Brain-enriched guanylate kinase-associated protein (BEGAIN), a component of the post-synaptic density protein complexes, contributes to physiological plasticity, learning and memory

doi:10.21203/rs.3.rs-3148125/v1

Download PDF

Article

Brain-enriched guanylate kinase-associated protein (BEGAIN), a component of the post-synaptic density protein complexes, contributes to physiological plasticity, learning and memory

https://doi.org/10.21203/rs.3.rs-3148125/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 12 Dec, 2023

Read the published version in Scientific Reports →

You are reading this latest preprint version

Brain-enriched guanylate kinase-associated protein (BEGAIN) is a neuropathic pain-related protein in the spinal dorsal horn that is highly enriched in the post-synaptic density (PSD) fraction. PSD proteins complexes containing N-methyl-d-aspartate (NMDA) receptors are known to be involved in synaptic plasticity in neuropathic pain. Since these PSD proteins are also participate in synaptic plasticity in learning and memory, BEGAIN is also expected to play a crucial role in this plasticity. To verify this, we first examined the distribution of BEGAIN in the brain. We found that BEGAIN was widely distributed in the brain and highly expressed in the dendritic regions of the hippocampus. Moreover, we also found that BEGAIN was concentrated in the PSD fraction of the hippocampus. Furthermore, immunoelectron microscopy confirmed that BEGAIN was localized at the asymmetric synapses. To determine the contribution of BEGAIN towards learning and memory, behavioral tests were performed using BEGAIN-knockout (KO) mice. Spatial reference memory and reversal learning in the Barns circular maze test along with contextual fear and cued fear memory in the contextual and cued fear conditioning test were significantly impaired in BEGAIN KO mice in comparison to the wild-type mice. Thus, this study reveals that BEGAIN is an excitatory postsynaptic molecule involved in learning and memory.

BEGAIN

post-synaptic density (PSD)

learning and memory

plasticity

The postsynaptic region of excitatory neurons is an assembly of more than 1000 proteins, including receptors, enzymes, scaffolding proteins, and structural proteins of the cytoskeleton^1–3. The proteins in this region are arranged as protein-protein interaction complexes, the organization of which is regulated by the action of activity-dependent signaling networks^4,5. These networks serve as important regulators of synaptic plasticity. We identified BEGAIN (brain enriched guanylate kinase-associated protein) as a neuropathic pain-related protein in the post-synaptic density (PSD) fraction of the spinal dorsal horn⁶. This BEGAIN was first identified by yeast two-hybrid system analysis as a molecule that interacts with the guanylate kinase (GK) domain of PSD-95⁷. We have shown that BEGAIN is highly expressed in the PSD fraction of spinal dorsal horn, and it functionally interacts with N-methyl-D-aspartate (NMDA) receptors, including the GluN2B subunit, in the state of neuropathic pain⁶.

Long-term potentiation (LTP), a phenomenon in which synaptic efficacy is enhanced in an activity-dependent manner, has been studied extensively in order to understand synaptic plasticity^8,9. LTP is considered a model for learning and memory. Inhibition or deletion of relevant proteins have been reported to inhibit LTP and impair learning and memory^4,10−12. Since LTP induction causes changes in the expression and phosphorylation of NMDA and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptors in the PSD, elucidating the protein composition and changes in their expression in the PSD will lead to a better understanding of the molecular mechanisms of synaptic plasticity, such as learning and memory^1,3,13−15. Phosphorylation of the tyrosine residue at 1472 of GluN2B has been reported to increase after LTP in the CA1 and amygdala. On the other hand, fear-dependent memory has been shown to be impaired by inhibiting phosphorylation at Y1472 of GluN2B in knock-in (GluN2B Y1472F-KI) mice^16,17. Inhibition of the interaction with α-actinin2, which anchors NMDARs to the synapse, alters the synaptic localization of GluN2B NMDARs. This interaction is attenuated in the GluN2B Y1472F-KI mice. In addition, effective calcium influx and downstream signaling are also reduced in the KI mice^17,18.

Central sensitization is a plastic change in the somatosensory nervous system in which inflammation or nerve injury enhances synaptic transmission and neuronal circuitry in the nociceptive pathway. Hyperalgesia and allodynia, symptoms of chronic pain, are observed in vivo as a result of central sensitization¹⁹. Although there are striking similarities in the molecular mechanisms of LTP and central sensitization, differences in synaptic plasticity exist between the two phenomena²⁰. Phosphorylation of Y1472 in GluN2B is increased during chronic pain, neuropathic pain with mechanical allodynia, and inhibition of its phosphorylation significantly suppresses mechanical allodynia^6,18,21,22. However, the downstream signaling of its phosphorylation in neuropathic pain is not fully understood. Moreover, its role in learning and memory also remains unknown. Therefore, understanding the molecular mechanisms underlying pathophysiological pain is not only important for discernment of neurological disorders such as chronic pain, but also for comprehension of physiological events and memory processes.

