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Sanfilippo disease is a lysosomal storage disorder from the group of mucopolysaccharidoses (MPS), characterized by storage of glycosaminoglycans (GAGs); thus, it is also called MPS type III. The syndrome is divided into 4 subtypes (MPS III A, B, C and D). Despite the storage of the same GAG, heparan sulfate (HS), the course of these subtypes can vary considerably. Here, we comprehensively evaluated the levels of protein aggregates (APP, β-amyloid, p-tau, α-synuclein, TDP43) in fibroblasts derived from patients with all MPS III subtypes, and tested whether lowering GAG levels results in a decrease in the levels of the investigated proteins and the number of aggregates they form. Elevated levels of APP, β-amyloid, tau, and TDP43 proteins were evident in all MPS III subtypes, and elevated levels of p-tau and α-synuclein were demonstrated in all subtypes except MPS IIIC. These findings were confirmed in the neural tissue of MPS IIIB mice. Fluorescence microscopy studies also indicated a high number of protein aggregates formed by β-amyloid and tau in all cell lines tested, and a high number of aggregates of p-tau, TDP43, and α-synuclein in all lines except MPS IIIC. Reduction of GAG levels by genistein led to the decrease of levels of all tested proteins and their aggregates except α-synuclein, indicating a relationship between GAG levels and those of some protein aggregates. This work describes for the first time the problem of deposited protein aggregates in all subtypes of Sanfilippo disease and suggests that GAGs are partly responsible for the formation of protein aggregates., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mucopolysaccharidoses (MPSs) are childhood diseases caused by inherited deficiencies in glycosaminoglycan degradation. Most MPSs involve neurodegeneration, which to date is untreatable. Currently, most therapeutic strategies aim at correcting the primary genetic defect. Among these strategies, gene therapy has shown great potential, although its clinical application is challenging. We have shown previously in an MPS-IIIA mouse model that the molecular tweezer (MT) CLR01, a potent, broad-spectrum anti-amyloid small molecule, inhibits secondary amyloid storage, facilitates amyloid clearance, and protects against neurodegeneration. Here, we demonstrate that combining CLR01 with adeno-associated virus (AAV)-mediated gene therapy, targeting both the primary and secondary pathologic storage in MPS-IIIA mice, results in a synergistic effect that improves multiple therapeutic outcomes compared to each monotherapy. Moreover, we demonstrate that CLR01 is effective therapeutically in mouse models of other forms of neuronopathic MPS, MPS-I, and MPS-IIIC. These strongly support developing MTs as an effective treatment option for neuronopathic MPSs, both on their own and in combination with gene therapy, to improve therapeutic efficacy and translation into clinical application., Competing Interests: Declaration of interests The compositions of matter (patent 1, application 2) and methods of use (applications 3 and 4) of CLR01 are protected by the patents and patent applications listed below. G.B. and T.S. are co-inventors on 1 and 2. A.F. and G.B. are co-inventors on 3 and 4., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described., Methods: Here, we isolated EVs secreted by the microglial cells after treatment with GAGs purified from urines of Sanfilippo patients (sfGAGs-EVs) or from age-matched healthy subjects (nGAGs-EVs) to explore the EVs' proteins and small RNA profiles using LC-MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following nGAGs- or sfGAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy., Results: Functional enrichment analysis for both proteomics and RNA sequencing data from sfGAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with sfGAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines., Conclusions: This study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies., (© 2024. The Author(s).)

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal inherited disease caused by mutations in gene encoding the lysosomal enzyme N-acetyl-alpha-glucosaminidase (NAGLU). These mutations result in reduced NAGLU activity, preventing it from catalyzing the hydrolysis of the glycosaminoglycan heparan sulfate (HS). There are currently no approved treatments for MPS IIIB. A novel approach in the treatment of lysosomal storage diseases is the use of pharmacological chaperones (PC). In this study, we used a drug repurposing approach to identify and characterize novel potential PCs for NAGLU enzyme. We modeled the interaction of natural and artificial substrates within the active cavity of NAGLU (orthosteric site) and predicted potential allosteric sites. We performed a virtual screening for both the orthosteric and the predicted allosteric site against a curated database of human tested molecules. Considering the binding affinity and predicted blood-brain barrier permeability and gastrointestinal absorption, we selected atovaquone and piperaquine as orthosteric and allosteric PCs. The PCs were evaluated by their capacity to bind NAGLU and the ability to restore the enzymatic activity in human MPS IIIB fibroblasts These results represent novel PCs described for MPS IIIB and demonstrate the potential to develop novel therapeutic alternatives for this and other protein deficiency diseases., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Mucopolysaccharidosis type IIIA (MPS IIIA) is a rare paediatric lysosomal storage disorder, caused by the progressive accumulation of heparan sulphate, resulting in neurocognitive decline and behavioural abnormalities. Anecdotal reports from paediatricians indicate a more severe neurodegeneration in MPS IIIA patients, following infection, suggesting inflammation as a potential driver of neuropathology. To test this hypothesis, we performed acute studies in which WT and MPS IIIA mice were challenged with the TLR3-dependent viral mimetic poly(I:C). The challenge with an acute high poly(I:C) dose exacerbated systemic and brain cytokine expression, especially IL-1β in the hippocampus. This was accompanied by an increase in caspase-1 activity within the brain of MPS IIIA mice with concomitant loss of hippocampal GFAP and NeuN expression. Similar levels of cell damage, together with exacerbation of gliosis, were also observed in MPS IIIA mice following low chronic poly(I:C) dosing. While further investigation is warranted to fully understand the extent of IL-1β involvement in MPS IIIA exacerbated neurodegeneration, our data robustly reinforces our previous findings, indicating IL-1β as a pivotal catalyst for neuropathological processes in MPS IIIA., (© 2024. The Author(s).)

MPS IIIC is a lysosomal storage disease caused by mutations in heparan-α-glucosaminide N-acetyltransferase (HGSNAT), for which no treatment is available. Because HGSNAT is a trans-lysosomal-membrane protein, gene therapy for MPS IIIC needs to transduce as many cells as possible for maximal benefits. All cells continuously release extracellular vesicles (EVs) and communicate by exchanging biomolecules via EV trafficking. To address the unmet need, we developed a rAAV-hHGSNAT EV vector with an EV-mRNA-packaging signal in the 3'UTR to facilitate bystander effects, and tested it in an in vitro MPS IIIC model. In human MPS IIIC cells, rAAV-hHGSNAT EV enhanced HGSNAT mRNA and protein expression, EV-hHGSNAT-mRNA packaging, and cleared GAG storage. Importantly, incubation with EVs led to hHGSNAT protein expression and GAG contents clearance in recipient MPS IIIC cells. Further, rAAV-hHGSNAT EV transduction led to the reduction of pathological EVs in MPS IIIC cells to normal levels, suggesting broader therapeutic benefits. These data demonstrate that incorporating the EV-mRNA-packaging signal into a rAAV-hHGSNAT vector enhances EV packaging of hHGSNAT-mRNA, which can be transported to non-transduced cells and translated into functional rHGSNAT protein, facilitating cross-correction of disease pathology. This study supports the therapeutic potential of rAAV EV for MPS IIIC, and broad diseases, without having to transduce every cell., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Heparan sulfate (HS) is degraded in lysosome by a series of glycosidases. Before the glycosidases can act, the terminal glucosamine of HS must be acetylated by the integral lysosomal membrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT). Mutations of HGSNAT cause HS accumulation and consequently mucopolysaccharidosis IIIC, a devastating lysosomal storage disease characterized by progressive neurological deterioration and early death where no treatment is available. HGSNAT catalyzes a unique transmembrane acetylation reaction where the acetyl group of cytosolic acetyl-CoA is transported across the lysosomal membrane and attached to HS in one reaction. However, the reaction mechanism remains elusive. Here we report six cryo-EM structures of HGSNAT along the reaction pathway. These structures reveal a dimer arrangement and a unique structural fold, which enables the elucidation of the reaction mechanism. We find that a central pore within each monomer traverses the membrane and controls access of cytosolic acetyl-CoA to the active site at its luminal mouth where glucosamine binds. A histidine-aspartic acid catalytic dyad catalyzes the transfer reaction via a ternary complex mechanism. Furthermore, the structures allow the mapping of disease-causing variants and reveal their potential impact on the function, thus creating a framework to guide structure-based drug discovery efforts., (© 2024. The Author(s).)

