Glycosaminoglycans In Lysosomes Research Articles

Synergistic effects of resveratrol and enzyme replacement therapy in the Mucopolysaccharidosis type I

Mucopolysaccharidosis type I (MPS I) is a rare genetic disorder caused by mutations in the IDUA gene, leading to alpha-L-iduronidase enzyme deficiency and resulting in the accumulation of glycosaminoglycans (GAG; heparan and dermatan sulfate) in lysosomes. The consequent GAG accumulation within cells leads to organ dysfunction and a range of debilitating symptoms. Enzyme replacement therapy (ERT) is the prevailing treatment, but its limitations (including high cost, time requirements, inefficiency in treatment of central nervous system (CNS), and immunogenicity) necessitate exploration of alternative therapeutic strategies. This research propose a novel approach leveraging the synergistic effects of ERT and resveratrol-induced autophagy. Resveratrol, with its immunomodulatory and GAG degradation-stimulating properties, holds a promise in mitigating immune responses triggered by ERT. Moreover, its ability to penetrate the blood–brain barrier presents a potential solution for addressing CNS manifestations. This study employed cells from MPS I patients to investigate the combined effects of resveratrol and the enzyme. Evaluation of the therapeutic impact involved assessing GAG accumulation, enzyme testing, and examining lysosome functionality and the autophagy process through fluorescence microscopy and Western blotting. The combined therapy stimulated the lysosomal mannose-6-phosphate receptor (M6PR) and lysosome biogenesis through the transcription factor EB (TFEB). Additionally, initial block of autophagy in autophagosome formation was relieved after the combined therapy and resveratrol alone. Together with increased enzyme activity through stimulation of the receptor, this synergistic therapy can be considered a new potential treatment for MPS I patients, improving their overall quality of life.

Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model.

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared withWT vehicle group) and genistein at concentrations of 25and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.

Mucopolysaccharidoses Differential Diagnosis by Mass Spectrometry-Based Analysis of Urine Free Glycosaminoglycans-A Diagnostic Prediction Model.

Impaired glycosaminoglycans (GAGs) catabolism may lead to a cluster of rare metabolic and genetic disorders called mucopolysaccharidoses (MPSs). Each subtype is caused by the deficiency of one of the lysosomal hydrolases normally degrading GAGs. Affected tissues accumulate undegraded GAGs in cell lysosomes and in the extracellular matrix, thus leading to the MPS complex clinical phenotype. Although each MPS may present with recognizable signs and symptoms, these may often overlap between subtypes, rendering the diagnosis difficult and delayed. Here, we performed an exploratory analysis to develop a model that predicts MPS subtypes based on UHPLC-MS/MS measurement of a urine free GAG profile (or GAGome). We analyzed the GAGome of 78 subjects (38 MPS, 37 healthy and 3 with other MPS symptom-overlapping disorders) using a standardized kit in a central-blinded laboratory. We observed several MPS subtype-specific GAGome changes. We developed a multivariable penalized Lasso logistic regression model that attained 91.2% balanced accuracy to distinguish MPS type II vs. III vs. any other subtype vs. not MPS, with sensitivity and specificity ranging from 73.3% to 91.7% and from 98.4% to 100%, depending on the predicted subtype. In conclusion, the urine GAGome was revealed to be useful in accurately discriminating the different MPS subtypes with a single UHPLC-MS/MS run and could serve as a reliable diagnostic test for a more rapid MPS biochemical diagnosis.

AIRWAY MANAGEMENT OF A CHILD WITH HUNTER'S SYNDROME

Hunter's syndrome, a very rare genetic disorder caused by a missing or malfunctioning enzyme iduronate-2-sulfatase, resulting in the accumulation of glycosaminoglycans in lysosomes. We report the airway management of an 8-year-old male child diagnosed as Hunters syndrome came with complaints of shortness of breath, lower respiratory tract infection and acute exacerbation of asthma. Admitted in pediatric ICU with severe wheeze and respiratory distress. Started on HFNC. Despite HFNC symptoms got worsened. So, planned for elective intubation. Child has large, protruded tongue, short neck. Difficult airway cart made ready. One attempt of video laryngoscopy and one attempt of normal laryngoscopy done. Unable to intubate so proceeded with tracheostomy as child was desaturating

Mucopolysaccharidoses: Cellular Consequences of Glycosaminoglycans Accumulation and Potential Targets.

