Proc. Nati. Acad. Sci. USA Vol. 89, pp. 2297-2301, March 1992 Medical Sciences

Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin (storage disease/GM2 ganglioside/brain cell culture/fl-hexosaminidase/endocytosis)

KOSTANTIN DOBRENIS*, ANSAMMA JOSEPHt, AND MARIO C. RATTAZZIt§ Department of Pediatrics, Biochemical Genetics Laboratory, North Shore University Hospital-Cornell University Medical College, Manhasset, NY 11030

Communicated by Elizabeth F. Neufeld, November 21, 1991 (received for review September 20, 1991)

neurons. The tetanus toxin (TT) binding sites present on neurons offer a potential alternative. The atoxic proteolytic fragment of TT, known as fragment C (TTC) or B-IIb, contains the holotoxin binding domain for polysialogangliosides that are abundant in neuronal membranes (11-17) and it is internalized (18-20); it has been suggested that TTC may prove useful as a neuronotropic carrier (11, 18). We now present evidence that, by conjugation to TTC, effective neuronal uptake and delivery to lysosomes of an exogenous lysosomal enzyme can be obtained.

ABSTRACT Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme fJ-N-acetylhexmlnidase A (Hex A; (3-N-acetyl-D-hexosaminide N-acetylhexmmnohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A coijugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These rmdings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement.

MATERIALS AND METHODS Preparation of TTC-Hex A. As previously reported (10), human placental Hex A (21) was derivatized with N-succinimidylpyridyldithiopropionate, and TTC (Calbiochem) was derivatized with 2-iminothiolane (modification of ref. 22); the dialyzed compounds were incubated for 30 min at 4°C in a 6:1 (TTC/Hex A) molar ratio to obtain mixed disulfides, followed by further dialysis in saline. Recovery was -50% by enzyme activity against 4-methylumbelliferyl N-acetylglucosaminide (4MUG) (23) (1 unit = 1 nmol of 4MUG cleaved per hr at 37°C), 4MUG 6-sulfate (24), and GM2 ganglioside [GalNAcI31--4(NeuAca2--*3)Galp1- 4Glc---Cer] (GM2) (25). TFC-Hex A preparations contained enzymatically active species with cationic charge/molecular weight higher than Hex A by nondenaturing PAGE and HPLC (Bio-Rad Bio-Sil TSK 400; apparent Mr, 150,000), immunoprecipitable with anti-TT antiserum, confirming coupling. Preparations were essentially free of nonmodified Hex A, and precipitated TTC was removed by centrifugation. Some remaining, soluble TTC was not removed, as purification resulted in deconjugation and enzyme loss. Neural Cell Cultures. Neuronal cell cultures were prepared (modification of ref. 26) by mechanical dissociation of day-15 embryonic Sprague-Dawley rat brains minus rhombencephalon and plating at 40,000 cells per cm2 in collagen-coated dishes with Dulbecco's modified Eagle's medium containing N1 additives (27) and 0.1 nM L-thyroxine. Only cells of neuronal morphology developed and were all Yr-binding positive, all galactocerebroside negative, and >98% glial fibrillary acidic protein (GFAP) negative [2 and 3 days in vitro (DIV)]; neuron-specific enolase (NSE) and/or neurofilament

Human lysosomal storage diseases result from genetic defects of hydrolases leading to accumulation of undegraded substrates. Many of these diseases are characterized by central nervous system (CNS) neuronal storage, untreatable neurodegeneration, and early death (1). In 1964, deDuve (2) proposed that the cellular endocytic pathway could be exploited to deliver exogenous enzyme to lysosomes to degrade stored substances. Although early enzyme replacement therapy trials were ineffective (3, 4), the recent success with nonneuronopathic Gaucher disease demonstrates that the approach is valid and that a key factor is efficient uptake of enzyme by target cells, as can be obtained by receptormediated endocytosis (5). In comparison, fluid-phase endocytosis is inefficient (6), requiring concentrations of extracellular enzyme not readily attainable in vivo. This is particularly true for CNS neurons, owing to their relative inaccessibility and low endocytic rate (7). Indeed, despite near normal activity of exogenous 8-N-acetylhexosaminidase A (Hex A; f3-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) obtained in GM2 gangliosidosis cat brain by blood-brain barrier permeabilization (8), the enzyme was detectable only in CNS nonneuronal cells (9). Cell culture experiments conflrmed low neuronal uptake of Hex A, consistent with fluid-phase endocytosis (10). Receptor systems that may undergo internalization with lysosomal delivery of ligands have not been well studied in

