Comparative analysis of brain pathology in heparan sulphate storing mucopolysaccharidoses.

The cause of neurodegeneration in MPS mouse models is the focus of much debate and what the underlying cause of disease pathology in MPS mice is. The timing of development of pathology and when this can be reversed or impacted is the key to developing suitable therapies in MPS. This study is the first of its kind to correlate the biochemical changes with the functional outcome as assessed using non-invasive behaviour testing across multiple mucopolysaccharidosis (MPS) mouse models. In the MPS brain, the primary lysosomal enzyme dysfunction leads to accumulation of primary glycosaminoglycans (GAGs) with gangliosides (GM2 and GM3) being the major secondary storage products. With a focus on the neuropathology, a time course experiment was conducted in MPS I, MPS IIIA, MPS VII (severe and attenuated models) in order to understand the relative timing and level of GAG and ganglioside accumulation and how this correlates to behaviour deficits. Time course analysis from 1 to 6 months of age was conducted on brain samples to assess primary GAG (uronic acid), ß-hexosaminidase enzyme activity and levels of GM2 and GM3 gangliosides. This was compared to a battery of non-invasive behaviour tests including open field, inverted grid, rotarod and water cross maze were assessed to determine effects on motor function, activity and learning ability. The results show that the GAG and ganglioside accumulation begins prior to the onset of detectable changes in learning ability and behaviour. Interestingly, the highest levels of GAG and ganglioside accumulation was observed in the MPS IIIA mouse despite having 3% residual enzyme activity. Deficits in motor function were clearly observed in the severe Gusmps/mps, which were significantly delayed in the attenuated Gustm(L175F)Sly model despite their minimal increase in detectable enzyme activity. This suggests that genotype and residual enzyme activity are not indicative of severity of disease pathology in MPS disease and there exists a window when there are considerable storage products without detectable functional deficits which may allow an alteration to occur with therapy.

Gene silencing of EXTL2 and EXTL3 as a substrate deprivation therapy for heparan sulphate storing mucopolysaccharidoses.

Neurological pathology is characteristic of the mucopolysaccharidoses (MPSs) that store heparan sulphate (HS) glycosaminoglycan (gag) and has been proven to be refractory to systemic therapies. Substrate deprivation therapy (SDT) using general inhibitors of gag synthesis improves neurological function in mouse models of MPS, but is not specific to an MPS type. We have investigated RNA interference (RNAi) as a method of targeting SDT to the HS synthesising enzymes, EXTL2 and EXTL3. Multiple shRNA molecules specific to EXTL2 or EXTL3 were designed and validated in a reporter gene assay, with four out of six shRNA constructs reducing expression by over 90%. The three EXTL2-specific shRNA constructs reduced endogenous target gene expression by 68, 32 and 65%, and decreased gag synthesis by 46, 50 and 27%. One EXTL3-specific shRNA construct reduced endogenous target gene expression by 14% and gag synthesis by 39%. Lysosomal gag levels in MPS IIIA and MPS I fibroblasts were also reduced by EXTL2 and EXTL3-specific shRNA. Incorporation of shRNAs into a lentiviral expression system reduced gene expression, and one EXTL2-specific shRNA reduced gag synthesis. These results indicate that deprivation therapy through shRNA-mediated RNAi has potential as a therapy for HS-storing MPSs.

Gastrointestinal pathology in a mouse model of mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in sulphamidase (NS), a lysosomal enzyme required for the degradation of heparan sulphate glycosaminoglycans (gags). The MPS IIIA mouse is a naturally occurring model that accurately reflects the human pathology and disease course. It displays primarily central nervous system pathology accompanied by widespread accumulation of gag in somatic tissues. MPS IIIA mice exhibit greater bodyweight gain than normal littermates and attain a higher mature bodyweight. In this study, gastrointestinal morphology and function was characterised in the IIIA mouse. Stomach and duodenum weight increased in MPS IIIA mice and duodenum length also increased. An increased submucosal thickness was observed in MPS IIIA intestine compared to normal mice and lysosomal storage of gag was observed in this region. Storage was also observed in the lamina propria of the villus tip. All other morphometric measurements including villus height and crypt depth fell within the normal range. The gastric emptying half-life of solid and liquid meals decreased with age in normal mice whereas the T(1/2) of solid meals did not alter with age in MPS IIA mice such that they were elevated above normal by 38 weeks of age. Sucrase activity was higher than normal in MPS IIIA at all ages tested. These abnormalities in GI structure and function observed in MPS IIIA may contribute to weight gain in this disorder.