Glial cells, namely microglia, astrocytes, and oligodendrocytes, play crucial roles in maintaining homeostasis in the central nervous system and orchestrating responses to injury, infection, and disease. Among the molecular regulators of glial function, cysteine cathepsins have emerged as key modulators of both physiological and pathological processes. These lysosomal peptidases are traditionally known for their housekeeping roles in protein degradation; however, accumulating evidence highlights their broader involvement in antigen presentation, microglial and astrocyte reactivity, inflammatory signalling, apoptosis, and myelination. Under normal conditions, cysteine cathepsins support essential functions in the central nervous system, including immune surveillance and tissue remodelling. Conversely, their dysregulation, characterized by overexpression, increased enzymatic activity, or mislocalization, can promote neuroinflammation and neurodegeneration, contributing to the pathogenesis of disorders such as Alzheimer’s disease and multiple sclerosis. This review provides a comprehensive synthesis specifically focused on the diverse roles of cysteine cathepsins across major glial cell types, systematically summarizing current knowledge in microglia, astrocytes, and oligodendrocytes. We emphasize their cell type-specific, context-dependent, protective, and deleterious functions. Furthermore, we discuss mechanistic links between cysteine cathepsin activity and neurodegenerative processes and evaluate the therapeutic potential and current limitations of selectively targeting glial cysteine cathepsins. A deeper understanding of the context-dependent dual roles of these enzymes in brain physiology and pathology is critical for designing targeted interventions that could mitigate neuroinflammation and neurodegeneration.

Neuronal differentiation into specific subtypes is crucial for nervous system development and function, guided by neurotrophic factors. γ-Enolase, a neuron-specific glycolytic enzyme, exhibits neurotrophic-like properties and supports neuronal differentiation; however, its role in specific neuronal subtypes remains unknown. Here, we investigate the role of γ-enolase in differentiation dopaminergic-, cholinergic-, and adrenergic-like neuronal cells. Our results demonstrate that γ-enolase expression is significantly upregulated in differentiated cells, with the highest expression observed in cholinergic-like neurons. Full-length γ-enolase, compared to its truncated form, promoted enhanced neurite outgrowth and increased β-tubulin, a cytoskeletal marker. Conversely, silencing endogenous γ-enolase significantly reduced neurite length, confirming its essential role in driving neuronal morphological maturation. Furthermore, a γ-enolase-derived peptide corresponding to the active C-terminus of γ-enolase significantly promoted neurite outgrowth and increased β-tubulin expression, particularly in cholinergic-like neuronal cells. Notably, γ-enolase activity is regulated by cathepsin X, a lysosomal peptidase that cleaves γ-enolase at its C-terminus, reducing its neurotrophic effects. Confocal microscopy revealed increased co-localization of γ-enolase and cathepsin X in differentiated neuronal cells, emphasizing their interaction in cholinergic-like neurons. Inhibiting cathepsin X preserved active γ-enolase, promoted neuronal differentiation, and altered cytoskeletal marker expression. These findings suggest an important role for γ-enolase in cholinergic-like neuronal cells and propose cathepsin X as a regulatory modulator of γ-enolase activity, suggesting novel therapeutic strategies for neuroregeneration.

Abstract Cysteine cathepsins are increasingly recognized as important regulators of lysosomal function and autophagy, two processes that help cancer cells survive under treatment-induced stress. In pancreatic ductal adenocarcinoma (PDAC), where gemcitabine is still widely used as first-line chemotherapy, growing evidence indicates that drug-induced autophagy contributes to the development of resistance. In this study, we asked whether autophagy triggered by gemcitabine depends on cysteine cathepsins and whether blocking these proteases can improve gemcitabine’s antitumor activity. We focused on cathepsins L, B, and V, major lysosomal proteases with established roles in proteolysis and emerging functions in survival signalling. Using three PDAC cell lines with distinct genetic backgrounds and chemosensitivity (PANC1, Capan2, BxPC3), we treated cells with sublethal LC30 and LC60 concentrations of gemcitabine to trigger stress responses without inducing extensive cell death, thereby allowing us to dissect adaptive autophagy from direct cytotoxicity. Using western blot analysis and immunocytochemistry, we examined expression, processing, and subcellular localization of cathepsins and key autophagy markers, and we quantified autophagic flux and lysosomal function in the presence or absence of selective inhibitors of cathepsins V, L, and B. In parallel, cell viability and apoptosis assays were used to assess treatment responses, and 3D patient-derived organoids and NK cell co-culture systems were employed to validate our findings in more physiologically relevant models. Our results show that gemcitabine-induced autophagy is at least partly dependent on cysteine cathepsin activity, and that pharmacologic inhibition of these enzymes interferes with autophagic processing and makes PDAC cells more sensitive to gemcitabine. We confirmed these findings in patient-derived organoids, where the combination of gemcitabine and cathepsin inhibition reduced tumour cell viability more effectively than either treatment alone. In addition, we tested how this combination affects immune recognition and found that natural killer (NK) cell–mediated killing was increased in gemcitabine- and cathepsin inhibitor–treated tumour cells. Overall, our data identify cathepsins L, B, and V as key modulators of gemcitabine-induced autophagy and immune susceptibility in PDAC and support their further exploration as therapeutic targets to overcome chemoresistance and improve clinical responses. Citation Format: Nika Mazej Jeram, Biljana Mileva Mileva Boshkoska, Aleš Tomažič, Stanislav Gobec, Damijan Knez, Janko Kos, Milica M. Perisic Nanut. Targeting cysteine cathepsins to boost gemcitabine in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7405.

Amyloid-β peptide (Aβ), a hallmark peptide in the pathology of Alzheimer's disease, together with the amyloid-β protein precursor, is increasingly associated with the disruption of cell adhesion. In addition to its well-characterized role in plaque formation and synaptic dysfunction, Aβ interacts with various adhesion molecules and extracellular matrix components, thereby impairing neuronal connectivity and integrity. We have shown that pretreatment of SH-SY5Y cells with Aβ42 fibrils affects cell adhesion; however, we did not observe this effect with Aβ42 monomers. Understanding the molecular mechanisms by which Aβ fibrils disrupt cell adhesion pathways may reveal new therapeutic approaches to prevent disease progression.

Pancreatic cancer remains one of the most lethal malignancies worldwide, with pancreatic ductal adenocarcinoma accounting for the vast majority of cases and characterized by extensive desmoplasia, immune exclusion, and resistance to systemic therapies. Increasing evidence implicates lysosomal cathepsins as important regulators of these defining features of pancreatic tumor biology. Cathepsin-dependent proteolysis and lysosome-associated signaling pathways contribute to extracellular matrix remodeling, regulate immune cell trafficking, and influence antigen processing and presentation. Beyond their classical degradative functions, cathepsins participate in stress-adaptive cellular programs linked to autophagy, metabolic regulation, and proteostasis, supporting tumor cell survival under hypoxic, nutrient-limited, and therapy-induced stress conditions. Within the tumor microenvironment, dysregulated cathepsin activity promotes immune evasion by reshaping cytokine networks, impairing effective antigen presentation, and reinforcing physical and functional barriers to cytotoxic T-cell infiltration. Collectively, these mechanisms position the lysosome–cathepsin system as a central regulator of proteolytic remodeling, immune exclusion, and adaptive therapy resistance in pancreatic cancer, highlighting its potential relevance for emerging combinatorial therapeutic strategies.

A significant amount of data about the different pharmacological activities of the established antimicrobial compound nitroxoline (8-hydroxy-5-nitroquinoline) is available in the scientific literature. On the other hand, its regioisomer 8-hydroxy-6-nitroquinoline was never characterised biochemically and the same also applies to their 1,2,3,4-tetrahydroquinoline analogues. Herein, we determined the influence of pyridine ring saturation and the position of the nitro group on various biochemical characteristics of compounds, such as metal-chelating properties, inhibition of methionine aminopeptidases (MetAPs) from Mycobacterium tuberculosis and human MetAP2, as well as antibacterial activities on Escherichia coli, Staphylococcus aureus, and Mycobacterium smegmatis. In addition, inhibition of endopeptidase and exopeptidase activities of cathepsin B was determined, together with the ability of new nitroxo-line analogues to reduce intracellular collagen IV degradation. Substantially different biological activities were observed for the 6-nitro regioisomer of nitroxoline, as well as for both of their partially saturated counterparts.

