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Afadin Regulates Puncta Adherentia Junction Formation and 学位論文題目 Presynaptic Differentiation in Hippocampal Neurons(アファディンは海 Title 馬ニューロンにおいてアドへレンスジャンクションの形成と前シナプ スの分化を調節している) 氏名 Toyoshima, Daisaku Author 専攻分野 博士（医学） Degree 学位授与の日付 2014-03-25 Date of Degree 公開日 2015-03-01 Date of Publication 資源タイプ Thesis or Dissertation / 学位論文 Resource Type 報告番号 甲第6188号 Report Number 権利 Rights JaLCDOI URL http://www.lib.kobe-u.ac.jp/handle_kernel/D1006188
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PDF issue: 2021-09-26
Afadin Regulates Puncta Adherentia Junction Formation and Presynaptic Differentiation in Hippocampal Neurons
アファディンは海馬ニューロンにおいてアドへレンスジャンクション の形成と前シナプスの分化を調節している
豊嶋大作, 萬代研二, 丸尾知彦、Irwan Supriyanto、 富樫英、井上貴仁、森正弘、高井義美
神戸大学大学院医学研究科医科学専攻 小児科学 （指導教員：飯島一誠 教授）
豊嶋大作
Key words: Afadin, puncta adherentia junction, presynapse, hippocampal neuron
Manuscript Click here to download Manuscript: Toyoshima_text_revised_2nd_final.pdf
1 Afadin Regulates Puncta Adherentia Junction Formation and
2 Presynaptic Differentiation in Hippocampal Neurons 3
4 Daisaku Toyoshima1,3,4, Kenji Mandai1,3, Tomohiko Maruo1,3, Irwan Supriyanto2,3,
5 Hideru Togashi1,3, Takahito Inoue1,3, Masahiro Mori2,3 & Yoshimi Takai1,3
6
7 1. Department of Biochemistry and Molecular Biology, Kobe University Graduate
8 School of Medicine, Kobe, Hyogo 650-0047, Japan.
9 2. Faculty of Health Sciences, Kobe University Graduate School of Health Sciences,
10 Kobe, Hyogo 654-0142, Japan.
11 3. CREST, Japan Science and Technology Agency, Kobe, Hyogo 650-0047, Japan.
12 4. Present address: Department of Pediatrics, Kobe University Graduate School of
13 Medicine, Kobe, Hyogo 650-0017, Japan.
14
15 Correspondence should be addressed to Y.T. ([email protected]), K.M.
16 ([email protected]).
17 1-5-6 Minatojimaminami-machi, Chuo-ku, Kobe, Hyogo 650-0047, Japan
18 Tel: +81-78-304-6034; Fax: +81-78-304-6035
19 1 Abstract
2
3 The formation and remodeling of mossy fiber-CA3 pyramidal cell synapses in the
4 stratum lucidum of the hippocampus are implicated in the cellular basis of learning and
5 memory. Afadin and its binding cell adhesion molecules, nectin-1 and nectin-3, together
6 with N-cadherin, are concentrated at puncta adherentia junctions (PAJs) in these synapses.
7 Here, we investigated the roles of afadin in PAJ formation and presynaptic differentiation
8 in mossy fiber-CA3 pyramidal cell synapses. At these synapses in the mice in which the
9 afadin gene was conditionally inactivated before synaptogenesis by using nestin-Cre
10 mice, the immunofluorescence signals for the PAJ components, nectin-1, nectin-3 and N-
11 cadherin, disappeared almost completely, while those for the presynaptic components,
12 VGLUT1 and bassoon, were markedly decreased. In addition, these signals were
13 significantly decreased in cultured afadin-deficient hippocampal neurons. Furthermore,
14 the interevent interval of miniature excitatory postsynaptic currents was prolonged in the
15 cultured afadin-deficient hippocampal neurons compared with control neurons, indicating
16 that presynaptic functions were suppressed or a number of synapse was reduced in the
17 afadin-deficient neurons. Analyses of presynaptic vesicle recycling and paired recordings
18 revealed that the cultured afadin-deficient neurons showed impaired presynaptic
19 functions. These results indicate that afadin regulates both PAJ formation and presynaptic
20 differentiation in most mossy fiber-CA3 pyramidal cell synapses, while in a considerable
21 population of these neurons, afadin regulates only PAJ formation but not presynaptic
22 differentiation.
23
2 1 Introduction
2
3 Synapses are specialized intercellular junctions that are indispensable for neuronal
4 transmission. Most excitatory synapses are formed on the heads of dendritic spines and
5 have asymmetric structures. Synapses contain at least two types of junctional structures:
6 synaptic junctions (SJs) and puncta adherentia junctions (PAJs) [1]. PAJs resemble
7 adherens junctions (AJs) of epithelial cells in their molecular architecture and are
8 regarded as mechanical adhesion sites between axons and their target dendrites, while SJs
9 function as sites of neurotransmission. SJs are associated both with synaptic vesicles
10 docked at the presynaptic active zones where Ca2+ channels are localized, and with
11 postsynaptic densities (PSDs) where specific neurotransmitter receptors are concentrated.
12 PAJs, in contrast, contain symmetrical paramembranous dense materials and are not
13 associated with synaptic vesicles. Both SJs and PAJs are highly developed as separate
14 clusters consisting of distinctive macromolecular complexes in mossy fiber-CA3
15 pyramidal cell synapses in the stratum lucidum of the hippocampus. The synapses in this
16 area undergo activity-dependent remodeling and reorganization, which is implicated in
17 the cellular basis of learning and memory [2]. However, the molecular mechanisms
18 underlying activity-dependent remodeling and reorganization are poorly understood.
19 Moreover, it is not fully understood how contacts between axons and dendrites are
20 initiated, or how presynaptic and postsynaptic components are recruited to the contact
21 sites to establish synapses.
22 Many cell-cell adhesion molecules (CAMs) are localized at synapses and are
23 implicated in synaptogenesis [3,4]. Among them, N-cadherin was shown to be localized
24 at the presynaptic and postsynaptic sides of PAJs, but not at SJs, in an adult cerebellar
25 nucleus [5]. Moreover, neuroligin 1 and neuroligin 2 were shown to be localized at the
26 PSDs of excitatory synapses in the neocortex and GABAA-receptor-containing inhibitory
3 1 postsynapses in the hippocampus, respectively [6-8]. Neurexins were shown to be
2 concentrated in presynaptic terminals in the pons and hippocampus [9]. Furthermore,
3 nectins were found to be co-localized with N-cadherin at PAJs, but not at SJs, at mossy
4 fiber-CA3 pyramidal cell synapses in the hippocampus [10]. Nectins comprise a family of
5 four members (nectin-1 nectin-2, nectin-3 and nectin-4) [11,12]. All members have three
6 immunoglobulin-like loops in their extracellular regions flanked by a single
7 transmembrane region. The cytoplasmic tail of nectins binds the filamentous actin (F-
8 actin)-binding protein, afadin. Although N-cadherin and afadin are symmetrically
9 localized at the presynaptic and postsynaptic sides of PAJs at mossy fiber-CA3 pyramidal
10 cell synapses, nectin-1 and nectin-3 are asymmetrically localized at the presynaptic and
11 postsynaptic sides of PAJs, respectively [10].
12 Afadin is homologous to a human AF-6 gene product [13,14]. AF-6 was originally
13 identified as the fusion partner of the ALL-1 gene involved in acute myeloid leukemia
14 with chromosome translocation [15]. In mice, rats and humans, afadin is encoded by an
15 Mllt4 gene that produces several translational products, presumably by alternative
16 splicing. The largest afadin protein, which is called l-afadin, binds F-actin through its F-
17 actin-binding domain in the C-terminus, but other short variants lack this domain. In the
18 brain, l-afadin and s-afadin, the latter of which is one of the short variants, are mainly
19 expressed [13]. Here, l-afadin is referred to simply as afadin. Afadin binds many proteins
20 through multiple domains, including two Ras-associated domains, a forkhead-associated
21 domain, a dilute domain, a PDZ domain, three proline-rich domains, and an F-actin-
22 binding domain from the N-terminus to the C-terminus. The afadin-binding proteins thus
23 far identified include α-catenin, p120ctn, ponsin, ADIP, LMO7, PLEKHA7, ZO-1, Rap1,
24 Rit, Rin, Eph receptors, neurexins, Jagged-1, JAM, SPA-1, Bcr, c-Src, LMO2, profilin,
25 and nArgBp2 [14,16].
26 We and another group generated the afadin straight knockout mouse lines [17,18].
4 1 Because systemic ablation of afadin caused early embryonic lethality, an afadin-floxed
2 mouse line was generated in which camk2a-Cre conditional ablation was utilized to
3 inactivate the afadin gene in the hippocampus after postnatal day 9 (P9) [19]. In the
4 mutant mice, the active zone protein, bassoon, and the postsynaptic density protein, PSD-
5 95, accumulate at mossy fiber-CA3 pyramidal cell synapses, but perforated PSDs tend to
6 be more frequently observed than in control mice. Because perforated PSDs are observed
7 in synapses that undergo remodeling [2], these results suggest that afadin is likely to
8 regulate the remodeling of synapses. However, the role of afadin in synaptogenesis
9 remains to be determined.
