GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived neuroligin-2 R215H mutation

GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived... Schizophrenia (SCZ) is a severe mental disorder characterized by delusion, hallucination, and cognitive deficits. We 215 215 have previously identified from schizophrenia patients a loss-of-function mutation Arg →His (R215H) of neuroligin 2 (NLGN2) gene, which encodes a cell adhesion molecule critical for GABAergic synapse formation and function. Here, we generated a novel transgenic mouse line with neuroligin-2 (NL2) R215H mutation. The single point mutation caused a significant loss of NL2 protein in vivo, reduced GABAergic transmission, and impaired hippocampal activation. Importantly, R215H KI mice displayed anxiety-like behavior, impaired pre-pulse inhibition (PPI), cognition deficits and abnormal stress responses, recapitulating several key aspects of schizophrenia-like behaviors. Our results demonstrate a significant impact of a single point mutation NL2 R215H on brain functions, providing a novel animal model for the study of schizophrenia and neuropsychiatric disorders. Keywords: Schizophrenia, GABA, Neuroligin-2, Mouse model, Mutation Introduction differentially locate to excitatory and inhibitory synapses Schizophrenia (SCZ) is a chronic neuropsychiatric dis- and are critical for the excitatory and inhibitory synapse for- order caused by both genetic and environmental factors. mation and function, respectively [9, 12, 35, 44, 51, 53, 59]. It is featured by long-standing delusion and hallucin- Neuroligin-3 locates at both type of synapses and ation (psychosis), and cognitive deficits [17, 27, 39]. SCZ contributes to both neurotransmission [7, 14, 57]. In recent is a highly heritable disorder [55] with a complex genetic years, genetic variants of neuroligin-1, neuroligin-3 and basis. Recent genomic studies identified a number of neuroligin-4 have been identified in autism patients [28, 43]. genetic variants associated with SCZ, including a group Mutations in proteins interacting with neuroligins such as of variants resided in the genes encoding synaptic adhe- Neurexin1, SHANK and MDGA have also been associated sion molecules that promoting synaptic development with autism and schizophrenia patients [6, 13, 31, 32]. Gen- and function such as IGSF9B, and NLGN4X [52]. etic mouse models based on these findings recapitulate sev- Neuroligins (NLGNs) are a family of synaptic adhesion eral aspects of patient symptoms, providing an entry point molecules highly expressed in the brain and are ligands for for the mechanistic study and drug development on psychi- another group of cell adhesion molecules neurexins atric disorders [2, 10, 14, 15, 29, 46, 50, 54, 57, 66]. (NRXNs) [26]. There are five neuroligin genes (neuroligin-1, We have previously reported several novel mutations of − 2, − 3, − 4, and − 5) in humans and four in mice (neuroli- NLGN2 from schizophrenia patients [56]. Among the gin 1–4). Neuroligin-1, − 2, and − 3 are close homologs be- NL2 mutants, we found that the R215H mutant protein tween human and mice. Neuroligin-1 and neuroligin-2 was retained in the endoplasmic reticulum (ER) and could not be transported to the cell membrane, resulting in a failure to interact with presynaptic neurexin and a loss of * Correspondence: gongchen@psu.edu Department of Biology, Huck Institutes of Life Sciences, Pennsylvania State function in GABAergic synapse assembly [56]. Based on University, University Park, PA 16802, USA these studies, we have now generated a transgenic mouse Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jiang et al. Molecular Brain (2018) 11:31 Page 2 of 11 line carrying the same NL2 R215H mutation to test its and was observed in a variety of brain regions (Fig. 1b, functional consequence in vivo. We demonstrate that the and quantified in Fig. 1c). In contrast, NL2 KO mice R215H knock-in (KI) mice show severe GABAergic defi- showed a complete absence of NL2 without any imma- cits and display not only anxiety-like behavior seen in glo- ture band at all (Fig. 1b, top row). Such difference of bal NL2 KO mice [1, 3, 61], but also impaired pre-pulse NL2 protein level between our KI mice and previous KO inhibition, cognitive deficits, and abnormal stress re- mice may underlie their functional difference reported sponses which are not reported in global NL2 KO mice. later. To test whether NL2 R215H mutation affects the Our results suggest that a single-point mutation R215H of expression of other NL family members, we examined NL2 can result in significant GABAergic deficits and con- the protein level of NL1 and NL3 in both NL2 R215H tribute to SCZ-like behaviors. This newly generated NL2 Het and KI mice but found no significant changes R215H KI mouse may provide a useful animal model for (Additional file 1: Figure S3). the studies of neuropsychiatric disorders including SCZ. To investigate the localization of NL2 R215H proteins inside the brain, we performed immunohistochemistry Results with NL2-specific antibodies and found a significant re- Generation of neuroligin-2 R215H mutant mice duction of NL2 puncta in R215H Het mice and almost Following our original discovery of a loss-of-function muta- absence of NL2 puncta in homozygous R215H KI mice tion R215H of NL2 in SCZ patients [56], we generated the (Fig. 1d-f). In WT mouse brains, NL2 formed numerous NL2 R215H mutant mice by introducing the same R215H postsynaptic puncta on cell soma and dendrites oppos- mutation into the exon 4 of Nlgn2 gene in the mouse gen- ing presynaptic vGAT puncta (Fig. 1d-f, puncta density 2 2 ome via homologous recombination (Fig. 1a). NL2 R215H 15.0 ± 0.9 per 100 μm , puncta size = 0.27 ± 0.01 μm ). heterozygotes were mated to obtain wild type (WT), het- The number and size of NL2 puncta were significantly erozygotes (referred here as Het mice), and homozygotes reduced in the NL2 R215H Het mouse brains (Fig. 1d-f, (referred here as KI mice) (Additional file 1:FigureS1a). Se- puncta density, 9.4 ± 1.6 per 100 μm , p < 0.01, puncta quencing analysis confirmed the R215H mutation in the size, 0.21± 0.02 μm , p = 0.02). Interestingly, in the NL2 KI mice (Additional file 1: Figure S1b). Mice carrying homozygous NL2 R215H KI mouse brains, only faint R215H mutation were born at a normal Mendelian rate NL2 signal was observed inside cell soma (Fig. 1d, right (Male mice: WT = 26.5%, Het = 52.9%, KI = 20.6%; Female columns, Additional file 1: Figure S4) and not coloca- mice: WT = 24.1%, Het = 52.8%, KI = 23.1%). Both R215H lized with vGAT, further suggesting that the NL2 R215H Het and KI mice were viable and fertile and did not exhibit proteins could not be transported to the cell membrane premature mortality. During development, we observed a [56]. To get a clear understanding of the physiological reduction of body weight in R215H KI mice comparing to role of NL2 R215H mutation in vivo, we focused our R215H Het and WT mice in large litters (litter size > 6, studies on the homozygous NL2 R215H KI mice in this Additional file 1: Figure S2a-b), but this phenomenon is not study. significant in small litters (litter size < 5, Additional file 1: Figure S2c). Body length and tail length is not significantly Reduced GABAergic synapse density in NL2 R215H KI different between genotypes (Additional file 1:Figure mice S2d-g). The mouse colony was maintained on a hybrid NL2 has been reported to form complex with genetic background to avoid the artificial phenotype con- gephyrin and collybistin at postsynaptic sites to recruit tributed by other homozygous genetic variants in a homo- GABA receptors [47]. Consistent with a substantial zygous inbred background. reduction of NL2 puncta in the KI mice, we detected a remarkable decrease of postsynaptic GABA recep- Reduction of neuroligin-2 protein level in NL2 R215H KI tor γ2 subunit and the scaffold protein gephyrin mice around cell soma in hippocampal regions (Fig. 2a). After obtaining the NL2 R215H Het and KI mice, we Quantitative analysis revealed that both the puncta first analyzed the NL2 protein expression level in the number and size of postsynaptic γ2 subunit and brain. We found that as early as postnatal 2 days, NL2 gephyrin decreased significantly in homozygous already showed substantial expression in the WT mice R215H KI mice (Fig. 2b-e), consistent with previous (Fig. 1b, top row). In NL2 R215H Het mice, the NL2 findings in NL2 KO mice [1, 19, 30, 47]. In addition protein level was about half of the WT mice; whereas in to postsynaptic changes, we also examined presynaptic R215H KI mice, the NL2 level was very low comparing marker vGAT (vesicular GABA transporter) and par- to the WT level, but with a clear band of lower molecu- valbumin (PV) positive GABAergic neurons that are lar weight representing non-glycosylated immature NL2 reported to be associated with SCZ patients [37]. Im- R215H protein [56, 65]. Such immature band of NL2 munohistochemistry analysis revealed that the number R215H protein persisted at adult stage in the KI mice, of PV neurons was not changed in the hippocampal Jiang et al. Molecular Brain (2018) 11:31 Page 3 of 11 Fig. 1 Generation and characterization of NL2 R215H mice. a A simplified diagram of NL2 R215H homologous recombination strategy. b Representative Western blot of NL2 protein expression at postnatal 2 days, 6 days, 21 days, and adult stage of WT, NL2 R215H Het, and NL2 R215H KI mice. GAPDH was used as internal control. c Quantification of NL2 protein expression level in littermates. WT, n = 6 mice, Het, n =5 mice, and KI, n = 6 mice. One-way ANOVA with post-hoc Tukey multi-comparison test was used for statistical analysis. d Representative images of NL2 postsynaptic puncta in WT, NL2 R215H Het and NL2 R215H KI mice. Images were taken at hippocampal CA1 region. Upper row scale bar = 10 μm. Bottom row scale bar = 5 μm. e, f Quantification of NL2 puncta number and size. Nine brain slices from 3 mice for each genotype were used for analysis. One-way ANOVA with post-hoc Tukey multi-comparison test was used for statistical analysis in (e). Student’s t-test was used for statistical analysis in (f). Data were shown as Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 area of the KI mice (Fig. 3a-d). However, both PV and Impaired GABAergic neurotransmission in NL2 R215H KI vGAT puncta number and size were significantly re- mice duced in the dentate granule cells (Fig. 3e-i), as well We next investigated the function of inhibitory neurotrans- as in the CA1/CA3 pyramidal cells in the KI mice mission in the R215H KI mice. Whole-cell patch-clamp re- (Additional file 1: Figure S5a-j). Consistently, we ob- cordings were performed on dentate granule cells in acute served a reduction of PV and vGAT protein level in brain slices of adult WT and homozygous R215H KI mice. the hippocampal tissue of KI mice (Additional file 1: We found that both the frequency and amplitude of mini- Figure S6a-b). In contrast, the excitatory presynaptic atureinhibitorypostsynapticcurrents (mIPSCs)weresig- marker vGluT1 was not altered in R215H KI mice nificantly decreased in the granule cells of R215H KI mice (Additional file 1: Figure S6c-e). These results suggest (Fig. 4a-d; Frequency: WT = 8.28 ± 2.21 Hz, KI = 3.98 ± that NL2 R215H mutation impaired both pre- and 0.78 Hz, p = 0.041; Median amplitude: WT = 41.3 ± 2.9 pA, post-synaptic GABAergic components. KI=32.7±1.8pA, p = 0.019; Student’s t-test). In contrast, Jiang et al. Molecular Brain (2018) 11:31 Page 4 of 11 Fig. 2 Reduced GABAergic postsynaptic components in the hippocampus of NL2 R215H KI mice. a Representative images of gephyrin, GABA receptor γ2 subunit and merged immunostaining in the granule cell layer of WT and R215H KI mice. b, c Quantification of gephyrin puncta number and size at granule cell soma region. d, e Quantification of γ2 puncta number and size at the same region as gephyrin. WT = 9 slices from 3 mice, R215H KI = 12 slices from 3 mice. Scale bar = 10 μm. Student’s t-test was used for analysis and data were shown as Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 there was no significant change of miniature excitatory although the total distance traveled was similar to the postsynaptic currents (mEPSCs) in the dentate granule cells WT mice (Fig. 5a-d). Consistently, in the elevated plus of R215H KI mice compared to WT mice (Fig. 4e-h). The maze test, the KI mice spent much less time in the open kinetics of both mIPSCs and mEPSCs were not altered arm compared to the WT mice, while the total travel (Additional file 1: Figure S7). These Results indicate that distance was also similar between the KI and WT mice NL2 R215H mutation is primarily affecting inhibitory (Fig. 5e-h). These results suggest that the R215H KI neurotransmission. mice display an increased level of anxiety while their locomotion activity is relatively normal. Behavioral deficits in NL2 R215H KI mice We next examined in R215H KI mice the acoustic The significant reduction of inhibitory neurotransmis- startle response and pre-pulse inhibition, a standard test sion in the NL2 R215H mutant mice prompted us to for the sensory motor gating function often assessed in further investigate whether such severe GABAergic defi- schizophrenia patients [5]. R215H KI mice showed a sig- cits will result in any behavioral deficits. We first per- nificant reduction in the startle response when stimu- formed open field test (10 min). We found that the KI lated at 100–120 dB (Fig. 5i). Furthermore, the pre-pulse mice spent significantly less time in the center region, inhibition was significantly impaired in the KI mice Jiang et al. Molecular Brain (2018) 11:31 Page 5 of 11 Fig. 3 Reduced GABAergic presynaptic components in the hippocampus of NL2 R215H KI mice. a Representative images of PV staining at the hippocampus in WT and R215H KI mice. Scale bar = 200 μm. b-d Quantification of PV-positive neurons at DG, CA2/3, and CA1 region. WT, n =14 slices / 5 mice; KI, n = 14 slices / 5 mice. e Representative images of PV, vGAT and merged immunostaining in the granule cell layer of WT and R215H KI mice. f, g Quantification of PV puncta number and size that targeted to the granule cell layer. h, i Quantification of vGAT puncta number and size that targeted to the same region. WT = 12 slices / 5 mice, R215H KI = 12 slices / 5 mice. Scale bar = 10 μm. Student’s t test was used for analysis and data were shown as Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 Fig. 4 NL2 R215H KI mice have decreased inhibitory synaptic transmission at the hippocampal region. a, b Representative traces of miniature inhibitory postsynaptic currents (mIPSCs) recorded from DG granule cells in hippocampal slices of WT (black) and R215H KI (red) mice. WT, n =14 cells / 4 mice; R215H KI, n = 18 cells / 4 mice. c, d Quantification of the mIPSC frequency and amplitude (Student’s t-test). e, f Representative traces of miniature excitatory postsynaptic currents (mEPSCs) in the DG region of hippocampal slices from WT (black) and R215H KI (red) mice. WT, n = 11 cells / 4 mice; KI, n = 12 cells / 3 mice. g, h Quantification of the mEPSC frequency and amplitude (Student’s t-test). Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 Jiang et al. Molecular Brain (2018) 11:31 Page 6 of 11 Fig. 5 NL2 R215H KI mice display schizophrenia-like behaviors. a Representative running track of WT and R215H KI mice (male) in an open field within 10 min duration. b The center time of WT and KI mice spent in the open field. c The frequency of WT and KI mice entering the center zone of the open field. d The total distance of WT and KI mice traveled in the open field test. e Representative running track of WT and R215H KI mice (male) in elevated plus maze for 5 min. White line indicates closed-arms. f The quantified time spent in the open-arms of WT and KI mice. g The time spent in the closed-arms of WT and KI mice. (h) The total distance traveled in the elevated plus maze test. a-h WT mice n = 11, KI mice n = 12,; Student’s t-test was used for analysis. i Startle response of WT and R215H KI mice (male) toward 80, 90, 100, 110, and 120 dB sound pulses. j Thepercentageofpre-pulse inhibition (PPI) toapre-pulse of 74 dB, 78dB, and 86dB. WT mice n = 12, KI mice n = 9. Two-way ANOVA with Sidak’s multiple comparison test was used for analysis. k Spontaneous Y maze test. WT mice n = 10, KI mice n =12, Student’s t-test. l Contextual fear conditioning test. R215H KI mice exhibit significant reduction of freezing time when placed back in the test chamber after 1–7 days of shock training (Two-way ANOVA with Sidak’s multiple comparison test, genotype F = 172.7, P < 0.0001, WT n =8, KI n =5). m Forced swim test. Freezing time were (1, 99) analyzed. WT mice n = 23, KI mice n = 16, Student’s t-test. Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 compared to the WT mice (Fig. 5j). Together, these defi- (Fig. 5k), indicating a working memory dysfunction. In the cits of R215H KI mice suggest that this new transgenic contextual fear conditioning test, while the KI mice were mouse model may recapitulate symptoms of schizophre- capable to associate the conditioning chamber with nia patients. foot-shock in the initial training, indicated by an increase of To further characterize the R215H KI mice, we investi- freezing state after foot-shock, they failed to retain the fear gated their cognitive functions by spontaneous Y maze and context memory in the followingdayswhentested(Fig. 5l), contextual fear conditioning test. In the Y maze test, we indicating an impaired hippocampal dependent cognitive found that R215H KI mice displayed a significant reduction function. Furthermore, we performed forced swim test to of spontaneous alternation compared to the WT mice investigate whether R215H KI mice have any Jiang et al. Molecular Brain (2018) 11:31 Page 7 of 11 depression-like behavior, because certain SCZ patients DG (Fig. 6f) regions in the KI mice. These results sug- display depression symptom. Interestingly, we observed gest that NL2 R215H KI mice had impaired hippocam- a reduction of freezing time in KI mice when perform- pal activation during acute stress. ing the forced swim test (Fig. 5m), consistent with our observation below that the KI mice show hyperactivity Discussion induced by acute stress. The behavioral data shown In the present study, we generated a unique mouse model above was all obtained from male mice, and the female carrying a single point mutation R215H of NLGN2 gene mice were also tested and exhibited the same trend that was originally identified from human schizophrenia (Additional file 1:FigureS8). patients. The NL2 R215H KI mice have impaired GABAergic synapse development, reduced inhibitory syn- Impaired hippocampal activation toward acute stress in aptic transmission, and decreased hippocampal activation NL2 R215H KI mice in response to stress. Moreover, the R215H KI mice dis- Schizophrenia is associated with abnormal response play anxiety-like behavior, impaired pre-pulse inhibition, to stress [60]. Stress is known to activate the cognitive deficits and abnormal stress response, partially hypothalamic-pituitary-adrenal axis (HPA axis) and recapitulating some of the core symptoms of schizophre- induce the hormone release of corticosterone (CORT) nia patients. These results suggest that this newly gener- into circulation [34, 41]. To investigate the stress re- ated R215H KI mouse line may provide a unique animal sponse of R215H KI mice, we put the WT and model for studying molecular mechanisms underlying R215H KI mice into restraining tubes for 1 hour as schizophrenia and related neuropsychiatric disorders. an acute stress test. We found that R215H KI mice struggled much more intensively for a long time and GABAergic and behavioral deficits in NL2 R215H KI mice excreted much more than the WT mice during the NL2 plays important roles in regulating perisomatic restraining test. After restraining, KI mice were more GABAergic synapse development, phasic GABAergic dirtyand stinkythan theWTmice(Fig. 6a). In ac- transmission, and neural excitability [1, 3, 9, 19, 24, 25, cordance, R215H KI mice showed a much higher 30, 40, 47, 59, 61]. Consistent with our previous in vitro level of CORT (384 ± 53 ng/ml) after restraining com- studies, the current in vivo work demonstrates that pared to the WT mice (215 ± 20 ng/ml). The baseline R215H mutation disrupts GABAergic synapse develop- level of CORT was similar between WT (49 ± 4 ng/ml) ment. Functionally, NL2 R215H mutation caused a re- and KI mice (36 ± 4 ng/ml) (Fig. 6b; p = 0.0035 after re- duction of both frequency and amplitude of inhibitory straint, Two way ANOVA followed with Sidak’spost hoc neurotransmission. These results suggest that the R215H test). These results suggest that R215H KI mice have KI mice display more GABAergic deficits than the re- hyperactive HPA response toward stress. ported NL2 KO mice [1, 9, 19, 30, 47], which might ex- Following the activation of HPA axis, hippocampus plain why our KI mice display more behavioral deficits will be activated as a negative feedback regulator and than the NL2 KO mice, such as PPI impairment, cogni- control the CORT level within normal range [23, 58]. To tive deficits, and abnormal stress response. Coinciden- examine the hippocampal activation in R215H KI mice tally, previous studies reported that NL3 R451C KI following the acute stress, we used a naïve cohort of mouse also displayed stronger phenotypes than the NL3 mice to perform the restraining test again. R215H KI KO mice [14, 16, 57, 64]. These evidences suggest that and WT mice were subjected to restraint for half an genetic mouse models based on mutations identified hour and then sacrificed after 2 h. Hippocampal activa- from patients may be more suitable than the germline tion was examined by assessing the expression level of KO mouse models for studying pathological mechanisms an immediate early gene cFos [42, 48]. At the baseline of human diseases, because of less compensation from level, very few cFos-positive neurons were detected in other genes in KI mice than in KO mice. the hippocampal regions in both WT and KI mice Behaviorally, NL2 R215H KI mice display an anxiety (Fig. 6c, top row). After stress, we observed a significant phenotype, which may be the result of decreased GABAer- increase of cFos-positive cells in the DG and CA2/3 re- gic inhibition [3, 11, 63]. Interestingly, R215H KI mice also gions of the hippocampus in WT mice (Fig. 6c, bottom show impaired startle responses and deficits in pre-pulse left). In contrast, the R215H KI mice showed much re- inhibition (PPI). Previous study in rats has reported that duced cFos-positive cells in the same regions of hippo- disturbance of PV neuron development in the hippocampal campus (Fig. 6c, bottom right). This is better illustrated DG region may cause reduction of PPI [21]. A recent study in the enlarged images showing the CA2/3 and DG re- also demonstrates that specific inhibition of PV neurons in gions of WT mice (Fig. 6d, top row) and KI mice (Fig. 6d, the ventral hippocampus results in a reduction of both star- bottom row). Quantitative analysis confirmed the reduc- tle response and PPI [45]. Consistent with these findings, tion of cFos-positive cells in both CA2/3 (Fig. 6e) and we demonstrateherethatour R215HKImice display a Jiang et al. Molecular Brain (2018) 11:31 Page 8 of 11 Fig. 6 Hippocampal neurons have impaired activation toward acute stress in NL2 R215H KI mice. a Typical appearance of WT and KI mice after restraining. b Quantified corticosteroid level of WT and KI mice at the baseline level and after 1 h restraining. 4 to 8 mice were used for each genotype at each condition, age 4 to 6 months. Two-way ANOVA with Sidak’smultiple comparison test wasused for analysis. c Upper row: representative images of baseline cFos immunoreactivity of WT and R215H KI hippocampus; bottom row: cFos immunoreactivity of WT and R215H KI hippocampus after restraining. d enlarged DG and CA2/3 region of WT and R215H mice after restraining. e Quantification of cFos-positive cells at DG granule cell layers. f Quantification of cFos positive cells at CA2/3 pyramidal cell layers. WT: n = 16 brain slices / 4 mice; R215H KI: n = 20 brain slices / 5 mice; scale bar = 200 μm. Student’s t-test was used for analysis. Data represent mean ± SEM; *P <0.05, **P < 0.01, ***P <0.001 significant reduction of PV innervation in the hippocam- Surprisingly, our former collaborator Dr. Chia-Hsiang pus, which may underlie the deficits of PPI. In contrast, the Chen’s group recently reported that their NL2 R215H KI NL2 KO mice lack PPI deficit, which might be related to an mice displayed an increased pre-pulse inhibition pheno- insufficient loss of PV innervation at hippocampal regions type [8]. However, because the startle response of their KI [61]. Besides PV neurons, CCK (cholecystokinin) neurons mice was not reported, it makes the data difficult to com- are another type of inhibitory neurons mainly innervate pare with ours. Additionally, they reported that their KI CA1/2/3 pyramidal cells and DG proximal dendrites. CCK mice didn’t express NL2 and resembled global NL2 KO neurons can release GABA to act on GABA receptor α2 mice, but they did not present the actual comparison with subunits that are known to mediate anxiolytic effect, or re- NL2 KO mice. In contrast, our R215H KI mice are clearly lease CCK to act on CCK2 receptors and induce anxiogenic different from the NL2 KO mice, because our KI mice effect [18]. It would be important to further investigate the showed small amount of NL2 expression, particularly dur- expression and functional alteration of CCK neurons in our ing early developmental stages. The low expression level R215H KI mice in future studies. of NL2 in our R215H KI mice distinguishes our KI mice Jiang et al. Molecular Brain (2018) 11:31 Page 9 of 11 from the NL2 KO mice, which showed completely absent Siu-Pok Yee’s team at the University of Connecticut expression of NL2 in our Western blot analysis. Further- Health Center. NL2 KO mice were purchased from Jack- more, our R215H KI mice also showed clear GABAergic son Laboratory (stock# 008139). The detailed procedures deficits as expected, but it is unknown whether their KI are described in the Additional file 1. mice have any GABAergic deficits or not [8]. All the experimental mice were group housed (2–3 Another interesting observation is that the R215H KI mice per cage) in home cages and lived at a constant mice are hyperactive after acute stress and are associated 25 °C in a 12 h light/dark cycle. Mice were given