Allogeneic stem cell transplantation does not improve neurological deficits in mucopolysaccharidosis type IIIA mice

Experimental Neurology 225 (2010) 445–454

Contents lists available at ScienceDirect

Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Allogeneic stem cell transplantation does not improve neurological deficits in mucopolysaccharidosis type IIIA mice Adeline A. Lau a,c,⁎, Hanan Hannouche a,c, Tina Rozaklis a,c, Sofia Hassiotis a,c, John J. Hopwood a,b,c, Kim M. Hemsley a,b,c a b c

Lysosomal Diseases Research Unit, SA Pathology at the Women's and Children's Hospital, Australia Adelaide University Department of Pediatrics, Australia Center for Stem Cell Research, The Robinson Institute, The University of Adelaide, Australia

a r t i c l e

i n f o

Article history: Received 8 April 2010 Revised 19 July 2010 Accepted 20 July 2010 Available online 27 July 2010 Keywords: Allogeneic stem cell transplantation Mucopolysaccharidosis Sanfilippo syndrome Lysosomal storage disease Mouse

a b s t r a c t Mucopolysaccharidosis type IIIA (MPS IIIA) is a neurodegenerative metabolic disorder caused by mutations in the N-sulfoglucosamine sulfohydrolase gene with resultant accumulation of partially degraded heparan sulfate (HS). Whilst allogeneic bone marrow transplantation (BMT) is indicated for several lysosomal storage disorders featuring neurodegeneration, its use in MPS III is highly controversial. Published evidence suggests that BMT does not improve cognitive function in MPS III patients. Despite this, patients continue to be transplanted in some centers. We therefore sought to determine the clinical effectiveness of BMT in a murine model of MPS IIIA. Pre-symptomatic young adult mice pre-conditioned with total body irradiation generated complete and stable donor-type chimerism. Whilst HS-derived disaccharides were reduced by up to 27% in the brain parenchyma, this was insufficient to decrease secondary cholesterol and GM3 ganglioside storage or permit clinical improvement. These results suggest that BMT is ineffective in its unmodified form and should not be considered as a treatment for MPS IIIA children. © 2010 Elsevier Inc. All rights reserved.

Introduction Mucopolysaccharidosis type IIIA (MPS IIIA), or Sanfilippo syndrome is a lysosomal storage disorder caused by mutations in the Nsulfoglucosamine sulfohydrolase (SGSH; E.C. 3.10.1.1) gene and results in the accumulation of partially-degraded heparan sulfate (HS) glycosaminoglycans. This disorder predominantly affects the central nervous system (CNS) and is characterized by progressive neurological decline in patients, including behavioral changes such as delayed development and regression of learned skills, hyperactivity, increased aggression and sleep disturbance (Neufeld and Muenzer, 2001). The predicted prevalence of MPS IIIA in Australia is 1 in 114,000 live births (Meikle et al., 1999) and, at present, no effective therapy is available.

Abbreviations: BMT, bone marrow transplantation; CNS, central nervous system; GFP, green fluorescent protein; HS, heparan sulfate; MPS IIIA, Mucopolysaccharidosis type IIIA; PBI, partial body irradiation; SGSH, N-sulfoglucosamine sulfohydrolase; TBI, total body irradiation. ⁎ Corresponding author. Lysosomal Diseases Research Unit, SA Pathology at the Women's and Children's Hospital, 72 King William Rd, North Adelaide, 5006, Australia. Fax: +61 8 8161 7100. E-mail addresses: [email protected] (A.A. Lau), [email protected] (H. Hannouche), [email protected] (T. Rozaklis), sofi[email protected] (S. Hassiotis), [email protected] (J.J. Hopwood), [email protected] (K.M. Hemsley). 0014-4886/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2010.07.024

Three naturally occurring MPS IIIA animal models exist, including a murine model (Bhaumik et al., 1999; Crawley et al., 2006) resulting from a missense D31N mutation in the catalytic site of SGSH (Bhattacharyya et al., 2001). The MPS IIIA mouse displays elevated concentrations of an HS-derived disaccharide, GlcNS-UA, from birth and cytoplasmic inclusions in neural and non-neural (e.g. hepatocytes, Kupffer) cells (Bhaumik et al., 1999; Crawley et al., 2006; King et al., 2006). Secondary accumulation of GM2 and GM3 gangliosides and unesterified cholesterol is also evident (McGlynn et al., 2004). MPS IIIA mice exhibit differences in behavior that mimic the human condition, such as impaired cognition, altered motor function and anxiety-related behaviors, making this an excellent model to evaluate therapeutic strategies (Crawley et al., 2006; Hemsley and Hopwood, 2005; Lau et al., 2008). Previous studies by our group have provided evidence demonstrating that if sufficient SGSH is delivered to the CNS of MPS IIIA mice by direct injection into the brain parenchyma (Savas et al., 2004) or the cerebrospinal fluid (Hemsley et al., 2007, 2008, 2009a), biochemical and histological correction of disease neuropathology can be achieved. The latter approach has also been applied to a large animal model of MPS IIIA, the Huntaway dog (Hemsley et al., 2009b). However, this is most likely a medium term approach due to the requirement for repeated infusions of enzyme and consequently long-term treatment strategies also need to be developed. Bone marrow-derived cells of the monocyte/macrophage lineage are capable of crossing the blood–brain barrier in lethally-irradiated, chimeric mice where they are able to differentiate into microglia, the

