Inhibition of cysteine protease cathepsin Lincreases the level and activity of lysosomal glucocerebrosidase.

The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L-KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L-mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans.

The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin (IrThy), which is secreted in the saliva of Ixodes ricinus ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.

Rational design of humanized antibody inhibitors for cathepsin S.

Cathepsin S (CTSS) is involved in pathogenesis of many human diseases. Inhibitors blocking its protease activity hold therapeutic potential. In comparison to small-molecule inhibitors, monoclonal antibodies capable of inhibiting CTSS enzymatic activity may possess advantageous pharmacological properties. Here we designed and produced inhibitory antibodies targeting human CTSS by genetically fusing the propeptide of procathepsin S (proCTSS) with antibodies in clinic. The resulting antibody fusions in full-length or fragment antigen-binding format could be stably expressed and potently inhibit CTSS proteolytic activity in high specificity. These fusion antibodies not only demonstrate a new approach for facile synthesis of antibody inhibitors against CTSS, but also represent novel anti-CTSS therapeutic candidates.

Whole genome identification of olive flounder (Paralichthys olivaceus) cathepsin genes: Provides insights into its regulation on biotic and abiotic stresses response.

Cathepsins are major lysosomal enzymes involved in essential physiological processes, including protein degradation, tissue differentiation, and innate or adaptive responses. Several kinds of cathepsins have been reported in teleost fishes, but no characterization have been performed for the inflammatory response of cathepsin family in olive flounder until now. In our current study, a total of 17 cathepsins in olive flounder were systematically identified and characterized. Phylogenetic analysis clearly indicated that the cathepsin genes was highly conserved. Analysis of structure and motifs exhibited high sequence similarity of cathepsin genes in olive flounder. Expression profiles of cathepsin genes in different tissues and developmental stages showed that cathepsins were temporally and spatially specific. RNA-seq analysis of bacteria and temperature stresses revealed that members of cathepsin were involved in inflammatory responses. Collectively, our findings would provide a further reference for understanding the molecular mechanisms of cathepsins in olive flounder.

Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage.

Cathepsin V (CTSV) is a cysteine cathepsin protease that plays a crucial role in extracellular matrix degradation. CTSV is correlated with poor prognosis in various cancers, but the underlying mechanism remains elusive. Here, we observed that CSTV is upregulated in lung cancer and is a poor prognosis factor for lung cancer. CTSV acts as a driver in the metastasis of lung cancer both in vitro and in vivo. CTSV promotes lung cancer metastasis by downregulating adhesion molecules, including fibronectin, E-cadherin, and N-cadherin. Our data revealed that CTSV functions by mediating the fragmentation of fibronectin, E-cadherin, and N-cadherin in cleavage, remodeling the extracellular matrix (ECM). The rationally designed antibody targeting CTSV blocks its cleaving ability towards fibronectin, E-cadherin, and N-cadherin, suppressing migration and invasion. Furthermore, we found that CTSV expression is negatively correlated with immune cell infiltration and immune scores and inhibits T cell activity. Targeting CTSV with specific antibodies effectively suppressed lung cancer metastasis in a mouse model. Our study demonstrates the critical role of CTSV in the immunity and metastasis of lung cancer, suggesting that the CTSV-targeting approach is a promising strategy for lung cancer.

NAIP/NLRC4 inflammasome participates in macrophage responses to Trypanosoma cruzi by a mechanism that relies on cathepsin-dependent caspase-1 cleavage.

Inflammasomes are large protein complexes that, once activated, initiate inflammatory responses by activating the caspase-1 protease. They play pivotal roles in host defense against pathogens. The well-established role of NAIP/NLRC4 inflammasome in bacterial infections involves NAIP proteins functioning as sensors for their ligands. However, recent reports have indicated the involvement of NLRC4 in non-bacterial infections and sterile inflammation, even though the role of NAIP proteins and the exact molecular mechanisms underlying inflammasome activation in these contexts remain to be elucidated. In this study, we investigated the activation of the NAIP/NLRC4 inflammasome in response to Trypanosoma cruzi, the protozoan parasite responsible for causing Chagas disease. This parasite has been previously demonstrated to activate NLRP3 inflammasomes. Here we found that NAIP and NLRC4 proteins are also required for IL-1ß and Nitric Oxide (NO) release in response to T. cruzi infection, with their absence rendering macrophages permissive to parasite replication. Moreover, Nlrc4 -/- and Nlrp3 -/- macrophages presented similar impaired responses to T. cruzi, underscoring the non-redundant roles played by these inflammasomes during infection. Notably, it was the live trypomastigotes rather than soluble antigens or extracellular vesicles (EVs) secreted by them, that activated inflammasomes in a cathepsins-dependent manner. The inhibition of cathepsins effectively abrogated caspase-1 cleavage, IL-1ß and NO release, mirroring the phenotype observed in Nlrc4 -/-/Nlrp3 -/- double knockout macrophages. Collectively, our findings shed light on the pivotal role of the NAIP/NLRC4 inflammasome in macrophage responses to T. cruzi infection, providing new insights into its broader functions that extend beyond bacterial infections.

