{'fraction_non_alphanumeric': 0.03012446825271782, 'fraction_numerical': 0.016291161178509532, 'mean_word_length': 4.914275065225494, 'pattern_counts': {'":': 0, '<': 2, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Microglial phagocytosis dysfunction is related to local neuronal activity in a genetic model of epilepsy

Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response in neurodegenerative diseases. Phagocytosis is very efficient in physiological conditions, as well as during apoptotic challenge induced by excitotoxicity or inflammation, but is impaired in mouse and human mesial temporal lobe epilepsy (MTLE). Here we extend our studies to a genetic model of progressive myoclonus epilepsy type 1 (EPM1) in mice lacking cystatin B (CSTB), an inhibitor of cysteine proteases involved in lysosomal proteolysis. We first demonstrated that microglial phagocytosis was impaired in the hippocampus in Cstb knock-out (KO) mice when seizures arise and hippocampal atrophy begins, at 1 month of age. To test if this blockage was related to the lack of Cstb in microglia, we used an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression but we found no significant effect in the phagocytosis of apoptotic cells. We then tested whether seizures were involved in the phagocytosis impairment, similar to MTLE, and analyzed Cstb KO mice before seizures begin, at postnatal day 14. Here, phagocytosis impairment was restricted to the granule neuron layer but not to the subgranular zone, where there are no active neurons. Furthermore, we observed apoptotic cells (both phagocytosed and not phagocytosed) in Cstb deficient mice at close proximity to active, cFos+ neurons and used mathematical modeling to demonstrate that the physical relationship between apoptotic cells and cFos+ neurons was specific for Cstb KO mice. These results suggest a complex crosstalk between apoptosis, phagocytosis and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, this data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis.


INTRODUCTION
Progressive myoclonus epilepsy 1 (EPM1) or Unverricht-Lundborg disease is a genetic neurodegenerative disease characterized by disabling myoclonus, epileptic seizures and ataxia. The disease is caused by biallelic loss-of-function mutations in the cystatin B gene (CSTB) encoding the cystatin B protein 1-3 , a protease inhibitor that regulates and limits the activity of nuclear and cytoplasmic cysteine proteases known as cathepsins 4,5 . Cstb knock-out (KO) mice recapitulate key clinical features of EPM1 5,6 , as they develop seizures at one month and progressive ataxia around six months of age 7-10 , consistent with findings in humans. In addition, Cstb KO mice present early alterations in the inflammatory response of microglia, the brain immune cells [11][12][13] . In addition to controlling the release of inflammatory mediators, microglia are also the brain professional macrophages 14 . We have recently reported that in both mouse and human mesial temporal lobe epilepsy (MTLE), microglial phagocytosis of apoptotic cells is impaired 15 , leading us to question whether the phagocytosis impairment would also occur in Cstb KO mice.
Phagocytosis is a highly regulated response that prevents the spillover of cytotoxic content that results from the cell death, is immunomodulatory, and actively participates in maintaining tissue homeostasis 14,16,17 . In physiological conditions, microglia are very efficient phagocytes, as their processes constantly scan the brain parenchyma and express a plethora of receptors for "find-me" and "eat-me" signals produced by apoptotic cells [18][19][20][21] . Microglia are similarly efficient facing increased apoptotic cell numbers generated during excitotoxicity and inflammation, as they use several strategies to cope with increased apoptosis: 1) recruitment of more phagocytic cells, 2) increase the number of apoptotic cells phagocytosed per microglial cell, and/or 3) increase the microglial numbers 15,22 . As a consequence, microglial phagocytosis is proportional to the number of apoptotic cells, i.e. microglial phagocytosis is tightly coupled to apoptosis 15 . However, during MTLE the hyperactivity of the neuronal network and the massive release of the "find-me" signal ATP during seizures interfered with the recognition by microglia, resulting in delayed dead cell clearance and inflammation 15 .
