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Intrathecal enzyme replacement therapy improves motor function and survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis
Molecular Genetics and Metabolism 116 (2015) 98–105
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Intrathecal enzyme replacement therapy improves motor function and survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis Jui-Yun Lu a,c, Hemanth R. Nelvagal d, Lingling Wang a,c, Shari G. Birnbaum b, Jonathan D. Cooper d, Sandra L. Hofmann a,c,⁎ a
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA c Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA d Pediatric Storage Disorders Laboratory, Department of Basic and Clinical Neuroscience, King's Health Partners Centre for Neurodegeneration, James Black Centre, Institute of Psychiatry, Psychology & Neuroscience, King's College London, 125 Coldharbour Lane, London SE5 9NU, UK b
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Article history: Received 7 April 2015 Received in revised form 9 May 2015 Accepted 10 May 2015 Available online 12 May 2015 Keywords: Enzyme replacement therapy Palmitoyl protein thioesterase Lysosomal storage disorder Batten disease Infantile neuronal ceroid lipofuscinosis
a b s t r a c t The neuronal ceroid lipofuscinoses (NCLs) are a group of related hereditary lysosomal storage disorders characterized by progressive loss of neurons in the central nervous system resulting in dementia, loss of motor skills, seizures and blindness. A characteristic intralysosomal accumulation of autofluorescent storage material occurs in the brain and other tissues. Three major forms and nearly a dozen minor forms of NCL are recognized. Infantile-onset NCL (CLN1 disease) is caused by severe deficiency in a soluble lysosomal enzyme, palmitoylprotein thioesterase-1 (PPT1) and no therapy beyond supportive care is available. Homozygous Ppt1 knockout mice reproduce the known features of the disease, developing signs of motor dysfunction at 5 months of age and death around 8 months. Direct delivery of lysosomal enzymes to the cerebrospinal fluid is an approach that has gained traction in small and large animal models of several other neuropathic lysosomal storage diseases, and has advanced to clinical trials. In the current study, Ppt1 knockout mice were treated with purified recombinant human PPT1 enzyme delivered to the lumbar intrathecal space on each of three consecutive days at 6 weeks of age. Untreated PPT1 knockout mice and wild-type mice served as additional controls. Four enzyme concentration levels (0, 2.6, 5.3 and 10.6 mg/ml of specific activity 20 U/mg) were administered in a volume of 80 μl infused over 8 min. Each group consisted of 16–20 mice. The treatment was well tolerated. Diseasespecific survival was 233, 267, 272, and 284 days for each of the four treatment groups, respectively, and the effect of treatment was highly significant (p b 0.0001). The timing of motor deterioration was also delayed. Neuropathology was improved as evidenced by decreased autofluorescent storage material in the spinal cord and a decrease in CD68 staining in the cortex and spinal cord. The improvements in motor function and survival are similar to results reported for preclinical studies involving other lysosomal storage disorders, such as CLN2/ TPP1 deficiency, for which intraventricular ERT is being offered in clinical trials. If ERT delivery to the CSF proves to be efficacious in these disorders, PPT1 deficiency may also be amenable to this approach. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The neuronal ceroid lipofuscinoses are a group of neurodegenerative disorders characterized by widespread accumulation of autofluorescent storage material in lysosomes and a progressive loss of neurons in the central nervous system [1]. Deficiency in the enzyme palmitoyl protein Abbreviations: ERT, enzyme replacement therapy; CLN1, ceroid lipofuscinosis, neuronal-1; CLN2, ceroid lipofuscinosis, neuronal-2; LSD, lysosomal storage disorder; MU6S-Palm-βGlc, 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucoside; NCL, neuronal ceroid lipofuscinosis; PPT1, palmitoyl-protein thioesterase-1; TPP1, tripeptidyl peptidase-1. ⁎ Corresponding author at: Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8593, USA. E-mail address: [email protected] (S.L. Hofmann).
http://dx.doi.org/10.1016/j.ymgme.2015.05.005 1096-7192/© 2015 Elsevier Inc. All rights reserved.
thioesterase-1 (PPT1; EC 3.1.2.22) causes the autosomal recessive storage disorder infantile neuronal ceroid lipofuscinosis, or CLN1 disease [2]. This enzyme is a small, globular hydrolase of the α/β type that removes fatty acids from cysteine residues in lipid-modified proteins during lysosomal protein degradation [3]. Over 60 mutations in the PPT1/CLN1 gene in NCL patients have been described [4]. Complete absence of enzyme activity results in severe neurodegeneration occurring in infancy and is characterized by progressive cognitive and motor deterioration, blindness, and seizures leading to premature death [2]. Childhood and adult onset cases are associated with missense mutations that allow for varying levels of residual enzyme activity in the range of 2–7% [5–7]. Hematopoietic stem cell transplantation has had no effect on the course of affected infants [8]. No effective treatment is available and
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affinity-purified using the AminoLink Plus Immobilization Kit (Thermo Scientific, #44894) following the manufacturer's instructions.
various approaches, including small molecule, enzyme replacement, and gene and cellular therapies are the subject of preclinical studies [9]. The Ppt1 knockout mouse demonstrates the major features associated with the human disease, including autofluorescent storage and manifestations such as seizures, decline in motor performance and reduced lifespan [10,11]. The mice live about 235 days in the absence of treatment [10,12]. We have previously shown that PPT1 administered weekly via the tail vein is effective in clearing visceral storage, but has no impact on motor deterioration or survival when the treatment is started after blood brain barrier closure in the mouse, which occurs at 3 weeks of age [13]. About a three-week survival advantage and delay in onset of motor symptoms was shown when treatment was started at birth, suggesting that the brain would be responsive to ERT if appropriate access could be achieved. In addition to infantile NCL, several of the other NCLs (CLN2, 5 and 10) are caused by deficiencies of soluble lysosomal enzymes that can be supplied exogenously to cells through the mannose 6-phosphate receptor pathway. Classic late-infantile NCL (CLN2 disease) is caused by deficiency in the soluble lysosomal protease, tripeptidyl peptidase-1 (TPP1). Treatment of TPP1 knockout mice with recombinant TPP1 enzyme (10 mg/ml) delivered to the cerebrospinal fluid via lumbar intrathecal injection (given on three consecutive days) in mice at 4 weeks of age prolonged lifespan of CLN2 knockout mice from 16 to 23 weeks [14]. Based on these encouraging results in mice, the approach was successfully applied to the TPP1-deficient dog model [15–17] and has progressed to a human clinical trial (ClinicalTrials.gov ID# NCT01907087). In the current study we have used the same protocol to treat Ppt1 knockout mice with human recombinant PPT1 and show a dose-dependent positive effect on motor function, brain pathology and survival.
All procedures were carried out under an Institutional Animal Care and Use Committee-approved protocol at the University of Texas Southwestern Medical Center. Ppt1 knockout mice [10] were maintained as homozygous breeding stock on a C57BL/6 background, housed in a barrier facility and received food and water ad libitum. Treatment groups were assigned randomly from littermates born within a 2–3 day window after timed mating and group housed. All assessments were carried out by individuals blinded with respect to treatment group and genotype. Concurrent groups of untreated Ppt1 knockout mice and wild-type C57BL/6 mice were maintained for comparison but were not included in the analysis for treatment effects. Each treatment group consisted of between 16 and 20 mice at about 6 weeks of age. Six weeks of age is in the presymptomatic period of Ppt1 knockout mice [13] and well after the permeability transition for lysosomal enzymes to reach the brain from the systemic circulation in the neonatal mouse [20]. For the enzyme replacement therapy, mice were anesthetized with isoflurane delivered through an inhalation system (EZ-Anesthesia Classic System, Braintree). Animals were shaved to expose the skin around the lumbar region and injected between vertebrae L5 and L6 using a 30-gauge needle (Becton Dickinson) oriented toward L4 as described [14]. The needle was connected to a 100 μl gas-tight Hamilton syringe (#81075) by a short length of silastic tubing (0.3 mm inner diameter, Dow Corning, #2415496). The dose (vehicle or enzyme) was administered at 10 μl per min using an NE-300 syringe pump (New Era Pump Systems).
