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Human Molecular Genetics Advance Access originally published online on August 19, 2008
Human Molecular Genetics 2008 17(22):3437-3445; doi:10.1093/hmg/ddn237
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Reversal of peripheral and central neural storage and ataxia after recombinant enzyme replacement therapy in {alpha}-mannosidosis mice

Judith Blanz1, Stijn Stroobants3, Renate Lüllmann-Rauch2, Willy Morelle4, Meike Lüdemann1, Rudi D'Hooge3, Helena Reuterwall5, Jean Claude Michalski4, Jens Fogh5, Claes Andersson5 and Paul Saftig1,*

1 Biochemical Institute 2 Anatomical Institute, University of Kiel, D-24098 Kiel, Germany 3 Laboratory of Biological Psychology, University of Leuven, B-3000 Leuven, Belgium 4 Unité Mixte de Recherche CNRS/USTL 8576, Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, France 5 Zymenex A/S, Roskildevej 12C, 3400 Hillerød, Denmark

* To whom correspondence should be addressed at: Biochemical Institute, Christian-Albrechts-University Kiel, Olshausenstr. 40, D-24098 Kiel, Germany. Tel: +49 4318802216; Fax: +49 4318802238; Email: psaftig{at}biochem.uni-kiel.de

Received July 3, 2008; Accepted August 7, 2008


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Despite the progress in the treatment of lysosomal storage disorders (LSDs) mainly by enzyme replacement therapy, only limited success was reported in targeting the appropriate lysosomal enzyme into the brain. This prevents efficient clearance of neuronal storage, which is present in many of these disorders including {alpha}-mannosidosis. Here we show that the neuropathology of a mouse model for {alpha}-mannosidosis can be efficiently treated using recombinant human {alpha}-mannosidase (rhLAMAN). After intravenous administration of different doses (25–500 U/kg), rhLAMAN was widely distributed among tissues, and immunohistochemistry revealed lysosomal delivery of the injected enzyme. Whereas low doses (25 U/kg) led to a significant clearance (<70%) in visceral tissues, higher doses were needed for a clear effect in central and peripheral nervous tissues. A distinct reduction (<50%) of brain storage required repeated high-dose injections (500 U/kg), whereas lower doses (250 U/kg) were sufficient for clearance of stored substrates in peripheral neurons of the trigeminal ganglion. Successful transfer across the blood-brain barrier was evident as the injected enzyme was found in hippocampal neurons, leading to a nearly complete disappearance of storage vacuoles. Importantly, the decrease in neuronal storage in the brain correlated with an improvement of the neuromotor disabilities found in untreated {alpha}-mannosidosis mice. Uptake of rhLAMAN seems to be independent of mannose-6-phosphate receptors, which is consistent with the low phosphorylation profile of the enzyme. These data suggest that high-dose injections of low phosphorylated enzymes might be an interesting option to efficiently treat LSDs with CNS involvement.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
{alpha}-Mannosidosis is an incurable lysosomal storage disorder (LSD) with autosomal recessive inheritance. It is caused by mutations in the lysosomal enzyme {alpha}-mannosidase (LAMAN), which is responsible for the lysosomal degradation of {alpha}1–2, {alpha}1–3 and {alpha}1–6 linked mannosides present in complex N-linked glycans (1,2). Mutations in LAMAN are heterogenous and affect enzyme activity or lysosomal sorting (3), leading to a dysfunction of this protein. Loss of LAMAN activity results in intralysosomal storage of neutral oligosaccharides carrying different mannosyl residues (Mannose2–Mannose9) in most visceral organs and in neurons of the peripheral (PNS) and central nervous system (CNS) (4,5). The symptoms of {alpha}-mannosidosis are heterogenous and characterized by hepatosplenomegaly, recurrent infections, impaired hearing, dysostosis multiplex and progressive mental retardation (4,5).

In the past, several approaches have been suggested for LSD treatment, including enzyme replacement therapy (ERT), bone marrow transplantation (BMT) and substrate reduction therapy. Despite the high cost and low permeability of the blood-brain barrier to lysosomal enzymes (6,7), ERT may remain the best therapeutic option for an effective treatment of many LSDs, possibly including {alpha}-mannosidosis. BMT effectively treated the visceral and neuronal pathology in {alpha}-mannosidosis cats (6), but showed varying efficacy and high mortality rate in {alpha}-mannosidosis patients (7–9).

Recently, we could show that ERT with human, murine and bovine recombinant LAMAN preparations was effective in the treatment of the visceral pathology of {alpha}-mannosidosis mice (10) that were obtained by targeted disruption of the mouse LAMAN gene (11). The human enzyme (rhLAMAN) proved to be most effective, and the first experiments suggested that high-dose injections of enzyme might even reduce neuronal storage in the brain (10,12–14). This promising effect prompted us to further investigate the mechanism of rhLAMAN entry in cells especially in those of the CNS, as well as the actual preclinical efficacy of ERT in {alpha}-mannosidosis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Effect on storage in visceral organs upon injections of different doses rhLAMAN
The suitable dosage of enzyme is a critical issue in ERT. In order to develop an effective therapy for {alpha}-mannosidosis patients, we investigated the effect on tissue carbohydrate storage after injection of different doses of recombinant human LAMAN (rhLAMAN) in adult {alpha}-mannosidosis mice. Therefore, mice were injected twice a week (interval 3.5 days) with phosphate-buffered saline (PBS) (mock treated) or 25, 100, 250 and 500 U rhLAMAN/kg body weight. Three to four days after the last injection, mice were perfused and the spleen, kidney, liver and brain were analysed for their carbohydrate storage. Thin layer chromatography (TLC) of sugar extracts from the brain showed no significant reduction of carbohydrate storage under this regimen (Fig. 1A), whereas in peripheral tissues like kidney (Fig. lA), spleen (Fig. lA) and liver (data not shown), low doses (25 U/kg) led to a major reduction of mannosyl-linked oligosaccharides (M2–M9). HPLC-based quantitative analyses (Supplementary Material, Fig. S1) revealed a more dose-dependent clearance in these tissues than visible by TLC. High doses (500 U/kg) were needed for a nearly complete reduction (>90%) of sugar storage. Ultrastructural analyses also displayed a dose-dependent decline in the number of storage vacuoles, which accumulate in cells of mock-treated knockout mice. After ERT, most of the storage vacuoles were absent, as demonstrated in the pancreas (Fig. 1B). To analyse the effect of intralysosomal storage on lysosomal function (15), we compared the expression of LAMP1 and the activity of the lysosomal hydrolase β-hexosaminidase in tissue extracts from wild-type and mock- and ERT-treated knockout mice. In comparison with wild-type, the liver, kidney and spleen of mock-treated knockouts displayed increased LAMP1 expression (Fig. 1C) and β-hexosaminidase activity (Fig. 1D). After ERT, the expression of both proteins was reduced, indicating a normalization of lysosomal function upon clearance of sugar storage.


Figure 1
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  		Figure 1. Lysosomal uptake, lysosomal normalization and reduction of storage after ERT with rhLAMAN. (A) TLC of neutral mannosyl-linked oligosaccharides (M2–M9) in tissues of KO mice injected 7 and 3.5 days prior to killing with different doses of rhLAMAN (25–500 U/kg; each dose n = 2). Mock (PBS) treated WT (n = 2) and KO mice (n = 2) served as controls. (B) Light microscopy displayed numerous storage vacuoles (arrowhead) in azinar cells (AZ) of the pancreas from mock but not ERT (2 x 500 U) treated KO mice. (C) Western blot analysis from tissue extracts (spleen, liver, kidney) from mock-treated WT and KO mice (n = 2, each group) or ERT (2 x 500 U/kg) treated KO mice (n = 2). In comparison with WT, LAMP1 is upregulated in mock- but not in ERT-treated KO mice. Actin staining served as a loading control (D) Measurement of β-hexoaminidase (β-hex) activity in tissue extracts. β-hex activity is upregulated in the liver, kidney and spleen of mock-treated KO mice. After ERT, β-hex levels are normalized. (E and F) Fluorescence microscopy of spleen sections (50 µm; free floating) from mock and ERT (2 x 500 U/kg) treated KO mice stained with an antibody specific for human LAMAN. LAMAN was found mainly in macrophages of the red pulpa (rP) where it colocalizes (inset) with LAMP1 (red). The absence of signal in mock-treated KO tissue indicates the specificity of the antibody. Nuclei are stained in blue (DAPI). KO_ERT=ERT-treated knockout mice; WT=mock-treated wild-type mice; KO*=mock-treated knockout mice. Scale bars: (B) 50 µm; (E and F) 200 µm.