Mucopolysaccharidoses (MPSs) constitute a heterogeneous group of lysosomal storage disorders characterized by the lysosomal accumulation of glycosaminoglycans (GAGs). Although lysosomal dysfunction is mainly affected, several cellular organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, and their related process are also impaired, leading to the activation of pathophysiological cascades. While supplying missing enzymes is the mainstream for the treatment of MPS, including enzyme replacement therapy (ERT), hematopoietic stem cell transplantation (HSCT), or gene therapy (GT), the use of modulators available to restore affected organelles for recovering cell homeostasis may be a simultaneous approach. This review summarizes the current knowledge about the cellular consequences of the lysosomal GAGs accumulation and discusses the use of potential modulators that can reestablish normal cell function beyond ERT-, HSCT-, or GT-based alternatives.

Modeling cartilage pathology in mucopolysaccharidosis VI using iPSCs reveals early dysregulation of chondrogenic and metabolic gene expression.

Mucopolysaccharidosis type VI (MPS VI) is a metabolic disorder caused by disease-associated variants in the Arylsulfatase B (ARSB) gene, resulting in ARSB enzyme deficiency, lysosomal glycosaminoglycan accumulation, and cartilage and bone pathology. The molecular response to MPS VI that results in cartilage pathology in human patients is largely unknown. Here, we generated a disease model to study the early stages of cartilage pathology in MPS VI. We generated iPSCs from four patients and isogenic controls by inserting the ARSB cDNA in the AAVS1 safe harbor locus using CRISPR/Cas9. Using an optimized chondrogenic differentiation protocol, we found Periodic acid-Schiff positive inclusions in hiPSC-derived chondrogenic cells with MPS VI. Genome-wide mRNA expression analysis showed that hiPSC-derived chondrogenic cells with MPS VI downregulated expression of genes involved in TGF-β/BMP signalling, and upregulated expression of inhibitors of the Wnt/β-catenin signalling pathway. Expression of genes involved in apoptosis and growth was upregulated, while expression of genes involved in glycosaminoglycan metabolism was dysregulated in hiPSC-derived chondrogenic cells with MPS VI. These results suggest that human ARSB deficiency in MPS VI causes changes in the transcriptional program underlying the early stages of chondrogenic differentiation and metabolism.

The Anesthetic Strategy for Patients with Mucopolysaccharidoses: A Retrospective Cohort Study.

Anesthesia for patients with mucopolysaccharidoses (MPS) is quite challenging due to vital systemic dysfunction following progressive accumulation of lysosomal glycosaminoglycans. Previous studies focused on perioperative difficult airway management under general anesthesia but rarely depicted the concern of choosing the size of the endotracheal tube (ETT) as well as neuraxial anesthesia. This study aimed to analyze the overall anesthetic management and related complications for a thorough anesthetic strategy. Within the study period from 2002 to 2021, each record of the anesthetic and perioperative quality assurance/improvement system for patients with a diagnosis of MPS at MacKay Memorial Hospital was retrospectively reviewed. A total of 51 individuals with 151 anesthesia for 163 interventions were cohort studied, and there were 136 general anesthesia and 15 neuraxial anesthesia. We found that the most common interventions for MPS patients were otolaryngological surgeries (49.6%). Additionally, a secured airway played a marked preference for the most general anesthesia (87.1%). The incidence of difficult intubation was 12.5%. In view of ETT size, a smaller than estimated size was used in MPS type II, III, IV, and VI patients and also in patients who received intubation with multiple attempts. However, a larger than estimated size of ETT was adopted whilst choosing cuffed ones. For neuraxial anesthesia, two failed spinal anesthesia procedures were converted to general anesthesia and 73 percent of the patients received perioperative sedation. In conclusion, through the individualized anesthetic strategy and build-up of an experienced team for airway management, high-quality anesthesia can be ensured in each patient.

Tralesinidase Alfa Enzyme Replacement Therapy Prevents Disease Manifestations in a Canine Model of Mucopolysaccharidosis Type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B; OMIM #252920) is a lethal, pediatric, neuropathic, autosomal recessive, and lysosomal storage disease with no approved therapy. Patients are deficient in the activity of N-acetyl-alpha-glucosaminidase (NAGLU; EC 3.2.150), necessary for normal lysosomal degradation of the glycosaminoglycan heparan sulfate (HS). Tralesinidase alfa (TA), a fusion protein comprised of recombinant human NAGLU and a modified human insulin-like growth factor 2, is in development as an enzyme replacement therapy that is administered via intracerebroventricular (ICV) infusion, thus circumventing the blood brain barrier. Previous studies have confirmed ICV infusion results in widespread distribution of TA throughout the brains of mice and nonhuman primates. We assessed the long-term tolerability, pharmacology, and clinical efficacy of TA in a canine model of MPS IIIB over a 20-month study. Long-term administration of TA was well tolerated as compared with administration of vehicle. TA was widely distributed across brain regions, which was confirmed in a follow-up 8-week pharmacokinetic/pharmacodynamic study. MPS IIIB dogs treated for up to 20 months had near-normal levels of HS and nonreducing ends of HS in cerebrospinal fluid and central nervous system (CNS) tissues. TA-treated MPS IIIB dogs performed better on cognitive tests and had improved CNS pathology and decreased cerebellar volume loss relative to vehicle-treated MPS IIIB dogs. These findings demonstrate the ability of TA to prevent or limit the biochemical, pathologic, and cognitive manifestations of canine MPS IIIB disease, thus providing support of its potential long-term tolerability and efficacy in MPS IIIB subjects.SIGNIFICANCE STATEMENTThis work illustrates the efficacy and tolerability of tralesinidase alfa as a potential therapeutic for patients with mucopolysaccharidosis type IIIB (MPS IIIB) by documenting that administration to the central nervous system of MPS IIIB dogs prevents the accumulation of disease-associated glycosaminoglycans in lysosomes, hepatomegaly, cerebellar atrophy, and cognitive decline.

Lysosomal storage diseases: mucopolysaccharidosis type I and II

Mucopolysaccharidosis (MPS) are a genetically heterogeneous group of rare monogenic metabolic diseases associated with hereditary insufficiency of lysosomal enzymes involved in the catabolism of glycosaminoglycans, or mucopolysaccharides. The pathogenesis of MPS is due to the accumulation of non-cleaved glycosaminoglycans in lysosomes, which can destroy cells. All MPS are characterized by a polysystemic manifestation, the simultaneous involvement of many organs and tissues in the pathological process, first of all, connective tissues, bones and cartilaginous. This review presents the epidemiology, clinical, biochemical, and molecular genetic characteristics of MPS types I and II, caused by the recessive mutations in the alpha-L-iduronidase and iduronate-2-sulfatase genes, respectively, and by the accumulation of dermatan and heparan sulfate. Each of these diseases is characterized by clinical polymorphism, especially observed in MPS I, which often manifests in a severe form of Hurler syndrome, but can also occur in a milder form of Scheie syndrome. Currently, there is an increased interest in MPS in the world due to the identification of the spectrum and frequencies of mutations in theIDUAandIDSgenes in various populations, including in Russia, and the practical availability of methods for individual molecular diagnostics. The description of the existing experimental models, their role in the study of the biochemical basis of the pathogenesis of these severe hereditary diseases and the development of various therapeutic approaches are given. Discusses the possibility of early diagnosis of MPS I and II types based on neonatal screening in order to increase the effectiveness of their prevention and treatment, as well as the advantages and disadvantages of the main approaches to the treatment of these serious diseases, such as hematopoietic stem cell transplantation, enzyme replacement and substrate-reducing therapy. A clinical example of a combination therapy for a severe form of mucopolysaccharidosis type I Hurler syndrome is presented

Deep intronic variant in the ARSB gene as the genetic cause for Maroteaux-Lamy syndrome (MPS VI).

