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Published as doi: 10.1096/fj.06-6169com.
 (The FASEB Journal. 2007;21:2520-2527.)
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Single-dose intracerebroventricular administration of galactocerebrosidase improves survival in a mouse model of globoid cell leukodystrophy

Wing C. Lee, Yuen K. Tsoi, Frederick J. Troendle, Michael W. DeLucia, Zeshan Ahmed, Chad A. Dicky, Dennis W. Dickson and Christopher B. Eckman1

Mayo Clinic College of Medicine, Departments of Pharmacology and Neuroscience, Jacksonville, Florida, USA

1Correspondence: Mayo Clinic College of Medicine, Birdsall Bldg. Rm. 327, 4500 San Pablo Rd., Jacksonville, Florida 32224, USA. E-mail: eckman{at}mayo.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a devastating, degenerative neurological disorder. It is inherited as an autosomal recessive trait caused by loss-of-function mutations in the galactocerebrosidase (GALC) gene. Previously, we have shown that peripheral injection of recombinant GALC, administered every other day, results in a substantial improvement in early clinical phenotype in the twitcher mouse model of GLD. While we did detect active enzyme in the brain following peripheral administration, most of the administered enzyme was localized to the periphery. Given the substantial central nervous system (CNS) involvement in this disease, we were interested in determining whether or not a single-dose administration of the recombinant enzyme directly to the CNS, which could potentially be achieved clinically, would result in any substantial improvement. Following intracerebroventricular (icv) administration of GALC we noted a significant, 16.5%, reduction in the GALC substrate psychosine, the abnormal accumulation of which is believed to play a pivotal role in the CNS pathology observed in this disease. Moreover, recombinant GALC was found not only in periventricular regions but also at sites distant to the injection such as the cerebral cortex and cerebellum. Most importantly, animals receiving a single icv dose of the enzyme at postnatal day 20 survived up to 51 days, which compares favorably to the control twitcher animals, which normally only live to postnatal day 40/42. These results indicate that even a single icv administration of the recombinant enzyme can have significant clinical impact and suggests that other lysosomal storage disorders with significant CNS involvement may similarly benefit.—Lee, W.C., Tsoi, Y.K., Troendle, F.J., DeLucia, M.W., Ahmed, Z., Dicky, C.A., Dickson, D.W., Eckman, C.B. Single dose intracerebroventricular administration of galactocerebrosidase improves survival in a mouse model of globoid cell leukodystrophy.


Key Words: krabbe disease • enzyme replacement therapy • psychosine • twitcher


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GLOBOID CELL LEUKODYSTROPHY (GLD), or Krabbe disease, is a devastating and rapidly progressing degenerative neurological disorder. It is an autosomal recessive disease caused by mutations in the galactocerebrosidase (GALC) gene that severely affect enzyme activity (1 2 3) . Clinically, the onset of classical GLD typically occurs between 3 to 6 months of age and is initially characterized by spasticity, irritability and hypersensitivity to external stimuli, followed by opisthotonic posturing, visual failure, hypertonic fits and loss of tendon reflexes, with death typically occurring usually before 2 yr of age (4) .

GALC enzymatic activity is localized predominantly in lysosomes, where it is essential for normal catabolism of certain galactolipids, including galactosylceramide, a major glycolipid of central and peripheral myelin, and psychosine, another product of UDP-galactose:ceramide galactosyl transferase (2 , 3) . Deficiency in GALC causes an abnormal accumulation of psychosine, which has been shown to be cytotoxic (5 6 7 8) , particularly in myelin-forming cells such as oligodendrocytes and Schwann cells, possibly through an inhibition of cytokinesis (9) and a triggering of apoptosis (10 11 12) . Loss of these myelin-forming cells causes demyelination in both central and peripheral nerves during early developmental stages (13 , 14) . Other pathological features of the disease include axonal loss, astrocytic gliosis, and infiltration of macrophages with characteristic galactosylceramide inclusions ("globoid cells") (15) .