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterized by neurological and skeletal pathologies caused by reduced activity of the lysosomal hydrolase, sulfamidase, and the subsequent primary accumulation of undegraded heparan sulfate (HS). Respiratory pathology is considered secondary in MPS IIIA and the mechanisms are not well understood. Changes in the amount, metabolism, and function of pulmonary surfactant, the substance that regulates alveolar interfacial surface tension and modulates lung compliance and elastance, have been reported in MPS IIIA mice. Here we investigated changes in lung function in 20-wk-old control and MPS IIIA mice with a closed and open thoracic cage, diaphragm contractile properties, and potential parenchymal remodeling. MPS IIIA mice had increased compliance and airway resistance and reduced tissue damping and elastance compared with control mice. The chest wall impacted lung function as observed by an increase in airway resistance and a decrease in peripheral energy dissipation in the open compared with the closed thoracic cage state in MPS IIIA mice. Diaphragm contractile forces showed a decrease in peak twitch force, maximum specific force, and the force-frequency relationship but no change in muscle fiber cross-sectional area in MPS IIIA mice compared with control mice. Design-based stereology did not reveal any parenchymal remodeling or destruction of alveolar septa in the MPS IIIA mouse lung. In conclusion, the increased storage of HS which leads to biochemical and biophysical changes in pulmonary surfactant also affects lung and diaphragm function, but has no impact on lung or diaphragm structure at this stage of the disease. NEW & NOTEWORTHY Heparan sulfate storage in the lungs of mucopolysaccharidosis type IIIA (MPS IIIA) mice leads to changes in lung function consistent with those of an obstructive lung disease and includes an increase in lung compliance and airway resistance and a decrease in tissue elastance. In addition, diaphragm muscle contractile strength is reduced, potentially further contributing to lung function impairment. However, no changes in parenchymal lung structure were observed in mice at 20 wk of age.

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs., (© 2024 International Society for Advancement of Cytometry.)

Lysosomal storage diseases (LSDs) are a group of monogenic diseases characterized by mutations in genes coding for proteins associated with the lysosomal function. Despite the monogenic nature, LSDs patients exhibit variable and heterogeneous clinical manifestations, prompting investigations into epigenetic factors underlying this phenotypic diversity. In this study, we focused on the potential role of epigenetic mechanisms in the pathogenesis of mucopolysaccharidosis IIIB (MPS IIIB) and mucopolysaccharidosis IVA (MPS IVA). We analyzed DNA methylation (5mC) and histone modifications (H3K14 acetylation and H3K9 trimethylation) in MPS IIIB and MPS IVA patients' fibroblasts and healthy controls. The findings revealed that global DNA hypomethylation is present in cell lines for both diseases. At the same time, histone acetylation was increased in MPS IIIB and MPS IVA cells in a donor-dependent way, further indicating a shift towards relaxed open chromatin in these MPS. Finally, the constitutive heterochromatin marker, histone H3K9 trimethylation, only showed reduced clustering in MPS IIIB cells, suggesting limited alterations in heterochromatin organization. These findings collectively emphasize the significance of epigenetic mechanisms in modulating the phenotypic variations observed in LSDs. While global DNA hypomethylation could contribute to the MPS pathogenesis, the study also highlights individual-specific epigenetic responses that might contribute to phenotypic heterogeneity. Further research into the specific genes and pathways affected by these epigenetic changes could provide insights into potential therapeutic interventions for these MPS and other LSDs., (© 2024. The Author(s).)

Background and Aim: Mucopolysaccharidosis type III (MPS III) is a rare autosomal recessive lysosomal storage disease (LSD) caused by a deficiency of lysosomal enzymes required for the catabolism of glycosaminoglycans (GAGs), mainly in the central nervous system. Trehalose has been proposed as a potential therapeutic agent to attenuate neuropathology in MPS III. We conducted a single- arm, open-label study to evaluate the efficacy of trehalose treatment in patients with MPS IIIA and MPS IIIB., Methods: Five patients with MPS III were enrolled. Trehalose was administrated intravenously (15 g/week) for 12 weeks. Health-related quality of life and cognitive function, serum biomarkers, liver, spleen, and lung imaging were assessed to evaluate trehalose efficacy at baseline and trial end (week 12)., Results: TNO-AZL Preschool children Quality of Life (TAPQOL) scores increased in all patients, and the mean scores for quality of life were increased after the intervention. Serum GAG levels were reduced in all treated patients (however, the differences were not statistically significant). Alanine aminotransferase (ALT) levels were reduced in all patients post-treatment (p=0.0039). The mean levels of aspartate transaminase (AST) were also decreased after 12 weeks of treatment with Trehalose. Decreased serum pro-oxidant-antioxidant balance and increased GPX activity were observed at the end of the study. Decreases in mean splenic length were observed, whereas the liver volume did not change., Conclusion: Improvements in health-related quality of life and serum biomarkers (GAGs, liver aminotransferase levels, antioxidant status), as well as liver and spleen size, were found following 3 months of trehalose administration in patients with MPS IIIA and MPS IIIB., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Mucopolysaccharidosis IIIC (MPSIIIC) is one of four Sanfilippo diseases sharing clinical symptoms of severe cognitive decline and shortened lifespan. The missing enzyme, heparan sulfate acetyl-CoA: α-glucosaminide-N-acetyltransferase (HGSNAT), is bound to the lysosomal membrane, therefore cannot cross the blood-brain barrier or diffuse between cells. We previously demonstrated disease correction in MPSIIIC mice using an Adeno-Associated Vector (AAV) delivering HGSNAT via intraparenchymal brain injections using an AAV2 derived AAV-truetype (AAV-TT) serotype with improved distribution over AAV9., Methods: Here, intraparenchymal AAV was delivered in sheep using catheters or Hamilton syringes, placed using Brainlab cranial navigation for convection enhanced delivery, to reduce proximal vector expression and improve spread., Results: Hamilton syringes gave improved AAV-GFP distribution, despite lower vector doses and titres. AAV-TT-GFP displayed moderately better transduction compared to AAV9-GFP but both serotypes almost exclusively transduced neurons. Functional HGSNAT enzyme was detected in 24-37% of a 140g gyrencephalic sheep brain using AAV9-HGSNAT with three injections in one hemisphere., Conclusions: Despite variabilities in volume and titre, catheter design may be critical for efficient brain delivery. These data help inform a clinical trial for MPSIIIC., (© 2023. The Author(s).)

