Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: A novel epilepsy model.

OBJECTIVE: To clarify the complex mechanism underlying epileptogeneis, a novel animal model was generated.
METHODS: In our previous research, we have generated a melanocyte-lineage mTOR hyperactivation mouse model (Mitf-M-Cre Tsc2 KO mice; cKO mice) to investigate mTOR pathway in melanogenesis regulation, markedly reduced skin pigmentation was observed. Very unexpectedly, spontaneous recurrent epilepsy was also developed in this mouse model.
RESULTS: Compared with control littermates, no change was found in either brain size or brain mass in cKO mice. Hematoxylin staining revealed no obvious aberrant histologic features in the whole brains of cKO mice. Histoimmunofluorescence staining and electron microscopy examination revealed markedly increased mTOR signaling and hyperproliferation of mitochondria in cKO mice, especially in the hippocampus. Furthermore, rapamycin treatment reversed these abnormalities.
CONCLUSIONS: This study suggests that our melanocyte-lineage mTOR hyperactivation mouse is a novel animal model of epilepsy, which may promote the progress of both epilepsy and neurophysiology research.

Introduction

Epilepsy affects over 70 million people worldwide[1], leading to adverse social, behavioral, health, and economic consequences. Although written records of epilepsy date back to 4000 BC, its pathophysiology remains incompletely understood[1]. As the complex mechanisms underlying epileptogenesis cannot be fully elucidated through human clinical studies, appropriate animal models are necessary.

Microphthalmia-associated transcription factor (Mitf)-M is expressed solely in neural crest-derived melanocytes[2]. Mitf-M–expressing cells are primarily found in the skin and hair follicles but also occur in other tissues, including the eyes, heart, meninges, and other brain tissues[3-6].

Skin-derived melanocytes offer a model system to investigate normal and pathological features of less accessible neurons because of their common origin and many similar signaling molecules and pathways[7]. Neurocutaneous syndromes, such as tuberous sclerosis complex (TSC), exhibit considerable overlap of dermatologic and neurologic manifestations, including epilepsy. In TSC, the prevalence of epilepsy is approximately 78%[8].

In our previous investigations of the mechanisms of melanogenesis, we constructed melanocyte-lineage Tsc2 (a pathogenic gene in TSC) knockout mice in which Cre recombinase was placed under the control of regulatory elements from the Mitf-M gene[9]. These mice presented with the anticipated skin hypopigmentation and unexpectedly developed spontaneous neural epileptic activity as well. In the current study, we confirmed hyperactivation of the mammalian target of rapamycin (mTOR) signaling pathway, abnormal neuronal excitability, and hyperproliferation of neuronal mitochondria in the brain of these animals. We herein suggest that this may be a useful mouse model for epilepsy research, providing novel insights into the mechanisms of seizure disorders.

Materials and methods

Animals

Tsc2flox/flox mice and Mitf-M-Cre mice were generated as described previously[9]. Melanocyte-specific Tsc2 knockout mice were generated by breeding Tsc2flox/flox mice with Mitf-M-Cre mice. Both lines maintained a C57BL/6 inbred background. The controls were littermates, either without cre or in a few cases, Mitf-M-cre;Tsc2flox/-. For rapamycin treatment, sirolimus was purchased from Selleck (Osaka, Japan) and dissolved in distilled water for oral administration at 2.285 mg/kg/day for 3 weeks (n = 5 mice/goup). All animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals, and the experimental protocol used in this study was approved by the Committee for Animal Experiments at Osaka University (Osaka, Japan).

EEG/EMG recording

Tsc2 cKO mice (male, 6 weeks old at the time of surgery) were instrumented with chronically implanted EEG/EMG electrodes according to previously published procedures[10].Briefly, a preamplifier (#8202) was surgically implanted in mice under isoflurane anesthesia. Mice (n = 3 mice/goup) were allown to recover from surgery for at least 12 h before recording was initiated. EEG/EMG data were recorded for a 24 h period using data acquisition system (#8200-K1-SE) and Sirenia Software (both from Neuroscience, inc).

Histology and immunohistochemistry analyses

Brain tissue samples (n = 5 mice/goup) were fixed in 10% formaldehyde and embedded in paraffin. Subsequently, 4-μm sections were either stained with hematoxylin for morphological examination or used for immunohistochemistry analysis. The following antibodies were used for immunohistochemistry: p-S6 (#4858, Cell Signaling Technology, Tokyo, Japan) at 1:100, c-FOS (ab208942, Abcam, Cambridge, UK) at 1:200, Parvalbumin (SAB4200545, Sigma) at 1:100, CaMKII-α (#11945, Cell Signaling Technology) at 1:100, COXIV (#459600, Invitrogen) at 1:200, GFAP (#12389, Cell Signaling Technology) at 1:100, and MAP2 (ab5392, Abcam) at 1:2000. The stained proteins were visualized using a Biozero confocal microscope (Keyence Co., Osaka, Japan).

Timm staining

For Timm staining, we intracardially perfused the mice (n = 5 mice/goup) with ice-cold 1% (w/v) sodium sulfide, followed by 4% paraformaldehyde. After removal from the body, the brain was post-fixed in 10% formaldehyde overnight and embedded in paraffin. We then created 10-μm thick sagittal sections and performed modified Timm staining, as previously described[11].

Electron microscopy examination

After dissection of the mouse brain (n = 5 mice/goup), hippocampal slices were prepared using a slicer (Narishige, ST-10, Tokyo, Japan), as previously described[12]. The slices were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer containing calcium chloride (pH 7.4) for 2 h and then washed three times with deionized distilled water. The samples were post-fixed in 1% OsO4 in phosphate-buffered saline for 1 h, and then dehydrated with a graded ethanol series and embedded in EPON. Ultra-thin sections (70–80 nm) were cut horizontally to the bottom of the dish, transferred to grids, dual-stained with uranyl acetate and lead citrate, and observed using a Hitachi H-7650 transmission electron microscope (Hitachi, Tokyo, Japan).

Primary culture of hippocampal pyramidal cells from adult mice

Primary neuronal cells were obtained from the hippocampus of 4-week-old wild-type and mutant mice (n = 5 mice/goup) as reported previously[13]. Briefly, the hippocampus was dissected and sliced into 0.5-mm sections in 2 mL HABG medium (40ml HA(HibernateTM-A Medium, Invitrogen, #A1247501; 0.8ml B27, Invitrogen, #17504; 0.1ml L-Glutamine, Invitrogen, #25030081)) at 4°C in a 35-mm-diameter dish using tissue slicer (Dosaka microslicer, Kyoto, Japan), removing the dentate gyrus to eliminate granule cells. The sections were digested with papain (2 mg/mL, Worthington, #LS003119 in HA-Ca, BrainBits LLC) at 30°C for 30 min. Cells were released by gentle trituration with a Pasteur pipette. Finally, primary neurons were separated using density-gradient centrifugation (OptiPrep, AXS, #1114542, XX). Cells were cultured in NeurobasalA/B27 medium (Invitrogen, #10888022 and #17504044) with L-Gin (Invitrogen, #25030149), growth factors (5 ng/mL mouse FGF2, Invitrogen, #PMG0034; 5 ng/mL mouse PDGF-BB, Invitrogen, #PMG0044), and gentamycin (Wako, #078–06061) for 1 week before the experiments.

