Introduction

The neuronal ceroid lipofuscinoses (NCLs), commonly referred to as Batten disease, are a group of inherited neurodegenerative diseases that are classified clinically by the age of onset^1,2. The two most rapidly progressing forms of NCL are each caused by deficiencies in lysosomal hydrolases^3,4,5. The earliest form of Batten disease, infantile neuronal ceroid lipofuscinosis (INCL), has an age of onset of around 1.5 years of age and symptoms include visual defects leading to blindness, seizures, motor deficits, mental decline, and early death^6,7,8. The genetic defect for INCL is due to mutations in the PPT1 gene, which codes for the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1)³. On the cellular level, PPT1 deficiency leads to the accumulation of autofluorescent storage material in many cell types, including neurons⁹. As INCL progresses, there is widespread neurodegeneration and severe brain atrophy^10,11. The extent of neuronal loss varies in different regions of the brain, with the most profound loss occurring in the cortex. A small-animal model of INCL was recently created by inactivating the murine PPT1 gene using a targeted gene disruption strategy¹². This murine model has many of the disease characteristics observed in children with INCL, including accumulation of autofluorescent material, brain atrophy, cortical thinning, seizures, and retinal deficits^12,13,14.

There is currently no effective treatment for children with INCL. However, numerous preclinical studies in murine models of other lysosomal storage diseases have demonstrated the potential of viral-mediated gene therapy to treat the CNS disease^{15,16,17,18,19,20,21,22}. The widespread correction observed in the CNS is due primarily to receptor-mediated uptake of the deficient lysosomal enzyme that is produced and secreted by the virally transduced cells^23,24. The secretion and subsequent uptake of lysosomal enzymes is commonly referred to as cross-correction²³. In principle, this receptor-mediated system will enable a small number of transduced cells that overexpress the deficient enzyme to correct a relatively large number of neighboring cells biochemically. Therefore, we set out to determine the potential of using a CNS-directed AAV-mediated gene therapy strategy to treat the disease in a murine model of INCL.

In a previous study, we determined the biochemical and histological consequences of CNS-directed AAV2-mediated gene transfer in the murine model of INCL²⁵. In that study, we demonstrated that recombinant human PPT1 expressed from an AAV2 vector (AAV2-PPT1) was able to cross-correct PPT1-deficient fibroblasts in vitro. We also showed that intracranial (ic) injection of rAAV-PPT1 into newborn Ppt1-deficient (INCL) mice was able both to decrease the accumulation of autofluorescent storage material and to increase the brain mass of treated mice²⁵. However, it was unclear if the reduction in storage material and the increased brain weights correlated with functional improvements.

Here we show that treatment of newborn Ppt1-deficient mice with AAV2-PPT1 results in improvements in several brain parameters, behavior, motor function, and interictal electroencephalographic (EEG) brain activity. Additionally, we show that the improvements in motor function and other behaviors are related to the dose and/or location of AAV2-PPT1 injected intracranially at birth. While our gene therapy strategy resulted in improvements in several functional characteristics of INCL, it was not able to improve all of the behavioral abnormalities, seizure frequency, or longevity in AAV2-PPT1-treated Ppt1-deficient animals.

Results and discussion

PPT1 Expression

Not surprisingly, the level of PPT1 activity in the brain varied for each of the three treatment groups. INCL mice given six ic injections at birth had the highest levels of PPT1-specific activity in the brain with an anterior peak of 109 nmol/mg/h and a posterior peak of 30 nmol/mg/h, which corresponds to 27 and 7.5% of normal levels, respectively (Fig. 1A). The four-injection group had a peak of PPT1 activity in the anterior cortex approximately 14% of normal and near the hippocampal injection site at approximately 5.5% of normal. The animals given only two ic injections had the lowest levels of PPT1-specific activity, with one anterior peak corresponding to approximately 4.5% of normal.

Figure 1.

