Live or Let Die in Neuroscience: The Story of the Proneurotrophins

Although it has been more than forty 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 sortilin to 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 and p75^NTR receptors and insure cell survival.

The neurotrophins are a family of soluble, basic growth factors which regulate neuronal development, maintenance, survival and death in the CNS and the PNS¹. They include nerve growth factor (NGF), the first member of the family to be discovered², brain derived growth factor³ (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4)⁴. 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 disulfide bonds⁵. They bind to two types of receptors: each neurotrophin binds to a specific tyrosine kinase (Trk) receptor (NGF to TrkA, BDNF and NT4 to TrkB and NT3 to TrkC)⁶, and all neurotrophins bind to the p75 neurotrophin receptor (p75^NTR) receptor⁷. The residuesinvolved in 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 plasticity⁸. However, binding of neurotrophin to the p75^NTR receptor leads to the activation of 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. NRIF, NRAGE and Schwann cell factor-1(SC-1) are involved in cell cycle arrest. The GTPase RhoA modulates neurite outgrowth. The activation of the NF-κB 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. Ultimately the final decision about a cell’s fate must take into account the ratio of Trk to p75^NTR receptors.

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 to aid in the folding of the mature protein¹¹ and to sort the mature neurotrophin into the various secretion pathways¹². The proneurotrophin is cleaved to the mature neurotrophin by various proteases: plasmin, furin, MP7, PC1/3, PC2, PC5/6, PC7, PACE4, which cut behind a pair or even a single basic residue¹⁰.

A paper by Lee et al. (2001) revealed a new role for the proneurotrophins¹³. They found that proNGF and not mature NGF is the ligand preferred by p75^NTR. Thus they postulated p75^NTR-induced cellular processes would be more efficiently induced by proNGF than by mature NGF. Indeed, they found that cleavage resistant proNGF (mutated at the cleavage site to insure that it retains the prodomain) induced apoptosis ten times more effectively than mature NGF in a vascular smooth muscle cell line expressing p75^NTR but not Trk receptors. The ability of proNGF to induce apoptosis was confirmed by work showing that proNGF induces p75^NTR mediated death of oligodendrocytes following spinal cord injury¹⁴. However, proNGF can also mediate survival. Rattenholl et al. (2001) have shown that proNGF was as effective as NGF in inducing survival of DRG neurons¹¹. Fahnestock et al. (2004) have shown that a mutated proNGF exhibits neurite outgrowth on both PC12 cells and mouse sympathetic cervical ganglions¹⁵.

ProNGF is not the only proneurotrophin shown to have biological activity mediated by binding to p75^NTR. Activation of p75^NTR by proBDNF has been shown to facilitate hippocampal long term depression¹⁶. Most interestingly, a mutation in proBDNF has been shown to correlate with memory deficits in humans¹⁷.

The finding 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 follicle¹⁸ underscores the biological significance of Lee et al.’s finding.

Expression of proBDNF has been found in many regions of the rat central nervous system including spinal cord, substantia nigra, amygdala, hypothalamus, cerebellum, hippocampus and cortex¹⁹. In the human cortex, proNGF appears to be the predominant isoform and is elevated in Alzheimer’s disease²⁰. In contrast, the level of proBDNF is decreased²¹.

Therefore, protease activity, controlling the balance between pro and mature neurotrophins becomes a major factor in the regulation of neurotrophin activity.

Several disease states, including stress and inflammation are known to affect the activity of proteases, and these also affect p75^NTR expression and function²².

Thus, the neurotrophin story becomes even more complex. Not only the ratio of Trk receptors to p75^NTR affects the cellular outcome, but also the ratio of mature to pro neurotrophin, controlled by protease activity.

Not all cells which express significant levels of p75^NTR undergo apoptosis in response to proNGF, suggesting that additional proteins are required. A solution to this dilemma was proposed in 2004 when sortilin was found to be a receptor for proneurotrophins but not mature neurotrophins²³. Sortilin is a 95 kDa member of the Vps10p domain receptor family and is expressed in a variety of tissues, notably brain, spinal tissue and muscle. ProNGF binds to sortilin via its prodomain, and to p75^NTR through residues in its mature domain. Thus a three membered complex between proNGF, sortilin and p75^NTR is proposed as the signaling intermediate required for proNGF mediated apoptosis. proBDNF also forms complexes with p75^NTR and sortilin to elicit apoptosis in neurons²⁴.

Although it has been more than forty 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 sortilin to 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 and p75^NTR receptors and insure cell survival.

Novel tools, such as antibodies to proneurotrophins, (Anti-proNGF Antibody (#ANT-005), Anti-proBDNF Antibody (#ANT-006)) and antibodies to neurotrophin receptors Mouse Anti-Rat p75 NGF Receptor (extracellular) Antibody (#AN-170), Mouse Anti-Rat p75 NGF Receptor (extracellular)-FITC Antibody (#AN-170-F), Anti-p75 NGF Receptor (extracellular) Antibody (#ANT-007), Anti-p75 NGF Receptor (extracellular)-FITC Antibody (#ANT-007-F), Anti-p75 NGF Receptor (intracellular) Antibody (#ANT-011) and Anti-Sortilin (extracellular) Antibody (ANT-009) 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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