Human Molecular Genetics, 2002, Vol. 11, No. 11 1251-1262
© 2002 Oxford University Press
Targeted disruption of the Epm2a gene causes formation of Lafora inclusion bodies, neurodegeneration, ataxia, myoclonus epilepsy and impaired behavioral response in mice
1Laboratory for Neurogenetics, 2Laboratory for Neurobiology of Emotion, 3Laboratory for Neural Architecture and 4Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan, 5Departamento de Neurociencias, Universidad Nacional Autónoma de México, Mexico, 6First Department of Pathology, Yamaguchi University School of Medicine, Yamaguchi, Japan, and 7Epilepsy Genetics/Genomics Laboratories and Comprehensive Epilepsy Program, UCLA School of Medicine and VA GLAHS West Los Angeles Medical Center, Los Angeles, CA, USA
Received January 16, 2002; Accepted March 19, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Mutations in the EPM2A gene encoding a dual-specificity phosphatase (laforin) cause Lafora disease (LD), a progressive and invariably fatal epilepsy with periodic acid–Schiff-positive (PAS+) cytoplasmic inclusions (Lafora bodies) in the central nervous system. To study the pathology of LD and the functions of laforin, we disrupted the Epm2a gene in mice. At two months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the absence of Lafora bodies. Dying neurons characteristically exhibit swelling in the endoplasmic reticulum, Golgi networks and mitochondria in the absence of apoptotic bodies or fragmentation of DNA. As Lafora bodies become more prominent at 4–12 months, organelles and nuclei are disrupted. The Lafora bodies, present both in neuronal and non-neural tissues, are positive for ubiquitin and advanced glycation end-products only in neurons, suggesting different pathological consequence for Lafora inclusions in neuronal tissues. Neuronal degeneration and Lafora inclusion bodies predate the onset of impaired behavioral responses, ataxia, spontaneous myoclonic seizures and EEG epileptiform activity. Our results suggest that LD is a primary neurodegenerative disorder that may utilize a non-apoptotic mechanism of cell death.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Lafora's progressive myoclonus epilepsy or Lafora disease (LD) is an autosomal recessive, invariably fatal disorder with pathognomonic periodic acid–Schiff-positive (PAS+) intracellular cytoplasmic inclusions (Lafora bodies) in the central nervous system, heart, muscle and liver (1–3). LD starts in early adolescence as stimulus-sensitive grand mal tonic–clonic, absence, visual and myoclonic seizures with electroencephalographic (EEG) 3–6 Hz spike-wave complexes. Rapid neurological deterioration that includes ataxia, dementia, psychosis, dysarthria, amaurosis, mutism, muscle wasting and respiratory failure leads to death between 17 and 30 years of age (2,3). LD, a rare disorder, occurs worldwide but is relatively frequent in the Mediterranean countries of Southern Europe, the Middle East, India, Pakistan and Northern Africa, and in cultures that practice consanguineous marriages (4–6). LD is caused by mutations in the EPM2A gene encoding a dual-specificity phosphatase named laforin (7–9). Laforin associates with polyribosomes and is expressed ubiquitously (7,9–11). The severity of LD symptoms, its rapid course and death at a young age underlie the important role laforin plays in cell biology.
