Article Figures & Data
Figures
- Fig. 1
Maturation of CTSB: zymogen conversion to active CTSB. Mature, active CTSB is generated from its inactive zymogen that is converted to the active CTSB enzyme. Preprocathepsin B is generated from its mRNA and its N-terminal signal sequence (SP) is removed by signal peptidase to result in procathepsin B. Procathepsin B undergoes autoproteolysis to remove the propeptide (Pro) to generate the mature CTSB. CTSB may also undergo additional processing into light and heavy chains linked by disulfide bonds. Cys108 (*) represents the active cysteine residue. These sequences of human CTSB were obtained from National Center for Biotechnology Information (NCBI) and UniProt databases.
- Fig. 2
CTSB expression in mouse brain regions. (A) Nissl stain of mouse brain. A coronal section of adult mouse brain was subject to Nissl staining (from the Allen Brain Institute (http://www.brain-map.org/). (B) CTSB mRNA expression in mouse brain. In situ hybridization of mouse brain sections was conducted with antisense mRNA to cathepsin B. The relative levels of CTSB mRNA expression are shown at high levels by shades of yellow to red (yellow, highest express); lower relative expression levels are shown in green to blue (blue, lowest level of expression). CTSB displays high expression of the hippocampus and cortex regions (adapted from Hook et al., 2015, DOI: 10.3389/fneur.2015.00178 indicating Frontiers as the original publisher).
- Fig. 3
Cathepsin B lysosomal leakage leads to cell death and neuroinflammation in behavioral deficits and neurodegeneration of neurologic disorders. CTSB is normally located within lysosomes. In numerous brain trauma and neurodegenerative disease conditions, lysosomal membrane permeabilization (LMP) results in translocation of CTSB to the cytosol. It is hypothesized that pathogenic cytosolic CTSB activates pathways for cell death and inflammation that result in behavioral deficits and neurodegeneration pathology. CTSB in the cytosol is involved in proteolysis to generate proapoptotic tBid and degrades antiapoptotic Bcl-xL to mediate cell death (Repnik and Turk, 2010; de Castro et al., 2016). Cytosolic CTSB activates production of IL-1β and IL-18 pro-inflammatory factors (Hentze et al., 2003; Bai et al., 2018; Campden and Zhang, 2019) that are released through the gasdermin D pore (GSDMD) (Tsuchiya et al., 2021). Cell death and inflammation result in neurodegeneration and behavioral deficits of numerous neurologic disease conditions.
- Fig. 4
APP-695 and APP-751/770 expression and processing in normal and transgenic mouse models of Alzheimer’s disease. (A) Mouse APP-695 (mAPP-695) isoform: (i) normal neuronal expression of mAPP-695 produces mouse Aβ (mAβ) and (ii) transgenic neuronal expression of human APP-695 (hAPP-695), driven by the PDGF promoter, produces human Aβ (hAβ). Panel (i) shows that in normal mouse brain, APP-695 is exclusively expressed in neurons for the production of amyloidogenic Aβ peptides, reported by several studies (Sandbrink et al., 1993; Rohan de Silva et al., 1997). APP-695 is the most abundant APP isoform expressed in the normal brain (Tanaka et al., 1989; Kang and Müller-Hill, 1990; Jacobsen et al., 1991; Rockenstein et al., 1995; Nalivaeva and Turner, 2013). Panel (ii) shows that in transgenic mice expressing hAPP-695 driven by the PDGF promoter, hAPP-695 is present in neurons and produces Aβ (Hook et al., 2009; Kindy et al., 2012; Hook et al., 2014b), which models the normal (nontransgenic) neuronal expression of hAPP-695 and production of Aβ. (B) Mouse APP-751/770 (mAPP-751/770) isoforms: (i) normal glia expression of mAPP-751/770) produces sAPPα and (ii) transgenic neuronal expression of hAPP-751/770, driven by the PDGF promoter, produces hAβ. Panel (i) shows that in normal mouse brain, APP-751/770 is expressed in glia cells (Sandbrink et al., 1993) and produces the nonamyloidogenic sAPPα fragment (Kametani et al., 1993; Nalivaeva and Turner, 2013). APP-751/770 is a minor APP isoform in the brain (Tanaka et al., 1989; Kang and Müller-Hill, 1990; Jacobsen et al., 1991; Rockenstein et al., 1995; Nalivaeva and Turner, 2013). Panel (ii) shows that in transgenic mice expressing hAPP-751/770 driven by the PDGF promoter, with deletions of segments within introns 6 and 7 and an insertion (4 bp) in intron 7 (Games et al., 1995; Rockenstein et al., 1995; Mucke et al., 2000; Mueller-Steiner et al., 2006; Wang et al., 2012), hAPP-751/770 is present in neurons and produces Aβ, which differs from the normal (nontransgenic) glia expression of hAPP-751/770 and production of sAPPα (Kametani et al., 1993; Sandbrink et al., 1993; Nalivaeva and Turner, 2013).