Maroteaux-Lamy syndrome (MPS-VI) is a rare autosomal-recessive disorder with a wide spectrum of clinical manifestations, ranging from an attenuated to a rapidly progressive disease. It is caused by variants in ARSB, which encodes the lysosomal arylsulfatase B (ARSB) enzyme, part of the degradation process of glycosaminoglycans in lysosomes. Over 220 variants have been reported so far, with a majority of missense variants. We hereby report two siblings of Bedouin origin with a diagnosis of MPS-VI. Western blots in patient fibroblasts revealed total absence of ARSB protein production. Complete sequencing of the coding region of ARSB did not identify a candidate disease-associated variant. However, deep sequencing of the noncoding region of ARSB by whole genome sequencing (WGS) revealed a c.1142+581A to G variant. The variant is located within intron 5 and fully segregated with the disease in the family. Determination of the genetic cause for these patients enabled targeted treatment by enzyme replacement therapy, along with appropriate genetic counseling and prenatal diagnosis for the family. These results highlight the advantage of WGS as a powerful tool, for improving the diagnostic rate of rare disease-causing variants, and emphasize the importance of studying deep intronic sequence variation as a cause of monogenic disorders.

THAP1 modulates oligodendrocyte maturation by regulating ECM degradation in lysosomes

Mechanisms controlling myelination during central nervous system (CNS) maturation play a pivotal role in the development and refinement of CNS circuits. The transcription factor THAP1 is essential for timing the inception of myelination during CNS maturation through a cell-autonomous role in the oligodendrocyte lineage. Here, we demonstrate that THAP1 modulates the extracellular matrix (ECM) composition by regulating glycosaminoglycan (GAG) catabolism within oligodendrocyte progenitor cells (OPCs). Thap1-/- OPCs accumulate and secrete excess GAGs, inhibiting their maturation through an autoinhibitory mechanism. THAP1 controls GAG metabolism by binding to and regulating the GusB gene encoding β-glucuronidase, a GAG-catabolic lysosomal enzyme. Applying GAG-degrading enzymes or overexpressing β-glucuronidase rescues Thap1-/- OL maturation deficits in vitro and in vivo. Our studies establish lysosomal GAG catabolism within OPCs as a critical mechanism regulating oligodendrocyte development.

GENE EDITING OF FIBROBLAST-LIKE SYNOVIOCYTES FROM MUCOPOLYSACCHARIDOSIS TYPE I MICE WITH THE CRISPR-CAS9 SYSTEM

Mucopolysaccharidosis type I (MPS I) is caused by alpha-L-iduronidase enzyme (IDUA) deficiency and lysosomal accumulation of glycosaminoglycans (GAGs). The accumulation of GAGs affects important cellular processes and triggers a cascade of responses altering cellular homeostasis and leading to multisystemic dysfunctions. In this disease, the bone/joint complications reduce mobility. Current treatments do not correct these complications. Therefore, it is necessary to look for new therapies. An alternative is gene editing using the CRISPRCas9 system. This work aims to evaluate the efficiency of cationic liposomes carrying plasmids with the CRISPRCas9 system plus a donor vector to replace IDUA in fibroblast-like synoviocytes. MPS I mouse fibroblast-like synoviocytes (FLS) were transfected with 1) Liposome/CRISPR+Donor IDUA or 2)Liposome/Donor IDUA or 3)Lipofectamine 3000® Lipofectamine/Donor IDUA or 4)Lipofectamine/CRISPR+Donor IDUA. The cells were maintained in culture and collected at 4 times (3, 7, 15, and 30 days) after transfection. IDUA activity and total proteins were measured at each of these times. The FLS from untreated MPS I and from normal mice were cultured in the same conditions and used as controls. The incubation of the complexes with FLS demonstrated high cell viability (over 70%) and no statistical difference (<i>p</i>>0.05) between treatments, and these results showed that the Liposome presented low cytotoxicity. At all times, the untreated FLS MPS I showed very low levels of IDUA. The treatment with Lipofectamine/CRISPR+Donor IDUA and Liposome/CRISPR+Donor IDUA always showed a significant increase in IDUA production (<i>p</i> < 0.0001 and <i>p</i><0.001). This increase was sustained for up to 30 days. The treatment with Lipofectamine/CRISPR+Donor IDUA reached an average of 4.0% (±0.26), of IDUA activity found in normal FLS, while the treatment with Liposome/CRISPR+Donor IDUA reached an average of 2.8% (±0.28). Both treatments were not significantly different from each other (<i>p</i>>0.05). Despite the enzyme levels not being normalized, due to cross-correction (enzyme exchange) between neighboring cells, an increase of 1% in the production of the enzyme could be enough to reduce the accumulation of glycosaminoglycans in lysosomes. This study demonstrates that the Liposome is appropriate for gene editing studies. New studies are being conducted in MPS I mice in vivo.

Gene Expression-Related Changes in Morphologies of Organelles and Cellular Component Organization in Mucopolysaccharidoses.

Mucopolysaccharidoses (MPS) are inherited metabolic diseases characterized by accumulation of incompletely degraded glycosaminoglycans (GAGs) in lysosomes. Although primary causes of these diseases are mutations in genes coding for enzymes involved in lysosomal GAG degradation, it was demonstrated that storage of these complex carbohydrates provokes a cascade of secondary and tertiary changes affecting cellular functions. Potentially, this might lead to appearance of cellular disorders which could not be corrected even if the primary cause of the disease is removed. In this work, we studied changes in cellular organelles in MPS fibroblasts relative to control cells. All 11 types and subtypes of MPS were included into this study to obtain a complex picture of changes in organelles in this group of diseases. Two experimental approaches were employed, transcriptomic analyses and electron microscopic assessment of morphology of organelles. We analyzed levels of transcripts of genes grouped into two terms included into the QuickGO database, ‘Cellular component organization’ (GO:0016043) and ‘Cellular anatomical entity’ (GO:0110165), to find that number of transcripts with significantly changed levels in MPS fibroblasts vs. controls ranged from 109 to 322 (depending on MPS type) in GO:0016043, and from 70 to 208 in GO:0110165. This dysregulation of expression of genes crucial for proper structures and functions of various organelles was accompanied by severe changes in morphologies of lysosomes, nuclei, mitochondria, Golgi apparatus, and endoplasmic reticulum. Interestingly, some observed changes occurred in all/most MPS types while others were specific to particular disease types/subtypes. We suggest that severe changes in organelles in MPS cells might arise from dysregulation of expression of a battery of genes involved in organelles’ structures and functions. Intriguingly, normalization of GAG levels by using recombinant human enzymes specific to different MPS types corrected morphologies of some, but not all, organelles, while it failed to improve regulation of expression of selected genes. These results might suggest reasons for inability of enzyme replacement therapy to correct all MPS symptoms, particularly if initiated at advanced stages of the disease.

Induced Pluripotent Stem Cells to Understand Mucopolysaccharidosis. I: Demonstration of a Migration Defect in Neural Precursors

Background: Mucopolysaccharidosis type I-Hurler (MPS1-H) is a severe genetic lysosomal storage disorder due to loss-of-function mutations in the IDUA gene. The subsequent complete deficiency of alpha l-iduronidase enzyme is directly responsible of a progressive accumulation of glycosaminoglycans (GAG) in lysosomes which affects the functions of many tissues. Consequently, MPS1 is characterized by systemic symptoms (multiorgan dysfunction) including respiratory and cardiac dysfunctions, skeletal abnormalities and early fatal neurodegeneration. Methods: To understand mechanisms underlying MPS1 neuropathology, we generated induced pluripotent stem cells (iPSC) from a MPS1-H patient with loss-of-function mutations in both IDUA alleles. To avoid variability due to different genetic background of iPSC, we established an isogenic control iPSC line by rescuing IDUA expression by a lentivectoral approach. Results: Marked differences between MPS1-H and IDUA-corrected isogenic controls were observed upon neural differentiation. A scratch assay revealed a strong migration defect of MPS1-H cells. Also, there was a massive impact of IDUA deficiency on gene expression (340 genes with an FDR < 0.05). Conclusions: Our results demonstrate a hitherto unknown connection between lysosomal degradation, gene expression and neural motility, which might account at least in part for the phenotype of MPS1-H patients.

The abnormal accumulation of heparan sulfate in patients with mucopolysaccharidosis prevents the elastolytic activity of cathepsin V

Mucopolysaccharidosis (MPS) are rare inherited diseases characterized by accumulation of lysosomal glycosaminoglycans, including heparan sulfate (HS). Patients exhibit progressive multi-visceral dysfunction and shortened lifespan mainly due to a severe cardiac/respiratory decline. Cathepsin V (CatV) is a potent elastolytic protease implicated in extracellular matrix (ECM) remodeling. Whether CatV is inactivated by HS in lungs from MPS patients remained unknown. Herein, CatV colocalized with HS in MPS bronchial epithelial cells. HS level correlated positively with the severity of respiratory symptoms and negatively to the overall endopeptidase activity of cysteine cathepsins. HS bound tightly to CatV and impaired its activity. Withdrawal of HS by glycosidases preserved exogenous CatV activity, while addition of Surfen, a HS antagonist, restored elastolytic CatV-like activity in MPS samples. Our data suggest that the pathophysiological accumulation of HS may be deleterious for CatV-mediated ECM remodeling and for lung tissue homeostasis, thus contributing to respiratory disorders associated to MPS diseases.

