LOTUS, an endogenous Nogo receptor antagonist, is involved in synapse and memory formation

The Nogo signal is involved in impairment of memory formation. We previously reported the lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of the Nogo receptor 1 that mediates the inhibition of axon growth and synapse formation. Moreover, we found that LOTUS plays an essential role in neural circuit formation and nerve regeneration. However, the effects of LOTUS on synapse formation and memory function have not been elucidated. Here, we clearly showed the involvement of LOTUS in synapse formation and memory function. The cultured hippocampal neurons derived from lotus gene knockout (LOTUS-KO) mice exhibited a decrease in synaptic density compared with those from wild-type mice. We also found decrease of dendritic spine formation in the adult hippocampus of LOTUS-KO mice. Finally, we demonstrated that LOTUS deficiency impairs memory formation in the social recognition test and the Morris water maze test, indicating that LOTUS is involved in functions of social and spatial learning and memory. These findings suggest that LOTUS affects synapse formation and memory function.


Results
LOTUS is distributed in the synapse region of cultured hippocampal neurons. NgR1 has been reported to be expressed at synapses and to negatively regulate synaptic morphology and density via the Nogo − NgR1 signal 10,12,14,15,28,29 . Here, first we examined the distribution of LOTUS in cultured hippocampal neurons using fluorescent immunostaining. We found that LOTUS was expressed in cellular regions that were costained with the Bassoon (a presynaptic marker) and postsynaptic density-95 (PSD-95, a postsynaptic marker), indicating that LOTUS is expressed in the synapse region of cultured hippocampal neurons (Fig. 1a − d). To assess the localization of LOTUS at the synapse, we compared LOTUS expression against that of Bassoon and PSD-95. We found that 33.5 ± 2.0% of PSD-95-positive puncta showed colocalization of PSD-95 and LOTUS, and 20.7 ± 3.1% of Bassoon-positive puncta showed colocalization of Bassoon and LOTUS (Fig. 1g). Next, we examined the localization of NgR1 and LOTUS at the post-synapse using fluorescent immunocytochemistry (Fig. 1e,f). We found that NgR1 was expressed in PSD-95-positive puncta, and that 28.6 ± 1.6% of PSD-95-positive puncta showed colocalization of LOTUS with NgR1. Moreover, 88.1 ± 6.1% of NgR1-positive, PSD-95-positive puncta showed colocalization of LOTUS with NgR1 (Fig. 1g). These findings suggest that LOTUS may be predominantly localized to the PSD-95-positive postsynapse and colocalizes with NgR1. Thus, LOTUS seems to be distributed in the synapse region of cultured hippocampal neurons.

Loss of LOTUS decreases synaptic density in cultured hippocampal neurons.
To investigate the function of LOTUS in synapse formation, we measured the synaptic density via simultaneous immunostaining of the postsynaptic marker PSD-95 and the presynaptic marker Bassoon in cultured hippocampal neurons. The number of positive staining deposits was measured ( Fig. 2a − d). The number of synapses was decreased in LOTUS-KO mice compared with WT mice (Fig. 2e). These data suggest that LOTUS contributes to synapse formation in cultured hippocampal neurons.

Loss of LOTUS decreases dendritic spine density in the hippocampus of adult mice. Because
LOTUS-KO mice showed a decrease in synaptic density in cultured hippocampal neurons (Fig. 2), next we investigated role of LOTUS in dendritic spine morphology in the adult hippocampus. Mice in which dendritic spines can be visualized were created using Thy1-EGFP mice. Thus, the spine density of EGFP-positive dendrites was examined in the hippocampus of adult Thy1-EGFP WT and Thy1-EGFP LOTUS-KO mice. The apical dendritic spine density in hippocampal CA1 pyramidal neurons was significantly decreased in LOTUS-KO mice compared with WT mice. In particular, the number of mushroom-type and thin-type spines was significantly decreased in LOTUS-KO mice. However, no difference was observed in the number of stubby-type spines in these mice (Fig. 3a,b). Similarly, the same measurement was performed in basal dendrite, which yielded similar results to those obtained for apical dendrites (Fig. 3c,d). These results indicate that loss of LOTUS reduces the number of thin and mushroom-type spines in hippocampal CA1 pyramidal neurons, as well as the total spine density, suggesting that LOTUS may contribute to hippocampal synapse formation.

