The lysosomal cysteine protease cathepsin B (CTSB) has been suggested as a biomarker for Alzheimer's disease (AD) because elevated serum CTSB in AD patients has been found to correlate with cognitive dysfunction. Furthermore, CTSB gene knockout (KO) in non-transgenic and transgenic AD animal models showed that elimination of CTSB improved memory deficits. However, conflicting CTSB KO results on amyloid-β (Aβ) pathology in transgenic AD models have been reported. The conflict is resolved here as likely being due to the different hAβPP transgenes used in the different AD mouse models. CTSB gene KO reduced wild-type (Wt) β-secretase activity, brain Aβ, pyroglutamate-Aβ, amyloid plaque, and memory deficits in models that used cDNA transgenes expressing hAβPP isoform 695. But in models that used mutated mini transgenes expressing hAβPP isoforms 751 and 770, CTSB KO had no effect on Wt β-secretase activity and slightly increased brain Aβ. All models expressed the AβPP transgenes in neurons. These conflicting results in Wt β-secretase activity models can be explained by hAβPP isoform specific cellular expression, proteolysis, and subcellular processing. CTSB KO had no effect on Swedish mutant (Swe) β-secretase activity in hAβPP695 and hAβPP751/770 models. Different proteolytic sensitivities for hAβPP with Wt versus Swe β-secretase site sequences may explain the different CTSB β-secretase effects in hAβPP695 models. But since the vast majority of sporadic AD patients have Wt β-secretase activity, the CTSB effects on Swe β-secretase activity are of little importance to the general AD population. As neurons naturally produce and process hAβPP isoform 695 and not the 751 and 770 isoforms, only the hAβPP695 Wt models mimic the natural neuronal hAβPP processing and Aβ production occurring in most AD patients. Significantly, these CTSB KO findings in the hAβPP695 Wt models demonstrate that CTSB participates in memory deficits and production of pyroglutamate-Aβ (pyroglu-Aβ), which provide rationale for future investigation of CTSB inhibitors in AD therapeutics development.

CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained its specificity for cathepsin B over other cysteine cathepsins, displayed irreversible inhibition, and inhibited diverse cleavages of peptide substrates of cathepsin B assessed by profiling mass spectrometry. Molecular docking suggested that pH-dependent ionic interactions of the C-terminal carboxylate of CA -074 occur with His110 and His111 residues in the S2 ' subsite of the enzyme at pH 4.6, but these interactions differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted by cellular esterases to CA-074) are used in biological studies to inhibit cathepsin B at both acidic and neutral pH locations, it is possible that adjusted levels of CA-074 or CA-074Me may be explored to differentially affect cathepsin B activity at these different pH values. Overall, the results of this study demonstrate the molecular, kinetic, and protease specificity features of CA-074 pH-dependent inhibition of cathepsin B.

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.

Cathepsin B is a cysteine protease that normally functions within acidic lysosomes for protein degradation, but in numerous human diseases, cathepsin B translocates to the cytosol having neutral pH where the enzyme activates inflammation and cell death. Cathepsin B is active at both the neutral pH 7.2 of the cytosol and the acidic pH 4.6 within lysosomes. We evaluated the hypothesis that cathepsin B may possess pH-dependent cleavage preferences that can be utilized for design of a selective neutral pH inhibitor by (1) analysis of differential cathepsin B cleavage profiles at neutral pH compared to acidic pH using multiplex substrate profiling by mass spectrometry (MSP-MS), (2) design of pH-selective peptide-7-amino-4-methylcoumarin (AMC) substrates, and (3) design and validation of Z-Arg-Lys-acyloxymethyl ketone (AOMK) as a selective neutral pH inhibitor. Cathepsin B displayed preferences for cleaving peptides with Arg in the P2 position at pH 7.2 and Glu in the P2 position at pH 4.6, represented by its primary dipeptidyl carboxypeptidase and modest endopeptidase activity. These properties led to design of the substrate Z-Arg-Lys-AMC having neutral pH selectivity, and its modification with the AOMK warhead to result in the inhibitor Z-Arg-Lys-AOMK. This irreversible inhibitor displays nanomolar potency with 100-fold selectivity for inhibition of cathepsin B at pH 7.2 compared to pH 4.6, shows specificity for cathepsin B over other cysteine cathepsins, and is cell permeable and inhibits intracellular cathepsin B. These findings demonstrate that cathepsin B possesses pH-dependent cleavage properties that can lead to development of a potent, neutral pH inhibitor of this enzyme.

