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Restoration of aspartoacylase activity in CNS neurons does not ameliorate motor deficits and demyelination in a model of Canavan disease
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doi:10.1016/j.ymthe.2005.01.006
Restoration of Aspartoacylase Activity in CNS Neurons Does Not Ameliorate Motor Deficits and Demyelination in a Model of Canavan Disease Matthias Klugmann,1,y,* Claudia B. Leichtlein,1,z C. Wymond Symes,1,z Tadao Serikawa,2 Deborah Young,1 Matthew J. During,1,3,y 1
Laboratory of Functional Genomics and Translational Neuroscience, Department of Molecular Medicine and Pathology, University of Auckland School of Medicine, 85 Park Road, Auckland, New Zealand 2 Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan 3 Department of Neurological Surgery, Weill Medical College of Cornell University, New York, NY 10021, USA *Present address: IZN, Neurobiologie, Universita¨t Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
y
To whom correspondence and reprint requests should be addressed. Fax: 0049 (0)6221 546700. E-mail: [email protected] or [email protected]. z
These authors contributed equally to this work.
Available online 8 February 2005
Canavan disease is an early onset leukodystrophy associated with psychomotor retardation, seizures, and premature death. This disorder is caused by mutations in the gene encoding the enzyme aspartoacylase (ASPA). Normally, ASPA is enriched in oligodendrocytes and ASPA deficiency results in elevated levels of its substrate molecule, N-acetylaspartate (NAA), brain edema, and dysmyelination. Using adeno-associated virus, we permanently expressed ASPA in CNS neurons of the tremor rat, a genetic model of Canavan disease, and examined the efficacy of the treatment by monitoring NAA metabolism, myelination, motor behavior, and seizures. Assessment of ASPA protein and enzyme activity in whole brain hemispheres showed restoration to normal levels as long as 6 months after treatment. This finding correlated with a reduction of NAA levels, along with a rescue of the seizure phenotype. However, gross brain pathology, such as dilated ventricles and spongiform vacuolization, was unchanged. Moreover, hypomyelination and motor deficits were not resolved by ASPA gene transfer. Our data suggest that NAA-mediated neuronal hyperexcitation but not oligodendrocyte dysfunction can be compensated for by neuronal ASPA expression. Key Words: adeno-associated virus, aspartoacylase, N-acetylaspartate, Canavan disease, demyelination, epilepsy, gene therapy, oligodendrocyte, leukodystrophy
INTRODUCTION Canavan disease (CD) is a recessive leukodystrophy generally characterized by gross white matter degeneration of the brain, psychomotor retardation, seizures, and premature death [1]. Histological abnormalities comprise vacuolization associated with gliosis, astrocytic edema, and progressive loss of oligodendrocytes [2], the myelinforming cells of the central nervous system (CNS). Mutations in the gene encoding the enzyme aspartoacylase (ASPA; EC 3.5.1.15) have been identified as the genetic cause in both Canavan disease and its rodent model, the tremor (tm) rat [3,4]. ASPA deacetylates its substrate N-acetylaspartate (NAA) to produce acetate and l-aspartate. The enzyme deficiency results in the body’s inability to break down NAA, which ultimately leads to
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the progressive accumulation of this amino acid in brain and urine. The tm rat is a natural Aspa-null mutant and exhibits movement tremors at 2 weeks of age [5]. At 8 weeks the tremors diminish but absence-like seizures, characterized by staring and 5- to 7-Hz spike-wave complexes in hippocampal electroencephalograms (EEGs), gradually appear [6]. These seizures can be inhibited by drugs that repress absence seizures in humans, hence the tm rat has served as a model of petit mal epilepsy [7]. Like children affected by CD, tm rats show severe CNS dysmyelination and vacuolization, while the periphery appears normal. The bipartite pathology, epilepsy and dysmyelination, is not fully understood, but current hypotheses for the etiology and progression of CD are related to NAA-
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dependant osmotic dysregulation [8] and a lack of acetyl groups for myelin lipid synthesis [9]. Under normal conditions, the NAA metabolism is remarkably compartmentalized. NAA is synthesized in neuronal mitochondria and microsomes [10] and degraded by ASPA, a cytosolic enzyme almost exclusively expressed in oligodendrocytes [11,12]. Although the physiological role and exact pathomechanisms of NAA remain elusive, the key unifying principle in both Canavan disease and the tremor rat is the lack of oligodendroglial ASPA. CD is a monogenic disease with a pathology localized to the CNS and no effective treatment exists. This provides a strong rationale for a gene replacement strategy. Indeed, the liposome-based introduction of the ASPA cDNA in a small cohort of affected children was the very first gene therapy approach for a neurodegenerative disease [13]. However, clinical changes were transient due to inadequacies of the vector delivery system. Somatic cell genetic engineering with viral vectors provides a versatile tool to express therapeutic genes [14], and a Phase I trial using recombinant adeno-associated virus serotype 2 (rAAV2) to introduce ASPA into CNS neurons, the source of NAA synthesis, has been approved [15]. However, a pilot study investigating the feasibility of this gene therapy approach in another CD model, the Aspa-knockout mouse [16], showed that interpretation of the data was hampered by the limited transduction frequency of AAV2 compared to other serotypes [17–19]. Furthermore, an adenovirus-based ASPA gene transfer into the brain of adult tm rats was inefficient to suppress seizures [20]. Interestingly, adult animals were used for these studies despite the early onset and severity of the disease. To address these limitations we evaluated the use of a novel chimeric rAAV1/2 system [21,22] to deliver the ASPA gene permanently into the brain of young tm rats. Here we report how ASPA expression in CNS neurons of tm rats affects NAA metabolism, myelination, motor behavior, and seizures activity and comment on the effectiveness of NAA-mediated gene therapy for CD.
RESULTS Restoration of ASPA Activity after Viral-Based Gene Transfer Prior to an application in vivo, we tested whether a hemagglutinin (HA) tag N-terminally added to recombinant ASPA would have an impact on enzyme activity. We transiently transfected HEK 293 cells with AAV expression plasmids encoding HA-ASPA, nontagged ASPA, and GFP driven by the 1.1-kb CMV enhancer/chicken h-actin promoter. We prepared and analyzed cell lysates 48 h later for ASPA expression and enzymatic activity. Interestingly, Western blot analysis of HA-ASPA-transfected cells, using anti-ASPA serum, revealed two immunoreactive species (not shown). One band, at 37 kDa, corresponded to the expected size of nontagged ASPA
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[12] and was detected in cells expressing the nontagged ASPA. The other species, at 38 kDa, was also detected by a HA antibody. These bands were absent in control lysate. From this it appeared that the HA tag was being cleaved from a subpopulation of recombinant ASPA through some form of posttranslational processing. We concluded that some unexpected posttranslational processing cleaved off the HA-tag. We did not detect ASPA enzyme activity in controls; however, expression of both ASPA plasmids yielded comparably high levels of activity (data not shown). Based on these results, and given the benefit of highly sensitive HA immunohistochemistry, we decided to use the vector expressing the HA-tagged ASPA for gene therapy in tremor rats. Tremor rat pathology becomes apparent after 2 weeks of age. As such, a therapeutic success likely correlates with both the extent of transduction and an intervention early in life. Therefore, we performed bilateral vector infusions into striatum and thalamus of weaned (age 21–24 days) tm rats (Fig. 1A). We sacrificed cohorts of wild-type and infused tm rats (n = 4 or 5) at different time points for biochemical analysis of the brains (Fig. 1B). In other groups, we examined motor performance or seizure activity. To assess the levels of introduced enzyme after AAVaspa transfer, we performed Western blot analysis of whole hemispheres from uninjected wild-type (wt) or AAVaspa- or AAVgfp-treated tremor rats (tmASPA or tmGFP; Fig. 1C). We detected endogenous ASPA, lacking in tmGFP brains, at 37 kDa in wt lysates, confirming previous studies [11,12]. We also observed this band in tmASPA brains, along with an additional band at 38 kDa, confirming our in vitro findings. Quantification of protein levels in tmASPA hemispheres at 5, 11, and 26 weeks of age showed 76, 197, and 606% of respective wt levels (Fig. 1D). Note that the absolute levels of introduced ASPA peaked between 5 and 11 weeks, to remain constant thereafter. This reflects the known profile of AAV-based gene expression in brain [23,24]. Interestingly, the apparent relative increase in 26-week-old tmASPA rats is due to an observed decline of expression in wild type rather than an increase in ectopic ASPA levels. Significantly, normal ASPA activity was restored or even exceeded after AAVaspa treatment at all time points (93, 146, and 142% of normal levels; Fig. 1E). Furthermore, expression and activity correlated directly in both wt (r 2 = 0.72, P b 0.001) and tmASPA (r 2 = 0.37, P b 0.05; Fig. 1F). Histological Findings Six months after intraparenchymal CNS-administration of AAVaspa, we assessed transduction by HA immunostaining. We observed robust expression of ectopic ASPA throughout the entire brain, except the cerebellum, extending several millimeters beyond the injection sites (Fig. 2A). Transgene expression was most abundant in cell bodies and neurites of striatum and thalamus. Moreover, we detected immunoreactivity in areas distant to the
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FIG. 1. Restoration of ASPA deficiency after multisite rAAV delivery. (A) Scheme of rAAV injection into the striatum (left) and thalamus (right). (B) Experimental time line. Shown is the type of intervention at a given age in weeks (wk). (C) Representative immunoblots showing endogenous ASPA and transgene expression: whole hemisphere week 5 brain lysates probed with (a) anti-ASPA sera, (b) anti-HA, or (c) anti-GFP. (D) ASPA expression levels in whole brain hemispheres at all three time points were quantified from immunoblot signals standardized to h-actin and presented relative to mean week 5 wild-type levels. (E) ASPA enzymatic activity in the same samples as used in (D). (F) Correlation between ASPA protein levels derived from (D) and enzyme activity from (E). The bold and thin trend lines represent correlation in wt and tmASPA, respectively. Bars represent mean values F SEM of 4 rats. Wt, uninjected wild type; tmASPA, AAV-ASPA-injected tremor rat; tmGFP, AAV-GFP-injected tremor rat. *P b 0.05, **P b 0.01, ***P b 0.001.