To test whether microglial phagocytosis was affected in other forms of epilepsy, such as EPM1, we analyzed microglial phagocytosis efficiency in Cstb KO mice. Most of the CSTB deficiency -related pathological changes have been reported in the cerebellum and the cortex 7,9-11 but here we focused on the dentate gyrus (DG) of the hippocampus, in whose subgranular zone (SGZ) there is ongoing production of newborn neurons through adulthood 23 . Most of these newborn neurons naturally undergo apoptosis and are immediately phagocytosed by microglia, allowing us to establish the baseline phagocytosis efficiency 22,24 . We here show hippocampal atrophy and microglial phagocytosis impairment at the age when seizures arise, at postnatal day 30 (P30).
We then studied whether this effect was related to a cell-autonomous effect on microglia, using an in vitro model of phagocytosis and siRNA-mediated depletion; or was related to altered neuronal function, by studying presymptomatic mice at P14, before seizures arise. We found that in the neurogenic region, the SGZ, phagocytosis was spared; in contrast, phagocytosis was specifically impaired in the granule neuron cell layer. We finally used direct analysis and mathematical modeling to determine that apoptotic cells were in fact in close proximity to cFos + , activated neurons, in the absence of seizures. Our data suggests a complex environmental signaling in Cstb KO mice that results in granule cell layer-specific microglial phagocytosis impairment and point towards microglia as a potential target of novel treatments aimed at reducing neuronal damage in epilepsy patients.

BV2 cell line. BV2 cells (Interlab Cell Line Collection San Martino-Instituto Scientifico
Tumori-Instituto Nazionale per la Ricerca sul Cancro), a cell line derived from raf/mycimmortalized rat neonatal microglia was used to perform the in vitro knock-down model of Cstb. BV2 cells were grown as an adherent culture in non-coated 90mm 2 Petri dishes in the presence of 10 mL of culture medium. The culture medium consisted in high glucose Dulbecco's Modified Eagle's Medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, GE Healthcare Hyclone) and 1% antibioticantimycotic (Gibco). When confluence was reached, cells were trypsinized (Trypsin-EDTA 0,5% no phenol red, Gibco) and replated at 1:5.
Red Fluorescent SH-SY5Y Cell Line (Vampire SH-SY5Y). The Vampire SH-SY5Y cell line was developed as a stable transfection of the SH-SY5Y cell line with the red fluorophore tFP602 (InnoProt, P20303). Cells were grown as an adherent culture in non-coated T-75 flasks in the presence of 10 mL of culture medium. The culture medium consisted in high glucose DMEM supplemented with 10% fetal bovine serum (FBS, GE Healthcare Hyclone) 1% antibiotic-antimycotic (Gibco) and 0.25mg/ml Geneticin (G418, Gibco) to select the transfected cells. When confluence was reached, cells were trypsinized (Trypsin-EDTA 0,5% no phenol red, Gibco) and replated at 1:3.
In vitro phagocytosis assay. The protocol for in vitro phagocytosis was adapted from 27 . Briefly, BV2 cells were transfected and phagocytosis was performed after 24 hours of expression. Phagocytosis was performed in high glucose DMEM, supplemented 1% antibiotic-antimycotic (Gibco) and 10% fetal bovine serum (FBS, GE Healthcare Hyclone) to ensure the presence of the complement molecules, such as C1q, that have been related to phagocytosis in vivo 28 and determine the immunomodulatory outcome of phagocytosis 29 . BV2 cells, transfected with either Cstb or scrambled siRNA, were fed for 1 and 4h with Vampire SH-SY5Y, previously treated with staurosporine (STP; 3µM, 4 h; Sigma-Aldrich) to induce apoptosis. The floating fraction of the dead cells was collected and added to the BV2 cells in a 1:1 proportion after their visualization and quantification with trypan blue in a Neubauer chamber.
Immunofluorescence. Six series of 50µm-thick sagittal sections per brain were sectioned using a Leica VT 1200S vibrating blade microtome (Leica Microsystem GmbH, Wetzlar, Germany). Immunostaining was performed following standard procedures 22,27 . Free floating vibratome sections were blocked in permeabilization solution (0.3% Triton X-100, 0.5% BSA in PBS; all from Sigma-Aldrich) for 2 h at room temperature (RT) with gentle shaking, and incubated overnight with the primary antibodies diluted in the permeabilization solution at 4°C. After primary antibody incubation, sections were intensively washed with 0.3% Triton X-100 in PBS and then incubated with secondary antibodies and DAPI (5mg/mL, Sigma-Aldrich) diluted in permeabilization solution for 2h at RT. After washing with PBS, the sections were mounted on glass slides with Dako Cytomation Fluorescent Mounting Medium.
BV2 cells were fixed for 10 min at RT in 4% PFA and then transferred to PBS.
Fluorescent immunostaining was performed following standard procedures 27,15 .
Coverslips with BV2 cells were blocked in 0.2% Triton X-100, 0.5% BSA in PBS for 1h at RT. The cells were then incubated overnight with primary antibodies in permeabilization solution (0.2% Triton X-100, 0.5% BSA in PBS). After overnight incubation, cells were rinsed in PBS and incubated in the secondary antibodies containing DAPI (5 mg/ml) in the permeabilization solution for 1 h at RT. After washing with PBS, primary cultures were mounted on glass slides with Dako Cytomation Fluorescent Mounting Medium. Rodhamine Red X or AlexaFluor 647 were purchased from Jackson Laboratories. Image analysis. All fluorescence immunostaining images were collected using a Leica SP8 laser-scanning microscope using a 40x oil-immersion objective and a z-step of 0.7µm. All images were imported into the Fiji distribution of ImageJ 30 in Tiff format.
Brightness, contrast, and background were adjusted equally for the entire image using the "brightness and contrast" and "levels" controls from the "image/adjustment" set of options without any further modification. For tissue sections, two to three 20µm-thick zstacks of each section containing the septal hippocampus from one vibratome series were analyzed. For BV2 cell cultures, over 4 -5 z-stacks were obtained per coverslip.

Phagocytosis analysis in vivo and in vitro.
Apoptotic cells were defined based on their nuclear morphology after DAPI staining as cells in which the chromatin structure (euchromatin and heterochromatin) was lost and appeared condensed and/or fragmented (pyknosis/ karyorrhexis). Phagocytosis was defined as the formation of an enclosed, three-dimensional pouch of microglial processes surrounding an apoptotic cell 22 . In tissue sections, the number of apoptotic cells, phagocytosed cells and microglia were estimated using unbiased stereology in the volume of the DG contained in the z-stack (determined by multiplying the thickness of the stack by the area of the DG at the center of the stack using ImageJ, Fiji). To obtain the total numbers, this density value was then multiplied by the volume of the septal hippocampus (spanning from -1 to -2.5 mm in the AP axes, from bregma; 6 slices in each of the 6 series), which was calculated using Fiji from Zeiss Axiovert epifluorescent microscope images collected at 20X. In vitro, the percentage of phagocytic microglia was defined as cells with pouches containing apoptotic Vampire-SH-SY5Y nuclei and/or RFP particles 27 Up to three independent reference genes were compared: L27A, which encodes a ribosomal protein of the 60S subunit 26 ; OAZ-1, which encodes ornithine decarboxylase antizyme, a rate-limiting enzyme in the biosynthesis of polyamines and recently validated as a reference gene in rat and human 33 ; and HPRT, which encodes hypoxanthine guanine phosphoribosyl transferase 34  image, the 3D coordinates of cFos + and apoptotic cells of the granule layer (GL) were visually identified and saved using the Point tool of the Fiji distribution of ImageJ. Then, we used these coordinates to estimate the cartesian distances of each apoptotic cell to its closest cFos + neuron (nearest neighbor, NN) and to the nearest border of the image stack (X, Y and Z borders). This direct analysis, however, was hindered by the fact that most apoptotic cells were closer to a border of the z-stack (the upper or bottom Z border in most cases) than they were to a cFos + neuron (Supplementary Figure 1).
Therefore, the inclusion criteria for analyzing apoptotic-cFos+ distances was: d cFosNN <d border (i.e., the calculated distance to cFos NN must be shorter than the distance to the closest border), and apoptotic-cFos+ distances that were further away than the distance to the closest border, which was the case for the vast majority of apoptotic cells, were discarded. We analyzed a total of 25 and 253 apoptotic cells collected from 2 series of sections from the GL of wild-type (WT) and KO mice, respectively. Out of these, only 5 phagocytosed and 15 non phagocytosed cells from 1 n=9 KO mice, and one cell of each type in n= 9 WT mice passed the inclusion criteria.