Cathepsin B (CatB), a lysosomal cysteine protease, is important to brain function and may have dual utility as a peripheral biomarker of moderate-severe traumatic brain injury (TBI). The present study determined levels of pro- and mature (mat) CatB protein as well as cysteine protease activity within the frontal cortex (FC; proximal injury site), hippocampus (HC; distal injury site), and cerebral spinal fluid (CSF) collected 1-7 days after craniotomy and penetrating ballistic-like brain injury (PBBI) in rats. Values were compared with naive controls. Further, the utility of CatB protein as a translational biomarker was determined in CSF derived from patients with severe TBI. Craniotomy increased matCatB levels in the FC and HC, and led to elevation of HC activity at day 7. PBBI caused an even greater elevation in matCatB within the FC and HC within 3-7 days. After PBBI, cysteine protease activity peaked at 3 days in the FC and was elevated at 1 day and 7 days, but not 3 days, in the HC. In rat CSF, proCatB, matCatB, and cysteine protease activity peaked at 3 days after craniotomy and PBBI. Addition of CA-074, a CatB-specific inhibitor, confirmed that protease activity was due to active matCatB in rat brain tissues and CSF at all time-points. In patients, CatB protein was detectable from 6 h through 10 days after TBI. Notably, CatB levels were significantly higher in CSF collected within 3 days after TBI compared with non-TBI controls. Collectively, this work indicates that CatB and its cysteine protease activity may serve as collective molecular signatures of TBI progression that differentially vary within both proximal and distal brain regions. CatB and its protease activity may have utility as a surrogate, translational biomarker of acute-subacute TBI.

Lysosomes are known to mediate neurite outgrowth in neurons. However, the principal lysosomal molecule controlling that outgrowth is unclear. We studied primary mouse neurons in vitro and found that they naturally develop neurite outgrowths over time and as they did so the lysosomal cysteine protease cathepsin B (CTSB) mRNA levels dramatically increased. Surprisingly, we found that treating those neurons with CA-074Me, which inhibits CTSB, prevented neurites. As that compound also inhibits another protease, we evaluated a N2a neuronal cell line in which the CTSB gene was deleted (CTSB knockout, KO) using CRISPR technology and induced their neurite outgrowth by treatment with retinoic acid. We found that CTSB KO N2a cells failed to produce neurite outgrowths but the wild-type (WT) did. CA-074Me is a cell permeable prodrug of CA-074, which is cell impermeable and a specific CTSB inhibitor. Neurite outgrowth was and was not suppressed in WT N2a cells treated with CA-074Me and CA-074, respectively. Lysosome-associated membrane glycoprotein 2-positive lysosomes traffic to the plasma cell membrane in WT but not in CTSB KO N(2)a cells. Interestingly, no obvious differences between WT and CTSB KO N2a cells were found in neurite outgrowth regulatory proteins, PI3K/AKT, ERK/MAPK, cJUN, and CREB. These findings show that intracellular CTSB controls neurite outgrowth and that it does so through regulation of lysosomal trafficking and remodeling in neurons. This adds valuable information regarding the physiological function of CTSB in neural development.

Investigations of Alzheimer's disease (AD), traumatic brain injury (TBI), and related brain disorders have provided extensive evidence for involvement of cathepsin B, a lysosomal cysteine protease, in mediating the behavioral deficits and neuropathology of these neurodegenerative diseases. This review integrates findings of cathepsin B regulation in clinical biomarker studies, animal model genetic and inhibitor evaluations, structural studies, and lysosomal cell biological mechanisms in AD, TBI, and related brain disorders. The results together indicate the role of cathepsin B in the behavioral deficits and neuropathology of these disorders. Lysosomal leakage occurs in AD and TBI, and related neurodegeneration, which leads to the hypothesis that cathepsin B is redistributed from the lysosome to the cytosol where it initiates cell death and inflammation processes associated with neurodegeneration. These results together implicate cathepsin B as a major contributor to these neuropathological changes and behavioral deficits. These findings support the investigation of cathepsin B as a potential drug target for therapeutic discovery and treatment of AD, TBI, and TBI-related brain disorders.

