A2MACROGLOBULIN DIFFERENTIALLY REGULATES LIGAND-DEPENDENT P75 AND TRKA RECEPTOR ACTIVATION EX VIVO AND IN RETINA

BARCELONA PF; SARAGOVI HU

Denver

Congreso; ARVO 2015 Annual Meeting (The Association for Research in Vision and Ophthalmology; 2015

The Association for Research in Vision and Ophthalmology

Purpose: In human glaucoma and human diabetic retinopathy, as well as in mouse models of these pathologies, a soluble protein α2Macroglobulin (α2M) is up-regulated in retina and vitreous. Intravitreal injection of α2M in a normal eye produces RGC death. Neutralization of α2M during glaucoma delays disease progression. Previously we showed ex vivo that α2M binds to the growth factor NGF making [α2M?NGF] complexes. The complex is not able to activate the neuroprotective activity of the NGF receptor TrkA. Prevention of neuroprotection may be a mechanism for α2M in pathological states.Here, we report that α2M also potentiates the neurotoxicity mediated by proNGF binding to its p75NTR receptor, and report on the mechanisms of action ex vivo and in vivo. Methods: We used solutions of α2M ± a small molecule antagonist of p75NTR, ± a biological antagonist of proNGF (neutralizing anti-proNGF mAb), or control vehicle. Agents were administrated intravitreally to adult mice. We studied RGC death and the mechanism of action of p75NTR and TrkA in vivo. 7 days after intravitreal injection, the activation of TrkA (p-TrkA) and adaptor proteins downstream (p-ERK1,2 and p-AKT) were quantified biochemically and neuronal survival was quantified by TUNEL assays. Protein stability assays of proNGF proteolysis were done ± Furin protease. The dimerization of TrkA was evaluated by cross-linking assays. The p75NTR?expressing Müller glial cell line (rMC) was stimulated by [α2M?proNGF] to enhance production of TNFα protein quantified by ELISA and TNFα mRNA quantified by RT-PCR. Results: We show in vivo that α2M induces neuronal death by (a) potentiation of proNGF:p75NTR activity and (b) by reduction of NGF:TrkA activity. Neutralization of proNGF or antagonism of the p75NTR prevent α2M induced RGC death. The mechanisms are as follows. (a) α2M binding to proNGF potentiates proNGF:p75NTR activity due to stabilization of proNGF and resistance to degradation. (b) α2M binding to NGF prevent TrkA dimerization and activation. Conclusions: α2macroglobulin is a protein that binds to and differentially regulates the function of proNGF and mature NGF. α2M is up-regulated in disease and could be exploited as a therapeutic target or as a tool to develop modifiers of neurotrophin signals.