Stephen Duplock

Effect of lysosomal storage on bis(monoacylglycero)phosphate

BMP [bis(monoacylglycero)phosphate] is an acidic phospholipid and a structural isomer of PG (phosphatidylglycerol), consisting of lysophosphatidylglycerol with an additional fatty acid esterified to the glycerol head group. It is thought to be synthesized from PG in the endosomal/lysosomal compartment and is found primarily in multivesicular bodies within the same compartment. In the present study, we investigated the effect of lysosomal storage on BMP in cultured fibroblasts from patients with eight different LSDs (lysosomal storage disorders) and plasma samples from patients with one of 20 LSDs. Using ESI-MS/MS (electrospray ionization tandem MS), we were able to demonstrate either elevations or alterations in the individual species of BMP, but not of PG, in cultured fibroblasts. All affected cell lines, with the exception of Fabry disease, showed a loss of polyunsaturated BMP species relative to mono-unsaturated species, and this correlated with the literature reports of lysosomal dysfunction leading to elevations of glycosphingolipids and cholesterol in affected cells, processes thought to be critical to the pathogenesis of LSDs. Plasma samples from patients with LSDs involving storage in macrophages and/or with hepatomegaly showed an elevation in the plasma concentration of the C(18:1)/C(18:1) species of BMP when compared with control plasmas, whereas disorders involving primarily the central nervous system pathology did not. These results suggest that the release of BMP is cell/tissue-specific and that it may be useful as a biomarker for a subset of LSDs.

Delivery of therapeutic protein for prevention of neurodegenerative changes: comparison of different CSF-delivery methods.

Injection of lysosomal enzyme into cisternal or ventricular cerebrospinal fluid (CSF) has been carried out in 11 lysosomal storage disorder models, with each study demonstrating reductions in primary substrate and secondary neuropathological changes, and several reports of improved neurological function. Whilst acute studies in mucopolysaccharidosis (MPS) type II mice revealed that intrathecally-delivered enzyme (into thoraco-lumbar CSF) accesses the brain, the impact of longer-term treatment of affected subjects via this route is unknown. This approach is presently being utilized to treat children with MPS types I, II and III. Our aim was to determine the efficacy of repeated intrathecal injection of recombinant human sulfamidase (rhSGSH) on pathological changes in the MPS IIIA dog brain. The outcomes were compared with those in dogs treated via intra-cisternal or ventricular routes. Control dogs received buffer or no treatment. Significant reductions in primary/secondary substrate levels in brain were observed in dogs treated via all routes, although the extent of the reduction differed regionally. Treatment via all CSF access points resulted in large reductions in microgliosis in superficial cerebral cortex, but only ventricular injection enabled amelioration in deep cerebral cortex. Formation of glutamic acid decarboxylase-positive axonal spheroids in deep cerebellar nuclei was prevented by treatment delivered via any route. Anti-rhSGSH antibodies in the sera of some dogs did not reduce therapeutic efficacy. Our data indicates the capacity of intra-spinal CSF-injected rhSGSH to circulate within CSF-filled spaces, penetrate into brain and mediate a significant reduction in substrate accumulation and secondary pathology in the MPS IIIA dog brain.

Selective reduction of bis(monoacylglycero)phosphate ameliorates the storage burden in a THP-1 macrophage model of Gaucher disease.

Bis(monoacylglycero)phosphate (BMP) assists lysosomal function by facilitating interaction of hydrolases and activator proteins with sphingolipid substrates. Impaired lysosomal degradation of the sphingolipid glucosylceramide (GC) occurs in Gaucher disease due to an inherited deficiency of acid β-glucosidase, with secondary BMP alterations. We investigated the nature of BMP accumulation and whether its correction reduced the storage burden in a THP-1 macrophage model of Gaucher disease. Using sucrose gradients and detergent solubility, 98% of BMP resided in the detergent-soluble membranes (DSM) rather than in the detergent-resistant membranes (DRM) where 73% of GC predominated. There was a 2-fold widespread elevation in BMP, including the saturated, mono- and polyunsaturated species. Linoleic acid in the culture media selectively reduced BMP from 4.2 nmol/mg to 0.49 nmol/mg (except 18:1/18:2) and prevented up to one third of GC, dihexosylceramide (DHC), and trihexosylceramide (THC) from accumulating. The 2-fold reduction in these sphingolipids occurred only in the DRM and did not reduce 18:1/16:0. However, once GC had accumulated, linoleic acid could not reverse it, DHC, or THC, despite effectively reducing BMP. These results imply a causative link for BMP in the pathobiology of Gaucher disease and demonstrate that linoleic acid can shield the cell from excessive substrate accumulation.

Liquid chromatography/electrospray ionisation–tandem mass spectrometry quantification of GM2 gangliosides in human peripheral cells and plasma

GM2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of GM2 gangliosides (GM2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify GM2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of GM2 was achieved using LC/ESI-MS/MS with d35GM1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all GM2 gangliosidosis patients were demarcated from controls by elevated GM2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in GM2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted β-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for GM2 gangliosidosis by allowing quantification of a reduction in the primary storage burden.

Glycosphingolipid analysis in a naturally occurring ovine model of acute neuronopathic Gaucher disease.

Gaucher disease arises from mutations in the β-glucocerebrosidase gene which encodes an enzyme required for the lysosomal catabolism of glucosylceramide. We have identified a naturally occurring mutation in the β-glucocerebrosidase gene in sheep that leads to Gaucher disease with acute neurological symptoms. Here we have examined the clinical phenotype at birth and subsequently quantified lipids in Gaucher lamb brain, in order to characterise the disorder. Enzyme activity assessments showed that a reduction in β-glucocerebrosidase activity to 1-5% of wild-type occurs consistently across newborn Gaucher lamb brain regions. We analyzed glucosylceramide, glucosylsphingosine, bis(monoacylglycero)phosphate and ganglioside profiles in brain, liver, and spleen, and observed 30- to 130-fold higher glucosylceramide, and 500- to 2000-fold higher glucosylsphingosine concentrations in Gaucher diseased lambs compared to wild-type. Significant increases of bis(monoacylglycero)phosphate and gangliosides [GM1, GM2, GM3] concentrations were also detected in the brain. As these glycosphingolipids are involved in many cellular events, an imbalance or disruption of the cell membrane lipid homeostasis would be expected to impair normal neuronal function. To our knowledge, this is the first detailed analysis of glycosphingolipids in various brain regions in a large animal model of neuronal disease, which permits the mechanistic investigation of lipid deregulation and their contribution to neurodegenerative process.

Low-dose, continual enzyme delivery ameliorates some aspects of established brain disease in a mouse model of a childhood-onset neurodegenerative disorder.

AIM To determine the capacity of continual low-dose lysosomal enzyme infusion into the cerebrospinal fluid of mucopolysaccharidosis type IIIA (MPS IIIA) mice to reverse established neurodegenerative disease. The rationale behind the study is that there is only limited animal model-derived evidence supporting treatment of symptomatic patients, principally because few studies have been designed to examine disease reversibility. METHODS Twelve-week old MPS IIIA mice were implanted with indwelling unilateral intra-ventricular cannulae. These were connected to subcutaneous mini-osmotic pumps infusing recombinant human sulphamidase. Pump replacement was carried out in some mice at 16-weeks of age, enabling treatment to continue for a further month. Control affected/unaffected mice received vehicle via the same method. Behavioural, neuropathological and biochemical parameters of disease were assessed. RESULTS Improvement in some, but not all, behavioural parameters occurred. Sulphamidase infusion mediated a statistically significant reduction in primary (heparan sulphate) and secondary (gangliosides GM2, GM3) substrate accumulation in the brain, with small reductions in micro- but not astro-gliosis. There was no change in axonal spheroid number. All mice developed a humoural response, however the antibodies were non-neutralising and no adverse clinical effects were observed. CONCLUSIONS Continual infusion of replacement enzyme partially ameliorates clinical, histological and biochemical aspects of MPS IIIA mice, when treatment begins at an early symptomatic stage.

Mass spectrometric quantification of glycogen to assess primary substrate accumulation in the Pompe mouse.

Glycogen storage in the α-glucosidase knockout((6neo/6neo)) mouse recapitulates the biochemical defect that occurs in the human condition; as such, this mouse serves as a model for the inherited metabolic deficiency of lysosomal acid α-glucosidase known as Pompe disease. Although this model has been widely used for the assessment of therapies, the time course of glycogen accumulation that occurs as untreated Pompe mice age has not been reported. To address this, we developed a quantitative method involving amyloglucosidase digestion of glycogen and quantification of the resulting free glucose by liquid chromatography/electrospray ionization-tandem mass spectrometry. The method was sensitive enough to measure as little as 0.1 μg of glycogen in tissue extracts with intra- and interassay coefficients of variation of less than 12%. Quantification of glycogen in tissues from Pompe mice from birth to 26 weeks of age showed that, in addition to the accumulation of glycogen in the heart and skeletal muscle, glycogen also progressively accumulated in the brain, diaphragm, and skin. Glycogen storage was also evident at birth in these tissues. This method may be particularly useful for longitudinal assessment of glycogen reduction in response to experimental therapies being trialed in this model.

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 (SgshKO) mouse model with ubiquitous Sgsh deletion, and compared the clinical and pathological phenotype with that of the spontaneous SgshD31N MPS-IIIA mouse model. Phenotypic deficits were noted in SgshKO mice prior to SgshD31N 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 SgshKO 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.