Sanfilippo syndrome comprises a group of four genetic diseases due to the lack or decreased activity of enzymes involved in heparan sulfate (HS) catabolism. HS accumulation in lysosomes and other cellular compartments results in tissue and organ dysfunctions, leading to a wide range of clinical symptoms including severe neurodegeneration. To date, no approved treatments for Sanfilippo disease exist. Here, we report the ability of N -substituted l-iminosugars to significantly reduce substrate storage and lysosomal dysfunctions in Sanfilippo fibroblasts and in a neuronal cellular model of Sanfilippo B subtype. Particularly, we found that they increase the levels of defective α- N -acetylglucosaminidase and correct its proper sorting toward the lysosomal compartment. Furthermore, l-iminosugars reduce HS accumulation by downregulating protein levels of exostosin glycosyltransferases. These results highlight an interesting pharmacological potential of these glycomimetics in Sanfilippo syndrome, paving the way for the development of novel therapeutic approaches for the treatment of such incurable disease.

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal storage disease caused by mutations in the gene that encodes the protein N-acetyl-glucosaminidase (NAGLU). Defective NAGLU activity results in aberrant retention of heparan sulfate within lysosomes leading to progressive central nervous system (CNS) degeneration. Intravenous treatment options are limited by the need to overcome the blood-brain barrier and gain successful entry into the CNS. Additionally, we have demonstrated that AAV8 provides a broader transduction area in the MPS IIIB mouse brain compared with AAV5, 9 or rh10. A triple-capsid mutant (tcm) modification of AAV8 further enhanced GFP reporter expression and distribution. Using the MPS IIIB mouse model, we performed a study using either intracranial six site or intracisterna magna injection of AAVtcm8-codon-optimized (co)-NAGLU using untreated MPS IIIB mice as controls to assess disease correction. Disease correction was evaluated based on enzyme activity, heparan sulfate storage levels, CNS lysosomal signal intensity, coordination, activity level, hearing and survival. Both histologic and enzymatic assessments show that each injection method results in supranormal levels of NAGLU expression in the brain. In this study, we have shown correction of lifespan and auditory deficits, increased CNS NAGLU activity and reduced lysosomal storage levels of heparan sulfate following AAVtcm8-coNAGLU administration and partial correction of NAGLU activity in several peripheral organs in the murine model of MPS IIIB., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by N-sulfoglucosamine sulfohydrolase (SGSH) deficiency. SGSH removes the sulfate from N-sulfoglucosamine residues on the nonreducing end of heparan sulfate (HS-NRE) within lysosomes. Enzyme deficiency results in accumulation of partially degraded HS within lysosomes throughout the body, leading to a progressive severe neurological disease. Enzyme replacement therapy has been proposed, but further evaluation of the treatment strategy is needed. Here, we used Chinese hamster ovary cells to produce a highly soluble and fully active recombinant human sulfamidase (rhSGSH). We discovered that rhSGSH utilizes both the CI-MPR and LRP1 receptors for uptake into patient fibroblasts. A single intracerebroventricular (ICV) injection of rhSGSH in MPS IIIA mice resulted in a tissue half-life of 9 days and widespread distribution throughout the brain. Following a single ICV dose, both total HS and the MPS IIIA disease-specific HS-NRE were dramatically reduced, reaching a nadir 2 weeks post dose. The durability of effect for reduction of both substrate and protein markers of lysosomal dysfunction and a neuroimmune response lasted through the 56 days tested. Furthermore, seven weekly 148 μg doses ICV reduced those markers to near normal and produced a 99.5% reduction in HS-NRE levels. A pilot study utilizing every other week dosing in two animals supports further evaluation of less frequent dosing. Finally, our dose-response study also suggests lower doses may be efficacious. Our findings show that rhSGSH can normalize lysosomal HS storage and markers of a neuroimmune response when delivered ICV., Competing Interests: Conflict of interest The authors were employees of BioMarin Pharmaceutical Inc at the time of the studies. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Lysosomal storage diseases result in various developmental and physiological complications, including cachexia. To study the causes for the negative energy balance associated with cachexia, we assessed the impact of sulfamidase deficiency and heparan sulfate storage on energy homeostasis and metabolism in a mouse model of type IIIa mucopolysaccharidosis (MPS IIIa, Sanfilippo A syndrome). At 12-weeks of age, MPS IIIa mice exhibited fasting and postprandial hypertriglyceridemia compared with wildtype mice, with a reduction of white and brown adipose tissues. Partitioning of dietary [ 3 H]triolein showed a marked increase in intestinal uptake and secretion, whereas hepatic production and clearance of triglyceride-rich lipoproteins did not differ from wildtype controls. Uptake of dietary triolein was also elevated in brown adipose tissue (BAT), and notable increases in beige adipose tissue occurred, resulting in hyperthermia, hyperphagia, hyperdipsia, and increased energy expenditure. Furthermore, fasted MPS IIIa mice remained hyperthermic when subjected to low temperature but became cachexic and profoundly hypothermic when treated with a lipolytic inhibitor. We demonstrated that the reliance on increased lipid fueling of BAT was driven by a reduced ability to generate energy from stored lipids within the depot. These alterations arose from impaired autophagosome-lysosome fusion, resulting in increased mitochondria content in beige and BAT. Finally, we show that increased mitochondria content in BAT and postprandial dyslipidemia was partially reversed upon 5-week treatment with recombinant sulfamidase. We hypothesize that increased BAT activity and persistent increases in energy demand in MPS IIIa mice contribute to the negative energy balance observed in patients with MPS IIIa., Competing Interests: Conflict of interest The University of California, San Diego and J. D. E. have a financial interest in TEGA Therapeutics, Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. B. E. T and C. A. G. are employees of TEGA Therapeutics, Inc., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

To evaluate a new approach to Mucopolysaccharidosis type IIIA (MPS-IIIA), work was initiated on primary fibroblasts from a well-known mouse model in which sulfamidase deficiency correlates with the accumulation of heparan sulfate - the hallmark of this disease. Once the culture of fibroblasts was established, we observed continuous proliferation with a rapid growth rate, loss of contact inhibition and late passage stability, corresponding to a spontaneously immortalized cell line. The presence of the single point D31N mutation was verified and both rapid and abundant intracellular accumulation of low molecular weight HS was observed, confirming both genotype and phenotype. This cell line is a potential in vitro model system for future studies of MPS-IIIA prior to employing animal models., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sanfilippo D syndrome (mucopolysaccharidosis type IIID) is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulfatase (GNS). A mouse model was generated by constitutive knockout of the Gns gene. We studied affected mice and controls at 12, 24, 36, and 48 weeks of age for neuropathological markers of disease in the somatosensory cortex, primary motor cortex, ventral posterior nuclei of the thalamus, striatum, hippocampus, and lateral and medial entorhinal cortex. We found significantly increased immunostaining for glial fibrillary associated protein (GFAP), CD68 (a marker of activated microglia), and lysosomal-associated membrane protein-1 (LAMP-1) in Sanfilippo D mice compared to controls at 12 weeks of age in all brain regions. Intergroup differences were marked for GFAP and CD68 staining, with levels in Sanfilippo D mice consistently above controls at all age groups. Intergroup differences in LAMP-1 staining were more pronounced in 12- and 24-week age groups compared to 36- and 48-week groups, as control animals showed some LAMP-1 staining at later timepoints in some brain regions. We also evaluated the somatosensory cortex, medial entorhinal cortex, reticular nucleus of the thalamus, medial amygdala, and hippocampal hilus for subunit c of mitochondrial ATP synthase (SCMAS). We found a progressive accumulation of SCMAS in most brain regions of Sanfilippo D mice compared to controls by 24 weeks of age. Cataloging the regional neuropathology of Sanfilippo D mice may aid in understanding the disease pathogenesis and designing preclinical studies to test brain-directed treatments., Competing Interests: Declaration of Competing Interest Dr. Dickson receives research support from Genzyme and M6P Therapeutics. Dr. Cooper receives research support from Regenexbio and Neurogene., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis type IIIB (MPS IIIB) is a lysosomal disease caused by mutations in the NAGLU gene encoding α-N-acetylglucosaminidase (NAGLU) which degrades heparan sulfate in lysosomes. Deficiency in NAGLU results in lysosomal accumulation of glycosaminoglycans (GAGs) and neurological symptoms. Currently, there is no effective treatment or cure for this disease. In this study, induced pluripotent stem cell lines were established from two MPS IIIB patient fibroblast lines and differentiated into neural stem cells and neurons. MPS IIIB neural stem cells exhibited NAGLU deficiency accompanied with GAG accumulation, as well as lysosomal enlargement and secondary lipid accumulation. Treatments with recombinant NAGLU, δ-tocopherol, and 2-hydroxypropyl-b-cyclodextrin significantly reduced the disease phenotypes in these cells. These results indicate the MPS IIIB neural stem cells and neurons have the disease relevant phenotype and can be used as a cell-based disease model system for evaluation of drug efficacy and compound screening for drug development., (Published by Elsevier Inc.)

