggested by Gallyas silver staining, was further characterized by extensive IHC and biochemical analyses. Controls included human tau transgenic mice (JNPL3) and another mouse model characterized by pronounced lysosomal dysfunction (Krabbe A). Additional experiments examined the effects of CatD inhibition on tau catabolism in vitro and in cultured neuroblastoma cells with inducible expression of human tau.
    Results: Deletion of CatD in hAPP transgenic mice triggers large increases in cerebral Aβ, manifesting as intense, exclusively intracellular aggregates; extracellular Aβ deposition, by contrast, is neither triggered by CatD deletion, nor affected in older, haploinsufficient mice. Unexpectedly, CatDKO mice were found to develop prominent tauopathy by just ~ 3 weeks of age, accumulating sarkosyl-insoluble, hyperphosphorylated tau exceeding the pathology in aged JNPL3 mice. CatDKO mice exhibit pronounced perinuclear Gallyas silver staining reminiscent of mature neurofibrillary tangles in human AD, together with widespread phospho-tau immunoreactivity. Striking increases in sarkosyl-insoluble phospho-tau (~ 1250%) are present in CatD-KO mice, but notably absent from Krabbe A mice collected at an identical antemortem interval. In vitro and in cultured cells, we show that tau catabolism is slowed by blockade of CatD proteolytic activity, including via competitive inhibition by Aβ42.
    Conclusions: Our findings support a major role for CatD in the proteostasis of both Aβ and tau in vivo. To our knowledge, CatD-KO mice are the only model to develop detectable Aβ acumulation and profound tauopathy in the absence of overexpression of hAPP or human tau with disease-associated mutations. Given that tauopathy emerges from disruption of CatD, which can itself be potently inhibited by Aβ42, our findings suggest that impaired CatD activity may represent a key mechanism linking amyloid accumulation and tauopathy in AD.
    Language English
    Publishing date 2023-10-23
    Publishing country United States
    Document type Preprint
    DOI 10.21203/rs.3.rs-3464352/v1
    Database MEDical Literature Analysis and Retrieval System OnLINE

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