ad libi- with impaired hippocampal activation. It has been reported tum access to food and water. Littermate or age and that robust neuron activation requires low background ac- gender matched mice were used for experiments. All tivity before stimulus [33, 49]. However, due to the reduc- animal care and experiments followed the Penn State tion of GABAergic inhibition in our R215H KI mice, the University IACUC protocol and NIH guidelines. background activity of hippocampal neurons may be chronically elevated, which will dampen further activation Biochemical measurements of the hippocampus by external stimulation [40]. The im- Protein levels were quantifiedusingtotalbrain homoge- paired activation of hippocampal neurons in R215H KI nates from 3 groups of adult male littermates- WT, hetero- mice may contribute to the abnormal stress response we zygous and homozygous. The western blot system used observed, as hippocampus acts like a “brake” during acute was the standard Bio-Rad mini protein electrophoresis sys- stress to prevent HPA axis from over activation [22]. Be- tem and the procedure followed the system manual. LiCOR sides hippocampus, sensitized HPA-axis involves brain re- Odyssey Clx was used for protein signal detection. The gions such as hypothalamus and amygdala. Loss of NL2 in antibodies used were Rb anti-Neuroligin 2 (1:1000, SYSY these areas could directly affect their GABAergic transmis- 129202), Rb anti-GAPDH (1:10000, Sigma G9545), and Gt sion and releasing of corticosteroids into the circulation, anti-Rb 800 (1:15000, P/N 925-32210, P/N 925-32211). De- which is worth of further investigation as well. tailed procedures are described in the Additional file 1. NL2 R215H mutation and schizophrenia Immunohistochemistry, image acquisition, and image It is well documented that schizophrenia patient’sshow im- analysis paired pre-pulse inhibition as an abnormal sensorimotor Mouse brain slices were prepared at 20–40 μM and reacted gating deficit [5, 20]. Many patients also have emotional with the primary antibodies Rb anti-Neuroligin 2 (1:1000, symptoms such as anxiety and depression [39]. Addition- SYSY129203), Ms. anti-Parvalbumin (1:1000, MAB1572), ally, patients are hypersensitive toward stress and certain GP anti-vGAT (1:1000, SYSY 131004), Gephyrin (1:1000, patients have been found with altered HPA axis function SYSY 147011), GABAaR γ2 (1:1000 SYSY 224003), and [4]. Intriguingly, R215H KI mice recapitulated these c-Fos (1:5000 Sigma F7799). The fluorescent secondary SCZ-like behaviors, suggesting a potential role of NL2 antibodies used were Gt anti-Rb 488, Gt anti-Ms Cy3, and R215H in the development of schizophrenia symptoms. Gt anti-GP 647. Images were taken with the Olympus Furthermore, reduction of PV expression and PV-positive FV1000 confocal microscope. The number of neurons and synapses is a prominent phenotype observed in SCZ pa- thedensity andsizeofsynapticpunctawereanalyzedwith tients [36–38, 62]. The R215H mutation KI mice also show the NIH ImageJ software (NIH, Bethesda, MD, USA). A de- a significant reduction of PV innervation, consistent with tailed description of the experimental procedures is in the the pathogenic deficit of SCZ patients. These GABAergic Additional file 1. deficits, together with cognition and PPI deficits manifested in the KI mice, support the hypothesis that GABA dysfunc- Slice electrophysiology tion makes an important contribution to the cognitive and Horizontal acute hippocampal slices were used for attention deficits of SCZ. Taken together, NLGN2 R215H whole-cell patch clamp recordings. Miniature inhibitory single point mutation has a significant impact on GABAer- or excitatory postsynaptic currents (mIPSCs or mEPSCs) gic synapse development and the pathogenesis of neuro- were pharmacologically isolated by including DNQX and psychiatric disorders. Our newly generated NL2 R215H KI APV or picrotoxin together with tetrodotoxin in artificial micemayprovideausefulmousemodelfor thestudyof cerebrospinal fluid. Details are in the Additional file 1. molecular mechanisms and drug development of neuro- psychiatric disorders including schizophrenia. Behavioral tests Overview Methods The mice for behavior tests were group housed by geno- NL2 R215H knock-in mice type. All tests were performed during 1 pm to 6 pm. The NL2 R215H knock-in mice were generated by hom- Four cohorts of mice were used: First cohort of mice ologous recombination in embryonic stem cells by Dr. was first tested for open field, elevated plus maze, and Y Jiang et al. Molecular Brain (2018) 11:31 Page 10 of 11 maze at 2–3 months old, and then tested for the startle Received: 27 February 2018 Accepted: 24 May 2018 response and pre-pulse inhibition at 3.5 months old. Second cohort of mice was used for contextual fear con- References ditioning test at 2–3 months old. Third cohort of mice 1. Babaev O, Botta P, Meyer E, Muller C, Ehrenreich H, Brose N, Luthi A, was used for restraining and corticosteroid serum level Krueger-Burg D. Neuroligin 2 deletion alters inhibitory synapse function and test at 4–6 months old. Fourth cohort of mice was tested anxiety-associated neuronal activation in the amygdala. Neuropharmacology. 2016;100:56–65. for forced swim at 3 months old. The open field test and 2. Baudouin SJ, Gaudias J, Gerharz S, Hatstatt L, Zhou K, Punnakkal P, Tanaka elevated maze data were analyzed by Noldus Ethovision KF, Spooren W, Hen R, De Zeeuw CI, et al. Shared synaptic pathophysiology XT 8.0 software. Y maze and forced swim tests were an- in syndromic and nonsyndromic rodent models of autism. Science. 2012; 338:128–32. alyzed with the researcher blind to genotype. Detailed 3. Blundell J, Tabuchi K, Bolliger MF, Blaiss CA, Brose N, Liu X, Sudhof TC, procedures are in Additional file 1. Powell CM. Increased anxiety-like behavior in mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2. Genes Brain Behav. 2009;8: 114–26. Additional file 4. Bradley AJ, Dinan TG. Review: a systematic review of hypothalamic-pituitary- adrenal axis function in schizophrenia: implications for mortality. J Psychopharmacol. 2010;24:91–118. Additional file 1: Supplemental information. (DOCX 6071 kb) 5. Braff DL, Grillon C, Geyer MA. Gating and habituation of the startle reflex in schizophrenic patients. Arch Gen Psychiatry. 1992;49:206–15. 6. Bucan M, Abrahams BS, Wang K, Glessner JT, Herman EI, Sonnenblick LI, Abbreviations Alvarez Retuerto AI, Imielinski M, Hadley D, Bradfield JP, et al. Genome-wide APV: D(−)-2-Amino-5-phosphonopentanoic acid; CORT: Corticosterone; analyses of exonic copy number variants in a family-based study point to DG: Dentate gyrus; DNQX: 6,7-dinitroquinoxaline-2,3-dione; ER: Endoplasmic novel autism susceptibility genes. PLoS Genet. 2009;5:e1000536. reticulum; HPA: Hypothalamic-pituitary-adrenal axis; KI: Knock in; KO: Knock 7. Budreck EC, Scheiffele P. Neuroligin-3 is a neuronal adhesion protein at out; mEPSC: Miniature excitatory postsynaptic current; mIPSC: Miniature GABAergic and glutamatergic synapses. Eur J Neurosci. 2007;26:1738–48. inhibitory postsynaptic current; NL-2: Neuroligin-2; NLGN: Neuroligin; 8. Chen CH, Lee PW, Liao HM, Chang PK. Neuroligin 2 R215H mutant mice NRXN: Neurexin; PPI: Pre-pulse inhibition; PV: Parvalbumin; manifest anxiety, increased prepulse inhibition, and impaired spatial SCZ: Schizophrenia; vGAT: Vesicular GABA transporter; WT: Wild type learning and memory. Front Psychiatry. 2017;8:257. 9. Chubykin AA, Atasoy D, Etherton MR, Brose N, Kavalali ET, Gibson JR, Sudhof Acknowledgements TC. Activity-dependent validation of excitatory versus inhibitory synapses by We would like to thank Dr. Thomas Fuchs for providing advices on neuroligin-1 versus neuroligin-2. Neuron. 2007;54:919–31. behavioral tests, Yuting Bai for providing initial genotyping support. We 10. Connor SA, Ammendrup-Johnsen I, Chan AW, Kishimoto Y, Murayama C, thank all members from Chen lab for thoughtful suggestions. Kurihara N, Tada A, Ge Y, Lu H, Yan R, et al. Altered cortical dynamics and cognitive function upon haploinsufficiency of the autism-linked excitatory synaptic suppressor MDGA2. Neuron. 2016;91:1052–68. Funding 11. Dalvi A, Rodgers RJ. GABAergic influences on plus-maze behaviour in mice. This study is supported by grants from NIH (MH092740 and MH083911) and Psychopharmacol. 1996;128:380–97. Charles H. “Skip” Smith Brain Repair Endowment Fund to G. C. 12. Dong N, Qi J, Chen G. Molecular reconstitution of functional GABAergic synapses with expression of neuroligin-2 and GABAA receptors. Mol Cell Neurosci. 2007;35:14–23. Availability of data and materials 13. Durand CM, Betancur C, Boeckers TM, Bockmann J, Chaste P, Fauchereau F, All data generated or analysed during this study are included in this Nygren G, Rastam M, Gillberg IC, Anckarsater H, et al. Mutations in the gene published article and its supplementary information files. encoding the synaptic scaffolding protein SHANK3 are associated with autism spectrum disorders. Nat Genet. 2007;39:25–7. Authors’ contributions 14. Etherton M, Foldy C, Sharma M, Tabuchi K, Liu X, Shamloo M, Malenka RC, DYJ performed most of the experiments and wrote the draft manuscript. GC Sudhof TC. Autism-linked neuroligin-3 R451C mutation differentially alters supervised the entire work and revised the manuscript. CF performed forced hippocampal and cortical synaptic function. Proc Natl Acad Sci U S A. 2011; swim test and measured body weight. CF and YH helped genotyping and 108:13764–9. Western blot. ZW helped electrophysiology recordings. SPY generated the 15. Etherton MR, Blaiss CA, Powell CM, Sudhof TC. Mouse neurexin-1alpha NL-2 R215H transgenic mice as a paid service. 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GABAergic deficits and schizophrenia-like behaviors in a mouse model carrying patient-derived neuroligin-2 R215H mutation