injection sites such as hippocampus and neocortex. As expected, the anti-ASPA antibody replicated the neuronal staining pattern of ectopic ASPA in tmASPA brains (not shown). Higher magnification revealed introduced ASPA even in fibers of the hind brain (Fig. 2B), most likely due to retrograde transportation of the vector [23]. Interestingly, neuritic swellings in both tmGFP and tmASPA brains became apparent by visualization of GFP fluorescence (not shown) and ASPA immunoreactivity (Fig. 2B), respectively. To assess the tropism of the AAV1/2 chimeric vectors in brain we performed double immunohistochemistry for the transgene and cell-type-specific markers. HA immunoreactivity was not present in carbonic anhydrase II (CAII)-positive oligodendrocytes, glial fibrillary acid protein (GFAP)-labeled astrocytes, or myelin basic protein (MBP)-positive myelin but rather in neurons identified by
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the marker NeuN as shown by laser confocal microscopy of the striatum in tmASPA brains (Figs. 2C–2F). This expression pattern confirmed previous reports on the neuronal tropism of the parental serotypes, AAV1 and AAV2 [23,24], but expectedly differs from the studies on endogenous ASPA known to be expressed in the cytoplasm of oligodendrocytes in wt rats [11,12]. A hallmark feature of CD and tm rat pathology is the progressive white matter vacuolization accompanied by enlargement of the ventricles [4,25]. Gross brain morphology appeared unchanged even in regions of most abundant transgene expression (Figs. 2G and 2H). Reduction of NAA Levels and Effects on Spontaneous Seizures Increased levels of NAA are believed to cause seizures due to neuronal overexcitation [4,26]. We assessed NAA
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FIG. 2. Brain morphology after rAAVaspa expression. (A) HA immunohistochemistry in sagittal section of a tmASPA brain. (B) Magnification of (A) showing HApositive fibers in the hind brain. Arrows point to axonal swellings. (C–F) Confocal overlays of HA immunohistochemistry in the striatum of tmASPA brains, in red, and (C) CAII, (D) MBP, (E) GFAP, or (F) NeuN in green. Ectopic ASPA is not expressed in the small cell bodies of oligodendrocytes labeled by CAII (C), in myelin (D), or in astrocytes (E) but is exclusively detected in NeuNpositive neurons (F, arrowheads). Note that N95% of neurons in (F) are transduced. (G, H) Histopathology in treated tm rats. The numbers of vacuoles (arrows) appear similar in H&E-stained thalamus of (G) tmASPA and (H) tmGFP brains. Shown are representative sections of n = 4 or 5 per group. Bars: 2 mm (A); 100 Am (B); 10 Am (C–F); 250 Am (G, H).
levels by NMR spectroscopy of perchloric acid extracts (Fig. 3). Although NAA levels were higher in both tmASPA and tmGFP relative to wt at week 5 (1.24 F 0.03 and 1.37 F 0.03 vs 0.92 F 0.02, respectively, P b 0.0001), the difference between the injected groups was significant ( P b 0.05), suggesting partially successful treatment by AAVaspa (Fig. 3B). Six weeks later, at week 11, when ectopic ASPA expression and associated enzyme activity in tmASPA were equal or in excess of wt levels, NAA was still elevated (1.16 F 0.03 vs. 0.91 F
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0.05 for wt, P b 0.001). Nonetheless, as observed at week 5, these levels were less than those in tmGFP (1.27 F 0.03; P b 0.05). Although our histological data pointed to a widely spread gene transfer, inherently, ASPA levels were highest in the plane around the injection sites. Therefore, we decided to analyze one 300-Am coronal slice per animal containing the region of the thalamic coordinates in the 26-week group. To exceed the threshold of detection robustly, we were required to pool the slices of individuals from the same group (n = 4) prior to
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FIG. 3. NAA measurement and seizure analysis. (A) Representative NMR spectra of perchloric acid lysates from whole hemispheres of wt (left) and tmGFP (right). (B and C) NAA levels presented as a ratio with creatine (Cr). For week 5 and week 11 time points (B), individual hemispheres (n = 4) were analyzed. At week 26 (C), tissue slices surrounding the thalamic injection site were combined (n = 4) and tested. (D) Representative hippocampal EEG recordings of spontaneous seizure activity in week 20 rats (n = 6–8 per group). Calibration in (D) 50 mV vertical, 5 s horizontal. (E) Average seizure occurrence quantified over 15-min sessions. EEGs were recorded three times per week, from week 18 to week 22. (F) Mean seizure length for the entire 4-week period. *P b 0.05, **P b 0.01, ***P b 0.001.
analysis. NAA levels in slices of wild type, tmASPA rat, and tmGFP rat were 0.85, 1.01, and 1.28, respectively (Fig. 3C). The minor variances within groups at previous time points strongly suggest a significant NAA reduction in AAVaspa brains at 26 weeks although the bpooled dataQ prohibited a statistical analysis. To address whether the reduced NAA levels after ASPA gene transfer could change the known seizure phenotype of aged tm rats, we monitored spontaneous absence-like seizures in both AAV treatment groups as 5- to 7-Hz polyspike discharges in hippocampal EEG recordings (Fig. 3D). Seizure occurrence at 18 and 19 weeks of age was reduced in AAVaspa rats (13.5 F 4.0 and 14.7 F 5.2) compared to controls (29.6 F 5.6 and 29.6 F 4.4; P b
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0.05). This trend was even more pronounced at later stages ( P b 0.01) as the seizure activity stayed constant in tmASPA but increased in tmGFP (Fig. 3E). Concomitantly, the duration of individual seizures after AAVaspa treatment was reduced (1.24 F 0.04 s vs. 1.63 F 0.03 s for tmGFP, P b 0.0001; Fig. 3F). Neuronal ASPA Expression Does Not Improve Myelin or Motor Deficits The pathology in CD and tm rats is characterized by hypomyelination [27]. We determined the amounts of myelin in whole hemispheres and found no differences between AAVaspa and AAVgfp treatment at any time point (Fig. 4A). As expected, the amount of myelin in
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FIG. 4. Myelin content and motor function. (A) Myelin measurements at all three time points. Shown is the myelin content in whole hemispheres from four rats per group. (B) Open field analysis shows immobility of both injection groups. (C) Gait analysis. Mean anterior–posterior distance between left and right hind paw (stride length) and medial–lateral deviation of left front and back paw derived from partially overlapping prints (paw spread). (D) Rotarod test. Shown are mean latencies (5 trials per rat) to fall off the stationary rod (phase 1), the constantly rotating (3 rpm) rod (phase 2), or the accelerating rod (phase 3). The maximum duration of an individual trial was 2 min (phases 1 and 2) or 7 min (phase 3). n = 8 for (B–D). *P b 0.05, **P b 0.01, ***P b 0.001.
