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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 IIIB (MPS IIIB, Sanfilippo syndrome type B) is caused by a deficiency in α-N-acetylglucosaminidase (NAGLU) activity, which leads to the accumulation of heparan sulfate (HS). MPS IIIB causes progressive neurological decline, with affected patients having an expected lifespan of approximately 20 years. No effective treatment is available. Recent pre-clinical studies have shown that intracerebroventricular (ICV) ERT with a fusion protein of rhNAGLU-IGF2 is a feasible treatment for MPS IIIB in both canine and mouse models. In this study, we evaluated the biochemical efficacy of a single dose of rhNAGLU-IGF2 via ICV-ERT in brain and liver tissue from Naglu -/- neonatal mice. Twelve weeks after treatment, NAGLU activity levels in brain were 0.75-fold those of controls. HS and β-hexosaminidase activity, which are elevated in MPS IIIB, decreased to normal levels. This effect persisted for at least 4 weeks after treatment. Elevated NAGLU and reduced β-hexosaminidase activity levels were detected in liver; these effects persisted for up to 4 weeks after treatment. The overall therapeutic effects of single dose ICV-ERT with rhNAGLU-IGF2 in Naglu -/- neonatal mice were long-lasting. These results suggest a potential benefit of early treatment, followed by less-frequent ICV-ERT dosing, in patients diagnosed with MPS IIIB., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis III (Sanfilippo syndrome, MPS III) is caused by lysosomal enzyme deficiency, which is a rare autosomal recessive hereditary disease. For now, there is no approved treatment for MPS III despite lots of efforts providing new vision of its molecular basis, as well as governments providing regulatory and economic incentives to stimulate the development of specific therapies. Those efforts and incentives attract academic institutions and industry to provide potential therapies for MPS III, including enzyme replacement therapies, substrate reduction therapies, gene and cell therapies, and so on, which were discussed in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mucopolysaccharidosis III B (MPS III-B) is a rare lysosomal storage disorder caused by deficiencies in Alpha-N-acetylglucosaminidase (NAGLU) for which there is currently no cure, and present treatment is largely supportive. Understanding the structure of NAGLU may allow for identification of novel therapeutic targets for MPS III-B. Here we describe the first crystal structure of human NAGLU, determined to a resolution of 2.3 Å. The crystal structure reveals a novel homotrimeric configuration, maintained primarily by hydrophobic and electrostatic interactions via domain II of three contiguous domains from the N- to C-terminus. The active site cleft is located between domains II and III. Catalytic glutamate residues, E316 and E446, are located at the top of the (α/β) 8 barrel structure in domain II. We utilized the three-dimensional structure of NAGLU to map several MPS III-B mutations, and hypothesize their functional consequences. Revealing atomic level structural information about this critical lysosomal enzyme paves the way for the design of novel therapeutics to target the underlying causes of MPS III-B., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Given the large and expanding quantity of publicly available sequencing data, it should be possible to extract incidence information for monogenic diseases from allele frequencies, provided one knows which mutations are causal. We tested this idea on a rare, monogenic, lysosomal storage disorder, Sanfilippo Type B (Mucopolysaccharidosis type IIIB). Sanfilippo Type B is caused by mutations in the gene encoding α-N-acetylglucosaminidase (NAGLU). There were 189 NAGLU missense variants found in the ExAC dataset that comprises roughly 60,000 individual exomes. Only 24 of the 189 missense variants were known to be pathogenic; the remaining 165 variants were of unknown significance (VUS), and their potential contribution to disease is unknown. To address this problem, we measured enzymatic activities of 164 NAGLU missense VUS in the ExAC dataset and developed a statistical framework for estimating disease incidence with associated confidence intervals. We found that 25% of VUS decreased the activity of NAGLU to levels consistent with Sanfilippo Type B pathogenic alleles. We found that a substantial fraction of Sanfilippo Type B incidence (67%) could be accounted for by novel mutations not previously identified in patients, illustrating the utility of combining functional activity data for VUS with population-wide allele frequency data in estimating disease incidence., Competing Interests: All authors are full time employees of BioMarin Pharmaceutical Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated naglu -/ - mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs., Abbreviations: ANOVA: analysis of variance; Atg7: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus.

Objective: Mucopolysaccharidosis IIIA or Sanfilippo disease type A is a progressive neurodegenerative disorder presenting in early childhood, caused by an inherited deficiency of the lysosomal hydrolase sulfamidase. New missense mutations, for which genotype-phenotype correlations are currently unknown, are frequently reported, hampering early prediction of phenotypic severity and efficacy assessment of new disease-modifying treatments. We aimed to design a method to determine phenotypic severity early in the disease course., Methods: Fifty-three patients were included for whom skin fibroblasts and data on disease course and mutation analysis were available. Patients were phenotypically characterized on clinical data as rapidly progressing or slowly progressing. Sulfamidase activity was measured in fibroblasts cultured at 37 °C and at 30 °C., Results: Sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a combination of known severe mutations remained below the limit of quantification under both culture conditions. In contrast, sulfamidase activity in fibroblasts from patients homozygous or compound heterozygous for a known mild mutation increased above the limit of quantification when cultured at 30 °C. With division on the basis of the patients' phenotype, fibroblasts from slowly progressing patients could be separated from rapidly progressing patients by increase in sulfamidase activity when cultured at 30 °C (p < 0.001, sensitivity = 96%, specificity = 93%)., Interpretation: Phenotypic severity strongly correlates with the potential to increase sulfamidase activity in fibroblasts cultured at 30 °C, allowing reliable distinction between patients with rapidly progressing or slowly progressing phenotypes. This method may provide an essential tool for assessment of treatment effects and for health care and life planning decisions. Ann Neurol 2017;82:686-696., (© 2017 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.)

