Proneurotrophins: The other half of the neurotrophin story

Although it has been more than fifty years since the neurotrophins were first discovered we are only now starting to appreciate the complexity of their true nature. These compounds lead a fascinating double life: as the proneurotrophin they bind to p75^NTR and set in motion diverse signaling pathways, which may lead to either apoptosis or cell survival, whereas in their incarnation as mature neurotrophins they bind to Trk receptors and insure cell survival.

The neurotrophins are a family of soluble, basic growth factor proteins which regulate neuronal development, maintenance, survival and death in the CNS and the PNS¹. They include NGF, the first member of the family to be discovered², BDNF³, NT-3 and NT 4⁴. Their molecular weights are 13-15 kDa , having isoelectric points between 9 and 10. All of the neurotrophins form noncovalently linked homodimers in solution, with three intrachain disulphide bonds⁵. They bind to two types of receptors: each neurotrophin binds to a specific Trk receptor (NGF to TrkA, BDNF and NT-4 to TrkB and NT-3 to TrkC)⁶, and all neurotrophins bind to the p75^NTRreceptor⁷. The residues involved in the binding of neurotrophin to Trk receptors are different than those involved in binding to p75^NTR.

Binding of a neurotrophin to its specific Trk receptor activates its tyrosine kinase, leading to the activation of PI3K (phosphatidylinositol-3-kinase), MAPK (mitogen activated protein kinase), and phospholipase C-γ pathways. Activation of these pathways leads to cell survival, differentiation, neurite growth, and activity dependent plasticity.8 However, binding of neurotrophin to the p75^NTR receptor leads to the activation of totally different pathways. p75^NTR lacks an intrinsic catalytic activity, so its signaling is mediated by interaction with various cytoplasmic interactors. NADE (p75^NTR NTR-associated cell death executor), NRIF (neurotrophin receptor-interacting factor) and NRAGE (neurotrophin receptor interacting MAGE homolog) are involved in the induction of cell death and SC-1 in cell cycle arrest. The GTPase RhoA modulates neurite outgrowth. The activation of the NF- B pathway mediates cell survival⁸. Thus binding to p75^NTR can either result in cell survival or cell death, depending on the particular cytoplasmic factor that has been recruited and the cellular context⁹.

The neurotrophins are synthesized as precursor proteins, 240-260 amino acids long, which are further processed until they are secreted as mature homodimeric proteins into the extracellular space¹⁰. It was thought that the roles of the prodomain were: 1) to aid in the folding of the mature protein¹¹ and 2) to sort the mature neurotrophin into the various secretion pathways¹². However, in the last few years a new role for the proneurotrophins has emerged. It has been found that the proneurotrophins bind to the p75^NTR receptor with a higher affinity than do mature neurotrophins¹³. In addition, it has been found that proNGF, not mature NGF, is the predominant isoform associated with a variety of cell types, including mast cells, sciatic nerve cells, thyroid gland, skeletal muscle, prostate gland, hippocampus and hair follicle. In the human cortex, proNGF appears to be the predominant isoform and is elevated in Alzheimer’s disease¹⁴. It has also been found that many commercial neurotrophins contain substantial amounts of proneurotrophins¹⁵, and the high concentrations at which they must be used in order to detect activity shed doubt on the identity of the substance actually causing that activity¹⁶. Therefore, many effects that had previously been attributed to neurotrophin action may be rightly attributed to the effects of the proneurotrophin instead.

The proneurotrophin is cleaved to the mature neurotrophin by various proteases: plasmin, furin, MP7, PC1/3, PC2, PC5/6, PC7 and PACE4, which cut behind a pair or even a single basic residue¹⁰. Stress and inflammation are known to upregulate protease expression and the expression of the p75^NTR receptor as well¹⁷. In order to investigate the effects of proneurotrophins on cells, it was neccesary to synthesize cleaveageresistant mutants, which exchanged those basic residues for amino acids not recognized by proteases.

It was found that mutated proNGF did not induced Trk phosphorylation even at concentrations five fold higher than those which induce phosphorylation by mature NGF. In contrast, treatment of cells with proNGF was at least ten times more effective than treatment with mature NGF in inducing apoptosis in a vascular smooth muscle cell line expressing p75^NTR but not Trk receptors¹³. Another recent paper shows that proNGF induces p75^NTR mediated death of oligodendrocytes following spinal cord injury¹⁸. ProBDNF has been shown to induce p75^NTR mediated neurite degeneration and apoptosis in sympathetic neurons¹⁹, and in vascular smooth muscle cells²⁰.

Although it has been more than fifty years since the neurotrophins were first discovered we are only now starting to appreciate the complexity of their true nature. These compounds lead a fascinating double life: as the proneurotrophin they bind to p75^NTR and set in motion diverse signaling pathways, which may lead to either apoptosis or cell survival, whereas in their incarnation as mature neurotrophins they bind to Trk receptors and insure cell survival.

Novel tools, such as antibodies to proneurotrophins, Anti-proNGF Antibody (#ANT-005) and antibodies to neurotrophin receptors, Mouse Anti-Rat p75 NGF Receptor (extracellular) Antibody (#AN-170) and Mouse Anti-Rat p75 NGF Receptor (extracellular)-FITC Antibody (#AN-170-F) will be essential for the numerous further studies which remain to be done in elucidating the control mechanisms which govern the balance between pro and mature neurotrophin, and ultimately the fate of the cell.

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