There is currently no therapeutic drug treatment for traumatic brain injury (TBI) despite decades of experimental clinical trials. This may be because the mechanistic pathways for improving TBI outcomes have yet to be identified and exploited. As such, there remains a need to seek out new molecular targets and their drug candidates to find new treatments for TBI. This review presents supporting evidence for cathepsin B, a cysteine protease, as a potentially important drug target for TBI. Cathepsin B expression is greatly up-regulated in TBI animal models, as well as in trauma patients. Importantly, knockout of the cathepsin B gene in TBI mice results in substantial improvements of TBI-caused deficits in behavior, pathology, and biomarkers, as well as improvements in related injury models. During the process of TBI-induced injury, cathepsin B likely escapes the lysosome, its normal subcellular location, into the cytoplasm or extracellular matrix (ECM) where the unleashed proteolytic power causes destruction via necrotic, apoptotic, autophagic, and activated glia-induced cell death, together with ECM breakdown and inflammation. Significantly, chemical inhibitors of cathepsin B are effective for improving deficits in TBI and related injuries including ischemia, cerebral bleeding, cerebral aneurysm, edema, pain, infection, rheumatoid arthritis, epilepsy, Huntington’s disease, multiple sclerosis, and Alzheimer’s disease. The inhibitor E64d is unique among cathepsin B inhibitors in being the only compound to have demonstrated oral efficacy in a TBI model and prior safe use in man and as such it is an excellent tool compound for preclinical testing and clinical compound development. These data support the conclusion that drug development of cathepsin B inhibitors for TBI treatment should be accelerated.

There are currently no effective therapeutic agents for traumatic brain injury (TBI), but drug treatments for TBI can be developed by validation of new drug targets and demonstration that compounds directed to such targets are efficacious in TBI animal models using a clinically relevant route of drug administration. The cysteine protease, cathepsin B, has been implicated in mediating TBI, but it has not been validated by gene knockout (KO) studies. Therefore, this investigation evaluated mice with deletion of the cathepsin B gene receiving controlled cortical impact TBI trauma. Results indicated that KO of the cathepsin B gene resulted in amelioration of TBI, shown by significant improvement in motor dysfunction, reduced brain lesion volume, greater neuronal density in brain, and lack of increased proapoptotic Bax levels. Notably, oral administration of the small-molecule cysteine protease inhibitor, E64d, immediately after TBI resulted in recovery of TBI-mediated motor dysfunction and reduced the increase in cathepsin B activity induced by TBI. E64d outcomes were as effective as cathepsin B gene deletion for improving TBI. E64d treatment was effective even when administered 8 h after injury, indicating a clinically plausible time period for acute therapeutic intervention. These data demonstrate that a cysteine protease inhibitor can be orally efficacious in a TBI animal model when administered at a clinically relevant time point post-trauma, and that E64d-mediated improvement of TBI is primarily the result of inhibition of cathepsin B activity. These results validate cathepsin B as a new TBI therapeutic target.

Pyroglutamate amyloid-beta peptides (pGlu-Abeta) are particularly pernicious forms of Abeta. pGlu-Abeta and full-length Abeta peptides accumulate in Alzheimer's disease (AD) brains, leading to severe memory deficits. pGlu-Abeta peptides are N-terminally truncated forms of full-length Abeta peptides with modification of the N-terminal glutamate to form pGlu-Abeta(3-40/42). The prominent presence of pGlu-Abeta in AD brains and involvment pGlu-Abeta to initiate formation of oligomeric neurotoxic Abeta forms may be key in AD. Our recent research indicates the key role of the alternative beta-secretase cathepsin B (CatB) in the production of pGlu-Abeta and Abeta (Hook et al., 2014, in press; Kindy et al., 2012), suggesting that inhibitors of CatB can reduce pGlu-Abeta and Abeta. Therefore, this study investigated the cysteine protease inhibitor E64d, that inhibits CatB, for its effectives in reducing Abeta peptide forms and improving memory deficits in APPLon AD mice, which express APP-695 and have the wild-type (wt) beta-secretase activity present in most AD patients. APPLon mice were administered E64d (oral), using E64d prepared in the food chow. Memory deficits were then assessed by the Morris water maze test. Brain tissue samples were measured for levels of pGlu-Abeta and flAbeta peptides by ELISAs, and amyloid plaque load was assessed by quantitative immunohistochemistry. E64d treatment reduced brain levels of pGlu-Abeta(3-40/42), flAbeta(1-40/42), and pGlu-Abeta/Abeta plaque load in APPLon mice. E64d treatment of APPLon mice with CatB gene knockout resulted in similar level of Abeta peptide reduction,. Notably, E64d resulted in substantial and significant improvement in memory deficits. Administration (oral) of the cysteine protease inhibitor E64d to APPLon mice resulted in decreased brain levels of pGlu-Abeta and flAbeta, decreased amyloid plaque load, and substantial improvement in memory deficits. E64d is known to be safe in patients, based on extensive clinical trials in Japan for muscular dystrophy. These data strongly suggest that the E64d type compound(s) will be useful as therapeutic drug candidates for treating AD patients.