normal rats increased with time [28]. Likewise, myelin levels increased in tmASPA or tmGFP rats but did not reach normal levels ( P b 0.01). There were no obvious neurological differences between tmASPA and tmGFP animals. Both groups showed the obvious signs of abnormal motor development, tremors, and ataxia. To evaluate possible subtle phenotypic changes after gene transfer, we examined the motor behavior of wt and treated tm rats in three different tests. General locomotion was determined in the open field test where tmASPA and tmGFP rats were dramatically less active than normal rats ( P b 0.0001, Fig. 4B). Neurological abnormalities like splayed legs and ataxia provided the rationale to evaluate the gait pattern. Fig. 4C shows that stride length and paw spread of either AAV-injected group were different from those of wild-type rats ( P b 0.0001). Also, the ability to balance on a rotating rod (rotarod) is impaired in tm rats [29]. In examining this, we conducted a multiphase rotarod protocol ascending in difficulty, to detect even subtle improvements (Fig. 4D). Compared to normal rats, both AAV groups performed similarly poorly in all phases ( P b 0.0001). Combined, these results show that the AAVaspa treatment did not improve motor impairment or the underlying demyelination.
DISCUSSION Canavan disease is one of the few examples of a neurological disease for which gene transfer has been tested in humans, primarily because the pathology is confined to the brain and there was a reasonable expectation that, via metabolic cooperativity, even low levels of transduction could result in phenotypic improvements [13,30]. Based
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on the idea that NAA is the toxic agent causing the CD pathology, it appeared reasonable to reduce its levels by introduction of ASPA into neurons, the location of NAA synthesis, and human experimentation has started [15]. Notably, a report on an AAV2-based ASPA gene transfer into the brain of Aspa-knockout mice failed to provide comprehensive biochemical or neurological data but showed limited transduction efficiency despite a multiple-site injection strategy [16]. Here, we have shown that AAV1/2-mediated transfer of ASPA into a large proportion of the brain and subsequent restoration of enzyme activity to normal levels does not significantly improve all neurological deficits in a genetic rat model of CD. Our data may have implications for human applications. We used a total of 5 billion AAV1/2 genomes per rat. In comparison, 900 billion AAV2 genomes per CD patient were proposed to be administered over six separate subcortical delivery sites in the context of a Phase I clinical trial [15]. Given the higher relative number of particles in the present study (approximately 500-fold difference in size between human and rat brain), the superior transduction efficiency of AAV1/2 over AAV2, and our use of the latest generation expression cassette [31], concerns may be raised regarding therapeutic benefits of the clinical trial. The reduction of NAA levels in tmASPA brains was most prominent in areas of highest transgenic protein expression (Fig. 2A). This finding was in line with the positive correlation between protein expression and enzyme activity (Fig. 1F). However, the decrease of NAA levels in whole brain hemispheres was less dramatic (Fig. 3B), suggesting inefficient metabolic cross-correction of deficient cells by transduced neurons [32] or that neurons
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are not the sole locus of NAA synthesis. In fact, in the normal brain, oligodendrocyte progenitors have low equilibrium concentrations of NAA, possibly caused by ASPA-mediated rapid turnover [10,33,34]. Hence, in our animal model, reduced neuronal NAA levels might have been counteracted by increased glial NAA pools. Moreover, our detailed neurological analyses did not reveal major improvement, except for the amelioration of the epileptic phenotype. We cannot rule out that the intervention may need to occur even earlier in life because some pathology is already present by the time of vector injection, AAV takes several weeks to express at maximum levels, and some severe and irreversible brain damage might have occurred prior gene transfer. To address this, future experiments will involve in utero gene transfer using AAV1/2 to investigate if the timing of the intervention rather than transduction of oligodendroglia is indeed limiting for the success of the therapy. However, the timing of our intervention, immediately postweaning, is consistent with the time of diagnosis for human Canavan disease. Hence, in terms of clinical relevance our timing of gene transfer is appropriate. NAA has been shown to induce seizures after intracerebroventricular administration to normal rats, probably by neuronal overexcitation through metabotropic glutamate receptors [26,35]. Here, we observed partial reduction of NAA in tmASPA brains resulting in modulation of both seizure length and frequency. Together, these data suggest that increased NAA levels in the tm rat brain greatly participate in the course of epilepsy. It might be tempting to speculate that NAA induces glial overexcitation in a similar fashion, and glutamate receptor-mediated toxicity in oligodendrocytes has been reported [36]. However, NAA does not appear to activate this pathway. This notion gets support by the fact that aged heterozygous carriers of the tm mutation exhibit seizures in the absence of dysmyelination [4]. It is not clear what actually causes the typical spongiform degeneration in homozygous mutants. Despite the diminished NAA levels in the thalamus of tmASPA rats (Fig. 3C), vacuolization remained unchanged. Moreover, this pathology is absent in aged carriers, indicating that oligodendrocyte impairment, and possibly death, is a prerequisite for vacuole formation. Motor deficits in the tm rat seem to be a function of ASPA deficiency in oligodendrocytes rather than a consequence of NAA build-up and were not successfully treated. Our biochemical analysis confirmed a relatively moderate extent of demyelination in aged tm rats [27], which is, somewhat surprisingly, paralleled by severe impairment of motor coordination and locomotion. Swollen astrocytes are a known feature of the CD pathology, but we noted axonal spheroids, throughout the CNS of both tmASPA and tmGFP (Fig. 2B). The nature of these swellings is uncertain and an axonal involvement has not yet been described for CD or its models. However,
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it is now widely accepted that axonal integrity and survival result from defects in the myelinating cells, whether myelin irregularities are apparent or not [37]. Further investigations on this were beyond the scope of this study but may contribute to the understanding of CD. This study might help to underscore the concept that for this disorder in particular, and others in which the deficient protein has such a highly segmented expression pattern confined to glia, vectors with specific tropism for such cells are likely to be needed to restore function fully.
MATERIALS AND METHODS Generation of recombinant adeno-associated virus. A fusion construct encoding the HA tag fused to the human ASPA cDNA was subcloned in a rAAV plasmid backbone containing the 1.1-kb CMV enhancer/chicken hactin (CBA) promoter, the woodchuck posttranscriptional regulatory element (WPRE), and the bovine growth hormone poly(A) (AAVaspa). The same rAAV-CBA-WPRE-bGH backbone carrying the Renilla gfp cDNA (Stratagene) was used as the control (AAVgfp). AAV chimeric vectors (virions containing a 1:1 ratio of AAV1 and AAV2 capsid proteins with AAV2 ITRs) were generated and purified using heparin-affinity columns as described [38]. AAV vector administration. Twenty-one- to twenty-four-day-old male and female tremor rats were anesthetized with ketamine and xylazine (84.4 and 12.7 mg/kg) before surgery. One microliter of either AAVaspa or AAVgfp (1.3 109 particles) was mixed with 0.5 Al of 20% mannitol, to enhance vector spread [39], and injected bilaterally into the striatum (+0.35 mm AP, F2.7 mm ML, 5.9 mm DV from bregma) and thalamus (3.2 mm AP, F2.2 mm ML, 5.9 mm DV from bregma) using a stereotaxic frame (Kopf Instruments). Vectors were infused at a rate of 66 nl/min using a microprocessor controlled minipump (World Precision Instruments). Brain tissue preparation for biochemical analysis. After removal of brain stem and cerebellum, brains were stored at 808C as individual hemispheres. Each right hemisphere was later thawed and homogenized in 0.25 M sucrose, 10 mM Hepes, pH 7.4 (20% w/v), to assess ASPA protein levels, enzymatic activity, and myelin content (see below). For NAA analysis of week 5 and week 11 brains, perchloric acid (12%) lysates of the left hemispheres were lyophilized, and the samples were redissolved in D2O spiked with 1 mM 3-(trimethylsilyl)tetradeutero sodium propionate (Sigma) and analyzed by nuclear proton magnetic resonance spectroscopy for NAA content. For the week 26 brain samples, one 300-Am coronal slice was dissected from the thalamic plane of each left hemisphere, combined with slices from brains of the same treatment group, and prepared for NMR. 1
H NMR spectroscopy. All spectra were obtained at 278C on a Bruker Avance DRX 400 NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) operating at a frequency of 400 MHz. One-pulse fully relaxed spectra were acquired with 908 pulses applied every 14 s with a spectral width of 6410.2 Hz and 64K resolution. For a satisfactory signal/noise ratio, 92 scans were accumulated and Fourier transformed. The creatine and NAA spectral peaks were identified by their known chemical shifts (3.04 and 2.02 ppm, respectively). NAA levels were presented as a ratio with creatine.