Our previous study confirmed that BEGAIN was increased in the PSD fraction of the spinal dorsal horn in the neuropathic pain, and this increase was suppressed in Y1472F-KI mice⁶. Since GluN2B-NMDA receptors are involved in learning and memory as well as chronic pain²³, we hypothesized that BEGAIN is also participate in learning and memory. On the other hand, its distribution, synaptic localization and role in the brain remained unclear. Therefore, the aim of this study was to confirm the distribution and synaptic localization of BEGAIN by histological analysis. In addition, to clarify the involvement of BEGAIN in learning and memory, we performed behavioral analysis using BEGAIN knockout (BEGAIN-KO) mice.

Generation of BEGAIN-iCre driver/-Venus reporter mouse

Previous immunohistochemical analysis could not identify BEGAIN-positive neurons as the BEGAIN protein tended to be localized in the neuronal projections in the spinal cord; however, its localization in the soma was also not detected⁶. To elucidate the BEGAIN-expressing neurons and the distribution of BEGAIN in the central nervous system (CNS), we generated BEGAIN-iCre-Venus mice. In these mice, transcription of improved Cre-recombinase (iCre) and tandemly triplicated Venus (3x Venus) were driven by the promoter of the BEGAIN gene (Fig. 1A). Unfortunately, BEGAIN-expressing cells could not be visualized by the fluorescent signal of the Venus protein (Fig. 1B lower), suggesting that the amount of Venus expression was not sufficient to visualize BEGAIN-expressing regions in the brain. Therefore, we crossed the driver mice with Ai9 reporter mice to visualize BEGAIN-positive neurons by fluorescence of tdTomato (Fig. 1B upper). TdTomato was detected as sufficient fluorescent signal for visualization in almost all the regions of coronal section from the brain, suggesting that BEGAIN expression was found to be widely distributed in the brain.

Distribution of BEGAIN in the hippocampus and the spinal dorsal horn

In our previous study, western blot analysis detected endogenous BEGAIN in the olfactory bulb, midbrain, hippocampus, and cerebrum. Extremely high expression of BEGAIN was detected in the hippocampus⁶. BEGAIN-iCre::Ai9 reporter mice showed the distribution of BEGAIN-positive neurons in the forebrain including the hippocampus (Fig. 2A and B). The tdTomato was detected in NeuN positive soma in the CA1 and DG, but not in the CA3 (Fig. 2B and C). In the spinal cord, tdTomato was highly expressed in the superficial laminae in the spinal dorsal horn (Fig. 2D and E), consistent with our previous immunohistochemical findings⁶. The BEGAIN-expressing neurons were identified in laminae II-III (Fig. 2E). The specificity of BGAIN signals was verified by negative labeling in the Ai9 reporter mouse line (Fig. 2F and G).

Detection of endogenous BEGAIN in the hippocampus.

To determine the distribution of BEGAIN in the brain, immunohistochemical analyses were performed. In our preliminary experiments, BEGAIN was not detected in the brain sections after perfusion of 4% paraformaldehyde (PFA) with and without antigen retrieval. Therefore, we used the fresh-frozen sections and performed antigen retrieval treatment, which resulted in immunohistochemical detection of endogenous BEGAIN widely in the brain. Relatively strong expression was detected in the cerebral cortex, striatum nucleus accumbens, and hippocampus (Fig. 3A), consistent with the distribution of tdTomato fluorescence in BEGAIN-iCre::Ai9 mice (Fig. 2A). Different labeling patterns were also noted in some regions. In the hippocampus, immunohistochemical signals were high in the dendritic layers of the CA1-CA3 regions (Fig. 3B), while tdTomato fluorescence signals were intense for granule cells in the dentate gyrus and mossy fibers running in the CA3 region (Fig. 2B). Moreover, immunohistochemical signals were considerably low in the olfactory bulb, where intense signals for tdTomato fluorescence was found in that of BEGAIN-iCre::Ai9 mice. The specificity of the immunohistochemical signals was verified by the lack of labeling in the BEGAIN-KO mice (insets in Fig. 3A and B).

Synaptic localization of BEGAIN protein in the hippocampus.

To determine whether BEGAIN was localized at excitatory, inhibitory, or both synapses in the dendritic area of CA, we performed double immunohistochemical analyses in the CA1 area with antibodies against VGluT1 and VIAAT, markers for the terminals of excitatory and inhibitory nerve endings, respectively (Fig. 3C and D). BEGAIN was well apposed to VGluT1-positive terminals (Fig. 3C), whereas BEGAIN signals were not associated with VIAAT-positive terminals (Fig. 3D). These results suggest that BEGAIN is selectively localized at excitatory synapses in the CA1. Western blot analysis with subcellular hippocampal fractions further confirmed that BEGAIN was highly concentrated in the the postsynaptic density (PSD) fraction, which was similar to PSD-95 as demonstrated by western blotting (Fig. 4). The PSD proteins are concentrated in the PSD to form a complex with receptors and signal transduction molecules and to orchestrate synapse transmission^1,3. Thus, post-embedding immunoelectron microscopy for BEGAIN and VIAAT was performed to clarify the synaptic localization of BEGAIN in the hippocampus. Immunogold particles for BEGAIN were often detected along the PSD at VIAAT-negative asymmetrical synapses in the wild-type, but not in the BEGAIN-KO mice (Fig. 5A, B and E). On the other hand, no significant labeling was not detected along the post synapse at VIAAT-labeled symmetrical synapses of both wild-type and BEGAIN-KO mice (Fig. 5C, D and E). Most immunogold particles for BEGAIN were distributed in the postsynaptic side from the midline of the synaptic cleft, peaking at 20–30 nm bin(Fig. 5F).

