N-butyldeoxynojirimycin treatment restores the innate fear response and improves learning in mucopolysaccharidosis IIIA mice

Molecular Genetics and Metabolism 118 (2016) 100–110

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N-butyldeoxynojirimycin treatment restores the innate fear response and improves learning in mucopolysaccharidosis IIIA mice Xenia Kaidonis a,b, Sharon Byers a,b,c, Enzo Ranieri a, Peter Sharp a, Janice Fletcher a,c, Ainslie Derrick-Roberts a,c,⁎ a b c

Department of Genetics and Molecular Pathology, SA Pathology (CYWHS site), North Adelaide, South Australia, Australia Department of Genetics, University of Adelaide, Adelaide, South Australia, Australia Department of Paediatrics, University of Adelaide, Adelaide, South Australia, Australia

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Article history: Received 18 February 2016 Received in revised form 11 April 2016 Accepted 11 April 2016 Available online 13 April 2016 Keywords: Ganglioside N-butyldeoxynojirimycin Mucopolysaccharidosis IIIA Behaviour Inflammation Substrate deprivation therapy

a b s t r a c t Mucopolysaccharidosis IIIA is a heritable neurodegenerative disorder resulting from the dysfunction of the lysosomal hydrolase sulphamidase. This leads to the primary accumulation of the complex carbohydrate heparan sulphate in a wide range of tissues and the secondary neuronal storage of gangliosides GM2 and GM3 in the brain. GM2 storage is associated with CNS deterioration in the GM2 gangliosidosis group of lysosomal storage disorders and may also contribute to MPS CNS disease. N-butyldeoxynojirimycin, an inhibitor of ceramide glucosyltransferase activity and therefore of ganglioside synthesis, was administered to MPS IIIA mice both prior to maximal GM2 and GM3 accumulation (early treatment) and after the maximum level of ganglioside had accumulated in the brain (late treatment) to determine if behaviour was altered by ganglioside level. Ceramide glucosyltransferase activity was decreased in both treatment groups; however, brain ganglioside levels were only decreased in the late treatment group. Learning in the water cross maze was improved in both groups and the innate fear response was also restored in both groups. A reduction in the expression of inflammatory gene Ccl3 was observed in the early treatment group, while IL1β expression was reduced in both treatment groups. Thus, it appears that NB-DNJ elicits a transient decrease in brain ganglioside levels, some modulation of inflammatory cytokines and a functional improvement in behaviour that can be elicited both before and after overt neurological changes manifest. Synopsis: NB-DNJ improves learning and restores the innate fear response in MPS IIIA mice by decreasing ceramide glucosyltransferase activity and transiently reducing ganglioside storage and/or modulating inflammatory signals. © 2016 Elsevier Inc. All rights reserved.

1. Introduction A deficiency in sulphamidase (EC 3.10.1.1) results in the subsequent lysosomal accumulation of the complex carbohydrate glycosaminoglycan (GAG) heparan sulphate (HS) and the lysosomal storage disorder (LSD) mucopolysaccharidosis type IIIA (MPS IIIA; OMIM #252900). MPS IIIA pathology manifests as progressive central nervous system (CNS) degeneration combined with relatively mild somatic disease [1]. To date, brain disease is untreatable by therapies aiming to introduce functional sulphamidase into the peripheral circulation, with a significant hurdle to treatment being the isolation of the CNS by the blood brain barrier (BBB) [1]. This has prompted the investigation of alternate strategies including substrate deprivation therapy (SDT), also termed substrate reduction therapy (SRT), which aims to decrease the synthesis Abbreviations: NB-DNJ, N-butyldeoxynojirimycin; SDT, substrate deprivation therapy; CNS, central nervous system; BBB, blood brain barrier; HPTLC, high performance thin layer chromatography; NPC, Niemann-Pick disease, type C; GFAP, glial fibrillary acidic protein. ⁎ Corresponding author at: Department of Genetics and Molecular Pathology, SA Pathology (CYWHS site), 72 King William Rd, North Adelaide 5006, South Australia, Australia. E-mail address: [email protected] (A. Derrick-Roberts).

http://dx.doi.org/10.1016/j.ymgme.2016.04.002 1096-7192/© 2016 Elsevier Inc. All rights reserved.

of HS to improve the balance between synthesis and degradation [2–6]. A number of SDT agents able to cross the BBB, including rhodamine B and genistein, have been effective at improving CNS pathology in mouse models of MPS II, MPS IIIA and MPS IIIB [5,7,8]. Genistein has since progressed to clinical trials, though with only minimal improvements observed in patients [3–6]. In addition to HS storage, the secondary accumulation of acidic glycosphingolipids (GSL) is observed in the MPS IIIA brain; specifically, the ganglioside species GM2 and GM3 [9–12]. While primary GM3 accumulation has not been attributed to any LSD, GM2 storage occurs in the GM2 gangliosidoses, Tay-Sachs and Sandhoff disease [13]. Similarities in neuronal morphology and pattern of neurodegeneration between the GM2 gangliosidoses and MPS suggests that gangliosides may be contributing to MPS brain disease [1,9,12–15]. Ganglioside targeted SDT may thus have a role to play in altering the progression of neurodegeneration in MPS IIIA. N-butyldeoxynojirimycin (NB-DNJ), also known as miglustat, is a small iminosugar inhibitor of glycosphingolipid (GSL) synthesis with the ability to cross the BBB [16–18]. Under the registered name Zavesca®, NB-DNJ is the first approved treatment for Niemann-Pick type C (NPC) and with a reduction in neurological disease observed in

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Fig. 1. Brain ganglioside levels and tissue weights in oral versus iv NB-DNJ treated mice. Gangliosides (A) were extracted from normal mouse brains treated with oral (black bars) or iv (grey bars) NB-DNJ. GM1 (d18:1/18:0), GM2 (d18:1/22:1), and GM3 (d18:1/18:0) levels were quantified by HPLC ESI-MS/MS against a d3-GM1 internal standard. Body and tissue weights (liver, kidney, spleen, heart, lung and brain) (B) were recorded post-mortem. Results are expressed as percentage of untreated normal mouse values presented as mean ± standard error. * Significantly different from untreated normal, p b 0.05 (one-way ANOVA, Tukey's HSD).

NPC patients [19,20]. In addition, Zavesca® is also approved for the treatment of mild to moderate type 1 Gaucher disease [21,22]. Clinical trials for other lysosomal storage disorders, including Gaucher type 3 and GM2 gangliosidoses Sandhoff and Tay-Sachs disease, show varied results depending on onset of disease, with some patients having stabilised or delayed neurological disease [23–27] and some having no improvement [24,28]. A preliminary 12 month clinical trial of NB-DNJ in MPS III patients reported no decrease in ganglioside levels or significant improvements in neurological symptoms despite the detection of NB-DNJ in patient cerebrospinal fluid [29]. However, the inclusion of patients of different ages, MPS III subtypes and disease stages resulted in varying degrees of functional damage prior to the initiation of NB-DNJ treatment [29], potentially impairing the ability to distinguish biochemical or functional effects of treatment. Animal models of MPS overcome these issues by providing a homogeneous starting group for testing the efficacy of NB-DNJ at altering MPS CNS pathology. NPC is similar to MPS IIIA in that the storage of gangliosides GM2 and GM3 is secondary; in the case of NPC to the primary storage of cholesterol [30]. The efficacy of NBDNJ in NPC supports the notion that reducing secondary ganglioside storage in the MPS IIIA brain may be beneficial in altering neurological disease. In this study, MPS IIIA mice were treated with NB-DNJ starting either at one month of age (early in the time-course of disease progression) or

at four months of age (late in the time-course of disease progression). Both groups were killed at six months of age and biochemical, enzymatic and functional outcomes were measured. 2. Materials and methods 2.1. Generation and identification of MPS IIIA mice All studies using animals were reviewed and approved by the author's institutional animal ethics committees. MPS IIIA mice and their normal littermates were bred from heterozygous parents and genotype determined as previously described [31]. 2.2. Oral versus intravenous route of NB-DNJ administration Normal mice treated orally (n = 5, 2F, 3 M) were fed with a daily intake of powdered chow, calculated at 5 g chow/30 g body weight/day, mixed with 4800 mg/kg/day NB-DNJ (Actelion, Switzerland and Sigma-Aldrich, USA), from four to five weeks of age. This dose was the highest previously described in the literature [17]. Normal mice treated with iv NB-DNJ (n = 5, 3F, 2 M) were injected via the tail vein with 0.4 μg/g body weight NB-DNJ in 0.9% sterile saline, three times a week, from four to eight weeks of age. Assuming a mouse blood

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volume of approximately 7% of body weight [32], this resulted in a blood concentration of 25 μM NB-DNJ. This concentration was within the range of 18 to 57 μM, previously described as therapeutically effective [17,18].