To evaluate neuronal activity and mitochondrial quantity, cells were fixed with 4% paraformaldehyde for 20 min and then processed for the detection of neuronal antigens. The primary antibody was MAP2 (ab5392, Abcam) at 1:2000.

Measurement of [Ca2+]i

[Ca2+]i in single cells was detected on the basis of fura-2 fluorescence intensity, as reported previuosly[14]. Briefly, neurons grown on coverslips were rinsed twice with artificial cerebrospinal fluid (ACSF; 127 mM NaCl, 1.5 mM KCl, 26 mM NaHCO3, 1.24 mM KH2PO4, 10 mM glucose, 1.4 mM MgSO4, 2.4 mM CaCl2; SIGMA) and incubated at 37°C for 45 min in the presence of fura-2 AM (fura-2 acetoxymethyl ester, DOJINDO, #CS23) with 1.25 mmol/L probenecid (SIGMA) and 0.03% Pluronic® F-127 (SIGMA) in carbogen-bubbled ACSF. After two washes with ACSF, cells were incubated for an additional 20 min in ACSF before imaging. The coverslips were transferred to a chamber and observed by microscopy (Nikon ECLIPCE E800). The excitation wavelengths for fura-2 were 340 and 380 nm, with emission at 505 nm. For the stimulation experiments, a range of K+ solutions were used: 10 mM, 30 mM, and 60 mM KCl. Fluorescence intensity was quantified using Metafluor software (Universal Imaging Corporation, West Chester, PA).

Epilepsy behavior

cKO mice and littermates (n = 5 mice/goup) were tested for seizure behavior during the night, because mice are nocturnal animals and more active at night. Spontaneous seizure and seizures induced by the ringing of a clock every 1 hour were video-recorded for 5 days. The behaviors of the mice were scored by two independent observers, who were blinded to their genotype. In the rapamycin treatment experiments, behavior analysis was performed after 3 weeks of oral sirolimus (Selleck) in distilled water.

Animal sacrifice

Mice were anaesthetized with a lethal dose of pentobarbital and sacrificed by intracardially perfusion using ice-cold 1% (w/v) sodium sulfide, followed by 4% paraformaldehyde. The brains were removed for primary culture of hippocampal pyramidal cells, measurement of [Ca2+]i, or post-fixed for 10% formaldehyde overnight and embedded in paraffin or cryoprotected in 30% sucrose/PBS for histologic analyses.

Statistical analyses

Data are presented as mean ± SD. Unpaired Student’s t-test (Microsoft Excel; Microsoft Corp., Redmond, WA) was used for comparisons between two groups. One-way ANOVA test, followed by Dunnett's post hoc test was used for multiple comparisons (Microsoft Excel). P-values <0.05 were considered statistically significant.

Results

Conditional Tsc2 deletion caused epilepsy

We generated Tsc2;Mitf-M-Cre (cKO) mice by knocking out Tsc2 in melanocyte-lineage cells under Mitf-M promoter regulation[9]. Progressive recurrent epilepsy, characterized by spontaneous adduction or flexion movements of the head, trunk, limbs, and tail for 20-60s, became apparent at 4–5 weeks of age (Fig 1A; S1 Video). Almost all cKO mice appeared to have this epilepsy-like phenotype, and seizure movements were easily triggered by changing environmental status, such as ringing a clock or moving the cage suddenly. The frequency and duration of seizure-like episodes increased with age.

Fig 1

Deletion of Tsc2 resulted in epilepsy in Tsc2 cKO mice without obvious histoarchitectural changes.

A. Images captured from video recordings, showing typical spontaneous epilepsy in a 6-week-old cKO mouse. B. EEG and EMG segments (300 s) showing normal electrography in a control (WT) mouse and typical electrographic epilepsy in a cKO mouse. C. Relative brain and body weight in cKO mice compared with control (WT) mice at 9 and 11 weeks of age. *p < 0.05 and **p < 0.01 versus WT mice, n = 5 in each group, unpaired t-test. D. Hematoxylin staining of murine brain tissue sections, Scale bars: 600 μm. Sizes of hippocampus, Cerebral cortex and Whole brain are shown in the right panel, **p < 0.01 versus WT mice, n = 5 in each group, unpaired t-test. Data in C and D are expressed as mean ± SD.

To further characterize these episodes, electrocorticographic activity was recorded for 6–12 hours using a digital video-EEG/EMG system (Neuroscience, inc) in cKO mice and control wild-type littermates at 6 weeks of age. Control mice showed well-organized background activity (under 100-μV spikes) during awake and at rest. By contrast, frequent (2~3 times/hour) high-amplitude sharp waves (above 300-μV spikes, over 10 seconds) were observed during awake in the cKO mice, it was accompanied with seizure-like convulsive movements determined by video recording (Fig 1B).

Macrocephaly has been previously reported in other neuronal cell–lineage cKO mice characterized by hyperactivation of the mTOR signaling pathway in neurons[15-20]. The macrocephaly was attributed to neuronal hypertrophy secondary to an autonomous increase in the nuclear and soma size of mTOR-hyperactivated neurons[15-20]. Interestingly, body weight decreased in cKO mice, and the relative brain weight (ratio of brain weight versus body weight) increased in cKO mice, indicated another form of macrocephaly (Fig 1C). Furthermore, hematoxylin staining revealed no aberrant histological features in the whole brains of cKO mice, however, the hippocampus increased in size compared with the control littermates (Fig 1D).

Hyperactivation of mTOR induced neural excitation in Tsc2 cKO mice

As a negative regulator of mTOR signaling, loss of TSC2 would be predicted to constitutive activation of mTOR [21] and subsequently phosphorylation of ribosomal protein S6 (pS6). Usually, p-S6 is regarded as a specific indicator of TSC2 loss and mTOR activation[22]. Marked hyperactivation of mTOR signaling was observed in the hippocampus, cerebral cortex, and thalamus of cKO mice, compared with control littermates (Fig 2A, panel a and b). Control mice exhibited little or no expression of pS6, especially in the hippocampus, whereas cKO mice exhibited a dramatic increase in pS6 in almost all of the hippocampus, from the dentate gyrus to the CA1 zone. To verify neuronal activity in cKO mice, immunohistochemistry staining was performed with anti-cFOS, a marker of neuronal excitability[23]. Similar to the mTOR activity (pS6) results, higher expression levels of cFOS were observed in the hippocampus, cerebral cortex, and thalamus of cKO mice, compared with control littermates (Fig 2A, panels c and d); the increase was most pronounced in the hippocampus and cerebral cortex. It could be possible that mTOR hyperactivation is inducing neuronal excitability.