Involvement of BEGAIN in learning and memory

To further pursue the contribution of BEGAIN in learning and memory, we conducted the Barnes circular maze test along with the contextual and cued fear conditioning test. In the learning process of the Barnes maze test, BEGAIN-KO mice showed slightly longer latency to 1st (escape box) than wild-type mice, but there were no significant differences in terms of the distance and the error to 1st (Fig. 6A). Probe test performed 24 h after the last training showed that the time spent around the hole by the BEGAIN-KO mice was significantly lesser compared to the wild-type mice (Fig. 6B). The BEGAIN-KO mice spent slightly less time around the target hole than the wild-type mice, but this was not significant at 1 month after the last training session (Fig. 6C). In addition, BEGAIN-KO mice showed an increase in distance, latency and error to 1st after the change to the opposite side of the escape box (Fig. 6D). These results indicate that BEGAIN is implicated in the spatial reference memory and reversal learning.

BEGAIN-KO mice showed a shorter freezing ratio than the wild-type mice during the conditioning period in the contextual and cued fear conditioning test (Fig. 7B left). However, the distance traveled by the BEGAIN-KO mice following the electric shock was not significantly different from that by the wild-type mice (Fig. 7A). This was in agreement with our previous study which showed that BEGAIN-KO did not alter the sensitivity to nociceptive stimuli under normal conditions⁶. After conditioning, the low freezing ratio was also demonstrated in the contexed testing at 1 and 28 days after conditioning (Fig. 7B middle and C left), and this phenotype was more pronounced after 28 days. These results suggest that BEGAIN is associated with contextual fear conditioning that associates chambers with electric shocks and their retention. In the cued testing with altered context, BEGAIN-KO mice demonstrated lower freezing ratio before cue tone at 1 and 28 days after conditioning. Moreover, significant differences in freezing were detected with cue tone at 28 days, but not at 1 day after training (Figs. 7B right and C right). These results suggest that BEGAIN is not involved in cued fear conditioning that associates sounds (cued tones) with electric shocks, but is associated with the retention of cued fear memory.

We demonstrated that BEGAIN is widely distributed in the brain and highly expressed in the hippocampus. In addition, it was also found that BEGAIN were distributed in the dendritic area, such as orient, radial and molecular layers in CA (Fig. 3A and B), with some co-localizing with NeuN (Sup. 1). Also, BEGAIN was observed in the molecular layer of DG, however the level of expression was lower than that in the CA (Fig. 3B). On the other hand, the signal of tdTomato in the granule cell layer, mossy fibers and bright layer was stronger than that in the orient, radial and molecular layers (Fig. 2B and E). This may be due to the fact that transport and degradation of BEGAIN and tdTomato are regulated by different mechanisms. Furthermore, BEGAIN mRNA was also detected in all the neurons in the brain and in the spinal cord in our experiments (data not shown) and in the Allen brain atlas (https://mouse.brain-map.org/gene/show/119936) by in situ hybridization. The tdTomato in BEG-iCre::Ai9 mice was specifically recognized in the superficial layer of the spinal cord, but not in the ventral horn (Fig. 2C and D), which was in accordance with our previous observation by immunostaining with anti-BEGAIN antibody⁶. These results infer that BEGAIN mRNA expression in the CNS is ubiquitous, but protein expression is limited. This indicates that translation, degradation, and localization of BEGAIN protein in the CNS may be regulated in a region-dependent manner. Further elucidation of these regulations may help to understand the tissue- and site-specific functions of BEGAIN.

Several receptors, scaffold proteins, and other signal transduction molecules concentrate and form the protein complex in the PSD, such as the NMDAR-complex^24,25. The architecture of PSD has been reported to be dynamic in response to synaptic activities such as learning and memory^5,26. PSD-95 is an abundant protein in the PSD, which binds directly to NMDARs via its PDZ domain². PSD-95 also forms multimeric complexes by itself and binds directly to other PSD proteins, thus, contributing to the formation of giant protein complexes in the PSD^2,11. Since BEGAIN forms a homodimer in the N-terminal region and interacts with the GK domain of PSD-95 in the C-terminal region^7,27, BEGAIN is also expected to act as a molecule contributing to complex formation in the PSD. In addition, we demonstrated that BEGAIN is localized at the asymmetrical synapses in the hippocampus, and more than 60% of immunogold particles for BEGAIN were located within 30–40 nm from the postsynaptic membrane (Fig. 5). GluN2 and PSD-95 are located 10–12 nm from the postsynaptic membrane, and GKAP, which binds to the GK domain of PSD-95 like BEGAIN, is located approximately 25–27 nm from the postsynaptic membrane²⁸. Thus, our results indicate that BEGAIN is a component of PSD protein complex. Deletion of PSD-95 leads to a reduction in the number of synapses containing glutamate receptors and defects in NMDAR transmission²⁹. Similarly, removal of GKAP also leads to loss of AMPAR-containing synapses and weakened synaptic transmission^30,31. In addition, BEGAIN-KO mice exhibited relatively longer delay of EPSCs between times to peak for NMDA receptor, which may have caused a reduction in the number of activated NMDAR during glutamatergic synaptic events in neuropathic pain⁶. Therefore, these previous reports together with our results suggest that BEGAIN modulates the number of synapses via NMDARs activity. In contrast, the GluN2 subunits of the hippocampal cPSD fraction of untreated (naive) BEGAIN-KO mice was slightly but not significantly reduced in comparison to wild-type mice (Sup. 2). This may have been because the effect of BEGAIN on NMDAR localization is partially dependent on synaptic plasticity during learning and memory.