To ensure that our z-stacks did not have an intrinsic bias in either of the cell types, we confirmed that both phagocytosed and non-phagocytosed apoptotic cells were located in z-stacks of similar thickness (23.4 ± 0.6 vs 24.3 ± 0.4 μm, respectively), and therefore had similar chance of detection.
Modelling apoptotic cell to cFos + neurons distances. To generate the model, we  3. Simulations. For each v3D grid and each of the four models, 10,000 randomizations were performed, and the Euclidean distance between each apoptotic cell and its nearest cFos + cell was computed in each randomization. Specifically, we performed 100 cell number (rn) randomizations, each followed by 100 cell location (rl) randomizations. In each rm, the number of cFos + and apoptotic cells in the grid was defined by different criteria according to the four different models detailed above. For each model and for each condition (WT or KO), the cumulative probability distribution of the NN distances between cFos and apoptotic cells was next calculated with 1 μm step and with 99% confidential intervals, by pooling data from the 10,000 simulations and the eight corresponding (WT or KO) v3D grids.
Statistical analysis. SigmaPlot (Systat Software) was used for statistical analysis.
Data were tested for normality and homoscedasticity. Two-sample experiments were analyzed by Student's t test and more than two-sample experiments by ANOVA. In two-way ANOVA, when interactions between factors were found, the analysis of the relevant variable was split into several one-way ANOVAs and Holm-Sidak method was used as a post hoc. Only p<0.05 is reported to be significant. Data are shown as mean ±SEM.

Microglial phagocytosis is uncoupled to apoptosis in Cstb KO mice at P30
To determine the apoptosis/phagocytosis dynamics in Cstb KO mice we first focused on analyzing hippocampal atrophy and neuronal apoptosis in the hippocampus in young mice at P30, when cerebellar apoptosis and cortical atrophy occur and seizures begin 7,9 We analyzed the granule cell population by immunostaining the dentate gyrus (DG) of both wild type (WT) and Cstb KO mice with the nuclear marker DAPI to assess apoptosis by karyorrhexis and/or pyknosis, and with Iba1 to visualize microglia ( Figure   1A). We found a significant increase in the total number of apoptotic cells ( Figure 1B) that was translated into a decrease in the total granule cell population in Cstb KO mice compared to WT mice (Fig 1C). However, the total granule cell density was similar in both genotypes, with a trend towards a reduction in the volume of the septal hippocampus (p=0.052), (Fig. 1D, E). This increased apoptosis and trend to decreased hippocampal volume in Cstb KO mice at P30 are consistent with the cerebellar and cortical atrophy previously described [9][10][11] .
Next, we analyzed microglial phagocytosis by measuring the phagocytic index (Ph Ph capacity compared to WT mice, with fewer non-phagocytic cells and more cells with one phagocytic pouch ( Figure 1G, H). Microglial numbers were also increased in Cstb KO mice compared to WT mice ( Figure 1I), leading to an increase in net phagocytosis (number of microglia multiplied by the Ph capacity). Nonetheless, this increase in net phagocytosis was insufficient to compensate for the increase in apoptosis, leading to a reduction in the Phagocytosis/Apoptosis coupling (ratio between net phagocytosis and total number of apoptotic cells; Figure 1J). Thus, despite the attempt to increase microglial numbers and Ph capacity, microglia were not able to remove the increasing numbers of dead cells in Cstb KO at P30.
As we had observed an increase in the total number of microglia ( Figure 1I), we then analyzed whether this effect was due to microglial proliferation using Ki67, a marker for proliferating cells that is expressed during all active phases of the cell cycle 35 (Figure 2 A, B). We observed a significant increase in proliferating microglia in Cstb KO mice compared to WT mice at P30 (Figure 2C). In addition, Cstb KO microglia presented a hypertrophic morphology (Figure 1A, 2A), which was reminiscent of the ameboid-like, multinucleated phenotype of microglia observed in the KA model of epilepsy 15;36 . To assess whether Cstb KO microglia also showed multinuclearity we quantified the percentage of cells with more than one nucleus ( Figure 1D). We found that while multinucleated cells were absent in control mice, they represented 13,1% ± 1,5 of microglia in Cstb KO mice, similar to what we observed 3d and 7d after KA injection 15 .