Because PAS-positive Lafora inclusions are polyglucosan and have been considered to be the hallmark and cause of epilepsy and neurologic deterioration, LD has been traditionally listed under the glycogen storage diseases (2,3). Only a few reports described the degenerative changes in the neuropile (3,12). Perhaps because analysis was restricted to brain and liver biopsy specimens and because a complete postmortem examination was carried out in only a few cases, no study has documented the early neuropathological changes that could define the primary events of the disease. We have generated laforin-deficient mice, and we describe here their primary neuropathologic event, behavioral changes and EEG epileptiform abnormalities.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Generation of Epm2a null mutants
We earlier demonstrated that the fourth exon of the EPM2A gene in humans encodes the dual-specificity phosphatase domain (DSPD) critical for the function of laforin (5). We therefore constructed a targeting vector in which a region of the Epm2a gene encoding the DSPD was replaced by a neomycin-resistance gene cassette (Fig. 1A). This deletion eliminates the coding sequence of the DSPD exon (Fig. 1B). Mice carrying the targeted allele were derived from two independent ES clones. Homozygous offspring for the deletion were viable and were produced in the expected Mendelian genotype ratio of 1 : 2 : 1 (+/+ : +/- : -/-, 115 : 245 : 99) (Fig. 1B). In mice, Epm2a is ubiquitously expressed (10). To define the effects of DSPD exon deletion on Epm2a expression, we compared the RNA extracted from brain and liver tissues of mutants with those of wild-type littermates. No Epm2a mRNA was detected in Epm2a-/- animals when the DSPD region or the coding exons occurring 5' to the deleted region was used as a probe in northern blot analyses (Fig. 1C). The absence of Epm2a transcripts in homozygous null mutants was further confirmed by reverse transcription–polymerase chain reaction (RT–PCR) analysis (Fig. 1D). Removal of the DSPD exon had therefore prevented expression or maturation of Epm2a transcript, and produced an Epm2a-null mutant.
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Null mutants develop Lafora inclusion bodies that are positive for ubiquitin and advanced glycation end-products
We examined the pathology of major organs of Epm2a-/- mice, their heterozygous and wild-type littermates from birth through 1 year of age. Sequential sectioning and staining of brains of Epm2a-/- littermates showed prominent (PAS+) inclusions (Lafora bodies) within neurons throughout the brain (Fig. 2B,D), an observation consistent with findings in LD patients (3). Application of the KM279 antibody, which had been raised against Lafora body extracts from cardiac muscle cells of a patient with LD (13), showed PAS+ inclusions to be antigenetically similar to human Lafora bodies (Fig. 2F,H,J,K). A few small Lafora bodies were observed as early as 2 months after birth. By 4 months, many regions of the brain were extensively loaded with immunoreactive Lafora bodies (Fig. 2J, K). Prominently affected areas include hippocampus, cerebellum, cerebral cortex, thalamus and brain stem (Fig. 2K), which roughly correlate with sites of laforin expression (11). Neuronal Lafora bodies were ubiquitin-positive (Fig. 3A–C), and also reacted with an antibody to advanced glycation end-products (AGEP) (Fig. 3D,E). AGEP are insoluble and non-degradable products that result from the interaction between reducing sugars and long-lived proteins (14–16). Positive reactions in Lafora bodies to antibodies against ubiquitin and AGEP indicate the presence of the polyglucosan protein complex.
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Lafora inclusions in non-neuronal tissues
Similarly to LD in human, Lafora bodies were also found in muscle and liver tissues of nine-month-old knockout mice (Fig. 3F,G,I). However, they were not apparent in non-neuronal tissues of four-month old null mutants. Moreover, Lafora bodies of liver and muscle tissues did not react with anti-ubiquitin or anti-AGEP antibody (Fig. 3L,M,N), suggesting different pathological consequence for Lafora inclusions in neuronal tissues. PAS+ and immunoreactive Lafora bodies were not detected in the neuronal or nonneuronal tissues of heterozygous and wild-type littermates (Fig. 3F,G,J).
