MicroRNA-1-associated effects of neuron-specific brain-derived neurotrophic factor gene deletion in dorsal root ganglia

MicroRNAs (miRNAs) regulate gene expression in physiological as well as in pathological processes, including chronic pain. Whether deletion of a gene can affect expression of the miRNAs that associate with the deleted gene mRNA remains elusive. We investigated the effects of brain-derived neurotrophic factor (Bdnf) gene deletion on the expression of miR-1 in dorsal root ganglion (DRG) neurons and its pain-associated downstream targets heat shock protein 60 (Hsp60) and connexin 43 (Cx43) in tamoxifen-inducible conditional knockout mice, Bdnf(fl/fl); Advillin-CreER(T2) (Bdnf cKO).

Our results indicate that deletion of Bdnf in DRG neurons leads to a temporary dysregulation of miR-1, suggesting an impairment of a presumable feedback loop between BDNF protein and its targeting miR-1. This appears to affect its downstream protein Hsp60 and as a consequence might influence the phenotype after inducible Bdnf gene deletion. While this appears to be a MEF2a-/SRF-independent and transient effect, expression levels of various other miRNAs may remain permanently altered.

Efficient Bdnf gene deletion was confirmed in DRG of Bdnf cKO mice by Real-Time qRT-PCR and ELISA 10days after completed tamoxifen treatment. In DRG, miR-1 expression was reduced 0.44-fold (p<0.05; Real-time qRT-PCR) in Bdnf cKO compared to floxed wildtype littermate control Bdnf(fl/fl) mice (WT). While Hsp60 protein expression was increased 1.85-fold (p<0.05; Western blot analysis), expression levels of Cx43 and the miR-1-associated transcription factors MEF2a and SRF remained unchanged. When analyzing Bdnf cKO mice 32days after complete tamoxifen treatment to investigate whether observed expression alterations remain permanently, we found no significant differences between Bdnf cKO and WT mice. However, miRNA microarray analysis revealed that 167 miRNAs altered (p<0.05) in DRG of these mice following Bdnf gene deletion. Conclusions: Our results indicate that deletion of Bdnf in DRG neurons leads to a temporary dysregulation of miR-1, suggesting an impairment of a presumable feedback loop between BDNF protein and its targeting miR-1. This appears to affect its downstream protein Hsp60 and as a consequence might influence the phenotype after inducible Bdnf gene deletion. While this appears to be a MEF2a-/SRF-independent and transient effect, expression levels of various other miRNAs may remain permanently altered.