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Abstract

Schizophrenia (SCZ) is a severe mental disorder characterized by delusion, hallucination, and cognitive deficits. We 215 215 have previously identified from schizophrenia patients a loss-of-function mutation Arg →His (R215H) of neuroligin 2 (NLGN2) gene, which encodes a cell adhesion molecule critical for GABAergic synapse formation and function. Here, we generated a novel transgenic mouse line with neuroligin-2 (NL2) R215H mutation. The single point mutation caused a significant loss of NL2 protein in vivo, reduced GABAergic transmission, and impaired hippocampal activation. Importantly, R215H KI mice displayed anxiety-like behavior, impaired pre-pulse inhibition (PPI), cognition deficits and abnormal stress responses, recapitulating several key aspects of schizophrenia-like behaviors. Our results demonstrate a significant impact of a single point mutation NL2 R215H on brain functions, providing a novel animal model for the study of schizophrenia and neuropsychiatric disorders. Keywords: Schizophrenia, GABA, Neuroligin-2, Mouse model, Mutation Introduction differentially locate to excitatory and inhibitory synapses Schizophrenia (SCZ) is a chronic neuropsychiatric dis- and are critical for the excitatory and inhibitory synapse for- order caused by both genetic and environmental factors. mation and function, respectively [9, 12, 35, 44, 51, 53, 59]. It is featured by long-standing delusion and hallucin- Neuroligin-3 locates at both type of synapses and ation (psychosis), and cognitive deficits [17, 27, 39]. SCZ contributes to both neurotransmission [7, 14, 57]. In recent is a highly heritable disorder [55] with a complex genetic years, genetic variants of neuroligin-1, neuroligin-3 and basis. Recent genomic studies identified a number of neuroligin-4 have been identified in autism patients [28, 43]. genetic variants associated with SCZ, including a group Mutations in proteins interacting with neuroligins such as of variants resided in the genes encoding synaptic adhe- Neurexin1, SHANK and MDGA have also been associated sion molecules that promoting synaptic development with autism and schizophrenia patients [6, 13, 31, 32]. Gen- and function such as IGSF9B, and NLGN4X [52]. etic mouse models based on these findings recapitulate sev- Neuroligins (NLGNs) are a family of synaptic adhesion eral aspects of patient symptoms, providing an entry point molecules highly expressed in the brain and are ligands for for the mechanistic study and drug development on psychi- another group of cell adhesion molecules neurexins atric disorders [2, 10, 14, 15, 29, 46, 50, 54, 57, 66]. (NRXNs) [26]. There are five neuroligin genes (neuroligin-1, We have previously reported several novel mutations of − 2, − 3, − 4, and − 5) in humans and four in mice (neuroli- NLGN2 from schizophrenia patients [56]. Among the gin 1–4). Neuroligin-1, − 2, and − 3 are close homologs be- NL2 mutants, we found that the R215H mutant protein tween human and mice. Neuroligin-1 and neuroligin-2 was retained in the endoplasmic reticulum (ER) and could not be transported to the cell membrane, resulting in a failure to interact with presynaptic neurexin and a loss of * Correspondence: gongchen@psu.edu Department of Biology, Huck Institutes of Life Sciences, Pennsylvania State function in GABAergic synapse assembly [56]. Based on University, University Park, PA 16802, USA these studies, we have now generated a transgenic mouse Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jiang et al. Molecular Brain (2018) 11:31 Page 2 of 11 line carrying the same NL2 R215H mutation to test its and was observed in a variety of brain regions (Fig. 1b, functional consequence in vivo. We demonstrate that the and quantified in Fig. 1c). In contrast, NL2 KO mice R215H knock-in (KI) mice show severe GABAergic defi- showed a complete absence of NL2 without any imma- cits and display not only anxiety-like behavior seen in glo- ture band at all (Fig. 1b, top row). Such difference of bal NL2 KO mice [1, 3, 61], but also impaired pre-pulse NL2 protein level between our KI mice and previous KO inhibition, cognitive deficits, and abnormal stress re- mice may underlie their functional difference reported sponses which are not reported in global NL2 KO mice. later. To test whether NL2 R215H mutation affects the Our results suggest that a single-point mutation R215H of expression of other NL family members, we examined NL2 can result in significant GABAergic deficits and con- the protein level of NL1 and NL3 in both NL2 R215H tribute to SCZ-like behaviors. This newly generated NL2 Het and KI mice but found no significant changes R215H KI mouse may provide a useful animal model for (Additional file 1: Figure S3). the studies of neuropsychiatric disorders including SCZ. To investigate the localization of NL2 R215H proteins inside the brain, we performed immunohistochemistry Results with NL2-specific antibodies and found a significant re- Generation of neuroligin-2 R215H mutant mice duction of NL2 puncta in R215H Het mice and almost Following our original discovery of a loss-of-function muta- absence of NL2 puncta in homozygous R215H KI mice tion R215H of NL2 in SCZ patients [56], we generated the (Fig. 1d-f). In WT mouse brains, NL2 formed numerous NL2 R215H mutant mice by introducing the same R215H postsynaptic puncta on cell soma and dendrites oppos- mutation into the exon 4 of Nlgn2 gene in the mouse gen- ing presynaptic vGAT puncta (Fig. 1d-f, puncta density 2 2 ome via homologous recombination (Fig. 1a). NL2 R215H 15.0 ± 0.9 per 100 μm , puncta size = 0.27 ± 0.01 μm ). heterozygotes were mated to obtain wild type (WT), het- The number and size of NL2 puncta were significantly erozygotes (referred here as Het mice), and homozygotes reduced in the NL2 R215H Het mouse brains (Fig. 1d-f, (referred here as KI mice) (Additional file 1:FigureS1a). Se- puncta density, 9.4 ± 1.6 per 100 μm , p < 0.01, puncta quencing analysis confirmed the R215H mutation in the size, 0.21± 0.02 μm , p = 0.02). Interestingly, in the NL2 KI mice (Additional file 1: Figure S1b). Mice carrying homozygous NL2 R215H KI mouse brains, only faint R215H mutation were born at a normal Mendelian rate NL2 signal was observed inside cell soma (Fig. 1d, right (Male mice: WT = 26.5%, Het = 52.9%, KI = 20.6%; Female columns, Additional file 1: Figure S4) and not coloca- mice: WT = 24.1%, Het = 52.8%, KI = 23.1%). Both R215H lized with vGAT, further suggesting that the NL2 R215H Het and KI mice were viable and fertile and did not exhibit proteins could not be transported to the cell membrane premature mortality. During development, we observed a [56]. To get a clear understanding of the physiological reduction of body weight in R215H KI mice comparing to role of NL2 R215H mutation in vivo, we focused our R215H Het and WT mice in large litters (litter size > 6, studies on the homozygous NL2 R215H KI mice in this Additional file 1: Figure S2a-b), but this phenomenon is not study. significant in small litters (litter size < 5, Additional file 1: Figure S2c). Body length and tail length is not significantly Reduced GABAergic synapse density in NL2 R215H KI different between genotypes (Additional file 1:Figure mice S2d-g). The mouse colony was maintained on a hybrid NL2 has been reported to form complex with genetic background to avoid the artificial phenotype con- gephyrin and collybistin at postsynaptic sites to recruit tributed by other homozygous genetic variants in a homo- GABA receptors [47]. Consistent with a substantial zygous inbred background. reduction of NL2 puncta in the KI mice, we detected a remarkable decrease of postsynaptic GABA recep- Reduction of neuroligin-2 protein level in NL2 R215H KI tor γ2 subunit and the scaffold protein gephyrin mice around cell soma in hippocampal regions (Fig. 2a). After obtaining the NL2 R215H Het and KI