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PlexinA3 Interacts with CRMP2 to Mediate Sema3A Signalling During Dendritic Growth in Cultured Cerebellar Granule Neurons
Journal Pre-proofs Research Article PlexinA3 interacts with CRMP2 to mediate Sema3A signalling during den‐ dritic growth in cultured cerebellar granule neurons Tao Jiang, Guowei Zhang, Yaozhong Liang, Zhenbin Cai, Zhi liang, Hongsheng Lin, Minghui Tan PII: DOI: Reference:
S0306-4522(20)30094-4 https://doi.org/10.1016/j.neuroscience.2020.02.008 NSC 19516
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Neuroscience
Received Date: Revised Date: Accepted Date:
4 September 2019 5 February 2020 6 February 2020
Please cite this article as: T. Jiang, G. Zhang, Y. Liang, Z. Cai, Z. liang, H. Lin, M. Tan, PlexinA3 interacts with CRMP2 to mediate Sema3A signalling during dendritic growth in cultured cerebellar granule neurons, Neuroscience (2020), doi: https://doi.org/10.1016/j.neuroscience.2020.02.008
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PlexinA3 interacts with CRMP2 to mediate Sema3A signalling during dendritic growth in cultured cerebellar granule neurons
Running title: PlexinA3/CRMP2-mediated dendritic growth in CGNs
Tao Jiang #, Guowei Zhang #, Yaozhong Liang #, Zhenbin Cai, Zhi liang, Hongsheng Lin *, Minghui Tan *
Department of Orthopaedics, the First Affiliated Hospital of Jinan University, Guangzhou 510630, China
# These authors contributed equally to this work.
* Corresponding to: Dr. Hongsheng Lin ([email protected]) and Dr. Minghui Tan ([email protected]), Department of Orthopaedics, the First Affiliated Hospital of Jinan University, Guangzhou 510630, China
Abbreviations: CGN, cerebellar granule neuron; CRMP2, collapsin response mediator protein 2; DIV, days in vitro; GST, glutathione S-transferase; RBD, Ras-binding domain; siRNA, short interfering RNA.
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Abstract Plexin family proteins mediate semaphorin signalling during dendritic arbour development. However, the role of PlexinA3 in the growth of dendrites of cultured cerebellar granule neurons is not known. We found that PlexinA3 colocalizes with CRMP2 (collapsin response mediator protein 2) in dendritic shafts. Immunoprecipitation and glutathione transferase pulldown assays showed that the intracellular Ras-binding domain of PlexinA3 directly interacts with CRMP2. PlexinA3 was necessary and sufficient for the growth of cerebellar granule neuron dendrites, as genetic knockdown of PlexinA3 reduced but its overexpression increased dendritic lengths and dendritic tip numbers. These increases were enhanced with CRMP2 overexpression and abolished with CRMP2 knockdown, indicating that CRMP2 is the downstream effector. Furthermore, PlexinA3/CRMP2 signalling contributed to Sema3A-controlled dendritic growth. Together, these data identify a novel PlexinA3/CRMP2 pathway in semaphorin-regulated growth of cultured cerebellar granule neuron dendrites.
Keywords CRMPs, plexins, protein interaction, dendritic development, semaphorin
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Introduction Dendrites, which integrate synaptic signals, are important for transmitting information within the nervous system. Dendritic arbours are established according to adhesive interactions, neuronal activity, and extracellular cues (Emoto K, 2012;Jan YN and Jan LY, 2003;Valnegri P et al., 2015). Sema3A, a secreted chemotrophic class 3 semaphorin, is involved in dendritic growth in new-born hippocampal (Li X et al., 2013) and cortical (Gonthier B et al., 2009) neurons and also influences dendritic patterning and synaptic maturation (Nakamura F et al., 2009;Shelly M et al., 2011;Yamashita N et al., 2014) and regulates axonal guidance, neuronal polarity, and axonal transport (Polleux F et al., 1998;Tran TS et al., 2009). Sema3A is a component of a signal transduction complex that includes ligand-binding subunit neuropilin 1 and a PlexinA family member (Takahashi T et al., 1999;Zhou Y et al., 2008). Sema3A and PlexinA regulate cytoskeleton remodelling in neurons via the Rho family GTPase Rac1 and actin-depolymerizing proteins such as cofilin. Semaphorins are involved in the development of cerebellar granule cells (CGNs) (Kerjan G et al., 2005). The plexin family proteins (including PlexinA, -B, -C, and -D) all have an extracellular semaphorin-binding domain, a transmembrane domain, and an intracellular domain involved in protein-protein interactions. The binding of Sema4D to PlexinB1 modulates M-Ras GAP activity to regulate actin-based dendrite remodelling in cortical neurons (Tasaka G et al., 2012). Sema3A and PlexinA2 signal through Fyn and Cdk5 to orient dendrites in the cerebral cortex (Sasaki Y et al., 2002). PlexinA2 modulates the migration of granule cells (Renaud J et al., 2008), and PlexinB2 controls the proliferation and differentiation of CGNs in studies of knockout mice (Friedel RH et al., 2007). PlexinA3, a receptor for Sema3A (Cheng HJ et al., 2001), is also involved in neurite extension and guidance (Negishi M et al., 2005), but its role in the development of dendrites in the cerebellum has not been explored. Collapsin response mediator protein 2 (CRMP2, also known as TOAD64, Ulip2, or DRP2) is widely expressed in the developing nervous system (Minturn JE et al., 1995) and regulates a variety of cellular functions, such as cell migration, neurite extension, and axon regeneration (Yoshimura T et al., 2005). CRMP2 interacts with presynaptic voltage-gated calcium channels to modulate neurotransmitter release (Brittain JM et al., 2009) but also controls the growth of axons of hippocampal neurons (Inagaki N et al., 2001) and regulates neurite growth and 3
polarity by modulating cytoskeletal dynamics (Fukata Y et al., 2002). CRMP2 is important for organizing the dendritic field and coordinates with CRMP1 to regulate dendrite development (Yamashita N et al., 2012). CRMP2 is inactivated by phosphorylation via Cdk5 and GSK-3 (Cole AR et al., 2004), and CRMP2 phosphorylation regulates the bifurcation of hippocampal CA1 pyramidal neuron dendrites (Niisato E et al., 2013). The CRMP family proteins have been implicated to facilitate semaphorin-mediated morphological changes in nonneuronal cells (Deo RC et al., 2004). However, the detailed scenario in neuronal cells needs to be elucidated. We previously found that CRMP2 is distributed in the dendrites of cultured CGNs, but not in the axons, and that it signals via GSK-3 during activity-induced dendritic growth (Tan M et al., 2013). In the present study, we demonstrate that CRMP2 colocalizes and interacts with PlexinA3, which is also enriched in the dendrites of cultured CGNs and is necessary and sufficient for their growth. We show that CRMP2 functions downstream of PlexinA3 to mediate Sema3A-regulated dendritic growth in CGNs.
