How scars shape the neural landscape: Key molecular mediators of TGF-β1’s anti-neuritogenic effects

Abstract

Following injury to the peripheral and central nervous systems, tissue levels of transforming growth factor (TGF)-β1 often increase, which is key for wound healing and scarring. However, active wound regions and scars appear to inhibit process outgrowth by regenerating neurons. We recently showed that corneal wound myofibroblasts block corneal nerve regeneration in vivo, and sensory neurite outgrowth in vitro in a manner that relies critically on TGF-β1. In turn, delayed, abnormal re-innervation contributes to long-term sensory dysfunctions of the ocular surface. Here, we exposed morphologically and biochemically-differentiated sensory neurons from the ND7/23 cell line to TGF-β1 to identify the intracellular signals regulating these anti-neuritogenic effects, contrasting them with those of Semaphorin(Sema)3A, a known inhibitor of neurite outgrowth. Neuronal morphology was quantified using phase-contrast imaging. Western blotting and specific inhibitors were then used to identify key molecular mediators. Differentiated ND7/23 cells expressed neuron-specific markers, including those involved in neurite extension and polarization. TGF-β1 increased phosphorylation of collapsin response mediator protein-2 (CRMP2), a molecule that is key for neurite extension. We now show that both glycogen synthase kinase (GSK)-3β and Smad3 modulate phosphorylation of CRMP2 after treatment with TGF-β1. GSK-3β appeared to exert a particularly strong effect, which could be explained by its ability to phosphorylate not only CRMP2, but also Smad3. In conclusion, TGF-β1’s inhibition of neurite outgrowth in sensory neurons appears to be regulated through a highly-conserved signaling pathway, which involves the GSK-3β/CRMP-2 loop via both canonical and non-canonical mechanisms. It is hoped that by defining the signaling pathways that control neurite outgrowth in wound environments, it will become possible to identify optimal molecular targets to promote re-innervation following injury.

Artificial spider silk supports and guides neurite extension in vitro

“…As the cells grew toward confluency, they occasionally grew on top of each other in multilayered structures. This is in line with the manufacturer’s description of the cell line and conforms to previous reports in which the cells were cultured on PDL, plastic, or glass surfaces 37 . However, in the L511 and L521 coated wells, the cells continued to grow as monolayers, also toward confluency, which suggests more efficient adhesion to the substrate (Figure 1A).…”

“…These cells are often used as a surrogate for peripheral sensory neurons since they extend long neurites and differentiate into polarized cells after addition of nerve growth factor and/or dbcAMP 37,55–58 . Moreover, differentiated ND7/23 cells express sensory neuron markers such as TrkA, Substance P, and CGRP, 37 which attest to their suitability for in vitro studies of peripheral nerve regeneration. We included several control substrates in order to benchmark the performance of NT2RepCT and VN‐NT2RepCT matrices, that is, PDL as it is the recommended substrate for ND7/23, L111, and L511 that have been reported to be important ECM proteins for neurite sprouting of adult DRG in vitro 14 and L521 that is present at neuromuscular junctions 59,60 .…”

“…In this study, we used a DRG cell line (ND7/23) as a model to study cell survival and neurite extension. These cells are often used as a surrogate for peripheral sensory neurons since they extend long neurites and differentiate into polarized cells after addition of nerve growth factor and/or dbcAMP 37,55–58 . Moreover, differentiated ND7/23 cells express sensory neuron markers such as TrkA, Substance P, and CGRP, 37 which attest to their suitability for in vitro studies of peripheral nerve regeneration.…”

“…This is in line with the manufacturer's description of the cell line and conforms to previous reports in which the cells were cultured on PDL, plastic, or glass surfaces. 37 However, in the L511 and L521 coated wells, the cells continued to grow as monolayers, also toward confluency, which suggests more efficient adhesion to the substrate (Figure 1A). The rate of the cell growth was similar for all coatings with no significant differences (Figure 1C).…”

Role of Transforming Growth Factor Beta in Peripheral Nerve Regeneration: Cellular and Molecular Mechanisms

“…As mentioned above, phenotypic changes of repair SCs mainly include dedifferentiation ( Jessen and Mirsky, 2019 ), secretion of neurotrophic factors ( Pandey and Mudgal, 2021 ), production of cytokines to recruit immune cells ( Qu et al, 2021 ), and changes in morphology ( Jessen and Mirsky, 2016 ). After nerve injury, it is shown that a large amount of TGF-β is secreted by macrophages ( Sulaiman and Gordon, 2009 ), SCs ( Li et al, 2015 ; Clements et al, 2017 ), fibroblasts ( Clements et al, 2017 ), and the neurons ( van Rossum et al, 2008 ; Li et al, 2015 ) at the site of injury ( Jeon and Huxlin, 2020 ). Meanwhile, the expression level of TGF-β receptor is significantly upregulated in SCs and neurons ( Fregnan et al, 2012 ).…”