Background: The autosomal recessive, neurodegenerative disorder mucopolysaccharidosis type IIIB (MPSIIIB) is caused by a deficiency of the lysosomal enzyme N-acetyl-α-glucosaminidase (NAGLU), resulting in accumulation of heparan sulfate. The disease spectrum comprises a severe, rapidly progressing (RP) phenotype and a more attenuated, slowly progressing (SP) phenotype. Previous studies showed significantly higher NAGLU activity in skin fibroblasts of SP patients when cultured at 30°C which may be relevant for development of novel therapeutic strategies. Here we report on the processes involved in this phenomenon., Methods: Fibroblasts from controls, one RP patient (homozygous for the p.R297* mutation) and three SP MPSIIIB patients (homozygous for the mutation p.S612G or p.R643C, or compound heterozygous for the mutations p.A72&#95;G79dup8 and p.R565Q) were cultured at temperatures ranging from 37°C to 27°C and harvested at different time points to assess NAGLU activity, mRNA and protein levels, and NAGLU glycosylation. Intracellular localization of wild-type and mutant mCherry-tagged NAGLU was analyzed by immunofluorescence., Results: In control fibroblasts NAGLU was present as a 85kDa precursor and a 82kDa mature form. In SP patients' fibroblasts cultured at 37°C, only the 85kDa form was detected. Culturing at lower temperatures resulted in higher NAGLU mRNA levels, increased levels of both precursor and mature NAGLU protein and improved processing. The formation of mature NAGLU corresponded with higher NAGLU activity levels., Conclusion: We show that the NAGLU protein consists of a precursor and a mature form and that in SP MPSIIIB patients' fibroblasts only the precursor protein is present at 37°C. Culturing at lower temperatures resulted in the formation of the mature, enzymatically active form, due to higher mRNA levels and improved processing., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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.

Sanfilippo B syndrome is a progressive neurological disorder caused by inability to catabolize heparan sulfate glycosaminoglycans. We studied neurobehavior in male Sanfilippo B mice and heterozygous littermate controls from 16 to 20 weeks of age. Affected mice showed reduced anxiety, with a decrease in the number of stretch-attend postures during the elevated plus maze (p=0.001) and an increased tendency to linger in the center of an open field (p=0.032). Water maze testing showed impaired spatial learning, with reduced preference for the target quadrant (p=0.01). In radial arm maze testing, affected mice failed to achieve above-chance performance in a win-shift working memory task (t-test relative to 50% chance: p=0.289), relative to controls (p=0.037). We found a 12.4% reduction in mean acetylcholinesterase activity (p<0.001) and no difference in choline acetyltransferase activity or acetylcholine in whole brain of affected male animals compared to controls. Cholinergic pathways are affected in adult-onset dementias, including Alzheimer disease. Our results suggest that male Sanfilippo B mice display neurobehavioral deficits at a relatively early age, and that as in adult dementias, they may display deficits in cholinergic pathways., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mucopolysaccharidosis type IIIC (MPSIIIC) is a severe lysosomal storage disease caused by deficiency in activity of the transmembrane enzyme heparan-α-glucosaminide N-acetyltransferase (HGSNAT) that catalyses the N-acetylation of α-glucosamine residues of heparan sulfate. Enzyme deficiency causes abnormal substrate accumulation in lysosomes, leading to progressive and severe neurodegeneration, somatic pathology and early death. There is no cure for MPSIIIC, and development of new therapies is challenging because of the unfeasibility of cross-correction. In this study, we generated a new mouse model of MPSIIIC by targeted disruption of the Hgsnat gene. Successful targeting left LacZ expression under control of the Hgsnat promoter, allowing investigation into sites of endogenous expression, which was particularly prominent in the CNS, but was also detectable in peripheral organs. Signs of CNS storage pathology, including glycosaminoglycan accumulation, lysosomal distension, lysosomal dysfunction and neuroinflammation were detected in 2-month-old animals and progressed with age. Glycosaminoglycan accumulation and ultrastructural changes were also observed in most somatic organs, but lysosomal pathology seemed most severe in liver. Furthermore, HGSNAT-deficient mice had altered locomotor and exploratory activity and shortened lifespan. Hence, this animal model recapitulates human MPSIIIC and provides a useful tool for the study of disease physiopathology and the development of new therapeutic approaches., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Purpose: To report longitudinal phenotypic findings in a patient with Sanfilippo syndrome type IIIA, harboring SGSH mutations, one of which is novel., Methods: Heparan-N-sulfatidase enzyme function testing in skin fibroblasts and white blood cells and SGSH gene sequencing were obtained. Clinical office examinations, examinations under anesthesia, electroretinogram, spectral domain optical coherence tomography (SD-OCT), and fundus photography were performed over a 5-year period., Results: Fundus examination revealed a progressive breadcrumb-like pigmentary retinopathy with perifoveal pigmentary involvement. SD-OCT showed loss of normal neuroretinal lamination and cystic macular changes responsive to treatment with carbonic anhydrase inhibitors. Electroretinography exhibited complex characteristics indicative of a generalized retinal rod > cone dysfunction with significant ON > OFF postreceptoral response compromise. Sequencing revealed compound heterozygous mutations in the SGSH gene, the novel c.88G > C (p.A30P) change and a second, previously reported one (c.734G > A, p.R245H)., Conclusions: We have identified ocular features of a patient with Sanfilippo syndrome type IIIA harboring a novel SGHS mutation that were not previously known to occur in this disease - namely, a progressive retinopathy with distinctive features, cystic macular changes responsive to carbonic anhydrase inhibitors, and complex electroretinographic abnormalities consistent with postreceptoral dysfunction. SD-OCT imaging revealed retinal lamination changes consistent with previously reported histologic studies. Both the SD-OCT and the electroretinogram changes appear attributable to intraretinal deposition of heparan sulfate.