In this study, we clarified that BEGAIN was highly concentrated in the hippocampus and specifically localized at the asymmetrical synapses in the CA1 (Figs. 4 and 5), thus, indicating the relevance of BEGAIN in learning and memory (Figs. 6 and 7). The memory retention for reference memory at 1 day after training and the fear memory (contextual fear conditioning and cued fear conditioning) at 28 days after conditioning were impaired in the BEGAIN-KO mice in comparison to the wild-type mice (Figs. 6 and 7). Retention of reference working memory was still reduced in BEGAIN-KO mice after 1 month of training, but the significant difference between wild-type and BEGAIN-KO mice observed at 1 day was not observed after 1 month (Fig. 6C). Based on the results of these behavioral analyses, it can be inferred that the effects of BEGAIN on long-term memory may be greater in fear memory than in spatial reference memory long-term. On the other hand, highest expression of BEGAIN in the hippocampus was confirmed (Figs. 2 and 3). The hippocampus has been reported to be important not only for both reference and fear memories³². The expression of BEGAIN in the amygdala was also demonstrated (Fig. 1). Further analysis of diverse behaviors and region-specific BEGAIN interacting molecules is needed to understand the function of BEGAIN, which is widely distributed in the forebrain.

The molecular mechanisms of chronic pain and memory share similarities, and many molecules are known to be involved in the plasticity of both, including PSD proteins such as glutamate receptors, kinases, and scaffold proteins, whose activities and interactions via post-translational modifications have been reported^{12,17,18,20,21,33,34}. NMDARs are common important molecules for synaptic plasticity in the spinal cord and hippocampus. Mechanical allodynia, a symptom of neuropathic pain, was suppressed by the NMDAR antagonist APV and the GluN2B-specific antagonist Ro25-6981^6,35,36. In addition, chronic intraventricular infusion of APV impairs spatial learning and LTP in pyramidal neurons³⁴. Furthermore, fear-related memory retention was impaired in the GluN2B Y1472F-KI mice¹⁷, and mechanical allodynia was also attenuated in the KI mice⁶. In addition, we demonstrated the impairment of learning and memory (Figs. 6 and 7) and attenuation of mechanical allodynia in the BEGAIN-KO mice⁶. The findings of the abovementioned studies along with our results suggest that BEGAIN, like GluN2B-NMDAR, is involved in synaptic plasticity for both learning and memory and neuropathic pain. In addition, conditional knockout of GluN1 in CA1 and DG has been reported to impair reversal learning, indicating that NMDAR activity is involved in reversal learning^37,38. BEGAIN localizes to PSD at glutamatergic synapses via direct binding to PSD-95 (Fig. 5)^7,28. Furthermore, inhibition of NMDAR reportedly suppresses BEGAIN expression in PSDs in the spinal dorsal horn and at the plasma membrane in the hippocampal neurons^6,27, suggesting that BEGAIN accumulates in PSDs during the activation of NMDAR in synaptic plasticity. Moreover, BEGAIN-KO mice also showed impairment of reversal learning (Fig. 6D). Considering these reports and our findings, it can be inferred that BEGAIN may interact with PSD complexes in the hippocampus and contribute to reversal learning via NMDAR activity. Thus, we conclude that BEGAIN is associated with physiological and pathological plasticity, including memory and neuropathic

pain.

Animals

BEGAIN-KO mice were generated using a gene targeting technique as previously reported6. BEGAIN-Cre-3xVenus mice were generated using the RENKA embryonic stem (ES) cell line derived from the C57BL/6N strain39. Improved Cre recombinase (iCre) and three tandem Venus genes (3 x Venus) were inserted into the second exon of the begain gene (Ex2, Fig. 1A). Homologous recombination of the gene between ES cells was detected using Southern blot analysis. Ai9 mice (B6. Cg-Gt (ROSA) 26Sor tm9 (CAG-tdTomato) Hze; Jackson Labs,Stock #007909) was obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All procedures described here were reviewed and approved by the Animal Care and Use Committee of Kansai medical university and National Institute for Physiological Sciences in Japan, and experiments were performed under the institutional guiding principles for the care and use of laboratory animals. All methods were performed in accordance with the ARRIVE guidelines (https://arriveguidelines.org).

Antibodies

Rabbit anti-BEGAIN against C-terminal 17 amino acid of mouse BEGAIN was used⁶. Commercially available antibodies against PSD-95 (abcam, Upstate Biotechnology), vinculin (abcam), VGluT1 (guinea pig, Nittobo Medical), VIAAT (guinea pig, Nittobo Medical), and NeuN (Millipore) were also used.