446

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

resident macrophage population of the brain (Hess et al., 2004; Simard and Rivest, 2004). Mildner and colleagues (2007) further defined the phenotype of this microglial precursor as Gr-1+CCR2+CX3CR1lo monocytes. Following migration into the CNS, donor-derived cells would be predicted to secrete therapeutic lysosomal enzyme within the host brain that can be taken up into neighboring cells by mannose-6-phosphate receptor-mediated endocytosis. Indeed, allogeneic stem cell transplantation (BMT) is indicated for several lysosomal storage disorders presenting with neurological signs, including MPS I, globoid cell leukodystrophy, metachromatic leukodystrophy, alpha-mannosidosis, and aspartylglucosaminuria (Krivit, 2004; Shapiro et al., 1995). However, in the few reports of BMT in Sanfilippo patients, neurological improvement was not observed despite successful engraftment and reconstitution of donor bone marrow with effective metabolic correction (Hoogerbrugge et al., 1995; Klein et al., 1995; Sivakumur and Wraith, 1999). The reason for the disparity in the response of the different lysosomal storage disorders to transplantation is unknown. Despite this, stem cellbased transplants continue to be performed in Sanfilippo children. In the most recent study, data on the cognitive outcome has yet to be published (Martin et al., 2006). This treatment approach has also been examined in a mouse model of Sanfilippo disease. Transplantation of neonatal MPS IIIB mice with unmodified bone marrow was found to improve some clinical parameters (e.g. auditory function) but not others (e.g. motor function) (Heldermon et al., 2010). This partial correction may be influenced by the low engraftment of donor cells obtained following transplantation in newborn pups (mean of 24.7%). Further, biochemical and histological improvements in adult MPS IIIB mice were only demonstrated following transplantation of gene-modified bone marrow donor cells over-expressing α-Nacetylglucosaminidase (Zheng et al., 2004). The effect of transplantation on neurological function was not reported. It is therefore critical to definitively determine whether BMT is beneficial for Sanfilippo patients. In the present study, we have undertaken a longterm, multi-time-point assessment of neurological status following allogeneic BMT in MPS IIIA mice. Our results demonstrate that this treatment does not arrest the clinical progression of disease or significantly improve neuropathology despite full reconstitution and widespread engraftment of donor-derived cells within the CNS. Materials and methods Mice Male and female C57BL/6-Tg(UBC-GFP)30Scha/J homozygous mice (8 –12 weeks) were used as donors (Schaefer et al., 2001). These mice express green fluorescent protein (GFP) under the control of the ubiquitin C promoter. Recipient mice were 3.5- to 4.5-week-old B6.Cg-Sgshmps3a (MPS IIIA) or unaffected (wild-type or heterozygous; subsequently referred to as ‘normal’) mice of the same genetic background (i.e. C57BL/6), from a breeding colony established and maintained at our institution (Crawley et al., 2006). The genotype of recipient mice was determined by PCR amplification of genomic DNA using published methods (Gliddon and Hopwood, 2004). All procedures were performed according to the protocols approved by the institutional Animal Ethics Committee in accordance with the Guidelines of the National Health and Medical Research Council of Australia. Transplantation Bone marrow cells were harvested from the femurs and tibiae of donor mice 3 days after intraperitoneal injection of 150 mg/kg 5fluorouracil (Sigma, MO, USA). The bones were flushed with DMEM containing 2% (v/v) fetal calf serum and antibiotics, using a 23G needle and filtered through a 100-μm cell strainer. Erythroid cells were lysed in 155 mM ammonium chloride, 10 mM potassium

hydrogen carbonate, and the nucleated cells were washed and resuspended in PBS with 30 U heparin/mL. Recipient mice were irradiated using a megavoltage linear accelerator as either a single fraction (9 Gy) or as two equal fractions (10 Gy or 11 Gy, separated by 3 to 4 h) at a dose rate of 2 Gy/min. The mice received either total body irradiation (TBI) or partial body irradiation (PBI), where the head of the mouse was excluded from the irradiation field. The mice then received 106 unfractionated donor bone marrow cells via the tail vein on the day of irradiation. Following transplant, recipient mice were housed in micro-isolator cages containing autoclaved bedding. The mice were fed autoclaved chow, a high-calorie nutrient drink (Ensure Plus, Abbott Nutrition, OH, USA) and acidified drinking water (2.8 N) ad libitum for up to 4 weeks post-transplant. The mice then reverted to regular municipal drinking water and standard chow for the remainder of the study. Behavioral tests Open field activity The exploratory locomotor activity of 15-week-old male mice was measured in a plastic enclosure (dimensions of 45 cm × 30 cm) divided into 15 zones in a 5 × 3 grid formation (9 cm × 10 cm each) (Hemsley and Hopwood, 2005; Lau et al., 2008). Testing was conducted in a random order between 0630 and 1000 h by an experimenter blinded to the treatment/genotype status of the mice. Mice were placed facing the wall in the corner closest to the operator and were allowed to explore the field for 3 min. Field2020 software (HVS Image) was used to track the path of each mouse. The field was cleaned with 70% ethanol between each test. Hind-limb gait Hind-limb gait was determined at 24 weeks of age (Hemsley and Hopwood, 2005). The rear paws were dipped into non-toxic food coloring and the mouse was allowed to freely walk down a walled corridor (10-cm width × 60-cm length × 15-cm height) towards a darkened goal box. The average length between footprints and the distance perpendicular to the direction of movement (gait width) was determined for at least three strides. The mean of two runs is presented. Elevated plus maze At 26 weeks of age, mice were placed into a cross-shaped maze consisting of two open arms (50 cm × 7 cm) and two walled arms (50 cm × 7 cm × 15 cm in height) and elevated to 50 cm above ground level (Lau et al., 2008). Mice were placed in the center square to face an open arm and were allowed to explore the maze for 5 min. The path of the mouse was tracked using Maze2020 software (HVS Image). The floor and internal walls were cleaned with 70% ethanol between tests. Necropsy and sample collection For biochemical analyses, mice were euthanased with carbon dioxide gas and then trans-cardially perfused with ice-cold PBS (pH 7.4) prior to the collection of somatic tissues as well as the brain and thoracic spinal cord. The brain was divided along the midline and into five 2-mm hemi-coronal slices, with slice 1 containing the olfactory bulb and slice 5 containing the cerebellum. In addition, the tibiae and femurs were flushed with 1 mL of ice-cold PBS and the bone marrow cells were separated from the extracellular fluid supernatant by centrifugation at 375g. Additional mice were transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde in PBS (pH 7.4) for histological assessment. Tissue samples were post-fixed overnight in the same solution, transferred to PBS, then cryoprotected in 30% sucrose in PBS overnight before being frozen in OCT compound (Tissue-Tek, Tokyo, Japan) using liquid nitrogen.

