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The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation Chikako Hayashi a, b, 1, Nobuharu Suzuki a, b, *, 1, Yo Mabuchi b, c, Naomi Kikura b, Yukina Hosoda a, Susana de Vega d, Chihiro Akazawa b, c, ** a
Department of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan Department of Biochemistry and Biophysics, Graduate School of Health Care Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan c Department of Biochemistry and Biophysics, Graduate School of Medical and Dental Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan d Department of Pathophysiology for Locomotive and Neoplastic Diseases and Department of Pathology and Oncology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2019 Accepted 1 December 2019 Available online xxx
Cell adhesion between oligodendrocytes and neuronal axons is a critical step for myelination that enables the rapid propagation of action potential in the central nervous system. Here, we show that the transmembrane protein teneurin-4 plays a role in the cell adhesion required for the differentiation of oligodendrocytes. We found that teneurin-4 formed molecular complexes with all of the four teneurin family members and promoted cell-cell adhesion. Oligodendrocyte lineage cells attached to the recombinant extracellular domain of all the teneurins and formed well-branched cell processes. In an axon-mimicking nanofibers assay, nanofibers coated with the recombinant teneurin-4 extracellular domain increased the differentiation of oligodendrocytes. Our results show that teneurin-4 binds to all teneurins through their extracellular domain, which facilitates the oligodendrocyte-axon adhesion, and promotes oligodendrocyte differentiation via its homophilic interaction. © 2019 Elsevier Inc. All rights reserved.
Keywords: Myelin Oligodendrocyte Cell adhesion Teneurin-4 Extracellular domain
1. Introduction In the central nervous system (CNS), oligodendrocytes (OLs) form myelin sheaths, composed of multi-lamellar sheets of their plasma membrane around neuronal axons that are required for the rapid propagation of action potential. During the postnatal
Abbreviations: CNS, central nervous system; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; MBP, myelin basic protein; ECD, extracellular domain; Ten-1, teneurin-1; Ten-2, teneurin-2; Ten-3, teneurin-3; Ten-4, teneurin-4; WT, wild-type; P, postnatal day; pTen, expression plasmid of the full-length teneurin; pTenECD, expression plasmid of the extracellular domain of teneurin; CHO, Chinese hamster ovary; LM, laminin. * Corresponding author. Department of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. ** Corresponding author. Department of Biochemistry and Biophysics, Graduate School of Medical and Dental Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. E-mail addresses: [email protected] (N. Suzuki), [email protected] (C. Akazawa). 1 These authors equally contributed to this work.
development, OL precursor cells (OPCs) contact and adhere to the targeted axons and subsequently differentiate into OLs that express myelin proteins, such as myelin basic protein (MBP) [1]. The OPC/OL interaction with axons promotes their survival as well [2]. It is known that the adhesion of OPCs/OLs to axons is mediated by the molecular complex of integrin a6b1-laminin-211 and F3/contactinL1 [3]. However, the significance of this molecular complex in vivo is still a matter of debate since the deficiency of these proteins does not, or does only temporally, exhibit effects on either generation or maintenance of OLs [4-7], indicating that there must be a compensatory mechanism by other critical protein(s) for the cell adhesion. Teneurins are type II transmembrane glycoproteins that consist of an N-terminal intracellular domain, a single-pass transmembrane domain, and a large extracellular domain (ECD) (~250 kDa) following an assembly of EGF-like repeats, Ig-like domain, NHL repeats/b-propeller domain, YD repeats/b-barrel domain, and toxin-like region [8]. In vertebrates, there are 4 teneurin family members (Ten-1 to -4). Teneurins are expressed in various tissues, including a high expression in the CNS. Recently,
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Please cite this article as: C. Hayashi et al., The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.002
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molecular functional studies of Ten-1, Ten-2, and Ten-3 have revealed that they homophilically or heterophilically bind between them or with latrophilins and promote neuronal cell adhesion for the target recognition and the formation of synapses between neural cells [9e13]. Ten-1, Ten-2, and Ten-3, however, are not expressed in glia cells [14]. Only Ten-4 is expressed in glia cells, among all the teneurins, including in OLs, and it acts as a regulator of CNS myelination [14,15]. In Ten-4 deficient (Ten-4 -/-) mice, a significant reduction in the number of OLs is observed due to inhibition of OL differentiation and survival, and myelination is substantially decreased in the CNS, especially in the spinal cord [15]. Ten-4 is expressed not only in OLs but also in neuronal cells and promotes cell processes formation through the activation of intracellular signaling molecules, such as focal adhesion kinase [15,16]. However, the role of the Ten-4 ECD, particularly in cell adhesion, has not been investigated. In this study, we attempted to elucidate the molecular function of Ten-4 ECD and found that Ten-4 ECD possessed cell adhesion activity by interacting with all teneurin ECDs. Further, the homophilic interaction of Ten-4 through its ECD promoted OL differentiation on nanofibers. Our results reveal that Ten-4 is a critical adhesion protein between OPCs/OLs and axons promoting OL differentiation.
transfected with pTenECDs was used, following the manufacturer’s instruction. Detailed methods are described in Supplementary Information. 2.6. Cell attachment assay The attachment activity of OL lineage cells from Sox10-Venus mice [17] was evaluated on rTenECD-coated wells. Additional procedures are described in Supplementary Information. 2.7. Primary culture of rat and mouse OPCs OPCs were prepared from spinal cords of P2 rat or P3 mouse pups using the shaken-off method as previously described [19] with some modifications. Procedures in detail are described in Supplementary Information. 2.8. Culture assays using rTenECD-coated wells and nanofibers The culture experiments of OLs were performed using rTenECDcoated wells and polycaprolactone nanofibers as previously described [20] with some modifications. Detailed methods are described in Supplementary Information.
2. Materials and methods 2.9. Statistical analyses 2.1. Animals Wild-type (WT) animals of postnatal day (P) 7 C57BL/6 mice and P2 Wistar rats were used for experiments of the immunoprecipitation and the isolation of primary OPCs, respectively. P3 mouse pups from the Sox10-Venus mouse line [17] and the Ten-4 -/mouse line [15] were used for the cell attachment assay and the culture experiment using nanofibers, respectively. All procedures for experimental animals were approved by the Institutional Animal Care and Use Committees of Tokyo Medical and Dental University (No. A2019-307A). 2.2. Immunoprecipitation The immunoprecipitation experiment of Ten-4 was carried out using tissue lysate from WT mouse spinal cords at P7, when is the stage of OL differentiation in WT and the onset of the defect in OL differentiation and survival in Ten-4 -/- spinal cord [15]. Detailed methods are described in Supplementary Information. 2.3. Plasmids construction The expression plasmids of the full-length transmembrane proteins (pTen) and the extracellular domain (pTenECD) of teneurins were prepared as previously described [16]. Additional information is provided in Supplementary Information. 2.4. Cell aggregation assay Cell aggregation assay was carried out by the hanging-drop method [18] using each pTen-transfected Chinese hamster ovary (CHO) cells. Following procedures are described in Supplementary Information. 2.5. Preparation of recombinant proteins of teneurin ECDs (rTenECDs) For the preparation of rTenECDs, the expression system of 293FreeStyle cells (ThermoFisher Scientific, Waltham, MA)
All of the experiments were independently performed at least 3 times. The statistics of the experimental results were analyzed as follows. The two-tailed Student’s t-test was used for the analyses between two groups in the experiments. We analyzed multiple groups compared with a control using one-way ANOVA followed by Dunnett’s post-hoc. The statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. 3. Results 3.1. Molecular complex formation of Ten-4 with teneurin family members in the spinal cord To explore the molecular function of the Ten-4 ECD, we searched for binding proteins to this domain. Immunoprecipitation of Ten-4 from WT spinal cord lysate was carried out using anti-Ten-4 antibody. Co-immunoprecipitated proteins were identified by shotgun mass spectrometry and were compared with precipitated proteins using normal IgG as a control. Extracellular proteins, extracellular membrane-associated proteins, and transmembrane proteins, which were obtained only in the immunoprecipitation using antiTen-4 antibody, were considered as candidates. Totally, 6 candidates of Ten-4 ECD binding proteins were obtained (Table 1). Ten-4 was one of the candidates, since Ten-4 homophilic binding possibly occurred. All of the 6 proteins were transmembrane proteins and no extracellular proteins, including extracellular membraneassociated proteins, were obtained. Interestingly, 4 of the 6 transmembrane proteins were the teneurin family members (Ten-1 to -4) (Table 1). The other two were the 11- and 8-times transmembrane proteins of ion exchanger and transporter, respectively (Table 1). In this study, we focused on Ten-4 interaction between the teneurin members due to a putative role in the cell adhesion between OLs and axons. Since all of the 4 teneurins are expressed in neurons, but only Ten-4 is expressed in glia cells, including OLs [14,15], we hypothesized that Ten-4 on OL lineage cells bound to Ten-1, Ten-2, Ten-3, or Ten-4 on axons homophilically or heterophilically through their ECDs to promote cell adhesion between these cells.
Please cite this article as: C. Hayashi et al., The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.002
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Table 1 Candidate binding proteins to Ten-4 ECD. Among co-immunoprecipitated proteins using anti-Ten-4 antibody, extracellular proteins, extracellular membraneassociated proteins, and transmembrane proteins were considered as candidates. Proteins precipitated using normal IgG as a control were excluded from the list. Unused is ProtScore reflecting the amount of total, unique peptide evidence related to a given protein from the result of the mass spectrometry. More than 2.00 of unused score is enough trustable for detection. The accession numbers from the National Center for Biotechnology Information (NCBI) database are indicated. No
Unused
Protein name
Accession
1 2 3 4 5 6
150.99 43.23 22.50 9.70 3.44 2.31
Teneurin-4 Teneurin-1 Solute carrier family 4 (anion exchanger), member 2 Teneurin-3 Teneurin-2 ATPase, Naþ/Kþ transporting, alpha 3 polypeptide
GI: GI: GI: GI: GI: GI:
3.2. Homophilic and heterophilic cell-cell adhesion activity of Ten-4 with all teneurins To examine whether Ten-4 possesses cell-cell adhesion activity, we prepared CHO cells overexpressing each teneurin and performed cell aggregation assay. In the control with mock plasmidexpressing cells, the number of cells in a cell aggregate was not changed 2 h after the assay was started (Fig. 1A and B: Mock Mock). When combinations of Ten-4-overexpressing cells with each teneurin-overexpressing cells were tested, the cell number in a cell aggregate was increased (Fig. 1A and B: Ten-1 Ten-4, Ten-2 Ten-4, Ten-3 Ten-4, and Ten-4 Ten-4). Interestingly, the most
124248484 7657413 32450722 81869788 81869787 148692350
significant increase of cell number was in the combination of Ten-4 with Ten-4 (Fig. 1B). We confirmed heterophilic cell binding using two different fluorescent dyes. The cell-cell adhesion aggregation data revealed that Ten-4 promoted cell-cell adhesion homophilically and heterophilically with the other teneurins.
