ERT is the current approach to treat MPSII patients. To investigate whether systemic application of IDS enzyme via ERT can deliver enzyme to the brain and therefore prevent or cure the CNS degeneration, we implemented different therapeutic approaches. These were designed to identify the minimal IDS dose and the longest treatment-interval time sufficient to treat the CNS phenotype of MPSII mice. Thus we proceeded as follows.

(A) A group of adult MPSII mice (aged 2 months; n = 21; seven mice per group) received human IDS doses of 10, 5 or 1.2 mg/kg in a volume of 300 μl via the tail vein, every other day (1–2), for a total period of 1 month. Groups of untreated, age-matched, MPSII (n = 5) and wild-type (n = 5) mice were used as controls.

During these treatments, IDS activity was measured in the plasma of all of the treated mice, every other month from T1 to T7 (months 1, 3, 5 and 7), at 4 h after the last injection. The IDS plasma activity was extremely high and was even higher than the activity measured in the wild-type control mice. Interestingly, also with the group D mice that received IDS for the longest time, after 7 months their mean plasma IDS activity was 8.7-fold higher than that of the wild-type control mice (Table 1). Moreover, in all of the groups of mice, the IDS activity measured 48 h after each injection was higher (from 4- to 11-fold, depending on treatments) than the levels in the untreated MPSII mice, but was also comparable to levels measured in the untreated wild-type mice (Table 2). These plasma IDS activities returned to the levels measured in the untreated MPSII mice 72 h after the last injections of each treatment protocol (data not shown). These data clearly show that the IDS enzyme is very stable, showing slow plasma clearance; indeed, it remains at high levels in the blood of the treated mice for a long time. Table 1.

Plasma IDS activities according to mouse treatment groups, 4 h after last treatment

The treated mice (groups A–D) were then sacrificed at different times 4 h after the final administration of IDS of each treatment protocol. In parallel, the groups of untreated MPSII and untreated wild-type mice were also sacrificed. The IDS activities were measured in homogenates from their brains and visceral tissues.

Group A showed brain IDS activities that decreased in parallel with the concentrations of IDS injected. Interestingly, with the administration of the lowest dose of IDS (1.2 mg/kg), some IDS activity was still detected in the brains of the treated MPSII mice (Fig. 1A). Surprisingly, the same IDS activities were detected in the group B mice, which received 1.2 mg/kg doses at longer time intervals, i.e. treatment every 4 and 7 days (Fig. 1B). The group C treatment showed that IDS can also be partially detected in MPSII mice of 7 months of age, even when these mice were treated once every 7 days (Fig. 1C). Finally, the MPSII mice in group D, which were treated with 1.2 mg/kg once every 7 days for a prolonged period of time, also showed some IDS activity in their brains (Fig. 1D). Figure 1.

IDS activities following ERT, measured in brain homogenates of untreated wild-type (wt; n = 5) and MPSII (Ids^y/-; n = 5) mice and treated MPSII mice sacrificed 4 h after the final IDS treatments (as indicated). (A) Group A (n = 7); (B) group B (n = 7); (C) group C (n = 7); (D) group D (n = 4). See text for details. Data are means ± standard deviations. P < 0.05 versus untreated MPSII control (ANOVA test).

Next, the brains and visceral tissues of the sacrificed mice were analyzed for GAG clearance by Alcian blue staining of the treated and untreated MPSII and wild-type mice. There was almost total clearance of GAG accumulation in the choroid plexus of the third and fourth ventricles and in the cortex and thalamus of all of groups of mice, with respect to the untreated MPSII mice (Fig. 2). Of note, even with the low dose of IDS administered over the longer time interval (1.2 mg/kg once every 7 days), there was good clearance of GAGs in all of the regions of the brain analyzed. Furthermore, there was clearance of GAGs when the mice were treated not just for 7 months, but also for only 1 month. Figure 2.

Representative GAG accumulation following ERT, measured by Alcian blue staining of sections of different brain regions of untreated MPSII (Ids^y/-) mice (3 and 10 months old) and groups A–D treated MPSII mice (as indicated). See text for details. Magnification, 20×.

We then wanted to determine whether we could measure IDS enzyme activity and GAG clearance after prolonged periods of time from the last IDS administration. Thus, to evaluate the residual IDS activity, a group of adult MPSII mice (aged 2 months; n = 4) were treated with 1.2 mg/kg IDS once every 7 days (group E) for 1 month, and 4 days after the final IDS injection, they were sacrificed. Groups of untreated, age-matched, MPSII and wild-type mice were used as controls.