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

Flow cytometry Peripheral blood was collected via cardiac puncture immediately after euthanasia and the percentage of GFP-expressing leukocytes was determined as a measure of engraftment. Red blood cells were removed from duplicate samples (50 μL whole blood) using BD FACS Lysing solution (BD Biosciences). Peripheral blood leukocytes were blocked with Intragam®P (CSL Ltd, Parkville, Australia) and immunostained for 5 min with PECy5-conjugated anti-CD45 (1:10 dilution; BD Biosciences, CA, USA). Cells were then washed with 0.5% (w/v) bovine serum albumin (Sigma, Sydney, Australia) in IsoFlow Sheath Flow (Beckman Coulter, CA, USA) and then analyzed on a FACScalibur

447

(Beckton Dickson, NJ, USA) equipped with CellQuest software (version 3.1). GlcNS-UA and SGSH activity measurement Samples were homogenized in 1 mL (samples in Fig. 1) or 0.5 mL (samples in Fig. 5) of 20 mM Tris, 500 mM sodium chloride, pH 7.2, and were then sonicated twice for 30 s each. Total protein was determined with a MicroBCA kit (Pierce, IL, USA). For liquid chromatographyelectrospray ionization tandem mass spectrometry-based measurement of the HS-derived disaccharide, GlcNS-UA, 50 μg (samples in Fig. 1) or 100 μg (samples in Fig. 5) of total protein were mixed

Fig. 1. Effect of pre-conditioning regime on reconstitution and HS-derived GlcNS-UA storage. (A) Bone marrow cells, bone marrow extracellular fluid (BMEF) and stromal cells were collected from wild-type (unfilled) or MPS IIIA mice (filled) at 4 weeks of age and the relative amount of HS-derived disaccharide storage was measured using tandem mass spectrometry (n = 5 mice per group). (B-F) MPS IIIA mice received various irradiation doses administered as a partial body irradiation (PBI) or total body irradiation (TBI) (n = 2–7 mice per group). The alignment of the irradiation field for PBI and TBI pre-conditioning are depicted in the schematic diagrams. (B) Donor-type chimerism was assessed at 4 weeks post-transplantation (8 weeks of age) by flow cytometric analysis of GFP-expressing peripheral blood leukocytes. The concomitant effect of transplantation on the relative level of HS-derived disaccharide storage is shown in (C) bone marrow extracellular fluid, (D) liver, (E) brain parenchyma slice 5 (sectioned as illustrated in the mouse brain diagram), and the (F) thoracic spinal cord from transplanted MPS IIIA mice, and compared to untreated normal and MPS IIIA mice. *P b 0.05, **P b 0.01, ***P b 0.001 compared to untreated MPS IIIA mice.

448

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

with 4-deoxy-L-threo-hex-4-enopyranosyluronic (1→3) N-acetylgalactosamine-4-sulfate as an internal standard (Sigma Aldrich, Australia) and derivatized with 1-phenyl-3-methyl-5-pyrazolone, as previously described (Hemsley et al., 2009a). Appropriate tissue matrix quality control samples were prepared from tissues collected from MPS IIIA mice and were included in each run to determine the intra-batch co-efficient of variation (%CV). The relative level of GlcNS-UA was determined by comparing the peak area of the derivatized GlcNS-UA to that of the internal standard. Figs. 1 and 5 show data from samples run in two different batches. For SGSH activity, samples were homogenized and sonicated as before and were then dialyzed overnight in 0.9% (w/v) sodium chloride at 4 °C. Samples (8.5 μL) were then mixed with 200 mM sodium acetate (pH 5.2; 3 μL) and 400 pmol of a tritiated tetrasaccharide substrate derived from heparin (0.5 μL) and incubated at 60 °C (Hopwood and Elliott, 1982). The reaction was quenched by the addition of 100 μL of 100 mM ammonium hydroxide and substrate and product were then quantified by high performance liquid chromatography. The threshold of detection under these conditions for this assay was 0.5 ng of recombinant human SGSH. Histological staining and imaging An experimenter blinded to genotype/treatment status undertook all procedures and post-staining image analyses using constant imaging and calibration parameters for each stain. Frozen brain sections (6 μm) in the sagittal plane were collected onto Superfrost Plus slides and stored at −70 °C. For GFP staining, the tissues were rehydrated in PBS and then blocked in 5% (v/v) normal donkey serum in PBS for 1 h at room temperature. GFP-expressing cells were then detected using a polyclonal anti-GFP antibody (Invitrogen cat. no. A6455; 1:10,000 dilution) and a Cy3-conjugated donkey anti-rabbit IgG secondary antibody (Jackson Immunoresearch cat. no. 711-165152, 1:600). GM3 ganglioside was immunodetected in frozen sections according to published methods (McGlynn et al., 2004) using an alternate source of monoclonal anti-GM3 antibody (Seikagaku Biobusiness Corporation cat. no. 370695; 1:750). Representative images were captured on an Olympus BX41 microscope fitted with an Olympus Colorview Soft Imaging System. The percentage of immunopositive (thresholded) staining in the inferior colliculus at the sagittal midline or in the amygdala, 2 mm lateral to the sagittal midline, was quantitated using AnalySIS Lifescience software (version 2.8, Build1235, Olympus Soft Imaging Solutions). Free cholesterol was also detected in frozen sections using filipin complex from Streptomyces filipinese (Sigma cat. no. F9765) resuspended in N,N dimethylformamide (10 mg/mL). Following rehydration in PBS, tissue sections were incubated with filipin complex diluted in PBS to a working concentration of 50 μg/mL for 1 h at room temperature, in the dark. Cells containing filipin-positive inclusions were semi-quantitated in the inferior colliculus and the amygdala. Stromal cell culture Bone marrow was flushed from the hind-limb bones of each normal or MPS IIIA mouse using 1 mL of 2% (v/v) fetal calf serum, 50 U/mL penicillin, 50 μg/mL streptomycin in DMEM. The cells were strained through a 100-μm nylon filter, rinsed with the same media as before and pelleted at 250 g. The cells were resuspended in 12 mL of 20% (v/v) fetal calf serum, 50 U/mL penicillin, 50 μg/mL streptomycin in DMEM, evenly distributed amongst the wells of a 6-well tray and cultured at 37 °C with 5% CO2. After 4 weeks of culture, the adherent stromal cells were washed twice in 1× PBS and detached with 0.25% trypsin, 1 mM EDTA in PBS. The cells were micro-centrifuged, resuspended in 20 mM Tris, 500 mM NaCl, pH 7.2, and processed as above to determine the relative level of GlcNS-UA.

