Biological Activity of Nerve Growth Factor Precursor Is Dependent upon Relative Levels of Its Receptors

Nerve growth factor (NGF) is produced as a precursor called pro-nerve growth factor (proNGF), which is secreted by many tissues and is the predominant form of NGF in the central nervous system. In Alzheimer disease brain, cholinergic neurons degenerate and can no longer transport NGF as efficiently, leading to an increase in untransported NGF in the target tissue. The protein that accumulates in the target tissue is proNGF, not the mature form. The role of this precursor is controversial, and both neurotrophic and apoptotic activities have been reported for recombinant proNGFs. Differences in the protein structures, protein expression systems, methods used for protein purification, and methods used for bioassay may affect the activity of these proteins. Here, we show that proNGF is neurotrophic regardless of mutations or tags, and no matter how it is purified or in which system it is expressed. However, although proNGF is neurotrophic under our assay conditions for primary sympathetic neurons and for pheochromocytoma (PC12) cells, it is apoptotic for unprimed PC12 cells when they are deprived of serum. The ratio of tropomyosin-related kinase A to p75 neurotrophin receptor is low in unprimed PC12 cells compared with primed PC12 cells and sympathetic neurons, altering the balance of proNGF-induced signaling to favor apoptosis. We conclude that the relative level of proNGF receptors determines whether this precursor exhibits neurotrophic or apoptotic activity.Nerve growth factor (NGF)³ regulates neuronal survival, neurite outgrowth, and differentiation in the peripheral and central nervous systems (1). The mature form of NGF forms a non-covalent homodimer and binds with high affinity (k_d ≈ 10⁻¹¹ m) to tropomyosin-related kinase A (TrkA) and with low affinity (k_d ≈ 10⁻⁹ m) to the common neurotrophin receptor p75^NTR (p75 neurotrophin receptor) (2). NGF promotes cell survival and growth in cells expressing TrkA through activation of the phosphatidylinositol 3-kinase/AKT pathway and the Ras/mitogen-activated protein kinase (MAPK) pathway (3, 4). p75^NTR plays diverse roles, ranging from cell survival to cell death depending on the cellular context in which it is expressed. Through activation of the NF-κB pathway, p75^NTR can contribute to cell survival in sensory neurons (5), it is involved in axonal growth via regulation of Rho activity (6), and it can interact with Trks to enhance neurotrophin affinity (at low concentration of ligand) and specificity of binding to Trks (7–9). High levels of p75^NTR expression can induce apoptosis when there are low levels of Trk or when Trk is absent (10, 11). Apoptosis occurs through increased ceramide production (12), activation of c-Jun N-terminal kinase (JNK1), and p53 (10, 13). p75^NTR requires a co-receptor called sortilin to induce cell death (14).NGF is produced as a precursor called pro-nerve growth factor (proNGF) (15). ProNGF is secreted by many tissues such as prostate cells, spermatids, hair follicles, oral mucosal keratinocytes, sympathetic neurons, cortical astrocytes, heart, and spleen (16–20). ProNGF is the predominant form of NGF in the central and peripheral nervous systems, whereas little or no mature NGF can be detected (21–24). In Alzheimer disease brain, retrograde transport from the cortex and hippocampus to basal forebrain cholinergic neurons is reduced as these neurons degenerate, with concomitant proNGF accumulation in the cortex and hippocampus (21, 23). This suggested that proNGF mediates biological activity besides its prodomain function of promoting protein folding and regulation of neurotrophin secretion (25–28). To study the role of proNGF protein in vitro, point mutations were inserted at the cleavage site used by furin, a proprotein convertase known to cleave proNGF (29), to minimize the conversion of proNGF to mature NGF. The resulting recombinant, cleavage-resistant proNGFs reportedly exhibit either apoptotic activity (30, 31) or neurotrophic activity (32, 33). These recombinant proteins differ in several ways (