Loss of LOTUS impairs hippocampus-dependent memory formation. To investigate whether
LOTUS deficiency affects learning and memory, we first evaluated the ability of social cognitive memory formation in WT and LOTUS-KO mice. The social recognition test is a behavioral analysis that evaluates hippocampal-dependent social cognitive memory formation in mice. In this experiment, a mature test mouse encounters a juvenile mouse as a stranger for 3 min; 24 h later, it is determined whether the test mouse remembers the juvenile mouse. A significant reduction in the investigation time was observed in WT mice (n = 9), while no significant decrease was observed in LOTUS-KO mice (n = 10) (Fig. 4a). Furthermore, the results of the recognition index, which indicates the ratio of the social investigation times during the Day2 and Day1, meant that LOTUS-KO mice showed a significantly worse recognition compared with WT mice (Fig. 4b), suggesting that LOTUS-KO mice have impaired memory compared with WT mice. Thus, these data show that loss of LOTUS impairs socialrecognition-related memory. Next, to evaluate the ability of spatial learning and memory, we performed the Morris water maze test, which is a behavioral analysis that evaluates hippocampal-dependent spatial memory formation in mice (Fig. 5a). In 6 days training, the escape latency was significantly longer in LOTUS-KO mice (n = 15) compared with WT mice (n = 13; two-way repeated-measures ANOVA). In addition, LOTUS-KO mice had significantly longer escape latency at Day 2 and Day 4 compared to WT mice (Student's unpaired t-test, Fig. 5b). In test 1, after training twice a day for 3 days, the WT and LOTUS-KO mice did not show any difference in the staying time in the target quadrant (TQ) (by χ 2 test, Fig. 5c), where the platform was set. In test 2, after training for 6 days, the staying time in the TQ was significantly increased in WT mice, whereas no difference was observed in LOTUS-KO mice (by χ 2 test, Fig. 5d). Furthermore, the ratio of time spent in the TQ during the probe test was not significantly different between WT and LOTUS-KO mice in Test 1, whereas a significant reduction in this ratio was observed in LOTUS-KO mice in Test 2 compared with WT mice (Fig. 5e). No difference was observed in the body weight and swimming speed of LOTUS-KO mice compared with WT mice (Fig. S2a,b). These results suggest that LOTUS-KO mice show an impairment of spatial learning and memory. Taken together, the results imply that loss of LOTUS impairs the ability to form hippocampus-dependent memory, such as social cognitive and spatial memories, and that LOTUS may be required for memory formation.