Current treatment options for GLD are mainly palliative, with the notable exception of bone marrow transplantation (BMT) (16 , 17) . However, the effectiveness of BMT is generally limited to presymptomatic infantile patients or those with late-onset GLD (18) . Recently, we have reported the results of a preclinical evaluation of direct enzyme replacement therapy (19) in the "twitcher" mouse model of GLD, which has been extensively used for studies of pathogenesis and for evaluating novel therapeutic approaches. The twitcher mouse is an enzymatically authentic model of GLD (20 , 21) carrying a G>A nonsense mutation in the GALC gene that results in complete deficiency of GALC activity (22 , 23) . The clinical phenotypes of these animals include failure to thrive, progressive limb weakness, and involuntary head tremor, and early death at about postnatal day (PND) 40/42 (20 , 21 , 24) . Peripheral GALC injection, administered intraperitoneally every other day, significantly improved most of the early clinical phenotypes normally found in these animals, such as motor performance and initial failure to thrive, and significantly increased the overall life span of treated animals (19) . While it is likely that much of the clinical benefit observed in the treated animals was due to peripheral improvements, we did observe some GALC activity in the brain and a reduction in psychosine levels. Given the substantial CNS involvement in this disease, we were interested in determining whether or not a single-dose administration of the recombinant enzyme directly to the CNS, something that might potentially be achieved clinically, could alone result in any significant improvement in the model. Thus, we examined the effect of a single, intracerebroventricular (icv) injection of the recombinant enzyme on psychosine clearance and survival in the twitcher mouse.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Production of GALC antibody and recombinant murine GALC protein
The affinity-purified anti-GALC polyclonal antibody CL1475 and recombinant murine GALC protein were prepared as described in (19) . For 30 µg GALC injection, a stock of recombinant GALC solution (10 mg/ml) was prepared in storage buffer (mannitol, 170 mM; sodium citrate, 50 mM; Tween 80, 100 mg/L) and stored in single-use aliquots at –80°C. The anti-GALC monoclonal antibody (CL13.1) was raised against recombinant human GALC protein and was prepared in conjunction with the Mayo Clinic Antibody Core Facility. Protein-A purified immunoglobulins from CL13.1 were used for immunofluorescence staining of brain sections.

Animals
The animal protocol used in this study has been approved by the Institutional Animal Care and Use Committee at the Mayo Clinic College of Medicine. Breeding pairs of twitcher heterozygotes and C57Bl/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The genotypes of pups born from the twitcher heterozygotes were determined by PCR (22) .

Intracerebroventricular injection
The GALC protein, or vehicle alone, was administered icv to anesthetized twitcher mice in a total volume of 3 µl. The mice were anesthetized by isoflurane (Abbott Lab, North Chicago, IL, USA) inhalation and placed in a stereotactic instrument (Stoelting Co., Wood Dale, IL, USA), positioned, and the incision site appropriately prepared. A small incision was made, and the skull was then exposed and cleaned and a small burr hole was drilled using a dental drill or Dremal rotary tool. The GALC protein, or vehicle, was injected using a Hamilton syringe with a 27-gauge needle (Reno, NV, USA) at a rate of 0.5 µl/min controlled by a syringe pump (Harvard Apparatus, Holliston, MA, USA) into the right lateral ventricle using the coordinates: 1 mm caudal to bregma, 1.3 mm lateral to sagittal suture, and 2 mm in depth. The incision was closed using Vet-Bond, and the mice were placed on an isothermal pad at 37°C and continuously observed following surgery until recovery. Topical analgesic was applied immediately following the surgical procedure and as needed thereafter.