Mucopolysaccharidoses (MPSs) are a group of inherited lysosomal storage disorders associated with the deficiency of lysosomal enzymes involved in glycosaminoglycan (GAG) degradation. The resulting cellular accumulation of GAGs is responsible for widespread tissue and organ dysfunctions. The MPS III, caused by mutations in the genes responsible for the degradation of heparan sulfate (HS), includes four subtypes (A, B, C, and D) that present significant neurological manifestations such as progressive cognitive decline and behavioral disorders. The established treatments for the MPS III do not cure the disease but only ameliorate non-neurological clinical symptoms. We previously demonstrated that the natural variant of the hepatocyte growth factor NK1 reduces the lysosomal pathology and reactivates impaired growth factor signaling in fibroblasts from MPS IIIB patients. Here, we show that the recombinant NK1 is effective in rescuing the morphological and functional dysfunctions of lysosomes in a neuronal cellular model of the MPS IIIB. More importantly, NK1 treatment is able to stimulate neuronal differentiation of neuroblastoma SK-NBE cells stable silenced for the NAGLU gene causative of the MPS IIIB. These results provide the basis for the development of a novel approach to possibly correct the neurological phenotypes of the MPS IIIB as well as of other MPSs characterized by the accumulation of HS and progressive neurodegeneration., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The majority of patients affected with lysosomal storage disorders (LSD) exhibit neurological symptoms. For mucopolysaccharidosis type IIIC (MPSIIIC), the major burdens are progressive and severe neuropsychiatric problems and dementia, primarily thought to stem from neurodegeneration. Using the MPSIIIC mouse model, we studied whether clinical manifestations preceding massive neurodegeneration arise from synaptic dysfunction. Reduced levels or abnormal distribution of multiple synaptic proteins were revealed in cultured hippocampal and CA1 pyramidal MPSIIIC neurons. These defects were rescued by virus-mediated gene correction. Dendritic spines were reduced in pyramidal neurons of mouse models of MPSIIIC and other (Tay-Sachs, sialidosis) LSD as early as at P10. MPSIIIC neurons also presented alterations in frequency and amplitude of miniature excitatory and inhibitory postsynaptic currents, sparse synaptic vesicles, reduced postsynaptic densities, disorganized microtubule networks, and partially impaired axonal transport of synaptic proteins. Furthermore, postsynaptic densities were reduced in postmortem cortices of human MPS patients, suggesting that the pathology is a common hallmark for neurological LSD. Together, our results demonstrate that lysosomal storage defects cause early alterations in synaptic structure and abnormalities in neurotransmission originating from impaired synaptic vesicular transport, and they suggest that synaptic defects could be targeted to treat behavioral and cognitive defects in neurological LSD patients.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.

Objective: To quantify changes in segmented brain volumes over 12 months in children with mucopolysaccharidosis types IIIA and IIIB (MPS IIIA and IIIB)., Methods: In order to establish suitable outcome measures for clinical trials, twenty-five children greater than 2 years of age were enrolled in a prospective natural history study of MPS IIIA and IIIB at Nationwide Children's Hospital. Data from sedated non-contrast brain 3 T MRIs and neuropsychological measures were reviewed from the baseline visit and at 12-month follow-up. No intervention beyond standard clinical care was provided. Age- and sex-matched controls were gathered from the National Institute of Mental Health Data Archive. Automated brain volume segmentation with longitudinal processing was performed using FreeSurfer., Results: Of the 25 subjects enrolled with MPS III, 17 children (4 females, 13 males) completed at least one MRI with interpretable volumetric data. The ages ranged from 2.8 to 13.7 years old (average 7.2 years old) at enrollment, including 8 with MPS IIIA and 9 with MPS IIIB. At baseline, individuals with MPS III demonstrated reduced cerebral white matter and corpus callosum volumes, but greater volumes of the lateral ventricles, cerebellar cortex, and cerebellar white matter compared to controls. Among the 13 individuals with MPS III with two interpretable MRIs, there were annualized losses or plateaus in supratentorial brain tissue volumes (cerebral cortex -42.10 ± 18.52 cm 3 /year [mean ± SD], cerebral white matter -4.37 ± 11.82 cm 3 /year, subcortical gray matter -6.54 ± 3.63 cm 3 /year, corpus callosum -0.18 ± 0.62 cm 3 /yr) and in cerebellar cortex (-0.49 ± 12.57 cm 3 /year), with a compensatory increase in lateral ventricular volume (7.17 ± 6.79 cm 3 /year). Reductions in the cerebral cortex and subcortical gray matter were more striking in individuals younger than 8 years of age. Greater cerebral cortex volume was associated with higher fine and gross motor functioning on the Mullen Scales of Early Learning, while greater subcortical gray matter volume was associated with higher nonverbal functioning on the Leiter International Performance Scale. Larger cerebellar cortex was associated with higher receptive language performance on the Mullen, but greater cerebellar white matter correlated with worse adaptive functioning on the Vineland Adaptive Behavioral Scales and visual problem-solving on the Mullen., Conclusions: Loss or plateauing of supratentorial brain tissue volumes may serve as longitudinal biomarkers of MPS III age-related disease progression compared to age-related growth in typically developing controls. Abnormally increased cerebellar white matter in MPS III, and its association with worse performance on neuropsychological measures, suggest the possibility of pathophysiological mechanisms distinct from neurodegeneration-associated atrophy that warrant further investigation., (Copyright © 2021. Published by Elsevier Inc.)

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N -sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh Δex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh Δex5-6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh Δex5-6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh Δex5-6 mutant larvae in a context-dependent manner. We expect the sgsh Δex5-6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.