Experimental procedures Cell culture CGNs from 7-day-old Sprague-Dawley rat pups were prepared as previously described (Tan M, et al., 2013) to high purity (Contestabile A, 2002). All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals of the NIH and approved by the Jinan University Institutional Animal Care and Use Committee. Briefly, neurons were isolated from dissected cerebella by using trypsin (Thermo Fisher Scientific, Rockford, IL), treated with DNase (Gibco BRL, Gaithersburg, MD), and cultured in BMEM containing 10% foetal bovine serum (Gibco BRL) and 25 mM KCl (Sigma-Aldrich, St. Louis, MO) at a density of 1.5 × 106 cells/ml. For immunocytochemistry analyses, neurons were plated on coverslips (Thermo Fisher Scientific). HEK293 cells were cultured in DMEM/F12 (Gibco BRL) supplemented with 10% foetal bovine serum
Constructs and transfection The full-length PlexinA3 gene was cloned from cDNA of rat CGNs, and truncated PlexinA3 fragments were subcloned into pEGFP-C1 plasmids (Clontech, Mountain View, CA). All 4
constructs were verified by sequencing. Two PlexinA3 small interfering RNA (siRNA) fragments, siPlexinA3-a and -b, were designed to target rat PlexinA3 (NM_001107581) with sequences
5-GUGUACAAGGGUAUUCCAUAC-3
and
5-
CACAAUUCGUGUUUGACAUCC-3, respectively. A nontargeting siRNA was used as a negative control for all siRNA transfections. CRMP2-encoding plasmids and siRNA fragments against rat CRMP2 were previously described (Tan M, et al., 2013). siRNAs were synthesized by GenePharma (Shanghai, China), and their efficacy and specificity were determined by transfection in HEK293 cells as previously described (Zhang J et al., 2017). CGNs were transfected via the calcium phosphate method within DIV2 (Zhang J et al., 2015) and cultured for another 3 days. Immunocytochemistry analyses were performed on CGNs after 5 days in vitro (DIV5).
Western blotting Western blotting analyses of cell lysates were as previously described (Tan M et al., 2013). Briefly, samples were separated by SDS-PAGE, transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA), and blocked for 1 h at room temperature with 5% skim milk in Tris-buffered saline. Membranes were probed overnight at 4°C with rabbit anti-CRMP2 (catalogue no. 9393; Cell Signal Technology, Danvers, MA), goat anti-PlexinA3 (catalogue no. AF4075; R&D systems, Minneapolis, MN), and anti-tubulin (Sigma-Aldrich) primary antibodies.
Horseradish
peroxidase-conjugated
secondary
antibodies
(Jackson
ImmunoResearch, West Grove, PA) were then applied and visualized via enhanced chemiluminescence (ECL kit; Pierce, Rockford, IL).
Fluorescence immunocytochemistry CGNs were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.1% Triton X-100 in Tris-buffered saline, blocked with 3% donkey serum and immunostaining was carried out as described before (Gong X et al., 2016) using anti-CRMP2, anti-PlexinA3, and anti-GFP (Abcam, Cambridge, UK) primary antibodies diluted 1:1,000 and Alexa Fluor 488 and 555 secondary antibodies (Molecular Probes, Leiden, the Netherlands) diluted 1:2,000. The coverslips were mounted using Fluro-Gel II with DAPI (Electron Microscopy Sciences, 5
Hatfield, PA) and imaged with an LSM 780 confocal microscope (Carl Zeiss, Oberkochen, Germany) with the pinhole set to 1 airy unit.
Glutathione S-transferase (GST) pulldown and immunoprecipitation GST pulldown assays were performed as described previously (Zhang J, et al., 2017). The expression of GST-tagged full-length and truncated PlexinA3 and CRMP2 in Top10F Escherichia coli cells (Invitrogen) was induced with 0.1 mM isopropyl-1-thio-β-Dgalactopyranoside (Roche Applied Science, Indianapolis, IN), and the proteins were extracted and purified by using glutathione agarose beads (Pierce). Cell lysates from CGNs were incubated with GST fusion proteins at 4°C overnight in a buffer containing 100 mM NaCl, 20 mM Tris-HCl, 5% glycerol (pH 7.0), 1% Triton X-100, and a cocktail of protease inhibitors consisting of 1 μM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.1 mg/ml benzamidine (Merck, Kenilworth, NJ). The pulldown sediments were separated and analysed by Western blotting. For immunoprecipitation, CGN lysates were prepared with buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 2.5 mM pyrophosphate, 1 mM glycerol phosphate, and a protease inhibitor mixture and incubated with 2 g ant-CRMP2 or anti-PlexinA3 antibodies. Immunoprecipitation was performed by incubating the mixture with 60 l precleared protein G/A agarose for 16 h at 4°C with continuous inversion. The immunocomplexes were pelleted by centrifugation, washed, and analysed by Western blotting.
CGN morphometry CGN morphometry was performed as described previously (Tan M, et al., 2013). Briefly, GFPexpressing CGNs were immunostained with antibodies against GFP, MAP2 (to label dendrites), and Tau-1 (to label axons) and imaged in a blinded manner with an Axio Observer Z1 (Carl Zeiss). At least 60 neurons for each experimental group were analysed with Image Pro Plus 6 software (Media Cybernetics, Silver Spring, MD).
Statistical analysis 6
Experiments were performed at least three times. Groups were compared via statistical analyses of variance with Bonferroni post hoc tests (for multiple comparisons) and Student’s t tests (for single comparisons). A P value of < 0.05 was considered statistically significant.
Results PlexinA3 colocalizes with CRMP2 in dendrites of cultured CGNs The distribution of PlexinA3 in CGNs was observed via immunofluorescence. PlexinA3 immunoreactivity was strong in dendritic shafts but weak in axons (Fig. 1). This pattern was consistent with the distribution of CRMP2 we observed previously in CGNs (Tan M, et al., 2013). Further examinations with confocal microscopy revealed that CRMP2 and PlexinA3 colocalized in dendrite shafts (Fig. 2). These data suggest that CRMP2 and PlexinA3 may interact in cultured CGNs.
PlexinA3 interacts directly with CRMP2 To determine whether CRMP2 and PlexinA3 interact, we performed coimmunoprecipitation assays. PlexinA3 was immunoprecipitated from cell lysates of developing CGNs (DIV5). Western blotting revealed the presence of CRMP2 in these immunoprecipitates (Fig. 3A). Conversely, PlexinA3 was detected in proteins immunoprecipitated with an antibody against CRMP2 (Fig. 3B), suggesting a direct interaction between CRMP2 and PlexinA3. This interaction was verified with a GST pulldown assay, as PlexinA3 was detected in the GSTCRMP2 pulldowns but not in the GST control (Fig. 3C). PlexinA3 is a large transmembrane protein (~220 kDa) containing extracellular and transmembrane domains and an intracellular region comprising an N-terminal segment, a C1 domain, Ras-binding domain (RBD), and C2 domain (Fig. 3D) (He H et al., 2009). As CRMP2 is a cytosolic protein, we targeted the intracellular domain of PlexinA3 to identify the region responsible for the interaction with CRMP2. To this end, we generated PlexinA3 fragments comprising the entire intracellular region, the N-terminal, C1, and RBD regions only, the Nterminal and C1 regions only and the RBD region only (Fig. 3E and F). A GST pulldown assay revealed that CRMP2 was present only with fragments containing the RBD, suggesting that this region of PlexinA3 is responsible for binding CRMP2. 7
PlexinA3 is sufficient and necessary for dendritic growth To examine the function of PlexinA3 in dendrites, we overexpressed the full-length protein or the intracellular region in cultured CGNs. Overexpression of full-length PlexinA3 promoted dendritic growth, significantly increasing the total dendritic length, whereas overexpression of the intracellular domain suppressed dendritic growth (Fig. 4). The inhibition of growth observed with expression of the intracellular domain may reflect a dominant negative effect on interactions with cytosolic proteins, such as CRMP2. In addition, we examined the effect of PlexinA3 knockdown. The knockdown efficiency of two siRNAs against PlexinA3 was determined in HEK293 cells by Western blotting (Fig. 5a and b) and in CGNs by immunostaining (Fig. 5c and d). Notably, knockdown of PlexinA3 reduced the overall dendritic length (Fig. 5e) and number of dendritic tips (Fig. 5f) of CGNs. Together, these results suggest that PlexinA3 is both sufficient and necessary for the growth of CGN dendrites.
PlexinA3 regulates dendritic growth via downstream CRMP2 We hypothesized that PlexinA3 regulates dendritic growth in CGNs via its direct interactions with CRMP2. To test this, we transfected CGNs with plasmids encoding PlexinA3 and/or CRMP2. Overexpression of PlexinA3 or CRMP2 promoted dendritic growth, with further increases in dendritic length and tip numbers when both were overexpressed (Fig. 6a and b). By contrast, CGNs transfected with siRNA against CRMP2 or PlexinA3 had impaired dendritic growth; however, an additive effect was not observed when both proteins were knocked down simultaneously (Fig. 6c and d). Moreover, knockdown of CRMP2 abolished the enhanced growth induced by PlexinA3 overexpression. Importantly, knockdown of PlexinA3 had no effect on the dendritic growth induced by CRMP2 overexpression (Fig. 6e and f). These data indicate that CRMP2 functions downstream of PlexinA3 to induce dendritic growth in CGNs.
PlexinA3/CRMP2 signalling mediates Sema3A-regulated dendritic growth We hypothesized that PlexinA3/CRMP2 signalling contributes to Sema3A-mediated dendritic growth in CGNs. The test this, we treated CGNs expressing GFP and/or siRNAs against CRMP2 or PlexinA3 with Sema3A recombinant protein. Immunocytochemistry revealed that 8
Sema3A-induced dendritic growth was significantly suppressed by knockdown of CRMP2 and/or PlexinA3 (Fig. 7). These data indicate that the PlexinA3/CRMP2 pathway is involved in Sema3A-regulated dendritic growth in CGNs.
Discussion Semaphorins, as well as brain-derived neurotrophic factor and Wnt signalling, are critical for dendritic development (McAllister AK et al., 1997;Polleux F et al., 2000;Yu X and Malenka RC, 2003). Sema3A recognizes neuropilin 1/PlexinA complexes to regulate dendritic growth, neuronal polarization, and synaptic formation and maturation during development (Pasterkamp RJ, 2012;Yoshida Y, 2012). Sema3A signals through Fyn and Cdk5 for dendrite orientation and growth cone collapse (Morita A et al., 2006;Sasaki Y, et al., 2002), and the phosphorylation of focal adhesion kinase by Cdk5 influences the complexity of neuronal dendrites (Vitagliano O et al., 2013). Furthermore, Sema3A signalling through Fyn and Src kinases influences the arborization of the basal dendrites of cortical neurons via protein tyrosine phosphatase δ (Nakamura F et al., 2017), and signalling through Farp1 mediates activity-dependent arborization hippocampal neuron dendrites of (Cheadle L and Biederer T, 2014). In this study, we show that Sema3A signals through PlexinA3 and CRMP2 to regulate dendritic growth in CGNs. Additionally, we demonstrate that the interaction of PlexinA3 and CRMP2 within dendritic shafts is necessary and sufficient for dendritic growth (Fig. 8). We previously showed that CRMP2 in CGN dendrites mediates activity-dependent dendritic outgrowth (Tan M, et al., 2013) and that CRMP4 in hippocampal neurons interacts with the actin cytoskeleton to mediate dendritic growth and maturation (Cha C et al., 2016). Here, we found that the CRMP2 colocalizes with PlexinA3 in the dendritic shaft and directly binds its RBD region. PlexinA3 is a large transmembrane protein whose function relies on protein interactions. For example, neuropilin 2/PlexinA3 receptors associate with GluA1 to mediate Sema3F-dependent homeostatic scaling in cortical neurons (Wang Q et al., 2017). A recent study showed that PlexinA2 complexes with CRMP2 when Nogo-A binds to Nogo receptor 1 to regulate corticospinal axon sprouting after trauma (Sekine Y et al., 2019). These studies and our own provide evidence that Plexin and CRMP proteins interact under very different physiological conditions. 9
CRMP proteins mediate Sema3A signalling; for example, Sema3A-regulated actin cytoskeleton remodelling is mediated by the interaction between CRMP1 and the amino- and carboxy-terminal domains of filamin-A (Nakamura F et al., 2014). Interactions with Spy1 disrupt the association of CRMP1 with actin during Sema3A-induced growth cone collapse and regeneration after sciatic nerve crush (Yao L et al., 2016). Yamane and colleagues (2017) reported that CRMP1 couples with Nav1.7 to mediate retrograde signalling of Sema3A in axons. Many such Sema3A-induced interactions are regulated by phosphorylation, such as the phosphorylation of CRMP2 by Cdk5 and GSK-3 during axon guidance (Uchida Y et al., 2005), phosphorylation of CRMP1 by Cdk5 during dendritic spine development in cultured cortical neurons (Yamashita N et al., 2007), and phosphorylation at Tyr32 of CRMP2 by Fyn in dorsal root ganglion neurons (Uchida Y et al., 2009). As CRMPs promote cytoskeleton assembly by binding to tubulin and actin (Fukata Y, et al., 2002;Tan M et al., 2015), phosphorylation interferes with this binding, leading to the dissociation of microtubules and actin filaments (Arimura N et al., 2005;Namekata K et al., 2012). In summary, we showed that CRMP2 colocalizes with PlexinA3 in the dendritic shafts of cultured CGNs. We demonstrated that PlexinA3 and CRMP2 interact directly and mediate Sema3A-regulated dendritic growth in cultured CGNs. Further investigation is needed to identify the corresponding phosphorylation status of CRMP2 and the regulation of upstream kinases during dendritic growth.
