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Loss of α1,6-Fucosyltransferase Decreases Hippocampal Long Term Potentiation

IMPLICATIONS FOR CORE FUCOSYLATION IN THE REGULATION OF AMPA RECEPTOR HETEROMERIZATION AND CELLULAR SIGNALING*
Open AccessPublished:May 15, 2015DOI:https://doi.org/10.1074/jbc.M114.579938
      Core fucosylation is catalyzed by α1,6-fucosyltransferase (FUT8), which transfers a fucose residue to the innermost GlcNAc residue via α1,6-linkage on N-glycans in mammals. We previously reported that Fut8-knock-out (Fut8−/−) mice showed a schizophrenia-like phenotype and a decrease in working memory. To understand the underlying molecular mechanism, we analyzed early form long term potentiation (E-LTP), which is closely related to learning and memory in the hippocampus. The scale of E-LTP induced by high frequency stimulation was significantly decreased in Fut8−/− mice. Tetraethylammonium-induced LTP showed no significant differences, suggesting that the decline in E-LTP was caused by postsynaptic events. Unexpectedly, the phosphorylation levels of calcium/calmodulin-dependent protein kinase II (CaMKII), an important mediator of learning and memory in postsynapses, were greatly increased in Fut8−/− mice. The expression levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the postsynaptic density were enhanced in Fut8−/− mice, although there were no significant differences in the total expression levels, implicating that AMPARs without core fucosylation might exist in an active state. The activation of AMPARs was further confirmed by Fura-2 calcium imaging using primary cultured neurons. Taken together, loss of core fucosylation on AMPARs enhanced their heteromerization, which increase sensitivity for postsynaptic depolarization and persistently activate N-methyl-d-aspartate receptors as well as Ca2+ influx and CaMKII and then impair LTP.
      Background: High expression levels of core fucosylated N-glycans in brain tissues remain unexplained.
      Results: Loss of core fucosylation enhanced AMPA receptor heteromerization and decreased long term potentiation.
      Conclusion: Core fucosylation is required for hippocampal long term potentiation.
      Significance: Core fucosylation may be very important for the neuronal synaptic plasticity that is required for learning and memory.

      Introduction

      Schizophrenia is a common, chronic, and severe brain disorder that ranks as one of the leading causes of disability worldwide because it afflicts 2% of the world's population (
      • Freedman R.
      Schizophrenia.
      ,
      • Stanta J.L.
      • Saldova R.
      • Struwe W.B.
      • Byrne J.C.
      • Leweke F.M.
      • Rothermund M.
      • Rahmoune H.
      • Levin Y.
      • Guest P.C.
      • Bahn S.
      • Rudd P.M.
      Identification of N-glycosylation changes in the CSF and serum in patients with schizophrenia.
      ). The disease is typically characterized by a loss of emotional expression and a lack of motivation. The etiology of schizophrenia is only partially understood, but multiepisode patients show significantly disturbed neuronal plasticity, suggesting that synaptic activity and connectivity are altered during the progression of the disease. Recently, cognitive impairments, such as deficits in learning and memory, have also been shown to be a fundamental feature of the disorder (
      • Savanthrapadian S.
      • Wolff A.R.
      • Logan B.J.
      • Eckert M.J.
      • Bilkey D.K.
      • Abraham W.C.
      Enhanced hippocampal neuronal excitability and LTP persistence associated with reduced behavioral flexibility in the maternal immune activation model of schizophrenia.
      ). Long term potentiation (LTP),
      The abbreviations used are: LTP
      long term potentiation
      E-LTP
      early form LTP
      AMPAR
      α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor
      CaMKII
      calcium/calmodulin-dependent protein kinase II
      GluA
      a subunit of AMPARs
      GluN
      a subunit of NMDARs
      HFS
      high frequency stimulation
      NMDAR
      N-methyl-d-aspartate receptor
      PSD
      postsynaptic density
      TARP
      transmembrane AMPA receptor regulatory protein
      TEA
      tetraethylammonium.
      a well established model based on the neurophysiological study of learning and memory, has been found to be an important mechanism that underlies synaptic changes and plasticity in schizophrenia (
      • Frantseva M.V.
      • Fitzgerald P.B.
      • Chen R.
      • Möller B.
      • Daigle M.
      • Daskalakis Z.J.
      Evidence for impaired long-term potentiation in schizophrenia and its relationship to motor skill learning.
      ,
      • Sanderson T.M.
      • Cotel M.C.
      • O'Neill M.J.
      • Tricklebank M.D.
      • Collingridge G.L.
      • Sher E.
      Alterations in hippocampal excitability, synaptic transmission and synaptic plasticity in a neurodevelopmental model of schizophrenia.
      ). The induction of LTP begins with the activation of one kind of ionotropic glutamate receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs), which induce the opening of N-methyl-d-aspartate receptors (NMDARs), which are another kind of glutamate receptor, and leads to calcium entry that initiates a biochemical cascade through the activation of CaMKII, thereby increasing the number of AMPARs in the postsynaptic density (PSD) area. The end product of this biological reaction is the long lasting potentiation of AMPAR-mediated excitatory postsynaptic current (
      • Lisman J.
      • Yasuda R.
      • Raghavachari S.
      Mechanisms of CaMKII action in long-term potentiation.
      ,
      • Kullmann D.M.
      • Lamsa K.P.
      Long-term synaptic plasticity in hippocampal interneurons.
      ).
      Previous research has focused on identifying genes or protein expression-level changes in schizophrenia and ascertaining the functional effects of those alterations. Recently, the post-translational modification, N-glycosylation, has become a new target of investigation for schizophrenia (
      • Khoury G.A.
      • Baliban R.C.
      • Floudas C.A.
      Proteome-wide post-translational modification statistics: frequency analysis and curation of the Swiss-Prot database.
      ). For example, abnormal N-glycans on AMPAR, NMDAR, and GABAA receptors were found in patients with schizophrenia (
      • Tucholski J.
      • Simmons M.S.
      • Pinner A.L.
      • Haroutunian V.
      • McCullumsmith R.E.
      • Meador-Woodruff J.H.
      Abnormal N-linked glycosylation of cortical AMPA receptor subunits in schizophrenia.
      ,
      • Mueller T.M.
      • Haroutunian V.
      • Meador-Woodruff J.H.
      N-Glycosylation of GABAA receptor subunits is altered in schizophrenia.
      • Tucholski J.
      • Simmons M.S.
      • Pinner A.L.
      • McMillan L.D.
      • Haroutunian V.
      • Meador-Woodruff J.H.
      N-Linked glycosylation of cortical N-methyl-d-aspartate and kainate receptor subunits in schizophrenia.
      ).
      The importance of fucosylation has been highlighted by the identification of the monogenetic inherited human disease, congenital disorder of glycosylation IIc (
      • Etzioni A.
      • Frydman M.
      • Pollack S.
      • Avidor I.
      • Phillips M.L.
      • Paulson J.C.
      • Gershoni-Baruch R.
      Brief report: recurrent severe infections caused by a novel leukocyte adhesion deficiency.
      ). Due to defective Golgi GDP-fucose transporter activity, patients present with important clinical symptoms that include mental and growth retardation and severe immunodeficiency (
      • Sturla L.
      • Puglielli L.
      • Tonetti M.
      • Berninsone P.
      • Hirschberg C.B.
      • De Flora A.
      • Etzioni A.
      Impairment of the Golgi GDP-l-fucose transport and unresponsiveness to fucose replacement therapy in LAD II patients.
      ). Interestingly, the phenotypes of the transporter deletion mice (
      • Hellbusch C.C.
      • Sperandio M.
      • Frommhold D.
      • Yakubenia S.
      • Wild M.K.
      • Popovici D.
      • Vestweber D.
      • Gröne H.J.
      • von Figura K.
      • Lübke T.
      • Körner C.
      Golgi GDP-fucose transporter-deficient mice mimic congenital disorder of glycosylation IIc/leukocyte adhesion deficiency II.
      ) are similar to α1,6-fucosyltransferase knock-out (Fut8−/−) mice (
      • Wang X.
      • Inoue S.
      • Gu J.
      • Miyoshi E.
      • Noda K.
      • Li W.
      • Mizuno-Horikawa Y.
      • Nakano M.
      • Asahi M.
      • Takahashi M.
      • Uozumi N.
      • Ihara S.
      • Lee S.H.
      • Ikeda Y.
      • Yamaguchi Y.
      • Aze Y.
      • Tomiyama Y.
      • Fujii J.
      • Suzuki K.
      • Kondo A.
      • Shapiro S.D.
      • Lopez-Otin C.
      • Kuwaki T.
      • Okabe M.
      • Honke K.
      • Taniguchi N.
      Dysregulation of TGF-β1 receptor activation leads to abnormal lung development and emphysema-like phenotype in core fucose-deficient mice.
      ), suggesting that core fucosylation is a key player among all fucosylations in mammals. Previously, we reported that the Fut8−/− mice exhibited multiple behavioral abnormalities, including decreased prepulse inhibition, increased locomotion, and impaired working memory with a schizophrenia-like phenotype (
      • Fukuda T.
      • Hashimoto H.
      • Okayasu N.
      • Kameyama A.
      • Onogi H.
      • Nakagawasai O.
      • Nakazawa T.
      • Kurosawa T.
      • Hao Y.
      • Isaji T.
      • Tadano T.
      • Narimatsu H.
      • Taniguchi N.
      • Gu J.
      α1,6-Fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.
      ). In fact, the unique structure of the core fucosylated N-glycans is highly expressed in brain tissues, and the expression patterns of N-glycans are altered during brain development (
      • Nakakita S.
      • Natsuka S.
      • Okamoto J.
      • Ikenaka K.
      • Hase S.
      Alteration of brain type N-glycans in neurological mutant mouse brain.
      ).
      To evaluate the effect of core fucosylation on LTP, we investigated synaptic strength and plasticity in the CA1 subregion of the hippocampus of Fut8−/− mice. We found that high frequency stimulation (HFS)-induced LTP was decreased in Fut8−/− mice. The molecular mechanism could have been caused by the aberrant heteromerization of AMPAR constitutively activating intracellular signaling, which may disturb LTP in Fut8−/− mice.

      Experimental Procedures

      Mice

      The Fut8−/− mice were generated as described previously (
      • Fukuda T.
      • Hashimoto H.
      • Okayasu N.
      • Kameyama A.
      • Onogi H.
      • Nakagawasai O.
      • Nakazawa T.
      • Kurosawa T.
      • Hao Y.
      • Isaji T.
      • Tadano T.
      • Narimatsu H.
      • Taniguchi N.
      • Gu J.
      α1,6-Fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.
      ). All experiments were conducted with female and 3–5-week-old Fut8−/− and wild-type (Fut8+/+) mice. The mice were housed in a temperature-controlled room with a 12-h dark/12-h light cycle and ad libitum feeding. This study was approved by the Institutional Animal Care and Use Committee of Tohoku Pharmaceutical University, Japan.

      Antibodies and Reagents

      Polyclonal antibodies against GluA1 and GluA2 (subunits of AMPAR) were obtained from Abcam; PSD95 was from Millipore; and GluA3, GluN2A, GluN2B (subunits of NMDAR), and CaMKII-α were from Cell Signaling. Rabbit monoclonal antibodies against GluN1, stargazin (TARP γ2), and synapsin-1 were from Cell Signaling. Monoclonal antibody against α-tubulin was from Sigma. Polyclonal antibodies against phosphorylated CaMKII at Thr-286, GluN2A at Tyr-1246, and GluN2B at Tyr-1472 all were obtained from Cell Signaling. Biotinylated Aleuria aurantia lectin was purchased from Seikagaku Corp. (Tokyo, Japan). Pholiota squarrosa lectin-Biotin conjugate agarose bead, which specifically recognizes core fucosylated N-glycans, was a generous gift from Dr. Kobayashi Yuka (J-Oil Mills, Inc., Tokyo, Japan). The magnetic protein A beads were from Invitrogen, and the avidin-biotin-agarose beads were from Millipore. Tetraethylammonium (TEA), kynurenic acid, and lidocaine all were obtained from Sigma. Glycine was purchased from Nacalai Tesque (Kyoto, Japan). l-Glutamic acid (glutamate), tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione, and d-(−)-2-amino-5-phosphonopentanoic acid were purchased from Wako (Osaka, Japan).

      Hippocampal Slice Preparation

      Hippocampal slices (400 μm) were prepared from 3–5-week-old mice decapitated under isoflurance anesthesia. For the dissection, the cutting solution contained 229 mm mannitol, 3 mm KCl, 26 mm NaHCO3, 1 mm H3PO4, 7 mm MgCl2, and 11 mm glucose, pH 7.4, along with a mixed gas that was 95% O2 and 5% CO2, 1 mm kynurenic acid, and 200 μm lidocaine. For TEA-induced LTP recording, a surgical cut was additionally made between CA3 and CA1 to prevent epileptiform bursting. Slices were incubated at 34 °C for 1 h with artificial cerebrospinal fluid, which contained 125 mm NaCl, 2.5 mm KCl, 25 mm NaHCO3, 1.25 mm NaH2PO4, 2 mm CaCl2, 1 mm MgCl2, 11 mm glucose, pH 7.4, with a mixed gas that was 95% O2 and 5% CO2. During the recording experiments, slices were superfused with an artificial cerebrospinal fluid flow (2 ml/min, 34 °C).

      Extracellular Recordings and Induction of LTP

      Field excitatory postsynaptic potentials were recorded in the stratum radiatum of the CA1 region using glass microelectrodes (tip diameters, 10 μm), which were filled with 1.78% Na2SO4 solution. A tungsten bipolar electrode was used to stimulate the Schaffer collateral axons and twin pulses at 200-μs duration and 50-ms intervals applied at a frequency of 0.033 Hz, with an intensity of less than 100 μA. The field excitatory postsynaptic potential was amplified and digitized using a conventional electrophysiological system (Axon Axopatch 200B plus Digidata1200B, Molecular Devices) and analyzed using Clampex version 10.2 software (Molecular Devices). A baseline recording was performed for at least 1 h. Then E-LTP was induced by applying a single 100-Hz train for 1 s at test strength. Voltage-dependent Ca2+ channel-dependent LTP was induced by applying 25 mm TEA for 10 min (
      • Ohta H.
      • Sakai S.
      • Ito S.
      • Ishizuka T.
      • Fukazawa Y.
      • Kemuriyama T.
      • Tandai-Hiruma M.
      • Mushiake H.
      • Sato Y.
      • Yawo H.
      • Nishida Y.
      Paired stimulation between CA3 and CA1 alters excitability of CA3 in the rat hippocampus.
      ).

      Ca2+ Imaging

      Primary neural cells were cultured as described previously (
      • Gu W.
      • Fukuda T.
      • Isaji T.
      • Hashimoto H.
      • Wang Y.
      • Gu J.
      α1,6-Fucosylation regulates neurite formation via the activin/phospho-Smad2 pathway in PC12 cells: the implicated dual effects of Fut8 for TGF-β/activin-mediated signaling.
      ). After culturing for 14 days on glass-bottom dishes (Greiner Bio-one), the cultured media were replaced by a fresh medium containing 2 μm Ca2+ indicator Fura-2/AM (Dojindo, Japan) at 37 °C for 1 h to load and sensitize the probe by cleaving its AM group with cytosolic esterase. Fura-2-loaded cells were washed with a balanced salt solution, pH 7.4, consisting of 20 mm HEPES, 5.5 mm glucose, 130 mm NaCl, 5.4 mm KCl, 0.8 μm MgSO4, and 1.8 μm CaCl2. Indirect responses were blocked via the application of 1 μm tetrodotoxin in all procedures (
      • Padjen A.L.
      • Smith P.A.
      The role of the electrogenic sodium pump in the glutamate afterhyperpolarization of frog spinal cord.
      ). Then cells were exposed to either 1 or 3 μm glutamate plus 10 μm glycine containing a balanced salt solution flow (1.75 ml/min, 34 °C) for 1 min, respectively, followed by a 3-min balanced salt solution flow wash. The AMPAR-mediated Ca2+ influx was blocked by an AMPA/kainate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione or d-(−)-2-amino-5-phosphonopentanoic acid, a competitive NMDA receptor antagonist. The images were acquired at emission wavelengths between 490 and 520 nm while being excited sequentially at 340 (red) and 380 (green) nm (exposure time, 0.1–2 s). The ratio of F340/F380, indicating an influx efficiency of Ca2+, was analyzed by using an image processor (Aqua Q, Hamamatsu, Japan) connected to a cooled CCD camera (Orca, Hamamatsu, Japan) (
      • Kanatsu Y.
      • Chen N.H.
      • Mitoma J.
      • Nakagawa T.
      • Hirabayashi Y.
      • Higashi H.
      Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation.
      ).

      Preparation of Hippocampus Extracts

      Hippocampi were obtained from 3–5-week-old mice decapitated under isoflurance anesthesia. For Western blot, tissues were homogenized with modified radioimmune precipitation assay buffer consisting of 50 mm Tris-HCl, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% SDS, 1% protease inhibitor mixture (Nacalai Tesque), and 1% phosphatase inhibitor mixture (Nacalai Tesque). For immunoprecipitation, tissues were homogenized with 50 mm Tris-HCl plus 1% protease inhibitor mixture. Membrane fractions were sedimented by centrifugation (105,000 × g, 1 h, 4 °C) and dissolved in 1% Triton X-100, 50 mm Tris-HCl, 1 mm EDTA, and 1% protease inhibitor mixture for 45 min in a 37 °C water bath. After a subsequent removal of the insoluble materials by centrifugation, the supernatants were obtained for immunoprecipitation.

      PSD Fractions

      All biochemical experiments were carried out either on ice or at 4 °C (
      • Morita I.
      • Kakuda S.
      • Takeuchi Y.
      • Itoh S.
      • Kawasaki N.
      • Kizuka Y.
      • Kawasaki T.
      • Oka S.
      HNK-1 glyco-epitope regulates the stability of the glutamate receptor subunit GluR2 on the neuronal cell surface.
      ). Dissected sections of mouse brains were homogenized with 10 volumes of buffer A (0.32 m sucrose, 10 mm Tris-HCl, 1 mm EDTA, and 1% protease inhibitor mixture) with 15 up and down strokes at 1,000 rpm. The homogenate was centrifuged at 1,000 × g for 10 min to remove the nuclear fraction and large debris. The supernatant (S1, postnuclear fraction) was centrifuged at 10,000 × g for 20 min to yield a crude synaptosome pellet (P2) and the supernatant (S2). The S2 was centrifuged at 105,000 × g for 60 min to yield a light membrane pellet (P3). The P2 fraction was lysed with hypo-osmotic (10% buffer A + 90% H2O) buffer and centrifuged at 25,000 × g for 30 min to yield the synaptosomal membrane fraction (LP1). The LP1 fraction was then suspended in 0.5% Triton X-100 in buffer A for 15 min and centrifuged at 105,000 × g for 1 h to yield the PSD fraction. All pellet fractions were dissolved in 0.5% SDS. The protein concentration of each fraction was adjusted to 2 μg/μl using a Pierce BCA protein assay kit (Thermo).

      Immunoblot Analysis and Immunoprecipitation

      Cells cultured under different conditions were washed with PBS and lysed with lysis buffer that contained 1% Triton X-100, 10 mm Tris-HCl, 150 mm NaCl, and 1% protease inhibitor mixture. Cell lysates were separated in SDS-PAGE gels under reducing conditions and were then transferred to PVDF membranes. For Western blot, the membranes were blocked in 5% dried skimmed milk for 1 h at room temperature and probed with specific primary antibodies followed by incubation with appropriate secondary antibodies that had been conjugated with HRP. Finally, specific proteins were visualized using an ECL system (Amersham Biosciences). These membranes were stripped and reprobed with an antibody against the corresponding total proteins to confirm equal loading. For lectin blot, the membranes were blocked in 3% BSA and then detected with A. aurantia lectin. The immunoreactive bands were visualized using a Vectastain ABC kit (Vector Laboratories).
      For the immunoprecipitation, all of the experiments were carried out on ice or at 4 °C; 2 μg of anti-GluA2 antibodies were attached to 5 μl of magnetic protein A beads in 100 μl of PBS plus 0.1% Triton X-100. Protein A beads were pelleted and incubated with the membrane lysates or cell lysates for 40 min with constant rotation. The beads were then washed three times with lysis buffer, and the immunoprecipitate was dissolved in 30 μl of SDS-PAGE sample solution.

      Statistical Analysis

      All of the electrophysiological data in this study are expressed as means ± S.E., and others are expressed as means ± S.D. Either a two-way analysis of variance or an unpaired Student's t test was used to analyze the statistical significance of these results.

      Results

      The Basal Synaptic Transmissions Were Increased, but HFS-induced LTP Was Decreased in Fut8−/− Mice

      Previously, we reported that the Fut8−/− mice exhibited a schizophrenia-like phenotype (
      • Fukuda T.
      • Hashimoto H.
      • Okayasu N.
      • Kameyama A.
      • Onogi H.
      • Nakagawasai O.
      • Nakazawa T.
      • Kurosawa T.
      • Hao Y.
      • Isaji T.
      • Tadano T.
      • Narimatsu H.
      • Taniguchi N.
      • Gu J.
      α1,6-Fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.
      ). Synaptic activity and connectivity played important roles during the progression of the schizophrenia (
      • Savanthrapadian S.
      • Wolff A.R.
      • Logan B.J.
      • Eckert M.J.
      • Bilkey D.K.
      • Abraham W.C.
      Enhanced hippocampal neuronal excitability and LTP persistence associated with reduced behavioral flexibility in the maternal immune activation model of schizophrenia.
      ). Therefore, we analyzed basal synaptic neurotransmission. First, we recorded the field excitatory postsynaptic potential (fEPSP) in the hippocampus CA1 area (Fig. 1A) and found that the responses to stimuli were more sensitive in Fut8−/− mice than in Fut8+/+ mice, which was confirmed by the input-output curve (Fig. 1B). However, the paired pulse ratio, which indicated presynaptic facilitation, showed no significant difference between Fut8+/+ and Fut8−/− mice (Fig. 1C). On the other hand, based on our previous results from the Y-maze task, we knew that learning and memory could be impaired in Fut8−/− mice (
      • Fukuda T.
      • Hashimoto H.
      • Okayasu N.
      • Kameyama A.
      • Onogi H.
      • Nakagawasai O.
      • Nakazawa T.
      • Kurosawa T.
      • Hao Y.
      • Isaji T.
      • Tadano T.
      • Narimatsu H.
      • Taniguchi N.
      • Gu J.
      α1,6-Fucosyltransferase-deficient mice exhibit multiple behavioral abnormalities associated with a schizophrenia-like phenotype: importance of the balance between the dopamine and serotonin systems.
      ). We further examined the formation of E-LTP, which is generally used as a primary experimental model of memory formation in neuronal circuits. E-LTP was triggered by the application of HFS, and we found that the scale of E-LTP was significantly decreased in Fut8−/− mice, compared with Fut8+/+ mice (Fig. 1D), but the TEA-induced (voltage-gated Ca2+ channel-dependent) LTP showed no significant difference (Fig. 1E), suggesting that the decline in the E-LTP in Fut8−/− mice was mainly caused by the events in postsynaptic neurons rather than in the presynaptic regions. Above all, Fut8−/− mice showed hyperactivity in basal synaptic transmissions and a decrease in E-LTP in postsynaptic neurons.
      Figure thumbnail gr1
      FIGURE 1.The HFS-induced LTP was decreased in Fut8−/− mice, but L-Ca2+ channel-dependent LTP was not. A, sample traces of field excitatory postsynaptic potential (fEPSP), induced by twin pulses at 200-μs duration and 50-ms intervals applied at a frequency of 0.033 Hz in 100 μA. B, ratio of stimulus intensity (input) to the amplitude of excitatory postsynaptic potential (output) (Fut8+/+ (N = 5, n = 12) and Fut8−/− (N = 6, n = 13)). Data represent the mean ± S.E. *, p < 0.05, two-way analysis of variance. C, statistical comparisons of paired pulse ratio by unpaired Student's t test (mean ± S.D. (error bars)): 1.71 ± 0.07 (N = 8, n = 20) in Fut8+/+ and 1.75 ± 0.15 (N = 9, n = 22) in Fut8−/−. D, HFS was delivered after 10 min of baseline recording. HFS-induced LTP was recorded for another 30 min (Fut8+/+ (N = 6, n = 12) and Fut8−/− (N = 6, n = 12)). Data represent the mean ± S.E. *, p < 0.05, two-way analysis of variance. E, 25 mm TEA was applied after baseline recording for 10 min. TEA-induced LTP was recorded for another 70 min. Fut8+/+ (N = 3, n = 7) and Fut8−/− (N = 3, n = 8). Data represent the mean ± S.E. (N, number of mice; n, number of recordings).

      Persistent Activation of CaMKII and Increased AMPARs in the PSD Area of Fut8−/− Mice

      To examine the events in the postsynaptic regions, we analyzed the activation levels of ionotropic glutamate receptors and CaMKII, which are mainly mediated in the induction and maintenance of E-LTP. We found that the CaMKII, a necessary component of the cellular machinery underlying learning and memory, was greatly activated in Fut8−/− mice (Fig. 2, A and B). Phosphorylation at Thr-286 of CaMKII plays essential roles in LTP, whereas LTP is attenuated in T286A (Thr-286 non-autophosphorylatable) mutant mice (
      • Ohno M.
      • Frankland P.W.
      • Silva A.J.
      A pharmacogenetic inducible approach to the study of NMDA/αCaMKII signaling in synaptic plasticity.
      ,
      • Giese K.P.
      • Fedorov N.B.
      • Filipkowski R.K.
      • Silva A.J.
      Autophosphorylation at Thr286 of the α calcium-calmodulin kinase II in LTP and learning.
      ). However, the expression of a constitutively active form of CaMKII into CA1 neurons enhances postsynaptic transmission and prevents further potentiation via synaptic stimulation (
      • Pettit D.L.
      • Perlman S.
      • Malinow R.
      Potentiated transmission and prevention of further LTP by increased CaMKII activity in postsynaptic hippocampal slice neurons.
      ,
      • Pi H.J.
      • Otmakhov N.
      • Lemelin D.
      • De Koninck P.
      • Lisman J.
      Autonomous CaMKII can promote either long-term potentiation or long-term depression, depending on the state of T305/T306 phosphorylation.
      ) because the CaMKII and LTP enhance synaptic transmission by the same mechanism (
      • Lledo P.M.
      • Hjelmstad G.O.
      • Mukherji S.
      • Soderling T.R.
      • Malenka R.C.
      • Nicoll R.A.
      Calcium/calmodulin-dependent kinase II and long-term potentiation enhance synaptic transmission by the same mechanism.
      ). Thus, to be consistent with those observations, we thought that the persistent activation of CaMKII might lead to a decrease in E-LTP in Fut8−/− mice.
      Figure thumbnail gr2
      FIGURE 2.Phosphorylated CaMKII levels were increased in Fut8−/− mice. A, comparison of the phosphorylation of CaMKII, GluN2A, and GluN2B in the hippocampus of Fut8+/+, Fut8+/−, and Fut8−/− mice. B, -fold P-CaMKII, P-GluN2A, and P-GluN2B expression levels are compared in Fut8+/+ mice. The quantitative data were obtained from three independent experiments. Data represent the mean ± S.D. (error bars). *, p < 0.05, unpaired Student's t test. C, the PSD fraction was isolated by ultracentrifuge as described under “Experimental Procedures” and blotted with anti-GluN2A, anti-GluA1, anti-GluA2, anti-GluA3, anti-γ2, anti-CaMKII, and PSD-95. P2, crude synaptosome fraction; P3, light membrane fraction. D, -fold of expression levels of GluN2A, GluA1, GluA2, GluA3, γ2, CaMKII, and PSD-95 in PSD fraction of Fut8−/− mice, compared with those in Fut8+/+ mice. The quantitative data were obtained from three independent experiments. Data represent the mean ± S.D. *, p < 0.05, unpaired Student's t test.
      Ionotropic glutamate receptors, which can be subdivided into NMDAR, AMPAR, and kainate receptors, mediate most excitatory neuronal transmission (
      • Dingledine R.
      • Borges K.
      • Bowie D.
      • Traynelis S.F.
      The glutamate receptor ion channels.
      ). Among these receptors, NMDAR channels have several unique features, including a voltage-sensitive block by extracellular Mg2+ and a high permeability to Ca2+ (
      • Cull-Candy S.
      • Brickley S.
      • Farrant M.
      NMDA receptor subunits: diversity, development and disease.
      ). The Mg2+ block acts as a molecular switch, with the removal of Mg2+ from the pore of the channel when postsynaptic cells are depolarized. The relief of the block leads to the Ca2+ influx through the NMDAR channel that regulates synaptic strength through Ca2+-activated signaling cascades. The phosphorylation of NMDAR is known to alter the channel properties (
      • Wang Y.T.
      • Yu X.M.
      • Salter M.W.
      Ca2+-independent reduction of N-methyl-d-aspartate channel activity by protein tyrosine phosphatase.
      ). Consistent with the activated CaMKII in Fut8−/− mice, in the present study, the expression levels of phosphorylated GluN2A at Tyr-1246 and GluN2B at Tyr-1472 were also increased (Fig. 2, A and B). The rise of intracellular Ca2+ level causes reversible inactivation through the phosphorylation of receptors to prevent more synaptic stimulation (
      • Medina I.
      • Filippova N.
      • Charton G.
      • Rougeole S.
      • Ben-Ari Y.
      • Khrestchatisky M.
      • Bregestovski P.
      Calcium-dependent inactivation of heteromeric NMDA receptor-channels expressed in human embryonic kidney cells.
      ). For example, phosphorylation of Tyr-1472, the major phosphorylation site by Fyn on GluN2B (
      • Takasu M.A.
      • Dalva M.B.
      • Zigmond R.E.
      • Greenberg M.E.
      Modulation of NMDA receptor-dependent calcium influx and gene expression through EphB receptors.
      ), disrupts its binding to an AP-2 adaptor, thereby resulting in the inhibition of GluN2B-mediated endocytosis.
      When postsynaptic cells receive stimulation, the activated CaMKII phosphorylates TARPs binding to PSD95, thereby regulating AMPAR numbers in the PSD area (
      • Lisman J.
      • Yasuda R.
      • Raghavachari S.
      Mechanisms of CaMKII action in long-term potentiation.
      ,
      • Payne H.L.
      The role of transmembrane AMPA receptor regulatory proteins (TARPs) in neurotransmission and receptor trafficking (Review).
      ). However, with the decay of LTP, the AMPARs leave the PSD area and prepare for the next stimulation (
      • Kullmann D.M.
      • Lamsa K.P.
      Long-term synaptic plasticity in hippocampal interneurons.
      ). The trafficking of AMPARs to regulate the number of receptors at the synapse plays a key role in various forms of synaptic plasticity. Interestingly, the expression levels of GluA1, GluA2, and GluA3, but not GluN2A and CaMKII, in the PSD area of brain tissues were increased in Fut8−/− mice (Fig. 2, C and D). Furthermore, Stargazin (γ2), the main member of the TARP family, accumulated in the PSD area and acted as auxiliary subunits of AMPARs (Fig. 2, C and D). The distribution of TARPs has been reported (
      • Payne H.L.
      The role of transmembrane AMPA receptor regulatory proteins (TARPs) in neurotransmission and receptor trafficking (Review).
      ), γ2 is abundant throughout the brain, and γ8 is mainly expressed in the hippocampus. Unfortunately, we failed to detect subunits of γ8 using commercially available antibody.

      The Enhanced Heteromerization of AMPARs in Fut8−/− Mice

      To investigate the underlying mechanism of the persistent activation of CaMKII in Fut8−/− mice, we analyzed the heteromerization of AMPARs. Among the ionotropic glutamate receptors that mediate fast excitatory synaptic transmission, most of the biochemical cascades were initiated by the AMPAR-mediated depolarization of postsynaptic cells. Because AMPARs are the primary requirement in the expression of LTP, the modulation of AMPARs could affect synaptic transmission. In fact, several studies have reported that the biological functions of AMPARs are affected by post-translational modification (
      • Jiang J.
      • Suppiramaniam V.
      • Wooten M.W.
      Posttranslational modifications and receptor-associated proteins in AMPA receptor trafficking and synaptic plasticity.
      ). For example, human natural killer (HNK1) glycoepitope regulates the stability of GluA2 on the neuronal cell surface and dendritic spine maturation (
      • Morita I.
      • Kakuda S.
      • Takeuchi Y.
      • Itoh S.
      • Kawasaki N.
      • Kizuka Y.
      • Kawasaki T.
      • Oka S.
      HNK-1 glyco-epitope regulates the stability of the glutamate receptor subunit GluR2 on the neuronal cell surface.
      ,
      • Kizuka Y.
      • Oka S.
      Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate.
      ). We found that there were no significant differences in either the total or the membrane expression levels of NMDARs and AMPARs in the hippocampi of the three genotypes of the mice (Fut8+/+, Fut8+/−, and Fut8−/−) (Figs. 2A, 3A, and 3C), whereas the core fucosylation on these receptors was completely blocked in Fut8−/− mice (Fig. 3B). Then the heteromerization of AMPARs was analyzed by using a pull-down assay. The association levels of GluA1/2 and GluA2/3 were greatly increased in Fut8−/− mice compared with that in Fut8+/+ mice (Fig. 3, C–E). Although it is well known that there are very few GluA1/3 complex formations in the hippocampus (
      • Wenthold R.J.
      • Petralia R.S.
      • Blahos 2nd, J.
      • Niedzielski A.S.
      Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons.
      ), the loss of core fucosylation could have altered the AMPAR complex formation. Thus, we checked the GluA1/3 complex formation as well as the interactions with GluA1. As shown in Fig. 3E, the levels of GluA1/3 complex formations could not be detected in the immunoprecipitates of either GluA1 (top panels) or GluA3 (bottom panels). Likewise, GluN1 expression was not detectable in either the GluA3 or GluA1 complexes. These data suggest that a loss of core fucosylation on AMPARs enhanced the heteromerization of their subunits, which might have subsequently depolarized the neuronal cell membrane to induce Ca2+ influx and CaMKII phosphorylation. Based on the observations in Fig. 3, C and E, we speculated that those heteromeric formations were mainly increased in the PSDs, as shown in Fig. 2C. Although the expression levels of homomeric receptors of membrane fractions in the KO mice were theoretically less than those in Fut8+/+ or Fut8+/− mice, it is quite difficult to completely exclude the possibility that those homomeric receptors were also increased in Fut8−/− mice. In fact, it has been reported that homomeric GluA1 is first recruited into PSD following glutamate stimulation (
      • Tanaka H.
      • Hirano T.
      Visualization of subunit-specific delivery of glutamate receptors to postsynaptic membrane during hippocampal long-term potentiation.
      ).
      Figure thumbnail gr3
      FIGURE 3.Enhanced AMPAR heteromerization in Fut8−/− mice. A, expression levels of GluN1 and GluAs between Fut8+/+, Fut8+/−, and Fut8−/− mice in hippocampus lysate. α-Tubulin was used as a loading control. B, comparison of core fucosylation levels on GluNs and GluAs between Fut8+/+ and Fut8−/− mice. The same amounts of hippocampus lysates were immunoprecipitated (IP) with P. squarrosa lectin-agarose and blotted with anti-GluNs and anti-GluAs. C, membrane proteins were isolated as described under “Experimental Procedures.” The same amounts of hippocampus membrane proteins were immunoprecipitated with anti-GluA2 and blotted with anti-GluA1, -GluA2, and -GluA3 (bottom panels). The total expression levels of GluA1, GluA2, and GluA3 and synapsin in hippocampus membranes were used as a loading control (top panels). D, -fold heteromerization levels of GluA1 and GluA2 (right) or GluA2 and GluA3 (left) were compared with those in Fut8+/+ mice, which were set as 1. The quantitative data were obtained from three independent experiments. Data represent the mean ± S.D. (error bars); *, p < 0.05, unpaired Student's t test. E, the same amounts of hippocampus membrane proteins were immunoprecipitated with anti-GluA1 (top panels) or anti-GluA3 (bottom panels) antibodies and then blotted with anti-GluA2, anti-GluN1, and anti-GluA3 or anti-GluA1 antibodies, respectively.
      To further confirm the effects of core fucosylation on the complex formation for AMPARs, we expressed recombinant mouse GluA1 and GluA2 heteromeric combinations in wild-type and Fut8-knock-out (Fut8-KO) 293T cells, which were generated using the CompoZr Knock-out Zinc Finger Nucleases (ZFN) kit (Sigma), which was confirmed by an A. aurantia lectin blot (Fig. 4A) and RT-PCR (Fig. 4B). The coimmunoprecipitation experiments showed that the association levels of GluA1 and GluA2 were much higher in Fut8-KO cells compared with that in the wild-type 293T cells (Fig. 4, C and D). Taken together, these results strongly suggested that the loss of core fucosylation on AMPARs enhanced their subunit complex formation in vitro.
      Figure thumbnail gr4
      FIGURE 4.Effect of core fucosylation on AMPAR heteromerization in 293T cells. A, the Fut8 gene was deleted by a KO technique using a CompoZr Knock-out Zinc Finger Nucleases (ZFN) kit (Sigma) in 293T cells according to the manufacturer's instructions. Equal amounts of 293T WT and Fut8-KO cell lysates were separated on 8% SDS-PAGE, and the membranes were probed with A. aurantia lectin, which specifically recognizes core fucose. α-Tubulin was used as a loading control. B, genomic DNA was extracted from 293T WT and Fut8-KO cells. The special primers were obtained from Sigma ZFN KIT and used to check for efficiency in the knock-out of Fut8. C, 293T WT and Fut8-KO cells were infected with GluA1 and GluA2 by Lipofectamine 2000 for 48 h. Total expression levels of GluA1 and GluA2 were imunoblotted with anti-GluA1 and anti-GluA2 antibodies (left panels). The same amounts of cell lysates were immunoprecipitated (IP) with anti-GluA2 (right panels) and blotted with anti-GluA1 and anti-GluA2 antibodies. D, -fold heteromerization levels of GluA1 and GluA2 in Fut8-KO cells compared with those in WT, which were set as 1. The quantitative data were obtained from three independent experiments. Data represent the mean ± S.D. (error bars); **, p < 0.05, Student's paired t test.

      AMPAR-mediated Ca2+ Influx Was Enhanced in Primary Neurons Obtained from Fut8−/− Mice

      Furthermore, to clarify the functions of the core fucosylation of AMPARs with respect to potentials, we examined the calcium influx in the 293T cells transfected with GluA1 alone compared with the cells co-transfected with GluA1 and GluA2. Unfortunately, when using the Fura-2 system, the induction of calcium influx upon glutamate stimulation was too small to measure in either the wild-type or the Fut8-KO 293T cells. Considering the possibility that an expression of NMDAR is required for the induction, we used primary cultured neurons as a model system to record the Ca2+ influx to examine the AMPAR and NMDAR potentials. Compared with primary neurons from Fut8+/+ and Fut8+/− mice, the Ca2+ influxes were significantly increased in the neurons from Fut8−/− mice (Fig. 5, A and B). To check whether the increased Ca2+ influx in Fut8−/− was mediated by an AMPAR-NMDAR sequential response (
      • Guo Z.Y.
      • Li C.Z.
      • Li X.J.
      • Wang Y.L.
      • Mattson M.P.
      • Lu C.B.
      The developmental regulation of glutamate receptor-mediated calcium signaling in primary cultured rat hippocampal neurons.
      ) (i.e. to determine whether AMPAR-induced postsynaptic depolarization can activate the NMDAR and then induce Ca2+ influx), both antagonists were added to the balanced salt solution bath flow. As shown in Fig. 5C, both 6-cyano-7-nitroquinoxaline-2,3-dione, an AMPAR blocker, and d-(−)-2-amino-5-phosphonopentanoic acid, a NMDAR inhibitor, could eliminate the difference in Ca2+ influx between Fut8+/+ and Fut8−/− mice, further suggesting that the loss of core fucosylation enhances the heteromerization of AMPARs and their downstream signaling in primary cultured neurons.
      Figure thumbnail gr5
      FIGURE 5.AMPAR-mediated Ca2+ influx was enhanced in primary neurons from Fut8−/− mice. A, primary neurons from Fut8+/+, Fut8+/−, and Fut8−/− mice cultured for 14 days in vitro. Cells were exposed to either 1 or 3 μm l-glutamic acid plus 10 μm glycine, respectively. The quantitative data were obtained from three independent experiments. One experiment comprised 40 cells in different fields of view. Data represent the mean ± S.E. An unpaired Student's t test was used for comparison of the peak reaction of independent groups; *, p < 0.05. B, a representative Ca2+ influx was visualized using an image processor connected to a cooled CCD camera. control, the image at baseline; 3 μm Glu: the image of the peak during the stimulation of 3 μm l-glutamic acid plus 10 μm glycine. C, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or d-(−)-2-amino-5-phosphonopentanoic acid (DAP-5) was used to block the AMPAR-NMDAR-mediated Ca2+ influx. The quantitative data were obtained from three independent experiments. One experiment comprised 40 cells in different fields of view. Data represent the mean ± S.E. Student's unpaired t test was used for comparison of the peak reaction of independent groups; *, p < 0.05.

      Discussion

      In the present study, we investigated the role of core fucosylation on E-LTP in the hippocampus and found that the HFS-induced LTP was dramatically decreased in Fut8−/− mice. A molecular mechanism could be postulated whereby the lack of core fucosylation in AMPARs results in aberrant heteromerization, which persistently activates cellular signaling, such as an increase in CaMKII phosphorylation levels at the basal level. Although the underlying molecular mechanism requires further study, it is reasonable to speculate that a persistent activation of CaMKII may interfere with some of the normal signal pathways (or feedback loops), which would decrease the response for LTP stimulation. In fact, the expression of a constitutively active form of CaMKII enhanced postsynaptic transmission and prevented further LTP (
      • Pettit D.L.
      • Perlman S.
      • Malinow R.
      Potentiated transmission and prevention of further LTP by increased CaMKII activity in postsynaptic hippocampal slice neurons.
      ). A similar phenomenon has also been observed in MAPK pathways. An activation of the MAPK/ERK cascade (short term) is commonly thought to be important for cell proliferation, but a persistently activated ERK (long term) reverses and down-regulates cell growth (
      • Booth A.
      • Trudeau T.
      • Gomez C.
      • Lucia M.S.
      • Gutierrez-Hartmann A.
      Persistent ERK/MAPK activation promotes lactotrope differentiation and diminishes tumorigenic phenotype.
      ,
      • Borrelli E.
      • Sawchenko P.E.
      • Evans R.M.
      Pituitary hyperplasia induced by ectopic expression of nerve growth factor.
      ).
      Social dysfunction related to the loss of neocortical excitation/inhibition balance is an important symptom in schizophrenia (
      • Yizhar O.
      • Fenno L.E.
      • Prigge M.
      • Schneider F.
      • Davidson T.J.
      • O'Shea D.J.
      • Sohal V.S.
      • Goshen I.
      • Finkelstein J.
      • Paz J.T.
      • Stehfest K.
      • Fudim R.
      • Ramakrishnan C.
      • Huguenard J.R.
      • Hegemann P.
      • Deisseroth K.
      Neocortical excitation/inhibition balance in information processing and social dysfunction.
      ). In the present study, we found an increase in synaptic transmission in the hippocampal CA1 area, which indicated that the balance of excitation/inhibition might be impaired in Fut8−/− mice. On the other hand, several studies have provided direct neurophysiological evidence for disrupted LTP-like plasticity in schizophrenia (
      • Frantseva M.V.
      • Fitzgerald P.B.
      • Chen R.
      • Möller B.
      • Daigle M.
      • Daskalakis Z.J.
      Evidence for impaired long-term potentiation in schizophrenia and its relationship to motor skill learning.
      ,
      • Sanderson T.M.
      • Cotel M.C.
      • O'Neill M.J.
      • Tricklebank M.D.
      • Collingridge G.L.
      • Sher E.
      Alterations in hippocampal excitability, synaptic transmission and synaptic plasticity in a neurodevelopmental model of schizophrenia.
      ,
      • Hasan A.
      • Nitsche M.A.
      • Rein B.
      • Schneider-Axmann T.
      • Guse B.
      • Gruber O.
      • Falkai P.
      • Wobrock T.
      Dysfunctional long-term potentiation-like plasticity in schizophrenia revealed by transcranial direct current stimulation.
      ). The evaluation of experimental LTP could be used to analyze the schizophrenia-like phenotype in Fut8−/− mice. It is well known that LTP is not a solitary phenomenon but contains various forms that can be categorized by the general mechanisms in vivo. For example, ionotropic glutamate receptors, metabotropic type glutamate receptors, and dopamine receptors (D1–D5) mediate LTP through different pathways (
      • Raymond C.R.
      LTP forms 1, 2 and 3: different mechanisms for the “long” in long-term potentiation.
      ). The LTP induced by HFS might be neither protein synthesis-dependent (D1–D5) nor metabotropic type glutamate receptor-dependent based on previous observations (
      • Raymond C.R.
      LTP forms 1, 2 and 3: different mechanisms for the “long” in long-term potentiation.
      ,
      • Raymond C.R.
      • Thompson V.L.
      • Tate W.P.
      • Abraham W.C.
      Metabotropic glutamate receptors trigger homosynaptic protein synthesis to prolong long-term potentiation.
      • Bengtson C.P.
      • Freitag H.E.
      • Weislogel J.M.
      • Bading H.
      Nuclear calcium sensors reveal that repetition of trains of synaptic stimuli boosts nuclear calcium signaling in CA1 pyramidal neurons.
      ). Therefore, in the present study, we mainly focused on the molecules mediated in the ionotropic glutamate receptor-dependent pathway, including the NMDAR, AMPAR, and CaMKII in postsynaptic regions.
      CaMKII activation plays essential roles in LTP because LTP induction that is produced by tetanus can be blocked by postsynaptic application of the peptide inhibitors of the kinase (
      • Hvalby O.
      • Hemmings Jr., H.C.
      • Paulsen O.
      • Czernik A.J.
      • Nairn A.C.
      • Godfraind J.M.
      • Jensen V.
      • Raastad M.
      • Storm J.F.
      • Andersen P.
      Specificity of protein kinase inhibitor peptides and induction of long-term potentiation.
      ) or by a knock-in mutation that encodes T286A in CaMKII α (
      • Giese K.P.
      • Fedorov N.B.
      • Filipkowski R.K.
      • Silva A.J.
      Autophosphorylation at Thr286 of the α calcium-calmodulin kinase II in LTP and learning.
      ). Based on these findings, we speculated that a decrease in recorded LTP in Fut8−/− mice could be accompanied by a dysfunction in the activation of CaMKII. Unexpectedly, the phosphorylation levels of CaMKII in Fut8−/− mice were significantly higher than those in Fut8+/+ mice (Fig. 2A). However, it is known that a constitutively active form of CaMKII is sufficient to augment synaptic strength and then prevents further tetanus-induced LTP (
      • Pettit D.L.
      • Perlman S.
      • Malinow R.
      Potentiated transmission and prevention of further LTP by increased CaMKII activity in postsynaptic hippocampal slice neurons.
      ). The hippocampal slices from Fut8−/− mice showed steeper input-output curves (Fig. 1C), which could strongly support potentiated synaptic transmission. Thus, we speculated that the absence of LTP in Fut8−/− mice could be due to a prior maximal activation rather than to a block of LTP. Altered hippocampal synaptic plasticity is likely to affect memory processing, and therefore any such pathology may contribute to the cognitive symptoms of schizophrenia. In a maternal immune activation animal model of schizophrenia, the enhanced persistence of dentate LTP has been associated with reduced behavioral flexibility, which may be related to the alteration of synaptic plasticity (
      • Savanthrapadian S.
      • Wolff A.R.
      • Logan B.J.
      • Eckert M.J.
      • Bilkey D.K.
      • Abraham W.C.
      Enhanced hippocampal neuronal excitability and LTP persistence associated with reduced behavioral flexibility in the maternal immune activation model of schizophrenia.
      ). In addition, Angelman disease, a form of intellectual disability, is associated with persistent activation of CaMKII, and a loss of LTP underlies this learning deficit in humans (
      • Weeber E.J.
      • Jiang Y.H.
      • Elgersma Y.
      • Varga A.W.
      • Carrasquillo Y.
      • Brown S.E.
      • Christian J.M.
      • Mirnikjoo B.
      • Silva A.
      • Beaudet A.L.
      • Sweatt J.D.
      Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome.
      ). These findings support our hypothesis that increased CaMKII activation in postsynaptic cells triggers LTP maximally and then prevents LTP induction, which may relate to the schizophrenia-like phenotype in Fut8−/− mice.
      The AMPAR-mediated postsynaptic depolarization can activate the NMDAR, as well as Ca2+ influx and CaMKII. Curiously, the phosphorylated forms of NMDARs were greatly increased in Fut8−/− mice, compared with those in wild type mice (Fig. 2). This seems contradictory because the phosphorylated NMDARs suppress CaMKII activation (
      • Chen B.S.
      • Roche K.W.
      Regulation of NMDA receptors by phosphorylation.
      ). However, we speculated that a persistent CaMKII activation could promote the phosphorylation of NMDARs via a novel negative feedback mechanism to inhibit CaMKII activation. Therefore, the activation of AMPAR might determine the persistent activation of CaMKII in Fut8−/− mice. The ability of AMPARs to form a complex regulates their biological functions (
      • Sans N.
      • Vissel B.
      • Petralia R.S.
      • Wang Y.X.
      • Chang K.
      • Royle G.A.
      • Wang C.Y.
      • O'Gorman S.
      • Heinemann S.F.
      • Wenthold R.J.
      Aberrant formation of glutamate receptor complexes in hippocampal neurons of mice lacking the GluR2 AMPA receptor subunit.
      ). Consistently, this heteromerization was greatly enhanced in the brain tissue of Fut8−/− mice, compared with that in Fut8+/+ mice, which was further confirmed by the 293T overexpression system. Taken together, these studies strongly suggest that the enhanced AMPAR heteromerization may play a key role in augmenting the synaptic strength in Fut8−/− mice.
      There are two major complex populations of AMPARs in adult hippocampal neurons: GluA1/2 and GluA2/3 (
      • Shi S.H.
      • Hayashi Y.
      • Petralia R.S.
      • Zaman S.H.
      • Wenthold R.J.
      • Svoboda K.
      • Malinow R.
      Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation.
      ). The GluA1/2 complex is recruited to dendritic spines in an activity-dependent manner, which is associated with CaMKII and requires an interaction with postsynaptic density proteins (
      • Kizuka Y.
      • Oka S.
      Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate.
      ,
      • Sans N.
      • Racca C.
      • Petralia R.S.
      • Wang Y.X.
      • McCallum J.
      • Wenthold R.J.
      Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway.
      ). In contrast, GluA2/3 appears at the synapse, and its delivery is dependent on an interaction with cytoskeletal proteins, such as the N-ethylmaleimide-sensitive factor, and, therefore, GluA2/3 neurons are activated in a constitutively synaptic stimulation-independent manner (
      • Lynch M.A.
      Long-term potentiation and memory.
      ). We focused on the function of the GluA1/2 complex in the present study because activity-dependent changes in the strength of excitatory synapses are the most important cellular mechanism for the plasticity of neuronal networks (
      • Derkach V.A.
      • Oh M.C.
      • Guire E.S.
      • Soderling T.R.
      Regulatory mechanisms of AMPA receptors in synaptic plasticity.
      ,
      • Malinow R.
      • Malenka R.C.
      AMPA receptor trafficking and synaptic plasticity.
      ). However, we did not exclude the possibility that the aberrant enhancement of the heteromerization of GluA2/3 might also contribute to a loss of LTP in Fut8−/− mice.
      Furthermore, in CA1 pyramidal neurons, which serve as a model for understanding LTP, most receptors are known to exist as GluA1/2 heteromers with only a minor contribution from GluA2/3 complexes (
      • Wenthold R.J.
      • Petralia R.S.
      • Blahos 2nd, J.
      • Niedzielski A.S.
      Evidence for multiple AMPA receptor complexes in hippocampal CA1/CA2 neurons.
      ,
      • Lu W.
      • Shi Y.
      • Jackson A.C.
      • Bjorgan K.
      • During M.J.
      • Sprengel R.
      • Seeburg P.H.
      • Nicoll R.A.
      Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach.
      ). The enhancement of the heteromerization of GluA2 with GluA1 and GluA3 should generate calcium-impermeable low conductivity AMPARs and reduce both the general currents and the chances of postsynaptic depolarizations, resulting in a reduction in the LTP observed in Fut8−/− mice. It is clear that our observations in the present study could not be explained by the preceding notion. Based on the observation shown in Fig. 1, the basal transmission was increased in Fut8−/− mice, which suggests that the changes mainly occurred in the postsynaptic region. Furthermore, the phosphorylation levels of CaMKII were increased in Fut8−/− mice. Therefore, we speculated that an aberrant increase in GluA1/2 and GluA2/3 heteromeric formation induces a hypersensitive response for glutamate, which leads to the induction of postsynaptic depolarization, a persistent activation of NMDAR, and an increase in CaMKII phosphorylation levels, which might reverse and decrease the response for LTP stimulation in Fut8−/− mice, as discussed above.
      The present study clearly demonstrated that a loss of core fucosylation could enhance the heteromerization of AMPARs and downstream signaling, but the molecular mechanism for the negative regulator of core fucosylation in the heteromerization remains unclear. Recently, two structural biology research groups analyzed the crystal structures of glycosylated FcγRIIIa and human core fucosylated or afucosylated Fc of IgG (
      • Ferrara C.
      • Grau S.
      • Jäger C.
      • Sondermann P.
      • Brünker P.
      • Waldhauer I.
      • Hennig M.
      • Ruf A.
      • Rufer A.C.
      • Stihle M.
      • Umaña P.
      • Benz J.
      Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose.
      ,
      • Mizushima T.
      • Yagi H.
      • Takemoto E.
      • Shibata-Koyama M.
      • Isoda Y.
      • Iida S.
      • Masuda K.
      • Satoh M.
      • Kato K.
      Structural basis for improved efficacy of therapeutic antibodies on defucosylation of their Fc glycans.
      ). Interestingly, core fucose depletion can increase the incidence of the active conformation of the Tyr-296 of Fc, thereby accelerating the high affinity heteromerization with its receptor. Those studies clearly revealed why a lack of core fucosylation on IgG1 can dramatically enhance antibody-dependent cellular cytotoxicity activity (
      • Shields R.L.
      • Lai J.
      • Keck R.
      • O'Connell L.Y.
      • Hong K.
      • Meng Y.G.
      • Weikert S.H.
      • Presta L.G.
      Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcγ RIII and antibody-dependent cellular toxicity.
      ,
      • Shinkawa T.
      • Nakamura K.
      • Yamane N.
      • Shoji-Hosaka E.
      • Kanda Y.
      • Sakurada M.
      • Uchida K.
      • Anazawa H.
      • Satoh M.
      • Yamasaki M.
      • Hanai N.
      • Shitara K.
      The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity.
      ). In addition, some important amino acid residues in the N-terminal domains of AMPARs reportedly influence their biological functions by regulating the balance between the homo- and heteromeric associations (
      • Rossmann M.
      • Sukumaran M.
      • Penn A.C.
      • Veprintsev D.B.
      • Babu M.M.
      • Greger I.H.
      Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers.
      ). It should be noted here that the heteromeric formation of AMPAR is believed to take place in the endoplasmic reticulum (
      • Greger I.H.
      • Khatri L.
      • Kong X.
      • Ziff E.B.
      AMPA receptor tetramerization is mediated by Q/R editing.
      ), whereas core fucosylation occurs in the medial Golgi apparatus. The present finding that a loss of core fucosylation could enhance the heteromerization of AMPARs reflect the possibility that the regulation of AMPAR heteromerization could be in the Golgi network or at the cell surface as well as in the endoplasmic reticulum. We speculated that the presence of core fucose could have regulated the strength of the interaction in AMPAR complexes.
      In fact, regulation of the AMPAR function is a complex process that involves many essential factors (
      • Derkach V.A.
      • Oh M.C.
      • Guire E.S.
      • Soderling T.R.
      Regulatory mechanisms of AMPA receptors in synaptic plasticity.
      ). For example, the TARP family members can associate with AMPARs and affect their expression levels in PSD, which would then regulate intracellular signaling for synaptic plasticity. Coincidentally, the higher expression levels of stargazin (TARP γ2) were increased in the PSD area in Fut8−/− mice (Fig. 2). The underlying molecular mechanism will require further study.
      Recently, Tucholski et al. (
      • Tucholski J.
      • Simmons M.S.
      • Pinner A.L.
      • Haroutunian V.
      • McCullumsmith R.E.
      • Meador-Woodruff J.H.
      Abnormal N-linked glycosylation of cortical AMPA receptor subunits in schizophrenia.
      ) reported that a high mannose type of N-glycans in AMPARs was significantly decreased in patients with schizophrenia, which, together with the results of the present study, further suggests that N-glycosylation can affect the functions of AMPARs. In conclusion, our study directly demonstrated the function of core fucosylation for learning and memory in hippocampal neurons, and the possible underlying mechanism for a schizophrenia-like phenotype was shown in Fut8−/− mice.

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