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Cathepsins are protease enzymes vital for normal physiological functions, such as digestion, coagulation, hormone secretion, bone resorption, apoptosis, autophagy, and both innate and adaptive immunity. Their altered expression and/or activity is associated with various pathological conditions, including inflammatory processes. In this study, we investigated the expression levels of cathepsins in muscle specimens collected from dogs affected by inflammatory myopathy (IM) of variable severity established through histopathological analysis. Samples collected from dogs affected by IM at mild, moderate, and severe stages and from healthy (control) dogs were analyzed for the expression profile of 35 proteases using a proteome profiler array. Among the other proteases, cathepsin B was upregulated to an extent depending on disease progression. By exploring the molecular mechanisms underlying the impact of cathepsin B on the disease, we found that the upregulation of cathepsin B in diseased tissues correlates with increased TGFβ-1 expression levels and elevated phosphorylation levels of the TGFβ-1 signaling mediator SMAD2/3. These results suggest that cathepsin B might be involved in the onset and progression of fibrosis commonly occurring in IM diseased dogs. Overall, our findings reveal that modulating cathepsin B activity may hold therapeutic potential for IM.

Cathepsins (CTSs) are ubiquitously expressed proteases normally found in the endolysosomal compartment where they mediate protein degradation and turnover. However, CTSs are also found in the cytoplasm, nucleus, and extracellular matrix where they actively participate in cell signaling, protein processing, and trafficking through the plasma and nuclear membranes and between intracellular organelles. Dysregulation in CTS expression and/or activity disrupts cellular homeostasis, thus contributing to many human diseases, including inflammatory and cardiovascular diseases, neurodegenerative disorders, diabetes, obesity, cancer, kidney dysfunction, and others. This review aimed to highlight the involvement of CTSs in inherited lysosomal storage disorders, with a primary focus to the emerging evidence on the role of CTSs in the pathophysiology of Mucopolysaccharidoses (MPSs). These latter diseases are characterized by severe neurological, skeletal and cardiovascular phenotypes, and no effective cure exists to date. The advance in the knowledge of the molecular mechanisms underlying the activity of CTSs in MPSs may open a new challenge for the development of novel therapeutic approaches for the cure of such intractable diseases.

Kidney disease is worldwide the 12th leading cause of death affecting 8-16% of the entire population. Kidney disease encompasses acute (short-lasting episode) and chronic (developing over years) pathologies both leading to renal failure. Since specific treatments for acute or chronic kidney disease are limited, more than 2 million people a year require dialysis or kidney transplantation. Several recent evidences identified lysosomal proteases cathepsins as key players in kidney pathophysiology. Cathepsins, originally found in the lysosomes, exert important functions also in the cytosol and nucleus of cells as well as in the extracellular space, thus participating in a wide range of physiological and pathological processes. Based on their catalytic active site residue, the 15 human cathepsins identified up to now are classified in three different families: serine (cathepsins A and G), aspartate (cathepsins D and E), or cysteine (cathepsins B, C, F, H, K, L, O, S, V, X, and W) proteases. Specifically in the kidney, cathepsins B, D, L and S have been shown to regulate extracellular matrix homeostasis, autophagy, apoptosis, glomerular permeability, endothelial function, and inflammation. Dysregulation of their expression/activity has been associated to the onset and progression of kidney disease. This review summarizes most of the recent findings that highlight the critical role of cathepsins in kidney disease development and progression. A better understanding of the signaling pathways governed by cathepsins in kidney physiopathology may yield novel selective biomarkers or therapeutic targets for developing specific treatments against kidney disease.

Mucopolysaccharidoses (MPSs) are inherited metabolic diseases caused by the deficiency of lysosomal enzymes needed to catabolize glycosaminoglycans (GAGs). Four therapeutic options are currently considered: enzyme replacement therapy, substrate reduction therapy, gene therapy, and hematopoietic stem cell transplantation. However, while some of them exhibit limited clinical efficacy and require high costs, others are still in development. Therefore, alternative treatments for MPSs need to be explored. Here we describe an innovative therapeutic approach based on the use of a recombinant protein that is able to bind the excess of extracellular accumulated heparan sulfate (HS). We demonstrate that this protein is able to reduce lysosomal defects in primary fibroblasts from MPS I and MPS IIIB patients. We also show that, by masking the excess of extracellular accumulated HS in MPS fibroblasts, fibroblast growth factor (FGF) signal transduction can be positively modulated. We, therefore, suggest the use of a competitive binding molecule for HS in MPSs as an alternative strategy to prevent the detrimental extracellular substrate storage.

