JOURNAL OF NEUROCHEMISTRY
| 2014
doi: 10.1111/jnc.12838
*Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto, Japan †Aging Regulation Research Team, Tokyo Metropolitan Institute of Gerontology, Itabashi, Tokyo, Japan ‡Department of Psychology, Faculty of Health Science, Kyoto Tachibana University, Yamashina, Kyoto, Japan §Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Wako, Saitama, Japan ¶Laboratory for Molecular Membrane Neuroscience, RIKEN Brain Science Institute, Wako, Saitama, Japan
Abstract Temporal lobe epilepsy (TLE) often becomes refractory, and patients with TLE show a high incidence of psychiatric symptoms, including anxiety and depression. Therefore, it is necessary to identify molecules that were previously unknown to contribute to epilepsy and its associated disorders. We previously found that the sialyltransferase ST3Gal IV is upregulated within the neural circuits through which amygdalakindling stimulation propagates epileptic seizures. In contrast, this study demonstrated that kindling stimulation failed to evoke epileptic seizures in ST3Gal IV-deficient mice. Furthermore, approximately 80% of these mice failed to show tonic–clonic seizures with stimulation, whereas all littermate wild-type mice
showed tonic–clonic seizures. This indicates that the loss of ST3Gal IV does not cause TLE in mice. Meanwhile, ST3Gal IVdeficient mice exhibited decreased acclimation in the open field test, increased immobility in the forced swim test, enhanced freezing during delay auditory fear conditioning, and sleep disturbances. Thus, the loss of ST3Gal IV modulates anxietyrelated behaviors. These findings indicate that ST3Gal IV is a key molecule in the mechanisms underlying anxiety – a side effect of TLE – and may therefore also be an effective target for treating epilepsy, acting through the same circuits. Keywords: amygdala, animal model, anxiety, sialyltransferase, temporal lobe epilepsy, thalamus. J. Neurochem. (2014) 10.1111/jnc.12838
Sialic acids are nine-carbon acidic monosaccharides that occur naturally at the ends of sugar chains attached to the surfaces of cells and soluble proteins, and mostly functions at the cell surface (Schnaar et al. 2014). In mammals, the highest concentration of sialic acids is found in the central nervous system (CNS), where they form an integral part of gangliosides, while sialic acids are abundant mainly in N- and O-linked glycoproteins in many tissues (Wang and Brand-Miller 2003; Chen and Varki 2010; Watanabe et al. 2010). The most abundant gangliosides in the adult CNS are GM1, GD1a, GDa1, GT1b, and GQ1b. These contain a ceramide lipid, a neutral tetrasaccharide core (Gal-beta1, 3GalNAc-beta1, 4Gal-beta1, 4Glc), and one or more sialic
acids, which are synthesized by the sequential action of a series of specific glycosyltransferases. The roles of gangliosides in the CNS have been elucidated based on studies in several human congenital disorders and mice models with Received April 30, 2014; revised manuscript received July 23, 2014; accepted July 23, 2014. Address correspondence and reprint requests to Keiko Kato, Faculty of Life Sciences, Kyoto Sangyo University, Motoyama, Kamigamo, Kita-ku, Kyoto 603-8555, Japan. E-mail:
[email protected] Abbreviations used: BLA, basolateral amygdala; CS, conditioned stimulus; EEG, electroencephalograpy; EMG, electromyograpy; GH, growth hormone; KO, knockout; TLE, temporal lobe epilepsy; US, unconditioned stimulus; WT, wild-type.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
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deficiency in the causative genes. Epilepsy is among the main disorders of human biosynthetic and lysosomal enzyme deficiencies. In humans, mutations in the GM2/GD2 synthase B4GALNT1 alter composition of gangliosides; a lack of GM2 and increase in levels of its precursor, GM3, cause spastic paraplegia, non-progressive cognitive impairment, and partial epileptic seizures (Harlalka et al. 2013). Although patients showed much more severe pathology than B4Galnt1-knockout (KO) mice, both patients and the KO mice had increased levels of GM3 ganglioside and showed symptoms accompanied by neurodegeneration (Sheikh et al. 1999; Harlalka et al. 2013). B4Galnt1-KO mice have reduced myelin-associated glycoprotein expression in the CNS and defective axon– myelin stability leading to progressive neuropathy. A recent study reported a mutation in the GM3 synthase ST3GAL V in ‘Salt & Pepper’ syndrome in human – an autosomal recessive condition characterized by neurocutaneous disorders including severe intellectual disability and epilepsy. Comprehensive glycomic analysis showed collateral alterations in N-linked, O-linked, and glycosphingolipid glycans in addition to a complete lack of GM3 ganglioside (Boccuto et al. 2014). Previous studies also reported that GM3 synthase deficiency was responsible for human earlyonset epilepsy syndrome (Simpson et al. 2004; Fragaki et al. 2013). The neurological symptoms of GM3 synthase deficiency are similar to those of ‘GM3 only’ observed in B4Galnt1/alpha 2, 8-sialyltransferase ST8Sia I-double KO (Kawai et al. 2001) and ‘no gangliosides’ in B4Galnt1/ ST3Gal V-double KO mice (Yamashita