Enzyme Replacement Improves Ataxic Gait and Central Nervous System Histopathology in a Mouse Model of Metachromatic Leukodystrophy
Authors: Ulrich Matzner, Renate Lüllmann-Rauch, Stijn Stroobants, Claes Andersson, Cecilia Weigelt, Carl Eistrup, Jens Fogh, Rudi D'Hooge, Volkmar Gieselmann
Journal: Molecular Therapy (2009)

Inherited deficiencies of lysosomal hydrolases cause lysosomal storage diseases (LSDs) that are characterized by a progressive multisystemic pathology and premature death. Repeated intravenous injection of the active counterpart of the deficient enzyme, a treatment strategy called enzyme replacement therapy (ERT), evolved as a clinical option for several LSDs without central nervous system (CNS) involvement. To assess the efficacy of long-term ERT in metachromatic leukodystrophy (MLD), an LSD with prevailing nervous system disease, we treated immunotolerant arylsulfatase A (ASA) knockout mice with 52 doses of either 4 or 50 mg/kg recombinant human ASA (rhASA). ERT was tolerated without side effects and improved disease manifestations in a dose-dependent manner. Dosing of 4 mg/kg diminished sulfatide storage in kidney and peripheral nervous system (PNS) but not the CNS, whereas treatment with 50 mg/kg was also effective in the CNS in reducing storage in brain and spinal cord by 34 and 45%, respectively. Histological analyses revealed regional differences in sulfatide clearance. While 70% less storage profiles were detectable, for example, in the hippocampal fimbria, the histopathology of the brain stem was unchanged. Both enzyme doses normalized the ataxic gait of ASA knockout mice, demonstrating prevention of nervous system dysfunctions that dominate early stages of MLD. Inherited deficiencies of lysosomal hydrolases cause lysosomal storage diseases (LSDs) that are characterized by a progressive multisystemic pathology and premature death. Repeated intravenous injection of the active counterpart of the deficient enzyme, a treatment strategy called enzyme replacement therapy (ERT), evolved as a clinical option for several LSDs without central nervous system (CNS) involvement. To assess the efficacy of long-term ERT in metachromatic leukodystrophy (MLD), an LSD with prevailing nervous system disease, we treated immunotolerant arylsulfatase A (ASA) knockout mice with 52 doses of either 4 or 50 mg/kg recombinant human ASA (rhASA). ERT was tolerated without side effects and improved disease manifestations in a dose-dependent manner. Dosing of 4 mg/kg diminished sulfatide storage in kidney and peripheral nervous system (PNS) but not the CNS, whereas treatment with 50 mg/kg was also effective in the CNS in reducing storage in brain and spinal cord by 34 and 45%, respectively. Histological analyses revealed regional differences in sulfatide clearance. While 70% less storage profiles were detectable, for example, in the hippocampal fimbria, the histopathology of the brain stem was unchanged. Both enzyme doses normalized the ataxic gait of ASA knockout mice, demonstrating prevention of nervous system dysfunctions that dominate early stages of MLD. IntroductionThe posttranslational modification of newly synthesized soluble lysosomal enzymes involves the phosphorylation of N-linked oligosaccharide side chains at one or more mannosyl residues.1Kornfeld S Mellman I The biogenesis of lysosomes.Annu Rev Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1232) Google Scholar The resulting mannose 6-phosphate (M6P) residues bind to mannose 6-phosphate receptors (MPRs) localized to the trans Golgi network. This interaction separates lysosomal enzymes from the secretory route and redirects them to the endosomal/lysosomal targeting pathway. MPRs also reside within the plasma membrane where they can bind extracellular ligands exposing M6P recognition markers. As a consequence, exogenous lysosomal enzymes are internalized and transported to the endosomal/lysosomal compartment as well. In case of an absence or inherited functional deficiency of a lysosomal enzyme, a condition leading to a fatal lysosomal storage disease (LSD), this peculiarity of the lysosomal sorting process can be exploited for treatment. The basic treatment concept is to supply the deficient cells with the active counterpart of the mutated enzyme from outside. In enzyme replacement therapy (ERT) this is accomplished by repeated injection of recombinantly expressed enzyme into the circulatory system (for a review see ref. 2Brady RO Schiffmann R Enzyme-replacement therapy for metabolic storage disorders.Lancet Neurol. 