Evaluation of neuroretina following i.v. or intra-CSF AAV9 gene replacement in mice with MPS IIIA, a childhood dementia.

BACKGROUND: Sanfilippo syndrome (mucopolysaccharidosis type IIIA; MPS IIIA) is a childhood dementia caused by inherited mutations in the sulfamidase gene. At present, there is no treatment and children with classical disease generally die in their late teens. Intravenous or intra-cerebrospinal fluid (CSF) injection of AAV9-gene replacement is being examined in human clinical trials; evaluation of the impact on brain disease is an intense focus; however, MPS IIIA patients also experience profound, progressive photoreceptor loss, leading to night blindness. AIM: To compare the relative efficacy of the two therapeutic approaches on retinal degeneration in MPS IIIA mice. METHODS: Neonatal mice received i.v. or intra-CSF AAV9-sulfamidase or vehicle and after 20 weeks, biochemical and histological evaluation of neuroretina integrity was carried out. RESULTS: Both treatments improved central retinal thickness; however, in peripheral retina, outer nuclear layer thickness and photoreceptor cell length were only significantly improved by i.v. gene replacement. Further, normalization of endo-lysosomal compartment size and microglial morphology was only observed following intravenous gene delivery. CONCLUSIONS: Confirmatory studies are needed in adult mice; however, these data indicate that i.v. AAV9-sulfamidase infusion leads to superior outcomes in neuroretina, and cerebrospinal fluid-delivered AAV9 may need to be supplemented with another therapeutic approach for optimal patient quality of life.

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.

Heterologous HSPC Transplantation Rescues Neuroinflammation and Ameliorates Peripheral Manifestations in the Mouse Model of Lysosomal Transmembrane Enzyme Deficiency, MPS IIIC.

Mucopolysaccharidosis III type C (MPS IIIC) is an untreatable neuropathic lysosomal storage disease caused by a genetic deficiency of the lysosomal N-acetyltransferase, HGSNAT, catalyzing a transmembrane acetylation of heparan sulfate. HGSNAT is a transmembrane enzyme incapable of free diffusion between the cells or their cross-correction, which limits development of therapies based on enzyme replacement and gene correction. Since our previous work identified neuroinflammation as a hallmark of the CNS pathology in MPS IIIC, we tested whether it can be corrected by replacement of activated brain microglia with neuroprotective macrophages/microglia derived from a heterologous HSPC transplant. Eight-week-old MPS IIIC (HgsnatP304L) mice were transplanted with HSPC from congenic wild type mice after myeloablation with Busulfan and studied using behavior test battery, starting from the age of 6 months. At the age of ~8 months, mice were sacrificed to study pathological changes in the brain, heparan sulfate storage, and other biomarkers of the disease. We found that the treatment corrected several behavior deficits including hyperactivity and reduction in socialization, but not memory decline. It also improved several features of CNS pathology such as microastroglyosis, expression of pro-inflammatory cytokine IL-1ß, and accumulation of misfolded amyloid aggregates in cortical neurons. At the periphery, the treatment delayed development of terminal urinary retention, potentially increasing longevity, and reduced blood levels of heparan sulfate. However, we did not observe correction of lysosomal storage phenotype in neurons and heparan sulfate brain levels. Together, our results demonstrate that neuroinflammation in a neurological lysosomal storage disease, caused by defects in a transmembrane enzyme, can be effectively ameliorated by replacement of microglia bearing the genetic defect with cells from a normal healthy donor. They also suggest that heterologous HSPC transplant, if used together with other methods, such as chaperone therapy or substrate reduction therapy, may constitute an effective combination therapy for MPS IIIC and other disorders with a similar etiology.

Novel gene-specific Bayesian Gaussian mixture model to predict the missense variants pathogenicity of Sanfilippo syndrome.

MPS III is an autosomal recessive lysosomal storage disease caused mainly by missense variants in the NAGLU, GNS, HGSNAT, and SGSH genes. The pathogenicity interpretation of missense variants is still challenging. We aimed to develop unsupervised clustering-based pathogenicity predictor scores using extracted features from eight in silico predictors to predict the impact of novel missense variants of Sanfilippo syndrome. The model was trained on a dataset consisting of 415 uncertain significant (VUS) missense NAGLU variants. Performance The SanfilippoPred tool was evaluated by validation and test datasets consisting of 197-labelled NAGLU missense variants, and its performance was compared versus individual pathogenicity predictors using receiver operating characteristic (ROC) analysis. Moreover, we tested the SanfilippoPred tool using extra-labelled 427 missense variants to assess its specificity and sensitivity threshold. Application of the trained machine learning (ML) model on the test dataset of labelled NAGLU missense variants showed that SanfilippoPred has an accuracy of 0.93 (0.86-0.97 at CI 95%), sensitivity of 0.93, and specificity of 0.92. The comparative performance of the SanfilippoPred showed better performance (AUC = 0.908) than the individual predictors SIFT (AUC = 0.756), Polyphen-2 (AUC = 0.788), CADD (AUC = 0.568), REVEL (AUC = 0.548), MetaLR (AUC = 0.751), and AlphMissense (AUC = 0.885). Using high-confidence labelled NAGLU variants, showed that SanfilippoPred has an 85.7% sensitivity threshold. The poor correlation between the Sanfilippo syndrome phenotype and genotype represents a demand for a new tool to classify its missense variants. This study provides a significant tool for preventing the misinterpretation of missense variants of the Sanfilippo syndrome-relevant genes. Finally, it seems that ML-based pathogenicity predictors and Sanfilippo syndrome-specific prediction tools could be feasible and efficient pathogenicity predictors in the future.

Characterization of early markers of disease in the mouse model of mucopolysaccharidosis IIIB.

BACKGROUND: Mucopolysaccharidosis (MPS) IIIB, also known as Sanfilippo Syndrome B, is a devastating childhood disease. Unfortunately, there are currently no available treatments for MPS IIIB patients. Yet, animal models of lysosomal storage diseases have been valuable tools in identifying promising avenues of treatment. Enzyme replacement therapy, gene therapy, and bone marrow transplant have all shown efficacy in the MPS IIIB model systems. A ubiquitous finding across rodent models of lysosomal storage diseases is that the best treatment outcomes resulted from intervention prior to symptom onset. Therefore, the aim of the current study was to identify early markers of disease in the MPS IIIB mouse model as well as examine clinically-relevant behavioral domains not yet explored in this model. METHODS: Using the MPS IIIB mouse model, we explored early developmental trajectories of communication and gait, and later social behavior, fear-related startle and conditioning, and visual capabilities. In addition, we examined brain structure and function via magnetic resonance imaging and diffusion tensor imaging. RESULTS: We observed reduced maternal isolation-induced ultrasonic vocalizations in MPS IIIB mice relative to controls, as well as disruption in a number of the spectrotemporal features. MPS IIIB also exhibited disrupted thermoregulation during the first two postnatal weeks without any differences in body weight. The developmental trajectories of gait were largely normal. In early adulthood, we observed intact visual acuity and sociability yet a more submissive phenotype, increased aggressive behavior, and decreased social sniffing relative to controls. MPS IIIB mice showed greater inhibition of startle in response to a pretone with a decrease in overall startle response and reduced cued fear memory. MPS IIIB also weighed significantly more than controls throughout adulthood and showed larger whole brain volumes and normalized regional volumes with intact tissue integrity as measured with magnetic resonance and diffusion tensor imaging, respectively. CONCLUSIONS: Together, these results indicate disease markers are present as early as the first two weeks postnatal in this model. Further, this model recapitulates social, sensory and fear-related clinical features. Our study using a mouse model of MPS IIIB provides essential baseline information that will be useful in future evaluations of potential treatments.

Identification and characterization of novel genetic variants in the first Chinese family of mucopolysaccharidosis IIIC (Sanfilippo C syndrome).

Mucopolysaccharidosis type IIIC (MPS IIIC) is one of inherited lysosomal storage disorders, caused by deficiencies in lysosomal hydrolases degrading acidic mucopolysaccharides. The gene responsible for MPS IIIC is HGSNAT, which encodes an enzyme that catalyses the acetylation of the terminal glucosamine residues of heparan sulfate. So far, few studies have focused on the genetic landscape of MPS IIIC in China, where IIIA and IIIB were the major subtypes. In this study, we utilized whole-exome sequencing (WES) to identify novel compound heterozygous variants in the HGSNAT gene from a Chinese patient with typical MPS IIIC symptoms: c.743G>A; p.Gly248Glu and c.1030C>T; p.Arg344Cys. We performed in silico analysis and experimental validation, which confirmed the deleterious pathogenic nature of both variants, as evidenced by the loss of HGSNAT activity and failure of lysosomal localization. To the best of our knowledge, the MPS IIIC is first confirmed by clinical, biochemical and molecular genetic findings in China. Our study thus expands the spectrum of MPS IIIC pathogenic variants, which is of importance to dissect the pathogenesis and to carry out clinical diagnosis of MPS IIIC. Moreover, this study helps to depict the natural history of Chinese MPS IIIC populations.