Although immune checkpoint inhibitors (ICI) have achieved undeniable success in the treatment of metastatic melanoma, primary and secondary resistance to ICI therapy continues to pose a significant challenge for melanoma patients. Around 40-60% of patients respond to immunotherapy, achieving long-term benefits, but over two-thirds experience immune-related adverse events (irAEs) with ICIs. About 10% show pseudoprogression, marked by transient inflammation, while up to 30% experience hyperprogression with rapid cancer advancement. Despite irAEs, treatment interruption often leads to better outcomes and prolonged survival. The lack of clinically validated predictive biomarkers remains a major factor contributing to the unpredictable effects of immunotherapy. Implementing biological markers in clinical practice is essential to enhance personalized treatment and predict responses. We have identified a protease inhibitor cystatin F as a potent down regulator of cytotoxic efficacy of cytotoxic T lymphocytes (CTLs). Its expression was shown to be increased in hypo responsive and exhausted CTLs but also in the cancer tissue in several types of cancer. In order to elucidate its role in CTLs of patients with melanoma and correlation its expression with response to treatment we have examined cystatin F expression, along with the CTL PD-1 expression and IFNγ in peripheral blood CTLs form patients with advanced melanoma (TNM classification, stage IIID and IV) receiving first-line therapy with ICIs. Study-specific data were collected at three different time points: up to 4 weeks before the start of treatment, at the 12th (+/−2 weeks) and 28th week (+/−2 weeks) and the expression of markers on whole blood preparations or isolated CTLs was analysed using flow cytometry. We characterized the cellular sources of cystatin F in tumor tissue using multicolor immunofluorescence staining and confocal analysis on formalin-fixed melanoma tissue samples. Our findings reveal cystatin F expression in predominantly in non-lymphoid cells within the tumor microenvironment. Analysis of cystatin F in peripheral blood CTLs shows higher expression in non-responders and patients with irAEs, suggesting its potential as a marker for enhancing personalized treatment and predicting treatment responses. Milica M. Perisic Nanut, Emanuela Senjor, Tanja Mesti, Simona Miceska, Janja Ocvirk, Janko Kos. Cystatin F changes in melanoma patients during therapy with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3215.

Research on rhenium complexes containing fac-tricarbonyl fragments has been on the rise in recent decades. Some complexes of this type exhibit advantageous properties that can be utilized in diagnostic and therapeutic applications. Herein, we report on the synthesis, structural characterization, solution speciation, and biological activity with mode of action studies of a new fac-tricarbonylrhenium-(I) complex with pyrithione ligand fac-[Re-(CO)3(pyrithionato)-(benzonitrile)] (4). In an attempt to prepare a stable rhenium-(I) complex with a pyrithionato ligand, several synthesis procedures were investigated and various products were discovered. In solution, the monodentate benzonitrile ligand can be replaced by a coordinating solvent molecule; however, the carbonyl ligands and pyrithione remain bound to the rhenium center. Complex 4 exhibited strong cytotoxic and antibacterial activity and effectively inhibited the growth of the HSV-2 virus. Additionally, complex 4 was also able to inhibit the cathepsin B enzyme (both its endo- and exopeptidase activities). In silico experiments confirmed that complex 4 can interact with cathepsin B near its active site, which may contribute to reduced enzymatic activity.