The graph shows the regional differences in mean PPT1-specific activity at 7 months of age in INCL mice given no (filled squares, n = 4), two (n = 6), four (n = 10), or six (n = 7) ic injections of AAV2-PPT1 at birth. PPT1-specific activity was measured in homogenates from 10 serial 1-mm coronal brain sections. Sections 1 through 10 represent activity from anterior to posterior, respectively. Positive immunostaining (brown) for PPT1 is observed in the (A) cortex, (B) hippocampus, and (C) cerebellum of an INCL mouse given six ic injections of AAV2-PPT1. Higher magnifications of these areas are shown (D–F). The staining in each area appears to be limited primarily to cells with characteristic neuronal morphology (G–I).

Full figure and legend (213K)

We performed immunohistochemical analysis of PPT1 expression on a six-injection INCL animal. We detected PPT1 immunoreactivity within neurons near each injection site, with no apparent staining of either astrocytes of microglia (Fig. 1B). We found localized areas of positive staining for PPT1 near the cortical and hippocampal injection sites, while only a few diverse PPT1-positive cells were scattered among different folia of the cerebellum. This localized expression was not surprising since it is known that, unlike AAV serotypes 1 and 5, AAV2 does not diffuse extensively throughout the brain^26,27.

Improved histologic and morphologic parameters

INCL mice have a significant increase in the level of autofluorescence in all regions of the brain at 7 months of age compared to WT mice (Fig. 2A). The level of autofluorescent storage material in several regions of the brain of INCL mice was reduced following as few as two or as many as six ic injections of AAV2-PPT1. We showed previously that a reduction in autofluorescent storage material correlated with a reduction in abnormal storage bodies observed on toluidine blue-stained histological sections²⁵. INCL mice that received six injections of AAV2-PPT1 had significantly decreased autofluorescence in every region examined compared to the untreated and two- or four-injection groups. However, the six-injection group still had significantly elevated levels of autofluorescent material compared to age-matched WT animals. We observed no reduction in autofluorescence within the cerebellum for either the two- or the four-injection groups. This was not unexpected since neither of these groups received injections into the cerebellum. Surprisingly, the two-injection group had lower levels of autofluorescence in several regions of the brain compared to the four-injection group. This could be due to the variability associated with both the newborn injection procedure and the sectioning.

Figure 2.

(A) INCL mice (-/-, n = 4) have a significant increase in the amount of autofluorescent storage material (AF) in the brain at 7 months of age compared to WT animals (+/+, n = 4). INCL mice given as few as two ic injections (n = 5) or as many as six ic injections (n = 5) of AAV2-PPT1 show a significant reduction in the level of autofluorescent storage material in several regions of the brain compared to untreated INCL mice. The injection site in the anterior cortex is identified on the horizontal axis, and all other distances reported are listed in reference to this site (see²⁵ for a diagram of the sampling sites). (B) There is a trend toward increased cortical volume in INCL animals receiving six ic injections of AAV2-PPT1; however, the mean was not significantly greater (P = 0.073) than that in untreated INCL mice. Autofluorescence and cortical volume measurements were performed on the same animals. (C) INCL mice (n = 26) have significantly reduced brain weights compared to normal (WT, n = 26) animals. Animals given four (n = 12) or six (n = 7) injections have significantly increased brain weights compared to untreated INCL animals. The brain weights of INCL animals given six injections of AAV2-PPT1 are significantly less than those of WT mice. Animals given two injections (n = 9) showed no significant increase in brain weight compared to untreated INCL mice.

Full figure and legend (461K)

INCL mice have a significant decrease in the volume of both the cortex and the striatum, but not the cerebellum, compared to WT mice at 7 months of age. Although there was a trend (P = 0.073) toward increased cortical volume in INCL mice that received six injections of AAV2-PPT1, none of the regions in any of the treatment groups showed a significant increase in volume (Fig. 2B).

With respect to total brain mass, there was a significant reduction in brain weight from 0.396 g in WT mice to 0.325 g in INCL mice at 7 months of age (Fig. 2C). INCL mice given four or six ic injections of AAV2-PPT1 at birth had significantly (P < 0.05) increased brain weights compared to untreated INCL mice. Mice that received either two injections of AAV2-PPT1 or four injections of AAV2-GFP (data not shown) at birth did not show a significant increase in brain weight compared to untreated INCL mice.