et al. 2005). The reports demonstrated that B4Galnt1/ST8Sia I- or B4Galnt1/ ST3Gal V-double KO show lethal auditory seizures or defective axon–myelin stability and small brain size, respectively. However, ST3Gal V-KO mice showed few neurological symptoms. Gangliosides of the 0-series (GM1b, GD1c, and GD1aplha) were formed in these mice, and might have compensated for the loss of the other gangliosides (Yamashita et al. 2003). On the other hand, deficiency of human GM3 synthase resulted in the formation of globo-series and neolactoseries, but no gangliosides of 0-series or GM3 were formed (Fragaki et al. 2013). Thus, human neurological disorders induced by a lack of GM3 ganglioside may be comparable to those observed in ‘no gangliosides’ mice. In human lysosomal enzyme deficiencies, GM1 and GM2 gangliosidosis results in epileptic seizures in addition to startle response, cognitive decline, ataxia, and progressive muscular atrophy, which were accompanied by neuronal degenerations. (Strømme et al. 2011; Patterson 2013). While only a single-gene mutation induces symptoms of human GM2 gangliosidosis, such as sodium/hydrogen exchanger 6 in X-linked Angelman-like syndrome and b-hexosaminidase A in Tay-Sachs disease, mice with single-gene mutation do not develop the disorders. Double-deficient mice with deficiency for sialidase 4 (Neu4) and b-hexosaminidase A
showed neuronal degeneration and epilepsy similar to that observed in human GM2 gangliosidosis (Seyrantepe et al. 2010). While the enzymes in mice might be better compensated than in humans, these data indicate that malfunctioning of enzymes in charge of biosynthesis and degradation of gangliosides are involved in severe neurological disorders including epilepsy. Epilepsy patients are at a greater risk for developing anxiety, depression, psychosis, and learning disorders (Dodrill 1986; Franks 2003; Motamedi and Meador 2003). In particular, patients with temporal lobe epilepsy (TLE) with foci in the amygdala, hippocampus, or surrounding cortex show a high incidence of depression and anxiety (Perini et al. 1996; Ekinci et al. 2009). Moreover, pharmacoresistance and simultaneous treatment using anti-epileptics and other drugs decrease treatment effectiveness and aggravate psychiatric problems (Glauser 2004; Hitiris et al. 2007). These clinical findings prompted examination of correlations between seizure activity and psychiatric symptoms in TLE animal models (Mortazavi et al. 2005; Gastens et al. 2008; M€ uller et al. 2009) and in genetic models of absence epilepsy (Midzyanovskaya et al. 2005). Few studies, however, have examined the mechanisms underlying connection between epilepsy and psychiatric symptoms. The amygdala participates in many behavioral and regulatory functions, including emotion, memory, social behaviors, autonomic functions, and neuroendocrine functions (Eichenbaum 2002). It receives sensory inputs from the thalamus, cortex, and olfactory bulb, and sends projections to the hypothalamus, brainstem, and other basal forebrain structures (Turner and Herkenham 1991). It is also reciprocally connected with the hippocampus (Witter and Amaral 2004). The pathway that interconnects the hippocampus, mammillary bodies, anterior thalamic nuclei, cingulate cortex, and entorhinal cortex (Papez 1937) forms the limbic system, which is known to be involved in emotional behaviors and TLE (Salzman and Fusi 2010). We previously focused on sialyltransferases as molecules that contribute to epilepsy, because sialyltransferases catalyze the addition of sialoglycans on glycoproteins and sphingolipids including gangliosides. We used the basolateral amygdala (BLA)-kindled mouse as a model of human TLE, because 50% of patients with refractory epilepsy are characterized as having TLE (Browne and Holmes 2000; Spencer 2002). We examined novel seizure-responsive sialyltransferase genes in BLA-kindled mice using sialyl motif sequences, and found increased sialyltransferase ST3Gal IV expression in neurons located within the limbic circuitry, including neurons in the subgranular layer of the dentate gyrus and anterior thalamic nuclei (Okabe et al. 2001; Matsuhashi et al. 2003). ST3Gal IV is one of the six sialyltransferases (ST3Gal I to VI) that generate Siaa2, 3Gal linkages. ST3Gal IV expression is region specific, and is strong noticeably in the thalamic sensory relay nuclei,
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
Sialyltransferase ST3Gal IV promotes epilepsy
including the medial and lateral geniculate, intergeniculate, and ventroposterior nuclei (Figure S1) (Matsuhashi et al. 2003). These thalamic regions send outputs to the amygdala and the thalamo-amygdala pathways, which play a role in anxiety (Turner and Herkenham 1991). Such findings led us to examine whether ST3Gal IV modulates epilepsy development and amygdala-dependent behaviors. Here, we investigated whether ST3Gal IV-deficient mice developed epilepsy or showed anxiety-related behaviors. To our knowledge, this study is the first in which mouse behavior associated with ST3Gal IV was studied. Our study findings indicate that the loss of ST3Gal IV results in anxiety-related behaviors but does not induce epilepsy.
Materials and methods Ethics statement and animals This study was carried out in accordance with the Guidelines for Proper Conduct of Animal Experiments published by the Science Council of Japan (2006), and all efforts were made to minimize suffering and the number of animals used. All procedures were performed in accordance with the institutional animal welfare committees of Kyoto Sangyo University (Approval No. 2010-28, 2012-08, and 2013-10). Two and four mice were bred in open-top (182 mm wide 9 260 mm long 9 128 mm high and 225 mm wide 9 338 mm long 9 140 mm high, respectively) plastic cages covered with stainless-steel wire grids. The cage floors were covered with sawdust that was changed twice weekly. Mice were housed in controlled temperature (22 5°C) and lighting conditions (12/12-h light/dark, with lights on at 08:00 hours). Food and water were available ad libitum. Surgical procedures were conducted under isoflurane anesthesia (Merck & Co., Inc., Tokyo, Japan). All behavioral experiments were performed between 08:00 and 12:00 hours. Generation of ST3Gal IV-deficient (KO) mice A genomic fragment was isolated from a C57Bl/6J strain library (BAC library in pBACe3.6, BAC Resources PAC). The targeting vector was constructed with a 10.5-kb FspI–SnaBI fragment on chromosome 9. A loxP (39-mer primer pairs) was inserted into an intron region between exons 2 and 3, and a loxP-frt-pgkneo (2.0-kb NotI–XhoI fragment) containing an SspI site was inserted into an intron region between exons 3 and 4. Then, the diphtheria toxin A fragment cDNA (a 1.2-kb NotI fragment of pMC1DTpA) (Yanagawa et al. 1999) was inserted behind a SnaBI site for negative selection. To generate the ST3Gal IV-deficient (KO) mice, a linearized targeting vector was transfected into MS12 embryonic stem cells by electroporation (ES, derived from C57Bl/6J mice). G418-resistant ES clones were screened from correct targeting of ST3Gal IV locus by Southern blotting with three probes. Targeted ES cells were injected into C57Bl/6J blastocysts to obtain chimeric mice, and chimeras were crossed to C57Bl/6J mice, resulting in offspring with germ-line transmission of the targeted locus that were interbred (targeted mice, C57BL/6-St3gal4, RBRC02284). Sperm from the targeted mice were used to fertilize C57Bl/6J eggs in vitro. A CAG promoter-flippase-poly A plasmid was then injected into the fertilized egg, and these 2-cell stage was transplanted into the uterus, which lead to deletion of the neo gene (C57BL/6-St3gal4Tg(CAG-flp)1Bsi, RBRC02287). We then deleted exon 3 of the ST3Gal IV gene (Fig. 1a) by injecting a CAG promoter-Cre-poly A plasmid into a fertilized egg of C57Bl/6J. The 2-cellstagewas transplanted into the uterus, which led to ST3Gal IV-deficient mice (C57BL/6St3gal4, RBRC02286) (KO allele in Fig. 1a). Mouse tails were screened for correct targeting of ST3Gal IV locus by Southern blotting with probe F (299 bp) in genome fragment produced by Ssp I, which was amplified by PCR reaction with 50 AGAGTACACTGTAACTGGTCTG-30 /50 -ACTCAGTGCCAGCT CTGTAGAT-30 (NT_039472.8) (Fig. 1a and b), probe I (220 bp, 50 GGAGACAGCCATGCAGGAGAAG-30 /50 -AACTTGAATTCCT CCTCCTTCT-30 ) in genome fragment produced by HindIII and probe N (235 bp, 50 -GTTAGTTAGCTGTTGCCCGGTT-30 /50 CTTGTTGGAGGCATCTGGATAG-30 ) in genome fragment produced by EcoRI. Genotyping analysis of mice was usually performed on tail genomic DNA with PCR using the following primers: 50 -AGGCAGGGGGTGTGTCTAGTTA-30 /50 -GTCTCTT GC CTCACCCTGGAA-30 . PCR product wild-type (WT) allele length is 996 bp; knockout-type allele is 649 bp; and floxed-type allele is 1227 bp (Fig. 1c). The following primers were used to screen for the targeted allele: 50 -CAGAGCCATGCCATCA AGGTTC-30 /50 -CAAGCTTGGATCCATAACTTCG-30 (688 bp). Finally, we confirmed that ST3Gal IV mRNA was absent in ST3Gal IV-deficient mice using real-time PCR with a forward primer on exon 3 and a reverse primer on exon 4 (Fig. 1d). Mice were backcrossed for at least 10 generations with C57Bl/6J mice. WT and ST3Gal IV-deficient mice used in all behavior experiments were littermate mice (9–15 weeks old) delivered from F1 mother and father. Quantitative real-time PCR After extraction from mice brain (10–11 weeks old, male), prefrontal cerebral cortex total RNA was prepared with RNeasy lipid tissue mini (Qiagen, Tokyo, Japan). Following total RNA (1 lg) transcription using M-MLV reverse transcriptase (Superscript III; Invitrogen, Tokyo, Japan), the quantitative PCR for growth hormone (GH) and insulin-like growth factor 1 (Igf1) mRNA was performed with TaqMan probes and TaqMan fast advanced master mix, using a StepOnePlus real-time PCR system (Applied Biosystems, Tokyo, Japan), and one cycle and 40 cycles of two-step amplification: 95°C 10 min (1 cycle), 95°C 15 s, and 60°C 1 min (40 cycles). The primer pairs and TaqMan probes for GH and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) were designed as follows: 50 -GGCTGCTGACACCTACAAAGAGTT-30 /50 -AGAAAGCAGCCTGGGCATT-30 /50 -FAM-CCCGAGGGACAGCG-30 (GH, 86 bp, 208–293 bp in NM008117) and 50 -GGAGCGA GACCCCACTAACA-30 /50 -GGCGGAGATGATGACCCTTT-30 / 50 -VIC-CCACGACATACTCAGCAC-30 (Gapdh, 136 bp, 278– 413 bp in NM008084) (Kato et al. 2009). TaqMan gene expression assay probe mix (Mm00439560_m1; Applied Biosystems) was used for Igf1 (77 bp, exon 2–3 boundary in NM01052). Quantitative PCR of ST3Gal IV cDNA was performed with primer pairs and SYBR Green I real-time PCR master mix (Thunderbird SYBR qPCR Mix; Toyobo, Osaka, Japan) using a StepOnePlus real-time PCR system. One cycle and 40 cycles of twostep amplification were performed: 95°C 10 min (1 cycle), 95°C 15 s, and 60°C 1 min (40 cycles). The following primer pairs were designed using Primer-Blast (Ye et al. 2012): 50 -CTGGCTCT
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
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Fig. 1 Generation of ST3Gal IV-KO mice. (a) Strategy to generate ST3Gal IV-deficient mice. (b and c) Genotyping analyses with Southern blotting (b) and PCR (c). (d) Quantitative real-time PCR of ST3Gal IV mRNA.
