MUCOPOLYSACCHARIDOSIS TYPE II:
PRELIMINARY DATA ON GLYCOSAMINOGLYCAN
LEVELS AND STRUCTURE IN MICE AT BASELINE
AND AFTER 6 WEEKS TREATMENT WITH ERT
Laura Rigon
1, Marika Salvalaio
1, Francesca Maccari
2, Fabio Galeot
2,
Veronica Mantovani
2, Orazio Gabrielli
3, Maurizio Scarpa
1, Nicola Volpi
2,
Rosella Tomanin
11 Department of Women’s and Children Health, University of Padova, Padova, Italy 2 Department of Life Science, University of Modena and Reggio Emilia, Modena, Italy
3 Department of Clinical Sciences, Polytechnic University of Marche, Ospedali Riuniti, Presidio
Salesi, Ancona, Italy
Corresponding author: Dr. Rosella Tomanin (rosella.tomanin@unipd.it)
INTRODUCTION
Mucopolysaccharidosis type II (or Hunter Syndrome) is an X-linked lysosomal storage disease due to the deficit of the iduronate 2-sulfatase (IDS) enzyme and to the consequent progressive accumulation of the glycosaminoglycans (GAGs) heparan- (HS) and dermatan-sulfate (DS), with multi-organ and multi-system involvement. Production of the human recombinant form of IDS (rhIDS) has allowed to develop a weekly lifelong enzyme replacement therapy (ERT) protocol. Many biological molecules have been proposed as potential biomarkers for evaluating the therapeutic efficacy in Mucopolysaccharidoses (MPSs) (Clarke et al,
Mol Genet Metab. 106(4):395-402, 2012). In most trials, efficacy was assessed by
the total content of urinary GAGs. However, the methods applied are not able to identify the structural composition of GAGs that, on the contrary, may result to be efficient biomarkers of disease progression and therapeutic efficacy.
XIX
CONGRESSO
NAZIONALE
METHODS
In vivo experiments:
6 knock-out mice for Ids (Ids-ko), 12 weeks old at the beginning of the treatment (t0), were intravenously injected once a week for 6 weeks with enzyme replacement therapy by using rhIDS at a dosage of 1 mg/kg. At the end of the treatment (t6), brain and liver were collected and snap frozen in liquid N2 for subsequent procedures of GAGs purification, isolation, structural characterization and quantification by CE-LIF.
In addition to treated animals, 6 wild-type and 6 Ids-ko untreated mice were sacrificed at 12 and 18 weeks of age as control.
Capillary electrophoresis-laser induced fluorescence (CE-LIF):
Structural characterization of HS, CS/DS and HA from brains and livers were performed by disaccharide composition, as reported in 2016 by Maccari et al.,
Metab Brain Dis. Isolated and purified GAGs, treated with chondroitinases or
submitted to heparinases treatment, were fluorotagged and analysed by CE-LIF. Standards, enzymatically treated as above reported, were used to calculate GAGs total content . The charge density of sulfated GAGs was calculated considering the presence and the percentages of sulfate groups for each disaccharide (see also
Maccari et al., Metab Brain Dis, 2016; Volpi et al., Nat Protoc 9:541–558, 2014). Total liver and urinary GAGs content analysis:
Total urinary GAGs content was determined using the standard protocol described by de Jong (de Jong et al., Clin Chem 38: 803–807, 1992) with modifications.
Total liver GAGs content was measured by using the standard Bjornsson’s protocol
(Bjornsson, Anal Biochem 210:282–291, 1993), with modifications previously
described (Friso et al., Biochim Biophys Acta 1782:574–580, 2008).
ABBREVIATIONS: CS = chondroitin sulfate DS = dermatan sulfate HA = hyaluronic acid or hyaluronate HS = heparan sulfate GAGs = glycosaminoglycans wt = wild-type mouse
Ids-ko = knock-out mouse for Ids gene
AIM
In this study we characterized glycosaminoglycans levels and structure in the brain and liver of the Ids knock-out mouse model, at 12 weeks of age and after 6 weeks treatment with recombinant human IDS (rhIDS) enzyme, by using the capillary electrophoresis-laser induced fluorescence (CE-LIF) technique.
LEGEND:
WT/NT/00 = untreated
wild-type mice at time 0 (12 weeks aged)
WT/NT/06 = untreated
wild-type mice at time 6 (18 weeks aged)
H/NT/00 = untreated Ids-ko
mice at time 0 (12 weeks aged)
H/NT/06 = untreated Ids-ko
mice at time 6 (18 weeks aged)
H/E/06 = Ids-ko mice after
6 weeks of treatment with rhIDS (18 weeks aged)
a b c d Figure 1: a) Cumulative CS and DS, b) HA, c) HS content reported in ng/mg tissue and d) CS/DS ratio in liver and brain of wild-type mice at t0 and t6, Ids-ko mice at t0 and t6, Ids-ko mice after 6 weeks treatment with rhIDS
Figure 2: a) CS+DS and b)
HS charge density
determined for liver and brain of wt at t0 and t6, Ids-ko mice at t0 and t6, Ids-ko mice after 6 weeks treatment with rhIDS
a b a b Figure 3: Total glycosaminoglycan content in a) liver and b) urine determined by standard procedure in wt at t0 and t6, Ids-ko mice at t0 and t6, Ids-ko mice after 6 weeks treatment with rhIDS
CONCLUSIONS
These preliminary data underline how by using a more sensitive technique of analysis, a clear separation of the GAGs pattern between wt and Ids-ko mice can be achieved. This results particularly important for the brain, where application of common biochemical techniques detects very low GAG levels in both animal types, thus limiting the use of GAGs analysis as possible biomarker of therapeutic efficacy. The extension of CE-LIF analysis to more accessible tissues could be therefore proposed for a potential monitoring of therapeutic efficacy.
Acknowledgment: This study was funded by MIUR, project PRIN 2012, Prot.
20122EK9SZ_II.
RESULTS
As expected, Ids-ko mice showed a heavy accumulation of HS, about 15 times higher than wt in the brain and up to 240 times in the liver (Fig. 1c). The overall HS charge density rose by 1.5 times only in the liver (Fig. 2b), but the sulfation pattern changed in both organs (data not shown). We also observed an increased CS+DS levels of about 2 times in the brain and 5 times in the liver (Fig. 1a), but an increased CS/DS ratio of about 22 times only in the liver (Fig. 1d). On the contrary, the HA levels is slightly reduced only in the oldest Ids-ko mice (Fig. 1b).
We also conducted the same analysis in Ids-ko mice treated with 1 mg/kg of rhIDS, once a week for 6 weeks. As expected, we observed in the liver a huge reduction of HS (20X vs untreated mice) (Fig. 1c) and also of CS+DS (Fig. 1a) and CS/DS (Fig. 1d). Instead, we did not observe a reduction of the different GAGs in the brain, confirming the enzyme inability to cross BBB. In this district a slight increase of CS/DS ratio, CS+DS and HA levels, and an about 40% increase of HS level, vs untreated ko mice, was observed (Fig. 1). On the opposite, the overall HS charge density is decreased 2.5X vs untreated ko and wt mice (Fig. 2b).
All these data confirmed the expected significant total GAGs reduction in liver and urine of Ids-ko treated mice, obtained by using standard techniques (Fig. 3). In addition, CE-LIF analysis provides a deeper comprehension of detailed GAG changes.
