Exogenous Modulation of Retinoic Acid Signaling Affects Adult RGC Survival in the Frog Visual System after Optic Nerve Injury.

After lesions to the mammalian optic nerve, the great majority of retinal ganglion cells (RGCs) die before their axons have even had a chance to regenerate. Frog RGCs, on the other hand, suffer only an approximately 50% cell loss, and we have previously investigated the mechanisms by which the application of growth factors can increase their survival rate. Retinoic acid (RA) is a vitamin A-derived lipophilic molecule that plays major roles during development of the nervous system. The RA signaling pathway is also present in parts of the adult nervous system, and components of it are upregulated after injury in peripheral nerves but not in the CNS. Here we investigate whether RA signaling affects long-term RGC survival at 6 weeks after axotomy. Intraocular injection of all-trans retinoic acid (ATRA), the retinoic acid receptor (RAR) type-α agonist AM80, the RARβ agonist CD2314, or the RARγ agonist CD1530, returned axotomized RGC numbers to almost normal levels. On the other hand, inhibition of RA synthesis with disulfiram, or of RAR receptors with the pan-RAR antagonist Ro-41-5253, or the RARβ antagonist LE135E, greatly reduced the survival of the axotomized neurons. Axotomy elicited a strong activation of the MAPK, STAT3 and AKT pathways; this activation was prevented by disulfiram or by RAR antagonists. Finally, addition of exogenous ATRA stimulated the activation of the first two of these pathways. Future experiments will investigate whether these strong survival-promoting effects of RA are mediated via the upregulation of neurotrophins

Axotomy-induced STAT3 activation is dependent on RA.

<p><b>A</b>. Representative Western blots of phosphorylated-STAT3 and total STAT3 from extracts of retinas from uncut animals, and animals at 1 week after axotomy. Approximate molecular weight in kDa is indicated. The order of some of the bands was rearranged to correspond to the bar chart below. <b>B.</b> Quantification of Western blots, standardized to control values. pSTAT3 levels are increased at 1 week after axotomy. Application of ATRA increases the levels of pSTAT3 further, but other RAR agonists do not. Inhibition of RA synthesis, or application of RAR antagonists, prevents the axotomy-induced increase in pSTAT3. One-way ANOVA followed by <i>post-hoc</i> Tukey-Kramer tests: * p<0.05, ** p<0.01, *** p<0.001, N = 4, 4, 3, 3, 3, 3, 3, 4, 4, and 3 pools, each of 2 animals and with duplicate measurements. Error bars represent the S.E.M.</p

Retinoic signaling antagonists reduce long-term survival of axotomized RGCs.

<p><b>A-D.</b> Representative fields of whole-mount retinas in which RGCs have been retrogradely labled with Texas Red dextran amine (TDA). <b>A.</b> Uncut normal retina. <b>B.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO (vehicle). <b>C.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RALDH inhibitor disulfiram (RALDH inhib). <b>D.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the pan-RAR antagonist Ro-41-5253 (RAR antag). <b>E.</b> Quantification of RGC percentage survival in the temporal region of the retina at 6 weeks after axotomy. Vehicle-treated retinas show a significant 40% decrease in RGC numbers. Treatment with RALDH inhibitor further reduces survival. Only the antagonist against RAR, not RARβ, further reduces survival. One-way ANOVA followed by <i>post-hoc</i> Tukey tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N = 4 animals in all cases. Error bars represent the S.E.M. Scale bar in A = 50 μm.</p

Axotomy-induced AKT activation is dependent on RA.

<p><b>A</b>. Representative Western blots of phosphorylated-AKT and total AKT from extracts of retinas from uncut animals, and animals at 1 week after axotomy. Approximate molecular weight in kDa is indicated. The order of some of the bands was rearranged to correspond to the bar chart below. <b>B.</b> Quantification of Western blots, standardized to control values. pAKT levels are increased at 1 week after axotomy. Application of ATRA or other RAR agonists do not significantly increase the levels of pAKT further. Inhibition of RA synthesis, or application of RAR antagonists, prevents the axotomy-induced increase in pAKT. One-way ANOVA followed by <i>post-hoc</i> Tukey tests: * p<0.05, ** p<0.01, *** p<0.001, N = 3 pools in all cases, each of 2 animals and with duplicate measurements. Error bars represent the S.E.M.</p

Retinoic signaling agonists promote long-term survival of axotomized RGCs.

<p><b>A-F.</b> Representative fields of whole-mount retinas in which RGCs have been retrogradely labled with Texas Red dextran amine (TDA). <b>A.</b> Uncut normal retina. <b>B.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO (vehicle). <b>C.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of all-trans retinoic acid (ATRA). <b>D.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RARα agonist AM80 (RARα agon). <b>E.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RARβ agonist CD2314 (RARβ agon). <b>F.</b> Retina from animal at 6 weeks after axotomy, eye injected with PBS and DMSO with the addition of the RARγ agonist CD1530 (RARγ agon). <b>G.</b> Quantification of RGC percentage survival in the temporal region of the retina at 6 weeks after axotomy. Vehicle-treated retinas show a significant 40% decrease in RGC numbers. Treatment with ATRA or RAR agonists rescues many of these RGCs. One-way ANOVA followed by <i>post-hoc</i> Tukey tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N = 4 animals in all cases. Error bars represent the S.E.M. Scale bar in A = 50 μm.</p

Increased activity of signaling pathways is localized to RGCs.

<p>Immunostaining was carried out against pMAPK, pAKT and pSTAT3 in frozen sections of retinas from uncut animals (A, E, I), animals at 1 week after the optic nerve was cut and the eyeball injected with vehicle (B, F, J), animals at 1 week after the optic nerve was cut and the eyeball injected with ATRA (C, G, K), and animals at 1 week after the optic nerve was cut and the eyeball injected with RALDH inhibitor (D, H, L). In A, the inner plexiform layer (IPL) and ganglion cell layer (GCL) are indicated. <b>A, E, I.</b> In control frog retinal sections, low levels of pMAPK, pAKT and pSTAT3 immunoreactivity are present in the GCL and the INL. <b>B, F, J.</b> One week after axotomy, immunostaining of all three proteins has increased in intensity, particularly in the cell bodies of RGCs of the GCL and their axons, and in the cells in the INL. <b>C, G, K.</b> One week after axotomy and ATRA treatment, immunostaining of all three proteins is intense in the cell bodies of RGCs of the GCL and their axons, and in the cells in the INL. <b>D, H, L.</b> One week after axotomy and RALDH inhibitor treatment, faint immunostaining of all three proteins is present in the GCL and the INL. Scale bar in A: 50 μm.</p