Genetic features of patients with MPS type IIIB: Description of five pathogenic gene variations.

BACKGROUND: There are four distinct forms of Sanfilippo syndrome (MPS type III), each of which is an autosomal lysosomal storage disorder. These forms are caused by abnormalities in one of four lysosomal enzymes. This study aimed to identify possible genetic variants that contribute to Sanfilippo IIIB in 14 independent families in Southwest Iran. METHODS: Patients were included if their clinical features and enzyme assay results were suggestive. The patients were subsequently subjected to Sanger Sequencing to screen for Sanfilippo-related genes. Additional investigations have been conducted using various computational analyses to determine the probable functional effects of diagnosed variants. RESULTS: Five distinct variations were identified in the NAGLU gene. This included two novel variants in two distinct families and three previously reported variants in 12 distinct families. All of these variations were recognized as pathogenic using the MutationTaster web server. In silico analysis showed that all detected variants affected protein structural stability; four destabilized protein structures, and the fifth variation had the opposite effect. CONCLUSION: In this study, two novel variations in the NAGLU gene were identified. The results of this study positively contribute to the mutation diversity of the NAGLU gene. To identify new disease biomarkers and therapeutic targets, precision medicine must precisely characterize and account for genetic variations. New harmful gene variants are valuable for updating gene databases concerning Sanfilippo disease variations and NGS gene panels. This may also improve genetic counselling for rapid risk examinations and disease surveillance.

[Natural history of mucopolysaccharidosis type III in a series of Colombian patients]. / Historia natural de la mucopolisacaridosis III en una serie de pacientes colombianos.

INTRODUCTION: Mucopolysaccharidosis type III (MPS III), also known as Sanfilippo syndrome, is a lysosomal storage disease with progressive neurodegenerative features, predominantly affecting the central nervous system. Diagnosis is based on clinical features, with neurodevelopmental and neuropsychiatric alterations taking precedence, including over phenotype alterations. The disease is confirmed by biochemical analysis to identify the type of glycosaminoglycans present, enzyme assay and molecular genetic studies. CASE REPORTS: A clinical description was performed for eight patients diagnosed with MPS III in Colombia. Their initial symptoms were related to developmental delay and behavioural disorders presenting between 3 and 8 years of age, associated in all cases with coarse facial features, thick eyebrows, hepatomegaly and progressive hearing loss. One of the patients presented cardiac anomalies; two presented focal epilepsy; and one presented optic atrophy. They all presented neuroimaging alterations, with evidence of parenchymal volume loss, corpus callosum atrophy and cortical thinning; the diagnosis was performed by biochemical glycosaminoglycan chromatography studies, and all patients have a confirmatory genetic study. CONCLUSIONS: MPS III is a challenge for diagnosis, particularly in its early stages and in patients in which the course of the disease is attenuated. This is due to its variable course, non-specific early neuropsychiatric symptoms, and the absence of obvious somatic features compared to other types of MPS. After a definitive diagnosis has been made, interdisciplinary care must be provided for the patient and their family, and support given for the treatment of physical symptoms, ensuring the best possible care and quality of life for the patient and their family, as the condition is neurodegenerative.