 
Cellular localization of rhLAMAN in peripheral tissues
To demonstrate efficient cellular uptake and lysosomal delivery of rhLAMAN, we performed immunohistochemistry on tissue sections of mock- and ERT-treated knockout mice. Therefore, mice were injected twice with 500 U/kg rhLAMAN and analysed 3.5 days after the last injection as endogenous levels of LAMAN activity were reached by this experimental set-up at least in the spleen (76.8% of WT level) and liver (96.1% of WT level) (Supplementary Material, Fig. S2). Using an antibody specific for rhLAMAN (10) (green) and the lysosomal marker protein LAMP1 (red), we demonstrated cellular uptake and lysosomal uptake in the spleen (Fig. 1E and F), liver and kidney (Supplementary Material, Fig. S3). In the spleen, rhLAMAN was localized mainly in the macrophages of the red pulpa, which are highly positive for LAMP1. In the liver, the enzyme was concentrated in sinus endothelial cells. Less enzyme was visible in Kupffer cells and hepatocytes. In the kidney (Supplementary Material, Fig. S3), the enzyme was found in glomeruli and in tubular structures such as the thick ascending limb where storage disappeared after ERT (Supplementary Material, Fig. S4).

Overcoming the blood-brain barrier with frequent high-dose treatment
After having evaluated the uptake and the benefit of rhLAMAN in visceral organs, we were interested in finding suitable conditions to efficiently cure {alpha}-mannosidosis mice of their lysosomal storage in the nervous system. As two injections of high doses did not reveal an obvious reduction of brain storage (Fig. 1A), we increased the frequency and injected mice four times with 250 U/kg and 500 U/kg, respectively. Central (brain) and peripheral nervous tissue (trigeminal ganglion) were then analysed for storage by chromatographical and histological methods. TLC analysis revealed a complete clearance in the tG after ERT with 250 U/kg (Fig. 2A), whereas in the brain, higher doses (500 U/kg) were needed for an obvious effect (Fig. 2B). After ERT with 500 U/kg, low levels of LAMAN activity (14.8% of WT level) were measured in brain lysates of injected knockout mice (Fig. 2C). Like in peripheral tissues (Fig. 1D), the clearance of lysosomal storage in the brain was reflected by a reduction of the β-hexosaminidase activity (Fig. 2C). To quantify residual carbohydrates in knockout brains before and after ERT, the respective sugar extracts were analysed by HPLC (Fig. 2D). Frequent injections of 500 U/kg led to a >50% reduction for all mannosyl-linked oligosaccharides (Man2–Man9) which accumulate in the brain of mock-treated mice. The highest reduction was found for Man4 (76%), Man7 (77%) and Man9 (75%). The sugar concentration in brains of ERT-treated knockouts was found to be highest for Man2–Man4 and lowest for Man7–Man9, indicating a sequential correction of long sugars before shorter carbohydrate species are hydrolyzed. ERT with 250 U/kg did not (Man2, Man3 and Man8) or only marginally (Man4–Man7 and Man9) reduce the amount of mannosyl-linked sugars (Fig. 2D).


Figure 2
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  		Figure 2. Dose-dependent ERT effect in the PNS and CNS. TLC analysis of sugar extracts from tG (A) and brain (B) from KO mice injected with high-dose rhLAMAN (250 and 500 U/kg, n = 2, each dose) 14, 11.5, 7 and 3.5 days prior to killing. (C) Enzyme activity measurements of LAMAN and β-hex in total brain lysates of mock-treated WT and KO mice and ERT-treated KO mice (4 x 500 U/kg) (n = 2, each group). Brain extracts of ERT-treated KO mice displayed 14% of WT LAMAN activity and lower levels of β-hex activity. (D) Quantification of mannosyl-linked oligosaccharides (M2–M9) based on high liquid phase chromatography. Mice were treated in the same way as described for (B) (WT, n = 5; KO*, n = 7; KO_ERT4 x250, n = 3; KO_ERT4 x500, n = 3). ERT with 500 U/kg led to a <50% reduction of brain sugar storage, whereas doses of 250 U/kg were not effective.

