An unexpected role of neurite outgrowth inhibitor A as regulator of tooth enamel formation

Abstract

Neurite outgrowth inhibitor A (Nogo-A) is a major player in neural development and regeneration and the target of clinical trials aiming at promoting the regeneration of the central nervous system upon traumatic and ischemic injury. In this work, we investigated the functions of Nogo-A during tooth development to determine its role in dental physiology and pathology. Using immunohistochemistry and in situ hybridization techniques, we showed that Nogo-A is highly expressed in the developing mouse teeth and, most specifically, in the ameloblasts that are responsible for the formation of enamel. Using both Nogo-A knockout and K14-Cre;Nogo-A fl/fl transgenic mice, we showed that Nogo-A deletion in the dental epithelium leads to the formation of defective enamel. This phenotype is associated with overexpression of a set of specific genes involved in ameloblast differentiation and enamel matrix production, such as amelogenin, ameloblastin and enamelin. By characterising the interactome of Nogo-A in the dental epithelium of wild-type and mutant animals, we found that Nogo-A directly interacts with molecules important for regulating gene expression, and its deletion disturbs their cellular localisation. Furthermore, we demonstrated that inhibition of the intracellular, but not cell-surface, Nogo-A is responsible for gene expression modulation in ameloblasts. Taken together, these results reveal an unexpected function for Nogo-A in tooth enamel formation by regulating gene expression and cytodifferentiation events.

References

[1.] OertleT, et al. . Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions. J. Neurosci., 2003, 23: 5393-5406

[2.] VoeltzGK, PrinzWA, ShibataY, RistJM, RapoportTA. A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell, 2006, 124: 573-586

[3.] FournierAE, GrandPreT, StrittmatterSM. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature, 2001, 409: 341-346

[4.] SchwabME, StrittmatterSM. Nogo limits neural plasticity and recovery from injury. Curr. Opin. Neurobiol., 2014, 27: 53-60

[5.] RamoO, et al. . NOGO-A/RTN4A and NOGO-B/RTN4B are simultaneously expressed in epithelial, fibroblast and neuronal cells and maintain ER morphology. Sci. Rep., 2016, 6

[6.] AkbikFV, BhagatSM, PatelPR, CaffertyWB, StrittmatterSM. Anatomical plasticity of adult brain is titrated by Nogo Receptor 1. Neuron, 2013, 77: 859-866

[7.] KempfA, et al. . The sphingolipid receptor S1PR2 is a receptor for Nogo-a repressing synaptic plasticity. PLoS Biol., 2014, 12: e1001763

[8.] KempfA, et al. . Control of Cell Shape, Neurite Outgrowth, and Migration by a Nogo-A/HSPG Interaction. Dev. Cell, 2017, 43: 24-34.e25

[9.] McDonaldCL, et al. . Nogo receptor is involved in the adhesion of dendritic cells to myelin. J. Neuroinflammation, 2011, 8

[10.] IobbiC, KorteM, ZagrebelskyM. Nogo-66 Restricts Synaptic Strengthening via Lingo1 and the ROCK2-Cofilin Pathway to Control Actin Dynamics. Cereb. Cortex, 2017, 27: 2779-2792

[11.] TheotokisP, GrigoriadisN. p75NTR and TROY: Uncharted Roles of Nogo Receptor Complex in Experimental Autoimmune Encephalomyelitis. Mol. Neurobiol., 2018, 55: 6329-6336

[12.] SahaN, KolevM, NikolovDB. Structural features of the Nogo receptor signaling complexes at the neuron/myelin interface. Neurosci. Res, 2014, 87: 1-7

[13.] PengWS, et al. . Distribution of paired immunoglobulin-like receptor B in the nervous system related to regeneration difficulties after unilateral lumbar spinal cord injury. Neural Regen. Res, 2015, 10: 1139-1146,

[14.] Kim, M. J., Kang, J. H., Theotokis, P., Grigoriadis, N. & Petratos, S. Can We Design a Nogo Receptor-Dependent Cellular Therapy to Target MS? Cells8, https://doi.org/10.3390/cells8010001 (2018).