Pyroglutamate amyloid-beta peptides (pGlu-Abeta) are particularly neurotoxic forms of Abeta, and they accumulate with full-length Abeta peptides in Alzheimer's disease (AD) brains. pGlu-Abeta peptides are N-terminally truncated forms of full-length Abeta peptides (flAbeta(1-40/42)) with modification of the N-terminal glutamate to form pGlu-Abeta(3-40/42). The significant presence of pGlu-Abeta in AD brains may be important in the development of AD because pGlu-Abeta initiates formation of oligomeric neurotoxic Abeta forms. Beta-secretase processing of amyloid precursor protein (APP) produces flAbeta(1-40/42), but it is not yet known whether the beta-secretase BACE1 or the alternative beta-secretase cathepsin B (CatB) participate in the production of pGlu-Abeta. Therefore, experiments examined the effects of gene knockout of these proteases on pGlu-Abeta and flAbeta brain levels, amyloid plaque load, and memory deficits in APPLon AD mice, which express APP-695 and have the wild-type (wt) β-secretase activity found in most AD patients. APPLon mice with knockout of the CatB or BACE1 gene were generated, and were assessed for memory deficits by the Morris water maze test. Brain extracts were measured for levels of pGlu-Abeta and flAbeta peptides by ELISAs, and amyloid plaque load was assessed by quantitative immunohistochemistry. Knockout of the CatB gene reduced brain levels of pGlu-Abeta(3-40/42), flAbeta(1-40/42), and pGlu-Abeta/Abeta plaque load in APPLon mice. Expression of the CatB gene increased levels of pGlu-Abeta and Abeta peptides, as well as amyloid plaque load. Substantial improvements in memory deficits resulted from knockout of CatB. But knockout of the BACE1 gene had no effect on levels of these Abeta forms and had no effect on memory in the APPLon mouse model of AD. CatB participates in the production of pGlu-Abeta and flAbeta, and the absence of the CatB gene improves memory deficits in the APPLon AD mouse model. These findings suggest that inhibitors targeting CatB will be useful in therapeutic treatment of AD patients.

Pyroglutamate amyloid-β peptides (pGlu-Aβ) are particularly pernicious forms of amyloid-β peptides (Aβ) present in Alzheimer's disease (AD) brains. pGlu-Aβ peptides are N-terminally truncated forms of full-length Aβ peptides (flAβ(1-40/42)) in which the N-terminal glutamate is cyclized to pyroglutamate to generate pGlu-Aβ(3-40/42). β-secretase cleavage of amyloid-β precursor protein (AβPP) produces flAβ(1-40/42), but it is not yet known whether the β-secretase BACE1 or the alternative β-secretase cathepsin B (CatB) participate in the production of pGlu-Aβ. Therefore, this study examined the effects of gene knockout of these proteases on brain pGlu-Aβ levels in transgenic AβPPLon mice, which express AβPP isoform 695 and have the wild-type (wt) β-secretase activity found in most AD patients. Knockout or overexpression of the CatB gene reduced or increased, respectively, pGlu-Aβ(3-40/42), flAβ(1-40/42), and pGlu-Aβ plaque load, but knockout of the BACE1 gene had no effect on those parameters in the transgenic mice. Treatment of AβPPLon mice with E64d, a cysteine protease inhibitor of CatB, also reduced brain pGlu-Aβ(3-42), flAβ(1-40/42), and pGlu-Aβ plaque load. Treatment of neuronal-like chromaffin cells with CA074Me, an inhibitor of CatB, resulted in reduced levels of pGlu-Aβ(3-40) released from the activity-dependent, regulated secretory pathway. Moreover, CatB knockout and E64d treatment has been previously shown to improve memory deficits in the AβPPLon mice. These data illustrate the role of CatB in producing pGlu-Aβ and flAβ that participate as key factors in the development of AD. The advantages of CatB inhibitors, especially E64d and its derivatives, as alternatives to BACE1 inhibitors in treating AD patients are discussed.