M2 macrophage-derived cathepsin S promotes peripheral nerve regeneration via fibroblast-Schwann cell-signaling relay.

BACKGROUND: Although peripheral nerves have an intrinsic self-repair capacity following damage, functional recovery is limited in patients. It is a well-established fact that macrophages accumulate at the site of injury. Numerous studies indicate that the phenotypic shift from M1 macrophage to M2 macrophage plays a crucial role in the process of axon regeneration. This polarity change is observed exclusively in peripheral macrophages but not in microglia and CNS macrophages. However, the molecular basis of axonal regeneration by M2 macrophage is not yet fully understood. Herein, we aimed to identify the M2 macrophage-derived axon regeneration factor. METHODS: We established a peripheral nerve injury model by transection of the inferior alveolar nerve (IANX) in Sprague-Dawley rats. Transcriptome analysis was performed on the injured nerve. Recovery from sensory deficits in the mandibular region and histological reconnection of IAN after IANX were assessed in rats with macrophage depletion by clodronate. We investigated the effects of adoptive transfer of M2 macrophages or M2-derived cathepsin S (CTSS) on the sensory deficit. CTSS initiating signaling was explored by western blot analysis in IANX rats and immunohistochemistry in co-culture of primary fibroblasts and Schwann cells (SCs). RESULTS: Transcriptome analysis revealed that CTSS, a macrophage-selective lysosomal protease, was upregulated in the IAN after its injury. Spontaneous but partial recovery from a sensory deficit in the mandibular region after IANX was abrogated by macrophage ablation at the injured site. In addition, a robust induction of c-Jun, a marker of the repair-supportive phenotype of SCs, after IANX was abolished by macrophage ablation. As in transcriptome analysis, CTSS was upregulated at the injured IAN than in the intact IAN. Endogenous recovery from hypoesthesia was facilitated by supplementation of CTSS but delayed by pharmacological inhibition or genetic silencing of CTSS at the injured site. Adoptive transfer of M2-polarized macrophages at this site facilitated sensory recovery dependent on CTSS in macrophages. Post-IANX, CTSS caused the cleavage of Ephrin-B2 in fibroblasts, which, in turn, bound EphB2 in SCs. CTSS-induced Ephrin-B2 cleavage was also observed in human sensory nerves. Inhibition of CTSS-induced Ephrin-B2 signaling suppressed c-Jun induction in SCs and sensory recovery. CONCLUSIONS: These results suggest that M2 macrophage-derived CTSS contributes to axon regeneration by activating SCs via Ephrin-B2 shedding from fibroblasts.

Protease-bound structure of Ricistatin provides insights into the mechanism of action of tick salivary cystatins in the vertebrate host.

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

Remarkable Effects of a Rhenium(I)-diselenoether Drug on the Production of Cathepsins B and S by Macrophages and their Polarizations.

BACKGROUND/OBJECTIVE: Tumor-associated macrophages (TAMs) produce an excessive amount of cysteine proteases, and we aimed to study the effects of anticancer rhenium(I)-diselenoether (Re-diSe) on the production of cathepsins B and S by macrophages. We investigated the effect of Re-diSe on lipopolysaccharides (LPS) induced M1 macrophages, or by interleukin 6 (IL-6) induced M2 macrophages. METHODS: Non-stimulated or prestimulated murine Raw 264 or human THP-1 macrophages were exposed to increasing concentrations of the drug (5, 10, 20, 50 and 100 µM) and viability was assayed by the MTT assay. The amount of cysteine proteases was evaluated by ELISA tests, the number of M1 and M2 macrophages by the expression of CD80 or CD206 biomarkers. The binding of Re-diSe with GSH as a model thiol-containing protein was studied by mass spectrometry. RESULTS: A dose-dependent decrease in cathepsins B and S was observed in M1 macrophages. There was no effect in non-stimulated cells. The drug induced a dramatic dose-dependent increase in M1 expression in both cells, significantly decreased the M2 expression in Raw 264 and had no effect in non-stimulated macrophages. The binding of the Re atom with the thiols was clearly demonstrated. CONCLUSION: The increase in the number of M1 and a decrease in M2 macrophages treated by Re-diSe could be related to the decrease in cysteine proteases upon binding of their thiol residues with the Re atom.

Targeted delivery of cathepsin-activatable near-infrared fluorescence probe for ultrahigh specific imaging of peritoneal metastasis.

Inadequate cytoreductive surgery (CRS) has been identified as a prognostic factor for poor patient outcomes in cases of peritoneal metastasis. While imaging probes are used to identify peritoneal metastasis to facilitate CRS, many of these probes exhibit high background signals, resulting in a significant delay in achieving a satisfactory tumor-to-normal ratio (TNR) due to prolonged clearance time. In this study, we designed a novel fluorescent probe named Tras-AA-Cy NH2, which enables the relatively rapid imaging of subcutaneous tumors and peritoneal tumors while maintaining a high TNR. Mechanistically, Tras-AA-Cy NH2 exhibits selective targeting towards the Human epidermal growth factor receptor 2 on the surface of cancer cells. Following internalization, it undergoes enzymatic cleavage catalyzed by the overexpressed cathepsin, leading to the subsequent release of near-infrared fluorophores. Consequently, Tras-AA-Cy NH2 achieved a TNR of 7.8 at 6 h and 21.4 at 24 h in subcutaneous tumor mice. Even after 522 h of in vivo circulation, the TNR remained above 5, indicating an ultralong imaging time window. It is noteworthy that Tras-AA-Cy NH2 has demonstrated successful utilization for peritoneal tumor-specific imaging and further affirmed its tumor tissue-specific recognition capability using human resected tissues. In summary, these findings underscore the rational design of Tras-AA-Cy NH2 for visualizing peritoneal tumors.

An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E.

The lack of suitable in vitro culture model has hampered research on wild-type (WT) human coronaviruses. While 3D tissue or organ cultures have been instrumental for this purpose, such models are challenging, time-consuming, expensive and require extensive cell culture adaptation and directed evolution. Consequently, high-throughput applications are beyond reach in most cases. Here we developed a robust A549 cell line permissive to a human coronavirus 229E (HCoV-229E) clinical isolate by transducing CD13 and transmembrane serine protease 2 (TMPRSS2), henceforth referred to as A549++ cells. This modification allowed for productive infection, and a more detailed analysis showed that the virus might use the TMPRSS2-dependent pathway but can still bypass this pathway using cathepsin-mediated endocytosis. Overall, our data showed that A549++ cells are permissive to HCoV-229E clinical isolate, and applicable for further studies on HCoV-229E infectiology. Moreover, this line constitutes a uniform platform for studies on multiple members of the Coronaviridae family.

Augmentation of Cathepsin Isoforms in Diabetic db/db Mouse Kidneys Is Associated with an Increase in Renal MARCKS Expression and Proteolysis.

The expression of the myristoylated alanine-rich C-kinase substrate (MARCKS) family of proteins in the kidneys plays an important role in the regulation of the renal epithelial sodium channel (ENaC) and hence overall blood pressure regulation. The function of MARCKS is regulated by post-translational modifications including myristoylation, phosphorylation, and proteolysis. Proteases known to cleave both ENaC and MARCKS have been shown to contribute to the development of high blood pressure, or hypertension. Here, we investigated protein expression and proteolysis of MARCKS, protein expression of multiple protein kinase C (PKC) isoforms, and protein expression and activity of several different proteases in the kidneys of diabetic db/db mice compared to wild-type littermate mice. In addition, MARCKS protein expression was assessed in cultured mouse cortical collecting duct (mpkCCD) cells treated with normal glucose and high glucose concentrations. Western blot and densitometric analysis showed less abundance of the unprocessed form of MARCKS and increased expression of a proteolytically cleaved form of MARCKS in the kidneys of diabetic db/db mice compared to wild-type mice. The protein expression levels of PKC delta and PKC epsilon were increased, while cathepsin B, cathepsin S, and cathepsin D were augmented in diabetic db/db kidneys compared to those of wild-type mice. An increase in the cleaved form of MARCKS was observed in mpkCCD cells cultured in high glucose compared to normal glucose concentrations. Taken together, these results suggest that high glucose may contribute to an increase in the proteolysis of renal MARCKS, while the upregulation of the cathepsin proteolytic pathway positively correlates with increased proteolysis of MARCKS in diabetic kidneys, where PKC expression is augmented.

The role of lysosomal peptidases in glioma immune escape: underlying mechanisms and therapeutic strategies.