Targeted Metabolomic Analysis of a Mucopolysaccharidosis IIIB Mouse Model Reveals an Imbalance of Branched-Chain Amino Acid and Fatty Acid Metabolism.

Mucopolysaccharidoses (MPSs) are inherited disorders of the glycosaminoglycan (GAG) metabolism. The defective digestion of GAGs within the intralysosomal compartment of affected patients leads to a broad spectrum of clinical manifestations ranging from cardiovascular disease to neurological impairment. The molecular mechanisms underlying the progression of the disease downstream of the genetic mutation of genes encoding for lysosomal enzymes still remain unclear. Here, we applied a targeted metabolomic approach to a mouse model of PS IIIB, using a platform dedicated to the diagnosis of inherited metabolic disorders, in order to identify amino acid and fatty acid metabolic pathway alterations or the manifestations of other metabolic phenotypes. Our analysis highlighted an increase in the levels of branched-chain amino acids (BCAAs: Val, Ile, and Leu), aromatic amino acids (Tyr and Phe), free carnitine, and acylcarnitines in the liver and heart tissues of MPS IIIB mice as compared to the wild type (WT). Moreover, Ala, Met, Glu, Gly, Arg, Orn, and Cit amino acids were also found upregulated in the liver of MPS IIIB mice. These findings show a specific impairment of the BCAA and fatty acid catabolism in the heart of MPS IIIB mice. In the liver of affected mice, the glucose-alanine cycle and urea cycle resulted in being altered alongside a deregulation of the BCAA metabolism. Thus, our data demonstrate that an accumulation of BCAAs occurs secondary to lysosomal GAG storage, in both the liver and the heart of MPS IIIB mice. Since BCAAs regulate the biogenesis of lysosomes and autophagy mechanisms through mTOR signaling, impacting on lipid metabolism, this condition might contribute to the progression of the MPS IIIB disease.

Expression of genes involved in apoptosis is dysregulated in mucopolysaccharidoses as revealed by pilot transcriptomic analyses.

Mucopolysaccharidoses (MPS), a group of lysosomal storage diseases (LSD), are inherited disorders caused by mutations in genes coding for enzymes involved in the degradation of glycosaminoglycans (GAGs). Therefore, accumulated GAGs in lysosomes lead to severe symptoms in patients and significantly shortened life span. Although GAG accumulation in cells is the primary cellular defect in MPS, recent reports indicated that severe changes in cellular processes occur there as secondary or tertiary effects, which may contribute significantly to the disease pathomechanism. Apoptosis is one of such process, while mechanisms leading to dysregulation of this process in MPS remain largely unknown. To learn about these mechanisms, we have performed transcriptomic studies using cultures of fibroblasts derived from patients suffering from all types and subtypes of MPS, and assessed genes related to apoptosis. We found that there are significant changes in expression levels of many such genes relative to control fibroblasts (Human Dermal Fibroblasts-adult cell line), and the number of down- or up-regulated transcripts was between 19 and 73 in different MPS types. We have identified apoptosis-related genes, which were considerably dysregulated in many MPS types, as well as those in which expression was significantly changed in specific MPS types. BNIP3, C1D, CLU, GPER1, KREMEN1, and PRKCD genes displayed the most changed expression profiles in most MPS types relative to control cells. Caspase 3/7 activity was increased in MPS IVA and IX. These results indicate that changes in apoptosis, observed in MPS, may arise, at least partially, from dysregulation of genes coding for proteins involved in this process.

Underestimated Aspect of Mucopolysaccharidosis Pathogenesis: Global Changes in Cellular Processes Revealed by Transcriptomic Studies.

Mucopolysaccharidoses (MPS), a group of inherited metabolic disorders caused by deficiency in enzymes involved in degradation of glycosaminoglycans (GAGs), are examples (and models) of monogenic diseases. Accumulation of undegraded GAGs in lysosomes was supposed to be the major cause of MPS symptoms; however, their complexity and variability between particular types of the disease can be hardly explained by such a simple storage mechanism. Here we show that transcriptomic (RNA-seq) analysis of the material derived from fibroblasts of patients suffering from all types and subtypes of MPS, supported by RT-qPCR results, revealed surprisingly large changes in expression of genes involved in various cellular processes, indicating complex mechanisms of MPS. Although each MPS type and subtype was characterized by specific changes in gene expression profile, there were genes with significantly changed expression relative to wild-type cells that could be classified as common for various MPS types, suggesting similar disturbances in cellular processes. Therefore, both common features of all MPS types, and differences between them, might be potentially explained on the basis of changes in certain cellular processes arising from disturbed regulations of genes’ expression. These results may shed a new light on the mechanisms of genetic diseases, indicating how a single mutation can result in complex pathomechanism, due to perturbations in the network of cellular reactions. Moreover, they should be considered in studies on development of novel therapies, suggesting also why currently available treatment methods fail to correct all/most symptoms of MPS. We propose a hypothesis that disturbances in some cellular processes cannot be corrected by simple reduction of GAG levels; thus, combined therapies are necessary which may require improvement of these processes.

Toward Engineering the Mannose 6-Phosphate Elaboration Pathway in Plants for Enzyme Replacement Therapy of Lysosomal Storage Disorders.

Mucopolysaccharidosis (MPS) I is a severe lysosomal storage disease caused by α-L-iduronidase (IDUA) deficiency, which results in accumulation of non-degraded glycosaminoglycans in lysosomes. Costly enzyme replacement therapy (ERT) is the conventional treatment for MPS I. Toward producing a more cost-effective and safe alternative to the commercial mammalian cell-based production systems, we have produced recombinant human IDUA in seeds of an Arabidopsis mutant to generate the enzyme in a biologically active and non-immunogenic form containing predominantly high mannose N-linked glycans. Recombinant enzyme in ERT is generally thought to require a mannose 6-phosphate (M6P) targeting signal for endocytosis into patient cells and for intracellular delivery to the lysosome. Toward effecting in planta phosphorylation, the human M6P elaboration machinery was successfully co-expressed along with the recombinant human IDUA using a single multi-gene construct. Uptake studies using purified putative M6P-IDUA generated in planta on cultured MPS I primary fibroblasts indicated that the endocytosed recombinant lysosomal enzyme led to substantial reduction of glycosaminoglycans. However, the efficiency of the putative M6P-IDUA in reducing glycosaminoglycan storage was comparable with the efficiency of the purified plant mannose-terminated IDUA, suggesting a poor in planta M6P-elaboration by the expressed machinery. Although the in planta M6P-tagging process efficiency would need to be improved, an exciting outcome of our work was that the plant-derived mannose-terminated IDUA yielded results comparable to those obtained with the commercial IDUA (Aldurazyme® (Sanofi, Paris, France)), and a significant amount of the plant-IDUA is trafficked by a M6P receptor-independent pathway. Thus, a plant-based platform for generating lysosomal hydrolases may represent an alternative and cost-effective strategy to the conventional ERT, without the requirement for additional processing to create the M6P motif.

Generation of human induced pluripotent stem cell line UNIGEi001-A from a 2-years old patient with Mucopolysaccharidosis type IH disease.

Mucopolysaccharidosis type I-Hurler (MPS1-H) is the most severe form of inherited metabolic diseases caused by mutations in the IDUA gene. The resulting deficiency of alpha L-iduronidase enzyme leads to a progressive accumulation of glycosaminoglycans in lysosomes which damages multiple organs and highly reduces life expectancy of affected children. Skin fibroblasts of a 2-year-old MPS1-H male, carrying two mutations in each IDUA alleles (H358_T364del; W402X), were reprogrammed into induced pluripotent stem cells (iPSCs) using the CytoTune-iPS Sendai Reprogramming method applying Yamanaka-factors (OCT4, SOX2, KLF4, c-MYC). iPSCs expressed pluripotency transcription factors while iPSC-derived embryoid bodies reveal markers of the three germ layers.