Statistics All data were assessed using GraphPad Prism software (v5.02) and are expressed as the mean + SEM. One-way ANOVA was performed for all behavioral data (open field, hind-limb gait, Elevated Plus Maze), SGSH activity data and the percentage of donor-type chimerism. Twoway ANOVA was utilized to assess GlcNS-UA data in the long-term study. Post-hoc analyses using the Bonferroni test were then applied to adjust for multiple comparisons between groups. Histological data were log-transformed Y = (Log Y + 1) and then examined using a two-way ANOVA and the Bonferroni test. Significance was considered to be P b 0.05. Results MPS IIIA bone marrow contains high levels of storage material at 4 weeks of age The relative level of GlcNS-UA was evaluated in the bone marrow compartment of wild-type or MPS IIIA mice at the age at which treatment was to be applied (Fig. 1A). Increased relative levels of HSderived disaccharide were detected in MPS IIIA bone marrow cells (162-fold normal; P = 0.0022), bone marrow extracellular fluid (514fold normal; P b 0.0005) and bone marrow stromal cells grown in culture for 4 weeks (33-fold normal; P b 0.0001). Partial body irradiation (head exclusion) yields low level donor cell engraftment and no effect on GlcNS-UA in the brain Pre-symptomatic male and female MPS IIIA mice were preconditioned with one of four irradiation protocols prior to transplantation with 106 normal-GFP donor cells. Pre-conditioning with PBI, where the head was excluded from the irradiation field, induced low levels of donor cell engraftment in peripheral blood leukocytes at 4 weeks post-transplant (Fig. 1B), with the highest mean percentage of GFP-expressing donor leukocytes measured in mice receiving a fractionated dose of 11 Gy (11.3 ± 3.7% GFP-expressing leukocytes). This replacement of up to 11% donor leukocytes had no impact on the relative GlcNS-UA levels in either bone marrow extracellular fluid or CNS tissues (brain parenchyma slice 5 and the thoracic spinal cord) in transplanted mice compared with age-matched untreated MPS IIIA animals (Fig. 1C, E, F). However, GlcNS-UA was reduced by up to 64% in the liver compared to untreated MPS IIIA mice (Fig. 1D). The intrabatch %CV of a quality control sample for each of the tissue matrices was: bone marrow cells, 7.3%; stromal cells, 10.3%; bone marrow extracellular fluid, 3.9%; liver, 11.9%; and brain, 8.5% (n = 5 for each). Total body irradiation mediates full chimerism but does not improve neurological function When MPS IIIA mice were pre-conditioned with TBI prior to transplant, they showed higher, statistically significant levels of donor-type chimerism with 85.3 ± 2.8% GFP-positive leukocytes at 4 weeks post-transplant (P b 0.0001). Significant reductions in the relative level of GlcNS-UA were evident in both the bone marrow compartment and the liver (69% and 80% reduction, respectively, compared to MPS IIIA untreated mice; P b 0.0001; P b 0.01). However, GlcNS-UA was not reduced in the brain or spinal cord of these same mice at 4 weeks post-transplant (P N 0.05 for both regions). In order to examine the long-term clinical efficacy of this protocol, we transplanted three cohorts of 4-week-old male mice using TBI preconditioning: normal recipients received normal donor cells (normal/ normal); MPS IIIA recipients received MPS IIIA donor cells (MPS IIIA/ MPS IIIA); and MPS IIIA recipients received normal donor cells (MPS IIIA/normal). Transplanted mice from the MPS IIIA/normal group gained weight during the first 8 days following transplant at a rate

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

449

similar to untreated normal mice (17% and 22% mean weight gain by day 8 compared to the day of transplant for MPS IIIA/normal and untreated normal mice, respectively; Fig. 2). Untreated MPS IIIA mice gained even more weight (40% mean weight gain). In contrast, the other two groups of transplanted mice lost weight over this same time period (normal/normal, 12% mean weight loss; MPS IIIA/MPS IIIA, 11% mean weight lost) and failed to efficiently reconstitute. Nodules were evident on the spleens of many of the transplanted mice and petechiae were also observed in the liver and/or brains of some mice, presumed to be due to thrombocytopenia. The remaining group (MPS IIIA mice transplanted with normal cells) continued to gain weight throughout the study. However, transplanted MPS IIIA mice were considerably smaller than age-matched untreated MPS IIIA mice. The effect of transplantation on neurological function was assessed in three different tests. Fifteen-week-old male mice (11 weeks posttransplant) and untreated age-matched normal and MPS IIIA male mice were assessed for locomotor and exploratory activity in the open field. Normal mice were significantly more active than MPS IIIA mice as determined by the total path length and number of cell entries in the open field (P b 0.05 for both measures; Fig. 3A, B). However, allogeneic BMT in pre-symptomatic MPS IIIA mice did not prevent the development of hypoactivity. The mice were then assessed for hind-limb gait abnormalities at 24 weeks of age (20 weeks post-treatment). Evaluation of stride width revealed a significantly narrower hind-limb gait width in both untreated and transplanted MPS IIIA mice when compared to untreated normal mice (P b 0.001; Fig. 3C). Similarly, when the pattern of activity and anxiety-related behaviors was examined in the Elevated Plus Maze test at 26 weeks of age (Supplementary Fig. 1), normal mice were found to be more active in the maze than both untreated and transplanted MPS IIIA mice as evidenced by increases in the path length. Despite the hypoactivity in both groups of MPS IIIA animals, they explored the open arms more frequently than age-matched normal controls, with a greater percentage of path length and time spent exploring the open arms. Statistical significance was not established, potentially due to the small group sizes in each of these measures (P N 0.05). Distribution of donor-derived cells and reconstitution of SGSH activity To investigate the degree of reconstitution in transplanted MPS IIIA mice, the presence of GFP-expressing, CD45-positive peripheral blood leukocytes was measured using flow cytometry. At 15 weeks post-transplant, an average of 96 ± 1% (n = 8; range of 88–99% GFP+/ CD45+ cells) of donor-derived cells had repopulated the peripheral blood of transplant recipients. Donor cell engraftment appeared stable, with an average of 93 ± 2% donor-derived leukocytes measured in additional mice at 25 weeks post-transplant (n = 8; range

Fig. 2. Body weight. Body weights of male MPS IIIA mice following pre-conditioning with TBI and transplantation at 4 weeks of age, and age-matched untreated wildtype and MPS IIIA control groups, were measured weekly (n = 16 mice per group until 15 weeks post-transplant; n = 8 mice per group from 16- to 25-week post-transplant).

