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In 2007, drawing inspiration from her previous experiments on chick embryos, Rita Levi-Montalcini, at the age of 98, proposed a new project, and a research group, in which I was included, was formed at the European Brain Research Institute (EBRI). Looking back on this experience, I can say that Professor Levi-Montalcini’s approach and the relationships she formed with my colleagues and me, contributed to my growth as a researcher. With her welcoming and warm-hearted disposition, she taught me how to consider other people’s ideas without prejudice, to reason and not to exclude any hypothesis. I also learned from her how to overcome those difficulties that are so frequent in the research field, always keeping in mind the starting point and looking toward the objective, with a factual optimism. I was just a young researcher and deeply flattered that a Nobel Laureate, with an incredible career and extraordinary life, treated me as her equal. My experience with Professor Levi-Montalcini has also provided me with a reliable path to follow, and when I encounter difficulties and challenges, I ask myself what would she have done. This approach has always helped me to move forward. Indeed, I believe the best way to celebrate Rita Levi-Montalcini as a woman in neuroscience is to recount how her exceptional example is a constant reminder as to why I have chosen to be a scientist. I hope she will always continue to be a source of inspiration for scientists in the future.

Supplemental material, sj-pdf-1-jbx-10.1177_24725552211000672 for A Quantitative Bioassay to Determine the Inhibitory Potency of NGF–TrkA Antagonists by Francesca Malerba, Bruno Bruni Ercole, Rita Florio and Antonino Cattaneo in SLAS Discovery

In the brain, the neurotrophin Nerve growth factor (NGF) regulates not only neuronal survival and differentiation, but also glial and microglial functions and neuroinflammation. NGF is known to regulate oligodendrogenesis, reducing myelination in the central nervous system (CNS). In this study, we found that NGF controls oligodendrogenesis by modulating the levels of miR-219a-5p, a well-known positive regulator of oligodendrocyte differentiation. We exploited an NGF-deprivation mouse model, the AD11 mice, in which the postnatal expression of an anti-NGF antibody leads to NGF neutralization and progressive neurodegeneration. Notably, we found that these mice also display increased myelination. A microRNA profiling of AD11 brain samples and qRT-PCR analyses revealed that NGF deprivation leads to an increase of miR-219a-5p levels in hippocampus and cortex and a corresponding down-regulation of its predicted targets. Neurospheres isolated from the hippocampus of AD11 mice give rise to more oligodendrocytes and this process is dependent on miR-219a-5p, as shown by decoy-mediated inhibition of this microRNA. Moreover, treatment of AD11 neurospheres with NGF inhibits miR-219a-5p up-regulation and, consequently, oligodendrocyte differentiation, while anti-NGF treatment of wild type (WT) oligodendrocyte progenitors increases miR-219a-5p expression and the number of mature cells. Overall, this study indicates that NGF inhibits oligodendrogenesis and myelination by down-regulating miR-219a-5p levels, suggesting a novel molecular circuitry that can be exploited for the discovery of new effectors for remyelination in human demyelinating diseases, such as Multiple Sclerosis.

In this article, we demonstrate and validate a new bioassay named the NTAB [NGF-TrkA (nerve growth factor-tropomyosin receptor kinase A) antagonist bioassay] for the determination of the inhibitory potency of NGF-TrkA antagonists, based on the inhibition of NGF-dependent proliferation of the human TF1 erythroleukemic cell line.It is well known that NGF holds great therapeutic potential due to its neurotrophic and neuroprotective properties. NGF is also involved in some pathways, however, principally driven by TrkA that, if not correctly regulated, can lead to unwanted pathological outcomes linked to pain, angiogenesis, and cancer.Indeed, there is an increasing interest, from a therapeutic perspective, in designing new effective molecules (antibodies, antibody fragments, or small molecules) able to inhibit the undesired NGF-TrkA pathway. For these reasons, there is an interest to develop functional cell-based assays for determination of the inhibition potency of compounds inhibiting the NGF-TrkA axis. The NTAB presents significant advantages over other published NGF-TrkA functional bioassays, for these reasons: (1) It is quantitative, (2) it measures a pure TrkA response, (3) it is simpler, (4) it is based on a natural biological response, and (5) it is easily scalable from a lab scale to an automated industrial assay.The NTAB assay was validated with a panel of well-characterized NGF-TrkA inhibitors, yielding characteristic dose-response curves, from which the relative strength of the inhibitors was quantitatively determined and used for comparisons. This new bioassay will be very useful to assist in the validation and prioritization of the best inhibitors among a large number of candidates.

The discovery of new biomarkers for Alzheimer's disease (AD) is essential for an accurate diagnosis, to conceive new strategies of treatments, and for monitoring the efficacy of potential disease-modifying therapies in clinical trials. proNGF levels in the cerebrospinal fluid (CSF) represent a promising diagnostic biomarker for AD, but its validation was hampered by the absence of a reliable immunoassay. In the literature, proNGF is currently measured in postmortem brain tissue by semiquantitative immunoblot. Here we describe the development and validation of a new method to measure proNGF in the CSF of living patients. This method, based on molecular size separation by capillary electrophoresis, is automated and shows a 40-fold increase in sensitivity with respect to the proNGF immunoblot, largely used in literature, and is robust, specific, and scalable to high-throughput. We have measured proNGF in the cerebrospinal fluid of 84 living patients with AD, 13 controls, and 15 subjective memory complaints (SMC) subjects. By comparing the proNGF levels in the three groups, we found a very significant difference between proNGF levels in AD samples compared with both controls and SMC subjects, while no significant difference was found between SMC and controls. Because of the development of this new immunoassay, we are ready to explore the potentiality of proNGF as a new biomarker for AD or subgroups thereof, as well as for other neurodegenerative diseases.

