Suppression of NK and CD8+ T cells reduces astrogliosis but accelerates cerebellar dysfunction and shortens life span in a mouse model of Sandhoff disease.

Sandhoff disease is an inherited lysosomal storage disease, resulting from the deficiency of lysosomal ß-hexosaminidase A and B enzyme activity. The Hexb-/- mouse model recapitulates human disease and leads to fatal neurodegeneration and neuroinflammation. IL-15 is important for the proliferation of NK, NK T, and CD8+ cytotoxic/memory T cells. In order to determine how changes to IL-15-dependent immune cell populations would alter the course of Sandhoff disease in mice, we generated a Hexb-/-Il-15-/- double knockout mouse and used motor behaviour tests, analyzed peripheral blood and brain leukocyte immunophenotypes, cytokine secretion, as well as examined markers of microgliosis, astrogliosis and apoptosis. Hexb-/-Il-15-/- mice had an accelerated neurodegenerative phenotype, and reached the humane endpoint at 118±3.5d, compared to Hexb-/- mice (127±2.2d). The performance of Hexb-/-Il-15-/- mice declined earlier than Hexb-/- mice on the rotarod and righting reflex motor behaviour tests. Hexb-/- mice had a significantly higher prevalence of pro-inflammatory monocytes in the blood relative to C57BL/6 mice, but this was unaltered by IL-15 deficiency. The prevalence of NK cells and CD8+ T cells in Il-15-/- and Hexb-/-Il-15-/- mice was decreased compared to wild type and Hexb-/- mice. While Hexb-/- mice displayed an increase in the prevalence of CD4+ and CD8+ T cells in brain leukocytes compared to C57BL/6 mice, there was a decrease in CD8+ T cells in Hexb-/-Il-15-/- compared to Hexb-/- mice. In addition, circulating IL-17 and IL-10 levels were significantly higher in Hexb-/-Il-15-/- mice, suggesting heightened inflammation compared to Hexb-/- mice. Interestingly, astrogliosis levels were significantly reduced in the cerebellum of Hexb-/-Il-15-/- mice compared to Hexb-/- mice while microgliosis was not affected in brains of Hexb-/-Il-15-/- mice. Our study demonstrated that IL-15 depletion dramatically reduced numbers of NK and CD8+ T cells as well as astrocytes but accelerated disease progression in Sandhoff mice. These results pointed to interactions between NK/CD8+ T cells and astrogliosis and potentially a protective role for NK/CD8+ T cells and/or astrocytes during disease progression. This observation supports the notion that expanding the IL-15-dependent NK and CD8+ T cells populations with IL-15 therapy may have therapeutic benefits for Sandhoff disease.

Long-term correction of Sandhoff disease following intravenous delivery of rAAV9 to mouse neonates.

G(M2) gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in ß-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb(-/-)) of the G(M2) gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or -LacZ and monitored for serum ß-hexosaminidase activity, motor function, and survival. Brain G(M2) ganglioside, ß-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB-treated SD mice survived until 43 weeks (P < 0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB-treated mice had a significant increase in brain ß-hexosaminidase activity, and a reduction in G(M2) ganglioside storage and neuroinflammation compared to adult SD-HexB- and SD-LacZ-treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other G(M2) gangliosidoses through early rAAV9 based systemic gene therapy.

Reversibility of neuropathology in Tay-Sachs-related diseases.

The GM2 gangliosidoses are progressive neurodegenerative disorders due to defects in the lysosomal ß-N-acetylhexosaminidase system. Accumulation of ß-hexosaminidases A and B substrates is presumed to cause this fatal condition. An authentic mouse model of Sandhoff disease (SD) with pathological characteristics resembling those noted in infantile GM2 gangliosidosis has been described. We have shown that expression of ß-hexosaminidase by intracranial delivery of recombinant adeno-associated viral vectors to young adult SD mice can prevent many features of the disease and extends lifespan. To investigate the nature of the neurological injury in GM2 gangliosidosis and the extent of its reversibility, we have examined the evolution of disease in the SD mouse; we have moreover explored the effects of gene transfer delivered at key times during the course of the illness. Here we report greatly increased survival only when the therapeutic genes are expressed either before the disease is apparent or during its early manifestations. However, irrespective of when treatment was administered, widespread and abundant expression of ß-hexosaminidase with consequent clearance of glycoconjugates, α-synuclein and ubiquitinated proteins, and abrogation of inflammatory responses and neuronal loss was observed. We also show that defects in myelination occur in early life and cannot be easily resolved when treatment is given to the adult brain. These results indicate that there is a limited temporal opportunity in which function and survival can be improved-but regardless of resolution of the cardinal pathological features of GM2 gangliosidosis, a point is reached when functional deterioration and death cannot be prevented.

NSAIDs increase survival in the Sandhoff disease mouse: synergy with N-butyldeoxynojirimycin.

The GM2 gangliosidoses are caused by incomplete catabolism of GM2 ganglioside in the lysosome, leading to progressive storage and a neurodegenerative clinical course. An inflammatory response (microglial activation, macrophage infiltration, oxidative damage) has been found to be a consequence of GM2 storage in the brain, although it remains unclear whether this contributes to pathogenesis or disease progression. In this study, we treated Sandhoff disease mice with nonsteroidal antiinflammatory drugs (indomethacin, aspirin, and ibuprofen) and antioxidants (L-ascorbic acid and alpha-tocopherol acetate). The treated mice lived significantly longer than untreated littermates (12-23%, p <0.0001) and showed a slower rate of disease progression (p <0.001). When aspirin treatment was combined with substrate reduction therapy, synergy resulted (11%, p <0.05) with a maximum improvement of 73% in survival (p <0.00001). This study demonstrates that inflammation contributes to disease progression and identifies antiinflammatory and antioxidant therapies as a potential adjunctive approach to slow the clinical course of this and related disorders.

Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease.

Sandhoff disease is a severe neurodegenerative glycosphingolipid (GSL) lysosomal storage disorder, currently without treatment options. One therapeutic approach under investigation is substrate reduction therapy (SRT). By partially inhibiting GSL biosynthesis, the impaired rate of GSL catabolism is balanced by a slower rate of influx of GSLs into the lysosome. In a previous study, we reported the beneficial effects of treating Sandhoff disease mice with the glucose analogue N-butyldeoxynojirimycin (NB-DNJ), a compound that inhibits the first step of GSL biosynthesis catalysed by the ceramide specific glucosyltransferase. NB-DNJ, however, exhibits adverse effects at high doses such as weight loss and GI tract distress (due to glucosidase inhibition). This might limit the therapeutic potential of NB-DNJ for treating diseases affecting the CNS where high dose therapy may be required to achieve therapeutic levels of the drug in the brain. In the present study, a more selective compound, the galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ), was evaluated in the Sandhoff disease mouse model. Treatment with NB-DGJ showed greater therapeutic efficacy than NB-DNJ with no detectable side effects. The ability to escalate the dose of NB-DGJ, leading to extended life expectancy and increased delay in symptom onset, demonstrates the greater therapeutic potential of NB-DGJ for the treatment of the human gangliosidoses.

Bone marrow transplantation prolongs life span and ameliorates neurologic manifestations in Sandhoff disease mice.

The GM2 gangliosidoses are a group of severe, neurodegenerative conditions that include Tay-Sachs disease, Sandhoff disease, and the GM2 activator deficiency. Bone marrow transplantation (BMT) was examined as a potential treatment for these disorders using a Sandhoff disease mouse model. BMT extended the life span of these mice from approximately 4.5 mo to up to 8 mo and slowed their neurologic deterioration. BMT also corrected biochemical deficiencies in somatic tissues as indicated by decreased excretion of urinary oligosaccharides, and lower glycolipid storage and increased levels of beta-hexosaminidase activity in visceral organs. Even with neurologic improvement, neither clear reduction of brain glycolipid storage nor improvement in neuronal pathology could be detected, suggesting a complex pathogenic mechanism. Histological analysis revealed beta-hexosaminidase-positive cells in the central nervous system and visceral organs with a concomitant reduction of colloidal iron-positive macrophages. These results may be important for the design of treatment approaches for the GM2 gangliosidoses.