NGF Expression in Reelin-Deprived Retinal Cells: A Potential Neuroprotective Effect

Abstract

We recently reported that increased NGF and p75(NTR) as well as decreased trkA(NGFR) characterized the Reelin-deprived (E-Reeler) retina, prospecting a potential contribution of NGF during E-Reeler retinogenesis. Herein, retinal ganglion cells (RGCs), glial cells and rod bipolar cells (RBCs) were isolated from E-Reeler retinas, and NGF, trkA(NGFR)/p75(NTR) expression and apoptosis were investigated. E-Reeler (n = 28) and E-control (n = 34) retinas were digested, and RGCs, glial cells and RBCs were isolated by the magnetic bead separation. Expression of NGF, trkA(NGFR), p75(NTR), Annexin V/PI and Bcl2/Bax was quantified by flow cytometry and validated by real-time PCR or WB. In E-Reeler retinas, NGF was significantly increased in RGCs and glial cells, p75(NTR) was increased in both RBCs and RGCs, and trkA(NGFR) was unchanged. In E-control retinas, NGF and p75(NTR) were expressed mainly in RBCs and RGCs and faintly in glial cells, while trkA(NGFR) was weakly expressed by RBCs and RGCs. In RBCs and RGCs, Annexin V expression was unchanged, while Bcl2 increased and Bax decreased selectively in E-Reeler RGCs. The data indicate that E-Reeler RBCs and RGCs overexpress NGF and p75(NTR) as a protective endogenous response to Reelin deprivation. The observation is strongly supported by the absence of apoptosis in both cell types.

Time-Dependent Nerve Growth Factor Signaling Changes in the Rat Retina During Optic Nerve Crush-Induced Degeneration of Retinal Ganglion Cells

“…Recently, Balzamino and coworkers (2015) showed that the levels of both NGF and p75 NTR , but not TrkA, are increased in RGCs and bipolar cells in reelin-deprived retinas, a condition characterized by impaired retinal structure and visual function [ 42 ]. The expression of p75 NTR has also been observed in post-mitotic RGCs in the developing retina [ 15 ], indicating a role for this receptor and its activated signaling in the retinal cell fate during development.…”

Changes in vitreal protein profile and retina mRNAs in Reeler mice: NGF, IL33 and Müller cell activation

“…Our previous studies highlighted the overexpression of NGF and NGF receptors (mRNA/protein) in Reeler retina at p14, p21 and particularly p28, postulating a potential compensatory NGF activity in Reelin deficiency [1,2]. Albeit Reelin deficient retina does not show any crucial inflammatory state, the presence of several cell-to-cell and cell-to-mediator events cannot be excluded during the neurodegenerative process that might include the expression and/or modulation of gene and related products [1,3].…”

“…At postnatal day (p) p14, p21 and p28, mice were anaesthetized by intraperitoneal injection of 2 mg/mL ketamine (0.2 mL/10 gr body-weight; Ketavet, Gellini Pharmaceutics, Italy) and 0.23 mg/mL medetomidine (0.24 mL/10 gr body-weight; Domitor, Orion Corp., Espoo, Finland) mixture. Sampling was carried out under a dissecting microscope (SMZ645; Nikon, Tokyo, Japan) equipped with cold-light optic fibers (PL2000 photonic; Axon, Vienna, Austria), as previously reported with slight modifications [2]. A corneal incision was produced and lens, retina and vitreous were collected in a microvial with separating membrane.…”

“…Vitreous and retina (n = 3/time-point; Reeler and WT mice) were diluted / extracted in modified RIPA Buffer (50mM Tris-HCl, 150mM NaCl, 1% Triton-X100, 5mM EDTA, 100mM NaF and 1mM PMSF; pH 7.5) and finally sonicated (VibraCell Sonics, Inc., Newtown, USA), according to a standard procedure [2]. Total proteins were quantified with Nanodrop Spectrophotometer (A1000, Celbio, Milan, Italy).…”

“…NGF and Reelin have been reported to take actively part in neurogenesis and retinogenesis [4–6]. NGF has been hypothesized to interact with Reelin by modulating neuronal migration, neurodevelopment and other physiological processes in the central nervous system and retina [1,2,7]. NGF activities encompass cell proliferation, cytoskeletal reorganization, migration, differentiation, survival and/or apoptosis [4,8].…”