Activation of a synapse weakening pathway by human Val66 but not Met66 pro-brain-derived neurotrophic factor (proBDNF).

This study describes a fundamental functional difference between the two main polymorphisms of the pro-form of brain-derived neurotrophic factor (proBDNF), providing an explanation as to why these forms have such different age-related neurological outcomes. Healthy young carriers of the Met66 form (present in ∼30% Caucasians) have reduced hippocampal volume and impaired hippocampal-dependent memory function, yet the same polymorphic population shows enhanced cognitive recovery after traumatic brain injury, delayed cognitive dysfunction during aging, and lower risk of late-onset Alzheimer's disease (AD) compared to those with the more common Val66 polymorphism. To examine the differences between the protein polymorphisms in structure, kinetics of binding to proBDNF receptors and in vitro function, we generated purified cleavage-resistant human variants. Intriguingly, we found no statistical differences in those characteristics. As anticipated, exogenous application of proBDNF Val66 to rat hippocampal slices dysregulated synaptic plasticity, inhibiting long-term potentiation (LTP) and facilitating long-term depression (LTD). We subsequently observed that this occurred via the glycogen synthase kinase 3ß (GSK3ß) activation pathway. However, surprisingly, we found that Met66 had no such effects on either LTP or LTD. These novel findings suggest that, unlike Val66, the Met66 variant does not facilitate synapse weakening signaling, perhaps accounting for its protective effects with aging.

Human ProNGF: biological effects and binding profiles at TrkA, P75NTR and sortilin.

Nerve growth factor (NGF) promotes cell survival via binding to the tyrosine kinase receptor A (TrkA). Its precursor, proNGF, binds to p75(NTR) and sortilin receptors to initiate apoptosis. Current disagreement exists over whether proNGF acts neurotrophically following binding to TrkA. As in Alzheimer's disease the levels of proNGF increase and TrkA decrease, it is important to clarify the properties of proNGF. Here, wild-type and cleavage-resistant mutated forms (M) of proNGF were engineered and their binding characteristics determined. M-proNGF and NGF bound to p75(NTR) with similar affinities, whilst M-proNGF had a lower affinity than NGF for TrkA. M-proNGF behaved neurotrophically, albeit less effectively than NGF. M-proNGF addition resulted in phosphorylation of TrkA and ERK1/2, and in PC12 cells elicited neurite outgrowth and supported cell survival. Conversely, M-proNGF addition to cultured cortical neurons initiated caspase 3 cleavage. Importantly, these biological effects were shown to be mediated by unprocessed M-proNGF. Surprisingly, binding of the pro region alone to TrkA, at a site other than that of NGF, caused TrkA and ERK1/2 phosphorylation. Our data show that M-proNGF stimulates TrkA to a lesser degree than NGF, suggesting that in Alzheimer brain the increased proNGF : NGF and p75(NTR) : TrkA ratios may permit apoptotic effects to predominate over neurotrophic effects.

Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.

The tyrosine kinase A (TrkA) receptor is a validated therapeutic intervention point for a wide range of conditions. TrkA activation by nerve growth factor (NGF) binding the second extracellular immunoglobulin (TrkAIg2) domain triggers intracellular signaling cascades. In the periphery, this promotes the pain phenotype and, in the brain, cell survival or differentiation. Reproducible structural information and detailed validation of protein-ligand interactions aid drug discovery. However, the isolated TrkAIg2 domain crystallizes as a ß-strand-swapped dimer in the absence of NGF, occluding the binding surface. Here we report the design and structural validation by nuclear magnetic resonance spectroscopy of the first stable, biologically active construct of the TrkAIg2 domain for binding site confirmation. Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF ( 1WWW .pdb), and the (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

TrkAd5: A novel therapeutic agent for treatment of inflammatory pain and asthma.

Elevated levels of nerve growth factor have been linked to the onset and persistence of many pain-related disorders and asthma. Described here are the design, expression, refolding, and purification of a monomeric (nonstrand-swapped) form of the binding domain of the nerve growth factor receptor, designated TrkAd5. We have shown that TrkAd5 produced recombinantly binds nerve growth factor with picomolar affinity. TrkAd5 has been characterized using a variety of biophysical and biochemical assays and is shown here to be stable in both plasma and urine. The palliative effects of TrkAd5 are demonstrated in animal models of inflammatory pain and allergic asthma. We conclude that TrkAd5 will prove effective in ameliorating both acute and chronic conditions where nerve growth factor acts as a mediator and suggest a role for its application in vivo as a novel therapeutic.