Lysosomal storage diseases (LSDs) are rare inherited metabolic diseases characterized by an abnormal accumulation of various toxic materials in the cells as a result of enzyme deficiencies leading to tissue and organ damage. Among clinical manifestations, cardiac diseases are particularly important in Pompe glycogen storage diseases (PD), in glycosphingolipidosis Fabry disease (FD), and mucopolysaccharidoses (MPS). Here, we evaluated the occurrence of aortopathy in knock out (KO) mouse models of three different LSDs, including PD, FD, and MPS IIIB. We measured the aortic diameters in 15 KO male mice, 5 for each LSD: 5 GLA-/- mice for FD, 5 NAGLU-/- mice for MPS IIIB, 5 GAA-/- mice for PD, and 15 wild type (WT) mice: 5 for each strain. In order to compare the aortic parameters between KO and WT mice deriving from the same colonies, different diameters were echocardiographically measured: aortic annulus, aortic sinus, sino-tubular junction, ascending aorta, aortic arch and descending aorta. Storage material content and aortic defects of the KO mice were also analyzed by histology, when available. Compared to their correspondent WT mice: GAA-/- mice showed greater diameters of ascending aorta (1.61mm vs. 1.11mm, p-value = 0.01) and descending aorta (1.17mm vs 1.02mm, p-value 0.04); GLA-/- mice showed greater diameters of aortic annulus (1.35mm vs. 1.22mm, p-value = 0.01), sinus of Valsalva (1.6mm vs. 1.38mm, p-value<0.01), ascending aorta (1.57mm vs. 1.34mm, p-value<0.01), aortic arch (1.36mm vs. 1.22mm, p-value = 0.03) and descending aorta (1.29mm vs. 1.11mm, p-value<0.01); NAGLU-/- mice showed greater diameters of sinus of Valsalva (1.46mm vs. 1.31mm, p-value = 0.05), ascending aorta (1.42mm vs. 1.29mm, p-value<0.01), aortic arch (1.34mm vs. 1.28mm, p-value<0.01) and descending aorta (1.18mm vs. 1.1mm, p-value 0.01). We evaluated for the first time the aortic diameters in 3 LSD mouse models and identified different aortopathy patterns, in concordance with recent human findings. Our results are relevant in view of using KO mouse models for efficiently testing the efficacy of new therapies on distinct cardiovascular aspects of LSDs.

Mucopolysaccharidosis IIIB (MPS IIIB) is an inherited metabolic disease due to deficiency of α-N-Acetylglucosaminidase (NAGLU) enzyme with subsequent storage of undegraded heparan sulfate (HS). The main clinical manifestations of the disease are profound intellectual disability and neurodegeneration. A label-free quantitative proteomic approach was applied to compare the proteome profile of brains from MPS IIIB and control mice to identify altered neuropathological pathways of MPS IIIB. Proteins were identified through a bottom up analysis and 130 were significantly under-represented and 74 over-represented in MPS IIIB mouse brains compared to wild type (WT). Multiple bioinformatic analyses allowed to identify three major clusters of the differentially abundant proteins: proteins involved in cytoskeletal regulation, synaptic vesicle trafficking, and energy metabolism. The proteome profile of NAGLU−/− mouse brain could pave the way for further studies aimed at identifying novel therapeutic targets for the MPS IIIB. Data are available via ProteomeXchange with the identifier PXD017363.

Lysosomal storage diseases (LSDs) are a group of metabolic diseases caused by inborn mutations of lysosomal enzymes, which lead to lysosome substrate accumulation in various cell types [...].Lysosomal storage diseases (LSDs) are a group of metabolic diseases caused by inborn mutations of lysosomal enzymes, which lead to lysosome substrate accumulation in various cell types [...].