Mucopolysaccharidosis IIIA (MPS IIIA) is a lysosomal storage disease with significant neurological and skeletal pathologies. Respiratory dysfunction is a secondary pathology contributing to mortality in MPS IIIA patients. Pulmonary surfactant is crucial to optimal lung function and has not been investigated in MPS IIIA. We measured heparan sulphate (HS), lipids and surfactant proteins (SP) in pulmonary tissue and bronchoalveolar lavage fluid (BALF), and surfactant activity in healthy and diseased mice (20 weeks of age). Heparan sulphate, ganglioside GM3 and bis(monoacylglycero)phosphate (BMP) were increased in MPS IIIA lung tissue. There was an increase in HS and a decrease in BMP and cholesteryl esters (CE) in MPS IIIA BALF. Phospholipid composition remained unchanged, but BALF total phospholipids were reduced (49.70%) in MPS IIIA. There was a reduction in SP-A, -C and -D mRNA, SP-D protein in tissue and SP-A, -C and -D protein in BALF of MPS IIIA mice. Captive bubble surfactometry showed an increase in minimum and maximum surface tension and percent surface area compression, as well as a higher compressibility and hysteresis in MPS IIIA surfactant upon dynamic cycling. Collectively these biochemical and biophysical changes in alveolar surfactant are likely to be detrimental to lung function in MPS IIIA.

There is currently no cure or effective treatment available for mucopolysaccharidosis type IIID (MPS IIID, Sanfilippo syndrome type D), a lysosomal storage disorder (LSD) caused by the deficiency of α- N -acetylglucosamine-6-sulfatase (GNS). The clinical symptoms of MPS IIID, like other subtypes of Sanfilippo syndrome, are largely localized to the central nervous system (CNS), and any treatments aiming to ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease need to be delivered across the blood-brain barrier. Here, we report a proof-of-concept enzyme replacement therapy (ERT) for MPS IIID using recombinant human α- N -acetylglucosamine-6-sulfatase (rhGNS) via intracerebroventricular (ICV) delivery in a neonatal MPS IIID mouse model. We overexpressed and purified rhGNS from CHO cells with a specific activity of 3.9 × 10 4 units/mg protein and a maximal enzymatic activity at lysosomal pH (pH 5.6), which was stable for over one month at 4 °C in artificial cerebrospinal fluid (CSF). We demonstrated that rhGNS was taken up by MPS IIID patient fibroblasts via the mannose 6-phosphate (M6P) receptor and reduced intracellular glycosaminoglycans to normal levels. The delivery of 5 μg of rhGNS into the lateral cerebral ventricle of neonatal MPS IIID mice resulted in normalization of the enzymatic activity in brain tissues; rhGNS was found to be enriched in lysosomes in MPS IIID-treated mice relative to the control. Furthermore, a single dose of rhGNS was able to reduce the accumulated heparan sulfate and β-hexosaminidase. Our results demonstrate that rhGNS delivered into CSF is a potential therapeutic option for MPS IIID that is worthy of further development.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24-30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.

Sanfilippo syndrome is an untreatable form of childhood-onset dementia. Whilst several therapeutic strategies are being evaluated in human clinical trials including i.v. delivery of AAV9-based gene therapy, an urgent unmet need is the availability of non-invasive, quantitative measures of neurodegeneration. We hypothesise that as part of the central nervous system, the retina may provide a window through which to 'visualise' degenerative lesions in brain and amelioration of them following treatment. This is reliant on the age of onset and the rate of disease progression being equivalent in retina and brain. For the first time we have assessed in parallel, the nature, age of onset and rate of retinal and brain degeneration in a mouse model of Sanfilippo syndrome. Significant accumulation of heparan sulphate and expansion of the endo/lysosomal system was observed in both retina and brain pre-symptomatically (by 3 weeks of age). Robust and early activation of micro- and macroglia was also observed in both tissues. There was substantial thinning of retina and loss of rod and cone photoreceptors by ~ 12 weeks of age, a time at which cognitive symptoms are noted. Intravenous delivery of a clinically relevant AAV9-human sulphamidase vector to neonatal mice prevented disease lesion appearance in retina and most areas of brain when assessed 6 weeks later. Collectively, the findings highlight the previously unrecognised early and significant involvement of retina in the Sanfilippo disease process, lesions that are preventable by neonatal treatment with AAV9-sulphamidase. Critically, our data demonstrate for the first time that the advancement of retinal disease parallels that occurring in brain in Sanfilippo syndrome, thus retina may provide an easily accessible neural tissue via which brain disease development and its amelioration with treatment can be monitored.

The cause of neurodegeneration in MPS mouse models is the focus of much debate and what the underlying cause of disease pathology in MPS mice is. The timing of development of pathology and when this can be reversed or impacted is the key to developing suitable therapies in MPS. This study is the first of its kind to correlate the biochemical changes with the functional outcome as assessed using non-invasive behaviour testing across multiple mucopolysaccharidosis (MPS) mouse models. In the MPS brain, the primary lysosomal enzyme dysfunction leads to accumulation of primary glycosaminoglycans (GAGs) with gangliosides (G M2 and G M3 ) being the major secondary storage products. With a focus on the neuropathology, a time course experiment was conducted in MPS I, MPS IIIA, MPS VII (severe and attenuated models) in order to understand the relative timing and level of GAG and ganglioside accumulation and how this correlates to behaviour deficits. Time course analysis from 1 to 6 months of age was conducted on brain samples to assess primary GAG (uronic acid), β-hexosaminidase enzyme activity and levels of G M2 and G M3 gangliosides. This was compared to a battery of non-invasive behaviour tests including open field, inverted grid, rotarod and water cross maze were assessed to determine effects on motor function, activity and learning ability. The results show that the GAG and ganglioside accumulation begins prior to the onset of detectable changes in learning ability and behaviour. Interestingly, the highest levels of GAG and ganglioside accumulation was observed in the MPS IIIA mouse despite having 3% residual enzyme activity. Deficits in motor function were clearly observed in the severe Gus mps/mps , which were significantly delayed in the attenuated Gus tm(L175F)Sly model despite their minimal increase in detectable enzyme activity. This suggests that genotype and residual enzyme activity are not indicative of severity of disease pathology in MPS disease and there exists a window when there are considerable storage products without detectable functional deficits which may allow an alteration to occur with therapy., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

The lack of the N-alpha-glucosaminidase (Naglu) is responsible for the incidence of a rare disease - mucopolysaccharidosis, type IIIB (MPS IIIB). To date, studies have been conducted based on cells derived from patients suffering from MPS or using in vivo MPS mouse models. These limitations have allowed for defining our research goal - to create and characterize the first in vitro murine cellular MPS IIIB model. In the current work we present a new, stable cell line with confirmed accumulation of glycosaminoglycans. The line stability was achieved by immortalization using a lentivirus carrying the T-antigens of SV40. The Naglu -/- cells were confirmed to produce no Naglu enzyme. To confirm the proper functioning of the in vitro MPS IIIB model, we determined the activity and expression of cystathionine γ-lyase, rhodanese and 3-mercaptopyruvate sulfurtransferase, as well as the level of low molecular-weight thiols (reduced and oxidized glutathione, cysteine and cystine). The results were referred to our earlier findings originating from the studies on the tissues of the Naglu -/- mice that were used to create the lines. The results obtained in the Naglu -/- cells were in accordance with the results found in the mouse model of MPS IIIB. It suggests that the presented murine Naglu -/- cell lines might be a convenient in vitro model of MPS IIIB., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

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.

Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid β progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis type IVA (MPS IVA, or Morquio syndrome type A) is an inherited metabolic lysosomal disease caused by the deficiency of the N-acetylglucosamine-6-sulfate sulfatase enzyme. The deficiency of this enzyme accumulates the specific glycosaminoglycans (GAG), keratan sulfate, and chondroitin-6-sulfate mainly in bone, cartilage, and its extracellular matrix. GAG accumulation in these lesions leads to unique skeletal dysplasia in MPS IVA patients. Clinical, radiographic, and biochemical tests are needed to complete the diagnosis of MPS IVA since some clinical characteristics in MPS IVA are overlapped with other disorders. Early and accurate diagnosis is vital to optimizing patient management, which provides a better quality of life and prolonged life-time in MPS IVA patients. Currently, enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are available for patients with MPS IVA. However, ERT and HSCT do not have enough impact on bone and cartilage lesions in patients with MPS IVA. Penetrating the deficient enzyme into an avascular lesion remains an unmet challenge, and several innovative therapies are under development in a preclinical study. In this review article, we comprehensively describe the current diagnosis, treatment, and management for MPS IVA. We also illustrate developing future therapies focused on the improvement of skeletal dysplasia in MPS IVA.

Lack of the N-alpha-acetylglucosaminidase gene is responsible for the occurrence of a rare disease - the Sanfilippo syndrome, type B. The result of this gene knock-out is accumulation of glycosaminoglycans (GAGs) - more specifically heparan sulfate - a sulfate rich macromolecule. The sulfur oxidative pathway is involved in the sulfate groups' turnover in the cells. In contrast, the non-oxidative sulfur pathway leads mostly to formation of sulfane sulfur-containing compounds. The aim of our research was to observe an interaction between MPS IIIB and non-oxidative sulfur metabolism. In this work, we examined selected tissues (livers, kidneys, hearts and spleens) of 3 month old mice with confirmed accumulation of GAGs. The activity and expression of three sulfurtransferases (components of non-oxidative sulfur metabolism): rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionine γ-lyase was determined, as well as the sulfane sulfur level and the level of other low molecular sulfur-containing compounds (reduced and oxidized glutathione, cysteine and cystine). In all tested tissues, the sulfane sulfur and/or sulfurtransferases' activities, as well as the cysteine content, underwent statistically significant changes. These correlations were also related to the sex of the tested animals. The obtained results indicated that accumulation of incompletely degraded GAGs in the tissues had affected the non-oxidative sulfur metabolism.

Mucopolysaccharidosis III type C is a lysosomal storage disorder caused by the accumulation of heparan sulfate in lysosomes. The disorder occurs due to Heparan Acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT) deficiency, an enzyme which typically catalyzes the transmembrane acetylation of heparan sulfate, a basement membrane component. When the gene encoding this enzyme is mutated, it cannot perform the processing of heparan sulfate, leading to un-acetylated heparan sulfate build-up in the lysosomes of cells, causing a storage disorder. This defect has been studied primarily in brain and liver cells, but its effect on the structural integrity of the glomerulus is poorly known. The present study focuses on the effect of Hgsnat gene inactivation and heparan sulfate toxicity on the integrity of the renal corpuscle. This cortical structure was chosen because of its abundance of basement membranes and heparan sulfate as well as the renal corpuscle's physiological importance in glomerular filtration. Light microscopy, electron microscopy, and immunocytochemistry of genetically modified mice revealed a buildup of lysosomes in the podocytes, suggesting that these cells are responsible for the processing of glomerular basement membranes.

Patients with the lysosomal storage disease mucopolysaccharidosis IIIA (MPSIIIA) lack the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), one of the many enzymes involved in degradation of heparan sulfate. Build-up of un-degraded heparan sulfate results in severe progressive neurodegeneration for which there is currently no treatment. Experimental gene therapies based on gene addition are currently being explored. Following preclinical evaluation in MPSIIIA mice, an adeno-associated virus vector of serotype rh10 designed to deliver SGSH and sulfatase modifying factor 1 (SAF301) was trialed in four MPSIIIA patients, showing good tolerance and absence of adverse events with some improvements in neurocognitive measures. This study aimed to improve SAF301 further by removing sulfatase modifying factor 1 (SUMF1) and assessing if expression of this gene is needed to increase the SGSH enzyme activity (SAF301b). Second, the murine phosphoglycerate kinase (PGK) promotor was exchanged with a chicken beta actin/CMV composite (CAG) promotor (SAF302) to see if SGSH expression levels could be boosted further. The three different vectors were administered to MPSIIIA mice via intracranial injection, and SGSH expression levels were compared 4 weeks post treatment. Removal of SUMF1 resulted in marginal reductions in enzyme activity. However, promotor exchange significantly increased the amount of SGSH expressed in the brain, leading to superior therapeutic correction with SAF302. Biodistribution of SAF302 was further assessed using green fluorescent protein (GFP), indicating that vector spread was limited to the area around the injection tract. Further modification of the injection strategy to a single depth with higher injection volume increased vector distribution, leading to more widespread GFP distribution and sustained expression, suggesting this approach should be adopted in future trials.

Heparan sulfate (HS) is a complex polysaccharide from the glycosaminoglycan (GAG) family that accumulates in tissues in several neurological lysosomal storage diseases known as mucopolysaccharidosis (MPS) disorders. The quantitation of HS in biological samples is important for studying MPS disorders but is very challenging because of its high molecular weight and heterogeneity. Recently, acid-catalyzed butanolysis followed by LC-MS/MS analysis has emerged as a promising method for the determination of HS. Butanolysis of HS produces fully desulfated disaccharide cleavage products which are detected by LC-MS/MS. Herein we describe the synthesis of butylated HS disaccharide standards and their use for determining the identity of major product peaks in LC-MS chromatograms from butanolysis of HS as well as the related GAGs heparin and heparosan. Furthermore, synthesis of a d 9 -labeled disaccharide internal standard enabled the development of a quantitative LC-MS/MS assay for HS. The assay was utilized for the analysis of MPS IIIA mouse brain tissues, revealing significant differences in abundance and in the regional accumulation of the various HS disaccharides in affected mice.

Background: Mucopolysaccharidosis type III (MPS III), or Sanfilippo syndrome, is caused by a deficiency in one of the four enzymes involved in the lysosomal degradation of heparan sulfate. Cardiac abnormalities have been observed in patients with all types of MPS except MPS IX, however few studies have focused on cardiac alterations in patients with MPS III., Methods: We reviewed medical records, echocardiograms, and electrocardiograms of 26 Taiwanese patients with MPS III (five with IIIA, 20 with IIIB, and one with IIIC; 14 males and 12 females; median age, 7.4 years; age range, 1.8-26.5 years). The relationships between age and each echocardiographic parameter were analyzed., Results: Echocardiographic examinations (n = 26) revealed that 10 patients (38%) had valvular heart disease. Four (15%) and eight (31%) patients had valvular stenosis or regurgitation, respectively. The most prevalent cardiac valve abnormality was mitral regurgitation (31%), followed by aortic regurgitation (19%). However, most of the cases of valvular heart disease were mild. Three (12%), five (19%) and five (19%) patients had mitral valve prolapse, a thickened interventricular septum, and asymmetric septal hypertrophy, respectively. The severity of aortic regurgitation and the existence of valvular heart disease, aortic valve abnormalities and valvular stenosis were all positively correlated with increasing age (p < 0.05). Z scores > 2 were identified in 0, 38, 8, and 27% of left ventricular mass index, interventricular septal end-diastolic dimension, left ventricular posterior wall end-diastolic dimension, and aortic diameter, respectively. Electrocardiograms in 11 patients revealed the presence of sinus arrhythmia (n = 3), sinus bradycardia (n = 2), and sinus tachycardia (n = 1). Six patients with MPS IIIB had follow-up echocardiographic data at 1.9-18.1 years to compare with the baseline data, which showed some patients had increased thickness of the interventricular septum, as well as more patients had valvular abnormalities at follow-up., Conclusions: Cardiac involvement in MPS III is less common and milder compared with other types of MPS. The existence of valvular heart disease, aortic valve abnormalities and valvular stenosis in the patients worsened with increasing age, reinforcing the concept of the progressive nature of this disease.