Fig. 3. Effect of transplantation in MPS IIIA mice on neurological signs. (A, B) MPS IIIA recipient mice were tested in the open field at 11 weeks post-transplant and compared to age-matched untreated normal and MPS IIIA groups (n = 14–16 mice per group). The (A) path length and (B) total number of cell entries was determined using Field2020 software. (C) Hind-limb gait was then assessed at 20 weeks post-transplant in these same groups of mice (n = 8 mice per group). The stride width was determined from at least three strides per track with two tracks assessed per mouse. *P b 0.05, **P b 0.01, ***P b 0.001 compared to untreated MPS IIIA mice.

84–98% GFP+/CD45+ cells). This resulted in significant increases in SGSH activity within the MPS IIIA bone marrow compartment at 25 weeks post-transplant (P b 0.01; Fig. 4H) with reconstitution of 67% of wild-type levels (range of 35–79%; n = 4 mice per group). In comparison, there was no increase in SGSH activity recorded within the brain parenchyma (P N 0.05; n = 4 mice per group; Fig. 4I). The spatial distribution of recruited donor-derived bone marrow cells from the bloodstream into the CNS following transplantation in lethally irradiated mice was analyzed by visualizing GFP-expressing donor cells in brain cryosections. By 15 weeks post-transplantation, many ramified, GFP-expressing cells were distributed throughout the brain parenchyma in the rostral–caudal plane at both lateral levels examined (0 and 2 mm from the midline; Fig. 4A–E). Donor-derived cells were abundant in all brain regions examined and appeared to be both free in the parenchyma and associated with the vasculature. Greater numbers of donor-derived cells were apparent in the periventricular regions of the brain compared to the brain parenchyma proper. The percentage of GFP-expressing donor cells in a defined area of medial cortex (437.24 μm × 327.76 μm) was calculated to be 11.8 ±

450

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

Fig. 4. Donor cell infiltration in the brain parenchyma and SGSH activity in transplanted mice. Cryo-sections of transplanted mouse brains were immunostained with a polyclonal rabbit anti-GFP primary antibody and detected with a Cy3-conjugated donkey anti-rabbit IgG secondary antibody. Representative images from chimeric mice demonstrate the widespread distribution of donor cells in the brain parenchyma in regions including the (A) olfactory bulb, (B) cerebral cortex, (C) inferior colliculus, (D) hippocampus, and (E) around the 4th ventricle. (F) GFP-expressing cells were not detected in the cortex of mice that did not undergo transplantation. Scale bar, 100 μm. (G) The percentage of GFP-expressing donor cells that had migrated to the cerebral cortex was quantitated at 15 or 25 weeks post-transplantation (n = 4 mice per group). SGSH activity was measured in (G) bone marrow and (H) brain parenchymal homogenates in mice at 25 weeks post-transplantation (n = 4 mice per group). **P b 0.01, ***P b 0.001 compared to untreated MPS IIIA mice. nd, not detected.

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

5.8% and 12.0 ± 2.9% at 15 and 25 weeks post-transplantation, respectively (Fig. 4G).

Effect of transplantation on primary and secondary storage compounds Small reductions in GlcNS-UA were seen in slice 5 of the brain parenchyma of transplanted MPS IIIA mice at 15 weeks posttransplant (19 weeks of age; 8% reduction; P b 0.05; Fig. 5). No differences were detected between treated and untreated MPS IIIA mice in brain slice 3 or the spinal cord at this age. After 25 weeks of treatment (29 weeks of age), significantly less GlcNS-UA was evident in both brain slices 3 and 5 in transplanted mice (22% and 27% reduction, respectively; P b 0.001). However, the relative levels of GlcNS-UA in transplanted MPS IIIA mice remained significantly elevated when compared to the wild-type amount of GlcNS-UA in the brain (294-fold and 170-fold wild-type levels in brain slices 3 and 5, respectively). Significant changes in GlcNS-UA were not observed in

451

the spinal cord of transplanted MPS IIIA mice at 25 weeks posttransplant. The intra-batch %CV was 2.8% (n = 8 replicates). Very little GM3 ganglioside immunoreactivity was observed in the amygdala and inferior colliculi of wildtype mice (Fig. 6). In contrast, considerable GM3 ganglioside staining was detected in untreated 19week-old MPS IIIA mice in these regions. There was no apparent increase in the amount of GM3 ganglioside staining in older MPS IIIA mice (29 weeks of age) when compared to 19 week-old MPS IIIA animals (P = 0.8 for both regions). Long-term studies (up to 25 weeks post-transplantation) in treated MPS IIIA mice showed no evidence of transplantation impacting on GM3 ganglioside storage (P N 0.05 for both regions). The treatment effect on filipin labelling of unesterified cholesterol storage was also evaluated in these same mice. Semi-quantification of cells containing filipin-labelled cholesterol inclusions in the amygdala revealed significantly more storage in untreated and transplanted MPS IIIA mice in both cohorts of mice (Table 1). Cholesterol storage in the inferior colliculi was also evident in transplanted and untreated MPS IIIA mice (Table 1). However, there were no differences in the number of cholesterol-containing cells between MPS IIIA treated and untreated mice in either brain region (P N 0.05). Filipin-positive inclusions were not detected in these brain regions or at either age in wildtype mice. Discussion

Fig. 5. Relative levels of HS-derived disaccharide in the CNS. The relative amount of GlcNS-UA storage in the brain parenchyma (slice 3 or 5, sectioned as depicted in the schematic diagram) and the thoracic spinal cord were assessed at 15 or 25 weeks posttreatment and compared to that in age-matched untreated controls using a tandem mass spectrometry. *P b 0.05, ***P b 0.001 compared to untreated MPS IIIA mice.