Discussion
Nogo signaling inhibits neurite outgrowth with growth cone collapse, thereby inhibiting axonal regeneration after injury in the central nervous system via NgR1 2,3,6,9 . MAIs such as Nogo-A, MAG, and OMgp are potent inhibitors of axon regrowth as ligands of NgR1 4,5 . NgR1 adopts a co-receptor structure with p75NTR and LINGO-1 or TROY and induces structural changes in the cytoskeleton through activation of RhoA 7  www.nature.com/scientificreports/ was reported that Nogo signaling reduces synaptic density in the hippocampus 14,15 . Furthermore, it was also reported that the β-amyloid protein (Aβ), which is believed to be a causal protein of Alzheimer's disease, also binds to NgR1 and reduces synaptic density, thereby inducing defective memory function 30 . In each case, NgR1 leads to the inhibition of synapse formation via the RhoA − ROCK signal. As an NgR1 antagonist, LGI1 has been reported to contribute to synapse formation by inhibiting Nogo signaling 18,[20][21][22] . Therefore, we hypothesized that LOTUS, an endogenous antagonist of NgR1, is also involved in synapse formation and memory formation. To address this issue, we first examined the location of LOTUS expression in cultured hippocampal neurons using fluorescent immunocytochemistry. The data showed that LOTUS was distributed along dendrites and in synapse regions of hippocampal neurons. It has been reported that inhibition of NgR1 by shRNA increased synaptic density in cultured hippocampal neurons 14 . Moreover, the application of Nogo to cultured hippocampal neurons has been reported to reduce synaptic density 10,11 . In the present study we observed LOTUS co-localizing with NgR1 at PSD-95-positive synaptic sites, indicating that the interaction between NgR1 and LOTUS in the synapses of hippocampal neurons may suppress Nogo signaling and affect synapse formation. This idea is supported by data showing that the loss of LOTUS decreases synapse density in cultured hippocampal neurons and decreases dendritic spine density in the adult hippocampus. Interestingly, LOTUS-KO mice exhibited a decreased density of thin-and mushroom-type spines compared with WT mice. These data suggest that LOTUS may influence synaptic morphology. As Nogo signaling is known to regulate actin dynamics 7 , it is possible that LOTUS plays a role in maintaining the synaptic actin assembly, supporting synaptic maturation and morphology.
It has also been reported that suppression of Nogo signaling increases hippocampal-dependent long-term memory function, and that enhancement of Nogo signaling decreases memory formation 12,15 . Based on these findings, we evaluated hippocampal-dependent long-term memory formation in LOTUS-KO mice; we found that social cognitive memory and spatial learning and memory were impaired in the absence of LOTUS in these animals, suggesting that LOTUS may contribute to hippocampus-dependent memory formation. Future studies of activity-dependent dendritic spine dynamics in LOTUS-KO mice are required to fully understand the contribution of LOTUS to synaptic plasticity.
It has been reported that the increase in Nogo signal 16,17,27 or the decrease of LOTUS expression 26 in the hippocampus according to aging causes memory impairment. Both processes may have a synergistic effect on age-dependent memory impairment. It would be interesting to examine whether LOTUS overexpression or the blockade of the decrease in LOTUS expression suppress the age-dependent memory impairment. Conversely, NgR1 has been reported as a receptor of the Aβ protein, a causative protein of Alzheimer's disease; moreover, Aβ binding to NgR1 suppresses synapse formation, and Aβ action through NgR1 may affect synaptic plasticity and cause memory impairment eventually 30,31 . Recently, we found that LOTUS also binds to the paired immunoglobulin-like receptor B (PirB) and suppresses Nogo-induced PirB function 32 . PirB also acts as an