Decreased neurodegeneration

Neurons that are undergoing degeneration typically become agyrophilic and stain intensely with silver. INCL mice exhibited widespread neurodegeneration throughout the brain at 7 months of age as indicated by intense silver staining (Fig. 3). In contrast, neurons of untreated WT mice have little or no affinity for silver. INCL mice that received either two or four injections of AAV2-PPT1 had qualitative decreases in silver staining compared to untreated INCL mice. Neurodegeneration in the anterior cortex of both two- and four-injection animals was less. The INCL mice given four ic injections, however, also showed qualitative reductions in silver staining in the anterior commissure, the lateral olfactory tract, and the CA1 region of the hippocampus. The decreased silver staining in the anterior cortex of the two-injection animals, and in both the anterior cortex and the hippocampus of the four-injection animals, corresponded to the respective injection sites. The decreased neurodegeneration in the white matter tracts of the four-injection animals could be due to axonal transport of the enzyme and subsequent rescue of those structures. It has been shown previously that lysosomal enzymes, including PPT1, can be transported axonally and correct CNS regions distant from the original injection site^14,28,29.

Figure 3.

AAV2-PPT1-treated INCL mice show less neurodegeneration in several areas of the brain. (A–D) Low-magnification images show the degree of silver staining (black) in 7-month-old WT, INCL (untreated MUT), two-injection INCL, and four-injection INCL mice. INCL mice given four injections showed less neurodegeneration in the anterior commissure (ac, arrowhead) and the lateral olfactory tract (lo, arrowhead) compared to untreated or two-injection INCL mice. (E–H) Less neurodegeneration and cortical thinning are observed in the anterior cortex of INCL mice given either two or four injections of AAV2-PPT1. (I–L) Reduced neurodegeneration in the CA1 region of the hippocampus (CA1, between the arrowheads) of PPT1-deficient animals is observed only in INCL mice given four injections of AAV2-PPT1.

Full figure and legend (508K)

Protective effects on behavioral functions

To determine if any of the histological or morphological changes corresponded to functional improvements in Ppt1-deficient mice, we examined the performance of treated Ppt1-deficient animals on several behavioral tests. The behavioral results presented are derived from tests performed when the mice were 7 months of age, a time when INCL mice have significant motor and behavioral deficits, but are not so severely affected as to be moribund. Untreated INCL mice had significant deficits compared to WT mice in each of the four behavioral tests (Fig. 4). INCL mice receiving four intracranial injections of a control AAV-GFP vector (n = 14) were indistinguishable from untreated INCL mice in all of the behavioral tests (data not shown). Similarly, INCL mice given two injections of AAV2-PPT1 were not significantly different from untreated mutant mice on any of the four behavioral tests.

Figure 4.

INCL mice treated with four (filled circles, n = 24) or six (open triangles, n = 12) injections of AAV2-PPT1 have significantly (P < 0.05) improved performance compared to untreated INCL mice (filled squares, n = 31) in (A) the continuously rotating rotarod and (B) the accelerating rotarod. There is no statistically significant difference between the six-injection INCL group and the WT animals (open squares, n = 24) in the accelerating rotarod assay (B). Similar improvements were seen in both (C) the pole and (D) the ledge tests, except that the four-injection group (AAV-PPT4) was not significantly different from INCL mice (-/-) in the ledge test. There is no significant improvement in the performance of INCL mice receiving two injections (AAV-PPT2, n = 10) of virus in any behavioral assay. There is no significant improvement in performance in any of the treatment groups in either (E) the total ambulations or (F) the center entries measurements during the open-field testing.

Full figure and legend (463K)

Untreated INCL mice were severely impaired in their ability to remain on the constant-speed and accelerating rotarods on all days and trials compared to WT mice (Fig. 4). In contrast, INCL mice given either four or six injections of AAV2-PPT1 at birth were significantly less impaired on the rotarod compared to untreated mutant animals (Figs. 4A and 4B). On the constant-speed rotarod, the performance of the six-injection group was significantly less impaired compared to the four-injection group, indicating further sparing of behavioral function with increasing dose. On the accelerating rotarod the performance of the six-injection group was not significantly different from the group of WT mice.

Similar to the rotarod results, the four- and six-injection INCL animals exhibited significantly less impairment on both the ledge and the pole tests (sensory motor coordination) compared to untreated or two-injection INCL mice (Figs. 4C and 4D). Interestingly, there were no significant effects of AAV2-PPT1 treatment on general activity levels as measured by total ambulations or on possible alterations in emotionality (e.g., increased anxiety-like behaviors) (Figs. 4E and 4F). Whether decreased activity levels reflect a general malaise from the illness and whether these mice also exhibit increased levels of anxiety-like behaviors remain to be confirmed in future studies. It is not clear why AAV2-PPT1 treatment spares certain INCL-induced functional deficits while others are unaffected by the treatment. The selective palliative effects of treatment are consistent with the limited diffusion of the virus/enzyme within the brain. In addition, it is important to note that INCL mice have profound retinal deficits¹⁴ that could dramatically affect performance on some of the behavioral tests. It is possible that some visual improvement may have occurred, which could account for some of the improved behavioral performance. Nevertheless, improvements in behavioral performance indicate efficacy, whether it be from improved cognitive function or improved visual function.

Electroencephalography

One prominent clinical feature of INCL is the development of progressive severe seizures. The original description of the INCL mouse noted the development of visually demonstrable spontaneous seizures starting at approximately 6 months of age¹². Here we characterize the seizure phenotype of INCL mice in more detail using both EEG and simultaneous video monitoring. These methods allowed us to confirm that stereotypical behavioral changes represented true seizure activity, obtain accurate values for seizure frequency, and analyze interictal EEG patterns.

Continuous monitoring showed that INCL mice display progressive changes in the interictal EEG pattern over time (Fig. 5). We developed a rating scale from 1 to 4 (1, normal; 4, severely abnormal) to quantify these changes (Figs. 5A and 5B). Wild-type animals had an average grade of 1.2. The interictal grades for untreated and AAV2-GFP-treated INCL mice were not significantly different; therefore, these groups were pooled. The mean interictal grade for untreated and AAV2-GFP-treated INCL animals combined was 2.84. INCL mice given four injections of AAV2-PPT1 at birth had a significantly improved average interictal grade of 2.21 compared to untreated and AAV2-GFP-treated INCL mice.

Figure 5.

(A) A graded scale from 1 to 4 was developed to quantify subtle changes in interictal EEG patterns in INCL mice. (B) There was a significant improvement in the interictal pattern (grade 2.21) in INCL mice (AAV-INCL) that received four injections of AAV2-PPT1 compared to untreated and AAV2-GFP-treated (grade 2.84) mice combined (INCL/GFP). However, that score was significantly different from that of WT mice (grade 1.2). (C) The EEG pattern of an INCL mouse during an observable seizure is shown. (D) Although the average number of seizures in INCL animals receiving four injections appeared to decrease compared to untreated INCL and AAV2-GFP-treated mice, the difference was not significant. No WT animal had a seizure (0) during any recording interval.

Full figure and legend (196K)

To determine if treatment with AAV2-PPT1 could also affect seizure frequency in INCL mice, we compared the average number of seizures in a 48-h period (Figs. 5C and 5D). We detected no seizures during any monitoring period in the WT animals. Seizure frequencies for untreated and AAV2-GFP-treated INCL mice at 7.5 months of age were not significantly different. The mean seizure frequency for untreated and AAV2-GFP-treated animals combined was 1.78 seizures/48 h, with 7 of 18 mice having at least one seizure during the monitoring period. INCL mice receiving four injections of AAV2-PPT1 at birth had an apparent decrease in seizure frequency with an average of 0.22 seizure/48 h, with only 1 of 9 animals having seizures during that period. However, this difference was not statistically significant (P = 0.126). The lack of significance in the seizure frequencies was due in part to the variability inherent when using animals on a mixed genetic background. Because of this, we are currently moving the PPT1 mutation onto the congenic C57BL/6 background to minimize the strain variations. Although the improvements in seizure phenotype in the INCL mice receiving four injections were minor, these data suggest that this debilitating clinical feature may respond to more effective therapies. In addition, EEG monitoring provides a powerful tool to evaluate further both the disease progression and the response to future therapies.