ASPA enzyme assay. Right hemisphere brain homogenate samples were diluted 1:3 with 3 mg/ml CaCl2 and the cytosolic fractions isolated by centrifugation (20,000g, 20 min, 48C). The enzyme activity test has been described elsewhere [40]. Western blotting. Ten-microgram samples of right hemisphere brain cytosol underwent separation by SDS–PAGE (12% gels) and transfer onto nitrocellulose membrane (Amersham). After blocking and probing with rat ASPA antiserum [12], monoclonal anti-HA antibody (Babco), or rat hrGFP antiserum, bound antibody was detected with secondary antibody
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conjugated to horseradish peroxidase (Santa Cruz) and an ECL chemiluminescence substrate (Amersham). Each membrane was reprobed with anti-h-actin (Abcam). Chemiluminescent signals were captured on film (Amersham) and quantified using the Quantity One image analysis system (Bio-Rad). ASPA signals were normalized to h-actin and presented as levels relative to week 5 wild type. Immunohistochemistry. The rostral–caudal extent of transgene expression of all animals used in behavioral experiments was assessed by HA immunohistochemistry. Processing of rat brain tissue for DAB detection and H&E staining has been described [41]. For fluorescent double labeling 40-Am coronal sections were washed in PBS–Triton, blocked in 4% horse serum/PBS, and incubated overnight with a combination of a monoclonal or polyclonal HA antibody (1:2000; Babco or Santa Cruz) and antibodies against GFAP (1:400; Sigma), MBP (1:500; Sigma), CAII (1:1000; kindly provided by S. Ghandour), or NeuN (1:200; Sigma). After washes, sections were incubated with anti-rabbit–Alexa488 and anti-mouse–Alexa594 antibodies (1:1000; Molecular Probes). Immunostaining was visualized using a Leica SP2 confocal microscope. Myelin measurements. Myelin membranes were isolated and purified as described [28]. Briefly, right hemisphere brain homogenate samples (0.6 ml) were layered onto 0.85 M sucrose (0.8 ml) and centrifuged (70,000g, 90 min, 48C). Myelin was extracted from the interface, osmotically shocked three times with H2O, and pelleted at 23,000g for 30 min at 48C. Following lyophilization the net weight was determined. EEG recordings of spontaneous seizures. At 17 weeks of age, rAAVinfused tm rats and wild-type controls were chronically implanted with an electrode in the hippocampus (4.5 mm AP, F2.0 mm ML, 3.2 mm DV from bregma). One week after electrode implantation, EEGs of the unrestrained animal were recorded twice a week, 15 min per session. One seizure was defined as a 5- to 7-Hz spike-wave-like complex lasting at least 1 s. Motor performance assays. In the open field test a 2-m-diameter circular field was divided into nine segments of equal size. Each rat was placed in the center of the field and monitored for a period of 5 min. The number of lines crossed within that time was recorded. For gait analysis, footprints were taken when walking forward in a 12-cm wide alley, after red and green food color was applied to the front and hind paws, respectively. Gait patterns were analyzed as described [42]. Both tests were conducted with week 13 rats. The rotarod test was performed with week 26 animals and utilized a motor-driven horizontal roller (Rotamex-4; Columbus Instruments). The rats were trained to stay on the rotarod using a threephase schedule. In phase 1 the animals had to perch on the stationary rod for 120 s. In phase 2, the rod was set to the constant speed of 3 rpm for 120 s. In phase 3, performance on the accelerating rod was monitored for 420 s as described previously [43]. In a series of five trials per animal, the time the rats remained on the roller was recorded. Statistics. Behavioral data, enzyme activity, and myelin and NAA measurements were analyzed by ANOVA with post hoc Fisher’s PLSD. An unpaired Student t test was used for Western blot and seizure data. Correlation analysis was performed with a two-tailed test.
ACKNOWLEDGMENTS We thank Michael Walker for excellent help with NMR. M.K. was supported by a European Molecular Biology Organization Fellowship and the Neurological Foundation of New Zealand. D.Y. is supported by a New Zealand Health Research Council Sir Charles Hercus Health Research Fellowship. RECEIVED FOR PUBLICATION DECEMBER 6, 2004; ACCEPTED JANUARY 6, 2005.
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N-Acetylaspartate in the vertebrate brain: metabolism and function. Neurochem. Res. 28: 941 – 953. 9. Chakraborty, G., Mekala, P., Yahya, D., Wu, G., and Ledeen, R. W. (2001). Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase. J. Neurochem. 78: 736 – 745. 10. Lu, Z. H., Chakraborty, G., Ledeen, R. W., Yahya, D., and Wu, G. (2004). NAcetylaspartate synthase is bimodally expressed in microsomes and mitochondria of brain. Brain Res. Mol. Brain Res. 122: 71 – 78. 11. Madhavarao, C. N., Moffett, J. R., Moore, R. A., Viola, R. E., Namboodiri, M. A., and Jacobowitz, D. M. (2004). Immunohistochemical localization of aspartoacylase in the rat central nervous system. J. Comp. Neurol. 472: 318 – 329. 12. Klugmann, M., et al. (2003). Identification and distribution of aspartoacylase in the postnatal rat brain. Neuroreport 14: 1837 – 1840. 13. Leone, P., et al. (2000). Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease. Ann. Neurol. 48: 27 – 38. 14. Janson, C. G., McPhee, S. W., Leone, P., Freese, A., and During, M. J. (2001). Viralbased gene transfer to the mammalian CNS for functional genomic studies. Trends Neurosci. 24: 706 – 712. 15. Janson, C., et al. (2002). Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum. Gene Ther. 13: 1391 – 1412. 16. Matalon, R., et al. (2003). Adeno-associated virus-mediated aspartoacylase gene transfer to the brain of knockout mouse for Canavan disease. Mol. Ther. 7: 580 – 587. 17. Davidson, B. L., et al. (2000). Recombinant adeno-associated virus type 2, 4, and 5 vectors: transduction of variant cell types and regions in the mammalian central nervous system. Proc. Natl. Acad. Sci. USA 97: 3428 – 3432. 18. Mastakov, M. Y., Baer, K., Kotin, R. M., and During, M. J. (2002). Recombinant adenoassociated virus serotypes 2- and 5-mediated gene transfer in the mammalian brain: quantitative analysis of heparin co-infusion. Mol. Ther. 5: 371 – 380. 19. Wang, C., Wang, C. M., Clark, K. R., and Sferra, T. J. (2003). Recombinant AAV serotype 1 transduction efficiency and tropism in the murine brain. Gene Ther. 10: 1528 – 1534. 20. Seki, T., et al. (2002). Adenoviral gene transfer of aspartoacylase into the tremor rat, a genetic model of epilepsy, as a trial of gene therapy for inherited epileptic disorder. Neurosci. Lett. 328: 249 – 252. 21. Richichi, C., et al. (2004). Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus. J. Neurosci. 24: 3051 – 3059. 22. Hauck, B., Chen, L., and Xiao, W. (2003). Generation and characterization of chimeric recombinant AAV vectors. Mol. Ther. 7: 419 – 425. 23. Burger, C., et al. (2004). Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 10: 302 – 317. 24. Xu, R., et al. (2001). Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes. Gene Ther. 8: 1323 – 1332. 25. Baslow, M. H. (2002). Evidence supporting a role for N-acetyl-L-aspartate as a molecular water pump in myelinated neurons in the central nervous system: an analytical review. Neurochem. Int. 40: 295 – 300. 26. Akimitsu, T., et al. (2000). Epileptic seizures induced by N-acetyl-L-aspartate in rats: in vivo and in vitro studies. Brain Res. 861: 143 – 150. 27. Kondo, A., Nagara, H., Akazawa, K., Tateishi, J., Serikawa, T., and Yamada, J. (1991). CNS pathology in the neurological mutant rats zitter, tremor and zitter–tremor double mutant (spontaneously epileptic rat, SER). Brain 114: 979 – 999. 28. Norton, W. T., and Poduslo, S. E. (1973). Myelination in rat brain: method of myelin isolation. J. Neurochem. 21: 749 – 757. 29. Asano, Y., Okaniwa, A., Serikawa, T., and Yamada, J. (1990). Delayed postnatal behavioral development in spontaneously epileptic rats and tremor rats, and poor operant performance in spontaneously epileptic rats. Jikken Dobutsu 39: 69 – 74. 30. Leone, P., Janson, C. G., McPhee, S. J., and During, M. J. (1999). Global CNS gene transfer for a childhood neurogenetic enzyme deficiency: Canavan disease. Curr. Opin. Mol. Ther. 1: 487 – 492. 31. Fitzsimons, H. L., Bland, R. J., and During, M. J. (2002). Promoters and regulatory elements that improve adeno-associated virus transgene expression in the brain. Methods 28: 227 – 236.
