Search

Nerve Growth Factor (NGF), the prototype of the neurotrophin family, is essential for maintenance and growth of different neuronal populations. The X-ray crystal structure of NGF has been known since the early ‘90s and shows a β-sandwich fold with extensive loops that are involved in the interaction with its binding partners. Understanding the dynamical properties of these loops is thus important for molecular recognition. We present here a combined solution NMR/molecular dynamics study which addresses the question of whether and how much the long loops of NGF are flexible and describes the N-terminal intrinsic conformational tendency of the unbound NGF molecule. NMR titration experiments allowed identification of a previously undetected epitope of the anti-NGF antagonist antibody aD11 which will be of crucial importance for future drug lead discovery. The present study thus recapitulates all the available structural information and unveils the conformational versatility of the relatively rigid NGF loops upon functional ligand binding.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aβ-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aβ oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

The homeostasis between mature neurotrophin NGF and its precursor proNGF is thought to be crucial in physiology and in pathological states. Therefore, the measurement of the relative amounts of NGF and proNGF could serve as a footprint for the identification of disease states, for diagnostic purposes. Since NGF is part of proNGF, their selective identification with anti-NGF antibodies is not straightforward. Currently, many immunoassays for NGF measurement are available, while the proNGF assays are few and not validated by published information. The question arises, as to whether the commercially available assays are able to distinguish between the two forms. Also, since in biological samples the two forms coexist, are the measurements of one species affected by the presence of the other? We describe experiments addressing these questions. For the first time, NGF and proNGF were measured together and tested in different immunoassays. Unexpectedly, NGF and proNGF were found to reciprocally interfere with the experimental outcome. The interference also calls into question the widely used NGF ELISA methods, applied to biological samples where NGF and proNGF coexist. Therefore, an immunoassay, able to distinguish between the two forms is needed. We propose possible ways forward, towards the development of a selective assay. In particular, the use of the well validated anti-NGF αD11 antibody in an alphaLISA assay with optimized incubation times would be a solution to avoid the interference in the measurement of a mixed sample containing NGF and proNGF. Furthermore, we explored the possibility of measuring proNGF in a biological sample. But the available commercial kit for the detection of proNGF does not allow the measurement of proNGF in mouse brain tissues. Therefore, we validated an SPR approach for the measurement of proNGF in a biological sample. Our experiments help in understanding the technical limits in the measurement of the NGF/proNGF ratio in biological samples, and propose concrete solutions towards the solution of this problem.

Background Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer's disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A “painless” human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the “painless” hNGF mutants has not been quantified. Objectives and Results Aiming at the therapeutic application of the “painless” hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency. Conclusions This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF “painless” mutants.

Background Growing evidence shows that, in vivo, the precursor of Nerve Growth Factor (NGF), proNGF, displays biological activities different from those of its mature NGF counterpart, mediated by distinct, and somewhat complementary, receptor binding properties. NGF and proNGF induce distinct transcriptional signatures in target cells, highlighting their different bioactivities. In vivo, proNGF and mature NGF coexist. It was proposed that the relative proNGF/NGF ratio is important for their biological outcomes, especially in pathological conditions, since proNGF, the principal form of NGF in Central Nervous System (CNS), is increased in Alzheimer’s disease brains. These observations raise a relevant question: does proNGF, in the presence of NGF, influence the NGF transcriptional response and viceversa? In order to understand the specific proNGF effect on NGF activity, depending on the relative proNGF/NGF concentration, we investigated whether proNGF affects the pattern of well-known NGF-regulated mRNAs. Results To test any influence of proNGF on pure NGF expression fingerprinting, the expression level of a set of candidate genes was analysed by qReal-Time PCR in rat adrenal pheochromocytoma cell line PC12, treated with a mixture of NGF and proNGF recombinant proteins, in different stoichiometric ratios. These candidates were selected amongst a set of genes well-known as being rapidly induced by NGF treatment. We found that, when PC12 cells are treated with proNGF/NGF mixtures, a unique pattern of gene expression, which does not overlap with that deriving from treatment with either proNGF or NGF alone, is induced. The specific effect is also dependent on the stoichiometric composition of the mixture. The proNGF/NGF equimolar mixture seems to partially neutralize the specific effects of the proNGF or NGF individual treatments, showing a weaker overall response, compared to the individual contributions of NGF and proNGF alone. Conclusions Using gene expression as a functional read-out, our data demonstrate that the relative availability of NGF and proNGF in vivo might modulate the biological outcome of these ligands.