Search Results (68)

Background: Lysosomal storage diseases (LSDs) are a genetically and clinically heterogeneous group of inborn errors of metabolism caused by variants in genes encoding lysosomal hydrolases, membrane proteins, activator proteins, or transporters. These disease-causing variants lead to enzymatic deficiencies and the progressive accumulation of undegraded substrates within lysosomes, disrupting cellular function across multiple organ systems. While classical phenotypes typically manifest in infancy or early childhood with severe multisystem involvement, a combination of advances in molecular diagnostics [particularly next-generation sequencing (NGS)] and improved understanding of disease heterogeneity have enabled the identification of attenuated forms characterised by residual enzyme activity and later-onset presentations. These milder phenotypes often evade early recognition due to nonspecific or isolated symptoms, resulting in significant diagnostic delays and missed therapeutic opportunities. Objectives/Methods: This study characterises the clinical, biochemical, and molecular profiles of 10 adult patients diagnosed with LSDs, all representing attenuated forms, and discusses them alongside a narrative review. Results: Enzyme activity, molecular data, and phenotypic assessments are described to explore genotype–phenotype correlations and identify diagnostic challenges. Conclusions: These findings highlight the variable expressivity and organ involvement of attenuated LSDs and reinforce the importance of maintaining clinical suspicion in adults presenting with unexplained cardiovascular, neurological, ophthalmological, or musculoskeletal findings. Enhanced recognition of atypical presentations is critical to facilitate earlier diagnosis, guide management, and enable cascade testing for at-risk family members. Full article

Mucolipidosis type II (ML II) is a rare and fatal disease of acid hydrolase trafficking. It is caused by pathogenic variants in the GNPTAB gene, leading to the absence of GlcNAc-1-phosphotransferase activity, an enzyme that catalyzes the first step in the formation of the mannose 6-phosphate (M6P) tag, essential for the trafficking of most lysosomal hydrolases. Without M6P, these do not reach the lysosome, which accumulates undegraded substrates. The lack of samples and adequate disease models limits the investigation into the pathophysiological mechanisms of the disease and potential therapies. Here, we report the generation and characterization of an ML II induced pluripotent stem cell (iPSC) line carrying the most frequent ML II pathogenic variant [NM_024312.5(GNPTAB):c.3503_3504del (p.Leu1168fs)]. Skin fibroblasts were successfully reprogrammed into iPSCs that express pluripotency markers, maintain a normal karyotype, and can differentiate into the three germ layers. Furthermore, ML II iPSCs showed a phenotype comparable to that of the somatic cells that originated them in terms of key ML II hallmarks: lower enzymatic activity of M6P-dependent hydrolases inside the cells but higher in conditioned media, and no differences in an M6P-independent hydrolase and accumulation of free cholesterol. Thus, ML II iPSCs constitute a novel model for ML II disease, with the inherent iPSC potential to become a valuable model for future studies on the pathogenic mechanisms and testing potential therapeutic approaches. Full article

Mutations in GNPTAB underlie mucolipidosis II and mucolipidosis III α/β, which are inherited lysosomal storage disorders caused by a defective UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine phosphotransferase. As a result, newly synthesized acid hydrolases fail to acquire Mannose-6-Phosphate (Man-6-P) sorting signals, or do so to a lesser extent, and exhibit an impaired trafficking to lysosomes. Interestingly, we found that GNPTAB knockout HeLa cells are resistant to several cytotoxic agents: doxorubicin, chloroquine, staurosporine and paclitaxel. While we detected an increased trapping of weak bases in the expanded lysosomal population of these cells, which could reduce the effect of doxorubicin and chloroquine; the decreased cell response to staurosporine and paclitaxel suggested the involvement of alternative resistance mechanisms. Indeed, further investigation revealed that the hyperactivation of the Insulin-like Growth Factor 1 Receptor (IGF1R) pathway is a central player in the apoptosis resistance exhibited by Man-6-P sorting deficient cells. Full article

Mucolipidosis type II is a very rare lysosomal disease affecting the UDP-GlcNAc N-acetylglucosamine-1-phosphotransferase enzyme, which catalyzes the synthesis of the targeting signal mannose 6-phosphate in lysosomal acid hydrolases. Its deficiency hinders the arrival of lysosomal enzymes to the lysosome, diminishing the multiple degradations of components that cells need to perform. Due to the low prevalence of this condition, available information is scarce. This article aims to deepen the understanding of the disease; clinical, biochemical, and proteomic data are analyzed. Three patients have been identified presenting GNPTAB pathogenic variants using whole exome sequencing. A biochemical profile for these patients has been carried out through quantification of glycosaminoglycans in urine samples and enzymatic analysis in dried blood spot (DBS) samples. Quantitative proteomic studies were performed. Results show how enzymatic assays in DBS can be used to diagnose this disease both during the neonatal period or in patients of more advanced age. Increased levels of acid sphingomyelinase, alpha-iduronidase, iduronidate 2-sulfatase, alpha-N-acetyl glucosaminidase, and beta-glucuronidase are found. Conclusion: this biochemical method could potentially improve early diagnosis. Proteomic data supporting these results reveal disrupted biochemical pathways, including the degradation of dermatan sulfate, heparan sulfate, and cellular cholesterol trafficking. Full article

Lysosomes are subcellular compartments characterised by an acidic pH, containing an ample variety of acid hydrolases involved in the recycling of biopolymers. Among these hydrolases, lysosomal proteases have merely been considered as end-destination proteases responsible for the digestion of waste proteins, trafficked to the lysosomal compartment through autophagy and endocytosis. However, recent reports have started to unravel specific roles for these proteases in the regulation of initially unexpected biological processes, both under physiological and pathological conditions. Furthermore, some lysosomal proteases are no longer restricted to the lysosomal compartment, as more novel non-canonical, extralysosomal targets are being identified. Currently, lysosomal proteases are accepted to play key functions in the extracellular milieu, attached to the plasma membrane and even in the cytosolic and nuclear compartments of the cell. Under physiological conditions, lysosomal proteases, through non-canonical, extralysosomal activities, have been linked to cell differentiation, regulation of gene expression, and cell division. Under pathological conditions, these proteases have been linked to cancer, mostly through their extralysosomal activities in the cytosol and nuclei of cells. In this review, we aim to provide a comprehensive summary of our current knowledge about the extralysosomal, non-canonical functions of lysosomal proteases, both under physiological and pathological conditions, with a particular interest in cancer, that could potentially offer new opportunities for clinical intervention. Full article

The existence of repeat breeder cows (RBCs) causes low reproductive performance. The causes of RBCs include low-quality oocytes and embryos, hormonal dysregulation, and unsuitable uterine environments. To improve unsuitable uterine conditions for RBCs, we focused on nobiletin (NOB), a natural citrus flavone with various beneficial roles. The role of NOB in bovine endometrial epithelial cells (BEECs) was examined. An analysis of BEECs showed that gene expression and altered pathways differed between the control and NOB treatment, with NOB regulating the pathways of steroid biosynthesis, lysosomal function, and inflammatory responses. NOB treatment significantly increased the number and activation of endosomes and lysosomes in BEECs. Moreover, we performed phagocytosis assays using fluorescence-conjugated lipopolysaccharide (LPS) with lysosomes in NOB-treated BEECs, which resulted in an increase in the co-localization of phagocytosed LPS with lysosomes. NOB treatment stimulated the mRNA expression of various lysosomal hydrolases, including cathepsin B and cathepsin K, and suppressed the gene expression of cytokines in inflammation-associated pathways (rheumatoid arthritis, the IL-17 signaling pathway, etc.). NOB significantly suppressed the LPS-induced mRNA expression of the inflammatory cytokine IL-8 and its secretion in BEECs. In conclusion, NOB activates the endosome–lysosomal system via phagocytosis to eliminate the bacterial component LPS and suppress inflammatory responses to defense mechanisms in BEECs. Full article

Heterozygous mutations or genetic variants in the GBA1 gene, which encodes for the β-glucocerebrosidase (GCase), a lysosomal hydrolase enzyme, may increase the risk of Parkinson’s disease (PD) onset. The heterozygous E326K form is one of the most common genetic risk factors for PD worldwide, but, to date, the underlying molecular mechanisms remain unclear. Here, we investigate the effect of the E326K on the structure, stability, dimerization process, and interaction mode with some proteins of the interactome of GCase using multiple molecular dynamics (MD) simulations at pH 5.5 and pH 7.0 to mimic the lysosomal and endoplasmic reticulum environments, respectively. The analysis of the MD trajectories highlights that the E326K mutation did not significantly alter the structural conformation of the catalytic dyad but significantly makes the structure of the dimeric complexes unstable, especially at lysosomal pH, potentially impacting the organization of the quaternary structure. Furthermore, the E326K mutation significantly impacts protein interactions by altering the binding mode with the activator Saposin C (SapC), reducing the binding affinity with the inhibitor α-Synuclein (α-Syn), and increasing the affinity for the Lysosomal integral membrane protein-2 (LIMP-2) transporter. Full article

Glycoside hydrolases have been implicated in a wide range of human conditions including lysosomal storage diseases. Consequently, many researchers have directed their efforts towards identifying new classes of glycoside hydrolase inhibitors, both synthetic and from natural sources. A large percentage of such inhibitors are reversible competitive inhibitors that bind in the active site often due to them possessing structural features, often a protonatable basic nitrogen atom, that mimic the enzymatic transition state. We report that mechanism-based small molecule galacto-like configured cyclohexenyl carbasugars form reversible covalent complexes with both α-galactosidase and β-galactosidase. In addition, we show that the β-galactosidase from Aspergillus oryzae reacts with three different carbasugar inhibitors, with three different second-order rate constants (k_inact/K_i), to give the same enzyme–carbasugar covalent intermediate. The surprising observation that the α-galacto-configured inhibitor covalently labels the A. oryzae β-galactosidase highlights the catalytic versatility of glycoside hydrolases. We expect that cyclohexenyl covalent inhibitors will become an important class of compounds in the chemical biologist’s tool box. Full article

Lysosomal storage diseases (LSDs) are caused by the deficient activity of a lysosomal hydrolase or the lack of a functional membrane protein, transporter, activator, or other protein. Lysosomal enzymes break down macromolecular compounds, which contribute to metabolic homeostasis. Stored, undegraded materials have multiple effects on cells that lead to the activation of autophagy and apoptosis, including the toxic effects of lyso-lipids, the disruption of intracellular Ca²⁺ ion homeostasis, the secondary storage of macromolecular compounds, the activation of signal transduction, apoptosis, inflammatory processes, deficiencies of intermediate compounds, and many other pathways. Clinical observations have shown that carriers of potentially pathogenic variants in LSD-associated genes and patients affected with some LSDs are at a higher risk of cancer, although the results of studies on the frequency of oncological diseases in LSD patients are controversial. Cancer is found in individuals affected with Gaucher disease, Fabry disease, Niemann-Pick type A and B diseases, alfa-mannosidosis, and sialidosis. Increased cancer prevalence has also been reported in carriers of a potentially pathogenic variant of an LSD gene, namely CLN3, SGSH, GUSB, NEU1, and, to a lesser extent, in other genes. In this review, LSDs in which oncological events can be observed are described. Full article

The study aimed to investigate late radiation-induced changes in the histology, ultrastructure, and activity of lysosomal enzymes in mouse liver exposed to ionizing radiation. The experiment was conducted on C57BL/6J male mice whose distal part of the liver was exposed occasionally to single doses of radiation (6 MV photons) during targeted heart irradiation; estimated doses delivered to analyzed tissue were 0.025 Gy, 0.25 Gy, 1 Gy, and 2 Gy. Tissues were collected 40 weeks after irradiation. We have observed that late effects of radiation have an adaptive nature and their intensity was dose-dependent. Morphological changes in hepatocytes included an increased number of primary lysosomes and autophagic vacuoles, which were visible in tissues irradiated with 0.25 Gy and higher doses. On the other hand, a significant increase in the activity of lysosomal hydrolases was observed only in tissues exposed to 2 Gy. The etiology of these changes may be multifactorial and result, among others, from unintentional irradiation of the distal part of the liver and/or functional interaction of the liver with an irradiated heart. In conclusion, we confirmed the presence of late dose-dependent ultrastructural and biochemical changes in mouse hepatocytes after liver irradiation in vivo. Full article

Mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome) is a rare autosomal recessive lysosomal storage disease (LSD) caused by deficiency of a hydrolase enzyme, N-acetylgalactosamine-6-sulfate sulfatase, and characterized clinically by mainly musculoskeletal manifestations. The mechanisms underlying bone involvement in humans are typically explored using invasive techniques such as bone biopsy, which complicates analysis in humans. We compared bone proteomes using DDA and SWATH-MS in wild-type and MPS IVA knockout mice (UNT) to obtain mechanistic information about the disease. Our findings reveal over 1000 dysregulated proteins in knockout mice, including those implicated in oxidative phosphorylation, oxidative stress (reactive oxygen species), DNA damage, and iron transport, and suggest that lactate dehydrogenase may constitute a useful prognostic and follow-up biomarker. Identifying biomarkers that reflect MPS IVA clinical course, severity, and progression have important implications for disease management. Full article