Fig 2

Hyperactivation of mTOR induced neural excitation in Tsc2 cKO mice.

A. Histoimmunostaining of whole brain sagittal sections from control (WT) mice (left panels) and cKO mice (right panels) at 5 weeks of age. p-S6 (upper panels) and c-FOS (bottom panels). The black rectangle outlines the area of hippocampus, cerebral cortex, and thalamus, and the detail is shown in the corresponding bottom panels. The circle shows representative p-S6 cytoplasmic and c-FOS nuclear positive staining. Scale bars: large bars, 600 μm; smaller bars, 200 μm. B. p-S6 and c-FOS positive rates (p-S6 or c-Fos-positive neuron cells versus all neuron cells) in the hippocampus, cerebral cortex, and thalamus. Data in C and D are expressed as mean ± SD. *P<0.05 versus WT mice, n = 5 in each group, unpaired t-test.

To quantify expression levels of pS6 and cFOS in the hippocampus, cerebral cortex, and thalamus, we counted the cFOS- and pS6-positive cells in these regions (Fig 2B). Almost 20% of hippocampal neurons exhibited increased mTOR activity (pS6 expression) in CKO mice (Fig 2B). Furthermore, excitability (cFOS expression) of hippocampal neuronal cells, increased dramatically from 9.7% in control mice to 69% in cKO mice (Fig 2B). The cerebral cortex exhibited lower mTOR activity than the hippocampus in cKO mice, and only slightly increased neuronal excitability compared with control mice. The thalamus in cKO mice exhibited a slight increase in mTOR activity but almost no change in neuronal excitability compared with control littermates. These data suggest that the neuronal abnormality of the hippocampus may be associated with the onset of the epilepsy phenotype in this mouse model.

Histologic changes were not observed in the hippocampal region of Tsc2Mitf-M cKO mice

A recent report indicated that the mTOR pathway regulates excitability of the hippocampal network through controlling the excitatory/inhibitory synaptic balance[24]. Therefore, we used immunofluorescence staining to examine excitatory neurons (using anti-CaMKII-a antibody) and inhibitory neurons (using anti-Parvalbumin antibody) in the mouse hippocampus (Fig 3A). Positive cells were counted, and the ratio of excitatory to inhibitory neurons was calculated (Fig 3B). No significant difference was observed in the excitatory/inhibitory synaptic balance between cKO mice and control littermates (Fig 3B). It suggests there might have some other players involved in seizure initiation and propagation, e.g. different interneuron subpopulations [25-27].

Fig 3

Histopathological analyses of the hippocampal region in Tsc2Mitf CKO mice.

A. Immunofluorescence staining showing excitatory (CaMKII-α) and inhibitory (Parvalbumin) neurons in the hippocampus. The insets show higher magnification of positive cells (arrowheads). B. Numbers of CaMKII-α-positive cells and Parvalbumin-positive cells were double-blind counted in 10 random fields per tissue section. Ratio of inhibitory to excitatory neurons were calculated (n = 5 mice). Data in B are expressed as mean ± SD. n.s. means no significance versus WT mice, unpaired t-test. C. Timm staining. The amount of mossy fiber sprouting is similar in cKO and control (WT) mice. Scale bars: A, 200 μm; C, 200 μm. n.s., not significant.

In 1989, Sutula et al. reported reorganization of mossy fiber axons, which projected abnormally into the dentate inner molecular layer in epilepsy[28]. This phenomenon, called mossy fiber sprouting, also appeared in a granule cell–lineage mTOR hyperactivation mouse model, suggesting that epilepsy might be associated with mTOR hyperactivation-induced neuronal restructuring[29]. As increased mTOR signaling was confirmed in the dentate gyrus of our Tsc2 cKO mice (Fig 2A, panel a), we further examined the status of mossy fibers in our cKO mice by Timm staining (Fig 3C). The mossy fiber tract was of normal thickness and exhibited no sprouting in cKO mice, compared with control littermates (Fig 3C). Together, these data suggest that restructuring of neuronal pathways, excitatory/inhibitory synaptic imbalance, and mossy fiber sprouting are not involved in the mechanism of epilepsy development in our cKO mice.

Deletion of Tsc2 induces mitochondrial hyperproliferation in the neurons of Tsc2 cKO mice

In neuronal cell-lineage transgenic mice, mTOR hyperactivity has been previously shown to induce many structural abnormalities associated with recurrent circuit formation, including hypertrophy of soma and dendrites, aberrant basal dendrites, impaired polarization, and enlarged axon tracts[20, 29, 30]. In the present study, we further investigated the ultrastructure of p-S6 high-expressed hippocampal pyramidal cells in the CA1 zone by electron microscopy (Fig 4A). Enlarged cell bodies and mitochondria were observed in the neurons of cKO mice. Somatic hypertrophy was confirmed in pyramidal cells of cKO mice (Fig 4A, upper panel). Furthermore, dramatic enlargement and hyperproliferation of mitochondria were observed in the pyramidal neurons of cKO mice (Fig 4A). Mitochondria increased nearly 5-fold in number and 2-fold in size, compared with neurons from control littermates (Fig 4B). To further confirm the increase in mitochondria, murine hippocampal tissue sections were assessed by histoimmunofluorescence staining with anti-COXIV antibody, a mitochondrial marker (Fig 4C). The results showed a substantial increase in the number of mitochondria, which corresponded to increased mTOR activity (anti-p-S6 binding), especially in the hippocampal CA1 region of cKO mice (Fig 4C).

Fig 4

Hyperproliferation of mitochondria in the neurons of Tsc2Mitf CKO mice.

A. Morphologic examination of p-S6 high-expressed hippocampal CA1 pyramidal cells by electron microscopy. Enlarged cell bodies and mitochondria were observed in the neurons of cKO mice. Bottom panels represent high-magnification images of the regions designated by squares. B. Quantification of mitochondria. The number of mitochondria increased more than 5-fold and the mitochondrial size increased more than 2-fold in neurons from cKO mice, compared with control (WT) mice. (n = 20 cells/mouse, 3 mice in each group). Data in B are expressed as mean ± SD, unpaired t-test versus WT mice. C. Immunofluorescence staining showed hyperactivation of mTOR (p-S6) with hyperproliferation of mitochondria (COXIV) in the hippocampus of cKO mice. Scale bars: A upper panel, 2 μm; A bottom panel, 500 nm; C, 200 μm.

