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. 2021 Jun 4;16(6):e0239111. doi: 10.1371/journal.pone.0239111

The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy

Anna Skupien-Jaroszek 1,#, Agnieszka Walczak 1,#, Iwona Czaban 1, Katarzyna Karolina Pels 1, Andrzej Antoni Szczepankiewicz 1, Katarzyna Krawczyk 1, Błażej Ruszczycki 1, Grzegorz Marek Wilczynski 1, Joanna Dzwonek 1,*, Adriana Magalska 1,2,*
Editor: Michal Hetman3
PMCID: PMC8177504  PMID: 34086671

Abstract

The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting—an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.

Introduction

The Brain-Derived Neurotrophic Factor (BDNF) is one of the most important neurotrophins in the brain. Acting via its synaptic receptor Tropomyosin-related kinase B (TrkB), BDNF is involved in neuronal differentiation, survival, and synaptic plasticity [1–3]. Thus, it plays an important role in the number of neurological and psychiatric disorders such as Parkinson’s disease [4], schizophrenia [5], depression [6], bipolar disease [7], and epilepsy [8–10].

Currently, it is known that BDNF is involved in the event of aberrant synaptic plasticity called mossy fiber sprouting, observed in temporal lobe epilepsy (TLE), which is one of the most common types of epilepsy in adults [11]. The level of both BDNF protein [12] and mRNA [13–15] were described to be elevated after seizures in the temporal lobe and hippocampi of epileptic patients. Experiments performed on animals and in vitro models showed that BDNF causes hypertrophy of granule neurons [16] and increased mossy fiber branching [17]. Moreover, intrahippocampal infusion of BDNF induced mild seizures with the development of mossy fiber sprouting [18]. Those findings support the involvement of BDNF in the aberrant synapse formation in TLE, however, underlying molecular mechanisms are still not clear.

The current trend in neuroscience is to look for mechanisms of neuronal functioning at a gene expression level. BDNF encoding gene is a so-called delayed, immediate-early gene induced in the later phase of neuronal activation [19]. It consists of 9 exons differentially expressed in humans, mice, and rats [20]. Regulation of BDNF expression after neuronal excitation has been quite well understood at the level of transcription factors and chromatin modifications. It is known that the 3’ end of the protein-coding exon is spliced to one of the eight of 5’ exons, which are controlled by their unique promoters [20–22]. Moreover, Bdnf can be epigenetically down-regulated through DNA methylation and histone deacetylation. The aforementioned epigenetic changes result in the recruitment of REST/NRSF complex [23] and MeCP2 [24,25] and chromatin remodeling. Conversely, the up-regulation of the gene can be triggered by DNA demethylation and/ or histone acetylation, which was already presented in both in vitro [20] and in vivo studies [26]. Importantly, the level of histone H3 acetylation at the Bdnf promoters IV and VI may underlie sustained up-regulation of transcription following chronic electroconvulsive shock [27]. Fukuchi et al. [28] showed that valproic acid, an inhibitor of histone deacetylases commonly used antiepileptic drug, increases expression of Bdnf under control of promoter I.

Studies of the last decade have shown that the genome is spatially organized within the nucleus [29]. Rearrangements of chromatin are involved in the regulation of gene expression, and the radial position of genes reflects their expression [30]. In differentiated cells, the nuclear periphery is a repressive environment, where heterochromatin is recruited to the nuclear lamina [31]. Artificial localization of gene at the nuclear periphery, by tethering to the inner nuclear membrane, is sufficient to induce silencing of its expression [32]. The role of chromatin structure in the regulation of gene expression in neurons remains still unexplored. Crepaldi and colleagues [33] showed that activity-dependent genes, including Bdnf, were repositioned to transcription factories after KCl induced depolarization in cultured cortical neurons. Moreover, in our previous studies [34] we showed that during neuronal excitation and epileptogenesis Bdnf alleles had been detached from the nuclear lamina and repositioned from the nuclear periphery toward the nuclear center. The observed phenomenon was associated with changes in Bdnf expression. However, it was not clear whether Bdnf repositioning had been a cause or a consequence of the gene transcriptional activity. Therefore, in the current study, we are addressing this interesting question.

Materials and methods

Animals

The experiments were performed on young, adult male Wistar rats, weighing 170–250 g, obtained from Mossakowski Medical Research Centre, Polish Academy of Sciences. Animals were kept under a 12 h light/dark cycle, with unlimited food and water supplies. All procedures were performed with the consent of the 1st Local Ethical Committee in Warsaw (Permission number LKE 306/2017).

Induction of seizures

Seizures were evoked by two doses of kainate (5 mg/kg, Sigma-Aldrich) (0.5% solution in saline, pH 7), administered intraperitoneally in 1 h intervals, and scored as described by Hellier et al [35]. The animals were taken for further studies regardless of whether they fulfilled the criterion of the full status epilepticus, or not [35]. To reduce kainate-induced mortality (up to 10%), diazepam (25mg/kg) was administrated intraperitoneally 3–6 hours after seizure onset. To monitor animal health, the rats were constantly observed by experimenters, for 6 hours after kainate injection, and 4 hours/day in the subsequent 4 weeks. All animals were euthanized with an overdose of pentobarbital sodium (Nembutal 150 mg/kg, i.p.) and perfused with 4% paraformaldehyde in PBS at the end of the experiments, and the brain tissues were collected.

Estimation of clinical and morphological traits

Clinical and morphological traits such as the intensity of seizures, ruffling of the fur, forelimb clonus, body, and head tremor, losing posture, immobility and aggression have been estimated for 6 hours after kainate injection, and 4 hours/day in subsequent 4 weeks. The intensity of seizures upon kainate treatment was scored according to the 6-grade modified Racine’s scale (0-lack of seizures to 5- fully developed tonic-clonic seizures with a loss of posture) [35]. Ruffling of the fur was scored on a 3-grade scale (0-lack of ruffling to 2-extensively ruffled fur). Forelimb clonus was scored according to a 7-grade scale (0-lack of clonus to 6-very strong forelimb clonus). Body and head tremor was scored according to a 6-grade scale (0-lack of tremor to 5-very strong tremor). Loss of posture was scored according to the 4-grade scale (0-lack of loss of posture to 4 constant loss of posture). Immobility was scored according to the 10-grade scale (0-completely active to 9-lack of activity). Aggression was scored on a 4-grade scale (0-lack of aggression to 3-extreme aggression). To minimize the bias, two independent observers performed the scoring of clinical parameters. Mossy fiber sprouting was verified by immunofluorescent staining for synaptoporin and scored on a 5-grade scale (0-lack of sprouting to 4-very strong sprouting). The mean synaptoporin intensity was measured for each individual using ImageJ. Measurements were done on confocal images in a specific area of the molecular layer of the dentate gyrus depicted in Fig 2B. Additionally, two independent observers estimated sprouting visually in the same brain area directly using a microscope.

Fig 2. Correlation between the intensity of sprouting and the percentage of the nuclei with Bdnf alleles at the nuclear periphery.

Fig 2

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A) Mossy fiber sprouting was verified by immunofluorescent staining for synaptoporin in the molecular layer of DG region of the hippocampus. Representative pictures of different levels of sprouting of animals at 4 weeks after administration of kainate are shown. B) Percentage of animals showing different levels of synaptoporin staining intensity scored in 5-grade scale (0-lack of sprouting- 4 very strong sprouting). C) Correlation between the levels of sprouting in the DG, 4 weeks from the administration of kainate, measured in 5- grade scale, and percentage of the nuclei with Bdnf alleles localized at the nuclear periphery. Scale Bar: 100 μm.

Primary neuronal hippocampal cultures and treatment

Primary neuronal cultures were prepared from the hippocampi of P0 rat brains as described previously [36]. Chemical long-term potentiation (cLTP) was initiated by stimulating the cells for 2 hours with 50 μM of picrotoxin, 50 μM forskolin, and 0.1 μM rolipram (all from Sigma-Aldrich). Transcription was inhibited by 2h exposure to 8mg/ml of Actinomycin D (Sigma-Aldrich). Histone deacetylases were inhibited by 12h treatment with 200 nM Trichotstatin A (Abcam) or 250 nM romidepsin (Cayman Chemical Company) for 2h.

Fluorescent in situ hybridization

Fluorescent in situ hybridization was performed according to the protocol of Cremer et al. (2008) [37] on 30 μm-thick brain cryosections of the 4% paraformaldehyde-perfused, 8 kainate-treated, and 4 control animals, as well as on 3 independent primary hippocampal neuronal cultures fixed with 4% paraformaldehyde at DIV 14 (day in vitro). As templates for Bdnf FISH probes, CH230-449H21 BAC obtained from Children’s Hospital Oakland Research Institute were used. Probes were verified on rat metaphase spreads. The probes were labeled using the standard nick-translation procedure. Biotinylated probes were detected using Alexa Fluor 488- conjugated avidin (Invitrogen), followed by FITC-conjugated rabbit anti-avidin antibody (Sigma-Aldrich).

Immunostaining

Immunostaining for synaptoporin was performed on 30 μm-thick brain cryosections of the 4% paraformaldehyde-perfused animal using standard immunofluorescent staining protocol [38]. 1 μg/ml of rabbit polyclonal anti synaptoporin (Synaptic Systems) antibody was used. The intensity of immunostaining was estimated using the 5-grade scale (0-no staining to 4-strong staining). The neuronal damage was examined by staining with Fluoro-Jade B (Millipore) according to the method of Schmued et al [39].

Image acquisition

Fluorescent specimens were examined under TCS SP8 confocal microscope (Leica) or Zeiss 800 confocal microscope (Zeiss), by sequential scanning of images, with a pixel size of 80 nm and axial spacing of 210 nm, using a PlanApo oil-immersion 63 (1.4 numerical aperture) objective.

Quantitative image analysis

The minimal distance from the nuclear periphery and Bdnf alleles in neuronal nuclei of animals was calculated using custom-written software, Segmentation Magick, described in Ruszczycki et al. [40]. For neuronal cultures, custom-written software Partseg was used, described in [41]. At least 120 nuclei, from 3 independent experiments were analyzed for each experimental variant.

Real-time reverse transcriptase-PCR for Bdnf mRNA

Total cellular RNA was isolated from three independent hippocampal primary cultures using RNeasy Mini Kit (Qiagen) according to the manufacturer’s procedure. 1 μg of RNA was subjected to RT reaction using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer’s protocol. PCR was performed using SYBR Green PCR Master Mix (Thermo Fischer Scientific). Forward and reverse primers, were respectively: 5-CCATAAGGACGCGGACTTGTAC and 5-AGACATGTTTGCGGCATCCAGG.

Statistical analysis

The correlation between sprouting intensity and percentage of BDNF alleles on the nuclear border was calculated with Anaconda Software Distribution (2020) (Anaconda Inc) retrieved from https://docs.anaconda.com/, using SciPy library.

The data for RT-PCR experiments were obtained from 3–5 independent batches of neurons and normalized to the control in each experiment. Statistical analysis of normalized data was performed using the Kruskal-Wallis group comparison, and the One-Sample T-test or Welch Two Sample t-test for pairwise comparison, with the use of R [42] retrieved from https://www.R-project.org/.

For the FISH experiments, the differences in the percentage of the gene at the nuclear periphery were analyzed using chi-square (all 4 groups together) and Fisher’s exact test for post hoc pairwise comparison. Since the contingency tables show precise counts, error bars were calculated as a standard deviation from the binomial distribution. The details are given in the Supplementary S1 File. The analysis was performed using GraphPad Prism software.

Results

Morphological traits of the kainate model of TLE and clinical implications of Bdnf repositioning in the hippocampal granule neurons

To verify the kainate model of TLE in rats, animals were carefully observed for 6 hours after a kainate injection, and 4 hours/day in the subsequent 4 weeks. The intensity of seizures has been assessed according to modified 6-grade Racine’s scale (0-lack of seizures- 5- fully developed tonic-clonic seizures with a loss of posture). Additionally, clinical traits such as fur ruffling, forelimb tonus, body and head tremor, loss of posture, immobility, and aggression have been rated. Moreover, hippocampal specimens from the aforementioned animals were analyzed for neuronal damage in the DG granule cell layer and CA3 pyramidal layer by Fluoro-jade B staining and mossy fiber sprouting in DG molecular layer by synaptoporin staining.

In our model, 60% of animals underwent status epilepticus (fully developed tonic-clonic seizures with a loss of posture, Fig 1A and Table 1 and S1 File), and the remaining animals showed moderate seizure symptoms like head tremors (wet dog shaking) and fur ruffling. In 4 weeks following administration of kainate 5 out of 10 animals showed fur ruffling, 5 out of 10 demonstrated forelimb tonus, all animals exhibited body and head tremor, 5 out of 10 showed loss of posture, 7 out of 10 –immobility, and 5 out of 10- aggression (for details see Table 1).

Fig 1. Kainate induced seizures and neuronal damage.

Fig 1

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A) Percentage of animals showing seizures after kainite administration. The intensity of seizure was scored according to the 6-grade modified Racine’s scale (0-lack of seizures—5- fully developed tonic-clonic seizures with loss of posture). B) Fluoro-jade B staining (depicted in green) showing neuronal damage in the CA3 region, but not the DG region of the hippocampus of kainite treated animals. Scale Bar: 100 μm.

Table 1. Estimation of the clinical and morphological traits in the kainate model of TLE.

animal ID 1B 1C 1G 2B 2C 2G 3G 3R 4G 4R
seizures (0–5) 2 5 5 5 4 5 2 5 5 2
% of nuclei with Bdnf at the perifery 43 53 29 29 53 55 65 33 24 50
sprouting level (0–4) 2 0 4 2 1 3 0 1 4 1
ruffling of fur (0–2) 0 1 1 0 0 0 0 2 1 1
forelimb tonus (0–6) 0 0 0 1 0 3 1 6 2 0
body/head tremor (0–5) 3 4 1 2 1 3 2 4 5 2
loss of posture (0–4) 2 3 0 2 1 0 0 0 0 1
inactivity (0–9) 1 0 1 1 0 0 1 7 1 1
agression (0–3) 1 0 1 0 0 1 0 0 3 1
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For all traits, scales are shown in brackets (see Materials and Methods for full details).

Fluoro-jade B staining showed no neuronal damage in the DG granule cell layer and extensive cell death in the CA3 pyramidal layer in all animals (Fig 1B). Extend of mossy fiber sprouting was estimated by the intensity of synaptoporin immunostaining (Figs 2A and 2B and S1). In 8 out of 10 animals, it was possible to distinguish mossy fibers stained against synaptoporin (scored from 0- lack of sprouting, to 1—weak sprouting to 4—very strong sprouting, in Figs 2A and 2B and S1 and Table 1), where 30% showed moderate to very strong sprouting (scored ≥3). The level of sprouting was independent of the intensity of the initial seizures (right after kainate injection). All tested animals showed behavioral and morphological traits of epilepsy within 4 weeks from kainite treatment.

In our previous work [34] using the same model of neuronal activation upon kainate-induced seizures, we have observed repositioning of the Bdnf alleles in hippocampal granule neurons. We have shown that transcriptionally inactive Bdnf is attached to a nuclear lamina and localized at the nuclear periphery, after neuronal activation it has repositioned towards the nuclear center. Here we observed, that the level of sprouting correlated (R = -0.67, Pearson correlation) with the percentage of the nuclei with Bdnf allele localized at the nuclear periphery (Fig 2C). In animals with the strongest level of sprouting, in less than 50% of nuclei Bdnf alleles were present at the nuclear rim, while lack of sprouting correlated with the higher percentage of nuclei showing Bdnf localized in proximity to the nuclear envelope. We have found the same correlation calculated for sprouting levels estimated visually by two independent observers (R = -0.70) Moreover, the intensity of sprouting correlated positively with the level of aggression (R = 0,75, Pearson correlation), animals with a higher level of sprouting tend to be more aggressive (supplementary S1 File). Unfortunately, we haven’t found any other correlations of behavioral traits neither with the level of sprouting nor the percentage of nuclei with Bdnf alleles on the nuclear periphery.

The causal relationship between Bdnf allele transcriptional activity and repositioning

To further investigate the cause of the intranuclear reposition of Bdnf and its relationship with BDNF transcription, we used an in vitro model of neuronal excitation based on hippocampal dissociated cultures and a chemical model of long-term potentiation (cLTP) [43–45]. cLTP evoked by picrotoxin, forskolin, and rolipram was proven to be non-toxic for neurons and to induce a program of gene expression similar to the one observed in a brain upon stimulation. We observed that 2 hours after the initiation of long-term potentiation, Bdnf expression was significantly (4 times) increased compared to the control (Fig 3A). At the same time, the Bdnf allele had repositioned toward the center of a cell nucleus (Figs 3B–3D and S2). In the control cells (treated with a solvent alone) the Bdnf alleles were most frequently positioned at a nuclear margin, with 76.4% of alleles located 350 nm or less from a nuclear border (Fig 3C and 3D, blue bars). This distance is an approximate microscope resolution limit, hence it has been chosen as an indicator of allele proximity to the nuclear border, as previously described [34]. In activated neurons, we observed a distinct repositioning of the Bdnf gene from the nucleus periphery towards the nucleus center (Fig 3C and 3D, orange bars). The percentage of Bdnf alleles localized closer than 350 nm to the nuclear margin was significantly lower than in the control group (62.9% Fisher’s exact tests). To verify whether Bdnf allele repositioning is a cause or a consequence of transcriptional activity, we performed experiments with the use of Actinomycin D, a potent inhibitor of RNA polymerase II. Preincubation with Actinomycin D for 2 hours was sufficient to inhibit Bdnf expression upon stimulation with cLTP (6 fold decrease compared to cLTP treatment, Fig 3A). Inhibition of transcription completely blocked Bdnf repositioning upon cLTP treatment (Figs 3B–3D and S2. The percentage of Bdnf alleles located at the nuclear periphery upon Actinomycin D and cLTP treatment was similar to control cells. Presented results show that transcriptional activity is a cause of Bdnf repositioning.

Fig 3. The causal relationship between Bdnf transcriptional activity and Bdnf’s allele repositioning.

Fig 3

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(A) The graph shows the expression of Bdnf normalized to control in the hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL, blue bar) or picrotoxin, forskolin, and rolipram (cLTP, orange bar), incubated for 2 hours with Actinomycin D and 2 hours with DMSO (ActD, green bar) or picrotoxin, forskolin, and rolipram (ActD+cLTP, red bar). Kruskal-Wallis group comparison: p<0.01, and the One-Sample T-test or Welch Two Sample t-test for pairwise comparison: * p<0.05, **p<0.01, *** p<0.001; error bars indicate standard error of the mean for 3 independent experiments (B) Representative picture of the nuclei of hippocampal neurons treated as described above. Hoechst 3342 staining for chromatin is shown in greyscale and segmentation of FISH signals for the Bdnf gene are shown in magenta and cyan. (C) Percentages of Bdnf alleles localized < 350 nm to the nuclear surface are shown (Chi-square test, all groups, p<0.001; Fisher’s exact tests * p<0.05, *** p<0.001, error bars indicate standard deviation of the binomial distribution). (D) Quantitative analysis of the intracellular positions of Bdnf alleles in the nuclei of hippocampal neurons treated and color-coded as in A. The minimal distance between the respective alleles and nucleus surface is presented in the normalized histogram.

Histone deacetylases are necessary for the attachment of Bdnf alleles to the nuclear lamina

Covalent modifications of chromatin were shown to be involved in the regulation of Bdnf gene expression [27,46,47]. Data indicate, that histone deacetylases (HDAC) play an important role in this process [48,49]. Therefore, we have raised a question of HDAC involvement in Bdnf repositioning observed after neuronal stimulation (Fig 4). Preincubation of hippocampal cultures with Trichostatin A, which is a commonly used HDAC paninhibitor, slightly increased Bdnf transcription (Fig 4A) and was sufficient to induce Bdnf allele repositioning toward the nuclear center (Figs 4B–4D and S3). The percentage of Bdnf alleles localized at the nuclear periphery was significantly lower upon TSA treatment compared to the control group. Induction of cLTP after Trichostatin A treatment did not decrease the percentage of alleles located at the nuclear periphery, in comparison to cLTP or TSA alone. The preincubation with a more specific HDAC inhibitor- romidepsin (Fig 5), selective for HDAC1 and HDAC2 deacetylases had no effect on Bdnf transcription (Fig 5A) and induced Bdnf allele repositioning only upon neuronal stimulation (Figs 5B–5D and S4). These results show again the causal relation between transcriptional activation of Bdnf and Bdnf allele’s repositioning towards the nucleus center.

Fig 4. The inhibition of histone deacetylases induces Bdnf transcription and repositioning independently from the neuronal stimulation.

Fig 4

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(A) The graph shows the expression of Bdnf normalized to control in the hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL, blue bar) or picrotoxin, forskolin, and rolipram (cLTP, orange bar), incubated for 12 hours with TSA and 2 hours with DMSO (TSA, cyan bar) or picrotoxin, forskolin, and rolipram (TSA+cLTP, magenta bar). Kruskal-Wallis group comparison p<0.01, and the One-Sample T-test or Welch Two Sample t-test for pairwise comparison: * p<0.05, **p<0.01, *** p<0.001; error bars indicate standard error of the mean for 5 independent experiments (B) Representative pictures of the nuclei of hippocampal neurons treated as described above. Hoechst 3342 staining for chromatin is shown in greyscale and segmentation of FISH signals for the Bdnf gene are shown in magenta and cyan. (C) Percentages of nuclei with the minimum distance between the respective alleles and nucleus surface < 350 nm are shown (Chi-square test, all groups, p<0.01; Fisher’s exact tests * p<0.05, *** p<0.001, error bars indicate standard deviation of the binomial distribution). (D) Quantitative analysis of the intracellular positions of Bdnf alleles in the nuclei of hippocampal neurons treated and color-coded as in A. The minimal distance between the respective alleles and nucleus surface is presented in the normalized histogram.

Fig 5. The inhibition of HDAC1/2 is not sufficient to induce Bdnf transcription and repositioning.

Fig 5

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(A) The graph shows the expression of Bdnf relative to Gapdh (normalized to control Bdnf level) in the hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL, blue bar) or picrotoxin, forskolin, and rolipram (cLTP, orange bar), incubated for 2 hours with 250 nM romidepsin and 2 hours with DMSO (romidepsin, light blue bar) or picrotoxin, forskolin, and rolipram (romidepsin+cLTP, violet bar). Kruskal-Wallis test for group comparison: p<0.01, One-Sample t-test or Welch two sample t-test for pairwise comparison: * p<0.05, *** p<0.001; error bars indicate standard error of the mean for 3 independent experiments (B) Representative picture of the nuclei of hippocampal neurons treated as described above. Hoechst 3342 staining for chromatin is shown in greyscale and segmentation of FISH signals for the Bdnf gene are shown in magenta and cyan. (C) Percentages of nuclei with the minimum distance between the respective alleles and nucleus surface < 350 nm are shown (Chi-square test, all groups, p<0.0001; Fisher’s exact tests: ** p<0.01, *** p<0.001, error bars indicate standard deviation of the binomial distribution). (D) Quantitative analysis of the intracellular positions of Bdnf alleles in the nuclei of hippocampal neurons treated and color-coded as in A. The minimal distance between the respective alleles and nucleus surface is presented in the normalized histogram.

Discussion

The three-dimensional organization of chromatin in the cell nucleus exhibits a higher-order regulation of gene expression [50–52]. The role of the nuclear lamina as a compartment regulating transcriptional activity is already quite well explored [53–55]. However, the knowledge of particular mechanisms, which are responsible for driving detachment of the genes from the nuclear lamina, is still vague [56].

To study the relationship between Bdnf repositioning, and morphological and clinical epileptic traits, we used the kainate model of epilepsy. This particular animal model was chosen because it resembles morphological traits of TLE such as mossy fiber sprouting in the dentate gyrus and neurodegeneration in CA1 and 3 regions of the hippocampus [57,58]. Further, to study mechanisms of Bdnf allele repositioning, we used in vitro model of neuronal stimulation. This model was chosen to avoid problems with the distribution of the Actinomycin D [59] through the blood-brain barrier and to decrease the number of sacrificed animals. Transcriptomic studies by Dabrowski and collaborators showed that the expression pattern in excited neurons in vivo is sustained in in vitro model [60]. Moreover, Szepesi and collaborators (2013) [43] showed that the induction of excitation by rolipram, forskolin, and picrotoxin induced a formation of dendritic spine protrusions, which resembled in vivo formation of new synapses.

The nuclear lamina is known as a transcriptionally repressive nuclear compartment [61]. Williams et al. (2007) [62] and Peric-Hupces et al. (2010) [63] showed a rearrangement of interactions between chromatin and the nuclear lamina, and an association of such phenomenon with transcription during differentiation of embryonic stem cells into neurons. However, the number of reports investigating a relationship between the rearrangement of chromatin architecture and transcription in fully differentiated neurons is very limited. In the presented study, using in vitro model, we confirmed our previous finding [34], where we showed that after neuronal excitation Bdnf alleles had repositioned toward the nuclear center. However, in comparison to the aforementioned paper, in the in vitro model, the percentage of alleles localized at the nuclear periphery was higher in the control cells, and the difference to stimulated neurons was smaller than in in vivo studies. Such discrepancy might be a result of a more uniform environment in cell culture, compared to in vivo situations, where spontaneous neuronal activity is present. Additionally, the kainate treatment, used in the in vivo studies, induces much stronger neuronal excitation than chemically induced LTP applied in vitro. In the presented study we showed that inhibition of transcription prevents Bdnf repositioning in neuronal nuclei after stimulation. It suggests that depolarization of the neuronal membrane is not sufficient for the detachment of Bdnf alleles, but the transcriptional activity itself is necessary for full repositioning. Our finding is consistent with the expertise presented by Crepaldi and colleagues (2013), who showed that in cultured cortical neurons depolarization induced by KCl stimulation evoked repositioning of activity-induced genes, including Bdnf, into transcription factories [33]. Moreover, this study showed that repositioning is controlled by transcription factor TFIIIC. Our current finding complements the previous results [34], where we showed the association of Bdnf repositioning and changes in transcription level during epileptogenesis. Both of those studies support the hypothesis that Bdnf allele repositioning acts as a kind of molecular memory of the cell to prepare neurons for future activation. The finding is also in agreement with the report by Ito and colleagues (2014) showing, that loss of three-dimensional architecture in neuronal nuclei leads to impaired transcription of several genes [64].

Surely, the presented mechanism is not the only one involved in the Bdnf repositioning. Presumably, several pathways must be orchestrated to activate Bdnf transcription and detachment of gene from the nuclear lamina, as well as its reposition toward the nuclear center. One possible mechanism may involve cohesins, which are well-known genome organizers [65] (and references therein). In Cornelia de Lange Syndrome, which belongs to cohesinopathies and is associated with epileptic seizures [66,67] acetylation of cohesins is impaired due to mutation in the gene encoding histone deacetylase HDAC8 [68]. Our results obtained with the use of inhibitors of histone deacetylases speak for this scenario. It has been shown that HDAC inhibitors induce hyperacetylation of more than 1700 proteins, which included chromatin remodelers, transcription factors, and protein kinases [69]. The trichostatin A- less specific but potent HDAC inhibitor, induced Bdnf transcription and repositioning even in the absence of neuronal activation. On the contrary, the more selective romidepsin, which inhibits mainly HDAC1 and 2, had no impact on both processes in silent neurons and upon stimulation. The cohesin-dependent mechanism may be at least in part responsible for Bdnf transcriptional activation, detachment, and repositioning, as CTCF, which acts in concert with cohesins [70] was shown to regulate the transcription of Bdnf [71].

Also, transcription factors such as Serum Response Factor (SRF) seem to be good candidates responsible for Bdnf repositioning. It is known that SRF can regulate Bdnf transcription [72] and its deletion leads to increased epileptogenesis and differential expression of more than 370 activity-induced genes including reduced transcription of Bdnf [73]. The disturbed balance of expression of inhibitory/excitatory regulators may contribute to the increased epileptogenesis, which was manifested by more frequent and acute spontaneous seizures in KO animals.

Furthermore, it was shown, that histone acetylation at the promoter region is necessary for activation of gene expression [74]. Histone deacetylases reverse this process and are taking part in creating a repressive zone by the nuclear lamina [75]. In a presented study we showed, that inhibition of histone deacetylases with paninhibitor Trichostatnin A leads to Bdnf transcription and repositioning. Similar activation of Bdnf gene transcription upon TSA treatment was shown for the Hek293 cell line [76]. Also, HDACs’ involvement in Bdnf expression is well established [28,48,77–80]. However, the exact mechanisms of HDACs’ participation in the higher-order mechanisms of transcriptional regulation of BDNF are unknown. One possible mechanism might involve Methyl-CpG-Binding protein 2 (MeCP2). Activation of neurons leads to the decreased CpG methylation of the Bdnf promoter region and dissociation of the MeCP2-mSin3A-HDAC1 silencing complex [25]. Besides, MeCP2, which expression is reduced upon TSA treatment [81], binds to the inner nuclear membrane lamin B receptor [82]. Therefore the association of transcriptionally inactive Bdnf to the nuclear lamina and its detachment upon TSA treatment may be dependent on MeCP2 protein. Mutations of the MeCP2 encoding gene are responsible for most cases of Rett Syndrome, a neurodevelopmental disorder, in which patients develop seizures [83].

Additionally, the process of Bdnf repositioning might involve actin-based molecular motors, since studies by Serebryannyy et al. (2016) [84] reported that actin regulates the function of HDAC1 and HDAC2 and also can be involved in gene expression by association with RNA polymerase II.

