Microglial MERTK eliminates phosphatidylserine‐displaying inhibitory post‐synapses

Article19 May 2021free access Source DataTransparent process Microglial MERTK eliminates phosphatidylserine-displaying inhibitory post-synapses Jungjoo Park Jungjoo Park orcid.org/0000-0002-0548-9736 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Yeeun Choi Yeeun Choi Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as second/third authors Search for more papers by this author Eunji Jung Eunji Jung Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as second/third authors Search for more papers by this author Seung-Hee Lee Seung-Hee Lee Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as senior authors Search for more papers by this author Jong-Woo Sohn Jong-Woo Sohn orcid.org/0000-0002-2840-4176 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as senior authors Search for more papers by this author Won-Suk Chung Corresponding Author Won-Suk Chung [email protected] orcid.org/0000-0003-1060-9007 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Jungjoo Park Jungjoo Park orcid.org/0000-0002-0548-9736 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Yeeun Choi Yeeun Choi Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as second/third authors Search for more papers by this author Eunji Jung Eunji Jung Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as second/third authors Search for more papers by this author Seung-Hee Lee Seung-Hee Lee Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as senior authors Search for more papers by this author Jong-Woo Sohn Jong-Woo Sohn orcid.org/0000-0002-2840-4176 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, KoreaThese authors contributed equally to this work as senior authors Search for more papers by this author Won-Suk Chung Corresponding Author Won-Suk Chung [email protected] orcid.org/0000-0003-1060-9007 Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea Search for more papers by this author Author Information Jungjoo Park1, Yeeun Choi1, Eunji Jung1, Seung-Hee Lee1, Jong-Woo Sohn1 and Won-Suk Chung *,1 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Korea **Corresponding author. Tel: +82 42 350 2624; E-mail: [email protected] The EMBO Journal (2021)40:e107121https://doi.org/10.15252/embj.2020107121 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat-me" signal that initiates glia-mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal-specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well-known "eat-me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post-synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post-synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post-synapse elimination. Moreover, we found that phosphatidylserine is used for microglia-mediated pruning of inhibitory post-synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat-me" signal for inhibitory post-synapse elimination. SYNOPSIS Neuronal-specific deletion of the flippase chaperone Cdc50a leads to exposure of phosphatidylserin on neuronal outer membranes causing specific loss of inhibitory post-synapses and seizures. Microglial phagocytosis via the phagocytic receptor MERTK promotes inhibitory post-synapse elimination in Cdc50a cKO brains. Inhibitory post-synapses in normal juvenile brains also use phosphatidylserine for synapse elimination, suggesting that phosphatidylserine exposure functions as an “eat-me” signal for microglia-dependent inhibitory post-synapse elimination. Neuronal Cdc50a deletion induces rapid lethality with appearance of audiogenic seizure. Neuronal Cdc50a deletion causes the specific loss of inhibitory post-synapses without affecting other synapses. Ablating microglia or deleting microglial Mertk rescues the loss of inhibitory post-synapses and seizure behaviors in Cdc50a cKO mice. Microglial Mertk deletion increases the number of phosphatidylserine-exposed inhibitory post-synapses in the wild-type juvenile brains. Introduction Synapse elimination, the process by which unnecessary synapses are selectively removed, occurs in the central nervous system during development and adult stages as well as in various neurological disorders (Chung & Barres, 2012). Recent studies have shown that microglia and astrocytes contribute to synapse elimination through complement and MERTK/MEGF10 phagocytic pathways, respectively (Stevens et al, 2007; Schafer