Surprisingly, 4 days after the last IDS administration, we still detected IDS activity in both the brains and the tissues of the treated mice; in most organs (brain, liver, kidney and spleen), this activity was higher than that for the untreated MPSII mice (Fig. 3A and B). GAG clearance was also seen to persist in the different brain areas and in the visceral tissues of these treated mice (Fig. 3C and D). Figure 3.

IDS activities and GAG accumulation following ERTin the brain and visceral tissue homogenates of 3-month-old mice sacrificed 4 days after the final IDS treatments. (A and B) IDS activities of untreated wild-type (wt; n = 5) and MPSII (Ids^y/-; n = 5) mice and treated MPSII mice (as indicated; n = 4). Data are means ± standard deviations. P < 0.05 versus untreated MPSII control (ANOVA test). (C and D) Representative GAG accumulation measured by Alcian blue staining of sections from untreated MPSII (Ids^y/-; 3 months old) mice and treated MPSII mice (as indicated) for different brain regions (C) and visceral tissues (D) (as indicated). Magnification, 20×.

We have recently shown that progressive accumulation of GAGs in lysosomes of neurons leads to severe vacuolization. In addition, we have seen diffuse neurodegeneration in the thalamus, cerebral cortex and brain stem, according to reduced neuron densities (decreased anti-NeuN signals) and to increased ubiquitin-expressing neurons. This was also associated with the triggering of apoptosis, as shown by TUNEL-positive signals in neurons of the thalamus, cerebral cortex and brain stem of MPSII mice (13).

Here, we saw a clear reduction in the lysosomal GAG accumulation, as showed by the anti-Lamp2 immunostaining that marks the lysosome storage that is massively evident in the untreated MPSII mice (Figs 4A and 5A). A clear amelioration of neurodegeneration was also evident in the treated mice in all of the groups, as revealed by reductions in anti-ubiquitin and TUNEL signals in neurons of the thalamus (Figs 4A and 5A) as well as in the cortex and brain stem (data not shown). Accordingly, we also noted increases in the neuron density (as anti-NeuN immunostaining) in the treated mice of groups A, B and D when compared with the untreated MPSII mice (Figs 4A and 5A). Figure 4.

Representative analysis of the rescue of CNS markers in the thalamus following ERT, with untreated wild-type (wt; 3 months old; A and B) and MPSII (Ids^y/-; 3 months old, A) mice and treated MPSII mice (A and B) sacrificed 4 h after the final IDS treatments in groups A andB, as indicated. (A) Immunohistochemistry (IHC) and immunofluorescence (IF) monitored according to (left to right): anti-Lamp2 IHC for GAG storage; anti-NeuN IF for neuron density; anti-ubiquitin IHC for ubiquitination; TUNEL assay for apoptosis; anti-GFAP IF for gliosis (all magnification 20×) and anti-CD68 IF for macrophage infiltration (magnification, 40×). See text for details. (B) Co-localization of the lysosomal marker Lamp-2 (anti-Lamp2 antibody) and the IDS protein (anti-hIDS antibody) as seen in the merge, as indicated. Magnification, 100×.

Ultrastructural analysis of thin sections from the cerebellum of wild-type mice revealed electron-dense membrane structures with morphological features of lysosomes in Purkinje cells (Fig. 6A). These lysosomal organelles were mainly electron-dense with a few translucent areas in the lumen, and they usually ranged from 300 to 1000 nm in diameter. In contrast, the cytoplasm of Purkinje cells from the MPSII mice contained large lysosome-like organelles (Fig. 6B), which in some cases reached even 3000 nm in diameter and were, on average, more than twice the diameter of those of the wild-type mice Purkinje cell lysosomes (Fig. 6D), as revealed by the morphometric analysis. The lumen of these organelles was filled with the heterogeneous material, part of which looks like ‘fuzzy flakes' that resemble GAGs. Taken together, these ultrastructural features suggest that the degradation processes in these organelles are impaired, which results in GAG storage. Figure 6.

Representative analysis (A–C) and quantification (D) of the ultrastructure of the lysosome-like organelles in the Purkinje cells in thin sections of cerebellum from untreated wild-type (wt; A and D) and MPSII (Ids^y/-; B and D) mice and treated MPSII mice (C and D) sacrificed 4 h after the final IDS treatments in group C, as indicated. In all cases, the Purkinje cells were sectioned through their central area (judged on the basis of the presence of the Golgi membranes; A–C, white arrows) to ensure most representative sampling and analysis. (A) Black arrowheads, small electron-dense lysosome-like organelles. (B) Black arrows, large vacuolar structures containing heterogeneous material. (C) Black arrowheads, significantly smaller vacuolar structures containing heterogeneous material; black arrows, lysosomes with regular size and morphology were also detected. Scale bar, 1000 nm. (D) Quantification of the lysosome diameter (average ± SD, n = 80 lysosomes) in sections of treated MPSII and control mice as indicated. P < 0.05 versus untreated MPSII control (ANOVA test).

