MPS IIIB Mice Research Articles

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

BackgroundMucopolysaccharidosis (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.MethodsUsing 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.ResultsWe 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.ConclusionsTogether, 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.

Elucidation of the pathogenesis of heparan sulfate in MPS IIIB mice using a membrane-tethered form of NAGLU

Elucidation of the pathogenesis of heparan sulfate in MPS IIIB mice using a membrane-tethered form of NAGLU

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.

Femoral Structure and Biomechanical Characteristics in Sanfilippo Syndrome Type-B Mice.

Sanfilippo syndrome Type-B, also known as mucopolysaccharidosis IIIB (MPS IIIB), accounts for approximately one-third of all Sanfilippo syndrome patients and is characterized by a similar natural history as Type-A. Patients suffer from developmental regression, bone malformation, organomegaly, GI distress, and profound neurological deficits. Despite human trials of enzyme replacement therapy (ERT) (SBC-103, AX250) in MPS IIIB, there is currently no FDA approved treatment and a few palliative options. The major concerns of ERT and gene therapy for the treatment of bone malformation are the inadequate biodistribution of the missing enzyme, N-acetyl-α-glucosaminidase (NAGLU), and that the skeleton is a poorly hit target tissue in ERT and gene therapy. Each of the four known human types of MPS III (A, B, C, and D) is usually regarded as having mild bone manifestations, yet it remains poorly characterized. This study aimed to determine bone mineral content (BMC), volumetric bone mineral density (vBMD), and biomechanical properties in femurs MPS IIIB C57BL/6 mice compared to phenotypic control C57BL/6 mice. Significant differences were observed in MPS IIIB mice within various cortical and cancellous bone parameters for both males and females (p < 0.05). Here, we establish some osteogenic manifestations of MPS IIIB within the mouse model by radiographic and biomechanical tests, which are also differentially affected by age and sex. This suggests that some skeletal features of the MPS IIIB mouse model may be used as biomarkers of peripheral disease correction for preclinical treatment of MPS IIIB.

Membrane-tethered form of NAGLU used to elucidate pathogenesis of heparan sulfate in MPS IIIB mice

Membrane-tethered form of NAGLU used to elucidate pathogenesis of heparan sulfate in MPS IIIB mice

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.

Site-specific modifications to AAV8 capsid yields enhanced brain transduction in the neonatal MPS IIIB mouse

Mucopolysaccharidosis type IIIB (MPS IIIB) is an autosomal recessive lysosomal disease caused by defective production of the enzyme α-N-acetylglucosaminidase. It is characterized by severe and complex central nervous system degeneration. Effective therapies will likely target early onset disease and overcome the blood–brain barrier. Modifications of adeno-associated viral (AAV) vector capsids that enhance transduction efficiency have been described in the retina. Herein, we describe for the first time, a transduction assessment of two intracranially administered adeno-associated virus serotype 8 variants, in which specific surface-exposed tyrosine (Y) and threonine (T) residues were substituted with phenylalanine (F) and valine (V) residues, respectively. A double-mutant (Y447 + 733F) and a triple-mutant (Y447 + 733F + T494V) AAV8 were evaluated for their efficacy for the potential treatment of MPS IIIB in a neonatal setting. We evaluated biodistribution and transduction profiles of both variants compared to the unmodified parental AAV8, and assessed whether the method of vector administration would modulate their utility. Vectors were administered through four intracranial routes: six sites (IC6), thalamic (T), intracerebroventricular, and ventral tegmental area into neonatal mice. Overall, we conclude that the IC6 method resulted in the widest biodistribution within the brain. Noteworthy, we demonstrate that GFP intensity was significantly more robust with AAV8 (double Y–F + T–V) compared to AAV8 (double Y–F). This provides proof of concept for the enhanced utility of IC6 administration of the capsid modified AAV8 (double Y–F + T–V) as a valid therapeutic approach for the treatment of MPS IIIB, with further implications for other monogenic diseases.

Proteomic Analysis of Mucopolysaccharidosis IIIB Mouse Brain.

Mucopolysaccharidosis IIIB (MPS IIIB) is an inherited metabolic disease due to deficiency of α-N-Acetylglucosaminidase (NAGLU) enzyme with subsequent storage of undegraded heparan sulfate (HS). The main clinical manifestations of the disease are profound intellectual disability and neurodegeneration. A label-free quantitative proteomic approach was applied to compare the proteome profile of brains from MPS IIIB and control mice to identify altered neuropathological pathways of MPS IIIB. Proteins were identified through a bottom up analysis and 130 were significantly under-represented and 74 over-represented in MPS IIIB mouse brains compared to wild type (WT). Multiple bioinformatic analyses allowed to identify three major clusters of the differentially abundant proteins: proteins involved in cytoskeletal regulation, synaptic vesicle trafficking, and energy metabolism. The proteome profile of NAGLU−/− mouse brain could pave the way for further studies aimed at identifying novel therapeutic targets for the MPS IIIB. Data are available via ProteomeXchange with the identifier PXD017363.