 
Clearance of storage in hippocampal neurons and in neurons of the trigeminal ganglion upon frequent high-dose treatment
As we were interested in finding out which cells of the PNS and CNS respond to ERT, we performed detailed histological and ultrastructural analyses on nervous tissues of wild-type (Fig. 3A, F and K) and mock- (Fig. 3B, G and L) and ERT- (4 x 250 U/kg, Fig. 3C and H; 4 x 500 U/kg, Fig. 3D–I and M) treated knockout mice. Different brain regions (cerebellum, medulla oblongata and forebrain), spinal cord and the trigeminal ganglion were analysed for the presence of storage vacuoles, 3.5 days after the last injection. Comparable to the TLC analysis (Fig. 2A), all tested ERT conditions led to a complete disappearance of storage vacuoles in neurons of the trigeminal ganglion (Fig. 3H–I). In the brain, the effect of ERT on neuronal storage showed regional and dose-dependent differences, and in most brain regions, ERT with 250 U/kg was not effective. The effect of ERT in the brain was most rewarding for the hippocampal neurons of the CA3 region where treatment with 500 U/kg (Fig. 3D) but not with 250 U/kg (Fig. 3C) led to an almost complete disappearance of storage vacuoles. Ultrastructural analysis of these neurons displayed reappearance of lipofuscin particles after ERT (Fig. 3M), which are present in wild-type (Fig. 3K) but not in mock-treated knockouts (Fig. 3L). In addition to the hippocampus, also neurons of the thalamic ventral posteromedial nucleus and within some nuclei of the medulla oblongata responded to ERT with 500 U/kg. However, in none of these nuclei a complete absence of storage vacuoles like in hippocampus was obtained. Although ERT with 250 U/kg did not effectively reduce storage in many brain regions, we observed a significant clearance in neurons of the gelatinous portion of the solitary nucleus, which is located immediately adjacent to the area postrema, a neurohemal region, which is devoid of a blood-brain barrier. In Purkinje cells and granular cells of the cerebellum and neurons of the cortical region of the forebrain, no clear effect was visible after ERT with 250 U/kg and 500 U/kg (data not shown). To evaluate the persistence of the observed ERT effect in the brain, we additionally examined knockout mice 12 days after the last injection with 500 U/kg (Fig. 3E–J). Interestingly, at this time point, storage vacuoles had fully reappeared in hippocampal neurons (Fig. 3E) but not in neurons of the trigeminal ganglion (Fig. 3J).


Figure 3
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  		Figure 3. Clearance in neuronal cells after high-dose treatment and improvement of neuromotor abilities. Light microscopy of hippocampal neurons (A–E) and the trigeminal ganglion (F–J). KO mice were injected four times with 250 U/kg or 500 U/kg (interval 3.5 days) and analysed 4 (A–D; F–I) and 12 days (d) after the last injection (E and J). Mock-injected WT (A, F and K) and KO mice (B, G and L) served as controls. In the CNS, clearance of storage was most rewarding for the CA3 region of the hippocampus. There, storage vacuoles (*) disappeared completely after ERT with 500 U/kg (D) but reappeared 12 days after the last injection (E). In the trigeminal ganglion, already doses of 250 U/kg led to a complete clearance 4 (H) and 12 days (J) after the last injection. (K–M) Ultrastructural analysis of hippocampal neurons displayed lipofuscin particles present in WT- (K) and ERT-treated KO (M) mice but not in mock-treated KO mice (L). Mock- and ERT-treated mice were also analysed for their neuromotor (treadmill; N and O) and exploratory (elevated plus maze, open field; P–U) abilities. On the treadmill, WT- and ERT-treated KO mice always performed better than mock-treated KO mice, as exemplified by increased error latencies during the training phase (N) and a decrease in the number of errors (O) during the challenge phase of the experiment. Exploratory tests did not reveal gross changes in KO mice after ERT. However, except for open field, path length (P) and number of counts in the elevated plus maze (U), ERT-treated KO mice tended to perform better than mock-treated KO mice (asterisks indicate significance of difference in comparison with the WT group: *P < 0.05, **P < 0.01 and ***P < 0.001; plus signs indicate significance of difference in comparison with the ERT KO group: +P < 0.05, ++P < 0.01, +++P < 0.001). Scale bars: 9A–J) 10 µ m; (K–M) 2 µ m.

 
Improvement of neurological symptoms in {alpha}-mannosidosis mice after ERT
Previous behavioural analyses revealed impairments in adult {alpha}-mannosidosis mice that resemble neuropsychopathological alterations in clinical {alpha}-mannosidosis (16,17). To investigate whether the observed clearance of neural storage after 500 U/kg ERT could reverse behavioural deficits in {alpha}-mannosidosis mice, we performed therapeutic studies using mock- and ERT-treated knockout and mock-treated wild-type mice. Mice were examined within 4 days after the last of four injections (interval 3.5 days) on a behavioural task battery that included tests for neuromotor (treadmill), exploratory (open field, elevated plus maze) and neurocognitive (water maze) abilities. During the training phase of the treadmill (Fig. 3N), mice showed an increase in error latencies over subsequent trials, reflecting neuromotor learning in all groups. However, during this training as well as during the challenge phase of the treadmill (Fig. 3O), mock-treated knockout mice performed consistently more poorly than wild-types (P < 0.01), demonstrating prominent ataxia in the knockout group. Notably, ERT knockout mice performed significantly better than mock-treated knockout mice (P < 0.01), at a level that was indistinguishable from the wild-type group. Conversely, we observed only marginal improvement of exploratory skills (Fig. 3P–U) and no changes in neurocognitive performance in {alpha}-mannosidosis mice after ERT (data not shown). We, therefore, conclude that under the chosen experimental regime, ERT led to a complete reversal of ataxic symptoms in {alpha}-mannosidosis mice, but did not or only marginally improve their neurocognitive and exploratory defects.

Uptake of rhLAMAN into hippocampal neurons upon high-dose treatment
With the present ERT analyses in {alpha}-mannosidosis mice, we demonstrate that lysosomal storage beyond the blood-brain barrier can be successfully reduced by frequent injections of high doses of rhLAMAN. This raised the question whether the sugar clearance in neuronal cells results from a direct uptake of rhLAMAN into these cells. To address this question, we injected knockout mice with a high dose of rhLAMAN (1000 U/kg) to ensure sufficient and detectable uptake of enzyme into the brain. Mice were perfused with PBS 8 h after injection to ensure that no enzyme remained in the circulation. Brains were prepared for immunohistochemistry and stained with an antibody specific for rhLAMAN (Fig. 4A and B). This immunohistological approach revealed a clear and prominent labelling of hippocampal neurons in ERT (Fig. 4B) but not in mock-treated knockout mice (Fig. 4A). Together with the enzyme activity measurements (Fig. 2C), the results suggest that the enzyme not only crossed the blood-brain barrier but was also taken up by hippocampal neurons where it directly reduced carbohydrate storage. We also looked for inflammatory processes in the brain of these mice and did not see any signs of inflammation (data not shown). A preserved integrity of the blood-brain barrier in {alpha}-mannosidosis mice has been demonstrated earlier (10).


Figure 4
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  		Figure 4. MPR-independent uptake of rhLAMAN in brain. (A and B) KO mice were injected with PBS, 8 h prior to perfusion (A) or rhLAMAN (1000 U; B) and 50 µm brain slices were stained for rhLAMAN by free-floating immunohistochemistry. Fluorescence microscopy revealed prominent labelling of hippocampal neurons in ERT but not in mock-treated knockouts. Control (WT) fibroblasts and cells deficient for MPR46 and/or MPR300 were incubated with 0.1 (C and D) and 0.5 mg/ml (D–F) rhLAMAN for 3 h at 37°C. The cells were then lysed to determine intracellular LAMAN activity (C and D) or fixed and stained for rhLAMAN by immunofluorescence (E and F). At low doses (0.1 mg; C), rhLAMAN is taken up mainly via the MPR300, whereas at higher doses (0.5 mg; D), the enzyme enters the cell independent of MPRS. Immunofluorescence studies of (E) WT and (F) MPR-deficient cells treated with 0.5 mg rhLAMAN demonstrate localization of rhLAMAN (red) in LAMP1 (green, inset) positive vesicles, indicating lysosomal delivery of the enzyme. Scale bars: (A and B) 50 µm; (E and F) 10 µm.

 
Mannose-6-phosphate receptor independent uptake of low phosphorylated rhLAMAN in fibroblast after high-dose treatment
It is well known from other ERT studies that the phosphorylation profile of recombinant lysosomal enzymes is an important factor for cellular uptake. To address this question, we analysed native PNGase F-released N-glycans from rhLAMAN for the presence of phosphate residues by ESI mass spectrometry. Using this technology, we failed to demonstrate any phosphorylated N-glycans (data not shown), indicating absent or very low phosphorylation of the enzyme. To correlate the phosphorylation status of our enzyme with mannose-6-phosphate (M6P) dependent uptake, we performed in vitro studies with rhLAMAN (0.1 mg/ml) using fibroblasts lacking either or both mannose-6-phosphate receptors (MPRs) (18,19) (Fig. 4C). The amount of uptake was determined by measuring LAMAN activity in cell lysates. In wild-type- and MPR46-deficient fibroblasts, only low amounts (<1%) of rhLAMAN were taken up. In both cell lines, the uptake was blocked in presence of M6P (85 and 71%, respectively). Fibroblasts lacking MPR300 did not show a significant uptake of LAMAN. From these in vitro experiments, we concluded that only marginal fractions of the purified rhLAMAN were phosphorylated and taken up into fibroblasts by the plasma membrane-bound MPR300 (19). This observation raised the question whether an MPR-independent uptake of rhLAMAN is relevant for the brain effect visible after high-dose treatment. Therefore, wild-type and MPR46/300 double-deficient fibroblasts were exposed to different doses of rhLAMAN (0.1 and 0.5 mg) (Fig. 4D). Additionally, we performed immunofluorescence studies to control for intracellular localization of the incorporated enzyme (Fig. 4E and F). These analyses revealed that rhLAMAN entered the cells and was delivered to the lysosomes independent of MPRs when used at higher (0.5 mg) but not at lower (0.1 mg) doses.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Most LSDs display neurological symptoms caused by lysosomal storage in cells of the CNS and PNS. ERT remains the method of choice for LSD treatment. Although effects of ERT on lysosomal storage in peripheral tissues are well accepted, the therapeutic enzyme is usually regarded as being unable to cross the blood-brain barrier. Thus, targeting the appropriate lysosomal enzyme to the CNS is a major issue in ERT effectiveness for LSD with CNS involvement, including {alpha}-mannosidosis. However, recent reports including the present study suggest that lysosomal enzymes may well be able to cross the blood-brain barrier and reduce neuronal storage even in adult mice. Deglycosylation of recombinant lysosomal enzyme (20), pharmacological manipulation with epinephrine (21), enzyme administration to neonates (22,23) and high-dose treatment (14) have all been shown to reduce lysosomal storage effectively in the CNS of mucopolysaccharidosis (MPS) type VII mice. The passage of phosphorylated lysosomal enzymes across the blood-brain barrier in newborn mice suffering from MPSVII (22,23) and MPSIIIA (24) has been suggested to be mediated by MPRs. These receptors are thought to be developmentally regulated and present only in the blood-brain barrier of neonatal mice (25). However, therapeutic effects on CNS storage in adult animal models were observed in several ERT studies at either high enzyme doses (10,12,13) or short post-treatment intervals (26). These studies provided histological and biochemical evidence for the reduction of CNS storage after ERT. In the present study, we showed dose-dependent effects on visceral and nervous system pathology in {alpha}-mannosidosis mice using low phosphorylated rhLAMAN. In contrast to neural cells, storage in peripheral tissues could be efficiently reduced (>70%) with low-dose injections (25 U/kg), whereas in neurons of the CNS, significant reduction of storage was only apparent after high-dose treatment (>250 U/kg). Quantification by HPLC analyses of residual sugars in the brain revealed >50% reduction of all sugar types in brains of knockout mice after 500 U ERT, whereas 250 U/kg ERT was not effective. This correlates with the histological analyses, which demonstrated an overall reduction of storage in different brain regions after 500 U/kg ERT. From these data, we conclude that a critical dose of >250 U/kg is needed to reach neuronal cells of the brain. In contrast to CNS neurons, peripheral neurons like those of the trigeminal ganglion are cleared already by doses <250 U/kg, due to their accessibility to larger molecules (27,28). Like in other ERT studies (20), the effect of ERT on brain pathology was most prominent in hippocampal neurons, which was probably due to an efficient uptake of rhLAMAN into these cells, as demonstrated by our immunohistological findings. Our study also presents the link between storage and disturbed lysosomal function, indicated by increased expression of LAMP1 and β-hexosaminidase activity in mock-treated knockout tissues. Both proteins are reduced after ERT in peripheral tissues as well as in the brain, suggesting a normalization of lysosomal function upon sugar clearance.

By using detailed gait analysis techniques that were efficient in detecting early signs of neuromotor impairment in mice with LSD (29), we have observed ataxic symptoms in {alpha}-mannosidosis mice, which improved significantly after high-dose ERT. However, their exploratory and cognitive impairments proved to be more intractable and benefit much less from the present ERT regime. Even though hippocampal neurons showed complete removal of storage, the present functional defects (e.g. in Morris water maze learning) might be due to neuropathology in telencephalic structures that may not experience or benefit from reduction in CNS storage. As we only used four subsequent high-dose injections of rhLAMAN, it cannot be excluded that longer treatment with high-dose rhLAMAN could also change these latter symptoms. Unfortunately, long-term schedules were precluded in our mouse model because of the induction of humoral immune responses by frequent injections of the recombinant enzyme.

As the integrity of the blood-brain barrier in {alpha}-mannosidosis mice has been demonstrated earlier (10) and no signs of inflammation were found in the brain of these mice (data not shown), we suggest that rhLAMAN reaches the brain by crossing the blood-brain barrier through a yet unknown mechanism. Several receptor systems, including the MPRs, are involved in the uptake and intracellular sorting of soluble lysosomal enzymes (30–32). However, there are a number of reasons to believe that the uptake of rhLAMAN does not rely on binding to these receptors in a M6P manner because (i) despite its low phosphorylation, the enzyme was taken up very fast and efficiently and led to a dose-dependent clearance in various tissues, including cells of the nervous system, and (ii) our in vitro studies revealed M6P-independent uptake of rhLAMAN into cells lacking both M6P receptors when offered at high doses. This supports the idea that either other receptors than MPRs or even micropinocytosis is responsible for the uptake of rhLAMAN in vitro and in vivo. It is tempting to speculate that an efficient binding to MPRs might even hinder sufficient uptake of lysosomal enzymes into the brain as similar observations were made for the neuronal uptake of β-glucoronidase in MPSVII mice (20). In this study, the authors demonstrated that deglycosylated β-glucoronidase reaches the brain more efficiently when compared with the phosphorylated enzyme, and these differences were explained through the prolonged half-life of dephosphorylated enzyme, which increases from 10 min to 18 h after deglycosylation (20). The authors speculate that if brain capillaries are exposed to prolonged, high levels of circulating enzyme, M6P-dependent uptake in adult brains is possible.

However, a long half-life of the therapeutic enzyme might not be necessarily the general mechanism for a better uptake into the brain as ERT studies with {alpha}-mannosidosis guinea pigs using another rhLAMAN preparation did not show a significant effect for the correction of the brain pathology even though the respective enzyme had a half-life of 53 h (12). Also in terms of phosphorylation, it is hard to predict the efficacy of the respective enzyme because recombinant human arylsulfatase A was shown to significantly improve the nervous system pathology and function in a mouse model for metachromatic leukodystrophy even though it was highly phosphorylated (13,33). These data underline the idea that there is not a unique uptake mechanism or defined degree of post-translational modification of the therapeutic lysosomal enzyme needed for an efficient correction of storage in the brain. It is likely that for each LSD, a different therapeutic strategy needs to be established.

From our studies, we conclude (i) that high-dose ERT in {alpha}-mannosidosis mice is necessary for treatment of storage beyond the blood-brain barrier and (ii) that despite the suggested dogma that lysosomal enzymes cannot cross the blood-brain barrier, high-dose injections of recombinant low-phosphorylated {alpha}-mannosidase might be an option to get sufficient amounts of enzyme into the brain to reduce neuronal storage and the associated behavioural deficits of {alpha}-mannosidosis mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
For more detailed information, see Supplementary Material, which is published as supporting information on the HMG website.

Expression and purification of the rhLAMAN in CHO cells
LAMAN cDNA under the control of the human CMV promoter was expressed in CHO cells deficient in dihydrofolate reductase. The CHO cells were cultured, processed and rhLAMAN was purified as described in (10).

Intravenous injection of recombinant LAMAN
LAMAN was injected into the tail vein in doses of 25–1000 U/kg body weight (1 U = 0.073 mg). Mock-injected mice received the same volume of PBS. Blood was taken from the retroorbital plexus 5 min after injection to control for the amount of injected enzyme.

Preparation of organ and cell extracts
Tissue extracts: Mice were usually analysed 3–4 days after the last injection. Mice were anaesthetized with 10 ml/kg Ketamin/Xylazin (Sigma) and perfused with PBS. Organs were homogenized at 4°C in 9 v/w lysis buffer (10 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100 with protease inhibitors (CompleteR, Roche)). Following sonication and incubation on ice for 30 min, the homogenate was cleared by centrifugation at 14 000g for 15 min at 4°C and the supernatant used for western blot analysis and enzyme activity measurements. Cell extracts: Cell pellets from in vitro uptake experiments were lysed in 100 µl lysis buffer (PBS, 0.5% Triton X-100 with protease inhibitors (CompleteR, Roche)) and treated as described above.

Western blot analysis
Equal amounts of protein were separated on SDS polyacrylamide gels, blotted onto nitrocellulose membranes and incubated with antibodies against rat {alpha}-LAMP-1 (1D4B, Pharmingen) and rabbit {alpha}-actin (Sigma). Secondary antibodies were conjugated to HRP (Chemicon) and detected by chemiluminescence (SuperSignalWest, Pierce).

Enzyme assays
LAMAN activity was determined as described in (10). For the β-hexosaminidase assay, 10 µ l of organ extracts were incubated in 100 µl of substrate (0.1 M sodium citrate pH 4.6, 0.08% NaN3, 0.4% BSA, 0.15% NaCl) and 10 mM p-nitrophenyl-2-acetamido-2-deoxy-β-D-glucopyranosid for 0.5–5 h at 37°C. Both enzyme assays were stopped after adding 1 ml of 0.4 M glycine/NaOH, pH 10.4. Absorbance was recorded at 405 nm (jukcy = 18500 M–1 cm–1).

Isolation of neutral oligosaccharides
Sugar extracts from tissue samples were extracted as described in (10). The sugar extracts were resuspended in HPLC grade water (1–4 ml/mg tissue) and analysed by chromatographical methods.

Chromatographical methods
Thin layer chromatography
The separation of mannose oligosaccharides by TLC was done as described in (10). A detailed description of the quantitative analysis of glycans in tissues is given in Supporting Materials and Methods, which is published as supporting information on the HMG website.

Histological examinations
Electron microscopy
Deeply anesthetized mice were perfused with 6% glutaraldehyde in 0.1 M phosphate buffer (PB) pH 7.4 supplemented with 1% Procain. Organs were stored in 3% glutaraldehyde in 0.1 M PB pH 7.4. Tissue samples were post-fixed with 2% osmium tetroxide, dehydrated and embedded in Araldite. Semi-thin sections were stained with toluidine blue. Ultrathin sections were processed according to standard techniques. Immunohistology: Tissues of mice perfused with 4% paraformaldehyde (PFA) in 0.1 M PB were stored in 30% sucrose. For immunohistochemistry, 50 µm free-floating cryo-sections (Leica microtome) were rinsed in 0.1 M PB and blocked in 0.1 M PB (+0.2% BSA, 4% NGS, 0.25% Triton X-100) for 2 h. After incubation with the first antibody (24–48 h, 4°C), the sections were rinsed with 0.1 M PB (+0.25% Triton X-100) and incubated with secondary fluorescent antibodies (Alexa Fluor 488 and 546, Molecular Probes). Immunofluorescence was examined using an Axiovert 200M microscope with the Axio Vision 4.6 software (Carl Zeiss, Jena, Germany). Primary antibodies used were rhLAMAN (10) and LAMP1 (1D4B, Pharmingen). Nuclei were stained with DAPI (Molecular Probes).

In vitro LAMAN uptake studies
Wild-type and M6P receptor-deficient fibroblasts (18,19) were seeded on 6 mm dishes or 12 mm glass plates, washed two times with PBS and starved in serum-free medium for 30 min. LAMAN was added (0.1 mg or 0.5 mg/ml in DMEM (+1% FCS)) and the cells incubated for 4 h at 37°C and 5% CO2. After endocytosis, the medium was collected, stored at –20°C and the cells chased in DMEM (+10% FCS, 1% PS) for 30 min. The cells on the 6 mm dishes were washed with ice cold PBS, scraped off and pelleted by centrifugation. Cell pellets were stored at –20°C. For immunofluorescence, cells on the 12 mm glass plates were fixed with 4% PFA, washed and stored in PBS, which was performed as described in (34).

Behavioural assays
A detailed description of the behavioural assays is found in Supporting Materials and Methods, which is published as supporting information on the HMG website.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This work was supported by the Fonds der Chemischen Industrie and the HUE-MAN consortium (European Commission FP VI Contract LSHM-CT-2006-018692).


   ACKNOWLEDGEMENTS
 
The technical assistance of Inez Götting, Katharina Stiebeling and Dagmar Niemeier (Kiel) is gratefully acknowledged. This publication does not necessarily represent the opinion of the European Community and the Community is not responsible for any use that might be made of data appearing in this publication.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

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