The mice in groups C and D underwent both the open-field and rotarod tests. These neurobehavioral tests were carried out only in these two groups of mice because the clear phenotype measured by these two tests can be seen only in MPSII mice from about 5 to 6 months of age (11). The ability to explore, the locomotion and the anxiety to move of the treated mice were determined through the open-field test. As shown in Figure 7, the performance in the open-field test showed both the vertical and horizontal activities to be fully corrected in the treated mice and to be comparable to the untreated wild-type mice (Fig. 7A, B, D and E). Figure 7.

Behavioral phenotypes following ERT, of 10-month-old untreated wild-type (wt; n = 5) and MPSII (Ids^y/-; n = 5) mice and treated MPSII mice (as indicated). (A–C) Group C mice (n = 7) after 3 months of treatment. (D–F) Group D mice (n = 7) after 7 months of treatment. See text for details. Data are means ± standard deviations for the open-field test (A, B, D and E) for the number of crossings (A and D) and rearings (B and E) and for the rotarod test (C and F) for latency time. P < 0.05 versus untreated MPSII control (ANOVA test).

Female heterozygous MPSII mice were used, as described previously (11).

Blood (50 µl) was collected in EDTA at different times after the injections (months 1, 3, 5 and 7; T1, T3, T5 and T7) of the treated mice and the untreated MPSII (Ids^y/-) and wild-type (control) mice. Injected human IDS (Elaprase) was kindly provided by Shire Human Genetic Therapies, Inc., Cambridge, MA, USA. The blood was centrifuged at 10 000×g for 10 min at 4°C, and the serum (supernatant) was used for enzyme assays. Tissues were collected at the end of each treatment from the treated mice and in parallel from the untreated MPSII and wild-type mice. The mice were anesthetized and sacrificed by cardiac perfusion: the left ventricle was cannulated, an incision was made in the right atrium and the mice were perfused with 40 ml of phosphate-buffered saline (PBS). The organs were collected, and half of each was fixed (for Alcian blue staining and immunohistochemistry or immunofluorescence), and the other half of each was frozen in dry ice before being processed for the IDS activity assay.

The tissues for analysis were homogenized in water. Serum and tissue protein concentrations were determined using the Bio-Rad colorimetric assay (Bio-Rad, Hercules, CA, USA). The IDS assay was performed as described previously (35): briefly, 50 µg total protein extract was incubated with 20 µl of the fluorogenic substrate, 4-methylumbelliferyl-α-iduronide-2-sulfate (Muscerdam Substrates), for 4 h at 37°C. Then, 10 µl purified α-iduronidase from rabbit liver (Muscerdam Substrates) and 40 µl McIlvain's buffer (0.4 m Na-phosphate/0.2 m Na-citrate, pH 4.5) were added to the reaction mixture, which was then incubated for an additional 24 h at 37°C. The reaction was stopped by adding carbonate stop buffer (0.5 m NaHCO₃/0.5 m Na₂CO₃, pH 10.7), and the fluorescence of the 4-methylumbelliferone liberated was measured using 365 nm excitation and 460 nm emission in a fluorimeter (BIO-RAD VersaFluor Fluorometer). The enzyme activities were expressed as nmol/4 h/mg protein, as calculated through the standard curve of the fluorogenic substrate, 4-methylumbelliferyl-α-iduronide (Sigma-Aldrich).

After the perfusion of the mice with PBS, the tissues were collected and fixed in methacarn solution (30% chloroform, 60% methanol and 10% acetic acid) for 24 h at 4°C. The next day, the tissues were embedded in paraffin (Sigma-Aldrich) after their dehydration through a 70–100% ethanol gradient. Finally, the tissues were sectioned into 7 µm thick serial sections. The tissue sections were stained with 1% Alcian blue (Sigma-Aldrich) in hydrochloric acid. The counterstaining was performed for 2 min with Nuclear-Fast red (Sigma-Aldrich).

Mice brains were collected after PBS perfusion and fixed with 10% neutral buffered formalin, pH 7.0, for 12 h at 4°C. Then the brains were embedded in paraffin (Sigma-Aldrich) and dehydrated through a 70–100% ethanol gradient. Immunohistochemistry and immunofluorescence analyses were performed on 7 µm thick serial sections. The specimens were incubated for 1 h with blocking solution [Tris-buffered saline, 0.2% Tween-20 (Sigma-Aldrich) and 10% normal horse serum, Vectastain Elite ABC kit] before incubation overnight with the primary antibodies.