Finally, we addressed whether the impairment in microglial phagocytosis was To directly assess the effect of microglial Cstb on phagocytosis, we set up an in vitro model of Cstb knock-down in the microglial cell line BV2 (Fig 3C-I). We transfected BV2 microglia with 6-carboxyfluorescein (FAM) labeled siRNAs against Cstb or a scrambled siRNA as a control (Figure 3C). We obtained a high transfection efficiency through a time course of 6, 24 and 48h ( Figure 3D) and validated the transcription down-regulation of the Cstb gene by RT-qPCR through the time course. We observed that the expression of Cstb was greatly reduced up to 48 hours (Figure 3E), whereas the expression of the related cathepsins was not affected by Cstb siRNA treatment ( Figure 3F). Control and Cstb KO microglia were then fed with apoptotic SHSY5Yvampire neurons for 1 and 4h, in which apoptosis was previously induced (Staurosporine 3µM, 4 hours) 15 ( Figure 3G). Microglia were stained with CD11b, apoptotic neurons constitutively expressed the red fluorescent protein and nuclei were stained with DAPI ( Figure 3H). We found no differences in phagocytosis in microglia treated with either scrambled or Cstb siRNA ( Figure 3I) 11 and in agreement we did not observe any changes in the number of active neurons, labelled with the immediate early gene cFos, whose expression is rapidly induced upon depolarization 40 ( Figure 4A-C). cFos + neurons were found at similar numbers in both WT and Cstb KO mice at P14, but in Cstb KO mice they were located exclusively in the GL and not in the SGZ, where radial neural stem cells and their immature progeny reside (Figure 4A, C).
However, we did notice that whereas in WT mice most apoptotic cells were found in the SGZ, in Cstb KO mice apoptotic cells were found mostly in the GL and in very close proximity to cFos + neurons (Figure 4B), prompting us to analyze separately GL and SGZ apoptosis and phagocytosis.
In the SGZ, we did not observe any significant difference in apoptosis nor phagocytosis between WT and Cstb KO mice at P14 (Figure 5A-F). In contrast, in the GL there were few apoptotic cells in WT mice and a 10-fold increase in Cstb KO mice (Figure 5D, E).
The majority of these GL apoptotic cells were not phagocytosed, resulting in a reduced GL Ph index in cstb KO mice compared to WT mice, at P14 (Figure 5F). Similar to the global effect we had observed before in the whole dentate gyrus at P30, we found that GL microglia increased their Ph capacity in Cstb KO mice compared to WT mice although this effect was insufficient to cope with the increased number of apoptotic cells (Figure 5G, H). In this early stage, we found no obvious morphological changes nor increases in microglial numbers ( Figure 5I). Overall, we found that the increase in apoptotic cells was not compensated by a sufficient increase in phagocytosis, resulting in an uncoupling between apoptosis and phagocytosis ( Figure 5J). Overall, these results demonstrate that the microglial phagocytosis impairment was specific of the GL and preceded seizure development in Cstb KO mice at P14. This data suggested that there could be functional differences between GL and SGZ that could explain the GLspecific increase in apoptosis and microglial phagocytosis impairment.

Apoptotic cells are in close proximity to active cFos + neurons
The close vicinity of apoptotic cells to GL cFos + neurons in P14 mice (Figure 4B), suggested that the phagocytosis efficiency of GL microglia could be related to neuronal activity, as neuronal hyperactivity during seizures prevents microglia from targeting apoptotic cells 15 . To address whether the proximity of cFos + neurons had an impact on apoptosis and phagocytosis, we directly estimated the distance to each phagocytosed and non-phagocytosed cell to the closest cFos + neuron (nearest neighbor, NN) ( Figure   6A). Because of the limitation imposed by the relatively small thickness of our z-stacks (border effect; see Methods for more detail), we only had a sufficient number of apoptotic cells in KO mice (n=15 phagocytosed and n=5 non-phagocytosed apoptotic cells from n=9 mice, from an initial set of 295 cells).