Glioblastoma is the most common primary malignant tumor of the central nervous system, which has the characteristics of strong invasion, frequent recurrence, and rapid progression. These characteristics are inseparable from the evasion of glioma cells from immune killing, which makes immune escape a great obstacle to the treatment of glioma, and studies have confirmed that glioma patients with immune escape tend to have poor prognosis. The lysosomal peptidase lysosome family plays an important role in the immune escape process of glioma, which mainly includes aspartic acid cathepsin, serine cathepsin, asparagine endopeptidases, and cysteine cathepsins. Among them, the cysteine cathepsin family plays a prominent role in the immune escape of glioma. Numerous studies have confirmed that glioma immune escape mediated by lysosomal peptidases has something to do with autophagy, cell signaling pathways, immune cells, cytokines, and other mechanisms, especially lysosome organization. The relationship between protease and autophagy is more complicated, and the current research is neither complete nor in-depth. Therefore, this article reviews how lysosomal peptidases mediate the immune escape of glioma through the above mechanisms and explores the possibility of lysosomal peptidases as a target of glioma immunotherapy.

Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range.

The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is, therefore, desirable to develop specific substrates for cathepsin B that measure its activity over broad pH ranges. Current substrates used to monitor cathepsin B activity consist of Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, but they lack specificity since they are cleaved by other cysteine cathepsins. Furthermore, Z-Arg-Arg-AMC monitors cathepsin B activity at neutral pH and displays minimal activity at acidic pH. Therefore, the purpose of this study was to design and validate specific fluorogenic peptide substrates that can monitor cathepsin B activity over a broad pH range from acidic to neutral pH conditions. In-depth cleavage properties of cathepsin B were compared to those of the cysteine cathepsins K, L, S, V, and X via multiplex substrate profiling by mass spectrometry at pH 4.6 and pH 7.2. Analysis of the cleavage preferences predicted the tripeptide Z-Nle-Lys-Arg-AMC as a preferred substrate for cathepsin B. Significantly, Z-Nle-Lys-Arg-AMC displayed the advantageous properties of measuring high cathepsin B specific activity over acidic to neutral pHs and was specifically cleaved by cathepsin B over the other cysteine cathepsins. Z-Nle-Lys-Arg-AMC specifically monitored cathepsin B activity in neuronal and glial cells which were consistent with relative abundances of cathepsin B protein. These findings validate Z-Nle-Lys-Arg-AMC as a novel substrate that specifically monitors cathepsin B activity over a broad pH range.

Cathepsin S deficiency improves muscle mass loss and dysfunction via the modulation of protein metabolism in mice under pathological stress conditions.

Cathepsin S (CTSS) is a widely expressed cysteinyl protease that has garnered attention because of its enzymatic and non-enzymatic functions under inflammatory and metabolic pathological conditions. Here, we examined whether CTSS participates in stress-related skeletal muscle mass loss and dysfunction, focusing on protein metabolic imbalance. Eight-week-old male wildtype (CTSS+/+ ) and CTSS-knockout (CTSS-/- ) mice were randomly assigned to non-stress and variable-stress groups for 2 weeks, and then processed for morphological and biochemical studies. Compared with non-stressed mice, stressed CTSS+/+ mice showed significant losses of muscle mass, muscle function, and muscle fiber area. In this setting, the stress-induced harmful changes in the levels of oxidative stress-related (gp91phox and p22phox ,), inflammation-related (SDF-1, CXCR4, IL-1ß, TNF-α, MCP-1, ICAM-1, and VCAM-1), mitochondrial biogenesis-related (PPAR-γ and PGC-1α) genes and/or proteins and protein metabolism-related (p-PI3K, p-Akt, p-FoxO3α, MuRF-1, and MAFbx1) proteins; and these alterations were rectified by CTSS deletion. Metabolomic analysis revealed that stressed CTSS-/- mice exhibited a significant improvement in the levels of glutamine metabolism pathway products. Thus, these findings indicated that CTSS can control chronic stress-related skeletal muscle atrophy and dysfunction by modulating protein metabolic imbalance, and thus CTSS was suggested to be a promising new therapeutic target for chronic stress-related muscular diseases.

Cathepsins and SARS-CoV-2 infection: From pathogenic factors to potential therapeutic targets.