Ultrastructural analysis: neuronal cell death in homozygous null mutants
To further understand the mechanism of cell death at 2 months and the pathological consequences of Lafora bodies in homozygous null mutants, we studied the fine structure of their neuropile (Fig. 4). Ultrastructural analysis in knockout mice brains revealed isolated or full rows of cerebellar Purkinje cells, hippocampal pyramidal and granular cells, and cerebral cortical pyramidal cells showing unequivocal features of somatic degeneration as early as 2 months of age (Fig. 4A–H). Distorted and shrunken cytoplasmic and nuclear membranes characterized these dark neurons (Fig. 4A). Most organelles in such cells showed disruption of their normal distribution, and their essential morphological features were lost. Dark mitochondria, dilated cisterni of endoplasmic reticulum, and displaced and collapsed Golgi apparatus were prominent and consistently observed changes (Fig. 4B). Neuronal processes and their organelles also showed disruption of structures (Fig. 4C–F). Longitudinal or transverse dendritic sections showed atrophic and distorted spines that were homogenously hyperdense. Some were shrunken and their organelles showed degenerative changes (Fig. 4C), whereas others were either hyperdense or vacuolated (Fig. 4D). Frequently, a normal synaptic bouton was impinging upon these degenerated dendrites (or upon their spines). Transverse sections of axons showed distortions and disruptions of myelin sheaths, with abnormal separation of lamellae, and a central, dense body with degenerated organelles (Fig. 4E). Axoplasm appeared watery and vacuoles formed. Mitochondrial cristae were no longer observed, and were replaced by fine granular material (Fig. 4F). Degenerating neurons were observed in EM preparations of 2-, 3-, 9- and 10-month-old knockout mice. Most degenerating neurons did not show Lafora inclusion bodies.
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There was no fragmentation or ‘blebbing’ of either nucleus or cytoplasm of degenerating cells. Repeated attempts to identify DNA fragmentation by in situ labeling with terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling (TUNEL) method failed to reveal a significant increase in the number of positive cells in the null mutant's brains when compared with wild-type and heterozygote littermates.
Ultrastructurally, Lafora inclusions in the knockout mice were very similar, if not identical, to electron-microscopic descriptions of Lafora bodies in humans (3). These inclusion bodies were formed by small, fragmented fibrillar material surrounded by abundant fine, punctiform particles that resembled ribosomes (Fig. 4G,H,J,K). Most inclusion bodies were relatively hypodense, but occasionally their density appeared darker. There was no limiting membrane surrounding these Lafora bodies. The appearance was very similar in experimental animals from all ages, as shown in 2-month-old (Fig. 4H) and 9-month-old null (Fig. 4K) mutants. In general, inclusion bodies showed variable sizes that increased in dimension with age. Figure 4G displays the beginning formation of a small Lafora body, within a neurite. Figure 4H is a high magnification of two intracytoplasmic Lafora bodies that barely displace mitochondria and other organelles. In contrast, in 9-month-old animals, the neuronal cytoplasm can be completely filled with larger Lafora inclusions that often displace the nucleus and organelles (Fig. 4J).
Epm2a null mutants show impaired behavioral response and progressive ataxia
In spite of the appearance of a few small Lafora inclusion bodies and signs of neurodegeneration on electron microscopy at 2 months, the Epm2a-/- mice appeared to grow and develop normally during the first 3 months. At 4 months, the magnitude of acoustic startle reflex in Epm2a-/- mice in response to a stimulus of standard intensity remained high throughout the whole test session (Fig. 5A). This suggests a deficit in habituation and/or enhanced excitability/diminished inhibition in central nervous system pathways. In the passive avoidance task, null mutants showed a deficit in retention (Fig. 5B). Taken together, these results indicate impaired behavioral response in 4-month-old null mutants due to inactivation of the Epm2a gene.
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At 9 months of age, muscular weakness and ataxia were grossly evident in null mutants. Rotarod and wire-hanging tasks showed significantly impaired motor coordination, balance and grip strength in 9-month-old Epm2a-/- mice when compared with wild-type littermates (Fig. 5C,D,E). Mean neuromuscular strength and endurance force exerted in a dynamometer by 9-month-old mutant mice were significantly less than that of wild-type littermates (Fig. 5F).