Tissue harvesting, processing, and immunohistochemistry
Mice were sacrificed by CO2 asphyxiation followed by perfusion with PBS, pH 7.4. Brains were rapidly dissected and cut into two hemibrains for different analyses. The left hemibrains were rapidly frozen on dry ice and stored at –80°C prior to homogenization for Western blot analysis, GALC substrate cleavage assays, and psychosine determination. The right hemibrains were immersion-fixed in neutral buffered 10% formalin for 16 h at room temperature and then processed for paraffin embedding using an autoprocessor (Shandon, Pittsburgh, PA, USA). Paraffin-embedded serial sections were cut sagittally at 5 µm. GALC antibody staining and microscopy examination of brain sections (Fig. 1 ) was performed as described previously (19) except for the immunohistofluorescence experiments (Figs. 2 and 4 ) in which a manual staining method was applied. Following deparaffinization and antigen retrieval steps, brain sections were blocked by 5% goat serum (Sigma, St. Louis, MO, USA) diluted in PBS for 1 h. Primary antibodies were incubated with brain sections for 2 h at room temperature and were diluted in the blocking solution as follows: anti-GALC antibodies (CL1475, 1 in 800; CL13.1, 1 in 6), neuronal marker—antineuronal nuclei, NeuN, mouse monoclonal antibody (Chemicon, Billerica, MA, USA) 1 in 800, microglial and macrophage marker—anti-ionized calcium binding adaptor molecule 1, IBA1, rabbit polyclonal antibody (Wako, Richmond, VA, USA) (1 in 800), oligodendrocyte marker—carbonic anhydrase II, CAII, rabbit polyclonal antibody (gift from Dr. Ghandour), 1 in 1600. The brain sections were then washed in PBS for 3 times, 10 min each. Secondary antibodies, Alexa Fluor 488 anti-mouse IgG and Alexa Fluor 568 anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA), were used at 1 in 500 dilutions and incubated with the sections for 1 h at room temperature. Finally, the sections were washed in PBS and coverslipped in Vectashield mounting medium containing dapi (Vector Lab, Burlingame, CA, USA). All procedures were carried out under a light-protected environment after secondary antibodies were added. The slides were examined using a Nikon Eclipse 80i fluorescence microscope, and the images were captured and edited using Metamorph software.


Figure 1
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  		Figure 1. Analysis of GALC distribution in brain regions proximal to the lateral ventricle after single-dose, icv-GALC injection in twitcher mice. Twitcher mice injected with vehicle (A) or GALC protein (B–F) were sacrificed 24 h postinjection. GALC positive staining (by CL1475 antibody) was located in various regions (B, low magnification) proximal to lateral ventricle, LV, as compared to no or very weak staining at these same regions found in the vehicle injected twitcher mouse (A, low magnification). Higher magnification of GALC positive staining in the periventricular region (C); fimbria hippocampus, Fi (D); corpus callosum, CC (E); hippocampus; and dentate gyrus, DG (F).


Figure 2
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  		Figure 2. Analysis of GALC distribution in cerebellum and brain stem after single icv-GALC injection in twitcher mice by GALC immunofluorescence staining. Twitcher mice injected with GALC protein were sacrificed 24 h postinjection. GALC positive staining (by CL1475 antibody) was located at the outer lining of cerebellar cortex (A, 20x), cerebellar fissure (B, 20x), and outer layer of brain stem (C, 20x; D, magnified view, 40x). No staining was observed in the same regions in vehicle-injected twitcher mice showing the specificity of the antibodies employed(data not shown).


Figure 3
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  		Figure 3. Analysis of GALC enzymatic activity in the brain homogenate of the single icv-GALC injected twitcher mice. A) Schematic diagram shows the approximate position of icv injection and the line of separation (dot-line) that defined the anterior and posterior parts of the injected brains for (B, C) enzymatic activity analysis. B) The homogenates from the anterior and posterior parts of the vehicle (Control) or GALC injected brain were analyzed by GALC substrate turnover reaction. All brains were perfused with PBS by cardiac puncture and harvested at 24 h postinjection. GALC enzymatic activity was similar in the anterior and posterior parts of the icv-GALC injected brains (n=6). C) Distribution of GALC enzymatic activity in the posterior and anterior parts of the brain as represent by the percentage of total activity resulting from the single GALC injection.