10 In the present study, we analyzed the role of afadin in PAJ formation and
11 presynaptic differentiation using a nestin-Cre mouse line, in which the nestin promoter
12 starts to operate around E10.5 [20]. These experiments revealed that afadin regulates PAJ
13 formation and presynaptic differentiation at mossy fiber-CA3 pyramidal cell synapses in
14 the stratum lucidum of the hippocampus.
15
5 1 Materials and Methods
2
3 Mice
4 The afadin-floxed mice [19] and nestin-Cre mice [21] were described previously. The
5 heterozygous mice carrying the afadin conditional allele are referred to as afadin+/f.
6 Genotyping was performed with a REDExtract-N-Amp Tissue PCR kit (Sigma). The
7 mutant and control samples were prepared from the same litter. The morning after coitus
8 and the day of birth were defined as E0.5 and P0, respectively. All animal experiments
9 were performed in strict accordance with the guidelines of the institution and approved by
10 the administrative panel on laboratory animal care of Kobe University. The protocol was
11 approved by the Committee on the Ethics of Animal Experiments of Kobe University
12 Graduate School of Medicine (Permit Number: P130205). All efforts were made to
13 minimize suffering.
14
15 Western blotting
16 Mouse forebrains were dissected, placed in tubes, frozen in liquid nitrogen, and stored at
17 −80°C until use. Tissues were homogenized with a Teflon-glass homogenizer in 20 mM
18 Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 1 mM
19 phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 1.5 µg/ml aprotinin. Then, 150
20 mM NaCl and 10% (wt/vol) glycerol were added to the homogenates. The homogenates
21 were centrifuged at 800 × g at 4°C for 10 min and the supernatants were collected.
22 Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad).
23 Protein lysates (20 µg each) were separated by SDS-PAGE, transferred to PVDF
24 membranes, and incubated with antibodies (Abs). Immunodetection was performed with
25 Immobilon Western (Millipore) and a LAS-4000 luminescent image analyzer (Fujifilm).
26
6 1 Immunohistochemistry and immunocytochemistry
2 For immunohistochemistry, deeply-anesthetized mice were perfused with an ice-cold
3 fixative composed of 2% paraformaldehyde, 4% sucrose, 1 mM sodium pyruvate, Hanks’
4 balanced salt solution (HBSS) containing 1 mM CaCl2 and 1 mM MgCl2 (Life
5 Technologies), 3 units/ml heparin sodium, and 10 mM HEPES (pH 7.3). The brains were
6 dissected and incubated in the same fixative at 4°C for 4 h, and then they were
7 dehydrated overnight in 30% sucrose, 1 mM sodium pyruvate, HBSS containing 1 mM
8 CaCl2 and 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The brains were placed in OCT
9 compound (Tissue Tek) and frozen on dry ice. Sections of 14-µm thickness were
10 mounted on glass slides and incubated at 62°C for 20 min in HistoVT One antigen
11 retrieval solution (Nacalai Tesque), and then blocked at room temperature for 20 min in
12 100 mM phosphate buffer (PB) (pH 7.4) containing 10% goat serum, 1% bovine serum
13 albumin, and 0.25% Triton X-100. The specimens were incubated at 4°C for 48 h with
14 primary Abs in CanGetSignal immunoreaction enhancer solution B (Toyobo). After
15 washing for 10 min three times in 100 mM PB containing 0.05% saponin, the samples
16 were incubated at 4°C for 24 h with secondary Abs and 1 µg/ml DAPI (Nacalai Tesque)
17 in the immunoreaction enhancer solution. After washing three times for 10 min in 100
18 mM PB containing 0.05% saponin, the samples were mounted in FluorSave reagent
19 (Merck) and observed with an LSM510 META confocal laser scanning microscope (Carl
20 Zeiss). For immunostaining of cultured hippocampal neurons, cells were fixed with the
21 above-mentioned fixative without heparin sodium at 37°C for 15 min. The fixed cells 22 were permeabilized at room temperature for 5 min with 0.25% Triton-X and 0.005%
23 Tween-20 in Tris-buffered saline (TBS) containing 1 mM CaCl2, and then blocked with
24 10% goat serum in TBS containing 0.005% Tween-20 and 1 mM CaCl2 at 37°C for 20 25 min. Then, the cells were incubated with primary Abs in the solution used for blocking at
26 4°C overnight. After washing 3 times for 5 min in 0.005% Tween-20 in TBS containing 1
7 1 mM CaCl2 at room temperature, the cells were incubated with Alexa Fluor-conjugated
2 secondary Abs (Life Technologies) at room temperature for 45 min. Maximum intensity
3 projection images were created from around 10 confocal images collected at a 0.4-µm
4 step along the z-axis with an LSM700 or LSM510 confocal laser scanning microscope
5 (Carl Zeiss) under exactly the same conditions for both control and afadin-deficient
6 neurons. The immunofluorescence signals for nectin-1, nectin-3, N-cadherin, β-catenin,
7 VGLUT1 and bassoon in cultured neurons were measured in synaptotagmin I –positive
8 punctae that located between 5 µm and 45 µm away from cell bodies along dendrites for
9 each genotype (20 punctae per neuron, totally 100 punctae for each genotype) and
10 subjected to statistical analysis.
11
12 Abs
13 Rabbit anti-l-afadin and rabbit anti-l/s-afadin were prepared as described [13]. The Abs
14 listed below were purchased from commercial sources: rat anti-nectin-1, clone 48-12
15 (MBL); rat anti-nectin-3, clone 103-A1 (MBL); mouse anti-N-cadherin, clone 32 (BD
16 Biosciences); rabbit anti-N-cadherin (Takara); rabbit anti-β-catenin (Sigma); rabbit anti-
17 synapsin I (Millipore); guinea pig anti-VGLUT1 (Millipore); mouse anti-bassoon (Enzo
18 Life Sciences); mouse anti-PSD-95, clone 7E3-1B8 (Enzo Life Sciences) and clone
19 K28/43 (NeuroMab); mouse anti-actin (clone C4) (Millipore); and chicken anti-MAP2
20 (Abcam). Alexa Fluor-conjugated secondary Abs (Life Technologies) were used for
21 immunohistochemistry and immunocytochemistry.
22
23 Cell culture
24 Hippocampal neuron cultures were prepared from E18.5 embryos, which were generated
25 by the breeding of afadinf/f and afadin+/f;nestin-Cre mice. To identify the mutants and
26 littermate controls, the embryos were set aside on ice in 1 mM sodium pyruvate, HBSS
8 1 without CaCl2 and MgCl2 (Life Technologies), and 10 mM HEPES (pH 7.3) for
2 approximately 3 h while genotyping. Dissociated hippocampal neurons were prepared as
3 described [22] with slight modifications. In brief, hippocampal neurons dissociated with
4 trypsin were plated at a density of 5–7 × 103 cells/cm2 on poly-L-lysine-coated coverslips
5 in Neurobasal medium (Life Technologies) containing B27 supplement (Life
6 Technologies) and GlutaMAX (Life Technologies), and cultured in a 5% CO2 incubator.
7 Neurons for electrophysiology experiments were initially cultured in MEM (Life
8 Technologies) containing 10% fetal bovine serum for 18 h. To label recycling synaptic
9 vesicles, cultured neurons were incubated with the rabbit polyclonal synaptotagmin I
10 luminal domain Ab (Synaptic Systems, #105 102, 1:50) in culture medium at 37°C for 30
11 min in 5% CO2. To quantify the immunofluorescence signal for synaptotagmin I uptake,
12 the intensities of sixty punctae along dendrites for each genotype were measured and
13 subjected to statistical analysis.
14
15 Electrophysiology
16 The hippocampal cultures at 14–21 days in vitro (DIV) were transferred to a recording
17 chamber and superfused with an external solution (pH 7.4) containing: 148.8 mM Na+,
+ − 2+ 2+ − 18 2.7 mM K , 149.2 mM Cl , 2.8 mM Ca , 2.0 mM Mg , 11.6 mM HCO3 , 0.4 mM
− 19 H2PO4 , 5.6 mM D-glucose, 0.01 mM gabazine, and 10 mg/l phenol red. The cultures
20 were kept at room temperature. Recordings were obtained from the cells held at –70 mV
21 with patch pipettes (2-5 MΩ) using an EPC 10 amplifier (HEKA Elektronik). Pipettes
22 were filled with a solution (pH 7.2) containing 135 mM K-gluconate, 5 mM KCl, 10 mM
23 HEPES, 1 mM EGTA, 2 mM Mg-ATP, 5 mM creatine phosphate, 0.4 mM GTP, and
24 0.07 mM CaCl2. The actual membrane potentials were corrected for the liquid junction
25 potential. Miniature excitatory postsynaptic currents (mEPSCs) were recorded in the
26 presence of tetrodotoxin (1 µM) added to the above-mentioned external solution.
9 1 mEPSCs were analyzed off-line using Synaptosoft mini analysis software. For recording
2 from synaptically-coupled pairs of neurons, presynaptic action potentials were evoked by
3 injecting depolarizing current (1 ms, 1.5 to 2 nA) at 0.1 Hz. Series resistance (typically
4 between 5 and 15 MΩ) was regularly monitored and cells were excluded if a change of
5 more than 20% occurred. Gabazine, ATP, CrP, EGTA and GTP were purchased from
6 Sigma/Fluka. For data acquisition, signals were filtered at 10 kHz and digitally recorded
7 using PATCHMASTER software (HEKA Elektronik).
8
9 Statistical analysis
10 Statistical analysis of the difference between mean values was performed with Student’s
11 two-tailed t test. The criterion for statistical significance was set at P < 0.05. All values
12 are reported as the mean ± s.e.m. 13
10 1 Results 2
3 Conditional ablation of afadin in the brain
4 In the present study, we used afadinf/f;nestin-Cre mice and their afadin+/f littermates as
5 controls. We first confirmed that afadin is indeed ablated in the afadin conditional
6 knockout (cKO) brain. Western blotting of P14 brain extracts demonstrated that both the
7 l-afadin and s-afadin proteins were almost lost by the conditional ablation, but the levels
8 of other synaptic components, including nectin-1, nectin-3, N-cadherin, β-catenin, PSD-
9 95, synapsin I, VGLUT1 and bassoon, were largely unaltered (Fig. 1A). These results
10 indicate that afadin expression was specifically ablated in the afadin cKO brain.
11
12 Decreased immunofluorescence signals for the PAJ CAMs at mossy fiber-CA3
13 pyramidal cell synapses in the afadin cKO brain
14 We next examined the role of afadin in the accumulation of nectin-1, nectin-3 and N-
15 cadherin at mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the
16 hippocampus. First, we confirmed by immunofluorescence microscopy that afadin
17 expression was ablated in this region in the afadin cKO brain (Fig. 1B). The
18 immunofluorescence signal for afadin was observed as dots that aligned along the
19 dendrites of the pyramidal cells at the control CA3 stratum lucidum, as described [10]. In
20 contrast, the signal for afadin was not detected in the same region of the afadin cKO brain.
21 These results indicate that the afadin expression was ablated at mossy fiber-CA3
22 pyramidal cell synapses in the afadin cKO brain.
23 We next examined the signals for nectin-1, nectin-3 and N-cadherin (Fig. 2),
24 which were observed as dots in the CA3 stratum lucidum of the control hippocampus as
25 previously described [10,23]. The distributions of these proteins were similar to that of
26 afadin. However, the signals for nectin-1, nectin-3 and N-cadherin disappeared almost
11 1 completely in the CA3 stratum lucidum of the afadin-deficient hippocampus. These
2 results indicate that afadin is required for the accumulation of the immunofluorescence
3 signals for the CAMs at mossy fiber-CA3 pyramidal cell synapses.
4
5 Decreased immunofluorescence signals for the PAJ CAMs at the synapses in
6 cultured afadin-deficient hippocampal neurons
7 To confirm the role of afadin in the accumulation of the immunofluorescence signals for
8 nectin-1, nectin-3 and N-cadherin at the synapses in vitro, hippocampal neuron cultures
9 were prepared from the afadinf/f;nestin-Cre embryos and control afadin+/f embryos. We
10 first confirmed that afadin was ablated in the hippocampal neurons from the
11 afadinf/f;nestin-Cre embryos. The immunofluorescence signal for afadin was observed as
12 dots along dendrites in the control neurons at 14 DIV (Fig. 3A). However, in the afadin-
13 deficient neurons, the signal for afadin was not observed. These results indicate that
14 afadin expression was indeed ablated in the cultured hippocampal neurons from the
15 afadinf/f;nestin-Cre embryos.
16 We then examined the effects of afadin ablation on the accumulation of nectin-1,
17 nectin-3, N-cadherin and β-catenin in the cultured hippocampal neurons. In the control
18 neurons, the signals for nectin-1, nectin-3, N-cadherin and β-catenin were observed as
19 dots along the dendrites at 14 DIV (Fig. 3B-F). However, in the afadin-deficient neurons,
20 the signals for nectin-1, nectin-3, N-cadherin and β-catenin decreased significantly. These
21 results indicate that afadin is required for the accumulation of the immunofluorescence
22 signals for the CAMs and β-catenin at the synapses in cultured hippocampal neurons.
23
24 Decreased immunofluorescence signals for the presynaptic components at mossy
25 fiber-CA3 pyramidal cell synapses of the afadin cKO brain
26 We next examined the role of afadin in the accumulation of the immunofluorescence
12 1 signals for the presynaptic components, VGLUT1 and bassoon, at mossy fiber-CA3
2 pyramidal cell synapses. VGLUT1 is a component of synaptic vesicles [24] and bassoon
3 is a component of the active zone [25]. The immunofluorescence signals for VGLUT1
4 and bassoon were observed as dots in the CA3 stratum lucidum of the control
5 hippocampus (Fig. 4). However, the signals for these proteins were markedly decreased,
6 but not abolished, in this region of the afadin-deficient hippocampus. These results
7 indicate that afadin is required for the accumulation of the immunofluorescence signals
8 for the presynaptic components at a considerable population of mossy fiber-CA3
9 pyramidal cell synapses.
10
11 Decreased immunofluorescence signals for the presynaptic components at synapses
12 in cultured afadin-deficient hippocampal neurons
13 Next, we confirmed the role of afadin in the accumulation of the immunofluorescence
14 signals for the presynaptic components at synapses in the cultured hippocampal neurons.
15 The immunofluorescence signals for VGLUT1 and bassoon were observed as dots along
16 the dendrites in control neurons at 14 DIV (Fig. 5). However, in the afadin-deficient
17 neurons, the signals for these proteins were decreased significantly but not abolished.
18 These results indicate that afadin is required for the accumulation of the
19 immunofluorescence signals for the presynaptic components at synapses in a
20 considerable population of cultured hippocampal neurons.
21
22 Decrease in functional synapses in cultured afadin-deficient hippocampal neurons
23 Thus far, we have shown that the immunofluorescence signals for the PAJ CAMs mostly
24 disappeared in mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the
25 the afadin cKO hippocampus and cultured afadin-deficient hippocampal neurons, while
26 the signals for the presynaptic markers decreased less extensively. From these results, we
13 1 could not distinguish between two possible mechanisms: 1) afadin is required for PAJ
2 formation and presynaptic differentiation; 2) afadin is required for the accumulation of
3 the presynaptic marker proteins at developing synapses. To address these issues, we
4 recorded mEPSCs in cultured hippocampal neurons. We detected mEPSCs in both the
5 control and afadin-deficient neurons, but the interevent interval was prolonged in the
6 afadin-deficient neurons compared with control neurons (Fig. 6). These results indicate
7 that presynaptic functions were suppressed or a number of synapse was reduced in the
8 afadin-deficient neurons. Therefore, it is likely that afadin is at least required for PAJ
9 formation and presynaptic differentiation in most mossy fiber-CA3 pyramidal cell
10 synapses.
11
12 Impaired presynaptic functions in cultured afadin-deficient hippocampal neurons
13 In the last set of experiments, we measured vesicle recycling and performed paired
14 recordings to ask whether presynaptic functions were impaired in cultured afadin-
15 deficient hippocampal neurons. Vesicle recycling was assessed by measuring
16 synaptotagmin I uptake in the cultured hippocampal neurons. The vesicle-recycling rate
17 per synapse was markedly decreased in the synapses of the afadin-deficient hippocampal
18 neurons compared with control neurons, indicating that presynaptic functions were
19 impaired in the mutant neurons (Fig. 7A, B). Analysis of paired recordings demonstrated
20 that the amplitude of the unitary EPSC was smaller in the cultured afadin-deficient
21 neurons than in the control neurons (Fig. 7C, D). Moreover, an increased paired-pulse
22 ratio was observed in the cultured afadin-deficient neurons, suggesting a decreased
23 release probability in these neurons (Fig. 7C, E). These results indicate that afadin is also
24 required for presynaptic functions in cultured hippocampal neurons.
25
14 1 Discussion
2 3 Here, we showed that the genetic ablation of afadin using nestin-Cre mice severely
4 decreased the accumulation of the immunofluorescence signals for PAJ components at
5 mossy fiber-CA3 pyramidal cell synapses in the stratum lucidum of the hippocampus. In
6 addition, we confirmed the similar effects of afadin in cultured hippocampal neurons and
7 further showed that the ablation of afadin impaired presynaptic functions. These results
8 indicate that afadin is required for PAJ formation in almost all mossy fiber-CA3
9 pyramidal cell synapses.
10 Afadin is ubiquitously expressed and consists of multiple domains, and no
11 structurally-related homologous proteins have been identified, suggesting that it plays
12 pivotal roles in multiple biological processes [13]. In fact, afadin deficiency leads to the
13 disruption of cell-cell junctions and cell polarity in neuroepithelial cells and causes early
14 embryonic lethality [17,18]. We previously showed that nectins first form cell–cell
15 adhesions and then recruit cadherins to the nectin-based cell–cell adhesion sites through
16 afadin, resulting in the formation of AJs, in both fibroblasts and epithelial cells [12].
17 Nectins bind afadin, which in turn binds α-catenin associated with cadherins through β-
18 catenin. These molecular linkages are required for the association of cadherins with
19 nectins. Therefore, it is likely that afadin cooperatively functions with nectins and
20 cadherins to form PAJs in mossy fiber-CA3 pyramidal cell synapses.
21 The major features of presynaptic differentiation include the formation of active
22 zones and the accumulation of synaptic vesicles in the presynaptic side of synapses. We
23 showed here that the genetic ablation of afadin considerably decreased the accumulation
24 of the immunofluorescence signals for the active zone component, bassoon, and the
25 synaptic vesicle component, VGLUT1, at mossy fiber-CA3 pyramidal cell synapses and
26 synapses in cultured hippocampal neurons. These results indicate that the active zones are
15 1 formed in an afadin-dependent manner in most mossy fiber-CA3 pyramidal cell synapses
2 in which PAJs are formed, while they are also formed in an afadin-independent manner in
3 a considerable population of these synapses in which PAJs might not be formed. Because
4 the residual synapses that were formed in afadin-deficient neurons showed impaired
5 recycling of synaptic vesicles and abnormal paired recordings, the active zones formed in
6 an afadin-independent manner may not be fully differentiated in their structures and
7 functions. Other CAMs such as neuroligins may be involved in the afadin-independent
8 formation of the active zones at mossy fiber-CA3 pyramidal cell synapses, but it is
9 currently unknown whether the active zones are formed in afadin-dependent and -
10 independent manners in the same or different synapses, how the active zones are formed
11 in afadin-dependent and -independent manners, or whether PAJs are formed in advance
12 and then the active zones are formed or vice versa.
13 We previously showed that the immunofluorescence signals for nectin-1 and
14 nectin-3 were almost completely lost in mossy fiber-CA3 pyramidal cell synapses in the
15 afadinf/f;camk2a-Cre hippocampus in which the afadin gene was inactivated in excitatory
16 neurons after synaptogenesis, and that the signals for N-cadherin and β-catenin were also
17 almost completely lost [19]. However, the immunofluorescence signal for bassoon and
18 PSD-95 were not altered there in the afadinf/f;camk2a-Cre hippocampus. These earlier
19 observations together with the present results indicate that afadin is required for PAJ
20 formation and presynaptic differentiation in mossy fiber-CA3 pyramidal cell synapses,
21 but is not required to maintain the accumulation of these components after the synapses
22 are formed. Interestingly, MLLT4/AFADIN expression was reduced in the postmortem
23 schizophrenic brain [26]. In addition, genetic ablation of nectin-1 or nectin-3 decreased
24 PAJ numbers in mossy fiber-CA3 pyramidal cell synapses [23]. Furthermore, mutations
25 in NECTIN-1 were shown to be a cause of mental retardation in humans [27]. Taken
26 together, these results indicate the importance of afadin and its binding proteins, nectin-1
16 1 and nectin-3, in both physiology and pathology.
2 It was shown that AF-6/afadin regulates the morphology of dendritic spines in
3 cultured hippocampal neurons [28]. Additionally, it was shown that afadin is required for
4 the maintenance of dendritic arborization and synapse number in cultured hippocampal
5 neurons [29]. These two studies employed afadin knockdown or overexpression of
6 mutant forms of afadin. These earlier observations indicate that afadin regulates
7 synaptogenesis through postsynaptic differentiation. Taken together with the present
8 results, it is likely that afadin is involved in synaptogenesis through both presynaptic and
9 postsynaptic differentiation. In contrast, it was recently shown by use of conditional
10 afadin-deficient mice with a Nex-Cre mouse line that the genetic ablation of afadin
11 neither changed the localizations of nectin-1 and nectin-3 in the CA1 region nor
12 perturbed the presynaptic functions in the CA3 region, although it was shown that
13 synapse number was decreased in the CA1 region of the afadin-deficient hippocampus
14 [30]. Some of these results are apparently inconsistent with our present results. The exact
15 reason for this inconsistency is not known, but it may be due to the different regions
16 analyzed in the hippocampus, the different promoters used for the Cre mice, the different
17 nectin Abs used, or the different samples used for electrophysiology experiments.
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21 1 Figure legends 2
3 Figure 1 Conditional ablation of afadin in the brain.
4 (A) Expression levels of various synaptic components in P14 forebrains. The indicated
5 synaptic proteins were analyzed by Western blotting. Twenty µg of protein lysates were
6 loaded in each lane. (B) Expression pattern of afadin in the CA3 stratum lucidum at P14.
7 Hippocampal sections were stained with the l-afadin Ab (red) and 4’,6-diamidino-2-
8 phenylindole dihydrochloride (DAPI) (blue). The signal for afadin in nuclei was likely
9 non-specific because it was not abolished by genetic ablation of afadin. The results
10 shown are representative of three independent experiments. SR, stratum radiatum; SL,
11 stratum lucidum; SP, stratum pyramidale. Control, afadin+/f; cKO, afadinf/f;nestin-Cre.
12 Scale bars, 25 µm.
13
14 Figure 2 Decreased immunofluorescence signals for the CAMs at the mossy fiber-
15 CA3 pyramidal cell synapses in the afadin cKO brain.
16 Coronal hippocampal sections at P14 were stained with the indicated Ab against nectin-1,
17 nectin-3 or N-cadherin (red) and DAPI (blue). The results shown are representative of
18 three independent experiments. SR, stratum radiatum; SL, stratum lucidum; SP, stratum
19 pyramidale. Control, afadin+/f; cKO, afadinf/f;nestin-Cre. Scale bars, 25 µm.
20
21 Figure 3 Decreased immunofluorescence signals for the CAMs at the synapses in
22 cultured afadin-deficient hippocampal neurons.
23 (A-E) Expression patterns of afadin, nectin-1, nectin-3, N-cadherin and β-catenin in the
24 cultured hippocampal neurons. The cultured neurons at 14 DIV were double-stained with
25 the MAP2 Ab and the indicated Ab against afadin, nectin-1, nectin-3, N-cadherin or β-
26 catenin. The results shown are representative of three independent experiments. Scale
22 1 bars, 5 µm. (F) Ratio of the intensity of punctae positive for nectin-1, nectin-3, N-
2 cadherin or β-catenin in the afadin-deficient neurons relative to control neurons. Punctae
3 that located between 5 µm and 45 µm away from cell bodies along dendrites were
4 analyzed (100 punctae from five neurons were analyzed for each genotype). Error bar,
5 s.e.m. Control, afadin+/f; cKO, afadinf/f;nestin-Cre.
6
7 Figure 4 Decreased immunofluorescence signals for the presynaptic components at
8 the mossy fiber-CA3 pyramidal cell synapses of the afadin cKO brain.
9 Coronal hippocampal sections at P14 were stained with the indicated Ab against
10 VGLUT1 or bassoon (red) and DAPI (blue). The results shown are representative of three
11 independent experiments. SR, stratum radiatum; SL, stratum lucidum; SP, stratum
12 pyramidale. Control, afadin+/f; cKO, afadinf/f;nestin-Cre. Scale bars, 25 µm.
13
14 Figure 5 Decreased immunofluorescence signals for the presynaptic components at
15 the synapses in cultured afadin-deficient hippocampal neurons.
16 (A, B) Expression patterns of VGLUT1 and bassoon in the cultured hippocampal neurons.
17 The cultured hippocampal neurons at 14 DIV were double-stained with the MAP2 Ab
18 and the indicated Ab against VGLUT1 or bassoon. The results shown are representative
19 of three independent experiments. Scale bars, 5 µm. (C) Ratio of the intensity of punctae
20 positive for VGLUT1 or bassoon in the afadin-deficient neurons relative to control
21 neurons. Punctae that located between 5 µm and 45 µm away from cell bodies along
22 dendrites were analyzed (100 punctae from five neurons were analyzed for each
23 genotype). Error bar, s.e.m. Control, afadin+/f; cKO, afadinf/f;nestin-Cre.
24
25 Figure 6 Decreased functional synapses in cultured afadin-deficient hippocampal
26 neurons.
23 1 (A) Example mEPSC traces. The vertical and horizontal bars denote 5 pA and 1 s,
2 respectively. (B) The average interevent interval of mEPSCs (n = 12 in both the control
3 and afadin cKO neurons). (C) Appearance of the cultured neurons used for analysis of
4 mEPSCs. The neuron density is similar between control and afadin cKO neurons. Scale
5 bar, 200 µm. Control, afadin+/f; cKO, afadinf/f;nestin-Cre.
6
7 Figure 7 Impaired presynaptic functions in cultured afadin-deficient hippocampal
8 neurons.
9 (A, B) Labeling of functional synapses in cultured live hippocampal neurons. (A)
10 Recycling synaptic vesicles labeled by incubation of cultured live hippocampal neurons
11 at 14 DIV with the synaptotagmin I luminal domain Ab. Scale bars, 1 µm. (B)
12 Quantification of the total integrated intensity of the internalized synaptotagmin I Ab.
13 Sixty punctae for each genotype were analyzed. (C-E) Modulation of presynaptic
14 vesicular release by afadin in cultured hippocampal neurons. (C) Examples of
15 postsynaptic responses evoked by paired action potentials in a presynaptic cell with a 50-
16 ms interval in control and afadin cKO neurons. Superimposed images for 15 recordings
17 of unitary EPSC (top), average unitary EPSC (middle), and action potentials (bottom). A
18 vertical scale bar denotes 20 pA for the top and middle rows and 5 mV for the bottom
19 row; a horizontal scale bar denotes 20 ms. (D) The average unitary amplitudes (n = 10
20 and 12 in control and afadin cKO neurons, respectively). (E) paired-pulse ratios (n = 8
21 and 11 in control and afadin cKO neurons, respectively). Error bars, s.e.m. Control,
22 afadin+/f; cKO, afadinf/f;nestin-Cre.
24 Figure 1 Click here to download high resolution image Figure 2 Click here to download high resolution image Figure 3 Click here to download high resolution image Figure 4 Click here to download high resolution image Figure 5 Click here to download high resolution image Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image