Purpose: Retinal degeneration is a common feature of several lysosomal storage disorders, including the mucopolysaccharidoses, a group of metabolic disorders that is characterized by widespread accumulation of glycosaminoglycans due to lysosomal enzyme dysfunction. We used a new mouse model of mucopolysaccharidosis IIIE to study the effect of Arylsulfatase G (ARSG) deficiency on retina integrity., Methods: The retina of Arsg knockout mice aged 1 to 24 months was studied by immunohistochemistry and Western blot analysis. Electron microscopic analyses were performed on retinas from 15- and 22-month-old animals. Photoreceptor and microglia cell numbers and retina thickness were determined to quantitatively characterize retinal degeneration in ARSG-deficient mice., Results: Arsg knockout mice showed a progressive degeneration of photoreceptor cells starting between 1 and 6 months of age, resulting in the loss of more than 50% of photoreceptor cells in 24-month-old mice. Photoreceptor loss was accompanied by reactive astrogliosis, reactive microgliosis that was evident in the outer but not inner retina, and elevated expression levels of some lysosomal proteins. Electron microscopic analyses of retinas revealed no evidence for the presence of storage vacuoles. Of note, expression of ARSG protein in wild-type mice was detectable only in the RPE which, however, appeared morphologically unaffected in knockout mice at the electron microscopic level., Conclusions: To our knowledge, this is the first study demonstrating that ARSG deficiency results in progressive photoreceptor degeneration and dysregulation of various lysosomal proteins.

Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina., (© The Author 2015. Published by Oxford University Press.)

Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the disease's main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Deficiency of arylsulfatase G (ARSG) leads to a lysosomal storage disease in mice resembling biochemical and pathological features of the mucopolysaccharidoses and particularly features of mucopolysaccharidosis type III (Sanfilippo syndrome). Here we show that Arsg KO mice share common neuropathological findings with other Sanfilippo syndrome models and patients, but they can be clearly distinguished by the limitation of most phenotypic alterations to the cerebellum, presenting with ataxia as the major neurological finding. We determined in detail the expression of ARSG in the central nervous system and observed highest expression in perivascular macrophages (which are characterized by abundant vacuolization in Arsg KO mice) and oligodendrocytes. To gain insight into possible mechanisms leading to ataxia, the pathology in older adult mice (>12 months) was investigated in detail. This study revealed massive loss of Purkinje cells and gliosis in the cerebellum, and secondary accumulation of glycolipids like GM2 and GM3 gangliosides and unesterified cholesterol in surviving Purkinje cells, as well as neurons of some other brain regions. The abundant presence of ubiquitin and p62-positive aggregates in degenerating Purkinje cells coupled with the absence of significant defects in macroautophagy is consistent with lysosomal membrane permeabilization playing a role in the pathogenesis of Arsg-deficient mice and presumably Sanfilippo disease in general. Our data delineating the phenotype of mucopolysaccharidosis IIIE in a mouse KO model should help in the identification of possible human cases of this disease., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Intracerebrospinal fluid (CSF) infusion of replacement enzyme is under evaluation for amelioration of disease-related symptoms and biomarker changes in patients with the lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA; www.clinicaltrials.gov ; NCT#01155778; #01299727). Determining the optimal dose/dose-frequency is important, given the invasive method for chronically supplying recombinant protein to the brain, the main site of symptom generation. To examine these variables, we utilised MPS IIIA Huntaway dogs, providing recombinant human sulphamidase (rhSGSH) to young pre-symptomatic dogs from an age when MPS IIIA dog brain exhibits significant accumulation of primary (heparan sulphate) and secondary (glycolipid) substrates. Enzyme was infused into CSF via the cisterna magna at one of two doses (3 mg or 15 mg/infusion), with the higher dose supplied at two different intervals; fortnightly or monthly. Euthanasia was carried out 24 h after the final injection. Dose- and frequency-dependent reductions in heparan sulphate were observed in CSF and deeper layers of cerebral cortex. When we examined the amount of immunostaining of the general endo/lysosomal marker, LIMP-2, or quantified activated microglia, the higher fortnightly dose resulted in superior outcomes in affected dogs. Secondary lesions such as accumulation of GM3 ganglioside and development of GAD-reactive axonal spheroids were treated to a similar degree by both rhSGSH doses and dose frequencies. Our findings indicate that the lower fortnightly dose is sub-optimal for ameliorating existing and preventing further development of disease-related pathology in young MPS IIIA dog brain; however, increasing the dose fivefold but halving the frequency of administration enabled near normalisation of disease-related biomarkers.

Enzyme replacement therapy for MPS IIIB (mucopolysaccharidosis type IIIB; also known as Sanfilippo B syndrome) has been hindered by inadequate mannose 6 phosphorylation and cellular uptake of rhNAGLU (recombinant human α-N-acetylglucosaminidase). We expressed and characterized a modified rhNAGLU fused to the receptor-binding motif of IGF-II (insulin-like growth factor 2) (rhNAGLU-IGF-II) to enhance its ability to enter cells using the cation-independent mannose 6-phosphate receptor, which is also the receptor for IGF-II (at a different binding site). RhNAGLU-IGF-II was stably expressed in CHO (Chinese-hamster ovary) cells, secreted and purified to apparent homogeneity. The Km and pH optimum of the fusion enzyme was similar to those reported for rhNAGLU. Both intracellular uptake and confocal microscopy suggested that MPS IIIB fibroblasts readily take up the fusion enzyme via receptor-mediated endocytosis that was inhibited significantly (P<0.001) by the monomeric IGF-II peptide. Glycosaminoglycan storage was reduced by 60% (P<0.001) to near background levels in MPS IIIB cells after treatment with rhNAGLU-IGF-II, with half-maximal correction at concentrations of 3-12 pM. A similar cellular uptake mechanism via the IGF-II receptor was also demonstrated in two different brain tumour-derived cell lines. Fusion of rhNAGLU to IGF-II enhanced its cellular uptake while maintaining enzymatic activity, supporting its potential as a therapeutic candidate for treating MPS IIIB.

A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG) activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB) due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG), in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB), a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in enzyme replacement therapy, gene therapy, and combination therapies.