Acknowledgments There is no conflict of interest to declare. This work was supported by the Pearl River S&T Nova Program of Guangzhou, China [no. 201806010011], and the National Natural Science Foundation of China [no. 31300885 and 81771331].
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Tasaka G, Negishi M, Oinuma I (2012), Semaphorin 4D/Plexin-B1-mediated M-Ras GAP activity regulates actinbased dendrite remodeling through Lamellipodin. J Neurosci 32:8293-8305. Tran TS, Rubio ME, Clem RL, Johnson D, Case L, Tessier-Lavigne M, Huganir RL, Ginty DD, et al. (2009), Secreted semaphorins control spine distribution and morphogenesis in the postnatal CNS. Nature 462:1065-1069. Uchida Y, Ohshima T, Sasaki Y, Suzuki H, Yanai S, Yamashita N, Nakamura F, Takei K, et al. (2005), Semaphorin3A signalling is mediated via sequential Cdk5 and GSK3beta phosphorylation of CRMP2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease. Genes Cells 10:165179. Uchida Y, Ohshima T, Yamashita N, Ogawara M, Sasaki Y, Nakamura F, Goshima Y (2009), Semaphorin3A signaling mediated by Fyn-dependent tyrosine phosphorylation of collapsin response mediator protein 2 at tyrosine 32. J Biol Chem 284:27393-27401. Valnegri P, Puram SV, Bonni A (2015), Regulation of dendrite morphogenesis by extrinsic cues. Trends Neurosci 38:439-447. Vitagliano O, Addeo R, D'Angelo V, Indolfi C, Indolfi P, Casale F (2013), The Bcl-2/Bax and Ras/Raf/MEK/ERK signaling pathways: implications in pediatric leukemia pathogenesis and new prospects for therapeutic approaches. Expert Rev Hematol 6:587-597. Wang Q, Chiu SL, Koropouli E, Hong I, Mitchell S, Easwaran TP, Hamilton NR, Gustina AS, et al. (2017), Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-Dependent Homeostatic Scaling in Cortical Neurons. Neuron 96:1084-1098 e1087. Yamashita N, Morita A, Uchida Y, Nakamura F, Usui H, Ohshima T, Taniguchi M, Honnorat J, et al. (2007), Regulation of spine development by semaphorin3A through cyclin-dependent kinase 5 phosphorylation of collapsin response mediator protein 1. J Neurosci 27:12546-12554. Yamashita N, Ohshima T, Nakamura F, Kolattukudy P, Honnorat J, Mikoshiba K, Goshima Y (2012), Phosphorylation of CRMP2 (collapsin response mediator protein 2) is involved in proper dendritic field organization. J Neurosci 32:1360-1365. Yamashita N, Usui H, Nakamura F, Chen S, Sasaki Y, Hida T, Suto F, Taniguchi M, et al. (2014), Plexin-A4dependent retrograde semaphorin 3A signalling regulates the dendritic localization of GluA2-containing AMPA receptors. Nat Commun 5:3424. Yao L, Liu YH, Li X, Ji YH, Yang XJ, Hang XT, Ding ZM, Liu F, et al. (2016), CRMP1 Interacted with Spy1 During the Collapse of Growth Cones Induced by Sema3A and Acted on Regeneration After Sciatic Nerve Crush. Mol Neurobiol 53:879-893. Yoshida Y (2012), Semaphorin signaling in vertebrate neural circuit assembly. Front Mol Neurosci 5:71. Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, Kaibuchi K (2005), GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity. Cell 120:137-149. Yu X, Malenka RC (2003), Beta-catenin is critical for dendritic morphogenesis. Nat Neurosci 6:1169-1177. Zhang J, Tan M, Yin Y, Ren B, Jiang N, Guo G, Chen Y (2015), Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis. Neural Plast 2015:371496. Zhang J, Yin Y, Ji Z, Cai Z, Zhao B, Li J, Tan M, Guo G (2017), Endophilin2 Interacts with GluA1 to Mediate AMPA Receptor Endocytosis Induced by Oligomeric Amyloid-beta. Neural Plast 2017:8197085. Zhou Y, Gunput RA, Pasterkamp RJ (2008), Semaphorin signaling: progress made and promises ahead. Trends Biochem Sci 33:161-170.
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Figures and legends Figure 1. PlexinA3 is distributed within dendrites in cultured cerebellar granule neurons (CGNs). CGNs were transfected with a plasmid encoding GFP after 2 days in vitro (DIV2). Immunocytochemistry was performed 3 days later for GFP (green) and PlexinA3 (red). White rectangles identify regions enlarged to show detail. 1, dendrite; 2, distal axon segment; 3, terminal part of the axon. Scale bar, 10 μm.
Figure 2. PlexinA3 colocalizes with CRMP2 in dendritic shafts. Immunocytochemistry for CRMP2 (green) and PlexinA3 (red) was performed with CGNs at DIV5. Hoechst 33258 was used to stain nuclei (blue). White arrows indicate colocalization (yellow in merged image) in the dendritic shafts. Scale bar, 10 μm. The right panels show the enlarged details. Figure 3. PlexinA3 interacts with CRMP2 via its RBD region. Cultured CGNs at DIV5 were subject to immunoprecipitation assays with PlexinA3 (A) and CRMP2 (B) antibodies, and the precipitates were processed for Western blotting with indicated antibodies. (C) Bacterial recombinant glutathione S-transferase (GST)-CRMP2 pulldown assay and Western blotting with CGN lysates. (D) Schematic representation of PlexinA3 domains that were used in GST vectors. (E, F) Proteins were purified and subjected to GST pulldown assays and Western blotting with a CRMP2 antibody.
Figure 4. PlexinA3 overexpression promotes dendritic growth in CGNs. CGNs were transfected at DIV2 with vector or plasmids encoding full-length PlexinA3 (PlexinA3-FL) or PlexinA3 intracellular region (PlexinA3-cyto) together with GFP. (A) Immunocytochemistry for GFP (green) was performed 3 days later to visualize dendrites (arrowheads) and axons (arrows). Representative images are shown. Scale bar, 5 μm. Quantification of total dendritic length (B) and numbers of tips (C). Shown are means and standard errors from more than four independent experiments; *P < 0.05, **P < 0.01.
