In many diseases, expression and ligand-dependent activity of the p75NTR receptor can promote pericyte and vascular dysfunction, inflammation, glial activation, and neurodegeneration. Diabetic retinopathy (DR) is characterized by all of these pathological events. However, the mechanisms by which p75NTR may be implicated at each stage of DR pathology remain poorly understood. Using a streptozotocin mouse model of diabetic retinopathy, we report that p75NTR is upregulated very early in glia and in pericytes to mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood–retina barrier breakdown, edema, and neuronal death. In a mouse model of oxygen-induced retinopathy, mimicking proliferative DR, p75NTR-dependent inflammation leads to ischemia and pathological angiogenesis through Semaphorin 3A. The acute use of antagonists of p75NTR or antagonists of the ligand proNGF suppresses each distinct phase of pathology, ameliorate disease, and prevent disease progression. Thus, our study documents novel disease mechanisms and validates druggable targets for diabetic retinopathy. SIGNIFICANCE STATEMENT Diabetic retinopathy (DR) affects an estimated 250 million people and has no effective treatment. Stages of progression comprise pericyte/vascular dysfunction, inflammation, glial activation, and neurodegeneration. The pathophysiology of each stage remains unclear. We postulated that the activity of p75NTR may be implicated. We show that p75NTR in glia and in pericytes mediate ligand-dependent induction of inflammatory cytokines, disruption of the neuro-glia-vascular unit, promotion of blood–retina barrier breakdown, edema, and neuronal death. p75NTR-promoted inflammation leads to ischemia and angiogenesis through Semaphorin 3A. Antagonists of p75NTR or antagonists of proNGF suppress each distinct phase of pathology, ameliorate disease, and prevent disease progression. Our study documents novel mechanisms in a pervasive disease and validates druggable targets for treatment.

Nerve growth factor (NGF) is generated from a precursor proNGF that is proteolytically procesed. NGF preferentially binds a trophic tyrosine kinase receptor TrkA, while proNGF binds a receptor p75^NTR that can have neurotoxic activity. Previously, we and others showed that a soluble protein α₂macroglobulin (α₂M) is neurotoxic. Toxicity is due in part because α₂M binds to NGF, and inhibits trophic activity presumably by preventing NGF binding to TrkA. However, the mechanisms remained unclear. Here, we show ex vivo and in vivo three mechanisms for α₂M neurotoxicity. First, unexpectedly the [α₂M•NGF] complexes do bind TrkA receptors, but do not induce TrkA dimerization or activation, resulting in deficient trophic support. Second, α₂M makes stable complexes with proNGF, conveying resistant to proteolysis that results in more proNGF and less NGF. Third, [α₂M•proNGF] complexes bind p75^NTR, and are more potent agonists than free proNGF, inducing TNFα production. Hence, α₂M regulates proNGF/p75^NTR positively and mature NGF/TrkA negatively, causing neuronal death ex vivo. These three mechanisms are operative in vivo, and α₂M causes neurodegeneration in a p75^NTR and proNGF–dependent manner. α₂M could be exploited as a therapeutic target, or as a modifier of neurotrophin signals.

In retinal proliferative diseases, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloprotease 2 (MMP‐2) and MMP‐9 also present increased levels of the protease inhibitor α ₂ ‐macroglobulin (α ₂ M) and its receptor, the low‐density lipoprotein receptor‐related protein 1 (LRP1). In the present work, we investigated whether the protease activated form of α ₂ M, α ₂ M∗, and LRP1 are involved with the MGC migratory process. By performing wound‐scratch migration and zymography assays, we demonstrated that α ₂ M∗ induced cell migration and proMMP‐2 activation in the human Müller glial cell line, MIO‐M1. This induction was blocked when LRP1 and MT1‐MMP were knocked down with siRNA techniques. Using fluorescence microscopy and biochemical procedures, we found that α ₂ M∗ induced an increase in LRP1 and MT1‐MMP accumulation in early endosomes, followed by endocytic recycling and intracellular distribution of MT1‐MMP toward cellular protrusions. Moreover, Rab11‐dominant negative mutant abrogated MT1‐MMP recycling pathway, cell migration, and proMMP‐2 activation induced by α ₂ M∗. In conclusion, α ₂ M∗, through its receptor LRP1, induces cellular migration of Müller glial cells by a mechanism that involves MT1‐MMP intracellular traffic to the plasma membrane by a Rab11‐dependent recycling pathway.—Barcelona, P. F., Jaldín‐Fincati, J. R., Sánchez, M. C.Chiabrando, G. A., Activated α2‐macroglobulin induces Müller glial cell migration by regulating MT1‐MMP activity through LRP1. FASEBJ. 27, 3181‐3197 (2013). www.fasebj.org