Myoclonic seizures and EEG epileptiform activity in null mutants
Nine-month-old Epm2a mutants also developed spontaneous myoclonic seizures with an approximately 80% penetrance (in 24 out of 30 animals observed). Ear and head spasms and shoulder jerks or minor shaking of the torso followed by an upward motion of the head at times forced the animal forward. Each episode lasted for a few seconds and usually consisted of one or two jerks. Frequency of seizures was measured by an automated seizure detection system (17). Several spontaneous seizures were observed during the animals' dark and light cycles, but the frequency was almost doubled during active (dark) periods. Scalp EEG and cortical electrography in mutant mice revealed frequent single spikes, repetitive spikes and sharp waves as well as spike and slow-wave formations that appeared synchronously or asynchronously in both cerebral hemispheres during awake and sleep periods (Fig. 6). Diffuse polyspikes followed by slow waves were also evident in the EEG during spontaneous myoclonic jerks (Fig. 6B). Awake cortical electrography also showed a reduction in the background frequency of the brain waves in the 9-month-old knockout mice (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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We have established a mouse model for LD through targeted disruption of the Epm2a gene. The phenotype resulting from the loss of the Epm2a gene in mice is comparable to the phenotype of human LD resulting from the same genetic defect. The Epm2a-/- mice are developmentally normal and fertile, but developed behavioral abnormalities at 4 months and signs of ataxia, and myoclonus epilepsy at 9 months of age. At 2 months of age, homozygous null mutants developed widespread degeneration of neurons, most of which occurred in the presence of only a few small Lafora inclusions. Mitochondrial, endoplasmic reticulum and Golgi apparatus swelling with lysis of cell membranes led to cell death. As Lafora bodies became more prominent at 4–12 months, organelles and nuclei were disrupted. Neuronal cell death and Lafora inclusion bodies predate impaired behavioral responses, ataxia, myoclonic seizures and EEG epileptiform activity. Our results suggest that LD is primarily a neurodegenerative disorder and provide evidence for laforin's critical role in neuronal survival.
Laforin deficiency in both humans and mice results in the formation of Lafora inclusions, progressive ataxia and myoclonic seizure. However, the mutant mice only developed spike and slow-wave formations, but not the regular and well-formed EEG difuse 3–6 Hz spike-wave complexes. Clinical absences or grand mal tonic–clonic seizures, which are almost invariably observed in LD patients, also did not develop in 1-year old mice. This suggests that additional factors (environmental or genetic) contribute to the appearance of grand mal and absence seizures in humans. Alternatively, such factors could reflect differences in the organization/functions of cortico–reticular–cortical pathways between mice and humans. As the null mutants analyzed in the present study were up to 1 year old, it is also possible that they will develop other elements of LD as they age. Such an extended study may also resolve the difference, if any, in the lifespan of the null mutants – an anticipated phenotype, but not seen in our 1-year-old mice colony.
Previous neuropathological studies for LD in humans have been limited to gross anatomical descriptions of autopsy results and did not characterize in detail the structural abnormalities. Moreover, these results in humans were complicated by chronic diphenylhydantoin treatment, which is known to produce cerebellar degeneration. Thus the contributions of chronic diphenylhydantoin treatment to the neuropathology found in LD were never resolved in humans. Our finding that the disease phenotype in knockout mice is associated with widespread neuronal degeneration earlier than had been reported in human LD provides new clues to the evolution and pathogenesis of LD. Neuronal cell death was found in all stages of null mutants analyzed (2- to 12-month old animals). However, a rigorous quantitative analysis for different age groups is necessary to understand the spatial and temporal difference in the number and distribution of degenerating neurons in null mutants' brains. Degenerating neurons exhibit cellular darkening, nuclear and cytoplasmic condensation, swelling of subcellular organelles, and structural abnormalities in the neuronal processes. However, the defining characteristics of apoptosis – the fragmentation or blebbing of both cytoplasm and nucleus – were not found in the degenerating neurons. Similarly, the absence of TUNEL-positive cells in null mutants' brains suggests that within degenerating neurons there is no DNA fragmentation, a prominent feature of apoptotic cells, and that cell death occurs without activation of apoptosis. It is noteworthy that morphologically very similar forms of cell death have been described previously in neural degeneration in Huntington disease patients and in Huntington model mouse (18). Further, it has been suggested that such forms of neuronal cell death may define a novel process that is morphologically and biochemically distinct from apoptosis (18). It is surprising that the loss of function of a normally ubiquitous protein, laforin, cause selective neuronal loss. One possibility is that laforin has a neuron-specific substrate whose activation/inactivation is critical for neuronal survival. The detection of cell death in laforin-deficient mice supports the interpretation that at least some of the symptoms of LD are initiated by neuronal death.
