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

ABSTRACT: BACKGROUND: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). RESULTS: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.

SUBMITTER: Neumann E

PROVIDER: S-EPMC6675923 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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

Neumann Elena E Brandenburger Timo T Santana-Varela Sonia S Deenen René R Köhrer Karl K Bauer Inge I Hermanns Henning H Wood John N JN Zhao Jing J Werdehausen Robert R

Molecular and cellular neurosciences 20160621

<h4>Background</h4>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 ...[more]

PMID: 27346077

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Project description:Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are two major members of the neurotrophin family. Using immunohistochemistry and in situ hybridization histochemistry, we examined the effect of L5 spinal nerve ligation (SPNL), a neuropathic pain model, on the expression of BDNF in the uninjured L4 dorsal root ganglion (DRG). After L5 SPNL, both immunoreactivity for BDNF and the hybridization intensity for BDNF mRNA increased mainly in the small- and medium-sized neurons. The percentage of BDNF mRNA-expressing neurons increased in the ipsilateral L4 DRG compared with the contralateral DRG from the third to 28th day after ligation. A significantly greater number of BDNF-immunoreactive neurons were observed in the ipsilateral L4 DRG than contralateral side 14 d after ligation. To test the contribution of BDNF to the thermal hyperalgesia produced in this model, we intrathecally injected anti-BDNF antibody at third day after ligation. This treatment clearly attenuated thermal hyperalgesia for a few hours. Almost all BDNF mRNA-expressing neurons coexpressed trkA, a high-affinity NGF receptor, mRNA. The percentage of BDNF mRNA-expressing cells of trkA cells significantly increased in the ipsilateral L4 DRG 14 d after ligation. Furthermore, we examined the contribution of NGF on this phenotypic change using ELISA, Northern blot analysis, and anti-NGF antibody. NGF content in the ipsilateral L4 DRG linearly increased and reached a statistical significant level 14 d after L5 SPNL. Moreover, at this time point, the increase in NGF mRNA was observed in the ipsilateral L5 DRG and sciatic nerve, but not in the ipsilateral L4 DRG or L4 spinal nerve. Local application of anti-NGF antibody to the L4 spinal nerve beside the L5 spinal nerve-ligation site prevented the development of thermal hyperalgesia for 5 d after ligation. Our data suggest that BDNF, which increased in the uninjured L4 DRG neurons, acts as a sensory neuromodulator in the dorsal horn and contributes to thermal hyperalgesia in this neuropathic pain model. The contribution of locally synthesized NGF to thermal hyperalgesia was also demonstrated. These dynamic alterations in the expression and content of BDNF and NGF in the uninjured DRG neurons might be involved in the pathomechanisms of neuropathic pain.

Project description:UnlabelledSafety sciences and the identification of chemical hazards have been seen as one of the most immediate practical applications of human pluripotent stem cell technology. Protocols for the generation of many desirable human cell types have been developed, but optimization of neuronal models for toxicological use has been astonishingly slow, and the wide, clinically important field of peripheral neurotoxicity is still largely unexplored. A two-step protocol to generate large lots of identical peripheral human neuronal precursors was characterized and adapted to the measurement of peripheral neurotoxicity. High content imaging allowed an unbiased assessment of cell morphology and viability. The computational quantification of neurite growth as a functional parameter highly sensitive to disturbances by toxicants was used as an endpoint reflecting specific neurotoxicity. The differentiation of cells toward dorsal root ganglia neurons was tracked in relation to a large background data set based on gene expression microarrays. On this basis, a peripheral neurotoxicity (PeriTox) test was developed as a first toxicological assay that harnesses the potential of human pluripotent stem cells to generate cell types/tissues that are not otherwise available for the prediction of human systemic organ toxicity. Testing of more than 30 chemicals showed that human neurotoxicants and neurite growth enhancers were correctly identified. Various classes of chemotherapeutic agents causing human peripheral neuropathies were identified, and they were missed when tested on human central neurons. The PeriTox test we established shows the potential of human stem cells for clinically relevant safety testing of drugs in use and of new emerging candidates.SignificanceThe generation of human cells from pluripotent stem cells has aroused great hopes in biomedical research and safety sciences. Neurotoxicity testing is a particularly important application for stem cell-derived somatic cells, as human neurons are hardly available otherwise. Also, peripheral neurotoxicity has become of major concern in drug development for chemotherapy. The first neurotoxicity test method was established based on human pluripotent stem cell-derived peripheral neurons. The strategies exemplified in the present study of reproducible cell generation, cell function-based test system establishment, and assay validation provide the basis for a drug safety assessment on cells not available otherwise.

Dataset Information

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

Publications

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

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