Molecular Mechanisms in Pathophysiology of Mucopolysaccharidosis and Prospects for Innovative Therapy.

Mucopolysaccharidoses (MPSs) are a group of inborn errors of the metabolism caused by a deficiency in the lysosomal enzymes required to break down molecules called glycosaminoglycans (GAGs). These GAGs accumulate over time in various tissues and disrupt multiple biological systems, including catabolism of other substances, autophagy, and mitochondrial function. These pathological changes ultimately increase oxidative stress and activate innate immunity and inflammation. We have described the pathophysiology of MPS and activated inflammation in this paper, starting with accumulating the primary storage materials, GAGs. At the initial stage of GAG accumulation, affected tissues/cells are reversibly affected but progress irreversibly to: (1) disruption of substrate degradation with pathogenic changes in lysosomal function, (2) cellular dysfunction, secondary/tertiary accumulation (toxins such as GM2 or GM3 ganglioside, etc.), and inflammatory process, and (3) progressive tissue/organ damage and cell death (e.g., skeletal dysplasia, CNS impairment, etc.). For current and future treatment, several potential treatments for MPS that can penetrate the blood-brain barrier and bone have been proposed and/or are in clinical trials, including targeting peptides and molecular Trojan horses such as monoclonal antibodies attached to enzymes via receptor-mediated transport. Gene therapy trials with AAV, ex vivo LV, and Sleeping Beauty transposon system for MPS are proposed and/or underway as innovative therapeutic options. In addition, possible immunomodulatory reagents that can suppress MPS symptoms have been summarized in this review.

Correction of symptoms of Huntington disease by genistein through FOXO3-mediated autophagy stimulation.

Huntington disease (HD) is a neurodegenerative disorder caused by a mutation in the HTT gene. The expansion of CAG triplets leads to the appearance of misfolded HTT (huntingtin) forming aggregates and leading to impairment of neuronal functions. Here we demonstrate that stimulation of macroautophagy/autophagy by genistein (4',5,7-trihydroxyisoflavone or 5,7-dihydroxy-3-(4-hydroxyphenyl)-4 H-1-benzopyran-4-one) caused a reduction of levels of mutated HTT in brains of HD mice and correction of their behavior as assessed in a battery of cognitive, anxiety and motor tests, even if the compound was administered after symptoms had developed in the animals. Biochemical and immunological parameters were also improved in HD mice. Studies on molecular mechanisms of genistein-mediated stimulation of autophagy in HD cells indicated the involvement of the FOXO3-related pathway. In conclusion, treatment with genistein stimulates the autophagy process in the brains of HD mice, leading to correction of symptoms of HD, suggesting that it might be considered as a potential drug for this disease. Combined with a very recently published report indicating that impaired autophagy may be a major cause of neurodegenerative changes, these results may indicate the way to the development of effective therapeutic approaches for different neurodegenerative diseases by testing compounds (or possibly combinations of compounds) capable of stimulating autophagy and/or unblocking this process.Abbreviations: CNS: central nervous system; EPM: elevated plus-maze; GOT1/ASPAT: glutamic-oxaloacetic transaminase 1, soluble; GPT/ALAT/ALT: glutamic pyruvic transaminase, soluble; HD: Huntington disease; HTT: huntingtin; IL: interleukin; mHTT: mutant huntingtin; NOR: novel object recognition; MWM: Morris water maze; OF: open field; ROS: reactive oxygen species; TNF: tumor necrosis factor.

Therapeutic potential of lithium chloride and valproic acid against neuronopathic types of mucopolysaccharidoses through induction of the autophagy process.

Mucopolysaccharidoses (MPS) are a group of inherited disorders, caused by mutations in the genes coding for proteins involved (directly or indirectly) in glycosaminoglycan (GAG) degradation. A lack or drastically decreased residual activity of a GAG-degrading enzyme leads to the storage of these compounds, thus damaging proper functions of different cells, including neurons. The disease leads to serious psycho-motor dysfunctions and death before reaching the adulthood. Until now, induction of the autophagy process was considered as one of the therapeutic strategies for treatment of diseases caused by protein aggregation (Alzheimer's, Parkinson's, and Huntington's diseases). However, this strategy has only been recently suggested as a potential therapy for MPS. In this work, we show that the pharmacological stimulation of autophagy, by using valproic acid and lithium chloride, led to accelerated degradation of accumulated GAGs. Cytotoxicity tests indicated the safety of the use of the investigated compounds. We observed an increased number of lysosomes and enhanced degradation of heparan sulfate (one of GAGs). Induction of the autophagy process was confirmed by measuring abundance of the marker proteins, including LC3-II. Moreover, inhibition of this process resulted in abolition of the valproic acid- and LiCl-mediated reduction in GAG levels. This is the first report on the possibility of using valproic acid and lithium chloride for reducing levels of GAGs in neuronopathic forms of MPS.

Molecular Trojan Horses for treating lysosomal storage diseases.

Lysosomal storage diseases (LSDs) are caused by monogenic mutations in genes encoding for proteins related to the lysosomal function. Lysosome plays critical roles in molecule degradation and cell signaling through interplay with many other cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes. Even though several strategies (i.e., protein replacement and gene therapy) have been attempted for LSDs with promising results, there are still some challenges when hard-to-treat tissues such as bone (i.e., cartilages, ligaments, meniscus, etc.), the central nervous system (mostly neurons), and the eye (i.e., cornea, retina) are affected. Consistently, searching for novel strategies to reach those tissues remains a priority. Molecular Trojan Horses have been well-recognized as a potential alternative in several pathological scenarios for drug delivery, including LSDs. Even though molecular Trojan Horses refer to genetically engineered proteins to overcome the blood-brain barrier, such strategy can be extended to strategies able to transport and deliver drugs to specific tissues or cells using cell-penetrating peptides, monoclonal antibodies, vesicles, extracellular vesicles, and patient-derived cells. Only some of those platforms have been attempted in LSDs. In this paper, we review the most recent efforts to develop molecular Trojan Horses and discuss how this strategy could be implemented to enhance the current efficacy of strategies such as protein replacement and gene therapy in the context of LSDs.

Clinical presentation of 13 children with alkaptonuria.

Until now, only a few studies have focused on the early onset of symptoms of alkaptonuria (AKU) in the pediatric population. This prospective, longitudinal study is a comprehensive approach to the assessment of children with recognized AKU during childhood. The study includes data from 32 visits of 13 patients (five males, eight females; age 4-17 years) with AKU. A clinical evaluation was performed with particular attention to eye, ear, and skin pigmentation, musculoskeletal complaints, magnetic resonance imaging (MRI), and ultrasound (US) imaging abnormalities. The cognitive functioning and adaptive abilities were examined. Molecular genetic analyses were performed. The most common symptoms observed were dark urine (13/13), followed by joint pain (6/13), and dark ear wax (6/13). In 4 of 13 patients the values obtained in the KOOS-child questionnaire were below the reference values. MRI and US did not show degenerative changes in knee cartilages. One child had nephrolithiasis. Almost half of the children with AKU (5/13) presented deficits in cognitive functioning and/or adaptive abilities. The most frequent HGD variants observed in the patients were c.481G>A (p.Gly161Arg) mutation and the c.240A>T (p.His80Gln) polymorphism. The newly described allele of the HGD gene (c.948G>T, p.Val316Phe) which is potentially pathogenic was identified.

Actin Cytoskeleton Polymerization and Focal Adhesion as Important Factors in the Pathomechanism and Potential Targets of Mucopolysaccharidosis Treatment.

The main approach used in the current therapy of mucopolysaccharidosis (MPS) is to reduce the levels of glycosaminoglycans (GAGs) in cells, the deposits considered to be the main cause of the disease. Previous studies have revealed significant differences in the expression of genes encoding proteins involved in many processes, like those related to actin filaments, in MPS cells. Since the regulation of actin filaments is essential for the intracellular transport of specific molecules, the process which may affect the course of MPSs, the aim of this study was to evaluate the changes that occur in the actin cytoskeleton and focal adhesion in cells derived from patients with this disease, as well as in the MPS I mouse model, and to assess whether they could be potential therapeutic targets for different MPS types. Western-blotting, flow cytometry and transcriptomic analyses were employed to address these issues. The levels of the key proteins involved in the studied processes, before and after specific treatment, were assessed. We have also analyzed transcripts whose levels were significantly altered in MPS cells. We identified genes whose expressions were changed in the majority of MPS types and those with particularly highly altered expression. For the first time, significant changes in the expression of genes involved in the actin cytoskeleton structure/functions were revealed which may be considered as an additional element in the pathogenesis of MPSs. Our results suggest the possibility of using the actin cytoskeleton as a potential target in therapeutic approaches for this disease.

Bone Growth Induction in Mucopolysaccharidosis IVA Mouse.

Mucopolysaccharidosis IVA (MPS IVA; Morquio A syndrome) is caused by a deficiency of the N-acetylgalactosamine-6-sulfate-sulfatase (GALNS) enzyme, leading to the accumulation of glycosaminoglycans (GAG), keratan sulfate (KS) and chondroitin-6-sulfate (C6S), mainly in cartilage and bone. This lysosomal storage disorder (LSD) is characterized by severe systemic skeletal dysplasia. To this date, none of the treatment options for the MPS IVA patients correct bone pathology. Enzyme replacement therapy with elosulfase alpha provides a limited impact on bone growth and skeletal lesions in MPS IVA patients. To improve bone pathology, we propose a novel gene therapy with a small peptide as a growth-promoting agent for MPS IVA. A small molecule in this peptide family has been found to exert biological actions over the cardiovascular system. This work shows that an AAV vector expressing a C-type natriuretic (CNP) peptide induces bone growth in the MPS IVA mouse model. Histopathological analysis showed the induction of chondrocyte proliferation. CNP peptide also changed the pattern of GAG levels in bone and liver. These results suggest the potential for CNP peptide to be used as a treatment in MPS IVA patients.

Roles of the Oxytocin Receptor (OXTR) in Human Diseases.

The oxytocin receptor (OXTR), encoded by the OXTR gene, is responsible for the signal transduction after binding its ligand, oxytocin. Although this signaling is primarily involved in controlling maternal behavior, it was demonstrated that OXTR also plays a role in the development of the nervous system. Therefore, it is not a surprise that both the ligand and the receptor are involved in the modulation of behaviors, especially those related to sexual, social, and stress-induced activities. As in the case of every regulatory system, any disturbances in the structures or functions of oxytocin and OXTR may lead to the development or modulation of various diseases related to the regulated functions, which in this case include either mental problems (autism, depression, schizophrenia, obsessive-compulsive disorders) or those related to the functioning of reproductive organs (endometriosis, uterine adenomyosis, premature birth). Nevertheless, OXTR abnormalities are also connected to other diseases, including cancer, cardiac disorders, osteoporosis, and obesity. Recent reports indicated that the changes in the levels of OXTR and the formation of its aggregates may influence the course of some inherited metabolic diseases, such as mucopolysaccharidoses. In this review, the involvement of OXTR dysfunctions and OXTR polymorphisms in the development of different diseases is summarized and discussed. The analysis of published results led us to suggest that changes in OXTR expression and OXTR abundance and activity are not specific to individual diseases, but rather they influence processes (mostly related to behavioral changes) that might modulate the course of various disorders. Moreover, a possible explanation of the discrepancies in the published results of effects of the OXTR gene polymorphisms and methylation on different diseases is proposed.

Decreased Levels of Chaperones in Mucopolysaccharidoses and Their Elevation as a Putative Auxiliary Therapeutic Approach.

Mucopolysaccharidoses (MPS) are rare genetic disorders belonging to the lysosomal storage diseases. They are caused by mutations in genes encoding lysosomal enzymes responsible for degrading glycosaminoglycans (GAGs). As a result, GAGs accumulate in lysosomes, leading to impairment of cells, organs and, consequently, the entire body. Many of the therapies proposed thus far require the participation of chaperone proteins, regardless of whether they are therapies in common use (enzyme replacement therapy) or remain in the experimental phase (gene therapy, STOP-codon-readthrough therapy). Chaperones, which include heat shock proteins, are responsible for the correct folding of other proteins to the most energetically favorable conformation. Without their appropriate levels and activities, the correct folding of the lysosomal enzyme, whether supplied from outside or synthesized in the cell, would be impossible. However, the baseline level of nonspecific chaperone proteins in MPS has never been studied. Therefore, the purpose of this work was to determine the basal levels of nonspecific chaperone proteins of the Hsp family in MPS cells and to study the effect of normalizing GAG concentrations on these levels. Results of experiments with fibroblasts taken from patients with MPS types I, II, IIIA, IIIB, IIIC, IID, IVA, IVB, VI, VII, and IX, as well as from the brains of MPS I mice (Idua-/-), indicated significantly reduced levels of the two chaperones, Hsp70 and Hsp40. Interestingly, the reduction in GAG levels in the aforementioned cells did not lead to normalization of the levels of these chaperones but caused only a slight increase in the levels of Hsp40. An additional transcriptomic analysis of MPS cells indicated that the expression of other genes involved in protein folding processes and the cell response to endoplasmic reticulum stress, resulting from the appearance of abnormally folded proteins, was also modulated. To summarize, reduced levels of chaperones may be an additional cause of the low activity or inactivity of lysosomal enzymes in MPS. Moreover, this may point to causes of treatment failure where the correct structure of the enzyme supplied or synthesized in the cell is crucial to lower GAG levels.

Activities of (Poly)phenolic Antioxidants and Other Natural Autophagy Modulators in the Treatment of Sanfilippo Disease: Remarkable Efficacy of Resveratrol in Cellular and Animal Models.

Sanfilippo disease, caused by mutations in the genes encoding heparan sulfate (HS) (a glycosaminoglycan; GAG) degradation enzymes, is a mucopolysaccharidosis (MPS), which is also known as MPS type III, and is characterized by subtypes A, B, C, and D, depending on identity of the dysfunctional enzyme. The lack of activity or low residual activity of an HS-degrading enzyme leads to excess HS in the cells, impairing the functions of different types of cells, including neurons. The disease usually leads to serious psychomotor dysfunction and death before adulthood. In this work, we show that the use of molecules known as dietary (poly)phenolic antioxidants and other natural compounds known as autophagy activators (genistein, capsaicin, curcumin, resveratrol, trehalose, and calcitriol) leads to accelerated degradation of accumulated HS in the fibroblasts of all subtypes of MPS III. Both the cytotoxicity tests we performed and the available literature data indicated that the use of selected autophagy inducers was safe. Since it showed the highest effectivity in cellular models, resveratrol efficacy was tested in experiments with a mouse model of MPS IIIB. Urinary GAG levels were normalized in MPS IIIB mice treated with 50 mg/kg/day resveratrol for 12 weeks or longer. Behavioral tests indicated complete correction of hyperactivity and anxiety in these animals. Biochemical analyses indicated that administration of resveratrol caused autophagy stimulation through an mTOR-independent pathway in the brains and livers of the MPS IIIB mice. These results indicate the potential use of resveratrol (and possibly other autophagy stimulators) in the treatment of Sanfilippo disease.

Apoptosis activation during Lagovirus europaeus/GI.2 infection in rabbits.

Rabbit Haemorrhagic Disease (RHD) is a severe disease caused by Lagovirus europaeus/GI.1 and GI.2. Immunological processes such as apoptosis are important factors involved in the pathogenesis of Rabbit Haemorrhagic Disease (RHD). The process of programmed cell death has been quite well characterized in infection with GI.1 strains, but apoptosis in infection with GI.2 strains has not been widely studied. This is particularly important as several studies have shown that significant differences in the host immune response are observed during infection with different strains of Lagovirus europaeus. In this study, we analyzed the gene expression, protein levels and activity of key apoptotic cell death factors in the spleen, kidney, lung, and heart of rabbits. As a result, we showed that there is a significant increase in caspase-3, Bax, Bcl2 and Bax/Bcl2 mRNA gene expression ratio in organs of infected animals. Our results show also increased levels of cleaved caspase-3, caspase-6 and PARP. Moreover, significant activity of caspase-3 was also detected. Our results indicate that caspase-3, caspase-6 and genes coding Bcl2 family proteins play a key role in the apoptotic response in Lagovirus europaeus/GI.2 infection in organs that are not the target of virus replication.

Biological aspects of phage therapy versus antibiotics against Salmonella enterica serovar Typhimurium infection of chickens.

