Kim M. Hemsley

Characterization of a C57BL/6 congenic mouse strain of mucopolysaccharidosis type IIIA

The original mucopolysaccharidosis type IIIA (MPS IIIA) mice were identified in a mixed background with contributions from four different strains. To ensure long-term stability and genetic homogeneity of this lysosomal storage disease (LSD) model, the aim of this study was to develop and characterize a C57BL/6 congenic strain. The B6.Cg-Sgsh(mps3a) strain compares favorably with the original mixed donor strain, exhibiting low liver sulfamidase activity and significant brain heparan sulfate-derived disaccharide elevation from birth. A rapid increase in brain disaccharide levels occurred after birth, with a plateau reached by 13 weeks of age at 110x the levels observed in brains of age-matched unaffected mice. Typical lysosomal inclusions were observed in cerebral cortical and cerebellar neurons and in liver hepatocytes and Kupffer cells. Ubiquitin-positive spheroids and GM(2)-ganglioside were also detected in brain. Using the Morris water maze in male mice, impaired memory and spatial learning was evident at 20 weeks of age in B6.Cg-Sgsh(mps3a) MPS IIIA mice. Other behavioral changes include motor, cognitive and sensory deficits, and aggression. Male B6.Cg-Sgsh(mps3a) MPS IIIA mice exhibited more behavioral abnormalities than B6.Cg-Sgsh(mps3a) MPS IIIA females, as observed previously in the original mixed background strain. Affected mice generally survive to 9 to 12 months of age, before death or euthanasia for humane reasons. Overall, minor differences were apparent between the new congenic and previously described mixed MPS IIIA strains. Availability of an in-bred strain will ensure more reproducible experimental outcomes thereby assisting in our goal of developing effective therapies for LSD with central nervous system disease.

Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease

Mucopolysaccharidosis type IIIA is a neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Absent or greatly reduced activity of sulphamidase, a lysosomal protein, results in intracellular accumulation of heparan sulphate. Subsequent neuroinflammation and neurodegeneration typify this and many other lysosomal storage disorders. We propose that intra‐cerebrospinal fluid protein delivery represents a potential therapeutic avenue for treatment of this and other neurodegenerative conditions; however, technical restraints restrict examination of its use prior to adulthood in mice. We have used a naturally‐occurring Mucopolysaccharidosis type IIIA mouse model to determine the effectiveness of combining intravenous protein replacement (1 mg/kg) from birth to 6 weeks of age with intra‐cerebrospinal fluid sulphamidase delivery (100 μg, fortnightly from 6 weeks) on behaviour, the level of heparan sulphate‐oligosaccharide storage and other neuropathology. Mice receiving combination treatment exhibited similar clinical improvement and reduction in heparan sulphate storage to those only receiving intra‐cerebrospinal fluid enzyme. Reductions in micro‐ and astrogliosis and delayed development of ubiquitin‐positive lesions were seen in both groups. A third group of intravenous‐only treated mice did not exhibit clinical or neuropathological improvements. Intra‐cerebrospinal fluid injection of sulphamidase effectively, but dose‐dependently, treats neurological pathology in Mucopolysaccharidosis type IIIA, even when treatment begins in mice with established disease.

Effect of high dose, repeated intra-cerebrospinal fluid injection of sulphamidase on neuropathology in mucopolysaccharidosis type IIIA mice.

Mucopolysaccharidosis type IIIA (MPS IIIA) is an inherited neurodegenerative lysosomal storage disorder characterized by progressive loss of learned skills, sleep disturbance and behavioural problems. Reduced activity of sulphamidase (N‐sulphoglucosamine sulphohydrolase; SGSH; EC 3.10.1.1) results in intracellular accumulation of heparan sulphate (HS), with the brain as the primary site of pathology. We have used a naturally occurring MPS IIIA mouse model to determine the effectiveness of SGSH replacement through the cerebrospinal fluid (CSF) to decrease neuropathology. This is a potential therapeutic option for patients with this disorder. Mice received intra‐CSF injections of recombinant human SGSH (30, 50 or 70 μg) fortnightly from 6 to 18 weeks of age, and the cumulative effect on neuropathology was examined and quantified. Anti‐SGSH antibodies detected in plasma at euthanasia did not appear to impact upon the health of the mice or the experimental outcome, with significant but region‐dependent and dose‐dependent reductions in an HS‐derived oligosaccharide observed in the brain and spinal cord using tandem mass spectrometry. SGSH infusion reduced the number of storage inclusions observed in the brain when visualized using electron microscopy, and this correlated with a significant decrease in the immunohistochemical staining of a lysosomal membrane marker. Reduced numbers of activated isolectin B4‐positive microglia and glial fibrillary acidic protein‐positive astrocytes were seen in many, but not all, brain regions. Significant reductions in the number of ubiquitin‐positive intracellular inclusions were also observed. These outcomes show the effectiveness of this method of enzyme delivery in reducing the spectrum of neuropathological changes in murine MPS IIIA brain.

Development of motor deficits in a murine model of mucopolysaccharidosis type IIIA (MPS-IIIA).

Mucopolysaccharidosis (MPS) type IIIA or Sanfilippo syndrome is a lysosomal storage disorder characterised by progressive neurological pathology. Patients exhibit aggression, disturbed sleep, hyperactivity and mental decline ultimately resulting in inanition and death. Recently, a mouse model of MPS-IIIA was discovered, and both the clinical signs and neuropathological changes mimic the human disease. This provides us with an opportunity to study the pathological progression of this disorder and to determine the efficacy of novel therapies, for at present all lysosomal storage disorders (LSD) affecting the brain are untreatable. Neuropathological changes have previously been described in areas of the brain involved in regulating motor function, therefore, in the present study we sought to determine whether quantifiable motor abnormalities were present in the MPS-IIIA mouse, and if so, at what age they became detectable. Tests of open-field locomotor activity, hindlimb gait, catalepsy, neuromuscular strength and negative geotaxis were administered to MPS-IIIA and normal male and female mice aged from 3 to 40 weeks of age. Significant changes in open-field activity were observed from 3 weeks in MPS-IIIA mice, whereas abnormalities in tests of gait, grip strength and in the assessment of the negative geotaxis response were observable from approximately 15 weeks of age. Behavioural changes were often detected in male MPS-IIIA mice before they appeared in females. Our observations provide insight into the chronology of pathological changes within the MPS-IIIA brain and this simple series of tests will prove useful in comparing mice following the administration of experimental therapies for this condition.

Effect of cisternal sulfamidase delivery in MPS IIIA Huntaway dogs - a proof of principle study.

Mucopolysaccharidosis type IIIA (MPS IIIA) results from lack of functional sulfamidase (SGSH), a lysosomal enzyme. Its substrate, heparan sulfate, and other secondarily-stored compounds subsequently accumulate primarily within the central nervous system (CNS), resulting in progressive mental deterioration and early death. Presently there is no treatment. As a potential therapeutic strategy, recombinant human sulfamidase (rhSGSH) was administered into the CSF (via the cerebellomedullary cistern) of three adult MPS IIIA dogs either twice with a 4 day interval, or weekly for up to 4 weeks. The dogs were euthanased 24 h post-injection along with one untreated unaffected and two MPS IIIA controls. We have examined the three dimensional pattern of distribution of enzyme in the CNS and its ability to reduce primary substrate storage. High concentrations of rhSGSH protein, with up to 39-fold normal enzyme activity levels were detected within widespread areas of the CNS. RhSGSH protein was also detectable by immunohistochemistry in neurons and glia in all three enzyme-treated dogs. In both weekly-treated dogs, relative levels of a heparan sulfate-derived disaccharide, measured using tandem mass spectrometry, were lower in many brain regions when compared to untreated MPS IIIA controls. A moderately severe meningitis was also present as well as antibodies to rhSGSH in CSF/plasma. These findings demonstrate proof of principle that MPS IIIA can be treated by intracisternal enzyme replacement warranting further experiments in animals tolerant to rhSGSH. This enzyme delivery method may represent a means of treating neuropathology in MPS IIIA and other lysosomal storage disorders affecting the CNS.

An animal model of extrapyramidal side effects induced by antipsychotic drugs: relationship with D2 dopamine receptor occupancy.

1. Muscle rigidity was assessed quantitatively and objectively as increases in electromyographic (EMG) activity (muscle rigidity) in the hindlimb muscles of the rat following subcutaneous administration of haloperidol, fluphenazine and thioridazine. 2. Behavioural changes were assessed as increases in the catalepsy score, defined as the time taken for an animal to move off an inclined grid. 3. Increased tonic EMG activity, or the presence of catalepsy was related to the level of occupancy of dopamine D2 receptors in the striatum and substantia nigra of the brain, measured using ex vivo quantitative autoradiography. 4. Increases in tonic EMG activity and the induction of catalepsy were associated with >80% occupancy of striatal and nigral D2 receptors by fluphenazine, while haloperidol increased tonic EMG activity at D2 occupancies of >57%. 5. Thioridazine at doses ranging from 1-15 mg/kg failed to increase EMG activity and occupied <61% of striatal D2 receptors. 6. Overall the findings support the hypothesis that muscle rigidity is observed when a threshold level of D2 receptors in the striatum and substantia nigra are occupied by antipsychotic drugs. 7. This conclusion is consistent with the results of positron emission tomography (PET) studies in humans, and those from our past studies in rats using raclopride, chlorpromazine and clozapine, in which a threshold of approximately 70% striatal and nigral D2 receptor occupancy has been demonstrated.