In Cstb KO mice, both phagocytosed and non-phagocytosed cells were found very close to the cFos + NN (3.0 ± 0.5 and 3.0 ± 0.6μm, respectively; non-significant difference; Figure 6B). To determine whether this short distance would be expected if cells were homogenously distributed through the thickness of our z-stacks, we calculate that 50% of the cells should be located at ¼ distance to either z-border (green-shaded area in Supplementary Figure 1), that is, at 5.9 ± 0.2 and 6.0 ± 0.1μm to the cFos + NN for phagocytosed and non-phagocytosed cells, respectively (Supplementary Figure 1). Thus, both phagocytosed and non-phagocytosed apoptotic cells in Cstb KO mice seem to be much closer (3μm) to active neurons than would be expected. Although we could not detect significant differences between phagocytosed and non-phagocytosed cells in Cstb KO mice, these results do suggest an unexpected relationship between neuronal activity and apoptosis.
To strengthen the analysis of distance between apoptotic cells and NN cFos + neurons and in both WT and KO mice and overcome the limitation of analyzing only 20 cells, we developed a mathematical model (see Methods for details). We created a virtual 3D model of the GL based on the spatial distribution and density of neurons, cFos + neurons and apoptotic cells (Supplementary Figure 1). In this model we reproduce the same experimental limitations imposed by the limited thickness of the z-stacks (see Methods) compared the size of the (XY) field of view and therefore no cells were discarded due to the border effect. Using this virtual 3D model we compared the entire experimental distribution of NN distance between cFos + and apoptotic cells to the distribution generated by the 10,000 simulations. Specifically, we developed four different models ( Figure 6C).
In the first model cFos + neurons were located in their original position (in the z-stack) and the position of the apoptotic cells was randomized, and their numbers were extrapolated from the experimental cell density (Figure 6C). For each apoptotic cell we performed 10,000 randomizations and calculated the distance to the NN cFos + cell.
Finally, the cumulative probability to encounter a cFos + cell within a given distance from an apoptotic cell was computed with its 99% confidence interval both in the WT-and KO-derived v3D grids (Figure 6D, E). In WT mice, the measured apoptotic-cFos + NN distance was within the modeled confidence interval up to a distance of 22μm, which matches the average z-stack thickness (24.2 ±0.4μm) and validates that our model is isotropic, i.e., does not depend on the orientation and therefore is within the range of the z-stack thickness. In contrast, in Cstb KO mice the measured percentage of NN cFos + -apoptotic cells between 17 and 24 μm was significantly higher than what observed both in the WT and Cstb KO models. For instance, in the Cstb KO model 20% of the apoptotic cells are found at less than 20μm distance from the cFos + NN, whereas in the real data from Cstb KO mice, 20% of the apoptotic cells were found at less than 16μm and 35% of the apoptotic cells are found at less than 20μm ( Figure   6E). This data suggested that as in our direct quantifications (Figure 6B), apoptotic cells were closer than expected to active cFos + neurons in Cstb KO mice.
Nonetheless, the modeled distance was significantly larger in KO than in WT mice ( Figure 6E). To further understand the role of other parameters, specifically cell number or location, on the model, we used other three different models ( Figure 6C):

Model 2 to test the impact of cFos + cell location, Model 3 to test cFos + cell density, and
Model 4 to test apoptotic cell density. We first tested the effect of cFos + cell location (Model 2), by randomly positioning both cFos + neurons and apoptotic cells ( Figure   6F1). In this model, there were no differences between the modeled NN distances between WT and KO. Importantly, in KO mice the difference between the measured and modeled apoptotic-cFos + NN distance was strongly reduced although it was still significant with a maximum around 20μm. The reduced difference between the measured and modeled NN distance in KO mice in model 2 suggest that the differences between the modeled NN distances in Model 1 originated from a differential distribution of cFos + cells between WT and KO.