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32. Hsich, G., Sena-Esteves, M., and Breakefield, X. O. (2002). Critical issues in gene therapy for neurologic disease. Hum. Gene Ther. 13: 579 – 604. 33. Urenjak, J., Williams, S. R., Gadian, D. G., and Noble, M. (1992). Specific expression of N-acetylaspartate in neurons, oligodendrocyte-type-2 astrocyte progenitors, and immature oligodendrocytes in vitro. J. Neurochem. 59: 55 – 61. 34. Urenjak, J., Williams, S. R., Gadian, D. G., and Noble, M. (1993). Proton nuclear magnetic resonance spectroscopy unambiguously identifies different neural cell types. J. Neurosci. 13: 981 – 989. 35. Yan, H. D., Ishihara, K., Serikawa, T., and Sasa, M. (2003). Activation by N-acetyl-Laspartate of acutely dissociated hippocampal neurons in rats via metabotropic glutamate receptors. Epilepsia 44: 1153 – 1159. 36. Matute, C., Alberdi, E., Domercq, M., Perez-Cerda, F., Perez-Samartin, A., and Sanchez-Gomez, M. V. (2001). The link between excitotoxic oligodendroglial death and demyelinating diseases. Trends Neurosci. 24: 224 – 230. 37. Popko, B. (2003). Myelin: not just a conduit for conduction. Nat. Genet. 33: 327 – 328.
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38. Klugmann, M., et al. (2005). AAV-mediated hippocampal expression of short and long Homer 1 proteins differentially affects cognition and seizure activity in adult rats. Mol. Cell. Neurosci. 28: 343 – 356. 39. Mastakov, M. Y., Baer, K., Xu, R., Fitzsimons, H., and During, M. J. (2001). Combined injection of rAAV with mannitol enhances gene expression in the rat brain. Mol. Ther. 3: 225 – 232. 40. Bhakoo, K. K., Craig, T. J., and Styles, P. (2001). Developmental and regional distribution of aspartoacylase in rat brain tissue. J. Neurochem. 79: 211 – 220. 41. Young, D., Lawlor, P. A., Leone, P., Dragunow, M., and During, M. J. (1999). Environmental enrichment inhibits spontaneous apoptosis, prevents seizures and is neuroprotective. Nat. Med. 5: 448 – 453. 42. Teunissen, C. E., et al. (2001). Behavioural correlates of striatal glial fibrillary acidic protein in the 3-nitropropionic acid rat model: disturbed walking pattern and spatial orientation. Neuroscience 105: 153 – 167. 43. Klugmann, M., et al. (1997). Assembly of CNS myelin in the absence of proteolipid protein. Neuron 18: 59 – 70.
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Restoration of Aspartoacylase Activity in CNS Neurons Does Not Ameliorate Motor Deficits and Demyelination in a Model of Canavan Disease Matthias Klugmann,1,y,* Claudia B. Leichtlein,1,z C. Wymond Symes,1,z Tadao Serikawa,2 Deborah Young,1 Matthew J. During,1,3,y 1
Laboratory of Functional Genomics and Translational Neuroscience, Department of Molecular Medicine and Pathology, University of Auckland School of Medicine, 85 Park Road, Auckland, New Zealand 2 Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan 3 Department of Neurological Surgery, Weill Medical College of Cornell University, New York, NY 10021, USA *Present address: IZN, Neurobiologie, Universita¨t Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.
y
To whom correspondence and reprint requests should be addressed. Fax: 0049 (0)6221 546700. E-mail: [email protected] or [email protected]. z
These authors contributed equally to this work.
Available online 8 February 2005
Canavan disease is an early onset leukodystrophy associated with psychomotor retardation, seizures, and premature death. This disorder is caused by mutations in the gene encoding the enzyme aspartoacylase (ASPA). Normally, ASPA is enriched in oligodendrocytes and ASPA deficiency results in elevated levels of its substrate molecule, N-acetylaspartate (NAA), brain edema, and dysmyelination. Using adeno-associated virus, we permanently expressed ASPA in CNS neurons of the tremor rat, a genetic model of Canavan disease, and examined the efficacy of the treatment by monitoring NAA metabolism, myelination, motor behavior, and seizures. Assessment of ASPA protein and enzyme activity in whole brain hemispheres showed restoration to normal levels as long as 6 months after treatment. This finding correlated with a reduction of NAA levels, along with a rescue of the seizure phenotype. However, gross brain pathology, such as dilated ventricles and spongiform vacuolization, was unchanged. Moreover, hypomyelination and motor deficits were not resolved by ASPA gene transfer. Our data suggest that NAA-mediated neuronal hyperexcitation but not oligodendrocyte dysfunction can be compensated for by neuronal ASPA expression. Key Words: adeno-associated virus, aspartoacylase, N-acetylaspartate, Canavan disease, demyelination, epilepsy, gene therapy, oligodendrocyte, leukodystrophy
INTRODUCTION Canavan disease (CD) is a recessive leukodystrophy generally characterized by gross white matter degeneration of the brain, psychomotor retardation, seizures, and premature death [1]. Histological abnormalities comprise vacuolization associated with gliosis, astrocytic edema, and progressive loss of oligodendrocytes [2], the myelinforming cells of the central nervous system (CNS). Mutations in the gene encoding the enzyme aspartoacylase (ASPA; EC 3.5.1.15) have been identified as the genetic cause in both Canavan disease and its rodent model, the tremor (tm) rat [3,4]. ASPA deacetylates its substrate N-acetylaspartate (NAA) to produce acetate and l-aspartate. The enzyme deficiency results in the body’s inability to break down NAA, which ultimately leads to
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the progressive accumulation of this amino acid in brain and urine. The tm rat is a natural Aspa-null mutant and exhibits movement tremors at 2 weeks of age [5]. At 8 weeks the tremors diminish but absence-like seizures, characterized by staring and 5- to 7-Hz spike-wave complexes in hippocampal electroencephalograms (EEGs), gradually appear [6]. These seizures can be inhibited by drugs that repress absence seizures in humans, hence the tm rat has served as a model of petit mal epilepsy [7]. Like children affected by CD, tm rats show severe CNS dysmyelination and vacuolization, while the periphery appears normal. The bipartite pathology, epilepsy and dysmyelination, is not fully understood, but current hypotheses for the etiology and progression of CD are related to NAA-
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dependant osmotic dysregulation [8] and a lack of acetyl groups for myelin lipid synthesis [9]. Under normal conditions, the NAA metabolism is remarkably compartmentalized. NAA is synthesized in neuronal mitochondria and microsomes [10] and degraded by ASPA, a cytosolic enzyme almost exclusively expressed in oligodendrocytes [11,12]. Although the physiological role and exact pathomechanisms of NAA remain elusive, the key unifying principle in both Canavan disease and the tremor rat is the lack of oligodendroglial ASPA. CD is a monogenic disease with a pathology localized to the CNS and no effective treatment exists. This provides a strong rationale for a gene replacement strategy. Indeed, the liposome-based introduction of the ASPA cDNA in a small cohort of affected children was the very first gene therapy approach for a neurodegenerative disease [13]. However, clinical changes were transient due to inadequacies of the vector delivery system. Somatic cell genetic engineering with viral vectors provides a versatile tool to express therapeutic genes [14], and a Phase I trial using recombinant adeno-associated virus serotype 2 (rAAV2) to introduce ASPA into CNS neurons, the source of NAA synthesis, has been approved [15]. However, a pilot study investigating the feasibility of this gene therapy approach in another CD model, the Aspa-knockout mouse [16], showed that interpretation of the data was hampered by the limited transduction frequency of AAV2 compared to other serotypes [17–19]. Furthermore, an adenovirus-based ASPA gene transfer into the brain of adult tm rats was inefficient to suppress seizures [20]. Interestingly, adult animals were used for these studies despite the early onset and severity of the disease. To address these limitations we evaluated the use of a novel chimeric rAAV1/2 system [21,22] to deliver the ASPA gene permanently into the brain of young tm rats. Here we report how ASPA expression in CNS neurons of tm rats affects NAA metabolism, myelination, motor behavior, and seizures activity and comment on the effectiveness of NAA-mediated gene therapy for CD.