Finally, in the presented study we attempted to investigate the involvement of Bdnf repositioning in the pathogenesis of TLE in rats. We observed that the percentage of nuclei with the Bdnf allele at the nuclear periphery is negatively correlated with the intensity of mossy fiber sprouting. This observation is consistent with an idea that BDNF takes part in sprouting events in epilepsy [17,18]. In addition the intensity of sprouting correlated positively with the increased level of aggression, which is one of the behavioral traits typical for TLE [85]. The Actinomycin D, which has been already used in treatments of several types of cancer [86–88], has blocked Bdnf repositioning. However, the potential use of Actinomycin D for the treatment of neurological disorders including epilepsy would be challenging due to difficulties of its distribution through the blood-brain barrier [59]. Taking into account all the results together, the presented study supports the idea of Simonato [89], that Bdnf can be a very good target for novel anti-epileptic therapies.

Conclusively, the presented results are consistent with the current trend in research on the pathogenesis of epilepsy and show that neuronal cell nuclei are interesting targets to search for mechanisms of epileptogenesis.

Supporting information

S1 Fig. The representative images of mossy fiber sprouting and Bdnf alleles positions within neuronal nuclei in the TLE model.

Mossy fiber sprouting was verified by immunofluorescent staining for synaptoporin (left panels, depicted in green) in the molecular layer of DG region of the hippocampus. Representative pictures from animals at 4 weeks after administration of kainate are shown. The right panels show images of nuclei of granular neurons acquired from the same animals. Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

(TIF)

Click here for additional data file. (4MB, tif)
S2 Fig. The causal relationship between Bdnf transcriptional activity and Bdnf’s allele repositioning.

The representative pictures of the nuclei of hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL) or picrotoxin, forskolin, and rolipram (cLTP), incubated for 2 hours with Actinomycin D and 2 hours with DMSO (ActD) or picrotoxin, forskolin, and rolipram (ActD+cLTP). Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

(TIF)

Click here for additional data file. (1.3MB, tif)
S3 Fig. The inhibition of histone deacetylases induces Bdnf repositioning independently from the neuronal stimulation.

The representative pictures of the nuclei of hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL) or picrotoxin, forskolin, and rolipram (cLTP), incubated for 12 hours with Trichostatin A and 2 hours with DMSO (TSA) or picrotoxin, forskolin, and rolipram (cLTP TSA). Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

(TIF)

Click here for additional data file. (1.3MB, tif)
S4 Fig. The inhibition of HDAC1/2 is not sufficient to induce Bdnf repositioning.

The representative pictures of the nuclei of hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL) or picrotoxin, forskolin, and rolipram (cLTP), incubated for 2 hours with romidepsin and 2 hours with DMSO (romidepsin) or picrotoxin, forskolin, and rolipram (cLTP romidepsin). Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

(TIF)

Click here for additional data file. (1.3MB, tif)
S1 File. Clinical correlates and statistical analysis for Figs 1–5.

(XLSX)

Click here for additional data file. (47.2KB, xlsx)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

JD was supported by the Polish National Science Centre grant No 2015/17/B/NZ4/02540, AS was supported by the Polish National Science Centre grant No 2018/29/B/NZ4/01473, AM was supported by the Polish National Science Centre grant No UMO-2015/18/E/ NZ3/00730. AAS was supported by the Polish National Science Centre grant No 2014/15/N/NZ3/04468. KKP was partially supported by the ETIUDA grant from the Polish National Science Centre no. UMO-2019/32/T/NZ4/00502. https://www.ncn.gov.pl The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Michal Hetman

5 Nov 2020

PONE-D-20-26926

An important role of the interplay between Bdnf transcription and histone acetylation in epileptogenesis

PLOS ONE

Dear Dr. Magalska,

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.

The reviewers have raised several important points, all of which should be addressed in your revision. I agree with the reviewers that the most critical issues include: 

1. providing adequate images to support all the conclusions

2. using more specific HDAC inhibition strategies than TSA

3. carefully revising/editing the  text to make sure it accurately describes the data/conclusions

5. providing sufficient methodological details on quantitations and statistics. 

     

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We look forward to receiving your revised manuscript.

Kind regards,

Michal Hetman

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

3. 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: Yes

Reviewer #2: Yes

**********

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

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. 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: Magalska et al. aim to uncover if repositioning of Bdnf alleles from the nuclear periphery to the nuclear center after chemically stimulation in rat neurons is a consequence of or cause of transcriptional activation of the Bdnf gene. Two methods of stimulation were used: an in vivo approach where kainate-induced seizures were used as a validated method to induce neuronal Bdnf transcription and an in vitro method of cLTP in primary rat neurons which has also been shown to induce Bdnf transcription. Overall, the evidence presented is insufficient and sometimes disagrees with the authors’ conclusions. The authors are clearly capable of producing the necessary data and figures based upon their previous publication in this area of study however for this manuscript, evidence is lacking. Stronger evidence is needed that may include additional imaging in primary neurons and a more specific HDAC inhibitor coupled with isoform-specific genetic silencing to demonstrate a role for HDACs. In Figure 2, it is unclear why the authors did not provide the microscopy FISH images from which the conclusions in Figure 2 were drawn in the main text. Lastly, in Figure 3, the images used contradict the authors’ conclusion (see below). This is an interesting and potentially fruitful area of research and the authors may therefore be given the opportunity to extensively re-work this study.

Major Points:

1. Please provide additional images that were used for quantification in supplementary data for figures 2, 3, and 4.

2. No data is shown for the quantification Bdnf allele repositioning in response to kainate-induced epileptogenesis, only a final plot of correlation of mossy fiber sprouting with the allele repositioning. Readers are unable to see how you obtained these results (Figure 2).

3. Organization of text needs to be edited. For example, under the subheading Figure 1, authors mention panels in Figure 2. Please edit the flow of the text to be more logical by moving all subheadings for Results to be sequential in the first part of the results and grouping all figure legends at the end with the corresponding figure.

4. The paragraph following “The causal relationship between Bdnf allele transcriptional activity and repositioning”: these images do not support the authors’ conclusion that, “Presented results show that transcriptional activity is a cause of Bdnf repositioning.” While actinomycin D inhibited transcription of Bdnf, repositioning of the Bdnf allele away from the periphery and to the center of the nucleus still occurs in its presence according to the representative images used. Please provide additional images used for quantification; the ones currently used are confusing and do not support the authors’ argument. The authors report “Inhibition of transcription completely blocked

5. Bdnf repositioning upon cLTP treatment (Fig 3B-D).” which I can’t see from these images as one allele is clearly located away from the periphery, identical to the cLTP alone treatment.

6. Please justify the use of Welch’s ANOVA for non homogenous variance by providing the statistics readouts from GraphPad Prism in supplemental data.

Minor Points:

1. Page 3: Revise “It is known that 3’ of the protein-coding exon is spliced to one of the eight of 5’ untranslated exons.” Doesn’t make sense, missing a word.

2. Figure 2B: 25.5% should be 25%

3. Figure 3D and 4D: please plot raw values with error bars. The way the data is currently presented does not account for sample size.

4. Page 11: Revise “The role of the nuclear lamina as a transcriptional activity regulating the compartment is already quite well explored (52-54).”

5. Please continue to revise overall grammar and sentence structure, especially in the discussion.

Reviewer #2: In this manuscript, the authors focused on their previous findings regarding the intranuclear repositioning of BDNF allele induced by kainate-induced neuronal activation. The authors investigated the correlation between the intranuclear repositioning of BDNF allele and clinical/morphological traits of kainate-induced epilepsy in particular. Furthermore, the authors used in vitro hippocampal cultures to investigate mechanisms of the repositioning of BDNF allele. Although this is an interesting work showing the correlation between higher-order epigenetic regulation of BDNF gene and epileptogenesis, several concerns lessen my enthusiasm for this paper. I listed my major points below.

1. The title of this manuscript is unlikely to represent this work appropriately. I think that one of the important points in this manuscript would be the interplay between the repositioning of BDNF allele and the seizure status. The title should be revised in order to represent this work, if possible.

2. In Figure 1A, the intensity of seizures was assessed by modified 6-grade Racine’s scale (from 0 to 5-grade). However, only 2 grades (white (5-grade?) and gray (2-grade?)) were shown in a pie chart (Fig. 1A). Is it correct? This pie chart seems not to reflect the data shown in Table 1, and therefore, the chart should be explained carefully.

3. The authors estimated the extend of mossy fiber sprouting that was based on the intensity of immunostaining with an anti-synaptoporin antibody. However, I could not understand how the extend of mossy fiber sprouting was classified into 5-grade scale (from level 0 to 4). The quantification method should be described.

4. I think that the result regarding the negative correlation between BDNF allele at the nuclear periphery and level of mossy fiber sprouting (Fig. 2C) is quite interesting. Is a similar correlation between BDNF allele at the nuclear periphery and intensity of seizures (assessed by modified 6-grade Racine’s scale) observed? And also, is a similar correlation between level of mossy fiber sprouting and intensity of seizures observed?

5. I think that the representative images shown in Figure 3B do not reflect the data shown in Figure 3C and D. Namely, the images seem to indicate that actinomycin D reduced the percentage of BDNF allele at the nuclear periphery in both control and cLTP-induced neurons. Furthermore, the images shown in Figure 3B closely resemble the images shown in Figure 4B (almost the same). The authors should revise the manuscript very carefully.

6. The authors show that HDAC inhibition was sufficient for the repositioning of BDNF allele toward the nuclear center, and they described the possibility of the involvement of HDAC1, 2, and 8, in the intranuclear repositioning of BDNF allele. In this work, the authors used TSA to inhibit HDACs. However, TSA is a pan-HDAC inhibitor. The authors’ proposal would be strengthened if they use class-specific HDAC inhibitors.

(Minor points)

1. How long were the cells treated with TSA? (In Method section, the cells were treated with TSA for 12 h. However, in Results section, the cells were treated with TSA for 2 h. Which is correct time?)

2. In Results section (page 8), “Here we observed, that the level of sprouting correlated...” should be “...the level of sprouting negatively correlated...”.

3. In Discussion section, the authors mentioned that the percentage of BDNF allele localized at the nuclear periphery in control hippocampal cells was higher than that in vivo model. They discuss that “Such discrepancy might be a result of a more uniform environment in cell culture compared to in vivo situation”. Spontaneous neuronal activity in vivo situation would be stronger than that in vitro cultured neurons, and this may be another possible reason.

4. In Discussion section, the authors mentioned that SRF may be a candidate factor responsible for the repositioning of BDNF allele. However, SRF deletion in mice leads to increased epileptogenesis, despite decreased expression of BDNF gene is observed in the SRF-deleted mice brain. The results obtained using SRF-deleted mice seem to contradict a series of previous reports showing that BDNF is involved in seizures.

5. In the end of Discussion section, the authors described that “HDAC activity might be a good target for the treatment of TLE”. Does it mean that HDAC activation might be beneficial for the treatment of TLE? I think that this should be clarified more.

6. In Figures 3D and 4D, error bars should be added.

**********

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Reviewer #1: Yes: Claes Wahlestedt

Reviewer #2: No

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PLoS One. 2021 Jun 4;16(6):e0239111. doi: 10.1371/journal.pone.0239111.r002

Author response to Decision Letter 0

5 May 2021

We are very grateful for all the comments and suggestions, which helped us improve our paper. We have changed the main text of manuscript accordingly.

Our detailed response to the editor's and reviewers’ comments is as follows:

Editor:

1. providing adequate images to support all the conclusions

The mistaken images were corrected and additional figures are presented in the supplementary materials.

2. using more specific HDAC inhibition strategies than TSA

Following the Editor’s and Reviewer’s comments, we have performed additional in vitro experiments with the use of romidepsin, a selective inhibitor of HDAC1 and 2. The results are presented in Fig.5 and Supplementary Fig.S5. For more details please see the response to the Rev.1 general comment and Rev.2 comment 6.

3. carefully revising/editing the text to make sure it accurately describes the data/conclusions

The text has been corrected accordingly.

5. providing sufficient methodological details on quantitations and statistics.

The detailed description of quantification and statistical analysis was included in the supplementary S1 file and in the Methods section.

Reviewer 1:

Magalska et al. aim to uncover if repositioning of Bdnf alleles from the nuclear periphery to the nuclear center after chemically stimulation in rat neurons is a consequence of or cause of transcriptional activation of the Bdnf gene. Two methods of stimulation were used: an in vivo approach where kainate-induced seizures were used as a validated method to induce neuronal Bdnf transcription and an in vitro method of cLTP in primary rat neurons which has also been shown to induce Bdnf transcription. Overall, the evidence presented is insufficient and sometimes disagrees with the authors’ conclusions. The authors are clearly capable of producing the necessary data and figures based upon their previous publication in this area of study however for this manuscript, evidence is lacking.

Stronger evidence is needed that may include additional imaging in primary neurons and a more specific HDAC inhibitor coupled with isoform-specific genetic silencing to demonstrate a role for HDACs. In Figure 2, it is unclear why the authors did not provide the microscopy FISH images from which the conclusions in Figure 2 were drawn in the main text. Lastly, in Figure 3, the images used contradict the authors’ conclusion (see below). This is an interesting and potentially fruitful area of research and the authors may therefore be given the opportunity to extensively re-work this study.

In agreement with the Reviewer’s comment, we performed additional in vitro experiments with the use of romidepsin which is an inhibitor of HDAC 1 and 2. The results are presented in Fig.5 and S5. Appropriate fragments are inserted into the Methods (line 130), the Results (lines 290-293), the Figures’ Legends (lines 314-329 and 682-687) and the Discussion (line 386-387) sections. Please find response to all other concerns below.

Major Concerns

1. Please provide additional images that were used for quantification in supplementary data for figures 2, 3, and 4.

Addressing the reviewer’s comment, we have provided additional images in Supplementary figures S2, S3, S4 and S5, describing experimental results shown on Figures 2, 3, 4, and a new Fig. 5 respectively.

2. No data is shown for the quantification Bdnf allele repositioning in response to kainate-induced epileptogenesis, only a final plot of correlation of mossy fiber sprouting with the allele repositioning. Readers are unable to see how you obtained these results (Figure 2).

Following the Reviewer’s remark, we have included the data used for the quantification of correlation in the Supplementary S1 file. The distance of BDNF alleles from the nuclear border was measured on 3D confocal images with the use of Segmentation Magic software, developed in our Laboratory (for further details please read: Ruszczycki, B.; Pels, K.K.; Walczak, A.; Zamłyńska, K.; Such, M.; Szczepankiewicz, A.A.; Hall, M.H.; Magalska, A.; Magnowska, M.; Wolny, A.; et al. Three-Dimensional Segmentation and Reconstruction of Neuronal Nuclei in Confocal Microscopic Images. Front. Neuroanat. 2019, 13, 81, doi:10.3389/fnana.2019.00081.).

The level of sprouting was measured using Image J, defined as a mean fluorescence intensity of synaptoporin immunostaining of the same region of interest in the molecular layer of dentate gyrus. Then, the intensity was ranked from 0 (minimal staining comparable with the untreated animals) to 4- the highest intensity in KA treated animals. The results are presented in the Table 1 (shown also below) and in the supplementary S1 file.