2. Materials and methods
2.4. Rotarod assessments of motor performance
2.1. Human recombinant PPT1
For motor coordination testing, mice were tested on a Rotarod (model 755, IITC Life Science Inc., Woodland Hills, CA) at 3, 5, 6, 7, 8 and 9 months of age and the latency to fall in seconds recorded. Trials were terminated after a maximum of 60 s. At each age, mice underwent a pre-test trial on a stationary rod, followed by two test trials on the constant speed Rotarod (3 rpm) for each of three consecutive days. The maximum latency to fall in the two test trials on the final day of testing was reported as the final outcome measure. A two-factor (treatment group and time of measurement) ordinary ANOVA model was used to analyze data for vehicle, 2.6, 5.3, and 10.6 mg/ml PPT1 dosing groups for months 3, 5, 6 and 7. Ordinary one-way ANOVA (treatment group) was used to analyze month 8 for the 2.6, 5.3 and 10.6 mg/ml treatment groups (as all mice in the vehicle group had died by month 8).
Human recombinant PPT1 was prepared from an overproducing Chinese Hamster Ovary (CHO) clonal cell line as described [18] except that the enzyme was exchanged into an artificial CSF buffer at the final step (aCSF, 148 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 0.9 mM MgCl2, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4, pH 7.2). Enzyme was concentrated using a stirred cell equipped with a YM-10 membrane (Millipore) at 4 °C. Solutions for injections were free of endotoxin (b 0.06 EU/ml) as determined using a limulus amebocyte lysate assay (Lonza, #50-648U). All injections in this study were from the same enzyme preparation. The specific activity of this lot was 20 U/mg (where 1 U = 1 μmol of 4-methylumbelliferyl-6thiopalmitoyl-β-D -glucoside (MU-6S-Palm-βGlc) hydrolyzed per minute [19]). 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucoside was obtained from Moscerdam Substrates. Mannose 6-phosphate receptor binding was 85% as determined by a column-binding assay [18]. All enzyme assays were conducted under conditions where the increase in fluorescent intensity was linear with respect to time and concentration and in comparison with known standards (purified PPT1 and 4-methylumbelliferone). Typically, each assay contained 20–50 μg of protein and incubations were carried out from 30 min to 1 h. Protein content was determined using the Dc protein assay (BioRad). Other reagents were obtained from Sigma-Aldrich unless otherwise noted.
2.3. Intrathecal mouse injections
2.5. Survival Mice were assessed weekly for body weight and more frequently for general health. Mice were sacrificed when a loss of greater than 10% of highest weight recorded was noted for two consecutive weeks, when animals could not right themselves, or were moribund (poorly responsive to tactile stimulation). The observer was blinded as to assignment of treatment groups. The Kaplan–Meier log-rank test for trend was used to determine whether there was an overall significant difference between the treatment groups. 2.6. Tissue processing, neuropathology and immunohistochemistry
2.2. Affinity-purified polyclonal antibodies Three New Zealand White rabbits were each immunized with 500 μg of purified human recombinant PPT1. The antigen was injected intradermally (final volume 1 ml) in Freund's complete adjuvant (Difco, #263910). An IgG fraction was prepared from preimmune serum by Protein A agarose chromatography. Human PPT1 antibody IgG was
Mice were anesthetized with Avertin and perfused transcardially with cold heparinized physiological saline followed by freshly prepared 4% formaldehyde in PBS, pH 7.4. Whole mice were then post-fixed in 4% formaldehyde for a further 48 h and transferred to 50 mM Tris. Spinal cords were separated from the brainstem just below the foramen magnum and dissected from the surrounding bone and musculature. Brains
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and spinal cords were cryoprotected in 30% sucrose in 50 mM TBS, pH 7.6 and 40 μm coronal sections were cut on a Microm HM430 Freezing Microtome (Microm International GmbH, Wallendorf, Germany). Cresyl violet staining and immunohistochemistry was performed as previously described [21,22]. For cresyl violet staining, a one in six series of coronal brain sections and a one in 24 series of spinal cord sections were mounted onto chrome-gelatin coated slides and allowed to dry overnight. Sections were then stained in 0.05% cresyl violet solution for 45 min at 60 °C for brain sections and 0.1% cresyl violet solution overnight at 60 °C for spinal cord sections, differentiated in alcohol, cleared in xylene and coverslipped in DPX. A one in six series of brain sections and a one in 24 series of spinal cord sections from each animal were immunostained free-floating for markers of astrocytosis (rabbit anti-GFAP, 1:8000 dilution, Dako Ltd, cat #Z0334) and microglial activation (rat anti-mouse CD68, 1:2000 dilution, AbD Serotec, cat #MCA1957). Sections were first quenched for endogenous peroxidase activity with 1% H2O2 in TBS, washed and then blocked in 15% normal serum (Vector Laboratories) diluted in TBS with 0.3% Triton-X100 (TBS-T). The normal serum was directed against the host species of the secondary antibody. After blocking, sections were incubated overnight at 4 °C in primary antibody diluted in 10% normal serum in TBS-T. Subsequently, sections were washed and incubated at room temperature in biotinylated secondary antibody (biotinylated swine anti-rabbit IgG, DAKO, cat #E0353; biotinylated rabbit anti-rat IgG, Vector Laboratories, cat #BA-4001), followed by washing and incubation in Vectastain Elite ABC solution (Vector Laboratories) before visualization with DAB (Sigma). Neuron counts were performed as previously described [21,22] on cresyl violet stained sections using StereoInvestigator software (MBF Bioscience, Williston, VT). Thresholding image analysis was carried out on GFAP and CD68 stained sections. Thirty non-overlapping images from three consecutive sections were captured from a defined region, with all parameters of light intensity, video camera setup and calibration settings kept constant. Images were analyzed using Image Pro Premier software (Media Cybernetics, Chicago, IL) by choosing an appropriate threshold that selected the foreground immunoreactivity above background. All analysis was done blinded to genotype and treatment status. An ordinary one-way ANOVA with a post test for linear trend was used to determine whether there was a dose-dependent treatment effect. The un-manipulated wild-type and Ppt1 knockout mice were not included in the analysis for treatment effect. For PPT1 activity assessment and immunohistochemistry, after heparin/PBS perfusion, the brain was harvested and sectioned sagittally; the right hemisphere of the brain was homogenized and assayed for PPT1 activity [18], the other half was post-fixed in 4% formaldehyde at room temperature for two days and then dehydrated, paraffin embedded, and sectioned according to standard histological protocol. Sections underwent antigen retrieval via heating in citrate buffer (BioGenex, cat# HK086-9K), for 10 min at 95 °C and were allowed to cool to room temperature, then were permeabilized with 0.3% Triton X-100 for 5 min. To quench endogenous peroxidase activity, sections were incubated for 30 min in 0.6% H2O2 and subsequently blocked for 30 min in commercially available 2.5% horse serum (Vector). Sections were incubated for 2 h at room temperature in affinity purified anti-hPPT1 polyclonal antibody (1.5 μg/ml), washed in PBS, followed by 30 min incubation in anti-rabbit peroxidase (Vector, cat# MP-7401), and then visualized with peroxidase substrate ImmPACT DAB (Vector, cat# SK-4105) according to the manufacturer's instructions. 3. Results and discussion 3.1. Effect of intrathecal ERT on motor performance Groups of 16–20 Ppt1 knockout mice at six weeks of age were randomly assigned to receive no injection or 0 (vehicle), 2.6, 5.3 or 10.6 mg/ml of human recombinant PPT1 injected at the L5/L6 lumbar
space on three consecutive days as shown in Fig. 1. Final outcome measures were motor performance as assessed by latency to fall from a Rotarod and disease-specific and overall survival. Concurrently maintained wild-type mice were tested for comparison. Rotarod performance using a constant speed paradigm was assessed as shown in Fig. 2. The maximum latency to fall on the third test day at each time point was used in the final analysis (maximum of 2 trials, 60 s trials) (Fig. 2A). Wild-type mice performed maximally (60 s) at each time point. Analysis was completed using a two-factor ANOVA model (treatment groups, 0, 2.6, 5.3 and 10.6 mg/ml dose; and time of measurement at 3, 5, 6, and 7 months). The effect of treatment was significant (p b 0.0001) as was interaction between time of measurement and treatment (p b 0.0001). Ordinary one-factor ANOVA was used to analyze data at month 8 for the 2.6, 5.3 and 10.6 mg/ml treatment groups (as no vehicle-only treated mice that could perform the test remained), and in this case, the effect was again significant (p b 0.01). 3.2. Effect of intrathecal ERT on survival Treatment on three consecutive days at 6 weeks of age with intrathecal PPT1 improved both disease-specific (Fig. 3A) and overall survival (Fig. 3B). For calculation of disease-specific survival, deaths prior to 150 days of age were censored to control for confounding secondary to technical problems related to the injections, as death prior to this age in Ppt1 knockout mice is very rare [10,23], a finding confirmed by overall survival of the un-manipulated Ppt1 knockout mice (Fig. 3B). As shown in Fig. 3A, disease-specific median survival was 233, 267, 272, and 284 days for mice receiving 0, 2.6, 5.3 and 10.6 mg/ml of PPT1, respectively. The effect of treatment was highly significant (p b 0.0001). The mean survival for uninjected Ppt1 knockout mice was 238 days, in concordance with previous studies (ranging from 231 to 255 days [13,24–28]) About 10–20% of mice in each group succumbed within several days of the injections, at the same rate regardless of whether injections contained PPT1 (Fig. 3B). (In fact, fewer mice died in the group receiving the highest dose of PPT1). These data suggest that the early deaths were related to injection-related trauma rather than to drug-related effects. Despite the early deaths, the effect of PPT1 treatment on overall survival was still also highly significant (p b 0.0001), and the early deaths had a negligible effect upon median survival, which was 231, 263, 262 and 282 days for mice receiving 0, 2.6, 5.3 and 10.6 mg/ml of PPT1, respectively. 3.3. Neuropathology In previous studies of the Ppt1 knockout mouse, the pathology was shown to proceed in an orderly progression, with astrocytosis detectable by GFAP staining in the first phase in months 1–4 followed by a wave of microglial activation which can be detected by F4/80 or CD68 staining during months 5–7 [21,22]. Autofluorescence accumulation progresses throughout the lifespan, and neuron loss is seen at late stages of disease. To assess the effects of intrathecal PPT1 on neuropathology, three mice chosen at random from each treatment group were sacrificed for detailed pathological examination of brain and spinal cord at 7.5 months of age (Figs. 4–7), a predetermined time when most Ppt1 knockout mice are near terminal. Three brain regions, S1 barrel field cortex (S1BF), medial and lateral ventroposterior nuclei of the thalamus (VPM/VPL) and the lateral geniculate nucleus (LGnD), and four spinal cord regions (dorsal and ventral regions of the cervical and lumbar cord) were examined for autofluorescence, CD68 and GFAP immunostaining, and by performing neuron counts on Nissl-stained sections. Because the mice were sacrificed 6 months from the time of the injections, it was anticipated that the greatest effects would be seen for the microglial marker (CD68 in this case) and this was indeed the case (Fig. 4). Strong dose-dependent treatment effects were seen in the somatosensory cortex and cervical and lumbar spinal cord of Ppt1 knockout mice (Fig. 4, A, D–F) with lesser effects in deep structures
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Inject PPT1 (lumbar spinal injection, PPT1 at 0, 2.6, 5.3 or 10.6 mg/ml) on three consecutive days, 16-20 mice/group
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Three mice/group sacrificed for pathology Fig. 1. Experimental design. Four groups of 16–20 Ppt1 knockout mice at six weeks of age were randomized to receive three consecutive daily lumbar spinal infusions of human recombinant PPT1 in 80 μl over 8 min at concentrations of 0, 2.6, 5.3 or 10.6 mg/ml. Rotarod testing was performed by a blinded observer at 3, 5, 6, 7, 8 and 9 months of age. Three mice from each group were sacrificed at 7.5 months for pathological examination. Unmanipulated groups of wild-type and Ppt1 knockout mice (n = 15) were tested for comparison.
additional mice receiving this dose as part of the treatment study were processed for analysis at terminal sacrifice (at 175, 205 and 248 days). As shown in Fig. 8, the decay of enzyme activity displayed two-phase kinetics, with fast (70%) and slow (30%) components. The half-life of the fast component was only about one day. Interestingly, about 20% of wild-type levels of PPT1 activity were present at 5 and 9 days after injection, and between 5–9% of wild-type activity was still present up to 248 days after injection, suggesting a stable pool of PPT1 persisting after the injection. (The activity was clearly attributable to exogenously
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such as thalamic nuclei and LGnD that did not reach statistical significance (Fig. 4, B–C). With respect to GFAP staining (which is a relatively early disease marker) (Fig. 5), animal-to-animal variability and a lowlevel astrocyte response to the infusion of human PPT1 precluded definitive conclusions. Nevertheless, two areas showed a significant reduction in GFAP staining, the VPM/VPL and dorsal cervical cord, and although similar effects were seen elsewhere, these did not reach statistical significance. Unbiased stereological neuron counts (Fig. 6) more clearly revealed dose-dependent treatment effects for the cervical dorsal and ventral spinal cord (Fig. 6, panels D and E). Neuron loss in the lumbar cord at this age was relatively small, and the effects of ERT administration were not significant. Finally, a significant decrease in autofluorescent storage material was demonstrated in the cervical and lumbar spinal cord (Fig. 7), which was somewhat surprising since 6 months had elapsed since treatment. These results suggest that either a pool of PPT1 persists in tissues longer than expected, or perhaps that the rate of accumulation of storage material is not uniform with respect to time, with a more pronounced effect resulting from earlier treatment. 3.4. Time course of PPT1 activity and immunoreactivity in brain after intrathecal injection In a separate study, three to four mice received injections of the highest concentration (10.6 mg/ml) of PPT1 and were sacrificed at 1, 3, 5, and 9 days post-injection and brains processed for PPT1 enzyme activity and immunohistochemistry. Furthermore, the brains of several
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Age (months) Fig. 2. Effect of IT ERT on latency to fall from a constant speed rotating rod (Rotarod). All tests were terminated after 60 s. Two trials were conducted daily on each of three consecutive days at 3, 5, 6, 7, 8, and 9 months of age. Maximum latency to fall on the third testing day is reported.
Fig. 3. Effect of IT ERT on disease-specific (A) and overall (B) survival. Kaplan–Meier plots analyzed using log-rank test for trend was significant for both disease-specific (p b 0.001) and overall survival (p b 0.001). Disease-specific mean survivals were 238, 233, 267, 272, and 284 days for control (uninjected), 0, 2.6, 5. 3, 10.6 mg/ml PPT1 injected groups, respectively. Overall mean survivals were 238, 231, 263, 262, and 282 days for control (uninjected), 0, 2.6, 5 .3, 10.6 mg/ml PPT1 injected groups, respectively.
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GFAP
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added PPT1, as the background measurements of control age-matched uninjected Ppt1 brain homogenates were no more than 3% of wildtype activity.) PPT1 immunohistochemistry (Fig. 9) showed immunoreactivity present in various brain regions, particularly in and around the Purkinje cell layer of the cerebellum, both within Purkinje cells and in smaller cells associated with Purkinje cells, and cell processes. Residual PPT1 immunoreactivity was still present at terminal sacrifice, mostly present as small clumps associated with non-neuronal cells in the Purkinje layer (Fig. 9D). While the immunoreactivity was best seen in the cerebellum, scattered rare punctate immunoreactivity was seen in the mid-brain and forebrain associated with non-neuronal cell bodies and cell processes (not shown).
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Fig. 4. Effect of IT ERT on CD68 immunohistochemical staining. Thresholding analysis of brain regions immunostained for microglial marker CD68 from three randomly selected 7.5 month-old mice from each group indicated are shown. Statistical significance is indicated for one-way ANOVA post-test for linear trend. (A) S1 barrel field cortex; (B) VPM/ VPL; (C) LGnD; (D) dorsal cervical spinal cord; (E) ventral cervical spinal cord; (F) dorsal lumbar spinal cord; (G) ventral lumbar spinal cord. Significant differences were seen for S1BF cortex (p = 0.0148) and highly significant differences for dorsal and ventral cervical and dorsal and ventral lumbar spinal cord (p = 0.0007, 0.0013, 0.0001, and 0.0010, respectively), but not significant for deep nuclear structures.
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Fig. 5. Effect of IT ERT on GFAP immunohistochemical staining. Thresholding analysis of brain regions immunostained for a marker for activated astrocytes, GFAP, from the three randomly selected 7.5 month-old mice from each group in Fig. 4 are shown. Statistical significance is indicated for ANOVA post-test for linear trend. (A) S1 barrel field cortex; (B) VPM/VPL; (C) LGnD; (D) dorsal cervical spinal cord; (E) ventral cervical spinal cord; (F) dorsal lumbar spinal cord; (G) ventral lumbar spinal cord. GFAP staining is an early marker of disease and showed inter-animal variability. No significant differences were recorded, with the exception of VPM/VPL (p = 0.0493) and dorsal cervical spinal cord (p = 0.001).
