Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages.

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.

Cathepsin D: Analysis of its potential role as an amyloid beta degrading protease.

Proteolysis catalyzed by the major lysosomal aspartyl protease cathepsin-D (CTSD) appears to be of pivotal importance for proteostasis within the central nervous system and in neurodegeneration. Neuronal Ceroid Lipofuscinosis (NCL) type 10 is caused by a lack of CTSD leading to a defective autophagic flow and pathological accumulation of proteins. We previously demonstrated a therapeutic-relevant clearance of protein aggregates after dosing a NCL10 mouse model with recombinant human pro-cathepsin-D (proCTSD). Similar results could be achieved in cells and mice accumulating α-synuclein. Prompted by these positive effects and our in vitro findings showing that cathepsin-D can cleave the Alzheimer's Disease (AD)-causing amyloid beta peptides (Aß), we envisaged that such a treatment with proCTSD could similarly be effective in clearance of potentially toxic Aß species. We demonstrated that CTSD is able to cleave human Aß1-42 by using liquid chromatography-mass spectrometry. Intracerebral dosing of proCTSD in a NCL10 (CTSD knockout) mouse model revealed uptake and processing of CTSD to its mature and active form. However, the re-addition of CTSD did not obviously affect intracellular APP processing or the generation of soluble APP and Aß-species. ProCTSD treated HEK cells in comparison with untreated cells were found to contain comparable levels of soluble and membrane bound APP and Aß-species. Also, the early intracranial application (P1 and P20) of proCTSD in the 5xFAD mouse model did not change Aß pathology, plaque number and plaque composition and neuroinflammation, however we observed an increased level of Aß1-42 in the CSF. Our data confirm proteolytic cleavage of human Aß1-42 by CTSD but exclude a prominent role of CTSD in APP processing and Aß degradation in our in vitro and in vivo models.

Recombinant pro-CTSD (cathepsin D) enhances SNCA/α-Synuclein degradation in α-Synucleinopathy models.

Parkinson disease (PD) is a neurodegenerative disorder characterized by the abnormal intracellular accumulation of SNCA/α-synuclein. While the exact mechanisms underlying SNCA pathology are not fully understood, increasing evidence suggests the involvement of autophagy as well as lysosomal deficiencies. Because CTSD (cathepsin D) has been proposed to be the major lysosomal protease involved in SNCA degradation, its deficiency has been linked to the presence of insoluble SNCA conformers in the brain of mice and humans as well as to the transcellular transmission of SNCA aggregates. We here postulate that SNCA degradation can be enhanced by the application of the recombinant human proform of CTSD (rHsCTSD). Our results reveal that rHsCTSD is efficiently endocytosed by neuronal cells, correctly targeted to lysosomes and matured to an enzymatically active protease. In dopaminergic neurons derived from induced pluripotent stem cells (iPSC) of PD patients harboring the A53T mutation within the SNCA gene, we confirm the reduction of insoluble SNCA after treatment with rHsCTSD. Moreover, we demonstrate a decrease of pathological SNCA conformers in the brain and within primary neurons of a ctsd-deficient mouse model after dosing with rHsCTSD. Boosting lysosomal CTSD activity not only enhanced SNCA clearance in human and murine neurons as well as tissue, but also restored endo-lysosome and autophagy function. Our findings indicate that CTSD is critical for SNCA clearance and function. Thus, enzyme replacement strategies utilizing CTSD may also be of therapeutic interest for the treatment of PD and other synucleinopathies aiming to decrease the SNCA burden.Abbreviations: aa: amino acid; SNCA/α-synuclein: synuclein alpha; APP: amyloid beta precursor protein; BBB: blood brain barrier; BF: basal forebrain; CBB: Coomassie Brilliant Blue; CLN: neuronal ceroid lipofuscinosis; CNL10: neuronal ceroid lipofuscinosis type 10; Corr.: corrected; CTSD: cathepsin D; CTSB: cathepsin B; DA: dopaminergic; DA-iPSn: induced pluripotent stem cell-derived dopaminergic neurons; dox: doxycycline; ERT: enzyme replacement therapy; Fx: fornix, GBA/ß-glucocerebrosidase: glucosylceramidase beta; h: hour; HC: hippocampus; HT: hypothalamus; i.c.: intracranially; IF: immunofluorescence; iPSC: induced pluripotent stem cell; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LSDs: lysosomal storage disorders; MAPT: microtubule associated protein tau; M6P: mannose-6-phosphate; M6PR: mannose-6-phosphate receptor; MB: midbrain; mCTSD: mature form of CTSD; neurofil.: neurofilament; PD: Parkinson disease; proCTSD: proform of CTSD; PRNP: prion protein; RFU: relative fluorescence units; rHsCTSD: recombinant human proCTSD; SAPC: Saposin C; SIM: structured illumination microscopy; T-insol: Triton-insoluble; T-sol: Triton-soluble; TEM: transmission electron microscopy, TH: tyrosine hydroxylase; Thal: thalamus.