Mucopolysaccharidoses type IIIA (MPS-IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate-2-sulphatase (IDS) and the blood-brain barrier (BBB)-binding domain (BD) from the Apolipoprotein B (ApoB-BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS-IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB-BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS-IIIA and other neurodegenerative LSDs., (Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

Cytotoxicity of laronidase (Aldurazyme(®)), employed in enzyme replacement therapy (ERT) for mucopolysaccharidosis type I (MPS I) and various siRNAs, tested previously in studies on substrate reduction therapy (SRT) for mucopolysaccharidoses, was tested. The enzyme did not cause any cytotoxic effects, and the siRNAs did not inhibit growth of most investigated cell lines. However, some cytotoxic effects of some tested siRNAs were observed in one MPS IIIA cell line. The efficacy of a combination of enzyme replacement therapy and siRNA-based substrate deprivation therapy was tested on three MPS I cell lines. Surprisingly, different results were obtained for different cell lines. The decrease of glycosaminoglycan storage in cells treated simultaneously with both methods was: (i) less pronounced than obtained with either of those methods used alone in one cell line, (ii) similar to that observed for enzyme replacement therapy in another cell line, and (iii) stronger than that obtained with either of the methods used alone in the third cell line. Therefore, it appears that the effects of various therapeutic methods may strongly depend on the features of the MPS cell line.

By providing access to affected neurons, human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease, defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system, causing relentless progression toward severe mental retardation. Partially digested proteoglycans, which affect fibroblast growth factor signaling, accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages, patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures, undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions, whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together, these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues, which possibly affect Golgi organization, cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development, once HS proteoglycan accumulation becomes prominent in the affected child brain.

The accumulation of heparan sulfate (HS) in lysosomes is the primary consequence of the enzyme defect (α-N-acetylglucosaminidase) in mucopolysaccharidosis type IIIB. This accumulation triggers a cascade of pathological events that progressively leads to CNS pathology. Here we examined the activation of the three major stress kinases in the neuronal tissue of a murine model of the disease. ERK1/2 was significantly higher in the cortex of 1-2-month-old affected animals compared with wild-type (Wt) littermates. Similarly, ERK1/2 was stimulated in neurons cultured from MPS IIIB mice. SAPK/JNK was also found to be activated in the cortex of 1-2-month-old affected animals compared with Wt subjects, and the same was found for cultured neurons. In contrast, the active form of p38MAPK was lower in the cortex of 1-month-old MPS IIIB mice compared with Wt animals, but no significant difference was found between the two p38MAPK analyzed in normal and affected neurons cultured in vitro. These data indicate the possible involvement of MAPK dysregulation in the early stage of MPS IIIB brain disease., (Copyright © 2011 Wiley-Liss, Inc.)

There is no treatment for the progressive neurodegenerative lysosomal storage disorder mucopolysaccharidosis type IIIA (MPS IIIA), which occurs due to a deficiency of functional N-sulfoglucosamine sulfohydrolase (SGSH), with subsequent accumulation of partially-degraded heparan sulfate and secondarily-stored compounds including GM2 and GM3 gangliosides and unesterified cholesterol. The brain is a major site of pathology and affected children exhibit progressive cognitive decline and early death. In the present study, six MPS IIIA dogs received intravenous recombinant human SGSH (rhSGSH) from birth to either 8 or 12 weeks of age (1 mg/kg, up to 5 mg), with subsequent intra-cerebrospinal fluid injection of 3 or 15 mg rhSGSH (or vehicle) on a weekly or fortnightly basis to 23 weeks of age. All dogs completed the protocol without incident, and there was no clinically-relevant cellular or humoral immune response to rhSGSH delivery. Immunohistochemistry demonstrated rhSGSH delivery to widespread regions of the brain, and tandem mass spectrometry revealed an apparent dose-dependent decrease in the relative level of a heparan sulfate-derived disaccharide, with near normalization of substrate in many brain regions at the higher dose. Secondarily-stored GM3 ganglioside and unesterified cholesterol, determined using histological methods, were also reduced in a dose-dependent manner, as was the number of activated microglia. We have demonstrated that pre-symptomatic treatment of this progressive neurodegenerative disorder via intra-cerebrospinal fluid injection of rhSGSH mediates highly significant reductions in neuropathology in this MPS IIIA model and clinical trials of this treatment approach in MPS IIIA patients are therefore indicated., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo syndrome) is a fatal inherited lysosomal storage disease accompanied by progressive neurologic degeneration. The gene underlying MPS IIIA, SGSH, encodes a lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (sulfamidase). Mutational analysis of a large cohort of MPS IIIA patients showed a correlation of the missense mutation p.Ser298Pro and a slowly progressive course of the disease. We report here on the expression of the mutant p.Ser298Pro sulfamidase in BHK cells retaining low residual activity. Pulse-chase experiments showed that rapid degradation is responsible for the low steady state level of the mutant protein. Processing and secretion of p.Ser298Pro sulfamidase suggests that small amounts of the newly synthesized enzyme are transported to lysosomes. Most of the mutant sulfamidase exits the endoplasmic reticulum for proteasomal degradation. The ability to predict the clinical course of MPS IIIA in patients with the p.Ser298Pro mutation, as well as the residual enzymatic activity, and the reduced stability of the mutant sulfamidase suggest that this subgroup of patients is especially well suited to early sulfamidase replacement therapy or treatment with selective pharmacological chaperones., (Copyright © 2011 Wiley-Liss, Inc.)

Background: Sanfilippo syndrome type B (MPS III B) is caused by a deficiency of α-N-acetylglucosaminidase enzyme, leading to accumulation of heparan sulfate within lysosomes and eventual progressive cerebral and systemic multiple organ abnormalities. However, little is known about the competence of the blood-brain barrier (BBB) in MPS III B. BBB dysfunction in this devastating disorder could contribute to neuropathological disease manifestations., Methodology/principal Findings: In the present study, we investigated structural (electron microscope) and functional (vascular leakage) integrity of the BBB in a mouse model of MPS III B at different stages of disease, focusing on brain structures known to experience neuropathological changes. Major findings of our study were: (1) endothelial cell damage in capillary ultrastructure, compromising the BBB and resulting in vascular leakage, (2) formation of numerous large vacuoles in endothelial cells and perivascular cells (pericytes and perivascular macrophages) in the large majority of vessels, (3) edematous space around microvessels, (4) microaneurysm adjacent to a ruptured endothelium, (6) Evans Blue and albumin microvascular leakage in various brain structures, (7) GM3 ganglioside accumulation in endothelium of the brain microvasculature., Conclusions/significance: These new findings of BBB structural and function impairment in MPS III B mice even at early disease stage may have implications for disease pathogenesis and should be considered in current and future development of treatments for MPS III B.