Mucopolysaccharidosis (MPS) IIIB is an inherited lysosomal storage disease caused by the deficiency of the enzyme α- N -acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The defective lysosomal clearance of undigested HS results in dysfunction of multiple tissues and organs. We recently demonstrated that the murine model of MPS IIIB develops cardiac disease, valvular abnormalities, and ultimately heart failure. To address the molecular mechanisms governing cardiac dysfunctions in MPS IIIB, we generated a model of the disease by silencing NAGLU gene expression in H9C2 rat cardiomyoblasts. NAGLU-depleted H9C2 exhibited accumulation of abnormal lysosomes and a hypertrophic phenotype. Furthermore, we found the specific activation of the epidermal growth factor receptor (EGFR), and increased phosphorylation levels of extracellular signal-regulated kinases (ERKs) in NAGLU-depleted H9C2. The inhibition of either EGFR or ERKs, using the selective inhibitors AG1478 and PD98059, resulted in the reduction of both lysosomal aberration and hypertrophy in NAGLU-depleted H9C2. We also found increased phosphorylation of c-Src and a reduction of the hypertrophic response in NAGLU-depleted H9C2 transfected with a dominant-negative c-Src. However, c-Src phosphorylation remained unaffected by AG1478 treatment, posing c-Src upstream EGFR activation. Finally, heparin-binding EGF-like growth factor (HB-EGF) protein was found overexpressed in our MPS IIIB cellular model, and its silencing reduced the hypertrophic response. These results indicate that both c-Src and HB-EGF contribute to the hypertrophic phenotype of NAGLU-depleted cardiomyoblasts by synergistically activating EGFR and subsequent signaling, thus suggesting that EGFR pathway inhibition could represent an effective therapeutic approach for MPS IIIB cardiac disease.

Postmortem cadaveric changes are commonly used to estimate the postmortem interval (PMI) in humans and animals. However, these modifications have been poorly investigated in animals of interest to veterinary forensic pathology. The aim of this study was to investigate the potential use of muscle proteins (desmin and dystrophin) as biomarkers for estimating the PMI in dogs. For this study, 10 dead adult dogs were evaluated for 4 days in a temperature-controlled room at 19 ± 1 °C. For each animal, at 3, 24, 48, 72, and 96 h after death, a 1 × 1 × 1 cm cube of muscle tissue was removed from the vastus lateralis and triceps brachii. Protein expression levels were analyzed by immunohistochemical examination and immunoblot analysis. The obtained results showed rapid dystrophin degradation, with complete disappearance at 72 h after death. In contrast, desmin-positive fibers and desmin protein bands detected by immunoblot were observed on all 4 days of observation. Our findings suggest the potential use of muscle proteins as biomarkers for estimating the PMI in dogs.

Mucopolysaccharidosis (MPS) IIIB is a lysosomal disease due to the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The disease is characterized by mild somatic features and severe neurological disorders. Very little is known on the cardiac dysfunctions in MPS IIIB. In this study, we used the murine model of MPS IIIB (NAGLU knockout mice, NAGLU(-/-)) in order to investigate the cardiac involvement in the disease. Echocardiographic analysis showed a marked increase in left ventricular (LV) mass, reduced cardiac function and valvular defects in NAGLU(-/-) mice as compared to wild-type (WT) littermates. The NAGLU(-/-) mice exhibited a significant increase in aortic and mitral annulus dimension with a progressive elongation and thickening of anterior mitral valve leaflet. A severe mitral regurgitation with reduction in mitral inflow E-wave-to-A-wave ratio was observed in 32-week-old NAGLU(-/-) mice. Compared to WT mice, NAGLU(-/-) mice exhibited a significantly lower survival with increased mortality observed in particular after 25 weeks of age. Histopathological analysis revealed a significant increase of myocardial fiber vacuolization, accumulation of HS in the myocardial vacuoles, recruitment of inflammatory cells and collagen deposition within the myocardium, and an increase of LV fibrosis in NAGLU(-/-) mice compared to WT mice. Biochemical analysis of heart samples from affected mice showed increased expression levels of cardiac failure hallmarks such as calcium/calmodulin-dependent protein kinase II, connexin43, α-smooth muscle actin, α-actinin, atrial and brain natriuretic peptides, and myosin heavy polypeptide 7. Furthermore, heart samples from NAGLU(-/-) mice showed enhanced expression of the lysosome-associated membrane protein-2 (LAMP2), and the autophagic markers Beclin1 and LC3 isoform II (LC3-II). Overall, our findings demonstrate that NAGLU(-/-) mice develop heart disease, valvular abnormalities and cardiac failure associated with an impaired lysosomal autophagic flux.