Predominant role of microglia in brain iron retention in Sanfilippo syndrome, a pediatric neurodegenerative disease.

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

Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10.

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB.

Electrophysiological and Histological Characterization of Rod-Cone Retinal Degeneration and Microglia Activation in a Mouse Model of Mucopolysaccharidosis Type IIIB.

Sanfilippo syndrome Type B or Mucopolysaccharidosis IIIB (MPS IIIB) is a neurodegenerative autosomal recessive lysosomal storage disorder in which patients suffer severe vision loss from associated retinopathy. Here we sought to study the underlying retinal functional and morphological changes associated with MPS IIIB disease progression using the established model of MPS IIIB, the B6.129S6-Naglu(tm1Efn)/J mouse line. Electroretinogram (ERG) was recorded from MPS IIIB and wild-type (WT) mice at the age of 28 and 46 weeks, and retinal tissues were subsequently collected for immunohistochemistry analysis. At the 28th week, rod a- and b-wave amplitudes were significantly diminished in MPS IIIB compared to WT mice. The cone a- and b-waves of MPS IIIB mice were not significantly different from those of the control at the 28th week but were significantly diminished at the 46th week, when MPS IIIB mice showed a major loss of rods and rod bipolar cells in both central and peripheral regions and a minor loss of cones in the periphery. Activation of microglia and neovascularization were also detected in the MPS IIIB retina. The new findings that cones and rod bipolar cells also undergo degeneration, and that retinal microglia are activated, will inform future development of therapeutic strategies.

Oxidative stress is independent of inflammation in the neurodegenerative Sanfilippo syndrome type B.

Mucopolysaccharidosis (MPS) type IIIB is a genetic deficiency of α-N-acetylglucosaminidase, inducing accumulation of partially degraded heparan sulfate (HS) oligosaccharides in tissues. In the central nervous system, this accumulation is associated with microglial activation, neurodegeneration, and oxidative stress. We have already shown that HS activates microglial cells through toll-like receptor 4 (TLR4) and triggers neuroinflammation. The present study investigates whether oxidative stress is a direct consequence of inflammation or is an independent event directly caused by HS accumulation. The present study addresses causative links between oxidative stress and inflammation by analyzing the corresponding markers in the cortex of control mice, MPSIIIB mice (with neuroinflammation), and double mutant TLR4 knockout MPSIIIB mice (without neuroinflammation at early stages). Results showed that, although inflammation was not present in the cortex of 10-day-old double mutant MPSIIIB/TLR4(-/-) mice, the enzymatic activity of total superoxide dismutase (SOD) was already greater than in control animals. Moreover, at 3 and 8 months of age, the total enzymatic activities of glutathione peroxidase, SOD, and carbonyl protein levels in the cortex of MPSIIIB/TLR4(-/-) mice were similar to those measured in MPSIIIB mice and were higher than those in controls. The results indicate that the oxidative stress present at a very early stage in the brain of MPSIIIB mice is not the consequence of neuroinflammation. Insofar as it has an impact on the development of neurological disease, reducing oxidative stress might prevent or slow the progression of MPSIIIB.

Intracerebroventricular enzyme replacement therapy with glycosylation-independent lysosomal targeted NAGLU leads to widespread enzymatic activity, reduction of lysosomal storage and of secondary defects in brain of mice with Sanfilippo syndrome type B