TITLE: Historia natural de la mucopolisacaridosis III en una serie de pacientes colombianos.Introducción. La mucopolisacaridosis de tipo III (MPS III), o síndrome de Sanfilippo, es un trastorno de almacenamiento lisosómico con características neurodegenerativas progresivas, predominante del sistema nervioso central. Su diagnóstico se basa en el cuadro clínico, y priman alteraciones en el neurodesarrollo y neuropsiquiátricas, incluso antes de la presencia de alteraciones fenotípicas. El análisis bioquímico para identificar el tipo de glucosaminoglucanos presente, la determinación enzimática y el estudio de genética molecular confirman la enfermedad. Casos clínicos. Se realiza la descripción clínica de ocho pacientes con diagnóstico de MPS III en Colombia, con síntomas iniciales en relación con retraso del desarrollo y trastornos comportamentales evidenciados entre los 3 y 8 años, asociado a facies toscas, cejas pobladas, hepatomegalia y pérdida auditiva progresiva en todos los casos. Uno de los pacientes presentó anomalías cardíacas; dos de ellos, epilepsia focal; y en uno se evidenció atrofia óptica. Todos presentaron alteraciones en las neuroimágenes con evidencia de pérdida del volumen parenquimatoso, atrofia del cuerpo calloso y adelgazamiento cortical; el diagnostico se realizó a través de estudios bioquímicos de cromatografía de glucosaminoglucanos y todos cuentan con un estudio genético confirmatorio. Conclusiones. La MPS III es un desafío diagnóstico, particularmente en pacientes con un curso atenuado de la enfermedad, debido al curso variable, síntomas neuropsiquiátricos tempranos inespecíficos y falta de características somáticas evidentes en comparación con otros tipos de MPS. Cuando se tiene el diagnóstico definitivo, es fundamental brindar atención interdisciplinaria para el paciente y la familia, y apoyar el tratamiento de los síntomas físicos, garantizando ofrecer el mejor cuidado posible y la mejor calidad de vida para el paciente y su familia, al tratarse de una condición neurodegenerativa.

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.

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.

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.

Drosophila melanogaster models of MPS IIIC (Hgsnat-deficiency) highlight the role of glia in disease presentation.

Sanfilippo syndrome (Mucopolysaccharidosis type III or MPS III) is a recessively inherited neurodegenerative lysosomal storage disorder. Mutations in genes encoding enzymes in the heparan sulphate degradation pathway lead to the accumulation of partially degraded heparan sulphate, resulting ultimately in the development of neurological deficits. Mutations in the gene encoding the membrane protein heparan-α-glucosaminide N-acetyltransferase (HGSNAT; EC2.3.1.78) cause MPS IIIC (OMIM#252930), typified by impaired cognition, sleep-wake cycle changes, hyperactivity and early death, often before adulthood. The precise disease mechanism that causes symptom emergence remains unknown, posing a significant challenge in the development of effective therapeutics. As HGSNAT is conserved in Drosophila melanogaster, we now describe the creation and characterisation of the first Drosophila models of MPS IIIC. Flies with either an endogenous insertion mutation or RNAi-mediated knockdown of hgsnat were confirmed to have a reduced level of HGSNAT transcripts and age-dependent accumulation of heparan sulphate leading to engorgement of the endo/lysosomal compartment. This resulted in abnormalities at the pre-synapse, defective climbing and reduced overall activity. Altered circadian rhythms (shift in peak morning activity) were seen in hgsnat neuronal knockdown lines. Further, when hgsnat was knocked down in specific glial subsets (wrapping, cortical, astrocytes or subperineural glia), impaired climbing or reduced activity was noted, implying that hgsnat function in these specific glial subtypes contributes significantly to this behaviour and targeting treatments to these cell groups may be necessary to ameliorate or prevent symptom onset. These novel models of MPS IIIC provide critical research tools for delineating the key cellular pathways causal in the onset of neurodegeneration in this presently untreatable disorder.

Repetitive, non-invasive imaging of neurodegeneration, and prevention of it with gene replacement, in mice with Sanfilippo syndrome.

Hampering assessment of treatment outcomes in gene therapy and other clinical trials in patients with childhood dementia is the lack of an objective, non-invasive measure of neurodegeneration. Optical coherence tomography (OCT) is a widely available, rapid, non-invasive, and quantitative method for examining the integrity of the neuroretina. Profound brain and retinal dysfunction occur in patients and animal models of childhood dementia, including Sanfilippo syndrome and we recently revealed a correlation between the age of onset and rate of progression of retinal and brain degeneration in sulfamidase-deficient Sanfilippo mice. The aim of the current study was to use OCT to visualise the discrete changes in retinal structure that occur during disease progression. A progressive decline in retinal thickness was readily observable in Sanfilippo mice using OCT, with differences seen in affected animals from 10-weeks of age. OCT applied to i.v. AAV9-sulfamidase-treated Sanfilippo mice enabled visualisation of improved retinal anatomy in living animals, an outcome confirmed via histology. Importantly, brain disease lesions were also ameliorated in treated Sanfilippo mice. The findings highlight the sensitivity, ease of repetitive use and quantitative capacity of OCT for detection of discrete changes in retinal structure and their prevention with a therapeutic. Combined with the knowledge that retinal and brain degeneration are correlated in Sanfilippo syndrome, OCT provides a window to the brain in this and potentially other childhood dementias.