Methods
Animals. C57BL/6 J mice were purchased from Charles River Co. (Japan, Inc.), and the lotus/crtac1b gene knockout mice (Acc. No. CDB0599K,(http://www.cdb.riken .jp/arg/mutan t%20mic e%20lis t.html) were generated as previously described 19 (http://www.cdb.riken .jp/arg/Metho ds.html). The heterozygous Thy1-EGFP mice were maintained by crossing with wild-type (WT) C57BL/6 J mice 34 . The mice were housed in a standard mouse facility and were provided autoclaved diet and water. Throughout the experimental procedures, all efforts were made to minimize the number of animals used and their suffering. The experimental procedures were approved by the institutional animal care and use ethical committee of Yokohama City University and were carried out in accordance with the recommended guidelines. The lotus/crtac1b mutants were assessed on the C57BL/6 J background.
Cell culture of hippocampal neurons. The hippocampal nerve cell primary culture method was partially modified from the original protocol 35 . The hippocampus was excised from embryos (E17.5) of WT and LOTUS-KO mice. Pregnant mice of each genotype were deeply anesthetized with isoflurane (Pfizer) and the embryos were removed. The hippocampus was dissected and dispersed using 0.25% trypsin and 100 µg/ml DNase at 37 °C for 12 min. Dispersed cells were immersed in a 24-well dish (Greiner Bio-One). The glass cover slips (φ, 12 mm; Matsunami) were coated with 10 μg/ml of polyethyleneimine and 10 μg/ml of laminin, and the surface was seeded with 0.5 × 10 5 cells/dish. Neurobasal medium (Gibco) containing 10% fetal bovine serum (Biowest) was used as the plating medium, and 1 × B-27 (Gibco), 1 × Glutamax (Gibco), and Neurobasal medium (Gibco) were used as the culture medium.
Analysis of synapse density in cultured hippocampal neurons. All fluorescence immunostaining images were acquired using a confocal microscope (TCS SP8; Leica) and the LasX software (Leica). The segment was imaged at 1-3 × magnification. All images were taken by using a resolution of 1024 × 1024 pixels with a z-step of 0.5 µm. Independently observable immunostaining with anti-PSD-95, anti-Bassoon, and anti-MAP2 antibodies was examined to identify the synapse sites (Fig. 2a,b). All captured images were subjected to a luminance histogram threshold with LAS X (luminance for PSD Analysis of dendritic spine density in the adult hippocampus. Mice (male, 2 months old) were deeply anesthetized with isoflurane (Pfizer) and perfused with 4% paraformaldehyde. The brain was then removed and fixed overnight in the same fixative. Subsequently, the fixed brain was immersed in 30% sucrose and stored at − 80 °C. Coronal Sects. (30 µm) were prepared using a cryostat. Fluorescence images were acquired using a confocal microscope (TCS SP8; Leica) equipped with a 63 × (NA, 1.4) oil-immersion objective and the LAS X software (Leica). Images were captured at a resolution of 512 × 512 pixels with a z-step of 0.5 µm. The confocal stack was semi-automatically analyzed with the Neuron Studio software 36 (http://resea rch.mssm.edu/ cnic/). Spine density was calculated as the number of spines divided by the length of the dendrite segment. Stubby spines were identified by a head-to-neck diameter ratio less than 1.1. Thin spines were determined by a head-to-neck diameter ratio greater than 1.1 and a maximum head diameter less than 0.35 µm. Mushroom spines were determined by a head-to-neck diameter ratio greater than 1.1 and a maximum head diameter greater than 0.35 µm. Spine density was quantified on the first branching site of apical or basal dendrites from hippocampal CA1 pyramidal neurons.
Behavioral tests. Before performing behavioral analysis, 3 min handling was performed for 5 days. The social recognition test and the Morris water maze test were performed using different mice, as described below.
Social recognition test. The social recognition test is a behavioral analysis that measures social cognitive memory, which is a hippocampal-dependent type of memory 37,38 . First, as a training session, juvenile mice (male, 2 − 3 weeks old) were used as strangers who had never met the test mature test mouse (male, 2 months old); the test mouse encountered the juvenile mouse for 3 min, during which the contact between the two mice via sniffing was measured as the time required for individual recognition. Twenty-four hours later, the investigation time was measured again in the same combination of mature and juvenile mice, as a test. When the investigation time at the time of testing was significantly decreased compared with that at the time of training, we considered that the mature test mouse remembered and recognized the juvenile mouse.
Morris water maze test. The Morris water maze test is used to examine whether a test mouse undergoes spatial learning 39 . The mice (male, 2 months old) were trained with two trials per day at an interval of 1 min for 6 days. The mice were trained at approximately the same time every day. In the probe test, at 24 h after training on days 3 and 6, the platform was removed, and the mice were allowed to swim for 1 min. Statistical analysis. The J-STAT software was used for statistical analysis. All data are expressed as the mean ± standard error. The colocalization ratio was analyzed by one-way ANOVA post hoc Tukey-Kramer (Fig. 1g). The escape latency of the Morris water maze was analyzed by two-way repeated ANOVA (Fig. 5b). The staying time in the TQ of the Morris water maze was analyzed by χ 2 test and one-way ANOVA with post-hoc Steel − Dwass test (Fig. 5c,d). Differences were considered significant at P < 0.05.