mice, we Quantitative analysis revealed that both the puncta first analyzed the NL2 protein expression level in the number and size of postsynaptic γ2 subunit and brain. We found that as early as postnatal 2 days, NL2 gephyrin decreased significantly in homozygous already showed substantial expression in the WT mice R215H KI mice (Fig. 2b-e), consistent with previous (Fig. 1b, top row). In NL2 R215H Het mice, the NL2 findings in NL2 KO mice [1, 19, 30, 47]. In addition protein level was about half of the WT mice; whereas in to postsynaptic changes, we also examined presynaptic R215H KI mice, the NL2 level was very low comparing marker vGAT (vesicular GABA transporter) and par- to the WT level, but with a clear band of lower molecu- valbumin (PV) positive GABAergic neurons that are lar weight representing non-glycosylated immature NL2 reported to be associated with SCZ patients [37]. Im- R215H protein [56, 65]. Such immature band of NL2 munohistochemistry analysis revealed that the number R215H protein persisted at adult stage in the KI mice, of PV neurons was not changed in the hippocampal Jiang et al. Molecular Brain (2018) 11:31 Page 3 of 11 Fig. 1 Generation and characterization of NL2 R215H mice. a A simplified diagram of NL2 R215H homologous recombination strategy. b Representative Western blot of NL2 protein expression at postnatal 2 days, 6 days, 21 days, and adult stage of WT, NL2 R215H Het, and NL2 R215H KI mice. GAPDH was used as internal control. c Quantification of NL2 protein expression level in littermates. WT, n = 6 mice, Het, n =5 mice, and KI, n = 6 mice. One-way ANOVA with post-hoc Tukey multi-comparison test was used for statistical analysis. d Representative images of NL2 postsynaptic puncta in WT, NL2 R215H Het and NL2 R215H KI mice. Images were taken at hippocampal CA1 region. Upper row scale bar = 10 μm. Bottom row scale bar = 5 μm. e, f Quantification of NL2 puncta number and size. Nine brain slices from 3 mice for each genotype were used for analysis. One-way ANOVA with post-hoc Tukey multi-comparison test was used for statistical analysis in (e). Student’s t-test was used for statistical analysis in (f). Data were shown as Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 area of the KI mice (Fig. 3a-d). However, both PV and Impaired GABAergic neurotransmission in NL2 R215H KI vGAT puncta number and size were significantly re- mice duced in the dentate granule cells (Fig. 3e-i), as well We next investigated the function of inhibitory neurotrans- as in the CA1/CA3 pyramidal cells in the KI mice mission in the R215H KI mice. Whole-cell patch-clamp re- (Additional file 1: Figure S5a-j). Consistently, we ob- cordings were performed on dentate granule cells in acute served a reduction of PV and vGAT protein level in brain slices of adult WT and homozygous R215H KI mice. the hippocampal tissue of KI mice (Additional file 1: We found that both the frequency and amplitude of mini- Figure S6a-b). In contrast, the excitatory presynaptic atureinhibitorypostsynapticcurrents (mIPSCs)weresig- marker vGluT1 was not altered in R215H KI mice nificantly decreased in the granule cells of R215H KI mice (Additional file 1: Figure S6c-e). These results suggest (Fig. 4a-d; Frequency: WT = 8.28 ± 2.21 Hz, KI = 3.98 ± that NL2 R215H mutation impaired both pre- and 0.78 Hz, p = 0.041; Median amplitude: WT = 41.3 ± 2.9 pA, post-synaptic GABAergic components. KI=32.7±1.8pA, p = 0.019; Student’s t-test). In contrast, Jiang et al. Molecular Brain (2018) 11:31 Page 4 of 11 Fig. 2 Reduced GABAergic postsynaptic components in the hippocampus of NL2 R215H KI mice. a Representative images of gephyrin, GABA receptor γ2 subunit and merged immunostaining in the granule cell layer of WT and R215H KI mice. b, c Quantification of gephyrin puncta number and size at granule cell soma region. d, e Quantification of γ2 puncta number and size at the same region as gephyrin. WT = 9 slices from 3 mice, R215H KI = 12 slices from 3 mice. Scale bar = 10 μm. Student’s t-test was used for analysis and data were shown as Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 there was no significant change of miniature excitatory although the total distance traveled was similar to the postsynaptic currents (mEPSCs) in the dentate granule cells WT mice (Fig. 5a-d). Consistently, in the elevated plus of R215H KI mice compared to WT mice (Fig. 4e-h). The maze test, the KI mice spent much less time in the open kinetics of both mIPSCs and mEPSCs were not altered arm compared to the WT mice, while the total travel (Additional file 1: Figure S7). These Results indicate that distance was also similar between the KI and WT mice NL2 R215H mutation is primarily affecting inhibitory (Fig. 5e-h). These results suggest that the R215H KI neurotransmission. mice display an increased level of anxiety while their locomotion activity is relatively normal. Behavioral deficits in NL2 R215H KI mice We next examined in R215H KI mice the acoustic The significant reduction of inhibitory neurotransmis- startle response and pre-pulse inhibition, a standard test sion in the NL2 R215H mutant mice prompted us to for the sensory motor gating function often assessed in further investigate whether such severe GABAergic defi- schizophrenia patients [5]. R215H KI mice showed a sig- cits will result in any behavioral deficits. We first per- nificant reduction in the startle response when stimu- formed open field test (10 min). We found that the KI lated at 100–120 dB (Fig. 5i). Furthermore, the pre-pulse mice spent significantly less time in the center region, inhibition was significantly impaired in the KI mice Jiang et al. Molecular Brain (2018) 11:31 Page 5 of 11 Fig. 3 Reduced GABAergic presynaptic components in the hippocampus of NL2 R215H KI mice. a Representative images of PV staining at the hippocampus in WT and R215H KI mice. Scale bar = 200 μm. b-d Quantification of PV-positive neurons at DG, CA2/3, and CA1 region. WT, n =14 slices / 5 mice; KI, n = 14 slices / 5 mice. e Representative images of PV, vGAT and merged immunostaining in the granule cell layer of WT and R215H KI mice. f, g Quantification of PV puncta number and size that targeted to the granule cell layer. h, i Quantification of vGAT puncta number and size that targeted to the same region. WT = 12 slices / 5 mice, R215H KI = 12 slices / 5 mice. Scale bar = 10 μm. Student’s t test was used for analysis and data were shown as Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 Fig. 4 NL2 R215H KI mice have decreased inhibitory synaptic transmission at the hippocampal region. a, b Representative traces of miniature inhibitory postsynaptic currents (mIPSCs) recorded from DG granule cells in hippocampal slices of WT (black) and R215H KI (red) mice. WT, n =14 cells / 4 mice; R215H KI, n = 18 cells / 4 mice. c, d Quantification of the mIPSC frequency and amplitude (Student’s t-test). e, f Representative traces of miniature excitatory postsynaptic currents (mEPSCs) in the DG region of hippocampal slices from WT (black) and R215H KI (red) mice. WT, n = 11 cells / 4 mice; KI, n = 12 cells / 3 mice. g, h Quantification of the mEPSC frequency and amplitude (Student’s t-test). Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 Jiang et al. Molecular Brain (2018) 11:31 Page 6 of 11 Fig. 5 NL2 R215H KI mice display schizophrenia-like behaviors. a Representative running track of WT and R215H KI mice (male) in an open field within 10 min duration. b The center time of WT and KI mice spent in the open field. c The frequency of WT and KI mice entering the center zone of the open field. d The total distance of WT and KI mice traveled in the open field test. e Representative running track of WT and R215H KI mice (male) in elevated plus maze for 5 min. White line indicates closed-arms. f The quantified time spent in the open-arms of WT and KI mice. g The time spent in the closed-arms of WT and KI mice. (h) The total distance traveled in the elevated plus maze test. a-h WT mice n = 11, KI mice n = 12,; Student’s t-test was used for analysis. i Startle response of WT and R215H KI mice (male) toward 80, 90, 100, 110, and 120 dB sound pulses. j Thepercentageofpre-pulse inhibition (PPI) toapre-pulse of 74 dB, 78dB, and 86dB. WT mice n = 12, KI mice n = 9. Two-way ANOVA with Sidak’s multiple comparison test was used for analysis. k Spontaneous Y maze test. WT mice n = 10, KI mice n =12, Student’s t-test. l Contextual fear conditioning test. R215H KI mice exhibit significant reduction of freezing time when placed back in the test chamber after 1–7 days of shock training (Two-way ANOVA with Sidak’s multiple comparison test, genotype F = 172.7, P < 0.0001, WT n =8, KI n =5). m Forced swim test. Freezing time were (1, 99) analyzed. WT mice n = 23, KI mice n = 16, Student’s t-test. Data represent mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 compared to the WT mice (Fig. 5j). Together, these defi- (Fig. 5k), indicating a working memory dysfunction. In the cits of R215H KI mice suggest that this new transgenic contextual fear conditioning test, while the KI mice were mouse model may recapitulate symptoms of schizophre- capable to associate the conditioning chamber with nia patients. foot-shock in the initial training, indicated by an increase of To further characterize the R215H KI mice, we investi- freezing state after foot-shock, they failed to retain the fear gated their cognitive functions by spontaneous Y maze and context memory in the followingdayswhentested(Fig. 5l), contextual fear conditioning test. In the Y maze test, we indicating an impaired hippocampal dependent cognitive found that R215H KI mice displayed a significant reduction function. Furthermore, we performed forced swim test to of spontaneous alternation compared to the WT mice investigate whether R215H KI mice have any Jiang et al. Molecular Brain (2018) 11:31 Page 7 of 11 depression-like behavior, because certain SCZ patients DG (Fig. 6f) regions in the KI mice. These results sug- display depression symptom. Interestingly, we observed gest that NL2 R215H KI mice had impaired hippocam- a reduction of freezing time in KI mice when perform- pal activation during acute stress. ing the forced swim test (Fig. 5m), consistent with our observation below that the KI mice show hyperactivity Discussion induced by acute stress. The behavioral data shown In the present study, we generated a unique mouse model above was all obtained from male mice, and the female carrying a single point mutation R215H of NLGN2 gene mice were also tested and exhibited the same trend that was originally identified from human schizophrenia (Additional file 1:FigureS8). patients. The NL2 R215H KI mice have impaired GABAergic synapse development, reduced inhibitory syn- Impaired hippocampal activation toward acute stress in aptic transmission, and decreased hippocampal activation NL2 R215H KI mice in response to stress. Moreover, the R215H KI mice dis- Schizophrenia is associated with abnormal response play anxiety-like behavior, impaired pre-pulse inhibition, to stress [60]. Stress is known to activate the cognitive deficits and abnormal stress response, partially hypothalamic-pituitary-adrenal axis (HPA axis) and recapitulating some of the core symptoms of schizophre- induce the hormone release of corticosterone (CORT) nia patients. These results suggest that this newly gener- into circulation [34, 41]. To investigate the stress re- ated R215H KI mouse line may provide a unique animal sponse of R215H KI mice, we put the WT and model for studying molecular mechanisms underlying R215H KI mice into restraining tubes for 1 hour as schizophrenia and related neuropsychiatric disorders. an acute stress test. We found that R215H KI mice struggled much more intensively for a long time and GABAergic and behavioral deficits in NL2 R215H KI mice excreted much more than the WT mice during the NL2 plays important roles in regulating perisomatic restraining test. After restraining, KI mice were more GABAergic synapse development, phasic GABAergic dirtyand stinkythan theWTmice(Fig. 6a). In ac- transmission, and neural excitability [1, 3, 9, 19, 24, 25, cordance, R215H KI mice showed a much higher 30, 40, 47, 59, 61]. Consistent with our previous in vitro level of CORT (384 ± 53 ng/ml) after restraining com- studies, the current in vivo work demonstrates that pared to the WT mice (215 ± 20 ng/ml). The baseline R215H mutation disrupts GABAergic synapse develop- level of CORT was similar between WT (49 ± 4 ng/ml) ment. Functionally, NL2 R215H mutation caused a re- and KI mice (36 ± 4 ng/ml) (Fig. 6b; p = 0.0035 after re- duction of both frequency and amplitude of inhibitory straint, Two way ANOVA followed with Sidak’spost hoc neurotransmission. These results suggest that the R215H test). These results suggest that R215H KI mice have KI mice display more GABAergic deficits than the re- hyperactive HPA response toward stress. ported NL2 KO mice [1, 9, 19, 30, 47], which might ex- Following the activation of HPA axis, hippocampus plain why our KI mice display more behavioral deficits will be activated as a negative feedback regulator and than the NL2 KO mice, such as PPI impairment, cogni- control the CORT level within normal range [23, 58]. To tive deficits, and abnormal stress response. Coinciden- examine the hippocampal activation in R215H KI mice tally, previous studies reported that NL3 R451C KI following the acute stress, we used a naïve cohort of mouse also displayed stronger phenotypes than the NL3 mice to perform the restraining test again. R215H KI KO mice [14, 16, 57, 64]. These evidences suggest that and WT mice were subjected to restraint for half an genetic mouse models based on mutations identified hour and then sacrificed after 2 h. Hippocampal activa- from patients may be more suitable than the germline tion was examined by assessing the expression level of KO mouse models for studying pathological mechanisms an immediate early gene cFos [42, 48]. At the baseline of human diseases, because of less compensation from level, very few cFos-positive neurons were detected in other genes in KI mice than in KO mice. the hippocampal regions in both WT and KI mice Behaviorally, NL2 R215H KI mice display an anxiety (Fig. 6c, top row). After stress, we observed a significant phenotype, which may be the result of decreased GABAer- increase of cFos-positive cells in the DG and CA2/3 re- gic inhibition [3, 11, 63]. Interestingly, R215H KI mice also gions of the hippocampus in WT mice (Fig. 6c, bottom show impaired startle responses and deficits in pre-pulse left). In contrast, the R215H KI mice showed much re- inhibition (PPI). Previous study in rats has reported that duced cFos-positive cells in the same regions of hippo- disturbance of PV neuron development in the hippocampal campus (Fig. 6c, bottom right). This is better illustrated DG region may cause reduction of PPI [21]. A recent study in the enlarged images showing the CA2/3 and DG re- also demonstrates that specific inhibition of PV neurons in gions of WT mice (Fig. 6d, top row) and KI mice (Fig. 6d, the ventral hippocampus results in a reduction of both star- bottom row). Quantitative analysis confirmed the reduc- tle response and PPI [45]. Consistent with these findings, tion of cFos-positive cells in both CA2/3 (Fig. 6e) and we demonstrateherethatour R215HKImice display a Jiang et al. Molecular Brain (2018) 11:31 Page 8 of 11 Fig. 6 Hippocampal neurons have impaired activation toward acute stress in NL2 R215H KI mice. a Typical appearance of WT and KI mice after restraining. b Quantified corticosteroid level of WT and KI mice at the baseline level and after 1 h restraining. 4 to 8 mice were used for each genotype at each condition, age 4 to 6 months. Two-way ANOVA with Sidak’smultiple comparison test wasused for analysis. c Upper row: representative images of baseline cFos immunoreactivity of WT and R215H KI hippocampus; bottom row: cFos immunoreactivity of WT and R215H KI hippocampus after restraining. d enlarged DG and CA2/3 region of WT and R215H mice after restraining. e Quantification of cFos-positive cells at DG granule cell layers. f Quantification of cFos positive cells at CA2/3 pyramidal cell layers. WT: n = 16 brain slices / 4 mice; R215H KI: n = 20 brain slices / 5 mice; scale bar = 200 μm. Student’s t-test was used for analysis. Data represent mean ± SEM; *P <0.05, **P < 0.01, ***P <0.001 significant reduction of PV innervation in the hippocam- Surprisingly, our former collaborator Dr. Chia-Hsiang pus, which may underlie the deficits of PPI. In contrast, the Chen’s group recently reported that their NL2 R215H KI NL2 KO mice lack PPI deficit, which might be related to an mice displayed an increased pre-pulse inhibition pheno- insufficient loss of PV innervation at hippocampal regions type [8]. However, because the startle response of their KI [61]. Besides PV neurons, CCK (cholecystokinin) neurons mice was not reported, it makes the data difficult to com- are another type of inhibitory neurons mainly innervate pare with ours. Additionally, they reported that their KI CA1/2/3 pyramidal cells and DG proximal dendrites. CCK mice didn’t express NL2 and resembled global NL2 KO neurons can release GABA to act on GABA receptor α2 mice, but they did not present the actual comparison with subunits that are known to mediate anxiolytic effect, or re- NL2 KO mice. In contrast, our R215H KI mice are clearly lease CCK to act on CCK2 receptors and induce anxiogenic different from the NL2 KO mice, because our KI mice effect [18]. It would be important to further investigate the showed small amount of NL2 expression, particularly dur- expression and functional alteration of CCK neurons in our ing early developmental