Subcellular fractionation of hippocampus

After anesthesia with isoflurane, 20–29 male 7-to 12-week-old mice from WT and BEGAIN-KO mice were killed, and their hippocampus were collected (wet weight approx. 1.0g). The hippocampus was homogenized in solution A [0.32 M sucrose, 1 mMNaHCO₃, 1 mM MgCl₂, 0.5 mM CaCl₂, PhosSTOP (Roche), and protease inhibitor cocktail (Nacalai Tesque)] with a Potter-Elvehjem homogenizer. After centrifugation of the homogenate at 800 x g for 10 min, the pellet (P1; nuclear) and supernatant (S1) were separated. The S1 fraction was centrifuged at 13,800 x g for 20 min. The supernatant (S2) and precipitate (P2) were collected as the cytosolic and the membrane fraction. The PSD fraction was prepared essentially as described by Carlin et al. (1980) with slight modifications²⁵. The P2 fraction was then suspended in solution B (0.32 M sucrose containing 1 mM NaHCO₃) and applied onto a discontinuous sucrose gradients composed of 3.4ml of 1.2 M, 3.4 ml of 1.0 M, and 3 ml of 0.85 M sucrose in1 mM NaHCO₃ in tubes. The tube was centrifuged at 82,500 x g for 120 min. The interface between 1.0 and 1.2M sucrose was collected and dissolved for 15 min with buffer C consisting of 0.5% Triton X-100 and 6 mM Tris-HCl (pH 8.0). For collection of the insoluble fraction, the interface fraction was centrifuged at 32,800 x g for 30min. The insoluble fraction was dissolved in 7 M urea, 2 M thiourea, 4% CHAPS, and 2% SDS and used as the PSD fraction.

Western blot analysis

All subcellular fractions from hippocampus were subjected to a single gel for SDS-PAGE (4–12% acrylamide), and the separated proteins were transferred to a polyvinylidene fluoride membrane. After blocking for 1 h at room temperature with 3% skim milk in TBS-T buffer consisting of 0.1% Triton X-100, 150 mM NaCl and 10 mM Tris-HCl (pH 7.5), the membrane was incubated at 4˚C overnight with anti-BEGAIN (1 µg/ml), anti-PSD-95 (1:1000) and anti-vinculin (1:10,000) antibodies. The membrane was washed with the TBS-T buffer and incubated for 1 h with horseradish peroxidase–conjugated goat anti-rabbit IgG (1:20,000; Invitrogene) or goat anti-mouse IgG (1:20,000; GE Healthcare). It was then washed four times with TBS-T buffer. Immunoreactivity was detected by use of an enhanced chemiluminescence detection kit (Chemi-Lumi One Super; Nacalai Tesque). Detection of several proteins, such as BEGAIN, PSD-95 and vinculin, in a single gel was performed sequentially. That is, the preceding antibody was stripped from the polyvinylidene fluoride membrane, which was then re-probed with other primary antibodies for the detection of the next protein.

Histochemistry

Animals were anesthetized by an intraperitoneal administration of sodium pentobarbital (50 mg/kg) and perfused with 4% paraformaldehyde (PFA) in 0.12 M sodium phosphate (PB, pH 7.4) for immunofluorescence for NeuN and tdTomato and 1%PFA in PB for post-embedding immunogold electron microscopy. After dissection, brain and spinal cord tissues were postfixed for 4 h in the same fixative at 4 ˚C and cryoprotected overnight in 30% (w/v) sucrose in PB. Sagittal and coronal sections of the brain and transverse sections of the spinal cord were cut on a cryostat or microtome (14 or 40 µm thick) and processed for immunohistochemistry with anti-NeuN antibody (1:500). For the detection of endogenous BEGAIN in the mouse brain, fresh frozen sections were cut on a cryostat (20 µm). The sections were stained with anti-BEGAIN anti-VGluT1, and anti-VIAAT antibodies (1 µg/ml each) as primary antibodies after fixation with 4% PFA in PB for 15 min and antigen retrieval at 95 ˚C for 30 min in Immnosaver solution (Nisshin). Sections were then incubated with Alexa Fluor 488 goat anti-mouse IgG as secondary antibody (1:300, Invitrogen) or with a mixture of Alexa 488- and Cy3-labeled species-specific secondary antibodies (1:200 dilution; Invitrogen; Jackson ImmunoResearch) for 1 to 2 h at room temperature. Images for fresh frozen sections were captured using a confocal laser scanning microscope equipped with 473 and 559 diode laser lines, and UPlanSApo (10×/0.40), UPlanSApo (20×/0.75) and PlanApoN (60×/1.4, oil immersion) objective lenses (FV1200; EVIDENT). Fluorescence images of tdTomato were captured using Zyla4.2 sCMOS camera (Andor) mounted onto an EVIDENT IX81(EVIDENT) and DragonFly500 spinning disk confocal imaging system (Oxford Instruments).

Electron immunohistochemistry

For post-embedding immunogold electron microscopy, microslicer sections (400 µm) were cryoprotected by immersion in 30% glycerol in PB for 30 min, and then frozen rapidly in liquid propane in an EM CPC unit (Leica Microsystems). Frozen sections were immersed in 0.5% uranyl acetate in methanol at − 90°C in an AFS freeze-substitution unit (Leica Microsystems), infiltrated at − 45°C with Lowicryl HM-20 resin (Electron Microscopy Sciences), which was then polymerized with ultraviolet light. Ultrathin sections were cut using an Ultracut ultramicrotome (Leica Microsystems), and mounted on nickel grids.