3.3. OPC/OL adhesion activity of the ECDs of teneurins As we hypothesized that teneurins on axonal membrane surface interacted with Ten-4 on OL lineage cells, we next analyzed whether the ECDs of teneurins promoted the attachment of OL lineage cells. We first prepared 4 different rTenECDs (rTen-1ECD,
Fig. 1. Cell-cell adhesion activity of Ten-4 with 4 teneurins. (A) Representative images of cell aggregations at 0 h and 2 h after incubation. Five different combinations were tested as indicated. Mock: mock plasmid-trasnfected CHO cells; Ten-1: Ten-1 expression plasmid-transfected CHO cells; Ten-2: Ten-2 expression plasmid-transfected CHO cells; Ten-3: Ten-3 expression plasmid-transfected CHO cells; Ten-4: Ten-4 expression plasmid-transfected CHO cells, Scale bar: 200 mm. (B) Quantitative analysis of cell aggregation assay. The number of cells in an aggregation was counted and categorized into 4 groups; single cell, 2-3, 4-6, and 7-9 cells in an aggregation. Triplicate experiments were independently performed (n ¼ 3). Error bars represent mean ± s.e.m. The two-tailed Student’s t-test was used for the statistical analysis in the experiments with two groups: *p < 0.05, **p < 0.01.
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rTen-2ECD, rTen-3ECD, and rTen-4ECD) using the 293 free-style expression system (Fig. 2A). For the attachment assay, a high purity of OL lineage cells was required because the attached cells were stained with crystal violet for quantification. Therefore, we utilized Sox10-Venus mice that express the fluorescence protein Venus driven by the Sox10 promoter, which is specifically expressed in OL lineage cells in the CNS [17], and purified OL lineage cells from P3 Sox10-Venus mouse spinal cords by flow cytometry. The collected OL lineage cells were seeded on rTenECD-coated wells and incubated for an hour. The results showed that all of rTenECDs had attachment activity of OL lineage cells (Fig. 2B and C). The attachment activity of rTen-1ECD, rTen-3ECD, and rTen-4ECD was higher than that of rTen-2ECD (Fig. 2B and C). To evaluate whether Ten-4 expressed in OL lineage cells affected the adhesion, soluble rTen4ECD was added to the assay. The cell attachment to all of rTenECDs was significantly inhibited by the addition of rTen-4ECD (Fig. 2B and C). These results indicated that all of the teneurins’ ECDs possessed adhesion activity of OL lineage cells and that the attachment to the ECDs was mediated, at least in part, by Ten-4 on the surface of OL lineage cells.
3.4. Promoted OL process formation and differentiation on the ECDs of teneurins To further test biological effects of rTenECDs on OL morphology and differentiation, we used rTenECD-coated wells and synthetic polycaprolactone nanofibers, mimicking axons. The rTenECD proteins were coated on flat bottom wells in a glass slide chamber and on the surface of the nanofibers. OPCs from rat spinal cords were plated and their differentiation was induced by changing the medium to the differentiation medium one day after plated. Rat OPCs/ OLs were used for these experiments because they differentiated
better and survived longer than murine OPCs/OLs in these assay conditions. Laminin (LM) was used as a control of process formation and differentiation [21]. On rTenECD-coated wells, OLs formed well-branched cellular processes with an intense expression signal of MBP, higher than on the LM-coated well (Fig. 3A). In the culture on rTenECD-coated nanofibers, MBP-positive OLs extended their branched processes along the nanofibers, covering larger areas than those on LM-nanofibers (Fig. 3B). Moreover, we found that rTen-1ECD and rTen-4ECD significantly increased the number of MBP-positive OLs, in comparison with LM (Fig. 3C). The number of MBP-positive OLs on rTen-2ECD and rTen-3ECD was also increased, but was not statistically different from those on the control (Fig. 3C). To further examine the requirement of Ten-4 in OLs, we used OPCs from Ten-4 -/- mice at P3 and their littermate WT mice as a control in this assay. Because murine OLs were less differentiated than rat OLs in this condition as mentioned above, immunostaining of O4, an earlier differentiation marker, was performed for evaluation of OL differentiation. A significant reduction in the number of Ten-4 -/- O4-positive OLs was observed on rTen-4ECDcoated nanofibers, compared with that of WT OLs (Fig. 3D). On rTen-1ECD-nanofibers, differentiated OLs were slightly decreased in the Ten-4 -/- culture, and this decrease was not significant. Ten4 -/- OLs were normally differentiated on LM-coated nanofibers (Fig. 3D). These results demonstrated that OL process formation was activated through the adhesion on the ECDs of all teneurins and that the homophilic interaction of Ten-4 promoted the differentiation of OLs.
4. Discussion In this study, we found that all of the four teneurin proteins formed molecular complexes with Ten-4 in the spinal cord tissue.
Fig. 2. Attachment activity of OPCs/OLs to rTenECDs. (A) Preparation of rTen-1ECD, rTen-2ECD, rTen-3ECD, and rTen-4ECD. Immunoblotting was performed using anti-V5 antibody to detect the proteins. Arrowhead: rTenECD proteins, CBB: Coomassie Brilliant Blue staining, IB: immunoblotting. (B) Representative images of attachment of OPCs/ OLs purified from the spinal cord tissue of Sox10-Venus mice at P3 to rTenECDs without (None) or with soluble rTen-4ECD (þSoluble rTen-4ECD) in the cell suspension. Scale bar: 200 mm. (C) Quantitative analysis of the attached cell number in the absence and presence of soluble rTen-4ECD. Triplicate or quadruplicate experiments were independently performed (n ¼ 3e4). Error bars represent mean ± s.e.m. One-way ANOVA followed by Dunnett’s post-hoc and the two-tailed Student’s t-test were used for the statistical analyses in the experiments with multiple samples (gray bars) relative to the control (black bar) and with two groups (gray and white bars), respectively: *p < 0.05, ***p < 0.001.
