Bidirectional regulation of P body formation mediated by eIF4F complex formation in sensory neurons

Abstract

Processing (P) bodies are RNA granules that comprise key cellular sites for the metabolism of mRNAs. In certain cells, including neurons, these RNA granules may also play an important role in storage of mRNAs in a translationally dormant state. Utilizing nerve growth factor (NGF) and interleukin 6 (IL6), which stimulate cap-dependent translation in sensory neurons, and adenosine monophosphate activated protein kinase (AMPK) activators, which inhibit cap-dependent translation, we have tested the hypothesis that cap-dependent translation is linked to P body formation in mammalian sensory neurons. Treatment with NGF and IL6 decreases, whereas metformin increases biochemical association of the P body marker and translational repressor/decapping activator Rck/p54/dhh1 with the 5'-mRNA-cap suggesting an ordered assembly of P bodies. Likewise, diverse AMPK activators enhance P body formation while NGF and IL6 decrease P bodies in sensory neurons. This bidirectional P body plasticity readily occurs in the axonal compartment of these neurons. These studies indicate that P body formation is intricately linked to cap-dependent translation in mammalian sensory neurons suggesting an important role for these organelles in the regulation of mRNA metabolism in the adult PNS.

Keywords: AMPK; P bodies; Translation initiation; Trigeminal ganglion; eIF4F; mRNA degradation.

Fig. 1

eIF4F complex formation is inversely related to m7GTP/eIF4E Rck binding. (A) Representative western blots of TG neurons co-treated with IL-6 (50 ng/ml) and NGF (20 ng/ml) resulting in enhanced association of eIF4A with m7GTP beads while decreasing the association of Rck. (B) In contrast, treatment of TG neurons with metformin (20 mM) reduced association of eIF4A while enhancing the association of Rck with m7GTP beads. Levels were standardized to eIF4E association with m7GTP beads in all conditions. *p < 0.05. n = 8 per condition.

Fig. 2

IL6 and NGF decrease while AMPK activators increase P body formation in TG neurons: Dcp2. Representative micrographs of TG neurons co-treated with IL-6 (50 ng/ml) and NGF (20 ng/ml) causing a reduction in Dcp2-labeled puncta. In contrast treatment with metformin (20 mM), A769662 (200 μM) and rapamycin (100 nM) resulted in an increased number of Dcp2-labeled puncta. The positive PDM values were used to generate a heat map image that visualizes intensity of Dcp2 expressed within neurons. ICQ% values are shown for each experimental condition to quantify Dcp2 intensity in neurons. Scale bar is30 μm. *p<0.05, **p<0.01 and ***p< 0.001. n= 10 per condition.

Fig. 3

IL6 and NGF decrease while AMPK activators increase P body formation in TG neurons: Rck. Representative micrographs of TG neurons co-treated with IL-6 (50 ng/ml) and NGF (20 ng/ml) causing a reduction in Rck-labeled puncta. In contrast treatment with metformin (20 mM), A769662 (200 μM) resulted in an increased number of Rck-labeled puncta. Rapamycin (100 nM) had no effect. The positive PDM values were used to generate a heat map image that visualizes intensity of Rck expressed within neurons. ICQ% values are shown for each experimental condition to quantify Rck intensity in neurons. Scale bar is 30 μm. **p < 0.01 and ***p < 0.001. n = 10 per condition.

Fig. 4

Robust regulation of P bodies in axons. Representative micrographs of TG neurons and their axons treated with vehicle, co-treated with IL-6 (50 ng/ml) and NGF(20ng/ml) or treated with metformin (20 mM). Positive (+) PDM images show axonal localization of P bodies in vehicle treated axons and a paucity of Dcp2 labeled puncta in NGF and IL6 treated culture. Metformin increases Dcp2 labeling in TG axons. Scale bar=30 μm.