Therapeutic lysosomal enzymes delivered to the cerebrospinal fluid have been studied in animal models for about a dozen lysosomal storage disorders (LSDs), and all of these studies have shown reduction in substrate accumulation, and in many cases, functional improvements (reviewed in [29]). In this paper we have shown that intrathecal enzyme replacement at a single time point (6 weeks) in PPT1 deficient mice improves motor performance and prolongs lifespan up to 7 weeks in a dose-dependent fashion. It should be noted that our experiments in mice used the large-volume injection protocol developed in the Lobel laboratory [14]. Although this protocol is not directly applicable to humans, a continuous infusion device is currently being used for
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Neuron Counts
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Fig. 6. Effect of IT ERT on neuron counts. Neuron counts from the three randomly selected 7.5 month-old mice from each group in Fig. 4 indicated are shown. (A) S1 barrel field cortex; (B) VPM/VPL; (C) LGnD; (D) dorsal cervical spinal cord; (E) ventral cervical spinal cord; (F) dorsal lumbar spinal cord; (G) ventral lumbar spinal cord. One-way ANOVA post-test linear test for trend was significant for dorsal and ventral spinal cord (p = 0.0012 and 0.0065 respectively). Note that neuron loss from the lumbar spinal cord in affected mice was minimal.
intrathecal administration of enzyme replacement therapy in humans (ClinicalTrials.gov #NCT00920647). The results we obtained are similar to those seen in a model of the late infantile form of Batten disease (CLN2 disease), in which a similar dosing of TPP1 enzyme led to a dose-dependent survival increase from 16 weeks to 23 weeks (seven weeks), which is comparable to our study (33 weeks to 40 weeks, also an increase of seven weeks) [14]. Another key issue in translating such advances is how often they need to be administered, and at present the treatment interval being tested in human CLN2/late infantile Batten disease is two weeks (NCT#01907087). Depending on the LSD, the treatment interval in other human studies can be longer; MPS II (NCT#00920647; #01506141) and MPS IIIA patients (NCT#01299727) is four weeks; in MPS I patients (NCT#00852358), the interval is initially four weeks, but after three injections the treatment interval is increased to three months. The finding of persistent PPT1 activity and immunoreactivity was unexpected, and the significance of this finding remains to be explored.
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Fig. 7. Effect of IT ERT on autofluorescence. Autofluorescence from the three randomly selected 7.5 month-old mice from each group indicated are shown. Statistical significance is indicated for ANOVA post-test for linear trend. (A) S1 barrel field cortex; (B) VPM/VPL; (C) LGnD; (D) cervical spinal cord; (E) lumbar spinal cord; (F) dorsal lumbar spinal cord. Differences were significant for cervical and lumbar spinal cord (p = 0.0102 and 0.0267 respectively).
No attempt to correlate areas containing residual enzyme with clearance of storage material was made in the current study, but this could be examined in a future prospective study.
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Time (days) Fig. 8. PPT1 activity in whole brain homogenate at varying times after intrathecal injection. Animals were given three daily infusions of hPPT1 (80 μl of 10.6 μg/μl enzyme delivered at 10 μl/min) and sacrificed at the times indicated. Cytosolic fractions of the brains were prepared and assayed for PPT1 activity. N = 4, 3, 3, and 4 for days 1, 3, 5, and 9 respectively and n = 1 for later time points.
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pre-injection A
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Fig. 9. Immunohistochemical staining for PPT1 in Ppt1 knockout mouse cerebellum before and at varying times after intrathecal injection. (A) Ppt1 knockout mouse, age 6 weeks, injected with aCSF (B–D) Ppt1 knockout mice, age 6 weeks, injected with 10 .6 mg/ml PPT1 as described under Materials and methods and sacrificed at 1 day (B), 3 weeks (C), or 5 months (D) after the injection. (E) Wild-type control mouse brain. Brown staining indicates PPT1 immunostaining (indicated by arrowheads). Bar, 20 μm.
We did not test for the presence of anti-human PPT1 antibodies that may develop in Ppt1 deficient mice as a result of enzyme administration. Although it is possible that such antibodies, if present, may have diminished the effect of the treatment, in a previous study we could not detect such antibodies in mice treated chronically with human recombinant PPT1 administered intravenously on a weekly schedule, and so similar data was not sought here. The treatment was seemingly well tolerated with equal morbidity in mice injected with PPT1 or vehicle, and no anaphylaxis or any overt increase in the neuroinflammatory response was observed. 4. Conclusion Human recombinant PPT1 administered to the cerebrospinal fluid via lumbar injection on three consecutive days at six weeks of age to Ppt1 deficient mice prolonged their survival by seven weeks without apparent undue toxicity. The survival and performance improvements are similar to those reported for other LSD models that affect the brain that are currently in clinical trials using this approach. These data provide a rationale for ERT being delivered to the cerebrospinal fluid in children with PPT1-deficient NCL. Acknowledgments The authors wish to thank Drs. Peter Lobel and David Sleat for advice in the experimental design, Dr. James Richardson and John Shelton of the UT Southwestern Molecular Pathology Core for PPT1 immunostaining and microscopy, and Lauren Peca for performing the behavioral tests. This work was funded by Taylor's Tale, the Batten Disease Support and Research Association, the Batten Disease Family Association, and a King's College London Graduate School International Studentship award to HRN. References [1] J.W. Mink, E.F. Augustine, H.R. Adams, F.J. Marshall, J.M. Kwon, Classification and natural history of the neuronal ceroid lipofuscinoses, J. Child Neurol. 28 (2013) 1101–1105. [2] J. Vesa, E. Hellsten, L.A. Verkruyse, L.A. Camp, J. Rapola, P. Santavuori, S.L. Hofmann, L. Peltonen, Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis, Nature 376 (1995) 584–587. [3] J.Y. Lu, S.L. Hofmann, Thematic review series: lipid posttranslational modifications. Lysosomal metabolism of lipid-modified proteins, J. Lipid Res. 47 (2006) 1352–1357. [4] M. Kousi, A.E. Lehesjoki, S.E. Mole, Update of the mutation spectrum and clinical correlations of over 360 mutations in eight genes that underlie the neuronal ceroid lipofuscinoses, Hum. Mutat. 33 (2012) 42–63. [5] A.K. Das, C.H. Becerra, W. Yi, J.Y. Lu, A.N. Siakotos, K.E. Wisniewski, S.L. Hofmann, Molecular genetics of palmitoyl-protein thioesterase deficiency in the U.S. J. Clin. Invest. 102 (1998) 361–370.