Ability change across multiple domains in mucopolysaccharidosis (Sanfilippo syndrome) type IIIA.

The objective of this paper is 1) to expand the scope of the domains previously published in a natural history study of Mucopolysaccharidosis IIIA (Sanfilippo syndrome type A) (MPS IIIA) and 2) to present evidence regarding the capacity of a new metric, Growth Scale Values (GSVs), in comparison with traditional metrics, to show changes in skills as assessed by the Bayley Scales of Infant Development -III (BSID-III) and the Vineland Adaptive Behavior Scales, Second Edition (VABS-II). We re-analyzed a cohort of 25 children, 20 with rapid progressing disease and 5 with slow progression, who had been followed over two years using the BSID-III, and the VABS-II. Previously findings were reported using age equivalent scores; now we are also presenting findings with GSVs. For the re-analysis, Language and Motor scores were added to the Cognitive scale on the BSID-III, and Domain- and Subdomain-level scores added to the Total VABS-II score (i.e., ABC Composite). We evaluated raw scores, age equivalent scores, and GSVs (and standard scores for the VABS-II only). Individual patient data can be found in the appendices to this publication. Results indicate that 1) Cognition as measured by GSVs was the most sensitive to decline; 2) GSVs showed significant decline in the range of 4 to 6 years of age; 3) For children under 4 years of age, positive growth occurs on most scales and most metrics, with the exception of language which slows somewhat earlier; 4) Other than the Cognitive scale, Receptive Language on the BSID-III and Receptive Communication on the VABS-II showed the most sensitivity to change; 5) Gross Motor skills showed the least decline over time and appeared to lack sensitivity to MPS IIIA motor concerns; and 6) No evidence for sensitivity to change for any metric was found in time intervals less than one year. We conclude that GSVs are a precise measurement of change to detect decline in function, and they are a valuable method for future clinical trials in MPS IIIA. Evidence continues to support cognition as a primary endpoint. Additional work is needed to identify sensitive measures of meaningful endpoints to families.

Late-onset mucopolysaccharidosis type IIIA mimicking Usher syndrome.

Mucopolysaccharidosis type IIIA (MPS IIIA or Sanfilippo syndrome type A) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants in the SGSH gene encoding N-sulfoglucosamine sulfohydrolase, an enzyme involved in the degradation of heparan sulfate. MPS IIIA is typically characterized by neurocognitive decline and hepatosplenomegaly with childhood onset. Here, we report on a 53-year-old male subject initially diagnosed with Usher syndrome for the concurrence of retinitis pigmentosa and sensorineural hearing loss. Clinical exome sequencing identified biallelic missense variants in SGSH, and biochemical assays showed complete deficiency of sulfamidase activity and increased urinary glycosaminoglycan excretion. Reverse phenotyping revealed left ventricle pseudo-hypertrophy, hepatosplenomegaly, bilateral deep white matter hyperintensities upon brain MRI, and decreased cortical metabolic activity by PET-CT. On neuropsychological testing, the proband presented only partial and isolated verbal memory deficits. This case illustrates the power of unbiased, comprehensive genetic testing for the diagnosis of challenging mild or atypical forms of MPS IIIA.

Disease pathology signatures in a mouse model of Mucopolysaccharidosis type IIIB.

Mucopolysaccharidosis type IIIB (MPS IIIB) is a rare and devastating childhood-onset lysosomal storage disease caused by complete loss of function of the lysosomal hydrolase α-N-acetylglucosaminidase. The lack of functional enzyme in MPS IIIB patients leads to the progressive accumulation of heparan sulfate throughout the body and triggers a cascade of neuroinflammatory and other biochemical processes ultimately resulting in severe mental impairment and early death in adolescence or young adulthood. The low prevalence and severity of the disease has necessitated the use of animal models to improve our knowledge of the pathophysiology and for the development of therapeutic treatments. In this study, we took a systematic approach to characterizing a classical mouse model of MPS IIIB. Using a series of histological, biochemical, proteomic and behavioral assays, we tested MPS IIIB mice at two stages: during the pre-symptomatic and early symptomatic phases of disease development, in order to validate previously described phenotypes, explore new mechanisms of disease pathology and uncover biomarkers for MPS IIIB. Along with previous findings, this study helps provide a deeper understanding of the pathology landscape of this rare disease with high unmet medical need and serves as an important resource to the scientific community.

An immune deficient mouse model for mucopolysaccharidosis IIIA (Sanfilippo syndrome).

Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation.