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.

Biomarkers for predicting disease course in Sanfilippo syndrome: An urgent unmet need in childhood-onset dementia.

Sanfilippo syndrome (MPS III) is an autosomal recessive inherited disorder causing dementia in children, following an essentially normal early developmental period. First symptoms typically include delayed language development, hyperactivity and/or insomnia from 2 years of age, followed by unremitting and overt loss of previously acquired skills. There are no approved treatments, and the median age of death is 18 years. Treatments under clinical trial demonstrate therapeutic benefit when applied pre-symptomatically in children diagnosed early through known familial inheritance risk. Newborn screening for Sanfilippo syndrome would enable pre-symptomatic diagnosis and optimal therapeutic benefit, however, many fold more patients with Sanfilippo syndrome are expected to be identified in the population than present with childhood dementia. Therefore, the capacity to stratify which Sanfilippo infants will need treatment in toddlerhood is necessary. While diagnostic methods have been developed, and continue to be refined, currently there are no tools or laboratory-based biomarkers available to provide pre-symptomatic prognosis. There is also a lack of progression and neurocognitive response-to-treatment biomarkers; disease stage and rate of progression are currently determined by age at symptom onset, loss of cerebral grey matter volume by magnetic resonance imaging and developmental quotient score for age. Robust blood-based biomarkers are an urgent unmet need. In this review, we discuss the development of biomarker assays for Sanfilippo based on the neuropathological pathways known to change leading into symptom onset and progression, and their performance as biomarkers in other neurodegenerative diseases. We propose that neural-derived exosomes extracted from blood may provide an ideal liquid biopsy to detect reductions in synaptic protein availability, and mitochondrial function. Furthermore, given the prominent role of neuroinflammation in symptom expression, glial fibrillary acidic protein detection in plasma/serum, alongside measurement of active brain atrophy by neurofilament light chain, warrant increased investigation for prognostic, progression and neurocognitive response-to-treatment biomarker potential in Sanfilippo syndrome and potentially other childhood dementias.

Filipin complex-reactive brain lesions: a cautionary tale.

OBJECTIVE: Filipin complex is an autooxidation-prone fluorescent histochemical stain used in the diagnosis of Niemann-Pick Disease Type C (NP-C), a neurodegenerative lysosomal storage disorder. It is also widely used by researchers examining the distribution and accumulation of unesterified cholesterol in cell and animal models of neurodegenerative diseases including NP-C and Sanfilippo syndrome (mucopolysaccharidosis IIIA; MPS IIIA). Recently, it has been suggested to be useful in studying Alzheimer's and Huntington's disease. Given filipin's susceptibility to photobleaching, we sought to establish a quantitative biochemical method for free cholesterol measurement. METHODS: Brain tissue from mice with MPS IIIA was stained with filipin. Total and free cholesterol in brain homogenates was measured using a commercially available kit and a quantitative LC-MS/MS assay was developed. Gangliosides GM1, GM2 and GM3 were also quantified using LC-MS/MS. RESULTS: As anticipated, the MPS IIIA mouse brain displayed large numbers of filipin-positive intra-cytoplasmic inclusions, presumptively endo-lysosomes. Challenging the prevailing dogma, however, we found no difference in the amount of free cholesterol in MPS IIIA mouse brain homogenates cf. control tissue, using either the fluorometric kit or LC-MS/MS assay. Filipin has previously been reported to bind to GM1 ganglioside, however, this lipid does not accumulate in MPS IIIA cells/tissues. Using a fluorometric assay, we demonstrate for the first time that filipin cross-reacts with both GM2 and GM3 gangliosides, explaining the filipin-reactive inclusions observed in MPS IIIA brain cells. CONCLUSION: Filipin is not specific for free cholesterol, and positive staining in any setting should be interpreted with caution.

Is SGSH heterozygosity a risk factor for early-onset neurodegenerative disease?

Lysosomal dysfunction may be an important factor in the pathogenesis of neurodegenerative disorders such as Parkinson's disease (PD). Heterozygous mutations in the gene encoding the lysosomal enzyme glucocerebrosidase (GBA1) have been found in PD patients, and some but not all mutations in other lysosomal enzyme genes, for example, NPC1 and MCOLN1 have been associated with PD. We have examined the behaviour and brain structure of mice carrying a D31N mutation in the sulphamidase (Sgsh) gene which encodes a lysosomal sulphatase. Female heterozygotes and wildtype mice aged 12-, 15-, 18- and 21-months of age underwent motor phenotyping and the brain was comprehensively evaluated for disease-associated lesions. Heterozygous mice exhibited impaired performance in the negative geotaxis test when compared with wildtype mice. Whilst the brain of Sgsh heterozygotes aged up to 21-months did not exhibit any of the gross features of PD, Alzheimer's disease or the neurodegenerative lysosomal storage disorders, for example, loss of striatal dopamine, reduced GBA activity, α-synuclein-positive inclusions, perturbation of lipid synthesis, or cerebellar Purkinje cell drop-out, we noted discrete structural aberrations in the dendritic tree of cortical pyramidal neurons in 21-month old animals. The overt disease lesions and resultant phenotypic changes previously described in individuals with heterozygous mutations in lysosomal enzyme genes such as glucocerebrosidase may be enzyme dependent. By better understanding why deficiency in, or mutant forms of some but not all lysosomal proteins leads to heightened risk or earlier onset of classical neurodegenerative disorders, novel disease-causing mechanisms may be identified.

ß-Mannosidosis in German Shepherd Dogs.

A neurological disease was investigated in 3 German Shepherd pups from the same litter that failed to grow normally, appeared stiff, were reluctant to move, and were deaf. They developed intermittent seizures and ataxia and had proprioceptive defects. Histopathology showed severe vacuolation of neurons, astrocytes in nervous tissue, renal tubular epithelial cells, and macrophages in nervous tissue, spleen, and liver. Vacuoles appeared empty with no storage material stained by periodic acid-Schiff (PAS) or Sudan black stains, leading to a diagnosis of a lysosomal storage disease and in particular an oligosaccharidosis. Biochemical and genomic studies showed that this was ß-mannosidosis, not previously diagnosed in dogs. A c.560T>A transition in exon 4 of the MANBA gene was found, which segregated in these and other family members in a manner consistent with it being the causative mutation of an autosomal recessive disease. This mutation led to substitution of isoleucine to asparagine at position 187 of the 885 amino acid enzyme, a change expected to have functional significance.

MPS-IIIA mice acquire autistic behaviours with age.

Mucopolysaccharidosis (MPS) type IIIA is an inherited, neurodegenerative lysosomal storage disorder resulting from mutations in the SGSH gene. Consequently, N-sulphoglucosamine sulphohydrolase enzyme activity is reduced resulting in impaired catabolism of heparan sulphate. After an asymptomatic period, patients typically show a progressive loss of cognitive and motor skills, with death often during the second decade of life. The diagnostic criteria of autism spectrum disorders (ASD) include impaired communication and social interactions, as well as displays of repetitive behaviours and fixed interests. Children with MPS-IIIA have been shown to exhibit decreased social communicative behaviours from approximately 3-4 years of age but behavioural stereotypies are mostly absent. In this study, we investigated whether a mouse model of MPS-IIIA exhibited ASD-like symptoms. The BTBR T+Itpr3tf/J inbred mouse model of autism was used as a positive control. Male MPS-IIIA and BTBR mice were less sociable compared with unaffected C57BL/6 male mice in the reciprocal social approach test administered at 20 weeks of age. Alternations in the frequency of social interactions was not evident at earlier stages of the disease course, suggesting an acquisition of ASD-like social behaviours. Stereotypical behaviours were not evident in male MPS-IIIA mice in the marble-burying test nor was the quality of nest constructed by mice affected. Collectively, these data suggest that MPS-IIIA mice acquire autistic social behaviours similar to the human condition, and thus they may be useful for elucidating symptom generating mechanisms and novel treatments for ASD.

Endo-lysosomal and autophagic dysfunction: a driving factor in Alzheimer's disease?

Alzheimer's disease (AD) is the most common cause of dementia, and its prevalence will increase significantly in the coming decades. Although important progress has been made, fundamental pathogenic mechanisms as well as most hereditary contributions to the sporadic form of the disease remain unknown. In this review, we examine the now substantial links between AD pathogenesis and lysosomal biology. The lysosome hydrolyses and processes cargo delivered by multiple pathways, including endocytosis and autophagy. The endo-lysosomal and autophagic networks are central to clearance of cellular macromolecules, which is important given there is a deficit in clearance of amyloid-ß in AD. Numerous studies show prominent lysosomal dysfunction in AD, including perturbed trafficking of lysosomal enzymes and accumulation of the same substrates that accumulate in lysosomal storage disorders. Examination of the brain in lysosomal storage disorders shows the accumulation of amyloid precursor protein metabolites, which further links lysosomal dysfunction with AD. This and other evidence leads us to hypothesise that genetic variation in lysosomal genes modifies the disease course of sporadic AD.

Slow, continuous enzyme replacement via spinal CSF in dogs with the paediatric-onset neurodegenerative disease, MPS IIIA.

Intra-cerebrospinal fluid (CSF) injection of recombinant human lysosomal enzyme is a potential treatment strategy for several neurodegenerative lysosomal storage disorders including Sanfilippo syndrome (Mucopolysaccharidosis type IIIA; MPS IIIA). Here we have utilised the MPS IIIA Huntaway dog model to compare the effectiveness of the repeated intermittent bolus injection strategy being used in the trials with an alternate approach; slow, continual infusion of replacement enzyme (recombinant human sulphamidase; rhSGSH) into the spinal CSF using a SynchroMed II® pump attached to a spinal infusion cannula. The ability of each enzyme delivery strategy to ameliorate lesions in MPS IIIA brain was determined in animals treated from ∼three- to six-months of age. Controls received buffer or no treatment. Significant reductions in heparan sulphate (primary substrate) were observed in brain samples from dogs treated via either cisternal or lumbar spinal CSF bolus injection methods and also in slow intra-spinal CSF infusion-treated dogs. The extent of the reduction differed regionally. Pump-delivered rhSGSH was less effective in reducing secondary substrate (GM3 ganglioside) in deeper aspects of cerebral cortex, and although near-amelioration of microglial activation was seen in superficial (but not deep) layers of cerebral cortex in both bolus enzyme-treated groups, pump-infusion of rhSGSH had little impact on microgliosis. While continual low-dose infusion of rhSGSH into MPS IIIA dog CSF reduces disease-based lesions in brain, it was not as efficacious as repeated cisternal or spinal CSF bolus infusion of rhSGSH over the time-frame of these experiments.

A novel conditional Sgsh knockout mouse model recapitulates phenotypic and neuropathic deficits of Sanfilippo syndrome.

Mucopolysaccharidosis (MPS) type IIIA, or Sanfilippo syndrome, is a neurodegenerative lysosomal storage disorder caused by a deficiency of the lysosomal enzyme N-sulfoglucosamine sulfohydrolase (SGSH), involved in the catabolism of heparan sulfate. The clinical spectrum is broad and the age of symptom onset and the degree of preservation of cognitive and motor functions appears greatly influenced by genotype. To explore this further, we generated a conditional knockout (Sgsh KO ) mouse model with ubiquitous Sgsh deletion, and compared the clinical and pathological phenotype with that of the spontaneous Sgsh D31N MPS-IIIA mouse model. Phenotypic deficits were noted in Sgsh KO mice prior to Sgsh D31N mice, however these outcomes did not correlate with any shift in the time of appearance nor rate of accumulation of primary (heparan sulfate) or secondary substrates (GM2/GM3 gangliosides). Other disease lesions (elevations in lysosomal integral membrane protein-II expression, reactive astrocytosis and appearance of ubiquitin-positive inclusions) were also comparable between affected mouse strains. This suggests that gross substrate storage and these neuropathological markers are neither primary determinants, nor good biomarkers/indicators of symptom generation, confirming similar observations made recently in MPS-IIIA patients. The Sgsh KO mouse will be a useful tool for elucidation of the neurological basis of disease and assessment of the clinical efficacy of new treatments for Sanfilippo syndrome.

Low-dose, continuous enzyme replacement therapy ameliorates brain pathology in the neurodegenerative lysosomal disorder mucopolysaccharidosis type IIIA.

Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained-release (osmotic pump-delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six-week-old MPS IIIA and unaffected mice were implanted with subcutaneous mini-osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part-way through the experimental period. We observed near-normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM 3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump-like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain. Clinical trials of repeated injection of replacement enzyme into CSF are underway in patients with the inherited neurodegenerative disorder mucopolysaccharidosis type IIIA. In this pre-clinical study, we examined an alternative approach - slow, continual infusion of enzyme using pumps. We observed significant reductions in substrate accumulation and other disease-based lesions in treated mouse brain. Thus, the strategy warrants consideration for testing in large animal models of MPS IIIA and also in other neurodegenerative lysosomal storage disorders.

Determination of the role of injection site on the efficacy of intra-CSF enzyme replacement therapy in MPS IIIA mice.

MPS IIIA is an inherited neurodegenerative lysosomal storage disorder characterized by cognitive impairment, sleep-wake cycle disturbance, speech difficulties, eventual mental regression and early death. Neuropathological changes include accumulation of heparan sulfate and glycolipids, neuroinflammation and degeneration. Pre-clinical animal studies indicate that replacement of the deficient enzyme, sulfamidase, via intra-cerebrospinal fluid (CSF) injection is a clinically-relevant treatment approach, reducing neuropathological changes and improving symptoms. Given that there are several routes of administration of enzyme into the CSF (intrathecal lumbar, cisternal and ventricular), determining the effectiveness of each injection strategy is crucial in order to provide the best outcome for patients. We delivered recombinant human sulfamidase (rhSGSH) to a congenic mouse model of MPS IIIA via each of the three routes. Mice were euthanized 24h or one-week post-injection; the distribution of enzyme within the brain and spinal cord parenchyma was investigated, and the impact on primary substrate levels and other pathological lesions determined. Both ventricular and cisternal injection of rhSGSH enable enzyme delivery to brain and spinal cord regions, with the former mediating large, statistically significant decreases in substrate levels and reducing microglial activation. The single lumbar CSF infusion permitted more restricted enzyme delivery, with no reduction in substrate levels and little change in other disease-related lesions in brain tissue. While the ventricular route is the most invasive of the three methods, this strategy may enable the widest distribution of enzyme within the brain, and thus requires further exploration.

Evaluation of enzyme dose and dose-frequency in ameliorating substrate accumulation in MPS IIIA Huntaway dog brain.

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

Disease stage determines the efficacy of treatment of a paediatric neurodegenerative disease.

Lysosomal storage disorders are a large group of inherited metabolic conditions resulting from the deficiency of proteins involved in lysosomal catabolism, with resulting accumulation of substrates inside the cell. Two-thirds of these disorders are associated with a neurodegenerative phenotype and, although few therapeutic options are available to patients at present, clinical trials of several treatments including lysosomal enzyme replacement are underway. Although animal studies indicate the efficacy of presymptomatic treatment, it is largely unknown whether symptomatic disease-related pathology and functional deficits are reversible. To begin to address this, we used a naturally-occurring mouse model with Sanfilippo syndrome (mucopolysaccharidosis type IIIA) to examine the effectiveness of intracisternal cerebrospinal fluid enzyme replacement in early, mid- and symptomatic disease stage mice. We observed a disease-stage-dependent treatment effect, with the most significant reductions in primary and secondary substrate accumulation, astrogliosis and protein aggregate accumulation seen in mucopolysaccharidosis type IIIA mice treated very early in the disease course. Affected mice treated at a symptomatic age exhibited little change in these neuropathological markers in the time-frame of the study. Microgliosis was refractory to treatment regardless of the age at which treatment was instigated. Although longer-term studies are warranted, these findings indicate the importance of early intervention in this condition.

Development of cerebellar pathology in the canine model of mucopolysaccharidosis type IIIA (MPS IIIA).

The temporal relationship between the onset of clinical signs in the mucopolysaccharidosis type IIIA (MPS IIIA) Huntaway dog model and cerebellar pathology has not been described. Here we sought to characterize the accumulation of primary (heparan sulfate) and secondary (G(M3)) substrates and onset of other changes in cerebellar tissues, and investigate the relationship to the onset of motor dysfunction in these animals. We observed that Purkinje cells were present in dogs aged up to and including 30.9 months, however by 40.9 months of age only ~12% remained, coincident with the onset of clinical signs. Primary and secondary substrate accumulation and inflammation were detected as early as 2.2 months and axonal spheroids were observed from 4.3 months in the deep cerebellar nuclei and later (11.6 months) in cerebellar white matter tracts. Degenerating neurons and apoptotic cells were not observed at any time. Our findings suggest that cell autonomous mechanisms may contribute to Purkinje cell death in the MPS IIIA dog.

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.

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.

Compromised transcription-mRNA export factor THOC2 causes R-loop accumulation, DNA damage and adverse neurodevelopment.

We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX (Transcription-Export) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37-38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37-38 deletion male (Thoc2Δ/Y) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2Δ/Y mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome.

The ovine Type II Gaucher disease model recapitulates aspects of human brain disease.

Acute neuronopathic (type II) Gaucher disease (GD) is a devastating, untreatable neurological disorder resulting from mutations in the glucocerebrosidase gene (GBA1), with subsequent accumulation of glucosylceramide and glucosylsphingosine. Patients experience progressive decline in neurological function, with onset typically within the first three-to-six months of life and premature death before two years. Mice and drosophila with GD have been described, however little is known about the brain pathology observed in the naturally occurring ovine model of GD. We have characterised pathological changes in GD lamb brain and compared the histological findings to those in GD patient post-mortem tissue, to determine the validity of the sheep as a model of this disease. Five GD and five age-matched unaffected lamb brains were examined. We observed significant expansion of the endo/lysosomal system in GD lamb cingulate gyrus however TPP1 and cathepsin D levels were unchanged or reduced. H&E staining revealed neurons with shrunken, hypereosinophilic cytoplasm and hyperchromatic or pyknotic nuclei (red neurons) that were also shrunken and deeply Nissl stain positive. Amoeboid microglia were noted throughout GD brain. Spheroidal inclusions reactive for TOMM20, ubiquitin and most strikingly, p-Tau were observed in many brain regions in GD lamb brain, potentially indicating disturbed axonal trafficking. Our findings suggest that the ovine model of GD exhibits similar pathological changes to human, mouse, and drosophila type II GD brain, and represents a model suitable for evaluating therapeutic intervention, particularly in utero-targeted approaches.

Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.

Lysosomal network abnormalities are an increasingly recognised feature of Alzheimer's disease (AD), which appear early and are progressive in nature. Sandhoff disease and Tay-Sachs disease (neurological lysosomal storage diseases caused by mutations in genes that code for critical subunits of ß-hexosaminidase) result in accumulation of amyloid-ß (Aß) and related proteolytic fragments in the brain. However, experiments that determine whether mutations in genes that code for ß-hexosaminidase are risk factors for AD are currently lacking. To determine the relationship between ß-hexosaminidase and AD, we investigated whether a heterozygous deletion of Hexb, the gene that encodes the beta subunit of ß-hexosaminidase, modifies the behavioural phenotype and appearance of disease lesions in App NL-G-F/NL-G-F (App KI/KI ) mice. App KI/KI and Hexb +/- mice were crossed and evaluated in a behavioural test battery. Neuropathological hallmarks of AD and ganglioside levels in the brain were also examined. Heterozygosity of Hexb in App KI/KI mice reduced learning flexibility during the Reversal Phase of the Morris water maze. Contrary to expectation, heterozygosity of Hexb caused a small but significant decrease in amyloid beta deposition and an increase in the microglial marker IBA1 that was region- and age-specific. Hexb heterozygosity caused detectable changes in the brain and in the behaviour of an AD model mouse, consistent with previous reports that described a biochemical relationship between HEXB and AD. This study reveals that the lysosomal enzyme gene Hexb is not haplosufficient in the mouse AD brain.

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.