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In 2007, drawing inspiration from her previous experiments on chick embryos, Rita Levi-Montalcini, at the age of 99, 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 towards 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.

In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications.

Frontotemporal dementia (FTD) is an extremely heterogeneous and complex neurodegenerative disease, exhibiting different phenotypes, genetic backgrounds, and pathological states. Due to these characteristics, and to the fact that clinical symptoms overlap with those of other neurodegenerative diseases or psychiatric disorders, the diagnosis based only on the clinical evaluation is very difficult. The currently used biomarkers help in the clinical diagnosis, but are insufficient and do not cover all the clinical needs. By the means of a new immunoassay, we have measured and analyzed the proNGF levels in 43 cerebrospinal fluids (CSF) from FTD patients, and compared the results to those obtained in CSF from 84 Alzheimer's disease (AD), 15 subjective memory complaints (SMC) and 13 control subjects. A statistically significant difference between proNGF levels in FTD compared to AD, SMC and controls subjects was found. The statistical models reveal that proNGF determination increases the accuracy of FTD diagnosis, if added to the clinically validated CSF biomarkers. These results suggest that proNGF could be included in a panel of biomarkers to improve the FTD diagnosis.

Multiple sclerosis (MS) is a chronic, progressive neurological disease characterized by early-stage neuroinflammation, neurodegeneration, and demyelination that involves a spectrum of heterogeneous clinical manifestations in terms of disease course and response to therapy. Even though several disease-modifying therapies (DMTs) are available to prevent MS-related brain damage-acting on the peripheral immune system with an indirect effect on MS lesions-individualizing therapy according to disease characteristics and prognostic factors is still an unmet need. Given that deregulated miRNAs have been proposed as diagnostic tools in neurodegenerative/neuroinflammatory diseases such as MS, we aimed to explore miRNA profiles as potential classifiers of the relapsing-remitting MS (RRMS) patients' prospects to gain a more effective DMT choice and achieve a preferential drug response. A total of 25 adult patients with RRMS were enrolled in a cohort study, according to the latest McDonald criteria before (pre-cladribine, pre-CLA; pre-ocrelizumab, pre-OCRE, time T0) and after high-efficacy DMTs, time T1, 6 months post-CLA ( = 10, 7 F and 3 M, age 39.0 ± 7.5) or post-OCRE ( = 15, 10 F and 5 M, age 40.5 ± 10.4) treatment. A total of 15 age- and sex-matched healthy control subjects (9 F and 6 M, age 36.3 ± 3.0) were also selected. By using Agilent microarrays, we analyzed miRNA profiles from peripheral blood mononuclear cells (PBMC). miRNA-target networks were obtained by miRTargetLink, and Pearson's correlation served to estimate the association between miRNAs and outcome clinical features. First, the miRNA profiles of pre-CLA or pre-OCRE RRMS patients compared to healthy controls identified modulated miRNA patterns (40 and seven miRNAs, respectively). A direct comparison of the two pre-treatment groups at T0 and T1 revealed more pro-inflammatory patterns in the pre-CLA miRNA profiles. Moreover, both DMTs emerged as being capable of reverting some dysregulated miRNAs toward a protective phenotype. Both drug-dependent miRNA profiles and specific miRNAs, such as miR-199a-3p, miR-29b-3p, and miR-151a-3p, emerged as potentially involved in these drug-induced mechanisms. This enabled the selection of miRNAs correlated to clinical features and the related miRNA-mRNA network. These data support the hypothesis of specific deregulated miRNAs as putative biomarkers in RRMS patients' stratification and DMT drug response.

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease (AD) treatment but the clinical application is hindered by its potent pro-nociceptive activity. Thus, to reduce systemic exposure that would induce pain, in recent clinical studies NGF was administered through an invasive intracerebral gene-therapy approach. Our group demonstrated the feasibility of a non-invasive intranasal delivery of NGF in a mouse model of neurodegeneration. NGF therapeutic window could be further increased if its nociceptive effects could be avoided altogether. In this study we exploit forms of NGF, mutated at residue R100, inspired by the human genetic disease HSAN V (Hereditary Sensory Autonomic Neuropathy Type V), which would allow increasing the dose of NGF without triggering pain. We show that \"painless\" hNGF displays full neurotrophic and anti-amyloidogenic activities in neuronal cultures, and a reduced nociceptive activity in vivo. When administered intranasally to APPxPS1 mice ( n = 8), hNGFP61S/R100E prevents the progress of neurodegeneration and of behavioral deficits. These results demonstrate the in vivo neuroprotective and anti-amyloidogenic properties of hNGFR100 mutants and provide a rational basis for the development of \"painless\" hNGF variants as a new generation of therapeutics for neurodegenerative diseases.

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, toward 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 toward the solution of this problem.

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. 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. 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.

Even though the number of patients suffering from Alzheimer’s Disease (AD) is rapidly growing worldwide, only a few symptomatic treatments have been approved for clinical use, pointing out the urgent need for more effective disease-modifying therapies that actually alter the progression of this neurodegenerative disorder which is characterized by co-occurence of both Amyloid beta (Aβ) and tau neuropathologies. Preclinical and clinical evidence suggests that a link between Aβ and tau drives the entire continuum of AD pathobiology. 12A12 is a monoclonal antibody (mAb) which offers neuroprotection into two transgenic lines of AD, including Tg2576 that overexpresses Swedish mutation (KM670/671NL) of Amyloid Precursor Protein (APP, isoform 695) and 3xTg (APP Swedish, MAPT P301L, and PSEN1 M146V), by targeting the 20-22kDa N-terminal tau fragments (NH 2 htau). In particular, acute (over 14 days with 4 doses), intravenous injection of 12A12mAb leads to significant improvement of cognitive, biochemical and histopathological AD signs in symptomatic 6-month-old Tg2576, a well-established transgenic mouse model that mimics the human amyloidosis with an age-dependent Aβ accumulation/aggregation and plaque deposition. Here, we report that Tg2576 mice, immunized with 12A12mAb at 6 months of age and returned to their home cage for additional 3 months, exhibit preserved spatial memory despite the anticipated interruption of antibody administration (discontinuous treatment). This enduring beneficial effect on memory deficit (up to 90 days after the last injection) is accompanied by normalization in the synaptic imbalance and microgliosis along with decrease of the most toxic A11-positive prefibrillar oligomers and inverse increase in 4kDa monomeric form(s) of Aβ 1–42. These findings reveal that: (i) soluble, pathogenetic tau specie(s) located at the N-terminal domain of protein early synergizes with Aβ in driving the progression of AD neuropathology; (ii) transient immunoneutralization of the NH 2 htau following short-term treatment with 12A12mAb exerts preventive, long-lasting neuroprotective effects, at least in part by interfering at “pre-plaque” stage with the progressive deposition of insoluble, fibrillar Aβ via a shift of its aggregation pathway into its less harmful, unaggregated monomeric forms. Taken together, these findings represent a strong rationale for the advancement of 12A12mAb to clinical stage aiming at preventing the Aβ-dependent neurodegeneration by lowering the cerebral levels of NH 2 htau in humans suffering from chronic, slow-progressing AD.

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.