Figure 4
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  		Figure 4. Analysis of cellular localization of GALC by double immunofluorescence staining. Twitcher mice injected with GALC protein were sacrificed 24 h postinjection. Cellular localization of GALC protein in neuronal (A–C), microglial (D–F), and oligodendrocytic (G–I) cells were analyzed by double immunofluorescence staining of GALC (CL1475 antibody)/NeuN for neurons, GALC (CL13.1 antibody)/IBA1 for microglial and macrophages, and GALC (CL13.1)/CAII for oligodendrocytes. Most of the GALC (green) we observed was found in NeuN-positive cells (red) particularly in the dentate gyrus (A) and hippocampus (B and C showing the CA1 and CA3 regions, respectively). Very little or no GALC staining was observed in IBA1-positive cells (red) in dentate gyrus (D), hippocampal white matter (E), and in the region between the lateral ventricle, and the CA3 region of the hippocampus (F). Some GALC (green) was localized in CAII-positive cells (red) in the dentate gyrus (I, annotated with white arrows) but was not apparent in either the fimbria hippocampus (G) or the hippocampal white matter (H). The blue color shown in (G–I) is dapi staining. All figures are shown at 20x, except (I), which is shown at 40x.

Processing of frozen tissues
Frozen hemibrain tissues for Western blot and GALC substrate cleavage assays were thawed on ice. To determine the distribution of GALC activity after a single icv injection, hemibrains from treated animals and control animals were dissected into anterior and posterior parts as shown in Fig. 3 A. The tissues were dounce homogenized in 4 volumes of modified RIPA buffer [50 mM Tris-HCl, pH 7.4; 150 mM sodium chloride; 1 mM EDTA; 1 mM sodium orthovanadate; 1 mM sodium fluoride; and Complete protease inhibitor cocktail tablet (1 tablet per 50 ml; Roche, Indianapolis, IN, USA)]. The homogenates were then centrifuged at 20,000 g for 1 h at 4°C. The supernatants were collected and used for subsequent assays. Protein concentration in the supernatant was determined using the BCA assay essentially as described by the manufacturer (Pierce, Rockford, IL, USA).

Western blot analysis
For an examination of GALC processing (Fig. 3D ), 50 µg of total protein for each sample was loaded per lane on a precast 10% Tris-glycine gel for SDS-PAGE (Bio-Rad, Hercules, CA, USA). Prestained protein standards were purchased from New England Biolabs (Ipswich, MA, USA). The subsequent general procedures were the same as described previously (19) . The Blotto blocking reagent for PVDF membranes was purchased from Pierce. The dilutions for primary antibody, CL1475, and secondary antibody, anti-rabbit horseradish peroxidase-linked antibody (Amersham Biosciences, Piscataway, NJ, USA) were 1:1000 and 1:2000, respectively.

GALC substrate cleavage assay
The method used was essentially similar to that described previously by Gal et al. (25) , as modified by us recently (19) . Briefly, supernatants of brain homogenates, each containing 50 µg total protein, were mixed with 10x reaction buffer, sodium taurocholate:oleic acid solution and substrate, and 2-hexadecanoylamino-4-nitrophenyl-ß-D-galactopyranoside (HNG), for 16 h at 37°C. After mixing with stop solution and ethanol, the reaction mixture was centrifuged at 20,000 g for 10 min at room temperature. The absorbance of the supernatant was measured at 410 nm. Calculation of GALC activity was based on the formula that 0.0712 absorbance units measured at 410 nm is equal to the presence of 1 nmol of hydrolyzed HNG product in a volume of 175 µl (25) .

Determination of brain psychosine level
The procedures of psychosine determination were the same as we have previously described (19) . Briefly, brain samples were spiked with the internal standard lactosylsphingosine. One percent of formic acid in methanol was added, and the samples were homogenized in a round-bottomed glass tube. The homogenate was allowed to stand at room temperature for 1 h, followed by centrifugation at 1800 g for 15 min at 4°C. The supernatant was collected and blown to dryness with nitrogen gas and resuspended in the methanol:formic acid solution. The material was sonicated for 15 min and centrifuged again. The supernatant was removed by syringe and filtered before analysis on an API 365 LC/MS system (Applied Biosystems, Foster City, CA, USA).