Neurons from Tsc2 cKO mice were more excitable when stimulated

Previous studies have reported that neuronal hyperexcitability does not account for spontaneous epileptic activity with loss of Tsc1 (another mutated gene associated with TSC), suggesting that network restructuring plays a more important role in epileptic activity[24, 31, 32]. However, no structural abnormalities of the hippocampal network were observed in our cKO mice (Fig 2); therefore, we investigated the autonomous excitability of pyramidal cells. We isolated pyramidal cells from the hippocampal CA zone and confirmed by immunofluorescence staining that the majority of isolated cells were pyramidal cells (labeled with anti-MAP2) (Fig 5A). Neuronal excitability was examined by calcium imaging with the calcium-sensitive dye fura-2 (Fig 5B). Cells were depolarized by KCl, a treatment that promotes calcium influx via voltage-gated calcium channels[33]. The response to KCl was much greater in cKO pyramidal cells (Fig 5B, No.1–10) than in control pyramidal cells, and some cKO cells demonstrated slow recovery of the calcium transient (Fig 5B, No.1–2). The explanation for these findings is unclear, but they suggest that calcium dynamics in the pyramidal cells of Tsc2 cKO mice are altered.

Fig 5

Neurons from Tsc2 cKO mice were more excitable than neurons from control mice.

A. Neurons were isolated from 4-week-old mice and cultured for 1 week. Immunofluorescence staining indicates that more than 80% of the isolated cells were neurons (GFAP, astrocytes; MAP2, neurons). B. Calcium imaging of cultured neurons, with corresponding traces shown at the bottom. Neurons from cKO mice respond to particularly low (10 mM) K+ stimulation. Scale bars: A, 100 μm; B, 100 μm. WT, wild-type.

Rapamycin revealed treatment effects

Finally, we assessed whether rapamycin treatment, which blocks the effects of mTOR, could prevent the development of abnormalities observed in our melanocyte-lineage mTOR hyperactivation mice. After 3 weeks of oral rapamycin, the epilepsy phenotype was dramatically improved both in frequency and duration of seizures (Fig 6A). Examination of mTOR activity (p-S6), neuronal excitability (c-FOS), and mitochondria (COXIV) demonstrated that rapamycin treatment downregulated mTOR signaling and promoted normalization of mitochondrial number and neuronal excitability (Fig 6B). The fluorescence intensity was quantified by ImageJ within the range of threshold limit [34] and showed same changes (Fig 6C).

Fig 6

Rapamycin treatment reduced seizures and number of mitochondria.

A. Frequency and duration of seizures (sz) in cKO mice in the absence or presence of rapamycin, n = 5 in each group. Data in A are expressed as mean ± SD. Unpaired t-test versus vehicle-treated mice. B. Histoimmunostaining analyses of mTOR signaling (p-S6), mitochondria (COXIV), and neuronal excitation (c-FOS). Scale bars: 100 μm. WT, wild-type. C. The fluorescence intensity was quantified by ImageJ. n = 5 in each group. Data in C are expressed as mean ± SD. One-way ANOVA test, followed by Dunnett's post hoc test for multiple comparisons (WT mice versus cKO mice; cKO mice versus Rapamycin-treated cKO mice (cKO_Rapa); WT mice versus cKO_Rapa mice) was performed and adjusted P values were calculated. *p < 0.05, **p < 0.01.

Discussion

In the present study, we found that mTOR hyperactivation resulting from loss of Tsc2 in Mitf-M-derived cell lineage was associated with the development of typical epilepsy, mitochondria hyperproliferation, and aberrant intracellular calcium dynamics. Our results showed a primary defect of mitochondrial biogenesis in pyramidal neurons, leading to dramatically enhanced sensitivity and aberrant synchronization, which may be involved in increased hippocampal network excitability.

mTOR signaling activates the transcription of genes for mitochondrial biogenesis, including the well-known master regulator, peroxisome-proliferator-activated receptor coactivator-1α (PGC-1α)[35]. Also, mTOR modulates mitochondrial activity by enhancing interaction between transcription factor yin-yang 1 and PGC-1α [36] or by directly modifying the expression of mitochondrial proteins[37-39]. Recently, mTOR has been found to control mitochondrial activity and biogenesis through 4E-BP-dependent translational regulation[40].

In our previous study[9], we detected swollen mitochondria in melanocytes in the presence of Tsc2 deletion. In the present study, we observed not only swelling but also hyperproliferation of mitochondria in neurons.

It has been previously reported that activation of mTOR suppresses local translation of the potassium channel Kv1.1[41], resulting in increased burst firing in neurons[42, 43]. Furthermore, a direct correlation has been demonstrated between seizures and Kv1.1 gene expression[44-46]. mTOR also regulates components of neuronal RNA granules called specific RNA-binding proteins, such as fragile X mental retardation protein[47], which are involved in dendritic mRNA localization. Another line of research has demonstrated the regulatory effects of mTOR in the synthesis of new proteins in dendrites, such as PSD95 and calcium/calmodulin-dependent protein kinase[48, 49]. In the present study, we found that cKO neurons are more sensitive to potassium stimulation than controls, which may be attributed to an aberrant potassium channel or abnormal cellular ion levels.

Mitochondria contribute to various cellular processes, including ATP production, intracellular calcium signaling, cell growth and differentiation, and generation of reactive oxygen species. Neurons are critically dependent on mitochondrial function to establish membrane excitability through neurotransmission and plasticity. Electrical activity of neurons is associated with calcium influx into the cells via calcium channels, such as voltage-operated channels, store-operated channels, receptor-operated channels, and non-selective cation channels. Intracellular accumulation of calcium stimulates Na+/Ca2+ exchange, which maintain ionic gradients to sustain neuronal excitability[50]. The dramatic increase in number of mitochondria observed in the present study may alter intracellular Ca2+ homeostasis, inducing hyperexcitability of neurons.

It is currently thought that a number of key molecular signaling cascades are involved in the hyperexcitability of brain tissue because controlled blocking of “master regulators” of these pathways may retard or even stop the epileptogenic process[51]. Candidate regulators that have emerged in recent years include mTOR, as well as FosB[52], p-ERK1/2[53], tropomyosin-related kinase B, brain-derived neurotrophic factor, Zn2+-dependent cascades, and neuron-restrictive silencer factor/repressor element 1-silencing transcription factor pathways[51]. Aberrant mTOR pathway signaling has been extensively characterized in genetically-determined epilepsy in patients with mutations in the Tsc1/2 genes in the context of TSC. This condition manifests primarily as highly-differentiated tumors or malformations in many different organs and epilepsy[15-20]. Because Tsc genes are negative regulators of mTOR, hyperactivation of the mTOR pathway because of Tsc gene mutations provides a rational mechanistic basis for abnormal cell growth and proliferation, causing tumors and other developmental lesions in TSC. Numerous transgenic mouse models of TSC have been developed by spontaneous or induced inactivation of the Tsc1 or Tsc2 genes in the neuronal cell lineage, which exhibit varying degrees of pathological brain abnormalities and evidence of neuronal hyperexcitability or seizures[15-20].