Please cite this article as: C. Hayashi et al., The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.002
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Fig. 3. OL process formation and differentiation on rTenECD-coated wells and nanofibers. Immunocytochemical images of MBP staining (green) on rTen-1ECD-, rTen-2ECD-, rTen-3ECD-, or rTen-4ECD-coated wells (A) and nanofibers (B). LM (laminin) was used as a control. DAPI (blue) was used for counting total cell numbers. Scale bar: 50 mm. (C) The number of MBP-positive differentiated OLs out of total cell numbers on the rTenECD-nanofibers. Quadruplicate experiments were independently performed (n ¼ 4). Error bars represent mean ± s.e.m. One-way ANOVA followed by Dunnett’s post-hoc was used for the statistical analysis in the experiments with multiple samples relative to the control: *p < 0.05. (D) The number of O4-positive differentiated OLs out of total cell numbers on the rTenECD-nanofibers. Triplicate experiments were independently performed (n ¼ 3). Error bars represent mean ± s.e.m. The two-tailed Student’s t-test was used for the statistical analysis in the experiments with two groups: *p < 0.05.
We also demonstrated that Ten-4 promoted both homophilic and heterophilic interaction between the teneurins for cell adhesion through their ECDs. All teneurin ECDs positively regulated cell process formation in OLs. However, in terms of the OL differentiation on axon-mimicking nanofibers, Ten-4 homophilic interaction was the most effective for the promotion of OL differentiation. With these results, we have demonstrated that either Ten-4 homophilic binding or its heterophilic binding to the other teneurin through their ECDs forms OPC/OL-axon adhesion and regulates cellular processes formation, however, for OL differentiation, Ten-4 homophilic interaction is required. In accordance with these results, it is known that the homophilic binding of most teneurins, including Drosophila teneurins, is more specific and functional than the heterophilic interactions, with the exception of the heterophilic binding of Ten-2 to latrophilins [9e12,22,23]. We have additionally demonstrated that Ten-4 ECD’s role in the OL-axon adhesion is unique among teneurins: the cell adhesion of other teneurins functions in neuron-neuron connectivity, such as target recognition and synapse formation, but not in OPC/OL-axon adhesion [9,12,13,23]. In addition, we identified solute carrier family 4 (anion exchanger), member 2 and ATPase, Naþ/Kþ transporting, alpha 3 polypeptide as putative binding partners of Ten-4 ECD. The biological relevance of these interactions will be analyzed in future studies. A number of in vitro experiments have shown that OPCs can
differentiate into OLs and survive for several days without axons in monoculture condition [19]. In vivo, however, the number of OPCs that can differentiate to OLs is restricted and OPCs/OLs that fail to contact axons are excluded by programmed cell death [2,24]. In addition, myelinating OLs survive extraordinarily long in vivo [25]. These evidences indicate that OL lineage cells require physical contact/adhesion to axons to receive axonal signal for their differentiation/maturation and survival. Previously, Laursen et al. demonstrated that the extracellular/transmembrane protein complex consisting of F3/contactin-L1 and integrin a6b1-laminin-211 facilitated the adhesion of OLs to axons and promoted OL survival and maturation/myelination [3]. However, the phenotypes in knockout mice of L1, F3/contactin, and integrin b1 resulted in less severe defects than expected. While the L1 knockout mice had malformations in the brain and the abnormality in corticospinal tract formation, no defects in OL differentiation or CNS myelination were observed [4]. The ablation of F3/contactin in mice resulted in a reduction in the number of OLs but only temporarily at P11, while the myelin structure was disruptive due to disorganization of the proteins at the node and paranode regions [7]. In addition, the conditional deletion of murine b1 integrin using either nestin-Cre or CNP-Cre expression showed no or mild effects on OL development and myelination in vivo, except for the region-specific reduction of the number of OLs in the cerebellum [5,6]. In contrast, Ten-4 deficient mice exhibited severe hypomyelination
Please cite this article as: C. Hayashi et al., The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.002
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due to a significant decrease of OLs, which resulted in a severe tremor phenotype [15]. From these in vivo observations and the results presented in this study, we demonstrate that Ten-4 is a key player among the cell adhesion proteins between OLs and axons for their differentiation/maturation and myelination. In summary, we have unveiled the role of Ten-4 as a cell adhesion protein in the OPC/OL-axon interaction for OL differentiation. Our findings will facilitate a better understanding of the molecular mechanism of myelination by OLs. The human Ten-4 gene (gene symbol: TENM4) is associated with some neurological and mental disorders, including essential tremor, bipolar disorder, and schizophrenia [26e28]. However, the pathogenesis of these disorders associated to the function of Ten-4 has not been elucidated. Since abnormalities in the CNS white matter are reported in these disorders, it is likely that oligodendroglial functions through Ten-4 are inhibited [29e31]. All together, our studies on Ten-4 will contribute to elucidate the pathogenesis of the related diseases and to the development of therapeutic reagents for these diseases.