[6] O.P. van Diggelen, S. Thobois, C. Tilikete, M.T. Zabot, J.L. Keulemans, P.A. van Bunderen, P.E. Taschner, M. Losekoot, Y.V. Voznyi, Adult neuronal ceroid lipofuscinosis with palmitoyl-protein thioesterase deficiency: first adult-onset patients of a childhood disease, Ann. Neurol. 50 (2001) 269–272. [7] H. Ramadan, A.S. Al-Din, A. Ismail, F. Balen, A. Varma, A. Twomey, R. Watts, M. Jackson, G. Anderson, E. Green, S.E. Mole, Adult neuronal ceroid lipofuscinosis caused by deficiency in palmitoyl protein thioesterase 1, Neurology 68 (2007) 387–388. [8] T. Lonnqvist, S.L. Vanhanen, K. Vettenranta, T. Autti, J. Rapola, P. Santavuori, U.M. Saarinen-Pihkala, Hematopoietic stem cell transplantation in infantile neuronal ceroid lipofuscinosis, Neurology 57 (2001) 1411–1416. [9] J.A. Hawkins-Salsbury, J.D. Cooper, M.S. Sands, Pathogenesis and therapies for infantile neuronal ceroid lipofuscinosis (infantile CLN1 disease), Biochim. Biophys. Acta 1832 (2013) 1906–1909. [10] P. Gupta, A.A. Soyombo, A. Atashband, K.E. Wisniewski, J.M. Shelton, J.A. Richardson, R.E. Hammer, S.L. Hofmann, Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 13566–13571. [11] N. Galvin, C. Vogler, B. Levy, A. Kovacs, M. Griffey, M.S. Sands, A murine model of infantile neuronal ceroid lipofuscinosis-ultrastructural evaluation of storage in the central nervous system and viscera, Pediatr. Dev. Pathol. 11 (2008) 185–192. [12] M.A. Griffey, D. Wozniak, M. Wong, E. Bible, K. Johnson, S.M. Rothman, A.E. Wentz, J.D. Cooper, M.S. Sands, CNS-directed AAV2-mediated gene therapy ameliorates functional deficits in a murine model of infantile neuronal ceroid lipofuscinosis, Mol. Ther. 13 (2006) 538–547. [13] J. Hu, J.Y. Lu, A.M. Wong, L.S. Hynan, S.G. Birnbaum, D.S. Yilmaz, B.M. Streit, E.M. Lenartowicz, T.C. Thompson, J.D. Cooper, S.L. Hofmann, Intravenous high-dose enzyme replacement therapy with recombinant palmitoyl-protein thioesterase reduces visceral lysosomal storage and modestly prolongs survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis, Mol. Genet. Metab. 107 (2012) 213–221. [14] S. Xu, L. Wang, M. El-Banna, I. Sohar, D.E. Sleat, P. Lobel, Large-volume intrathecal enzyme delivery increases survival of a mouse model of late infantile neuronal ceroid lipofuscinosis, Mol. Ther. 19 (2011) 1842–1848. [15] B.R. Vuillemenot, M.L. Katz, J.R. Coates, D. Kennedy, P. Tiger, S. Kanazono, P. Lobel, I. Sohar, S. Xu, R. Cahayag, S. Keve, E. Koren, S. Bunting, L.S. Tsuruda, C.A. O'Neill, Intrathecal tripeptidyl-peptidase 1 reduces lysosomal storage in a canine model of late infantile neuronal ceroid lipofuscinosis, Mol. Genet. Metab. 104 (2011) 325–337. [16] M.L. Katz, J.R. Coates, C.M. Sibigtroth, J.D. Taylor, M. Carpentier, W.M. Young, F.A. Wininger, D. Kennedy, B.R. Vuillemenot, C.A. O'Neill, Enzyme replacement therapy attenuates disease progression in a canine model of late-infantile neuronal ceroid lipofuscinosis (CLN2 disease), J. Neurosci. Res. 92 (2014) 1591–1598. [17] P. Calias, M. Papisov, J. Pan, N. Savioli, V. Belov, Y. Huang, J. Lotterhand, M. Alessandrini, N. Liu, A.J. Fischman, J.L. Powell, M.W. Heartlein, CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and mouse: implications for neurological outcomes of lysosomal storage disorder, PLoS One 7 (2012) e30341. [18] J.Y. Lu, J. Hu, S.L. Hofmann, Human recombinant palmitoyl-protein thioesterase-1 (PPT1) for preclinical evaluation of enzyme replacement therapy for infantile neuronal ceroid lipofuscinosis, Mol. Genet. Metab. 99 (2010) 374–378. [19] O.P. van Diggelen, J.L. Keulemans, B. Winchester, I.L. Hofman, S.L. Vanhanen, P. Santavuori, Y.V. Voznyi, A rapid fluorogenic palmitoyl-protein thioesterase assay: pre- and postnatal diagnosis of INCL, Mol. Genet. Metab. 66 (1999) 240–244. [20] A. Urayama, J.H. Grubb, W.S. Sly, W.A. Banks, Developmentally regulated mannose 6-phosphate receptor-mediated transport of a lysosomal enzyme across the blood–brain barrier, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 12658–12663. [21] E. Bible, P. Gupta, S.L. Hofmann, J.D. Cooper, Regional and cellular neuropathology in the palmitoyl protein thioesterase-1 null mutant mouse model of infantile neuronal ceroid lipofuscinosis, Neurobiol. Dis. 16 (2004) 346–359.
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Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Intrathecal enzyme replacement therapy improves motor function and survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis Jui-Yun Lu a,c, Hemanth R. Nelvagal d, Lingling Wang a,c, Shari G. Birnbaum b, Jonathan D. Cooper d, Sandra L. Hofmann a,c,⁎ a
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA Department of Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA c Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA d Pediatric Storage Disorders Laboratory, Department of Basic and Clinical Neuroscience, King's Health Partners Centre for Neurodegeneration, James Black Centre, Institute of Psychiatry, Psychology & Neuroscience, King's College London, 125 Coldharbour Lane, London SE5 9NU, UK b
a r t i c l e
i n f o
Article history: Received 7 April 2015 Received in revised form 9 May 2015 Accepted 10 May 2015 Available online 12 May 2015 Keywords: Enzyme replacement therapy Palmitoyl protein thioesterase Lysosomal storage disorder Batten disease Infantile neuronal ceroid lipofuscinosis
a b s t r a c t The neuronal ceroid lipofuscinoses (NCLs) are a group of related hereditary lysosomal storage disorders characterized by progressive loss of neurons in the central nervous system resulting in dementia, loss of motor skills, seizures and blindness. A characteristic intralysosomal accumulation of autofluorescent storage material occurs in the brain and other tissues. Three major forms and nearly a dozen minor forms of NCL are recognized. Infantile-onset NCL (CLN1 disease) is caused by severe deficiency in a soluble lysosomal enzyme, palmitoylprotein thioesterase-1 (PPT1) and no therapy beyond supportive care is available. Homozygous Ppt1 knockout mice reproduce the known features of the disease, developing signs of motor dysfunction at 5 months of age and death around 8 months. Direct delivery of lysosomal enzymes to the cerebrospinal fluid is an approach that has gained traction in small and large animal models of several other neuropathic lysosomal storage diseases, and has advanced to clinical trials. In the current study, Ppt1 knockout mice were treated with purified recombinant human PPT1 enzyme delivered to the lumbar intrathecal space on each of three consecutive days at 6 weeks of age. Untreated PPT1 knockout mice and wild-type mice served as additional controls. Four enzyme concentration levels (0, 2.6, 5.3 and 10.6 mg/ml of specific activity 20 U/mg) were administered in a volume of 80 μl infused over 8 min. Each group consisted of 16–20 mice. The treatment was well tolerated. Diseasespecific survival was 233, 267, 272, and 284 days for each of the four treatment groups, respectively, and the effect of treatment was highly significant (p b 0.0001). The timing of motor deterioration was also delayed. Neuropathology was improved as evidenced by decreased autofluorescent storage material in the spinal cord and a decrease in CD68 staining in the cortex and spinal cord. The improvements in motor function and survival are similar to results reported for preclinical studies involving other lysosomal storage disorders, such as CLN2/ TPP1 deficiency, for which intraventricular ERT is being offered in clinical trials. If ERT delivery to the CSF proves to be efficacious in these disorders, PPT1 deficiency may also be amenable to this approach. © 2015 Elsevier Inc. All rights reserved.
1. Introduction The neuronal ceroid lipofuscinoses are a group of neurodegenerative disorders characterized by widespread accumulation of autofluorescent storage material in lysosomes and a progressive loss of neurons in the central nervous system [1]. Deficiency in the enzyme palmitoyl protein Abbreviations: ERT, enzyme replacement therapy; CLN1, ceroid lipofuscinosis, neuronal-1; CLN2, ceroid lipofuscinosis, neuronal-2; LSD, lysosomal storage disorder; MU6S-Palm-βGlc, 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucoside; NCL, neuronal ceroid lipofuscinosis; PPT1, palmitoyl-protein thioesterase-1; TPP1, tripeptidyl peptidase-1. ⁎ Corresponding author at: Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8593, USA. E-mail address: [email protected] (S.L. Hofmann).
http://dx.doi.org/10.1016/j.ymgme.2015.05.005 1096-7192/© 2015 Elsevier Inc. All rights reserved.
thioesterase-1 (PPT1; EC 3.1.2.22) causes the autosomal recessive storage disorder infantile neuronal ceroid lipofuscinosis, or CLN1 disease [2]. This enzyme is a small, globular hydrolase of the α/β type that removes fatty acids from cysteine residues in lipid-modified proteins during lysosomal protein degradation [3]. Over 60 mutations in the PPT1/CLN1 gene in NCL patients have been described [4]. Complete absence of enzyme activity results in severe neurodegeneration occurring in infancy and is characterized by progressive cognitive and motor deterioration, blindness, and seizures leading to premature death [2]. Childhood and adult onset cases are associated with missense mutations that allow for varying levels of residual enzyme activity in the range of 2–7% [5–7]. Hematopoietic stem cell transplantation has had no effect on the course of affected infants [8]. No effective treatment is available and
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affinity-purified using the AminoLink Plus Immobilization Kit (Thermo Scientific, #44894) following the manufacturer's instructions.
various approaches, including small molecule, enzyme replacement, and gene and cellular therapies are the subject of preclinical studies [9]. The Ppt1 knockout mouse demonstrates the major features associated with the human disease, including autofluorescent storage and manifestations such as seizures, decline in motor performance and reduced lifespan [10,11]. The mice live about 235 days in the absence of treatment [10,12]. We have previously shown that PPT1 administered weekly via the tail vein is effective in clearing visceral storage, but has no impact on motor deterioration or survival when the treatment is started after blood brain barrier closure in the mouse, which occurs at 3 weeks of age [13]. About a three-week survival advantage and delay in onset of motor symptoms was shown when treatment was started at birth, suggesting that the brain would be responsive to ERT if appropriate access could be achieved. In addition to infantile NCL, several of the other NCLs (CLN2, 5 and 10) are caused by deficiencies of soluble lysosomal enzymes that can be supplied exogenously to cells through the mannose 6-phosphate receptor pathway. Classic late-infantile NCL (CLN2 disease) is caused by deficiency in the soluble lysosomal protease, tripeptidyl peptidase-1 (TPP1). Treatment of TPP1 knockout mice with recombinant TPP1 enzyme (10 mg/ml) delivered to the cerebrospinal fluid via lumbar intrathecal injection (given on three consecutive days) in mice at 4 weeks of age prolonged lifespan of CLN2 knockout mice from 16 to 23 weeks [14]. Based on these encouraging results in mice, the approach was successfully applied to the TPP1-deficient dog model [15–17] and has progressed to a human clinical trial (ClinicalTrials.gov ID# NCT01907087). In the current study we have used the same protocol to treat Ppt1 knockout mice with human recombinant PPT1 and show a dose-dependent positive effect on motor function, brain pathology and survival.