Physiological and anatomical studies have produced conflicting findings regarding mTOR hyperactivation-induced neurological abnormalities. For example, some research reported reduced dendritic spine density after Tsc deletion[54], whereas another study indicated that the density increased[20]. As Tsc deletion produces a neurodevelopmental disorder, these discrepancies may depend on the role of mTOR in different types of cells and at different stages of development.

Until now, rodent models of spontaneous recurrent epilepsy have been generated by chemoconvulsants (primarily pilocarpine and kainic acid), neonatal hypoxia, traumatic brain injuries, electrical stimulation or genetic manipulations [55]. However, none of these models provide cell-type specificity in the brain[55]. By contrast, our mouse model involves Tsc knockout in specific Mitf-M-lineage cells.

In previous research regarding mTOR-associated epilepsy, structural abnormalities of neurons have been considered the primary etiologic factor. In the present study, we generated a novel epilepsy mouse model based on mTOR hyperactivation. The model is characterized by abnormal mitochondria, which may be responsible for the development of epilepsy by directly upregulating neuronal excitability. This model may be used to facilitate the development of new therapeutic interventions for seizure disorders.

Video recording of typical epilepsy.

(MP4)

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22 Oct 2019

PONE-D-19-25631

Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: a novel epilepsy model

PLOS ONE

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Reviewer #1: 1) In material and methods, you should precise the total number of animals used in your experiment.

2) “The fluorescence intensity was quantified by ImageJ and showed same changes (Fig. 6C).” Could please explain the method of quantification by ImageJ?

3) In Fig. 3A, 3C, 4A and 4C, the scale bars are different in cKO vs WT. Why?

4) It would be preferable to have some sentences about electrographic seizures resumed in a single paragraph. Particularly:

• you should deeply precise how their activity different from baseline activity (duration, rhythms, frequency range and amplitude)

• you should also precise how you define the onset of the seizure: shape of the first part of the seizure, occurrence …

• finally, you should explain how you characterize the end of the seizure: return to baseline activity, post-ictal depression...

5) In results, you reported that “A recent report indicated that the mTOR pathway regulates excitability of the hippocampal network through controlling the excitatory/inhibitory synaptic balance (19). Therefore, we used immunofluorescence staining to examine excitatory neurons (using anti-CaMKII-a antibody) and inhibitory neurons (using anti-Parvalbumin antibody) in the mouse hippocampus (Fig. 3A). Positive cells were counted, and the ratio of excitatory to inhibitory neurons was calculated (Fig. 3B). No significant difference was observed in the excitatory/inhibitory synaptic balance between cKO mice and control littermates (Fig. 3B).” In this regard, I think you should deeply discuss the crucial importance of different interneuron subpopulations in seizure initiation and propagation (refer to “de Curtis, M., and Avoli, M. (2016). GABAergic networks jump-start focal seizures. Epilepsia”), but also in seizure modulation (refer to "Khoshkhoo, S., Vogt, D., and Sohal, V. S. (2017). Dynamic, Cell-Type-Specific Roles for GABAergic Interneurons in a Mouse Model of Optogenetically Inducible Seizures. Neuron"). Moreover, only specific interneuron subpopulations but not others seem to be involved in the anticonvulsant effects in other models of seizures (refer to “Lucchi et al. (2017). Involvement of PPARγ in the anticonvulsant activity of EP-80317, a ghrelin receptor antagonist. Frontiers in Pharmacology”.)

6) In the discussion, the comparison between mTOR and other markers of neuronal activation used in literature should be done. For instance, you could refer to “Yutsudo et al. (2013). fosB-null mice display impaired adult hippocampal neurogenesis and spontaneous epilepsy with depressive behavior. Neuropsychopharmacology” and to “Giordano et al. (2016). Progressive seizure aggravation in the repeated 6-hz corneal stimulation model is accompanied by marked increase in hippocampal p-ERK1/2 immunoreactivity. Frontiers in Cellular Neuroscience”.

Reviewer #2: In the current Ms, the authors investigate further their melanocyte-lineage mTOR hyperactivation mouse model and try to describe it as a new model of epilepsy.

This Ms provides very important data to the field of epilepsy research. However, after reading the title of this Ms, I was quite disappointed when I realised that the authors did not try any classical antiepileptic drug on their supposedly new model of epilepsy. Has this been considered?

I have some other concerns listed below.

A) The “Materials and Methods” section lacks sufficient information.

To give few examples:

1) This is not easy to find the number of animals used for each protocol.

2) I could not find a clear description of how the animals were sacrified or anesthetized. This comment applies for almost all sections.

P5 Ln84 “Histology and immunohistochemistry analyses”: How animals are sacrificed before the brains being fixed?

P6Ln95: “Timm staining”: How were the rats anesthetized?

P6LnLn100: “Electron microscopy examination”: How were the rats sacrificed?

3) P7LnLn110: “Primary culture of hippocampal pyramidal cells from adult mice”: How were the slices cut?

4) P7Ln122: Measurement of [Ca2+]i: Please could you check the composition of your ACSF? What was the osmolarity of this solution?

B) In the discussion at LnP310: “Until now, rodent models of spontaneous recurrent epilepsy have been generated by chemoconvulsants (primarily pilocarpine and kainic acid), neonatal hypoxia, traumatic brain injuries or electrical stimulation(44).” Please note that genetic models of epilepsy are also available to study some types of epilepsy.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Dec 2019

COMMENTS FROM REVIEWER #1:

COMMENT: 1. In material and methods, you should precise the total number of animals used in your experiments.

RESPONSE: Thank you for your careful reading and valuable comment. As requested, we have added the description about the number of mice we used in each experiment in Materials and methods section in red with underline.

COMMENT: 2. “The fluorescence intensity was quantified by ImageJ and showed same changes (Fig. 6C).” Could please explain the method of quantification by ImageJ?

RESPONSE: Thank you for this important comment. As suggested, we have added the description of quantification method as indicated below.

(manuscript, Results section, page 14, line 261-262, in red with underline).

The fluorescence intensity was quantified by ImageJ within the range of threshold limit (Jensen EC. Quantitative analysis of histological staining and fluorescence using ImageJ. Anat Rec (Hoboken). 2013 Mar;296(3):378-81).

COMMENT: 3. In Fig. 3A, 3C, 4A and 4C, the scale bars are different in cKO vs WT. Why?