Mucoplysaccharydosis IIIA (MPSIIIA) is the most severe form of Sanfilippo syndrome. Skin fibroblasts from a MPSIIIA compound heterozygous (E447K/R245H) patient were nucleofected with four OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-Myc, LIN28, BCL-xL and shp53. The two iPSCs lines generated carry both sulfamidase enzyme (SGSH) mutations, are free of plasmid integration, have normal karyotype, express pluripotency-associated markers and are able to differentiate into the three germ layers., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Metabolomics represent a valuable tool to recover biological information using body fluids and may help to characterize pathophysiological mechanisms of the studied disease. This approach has not been widely used to explore inherited metabolic diseases. This study investigates mucopolysaccharidosis type III (MPS III). A thorough and holistic understanding of metabolic remodeling in MPS III may allow the development, improvement and personalization of patient care., Methods: We applied both targeted and untargeted metabolomics to urine samples obtained from a French cohort of 49 patients, consisting of 13 MPS IIIA, 16 MPS IIIB, 13 MPS IIIC, and 7 MPS IIID, along with 66 controls. The analytical strategy is based on ultra-high-performance liquid chromatography combined with ion mobility and high-resolution mass spectrometry. Twenty-four amino acids have been assessed using tandem mass spectrometry combined with liquid chromatography. Multivariate data modeling has been used for discriminant metabolite selection. Pathway analysis has been performed to retrieve metabolic pathways impairments., Results: Data analysis revealed distinct biochemical profiles. These metabolic patterns, particularly those related to the amino acid metabolisms, allowed the different studied groups to be distinguished. Pathway analysis unveiled major amino acid pathways impairments in MPS III mainly arginine-proline metabolism and urea cycle metabolism., Conclusion: This represents one of the first metabolomics-based investigations of MPS III. These results may shed light on MPS III pathophysiology and could help to set more targeted studies to infer the biomarkers of the affected pathways, which is crucial for rare conditions such as MPS III.

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) has a variable age of onset and variable rate of progression. However, information regarding the natural history of this disorder in Asian populations is limited. A retrospective analysis was carried out for 28 patients with MPS III (types IIIA [n = 3], IIIB [n = 23], and IIIC [n = 2]; 15 males and 13 females; median age, 8.2 years; age range, 2.7-26.5 years) seen in six medical centers in Taiwan from January 1996 through October 2017. The median age at confirmed diagnosis was 4.6 years. The most common initial symptom was speech delay (75%), followed by hirsutism (64%) and hyperactivity (54%). Both z scores for height and weight were negatively correlated with age (r = -.693 and -0.718, respectively; p < .01). The most prevalent clinical manifestations were speech delay (100%) and intellectual disability (100%), followed by hirsutism (93%), hyperactivity (79%), coarse facial features (68%), sleep disorders (61%), and hepatosplenomegaly (61%). Ten patients (36%) had epilepsy, and the median age at the first seizure was 11 years. Thirteen patients (46%) experienced at least one surgical procedure. At the time of the present study, 7 of the 28 patients had passed away at the median age of 13.0 years. Molecular studies showed an allelic heterogeneity without clear genotype and phenotype correlations. MPS IIIB is the most frequent subtype among MPS III in the Taiwanese population. An understanding of the natural history of MPS III may allow early diagnosis and timely management of the disease facilitating better treatment outcomes., (© 2018 Wiley Periodicals, Inc.)

Neuroinflammation and iron accumulation are hallmarks of a variety of adult neurodegenerative diseases. In Sanfilippo syndrome (mucopolysaccharidosis type III, MPSIII, a pediatric neurodegenerative disease that shares some features with adult neurodegenerative diseases), the progressive accumulation of heparan sulfate oligosaccharides (HSOs) induces microglia and astrocytes to produce pro-inflammatory cytokines leading to severe neuroinflammation. The objectives of the present study were (1) to measure the local iron concentration and to assess iron metabolism in the brain of a MPSIIIB murine model and (2) to identify the brain cells involved in this accumulation. We found that iron accumulation in MPSIIIB mice primarily affected the cerebral cortex where hepcidin levels were higher than in wild-type mice, and increased with aging. This increase was correlated with low expression of ferroportin 1 (FPN1), and thus brain iron retention. Moreover, we showed in vitro that HSOs are directly responsible for the production of hepcidin and the relative decrease in FPN1 expression when added to cultures of microglia and, to a lesser extent, to cultures of astrocytes. In contrast, no significant differences were observed in neurons. Hepcidin induction results from activation of the TLR4 pathway and STAT3 signaling, and leads to iron retention within microglia. Our results show that microglia have a key role in cerebral hepcidin overexpression and thus in the brain iron accumulation observed in the MPSIIIB model., (© 2018 Wiley Periodicals, Inc.)

Sulfamidase (SGSH) deficiency causes mucopolysaccharidosis type IIIA (MPS IIIA), a lysosomal storage disease (LSD) that affects the CNS. In earlier work in LSD mice and dog models, we exploited the utility of adeno-associated viruses (AAVs) to transduce brain ventricular lining cells (ependyma) for secretion of lysosomal hydrolases into the cerebrospinal fluid (CSF), with subsequent distribution of enzyme throughout the brain resulting in improved cognition and extending lifespan. A critical feature of this approach is efficient secretion of the expressed enzyme from transduced cells, for delivery by CSF to nontransduced cells. Surprisingly, we found that SGSH was poorly secreted from cells, resulting in retention of the expressed product. Using site-directed mutagenesis of native SGSH, we identified an improved secretion variant that also displayed enhanced uptake properties that were mannose-6-phosphate receptor independent. In studies in MPS IIIA-deficient mice, ependymal transduction with AAVs expressing variant SGSH improved spatial learning and reduced memory deficits, substrate accumulation, and astrogliosis. Secondary lysosomal enzyme elevations in the CSF and brain parenchyma were also resolved. In contrast, ependymal transduction with AAVs expressing wild-type SGSH had significantly lower CSF SGSH levels and limited impacts on behavior. These results demonstrate the utility of a previously undescribed SGSH variant for improved MPS IIIA brain gene therapy., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Sanfilippo type B syndrome (mucopolysac-charidosis type IIIB; MPS IIIB) is an autosomal recessive lysosomal storage disorder. It is caused by a critically reduced α-2-acetamido-2-deoxy-D-glucoside acetamidodeoxy glucohydrolase (α-N-acetylglucosaminidase or NAGLU) activity. Recently, an autosomal recessive disorder of skeletal dysplasia associated with CYP26B1 was reported in three families, in which the patients were all homozygous variations. However, the co-occurrence of two rare diseases in a person is very rare. Here, we reported one patient with two novel pathogenic missense variations in NAGLU and CYP26B1., Case Presentation: We found an infant with biallelic variation both in NAGLU-compound heterozygous c.1843C > T (p. R615C) and c.1224C > A (p. H408Q) as well as in CYP26B1-compound heterozygous c.529G > A (p. E177K) and c.525C > A (p. H175Q). All variations were novel but predicted pathogenicity according to American College of Medical Genetics and Genomics (ACMG) guidelines. The main phenotypes of the infant were quite different from those previously reported, and some were combinations of the two rare diseases, including epilepsy, early onset epileptic encephalopathy, hypermyotonia, skull deformity, dilatation of the lateral ventricles and premature closure of fontanel. His NAGLU enzyme activity was significantly decreased., Conclusions: NAGLU and CYP26B1 mutations were related to MPS IIIB and skeletal dysplasia, respectively. Here, we first reported the pathogenic mutations of two genes concurrent in one patient, which not only expands the phenotype and genotype spectra of NAGLU and CYP26B1, but more importantly indicates the possibility of simultaneous occurrence of two rare diseases in one patient. This interesting finding should be attributed to the use of whole exome sequencing (WES), which indicates that we should be aware of the importance of WES in diagnosing rare diseases.