Ultrathin sections on nickel grids were etched with saturated sodium-ethanolate solution for 1–5 s and treated with successive solutions, as follows: sections were incubated in 50 mM glycine in incubation solution [0.01% Triton X-100 in Tris-buffered saline, pH 7.4 (TTBS)] for 10 min. For double labeling against VIAAT and BEGAIN, sections were incubated in blocking solution containing 2% normal goat serum, followed by guinea pig anti-VIAAT antibody (20 µg/ml) diluted in 2% normal goat serum in TTBS overnight and then colloidal gold-conjugated (20 nm) anti guinea pig IgG in blocking solution for 2 h. After extensive washing in distilled water, sections were incubated in blocking solution containing 2% normal guineapig serum (Nichirei) in TTBS for 10 min, rabbit anti-BEGAIN antibody diluted with 2% normal guineapig serum in TTBS overnight, and colloidal gold-conjugated (10 nm) anti-rabbit IgG (1:100, British BioCell International) in blocking solution for 2 h. After extensive washing in distilled water, sections were fixed with 2% OsO₄ for 15 min, and stained with 5% uranyl acetate/40% ethanol for 90 s and Reynold's lead citrate solution for 60 s. Photographs were taken from the molecular layer of the cerebellum with an H-7100 electron microscope (Hitachi).

For quantitative analysis, the density of postsynaptic membrane-associated immunogold particles, which were defined as those < 20 nm from the cell membrane, were counted on electron micrographs and analyzed using MetaMorph software (Molecular Devices). The vertical distribution at synapses was examined by sampling synaptic profiles whose presynaptic and postsynaptic membranes were cut perpendicularly to the plane of the synaptic cleft, and by measuring the distance from the midline of the synaptic cleft to the center of an immunogold particle.

Behavioral studies

Behavioral tests were carried out with 24 wild-type and 24 BEGAIN-KO male mice, which were obtained by heterozygous intercrossing breeding. Four-to-five week-old mice were housed four (two pairs of wild-type and BEGAIN-KO mice) per cage in a room with a 12-hr light/ dark cycle and with access to food and water ad libitum. Behavioral testing was performed between 9:00 a.m. and 6:00 p.m. Raw data of the behavioral test and the information about each mouse are open on a public database “Mouse Phenotype Database” (http://www.mouse-phenotype.org/).

Barnes circular maze test

The maze apparatus consisted of a white circular disk 1.0 m in diameter with 12 holes equally spaced around the perimeter, which was elevated 75 cm from the floor. A black escape box (17 x 13 x 7 cm) was located under one of the holes as the target (O’Hara & Co.). The maze was rotated daily, with the spatial location of the target unchanged with respect to the distal visual room cues, to prevent a bias based on olfactory or the proximal cues within the maze. Three trials per day were conducted every day until the total number of trials reached 15. After 1 day and 1 month, a probe trial without the escape box was conducted to confirm that this spatial task was acquired based on navigation by distal environment room cues.

Contextual and cued fear conditioning test

Each mouse was placed in a transparent acrylic chamber (33 × 25 × 28 cm) with a stainless-steel grid floor (0.2 cm-diameter, spaced 0.5 cm apart; O’Hara & Co.) and was allowed to explore freely for 2 min. Subsequently, a 55-dB white noise, which served as the conditioning stimulus (CS), was presented for 30 s. During the last 2 s of CS presentation, a mild foot shock (0.3 mA, 2 s), which served as the unconditioning stimulus (US), was presented. Two more CS-US pairings were presented with a 2-min inter-stimulus interval. Twenty-four hours after the conditioning, a context test was conducted in the same chamber. A cued test with altered context was then performed in a triangular chamber (33 × 29 × 32 cm) made of white opaque plastic, which was located in a different room. In each test, freezing percentage and distance traveled were calculated automatically by using ImageFZ software (see Section, “Image analysis for behavioral tests”).

Image analysis and statistical analysis for behavioral tests

The application software used for the behavioral studies were based on the public domain NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/) and ImageJ program (http://rsb.info.nih.gov/ij/), which were modified for each test (available through O’Hara & Co.). ImageFZ⁴⁰ is freely available at the following URL: http://www.mouse-phenotype.org/software.html. Statistical analyses for behavioral studies were conducted with StatView (SAS Institute). Data were analyzed by one- way ANOVA and two-way repeated measures ANOVA. Values in graphs were expressed as the mean ± SEM.

Acknowledgements: This work was supported in part by the JSPS KAKENHI (Grant Numbers 16K09001, 19K07856, and 22H02960), and also partly supported by the AMED under Grant Numbers 21ek0610027h0001, 22ek0610027h0002 and 23ek0610027h0003. We gratefully acknowledge JSPS KAKENHI (Grant Number JP16H06280), Grant-in-Aid for Scientific Research on Innovative Areas - Platforms for Advanced Technologies and Research Resources “Advanced Bioimaging Support”, Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network, CBSN) from the Ministry of Education, Science, Sports and Culture of Japan for support, and JSPS KAKENHI (Grant Number JP16H06276; AdAMS). We thank Dr. K Nishida for critical comments regarding the manuscript. We also thank R Natsume and other members of Niigata University for their help in mice generation.