- Fig. 5
The consilience of CTSB KO data in neurologic disorders modeled in mice demonstrate CTSB-dependent behavioral deficits and pathology. Evidence for elevation of CTSB in models of brain disorders and amelioration of behavioral deficits and neuropathology by CTSB gene knockout in these models is summarized in this figure. (A) Elevation of CTSB expression results in several behavioral deficits and pathology in several neurologic disorders modeled in mice. Increased levels of CTSB in the brain occurs in numerous neurologic disorders modeled in mice (Table 2). The elevated CTSB in the animal models of brain disorders parallels the increased CTSB found in numerous patients with clinical neurologic disease (Table 1). (B) CTSB gene KO results in substantial improvements in behavioral deficits and pathology of several neurologic disorders modeled in mice. The consilience of results of CTSB gene KO studies in numerous animal models of neurologic disease demonstrate that the absence of CTSB results in substantial improvements in behavioral deficits and pathology (Tables 3 and 4).
Tables
Clinical Condition Biofluid or Tissue CTSB Regulation Features Reference AD Brain cortex ↑ CTSB protein increased by 18-fold Batkulwar et al., 2018 AD Brain ↑ High CTSB protein and proteolytic activity abnormally localized at amyloid plaques in brain Cataldo and Nixon, 1990 AD Serum ↑ Increased CTSB correlates with cognitive deficits Sun et al., 2015 AD CSF ↑ Increased CTSB protein Sundelof et al., 2010; Armstrong et al., 2014 AD CSF ↑ Increased CTSB protein in AD analyzed by proteomics Zhang et al., 2005 AD Plasma ↑ Elevated CTSB protein in mild and severe AD by 50%–80% above controls Morena et al., 2017 Periodontitis linked to AD Serum ↑ Increased CTSB levels by 43% Rong et al., 2020 HIV Brain and plasma monocytes ↑ Elevated CTSB Rodriguez-Franco et al., 2012; Cantres-Rosario et al., 2013 ALS Spinal cord ↑ Increased CTSB expression and protein Kikuchi et al., 2003; Dangond et al., 2004; Offen et al., 2009; Saris et al., 2013 Severe trauma Plasma ↑ CTSB activity was elevated 5- to 6-fold in severe trauma leading to organ failure Assfalg-Machleidt et al., 1990; Jochum et al., 1993 Traumatic brain injury CSF ↑ Elevated CTSB protein by twofold Boutte et al., 2020 Multiple trauma Plasma ↑ Elevated CTSB associated with trauma and sepsis Jochum et al., 1993 Vascular pathology Brain vascular endothelium ↑ Elevated CTSB levels Aoki et al., 2008 Guillain-Barre syndrome CSF ↑ Elevated CTSB activity Nagai et al., 2000 Aging CSF ↑ Increased CTSB protein correlated positively with age Nilsson et al., 2013 Animal Model Species, Age CTSB Regulation CTSB mRNA, Protein, or Activity Tissue References Alzheimer’s disease, 5XFAD model Mouse, 12 months ↑ Elevated gene expression Brain Bouter et al., 2014 Alzheimer’s disease, APPSwe/PS1 Mouse, 12 months ↑ Increased protein Brain Sun et al., 2015 Periodontitis, Alzheimer’s disease Mouse, 12 months ↑ Increased protein Hippocampus brain region Wu et al., 2017 TBI trauma Mouse, 3–5 months, and rat, 2–3 months ↑ Elevated mRNA, protein and proteolytic activity Brain Natale et al., 2003; Zhang et al., 2006; Luo et al., 2010; Sun et