HSD17B1 is a steroid metabolising enzyme. We have previously generated knockout mice that had the entire coding region of Hsd17b1 replaced with lacZ-neo cassette (Hsd17b1-LacZ/Neo mice). This resulted in a 90% reduction of HSD17B1 activity, associated with severe subfertility in the knockout females. The present study indicates that Hsd17b1-LacZ/Neo male mice have a metabolic phenotype, including reduced adipose mass, increased lean mass and lipid accumulation in the liver. During the characterisation of this metabolic phenotype, it became evident that the expression of the Naglu gene, located closely upstream of Hsd17b1, was severely reduced in all tissues analysed. Similar results were obtained from Hsd17b1-LacZ mice after removing the neo cassette from the locus or by crossing the Hsd17b1-LacZ/Neo mice with transgenic mice constitutively expressing human HSD17B1. The deficiency of Naglu caused the accumulation of glycosaminoglycans in all studied mouse models lacking the Hsd17b1 gene. The metabolic phenotypes of the Hsd17b1 knockout mouse models were recapitulated in Naglu knockout mice. Based on the data we propose that the Hsd17b1 gene includes a regulatory element controlling Naglu expression and the metabolic phenotype in mice lacking the Hsd17b1 genomic region is caused by the reduced expression of Naglu rather than the lack of Hsd17b1.

The monogenic defects in specific lysosomal enzymes in mucopolysaccharidosis (MPS) III lead to lysosomal storage of glycosaminoglycans and complex CNS and somatic pathology, for which the detailed mechanisms remain unclear. In this study, serum samples from patients with MPS IIIA (age 2-9 yr) and MPS IIIB (2-13 yr) and healthy controls (age 2-9 yr) were assayed by global metabolomics profiling of 658 metabolites using mass spectrometry. Significant alterations were detected in 423 metabolites in all MPS III patients, of which 366 (86.5%) decreased and 57 (13.5%) increased. Similar profiles were observed when analyzing data from MPS IIIA and MPS IIIB samples separately, with only limited age variations in 36 metabolites. The observed metabolic disturbances in MPS III patients involve virtually all major pathways of amino acid (101/150), peptide (17/21), carbohydrate (19/23), lipid (221/325), nucleotide (15/25), energy (8/9), vitamins and co-factors (8/21), and xenobiotics (34/84) metabolism. Notably, detected serum metabolite decreases involved all key amino acids, all major neurotransmitter pathways, and broad neuroprotective compounds. The elevated metabolites are predominantly lipid derivatives, and also include cysteine metabolites and a fibrinogen peptide fragment, consistent with the status of oxidative stress and inflammation in MPS III. This study demonstrates that the lysosomal glycosaminoglycans storage triggers profound metabolic disturbances in patients with MPS III disorders, leading to severe functional depression of virtually all metabolic pathways, which emerge early during the disease progression. Serum global metabolomics profiling may provide an important and minimally invasive tool for better understanding the disease mechanisms and identification of potential biomarkers for MPS III.

Mucopolysaccharidosis (MPS) type IIIA, or Sanfilippo syndrome, is a neurodegenerative lysosomal storage disorder caused by a deficiency of the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), involved in the catabolism of heparan sulfate. The clinical spectrum is broad and the age of symptom onset and the degree of preservation of cognitive and motor functions appears greatly influenced by genotype. To explore this further, we generated a conditional knockout (Sgsh KO ) mouse model with ubiquitous Sgsh deletion, and compared the clinical and pathological phenotype with that of the spontaneous Sgsh D31N MPS-IIIA mouse model. Phenotypic deficits were noted in Sgsh KO mice prior to Sgsh D31N mice, however these outcomes did not correlate with any shift in the time of appearance nor rate of accumulation of primary (heparan sulfate) or secondary substrates (GM2/GM3 gangliosides). Other disease lesions (elevations in lysosomal integral membrane protein-II expression, reactive astrocytosis and appearance of ubiquitin-positive inclusions) were also comparable between affected mouse strains. This suggests that gross substrate storage and these neuropathological markers are neither primary determinants, nor good biomarkers/indicators of symptom generation, confirming similar observations made recently in MPS-IIIA patients. The Sgsh KO mouse will be a useful tool for elucidation of the neurological basis of disease and assessment of the clinical efficacy of new treatments for Sanfilippo syndrome.

Background: Mucopolysaccharidosis type III is a progressive, neurodegenerative lysosomal storage disorder for which there is currently no effective therapy. Though numerous potential therapies are in development, there are several challenges to conducting clinical research in this area. We seek to make recommendations on the approach to clinical research in MPS III, including the selection of outcome measures and trial endpoints, in order to improve the quality and impact of research in this area., Results: An international workshop involving academic researchers, clinical experts and industry groups was held in June 2015, with presentations and discussions on disease pathophysiology, biomarkers, potential therapies and clinical outcome measures. A set of recommendations was subsequently prepared by a working group and reviewed by all delegates. We present a series of 11 recommendations regarding the conduct of clinical research, outcome measures and management of natural history data in Mucopolysaccharidosis type III., Conclusions: Improving the quality of clinical research in Mucopolysaccharidosis type III will require an open, collaborative and systematic approach between academic researchers, clinicians and industry. Natural history data should be published as soon as possible and ideally collated in a central repository. There should be agreement on outcome measures and instruments for evaluation of clinical outcomes to maximise the effectiveness of current and future clinical research.

Intra-cerebrospinal fluid (CSF) injection of recombinant human lysosomal enzyme is a potential treatment strategy for several neurodegenerative lysosomal storage disorders including Sanfilippo syndrome (Mucopolysaccharidosis type IIIA; MPS IIIA). Here we have utilised the MPS IIIA Huntaway dog model to compare the effectiveness of the repeated intermittent bolus injection strategy being used in the trials with an alternate approach; slow, continual infusion of replacement enzyme (recombinant human sulphamidase; rhSGSH) into the spinal CSF using a SynchroMed II® pump attached to a spinal infusion cannula. The ability of each enzyme delivery strategy to ameliorate lesions in MPS IIIA brain was determined in animals treated from ∼three- to six-months of age. Controls received buffer or no treatment. Significant reductions in heparan sulphate (primary substrate) were observed in brain samples from dogs treated via either cisternal or lumbar spinal CSF bolus injection methods and also in slow intra-spinal CSF infusion-treated dogs. The extent of the reduction differed regionally. Pump-delivered rhSGSH was less effective in reducing secondary substrate (G M3 ganglioside) in deeper aspects of cerebral cortex, and although near-amelioration of microglial activation was seen in superficial (but not deep) layers of cerebral cortex in both bolus enzyme-treated groups, pump-infusion of rhSGSH had little impact on microgliosis. While continual low-dose infusion of rhSGSH into MPS IIIA dog CSF reduces disease-based lesions in brain, it was not as efficacious as repeated cisternal or spinal CSF bolus infusion of rhSGSH over the time-frame of these experiments.