Abbreviations: CNS, central nervous system; Hex A, f-Nacetylhexosaminidase A; TT, tetanus toxin; TTC, tetanus toxin fragment C; 4MUG, 4-methylumbelliferyl N-acetylglucosaminide; GFAP, glial fibrillary acidic protein; DIV, days in vitro; NSE, neuron-specific enolase. *Present address: Department of Neuroscience, Albert Einstein College of Medicine, Rose F. Kennedy Center, 1410 Pelham Parkway South, Bronx, NY 10461. tPresent address: Department of Radiation Oncology, Long Island Jewish Medical Center, 270-05 76th Street, New Hyde Park, NY 11042. *Present address: Molecular and Medical Genetics, Mount Sinai School of Medicine, Box 1203, 1 Gustave Levy Place, New York, NY 10029. §To whom reprint requests should be addressed at t.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2297

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detectable in 42% and 71% of the cells by 3 and 4 DIV, respectively, consistent with neuronal identity. These cultures were used at 2-3 DIV, when the number of processbearing neurons was highest. Rat mixed neural cell-type cultures, similarly prepared but with 10% fetal bovine serum at plating, contained large morphologically identifiable NSE-positive neurons and GFAPpositive astrocytes, and some small glia (TT-binding negative and/or GFAP positive) of neuronal morphology (28). Feline mixed neural cell-type cultures were prepared (29) from cerebral cortex of 1- to 3-day postnatal normal and GM2 gangliosidosis cats (Felis catus). By immunofluorescence, lysosomally stored GM2 was detectable in most neurons and in some glial cells only in affected cat cultures (29). TTC-Hex A Cell Binding and Uptake Experiments. Enzymes were diluted in growth medium (excluding serum and thyroxine) to 50 nM, at which concentration low levels of Hex A uptake were consistently detectable. This concentration assumed complete enzyme protein recovery in the TTC-Hex A preparation since precise yield could not be determined because of residual TTC. After enzyme incubation and at least three washes, cultures were fixed/ permeabilized with 5% (vol/vol) glacial acetic acid/95% (vol/vol) ethanol (10 min; -20°C) and enzymes were detected by immunofluorescence. In two experiments, fixation was by 40 g of paraformaldehyde per liter in 0.01 M phosphatebuffered saline (PBS)/0.05 M sucrose (5 min; 24°C). Rocket Immunoelectrophoresis Assay. Enzyme-incubated rat neuronal cultures were extensively washed and mechanically harvested. Cell pellets were frozen/thawed and sonicated in 0.1 M phosphate buffer (pH 7.2) with 10 g of saponin per liter, 20 g of taurodeoxycholate per liter, and 50 mM 2-mercaptoethanol to solubilize and uncouple Hex A from fTC. The high-speed (40,000 x g; 30 min; 4°C) supernatant was treated with Biobeads SM-2 (Bio-Rad) to remove detergents. Extract dilutions and Hex A standards (0.34-1.4 fmol or 3.5-350 fmol) were electrophoresed (210 V-hr; Pharmacia Phast System) in 50 x 50 x 0.5 mm gels [20 g of agarose per liter in 0.08 M Tris citrate buffer (pH 8.6) with 30 g ofPEG 6000 per liter] containing anti-human Hex A (1:32,000 or 1:6000 dilution). Washed gels were blotted with Celiogel (Chemetron, Milan) soaked with 4MUG (2 hr; 37°C). Interpolated enzyme concentrations were averaged from three to eight fluorescent rockets per extract and from two extracts (each from 2 x 35