Longevity

Children with INCL have a dramatically reduced life span of approximately 5–10 years of age. INCL mice also have a dramatically reduced life span of approximately 36 weeks¹². Although INCL mice that received four or six intracranial injections of AAV2-PPT1 at birth had significant improvements in a number of parameters, most notably in behavior, there was no significant increase in life span compared to untreated INCL mice (Fig. 6). There are a number of factors that could account for this incomplete response. It is possible, perhaps likely, that the level and distribution of PPT1 expression were not sufficient to correct the disease. The development of AAV vectors with different pseudotypes and tropisms, for example AAV1 and AAV5, may provide higher levels of expression and more widespread dissemination of the vectors^26,27. Other routes of administration such as intrathecal or intraventricular may also increase efficacy^30,31.

Figure 6.

INCL mice given either four (n = 21) or six (n = 5) injections of AAV2-PPT1 did not show any statistically significant increase in life span compared to untreated INCL (n = 19) or AAV2-GFP-treated INCL (n = 15) mice. No WT mice (n = 11) died during the course of the 1-year study.

Full figure and legend (86K)

Storage material is present in many other tissues of the INCL mouse (Vogler et al., manuscript in preparation). Since the therapy in the current study was directed to the CNS, the disease in peripheral sites probably remained uncorrected and may have contributed to the limited clinical response. We are currently performing experiments targeting both the systemic and the CNS disease to address this issue. Another contributing factor to the clinical progression of disease could be inflammation. Chronic CNS inflammation has been observed in several models of lysosomal storage disease^32,33 and has been shown to contribute significantly to the progression of disease in the murine model of Sandhoff disease³⁴. Progressive astrocytosis (GFAP staining) and macrophage activation (CD11b staining) have also been observed in the CNS of the INCL mouse (Cooper et al., manuscript in preparation). We are currently determining if AAV2-mediated gene therapy reduces CNS inflammation in the INCL mouse. Finally, it is possible that immune reactions against PPT1 decreased the efficacy. However, we believe this is unlikely. Previous studies of AAV-mediated gene therapy initiated during the newborn period have shown that those animals do not mount a humoral response to the transgene product¹⁷ and have stable levels of expression for more than 1 year¹⁸. The lack of a humoral response when therapy is initiated in newborn animals is likely due to tolerization.

Taken together, these data suggest that early treatment of INCL using gene transfer techniques can be efficacious; however, higher levels or a broader distribution of PPT1 expression, or both, will be required for more complete correction of this inherited neurodegenerative disease.

Methods

Ppt1-deficient mice and intracranial Injections

The Ppt1-deficient (INCL) mice used in this study were originally created through a targeted disruption strategy that eliminates the last exon of the murine Ppt1 gene¹². AAV2-PPT1-treated mice were given two, four, or six ic injections of 0.5 l AAV2-PPT1 as newborns (1–3 days of age). Two-injection animals received one injection per hemisphere in the anterior cortex. Four-injection animals received one injection per hemisphere in each anterior cortex and the hippocampus. Six-injection animals received one injection per hemisphere in each anterior cortex, hippocampus, and cerebellum. The coordinates for all of the injection sites were measured from the bregma and were as follows: anterior cortex 1 mm caudal and 2 mm lateral, hippocampus 3.5 mm caudal and 2 mm lateral, and cerebellum 6 mm caudal and 2 mm lateral. All injections were done at a depth of 1.5 mm from the skull surface. All animal procedures were carried out in accordance with NIH guidelines and the Institutional Animal Care and Use Committee regulations of Washington University.