Figure 5. Knockdown of PlexinA3 inhibits dendritic growth in CGNs. (A) The efficiency 14
of PlexinA3 siRNAs was determined in HEK293 cells cotransfected with PlexinA3-V5encoding plasmids. Western blotting was performed with V5 antibody; NC, nontargeting siRNA control. (B) Relative expression levels of PlexinA3. (C) CGNs were transfected at DIV2 with siRNAs against PlexinA3 and stained for GFP (green), PlexinA3 (red) and Hoechst for nuclei (blue). Representative images are shown. Scale bar, 10 μm. (D) Percentages of GFPexpressing neurons positive for PlexinA3. Quantification of total dendritic length (E) and numbers of dendritic tips (F). Shown are means and standard errors from more than four independent experiments; *P < 0.05, **P < 0.01.
Figure 6. PlexinA3 regulates dendritic growth via CRMP2. CGNs were transfected at DIV2 with plasmids encoding PlexinA3 and/or CRMP2 (A, B), with siRNAs against PlexinA3 and/or CRMP2 (C, D), or with plasmids encoding PlexinA3 with or without siRNA against CRMP2 or encoding CRMP2 with or without siRNA against PlexinA3 (E, F). The total dendritic lengths and total numbers of dendritic tips were measured. Shown are means and standard errors from more than three independent experiments. *P or #P < 0.05; n.s., no statistical significance.
Figure 7. PlexinA3/CRMP2 signalling mediates Sema3A-regulated dendritic growth in CGNs. CGNs were transfected at DIV2 with indicated siRNAs and treated with Sema3A protein (500 ng/mL) for 3 days. (A) Representative images. Scale bar, 10 μm. Quantification of total dendritic length (B) and numbers of dendritic tips (C). Shown are means and standard errors from more than three independent experiments; *P or #P < 0.05; n.s., no statistical significance.
Figure 8. Model for PlexinA3/CRMP2-mediated dendritic growth in CGNs. Growth factors such as Sema3A activate PlexinA3; the intracellular region of PlexinA3 interacts with CRMP2 to influence cytoskeleton remodelling that leads to dendritic growth in cultured CGNs.
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Highlights
PlexinA3 colocalizes and interacts with CRMP2 via RBD region in dendrites of CGNs,
PlexinA3 is necessary and sufficient for dendritic growth of CGNs;
CRMP2 functions as downstream of PlexinA3
PlexinA3/CRMP2 signalling is responsible for Seme3A-mediated dendritic growth.
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S0306-4522(20)30094-4 https://doi.org/10.1016/j.neuroscience.2020.02.008 NSC 19516
To appear in:
Neuroscience
Received Date: Revised Date: Accepted Date:
4 September 2019 5 February 2020 6 February 2020
Please cite this article as: T. Jiang, G. Zhang, Y. Liang, Z. Cai, Z. liang, H. Lin, M. Tan, PlexinA3 interacts with CRMP2 to mediate Sema3A signalling during dendritic growth in cultured cerebellar granule neurons, Neuroscience (2020), doi: https://doi.org/10.1016/j.neuroscience.2020.02.008
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© 2020 Published by Elsevier Ltd on behalf of IBRO.
PlexinA3 interacts with CRMP2 to mediate Sema3A signalling during dendritic growth in cultured cerebellar granule neurons
Running title: PlexinA3/CRMP2-mediated dendritic growth in CGNs
Tao Jiang #, Guowei Zhang #, Yaozhong Liang #, Zhenbin Cai, Zhi liang, Hongsheng Lin *, Minghui Tan *
Department of Orthopaedics, the First Affiliated Hospital of Jinan University, Guangzhou 510630, China
# These authors contributed equally to this work.
* Corresponding to: Dr. Hongsheng Lin ([email protected]) and Dr. Minghui Tan ([email protected]), Department of Orthopaedics, the First Affiliated Hospital of Jinan University, Guangzhou 510630, China
Abbreviations: CGN, cerebellar granule neuron; CRMP2, collapsin response mediator protein 2; DIV, days in vitro; GST, glutathione S-transferase; RBD, Ras-binding domain; siRNA, short interfering RNA.
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Abstract Plexin family proteins mediate semaphorin signalling during dendritic arbour development. However, the role of PlexinA3 in the growth of dendrites of cultured cerebellar granule neurons is not known. We found that PlexinA3 colocalizes with CRMP2 (collapsin response mediator protein 2) in dendritic shafts. Immunoprecipitation and glutathione transferase pulldown assays showed that the intracellular Ras-binding domain of PlexinA3 directly interacts with CRMP2. PlexinA3 was necessary and sufficient for the growth of cerebellar granule neuron dendrites, as genetic knockdown of PlexinA3 reduced but its overexpression increased dendritic lengths and dendritic tip numbers. These increases were enhanced with CRMP2 overexpression and abolished with CRMP2 knockdown, indicating that CRMP2 is the downstream effector. Furthermore, PlexinA3/CRMP2 signalling contributed to Sema3A-controlled dendritic growth. Together, these data identify a novel PlexinA3/CRMP2 pathway in semaphorin-regulated growth of cultured cerebellar granule neuron dendrites.
Keywords CRMPs, plexins, protein interaction, dendritic development, semaphorin
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Introduction Dendrites, which integrate synaptic signals, are important for transmitting information within the nervous system. Dendritic arbours are established according to adhesive interactions, neuronal activity, and extracellular cues (Emoto K, 2012;Jan YN and Jan LY, 2003;Valnegri P et al., 2015). Sema3A, a secreted chemotrophic class 3 semaphorin, is involved in dendritic growth in new-born hippocampal (Li X et al., 2013) and cortical (Gonthier B et al., 2009) neurons and also influences dendritic patterning and synaptic maturation (Nakamura F et al., 2009;Shelly M et al., 2011;Yamashita N et al., 2014) and regulates axonal guidance, neuronal polarity, and axonal transport (Polleux F et al., 1998;Tran TS et al., 2009). Sema3A is a component of a signal transduction complex that includes ligand-binding subunit neuropilin 1 and a PlexinA family member (Takahashi T et al., 1999;Zhou Y et al., 2008). Sema3A and PlexinA regulate cytoskeleton remodelling in neurons via the Rho family GTPase Rac1 and actin-depolymerizing proteins such as cofilin. Semaphorins are involved in the development of cerebellar granule cells (CGNs) (Kerjan G et al., 2005). The plexin family proteins (including PlexinA, -B, -C, and -D) all have an extracellular semaphorin-binding domain, a transmembrane domain, and an intracellular domain involved in protein-protein interactions. The binding of Sema4D to PlexinB1 modulates M-Ras GAP activity to regulate actin-based dendrite remodelling in cortical neurons (Tasaka G et al., 2012). Sema3A and PlexinA2 signal through Fyn and Cdk5 to orient dendrites in the cerebral cortex (Sasaki Y et al., 2002). PlexinA2 modulates the migration of granule cells (Renaud J et al., 2008), and PlexinB2 controls the proliferation and differentiation of CGNs in studies of knockout mice (Friedel RH et al., 2007). PlexinA3, a receptor for Sema3A (Cheng HJ et al., 2001), is also involved in neurite extension and guidance (Negishi M et al., 2005), but its role in the development of dendrites in the cerebellum has not been explored. Collapsin response mediator protein 2 (CRMP2, also known as TOAD64, Ulip2, or DRP2) is widely expressed in the developing nervous system (Minturn JE et al., 1995) and regulates a variety of cellular functions, such as cell migration, neurite extension, and axon regeneration (Yoshimura T et al., 2005). CRMP2 interacts with presynaptic voltage-gated calcium channels to modulate neurotransmitter release (Brittain JM et al., 2009) but also controls the growth of axons of hippocampal neurons (Inagaki N et al., 2001) and regulates neurite growth and 3
polarity by modulating cytoskeletal dynamics (Fukata Y et al., 2002). CRMP2 is important for organizing the dendritic field and coordinates with CRMP1 to regulate dendrite development (Yamashita N et al., 2012). CRMP2 is inactivated by phosphorylation via Cdk5 and GSK-3 (Cole AR et al., 2004), and CRMP2 phosphorylation regulates the bifurcation of hippocampal CA1 pyramidal neuron dendrites (Niisato E et al., 2013). The CRMP family proteins have been implicated to facilitate semaphorin-mediated morphological changes in nonneuronal cells (Deo RC et al., 2004). However, the detailed scenario in neuronal cells needs to be elucidated. We previously found that CRMP2 is distributed in the dendrites of cultured CGNs, but not in the axons, and that it signals via GSK-3 during activity-induced dendritic growth (Tan M et al., 2013). In the present study, we demonstrate that CRMP2 colocalizes and interacts with PlexinA3, which is also enriched in the dendrites of cultured CGNs and is necessary and sufficient for their growth. We show that CRMP2 functions downstream of PlexinA3 to mediate Sema3A-regulated dendritic growth in CGNs.