For immunohistochemistry analyses of Lamp2 and ubiquitin of the paraffin-embedded, formalin-fixed brains, the avidin-biotin complex (ABC) method was used (Vectastain Elite ABC kit). A monoclonal rat antibody against murine Lamp2 (diluted 1:100; Santa Cruz Biotechnology, Inc., CA, USA) and a polyclonal rabbit antibody against murine ubiquitin (diluted 1:50; Abcam, Cambridge, UK) were used. Then, secondary biotinylated horse anti-rat and anti-rabbit IgG and streptavidin–biotin–peroxidase complex (Vectastain Elite ABC kit) were used for 1 h of incubation (for anti-Lamp2 and ubiquitin). The color was developed using the ABC Elite Vector Staining kit and the horseradish peroxidase substrate (Vector Laboratories, Inc., Burlingame, CA, USA). For the detection of apoptotic cells in the brain sections, TUNEL staining kits (Chemicon International) were used, according to the manufacturer's instructions.

Immunofluorescence analyses were carried out for the detection of NeuN, GFAP and CD68 (36,37) and for the co-localization signal of Lamp2-hIDS. A monoclonal mouse antibody against murine NeuN (diluted 1:200; Abcam), a monoclonal rabbit antibody against murine GFAP (diluted 1:400; Sigma-Aldrich), a monoclonal rat antibody against murine CD68 (diluted 1:250; AbD Serotec) and an antibody against human IDS (hIDS, Shire Pharmaceuticals, Boston, MA, USA) were used for the detection of co-localization signals between Lamp2-hIDS. After washing, the sections were incubated for 1 h with secondary antibodies (Molecular Probes, Invitrogen, CA, USA). Stained sections were mounted with Vectashield with DAPI (Vector Laboratories, Inc.).

The cerebellum was excised from 10-month-old treated mice and untreated MPSII and wild-type mice and fixed in 1% glutaraldehyde in 0.2 m HEPES buffer. Then small blocks of the cerebellum tissue were cut and post-fixed in uranyl acetate and in OsO₄. After dehydration through a graded series of ethanol, the tissue samples were cleared in propylene oxide, embedded in the Epoxy medium (Epon 812) and polymerized at 60°C for 72 h. From each sample, semi-thin sections were cut with a Leica EM UC6 ultramicrotome and mounted on glass slides for light microscopic inspection to identify the Purkinje and granular cell layers. Ultrathin (70 nm thick) sections of the area of interest were obtained. Electron microscopic images were acquired from thin sections using an FEI Tecnai-12 electron microscope equipped with an ULTRA VIEW CCD digital camera (FEI, Einhoven, The Netherlands). Quantification of the lysosome-like organelle dimensions was performed using the AnalySIS software (Soft Imaging Systems GmbH, Munster, Germany).

Urine from individual mice was collected in metabolic cages at the end of each treatment and from the untreated MPSII and wild-type mice. GAG levels in the urine were determined using the dimethylmethylene-blue-based spectrophotometry of GAGs (38). These were normalized to the creatinine content. Urine creatinine was measured using Creatinine Assay kits (Quidel Corporation, San Diego, CA, USA). Absorbance was read at 490 nm. Urinary GAG was expressed as milligram GAG/milligram creatinine.

The motor and the exploratory behaviors of treated mice were assessed in an acrylic open arena, as described previously (39). The open-field test was performed (between 09:00 and 11:00 h) using the open-field apparatus (60 cm × 60 cm × 40 cm). The floor of the wooden arena was divided equally into squares marked by red lines. In this test, the treated mice and the untreated MPSII and wild-type mice were placed individually in the center of the arena and allowed to explore freely. The number of crossings (squares crossed with all paws) and rearings (rising of the front paws) was recorded during the test period of 10 min. This apparatus was cleaned with a detergent and dried after occupancy by each mouse.

The rotarod test is designed to assess the sensory motor coordination, balance, equilibrium and motor learning. The treated mice and the untreated MPSII and wild-type mice were placed on top of the rotating rod facing away from the experimenter, in the orientation opposite to that of the rod rotation. The latency times for the mice to fall from the rod were recorded automatically by the apparatus. For the first day of the test, the mice were placed on the rotating rod set at the steady slow speed of 4 rpm and trained to remain on the rod for 60 s. After this habituation trial, each mouse was tested in four trials per day, for 3 consecutive days, with an inter-trial interval of 30 min. The rotarod apparatus (Ugo Basile, Italy) accelerates gradually from 4to 40 rpm. The cut-off time was 600 s for the trials.

The statistical significance was determined for the measurements compared by the analysis of variance (ANOVA) test.