GABA, the main inhibitory neurotransmitter in the adult brain, depolarizes and excites immature neurons because of an initially higher intracellular chloride concentration [Cl−]i due to the delayed expression of the chloride exporter KCC2 at birth. Depolarization-induced calcium rise via NMDA receptors and voltage-dependent calcium channels is instrumental in shaping neuronal circuits and in controlling the excitatory (E)/inhibitory (I) balance in selective brain areas. An E/I imbalance accounts for cognitive impairment observed in several neuropsychiatric disorders. The aim of this review is to summarize recent data on the mechanisms by which alterations of GABAergic signaling alter the E/I balance in cortical and hippocampal neurons in Alzheimer’s disease (AD) and the role of cation-chloride co-transporters in this process. In particular, we discuss the NGF and AD relationship and how mice engineered to express recombinant neutralizing anti-NGF antibodies (AD11 mice), which develop a neurodegenerative pathology reminiscent of that observed in AD patients, exhibit a depolarizing action of GABA due to KCC2 impairment. Treating AD and other forms of dementia with bumetanide, a selective NKCC1 antagonist, contributes to re-establishing a proper E/I balance in selective brain areas, leading to amelioration of AD symptoms and the slowing down of disease progression.

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease. However, the development of an NGF-based therapy is limited by its potent pain activity. We have developed a “painless” derivative form of human NGF (NGF61/100), characterized by identical neurotrophic properties but a reduced nociceptive sensitization activity in vivo. Here we characterized the response of rat dorsal root ganglia neurons (DRG) to the NGF derivative NGF61/100, in comparison to that of control NGF (NGF61), analyzing the expression of noxious pro-nociceptive mediators. NGF61/100 displays a neurotrophic activity on DRG neurons comparable to that of control NGF61, despite a reduced activation of PLCγ, Akt and Erk1/2. NGF61/100 does not differ from NGF61 in its ability to up-regulate Substance P (SP) and Calcitonin Gene Related Peptide (CGRP) expression. However, upon Bradykinin (BK) stimulation, NGF61/100-treated DRG neurons release a much lower amount of SP and CGRP, compared to control NGF61 pre-treated neurons. This effect of painless NGF is explained by the reduced up-regulation of BK receptor 2 (B2R), respect to control NGF61. As a consequence, BK treatment reduced phosphorylation of the transient receptor channel subfamily V member 1 (TRPV1) in NGF61/100-treated cultures and induced a significantly lower intracellular Ca2+ mobilization, responsible for the lower release of noxious mediators. Transcriptomic analysis of DRG neurons treated with NGF61/100 or control NGF allowed identifying a small number of nociceptive-related genes that constitute an “NGF pain fingerprint”, whose differential regulation by NGF61/100 provides a strong mechanistic basis for its selective reduced pain sensitizing actions.

Nerve growth factor (NGF) is a therapeutic candidate for Alzheimer’s disease, but must be administered intraparenchymally owing to its pain-inducing activity. Capsoni et al. develop human painless NGF (hNGFp) and show that intranasal administration results in widespread biodistribution in the brains of Alzheimer’s disease mice, with potent anti-amyloidogenic effects., Nerve growth factor is a therapeutic candidate for Alzheimer’s disease. Due to its pain-inducing activity, in current clinical trials nerve growth factor is delivered locally into the brain by neurosurgery, but data on the efficacy of local nerve growth factor delivery in decreasing amyloid-β deposition are not available. To reduce the nerve growth factor pain-inducing side effects, thus avoiding the need for local brain injection, we developed human painless nerve growth factor (hNGFp), inspired by the human genetic disease hereditary sensory and autonomic neuropathy type V. hNGFp has identical neurotrophic potency as wild-type human nerve growth factor, but a 10-fold lower pain sensitizing activity. In this study we first mimicked, in the 5xFAD mouse model, the intraparenchymal delivery of hNGFp used in clinical trials and found it to be ineffective in decreasing amyloid-β plaque load. On the contrary, the same dose of hNGFp delivered intranasally, which was widely biodistributed in the brain and did not induce pain, showed a potent anti-amyloidogenic action and rescued synaptic plasticity and memory deficits. We found that hNGFp acts on glial cells, modulating inflammatory proteins such as the soluble TNFα receptor II and the chemokine CXCL12. We further established that the rescuing effect by hNGFp is mediated by CXCL12, as pharmacological inhibition of CXCL12 receptor CXCR4 occludes most of hNGFp effects. These findings have significant therapeutic implications: (i) we established that a widespread exposure of the brain is required for nerve growth factor to fully exert its neuroprotective actions; and (ii) we have identified a new anti-neurodegenerative pathway as a broad target for new therapeutic opportunities for neurodegenerative diseases.

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.

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.