In addition to the PAS reactivity, biochemical studies in humans have indicated that Lafora bodies are principally composed of polysaccharides (19,20). A recent report demonstrates that laforin contains a functional carbohydrate-binding domain at its N terminus that targets laforin to glycogen (21). This raises an interestingly possibility, supported by recent studies by Raben et al. (22). By targeting the glycogen complex in vivo, laforin as a dual-specificity phosphatase may regulate proteins, such as glycogen synthase, that are involved in glycogen synthesis. Loss of function of a mutated laforin would lead to an overdrive in glycogen synthesis and the accumulation of polyglucosan as Lafora bodies. Alternatively, laforin may mediate the disposition of Lafora bodies by directly binding to these inclusions. Polyglucosan bodies are known to be produced normally in neuronal cells and cleared via axons into the cerebrospinal fluid (23). Thus, laforin may be involved in the inter- and intracellular migration of polyglucosan, and its loss of function would result in polyglucosan accumulation as Lafora bodies. Our observations on PAS+ inclusions increasing in dimension with age in laforin-deficient mice are consistent with both propositions. Our knockout mouse model provides a valid model to test both of these hypotheses.
Are Lafora bodies toxic to neuronal cells? Since the majority of degenerating neurons do not contain Lafora bodies and not all cells that contain Lafora inclusions degenerate, the formation of Lafora bodies may not necessarily lead to neuronal cell death. However, a causative role for Lafora inclusion in neuronal dysfunction cannot be excluded. Inclusions harboring ubiquitin and/or AGEP modified proteins have been implicated in the pathogenesis of a number of degenerative diseases (24–30). Lafora inclusions may thus induce neuronal stress and neurotoxicity due to a diminished ubiquitin proteolytic system (27,30,31) and/or by generation of reactive oxygen species through an interaction between AGEP and RAGE (receptor for advanced glycation end-products) (32–35). Importantly, the early onset of both neurodegeneration and Lafora inclusion in Epm2a-knockout mice predates behavioral abnormalities, ataxia and epileptic myoclonias by many months. Any attempt to treat LD in humans must therefore start at the presymptomatic stages of the disease in early childhood. The Epm2a-/- mouse model of human LD, with its progressive and profound neurologic disturbances and pathology, should be critical in developing treatment such as gene therapy.
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METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Generation of Epm2a-deficient mice
We deleted the dual-specificity phosphatase domain (DSPD) coding region of the Epm2a gene (Fig. 1A). A 15 kb genomic clone B6LDM8 identified in a previous study (7) was used to make a targeting construct. A 2.0 kb HindIII–EcoRV fragment and a 3.0 kb EcoRI–HindIII fragment of the B6LDM8 clone were subcloned respectively into the HindIII and SmaI sites and EcoRI–HindIII sites of the plasmid vector pBluescript IISK+ (Stratagene Inc., La Jolla, CA). Subsequently, the 2.0 kb HindIII–EcoRV fragment was excised by HindIII and BamHI digestion and cloned into HindIII and BamHI sites of a targeting vector, generating the 5' arm of the construct (Fig. 1A). The 3' arm was generated by inserting the 3.0 kb Eco/RI–HindIII fragment into the EcoRI and HindIII sites, thus creating the neomycin phosphotransferease (neo) targeting cassette (Fig. 1A). Another negative-selection cassette (DTA), which encodes the diphtheria toxin A-fragment under the control of the HSV-tk (herpes simplex virus thymidine kinase gene) promoter, was included in the targeting construct to allow enrichment for the correctly targeted embryonic stem (ES) cell clones.