The primary pathology in mucopolysaccharidosis (MPS) IIIB is lysosomal storage of heparan sulfate (HS) glycosaminoglycans, leading to complex neuropathology and dysfunction, for which the detailed mechanisms remain unclear. Using antibodies that recognize specific HS glycoforms, we demonstrate differential cell-specific and domain-specific lysosomal HS-GAG distribution in MPS IIIB mouse brain. We also describe a novel neuron-specific brain HS epitope with broad, non-specific increase in the expression in all neurons in MPS IIIB mouse brain, including cerebellar granule neurons, which do not exhibit lysosomal storage pathology. This suggests that biosynthesis of certain HS glycoforms is enhanced throughout the CNS of MPS IIIB mice. Such a conclusion is further supported by demonstration of increased expression of multiple genes encoding enzymes essential in HS biosynthesis, including HS sulfotransferases and epimerases, as well as FGFs, for which HS serves as a co-receptor, in MPS IIIB brain. These data suggest that lysosomal storage of HS may lead to the increase in HS biosyntheses, which may contribute to the neuropathology of MPS IIIB by exacerbating the lysosomal HS storage.

Heparin acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferase, EC 2.3.1.78) is an integral lysosomal membrane protein containing 11 transmembrane domains, encoded by the HGSNAT gene. Deficiencies of N-acetyltransferase lead to mucopolysaccharidosis IIIC. We demonstrate that contrary to a previous report, the N-acetyltransferase signal peptide is co-translationally cleaved and that this event is required for its intracellular transport to the lysosome. While we confirm that the N-acetyltransferase precursor polypeptide is processed in the lysosome into a small amino-terminal alpha- and a larger ß- chain, we further characterize this event by identifying the mature amino-terminus of each chain. We also demonstrate this processing step(s) is not, as previously reported, needed to produce a functional transferase, i.e., the precursor is active. We next optimize the biochemical assay procedure so that it remains linear as N-acetyltransferase is purified or protein-extracts containing N-acetyltransferase are diluted, by the inclusion of negatively charged lipids. We then use this assay to demonstrate that the purified single N-acetyltransferase protein is both necessary and sufficient to express transferase activity, and that N-acetyltransferase functions as a monomer. Finally, the kinetic mechanism of action of purified N-acetyltransferase was evaluated and found to be a random sequential mechanism involving the formation of a ternary complex with its two substrates; i.e., N-acetyltransferase does not operate through a ping-pong mechanism as previously reported. We confirm this conclusion by demonstrating experimentally that no acetylated enzyme intermediate is formed during the reaction.

Objective: Mucopolysaccharidosis (MPS) IIIA (Sanfilippo syndrome type A) is a lysosomal storage disorder caused by deficiency of the enzyme sulfamidase. Information on the natural course of MPS IIIA is scarce, but is much needed in view of emerging therapies., Methods: Clinical history and molecular defects of all 110 MPS IIIA patients identified by enzymatic studies in the Netherlands were collected and included in this study., Results: First clinical signs, mainly consisting of delayed speech development and behavioral problems, were noted between the ages of 1 and 6 years. Other symptoms included sleeping and hearing problems, recurrent upper airway infections, diarrhea, and epilepsy. The clinical course varied remarkably and could be correlated with the molecular defects. The frequent pathogenic mutations p.R245H, p.Q380R, p.S66W, and c.1080delC were associated with the classical severe phenotype. Patients compound heterozygous for the p.S298P mutation in combination with 1 of the mutations associated with the classical severe phenotype had a significantly longer preservation of psychomotor functions and a longer survival. Two patients homozygous for the p.S298P mutation, and 4 patients from 3 families heterozygous for 3 missense variants not reported previously (p.T421R, p.P180L, and p.L12Q), showed a remarkably attenuated phenotype., Interpretation: We report the natural history and mutational analysis in a large unbiased cohort of MPS IIIA patients. We demonstrate that the clinical spectrum of MPS IIIA is much broader than previously reported. A significant genotype-phenotype correlation was established in this cohort.

Background: Finding efficient central nervous system (CNS) delivery approaches has been the major challenge facing therapeutic development for treating diseases with global neurological manifestation, such as mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease, caused by autosomal recessive defect of alpha-N-acetylglucosaminidase (NaGlu). Previously, we developed an approach, intracisternal (i.c.) injection, to deliver recombinant adeno-associated viral (rAAV) vector to the CNS of mice, leading to a widespread periventricular distribution of transduction., Methods: In the present study, we delivered rAAV2 vector expressing human NaGlu into the CNS of MPS IIIB mice by an i.c. injection approach, to test its therapeutic efficacy and feasibility for treating the neurological manifestation of the disease., Results: We demonstrated significant functional neurological benefits of a single i.c. vector infusion in adult MPS IIIB mice. The treatment slowed the disease progression by mediating widespread recombinant NaGlu expression in the CNS, resulting in the reduction of brain lysosomal storage pathology, significantly improved cognitive function and prolonged survival. However, persisting motor function deficits suggested that pathology in areas outside the CNS contributes to the MPS IIIB behavioral phenotype. The therapeutic benefit of i.c. rAAV2 delivery was dose-dependent and could be attribute solely to the CNS transduction because the procedure did not lead to detectable transduction in somatic tissues., Conclusions: A single IC rAAV2 gene delivery is functionally beneficial for treating the CNS disease of MPS IIIB in mice. It is immediately clinically translatable, with the potential of improving the quality of life for patients with MPS IIIB.

Mucopolysaccharidosis (MPS) IIIC is an autosomal recessive lysosomal storage disorder caused by a deficiency in heparan acetyl CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT). The characteristic feature is the deterioration of the central nervous system, but other symptoms may include coarse facies, developmental delay, macrocrania and motor retardation. HGSNAT is localised to the lysosomal membrane and catalyses a transmembrane acetylation in which the terminal glucosamine residue of heparan sulphate acquires an acetyl group, thus forming N-acetylglucosamine. 54 variants of the HGSNAT gene have been identified in MPS IIIC patients thus far, 22 of which are missense mutations. In this study, 20 of the latter were introduced into the cDNA of HGSNAT, and the resultant derivatives were exogenously expressed in cell culture. Transfection of 16 of these resulted in the synthesis of negligible HGSNAT protein and activity. The levels and function of the remaining 4 mutants, however, were similar to those of exogenously expressed wild-type HGSNAT. Interestingly, c.1209G>T (p.W403C), which is present in a variant classified in the former category, has only been sequenced in alleles also possessing c.1843G>A (p.A615T), which independently has a negligible effect on HGSNAT expression. This report suggests that these may function together to abolish HGSNAT activity., ((c) 2010 Wiley-Liss, Inc.)