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Declaration of competing interest The authors declare no competing financial interests. Acknowledgements We thank Dr. Yoshihiko Yamada (NIDCR, NIH, Bethesda, U.S.A.), Dr. Hideyuki Okano (Keio Univ., Tokyo, Japan), and Dr. Shinsuke Shibata (Keio Univ., Tokyo, Japan) for providing and maintaining the Ten-4 deficient mouse line, and Dr. Atsuyuki Yamataka and Dr. Katsumi Miyahara (Juntendo Univ., Tokyo, Japan) for maintaining the Sox10-Venus mouse line. We also thank Dr. Kenneth Yamada and Dr. Will Daley (NIDCR, NIH, Bethesda, U.S.A.) for their technical advice for the cell aggregation assay. We additionally thank Dr. Minami Ito (TMDU, Tokyo, Japan) for his advice for the statistical analyses, and Dr. Eriko Grace Suto, Ms. Mai Hyodo, Ms. Yuri Ueki, and Ms. Momoka Ota (TMDU, Tokyo, Japan) for their technical support. This work was supported by the Grant-in-Aid for Scientific Research (C) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) in Japan (16K09667) (N.S. and C.A.) and the Grant-in-Aid for Young Scientists of the MEXT in Japan (25860701) (N.S.). Appendix A. Supplementary data
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The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation Chikako Hayashi a, b, 1, Nobuharu Suzuki a, b, *, 1, Yo Mabuchi b, c, Naomi Kikura b, Yukina Hosoda a, Susana de Vega d, Chihiro Akazawa b, c, ** a
Department of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan Department of Biochemistry and Biophysics, Graduate School of Health Care Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan c Department of Biochemistry and Biophysics, Graduate School of Medical and Dental Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan d Department of Pathophysiology for Locomotive and Neoplastic Diseases and Department of Pathology and Oncology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2019 Accepted 1 December 2019 Available online xxx
Cell adhesion between oligodendrocytes and neuronal axons is a critical step for myelination that enables the rapid propagation of action potential in the central nervous system. Here, we show that the transmembrane protein teneurin-4 plays a role in the cell adhesion required for the differentiation of oligodendrocytes. We found that teneurin-4 formed molecular complexes with all of the four teneurin family members and promoted cell-cell adhesion. Oligodendrocyte lineage cells attached to the recombinant extracellular domain of all the teneurins and formed well-branched cell processes. In an axon-mimicking nanofibers assay, nanofibers coated with the recombinant teneurin-4 extracellular domain increased the differentiation of oligodendrocytes. Our results show that teneurin-4 binds to all teneurins through their extracellular domain, which facilitates the oligodendrocyte-axon adhesion, and promotes oligodendrocyte differentiation via its homophilic interaction. © 2019 Elsevier Inc. All rights reserved.
Keywords: Myelin Oligodendrocyte Cell adhesion Teneurin-4 Extracellular domain
1. Introduction In the central nervous system (CNS), oligodendrocytes (OLs) form myelin sheaths, composed of multi-lamellar sheets of their plasma membrane around neuronal axons that are required for the rapid propagation of action potential. During the postnatal
Abbreviations: CNS, central nervous system; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; MBP, myelin basic protein; ECD, extracellular domain; Ten-1, teneurin-1; Ten-2, teneurin-2; Ten-3, teneurin-3; Ten-4, teneurin-4; WT, wild-type; P, postnatal day; pTen, expression plasmid of the full-length teneurin; pTenECD, expression plasmid of the extracellular domain of teneurin; CHO, Chinese hamster ovary; LM, laminin. * Corresponding author. Department of Molecular and Cellular Biology, Graduate School of Medical and Dental Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. ** Corresponding author. Department of Biochemistry and Biophysics, Graduate School of Medical and Dental Sciences, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan. E-mail addresses: [email protected] (N. Suzuki), [email protected] (C. Akazawa). 1 These authors equally contributed to this work.
development, OL precursor cells (OPCs) contact and adhere to the targeted axons and subsequently differentiate into OLs that express myelin proteins, such as myelin basic protein (MBP) [1]. The OPC/OL interaction with axons promotes their survival as well [2]. It is known that the adhesion of OPCs/OLs to axons is mediated by the molecular complex of integrin a6b1-laminin-211 and F3/contactinL1 [3]. However, the significance of this molecular complex in vivo is still a matter of debate since the deficiency of these proteins does not, or does only temporally, exhibit effects on either generation or maintenance of OLs [4-7], indicating that there must be a compensatory mechanism by other critical protein(s) for the cell adhesion. Teneurins are type II transmembrane glycoproteins that consist of an N-terminal intracellular domain, a single-pass transmembrane domain, and a large extracellular domain (ECD) (~250 kDa) following an assembly of EGF-like repeats, Ig-like domain, NHL repeats/b-propeller domain, YD repeats/b-barrel domain, and toxin-like region [8]. In vertebrates, there are 4 teneurin family members (Ten-1 to -4). Teneurins are expressed in various tissues, including a high expression in the CNS. Recently,
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molecular functional studies of Ten-1, Ten-2, and Ten-3 have revealed that they homophilically or heterophilically bind between them or with latrophilins and promote neuronal cell adhesion for the target recognition and the formation of synapses between neural cells [9e13]. Ten-1, Ten-2, and Ten-3, however, are not expressed in glia cells [14]. Only Ten-4 is expressed in glia cells, among all the teneurins, including in OLs, and it acts as a regulator of CNS myelination [14,15]. In Ten-4 deficient (Ten-4 -/-) mice, a significant reduction in the number of OLs is observed due to inhibition of OL differentiation and survival, and myelination is substantially decreased in the CNS, especially in the spinal cord [15]. Ten-4 is expressed not only in OLs but also in neuronal cells and promotes cell processes formation through the activation of intracellular signaling molecules, such as focal adhesion kinase [15,16]. However, the role of the Ten-4 ECD, particularly in cell adhesion, has not been investigated. In this study, we attempted to elucidate the molecular function of Ten-4 ECD and found that Ten-4 ECD possessed cell adhesion activity by interacting with all teneurin ECDs. Further, the homophilic interaction of Ten-4 through its ECD promoted OL differentiation on nanofibers. Our results reveal that Ten-4 is a critical adhesion protein between OPCs/OLs and axons promoting OL differentiation.
transfected with pTenECDs was used, following the manufacturer’s instruction. Detailed methods are described in Supplementary Information. 2.6. Cell attachment assay The attachment activity of OL lineage cells from Sox10-Venus mice [17] was evaluated on rTenECD-coated wells. Additional procedures are described in Supplementary Information. 2.7. Primary culture of rat and mouse OPCs OPCs were prepared from spinal cords of P2 rat or P3 mouse pups using the shaken-off method as previously described [19] with some modifications. Procedures in detail are described in Supplementary Information. 2.8. Culture assays using rTenECD-coated wells and nanofibers The culture experiments of OLs were performed using rTenECDcoated wells and polycaprolactone nanofibers as previously described [20] with some modifications. Detailed methods are described in Supplementary Information.
2. Materials and methods 2.9. Statistical analyses 2.1. Animals Wild-type (WT) animals of postnatal day (P) 7 C57BL/6 mice and P2 Wistar rats were used for experiments of the immunoprecipitation and the isolation of primary OPCs, respectively. P3 mouse pups from the Sox10-Venus mouse line [17] and the Ten-4 -/mouse line [15] were used for the cell attachment assay and the culture experiment using nanofibers, respectively. All procedures for experimental animals were approved by the Institutional Animal Care and Use Committees of Tokyo Medical and Dental University (No. A2019-307A). 2.2. Immunoprecipitation The immunoprecipitation experiment of Ten-4 was carried out using tissue lysate from WT mouse spinal cords at P7, when is the stage of OL differentiation in WT and the onset of the defect in OL differentiation and survival in Ten-4 -/- spinal cord [15]. Detailed methods are described in Supplementary Information. 2.3. Plasmids construction The expression plasmids of the full-length transmembrane proteins (pTen) and the extracellular domain (pTenECD) of teneurins were prepared as previously described [16]. Additional information is provided in Supplementary Information. 2.4. Cell aggregation assay Cell aggregation assay was carried out by the hanging-drop method [18] using each pTen-transfected Chinese hamster ovary (CHO) cells. Following procedures are described in Supplementary Information. 2.5. Preparation of recombinant proteins of teneurin ECDs (rTenECDs) For the preparation of rTenECDs, the expression system of 293FreeStyle cells (ThermoFisher Scientific, Waltham, MA)
All of the experiments were independently performed at least 3 times. The statistics of the experimental results were analyzed as follows. The two-tailed Student’s t-test was used for the analyses between two groups in the experiments. We analyzed multiple groups compared with a control using one-way ANOVA followed by Dunnett’s post-hoc. The statistical significance was defined as *p < 0.05, **p < 0.01, and ***p < 0.001. 3. Results 3.1. Molecular complex formation of Ten-4 with teneurin family members in the spinal cord To explore the molecular function of the Ten-4 ECD, we searched for binding proteins to this domain. Immunoprecipitation of Ten-4 from WT spinal cord lysate was carried out using anti-Ten-4 antibody. Co-immunoprecipitated proteins were identified by shotgun mass spectrometry and were compared with precipitated proteins using normal IgG as a control. Extracellular proteins, extracellular membrane-associated proteins, and transmembrane proteins, which were obtained only in the immunoprecipitation using antiTen-4 antibody, were considered as candidates. Totally, 6 candidates of Ten-4 ECD binding proteins were obtained (Table 1). Ten-4 was one of the candidates, since Ten-4 homophilic binding possibly occurred. All of the 6 proteins were transmembrane proteins and no extracellular proteins, including extracellular membraneassociated proteins, were obtained. Interestingly, 4 of the 6 transmembrane proteins were the teneurin family members (Ten-1 to -4) (Table 1). The other two were the 11- and 8-times transmembrane proteins of ion exchanger and transporter, respectively (Table 1). In this study, we focused on Ten-4 interaction between the teneurin members due to a putative role in the cell adhesion between OLs and axons. Since all of the 4 teneurins are expressed in neurons, but only Ten-4 is expressed in glia cells, including OLs [14,15], we hypothesized that Ten-4 on OL lineage cells bound to Ten-1, Ten-2, Ten-3, or Ten-4 on axons homophilically or heterophilically through their ECDs to promote cell adhesion between these cells.
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Table 1 Candidate binding proteins to Ten-4 ECD. Among co-immunoprecipitated proteins using anti-Ten-4 antibody, extracellular proteins, extracellular membraneassociated proteins, and transmembrane proteins were considered as candidates. Proteins precipitated using normal IgG as a control were excluded from the list. Unused is ProtScore reflecting the amount of total, unique peptide evidence related to a given protein from the result of the mass spectrometry. More than 2.00 of unused score is enough trustable for detection. The accession numbers from the National Center for Biotechnology Information (NCBI) database are indicated. No
Unused
Protein name
Accession
1 2 3 4 5 6
150.99 43.23 22.50 9.70 3.44 2.31
Teneurin-4 Teneurin-1 Solute carrier family 4 (anion exchanger), member 2 Teneurin-3 Teneurin-2 ATPase, Naþ/Kþ transporting, alpha 3 polypeptide
GI: GI: GI: GI: GI: GI:
3.2. Homophilic and heterophilic cell-cell adhesion activity of Ten-4 with all teneurins To examine whether Ten-4 possesses cell-cell adhesion activity, we prepared CHO cells overexpressing each teneurin and performed cell aggregation assay. In the control with mock plasmidexpressing cells, the number of cells in a cell aggregate was not changed 2 h after the assay was started (Fig. 1A and B: Mock Mock). When combinations of Ten-4-overexpressing cells with each teneurin-overexpressing cells were tested, the cell number in a cell aggregate was increased (Fig. 1A and B: Ten-1 Ten-4, Ten-2 Ten-4, Ten-3 Ten-4, and Ten-4 Ten-4). Interestingly, the most
124248484 7657413 32450722 81869788 81869787 148692350
significant increase of cell number was in the combination of Ten-4 with Ten-4 (Fig. 1B). We confirmed heterophilic cell binding using two different fluorescent dyes. The cell-cell adhesion aggregation data revealed that Ten-4 promoted cell-cell adhesion homophilically and heterophilically with the other teneurins.
3.3. OPC/OL adhesion activity of the ECDs of teneurins As we hypothesized that teneurins on axonal membrane surface interacted with Ten-4 on OL lineage cells, we next analyzed whether the ECDs of teneurins promoted the attachment of OL lineage cells. We first prepared 4 different rTenECDs (rTen-1ECD,
Fig. 1. Cell-cell adhesion activity of Ten-4 with 4 teneurins. (A) Representative images of cell aggregations at 0 h and 2 h after incubation. Five different combinations were tested as indicated. Mock: mock plasmid-trasnfected CHO cells; Ten-1: Ten-1 expression plasmid-transfected CHO cells; Ten-2: Ten-2 expression plasmid-transfected CHO cells; Ten-3: Ten-3 expression plasmid-transfected CHO cells; Ten-4: Ten-4 expression plasmid-transfected CHO cells, Scale bar: 200 mm. (B) Quantitative analysis of cell aggregation assay. The number of cells in an aggregation was counted and categorized into 4 groups; single cell, 2-3, 4-6, and 7-9 cells in an aggregation. Triplicate experiments were independently performed (n ¼ 3). Error bars represent mean ± s.e.m. The two-tailed Student’s t-test was used for the statistical analysis in the experiments with two groups: *p < 0.05, **p < 0.01.
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rTen-2ECD, rTen-3ECD, and rTen-4ECD) using the 293 free-style expression system (Fig. 2A). For the attachment assay, a high purity of OL lineage cells was required because the attached cells were stained with crystal violet for quantification. Therefore, we utilized Sox10-Venus mice that express the fluorescence protein Venus driven by the Sox10 promoter, which is specifically expressed in OL lineage cells in the CNS [17], and purified OL lineage cells from P3 Sox10-Venus mouse spinal cords by flow cytometry. The collected OL lineage cells were seeded on rTenECD-coated wells and incubated for an hour. The results showed that all of rTenECDs had attachment activity of OL lineage cells (Fig. 2B and C). The attachment activity of rTen-1ECD, rTen-3ECD, and rTen-4ECD was higher than that of rTen-2ECD (Fig. 2B and C). To evaluate whether Ten-4 expressed in OL lineage cells affected the adhesion, soluble rTen4ECD was added to the assay. The cell attachment to all of rTenECDs was significantly inhibited by the addition of rTen-4ECD (Fig. 2B and C). These results indicated that all of the teneurins’ ECDs possessed adhesion activity of OL lineage cells and that the attachment to the ECDs was mediated, at least in part, by Ten-4 on the surface of OL lineage cells.
3.4. Promoted OL process formation and differentiation on the ECDs of teneurins To further test biological effects of rTenECDs on OL morphology and differentiation, we used rTenECD-coated wells and synthetic polycaprolactone nanofibers, mimicking axons. The rTenECD proteins were coated on flat bottom wells in a glass slide chamber and on the surface of the nanofibers. OPCs from rat spinal cords were plated and their differentiation was induced by changing the medium to the differentiation medium one day after plated. Rat OPCs/ OLs were used for these experiments because they differentiated
better and survived longer than murine OPCs/OLs in these assay conditions. Laminin (LM) was used as a control of process formation and differentiation [21]. On rTenECD-coated wells, OLs formed well-branched cellular processes with an intense expression signal of MBP, higher than on the LM-coated well (Fig. 3A). In the culture on rTenECD-coated nanofibers, MBP-positive OLs extended their branched processes along the nanofibers, covering larger areas than those on LM-nanofibers (Fig. 3B). Moreover, we found that rTen-1ECD and rTen-4ECD significantly increased the number of MBP-positive OLs, in comparison with LM (Fig. 3C). The number of MBP-positive OLs on rTen-2ECD and rTen-3ECD was also increased, but was not statistically different from those on the control (Fig. 3C). To further examine the requirement of Ten-4 in OLs, we used OPCs from Ten-4 -/- mice at P3 and their littermate WT mice as a control in this assay. Because murine OLs were less differentiated than rat OLs in this condition as mentioned above, immunostaining of O4, an earlier differentiation marker, was performed for evaluation of OL differentiation. A significant reduction in the number of Ten-4 -/- O4-positive OLs was observed on rTen-4ECDcoated nanofibers, compared with that of WT OLs (Fig. 3D). On rTen-1ECD-nanofibers, differentiated OLs were slightly decreased in the Ten-4 -/- culture, and this decrease was not significant. Ten4 -/- OLs were normally differentiated on LM-coated nanofibers (Fig. 3D). These results demonstrated that OL process formation was activated through the adhesion on the ECDs of all teneurins and that the homophilic interaction of Ten-4 promoted the differentiation of OLs.
4. Discussion In this study, we found that all of the four teneurin proteins formed molecular complexes with Ten-4 in the spinal cord tissue.
Fig. 2. Attachment activity of OPCs/OLs to rTenECDs. (A) Preparation of rTen-1ECD, rTen-2ECD, rTen-3ECD, and rTen-4ECD. Immunoblotting was performed using anti-V5 antibody to detect the proteins. Arrowhead: rTenECD proteins, CBB: Coomassie Brilliant Blue staining, IB: immunoblotting. (B) Representative images of attachment of OPCs/ OLs purified from the spinal cord tissue of Sox10-Venus mice at P3 to rTenECDs without (None) or with soluble rTen-4ECD (þSoluble rTen-4ECD) in the cell suspension. Scale bar: 200 mm. (C) Quantitative analysis of the attached cell number in the absence and presence of soluble rTen-4ECD. Triplicate or quadruplicate experiments were independently performed (n ¼ 3e4). Error bars represent mean ± s.e.m. One-way ANOVA followed by Dunnett’s post-hoc and the two-tailed Student’s t-test were used for the statistical analyses in the experiments with multiple samples (gray bars) relative to the control (black bar) and with two groups (gray and white bars), respectively: *p < 0.05, ***p < 0.001.