2.3. NB-DNJ in vivo trial MPS IIIA mice were injected via the tail vein, three times per week, either from four weeks to six months of age (early treatment group)

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(n = 8, 4F, 4 M) or from four months to six months of age (late treatment group) (n = 6, 3F, 3 M) with 0.8 μg/g body weight NB-DNJ in 0.9% sterile saline. Normal mice were also treated from four weeks to six months of age (n = 2, 2F) with the same dose of NB-DNJ. Mice were compared to age-matched normal untreated (n = 6, 3F, 3 M) and MPS IIIA untreated (n = 9, 4F, 5 M) mice. 2.4. Behaviour testing Exploratory behaviour and the innate fear response were assessed using the open field test [33,34] using an automated animal monitoring system (Harvard Apparatus) and Versamax 4.20 software (AccuScan Instruments, Inc., USA, 2005). Total distance travelled, rearing events and the proportion of activity in the centre versus the margin of the apparatus were determined. Spatial learning was assessed in the water cross maze as previously described [5,33,34]. Escape latency, the number of incorrect entries and the proportion of correct entries were recorded. 2.5. Ceramide glucosyltransferase assay Brain tissue was homogenised in 2.5 volumes of 0.1% (v/v) Triton X100, centrifuged at 8500 ×g, and the supernatants recovered. Ceramide glucosyltransferase activity was measured as previously described [35] with the use of phosphatidylcholine instead of lecithin to form liposomes and an increase in incubation time to 24 h. The fluorescent substrate C6-NBD-ceramide (50 μg, Invitrogen, USA) and L-αphosphatidylcholine (500 μg, Sigma-Aldrich, USA) were mixed in ethanol and the solvent evaporated, before being sonicated in 1 mL deionised water to form liposomes. The assay mix as described in [35] was incubated at 30 °C for 24 h. After incubation lipids were extracted with 20 volumes of 2:1 (v/v) chloroform/methanol using the Folch method [36] as described previously [33]. Free C6-NBD-ceramide was separated from glucose bound C6-NBD-ceramide by high performance thin layer chromatography (HPTLC), on 10 × 20 cm HPTLC pre-coated silica gel plates (Merck, Germany), in 65:25:4 (v/v/v) chloroform/methanol/deionised water and visualised by UV illumination. Bands representing C6-NBD-glucosylceramide (i.e. bands larger than unincorporated C6-NBD-ceramide) were scraped from the plate and resuspended in 5 mL 2:1 (v/v) chloroform/methanol and the fluorescence was measured at λex 466 and λem 539 nm using a Victor 3 multiplate reader. 2.6. Extraction of brain gangliosides and quantitation using mass spectrometry Gangliosides were isolated using a modified Folch method [36,37] combined with solid phase extraction according to Williams et al. [36, 37] as previously described [33] and quantified by electrospray ionization tandem mass spectrometry. GM1 (d18:1/18:0), GM2 (d18:1/22:1), GM3 (d18:1, 18:0), and d3-GM1 (d18:1, 18:0) were quantified against a d3-GM1 internal standard and normalised to tissue weight as previously described [33]. Results are calculated as nmol/g wet tissue weight and expressed as a percentage of untreated normal values. 2.7. Organic phase lipid analysis Organic lipids were separated by TLC on 10 × 20 cm HPTLC precoated silica gel plates (Merck, Germany). Simple lipids were analysed using a solvent system of hexane/diethylether/acetic acid (85:15:1, v/