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disease that is caused by a deficiency of the lysosomal enzyme alpha-N-acetylglucosaminidase (NAGLU). Over 100 different mutations in the NAGLU gene have been identified in Sanfilippo syndrome type B patients; however, no large deletions have been reported. Here we present the first case of a large homozygous intragenic NAGLU gene deletion identified in an affected child of consanguineous parents. Long range and multiplex PCR methods were used to characterize this deletion which encompasses exons 3 and 4 and is 1146 base pairs long. We propose that Alu element-mediated unequal homologous recombination between an Alu-Y in intron 2 and an Alu-Sx in intron 4 is the likely mechanism for this deletion, thereby contributing further insight into the molecular etiology of this disorder and providing additional evidence of its allelic heterogeneity., ((c) 2010 Elsevier Inc. All rights reserved.)

Pluripotent stem cells, including human embryonic stem cells and induced pluripotent stem cells, have generated much excitement about their prospects for use in cell transplantation therapies. This is largely attributable to their virtually unlimited growth potential, their ability to be precisely genetically altered in culture, and their utility for forming differentiated cell populations with potential clinical applications. Lysosomal storage diseases such as Sanfilippo syndrome (MPS-IIIA) represent ideal candidate diseases for the evaluation of cell therapies in the central nervous system (CNS). These diseases exhibit widespread pathology yet result from a single gene deficiency, in the case of Sanfilippo syndrome the lysosomal enzyme sulfamidase. The aim of this study was to investigate mouse embryonic stem (ES) cell-derived glial precursor cells as a vehicle for sulfamidase delivery in the MPS-IIIA mouse brain. In this study we have created a mouse ES cell line genetically modified to stably express and secrete high levels of human sulfamidase and a protocol for the in vitro derivation of large numbers glial precursors from ES cells. Differentiation of sulfamidase-expressing ES cells resulted in cell populations with sustained secretion of high levels of sulfamidase, comprised primarily of glial precursor cells with minor contaminants of other neural cell phenotypes but not residual pluripotent cells. CNS implantation studies demonstrated that ES cell-derived glial precursor cells formed using this differentiation method were able to engraft and survive for at least 12 weeks following implantation. The percentage of engraftment was quantified in different regions of the brain in 2-, 4-, and 8-week-old normal and MPS-IIIA mice. No teratomas were observed in any of the cell-transplanted animals. The results of this study support the further investigation of sulfamidase-expressing glial precursor cells as a vehicle for delivery of deficient enzyme into the CNS of MPS-IIIA mice.

Sanfilippo syndrome type B (MPS III B) is caused by a deficiency of alpha-N-acetylglucosaminidase enzyme (Naglu), leading to accumulation of heparan sulfate (HS), a glycosaminoglycan (GAG), within lysosomes and to eventual progressive cerebral and systemic multiple organ abnormalities. Treatment of MPS patients is mainly supportive and enzyme replacement cell therapy shows promise for treating this disease. One new approach for potential treatment of MPS III B is human umbilical cord blood (hUCB) cell transplantation. Recently, we demonstrated that administration of hUCB cells into the cerebral ventricle of presymptomatic Naglu mice had a beneficial effect, probably due to enzyme delivery into the enzyme-deficient mutant mice. However, administration of these cells into the systemic circulation of mutant mice could be more advantageous and may lead to new strategies of enzyme replacement for Sanfilippo. The aim of this study was to determine the effect of intravenous administration of hUCB cells into a mouse model of Sanfilippo Syndrome type B. The major findings in our study were that hUCB cell administration improved behavioral outcomes (decreased hyper/stereotypical activity and improved cognitive function). Cells widely distribute within and outside the CNS and intraparenchymally migrate. Administered cells have an antiinflammatory effect (Th2-associated cytokines) in the brain and reduce heparan sulfate accumulation in the liver and spleen. Our results demonstrate the advantages of intravenously administering hUCB cells into a mouse model of Sanfilippo Syndrome type B, the advantages probably a result of Naglu delivery to enzyme-deficient organs., (Copyright 2009 Wiley-Liss, Inc.)

Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N-acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by alpha-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death, causing neurodegeneration and severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select&#95;db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT.

Mucopolysaccharidosis III (MPS III) has four forms (A-D) that result from buildup of an improperly degraded glycosaminoglycan in lysosomes. MPS IIIB is attributable to the decreased activity of a lysosomal alpha-N-acetylglucosaminidase (NAGLU). Here, we describe the structure, catalytic mechanism, and inhibition of CpGH89 from Clostridium perfringens, a close bacterial homolog of NAGLU. The structure enables the generation of a homology model of NAGLU, an enzyme that has resisted structural studies despite having been studied for >20 years. This model reveals which mutations giving rise to MPS IIIB map to the active site and which map to regions distant from the active site. The identification of potent inhibitors of CpGH89 and the structures of these inhibitors in complex with the enzyme suggest small-molecule candidates for use as chemical chaperones. These studies therefore illuminate the genetic basis of MPS IIIB, provide a clear biochemical rationale for the necessary sequential action of heparan-degrading enzymes, and open the door to the design and optimization of chemical chaperones for treating MPS IIIB.

Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is an autosomal recessive disorder, caused by a deficiency in one of the four enzymes involved in the lysosomal degradation of the glycosaminoglycan heparan sulfate. Based on the enzyme deficiency, four different subtypes, MPS IIIA, B, C, and D, are recognized. The genes encoding these four enzymes have been characterized and various mutations have been reported. The probable diagnosis of all MPS III subtypes is based on increased concentration of heparan sulfate in the urine. Enzymatic assays in leukocytes and/or fibroblasts confirm the diagnosis and allow for discrimination between the different subtypes of the disease. The clinical course of MPS III can be divided into three phases. In the first phase, which usually starts between 1 and 4 years of age, a developmental delay becomes apparent after an initial normal development during the first 1-2 years of life. The second phase generally starts around 3-4 years and is characterized by severe behavioural problems and progressive mental deterioration ultimately leading to severe dementia. In the third and final stage, behavioural problems slowly disappear, but motor retardation with swallowing difficulties and spasticity emerge. Patients usually die at the end of the second or beginning of the third decade of life, although survival into the fourth decade has been reported. Although currently no effective therapy is yet available for MPS III, several promising developments raise hope that therapeutic interventions, halting the devastating mental and behavioural deterioration, might be feasible in the near future.

Mucopolysaccharidosis type IIIB (Sanfilippo B disease) is a rare autosomal recessive disorder caused by defective alpha-N-acetylglucosaminidase (NAGLU). We examined the NAGLU gene in 11 MPS IIIB Portuguese patients, having identified five novel (M1K, W147X, G304V, S522P, and R533X) and four previously reported mutations (W168X, R234C, R565W and R643C). R234C attained the high prevalence of 32% of the mutated alleles. Because R234C had already been reported to be common in Spanish patients, a haplotypic analysis was conducted to address the question of its origin in the Iberian Peninsula. Three neutral markers were studied that allowed for the identification of the probable founder haplotype (174-234-G) on which R234C arose. The sharing of the ancestral haplotype by Portuguese and Spanish patients clearly implied a common origin of the mutation in Iberia, through an event that was inferred to have been rather recent. Therefore, the reconstructed history of R234C explains the high incidence of the mutation in Iberian patients with Sanfilippo B disease.

Mucopolysaccharidosis IIIC (MPS IIIC, Sanfilippo C syndrome) is a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). We performed a clinical study on 29 Dutch MPS IIIC patients and determined causative mutations in the recently identified HGSNAT gene. Psychomotor development was reported to be normal in all patients during the first year of life. First clinical signs were usually noted between 1 and 6 years (mean 3.5 years), and consisted of delayed psychomotor development and behavioral problems. Other symptoms included sleeping and hearing problems, recurrent infections, diarrhoea and epilepsy. Two sisters had attenuated disease and did not have symptoms until the third decade. Mean age of death was 34 years (range 25-48). Molecular analysis revealed mutations in both alleles for all patients except one. Altogether 14 different mutations were found: two splice site mutations, one frame shift mutation due to an insertion, three nonsense mutations and eight missense mutations. Two mutations, p.R344C and p.S518F, were frequent among probands of Dutch origin representing 22.0% and 29.3%, respectively, of the mutant alleles. This study demonstrates that MPS IIIC has a milder course than previously reported and that both severity and clinical course are highly variable even between sibs, complicating prediction of the clinical phenotype for individual patients. A clear phenotype-genotype correlation could not be established, except that the mutations p.G262R and p.S539C were only found in two sisters with late-onset disease and presumably convey a mild phenotype.

Background: Mucopolysaccharidosis (MPS) IIIB (Sanfilippo Syndrome type B) is caused by a deficiency in the lysosomal enzyme N-acetyl-glucosaminidase (Naglu). Children with MPS IIIB develop disturbances of sleep, activity levels, coordination, vision, hearing, and mental functioning culminating in early death. The murine model of MPS IIIB demonstrates lysosomal distention in multiple tissues, a shortened life span, and behavioral changes., Principal Findings: To more thoroughly assess MPS IIIB in mice, alterations in circadian rhythm, activity level, motor function, vision, and hearing were tested. The suprachiasmatic nucleus (SCN) developed pathologic changes and locomotor analysis showed that MPS IIIB mice start their daily activity later and have a lower proportion of activity during the night than wild-type controls. Rotarod assessment of motor function revealed a progressive inability to coordinate movement in a rocking paradigm. Purkinje cell counts were significantly reduced in the MPS IIIB animals compared to age matched controls. By electroretinography (ERG), MPS IIIB mice had a progressive decrease in the amplitude of the dark-adapted b-wave response. Corresponding pathology revealed shortening of the outer segments, thinning of the outer nuclear layer, and inclusions in the retinal pigmented epithelium. Auditory-evoked brainstem responses (ABR) demonstrated progressive hearing deficits consistent with the observed loss of hair cells in the inner ear and histologic abnormalities in the middle ear., Conclusions/significance: The mouse model of MPS IIIB has several quantifiable phenotypic alterations and is similar to the human disease. These physiologic and histologic changes provide insights into the progression of this disease and will serve as important parameters when evaluating various therapies.

Mucopolysaccharidosis IIIB, an autosomal recessive lysosomal storage disorder of heparan sulfate caused by mutations in the alpha-N-acetylglucosaminidase (NAGLU) gene, was recently discovered in cattle. Clinical signs include progressive ataxia, stumbling gait, swaying and difficulty in balance and walking. These clinical signs are usually first observed at approximately 2 years of age and then develop progressively over the lifespan of the animals. Affected bulls were found to be homozygous for the missense mutation E452K (c.1354G > A). The availability of mutational analysis permits screening for the NAGLU mutation to eradicate this mutation from the cattle breeding population.

Sanfilippo syndrome type D is an autosomal recessive lysosomal storage disease that is caused by a deficiency of N-acetylglucosamine-6-sulphatase, one of the enzymes involved in the catabolism of heparan sulphate. Only 15 patients have been described in the literature and just two mutations have been reported to date. We present the clinical, biochemical and molecular analysis of two Italian Sanfilippo D families. Novel homozygous mutations were identified in the affected patients from each family: a large intragenic deletion of 8723 bp encompassing exons 2 and 3 in family 1 and a nonsense mutation, Q272X, in family 2. The deletion is the first large intragenic deletion to be reported in any of the four Sanfilippo subtypes, including Sanfilippo type C in which the gene has recently been identified.

