Integrin Activation Promotes Axon Growth on Inhibitory Chondroitin Sulfate Proteoglycans by Enhancing Integrin Signaling

Abstract

Chondroitin sulfate proteoglycans (CSPGs) are upregulated after CNS lesions, where they inhibit axon regeneration. In order for axon growth and regeneration to occur, surface integrin receptors must interact with surrounding extracellular matrix molecules. We have explored the hypothesis that CSPGs inhibit regeneration by inactivating integrins and that forcing integrins into an active state might overcome this inhibition. Using cultured rat sensory neurons, we show that the CSPG aggrecan inhibits laminin-mediated axon growth by impairing integrin signaling via decreasing phosphorylated FAK (pFAK) and pSrc levels, without affecting surface integrin levels. Forcing integrin activation and signaling by manganese or an activating antibody TS2/16 reversed the inhibitory effect of aggrecan on mixed aggrecan/ laminin surfaces, and enhanced axon growth from cultured rat sensory neurons (manganese) and human embryonic stem cell-derived motoneurons (TS2/16). The inhibitory effect of Nogo-A can also be reversed by integrin activation. These results suggest that inhibition by CSPGs can act via inactivation of integrins, and that activation of integrins is a potential method for improving axon regeneration after injury.

Kindlin-1 Enhances Axon Growth on Inhibitory Chondroitin Sulfate Proteoglycans and Promotes Sensory Axon Regeneration

“…In addition, kindlin-1-treated animals showed faster sensory recovery. These results are consistent with several earlier reports that integrin activation could enhance axon growth from neurons in vitro, even when cultured in the presence of inhibitory molecules such as amino-Nogo or aggrecan (Ivins et al, 2000;Lein et al, 2000;Hu and Strittmatter, 2008;Tan et al, 2011), and also with our demonstration that expression of a tenascin-binding integrin can promote axon regeneration (Andrews et al, 2009). CNS injuries cause an upregulation of myelin-related inhibitors (e.g., Nogo, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein) (McKerracher et al, 1994;Mukhopadhyay et al, 1994;GrandPré et al, 2000GrandPré et al, , 2002Prinjha et al, 2000;Kottis et al, 2002;Wang et al, 2002) and CSPGs (e.g., aggrecan, brevican, phosphacan, versican), which impair regenerative responses (Jaworski et al, 1999;McKeon et al, 1999;Asher et al, 2000;Afshari et al, 2010).…”

“…In addition to promoting axon growth, kindlin-1-transfected neurons had many short, actin-containing branches arising from the axons, mirroring the localization of endogenous kindlin-1 around the cell periphery, cell-cell junctions, and focal adhesions (Siegel et al, 2003;Herz et al, 2006;Ussar et al, 2006;Lai-Cheong et al, 2008). The fact that kindlin-1 expression only enhances axon growth in the presence of inhibitory aggrecan is consistent with the idea that aggrecan impairs integrin signaling, which can be restored by integrin activation (Tan et al, 2011). This is important in axon regeneration, since CSPG upregulation usually follows a CNS injury (Jaworski et al, 1999;McKeon et al, 1999;Asher et al, 2000;Afshari et al, 2010).…”

“…This strategy was very successful with PC12 cells and DRG in vitro but virus-mediated transduction of neurons in vivo only modestly improved axon regeneration, to a lesser extent than in the present experiments (Andrews et al, 2009). This result, together with those discussed above, suggest that Nogo-A and CSPGs in the injury site would have inactivated the ␣9 integrin transduced into sensory axons in our previous experiment (Hu and Strittmatter, 2008;Andrews et al, 2009;Tan et al, 2011). In the present experiments, the integrins expressed by DRG neurons, although activated, were not well matched to the molecules in the environment surrounding their cut axons.…”

“…Their functions are regulated by a conformational change in the extracellular domain from a low-affinity to a high-affinity state (also called "inside-out " signaling or integrin activation) and "outside-in " signaling (the signaling cascades propagated intracellularly following integrin-ligand binding). Enhancing ligand-binding affinity via integrin activation promotes neurite outgrowth from cultured CNS and PNS neurons (Ivins et al, 2000;Lein et al, 2000;Lemons and Condic, 2006;Tan et al, 2011). Crucially, integrin activation also enhances the ability of neurons to extend axons in the presence of inhibitory molecules such as amino-Nogo and the chondroitin sulfate proteoglycan (CSPG) aggrecan, which can both inactivate axonal integrins (Hu and Strittmatter, 2008;Tan et al, 2011) and are highly expressed after a CNS injury (Liu et al, 2006).…”

Laminin/β1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization

“…npg adhesion molecules are involved in initial neuritogenesis [20], neurite outgrowth and regeneration [21], axon path finding [22], neuronal positioning [23][24][25], as well as synaptic development and plasticity [26]. However, whether and how integrin-mediated cell adhesion is involved in neuronal polarization is unknown.…”