Determination of survivability
Mice were maintained until becoming moribund in accordance with the acceptable practice of laboratory animal care without forced feeding or other substantial interventions. The date the animals became moribund was recorded as the last day of survival, and the animals were subsequently sacrificed for analysis.

Body weight assessment
Mice were weighed by using a triple beam balance every other day, at the same time each day, beginning on the day of icv injection and ending when the animals became moribund.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ICV injection of GALC in twitcher mice results in widespread distribution in CNS, cellular uptake and processing, and reduction in psychosine accumulation
To determine the biodistribution of GALC in the CNS on unilateral icv administration, 30 µg of recombinant GALC was injected at PND 20 into the right lateral ventricle of twitcher mice. Control twitcher mice were injected with vehicle alone. The mice were sacrificed 24 h later, and immunohistochemical analysis was performed to examine the distribution of GALC in the brain. As expected, we observed dense, positive staining in the regions near the injection site of the treated animals (Fig. 1B ) with no staining observed in the vehicle control animals (Fig. 1A ), indicating that the staining observed was due directly to the recombinant enzyme injected. In addition to periventricular staining (Fig. 1C ), we readily observed staining in the fimbria hipocampus (Fig. 1D ) as well as at more distal sites such as the corpus callosum (Fig. 1E ), dentate gyrus, and hippocampus (Fig. 1F ). GALC staining was also detected in the lining of cerebellar cortex (Fig. 2A ), cerebellar fissures (Fig. 2B ), and in the outer layer of brain stem (Fig. 2C, D ). Again, no staining was observed in the vehicle control animals indicating the specificity of the antibody (data not shown).

To confirm the fairly widespread biodistribution of GALC to sites distal from the injection site, we analyzed GALC enzymatic activity in homogenates obtained from anterior portions of the left (contralateral) hemibrain containing the cerebral cortex and compared that to enzymatic activity found in the posterior portion of the hemibrain containing the cerebellum and brain stem (Fig. 3) . Following injection, GALC enzymatic activity rose from essentially undetectable levels in the vehicle-treated twitcher mice to ~0.5 nmol/mg/h (Fig. 3B ). Consistent with the immunohistochemical analysis, we observed similar levels of activity in both the anterior and posterior brain homogenates, when the tissues were extracted under the same weight-to-volume ratio in homogenization buffer (Fig. 3B ). Given the weight differences between the anterior and posterior portions of the brain, this translates to ~75% of the total enzymatic activity detected being localized to the anterior with ~25% being localized to the posterior (Fig. 3C ). Collectively, these data, coupled with the immunohistochemical and immunofluorescent analysis performed, indicate fairly widespread distribution of the recombinant enzyme following unilateral icv injection.

To study the cellular localization of GALC following icv injection, we performed double immunohistofluorescence staining using antibodies against GALC (green) and several cell specific antibodies (red) (Fig. 4) . Consistent cellular colocalization with GALC and the antineuronal nuclei (NeuN) antibody was observed in the dentate gyrus (Fig. 4A ) and hippocampus (Fig. 4B-C ) with the subcellular staining pattern for GALC being consistent with a lysosomal/endosomal distribution. To assess microglial and macrophage colocalization, we examined costaining with GALC and the ionized calcium-binding adaptor molecule 1 (IBA1) (Fig. 4D-F ). Very little costaining was observed, indicating that these cells do not likely serve as a major source of uptake for the recombinant enzyme. Given that we observed GALC in several predominant white matter structures such as the fimbria hippocampus (Fig. 1D ), hippocampal white matter (Fig. 1F ), and corpus callosum (Fig. 1E ), we suspected to find GALC taken up by oligodendrocytes in these structures. To our surprise, however, we observed very little GALC in cells costained with the oligodendrocyte marker, carbonic anhydrase II (CAII), in the fimbria (Fig. 4G ), hippocampal white matter (Fig. 4H ), and corpus callosum (not shown). However, some GALC-positive oligodendrocytes were found in the dentate gyrus (Fig. 4I , white arrows). The reason for this apparent regional difference in GALC uptake into oligodendrocytes is not clear. Overall, our data suggests that GALC is preferentially taken up into neuronal cells when it is administered icv.