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v/v) for 30 min. Bands were visualised by spraying with 75% H2SO4 and developing at 120 °C for 10–15 min. Glycolipids in lower phase lipid extracts, including cerebroside, triglycosylceramide and sulphatide, were run in chloroform/methanol/deionised water (65:25:4, v/v/v) and visualised using 2 M orcinol in 75% H2SO4 spray developed at 120 °C for 5 min. Phospholipids including cardiolipin (CL), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylcholine (PC) and sphingomyelin (SM) were run in chloroform/methanol/deionised water (65:25:4, v/ v/v) and visualised using 0.025% (w/v) phosphomolymbic acid, 0.05% (v/v) H2SO4 and 10% (v/v) deionised water in glacial acetic acid and developed at 100 °C for 2 min. Densitometry was performed to determine the relative amount of each lipid using the UVitec gel documentation system (UK) and UVI photoMW software (version 11.01). 2.8. GAG measurement Brain tissue was homogenised in 2.5 volumes of 0.1% (v/v) Triton X100 (1:2.5 w/v), GAGs were isolated as previously described [6,33,34] and GAG levels measured using a uronic acid assay [38]. 2.9. Immunohistochemistry Immunohistochemistry of glial fibrillary acidic protein (GFAP) was performed on 10 μm frozen brain sections on superfrost plus microscope slides (Menzel-Glaser, Germany). Slides were defrosted at room temperature, fixed in 10% neutral buffered formalin at room temperature for 30 min and washed three times in PBS at room temperature. Antigen retrieval was performed in 1% (w/v) SDS at room temperature for five minutes before blocking in 2% (w/v) BSA at 4 °C for one hour. Incubation in rabbit anti-GFAP primary antibody (Dako, Denmark) (diluted 1:50 in blocking solution) was performed at 37 °C for 30 min followed by incubation with anti-rabbit FITC conjugated secondary antibody (Silenus Labs, Australia) (diluted 1:1000 in blocking solution) at room temperature for one hour. Slides were mounted in ProLong Gold antifade reagent with DAPI (Invitrogen, USA). Staining was viewed using an upright fluorescence microscope and photographed at a magnification of 40×. 2.10. Real-time PCR Brain tissue was homogenised in TRIzol (Invitrogen, USA) reagent, total RNA extracted and RNA quantified using a nanodrop 1000 spectrophotometer (Thermo Scientific, USA). cDNA was generated from 1 μg of total RNA using the QiagenQuantiTec reverse transcription kit. The expression levels of ten inflammatory mediator genes were determined: glial fibrillary acidic protein (Gfap), tumor necrosis factor family receptor superfamily member 1a (Tnfrs1α), chemokine (C\\C motif) ligand 3 (Ccl3; Mip1a), interleukin 1 beta (Il1β), transforming growth factor beta 1 (Tgfβ1), CD68 antigen (Cd68), interferon gamma (Ifnγ), synuclein alpha (αSyn), cathepsin B (Ctsb) and tumor necrosis factor alpha (Tnfα) (Supplementary Table 1). These genes were chosen for analysis because they are upregulated in murine MPS IIIA brain as well as murine MPS I, MPS IIIB and MPS VII brain. [39–44]. Forward and reverse primers for each gene of interest were chosen using NCBI primer-BLAST (www. ncbi.nlm.nih.gov/tools/primer-blast), ensuring that primers crossed an exon-exon boundary and were specific to the gene of interest (Supplementary Table 1). PCR was carried out on an Applied Biosystems 7300 thermocycler using SYBR green PCR master mix and normalised to

Fig. 2. Effect of early and late NB-DNJ treatment on ceramide glucosyltransferase activity and ganglioside levels. Ceramide glucosyltransferase enzyme activity (A) and ganglioside levels (B-D) in six month old untreated normal, NB-DNJ treated normal, untreated MPS IIIA and early and late NB-DNJ treated MPS IIIA mouse brain samples. Results are expressed as percentage of untreated normal mice and values presented as mean ± standard error except for the treated normal group which is the average of 2 values. *Significantly different from untreated normal, p b 0.05 (one-way ANOVA, Tukey's HSD). Gangliosides were extracted from six month old untreated normal, NB-DNJ treated normal, untreated MPS IIIA and early and late NB-DNJ treated MPS IIIA mouse brains. GM1 (d18:1/18:0) (B), GM2 (d18:1/22:1) (C) and GM3 (d18:1/18:0) (D) levels were quantified by HPLC ESI-MS/MS against a d3-GM1 internal standard. Results are expressed as percentage of untreated normal mice and values presented as mean ± standard error except for the treated normal group which is the average of 2 values. *Significant difference from normal, p b 0.05 (one-way ANOVA, Tukey's HSD). #Significant difference from untreated MPS IIIA, p b 0.05 (one-way ANOVA, Tukey's HSD).

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Fig. 3. Organic lower phase lipids in the NB-DNJ treated brain. Organic lower phase lipids extracted from the brains of six month old untreated normal (black bars), untreated MPS IIIA (white bars), early (diagonal lines) and late (horizontal lines) treated MPS IIIA mice using the Folch method were separated by HPTLC, and visualised with stains specific to each lipid type. Densitometry was used to determine relative percentages of simple (A) glycolipids (B) and phospholipids (C) (CL = cardiolipin, PA = phosphatidic acid, PE = phosphatidylethanolamine, PI = phosphatidylinositol, PC = phosphatiylcholine and SM = sphingomyelin). Data is expressed as mean ± standard error.

cyclophilin A (Cypa). The 2-ΔΔCt method was used to calculate the fold change in gene expression [45]. 2.11. Statistical analysis Statistical analysis was performed using SigmaStat 3.0 by one-way analysis of variance (ANOVA) with Tukey's HSD post hoc test where appropriate. 3. Results 3.1. Ganglioside levels and body and tissue weights differed with oral versus iv route of NB-DNJ administration Oral (4800 mg/kg/day NB-DNJ) or iv (0.4 μg/g body weight) NB-DNJ was administered to normal mice. Both modes of administration were initiated at four weeks of age. Mice treated with iv NB-DNJ were treated for a period of four weeks, however the oral treatment was terminated for ethical reasons after one week due to poor temperature regulation and weight loss. Oral treatment for one week resulted in a reduction in brain GM1, GM2, and GM3 levels by 15%, 20% and 42% respectively, with the decrease in GM3 reaching statistical significance (Fig. 1A). Intravenous treatment of normal mice for four weeks also resulted in a reduction in brain GM1, GM2, and GM3 levels by 39%, 31% and 42% respectively, with the

decreases in GM1 and GM3 reaching statistical significance (Fig. 1A). Body weight of mice treated with oral NB-DNJ was significantly less than that of untreated mice at 63% of untreated values (Fig. 1B), consistent with published data [18, 46]. Liver, kidney, spleen, heart, lung and brain weights were also significantly less than untreated mice at 64%, 74%, 22%, 67%, 75% and 96% of untreated weights respectively (Fig. 1B). However, the body and tissue weights of iv treated mice were no different from untreated mice (Fig. 1B). Based on these results MPS IIIA mice were treated with iv NB-DNJ with treatment starting at four weeks (early treatment group) or four months (late treatment group) of age and continuing until mice were six months of age. 3.2. GSL synthesis was reduced by early and late NB-DNJ treatment but GSL levels were only reduced with late treatment A significant decrease in brain ceramide glucosyltransferase activity was observed in both early and late treatment groups compared to untreated MPS IIIA mice (41% and 22% reduction respectively; Fig. 2A), but did not differ significantly between the treatment groups. Brain GM1 levels did not differ between normal, untreated MPS IIIA and the early treatment group, but was significantly reduced in the late treatment group (Fig. 2B). GM2 was significantly raised to 475% of normal in untreated MPS IIIA, and while this did not change with early NB-DNJ treatment (461% of normal; Fig. 2C), it decreased significantly to 249% of normal in the brains of late treated MPS IIIA mice.

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GM3 was significantly elevated to 512% of normal in untreated MPS IIIA brains and this did not change significantly with early NB-DNJ treatment (484% of normal; Fig. 2D). GM3 brain levels were decreased by 235% in late treated MPS IIIA mouse brains, but this decrease did not reach statistical significance compared to untreated MPS IIIA levels. 3.3. Brain organic phase lipid levels and GAG levels were unchanged with NB-DNJ treatment In order to demonstrate that NB-DNJ treatment was specific to the inhibition of GSLs, analysis of brain organic phase lipids and glycosaminoglycans was performed. Simple, phospho- and glyco- lipids were separated by HPTLC and visualised using stains specific to each lipid type. No alteration in lipid level was observed between untreated normal mice, untreated MPS IIIA mice and either of the treatment groups (Fig. 3). Untreated, early and late treated MPS IIIA brain GAG levels were significantly higher than normal (333 ± 9%, 280 ± 36% and 336 ± 33% of normal, respectively) and no significant difference was noted between treatment groups and untreated MPS IIIA mice. 3.4. Behavioural changes were observed with NB-DNJ treatment of MPS IIIA mice. Activity in the open field test was determined at two, four and six months of age in the early treatment group and at six months of age

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in the late treatment group. Total distance travelled and rearing events were not significantly different to normal in untreated MPS IIIA mice and treatment had no effect (Fig. 4A and B). However, percentage time spent in the centre and percentage centre distance travelled was significantly greater in untreated MPS IIIA mice compared to normal mice from four months of age (307% and 202% of normal for time and distance, respectively), and this was significantly reduced with early NB-DNJ treatment (Fig. 4C and D). At six months of age, both percentage centre distance and percentage centre time for early and late treated MPS IIIA mice was significantly reduced compared with untreated MPS IIIA (Fig. 4C and D). However, the increase in untreated MPS IIIA centre distance over untreated normal was no longer significant (Fig. 4C)). Treatment of normal mice with NB-DNJ had no effect on activity in the open field. Spatial learning was assessed in the water cross-maze test at six months of age in both treatment groups and controls. Untreated MPS IIIA mice exhibited a significant decrease in correct entries (Fig. 5A), a significant increase in the number of incorrect entries (Fig. 5B) and a significant increase in escape latency (Fig. 5C) compared to untreated normal controls. All parameters returned to the normal range in both treatment groups. Treatment of normal mice with NB-DNJ had no effect on learning in the water cross-maze compared to untreated normal controls.

Fig. 4. Open field with NB-DNJ treatment. Mice were placed in the front left hand corner of the open field apparatus and distance travelled (A), rearing events (B), percentage of overall distance travelled in the central zone (C) and percentage of time spent in the central zone (D) were recorded. Results were expressed as mean ± standard error except for the treated normal group which is the average of 2 values. Normal (black bars), treated normal (grey bars), MPS IIIA (white bars) and early (diagonal lines) and late (horizontal lines) treated MPS IIIA. *Significant difference between groups, p b 0.05 (one-way ANOVA, Tukey's HSD).

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3.5. NB-DNJ treatment modulated brain inflammation An overall increase in inflammatory gene expression was observed in the MPS IIIA brain compared to normal (Fig. 6A). This increase was significant for Ccl3, Gfap, Cd68, Il1β and Tgfb1, which were 5.1, 5.5, 5.8, 3.9 and 1.5 fold greater than normal, respectively. In general there was a suppression of inflammatory gene expression with NB-DNJ treatment, which in many cases, was greater with early (longer term) than with late (short-term) treatment. Ccl3 expression in MPS IIIA brain decreased significantly with early (by 2.9 fold) but not late NB-DNJ treatment. Gfap expression in MPS IIIA brain was reduced with early and late treatment

(by 2.7 and 2.3 fold, respectively). Il1β expression in MPS IIIA brain was decreased significantly with both early and late NB-DNJ treatment (by 1.6 and 1.3 fold, respectively). Tgfb1expression in MPS IIIA brain was reduced with both early and late NB-DNJ treatment (by 0.9 and 0.4 fold, respectively). Cd68 expression in MPS IIIA brain was not affected by either early or late treatment. Astroglial activation was demonstrated with GFAP-targeted immunohistochemistry on formalin-fixed, frozen, sagittal sections of six month old normal, MPS IIIA and both early- and late- NB-DNJ treated MPS IIIA mouse brain. Very little GFAP staining was observed on normal brain sections (Fig. 6B and F). Untreated (Fig. 6C and G), early (Fig. 6D

Fig. 5. Water cross-maze with NB-DNJ treatment. At six months of age, mice were placed in a cross shaped pool filled with opaque water, and used constant visual cues to locate a submerged platform. Correct entries (A), incorrect entries (B) and escape latency (C) were calculated as a percentage of untreated normal values and expressed as mean ± standard error except for the treated normal group which is the average of 2 values. Treated normal (grey bars), MPS IIIA (white bars) and early (diagonal lines) and late (horizontal lines) treated MPS IIIA. *Significant difference from untreated normal, p b 0.05 (one-way ANOVA, Tukey's HSD).

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Fig. 6. Inflammatory gene expression and GFAP localisation with NB-DNJ treatment. Total RNA was extracted from normal, MPS IIIA (white bars) and both early (diagonal lines) and late treated (horizontal lines) MPS IIIA mouse brain tissue and used to make cDNA. Real time PCR was used to measure Ccl3, a Syn, Tnf, Tnfrsf1α, Gfap, Cd68, Il1β, Ifnγ, Tgfβ1 and Ctsb expression, which was normalised to Cypα expression levels (A). The fold change as compared to expression in normal brain was calculated and expressed as mean ± SEM. Significant differences from normal (*) or untreated MPS IIIA (#) are indicated, p b 0.05 (two-way ANOVA, Tukey's HSD). GFAP localisation was determined by specific antibody staining on formalin fixed frozen sections. Representative sections are shown for normal (B and F), untreated MPS IIIA (C and G), early treated MPS IIIA (D and H) and late treated MPS IIIA (E and I) for cerebral hemisphere and cerebellum sections, respectively. Merged images of DAPI and GFAP are shown at a magnification of 40×. Scale bar indicates 100 μm.

and H)- and late-treated (Fig. 6E and I) MPS IIIA brains all stained positively for GFAP in both the cerebellum and cerebral hemisphere. There appeared to be less GFAP staining in the cerebral hemisphere of both early- and late- treatment groups compare to untreated MPS IIIA, which was consistent with Gfap expression levels. In contrast the levels of GFAP staining in the cerebellum was not affected by either early or late treatment. 4. Discussion Inhibition of ganglioside synthesis using NB-DNJ is currently in clinical use for the non-neuronal form of Gaucher disease [22], in which ganglioside is the primary storage product in the lysosome. NB-DNJ is also prescribed for NPC patients who exhibit CNS symptoms [21], a lysosomal storage disorder in which ganglioside storage is secondary to cholesterol storage. In this study we assessed the efficacy of NB-DNJ to alter neurological pathology in murine MPS IIIA, another lysosomal storage disorder in which ganglioside is a secondary storage product, in this case to glycosaminoglycan. The modulation of neurological symptoms in NPC suggests that ganglioside targeted SDT may be effective in altering neuronal symptoms of MPS III. Typically, NB-DNJ is administered orally, however, we used iv administration in this study as much smaller amounts of drug could be used and gastrointestinal side effects and associated weight loss could

be avoided [17]. Comparable decreases in GM2 and GM3 ganglioside levels were observed with both routes of administration with a slightly greater decrease in GM1 levels observed with iv treatment and the iv route of administration was chosen for the in vivo study. This is the first report of intravenous administration of NB-DNJ in mice. NB-DNJ treatment was initiated at two different ages, representing different stages in ganglioside accumulation in the murine MPS IIIA brain. At one month of age MPS IIIA brain ganglioside accumulation has not reached its maximum level, with GM2 and GM3 levels at 64% and 60% of maximal values, respectively. At four months of age both GM2 and GM3 accumulation are at maximum levels (manuscript in preparation). Ceramide glucosyltransferase activity was reduced in both treatment groups confirming the efficacy of NB-DNJ. However, brain ganglioside levels were reduced only in the late treatment group. Together with the evidence that NB-DNJ reduces the amount of all gangliosides in the short term (Fig. 1), this suggests that the suppression of enzyme activity and reduction in ganglioside synthesis delays accumulation of ganglioside but that storage continues to increase with time. NB-DNJ treatment was specific to gangliosides as the level of GAG and other brain lipids was unaffected. Our observation that ganglioside levels were unchanged in the early treatment group is consistent with murine Sandhoff disease studies in which gangliosides were decreased with short term, but not with long term, NB-DNJ use [47–49] and murine NPC studies in which increased glucosylceramide levels were

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observed [50]. In addition to inhibiting the activity of ceramide glucosyltransferase activity which catalyses the first step in glycospingolipid synthesis, NB-DNJ also inhibits the activity of lysosomal β–glucosidase and non-lysosomal β–glucosidase 2 [51–53] the 2 enzymes that catalyse the reverse reaction. NB-DNJ can also act as a chaperone for mutated lysosomal β–glucosidase [54]. The long-term level of brain ganglioside in the presence of NB-DNJ will be dependent upon NB-DNJ concentration and its relative effect on synthetic and degradative enzymes and it maybe that the GSL metabolic cascade homeostasis is recovered after a period of inhibition by NB-DNJ. Despite the persistence of brain ganglioside levels, significant behavioural improvements were observed with NB-DNJ treatment in this study. Restoration of the innate fear response that causes mice to explore the peripheral zone of the apparatus preferentially over the central zone [55] was the most obvious change observed in treated MPS IIIA mice. The loss of the fear response has previously been shown to resolve in MPS IIIB mice with the administration of genistein, a GAG targeting SDT agent [8]. However, as decreasing GAG levels are also associated with a decrease in brain GM2 and GM3, it is unclear which of these stored substrates correlates to the loss of the fear response [8,56–58]. We have demonstrated that targeting gangliosides alone also increases mouse aversion to open spaces, supporting the contention that GM2 and GM3 contribute to this behaviour. Furthermore, improvements in the fear response of MPS IIIA-affected mice were observed in both early and late NB-DNJ treated animals, suggesting that ganglioside accumulation is reversible, independent of the extent of storage. In addition to the restoration of the innate fear response, both early and late NB-DNJ treated mice were indistinguishable from normal in learning the water cross-maze task suggesting that NB-DNJ could be effective in altering this aspect of CNS pathology in MPS patients. Notably, overall activity as measured by total distance travelled and rearing events in the open field test did not change with NB-DNJ treatment (Fig 5A and 5B). The lack of change in distance travelled is consistent with reports in NB-DNJ treated NPC and GM1 gangliosidosis mice, although rearing events increased in these latter mice [50,59]. Increases in overall ambulatory activity are only observed in Sandhoff mice treated with NB-DNJ and this appears to be age dependent [48,49,60] and it is unclear if this represents a delay in progression of disease pathology or alternatively hyperactivity as a result of NB-DNJ treatment. NB-DNJ has been shown to modulate inflammatory cytokine expression in LSD mouse models [17,59,61,62] and the inflammatory gene expression and astroglial activation data presented here supports the contention that MPS IIIA brain inflammation is reduced with NB-DNJ treatment. Both early and late treatment gave similar results, with early treatment producing some greater changes compared to late treatment. Cd68, expressed by monocytes, granulocytes and activated T-cells, was the only gene to be raised in the MPS IIIA brain and to be unchanged by either early or late NB-DNJ treatment, although NB-DNJ treatment has been demonstrated previously to decrease CD68 staining in the NPC mouse thalamus and Sandhoff mouse cerebellum and brainstem [47,50]. The reason for these differences may have been that the analysis of gene expression in this study combined different regions of the brain. This could be similar to previous studies in NPC where a significant reduction in CD68 was observed in NPC at 1.5 and 2 months of age but this was no longer significant at 2.5 months of age [50]. In addition to this, a recent study in MPS IIIB showed that administration of a fusion protein of NAGLU-IGFII decreased CD68 protein levels to 72% of normal levels but other markers were normalised [63]. This suggests that there is a different mechanism of microglial activation in different murine models of LSD which could contribute to the differences seen in CD68. Given the significant increase in MPS IIIA brain Gfap expression and its reduction with both early and late treatment, GFAP staining of sagittal brain sections was also conducted. This was consistent with gene expression, demonstrating reduced GFAP staining in the cerebral hemisphere in brains from treated mice. Reduced brain GFAP staining

with NB-DNJ treatment has also been shown previously in Sandhoff mice [47]. This again supports an anti-inflammatory role for NB-DNJ, in addition to its known role in the inhibition of ceramide glucosyltransferase. Thus, the anti-inflammatory role of NB-DNJ may have contributed to the behavioural improvements observed with treatment of MPS IIIA mice. A 12 month clinical trial in MPS III patients demonstrated the presence of NB-DNJ in patient cerebrospinal fluid (CSF), while no decrease in ganglioside levels or significant improvements in neurological symptoms were observed [29]. This is consistent with our results that ganglioside levels were reduced only in the late treatment group and only for GM2, suggesting a transient response that may have been missed in the human study. The accurate assessment of the effect of treatment on MPS patient brain disease is difficult. Patient behaviour, including hyperactivity, distractibility, aggression, anxiety and visual impairment, makes carrying out assessment procedures challenging [64]. The inclusion of patients of different ages, disease stages and enzyme deficiencies, with varying degrees of functional damage prior to the initiation of NB-DNJ treatment [29], further compounds the challenge of assessment. The MPS IIIA mouse provides a useful model for the determination of the effect of NB-DNJ on MPS IIIA behaviour, where patient assessment is difficult, and would allow further investigation into the dynamics of ganglioside reduction and delayed accumulation. Studies in mice are also possible before the onset of clinical symptoms to assess the NB-DNJ effect of treatment on prevention of disease (early treatment equivalent), and these can be compared to reversal of established pathology (late treatment equivalent). The lack of improvement seen in human studies could still be due to disease being too advanced before the initiation of treatment. The MPS IIIA mouse model retains 3% residual enzyme activity, meaning that it is possible that only mild-moderate severity patients would see in improvement in disease pathology in the clinic. The residual enzyme activity of patients was not reported in the human trial [29]. We have shown that NB-DNJ is effective in improving murine MPS IIIA learning ability and the innate fear response and that this is not dependent upon reduction in brain GAG levels. This suggests that it is worth pursuing NB-DNJ treatment of MPS patients with CNS involvement, despite poor treatment outcomes in the first clinical trial of this kind by Guffon et al. [29]. Questions arising from the clinical trial and MPS IIIA mouse study include the timeframe over which NB-DNJ remains effective at decreasing brain ganglioside levels. Whether enzyme activity and ganglioside levels can be further manipulated by an intermittent treatment regimen are important to our understanding of the potential role of NB-DNJ in the treatment of neurological symptoms of MPS. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ymgme.2016.04.002.

Acknowledgements This study was supported by grants from the National Health and Medical Research Council (565080) of Australia, The National MPS Society, and the Women's and Children's Hospital Research Foundation. A portion of the NB-DNJ used in this study was a gift from Actelion pharmaceuticals (Switzerland).

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