The glycolytic enzyme γ-enolase is a highly specific neuronal marker that is known to replace ubiquitously expressed α-enolase in the brain. Moreover, γ-enolase has been shown to exert neurotrophic activity, which is regulated by cathepsin X, a lysosomal peptidase. This study investigates the role of γ-enolase and its regulation by cathepsin X during the differentiation of oligodendrocytes, which are essential for normal brain function. We established a differentiation protocol for the human oligodendroglioma (HOG) cell line and demonstrated for the first time that an α- to γ-enolase switch occurs during HOG cell differentiation. This switch was confirmed by the expression of specific markers underscoring the role of γ-enolase in oligodendrocyte differentiation. Moreover, γ-enolase overexpression enhanced oligodendrocyte differentiation, while silencing of γ-enolase by siRNA significantly decreased maturation marker. Further, the regulatory role of cysteine peptidase cathepsin X on γ-enolase function was found. Silencing cathepsin X significantly changed cell morphology, enhanced oligodendrocyte differentiation, altered the expression of oligodendrocyte markers, and increased levels of the active form of γ-enolase. Inhibiting cathepsin X similarly changed cell morphology and enhanced oligodendrocyte differentiation. These findings suggest that cathepsin X modulates γ-enolase activity and thereby influences oligodendrocyte differentiation and thus neuronal function.

Objective Natural killer (NK) cells are the largest innate lymphocyte subset with potent antitumour and antiviral functions. However, clinical utilisation of human NK cells is hampered due to a lack of reliable methods to augment their antitumour potential. We demonstrated technology in which human NK cells were cocultured with osteoclasts in the presence of probiotic bacteria. This approach significantly augmented the antitumour cytotoxicity and polyfunctionality of human NK cells, resulting in the generation of supercharged NK (sNK) cells.Methods and analysis We explored the proteomic, transcriptomic and functional characterisation of sNK cells using cell imaging, flow cytometric analysis, 51-chromium release cytotoxicity assay, ELISA, ELIspot, IsoPLexis single-cell secretome analysis, proteomic analysis, RNA analysis, western blot and enzyme kinetics.Results We found that sNK cells were less susceptible to split anergy and tumour-induced exhaustion. Proteomic analyses revealed that sNK cells significantly increased their cell motility and proliferation. Single-cell transcriptomes uncovered sNK cells undertaking a unique differentiation trajectory and turning on STAT1, JUN, BHLHE40, ELF1, MAX and MYC regulons essential for augmenting antitumour effector functions and proliferation, respectively. Both proteomic and single-cell transcriptomes revealed that an increase in Cathepsin C helped to augment the quantity and function of Granzyme B.Conclusions These results support that this unique method produces potent NK cells for clinical utilisation and delineate the molecular mechanisms associated with this process.

Enolase is well-known for its role in glycolysis but also plays other roles in the central nervous system, including neuronal survival, differentiation, and axonal regeneration. Here, we investigated α- and γ-enolase expression patterns and their association with cathepsin X in distinct SH-SY5Y cell phenotypes. Enriched substrate-adherent S-type cells are characterized by large, flat morphology with extensive cytoplasm and higher expression of vimentin, while neuroblastic N-type are recognized by neurite extensions and higher expression of B-cell lymphoma 2 (Bcl-2) and growth-associated protein-43. We demonstrated that γ-enolase expression was specific to N-type cells, whereas α-enolase expression was not phenotype-specific. Moreover, a shift from ubiquitously expressed α-enolase to neuron-specific γ-enolase was observed during the enrichment and differentiation. Additionally, cathepsin X exhibited higher proteolytic activity in S-type cells. Inhibition of cathepsin X with AMS36 promoted differentiated cell morphology and increased expression of the active form of γ-enolase. Furthermore, AMS36 altered the expression of vimentin and Bcl-2, indicating a regulatory role in neuronal differentiation. Furthermore, AMS36 activated extracellular signal-regulated kinase 1/2 in N-type cells and enhanced the association between γ-enolase and tyrosine receptor kinase in both, suggesting a link between cathepsin X/γ-enolase and the key signaling pathways of differentiation. Our findings underscore the multifaceted role of enolase isoforms in SH-SY5Y cell differentiation, with α-enolase and γ-enolase showing distinct expression patterns in S- and N-type cells. The expression and activity of cathepsin X in S-type cells, along with its regulatory impact on γ-enolase in N-type cells, highlight the importance of these proteins in neuronal differentiation. The roles of α-enolase, γ-enolase, and cathepsin X in enriched and differentiated SH-SY5Y cell populations. The enrichment and differentiation of SH-SY5Y cells resulted in two distinct cell phenotypes: S-type and N-type cells. S-type cells were characterized by an epithelial-like morphology, the presence of vimentin, lower γ-enolase expression, and higher cathepsin X expression. N-type cells were characterized by a neuron-like morphology, GAP-43 and Bcl-2 expression, higher γ-enolase expression, and lower cathepsin X expression. Both phenotypes expressed α-enolase. The cathepsin X inhibitor AMS36 promoted SH-SY5Y cell differentiation and enrichment into S- and N-type cells. AMS36-treated S-type cells exhibited decreased vimentin levels and increased active γ-enolase levels, indicating enhanced differentiation. AMS36-treated N-type cells exhibited decreased Bcl-2 levels, indicating further differentiation. These results highlight the differential protein expression and activity between S- and N-type cells. Furthermore, they highlight the modulatory effects of AMS36, emphasizing its potential role in promoting differentiation and altering protein expression profiles.