We then tested the effect of cFos + density by creating a Model 3 in which cFos + cells were positioned randomly in the v3D grid and their number was estimated from the experimentally measured cFos density rescaled by the number of cells in the grid, whereas apoptotic cells were modeled randomly as above ( Figure 6F2). The results of Model 3 were similar to Model 2, with no differences between modeled and measured NN distances for WT and KO, suggesting that the number of cFos + cells was not related to the differential effect found in Model 1. Finally, we tested the effect of different density of apoptotic cells in the WT and KO modeled distances in Model 4. For this, we went back to use the real distribution of cFos+ cells (as in Model 1) and positioned the apoptotic cells randomly but with an inverted density: WT model using KO apoptotic cell density and viceversa ( Figure 6F3). The results of Model 4 were similar to Model 1: the modeled NN distance was larger in KO than in WT, and apoptotic cells were found closer to active neurons than expected in KO mice. Overall, this data suggests that while Cstb KO mice have similar cFos + density and intensity at P14 ( Figure 4C) and do not have yet seizures 11 , they have an abnormal distribution of cFos + active neurons that results in a closer distance to apoptotic cells (Figure 6B,C).

These results suggest a very close relationship between abnormal neuronal activity in
Cstb KO mice and apoptotic cells. Indirectly, they also suggest a relationship with the phagocytosis impairment found in Ctsb KO mice, because the number of apoptotic cells observed is the net result between apoptosis induction and phagocytosis removal. .

DISCUSSION
In this paper, we show for the first time that microglial phagocytosis of apoptotic cells is  47 . Thus, granule cell death and hippocampal atrophy start early in the brains of Cstb KO mice and exhibit a progressive nature.

KO mice
In addition to the neuronal damage, other cell types have also been involved in the pathology of EPM1. Some of the earliest changes pointed towards an altered inflammatory response by microglia, the brain resident macrophages 11,13 . Here we

Microglial phagocytosis disruption is not due to the cell autonomous lack of CSTB
The microglial phagocytosis impairment could be mechanistically related to the lack of Cstb in microglia and/or in other brain cell types. Indeed, we here show that Cstb expression was not restricted to microglia in the brain, suggesting that cell autonomous The close relationship between apoptotic cells and active neurons in Cstb KO mice is supported by both direct measurements and the virtual 3D model but does not provide evidence that this relationship affects phagocytosis. Nonetheless, it is important to note that the net apoptosis observed is in part the result of the phagocytosis dynamics: the number of apoptotic cells at any given time depends as much on the input (apoptosis induction) as on the output (removal by phagocytosis) 14 . Therefore, our data shows an overall impairment of microglial phagocytosis in the GL of Cstb KO mice and suggests a complex scenario in which sub-seizure local neuronal activity may affect the clearance of apoptotic cells by microglia.
In addition, other pathophysiological mechanisms may participate in the phagocytosis impairment observed in Cstb KO mice. For instance, one possibility is that intrinsic differences between GL and SGZ microglia explain why the impairment is restricted to the GL. SGZ microglia are likely "trained" for phagocytosis, as they have been exposed to apoptotic cells during the postnatal period, whereas GL microglia is not. This of keratan sulfate polysaccharides (epitope 5D4), whose overexpression is related to a higher ex vivo phagocytosis of zymosan particles possibly related to the engulfment of synapses 52 . Another potential candidate is the complement system, which is used by microglia to facilitate recognition (opsonization) of apoptotic cells 28 as well as synapses 53 , and is progressively activated in rodent and human epilepsy 54,55 , including Cstb KO mice 37,56 . Nonetheless, it is beyond the scope of this paper to test the role of complement or keratan sulfate and future studies will decipher the underlying mechanisms of microglial phagocytosis impairment in Cstb KO mice.
In summary, we here extend our initial observations that microglial phagocytosis impairment is an early feature of mouse and human MTLE and show that it also occurs in a genetic model of EMP1 by Cstb deficiency. We also provide an unexpected link between phagocytosis impairment, accumulation of apoptotic cells and local neuronal activity in these mice that further supports the suggestion that both abnormal local neuronal activity (in presymptomatic Cstb KO mice) and network hyperactivity (in MTLE mice) regulate the efficiency of microglial phagocytosis. Phagocytosis is an essential component of the brain regenerative response and therefore, future therapies for epilepsy patients should be aimed not only at reducing neuronal death but also at harnessing microglial phagocytosis.