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GGTCCTTGTTGT-30 /50 -CCCTGGAAGCATGGCTCTTT-30 (ST3Gal IV, 113 bp, 155–267 bp in AB061305). Preparation of kindled mice Kindled mice were prepared as described previously (Kato et al. 2001). The stimulating tungsten electrode was implanted in the right BLA (anterior 2.0 mm, lateral 3.0 mm, ventral 4.5 mm from the bregma), and the anode screw electrode was implanted on the left side of the skull so that it was in continuation with the subdural space (anterior 2.0 mm, lateral 1.5 mm from the bregma). Unrestrained conscious mice (9-week-old males) received a biphasic square wave pulse (480 lA; 60 Hz, 200 ls duration, for 2 s) once a day. Seizures were monitored with the following modified classification of Racine’s criteria (Racine 1972; Kato et al. 2001): stage 1, mouth and facial movement; stage 2, forelimb clonus and a duration of afterdischarges greater than 5 s; stage 3, forelimb clonus and a freezing duration greater than 15 s; stage 4, tonic–clonic seizures and tail held in up position; and stage 5, inability to stand or maintain posture, or falling over. Mice were considered to show tonic–clonic seizures if their seizures were categorized as a stage greater than four and afterdischarges were observed. Behavioral tests Behavioral tests were conducted on the ST3Gal IV-KO mice and the littermate WT mice (male) at 9–13 weeks of age. Animals were acclimated to being alone in a separate waiting room at least 30 min before the behavioral sessions. Behavioral tests were performed between 08:00 and 12:00 hours by an experimenter blinded to the mouse genotype. Open-field test The open-field test was performed with mice in a 49-cm squareshaped arena (height, 19.5 cm; ceiling height was 125 cm above the field floor). In a single trial, the mouse was placed in the field for
5 min and then returned to the home cage for 5 min. Three trials were performed on each mouse. A fluorescent lamp was set to 350 lux in the first and second trials and 560 lux in the third trial. Each trial was recorded with a camera mounted 47 cm above the field floor and analyzed automatically with Time OFCR4 (O’hara and Co., Ltd, Tokyo, Japan). Forced swim test A clear tank (diameter: 11.3 cm, height: 22.3 cm) was filled with water (24°C) to a height that was 15 cm from the floor; the water was changed between each trial. The tank was placed into a box containing a fluorescent lamp and illumination was set to 1010 lux. Trials were 6 min in duration, and all data were collected and analyzed with Time FZ1 (O’hara and Co., Ltd). Immobility times of more than 1 s were measured. Tail suspension test Mice were lifted by their tail 26.5 cm above the floor and placed in a box containing a fluorescent lamp (illumination was set to 1010 lux). Trials were 6 min in duration, and all data were collected and analyzed with Time FZ1 (O’hara and Co., Ltd). Immobility times of more than 1 s were measured. Fear conditioning test Auditory fear conditioning was performed with mice in a clear, rectangle-shaped arena (17 cm wide 9 10 cm deep 9 10 cm high). The arena was fitted with a metal grid floor through which scrambled foot shocks could be delivered as unconditioned stimuli (US; 0.5 mA, 1 s; O’hara and Co., Ltd). The arena was placed into an acoustic isolation box (50 dB background noise level, 220 lux with a light-emitting diode), and a speaker positioned on the right side of the chamber delivered the auditory conditioning stimuli (10 KHz, 70 dB, 10 s). On the day of training, each mouse was placed in the clear square-shaped arena and allowed to explore for
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
Sialyltransferase ST3Gal IV promotes epilepsy
60 s. The conditioned stimulus (CS), a 70-dB tone, was presented for 10 s. The US was delivered during the final 1 s of the CS presentation. The CS and US were delivered automatically with a 20-s interval using a tone generator and shock controller. A freezing time of at least 2 s was measured over a 160-s period. Mice were returned to their home cages after conditioning. Twenty-four hours later, the contextual fear conditioning test was performed in the acoustic isolation box, where mice were placed for 5 min in the same clear square-shaped arena, without delivering CS or US. Freezing times of more than 2 s were measured over a 5-min period. Mice were returned to their cages, and 24 h later, they were subjected to a cued test. In the cued test, mice were placed in a novel, solid gray, square-shaped arena illuminated at 50 lux, which they were allowed to explore for 60 s. The CS was presented for 30 s, and the mice were again allowed to explore for 90 s. Freezing times of more than 2 s were measured over a 3-min period. All data were collected and analyzed with Time FZ1. Sleep recording In implantation of electroencephalographic (EEG) and electromyographic (EMG) electrodes, mice (8-week-old males) were anesthetized with isoflurane and implanted with EEG and EMG electrodes. The cranium was visualized and two burr holes were made with a dental drill. Stainless-steel screws were inserted into the holes (anterior 1.0 mm, lateral 1.5 mm from the lambda; anterior 1.0 mm, lateral 1.5 mm from the bregma) to record EEG signals bilaterally in the subdural space. Pairs of urethane-coated stainless-steel wires with exposed ends were inserted into the trapezius muscles to record EMG signals. All electrodes were connected with the skull via a 3-mm square plastic plate with Vetbond (3M, Setagaya, Japan) and dental acrylic. After surgery, mice were allowed 10 days to recover, and following their recovery, they were placed in an experimental cage for habituation for 2 days. In classification of sleep, sleep–awake states were monitored for 26 h with EEG and EMG signals, which were recorded using Vital Recorder and a video system (Kissei Comtec Co., Ltd., Nagano, Japan) with a camera that detected both light and infrared rays to observe mice in both light and dark conditions. Recordings were performed under a 12/12-h dark/light cycle (180 lux), with lights on at 08:00 hours. Mice were habituated for 2 days to a counterbalanced, lightweight cable connected to a low-torque commutator (Biotex, Kyoto, Japan) affixed to the center of the top of the cage (Huang et al. 2005). EEG and EMG data were amplified and filtered to 0.5–20 and 20–128 Hz, respectively, and then digitized at a sampling rate of 128 Hz and recorded using SleepSign ver. 2.0 (Kissei Comtec Co., Ltd.). In an offline analysis, EEG patterns were categorized as 0.5–4 Hz (d), 4–9 Hz (h), or 9–13 Hz (a). The EEG and EMG data were classified by the sleep/awake stage, including non-rapid eye movement (REM) sleep, REM sleep, and wakefulness, and were extracted in 4-s epochs using SleepSign ver. 2.0. We analyzed the data recorded over a 24-h period beginning at 12:00 and ending at 12:00 hours on the following day. Statistics Descriptive statistics are indicated as mean SEM; p ≤ 0.05 was considered statistically significant. All statistical analyses were
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performed with Excel and GraphPad Prism 6 (MDF; Kotoku, Tokyo, Japan).
Results Deficiency of ST3Gal IV fails to evoke epileptic seizures in mice We applied repeated kindling stimulation in the BLA of both ST3Gal IV-KO mice and the littermate WT mice once a day for 18 days (Fig. 2). Consistent with previous studies (Okabe et al. 2001; Matsuhashi et al. 2003), we observed that the stimulation induced seizures in the WT mice. In the ST3Gal IVKO mice, however, a two-way repeated-measures ANOVA (genotype 9 day) revealed that kindling stimulation evoked fewer afterdischarge spikes [F(1, 10) = 5.39, p = 0.043] (Fig. 2a), and tended to induce shorter afterdischarge durations in ST3Gal IV-KO mice than in WT mice [F(1, 10) = 2.87, p = 0.121] (Fig. 2b). Thus, afterdischarges were almost eliminated in ST3Gal IV-KO mice. Elimination of afterdischarges was confirmed with dispersion diagrams with the number of spikes (p = 0.010 in Mann–Whitney U-test) and the duration (p = 0.017) on day 18 when the 18th stimulation was applied (Fig. 3b and c). Furthermore, compared to WT mice, ST3Gal IV-KO mice showed decreased durations in freezing behaviors [F(1, 10) = 5.85, p = 0.036] (Fig. 2c), and little elevation in the stages of seizure severity [F(1, 10) = 9.99, p = 0.010] (Fig. 2d). The Bonferroni multiple comparisons test confirmed significant differences in the number of afterdischarge spikes, afterdischarge and freezing durations, and progression of stages between ST3Gal IV-KO and WT mice on days 15–18 (shown as asterisks and hashes in Fig. 2a–d), and these differences were the most obviouson day 18 (Fig. 3b–e). On day 18, 100%of WT mice showed tonic–clonic seizures, whereas 17% of ST3Gal IV-KO mice showed tonic–clonic seizures (Fig. 3f), among which one ST3Gal IV-KO mouse that exhibited afterdischargelike EEGs, which contained spikes of smaller amplitude (lV) than those in WT mice, showed tonic–clonic seizures (data not shown). ST3Gal IV-deficient mice exhibit anxiety-related behaviors We used several standard behavioral tests, including the open-field test, forced swim test, tail suspension test, and fear conditioning test, as well as sleep patterns in light–dark cycles. The performances in these tests were compared between ST3Gal IV-KO and WT mice. In the open-field test, there was no difference between ST3Gal IV-KO and WT mice for the total distances walked during the three trials of 5-min test periods using a two-way ANOVA [F(1, 51) = 0.0001, p = 0.991] (Fig. 4a). The frequency of entry into the center area, however, was significantly higher for WT mice than for ST3Gal IV-KO mice [F (1, 51) = 5.86, p = 0.019] (Fig. 4b). The frequency of entry into the center area was also compared among subsequent
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
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Fig. 2 ST3Gal IV-knockout (KO) mice fail to show epileptic seizures. (a–d) ST3Gal IV deficiency prevented the development of seizures. Unrestrained conscious mice at 9 weeks of age received kindling stimulation in the basolateral amygdala (BLA) once a day for 18 days to investigate the development of epileptic seizures. Vertical lines indicate the number of afterdischarge spikes (a), duration (s) of the afterdischarge period (b), freezing times (s) (c), and transition among stages (d). These four items were recorded for 20 min following kindling stimulation; each plot in the graph represents the mean SEM. Significance with a two-way repeated measures ANOVA (genotype 9 day) according to the Bonferroni multiple comparisons test is ***p < 0.001; indicated (#p < 0.0001; **p < 0.01; *p < 0.05).
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trials of ST3Gal IV-KO and WT mice using a one-way ANOVA [F(5, 51) = 2.68, p = 0.032]. This analysis revealed that WT mice showed a 1.6-fold increase in the frequency of entry into the center area from the first to second trial (Fisher’s LSD, p = 0.026), whereas ST3Gal IV-KO mice showed little difference in the frequency of entry between trials (Fisher’s LSD, p = 0.175). These results indicate that ST3Gal IV-KO mice poorly acclimated to the center area. Meanwhile, when we increased luminance from 350 lux in the second trial to 560 lux in the third trial, both ST3Gal IV-KO and WT mice showed a decreased frequency of entry into the center area. Both mice might have recognized the center area illuminated with 560 lux as a novel condition. The forced swim (Fig. 4c) and tail suspension tests (Fig. 4d) were performed in a single 6-min session to compare ‘behavioral despair’ between ST3Gal IV-KO and WT mice. To compare the performance in these tests between ST3Gal IV-KO and WT mice, we used a two-way repeated measures ANOVA (genotype 9 min) for immobility per minute [F(1, 13) = 3.16, p = 0.099 in forced swim test; F(1, 8) = 14.55, p = 0.005 in tail suspension test] and the Mann–Whitney U-test for total immobility (%) (forced swim test, p = 0.049; tail suspension test, p = 0.008). This analysis indicated a difference in immobility between ST3Gal IV-KO and WT mice in the forced swim test and the tail suspension test. The mean duration of total immobility (%) exhibited a 1.5-fold increased immobility in the forced swim test and a 1.1-fold decreased immobility in the tail suspension test. Hence, ST3Gal IV-KO mice showed increased ‘swimming despair’ compared to WT mice, but showed decreased ‘suspension despair.’