et al, 2012; Chung et al, 2013; Lee et al, 2020). Interestingly, microglia have been shown to use the complement cascade, and eliminate either excitatory or inhibitory synapses during development and under disease conditions (Stevens et al, 2007; Schafer et al, 2012; Hong et al, 2016; Lui et al, 2016; Sekar et al, 2016; Vasek et al, 2016). However, the identity of the “eat-me” signal that allows these glial phagocytic receptors to recognize specific synapses for subsequent elimination is still unclear. Moreover, whether microglia use other molecular mechanism than the complement cascade for eliminating synapses and whether excitatory and inhibitory synapses utilize the same or distinct mechanisms for presenting “eat-me” signals are unknown. Phosphatidylserine (PS) is a phospholipid that predominantly resides on the inner plasma membrane under normal physiological conditions. When cells undergo stress or receive apoptotic stimuli, PS flips to the outer plasma membrane, where it functions as an “eat-me” signal for subsequent engulfment by neighboring phagocytes (Segawa & Nagata, 2015). Interestingly, recent studies have also shown that PS can be exposed transiently in restricted portion of the plasma membrane, such as neuronal processes (Sapar et al, 2018) or excitatory pre-synapses (Li et al, 2020; Scott-Hewitt et al, 2020), suggesting their presentation can be controlled locally within the cell. Among the upstream enzymes that control PS presentation in the inner or outer plasma membrane, the flippases, type IV P-type adenosine triphosphatases (P4-ATPases), were shown to use adenosine triphosphate (ATP) to translocate PS from the outer to the inner plasma membrane. Although many functionally redundant P4-ATPases are expressed in various tissues, most P4-ATPase family members require the activity of the flippase chaperone cell cycle control protein 50A (CDC50A) for proper localization on the plasma membrane (Segawa et al, 2014; Segawa & Nagata, 2015). Previous in vitro studies have shown that a Cdc50a null mutation causes constitutive PS exposure on the outer plasma membrane without inducing general apoptosis and that PS exposure is sufficient to initiate phagocytic engulfment by macrophages (Segawa et al, 2014). However, although various P4-ATPases and CDC50A are highly expressed in the mammalian brain (Coleman & Molday, 2011; Zhang et al, 2017), their potential roles in synapse elimination have not yet been studied. Here, by conditionally deleting Cdc50a in mature neurons, we reveal that CDC50A-dependent PS exposure is required for eliminating inhibitory post-synapses and generates excessive excitability with appearance of seizure. Surprisingly, CDC50A deletion induces PS exposure preferentially in neuronal soma, thereby inducing engulfment of inhibitory post-synapses by microglial phagocytosis, without affecting excitatory synapses or inhibitory pre-synapses. Importantly, ablating microglia or deleting microglial Mertk, a critical phagocytic receptor, rescues the loss of inhibitory post-synapses and seizure phenotype in Cdc50a-deleted mice. These data indicate that microglial MERTK phagocytic pathway can specifically phagocytose and eliminate PS-exposed inhibitory post-synapses without damaging other cellular membranes. Moreover, we find that inhibitory post-synapses in normal brains also use PS for synapse pruning by microglia, suggesting PS may serve as a general “eat-me” signal for inhibitory post-synapse elimination. Results Audiogenic seizure phenotype of Cdc50a cKO mice In the mammalian brains, various P4-ATPases are expressed, potentially performing redundant functions (Andersen et al, 2016). Therefore, to understand the roles of PS in synapse elimination, we decided to acutely delete Cdc50a and block the activity of the P4-ATPase family members in mature neurons. First, we investigated whether CDC50A is expressed in neurons in the mouse brain. In heterozygous Cdc50a knockout-first allele mice, in which the lacZ gene is knocked in at the Cdc50a locus, we found that β-galactosidase was highly localized in both CAMKII-positive and GAD67-positive neurons (Fig 1A), indicating that Cdc50a is expressed by both excitatory and inhibitory neurons. To generate loxP-floxed Cdc50a (Cdc50afl/+) mice, we crossed mice with the Cdc50a knockout-first allele with Act-FLPe mice to remove FRT sites. Then, the mice with the Cdc50afl/+ allele