RESPONSE: Thank you very much for the careful reading of our manuscript. Because the hippocampus is bigger than WT (as shown in Fig 1C), in Figure 3A, 3C and 4C, to put the whole hippocampus of cKO into the same frame size to WT, images of WT and cKO were shown in different magnification with their different scales respectively. And the hippocampal CA1 pyramidal cells of cKO is also a little bit bigger than WT, in Figure 4A, to put the whole hippocampal CA1 pyramidal cell of cKO into the same frame size to WT, images of WT and cKO were shown in different magnification with their different scales respectively.

COMMENT: 4. It would be preferable to have some sentences about electrographic seizures resumed in a single paragraph. Particularly:

• you should deeply precise how their activity different from baseline activity (duration, rhythms, frequency range and amplitude)

• you should also precise how you define the onset of the seizure: shape of the first part of the seizure, occurrence …

• finally, you should explain how you characterize the end of the seizure: return to baseline activity, post-ictal depression...

RESPONSE: Thank you for your kind advice. According to your suggestion, we have modified the description as indicated below (manuscript, results section, page 9, line 160-166, in red with underline).

To further characterize these episodes, electrocorticographic activity was recorded for 6-12 hours using a digital video-EEG/EMG system (Neuroscience, inc) in cKO mice and control wild-type littermates at 6 weeks of age. Control mice showed well-organized background activity (under 100-µV spikes) during awake and at rest. By contrast, frequent (2~3 times/hour) high-amplitude sharp waves (above 300-µV spikes, over 10 seconds) were observed during awake in the cKO mice, it was accompanied with seizure-like convulsive movements determined by video recording (Fig 1B).

COMMENT: 5. In results, you reported that “A recent report indicated that the mTOR pathway regulates excitability of the hippocampal network through controlling the excitatory/inhibitory synaptic balance (19). Therefore, we used immunofluorescence staining to examine excitatory neurons (using anti-CaMKII-a antibody) and inhibitory neurons (using anti-Parvalbumin antibody) in the mouse hippocampus (Fig. 3A). Positive cells were counted, and the ratio of excitatory to inhibitory neurons was calculated (Fig. 3B). No significant difference was observed in the excitatory/inhibitory synaptic balance between cKO mice and control littermates (Fig. 3B).” In this regard, I think you should deeply discuss the crucial importance of different interneuron subpopulations in seizure initiation and propagation (refer to “de Curtis, M., and Avoli, M. (2016). GABAergic networks jump-start focal seizures. Epilepsia”), but also in seizure modulation (refer to "Khoshkhoo, S., Vogt, D., and Sohal, V. S. (2017). Dynamic, Cell-Type-Specific Roles for GABAergic Interneurons in a Mouse Model of Optogenetically Inducible Seizures. Neuron"). Moreover, only specific interneuron subpopulations but not others seem to be involved in the anticonvulsant effects in other models of seizures (refer to “Lucchi et al. (2017). Involvement of PPARγ in the anticonvulsant activity of EP-80317, a ghrelin receptor antagonist. Frontiers in Pharmacology”.) RESPONSE: Thank you for this helpful advice. According to your suggestion, we have added these three references (25-27) to our manuscript and modified the description as indicated below (manuscript, results section, page 11, line 203-211, in red with underline). “A recent report indicated that the mTOR pathway regulates excitability of the hippocampal network through controlling the excitatory/inhibitory synaptic balance(24). Therefore, we used immunofluorescence staining to examine excitatory neurons (using anti-CaMKII-a antibody) and inhibitory neurons (using anti-Parvalbumin antibody) in the mouse hippocampus (Fig 3A). Positive cells were counted, and the ratio of excitatory to inhibitory neurons was calculated (Fig 3B). No significant difference was observed in the excitatory/inhibitory synaptic balance between cKO mice and control littermates (Fig 3B). It suggests there might have some other players involved in seizure initiation and propagation, e.g. different interneuron subpopulations (25-27).”

COMMENT: 6. In the discussion, the comparison between mTOR and other markers of neuronal activation used in literature should be done. For instance, you could refer to “Yutsudo et al. (2013). fosB-null mice display impaired adult hippocampal neurogenesis and spontaneous epilepsy with depressive behavior. Neuropsychopharmacology” and to “Giordano et al. (2016). Progressive seizure aggravation in the repeated 6-hz corneal stimulation model is accompanied by marked increase in hippocampal p-ERK1/2 immunoreactivity. Frontiers in Cellular Neuroscience”.

RESPONSE: Thank you for this valuable comment. we have added these three references (52, 53) to our manuscript and modified the description as indicated below (manuscript, discussion section, page 16, line 304-307, in red with underline).

“Candidate regulators that have emerged in recent years include mTOR, as well as FosB(52), p-ERK1/2(53), tropomyosin-related kinase B, brain-derived neurotrophic factor, Zn2+-dependent cascades, and neuron-restrictive silencer factor/repressor element 1-silencing transcription factor pathways(51).”

COMMENTS FROM REVIEWER #2:

COMMENT: In the current Ms, the authors investigate further their melanocyte-lineage mTOR hyperactivation mouse model and try to describe it as a new model of epilepsy. This Ms provides very important data to the field of epilepsy research.

RESPONSE: Thank you for your careful reading and thank for your interest and compliments on our study.

COMMENT: However, after reading the title of this Ms, I was quite disappointed when I realised that the authors did not try any classical antiepileptic drug on their supposedly new model of epilepsy. Has this been considered?

RESPONSE: Thank you for your kind comment. We have tried to treat our mice using the classical mTOR inhibitor-Rapamycin, after 3 weeks of oral rapamycin treatment, epilepsy phenotype was dramatically improved both in frequency and duration of seizures (Fig 6A), however, we have not treated with some other classical clinical antiepileptic drugs. We would like to investigate that in our future study and report that in our next paper.

COMMENT: I have some other concerns listed below.

A) The “Materials and Methods” section lacks sufficient information.

RESPONSE: Thank you for your careful reading and valuable comment. As requested, we have modified our manuscript according to your suggestions.

COMMENT: 1. This is not easy to find the number of animals used for each protocol.

RESPONSE: Thank you for your careful reading and valuable comment. As requested, we have added the description about the number of mice we used in each experiment in Materials and methods section in red with underline.

COMMENT: 2. I could not find a clear description of how the animals were sacrified or anesthetized. This comment applies for almost all sections. P5 Ln84 “Histology and immunohistochemistry analyses”: How animals are sacrificed before the brains being fixed? P6Ln95: “Timm staining”: How were the rats anesthetized? P6LnLn100: “Electron microscopy examination”: How were the rats sacrificed?

RESPONSE: Thank you for your important comments. As requested, we have added the description of animal sacrifice as indicated below.

(manuscript, Methods section, page 8, line 140-144, in red with underline).

“Mice were anaesthetized with a lethal dose of pentobarbital and sacrificed by intracardially perfusion using ice-cold 1% (w/v) sodium sulfide, followed by 4% paraformaldehyde. The brains were removed, post-fixed for 10% formaldehyde overnight and embedded in paraffin or cryoprotected in 30% sucrose/PBS.”

COMMENT: 3. P7LnLn110: “Primary culture of hippocampal pyramidal cells from adult mice”: How were the slices cut?

RESPONSE: Thank you for your important comments. As requested, we have added the description about the slice cut as indicated below.

(manuscript, Methods section, page 7, line 106-116, in red with underline).

“Primary neuronal cells were obtained from the hippocampus of 4-week-old wild-type and mutant mice (n = 5 mice/goup) as reported previously(13). Briefly, the hippocampus was dissected and sliced into 0.5-mm sections using tissue slicer (Dosaka microslicer, Kyoto, Japan), removing the dentate gyrus to eliminate granule cells. The sections were digested with papain (2 mg/mL, Worthington, #LS003119 in HA-Ca, BrainBits LLC) at 30°C for 30 min. Cells were released by gentle trituration with a Pasteur pipette. Finally, primary neurons were separated using density-gradient centrifugation (OptiPrep, AXS, #1114542, XX). Cells were cultured in NeurobasalA/B27 medium (Invitrogen, #10888022 and #17504044) with L-Gin (Invitrogen, #25030149), growth factors (5 ng/mL mouse FGF2, Invitrogen, #PMG0034; 5 ng/mL mouse PDGF-BB, Invitrogen, #PMG0044), and gentamycin (Wako, #078-06061) for 1 week before the experiments.”

COMMENT: 4. P7Ln122: Measurement of [Ca2+]i: Please could you check the composition of your ACSF? What was the osmolarity of this solution?

RESPONSE: Thank you for your careful reading and the important comment. Regret for this mistake, as suggested, we have added the reference (14) in our manuscript and revised our mistake in the concentration of NaCl (revised 45mM to 127 mM NaCl).

(manuscript, Methods section, page 7, line 121-126, in red with underline).

[Ca2+]i in single cells was detected on the basis of fura-2 fluorescence intensity, as reported previuosly(14). Briefly, neurons grown on coverslips were rinsed twice with artificial cerebrospinal fluid (ACSF; 127 mM NaCl, 1.5 mM KCl, 26 mM NaHCO3, 1.24 mM KH2PO4, 10 mM glucose, 1.4 mM MgSO4, 2.4 mM CaCl2; SIGMA) and incubated at 37°C for 45 min in the presence of fura-2 AM (fura-2 acetoxymethyl ester, DOJINDO, #CS23) with 1.25 mmol/L probenecid (SIGMA) and 0.03% Pluronic® F-127 (SIGMA) in ACSF.

COMMENT: B) In the discussion at LnP310: “Until now, rodent models of spontaneous recurrent epilepsy have been generated by chemoconvulsants (primarily pilocarpine and kainic acid), neonatal hypoxia, traumatic brain injuries or electrical stimulation(44).” Please note that genetic models of epilepsy are also available to study some types of epilepsy.

RESPONSE: Thank you for the important comment. According to your suggestion, we have modified the description as indicated below.

(marked manuscript, Discussion section, page 18, line 324-326, in red with underline). “Until now, rodent models of spontaneous recurrent epilepsy have been generated by chemoconvulsants (primarily pilocarpine and kainic acid), neonatal hypoxia, traumatic brain injuries, electrical stimulation or genetic manipulations (55).”

Submitted filename: Point-by-point response cover letter.docx

Click here for additional data file.

20 Dec 2019

PONE-D-19-25631R1

Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: a novel epilepsy model

PLOS ONE

Dear Dr. Wataya-Kaneda,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

We would appreciate receiving your revised manuscript by Feb 03 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Giuseppe Biagini, MD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have adequately addressed my comments raised in a previous round of review and I feel that this manuscript is now acceptable for publication.

Reviewer #2: The Ms has improved following the authors's reply to the reviewers's comments. However in my opinion, supplemental information particularly in the method section, are needed to make this Ms a little bit clearer.

1) Primary culture of hippocampal pyramidal cells from adult mice: Please could you precise in which media the slices were cut?

2) Measurement of [Ca2+]i: Please could you precise if the ACSF was "bubbled" with carbogen?

3) Animal sacrifice: one can read "Mice were anaesthetized with a lethal dose of pentobarbital and sacrificed by intracardially perfusion using ice-cold 1% (w/v) sodium sulfide, followed by 4% paraformaldehyde." I am a little bit confused. Did the authors perform Primary culture of hippocampal pyramidal cells and measurement of [Ca2+]i on cells from animals that have been perfused with 4% paraformaldehyde?

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

7 Jan 2020

COMMENTS FROM REVIEWER #1:

“The authors have adequately addressed my comments raised in a previous round of review and I feel that this manuscript is now acceptable for publication.”

RESPONSE: Thank you for your careful reading and valuable suggestions for revising and improving our manuscript.

COMMENTS FROM REVIEWER #2:

“The Ms has improved following the authors's reply to the reviewers's comments. However, in my opinion, supplemental information particularly in the method section, are needed to make this Ms a little bit clearer.”

RESPONSE: Thank you for your careful reading and important suggestions. We have revised our manuscript according to your comments and suggestions.

COMMENT: 1. “Primary culture of hippocampal pyramidal cells from adult mice: Please could you precise in which media the slices were cut?”

RESPONSE: Thank you for your important comments. As requested, we have added the description about the medium as indicated below.

(Manuscript with Track Changes, Methods section, page 7, line 106-111, in red with underline).

“Primary neuronal cells were obtained from the hippocampus of 4-week-old wild-type and mutant mice (n = 5 mice/goup) as reported previously(13). Briefly, the hippocampus was dissected and sliced into 0.5-mm sections in 2 mL HABG medium (40ml HA(HibernateTM-A Medium, Invitrogen, #A1247501; 0.8ml B27, Invitrogen, #17504; 0.1ml L-Glutamine, Invitrogen, #25030081)) at 4°C in a 35-mm-diameter dish using tissue slicer (Dosaka microslicer, Kyoto, Japan), removing the dentate gyrus to eliminate granule cells.”

COMMENT: 2. “Measurement of [Ca2+]i: Please could you precise if the ACSF was "bubbled" with carbogen?”

RESPONSE: Thank you for your important comments. In our experiments, ACSF has been bubbled with carbogen. As requested, we have added the description as indicated below.

(Manuscript with Track Changes, Methods section, page 8, line 124-129, in red with underline).