RESULTS Restoration of ASPA Activity after Viral-Based Gene Transfer Prior to an application in vivo, we tested whether a hemagglutinin (HA) tag N-terminally added to recombinant ASPA would have an impact on enzyme activity. We transiently transfected HEK 293 cells with AAV expression plasmids encoding HA-ASPA, nontagged ASPA, and GFP driven by the 1.1-kb CMV enhancer/chicken h-actin promoter. We prepared and analyzed cell lysates 48 h later for ASPA expression and enzymatic activity. Interestingly, Western blot analysis of HA-ASPA-transfected cells, using anti-ASPA serum, revealed two immunoreactive species (not shown). One band, at 37 kDa, corresponded to the expected size of nontagged ASPA
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[12] and was detected in cells expressing the nontagged ASPA. The other species, at 38 kDa, was also detected by a HA antibody. These bands were absent in control lysate. From this it appeared that the HA tag was being cleaved from a subpopulation of recombinant ASPA through some form of posttranslational processing. We concluded that some unexpected posttranslational processing cleaved off the HA-tag. We did not detect ASPA enzyme activity in controls; however, expression of both ASPA plasmids yielded comparably high levels of activity (data not shown). Based on these results, and given the benefit of highly sensitive HA immunohistochemistry, we decided to use the vector expressing the HA-tagged ASPA for gene therapy in tremor rats. Tremor rat pathology becomes apparent after 2 weeks of age. As such, a therapeutic success likely correlates with both the extent of transduction and an intervention early in life. Therefore, we performed bilateral vector infusions into striatum and thalamus of weaned (age 21–24 days) tm rats (Fig. 1A). We sacrificed cohorts of wild-type and infused tm rats (n = 4 or 5) at different time points for biochemical analysis of the brains (Fig. 1B). In other groups, we examined motor performance or seizure activity. To assess the levels of introduced enzyme after AAVaspa transfer, we performed Western blot analysis of whole hemispheres from uninjected wild-type (wt) or AAVaspa- or AAVgfp-treated tremor rats (tmASPA or tmGFP; Fig. 1C). We detected endogenous ASPA, lacking in tmGFP brains, at 37 kDa in wt lysates, confirming previous studies [11,12]. We also observed this band in tmASPA brains, along with an additional band at 38 kDa, confirming our in vitro findings. Quantification of protein levels in tmASPA hemispheres at 5, 11, and 26 weeks of age showed 76, 197, and 606% of respective wt levels (Fig. 1D). Note that the absolute levels of introduced ASPA peaked between 5 and 11 weeks, to remain constant thereafter. This reflects the known profile of AAV-based gene expression in brain [23,24]. Interestingly, the apparent relative increase in 26-week-old tmASPA rats is due to an observed decline of expression in wild type rather than an increase in ectopic ASPA levels. Significantly, normal ASPA activity was restored or even exceeded after AAVaspa treatment at all time points (93, 146, and 142% of normal levels; Fig. 1E). Furthermore, expression and activity correlated directly in both wt (r 2 = 0.72, P b 0.001) and tmASPA (r 2 = 0.37, P b 0.05; Fig. 1F). Histological Findings Six months after intraparenchymal CNS-administration of AAVaspa, we assessed transduction by HA immunostaining. We observed robust expression of ectopic ASPA throughout the entire brain, except the cerebellum, extending several millimeters beyond the injection sites (Fig. 2A). Transgene expression was most abundant in cell bodies and neurites of striatum and thalamus. Moreover, we detected immunoreactivity in areas distant to the
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FIG. 1. Restoration of ASPA deficiency after multisite rAAV delivery. (A) Scheme of rAAV injection into the striatum (left) and thalamus (right). (B) Experimental time line. Shown is the type of intervention at a given age in weeks (wk). (C) Representative immunoblots showing endogenous ASPA and transgene expression: whole hemisphere week 5 brain lysates probed with (a) anti-ASPA sera, (b) anti-HA, or (c) anti-GFP. (D) ASPA expression levels in whole brain hemispheres at all three time points were quantified from immunoblot signals standardized to h-actin and presented relative to mean week 5 wild-type levels. (E) ASPA enzymatic activity in the same samples as used in (D). (F) Correlation between ASPA protein levels derived from (D) and enzyme activity from (E). The bold and thin trend lines represent correlation in wt and tmASPA, respectively. Bars represent mean values F SEM of 4 rats. Wt, uninjected wild type; tmASPA, AAV-ASPA-injected tremor rat; tmGFP, AAV-GFP-injected tremor rat. *P b 0.05, **P b 0.01, ***P b 0.001.
injection sites such as hippocampus and neocortex. As expected, the anti-ASPA antibody replicated the neuronal staining pattern of ectopic ASPA in tmASPA brains (not shown). Higher magnification revealed introduced ASPA even in fibers of the hind brain (Fig. 2B), most likely due to retrograde transportation of the vector [23]. Interestingly, neuritic swellings in both tmGFP and tmASPA brains became apparent by visualization of GFP fluorescence (not shown) and ASPA immunoreactivity (Fig. 2B), respectively. To assess the tropism of the AAV1/2 chimeric vectors in brain we performed double immunohistochemistry for the transgene and cell-type-specific markers. HA immunoreactivity was not present in carbonic anhydrase II (CAII)-positive oligodendrocytes, glial fibrillary acid protein (GFAP)-labeled astrocytes, or myelin basic protein (MBP)-positive myelin but rather in neurons identified by
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the marker NeuN as shown by laser confocal microscopy of the striatum in tmASPA brains (Figs. 2C–2F). This expression pattern confirmed previous reports on the neuronal tropism of the parental serotypes, AAV1 and AAV2 [23,24], but expectedly differs from the studies on endogenous ASPA known to be expressed in the cytoplasm of oligodendrocytes in wt rats [11,12]. A hallmark feature of CD and tm rat pathology is the progressive white matter vacuolization accompanied by enlargement of the ventricles [4,25]. Gross brain morphology appeared unchanged even in regions of most abundant transgene expression (Figs. 2G and 2H). Reduction of NAA Levels and Effects on Spontaneous Seizures Increased levels of NAA are believed to cause seizures due to neuronal overexcitation [4,26]. We assessed NAA
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FIG. 2. Brain morphology after rAAVaspa expression. (A) HA immunohistochemistry in sagittal section of a tmASPA brain. (B) Magnification of (A) showing HApositive fibers in the hind brain. Arrows point to axonal swellings. (C–F) Confocal overlays of HA immunohistochemistry in the striatum of tmASPA brains, in red, and (C) CAII, (D) MBP, (E) GFAP, or (F) NeuN in green. Ectopic ASPA is not expressed in the small cell bodies of oligodendrocytes labeled by CAII (C), in myelin (D), or in astrocytes (E) but is exclusively detected in NeuNpositive neurons (F, arrowheads). Note that N95% of neurons in (F) are transduced. (G, H) Histopathology in treated tm rats. The numbers of vacuoles (arrows) appear similar in H&E-stained thalamus of (G) tmASPA and (H) tmGFP brains. Shown are representative sections of n = 4 or 5 per group. Bars: 2 mm (A); 100 Am (B); 10 Am (C–F); 250 Am (G, H).