al., 2013; Hook et al., 2014a; Boutte et al., 2020 Trauma spinal cord contusion Rat, 2–3 months ↑ Increased mRNA, protein, and activity Brain Ellis et al., 2004, 2005 Trauma surgery Mouse, ∼3 months ↑ Elevated activity in extracellular matrix Intestine Vreemann et al., 2009 Subarachnoid hemorrhage Rat, ∼3–4 months ↑ Increased protein Brain Yu et al., 2014; Wang et al., 2015 Brain aneurysm Rat, 2 months at time of injury, and 5 months for analysis ↑ Elevated mRNA and activity Cerebral aneurysm walls Aoki et al., 2008 ALS (amyotrophic lateral sclerosis) Mouse, 2–4 months ↑ Increased mRNA Spinal cord motoneurons Ferraiuolo et al., 2007; Offen et al., 2009 Excitatory epilepsy Rat, ∼1 month ↑ Increased protein Brain and spinal cord Ni et al., 2013 Excitotoxicity, Huntington’s disease Rat, 2–3 months ↑ Elevated protein Brain Wang et al., 2006 Ischemia, acute Rat, 2–3 months ↑ Elevated protein and activity Brain Tsubokawa et al., 2006 Ischemia Monkey, adult, and rat, 2–3 months ↑ Increased protein and activity Brain Seyfried et al., 1997; Yamashima et al., 1998; Tsuchiya et al., 1999; Tsubokawa et al., 2006 Hypoxia/ischemia, neonatal Mouse, neonatal ↑ Increased mRNA and enzyme protein Microglia hippocampus Ni et al., 2015 Meningitis brain infection Mousea ↑ Increased proteolytic activity Human THP-1 cells Hoegen et al., 2011 Sepsis infection Rat, 2 months or 1 month ↑ Increased proteolytic activity Skeletal muscle Ruff and Secrist, 1984; Hummel et al., 1988 Inflammation, aging Mouse, 12 months, and 2, 10, 20 months ↑ Increased mRNA and protein Brain Wu et al., 2017; Ni et al., 2019 Inflammatory pain Mouse, ∼1 month ↑ Elevated protein Spinal cord Sun et al., 2012 aAge not indicated.
- TABLE 3
Cathepsin B Knockout Improves Behavioral Deficits in Animal Models of Neurologic Disorders
Neurologic Disease Mouse Model, Age, Sex Cathepsin B Knockout in the Neurologic Disease Model Improves Outcomes References Behaviors Cell Pathology Biomarkers Traumatic brain injury: controlled cortical impact mouse model, 15–28 weeks old, male ↓Neuromotor deficits ↓Brain tissue loss, ↓cell death in hippocampus ↓Proapoptotic Bax Hook et al., 2014a Hypoxia-ischemia: neonatal HI mouse model, neonatal, sex not specified nd ↓Cell death in hippocampus, ↓neuroinflammation ↓NF-κB to reduce inflammatory cytokines Ni et al., 2015 Epilepsy: EPM1, cystatin B KO mouse model, 2, 4, and 8 months old, sex not specified No effect on seizures ↓Cell death in neuronal granule cells nd Houseweart et al., 2003 Multiple sclerosis: EAE mouse model, 8–10 weeks old, female ↑Clinical score, ↑ time of disease onset ↓Infiltrating immunologic cells, ↓antigen presentation nd Allan and Yates, 2015 Inflammatory pain: CFA model, 5 weeks old, male ↓Allodynia behavioral test for pain ↓Microglia cell size morphology, ↓decreased microglia extensions Blockade of CFA-induced increase in IL-1β, IL-18, and COX-2 Sun et al., 2012 Tolerance to opioid: chronic morphine antinociceptive tolerance, 10 weeks old, male Prevents opiate tolerance, ↓pain assessed by thermal hot plate test ↓Elevation in excitatory postsynaptic potential ↓Glutamate release from spinal neurons Hayashi et al., 2014 nd, not determined.