Alzheimer’s disease is a neurodegeneration with protein deposits, altered proteolysis, and inflammatory and oxidative processes as major hallmarks. Despite the continuous search for potential therapeutic treatments, no cure is available to date. The use of natural molecules as adjuvants in the treatment of Alzheimer’s disease is a very promising strategy. In this regard, ginsenosides from ginseng root show a variety of biological effects. Here, we dissected the role of ginsenosides Rg1 and Rg2 in modulating autophagy and oxidative stress in neuroblastoma cells overexpressing Aβ(1-42). Key hallmarks of these cellular processes were detected through immunomethods and fluorometric assays. Our findings indicate that ginsenosides are able to upregulate autophagy in neuronal cells as demonstrated by increased levels of LC3II and Beclin-1 proteins and decreased amounts of p62. Simultaneously, an activation of lysosomal hydrolases was observed. Furthermore, autophagy activation promoted the clearance of Aβ(1-42). Rg1 and Rg2 also reduced oxidative stress sources and macromolecule oxidation, promoting NRF2 nuclear translocation and the expression of antioxidant enzymes. Our data further clarify the mechanisms of action of Rg1 and Rg2, indicating new insights into their role in the management of disorders like Alzheimer’s disease. Full article

Gaucher disease (GD) is a rare genetic metabolic disorder characterized by a dysfunction of the lysosomal glycoside hydrolase glucocerebrosidase (GCase) due to mutations in the gene GBA1, leading to the cellular accumulation of glucosylceramide (GlcCer). While most of the current research focuses on the primary accumulated material, lesser attention has been paid to secondary storage materials and their reciprocal intertwining. By using a novel approach based on flow cytometry and fluorescent labelling, we monitored changes in storage materials directly in fibroblasts derived from GD patients carrying N370S/RecNcil and homozygous L444P or R131C mutations with respect to wild type. In L444P and R131C fibroblasts, we detected not only the primary accumulation of GlcCer accumulation but also a considerable secondary increase in GM1 storage, comparable with the one observed in infantile patients affected by GM1 gangliosidosis. In addition, the ability of a trivalent trihydroxypiperidine iminosugar compound (CV82), which previously showed good pharmacological chaperone activity on GCase enzyme, to reduce the levels of storage materials in L444P and R131C fibroblasts was tested. Interestingly, treatment with different concentrations of CV82 led to a significant reduction in GM1 accumulation only in L444P fibroblasts, without significantly affecting GlcCer levels. The compound CV82 was selective against the GCase enzyme with respect to the β-Galactosidase enzyme, which was responsible for the catabolism of GM1 ganglioside. The reduction in GM1-ganglioside level cannot be therefore ascribed to a direct action of CV82 on β-Galactosidase enzyme, suggesting that GM1 decrease is rather related to other unknown mechanisms that follow the direct action of CV82 on GCase. In conclusion, this work indicates that the tracking of secondary storages can represent a key step for a better understanding of the pathways involved in the severity of GD, also underlying the importance of developing drugs able to reduce both primary and secondary storage-material accumulations in GD. Full article

Recent data described that patients with lysosomal storage disorders (LSDs) may have clinical schizophrenia (SCZ) features. Disruption of lipid metabolism in SCZ pathogenesis was found. Clinical features of schizophrenia (SCZ) have been demonstrated in patients with several lysosomal storage disorders (LSDs). Taking into account the critical role of lysosomal function for neuronal cells’ lysosomal dysfunction could be proposed in SCZ pathogenesis. The current study analyzed lysosomal enzyme activities and the alpha-synuclein level in the blood of patients with late-onset SCZ. In total, 52 SCZ patients with late-onset SCZ, 180 sporadic Parkinson’s disease (sPD) patients, and 176 controls were recruited. The enzymatic activity of enzymes associated with mucopolysaccharidosis (alpha-L-Iduronidase (IDUA)), glycogenosis (acid alpha-glucosidase (GAA)) and sphingolipidosis (galactosylceramidase (GALC), glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase)) and concentration of lysosphingolipids (hexosylsphingosine (HexSph), globotriaosylsphingosine (LysoGb3), and lysosphingomyelin (LysoSM)) were measured using LC-MS/MS. The alpha-synuclein level was estimated in magnetically separated CD45+ blood cells using the enzyme-linked immunosorbent assay (ELISA). Additionally, NGS analysis of 11 LSDs genes was conducted in 21 early-onset SCZ patients and 23 controls using the gene panel PGRNseq-NDD. Decreased ASMase, increased GLA activities, and increased HexSpn, LysoGb3, and LysoSM concentrations along with an accumulation of the alpha-synuclein level were observed in late-onset SCZ patients in comparison to the controls (p < 0.05). Four rare deleterious variants among LSDs genes causing mucopolysaccharidosis type I (IDUA (rs532731688, rs74385837) and type III (HGSNAT (rs766835582)) and sphingolipidosis (metachromatic leukodystrophy (ARSA (rs201251634)) were identified in five patients from the group of early-onset SCZ patients but not in the controls. Our findings supported the role of sphingolipid metabolism in SCZ pathogenesis. Aberrant enzyme activities and compounds of sphingolipids associated with ceramide metabolism may lead to accumulation of alpha-synuclein and may be critical in SCZ pathogenesis. Full article

Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short. Full article