Animal ID selected area in pixels the mean synaptoporin intensity the sprouting level

1B 10207,201 10,127 2

2B 10009,387 11,571 2

1G 9940,673 30,554 4

2G 10325,890 16,044 3

3G 10197,368 4,345 0

4G 10523,704 25,694 4

1C 10276,725 4,198 0

2C 10506,294 5,151 1

3R 9447,584 5,674 1

4R 10484,141 5,444 1

And intensity scores:

The mean synaptoporin intensity scale (0-255 grey scale) The level of sprouting

0 to 4 0

5 to 9 1

10 to 15 2

16 to 20 3

above 21 4

Additionally two independent observers estimated sprouting visually in the same brain area directly using a microscope. The sprouting scores and correlation scores are included in supplementary S1 file.

In the Supplementary Figure S2, we have included the representative images for each analyzed animal of the synaptoporin immunostaining and BDNF alleles positioning within the cell nuclei.

3. Organization of text needs to be edited. For example, under the subheading Figure 1, authors mention panels in Figure 2. Please edit the flow of the text to be more logical by moving all subheadings for Results to be sequential in the first part of the results and grouping all figure legends at the end with the corresponding figure.

The text was corrected following the Reviewer’s comment.

4. The paragraph following “The causal relationship between Bdnf allele transcriptional activity and repositioning”: these images do not support the authors’ conclusion that, “Presented results show that transcriptional activity is a cause of Bdnf repositioning.” While actinomycin D inhibited transcription of Bdnf, repositioning of the Bdnf allele away from the periphery and to the center of the nucleus still occurs in its presence according to the representative images used. Please provide additional images used for quantification; the ones currently used are confusing and do not support the authors’ argument. The authors report “Inhibition of transcription completely blocked

And

5. Bdnf repositioning upon cLTP treatment (Fig 3B-D).” which I can’t see from these images as one allele is clearly located away from the periphery, identical to the cLTP alone treatment.

The images representing localization of BDNF alleles in cell nuclei were swapped by mistake. Responding to the Reviewer’s comment, we have corrected the figures by introducing proper images. Additionally, in the Supplementary Figures S3-S5, we have included more representative images of nuclei used for the calculations, for all the treatments.

6. Please justify the use of Welch’s ANOVA for non homogenous variance by providing the statistics readouts from GraphPad Prism in supplemental data.

In the previous version of the manuscript, we used Welch’s ANOVA statistical analysis, since the variances calculated with Bartlet test were not homogenous. In the present version of the manuscript improved, after addressing Reviewer’s remarks, we have included new data for more specific histone deacetylases inhibitor, romidepsin. We decided to normalize data to the control (DMSO treatment) to compare the obtained data from the all in vitro experiments. Therefore, we used Kruskal-Wallis test for group comparisons and One Sample T-test for comparison whether the normalized value differs from the unity, or Welch Two sample T-test for pairwise comparisons between treatment groups. All raw data and results of statistical analysis are shown in supplementary S1 file.

Minor Points:

7. Page 3: Revise “It is known that 3’ of the protein-coding exon is spliced to one of the eight of 5’ untranslated exons.” Doesn’t make sense, missing a word.

The text was corrected to: It is known that 3’ end of the protein-coding exon is spliced to one of the eight of 5’ exons, which are controlled by their unique promoters (20-22).

8. Figure 2B: 25.5% should be 25%

To fulfill the Reviewer’s comments, we have reanalyzed the data obtained from the KA treated animals and increased the number of animals to 10. Therefore all the percentages had changed.

9. Figure 3D and 4D: please plot raw values with error bars. The way the data is currently presented does not account for sample size.

Addressing the Reviewer’s remark, we introduced the error bars in the Figures 2,3-4 and in the new Fig.5. All raw data used for statistical analysis are shown in supplementary S1 file.

10. Page 11: Revise “The role of the nuclear lamina as a transcriptional activity regulating the compartment is already quite well explored (52-54).”

We corrected the sentence to: The role of the nuclear lamina as a compartment regulating the transcriptional activity is already quite well explored (52-54).

11. Please continue to revise overall grammar and sentence structure, especially in the discussion.

Following the Reviewer’s suggestion, the text was subjected to extensive language corrections.

Reviewer #2:

In this manuscript, the authors focused on their previous findings regarding the intranuclear repositioning of BDNF allele induced by kainate-induced neuronal activation. The authors investigated the correlation between the intranuclear repositioning of BDNF allele and clinical/morphological traits of kainate-induced epilepsy in particular. Furthermore, the authors used in vitro hippocampal cultures to investigate mechanisms of the repositioning of BDNF allele. Although this is an interesting work showing the correlation between higher-order epigenetic regulation of BDNF gene and epileptogenesis, several concerns lessen my enthusiasm for this paper. I listed my major points below.

Major Points

1. The title of this manuscript is unlikely to represent this work appropriately. I think that one of the important points in this manuscript would be the interplay between the repositioning of BDNF allele and the seizure status. The title should be revised in order to represent this work, if possible.

In agreement with the Reviewer’s comment, we modified the title to: “The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy”

2. In Figure 1A, the intensity of seizures was assessed by modified 6-grade Racine’s scale (from 0 to 5-grade). However, only 2 grades (white (5-grade?) and gray (2-grade?)) were shown in a pie chart (Fig. 1A). Is it correct? This pie chart seems not to reflect the data shown in Table 1, and therefore, the chart should be explained carefully.

Addressing the Reviewer’s comments, we have revised the results carefully. We have repeated the synaptoporin immunostaining and included animals 2C and 4G, previously excluded from the analysis because of technical problems with synaptoporin staining. The animals observed in our kainate experiments developed seizures of grade 2, 4 and 5, in the agreement with the data presented below, Table 1 and supplementary S1 file.

Animal ID seizures level

1B I blue 2

1C I black 5

1G I green 5

2B II blue 5

2C II black 4

2G II green 5

3G III green 2

3R III red 5

4G IV green 5

4R IV red 2

3. The authors estimated the extend of mossy fiber sprouting that was based on the intensity of immunostaining with an anti-synaptoporin antibody. However, I could not understand how the extend of mossy fiber sprouting was classified into 5-grade scale (from level 0 to 4). The quantification method should be described.

The intensity of immunostaining was calculated with the use of ImageJ. Then, the mean fluorescence intensity was assessed using 5-grade scale, describing the level of sprouting according to the intervals presented in the following Tables, that were also included in the supplementary S1 file

The mean synaptoporin intensity scale (0-255 grey scale) The level of sprouting

0 to 4 0

5 to 9 1

10 to 15 2

16 to 20 3

above 21 4

Animal ID selected area in pixels the mean synaptoporin intensity the sprouting level

1B 10207,201 10,127 2

2B 10009,387 11,571 2

1G 9940,673 30,554 4

2G 10325,890 16,044 3

3G 10197,368 4,345 0

4G 10523,704 25,694 4

1C 10276,725 4,198 0

2C 10506,294 5,151 1

3R 9447,584 5,674 1

4R 10484,141 5,444 1

Additionally, two independent observers estimated sprouting visually in the same brain area directly using a microscope. The sprouting scores and correlation scores are included in supplementary S1 file.

4. I think that the result regarding the negative correlation between BDNF allele at the nuclear periphery and level of mossy fiber sprouting (Fig. 2C) is quite interesting. Is a similar correlation between BDNF allele at the nuclear periphery and intensity of seizures (assessed by modified 6-grade Racine’s scale) observed? And also, is a similar correlation between level of mossy fiber sprouting and intensity of seizures observed?

We have calculated correlations suggested by the Reviewer including additional animals and newly obtained data. The calculated Pearson’s correlations are enlisted below and included in supplementary S1 file. We have found one additional significant positive correlation between the level of sprouting and aggression. The increase in the aggressive behavior has frequently been reported in association with the development of temporal lobe epilepsy. However no significant correlation was found between the intensity of initial seizures and sprouting or seizure and the percentage of nuclei with the Bdnf alleles localized on the nuclear periphery. We decided to show correlations for sprouting level measured with Image J and estimated subjectively by two independent observers as obtained results were very similar.