All procedures were carried out under an Institutional Animal Care and Use Committee-approved protocol at the University of Texas Southwestern Medical Center. Ppt1 knockout mice [10] were maintained as homozygous breeding stock on a C57BL/6 background, housed in a barrier facility and received food and water ad libitum. Treatment groups were assigned randomly from littermates born within a 2–3 day window after timed mating and group housed. All assessments were carried out by individuals blinded with respect to treatment group and genotype. Concurrent groups of untreated Ppt1 knockout mice and wild-type C57BL/6 mice were maintained for comparison but were not included in the analysis for treatment effects. Each treatment group consisted of between 16 and 20 mice at about 6 weeks of age. Six weeks of age is in the presymptomatic period of Ppt1 knockout mice [13] and well after the permeability transition for lysosomal enzymes to reach the brain from the systemic circulation in the neonatal mouse [20]. For the enzyme replacement therapy, mice were anesthetized with isoflurane delivered through an inhalation system (EZ-Anesthesia Classic System, Braintree). Animals were shaved to expose the skin around the lumbar region and injected between vertebrae L5 and L6 using a 30-gauge needle (Becton Dickinson) oriented toward L4 as described [14]. The needle was connected to a 100 μl gas-tight Hamilton syringe (#81075) by a short length of silastic tubing (0.3 mm inner diameter, Dow Corning, #2415496). The dose (vehicle or enzyme) was administered at 10 μl per min using an NE-300 syringe pump (New Era Pump Systems).
2. Materials and methods
2.4. Rotarod assessments of motor performance
2.1. Human recombinant PPT1
For motor coordination testing, mice were tested on a Rotarod (model 755, IITC Life Science Inc., Woodland Hills, CA) at 3, 5, 6, 7, 8 and 9 months of age and the latency to fall in seconds recorded. Trials were terminated after a maximum of 60 s. At each age, mice underwent a pre-test trial on a stationary rod, followed by two test trials on the constant speed Rotarod (3 rpm) for each of three consecutive days. The maximum latency to fall in the two test trials on the final day of testing was reported as the final outcome measure. A two-factor (treatment group and time of measurement) ordinary ANOVA model was used to analyze data for vehicle, 2.6, 5.3, and 10.6 mg/ml PPT1 dosing groups for months 3, 5, 6 and 7. Ordinary one-way ANOVA (treatment group) was used to analyze month 8 for the 2.6, 5.3 and 10.6 mg/ml treatment groups (as all mice in the vehicle group had died by month 8).
Human recombinant PPT1 was prepared from an overproducing Chinese Hamster Ovary (CHO) clonal cell line as described [18] except that the enzyme was exchanged into an artificial CSF buffer at the final step (aCSF, 148 mM NaCl, 3 mM KCl, 1.4 mM CaCl2, 0.9 mM MgCl2, 0.8 mM Na2HPO4, 0.2 mM NaH2PO4, pH 7.2). Enzyme was concentrated using a stirred cell equipped with a YM-10 membrane (Millipore) at 4 °C. Solutions for injections were free of endotoxin (b 0.06 EU/ml) as determined using a limulus amebocyte lysate assay (Lonza, #50-648U). All injections in this study were from the same enzyme preparation. The specific activity of this lot was 20 U/mg (where 1 U = 1 μmol of 4-methylumbelliferyl-6thiopalmitoyl-β-D -glucoside (MU-6S-Palm-βGlc) hydrolyzed per minute [19]). 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucoside was obtained from Moscerdam Substrates. Mannose 6-phosphate receptor binding was 85% as determined by a column-binding assay [18]. All enzyme assays were conducted under conditions where the increase in fluorescent intensity was linear with respect to time and concentration and in comparison with known standards (purified PPT1 and 4-methylumbelliferone). Typically, each assay contained 20–50 μg of protein and incubations were carried out from 30 min to 1 h. Protein content was determined using the Dc protein assay (BioRad). Other reagents were obtained from Sigma-Aldrich unless otherwise noted.
2.3. Intrathecal mouse injections
2.5. Survival Mice were assessed weekly for body weight and more frequently for general health. Mice were sacrificed when a loss of greater than 10% of highest weight recorded was noted for two consecutive weeks, when animals could not right themselves, or were moribund (poorly responsive to tactile stimulation). The observer was blinded as to assignment of treatment groups. The Kaplan–Meier log-rank test for trend was used to determine whether there was an overall significant difference between the treatment groups. 2.6. Tissue processing, neuropathology and immunohistochemistry
2.2. Affinity-purified polyclonal antibodies Three New Zealand White rabbits were each immunized with 500 μg of purified human recombinant PPT1. The antigen was injected intradermally (final volume 1 ml) in Freund's complete adjuvant (Difco, #263910). An IgG fraction was prepared from preimmune serum by Protein A agarose chromatography. Human PPT1 antibody IgG was
Mice were anesthetized with Avertin and perfused transcardially with cold heparinized physiological saline followed by freshly prepared 4% formaldehyde in PBS, pH 7.4. Whole mice were then post-fixed in 4% formaldehyde for a further 48 h and transferred to 50 mM Tris. Spinal cords were separated from the brainstem just below the foramen magnum and dissected from the surrounding bone and musculature. Brains
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and spinal cords were cryoprotected in 30% sucrose in 50 mM TBS, pH 7.6 and 40 μm coronal sections were cut on a Microm HM430 Freezing Microtome (Microm International GmbH, Wallendorf, Germany). Cresyl violet staining and immunohistochemistry was performed as previously described [21,22]. For cresyl violet staining, a one in six series of coronal brain sections and a one in 24 series of spinal cord sections were mounted onto chrome-gelatin coated slides and allowed to dry overnight. Sections were then stained in 0.05% cresyl violet solution for 45 min at 60 °C for brain sections and 0.1% cresyl violet solution overnight at 60 °C for spinal cord sections, differentiated in alcohol, cleared in xylene and coverslipped in DPX. A one in six series of brain sections and a one in 24 series of spinal cord sections from each animal were immunostained free-floating for markers of astrocytosis (rabbit anti-GFAP, 1:8000 dilution, Dako Ltd, cat #Z0334) and microglial activation (rat anti-mouse CD68, 1:2000 dilution, AbD Serotec, cat #MCA1957). Sections were first quenched for endogenous peroxidase activity with 1% H2O2 in TBS, washed and then blocked in 15% normal serum (Vector Laboratories) diluted in TBS with 0.3% Triton-X100 (TBS-T). The normal serum was directed against the host species of the secondary antibody. After blocking, sections were incubated overnight at 4 °C in primary antibody diluted in 10% normal serum in TBS-T. Subsequently, sections were washed and incubated at room temperature in biotinylated secondary antibody (biotinylated swine anti-rabbit IgG, DAKO, cat #E0353; biotinylated rabbit anti-rat IgG, Vector Laboratories, cat #BA-4001), followed by washing and incubation in Vectastain Elite ABC solution (Vector Laboratories) before visualization with DAB (Sigma). Neuron counts were performed as previously described [21,22] on cresyl violet stained sections using StereoInvestigator software (MBF Bioscience, Williston, VT). Thresholding image analysis was carried out on GFAP and CD68 stained sections. Thirty non-overlapping images from three consecutive sections were captured from a defined region, with all parameters of light intensity, video camera setup and calibration settings kept constant. Images were analyzed using Image Pro Premier software (Media Cybernetics, Chicago, IL) by choosing an appropriate threshold that selected the foreground immunoreactivity above background. All analysis was done blinded to genotype and treatment status. An ordinary one-way ANOVA with a post test for linear trend was used to determine whether there was a dose-dependent treatment effect. The un-manipulated wild-type and Ppt1 knockout mice were not included in the analysis for treatment effect. For PPT1 activity assessment and immunohistochemistry, after heparin/PBS perfusion, the brain was harvested and sectioned sagittally; the right hemisphere of the brain was homogenized and assayed for PPT1 activity [18], the other half was post-fixed in 4% formaldehyde at room temperature for two days and then dehydrated, paraffin embedded, and sectioned according to standard histological protocol. Sections underwent antigen retrieval via heating in citrate buffer (BioGenex, cat# HK086-9K), for 10 min at 95 °C and were allowed to cool to room temperature, then were permeabilized with 0.3% Triton X-100 for 5 min. To quench endogenous peroxidase activity, sections were incubated for 30 min in 0.6% H2O2 and subsequently blocked for 30 min in commercially available 2.5% horse serum (Vector). Sections were incubated for 2 h at room temperature in affinity purified anti-hPPT1 polyclonal antibody (1.5 μg/ml), washed in PBS, followed by 30 min incubation in anti-rabbit peroxidase (Vector, cat# MP-7401), and then visualized with peroxidase substrate ImmPACT DAB (Vector, cat# SK-4105) according to the manufacturer's instructions. 3. Results and discussion 3.1. Effect of intrathecal ERT on motor performance Groups of 16–20 Ppt1 knockout mice at six weeks of age were randomly assigned to receive no injection or 0 (vehicle), 2.6, 5.3 or 10.6 mg/ml of human recombinant PPT1 injected at the L5/L6 lumbar
space on three consecutive days as shown in Fig. 1. Final outcome measures were motor performance as assessed by latency to fall from a Rotarod and disease-specific and overall survival. Concurrently maintained wild-type mice were tested for comparison. Rotarod performance using a constant speed paradigm was assessed as shown in Fig. 2. The maximum latency to fall on the third test day at each time point was used in the final analysis (maximum of 2 trials, 60 s trials) (Fig. 2A). Wild-type mice performed maximally (60 s) at each time point. Analysis was completed using a two-factor ANOVA model (treatment groups, 0, 2.6, 5.3 and 10.6 mg/ml dose; and time of measurement at 3, 5, 6, and 7 months). The effect of treatment was significant (p b 0.0001) as was interaction between time of measurement and treatment (p b 0.0001). Ordinary one-factor ANOVA was used to analyze data at month 8 for the 2.6, 5.3 and 10.6 mg/ml treatment groups (as no vehicle-only treated mice that could perform the test remained), and in this case, the effect was again significant (p b 0.01). 3.2. Effect of intrathecal ERT on survival Treatment on three consecutive days at 6 weeks of age with intrathecal PPT1 improved both disease-specific (Fig. 3A) and overall survival (Fig. 3B). For calculation of disease-specific survival, deaths prior to 150 days of age were censored to control for confounding secondary to technical problems related to the injections, as death prior to this age in Ppt1 knockout mice is very rare [10,23], a finding confirmed by overall survival of the un-manipulated Ppt1 knockout mice (Fig. 3B). As shown in Fig. 3A, disease-specific median survival was 233, 267, 272, and 284 days for mice receiving 0, 2.6, 5.3 and 10.6 mg/ml of PPT1, respectively. The effect of treatment was highly significant (p b 0.0001). The mean survival for uninjected Ppt1 knockout mice was 238 days, in concordance with previous studies (ranging from 231 to 255 days [13,24–28]) About 10–20% of mice in each group succumbed within several days of the injections, at the same rate regardless of whether injections contained PPT1 (Fig. 3B). (In fact, fewer mice died in the group receiving the highest dose of PPT1). These data suggest that the early deaths were related to injection-related trauma rather than to drug-related effects. Despite the early deaths, the effect of PPT1 treatment on overall survival was still also highly significant (p b 0.0001), and the early deaths had a negligible effect upon median survival, which was 231, 263, 262 and 282 days for mice receiving 0, 2.6, 5.3 and 10.6 mg/ml of PPT1, respectively. 3.3. Neuropathology In previous studies of the Ppt1 knockout mouse, the pathology was shown to proceed in an orderly progression, with astrocytosis detectable by GFAP staining in the first phase in months 1–4 followed by a wave of microglial activation which can be detected by F4/80 or CD68 staining during months 5–7 [21,22]. Autofluorescence accumulation progresses throughout the lifespan, and neuron loss is seen at late stages of disease. To assess the effects of intrathecal PPT1 on neuropathology, three mice chosen at random from each treatment group were sacrificed for detailed pathological examination of brain and spinal cord at 7.5 months of age (Figs. 4–7), a predetermined time when most Ppt1 knockout mice are near terminal. Three brain regions, S1 barrel field cortex (S1BF), medial and lateral ventroposterior nuclei of the thalamus (VPM/VPL) and the lateral geniculate nucleus (LGnD), and four spinal cord regions (dorsal and ventral regions of the cervical and lumbar cord) were examined for autofluorescence, CD68 and GFAP immunostaining, and by performing neuron counts on Nissl-stained sections. Because the mice were sacrificed 6 months from the time of the injections, it was anticipated that the greatest effects would be seen for the microglial marker (CD68 in this case) and this was indeed the case (Fig. 4). Strong dose-dependent treatment effects were seen in the somatosensory cortex and cervical and lumbar spinal cord of Ppt1 knockout mice (Fig. 4, A, D–F) with lesser effects in deep structures
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Three mice/group sacrificed for pathology Fig. 1. Experimental design. Four groups of 16–20 Ppt1 knockout mice at six weeks of age were randomized to receive three consecutive daily lumbar spinal infusions of human recombinant PPT1 in 80 μl over 8 min at concentrations of 0, 2.6, 5.3 or 10.6 mg/ml. Rotarod testing was performed by a blinded observer at 3, 5, 6, 7, 8 and 9 months of age. Three mice from each group were sacrificed at 7.5 months for pathological examination. Unmanipulated groups of wild-type and Ppt1 knockout mice (n = 15) were tested for comparison.
additional mice receiving this dose as part of the treatment study were processed for analysis at terminal sacrifice (at 175, 205 and 248 days). As shown in Fig. 8, the decay of enzyme activity displayed two-phase kinetics, with fast (70%) and slow (30%) components. The half-life of the fast component was only about one day. Interestingly, about 20% of wild-type levels of PPT1 activity were present at 5 and 9 days after injection, and between 5–9% of wild-type activity was still present up to 248 days after injection, suggesting a stable pool of PPT1 persisting after the injection. (The activity was clearly attributable to exogenously
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such as thalamic nuclei and LGnD that did not reach statistical significance (Fig. 4, B–C). With respect to GFAP staining (which is a relatively early disease marker) (Fig. 5), animal-to-animal variability and a lowlevel astrocyte response to the infusion of human PPT1 precluded definitive conclusions. Nevertheless, two areas showed a significant reduction in GFAP staining, the VPM/VPL and dorsal cervical cord, and although similar effects were seen elsewhere, these did not reach statistical significance. Unbiased stereological neuron counts (Fig. 6) more clearly revealed dose-dependent treatment effects for the cervical dorsal and ventral spinal cord (Fig. 6, panels D and E). Neuron loss in the lumbar cord at this age was relatively small, and the effects of ERT administration were not significant. Finally, a significant decrease in autofluorescent storage material was demonstrated in the cervical and lumbar spinal cord (Fig. 7), which was somewhat surprising since 6 months had elapsed since treatment. These results suggest that either a pool of PPT1 persists in tissues longer than expected, or perhaps that the rate of accumulation of storage material is not uniform with respect to time, with a more pronounced effect resulting from earlier treatment. 3.4. Time course of PPT1 activity and immunoreactivity in brain after intrathecal injection In a separate study, three to four mice received injections of the highest concentration (10.6 mg/ml) of PPT1 and were sacrificed at 1, 3, 5, and 9 days post-injection and brains processed for PPT1 enzyme activity and immunohistochemistry. Furthermore, the brains of several
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Fig. 3. Effect of IT ERT on disease-specific (A) and overall (B) survival. Kaplan–Meier plots analyzed using log-rank test for trend was significant for both disease-specific (p b 0.001) and overall survival (p b 0.001). Disease-specific mean survivals were 238, 233, 267, 272, and 284 days for control (uninjected), 0, 2.6, 5. 3, 10.6 mg/ml PPT1 injected groups, respectively. Overall mean survivals were 238, 231, 263, 262, and 282 days for control (uninjected), 0, 2.6, 5 .3, 10.6 mg/ml PPT1 injected groups, respectively.
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added PPT1, as the background measurements of control age-matched uninjected Ppt1 brain homogenates were no more than 3% of wildtype activity.) PPT1 immunohistochemistry (Fig. 9) showed immunoreactivity present in various brain regions, particularly in and around the Purkinje cell layer of the cerebellum, both within Purkinje cells and in smaller cells associated with Purkinje cells, and cell processes. Residual PPT1 immunoreactivity was still present at terminal sacrifice, mostly present as small clumps associated with non-neuronal cells in the Purkinje layer (Fig. 9D). While the immunoreactivity was best seen in the cerebellum, scattered rare punctate immunoreactivity was seen in the mid-brain and forebrain associated with non-neuronal cell bodies and cell processes (not shown).
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Fig. 4. Effect of IT ERT on CD68 immunohistochemical staining. Thresholding analysis of brain regions immunostained for microglial marker CD68 from three randomly selected 7.5 month-old mice from each group indicated are shown. Statistical significance is indicated for one-way ANOVA post-test for linear trend. (A) S1 barrel field cortex; (B) VPM/ VPL; (C) LGnD; (D) dorsal cervical spinal cord; (E) ventral cervical spinal cord; (F) dorsal lumbar spinal cord; (G) ventral lumbar spinal cord. Significant differences were seen for S1BF cortex (p = 0.0148) and highly significant differences for dorsal and ventral cervical and dorsal and ventral lumbar spinal cord (p = 0.0007, 0.0013, 0.0001, and 0.0010, respectively), but not significant for deep nuclear structures.
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Fig. 5. Effect of IT ERT on GFAP immunohistochemical staining. Thresholding analysis of brain regions immunostained for a marker for activated astrocytes, GFAP, from the three randomly selected 7.5 month-old mice from each group in Fig. 4 are shown. Statistical significance is indicated for ANOVA post-test for linear trend. (A) S1 barrel field cortex; (B) VPM/VPL; (C) LGnD; (D) dorsal cervical spinal cord; (E) ventral cervical spinal cord; (F) dorsal lumbar spinal cord; (G) ventral lumbar spinal cord. GFAP staining is an early marker of disease and showed inter-animal variability. No significant differences were recorded, with the exception of VPM/VPL (p = 0.0493) and dorsal cervical spinal cord (p = 0.001).
Therapeutic lysosomal enzymes delivered to the cerebrospinal fluid have been studied in animal models for about a dozen lysosomal storage disorders (LSDs), and all of these studies have shown reduction in substrate accumulation, and in many cases, functional improvements (reviewed in [29]). In this paper we have shown that intrathecal enzyme replacement at a single time point (6 weeks) in PPT1 deficient mice improves motor performance and prolongs lifespan up to 7 weeks in a dose-dependent fashion. It should be noted that our experiments in mice used the large-volume injection protocol developed in the Lobel laboratory [14]. Although this protocol is not directly applicable to humans, a continuous infusion device is currently being used for
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Fig. 6. Effect of IT ERT on neuron counts. Neuron counts from the three randomly selected 7.5 month-old mice from each group in Fig. 4 indicated are shown. (A) S1 barrel field cortex; (B) VPM/VPL; (C) LGnD; (D) dorsal cervical spinal cord; (E) ventral cervical spinal cord; (F) dorsal lumbar spinal cord; (G) ventral lumbar spinal cord. One-way ANOVA post-test linear test for trend was significant for dorsal and ventral spinal cord (p = 0.0012 and 0.0065 respectively). Note that neuron loss from the lumbar spinal cord in affected mice was minimal.
intrathecal administration of enzyme replacement therapy in humans (ClinicalTrials.gov #NCT00920647). The results we obtained are similar to those seen in a model of the late infantile form of Batten disease (CLN2 disease), in which a similar dosing of TPP1 enzyme led to a dose-dependent survival increase from 16 weeks to 23 weeks (seven weeks), which is comparable to our study (33 weeks to 40 weeks, also an increase of seven weeks) [14]. Another key issue in translating such advances is how often they need to be administered, and at present the treatment interval being tested in human CLN2/late infantile Batten disease is two weeks (NCT#01907087). Depending on the LSD, the treatment interval in other human studies can be longer; MPS II (NCT#00920647; #01506141) and MPS IIIA patients (NCT#01299727) is four weeks; in MPS I patients (NCT#00852358), the interval is initially four weeks, but after three injections the treatment interval is increased to three months. The finding of persistent PPT1 activity and immunoreactivity was unexpected, and the significance of this finding remains to be explored.
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Fig. 7. Effect of IT ERT on autofluorescence. Autofluorescence from the three randomly selected 7.5 month-old mice from each group indicated are shown. Statistical significance is indicated for ANOVA post-test for linear trend. (A) S1 barrel field cortex; (B) VPM/VPL; (C) LGnD; (D) cervical spinal cord; (E) lumbar spinal cord; (F) dorsal lumbar spinal cord. Differences were significant for cervical and lumbar spinal cord (p = 0.0102 and 0.0267 respectively).
No attempt to correlate areas containing residual enzyme with clearance of storage material was made in the current study, but this could be examined in a future prospective study.
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Time (days) Fig. 8. PPT1 activity in whole brain homogenate at varying times after intrathecal injection. Animals were given three daily infusions of hPPT1 (80 μl of 10.6 μg/μl enzyme delivered at 10 μl/min) and sacrificed at the times indicated. Cytosolic fractions of the brains were prepared and assayed for PPT1 activity. N = 4, 3, 3, and 4 for days 1, 3, 5, and 9 respectively and n = 1 for later time points.
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pre-injection A
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Fig. 9. Immunohistochemical staining for PPT1 in Ppt1 knockout mouse cerebellum before and at varying times after intrathecal injection. (A) Ppt1 knockout mouse, age 6 weeks, injected with aCSF (B–D) Ppt1 knockout mice, age 6 weeks, injected with 10 .6 mg/ml PPT1 as described under Materials and methods and sacrificed at 1 day (B), 3 weeks (C), or 5 months (D) after the injection. (E) Wild-type control mouse brain. Brown staining indicates PPT1 immunostaining (indicated by arrowheads). Bar, 20 μm.
We did not test for the presence of anti-human PPT1 antibodies that may develop in Ppt1 deficient mice as a result of enzyme administration. Although it is possible that such antibodies, if present, may have diminished the effect of the treatment, in a previous study we could not detect such antibodies in mice treated chronically with human recombinant PPT1 administered intravenously on a weekly schedule, and so similar data was not sought here. The treatment was seemingly well tolerated with equal morbidity in mice injected with PPT1 or vehicle, and no anaphylaxis or any overt increase in the neuroinflammatory response was observed. 4. Conclusion Human recombinant PPT1 administered to the cerebrospinal fluid via lumbar injection on three consecutive days at six weeks of age to Ppt1 deficient mice prolonged their survival by seven weeks without apparent undue toxicity. The survival and performance improvements are similar to those reported for other LSD models that affect the brain that are currently in clinical trials using this approach. These data provide a rationale for ERT being delivered to the cerebrospinal fluid in children with PPT1-deficient NCL. Acknowledgments The authors wish to thank Drs. Peter Lobel and David Sleat for advice in the experimental design, Dr. James Richardson and John Shelton of the UT Southwestern Molecular Pathology Core for PPT1 immunostaining and microscopy, and Lauren Peca for performing the behavioral tests. This work was funded by Taylor's Tale, the Batten Disease Support and Research Association, the Batten Disease Family Association, and a King's College London Graduate School International Studentship award to HRN. References [1] J.W. Mink, E.F. Augustine, H.R. Adams, F.J. Marshall, J.M. Kwon, Classification and natural history of the neuronal ceroid lipofuscinoses, J. Child Neurol. 28 (2013) 1101–1105. [2] J. Vesa, E. Hellsten, L.A. Verkruyse, L.A. Camp, J. Rapola, P. Santavuori, S.L. Hofmann, L. Peltonen, Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis, Nature 376 (1995) 584–587. [3] J.Y. Lu, S.L. Hofmann, Thematic review series: lipid posttranslational modifications. Lysosomal metabolism of lipid-modified proteins, J. Lipid Res. 47 (2006) 1352–1357. [4] M. Kousi, A.E. Lehesjoki, S.E. Mole, Update of the mutation spectrum and clinical correlations of over 360 mutations in eight genes that underlie the neuronal ceroid lipofuscinoses, Hum. Mutat. 33 (2012) 42–63. [5] A.K. Das, C.H. Becerra, W. Yi, J.Y. Lu, A.N. Siakotos, K.E. Wisniewski, S.L. Hofmann, Molecular genetics of palmitoyl-protein thioesterase deficiency in the U.S. J. Clin. Invest. 102 (1998) 361–370.
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