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Fig. 3. OL process formation and differentiation on rTenECD-coated wells and nanofibers. Immunocytochemical images of MBP staining (green) on rTen-1ECD-, rTen-2ECD-, rTen-3ECD-, or rTen-4ECD-coated wells (A) and nanofibers (B). LM (laminin) was used as a control. DAPI (blue) was used for counting total cell numbers. Scale bar: 50 mm. (C) The number of MBP-positive differentiated OLs out of total cell numbers on the rTenECD-nanofibers. Quadruplicate experiments were independently performed (n ¼ 4). Error bars represent mean ± s.e.m. One-way ANOVA followed by Dunnett’s post-hoc was used for the statistical analysis in the experiments with multiple samples relative to the control: *p < 0.05. (D) The number of O4-positive differentiated OLs out of total cell numbers on the rTenECD-nanofibers. Triplicate experiments were independently performed (n ¼ 3). Error bars represent mean ± s.e.m. The two-tailed Student’s t-test was used for the statistical analysis in the experiments with two groups: *p < 0.05.
We also demonstrated that Ten-4 promoted both homophilic and heterophilic interaction between the teneurins for cell adhesion through their ECDs. All teneurin ECDs positively regulated cell process formation in OLs. However, in terms of the OL differentiation on axon-mimicking nanofibers, Ten-4 homophilic interaction was the most effective for the promotion of OL differentiation. With these results, we have demonstrated that either Ten-4 homophilic binding or its heterophilic binding to the other teneurin through their ECDs forms OPC/OL-axon adhesion and regulates cellular processes formation, however, for OL differentiation, Ten-4 homophilic interaction is required. In accordance with these results, it is known that the homophilic binding of most teneurins, including Drosophila teneurins, is more specific and functional than the heterophilic interactions, with the exception of the heterophilic binding of Ten-2 to latrophilins [9e12,22,23]. We have additionally demonstrated that Ten-4 ECD’s role in the OL-axon adhesion is unique among teneurins: the cell adhesion of other teneurins functions in neuron-neuron connectivity, such as target recognition and synapse formation, but not in OPC/OL-axon adhesion [9,12,13,23]. In addition, we identified solute carrier family 4 (anion exchanger), member 2 and ATPase, Naþ/Kþ transporting, alpha 3 polypeptide as putative binding partners of Ten-4 ECD. The biological relevance of these interactions will be analyzed in future studies. A number of in vitro experiments have shown that OPCs can
differentiate into OLs and survive for several days without axons in monoculture condition [19]. In vivo, however, the number of OPCs that can differentiate to OLs is restricted and OPCs/OLs that fail to contact axons are excluded by programmed cell death [2,24]. In addition, myelinating OLs survive extraordinarily long in vivo [25]. These evidences indicate that OL lineage cells require physical contact/adhesion to axons to receive axonal signal for their differentiation/maturation and survival. Previously, Laursen et al. demonstrated that the extracellular/transmembrane protein complex consisting of F3/contactin-L1 and integrin a6b1-laminin-211 facilitated the adhesion of OLs to axons and promoted OL survival and maturation/myelination [3]. However, the phenotypes in knockout mice of L1, F3/contactin, and integrin b1 resulted in less severe defects than expected. While the L1 knockout mice had malformations in the brain and the abnormality in corticospinal tract formation, no defects in OL differentiation or CNS myelination were observed [4]. The ablation of F3/contactin in mice resulted in a reduction in the number of OLs but only temporarily at P11, while the myelin structure was disruptive due to disorganization of the proteins at the node and paranode regions [7]. In addition, the conditional deletion of murine b1 integrin using either nestin-Cre or CNP-Cre expression showed no or mild effects on OL development and myelination in vivo, except for the region-specific reduction of the number of OLs in the cerebellum [5,6]. In contrast, Ten-4 deficient mice exhibited severe hypomyelination
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due to a significant decrease of OLs, which resulted in a severe tremor phenotype [15]. From these in vivo observations and the results presented in this study, we demonstrate that Ten-4 is a key player among the cell adhesion proteins between OLs and axons for their differentiation/maturation and myelination. In summary, we have unveiled the role of Ten-4 as a cell adhesion protein in the OPC/OL-axon interaction for OL differentiation. Our findings will facilitate a better understanding of the molecular mechanism of myelination by OLs. The human Ten-4 gene (gene symbol: TENM4) is associated with some neurological and mental disorders, including essential tremor, bipolar disorder, and schizophrenia [26e28]. However, the pathogenesis of these disorders associated to the function of Ten-4 has not been elucidated. Since abnormalities in the CNS white matter are reported in these disorders, it is likely that oligodendroglial functions through Ten-4 are inhibited [29e31]. All together, our studies on Ten-4 will contribute to elucidate the pathogenesis of the related diseases and to the development of therapeutic reagents for these diseases.
[8]
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Declaration of competing interest The authors declare no competing financial interests. Acknowledgements We thank Dr. Yoshihiko Yamada (NIDCR, NIH, Bethesda, U.S.A.), Dr. Hideyuki Okano (Keio Univ., Tokyo, Japan), and Dr. Shinsuke Shibata (Keio Univ., Tokyo, Japan) for providing and maintaining the Ten-4 deficient mouse line, and Dr. Atsuyuki Yamataka and Dr. Katsumi Miyahara (Juntendo Univ., Tokyo, Japan) for maintaining the Sox10-Venus mouse line. We also thank Dr. Kenneth Yamada and Dr. Will Daley (NIDCR, NIH, Bethesda, U.S.A.) for their technical advice for the cell aggregation assay. We additionally thank Dr. Minami Ito (TMDU, Tokyo, Japan) for his advice for the statistical analyses, and Dr. Eriko Grace Suto, Ms. Mai Hyodo, Ms. Yuri Ueki, and Ms. Momoka Ota (TMDU, Tokyo, Japan) for their technical support. This work was supported by the Grant-in-Aid for Scientific Research (C) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) in Japan (16K09667) (N.S. and C.A.) and the Grant-in-Aid for Young Scientists of the MEXT in Japan (25860701) (N.S.). Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.002.
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Please cite this article as: C. Hayashi et al., The extracellular domain of teneurin-4 promotes cell adhesion for oligodendrocyte differentiation, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.002