Treatment for mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome type B) is hampered because recombinant alpha-N-acetylglucosaminidase (NAGLU) contains little/no mannose 6-phosphate. We developed a fusion protein of insulin-like growth factor 2 (IGF2) and NAGLU (rhNAGLU-IGF2). IGF2 is a natural ligand of the mannose-6phosphate receptor, and thus provides glycosylation-independent lysosomal targeting. Purified rhNAGLU-IGF2 is active in biochemical and cell-based assays (see Abstract: Aoyagi-Scharber, et al.). To evaluate rhNAGLU-IGF2 in vivo, adult (16 wk) MPS IIIB mice were catheterized into the left ventricle and given 4 twice-weekly injections of vehicle or rhNAGLU-IGF2 (100 μg). Mice were harvested 1d, 7d, 14d, and 28d after the last injection (n= 4-8 per group). Vehicle-treated heterozygotes were used as controls. Forebrain NAGLU activity ranged from 100x (1d, p b 0.001), 400x (7d, p = 0.001), 30x (14d, p = 0.9), and 130x fold (28d, p = 0.002) in rhNAGLU-IGF2 treated mice vs. vehicle-treated heterozygotes. Hexosaminidase activity was reduced by 26% (1d), 44% (7d), 51% (14d), and 57% (28d) in rhNAGLU-IGF2 treated vs vehicle-treated MPS IIIB mice (p b 0.001). Brain heparan sulfate was reduced from 228 pmol/ 5 μg brain (in vehicle-treated MPS IIIB) to 79.3 (1d), 24.8 (7d), 20.8 (14d), 17.4 (28d) in rhNAGLU-IGF2-treatedMPS IIIBmice (p b 0.001), vs 10.5 in vehicle-treated heterozygotes. In multiple brain regions, robust neuronal uptake was evident by immunostaining with NAGLU antibody and reduced LAMP1 signal with rhNAGLU-IGF2 treatment. In a separate experiment using 8 week-old mice, secondary accumulations were measured 1d after the last injection. Neuronal accumulations of SCMAS, glypican 5, amyloid beta and P-GSK3 beta in themedial entorhinal cortex and of P-tau in the dentate gyrus were reduced to the control level of these proteins. Accumulation of CD68 in activated microglia in the somatosensory cortex was reduced about half-way to control. In summary, we achieved broad distribution of NAGLU and improvement in neuropathology with short-term intraventricular rhNAGLU-IGF2. Glycosylation-independent lysosomal targeting may permit development of superior therapy for Sanfilippo syndrome type B. Support from NIH/NINDS 1R21NS078314-01A1 and BioMarin Pharmaceutical Inc.

332. Differences in AAV Serotype Dependent Distribution and Tropism Exist within the CNS of Wild Type and Mutant Mice in the MPSIIIB Mouse Model

332. Differences in AAV Serotype Dependent Distribution and Tropism Exist within the CNS of Wild Type and Mutant Mice in the MPSIIIB Mouse Model

Disease correction by combined neonatal intracranial AAV and systemic lentiviral gene therapy in Sanfilippo Syndrome type B mice

Mucopolysaccharidosis type IIIB (MPS IIIB) or Sanfilippo Syndrome type B is a lysosomal storage disease resulting from the deficiency of N-acetyl glucosaminidase (NAGLU) activity. We previously showed that intracranial adeno-associated virus (AAV) -based gene therapy results in partial improvements of several aspects of the disease. In an attempt to further correct the disease, MPS IIIB mice were treated at 2–4 days of age with intracranial AAV2/5-NAGLU (IC-AAV), intravenous lentiviral-NAGLU (IV-LENTI) or the combination of both (BOTH). The BOTH group had the most complete biochemical and histological improvements of any treatment group. Compared to untreated MPS IIIB animals, all treatments resulted in significant improvements in motor function (rotarod) and hearing (auditory-evoked brainstem response). In addition, each treatment group had a significantly increased median life span compared to the untreated group (322 days). The combination arm had the greatest increase (612 days), followed by IC-AAV (463 days) and IV-LENTI (358 days). Finally, the BOTH group had nearly normal circadian rhythm measures with improvement in time to activity onset. In summary, targeting both the systemic and central nervous system disease of MPS IIIB early in life appears to be the most efficacious approach for this inherited metabolic disorder.

Differences in AAV serotype dependent distribution and tropism within the CNS of wild type and mutant mice in the MPS IIIB mouse model

Differences in AAV serotype dependent distribution and tropism within the CNS of wild type and mutant mice in the MPS IIIB mouse model

Accelerated clinical disease and pathology in mucopolysaccharidosis type IIIB and GalNAc transferase double knockout mice

Mucopolysaccharidosis type IIIB (MPS IIIB) is a neuropathic lysosomal storage disorder (LSD) resulting from an inherited deficiency of N-acetyl-α-d-glucosaminidase (Naglu) activity, an enzyme required to degrade the glycosaminoglycan heparan sulfate (HS). A deficiency in Naglu activity leads to lysosomal accumulation of HS as a primary storage substrate, and the gangliosides GM2 and GM3 as secondary accumulation products. To test the effect on neuropathogenesis of ganglioside accumulation, we bred mice deficient in both Naglu and GalNaAcT activities. The latter is the enzyme required for synthesis of GM2 and other complex gangliosides. Contrary to our expectation and to double knockout (DKO) studies where GalNAcT was knocked out in combination with other LSDs, our DKO mice showed a drastically shortened lifespan (24.5±1.4weeks, versus 50.5±0.9weeks (MPS IIIB), and 38.6±1.2weeks (GalNAcT)). To confirm that HS storage was the primary element resulting in the accelerated disease in our DKO mice, and not a locus tightly linked to the Naglu gene, we replicated our study with MPS IIIA mice, and found a virtually identical result (27.5±1.8weeks, versus 53.8±1.6weeks). All DKO mice showed motor signs of hind limb ataxia and hyper-extension, which were not seen in single KO or normal mice. At approximately 5months of age, the MPS IIIB–DKO showed a unique pattern of vacuolization and nerve fiber degeneration in the corpus callosum, seen only in the DKO mice, as well as the relatively early intracytoplasmic vacuolation of many neurons and glia characteristic of the MPS IIIB mice. We analyzed motor performance on a rocking Rota-Rod beginning at 3months of age. The MPS IIIA–DKO and MPS IIIB–DKO mice showed impaired performance and were statistically different from all parental lines. In particular, the MPS IIIB–DKO mice were significantly different from the parent MPS IIIB strains at 3, 5, and 6months (p≤0.0245). In conclusion we identified an accelerated phenotype associated with MPS IIIB within a DKO model system which showed white matter changes, with attendant performance deficits and a drastically shortened lifespan. This was in stark contrast to our expectations of a salutary response to the elimination of GM2. Despite this, the accelerated pathology and clinical signs represent a potentially improved system to study MPS IIIB neuropathogenesis as well as the role of complex gangliosides in normal CNS function.