Experimental procedures Cell culture CGNs from 7-day-old Sprague-Dawley rat pups were prepared as previously described (Tan M, et al., 2013) to high purity (Contestabile A, 2002). All procedures involving animals were in accordance with the Guide for the Care and Use of Laboratory Animals of the NIH and approved by the Jinan University Institutional Animal Care and Use Committee. Briefly, neurons were isolated from dissected cerebella by using trypsin (Thermo Fisher Scientific, Rockford, IL), treated with DNase (Gibco BRL, Gaithersburg, MD), and cultured in BMEM containing 10% foetal bovine serum (Gibco BRL) and 25 mM KCl (Sigma-Aldrich, St. Louis, MO) at a density of 1.5 × 106 cells/ml. For immunocytochemistry analyses, neurons were plated on coverslips (Thermo Fisher Scientific). HEK293 cells were cultured in DMEM/F12 (Gibco BRL) supplemented with 10% foetal bovine serum
Constructs and transfection The full-length PlexinA3 gene was cloned from cDNA of rat CGNs, and truncated PlexinA3 fragments were subcloned into pEGFP-C1 plasmids (Clontech, Mountain View, CA). All 4
constructs were verified by sequencing. Two PlexinA3 small interfering RNA (siRNA) fragments, siPlexinA3-a and -b, were designed to target rat PlexinA3 (NM_001107581) with sequences
5-GUGUACAAGGGUAUUCCAUAC-3
and
5-
CACAAUUCGUGUUUGACAUCC-3, respectively. A nontargeting siRNA was used as a negative control for all siRNA transfections. CRMP2-encoding plasmids and siRNA fragments against rat CRMP2 were previously described (Tan M, et al., 2013). siRNAs were synthesized by GenePharma (Shanghai, China), and their efficacy and specificity were determined by transfection in HEK293 cells as previously described (Zhang J et al., 2017). CGNs were transfected via the calcium phosphate method within DIV2 (Zhang J et al., 2015) and cultured for another 3 days. Immunocytochemistry analyses were performed on CGNs after 5 days in vitro (DIV5).
Western blotting Western blotting analyses of cell lysates were as previously described (Tan M et al., 2013). Briefly, samples were separated by SDS-PAGE, transferred to nitrocellulose membranes (Invitrogen, Carlsbad, CA), and blocked for 1 h at room temperature with 5% skim milk in Tris-buffered saline. Membranes were probed overnight at 4°C with rabbit anti-CRMP2 (catalogue no. 9393; Cell Signal Technology, Danvers, MA), goat anti-PlexinA3 (catalogue no. AF4075; R&D systems, Minneapolis, MN), and anti-tubulin (Sigma-Aldrich) primary antibodies.
Horseradish
peroxidase-conjugated
secondary
antibodies
(Jackson
ImmunoResearch, West Grove, PA) were then applied and visualized via enhanced chemiluminescence (ECL kit; Pierce, Rockford, IL).
Fluorescence immunocytochemistry CGNs were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.1% Triton X-100 in Tris-buffered saline, blocked with 3% donkey serum and immunostaining was carried out as described before (Gong X et al., 2016) using anti-CRMP2, anti-PlexinA3, and anti-GFP (Abcam, Cambridge, UK) primary antibodies diluted 1:1,000 and Alexa Fluor 488 and 555 secondary antibodies (Molecular Probes, Leiden, the Netherlands) diluted 1:2,000. The coverslips were mounted using Fluro-Gel II with DAPI (Electron Microscopy Sciences, 5
Hatfield, PA) and imaged with an LSM 780 confocal microscope (Carl Zeiss, Oberkochen, Germany) with the pinhole set to 1 airy unit.
Glutathione S-transferase (GST) pulldown and immunoprecipitation GST pulldown assays were performed as described previously (Zhang J, et al., 2017). The expression of GST-tagged full-length and truncated PlexinA3 and CRMP2 in Top10F Escherichia coli cells (Invitrogen) was induced with 0.1 mM isopropyl-1-thio-β-Dgalactopyranoside (Roche Applied Science, Indianapolis, IN), and the proteins were extracted and purified by using glutathione agarose beads (Pierce). Cell lysates from CGNs were incubated with GST fusion proteins at 4°C overnight in a buffer containing 100 mM NaCl, 20 mM Tris-HCl, 5% glycerol (pH 7.0), 1% Triton X-100, and a cocktail of protease inhibitors consisting of 1 μM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.1 mg/ml benzamidine (Merck, Kenilworth, NJ). The pulldown sediments were separated and analysed by Western blotting. For immunoprecipitation, CGN lysates were prepared with buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 2.5 mM pyrophosphate, 1 mM glycerol phosphate, and a protease inhibitor mixture and incubated with 2 g ant-CRMP2 or anti-PlexinA3 antibodies. Immunoprecipitation was performed by incubating the mixture with 60 l precleared protein G/A agarose for 16 h at 4°C with continuous inversion. The immunocomplexes were pelleted by centrifugation, washed, and analysed by Western blotting.
CGN morphometry CGN morphometry was performed as described previously (Tan M, et al., 2013). Briefly, GFPexpressing CGNs were immunostained with antibodies against GFP, MAP2 (to label dendrites), and Tau-1 (to label axons) and imaged in a blinded manner with an Axio Observer Z1 (Carl Zeiss). At least 60 neurons for each experimental group were analysed with Image Pro Plus 6 software (Media Cybernetics, Silver Spring, MD).
Statistical analysis 6
Experiments were performed at least three times. Groups were compared via statistical analyses of variance with Bonferroni post hoc tests (for multiple comparisons) and Student’s t tests (for single comparisons). A P value of < 0.05 was considered statistically significant.
Results PlexinA3 colocalizes with CRMP2 in dendrites of cultured CGNs The distribution of PlexinA3 in CGNs was observed via immunofluorescence. PlexinA3 immunoreactivity was strong in dendritic shafts but weak in axons (Fig. 1). This pattern was consistent with the distribution of CRMP2 we observed previously in CGNs (Tan M, et al., 2013). Further examinations with confocal microscopy revealed that CRMP2 and PlexinA3 colocalized in dendrite shafts (Fig. 2). These data suggest that CRMP2 and PlexinA3 may interact in cultured CGNs.
PlexinA3 interacts directly with CRMP2 To determine whether CRMP2 and PlexinA3 interact, we performed coimmunoprecipitation assays. PlexinA3 was immunoprecipitated from cell lysates of developing CGNs (DIV5). Western blotting revealed the presence of CRMP2 in these immunoprecipitates (Fig. 3A). Conversely, PlexinA3 was detected in proteins immunoprecipitated with an antibody against CRMP2 (Fig. 3B), suggesting a direct interaction between CRMP2 and PlexinA3. This interaction was verified with a GST pulldown assay, as PlexinA3 was detected in the GSTCRMP2 pulldowns but not in the GST control (Fig. 3C). PlexinA3 is a large transmembrane protein (~220 kDa) containing extracellular and transmembrane domains and an intracellular region comprising an N-terminal segment, a C1 domain, Ras-binding domain (RBD), and C2 domain (Fig. 3D) (He H et al., 2009). As CRMP2 is a cytosolic protein, we targeted the intracellular domain of PlexinA3 to identify the region responsible for the interaction with CRMP2. To this end, we generated PlexinA3 fragments comprising the entire intracellular region, the N-terminal, C1, and RBD regions only, the Nterminal and C1 regions only and the RBD region only (Fig. 3E and F). A GST pulldown assay revealed that CRMP2 was present only with fragments containing the RBD, suggesting that this region of PlexinA3 is responsible for binding CRMP2. 7
PlexinA3 is sufficient and necessary for dendritic growth To examine the function of PlexinA3 in dendrites, we overexpressed the full-length protein or the intracellular region in cultured CGNs. Overexpression of full-length PlexinA3 promoted dendritic growth, significantly increasing the total dendritic length, whereas overexpression of the intracellular domain suppressed dendritic growth (Fig. 4). The inhibition of growth observed with expression of the intracellular domain may reflect a dominant negative effect on interactions with cytosolic proteins, such as CRMP2. In addition, we examined the effect of PlexinA3 knockdown. The knockdown efficiency of two siRNAs against PlexinA3 was determined in HEK293 cells by Western blotting (Fig. 5a and b) and in CGNs by immunostaining (Fig. 5c and d). Notably, knockdown of PlexinA3 reduced the overall dendritic length (Fig. 5e) and number of dendritic tips (Fig. 5f) of CGNs. Together, these results suggest that PlexinA3 is both sufficient and necessary for the growth of CGN dendrites.
PlexinA3 regulates dendritic growth via downstream CRMP2 We hypothesized that PlexinA3 regulates dendritic growth in CGNs via its direct interactions with CRMP2. To test this, we transfected CGNs with plasmids encoding PlexinA3 and/or CRMP2. Overexpression of PlexinA3 or CRMP2 promoted dendritic growth, with further increases in dendritic length and tip numbers when both were overexpressed (Fig. 6a and b). By contrast, CGNs transfected with siRNA against CRMP2 or PlexinA3 had impaired dendritic growth; however, an additive effect was not observed when both proteins were knocked down simultaneously (Fig. 6c and d). Moreover, knockdown of CRMP2 abolished the enhanced growth induced by PlexinA3 overexpression. Importantly, knockdown of PlexinA3 had no effect on the dendritic growth induced by CRMP2 overexpression (Fig. 6e and f). These data indicate that CRMP2 functions downstream of PlexinA3 to induce dendritic growth in CGNs.
PlexinA3/CRMP2 signalling mediates Sema3A-regulated dendritic growth We hypothesized that PlexinA3/CRMP2 signalling contributes to Sema3A-mediated dendritic growth in CGNs. The test this, we treated CGNs expressing GFP and/or siRNAs against CRMP2 or PlexinA3 with Sema3A recombinant protein. Immunocytochemistry revealed that 8
Sema3A-induced dendritic growth was significantly suppressed by knockdown of CRMP2 and/or PlexinA3 (Fig. 7). These data indicate that the PlexinA3/CRMP2 pathway is involved in Sema3A-regulated dendritic growth in CGNs.
Discussion Semaphorins, as well as brain-derived neurotrophic factor and Wnt signalling, are critical for dendritic development (McAllister AK et al., 1997;Polleux F et al., 2000;Yu X and Malenka RC, 2003). Sema3A recognizes neuropilin 1/PlexinA complexes to regulate dendritic growth, neuronal polarization, and synaptic formation and maturation during development (Pasterkamp RJ, 2012;Yoshida Y, 2012). Sema3A signals through Fyn and Cdk5 for dendrite orientation and growth cone collapse (Morita A et al., 2006;Sasaki Y, et al., 2002), and the phosphorylation of focal adhesion kinase by Cdk5 influences the complexity of neuronal dendrites (Vitagliano O et al., 2013). Furthermore, Sema3A signalling through Fyn and Src kinases influences the arborization of the basal dendrites of cortical neurons via protein tyrosine phosphatase δ (Nakamura F et al., 2017), and signalling through Farp1 mediates activity-dependent arborization hippocampal neuron dendrites of (Cheadle L and Biederer T, 2014). In this study, we show that Sema3A signals through PlexinA3 and CRMP2 to regulate dendritic growth in CGNs. Additionally, we demonstrate that the interaction of PlexinA3 and CRMP2 within dendritic shafts is necessary and sufficient for dendritic growth (Fig. 8). We previously showed that CRMP2 in CGN dendrites mediates activity-dependent dendritic outgrowth (Tan M, et al., 2013) and that CRMP4 in hippocampal neurons interacts with the actin cytoskeleton to mediate dendritic growth and maturation (Cha C et al., 2016). Here, we found that the CRMP2 colocalizes with PlexinA3 in the dendritic shaft and directly binds its RBD region. PlexinA3 is a large transmembrane protein whose function relies on protein interactions. For example, neuropilin 2/PlexinA3 receptors associate with GluA1 to mediate Sema3F-dependent homeostatic scaling in cortical neurons (Wang Q et al., 2017). A recent study showed that PlexinA2 complexes with CRMP2 when Nogo-A binds to Nogo receptor 1 to regulate corticospinal axon sprouting after trauma (Sekine Y et al., 2019). These studies and our own provide evidence that Plexin and CRMP proteins interact under very different physiological conditions. 9
CRMP proteins mediate Sema3A signalling; for example, Sema3A-regulated actin cytoskeleton remodelling is mediated by the interaction between CRMP1 and the amino- and carboxy-terminal domains of filamin-A (Nakamura F et al., 2014). Interactions with Spy1 disrupt the association of CRMP1 with actin during Sema3A-induced growth cone collapse and regeneration after sciatic nerve crush (Yao L et al., 2016). Yamane and colleagues (2017) reported that CRMP1 couples with Nav1.7 to mediate retrograde signalling of Sema3A in axons. Many such Sema3A-induced interactions are regulated by phosphorylation, such as the phosphorylation of CRMP2 by Cdk5 and GSK-3 during axon guidance (Uchida Y et al., 2005), phosphorylation of CRMP1 by Cdk5 during dendritic spine development in cultured cortical neurons (Yamashita N et al., 2007), and phosphorylation at Tyr32 of CRMP2 by Fyn in dorsal root ganglion neurons (Uchida Y et al., 2009). As CRMPs promote cytoskeleton assembly by binding to tubulin and actin (Fukata Y, et al., 2002;Tan M et al., 2015), phosphorylation interferes with this binding, leading to the dissociation of microtubules and actin filaments (Arimura N et al., 2005;Namekata K et al., 2012). In summary, we showed that CRMP2 colocalizes with PlexinA3 in the dendritic shafts of cultured CGNs. We demonstrated that PlexinA3 and CRMP2 interact directly and mediate Sema3A-regulated dendritic growth in cultured CGNs. Further investigation is needed to identify the corresponding phosphorylation status of CRMP2 and the regulation of upstream kinases during dendritic growth.
Acknowledgments There is no conflict of interest to declare. This work was supported by the Pearl River S&T Nova Program of Guangzhou, China [no. 201806010011], and the National Natural Science Foundation of China [no. 31300885 and 81771331].
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Figures and legends Figure 1. PlexinA3 is distributed within dendrites in cultured cerebellar granule neurons (CGNs). CGNs were transfected with a plasmid encoding GFP after 2 days in vitro (DIV2). Immunocytochemistry was performed 3 days later for GFP (green) and PlexinA3 (red). White rectangles identify regions enlarged to show detail. 1, dendrite; 2, distal axon segment; 3, terminal part of the axon. Scale bar, 10 μm.
Figure 2. PlexinA3 colocalizes with CRMP2 in dendritic shafts. Immunocytochemistry for CRMP2 (green) and PlexinA3 (red) was performed with CGNs at DIV5. Hoechst 33258 was used to stain nuclei (blue). White arrows indicate colocalization (yellow in merged image) in the dendritic shafts. Scale bar, 10 μm. The right panels show the enlarged details. Figure 3. PlexinA3 interacts with CRMP2 via its RBD region. Cultured CGNs at DIV5 were subject to immunoprecipitation assays with PlexinA3 (A) and CRMP2 (B) antibodies, and the precipitates were processed for Western blotting with indicated antibodies. (C) Bacterial recombinant glutathione S-transferase (GST)-CRMP2 pulldown assay and Western blotting with CGN lysates. (D) Schematic representation of PlexinA3 domains that were used in GST vectors. (E, F) Proteins were purified and subjected to GST pulldown assays and Western blotting with a CRMP2 antibody.
Figure 4. PlexinA3 overexpression promotes dendritic growth in CGNs. CGNs were transfected at DIV2 with vector or plasmids encoding full-length PlexinA3 (PlexinA3-FL) or PlexinA3 intracellular region (PlexinA3-cyto) together with GFP. (A) Immunocytochemistry for GFP (green) was performed 3 days later to visualize dendrites (arrowheads) and axons (arrows). Representative images are shown. Scale bar, 5 μm. Quantification of total dendritic length (B) and numbers of tips (C). Shown are means and standard errors from more than four independent experiments; *P < 0.05, **P < 0.01.
Figure 5. Knockdown of PlexinA3 inhibits dendritic growth in CGNs. (A) The efficiency 14
of PlexinA3 siRNAs was determined in HEK293 cells cotransfected with PlexinA3-V5encoding plasmids. Western blotting was performed with V5 antibody; NC, nontargeting siRNA control. (B) Relative expression levels of PlexinA3. (C) CGNs were transfected at DIV2 with siRNAs against PlexinA3 and stained for GFP (green), PlexinA3 (red) and Hoechst for nuclei (blue). Representative images are shown. Scale bar, 10 μm. (D) Percentages of GFPexpressing neurons positive for PlexinA3. Quantification of total dendritic length (E) and numbers of dendritic tips (F). Shown are means and standard errors from more than four independent experiments; *P < 0.05, **P < 0.01.
Figure 6. PlexinA3 regulates dendritic growth via CRMP2. CGNs were transfected at DIV2 with plasmids encoding PlexinA3 and/or CRMP2 (A, B), with siRNAs against PlexinA3 and/or CRMP2 (C, D), or with plasmids encoding PlexinA3 with or without siRNA against CRMP2 or encoding CRMP2 with or without siRNA against PlexinA3 (E, F). The total dendritic lengths and total numbers of dendritic tips were measured. Shown are means and standard errors from more than three independent experiments. *P or #P < 0.05; n.s., no statistical significance.
Figure 7. PlexinA3/CRMP2 signalling mediates Sema3A-regulated dendritic growth in CGNs. CGNs were transfected at DIV2 with indicated siRNAs and treated with Sema3A protein (500 ng/mL) for 3 days. (A) Representative images. Scale bar, 10 μm. Quantification of total dendritic length (B) and numbers of dendritic tips (C). Shown are means and standard errors from more than three independent experiments; *P or #P < 0.05; n.s., no statistical significance.
Figure 8. Model for PlexinA3/CRMP2-mediated dendritic growth in CGNs. Growth factors such as Sema3A activate PlexinA3; the intracellular region of PlexinA3 interacts with CRMP2 to influence cytoskeleton remodelling that leads to dendritic growth in cultured CGNs.
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Highlights
PlexinA3 colocalizes and interacts with CRMP2 via RBD region in dendrites of CGNs,
PlexinA3 is necessary and sufficient for dendritic growth of CGNs;
CRMP2 functions as downstream of PlexinA3
PlexinA3/CRMP2 signalling is responsible for Seme3A-mediated dendritic growth.
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