The targeting vector linearized with NotI was electroporated into approximately 5x107 E14 ES cells with a Gene-Pulser (Bio-Rad Laboratories, Hercules, CA) at 3 µF and 800 V. Transfected cells were plated on neomycin-resistant, mitomycin C-treated mouse embryonic feeder cells (MEF) in a 10 cm dish. One day after plating, positive selection was performed in the presence of Geneticin (G418; 125 µg/ml). Resistant clones were picked at day 9 and subsequently expanded into 24-well plates preseeded with MEF. BamHI-digested genomic DNA from individual clones was analyzed by Southern blot with a 1.5 kb probe derived from the genomic sequence immediately upstream of the targeting vector (Probe A; Fig. 1A). The probe was generated by subcloning the 1.5 kb BamHI–HindIII fragment into a pBluescript II SK+ vector. Positive clones were further confirmed by hybridizing the EcoRV-digested genomic DNA to a 3-end probe (Probe C, 
1.5 kb BamHI–EcoRV fragment; Fig. 1A). Approximately 15 ES cells from each of the two targeted clones (S31 and S128) were injected into C57BL/6J blastocysts. An average of 15 injected blastocysts were transferred into pseudopregnant female recipient ICR mice. Both ES clones produced several chimeras with greater than 50% agouti coat color. Eight (four for each clone) male chimeras were bred to C57BL/6J females to allow for the detection of germline transmission by the agouti coat color marker. Thirty-eight percent of the agouti offspring (92 of 237) were positive for the insertion, as determined by Southern blot analysis. Heterozygous mice were crossed to generate animals homozygous for the insertion. All observations reported here were made using the F2 generation, mixed C57BL/6 and 129Svj genetic background animals from lines derived from ES clones S31 and S128. While histological and EEG/EcoG examinations were done on both lines, behavioral analysis was restricted to a single line (S31 only).
Expression analysis
Liver and brain tissues were dissected from 2-month-old mice, and RNA was extracted as described previously (11). Six micrograms of poly(A) RNA was loaded per lane on 1% agarose formaldehyde gel, and the size-separated RNA was transferred to a nylon membrane (Biodyne; Pall Bio Support, East Hills, NY). For hybridization, DNA probes were labeled with [
-32P]dCTP and hybridized overnight in ExpressHyb hybridization solution (Clontech, Palo Alto, CA) as recommended by the manufacturer. The blots were washed finally in 0.1xSSC/0.1% SDS at 55°C and exposed to X-ray film at -80°C. For RT–PCR, cDNA was synthesized using the ThermoScript RT–PCR system (Life Technologies, Grand Island, NY).
Light microscopy and immunohistochemistry
PAS and immunohistochemical stainings were done as described previously (36). Sections were reacted with biotinylated IgM monoclonal mouse antibody KM279 and visualized using peroxidase-labeled streptavidin conjugate (Dako, Copenhagen). For double labeling, sections were reacted simultaneously with mouse monoclonal KM279 and rabbit polyclonal anti-ubiquitin antibodies (Dako), followed by anti-mouse IgG–rodamine and anti-rabbit IgG–FITC conjugates, and the fluorescence was observed by a confocal laser scanning microscope. The monoclonal anti-AGEP IgG was obtained commercially (TransGenic Inc., Kumamoto, Japan). TUNEL was performed on paraffin sections using the DeadEND Colorimetric Apoptosis Detection System as described by the manufacturer (Promega corporation, Madison, WI).
Electron microscopy
Different age groups of mice were perfused intracardially through the left ventricle with an ice-cold solution of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. The perfusions were followed by a 24-hour immersion fixation at 4°C in the same solution. Tissue samples were harvested, embedded in both cross and longitudinal orientations, and processed for electron microscopic analyses.
Startle response test
In the acoustic startle test, mice were exposed to 40 ms broadband 110 dB burst (pulse trials) with an intertrial interval of 20 s (TIK Co. Ltd, Tokyo). Forty pulse trials were administered in total. The startle response was recorded for 1 s starting with the onset of the startle stimulus.
Step-through passive avoidance test
During the acquisition trial, animals were placed in the non-preferred bright chamber of a two-chamber apparatus (Ohara Co. Ltd, Tokyo, Japan). When animals entered the dark side, the door between the chambers was shut and the animal received a 0.4 mA, 5 s shock. After 10 s, the mice were returned to their home cage. Mice were tested 24 hours after the training, and the latency to enter to the dark chamber during the retention trial was recorded.
Rotarod test
A 3 cm diameter rotarod (Rota-Rod treadmill; Ugo Basile, Comerio, Italy) consisting of a plastic roller flanked by two round plastic plates was used for testing neuromuscular abnormalities. Mice were placed on a stationary cylinder. After 3 s, the rotarod was switched on, rotating evenly at a speed of 20 r.p.m. The ability of the mice to stay on the rotarod was timed until they fell, with a maximum cutoff of 200 s. Mice were tested for three trials on succeeding days, and the fourth trial was done on day 6, after a 2-day rest.
Hanging wire test
The ability to hang upside-down from a wire screen was tested as described previously (37). After naive mice were placed on a modified wire cover to a rat tub cage, the cover was waved gently for three times and immediately turned upside down. Latency to time of falling from the cover was recorded. Mice that fell within 10 s were given a second trial and those that did not fall during the 60 s trial period were removed and given a maximum score of 60 s.
Test for muscle strength
Grip strength of the forepaws was assessed at the point when mice released a horizontal strain gauge grasped by both front paws as a result of gentle traction applied to the tail. The gauge was connected to a gram dynamometer (Ohba Siki, Tokyo) that measured the force exerted.
Myoclonic seizure activity
Groups of animals (30 for each genotype) were monitored by constant videotaping using an automated seizure detection system (COBAS; GE Marquette Medical System, Kyoto) (17) for three consecutive days. Occurrence and frequency of myoclonic jerks were scored for each animal during dark and light periods separately.
EEG and ECoG recordings
Brain waves were recorded in two ways. (i) Electroencephalography (EEG): ten null mutant mice and accompanying heterozygous and wild-type littermates received 10–20 mg/kg of intraperitoneal sodium pentobarbital to induce stage 1 and 2 sleep. The scalp EEG was then recorded for 60–120 minutes using subdermal needle electrodes and a Caldwell digitized EEG. (ii) Electrocorticography (ECoG): six null mutants and four wild-type littermates were used for the EcoG recording. Under sodium pentobarbital anesthesia (40 mg/kg) five stainless screw electrodes (0.95 mm in diameter), directed toward and over frontal and occipital cortices of both cerebral hemispheres, were implanted in the skull. After a recovery period of 7 days, ECoG was recorded using amplifiers and MacLab system. Cortical recording was made on awake animals freely moving in the test box for a period of 20 minutes.
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ACKNOWLEDGEMENTS |
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We thank K. Shoda, N. Nishiyama, Y. Onodera, C. Uchikawa, A. Rondan, F. Pasos, J. Espinosa and P. Aley for technical support, and M. Hooper for the E14 ES cell line. This study was supported in part by a grant from CURE (Citizens United for Research in Epilepsy) and the Lafora Disease associations of Sweden (Vera Faludi) and Quebec (Audette Malenfant) to A.V.D.-E.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Laboratory for Neurogenetics, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel: +81 48 467 9703, Fax: +81 48 467 7095; Email: yamakawa{at}brain.riken.go.jp
Correspondence may also be addressed to: Antonio V. Delgado-Escueta, MD, Epilepsy Genetics/Genomics Laboratories, Comprehensive Epilepsy Program, VA GLAHC Medical Center, 11301 Wilshire Blvd., Los Angeles, CA 90073, USA. Tel: +1 310 268 3129; Fax: +1 310 268 4937; Email: escueta{at}ucla.edu
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