Microglia-Derived Pronerve Growth Factor Promotes Photoreceptor Cell Death in Culture

Abstract

Abstract: : Purpose: Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its role in photoreceptor cell death in retinal disease. Methods: Microglial conditioned media (MGCM) collected in the presence or absence of bovine serum albumin (BSA) or protease inhibitors (PI) were assayed for NGF and proNGF levels; and their effects on survival of cultured 661w photoreceptor cells. The expression and cellular distribution of NGF and proNGF were also determined in RCS dystrophic rat retinas. Results: MGCM exhibited a single NGF-reactive band of ~66 kDa in immunoblots; but also produced ~32 kDa NGF-reactive band in MGCM/BSA and MGCM/PI. MGCM/BSA, but not MGCM alone nor NGFß, promoted photoreceptor cell death and this was reversed by immunodepletion with an anti-NGF IgG or anti-proNGF IgG. A polyclonal antiserum against microglial cells (RMG) reactive with recombinant proNGF protein also labeled a solitary ~32 kDa band in MGCM/BSA and MGCM/PI, and suppressed their toxicity on photoreceptor cells. The ~66 kDa NGF-band was not reactive with RMG. Gel filtration chromatography isolated the RMG reactivity and microglia-derived toxicity to MGCM/PI fractions containing ~32 kDa, but not ~66 kDa, NGF-reactive proteins. RMG, similarly labeled a ~32 kDa band in RCS dystrophic retinas but not in age-match genetic control retinas. RMG staining localized primarily to activated microglia among degenerating photoreceptor cells in dystrophic retinas and to few vitreal hyalocytes in both normal and dystrophic eyes. The increased proNGF levels in dystrophic retinas were verified in relative PCR and Southern blot. MGCM/BSA upregulated p75NTR expression and induced cell death in p75NTR+/trkA- photoreceptor cells and these were reversed by RMG or by a p75NTR neutralizing antiserum, respectively. Conclusions: Our study shows that a ~32 kDa proNGF protein released by activated microglia promoted photoreceptor cell death in vitro. Moreover, our study suggests that defective post-translational processing of proNGF, and perhaps of other proneurotrophins, might be involved in photoreceptor degeneration in retinal dystrophy.

Keywords: cell death/apoptosis • microglia • growth factors/growth factor receptors