were subsequently crossed with Thy1-CreERT2, -EYFP mice to generate inducible neuron-specific Cdc50a conditional KO (cdc50a cKO) mice (Fig EV1A). To delete Cdc50a specifically in mature neurons, we injected oil or 4-hydroxytamoxifen (4-OHT) into 1-month-old control and cdc50a cKO mice (Fig EV1B). Unlike control mice, Cdc50a cKO mice showed rapid lethality within 2 weeks after 4-OHT injection (Fig 1B) without significant changes in body weight or brain morphology (Fig EV1C and D). Interestingly, there was no difference in the expression of cleaved caspase-3 (cCas3), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining or the number of NeuN-positive neuronal cells between the brains of Cdc50a cKO and those of control mice (Figs 1C and EV1E–G). We also examined the axonal processes of Thy1-CreERT2, -EYFP-positive motor neurons in the diaphragm muscles and neuronal morphology of sparsely labeled Thy1-CreERT2, -EYFP-positive neurons with a floxed tdTomato (Rosa26-CAG-tdTomato) reporter, and found that they were unaltered in Cdc50a cKO mice (Fig EV1H and J). These data indicate that acute deletion of neuronal CDC50A induces rapid lethality in mice without inducing cell death. Figure 1. Neuronal Cdc50a deletion induces rapid lethality and audiogenic seizures A. Representative confocal z-stack images of co-immunostaining for β-galactosidase (green) and CAMKII (red, upper) or GAD67 (magenta, lower) in neurons from the brains of Cdc50a knockout-first allele (Cdc50a lacZ/+) mice. Scale bar, 20 μm. B. Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, ***P = 0.0003). C. Schematic diagram of the experiment. Mice of both genotypes were sacrificed 13 days after oil or 4-OHT injection. Representative confocal z-stack images of cleaved caspase-3 (cCas3, red)- and Thy1-CreERT2, -EYFP (green)-expressing cells from control and Cdc50a cKO mice. Scale bar, 20 μm. D. Schematic diagram of behavioral tests for measuring audiogenic seizures (0: normal behavior, 1: wild running, 2: seizure, and 3: respiratory arrest and death) following auditory stimuli. E, F. Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz or white noise (WN) at 70, 80, 90, or 100 dB) in control (E) and Cdc50a cKO (F) mice (n = 8 for each group). G. Representative confocal z-stack images showing cFos (magenta)-positive neurons from control and Cdc50a cKO mice (CTX: cortex, HP: hippocampus, and IC: inferior colliculus). Scale bar, 20 μm. H. Bar graphs showing the relative area of cFos-positive cells in the brains of control and Cdc50a cKO mice (n = 5 for each group, **P = 0.0030, n.s., not significant). Data information: The individual dots indicate experimental replicates (3 to 5 images were taken from 2 brain slices per an animal, H). The data are the mean ± SEM and were analyzed by the log-rank (Mantel-Cox) test (B) or two-tailed unpaired t-test (H). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Acute neuronal Cdc50a deletion does not induce cell death Schematic diagram of the control and Cdc50a cKO mice. For control and Cdc50a cKO mice, Thy1-creERT2, -EYFP;Cdc50afl/fl mice were injected with oil or 4-OHT at 1 month of age, respectively. Schematic diagram of the experiment. Bar graphs showing relative protein expression of CDC50A in the IC (inferior colliculus) of control and Cdc50a cKO mice (n = 3 for each group, *P = 0.0127). Please see Fig EV1 source data. Bar graphs showing the body weight changes in control and Cdc50a cKO mice (n = 8 for each group, n.s., not significant). Representative images of Thy1-creERT2, -EYFP (green)-expressing neurons in sagittal brain sections from control and Cdc50a cKO mice. Scale bar, 100 μm. Bar graphs showing the area of cCas3-positive cells in the CTX (cortex), HP (hippocampus), and IC of control and Cdc50a cKO mice (n = 5 for each group). Bar graphs showing the area of NeuN-positive cells in the CTX, HP, and IC of control and Cdc50a cKO mice (n = 4 for each group). Representative confocal z-stack images of TUNEL staining (red) and DAPI (blue) in Thy1-CreERT2, -EYFP (green)-expressing IC neurons. Scale bar, 40 μm. Representative confocal z-stack images of Thy1-creERT2, -EYFP (green)-expressing axons and neuromuscular junctions of motor neurons in the diaphragms of control and Cdc50a cKO mice. Scale bar, 100 μm. Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz, or white noise (WN) at 70, 80, 90, or 100 dB) in Cre-negative Cdc50afl/fl mice injected with 4-OHT (n = 5). Schematic diagram of the experiments and representative confocal z-stack images of tdTomato (red)-positive HP neurons in control and Cdc50a cKO mice after low-dose tamoxifen injection. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm. Representative confocal z-stack images showing Cre-mediated recombination in Thy1-CreERT2, -EYFP;Cdc50afl/fl;Rosa26-CAG-tdTomatofl/+ mice after low-dose tamoxifen injection. tdTomato (red) expression in Thy1-CreERT2, -EYFP (green)-expressing CTX, HP, and IC (CTX: cortex, HP: hippocampus, IC: inferior colliculus) neurons. Scale bar, 100 μm. Bar graphs showing the percentage of EYFP and tdTomato double-positive neurons relative to EYFP-positive neurons in the CTX, HP, and IC (n = 3 for each group, P**** < 0.0001). Data information: The individual dots indicate experimental replicates (3–5 images were taken from 2 brain slices per an animal, E, F, and L). The data are the mean ± SEM and were analyzed by two-tailed unpaired t-test (B, C, E, and F) or one-way ANOVA with Tukey’s test (L). Source data are available online for this figure. Download figure Download PowerPoint Unexpectedly, while monitoring the behaviors of Cdc50a cKO mice, we found they showed convulsive seizures upon exposure to a loud noise (Movie EV1). To test whether Cdc50a cKO mice develop seizures in response to sounds, we provided auditory stimuli and scored seizure behaviors (0: normal behavior, 1: wild running, 2: seizure, and 3: tonic–clonic seizures with loss of righting reflex, Movie EV2). Interestingly, we found that unlike control mice, Cdc50a cKO mice showed severe audiogenic seizures (wild running and seizures) at a high frequency between 10 and 20 kHz (Figs 1D–F and EV1I). In agreement with the audiogenic seizure phenotype, neuronal activity as measured by cFos protein expression, was significantly upregulated in the inferior colliculus (IC) (Fig 1G and H), the principal midbrain nucleus of the auditory pathway (Lee & Sherman, 2010) and primary brain region for audiogenic seizure (Faingold et al, 1992; Snyder-Keller & Pierson, 1992; Yang et al, 2001; Faingold, 2002), in Cdc50a cKO mice compared with control mice 13 days after 4-OHT injection. The observed audiogenic seizure phenotype and cFos upregulation in the IC are likely due to strong Thy1 promoter activity in IC neurons. When we sparsely labeled Thy1-CreERT2, -EYFP-positive neurons with a floxed tdTomato (Rosa26-CAG-tdTomato) reporter by injecting low doses of tamoxifen, we found more frequent and robust genetic recombination in the IC than in other brain regions, such as the cortex and hippocampus (Fig EV1K and L). Inhibitory post-synapse loss in Cdc50a cKO brains Next, to test whether audiogenic seizures in Cdc50a cKO mice are due to synaptic imbalance (Fritschy, 2008), we measured the number of excitatory and inhibitory synapses in various brain regions. Interestingly, we found that the number of VGLUT1/2- and PSD95-positive excitatory synapses was not changed in Cdc50a KO mice compared with control mice (Figs 2A and C and EV2A and B). Moreover, there was no significant difference in the number of VGAT-positive inhibitory pre-synapses (Figs 2B and D). However, surprisingly, we found that the number of Gephyrin-positive inhibitory post-synapses was significantly lower in Cdc50a cKO mice than in control mice in the cortex, hippocampus, and IC (Figs 2B and D and EV2C and D). Due to the reduced number of Gephyrin-positive inhibitory post-synapses, the total number of inhibitory synapses containing both Gephyrin and VGAT was significantly reduced (Fig 2D). Figure 2. Neuronal Cdc50a deletion induces loss of inhibitory post-synapses A, B. Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. C, D. Bar graphs showing the numbers of excitatory (C, PSD95 and VGLUT1) and inhibitory (D, Gephyrin and VGAT) synapses in the CTX, HP, and IC of control and Cdc50a cKO mice (n = 5 for each group, for excitatory synapses: n.s., not significant, for inhibitory synapses: CTX, ***P = 0.0001, **P = 0.0023, HP, **P = 0.0038, IC, **P = 0.0023, ****P < 0.0001). E. Representative confocal z-stack images of GABAA receptor α (red) and β (green) staining in the IC in control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. F. Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms. G. Frequency–current (F-I) curve of the IC of control and Cdc50a cKO mice. The number of spikes per 500 ms was measured and averaged for each current (each, n = 9 and 13 cells, 300 pA: *P = 0.0203, 350 pA: *P = 0.0133, 400 pA **P = 0.0098, 450 pA: **P = 0.0072). Data information: The individual dots indicate experimental replicates (3–5 images were taken from 2 brain slices per an animal, C and D). The data are the mean ± SEM and were analyzed by two-tailed unpaired t-test (C and D) or two-way ANOVA (G). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Neuronal Cdc50a deletion reduces the number of inhibitory synapses without affecting excitatory synapses A. Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT2 (red)-positive excitatory synapses in the CTX and IC (CTX: cortex and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm. B. Bar graphs showing the number of excitatory (PSD95- and VGLUT2-positive) synapses in the CTX, HP (hippocampus), and IC of control and Cdc50a cKO mice (n = 5 for each group). C. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in control and Cre-negative Cdc50afl/fl mice injected with 4-OHT. Scale bar, 10 μm. D. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control and Cre-negative Cdc50afl/fl mice injected with 4-OHT (n = 3 to 5 for each group, n.s., not significant). E. Representative confocal z-stack images of GABAA receptor γ (green), glycine receptor (cyan), and gephyrin (red) in the IC of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. F. Bar graphs showing the relative inhibitory synaptic density in the IC of control and Cdc50a cKO mice (n = 4 for each group, *P = 0.0123, **P = 0.0042, ****P < 0.0001). G. Representative confocal z-stack images of GABAA receptor α (red), Gephyrin (green), and VGAT (magenta) in the IC of control and Cdc50a cKO mice. The white circles indicate colocalization of GABAA receptor α with Gephyrin and VGAT. Scale bar, 20 μm. H. Representative Western blot images of synaptic proteins (Homer, Synapsin, VGAT, Gephyrin, GABAAR β, GABAAR γ, and Glycine R) and GAPDH in the IC of control and Cdc50a cKO mice. Please see Fig EV2 source data. I. Bar graphs showing the quantification of the Western blot data of synaptic proteins (Homer, Synapsin, VGAT, Gephyrin, GABAAR β, GABAAR γ, and Glycine R) for the IC of control and Cdc50a cKO mice (n = 3 for each group, Gephyrin, *P = 0.0240 and Glycine receptor, *P = 0.0412). J–L. Resting membrane potential (RMP, J), input resistance (IR, K), and cell capacitance (L) of the IC of control and Cdc50a cKO mice (each, n = 13 cells). M. Frequency-current (F-I) curve of the IC of control and Cdc50a cKO mice with kainate (KA) and picrotoxin (PTX). The number of spikes per 500 ms was measured and averaged for each current (each, n = 10 and 11 cells). Data information: The individual dots indicate experimental replicates (3–5 images were taken from 2 brain slices per an animal, B, D, and F). The data are the mean ± SEM and were analyzed by two-tailed unpaired t-test. Source data are available online for this figure. Download figure Download PowerPoint Gephyrin is a critical inhibitory post-synaptic element that binds and stabilizes both glycine and γ-aminobutyric acid type A (GABAA) receptors for inhibitory synaptic transmission (Brickley & Mody, 2012). We found significantly fewer glycinergic and GABAergic post-synapses, as detected by glycine receptor and GABAA receptor γ subunit antibodies, respectively, in Cdc50a cKO mice than in control mice (Fig EV2E–I). However, GABAA receptor α and β subunits, which are also localized in the extrasynaptic membrane as well (Choii & Ko, 2015), appeared to be intact in Cdc50a cKO mice (Figs 2E and EV2G–I), suggesting that Cdc50a deletion in neurons specifically reduces the number of inhibitory post-synapses without disrupting neuronal membranes. Importantly, when we evaluated the basic electrophysiological properties of IC neurons from control and Cdc50a cKO mice, we found that there were no significant changes in resting membrane potential (RMP), input resistance (IR), and cell capacitance (Fig EV2J–L), showing that membrane integrity was unaffected in Cdc50a cKO neurons. However, when we measured the frequency–current relationship (F-I curve) by holding the membrane potential at −60 mV and injecting the current from +50 pA to +450 pA for 500 ms each, there were significantly more spikes in Cdc50a cKO neurons than in control neurons (Fig 2F and G), suggesting