2004; 3: 752-756Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Upon cellular uptake and delivery to the lysosome, the substituted enzyme becomes functionally integrated into the lysosomal degradation pathways, hydrolyzes the accumulated substrate(s), and thereby compensates the catabolic defect.Preclinical and clinical ERT studies revealed that efficient treatment can be compromised by a variety of factors. These include an immune response to the therapeutic enzyme,3Matzner U Matthes F Weigelt C Andersson C Eistrup C Fogh J et al.Non-inhibitory antibodies impede lysosomal storage reduction during enzyme replacement therapy of a lysosomal storage disease.J Mol Med. 2008; 86: 433-442Crossref PubMed Scopus (29) Google Scholar,4Wang J Lozier J Johnson G Kirshner S Verthelyi D Pariser A et al.Neutralizing antibodies to therapeutic enzymes: considerations for testing, prevention and treatment.Nat Biotechnol. 2008; 26: 901-908Crossref PubMed Scopus (126) Google Scholar the persistance of already established cellular degeneration and organ dysfunctions,5Walkley SU Neurobiology and cellular pathogenesis of glycolipid storage diseases.Philos Trans R Soc Lond B Biol Sci. 2003; 358: 893-904Crossref PubMed Scopus (44) Google Scholar,6Hollak CE Vedder AC Linthorst GE Aerts JM Novel therapeutic targets for the treatment of Fabry disease.Expert Opin Ther Targets. 2007; 11: 821-833Crossref PubMed Scopus (16) Google Scholar inefficient cellular uptake of substituted enzyme,7Raben N Danon M Gilbert AL Dwivedi S Collins B et al.Enzyme replacement therapy in the mouse model of Pompe disease.Mol Genet Metab. 2003; 80: 159-169Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar and a poor accessibility of affected tissues.8Prows CA Sanchez N Daugherty C Grabowski GA Gaucher disease: enzyme therapy in the acute neuronopathic variant.Am J Med Genet. 1997; 71: 16-21Crossref PubMed Scopus (76) Google Scholar In the latter regard, the blood-brain barrier (BBB) is a major issue because it prevents efficient transfer of systemically administered enzyme from the circulation to the brain parenchyma. Due to the low permeability of the BBB for lysosomal enzymes, the central nervous system (CNS) disease that prevails in the majority of LSDs, has been believed to be resistant to ERT.This view has been challenged by recent preclinical ERT studies demonstrating clearance of CNS storage in mouse models of aspartylglucosaminuria,9Dunder U Kaartinen V Valtonen P Väänänen E Kosma VM Heisterkamp N et al.Enzyme replacement therapy in a mouse model of aspartylglycosaminuria.FASEB J. 2000; 14: 361-367Crossref PubMed Scopus (57) Google Scholar α-mannosidosis,10Roces DP Lüllmann-Rauch R Peng J Balducci C Andersson C Tollersrud O et al.Efficacy of enzyme replacement therapy in alpha-mannosidosis mice: a preclinical animal study.Hum Mol Genet. 2004; 13: 1979-1988Crossref PubMed Scopus (79) Google Scholar,11Blanz J Stroobants S Lüllmann-Rauch R Morelle W Lüdemann M D'Hooge R et al.Reversal of peripheral and central neural storage and ataxia after recombinant enzyme replacement therapy in alpha-mannosidosis mice.Hum Mol Genet. 2008; 17: 3437-3445Crossref PubMed Scopus (54) Google Scholar and Sly disease.12Vogler C Levy B Grubb JH Galvin N Tan Y Kakkis E et al.Overcoming the blood-brain barrier with high-dose enzyme replacement therapy in murine mucopolysaccharidosis VII.Proc Natl Acad Sci USA. 2005; 102: 14777-14782Crossref PubMed Scopus (154) Google Scholar In all three models, ERT led to significant levels of therapeutic enzyme in brain. It is still unclear which parameters favor the transfer of enzyme into the CNS of these animals. ERT also alleviated CNS storage of an arylsulfatase A (ASA) knockout mouse model of metachromatic leukodystrophy (MLD).13Matzner U Herbst E Hedayati KK Lüllmann-Rauch R Wessig C Schröder S et al.Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy.Hum Mol Genet. 2005; 14: 1139-1152Crossref PubMed Scopus (128) Google Scholar Due to the significant improvement of the nervous system pathology and function, the main site of disease in MLD,14von Figura K Gieselmann V Jaeken J Metachromatic leukodystrophy. In: Scriver, CR, Beaudet, AL, Sly, WS, Valle, D, Childs, B, Kinzler, KW, Vogelstein, B (eds). The Metabolic and Molecular Bases of Inherited Disease. Mc Graw-Hill, New York2001: 3695-3724Google Scholar ERT was suggested as a viable treatment option for the human disorder. Thus far, the determination of the full therapeutic potential of ERT in mice was, however, prevented by a progressive immune response to the repeatedly injected human enzyme.3Matzner U Matthes F Weigelt C Andersson C Eistrup C Fogh J et al.Non-inhibitory antibodies impede lysosomal storage reduction during enzyme replacement therapy of a lysosomal storage disease.J Mol Med. 2008; 86: 433-442Crossref PubMed Scopus (29) Google Scholar To circumvent this limitation of the conventional ASA knockout mouse, we have recently constructed an immunotolerant ASA knockout mouse line that does not express antibodies to repeatedly injected recombinant human ASA (rhASA) due to lifelong expression of an active site mutant of human ASA from a stably integrated transgene.15Matzner U Matthes F Herbst E Lüllmann-Rauch R Callaerts-Vegh Z D'Hooge R et al.Induction of tolerance to human arylsulfatase a in a mouse model of metachromatic leukodystrophy.Mol Med. 2007; 13: 471-479Crossref PubMed Google Scholar This study was designed to evaluate the full therapeutic potential of ERT for ASA knockout mice by treating 6–7-week-old immunotolerant ASA knockout mice 2 times a week for 26 weeks (52 injections). To evaluate a possible dose dependency of therapeutic effects, we treated two cohorts of mice: One was treated with a lower enzyme dose (4 mg/kg) and another with a higher dose (50 mg/kg).ResultsAbsence of side effectsSix-to-seven-week-old mice (26 per group) were treated for 26 weeks with two weekly bolus injections of either 4 or 50 mg/kg rhASA into the tail vein. Controls were mock treated by injection of buffer. Possible side effects were monitored by visual inspection of cage behavior, food and water consumption, and measurement of body weight. During treatment weeks 13 and 26, hematological parameters (hemoglobin concentration, corpuscular indexes, blood chemistry, coagulation) and urinary parameters (pH, specific gravity, concentration of analytes) were analyzed. After necropsy organs were dissected, weighed, and histologically analyzed. None of the evaluated parameters showed any alteration compared to mock-treated controls (not shown). Most important, hypersensitivity reactions and mortality, which limited the treatment of conventional ASA knockout mice to 4 weeks,13Matzner U Herbst E Hedayati KK Lüllmann-Rauch R Wessig C Schröder S et al.Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy.Hum Mol Genet. 2005; 14: 1139-1152Crossref PubMed Scopus (128) Google Scholar were absent throughout the entire treatment period. Anti-rhASA antibodies were undetectable in all but 2 animals that showed low, but significant titers (not shown), and were excluded from subsequent analyses.Lipid analysisCholesterol, sphingomyelin, and sulfatide levels of kidney, peripheral nerves (plexus brachialis, nervus ischiadicus), spinal cord, and total brain were analyzed by thin layer chromatography (TLC). To quantify sulfatide reduction, ASA knockout mice treated with 4 or 50 mg/kg rhASA were compared with age-matched mock-treated knockout mice and untreated wild-type mice. TLC revealed significant differences in the sulfatide concentrations between mock-treated ASA knockout mice and wild-type controls. Thus, sulfatide levels were increased around 1.4-fold in peripheral nervous system (PNS) and CNS and >15-fold in kidney (Figure 1). Compared to mock treatment with buffer, treatment with 4 mg/kg rhASA reduced excess sulfatide in kidneys of ASA knockout mice by 21% (Figure 1a) and in peripheral nerves by 50 to 62% (Figure 1b,c) on average. In contrast to peripheral tissues, no significant decline was detectable in the CNS (Figure 1d,e). Treatment with 50 mg/kg rhASA was more effective than low-dose treatment in all tissues under investigation. It diminished excess sulfatide in kidney by 50% (Figure 1a) and in peripheral nerves by 63 to 84% (Figure 1b,c). Most important, the mean sulfatide storage was also significantly reduced in total brain and spinal cord where 34 and 45% of excess sulfatide had disappeared, respectively (Figure 1d,e).Histological investigationsSulfatide storage patterns were analyzed in kidney and several parts of the CNS (inner ear, spinal cord, brain stem, hippocampal fimbria) from three ASA knockout mice treated with 50 mg/kg rhASA, three mock-treated ASA knockout mice, and one age-matched wild-type mouse. Wild-type organs showed virtually no alcianophilic structures (shown for kidney in Figure 2c). Because TLC had revealed no reduction of sulfatide levels in the CNS of low-dose–treated mice, mice treated with 4 mg/kg were not histologically examined.Figure 2Sulfatide storage in kidney. (a,b) 100 µm slices through corresponding regions of kidneys from arylsulfatase A (ASA) knockout mice that were either mock treated with buffer (a) or enzyme treated with 50 mg/kg recombinant human ASA (b). Representative examples are shown of descending thin limbs (DTLs) of long loops in the inner stripe of outer medulla. Sulfatide storage material appears blue. (c–e) Semithin sections through DTLs and thick ascending limbs (TALs) stained with toluidine blue that stains storage material dark blue to purple. Corresponding regions of a wild-type control mouse (c), a mock-treated ASA knockout mouse (d), and an ASA knockout mouse treated with 50 mg/kg recombinant human ASA (e) are shown. Bars correspond to 20 µm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the kidneys of mock-treated ASA knockout mice the distribution of alcianophilic material was the same as described previously16Lüllmann-Rauch R Matzner U Franken S Hartmann D Gieselmann V Lysosomal sulfoglycolipid storage in the kidneys of mice deficient for arylsulfatase A (ASA) and of double-knockout mice deficient for ASA and galactosylceramide synthase.Histochem Cell Biol. 2001; 116: 161-169Crossref PubMed Scopus (25) Google Scholar with the thick ascending limbs (TALs) and the descending thin limbs of long loops being the most severely affected parts of the nephron. In the enzyme-treated ASA knockout mice a clear reduction of alcianophilic material was observed in the descending thin limb (Figure 2). In the TAL such a reduction was not obvious.In 100 µm slices from the CNS of the present knockout mice several populations of alcianophilic profiles were observed. The larger ones usually corresponded to sulfatide-storing neuronal perikarya (shown for spinal cord in Figure 3b,d). Large sulfatide-storing microglial cells (phagocytes), which are a regular occurrence in the CNS of 13–24-month-old ASA knockouts,13Matzner U Herbst E Hedayati KK Lüllmann-Rauch R Wessig C Schröder S et al.Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy.Hum Mol Genet. 2005; 14: 1139-1152Crossref PubMed Scopus (128) Google Scholar,17Wittke D Hartmann D Gieselmann V Lüllmann-Rauch R Lysosomal sulfatide storage in the brain of arylsulfatase A-deficient mice: cellular alterations and topographic distribution.Acta Neuropathol. 2004; 108: 261-271Crossref PubMed Scopus (52) Google Scholar were not seen in the present 7-month-old knockout mice, except for the hippocampal fimbria as described below. The smaller alcianophilic profiles, according to the earlier experience with ASA knockouts, most likely corresponded mainly to oligodendrocytes. In the spinal cord of rhASA-treated knockout mice, the overall density of alcianophilic particles appears to be reduced particularly in the ventral horn and in the white matter (Figure 3a,c). This could be verified by morphometric evaluation, which revealed a significant reduction of the mean number of storage profiles by 22% (P = 0.011; Figure 4a). The brain stem was examined at a defined level (Figure 3e–h) comprising the vestibular nuclei, lateral cerebellar nucleus, the intramedullary portion of the facial nerve, and the ventral cochlear nucleus. Alcianophilic material was mainly seen in neuronal perikarya (Figure 3f,h). Enzyme treatment failed to yield an obvious reduction of the storage material. The hippocampal fimbria (Figure 3i–l) is one of the central white matter regions where, according to previous experience, large alcianophilic phagocytes occur already in relatively young ASA knockout mice. Therefore, this portion was investigated in order to test whether or not enzyme treatment had an influence on the sulfatide storage within these cells. In 5 µm paraffin sections, large profiles corresponding to sulfatide-storing phagocytes and smaller profiles corresponding to oligodendrocytes were visible (Figure 3i,j). After enzyme treatment the number of large and small alcianophilic particles appeared to be reduced. Morphometric evaluation confirmed the histological observations by demonstrating 73% less alcianophilic profiles in ERT-treated mice (P = 0.00006; Figure 4b). It has to be mentioned that some large profiles typical of phagocytes, albeit significantly reduced in number, were still detectable after treatment (Figure 3l).Figure 3Sulfatide storage in the central nervous system. Corresponding sections from mock-treated arylsulfatase A (ASA) knockout mice (mock) and knockout mice treated with 50 mg/kg recombinant human ASA (ERT) are compared at low (left panel) and high resolution (right panel). Bars correspond to 100 (left panel) amd 50 µm (right panel), respectively. (a–d) Spinal cord. 100-µm slices (a,c) and paraffin sections (b,d). The large particles in the gray matter are seen to correspond to sulfatide-storing neuronal perikarya (arrows). (e–h) Brain stem (100-µm slices). The vestibular (Ve) and the lateral cerebellar nucleus (CbN) are positively stained due to sulfatide storage in the neurons as seen at higher resolution. (i–l) Hippocampal fimbria (paraffin sections).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Morphometric analyses of sulfatide storage in the central nervous system of mock-treated arylsulfatase A (ASA) knockout mice (ASA–/– mock), ASA knockout mice treated with 50 mg/kg recombinant human ASA (ASA–/– 50 mg/kg), and wild-type control mice (ASA+/+) are compared. Bars represent means ± SDs. Statistically significant differences between mock- and enzyme-treated ASA knockout mice are indicated by asterices (for P values see text). (a) Number of alcianophilic profiles in the gray matter of the cervical spinal cord per test area (40,000 µm2), n = 3. (b) Number of alcianophilic profiles in the hippocampal fimbria per test area (40,000 µm2), n = 6. (c) Number of ganglion cells in the spiral ganglion per test area (10,000 µm2), n = 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In the inner ear of ASA knockout mice the main features are severe sulfatide storage in the perikarya of the spiral and vestibular ganglia and rapid loss of spiral ganglion cells between the 4th and 8th month of life.18D'Hooge R Coenen R Gieselmann V Lüllmann-Rauch R De Deyn PP Decline in brainstem auditory-evoked potentials coincides with loss of spiral ganglion cells in arylsulfatase A-deficient mice.Brain Res. 1999; 847: 352-356Crossref PubMed Scopus (27) Google Scholar,19Coenen R Gieselmann V Lüllmann-Rauch R Morphological alterations in the inner ear of the arylsulfatase A-deficient mouse.Acta Neuropathol. 2001; 101: 491-498PubMed Google Scholar In this study, the cochleae were examined in 5 µm paraffin sections (preembedding incubation with alcian blue) and the density of spiral ganglion cells related to the basal turn was evaluated quantitatively. The findings were similar as in previous studies. There was no clear difference between mock- and enzyme-treated mice as far as sulfatide storage and loss of ganglion cells are concerned (Figure 4c).Behavioral studiesASA knockout mice develop progressive motor coordination impairments and ataxia leading to severe behavioral deficits in the second year of life.20Hess B Saftig P Hartmann D Coenen R Lüllmann-Rauch R Goebel HH et al.Phenotype of arylsulfatase A-deficient mice: relationship to human metachromatic leukodystrophy.Proc Natl Acad Sci USA. 1996; 93: 14821-14826Crossref PubMed Scopus (202) Google Scholar,21D'Hooge R Hartmann D Manil J Colin F Gieselmann V De Deyn PP Neuromotor alterations and cerebellar deficits in aged arylsulfatase A-deficient transgenic mice.Neurosci Lett. 1999; 273: 93-96Crossref PubMed Scopus (47) Google Scholar,22D'Hooge R Van Dam D Franck F Gieselmann V De Deyn PP Hyperactivity, neuromotor defects, and impaired learning and memory in a mouse model for metachromatic leukodystrophy.Brain Res. 2001; 907: 35-43Crossref PubMed Scopus (39) Google Scholar To determine genotype- and treatment-related effects in the 7-month-old mice of this study, ataxia and other manifestations of motor incoordination were assessed with treadmill and swimming performance tests in immunotolerant ASA knockout mice treated with 4 or 50 mg rhASA/kg, mock-treated knockout mice, and wild-type controls (6 mice per group).In treadmill experiments mock-treated ASA knockout mice showed impaired gait relative to wild-type mice in all test conditions as indicated by the requirement of earlier or more frequent stimulation (Figure 5a,b). Compared to mock-treated controls, performance of low- and high-dose–treated mice was substantially improved and virtually indistinguishable from the performance of wild-type mice. In the four test trials, for example, mock-treated ASA knockout mice received 5.8-foot shocks on average, whereas 2.2, 2.4, and 2.7 shocks were sufficient for low- and high-dose–treated mice and wild-type controls, respectively (Figure 5b). This indicated a large number of errors in the mock-treated ASA knockouts compared to the other groups. Post hoc analysis revealed significantly improved performance in low- (P < 0.01) and high-dose–treated ASA knockout mice (P < 0.05) compared to mock-treated ASA knockout mice.Figure 5Behavioral analysis of treated mice and controls. (a) Treadmill experiment: latency to the second footshock in the training phase (velocity 19 cm/s, slope 5 °). The differences between the experimental groups were not significant. Open circle: mock-treated arylsulfatase A (ASA) knockout mice, open diamond: ASA knockout mice treated with 4 mg/kg recombinant human (rhASA), open square: ASA knockout mice treated with 50 mg/kg rhASA, and closed triangle: wild-type mice. Data points represent means of n = 6 animals per group. (b) Treadmill experiment: number of foot shocks in the test phase at different combinations of velocity and slope as indicated (same symbols and group sizes as in a). The statistical analysis revealed significant differences between mock- and enzyme-treated animals (see text). (c) Stride lengths of mock-treated ASA knockout mice (open bars) and wild-type mice (closed bars) measured at different combinations of velocity and slope. For each mouse and condition, the stride lengths of the four paws was averaged. Bars represent means ± SDs of n = 6 mice per group. (d) Stride lengths of individual paws at a treadmill velocity of 22 cm/s and a slope of 10 °. LF: left front paw, RF: right front paw, LH: left hind paw, RH: right hind paw, open bar: mock-treated ASA knockout mice, diagonally hatched bars: ASA knockout mice treated with 4 mg/kg rhASA, horizontally hatched bars: ASA knockout mice treated with 50 mg/kg rhASA, and closed bars: wild-type mice. Bars represent means ± SDs of n = 6 mice per group. (e) Standard deviations of the stride lengths measured at a velocity of 16 cm/s and a slope of 0 ° (same abbreviations, symbols, and group sizes as in d). (f) Mean swimming velocity during 100 s of free swimming. Bars represent means ± SDs of n = 6 mice per group. *P = 0.05, **P < 0.01 (compared to mock-treated knockout mice).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The analysis of the gait pattern revealed increased stride lengths for mock-treated ASA knockout mice, particularly, when they were challenged to walk fast and/or uphill (Figure 5c). Treatment with 4 and 50 mg/kg rhASA led to a normalization of the mean stride lengths (Figure 5d). However, despite strong trends the differences did not reach statistical significance for any of the four combinations of velocity and slope. When the stride lengths measured under the four conditions were combined, the stride lengths of the right hind paw was significantly reduced (P < 0.05).Also the variation of the stride lengths is increased in mock-treated ASA knockout mice when compared to wild-type controls. In contrast to the absolute stride lengths, the stride lengths variation is, however, highest during horizontal walking at low speed (16 cm/s and 0 °). Under these conditions, the mean value for the four paws of mock-treated ASA knockout mice was increased by 38% compared to wild-type controls (Figure 5e). Treatment with 4 or 50 mg/kg rhASA led to a reduction of the increased stride length variation to near normal values (Figure 5e). When the values measured at the four different conditions were combined, mock-treated ASA knockout mice showed significantly increased standard deviations of stride lengths for all paws (P < 0.01 or 0.05). High-dose–treated ASA knockout mice displayed a significantly decreased standard deviations of the left hind paw stride length in comparison to mock-treated controls (P < 0.05). For the other paws strong trends for reduced variation were detectable.The correlation of left front/hind distance to right front/hind distance was taken as a second and independent measure for the uniformity of the gait. The correlation coefficient is high if the distance between the print of the left front paw and the following placement of the left hind paw is very similar to the analogous distance between the right front and hind paw prints. As a consequence of their gait abnormalities, most combinations of velocity and slope revealed decreased correlation values for mock-treated ASA knockout mice compared to wild-type controls (Table 1). Treatment with 50 mg/kg led to an increase of all correlation coefficients. In contrast to high-dose treatment, no clear effect of low-dose treatment was detectable at any condition.Table 1Correlation of right to left front/hind distances depending on trial conditionsASA-/-ASA-/-ASA-/-ASA+/+Mock4 mg/kg50 mg/kg16 cm/s and 0 °aVelocity and slope.0.380.250.83*Velocity and slope.bValues >0.8 are printed in bold. Asterices indicate significance:0.91*Velocity and slope.16 cm/s and 10 °0.770.750.85*P < 0.05,0.7322 cm/s and 0 °0.530.650.790.92**P < 0.01.22 cm/s and 10 °-0.110.310.600.95**Values >0.8 are printed in bold. Asterices indicate significance:Abbreviations: ASA-/-, arylsulfatase A knockout mouse; ASA+/+, ASA wild-type control.a Velocity and slope.b Values >0.8 are printed in bold. Asterices indicate significance:* P < 0.05,** P < 0.01. Open table in a new tab Mock-treated ASA knockout mice swam significantly slower than wild-type controls (Figure 5f, P < 0.01). On average, the velocity of mock-treated ASA knockout mice was reduced by 26%. The mean velocity was increased by 6 and 20% after low- and high-dose treatment, respectively. The difference between mock-treated and high-dose–treated ASA knockout mice was at the edge of statistical significance (P = 0.05).DiscussionWe have shown in a previous proof-of-concept study that short-term ERT of conventional ASA knockout mice with a dose of 20 mg/kg rhASA improves the nervous system histopathology and function.13Matzner U Herbst E Hedayati KK Lüllmann-Rauch R Wessig C Schröder S et al.Enzyme replacement improves nervous system pathology and function in a mouse model for metachromatic leukodystrophy.Hum Mol Genet. 2005; 14: 1139-1152Crossref PubMed Scopus (128) Google Scholar In these trials the number of injections was limited to four because mice developed a strong immunological response to the rhASA. In the present study we used immunotolerant mice that allow to assess the benefits of long-term treatment. In addition, to reveal possible dose-dependent effects, we chose to treat young presymptomatic mice with two clearly distinct enzyme doses of 4 and 50 mg/kg body weight two times a week for 26 weeks, respectively.As determined by TLC, treatment with 50 mg rhASA/kg led to a substantial reduction of excess sulfatide in all tissues analyzed (Figure 1). Most important, sulfatide storage was reduced by up to 84% in the PNS and by up to 45% in