levels by NMR spectroscopy of perchloric acid extracts (Fig. 3). Although NAA levels were higher in both tmASPA and tmGFP relative to wt at week 5 (1.24 F 0.03 and 1.37 F 0.03 vs 0.92 F 0.02, respectively, P b 0.0001), the difference between the injected groups was significant ( P b 0.05), suggesting partially successful treatment by AAVaspa (Fig. 3B). Six weeks later, at week 11, when ectopic ASPA expression and associated enzyme activity in tmASPA were equal or in excess of wt levels, NAA was still elevated (1.16 F 0.03 vs. 0.91 F
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0.05 for wt, P b 0.001). Nonetheless, as observed at week 5, these levels were less than those in tmGFP (1.27 F 0.03; P b 0.05). Although our histological data pointed to a widely spread gene transfer, inherently, ASPA levels were highest in the plane around the injection sites. Therefore, we decided to analyze one 300-Am coronal slice per animal containing the region of the thalamic coordinates in the 26-week group. To exceed the threshold of detection robustly, we were required to pool the slices of individuals from the same group (n = 4) prior to
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FIG. 3. NAA measurement and seizure analysis. (A) Representative NMR spectra of perchloric acid lysates from whole hemispheres of wt (left) and tmGFP (right). (B and C) NAA levels presented as a ratio with creatine (Cr). For week 5 and week 11 time points (B), individual hemispheres (n = 4) were analyzed. At week 26 (C), tissue slices surrounding the thalamic injection site were combined (n = 4) and tested. (D) Representative hippocampal EEG recordings of spontaneous seizure activity in week 20 rats (n = 6–8 per group). Calibration in (D) 50 mV vertical, 5 s horizontal. (E) Average seizure occurrence quantified over 15-min sessions. EEGs were recorded three times per week, from week 18 to week 22. (F) Mean seizure length for the entire 4-week period. *P b 0.05, **P b 0.01, ***P b 0.001.
analysis. NAA levels in slices of wild type, tmASPA rat, and tmGFP rat were 0.85, 1.01, and 1.28, respectively (Fig. 3C). The minor variances within groups at previous time points strongly suggest a significant NAA reduction in AAVaspa brains at 26 weeks although the bpooled dataQ prohibited a statistical analysis. To address whether the reduced NAA levels after ASPA gene transfer could change the known seizure phenotype of aged tm rats, we monitored spontaneous absence-like seizures in both AAV treatment groups as 5- to 7-Hz polyspike discharges in hippocampal EEG recordings (Fig. 3D). Seizure occurrence at 18 and 19 weeks of age was reduced in AAVaspa rats (13.5 F 4.0 and 14.7 F 5.2) compared to controls (29.6 F 5.6 and 29.6 F 4.4; P b
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0.05). This trend was even more pronounced at later stages ( P b 0.01) as the seizure activity stayed constant in tmASPA but increased in tmGFP (Fig. 3E). Concomitantly, the duration of individual seizures after AAVaspa treatment was reduced (1.24 F 0.04 s vs. 1.63 F 0.03 s for tmGFP, P b 0.0001; Fig. 3F). Neuronal ASPA Expression Does Not Improve Myelin or Motor Deficits The pathology in CD and tm rats is characterized by hypomyelination [27]. We determined the amounts of myelin in whole hemispheres and found no differences between AAVaspa and AAVgfp treatment at any time point (Fig. 4A). As expected, the amount of myelin in
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FIG. 4. Myelin content and motor function. (A) Myelin measurements at all three time points. Shown is the myelin content in whole hemispheres from four rats per group. (B) Open field analysis shows immobility of both injection groups. (C) Gait analysis. Mean anterior–posterior distance between left and right hind paw (stride length) and medial–lateral deviation of left front and back paw derived from partially overlapping prints (paw spread). (D) Rotarod test. Shown are mean latencies (5 trials per rat) to fall off the stationary rod (phase 1), the constantly rotating (3 rpm) rod (phase 2), or the accelerating rod (phase 3). The maximum duration of an individual trial was 2 min (phases 1 and 2) or 7 min (phase 3). n = 8 for (B–D). *P b 0.05, **P b 0.01, ***P b 0.001.
normal rats increased with time [28]. Likewise, myelin levels increased in tmASPA or tmGFP rats but did not reach normal levels ( P b 0.01). There were no obvious neurological differences between tmASPA and tmGFP animals. Both groups showed the obvious signs of abnormal motor development, tremors, and ataxia. To evaluate possible subtle phenotypic changes after gene transfer, we examined the motor behavior of wt and treated tm rats in three different tests. General locomotion was determined in the open field test where tmASPA and tmGFP rats were dramatically less active than normal rats ( P b 0.0001, Fig. 4B). Neurological abnormalities like splayed legs and ataxia provided the rationale to evaluate the gait pattern. Fig. 4C shows that stride length and paw spread of either AAV-injected group were different from those of wild-type rats ( P b 0.0001). Also, the ability to balance on a rotating rod (rotarod) is impaired in tm rats [29]. In examining this, we conducted a multiphase rotarod protocol ascending in difficulty, to detect even subtle improvements (Fig. 4D). Compared to normal rats, both AAV groups performed similarly poorly in all phases ( P b 0.0001). Combined, these results show that the AAVaspa treatment did not improve motor impairment or the underlying demyelination.
DISCUSSION Canavan disease is one of the few examples of a neurological disease for which gene transfer has been tested in humans, primarily because the pathology is confined to the brain and there was a reasonable expectation that, via metabolic cooperativity, even low levels of transduction could result in phenotypic improvements [13,30]. Based
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on the idea that NAA is the toxic agent causing the CD pathology, it appeared reasonable to reduce its levels by introduction of ASPA into neurons, the location of NAA synthesis, and human experimentation has started [15]. Notably, a report on an AAV2-based ASPA gene transfer into the brain of Aspa-knockout mice failed to provide comprehensive biochemical or neurological data but showed limited transduction efficiency despite a multiple-site injection strategy [16]. Here, we have shown that AAV1/2-mediated transfer of ASPA into a large proportion of the brain and subsequent restoration of enzyme activity to normal levels does not significantly improve all neurological deficits in a genetic rat model of CD. Our data may have implications for human applications. We used a total of 5 billion AAV1/2 genomes per rat. In comparison, 900 billion AAV2 genomes per CD patient were proposed to be administered over six separate subcortical delivery sites in the context of a Phase I clinical trial [15]. Given the higher relative number of particles in the present study (approximately 500-fold difference in size between human and rat brain), the superior transduction efficiency of AAV1/2 over AAV2, and our use of the latest generation expression cassette [31], concerns may be raised regarding therapeutic benefits of the clinical trial. The reduction of NAA levels in tmASPA brains was most prominent in areas of highest transgenic protein expression (Fig. 2A). This finding was in line with the positive correlation between protein expression and enzyme activity (Fig. 1F). However, the decrease of NAA levels in whole brain hemispheres was less dramatic (Fig. 3B), suggesting inefficient metabolic cross-correction of deficient cells by transduced neurons [32] or that neurons
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are not the sole locus of NAA synthesis. In fact, in the normal brain, oligodendrocyte progenitors have low equilibrium concentrations of NAA, possibly caused by ASPA-mediated rapid turnover [10,33,34]. Hence, in our animal model, reduced neuronal NAA levels might have been counteracted by increased glial NAA pools. Moreover, our detailed neurological analyses did not reveal major improvement, except for the amelioration of the epileptic phenotype. We cannot rule out that the intervention may need to occur even earlier in life because some pathology is already present by the time of vector injection, AAV takes several weeks to express at maximum levels, and some severe and irreversible brain damage might have occurred prior gene transfer. To address this, future experiments will involve in utero gene transfer using AAV1/2 to investigate if the timing of the intervention rather than transduction of oligodendroglia is indeed limiting for the success of the therapy. However, the timing of our intervention, immediately postweaning, is consistent with the time of diagnosis for human Canavan disease. Hence, in terms of clinical relevance our timing of gene transfer is appropriate. NAA has been shown to induce seizures after intracerebroventricular administration to normal rats, probably by neuronal overexcitation through metabotropic glutamate receptors [26,35]. Here, we observed partial reduction of NAA in tmASPA brains resulting in modulation of both seizure length and frequency. Together, these data suggest that increased NAA levels in the tm rat brain greatly participate in the course of epilepsy. It might be tempting to speculate that NAA induces glial overexcitation in a similar fashion, and glutamate receptor-mediated toxicity in oligodendrocytes has been reported [36]. However, NAA does not appear to activate this pathway. This notion gets support by the fact that aged heterozygous carriers of the tm mutation exhibit seizures in the absence of dysmyelination [4]. It is not clear what actually causes the typical spongiform degeneration in homozygous mutants. Despite the diminished NAA levels in the thalamus of tmASPA rats (Fig. 3C), vacuolization remained unchanged. Moreover, this pathology is absent in aged carriers, indicating that oligodendrocyte impairment, and possibly death, is a prerequisite for vacuole formation. Motor deficits in the tm rat seem to be a function of ASPA deficiency in oligodendrocytes rather than a consequence of NAA build-up and were not successfully treated. Our biochemical analysis confirmed a relatively moderate extent of demyelination in aged tm rats [27], which is, somewhat surprisingly, paralleled by severe impairment of motor coordination and locomotion. Swollen astrocytes are a known feature of the CD pathology, but we noted axonal spheroids, throughout the CNS of both tmASPA and tmGFP (Fig. 2B). The nature of these swellings is uncertain and an axonal involvement has not yet been described for CD or its models. However,
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it is now widely accepted that axonal integrity and survival result from defects in the myelinating cells, whether myelin irregularities are apparent or not [37]. Further investigations on this were beyond the scope of this study but may contribute to the understanding of CD. This study might help to underscore the concept that for this disorder in particular, and others in which the deficient protein has such a highly segmented expression pattern confined to glia, vectors with specific tropism for such cells are likely to be needed to restore function fully.