Analysis of cathepsin B and cathepsin L treatment to clear toxic lysosomal protein aggregates in neuronal ceroid lipofuscinosis.

Proteolysis mediated by lysosomal cathepsin proteases maintains a physiological flow in autophagy, phagocytosis and endocytosis. Neuronal Ceroid Lipofuscinosis (NCL) is a childhood neurodegenerative disorder characterized by disturbed autophagic flow and pathological accumulation of proteins. We demonstrated a therapeutic clearance of protein aggregates after dosing NCL10 mice with recombinant human pro-cathepsin-D. Prompted by these results and speculating that cathepsins may act in a redundant and in an hierarchical manner we envisaged that a treatment with human recombinant cysteine proteases pro-cathepsin-L (proCTSL) and pro-cathepsin-B (proCTSB) could similarly be used to induce protein degradation. Both enzymes were taken up by mannose 6-phosphate receptor- and LRP-receptor-mediated endocytosis and processed to the lysosomal mature cathepsins. In murine NCL10 astrocytes an abnormal increase in LAMP1 and saposin expression was revealed. Although proCTSB application did not improve this phenotype, proCTSL treatment led to reduced saposin-C levels in this model as well as in an acute brain slice model. Intracerebral dosing in a NCL10 mouse model revealed cellular and lysosomal uptake of both enzymes. Only proCTSL mildly reduced saposin-C levels and attenuated reactive astrogliosis. Application of both proteases did not improve weight loss and mortality of mutant mice. Our data reveal that although recombinant lysosomal proteases can be efficiently delivered to neuronal lysosomes cysteine proteases are less efficient in protein aggregates clearance as compared to the cathepsin-D treatment. Our data including in vitro degradation assays support the idea that bulk proteolysis requires a hierarchical process in which both aspartyl and cysteine hydrolases play a role.

Deletion of Von Hippel-Lindau Interferes with Hyper Osmolality Induced Gene Expression and Induces an Unfavorable Gene Expression Pattern.

Loss of von Hippel-Lindau (VHL) protein function can be found in more than 90% of patients with clear cell renal carcinoma (ccRCC). Mice lacking Vhl function in the kidneys have urine concentration defects due to postulated reduction of the hyperosmotic gradient. Hyperosmolality is a kidney-specific microenvironment and induces a unique gene expression pattern. This gene expression pattern is inversely regulated in patients with ccRCC with consequences for cancer-specific survival. Within this study, we tested the hypothesis if Vhl function influences the hyperosmolality induced changes in gene expression. We made use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to inhibit functional Vhl expression in murine collecting duct cell line. Loss of Vhl function induced morphological changes within the cells similar to epithelial to mesenchymal transition like phenotype. Vhl-deficient cells migrated faster and proliferated slower compared to control cells. Gene expression profiling showed significant changes in gene expression patterns in Vhl-deficient cells compared to control cells. Several genes with unfavorable outcomes showed induced and genes with favorable outcomes for patients with renal cancer reduced gene expression level. Under hyperosmotic condition, the expression of several hyperosmolality induced genes, with favorable prognostic value, was downregulated in cells that do not express functional Vhl. Taken together, this study shows that Vhl interferes with hyperosmotic signaling pathway and hyperosmolality affected pathways might represent new promising targets.

P4HA1: A single-gene surrogate of hypoxia signatures in oral squamous cell carcinoma patients.

BACKGROUND AND PURPOSE: Hypoxia gene expression signatures are of high prognostic value for head and neck cancer patients. Recently, the prognostic information of a multiple-gene hypoxia signature was found to be provided by the mRNA level of P4HA1 alone (Tawk et al., 2016). Therefore, we studied the prognostic value of P4HA1 in an independent cohort of oral squamous cell carcinoma (OSCC) patients. MATERIAL AND METHODS: Frozen tumor samples of 118 adult OSCC patients were analysed for P4HA1 mRNA level by quantitative real-time TaqMan™ PCR analysis. Kaplan-Meier analysis and Cox's regression analysis were performed to characterize the prognostic impact of P4HA1 mRNA level in OSCC patients. RESULTS: The analyzed patient cohort was divided into four subgroups according to the quartiles of the P4HA1 mRNA levels. The highest intratumoral P4HA1 mRNA level was significantly correlated with a poor overall survival (RR = 2.2; P = 0.04) and an increased risk of locoregional recurrence (RR = 4.8; P = 0.02). In patients who received radiotherapy (n = 82) highest intratumoral P4HA1 mRNA level was significantly correlated with a poor overall survival (RR = 3.4; P = 0.01) and an increased risk of locoregional recurrence (RR = 10.3; P = 0.005). Moreover, significant correlations between the P4HA1 mRNA level and the mRNA level of several EMT and stem cell markers were found. CONCLUSIONS: A high P4HA1 mRNA level, as a single-gene surrogate of hypoxia, is an independent prognostic marker for the overall survival and locoregional recurrence of OSCC patients.