We have evaluated the impact of allogeneic transplantation of mononuclear bone marrow cell transplantation on neurological outcome in MPS IIIA. TBI and transplantation of young MPS IIIA mice with wild-type bone marrow generated stable chimeras for at least 25 weeks post-treatment. Whilst apparently large numbers of donor cells made their way into the host brain and we observed small, but significant, reductions in primary storage (GlcNS-UA) in brain parenchyma, neurological function was not improved. These findings would seem to confirm and support the lack of neurological benefit reported in Sanfilippo patients receiving bone marrow transplants (Hoogerbrugge et al., 1995; Klein et al., 1995; Shapiro et al., 1995), and would argue against further transplantation of MPS III patients. Although Sanfilippo patients have also undergone transplantation with umbilical cord blood cells (Martin et al., 2006), the effect of treatment on neurological function has not been reported thus far. Whether this alternative source of cells is more likely to result in improved clinical outcomes is uncertain but possible. We are unable to exclude any impact of irradiation on the behavioral phenotype of the transplanted MPS IIIA mice as the two control groups for this (normal/normal and MPS IIIA/MPS IIIA) exhibited graft failure, possibly due to increased susceptibility to bacteremia as a result of slower reconstitution of blood leukocytes (Duran-Struuck et al., 2008). Subsequent experiments have demonstrated that these groups can be successfully transplanted when lower irradiation doses are employed (data not shown). PBI was ineffective in generating stable macro-chimeras, potentially due to the exclusion of the thymus from the irradiation field. We have therefore used what are likely to be favorable conditions for the immigration of donor bone marrow-derived cells into the brain of the recipient. Two recent publications have highlighted that the degree of infiltrating bone marrow-derived microglia is increased by TBI (Ajami et al., 2007; Mildner et al., 2007). Using a parabiosis model with no irradiation pre-conditioning, little or no microglia progenitor cell recruitment to the CNS was evident in the parabiotic partner (Ajami et al., 2007). This model joins the circulatory system of a GFP-positive mouse to that of a GFP-negative mouse to generate a chimeric mouse whilst maintaining an intact blood–brain barrier. Likewise, bone marrow-derived cells did not contribute to the microglial population of chimeric mice pre-conditioned with irradiation with the head excluded from the irradiation zone (Mildner et al.,

452

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

Fig. 6. Quantitation of GM3 ganglioside immunoreactivity. The effect of treatment on secondary storage of GM3 ganglioside was assessed within the brain. (A,B) GM3 ganglioside accumulating intracellularly in the amygdala and inferior colliculus was not reduced by transplantation (n = 4 mice per group). (C) Representative immunostained images from the amygdala or inferior colliculus of normal untreated, MPS IIIA untreated or MPS IIIA transplanted mice at 15 or 25 weeks post-treatment. ***P b 0.001 compared to untreated MPS IIIA mice. Original magnification ×200.

2007). The authors speculated that irradiation may temporarily disrupt the blood–brain barrier as well as induce the production of the chemokine CCL2, the ligand of CCR2, which may aid in the recruitment of CCR2-expressing bone marrow-derived myeloid cells to the adult CNS (Mildner et al., 2007). Furthermore, it has been

demonstrated that neurodegeneration and neuroinflammation in diseases such as metachromatic leukodystrophy and Parkinson's disease results in increased migration of bone marrow-derived cells to the CNS (Biffi et al., 2006; Rodriguez et al., 2007). Thus, it is possible that the rate of bone marrow-derived cell migration to the CNS may

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

453

Table 1 Effect of BMT on cholesterol storage. Filipin labelling of unesterified cholesterol in the amygdala and inferior colliculus was semi-quantitatively assessed and scored using the following scale: absent (−), low (+), moderate (++), abundant (+++). The number of filipin labelled cells in each brain regions was also scored (indicated in parentheses): no labelled cells (0), few labelled cells (1), moderate numbers of labelled cells (2), high numbers of labelled cells (3). Amygdala

Normal untreated MPS IIIA untreated MPS IIIA transplanted

Inferior colliculus

15 weeks

25 weeks

15 weeks

25 weeks

− (0) ++ / +++ (2–3) ++ / +++ (2.5–3)

− (0) +++ (3) +++ (3)

− (0) ++ / +++ (2.5–3) + / +++ (1.5–3)

− (0) ++ / +++ (2.5–3) ++ / +++ (2.5–3)

be elevated in MPS IIIA mice relative to the migration in wild-type mice due to the neuroinflammatory and neurodegenerative components of disease (Hemsley et al., 2008; Savas et al., 2004). Several strategies may improve the efficacy of bone marrowmediated therapies in MPS IIIA. First, given the significant HS storage burden present at the time of treatment, transplantation may need to be initiated earlier in the disease course (i.e. at birth or in utero) similar to the experiments undertaken in neonatal MPS IIIB mice (Heldermon et al., 2010), using transplantation conditions that mediate full donor-type chimerism. The rate of infiltration of donor cells into the brain is slower than in peripheral organs (Kennedy and Abkowitz, 1997), further delaying the time until sufficient quantities of therapeutic enzyme are delivered within the CNS. This hypothesis is supported by the improved cognition observed in Krabbe disease babies transplanted prior to the development of symptoms, compared to the poor outcomes in Krabbe children who were symptomatic at the time of transplant (Escolar et al., 2005). Further support comes from studies where BMT performed in neonatal MPS VII mice was more effective than when applied to adult MPS VII mice (Birkenmeier et al., 1991; Sands et al., 1993). Second, the secretion of active SGSH from the unmodified donor-derived cells may be inadequate to treat the neurodegeneration in the recipient MPS IIIA mice and may require ex vivo gene modification to generate superphysiological SGSH levels in the transplanted cell population. This approach has provided a neurological benefit in two X-linked adrenoleukodystrophy boys who underwent transplantation of CD34+ hematopoietic stem cells modified to over-express the ABCD1 gene using a lentiviral vector (Cartier et al., 2009). Considering that 11% donor engraftment was unable to mediate reductions in HS storage in MPS IIIA bone marrow, this disease may require much higher levels of replacement enzyme compared to other lysosomal storage disorders such as Gaucher disease, where 7% engraftment was sufficient for normalization of disease pathology in transplanted mice (Enquist et al., 2009). Combining these two strategies may increase the likelihood of BMTmediated therapies altering the disease course in MPS IIIA. Taken together, our results suggest that transplantation with wild-type bone marrow in pre-symptomatic MPS IIIA mice does not improve the clinical symptoms and should not be undertaken in Sanfilippo patients. Further studies will be required to develop an effective bone marrow-mediated therapy for MPS IIIA. Authorship KMH, JJH and AAL designed research; AAL, KMH, HH, SH and TR performed research; AAL, KMH, JJH, SH and TR analyzed data; and AAL, KMH wrote the paper. Conflict of interest statement The authors declare no competing financial interests. Acknowledgments We thank the Department of Radiation Oncology at the Royal Adelaide Hospital for irradiating the mice; Dr Allison Crawley for

assistance with the intravenous injections; Ms Amanda Luck for breeding the experimental mice; Ms Barbara King, Dr Tomas Rozek and Dr Maria Fuller for assistance with the mass spectrometry; Ms Noor Shamsani for determining the threshold of the SGSH activity assay; the Children, Youth and Women's Health Service Animal House staff for the routine care of the mice; the Department of Hematology for access to the flow cytometer. This work was supported by the National Health and Medical Research Council of Australia (grant 453471) to JJH and KMH.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.expneurol.2010.07.024.

References Ajami, B., Bennett, J.L., Krieger, C., Tetzlaff, W., Rossi, F.M., 2007. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat. Neurosci. 10, 1538–1543. Bhattacharyya, R., Gliddon, B., Beccari, T., Hopwood, J.J., Stanley, P., 2001. A novel missense mutation in the lysosomal sulfamidase is the basis of MPS III A in a spontaneous mouse mutant. Glycobiology 11, 99–103. Bhaumik, M., Muller, V.J., Rozaklis, T., Johnson, L., Dobrenis, K., Bhattacharyya, R., Wurzelmann, S., Finamore, P., Hopwood, J.J., Walkley, S.U., Stanley, P., 1999. A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology 9, 1389–1396. Biffi, A., Capotondo, A., Fasano, S., del Carro, U., Marchesini, S., Azuma, H., Malaguti, M.C., Amadio, S., Brambilla, R., Grompe, M., Bordignon, C., Quattrini, A., Naldini, L., 2006. Gene therapy of metachromatic leukodystrophy reverses neurological damage and deficits in mice. J. Clin. Invest. 116, 3070–3082. Birkenmeier, E.H., Barker, J.E., Vogler, C.A., Kyle, J.W., Sly, W.S., Gwynn, B., Levy, B., Pegors, C., 1991. Increased life span and correction of metabolic defects in murine mucopolysaccharidosis type VII after syngeneic bone marrow transplantation. Blood 78, 3081–3092. Cartier, N., Hacein-Bey-Abina, S., Bartholomae, C.C., Veres, G., Schmidt, M., Kutschera, I., Vidaud, M., Abel, U., Dal-Cortivo, L., Caccavelli, L., Mahlaoui, N., Kiermer, V., Mittelstaedt, D., Bellesme, C., Lahlou, N., Lefrere, F., Blanche, S., Audit, M., Payen, E., Leboulch, P., l'Homme, B., Bougneres, P., Von Kalle, C., Fischer, A., Cavazzana-Calvo, M., Aubourg, P., 2009. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science 326, 818–823. Crawley, A.C., Gliddon, B.L., Auclair, D., Brodie, S.L., Hirte, C., King, B.M., Fuller, M., Hemsley, K.M., Hopwood, J.J., 2006. Characterization of a C57BL/6 congenic mouse strain of mucopolysaccharidosis type IIIA. Brain Res. 1104, 1–17. Duran-Struuck, R., Hartigan, A., Clouthier, S.G., Dyson, M.C., Lowler, K., Gatza, E., Tawara, I., Toubai, T., Weisiger, E., Hugunin, K., Reddy, P., Wilkinson, J.E., 2008. Differential susceptibility of C57BL/6NCr and B6.Cg-Ptprca mice to commensal bacteria after whole body irradiation in translational bone marrow transplant studies. J. Transl. Med. 6, 10. Enquist, I.B., Nilsson, E., Mansson, J.E., Ehinger, M., Richter, J., Karlsson, S., 2009. Successful low-risk hematopoietic cell therapy in a mouse model of type 1 Gaucher disease. Stem Cells 27, 744–752. Escolar, M.L., Poe, M.D., Provenzale, J.M., Richards, K.C., Allison, J., Wood, S., Wenger, D.A., Pietryga, D., Wall, D., Champagne, M., Morse, R., Krivit, W., Kurtzberg, J., 2005. Transplantation of umbilical-cord blood in babies with infantile Krabbe's disease. N. Engl. J. Med. 352, 2069–2081. Gliddon, B.L., Hopwood, J.J., 2004. Enzyme-replacement therapy from birth delays the development of behavior and learning problems in mucopolysaccharidosis type IIIA mice. Pediatr. Res. 56, 65–72. Heldermon, C.D., Ohlemiller, K.K., Herzog, E.D., Vogler, C., Qin, E., Wozniak, D.F., Tan, Y., Orrock, J.L., Sands, M.S., 2010. Therapeutic efficacy of bone marrow transplant, intracranial AAV-mediated gene therapy, or both in the mouse model of MPS IIIB. Mol. Ther. 18, 873–880. Hemsley, K.M., Hopwood, J.J., 2005. Development of motor deficits in a murine model of mucopolysaccharidosis type IIIA (MPS-IIIA). Behav. Brain Res. 158, 191–199.