stages. The low expression level R215H KI mice in future studies. of NL2 in our R215H KI mice distinguishes our KI mice Jiang et al. Molecular Brain (2018) 11:31 Page 9 of 11 from the NL2 KO mice, which showed completely absent Siu-Pok Yee’s team at the University of Connecticut expression of NL2 in our Western blot analysis. Further- Health Center. NL2 KO mice were purchased from Jack- more, our R215H KI mice also showed clear GABAergic son Laboratory (stock# 008139). The detailed procedures deficits as expected, but it is unknown whether their KI are described in the Additional file 1. mice have any GABAergic deficits or not [8]. All the experimental mice were group housed (2–3 Another interesting observation is that the R215H KI mice per cage) in home cages and lived at a constant mice are hyperactive after acute stress and are associated 25 °C in a 12 h light/dark cycle. Mice were given ad libi- with impaired hippocampal activation. It has been reported tum access to food and water. Littermate or age and that robust neuron activation requires low background ac- gender matched mice were used for experiments. All tivity before stimulus [33, 49]. However, due to the reduc- animal care and experiments followed the Penn State tion of GABAergic inhibition in our R215H KI mice, the University IACUC protocol and NIH guidelines. background activity of hippocampal neurons may be chronically elevated, which will dampen further activation Biochemical measurements of the hippocampus by external stimulation [40]. The im- Protein levels were quantifiedusingtotalbrain homoge- paired activation of hippocampal neurons in R215H KI nates from 3 groups of adult male littermates- WT, hetero- mice may contribute to the abnormal stress response we zygous and homozygous. The western blot system used observed, as hippocampus acts like a “brake” during acute was the standard Bio-Rad mini protein electrophoresis sys- stress to prevent HPA axis from over activation [22]. Be- tem and the procedure followed the system manual. LiCOR sides hippocampus, sensitized HPA-axis involves brain re- Odyssey Clx was used for protein signal detection. The gions such as hypothalamus and amygdala. Loss of NL2 in antibodies used were Rb anti-Neuroligin 2 (1:1000, SYSY these areas could directly affect their GABAergic transmis- 129202), Rb anti-GAPDH (1:10000, Sigma G9545), and Gt sion and releasing of corticosteroids into the circulation, anti-Rb 800 (1:15000, P/N 925-32210, P/N 925-32211). De- which is worth of further investigation as well. tailed procedures are described in the Additional file 1. NL2 R215H mutation and schizophrenia Immunohistochemistry, image acquisition, and image It is well documented that schizophrenia patient’sshow im- analysis paired pre-pulse inhibition as an abnormal sensorimotor Mouse brain slices were prepared at 20–40 μM and reacted gating deficit [5, 20]. Many patients also have emotional with the primary antibodies Rb anti-Neuroligin 2 (1:1000, symptoms such as anxiety and depression [39]. Addition- SYSY129203), Ms. anti-Parvalbumin (1:1000, MAB1572), ally, patients are hypersensitive toward stress and certain GP anti-vGAT (1:1000, SYSY 131004), Gephyrin (1:1000, patients have been found with altered HPA axis function SYSY 147011), GABAaR γ2 (1:1000 SYSY 224003), and [4]. Intriguingly, R215H KI mice recapitulated these c-Fos (1:5000 Sigma F7799). The fluorescent secondary SCZ-like behaviors, suggesting a potential role of NL2 antibodies used were Gt anti-Rb 488, Gt anti-Ms Cy3, and R215H in the development of schizophrenia symptoms. Gt anti-GP 647. Images were taken with the Olympus Furthermore, reduction of PV expression and PV-positive FV1000 confocal microscope. The number of neurons and synapses is a prominent phenotype observed in SCZ pa- thedensity andsizeofsynapticpunctawereanalyzedwith tients [36–38, 62]. The R215H mutation KI mice also show the NIH ImageJ software (NIH, Bethesda, MD, USA). A de- a significant reduction of PV innervation, consistent with tailed description of the experimental procedures is in the the pathogenic deficit of SCZ patients. These GABAergic Additional file 1. deficits, together with cognition and PPI deficits manifested in the KI mice, support the hypothesis that GABA dysfunc- Slice electrophysiology tion makes an important contribution to the cognitive and Horizontal acute hippocampal slices were used for attention deficits of SCZ. Taken together, NLGN2 R215H whole-cell patch clamp recordings. Miniature inhibitory single point mutation has a significant impact on GABAer- or excitatory postsynaptic currents (mIPSCs or mEPSCs) gic synapse development and the pathogenesis of neuro- were pharmacologically isolated by including DNQX and psychiatric disorders. Our newly generated NL2 R215H KI APV or picrotoxin together with tetrodotoxin in artificial micemayprovideausefulmousemodelfor thestudyof cerebrospinal fluid. Details are in the Additional file 1. molecular mechanisms and drug development of neuro- psychiatric disorders including schizophrenia. Behavioral tests Overview Methods The mice for behavior tests were group housed by geno- NL2 R215H knock-in mice type. All tests were performed during 1 pm to 6 pm. The NL2 R215H knock-in mice were generated by hom- Four cohorts of mice were used: First cohort of mice ologous recombination in embryonic stem cells by Dr. was first tested for open field, elevated plus maze, and Y Jiang et al. Molecular Brain (2018) 11:31 Page 10 of 11 maze at 2–3 months old, and then tested for the startle Received: 27 February 2018 Accepted: 24 May 2018 response and pre-pulse inhibition at 3.5 months old. Second cohort of mice was used for contextual fear con- References ditioning test at 2–3 months old. Third cohort of mice 1. Babaev O, Botta P, Meyer E, Muller C, Ehrenreich H, Brose N, Luthi A, was used for restraining and corticosteroid serum level Krueger-Burg D. Neuroligin 2 deletion alters inhibitory synapse function and test at 4–6 months old. Fourth cohort of mice was tested anxiety-associated neuronal activation in the amygdala. Neuropharmacology. 2016;100:56–65. for forced swim at 3 months old. The open field test and 2. Baudouin SJ, Gaudias J, Gerharz S, Hatstatt L, Zhou K, Punnakkal P, Tanaka elevated maze data were analyzed by Noldus Ethovision KF, Spooren W, Hen R, De Zeeuw CI, et al. Shared synaptic pathophysiology XT 8.0 software. Y maze and forced swim tests were an- in syndromic and nonsyndromic rodent models of autism. Science. 2012; 338:128–32. alyzed with the researcher blind to genotype. Detailed 3. Blundell J, Tabuchi K, Bolliger MF, Blaiss CA, Brose N, Liu X, Sudhof TC, procedures are in Additional file 1. Powell CM. Increased anxiety-like behavior in mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2. Genes Brain Behav. 2009;8: 114–26. Additional file 4. Bradley AJ, Dinan TG. Review: a systematic review of hypothalamic-pituitary- adrenal axis function in schizophrenia: implications for mortality. J Psychopharmacol. 2010;24:91–118. Additional file 1: Supplemental information. (DOCX 6071 kb) 5. Braff DL, Grillon C, Geyer MA. Gating and habituation of the startle reflex in schizophrenic patients. Arch Gen Psychiatry. 1992;49:206–15. 6. Bucan M, Abrahams BS, Wang K, Glessner JT, Herman EI, Sonnenblick LI, Abbreviations Alvarez Retuerto AI, Imielinski M, Hadley D, Bradfield JP, et al. Genome-wide APV: D(−)-2-Amino-5-phosphonopentanoic acid; CORT: Corticosterone; analyses of exonic copy number variants in a family-based study point to DG: Dentate gyrus; DNQX: 6,7-dinitroquinoxaline-2,3-dione; ER: Endoplasmic novel autism susceptibility genes. PLoS Genet. 2009;5:e1000536. reticulum; HPA: Hypothalamic-pituitary-adrenal axis; KI: Knock in; KO: Knock 7. Budreck EC, Scheiffele P. Neuroligin-3 is a neuronal adhesion protein at out; mEPSC: Miniature excitatory postsynaptic current; mIPSC: Miniature GABAergic and glutamatergic synapses. Eur J Neurosci. 2007;26:1738–48. inhibitory postsynaptic current; NL-2: Neuroligin-2; NLGN: Neuroligin; 8. Chen CH, Lee PW, Liao HM, Chang PK. Neuroligin 2 R215H mutant mice NRXN: Neurexin; PPI: Pre-pulse inhibition; PV: Parvalbumin; manifest anxiety, increased prepulse inhibition, and impaired spatial SCZ: Schizophrenia; vGAT: Vesicular GABA transporter; WT: Wild type learning and memory. Front Psychiatry. 2017;8:257. 9. Chubykin AA, Atasoy D, Etherton MR, Brose N, Kavalali ET, Gibson JR, Sudhof Acknowledgements TC. Activity-dependent validation of excitatory versus inhibitory synapses by We would like to thank Dr. Thomas Fuchs for providing advices on neuroligin-1 versus neuroligin-2. Neuron. 2007;54:919–31. behavioral tests, Yuting Bai for providing initial genotyping support. We 10. 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Molecular BrainSpringer Journals

Published: Jun 1, 2018

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