Phage therapy is a promising alternative treatment of bacterial infections in human and animals. Nevertheless, despite the appearance of many bacterial strains resistant to antibiotics, these drugs still remain important therapeutics used in human and veterinary medicine. Although experimental phage therapy of infections caused by Salmonella enterica was described previously by many groups, those studies focused solely on effects caused by bacteriophages. Here, we compared the use of phage therapy (employing a cocktail composed of two previously isolated and characterized bacteriophages, vB_SenM-2 and vB_Sen-TO17) and antibiotics (enrofloxacin and colistin) in chickens infected experimentally with S. enterica serovar Typhimurium. We found that the efficacies of both types of therapies (i.e. the use of antibiotics and phage cocktail) were high and very similar to one another when the treatment was applied shortly (one day) after the infection. Under these conditions, S. Typhimurium was quickly eliminated from the gastrointestinal tract (GIT), to the amount not detectable by the used methods. However, later treatment (2 or 4 days after detection of S. Typhimurium in chicken feces) with the phage cocktail was significantly less effective. Bacteriophages remained in the GIT for up to 2-3 weeks, and then were absent in feces and cloaca swabs. Interestingly, both phages could be found in various organs of chickens though with a relatively low abundance. No development of resistance of S. Typhimurium to phages or antibiotics was detected during the experiment. Importantly, although antibiotics significantly changed the GIT microbiome of chickens in a long-term manner, analogous changes caused by phages were transient, and the microbiome normalized a few weeks after the treatment. In conclusion, phage therapy against S. Typhimurium infection in chickens appeared as effective as antibiotic therapy (with either enrofloxacin or colistin), and less invasive than the use the antibiotics as fewer changes in the microbiome were observed.

Molecular Mechanism of Induction of Bone Growth by the C-Type Natriuretic Peptide.

The skeletal development process in the body occurs through sequential cellular and molecular processes called endochondral ossification. Endochondral ossification occurs in the growth plate where chondrocytes differentiate from resting, proliferative, hypertrophic to calcified zones. Natriuretic peptides (NPTs) are peptide hormones with multiple functions, including regulation of blood pressure, water-mineral balance, and many metabolic processes. NPTs secreted from the heart activate different tissues and organs, working in a paracrine or autocrine manner. One of the natriuretic peptides, C-type natriuretic peptide-, induces bone growth through several mechanisms. This review will summarize the knowledge, including the newest discoveries, of the mechanism of CNP activation in bone growth.

Promoter considerations in the design of lentiviral vectors for use in treating lysosomal storage diseases.

More than 50 lysosomal storage diseases (LSDs) are associated with lysosomal dysfunctions with the frequency of 1:5,000 live births. As a result of missing enzyme activity, the lysosome dysfunction accumulates undegraded or partially degraded molecules, affecting the entire body. Most of them are life-threatening diseases where patients could die within the first or second decade of life. Approximately 20 LSDs have the approved treatments, which do not provide the cure for the disorder. Therefore, the delivery of missing genes through gene therapy is a promising approach for LSDs. Over the years, ex vivo lentiviral-mediated gene therapy for LSDs has been approached using different strategies. Several clinical trials for LSDs are under investigation.Ex vivo lentiviral-mediated gene therapy needs optimization in dose, time of delivery, and promoter-driven expression. Choosing suitable promoters seems to be one of the important factors for the effective expression of the dysfunctional enzyme. This review summarizes the research on therapy for LSDs that has used different lentiviral vectors, emphasizing gene promoters.

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.

Impaired ion homeostasis as a possible associate factor in mucopolysaccharidosis pathogenesis: transcriptomic, cellular and animal studies.

Mucopolysaccharidoses (MPS) are a group of diseases caused by mutations resulting in deficiencies of lysosomal enzymes which lead to the accumulation of partially undegraded glycosaminoglycans (GAG). This phenomenon causes severe and chronic disturbances in the functioning of the organism, and leads to premature death. The metabolic defects affect also functions of the brain in most MPS types (except types IV, VI, and IX). The variety of symptoms, as well as the ineffectiveness of GAG-lowering therapies, question the early theory that GAG storage is the only cause of these diseases. As disorders of ion homeostasis increasingly turn out to be co-causes of the pathogenesis of various human diseases, the aim of this work was to determine the perturbations related to the maintenance of the ion balance at both the transcriptome and cellular levels in MPS. Transcriptomic studies, performed with fibroblasts derived from patients with all types/subtypes of MPS, showed extensive changes in the expression of genes involved in processes related to ion binding, transport and homeostasis. Detailed analysis of these data indicated specific changes in the expression of genes coding for proteins participating in the metabolism of Ca2+, Fe2+ and Zn2+. The results of tests carried out with the mouse MPS I model (Idua-/-) showed reductions in concentrations of these 3 ions in the liver and spleen. The results of these studies indicate for the first time ionic concentration disorders as possible factors influencing the course of MPS and show them as hypothetical, additional therapeutic targets for this rare disease.