“Briefly, neurons grown on coverslips were rinsed twice with artificial cerebrospinal fluid (ACSF; 127 mM NaCl, 1.5 mM KCl, 26 mM NaHCO3, 1.24 mM KH2PO4, 10 mM glucose, 1.4 mM MgSO4, 2.4 mM CaCl2; SIGMA) and incubated at 37°C for 45 min in the presence of fura-2 AM (fura-2 acetoxymethyl ester, DOJINDO, #CS23) with 1.25 mmol/L probenecid (SIGMA) and 0.03% Pluronic® F-127 (SIGMA) in carbogen-bubbled ACSF.”

COMMENT: 3. “Animal sacrifice: one can read "Mice were anaesthetized with a lethal dose of pentobarbital and sacrificed by intracardially perfusion using ice-cold 1% (w/v) sodium sulfide, followed by 4% paraformaldehyde." I am a little bit confused. Did the authors perform Primary culture of hippocampal pyramidal cells and measurement of [Ca2+]i on cells from animals that have been perfused with 4% paraformaldehyde?”

RESPONSE: Thank you for your careful reading and the important comment. We have added the information as indicated below.

(Manuscript with Track Changes, Methods section, page 8, line 143-147, in red with underline).

“Mice were anaesthetized with a lethal dose of pentobarbital and sacrificed by intracardially perfusion using ice-cold 1% (w/v) sodium sulfide, followed by 4% paraformaldehyde. The brains were removed for primary culture of hippocampal pyramidal cells, measurement of [Ca2+]i, or post-fixed for 10% formaldehyde overnight and embedded in paraffin or cryoprotected in 30% sucrose/PBS for histologic analyses.”

Submitted filename: Response to reviewers.docx

Click here for additional data file.

9 Jan 2020

PONE-D-19-25631R2

Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: a novel epilepsy model

PLOS ONE

Dear Dr Wataya-Kaneda,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Specifically, I invite you to complete and correct description of statistical analysis because:

- results of statistical analysis are not detailed in results, and should be better illustrated in legends to figures;

- in methods, I read that you used "two-way analysis of variance", but the post hoc test is not indicated.

We would appreciate receiving your revised manuscript by Feb 23 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Giuseppe Biagini, MD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

9 Jan 2020

Prof. Giuseppe Biagini,

Chief of Editor

PLOS ONE

January 9th, 2020

Dear Prof. Giuseppe Biagini,

Thank you very much for your e-mail of January/09/2020 with regard to our manuscript (PONE-D-19-25631R2) entitled “Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: a novel epilepsy model” together with the comments from the editor and reviewer. We appreciate the editors and reviewers very much for their positive and constructive comments and suggestions on our manuscript. We have revised our manuscript accordingly. The alterations are referred in this response letter.

We hope that the revised manuscript meets with your approval.

Sincerely,

Mari Wataya-Kaneda, M.D., Ph.D.

Department of Dermatology, Course of Integrated Medicine, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, 565-0872, Japan

E-mail: mkaneda@derma.med.osaka-u.ac.jp

Tel: +81 6 6879 3031. Fax: +81 6 6879 3039

COMMENTS FROM EDITOR:

“Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Specifically, I invite you to complete and correct description of statistical analysis.”

RESPONSE: Thank you for your important suggestions. We have carefully checked our manuscript again and corrected descriptions of statistical analysis in our revised manuscript.

COMMENT: 1. “results of statistical analysis are not detailed in results, and should be better illustrated in legends to figures”

RESPONSE: Thank you for your kind advice. As requested, we have added the detail in legends to figures as indicated below.

(Manuscript with Track Changes, Figure Legend section, page 24-26, line 488-549, in red with underline).

“Figure 1. Deletion of Tsc2 resulted in epilepsy in Tsc2Mitf-M cKO mice without obvious histoarchitectural changes.

Figure 2. Hyperactivation of mTOR induced neural excitation in Tsc2Mitf-M cKO mice.

Figure 3. Histopathological analyses of the hippocampal region in Tsc2Mitf CKO mice.

Figure 4. Hyperproliferation of mitochondria in the neurons of Tsc2Mitf CKO mice.

Figure 5. Neurons from Tsc2Mitf -M cKO mice were more excitable than neurons from control mice.

Figure 6. Rapamycin treatment reduced seizures and number of mitochondria.

Supplementary information

S1 video. Video recording of typical epilepsy.”

COMMENT: 2. “in methods, I read that you used "two-way analysis of variance", but the post hoc test is not indicated.”

RESPONSE: Thank you for your careful reading and the important comment. Regret for this mistake, in our experiment, in Figure 6C, the differences of WT mice versus cKO mice, cKO mice versus Rapamycin-treated cKO mice, and WT mice versus cKO_Rapa mice were calculated using One-way ANOVA test, followed by Dunnett's post hoc test. As suggested, the description have been corrected in our revised manuscript as indicated below.

(Manuscript with Track Changes, Figure Legend section, page 26, line 537-547, in red with underline; Methods section, page 9, line 148-152, in red with underline).

“Figure 6. Rapamycin treatment reduced seizures and number of mitochondria.

Frequency and duration of seizures (sz) in cKO mice in the absence or presence of rapamycin, n = 5 in each group. Data in A are expressed as mean ± SD. Unpaired t-test versus vehicle-treated mice. B. Histoimmunostaining analyses of mTOR signaling (p-S6), mitochondria (COXIV), and neuronal excitation (c-FOS). Scale bars: 100 µm. WT, wild-type. C. The fluorescence intensity was quantified by ImageJ. n = 5 in each group. Data in C are expressed as mean ± SD. One-way ANOVA test, followed by Dunnett's post hoc test for multiple comparisons (WT mice versus cKO mice; cKO mice versus Rapamycin-treated cKO mice (cKO_Rapa); WT mice versus cKO_Rapa mice) was performed and adjusted P values were calculated. *p < 0.05, **p < 0.01.”

(Manuscript with Track Changes, Figure Legend section, page 26, line 537-547, in red with underline; Methods section, page 9, line 148-152, in red with underline).

“Statistical analyses

Data are presented as mean ± SD. Unpaired Student’s t-test (Microsoft Excel; Microsoft Corp., Redmond, WA) was used for comparisons between two groups. One-way ANOVA test, followed by Dunnett's post hoc test was used for multiple comparisons (Microsoft Excel). P-values <0.05 were considered statistically significant.”

Submitted filename: Response to reviewers.docx

Click here for additional data file.

10 Jan 2020

Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: a novel epilepsy model

PONE-D-19-25631R3

Dear Dr. Wataya-Kaneda,

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

16 Jan 2020

PONE-D-19-25631R3

Epilepsy in a melanocyte-lineage mTOR hyperactivation mouse model: a novel epilepsy model

Dear Dr. Wataya-Kaneda:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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