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Proc. Natl. Acad Sci. USA 89 (1992)

mm dish cultures) per condition. Control cultures had no detectable cross-reactivity. For statistical testing, data were transformed to the square root to stabilize variances. GM2 Ganglioside Storage Depletion Experiments. Feline GM2 gangliosidosis cortical cell cultures were incubated with equally diluted enzyme or carrier (saline) alone in growth medium. To detect stored GM2 and TT-binding cells, cultures were then washed and chased for 1 hr, incubated with TT (10 ,ug/ml) (Calbiochem) (1 hr; 40C), washed, fixed/permeabilized with acetone (10 min; 40C), and double immunolabeled. In the event that TTC-Hex A continued to occupy surface binding sites, TT-binding cell types could still be identified because the antiserum to TT also recognized TTC-Hex A. The proportions of cells positive for immunoreactive GM2 storage were determined and converted to the arc sin square root for statistical analysis. To detect GM2 and NSE, enzyme-incubated cultures were washed, fixed with 40 g of paraformaldehyde per liter, 1 g of picric acid per liter in 0.01 M PBS/0.05 M sucrose, pH 7.4 (45 min; 40C), incubated with antiserum to NSE, permeabilized with saponin (40 tug/ml), and incubated with antibody to GM2 and with secondary antibodies in saponin. Biochemical quantitation of neuronal GM2 was not possible as purified cat neuronal cultures could not be obtained. Immunotluorescence. Primary antibodies included mouse monoclonal antibodies to neurofilament (clones NR4 and NN18) (30, 31) and GFAP (28, 30) from Boehringer Mannheim; human antiserum to TT (1:100-1:150 dilution) (28) from Cutter; rabbit anti-human NSE antibody (32) from Accurate Chemicals, Westbury, NY; rabbit anti-galactocerebroside antibody (1:100) (28) from J. deVellis; highly specific monoclonal mouse anti-GM2 antibody (1:25) (33) from P. 0. Livingston; and species-specific rabbit anti-human Hex A antibody (1:301:60) prepared by us (34). By immunoprecipitation, Hex A and TTC-Hex A had the same equivalence point with antiserum to Hex A. Commercial, species-specific, and immunoglobulinspecific goat secondary antibodies included fluorescein, rhodamine, and Texas Red conjugates. When double labeling for NSE and Hex A, using rabbit antiserum for both, cultures were treated sequentially with primary and secondary (fluorescein) antibodies for enzyme detection, with goat anti-rabbit IgG, and with primary and secondary (Texas Red) antibodies for NSE detection. Enzyme uptake was identified by fluorescent granules as distinct from NSE-positive staining that labeled the entire perikaryoplasm. All incubations were for

FIG. 1. Cell-surface binding test of TTC-Hex A and Hex A in rat brain mixed cell-type cultures. Sister cultures were incubated at

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27 DIV with SO nM TTC-Hex A (A and B) or Hex A (C and D) in medium with 25 mM Hepes for 1 hr at 40C, then washed on ice, fixed with acid/alcohol, and processed by indirect immunofluorescence using antiserum to human Hex A and fluorescein-conjugated secondary antibody. (A and C) Epifluorescence. (B and D) Matching phase contrast. The only surface binding seen is of TTC-Hex A to cells of neuronal morphology (A). Similar results were obtained with paraformaldehyde fixation without permeabilization. (Bar = 50 gm.)

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FIG. 2. Uptake of TIC-Hex A and Hex A by cultured rat brain cells. Mixed cell-type cultures (29 DIV) were incubated at 370C for 17 hr with 50 nM TTC-Hex A (A and B) or Hex A (C and D) in medium, then washed for 1 hr and fixed, and enzyme was detected as described in Fig. 1. (A and C) Epifluorescence. (B and D) Matching phase contrast. Both fields are centered on a large neuron. No neuronal uptake of Hex A is detectable; but for some granules evident in surrounding glia, only diffuse nonspecific staining is present (C), indistinguishable from that in sister cultures incubated without enzyme (data not shown). (A) Abundant neuronal uptake of TTC-Hex A is detectable as granular and vesicular, perikaryoplasmic staining. Positive granules were visible throughout the depth of the cell body. Some TTC-Hex A is still also present on the neuronal surface. (Bar = 20 tum.)

0.5-1 hr at room temperature. Controls included omission of antigen or antibody or substitution with nonimmune serum. Two to four sister coverslip cultures were used per condition. Coverslips were mounted with 20 g of phenylenediamine per liter in PBS (pH 8.0)/glycerol (1:9; vol/vol).

RESULTS After 40C incubation of rat brain mixed cell-type cultures with the enzymes, Hex A was undetectable by immunofluorescence while TTC-Hex A was detectable only on cells of neuronal morphology (Fig. 1). When cold TTC-Hex A incua)

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degree of TT/TTC surface binding. Feline GM2 gangliosidosis cerebral cortex cell cultures (90 DIV) were incubated for 21 hr at 37°C with and without 50 nM enzyme (equivalent to 67,000 units/ml), incubated with TI, and double immunolabeled for TT/TTC surface binding and GM2. Rounded process-bearing cells were categorized as negative (-), faintly positive (+), or strongly positive (+ +) for TT/TTC binding and were scored as positive or negative for GM2 storage (visible as immunofluorescent granules; e.g., see Fig. 4B). Each bar represents the mean (SEM < 13%) from six counts of 50-100 cells each. TTC-Hex A has reduced the percentage of cells that are GM2 positive significantly more than Hex A has, and the magnitude of the effect is dependent on the cell type (two-way analysis of variance, P < 0.0001; interaction effect, P < 0.005).

bation was followed by washing and a 370C enzyme-free incubation for 2, 4, and 6 hr, immunofluorescent staining of enzyme was increasingly evident over time in neuronal perikarya in a granular and vesicular form, indicating endocytosis of surface-bound TTC-Hex A. In all of 10 immunofluorescence experiments in which mixed cell-type cultures were incubated with enzymes at 370C, the amount of endocytosed$ TTC-Hex A was greater than that of Hex A in cells of neuronal morphology but not in nonneuronal flat cells (Fig. 2). The majority of neuron-like cells demonstrated abundant uptake of TTC-Hex A and little or no Hex A uptake. The results were consistent regardless of culture age (7-53 DIV) and time of incubation (6-23 hr). To exclude neuron-like glia from our evaluations, two uptake experiments were conducted on mixed cell-type cultures in which double immunolabeling was used to identify neurons by NSE staining, and three experiments were conducted with rat neuronal cultures. The difference in enzyme uptake by neurons was even greater than that by the neuron-like cell population, for on the average TTC-Hex A uptake was higher, and Hex A uptake was lower. In a control experiment, Hex A uptake was not enhanced in the presence of

thiolated, free TTC. By immunoelectrophoretic quantitative analysis of rat neuronal cultures exposed to enzyme (44 nM; 18 hr; 37°C) followed by washing and a 2.5-hr incubation without enzyme to deplete surface-bound TTC-Hex A, the TTC-Hex A group contained -16 times more enzyme than was found in the Hex A group [2.6 + 0.48 (SEM) vs. 0.15 ± 0.021 pmol per 35-mm dish culture (P < 0.01, Student's t test, unpaired, one-tailed)]. In a subsequent immunofluorescence experiment with 37°C TTC-Hex A incubation and various enzyme-free chase times, some surface-bound enzyme was still evident after a 3-hr chase but was virtually absent after a 24-hr chase, while internalized enzyme was still detectable. With similar immunofluorescence results, in a second immunoelectrophoretic

IEndocytosed enzyme was defined as granular and vesicular immu-

nofluorescent staining in a perikaryoplasmic distribution, as shown in Fig. 2A. This pattern is consistent with endosomal/lysosomal localization, and such staining was not present using paraformaldehyde fixation without methanol permeabilization (2 min; -20°C) before incubation with antibodies, further indicating that it represented internalized enzyme.

