AAV gene replacement therapy for treating MPS IIIC: Facilitating bystander effects via EV-mRNA cargo.

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-hHGSNATEV 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-hHGSNATEV 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-hHGSNATEV 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 rAAVEV for MPS IIIC, and broad diseases, without having to transduce every cell.

Systemic immune challenge exacerbates neurodegeneration in a model of neurological lysosomal disease.

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-1ß 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-1ß involvement in MPS IIIA exacerbated neurodegeneration, our data robustly reinforces our previous findings, indicating IL-1ß as a pivotal catalyst for neuropathological processes in MPS IIIA.

Structural and mechanistic insights into a lysosomal membrane enzyme HGSNAT involved in Sanfilippo syndrome.

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.

Identification of Orthosteric and Allosteric Pharmacological Chaperones for Mucopolysaccharidosis Type IIIB.

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.

Mucopolysaccharidosis (MPS IIIA) mice have increased lung compliance and airway resistance, decreased diaphragm strength, and no change in alveolar structure.

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.

Investigation on lysosomal accumulation by a quantitative analysis of 2D phase-maps in digital holography microscopy.

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.

Evidence of epigenetic landscape shifts in mucopolysaccharidosis IIIB and IVA.

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.

Intraparenchymal convection enhanced delivery of AAV in sheep to treat Mucopolysaccharidosis IIIC.

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.

N-Substituted l-Iminosugars for the Treatment of Sanfilippo Type B Syndrome.

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.

Disease correction in mucopolysaccharidosis type IIIB mice by intraparenchymal or cisternal delivery of a capsid modified AAV8 codon-optimized NAGLU vector.

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.

Intracerebroventricular dosing of N-sulfoglucosamine sulfohydrolase in mucopolysaccharidosis IIIA mice reduces markers of brain lysosomal dysfunction.

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.

Impaired mitophagy in Sanfilippo a mice causes hypertriglyceridemia and brown adipose tissue activation.

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 [3H]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.

Sanfilippo syndrome type A: early cardiac involvement of two patients with cardiac manifestations.

PURPOSE: To report two unusual presentations of mucopolysaccharidosis type III (Sanfilippo syndrome) and provide evidence for the cardiac involvement. PATIENTS AND METHODS: We report two siblings with cardiac involvement that were diagnosed in childhood with Sanfilippo A Syndrome (SAS). All patients' diagnosis was confirmed by the excess of heparan sulfate in the urine and the reduction of heparan sulfamidase protein activity. The heart specimens were studied. RESULTS: We report two sibling patients (15-years-old female and 12-years-old female) occurring in sisters both with onset in childhood with no neurological, ophthalmic, hepatic symptoms or coarsening of features as classically described. Both patients underwent bilateral hip arthroplasty in their early 30`s. The older sister had an orthotopic heart transplant because of end-stage heart failure of her cardiomyopathy at the age of 45. She is alive and well. The youngest sister died due to heart failure before a transplantation took place. In the two siblings a thin right ventricular free wall was seen, which triggered the differential diagnosis with arrhythmogenic right ventricular cardiomyopathy or lamin A/C cardiomyopathy. CONCLUSIONS: Early recognition of solitary or mainly cardiac involvement is essential for patients with mucopolysaccharidosis type III (SAS).

Neuropathology of murine Sanfilippo D syndrome.

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.

Disease modeling for Mucopolysaccharidosis type IIIB using patient derived induced pluripotent stem cells.

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.

Competitive binding of extracellular accumulated heparan sulfate reduces lysosomal storage defects and triggers neuronal differentiation in a model of Mucopolysaccharidosis IIIB.

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.

Early defects in mucopolysaccharidosis type IIIC disrupt excitatory synaptic transmission.

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.

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.

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.

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA.

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 sgshΔex5-6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5-6 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 sgshΔex5-6 zebrafish is largely dependent on interleukin-1ß and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgshΔex5-6 mutant larvae in a context-dependent manner. We expect the sgshΔex5-6 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.

Longitudinal MRI brain volume changes over one year in children with mucopolysaccharidosis types IIIA and IIIB.

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 cm3/year [mean ± SD], cerebral white matter -4.37 ± 11.82 cm3/year, subcortical gray matter -6.54 ± 3.63 cm3/year, corpus callosum -0.18 ± 0.62 cm3/yr) and in cerebellar cortex (-0.49 ± 12.57 cm3/year), with a compensatory increase in lateral ventricular volume (7.17 ± 6.79 cm3/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.