Previous studies have shown that extracellular, secreted GALC is normally taken up by neighboring cells through both mannose-6-phosphate receptor dependent and independent pathways (26 , 27) . On internalization the enzyme is believed to undergo proteolytic digestion to an active form in lysosomes (28) . To determine if the recombinant enzyme was similarly processed, we performed Western blot analysis using an N-terminal antibody against GALC, CL1475 (19) . Consistent with the enzyme being appropriately processed, we observed a reduction in the apparent size of the recombinant enzyme from ~80 to ~55 kDa in the treated animals, which is identical to the apparent molecular weight of endogenous processed GALC detected in tissues of wild-type mice using the same antibody (data not shown). The presence of processed GALC strongly suggests that the recombinant enzyme has been trafficked to lysosomes, where it is likely to be functional. To directly determine the functional significance, we next examined the levels of the GALC substrate psychosine, a cytotoxic substrate that abnormally accumulates in the brains of twitcher mice. As shown in Fig. 5 , analysis of psychosine levels showed a significant (16.5%) decrease (P=0.0079) in psychosine accumulation in the brain following unilateral icv injection of recombinant GALC. Collectively, these data indicate that GALC is not only widely distributed following injection, but it is also appropriately processed and is physiologically active.


Figure 5
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  		Figure 5. Analysis of psychosine clearance followed by single icv-GALC administration in the twitcher mice. The hemibrain for psychosine analysis was perfused with PBS and harvested 24 h postinjection. Psychosine level in the twitcher mice injected with GALC was reduced by 16.5% (Mann-Whitney nonparametric analysis: P=0.0079, n=5) as compared to the vehicle injected control.

ICV injection of GALC improves the survival of the twitcher mouse model of GLD
Twitcher mice normally die at approximately PND 40/42. To determine if icv administration of GALC can improve survival, we examined survival following a single unilateral injection at three different doses: 3 µg, 6 µg, and 30 µg. Following treatment, the animals showed an apparent dose-dependent increase in survivability (Fig. 6 ) with animals at the highest dose surviving to ~50 days.


Figure 6
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  		Figure 6. Analysis of the life span of twitcher mice with single icv-GALC administration. Twitchers at around PND 20 (±2 days) were injected with vehicle or different amounts of GALC protein (3, 6, and 30 µg) only for single time. Data points represent the day that the animal became moribund and had to be sacrificed. All the GALC-treated groups had significantly longer life span than the control group. (Mann-Whitney nonparametric analysis: GALC 3 µg vs. control, P=0.0087; GALC 6 µg vs. control, P=0.0022). No statistical significance was found between the GALC 6 µg and the GALC 3 µg groups. (Mann-Whitney nonparametric analysis, P=0.3095)

Twitcher mice normally show a significant failure to thrive in early life when compared to their wild-type littermates. To determine if the treatment can improve the early failure to thrive that is characteristic of this model, we examined growth curves of the animals injected with vehicle, 3 µg GALC or 6 µg GALC. As shown in Fig. 7 A, twitcher mice typically gain weight, albeit at a much slower rate than wild-type mice (19) , until they reach a peak at approximately PND 32, which is normally followed by a precipitous decline in weight until the animals become moribund. Single-dose icv injection of recombinant GALC had very little effect on the animals’ failure to thrive with no significant changes observed at 3 µg (Fig. 7B, D ), and only a slight change observed at 6 µg (10%, P=0.04) (Fig. 7C, D ) in the average maximum weight gain during the measuring period. This compares relatively poorly to the effect we previously observed in animals that were treated peripherally with recombinant enzyme (19) . For comparison, we saw an ~48% increase in the average maximal weight gain following chronic peripheral dosing of the enzyme, which closely mirrors that found in normal mice during the same time period, indicating almost a complete attenuation in early failure to thrive in the peripherally treated mice (19) . These data suggest that the failure to thrive may be mediated more by peripheral effects, although we cannot rule out the possibility that chronic icv administration of the enzyme, as opposed to the single-dose treatment we performed in this study, might have resulted in a better attenuation in the early failure to thrive.


