Enamel is the hard tissue on the outermost layer of teeth. It is mainly composed of hydroxyapatite crystals and is highly mineralized. Its formation is a complex process involving the secretion of related proteins, mineralization, and the degradation of proteins by proteases. Enamel is crucial for oral health. Defects in enamel increase the risk of oral diseases such as dental caries. Therefore, an in-depth study of the formation mechanism of enamel is of great significance.

Neurite outgrowth inhibitor A (Nogo-A), encoded by the Reticulon-4 (RTN4) gene, has multiple isoforms. It is mainly located in the endoplasmic reticulum membrane and can regulate ER curvature. Some are also on the cell surface and function by interacting with multiple receptor complexes. Nogo-A is widely known for its role in neural development and regeneration. However, its function in enamel formation needs further exploration.

Recently, Martin E. Schwab and Thimios A. Mitsiadis's team from Switzerland published an article titled "Neurite Outgrowth Inhibitor A in Tooth Enamel Formation: Unraveling the Mechanism and Potential Role" in the International Journal of Oral Science. This study investigated the role of Nogo-A in tooth development and discovered its crucial role in enamel formation, providing a new perspective for understanding the mechanism of enamel formation.

Materials and Methods

The research adheres to Swiss laws and regulations for animal handling. Mice are housed in specific pathogen-free conditions with controlled environments. Nogo-A KO and other relevant mouse strains are utilized. Immunohistochemistry, immunofluorescent staining, and in situ hybridization techniques are employed for histological analyses. Scanning electron microscopy is used to analyze enamel structure. Transmission electron microscopy is employed for ameloblast analysis after sample decalcification and specific post-fixation and embedding steps. RNA sequencing is carried out on dental epithelium samples from newborn pups. Immunoprecipitation and mass spectrometry are used to characterize the Nogo-A interactome. Nuclear/cytoplasm differential mass spectrometry is performed on dental epithelium samples from specific pups. LS8 cell culture and analysis are conducted with specific culture conditions, antibody-mediated inhibition, and shRNA-mediated knockdown experiments.

Results

They first observed the expression of Nogo-A in several developing orofacial organs, including teeth. At E13.5, Nogo-A was low in molars but strong in trigeminal axons. From E16.5, it's detectable in molar epithelium and mesenchyme. At E18.5, it's highly expressed in pre-odontoblasts and pre-ameloblasts. Postnatally, it persisted in odontoblasts and ameloblasts, while it was low in other dental epithelium cells. At postnatal day 6, it was high in ameloblasts then disappeared (Fig. 1a-d). A similar expression pattern was observed in the incisors (Fig. 1h-l). Overall, Nogo-A has specific expression patterns in teeth at different stages.

Fig. 1 Nogo-A expression during tooth development.

Next, they investigated the role of Nogo-A in tooth development by using Nogo-A KO mice and K14; Nogo-A mice (where the K14 drivers led to the selective deletion of Nogo-A in the dental epithelium). In the Nogo-A KO mice, the enamel structure was altered and the amount of iron in the enamel was reduced (Fig. 2a-i). In K14; Nogo-A mice, Nogo-A deficiency in tooth epithelial cells led to enamel defects, and histologic analysis showed impaired intercellular adhesion of enamel-forming cells (Fig. 2j-s). This suggests that Nogo-A is important for enamel formation and tooth epithelial function.

Fig. 2 Nogo-A deletion leads to the formation of defective enamel.

To investigate the function of Nogo-A in the dental epithelium, they first analyzed the expression of Nogo-A in deeper detail. Double immunostaining against Nogo-A and E-Cadherin, a marker of the ameloblasts membrane, was performed, and the results suggested that Nogo-A in ameloblasts was localized at the ER and the border of the ER with the nuclear membrane (Fig. 3a-e).

Then, by isolating dental epithelium and performing immunoprecipitation and mass spectrometry analysis, 163 proteins were identified as Nogo-A putative interactors, divided into four main functional hubs (Fig. 3f). Nuclear vs cytoplasm fractionation and proteomics analysis on ameloblasts from K14; Nogo-A pups and controls showed alterations in protein distribution. Some proteins were enriched in the cytoplasm and depleted in the nucleus or vice versa in Nogo-A-deleted ameloblasts (Fig. 3g, h). These findings suggest that deletion of Nogo-A affects protein localization and transport between cytoplasm and nucleus.

Fig. 3 Nogo-A interacts with proteins involved in gene regulation, and Nogo-A deletion affects protein nuclear/cytoplasmic localization.

Based on the above results, they investigated whether Nogo-A deletion could induce relevant gene expression alterations. The dental epithelium from the lower incisors of K14; Nogo-A PN0 pups and control littermate were isolated and their transcriptome was analyzed by RNA sequencing (Fig. 4a). It was found that deletion of Nogo-A led to a significant deregulation of a high number of genes, including downregulation of genes for cell adhesion and some Wnt pathway members, upregulation of genes for ion transport and ameloblast differentiation markers (Fig. 4b-d). These results indicate that Nogo-A regulates the expression of genes encoding proteins required for ameloblast differentiation and enamel formation.

Fig. 4 Nogo-A regulates the expression of gene clusters.

Finally, they set out to distinguish between a signaling or a cell-autonomous role of Nogo-A, by employing LS8 mouse cells, a highly representative line of preameloblasts. Double immunofluorescent staining showed Nogo-A and Hsp90 were expressed and colocalized intracellularly (Fig. 5a). By selectively staining for extracellular membrane-localized Nogo-A, it was found that only a small portion was on the plasma membrane and most Nogo-A was intracellular (Fig. 5b–d). Further inhibition experiments were conducted to assess the effect of Nogo-A on the expression of the ameloblast differentiation markers Amelx, Ambn, Enam, and Klk4 (Fig. 5e). It was showed that the intracellular Nogo-A, and not the cell membrane Nogo-A, is responsible for the modulation of the expression of genes important for ameloblast differentiation.

Fig. 5 Inhibition of intracellular-, and not extracellular membrane-bound, Nogo-A is responsible for gene expression modulation.

Conclusion

Fig. 6 Hypothetical mode of action of Nogo-A in the dental epithelium.

In conclusion, this study reveals an unexpected role of Nogo-A in tooth enamel formation. Various experiments show that Nogo-A is crucial for proper enamel formation. Its deletion causes defective enamel, associated with gene and protein changes. Intracellular Nogo-A modulates genes for ameloblast differentiation and enamel production. This research expands our understanding of Nogo-A and provides insights into tooth development and enamel formation.

• Amelogenin, Human
• Animal Model Establishment
• Cell Culture