MATERIALS AND METHODS Generation of recombinant adeno-associated virus. A fusion construct encoding the HA tag fused to the human ASPA cDNA was subcloned in a rAAV plasmid backbone containing the 1.1-kb CMV enhancer/chicken hactin (CBA) promoter, the woodchuck posttranscriptional regulatory element (WPRE), and the bovine growth hormone poly(A) (AAVaspa). The same rAAV-CBA-WPRE-bGH backbone carrying the Renilla gfp cDNA (Stratagene) was used as the control (AAVgfp). AAV chimeric vectors (virions containing a 1:1 ratio of AAV1 and AAV2 capsid proteins with AAV2 ITRs) were generated and purified using heparin-affinity columns as described [38]. AAV vector administration. Twenty-one- to twenty-four-day-old male and female tremor rats were anesthetized with ketamine and xylazine (84.4 and 12.7 mg/kg) before surgery. One microliter of either AAVaspa or AAVgfp (1.3 109 particles) was mixed with 0.5 Al of 20% mannitol, to enhance vector spread [39], and injected bilaterally into the striatum (+0.35 mm AP, F2.7 mm ML, 5.9 mm DV from bregma) and thalamus (3.2 mm AP, F2.2 mm ML, 5.9 mm DV from bregma) using a stereotaxic frame (Kopf Instruments). Vectors were infused at a rate of 66 nl/min using a microprocessor controlled minipump (World Precision Instruments). Brain tissue preparation for biochemical analysis. After removal of brain stem and cerebellum, brains were stored at 808C as individual hemispheres. Each right hemisphere was later thawed and homogenized in 0.25 M sucrose, 10 mM Hepes, pH 7.4 (20% w/v), to assess ASPA protein levels, enzymatic activity, and myelin content (see below). For NAA analysis of week 5 and week 11 brains, perchloric acid (12%) lysates of the left hemispheres were lyophilized, and the samples were redissolved in D2O spiked with 1 mM 3-(trimethylsilyl)tetradeutero sodium propionate (Sigma) and analyzed by nuclear proton magnetic resonance spectroscopy for NAA content. For the week 26 brain samples, one 300-Am coronal slice was dissected from the thalamic plane of each left hemisphere, combined with slices from brains of the same treatment group, and prepared for NMR. 1
H NMR spectroscopy. All spectra were obtained at 278C on a Bruker Avance DRX 400 NMR spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) operating at a frequency of 400 MHz. One-pulse fully relaxed spectra were acquired with 908 pulses applied every 14 s with a spectral width of 6410.2 Hz and 64K resolution. For a satisfactory signal/noise ratio, 92 scans were accumulated and Fourier transformed. The creatine and NAA spectral peaks were identified by their known chemical shifts (3.04 and 2.02 ppm, respectively). NAA levels were presented as a ratio with creatine.
ASPA enzyme assay. Right hemisphere brain homogenate samples were diluted 1:3 with 3 mg/ml CaCl2 and the cytosolic fractions isolated by centrifugation (20,000g, 20 min, 48C). The enzyme activity test has been described elsewhere [40]. Western blotting. Ten-microgram samples of right hemisphere brain cytosol underwent separation by SDS–PAGE (12% gels) and transfer onto nitrocellulose membrane (Amersham). After blocking and probing with rat ASPA antiserum [12], monoclonal anti-HA antibody (Babco), or rat hrGFP antiserum, bound antibody was detected with secondary antibody
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conjugated to horseradish peroxidase (Santa Cruz) and an ECL chemiluminescence substrate (Amersham). Each membrane was reprobed with anti-h-actin (Abcam). Chemiluminescent signals were captured on film (Amersham) and quantified using the Quantity One image analysis system (Bio-Rad). ASPA signals were normalized to h-actin and presented as levels relative to week 5 wild type. Immunohistochemistry. The rostral–caudal extent of transgene expression of all animals used in behavioral experiments was assessed by HA immunohistochemistry. Processing of rat brain tissue for DAB detection and H&E staining has been described [41]. For fluorescent double labeling 40-Am coronal sections were washed in PBS–Triton, blocked in 4% horse serum/PBS, and incubated overnight with a combination of a monoclonal or polyclonal HA antibody (1:2000; Babco or Santa Cruz) and antibodies against GFAP (1:400; Sigma), MBP (1:500; Sigma), CAII (1:1000; kindly provided by S. Ghandour), or NeuN (1:200; Sigma). After washes, sections were incubated with anti-rabbit–Alexa488 and anti-mouse–Alexa594 antibodies (1:1000; Molecular Probes). Immunostaining was visualized using a Leica SP2 confocal microscope. Myelin measurements. Myelin membranes were isolated and purified as described [28]. Briefly, right hemisphere brain homogenate samples (0.6 ml) were layered onto 0.85 M sucrose (0.8 ml) and centrifuged (70,000g, 90 min, 48C). Myelin was extracted from the interface, osmotically shocked three times with H2O, and pelleted at 23,000g for 30 min at 48C. Following lyophilization the net weight was determined. EEG recordings of spontaneous seizures. At 17 weeks of age, rAAVinfused tm rats and wild-type controls were chronically implanted with an electrode in the hippocampus (4.5 mm AP, F2.0 mm ML, 3.2 mm DV from bregma). One week after electrode implantation, EEGs of the unrestrained animal were recorded twice a week, 15 min per session. One seizure was defined as a 5- to 7-Hz spike-wave-like complex lasting at least 1 s. Motor performance assays. In the open field test a 2-m-diameter circular field was divided into nine segments of equal size. Each rat was placed in the center of the field and monitored for a period of 5 min. The number of lines crossed within that time was recorded. For gait analysis, footprints were taken when walking forward in a 12-cm wide alley, after red and green food color was applied to the front and hind paws, respectively. Gait patterns were analyzed as described [42]. Both tests were conducted with week 13 rats. The rotarod test was performed with week 26 animals and utilized a motor-driven horizontal roller (Rotamex-4; Columbus Instruments). The rats were trained to stay on the rotarod using a threephase schedule. In phase 1 the animals had to perch on the stationary rod for 120 s. In phase 2, the rod was set to the constant speed of 3 rpm for 120 s. In phase 3, performance on the accelerating rod was monitored for 420 s as described previously [43]. In a series of five trials per animal, the time the rats remained on the roller was recorded. Statistics. Behavioral data, enzyme activity, and myelin and NAA measurements were analyzed by ANOVA with post hoc Fisher’s PLSD. An unpaired Student t test was used for Western blot and seizure data. Correlation analysis was performed with a two-tailed test.
ACKNOWLEDGMENTS We thank Michael Walker for excellent help with NMR. M.K. was supported by a European Molecular Biology Organization Fellowship and the Neurological Foundation of New Zealand. D.Y. is supported by a New Zealand Health Research Council Sir Charles Hercus Health Research Fellowship. RECEIVED FOR PUBLICATION DECEMBER 6, 2004; ACCEPTED JANUARY 6, 2005.
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