Nerve growth factor induces neurite outgrowth of PC12 cells by promoting Gβγ-microtubule interaction

Background: Assembly and disassembly of microtubules (MTs) is critical for neurite outgrowth and differentiation. Evidence suggests that nerve growth factor (NGF) induces neurite outgrowth from PC12 cells by activating the receptor tyrosine kinase, TrkA. G protein-coupled receptors (GPCRs) as well as heterotrimeric G proteins are also involved in regulating neurite outgrowth. However, the possible connection between these pathways and how they might ultimately converge to regulate the assembly and organization of MTs during neurite outgrowth is not well understood. Results: Here, we report that Gβγ, an important component of the GPCR pathway, is critical for NGF-induced neuronal differentiation of PC12 cells. We have found that NGF promoted the interaction of Gβγ with MTs and stimulated MT assembly. While Gβγ-sequestering peptide GRK2i inhibited neurite formation, disrupted MTs, and induced neurite damage, the Gβγ activator mSIRK stimulated neurite outgrowth, which indicates the involvement of Gβγ in this process. Because we have shown earlier that prenylation and subsequent methylation/demethylation of γ subunits are required for the Gβγ-MTs interaction in vitro, small-molecule inhibitors (L-28 and L-23) targeting prenylated methylated protein methyl esterase (PMPMEase) were tested in the current study. We found that these inhibitors disrupted Gβγ and ΜΤ organization and affected cellular morphology and neurite outgrowth. In further support of a role of Gβγ-MT interaction in neuronal differentiation, it was observed that overexpression of Gβγ in PC12 cells induced neurite outgrowth in the absence of added NGF. Moreover, overexpressed Gβγ exhibited a pattern of association with MTs similar to that observed in NGF-differentiated cells. Conclusions: Altogether, our results demonstrate that βγ subunit of heterotrimeric G proteins play a critical role in neurite outgrowth and differentiation by interacting with MTs and modulating MT rearrangement. Electronic supplementary material The online version of this article (doi:10.1186/s12868-014-0132-4) contains supplementary material, which is available to authorized users

Quantification of In Situ Denitrification Rates in Groundwater Below an Arable and a Grassland System

peer-reviewedUnderstanding denitrification rates in groundwater ecosystems can help predict where agricultural reactive nitrogen (N) contributes to environmental degradation. In situ groundwater denitrification rates were determined in subsoil, at the bedrock-interface and in bedrock at two sites, grassland and arable, using an in situ ‘push-pull’ method with 15N labelled nitrate (NO3--N). Measured groundwater denitrification rates ranged from 1.3 to 469.5 µg N kg-1d-1. Exceptionally high denitrification rates observed at the bedrock-interface at grassland site (470±152µg N kg-1d-1; SE, standard error) suggest that deep groundwater can serve as substantial hotspots for NO3--N removal. However, denitrification rates at the other locations were low and may not substantially reduce NO3--N delivery to surface waters. Denitrification rates were negatively correlated with ambient dissolved oxygen (DO), redox potential (Eh), ks and NO3- (all p-values p<0.01) and positively correlated with SO42- (p<0.05). Higher mean N2O/(N2O+N2) ratios at arable (0.28) site than the grassland (0.10) revealed that arable site has higher potential to indirect N2O emissions. Identification of areas with high and low denitrification and related site parameters can be a tool to manage agricultural N to safeguard the environment.Department of Agriculture and Food, Ireland - Research Stimulus Fund Programme (Grant RSF 06383