Upregulation of reggie-1/flotillin-2 promotes axon regeneration in the rat optic nerve in vivo and neurite growth in vitro

Abstract

The ability of fish retinal ganglion cells (RGCs) to regenerate their axons was shown to require the re-expression and function of the two proteins reggie-1 and -2. RGCs in mammals fail to upregulate reggie expression and to regenerate axons after lesion suggesting the possibility that induced upregulation might promote regeneration. In the present study, RGCs in adult rats were induced to express reggie-1 by intravitreal injection of adeno-associated viral vectors (AAV2/1) expressing reggie-1 (AAV.R1-EGFP) 14d prior to optic nerve crush. Four weeks later, GAP-43-positive regenerating axons had crossed the lesion and grown into the nerve at significantly higher numbers and length (up to 5mm) than the control transduced with AAV.EGFP. Consistently, after transduction with AAV.R1-EGFP as opposed to AAV.EGFP, primary RGCs in vitro grew long axons on chondroitin sulfate proteoglycan (CSPG) and Nogo-A, both glial cell-derived inhibitors of neurite growth, suggesting that reggie-1 can provide neurons with the ability to override inhibitors of neurite growth. This reggie-1-mediated enhancement of growth was reproduced in mouse hippocampal and N2a neurons which generated axons 40-60% longer than their control counterparts. This correlates with the reggie-1-dependent activation of Src and PI3 kinase (PI3K), of the Rho family GTPase Rac1 and downstream effectors such as cofilin. This increased growth also depends on TC10, the GTPase involved in cargo delivery to the growth cone. Thus, the upregulation of reggie-1 in mammalian neurons provides nerve cells with neuron-intrinsic properties required for axon growth and successful regeneration in the adult mammalian CNS.

Flotillins in intercellular adhesion – from cellular physiology to human diseases

“…F-actin modulates flotillin function in leukocytes because disruption of F-actin prevents chemoattractant-induced formation of large flotillin scaffolds at the cell rear (Affentranger et al, 2011;Langhorst et al, 2007;Rossy et al, 2009). Flotillins could also be involved in remodeling of the F-actin cytoskeleton and in Rho GTPase activation (Koch et al, 2013;Langhorst et al, 2008a;Munderloh et al, 2009;Neumann-Giesen et al, 2007). Moreover, in neutrophils, flotillin microdomains associate with the actin cortical cytoskeleton and regulate the activity of myosin II (Ludwig et al, 2010), a key mediator of cortical tension.…”

Can mammalian vision be restored following optic nerve degeneration?

“…48 The ability of fish RGC axons to regenerate requires the reexpression of the proteins reggie-1 and -2, but mammalian RGCs fail to upregulate reggie expression and regenerate axons after a lesion, 49 but adult rat RGCs will regenerate following a lesion if reggie-1 expression is induced 14 days prior to ONI. 49 Since RGCs grow long axons on CSPG and Nogo-A, this suggests that reggie-1 provides neurons the ability to override inhibitors of neurite growth. 49 The inhibition of axon regeneration in mice by the rho pathway can be neutralized by the enzyme C3, allowing significant axon regeneration after optic nerve transection.…”

“…49 Since RGCs grow long axons on CSPG and Nogo-A, this suggests that reggie-1 provides neurons the ability to override inhibitors of neurite growth. 49 The inhibition of axon regeneration in mice by the rho pathway can be neutralized by the enzyme C3, allowing significant axon regeneration after optic nerve transection. 50 The injection of cyclic adenosine monophosphate (cAMP) into the vitreous of mice a day after the crush injury also induces significant axon regeneration throughout an optic nerve crush site.…”

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

“…Reggies were also needed for growth cone formation and elongation (Koch et al, 2013;Munderloh et al, 2009) and the targeted delivery of N-cadherin as one of many growth and guidance receptors (Bodrikov et al, 2011) in elongating neurites. The Rab11/ARF-6 dependent recycling of integrins was demonstrated by Eva and colleagues as an essential molecular mechanism for the regeneration of axons in mammals (Eva et al, 2010(Eva et al, , 2012.…”