Search

IntroductionFrontotemporal 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.MethodsBy 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.ResultsA 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.DiscussionThese results suggest that proNGF could be included in a panel of biomarkers to improve the FTD diagnosis.

Background and objectivesMultiple 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.MethodsA 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 (n = 10, 7 F and 3 M, age 39.0 ± 7.5) or post-OCRE (n = 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.ResultsFirst, 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.DiscussionThese data support the hypothesis of specific deregulated miRNAs as putative biomarkers in RRMS patients’ stratification and DMT drug response.

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.

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.

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.