Glycosaminoglycans as Biomarkers for Mucopolysaccharidoses and Other Disorders.

Glycosaminoglycans (GAGs) are present in proteoglycans, which play critical physiological roles in various tissues. They are known to be elevated in mucopolysaccharidoses (MPS), a group of rare inherited metabolic diseases in which the lysosomal enzyme required to break down one or more GAG is deficient. In a previous study, we found elevation of GAGs in a subset of patients without MPS. In the current study, we aim to investigate serum GAG levels in patients with conditions beyond MPS. In our investigated samples, the largest group of patients had a clinical diagnosis of viral or non-viral encephalopathy. Clinical diagnoses and conditions also included epilepsy, fatty acid metabolism disorders, respiratory and renal disorders, liver disorders, hypoglycemia, developmental disorders, hyperCKemia, myopathy, acidosis, and vomiting disorders. While there was no conclusive evidence across all ages for any disease, serum GAG levels were elevated in patients with encephalopathy and some patients with other conditions. These preliminary findings suggest that serum GAGs are potential biomarkers in MPS and other disorders. In conclusion, we propose that GAGs elevated in blood can be used as biomarkers in the diagnosis and prognosis of various diseases in childhood; however, further designed experiments with larger sample sizes are required.

Potential of genistein-induced autophagy in the treatment of neurodegenerative diseases / Potencjal autofagii indukowanej przez genisteine w leczeniu chorób neurodegeneracyjnych.

Development of therapies for neurodegenerative diseases, disorders characterized by progressing loss of neurons, is a great challenge for current medicine. Searching for drugs for these diseases is being proceeded in many laboratories in the world. To date, several therapeutical strategies have been proposed which, however, are either of insufficient efficacy or at the early preclinical stages. One of the newest concepts is elevated efficiency of degradation of protein aggregates which are causes of 70% of these diseases. Autophagy, i.e. lysosomal degradation of macromolecules, is a process which could be employed in such a strategy Searching for a compound which would not only stimulate autophagy but also reveal safety in a long-term usage and be able to cross the blood-brain-barrier led to studies on one of flavonoids, genistein which occurs at high concentrations in soy. Experiments with this compound indicated its enormous efficiency in removing protein aggregated formed by beta-amyloid, hyperphosphorylated tau protein, and mutant huntingtin. Moreover, using animal models of these diseases, correction of cognitive and motoric symptoms was demonstrated. Considering safety of genistein as well as its ability to crossing the blood-brain-barrier, one may assume that this molecule is a candidate for an effective drug in therapies of not only Alzheimer disease and Huntington disease, but also other disorders caused be protein aggregates. In this article, recent results of studies on the use of genistein in different models of neurodegenerative diseases are summarized, with special emphasis on its autophagy-dependent action.

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.

Ferroptosis and Its Modulation by Autophagy in Light of the Pathogenesis of Lysosomal Storage Diseases.

Ferroptosis is one of the recently described types of cell death which is dependent on many factors, including the accumulation of iron and lipid peroxidation. Its induction requires various signaling pathways. Recent discovery of ferroptosis induction pathways stimulated by autophagy, so called autophagy-dependent ferroptosis, put our attention on the role of ferroptosis in lysosomal storage diseases (LSD). Lysosome dysfunction, observed in these diseases, may influence ferroptosis efficiency, with as yet unknown consequences for the function of cells, tissues, and organisms, due to the effects of ferroptosis on physiological and pathological metabolic processes. Modulation of levels of ferrous ions and enhanced oxidative stress, which are primary markers of ferroptosis, are often described as processes associated with the pathology of LSD. Inhibition of autophagy flux and resultant accumulation of autophagosomes in neuronopathic LSD may induce autophagy-dependent ferroptosis, indicating a considerable contribution of this process in neurodegeneration. In this review article, we describe molecular mechanisms of ferroptosis in light of LSD, underlining the modulation of levels of ferroptosis markers in these diseases. Furthermore, we propose a hypothesis about the possible involvement of autophagy-dependent ferroptosis in these disorders.