No abstract

Mucopolysaccharide diseases (MPS) are caused by deficiency of glycosaminoglycan (GAG) degrading enzymes, leading to GAG accumulation. Neurodegenerative MPS diseases exhibit cognitive decline, behavioural problems and shortened lifespan. We have characterised neuropathological changes in mouse models of MPSI, IIIA and IIIB to provide a better understanding of these events.Wild-type (WT), MPSI, IIIA and IIIB mouse brains were analysed at 4 and 9 months of age. Quantitative immunohistochemistry showed significantly increased lysosomal compartment, GM2 ganglioside storage, neuroinflammation, decreased and mislocalised synaptic vesicle associated membrane protein, (VAMP2), and decreased post-synaptic protein, Homer-1, in layers II/III-VI of the primary motor, somatosensory and parietal cortex. Total heparan sulphate (HS), was significantly elevated, and abnormally N-, 6-O and 2-O sulphated compared to WT, potentially altering HS-dependent cellular functions. Neuroinflammation was confirmed by significantly increased MCP-1, MIP-1α, IL-1α, using cytometric bead arrays. An overall genotype effect was seen in all parameters tested except for synaptophysin staining, neuronal cell number and cortical thickness which were not significantly different from WT. MPSIIIA and IIIB showed significantly more pronounced pathology than MPSI in lysosomal storage, astrocytosis, microgliosis and the percentage of 2-O sulphation of HS. We also observed significant time progression of all genotypes from 4–9 months in lysosomal storage, astrocytosis, microgliosis and synaptic disorganisation but not GM2 gangliosidosis. Individual genotype*time differences were disparate, with significant progression from 4 to 9 months only seen for MPSIIIB with lysosomal storage, MPSI with astrocytocis and MPSIIIA with microgliosis as well as neuronal loss. Transmission electron microscopy of MPS brains revealed dystrophic axons, axonal storage, and extensive lipid and lysosomal storage. These data lend novel insight to MPS neuropathology, suggesting that MPSIIIA and IIIB have more pronounced neuropathology than MPSI, yet all are still progressive, at least in some aspects of neuropathology, from 4–9 months.

Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-β. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.

Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo disease) is a neurodegenerative disorder caused by a deficiency in the lysosomal enzyme sulfamidase (SGSH), catabolizing heparan sulfate (HS). Affected children present with severe behavioral abnormalities, sleep disturbances, and progressive neurodegeneration, leading to death in their second decade. MPS I, a similar neurodegenerative disease accumulating HS, is treated successfully with hematopoietic stem cell transplantation (HSCT) but this treatment is ineffectual for MPS IIIA. We compared HSCT in MPS IIIA mice using wild-type donor cells transduced ex vivo with lentiviral vector-expressing SGSH (LV-WT-HSCT) versus wild-type donor cell transplant (WT-HSCT) or lentiviral-SGSH transduced MPS IIIA cells (LV-IIIA-HSCT). LV-WT-HSCT results in 10% of normal brain enzyme activity, near normalization of brain HS and GM2 gangliosides, significant improvements in neuroinflammation and behavioral correction. Both WT-HSCT and LV-IIIA-HSCT mediated improvements in GM2 gangliosides and neuroinflammation but were less effective at reducing HS or in ameliorating abnormal HS sulfation and had no significant effect on behavior. This suggests that HS may have a more significant role in neuropathology than neuroinflammation or GM2 gangliosides. These data provide compelling evidence for the efficacy of gene therapy in conjunction with WT-HSCT for neurological correction of MPS IIIA where conventional transplant is ineffectual.

BackgroundNeurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein.Methodology/Principal FindingsWe report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed.Conclusions/SignificanceGenistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.

The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass. Moreover, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes in foetal and adult liver can clonally expand, suggesting that hepatoblasts/hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.

Hematopoietic stem cell gene therapy for neurological disorders relies on transmigration of donor-derived monocytes to the brain, where they can engraft as microglia and deliver therapeutic proteins. Many mouse studies use whole-body irradiation to investigate brain transmigration pathways, but chemotherapy is generally used clinically. The current evidence for transmigration to the brain after chemotherapy is conflicting. We compared hematopoietic donor cell brain engraftment after bone marrow (BM) transplants in busulfan- or irradiation-conditioned mice. Significantly more donor-derived microglial cells engrafted posttransplant in busulfan-conditioned brain compared with the irradiated, in both the short and long term. Although total Iba-1(+) microglial content was increased in irradiated brain in the short term, it was similar between groups over long-term engraftment. MCP-1, a key regulator of monocyte transmigration, showed long-term elevation in busulfan-conditioned brain, whereas irradiated brains showed long-term elevation of the proinflammatory chemokine interleukin 1α (IL-1α), with increased in situ proliferation of resident microglia, and significant increases in the relative number of amoeboid activated microglia in the brain. This has implications for the choice of conditioning regimen to promote hematopoietic cell brain engraftment and the relevance of irradiation in mouse models of transplantation.