In the delay fear conditioning tests, ST3Gal IV-KO mice and WT mice were trained with a two-shock conditioning session (Fig. 4e). We used a two-way repeated measures ANOVA (genotype 9 time) with the Bonferroni multiple comparisons test for freezing per period (s) and the Mann– Whitney U-test for total freezing (%) to compare the performances in conditioning (Fig. 4e), context (Fig. 4f), and tone (Fig. 4g) tests between ST3Gal IV-KO and WT mice. The two groups showed similar responses to the shocks both initially [F(1, 18) = 0.40, p = 0.537 in ANOVA; p = 0.311 in Mann–Whitney U-test] (Fig. 4e) and 24 h after the conditional training [F(1, 18) = 0.02, p = 0.894 in ANOVA; p = 0.242 in Mann–Whitney U-test] (Fig. 4f). These data suggest that ST3Gal IV did not alter their foot shock sensitivity, sensory processing, or freezing behaviors during initial conditioning or contextual tests. However, ST3Gal IVKO mice showed greater freezing behavior during the tone test than WT mice [F(1, 18) = 9.84, p = 0.006 in ANOVA] (Fig. 4g). The mean duration of freezing (%) for the 30-s period between 90 and 120 s was 2.2 times longer in ST3Gal IV-KO mice (Bonferroni multiple comparisons test, p < 0.0001, hash in Fig. 4g). ST3Gal IV-KO mice also showed a 1.9-fold increase in total freezing times (%) compared with WT mice in the tone test (p = 0.009 in Mann–Whitney U-test) (Fig. 4g). These results indicate that ST3 Gal IV-KO mice experienced increased anxiety that was enhanced by an auditory cue. EEG activity was recorded for 24 h in unanesthetized, freely moving mice with electrodes implanted bilaterally in the subdural space. The graphs presented in Fig. 5(a) indicate the percentage (%) of each group at 1-h intervals
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
Sialyltransferase ST3Gal IV promotes epilepsy
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Fig. 3 ST3Gal IV deficiency prevented the onset of tonic–clonic seizures. (a) Representative electroencephalographic (EEG) traces in ST3Gal IV-knockout (KO) mice and the littermate wild-type (WT) mice that received amygdala-kindling stimulations (arrow) on day 18. ‘YES’ and ‘NO’ indicate the appearance and disappearance of tonic–clonic seizures, respectively. WT mice show representative afterdischarge. (b–e) Dispersion diagram of epileptic behaviors of ST3Gal IV-KO and wild-type (WT) mice on day 18. Vertical lines
indicate the number of afterdischarge spikes (b), durations of the afterdischarge period (c), freezing times (d), and transition among stages (e). Lines represent mean SEM, KO: 121.67 100.27, WT: 510.83 93.54 (b); KO: 17.17 13.88 s, WT: 50.33 11.15 s (c); KO: 26.83 16.51 s, WT: 80.83 11.88 s (d); KO: 2.08 0.64, WT: 4.91 0.08 (e). Mann–Whitney U-test (one-tailed test) p-values are shown in each figure. (f) Number of mice (%) showing tonic–clonic seizures on day 18.
over a 24-h period that were classified as awake, in REM sleep or in non-REM sleep. The representative sleep cycles demonstrated that WT mice showed sleep patterns including siesta-like sleep between 00:00 and 04:00 hours, which was not observed in ST3Gal IV-KO (Fig. 5a). Furthermore, compared to WT mice, ST3Gal IV-KO mice showed a 3.6-fold decrease in the mean duration of the REM period (%) (p = 0.050, Fig. 5b). On the other hand, there was no difference between ST3Gal IV-KO and WT mice in awake (p = 0.200, Fig. 5c) or non-REM (p = 0.450, Fig. 5d) sleep periods. These results indicate that deficiency of ST3Gal IV disturbed normal sleep patterns in mice.
and ST3Gal IV mRNA levels in the neural circuits that were activated with kindling stimulation during epileptogenesis (Matsuhashi et al. 2003; Kato et al. 2009); however, there was no difference in Igf1 expression in the brain during epileptogenesis or following epilepsy onset (Kruskal–Wallis test, p = 0.553, data not shown).
GH mRNA levels depend on the quantity of ST3Gal IV in the mouse brain We demonstrated that brain GH and Igf1 mRNA concentrations in ST3Gal IV-KO mice were 2.8 and 2.2 times less than that observed in littermate WT mice (p = 0.030 and 0.040 in Mann–Whitney U-test, Fig. 6a and b), respectively. On the other hand, we previously revealed an increase in GH mRNA
Discussion Kindling stimulation was applied to the BLA in TLE mice, which was propagated from the BLA to the hippocampus, anterior thalamus, and apical cortex, including the cingulate cortex (Figure S2) (Meibach and Siegel 1977; Price 1995; Gemmell and O’Mara 2002; Witter and Amaral 2004). The stimulation elicited increased ST3Gal IV expression in anterior thalamic neurons and induced epileptic seizures (Matsuhashi et al. 2003). In contrast, ST3Gal IV-KO mice did not develop epilepsy and almost all ST3Gal IV-KO mice showed no or slight symptoms on day 18 following repeated kindling stimulation once a day (Figs 2 and 3).