IgA vasculitis nephritis (IgAVN) manifests in up to 84

IntroductionGlioblastoma (GBM) is a highly invasive brain tumor with limited treatment options and poor prognosis. Natural killer (NK) cells are key effectors of antitumor immunity, capable of eliminating cancer stem-like cells. However, GBM creates an immunosuppressive microenvironment that limits NK cell function. Here, we identify cystatin F as an immunosuppressive factor involved in regulating NK cell granule-mediated cytotoxicity.MethodsWe analyzed cystatin F expression in GBM and its correlation with immune exhaustion markers. NK cell activity was compared between GBM patients and healthy donors. In vitro co-cultures of cystatin F-expressing microglial cells and glioblastoma stem-like cells were used to assess NK cell function. To block cystatin F activation from dimeric to active monomeric form, a small-molecule inhibitor of cathepsin V, the activating protease, was applied.ResultsCystatin F expression correlated with immune exhaustion and suppression markers in GBM. NK cells from patients showed reduced cytotoxicity compared to healthy donors. Co-cultures confirmed that cystatin F-expressing microglia impaired NK cell cytotoxicity, while inhibition of cathepsin V restored NK cell function in standard cytotoxicity assays, 3D spheroids, and microfluidic perfused models.DiscussionThese results indicate that cystatin F mediates NK cell suppression in GBM. Targeting its activation enhances NK cell cytotoxicity, offering a potential strategy to improve NK-based immunotherapy for glioblastoma.

Enolase, a multifunctional protein with diverse isoforms, has generally been recognized for its primary roles in glycolysis and gluconeogenesis. The shift in isoform expression from α-enolase to neuron-specific γ-enolase extends beyond its enzymatic role. Enolase is essential for neuronal survival, differentiation, and the maturation of neurons and glial cells in the central nervous system. Neuron-specific γ-enolase is a critical biomarker for neurodegenerative pathologies and neurological conditions, not only indicating disease but also participating in nerve cell formation and neuroprotection and exhibiting neurotrophic-like properties. These properties are precisely regulated by cysteine peptidase cathepsin X and scaffold protein γ1-syntrophin. Our findings suggest that γ-enolase, specifically its C-terminal part, may offer neuroprotective benefits against neurotoxicity seen in Alzheimer's and Parkinson's disease. Furthermore, although the therapeutic potential of γ-enolase seems promising, the effectiveness of enolase inhibitors is under debate. This paper reviews the research on the roles of γ-enolase in the central nervous system, especially in pathophysiological events and the regulation of neurodegenerative diseases.