Correlation: The sprouting level Image J vs % of nuclei with Bdnf loci on the perifery

(Pearson R= -0.6747943402484425, p-value= 0.03230502682762817)

Correlation: The subjective Sprouting vs % of nuclei with Bdnf loci on the perifery

(Pearson R= -0.7086823744896321, p-value= 0.02177595135907097)

Correlation: The Sprouting level Image J vs agression

(Pearson R=0.7460317460317462, p-value= 0.01321972976110183

Correlation: % of nuclei with Bdnf loci on the perifery vs Seizures

(Pearson R= -0.5272596866202862, p-value= 0.11731880152677869)

Correlation: The sprouting level vs Seizures

(Pearson R= 0.4259177099999599, p-value= 0.2197183529541219)

5. I think that the representative images shown in Figure 3B do not reflect the data shown in Figure 3C and D. Namely, the images seem to indicate that actinomycin D reduced the percentage of BDNF allele at the nuclear periphery in both control and cLTP-induced neurons. Furthermore, the images shown in Figure 3B closely resemble the images shown in Figure 4B (almost the same). The authors should revise the manuscript very carefully.

The images representing localization of BDNF alleles in cell nuclei were swapped by mistake. In agreement with the Reviewer’s comment, we corrected the figures by introducing proper images. Additionally, we have included more representative images of nuclei used for the calculations in supplementary figures S3 and S4.

6. The authors show that HDAC inhibition was sufficient for the repositioning of BDNF allele toward the nuclear center, and they described the possibility of the involvement of HDAC1, 2, and 8, in the intranuclear repositioning of BDNF allele. In this work, the authors used TSA to inhibit HDACs. However, TSA is a pan-HDAC inhibitor. The authors’ proposal would be strengthened if they use class-specific HDAC inhibitors.

In agreement with the Reviewer’s criticism, we performed additional in vitro experiments with the use of romidepsin which is an inhibitor of HDAC 1 and 2. The results are presented in Fig.5 and S5. Appropriate fragments are inserted into the Methods (line 130), the Results (lines 290-293), the Figures’ Legends (lines 314-329 and 682-687) and the Discussion (line 386-387) sections.

(Minor points)1. How long were the cells treated with TSA? (In Method section, the cells were treated with TSA for 12 h. However, in Results section, the cells were treated with TSA for 2 h. Which is correct time?)

Cells were incubated with TSA for 12 h. The mistake in the text was corrected.

1. In Results section (page 8), “Here we observed, that the level of sprouting correlated...” should be “...the level of sprouting negatively correlated...”.

The text was corrected to: “Here we observed, that the level of sprouting negatively correlated (R= -0.67, Pearson correlation) with the percentage of the nuclei with Bdnf allele localized at the nuclear periphery (Fig. 2 C).”

2. In Discussion section, the authors mentioned that the percentage of BDNF allele localized at the nuclear periphery in control hippocampal cells was higher than that in vivo model. They discuss that “Such discrepancy might be a result of a more uniform environment in cell culture compared to in vivo situation”. Spontaneous neuronal activity in vivo situation would be stronger than that in vitro cultured neurons, and this may be another possible reason.

Thank you for a great comment, We have included it in the text, which now sounds:

“Such discrepancy might be a result of more uniform environment in cell culture, compared to in vivo situation, where spontaneous neuronal activity is present.”

3. In Discussion section, the authors mentioned that SRF may be a candidate factor responsible for the repositioning of BDNF allele. However, SRF deletion in mice leads to increased epileptogenesis, despite decreased expression of BDNF gene is observed in the SRF-deleted mice brain. The results obtained using SRF-deleted mice seem to contradict a series of previous reports showing that BDNF is involved in seizures.

Thank you for this interesting comment. In the mentioned publication of Kuzniewska et al. SRF deletion induced differential expression of more than 370 activity-induced genes. These included regulators of both inhibitory and excitatory pathways. Therefore the increased epileptogenesis is rather a result of an imbalance between the inhibitory and excitatory pathways, than a reduced level of BDNF observed in SRF KO animals.

We have changed this part of the Discussion to explain better the results obtained by Kuzniewska et al.

5. In the end of Discussion section, the authors described that “HDAC activity might be a good target for the treatment of TLE”. Does it mean that HDAC activation might be beneficial for the treatment of TLE? I think that this should be clarified more.

We have removed this statement from the Discussion as our experiments with a selective inhibitor of HDAC1 and 2 showed no change in Bdnf transcription and positioning neither in silent nor activated neurons. The trichostatin A, which inhibits a broad spectrum of HDACs might induce Bdnf transcription and repositioning via chromatin silencer- MeCP2, which reduced levels are observed upon TSA treatment. We have included this possibility in the Discussion.

6. In Figures 3D and 4D, error bars should be added.

Following the Reviewer’s remark, we introduced the error bars in the figures 3, 4 and in the new Fig.5. All data used for statistical analysis are shown in supplementary S1 file.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file. (39.9KB, docx)

Decision Letter 1

Michal Hetman

18 May 2021

The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy

PONE-D-20-26926R1

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Acceptance letter

Michal Hetman

24 May 2021

PONE-D-20-26926R1

The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy

Dear Dr. Magalska:

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. The representative images of mossy fiber sprouting and Bdnf alleles positions within neuronal nuclei in the TLE model.

    Mossy fiber sprouting was verified by immunofluorescent staining for synaptoporin (left panels, depicted in green) in the molecular layer of DG region of the hippocampus. Representative pictures from animals at 4 weeks after administration of kainate are shown. The right panels show images of nuclei of granular neurons acquired from the same animals. Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

    (TIF)

    Click here for additional data file. (4MB, tif)
    S2 Fig. The causal relationship between Bdnf transcriptional activity and Bdnf’s allele repositioning.

    The representative pictures of the nuclei of hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL) or picrotoxin, forskolin, and rolipram (cLTP), incubated for 2 hours with Actinomycin D and 2 hours with DMSO (ActD) or picrotoxin, forskolin, and rolipram (ActD+cLTP). Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

    (TIF)

    Click here for additional data file. (1.3MB, tif)
    S3 Fig. The inhibition of histone deacetylases induces Bdnf repositioning independently from the neuronal stimulation.

    The representative pictures of the nuclei of hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL) or picrotoxin, forskolin, and rolipram (cLTP), incubated for 12 hours with Trichostatin A and 2 hours with DMSO (TSA) or picrotoxin, forskolin, and rolipram (cLTP TSA). Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

    (TIF)

    Click here for additional data file. (1.3MB, tif)
    S4 Fig. The inhibition of HDAC1/2 is not sufficient to induce Bdnf repositioning.

    The representative pictures of the nuclei of hippocampal neurons incubated for 2 hours with DMSO vehicle (CTRL) or picrotoxin, forskolin, and rolipram (cLTP), incubated for 2 hours with romidepsin and 2 hours with DMSO (romidepsin) or picrotoxin, forskolin, and rolipram (cLTP romidepsin). Hoechst 3342 staining for chromatin is shown in a grey scale and segmentation of FISH signals for Bdnf gene are shown in magenta and cyan.

    (TIF)

    Click here for additional data file. (1.3MB, tif)
    S1 File. Clinical correlates and statistical analysis for Figs 1–5.

    (XLSX)

    Click here for additional data file. (47.2KB, xlsx)
    Attachment

    Submitted filename: Response to reviewers.docx

    Click here for additional data file. (39.9KB, docx)

    Data Availability Statement

    All relevant data are within the manuscript and its Supporting Information files.

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