AAV2-PPT1 production

The recombinant AAV2 vector, AAV2-PPT1, has been described in detail previously^16,17,25,35. Virus used in this study was produced at the Vector Core Facility at the University of Florida using previously published techniques^36,37. The particle number (3.83 10¹¹/ml) and infectious units (2.5 10¹⁰/ml) were determined by slot-blot analysis and infectious center assay, respectively.

Biochemical analyses, autofluorescence measurements, and brain morphometry

PPT1-specific activity was measured in homogenates from serial 1-mm coronal sections of brain from 7-month-old animals as previously described^25,38. Autofluorescence was measured in 40-m-thick coronal sections of brain as previously described²⁵. Briefly, six regions of the brain were assayed for the level of autofluorescence: (1) anterior cortex injection site, (2) cerebellar injection site, (3) 2 mm rostral to the anterior injection site, (4) 2 mm caudal to the anterior injection site, (5) 4 mm caudal to the anterior injection site, and (6) striatum (see²⁵ for diagram of sampling sites). Statistical analysis was done by determining the average for each region and treatment group and then performing ANOVA with post hoc Bonferroni. At 7 months of age the brains from experimental and control mice were harvested and weighed as previously described²⁵. Unbiased stereological estimates of the regional volume of the cortex, hippocampus, and cerebellum were obtained from a one-in-six series of Nissl-stained coronal sections using StereoInvestigator software (Microbrightfield, Inc., Williston, VT, USA), as described previously^13,25.

Immunohistochemistry and neurodegeneration staining

Brain sections were incubated with rabbit anti-rat PPT1 primary antiserum (kind gift from Dr. S. Hofmann, University of Texas Southwestern) at 4°C overnight. The sections were then incubated with biotinylated goat anti-rabbit followed by Vectastain ABC Elite (both from Vector). The staining was visualized using 0.05% DAB (Sigma D55905). For neurodegeneration staining, Ppt1-deficient, normal, and AAV2-PPT1-treated mice given either two or four ic injections were deeply anesthetized and then perfused by transcardiac approach with 4% paraformaldehyde in sodium cacodylate buffer (pH 7.4) at 7 months of age (insufficient numbers of six-injection animals were available for silver staining). Thirty-five-micrometer serial coronal sections of brain were obtained and every 12th section was stained according to a modified amino cupric silver method developed by DeOlmos (Neuroscience Associates, Knoxville, TN, USA)^39,40.

Behavioral testing

Behavioral tests were performed on 7-month-old untreated INCL (n = 31) and WT (n = 24) mice, AAV2-GFP-treated INCL (four ic injections, n = 12) mice, and AAV2-PPT1-treated INCL mice given two (n = 10), four (n = 24), or six ic injections (n = 12). The AAV2-GFP-treated INCL mice were not significantly different in performance from untreated INCL animals in any of the tests.

Rotarod

The rotarod (Columbus Instruments, Columbus, OH, USA) test was used to evaluate motor coordination and balance. Continuously rotating (speed 2.5 rpm, max 60 s) and accelerating (constant acceleration over 3 min from 2.5 to 10.5 rpm) rotarod paradigms were used. The protocol used in this study was similar to that described previously⁴¹. This protocol involved three training sessions, with a 3-day span in between each training session. The mice were then given one trial on the stationary rod and two trials on each of the continuously rotating and accelerating rotarods per training session. Performance on the rotarod was scored as time spent on the rod before falling for each condition.

Pole test

The pole test can be used as a general measure of ataxia and specifically measures the coordination between forelimbs and hind limbs. The protocol used in this study is similar to that described previously^42,43. Performance on this test was scored as the time it took the mouse to climb down and reach the bottom of the pole. The performance of the mice was evaluated during two different trials, one each on 2 consecutive days. A maximal time of 120 s was allowed for the mouse to perform the test, and if the mouse fell from the pole during the test it was given the maximum score.