Coronavirus disease-19 (COVID-19) is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. The COVID-19 pandemic began in March 2020 and has wrought havoc on health and economic systems worldwide. Efficacious treatment for COVID-19 is lacking: Only preventive measures as well as symptomatic and supportive care are available. Preclinical and clinical studies have indicated that lysosomal cathepsins might contribute to the pathogenesis and disease outcome of COVID-19. Here, we discuss cutting-edge evidence on the pathological roles of cathepsins in SARS-CoV-2 infection, host immune dysregulations, and the possible underlying mechanisms. Cathepsins are attractive drug targets because of their defined substrate-binding pockets, which can be exploited as binding sites for pharmaceutical enzyme inhibitors. Accordingly, the potential modulatory strategies of cathepsin activity are discussed. These insights could shed light on the development of cathepsin-based interventions for COVID-19.

Enhancement of Inhibitory Activity by Combining Allosteric Inhibitors Putatively Binding to Different Allosteric Sites on Cathepsin K.

BACKGROUND: Cathepsin K, which is involved in bone resorption, is a good target for treating osteoporosis, but no clinically approved medicine has been developed. Recently, allosteric inhibitors with high specificity and few side effects have been attracting attention for use in new medicines. METHODS: Cathepsin K inhibitors were isolated from the methanol extract of Chamaecrista nomame (Leguminosae) using cathepsin K inhibition activity-assisted multi-step chromatography. Standard kinetic analysis was employed to examine the mechanism of cathepsin K inhibition when an isolated inhibitor and its derivative were used. The allosteric binding of these cathepsin K inhibitors was supported by a docking study using AutoDock vina. Combinations of allosteric cathepsin K inhibitors expected to bind to different allosteric sites were examined by means of cathepsin K inhibition assay. RESULTS: Two types of cathepsin K inhibitors were identified in the methanol extract of Chamaecrista nomame. One type consisted of cassiaoccidentalin B and torachrysone 8-ß-gentiobioside, and inhibited both cathepsin K and B with similar inhibitory potential, while the other type of inhibitor consisted of pheophytin a, and inhibited cathepsin K but not cathepsin B, suggesting that pheophytin a binds to an allosteric site of cathepsin K. Kinetic analysis of inhibitory activity suggested that pheophytin a and its derivative, pheophorbide b, bind allosterically to cathepsin K. This possibility was supported by a docking study on cathepsin K. The cathepsin K inhibitory activity of pheophytin a and pheophorbide b was enhanced by combining them with the allosteric inhibitors NSC 13345 and NSC94914, which bind to other allosteric sites on cathepsin K. CONCLUSIONS: Different allosteric inhibitors that bind to different sites in combination, as shown in this study, may be useful for designing new allosteric inhibitory drugs with high specificity and few side effects.

Serum Cathepsin S Levels Do Not Show Alterations in Different Clinical, Neuropathological, or Genetic Subtypes of Frontotemporal Dementia Patients nor in Comparison to Healthy Control Individuals.

Frontotemporal dementia (FTD) can manifest as diverse clinical phenotypes and is frequently caused by mutations in different genes, complicating differential diagnosis. This underlines the urgent need for valid biomarkers. Altered lysosomal and immune functions proposedly contribute to FTD pathogenesis. Cathepsins, including cathepsin S, are enzymes preferentially expressed in brain in microglia, which influence lysosomal and immune function. Here, we examined whether alterations in serum cathepsin S levels associate with specific clinical, genetic, or neuropathological FTD subgroups, but no such alterations were observed. However, further research on other lysosomal proteins may reveal new biologically relevant biomarkers in FTD.

Cathepsin-L Secreted by High-Quality Bovine Embryos Exerts an Embryotrophic Effect In Vitro.

While human in vitro embryo production is generally performed individually, animal models have shown that culturing embryos in groups improves blastocyst yield and quality. Paracrine embryotrophins could be responsible for this improved embryo development, but their identity remains largely unknown. We hypothesize that supplementation of embryotrophic proteins to a culture medium could be the key to improve individual embryo production. In this study, proteomics screening of culture media conditioned by bovine embryos revealed cathepsin-L as being secreted by both excellent- and good-quality embryos, while being absent in the medium conditioned by poor-quality embryos. The embryotrophic role of cathepsin-L was explored in vitro, whereby bovine zygotes were cultured individually for 8 days with or without cathepsin-L. Preliminary dose-response experiments pointed out 100 ng/mL as the optimal concentration of cathepsin-L in embryo culture medium. Supplementation of cathepsin-L to individual culture systems significantly improved blastocyst development and quality in terms of blastocoel formation at day 7, and the hatching ratio and apoptotic cell ratio at day 8, compared to the control. Taken together, cathepsin-L acts as an important embryotrophin by increasing embryo quality, and regulating blastulation and hatching in bovine in vitro embryo production.