Cathepsins, a family of lysosomal peptidases, play a crucial role in maintaining cellular homeostasis by regulating protein turnover and degradation as well as many specific regulatory actions that are important for proper cell function and human health. Alterations in the activity and expression of cathepsins have been observed in many diseases such as cancer, inflammation, neurodegenerative disorders, bone remodelling-related conditions and others. These changes are not exclusively harmful, but rather appear to be a compensatory response on the lack of one cathepsin in order to maintain tissue integrity. The upregulation of specific cathepsins in response to the inhibition or dysfunction of other cathepsins suggests a fine-tuned system of proteolytic balance and understanding the compensatory role of cathepsins may improve therapeutic potential of cathepsin's inhibitors. Selectively targeting one cathepsin or modulating their activity could offer new treatment strategies for a number of diseases. This review emphasises the need for comprehensive research into cathepsin biology in the context of disease. The identification of the specific cathepsins involved in compensatory responses, the elucidation of the underlying molecular mechanisms and the development of targeted interventions could lead to innovative therapeutic approaches.

We describe the development of quinolylnitrones（QNs） as multifunctional ligands inhibiting cholinesterases（ChEs: acetylcholinesterase and butyrylcholinesterase—h BChE） and monoamine oxidases（hMAO-A/B） for the therapy of neurodegenerative diseases. We identified QN 19, a simple, low molecular weight nitrone, that is readily synthesized from commercially available 8-hydroxyquinoline-2-carbaldehyde. Quinolylnitrone 19 has no typical pharmacophoric element to suggest ChE or MAO inhibition, yet unexpectedly showed potent inhibition of h BChE（IC50= 1.06 ± 0.31 nmol/L） and h MAO-B(IC 50 =4.46±0.18 μmol/L). The crystal structures of 19 with hBChE and hMAO-B provided the structural basis for potent binding, which was further studied by enzyme kinetics. Compound 19 acted as a free radical scavenger and biometal chelator, crossed the blood—brain barrier, was not cytotoxic, and showed neuroprotective properties in a 6-hydroxydopamine cell model of Parkinson’s disease. In addition, in vivo studies showed the anti-amnesic effect of 19 in the scopolamine-induced mouse model of AD without adverse effects on motoric function and coordination. Importantly, chronic treatment of double transgenic APPswe-PS1δE9 mice with 19 reduced amyloid plaque load in the hippocampus and cortex of female mice, underscoring the disease-modifying effect of QN 19.

Introduction and methods:In this study we report that sequential treatment of supercharged NK (sNK) cells with either chemotherapeutic drugs or check-point inhibitors eliminate both poorly differentiated and well differentiated tumors in-vivo in humanized-BLT mice.Background and results:sNK cells were found to be a unique population of activated NK cells with genetic, proteomic, and functional attributes that are very different from primary untreated or IL-2 treated NK cells. Furthermore, NK-supernatant differentiated or well-differentiated oral or pancreatic tumor cell lines are not susceptible to IL-2 activated primary NK cell-mediated cytotoxicity; however, they are greatly killed by the CDDP and paclitaxel in in-vitro assays. Injection of one dose of sNK cells at 1 million cells per mouse to aggressive CSC-like/poorly differentiated oral tumor bearing mice, followed by an injection of CDDP, inhibited tumor weight and growth, and increased IFN-γ secretion as well as NK cell-mediated cytotoxicity substantially in bone marrow, spleen and peripheral blood derived immune cells. Similarly, the use of check point inhibitor anti-PD-1 antibody increased IFN-γ secretion and NK cell-mediated cytotoxicity, and decreased the tumor burden in-vivo, and tumor growth of resected minimal residual tumors from hu-BLT mice when used sequentially with sNK cells. The addition of anti-PDL1 antibody to poorly differentiated MP2, NK-differentiated MP2 or well-differentiated PL-12 pancreatic tumors had different effects on tumor cells depending on the differentiation status of the tumor cells, since differentiated tumors expressed PD-L1 and were susceptible to NK cell mediated ADCC, whereas poorly differentiated OSCSCs or MP2 did not express PD-L1 and were killed directly by the NK cells.Conclusions:Therefore, the ability to target combinatorially clones of tumors with NK cells and chemotherapeutic drugs or NK cells with checkpoint inhibitors at different stages of tumor differentiation may be crucial for successful eradication and cure of cancer. Furthermore, the success of check point inhibitor PD-L1 may relate to the levels of expression on tumor cells.

Peptidases represent a large family of hydrolases present in all living organisms, which catalyze the degradation of peptide bonds in different biological processes