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Mucopolysaccharidosis IIIC (MPS IIIC), or Sanfilippo C, represents the only MPS disorder in which the responsible gene has not been identified; however, the gene has been localized to the pericentromeric region of chromosome 8. In an ongoing proteomics study of mouse lysosomal membrane proteins, we identified an unknown protein whose human homolog, TMEM76, was encoded by a gene that maps to 8p11.1. A full-length mouse expressed sequence tag was expressed in human MPS IIIC fibroblasts, and its protein product localized to the lysosome and corrected the enzymatic defect. The mouse sequence was used to identify the full-length human homolog (HGSNAT), which encodes a protein with no homology to other proteins of known function but is highly conserved among plants and bacteria. Mutational analyses of two MPS IIIC cell lines identified a splice-junction mutation that accounted for three mutant alleles, and a single base-pair insertion accounted for the fourth.

Numerous data support passage of maternal cells into the fetus during pregnancy in both human and animal models. However, functional benefits of maternal microchimerism in utero are unknown. The current study attempted to take advantage of this route for prenatal delivery of alpha-N-acetylglucosaminidase (Naglu) enzyme into the enzyme-deficient mouse model of Sanfilippo syndrome type B (MPS III B). Enzymatically sufficient mononuclear cells from human umbilical cord blood (MNC hUCB) were intravenously administered into heterozygote females modeling MPS III B on the 5th day of pregnancy during blastocyst implantation. The major findings were 1) administered MNC hUCB cells transmigrated and diffused into the embryos (E12.5); 2) some transmigrated cells expressed CD34 and CD117 antigens; 3) transmigrated cells were found in both the maternal and embryonic parts of placentas; 4) transmigrated cells corrected Naglu enzyme activity in all embryos; 5) administered MNC hUCB cells were extensively distributed in the organs and the blood of heterozygote mothers at one week after transplantation. Results indicate that prenatal delivery of Naglu enzyme by MNC hUCB cell administration into mothers of enzyme-deficient embryos is possible and may present a significant opportunity for new biotechnologies to treat many inherited disorders.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder resulting from sulfamidase deficiency, which leads to accumulation of heparan sulfate within lysosomes. We have determined the time-course of accumulation of a disaccharide [hexosamine-N-sulfate[alpha-1,4]hexuronic acid; HNS-UA] marker of heparan sulfate storage within the brain, liver, and spleen of a naturally occurring mouse model of MPS IIIA. HNS-UA is detectable in the brain of affected mice on the day of birth, when it is significantly increased compared to normal control mice. As mice age, this compound steadily accumulates until a plateau is reached at approximately 20-weeks. A similar rate of accumulation of HNS-UA is seen in the liver and spleen of affected mice. Intracerebral delivery of recombinant human sulfamidase reduced the amount of HNS-UA present in segments of the brain receiving the correcting enzyme, thus demonstrating the effectiveness of enzyme replacement therapy within the central nervous system of affected mice. This finding therefore provides evidence for the use of the disaccharide HNS-UA to monitor the effect of therapies for this condition in humans, when treatment strategies are devised.

Lysosomal storage disorders are rare, inherited diseases caused by a deficiency of a specific, lysosomal enzyme. In the case of mucopolysaccharidosis type IIIA, a lack of active sulfamidase enzyme results in heparan sulfate accumulation, severe and progressive neurological deficits, and usually premature death. Embryonic stem cells can be genetically modified to overexpress lysosomal enzymes, providing a renewable reservoir of cells that can be readily expanded in culture. Screening clonal lines of embryonic stem cells for desirable properties such as high levels and maintenance of enzyme activity throughout terminal differentiation to neural phenotypes theoretically provides a reproducible population of cells that can be fully characterized in vitro before implantation within the central nervous system in animal models of lysosomal storage disorders.

Genetic deficiency of the lysosomal enzyme, acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase), which catalyses the transmembrane acetylation of heparan sulfate results in severe neurodegenerative disease, mucopolysaccharidosis IIIC. N-Acetyltransferase has never been characterized structurally and its gene has never been identified. We combined traditional methods of enzyme purification with organellar proteomics, isolating lysosomal membranes from mouse liver using differential centrifugation and osmolysis, followed by detergent extraction and purification of N-acetyltransferase by liquid chromatography. Partially purified enzyme had a molecular mass of 240 kDa and pI of 7.4 by gel filtration and chromatofocusing. Its specific activity varied with protein concentration typical of oligomeric enzymes or multienzyme complexes. Incubation of N-acetyltransferase with acetyl[14C]CoA in the absence of the acceptor of the acetyl group resulted in radioactive labeling of a 120-kDa polypeptide, suggesting that it represents a subunit containing the enzyme active site. Furthermore, following acetyl[14C]-labeling, the 120-kDa protein was present in the lysosomal membranes purified from the normal human skin fibroblasts but absent in those from the skin fibroblasts of MPS IIIC patients.

The Sanfilippo syndrome type B (mucopolysaccharidosis IIIB) is an autosomal recessive disorder due to mutations in the gene encoding NAGLU (alpha-N-acetylglucosaminidase), one of the enzymes required for the degradation of the GAG (glycosaminoglycan) heparan sulphate. No therapy exists for affected patients. We have shown previously the efficacy of lentiviral-NAGLU-mediated gene transfer in correcting in vitro the defect on fibroblasts of patients. In the present study, we tested the therapy in vivo on a knockout mouse model using intravenous injections. Mice (8-10 weeks old) were injected with one of the lentiviral doses through the tail vein and analysed 1 month after treatment. A single injection of lentiviral-NAGLU vector resulted in transgene expression in liver, spleen, lung and heart of treated mice, with the highest level reached in liver and spleen. Expression of 1% normal NAGLU activity in liver resulted in a 77% decrease in the GAG content; more remarkably, an expression of 0.16% normal activity in lung was capable of decreasing the GAG level by 29%. Long-term (6 months) follow up of the gene therapy revealed that the viral genome integration persisted in the target tissues, although the real-time PCR analysis showed a decrease in the vector DNA content with time. Interestingly, the decrease in GAG levels was maintained in liver, spleen, lung and heart of treated mice. These results show the promising potential and the limitations of lentiviral-NAGLU vector to deliver the human NAGLU gene in vivo.