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
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(b)
(c)
(d)
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Fig. 4 The behavioral phenotype of ST3Gal IV-knockout (KO) mice is indicative of increased anxiety- and depression-like behaviors. (a and b) Ambulation of ST3Gal IV-KO and wild-type (WT) mice was observed in the open-field test. Mice entered the arena for 5 min and then the home cage for 5 min in 3 iterations (1st- to 3rd-E, first to third entry). Illumination was 350 lux in the first and second entries and 560 lux in the third entry. The bar graph (mean SEM) shows the mean total distance (cm); mean distance travelled in the arena over a 5-min duration (a), and the 15% center area; the duration within a 5-min period in which the mouse entered and stood in the 15% center area (b). The p-value obtained with a 2-way ANOVA is shown. (c and d) An immobility time of more than 1 s was scored during minutes 1–6 of the forced swim test (c) and tail suspension
tests (d) to compare ‘behavioral despair’ between ST3Gal IV-KO and WT mice. The line graph (mean SEM) shows immobility time in a single 6-min session and the bar graph shows total immobility for 6 min. (e–g) Mice were trained with a two-shock conditioning session on day 1 (e), and were subjected to a contextual fear test on day 2 (f) and to the tone fear test on day 3 (g). A freezing time of more than 2 s was scored. The line graph (mean SEM) shows % summation of freezing time for each period (s) and the bar graph shows % total freezing time for 160 s (e), 300 s (f), and 180 s (g). To obtain the p-value, a 2-way repeated measures ANOVA (genotype 9 min or second) for immobility or freezing per time and the Mann–Whitney U-test (one-tailed test) p-value for total immobility or freezing (%) were used in (c–g). #p < 0.0001.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
Sialyltransferase ST3Gal IV promotes epilepsy
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(a)
Fig. 5 ST3Gal IV-knockout (KO) mice show a disturbance in sleep patterns. (a) Representative sleep patterns of ST3Gal IVKO and wild-type (WT) mice. (b–d) Comparison of sleep patterns between ST3Gal IV-KO and WT mice. Vertical line shows the percentage of awake, rapid eye movement (REM), and non-REM periods at 1-h intervals. Horizontal line shows time over 24 h. The bar graph (mean SEM) indicates percentages for REM (b), awake (c), and non-REM periods (d); Mann– Whitney U-test (one-tailed test) p-values are shown.
(b)
These findings demonstrate that ST3Gal IV contributes to seizures. The EL/Suz (EL) mouse is another model of multifactorial TLE (Suzuki and Nakamoto 1982; Leussis and Heinrichs 2006). Tail suspension induces severe immobility in EL mice compared to that in other mouse strains (Leussis and Heinrichs 2007), and the routine tail suspension elicits seizures in EL mice (Leussis and Heinrichs 2006). Contrary to EL mice that show seizures, ST3Gal IV-KO mice showed decreased immobility with tail suspension (Fig. 4d) and were without seizures (Figs 2 and 3). Previous studies have identified psychiatric symptoms in TLE animal models (Ma and Leung 2004; Midzyanovskaya et al. 2005; Mortazavi et al. 2005; Gastens et al. 2008; Zhang et al. 2008; M€ uller et al. 2009). This suggests that ‘suspension despair’ is a comorbidity in epilepsy that depends on the presence of ST3Gal IV. Hence, there is a high possibility that the developmental mechanisms of TLE were similar between EL and kindled mice. Furthermore, ST3Gal IV-KO mice did not show ‘suspension despair’ or seizures, which reinforces ST3Gal IV involvement in TLE. Mice receive a mild anxiogenic stimulus if they enter an open field. In this study, ST3Gal IV-KO mice entered the center of the arena less often than WT mice, which indicates a decrease in both exploratory behavior and acclimation in ST3Gal IV-KO mice. Mice deficient for several ganglioside
(c)
(d)
biosynthetic enzyme genes have also been tested under mild anxiogenic stimulation, including an open-field test. Concretely, B4galnt1-KO mice with GM3 accumulation showed increase of spontaneous locomotion activity (Pan et al. 2005); ST3Gal V-KO mice that form 0-series gangliosides (GM1b, GD1c, and GD1aplha) showed increased levels in
(a)
(b)
Fig. 6 Deficiency of ST3Gal IV reduces growth hormone (GH) and insulin-like growth factor 1 (Igf1) mRNAs in the mouse brain. The bar graph (mean SEM) shows arbitrary units of GH mRNA (a) and Igf1 mRNA (b) relative to glyceraldehyde 3-phosphate dehydrogenase (Gapdh) mRNA in the apical part of the cerebral cortex using quantitative real-time PCR. Mann–Whitney U-test (one-tailed test) p-values are shown.
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spontaneous locomotion, Y-maze, and elevated plus maze tests (Niimi et al. 2011); and solute carrier family 9 (sodium/ hydrogen exchanger) member 6 (Slc9a6)-KO mice with GM2 gangliosidosis exhibited increased activity in the open field (Strømme et al. 2011). Thus, B4galnt1-, ST3Gal V-, and Slc9a6-KO mice showed significant hyperactivity. When ceramide synthase 1 (CerS1)-KO mice with a decreased level of major gangliosides (GM1, GD1a, GD1b, and GT1b) received the open-field and Y-maze tests, CerS1-KO mice exhibited significantly decreased levels in locomotion, Y-maze performance, and investigation of novel objects, indicating hypoactivity and low exploration. However, these mice also showed increased center access (Ginkel et al. 2012). As an increase in activity or time spent at the center of the open field is generally interpreted as decreased anxietylike behavior (Bailey and Crawley 2009), the contradiction among behaviors still remains in CerS1-KO mice. Finally, behaviors in mice deficient for the above ganglioside-related enzyme were not identical with those in ST3Gal IV-KO mice. Six species of alpha2,3-sialyltranseferases (ST3Gal I to VI) have been examined to determine whether they act as enzymes for combinational ganglioside biosynthesis (Kolter et al. 2002; Sturgill et al. 2012). ST3Gal V is a GM3 synthase (Kolter et al. 2002). Sturgill et al. (2012) reported that brain gangliosides from ST3Gal II/ ST3Gal III-doubleKO mice lacked GT1b and GQ1b, had diminished GD1a, and predominantly more GM1 and GD1b masses. Thus, ST3Gal II and ST3Gal III contribute to GD1a and GT1b ganglioside biosynthesis in vivo and as a result, loss of ST3Gal II and ST3Gal III caused small brain size and early dysreflexia that is caused by defective axon–myelin stability in mice (Sturgill et al. 2012). On the other hand, composition of gangliosides in TLE rodent models was different from that in ST3Gal II/ ST3Gal III-double KO mice; seizures significantly reduced the major brain ganglioside (GM1, GD1a, GD1b, and GT1b) levels (de Freitas et al. 2010) and increased GQ1b levels (Kato et al. 2008). While seizures were associated with tremendous up-regulation of ST3Gal IV expression (Okabe et al. 2001; Matsuhashi et al. 2003), there is little evidence that ST3Gal IV participates in combinatorial ganglioside biosynthesis. If ST3Gal IV could interact with gangliosides in neurons and exhibit enzyme activity, it still leave possibility that the gangliosides absent in ST3Gal II/ ST3Gal III-double KO mice might be replenished. We selected the forced swim and fear conditioning tests to examine the responses of ST3Gal IV-KO mice to anxiogenic stressors. The forced swim test has been used to examine antidepressant activity, and antidepressants are known to act on the amygdala (Borsini and Meli 1988). Lesions in the amygdala, especially in the BLA, attenuate anxiety- and depression-related behaviors in animals (White and Price 1993; Woolley et al. 2006) and humans
(Etkin and Wager 2007). Hence, the BLA is among the most critical regions involved in anxiety and depression. ST3Gal IV-KO mice showed elevated immobility in the forced swim test (Fig. 4c), which suggests that ST3Gal IV-KO mice experience an increase in depression-related emotions. Thus, epilepsy treatments targeting ST3Gal IV might have side effects related to attention and depression. The amygdala also plays a critical role in fear conditioning tests (Goosens and Maren 2001) including contextual and delay conditioning (Raybuck and Lattal 2011). In this study, we observed no difference in contextual fear between ST3Gal IV-KO and WT mice. However, ST3Gal IV-KO mice demonstrated tone fear. When delay conditioning is performed with an auditory tone, neurons in the medial geniculate nucleus (MGN) and auditory cortex transmit auditory information to the lateral amygdaloid nucleus (Figure S2) (LeDoux et al. 1991; Quirk et al. 1997). MGN neurons in the adult brain express ST3Gal IV mRNA (Figure S1) (Matsuhashi et al. 2003), and therefore might modulate auditory fear by regulating ST3Gal IV expression. A human epidemiological study indicated that both TLE (Bazil et al. 2000) and depression (Kupfer 1976) decrease REM sleep, but the mechanisms for these relationships remain unknown. ST3Gal IV-KO mice that showed increased anxiety behaviors also showed decreased REM sleep (Fig. 5). This suggests that the ST3Gal IV-KO mouse is a viable model for investigating sleep disturbance. ST3Gal IV-KO mice that exhibited no seizure activity with kindling stimulation showed decreased GH and Igf1 mRNA expression in the brain (Fig. 6). This finding indicates that the amount of brain GH mRNA depends on the quantity of brain ST3Ga IV mRNA in mice. Igf1 is up-regulated in the liver, brain, and vasculature in response to GH stimulation (Perrini et al. 2010). GH down-regulation mediated by ST3Gal IV deficiency, therefore, could modulate Igf1 expression in the brain. On the other hand, epileptic seizures increased GH production (Kato et al. 2009), but did not affect Igf1 expression in the brain (Kruskal–Wallis test, p = 0.553, K. Kato, unpublished result). Previous GH proliferative analyses revealed that this activity curve has a bell-shaped and dose-dependent pattern that is consistent with the sequential formation of 1 GH molecule and 2 receptors in immune functions (Postel-Vinay et al. 1997; Uchida et al. 1999). This theory suggests that GH upregulation in the brain following kindled seizures might be inadequate to induce Igf1 expression via GH signaling. This study indicates that the loss of ST3Gal IV protects against TLE in mice. Furthermore, the present findings suggest that medications that inactivate neurons participating in ST3Gal IV up-regulation, or that directly inhibit ST3Gal IV, might be effective treatments against TLE. On the other hand, ST3Gal IV-KO mice might show side effects such as
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 10.1111/jnc.12838
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anxiety, depression, and reduced REM sleep. An improved understanding of the mechanisms that mediate these anxietyrelated behaviors may provide insights into the neural systems associated with epilepsy.
Acknowledgments and conflict of interest disclosure This work was supported by Kyoto Sangyo University Research Grants (E1106); the Ministry of Education, Culture, Sports, Science, and Technology (22580338); and Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST). We thank Ms Yuko Watanabe for assisting in the paperwork, and Mr Hirotaka Nishizaki and Ms Asami Miyauchi for assisting in the experiments. Ms Shigeko Yamada participated as an undergraduate student in the early stages of this research. The authors declare no competing financial interests. All experiments were conducted in compliance with the ARRIVE guidelines.
Supporting information Additional supporting information may be found in the online version of this article at the publisher's web-site: Figure S1. Distribution of ST3Gal IV mRNA in the mouse brain. Figure S2. Neural pathways between the thalamus and amygdala underlying epileptic seizures and tone fear.
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