Open field locomotor activity and anxiety-related behaviors in mucopolysaccharidosis type IIIA mice.

Mucopolysaccharidosis (MPS) IIIA, or Sanfilippo syndrome, is a lysosomal storage disorder characterized by severe and progressive neuropathology. Following an asymptomatic period, patients may present with sleep disturbances, cognitive decline, aggressive tendencies and hyperactivity. A naturally-occurring mouse model of MPS IIIA also exhibits many of these behavioral features and has been recently back-crossed onto a C57BL/6 genetic background. To more thoroughly characterize the behavioral phenotype of congenic MPS IIIA mice, we assessed exploratory activity and unconditioned anxiety-related behavior in the elevated plus maze (EPM) and open field locomotor activity. Although MPS IIIA male mice were less active in the EPM at 18 and 20 weeks of age, they were more likely to explore the open arms than their normal counter-parts suggesting reduced anxiety. Repeated EPM testing reduced exploration of the open arms in MPS IIIA mice. In the open field test, significant reductions in activity were evident in naïve-tested male MPS IIIA mice from 10 weeks of age. Female normal and MPS IIIA mice displayed similar exploratory activity in the open field test. These differences in anxiety and locomotor activity will allow us to evaluate the efficacy of therapeutic regimes for MPS IIIA as a forerunner to developing safe and effective therapies for Sanfilippo patients.

Enzyme replacement reduces neuropathology in MPS IIIA dogs

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

Impact of high-dose, chemically modified sulfamidase on pathology in a murine model of MPS IIIA

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative lysosomal storage disorder that results from a deficiency of sulfamidase (N-sulfoglucosamine sulfohydrolase), with consequential accumulation of its substrate, partially degraded heparan sulfate. Conventional doses (e.g. 1mg/kg) of intravenously delivered recombinant human sulfamidase (rhSGSH) do not improve neuropathology in MPS IIIA mice due to an inability to traverse the blood-brain barrier; however high-dose treatment or administration of enzyme that has been chemically modified to remove mannose-6-phosphate glycans has been shown to reduce neuropathology in related animal models. We have combined these approaches to evaluate the ability of 1, 5, 10 or 20mg/kg of similarly chemically modified or unmodified rhSGSH to reduce neuropathology following repeated intravenous delivery to adult MPS IIIA mice. rhSGSH was detected in brain homogenates from mice treated with all doses of modified rhSGSH and those receiving the two higher doses of unmodified rhSGSH, albeit at significantly lower levels. Immunohistochemically, rhSGSH visualized in the brain was localized to the endothelium, meninges and choroid plexus, with no convincing punctate intra-neuronal staining seen. This presumably underlies the failure of the treatment to reduce the relative level of a heparan sulfate-derived oligosaccharide (GlcNS-UA), or secondarily stored substrates that accumulate in MPS IIIA brain cells. However, modification of rhSGSH significantly increased its effectiveness in degrading GlcNS-UA in non-CNS tissues, potentially as a result of its reduced plasma clearance. If this observation is generally applicable, chemical modification may permit the use of significantly lower doses of lysosomal enzymes in patients currently receiving intravenous enzyme replacement therapy.

Allogeneic stem cell transplantation does not improve neurological deficits in mucopolysaccharidosis type IIIA mice

Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative metabolic disorder caused by mutations in the N-sulfoglucosamine sulfohydrolase gene with resultant accumulation of partially degraded heparan sulfate (HS). Whilst allogeneic bone marrow transplantation (BMT) is indicated for several lysosomal storage disorders featuring neurodegeneration, its use in MPS III is highly controversial. Published evidence suggests that BMT does not improve cognitive function in MPS III patients. Despite this, patients continue to be transplanted in some centers. We therefore sought to determine the clinical effectiveness of BMT in a murine model of MPS IIIA. Pre-symptomatic young adult mice pre-conditioned with total body irradiation generated complete and stable donor-type chimerism. Whilst HS-derived disaccharides were reduced by up to 27% in the brain parenchyma, this was insufficient to decrease secondary cholesterol and GM3 ganglioside storage or permit clinical improvement. These results suggest that BMT is ineffective in its unmodified form and should not be considered as a treatment for MPS IIIA children.