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(i) The atoxic fragment retains affinity for polysialogangliosides abundant in neuronal membranes (11-17); thus, a large number of binding sites are available for TTC adducts. Adducts can be obtained by thiol addition and mixed disulfide formation without loss of ganglioside binding (22). Similarly, TTC-Hex A showed the same specific cell-surface binding patterns (Fig. 1) as found with TT (28) and TTC (35), consistent with retention of ganglioside binding. Furthermore, this synthetic approach is compatible with retention of the activity of Hex A, a labile enzyme. (ii) Neuronal surface-bound TTC is internalized and has been localized to somatic cytoplasmic inclusions after retrograde axonal transport (18-20). Similarly, the results of our experiments on TTC-Hex A cold-incubated, warm-chased cultures are consistent with TTC-mediated, adsorptive endocytic uptake. This mechanism is more efficient than fluidphase endocytosis (6) by which neurons take up Hex A (10); enhanced uptake was confirmed in our 37°C incubation studies. Accurate quantitation of internalized TTC-Hex A was complicated by the persistence of TTC-Hex A on the neuronal surface. Nevertheless, the difference in uptake of TTC-Hex A and Hex A is significant, since extending the chase time and thereby decreasing the surface-bound fraction did not decrease the ratio of TTC-Hex A to Hex A measured in neuronal extracts. (iii) The extent to which TTC reaches secondary lysosomes is unclear (19). Indeed, TTC could not undergo transneuronal transfer (20, 36) from a lysosomal compartment regarded as terminal (37, 38). However, the enhanced depletion of GM2 immunoreactivity in neurons exposed to TTC-Hex A demonstrates that enzyme is delivered to secondary lysosomes. Internalized TTC-Hex A may reach secondary lysosomes by diverging from the route that recycles TTC to the surface for transneuronal transfer. By analogy, TT undergoes transneu-

experiment using a 25-hr chase (after 15 hr, 32 nM enzyme incubation), differences remained significant: homogenates contained -40 times more TTC-Hex A than Hex A (0.78 ± 0.38 vs. 0.01& ± 0.0095 pmol per dish; P < 0.05). GM2 gangliosidosis cat cerebral cortex cell cultures were used to assess the effect of TTC-Hex A and Hex A on lysosomal storage. By immunofluorescence, enzyme uptake was similar to that in rat cultures. Comparing the degradative effectiveness, TTC-Hex A reduced GM2 immunoreactivity more than Hex A in the TT/TTC binding (neuron containing) and not in the non-TT/TTC binding (nonneuronal) cell population, suggesting that TTC-mediated enhanced uptake of enzyme led to enhanced GM2 degradation (Fig. 3). Similarly, after overnight enzyme treatment, and double immunolabeling for NSE and GM2 (Fig. 4), a significant decline in the percentage of neurons that were GM2 positive was evident with TTC-Hex A and not with Hex A (Fig. 5A). In addition, the number and intensity of GM2-positive granules appeared lower in TTC-Hex A- than in control and Hex A-treated neurons. When cultures were dosed twice over a period of 3 days, virtually no neurons were GM2 positive in TTC-Hex A-treated cultures on day 4 (Fig. 4), while in Hex A-treated cultures, the majority remained GM2 positive (Fig. SB) and were qualitatively similar to controls.

DISCUSSION Our data demonstrate that efficient delivery of an exogenous lysosomal enzyme to neuronal lysosomes can be obtained by covalent coupling to TTC, with effective degradation of stored substrate. These results have a direct bearing on the treatment of neurodegenerative lysosomal storage diseases by enzyme replacement. Several points are relevant to the use of TTC as a neuron-

specific, lysosomotropic ligand. c

FIG. 4. Double immunofluorescence staining to identify NSEpositive neurons and detect stored GM2 after treatment of GM2 gan-

gliosidosis neural cultures with and without TTC-Hex A. After incubation (as described for Fig. SB), cultures were fixed with paraformaldehyde/picric acid, permeabilized with saponin, and double immunolabeled for NSE (Texas Red-conjugated secondary antibody) and GM2 (fluoresceinconjugated secondary antibody).

F

-W

A field from cultures not treated (A-C) and treated (D-F) with TTC-Hex A is shown [epifluorescence for Texas Red (A and D) and fluorescein (B and E); (C and F) phase contrast]. Neuronal perinuclear fluorescent granules (at different planes of focus) indicating lysosomal, stored GM2 are evident in the absence of enzyme treatment (B) but are undetectable after treatment with TTC-Hex A (E). (Bar = 20 Am.)

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Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 5. Percentage of NSE-positive neurons positive for GM2 storage in GM2 gangliosidosis neural cultures after TTC-Hex A (T), Hex A (H), or no enzyme (C) treatment. Enzyme stocks were diluted to 50,000 units/ml in medium. After treatments, cultures were fixed and immunolabeled as described for Fig. 4, and NSE-positive neurons (n = 56-333 per treatment) were scored (double blind) as either positive or negative for GM2-positive granules. (A) Cultures (79 DIV) were incubated for 22 hr with one dose. x2 analysis: T vs. H vs. C, P < 0.05; H vs. C and T vs. H, P> 0.05; T vs. C, P < 0.02. (B) Cultures (60 DIV) were incubated for 4 days with an enzyme dose given on day 1 and again on day 3. x2 analysis: T vs. H vs. C, P < 0.0001; H vs. C, P < 0.02; T vs. C and T vs. H, P < 0.0001.

ronal transport (36) but has also been localized to lysosomes (39). Another possibility is that Hex A reaches lysosomes after release from membrane-bound TTC by disulfide reduction in endosomes (40) while any uncleaved adduct could recycle to the plasma membrane (6, 37, 38). Both hypotheses are consistent with the apparent persistence of TTC-Hex A on the neuronal surface. (iv) TTC conjugates undergo retrograde axonal transport (19, 41-43) and may undergo transneuronal transfer (19); this would facilitate distribution to distant neurons of enzyme introduced into the CNS and may allow delivery to the CNS from the periphery (43). In experiments with GM2 gangliosidosis cats, by reversible blood-brain barrier permeabilization, we attained brain tissue concentrations of Hex A (8) comparable to those at which TTC-Hex A virtually eliminated detectable storage of GM2 in vitro. Cell uptake and catabolic and therapeutic effectiveness of TTC-Hex A in the CNS can now be tested in this model (8), preferably by using the hyperosmotic method currently applied to humans (44). Repeated infusion of Hex A (4) and other lysosomal enzymes (5, 45) in patients has not resulted in significant immune reactions. Smaller ganglioside binding domains of TTC (15, 35) and anti-idiotypic antibodies (46) could be used to circumvent TTC immunogenicity and the presence of anti-TT/TTC antibodies. While in vivo studies are required, our findings indicate that TTC is an effective vector for the delivery of enzymes to neuronal lysosomes, potentially applicable to the treatment of lysosomal storage diseases with neurologic involvement. We thank P. Livingston and J. deVellis for kindly providing antibodies to GM2 and galactocerebroside, respectively, M. E. Lesser for assistance with the statistical analysis, P. Sherman for technical assistance, and M. Feeney for typing the manuscript. This work was supported in part by National Institutes of Health Grants NS 21404 and RR 05924. 1. Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D., eds. (1989) The Metabolic Basis of Inherited Diseases (McGraw-Hill, New York), Vol. 2, Ed. 6, pp. 1565-1839. 2. deDuve, C. (1964) Fed. Proc. Fed. Am. Soc. Exp. Biol. 23, 1045-1049. 3. Tager, J. M., Hamers, N. M., Schram, A. W., van den Berg, F. A., Rietra, P. J., Loonen, C., Koster, J. F. & Slee, R. (1980) in Enzyme

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