454

A.A. Lau et al. / Experimental Neurology 225 (2010) 445–454

Hemsley, K.M., King, B., Hopwood, J.J., 2007. Injection of recombinant human sulfamidase into the CSF via the cerebellomedullary cistern in MPS IIIA mice. Mol. Genet. Metab. 90, 313–328. Hemsley, K.M., Beard, H., King, B.M., Hopwood, J.J., 2008. Effect of high dose, repeated intra-CSF injection of sulphamidase on neuropathology in MPS IIIA mice. Genes Brain Behav. 7, 740–753. Hemsley, K.M., Luck, A.J., Crawley, A.C., Hassiotis, S., Beard, H., King, B., Rozek, T., Rozaklis, T., Fuller, M., Hopwood, J.J., 2009a. Examination of intravenous and intra-CSF protein delivery for treatment of neurological disease. Eur. J. Neurosci. 29, 1197–1214. Hemsley, K.M., Norman, E.J., Crawley, A.C., Auclair, D., King, B., Fuller, M., Lang, D.L., Dean, C.J., Jolly, R.D., Hopwood, J.J., 2009b. Effect of cisternal sulfamidase delivery in MPS IIIA Huntaway dogs-A proof of principle study. Mol. Genet. Metab. 98, 383–392. Hess, D.C., Abe, T., Hill, W.D., Studdard, A.M., Carothers, J., Masuya, M., Fleming, P.A., Drake, C.J., Ogawa, M., 2004. Hematopoietic origin of microglial and perivascular cells in brain. Exp. Neurol. 186, 134–144. Hoogerbrugge, P.M., Brouwer, O.F., Bordigoni, P., Ringden, O., Kapaun, P., Ortega, J.J., O'Meara, A., Cornu, G., Souillet, G., Frappaz, D., et al., 1995. Allogeneic bone marrow transplantation for lysosomal storage diseases. Lancet 345, 1398–1402. Hopwood, J.J., Elliott, H., 1982. Diagnosis of Sanfilippo A syndrome by estimation of sulphamidase activity using a radiolabelled tetrasaccharide substrate. Clin. Chim. Acta 123, 241–250. Kennedy, D.W., Abkowitz, J.L., 1997. Kinetics of central nervous system microglial and macrophage engraftment—analysis using a transgenic bone marrow transplantation model. Blood 90, 986–993. King, B., Savas, P., Fuller, M., Hopwood, J., Hemsley, K., 2006. Validation of a heparan sulfate-derived disaccharide as a marker of accumulation in murine mucopolysaccharidosis type IIIA. Mol. Genet. Metab. 87, 107–112. Klein, K., Krivit, W., Whitley, C., Peters, C., Cool, V., Fuhrman, M., de Alarcon, P., Klemperer, M., Miller, L., Nelson, R., Henslee-Downey, J., Chang, P., Wraith, J., Lockman, L., Shapiro, E., 1995. Poor cognitive outcome of eleven children with Sanfilippo syndrome after bone marrow transplantation and successful engraftment. Bone Marrow Transplant. 15, S176–S181. Krivit, W., 2004. Allogeneic stem cell transplantation for the treatment of lysosomal and peroxisomal metabolic diseases. Springer Semin. Immunopathol. 26, 119–132. Lau, A.A., Crawley, A.C., Hopwood, J.J., Hemsley, K.M., 2008. Open field locomotor activity and anxiety-related behaviors in mucopolysaccharidosis type IIIA mice. Behav. Brain Res. 91, 130–136. Martin, P.L., Carter, S.L., Kernan, N.A., Sahdev, I., Wall, D., Pietryga, D., Wagner, J.E., Kurtzberg, J., 2006. Results of the cord blood transplantation study (COBLT):

outcomes of unrelated donor umbilical cord blood transplantation in pediatric patients with lysosomal and peroxisomal storage diseases. Biol. Blood Marrow Transplant. 12, 184–194. McGlynn, R., Dobrenis, K., Walkley, S.U., 2004. Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolysaccharide storage disorders. J. Comp. Neurol. 480, 415–426. Meikle, P.J., Hopwood, J.J., Clague, A.E., Carey, W.F., 1999. Prevalence of lysosomal storage disorders. JAMA 281, 249–254. Mildner, A., Schmidt, H., Nitsche, M., Merkler, D., Hanisch, U.K., Mack, M., Heikenwalder, M., Bruck, W., Priller, J., Prinz, M., 2007. Microglia in the adult brain arise from Ly-6C(hi)CCR2(+) monocytes only under defined host conditions. Nat. Neurosci. 10, 1544–1553. Neufeld, E.F., Muenzer, J., 2001. The mucopolysaccharidoses. In: Scriver, C.R. (Ed.), The metabolic and molecular bases of inherited diseases. McGraw-Hill, New York, pp. 3421–3452. Rodriguez, M., Alvarez-Erviti, L., Blesa, F.J., Rodriguez-Oroz, M.C., Arina, A., Melero, I., Ramos, L.I., Obeso, J.A., 2007. Bone-marrow-derived cell differentiation into microglia: a study in a progressive mouse model of Parkinson's disease. Neurobiol. Dis. 28, 316–325. Sands, M.S., Barker, J.E., Vogler, C., Levy, B., Gwynn, B., Galvin, N., Sly, W.S., Birkenmeier, E., 1993. Treatment of murine mucopolysaccharidosis type VII by syngeneic bone marrow transplantation in neonates. Lab. Invest. 68, 676–686. Savas, P.S., Hemsley, K.M., Hopwood, J.J., 2004. Intracerebral injection of sulfamidase delays neuropathology in murine MPS-IIIA. Mol. Genet. Metab. 82, 273–285. Schaefer, B.C., Schaefer, M.L., Kappler, J.W., Marrack, P., Kedl, R.M., 2001. Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo. Cell. Immunol. 214, 110–122. Shapiro, E.G., Lockman, L.A., Balthazor, M., Krivit, W., 1995. Neuropsychological outcomes of several storage diseases with and without bone marrow transplantation. J. Inherit. Metab. Dis. 18, 413–429. Simard, A.R., Rivest, S., 2004. Bone marrow stem cells have the ability to populate the entire central nervous system into fully differentiated parenchymal microglia. FASEB J. 18, 998–1000. Sivakumur, P., Wraith, J.E., 1999. Bone marrow transplantation in mucopolysaccharidosis type IIIA: a comparison of an early treated patient with his untreated sibling. J. Inherit. Metab. Dis. 22, 849–850. Zheng, Y., Ryazantsev, S., Ohmi, K., Zhao, H.Z., Rozengurt, N., Kohn, D.B., Neufeld, E.F., 2004. Retrovirally transduced bone marrow has a therapeutic effect on brain in the mouse model of mucopolysaccharidosis IIIB. Mol. Genet. Metab. 82, 286–295.