that neuronal excitability was increased by Cdc50a deletion. Interestingly, this difference in excitability was significantly diminished by applying both kainate (KA) and picrotoxin (PTX), AMPA/kainate agonist and GABAA receptor antagonist, respectively (Fig EV2M). Thus, our data suggest that increased excitability of Cdc50a cKO IC neurons to sound-evoked stimuli is due to synaptic imbalance and may underlie audiogenic seizure behaviors in Cdc50a cKO mice. PS exposure in Cdc50a cKO neuronal somas Why are only inhibitory post-synapses affected when Cdc50a is deleted from neurons? When we expressed Cdc50a-mCherry in neurons in vitro and in vivo, we found that CDC50A-mCherry remained in somas of neurons instead of spreading to their processes (Fig 3A and B). Nissl staining of rough endoplasmic reticulum (rER) revealed that CDC50A-mCherry was localized to the rER, as previously suggested (Byrne & Roberts, 2009). Since rER resides mostly within the cell soma without extending to the distal processes of neurons (Ramirez & Couve, 2011), we hypothesized that Cdc50a cKO neurons might expose PS preferentially in the neuronal soma. To test our hypothesis, we cultured cortical neurons from Cdc50afl/fl mice in vitro and transfected them with hSyn-mCherry or hSyn-Cre-p2a-dTomato to generate control and Cdc50a cKO neurons, respectively (Fig 3C). After deleting Cdc50a, we monitored the extent of PS exposure on the outer plasma membranes of neurons by utilizing polarity-sensitive indicator of viability (pSIVA), a probe that reversibly binds PS and emits green fluorescence (Kim et al, 2010). Surprisingly, after hSyn-Cre-p2a-dTomato transfection, we found that PS exposure preferentially occurred in the soma first in Cdc50a cKO neurons (Fig 3D). Next, to detect this preferential PS exposure in the brains of Cdc50a cKO mice, we adopted the secreted Annexin V (secA5) system (van Ham et al, 2010), in which neighboring cells, in our case astrocytes, express and secrete Annexin V fused with mCherry (Fig 3E). AAV9-GFAP-secA5-mCherry was injected to allow secA5-mCherry to be expressed by and secreted from astrocytes, enabling the detection of PS-exposed material in the brain (Fig 3E). Consistent with our in vitro data, we found that secA5 puncta were mostly localized in the neuronal soma rather than in processes in the brains of Cdc50a cKO mice (Fig 3F), suggesting that the neuronal soma is the major site of PS exposure when Cdc50a is deleted. Importantly, we also found that the number of Gephyrin-positive inhibitory post-synapses was significantly lower in the somas and proximal dendrites, but not in the distal dendrites, in the hippocampal CA1 region in Cdc50a cKO mice (Fig 3G and H). Thus, since neuronal somas harbor many more inhibitory post-synapses than other types of synapses (Keith & El-Husseini, 2008; Kubota et al, 2016), our data suggest that preferential PS exposure in the somas of Cdc50a-deleted neurons is responsible for the specific loss of inhibitory post-synapses. Figure 3. Neuronal Cdc50a deletion induces PS exposure preferentially in the cell soma Schematic diagram of the in vitro CDC50A-mCherry expression experiment. After 6 days in vitro (DIV), cortical neurons were transfected with CAG-GFP and CAG-mCherry or CAG-GFP and CAG-Cdc50a-mCherry. Representative confocal z-stack images of the rER (Nissl staining, cyan) with GFP (green) and mCherry/CDC50A-mCherry (red). The dashed line represents where the high magnified insert was taken from. Scale bars, 50 (left), 20 (middle), and 5 μm (right). Schematic diagram of in vivo CDC50A-mCherry expression experiment. Neonatal (P0) wild-type mice were injected with AAV9-CAG-Cdc50a-mCherry. Representative confocal z-stack images of the rER (Nissl staining, green) and CDC50A-mCherry (red) in the CTX (cortex). The dashed line represents where the high magnified insert was taken from. Scale bars, 20 μm (upper) and 5 μm (lower). Schematic diagram of the experiment in which PS exposure was detected in control and Cdc50a cKO neurons in vitro. After 6 DIV, cortical Cdc50afl/fl neurons were transfected with either hSyn-mCherry or hSyn-Cre-p2a-dTomato to generate control and Cdc50a cKO neurons, respectively. Representative live confocal z-stack images showing PS exposure (pSIVA, green) in mCherry/dTomato (red)-expressing neurons. Scale bar, 20 μm. The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall P