Brain-Derived Neurotrophic Factor (BDNF) has attracted increasing interest as potential biomarker to support the diagnosis or monitor the efficacy of therapies in brain disorders. Circulating BDNF can be measured in serum, plasma or whole blood. However, the use of BDNF as biomarker is limited by the poor reproducibility of results, likely due to the variety of methods used for sample collection and BDNF analysis. To overcome these limitations, using sera from 40 healthy adults, we compared the performance of five ELISA kits (Aviscera-Bioscience, Biosensis, Millipore-ChemiKineTM, Promega-Emax®, R&D-System-Quantikine®) and one multiplexing assay (Millipore-Milliplex®). All kits showed 100% sample recovery and comparable range. However, they exhibited very different inter-assay variations from 5% to 20%. Inter-assay variations were higher than those declared by the manufacturers with only one exception which also had the best overall performance. Dot-blot analysis revealed that two kits selectively recognize mature BDNF, while the others reacted with both pro-BDNF and mature BDNF. In conclusion, we identified two assays to obtain reliable measurements of human serum BDNF, suitable for future clinical applications.

Background: Brain-derived neurotrophic factor is encoded by multiple transcripts with specific brain localization. Results: BDNF transcripts display a different ability to translate, basally and after stimulation. Conclusion: BDNF mRNA variants represent a "quantitative code" to regulate expression of the protein.Significance: This cell-based assay allows identification of compounds able to modulate BDNF variants having local effects in specific brain regions.

Chronic psychosocial stress at workplace is an important factor in the development of physical and mental illness. Objective biological measures of chronic stress are still lacking, but inflammatory response and growth factors are increasingly considered as potential stress biomarkers. Therefore, we investigated the relationship between psychophysical strain and serum levels of 48 chemokines, cytokines and growth factors measured using a multiplex immunoassay, and serum brain-derived neurotrophic factor (BDNF) measured by ELISA. Severity of psychophysical strain was scored in 115 healthy hospital workers using specific scales for anxiety, depression-like emotion, gastrointestinal or cardiac disturbances, and ergonomic dysfunction. Multivariate analysis revealed that higher anxiety scale scores were correlated with lower serum chemokine C-C motif ligand-2 (CCL2/MCP-1), chemokine C-C motif ligand-5 (CCL5/RANTES), chemokine C-C motif ligand-27 (CCL27/CTACK), chemokine C-C motif ligand-11 (CCL11/Eotaxin) and interleukin-6 (IL-6); gastrointestinal disturbances correlated with increased levels of interleukin-17 (IL-17) and reduced CCL11/Eotaxin, CCL27/CTACK and CCL2/MCP-1; while cardiac dysfunctions associate only to reduced CCL27/CTACK, and ergonomic dysfunction correlated with increased BDNF and reduced CCL11/Eotaxin and CCL5/RANTES. Thus, these 7 serum factors may provide a distinct signature for each different stress-related psychophysical outcome giving indications on individual vulnerabilities.

In mammals, an array of MEF2C proteins is generated by alternative splicing (AS), yet specific functions have not been ascribed to each isoform. Teleost fish possess two MEF2C paralogues, mef2ca and mef2cb. In zebrafish, the Mef2cs function to promote cardiomyogenic differentiation and myofibrillogenesis in nascent skeletal myofibers. We found that zebrafish mef2ca and mef2cb are alternatively spliced in the coding exons 4–6 region and these splice variants differ in their biological activity. Of the two, mef2ca is more abundantly expressed in developing skeletal muscle, its activity is tuned through zebrafish development by AS. By 24 hpf, we found the prevalent expression of the highly active full length protein in differentiated muscle in the somites. The splicing isoform of mef2ca that lacks exon 5 (mef2ca 4–6), encodes a protein that has 50% lower transcriptional activity, and is found mainly earlier in development, before muscle differentiation. mef2ca transcripts including exon 5 (mef2ca 4–5–6) are present early in the embryo. Over-expression of this isoform alters the expression of genes involved in early dorso-ventral patterning of the embryo such as chordin, nodal related 1 and goosecoid, and induces severe developmental defects. AS of mef2cb generates a long splicing isoform in the exon 5 region (Mef2cbL) that predominates during somitogenesis. Mef2cbL contains an evolutionarily conserved domain derived from exonization of a fragment of intron 5, which confers the ability to induce ectopic muscle in mesoderm upon over-expression of the protein. Taken together, the data show that AS is a significant regulator of Mef2c activity.

Drug-resistance to chemotherapics in aggressive neuroblastoma (NB) is characterized by enhanced cell survival mediated by TrkB and its ligand, brain-derived neurotrophic factor (BDNF); thus reduction in BDNF levels represent a promising strategy to overcome drugresistance, but how chemotherapics regulate BDNF is unknown. Here, cisplatin treatment in SK-N-BE neuroblastoma upregulated multiple BDNF transcripts, except exons 5 and 8 variants. Cisplatin increased BDNF mRNA and protein, and enhanced translation of a firefly reporter gene flanked by BDNF 5′UTR exons 1, 2c, 4 or 6 and 3′UTR-long. To block BDNF translation we focused on aurora kinases inhibitors which are proposed as new chemotherapeutics. NB cell survival after 24 h treatment was 43% with cisplatin, and 22% by cisplatin+aurora kinase inhibitor PHA-680632, while the aurora kinases inhibitor alone was less effective; however the combined treatment induced a paradoxical increase of BDNF in surviving cells with strong translational activation of exon6-3′UTR-long transcript, while translation of BDNF transcripts 1, 2C and 4 was suppressed. In conclusion, combined cisplatin and aurora kinase inhibitor treatment increases cell death, but induces BDNF overproduction in surviving cells through an aurora kinase-independent mechanism.