Author contributions: T.K. K.K, K.T and M.A, contributed to experimental design, performed experiments, interpreted the results, A.Y. contributed to performed experiments, T.M, K.S, M.W, S.I and T.K. contributed to supported experiments and interpreted the results, and T.K. K.K contributed to wrote the manuscript.

Data availability statement: The row data of the behavioral tests are accessible on the public database “Mouse Phenotype Database” (http://www.mouse-phenotype.org/).

Competing interests statements: The authors have no competing interests to disclose.

Boeckers, T. M. The postsynaptic density. Cell Tissue Res 326, 409-422, doi:10.1007/s00441-006-0274-5 (2006).
Feng, W. & Zhang, M. Organization and dynamics of PDZ-domain-related supramodules in the postsynaptic density. Nat Rev Neurosci 10, 87-99, doi:10.1038/nrn2540 (2009).
Sheng, M. & Hoogenraad, C. C. The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu Rev Biochem 76, 823-847, doi:10.1146/annurev.biochem.76.060805.160029 (2007).
Lau, C. G. & Zukin, R. S. NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat Rev Neurosci 8, 413-426, doi:10.1038/nrn2153 (2007).
Sheng, M. & Kim, M. J. Postsynaptic signaling and plasticity mechanisms. Science 298, 776-780, doi:10.1126/science.1075333 (2002).
Katano, T. et al. Involvement of Brain-Enriched Guanylate Kinase-Associated Protein (BEGAIN) in Chronic Pain after Peripheral Nerve Injury. eNeuro 3, doi:10.1523/ENEURO.0110-16.2016 (2016).
Deguchi, M. et al. BEGAIN (brain-enriched guanylate kinase-associated protein), a novel neuronal PSD-95/SAP90-binding protein. J Biol Chem 273, 26269-26272 (1998).
Malenka, R. C. & Bear, M. F. LTP and LTD: an embarrassment of riches. Neuron 44, 5-21, doi:10.1016/j.neuron.2004.09.012 (2004).
Bannerman, D. M. et al. Hippocampal synaptic plasticity, spatial memory and anxiety. Nat Rev Neurosci 15, 181-192, doi:10.1038/nrn3677 (2014).
Lim, I. A., Merrill, M. A., Chen, Y. & Hell, J. W. Disruption of the NMDA receptor–PSD-95 interaction in hippocampal neurons with no obvious physiological short-term effect. Neuropharmacology 45, 738-754, doi:10.1016/s0028-3908(03)00276-4 (2003).
Kim, E. & Sheng, M. PDZ domain proteins of synapses. Nat Rev Neurosci 5, 771-781, doi:10.1038/nrn1517 (2004).
Li, J. et al. Long-term potentiation modulates synaptic phosphorylation networks and reshapes the structure of the postsynaptic interactome. Sci Signal 9, rs8, doi:10.1126/scisignal.aaf6716 (2016).
Cheng, D. et al. Relative and absolute quantification of postsynaptic density proteome isolated from rat forebrain and cerebellum. Mol Cell Proteomics 5, 1158-1170, doi:10.1074/mcp.D500009-MCP200 (2006).
Collins, M. O. et al. Proteomic analysis of in vivo phosphorylated synaptic proteins. J Biol Chem 280, 5972-5982, doi:10.1074/jbc.M411220200 (2005).
Katano, T. et al. N-ethylmaleimide-sensitive fusion protein (NSF) is involved in central sensitization in the spinal cord through GluR2 subunit composition switch after inflammation. Eur J Neurosci 27, 3161-3170, doi:10.1111/j.1460-9568.2008.06293.x (2008).
Nakazawa, T. et al. Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor. J Biol Chem 276, 693-699, doi:10.1074/jbc.M008085200 (2001).
Nakazawa, T. et al. NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity. EMBO J 25, 2867-2877, doi:10.1038/sj.emboj.7601156 (2006).
Matsumura, S. et al. Impairment of CaMKII activation and attenuation of neuropathic pain in mice lacking NR2B phosphorylated at Tyr1472. Eur J Neurosci 32, 798-810, doi:10.1111/j.1460-9568.2010.07348.x (2010).
Latremoliere, A. & Woolf, C. J. Central sensitization: a generator of pain hypersensitivity by central neural plasticity. J Pain 10, 895-926, doi:10.1016/j.jpain.2009.06.012 (2009).
Ji, R. R., Kohno, T., Moore, K. A. & Woolf, C. J. Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci 26, 696-705, doi:10.1016/j.tins.2003.09.017 (2003).
Katano, T. et al. Involvement of spinal phosphorylation cascade of Tyr1472-NR2B, Thr286-CaMKII, and Ser831-GluR1 in neuropathic pain. Neuropharmacology 60, 609-616, doi:10.1016/j.neuropharm.2010.12.005 (2011).
Qu, X. X. et al. Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain. Exp Neurol 215, 298-307, doi:10.1016/j.expneurol.2008.10.018 (2009).
Perez-Otano, I. & Ehlers, M. D. Homeostatic plasticity and NMDA receptor trafficking. Trends Neurosci 28, 229-238, doi:10.1016/j.tins.2005.03.004 (2005).
Husi, H., Ward, M. A., Choudhary, J. S., Blackstock, W. P. & Grant, S. G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes. Nat Neurosci 3, 661-669, doi:10.1038/76615 (2000).
Carlin, R. K., Grab, D. J., Cohen, R. S. & Siekevitz, P. Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities. J Cell Biol 86, 831-845 (1980).
Carroll, R. C. & Zukin, R. S. NMDA-receptor trafficking and targeting: implications for synaptic transmission and plasticity. Trends Neurosci 25, 571-577, doi:10.1016/s0166-2236(02)02272-5 (2002).
Yao, I., Iida, J., Nishimura, W. & Hata, Y. Synaptic and nuclear localization of brain-enriched guanylate kinase-associated protein. J Neurosci 22, 5354-5364, doi:20026505 (2002).
Valtschanoff, J. G. & Weinberg, R. J. Laminar organization of the NMDA receptor complex within the postsynaptic density. J Neurosci 21, 1211-1217, doi:10.1523/JNEUROSCI.21-04-01211.2001 (2001).
Levy, J. M., Chen, X., Reese, T. S. & Nicoll, R. A. Synaptic Consolidation Normalizes AMPAR Quantal Size following MAGUK Loss. Neuron 87, 534-548, doi:10.1016/j.neuron.2015.07.015 (2015).
Hung, A. Y. et al. Smaller dendritic spines, weaker synaptic transmission, but enhanced spatial learning in mice lacking Shank1. J Neurosci 28, 1697-1708, doi:10.1523/JNEUROSCI.3032-07.2008 (2008).
Shin, S. M. et al. GKAP orchestrates activity-dependent postsynaptic protein remodeling and homeostatic scaling. Nat Neurosci 15, 1655-1666, doi:10.1038/nn.3259 (2012).
Anagnostaras, S. G., Gale, G. D. & Fanselow, M. S. Hippocampus and contextual fear conditioning: recent controversies and advances. Hippocampus 11, 8-17, doi:10.1002/1098-1063(2001)11:1<8::AID-HIPO1015>3.0.CO;2-7 (2001).
Abe, T. et al. Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain. Eur J Neurosci 22, 1445-1454, doi:10.1111/j.1460-9568.2005.04340.x (2005).
Morris, R. G. Synaptic plasticity and learning: selective impairment of learning rats and blockade of long-term potentiation in vivo by the N-methyl-D-aspartate receptor antagonist AP5. J Neurosci 9, 3040-3057 (1989).
Zhang, G. F. et al. alpha2delta-1 Upregulation in Primary Sensory Neurons Promotes NMDA Receptor-Mediated Glutamatergic Input in Resiniferatoxin-Induced Neuropathy. J Neurosci 41, 5963-5978, doi:10.1523/JNEUROSCI.0303-21.2021 (2021).
Mihara, Y. et al. Involvement of spinal NR2B-containing NMDA receptors in oxaliplatin-induced mechanical allodynia in rats. Mol Pain 7, 8, doi:10.1186/1744-8069-7-8 (2011).
Bannerman, D. M. et al. Dissecting spatial knowledge from spatial choice by hippocampal NMDA receptor deletion. Nat Neurosci 15, 1153-1159, doi:10.1038/nn.3166 (2012).
Tsien, J. Z., Huerta, P. T. & Tonegawa, S. The Essential Role of Hippocampal CA1 NMDA Receptor–Dependent Synaptic Plasticity in Spatial Memory. Cell 87, 1327-1338, doi:10.1016/s0092-8674(00)81827-9 (1996).
Mishina, M. & Sakimura, K. Conditional gene targeting on the pure C57BL/6 genetic background. Neurosci Res 58, 105-112, doi:10.1016/j.neures.2007.01.004 (2007).
Shoji, H., Takao, K., Hattori, S. & Miyakawa, T. Contextual and cued fear conditioning test using a video analyzing system in mice. J Vis Exp, doi:10.3791/50871 (2014).

No competing interests reported.

Sup.pdf

Download PDF

Journal Publication

published 12 Dec, 2023

Read the published version in Scientific Reports →

Editorial decision: Major revision
24 Aug, 2023
Reviewers agreed at journal
12 Aug, 2023
Reviews received at journal
11 Aug, 2023
Reviewers agreed at journal
31 Jul, 2023
Reviewers invited by journal
26 Jul, 2023
Editor assigned by journal
26 Jul, 2023
Editor invited by journal
23 Jul, 2023
Submission checks completed at journal
23 Jul, 2023
First submitted to journal
07 Jul, 2023

You are reading this latest preprint version

Brain-enriched guanylate kinase-associated protein (BEGAIN), a component of the post-synaptic density protein complexes, contributes to physiological plasticity, learning and memory

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Results

Generation of BEGAIN-iCre driver/-Venus reporter mouse

Distribution of BEGAIN in the hippocampus and the spinal dorsal horn

Involvement of BEGAIN in learning and memory

Discussion

Materials & Methods

Animals

Antibodies

Subcellular fractionation of hippocampus

Western blot analysis

Histochemistry

Electron immunohistochemistry

Behavioral studies

Barnes circular maze test

Contextual and cued fear conditioning test

Image analysis and statistical analysis for behavioral tests

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1