Elucidation of Aβ-lowering agents that inhibit processing of the wild-type (WT) β-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical ...approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of β-secretase activity in regulated secretory vesicles that produce β-amyloid (Aβ). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT β-secretase site but not the rare Swedish mutant β-secretase site. In vivo treatment of London APP mice, expressing the WT β-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Aβ40 and Aβ42, and reduced C-terminal β-secretase fragment derived from APP by β-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant β-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Aβ in an AD animal model expressing the WT β-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.
The human tau protein is implicated in a wide range of neurodegenerative "tauopathy" diseases, consisting of Alzheimer's disease (AD) and frontotemporal lobar degeneration which includes progressive ...supranuclear palsy, corticobasal degeneration, Pick's disease, and FTLD-tau (frontotemporal dementia with parkinsonism caused by MAPT mutations). Tau gene transcripts in the human brain undergo alternative splicing to yield 6 different tau protein isoforms that are expressed in different ratios in neurodegeneration which result in tau pathology of paired-helical filaments, neurofibrillary tangles, and tau fibrillar aggregates with detrimental microtubule destabilization. Protease-mediated tau truncation is an important post-translational modification (PTM) which drives neurodegeneration in a tau fragment-dependent manner. While numerous tau fragments have been identified, knowledge of the proteolytic steps that convert each parent tau isoform into specific truncated tau fragments has not yet been fully defined. An improved understanding of the relationships between tau isoforms and their proteolytic processing to generate neurotoxic tau fragments is important to the field. This review evaluates tau isoform expression patterns including PTMs and mutations that influence proteolysis of tau to generate toxic fragments that drive cognitive deficits in AD and other tauopathy models. This assessment identifies the gap in the field on understanding the details of proteolytic steps used to convert each tau isoform into fragments. Knowledge of the processing mechanisms of tau isoforms can lead to new protease targeted drug strategies to prevent the formation of toxic tau fragments in tauopathy neurodegenerative diseases.
Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated
gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion ...of the
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
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
KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no
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
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 (
) gene in neurological and aging mouse models of brain disorders. Mice lacking the
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.
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.
•Cathepsin B is elevated in human Alzheimer's disease (AD) and traumatic brain injury (TBI) patients, and correlates with behavioral and injury outcomes•Inhibition of cathepsin B in AD and TBI animal models results in alleviation of cognitive and behavioral deficits with improvement in neuropathology•Preprocathepsin B undergoes processing to generate mature cathepsin B, which can be selectively inhibited by CA-074•The occluding loop of cathepsin B regulates dipeptidylcarboxypeptidase and endopeptidase activities of cathepsin B•Lysosomal leakage of cathepsin B to the cytosol participates in cell death and inflammation of AD, TBI, and related brain disorders
Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass ...spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated k cat and K M for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine-AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine-AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.
Cathepsin protease genes are necessary for protein homeostasis in normal brain development and function. The diversity of the 15 cathepsin protease activities raises the question of what are the ...human brain expression profiles of the cathepsin genes during development from prenatal and infancy to childhood, adolescence, and young adult stages. This study, therefore, evaluated the cathepsin gene expression profiles in 16 human brain regions during development by quantitative RNA-sequencing data obtained from the Allen Brain Atlas resource. Total expression of all cathepsin genes was the lowest at the early prenatal stage which became increased at the infancy stage. During infancy to young adult phases, total gene expression was similar. Interestingly, the rank ordering of gene expression among the cathepsins was similar throughout the brain at the age periods examined, showing (a) high expression of cathepsins B, D, and F; (b) moderate expression of cathepsins A, L, and Z; (c) low expression of cathepsins C, H, K, O, S, and V; and (d) very low expression of cathepsins E, G, and W. Results show that the human brain utilizes well-defined, balanced patterns of cathepsin gene expression throughout the different stages of human brain development. Knowledge gained by this study of the gene expression profiles of lysosomal cathepsin proteases among human brain regions during normal development is important for advancing future investigations of how these cathepsins are dysregulated in lysosomal-related brain disorders that affect infants, children, adolescents, and young adults.
Neurotoxic β-amyloid (Aβ) peptides participate in Alzheimer’s disease (AD); therefore, reduction of Aβ generated from APP may provide a therapeutic approach for AD. Gene knockout studies in ...transgenic mice producing human Aβ may identify targets for reducing Aβ. This study shows that knockout of the cathepsin B gene in mice expressing human wild-type APP (hAPPwt) results in substantial decreases in brain Aβ40 and Aβ42 by 67% and decreases in levels of the C-terminal β-secretase fragment (CTFβ) derived from APP. In contrast, knockout of cathepsin B in mice expressing hAPP with the rare Swedish (Swe) and Indiana (Ind) mutations had no effect on Aβ. The difference in reduction of Aβ in hAPPwt mice, but not in hAPPSwe/Ind mice, shows that the transgenic model can affect cathepsin B gene knockout results. Since most AD patients express hAPPwt, these data validate cathepsin B as a target for development of inhibitors to lower Aβ in AD.
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 N2a 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.
Lysosomes were trafficked to the cell membrane in N2a cells during neurite outgrowth initiation; however, Cathepsin B (CTSB) inhibition induced a cytosol distribution of lysosomes and neurite outgrowth blockage. We proposed that intracellular CTSB controls neurite outgrowth and that it does so through regulation of lysosomal trafficking and remodeling in neurons.
Schizophrenia is a devastating psychiatric illness that detrimentally affects a significant portion of the worldwide population. Aging of schizophrenia patients is associated with reduced longevity, ...but the potential biological factors associated with aging in this population have not yet been investigated in a global manner. To address this gap in knowledge, the present study assesses proteomics and metabolomics profiles in the plasma of subjects afflicted with schizophrenia compared to non-psychiatric control patients over six decades of life. Global, unbiased analyses of circulating blood plasma can provide knowledge of prominently dysregulated molecular pathways and their association with schizophrenia, as well as features of aging and gender in this disease. The resulting data compiled in this study represent a compendium of molecular changes associated with schizophrenia over the human lifetime. Supporting the clinical finding of schizophrenia's association with more rapid aging, both schizophrenia diagnosis and age significantly influenced the plasma proteome in subjects assayed. Schizophrenia was broadly associated with prominent dysregulation of inflammatory and metabolic system components. Proteome changes demonstrated increased abundance of biomarkers for risk of physiologic comorbidities of schizophrenia, especially in younger individuals. These findings advance our understanding of the molecular etiology of schizophrenia and its associated comorbidities throughout the aging process.
Neuropeptides are essential for cell-cell communication in neurological and endocrine physiological processes in health and disease. While many neuropeptides have been identified in previous studies, ...the resulting data has not been structured to facilitate further analysis by tandem mass spectrometry (MS MS), the main technology for high-throughput neuropeptide identification. Many neuropeptides are difficult to identify when searching MS MS spectra against large protein databases because of their atypical lengths (e.g. shorter longer than common tryptic peptides) and lack of tryptic residues to facilitate peptide ionization fragmentation. NeuroPedia is a neuropeptide encyclopedia of peptide sequences (including genomic and taxonomic information) and spectral libraries of identified MS MS spectra of homolog neuropeptides from multiple species. Searching neuropeptide MS MS data against known NeuroPedia sequences will improve the sensitivity of database search tools. Moreover, the availability of neuropeptide spectral libraries will also enable the utilization of spectral library search tools, which are known to further improve the sensitivity of peptide identification. These will also reinforce the confidence in peptide identifications by enabling visual comparisons between new and previously identified neuropeptide MS MS spectra.
Availability:
http://proteomics.ucsd.edu/Software/NeuroPedia.html
Contact:
bandeira@ucsd.edu
Supplementary information:
Supplementary materials are available at Bioinformatics online.
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