32. Normalized Survival and Permanent Restoration of NAGLU Activity in the CNS, PNS and Somatic Tissues in MPS IIIB Mice after a Single Intravenous rAAV9-hNAGLU Gene Delivery

32. Normalized Survival and Permanent Restoration of NAGLU Activity in the CNS, PNS and Somatic Tissues in MPS IIIB Mice after a Single Intravenous rAAV9-hNAGLU Gene Delivery

Defects in the medial entorhinal cortex and dentate gyrus in the mouse model of Sanfilippo syndrome type B.

Sanfilippo syndrome type B (MPS IIIB) is characterized by profound mental retardation in childhood, dementia and death in late adolescence; it is caused by deficiency of α-N-acetylglucosaminidase and resulting lysosomal storage of heparan sulfate. A mouse model, generated by homologous recombination of the Naglu gene, was used to study pathological changes in the brain. We found earlier that neurons in the medial entorhinal cortex (MEC) and the dentate gyrus showed a number of secondary defects, including the presence of hyperphosphorylated tau (Ptau) detected with antibodies raised against Ptau in Alzheimer disease brain. By further use of immunohistochemistry, we now show staining in neurons of the same area for beta amyloid, extending the resemblance to Alzheimer disease. Ptau inclusions in the dentate gyrus of MPS IIIB mice were reduced in number when the mice were administered LiCl, a specific inhibitor of Gsk3β. Additional proteins found elevated in MEC include proteins involved in autophagy and the heparan sulfate proteoglycans, glypicans 1 and 5, the latter closely related to the primary defect. The level of secondary accumulations was associated with elevation of glypican, as seen by comparing brains of mice at different ages or with different mucopolysaccharide storage diseases. The MEC of an MPS IIIA mouse had the same intense immunostaining for glypican 1 and other markers as MPS IIIB, while MEC of MPS I and MPS II mice had weak staining, and MEC of an MPS VI mouse had no staining at all for the same proteins. A considerable amount of glypican was found in MEC of MPS IIIB mice outside of lysosomes. We propose that it is the extralysosomal glypican that would be harmful to neurons, because its heparan sulfate branches could potentiate the formation of Ptau and beta amyloid aggregates, which would be toxic as well as difficult to degrade.

Hyperactive behaviour in the mouse model of mucopolysaccharidosis IIIB in the open field and home cage environments

Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder characterized by severe behavioural disturbances and progressive loss of cognitive and motor function. There is no effective treatment, but behavioural testing is a valuable tool to assess neurodegeneration and the effect of novel therapies in mouse models of disease. Several groups have evaluated behaviour in this model, but the data are inconsistent, often conflicting with patient natural history. We hypothesize that this discrepancy could be due to differences in open field habituation and home cage behaviour. Eight-month-old wild-type and MPS IIIB mice were tested in a 1-h open field test, performed 1.5 h after lights on, and a 24-h home cage behaviour test performed after 24 h of acclimatization. In the 1-h test, MPS IIIB mice were hyperactive, with increased rapid exploratory behaviour and reduced immobility time. No differences in anxiety were seen. Over the course of the test, differences became more pronounced with maximal effects at 1 h. The 24-hour home cage test was less reliable. There was evidence of increased hyperactivity in MPS IIIB mice, however, immobility was also increased, suggesting a level of inconsistency in this test. Performance of open field analysis within 1-2 h after lights on is probably critical to achieving maximal success as MPS IIIB mice have a peak in activity around this time. The open field test effectively identifies hyperactive behaviour in MPS IIIB mice and is a significant tool for evaluating effects of therapy on neurodegeneration.