Figure 7
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  		Figure 7. Growth curves analysis of twitcher mice injected with different amount of GALC via icv route. Same animals injected either with (A) vehicle or (B) 3 µg GALC or (C) 6 µg GALC at around PND 20 (±2 days) for the survivability study (Fig. 4) were also analyzed for the body weight gain. The recording of the data points began on the day of injection and ended when the animal became moribund. A general drop in the body weight occurred in almost all the twitcher mice right after the icv injection. The body weight recovered in ~3 days and continued to increase until it reached the peak body weight just before the precipitous decline that led to die. D) The maximal percentage weight gain was calculated by the difference between the highest and lowest point of the curve as a percentage to the lowest point body weight value. As from the result, the GALC 6 µg group showed a significant increase in the maximal % weight gain, while the GALC 3 µg group showed a trend toward significant (Mann-Whitney nonparametric analysis: GALC 6 µg vs. control, P=0.0411; GALC 3 µg vs. control, P=0.4848).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our earlier study of peripherally administered ERT in twitcher mice, we could show improvements in most of the early clinical phenotypes apparent in these animals such as gait abnormalities, an initial failure to thrive, and reduced life span (19) . While much of the benefits we observed in that study may be due to peripheral improvements, we did observe small but readily detectable levels of GALC activity in the brain that was associated with a reduction in psychosine. Given the substantial CNS involvement in this disease, we were interested in determining the effect of single-dose administration of the recombinant GALC to the CNS by icv injection, something that could potentially be achieved clinically. Twenty-four hours postinjection, we found recombinant GALC immunostaining at sites considerably distal to the injection site including the outer layer of brain stem. This immunostaining was accompanied by an increase in GALC activity and a decrease in the GALC substrate, psychosine. More importantly, and somewhat unexpectedly, the single-dose injection at PND20 resulted in a dose-dependent increase in life span similar to that we previously observed with chronic peripheral administration of the enzyme.

Previous studies have demonstrated that GALC secreted from normal or over-expressing cells can be taken up by different types of GALC-deficient cells including oligodendrocytes, astrocytes, and Schwann cells in vitro (26 , 27 , 29) . In a very recent study, Duncan and colleagues have shown that twitcher-derived oligodendrocyte progenitor cells can take up GALC when transplanted into the spinal cord of myelin-deficient shiverer mice (30) , indicating that this uptake can also take place in vivo. In this study, colabeling indicates that much of the recombinant enzyme appears to colocalize with the neuronal marker Neu-N. However, costaining was also observed in CAII positive oligodendrocytes, at least in the dentate gyrus (Fig. 4I ) indicating that these cells can also take up the recombinant enzyme.

Despite the advancement in using hematopoietic stem cell transplantation to treat GLD patients before symptomatic onset (16 , 31) , no significant treatment options are available for symptomatic GLD patients, which account for most cases without family history of the disease. Our data here, and our previous data (19) , demonstrate that enzyme replacement therapy, either by single-dose administration to the CNS or through more chronic systemic administration, can result in significant clinical improvement even when initiated after symptoms become apparent in the twitcher model. Thus, ERT has the potential to provide benefit to patients presenting postsymptomatically and for whom only palliative care is currently available. Further, the lessons learned from ERT in Krabbe disease are likely to be directly applicable to other lysosomal storage disorders with significant CNS pathology.


   ACKNOWLEDGMENTS
 
We thank Dr. Ghandour M. S. for providing the antibody against carbonic anhydrase II protein as a gift in the current study, and the Mayo Antibody Lore Facility for assistance in producing the GALC monoclonal antibody.

Received for publication October 3, 2006. Accepted for publication March 1, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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