Animal Model hAPP Isotype CTSB Knockout Improves Memory Deficits and Outcomes in hAPP AD Models References Isotype, Cell Type β-Secretase Site γ-Secretase Site Memory Deficits Cellular and Biomarker Features Pathology Aging Mouse APP isoforms of 695 in neurons and isoforms 751 and 770 in glia WT WT ↓Memory deficits ↓Activated macrophages, ↓inflammatory cytokines, ↓oxidative stress, ↑long-term potentiation nd Terada et al., 2010; Ni et al., 2019 Periodontitis AD Mouse APP isoforms of 695 in neurons and 751 and 770 in glia WT WT ↓Memory deficits ↓Activated macrophages
↓Aβ (1-42), ↓inflammatory cytokinesnd Wu et al., 2017 AD hAPP-WT-695, neuronal expression (PDGF promoter) WT WT n/a ↓Aβ (1–42) by ∼70%
↓β (1–40) by ∼70%
↓CTFβ by 40%, ↑sAPPα by 60%, ↓WT β-secretase activityn/a Hook et al., 2009 AD hAPP-WT-Lon-695, neuronal expression (PDGF promoter) WT Lon, V717I ↓Memory deficits ↓Aβ(1–40) by 85%
↓Aβ(1–42) by 87%
↓pGluAβ(3–40) by 65%
↓pGluAβ(3–42) by 92%
↓CTFβ by 60%
↑sAβPPα by 60%
↓WT β-secretase activity↓Aβ plaque by 85%, ↓pGluAβ plaque by 46% Kindy et al., 2012; Hook et al., 2014b AD hAPP-Swe-Lon-695, neuronal expression (PDGF promoter) Swe, K670N/M671L Lon, V717I No effect on memory deficits No effects on Aβ(1–42), CTFβ, or APPα No effect on amyloid plaque Kindy et al., 2012 AD hAPP-Swe-Ind-695, neuronal expression (PDGF promoter) Swe K670N/M671L Ind, V717F n/a No effects on Aβ, CTFβ, or sAPPα n/a Hook et al., 2009 AD hAPP-Swe-Ind-751/770, neuronal expression (PDGF promoter), J20 line, introns modified, PDAPP) Swe Ind nd No change in fIAPP, β-CTF, α-sAPP, or α-CTF
↑Aβ(1–42)/Aβ(1–x) ratio by ∼25%Elevated plaque load Mueller-Steiner et al., 2006 AD hAPP-751/770, neuronal expression (PDGF promoter, I63 line, introns modified, PDAPP) WT WT nd No change in hippocampal Aβ42, ↑cortical Aβ42 by 12% nd Wang et al., 2012 nd, not determined; n/a, not applicable.
In this issue
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- Article
- Abstract
- I. Introduction
- II. Cathepsin B is Elevated in Human Neurologic Disorders
- III. Cathepsin B Genetic Mutations Increase Cathepsin B and Cause Human Diseases
- IV. Cathepsin B Is Elevated in Animal Models of Neurologic Diseases
- V. Cathepsin B Neurobiology
- VI. Gene Knockout Mice for Target Validation and Drug Discovery
- VII. Cathepsin B Knockout Mice Are Generally Indistinguishable from Wild-Type Healthy Animals
- VIII. Cathepsin B Knockout Improves Behavioral Deficits in Neurologic Disease Animal Models
- IX. Cathepsin B Upregulation Is a Common Response in Neurologic Disorders and Causes Cellular Pathology by Multiple Specific Mechanisms
- X. Summary and Conclusion: Cathepsin B Knockout Data Validates Cathepsin B as a Drug Target for Development of Cathepsin B Inhibitors as Potentially New Therapeutics for Neurologic Disorders
- XI. Significance
- Acknowledgments
- Authorship Contributions
- Footnotes
- Abbreviations
- References
- Figures & Data
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