Cathepsin B Gene Knockout Improves Behavioral Deficits and Reduces Pathology in Models of Neurologic Disorders

Abstract

Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated CTSB gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion of the CTSB gene resulted in significant improvements in behavioral deficits, neuropathology, and/or biomarkers in traumatic brain injury, ischemia, inflammatory pain, opiate tolerance, epilepsy, aging, transgenic Alzheimer's disease (AD), and periodontitis AD models as shown in 12 studies. One study found beneficial effects for double CTSB and cathepsin S KO mice in a multiple sclerosis model. Transgenic AD models using amyloid precursor protein (APP) mimicking common sporadic AD in three studies showed that CTSB KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no CTSB KO effect, and two studies used highly engineered APP constructs and reported slight increases in a biomarker. In clinical studies, all reports found that CTSB enzyme was upregulated in diverse neurologic disorders, including AD in which elevated CTSB was positively correlated with cognitive dysfunction. In a wide range of neurologic animal models, CTSB was also upregulated and not downregulated. Further, human genetic mutation data provided precedence for CTSB upregulation causing disease. Thus, the consilience of data is that CTSB gene KO results in improved brain dysfunction and reduced pathology through blockade of CTSB enzyme upregulation that causes human neurologic disease phenotypes. The overall findings provide strong support for CTSB as a rational drug target and for CTSB inhibitors as therapeutic candidates for a wide range of neurologic disorders. SIGNIFICANCE STATEMENT: This review provides a comprehensive compilation of the extensive data on the effects of deleting the cathepsin B (CTSB) gene in neurological and aging mouse models of brain disorders. Mice lacking the CTSB gene display improved neurobehavioral deficits, reduced neuropathology, and amelioration of neuronal cell death and inflammatory biomarkers. The significance of the compelling CTSB evidence is that the data consilience validates CTSB as a drug target for discovery of CTSB inhibitors as potential therapeutics for treating numerous neurological diseases.

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).