Risk factors for Alzheimer's disease (AD) include ischemic stroke (IS) and traumatic brain injury (TBI). Unfortunately, there currently are no effective neurological treatments for these conditions or interventions that prevent their downstream consequences from increasing the risk for AD. Such treatments may be possible if compounds are identified that affect a common brain mechanism and improve neurological outcomes in AD, IS, and TBI animal models. E64d is a cysteine protease inhibitor that has been shown to be orally effective in AD animal models for improving memory and reducing beta-amyloid, via inhibition of cathepsin B (Hook, 2011, J Alz Dis 26:387; Kindy, 2012, J Alz Dis 29:827). IS increases cathepsin B activity and E64d administration results in significant neuroprotection in IS animal models (Tsubokawa, 2006, J Neurosci Res 84:832). TBI also results in activation of cathepsin B and neuronal degeneration with apoptosis (Luo, 2010, J Neurosc Res 88:2847). This study evaluated the effects of deleting the cathepsin B gene and of oral E64d treatment post-trauma in a TBI mouse model. A controlled cortical impact TBI mouse model was used to study the effect of deleting the cathepsin B gene, or orally treating wild-type (wt) and cathepsin B deficient mice with E64d, on neuromotor dysfunction, as evaluated by the rotarod test, brain pathology and bax, a pro-apoptotic biomarker, in the week following trauma. Knockout of the cathepsin B gene resulted in significantly improved neuromotor function, alleviation of brain pathology, and reduced Bax levels relative to wt mice. E64d treatment of cathepsin B deficient or wt mice resulted in even better outcomes than that of the untreated cathepsin B deficient mice. Cathepsin B is an important target for TBI drug development and oral E64d administration is effective at improving TBI outcomes through inhibiting cathepsin B activity. E64d is rare in that it has been shown effective in AD, IS and now TBI animal models. Importantly, because E64d has been previously shown safe to use in man (Imahori, 1985, Rinsho Yakuri 16:749), E64d, or its derivatives, have great potential for AD prevention as AD, IS and TBI therapeutic agents.

Amyloid- β peptides (Aβ) participate in the development of Alzheimer's disease (AD). AD brains accumulate multiple forms of Aβ peptides, composed of Aβ(1–40/42) with pyroglutamate-Aβ(3–40/42) (pGlu-A β) present as a major portion of Aβ. Several studies estimate that pGlu-A β comprises approximately 50% or more of total A β (Saido et al., 1995; Kuo et al., 1997). The pGlu-Aβ(3–40/42) is hypothesized to facilitate the oligomerization of Aβ(1–40/42) (Schilling et al., 2006) to result in neurotoxicity. The formation of pGlu-Aβ(3–40/42) is produced from the substrate Aβ(3–40/42), which may be generated by a beta-secretase-dependent or -independent mechanism. Cathepsin B has been shown to generate A β(1–40/42) from AD mice expressing APP with the wild-type beta-secretase site (Kindy et al., 2012) expressed in the majority of AD patients. This study, therefore, addresses the role of cathepsin B, possessing wild-type beta-secretase function, in producing pGlu-Aβ(3–40/42). Knockout of the cathepsin B gene was conducted in the APP/Lon mouse model of AD. The APP/Lon mice were also treated with E64d, inhibitor of cathepsin B, by oral administration. Brains were then assessed for levels of pGlu-Aβ(3–40/42) and Aβ(1–40/42), amyloid plaque; animals were evaluated for memory function by the Morris water maze test. Knockout of the cathepsin B gene in the APP/Lon AD mouse model, expressing APP with the wild-type beta-secretase site, was assessed. Knockout of the cathepsin gene resulted in a significant reduction in brain pGlu-Aβ(3–40/42) as well as Aβ(1–40/42). Chemical inhibition of cathepsin B, conducted by oral administration of the E64d inhibitor, in the APP/Lon mice reduced brain pGlu-Aβ(3–40/42) and Aβ(1–40/42). Also, cathepsin B inhibition and gene knockout reduced brain amyloid plaque accumulation and improved memory function in the APP/Lon mice. Cellular studies show that pGlu-Aβ(3–40) is secreted from the regulated secretory pathway of neuronal-like chromaffin cells, and inhibition of cathepsin B (with CA074Me) reduces pGlu-A β(3–40). Results from knockout of the cathepsin B gene in the APP/Lon AD mouse model show that cathepsin B participates in the production of pGlu-Aβ(3–40/42), as well as A β(1–40/42). Cathepsin B inhibitors will likely be effective at reducing neurotoxic pGlu-Aβ(3–40/42), as well as Aβ(1–40/42), for improvement of memory function in AD.