[15.] KempfA, SchwabME. Nogo-A represses anatomical and synaptic plasticity in the central nervous system. Physiol. (Bethesda), 2013, 28: 151-163

[16.] SchmandkeA, SchmandkeA, SchwabME. Nogo-A: Multiple Roles in CNS Development, Maintenance, and Disease. Neuroscientist, 2014, 20: 372-386

[17.] PernetV, SchwabME. The role of Nogo-A in axonal plasticity, regrowth and repair. Cell Tissue Res, 2012, 349: 97-104

[18.] HuntD, CoffinRS, PrinjhaRK, CampbellG, AndersonPN. Nogo-A expression in the intact and injured nervous system. Mol. Cell Neurosci., 2003, 24: 1083-1102

[19.] WahlAS, et al. . Neuronal repair. Asynchronous therapy restores motor control by rewiring of the rat corticospinal tract after stroke. Science, 2014, 344: 1250-1255

[20.] FreundP, et al. . Nogo-A-specific antibody treatment enhances sprouting and functional recovery after cervical lesion in adult primates. Nat. Med., 2006, 12: 790-792

[21.] WalchliT, et al. . Nogo-A is a negative regulator of CNS angiogenesis. Proc. Natl Acad. Sci. USA, 2013, 110: E1943-E1952

[22.] WalchliT, et al. . Nogo-A regulates vascular network architecture in the postnatal brain. J. Cereb. Blood Flow. Metab., 2017, 37: 614-631

[23.] RustR, et al. . Nogo-A targeted therapy promotes vascular repair and functional recovery following stroke. Proc. Natl Acad. Sci. USA, 2019, 116: 14270-14279

[24.] ZhangN, CuiY, LiY, MiY. A Novel Role of Nogo Proteins: Regulating Macrophages in Inflammatory Disease. Cell Mol. Neurobiol., 2022, 42: 2439-2448

[25.] Pirenne, L. et al. Trigeminal ganglion and tooth innervation modifications following genetic and pharmacological Nogo-A inhibition. bioRxiv, (2024).

[26.] Lai, C. F. et al. Nogo-A Regulates the Fate of Human Dental Pulp Stem Cells toward Osteogenic, Adipogenic, and Neurogenic Differentiation. Cells11, https://doi.org/10.3390/cells11213415 (2022).

[27.] MitsiadisTA, GrafD. Cell fate determination during tooth development and regeneration. Birth Defects Res C. Embryo Today, 2009, 87: 199-211

[28.] BartlettJD. Dental enamel development: proteinases and their enamel matrix substrates. ISRN Dent., 2013, 2013

[29.] Pagella, P., Porcheri, C. & Mitsiadis, T. A. Exploiting teeth as a model to study basic features of signaling pathways. Biochem Soc Trans. (2020).

[30.] SimonenM, et al. . Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron, 2003, 38: 201-211

[31.] HuberAB, WeinmannO, BrosamleC, OertleT, SchwabME. Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions. J. Neurosci., 2002, 22: 3553-3567

[32.] DassuleHR, LewisP, BeiM, MaasR, McMahonAP. Sonic hedgehog regulates growth and morphogenesis of the tooth. Development, 2000, 127: 4775-4785

[33.] BartlettJD, YamakoshiY, SimmerJP, NanciA, SmithCE. MMP20 cleaves E-cadherin and influences ameloblast development. Cells Tissues Organs, 2011, 194: 222-226

[34.] SchwabM. Functions of Nogo proteins and their receptors in the nervous system. Nat. Rev. Neurosci., 2010, 11: 799-811

[35.] SarkarJ, et al. . Comparison of two mouse ameloblast-like cell lines for enamel-specific gene expression. Front Physiol., 2014, 5: 277

[36.] ZouJ, GuoY, GuettoucheT, SmithDF, VoellmyR. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell, 1998, 94: 471-480

[37.] Al HalabiahH, DelezoideAL, CardonaA, MoalicJM, SimonneauM. Expression pattern of NOGO and NgR genes during human development. Gene Expr. Patterns, 2005, 5: 561-568

[38.] Cantu, C. et al. A cytoplasmic role of Wnt/beta-catenin transcriptional cofactors Bcl9, Bcl9l, and Pygopus in tooth enamel formation. Sci. Signal10, https://doi.org/10.1126/scisignal.aah4598 (2017).

[39.] GibsonCW, et al. . Amelogenin-deficient mice display an amelogenesis imperfecta phenotype. J. Biol. Chem., 2001, 276: 31871-31875

[40.] RajparMH, HarleyK, LaingC, DaviesRM, DixonMJ. Mutation of the gene encoding the enamel-specific protein, enamelin, causes autosomal-dominant amelogenesis imperfecta. Hum. Mol. Genet, 2001, 10: 1673-1677

[41.] WrightJT, et al. . Human and mouse enamel phenotypes resulting from mutation or altered expression of AMEL, ENAM, MMP20 and KLK4. Cells Tissues Organs, 2009, 189: 224-229

[42.] ChunYP, et al. . Overexpression of ameloblastin in secretory ameloblasts results in demarcated, hypomineralized opacities in enamel. Front Physiol., 2023, 14

[43.] MiS, et al. . LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat. Neurosci., 2004, 7: 221-228

[44.] KiselevaE, MorozovaKN, VoeltzGK, AllenTD, GoldbergMW. Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth. J. Struct. Biol., 2007, 160: 224-235

[45.] HatakeyamaJ, et al. . The ER structural protein Rtn4A stabilizes and enhances signaling through the receptor tyrosine kinase ErbB3. Sci. Signal, 2016, 9: ra65

[46.] MutoT, et al. . Differential expression of syndecan isoforms during mouse incisor amelogenesis. J. Med Invest, 2007, 54: 331-339

[47.] Mahler ConvenorM, et al. . FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Lab Anim., 2014, 48: 178-192

[48.] VajdaF, et al. . Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve. Cell Death Differ., 2015, 22: 323-335

[49.] MitsiadisTA, HirsingerE, LendahlU, GoridisC. Delta-notch signaling in odontogenesis: correlation with cytodifferentiation and evidence for feedback regulation. Dev. Biol., 1998, 204: 420-431

[50.] HatakeyamaM, et al. . SUSHI: an exquisite recipe for fully documented, reproducible and reusable NGS data analysis. BMC Bioinforma., 2016, 17

[51.] ChenS, ZhouY, ChenY, GuJ. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34: i884-i890

[52.] DobinA, et al. . STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 2013, 29: 15-21

[53.] ZerbinoDR, et al. . Ensembl 2018. Nucleic Acids Res., 2018, 46: D754-D761

[54.] LiaoY, SmythGK, ShiW. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res., 2013, 41: e108

[55.] RobinsonMD, McCarthyDJ, SmythG. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 2010, 26: 139-140

[56.] YuG, WangLG, HanY, HeQY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS, 2012, 16: 284-287

[57.] GoldsteinLD, et al. . Prediction and Quantification of Splice Events from RNA-Seq Data. PLoS One, 2016, 11: e0156132

[58.] WisniewskiJR, ZougmanA, NagarajN, MannM. Universal sample preparation method for proteome analysis. Nat. Methods, 2009, 6: 359-362

[59.] FascellaroG, et al. . Comprehensive Proteomic Analysis of Nitrogen-Starved Mycobacterium smegmatis Deltapup Reveals the Impact of Pupylation on Nitrogen Stress Response. J. Proteome Res., 2016, 15: 2812-2825

[60.] Barkow-OesterreicherS, TurkerC, PanseC. FCC - An automated rule-based processing tool for life science data. Source Code Biol. Med., 2013, 8

[61.] KellerA, NesvizhskiiAI, KolkerE, AebersoldR. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem., 2002, 74: 5383-5392

[62.] NesvizhskiiAI, KellerA, KolkerE, AebersoldR. A statistical model for identifying proteins by tandem mass spectrometry. Anal. Chem., 2003, 75: 4646-4658

[63.] The Gene Ontology, C.. The Gene Ontology Resource: 20 years and still GOing strong. Nucleic Acids Res., 2019, 47: D330-D338

[64.] AshburnerM, et al. . Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet, 2000, 25: 25-29

[65.] CantuC, et al. . The Pygo2-H3K4me2/3 interaction is dispensable for mouse development and Wnt signaling-dependent transcription. Development, 2013, 140: 2377-2386

[66.] HughesCS, et al. . Ultrasensitive proteome analysis using paramagnetic bead technology. Mol. Syst. Biol., 2014, 10: 757

[67.] LeutertM, Rodriguez-MiasRA, FukudaNK, VillenJ. R2-P2 rapid-robotic phosphoproteomics enables multidimensional cell signaling studies. Mol. Syst. Biol., 2019, 15

[68.] Turker, C. et al. B-Fabric: The Swiss Army Knife for Life Sciences. EDBT ‘10: Proceedings of the 13th International Conference on Extending Database Technology, https://doi.org/10.1145/1739041.1739135 (2010).

[69.] CoxJ, MannM. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol., 2008, 26: 1367-1372

[70.] RitchieME, et al. . limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res., 2015, 43: e47

[71.] VizcainoJA, et al. . 2016 update of the PRIDE database and its related tools. Nucleic Acids Res., 2016, 44: 11033

[72.] GrantCE, BaileyTL, NobleWS. FIMO: scanning for occurrences of a given motif. Bioinformatics, 2011, 27: 1017-1018

[73.] DailyK, PatelVR, RigorP, XieX, BaldiP. MotifMap: integrative genome-wide maps of regulatory motif sites for model species. BMC Bioinforma., 2011, 12

[74.] SchindelinJ, et al. . Fiji: an open-source platform for biological-image analysis. Nat. Methods, 2012, 9: 676-682

Funding

Swiss National Science Foundation (SNSF) Swiss Dental Association (SSO)