Pharmacological Profile of Brain-derived Neurotrophic Factor (BDNF) Splice Variant Translation Using a Novel Drug Screening Assay A QUANTITATIVE CODE

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. The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5- and 3UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5- and 3UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5UTR or a 3UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent a quantitative code for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests.