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Section MOLECULAR NEUROSCIENCE
Post-Translational Control of Vesicular Release by NO
Julie-Myrtille Bourgognon* and Joern Steinert
MRC Toxicology Unit, UK. * Presenting e-mail: [email protected]
Nitric oxide (NO) signalling is implicated in several neurodegenerative diseases through induction of high NO release. However, its exact contribution to degeneration remains elusive due to the complexity of downstream nitrergic targets. High levels of NO can induce post-translational modifications which are associated with neuronal degeneration 1,2. NO reacts with superoxide anions to form cytotoxic peroxynitrite which in turn leads to 3-Nitrotyrosination with largely detrimental changes in protein function. Additionally, NO signalling alters protein function through S-nitrosylation To date, little is known as to what extent these post-translational modifications contribute to or exacerbate neuronal dysfunction. We use glutamatergic synapses as a model system to identify novel nitrergic signalling pathways to correlate protein modifications with functional changes.
The Drosophila neuromuscular junction was used to characterise nitrergic effects employing electrophysiological methods. Two-electrode-voltage-clamp (TEVC) analyses were carried out in HL-3 solution using sharp electrodes (20-30MQ). Data denote mean±SEM (n-number) with *p<0.05 indicating statistical significance (t-test, ANOVA). Evoked excitatory junctional currents (eEJC) amplitudes and quantal content (QC) were strongly reduced following NO exposure for >40min (eEJC: Ctrl: 119±7nA (22) vs NO: 62±8nA* (14); QC: Ctrl: 189±12 vs NO: 104±12*) suggesting a reduction in presynaptic release. Cumulative postsynaptic current analysis (500ms 50Hz train) further showed a reduced number of release-ready vesicles following NO exposure (Ctrl: 276±21 (22) vs NO: 108±19* (14)). Fluctuation analysis estimating the number of available release sites further confirmed a strong reduction under NO conditions. The above NO effects were detected following inhibition of the soluble guanylyl cyclase (sGC) and absent in the presence of N-ethylmaleimide which prevents the formation of S-nitrosothiols suggesting that NO modulates release via post-translational modifications. This interpretation is also supported by the findings that nitric oxide synthase (NOS) KO NMJs showed a strongly enhanced synaptic release and larger available vesicle pools. Importantly, enhancing presynaptic S-Nitrosoglutathione reductase (GSNOR) or glutamate-cysteine ligase (GCLC) enzyme activities, by overexpression (OE), prevented the above nitrergic effects. Both pathways favour denitrosylation by reducing S-nitro-sothiols and elevating cellular glutathione, respectively.
Together, our data suggest that NO can modify synaptic signalling possibly via inducing post-translational protein modifications. This data interpretation is supported by the notion that sGC inhibition is ineffective but modulation of neuronal nitrosylation pathways impacts on synaptic physiology implying presynaptic actions of NO in a sGC-in-dependent manner. The data extends our understanding of NO signalling, potentially leading to the identification of putative targets disease.
Acknowledgements
This study was funded by the MRC (JS) and The Henry Smith Charity (JB). References
1. Steinert, J. R., Chernova, T. & Forsythe, I. D. The Neuroscientist 16, 435-52, (2010).
2. Nakamura, T. et al. Neurobiology of disease, (2015).
Dynamic Control of Neural Progenitor Fates in the Developing Neocortex
Debra Silver*
Duke University School of Medicine, USA. * Presenting e-mail: [email protected]
Our laboratory studies neurogenesis, the process whereby neural progenitors generate neurons of the cerebral cortex during embryonic development. Our long-term objective is to help broaden our fundamental understanding of how the brain is built, how stem cells behave, and the etiology of neurodevelopmental diseases. The lab employs genetic,
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26 Opera Med Physiol 2016 Vol. 2 (S1)