Ledge test

The ledge test is sensitive to defects in sensory motor capability and is particularly useful in animals with an ataxic phenotype^44,45. Performance on the ledge test was determined by how long a mouse could maintain its balance on a 0.75-cm-wide Plexiglas ledge without falling. A maximum time of 60 s was allowed for this task.

Open-field behavior test

In this test mice were placed in transparent (47.6 25.4 20.6-cm high) polystyrene enclosures for a 1-h period, as described previously⁴⁶. A high-resolution photobeam system (Motor Monitor; Hamilton-Kinder, LLC, Poway, CA, USA) was used to evaluate the general activity levels and possible emotionality of the mice. This test analyzed different variables, including total ambulations and entries into the center of the apparatus.

Statistical analyses

Behavioral data in this study were analyzed by ANOVA models. Two between-subjects variables of genotype and gender were used, and one within-subjects variable of trial was used. To ensure that sphericity/compound symmetry assumptions of the ANOVA model were not violated, we used the Huynh–Feldt adjustment of levels when within-subject effects contained more than two levels. For multiple comparisons, the Bonferroni correction was used to maintain levels as prescribed.

Electroencephalography

Seven-and-one-half-month-old mice were monitored for interictal EEG activity and clinical-electrographic seizures by video/EEG recording. Epidural screw electrodes were surgically placed under halothane anesthesia to allow continuous EEG recordings. Two skull electrodes (2 mm to the right and left of midline, 1–2 mm posterior to bregma) were referenced to a midfrontal bone electrode (3 mm anterior to bregma). Animals were allowed to recover for at least 2 days before recording. EEG and video data were collected during a single, continuous 48-h monitoring session. EEG data were acquired with Grass P-100 EEG amplifiers, an Axon Digidata 1320 analog-to-digital converter, and Axoscope software on a personal computer. Simultaneous digital video data were also stored on a computer with a Sanyo Day–Night camera and a Darim MG-100 MPEG video capture card.

For analysis of seizures, the entire 48-h EEG record was reviewed and electrographic seizures were identified by their characteristic pattern of discrete periods of repetitive spike discharges that evolved in amplitude and frequency lasting at least 10 s and were typically followed by voltage suppression (see Fig. 5C). Average seizure frequency (number of seizures/48-h period) and number of animals with seizures were calculated for each treatment group. Statistical analysis was performed using Kruskal–Wallis nonparametric ANOVA.

For analysis of the interictal EEG background, 1-min segments obtained from every 4 h of the 48-h EEG record were randomly selected and graded with the following scale: grade 1 (normal)—normal background rhythm with no epileptiform spikes, grade 2 (mildly abnormal)—mostly normal background with some epileptiform spikes, grade 3 (moderately abnormal)—mostly abnormal background with frequent epileptiform spikes, grade 4 (severely abnormal)—burst–suppression pattern (see Fig. 5A). There was no significant difference in interictal score between INCL and AAV2-GFP-injected animals. A mean interictal EEG grade was calculated for the WT, INCL, and control groups combined and the AAV2-PPT1-treated groups. Statistical comparisons were made using ANOVA with Tukey–Kramer multiple comparison post hoc test.

Four WT mice and nine mice per group of untreated INCL and four-injection AAV2-PPT1- and four-injection AAV2-GFP-treated INCL mice were included as part of the EEG study. Insufficient numbers of two- or six-injection animals were available for EEG analysis.

Longevity

Following intracranial injection, AAV2-GFP-treated, AAV2-PPT1-treated, INCL, and WT mice remained unmanipulated for the duration of this study. The cumulative survival was measured and statistical significance was determined by Kaplan–Meier analysis.

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Acknowledgements

This work was supported in part by Grants NS043205 (M.S.S.) and NS41930 (J.D.C.) from the National Institutes of Health. The following nonprofit organizations also contributed financially to this work: the Batten Disease Support and Research Association (M.S.S. and E.B.), the Natalie Fund (J.D.C.), the Batten Disease Family Association (J.D.C.), and the Remy Fund (J.D.C.).